WO2009094592A2 - Base génétique de la maladie d'alzheimer et diagnostic et traitement de cette dernière - Google Patents

Base génétique de la maladie d'alzheimer et diagnostic et traitement de cette dernière Download PDF

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WO2009094592A2
WO2009094592A2 PCT/US2009/031909 US2009031909W WO2009094592A2 WO 2009094592 A2 WO2009094592 A2 WO 2009094592A2 US 2009031909 W US2009031909 W US 2009031909W WO 2009094592 A2 WO2009094592 A2 WO 2009094592A2
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related disease
gene
expression
agent
polypeptide
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David A. Cox
Erica Beilharz
Karel Konvicka
Gerard D. Schellenberg
Eric Larson
Yiping Zhan
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Perlegen Sciences, Inc.
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    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations

Definitions

  • the method comprises determining the total number of resistance and susceptibility alleles in a polymorphic profile, whereby the number of susceptibility alleles and/or the ratio of susceptibility alleles to resistance alleles provides an indication of whether the individual has or is at risk of developing an AD-related disease, or the likelihood of developing an AD-related disease at an early age.
  • a ratio of resistance to susceptibility alleles of less than a threshold value can be an indication that the individual is at high or low risk of developing Alzheimer's disease.
  • a threshold value may be determined according to the methods provided in USSN 60/648,957, filed January 31, 2005; USSN 11/344,975, filed January 31, 2006, and PCT application no. US2006/003384, filed January 31, 2006.
  • Any of the above methods can include informing the patient or a relative thereof of presence or susceptibility to an AD-related disease, performing a secondary test for an AD- related disease, such as determimng mental activity by a psychometric measure or taking a
  • any of the above methods can also involve determining at least one susceptibility allele not in or within 40 kb of TOMM40 or APOCl, or not in or within 40 kb of PVRL2, TOMM40, APOCl, APOC4, BCAM, APOC2, or CLPTMl
  • the agent is selected from the group consisting of: an antibody, small molecule or natural product that specifically binds to a protein encoded by a gene selected from the group; a zinc finger protein that modulates expression of a gene selected from the group; or an siRNA, antisense RNA, RNA complementary to a regulatory sequence, or ribozyme that inhibits expression of a gene selected from the group.
  • the method also involves monitoring a sign or symptom of the AD-related disease in the patient responsive to the administration.
  • the method involves administering a second agent effective to effect treatment or prophylaxis (which includes delaying age-of-onset) of the AD-related disease.
  • the patient is human.
  • the disease is late-onset Alzheimer's disease.
  • the group can further comprise other genes shown in Tables A, B, C, and/or D, e.g., PSEN2, APP, HDAC4, OLFMl, RAB12, KIAA0802, CLYBL, ZIC5, LOC728155, LOC727827, FARPl, RNFl 13B, EXOC2, LOC642335, ZNF366, LOC389300, LOC644154, and LOC645932.
  • the group can exclude APOE, APOCl, PVRL2, TOMM40, CLPTMl, APOC2, APOC4, APP, or PSEN2.
  • the group can further comprise other genes shown in Tables A, B, C, and/or D, e.g., PSEN2, APP, HDAC4, OLFMl, RAB12, KIAA0802, CLYBL, ZIC5, LOC728155, LOC727827, FARPl, RNFl 13B, EXOC2, LOC642335, ZNF366, LOC389300, LOC644154, and LOC645932.
  • the group can exclude APOE, APOCl, PVRL2, TOMM40, CLPTMl , AP0C2, APOC4, APP, or PSEN2.
  • the primary screen measures binding of the agent to the protein.
  • the primary screen measures capacity of the agent to agonize or antagonize the protein.
  • the group can further comprise other genes shown in Tables A, B, C, and/or D, e.g., PSEN2, APP, HDAC4, OLFMl, RAB12, KIAA0802, CLYBL, ZIC5, LOC728155, LOC727827, FARPl, RNF113B, EXOC2, LOC642335, ZNF366, LOC389300, LOC644154, and LOC645932.
  • the group can exclude APOE, APOCl, PVRL2, TOMM40, CLPTMl, APOC2, AP0C4, APP, or PSEN2.
  • the segment is within a gene selected from the group consisting of APOE, APOCl, PVRL2, TOMM40, CLPTMl, AP0C2, APOC4, BCAM, LOC728050, NEUR0G3, C10ORF35, LOC729099, ND3, and ND4.
  • an AD-related disease is a neurodegenerative disease the affects neurons in the brain.
  • An AD-related disease may be e.g. a condition that is a risk factor for developing AD, or may be a condition for which AD is a risk factor, or both.
  • Many AD-related diseases are characterized by related pathology of amyloid deposits of a protein that stain with Congo red dye and/or related neurodegenerative symptoms.
  • Resistance to AD or related disease means that a subject has a significantly lower risk of developing the disease than the average risk of an age- and sex-matched individual from the general population. Resistance can also mean that a subject is likely to exhibit a significantly later age-of-onset than the average age-of-onset of an age-, and sex-matched individual from the general population who will eventually develop the disease.
  • AD nucleic acid or "AD-associated genomic region” means a nucleic acid, or fragment, derivative, variant, polymorphism, or complement thereof, associated with resistance or susceptibility to AD-related disease or age-of-onset thereof, including, for example, at least one or more AD polymorphisms, genomic regions spanning 10 kb immediately upstream and 10 kb immediately downstream of an AD polymorphism, coding and non-coding regions of an associated gene, and/or genomic regions spanning 10 kb immediately upstream and 10 kb immediately downstream of an associated gene, and variants thereof.
  • the term also includes nucleic acids similarly related to genes in an associated gene pathway.
  • An AD nucleic acid may also be an "associated genomic region" when it is found within the genome of an organism.
  • associated gene or "associated gene region” or “AD gene” refers to a gene, a genomic region 10 kb upstream and 10 kb downstream of such gene, or regulatory regions that modulate the expression of such gene, comprising at least a portion of one of the polymorphic regions identified in Tables A, B, C, D, and/or E, and all associated gene products (e.g., isoforms, splicing variants, and/or modifications, derivatives, etc.)
  • the sequence of an AD gene in an individual may contain one or more reference or alternate alleles, may contain a combination of reference and alternate alleles, or may contain alleles in linkage disequilibrium with one or more of the polymorphic regions identified in Tables A, B, C, D, and/or E.
  • a nucleic acid includes analogs (e.g., phosphorothioates, phosphoramidates, methyl phosphonate, chiral-methyl phosphonates, 2-O-methyl ribonucleotides) or modified nucleic acids (e.g., modified backbone residues or linkages) or nucleic acids that are combined with carbohydrates, lipids, protein or other materials, or peptide nucleic acids (PNAs) (e.g., chromatin, ribosomes, transcriptosomes, etc.)
  • PNAs peptide nucleic acids
  • a nucleic acid can include one or more polymorphisms, variations or mutations (e.g., SNPs, insertions, deletions, inversions, translocations, etc.)
  • nucleic acids include oligonucleotides, nucleotides, polynucleotides, nucleic acid sequences, genomic sequences, antisense nucleic acids, DNA regions, probes,
  • an allele of a polymorphism that is identical to a reference sequence is referred to as a "reference allele” and an allele of a polymorphism that is different from a reference sequence is referred to as an "alternate allele", or sometimes a “variant allele”.
  • an "alternate allele” may be found in a reference sequence and a “reference allele” may not.
  • the designation of a "reference allele” and an “alternate allele” need not be based on any particular reference different alleles of a polymorphism.
  • probes refers to nucleic acids that can hybridize, m whole or in part, in a base-specific manner to a complementary strand
  • primer refers to a smgle-stranded nucleic acid that acts as a point of initiation of template-directed DNA synthesis (e g , PCR primers)
  • probe refers to a single-stranded nucleic acid designed to hyb ⁇ dize to a target nucleic acid
  • hybridization of the probe to a sample nucleic acid may be used to purify a target nucleic acid within the sample, or detection of hybridization (or lack thereof) of the probe to a sample nucleic acid may be used to determine the presence (or absence) of a target nucleic acid in the sample
  • smgle-stranded probes and p ⁇ mers are primarily discussed herein, the present invention is not limited to such probes and primers, double-stranded or partially
  • specific hybridization refers to the ability of a first nucleic acid to bind, duplex or hybndize to a second nucleic acid in a manner such that the second nucleic acid can be identified or distinguished from other components of a mixture (e g , cellular extracts, genomic DNA, etc ) In certain embodiments, specific hybridization is performed under stringent conditions
  • haplotype block refers to a region of a chromosome that contains one or more polymorphic sites (e.g., 1-10) that tend to be inherited together (i.e., are in linkage disequilibrium) (see Patil, et al., Science, 294:1719-1723 (2001); US 20030186244)).
  • polymorphic sites e.g., 1-10
  • the invention provides a collection of polymorphic sites having resistance and susceptibility alleles associated with resistance or susceptibility (including age-of-onset) to Alzheimer's disease and other AD-related diseases, particularly the most common form of Alzheimer's disease, known as late-onset Alzheimer's disease (LOAD).
  • the polymorphic sites were identified by analyzing a sampling of polymorphic sites throughout the human genome in a population having late-onset disease and a control population.
  • This application hereby incorporates the following applications by reference in their entireties for all purposes: USSN 60/648,957, filed January 31, 2005; USSN 11/344,975, filed January 31, 2006, and PCT application no. US2006/003384, filed January 31, 2006.
  • Late-onset AD is the most common form of AD. It usually appears after a person reaches the age of 65. Late-onset AD strikes almost half of all people over the age of 85 and may or may not be hereditary. Late-onset AD is also called sporadic AD. Late-onset Alzheimer's, has a subtler and less clearly understood inheritance pattern.
  • the cholesterol- processing protein called apolipoprotein E (APOE) is a susceptibility gene that occurs in three different alleles: APOE-e4, APOE-e3, and APOE-e2. ApoE-e3 is the most common form and APOE-e2 is the least common. People with one copy of APOE-e4 have an increased chance of developing Alzheimer's, and people with two copies are at even higher risk.
  • APOC4 is encoded by apolipoprotein (apo)C4 gene located at 19ql3.2, and which is a member of the apolipoprotein gene family. It is expressed in the liver and has a predicted protein structure characteristic of the other genes in this family. Apo C4 is a 3.3-kb gene consisting of 3 exons and 2 introns; it is located 0.5 kb 5' to the apoC2 gene.
  • apoC4 apolipoprotein (apo)C4 gene located at 19ql3.2, and which is a member of the apolipoprotein gene family. It is expressed in the liver and has a predicted protein structure characteristic of the other genes in this family.
  • Apo C4 is a 3.3-kb gene consisting of 3 exons and 2 introns; it is located 0.5 kb 5' to the apoC2 gene.
  • NEUROG3 (neurogenin 3) is encoded by the NEUROG3 gene located at 10q21.3, and belongs to a family of basic helix-loop-helix transcription factors involved in the determination of neural precursor cells in the neuroectoderm.
  • Table A column 4, entitled “POSITION,” provides the nucleotide position in the NCBI Build 36.2 contig of the "N" position in the assayed sequence provided in Table C, if available.
  • Table A, column 8, entitled “REP_GLM_P_VALUE,” identifies p-value of a chi- square statistic, representing the significance of the difference in a logistic regression comparing a model with genotype terms to one without This statistic is computed over the replication case and control samples
  • Table A, column 17, entitled “REF_AA,” provides the alternate amino acid residue if SNP Allele 1 causes a change in the amino acid sequence with respect to the reference ammo acid
  • Table A, column 18, entitled “ALT_AA,” provides the alternate ammo acid residue if SNP Allele 2 causes a change in the amino acid sequence with respect to the reference ammo acid
  • Table B, column 12, entitled “SYNONYMOUS,” indicates whether the SNP alleles code for different amino acids in an encoded protein, "yes” indicates the two alleles encode the same protein sequence, and “no” indicates that the two alleles encode different amino acids at the same position in the resulting protein.
  • “outsideCodingRegion” indicates the SNP occurs outside the coding region of a gene.
  • “unknown_poor_alignment_at_snp_pos” indicates the synonymous nature of the SNP alleles cannot be determined due to poor local alignment. There is no entry for those SNP positions that do not occur within an exon.
  • Table E, column 11 entitled “Assayed sequence” is the 29-mer (SNPs) or 30-mer (DIPs) that was used to assay the SNP on a microarray, with an ambiguity character “N” representing the SNP or DIP at the middle base.
  • nucleic acids herein are associated with AD or age-of- onset thereof
  • Nucleic acids associated with resistance to AD comprise at least one allele associated with resistance to AD or an allele in linkage disequilibrium with (e g , in a haplotype pattern with) an allele associated with resistance to AD
  • Nucleic acids associated with susceptibility to AD comprise at least one allele associated with susceptibility to AD or an allele in linkage disequilibrium with (e g , in a haplotype pattern with) an allele associated with susceptibility to AD
  • a nucleic acid associated with resistance to AD is one that is expressed differently in individuals having a phenotype of resistance to AD as compared to individuals having who do not have a phenotype of resistance to AD, or a nucleic acid having one or more alleles associated with resistance to AD
  • a nucleic acid associated with resistance to AD is one that can specifically hybridize to a genomic region having one or more alleles of polymorphism
  • Conditions for nucleic acid hybridization vary depending on the buffers used, length of nucleic acids, ionic strength, temperature, etc.
  • stringent conditions refers to the incubation and wash conditions (e.g., conditions of temperature and buffer concentration) that permit hybridization of a first nucleic acid to a second nucleic acid.
  • the first nucleic acid may be perfectly (e.g. 100%) complementary to the second or may share some degree of complementarity, which is less than perfect (e.g., more than 70%, 75%, 85%, or 95%).
  • certain high stringency conditions can be used which distinguish perfectly complementary nucleic acids from those less complementary, even those having only a single base mismatch.
  • hybridization conditions By varying hybridization conditions from a level of stringency at which no hybridization occurs to a level at which hybridization is observed, conditions which will allow a given sequence to hybridize (e.g., selectively) with the most similar sequences in the sample can be determined. Exemplary conditions are described in Krause, et al., Methods in Enzymology, (1991) 200:546-556 and in Ausubel, et al., "Current Protocols in Molecular Biology", (John Wiley & Sons 1998), which describes the determination of washing conditions for moderate or low stringency conditions. Washing is the step in which conditions are usually set so as to determine a minimum level of complementarity of the hybrids.
  • an AD nucleic acid may be labeled and used to screen a genomic or cDNA library constructed from mKNA obtained from the organism of interest.
  • Hybridization conditions will be of a lower stringency when the cDNA library was derived from an organism different from the type of organism from which the labeled nucleic acid was derived.
  • Such lower stringency conditions vary predictably depending on the specific organisms from which the library and the labeled nucleic acids are derived. For guidance regarding such conditions see, for example, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N. Y.; and Ausubel et al. (1989) Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N. Y. 1. Probes and Primers
  • the antisense nucleic acids may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'
  • oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier, et al, (1987) Nucl. Acids Res. 15:6625-6641).
  • the oligonucleotide is a 2'-O- methylribonucleotide (Inoue, et al., (1987) Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue, et al., (1987) FEBS Lett. 215:327-330).
  • Antisense nucleic acids may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
  • an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate oligonucleotides may be synthesized by the method of Stein, et al. (1988) Nucl. Acids Res. 16:3209
  • methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports Sarin, et al., (1988) Proc. Natl. Acad. Sci. USA 85:7448-7451, etc.
  • Ribozyme molecules designed to catalytically cleave target mRNA transcripts can also be used to prevent translation of such mRNA. See, e.g., WO 90/11364; Sarver, et al., (1990) Science 247: 1222-1225.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. See Rossi, (1994) Current Biology 4:469-471. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme to complementary target RNA, followed by an endonucleolytic cleavage event. The composition of ribozyme molecules must have one or more sequences complementary to the target mRNA and must include the well known catalytic sequence responsible for mRNA cleavage. See, e.g., U.S. Pat. No. 5,093,246.
  • RNA endoribonucleases also known as "Cech-type ribozymes," such as the one which occurs naturally in Tetrahymena thermophila (known as the IVS, or L- 19 IVS RNA) and which has been extensively described in Zaug, et al., (1984) Science 224:574-578; Zaug and Cech, (1986) Science 231:470-475; Zaug, et al., (1986) Nature 324:429-433; PCT Publication No. WO 88/04300; Been and Cech, (1986) Cell 47:207-216.
  • Ceech-type ribozymes such as the one which occurs naturally in Tetrahymena thermophila (known as the IVS, or L- 19 IVS RNA) and which has been extensively described in Zaug, et al., (1984) Science 224:574-578; Zaug and Cech, (1986) Science 231:470-475; Zaug, et al., (19
  • the nucleic acids herein are used to over-express polypeptides associated with resistance to AD-related disease.
  • the nucleic acids herein are used to underexpress polypeptides associated with susceptibility to AD-related disease.
  • a nucleic acid encoding the polypeptide of interest can be ligated to a regulatory sequence that can drive the expression of the polypeptide in the animal cell type of interest at a level that is higher than expression in the absence of such a construct.
  • regulatory regions are well known.
  • a non-coding nucleic acid e.g., an intron or a regulatory nucleic acid
  • vectors e.g., non-episomal mammalian vectors
  • vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors e.g., expression vectors
  • a preferable expression vector is a plasmid, an artificial chromosome, a cosmid or a viral vector.
  • regulatory sequences are described, for example, in Goeddel, “Gene Expression Technology Methods in Enzymology” (1990) 185, Academic Press, San Diego, CA Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e g , tissue-specific regulatory sequences)
  • transformation and transfection refer to a variety of art- recognized techniques for introducing a foreign nucleic acid molecule (e g , DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAF-dextran- mediated transfection, hpofection, or electroporation Suitable methods for transforming or transfectmg host cells can be found in Sambrook, et al and other laboratory manuals For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome To identify and select these integrants, a gene that encodes a selectable marker is generally introduced into the host cells along with the gene of interest Preferred selectable markers include those that confer resistance to drugs Nucleic acid molecules encoding a selectable marker can be introduced into a host cell on the same vector as the nucleic acids or can be introduced on a separate vector Cells stably transfected with the introduced nucle
  • Such vectors and host- expression vector systems are well known in the art and are further described in, e.g., Ruther et al. (1983), EMBO J. 2:1791; Inouye & Inouye (1985) Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster (1989) J. Biol. Chem. 264:5503-5509; Smith et al. (1983) J. Virol. 46:584; Smith, U.S. patent no. 4,215,051 ; Logan & Shenk (1984) Proc. Natl. Acad. Sci. USA 81 :3655-3659; Bittner et al. (1987) Methods in Enzymol.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal include a transgene.
  • Other examples of transgenic animals include, for example, non-human primates, sheep, dogs, cows, goats, chickens and amphibians.
  • a transgene is an exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • a polypeptide associated with AD-related disease can be recombinantly produced using an expression vector having a non-coding regulatory region associated with resistance to AD, operably linked to an AD polypeptide.
  • the expression vector is introduced into a host cell under conditions appropriate for expression.
  • the polypeptide can then be isolated from the host cell using standard protein purification techniques.
  • a similar method can be used to produce a polypeptide associated with susceptibility to AD-related disease.
  • a non-coding nucleic acid or nucleic acid outside coding region which is associated with susceptibility to AD-related disease, operably linked to an associated genomic region coding sequence can be inserted into a host cell under conditions appropriate for protein synthesis.
  • the resulting polypeptide associated with susceptibility to AD-related disease is then collected and purified.
  • polypeptides are purified. There are various degrees of purity. While a polypeptide can be purified to homogeneity, preparations in which a polypeptide is not purified to homogeneity are also useful where the polypeptide retains a desired function even in the presence of considerable amount of other components. In some embodiments, polypeptides are substantially free of cellular material which includes preparations of a polypeptide having less than about 30% (dry weight) other polypeptides (e.g., contaminating polypeptides), less than about 20% other polypeptides, less than about 10% other polypeptides, or less than about 5% other polypeptides.
  • culture medium represents less than about 20% of the volume of the polypeptide preparation, preferably less than about 10% of the volume of the polypeptide preparation or more preferably less than about 5% of the volume of the polypeptide preparation.
  • Polypeptides that are substantially free of chemical precursors or other chemicals generally include those that are separated from chemicals that are involved in its synthesis.
  • polypeptides may have a lower degree of sequence homology but are still able to perform one or more of the same functions.
  • Conservative substitutions that can maintain the same function include replacements among aliphatic amino acids methionine, valine, leucine and isoleucine; interchange of the hydroxyl residues serine and threonine; exchange of acidic residues aspartic and glutamic acids; substitution between amide residues asparagine and glutamine, exchange between basic residues lysine and argmine, and replacements among aromatic residues phenylalanin, tyrosine and tryptophan. Alanine and glycine may also result in conservative substitutions.
  • Other types of fusion polypeptides include enzymatic fusion polypeptides, for example ⁇ -galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions and Ig fusions. Fusion polypeptides, especially poly-His fusions, can facilitate the purification of recombinant polypeptide. In some host cells, such as mammalian cells, expression and secretion of an AD polypeptide can be increased using a heterologous signal sequence.
  • any of the polypeptides herein, or fragments, derivatives, or complements thereof, can be used as an immunogen (e.g. epitope) to generate polypeptide-specific antibodies.
  • Antibodies can be used to detect, isolate and inhibit the activity of one or more AD polypeptides.
  • an AD polypeptide or a fragment thereof is used as an epitope.
  • an epitope is at least 6 amino acids, at least 9 amino acids, at least 20 amino acids, at least 40 amino acids, or at least 80 amino acids in length.
  • Polyclonal antibodies are prepared by immunizing a suitable subject (e.g., goats, rabbits, rats, mice or humans) with a desired antigen.
  • a suitable subject e.g., goats, rabbits, rats, mice or humans
  • the antibody titer in the immunized subject can be monitored over time using methods known in the art, such as by using an enzyme linked immunosorbent assay (ELISA).
  • ELISA enzyme linked immunosorbent assay
  • the antibodies can then be isolated from the subject (e.g., from blood) and further purified using techniques, such as protein A chromatography, to obtain the IgG fraction.
  • any of the antibodies can further be coupled to a substance (label) for detection of a polypeptide-antibody binding complex.
  • labels include, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, or radioactive materials
  • suitable enzymes include, for example, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase
  • suitable prosthetic group complexes include, for example, streptavidin/biotin and avidm/biotin
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorot ⁇ azinylamme fluorescein, dansyl chloride or phycoerythrin
  • An example of a luminescent material is luminol
  • bioluminescent materials include luciferase, luciferin and aequo ⁇ n
  • suitable radioactive material include 1251,
  • the polymorphic profile of an individual can be scored by comparison with the polymorphic forms associated with resistance or susceptibility to Alzheimer's disease occurring at each site as shown in Table E.
  • the comparison can be performed on at least 1, 2, 5, 10, 25, 50, or all of the polymorphic sites, and optionally, others in linkage disequilibrium with them.
  • the polymorphic sites can be analyzed in combination with other polymorphic sites. However, the total number of polymorphic sites analyzed is usually fewer than 10,000, 1000, 100, 50 or 25.
  • polymorphic profile from a sample at the scene of a crime is compared with that of a suspect.
  • a match between the two is evidence that the suspect in fact committed the crime, whereas lack of a match excludes the suspect.
  • the present polymorphic sites can be used in such methods, as can other polymorphic sites in the human genome.
  • Detection of presence or increased level of one or more nucleic acids, or fragments, derivatives, variants or complements thereof, associated with resistance to AD-related disease is a prognostic and diagnostic for resistance to AD-related disease.
  • detection of presence or increased level of one or more nucleic acids, or fragments, derivatives, variants or complements thereof, associated with susceptibility to AD-related disease is a prognostic and diagnostic for susceptibility to AD-related disease.
  • detection of the presence of a genetic variant e.g., SNP
  • detection of a variant correlated with resistance may be indicative of a healthy state.
  • Alternative diagnostic and prognostic methods employ amplification of target nucleic acids associated with resistance or susceptibility to AD-related disease, e.g., by PCR. This is especially useful for target nucleic acids present in very low quantities.
  • amplification of target nucleic acids associated with resistance to AD-related disease indicates their presence and is a prognostic and diagnostic of resistance to AD-related disease.
  • amplification of target nucleic acids associated with susceptibility to AD-related disease indicates their presence and is a prognostic and diagnostic of susceptibility to AD-related disease.
  • microarrays can generally be produced using mechanical synthesis methods or light directed synthesis methods that incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods See Fodor et al , (1991) Science 251 767-777, and U S Pat No 5,424,186 Techniques for the mechanical synthesis of microarrays are described in, for example, U S Pat No 5,384,261
  • target nucleic acid sequences that include one or more previously identified variants or polymorphisms are amplified and labeled by well-known techniques, such as attachment of a fluorescent moiety or using labeled primers during amplification (e g PCR)
  • Primers that are complementary to both strands of the target sequence one p ⁇ mer complementary to one strand upstream and the other pnmer complementary to the other strand downstream from a variant or polymorphism
  • Asymmetric PCR techniques may be used.
  • Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect wild type or mutant peptides through the use of a radioimmunoassay. See Weintraub, B , "Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay
  • Kits useful in diagnosis and prognosis include reagents comprising, for example, instructions for use and analysis; means for collecting a tissue or cell sample; nucleic acid probes or primers (e.g., for amplification, reverse transcriptase and detection); labels (e.g., for nucleic acids or proteins); microarrays, gels, membranes or other detection apparati; restriction enzymes (e.g., for RFLP analysis); allele-specific probes; antisense nucleic acids; antibodies; and other protein binding probes, any of which may be labeled
  • the invention provides methods to identify agents potentially useful in diagnosis, prognosis, prophylaxis or treatment of an AD-related disease, including the likelihood of developing an AD-related disease at an early age. Agents are tested for their capacity to modulate expression or activity of a gene selected from the group consisting of APOE, APOCl, PVRL2, TOMM40, CLPTMl, AP0C2, AP0C4, BCAM, LOC728050, NEUR0G3, C10ORF35, LOC729099, ND3, and ND4.
  • agents include, but are not limited to: transcription factors, binding molecules, antisense nucleic acids, PNAs, mimetics, small or large organic or inorganic molecules, polypeptides (e.g., soluble peptides, or Ig-tailed fusion peptides), antibodies, as described above, (e.g., monoclonal, polyclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, Fab, F(ab')2, Fab expression library fragments, and epitope-binding fragments thereof), fusion proteins, prodrugs, drugs in trials, previously approved drugs for AD, drugs developed for indications other than AD, and any fragments, derivatives, variants or complements of any of the above.
  • polypeptides e.g., soluble peptides, or Ig-tailed fusion peptides
  • antibodies as described above, (e.g., monoclonal, polyclonal, humanized, anti-idiotypic, chimeric or single chain antibodies
  • Combinatorial libraries can be produced for many types of agents that can be 5 synthesized in a step-by-step fashion.
  • agents include polypeptides, proteins, nucleic acids, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N- substituted glycines and oligocarbamates.
  • ESL encoded synthetic libraries
  • Peptide libraries can also be generated by phage display methods. See, e.g., Devlin, WO 91/18980. Compounds to be screened can also be obtained from governmental or private sources, including, e.g., the National Cancer Institute's (NCI)
  • the target site is within the structural gene.
  • the zinc finger protein is linked to a transcriptional repressor, such as the KRAB repression domain from the human KOX-I protein (Thiesen et al., New Biologist 2, 363-374 (1990); Margolin et al., Proc. Natl. Acad. Sci. USA 91, 4509-4513 (1994); Pengue et al., Nucl. Acids Res. 22:2908-2914 (1994); Witzgall et al., Proc. Natl. Acad. Sci. USA 91,
  • agents that modulate the expression of coding AD nucleic acids by interacting with an AD regulatory region e.g., enhancers, introns, 5' and 3' untranslated regions (e.g., promoters) and uORF's
  • AD regulatory region e.g., enhancers, introns, 5' and 3' untranslated regions (e.g., promoters) and uORF's
  • agents that modulate transcription or translation of nucleic acids herein can be identified by contacting a solution containing non-coding nucleic acids associated with AD- related disease operably linked to a reporter gene with a test agent.
  • a protein microchip may be used to identify polypeptides that bind to AD polypeptides or any other polypeptide herein
  • a protein microchip or microarray is provided having one or more protein complexes and/or antibodies selectively lmmunoreactive with a polypeptide of interest Protein microarrays are becoming
  • Protein microarrays can be prepared in a number of methods known in the art An example of a suitable method is that disclosed in MacBeath and Schreiber, (2002) Science, 289 1760-1763 Essentially, glass microscope slides are treated with an aldehyde-containing silane reagent (SuperAldehyde Substrates purchased from TeleChem International, Cupertino, Calif) Nanohter volumes of protein samples in a phosphate-buffered salme with
  • a substrate or chip base is covered with one or more layers of thin organic film to eliminate any surface defects, insulate proteins from the base materials, and to ensure uniform protein array.
  • a plurality of protein-capturing agents e.g., antibodies, peptides, etc.
  • Proteins or protein complexes can then be bound to the capturing agents forming a protein microarray.
  • the protein microchips are kept in flow chambers with an aqueous solution.
  • the protein microarrays herein can also be made by the method disclosed in PCT
  • complex formation in a reaction mixture containing a test agent and AD polypeptide may be compared to complex formation in a reaction mixture containing the test agent and a second AD polypeptide that is encoded by a different nucleic acid sequence than the first AD polypeptide.
  • the first and second AD polypeptides are encoded by different alleles of the same gene. This comparison can be important in those cases in which it is desirable to identify agents that disrupt interaction of a particular AD polypeptide.
  • test agent to bind to an AD polypeptide, a cellular or extracellular binding agent, or a complex thereof can be assessed, for example, by coupling a test agent with a radioisotope or enzymatic label such that binding of the test agent to the AD polypeptide, binding agent, or complex thereof can be determined by detecting the label (e.g., 1251, 35S, 14C, or 3H) either directly or indirectly (e.g., by direct counting of radio emission or by scintillation counting).
  • label e.g., 1251, 35S, 14C, or 3H
  • test agents can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase or luciferase and the enzymatic label can be detected by determination of conversion of an appropriate substrate to a product.
  • Non-peptide agents or small molecules are generally preferred because they are more readily absorbed after oral administration and have fewer potential antigenic determinants. Small molecules are also more likely to cross the blood brain barrier than larger protein-based pharmaceuticals. Methods for screening small molecule libraries for candidate protein-binding molecules are well known in the art and may be employed to identify molecules that modulate (e.g., through direct or indirect interaction) one or more of the AD polypeptides herein. Briefly, AD polypeptides may be immobilized on a substrate and a solution including the small molecules is contacted with the AD polypeptide under conditions that are permissive for binding. The substrate is then washed with a solution that substantially reflects physiological conditions to remove unbound or weakly bound small molecules.
  • the DOCK3.5 is used to screen for small molecules that interact with AD polypeptides, preferably the binding pocket of an AD polypeptide.
  • a "negative image" of the binding pocket on a protein surface is created. The image is created by the computational equivalent of placing atom-sized spheres into the binding pocket.
  • a representative set of spheres are identified by DOCK3.5 that fit extremely well into the binding pocket.
  • the generated spheres constitute an irregular grid that is matched to the atomic centers of potential ligands.
  • the list of atom centers, or more conveniently the matrix of interatomic distances linking these atom centers forms a useful description of the binding site.
  • the matrix of interatomic distances for the putative ligand is also made. The best mutual overlap of the two matrices is sought. This alignment specifies the orientation of the ligand relative to the negative image of the protein and thus docks the ligand into the protein's binding pocket.
  • Non-peptide agents or small molecule libraries can be prepared by a synthetic approach, but recent advances in biosynthetic methods using enzymes may enable one to prepare chemical libraries that are otherwise difficult to synthesize chemically.
  • Small molecule libraries can also be obtained from various commercial entities, for example, SPECS and BioSPEC B.V. (Rijswijk, the Netherlands), Chembridge Corporation (San Diego, California), Comgenex USA Inc., (Princeton, N.J.), Maybridge Chemical Ltd. (Cornwall, U.K.), and Asinex (Moscow, Russia).
  • small molecule libraries can be screening in a high throughput manner to identify one or more agents
  • a high throughput screening assay for small molecules that was disclosed in Stockwell, B R et al , Chem & Bio , (1999) 6 71-83, is a miniaturized cell-based assay for monitoring biosynthetic processes such as DNA synthesis and post-translational processes 7 Immobilization Assays
  • a substrate can be any vessel suitable for containing the reactants
  • substrates include microtrter plates, test tubes, and micro-cent ⁇ fuge tubes
  • agents that bind a polypeptide of interest can be detected by anchoring either the polypeptide of interest (e g , any polypeptide herein) or the test agent (e g , antibody) to a substrate (e g , microtiter plates) and then detecting complexes of the polypeptide of interest and test agent anchored to the substrate at the end of the reaction Where the polypeptide of interest is anchored and the test agent is not anchored, the test agent can be labeled, either directly or indirectly
  • the polypeptide or other components of the assay maybe label
  • an assay performed in liquid phase has the pre-formed complexes of the AD polypeptides and the cellular or extracellular binding agents prepared such that either the polypeptide or the binding agents are labeled, but the signal generated from the label is eliminated or diminished due to complex formation.
  • the addition of a test agent that competes with and displaces one of the species from the pre-formed complex results in the generation of a signal above background.
  • the cellular or extracellular binding agent is then added in the presence or absence of the test agent in a manner that allows interaction and binding to occur. At the end of the reaction period, unbound material is washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components.
  • the interaction between the AD polypeptide and the cellular or extracellular binding agents is detected by measuring the amount of radioactivity that remains associated with the beads. A successful inhibition of the interaction by the test agent will result in a decrease in measured radioactivity.
  • the GST bound AD polypeptide fusion product and the interactive cellular or extracellular binding agent can be mixed together in liquid in the absence of the solid glutathione-agarose beads.
  • the test agent is added either during or after the binding agent is allowed to interact with the GST-fusion polypeptide. This mixture is then added to the glutathione-agarose beads and unbound material is washed away.
  • the extent of inhibition of the binding agent interaction can be detected by adding the labeled antibody and comparing the radioactivity associated with the beads to that of a control reaction (e.g., lacking test agent)
  • compositions can be formulated in accordance with the routine procedures adapted for administration to human beings. Often, pharmaceutical compositions are formulated with an acceptable carrier or excipient. See Remington's Pharmaceutical Sciences, Gennaro, A., (ed., Mack Publishing Co. 1990).
  • compositions can include, if desired, auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active agents.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active agents.
  • vitamins examples include components of the vitamin B complex, such as vitamin Bl, B2, B6, B12, biotin, niacin, pantothenic acid, folic acid, adenine, choline, adenosine phosphate, orotic acid, pangamic acid, carnitine, 4-aminobenzoic acid, myo-inositol, liponic acid and/or amygdaline.
  • vitamin Bl also known as thiamin
  • thiamin is converted into thiamin-pyrophosphate, a coenzyme in a number of reactions in which C-C bonds are cleaved. It can also be added as thiamin hydrochloride.
  • Vitamin B2 also known as riboflavin, is reabsorbed in the small intestines, converted into FMN (flavin mononucleotide) and, in the liver, into FAD (flavin-adenine-dinucleotide), both of which are coenzymes in redox reactions
  • Vitamin B6 also known as pyndoxal, pyrodoxin and py ⁇ doxamme, is a component of pyndoxal-5 -phosphate, which is a co factor m glycogen degradation and in ammo acid metabolism, e g as a coenzyme of decarboxylases
  • Vitamin B 12 also known as cyanocobalamine, has a complex structure and is a component of cobalamme-coenzymes, with methyl-cobalamine and cobalamide, e g , being involved in rearrangements with hydrogen migration Biotin, also known as
  • compositions used for intravenous administration are typically solutions in sterile isotonic aqueous buffer. Where necessary, the compositions may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water.
  • sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carver material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., pressurized air.
  • a pressurized volatile, normally gaseous propellant e.g., pressurized air.
  • AD-related diseases e.g., LOAD
  • memory loss for simple things like friends' names, commonly used phone numbers, or what month it is and how to get to a familiar place; misplacement of things more often than usual; loss of train of thought when speaking; repeating things often; feeling more suspicious, cautious, or anxious; loss of interest in things that used to be enjoyable; and feeling of stress when making decisions.
  • a more definitive indication of AD-related disease may require a biopsy.
  • the agents and pharmaceutical compositions may be administered or coadministered orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery (for example by catheter or stent), subcutaneously, intraadiposally, intraarticularly, or intrathecally.
  • the compounds and/or compositions may also be administered or co-administered in slow release dosage forms. Other suitable methods include gene therapy using rechargeable or biodegradable devices, particle acceleration devices ("gene guns") and slow release polymeric devices.
  • the pharmaceutical compositions herein can also be administered as part of a combinatorial therapy with other agents.
  • one or more nucleic acids associated with resistance to AD-related diesease may be inserted into appropriate cells within a patient, using vectors such as adenovirus, adeno-associated virus and retrovirus vectors. Nucleic acids can also be introduced into cells via particles, such as liposomes. Other techniques for direct administration involve stereotactic delivery of such sequences to the site of the cells in which the sequences are to be expressed.
  • a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
  • the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180 dated Apr. 16, 1992; WO 92/22635 dated Dec. 23, 1992; WO92/20316 dated Nov. 26, 1992; WO93/14188 dated JuI. 22, 1993; WO 93/20221 dated Oct. 14, 1993).
  • Additional methods that may be utilized to increase or decrease the overall level of expression of an AD nucleic acid include using targeted homologous recombination methods to modify the expression characteristics of an endogenous sequence in a cell or microorganism by inserting a heterologous DNA regulatory element such that the inserted regulatory element is operatively linked with the endogenous sequence in question.
  • Targeted homologous recombination can thus be used to activate transcription of an endogenous nucleic acid that is transcriptionally silent, (e.g., not normally expressed or expressed at very low levels), to silence the transcription of an endogenous nucleic acid that is transcriptionally active, or to enhance or decrease the expression of an endogenous sequence that is normally expressed.
  • the overall level of expression of polypeptides associated with resistance to AD may be increased by the introduction of cells that express such polypeptides associated with resistance to AD, preferably autologous cells, into a patient at positions and in numbers which are sufficient to prevent or ameliorate symptoms or conditions associated with AD- related disease.
  • Such cells may be either recombinant or non-recombinant.
  • such cells are healthy brain cells.
  • the cells to be administered are non-autologous cells, they can be administered using well-known techniques that prevent a host immune response against the introduced cells from developing.
  • the cells may be introduced in an encapsulated form that, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.
  • the amounts of therapeutic agents or compositions to be administered can vary, according to determinations made by one of skill, but preferably are in amounts effective to create reduce or reverse AD-related disease symptoms.
  • Treatment compositions and dosages can be specifically tailored to each situation based on an individual patient's pharmacogenomics (response to a drug), phenotype, genotype and the compositions used for treatment. Preferably, for co-administration, the total amounts are less than the total amounts for each pharmaceutical compound added together.
  • appropriate release times can vary, but preferably should last from about 1 hour to about 6 months, most preferably from about 1 week to about 4 weeks.
  • Formulations for slow release dosage can vary as determinable by one of skill, according to the particular situation and as generally taught herein.
  • a dose can also be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (the concentration of the test 5 compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.
  • kits The combination of therapeutic agents may be used in the form of kits.
  • the arrangement and construction of such kits is conventionally known.
  • kits may include 10 containers for containing the inventive combination of therapeutic agents and/or compositions, and/or other apparatus for administering the inventive combination of therapeutic agents and/or compositions.
  • LOAD is a devastating neurodegenerative disease, characterized by the formation of pathogenic plaques in the brain, which affects as many as 10% of people 65 or older.
  • 25 disease is complex, and is likely to involve the interaction of a number of genes.
  • Apolipoprotein E has been reported to increase the risk of LOAD and lower the age of onset (Corder, E. H. et al. Science 261, 921-3 (1993); Fairer, L. A. et al. Jama 278, 1349- 56 (1997); Saunders, A. M. et al. Neurology 43, 1467-72 (1993)).
  • linkage peaks have been reported on chromosomes 9, 10, and 12 ( Kehoe, P. et al. Hum MoI Genet 8, 237-
  • a whole-genome association study was performed in a total of 800 unrelated case and control samples, using a dense set of SNP markers (approximately 1.5 million) that cover the entire genome. To individually screen such a large number of SNPs in all 800 samples would be prohibitive. Therefore, a two-stage approach was used. First a screen for associations using pooled sample sets was performed. Allele frequency differences between the case and control sample pools was estimated, and the estimated allele frequency differences were used to select a subset of SNPs for further evaluation. This subset, which may have contained false positives in addition to true positives, was genotyped in the individual case and control samples, and the exact allele frequency differences between the populations was calculated. SNPs showing significant association with LOAD in the original sample set were analyzed in a second sample set, to verify their association.
  • NIMH Center for Genetics Initiative The NIMH collection contains multiplex families ascertained with two or more living related individuals with AD. Individuals were evaluated clinically, with longitudinal follow-up, and diagnoses were confirmed at autopsy. We used the following criteria to select a subset of individuals with LOAD.
  • oligonucleotide arrays designed such that each SNP was interrogated by forty distinct 25 bp probes. These forty features consisted of four sets often features, corresponding to the forward and reverse strands of the two SNP alleles (reference and alternate). Each set often features consisted of two sets of five features, with offsets of -2, -1, 0, +1, and +2 bases between the center of the 25 bp probe and the SNP position. For each offset, we tiled one perfect-match feature and one mismatch feature (complement of the perfect match) at the central position of the probe. Thus, for each allele there were a total of ten perfect-match probes and ten mismatch probes. The oligonucleotide features necessary to query the 267,852 SNPs studied here were arrayed on three distinct array designs. Example 6 - Generation of Pools
  • each pool contained 100 samples, randomly selected from either the cases or controls, with each sample present in just one pool. Equimolar amounts (600 ng) of each sample were transferred into one of the eight pools robotically. Each pool was then re-quantified by PicoGreen assay and diluted to a standard concentration for use as a PCR template.
  • delta p-hat the estimated allele frequency difference between case and control pools, termed delta p-hat.
  • the fluorescence intensities of the reference and alternate perfect- match features on the arrays correlate with the concentration of the corresponding SNP allele in the DNA sample.
  • Our estimates of allele frequency, p-hat were computed from ratios of trimmed means of intensities of the perfect-match features, after subtracting a measure of background computed from trimmed means of intensities of mismatch features. The case pool ⁇ -hats and control pool p-hats were separately averaged, and the delta p-hat was calculated.
  • the aim of the pooled genotyping is to enrich for significant SNPs, so that we reduce the number of SNPs requiring genotyping in each of the individual samples We expect a large number of the SNPs selected for individual genotyping on the basis of the pooled genotypmg results to be false positives Thus it is important to show that amongst these false positives we have captured a large fraction of the SNPs that we are looking for - those with true allele frequency differences between the case and control populations. That is, is the pooled genotyping powerful enough to identify SNPs with significant allele frequency differences between the case and control pools, even if the differences are relatively small. In this experiment we used criteria that resulted in the selection of approximately
  • the PCRs were performed in 384-well plates containing DNA template (IOng) and PCR cocktail (1.47 ⁇ l 1OX AK2 buffer (0.5M Trizma, 0.14M ammonium sulfate, and 27mM MgC12), 0.03M tricine, 0.67 ⁇ l MasterAmp 1OX PCR Enhancer (Epicentre, Madison, WI), 3.9% DMSO, 0.05M KCl, dNTPs (0.54 mM each), PCR primers (0.42 pmol/ ⁇ l/primer), and - ⁇ 2X Titanium Taq polymerase (BD Biosciences, Palo Alto, CA)).
  • the PCR plates were sealed prior to PCR. Short-range PCR was performed for approximately three hours.
  • the thermocycler hlock was allowed to reach 90 0 C before the PCR plates were placed in the thermocycler.
  • the thermocycler program used for short-range PCR is identified in Table 12-1 :
  • the plates were removed from the thermocycler and were pooled as described infra. (At this point, the plates could also have been stored at -20 0 C for an extended period, if so desired.)
  • Hybridization buffer 1.5M TMACL (tetramethylammonium chloride), 5mM Tris (pH 7.8 or 8.0), 0.005% Triton X-100, 26 pM b-948 control oligo (Genset, La Jolla, CA), and 0.05 mg/ml HS (herring sperm) DNA
  • TMACL tetramethylammonium chloride
  • Tris pH 7.8 or 8.0
  • Triton X-100 0.005% Triton X-100
  • 26 pM b-948 control oligo Geneset, La Jolla, CA
  • HS herring sperm
  • the mixture containing the denatured samples and hybridization buffer was transferred to a prewarmed microarray.
  • the array was sealed, returned to the 5O 0 C hybridization oven, and rotated at 20 r.p.m. overnight (14-19 hours). After the overnight incubation, the array was stained, washed and scanned as described below.
  • the microarray was removed from the hybridization oven and the sample was removed and stored at -2O 0 C. Then, the microarray was washed l-2x with 200 ⁇ l of IX MES/0.01% Triton X-IOO. The microarray was inverted several times to ensure that the wash solution moved freely over the surface of the microarray prior to removing the wash solution by vacuum suction.
  • the "Third Stain Solution” (174 ⁇ l of IX MES/0.01% Triton X-100, 25 ⁇ l of 20 mg/ml acetylated BSA, and 1 ⁇ l of 0.2 mg/ml streptavidin Cy-chrome) was added to each microarray.
  • the microarray was inverted several times to ensure that the Third Stain Solution moved freely over the surface of the microarray.
  • the microarray was rotated at 25 r.p.m. for 15 minutes at room temperature.
  • the microarray was washed with IX MES/0.01% Triton X-100 wash solution in a RevD Fluidics Station. When the wash was finished the microarray was removed from the fluidics station and the IX MES/0.01% Triton X-100 wash solution was removed by vacuum suction.
  • a wash solution of 6X SSPE/0.01% Triton X-100 was added to the microarray.
  • the microarray was inverted several times to ensure that the 6X SSPE/0.01% Triton X-100 moved freely over the surface of the microarray before it was removed by vacuum suction.
  • a wash solution of 0.2X SSPE/0.005% Triton X-IOO that had been prewarmed to 37 0 C was added to the microarray, which was then incubated at 37 0 C for 30 minutes.
  • the 0.2X SSPE/0.005% Triton X-IOO was removed by vacuum suction and a solution of IX MES/0.01% Triton X-100 was added to the microarray.
  • microarray was then inverted several times before the IX MES/0.01% Triton X-100 was removed by vacuum suction. Finally, fresh IX MES/0.01% Triton X-100 was added to the microarray, which was wrapped in foil prior to storage at 4 0 C or scanning of the microarray.
  • Example 17 On the same days the microarrays were stained and washed, they were scanned using an arc scanner. After scanning, the microarrays were removed from the scanner, wrapped in foil and stored at 4°C. The scan files generated by the scanner were then analyzed by software programs designed to interpret intensity data from microarrays. For the pooled genotyping, this software allowed discrimination of hybridization patterns that distinguished the case pools from the control pools. The data were analyzed according to the methods disclosed in the following U.S. patent applications, all of which are assigned to the assignee of the present applications: U.S. provisional patent application no. 60/460,329, filed on April 3, 2003, entitled “Apparatus and Methods for Analyzing and Characterizing Nucleic Acid Sequences"; and U.S. patent application no. 10/768,788, filed January 30, 2004, entitled “Apparatus and Methods for Analyzing and Characterizing Nucleic Acid Sequences”.
  • Nucleic acids that were identified as strongly associated with the case or control group based on the pooled genotyping analysis were reanalyzed by genotyping individual samples for those potentially associated nucleic acids, as described below. As such, individual genotyping was performed on approximately 30,000 (-2%) of the original 1.7 million SNPs. For the individual genotyping, the software assigned genotypes at each SNP position for each individual in the case and control groups. The data were analyzed according to the methods disclosed in the following U.S. patent applications, all of which are assigned to the assignee of the present applications: U.S. patent application no. 10/351,973, filed January 27, 2003, entitled “Apparatus and Methods for Determining Individual Genotypes"; and U.S. patent application no. 10/786,475, filed February 24, 2004, entitled “Improvements to Analysis Methods for Individual Genotyping.”
  • SNPs for the following IG were selected from 4 different sources.
  • SNPs in chromosomal regions of interest on chromosomes 10, 12 and 19 were identified and provided. In particular, 3656 SNPs were selected from a region between positions 60,000,000 and 95,000,000 on chromosome 10, 1173 SNPs were selected from a region between positions 2,000,000 and 10,000,000 on chromosome 12, and 50 SNPs were selected from a region between positions 50,000,000 and 50,200,000 on chromosome 19.
  • the custom IG (individual genotyping) chip contained 25,990 SNPs and was composed of SNPs selected from PG, representative SNPs for candidate genes on chromosomes 10, 12 and 19, 311 stratification SNPs and SNPs that failed amplification on PG chips
  • the SNPs were catego ⁇ zed into categories by first assigning all SNPs that were selected as part of the candidate gene region to the "candidate gene regions" category (4879 SNPs), then from the remaining SNPs all SNPs that were included on the chip due to the unamphfied 10 p ⁇ mer plates in PG were assigned to "10 missing primer plates" category (970 SNPs), then again from the remaining SNPs after the above selections all SNPs that were included on the chip by selection from PG were assigned to the "pooled” category (19969 SNPs) and the remaining stratification SNPs were assigned to the "extra” category (172 SNPs) Due to a significant overlap between SNPs selected from PG and the stratification SNPs the "
  • SNPs were filtered for obvious genotyping problems in round 1 and replication.
  • SNPs that have call rate in replication ⁇ 80 or have any of the 3 chi-square p-values measuring allele frequency differences between cases in the 3 different protocol IG analyses ⁇ 0.05/25990 or have any of the 3 similar chi- square p-value for controls ⁇ 0.05/25990 were labeled with is_replication_ok 0 indicating likely problem with replication genotypes.
  • the 799 PG samples were individually genotyped on this custom chip as part of round 1 IG.
  • the individual genotyping of the PG samples was done in two phases. In the first phase, we have included for each sample its best pass rate scan and a redo scan (if available) that had pass rate within 10% of the best scan pass rate. This genotyping was used to select a set of canonical scans for the samples. The best call rate scan for each sample was selected, however samples with scans that had best scan call rate ⁇ 80% were excluded. This set of 787 scans (784 NIA samples + 3 CEPH samples) was genotyped in the second phase. The second phase yielded the final genotypes for the PG samples on the IG chip.
  • Table 18-2 provides the Y chromosome call rate and the X chromosome heterozygosity of the 6 excluded samples: Table 18-2
  • K ⁇ r( ⁇ p) (p, + P 11 - 2P 1 1 J -J- + 2n ⁇ 2n C i
  • ⁇ 1 is the overall population prevalence of the arbitrary designated "1" allele
  • " is the fraction of samples that have two copies of allele "1”
  • c and n ⁇ are the number of case and control samples, respectively.
  • the stratified trend score was computed as follows: ⁇ s ⁇ lralat ⁇
  • the association of phenotype with the SNP genotypes was evaluated using logistic regressions and ANOVA tests.
  • the genotype-phenotype association was computed using an ANOVA test between a simple model that includes possible covariates but does not include genotype-related terms and a model that includes these covariates, genotype and genotype- covariate interaction terms.
  • the simple model involved gender, age at which the individual was diagnosed with Alzheimer disease or be disease free (called age from here on) and the population fractional ancestry (PFA) inferred from STRUCTURE.
  • the second more complex model with genotype had additional terms consisting of genotype, gender-genotype and age- genotype interaction.
  • the gender-genotype interaction term models possible differences in genotype effect between males and females and the age-genotype interaction models possible differences in genotype effect at early and late ages.
  • the trend scores were corrected using GC correction.
  • a set of SNPs that is unbiased in respect to the inflation of small association p-values between cases and controls was used to compute the Genomic Control (GC) variance inflation factor.
  • the SNPs that were included in the IG either by selection from pooled SNPs (see section above) or as representative SNPs for the selected candidate gene regions, were excluded from the set that was used to evaluate the GC variance inflation factor for both the round 1 and combined sample analyses.
  • SNPs that were selected from PG are expected to show inflation of small p-values due to either population structure or random sampling differences or true associations with phenotype between cases and controls among the PG samples.
  • SNPs therefore will show inflation of small p- values in IG if the samples that were used in the PG are included in the analysis and thus these SNPs were excluded from the GC variance inflation analysis of round 1 and combined sample set.
  • the candidate gene region SNP representatives also have a non-neutral prior probability to show association between cases and controls and thus were also excluded from the GC variance inflation analysis for round 1 and combined set of samples.
  • the unbiased set of SNPs consisting of stratification SNPs and SNPs that were added due to failure of amplification in the PG were analyzed for GC variance inflation for round 1 and combined sample set after filtering for call rate > 80% and for SNPs that do show all 3 genotypes (homozygous reference and alternate and heterozygous).
  • FWER Familywise error rate
  • 1000 case - control assignment permutations were created while conserving each sample's stratum assignment. These permutations were used to compute 1000 stratified trend scores for each SNP.
  • the FWER was computed as the number of permutations that yielded any stratified trend score higher than the SNPs' stratified trend score, divided by the total number of permutations (1000 in this case).
  • the FWER reflects any LD structure among the SNPs, because the LD structure is preserved in permutations and because the FWER is the probability of making one or more Type 1 errors in the set of tests at different significance levels. These p-values are superior to Bonferroni corrected p-values which do correctly represent the number of independent tests.
  • the FWER scores also represent exact p-values with no assumptions on the parametric distribution of the stratified trend scores.
  • FDR False Discovery Rate
  • the "expected number of significant SNPs” is just the number of false positives that we would expect at the given level of statistical significance (i.e. 18576*significance cutoff). It is obvious that the PG must have enriched for large case-control differences. Only SNPs that were selected from PG and have call rate > 80% were counted. There are 18,576 of such SNPs.
  • the IG results were used to estimate the PG power.
  • the power was evaluated over SNPs that were selected for the IG chip independent of the PG results (the candidate gene SNPs, and the stratification SNPs), but that were also analyzed in the PG study. Therefore the estimate gives us a true unbiased estimate of the power since the set of SNPs is an unbiased subset from the PG chips.
  • This set of SNPs was further filtered for call rate > 80% and Hardy Weinberg Equilibrium p-value > 0.0001 for both cases and controls to ensure good genotyping and therefore an accurate estimate of the allele frequency of cases and controls in the IG.
  • Table 18-4 gives the estimates of power (in %) to select from PG SNPs with varying allele frequency differences using different cutoffs for the corrected t-test empirical p-value:
  • Standard trend scores and protocol-stratified trend scores were computed and corrected using GC variance inflation factors (for details see Methods section)
  • a variance inflation factor of 1 099 was computed from the mean trend scores and 1 086 from the protocol-stratified trend scores from all autosomal SNPs after applying a filter for call rate > 80% in all 3 protocol IG analyses, for SNPs that have all 3 genotype clusters, for all 3 chisq tests for allele frequency differences in controls between different replication protocols > 0 05/25990 and also the similar 3 chisq tests for cases > 0 05/25990 (20,015 SNPs after the filtering)
  • the variance inflation factor revealed minimal population structure or sampling differences between cases and controls and the STRUCTURE program confirmed that the population fractional ancestry distributions are very similar tor cases and controls (see above)
  • Standard trend scores and trend scores stratified on the four IG analyses were computed.
  • the stratification on the IG analyses ensures that the case-control imbalances in the replication set done with 3 different lab protocols are handled correctly after being genotyped separately and also that the replication and round 1 genotypes are correctly combined. Since we have seen some population structure differences between round 1 and replication, the trend score stratification corrects for these and other possible confounding differences between round 1 and replication. Both sets of trend scores were corrected using GC correction.
  • the GC variance inflation factor computed over all autosomal SNPs in the "10 missing primer plates" and stratification SNPs that did have all 3 genotype clusters, had call rates in all 4 IG analyses > 80% and had all 3 chisq tests for allele frequency differences in controls between different replication protocols > 0.05/25990 and also the similar 3 chisq tests for cases > 0.05/25990 (961 SNPs after the filtering). This stringent selection ensures that all SNPs that are used for the computation of GC variance inflation factor are well genotyped.
  • the GC variance inflation factor was 1.078 for trend scores and 1.088 for the stratified trend scores.
  • the logistic regression was not stratified on the three different protocols that were genotyped separately with a fourth stratum for the round 1 samples, therefore a filter must be applied to make sure that the genotype clustering is consistent between the 3 replication protocols.
  • the chi-square test for the allele frequency differences for controls between the 3 different genotyping analyses and the similar test for cases will reveal the inconsistencies in replication. Therefore when looking at the logistic regression ANOVA p-values a filter must be applied to eliminate SNPs with any of the above mentioned six chi-square p-values ⁇ 0.05/25990.
  • SNP ID no. 4813803 An additional SNP is significant after Bonferroni correction in the combined sample analysis using GC corrected stratified trend score (SNP ID no. 4813803). This additional SNP is in the intron of APOE and upstream from APOCl and downstream from TOMM40 on chromosome 19. All of the 7 SNPs are also significant after Bonferroni correction using the logistic regression ANOVA test.
  • the replication samples provide an unbiased estimate of the FDR q-values for the "pooled" SNPs.
  • Table 18-6 shows the distribution of SNPs that have FDR q-values ⁇ 50% for the stratified GC corrected trend scores:
  • Example 19 In this Example, an additional 433 cases and 473 controls (“replication samples”) were individually genotyped at the same 25,990 SNPs, and these genotypes along with the individual genotypes of the original 400 cases and 400 controls were used to compute a final set of delta P values. These additional samples come from two sets of replication samples. The first set consisted of 222 cases (non-familial LOAD patients) and 191 controls. These were clinic-based cases and controls. All controls were evaluated using a neuropsychological battery of tests.
  • Table 19-2 columns 1 and 3 provide references for a SNP position.
  • Table 19-2, column 2 provides the chromosome containing a SNP.
  • Columns 4, 6, and 8 are different statistical analyses used to determine significance of the delta P values.
  • Column 8 lists p- values from the Cochran-Armitage trend test (Freidlin et al., Human Heredity 2002;53 : 146- 152).
  • Column 4 lists p- values from the trend test that have been corrected for population stratification.
  • Column 6 lists Chi-squared p-values.
  • Column 7 lists Hardy- Weinberg Equilibrium chi-squared p-values computed from the allele frequencies of the controls.
  • Column 5 lists delta P values (the difference in frequency of a reference allele between cases and controls).
  • Table 19-3 lists shows the identity of the reference allele used in calculating the delta p values shown in Table 19-2.
  • the total number of SNPs we attempted to genotype in round 2 IG is 50707. These include 311 stratification SNPs and 202 SNPs previously found to be on top of the list based on an analysis of the first round IG data on essentially the same samples The rest of the SNPs were selected based on pooled genotypmg results of ⁇ 1 6 million tested SNPs
  • the number of SNPs m the genotype report is 40432 (before any SNPs with bad genotype clustering were manually eliminated) Based on manual inspection of the genotype clustering of the top 160 SNPs in the trend test for all Caucasian samples, 16 SNPs with obvious bad clustering were identified These SNPs are excluded from further analysis Unless indicated otherwise, further analysis does not include any of the previously identified 202 SNPs 285 out of 311 stratification SNPs are included m the genotype report After eliminating the 202 previously identified SNPs and the 16 SNPs with bad genotype clustering, there were 40229 SNPs left for analysis
  • the stratification SNPs are unbiased as far as variance inflation is concerned
  • the mean trend test statistic for stratification SNPs is 1 131 for all Caucasian samples
  • the 95% confidence interval for this mean is [0 835, 1 165] as obtained from simulation given the number of stratification SNPs Therefore, the 1 131 value is not large enough for us to rule out our null hypothesis that our variance inflation factor is 1
  • the test statistics for the trend test scores of the 40229 SNPs for the pg samples are not expected to follow the chi-squared distribution with 1 degree of freedom since most of the SNPs are selected from pooled genotyping data.
  • the QQ plot in Figure 20-1 simply shows that the selected SNPs are indeed enriched for allele frequency differences between cases and controls in the pg samples.
  • the QQ plot of the trend test statistics for the non-pg Caucasian samples indicates that there is a small inflation of test statistic, with a mean test statistic value of 1.036.
  • Figure 20-2 QQ plot of the trend test scores for Caucasian non-pg sample data
  • the QQ plot for the test statistics is shown in Figure 20-2.
  • the largest test score is 18.84, which corresponds to a P value of 1.4e-5.
  • Association test of all Caucasian sample data is shown in Figure 20-2.
  • Figure 20-3 QQ plot of the trend test scores for all Caucasian sample data
  • Association test of the Caucasian non-pg sample data The QQ plot for statistics obtained from testing the two genotype-containing terms is shown in Figure 20-5. This analysis did not identify any SNPs to be genome-wide significant after correcting for the number of SNPs analyzed.
  • Figure 20-6 Maximum F statistics (df 1,1674) obtained in haplotype trend regression tests for different sliding windows Window sizes are shown in legend. Only haplotype alleles with at least 5% allele frequencies are tested. Missing values arise from situations where none of the haplotype alleles in a sliding window has a high enough allele frequency to be tested.
  • haplotype analysis was performed for the chromosomal region of chromosome 19 near the APOE gene.
  • Haplotype trend regression tests were performed for sliding window sizes 1-15 for 61 SNPs with positions in the range between 49131974 and
  • the maximum F statistic values for each haplotype sliding window are shown in Figure 21-1.
  • the F statistics obtained for window sizes of 1 are very similar to the chi- squared statistics obtained from trend tests, reflecting results of single SNP association tests.
  • haplotype window sizes greater than one the most significantly associated positions are closer to the APOE gene.
  • SNP #38 (snp id 4813803), which is in gene APOE, is included in every one of these haplotypes (Table 21-2).
  • Table 21-1 The F statistics from haplotype trend regression tests for window size 1. All SNPs with an F statistic larger than 10 (an arbitrary high cutoff value) happen to be adjacent to each other, and they are all listed in this table ordered by their positions.
  • Figure 21-1 Maximum F statistics (df 1,1673) obtained in haplotype trend regression tests for different sliding windows. Window sizes are shown in legend. Only haplotype alleles with at least 5% allele frequencies are tested Missmg values arise from situations where none of the haplotype alleles in a sliding window has a high enough allele frequency to be tested.
  • haplotypes were examined for three associated genes, PSEN2, APP, and HDAC4.
  • the haplotype blocks were obtained with Haploview with the default settings using all 1713 samples.
  • the haplotype block containing the most significant SNP (in pooled genotyping samples) in or around the gene is shown. Only haplotype alleles with frequencies larger than 5% are inspected here.
  • the haplotype allele frequencies are obtained by fastPHASE based on all 1713 samples (allele frequencies obtained from Haploview are extremely similar).
  • the SNP genotype shown in underlined bold font is the risky allele
  • the genotype shown in underlined italic font is the protective allele. Additional data for this analysis is provided in the file
  • [2 kb haplotype block, contains SNPs 4010865, 4010866, and 4232917 (arbitrary SNP index
  • SNP #15-17 and three major haplotype alleles (SNP #15-17, the first SNP is 4010865)]
  • a chi-squared statistic was obtained for each SNP by testing the following two nested logistic regression models: early_onset - PCl, and early_onset ⁇ PCl + genotype, where genotype is coded as 0, 1, or 2.
  • the histogram of P values of all analyzed 9724 SNPs included in IG due to pooled genotyping analysis results (PG) and of all 307 analyzed stratification SNPs are shown in Figure 23-3. There is clearly an enrichment of small P values for the PG SNPs.
  • the mean test statistic for the 307 stratification SNPs is 1.075, which is well within the 95% confidence interval of (0.849, 1.164) obtained by simulation based on the null distribution.
  • An F statistic is obtained for each SNP by testing the following two nested linear regression models age_of_onset ⁇ PCl, and age_of_onset ⁇ PCl + genotype, where genotype is coded as 0, 1, or 2
  • PG 1 indicates that SNP is selected based on pooled genotyping analysis results, 0 otherwise
  • STRAT 1 indicates that SNP belongs to the 311 stratification SNP set, 0 otherwise
  • SEX 1 indicates that SNP belongs to the 72 X-linked QC SNP set, 0 otherwise
  • the D4 allele of the APOE gene (accession NC_000019.8, position 50100879 to 50104490 according to NCBI Build 36.2) is known to be associated with early age of onset for Alzheimer's.
  • SNPs analyzed in pooled genotyping there are 34 SNPs within 50kb of the APOE gene. Five of them have empirical P values less than the cutoff (0.02940) for being included in individual genotyping (Table 23-2).
  • the "use_for_case_control_analysis” column specifies whether a sample is included in the analysis. If a sample is labeled as Caucasian and is not one of the 3 excluded samples (90C02115, 90C03354, and CBT82001), the use_for_case_control_analysis value is 1. There are 1675 such samples.
  • the "use_for_age_of onset analysis” column specifies whether a sample is included in the current age of onset analysis.
  • a sample is a case, and it has a use_for_case_control_analysis value of 1, and it is not sample ARC30679 (with inconsistent age of onset info), then the use for age_of_onset value is 1.
  • the "g[#]" columns are genotypes for the samples. "0" means homozygous reference; “1” means heterozygous, and “2” means homozygous alternate. Missing values are possible, gl is a haplotype allele genotype, and is the best estimate of the allele genotype obtained by the fastPHASE software. All other genotypes are for single SNPs. g2-g9 are the genotypes of all single SNPs for F test statistics of larger than 50.
  • SNPs are all SNPs in the TOMM40/APOE region with individual genotype data in the study comprising pooled genotyping of ⁇ 1.6 million SNPs. Tests were carried out to further test the association between late-onset Alzheimer's disease and the three SNPs in each of PSEN2, APP, and HDAC4, in part by excluding early- onset cases from the analysis, and examples of the results are found on Tables 24-1, 24-2, 24- 3, 24-4, and 24-5. The 717 “pg” samples include 358 cases and 359 controls, and the 958 "npg” samples include 478 cases and 480 controls. Table 24-1
  • the 683 "pg” samples with age-of-onset below 65 include 324 cases and 359 controls, and the 832 "npg” samples with age-of-onset above 65 include 370 cases and 462 controls.
  • the 504 "pg” samples with age-of-onset below 70 include 225 cases and 279 controls, and the 625 "npg” samples with age-of-onset above 70 include 255 cases and 370 controls.
  • Table 24-7 provides analyses for testing an association between genotype and age-of- onset. Only data for 835 case samples were used for the analysis. The association was tested using linear models. The genotype was treated either as a numeric variable (Test 1) (i.e., assuming linear effects on the age-of-onset), or as a factor (Test 2) (allowing the 3 genotypes to have 3 different effect sizes on age-of-onset). The null hypothesis was that the age-of-onset in the case samples was independent of the genotype.

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Abstract

La présente invention concerne une série de sites polymorphes conférant résistance ou prédisposition à la maladie d'Alzheimer et à d'autres maladies apparentées. Ces sites peuvent être utilisés dans le cadre de procédés de diagnostic et de traitement de la maladie d'Alzheimer et d'autres maladies apparentées.
PCT/US2009/031909 2008-01-23 2009-01-23 Base génétique de la maladie d'alzheimer et diagnostic et traitement de cette dernière WO2009094592A2 (fr)

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US10106602B2 (en) 2012-01-27 2018-10-23 AbbVie Deutschland GmbH & Co. KG Isolated monoclonal anti-repulsive guidance molecule A antibodies and uses thereof
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