US20040005666A1

US20040005666A1 - Methods and reagents for modulating cholesterol levels

Info

Publication number: US20040005666A1
Application number: US10/452,510
Authority: US
Inventors: Michael Hayden; Angela Brooks-Wiison; Simon Pimstone
Original assignee: Individual
Current assignee: Individual
Priority date: 1999-03-15
Filing date: 2003-06-02
Publication date: 2004-01-08
Also published as: US6617122B1; CA2367955A1; ATE364696T1; AU784108B2; WO2000055318A2; US20110065147A1; WO2000055318A3; AU3832700A; US8715968B2; US20040058869A1; WO2000055318A8; US20040157250A1; EP1100895B1; DE60035163T2; US20040185508A1; US8067219B2; JP4726302B2; EP1100895A2; JP2003513611A; DE60035163D1

Abstract

The invention features ABC1 nucleic acids and polypeptides for the diagnosis and treatment of abnormal cholesterol regulation. The invention also features methods for identifying compounds for modulating cholesterol levels in an animal (e.g., a human).

Description

This application claims priority from U.S. Provisional Application No. 60/124,702, filed Mar. 15, 1999, U.S. Provisional Application No. 60/138,048, filed Jun. 8, 1999, U.S. Provisional Application No. 60/139,600, filed Jun. 17, 1999, and U.S. Provisional Application No. 60/151,977, filed Sep. 1, 1999.[0001]

BACKGROUND OF THE INVENTION

Low HDL cholesterol (HDL-C), or hypoalphalipoproteinemia, is a blood lipid abnormality which correlates with a high risk of cardiovascular disease (CVD), in particular coronary artery disease (CAD), but also cerebrovascular disease, coronary restenosis, and peripheral vascular disease. HDL, or ‘good cholesterol’ levels are influenced by both environmental and genetic factors.

Epidemiological studies have consistently demonstrated that plasma HDL-C) concentration is inversely related to the incidence of CAD. HDL-C levels are a strong graded and independent cardiovascular risk factor. Protective effects of an elevated HDL-C persist until 80 years of age. A low HDL-C is associated with an increased CAD risk even with normal (<5.2 mmol/l) total plasma cholesterol levels. Coronary disease risk is increased by 2% in men and 3% in women for every 1 mg/dL (0.026 mmol/l) reduction in HDL-C and in the majority of studies this relationship is statistically significant even after adjustment for other lipid and non-lipid risk factors. Decreased HDL-C levels are the most common lipoprotein abnormality seen in patients with premature CAD. Four percent of patients with premature CAD with have an isolated form of decreased HDL-C levels with no other lipoprotein .abnormalities while 25% have low HDL levels with accompanying hypertriglyceridemia.

Even in the face of other dyslipidemias or secondary factors, HDL-C levels are important predictors of CAD. In a cohort of diabetics, those with isolated low HDL cholesterol had a 65% increased death rate compared to diabetics with normal HDL cholesterol levels (>0.9 mmol/l). Furthermore, it has been shown that even within high risk populations, such as those with familial hypercholesterolemia, HDL cholesterol level is an important predictor of CAD. Low HDL cholesterol levels thus constitute a major, independent, risk for CAD.

These findings have led to increased attention to HDL cholesterol levels as a focus for treatment, following the recommendations of the National Cholesterol Education Program. These guidelines suggest that HDL cholesterol values below 0.9 mmol/l confer a significant risk for men and women. As such, nearly half of patients with CAD would have low HDL cholesterol. It is therefore crucial that we obtain a better understanding of factors which contribute to this phenotype. In view of the fact that pharmacological intervention of low HDL cholesterol levels has so far proven unsatisfactory, it is also important to understand the factors that regulate these levels in the circulation as this understanding may reveal new therapeutic targets.

Absolute levels of HDL cholesterol may not always predict risk of CAD. In the case of CETP deficiency, individuals display an increased risk of developing CAD, despite increased HDL cholesterol levels. What seems to be important in this case is the functional activity of the reverse cholesterol transport pathway, the process by which intracellular cholesterol is trafficked out of the cell to acceptor proteins such as ApoAI or HDL. Other important genetic determinants of HDL cholesterol levels, and its inverse relation with CAD, may reside in the processes leading to HDL formation and intracellular cholesterol trafficking and efflux. To date, this process is poorly understood, however, and clearly not all of the components of this pathway have been identified. Thus, defects preventing proper HDL-mediated cholesterol efflux may be important predictors of CAD. Therefore it is critical to identify and understand novel genes involved in the intracellular cholesterol trafficking and efflux pathways.

HDL particles are central to the process of reverse cholesterol transport and thus to the maintenance of tissue cholesterol homeostasis. This process has multiple steps which include the binding of HDL to cell surface components, the acquisition of cholesterol by passive absorption, the esterification of this cholesterol by LCAT and the subsequent transfer of esterified cholesterol by CETP, to VLDL and chylomicron remnants for liver uptake. Each of these steps is known to impact the plasma concentration of HDL.

Changes in genes for ApoAI-CIII, lipoprotein lipase, CETP, hepatic lipase, and LCAT all contribute to determination of HDL-C levels in humans. One rare form of genetic HDL deficiency is Tangier disease (TD), diagnosed in approximately 40 patients world-wide, and associated with almost complete absence of HDL cholesterol (HDL-C) levels (listed in OMIM as an autosomal recessive trait (OMIM 205400)). These patients have very low HDL cholesterol and ApoAI levels, which have been ascribed to hypercatabolism of nascent HDL and ApoAI, due to a delayed acquisition of lipid and resulting failure of conversion to mature HDL. TD patients accumulate cholesterol esters in several tissues, resulting in characteristic features, such as enlarged yellow tonsils, hepatosplenomegaly, peripheral neuropathy, and cholesterol ester deposition in the rectal mucosa. Defective removal of cellular cholesterol and phospholipids by ApoAI as well as a marked deficiency in HDL mediated efflux of intracellular cholesterol has been demonstrated in TD fibroblasts. Even though this is a rare disorder, defining its molecular basis could identify pathways relevant for cholesterol regulation in the general population. The decreased availability of free cholesterol for efflux in the surface membranes of cells in Tangier Disease patients appears to be due to a defect in cellular lipid metabolism or trafficking. Approximately 45% of Tangier patients have signs of premature CAD, suggesting a strong link between decreased cholesterol efflux, low HDL cholesterol and CAD. As increased cholesterol is observed in the rectal mucosa of persons with TD, the molecular mechanism responsible for TD may also regulate cholesterol adsorption from the gastrointestinal (GI) tract.

A more common form of genetic HDL deficiency occurs in patients who have low plasma HDL cholesterol usually below the 5th percentile for age and sex (OMIM 10768), but an absence of clinical manifestations specific to Tangier disease (Marcil et al., Arterioscler. Thromb. Vasc. Biol. 19:159-169, 1999; Marcil et al., Arterioscler. Thromb. Vasc. Biol. 15:1015-1024, 1995). These patients have no obvious environmental factors associated with this lipid phenotype, and do not have severe hypertriglyceridemia nor have known causes of severe HDL deficiency (mutations in ApoAI, LCAT, or LPL deficiency) and are not diabetic. The pattern of inheritance of this condition is most consistent with a Mendelian dominant trait (OMIM 10768).

The development of drugs that regulate cholesterol metabolism has so far progressed slowly. Thus, there is a need for a better understanding of the genetic components of the cholesterol efflux pathway. Newly-discovered components can then serve as targets for drug design.

Low HDL levels are likely to be due to multiple genetic factors. The use of pharmacogenomics in the aid of designing treatment tailored to the patient makes it desirable to identify polymorphisms in components of the cholesterol efflux pathway. An understanding of the effect of these polymorphisms on protein function would allow for the design of a therapy that is optimal for the patient.

SUMMARY OF THE INVENTION

In a first aspect, the invention features a substantially pure ABC1 polypeptide having ABC1 biological activity. Preferably, the ABC1 polypeptide is human ABC1 (e.g., one that includes amino acids 1 to 60 or amino acids 61 to 2261 of SEQ ID NO: 1). In one preferred embodiment, the ABC1 polypeptide includes amino acids 1 to 2261 of SEQ ID NO: 1.

Specifically excluded from the polypeptides of the invention are the polypeptide having the exact amino acid sequence as GenBank accession number CM10005.1 and the nucleic acid having the exact sequence as AJ012376.1. Also excluded is protein having the exact amino acid sequence as GenBank accession number X75926.

In a related aspect, the invention features a substantially pure ABC1 polypeptide that includes amino acids 1 to 2261 of SEQ ID NO: 1.

In another aspect, the invention features a substantially pure nucleic acid molecule encoding an ABC1 polypeptide having ABC1 biological activity (e.g., a nucleic acid molecule that includes nucleotides 75 to 254 or nucleotides 255 to 6858 of SEQ ID NO: 2). In one preferred embodiment, the nucleic acid molecule includes nucleotides 75 to 6858 of SEQ ID NO: 2.

In a related aspect, the invention features an expression vector, a cell, or a non-human mammal that includes the nucleic acid molecule of the invention.

In yet another aspect, the invention features a substantially pure nucleic acid molecule that includes nucleotides 75 to 254 of SEQ ID NO: 2, nucleotides 255 to 6858 of SEQ ID NO: 2, or nucleotides 75 to 6858 of SEQ ID NO: 2.

In still another aspect, the invention features a substantially pure nucleic acid molecule that includes at least fifteen nucleotides corresponding to the 5′ or 3′ untranslated region from a human ABC1 gene. Preferably, the 3′ untranslated region includes nucleotides 7015-7860 of SEQ ID NO: 2.

In a related aspect, the invention features a substantially pure nucleic acid molecule that hybridizes at high stringency to a probe comprising nucleotides 7015-7860 of SEQ ID NO: 2.

In another aspect, the invention features a method of treating a human having low HDL cholesterol or a cardiovascular disease, including administering to the human an ABC1 polypeptide, or cholesterol-regulating fragment thereof, or a nucleic acid molecule encoding an ABC1 polypeptide, or cholesterol-regulating fragment thereof. In a preferred embodiment, the human has a low HDL cholesterol level relative to normal. Preferably, the ABC1 polypeptide is wild-type ABC1, or has a mutation that increases its stability or its biological activity. A preferred biological activity is regulation of cholesterol.

In a related aspect, the invention features a method of preventing or treating cardiovascular disease, including introducing into a human an expression vector comprising an ABC1 nucleic acid molecule operably linked to a promoter and encoding an ABC1 polypeptide having ABC1 biological activity.

In another related aspect, the invention features a method of preventing or ameliorating the effects of a disease-causing mutation in an ABC1 gene, including introducing into a human an expression vector comprising an ABC1 nucleic acid molecule operably linked to a promoter and encoding an ABC1 polypeptide having ABC1 biological activity.

In still another aspect, the invention features a method of treating or preventing cardiovascular disease, including administering to an animal (e.g., a human) a compound that mimes the activity of wild-type ABC1 or modulates the biological activity of ABC1.

One preferred cardiovascular disease that can be treated using the methods of the invention is coronary artery disease. Others include cerebrovascular disease and peripheral vascular disease.

The discovery that the ABC1 gene and protein are involved in cholesterol transport that affects serum HDL levels allows the ABC1 protein and gene to be used in a variety of diagnostic tests and assays for identification of HDL-increasing or CVD-inhibiting drugs. In one family of such assays, the ability of domains of the ABC1 protein to bind ATP is utilized; compounds that enhance this binding are potential HDL-increasing drugs. Similarly, the anion transport capabilities and membrane pore-forming functions in cell membranes can be used for drug screening.

ABC1 expression can also serve as a diagnostic tool for low HDL or CVD; determination of the genetic subtyping of the ABC1 gene sequence can be used to subtype low HDL individuals or families to determine whether the low HDL phenotype is related to ABC1 function. This diagnostic process can lead to the tailoring of drug treatments according to patient genotype (referred to as pharmacogenomics), including prediction of the patient's response (e.g., increased or decreased efficacy or undesired side effects upon administration of a compound or drug.

Antibodies to an ABC1 polypeptide can be used both as therapeutics and diagnostics. Antibodies are produced by immunologically challenging a B-cell-containing biological system, e.g., an animal such as a mouse, with an ABC1 polypeptide to stimulate production of anti-ABC1 protein by the B-cells, followed by isolation of the antibody from the biological system. Such antibodies can be used to measure ABC1 polypeptide in a biological sample such as serum, by contacting the sample with the antibody and then measuring immune complexes as a measure of the ABC1 polypeptide in the sample. Antibodies to ABC1 can also be used as therapeutics for the modulation of ABC1 biological activity.

Thus, in another aspect, the invention features a purified antibody that specifically binds to ABC1.

In yet another aspect, the invention features a method for determining whether a candidate compound modulates ABC1 biological activity, comprising: (a) providing an ABC1 polypeptide; (b) contacting the ABC1 polypeptide with the candidate compound; and (c) measuring ABC1 biological activity, wherein altered ABC1 biological activity, relative to an ABC1 polypeptide not contacted with the compound, indicates that the candidate compound modulates ABC1 biological activity. Preferably, the ABC1 polypeptide is in a cell or is in a cell-free assay system.

In still another aspect, the invention features a method for determining whether a candidate compound modulates ABC1 expression. The method includes (a) providing a nucleic acid molecule comprising an ABC1 promoter operably linked to a reporter gene; (b) contacting the nucleic acid molecule with the candidate compound; and (c) measuring reporter gene expression, wherein altered reporter gene expression, relative to a nucleic acid molecule not contacted with the compound, indicates that the candidate compound modulates ABC1 expression.

In another aspect, the invention features a method for determining whether candidate compound is useful for modulating cholesterol levels, the method including the steps of: (a) providing an ABC1 polypeptide; (b) contacting the polypeptide with the candidate compound; and (c) measuring binding of the ABC1 polypeptide, wherein binding of the ABC1 polypeptide indicates that the candidate compound is useful for modulating cholesterol levels.

In a related aspect, the invention features method for determining whether a candidate compound mimics ABC1 biological activity. The method includes (a) providing a cell that is not expressing an ABC1 polypeptide; (b) contacting the cell with the candidate compound; and (c) measuring ABC1 biological activity of the cell, wherein altered ABC1 biological activity, relative to a cell not contacted with the compound, indicates that the candidate compound modulates ABC1 biological activity. Preferably, the cell has an ABC1 null mutation. In one preferred embodiment, the cell is in a mouse or a chicken (e.g., a WHAM chicken) in which its ABC1 gene has been mutated.

In still another aspect, the invention features a method for determining whether a candidate compound is useful for the treatment of low HDL cholesterol. The method includes (a) providing an ABC transporter (e.g., ABC1); (b) contacting the transporter with the candidate compound; and (c) measuring ABC transporter biological activity, wherein increased ABC transporter biological activity, relative to a transporter not contacted with the compound, indicates that the candidate compound is useful for the treatment of low HDL cholesterol. Preferably the ABC transporter is in a cell or a cell free assay system.

In yet another aspect, the invention features a method for determining whether candidate compound is useful for modulating cholesterol levels. The method includes (a) providing a nucleic acid molecule comprising an ABC transporter promoter operably linked to a reporter gene; (b) contacting the nucleic acid molecule with the candidate compound; and (c) measuring expression of the reporter gene, wherein increased expression of the reporter gene, relative to a nucleic acid molecule not contacted with the compound, indicates that the candidate compound is useful for modulating cholesterol levels.

In still another aspect, the invention features a method for determining whether a candidate compound increases the stability or decreases the regulated catabolism of an ABC transporter polypeptide. The method includes (a) providing an ABC transporter polypeptide; (b) contacting the transporter with the candidate compound; and (c) measuring the half-life of the ABC transporter polypeptide, wherein an increase in the half-life, relative to a transporter not contacted with the compound, indicates that the candidate compound increases the stability or decreases the regulated catabolism of an ABC transporter polypeptide. Preferably the ABC transporter is in a cell or a cell free assay system.

In a preferred embodiment of the screening methods of the present invention, the cell is in an animal. The preferred ABC transporters are ABC1, ABC2, ABCR, and ABC8, and the preferred biological activity is transport of cholesterol (e.g., HDL cholesterol or LDL cholesterol) or interleukin-1, or is binding or hydrolysis of ATP by the ABC1 polypeptide.

Preferably, the ABC1 polypeptide used in the screening methods includes amino acids 1-60 of SEQ ID NO: 1. Alternatively, the ABC1 polypeptide can include a region encoded by a nucleotide sequence that hybridizes under high stringency conditions to nucleotides 75 to 254 of SEQ ID NO: 2.

In another aspect, the invention features a method for determining whether a patient has an increased risk for cardiovascular disease. The method includes determining whether an ABC1 gene of the patient has a mutation, wherein a mutation indicates that the patient has an increased risk for cardiovascular disease.

In related aspect, the invention features a method for determining whether a patient has an increased risk for cardiovascular disease. The method includes determining whether an ABC1 gene of the patient has a polymorphism, wherein a polymorphism indicates that the patient has an increased risk for cardiovascular disease.

In another aspect, the invention features a method for determining whether a patient has an increased risk for cardiovascular disease. The method includes measuring ABC1 biological activity in the patient, wherein increased or decreased levels in the ABC1 biological activity, relative to normal levels, indicates that the patient has an increased risk for cardiovascular disease.

In still another aspect, the invention features a method for determining whether a patient has an increased risk for cardiovascular disease. The method includes measuring ABC1 expression in the patient, wherein decreased levels in the ABC1 expression relative to normal levels, indicates that the patient has an increased risk for cardiovascular disease. Preferably, the ABC1 expression is determined by measuring levels of ABC1 polypeptide or ABC1 RNA.

In another aspect, the invention features a non-human mammal having a transgene comprising a nucleic acid molecule encoding a mutated ABC1 polypeptide. In one embodiment, the mutation is a dominant-negative mutation.

In a related aspect, the invention features a non-human mammal, having a transgene that includes a nucleic acid molecule encoding an ABC1 polypeptide having ABC1 biological activity.

In another related aspect, the invention features a cell from a non-human mammal having a transgene that includes a nucleic acid molecule encoding an ABC1 polypeptide having ABC1 biological activity.

In still another aspect, the invention features a method for determining 1o whether a candidate compound decreases the inhibition of a dominant-negative ABC1 polypeptide. The method includes (a) providing a cell expressing a dominant-negative ABC1 polypeptide; (b) contacting the cell with the candidate compound; and (c) measuring ABC1 biological activity of the cell, wherein an increase in the ABC1 biological activity, relative to a cell not contacted with the compound, indicates that the candidate compound decreases the inhibition of a dominant-negative ABC1 polypeptide.

By “polypeptide” is meant any chain of more than two amino acids, regardless of post-translational modification such as glycosylation or phosphorylation.

By “substantially identical” is meant a polypeptide or nucleic acid exhibiting at least 50%, preferably 85%, more preferably 90%, and most preferably 95% identity to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.

Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). This software program matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

By “high stringency conditions” is meant hybridization in 2×SSC at 40_C with a DNA probe length of at least 40 nucleotides. For other definitions of high stringency conditions, see F. Ausubel et al., Current Protocols in Molecular Biology, pp. 6.3.1-6.3.6, John Wiley & Sons, New York, N.Y., 1994, hereby incorporated by reference.

By “substantially pure polypeptide” is meant a polypeptide that has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the polypeptide is an ABC1 polypeptide that is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, pure. A substantially pure ABC1 polypeptide may be obtained, for example, by extraction from a natural source (e.g., a pancreatic cell), by expression of a recombinant nucleic acid encoding a ABC1 polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

A polypeptide is substantially free of naturally associated components when it is separated from those contaminants that accompany it in its natural state. Thus, a polypeptide which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include those which naturally occur in eukaryotic organisms but are synthesized in E. coli or other prokaryotes.

By “substantially pure nucleic acid” is meant nucleic acid that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid of the invention is derived, flank the nucleic acid. The term therefore includes, for example, a recombinant nucleic acid that is incorporated into a vector; into an autonomously replicating plasmid or virus; into the genomic nucleic acid of a prokaryote or a eukaryote cell; or that exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant nucleic acid that is part of a hybrid gene encoding additional polypeptide sequence.

By “modulates” is meant increase or decrease. Preferably, a compound that modulates cholesterol levels (e.g., HDL-cholesterol levels, LDL-cholesterol levels, or total cholesterol levels), or ABC1 biological activity, expression, stability, or degradation does so by at least 10%, more preferably by at least 25%, and most preferably by at least 50%.

By “purified antibody” is meant antibody which is at least 60%, by weight, free from proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, antibody. A purified antibody may be obtained, for example, by affinity chromatography using recombinantly- produced protein or conserved motif peptides and standard techniques.

By “specifically binds” is meant an antibody that recognizes and binds to, for example, a human ABC1 polypeptide but does not substantially recognize and bind to other non-ABC1 molecules in a sample, e.g., a biological sample, that naturally includes protein. A preferred antibody binds to the ABC1 polypeptide sequence of FIG. 9A (SEQ ID NO: 1).

By “polymorphism” is meant that a nucleotide or nucleotide region is characterized as occurring in several different forms. A “mutation” is a form of a polymorphism in which the expression level, stability, function, or biological activity of the encoded protein is substantially altered.

By “ABC transporter” or “ABC polypeptide” is meant any transporter that hydrolyzes ATP and transports a substance across a membrane. Preferably, an ABC transporter polypeptide includes an ATP Binding Cassette and a transmembrane region. Examples of ABC transporters include, but are not limited to, ABC1, ABC2, ABCR, and ABC8.

By “ABC1 polypeptide” is meant a polypeptide having substantial identity to an ABC1 polypeptide having the amino acid sequence of SEQ ID NO: 1.

By “ABC biological activity” or “ABC1 biological activity” is meant hydrolysis or binding of ATP, transport of a compound (e.g., cholesterol, interleukin-1) or ion across a membrane, or regulation of cholesterol or phospholipid levels (e.g., either by increasing or decreasing HDL-cholesterol or LDL-cholesterol levels).

The invention provides screening procedures for identifying therapeutic compounds (cholesterol-modulating or anti-CVD pharmaceuticals) which can be used in human patients. Compounds that modulate ABC biological activity (e.g., ABC1 biological activity) are considered useful in the invention, as are compounds that modulate ABC concentration, protein stability, regulated catabolism, or its ability to bind other proteins or factors. In general, the screening methods of the invention involve screening any number of compounds for therapeutically active agents by employing any number of in vitro or in vivo experimental systems. Exemplary methods useful for the identification of such compounds are detailed below.

The methods of the invention simplify the evaluation, identification and development of active agents for the treatment and prevention of low HDL and CVD. In general, the screening methods provide a facile means for selecting natural product extracts or compounds of interest from a large population which are further evaluated and condensed to a few active and selective materials. Constitutes of this pool are then purified and evaluated in the methods of the invention to determine their HDL-raising or anti-CVD activities or both.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are schematic illustrations showing two pedigrees with Tangier Disease, (TD-1 and TD-2). Square and circle symbols represent males and females, respectively. Diagonal lines are placed through the symbols of all deceased individuals. A shaded symbol on both alleles indicates the probands with Tangier Disease. Individuals with half shaded symbols have HDL-C levels at or below the 10th percentile for age and sex, while those with quarter shaded symbols have HDL-C between the 11th and 20th percentiles. [0063]
Each individual's ID number, age at the time of lipid measurement, triglyceride level and HDL cholesterol level followed by their percentile ranking for age and sex are listed below the pedigree symbol. Markers spanning the 9q31.1 region are displayed to the left of the pedigree. The affected allele is represented by the darkened bars which illustrate the mapping of the limits of the shared haplotype region as seen in FIG. 3. Parentheses connote inferred marker data, questions marks indicate unknown genotypes, and large arrows show the probands. [0064]
FIG. 1C shows ApoAI (10 μg/mL) -mediated cellular cholesterol efflux in control fibroblasts (n=5, normalized to 100%) and two subjects with Tangier disease (TD). Cells were [0065] ³H-cholesterol (0.2 □Ci/mL) labeled during growth and cholesterol (20 □g/mL) loaded in growth arrest. Cholesterol efflux is determined as ³H medium/(³H cell+³H medium)
FIGS. [0066] 2A-2D are schematic illustrations showing four French Canadian pedigrees with FHA (FHA-1 to −4). The notations are as in FIG. 1. Exclamation points on either side of a genotype (as noted in Families FHA-3 and FHA-4) are used when the marker data appears to be inconsistent due to potential microsatellite repeat expansions. A bar that becomes a single thin line suggests that the haplotype is indeterminate at that marker.
FIGS. [0067] 3A-3E are a schematic illustration showing a genetic and physical map of 9q31 spanning 35 cM. FIG. 3A: YACs from the region of 9q22-34 were identified and a YAC contig spanning this region was constructed. FIG. 3B: A total of 22 polymorphic CA microsatellite markers were mapped to the contig and used in haplotype analysis in TD-1 and TD-2. FIG. 3C: The mutant haplotypes for probands in TD-1 and -2 indicate a significant region of homozygosity in TD-2, while the proband in TD-1 has 2 different mutant haplotypes. The candidate region can be narrowed to the region of homozygosity for CA markers in proband 2. A critical crossover at D9S1690 in TD-1 (A)* also provides a centromeric boundary for the region containing the gene. Three candidate genes in this region (ABC1, LPA-R and RGS-3) are shown. FIG. 3D: Meiotic recombinations in the FHA families (A-H) refine the minimal critical region to 1.2 cM between D9S277 and D9S1866. The heterozygosity of the TD-2 proband at D9S127, which ends a continuous region of homozygosity in TD-2, further refines the region to less than 1 cM. This is the region to which ABC1 has been mapped. FIG. 3E: Isolated YAC DNA and selected markers from the region were used to probe high-density BAC grid filters, selecting BACs which via STS-content mapping produced an 800 Kb contig. Four BACs containing ABC1 were sequenced using high-throughput methods.
FIG. 4A shows sequence of one mutation in family TD-1. Patient 111-01 is heterozygous for a T to C transition at [0068] nucleotide 4503 of the cDNA; the control is homozygous for T at this position. This mutation corresponds to a cysteine to arginine substitution in the ABC1 protein (C1477R).
FIG. 4B shows the amino acid sequence conservation of residue 1477 in mouse and human, but not a related [0069] C. elegans gene. A change from cysteine to arginine likely has an important effect on the protein secondary and tertiary structure, as noted by its negative scores in most substitution matrices (Schuler et al., A Practical Guide to the Analysis of Genes and Proteins, eds. Baxevanis, A. D. & Ouellette, B. F. F. 145:171, 1998). The DNA sequences of the normal and mutant genes are shown above and below the amino acid sequences, respectively.
FIG. 4C shows the segregation of the T4503C mutation in TD-1. The presence of the T4503C mutation (+) was assayed by restriction enzyme digestion with Hgal, which cuts only the mutant (C) allele (0). Thus, in the absence of the mutation, only the 194 bp PCR product (amplified between ø and Ø) is observed, while in its presence the PCR product is cleaved into fragments of 134 bp and 60 bp. The proband (individual 111.01) was observed to be heterozygous for this mutation (as indicated by both the 194 bp and 134 bp bands), as were his daughter, father, and three paternal cousins. A fourth cousin and three of the father's siblings were not carriers of this mutation. [0070]
FIG. 4D shows Northern blot analysis with probes spanning the complete ABC1 gene reveal the expected ˜8 Kb transcript and, in addition, a −3.5 kb truncated transcript only seen in the proband TD-1 and not in TD-2 or control. This was detected by probes spanning exons 1-49 (a), 1-41 (b), 1-22 (c), and 23-29 (d), but not with probes spanning exons 30-41 (e) or 42-49 (f). [0071]
FIG. 5A shows the sequence of the mutation in family TD-2. Patient IV-10 is homozygous for an A to G transition at [0072] nucleotide 1864 of the cDNA (SEQ ID NO: 2); the control is homozygous for A at this position. This mutation corresponds to a glutamine to arginine substitution in the ABC1 protein (Q597R).
FIG. 5B shows that the glutamine amino acid, which is mutated in the TD-2 proband, is conserved in human and mouse ABC1 as well as in an ABC orthologue from [0073] C. elegans, revealing the specific importance of this residue in the structure/function of this ABC protein in both worms and mammals. The DNA sequences of the normal and mutant proteins are shown above and below the amino acid sequences, respectively.
FIG. 5C shows the segregation of the A1864G mutation in TD-2. The presence of the A1864G mutation (indicated by +) was assayed by restriction enzyme digestion with Acil. The 360 bp PCR product has one invariant Acil recognition site (Ö), and a second one is created by the Al 864G mutation. The wild-type allele is thus cleaved to fragments of 215 bp and 145 bp, while the mutant allele (G-allele) is cleaved to fragments of 185 bp, 145 bp and 30 bp. The proband (individual IV-10), the product of a consanguineous mating, was homozygous for the A1864G mutation (+/+), as evidenced by the presence of only the 185 bp and 145 bp bands, while four other family members for whom DNA was tested are heterozygous carriers of this mutation (both the 215 bp and 185 bp fragments were present). Two unaffected individuals (−/−), with only the 215 bp and 145 bp bands are shown for comparison. [0074]
FIG. 6A shows a sequence of the mutation in family FHA-1. Patient 111-01 is heterozygous for a deletion of nucleotides 2151-2153 of the CDNA (SEQ ID NO: 2). This deletion was detected as a superimposed sequence starting at the first nucleotide after the deletion. This corresponds to deletion of [0075] leucine 693 in the ABC1 protein (SEQ ID NO: 1).
FIG. 6B is an alignment of the human and mouse wild-type amino acid sequences, showing that the human and mouse sequences are identical in the vicinity of L693. L693 is also conserved in [0076] C. elegans. This highly conserved residue lies within a predicted transmembrane domain. The DNA sequences of the normal and mutant proteins are shown above and below the amino acid sequences, respectively.
FIG. 6C shows segregation of the L693 mutation in FHA-1, as assayed by Earl restriction digestion. Two invariant Earl restriction sites (indicated by 0) are present within the 297 bp PCR product located between the horizontal arrows (øØ) while a third site is present in the wild-type allele only. The presence of the mutant allele is thus distinguished by the presence of a 210 bp fragment (+), while the normal allele produces a 151 bp fragment (−). The proband of this family (III.01) is heterozygous for this mutation, as indicated by the presence of both the 210 and 151 bp bands. [0077]
FIG. 6D shows a sequence of the mutation in family FHA-3. Patient 111-01 is heterozygous for a deletion of nucleotides 5752-5757 of the cDNA (SEQ ID NO: 2). This deletion was detected as a superimposed sequence starting at the first nucleotide after the deletion. This corresponds to deletion of glutamic acid 1893 and aspartic acid 1894 in the ABC1 protein (SEQ ID NO: 1). [0078]
FIG. 6E is an alignment of the human and mouse wild-type amino acid sequences, showing that the human and mouse sequences are identical in the vicinity of 5752-5757. This region is highly conserved in [0079] C. elegans. The DNA sequences of the normal and mutant proteins are shown above and below the amino acid sequences, respectively.
FIG. 6F shows a sequence of the mutation in family FHA-2. Patient 111-01 is heterozygous for a for a C to T transition at nucleotide 6504 of the cDNA (SEQ ID NO: 2). This alteration converts an arginine at position 2144 of SEQ ID NO: 1 to a STOP codon, causing truncation of the last 118 amino acids of the ABC1 protein. [0080]
FIGS. 7A and 7B show cholesterol efflux from human skin fibroblasts treated with ABC1 antisense oligonucleotides. Fibroblasts from a control subject were labeled with [0081] ³H cholesterol (0.2 □Ci/mL) during growth for 48 hours and transfected with 500 nM ABC1 antisense AN-1 (5′-GCA GAG GGC ATG GCT TTA TTT G-3′; SEQ ID NO: 3) with 7.5 μg lipofectin for 4 hours. Following transfection, cells were cholesterol loaded (20 μg/mL) for 12 hours and allowed to equilibrate for 6 hours. Cells were either then harvested for total RNA and 10 μg was used for Northern blot analysis. Cholesterol efflux experiments were carried out as described herein. FIG. 7A: AN-1 was the oligonucleotide that resulted in a predictable decrease in ABC1 RNA transcript levels. FIG. 7B: A double antisense transfection method was used. In this method, cells were labeled and transfected with AN-1 as above, allowed to recover for 20 hours, cholesterol loaded for 24 hours, and then re-transfected with AN-1. Twenty hours after the second transfection, the cholesterol efflux as measured. A -50% decrease in ABC1 transcript levels was associated with a significant decrease in cholesterol efflux intermediate between that seen in wild-type and TD fibroblasts.
FIG. 7C shows show cholesterol efflux from human skin fibroblasts treated with antisense oligonucleotides directed to the region encoding the amino-terminal 60 amino acids. Note that the antisense oligonucleotide AN-6, which is directed to the previously unrecognized translation start site, produces a substantial decrease in cellular cholesterol efflux. [0082]
FIG. 8 is a schematic illustration showing predicted topology, mutations, and polymorphisms of ABC1 in Tangier disease and FHA. The two transmembrane and ATP binding domains are indicated. The locations of mutations are indicated by the arrows with the amino acid changes, which are predicted from the human ABC1 cDNA sequence. These mutations occur in different regions of the ABC1 protein. [0083]
FIG. 9A shows the amino acid sequence of the human ABC1 protein (SEQ ID NO: 1). [0084]
FIGS. [0085] 9B-9E show the nucleotide sequence of the human ABC1 cDNA (SEQ ID NO: 2).
FIG. 10 shows the 5′ and 3′ nucleotide sequences suitable for use as 5′ and 3′ PCR primers, respectively, for the amplification of the indicated ABC1 exon. [0086]
FIG. 11 shows a summary of alterations found in ABC1, including sequencing errors, mutations, and polymorphisms. [0087]
FIG. 12 shows a series of genomic contigs (SEQ ID NOS. 14-29) containing the ABC1 promoter (SEQ ID NO: 14), as well as exons 1-49 (and flanking intronic sequence) of ABC1. The exons (capitalized letters) are found in the contigs as follows: SEQ ID NO: 14—[0088] exon 1; SEQ ID NO: 15—exon 2; SEQ ID NO: 16—exon 3; SEQ ID NO: 17—exon 4; SEQ ID NO: 18—exon 5; SEQ ID NO: 19—exon 6; SEQ ID NO: 20— exons 7 and 8; SEQ ID NO: 21—exons 9 through 22; SEQ ID NO: 22—exons 23 through 28; SEQ ID NO: 23—exon 29; SEQ ID NO: 24— exons 30 and 31; SEQ ID NO: 25—exon 32; SEQ ID NO: 26—exons 33 through 36; SEQ ID NO: 27—exons 37 through 41; SEQ ID NO: 28—exons 42-45; SEQ ID NO: 29—exons 46-49.
FIG. 13 is a series of illustrations showing that the amino-terminal 60 amino acid region of ABC1 is protein-coding. Lysates of normal human fibroblasts were immunoblotted in parallel with a rabbit polyclonal antibody to amino acids 1-20 of human ABC1 (1); a rabbit polyclonal antibody to amino acids 1430-1449 of human ABC1 (2); and a mouse monoclonal antibody to amino acids 2236-2259 of human ABC1. The additional bands detected in [0089] lane 2 may be due to a lack of specificity of that antibody or the presence of degradation products of ABC1.
FIG. 14 is a schematic illustration showing that the WHAM chicken contains a non-conservative substitution (G265A) resulting in an amino acid change (E89K). [0090]
FIG. 15 is a schematic illustration showing that the mutation in the WHAM chicken is at an amino acid that is conserved among human, mouse, and chicken. [0091]
FIG. 16 show a summary of locations of consensus transcription factor binding sites in the human ABC1 promoter (nucleotides 1-8238 of SEQ ID NO: 14). The abbreviations are as follows: PPRE=peroxisome proliferator-activated receptor. SRE=steroid response element-binding protein site. ROR=RAR-related orphan receptor.[0092]

DETAILED DESCRIPTION

Genes play a significant role influencing HDL levels. Tangier disease (TD) was the first reported genetic HDL deficiency. The molecular basis for TD is unknown, but has been mapped to 9q31 in three families. We have identified two additional probands and their families, and confirmed linkage and refined the locus to a limited genomic region. Mutations in the ABC1 gene accounting for all four alleles in these two families were detected. A more frequent cause of low HDL levels is a distinct disorder, familial HDL deficiency (FHA). On the basis of independent linkage, meiotic recombinants and disease associated haplotypes, FHA was localized to a small genomic region encompassing the ABC1 gene. A mutation in a conserved residue in ABC1 segregated with FHA. Antisense reduction of the ABC1 transcript in fibroblasts was associated with a significant decrease in cholesterol efflux. [0093]
Cholesterol is normally assembled with intracellular lipids and secreted, but in TD the process is diverted and cholesterol is degraded in lysosomes. This disturbance in intracellular trafficking of cholesterol results in an increase in intracellular cholesterol ester accumulation associated with morphological changes of lysosomes and the Golgi apparatus and cholesteryl ester storage in histiocytes, Schwann cells, smooth muscle cells, mast cells and fibroblasts. [0094]
The clinical and biochemical heterogeneity in patients with TD has led to the possibility that genetic heterogeneity may also underlie this disorder. Considering this, we initially performed linkage analysis on these two families of different ancestries (TD-1 is Dutch, TD-2 is British; Frohlich et al., Clin. Invest. Med. 10:377-382, 1987) and confirmed that the genetic mutations underlying TD in these families were localized to the same 9q31 region, to which a large family with TD had been assigned (Rust et al., Nature Genetics 20:96-98, 1998). Detailed haplotype analysis, together with the construction of a physical map, refined the localization of this gene. Mutations in the ABC1 gene were found in TD. [0095]
FHA is much more common than TD, although its precise frequency is not known. While TD has been described to date in only 40 families, we have identified more than 40 FHA families in the Netherlands and Quebec alone. After initial suggestions of linkage to 9q31, thirteen polymorphic markers spanning approximately 10 cM in this region were typed and demonstrated the highest LOD score at D9S277. Analysis of the homozygosity of markers in the TD-2 proband, who was expected to be homozygous for markers close to TD due to his parents' consanguinity, placed the TD gene distal to D95127. Combined genetic data from TD and FHA families pointed to the same genomic segment spanning approximately 1,000 kb between D9S127 and D9S1866. The ABC1 transporter gene was contained within the minimal genomic region. RT-PCR analysis in one family demonstrated a deletion of leucine at residue 693 (693) in the first transmembrane domain of ABC1, which segregated with the phenotype of HDL deficiency in this family. [0096]
ABC1 is part of the ATP-binding cassette (ABC transporter) superfamily, which is involved in energy-dependent transport of a wide variety of substrates across membranes (Dean et al., Curr. Opin. Gen. Dev. 5:779-785, 1995). These proteins have characteristic motifs conserved throughout evolution which distinguish this class of proteins from other ATP binding proteins. In humans these genes essentially encode two ATP binding segments and two transmembrane domains (Dean et al., Curr. Opin. Gen. Dev. 5:779-785, 1995). We have now shown that the ABC1 transporter is crucial for intracellular cholesterol transport. [0097]
We have demonstrated that reduction of the ABC1 transcript using oligonucleotide antisense approaches results in decreased efflux, clearly demonstrating the link between alterations in this gene and its functional effects. TD and FHA now join the growing list of genetic diseases due to defects in the ABC group of proteins including cystic fibrosis (Zielenski, et al., Annu. Rev. Genet. 29:777-807, 1995), adrenoleukodystrophy (Mosser et al., Nature 361: 726-730, 1993), Zellweger syndrome (Gärtner et al., Nat. Genet. 1:23, 1992), progressive familial intrahepatic cholestatis (Bull et al., Nat. Genet. 18:219-224, 1998), and different eye disorders including Stargardt disease (Allikmets et al., Nat. Genet.15:236-246, 1997), autosomal recessive retinitis pigmentosa (Allikmets et al., Science 277:1805-1807, 1997), and cone-rod dystrophy (Cremers et al., Hum. Mol. Genet. 7:355-362, 1998). [0098]
Patients with TD have been distinguished from patients with FHA on the basis that Tangier disease was an autosomal recessive disorder (OMIM 20540) while FHA is inherited as an autosomal dominant trait (OMIM 10768). Furthermore, patients with TD have obvious evidence for intracellular cholesterol accumulation which is not seen in FHA patients. It is now evident that heterozygotes for TD do have reduced HDL levels and that the same mechanisms underlie the HDL deficiency and cholesterol efflux defects seen in heterozygotes for TD as well as FHA. Furthermore, the more severe phenotype in TD represents loss of function from both alleles of the ABC1 gene. [0099]
ABC1 is activated by protein kinases, presumably via phosphorylation, which also provides one explanation for the essential role of activation of protein kinase C in promoting cholesterol efflux (Drobnick et al., Arterioscler. Thromb. Vasc. Biol. 15: 1369-1377, 1995). Brefeldin, which inhibits trafficking between the endoplasmic reticulum and the Golgi, significantly inhibits cholesterol efflux, essentially reproducing the effect of mutations in ABC1, presumably through the inhibition of ABC1 biological activity. This finding has significance for the understanding of mechanisms leading to premature atherosclerosis. TD homozygotes develop premature coronary artery disease, as seen in the proband of TD-1 (111-01) who had evidence for coronary artery disease at 38 years. This is particular noteworthy as TD patients, in addition to exhibiting significantly reduced HDL, also have low LDL cholesterol, and yet they develop atherosclerosis despite this. This highlights the importance of HDL intracellular transport as an important mechanism in atherogenesis. There is significant evidence that heterozygotes for TD are also at increased risk for premature vascular disease (Schaefer et al., Ann. Int. Med. 93:261-266, 1980; Serfaty-Lacrosniere et al., Atherosclerosis 107:85-98, 1994). There is also preliminary evidence for premature atherosclerosis in some probands with FHA (FIG. 2B), e.g., the proband in FHA-2 (111-01) had a coronary artery bypass graft at 46 years while the proband in FHA-3 (FIG. 2C) had evidence for CAD around 50 years of age. The TD-1 proband had more severe efflux deficiency than the TD-2 proband (FIG. 1C). Interestingly, the TD-2 proband had no evidence for CAD by 62 when he died of unrelated causes, providing preliminary evidence for a relationship between the degree of cholesterol efflux (mediated in part by the nature of the mutation) and the likelihood of atherosclerosis. [0100]
The ABC1 gene plays a crucial role in cholesterol transport and, in particular, intracellular cholesterol trafficking in monocytes and fibroblasts. It also appears to play a significant role in other tissues such as the nervous system, GI tract, and the cornea. Completely defective intracellular cholesterol transport results in peripheral neuropathy, corneal opacities, and deposition of cholesterol esters in the rectal mucosa. [0101]
HDL deficiency is heterogeneous in nature. The delineation of the genetic basis of TD and FHA underlies the importance of this particular pathway in intracellular cholesterol transport, and its role in the pathogenesis of atherosclerosis. Unraveling of the molecular basis for TD and FHA defines a key step in a poorly defined pathway of cholesterol efflux from cells and could lead to new approaches to treatment of patients with HDL deficiency in the general population. [0102]
HDL has been implicated in numerous other biological processes, including but not limited to: prevention of lipoprotein oxidation; absorption of endotoxins; protection against [0103] Trypanosoma brucei infection; modulation of endothelial cells; and prevention of platelet aggregation (see Genest et al., J. Invest. Med. 47: 31-42, 1999, hereby incorporated by reference). Any compound that modulates HDL levels may be useful in modulating one or more of the foregoing processes. The present discovery that ABC1 functions to regulate HDL levels links, for the first time, ABC1 with the foregoing processes.
The following examples are to illustrate the invention. They are not meant to limit the invention in any way. [0104]
Analysis of TD Families [0105]
Studies of Cholesterol Efflux [0106]
Both probands had evidence of marked deficiency of cholesterol efflux similar to that previously demonstrated in TD patients (FIG. 1C). TD-1 is of Dutch descent while TD-2 is of British descent. [0107]
Linkage Analysis and Establishment of a Physical Map [0108]
Multiple DNA markers were genotyped in the region of 9q31 to which linkage to TD had been described (Rust et al., Nat. Genet. 20, 96-98, 1998). Two point linkage analysis gave a maximal peak LOD score of 6.49 at D9S1832 (Table 1) with significant evidence of linkage to all markers in a ˜10 cM interval. Recombination with the most proximal marker, D9S1690 was seen in 11-09 in Family TD-1 (A* in FIG. 3D) providing a centromeric boundary for the disease gene. Multipoint linkage analysis of these data did not increase the precision of the positioning of the disease trait locus. [0109]
A physical map spanning approximately 10 cM in this region was established with the development of a YAC contig (FIG. 3A). In addition, 22 other polymorphic multi-allelic markers which spanned this particular region were mapped to the contig (FIG. 3B) and a subset of these were used in construction of a haplotype for further analysis (FIGS. 1A and 1B; Table 2). The condensed haplotype in these families is shown in FIGS. 1A and 1B. [0110]
While the family of Dutch decent did not demonstrate any consanguinity, the proband in TD-2 was the offspring of a first-cousin consanguineous marriage (FIG. 1B). We postulated, therefore, that it was most likely that this proband would be homozygous for the mutation while the proband in the Dutch family was likely to be a compound heterozygote. The Dutch proband shows completely different mutation bearing haplotypes, supporting this hypothesis (FIG. 3C). [0111]

The TD-2 proband was homozygous for all markers tested (FIG. 1B) distal to D9S127 but was heterozygous at D9S127 and DNA markers centromeric to it (FIG. 3C). This suggested that the gene for TD was likely located to the genomic region telomeric of D9S127 and encompassed by the markers demonstrating homozygosity (FIG. 3B).

TABLE 1


Two Point Linkage Analysis of TD-1 and TD-2

LOD Score at recombination fraction

Marker Locus

	0	0.01	0.05	0.10	0.20	0.30	0.40

D9S1690	−infini	4.25	4.52	4.26	3.39	2.30	1.07
D9S277	6.22	6.11	5.67	5.10	3.90	2.60	1.17
D9S1866	4.97	4.87	4.49	4.00	2.96	1.85	0.70
D9S1784	5.50	5.40	5.00	4.47	3.36	2.17	0.92
D9S1832	6.49	6.37	5.91	5.31	4.05	2.69	21,21
D9S1677	4.60	4.51	4.18	3.76	2.88	1.93	0.93

TABLE 2


Microcrystalline markers used in this study

		Hetero-	Number	Allele frequency^t
Genetic Markers	Type	zygocity	of alleles	size.bp (proportion

D9S283	CA	0.80	10	179(0.04); 181(0.34); 183(0.19); 185(0.20);
				189(0.05); 193(0.04); 197(0.07); 199(0.02);
				201(0.04); 203(0.04)
D9S176	CA	0.82	9	129(0.03); 131(0.06); 133(0.26); 135(0.12);
				137(0.25); 139(0.03); 141(0.01); 145(0.05);
				147(0.05)
D9S1690	CA	0.79	8	225(0.36); 227(0.14); 229(0.04); 231(0.12);
				233(0.05); 235(0.16); 237(0.05); 239(0.05)
D9S277	CA	0.89	15	167(0.17); 171(0.02); 173(0.15); 175(0.11);
				177(0.07); 179(0.04); 181(0.17); 183(0.06);
				185(0.02); 187(0.02); 189(0.13); 191(0.13);
				193(0.02); 197(0.00); 199(0.00)
D9S127	CA	0.72	6	149(0.11); 151(0.07); 153(0.25); 155(0.03);
				157(0.45); 159(0.06)
D9S306	CA	0.87	13	102(0.06); 104(0.01); 110(0.03); 112(0.08);
				114(0.16); 116(0.15); 118(0.11); 120(0.23);
				122(0.06); 124(0.06); 126(0.03); 134(0.02);
				136(0.01)
D9S1866	CA	0.62	11	248(0.06); 252(0.04); 254(0.01); 256(58);
				258(0.03); 260(0.06); 262(0.02); 264(0.12);
				266(0.06); 268(0.03); 270(0.01)
D9S1784	CA	0.86	15	174(0.10); 176(0.02); 178(0.00); 180(0.08);
				182(0.11); 184(0.22); 136(0.15); 158(0.06);
				190(0.04); 192(0.07); 194(0.08); 196(0.07);
				198(0.01); 200(0.01); 202(0.01)
AFMa107xf9	CA	n.a.	n.a.	n.a.
D9S2170	CA	n.a.	n.a.	n.a.
D9S2171	CA	n.a.	n.a.	n.a.
D9S2107	CA	0.63	5	n.a.
D9S172	CA	0.54	5	291(0.00); 297(0.05); 299(0.032); 303(0.62);
				305(0.02)
D9S2109	CA	0.51	3	1(0.42); 2(0.56); 3(0.02)
D9S1832	CA	0.88	12	161(0.04); 163(0.02); 167(0.02); 169(0.04);
				171(0.10); 173(0.09); 175(0.15); 177(0.28);
				179(0.19); 181(0.04); 183(0.01); 185(0.01)
D9S1835	CA	0.48	4	110(0.02); 112(0.23); 116(0.68); 118(0.07);
D9S1801	CA	0.77	10	166(0.10); 172(0.04); 174(0.02); 182(0.02);
				184(0.19); 186(0.40); 188(0.15); 190(0.04);
				192(0.02); 194(0.02)
D9S261	CA	0.63	7	90(0.02); 92(0.52); 94(0.02); 98(0.02);
				100(0.10); 102(0.04); 104(0.08)
D9S160	CA	0.62	6	136(0.25); 138(0.53); 140(0.01); 142(0.12);
				144(0.00); 146(0.07)
D9S1677	CA	0.81	10	251(0.27); 257(0.27); 259(0.07); 261(0.09);
				263(0.27); 265(0.14); 267(0.02); 267(0.02);
				271(0.04); 273(0.02)
D9S279	CA	0.78	6	244(0.09); 246(0.18); 248(0.29); 250(0.29);
				252(0.07); 254(0.09)
D9S275	CA	0.62	4	190(0.31); 196(0.07); 198(0.52); 200(0.09)

Mutation Detection [0114]
Based on the defect in intracellular cholesterol transport in patients with TD, we reviewed the EST database for genes in this region which might be relevant to playing a role in this process. One gene that we reviewed as a candidate was the lysophosphatidic acid (LPA) receptor (EDG2) which mapped near D9S1801 (FIG. 3C). This receptor binds LPA and stimulates phospholipase-C (PLC), and is expressed in fibroblasts. It has previously been shown that the coordinate regulation of PLC that is necessary for normal HDL3 mediated cholesterol efflux is impaired in TD (Walter et al., J. Clin. Invest. 98:2315-2323, 1996). Therefore this gene represented an excellent candidate for the TD gene. Detailed assessment of this gene, using Northern blot and RT-PCR and sequencing analysis, revealed no changes segregating with the mutant phenotype in this family, in all likelihood excluding this gene as the cause for TD. Polymorphisms were detected, however, in the RT-PCR product, indicating expression of transcripts from both alleles. [0115]
The second candidate gene (RGS3) encodes a member of a family regulating G protein signaling which could also be involved in influencing cholesterol efflux (Mendez et al., Trans. Assoc. Amer. Phys. 104:48-53, 1991). This gene mapped 0.7 cM telomeric to the LPA-receptor (FIG. 3C), and is expressed in fibroblasts. It was assessed by exon-specific amplification, as its genomic organization was published (Chafterjee et al., Genomics 45:429-433, 1997). No significant sequence changes were detected. [0116]
The ABC1 transporter gene had previously been mapped to 9q31, but its precise physical location had not been determined (Luciani et al., Genomics 21:150-159, 1994). The ABC1 gene is a member of the ATP binding cassette transporters which represents a super family of highly conserved proteins involved in membrane transport of diverse substrates including amino acids, peptides, vitamins and steroid hormones (Luciani et al., Genomics 21:150-159, 1994; Dean et al., Curr. Opin. Gen. Dev. 5:779-785, 1995). Primers to the 3′ UTR of this gene mapped to YACs spanning D9S306 (887-B2 and 930-D3) compatible with it being a strong candidate for TD. We initiated large scale genomic sequencing of BACs spanning approximately 800 kb around marker D9S306 ([0117] BACs 269, 274, 279 and 291) (FIG. 3E). The ABC1 gene was revealed encompassing 49 exons and a minimum of 75 Kb of genomic sequence. In view of the potential function of a gene in this family as a cholesterol transporter, its expression in fibroblasts and localization to the minimal genomic segment underlying TD, we formally assessed ABC1 as a candidate.
Patient and control total fibroblast RNA was used in Northern blot analysis and RT-PCR and sequence analyses. RT-PCR and sequence analysis of TD-1 revealed a heterozygous T to C substitution (FIG. 4A) in the TD-1 proband, which would result in a substitution of arginine for cysteine at a conserved residue between mouse and man (FIG. 4B). This mutation, confirmed by sequencing [0118] exon 30 of the ABC1 gene, exhibited complete segregation with the phenotype on one side of this family (FIG. 4C). This substitution creates a Hgal site, allowing for RFLP analysis of amplified genomic DNA and confirmation of the mutation (FIG. 4C). The point mutation in exon 30 was not seen on over 200 normal chromosomes from unaffected persons of Dutch decent, and 250 chromosomes of Western European decent, indicating it is unlikely to be a polymorphism. Northern blot analysis of fibroblast RNA from this patient, using a cDNA encompassing exons 1 to 49 of the gene, revealed a normal sized ˜8 Kb transcript and a truncated mutant transcript which was not visible in control RNA or in RNA from other patients with HDL deficiency (FIG. 4D). Additionally, Northern blot analysis using clones encompassing discrete regions of the cDNA revealed that the mutant transcript was detected with a cDNA compassing exons 1 to 49 (a), 1 to 41 (b), 1 to 22 (c), much more faintly with a probe spanning exon 23 to 29 (d) and not seen with probes encompassing exons 30 to 42 (e), but not seen with cDNA fragment spanning exons 30 to 49 (f). This was repeated on multiple filters with control RNA, RNA from other patients with HDL deficiency and the other TD proband, and only in TD-1 was the truncated transcript observed. Sequence analysis of the coding region did not reveal an alteration in sequence that could account for this finding. Furthermore, DNA analysis by Southern blot did not reveal any major rearrangements. Completion of exon sequencing in genomic DNA showed that this mutation was a G to C transversion at position (+1) of intron 24, (FIG. 11) affecting a splice donor site and causing aberrant splicing.
RT-PCR analysis of fibroblast RNA encoding the ABC1 gene from the proband in TD-2 (FIG. 1B) revealed a homozygous nucleotide change of A to G at [0119] nucleotide 1864 of SEQ ID NO: 2 in exon 13 (FIG. 5A), resulting in a substitution of arginine for glutamine at residue 597 of SEQ ID NO: 1 (FIG. 5B), occurring just proximal to the first predicted transmembrane domain of ABC1 (FIG. 8) at a residue conserved in mouse and as well as a C. elegans homolog. This mutation creates a second Acil site within exon 13. Segregation analysis of the mutation in this family revealed complete concordance between the mutation and the low HDL phenotype as predicted (FIG. 5C). The proband in TD-2 is homozygous for this mutation, consistent with our expectation of a disease causing mutation in this consanguineous family.
Analysis of FHA families [0120]
Linkage Analysis and Refinement of the Minimal Genomic Region Containing the Gene for FHA [0121]

Data from microsatellite typing of individual family members from the four pedigrees of French Canadian origin were analyzed (FIG. 2). A maximum LOD score of 9.67 at a recombination fraction of 0.0 was detected at D9S277 on chromosome 9q31 (FIG. 3; Table 3). Thereafter, 22 markers were typed in a region spanning 10 cM around this locus in these families (FIGS. 2 and 3). The frequency for these markers were estimated from a sample of unrelated and unaffected subjects of French ancestry (Table 2).

TABLE 3


Two Point Linkage Analysis of FHA

LOD Score at recombination fraction

Marker Locus

	0	0.01	0.05	0.10	0.20	0.30	0.40

D9S283	−infini	−2.57	0.51	1.48	1.84	1.48	0.76
D9S176	−infini	1.42	3.07	3.39	3.05	2.22	1.12
D9S1690	−infini	3.11	4.04	4.04	3.33	2.24	0.96
D9S277	9.67	9.51	8.89	8.06	6.29	4.30	2.10
D9S306	5.60	5.51	5.13	4.62	3.55	2.36	1.11
D9S1866	−infini	7.24	7.35	6.87	5.50	3.82	1.91
D9S1784	−infini	8.85	7.76	9.03	7.09	4.78	2.25
D9S172	−infini	2.63	3.00	2.87	2.26	1.50	0.67
D9S1832	−infini	5.20	5.97	5.75	4.59	3.02	1.30
D9S1801	0.14	0.13	0.11	0.09	0.06	0.03	0.01
D9S1677	−infini	7.83	7.90	7.38	5.90	4.08	2.01
D9S279	−infini	3.43	3.80	3.66	3.01	2.12	1.05
D9S275	−infini	2.57	2.98	2.91	2.41	1.69	0.81

TD and FHA have thus far been deemed distinct with separate clinical and biochemical characteristics. Even though the genes for these disorders mapped to the same region, it was uncertain whether FHA and TD were due to mutations in the same gene or, alternatively, due to mutations in genes in a similar region. [0123]
Refinement of the region containing the gene for FHA was possible by examining haplotype sharing and identification of critical recombination events (FIG. 2). Seven separate meiotic recombination events were seen in these families (“A” through “G” in FIGS. 2 and 3), clearly indicating that the minimal genomic region containing the potential disease gene was a region of approximately 4.4 cM genomic DNA spanned by marker D9S1690 and D9S1866 (FIGS. 2 and 3). This region is consistent with the results of two point linkage analysis which revealed maximal LOD scores with markers D9S277 and D9S306 and essentially excluded the region centromeric to D9S1690 or telomeric to D9S1866. An 8[0124] ^thmeiotic recombination event (“H” in FIG. 3) further refined the FHA region to distal to D9S277.
As described herein, the ABC1 gene mapped within this interval. The overlapping genetic data strongly suggested that FHA may in fact be allelic to TD. Utilization of sets of genetic data from FHA and TD provided a telomeric boundary at D9S1866 (meiotic recombinant) (FIG. 3D) and a centromeric marker at D9S127 based on the homozygosity data of TD-2. This refined the locus to approximately 1 Mb between D9S127 and D9S1866. The ABC1 gene mapped within this minimal region (FIG. 3E). [0125]
Mutation Detection in FHA [0126]
Mutation assessment of the ABC1 gene was undertaken in FHA-1 (FIG. 2A). Using primers that spanned overlapping segments of the mRNA we performed RT-PCR analysis and subjected these fragments to mutational analysis. A deletion of three nucleotides is evident in the RT-PCR sequence of FHA-1 III.01 (FIG. 6A), resulting in a loss of nucleotides 2151-2153 of SEQ ID NO: 2 and deletion of a leucine (L693) at [0127] amino acid position 693 of SEQ ID NO: 1 (FIG. 6A). This leucine is conserved in mouse and C. elegans (FIG. 6B). The alteration was detected in the RT-PCR products as well as in genomic sequence from exon 14 specific amplification. This mutation results in a loss of an Earl restriction site. Analysis of genomic DNA from the family indicated that the mutation segregated completely with the phenotype of HDL deficiency. The loss of the Earl site results in a larger fragment being remaining in persons heterozygous for this mutation (FIG. 6C). This mutation maps to the first putative transmembrane domain of ABC1 (FIG. 8) and was not seen in 130 chromosomes from persons of French Canadian descent nor seen in over 400 chromosomes from persons of other Western European ancestry.
A mutation has also been found in patient genomic DNA in pedigree FHA-3 from Quebec. The alteration, a 6 bp deletion of nucleotides 5752-5757 of SEQ ID NO: 2 within [0128] exon 41, results in a deletion of amino acids 1893 (Glu) and 1894 (Asp) of SEQ ID NO: 1. The deletion was detected as a double, superimposed, sequence starting from the point of the deletion (FIG. 6D), and was detected in sequence reads in both directions. The deletion can be detected on 3% agarose or 10% polyacrylamide gels, and segregates with disease in FHA-3. It was not seen in 128 normal chromosomes of French-Canadian origin or in 434 other control chromosomes. Amino acids 1893 and 1894 are in a region of the ABC1 protein that is conserved between human, mouse, and C. elegans (FIG. 6E), implying that it is of functional importance.
An additional mutation has been found in patient genomic DNA in pedigree FHA-2 from Quebec (FIG. 6F). The alteration, a C to T transition at position 6504 of SEQ ID NO: 2, converts an arginine at position 2144 of SEQ ID NO: 1 to a STOP codon, causing truncation of the last 118 amino acids of the ABC1 protein. This alteration segregates with disease in family FHA-2. [0129]
A summary of all mutations and polymorphisms found in ABC1 is shown in FIG. 11. Each variant indicated as a mutation segregates with low HDL in its family, and was not seen in several hundred control chromosomes. [0130]
Functional Relationship Between Changes in ABC1 Transcript Levels and Cholesterol Efflux [0131]
Antisense approaches were undertaken to decrease the ABC1 transcript and assess the effect of alteration of the transcript on intracellular cholesterol transport. The use of antisense primers to the 5′ end of ABC1 clearly resulted in a decrease to approximately 50% of normal RNA levels (FIG. 7A). This would be expected to mimic in part the loss of function due to mutations on one allele, similar to that seen in heterozygotes for TD and patients with FHA. Importantly, reduction in the mRNA for the ABC1 gene resulted in a significant reduction in cellular cholesterol efflux (FIG. 7B), further establishing the role of this protein in reverse cholesterol transport and providing evidence that the mutations detected are likely to constitute loss of function mutations. Furthermore, these data support the functional importance of the first 60 amino acids of the protein. Antisense oligonucleotide AN-6 is directed to the [0132] novel start codon 5′ to the one indicated in AJ012376.1; this antisense oligonucleotide effectively suppresses efflux.
The above-described results were obtained using the following materials and methods. [0133]
Patient Selection [0134]
The probands in TD families had previously been diagnosed as suffering from TD based on clinical and biochemical data. Study subjects with FHA were selected from the Cardiology Clinic of the Clinical Research Institute of Montreal. The main criterion was an HDL-C level <5th percentile for age and gender, with a plasma concentration of triglycerides <95th percentile in the proband and a first-degree relative with the same lipid abnormality. In addition, the patients did not have diabetes. [0135]
Biochemical Studies [0136]
Blood was withdrawn in EDTA-containing tubes for plasma lipid, lipoprotein cholesterol, ApoAI, and triglyceride analyses, as well as storage at. [0137]
−80° C. Leukocytes were isolated from the buffy coat for DNA extraction. [0138]
Lipoprotein measurement was performed on fresh plasma as described elsewhere (Rogler et al., Arterioscler. Thromb. Vasc. Biol. 15:683-690, 1995). The laboratory participates and meets the criteria of the Lipid Research Program Standardization Program. Lipids, cholesterol and triglyceride levels were determined in total plasma and plasma at density d<1.006 g/mL (obtained after preparative ultracentrifugation) before and after precipitation with dextran manganese. Apolipoprotein measurement was performed by nephelometry for ApoB and ApoAI. [0139]
Linkage Analysis [0140]
Linkage between the trait locus and microsatellite loci was analyzed using the FASTLINK version (4.0 P). FASTLINK/MLINK was used for two-point linkage analysis assuming an autosomal dominant trait with complete penetrance. In FHA and TD heterozygotes, the phenotype was HDL deficiency <5th percentile for age and sex. The disease allele frequency was estimated to be 0.005. Marker allele frequencies were estimated from the genotypes of the founders in the pedigrees using NEWPREP. Multipoint linkage analysis was carried out using FASTLINK/LINKMAP. [0141]
Genomic Clone Assembly and Physical Map Construction of the 9q31 Region [0142]
Using the Whitehead Institute/MIT Center for Genome Research map as a reference, the genetic markers of interest at 9q31 were identified within YAC contigs. Additional markers that mapped to the approximate 9q31 interval from public databases and the literature were then assayed against the YAC clones by PCR and hybridization analysis. The order of markers was based on their presence or absence in the anchored YAC contigs and later in the BAC contig. Based on the haplotype analysis, the region between D9S277 and D9S306 was targeted for higher resolution physical mapping studies using bacterial artificial chromosomes (BACs). BACs within the region of interest were isolated by hybridization of DNA marker probes and whole YACs to high-density filters containing clones from the RPCI-11 human BAC library (FIG. 3). [0143]
Sequence Retrieval and Alignment [0144]
The human ABC1 mRNA sequence was retrieved from GenBank using the Entrez nucleotide query (Baxevanis et al., A Practical Guide to the Analysis of Genes and Proteins, eds. Baxevanis, A. D. & Ouellette, B. F. F. 98:120, 1998) as GenBank accession number AJ012376.1. The version of the protein sequence we used as wild-type (normal) was CAA10005.1. [0145]
We identified an additional 60 amino acids in-frame with the previously-believed start methionine (FIG. 9A). Bioinformatic analysis of the additional amino acids indicates the presence of a short stretch of basic amino acid residues, followed by a hydrophobic stretch, then several polar residues. This may represent a leader sequence, or another transmembrane or membrane-associated region of the ABC1 protein. In order to differentiate among the foregoing possibilities, antibodies directed to the region of amino acids 1-60 are raised against and used to determine the physical relationship of amino acids 1-60 in relation to the cell membrane. Other standard methods can also be employed, including, for example, expression of fusion proteins and cell fractionation. [0146]
We also identified six errors in the previously-reported nucleotide sequence (at positions 839, 4738, 5017, 5995, 6557, and 6899 of SEQ ID NO: 2; FIG. 11). Hence, the sequence of the ABC1 polypeptide of FIG. 9A differs from CAA10005.1 as follows: Thr_IIe at position 1554; Pro_Leu at position 1642; Arg_Lys at position 1973; and Pro_Leu at position 2167. We also identified 5′ and 3′ UTR sequence (FIGS. [0147] 9B-9E).
The mouse ABC1 sequence used has accession number X75926. It is very likely that this mouse sequence is incomplete, as it lacks the additional 60 amino acids described herein for human ABC1. [0148]
Version 1.7 of ClustalW was used for multiple sequence alignments with BOXSHADE for graphical enhancement (www.isrec.isb-sib.ch:8080/software/BOX_form.html) with the default parameter. A [0149] Caenorhabditis elegans ABC1 orthologue was identified with BLAST (version 2.08) using CAA1005.1 (see above) as a query, with the default parameter except for doing an organism filter for C. elegans. The selected protein sequence has accession version number MC69223.1 with a score of 375, and an E value of 103.
Genomic DNA Sequencing [0150]
BAC DNA was extracted from bacterial cultures using NucleoBond Plasmid Maxi Kits (Clontech, Palo Alto, Calif.). For DNA sequencing, a sublibrary was first constructed from each of the BAC DNAs (Rowen et al., Automated DNA Sequencing and Analysis, eds. Adams, M. D., Fields, C. & Venter, J. C., 1994). In brief, the BAC DNA was isolated and randomly sheared by nebulization. The sheared DNA was then size fractionated by agarose gel electrophoresis and fragments above 2 kb were collected, treated with Mung Bean nuclease followed by T4 DNA polymerase and klenow enzyme to ensure blunt-ends, and cloned into SmaI-cut M13 mp19. Random clones were sequenced with an AB1373 or 377 sequencer and fluorescently labeled primers (Applied BioSystems, Foster City, Calif.). DNAStar software was used for gel trace analysis and contig assembly. All DNA sequences were examined against available public databases primarily using BLASTn with RepeatMasker (University of Washington). [0151]
Reverse transcription (RT)-PCR amplification and sequence analysis [0152]
Total RNA was isolated from the cultured fibroblasts of TD and FHA patients, and reverse transcribed with a CDS primer containing oligo d(T)18 using 250 units of SuperScript II reverse transcriptase (Life Technologies, Inc., Rockville, Md.) as described (Zhang et al., J. Biol. Chem. 27:1776-1783, 1996). cDNA was amplified with Taq DNA polymerase using primers derived from the published human ABC1 cDNA sequence (Luciani et al., Genomics 21:150-159, 1994). Six sets of primer pairs were designed to amplify each cDNA sample, generating six DNA fragments which are sequentially overlapped covering 135 to 7014 bp of the full-length human ABC1 cDNA. The nucleotides are numbered according to the order of the published human cDNA sequence (AJ012376.1). Primer pairs (1): 135-158 (f) and 1183-1199 (r); (2): 1080-1107 (f) and 2247-2273 (r); (3): 2171-2197 (f) and 3376-3404 (r); (4): 3323-3353 (f) and 4587-4617 (r); (5) 4515-4539 (f) and 5782-5811 (r); (6): 5742-5769 (f) and 6985-7014 (r). RT-PCR products were purified by Qiagen spin columns. Sequencing was carried out in a Model 373A Automated DNA sequencer (Applied Biosystems) using Taq di-deoxy terminator cycle sequencing and Big Dye Kits according to the manufacturer's protocol. [0153]
Northern Blot Analysis [0154]
Northern transfer and hybridizations were performed essentially as described (Zhang et al., J. Biol. Chem. 27:1776-1783, 1996). Briefly, 20 μg of total fibroblast RNA samples were resolved by electrophoresis in a denaturing agarose (1.2%; w/v) gel in the presence of 7% formaldehyde, and transferred to nylon membranes. The filters were probed with [0155] ³²P-labeled human ABC1 cDNA as indicated. Pre-hybridization and hybridizations were carried out in an ExpressHyb solution (ClonTech) at 68° C. according to the manufacturer's protocol.
Detection of the Mutations in TD [0156]
Genotyping for the T4503C and A1864G variants was performed by PCR amplification of [0157] exon 30 followed by restriction digestion with Hgal and amplification of exon 13 followed by digestion with Acil, respectively. PCR was carried out in a total volume of 50 μL with 1.5 mM MgCI₂, 187.5 nM of each dNTP, 2.5U Taq polymerase and 15 pmol of each primer (forward primer in exon 30: 5′-CTG CCA GGC AGG GGA GGA AGA GTG-3′ (SEQ ID NO: 4); reverse primer spanning the junction of exon 30 and intron 30: 5′-GAA AGT GAC TCA CTT GTG GAG GA-3′ (SEQ ID NO: 5); forward primer in intron 12: 5′-AAA GGG GCT TGG TM GGG TA-3′ (SEQ ID NO: 6); reverse in intron 13: 5′-CAT GCA CAT GCA CAC ACA TA-3′ (SEQ ID NO: 7)). Following an initial denaturation of 3 minutes at 95° C., 35 cycles consisting of 95° C. 10 seconds, 58° C. 30 seconds, 72° C. 30 seconds were performed, with a final extension of 10 minutes at 72° C. For detection of the T4503C mutation, 15 μL of exon 30 PCR product was incubated with 4 U Hgal in a total volume of 25 μL, for 2 hours at 37° C., and the resulting fragments were separated on a 1.5% agarose gel. The presence of the T4503C mutation creates a restriction site for Hgal, and thus the 194 bp PCR product will be cut into fragments of 134 and 60 bp in the presence of the T4503C variant, but not in its absence. For detection of the A1864G mutation, 15 μL of exon 13 PCR products were digested with 8 U Acil for three hours at 37° C. Products were separated on 2% agarose gels. The presence of the A1864G mutation creates a second Acil site within the PCR product. Thus, the 360 bp PCR product is cleaved into fragments of 215 bp and 145 bp on the wild-type allele, but 185 bp, 145 bp and 30 bp on the mutant allele. Detection of mutation in FHA Genotyping for the 693 variant was performed by PCR amplification of exon 14 followed by restriction enzyme digestion with Earl. PCR was carried out in a total volume of 80 μL with 1.5 mM MgCI₂, 187.5 nM of each dNTP, 2.5 U Taq polymerase and 20 μmol of each primer (forward primer in exon 14: 5′- CTT TCT GCG GGT GAT GAG CCG GTC AAT-3′ (SEQ ID NO: 8); reverse primer in intron 14: 5′-CCT TAG CCC GTG TTG AGC TA-3′ (SEQ ID NO: 9)). Following an initial denaturation of 3 minutes at 95° C., 35 cycles consisting of 95° C. 10 seconds, 55° C. 30 seconds, 72° C. 30 seconds were performed, with a final extension of 10 minutes at 72° C. Twenty microliters of PCR product was incubated with 4 U Earl in a total volume of 25 μL, for two hours at 37° C., and the fragments were separated on a 2% agarose gel. The presence of the 693 mutation destroys a restriction site for Earl, and thus the 297 bp PCR product will be cut into fragments of 151 bp, 59 bp, 48 bp and 39 bp in the presence of a wild-type allele, but only fragments of 210 bp, 48 bp and 39 bp in the presence of the deletion.
A 6 bp deletion encompassing nucleotides 5752-5757 (inclusive), was detected in [0158] exon 41 in the proband of family FHA-3 by genomic sequencing using primers located within the introns flanking this exon. Genotyping of this mutation in family FHA-3 and controls was carried out by PCR with forward (5′-CCT GTA MT GCA MG CTA TCT CCT CT-3′ (SEQ ID NO: 10)) and reverse primers (5′-CGT CM CTC CTT GAT TTC TM GAT GT (SEQ ID NO: 11)) located near the 5′ and 3′ ends of exon 41, respectively. Each PCR was carried out as for the genotyping of the 693 variant, but with annealing temperature of 58° C. Twenty microliters of PCR product was resolved on 3% agarose or 10% acrylamide gels. The wild type allele was detected as a 117 bp band and the mutant allele as a 111 bp band upon staining with ethidium bromide.
A C to T transition was detected at nucleotide 6504 in genomic DNA of the proband of family FHA-2. It was detectable as a double C and T peak in the genomic sequence of [0159] exon 48 of this individual, who is heterozygous for the alteration. This mutation, which creates a STOP codon that results in truncation of the last 118 amino acids of the ABC1 protein, also destroys an RsaI restriction site that is present in the wild type sequence. Genotyping of this mutation in family FHA-2 and controls was carried out by PCR with forward (5′-GGG TTC CCA GGG TTC AGT AT-3′) (SEQ ID NO: 12)) and reverse (5′-GAT CAG GM TTC MG CAC CM-3′) (SEQ ID NO: 13)) primers directed to the intronic sequences flanking exon 48. PCR was done as for the 693 variant. Fifteen microliters of PCR product was digested with 5 Units of RsaI at 37° C. for two hours and the digestion products resolved on 1.5% agarose gels. The mutant allele is detected as an uncut 436 bp band. The normal sequence is cut by RsaI to produce 332 and 104 bp bands.
Cell Culture [0160]
Skin fibroblast cultures were established from 3.0 mm punch biopsies of the forearm of FHD patients and healthy control subjects as described (Marcil et al., Arterioscler. Thromb. Vasc. Biol. 19:159-169, 1999). [0161]
Cellular Cholesterol Labeling and Loading [0162]
The protocol for cellular cholesterol efflux experiments was described in detail elsewhere (Marcil et al., Arterioscler. Thromb. Vasc. Biol. 19:159-169, 1999). The cells were [0163] ³H-cholesterol labeled during growth and free cholesterol loaded in growth arrest.
Cholesterol Efflux Studies [0164]
Efflux studies were carried out from 0 to 24 hours in the presence of purified ApoAI (10 μg protein/mL medium). Efflux was determined as a percent of free cholesterol in the medium after the cells were incubated for specified periods of time. All experiments were performed in triplicate, in the presence of cells from one control subject and the cells from the study subjects to be examined. All results showing an efflux defect were confirmed at least three times. [0165]
Oligonucleotide synthesis [0166]

Eight phosphorothioate deoxyoligonucleotides complementary to various regions of the human ABC1 cDNA sequence were obtained from GIBCO BRL. The oligonucleotides were purified by HPLC. The sequences of the antisense oligonucleotides and their location are listed. One skilled in the art will recognize that other ABC1 antisense sequences can also be produced and tested for their ability to decrease ABC1-mediated cholesterol regulation.



			%
			con-
Name	Seguence (5′-3′)	mRNA target	trol

AN-1	GCAGAGGGCATGGCTTTATTTG (SEQ ID NO: 3)	AUG codon	46

AN-2	GTGTTCCTGCAGAGGGCATG (SEQ ID NO: 30)	AUG codon	50

AN-3	CACTTCCAGTAACAGCTGAC (SEQ ID NO: 31)	5′-Untranslated	79

AN-4	CTTTGCGCATGTCCTTCATGC (SEQ ID NO: 32)	Coding	80

AN-5	GACATCAGCCCTCAGCATCTT (SEQ ID NO: 33)	Coding	120

AN-6:	CAACAAGCCATGTTCCCTC (SEQ ID NO: 34)	Coding

AN-7:	CATGTTCCCTCAGCCAGC (SEQ ID NO: 35)	Coding

AN-8:	CAGAGCTCACAGCAGGGA C (SEQ ID NO: 36)	Coding

Cell Transfection With Antisense Oligonucleotides [0168]
Cells were grown in 35 mm culture dishes until 80% confluent, then washed once with DMEM medium (serum and antibiotics free). One milliliter of DMEM (serum and antibiotics free) containing 500 nM antisense oligonucleotides and 5 μg/ml or 7.5 μg/ml of lipofectin (GIBCO BRL) were added to each well according to the manufacturer's protocol. The cells were incubated at 37° C. for 4 hours, and then the medium was replaced by DMEM containing 10% FCS. Twenty-four hours after the transfection, the total cell RNA was isolated. Ten micrograms of total RNA was resolved on a 1% of agarose-formaldehyde gel and transferred to nylon membrane. The blot was hybridized with γ-[0169] ³²p dCTP labeled human ABC1 cDNA overnight at 68° C. The membrane was subsequently exposed to x-ray film. The hybridizing bands were scanned by optical densitometry and standard to 28S ribosome RNA.
Cholesterol efflux with anti-ABC1 oligonucleotides Human skin fibroblasts were plated in 6-well plates. The cells were labeled with [0170] ³H-cholesterol (0.2 μCi /ml) in DMEM with 10% FBS for two days when the cell reached 50% confluence. The cells were then transfected with the antisense ABC1 oligonucleotides at 500 nM in DMEM (serum and antibiotic free) with 7.5 μg/ml Lipofectin (GIBCO BRL) according to the manufacturer's protocol. Following the transfection, and the cells were loaded with nonlipoprotein (20 μg/ml) for 12 hours in DMEM containing 2 mg/ml BSA without serum. The cellular cholesterol pools were then allowed to equilibrate for 6 hours in DMEM-BSA. The cholesterol efflux mediated by ApoAI (10 μg/ml, in DMEM-BSA) were then carried out which is 48 hours after transfection.
Radiolabeled cholesterol released into the medium is expressed as a percentage of total [0171] ³H-cholesterol per well (medium +cell ). Results are the mean +/−SD of triplicate dishes.
Determination of Genomic Structure of the ABC1 Gene [0172]
Most splice junction sequences were determined from genomic sequence generated from BAC clones spanning the ABC1 gene. More than 160 kb of genomic sequence were generated. Genomic sequences were aligned with cDNA sequences to identify intron/exon boundaries. In some cases, long distance PCR between adjacent exons was used to amplify intron/exon boundary sequences using amplification primers designed according to the cDNA sequence. [0173]
Functionality of the Newly-Discovered 60 Amino Acids at the N-Terminus [0174]
Antisense Experiments [0175]
Phosphorothioate antisense oligonucleotides were designed to be complementary to the regions of the cDNA near newly discovered translation start site. AN-6 and AN-7 both overlap the initiator methionine codon; this site is in the middle of oligonucleotide AN-6. AN-8 is complementary to the very 5′ end of the ABC1 cDNA. Antisense oligonucleotide AN-1 is complementary to the region of the ABC1 cDNA corresponding to the site identified as the ABC1 initiator methionine in AJ012376. FIG. 7C shows that antisense oligonucleotide AN-6 interferes with cellular cholesterol efflux in normal fibroblasts to the same extent as does antisense oligonucleotide AN-1. Transfection with either of these antisense oligonucleotides results in a decrease in cellular cholesterol efflux almost as severe as that seen in FHA cells. In general, antisense oligonucleotides complementary to coding sequences, especially near the 5‘end of a gene’s coding sequence, are expected to be more effective in decreasing the effective amount of transcript than are oligonucleotides directed to more 3′ sequences or to non-coding sequences. The observation that AN-6 depresses cellular cholesterol efflux as effectively as AN-1 implies that both of these oligonucleotides are complementary to ABC1 coding sequences, and that the [0176] amino terminal 60 amino acids are likely to be contained in ABC1 protein. In contrast, the ineffectiveness of AN-8 shows that it is likely to be outside the protein coding region of the transcript, as predicted by presence of an in-frame stop codon between the initiator methionine and the region targeted by AN-8.
Antibody Experiments [0177]
Polyclonal and monoclonal antibodies have been generated using peptides corresponding to discrete portions of the ABC1 amino acid sequence. One of these, 20-amino acid peptide #2 (Pep2: CSVRLSYPPYEQHECHFPNKA (SEQ ID NO: 37), in which the N-terminal cysteine was added to facilitate conjugation of the peptide) corresponds to a protein sequence within the 60 amino-terminal amino acids of the newly-discovered ABC1 protein sequence. The peptide was coupled to the KLH carrier protein and 300 □g injected at three intervals into two Balb/c mice over a four week period. The spleen was harvested from the mouse with the highest ELISA-determined immune response to free peptide, and the cells fused to NS-1 myeloma cells by standard monoclonal antibody generation methods. Positive hybridomas were selected first by ELISA and then further characterized by western blotting using cultured primary human fibroblasts. Monoclonal cell lines producing a high antibody titre and specifically recognizing the 245 kD human ABC1 protein were saved. The same size ABC1 protein product was detected by antibodies directed to four other discrete regions of the same protein. The 245 kD band could be eliminated in competition experiments with appropriate free peptide, indicating that it represents ABC1 protein (FIG. 13). [0178]
The foregoing experiments indicate that ABC1 protein is detected not only by antibodies corresponding to amino acid sequences within the previously-described ABC1 amino acid sequence, but also by the Pep2 monoclonal antibody that recognizes an epitope within the newly-discovered N-[0179] terminal 60 amino acids. The N-terminal 60 amino acid region is therefore coding, and is part of the ABC1 protein.
The epitope recognized by the Pep2 monoclonal antibody is also conserved among human, mouse, and chicken. Liver tissues from these three species employed in a Western blot produced an ABC1 band of 245 kD when probed with the Pep2 monoclonal antibody. This indicates that the 60 amino acid N-terminal sequence is part of the ABC1 coding sequence in humans, mice, and chickens. Presence of this region is therefore evolutionarily conserved and likely to be of important functional significance for the ABC1 protein. [0180]
Bioinformatic Analyses of ABC1 Protein Sequences [0181]

Transmembrane prediction programs indicate 13 transmembrane (TM) regions, the first one being between amino acids 26 and 42 (psort.nibb.ac.jp:8800/psort/helpwww2.ealom). The tentative number of TM regions for the threshold 0.5 is 13. (INTEGRAL Likelihood =−7.75 Transmembrane 26-42). The other 12 TM range in value between −0.64 and -12 (full results below). It is therefore very likely that the newly-discovered 60 amino acids contain a TM domain, and that the amino end of ABC1 may be on the opposite side of the membrane than originally thought.



ALOM: TM region allocation

	Init position for calculation: 1
	Tentative number of TMs for the threshold 0.5: 13
	INTEGRAL Likelihood = −7.75 Transmembrane 26-42
	INTEGRAL Likelihood = −3.98 Transmembrane 640-656
	INTEGRAL Likelihood = −8.70 Transmembrane 690-706
	INTEGRAL Likelihood = −9.61 Transmembrane 717-733
	INTEGRAL Likelihood = −1.44 Transmembrane 749-765
	INTEGRAL Likelihood = −0.64 Transmembrane 771-787
	INTEGRAL Likelihood = −1.28 Transmembrane 1041-1057
	INTEGRAL Likelihood = −12.79 Transmembrane 1351-1367
	INTEGRAL Likelihood = −8.60 Transmembrane 1661-1677
	INTEGRAL Likelihood = −6.79 Transmembrane 1708-1724
	INTEGRAL Likelihood = −3.40 Transmembrane 1737-1753
	INTEGRAL Likelihood = −1.49 Transmembrane 1775-1791
	INTEGRAL Likelihood = −8.39 Transmembrane 1854-1870
	PERIPHERAL Likelihood = 0.69 (at 1643)
	ALOM score: −12.79 (number of TMSs: 13)

There does not appear to be an obvious cleaved peptide, so this first 60 amino acid residues are not likely to be cleaved, and are therefore not specifically a signal/targeting sequence. No other signals (e.g., for targeting to specific organelles) are apparent. [0183]
Agonists and Antagonists [0184]
Useful therapeutic compounds include those which modulate the expression, activity, or stability of ABC1. To isolate such compounds, ABC1 expression, biological activity, or regulated catabolism is measured following the addition of candidate compounds to a culture medium of ABC1-expressing cells. Alternatively, the candidate compounds may be directly administered to animals (for example mice, pigs, or chickens) and used to screen for their effects on ABC1 expression. [0185]
In addition its role in the regulation of cholesterol, ABC1 also participates in other biological processes for which the development of ABC1 modulators would be useful. In one example, ABC1 transports interleukin-1β (IL-1β) across the cell membrane and out of cells. IL-1β is a precursor of the inflammatory response and, as such, inhibitors or antagonists of ABC1 expression or biological activity may be useful in the treatment of any inflammatory disorders, including but not limited to rheumatoid arthritis, systemic lupus erythematosis (SLE), hypo- or hyper-thyroidism, inflammatory bowel disease, and diabetes mellitus. In another example, ABC1 expressed in macrophages has been shown to be engaged in the engulfinent and clearance of dead cells. The ability of macrophages to ingest these apoptotic bodies is impaired after antibody-mediated blockade of ABC1. Accordingly, compounds that modulate ABC1 expression, stability, or biological activity would be useful for the treatment of these disorders. [0186]
ABC1 expression is measured, for example, by standard Northern blot analysis using an ABC1 nucleic acid sequence (or fragment thereof as a hybridization probe, or by Western blot using an anti-ABC1 antibody and standard techniques. The level of ABC1 expression in the presence of the candidate molecule is compared to the level measured for the same cells, in the same culture medium, or in a parallel set of test animals, but in the absence of the candidate molecule. ABC1 activity can also be measured using the cholesterol efflux assay. [0187]
Transcriptional Regulation of [0188] ABC 1 Expression
ABC1 mRNA is increased approximately 8-fold upon cholesterol loading. This increase is likely controlled at the transcriptional level. Using the promoter sequence described herein, one can identify transcription factors that bind to the promoter by performing, for example, gel shift assays, DNAse protection assays, or in vitro or in vivo reporter gene-based assays. The identified transcription factors are themselves drug targets. In the case of ABC1, drug compounds that act through modulation of transcription of ABC1 could be used for HDL modulation, atherosclerosis prevention, and the treatment of cardiovascular disease. For example, using a compound to inhibit a transcription factor that represses ABC1 would be expected to result in up-regulation of ABC1 and, therefore, HDL levels. In another example, a compound that increases transcription factor expression or activity would also increase ABC1 expression and HDL levels. [0189]
Transcription factors known to regulate other genes in the regulation of apolipoprotein genes or other cholesterol- or lipid-regulating genes are of particular relevance. Such factors include, but are not limited to, the steroid response element binding proteins (SREBP-1 and SREBP-2), the PPAR (peroxisomal proliferation-activated receptor) transcription factors. Several consensus sites for certain elements are present in the sequenced [0190] region 5′ to the ABC1 gene (FIG. 16) and are likely to modulate ABC1 expression. For example, PPARs may alter transcription of ABC1 by mechanisms including heterodimerization with retinoid X receptors (RXRs) and then binding to specific proliferator response elements (PPREs). Examples of such PPARs include PPARα, β, γ and δ. These distinct PPARs have been shown to have transcriptional regulatory effects on different genes. PPARδ is expressed mainly in liver, whereas PPARδ is expressed in predominantly in adipocytes. Both PPARα and PPARδ are found in coronary and carotid artery atherosclerotic plaques and in endothelial cells, smooth muscle cells, monocytes and monocyte-derived macrophages. Activation of PPAR□ results in altered lipoprotein metabolism through PPARα's effect on genes such as lipoprotein lipase (LPL), apolipoprotein CIII (apo CIII) and apolipoprotein AI (apo AI) and AII (apo AII). PPAR□ activation results in overexpression of LPL and apoA-I and apoA-II, but inhibits the expression of apo CIII. PPAR□ activation also inhibits inflammation, stimulates lipid oxidation and increases the hepatic uptake and esterification of free fatty acids (FFA's). PPARE□ and PPAR□ activation may inhibit nitric oxide (NO) synthase in macrophages and prevent interleukin-1 (IL-1) induced expression of IL-6 and cyclo-oxygenase-2 (COX-2) and thrombin induced endothelin-1 expression secondary to negative transcriptional regulation of NF-KB and activation of protein-1 signaling pathway. It has also been shown that PPAR□ induces apoptosis in monocyte-derived macrophages through the inhibition of NF-KB activity.
Activation of PPARα can be achieved by compounds such as fibrates, β-estradiol, arachidonic acid derivatives, WY-14,643 and LTB4 or 8(s)HETE. PPARγ activation can be achieved through compounds such as thiozolidinedione antidiabetic drugs, 9-HODE and 13-HODE. Additional compounds such as nicotinic acid or HMG CoA reductase inhibitors may also alter the activity of PPARs. [0191]
Compounds which alter activity of any of the PPARs (e.g., PPARα or PPARγ) may have an effect on ABC1 expression and thereby could affect HDL levels, atherosclerosis and risk of CAD. PPARs are also regulated by fatty acids (including modified fatty acids such as 3 thia fatty acids), leukotrienes such as leukotriene B4 and prostaglandin J2, which is a natural activator/ligand for PPARγ. Drugs that modulate PPARs may therefore have an important effect on modulating lipid levels (including HDL and triglyceride levels) and altering CAD risk. This effect could be achieved through the modulation of ABC1 gene expression. Drugs may also effect ABC1 gene expression and thereby HDL levels, by an indirect effect on PPARs via other transcriptional factors such as adipocyte differentiation and determination factor-1 (ADD-1) and sterol regulatory element binding protein-1 and 2 (SREBP-1 and 2). Drugs with combined PPARa and PPARγ agonist activity or PPARα and PPARγ agonists given in combination for example, may increase HDL levels even more. [0192]
A PPAR binding site (PPRE element) is found 5′ to the ABC1 gene (nucleotides 2150 to 2169 of SEQ ID NO: 14). Like the PPRE elements found in the C-ACS, HD, CYP4A6 and ApoA-I genes, this PPRE site is a trimer related to the PPRE consensus sequence. Partly because of its similarity in the number and arrangement of repeats in this PPAR binding site, this element in particular is very likely to be of physiological relevance to the regulation of the ABC1 gene. [0193]
Additional transcription factors which may also have an effect in modulating ABC1 gene expression and thereby HDL levels, atherosclerosis and CAD risk include; REV-ERBA, SREBP-1 & 2, ADD-1, EBPα, CREB binding protein, P300, [0194] HNF 4, RAR, LXR, and RORα. Additional degenerate binding sites for these factors can be found through examination of the sequence in SEQ ID NO: 14.
Additional Utility of ABC1 Polypeptides, Nucleic Acids, and Modulators [0195]
ABC1 may act as a transporter of toxic proteins or protein fragments (e.g., APP) out of cells. Thus, ABC1 agonists/upregulators may be useful in the treatment of other disease areas, including Alzheimer's disease, Niemann-Pick disease, and Huntington's disease. [0196]
ABC transporters have been shown to increase the uptake of long chain fatty acids from the cytosol to peroxisomes and, moreover, to play a role in □-oxidation of very long chain fatty acids. Importantly, in x-linked adrenoleukodystrophy (ALD), fatty acid metabolism is abnormal, due to defects in the peroxisomal ABC transporter. Any agent that upregulates ABC transporter expression or biological activity may therefor be useful for the treatment of ALD or any other lipid disorder. [0197]
ABC1 is expressed in macrophages and is required for engulfment of cells undergoing programmed cell death. The apoptotic process itself, and its regulation, have important implications for disorders such as cancer, one mechanism of which is failure of cells to undergo cell death appropriately. ABC1 may facilitate apoptosis, and as such may represent an intervention point for cancer treatment. Increasing ABC1 expression or activity or otherwise up-regulating ABC1 by any method may constitute a treatment for cancer by increasing apoptosis and thus potentially decreasing the aberrant cellular proliferation characterized by this disease. Conversely, down-regulation of ABC1 by any method may provide opportunity for decreasing apoptosis and allowing increased proliferation of cells in conditions where cell growth is limited. Such disorders include but are not limited to neurodeficiencies and neurodegeneration, and growth disorders. ABC1 could, therefore, potentially be used as a method for identification of compounds for use in the treatment of cancer, or in the treatment of degenerative disorders. [0198]
Agents that have been shown to inhibit ABC1 include, for example, the anti-diabetic agents glibenclamide and glyburide, flufenamic acid, diphenylamine-2-carbonic acid, sulfobromophthalein, and DIDS. [0199]
Agents that upregulate ABC1 expression or biological activity include but are not limited to protein kinase A, protein kinase C, vanadate, okadaic acid, and IBMX1. [0200]
Those in the art will recognize that other compounds can also modulate ABC1 biological activity, and these compounds are also in the spirit of the invention. [0201]
Drug Screens Based on the ABC1 Gene or Protein [0202]
The ABC1 protein and gene can be used in screening assays for identification of compounds which modulate its activity and may be potential drugs to regulate cholesterol levels. Useful ABC1 proteins include wild-type and mutant ABC1 proteins or protein fragments, in a recombinant form or endogenously expressed. Drug screens to identify compounds acting on the ABC1 expression product may employ any functional feature of the protein. In one example, the phosphorylation state or other post-translational modification is monitored as a measure of ABC1 biological activity. ABC1 has ATP binding sites, and thus assays may wholly or in part test the ability of ABC1 to bind ATP or to exhibit ATPase activity. ABC1, by analogy to similar proteins, is thought to be able to form a channel-like structure; drug screening assays could be based upon assaying for the ability of the protein to form a channel, or upon the ability to transport cholesterol or another molecule, or based upon the ability of other proteins bound by or regulated by ABC1 to form a channel. Alternatively, phospholipid or lipid transport can also be used as measures of ABC1 biological activity. [0203]
There is evidence that, in addition to its role as a regulator of cholesterol levels, ABC1 also transports anions. Functional assays could be based upon this property, and could employ drug screening technology such as (but not limited to) the ability of various dyes to change color in response to changes in specific ion concentrations in such assays can be performed in vesicles such as liposomes, or adapted to use whole cells. [0204]
Drug screening assays can also be based upon the ability of ABC1 or other ABC transporters to interact with other proteins. Such interacting proteins can be identified by a variety of methods known in the art, including, for example, radioimmunoprecipitation, co-immunoprecipitation, co-purification, and yeast two-hybrid screening. Such interactions can be further assayed by means including but not limited to fluorescence polarization or scintillation proximity methods. Drug screens can also be based upon functions of the ABC1 protein deduced upon X-ray crystallography of the protein and comparison of its 3-D structure to that of proteins with known functions. Such a crystal structure has been determined for the prokaryotic ABC family member HisP, histidine permease. Drug screens can be based upon a function or feature apparent upon creation of a transgenic or knockout mouse, or upon overexpression of the protein or protein fragment in mammalian cells in vitro. Moreover, expression of mammalian (e.g., human) ABC1 in yeast or [0205] C. elegans allows for screening of candidate compounds in wild-type and mutant backgrounds, as well as screens for mutations that enhance or suppress an ABC1-dependent phenotype. Modifier screens can also be performed in ABC1 transgenic or knock-out mice.
Additionally, drug screening assays can also be based upon ABC1 functions deduced upon antisense interference with the gene function. Intracellular localization of ABC1, or effects which occur upon a change in intracellular localization of the protein, can also be used as an assay for drug screening. Immunocytochemical methods will be used to determine the exact location of the ABC1 protein. [0206]
Human and rodent ABC1 protein can be used as an antigen to raise antibodies, including monoclonal antibodies. Such antibodies will be useful for a wide variety of purposes, including but not limited to functional studies and the development of drug screening assays and diagnostics. Monitoring the influence of agents (e.g., drugs, compounds) on the expression or biological activity of ABC1 can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase ABC1 gene expression, protein levels, or biological activity can be monitored in clinical trails of subjects exhibiting altered ABC1 gene expression, protein levels, or biological activity. Alternatively, the effectiveness of an agent determined by a screening assay to modulate ABC1 gene expression, protein levels, or biological activity can be monitored in clinical trails of subjects exhibiting decreased altered gene expression, protein levels, or biological activity. In such clinical trials, the expression or activity of ABC1 and, preferably, other genes that have been implicated in, for example, cardiovascular disease can be used to ascertain the effectiveness of a particular drug. [0207]
For example, and not by way of limitation, genes, including ABC1, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates ABC1 biological activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on cholesterol levels or cardiovascular disease, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of ABC1 and other genes implicated in the disorder. The levels of gene expression can be quantified by Northern blot analysis or RT-PCR, or, alternatively, by measuring the amount of protein produced, by one of a number of methods known in the art, or by measuring the levels of biological activity of ABC1 or other genes. In this way, the gene expression can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points, during, treatment of the individual with the agent. [0208]
In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) including the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of an ABC1 protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the ABC1 protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the ABC1 protein, mRNA, or genomic DNA in the pre-administration sample with the ABC1 protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of ABC1 to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of ABC1 to lower levels than detected. [0209]
The ABC1 gene or a fragment thereof can be used as a tool to express the protein in an appropriate cell in vitro or in vivo (gene therapy), or can be cloned into expression vectors which can be used to produce large enough amounts of ABC1 protein to use in in vitro assays for drug screening. Expression systems which may be employed include baculovirus, herpes virus, adenovirus, adeno-associated virus, bacterial systems, and eucaryotic systems such as CHO cells. Naked DNA and DNA-liposome complexes can also be used. [0210]
Assays of ABC1 activity includes binding to intracellular interacting proteins; interaction with a protein that up-regulates ABC1 activity; interaction with HDL particles or constituents; interaction with other proteins which facilitate interaction with HDL or its constituents; and measurement of cholesterol efflux. Furthermore, assays may be based upon the molecular dynamics of macromolecules, metabolites and ions by means of fluorescent-protein biosensors. Alternatively, the effect of candidate modulators on expression or activity may be measured at the level of ABC1 protein production using the same general approach in combination with standard immunological detection techniques, such as Western blotting or immunoprecipitation with an ABC1-specific antibody. Again, useful cholesterol-regulating or anti-CVD therapeutic modulators are identified as those which produce an change in ABC1 polypeptide production. Agonists may also affect ABC1 activity without any effect on expression level. [0211]
Candidate modulators may be purified (or substantially purified) molecules or may be one component of a mixture of compounds (e.g., an extract or supernatant obtained from cells). In a mixed compound assay, ABC1 expression is tested against progressively smaller subsets of the candidate compound pool (e.g., produced by standard purification techniques, e.g., HPLC or FPLC; Ausubel et al.) until a single compound or minimal compound mixture is demonstrated to modulate ABC1 expression. [0212]
Agonists, antagonists, or mimetics found to be effective at modulating the level of cellular ABC1 expression or activity may be confirmed as useful in animal models (for example, mice, pigs, rabbits, or chickens). For example, the compound may ameliorate the low HDL levels of mouse or chicken hypoalphalipoproteinemias. [0213]
A compound that promotes an increase in ABC1 expression or activity is considered particularly useful in the invention; such a molecule may be used, for example, as a therapeutic to increase the level or activity of native, cellular ABC1 and thereby treat a low HDL condition in an animal (for example, a human). [0214]
One method for increasing ABC biological activity is to increase the stabilization of the ABC protein or to prevent its degradation. Thus, it would be useful to identify mutations in an ABC polypeptide (e.g., ABC1) that lead to increased protein stability. These mutations can be incorporated into any protein therapy or gene therapy undertaken for the treatment of low HDL-C or any other condition resulting from loss of ABC1 biological activity. Similarly, compounds that increase the stability of a wild-type ABC polypeptide or decrease its catabolism may also be useful for the treatment of low HDL-C or any other condition resulting from loss of ABC1 biological activity. Such mutations and compounds can be identified using the methods described herein. [0215]
In one example, cells expressing an ABC polypeptide having a mutation are transiently metabolically labeled during translation and the half-life of the ABC polypeptide is determined using standard techniques. Mutations that increase the half-life of an ABC polypeptide are ones that increase ABC protein stability. These mutations can then be assessed for ABC biological activity. They can also be used to identify proteins that affect the stability of ABC1 mRNA or protein. One can then assay for compounds that act on these factors or on the ability of these factors to bind ABC1. [0216]
In another example, cells expressing wild-type ABC polypeptide are transiently metabolically labeled during translation, contacted with a candidate compounds, and the half-life of the ABC polypeptide is determined using standard techniques. Compounds that increase the half-life of an ABC polypeptide are useful compounds in the present invention. [0217]
If desired, treatment with an agonist of the invention may be combined with any other HDL-raising or anti-CVD therapies. [0218]
It is understood that, while ABC1 is the preferred ABC transporter for the drug screens described herein, other ABC transporters can also be used. The replacement of ABC1 with another ABC transporter is possible because it is likely that ABC transporter family members, such as ABC2, ABCR, or ABC8 will have a similar mechanism of regulation. [0219]
Exemplary assays are described in greater detail below. [0220]
Protein-Based Assays [0221]
ABC1 polypeptide (purified or unpurified) can be used in an assay to determine its ability to bind another protein (including, but not limited to, proteins found to specifically interact with ABC1). The effect of a compound on that binding is then determined. [0222]
Protein Interaction Assays [0223]
ABC1 protein (or a polypeptide fragment thereof or an epitope-tagged form or fragment thereof) is harvested from a suitable source (e.g., from a prokaryotic expression system, eukaryotic cells, a cell-free system, or by immunoprecipitation from ABC1-expressing cells). The ABC1 polypeptide is then bound to a suitable support (e.g., nitrocellulose or an antibody or a metal agarose column in the case of, for example, a his-tagged form of ABC1). Binding to the support is preferably done under conditions that allow proteins associated with ABC1 polypeptide to remain associated with it. Such conditions may include use of buffers that minimize interference with protein-protein interactions. The binding step can be done in the presence and absence of compounds being tested for their ability to interfere with interactions between ABC1 and other molecules. If desired, other proteins (e.g., a cell lysate) are added, and allowed time to associate with the ABC polypeptide. The immobilized ABC1 polypeptide is then washed to remove proteins or other cell constituents that may be non-specifically associated with it the polypeptide or the support. The immobilized ABC1 polypeptide is then dissociated from its support, and so that proteins bound to it are released (for example, by heating), or, alternatively, associated proteins are released from ABC1 without releasing the ABC1 polypeptide from the support. The released proteins and other cell constituents can be analyzed, for example, by SDS-PAGE gel electrophoresis, Western blotting and detection with specific antibodies, phosphoamino acid analysis, protease digestion, protein sequencing, or isoelectric focusing. Normal and mutant forms of ABC1 can be employed in these assays to gain additional information about which part of ABC1 a given factor is binding to. In addition, when incompletely purified polypeptide is employed, comparison of the normal and muatant forms of the protein can be used to help distinguish true binding proteins. [0224]
The foregoing assay can be performed using a purified or semipurified protein or other molecule that is known to interact with ABC1. This assay may include the following steps. [0225]
1. Harvest ABC1 protein and couple a suitable fluorescent label to it; [0226]
2. Label an interacting protein (or other molecule) with a second, different fluorescent label. Use dyes that will produce different quenching patterns when they are in close proximity to each other vs. when they are physically separate (i.e., dyes that quench each other when they are close together but fluoresce when they are not in close proximity); [0227]
3. Expose the interacting molecule to the immobilized ABC1 in the presence or absence of a compound being tested for its ability to interfere with an interaction between the two; and [0228]
4. Collect fluorescent readout data. [0229]
Another assay is includes Fluorescent Resonance Energy Transfer (FRET) assay. This assay can be performed as follows. 1. Provide ABC1 protein or a suitable polypeptide fragment thereof and couple a suitable FRET donor (e.g., nitro-benzoxadiazole (NBD)) to it; [0230]
2. Label an interacting protein (or other molecule) with a FRET acceptor (e.g., rhodamine); [0231]
3. Expose the acceptor-labeled interacting molecule to the donor-labeled ABC1 in the presence or absence of a compound being tested for its ability to interfere with an interaction between the two; and [0232]
4. Measure fluorescence resonance energy transfer. [0233]
Quenching and FRET assays are related. Either one can be applied in a given case, depending on which pair of fluorophores is used in the assay. [0234]
Membrane Permeability Assay [0235]
The ABC1 protein can also be tested for its effects on membrane permeability. For example, beyond its putative ability to translocate lipids, ABC1 might affect the permeability of membranes to ions. Other related membrane proteins, most notably the cystic fibrosis transmembrane conductance regulator and the sulfonylurea receptor, are associated with and regulate ion channels. [0236]
ABC1 or a fragment of ABC1 is incorporated into a synthetic vesicle, or, alternatively, is expressed in a cell and vesicles or other cell sub-structures containing ABC1 are isolated. The ABC1-containing vesicles or cells are loaded with a reporter molecule (such as a fluorescent ion indicator whose fluorescent properties change when it binds a particular ion) that can detect ions (to observe outward movement), or alternatively, the external medium is loaded with such a molecule (to observe inward movement). A molecule which exhibits differential properties when it is inside the vesicle compared to when it is outside the vesicle is preferred. For example, a molecule that has quenching properties when it is at high concentration but not when it is at another low concentration would be suitable. The movement of the charged molecule (either its ability to move or the kinetics of its movement) in the presence or absence of a compound being tested for its ability to affect this process can be determined. [0237]
In another assay, membrane permeability is determined electro-physiologically by measuring ionic influx or efflux mediated by or modulated by ABC1 by standard electrophysiological techniques. A suitable control (e.g., TD cells or a cell line with very low endogenous ABC1 expression) can be used as a control in the assay to determine if the effect observed is specific to cells expressing ABC1. [0238]
In still another assay, uptake of radioactive isotopes into or out of a vesicle can be measured. The vesicles are separated from the extravesicular medium and the radioactivity in the vesicles and in the medium is quantitated and compared. [0239]
Nucleic Acid-Based Assays [0240]
ABC1 nucleic acid may be used in an assay based on the binding of factors necessary for ABC1 gene transcription. The association between the ABC1 DNA and the binding factor may be assessed by means of any system that discriminates between protein-bound and non-protein-bound DNA (e.g., a gel retardation assay). The effect of a compound on the binding of a factor to ABC1 DNA is assessed by means of such an assay. In addition to in vitro binding assays, in vivo assays in which the regulatory regions of the ABC1 gene are linked to reporter genes can also be performed. [0241]
Assays Measuring ABC1 Stability [0242]
A cell-based or cell-free system can be used to screen for compounds based on their effect on the half-life of ABC1 mRNA or ABC1 protein. The assay may employ labeled mRNA or protein. Alternatively, ABC1 mRNA may be detected by means of specifically hybridizing probes or a quantitative PCR assay. Protein can be quantitated, for example, by fluorescent antibody-based methods. [0243]
In Vitro mRNA Stability Assay [0244]
1. Isolate or produce, by in vitro transcription, a suitable quantity of ABC1 mRNA; [0245]
2. Label the ABC1 mRNA; [0246]
3. Expose aliquots of the mRNA to a cell lysate in the presence or absence of a compound being tested for its ability to modulate ABC1 mRNA stability; [0247]
4. Assess intactness of the remaining mRNA at suitable time points. [0248]
In Vitro Protein Stability Assay [0249]
1. Express a suitable amount of ABC1 protein; [0250]
2. Label the protein; [0251]
3. Expose aliquots of the labeled protein to a cell lysate in the presence or absence of a compound being tested for its ability to modulate ABC1 protein stability; [0252]
4. Assess intactness of the remaining protein at suitable time points [0253]
In Vivo mRNA or Protein Stability Assay [0254]
1. Incubate cells expressing ABC1 mRNA or protein with a tracer (radiolabeled ribonucleotide or radiolabeled amino acid, respectively) for a very brief time period (e.g., five minutes) in the presence or absence of a compound being tested for its effect on mRNA or protein stability; [0255]
2. Incubate with unlabeled ribonucleotide or amino acid; and [0256]
3. Quantitate the ABC1 mRNA or protein radioactivity at time intervals beginning with the start of [0257] step 2 and extending to the time when the radioactivity in ABC1 mRNA or protein has declined by approximately 80%. It is preferable to separate the intact or mostly intact mRNA or protein from its radioactive breakdown products by a means such as gel electrophoresis in order to quantitate the mRNA or protein.
Assays Measuring Inhibition of Dominant Negative Activity [0258]
Mutant ABC1 polypeptides are likely to have dominant negative activity (i.e., activity that interferes with wild-type ABC1 function). An assay for a compound that can interfere with such a mutant may be based on any method of quantitating normal ABC1 activity in the presence of the mutant. For example, normal ABC1 facilitates cholesterol efflux, and a dominant negative mutant would interfere with this effect. The ability of a compound to counteract the effect of a dominant negative mutant may be based on cellular cholesterol efflux, or on any other normal activity of the wild-type ABC1 that was inhibitable by the mutant. [0259]
Assays Measuring Phosphorylation [0260]
The effect of a compound on ABC1 phosphorylation can be assayed by methods that quantitate phosphates on proteins or that assess the phosphorylation state of a specific residue of a ABC1. Such methods include but are not limited to [0261] ³²P labelling and immunoprecipitation, detection with antiphosphoamino acid antibodies (e.g., antiphosphoserine antibodies), phosphoamino acid analysis on 2-dimensional TLC plates, and protease digestion fingerprinting of proteins followed by detection of ³²P-labeled fragments.
Assays Measuring Other Post-Translational Modifications [0262]
The effect of a compound on the post-translational modification of ABC1 is based on any method capable of quantitating that particular modification. For example, effects of compounds on glycosylation may be assayed by treating ABC1 with glycosylase and quantitating the amount and nature of carbohydrate released. [0263]
Assays Measuring ATP Binding [0264]
The ability of ABC1 to bind ATP provides another assay to screen for compounds that affect ABC1. ATP binding can be quantitated as follows. [0265]
1. Provide ABC1 protein at an appropriate level of purity and reconstitute it in a lipid vesicle; [0266]
2. Expose the vesicle to a labeled but non-hydrolyzable ATP analog (such as gamma [0267] ³⁵S-ATP) in the presence or absence of compounds being tested for their effect on ATP binding. Note that azido-ATP analogs can be used to allow covalent attachment of the azido-ATP to protein (by means of U.V. light), and permit easier quantitation of the amount of ATP bound to the protein.
3. Quantitate the amount of ATP analog associated with ABC1 [0268]
Assays Measuring ATPase Activity [0269]
Quantitation of the ATPase activity of ABC1 can also be assayed for the effect of compounds on ABC1. This is preferably performed in a cell-free assay SO as to separate ABC1 from the many other ATPases in the cell. An ATPase assay may be performed in the presence or absence of membranes, and with or without integration of ABC1 protein into a membrane. If performed in a vesicle-based assay, the ATP hydrolysis products produced or the ATP hydrolyzed may be measured within or outside of the vesicles, or both. Such an assay may be based on disappearance of ATP or appearance of ATP hydrolysis products. [0270]
For high-throughput screening, a coupled ATPase assay is preferable. For example, a reaction mixture containing pyruvate kinase and lactate dehydrogenase can be used. The mixture includes phosphoenolpyruvate (PEP), nicotinamide adenine dinucleotide (NAD+), and ATP. The ATPase activity of ABC1 generates ADP from ATP. The ADP is then converted back to ATP as part of the pyruvate kinase reaction. The product, pyruvate, is then converted to lactate. The latter reaction generates a colored quinone (NADH) from a colorless substrate (NAD+), and the entire reaction can be monitored by detection of the color change upon formation of NADH. Since ADP is limiting for the pyruvate kinase reaction, this coupled system precisely monitors the ATPase activity of [0271] ABC 1.
Assays Measuring Cholesterol Efflux [0272]
A transport-based assay can be performed in vivo or in vitro. For example, the assay may be based on any part of the reverse cholesterol transport process that is readily re-created in culture, such as cholesterol or phospholipid efflux. Alternatively, the assay may be based on net cholesterol transport in a whole organism, as assessed by means of a labeled substance (such as cholesterol). [0273]
For high throughput, fluorescent lipids can be used to measure ABC1-catalyzed lipid efflux. For phospholipids, a fluorescent precursor, C6-NBD-phosphatidic acid, can be used. This lipid is taken up by cells and dephosphorylated by phosphatidic acid phosphohydrolase. The product, NBD-diglyceride, is then a precursor for synthesis of glycerophospholipids like phosphatidylcholine. The efflux of NBD-phosphatidylcholine can be monitored by detecting fluorescence resonance energy transfer (FRET) of the NBD to a suitable acceptor in the cell culture medium. This acceptor can be rhodamine-labeled phosphatidylethanolamine, a phospholipid that is not readily taken up by cells. The use of short-chain precursors obviates the requirement for the phospholipid transfer protein in the media. For cholesterol, NBD-cholesterol ester can be reconstituted into LDL. The LDL can efficiently deliver this lipid to cells via the LDL receptor pathway. The NBD-cholesterol esters are hydrolyzed in the lysosomes, resulting in NBD-cholesterol that can now be transported back to the plasma membrane and efflux from the cell. The efflux can be monitored by the aforementioned FRET assay in which NBD transfers its fluorescence resonance energy to the rhodamine-phosphatidylethanoline acceptor. [0274]
Animal Model Systems [0275]
Compounds identified as having activity in any of the above-described assays are subsequently screened in any available animal model system, including, but not limited to, pigs, rabbits, and WHAM chickens. Test compounds are administered to these animals according to standard methods. Test compounds may also be tested in mice bearing mutations in the ABC1 gene. Additionally, compounds may be screened for their ability to enhance an interaction between ABC1 and any HDL particle constituent such as ApoAI, ApoAII, or ApoE. [0276]
The Cholesterol Efflux Assay as a Drug Screen [0277]
The cholesterol efflux assay measures the ability of cells to transfer cholesterol to an extracellular acceptor molecule and is dependent on ABC1 function. In this procedure, cells are loaded with radiolabeled cholesterol by any of several biochemical pathways (Marcil et al., Arterioscler. Thromb. Vasc. Biol. 19:159-169, 1999). Cholesterol efflux is then measured after incubation for various times (typically 0 to 24 hours) in the presence of HDL3 or purified ApoAI. Cholesterol efflux is determined as the percentage of total cholesterol in the culture medium after various times of incubation. ABC1 expression levels and/or biological activity are associated with increased efflux while decreased levels of ABC1 are associated with decreased cholesterol efflux. [0278]
This assay can be readily adapted to the format used for drug screening, which may consist of a multi-well (e.g., 96-well) format. Modification of the assay to optimize it for drug screening would include scaling down and streamlining the procedure, modifying the labeling method, using a different cholesterol acceptor, altering the incubation time, and changing the method of calculating cholesterol efflux. In all these cases, the cholesterol efflux assay remains conceptually the same, though experimental modifications may be made. A transgenic mouse overexpressing ABC1 would be expected to have higher than normal HDL levels. [0279]
Knock-Out Mouse Model [0280]
An animal, such as a mouse, that has had one or both ABC1 alleles inactivated (e.g., by homologous recombination) is likely to have low HDL-C levels, and thus is a preferred animal model for screening for compounds that raise HDL-C levels. Such an animal can be produced using standard techniques. In addition to the initial screening of test compounds, the animals having mutant ABC1 genes are useful for further testing of efficacy and safety of drugs or agents first identified using one of the other screening methods described herein. Cells taken from the animal and placed in culture can also be exposed to test compounds. HDL-C levels can be measured using standard techniques, such as those described herein. [0281]
WHAM Chickens: An Animal Model For Low HDL Cholesterol [0282]
Wisconsin Hypo-Alpha Mutant (WHAM) chickens arose by spontaneous mutation in a closed flock. Mutant chickens came to attention through their a Z-linked white shank and white beak phenotype referred to as ‘recessive white skin’ (McGibbon, 1981) and were subsequently found to have a profound deficiency of HDL (Poernama et al., 1990). [0283]
This chicken low HDL locus (Y) is Z-linked, or sex-linked. (In birds, females are ZW and males are ZZ). Genetic mapping placed the Y locus on the long arm of the Z chromosome (Bitgood, 1985), proximal to the ID locus (Bitgood, 1988). Examination of current public mapping data for the chicken genome mapping project, ChickMap (maintained by the Roslin Institute; http://www.ri.bbsrc.ac.uk/ chickmap/ChickMapHomePage.html) showed that a region of synteny with [0284] human chromosome 9 lies on the long arm of the chicken Z chromosome (Zq) proximal to the ID locus. Evidence for this region of synteny is the location of the chicken aldolase B locus (ALDOB) within this region. The human ALDOB locus maps to chromosome 9q22.3 (The Genome Database, hftp://gdbwww.gdb.org/), not far from the location of human ABC1. This comparison of maps showed that the chicken Zq region near chicken ALDOB and the human 9q region near human ALDOB represent a region of synteny between human and chicken.
Since a low HDL locus maps to the 9q location in humans and to the Zq region in chickens, these low HDL loci are most probably located within the syntenic region. Thus we predicted that ABC1 is mutated in WHAM chickens. In support of this, we have identified an E_K mutation at a position that corresponds to [0285] amino acid 89 of human ABC1 (FIGS. 14 and 15). This non-conservative substitution is at a position that is conserved among human, mouse, and chicken, indicating that it is in a region of the protein likely to be of functional importance.
Discovery of the WHAM mutation in the amino-terminal portion of the ABC1 protein also establishes the importance of the amino-terminal region. This region may be critical because of association with other proteins required to carry out cholesterol efflux or related tasks. It may be an important regulatory region (there is a phosphorylation site for casein kinase near the mutated residue), or it may help to dictate a precise topological relationship with cellular membranes (the N-[0286] terminal 60 amino acid region contains a putative membrane-spanning or membrane-associated segment).
The amino-terminal region of the protein (up to the first 6-TM region at approximately amino acid 639) is an ideal tool for screening factors that affect ABC1 activity. It can be expressed as a truncated protein in ABC1 wild type cells in order to test for interference of the normal ABC1 function by the truncated protein. If the fragment acts in a dominant negative way, it could be used in immunoprecipitations to identify proteins that it may be competing away from the normal endogenous protein. [0287]
The C-terminus also lends itself to such experiments, as do the intracellular portions of the molecule, expressed as fragments or tagged or fusion proteins, in the absence of transmembrane regions. [0288]
Since it is possible that there are several genes in the human genome which affect cholesterol efflux, it is important to establish that any animal model to be used for a human genetic disease represents the homologous locus in that animal, and not a different locus with a similar function. The evidence above establishes that the chicken Y locus and the [0289] human chromosome 9 low HDL locus are homologous. WHAM chickens are therefore an important animal model for the identification of drugs that modulate cholesterol efflux.
The WHAM chickens' HDL deficiency syndrome is not, however, associated with an increased susceptibility to atherosclerosis in chickens. This probably reflects the shorter lifespan of the chicken rather than an inherent difference in the function of the chicken ABC1 gene compared to the human gene. We propose the WHAM chicken as a model for human low HDL for the development and testing of drugs to raise HDL in humans. Such a model could be employed in several forms, through the use of cells or other derivatives of these chickens, or by the use of the chickens themselves in tests of drug effectiveness, toxicity, and other drug development purposes. [0290]
Therapy [0291]
Compounds of the invention, including but not limited to, ABC1 polypeptides, ABC1 nucleic acids, other ABC transporters, and any therapeutic agent that modulates biological activity or expression of ABC1 identified using any of the methods disclosed herein, may be administered with a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer such compositions to patients. Although intravenous administration is preferred, any appropriate route of administration may be employed, for example, perenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration. Therapeutic formulations may be in the form of liquid solutions or suspension; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols. [0292]
Methods well known in the art for making formulations are found in, for example, [0293] Remington: The Science and Practice of Pharmacy, (19th ed.) ed. A.R. Gennaro AR., 1995, Mack Publishing Company, Easton, Pa. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for agonists of the invention include ethylenevinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, or example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
Compounds [0294]
In general, novel drugs for the treatment of aberrant cholesterol levels and/or CVD are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the field or drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods. [0295]
In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their HDL-raising and anti-CVD activities should be employed whenever possible. [0296]
When a crude extract is found to have cholesterol-modulating or anti-CVD activities or both, further fractionation of the positive lead extract is necessary to isolate chemical constituent responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract having cholesterol-modulating or anti-CVD activities. The same in vivo and in vitro assays described herein for the detection of activities in mixtures of compounds can be used to purify the active component and to test derivatives thereof. Methods of fractionation and purification of such heterogeneous extracts are known in the art. If desired, compounds shown to be useful agents for the treatment of pathogenicity are chemically modified according to methods known in the art. Compounds identified as being of therapeutic value are subsequently analyzed using any standard animal model of diabetes or obesity known in the art. [0297]
It is understood that compounds that modulate activity of proteins that modulate or are modulated by ABC1 are useful compounds for modulating cholesterol levels. Exemplary compounds are provided herein; others are known 10 in the art. [0298]
Compounds that are structurally related to cholesterol, or that mimic ApoAI or a related apolipoprotein, and increase ABC1 biological activity are particularly useful compounds in the invention. Other compounds, known to act on the MDR protein, can also be used or derivatized and assayed for their ability to increase ABC1 biological activity. Exemplary MDR modulators are PSC833, bromocriptine, and cyclosporin A. [0299]
Screening Patients Having Low HDL-C [0300]
ABC1 expression, biological activity, and mutational analysis can each serve as a diagnostic tool for low HDL; thus determination of the genetic subtyping of the ABC1 gene sequence can be used to subtype low HDL individuals or families to determine whether the low HDL phenotype is related to ABC1 function. This diagnostic process can lead to the tailoring of drug treatments according to patient genotype, including prediction of side effects upon administration of HDL increasing drugs (referred to herein as pharmacogenomics). Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual is examined to determine the ability of the individual to respond to a particular agent). [0301]
Agents, or modulators which have a stimulatory or inhibitory effect on ABC1 biological activity or gene expression can be administered to individuals to treat disorders (e.g., cardiovascular disease or low HDL cholesterol) associated with aberrant ABC1 activity. In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual may be considered. Differences in efficacy of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of ABC1 protein, expression of ABC1 nucleic acid, or mutation content of ABC1 genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual. [0302]
Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons (Eichelbaum, M., Clin. Exp. Pharmacol. Physiol., 23:983-985, 1996; Linder, M. W., Clin. Chem., 43:254-266, 1997). In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). Altered drug action may occur in a patient having a polymorphism (e.g., an single nucleotide polymorphism or SNP) in promoter, intronic, or exonic sequences of ABC1. Thus by determining the presence and prevalence of polymorphisms allow for prediction of a patient's response to a particular therapeutic agent. In particular, polymorphisms in the promoter region may be critical in determining the risk of HDL deficiency and CVD. [0303]
In addition to the mutations in the ABC1 gene described herein, we have detected polymorphisms in the human ABC1 gene (FIG. 11). These polymorphisms are located in promoter, intronic, and exonic sequence of ABC1. Using standard methods, such as direct sequencing, PCR, SSCP, or any other polymorphism-detection system, one could easily ascertain whether these polymorphisms are present in a patient prior to the establishment of a drug treatment regimen for a patient having low HDL, cardiovascular disease, or any other ABC1-mediated condition. It is possible that some these polymorphisms are, in fact, weak mutations. Individuals harboring such mutations may have an increased risk for cardiovascular disease; thus, these polymorphisms may also be useful in diagnostic assays. [0304]
Association Studies of ABC1 Gene Variants and HDL Levels or Cardiovascular Disease [0305]
The following polymorphisms have been examined for their effect on cholesterol regulation and the predisposition for the development of cardiovascular disease. [0306]
Substitution of G for A at nucleotide -1045 [G(-1045)A]. This variant is in complete linkage disequilibrium with the variant at −738 in the individuals we have sequenced, and thus any potential phenotypic effects currently attributed to the variant at −738 may at least in part be due to changes at this site. [0307]
Substitution of G for A at nucleotide—738 [G(-738)A]. This variant has been found at very high frequencies in populations selected for low HDL cholesterol or premature coronary artery disease. [0308]
Insertion of a G nucleotide at position—4 [G ins (-4)]. This variant has been associated with less coronary artery disease in its carriers than in non-carriers. [0309]
Substitution of a C for G at nucleotide—57 [G(-57)C]. This variant is in complete linkage disequilibrium with the variant at −4 in the individuals we have sequenced, and thus the phenotypic effects currently attributed to the variant at −4 may at least in part be due to changes at this site. [0310]
Substitution of A for G at nucleotide 730 (R219K). We have found carriers to have significantly less cardiovascular disease. [0311]
Substitution of C for T at nucleotide 1270 (V399A). Within the French Canadian population, this variant has only been found in individuals from the low HDL population. It has also been seen in individuals with low HDL or premature coronary artery disease in individuals of Dutch ancestry. [0312]
Substitution of A for G at nucleotide 2385 (V771M). This variant has been found at an increased frequency in a Dutch population selected for low HDL and at an increased frequency in a population selected for premature coronary artery disease compared to a control Dutch population, indicating carriers of this variant may have reduced HDL and an increased susceptibility to coronary artery disease. [0313]
Substitution of C for A at nucleotide 2394 (T774P). This variant has been seen at lower frequencies in populations with coronary artery disease or low HDL than in individuals without. [0314]
Substitution of C for G at nucleotide 2402 (K776N). This variant has been found at a significantly lower frequency (0.56% vs. 2.91%, p=0.02) in a coronary artery disease population vs. a control population of similar Dutch background. [0315]
Substitution of C for G at nucleotide 3590 (E 1172D). This variant is seen at lower frequencies in individuals with low HDL and in some populations with premature coronary artery disease. [0316]
Substitution of A for G at nucleotide 4384 (R1587K). This variant has been found at decreased frequencies in the ⅓ of individuals with the highest HDL levels in our large Dutch coronary artery disease population (p=0.036), at increased frequencies in those with HDL cholesterol <0.9 mmol/L (p<0.0001) and at decreased frequencies in the cohorts with HDL cholesterol >1.4 mmol/L in both this population (p=0.02) and the Dutch control population (p=0.003). [0317]
Substitution of G for C at nucleotide 5266 (S1731C). Two FHA individuals who have this variant on the other allele have much lower HDL cholesterol (0.155+0.025) than the FHA individuals in the family who do not have this variant on the other allele (0.64+0.14, p=0.0009). This variant has also been found in one general population French Canadian control with HDL at the 8th percentile (0.92) and one French Canadian individual from a population selected for low HDL and coronary disease (0.72). [0318]
Substitution of G for A at nucleotide -1113 [A(-1113)G]. This variant has been seen at varying frequencies in populations distinguished by their HDL levels. [0319]
Additional polymorphisms that may be associated with altered risk for cardiovascular disease or altered cholesterol levels are as follows: [0320]
Substitution of G for A at nucleotide 2723 (1883M). This variant has been seen at a much higher frequency in individuals of Dutch ancestry with premature coronary artery disease. [0321]
Insertion of 4 nucleotides (CCCT) at position −1181. [0322]
Substitution of C for A at nucleotide -479 (linkage disequilibrium with -518). [0323]
Substitution of G for A at nucleotide -380. [0324]
Other Embodiments [0325]
All publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication was specifically and individually indicated to be incorporated by reference. [0326]
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations following, in general, the principles of the invention and including such departures from the present disclosure within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth. [0327]

0

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 287

<210> SEQ ID NO 1

<211> LENGTH: 2261

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 1

Met Ala Cys Trp Pro Gln Leu Arg Leu Leu Leu Trp Lys Asn Leu Thr

1 5 10 15

Phe Arg Arg Arg Gln Thr Cys Gln Leu Leu Leu Glu Val Ala Trp Pro

20 25 30

Leu Phe Ile Phe Leu Ile Leu Ile Ser Val Arg Leu Ser Tyr Pro Pro

35 40 45

Tyr Glu Gln His Glu Cys His Phe Pro Asn Lys Ala Met Pro Ser Ala

50 55 60

Gly Thr Leu Pro Trp Val Gln Gly Ile Ile Cys Asn Ala Asn Asn Pro

65 70 75 80

Cys Phe Arg Tyr Pro Thr Pro Gly Glu Ala Pro Gly Val Val Gly Asn

85 90 95

Phe Asn Lys Ser Ile Val Ala Arg Leu Phe Ser Asp Ala Arg Arg Leu

100 105 110

Leu Leu Tyr Ser Gln Lys Asp Thr Ser Met Lys Asp Met Arg Lys Val

115 120 125

Leu Arg Thr Leu Gln Gln Ile Lys Lys Ser Ser Ser Asn Leu Lys Leu

130 135 140

Gln Asp Phe Leu Val Asp Asn Glu Thr Phe Ser Gly Phe Leu Tyr His

145 150 155 160

Asn Leu Ser Leu Pro Lys Ser Thr Val Asp Lys Met Leu Arg Ala Asp

165 170 175

Val Ile Leu His Lys Val Phe Leu Gln Gly Tyr Gln Leu His Leu Thr

180 185 190

Ser Leu Cys Asn Gly Ser Lys Ser Glu Glu Met Ile Gln Leu Gly Asp

195 200 205

Gln Glu Val Ser Glu Leu Cys Gly Leu Pro Arg Glu Lys Leu Ala Ala

210 215 220

Ala Glu Arg Val Leu Arg Ser Asn Met Asp Ile Leu Lys Pro Ile Leu

225 230 235 240

Arg Thr Leu Asn Ser Thr Ser Pro Phe Pro Ser Lys Glu Leu Ala Glu

245 250 255

Ala Thr Lys Thr Leu Leu His Ser Leu Gly Thr Leu Ala Gln Glu Leu

260 265 270

Phe Ser Met Arg Ser Trp Ser Asp Met Arg Gln Glu Val Met Phe Leu

275 280 285

Thr Asn Val Asn Ser Ser Ser Ser Ser Thr Gln Ile Tyr Gln Ala Val

290 295 300

Ser Arg Ile Val Cys Gly His Pro Glu Gly Gly Gly Leu Lys Ile Lys

305 310 315 320

Ser Leu Asn Trp Tyr Glu Asp Asn Asn Tyr Lys Ala Leu Phe Gly Gly

325 330 335

Asn Gly Thr Glu Glu Asp Ala Glu Thr Phe Tyr Asp Asn Ser Thr Thr

340 345 350

Pro Tyr Cys Asn Asp Leu Met Lys Asn Leu Glu Ser Ser Pro Leu Ser

355 360 365

Arg Ile Ile Trp Lys Ala Leu Lys Pro Leu Leu Val Gly Lys Ile Leu

370 375 380

Tyr Thr Pro Asp Thr Pro Ala Thr Arg Gln Val Met Ala Glu Val Asn

385 390 395 400

Lys Thr Phe Gln Glu Leu Ala Val Phe His Asp Leu Glu Gly Met Trp

405 410 415

Glu Glu Leu Ser Pro Lys Ile Trp Thr Phe Met Glu Asn Ser Gln Glu

420 425 430

Met Asp Leu Val Arg Met Leu Leu Asp Ser Arg Asp Asn Asp His Phe

435 440 445

Trp Glu Gln Gln Leu Asp Gly Leu Asp Trp Thr Ala Gln Asp Ile Val

450 455 460

Ala Phe Leu Ala Lys His Pro Glu Asp Val Gln Ser Ser Asn Gly Ser

465 470 475 480

Val Tyr Thr Trp Arg Glu Ala Phe Asn Glu Thr Asn Gln Ala Ile Arg

485 490 495

Thr Ile Ser Arg Phe Met Glu Cys Val Asn Leu Asn Lys Leu Glu Pro

500 505 510

Ile Ala Thr Glu Val Trp Leu Ile Asn Lys Ser Met Glu Leu Leu Asp

515 520 525

Glu Arg Lys Phe Trp Ala Gly Ile Val Phe Thr Gly Ile Thr Pro Gly

530 535 540

Ser Ile Glu Leu Pro His His Val Lys Tyr Lys Ile Arg Met Asp Ile

545 550 555 560

Asp Asn Val Glu Arg Thr Asn Lys Ile Lys Asp Gly Tyr Trp Asp Pro

565 570 575

Gly Pro Arg Ala Asp Pro Phe Glu Asp Met Arg Tyr Val Trp Gly Gly

580 585 590

Phe Ala Tyr Leu Gln Asp Val Val Glu Gln Ala Ile Ile Arg Val Leu

595 600 605

Thr Gly Thr Glu Lys Lys Thr Gly Val Tyr Met Gln Gln Met Pro Tyr

610 615 620

Pro Cys Tyr Val Asp Asp Ile Phe Leu Arg Val Met Ser Arg Ser Met

625 630 635 640

Pro Leu Phe Met Thr Leu Ala Trp Ile Tyr Ser Val Ala Val Ile Ile

645 650 655

Lys Gly Ile Val Tyr Glu Lys Glu Ala Arg Leu Lys Glu Thr Met Arg

660 665 670

Ile Met Gly Leu Asp Asn Ser Ile Leu Trp Phe Ser Trp Phe Ile Ser

675 680 685

Ser Leu Ile Pro Leu Leu Val Ser Ala Gly Leu Leu Val Val Ile Leu

690 695 700

Lys Leu Gly Asn Leu Leu Pro Tyr Ser Asp Pro Ser Val Val Phe Val

705 710 715 720

Phe Leu Ser Val Phe Ala Val Val Thr Ile Leu Gln Cys Phe Leu Ile

725 730 735

Ser Thr Leu Phe Ser Arg Ala Asn Leu Ala Ala Ala Cys Gly Gly Ile

740 745 750

Ile Tyr Phe Thr Leu Tyr Leu Pro Tyr Val Leu Cys Val Ala Trp Gln

755 760 765

Asp Tyr Val Gly Phe Thr Leu Lys Ile Phe Ala Ser Leu Leu Ser Pro

770 775 780

Val Ala Phe Gly Phe Gly Cys Glu Tyr Phe Ala Leu Phe Glu Glu Gln

785 790 795 800

Gly Ile Gly Val Gln Trp Asp Asn Leu Phe Glu Ser Pro Val Glu Glu

805 810 815

Asp Gly Phe Asn Leu Thr Thr Ser Val Ser Met Met Leu Phe Asp Thr

820 825 830

Phe Leu Tyr Gly Val Met Thr Trp Tyr Ile Glu Ala Val Phe Pro Gly

835 840 845

Gln Tyr Gly Ile Pro Arg Pro Trp Tyr Phe Pro Cys Thr Lys Ser Tyr

850 855 860

Trp Phe Gly Glu Glu Ser Asp Glu Lys Ser His Pro Gly Ser Asn Gln

865 870 875 880

Lys Arg Ile Ser Glu Ile Cys Met Glu Glu Glu Pro Thr His Leu Lys

885 890 895

Leu Gly Val Ser Ile Gln Asn Leu Val Lys Val Tyr Arg Asp Gly Met

900 905 910

Lys Val Ala Val Asp Gly Leu Ala Leu Asn Phe Tyr Glu Gly Gln Ile

915 920 925

Thr Ser Phe Leu Gly His Asn Gly Ala Gly Lys Thr Thr Thr Met Ser

930 935 940

Ile Leu Thr Gly Leu Phe Pro Pro Thr Ser Gly Thr Ala Tyr Ile Leu

945 950 955 960

Gly Lys Asp Ile Arg Ser Glu Met Ser Thr Ile Arg Gln Asn Leu Gly

965 970 975

Val Cys Pro Gln His Asn Val Leu Phe Asp Met Leu Thr Val Glu Glu

980 985 990

His Ile Trp Phe Tyr Ala Arg Leu Lys Gly Leu Ser Glu Lys His Val

995 1000 1005

Lys Ala Glu Met Glu Gln Met Ala Leu Asp Val Gly Leu Pro Ser Ser

1010 1015 1020

Lys Leu Lys Ser Lys Thr Ser Gln Leu Ser Gly Gly Met Gln Arg Lys

1025 1030 1035 1040

Leu Ser Val Ala Leu Ala Phe Val Gly Gly Ser Lys Val Val Ile Leu

1045 1050 1055

Asp Glu Pro Thr Ala Gly Val Asp Pro Tyr Ser Arg Arg Gly Ile Trp

1060 1065 1070

Glu Leu Leu Leu Lys Tyr Arg Gln Gly Arg Thr Ile Ile Leu Ser Thr

1075 1080 1085

His His Met Asp Glu Ala Asp Val Leu Gly Asp Arg Ile Ala Ile Ile

1090 1095 1100

Ser His Gly Lys Leu Cys Cys Val Gly Ser Ser Leu Phe Leu Lys Asn

1105 1110 1115 1120

Gln Leu Gly Thr Gly Tyr Tyr Leu Thr Leu Val Lys Lys Asp Val Glu

1125 1130 1135

Ser Ser Leu Ser Ser Cys Arg Asn Ser Ser Ser Thr Val Ser Tyr Leu

1140 1145 1150

Lys Lys Glu Asp Ser Val Ser Gln Ser Ser Ser Asp Ala Gly Leu Gly

1155 1160 1165

Ser Asp His Glu Ser Asp Thr Leu Thr Ile Asp Val Ser Ala Ile Ser

1170 1175 1180

Asn Leu Ile Arg Lys His Val Ser Glu Ala Arg Leu Val Glu Asp Ile

1185 1190 1195 1200

Gly His Glu Leu Thr Tyr Val Leu Pro Tyr Glu Ala Ala Lys Glu Gly

1205 1210 1215

Ala Phe Val Glu Leu Phe His Glu Ile Asp Asp Arg Leu Ser Asp Leu

1220 1225 1230

Gly Ile Ser Ser Tyr Gly Ile Ser Glu Thr Thr Leu Glu Glu Ile Phe

1235 1240 1245

Leu Lys Val Ala Glu Glu Ser Gly Val Asp Ala Glu Thr Ser Asp Gly

1250 1255 1260

Thr Leu Pro Ala Arg Arg Asn Arg Arg Ala Phe Gly Asp Lys Gln Ser

1265 1270 1275 1280

Cys Leu Arg Pro Phe Thr Glu Asp Asp Ala Ala Asp Pro Asn Asp Ser

1285 1290 1295

Asp Ile Asp Pro Glu Ser Arg Glu Thr Asp Leu Leu Ser Gly Met Asp

1300 1305 1310

Gly Lys Gly Ser Tyr Gln Val Lys Gly Trp Lys Leu Thr Gln Gln Gln

1315 1320 1325

Phe Val Ala Leu Leu Trp Lys Arg Leu Leu Ile Ala Arg Arg Ser Arg

1330 1335 1340

Lys Gly Phe Phe Ala Gln Ile Val Leu Pro Ala Val Phe Val Cys Ile

1345 1350 1355 1360

Ala Leu Val Phe Ser Leu Ile Val Pro Pro Phe Gly Lys Tyr Pro Ser

1365 1370 1375

Leu Glu Leu Gln Pro Trp Met Tyr Asn Glu Gln Tyr Thr Phe Val Ser

1380 1385 1390

Asn Asp Ala Pro Glu Asp Thr Gly Thr Leu Glu Leu Leu Asn Ala Leu

1395 1400 1405

Thr Lys Asp Pro Gly Phe Gly Thr Arg Cys Met Glu Gly Asn Pro Ile

1410 1415 1420

Pro Asp Thr Pro Cys Gln Ala Gly Glu Glu Glu Trp Thr Thr Ala Pro

1425 1430 1435 1440

Val Pro Gln Thr Ile Met Asp Leu Phe Gln Asn Gly Asn Trp Thr Met

1445 1450 1455

Gln Asn Pro Ser Pro Ala Cys Gln Cys Ser Ser Asp Lys Ile Lys Lys

1460 1465 1470

Met Leu Pro Val Cys Pro Pro Gly Ala Gly Gly Leu Pro Pro Pro Gln

1475 1480 1485

Arg Lys Gln Asn Thr Ala Asp Ile Leu Gln Asp Leu Thr Gly Arg Asn

1490 1495 1500

Ile Ser Asp Tyr Leu Val Lys Thr Tyr Val Gln Ile Ile Ala Lys Ser

1505 1510 1515 1520

Leu Lys Asn Lys Ile Trp Val Asn Glu Phe Arg Tyr Gly Gly Phe Ser

1525 1530 1535

Leu Gly Val Ser Asn Thr Gln Ala Leu Pro Pro Ser Gln Glu Val Asn

1540 1545 1550

Asp Ala Ile Lys Gln Met Lys Lys His Leu Lys Leu Ala Lys Asp Ser

1555 1560 1565

Ser Ala Asp Arg Phe Leu Asn Ser Leu Gly Arg Phe Met Thr Gly Leu

1570 1575 1580

Asp Thr Arg Asn Asn Val Lys Val Trp Phe Asn Asn Lys Gly Trp His

1585 1590 1595 1600

Ala Ile Ser Ser Phe Leu Asn Val Ile Asn Asn Ala Ile Leu Arg Ala

1605 1610 1615

Asn Leu Gln Lys Gly Glu Asn Pro Ser His Tyr Gly Ile Thr Ala Phe

1620 1625 1630

Asn His Pro Leu Asn Leu Thr Lys Gln Gln Leu Ser Glu Val Ala Leu

1635 1640 1645

Met Thr Thr Ser Val Asp Val Leu Val Ser Ile Cys Val Ile Phe Ala

1650 1655 1660

Met Ser Phe Val Pro Ala Ser Phe Val Val Phe Leu Ile Gln Glu Arg

1665 1670 1675 1680

Val Ser Lys Ala Lys His Leu Gln Phe Ile Ser Gly Val Lys Pro Val

1685 1690 1695

Ile Tyr Trp Leu Ser Asn Phe Val Trp Asp Met Cys Asn Tyr Val Val

1700 1705 1710

Pro Ala Thr Leu Val Ile Ile Ile Phe Ile Cys Phe Gln Gln Lys Ser

1715 1720 1725

Tyr Val Ser Ser Thr Asn Leu Pro Val Leu Ala Leu Leu Leu Leu Leu

1730 1735 1740

Tyr Gly Trp Ser Ile Thr Pro Leu Met Tyr Pro Ala Ser Phe Val Phe

1745 1750 1755 1760

Lys Ile Pro Ser Thr Ala Tyr Val Val Leu Thr Ser Val Asn Leu Phe

1765 1770 1775

Ile Gly Ile Asn Gly Ser Val Ala Thr Phe Val Leu Glu Leu Phe Thr

1780 1785 1790

Asp Asn Lys Leu Asn Asn Ile Asn Asp Ile Leu Lys Ser Val Phe Leu

1795 1800 1805

Ile Phe Pro His Phe Cys Leu Gly Arg Gly Leu Ile Asp Met Val Lys

1810 1815 1820

Asn Gln Ala Met Ala Asp Ala Leu Glu Arg Phe Gly Glu Asn Arg Phe

1825 1830 1835 1840

Val Ser Pro Leu Ser Trp Asp Leu Val Gly Arg Asn Leu Phe Ala Met

1845 1850 1855

Ala Val Glu Gly Val Val Phe Phe Leu Ile Thr Val Leu Ile Gln Tyr

1860 1865 1870

Arg Phe Phe Ile Arg Pro Arg Pro Val Asn Ala Lys Leu Ser Pro Leu

1875 1880 1885

Asn Asp Glu Asp Glu Asp Val Arg Arg Glu Arg Gln Arg Ile Leu Asp

1890 1895 1900

Gly Gly Gly Gln Asn Asp Ile Leu Glu Ile Lys Glu Leu Thr Lys Ile

1905 1910 1915 1920

Tyr Arg Arg Lys Arg Lys Pro Ala Val Asp Arg Ile Cys Val Gly Ile

1925 1930 1935

Pro Pro Gly Glu Cys Phe Gly Leu Leu Gly Val Asn Gly Ala Gly Lys

1940 1945 1950

Ser Ser Thr Phe Lys Met Leu Thr Gly Asp Thr Thr Val Thr Arg Gly

1955 1960 1965

Asp Ala Phe Leu Asn Lys Asn Ser Ile Leu Ser Asn Ile His Glu Val

1970 1975 1980

His Gln Asn Met Gly Tyr Cys Pro Gln Phe Asp Ala Ile Thr Glu Leu

1985 1990 1995 2000

Leu Thr Gly Arg Glu His Val Glu Phe Phe Ala Leu Leu Arg Gly Val

2005 2010 2015

Pro Glu Lys Glu Val Gly Lys Val Gly Glu Trp Ala Ile Arg Lys Leu

2020 2025 2030

Gly Leu Val Lys Tyr Gly Glu Lys Tyr Ala Gly Asn Tyr Ser Gly Gly

2035 2040 2045

Asn Lys Arg Lys Leu Ser Thr Ala Met Ala Leu Ile Gly Gly Pro Pro

2050 2055 2060

Val Val Phe Leu Asp Glu Pro Thr Thr Gly Met Asp Pro Lys Ala Arg

2065 2070 2075 2080

Arg Phe Leu Trp Asn Cys Ala Leu Ser Val Val Lys Glu Gly Arg Ser

2085 2090 2095

Val Val Leu Thr Ser His Ser Met Glu Glu Cys Glu Ala Leu Cys Thr

2100 2105 2110

Arg Met Ala Ile Met Val Asn Gly Arg Phe Arg Cys Leu Gly Ser Val

2115 2120 2125

Gln His Leu Lys Asn Arg Phe Gly Asp Gly Tyr Thr Ile Val Val Arg

2130 2135 2140

Ile Ala Gly Ser Asn Pro Asp Leu Lys Pro Val Gln Asp Phe Phe Gly

2145 2150 2155 2160

Leu Ala Phe Pro Gly Ser Val Leu Lys Glu Lys His Arg Asn Met Leu

2165 2170 2175

Gln Tyr Gln Leu Pro Ser Ser Leu Ser Ser Leu Ala Arg Ile Phe Ser

2180 2185 2190

Ile Leu Ser Gln Ser Lys Lys Arg Leu His Ile Glu Asp Tyr Ser Val

2195 2200 2205

Ser Gln Thr Thr Leu Asp Gln Val Phe Val Asn Phe Ala Lys Asp Gln

2210 2215 2220

Ser Asp Asp Asp His Leu Lys Asp Leu Ser Leu His Lys Asn Gln Thr

2225 2230 2235 2240

Val Val Asp Val Ala Val Leu Thr Ser Phe Leu Gln Asp Glu Lys Val

2245 2250 2255

Lys Glu Ser Tyr Val

2260

<210> SEQ ID NO 2

<211> LENGTH: 7860

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 2

gtccctgctg tgagctctgg ccgctgcctt ccagggctcc cgagccacac gctgggggtg 60

ctggctgagg gaacatggct tgttggcctc agctgaggtt gctgctgtgg aagaacctca 120

ctttcagaag aagacaaaca tgtcagctgt tactggaagt ggcctggcct ctatttatct 180

tcctgatcct gatctctgtt cggctgagct acccacccta tgaacaacat gaatgccatt 240

ttccaaataa agccatgccc tctgcaggaa cacttccttg ggttcagggg attatctgta 300

atgccaacaa cccctgtttc cgttacccga ctcctgggga ggctcccgga gttgttggaa 360

actttaacaa atccattgtg gctcgcctgt tctcagatgc tcggaggctt cttttataca 420

gccagaaaga caccagcatg aaggacatgc gcaaagttct gagaacatta cagcagatca 480

agaaatccag ctcaaacttg aagcttcaag atttcctggt ggacaatgaa accttctctg 540

ggttcctgta tcacaacctc tctctcccaa agtctactgt ggacaagatg ctgagggctg 600

atgtcattct ccacaaggta tttttgcaag gctaccagtt acatttgaca agtctgtgca 660

atggatcaaa atcagaagag atgattcaac ttggtgacca agaagtttct gagctttgtg 720

gcctaccaag ggagaaactg gctgcagcag agcgagtact tcgttccaac atggacatcc 780

tgaagccaat cctgagaaca ctaaactcta catctccctt cccgagcaag gagctggctg 840

aagccacaaa aacattgctg catagtcttg ggactctggc ccaggagctg ttcagcatga 900

gaagctggag tgacatgcga caggaggtga tgtttctgac caatgtgaac agctccagct 960

cctccaccca aatctaccag gctgtgtctc gtattgtctg cgggcatccc gagggagggg 1020

ggctgaagat caagtctctc aactggtatg aggacaacaa ctacaaagcc ctctttggag 1080

gcaatggcac tgaggaagat gctgaaacct tctatgacaa ctctacaact ccttactgca 1140

atgatttgat gaagaatttg gagtctagtc ctctttcccg cattatctgg aaagctctga 1200

agccgctgct cgttgggaag atcctgtata cacctgacac tccagccaca aggcaggtca 1260

tggctgaggt gaacaagacc ttccaggaac tggctgtgtt ccatgatctg gaaggcatgt 1320

gggaggaact cagccccaag atctggacct tcatggagaa cagccaagaa atggaccttg 1380

tccggatgct gttggacagc agggacaatg accacttttg ggaacagcag ttggatggct 1440

tagattggac agcccaagac atcgtggcgt ttttggccaa gcacccagag gatgtccagt 1500

ccagtaatgg ttctgtgtac acctggagag aagctttcaa cgagactaac caggcaatcc 1560

ggaccatatc tcgcttcatg gagtgtgtca acctgaacaa gctagaaccc atagcaacag 1620

aagtctggct catcaacaag tccatggagc tgctggatga gaggaagttc tgggctggta 1680

ttgtgttcac tggaattact ccaggcagca ttgagctgcc ccatcatgtc aagtacaaga 1740

tccgaatgga cattgacaat gtggagagga caaataaaat caaggatggg tactgggacc 1800

ctggtcctcg agctgacccc tttgaggaca tgcggtacgt ctgggggggc ttcgcctact 1860

tgcaggatgt ggtggagcag gcaatcatca gggtgctgac gggcaccgag aagaaaactg 1920

gtgtctatat gcaacagatg ccctatccct gttacgttga tgacatcttt ctgcgggtga 1980

tgagccggtc aatgcccctc ttcatgacgc tggcctggat ttactcagtg gctgtgatca 2040

tcaagggcat cgtgtatgag aaggaggcac ggctgaaaga gaccatgcgg atcatgggcc 2100

tggacaacag catcctctgg tttagctggt tcattagtag cctcattcct cttcttgtga 2160

gcgctggcct gctagtggtc atcctgaagt taggaaacct gctgccctac agtgatccca 2220

gcgtggtgtt tgtcttcctg tccgtgtttg ctgtggtgac aatcctgcag tgcttcctga 2280

ttagcacact cttctccaga gccaacctgg cagcagcctg tgggggcatc atctacttca 2340

cgctgtacct gccctacgtc ctgtgtgtgg catggcagga ctacgtgggc ttcacactca 2400

agatcttcgc tagcctgctg tctcctgtgg cttttgggtt tggctgtgag tactttgccc 2460

tttttgagga gcagggcatt ggagtgcagt gggacaacct gtttgagagt cctgtggagg 2520

aagatggctt caatctcacc acttcggtct ccatgatgct gtttgacacc ttcctctatg 2580

gggtgatgac ctggtacatt gaggctgtct ttccaggcca gtacggaatt cccaggccct 2640

ggtattttcc ttgcaccaag tcctactggt ttggcgagga aagtgatgag aagagccacc 2700

ctggttccaa ccagaagaga atatcagaaa tctgcatgga ggaggaaccc acccacttga 2760

agctgggcgt gtccattcag aacctggtaa aagtctaccg agatgggatg aaggtggctg 2820

tcgatggcct ggcactgaat ttttatgagg gccagatcac ctccttcctg ggccacaatg 2880

gagcggggaa gacgaccacc atgtcaatcc tgaccgggtt gttccccccg acctcgggca 2940

ccgcctacat cctgggaaaa gacattcgct ctgagatgag caccatccgg cagaacctgg 3000

gggtctgtcc ccagcataac gtgctgtttg acatgctgac tgtcgaagaa cacatctggt 3060

tctatgcccg cttgaaaggg ctctctgaga agcacgtgaa ggcggagatg gagcagatgg 3120

ccctggatgt tggtttgcca tcaagcaagc tgaaaagcaa aacaagccag ctgtcaggtg 3180

gaatgcagag aaagctatct gtggccttgg cctttgtcgg gggatctaag gttgtcattc 3240

tggatgaacc cacagctggt gtggaccctt actcccgcag gggaatatgg gagctgctgc 3300

tgaaataccg acaaggccgc accattattc tctctacaca ccacatggat gaagcggacg 3360

tcctggggga caggattgcc atcatctccc atgggaagct gtgctgtgtg ggctcctccc 3420

tgtttctgaa gaaccagctg ggaacaggct actacctgac cttggtcaag aaagatgtgg 3480

aatcctccct cagttcctgc agaaacagta gtagcactgt gtcatacctg aaaaaggagg 3540

acagtgtttc tcagagcagt tctgatgctg gcctgggcag cgaccatgag agtgacacgc 3600

tgaccatcga tgtctctgct atctccaacc tcatcaggaa gcatgtgtct gaagcccggc 3660

tggtggaaga catagggcat gagctgacct atgtgctgcc atatgaagct gctaaggagg 3720

gagcctttgt ggaactcttt catgagattg atgaccggct ctcagacctg ggcatttcta 3780

gttatggcat ctcagagacg accctggaag aaatattcct caaggtggcc gaagagagtg 3840

gggtggatgc tgagacctca gatggtacct tgccagcaag acgaaacagg cgggccttcg 3900

gggacaagca gagctgtctt cgcccgttca ctgaagatga tgctgctgat ccaaatgatt 3960

ctgacataga cccagaatcc agagagacag acttgctcag tgggatggat ggcaaagggt 4020

cctaccaggt gaaaggctgg aaacttacac agcaacagtt tgtggccctt ttgtggaaga 4080

gactgctaat tgccagacgg agtcggaaag gattttttgc tcagattgtc ttgccagctg 4140

tgtttgtctg cattgccctt gtgttcagcc tgatcgtgcc accctttggc aagtacccca 4200

gcctggaact tcagccctgg atgtacaacg aacagtacac atttgtcagc aatgatgctc 4260

ctgaggacac gggaaccctg gaactcttaa acgccctcac caaagaccct ggcttcggga 4320

cccgctgtat ggaaggaaac ccaatcccag acacgccctg ccaggcaggg gaggaagagt 4380

ggaccactgc cccagttccc cagaccatca tggacctctt ccagaatggg aactggacaa 4440

tgcagaaccc ttcacctgca tgccagtgta gcagcgacaa aatcaagaag atgctgcctg 4500

tgtgtccccc aggggcaggg gggctgcctc ctccacaaag aaaacaaaac actgcagata 4560

tccttcagga cctgacagga agaaacattt cggattatct ggtgaagacg tatgtgcaga 4620

tcatagccaa aagcttaaag aacaagatct gggtgaatga gtttaggtat ggcggctttt 4680

ccctgggtgt cagtaatact caagcacttc ctccgagtca agaagttaat gatgccatca 4740

aacaaatgaa gaaacaccta aagctggcca aggacagttc tgcagatcga tttctcaaca 4800

gcttgggaag atttatgaca ggactggaca ccagaaataa tgtcaaggtg tggttcaata 4860

acaagggctg gcatgcaatc agctctttcc tgaatgtcat caacaatgcc attctccggg 4920

ccaacctgca aaagggagag aaccctagcc attatggaat tactgctttc aatcatcccc 4980

tgaatctcac caagcagcag ctctcagagg tggctctgat gaccacatca gtggatgtcc 5040

ttgtgtccat ctgtgtcatc tttgcaatgt ccttcgtccc agccagcttt gtcgtattcc 5100

tgatccagga gcgggtcagc aaagcaaaac acctgcagtt catcagtgga gtgaagcctg 5160

tcatctactg gctctctaat tttgtctggg atatgtgcaa ttacgttgtc cctgccacac 5220

tggtcattat catcttcatc tgcttccagc agaagtccta tgtgtcctcc accaatctgc 5280

ctgtgctagc ccttctactt ttgctgtatg ggtggtcaat cacacctctc atgtacccag 5340

cctcctttgt gttcaagatc cccagcacag cctatgtggt gctcaccagc gtgaacctct 5400

tcattggcat taatggcagc gtggccacct ttgtgctgga gctgttcacc gacaataagc 5460

tgaataatat caatgatatc ctgaagtccg tgttcttgat cttcccacat ttttgcctgg 5520

gacgagggct catcgacatg gtgaaaaacc aggcaatggc tgatgccctg gaaaggtttg 5580

gggagaatcg ctttgtgtca ccattatctt gggacttggt gggacgaaac ctcttcgcca 5640

tggccgtgga aggggtggtg ttcttcctca ttactgttct gatccagtac agattcttca 5700

tcaggcccag acctgtaaat gcaaagctat ctcctctgaa tgatgaagat gaagatgtga 5760

ggcgggaaag acagagaatt cttgatggtg gaggccagaa tgacatctta gaaatcaagg 5820

agttgacgaa gatatataga aggaagcgga agcctgctgt tgacaggatt tgcgtgggca 5880

ttcctcctgg tgagtgcttt gggctcctgg gagttaatgg ggctggaaaa tcatcaactt 5940

tcaagatgtt aacaggagat accactgtta ccagaggaga tgctttcctt aacaaaaata 6000

gtatcttatc aaacatccat gaagtacatc agaacatggg ctactgccct cagtttgatg 6060

ccatcacaga gctgttgact gggagagaac acgtggagtt ctttgccctt ttgagaggag 6120

tcccagagaa agaagttggc aaggttggtg agtgggcgat tcggaaactg ggcctcgtga 6180

agtatggaga aaaatatgct ggtaactata gtggaggcaa caaacgcaag ctctctacag 6240

ccatggcttt gatcggcggg cctcctgtgg tgtttctgga tgaacccacc acaggcatgg 6300

atcccaaagc ccggcggttc ttgtggaatt gtgccctaag tgttgtcaag gaggggagat 6360

cagtagtgct tacatctcat agtatggaag aatgtgaagc tctttgcact aggatggcaa 6420

tcatggtcaa tggaaggttc aggtgccttg gcagtgtcca gcatctaaaa aataggtttg 6480

gagatggtta tacaatagtt gtacgaatag cagggtccaa cccggacctg aagcctgtcc 6540

aggatttctt tggacttgca tttcctggaa gtgttctaaa agagaaacac cggaacatgc 6600

tacaatacca gcttccatct tcattatctt ctctggccag gatattcagc atcctctccc 6660

agagcaaaaa gcgactccac atagaagact actctgtttc tcagacaaca cttgaccaag 6720

tatttgtgaa ctttgccaag gaccaaagtg atgatgacca cttaaaagac ctctcattac 6780

acaaaaacca gacagtagtg gacgttgcag ttctcacatc ttttctacag gatgagaaag 6840

tgaaagaaag ctatgtatga agaatcctgt tcatacgggg tggctgaaag taaagaggaa 6900

ctagactttc ctttgcacca tgtgaagtgt tgtggagaaa agagccagaa gttgatgtgg 6960

gaagaagtaa actggatact gtactgatac tattcaatgc aatgcaattc aatgcaatga 7020

aaacaaaatt ccattacagg ggcagtgcct ttgtagccta tgtcttgtat ggctctcaag 7080

tgaaagactt gaatttagtt ttttacctat acctatgtga aactctatta tggaacccaa 7140

tggacatatg ggtttgaact cacacttttt tttttttttt tgttcctgtg tattctcatt 7200

ggggttgcaa caataattca tcaagtaatc atggccagcg attattgatc aaaatcaaaa 7260

ggtaatgcac atcctcattc actaagccat gccatgccca ggagactggt ttcccggtga 7320

cacatccatt gctggcaatg agtgtgccag agttattagt gccaagtttt tcagaaagtt 7380

tgaagcacca tggtgtgtca tgctcacttt tgtgaaagct gctctgctca gagtctatca 7440

acattgaata tcagttgaca gaatggtgcc atgcgtggct aacatcctgc tttgattccc 7500

tctgataagc tgttctggtg gcagtaacat gcaacaaaaa tgtgggtgtc tccaggcacg 7560

ggaaacttgg ttccattgtt atattgtcct atgcttcgag ccatgggtct acagggtcat 7620

ccttatgaga ctcttaaata tacttagatc ctggtaagag gcaaagaatc aacagccaaa 7680

ctgctggggc tgcaactgct gaagccaggg catgggatta aagagattgt gcgttcaaac 7740

ctagggaagc ctgtgcccat ttgtcctgac tgtctgctaa catggtacac tgcatctcaa 7800

gatgtttatc tgacacaagt gtattatttc tggctttttg aattaatcta gaaaatgaaa 7860

<210> SEQ ID NO 3

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 3

gcagagggca tggctttatt tg 22

<210> SEQ ID NO 4

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 4

ctgccaggca ggggaggaag agtg 24

<210> SEQ ID NO 5

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 5

gaaagtgact cacttgtgga gga 23

<210> SEQ ID NO 6

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 6

aaaggggctt ggtaagggta 20

<210> SEQ ID NO 7

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 7

catgcacatg cacacacata 20

<210> SEQ ID NO 8

<211> LENGTH: 27

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 8

ctttctgcgg gtgatgagcc ggtcaat 27

<210> SEQ ID NO 9

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 9

ccttagcccg tgttgagcta 20

<210> SEQ ID NO 10

<211> LENGTH: 26

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 10

cctgtaaatg caaagctatc tcctct 26

<210> SEQ ID NO 11

<211> LENGTH: 26

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 11

cgtcaactcc ttgatttcta agatgt 26

<210> SEQ ID NO 12

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 12

gggttcccag ggttcagtat 20

<210> SEQ ID NO 13

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 13

gatcaggaat tcaagcacca a 21

<210> SEQ ID NO 14

<211> LENGTH: 10545

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)...(10545)

OTHER INFORMATION: n = a, t, c, o g

<400> SEQUENCE: 14

acctcttata gaatgataga attcctctgg aatgattgga taacttcatt tcatccttga 60

cttttacctt ggaggatttc ttaccccttt tggcttctca aatttgacta ttaaaatgtt 120

gcctttaaaa ataggaacac agtttcaggg gggagtacca gcccatgacc cttctgcaag 180

gccccctaac tcaaggtagt ttccctggaa ctgtggttta tggaatgttt caggagtgtg 240

aggaggtata atttaaggct gtcctagcaa ggataccctt aaggatagag ggcccagtag 300

catctggagg ccagaaaagt taaactgagg cagtcagatt agcttcaggc tcaattaagc 360

tgatgggtca gcctgggaga aattgcagga tgactctcaa tatcccctcc cacccccaca 420

gcagccacga tctgtctgtc tttaatcatg ggtgcagtga acctgttctt tccaggtgtc 480

ttggccttca gtaaccttgt taggcttgtc cctgaacgtg gctaccgatc caaagacaca 540

tgatcagaga ggcaattaga gaacagacct tttccaaagc aagcatgttc tgttgggctt 600

agaagtttca tgtcctaata ttataggacc ctgtgcatct ctctggagat gaggcacatg 660

agtcatatct gtgattcttg cttttgtgtc aacatctcat gaataggcaa tcagagcttt 720

ggcaccaatg tattttcagt tcatatctga tgtagttaaa tccacctcct gctttgtagt 780

ttactggcaa gctgtttttg atataagaca tctagaacac tgtaaatata taacattttt 840

atttgtctat tatacctcaa ttacgaaaaa gacatctaga agcaacctca tcaagagaga 900

tactgaggcc gggcatggta gctcacactt gcaatcccat tactttggga ggctgaggca 960

ggtagatcac ttgaggtcaa gagtttgaaa ccagcctggc caacatgttg aaaccctgtc 1020

tctattaaaa atacaaaaaa gttagctggg cttggtggtg ggcacctgta atcccagcta 1080

ctccggaggc tgaggcagga gaatcacttg aacctgggag gcagaggttg cagtgagctg 1140

agatcacacc actgcactcc aacctgggca ccagagtgag attacatcta aaaaataaaa 1200

taaagtaata aaaaagagag atattgatag ctgttgttgg aaatttcaac ttccatctca 1260

cttctggtaa ctttttggaa gtttgttgaa caaagtggaa tacacgcaca tacacacaca 1320

cacatactct cttgtttgtt taaggtttaa tgaaatagct gtcatataat cactgttttt 1380

gaaagaggag aattagttgc tatctgtaca ttttgggtat gtgaactatt tggatagaac 1440

tctgagaaat gcattcagaa caacaaacaa aatcatagga gaaatagcta agtgggaagg 1500

ggcatataag agttgttgaa aaagttattt cttgagaaac cagctctaat gctaggcaag 1560

tcacttgctt tgggggaggc ctcagcttct ctgtctataa gattgcagca ggggtgtagt 1620

gggaatgagt cttcaacatt ccaagagatt ttatctacta atacgacagt caaatggagc 1680

atgactttgt ggaagcctct cctcttccac ccagaggggc caatttctct gtcccagtga 1740

gatgttgaca cttgtatgat ccctgcttgg agacttccct cttctggaac ctgccctggc 1800

tcaggcatga gggctgactg tcacccttcg ataggagccc agcactaaag ctcatgtgtt 1860

ggcagtgttc ttgcgggaag gaaaaagacc agccagccca tttgttactg cacaagcaaa 1920

cagcttctgg tagctgtaca gatacatgca ctttctttcc tcactgtgtt tccatagaca 1980

gatttagtgc tgtagaagag tagagggcag tcacgggaag gagttcctgt ttttcttttg 2040

gctatgccaa atggggaaaa atcctcctat cttgtctttt tagtgtcatc ctctctcccc 2100

ttttcttctt ctttataatt ctcatctctc atctctcctg gaaatgtgca tgtcaagttc 2160

aaaagggcac aatgttttgg tgaggaagag gtgggagaac acgtgccagg tgctaactag 2220

ggtcatcatt tcccccttca cagccagctt cctgtgaatg tgtgtgtgtg tgtgtgtgtg 2280

tgtgtgtgtg tgtgtgtgtg tgtgtatttc ttttgccagc atcactgaat ctgtctgctg 2340

tctggtattc caggttttgg tttagggaaa agtaaaagta attttataat cccagctgtc 2400

atttaagcca cccctttgtg ggtagcatat ggtccactct ctcagttcat tgtcctaaag 2460

atgcttcatc agaaaggaat aacttccacc ccgttactct ctgtcccctt actctgcttt 2520

atttttcttc gtcaatccta ccaccaccac ccactgtttg aacaacccac tattatttgt 2580

ctgtttccca tccctggtag aataggagcc ccatgaatga aggaactttg cttctgttgt 2640

tcaccactga atctctaagg tatggaacac acctggcatg tgataggcac tcgataaata 2700

tttgttgtgg ctcatgggca ccttgcagag ttaaggctgc agttgtttgt ggaatttata 2760

agtggtaatg aatatttatc tactattcct cttccaaggc gatcacacaa taatcaggct 2820

ttacactatc cagttcttag gtcttccaag ttatgacttg tgaggtatgt taattatgat 2880

aatagaaggc agtttatttg gttcagattt attgatgtgt aatttaccac agtaagactt 2940

cccctttaca aaagtatgat gagttttgac aaatggatac acatgtgtat ctaccactgc 3000

catgctcctt ttcagtctgt cgtcccctcc acccatgacc actggtcacc actgcagtga 3060

tttctgtccc cttcatttca ccttttccag aatgtcatat aaatggaatc atgcagtatg 3120

tagttttttg tgtctggctt atttttctta gcattaggct tttgggattc atccaggttg 3180

tcgcatgtaa cagtagctta ttccttttta tggctgagta agtgtcccag ttttatttat 3240

atatttattt atgaggaggt gtctcactct gtcacccagg ctggagtgcg gtagcgcgat 3300

ctcagctcac tgcaacctcc gcctcccagg ttcaagcaat tctcctgcct cctgagtagc 3360

tgggattaca ggcacccacc gccacgccca actaattttt atatttttag tagagatggg 3420

gtttcaccat gttggccagg ctgatctcaa actcttgacc tcaggtgatc cgcccacctc 3480

tggctcccaa agtgctagga ttacaggcat gagccactgt gcccagcccc agttttattt 3540

attcaccagt tgatggtctt ttcgacaact aattgtttcc agtttttggc tattctgtat 3600

aaggcttcta taaatattca caaataccta ggatgggatg actgggtcat ataatagtac 3660

tgtataacct tagcagaaac tgtcaaacta ttttccaaag tggctcttcc attttacaat 3720

tccacagtgt attgagtccc agtgtctcca tacacatgct agcactttta atatttaatt 3780

tagtgggtat gtaatgatat ctcattgtgg ttttaatttg catttctctg cagctaatga 3840

tgagtgtttc tgcttatttg ggaaggtttt aatttagcag tctgttgtat tctgtagata 3900

ttaataactt caaaatatca gtggcatttg cagttaaaat ttccttaaaa aattggccaa 3960

aggtttccag cagtcacttc tgccatgccc aaactgtatg aaacaaggct gaggtgtgga 4020

gattgtcaca ttttggcaag gagtgatcca cttgggtgac tgatgagacc cagagagcgt 4080

acgcctcggg cttgagggtg aggacgggcg ggaagtcgac tgcatggccc tgctggcctt 4140

gggaggctgc ccagtcctta gctaaagctg gcagttatgg gaaacagact tagattctat 4200

tacgtttttc aggatgtccc aggagtcacc tgggaagctc agcagtcctt tgtgactttc 4260

aagcatatgg tagaagctgc tgaacacaga gctccctctt tggggataat ttgcccaaat 4320

catttaatca ggcttgagaa atgagttacc acaggtccag gagtgctgcc acccttgaat 4380

tctgacaccc tatttctcct atccgtctct taattaatta agcagacatc cccaagtgct 4440

tacgacaagc caggaccctt ttgcatacta aggaaaacag ggatgaagga aacagaaatg 4500

gtctctgctc tgactcagaa ggtagaaatc ctctttccca gccaagtctt cctagggagc 4560

acgtaggaag ggctctgaac ccacgtgtca gttgcagggg aggatatcag gaaaggacat 4620

tgaagaagtg gagacctaag tttgagacct aggcattagc caggctagca gtgcttgaaa 4680

aagtgtctta ggacaagaga actcaccagt gaagtcccag tggtaggaga gcgtgcagca 4740

tattctgagc ctgtatacac atctccaggg cattgcttag caggtgggga gtggcaagag 4800

agtaggctgg agtcacagaa gggaggccag gtagaccttg gtgagcactg gactctatgt 4860

tcaggtgctg aggagctggc aaaaggtttt aagtcgggga gaggcatgtt cagatatttg 4920

gtctagctga gtaactttgg gtgctctgtg acaaatggtt gggagaccag tgaggtggca 4980

gttgcggtca tctaggagca ggatcagagt ggcctattga ctgggatgac tgtgaagtgg 5040

gatcctttcc agccagtaac tggaaatgtg tatgagggca gaagtgagtg tactgcattt 5100

gaaacattga gaaatctagt acatagtact gtctctttta tatctttttt tttttttttt 5160

ttgattttgg tttgtttgtt cactaacttg gaaaactgat gtggaaatgt ccctttggct 5220

tcagttacct gagcagaagg ggccgggcat tgccaaactc tcctcttagg acagaattgc 5280

tcccagtatt gatcattgtg ttctgagttg ggggagcaaa ttgtgcagga ggccaggtca 5340

gtgccaaggt gggtgggagg aattggagca ggaagcttgc ctaagtgtgc ccagcaaagc 5400

cacggtagaa ctttctactg tggctctatg ctacttctta gcaaccttct ccatgtgctt 5460

cctggagagt ccttggagtc agaacctttt tcttgaaacc cagacacttt acttccaaga 5520

aaatgctgtc caagaaaact catccttccc ttcttctcat gaacgttgtg tagaggtgtg 5580

tcttctcttc ctttgagctt ttccactcag ggtttagggg aggtgatatt ctatatttgg 5640

gtttggctct gggtactgca acactaggct attaagattt catccttact gctttgcccc 5700

tcctatcttt ccagaaaccc acaatggatt tgctagaaat aatggaacgt cctgtttgga 5760

caggatataa ccatttctca gctagaggat attgttggaa tgaagaaaga taaatgggga 5820

gaagggaact cacattgctt tggcacttaa attaagccat gtactgtgtt gggaaattat 5880

ttatattatc tcgttgaatc cacagtagaa cacagttgaa caccatacaa ggtaagtatt 5940

gtcatcctta ttttaccatg aggaaattga tgcttagaga gcataaagcc ttggccaggg 6000

gcacatagtt gggaagccgg ggctaattca tgcctgggct ctttctgata gttttccttt 6060

tttaattgtc ccctcctcat tgttaccttg gggatttcaa gagattcatg tagcttctaa 6120

atcaacgaac tgattcctgg agagcagctt ctgtatgaga aaaatctagc taattattta 6180

tttcagtgtc tctggaatgc aagctctgtc ctgagccact tagaaaacaa tttgggatga 6240

caagcatgtg tctcacaatg ctgctctggt tgccagtgct gtgctgccag ttgtcatctt 6300

tgaacaaact gatgcagtgc tggtttaact cttcctcttt ttggagtaag aaactttgga 6360

ggcctgtgtc cttctagaag tttgctgagc aaatggtaag gaaaagaaat aggtcctaag 6420

gcttgactat ttcagagaat ttcttgattt attggactgt caatgaatga attggaatac 6480

atagtggtag gctgtctttt cttctcagac actgcaattt cctccaatct cttgactttt 6540

ctagaagttt taatccaagt ccttgttggg tggtagataa aagggtattg ttctactaga 6600

gactgacctt ggcatggaga tctcatttgg actcacagat ttctagtcta gcgcttggtt 6660

ttgtatccat acctcgctac tgcattctta gttccttctg ctccttgttc ctcatgccca 6720

gtgtcccacc ctacccttgc ccctactcct ctagaggcca cagtgattca ctgagccatt 6780

tcataagcac agctaggaga gttcatggct accaagtgcc agcagggccg aattttcacc 6840

tgtgtgtcct cccttccatt tttcatcttc tgccccctcc ccagctttaa ctttaatata 6900

actacttggg actattccag cattaaataa gggtaactgc tggatgggtg gctgggatac 6960

acagaatgta gtatcccttg ttcacgagaa gaccttcttg ccctagcatg gcaaacagtc 7020

ctccaaggag gcacctgtga cacccaacgg agtagggggg cggtgtgttc aggtgcaggt 7080

ggaacaaggc cagaagtgtg catatgtgct gaccatggga gcttgtttgt cggtttcaca 7140

gttgatgccc tgagcctgcc atagcagact tgtttctcca tgggatgctg ttttctttcc 7200

agagacacag cgctagggtt gtcctcatta cctgagagcc aggtgtcggt agcattttct 7260

tggtgtttac tcacactcat ctaaggcacg ttgtggtttt ccagattagg aaactgcttt 7320

attgatggtg cttttttttt ttttttttga gacagagtct cgctctgtcg ccatgctgga 7380

gtgtagtggc acaatcttgg ctcactgcac ctccgcctgc caggttcagc gattctcctg 7440

cctcagcctc ccaagtagct gggactacag gtgcctgcca ccatgcccag ctaatttttg 7500

tatttttagt agagacgggg tttcaccgta ttggctagga tggtctcgat ttcttgacct 7560

cgtgatccgc ctgcctcggc ctcccaaagt gctgggatta taggcttgag ccaccacgcc 7620

tggccgatgg tgctttttat catttgaagg actcagttgt ataacccact gaaaattagt 7680

atgtaaggaa gttcagggaa tagtataagt cactccaggc ttgaggcaaa atttacaaat 7740

gctgctgact ttgtatgtaa ggggaggcat tttcttagaa aagagaggta ggtctctggg 7800

attccagtat gccatttcca tcctcagtgt ttttggccac ctgagagagg tctattttca 7860

gaaatgcatt cttcattccc agatgataac atctatagaa ctaaaatgat taggaccata 7920

acacgtagct cctagcctgc tgtcggaaca cctcccgagt ccctctttgt gggtgaaccc 7980

agaggctggg agctggtgac tcatgatcca ttgagaagca gtcatgatgc agagctgtgt 8040

gttggaggtc tcagctgaga gggctggatt agcagtcctc attggtgtat ggctttgcag 8100

caataactga tggctgtttc ccctcctgct ttatctttca gttaatgacc agccacggcg 8160

tccctgctgt gagctctggc cgctgccttc cagggctccc gagccacacg ctgggggtgc 8220

tggctgaggg aacatggctt gttggcctca gctgaggttg ctgctgtgga agaacctcac 8280

tttcagaaga agacaaacag taagcttggg tttttcagca gcggggggtt ctctcatttt 8340

ttctttgtgg ttttgagttg gggattggag gagggaggga gggaaggaag ctgtgttggt 8400

tttcacacag ggattgatgg aatctggctc ttatggacac agaactgtgt ggtccggata 8460

tggcatgtgg cttatcatag agggcagatt tgcagccagg tagaaatagt agctttggtt 8520

tgtgctactg cccaggcatg agttctgatc cctaggacct ggctccgaat cgcccctgag 8580

caccccactt tttccttttg ctgcagccct gggaccacct ggctctccaa aagcccctaa 8640

tgggcccctg tatttctgga agctgtgggt gaagtgagtt agtggcccca ctcttagaga 8700

tcaatactgg gtatcttggt gtcaatctgg attctttcct tcaggcctgg aggaatataa 8760

taactgagac ttgttttatt tctgcagagg gttctaagcc attcacttcc cagatgggcc 8820

aataatgctt tgagtaatct ggagatcatc tttaatgcgc aggtgaatgg aactcttcca 8880

cagagggatg tgagggctgt agagcagagt gaactccctg aaactcagac gtcagctctt 8940

tgtctctcta tctctgaaca cccttcctta gagatcccat ctctaggatg catttctctg 9000

tagttagttt ctaagtctct tgttcctgtt ctgcctttat ttttttttcc tggattctaa 9060

gccagtatcc ccacttggct gtcttaatgt agcttaacat gtctgtaatc aaaatgatca 9120

tctttctgag attcaaaggg ctataaggga ctttggagag aatttcattc agttttcctc 9180

aaactagaat aatgcttgca ctgtctgtaa aagaacaaaa gtgtcaaagc atccttttgt 9240

tcactaaatt tcctttttta ttatagtgtt acttaaatat taggaagtta aaagtaggta 9300

taaacttctt ataggctgtt attatacaac tatatgaccc atacatattt acaaattaag 9360

tgcagccaaa attgcaaaat caataccatt caaattaata ccttaaatgt ggtgaggcag 9420

ctgttgttca actgaaacca aattataagt tgcatggcag taaatgctat catgctgatc 9480

attttgagtt tggccagtct atattatcat gtgctaatga ttgaattctc cacccatttt 9540

tctacttgta tgaccttaat ttgatggcac ctgttccatc ctcatgagtt tgctacaatt 9600

atactggtgc caacacaatc ataaacacaa atataaactt gggctttgaa atcttgtgcc 9660

agaacttggc tttaaagtaa gcatttaaaa aatccatatg tgtttattag actttgttta 9720

gatgactgtt gaaatgaaaa caaagtgttt aaaatcctct tagagaactt aaatataatc 9780

cctcagcaat atgtatacag atcttccttt gagaaaaact gattgtgttc agcctctcat 9840

gttacaaatg gggaacctga attctgaggt ctctagtgag agaacaggga ctggaatctg 9900

tggatcctat ctgttttaat aataattgta aagtataata gataatatta tattaaaaag 9960

agagnnnnnn acacttagaa tgagcttcca tgtgtgaggc actaactgat taggcattat 10020

taactagatt tattcctttt aaggccccgc gatgtactgt tatttccaca tgttgtagct 10080

ggggaacgtg ctactcagag aggttaagta acttgtctga ggtccacacc actaacaagg 10140

agcacaggta gggttcaaat ccagataatc tgactttgga gctggcactc taactcaatg 10200

tgcctaatcg cttttcagtg gtgtcattat tttgcctatt ctccatctga gaatattgaa 10260

gtttctgact ccttccttgc ctttctccct gcctcccgtg gttatcccca ggtcttggtg 10320

ttccagtcct ctatgtccgt ccttactctt attcctttgc tacagtgtga tccagggctc 10380

ctgcccttct tatcctggta gagggggccc acttgctggg aaattgtctc cgccatggtt 10440

tatccatgtt gtgtgtccat tagtgagtag tgggaagaat catatcatgt tggcaatgaa 10500

aggggggcta tggctctggg gtagtctagt ctgaactctt atttt 10545

<210> SEQ ID NO 15

<211> LENGTH: 4736

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 15

cttttttttt tttttttttt tttttttttt tgaggtgaag tctcactctg ttgcccaggc 60

tggagtgcaa tggagcgatc ttggctcacc ccaacctctg tctcctgggt tcaaacagtt 120

ctcctgcctc agcctcccga gtagctggga ttacaggctc ccgccaccat gcccagctat 180

ttttttgtat tttcagtaga gatggggttt cacccttttg accaggctgg tcttgaactc 240

ctgacctcat gatcaaccca cctcagcctc ccaaagtgct gggattacag gtgtgagcca 300

ccacgcccgg cctcataagt attttctaaa tttatttaca gtcatgccat ttaaaaggaa 360

agttgtattc ctgtctttgt taatatttat aagtgatttt attcagctac aagcttggaa 420

tggcatataa ttttgtattc tgcttttttc acttaatatt acatggctaa tgatttctgt 480

gtttcataaa cattattctg atgatggcat gatatattgt tgagtacatg taccataatt 540

gaatcatttc cctattgcta tgcaattaag ttgtttccaa tattttgcaa ttataatgtt 600

tcaatgaatg aataacttta tgcatatagc tttttgatat cttaagttca gtttcctagg 660

atgaatttcc aggaatagta attgggcaaa tgggataaac atgactcttg aatacgtatt 720

gttaacattg ctttcccaaa gggctcaact gatttatatt tccgtgttca ttatctttta 780

aaccagctca tttactcacc aaacattttt aaagccatta tcatgtggta ggcttagtaa 840

gaagaaagtg accctaaggg agaagcttat atataaatag ggtccctggt gtaccaagtg 900

ctgatacaga cacaaagtac ctggggaaat tgagatgagg gagtcctggc tcagctggga 960

gaaaagttca ttttcataga gtcatggttt tgttctttgg cagaaagaaa attgctttct 1020

tccccacccc cacccccagc tttattgagg tataattgac aaataaaaat tgtatatctt 1080

taagatatgc aatgtgatat atatgtatat ctcaacttaa aaaataagct acagaataaa 1140

aaggtgtttg ctattaaaaa aaaagaaaag gctgaatgtc attcccaagc ttggaaattt 1200

gagtatgttg cctctttggg attatttaca gaaatattag caagaccagc cccatctttg 1260

gtcttgagta ctccactgtc agcatgcttt cttccagaga gggatccatt tgcctttatt 1320

tttcattctg ttgtgccgtc tatgcaaact attcttgata gttttatggt aacagtgttt 1380

ttttgttcca tgagataaat ttatacatgc tcattgtgga aaatttagaa aagacaggaa 1440

agtattaaaa acatcmcytt tttttttttt tttttttttt tttttttamg cagacagagt 1500

cttgctctgt cgcccaggcc ggagtgcagt ggcgtgatct cagctcacag caacctccgc 1560

ttcccaggtt taagtgattc tcctgcctca gcctcccaag tagctgggag tacaggcatg 1620

caccaccacg cccggctaat tttgtatttt tagtagagat ggggtttcac catgttggcc 1680

aggctggtct caaactcctg acctcaggtg atccgcctgc cttggcctcg caaagttctg 1740

ggattatagg caggagccac tgcgccagcc acacctacgt tcttatcatc ctagtacatc 1800

cactgtcatt atcttgctgt atttccttct gcccagtctc actctgatca tgcagtggcg 1860

tgatcatgca gtgatctcgg ctcactgcaa cctaggcctt ctgggttcga gtgattctcc 1920

tgccttagcc tcctgggttc aagtgattct cttgccttgg cctcccaagt agctgggatt 1980

acaggcatac acccccatgc ccatctaatt tttgtatttt tagtagacac agcgtttcac 2040

taaaattttg tatttttagt agagatgggg tttcaccatg ttggccaggc tggtctccaa 2100

ctcctgacct caggtgatcc gcctgccttg gcctcacaaa gtgattacag gcatgagcca 2160

ctgcatccat cgccaaaaag attttttaaa agagtttaat gtagaaccat atcaaaggtc 2220

tttggaaata aaaaacagtt ttttaaaaat atcagaaata aaacaacaaa taaataaata 2280

aataaaaaca cccaaaacaa tctgaagcac gagcacctag cagaaaggtt caattatgat 2340

ctattcatag agtggaatat caagtagaca ttacaggaca tgttttaaga ttatatttta 2400

tgtcatggga aatgctctcc cagtatgatg ttaaatgaaa aaacagaata caaaagtata 2460

tatgctgcat agtctcaata ttgtagagaa aaaatattat ttatgtatgc atgaaaaaag 2520

acaaaagatg ttaacagaga tccattgtta cttcagttta ctagggattg tctctgggag 2580

gtaggattaa ggtgatttat atttaccttt ttaaactttt ctgtattttt ttattttcaa 2640

attttccata aaaatataag gacttgaaga tcaagaaaaa atttctgctt tggctcagtg 2700

cagtcgtcac gcctgtaatc ccagcagttt gggagcccta ggggagagga tcacttgaac 2760

ccaagagttt gacgttccag tgagctatga tctccggatc gtaccgcctg gacgatggag 2820

caagaccctg tctcaaaaaa aaaaatcttt gctttttttt tttgtttgtt tttgagacgg 2880

agtctctctc tgttgcccca gctggagtac agtggcacaa tctcagctca ccgcaacctc 2940

tgcctcctgg gttcaagcga ttctcttgcc tcagcctccc aagtacctgg gattccatgc 3000

acccaccact atgcccagct acttttttgt attttcagta gagacagggt ttcaccatgt 3060

tggccaggct ggtctcgaat tcctgacctc agctgatcca ccggccttgg cctcccaaag 3120

tgctgggatt acaggcatga gccactgtgc ccagcccaat cttttgcttt ttttaaaaaa 3180

agaagacaaa aagggatttt ataccagtat tatcttggct gtgtgactct gaagccacag 3240

ttgtaagtta taattactct gaaacacaag gccctgtgac tcttttgggc tctttggtgt 3300

ttatcttgat tacaacgttg gaatatagaa atgaaaggaa tgggagaggt gatagacttc 3360

aggcagtgta actagttgtc tgaacactac tggctcaatt atattgtgtc tagtgatttc 3420

catcttgtcc gtctgctaat ttatcgcctg gtaactcact gaggcagggt tttcctttgg 3480

agaaacctca ttgttttaac cagtgtatca tgcttgttta gaagttcaat gatcttttta 3540

actcatcgga gaagatgatg accagacctg gacagatggg gaaggacttt gcactctctc 3600

tttacagtcc tgagtgcaca caggtcaata tggaactatg tgtgaatttt cattgtcttt 3660

gagagccctc ttctctgccc catagggagc agctttgtgt gcaattagag gagcaagggt 3720

tgtgtgtatt tagcacagca ggttggcctg gtcctctcct ctcaacatag tcaccacata 3780

cctggcacta tgctaaggct gggaatgcag acagatgggt gcctgctttc agagtgctca 3840

atgtgctgag gaagccagca acagaaacag atgatttcag gagctccagg aaaatgctac 3900

aggaggagtg tgcctgggtt actggagtag cacaggagga gggcttctag ctcaggctga 3960

gattttagta aaggaaatta tgccacgatg aatcctgaag aatgaataga agtgaaccag 4020

ataaagcacg ataggaagca tcttccctta cctaagggaa gacacagagg tatatggaat 4080

ggtatgttaa aaggttggga ctccaaacag ttctgttaaa gcttagagag tggtgggaga 4140

gactggagaa gttgattaat tagtaaatga agttgtctgt ggatttccca gatcccagtg 4200

gcattggata tccatattat ttttaaattt acagtgttct atcttatttc ccactcagtg 4260

tcagctgctg ctggaagtgg cctggcctct atttatcttc ctgatcctga tctctgttcg 4320

gctgagctac ccaccctatg aacaacatga atgtaagtaa ctgtggatgt tgcctgagac 4380

tcaccaatgg cagggaaaat ccaggcaatt aacgtgggct aaattggact tttccaaaga 4440

tgctgtcttt gggaaacatc acacatgctt tggatcagaa aacctaggct tctaatttgt 4500

tgataaggca tgaactcagg agactgtttt cagtcctagt gaatggtgat aattgtaatt 4560

ataacagtag acaacatctc ttttacacat tttaaatcat gaaaatagaa taaccttact 4620

gataatttta gaaagtggtg attaaaagca catttaagat aatgccttaa cacctagtct 4680

tttccatatg catgatgtct taatcacaca ttgcaaatca tggaacacag aatttt 4736

<210> SEQ ID NO 16

<211> LENGTH: 4768

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 16

atcttacaat cacagtcttt ctcttagggc tgggctcagt gggtggattg acactgcaga 60

aatggccaga tctaaaggat caacatttac gtagctggga aatgtagctg ggacttcagt 120

ttcactgccc tagtgatttt tcctaccact aagcagctca gtccataccc ctacgagacc 180

cacaagctta tgagatactg ttcttccagg aaagcagtgg ggccagggcc accttttaat 240

tgtgtttctt ggcctggtcc catctttctc acaatatata gcaacagtta tttacttgct 300

gattttctaa tgcacatcac acatagtcat attaaacaca cacacacaca cacacacaca 360

cacacacccc tcaagaaaca ttttctgaga cgtgatttcc tgatttcatc aaaaaagaaa 420

agagcgggcc aggcacagtg ggaagtcaag gtgggtggat cacttgaggt caggagtttg 480

aaaccagcct ggccaacacg gtggaacctc gtctctacta aaaatacaaa aattagccag 540

gcgtggtggc gcacacctgt aatcccagct actggggagg ctgaggcagg agaattgctt 600

caacctgcga ggctgaggtt gcagtgagcc gagattgcgc cattgcactc cagcctgggc 660

aacagagtga gactctgtct caaaaaaaaa aaaaaaaaaa aaagcataaa ctgaaattta 720

tatgcaattt atatgcctgt gagataattc tgttttctct tttggaaccc caaagagatt 780

tttttgattg atgagcaaat acattttaga ttttatttaa gcattatgcc aagcaccact 840

gaagtataag tttcaagggc aaactcagtt ttttcatcta ctagacgaat gattttctgg 900

aatgattaca agcaggcaag atggtgtagt ggaaatagca aatgtcttcg gcatcagaca 960

agttggggtt tgtttgtatc ctgcctctgc ccttcaccga ggttgtgatc ttgggcagat 1020

tgttgagttt taacctagat tcctctgact ccagatcata aattttcaga aaagttctga 1080

aattcttgta tatactgatg gtaaatgaga cttttcctta catctatgca cttctttgtt 1140

tgtttgtttt gagatggtct tgctctgttg cccagactgg agtgcagtag tgcaatctcc 1200

gctcactaca atgtctgcct cccaggttcc agtgagcctc ctgcctcagc ctcccaaata 1260

gctgagacta caggcatgtg ccaccacgtc cggctaattt ttgtattttt agtagagaca 1320

gggttttgcc atgttgacca cactggtctc gaactcctgg cctcaggtga ttcgcccgcc 1380

tcagcctccc aaagtgctgg gattacaggc atgagccacc atgcccggcc atatccatgc 1440

acttcttgca accttacctt cttttctcat caccctccag ggacctagtt ggaagagcag 1500

agttaaaagt taaggtgaaa cttggagagg tgtcttgtcc ctaggaacaa aggactggtt 1560

tgaaattctc tgtaaatctt ccccagttca aaccagagtt atcaaggtct taaaaacttc 1620

cctgggtcct gagagcccat tatattattt acttgtcttc ctgtacaccc actgcctagt 1680

cctgatccta cttttgtttg caaataggat ggggcacaac gtacaaggaa gggcctttgc 1740

cacccctgct aagggataac ctgaaatacc ttcaccatca ctgccctgtg ctgcttttca 1800

cctatgccag tctgtctaca gtgccagtgt ctcctggcat tgaaagggga gaatcttttg 1860

gtcctttgag tatttggttg ggttacataa atctccctga atgaagagca gctgacttag 1920

gcaaggggcc ttgtttggtt ttccttgaac tattaacagg aagataggga gattaactgt 1980

gtaaatgttc aataggccag agtccctgca gagggtggcc acagtgatca gatcttatca 2040

catccttgct ttgggtgttg cctctctggt tggagtatgg atagaaaaga aagaaagacc 2100

ctatattgaa atgcaaagtg cagcaagtcc tgactttgga ttaacttctc agcccatttg 2160

catgaaaata aaaagatgaa taaaacaagg ttcccacttt ggagggaggt ggtagctgtg 2220

agatggaagg agtgttcctg ctgggcaaca gcagagtaag tgctggggta gattcactcc 2280

cacagtgcct ggaaaatcct cataggctca tttgttgagt ctttgtccta caccaggcac 2340

tctgcaaaaa cgctttgcct gcaaggtctc atgcgatgct caccacagct ctgtgaagtt 2400

aattgtactt ttatcaccat tttacagatg agaaaactga gggtatgggg tcaatgactt 2460

ggctaaagtc actgcttagc aagctgcagg gactggatgt gaattccaat tggtttgact 2520

ccaaagcctg tgaagctact tgttcttcac cacctagagc tgtggttctt gataactgtg 2580

aactcttttg gggtcacaaa tagccctgag aatatgatag aagcaggagc tctggccttt 2640

ctgtccatac ctgaacaggt ccttgggtta agagcccctc gtccagggcc tattaatctt 2700

gatcctcata agcagcatcc atgtattacg gccgcaaacc aaactgtgcc agaccgaatc 2760

ctaggaccaa gcccaaatat gtcccatcat ccttttggta agaagctcat tgtaagaaag 2820

aaagaggaga gcaagaggat gacctagtgc atggggcctc attgttttaa ttagtgacaa 2880

aacaacaata ataacaacaa aacccccgaa gcttcacaga tgacatcaga ccccaagcct 2940

gtgtgttttt caggtgccct tgaggagctt tgtagctggc agaggaggtg aaactgacaa 3000

atgtttggca gatggaggag agtaccagag gggtttgaga tgagctaaat tccaatctaa 3060

ccgcagtgtt gaggaagagg cttggattgg gaccatggag atgggggttc tactcccagt 3120

cacgccagct gactttgcga gtgttctttg tcagtcactt tatcttattt tatttatttt 3180

tatttttttg aaatggagtt tcgctcttgt cgcccaggct ggagtgaaat ggcgcgatct 3240

tggctcactg caacctcccc ctcctgagtt caagcgattc tcctgcctca gcctccagag 3300

tacctgggat tacaggcgcc tgccaccaag cccatcgaat ttttgtatgc ttagtagaga 3360

cagggtttcg ccatgttggc cagggtggtc ttgaactcct gacctcaggt gatccgccca 3420

ccttggcctc ccaaagtgct gggattacag gcgcgagcca ctgtgcccag cccacttcat 3480

cttaccgtag ttacctcctt agagtatgaa aaaataggct tagggcatcc ccaagtcccc 3540

tctatgtctg agagctgagg ctggctgtca aagaggaact aaggatgcca gggactttct 3600

gcttaggacc cctctcatca cttctccaac gctggtatca tgaaccccat tctacagatg 3660

atgtccacta gattaagaat ggcatgtgag gccaagtttc cacctgagag tcagttttat 3720

tcagaagaga caggtctctg ggatgtgggg aatgggacgg acagacttgg catgaagcat 3780

tgtataaatg gagcctcaaa atcgcttcag ggaattaatg tttctccctg tgtttttcta 3840

ctcctcgatt tcaacaggcc attttccaaa taaagccatg ccctctgcag gaacacttcc 3900

ttgggttcag gggattatct gtaatgccaa caacccctgt ttccgttacc cgactcctgg 3960

ggaggctccc ggagttgttg gaaactttaa caaatccatg taagtatcag atcaggtttt 4020

ctttccaaac ttgtcagtta atccttttcc ttcctttctt gtcctctgga gaattttgaa 4080

tggctggatt taagtgaagt tgtttttgta aatgcttgtg tgatagagtc tgcagaatga 4140

gggaagggag aattttggag aatttggggt atttggggta tccatcacct cgagtattta 4200

tcatttctgt atgttgtgaa catttcaagt cctgtctgct agctattttg gaatatacta 4260

tatgttgtta atgatatcat gcagcagacg tgcatctgaa tgggctggct ctaggagcta 4320

gagggtaggg gctggcacaa agatgcatgc tggaagggtc cttgcccata agaagcttac 4380

agccaaggct aggggagttc tgtcttctct gcatcaggtc acctctctca cctctgtcac 4440

tgccccatca gactacaatg tctgcaggtc tttctcccct gagtgtgagc tccctgagca 4500

aagcaggatg ctgccccttc cctttgtatt ccttgctcct tgcttcagtg cctgtacata 4560

agtatgggca taataagtgt cccccaaatg agacattgag gattcttcaa atgcacagga 4620

ccgtgatgtg agttaggacg gagtaaggac gatgggatgt ggctcatgac aatcctgagg 4680

aagctgcagc tgcggcacgc agggccacac tgtcatgttc atggacccta gactggcttt 4740

gtagcctcca tgggcccctt ccatacac 4768

<210> SEQ ID NO 17

<211> LENGTH: 1295

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 17

tcatgactgc cattggtata aagatgaata taatccagac cagattcatg attattcata 60

catttttagt gtattaactt ttaattctgc ttttaaaata aattaaaaca ttctaatatg 120

cccttaagag tatcccagcc caggccactg agcctactgt ggttcatgga taagtttgcc 180

cctgggggca tgtgtgtgca tgcatgtgtg tgcacatgca tgatgagccg ggccttgaag 240

ggtggtaaga tttgggtgtg tagaccaatg gagaaaggca tttggggcag tgatgatggg 300

tgggggaggg aacatggtga tgaatggagc tgggtgtggg gagccatggg agtgggttag 360

ggccagcctg tggaggacct gggagccagg ctgagttcta tgcacttggc agtcacttct 420

gtaaagcagc agaggcagtt ggcctagcta aagcctttcg ccttttcttg caccctttac 480

agtgtggctc gcctgttctc agatgctcgg aggcttcttt tatacagcca gaaagacacc 540

agcatgaagg acatgcgcaa agttctgaga acattacagc agatcaagaa atccagctca 600

agtaagtaaa aaccttctct gcatccgttt ataattggaa attgacctgc accagggaaa 660

agagtagccc aggtgtctgg ggcttgttcc cattagatct tccccaaggg gtttttctcc 720

ttggtggctg gcctgtgggg cccctctcca ggaggcattg gtgaagaaac taggggagct 780

ggttgccaca gacagtgatg tactaatctt ctctgggaag acagaagaaa agtccccagg 840

gaagaatact acagacttgg ccttagggac agctaggggt gcagattgct gccaactgca 900

ttttttctga agttggccat atggttgcag tgaatggatt tatagacaga gtatttctgt 960

gcatataaga gcaattacag ttgtaagttg atatggataa gtgaaagtta agcacttctt 1020

tctaaaaaga gaatgcaatt cattttcccc taatcatttc aattagtctg atgggcattt 1080

gaacttgttg tctttaaaaa gtgaaatctt tacctctgat ctggtaagta tccaggcaat 1140

ttcttgtgtg ccacccagga ggtatctggg gagtgggcat tttctgactg aggcattggc 1200

tgccatagca tcagagcagc cttccaggca gtggcctggc aaggggacag aggctggtgg 1260

gagcagctgg ctgagtgcag ccagtaatgg catgt 1295

<210> SEQ ID NO 18

<211> LENGTH: 2188

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 18

agctctccag gtgattctga tgcatactta agtttgagaa ccattgcttg ttttgcatta 60

aacaggagat tagtctctgc agcttgtggg aataaagctt taaatctctc caattttagc 120

tctgtgaaaa ggcagtgggg agacaggaat gaacggacta gtgccacaaa gctcaggtgg 180

ggtgggtgag atcatttaga agagaaagac cgggcatggt ggctcacgcc tgtactgtca 240

gcactttggg aggccaaggc aggttggatc acaaggtcag gagtttgaga ccagcctgcc 300

tatcatggtg aaaccctgtc tgtactaaag ataaaaaaaa aaaaatttgc cagtcatggt 360

gatgcatacc tgtaatccca gctactcggg aggctgaggc aggagaatct cttgaacccg 420

ggaggcgggg gttgcagtga gctgagattc caccattgca ctccaaccta ggtgacaggg 480

tgagactccg tctcaaaata aaaaaaaaaa aagaaaagga aaggctgtgt gtgtgtgtat 540

gtgtgtgtgt gtgtgtgtgt gtgtgtgtaa cagcaccatc acactgtttg agttgaggag 600

cacatgctga gtgtggctca acatgttacc agaaagcaat attttcatgc ctctcctgat 660

atggcgatgc tcccctatct cattcctgtg tgtgtttagc caggcaactg ttgatcatca 720

atattatgat aacgtttctc cactgtccca ttgtgcccac tttttttttt tttttgagtt 780

acttactaaa taaaaataaa acactatttc tcaatagact tgaagcttca agatttcctg 840

gtggacaatg aaaccttctc tgggttcctg tatcacaacc tctctctccc aaagtctact 900

gtggacaaga tgctgagggc tgatgtcatt ctccacaagg taagctgatg cctccagctt 960

cctcagtagg gctgatggca attacgttgt gcagctactg gaaagaaatg aataaaccct 1020

tgtccttgta atggtggtga aggggaggga ggtagtttga atacaacttc acttaatttt 1080

acttccctat tcaggcagga attgccaaac catccaggag tggaatatgc aacctggcgt 1140

catgggccag ctggttaaaa taaaattgat ttctggctta tcacttggca tttgtgatga 1200

tttcctccta caagggatac attttaagtt gagttaaact taaaaaatat tcacagttct 1260

gaggcaataa ccgtggttaa gggttattga tctggaggag ctctgtctaa aaaattgagg 1320

acaggagact ttagacaagg gtgtatttgg agacttttaa gaattttata aaataagggc 1380

tggacgcagt ggcactgagt tgagaactgt tgcttgcttt gcattaaata ggagatcagt 1440

ccctgcagct tgtgggaata aggctttaaa tctctccaat tttagctctg tgagatggca 1500

ctggggaaac agaaatgaac ggactagtgt cacaaagctc aggtgggatg gacgagatca 1560

cttcaaaggt ctgtaatccc acgtctataa tcccagcact ttgggaggcc aaggcgggaa 1620

aatcacttga ggtcaggagt tcgagaccat cctggccaac aatgcaaagc ctgtctctac 1680

taaaaatatg aaaattagct cagcgtggtg gcatgctcct gtagtcccag ctactcgtga 1740

ggctgagaca ggagaatcgt ttgaacctgg gaggcggagg ttgcagtgag ccaatatcac 1800

gccattgcac tccagcctgg ctgacagagt gagactccat ctcaaaaaaa aaaaaaaaaa 1860

aagaatttta taaaatcagg aaataatatt agtgtttatg ttgaatttta actttagaat 1920

catagaaaac ttcctctggc atcattatta gacagctctt gtgcagtggg tagcaccaga 1980

cccagcttgc atggttattg atttttcaga gacacttttt gagcttattc tctggcagaa 2040

aggggaactg cttcctcccc tatctcgtgt ctgcatacta gcttgtcttt acaagaagca 2100

gaagtagtgg aaatgtttat tcttgaaaat aagctttttg cttcacatga tctagaattt 2160

ttaaaattag aaaaatgtgc ttactgcg 2188

<210> SEQ ID NO 19

<211> LENGTH: 1183

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)...(1183)

<223> OTHER INFORMATION: n = a, t, c, or g

<400> SEQUENCE: 19

agtaaaatgg agaattccaa attctgaaat tgttagaaca tagttctgtg tcttagttaa 60

atatcgacac ttacagataa atagcataaa tgctttctcc ccatatttca gcccagtcct 120

acttaaagac aacataaatt gcaaaatagt gaggatgttg ttcatctaat aaaagtggtt 180

ccaggaattc agactctgga ttcctgtttg ccaaatcatg tgtcccactc ttaagaaaac 240

gagttggact ntggattttt ctttgcaaga gggacaagag tgtgggagat actgagttaa 300

tgcaacttgc aggttttaag tgtcctgtca ttgtgccttg tgctttgata cattctgagt 360

ttcagtaaag agacctgatg cattggactg ttgcaatgga acctgtttta agatcttcaa 420

agctgtattg atatgaagtt ctccaaaaga cttcaaggac ccagcttcca atcttcataa 480

tcctcttgtg cttgtctctc tttgcatgaa atgcttccag gtatttttgc aaggctacca 540

gttacatttg acaagtctgt gcaatggatc aaaatcagaa gagatgattc aacttggtga 600

ccaagaagtt tctgagcttt gtggcctacc aagggagaaa ctggctgcag cagagcgagt 660

acttcgttcc aacatggaca tcctgaagcc aatcctggtg agtagacttg ctcactggag 720

aaacttcaag cactaatgct ttcggaatgt gaggcttttc cttggacagc atgactttgt 780

tttgtagaaa agtacggctg gctgggagtt tgtgatataa tttagttcag tggtattcta 840

agtgttctta gtgttctttc agacttttgg gccatctccc aaagggtgaa tgggaagaat 900

aagctgggtg tggctgagtt taagccaaaa gttttttgtg cttgtttcaa tcagagaaga 960

cctgcttttt catgttttta ctattataat actaagcaag agctcatttg aaaacagagt 1020

tcttcatatt taaaaaaaaa aagtcttgaa accattgatg ggaagatgga tatctattta 1080

tgtttaaaaa cccatcataa agatgacatt gtgggctgtc acagttggaa ggccctggaa 1140

ttagatgaga ccacactatt tagcttactt agtaataaca ttg 1183

<210> SEQ ID NO 20

<211> LENGTH: 8981

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 20

ccgtttggca aatgctcagt aaaagaaaag ggttagaagg ggagaaaggc attttatccc 60

aagccttcag gaatcaggat gaggatgtct tcaccttgtg gtggggagta attatacaat 120

tagagacagc acattggagt gtggctgata tgctgtgtga tgatagctct agctctctgc 180

ctagcagagg aaggacattt caatagaaga aaaagtttaa gaccttgccg agaaacagag 240

aaaggatgtt tgtcttttta agaagttgaa aaccctgttt gcagacaaaa gccctccagt 300

tttggcagta aactttcatg caagggaaga aaaaggcagg ggatgacatt gttgacaatt 360

gtgaggaatt accatgtgcc aggcactgtg cgaggggctt tgtacatatc ctctagtttt 420

agtgcttata aaaactctgt gatatgtgca cagcatttta aactttgctg catagtcgag 480

aaaatggaag gatggggaat ttgagtcatt tgcccagggt tctatagcta ccccaggttc 540

ccatgactgg agaattgggg cacagggtgg cgggggagag tgagtgacaa gaatcctaac 600

aatcttattt ccattgagtc cttataaaag aagtggatta actaccacgt ttttaagttt 660

ttcttaaatt taggttatgt ggatctggcg tttcttgttt tgtcctgggt ttgttttgtt 720

tttgctatgc tgtcttgaac atctgtcatc ttgtaggcct aacggtaaac acaaaaacac 780

tttacctcct atagctttca attaagatct ctcagtttgt gtttgtaata gttttccagg 840

caagttctcc ctaggttcgg cttctagtgt gttaaccttt agttataaag tgaacccaaa 900

gagagaaagt agaaacaaaa cacctcacct gtttttgctc atgaattact ctctatggaa 960

ggaacaatca tgaacacctc tgcgtatcac agaggcctat ctgagtctga cgtttaaggg 1020

agaccgcgta ggtccctttg aggactgtga atgtgggagt cctgggactc tggtgaagaa 1080

cccgttccag aagagatgaa tgagctggac aagttctttc atagaacctt taggcaggtt 1140

ttcttagaaa tgcacattga ggattatgct tggatattgt gatgatcaga atgatactca 1200

atcccttctg catttggaat tctctttgaa agaaaacatc ccaggcagct atttctcaga 1260

gatagtgagt cccagccact tctagacatt ttcttgtgta gtctacatta taatttcaca 1320

gcagtctctg atatgacaaa tgtcaaaata gcccaacctt ctctaaactt cagagatgtc 1380

tgatatgata ttgaataaaa caatgctcat agaaacatca agaaaggtgg attttccctg 1440

gatacttttt tcctgcttga caaataacag tgaagaaact gatctcacgt ctttttctct 1500

ttggaagcct gaacactcag aacccaactt gaggctcctc agctatagca attctgactt 1560

cacagtctgt aaattattgt tctttttttt ctttagctta tgctttctgc cctaatttat 1620

cttttccctg ttctaatgaa ttattgtcct atatctgctg tgcagttagg tgacatataa 1680

cagcaattaa atatatgaat tggtacatat aaagatttga ctaaaactcg atgtaaaaat 1740

aagtgttcta cattcaattt ccagtgttag aaacagtgct gacttgaaca gagtgacaga 1800

attccatctt tccctatttt tgacagcttt aaactttata ttttcttcct ttcttgtgag 1860

ccgtcattaa cttgtttctc aaagccattc ccgtattacc catcttgcag acgcagacag 1920

atttgggaat ttgcggtcag agttgtattg gacacatccc cccagcccac atgagatcct 1980

tttaatctat tgcatattaa ctagttttaa gtacaatatt cctacttcat ttaaaaccat 2040

taatcaaaga atgagtttga aaatgaacaa aatgcaaact tacagttaga aataattgta 2100

gtgtctttag ttttggttag gagtcggttt cttgtttgtt aaactcaaga ttgtgaacag 2160

ttttaattca cttgtttatt tccaatagag atttcaggtt tacatttgaa ttcagaaaca 2220

aagttttctt tctcattaca gagaacacta aactctacat ctcccttccc gagcaaggag 2280

ctggccgaag ccacaaaaac attgctgcat agtcttggga ctctggccca ggaggtaagt 2340

tgtgtctttc cagtaccagg aagcggatca tccactgtat cagtattttc attcctgagt 2400

ctggcaagag gtccttttga gttgaatatc acatgggatg taatatcaat tttcaaagta 2460

taagtgatgt aaacaataat gttttgattt ccttatttta gaaatgaaga aacctaaaac 2520

tcatagatgt ctcagagcta attggttagt ggctaacagc tggatatcta gtttagaacc 2580

ttctccattt tttctttttg cccctaggta atcatacatt tgtaaagagg agaattatct 2640

ctgccactgc ccatgcactg cttttgtctg accagcaatt tctccatatt gcttcttcag 2700

tagcaaggcc aatcatttta ccaacacaca tgcttgctaa ctaacaggaa taacgtggta 2760

cccctaattc agccctttcc cttgaaagca tctggcttct gaggttcaac tatgggaata 2820

tggtctctta atgaacatta agttgagttt gccttttagg tccacatgtt gacaaatgta 2880

tcagagtaat ctctgtccta ggatcagagg gcctgtaggc acttgcaaaa gcagttagct 2940

ctgactccca gccagtgcac actccacctt tctgactccc agccttgtct caaattaggc 3000

ttggaagcga ggaactgtct ggtgtccccc agcataggaa gctgagccag ggggcagtgc 3060

tcacaaacaa tacagacttt aacgtgtagg atattggaaa ataataattt gtggggaaat 3120

tgtctcagac ttggtccacc cttattttta gctgcttctc taatccgttt ttcttttttt 3180

ggtgcttgta tctaacctac ccattttttg gtgcttgcat cattttttca aatatcaaaa 3240

acgaacttta tgttttctaa caatgaaagt attgcatgtt cattgtggaa aatgctgaag 3300

acttggaaaa tacaaaaatg ctgagatcaa acactattga tacgttagtg tatttcttcc 3360

tgtcctgttc tactttcttt ctttgaattc tgctcacgtg tttctgactg atgaggtctg 3420

acttttgggt tccttttcca gaggagaagc cttctttcag cttgccattt gttaccctgg 3480

ttatgaaggc tggtaacctt ttttactagg tagagaagct ggaccaactg gggttcttcc 3540

agggggagaa tgagaaagag aaactgtttt gcaagtccgt agctatttct ctagggccct 3600

gttagctgac attgacatgc cttgcattgc tctgcagatc ccctcgcagc cctctgtccc 3660

ttgttcattt ctggccttag agaaagcaaa gcagggtctg taacagggga ggctgcctct 3720

aaactcaggg tttggttaca gctgttttca cttacatcac tggccctggt tttttttttt 3780

tttctggcat taaaaaaaaa aattggaagc aggtgatgtt cccattgctg atgtggtgga 3840

aactctccaa gtgaacaata tacgtttttc ttggcagctg tttcttgtgc cctgcttgct 3900

cctggtccag gacaagcaag gaccatctgc ctctttcaat agaacacctc cagatccctt 3960

tgatcaaaag ttactcattg tctgacttgc tatttctgtg agataaatgg gagaagatca 4020

ataaatgcac ttgtttgtcc agtcagcgtg tggaaagttg ataattttga ccaaagcaca 4080

accctgaaag gaaaagaaaa agggagtgaa tgtcttctga gaagctgcct aggttcagac 4140

agtgtcaccc atttccctgt atgctccaca tgacaaacct gagtgggtct catcatgtcc 4200

attttgcaga tggcaccaag gctcagaaag gttaggcaac ttttccagtc acccaatgag 4260

ttaattgaca aaactgggat tcaaacccag aactgttgga ttccaaagcc tgtgttgttg 4320

cctgcttcgt gaaaaactcc agtagcgact ggaatagaaa ggagaacctt ccaagaaaga 4380

aaatacgcac tagcagaacc tggaaattgg gaggaaatga ggacttgagg aataagatga 4440

atgaaagctg acctgagttt cacatctggg tgatgggaag ggaggacagg gaggcagcat 4500

ctcagatgtc cacccagcac cgaccagctg cctggcattg ctaggtgttg aggactcagc 4560

agtgaacacg ctaacttctc tgctttcttg gggcacgtat agggtgagag acagaaacaa 4620

acaggtcagt gtacaatgcc acaggaggga tatatgcagt gaagaaaaag cagggtaagg 4680

ggcatagagc atgagaaggt gcttttttta aaggggktga ttaggaaagc tctctctaag 4740

gtgacagttg gacctgaagg agatgatagc atgtctgtgg tgagggaagg aaactccgaa 4800

caggaagaat ggcagataca aagacattga tgctagagca tgcctaagga atgtgtttaa 4860

ggaccaggga aagtgagcaa gtggtggggg gaggagagga gctcagagca ggaggaggtg 4920

agtgccatac aggcctggca agactttgga ttcctgctgg gtgagatgag aatccagcgg 4980

agggcttgag ggaggggaca tgatgtgatc tagagtttag actgtttaca ctctggttgt 5040

tgggttgaga agagactggg atgggggaaa gggaggacaa aggacattgt gctggattga 5100

gaaagcagta agtcagtttc attcattcac tcaaccgatg atgttcaaat accaccatca 5160

tccgtgggct aaaggatgaa gagccatccc tccctgagag tcaggaagca cttcccagat 5220

aaagtttgga gtgtgagctg aggtgtagga gaaagagtaa gagtttaccc ctgaaacggg 5280

tgctgggaag agtcaatagt ttggaataac tcaataattt atggtgcttc tttagaaaga 5340

tttgctggct ttatgtggga agaaatttkt ttttttgatt ggggagtggt gggttggtgg 5400

tgaggctgcc tgtggaaaga gaagtgagtg ttttgactca ctgttattta aaaatctcta 5460

gggctgttcc aataagcaac aaaaggcaaa atggcctggt tctctgtccc ctttctgtct 5520

gtatgcctcg tacaggttat gaaaagaaaa agttgggaaa agctgtccac ctcacctaat 5580

tgtgttcttg tggagtgtgc tagatgcccc ctctctggag aaaaaaaatc cttgtggcct 5640

ctgacccacc tctggagagc ctagttccct tctggaggca gaaggcaaag cttaggacct 5700

agagagtgct ggaccacgcc actcacagga accagcaggc tgtgaggttg aaagctaggc 5760

atatggagct ttccaggctg ggtgcagggc ctcgtggccc ttcccctccc ctctgtgctc 5820

tatagctcag tcttcccagg cggtgtgaac acgcagtgac atttccagga atacagggat 5880

ttattaatga tttcttgtga aatgtttgga aatacaaagt actctataaa tatttcataa 5940

tagcattggg gctgagaact ccacaaagtg ccggaataca tttgcatgta agacagaacg 6000

ctgcctgggt cattgatgcc tgttgagtgg cagtcacaga cactgcctag ggtttctgac 6060

tcacgctgtt gggactgttc tatgcagggc accctcttgt gtggcatagg atttgtgcct 6120

caccacacac tgttgtagct ttgctgtctt gatgatgagt agagggcagt gtccaggcca 6180

tggtataagc atctactgcc ccccagggtt accaaaacca agccaagttg tgtctcagcg 6240

agctccgtga agcatggaga agttgagtac tcagagacat gacgtgactt ttcaaaggct 6300

gtaagctgac gagggacata gctagggttc agacttgagt ttttcttttt ctttttcttt 6360

ttcttttttt tttaagactg agtcttgctt ttgtcgccca ggctggattg cagtggtgct 6420

tggctcactg caacctctgc ctcccgggtt caagcaattc tcctgcctca gcctccccag 6480

tagctgggat tacaggcacc tgccaccatg cctggccaac atttttgtat ttttttagta 6540

gagatggggt ttcaccatgt tggccaggct ggtcttgaac tcctgacctc aggtgatcca 6600

cccgcctcga cctcccaaag tactgggatt acaggtgtga gccactgcac ccggcccaga 6660

ctcgagtttt tcatcttaat gctttttcat tgcctgacac tttactgaga ccaagatagg 6720

gaacttcaca tacagtacct tttctcccaa ggcggaagag ggctgttcaa tttctacact 6780

agagttcggg gagttttaga aatgagtcag ttatcgagga tgagagcagt tcctgatagg 6840

ctcaaccaca atgagatgta gctgttcaga gaaagcattc ttttatctat aaactggaag 6900

ataatcccgg tgaaacgaag cccagcccca ggggcttcac taactccagg ctgtgcttct 6960

caaactttag tgagcatagg aatcacctgg gcatcttgtg aagctgtaga tttgaattct 7020

gcaggtcggc agaggggtct cagaatccgc atttccaaca atgtctccag taatgctgat 7080

gctgctcgtc cctggaccac agattgggta gccaggttct ggcaagctca tcccaaggct 7140

ttgagatgac atcagacaaa atatgttctg ggacatggct tttgagaggt caagaaaata 7200

agatgtttct ttctcttctc atccccaacc cttgcactgc ccttttctcc cttcccctac 7260

cctcctttct gtccccatcc ctgacgccag ctgttcagca tgagaagctg gagtgacatg 7320

cgacaggagg tgatgtttct gaccaatgtg aacagctcca gctcctccac ccaaatctac 7380

caggctgtgt ctcgtattgt ctgcgggcat cccgagggag gggggctgaa gatcaagtct 7440

ctcaactggt atgaggacaa caactacaaa gccctctttg gaggcaatgg cactgaggaa 7500

gatgctgaaa ccttctatga caactctaca agtgagtgtc catgcagacc ccagccctgt 7560

ccccaacccc atccctccct tagttctggc cttggcctgt gtcatctcct ccctctgtag 7620

cagcgttaga tgtctacatg cccatttgcc caccagactg agctcttcct agaggagaga 7680

ggcttctctt gaatagctac ctgtccccag ttctctgaat gcagcctggc acatctcagg 7740

tgcacagtag tgtttatcaa tggaatgaat gattgacagc caaccttctg gttttctggg 7800

ggatgtggaa gggtggcttc cagggtgatc aagaatgaga taatggcaga aggacaaatc 7860

ctgcaagatc tcacttatat atggaatata tgtaaggtag aaagtgtcag tttcacatga 7920

tgaataagtt cctgggatct tgatgtacat cgtgatgact atagttagta acactgtata 7980

gtatacttga aatttgctaa gagagtagat ccgaagtgtt cacactacac aaaaaaggca 8040

actatgaggt gatggattta ttaacagctt gattgtggtg atccttttac aaagtataca 8100

tatattaaaa catcacattg tataccttaa atatatacaa tttttatttg tcagttgtaa 8160

ctcaaaaaag ctagaaaagc atttttaaaa aggatgatgt actggtctta atattaccat 8220

tgagataagc tttataataa cataaaaaga aataacagta atgataatag caacaacaac 8280

aacaacaaag aactaacatt taagtagaat ttcttgtgca ctgtgcattc tgtttaagtt 8340

atctcatttt accctcatga taacctgcag ggaagattct ttaaccccac atttcatagg 8400

ctcagagagg ttaagtgcct tggttagagc cacatcagag ttaatccaca agagccagga 8460

ttcaagccca aatctgcctg gatctgtgct ctctaagata actgttagtg gtggcgtgtg 8520

tgttctcaca ctcagacatt tgatctgccc tttgtttccc attcttagct gcaaggcagt 8580

gttaaagaac cctgtgtctc catatccact ccccacactt aagcactttt gtgggcccgt 8640

gtgccgtatg cctcgtggca gcagggatcc aatgtcacag ttttaggcag tggcatcctt 8700

ttccttgaaa acttgatgca ggggaacctt tctccatttc caaccacagg tgtgtctttc 8760

agacactgag tgaggcaggt tttgtacttt attgtaacac aagaaccttt tcttctctgg 8820

agtaaagcac tccagacatt cgcaagttgc tttacaagcc ttaaaaggat ggtattgtag 8880

gcaactttaa ttaaatccca tctcctcctc tcccccagct tgcaagttga cccaaggaag 8940

ccttcatttc catgacagac ttaattgtga gggcatcctc a 8981

<210> SEQ ID NO 21

<211> LENGTH: 20284

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)...(20284)

<223> OTHER INFORMATION: n = a, t, c, or g

<400> SEQUENCE: 21

actgtgttag caaggatggt ctcgatctcc tgacctcgtg atccgcctgt atcggcctcc 60

caaagtgctg ggattacagg cgtgaaccac tgcgccctgt tgagaatttt tttttttttt 120

tttgggagaa agagtttcgc tcttgttgcc cgggctagag tgcagtgaca caatctcggc 180

tcactgcaac ctctgcctcc tgggttcaag caattctcct gcctcagcct catgcgtcac 240

cacgcccagc taattttgta tttttagtag agacagggtt tctccatgtt ggtcaggctg 300

gtctcgaact cccaacctca ggtggttcgc ccgccttggc ctcccaaagt gctgggattg 360

caggcatgag ccactgcgcc cagccccaaa ttttggtttt tgcttgaaaa ctgaggtctg 420

aattcagcct tctggttgcc cctcaagagt cagtttaaat gttggtcatg ttagttgtca 480

gtgaaaacaa tggtgaggct ggcatgagag tgtgaatctg gatgggaggg cttgtgcttc 540

atgaaaacat ttttccagat cagctcagtc gtgagttatc cgtcattgac gttataataa 600

gctctgatta tttatcaagc atcattcttt atagatatct cagtttaatc tgagataatc 660

ttctccacat ctctccacat agatgttatg aattttactt ttacagagga gccaactgag 720

gctcagataa gttacttatt atatgactag tagtggtaga gctggggttt caactaagaa 780

ctctctggct ccaaagccct tgtaagtttc tatcagtata tgaccatgca tatgagcatt 840

tgtctctcct cttcttcata gctccttact gcaatgattt gatgaagaat ttggagtcta 900

gtcctctttc ccgcattatc tggaaagctc tgaagccgct gctcgttggg aagatcctgt 960

atacacctga cactccagcc acaaggcagg tcatggctga ggtaagctgc ccccagccca 1020

agactccctc cccagaatct ccccagaact gggggcaaaa aactcaaggt agcttcagag 1080

gtgtgcgcta agtatactca cggctcttct ggaattccca gagtgaaaac ctcaagtctg 1140

atgcagacca gagctgggcc agctccccag tcgtgggtat agaatcatag ttacaagcag 1200

gcatttcttg gggatgggga ggactggcac agggctgctg tgatggggta tcttttcagg 1260

gaggagccaa acgctcattg tctgtgcttc tcctcctttt tctgcggtcc ctggctcccc 1320

acctgactcc aggtgaacaa gaccttccag gaactggctg tgttccatga tctggaaggc 1380

atgtgggagg aactcagccc caagatctgg accttcatgg agaacagcca agaaatggac 1440

cttgtccggg tgagtgtccc tcccattatt accatgtgcc tgcttgatac tggagaggtg 1500

agtttctggt cactttccca ggtgtgagtg aggtgagaat tctttcagtt tatctagctg 1560

ggggaatgta gtgagcatag ctaaagtcac agggcaccac ctctccagaa gtacaggcca 1620

tggtgcagag ataacgctgt gcatatcagc atccatgcca ctcacggtca aatagcagtt 1680

ttctgcaaaa cttagtgagg gctggtgttt ggaagtggag ttgagtaatt gcagtaccct 1740

attttccttt ttgctgcagc ctctcagcca gccacagcat ctccctgtgt cttggtaggt 1800

tttggaaaga agtgtgggag caaaagcatg atgttacatg tagactggcc tgagatactc 1860

attctcaggg cactgtgtga atgatgagct gctgttactg tgtggagggg aaatgcactt 1920

agtgcttcag agccacttga aagggataag tgctctagag acaattgggt tcaaatgtgg 1980

agcaggctga gcaagaacag aatgtctcct ttgcctgagc ctgagtgctg ttaatcacat 2040

cttcctgcct tgggctgagt tagagaatca ttagactatt tcctgtttcc atggtgaggg 2100

aggcctcttc cttttgtctc tgctcccctt aagaagcagg tgaggatttt gccaggtttc 2160

ttgttttgaa ccttattgac tttaagggcg gctgggtttt agagactgta cctacctagg 2220

gggaacactt ccgaagttta ggactattcc ctgatccgct gggaggcagg ttactgagga 2280

agtcccttta aaaacaaagg agtttatact gagaaaagca taaacagtga tttgtatgga 2340

ttcacactga ctaatatagc tcatgccatt aaagtggggt ctcttctcta aaggagggtt 2400

atatgatcta gccccgtaga cctaagtgtg gtttcagacc tgttcttcct ggtcctctcc 2460

ttggaatcca tatttctact agttggactt tttctgtttg tctggctctc agaggattat 2520

aggaggccct gtgaagtgac tcagtgaatt ttgatttgtg ggcaagtaga tggttcccta 2580

gtctgaaatt gactttgcct taggtgcttc aattcttcat aagctcccag ttcttaaagg 2640

acaagatcct tgtaaacatg gcaatggcat tcattaggaa tctagctggg aaaatccagt 2700

gtgtatgctt ggaaatgagg gatctggggc tggagagaaa ggcatgggca tgccttggag 2760

ggacttgtgt gtcaagctga ggacctttac tttaagctct aggggaccag gcaaggggag 2820

atgtagatac gttactctga tggggtggat gaattgaaga aggatgaggc aagaatgaag 2880

gcagagacca gggaggaggc tctccaagtg gccaaggcat aaagcaagaa atgaggcctg 2940

gtgactgctt agtggcagag cagtgaaaga gagggaggca tcaaagtgag tctcgatttc 3000

tagctgggtg ggtggtagcg atgtccagta ggccagtggc tactgaggtc tgcagtggag 3060

gagggtggtt gggctggaga cagatgatga gggagtcatc agcctgtggg tggaagaaaa 3120

gggaacctct tccaactgtt ttctttgctt cttccctctc tttctctttt tttttttttt 3180

tggacagagt cttgctctgt cacccaggct gaaatgcagt ggcatgatct tggctcacca 3240

cagcctccgc ctcctgggtt caagcaattc tcctgtctca gcctccagag tagctgggat 3300

tacaggcaca tatcactgtg cccggctaat ttttgtattt tcagtggaga tgggatttca 3360

ccatgttggt cgggctggaa tgaactcctg acctcaagtg atccacctgc ctcagcctcc 3420

caaagtgttg ggattacagg catgagccac cgcgcccggc ctttcttccc tctcttaaag 3480

agtgtttatt taattccaca aacatgagct tgtcaccccc tgtagcctgg catctcctac 3540

acgaggtgat ggctgaggct tctgcttctg ctggggtagc tctgatcttt ctgctttctc 3600

tggcactgtc tacccatgtt gcctcacccc acaggtccca gggcacctct ctcgggcaag 3660

tcttggaacc ctctgacact gatttgctct cttttctgag ctgcttttag ccacccatcc 3720

tcgggacctg ttttctctct gcctccaccc ctgcgggcag tcttaggtct cctgcccctc 3780

acgagcaccc cagagaggcc acgtgctcag tgatctcagt gggcgcatct ttctagtctt 3840

gctattcttt ttggccatgt tgttcagaaa ccatactggg cagggccgac ttcaccctaa 3900

aggctgcgtc tcttcactct gcttttgttt gttccaaata aagtggcttc agaattgcta 3960

accctagcct ctgtgaactt gtgaggtaca attttgtgtc tgttatgtta acaaaaatac 4020

atacatacct tcctggtgat ggtataaatt gctattctct attggaaagc aatttggaat 4080

gaaaatttaa agaaccattt taaaatatgc tatcctgcgt acctccattc cacccacccc 4140

cagggatgta gcctactgaa ataattttaa agaagtcacc atatgagaga aaatgttatt 4200

gctatattgt tattgtgaga aattggaaat agactaaatg ttcagcacta taggaataat 4260

taatgaaatt acatatactc tatacaatca ttatgctgcc attgaaataa taaatacaaa 4320

ggcgcaaggg gggaaaagct tataatgtta gtgaaactaa gactgatttt tttataaagc 4380

agcagttttc agacccttgg agactccaat tcggtagaac cagagcttca tcttctctgt 4440

cgaagctgtg acaggagttg caaatgcctc tcctttttgc tgagtttgca gctgctgttt 4500

ttccggcagc acatctgtgc aggcctctgc ctcggcccct ctggatctgc tgattgagca 4560

gcggattgat ctgtccttct ctttcgtgtt gacccatgtg aggaaccaac tggcaaggga 4620

acaagaaatg gaaataggcc tcctttgcat catgacctgt acatcctgca attggaaaag 4680

attgtacttt agttggttta accagcagca ttatttttct aaactaagca gtaagaagga 4740

attaggtttt atgtgggatc aacagactgg gtctcaaaag aggaaggtga tagaacacag 4800

tggggagggg gaggtgcact agaaacagag ggcctatgct ttcattctgg ctttgctact 4860

taatagctgt gtgacccaat cttagagact taacctctct gaacttccat tttctcatgt 4920

ataaaatggg aaatattaaa ggatactcac tgggctggtg gcttgtgcct gtaatcccag 4980

cacttgggga ggttgaggtg ggaggatcac ttgagcccag gtgttcaaga ccagcccagg 5040

caacatggca agactctgtc tctatgaaaa aattaaaaat tagccaggtg tggtggtgtg 5100

cacctgtagt cttagctact tggtaggctg agatgggagg atcacttggg cttgggaggt 5160

caaggctgcg gtgagctgtg attccatcac tgcactccag cccgggcggc agagcgagac 5220

actgaatcca aacgacaaca acaacaaaag gcaaaaaaat aaaagtgccc tctttatgga 5280

gttgtgtaag gtgaagcata tacactattc aacatagtaa ctatataaag gaagtattgt 5340

tgttgttact gtagttaata ccattaagtg agatgtttcg tatagtggaa agcacatgga 5400

ctctgaattc agactggtct gactttgagt ctcagctcca catctagtaa tactatgacc 5460

aagccctggt taaaatcatg tttttttttc ttcagcctca gtcttctcac atataaaata 5520

gggacactgt catttacctc agttttctgt gaggataaaa caacgacagt gtatatgcaa 5580

gtattttgta aattttgtag tgctcctcaa gatttagttg gtgtttacta cttgtacttt 5640

ctcactggaa tggcagatgc tgttggacag cagggacaat gaccactttt gggaacagca 5700

gttggatggc ttagattgga cagcccaaga catcgtggcg tttttggcca agcacccaga 5760

ggatgtccag tccagtaatg gttctgtgta cacctggaga gaagctttca acgagactaa 5820

ccaggcaatc cggaccatat ctcgcttcat ggaggtgaat ctgttgctgg gatcatttag 5880

aaaagactta acggcttctt tctctgagac gttacaataa ggttcaggca ggaggcaagt 5940

ttagaaataa tgtatagtct catttacaaa actatccctc aagcctaaca caggatttga 6000

taacaaaagg cacttaataa atgttagttg agtggttgaa tgagtaaata aactctagct 6060

ttagtaaatt aactctagct tattctatat aggctcaaga gaatatttct acccattttc 6120

ttctaggttt tcctatctca gtgactaatg gtagcaaagc attcccttaa aaaggcatta 6180

tttgtgaaac ttayctaaaa tcgaattcgg gtccaattaa atttttgaaa ttttatatta 6240

aaaattatat tagtagggat gggtaagagg tgttttggtc tggttggttg gttagttgct 6300

atgactcaga attgctaaga aaacagaaaa gtaagataag atcattgttt taacctcttt 6360

tcctccacaa aatcaataaa taacatatcc ctaaattact cttagaattt ctcttaaatt 6420

gcagtgaaaa accaaaatcc ttcattcttg gttgaaggtt ggaaaactac gttagagagg 6480

attagagaga gaggatgagc aatcgtgtag tcagcccttg cctcctagtg taggatttgt 6540

ctcagccact gcttgttgtc ctggctgcca acgttctcat gaaggctgtt cttctatcag 6600

tgtgtcaacc tgaacaagct agaacccata gcaacagaag tctggctcat caacaagtcc 6660

atggagctgc tggatgagag gaagttctgg gctggtattg tgttcactgg aattactccm 6720

rgcagcattg agctgcccca tcatgtcaag tacaagatcc gaatggacat tgacaatgtg 6780

gagaggacaa ataaaatcaa ggatgggtaa gtggaatccc atcacaccag cctggtcttg 6840

gggaggtcca gagcacctat tatattagga caagaggtac tttattttaa ctaaaaattt 6900

ggtagaaatt tcaacaacaa caaaaaaact caacttggtg tcatgatttt ggtgaaattg 6960

gtacatgact tgctggaagg tttttcatag gtcataaaat aacagtatct tttgatttag 7020

catttctact caagggaatt aattccagga attttggtgg caggcacctg taatcccagc 7080

tactcgggag gctgaggcag gagaattgct tgaacccagg aggcagaggt tgcagtgagc 7140

taagatcgca tcattgcact cccgcctggg caataagagt gaaactccat ctcaaaaaaa 7200

aaaaagatac aaaaatagaa aaaggggctt ggtaagggta gtagggtttt gggcaatttt 7260

tttttttttt ttttttttta ttgtatggtt ctaaaggaat ggttgattac ctgtggtttg 7320

gttttaggta ctgggaccct ggtcctcgag ctgacccctt tgaggacatg cggtacgtct 7380

gggggggctt cgcctacttg caggatgtgg tggagcaggc aatcatcagg gtgctgacgg 7440

gcaccgagaa gaaaactggt gtctatatgc aacagatgcc ctatccctgt tacgttgatg 7500

acatgtaagt tacctgcaag ccactgtttt taaccagttt atactgtgcc agatgggggt 7560

gtatatatgt gtgtgcatgt gcatgcatgt gtgaatgatc tggaaataag atgccagatg 7620

taagttgtca acagttgcag ccacatgaca gacatagata tatgtgcaca cactagtaaa 7680

cctctttcct tctcatccat ggttgccact tttatctttt tatttttatt tttttttttg 7740

agatggagtc tcgctctgac gcccaggctg gagtgcagtg gctcgatctc ggctcactgc 7800

aacctttgcc tcccgggttc aagctattct cctgcctcag cctccacagt agctgggact 7860

acaggctcat gctgccacgc ccggctgact ttttgtattt tagtagagac gaggtttcac 7920

catgttaccc aggctagact tcaactcctg agctcaggca atccaccctc cttggcctcc 7980

caaagtgctg ggattacagg tgtgagccac tgcacccagc ccaccacttt aattttttac 8040

actctaccct tttggtcaaa atttgctcaa tctgcaagct taaaatgtgt catgacaaac 8100

acatgcaagc acatactcac acatagatgc agaaacagcg tctaaactta taaaagcaca 8160

gtttatgtaa atgtgtgcac ttcttctccc taggtggtaa accacatttc aaaacaaccc 8220

aaataaaact gaacaaagct tcttcctctt agacttttta gaaaatcttt cagtgctgag 8280

tcactaagct gccaagttct cattgtggga actatgcctt tggatgtaat gatttcttct 8340

aagacaatgg gcggaggtgt agttattgca gacatctgaa atatgtaatg tttcttccag 8400

attctggaaa ttctcttatt ctctgtggtt ggtggtggtg gtgggatgtg tgtgtgtgtg 8460

tgtgtgtgtg tgtgtgtgtg tgtgtaggga tcaggatgcg ggaggagctg ggttctgctt 8520

gtattggttc tctgttttgc attgaatagt gtgtttcctt gtatggctat ctatagcttt 8580

tcaaggtcac cagaaattat cctgtttttc accttctaaa caattagctg gaatttttca 8640

aaggaagact tttacaaaga cccctaagct aaggtttact ctagaaagga tgtcttaaga 8700

cagggcacag gagttcagag gcattaagag ctggtgcctg ttgtcatgta gtgagtatgt 8760

gcctacatgg taaagctttg acgtgaacct caagttcagg gtccaaaatc tgtgtgcctt 8820

tttactttgc acatctgcat tttctattct agcttggaat ctgaaacatt gacaagagct 8880

gcctgaaatg tatgtctgtg gtgtgattag agttacgata agcaagtcaa tagtgagatg 8940

accttggaga tgttgaactt ttgtgagaga atgagttgtt tttttgtttt ggtttttagt 9000

actttaacat aatctacctt tagtttaagt atcgctcaca gttacctagt tactgaagca 9060

agcccccaaa gaaatttggt ttggcaacac tttgttagcc tcgtttttct ctctacattg 9120

cattgctcgt gaagcattgg atcatacgta catttcagag tctagagggc ctgtccttct 9180

gtggcccaga tgtggtgctc cctctagcat gcaggctcag aggccttggc ccatcaccct 9240

ggctcacgtg tgtctttctt tctccccttg tccttccttg gggcctccag ctttctgcgg 9300

gtgatgagcc ggtcaatgcc cctcttcatg acgctggcct ggatttactc agtggctgtg 9360

atcatcaagg gcatcgtgta tgagaaggag gcacggctga aagagaccat gcggatcatg 9420

ggcctggaca acagcatcct ctggtttagc tggttcatta gtagcctcat tcctcttctt 9480

gtgagcgctg gcctgctagt ggtcatcctg aaggtaaggc agcctcactc gctcttccct 9540

gccaggaaac tccgaaatag ctcaacacgg gctaagggag gagaagaaga aaaaaaatcc 9600

aagcctctgg tagagaaggg gtcatacctg tcatttcctg caatttcatc catttatagt 9660

tggggaaagt gaggcccaga gaggggcagt gacttgccca aggtcaaccc agccgggtag 9720

cagctaagta ggatgagagt gcagggttca tgctttccag ataaccacat gctcaactgt 9780

gccatgctgt ctcattggta gtggttcatg gcagcatctg aaagctattt attttcttag 9840

atatattggg tggcgattct tcctaagttt ctaagaacaa taatcagaag gatatatatt 9900

gttgcaggtt agactgtctg gaagcagagg ctgaaataga gtttgatgta tgggtattta 9960

tgagggctca atacctatgg aagagatatg gaagatgcag gattgggcag agggaggagt 10020

tgaactgtga tatagggcca accccgtggg gcactctaga gaatatgcag cttgttggag 10080

ttgttcttca tcgagctgaa acatccagcc ctttgtgctc ccccaaggcc tccctcctga 10140

caccacctac ctcagccctc tcaatcaatc actggatgtg ggctgccctg ggaaggtcgt 10200

gccccagggc ctacatggct ctctgctgct gtgacaaacc cagagttgct gatgcctgag 10260

gccgtctact gacagctggg caacaaggct tccctgaatg gggactctgg gcagtgcagt 10320

tttgtgtctg aaccatacat taatatattt atatccgaat tttctttctc tgcaagcatt 10380

tcatataaag acacatcagg taaaaataaa tgtttttgaa gcaaaaggag tacaaagaga 10440

taagaactaa ctaatttaat actagttacc atctgttaca aatagttcct actgattgcc 10500

aaggactgtt taaacacatc acatgggctt cttcttctat cctcactaac ccttttaaca 10560

gacaaggaaa tgaggctcag gaaggtcaag gactttattg aggttccaca gtaggataca 10620

gttcttgcta aaagcaaccc ctccctcatg ctctgttatc taactgcaag gggaaggtca 10680

gtggcagagg tagtggtccc atggttggtg cataagagct gctctgagac aactgcatgc 10740

tggtgggtcc tgcagacatg tacccatcag ccggagatag gctcaaaata tccacaagag 10800

tttggatgat tgtgggaatg cagaatccat ggtgatcaag agggaaagtc aagttgcctg 10860

gccattttcc ttggctttta gacagaaaag ttacgtggga tattatctcc cacagctctt 10920

ctgtggtgcc accagtcata gtccttatat aaggagaaac cagttgaaat tacctattga 10980

agaaacaaag agcaaactcg cccactgaaa tgcgtagaaa gccctggact ctgttgtatt 11040

cataactctg ccattatttt tctgcgtagt tttgggtaag tcacttatct tctttaggat 11100

ggtaatgatc agttgcctca tcagaaagat gaacagcatt acgcctctgc attgtctcta 11160

acatgagtag gaataaaccc tgtctttttt ctgtagatca tacaagtgag tgcttgggat 11220

tgttgaggca gcacatttga tgtgtctctt ccttcccagt taggaaacct gctgccctac 11280

agtgatccca gcgtggtgtt tgtcttcctg tccgtgtttg ctgtggtgac aatcctgcag 11340

tgcttcctga ttagcacact cttctccaga gccaacctgg cagcagcctg tgggggcatc 11400

atctacttca cgctgtacct gccctacgtc ctgtgtgtgg catggcagga ctacgtgggc 11460

ttcacactca agatcttcgc tgtgagtacc tctggccttt cttcagtggc tgtaggcatt 11520

tgaccttcct ttggagtccc tgaataaaag cagcaagttg agaacagaag atgattgtct 11580

tttccaatgg gacatgaacc ttagctctag attctaagct ctttaagggt aagggcaagc 11640

attgtgtttt attaaattgt ttacctttag tcttctcagt gaatcctggt tgaattgaat 11700

tgaatggaat ttttccgaga gccagactgc atcttgaact gggctgggga taaatggcat 11760

tgaggaatgg cttcaggcaa cagatgccat ctctgccctt tatctcccag ctctgttggc 11820

tatgttaagc tcatgacaaa ccaaggccac aaatagaact gaaaactctt gatgtcagag 11880

atgacctctc ttgtcttcct tgtgtccagt atggtgtttt gcttgagtaa tgttttctga 11940

actaagcaca actgaggagc aggtgcctca tcccacaaat tcctgacttg gacacttcct 12000

tccctcgtac agagcagggg gatatcttgg agagtgtgtg agcccctaca agtgcaagtt 12060

gtcagatgtc cccaggtcac ttatcaggaa agctaagagt gactcatagg atgctcctgt 12120

tgcctcagtc tgggcttcat aggcatcagc agccccaaac aggcacctct gatcctgagc 12180

catccttggc tgagcaggga gcctcagaag actgtgggta tgcgcatgtg tgtgggggaa 12240

caggattgct gagccttggg gcatctttgg aaacataaag ttttaaaagt tttatgcttc 12300

actgtatatg catttctgaa atgtttgtat ataatgagtg gttacaaatg gaatcatttt 12360

atatgttact tggtagccca ccactcccta aagggactct ataggtaaat actacttctg 12420

caccttatga ttgatccatt ttgcaaattc aaatttctcc aggtataatt tacactagaa 12480

gagatagaaa aatgagactg accaggaaat ggataggtga ctttgcctgt ttctcacaga 12540

gcctgctgtc tcctgtggct tttgggtttg gctgtgagta ctttgccctt tttgaggagc 12600

agggcattgg agtgcagtgg gacaacctgt ttgagagtcc tgtggaggaa gatggcttca 12660

atctcaccac ttcggtctcc atgatgctgt ttgacacctt cctctatggg gtgatgacct 12720

ggtacattga ggctgtcttt ccaggtacac tgctttgggc atctgtttgg aaaatatgac 12780

ttctagctga tgtcctttct ttgtgctaga atctctgcag tgcatgggct tccctgggaa 12840

gtggtttggg ctatagatct atagtaaaca gatagtccaa ggacaggcag ctgatgctga 12900

aagtacaatt gtcactactt gtacagcact tgtttcttga aaactgtgtg ccaggcagca 12960

tgcaaaatgt tttatacaca ttgcttcatt taattctcac aaggctactc tgaagtagtt 13020

actataataa ccagcaattt tcaaatgaga gaactgtgac tcaaagacgt taagtaacca 13080

gctttggtca cacaactgtt aaatgttggt acgtggaggt gaatccactt cggttacact 13140

gggtcaataa gcccaggcga atcctcccaa tgctcaccca attctgtatt tctgtgtcct 13200

cagagggggt acaactagga gaggttctgt ttcctgagta caggttgtta ataattaaat 13260

atactagctc taaggcctgc ctgtgattta attagcattc aataaaaatt catgttgaat 13320

ttttctttag tacttctttc ttaatataat acatcttctt gaccaagtcc aagaggaacc 13380

tgcgttggac agttttcata tgagatcaaa ttctgagaga gcaagattta accctttttg 13440

gttcaccttc tgatcctccc ctaaggaggt atacatgaaa tatttattac tcctgcctga 13500

acttctttca ttgaatatgc aattttgcag catgcagatt ctggatttaa attctgagtc 13560

ttaacttact ggctgaggga ccttggatag gctccttatc cctcagtttc ctcatctcta 13620

aaatggggat ggcacctgcc ccgtgggttg ttggaaggac ttacagaggt gcagaatgta 13680

cgttgtacat agcaggtttc agcaaatgtt agctccctct ttccccacat ccattcaaat 13740

ctgttccttc tccaaaggat gtgtcaagga ggaaatggac ctggctggga aaccctcaga 13800

atactgggat gatgctgagc ttggctcata cctgtgcttt gctttcaggc cagtacggaa 13860

ttcccaggcc ctggtatttt ccttgcacca agtcctactg gtttggcgag gaaagtgatg 13920

agaagagcca ccctggttcc aaccagaaga gaatgtcaga aagtaagtgc tgttgacctc 13980

ctgctctttc tttaacctag tgctgctgcc tctgctaact gttgggggca agcgatgtct 14040

cctgcctttc taaaagactg tgaaaccact ccaggggcag agaaatcaca tgcagtgtcc 14100

ctttccaaat cctcccatgc catttatgtc caatgctgtt gacctattgg gagttcacgg 14160

tctcgatccc tgagggacat tttctttgtt gtcttggctt ctagaagagt atcttttact 14220

tgccccctcc caaacacaca tttcatggtc tcctaacaag ctagaagaaa gaggtaaaga 14280

caagcgtgat tgtggaacca tagcctcgct gcctgcctgt gacatggtga cctgtgtatc 14340

agcctgtgtg ggctgagacc aagtggctac cacagagctc agcctatgct tcataatgta 14400

atcattaccc agatccctaa tcctctcttg gctcttaact gcagacagag atgtccacag 14460

ctcatcaaag gctctgcttc tgggttcttt gtgcttagag tggcttccta aatatttaat 14520

aggtcccttt tctgccagtc tcttctgtgc ccatcccctg attgcccttg gtaaaagtat 14580

gatgcccctt agtgtagcac gcttgcctgc tgttcctaat catcttctcc tacctcctct 14640

ttacacctag ctcctgtttc agtcacctag aaatgctcac agtcgctgga atatgtcatg 14700

ttcttccaca cctccatgcc tttgtaggta ctgtttgctc tcacaggaga actttctctc 14760

taacttgcct atcttctcaa ctcctccttt ctctccaaga tctagttccg gatcccctcc 14820

cctgagcatc cctccttggt tctcaggtag tcagtcactc tctgccctga acttccatgg 14880

cacgtgaaag aaaatctttt tattttaaaa caattacaga ctcacaagaa gtaatacaaa 14940

ttacatgagg gggttccctt aaacctttca tccagtttcc ccaatggtag cagcatgtgt 15000

aactgtagaa tagtatcaaa accatgaaat tgacataggt acaattcaca aaccttcttc 15060

agatttcact agctttatgt gcgctcattt gtgtgtgtgt gtgcgtattt agttctatgc 15120

aattttatca tgtgtgaatt catgtaatta ctagctcagt caagctgcag aaatatctca 15180

ttgtcacaaa gctccttcat gctacccctt aatggccaca gccacctccc ttcttcctca 15240

gttcctgaca cctgtcaacc actaatgcgt tcctcgtttt tacagtttta ttatttctag 15300

aatgttacat aaatggaacc atacagtagg tatccttttg atactggctt tttttttttt 15360

ttcactcagc agtattccct tagatctatc caagttgtgt gtgtcaacag ttcattcctc 15420

ttcactgctg agtagtgttc cctgggaggg gtgtatcaca gttccatggc atttttagat 15480

gtatttttta aacagctttc agcatcctct attttaattg ttcatcaagt cctttttccc 15540

aatagactct gaatgctcct ttatcatcgt attcccatca ccaacatcag tacccaaata 15600

ggccctaaat aaacatttat agcctcctgc ctgcctgaga aaccagggtg gacatggaga 15660

gaaggcactt ctgaaagttc aagcgcagtg csctgtgtcc ttacactcca ctcctcagtg 15720

ctttctgtgg gttcatttct gtcttctctc ctgtcacagt ctgcatggag gaggaaccca 15780

cccacttgaa gctgggcgtg tccattcaga acctggtaaa agtctaccga gatgggatga 15840

aggtggctgt cgatggcctg gcactgaatt tttatgaggg ccagatcacc tccttcctgg 15900

gccacaatgg agcggggaag acgaccacca tgtaagaaga gggtgtggtt cccgcagaat 15960

cagccacagg agggttctgc agtagagtta gaaatttata ccttaggaaa ccatgctgat 16020

ccctgggcca agggaaggag cacatgagga gttgccgaat gtgaacatgt tatctaatca 16080

tgagtgtctt tccacgtgct agtttgctag atgttatttc ttcagcctaa aacaagctgg 16140

ggcctcagat gacctttccc atgtagttca cagaattctg cagtggtctt ggaacctgca 16200

gccacgaaaa gatagattac atatgttgga gggagttggt aattcccagg aactctgtct 16260

ctaagcagat gtgagaagca cctgtgagac gcaatcaagc tgggcagctg gcttgattgc 16320

cttccctgcg acctcaagga ccttacagtg ggtagtatca ggaggggtca ggggctgtaa 16380

agcaccagcg ttagcctcag tggcttccag cacgattcct caaccattct aaccattcca 16440

aagggtatat ctttgggggg tgacattctt ttcctgtttt ctttttaatc tttttttaaa 16500

acatagaatt aatatattat gagcttttca gaagattttt aaaaggcagt cagaaatcct 16560

actacctaac acaaaaattg tttttatctt tgaataatat gttcttgttt gtccattttc 16620

catgcatgcg atgttaggca tacaaaatac attttttaaa gaatactttc attgcaaatt 16680

ggaaacttcg tttaaaaaat gctcatacta aaattggcat ttctaaccca taggcccact 16740

tgtagttatt taccgaagca aaaggacagc tttgctttgt gtgggtctgg tagggttcat 16800

tagaaaggaa tgggggcggt gggagggttg gtgttctgtt ctctctgcag actgaatgga 16860

gcatctagag ttaagggtag gtcaaccctg acttctgtac ttctaaattt ttgtcctcag 16920

gtcaatcctg accgggttgt tccccccgac ctcgggcacc gcctacatcc tgggaaaaga 16980

cattcgctct gagatgagca ccatccggca gaacctgggg gtctgtcccc agcataacgt 17040

gctgtttgac atgtgagtac cagcagcacg ttaagaatag gccttttctg gatgtgtgtg 17100

tgtcatgcca tcatgggagg agtgggactt aagcatttta ctttgctgtg tttttgtttt 17160

ttcttttttt cttttttatt tttttgagat ggagtctcgc tctgtagcca ggctggactg 17220

tagtggcgcg atctcggctc actgcaacct tggcctccca ggttcaagcg attctcctgc 17280

ctcagcctcc cgagtagctg ggactctagg cacacaccac catgcccagc taatttttgt 17340

gtttttagta gagacggggt ttcaccatgt tggccaggat ggtctcaatg tcttgacctc 17400

gtgatccgcc cacctcggtc tcccaaagtg ctgggaacac aggcatgagc cactgtgtct 17460

ggccacattt tactttcttt gaatatggca ggctcacctc cgtgaacacc ttgagaccta 17520

gttgttcttt gattttagga gaagtgggag gtgaatggtt gagctgtaga ggtgacatca 17580

gcccagccag tggatggggg cttgggaaac attgcttccc attattgtca tgctggaggg 17640

ccctttagcc catcctctcc ccccgccacc ctccttattg aggcctggag cagacttccc 17700

agacctggta gtgcttcagg gccctggtat gatggaccta tatttgctgc ttaagacatt 17760

tgctcccact caggttgtcc catcagccat aaggccccca gggagcccgt gtgatggagc 17820

agagagagac ctgagctctg caatcttggg caaggctttt cccttatgtt tcttcttatc 17880

taaagtgaac agctggggct catgtgctcc ctcctcatct aaagtgaaca catggggctc 17940

atgtgcaggg tcctccccgc tttcagagcc tgaggtcccc tgaggctcag gaaggctgct 18000

ccaggtgagt gccgagctga cttcttggtg gacgtgctgt ggggacagcc cattaaagac 18060

cacatcttgg ggccctgaaa ttgaaagttg taactgcctg gtgcatggtg gccaggcctg 18120

ctggaaacag gttggaagcg atctgtcacc tttcactttg atttcctgag cagctcatgt 18180

ggttgctcac tgttgttcta ccttgaatct tgaagattat ttttcagaaa ttgataaagt 18240

tattttaaaa agcacgggga gagaaaaata tgcccattct catctgttct gggccagggg 18300

acactgtatt ctggggtatc cagtagggcc cagagctgac ctgcctccct gtccccaggc 18360

tgactgtcga agaacacatc tggttctatg cccgcttgaa agggctctct gagaagcacg 18420

tgaaggcgga gatggagcag atggccctgg atgttggttt gccatcaagc aagctgaaaa 18480

gcaaaacaag ccagctgtca ggtgcggccc agagctacct tccctatccc tctcccctcc 18540

tcctccggct acacacatgc ggaggaaaat cagcactgcc ccagggtccc aggctgggtg 18600

cggttggtaa cagaaacttg tccctggctg tgcccctagg tcctctgcct tcactcactg 18660

tctggggctg gtcctggagt ttgtcttgct ctgttttttt gtaggtggaa tgcagagaaa 18720

gctatctgtg gccttggcct ttgtcggggg atctaaggtt gtcattctgg atgaacccac 18780

agctggtgtg gacccttact cccgcagggg aatatgggag ctgctgctga aataccgaca 18840

aggtgcctga tgtgtattta ttctgagtaa atggactgag agagagcggg gggcttttga 18900

gaagtgtggc tgtatctcat ggctaggctt ctgtgaagcc atgggatact cttctgttak 18960

cacagaagag ataaagggca ttgagactga gattcctgag aggagatgct gtgtctttat 19020

tcatcttttt gtccccaaca tggtgcacta aatttatggt tagttgaaag ggtggatgct 19080

taaatgaatg gaagcggaga ggggcaggaa gacgattggg ctctctggtt agagatctga 19140

tgtggtacag tatgaggagc acaggcaggc ttggagccaa ctctggcttg gccctgagac 19200

attgggaaag tcacaacttg cctcaccttc tttgccgata ataatagtgg tgcgttacct 19260

catagaggat taaattaaat gagaatgcac acaaaccacc tagcacaatg cctggcatat 19320

agcaagttcc caaataaaat gcgtactgtt cttacctctg tgaggatgtg gtacctatat 19380

atacaaagct ttgccattct aggggtcata gccatacagg gtgaaaggtg gcttccaggt 19440

ctcttccagt gcttacccct gctaatatct ctctagtccc tgtcactgtg acaaatcaga 19500

actgagaggc ctcacctgtc ccacatcctt gtgtttgtgc ctggcaggcc gcaccattat 19560

tctctctaca caccacatgg atgaagcgga cgtcctgggg gacaggattg ccatcatctc 19620

ccatgggaag ctgtgctgtg tgggctcctc cctgtttctg aagaaccagc tgggaacagg 19680

ctactacctg accttggtca agaaagatgt ggaatcctcc ctcagttcct gcagaaacag 19740

tagtagcact gtgtcatacc tgaaaaaggt gagctgcagt cttggagctg ggctggtgtt 19800

gggtctgggc agccaggact tgctggctgt gaatgatttc tccatctcca ccccttttgc 19860

catgttgaaa ccaccatctc cctgctctgt tgcccctttg aaatcatatc atacttaagg 19920

catggaaagc taaggggccc tctgctccca ttgtgctagt tctgttgaat cccgttttcc 19980

ttttcctatg aggcacanag agtgatggag aaggtcctta gaggacatta ttatgtcaaa 20040

gaaaagagac ttgtcaagag gtaagagcct tggctacaaa tgacctggtc gttcctgctc 20100

attacttttc aatctcattg accttaactt ttaaactata aaacagccaa tatttattag 20160

gcactgattt catgccagag acactctggg cattgaaaga aagtaatgat aatagttaat 20220

tttatatagc gttgttacca tttcaacctt tttttttttt taacctctat catctcaatt 20280

aaag 20284

<210> SEQ ID NO 22

<211> LENGTH: 7052

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 22

gtgaacacac attaaagcat gagaagcatg aactagacat gtagccaggt aaaggccttg 60

ctgagatggt tggcaaaggc ctcattgcag cattcattgg caggccacag ttcttttggc 120

agctctgctt cctgaccttt caccctcagg aagcgaggct gttcacacgg cacacacatg 180

ccagacaggg tcctctgaag ccacggctgc cagtgcatgt gtcccaggga aagctttttc 240

ctttagttct cacacaacag agcttcttgg aagccctccc cggcgaaggt gctggtggct 300

ctgccttgct ccgtccctga cccgttctca cctccttctt tgccatcagg aggacagtgt 360

ttctcagagc agttctgatg ctggcctggg cagcgaccat gagagtgaca cgctgaccat 420

cggtaaggac tctggggttt cttattcagg tggtgcctga gcttccccca gctgggcaga 480

gtggaggcag aggaggagag gtgcagaggc tggtggcgct gactcaaggt ttgctgctgg 540

gctggggctg ggtggctgcg ggggtgggag cagcttggtg gcgggttggc ctaatgcttg 600

ctggggtgcc tggggctcgg tttgggagct agcagggcag tgtcccagag agctgagatg 660

attggggttt ggggaatccc ttaggggagt ggacactgaa taccagggat gaggagctga 720

gggccaagcc aggagggtgg gatttgagct tagtacataa gaagagtgag agcccaggag 780

atgaggaaca gccttccaga tttttcttgg gtagcgtgtg taggaggcca gtgtcaccag 840

tagcatatgt ggaacagaag tcttgaccct tgctatctct gcctagtcct aatggctggc 900

ttttcccagg aaggcttctg cttccatgga ctgttagatt aaccctttat ttaggtaaat 960

gagggaacct actttataag cataggaaag ggtgaagaat cttttaagat tcctttactc 1020

aagttttctt ttgaagaatc ccagagctta ggcaatagac accagacttt gagcctcagt 1080

tatccattca cccatccacc cacccaccca cccatccttc catcctccca tcctcccatt 1140

cacccatcca cccatccagc tgtccaccca ttctacactg agtacctata atgtgcctgg 1200

ctttggtgat acaaaggtga ataagacata gtcctttcct ttgcccccaa ccctcagacc 1260

agagatgaac atgtggaatg acctaaacac ctggaacagg tgtggtgtat gagcggcagg 1320

cctctgatga gagggtgggg gatggccagc cctcactccg aagcccctct gagttgattg 1380

agccatcttt gcattctggt cctgcagatg tctctgctat ctccaacctc atcaggaagc 1440

atgtgtctga agcccggctg gtggaagaca tagggcatga gctgacctat gtgctgccat 1500

atgaagctgc taaggaggga gcctttgtgg aactctttca tgagattgat gaccggctct 1560

cagacctggg catttctagt tatggcatct cagagacgac cctggaagaa gtaagttaag 1620

tggctgactg tcggaatata tagcaaggcc aaatgtccta aggccagacc agtagcctgc 1680

attgggagca ggattatcat ggagttagtc attgagtttt taggtcatcg acatctgatt 1740

aatgttggcc ccagtgagcc atttaagatg gtagtgggag atagcaggaa agaagtgttt 1800

tcctctgtac cacagtacat gcctgagatt tgtgtgttga aaccagtggt acctaacaca 1860

tttacatccc aaccttaaac tcctatgcac ttatttaccc tttaatgagc ctctttactt 1920

aagtacagtg kgaggaacag cggcatcagg atcacttggg aacttgttag aaattcagca 1980

acttgggccc agctcagacc tactgaatca gaatcaggag caattctctg gtgtgactgt 2040

gtcacagcca ggtatcaact ggattctcat acataggaaa tgacaaacgt ttatggatgg 2100

atagtctact tgtgccaggt gctgagattt gttttttgtt ttttgatttt tttttaatca 2160

ctgtgacctc atttaattct caaaaaaaga tgaaaaaatg aacactcagg aatgctgaca 2220

tgagattcag aatcaggggt ttggggcttc aaagtccatc ctctctttat ccatgtaatg 2280

cctcccctta gagatacaac atcacagacc ttgaaggctg aaggggatat aaaagctgtc 2340

tggccaagtg gtctccaagc ttgacagtgc agcagaatca cctggggata ttattaaaaa 2400

taaacatact aaggtttggc ttcagggcct gtgaatcaga atttctggag gtgaggcctt 2460

gaagtctgta tttctattgc atactttgga cacagtggtc tatagactag agtttggaaa 2520

tgattgcgct cattcagatt ctcttctgat gtttgaattg ctgccatcat atttctagtg 2580

ctctatttcc tcctgctcat tctgtcttgg ataacttatc atagtactag cctactcaaa 2640

gatttagagc cacagtcctg aaagaagcca cttgactcat tccctgtagg ttcagaataa 2700

atttcttctg cgcagtgtct gtcatagctt tttttaaatt tttttttatt tttgatgaga 2760

ctggagtttt gctcttattg cccaagctgg agtgcagtgg tgcgattttg gctcactgca 2820

acctccacct cccaggttca agcgattctc ctgcctcagc ctcccaagta gctgagatta 2880

caagcatgtg ctaccacgcc cagctaattt tgtattttta gtagagatgg gttttatcca 2940

tgttggtcag gctggtctcg agctccagac ctcaggtgat ctgcccgcct cggcctccca 3000

aagtgctggg attataggcc tgagccacag cgctcagcca taactttaat ttgaaaatga 3060

ttgtctagct tgatagctct caccactgag gaaatgttct ctggcaaaaa cggcttctct 3120

cccaggtaac tctgagaaag tgttattaag aaatgtggct tctactttct ctgtcttacg 3180

gggctaacat gccactcagt aatataataa tcgtggcagt ggtgactact ctcgtaatgt 3240

tggtgcttat aatgttctca tctctctcat tttccagata ttcctcaagg tggccgaaga 3300

gagtggggtg gatgctgaga cctcaggtaa ctgccttgag ggagaatggc acacttaaga 3360

tagtgccttc tgctggcttt ctcagtgcac gagtattgtt cctttccctt tgaattgttc 3420

tattgcattc tcatttgtag agtgtaggtt tgttgcagat ggggaaggtt tgttttgttg 3480

taaataaaat aaagtatggg attctttcct tgtgccttca gatggtacct tgccagcaag 3540

acgaaacagg cgggccttcg gggacaagca gagctgtctt cgcccgttca ctgaagatga 3600

tgctgctgat ccaaatgatt ctgacataga cccaggtctg ttagggcaag atcaaacagt 3660

gtcctactgt ttgaatgtga aattctctct catgctctca cctgttttct ttggatggcc 3720

tttagccaag gtgatagatc cctacagagt ccaaagagaa gtgaggaaat ggtaaaagcc 3780

acttgttctt tgcagcatcg tgcatgtgat caaacctgaa agagcctatc catatcactt 3840

cctttaaaga cataaagatg gtgcctcaat cctctgaacc catgtattta ttatcttttc 3900

tgcggggtcc tagtttcttg tatacattag gtgtttaatt gttgaacaaa tattcattcg 3960

agtagatgag tgattttgaa agagtcagaa aggggaattt gctgttagag ttaattgtac 4020

cctaagactt agatatttga ggctgggcat ggtggctcat gccagtaatc ccagcgcttt 4080

gagaggctga ggtgggtaga tcacctgagg tcaggagttt gagaccagtc tgaccaacaa 4140

ggtgaaaccc cgtctctact aaatacaaaa aattagccga gtgtggtggc acatgcctgt 4200

catcccagct acttgggagg ctgaggcagg agaatcgctt gaacccagga ggcagaggtt 4260

gcagtcagcc acggttgcgc cattgcactc cagactgggc aacaagagtg aaaactccat 4320

ctcaaaaaag aaaaaaaaag aattagatat tttggatgag tgtgtctttg tgtgtttaac 4380

tgagatggag aggagagcta agacatcaaa caaatattgt taagatgtaa aagcacatca 4440

gttaggtatc attagtttag gacaaggatt tctagaaaat ttttaggaac agaaaacttt 4500

ccagttctct cacccctgct caaagagtgt atggctctta cattatatat aactgcctga 4560

cttcatacag tatcagtact tagatcattt gaaatgtgtc cacgttttac caaaatataa 4620

tagggtgaga agctgagatg ctaattgcca ttgtgtattc tcaaatatgt caagctacgt 4680

acatggcctg tttcatagag tagtctataa gaaattgatg acttgattca tccgaatggc 4740

tggctgtaac acctggttac gcatgaacac ctcttttcag ttgtctcaag acacctttct 4800

tttctgtact tatcagacaa ggactgaaag gcagagactg ctactgttag acattttgag 4860

tcaagctttt ccttggacat agctttgtca tgaaagccct ttacttctga gaaacttcta 4920

gcttcagaca catgccttca agatagttgt tgaagacacc agaagaagga gcatggcaat 4980

gccgaaaaca cctaagataa taggtgacct tcagtgttgg cttcttgcag aatccagaga 5040

gacagacttg ctcagtggga tggatggcaa agggtcctac caggtgaaag gctggaaact 5100

tacacagcaa cagtttgtgg cccttttgtg gaagagactg ctaattgcca gacggagtcg 5160

gaaaggattt tttgctcagg tgagacgtgc tgttttcgcc agagactctg gcttcatggg 5220

tgggctgcag gctctgtgac cagtgaaggc aggatagcat cctggtcaag atatggatgc 5280

cggagccaga tttatctgta tttcaatccc agttctattc cttgccagtt gtgtatccgc 5340

tggcaagtta cttctctatg cctcaatctc ctcatctgta aaatggggat aataatatta 5400

cctgcaatac agggttgtta cgaaaataaa aatgaatagg tgcttagaat ggggcctgac 5460

attagtaagt gcttagtttt gtgtgtgtat atgttatttt tattttggag gagaacataa 5520

aaaggacaaa gtgtagaaaa actggttggg tgtattcagc tgtcataaca tgagagttgt 5580

tatgcccaga tgcacttgac atgtgaattt attagaaaca tgatttttct ctgagttgat 5640

gtttaactca aactgataga aaagataggt cagaatatag ttggccaaca gagaagactt 5700

gttagactat tgtctgcatg tcagtgtttg catgctaact tgcttagtta gaaaggttaa 5760

attttttcac tctataaaat caagaaatat agagaaaagg tctgcagaga gtctttcatt 5820

tgatgatgtg gatattgtta agagcgggag tttggagcat acagagctca agttgaatcc 5880

tgactttgct acttattggc tatatgacct tgggcaagct gcttagtctc tctgatcctc 5940

agttaccttt gtttgttgat gatgaccatt gataacacaa ccataaataa tgacaacata 6000

gagatagttc tcattatagt agttgttata cagaattatt cactcaatgt taattttctg 6060

cattgaaatc ccagaacatt agaattgggg gcattatttg aatctttaag gttataagga 6120

atacatttct cagcaataaa tggaaggagt tttgggttaa cttataaagt atacccaagt 6180

catttttttt cagagaagat atggtagaaa gtcttaggag gttgaagaag gaattggata 6240

tttattcttt ctgagactat catgggagat aatgactatg gttgtccatg attggagccg 6300

ttgctgtaga gttggtttta ttatagtgta ggatttgaat gggccatgtg ttctcagacc 6360

tcagaataaa aagagaaaac tgaggccagt ggggagcgtg acttcacatg ggtacacttg 6420

tgctagagac agaaccagga ttcaggactt ctggctcctg gtcctgggtt catggcccaa 6480

tgtagtcttt ctcagtcttc aggaggagga agggcaggac ccagtgttct gagtcaccct 6540

gaatgtgagc actatttact tcgtgaactt cttggcttag tgcctctgcc aggtggccat 6600

aacctctggc cttgtgttgc cagagaaaag gtttagtttt caggctccat tgcttcccag 6660

ctgccaagaa tgccttggtg cagcacagtc ataggccctg cattcctcat tgccgtgctg 6720

gttggtcggg gaggtgggct ggactcgtag ggatttgccc cttggccttg tttctaacac 6780

ttgccgtttc ctgctgtccc cctgccccct ccactgcctg ggtaaagatt gtcttgccag 6840

ctgtgtttgt ctgcattgcc cttgtgttca gcctgatcgt gccacccttt ggcaagtacc 6900

ccagcctgga acttcagccc tggatgtaca acgaacagta cacatttgtc aggtatgttt 6960

gtcttctaca tcccaggagg gggtaagatt cgagcagacc aaagatgttt acgagggcca 7020

agggaatgga cttcagaatt acacggtgga at 7052

<210> SEQ ID NO 23

<211> LENGTH: 2534

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 23

gggaagcatt taaaaaaaaa aaagtatata tatatatata tatatatata tgtaatgtga 60

attggcctct ttttctctaa gcccacattt tcttcttaca tagttcaggt ttactttatt 120

ttttcctttc cggctgctga ccctgtattg cccgtagttg tggaacatag catgtgtttg 180

tgacctgtgc ctgttatttt tgtgctttct agttgtgcat gcaaagagta caaagttttc 240

ttgccctttc ttggaaaatc ctgcttgtct gtgccaaagg gataattgtg aaagcacttt 300

tgaaatactt aatgagttga ttttcttcaa attaaaaaaa atatataaat gtatatgtgt 360

atgtacatgt gtgtacacat acacaccttt atacatacag cccatttaaa acaagctcca 420

ctttggagtg ctctacgtca ccctgatgcc gaatacaggg ccagagtctg agatccttct 480

gggtggtttc tgtgttttgt tcatttctgt tttaagagcc tgtcacagag aaatgcttcc 540

taaaatgttt aatttataaa aacattttta tctctcgatt actggtttta atgaattact 600

aagctggctg cctctcatgt acccacagca atgatgctcc tgaggacacg ggaaccctgg 660

aactcttaaa cgccctcacc aaagaccctg gcttcgggac ccgctgtatg gaaggaaacc 720

caatcccgtg agtgccactt tagccataag cagggcttct tgtgcttgtt gcctggtttg 780

atttctaata tgctgcattt atcaactgca tgccacattg tgaccgccag catttgccct 840

ttgaattatt attatgtttt atttacaaaa agcgaaggta gtaaccgaac taaattatct 900

aggaacaaac gtttggagag tcttctaaca ccgyscaaag cacgtcatta cagacatttg 960

tttactgatt tagaacctta atatttaatt taaatacgca ctttacactt actgatgaaa 1020

tgcttttcct ttctttctct cccagcccct gtacttaagt gcttcaatag gctctcatta 1080

tatatgattt ttaggttttg cttatcagct tcttcgcttt tataatctga aaagatggca 1140

tatgaatttt tataaaaagg gacactttct tcttctcaaa ttgtatattt ttattgtact 1200

ttccttcaaa accccctttt aaaaagtaag cagtggataa ataaattcag tgaagcatcc 1260

atatgaccct taagtgagtg taggggaagg gaggtcacca gatcactgtg agtgaagatg 1320

gtggagaggt gaggatctta tgaggccgtg ctcaaggctg gtagaggtgg gttagtgttt 1380

ccaggtttag gcagaatctc agctgaggtc atgaaacaac agtgatctct gaaaaattat 1440

ggcaaggtgg gaaggtgctg gagaattgga gagggggcaa acttgacttt caagtttcaa 1500

tgggaagata ggtgactctg cacaccacag aacagtgagc atgataacct gtttatacaa 1560

ggttctagag cagatttcta aatggatagc tactgtgtgc ttgtttgttc ttaattagta 1620

ttggatagtt actaaatact tgttagtact tagtacataa tgggtggtaa atcctagcag 1680

ctaatattgg ttcccaaata accagatgac aaggatagag aaggacacag acacggccta 1740

tctggatttc atggtgcctt tgattttcca catgaaggtt gtgtagggaa gatagaagca 1800

tgagatgaga tgataatata gttatctgga ttcatcactg gccagctgaa ccatatgaac 1860

tcatggattg atgctagctt aggaaggctc tgtaggagcc agaactgggc tgagagccag 1920

cccatagaga caaaagaggc ccggccctga catcagaggg ttcaaacatg atgtctgagc 1980

cccacctaca gtctgccgga ggtggttgga aggaagagcc tttatcctta caattcttac 2040

tgaaattcaa atttttaggt tttgcaaaaa aatggtggac ctgaaggaaa tttgacagga 2100

gcatgtctca gctgtattta aatttgtctc agccaatccc cttttgaatg ttcagagtgt 2160

aagcttcagg agggcagcgc gtcttagtgt gacttttctg gtcagttcag gtgctttaag 2220

gagacaatta gagatcaatc tggaaaactt catttgaatt tttaatacat aagaaaacaa 2280

taagaaatag ttaaaaatat atatttatat aatatatata tgtgtgtgtg tgtgtgtgtg 2340

tgtgtgtgtg tatatatata tatattttat ttatttattt ttttttgaga tggagtctcg 2400

ctctgttgcc caggctggag tgcagtggct caatcttggc tcactgccac ctctgcctcc 2460

caggttcaag tgattctcct acctcagcct cctgagtagc tgggattaca agcatgtgcc 2520

accacactgg ctaa 2534

<210> SEQ ID NO 24

<211> LENGTH: 2841

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 24

tcttgccagt ctctactcat ttttcagcac atcgagcata agatccagac tctttcccag 60

gcctctctca tctggctcct ctcctcctcc tttatcatta ctcttcttcg tagcttatcc 120

tactccagcc atgctgtctt cctattattc ctaaaaarta gaaatgcatt tcttcctagg 180

gcctttgtac ctgcacttgc catcgctttt gctcagaatg ttctttttgc caagcttttg 240

cccagcttgt tctccatcat tgttatgttt tggctgaaat gtcttctctt agtaggttca 300

ttctccccag tcactgtctt tttattttgc tttattttgg gccatctaag gttatcttat 360

tagtgtattt gttgttcgtc tcctccatgg gcatacacct ccatgaaggc aggtattttc 420

accttaggcc ctcgaatata ctggacagca tctggcacgt agtagatgct caacgaatgt 480

ttgttgtgtg agcaaatggt tggttgattg gattgaactg agttcagtat gtaaatattt 540

agggcctctt tgcattctat tttacttatg tataaaatga tacataatga tgatataaat 600

gatgtcacag tgtacaaggc tgttgtggga tcaagcaatc aaatgagatc atgcttgtct 660

tttccaaatg gtgagggaat agatgcatgt ttgtggttgt tacggaatga tcctgtgctc 720

ctgaggcaac agaaaggcca ggccatctct ggtaatccta ctcttgctgt cttccctttg 780

cagagacacg ccctgccagg caggggagga agagtggacc actgccccag ttccccagac 840

catcatggac ctcttccaga atgggaactg gacaatgcag aacccttcac ctgcatgcca 900

gtgtagcagc gacaaaatca agaagatgct gcctgtgtgt cccccagggg caggggggct 960

gcctcctcca caagtgagtc actttcaggg ggtgattggg cagaaggggt gcaggatggg 1020

ctggtagctt ccgcttggaa gcaggaatga gtgagatatc atgttgggag ggtctgtttc 1080

agtctttttt gttttttgtt tttttttctg aggcggagtc ttgctctgtc gcccaggctg 1140

gagtgctgtg gcatgatctt gcctcactgc aacctccacc tcccaggttc aagcgattct 1200

cctgcctcag cctcctgagt agctgggatt acaggcacgc accaccatgt ctggctaatt 1260

tttgtgtttt tagtagagat agggtttcgc cgtgttggct aggctggtct ggaattcctg 1320

acctcaggtg atccacccgc ctcggcctcc caaagtgctg ggattacagg cgtgagccac 1380

tacgcccagc cctgtttcag tctttaactc gcttcttgtc ataagaaaaa gcatgtgagt 1440

tttgagggga gaaggtttgg accacactgt gcccatgcct gtcccacagc agtaaagtca 1500

caggacagac tgtggcaggc ctggcttcca atcttggctc tgcaacaaat gagctggtag 1560

cctttgacag gcctgggcct gtttcttcac ctctgaatta gggaggctgg accagaaaac 1620

tcctgtggat cttgtcaact ctggtattct tagagactct gtttgggaag gagtcctgag 1680

ccattttttt tttcttgaga atttcaggaa gaggagtgct tatgatagct ctctgctgct 1740

tttatcagca accaaattgc aggatgagga caagcaattc taaatgagta caggaactaa 1800

aagaaggctt ggttaccact cttgaaaata atagctagtc caggtgcggg gtggctcaca 1860

cctgtaatct cagtattttg ggatgccgag gtggactgat cacctaaggt caggagttcg 1920

aaaccagctt ggccaatgtg gcgaaaccct gtctctacta aaaattcaaa aattagccag 1980

gcatggtggc acatgcctgt aatcccagtt acttgggagg ctgaagcagg agaattgctt 2040

gaacctggga ggtggaggtc gcagggagcc aaaattgcgc cactgtactc cagcctgagc 2100

aacacagcaa aactccatat caaaaaataa aatgaataaa ataacagcta atctagtcat 2160

cagtataact ccagtgaaca gaagatttat taggcatagt gaatgatggt gcttcctaaa 2220

aatctcttga ctacaaagaa tctcatttca atgtttattg tttagatgtt cagaataaat 2280

tcttgggaaa gaccttggct tggtgtaagt gaattaccag tgccgagggc agggtgaacc 2340

aagtctcagt gctggttgac tgagggcagt gtctgggacc tgtagtcagg tttccggtca 2400

cactgtggac atggtcactg ttgtccttga tttgttttct gtttcaattc ttgtctataa 2460

agacccgtat gcttggtttt catgtgatga cagagaaaac aaaacactgc agatatcctt 2520

caggacctga caggaagaaa catttcggat tatctggtga agacgtatgt gcagatcata 2580

gccaaaaggt gactttttac taaacttggc ccctgcctta ttattactaa ttagaggaat 2640

taaagaccta caaataacag actgaaacag tgggggaaat gccagattat ggcctgattc 2700

tgtctattgg aagtttagga tattatccca aactagaaaa gatgacgaga gggactgtga 2760

acattcagtt gtcagcttca aggctgaggc agcctggtct agaatgaaaa tagaaatgga 2820

ttcaacgtca aattttgcca c 2841

<210> SEQ ID NO 25

<211> LENGTH: 852

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 25

gcatgctgga gtgatagtga ccatgagttt ctaagaaaga agcataattt ctccatatgt 60

catccacaat tgaaatatta ttgttaattg aaaaagcttc taggccaggc acggtggctc 120

atgcctgtaa tcccagcact ttaggagcca aggcgggtgg atcacttgag gtcaggagtt 180

tgagaccagc ctggccaaca tggggaaacc ctgtctctac taaaaataca aaataagctg 240

ggcgtggtgg tgcgtgcctg taatcccagc tacttgggag gctgaggcag gagaactgct 300

tgaatctggg aggcggaggt tgcagtgagc tgagttcatg ccattgcatt ccagcctggg 360

caacaagagc gaaaccatct cccaaaagaa aaaaaaaaga aagaaaaagc ttctagtttg 420

gttacatctt ggtctataag gtggtttgta aattggttta acccaaggcc tggttctcat 480

ataagtaata gggtatttat gatggagaga aggctggaag aggcctgaac acaggcttct 540

tttctctagc acaaccctac aaggccagct gattctaggg ttatttctgt ccgttcctta 600

tatcctcagg tggatattta ctccttttgc atcattagga ataggctcag tgctttcttt 660

gaactgattt tttgtttctt tgtctctgca gcttaaagaa caagatctgg gtgaatgagt 720

ttaggtaagt tgctgtcttt ctggcacgtt tagctcaggg ggaggatggt gttgtaggtg 780

tgcttggatt gaagaaagcc ttggggattg tttgtcactc acacacttgt gggtgccatc 840

tcactgtgag ga 852

<210> SEQ ID NO 26

<211> LENGTH: 6289

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 26

gctttataga gtttctgcct agagcatcat ggctcagtgc ccagcagccc ctccagaggc 60

ctctgaatat ttgatatact gatttccttg aggagaatca gaaatctcct gcaggtgtct 120

agggatttca agtaagtagt gttgtgaggg gaatacctac ttgtactttc cccccaaacc 180

agattcccga ggcttcttaa ggactcaagg acaatttcta ggcatttagc acgggactaa 240

aaaggtctta gaggaaataa gaagcgccaa aaccatctct ttgcactgta tttcaaccca 300

tttgtccttc tgggttttga aggaacaggt gggactgggg acagaagagt tcttgaagcc 360

agtttgtcca tcatggaaaa tgagataggt gatgtggcta cgtcaggggg cccgaaggct 420

ccttgttact gatttccgtc ttttctctct gccttttccc caagggccag gacccctgga 480

tctctgggca gagcagacgc aggcccctat aatagccctc atgctagaaa ggagccggag 540

cctgtgtata aggccagcgc agcctactct ggacagtgca gggttcccac tctcccaact 600

ccccatctgc ttgcctccag acccacattc acacacgagc cactgggttg gaggagcatc 660

tgtgagatga aacaccattc tttcctcaat gtctcagcta tctaactgtg tgtgtaatca 720

ggccaggtcc tccctgctgg gcagaaacca tgggagttaa gagattgcca acatttatta 780

gaggaagctg acgtgtaact tctgaggcaa aatttagccc tcctttgaac aggaatttga 840

ctcagtgaac cttgtacaca ctcgcactga gtctgctgct gatgatactg tgcaccccac 900

tgtctgggtt ttaatgtcag gctgttcttt taggtatggc ggcttttccc tgggtgtcag 960

taatactcaa gcacttcctc cgagtcaaga agttaatgat gccatcaaac aaatgaagaa 1020

acacctaaag ctggccaagg taaaatatct atcgtaagat gtatcagaaa aatgggcatg 1080

tagctgctgg gatataggag tagttggcag gttaaacgga tcacctggca gctcattgtt 1140

ctgaatatgt tggcatacag agccgtcttt ggcatttagc gatttgagcc agacaaaact 1200

gaattactta gttgtacgtt taaaagtgta ggtcaaaaac aaatccagag gccaggagct 1260

gtggctcatg cctgtaatcc tagcactttg ggaggctgaa gcgggtggat cacttgaggt 1320

caggagttcg agaccagcct ggcctacatg acaaaacccc gtatctacta aaaatacaaa 1380

aaaattagct gggcttggtg gcacacacct gtaatcccag ctacttggga ggctgaggca 1440

ggagaattgc ttgaaccctg taggaagagg ttgtagtgag ccaagatcgc accgttgcac 1500

tccagcctgg gcaacaagag caaaactcca tctcaaaaaa caaattaaat ccagagattt 1560

aaaagctctc agaggctggg cgcggtggct tacacctgtt atcccagcat tttgggatgc 1620

cgaggcgggc aaagcacaag gtcaggagtt tgagaccagc ctggccaaca tagtgaaacc 1680

ctgtctctgc taaaaacata gaaaaattag ccgggcatgg tggcgtgcgc ctgtaatccc 1740

agctactcgg gaggctgagg tgagagaatt rcttgaaccc gggaggcgga ggttgcagtg 1800

agcccagatt gcaccactgc actccagcct gggcgacaga gcaagactcc atctcaaaaa 1860

aagctctcag aacaaccagg tttacaaatt tggtcagttg gtaaataaac tgggtttcaa 1920

acatactttg ctgaaayaat cactgactaa ataggaaatg aatctttttt tttttttttt 1980

taagctggca agctggtctg taggacctga taagtactca cttcatttct ctgtgtctca 2040

ggtttcccat ttttaggtga gaattaaggg gctctgataa aacagaccct aggattgtgg 2100

acagcagtga tagtcctaga gtccacaagt ctgcttttga gtgatgggcc catgtatctg 2160

gcacatctgc aggcagagcg tggttctggc tcttcagatg atgccggtgg agcactttga 2220

ggagtcctca ccccaccgtg ataaccagac attaaaatct tggggctttg catcccagga 2280

tttctctgtg attccttcta gacttgtggc atcatggcag catcactgct gtagatttct 2340

agtcacttgg ttctcaggag ccgtttattt aatggcttca catttaattt cagtgaacaa 2400

ggtagtggca ttgctcttca cagggccgtc ctgttgtcca caggttccag attgactgtt 2460

gccccttatc tatgtgaaca gtcacaactg aggcaggttt ctgttgttta caggacagtt 2520

ctgcagatcg atttctcaac agcttgggaa gatttatgac aggactggac accagaaata 2580

atgtcaaggt aaaccgctgt ctttgttcta gtagcttttt gatgaacaat aatccttatg 2640

tttcctggag tactttcaac tcatggtaaa gttggcaggg gcattcacaa cagaaaagag 2700

caaactatta actttaccag tgaggcagta cggtgtagtg tagtgattca gagaatttgc 2760

tttgccacca gacataccag gtaaccttga ctaagttact taacctatct aaacctcagt 2820

tycctcatct gtgaaatgga gacagtaatc atagctattt ccaaactgtt gtgagaattc 2880

aatgagttaa aggtataagg tcctcaccac agcgcctgcc cacatagtca gtgatcacta 2940

tgtcctgaac actgtaatta cttcgccata ttctctgatc atagtgtttt gccttggtat 3000

gtgactagaa tttctttctg aggtttatgg gcatggttgg tgggtatgca cctgcctgca 3060

ggagcccggt ttgggggcat taccttgtac ctggtatgtt ttctttcagg tgtggttcaa 3120

taacaagggc tggcatgcaa tcagctcttt cctgaatgtc atcaacaatg ccattctccg 3180

ggccaacctg caaaagggag agaaccctag ccattatgga attactgctt tcaatcatcc 3240

cctgaatctc accaagcagc agctctcaga ggtggctctg taagtgtggc tgtgtctgta 3300

tagatggagt ggggcaaggg agagggttat ggagaagggg agaaaaatgt gaatctcatt 3360

gtaggggaac agctgcagag accgttatat tatgataaat ctggattgat ccaggctctg 3420

ggcagaagtg ataagtttac gaattggctg gttgggcttc ttgaactgca gaagagaaaa 3480

tgacactgat atgtaaaaat cgtaacattt agtgaattca tataaagtga gttcaaaaat 3540

tgttaattaa attataattt aattataagt gtttaatcag tttgatttgt ttaaaaacca 3600

ctgttttaaa tttggtggaa tatgttttta ttagcttgta tctttaattc ctaaattaag 3660

ctgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gaagtttaaa 3720

gccaggatga gctagtttaa agtatgcagc ctttggagtc atacagatct gggtttgaat 3780

ctggtctcta aactttatag atgtatgata ttaaatgagg cagttcatgt aaattgccaa 3840

gcccagcact cagcacagag ttgatatttc acacacatta gatacctttc ctgtatgtgg 3900

agcatggcag ttcctgtttc tgctttactc ctacaggata ctaatatagg acactaggat 3960

ctttatacca agaccccatg taatgggctt atgagaccat tcttcttata aaaatctgac 4020

agaatttttg tatgtgttag atcaataggc tgcatactgt tattttcaag ttgatttaca 4080

gccagaaata ttaatttatt tgagtagtta cagagtaata tttctgctct catttagttt 4140

tcaagcccca ctagtccttt gtgtgtgaaa atttacaact tactgctctt acaaggtcat 4200

gaacagtgga ccaaagtgaa tgccattaac cactctgact tccttcatta gttttattgt 4260

gacagtggac tcttttgacc tcagtaatac cagtttggca tttacattgt catattttta 4320

gacttaaaaa tgatcatctt aaccctgaat aaaatgtgtc tggtgaacag atgtttttcc 4380

ttggctgtgc ctcagatatc tctgtgtgtg tgtacgtgtg tgtttgtctg tgtgtccatg 4440

tcctcactga ttgagcccta actgcatcaa agacccctca gattttcaca cgctttttct 4500

ctccaggatg accacatcag tggatgtcct tgtgtccatc tgtgtcatct ttgcaatgtc 4560

cttcgtccca gccagctttg tcgtattcct gatccaggag cgggtcagca aagcaaaaca 4620

cctgcagttc atcagtggag tgaagcctgt catctactgg ctctctaatt ttgtctggga 4680

tatggtaagg acacaggcct gctgtatctt tctgatgtct gtcagggcca tggattgata 4740

tggataagaa agaaagagct ctggctatca tcaggaaatg ttccagctac tctaaagatg 4800

tatgaaaaag aaatagccag aggcaggtga tcactttcat gacaccaaac acagcattgg 4860

gtaccagagt tcatgtcaca ccagagggaa aattctgtac acaatgatga aaattaatac 4920

cactaccact taagttccta tgtgacaact ttcccaagaa tcagagagat acaagtcaaa 4980

actccaagtc aatgcctcta acttctctga tgggttttaa cctccagagt cagaatgttc 5040

tttgccttac taggaaagcc atctgtcatt tagaaaactc tgtacatttt atcagcagct 5100

tatccatcca ttgcaaatat tgtttttgtg ccasccacaa tatattgctt ctatttggac 5160

caatatgggg gatttgaagg aattctgaag ttctaattat atttcaactc tactttacaa 5220

tatctccctg aaatatatct ccctgtaact tctattaatt ataagctaca cagagcaaat 5280

ctaattcttc tcccaccgaa caagtccctg gatatttaaa aataactctc atactctcat 5340

ttaacctgag tattacccag ataagatgat atatgagaat acaccttgta acctccgaag 5400

cactgtacaa atgtgagcaa tgatggtgga gatgatgatg agatctttgc tgtttatacc 5460

aagcccctta gactgtgtca ctcttctgat ccggttgtcc ttgtatggcc atgctgtata 5520

ttgtgaatgt cccgttttca aaagcaaagc caagaattaa ccttgtgttc aggctgtggt 5580

ctgaatggtt atgggtccag agggagttga tctttagctc acacttctat tactgcagca 5640

caaagatttt gcattttgga aggagcaccg tcttactggc aacttagtgg taaaccaaaa 5700

cctccatttc acacaaatga ttgtgaaatt cgggtctcct tcattctata caaattcatt 5760

tgattttttt gaaactaaac tttatattta tccatattaa attacatggg ttttattttt 5820

gttttatctt gattcagtaa ttactccttt cagtaaacac agactgagtg ctgtgtgtct 5880

gacttatgcc aggcataggt gattcagaga tgaaaggtca agtccctgaa cccatctctt 5940

gtcttcctgg gtattatctg tccctccctg ctttagagct cctgaaattt gctagaagca 6000

tgtcttcatc taagttgttg ataaacacat caagtaggat tggactgagg cagagccctg 6060

tagtctgaag ctgcagttct tctagcggct gacaagcccc actatcactt ccctgctggt 6120

gctttgctct gccagctgtg aattctcata attgtcctat cgtcaagtct ttatttctgc 6180

attttactgc ttgatacact gtcaggacag actttaaaat tattctcagt gcgatgaaac 6240

aattctgaca ttcatgttat gagcagttac ctcataaata gattacatg 6289

<210> SEQ ID NO 27

<211> LENGTH: 4244

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 27

aaattactct gactgggaat ccatcgttca gtaagtttac tgagtgtgac accttggctt 60

gactgttgga aagacagaaa gggcatgtag tttataaaat cagccaaggg gaaaatgctt 120

gtcaaaatgt attgtcgggt attttgatta atagtttatg tggcttcatt aattcagagt 180

tactctccaa tatgtttatc tgccctttct tgtctgataa tggtgaaaac ttgtgtgatg 240

cattgtatat ttgatttagg ggtgaactgg atgtctttgt tttcactttt agtgcaatta 300

cgttgtccct gccacactgg tcattatcat cttcatctgc ttccagcaga agtcctatgt 360

gtcctccacc aatctgcctg tgctagccct tctacttttg ctgtatgggt aagtcacctc 420

tgagtgaggg agctgcacag tggataaggc atttggtgcc cagtgtcaga aggagggcag 480

ggactctcag tagacactta tctttttgtg tctcaacagg tggtcaatca cacctctcat 540

gtacccagcc tcctttgtgt tcaagatccc cagcacagcc tatgtggtgc tcaccagcgt 600

gaacctcttc attggcatta atggcagcgt ggccaccttt gtgctggagc tgttcaccga 660

caatgtgagt catgcagaga gaacactcct gctgggatga gcatctctgg gagccagagg 720

acagtgttta attgtgatct tattccactt gtcagtggta ttgacactgc tgactgcctt 780

gtcctgtctt cagagtctgt cttccctgag aaggcaaagc acctttcttt cttgctgtgc 840

cttacatttt gctggtcaag cctttcagtt tcttttgaca gtttttttta cttctttctt 900

ttttcaatgt tgctcttacc aagagtagct cctctgcctt ccactttaca catgagagct 960

gggcgacgca ttcagtccta aggcttttac catcacctct cttggtgttt ttattgtcat 1020

ctctaagatc aatgccttta gccttgatca taaccttgaa ctctaatctc aaattctcac 1080

ttgcctagtg gattgctcca tttagatagt atatagatac cccaacctgg atatgtccta 1140

gttttctttc cccttggaac ttaatgcttt tcttgccatc cctgtcacac tcagtggcac 1200

taccatccac tcggttgccc aagctggctc ttagagttat cctagatgct tgctttgctg 1260

ttgcagattt cccacattca actggttatg ttgtcagttc ttccaggtat ggacctctaa 1320

aataaggctt cctctccatt ccggttgtca ttgcctttgt ccaaacacag cacacaaggc 1380

cttttacagt tgcacaactc ttcctgtcca tacccaccac accctttccc agctgtaagc 1440

ttcagatgag ttgcctccaa ccaccatgct cctgtaggcc tggcttgaaa tgcccttctt 1500

ctgtcacagg gtctggtagt atatcccttg cccttcaaga tttagctaaa atgtgaagct 1560

ttccttacct gctgggaggt gttctctctt ttctctgtgc tctcagagtc cttagtccat 1620

gcctccagta caacgtacat ccacttacat ggtaatttcc tgtttacata cttttcctac 1680

tcggagtgga gtctgtttct taataatttt gcctctccca tgccctagca cagtgcatcc 1740

agcgtatagc cccttattca gttggtagat atttggccac tgttgccttg tgggatcata 1800

agttctgatg tatttgagaa gaatttctaa aattctgaca aaatcctgaa actcaaatat 1860

tgacccagac atgagcaatt tgcttttcaa atgctaaggg atttttaatg gatttgcttt 1920

aattaaatct agcctgtttc taagctttat tcattatttc tccatactca gagcatttct 1980

ccagattttc taaagaatag aattttattg ctacatatca tcagctatgc ctgctgctat 2040

ttaattggta tctgaattaa aaggtctggt ttgtccctag agaatcaaat tttttcttca 2100

ctcccatatt tcagaacttg atacattttt aggataaacc atgaatgaca cccgtttctt 2160

ctccctcacc ctcccttccc tcccattttt tttttttttt ttttttagaa gctgaataat 2220

atcaatgata tcctgaagtc cgtgttcttg atcttcccac atttttgcct gggacgaggg 2280

ctcatcgaca tggtgaaaaa ccaggcaatg gctgatgccc tggaaaggtt tggtgagtga 2340

agcagtggct gtaggatgct ttaatggaga tggcactctg cataggcctt ggtaccctga 2400

actttgtttt ggaaagaagc aggtgactaa gcacaggatg ttcccccacc cccatgccca 2460

gtgacagggc tcatgccaac acagctggtt gtggcatggg ttttgtgaca caaccatttg 2520

tctgtgtctc tgatagcatt gagaaaagtg aaagggcagt tttgaaggta aggaaaatag 2580

tgttatttgc ttggatccac tggctcatgc cactgtctgg gttggttaga agcactggaa 2640

aagtcaaacc ataactttga gaattaggtg atcagggaat cagaaggaaa gatgcaaact 2700

ttggctcttt taggcgaatc atgtgcctgc agatgaggtc atttattatc ttttacacag 2760

tctataaaat tataatgtat tacatctttt tctaccttta gaatggttaa aaatatttct 2820

ccggtagcca tatgattatt attcatccat tagataatat agtcaaatgg gccatgttat 2880

ttactgttca tagaagaggg gctttttgca acttgggcta caaaggagat atgtaaggaa 2940

tttaaggaat ggttacatgg aactagattt aattgaatct agtggtttaa ttgattcact 3000

aggatatatg ctactgaaag gggaatctgc ttaaagtgct ttctgatatt tattattact 3060

aaaacttaga atttattaaa aatactgact gtgaaaatta cttgggtcgt ttgccttttt 3120

aaaaggattt ttggcatgtc tcattaaaaa aagaaatact agatatcttc agtgaagtta 3180

caaatcgaat acacattggc tctgaaattc tgattgatac tgggtcataa aaagttttcc 3240

caaatcagac ttggaaagtg atcactctct tgttactctt ttttccttgt catgggtgat 3300

agccatttgt gtttattgga agatcggtga attttaagga acataggccc aaatttgagg 3360

aagggccatg gtttttgatc cctccattct gaccggatct ctgcattgtg tctactaggg 3420

gagaatcgct ttgtgtcacc attatcttgg gacttggtgg gacgaaacct cttcgccatg 3480

gccgtggaag gggtggtgtt cttcctcatt actgttctga tccagtacag attcttcatc 3540

aggcccaggt gagctttttc ttagaacccg tggagcacct ggttgagggt cacagaggag 3600

gcgcacaggg aaacactcac caatgggggt tgcattgaac tgaactcaaa atatgtgata 3660

aaactgattt tcctgatgtg ggcatcccgc agccccctcc ctgcccatcc tggagactgt 3720

ggcaagtagg ttttataata ctacgttaga gactgaatct ttgtcctgaa aaatagtttg 3780

aaaggttcat ttttcttgtt ttttccccca agacctgtaa atgcaaagct atctcctctg 3840

aatgatgaag atgaagatgt gaggcgggaa agacagagaa ttcttgatgg tggaggccag 3900

aatgacatct tagaaatcaa ggagttgacg aaggtgagag agtacaggtt acaatagctc 3960

atcttcagtt tttttcagct ttatgtgctg taacccagca gtttgctgac ttgcttaata 4020

aaagggcatg tgttcccaaa atgtacatct ataccaaggt tctgtcaatt ttattttaaa 4080

aacaccatgg agacttctta aagaattctt actgagaatt cttttgtgat atgaattccc 4140

attctcgaat actttggttt tatatgctta catttatgtg ttagttatta aaacatacta 4200

atattgtata tctagtcaaa ctgagtagag agataatggt gatt 4244

<210> SEQ ID NO 28

<211> LENGTH: 5023

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 28

ttttaaaata cctgcaatac atatatatgt tgaatagatg aaaaattatg tagatgataa 60

tgaatgatac ggttctaaaa agacaggtta aaaagtaagt tcacttttat tttgagcttc 120

agaatcattc agaagccagt cgccacaaac gcagaccaag gctcttggca catcaaatat 180

gcctatggct tagggttatt gacaagtctt atgttgcagt gtatgtggtt tatagtcctg 240

ccttccacag ttgcttggga gagctgtgag tcactgaggc ttatgaatgt ttacattttg 300

tttgttgcag atatatagaa ggaagcggaa gcctgctgtt gacaggattt gcgtgggcat 360

tcctcctggt gaggtaaaga cactttgtct atattgcgtt tgtccctatt agttcagact 420

atctctaccc aatcaagcaa cgatgctcgt taagaggtaa aagtggattt taaaggcttc 480

tgtatttatg ccaggatgga gcaattagtc atcgagaaga gagggaccct gtatgtcaag 540

agaatgattt cagagaatcc aatacaattt aagaaaaagc atggggctgg gcgcagtgat 600

tcactcctgt aatcccagca ctttgggagg ccgaggtggg cggactcacg aggtcaggag 660

attgagacca tcctggccaa catggtgaaa ccccatctct actataaata caaaaattag 720

ctgggcatag tagtgcattc ctgtagtccc agctactcgg gaggctgagg caggagaatt 780

gcttgaacct aggaggggga ggttgcccag attgcgctgc tgcactccag cctggtgaca 840

gagtgagact catgtcaaca acaaaaacag aaaaagcacg cacatctaaa acatgctttt 900

gtgatccatt tgggatggtg atgacattca aatagttttt taaaaataga ttttctcctt 960

tctggtttcc gtttgtgttc ttttatgccc ttttgccaga gtaggtggtg caatttggct 1020

agctggcttt cattactgtt tttcacacat taactttggc ctcaacttga caactcaaat 1080

aatatttata aatacagcca cacttaaaat ggtcccatta tgaaatacat atttaaatat 1140

ctatacgatg tgttaaaacc aagaaaatat ttgattcttc tctgatattt aagaattgaa 1200

ggtttgaggt agttacgtgt taggggcatt tatattcatg tttttagagt ttgcttatac 1260

aacttaatct ttccttttca gtgctttggg ctcctgggag ttaatggggc tggaaaatca 1320

tcaactttca agatgttaac aggagatacc actgttacca gaggagatgc tttccttaac 1380

aaaaataggt gagaaaagaa gtggcttgta ttttgctgca aagactttgt ttttaattta 1440

tttaaagaaa taggttgtta tttttgatta cagtggtatt tttagagttc ataaaaatgt 1500

tgaaatatag taaagggtaa agaagcacat aaaatcatcc atgatttcaa tatctagaga 1560

taatcacaat ttacatttcc tttcagtctc attctcttct tttaacagct ttattcaggt 1620

ataatttaca tacaatataa tttgcttgtt ttttaagagt ataatttagt gatttttggt 1680

aaattgagag ttttgcaacc atcaccacaa tccagtttta gaacttttcc atcaccccac 1740

atctgtctta tatacacata taaatgtgcc atacaattga gatcatactg tatgtagaat 1800

ttaaaattag tttttattgt taatgagtgt attatgaata tttcccagtg ggttacattt 1860

cctaagatgt ggaattttac attgctacat aaaatccccc tatgtacatg tacctataat 1920

ttatttaata aattccttat aaatgttgga cacattagtt tccatttttc actatgtaaa 1980

tatgtccctg tatacatctt ttattatttc ctcaggaaca attcctacaa agtaaattgc 2040

cctctctaaa gagcatacaa attgactgag ccaccgttag gccattttct gagactgcac 2100

aggtcacaaa gcaatctgat ctttgggaat acagctacat tttataggct tcttagataa 2160

tgttactcta agtactttaa atatgtgggg cttctctggg cttttttttt tttgagacgg 2220

agtttcactc ttactgccca ggctggagag caatggcgcg accttggctc actgcaacct 2280

ccgcctccca ggttcaagcg attctcctgc ctcagcctcc tgagtagctg agattacagg 2340

tgcccgccac aatgcctgcc taattttttt gtattttcag tagagatggg gtttcaccat 2400

gttggccaga ctggtctcga gctcctgacc tcaggtgatc cacctgcctc agcctcccaa 2460

agttctggga ttacaggcat gagccactgc gcccggcttc tctggactta ttatgtggag 2520

agatagtaca aggcagtggc tttcagagtt ttttgaccat gaccgttgtg ggaaatacat 2580

tttatatctc aacctagtat gtacacacag acatgtagac acatgtataa cctaaagttt 2640

cataaagcag tacctactgt tactaattgt agtgcactct gctatttctt attctacctt 2700

atactgcgtc attaaaaaag tgctggtcat gacccactaa atttatttcc caaaccacta 2760

atgaacaatg actcacaatt tgaacacact ggacaggggg atagccaata aaattgaaaa 2820

gagcaaggaa attaatgtat tcatgatctc ctctcctgtc tcttacattt ttgcagtagc 2880

aatgtaaagg aatcctaaga gaacagacat tctgggaata gcaggcctag cgctgcacaa 2940

ctgctttcct aggcttgctc ctagtaccaa gctcctgacg catatagcag tggcagtaat 3000

aaccagccca tagtaaggtt tgtcacaggg actggttgta agaactgatt tgrttggtat 3060

agctgtgagg gcctggcacg gtgtccacgt gtgcctcaat cctaattctg aaaaaggctg 3120

accctggggg tgctaattag atacacagag aggaatgaat gctgccagaa ggccaagttc 3180

atggcaatgc cgctgtggct gaggtgcagt catcagtctg gaacgtgaac actgaacttc 3240

tctcacatgt gattcttcac ttgactggct tcatagaacc ccaaagccac cccaccacca 3300

cataaattgt gtctctaggt tctgtgttgc tcacactcaa aatttctggg ccttctcatt 3360

tggtgcatgt gaatggtgca tatgagtgaa gtctaggatg gggccttagc gttaaagccc 3420

tggggtagtg tgactgagat tgttggtaaa gaatgtgcag tggttggcat gacctcagaa 3480

attctgaaat gggactgcac ctgcagactg aagtgttcag agagccaggg aggtgcaagg 3540

actggggagg gtagaggcag gaaccctgcc tgccaggaag agctagcatc ctgggggcag 3600

aaaggctgtg ctttcaagta gcagcagatg tattggtatc tttgtaatgg agaagcatac 3660

tttacaggaa cattaggcca gattgtctaa ccagagtatc tctacctgct taaaatctaa 3720

gtagttttct tgtcctttgc agtatcttat caaacatcca tgaagtacat cagaacatgg 3780

gctactgccc tcagtttgat gccatcacag agctgttgac tgggagagaa cacgtggagt 3840

tctttgccct tttgagagga gtcccagaga aagaagttgg caaggtactg tgggcacctg 3900

aaagccagcc tgtctccttt ggcatcctga caatatatac cttatggctt ttccacacgc 3960

attgacttca ggctgttttt cctcatgaat gcagcagcac aaaatgctgg ttctttgtat 4020

ctgctttcag ggtggaaacc tgtaacggtg gtggggcagg gctgggtggg cagagaggga 4080

gtgctgctcc caccacacga gtcccttctc cctgctttgg ctcctcacca gttgtcaggt 4140

tatgattata gaatctagtc ctactcagtg aaagaacttt catacatgta tgtgtaggac 4200

agcatgataa aattcccaag ccagaccaaa gtcaaggtgc tttttatcac tgtaggttgg 4260

tgagtgggcg attcggaaac tgggcctcgt gaagtatgga gaaaaatatg ctggtaacta 4320

tagtggaggc aacaaacgca agctctctac agccatggct ttgatcggcg ggcctcctgt 4380

ggtgtttctg gtgagtataa ctgtggatgg aaaactgttg ttctggcctg agtggaaaac 4440

atgactgttc aaaagtccta tatgtccagg gctgttgtat gattggcttg tcttccccca 4500

gggacagcag agcaaccttg gaaaagcaga gggaagcttc tcccttggca cacactgggg 4560

tggctgtacc atgcctgcag atgctcccaa atagaggcac tccaagcact ttgtttctta 4620

gcgtgattga ggctggatat gtgatttgat ctttctctgg aacattcttt ctaatcatct 4680

ttgtgttcat tccctgaaaa tgaagagtgt ggacacagct ttaaaatccc caaggtagca 4740

actaggtcat agttccttac acacggatag atgaaaaaca gatcagactg ggaagtggcc 4800

cttgaccttt tttcttctgt agataagagc attgatgtta ttacgggaag aagcctttga 4860

ggcttttatg tattccacct cggtctggaa tttgtttctg taaggctaac agttgcaata 4920

tactagggta atctgagtga gctggaatta aaaaaaaaaa ggaatttcac cccaatctta 4980

tactgacttc aatagaggtt tcagacaaaa agttgttttg tat 5023

<210> SEQ ID NO 29

<211> LENGTH: 5138

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)...(5138)

<223> OTHER INFORMATION: n = a, t, c, or g

<400> SEQUENCE: 29

gccnngttn aaaangaaaa tttnnnnnaa attnaanntt annggngnnn tttccccaga 60

aaaacnaaa angatttccn cccngggggg ncccccnant cnaaaaggcc ccncttnttt 120

nggngaggg aaagnttttt ttggaatttt taatttttgg tcccccaaaa cctattattg 180

gaatttaat tacataaaaa agtactcaga atatttgagt ttcctgcatc aataagacat 240

tataataat gaccttgttt acaaatgaat ttgaaagtta ctctaattct ttgattcatc 300

agaaataac tagaatggca agttaaaatt taagctgttt caaagatgct tctgcattta 360

aaacaaatt tatctttgat tttttttccc cccagcaaat aagacttatt ttattctaat 420

acaggatga acccaccaca ggcatggatc ccaaagcccg gcggttcttg tggaattgtg 480

cctaagtgt tgtcaaggag gggagatcag tagtgcttac atctcatagg tccgtagtaa 540

gtcttgggt tcctcactgt gggatgtttt aactttccaa gtagaatatg cgatcatttt 600

taaaaatta gaaaatacag aaaagcaaag agtaaaacaa ttattacctg aaattatata 660

gcatattct tacaaaaatg caagcccagt ataaatactg ctctttttca cttaatatat 720

gtaaacatt attccaagtc agtgcattta ggtgtcattt cttatagctg gatagtattc 780

attaggata tactcttatt taactattcc cccttttgta gacatttgga ttatttccaa 840

ttgttcaca attgtaaaca ccactacact gaacagcatc atccctatat ccacatgtac 900

tgtaacaga atacaattcc ctaggaagct ggaatgctgg aagtcatggt gatgttctca 960

ggttacaga gaatctctct aaaactaaaa cctctttctg ttttaccgca gtatggaaga 1020

tgtgaagct ctttgcacta ggatggcaat catggtcaat ggaaggttca ggtgccttgg 1080

agtgtccag catctaaaaa ataggtaata aagataattt ctttgggata gtgcctagtg 1140

gaaggcttg atatttattc ttttgtgagt atataaatgg tgcctctaaa ataaagggaa 1200

taaaactga gcaaaacagt atagtggaaa gaatgagggc tttgaagtcc gaactgcatt 1260

aaattctgt ctttaccatt tactggttct gtgactcttg ggcaagttac ttaactactg 1320

aagagttag tttccctgga agatctacct cctagctttg tgctatagat gaaatgaaaa 1380

aatttacat gtgccagtac tggtgagagc gcaagctttg gagtcaaaca caaatgggtt 1440

gcatcctgg ccctaccaat tatgagctct gagccatggg caagtgacta actccctggg 1500

ctcagtttc tctgtaacat ctgtcagact tcatgggtcc aggtgaggat taaaggagat 1560

atgtattta cagcacatgg catggtgctt cacataaaat aagtatttag taaatgataa 1620

tggttcctt ctctcagaaa cttatttctg ggcctgccag gggccgccct ttttcatggc 1680

caagttggg ttcccagggt tcagtattct tttaaatagt tttctggaga tcctccattt 1740

ggtattttt tcctgctttc aggtttggag atggttatac aatagttgta cgaatagcag 1800

gtccaaccc ggacctgaag cctgtccagg atttctttgg acttgcattt cctggaagtg 1860

tcyaaaaga gaaacaccgg aacatgctac aataccagct tccatcttca ttatcttctc 1920

ggccaggat attcagcatc ctctcccaga gcaaaaagcg actccacata gaagactact 1980

tgtttctca gacaacactt gaccaagtaa gctttgagtg tcaaaacaga tttacttctc 2040

gggtgtgga ttcctgcccc gacactcccg cccataggtc caagagcagt ttgtatcttg 2100

attggtgct tgaattcctg atctactatt cctagctatg ctttttacta aacctctctg 2160

acctgaaaa gggagatgat gcctatgtac tctataggat tattgtgaga atttactgta 2220

taataacca taaaaactac catttagtga gcacctacca tgggccaggc attttacttg 2280

tgcctaatc ctatttaaat tagataaaaa agtaccaaat aggtcctgac acttaagaag 2340

actcagtaa atattttctt ccctcttccc tttaatcaag accgtatgtg ccaaagtaaa 2400

ggatgactg agcagttggt gatgtagggg tggggggcga tatagaaagt cagtttttgg 2460

cgggcgtgg tggctcatgc ctgtaatccc agcactttgg gaggctgagg agcaggcaga 2520

catgaggtc aggagatcca gataatcctg gccaacaggg tgaaaccccg tctctactaa 2580

aatacaaaa attagctggg catggtggtg cgcacttgta gtcccagcta cttgcgaggc 2640

gaggcagga gaattgctcg aacccaggag gtggaggtta cagtgagcca aggtctcgcc 2700

ctgcactcc agcctgggga cagagcaaga ccccatttca aggggggaaa aaaagtctat 2760

tttaagttg ttattgcttt tttcaagtat tcttccctcc ttcacacaca gttttctagt 2820

aatccattt atgtaattct gtatgctcct acttgaccta atttcaacat ctggaaaaat 2880

gaactagaa taaagaatga gcaagttgag tggtatttat aaaggtccat cttaatcttt 2940

aacaggtat ttgtgaactt tgccaaggac caaagtgatg atgaccactt aaaagacctc 3000

cattacaca aaaaccagac agtagtggac gttgcagttc tcacatcttt tctacaggat 3060

agaaagtga aagaaagcta tgtatgaaga atcctgttca tacggggtgg ctgaaagtaa 3120

gaggaacta gactttcctt tgcaccatgt gaagtgttgt ggagaaaaga gccagaagtt 3180

atgtgggaa gaagtaaact ggatactgta ctgatactat tcaatgcaat gcaattcaat 3240

caatgaaaa caaaattcca ttacaggggc agtgcctttg tagcctatgt cttgtatggc 3300

ctcaagtga aagacttgaa tttagttttt tacctatacc tatgtgaaac tctattatgg 3360

acccaatgg acatatgggt ttgaactcac actttttttt ttttttttgt tcctgtgtat 3420

ctcattggg gttgcaacaa taattcatca agtaatcatg gccagcgatt attgatcaaa 3480

tcaaaaggt aatgcacatc ctcattcact aagccatgcc atgcccagga gactggtttc 3540

cggtgacac atccattgct ggcaatgagt gtgccagagt tattagtgcc aagtttttca 3600

aaagtttga agcaccatgg tgtgtcatgc tcacttttgt gaaagctgct ctgctcagag 3660

ctatcaaca ttgaatatca gttgacagaa tggtgccatg cgtggctaac atcctgcttt 3720

attccctct gataagctgt tctggtggca gtaacatgca acaaaaatgt gggtgtctcc 3780

ggcacggga aacttggttc cattgttata ttgtcctatg cttcgagcca tgggtctaca 3840

ggtcatcct tatgagactc ttaaatatac ttagatcctg gtaagaggca aagaatcaac 3900

gccaaactg ctggggctgc aactgctgaa gccagggcat gggattaaag agattgtgcg 3960

tcaaaccta gggaagcctg tgcccatttg tcctgactgt ctgctaacat ggtacactgc 4020

tctcaagat gtttatctga cacaagtgta ttatttctgg ctttttgaat taatctagaa 4080

atgaaaaga tggagttgta ttttgacaaa aatgtttgta ctttttaatg ttatttggaa 4140

tttaagttc tatcagtgac ttctgaatcc ttagaatggc ctctttgtag aaccctgtgg 4200

atagaggag tatggccact gcccactatt tttattttct tatgtaagtt tgcatatcag 4260

catgactag tgcctagaaa gcaatgtgat ggtcaggatc tcatgacatt atatttgagt 4320

tctttcaga tcatttagga tactcttaat ctcacttcat caatcaaata ttttttgagt 4380

tatgctgta gctgaaagag tatgtacgta cgtataagac tagagagata ttaagtctca 4440

tacacttcc tgtgccatgt tattcagctc actggtttac aaatataggt tgtcttgtgg 4500

tgtaggagc ccactgtaac aatactgggc agcctttttt tttttttttt taattgcaac 4560

atgcaaaag ccaagaaagt ttaagggtca caagtctaaa caatgaattc ttcaacaggg 4620

aaacagcta gcttgaaaac ttgctgaaaa acacaacttg tgtttatggc atttagtacc 4680

tcaaataat tggctttgca gatattggat accccattaa atctgacagt ctcaaatttt 4740

catctcttc aatcactagt caagaaaaaa tataaaaaca acaaatactt ccatatggag 4800

atttttcag agttttctaa cccagtctta tttttctagt cagtaaacat ttgtaaaaat 4860

ctgtttcac taatacttac tgttaactgt cttgagagaa aagaaaaata tgagagaact 4920

ttgtttggg gaagttcaag tgatctttca atatcattac taacttcttc cactttttcc 4980

gaatttgaa tattaacgct aaaggtgtaa gacttcagat ttcaaattaa tctttctata 5040

tttttaaat ttacagaata ttatataacc cactgctgaa aaagaaacaa atgattgttt 5100

agaagttaa aggtcaatat tgattttaaa atattaag 5138

<210> SEQ IDNO 30

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 30

gtgttcctgc agagggcatg 20

<210> SEQ ID NO 31

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 31

cacttccagt aacagctgac 20

<210> SEQ ID NO 32

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 32

ctttgcgcat gtccttcatg c 21

<210> SEQ ID NO 33

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 33

gacatcagcc ctcagcatct t 21

<210> SEQ ID NO 34

<211> LENGTH: 19

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 34

caacaagcca tgttccctc 19

<210> SEQ ID NO 35

<211> LENGTH: 18

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 35

catgttccct cagccagc 18

<210> SEQ ID NO 36

<211> LENGTH: 19

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 36

cagagctcac agcagggac 19

<210> SEQ ID NO 37

<211> LENGTH: 21

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 37

Cys Ser Val Arg Leu Ser Tyr Pro Pro Tyr Glu Gln His Glu Cys His

1 5 10 15

Phe Pro Asn Lys Ala

20

<210> SEQ ID NO 38

<211> LENGTH: 14

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 38

gcctgtgtgt cccc 14

<210> SEQ ID NO 39

<211> LENGTH: 14

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)...(14)

<223> OTHER INFORMATION: n = t or c

<400> SEQUENCE: 39

gcctgtgngt cccc 14

<210> SEQ ID NO 40

<211> LENGTH: 45

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 40

aagaagatgc tgcctgtgtg tcccccaggg gcaggggggc tgcct 45

<210> SEQ ID NO 41

<211> LENGTH: 15

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 41

Lys Lys Met Leu Pro Val Cys Pro Pro Gly Ala Gly Gly Leu Pro

1 5 10 15

<210> SEQ ID NO 42

<211> LENGTH: 15

<212> TYPE: PRT

<213> ORGANISM: Mus musculus

<400> SEQUENCE: 42

Lys Lys Met Leu Pro Val Cys Pro Pro Gly Ala Gly Gly Leu Pro

1 5 10 15

<210> SEQ ID NO 43

<211> LENGTH: 15

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 43

Lys Lys Met Leu Pro Val Arg Pro Pro Gly Ala Gly Gly Leu Pro

1 5 10 15

<210> SEQ ID NO 44

<211> LENGTH: 5

<212> TYPE: PRT

<213> ORGANISM: Caenorhabditis elegans

<400> SEQUENCE: 44

Leu Leu Gly Gly Ser

1 5

<210> SEQ ID NO 45

<211> LENGTH: 45

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 45

aagaagatgc tgcctgtgcg tcccccaggg gcaggggggc tgcct 45

<210> SEQ ID NO 46

<211> LENGTH: 14

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 46

gcctacttgc agga 14

<210> SEQ ID NO 47

<211> LENGTH: 14

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 47

gcctacttgc ggga 14

<210> SEQ ID NO 48

<211> LENGTH: 45

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 48

tgggggggct tcgcctactt gcaggatgtg gtggagcagg caatc 45

<210> SEQ ID NO 49

<211> LENGTH: 15

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 49

Trp Gly Gly Phe Ala Tyr Leu Gln Asp Val Val Glu Gln Ala Ile

1 5 10 15

<210> SEQ ID NO 50

<211> LENGTH: 15

<212> TYPE: PRT

<213> ORGANISM: Mus musculus

<400> SEQUENCE: 50

Trp Gly Gly Phe Ala Tyr Leu Gln Asp Val Val Glu Gln Ala Ile

1 5 10 15

<210> SEQ ID NO 51

<211> LENGTH: 15

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 51

Trp Gly Gly Phe Ala Tyr Leu Arg Asp Val Val Glu Gln Ala Ile

1 5 10 15

<210> SEQ ID NO 52

<211> LENGTH: 12

<212> TYPE: PRT

<213> ORGANISM: Caenorhabditis elegans

<400> SEQUENCE: 52

Phe Met Thr Val Gln Arg Ala Val Asp Val Ala Ile

1 5 10

<210> SEQ ID NO 53

<211> LENGTH: 45

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 53

tgggggggct tcgcctactt gcgggatgtg gtggagcagg caatc 45

<210> SEQ ID NO 54

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)...(25)

<223> OTHER INFORMATION: n is a, t, c, or g.

<400> SEQUENCE: 54

tcattcctct tgtnngcncn gnncn 25

<210> SEQ ID NO 55

<211> LENGTH: 45

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 55

agtagcctca ttcctcttct tgtgagcgct ggcctgctag tggtc 45

<210> SEQ ID NO 56

<211> LENGTH: 15

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 56

Ser Ser Leu Ile Pro Leu Leu Val Ser Ala Gly Leu Leu Val Val

1 5 10 15

<210> SEQ ID NO 57

<211> LENGTH: 15

<212> TYPE: PRT

<213> ORGANISM: Mus musculus

<400> SEQUENCE: 57

Ser Ser Leu Ile Pro Leu Leu Val Ser Ala Gly Leu Leu Val Val

1 5 10 15

<210> SEQ ID NO 58

<211> LENGTH: 14

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 58

Ser Ser Leu Ile Pro Leu Val Ser Ala Gly Leu Leu Val Val

1 5 10

<210> SEQ ID NO 59

<211> LENGTH: 15

<212> TYPE: PRT

<213> ORGANISM: Caenorhabditis elegans

<400> SEQUENCE: 59

Ile Asn Tyr Ala Lys Leu Thr Phe Ala Val Ile Val Leu Thr Ile

1 5 10 15

<210> SEQ ID NO 60

<211> LENGTH: 42

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 60

agtagcctca ttcctcttgt gagcgctggc ctgctagtgg tc 42

<210> SEQ ID NO 61

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)...(25)

<223> OTHER INFORMATION: n is a, t, c, or g.

<400> SEQUENCE: 61

tgatgaagat gananncngn ngcga 25

<210> SEQ ID NO 62

<211> LENGTH: 36

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 62

aatgatgaag atgaagatgt gaggcgggaa agacag 36

<210> SEQ ID NO 63

<211> LENGTH: 12

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 63

Asn Asp Glu Asp Glu Asp Val Arg Arg Glu Arg Gln

1 5 10

<210> SEQ ID NO 64

<211> LENGTH: 12

<212> TYPE: PRT

<213> ORGANISM: Mus musculus

<400> SEQUENCE: 64

Asn Asp Glu Asp Glu Asp Val Arg Arg Glu Arg Gln

1 5 10

<210> SEQ ID NO 65

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 65

Asn Asp Glu Asp Val Arg Arg Glu Arg Gln

1 5 10

<210> SEQ ID NO 66

<211> LENGTH: 15

<212> TYPE: PRT

<213> ORGANISM: Caenorhabditis elegans

<400> SEQUENCE: 66

Asp Glu Arg Asp Val Glu Asp Ser Asp Val Ile Ala Glu Lys Ser

1 5 10 15

<210> SEQ ID NO 67

<211> LENGTH: 30

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 67

aatgatgaag atgtgaggcg ggaaagacag 30

<210> SEQ ID NO 68

<211> LENGTH: 14

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 68

agttgtacga atag 14

<210> SEQ ID NO 69

<211> LENGTH: 14

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)...(14)

<223> OTHER INFORMATION: n is t or c.

<400> SEQUENCE: 69

agttgtanga atag 14

<210> SEQ ID NO 70

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 70

ggctggatta gcagtcctca 20

<210> SEQ ID NO 71

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 71

ggatttccca gatcccagtg 20

<210> SEQ ID NO 72

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 72

gacagacttg gcatgaagca 20

<210> SEQ ID NO 73

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 73

gcacttggca gtcacttctg 20

<210> SEQ ID NO 74

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 74

cgtttctcca ctgtcccatt 20

<210> SEQ ID NO 75

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 75

acttcaagga cccagcttcc 20

<210> SEQ ID NO 76

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 76

tcggtttctt gtttgttaaa ctca 24

<210> SEQ ID NO 77

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 77

tcccaaggct ttgagatgac 20

<210> SEQ ID NO 78

<211> LENGTH: 19

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 78

ggctccaaag cccttgtaa 19

<210> SEQ ID NO 79

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 79

gctgctgtga tggggtatct 20

<210> SEQ ID NO 80

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 80

tttgtaaatt ttgtagtgct cctca 25

<210> SEQ ID NO 81

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 81

tagtcagccc ttgcctccta 20

<210> SEQ ID NO 82

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 82

aaaggggctt ggtaagggta 20

<210> SEQ ID NO 83

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 83

gatgtggtgc tccctctagc 20

<210> SEQ ID NO 84

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 84

caagtgagtg cttgggattg 20

<210> SEQ ID NO 85

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 85

gcaaattcaa atttctccag g 21

<210> SEQ ID NO 86

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 86

tcaaggagga aatggacctg 20

<210> SEQ ID NO 87

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 87

ctgaaagttc aagcgcagtg 20

<210> SEQ ID NO 88

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 88

tgcagactga atggagcatc 20

<210> SEQ ID NO 89

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 89

gccaggggac actgtattct 20

<210> SEQ ID NO 90

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 90

aggtcctctg ccttcactca 20

<210> SEQ ID NO 91

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 91

ccagtgctta cccctgctaa 20

<210> SEQ ID NO 92

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 92

cacacaacag agcttcttgg a 21

<210> SEQ ID NO 93

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 93

acctggaaca ggtgtggtgt 20

<210> SEQ ID NO 94

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 94

gggctaacat gccactcagt a 21

<210> SEQ ID NO 95

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 95

gtttgttgca gatggggaag 20

<210> SEQ ID NO 96

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 96

caccagaaga aggagcatgg 20

<210> SEQ ID NO 97

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 97

ctggactcgt agggatttgc 20

<210> SEQ ID NO 98

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 98

gcctgtcaca gagaaatgct t 21

<210> SEQ ID NO 99

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 99

ttacggaatg atcctgtgct c 21

<210> SEQ ID NO 100

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 100

agtcaggttt ccggtcacac 20

<210> SEQ ID NO 101

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 101

ccgttcctta tatcctcagg tg 22

<210> SEQ ID NO 102

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 102

ccttgtacac actcgcactg a 21

<210> SEQ ID NO 103

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 103

tgttgtccac aggttccaga 20

<210> SEQ ID NO 104

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 104

tgaggtttat gggcatggtt 20

<210> SEQ ID NO 105

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 105

atgtttttcc ttggctgtgc 20

<210> SEQ ID NO 106

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 106

atctgccctt tcttgtctga 20

<210> SEQ ID NO 107

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 107

agggagctgc acagtggata 20

<210> SEQ ID NO 108

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 108

tcactcccat atttcagaac ttga 24

<210> SEQ ID NO 109

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 109

tgtttattgg aagatcggtg aa 22

<210> SEQ ID NO 110

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 110

cgttagagac tgaatctttg tcctg 25

<210> SEQ ID NO 111

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 111

agtcctgcct tccacagttg 20

<210> SEQ ID NO 112

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 112

ggtagttacg tgttaggggc a 21

<210> SEQ ID NO 113

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 113

caggaacatt aggccagatt g 21

<210> SEQ ID NO 114

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 114

catgtatgtg taggacagca tga 23

<210> SEQ ID NO 115

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 115

ctgtttcaaa gatgcttctg c 21

<210> SEQ ID NO 116

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 116

cctaggaagc tggaatgctg 20

<210> SEQ ID NO 117

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 117

gggttcccag ggttcagtat 20

<210> SEQ ID NO 118

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 118

cttgacctaa tttcaacatc tgg 23

<210> SEQ ID NO 119

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 119

atccccaact caaaaccaca 20

<210> SEQ ID NO 120

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 120

aagtccaatt tagcccacgt t 21

<210> SEQ ID NO 121

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 121

ccagccattc aaaattctcc 20

<210> SEQ ID NO 122

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 122

ggtgcaggtc aatttccaat 20

<210> SEQ ID NO 123

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 123

ccccttcacc accattacaa 20

<210> SEQ ID NO 124

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 124

tgtccaagga aaagcctcac 20

<210> SEQ ID NO 125

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 125

aggacctctt gccagactca 20

<210> SEQ ID NO 126

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 126

aggagatgac acaggccaag 20

<210> SEQ ID NO 127

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 127

cgcacacctc tgaagctacc 20

<210> SEQ ID NO 128

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 128

acctcactca cacctgggaa 20

<210> SEQ ID NO 129

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 129

gcctcctgcc tgaaccttat 20

<210> SEQ ID NO 130

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 130

caaaatcatg acaccaagtt gag 23

<210> SEQ ID NO 131

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 131

catgcacatg cacacacata 20

<210> SEQ ID NO 132

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 132

ccttagcccg tgttgagcta 20

<210> SEQ ID NO 133

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 133

tgcttttatt cagggactcc a 21

<210> SEQ ID NO 134

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 134

cccatgcact gcagagattc 20

<210> SEQ ID NO 135

<211> LENGTH: 19

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 135

aaggcaggag acatcgctt 19

<210> SEQ ID NO 136

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 136

gggatcagca tggtttccta 20

<210> SEQ ID NO 137

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 137

gcttaagtcc cactcctccc 20

<210> SEQ ID NO 138

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 138

attttcctcc gcatgtgtgt 20

<210> SEQ ID NO 139

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 139

tcacagaagc ctagccatga 20

<210> SEQ ID NO 140

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 140

aacagagcag ggagatggtg 20

<210> SEQ ID NO 141

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 141

tctgcacctc tcctcctctg 20

<210> SEQ ID NO 142

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 142

actggggcca acattaatca 20

<210> SEQ ID NO 143

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 143

cttccccatc tgcaacaaac 20

<210> SEQ ID NO 144

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 144

gctaaaggcc atccaaagaa 20

<210> SEQ ID NO 145

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 145

tcaagtgcat ctgggcataa 20

<210> SEQ ID NO 146

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 146

tctgaagtcc attcccttgg 20

<210> SEQ ID NO 147

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 147

caatgtggca tgcagttgat 20

<210> SEQ ID NO 148

<211> LENGTH: 19

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 148

gaagctacca gcccatcct 19

<210> SEQ ID NO 149

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 149

catttccccc actgtttcag 20

<210> SEQ ID NO 150

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 150

ccaaggcttt cttcaatcca 20

<210> SEQ ID NO 151

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 151

gatccgttta acctgccaac 20

<210> SEQ ID NO 152

<211> LENGTH: 19

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 152

atgcccctgc caactttac 19

<210> SEQ ID NO 153

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 153

ctctgcagct gttcccctac 20

<210> SEQ ID NO 154

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 154

tatcaatcca tggccctgac 20

<210> SEQ ID NO 155

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 155

agagtccctg ccctccttct 20

<210> SEQ ID NO 156

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 156

aaggcagtca gcagtgtcaa 20

<210> SEQ ID NO 157

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 157

ggggaacatc ctgtgcttag 20

<210> SEQ ID NO 158

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 158

ccattggtga gtgtttccct 20

<210> SEQ ID NO 159

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 159

agtcagcaaa ctgctgggtt 20

<210> SEQ ID NO 160

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 160

attgctccat cctggcataa 20

<210> SEQ ID NO 161

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 161

tcatggatga ttttatgtgc ttc 23

<210> SEQ ID NO 162

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 162

gcgtgtggaa aagccataag 20

<210> SEQ ID NO 163

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 163

gccaatcata caacagccct 20

<210> SEQ ID NO 164

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 164

tgatcgcata ttctacttgg aaa 23

<210> SEQ ID NO 165

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 165

tccctttatt ttagaggcac ca 22

<210> SEQ ID NO 166

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 166

gatcaggaat tcaagcacca a 21

<210> SEQ ID NO 167

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 167

tgggttccat aatagagttt caca 24

<210> SEQ ID NO 168

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 168

tgtcagctgt tactggaagt gg 22

<210> SEQ ID NO 169

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 169

tgtcagctgc tgctggaagt gg 22

<210> SEQ ID NO 170

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 170

aggagctggc cgaagccaca a 21

<210> SEQ ID NO 171

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 171

aggagctggc tgaagccaca a 21

<210> SEQ ID NO 172

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 172

aatgatgcca ccaaacaaat g 21

<210> SEQ ID NO 173

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 173

aatgatgcca tcaaacaaat g 21

<210> SEQ ID NO 174

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 174

gaggtggctc cgatgaccac a 21

<210> SEQ ID NO 175

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 175

gaggtggctc tgatgaccac a 21

<210> SEQ ID NO 176

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 176

ttccttaaca gaaatagtat c 21

<210> SEQ ID NO 177

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 177

ttccttaaca aaaatagtat c 21

<210> SEQ ID NO 178

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 178

ggaagtgttc caaaagagaa a 21

<210> SEQ ID NO 179

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 179

ggaagtgttc taaaagagaa a 21

<210> SEQ ID NO 180

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 180

agtaaagagg gactagactt t 21

<210> SEQ ID NO 181

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 181

agtaaagagg aactagactt t 21

<210> SEQ ID NO 182

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 182

gcctacttgc aggatgtggt g 21

<210> SEQ ID NO 183

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 183

gcctacttgc gggatgtggt g 21

<210> SEQ ID NO 184

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 184

cctcattcct cttcttgtga gcg 23

<210> SEQ ID NO 185

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 185

cctcattcct cttgtgagcg 20

<210> SEQ ID NO 186

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 186

gcaggactac gtgggcttca c 21

<210> SEQ ID NO 187

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 187

gcaggactac atgggcttca c 21

<210> SEQ ID NO 188

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 188

aaaagtctac cgagatggga t 21

<210> SEQ ID NO 189

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 189

aaaagtctac tgagatggga t 21

<210> SEQ ID NO 190

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 190

ggccagatca cctccttcct g 21

<210> SEQ ID NO 191

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 191

ggccagatca tctccttcct g 21

<210> SEQ ID NO 192

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 192

acacaccaca tggatgaagc g 21

<210> SEQ ID NO 193

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 193

acacaccaca cggatgaagc g 21

<210> SEQ ID NO 194

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 194

cctggaagaa gtaagttaag t 21

<210> SEQ ID NO 195

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 195

cctggaagaa ctaagttaag t 21

<210> SEQ ID NO 196

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 196

gctgcctgtg tgtcccccag g 21

<210> SEQ ID NO 197

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 197

gctgcctgtg cgtcccccag g 21

<210> SEQ ID NO 198

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 198

tagccattat ggaattactg ct 22

<210> SEQ ID NO 199

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 199

tagccattat caattactgc t 21

<210> SEQ ID NO 200

<211> LENGTH: 26

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 200

gatgaagatg aagatgtgag gcggga 26

<210> SEQ ID NO 201

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 201

gatgaagatg tgaggcggga 20

<210> SEQ ID NO 202

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 202

aatagttgta cgaatagcag g 21

<210> SEQ ID NO 203

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 203

aatagttgta tgaatagcag g 21

<210> SEQ ID NO 204

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 204

acacgctggg ggtgctggct g 21

<210> SEQ ID NO 205

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 205

acacgctggg cgtgctggct g 21

<210> SEQ ID NO 206

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 206

gaccagccac ggcgtccctg 20

<210> SEQ ID NO 207

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 207

gaccagccac gggcgtccct g 21

<210> SEQ ID NO 208

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 208

cattttctta gaaaagagag gt 22

<210> SEQ ID NO 209

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 209

cattttctta gagaagagag gt 22

<210> SEQ ID NO 210

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 210

gaaaattagt atgtaaggaa g 21

<210> SEQ ID NO 211

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 211

gaaaattagt ctgtaaggaa g 21

<210> SEQ ID NO 212

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 212

cctccgcctg ccaggttcag cgatt 25

<210> SEQ ID NO 213

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 213

cctccgcctg ccgggttcag cgatt 25

<210> SEQ ID NO 214

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 214

tatgtgctga ccatgggagc ttgtt 25

<210> SEQ ID NO 215

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 215

tatgtgctga ccgtgggagc ttgtt 25

<210> SEQ ID NO 216

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 216

gtgacaccca acggagtagg g 21

<210> SEQ ID NO 217

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 217

gtgacaccca gcggagtagg g 21

<210> SEQ ID NO 218

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 218

agtatccctt gttcacgaga a 21

<210> SEQ ID NO 219

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 219

agtatccctc ccttgttcac gagaa 25

<210> SEQ ID NO 220

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 220

ctgggttcct gtatcacaac c 21

<210> SEQ ID NO 221

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 221

ctgggttcct atatcacaac c 21

<210> SEQ ID NO 222

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 222

ggcctaccaa gggagaaact g 21

<210> SEQ ID NO 223

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 223

ggcctaccaa aggagaaact g 21

<210> SEQ ID NO 224

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 224

tttaaagggg gtgattagga 20

<210> SEQ ID NO 225

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 225

tttaaagggg ttgattagga 20

<210> SEQ ID NO 226

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 226

gaagaaattt gtttttttga tt 22

<210> SEQ ID NO 227

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 227

gaagaaattt ttttttttga tt 22

<210> SEQ ID NO 228

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 228

gcgggcatcc cgagggaggg g 21

<210> SEQ ID NO 229

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 229

gcgggcatcc tgagggaggg g 21

<210> SEQ ID NO 230

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 230

agggaggggg gctgaagatc a 21

<210> SEQ ID NO 231

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 231

agggaggggg actgaagatc a 21

<210> SEQ ID NO 232

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 232

aggagccaaa cgctcattgt 20

<210> SEQ ID NO 233

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 233

aggagccaaa gcgctcattg t 21

<210> SEQ ID NO 234

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 234

aagccactgt ttttaaccag t 21

<210> SEQ ID NO 235

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 235

aagccactgt atttaaccag t 21

<210> SEQ ID NO 236

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 236

cgtgggcttc acactcaaga t 21

<210> SEQ ID NO 237

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 237

cgtgggcttc ccactcaaga t 21

<210> SEQ ID NO 238

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 238

tcacactcaa gatcttcgct g 21

<210> SEQ ID NO 239

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 239

tcacactcaa catcttcgct g 21

<210> SEQ ID NO 240

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 240

gcagcctcac ccgctcttcc c 21

<210> SEQ ID NO 241

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 241

gcagcctcac tcgctcttcc c 21

<210> SEQ ID NO 242

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 242

agaagagaat atcagaaatc t 21

<210> SEQ ID NO 243

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 243

agaagagaat gtcagaaatc t 21

<210> SEQ ID NO 244

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 244

gcgcagtgcc ctgtgtcctt a 21

<210> SEQ ID NO 245

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 245

gcgcagtgcg ctgtgtcctt a 21

<210> SEQ ID NO 246

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 246

gatctaaggt tgtcattctg g 21

<210> SEQ ID NO 247

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 247

gatctaaggt ggtcattctg g 21

<210> SEQ ID NO 248

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 248

ctcttctgtt agcacagaag aga 23

<210> SEQ ID NO 249

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 249

ctcttctgtt atcacagaag aga 23

<210> SEQ ID NO 250

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 250

cattctaggg atcatagcca t 21

<210> SEQ ID NO 251

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 251

cattctaggg gtcatagcca t 21

<210> SEQ ID NO 252

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 252

aagtacagtg ggaggaacag cg 22

<210> SEQ ID NO 253

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 253

aagtacagtg tgaggaacag cg 22

<210> SEQ ID NO 254

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 254

attcctaaaa aatagaaatg ca 22

<210> SEQ ID NO 255

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 255

attcctaaaa agtagaaatg ca 22

<210> SEQ ID NO 256

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 256

ggcccctgcc ttattattac t 21

<210> SEQ ID NO 257

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 257

ggcccctgcc gtattattac t 21

<210> SEQ ID NO 258

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 258

tgagagaatt acttgaaccc gg 22

<210> SEQ ID NO 259

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 259

tgagagaatt gcttgaaccc gg 22

<210> SEQ ID NO 260

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 260

tttgctgaaa caatcactga c 21

<210> SEQ ID NO 261

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 261

tttgctgaaa taatcactga c 21

<210> SEQ ID NO 262

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 262

aacctcagtt ccctcatctg tg 22

<210> SEQ ID NO 263

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 263

aacctcagtt tcctcatctg tg 22

<210> SEQ ID NO 264

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 264

ctggacacca gaaataatgt c 21

<210> SEQ ID NO 265

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 265

ctggacacca aaaataatgt c 21

<210> SEQ ID NO 266

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 266

tcctatgtgt cctccaccaa t 21

<210> SEQ ID NO 267

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 267

tcctatgtgt gctccaccaa t 21

<210> SEQ ID NO 268

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 268

aagaagtggc ttgtattttg c 21

<210> SEQ ID NO 269

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 269

aagaagtggc ctgtattttg c 21

<210> SEQ ID NO 270

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 270

aactgatttg attggtatag ctg 23

<210> SEQ ID NO 271

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 271

aactgatttg gttggtatag ctg 23

<210> SEQ ID NO 272

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 272

cagggtccaa cccggacctg a 21

<210> SEQ ID NO 273

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 273

cagggtccaa tccggacctg a 21

<210> SEQ ID NO 274

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 274

ttgggaggct aaggcaggag aa 22

<210> SEQ ID NO 275

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 275

ttgggaggct gaggcaggag aa 22

<210> SEQ ID NO 276

<211> LENGTH: 15

<212> TYPE: DNA

<213> ORGANISM: Gallus gallus

<400> SEQUENCE: 276

accaggggaa tctcc 15

<210> SEQ ID NO 277

<211> LENGTH: 15

<212> TYPE: DNA

<213> ORGANISM: Gallus gallus

<400> SEQUENCE: 277

accagggaaa tctcc 15

<210> SEQ ID NO 278

<211> LENGTH: 45

<212> TYPE: DNA

<213> ORGANISM: Gallus gallus

<400> SEQUENCE: 278

cgctacccaa caccagggga atctcctggt attgttggaa acttc 45

<210> SEQ ID NO 279

<211> LENGTH: 15

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 279

Arg Tyr Pro Thr Pro Gly Glu Ala Pro Gly Val Val Gly Asn Phe

1 5 10 15

<210> SEQ ID NO 280

<211> LENGTH: 15

<212> TYPE: PRT

<213> ORGANISM: Mus musculus

<400> SEQUENCE: 280

Arg Tyr Pro Thr Pro Gly Glu Ala Pro Gly Val Val Gly Asn Phe

1 5 10 15

<210> SEQ ID NO 281

<211> LENGTH: 15

<212> TYPE: PRT

<213> ORGANISM: Gallus gallus

<400> SEQUENCE: 281

Arg Tyr Pro Thr Pro Gly Glu Ser Pro Gly Ile Val Gly Asn Phe

1 5 10 15

<210> SEQ ID NO 282

<211> LENGTH: 15

<212> TYPE: PRT

<213> ORGANISM: Gallus gallus

<400> SEQUENCE: 282

Arg Tyr Pro Thr Pro Gly Lys Ser Pro Gly Ile Val Gly Asn Phe

1 5 10 15

<210> SEQ ID NO 283

<211> LENGTH: 45

<212> TYPE: DNA

<213> ORGANISM: Gallus gallus

<400> SEQUENCE: 283

cgctacccaa caccagggaa atctcctggt attgttggaa acttc 45

<210> SEQ ID NO 284

<211> LENGTH: 19

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 284

gcgtcaggga tggggacag 19

<210> SEQ ID NO 285

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 285

gcgtcaggga ttggggacag 20

<210> SEQ ID NO 286

<211> LENGTH: 17

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 286

ccacttcggt ctccatg 17

<210> SEQ ID NO 287

<211> LENGTH: 17

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 287

ccacttcgat ctccatg 17

Claims

What is claimed is:

1. An isolated ABCA1 polypeptide having ABCA1 biological activity and comprising an amino acid sequence with at least 50% identity to SEQ ID NO: 1.

2. The isolated ABCA1 polypeptide of claim 1 wherein said ABCA1 polypeptide is at least 75% pure by weight.

3. The isolated ABCA1 polypeptide of claim 1 wherein said ABCA1 polypeptide is at least 90% pure by weight.

4. The isolated ABCA1 polypeptide of claim 1 wherein said ABCA1 polypeptide is at least 99% pure by weight.

5. The isolated ABCA1 polypeptide of claim 1 wherein said percent identity is at least 85%.

6. The isolated ABCA1 polypeptide of claim 1 wherein said percent identity is at least 90%.

7. The isolated ABCA1 polypeptide of claim 1 wherein said percent identity is at least 95%.

8. The isolated ABCA1 polypeptide of claim 1 wherein said polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

9. The isolated ABCA1 polypeptide of claim 1 wherein said ABCA1 polypeptide is a mammalian ABCA1.

10. The isolated ABCA1 polypeptide of claim 1 wherein said polypeptide is chicken ABCA1.

11. The isolated ABCA1 polypeptide of claim 9 wherein said polypeptide is mouse ABCA1.

12. The isolated ABCA1 polypeptide of claim 9 wherein said polypeptide is human ABCA1.

13. The isolated ABCA1 polypeptide of claim 1, wherein said polypeptide comprises amino acids 1 to 60 of SEQ ID NO: 1.

14. The isolated ABCA1 polypeptide of claim 1, wherein said polypeptide comprises amino acids 61 to 2261 of SEQ ID NO: 1.

15. The isolated ABCA1 polypeptide of claim 1, wherein said polypeptide comprises amino acids 1 to 2261 of SEQ ID NO: 1.

16. The isolated ABCA1 polypeptide of claim 1 wherein said polypeptide is a recombinant polypeptide.

17. The isolated ABCA1 polypeptide of claim 1 wherein said polypeptide is derived from a natural source.

18. The isolated ABCA1 polypeptide of claim 1 wherein said biological activity is lipid transport across a membrane.

19. The isolated ABCA1 polypeptide of claim 19 wherein said lipid is member selected from the group consisting of cholesterol and phospholipid.

20. The isolated ABCA1 polypeptide of claim 1 wherein said biological activity is transport of an ion across a membrane.

21. The isolated ABCA1 polypeptide of claim 1 wherein said biological activity is transport of interleukin-1 across a membrane.

22. An isolated ABCA1 polypeptide comprising amino acids 1 to 60 of SEQ ID NO: 1.

23. An isolated ABCA1 polypeptide comprising amino acids 61 to 2261 of SEQ ID NO: 1.

24. An isolated ABCA1 polypeptide comprising amino acids 1 to 2261 of SEQ ID NO: 1.

25. An isolated polynucleotide comprising a nucleotide sequence having at least 50% identity to SEQ ID NO: 1 and that encodes an ABCA1 polypeptide having ABCA1 biological activity.

26. The isolated polynucleotide of claim 25 wherein said polynucleotide is at least 75% pure by weight.

27. The isolated polynucleotide of claim 25 wherein said polynucleotide purity is at least 90% pure by weight.

28. The isolated polynucleotide of claim 25 wherein said polynucleotide is at least 99% pure by weight.

29. The isolated polynucleotide of claim 25 wherein said polynucleotide is at least 99% pure by weight.

30. The isolated polynucleotide of claim 25 wherein said percent identity is at least 85%.

31. The isolated polynucleotide of claim 25 wherein said percent identity is at least 90%.

32. The isolated polynucleotide of claim 25 wherein said percent identity is at least 95%.

33. The isolated polynucleotide of claim 25 wherein said polynucleotide acid comprises the nucleotide sequence of SEQ ID NO: 2.

34. The isolated polynucleotide of claim 25 wherein said polynucleotide acid hybridizes at high stringency conditions to nucleotides 75 to 254 of SEQ ID NO: 2.

35. The isolated polynucleotide of claim 25 wherein said polynucleotide comprises nucleotides 75 to 254 of SEQ ID NO: 2.

36. The isolated polynucleotide of claim 25 wherein said polynucleotide comprises nucleotides 255 to 6858 of SEQ ID NO: 2.

37. The isolated polynucleotide of claim 25 wherein said polynucleotide comprises nucleotides 75 to 6857 of SEQ ID NO: 2.

38. The isolated polynucleotide of claim 25 wherein said polynucleotide comprises nucleotides 75 to 254 of SEQ ID NO: 2.

39. The isolated polynucleotide of claim 25 wherein said polynucleotide comprises nucleotides 75 to 6857 of SEQ ID NO: 2.

40. The isolated polynucleotide of claim 25 that comprises nucleotides 255 to 6857 of SEQ ID NO: 2.

41. A substantially pure polynucleotide encoding the ABCA1 polypeptide of claim 1.

42. An expression vector comprising the polynucleotide of claim 25.

43. An expression vector comprising the polynucleotide of claim 41.

44. A recombinant cell genetically engineered to express the polynucleotide of claim 25.

45. The recombinant cell of claim 44 wherein said cell does not express said polynucleotide absent said genetic engineering.

46. A recombinant cell expressing the polynucleotide of claim 41.

47. The recombinant cell of claim 46 wherein said cell does not express said polynucleotide absent said genetic engineering.

48. A method of making an ABCA1 polypeptide comprising expressing said polypeptide from the recombinant cell of claim 44.

49. A method of making an ABCA1 polypeptide comprising expressing said polypeptide from the recombinant cell of claim 46.

50. A transgenic non-human animal that expresses the polynucleotide of claim 25.

51. A transgenic non-human animal that expresses the polynucleotide of claim 41.

52. A substantially pure polynucleotide comprising at least thirty consecutive nucleotides contained in nucleotides 7015-7860 of SEQ ID NO: 2.

53. The substantially pure polynucleotide of claim 52 wherein said polynucleotide comprises nucleotides 7015-7860 of SEQ ID NO: 2.

54. A substantially pure polynucleotide that hybridizes at high stringency to a probe comprising nucleotides 7015-7860 of SEQ ID NO: 2.

55. An oligonucleotide comprising nucleotides 75 to 254 of SEQ ID NO: 2.

56. The polynucleotide of claim 25 wherein said polynucleotide is a cDNA.

57. The polynucleotide of claim 41 wherein said polynucleotide is a cDNA.

58. A cell engineered to contain in its membrane the ABCA1 polypeptide of claim 1 wherein said cell does not contain said polypeptide absent said engineering.

59. The cell of claim 58 wherein said engineering is genetic engineering.

60. The cell of claim 58 wherein said cell is a recombinant cell.

61. The cell of claim 58 wherein said cell is a mammalian cell.

62. The cell of claim 61 wherein said mammalian cell is a human cell.

63. A cell engineered to contain in its membrane the ABCA1 polypeptide of claim 22 wherein said cell does not contain said polypeptide absent said engineering.

64. A cell engineered to contain in its membrane the ABCA1 polypeptide of claim 23 wherein said cell does not contain said polypeptide absent said engineering.

65. A cell engineered to contain in its membrane the ABCA1 polypeptide of claim 24 wherein said cell does not contain said polypeptide absent said engineering.