WO2003061583A9 - Digenic mutations associated with severe insulin resistance and type 2 diabetes and their use in the diagnosis and treatment of diabetes - Google Patents

Digenic mutations associated with severe insulin resistance and type 2 diabetes and their use in the diagnosis and treatment of diabetes

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Publication number
WO2003061583A9
WO2003061583A9 PCT/US2003/001625 US0301625W WO03061583A9 WO 2003061583 A9 WO2003061583 A9 WO 2003061583A9 US 0301625 W US0301625 W US 0301625W WO 03061583 A9 WO03061583 A9 WO 03061583A9
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protein
diabetes
seq
polynucleotide
sample
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PCT/US2003/001625
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French (fr)
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WO2003061583A2 (en
WO2003061583A3 (en
Inventor
Ines Barroso
Alan J Schafer
Stephen O O'rahilly
Nicholas J Wareham
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Incyte Genomics Inc
Ines Barroso
Alan J Schafer
Stephen O O'rahilly
Nicholas J Wareham
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Application filed by Incyte Genomics Inc, Ines Barroso, Alan J Schafer, Stephen O O'rahilly, Nicholas J Wareham filed Critical Incyte Genomics Inc
Priority to AU2003205230A priority Critical patent/AU2003205230A1/en
Publication of WO2003061583A2 publication Critical patent/WO2003061583A2/en
Publication of WO2003061583A9 publication Critical patent/WO2003061583A9/en
Publication of WO2003061583A3 publication Critical patent/WO2003061583A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates to the use of mutations in PPAR ⁇ and PPP1R3A and its encoding polynucleotides to diagnose, to stage, to treat, or to monitor the progression or treatment of diabetes, including insulin resistance and type 2 diabetes.
  • Type 2 diabetes is a serious health problem in the Western world. It arises when resistance to the glucose-lowering effects of insulin combines with impaired insulin secretion to raise the levels of glucose in the blood beyond the normal range.
  • PPAR ⁇ peroxisome proliferator-activated receptor gamma
  • PPP1R3A a muscle-specific regulatory subunit of protein phosphatase-1 (PP1).
  • PPAR ⁇ is a key regulator of adipocyte differentiation and a target for a new class of antidiabetic drugs, thiazolidinediones; highly specific ligands for PPAR ⁇ that act to increase insulin sensitivity (Lehman et al (1995) J Biol Chem 270:12953-12956; Day, C (1999) Diabetic Med 16:179- 192).
  • the requirement for PPAR ⁇ in m-imtaining normal insulin sensitivity in humans was further confirmed in a key study by Barroso et al identifying two loss-of-function mutations in PPAR ⁇ associated with severe insulin resistance (SIR) and diabetes mellitus in humans (Barroso et al (1999) Nature 402:880-883).
  • PPP1R3A is a key molecule in the regulation of glycogen synthesis, the muscle and liver mode of glucose storage. Acting in part through PPP1R3 A, insulin activates glycogen synthase by the activation of PP1 which, in turn, activates glycogen synthase through its dephosphorylation. Impaired insulin-stimulated glycogen synthesis is a characteristic feature of type 2 diabetes , however, the potential role of PPP1R3 A in participating and leading to this impairment is uncertain.
  • Retinitis pigmentosa is caused by mutations in ROM1 and RDS, which encode subunits of an oligomeric transmembrane protein complex in photoreceptor discs (Goldberg, A.F. and Molday, R.S. (1996) Proc Natl Acad Sci USA 93:13726-13730).
  • Junctional epidermolysis bullosa results from mutations in laminin 5 and collagen XVII, two of the components of the hemidesmosome-anchoring filament in skin (Floeth, M and Bruckner-Tuderman, L (1999) Am J Hum Genet 65:1520-1537).
  • Bardet-Biedl syndrome may be a complex trait requiring three mutant alleles in at least two genes to manifest the phenotype (Katsanis et al (2001) Science 293:2256-2259).
  • the present invention provides compositions and their methods of use in the diagnosis, prevention and/or treatment of diabetes, including insulin resistance and type 2 diabetes.
  • the invention is based on the discovery two mutations in the genes encoding PPAR ⁇ and PPP1R3A, designated PPAR ⁇ FS and PPP1R3 AFS, respectively, which are useful to diagnose, stage, treat, or to monitor the progression or treatment of a diabetes, including insulin resistance and type 2 diabetes.
  • the invention provides a method for the diagnosis of diabetes in a subject by genotyping of a subject's DNA to detect the presence of the two mutations for PPAR ⁇ FS and PPP1R3AFS in a sample of genomic DNA from the subject.
  • the invention also provides a method for diagnosing a predisposition or a risk factor for development of diabetes in a subject by genotyping of a subject's DNA to detect the presence of the mutation for PPP1R3AFS, alone.
  • the invention provides an isolated polynucleotide comprising a nucleic acid sequence encoding a protein having the amino acid sequence of SEQ ED NO:l (PPAR ⁇ FS) or SEQ ID NO:2 (PPP1R3AFS,).
  • the invention also provides an isolated polynucleotide or the complement thereof comprising a nucleic acid sequence of SEQ ID NO:3 (encoding SEQ ID NO:l) or SEQ ID NO:4 (encoding SEQ ED NO:2), a cell transformed with the polynucleotide encoding PPAR ⁇ FS or
  • PPP1R3AFS a composition comprising the polynucleotide encoding PPAR ⁇ FS or PPP1R3AFS, and a labeling moiety, and an array element comprising the polynucleotide encoding PPAR ⁇ FS or PPP1R3AFS.
  • the invention provides a vector containing the polynucleotide encoding PPAR ⁇ FS or PPP1R3AFS, a host cell containing the vector and a method for using the polynucleotide to make the protein, the method comprising culturing the host cell containing the vector containing the polynucleotide encoding the protein under conditions for expression and recovering the protein from the host cell culture.
  • the invention further provides a composition, a substrate or a probe comprising the polynucleotide, or complement thereof, which can be used in methods of detection, screening, and purification.
  • the probe is a single-stranded complementary RNA or DNA molecule.
  • the invention provides a method for using a nucleic acid probe to detect the differential expression of a nucleic acid in a sample comprising hybridizing a probe to the nucleic acids, thereby forming hybridization complexes and comparing hybridization complex formation with a standard, wherein the comparison indicates the differential expression of the nucleic acid in the sample.
  • the method of detection further comprises amplifying the nucleic acids of the sample prior to hybridization.
  • the method showing the coexpression of the polynucleotides encoding PPAR ⁇ FS and PPP1R3AFS is diagnostic of type 2 diabetes .
  • the invention provides a purified protein comprising SEQ ID NO:l or SEQ ID NO:2.
  • the invention further provides a method for detecting expression of a protein having the amino acid sequence of SEQ ID NO:l or SEQ ID NO:2 in a sample, the method comprising performing an assay to determine the amount of the protein in a sample; and comparing the amount of protein to a standard, thereby detecting expression of the protein in the sample.
  • the assay is selected from affinity chromatography, antibody arrays, enzyme-linked immunosorbent assays, fluorescence- activated cell sorting, protein arrays, radioimmunoassays, and 2D-PAGE in conjunction with scintillation counting, high performance hquid chromatography, mass spectrometry or western analysis.
  • the assay detects the expression of SEQ ID NO: 1 in the sample and is diagnostic of a predisposition to diabetes.
  • the assay detects the coexpression of SEQ ID NO:l and SEQ ID NO:2 and is diagnostic of the presence of type 2 diabetes.
  • the invention provides a method for using a protein to screen a library or a plurality of molecules or compounds to identify at least one ligand, the method comprising combining the protein with the molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein.
  • the molecules or compounds are selected from agonists, antagonists, DNA molecules, small drug molecules, immunoglobulins, inhibitors, rnimetics, multispecific molecules, peptides, peptide nucleic acids, pharmaceutical agent, proteins, and RNA molecules.
  • the ligand is used to treat a subject with diabetes.
  • the invention further provides an agonist which specifically binds PPP1R3A.
  • the invention yet further provides a small drug molecule which specifically binds PPP1R3 A.
  • the invention also provides a method for testing ligand for effectiveness as an agonist or antagonist comprising exposing a sample comprising the protein to the molecule or compound, and detecting agonist or antagonist activity in the sample.
  • the invention provides an agonist that specifically binds the protein, and a composition comprising the agonist and a pharmaceutical carrier.
  • the invention further provides a pharmaceutical agent or a small drag molecule that specifically binds the protein.
  • the invention provides a method of treating diabetes comprising administering to a patient in need of such treatment an effective amount of an agonist of PPP1R3 A.
  • Figure 1 illustrates the effects of the mutations in PPAR ⁇ and PPP1R3 A on truncation of the proteins.
  • PP1C/GBD PP1 catalytic subunit and glycogen binding domains;
  • SRBD sarcoplasmic reticulum-binding domain.
  • Figure 2 illustrates a family pedigree, Family A, and the concordance of features related to severe insulin resistance and type 2 diabetes and the presence of PPAR ⁇ and PPP1R3 A mutations.
  • Solid coloring in individual family members indicates the presence of the phenotypes: upper left quadrant, acanthosis nigricans + hyperinsulinemia; upper right, diabetes; lower left, dyslipidemia; lower right, hypertension.
  • Figure 3 represents fasting plasma insulin concentrations (Y axis) plotted against body mass index (BMI; X axis) for family A members.
  • the solid line represents the log-linear regression line between FI and BMI in 1121 participants in the MRC Ely population-based cohort study (Williams et al. (1994) Diabetic Medicine 12:30-35).
  • the 95% confidence intervals (broken lines) include 95% of the individuals at any given BMI.
  • Figure 4 illustrates a Family B pedigree, the presence of the R3 mutation, and the incidence of phenotypes related to diabetes. Abbreviations and the presence of various phenotypes are the same as described for Figure 2.
  • Figure 5 A shows the results of an assay to measure DNA binding properties of the FS mutation in either PPAR ⁇ l (FS ⁇ l) or PPAR ⁇ 2 (FS ⁇ 2) compared with the corresponding wild type gene products (WT ⁇ l and WY ⁇ 2, respectively).
  • the DNA binding assay is described in Example II.
  • the open arrow indicates the location of the PPAR ⁇ -RXR heterodimer, and the solid arrow denotes the unbound probe.
  • RL reticulocyte lysate;
  • RXR retinoid X receptor.
  • Figure 5B shows 35 S- labeled in vitro translated wildtype and mutant PPAR ⁇ l and PPAR ⁇ 2.
  • Figure 6 shows the results of receptor mediated transactivation in 293 EBNA cells transfected with either PPAR ⁇ wild type isoforms (WT ⁇ l or WT ⁇ 2), or mutant WT ⁇ (FS ⁇ l or FS ⁇ 2) together with a reporter gene, (PPARE) 3 TKLUC, in the presence of increasing concentrations of the thiazolidinedione, rosiglitazone.
  • Vector empty expression vector + reporter gene. Results are expressed as % of the maximum transactivation obtained with WT ⁇ l.
  • Figure 7 shows the results of cotransfection of 293 EBNA cells with 100 ng each of wild type + wild type PPAR ⁇ (WT + WT) or wild type PPAR ⁇ + mutant PPAR ⁇ (WT + FS) together with the reporter gene, (PPARE) 3 TKLUC, and consequent effects on transactivation as determined in Figure 6. Results are expressed as % of the maximum transactivation obtained with WT ⁇ l.
  • Figures 8 shows western blots of whole cell lysates from CHO cells transiently transfected with HA-tagged wild type PPP1R3 A (WT) or PPP1R3AFS (FS).
  • Figure 8A shows the western blot analyzed using a sheep monoclonal N-terminal PPP1R3A antibody.
  • Figure 8B shows the results of a western blot in which the cell lysates were first immunoprecipitated with anti-HA antibody (Santa Cruz Biotechnology, Inc, Santa Cruz, CA) followed by western blot with an anti-PPlC antibody (Santa Cruz Biotechnology).
  • Figure 9 shows the results of immunofluoresence microscopy of CHO cells transfected with HA-tagged wild type PPP1R3A (WT) or PPP1R3AFS (FS).
  • WT wild type PPP1R3A
  • FS PPP1R3AFS
  • Table 1 summarizes clinical and biochemical characteristics of the frameshift mutation carriers for both PPAR ⁇ FS and PPP1R3 AFS in both Family A and Family B members.
  • HDL high-density lipoprotein
  • NEFA non-esterified fatty acids
  • IMCL intramyocellular lipid.
  • antibody refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic determinant.
  • Antibodies that bind PPAR ⁇ FS or PPP1R3AFS polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide or oligopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
  • RNA e.g., a mouse, a rat, or a rabbit
  • Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
  • antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
  • PPAR ⁇ FS refers to a PPAR ⁇ mutation that is the same or highly homologous (>85%) to the amino acid sequence of SEQ ID NO:l or the polynucleotide sequence of SEQ ID NO:3 obtained from any species including bovine, ovine, porcine, murine, equine, and preferably the human species, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • PPP1R3AFS refers to a PPP1R3A mutation that is the same or highly homologous (>85%) to the amino acid sequence of SEQ ID NO:2 or the polynucleotide sequence of SEQ ID NO:4 obtained from any species including bovine, ovine, porcine, murine, equine, and preferably the human species, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • nucleic acid of the Sequence Listing refers to a nucleic acid molecule which is completely complementary over its full length and which will hybridize to a nucleic acid molecule under conditions of high stringency.
  • composition comprising a given polynucleotide and a “composition comprising a given polypeptide” can refer to any composition containing the given polynucleotide or polypeptide.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotides encoding PPAR ⁇ FS or PPP1R3 AFS or fragments of PPAR ⁇ FS or PPP1R3 AFS may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
  • salts e.g., NaCl
  • detergents e.g., sodium dodecyl sulfate; SDS
  • other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
  • Constant amino acid substitutions are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions.
  • the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
  • Val lie, Leu, Thr Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • a “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
  • “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, dete ⁇ riined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
  • An "expression profile” is a representation of gene expression in a sample.
  • a nucleic acid expression profile may be produced, for example, using sequencing, hybridization, or amplification (quantitative PCR) technologies and mRNAs or cDNAs from a sample.
  • a protein expression profile although time delayed, mirrors the nucleic acid expression profile and may use antibody or protein arrays, enzyme-linked immunoadsorbent assays, fluorescence-activated cell sorting, spatial immobilization such as 2D-PAGE in conjunction with a scintillation counter, mass spectrophotometry, or western analysis or affinity chromatography, to detect protein expression in a sample.
  • the nucleic acids, proteins, or antibodies may be used in solution or attached to a substrate, and their detection is based on methods and labeling moieties well known in the art.
  • Expression profiles may also be evaluated by methods such as electronic northern analysis, guilt-by-association, and transcript imaging. Expression profiles produced using any of the above methods may be compared with expression profiles produced using normal or diseased tissues.
  • the correspondence between mRNA and protein expression has been discussed by Zweiger (2001, Transducing the Genome. McGraw-Hill, San Francisco, CA) and Glavas et al. (2001; T cell activation upregulates cyclic nucleotide phosphodiesterases 8A1 and 7A3, Proc Natl Acad Sci 98:6319-6342) among others.
  • Genotyping refers to the determination of the specific nucleotide- base sequence composition of alternative forms of DNA, at specific locations within each of the chromosome pairs in a cell or in an organism as a whole.
  • hybridization complex refers to a complex formed between two nucleic acids by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (e.g., C 0 t or Rot analysis) or formed between one nucleic acid present in solution and another nucleic acid immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
  • Identity refers to the quantification (usually percentage) of nucleotide or residue matches between at least two sequences aligned using a standardized algorithm such as Smith- Waterman alignment (Smith and Waterman (1981) J Mol Biol 147:195-197), CLUSTALW (Thompson et al. (1994) Nucleic Acids Res 22:4673-4680), or BLAST2 (Altschul (1997, supra).
  • BLAST2 maybe used in a standardized and reproducible way to insert gaps in one of the sequences in order to optimize alignment and to achieve a more meaningful comparison between them.
  • Similarity uses the same algorithms but takes conservative substitution of residues into account. In proteins, similarity exceeds identity in that substitation, for example, of a valine for a leucine or isoleucine, is counted in calculating the reported percentage. Substitutions which are considered to be conservative are well known in the art.
  • an “immunogenic fragment” is a polypeptide or oligopeptide fragment of PPAR ⁇ FS or PPP1R3AFS which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal
  • immunogenic fragment also includes any polypeptide or oligopeptide fragment of PPAR ⁇ FS or PPP1R3AFS which is useful in any of the antibody production methods disclosed herein or known in the art.
  • Labeling moiety refers to any reporter molecule including radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, substrates, cofactors, inhibitors, or magnetic particles than can be attached to or incorporated into a polynucleotide, protein, or antibody.
  • conjugation techniques include both direct synthesis and chemical conjugation, particularly to amines, thiols and other side groups which may be present.
  • Visible labels and dyes include but are not limited to anthocyanins, ⁇ glucuronidase, biotin, BIODIPY, Coomassie blue, Cy3 and Cy5, 4,6-diamidino- 2-phenylindole (DAPI), digoxigenin, fluorescein, FTTC, gold, green fluorescent protein (GFP), nssamine, luciferase, phycoerythrin, rhodamine, spyro red, silver, streptavidin, and the like.
  • Radioactive markers include radioactive forms of hydrogen, iodine, phosphorous, sulfur, and the like.
  • Ligand refers to any agent, molecule, or compound which will bind specifically to a polynucleotide or to an epitope of a protein. Such ligands stabilize or modulate the activity of polynucleotides or proteins and may be composed of inorganic and/or organic substances including minerals, cofactors, nucleic acids, proteins, carbohydrates, fats, and lipids.
  • microarray refers to an arrangement of a plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.
  • element and “array element” refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microarray.
  • modulate refers to a change in the activity of PPAR ⁇ or PPP1R3A. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of PPAR ⁇ or PPP1R3A.
  • a "multispecific molecule” can bind with at least two different binding specificities to at least two different molecules or two different sites on a molecule.
  • Antibodies can perform as multispecific molecules in that they can bind to both a target protein and a pharmaceutical agent.
  • nucleic acid and nucleic acid sequence refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
  • Oligonucleotide refers a single-stranded molecule from about 18 to about 60 nucleotides in length which may be used in hybridization or amplification technologies or in regulation of replication, transcription or translation. Equivalent terms are amplicon, amplimer, primer, and oligomer.
  • “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
  • a “pharmaceutical agent” may be an antibody, an antisense molecule, a multispecific molecule, a peptide, a protein, a radionuclide, a small drug molecule, a cytospecific or cytotoxic drug such as abrin, actinomyosin D, cisplatin, crotin, doxorubicin, 5-fluorouracil, methotrexate, ricin, vincristine, vinblastine, or any combination of these elements.
  • Post-translational modification of a protein can involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and the like. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cellular location, cell type, pH, enzymatic milieu, and the like.
  • Probe refers to polynucleotides encoding PPAR ⁇ FS or PPP1R3AFS, their complements, or fragments thereof, which are used to detect identical, allelic or related polynucleotides.
  • Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
  • Probes are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Protein refers to a polypeptide or any portion thereof.
  • a "portion" of a protein refers to that length of amino acid sequence which would retain at least one biological activity, a domain identified by PFAM or PRINTS analysis or an antigenic dete ⁇ ninant of the protein identified using Kyte-Doolittle algorithms of the PROTEAN program (DNASTAR, Madison WI).
  • An "oligopeptide” is an amino acid sequence from about five residues to about 15 residues that is used as part of a fusion protein to produce an antibody.
  • a "recombinant nucleic acid” is a nucleic acid that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook and Russell (supra).
  • the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
  • a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence.
  • Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
  • such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
  • a “regulatory element” refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
  • Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
  • RNA equivalent in reference to a DNA molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • sample is used in its broadest sense as containing nucleic acids, proteins, and antibodies.
  • a sample may comprise a bodily fluid such as ascites, blood, cerebrospinal fluid, lymph, semen, sputum, urine and the like; the soluble fraction of a cell preparation, or an aliquot of media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue, a tissue biopsy, or a tissue print; buccal cells, skin, hair, a hair follicle; and the like.
  • binding and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
  • substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably at least about 75% free, and most preferably at least about 90% free from other components with which they are naturally associated.
  • substitution refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
  • Substrate refers to any rigid or semi-rigid support to which polynucleotides, proteins, or antibodies are bound and includes magnetic or nonmagnetic beads, capillaries or other tubing, chips, fibers, filters, gels, membranes, plates, polymers, slides, wafers, and micro particles with a variety of surface forms including channels, columns, pins, pores, trenches, and wells.
  • Transformation describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell.
  • the method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment.
  • transformed cells includes stable transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating phasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
  • a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by micro injection or by infection with a recombinant virus.
  • the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002) Science 295:868-872).
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
  • the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals.
  • the isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook and Russell (supra).
  • “Variant” refers to molecules that are recognized variations of a protein or the polynucleotides that encode it. Splice variants may be determined by BLAST score, wherein the score is at least 100, and most preferably at least 400. Allelic variants have a high percent identity to the cDNAs and may differ by about three bases per hundred bases. "Single nucleotide polymorphism” (SNP) refers to a change in a single base as a result of a substitution, insertion or deletion. The change may be conservative (purine for purine) or non-conservative (purine to pyrimidine) and may or may not result in a change in an encoded amino acid or its secondary, tertiary, or quaternary structure. THE INVENTION
  • the mutation (R3) was present in the grandmother (Iii), in all five individuals with severe insulin resistance and in one other relative (Elii).
  • all five family members with severe insulin resistance, and no other family members were doubly heterozygous with respect to the two frameshift mutations of these two unlinked genes.
  • the doubly heterozygous individuals were variably affected by additional features of syndrome X, a condition characterized by obesity, insulin resistance, dyslipidemia, hypertriglyceridemia, and high LDL (low-density lipoprotein)/low HDL (Hauler, H. (2002) Eur J Clin Nut 56:S25-S29).
  • Dyslipidemia is defined by triglycerides > 2mmol/L and high- density lipoprotein (HDL) ⁇ lmmol/L.
  • Figure 3 shows that fasting insulin levels in the singly heterozygous and wildtype family members were within the normal range.
  • the double heterozygotes showed extreme hyperinsulinemia and, to a variable extent, diabetes, hyperlipidemia and hypertension ( Figure 2 and Table 1).
  • diabetes, hypertension or dyslipidemia were also present in some other members of the kindred, these phenotypes do not seem to require mutations in both PPAR ⁇ and PPP1R3A.
  • the PPAR ⁇ FS frameshift mutation was not detected in any other individuals, whereas one Europid individual carried the same heterozygous frameshift mutation of PPP1R3A that was found in family A.
  • This individual (iii, family B; Figure 4) presented with acanthosis nigricans at age 20 years. He had a body mass index (BMI) of 36.5 kg m 2 and a fasting insulin level of 437 pmol i 1 (normal ⁇ 80 ⁇ mol/1).
  • Figure 5 shows the results of a DNA binding assay for wild type PPAR ⁇ l and PPAR ⁇ 2 and the corresponding mutant isoforms when complete with RXR using an electrophoretic mobility shift assay. Unlike their wildtype counterparts (WT ⁇ l and WT ⁇ 2), neither mutant PPAR ⁇ isoforms (FS ⁇ l or FS ⁇ 2) formed heterodimeric complexes when co-incubated with a radio labeled probe encoding the acyl-CoA. oxidase, PPARE ( Figure 5A).
  • Figure 8A shows the presence of the truncated PPP1R3A mutant protein in CHO cells transfected with the FS mutation and compared with cells likewise transfected with the wildtype gene.
  • the mutant gene produces a protein of the expected size, approximately 83 kDa.
  • the wildtype PPP1R3A undergoes rapid proteolysis to produce proteolytic fragments of a similar size.
  • Figure 8B demonstrates that when the cell lysate from either PPP1R3A (WT) or PPP1R3AFS (FS) transfected cells is immunoprecipitated with anti-HA and then western blotted with anti-PPIC antibody, the truncated PPP1R3AFS protein interacts with PP1C with an efficiency similar to PPP1R3A.
  • Figure 9 shows a comparison of the intracellular distribution for wildtype PPP1R3A and wildtype PPP1R3 AFS. The results show a significant fraction of wildtype PPP1R3 A localized to intracellular membranes, as expected. However, the mutant PPP1R3 AFS protein is almost exclusively cytosolic.
  • PPP1R3 A alters the function of the gene, e.g. , loss of the SRBD binding domain ( Figure IB).
  • the truncated PPP1R3A mutant protein can still interact with the catalytic subunit, PP1C, it may actively interfere with the normal function of that subunit. Therefore, modulation of the activity of the PPP1R3A gene by any means, such as a small molecule drug, in particular an agonist of PPP1R3 A, could be an effective means of treating diabetes.
  • protein STOP mutation protein STOP mutation
  • EPIC-Norfolk Cohort Study a The EPIC-Norfolk study is a population-based cohort study involving 25,000 individuals recruited from general practices throughout Norfolk, UK. Prevalent and incident cases of diabetes were included in the analysis. Cases were ascertained from multiple sources including primary care registers, hospital clinics, self-report and reported medication. Controls were selected from the same cohort study and were individually matched for age, sex and General Practice. Controls were excluded if they had a HbAlc >6.0%. b. 512 cases were considered with 516 controls. The frequency of the PPP1R3AFS mutation was lower than in the previous study, and similar in type 2 diabetics (0.78%) and controls (0.97%).
  • Methods for sequencing nucleic acids are well known in the art and may be used to practice any of the embodiments of the invention. These methods employ enzymes such as the Klenow fragment of DNA polymerase I, SEQUENASE, Taq DNA polymerase and thermostable T7 DNA polymerase (Amersham Biosciences (APB), Piscataway NJ), or combinations of polymerases and proofreading exonucleases (Invitrogen, Carlsbad CA).
  • enzymes such as the Klenow fragment of DNA polymerase I, SEQUENASE, Taq DNA polymerase and thermostable T7 DNA polymerase (Amersham Biosciences (APB), Piscataway NJ), or combinations of polymerases and proofreading exonucleases (Invitrogen, Carlsbad CA).
  • Sequence preparation is automated with machines such as the MICROLAB 2200 system (Hamilton, Reno NV) and the DNA ENGINE thermal cycler (MJ Research, Watertown MA) and sequencing, with the PRISM 3700, 377 or 373 DNA sequencing systems (ABI) or the MEGABACE 1000 DNA sequencing system (APB).
  • the nucleic acid sequences of the cDNAs presented in the Sequence Listing were prepared by such automated methods and may contain occasional sequencing errors and unidentified nucleotides, designated with an N, that reflect state-of-the-art technology at the time the cDNA was sequenced.
  • Vector, linker, and polyA sequences were masked using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis.
  • Ns and SNPs can be verified either by resequencing the cDNA or using algorithms to compare multiple sequences that overlap the area in which the Ns or SNP occur. Both of these techniques are well known to and used by those skilled in the art.
  • the sequences may be analyzed using a variety of algorithms described in Ausubel et al. (1997; Short Protocols in Molecular Biology, John Wiley & Sons, New York NY, unit 1.1) and in Meyers (1995; Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853). Shotgun sequencing may also be used to complete the sequence of a particular cloned insert of interest. Shotgun strategy involves randomly breaking the original insert into segments of various sizes and cloning these fragments into vectors.
  • the fragments are sequenced and reassembled using overlapping ends until the entire sequence of the original insert is known.
  • Shotgun sequencing methods are well known in the art and use thermostable DNA polymerases, heat-labile DNA polymerases, and primers chosen from representative regions flanking the cDNAs of interest. Incomplete assembled sequences are inspected for identity using various algorithms or programs such as CONSED (Gordon (1998) Genome Res 8:195-202) which are well known in the art. Contaminating sequences, including vector or chimeric sequences, can be removed, and deleted sequences can be restored to complete the assembled, finished sequences.
  • CONSED Gibdon (1998) Genome Res 8:195-202
  • sequences of the invention may be extended using various PCR-based methods known in the art.
  • the XL-PCR kit (ABI)
  • nested primers and cDNA or genomic DNA libraries may be used to extend the nucleic acid sequence.
  • primers may be designed using software, such as LOLIGO primer analysis software (Molecular Biology Insights, Cascade CO) to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to a target molecule at temperatures from about 55C to about 68C.
  • genomic, rather than cDNA libraries are used.
  • a probe may be designed or derived from unique regions such as the 5' regulatory region or from a nonconserved region (i.e., 5 ' or 3 'of the nucleotides encoding the conserved catalytic domain of the protein) and used in protocols to identify naturally occurring molecules encoding the PPP1R3AFS, allelic variants, or related molecules.
  • the probe may be DNA or RNA, may be single-stranded, and should have at least 50% sequence identity to any of the nucleic acid sequences, SEQ ID NOs:3 or 4.
  • Hybridization probes may be produced using oligolabeling, nick-translation, end-labeling, or PCR amplification in the presence of a reporter molecule.
  • a vector containing the nucleic acid or a fragment thereof may be used to produce an mRNA probe in vitro by addition of an RNA polymerase and labeled nucleotides. These procedures may be conducted using kits such as those provided by APB.
  • the stringency of hybridization is determined by G+C content of the probe, salt concentration, and temperature. In particular, stringency can be increased by reducing the concentration of salt or raising the hybridization temperature.
  • Hybridization can be performed at low stringency with buffers, such as 5xSSC with 1% sodium dodecyl sulfate (SDS) at 60C, which permits the formation of a hybridization complex between nucleic acid sequences that contain some mismatches. Subsequent washes are performed at higher stringency with buffers such as 0.2xSSC with 0.1% SDS at either 45C (medium stringency) or 68C (high stringency). At high stringency, hybridization complexes will remain stable only where the nucleic acids are completely complementary. In some membrane-based hybridizations, from about 35% to about 50% formalize can be added to the hybridization solution to reduce the temperature at which hybridization is performed.
  • buffers such as 5xSSC with 1% sodium dodecyl sulfate (SDS) at 60C, which permits the formation of a hybridization complex between nucleic acid sequences that contain some mismatches.
  • buffers such as 0.2xSSC with 0.1% SDS at either 45C (
  • Arrays may be prepared and analyzed using methods well known in the art. Oligonucleotides or cDNAs may be used as hybridization probes or targets to monitor the expression level of large numbers of genes simultaneously or to identify genetic variants, mutations, and single nucleotide polymorphisms. Arrays may be used to determine gene function; to understand the genetic basis of a condition, disease, or disorder; to diagnose a condition, disease, or disorder; and to develop and monitor the activities of therapeutic agents. (See, e.g., USN 5,474,796; Schena et al. (1996) Proc Natl Acad Sci 93:10614-10619; Heller et al. (1997) Proc Natl Acad Sci 94:2150-2155; USN 5,605,662.)
  • Hybridization probes are also useful in mapping the naturally occurring genomic sequence.
  • the probes may be hybridized to a particular chromosome, a specific region of a chromosome, or an artificial chromosome construction.
  • Such constructions include human artificial chromosomes , yeast artificial chromosomes, bacterial artificial chromosomes, bacterial PI constructions, or the cDNAs of libraries made from single chromosomes.
  • QPCR is a method for quantifying a nucleic acid molecule based on detection of a fluorescent signal produced during PCR amplification (Gibson et al. (1996) Genome Res 6:995-1001; Heid et al. (1996) Genome Res 6:986-994).
  • Amplification is carried out on machines such as the PRISM 7700 detection system (ABI) which consists of a 96-well thermal cycler connected to a laser and charge-coupled device (CCD) optics system.
  • BCI PRISM 7700 detection system
  • CCD charge-coupled device
  • the probe which is designed to anneal between the standard forward and reverse PCR primers, is labeled at the 5' end by a flourogenic reporter dye such as 6-carboxyfluorescein (6-FAM) and at the 3 'end by a quencher molecule such as
  • TAMRA 6-carboxy-tetrame yl-rhodamine
  • a cycle threshold (C- ) value representing the cycle number at which the PCR product crosses a fixed threshold of detection is determined by the instrument software.
  • the C ⁇ is inversely proportional to the copy number of the template and can therefore be used to calculate either the relative or absolute initial concentration of the nucleic acid molecule in the sample.
  • the relative concentration of two different molecules can be calculated by determining their respective . values (comparative C ⁇ method).
  • the absolute concentration of the nucleic acid molecule can be calculated by constructing a standard curve using a housekeeping molecule of known concentration. The process of calculating O- values, preparing a standard curve, and determining starting copy number is performed using SEQUENCE DETECTOR 1.7 software (ABI). Expression
  • Any one of a multitude of polynucleotides encoding PPAR ⁇ FS or PPP1R3 AFS may be cloned into a vector and used to express the protein, or portions thereof, in host cells.
  • the nucleic acid sequence can be engineered by such methods as DNA shuffling (USPN 5,830,721) and site-directed mutagenesis to create new restriction sites, alter glycosylation patterns, change codon preference to increase expression in a particular host, produce splice variants, extend half-life, and the like.
  • the expression vector may contain transcriptional and translational control elements (promoters, enhancers, specific initiation signals, and polyadenylated 3' sequence) from various sources which have been selected for their efficiency in a particular host.
  • the vector, cDNA, and regulatory elements are combined using in vitro recombinant DNA techniques, synthetic techniques, and/or in vivo genetic recombination techniques well known in the art and described in Sambrook (supra, ch. 4, 8, 16 and 17).
  • a variety of host systems may be transformed with an expression vector. These include, but are not limited to, bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems transformed with baculovirus expression vectors or plant cell systems transformed with expression vectors containing viral and/or bacterial elements (Ausubel supra, unit 16).
  • an adenovirus transcriptional/ translational complex may be utilized. After sequences are ligated into the El or E3 region of the viral genome, the infective virus is used to transform and express the protein in host cells.
  • the Rous sarcoma virus enhancer or SV40 or EBV-based vectors may also be used for high-level protein expression.
  • Routine cloning, subcloning, and propagation of nucleic acid sequences can be achieved using the multifunctional pBLUESCRIPT vector (Stratagene, La Jolla CA) or pSPORTl plasmid (Invitrogen). Introduction of a nucleic acid sequence into the multiple cloning site of these vectors disrupts the lacZ gene and allows colorimetric screening for transformed bacteria. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
  • the vector can be stably transformed into cell lines along with a selectable or visible marker gene on the same or on a separate vector. After transformation, cells are allowed to grow for about 1 to 2 days in enriched media and then are transferred to selective media. Selectable markers, antimetabolite, antibiotic, or herbicide resistance genes, confer resistance to the relevant selective agent and allow growth and recovery of cells which successfully express the introduced sequences. Resistant clones identified either by survival on selective media or by the expression of visible markers may be propagated using culture techniques. Visible markers are also used to estimate the amount of protein expressed by the introduced genes. Verification that the host cell contains the desired poynucleotide is based on DNA-DNA or DNA-RNA hybridizations or PCR amplification.
  • the host cell may be chosen for its ability to modify a recombinant protein in a desired fashion. Such modifications include acetylation, carboxylation, glycosylation, phosphorylation, lipidation, acylation and the like. Post-translational processing which cleaves a "prepro" form may also be used to specify protein targeting, folding, and/or activity. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities may be chosen to ensure the correct modification and processing of the recombinant protein.
  • Heterologous moieties engineered into a vector for ease of purification include glutathione S- transferals (GST), 6xHis, FLAG, MRC, and the like.
  • GST and 6-His are purified using affinity matrices such as immobilized glutathione and metal-chelate resins, respectively.
  • FLAG and MRC are purified using monoclonal and polyclonal antibodies.
  • a sequence encoding a proteolytic cleavage site may be part of the vector located between the protein and the heterologous moiety. Methods for recombinant protein expression and purification are discussed in Isabel (supra, unit 16). Protein Identification
  • proteins are separated using two-dimensional gel electrophoresis (2-DE), 2) selected proteins are excised from the gel and digested with a protease to produce a set of peptides; and 3) the peptides are subjected to mass spectral analysis to derive peptide ion mass and spectral pattern information.
  • MS information is used to identify the protein by comparing it with information in a protein database (Shevenko et al. (1996) Proc Natl Acad Sci 93:14440-14445).
  • Proteins are separated by 2DE employing isoelectric focusing (EEF) in the first dimension followed by SDS-PAGE in the second dimension.
  • EEF isoelectric focusing
  • an immobilized pH gradient strip is useful to increase reproducibility and resolution of the separation.
  • Alternative techniques may be used to improve resolution of very basic, hydrophobic, or high molecular weight proteins.
  • the separated proteins are detected using a stain or dye such as silver stain, Coomassie blue, or spyro red (Molecular Probes, Eugene OR) that is compatible with MS.
  • Gels may be blotted onto a PVDF membrane for western analysis and optically scanned using a STORM scanner (APB) to produce a computer-readable output which is analyzed by pattern recognition software such as MELANIE (GeneBio, Geneva, Switzerland).
  • the software annotates individual spots by assigning a unique identifier and calculating their respective x,y coordinates, molecular masses, isoelectric points, and signal intensity.
  • Individual spots of interest such as those representing differentially expressed proteins, are excised and proteolytically digested with a site- specific protease such as trypsin or chymotrypsin, singly or in combination, to generate a set of small peptides, preferably in the range of 1-2 kDa.
  • a site-specific protease such as trypsin or chymotrypsin, singly or in combination
  • samples Prior to digestion, samples may be treated with reducing and alkylatmg agents, and following digestion, the peptides are then separated by liquid chromatography or capillary electrophoresis and analyzed using MS.
  • MS converts components of a sample into gaseous ions, separates the ions based on their mass-to-charge ratio, and determines relative abundance.
  • a MA -DI-TOF Microx Assisted Laser Desorption Ionization-Time of Flight
  • ESI Electron ray
  • TOF-TOF Time of Flight Time of Flight machines
  • analytical programs such as TURBOSEQUEST software (Finnigan, San Jose CA)
  • the MS data is compared against a database of theoretical MS data derived from known or predicted proteins. A rninimum match of three peptide masses is used for reliable protein identification. If additional information is needed for identification, Tandem-MS may be used to derive information about individual peptides.
  • tandem-MS a first stage of MS is performed to determine individual peptide masses. Then selected peptide ions are subjected to fragmentation using a technique such as collision induced dissociation (CID) to produce an ion series. The resulting fragmentation ions are analyzed in a second round of MS, and their spectral pattern may be used to determine a short stretch of amino acid sequence (Dancik et al (1999) J Comput Biol 6:327-342).
  • CID collision induced dissociation
  • Proteins or portions thereof may be produced not only by recombinant methods, but also by using chemical methods well known in the art.
  • Solid phase peptide synthesis may be carried out in a batchwise or continuous flow process which sequentially adds ⁇ -amino- and side chain-protected amino acid residues to an insoluble polymeric support via a linker group.
  • a linker group such as me ylamine- derivatized polyethylene glycol is attached to poly(styrene-co-divinylbenzene) to form the support resin.
  • the amino acid residues are N- ⁇ -protected by acid labile Boc (t-butyloxycarbonyl) or base-labile Fmoc (9-fluorenylmethoxycarbonyl).
  • the carboxyl group of the protected amino acid is coupled to the amine of the linker group to anchor the residue to the solid phase support resin.
  • Trifluoroacetic acid or piperidine are used to remove the protecting group in the case of Boc or Fmoc, respectively.
  • Each additional amino acid is added to the anchored residue using a coupling agent or pre-activated amino acid derivative, and the resin is washed.
  • the full length peptide is synthesized by sequential deprotection, coupling of derivitized amino acids, and washing with dichloromethane and/or N, N-dimethylformamide. The peptide is cleaved between the peptide carboxy terminus and the linker group to yield a peptide acid or amide.
  • Antibodies or immunoglobulins (Ig) are components of immune response expressed on the surface of or secreted into the circulation by B cells.
  • the prototypical antibody is a tetramer composed of two identical heavy polypeptide chains (H-chains) and two identical light polypeptide chains (L-chains) interlinked by disulfide bonds which binds and neutralizes foreign antigens. Based on their H-chain, antibodies are classified as IgA, IgD, IgE, IgG or IgM.
  • the most common class, IgG is tetrameric while other classes are variants or multimers of the basic structure.
  • Antibodies are described in terms of their two functional domains. Antigen recognition is mediated by the Fab (antigen binding fragment) region of the antibody, while effector functions are mediated by the Fc (crystallizable fragment) region. The binding of antibody to antigen triggers destruction of the antigen by phagocytic white blood cells such as macrophages and neutrophils. These cells express surface Fc receptors that specifically bind to the Fc region of the antibody and allow the phagocytic cells to destroy antibody-bound antigen. Fc receptors are single-pass transmembrane glycoproteins containing about 350 amino acids whose extracellular portion typically contains two or three Ig domains (Sears et al. (1990) J Immunol 144:371-378). Preparation and Screening of Antibodies
  • mice including mice, rats, rabbits, goats, llamas, camels, and human cell lines may be immunized by injection with an antigenic determinant.
  • Adjuvants such as Freund's, mineral gels, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemacyanin (KLH; Sigma- Aldrich), and dinitrophenol may be used to increase immunological response.
  • BCG Bacilli Calmette-Guerin
  • Corvnebacterium parvum increase response.
  • the antigenic determinant may be an oligopeptide, peptide, or protein.
  • Chimeric antibodies may be produced by techniques such as splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity (Morrison et al. (1984) Proc Natl Acad Sci 81:6851-6855; Neuberger et al. (1984) Nature 312:604-608; and Takeda et al. (1985) Nature 314:452-454).
  • techniques described for antibody production may be adapted, using methods known in the art, to produce specific, single chain antibodies.
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton (1991) Proc Natl Acad Sci 88:10134-10137).
  • Antibody fragments which contain specific binding sites for an antigenic determinant may also be produced.
  • fragments include, but are not limited to, F(ab ⁇ )2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries maybe constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al. (1989) Science 246:1275-1281).
  • Antibodies may also be produced by inducing production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al. (1989; Proc Natl Acad Sci 86:3833-3837) or Winter et al. (1991; Nature 349:293-299).
  • a protein may be used in screening assays of phagemid or B-lymphocyte immunoglobulin libraries to identify antibodies having a desired specificity. Numerous protocols for competitive binding or immunoassays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.
  • K a association constant
  • High-affinity antibody preparations with K a ranging from about 10 9 to 10 12 IVmole are commonly used in immunoassays in which the protein-antibody complex must withstand rigorous manipulations.
  • Low-affinity antibody preparations with K. ranging from about 10 6 to 10 7 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of the protein, preferably in active form, from the antibody (Catty (1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington DC; Eiddell and Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
  • polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications.
  • a polyclonal antibody preparation containing about 5-10 mg specific antibody/ml is generally employed in procedures requiring precipitation of protein-antibody complexes. Procedures for making antibodies, evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are discussed in Catty (supra) and Ausubel (supra-) pp. 11.1-11.31.
  • DIAGNOSTICS The presence of the mutations for PPP1R3 AFS and PPAR ⁇ FS in DNA as detected by genotyping of DNA may be used to diagnose diabetes, including type 2 diabetes. In addition, the presence of the PPP1R3 AFS mutation in DNA as determined by genotyping may be used to diagnose a predisposition to, or a risk factor for, insulin resistance and diabetes.
  • the co-expression of PPP1R3AFS and PPAR ⁇ FS may also be detected using PPP1R3AFS and PPAR ⁇ FS, a polynucleotide encoding PPP1R3AFS or PPAR ⁇ FS, or an antibody that specifically binds PPP1R3AFS or PPAR ⁇ FS, and at least one of the assays described below to diagnose severe insulin resistance and diabetes, including type 2 diabetes.
  • the expression of PPP1R3AFS detected using any of the compositions for PPP1R3AFS noted above, and at least one of the assays described below can be used to diagnose a predisposition, or a risk factor for the development of diabetes.
  • reporter molecules and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid, amino acid, and antibody assays. Synthesis of labeled molecules may be achieved using kits such as those supplied by Promega (Madison WI) or APB for incorporation of a labeled nucleotide such as 32 P-dCTP (APB), Cy3-dCTP or Cy5-dCTP (Qiagen-
  • Nucleotides and amino acids may be directly labeled with a variety of substances including fluorescent, chemiluminescent, or chromogenic agents, and the like, by chemical conjugation to amines, thiols and other groups present in the molecules using reagents such as BIODIPY or FTTC (Molecular Probes). Nucleic Acid Assays
  • the polynucleotides, cDNAs, fragments, oligonucleotides, complementary PNAs, and peptide nucleic acids (PNA) may be used to detect and quantify differential gene expression for diagnosis of a disorder.
  • Disorders associated with such differential expression of the nucleic acids of the invention include diabetes.
  • the diagnostic assay may use hybridization or amplification technology to compare gene expression in a biological sample from a patient to standard samples in order to detect differential gene expression. Qualitative or quantitative methods for this comparison are well known in the art.
  • An expression profile comprises the expression of a plurality of polynucleotides or protein as measured using standard assays with a sample.
  • the polynucleotides, proteins or antibodies of the invention may be used as elements on a array to produce an expression profile.
  • the array is used to diagnose or monitor the progression of disease.
  • An expression profile comprises the expression of a plurality of polynucleotides or proteins as measured using standard assays with a sample.
  • the polynucleotides, proteins or antibodies of the invention may be used as elements in the assay to produce the expression profile.
  • an array upon which the elements are immobilized is used to diagnose, stage or monitor the progression or treatment of a disorder.
  • the polynucleotides, proteins or antibodies may be labeled using standard methods and added to a biological sample from a patient under conditions for the complex formation. After an incubation period, the sample is washed, and the amount of label (or signal) associated with each complexes is quantified and compared with a standard value. If the amount of complex formation in the patient sample is altered in comparison to normal or disease standards, then complex formation can be used to indicate the presence of a disorder.
  • normal and disease profiles are established. This is accomplished by combining a sample taken from a normal subject, either animal or human, with a polynucleotide under conditions for complex formation to occur. Standard complex formation may be quantified by comparing the values obtained using samples from normal subjects with values from an experiment in which a known amount of a purified control is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who were diagnosed with a particular condition, disease, or disorder. Deviation from standard values toward those associated with a particular disorder is used to diagnose or stage that disorder.
  • a disorder By analyzing changes in patterns of gene expression, a disorder can be diagnosed earlier, sometimes even before the patient is symptomatic.
  • the invention can be used to formulate a prognosis and to design a treatment regimen.
  • the invention can also be used to monitor the efficacy of treatment or to establish a dosage that causes a change in the expression profile indicative of successful treatment.
  • the expression profile is employed to improve the treatment regimen so that expression patterns associated with the onset of undesirable side effects are avoided. This approach may be more sensitive and rapid than waiting for the patient to show inadequate improvement, or to manifest side effects, before altering the course of treatment.
  • animal models which mimic a human disease can be used to characterize expression profiles associated with a particular condition, disease, or disorder; or treatment of the condition, disease, or disorder.
  • Novel treatment regimens may be tested in these animal models using an expression profile over time.
  • an expression profile may be used with cell cultures or tissues removed from animal models to rapidly screen large numbers of candidate drug molecules, looking for ones that produce an expression profile similar to those of known therapeutic drugs, with the expectation that molecules with the same expression profile will likely have similar therapeutic effects.
  • the invention provides the means to rapidly determine the molecular mode of action of a drug.
  • Such expression profiles may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies or in clinical trials or to monitor the treatment of an individual patient. Once the presence of a condition is established and a treatment protocol is initiated, expression may be analyzed on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in a normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to years. Protein and Antibody Assays
  • Antibodies which specifically bind PPP1R3AFS or PPAR ⁇ FS maybe used to candidate those proteins.
  • Immunological methods for detecting and measuring complex formation as a measure of protein expression using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include antibody or protein arrays, ELISA, FACS, spatial immobilization such as 2D- PAGE and SC, HPLC or MS, RLAs and western analysis.
  • Such immunoassays typically involve the measurement of complex formation between the protein and its specific antibody.
  • a two-site, monoclonal-based immunoassay utilizing antibodies reactive to two non-interfering epitopes is preferred, but a competitive binding assay may be employed (Pound (1998) Immunochemical Protocols, Humana Press, Totowa NJ).
  • Normal or standard values for protein expression are estabhshed by combining body fluids or cell extracts taken from a normal mammalian or human subject with specific antibodies to a protein under conditions for complex formation.
  • Standard values for complex formation in normal and diseased tissues are established by various methods, often photometric means. Then complex formation as it is expressed in a subject sample is compared with the standard values. Deviation from the normal standard and toward the diseased standard provides parameters for disease diagnosis or prognosis while deviation away from the diseased and toward the normal standard may be used to evaluate treatment efficacy.
  • Antibody arrays allow the development of techniques for high-throughput screening of recombinant antibodies.
  • Such methods use robots to pick and grid bacteria containing antibody genes, and a filter-based ELIZA to screen and identify clones that express antibody fragments. Because liquid handling is eliminated and the clones are arrayed from master stocks, the same antibodies can be spotted multiple times and screened against multiple antigens simultaneously. Antibody arrays are highly useful in the identification of differentially expressed proteins. See de Wild et al. (2000) Nature Biotechnol 18:989-94. THERAPEUTICS
  • a pharmaceutical agent such as an agonist, transcription factor or a small drug molecule that specifically binds the protein and increases its expression or activity may be administered to a subject in need of such treatment.
  • a pharmaceutical composition comprising an agonist, transcription factor or a small drug molecule and a pharmaceutical carrier may be administered to a subject to treat decreased expression or activity associated with the endogenous protein.
  • an antibody that specifically binds the protein can act as a carrier to effect delivery.
  • a vector expressing the encoding polynucleotide, or fragments thereof may be administered to a subject to treat the disorder.
  • any of the polynucleotides, complementary molecules, or fragments thereof, proteins or portions thereof, vectors delivering these nucleic acid molecules or expressing the proteins, therapeutic antibodies, and ligands binding the polynucleotide or protein may be administered in combination with other therapeutic agents. Selection of the agents for use in combination therapy may be made by one of ordinary skill in the art according to conventional pharmaceutical principles. A combination of therapeutic agents may act synergistically to affect treatment of a particular disorder at a lower dosage of each agent.
  • Gene expression may be modified by designing complementary or antisense molecules (DNA, RNA, or PNA) to the control, 5', 3', or other regulatory regions of the gene encoding PPP1R3AFS or PPAR ⁇ FS. Oligonucleotides designed to inhibit transcription initiation are preferred. Similarly, inhibition can be achieved using triple helix base-pairing which inhibits the binding of polymerase, transcription factors, or regulatory molecules (Gee et al. In: Huber and Carr (1994) Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-177). A complementary molecule may also be designed to block translation by preventing binding between ribosomes and mRNA.
  • a library or plurality of polynucleotides may be screened to identify those which specifically bind a regulatory, nontranslated sequence.
  • Ribosomes, enzymatic RNA molecules may also be used to catalyze the specific cleavage of
  • RNA Ribonucleic acid
  • the mechanism of ribosome action involves sequence-specific hybridization of the ribosome molecule to complementary target RNA followed by endonucleolytic cleavage at sites such as GUN, Glu, and GUN. Once such sites are identified, an oligonucleotide with the same sequence may be evaluated for secondary structural features which would render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing their hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • RNA molecules may be modified to increase intracellular stability and half-life by addition of flanking sequences at the 5' and/or 3' ends of the molecule or by the use of phosphorothioate or 2' O-methyl rather than phosphodiesterases linkages within the backbone of the molecule. Modification is inherent in the production of PNAs and can be extended to other nucleic acid molecules.
  • RNA Interference Either the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, or the modification of adenine, cytidine, guanine, thymine, and uridine with acetyl-, methyl-, thio- groups renders the molecule more resistant to endogenous endonucleases.
  • RNA interference also known as double-stranded RNA (dsRNA)-induced gene silencing
  • dsRNA double-stranded RNA
  • shRNAs short hairpin RNAs
  • RISC multicomponent nuclease complex
  • Dicer RNAse HI family nuclease
  • Transient infection of cells with RNAs capable of interference can bypass the need for Dicer and result in silencing of a gene for the lifespan of the introduced RNAs, usually from about 2 to about 7 days.
  • the RANi pathway is believed to have evolved in early eucaryote as a cell-based immunity against viral and genetic parasites. It is considered, however, to have great potential as a method for identifying gene function, particularly in signal transduction disorders (such as insulin resistance and type 2 diabetes), and for providing a highly specific means for treating such disorders.
  • polynucleotides of the invention can be used in gene therapy, polynucleotides can be delivered ex vivo to target cells, such as cells of bone marrow. Once stable integration and transcription and or translation are confirmed, the bone marrow maybe reintroduced into the subject. Expression of the protein encoded by the polynucleotide may correct a disorder associated with mutation of a normal sequence, reduction or loss of an endogenous target protein, or overepression of an endogenous or mutant protein.
  • polynucleotides may be delivered in vivo using vectors such as retro virus, adenovirus, Aden-associated virus, herpes simplex virus, and bacterial plastics.
  • Non- viral methods of gene delivery include cationic liposomes, polylysine conjugates, artificial viral envelopes, and direct injection of DNA (Anderson (1998) Nature 392:25-30; Dachs et al. (1997) Oncol Res 9:313-325; Chu et a]. (1998) J Mol Med 76(3-4):184-192; Weiss et al. (1999) CeU Mol Life Sci 55(3):334-358; Agrawal (1996) Antisense Therapeutics. Humana Press, Totowa NJ; and August et al. (1997) Gene Therapy (Advances in Pharmacology, Vol. 40), Academic Press, San Diego CA). Screening and Purification Assays
  • the polynucleotide encoding PPP1R3AFS or PPAR ⁇ FS may be used to screen a library or a plurality of molecules or compounds for specific binding affinity.
  • the libraries maybe antisense molecules, artificial chromosome constructions, branched nucleic acid molecules, DNA molecules, peptides, peptide nucleic acid, proteins such as transcription factors, enhancers, or repressions, RNA molecules, ribosomes, and other ligands which regulate the activity, replication, transcription, or translation of the endogenous gene.
  • the assay involves combining a polynucleotide with a library or plurality of molecules or compounds under conditions allowing specific binding, and detecting specific binding to identify at least one molecule which specifically binds the polynucleotide.
  • the polynucleotide of the invention may be incubated with a plurality of purified molecules or compounds and binding activity determined by methods well known in the art, e.g., a gel-retardation assay (USN 6,010,849) or a reticulocyte lysate transcriptional assay.
  • the polynucleotide may be incubated with nuclear extracts from biopsies and/or cultured cells and tissues. Specific binding between the polynucleotide and a molecule or compound in the nuclear extract is initially determined by gel shift assay and may be later confirmed by recovering and raising antibodies against that molecule or compound.
  • the polynucleotide may be used to purify a molecule or compound using affinity chromatography methods well known in the art.
  • the polynucleotide is chemically reacted with cyanogen bromide groups on a polymeric resin or gel. Then a sample is passed over and reacts with or binds to the polynucleotide. The molecule or compound which is bound to the polynucleotide may be released from the polynucleotide by increasing the salt concentration of the flow- through medium and collected.
  • the protein or a portion thereof may be used to purify a ligand from a sample.
  • a method for using a protein to purify a ligand would involve combining the protein with a sample under conditions to allow specific binding, detecting specific binding between the protein and ligand, recovering the bound protein, and using a chaotropic agent to separate the protein from the purified ligand.
  • PPP1R3 A may be used to screen a plurality of molecules or compounds in any of a variety of screening assays.
  • the portion of the protein employed in such screening may be free in solution, affixed to an biotic or biotic substrate (e.g. borne on a cell surface), or located infra cellularly.
  • viable or fixed prokaryotic host cells that are stable transformed with recombinant nucleic acids that have expressed and positioned a peptide on their cell surface can be used in screening assays. The cells are screened against a plurality or libraries of ligands, and the specificity of binding or formation of complexes between the expressed protein and the ligand can be measured.
  • the assay may be used to identify agonists, antagonists, antibodies, DNA molecules, small drug molecules, immunoglobulins, inhibitors, mimetics, peptides, peptide nucleic acids, proteins, and RNA molecules or any other ligand, which specifically binds the protein.
  • this invention contemplates a method for high throughput screening using very small assay volumes and very small amounts of test compound as described in USN 5,876,946, incorporated herein by reference. This method is used to screen large numbers of molecules and compounds via specific binding.
  • this invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding the protein specifically compete with a test compound capable of binding to the protein. Molecules or compounds identified by screening may be used in a mammalian model system to evaluate their toxicity or therapeutic potential.
  • compositions may be formulated and administered, to a subject in need of such treatment, to attain a therapeutic effect.
  • Such compositions contain the instant protein, agonists, antagonists, small drug molecules, immunoglobulins, inhibitors, mimetics, multispecific molecules, peptides, peptide nucleic acids, pharmaceutical agent, proteins, and RNA molecules.
  • Compositions may be manufactured by conventional means such as mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing.
  • compositions may be provided as a salt, formed with acids such as hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic, or as a lyophilized powder which may be combined with a sterile buffer such as saline, dextrose, or water.
  • acids such as hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic
  • a sterile buffer such as saline, dextrose, or water.
  • auxiliaries or excipients which facilitate processing of the active compounds.
  • Auxiliaries and excipients may include coatings, fillers or binders including sugars such as lactose, sucrose, mannitol, glycerol, or sorbitol; starches from corn, wheat, rice, or potato; proteins such as albumin, gelatin and collagen; cellulose in the form of hydroxypropylmethyl-cellulose, methyl cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; lubricants such as magnesium stearate or talc; disintegrating or solubilizing agents such as the, agar, alginic acid, sodium alginate or cross-linked polyvinyl pyrrolidone; stabilizers such as carbopol gel, polyethylene glycol, or titanium dioxide; and dyestuffs or pigments added for identify the product or to characterize the quantity of active compound or dosage.
  • sugars such as lactose, sucrose, mannitol, glycerol, or sorbitol
  • compositions may be administered by any number of routes including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal.
  • parenteral administration may be formulated in aqueous, physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Suspensions for injection may be aqueous, containing viscous additives such as sodium carboxymethyl cellulose or dextran to increase the viscosity, or oily, containing lipophilic solvents such as sesame oil or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes.
  • Penetrants well known in the art are used for topical or nasal administration. Toxicity and Therapeutic Efficacy
  • a therapeutically effective dose refers to the amount of active ingredient which ameliorates symptoms or condition.
  • a therapeutically effective dose can be estimated from cell culture assays using normal and neoplastic cells or in animal models.
  • Therapeutic efficacy, toxicity, concentration range, and route of administration maybe determined by standard pharmaceutical procedures using experimental animals.
  • the therapeutic index is the dose ratio between therapeutic and toxic effects--LD50 (the dose lethal to 50% of the population)/ED50 (the dose therapeutically effective in 50% of the population)— and large therapeutic indices are preferred.
  • Dosage is within a range of circulating concentrations, includes an ED50 with little or no toxicity, and varies depending upon the composition, method of delivery, sensitivity of the patient, and route of administration. Exact dosage will be determined by the practitioner in light of factors related to the subject in need of the treatment.
  • Dosage and administration are adjusted to provide active moiety that maintains therapeutic effect.
  • Factors for adjustment include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
  • Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular composition.
  • Normal dosage amounts may vary from 0.1 ⁇ g, up to a total dose of about 1 g, depending upon the route of administration.
  • the dosage of a particular composition maybe lower when administered to a patient in combination with other agents, drugs, or hormones.
  • Guidance as to particular dosages and methods of delivery is provided in the pharmaceutical literature. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Mack Publishing, Easton PA). Model Systems
  • Animal models may be used as bioassays where they exhibit a phenotypic response similar to that of humans and where exposure conditions are relevant to human exposures. Mammals are the most common models, and most infectious agent, cancer, drug, and toxicity studies are performed on rodents such as rats or mice because of low cost, availability, lifespan, gestation period, numbers of progeny, and abundant reference literature. Inbred and outbred rodent strains provide a convenient model for investigation of the physiological consequences of under- or over-expression of genes of interest and for the development of methods for diagnosis and treatment of diseases. A mammal inbred to over-express a particular gene (for example, secreted in milk) may also serve as a convenient source of the protein expressed by that gene. Toxicology
  • Toxicology is the study of the effects of agents on living systems. The majority of toxicity studies are performed on rats or mice. Observation of qualitative and quantitative changes in physiology, behavior, hemostatic processes, and lethality in the rats or mice are used to generate a toxicity profile and to assess consequences on human health following exposure to the agent. Genetic toxicology identifies and analyzes the effect of an agent on the rate of endogenous, spontaneous, and induced genetic mutations. Genotoxic agents usually have common chemical or physical properties that facilitate interaction with nucleic acids and are most harmful when chromosomal aberrations are transmitted to progeny.
  • Toxicological studies may identify agents that increase the frequency of structural or functional abnormalities in the tissues of the progeny if administered to either parent before conception, to the mother during pregnancy, or to the developing organism. Mice and rats are most frequently used in these tests because their short reproductive cycle allows the production of the numbers of organisms needed to satisfy statistical requirements.
  • Acute toxicity tests are based on a single administration of an agent to the subject to determine the symptom ology or lethality of the agent. Three experiments are conducted: 1) an initial dose-range- finding experiment, 2) an experiment to narrow the range of effective doses, and 3) a final experiment for establishing the dose-response curve.
  • Subchronic toxicity tests are based on the repeated administration of an agent. Rat and dog are commonly used in these studies to provide data from species in different families. With the exception of carcinogenesis, there is considerable evidence that daily administration of an agent at high-dose concentrations for periods of three to four months will reveal most forms of toxicity in adult animals.
  • Transgenic rodents that over-express or under-express a gene of interest may be inbred and used to model human diseases or to test therapeutic or toxic agents.
  • the introduced gene may be activated at a specific time in a specific tissue type during fetal or postnatal development. Expression of the transcend is monitored by analysis of phenotype, of tissue-specific mRNA expression, or of serum and tissue protein levels in transgenic animals before, during, and after challenge with experimental drug therapies.
  • Embryonic (ES) stem cells isolated from rodent embryos retain the ability to form embryonic tissues. When ES cells are placed inside a carrier embryo, they resume normal development and contribute to tissues of the live-born animal. ES cells are the preferred cells used in the creation of experimental knockout and knocking rodent strains.
  • Mouse ES cells such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and are grown under culture conditions well known in the art. Vectors used to produce a transgenic strain contain a disease gene candidate and a marker gene, the latter serves to identify the presence of the introduced disease gene.
  • the vector is transformed into ES cells by methods well known in the art, and transformed ES cells are identified and micro injected into mouse cell blastocyst such as those from the C57BL/6 mouse strain.
  • the blastocyst are surgically transferred to pseudo pregnant dams, and the resulting chimeric progeny are genotype and bred to produce heterozygous or homozygous strains.
  • ES cells derived from human blastocyst may be manipulated in vitro to differentiate into at least eight separate cell lineages.
  • Lineages are used to study the differentiation of various cell types and tissues in vitro, and they include endoderm, mesoderm, and ectodermal cell types which differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes. Knockout Analysis
  • a region of a gene is enzymatically modified to include a non- mammalian gene such as the neomycin phosphotransferase gene (neo; Capecchi (1989) Science 244:1288-1292).
  • the modified gene is transformed into cultured ES cells and integrates into the endogenous genome by homologous recombination. The inserted sequence disrupts transcription and translation of the endogenous gene.
  • Transformed cells are injected into rodent blastalae, and the blastalae are implanted into pseudo pregnant dams.
  • Transgenic progeny are crossbred to obtain homozygous inbred lines which lack a functional copy of the mammalian gene.
  • the mammalian gene is a human gene.
  • Knockin Analysis ES cells can be used to create knocking humanized animals (pigs) or transgenic animal models
  • NHPs are the first choice test animal.
  • NHPs and individual humans exhibit differential sensitivities to many drugs and toxins and can be classified as a range of phenotypes from "extensive metabolites” to "poor metabolites” of these agents.
  • the polynucleotides which encode the protein maybe used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of polynucleotides that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
  • Genomic DNA from subjects was randomly pre-amplified in a primer extension pre-amplification (PEP) reaction (Zhang et al (1992) Proc Natl Acad Sci USA 89:547-5851). All coding eons and splice junctions of PPAR ⁇ transcripts and PPP1R3 A were amplified by PCR from PEP DNA with gene-specific primers (primer sequences available upon request). PCR products were studied using single-stranded conformation polymorphism analysis and direct sequencing of all abnormal conformers (Thorpe et al.(1999) Immunogenetics 49:256-265). The PPP1R3A frameshift mutation was screened in participants in two independent, population-based, case-control studies in East Yale, UK.
  • PPP primer extension pre-amplification
  • the Cambridgeshire Case Control population consists of a collection of 517 type 2 diabetics and 517 matched controls.
  • the cases were a random sample of Europid men and women with type 2 diabetes, aged 47-75 years from a population-based diabetes register in a geographically defined region of Cambridgeshire, UK.
  • the presence of type 2 diabetes was assumed if the onset of diabetes was after the age of 30 y and insulin therapy was not used in the first year after diagnosis.
  • the EPIC-Norfolk study is a population-based cohort study involving 25,000 individuals recruited from general practices throughout Norfolk, UK. Prevalent and incident cases of diabetes were included in the analysis. Cases were ascertained from multiple sources including primary care registers, hospital clinics, self-report and reported medication.
  • Receptor binding of proteins to DNA was assessed in electrophoretic mobility supershift assays as described in Collingwood et al (1994 Mol Endocrinol 8:1262-1277) using 35 S-labeled, in vitro translated receptors quantified by SDS-PAGE analysis and a 32 P-labeled oligonucleotide duplex corresponding to the PPARE derived from the acyl-CoA. oxidase gene (Zamia et al. (1995) Genes Dev 11:835-846).
  • Transfections of 293 EBNA cells were carried out in 24-well plates using 500 ng of (PPARE) 3 TKLUC and 100 ng of receptor expression vector (wildtype, frameshift mutant, or empty vector, pcDNA3) (Invitrogen Corp, Carlsbad, CA) Using the calcium phosphate method (Collingwood et al, supra). Luciferase values were normalized to ⁇ -galactosidase activity from the internal control phasmid, Bos ⁇ gal (Collingwood et al., supra), and represent the mean +/- sem. of at least three independent experiments each performed in triplicate. IV Immunofluorescence Microscopy
  • CHO cells were transiently transfected (EUGENE; Roche Diagnostics, Ltd, East Canal, UK) with N-terminal, HA-tagged expression vectors containing wildtype or mutated PPP1R3A.
  • PACCMV.pKpA-HA-PPPlR3A was a gift from P. Cohen (Rasmussen, SK (2000) Diabetologia 43:718- 722).
  • Cells were fixed in 3% paraformaldehyde/0.05% gluteraldehyde in 100 mM potassium HEPES/3 mM MgCls buffer (pH 7.5) for 15 min., treated with 0.5% borohydride/PBS for 10 min., and then blocked and permeabilized in 1% BSA/0.1% saponin for 20 min. When permeabilizing cells before fixation, the cells were incubated for 5 min. in 0.5% saponin in 80 mm potassium PIPES/5 mM EGTA/1 mM MgCl 2 (pH 6.8) at room temperature.
  • the assay was automated using the AutoDELFIA immunoassay system (Pekin Elmer Life Sciences).
  • Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIRG), and Genethon are used to determine if any of the polynucleotides presented in the Sequence Listing have been mapped. Any of the fragments of the polynucleotide encoding PPP1R3AFS or PPAR ⁇ FS that have been mapped result in the assignment of all related regulatory and coding sequences to the same location.
  • the genetic map locations are described as ranges, or intervals, of human chromosomes. The map position of an interval, in cm (which is roughly equivalent to 1 megabase of human DNA), is measured relative to the
  • the polynucleotides are applied to a substrate by one of the following methods.
  • a mixture of polynucleotides is fractionated by gel electrophoresis and transferred to a nylon membrane by capillary
  • the polynucleotides are individually ligated to a vector and inserted into bacterial host cells to form a library.
  • the polynucleotides are then arranged on a substrate by one of the following methods.
  • bacterial cells containing individual clones are robotically picked and arranged on a nylon membrane.
  • the membrane is placed on LB agar containing selective agent (carbenicillin, kanamycin, ampicillin, or chloramphenicol depending on the vector used) and incubated at
  • the membrane is removed from the agar and consecutively placed colony side up in 10% SDS, denaturing solution (1.5 M NaCl, 0.5 M NaOH. ), neutralizing solution (1.5 M NaCl, 1 M Tris, pH 8.0), and twice in 2xSSC for 10 min each.
  • the membrane is then UV irradiated in a STRATALENKER UN-cross linker (Stratagene).
  • polynucleotides are amplified from bacterial vectors by thirty cycles of
  • PCR amplification increases a starting concentration of 1-2 ng nucleic acid to a final quantity greater than 5 ⁇ g.
  • Amplified nucleic acids from about 400 bp to about 5000 bp in length are purified using SEPHACRYL-400 beads (APB). Purified nucleic acids are arranged on a nylon membrane manually or using a dot/slot blotting manifold and suction device and are immobilized by denataration, neutralization, and UV irradiation as described
  • Purified nucleic acids are robotically arranged and immobilized on polymer-coated glass slides using the procedure described in US ⁇ 5,807,522.
  • Polymer-coated slides are prepared by cleaning glass microscope slides (Corning Life Sciences) by ultrasound in 0.1% SDS and acetone, etching in 4% hydrofluoric acid (VWR. Scientific Products, West Chester PA), coating with 0.05% aminopropyl silane (Sigma Aldrich) in 95% ethanol, and curing in a HOC oven. The slides are washed extensively with
  • nucleic acids are arranged on the slide and then immobilized by exposing the array to UV irradiation using a STRATA LINKER UV-cross linker (Stratagene). Arrays are then washed at room temperature in 0.2% SDS and rinsed three times in distilled water. Non-specific binding sites are blocked by incubation of arrays in 0.2% casein in phosphate buffered saline (PBS; Tropix, Bedford MA) for 30 min at 60C; then the arrays are washed in 0.2% SDS and rinsed in distilled water as before. Probe Preparation for Membrane Hybridization
  • Hybridization probes derived from the polynucleotides of the Sequence Listing are employed for screening cDNAs, mRNAs, or genomic DNA in membrane-based hybridizations. Probes are prepared by diluting the polynucleotides to a concentration of 40-50 ng in 45 ⁇ l TE buffer, denaturing by heating to 100C for five min, and briefly centrifuging. The denatured polynucleotide is then added to a REDEPRIME tube (APB), gently mixed until blue color is evenly distributed, and briefly centrifuged. Five ⁇ l of [ 32 P]dCTP is added to the tube, and the contents are incubated at 37C for 10 min.
  • APB REDEPRIME tube
  • probe is purified from unincorporated nucleotides using a PROBEQUANT G-50 microcolumn (APB).
  • APB PROBEQUANT G-50 microcolumn
  • the purified probe is heated to 100C for five min, snap cooled for two min on ice, and used in membrane-based hybridizations as described below.
  • Hybridization probes derived from mRNA isolated from samples are employed for screening polynucleotides of the Sequence Listing in array-based hybridizations. Probe is prepared using the
  • GEMbright kit (Incyte Genomics) by diluting mRNA to a concentration of 200 ng in 9 ⁇ l TE buffer and adding 5 ⁇ l 5x buffer, 1 ⁇ l 0.1 M DTT, 3 ⁇ l Cy3 or Cy5 labeling mix, 1 ⁇ l RNAse inhibitor, 1 ⁇ l reverse transcriptase, and 5 ⁇ l lx yeast control mRNAs.
  • yeast control mRNAs are synthesized by in vitro transcription from noncoding yeast genomic DNA (W. Lei, unpublished).
  • one set of control mRNAs at 0.002 ng, 0.02 ng, 0.2 ng, and 2 ng are diluted into reverse transcription reaction mixture at ratios of 1:100,000, 1:10,000, 1:1000, and 1:100 (w/w) to sample mRNA respectively.
  • a second set of control mRNAs are diluted into reverse transcription reaction mixture at ratios of 1:3, 3:1, 1:10, 10:1, 1:25, and 25:1 (w/w).
  • the reaction mixture is mixed and incubated at 37C for two hr.
  • the reaction mixtare is then incubated for 20 min at 85C, and probes are purified using two successive CHROMA SPEN+TE 30 columns (Clontech, Palo Alto CA).
  • Purified probe is ethanol precipitated by diluting probe to 90 ⁇ l in DEPC-treated water, adding 2 ⁇ l lmg/ml glycogen, 60 ⁇ l 5 M sodium acetate, and 300 ⁇ l 100% ethanol.
  • the probe is centrifuged for 20 min at 20,800xg, and the pellet is resuspended in 12 ⁇ l resuspension buffer, heated to 65C for five min, and mixed thoroughly. The probe is heated and mixed as before and then stored on ice. Probe is used in high density array-based hybridizations as described below.
  • Membrane-based Hybridization as described below.
  • Membranes are pre-hybridized in hybridization solution containing 1% Sarkosyl and lx high phosphate buffer (0.5 M NaCl, 0.1 M Na 2 HP0 4 , 5 mM EDTA, pH 7) at 55C for two hr.
  • the probe diluted in 15 ml fresh hybridization solution, is then added to the membrane.
  • the membrane is hybridized with the probe at 55C for 16 hr.
  • the membrane is washed for 15 min at 25C in ImM Tris (pH 8.0), 1% Sarkosyl, and four times for 15 min each at 25C in ImM Tris (pH 8.0).
  • XOMAT-AR film Eastman Kodak, Rochester NY is exposed to the membrane overnight at -70C, developed, and examined visually.
  • Probe is heated to 65C for five min, centrifuged five min at 9400 rpm in a 5415C microcentrifuge (Eppendorf Scientific, Westbury NY), and then 18 ⁇ l is aliquoted onto the array surface and covered with a coverslip.
  • the arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide.
  • the chamber is kept at 100% humidity internally by the addition of 140 ⁇ l of 5xSSC in a corner of the chamber.
  • the chamber containing the arrays is incubated for about 6.5 hr at 60C.
  • the arrays are washed for 10 min at 45C in lxSSC, 0.1% SDS, and three times for 10 min each at 45C in O.lxSSC, and dried.
  • Hybridization reactions are performed in absolute or differential hybridization formats.
  • absolute hybridization format probe from one sample is hybridized to array elements, and signals are detected after hybridization complexes form. Signal strength correlates with probe mRNA levels in the sample.
  • differential hybridization format differential expression of a set of genes in two biological samples is analyzed. Probes from the two samples are prepared and labeled with different labeling moieties. A mixture of the two labeled probes is hybridized to the array elements, and signals are examined under conditions in which the emissions from the two different labels are individually detectable. Elements on the array that are hybridized to equal numbers of probes derived from both biological samples give a distinct combined fluorescence (Shalon WO95/35505).
  • Hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
  • the excitation laser light is focused on the array using a 20X microscope objective (Nikon, Melville NY).
  • the slide containing the array is placed on a computer- controled X-Y stage on the microscope and raster-scanned past the objective with a resolution of 20 micrometers.
  • the two fluorophores are sequentially excited by the laser.
  • Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Filters positioned between the array and the photomultiplier tabes are used to separate the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5. The sensitivity of the scans is calibrated using the signal intensity generated by the yeast control mRNAs added to the probe mix. A specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000.
  • the output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (A D) conversion board (Analog Devices, Norwood MA) installed in an IBM-compatible PC computer.
  • the digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
  • the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using the emission spectrum for each fluorophore.
  • a grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid.
  • the fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal.
  • the software used for signal analysis is the GEMTOOLS program (Incyte Genomics). VIII Complementary Mol
  • Antisense molecules complementary to the cDNA are used to detect or inhibit gene expression. Detection is described in Example VH.
  • the complementary molecule is designed to bind to the most unique 5' sequence and includes nucleotides of the 5' UTR upstream of the initiation codon of the open reading frame.
  • Complementary molecules include genomic sequences (such as enhancers or introns) and are used in triple helix base pairing to compromise the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • a complementary molecule is designed to prevent ribosomal binding to the mRNA encoding the protein.
  • Complementary molecules are placed in expression vectors and used to transform a cell line to test efficacy; into an organ, tumor, synovial cavity, or the vascular system for transient or short term therapy; or into a stem cell, zygote, or other reproducing lineage for long term or stable gene therapy.
  • Transient expression lasts for a month or more with a non-replicating vector and for three months or more if elements for inducing vector replication are used in the transformation/expression system.
  • Stable transformation of dividing cells with a vector encoding the complementary molecule produces a transgenic cell line, tissue, or organism (USPN 4,736,866). Those cells that assimilate and replicate sufficient quantities of the vector to allow stable integration also produce enough complementary molecules to compromise or entirely eliminate activity of the cDNA encoding the protein.
  • an antibody array can be used to study protein-protein interactions and phosphorylation.
  • a variety of protein ligands are immobilized on a membrane using methods well known in the art. The array is incubated in the presence of cell lysate until proteimantibody complexes are formed. Proteins of interest are identified by exposing the membrane to an antibody specific to the protein of interest.
  • a protein of interest is labeled with digoxigenin (DIG) and exposed to the membrane; then the membrane is exposed to anti-DIG antibody which reveals where the protein of interest forms a complex.
  • DIG digoxigenin
  • the identity of the proteins with which the protein of interest interacts is determined by the position of the protein of interest on the membrane.
  • Antibody arrays can also be used for high-throughput screening of recombinant antibodies. Bacteria containing antibody genes are robotically-picked and griddled at high density (up to 18,342 different double-spotted clones) on a filter. Up to 15 antigens at a time are used to screen for clones to identify those that express binding antibody fragments. These antibody arrays can also be used to identify proteins which are differentially expressed in samples (de Wildt, supra)
  • the polynucleotide, protein, or antibody is labeled with a nucleotide such as 32 P-dCTP, Cy3- dCTP, or Cy5-dCTP, an amino acid such as 35 S-methionine, or reagents such as BIODEPY or E-TTC (Molecular Probes, Eugene OR). Kits for direct synthesis or chemical conjugation are supplied by companies such as APB, Invitrogen, Promega, or Qiagen. Libraries of candidate molecules ors compounds previously arranged on a substrate are incubated in the presence of labeled polynucleotide or protein.
  • the substrate After incubation under conditions for either a nucleic acid or amino acid sequence, the substrate is washed, and any position on the substrate retaining label, which indicates specific binding or complex formation, is assayed, and the ligand is identified. Data obtained using different concentrations of the nucleic acid or protein are used to calculate affinity between the labeled nucleic acid or protein and the bound molecule.
  • a yeast two-hybrid system MATCHMAKER LexA Two-Hybrid system (Clontech Laboratories), is used to screen for peptides that bind the protein of the invention.
  • a polynucleotide encoding the protein is inserted into the multiple cloning site of a pLexA vector, ligated, and transformed into E. coh.
  • cDNA prepared from mRNA, is inserted into the multiple cloning site of a pB42AD vector, ligated, and transformed into E. coli to construct a cDNA library.
  • the pLexA plasmid and pB42AD- cDNA library constructs are isolated from E.
  • Transformed yeast cells are plated on synthetic dropout (SD) media lacking histidine (-His), tryptophan (-Trp), and uracil (-Ura), and incubated at 30C until the colonies have grown up and are counted.
  • SD synthetic dropout
  • the colonies are pooled in a minimal volume of lx TE (pH 7.5), replated on SD/-His/-I-eu/-Trp/-Ura media supplemented with 2% galactose (Gal), 1% raffinose (Raf), and 80 mg/ml 5-bromo-4-chloro-3-indolyl ⁇ -d-galactopyranoside (X-Gal), and subsequently examined for growth of blue colonies. Interaction between expressed protein and cDNA fusion proteins activates expression of a LEU2 reporter gene in EGY48 and produces colony growth on media lacking leucine (-Leu).
  • Interaction also activates expression of ⁇ -galactosidase from the p8op-lacZ reporter construct that produces blue color in colonies grown on X-Gal Positive interactions between expressed protein and cDNA fusion proteins are verified by isolating individual positive colonies and growing them in SD/-Trp/-Ura liquid medium for 1 to 2 days at 30C. A sample of the culture is plated on SD/-Trp/-Ura media and incubated at 30C until colonies appear. The sample is replica-plated on SD/-Trp/-Ura and SD/-His/-Trp/-Ura plates.
  • Body fat was quantified by magnetic resonance imaging (MRl) as described previously ⁇ !.
  • HOMA homeostasis model assessment
  • ⁇ IMCL reference values represent mean and SD of measurements from 76 control subjects (unpublished observations EL Thomas and JD Bell)

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Abstract

The invention provides mutated genes for peroxisome proliferator-activated receptor gamma (PPARϜ ) and the glycogen-associated regulatory subunit of protein phosphatase-1 (PPP1R3A), PPARϜ FS and PPP1R3AFS, respectively, and polynucleotides and proteins of PPARϜ FS and PPP1R3AFS that are expressed in diabetes. It also provides for the use of the DNA mutation, the protein, a polynucleotide encoding the protein, and antibodies that specifically bind the protein in various methods to diagnose, stage, treat, or monitor the treatment of diabetes.

Description

DIGENIC MUTATIONS ASSOCIATED WITH SEVERE INSULIN RESISTANCE AND TYPE 2 DIABETES AND THEIR USE IN THE DIAGNOSIS AND TREATMENT OF
DIABETES This application claims the benefit of Provisional Application Serial No. 60/350,405, filed 18
January 2002, all of which application is hereby incorporated by reference herein.
FIELD OF THE INVENTION This invention relates to the use of mutations in PPARγ and PPP1R3A and its encoding polynucleotides to diagnose, to stage, to treat, or to monitor the progression or treatment of diabetes, including insulin resistance and type 2 diabetes.
BACKGROUND OF THE INVENTION Type 2 diabetes is a serious health problem in the Western world. It arises when resistance to the glucose-lowering effects of insulin combines with impaired insulin secretion to raise the levels of glucose in the blood beyond the normal range. Recent studies on the molecular basis of insulin resistance have focused on two particular molecules involved in glucose metabolism; peroxisome proliferator-activated receptor gamma (PPARγ), and PPP1R3A, a muscle-specific regulatory subunit of protein phosphatase-1 (PP1).
PPARγ is a key regulator of adipocyte differentiation and a target for a new class of antidiabetic drugs, thiazolidinediones; highly specific ligands for PPARγ that act to increase insulin sensitivity (Lehman et al (1995) J Biol Chem 270:12953-12956; Day, C (1999) Diabetic Med 16:179- 192). The requirement for PPARγ in m-imtaining normal insulin sensitivity in humans was further confirmed in a key study by Barroso et al identifying two loss-of-function mutations in PPARγ associated with severe insulin resistance (SIR) and diabetes mellitus in humans (Barroso et al (1999) Nature 402:880-883). PPP1R3A is a key molecule in the regulation of glycogen synthesis, the muscle and liver mode of glucose storage. Acting in part through PPP1R3 A, insulin activates glycogen synthase by the activation of PP1 which, in turn, activates glycogen synthase through its dephosphorylation. Impaired insulin-stimulated glycogen synthesis is a characteristic feature of type 2 diabetes , however, the potential role of PPP1R3 A in participating and leading to this impairment is uncertain. Although previous studies have identified a number of polymorphisms in PPP1R3 A, these studies have given conflicting results in terms of their association with insulin resistance and type 2 diabetes (Hansen et al 1995 Hum Mol Genet 4:1313-1320; Xia et al (1998) Diabetes 47:1519-1524; Shen et al 1998, Diabetes Care 21:1086-1089; and Suzuki and Lanner (2001) Mol Cell Biol 21:2683-2694).
Digenic inheritance has been attributed to a number of human inherited conditions. Retinitis pigmentosa is caused by mutations in ROM1 and RDS, which encode subunits of an oligomeric transmembrane protein complex in photoreceptor discs (Goldberg, A.F. and Molday, R.S. (1996) Proc Natl Acad Sci USA 93:13726-13730). Junctional epidermolysis bullosa (JEB) results from mutations in laminin 5 and collagen XVII, two of the components of the hemidesmosome-anchoring filament in skin (Floeth, M and Bruckner-Tuderman, L (1999) Am J Hum Genet 65:1520-1537). Finally, it has been suggested that Bardet-Biedl syndrome (BBS) may be a complex trait requiring three mutant alleles in at least two genes to manifest the phenotype (Katsanis et al (2001) Science 293:2256-2259).
Although no previous human examples of digenic inheritance of human insulin resistance or type 2 diabetes have been described, a number of experimental genetic manipulations in murine models have established the principle that such gene-gene interaction might result in metabolic disorders. Thus Bruning et al. (1997, Cell 8:561-572) demonstrated that while mice heterozygous for the insulin receptor or insulin receptor substrate-1 (IRS-1) knockouts had minor abnormalities, doubly heterozygous animals were markedly insulin resistant and had a high incidence of diabetes. Similarly, the crossing of IRS-1 and glucokinase knockout mice produced a digenic model of type 2 diabetes (Terauchi et al (1997) J Clin Invest 99:861-866). While previous reports involving human digenic diseases involved direct protein-protein interactions between the two mutant proteins, there is evidence in insulin regulation and diabetes that cooperativity between genes in separate insulin-sensitive tissues may occur. Both skeletal muscle and adipose tissue are involved in insulin-stimulated nutrient storage and may communicate by poorly understood mechanisms (Birnbaum, M.J. (2001) Nature 409:672-673). Moreover, the development of muscle insulin resistance in fat-specific Glut4 knockout mice provides in vivo evidence of such an interaction between fat and muscle (Abel et al. (2001) Nature 409:729-733).
The present invention provides compositions and their methods of use in the diagnosis, prevention and/or treatment of diabetes, including insulin resistance and type 2 diabetes.
SUMMARY OF THE INVENTION
The invention is based on the discovery two mutations in the genes encoding PPARγ and PPP1R3A, designated PPARγFS and PPP1R3 AFS, respectively, which are useful to diagnose, stage, treat, or to monitor the progression or treatment of a diabetes, including insulin resistance and type 2 diabetes. The invention provides a method for the diagnosis of diabetes in a subject by genotyping of a subject's DNA to detect the presence of the two mutations for PPARγFS and PPP1R3AFS in a sample of genomic DNA from the subject. The invention also provides a method for diagnosing a predisposition or a risk factor for development of diabetes in a subject by genotyping of a subject's DNA to detect the presence of the mutation for PPP1R3AFS, alone. The invention provides an isolated polynucleotide comprising a nucleic acid sequence encoding a protein having the amino acid sequence of SEQ ED NO:l (PPARγFS) or SEQ ID NO:2 (PPP1R3AFS,). The invention also provides an isolated polynucleotide or the complement thereof comprising a nucleic acid sequence of SEQ ID NO:3 (encoding SEQ ID NO:l) or SEQ ID NO:4 (encoding SEQ ED NO:2), a cell transformed with the polynucleotide encoding PPARγFS or
PPP1R3AFS, a composition comprising the polynucleotide encoding PPARγFS or PPP1R3AFS, and a labeling moiety, and an array element comprising the polynucleotide encoding PPARγFS or PPP1R3AFS.
The invention provides a vector containing the polynucleotide encoding PPARγFS or PPP1R3AFS, a host cell containing the vector and a method for using the polynucleotide to make the protein, the method comprising culturing the host cell containing the vector containing the polynucleotide encoding the protein under conditions for expression and recovering the protein from the host cell culture. The invention further provides a composition, a substrate or a probe comprising the polynucleotide, or complement thereof, which can be used in methods of detection, screening, and purification. In one aspect, the probe is a single-stranded complementary RNA or DNA molecule. The invention provides a method for using a nucleic acid probe to detect the differential expression of a nucleic acid in a sample comprising hybridizing a probe to the nucleic acids, thereby forming hybridization complexes and comparing hybridization complex formation with a standard, wherein the comparison indicates the differential expression of the nucleic acid in the sample. In one aspect, the method of detection further comprises amplifying the nucleic acids of the sample prior to hybridization. In another aspect, the method showing the coexpression of the polynucleotides encoding PPARγFS and PPP1R3AFS is diagnostic of type 2 diabetes .
The invention provides a purified protein comprising SEQ ID NO:l or SEQ ID NO:2. The invention further provides a method for detecting expression of a protein having the amino acid sequence of SEQ ID NO:l or SEQ ID NO:2 in a sample, the method comprising performing an assay to determine the amount of the protein in a sample; and comparing the amount of protein to a standard, thereby detecting expression of the protein in the sample. In a one aspect, the assay is selected from affinity chromatography, antibody arrays, enzyme-linked immunosorbent assays, fluorescence- activated cell sorting, protein arrays, radioimmunoassays, and 2D-PAGE in conjunction with scintillation counting, high performance hquid chromatography, mass spectrometry or western analysis. In a second aspect the assay detects the expression of SEQ ID NO: 1 in the sample and is diagnostic of a predisposition to diabetes. In a third aspect, the assay detects the coexpression of SEQ ID NO:l and SEQ ID NO:2 and is diagnostic of the presence of type 2 diabetes.
The invention provides a method for using a protein to screen a library or a plurality of molecules or compounds to identify at least one ligand, the method comprising combining the protein with the molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein. In one aspect, the molecules or compounds are selected from agonists, antagonists, DNA molecules, small drug molecules, immunoglobulins, inhibitors, rnimetics, multispecific molecules, peptides, peptide nucleic acids, pharmaceutical agent, proteins, and RNA molecules. In another aspect, the ligand is used to treat a subject with diabetes. The invention further provides an agonist which specifically binds PPP1R3A. The invention yet further provides a small drug molecule which specifically binds PPP1R3 A. The invention also provides a method for testing ligand for effectiveness as an agonist or antagonist comprising exposing a sample comprising the protein to the molecule or compound, and detecting agonist or antagonist activity in the sample.
The invention provides an agonist that specifically binds the protein, and a composition comprising the agonist and a pharmaceutical carrier. The invention further provides a pharmaceutical agent or a small drag molecule that specifically binds the protein. The invention provides a method of treating diabetes comprising administering to a patient in need of such treatment an effective amount of an agonist of PPP1R3 A.
BRIEF DESCRIPTION OF THE FIGURES AND TABLE
Figure 1 illustrates the effects of the mutations in PPARγ and PPP1R3 A on truncation of the proteins. Figure 1A shows the frameshift premature stop mutation (A533AAAiT)fs.l85(stop 186) in PPARγ resulting in a mutation of K = lysine to M = methionine at amino acid position 185 of PPARγ within the second zinc-finger (Zn) of the DNA-binding domain (DBD) together with a mutation of S = serine to stop codon (X) at position 186 of PPARγ. Figure IB shows the frameshift/prematare stop mutation (C1984AG)fs.662(stop 668) in PPP1R3A resulting in mutation of an N = asparagine to stop codon at amino acid position 668 of PPP1R3A. PP1C/GBD = PP1 catalytic subunit and glycogen binding domains; SRBD = sarcoplasmic reticulum-binding domain.
Figure 2 illustrates a family pedigree, Family A, and the concordance of features related to severe insulin resistance and type 2 diabetes and the presence of PPARγ and PPP1R3 A mutations. Solid coloring in individual family members indicates the presence of the phenotypes: upper left quadrant, acanthosis nigricans + hyperinsulinemia; upper right, diabetes; lower left, dyslipidemia; lower right, hypertension. Other abbreviations are: + = wild Type gene; P = PPARγ mutation; R3 = PPP1R3 A mutation; BMI = Body Mass Index; FI = fasting plasma insulin concentration (pmol/L).
Figure 3 represents fasting plasma insulin concentrations (Y axis) plotted against body mass index (BMI; X axis) for family A members. The solid line represents the log-linear regression line between FI and BMI in 1121 participants in the MRC Ely population-based cohort study (Williams et al. (1994) Diabetic Medicine 12:30-35). The 95% confidence intervals (broken lines) include 95% of the individuals at any given BMI.
Figure 4 illustrates a Family B pedigree, the presence of the R3 mutation, and the incidence of phenotypes related to diabetes. Abbreviations and the presence of various phenotypes are the same as described for Figure 2.
Figure 5 A shows the results of an assay to measure DNA binding properties of the FS mutation in either PPARγl (FSγl) or PPARγ2 (FSγ2) compared with the corresponding wild type gene products (WTγl and WYγ2, respectively). The DNA binding assay is described in Example II. The open arrow indicates the location of the PPARγ-RXR heterodimer, and the solid arrow denotes the unbound probe. RL = reticulocyte lysate; RXR = retinoid X receptor. Figure 5B shows 35S- labeled in vitro translated wildtype and mutant PPARγl and PPARγ2.
Figure 6 shows the results of receptor mediated transactivation in 293 EBNA cells transfected with either PPARγ wild type isoforms (WTγl or WTγ2), or mutant WTγ (FSγl or FSγ2) together with a reporter gene, (PPARE)3TKLUC, in the presence of increasing concentrations of the thiazolidinedione, rosiglitazone. Vector = empty expression vector + reporter gene. Results are expressed as % of the maximum transactivation obtained with WTγl.
Figure 7 shows the results of cotransfection of 293 EBNA cells with 100 ng each of wild type + wild type PPARγ (WT + WT) or wild type PPARγ + mutant PPARγ (WT + FS) together with the reporter gene, (PPARE)3TKLUC, and consequent effects on transactivation as determined in Figure 6. Results are expressed as % of the maximum transactivation obtained with WTγl.
Figures 8 shows western blots of whole cell lysates from CHO cells transiently transfected with HA-tagged wild type PPP1R3 A (WT) or PPP1R3AFS (FS). Figure 8A shows the western blot analyzed using a sheep monoclonal N-terminal PPP1R3A antibody. Figure 8B shows the results of a western blot in which the cell lysates were first immunoprecipitated with anti-HA antibody (Santa Cruz Biotechnology, Inc, Santa Cruz, CA) followed by western blot with an anti-PPlC antibody (Santa Cruz Biotechnology).
Figure 9 shows the results of immunofluoresence microscopy of CHO cells transfected with HA-tagged wild type PPP1R3A (WT) or PPP1R3AFS (FS). Cells were either treated with saponin to release cytosolic PPP1R3A (+ saponin; right panel), or untreated (- saponin; left panel), then fully permeabilized and labeled with anti-HA prior to fixation.
Table 1 summarizes clinical and biochemical characteristics of the frameshift mutation carriers for both PPARγFS and PPP1R3 AFS in both Family A and Family B members. HDL, high-density lipoprotein; NEFA, non-esterified fatty acids; IMCL, intramyocellular lipid. DESCRIPTION OF THE INVENTION
It is understood that this invention is not limited to the particular machines, materials and methods described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to limit the scope of the present invention which will be limited only by the appended claims. As used herein, the singular forms "a", "an", and "the" may include plural reference unless the context clearly dictates otherwise. For example, a reference to "a host cell" includes a plurality of such host cells known to those skilled in the art.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Definitions
The term "antibody" refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding an epitopic determinant. Antibodies that bind PPARγFS or PPP1R3AFS polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protem may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody. "PPARγFS "refers to a PPARγ mutation that is the same or highly homologous (>85%) to the amino acid sequence of SEQ ID NO:l or the polynucleotide sequence of SEQ ID NO:3 obtained from any species including bovine, ovine, porcine, murine, equine, and preferably the human species, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
"PPP1R3AFS" refers to a PPP1R3A mutation that is the same or highly homologous (>85%) to the amino acid sequence of SEQ ID NO:2 or the polynucleotide sequence of SEQ ID NO:4 obtained from any species including bovine, ovine, porcine, murine, equine, and preferably the human species, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
The "complement" of a nucleic acid of the Sequence Listing refers to a nucleic acid molecule which is completely complementary over its full length and which will hybridize to a nucleic acid molecule under conditions of high stringency.
A "composition comprising a given polynucleotide" and a "composition comprising a given polypeptide" can refer to any composition containing the given polynucleotide or polypeptide. The composition may comprise a dry formulation or an aqueous solution. Compositions comprising polynucleotides encoding PPARγFS or PPP1R3 AFS or fragments of PPARγFS or PPP1R3 AFS may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
"Conservative amino acid substitutions" are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. The table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions. Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gin, His
Asp Asn, Glu
Cys Ala, Ser
Gin Asn, Glu, His
Glu Asp, Gin, His
Gly Ala
His Asn, Arg, Gin, Glu lie Leu, Val
Leu He, Val
Lys Arg, Gin, Glu
Met Leu, lie
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val lie, Leu, Thr Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain. A "deletion" refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide. "Differential expression" refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, deteπriined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
An "expression profile" is a representation of gene expression in a sample. A nucleic acid expression profile may be produced, for example, using sequencing, hybridization, or amplification (quantitative PCR) technologies and mRNAs or cDNAs from a sample. A protein expression profile, although time delayed, mirrors the nucleic acid expression profile and may use antibody or protein arrays, enzyme-linked immunoadsorbent assays, fluorescence-activated cell sorting, spatial immobilization such as 2D-PAGE in conjunction with a scintillation counter, mass spectrophotometry, or western analysis or affinity chromatography, to detect protein expression in a sample. The nucleic acids, proteins, or antibodies may be used in solution or attached to a substrate, and their detection is based on methods and labeling moieties well known in the art. Expression profiles may also be evaluated by methods such as electronic northern analysis, guilt-by-association, and transcript imaging. Expression profiles produced using any of the above methods may be compared with expression profiles produced using normal or diseased tissues. The correspondence between mRNA and protein expression has been discussed by Zweiger (2001, Transducing the Genome. McGraw-Hill, San Francisco, CA) and Glavas et al. (2001; T cell activation upregulates cyclic nucleotide phosphodiesterases 8A1 and 7A3, Proc Natl Acad Sci 98:6319-6342) among others.
"Genotyping" refers to the determination of the specific nucleotide- base sequence composition of alternative forms of DNA, at specific locations within each of the chromosome pairs in a cell or in an organism as a whole.
The term "hybridization complex" refers to a complex formed between two nucleic acids by virtue of the formation of hydrogen bonds between complementary bases. A hybridization complex may be formed in solution (e.g., C0t or Rot analysis) or formed between one nucleic acid present in solution and another nucleic acid immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
"Identity" as applied to sequences, refers to the quantification (usually percentage) of nucleotide or residue matches between at least two sequences aligned using a standardized algorithm such as Smith- Waterman alignment (Smith and Waterman (1981) J Mol Biol 147:195-197), CLUSTALW (Thompson et al. (1994) Nucleic Acids Res 22:4673-4680), or BLAST2 (Altschul (1997, supra). BLAST2 maybe used in a standardized and reproducible way to insert gaps in one of the sequences in order to optimize alignment and to achieve a more meaningful comparison between them. "Similarity" uses the same algorithms but takes conservative substitution of residues into account. In proteins, similarity exceeds identity in that substitation, for example, of a valine for a leucine or isoleucine, is counted in calculating the reported percentage. Substitutions which are considered to be conservative are well known in the art.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of PPARγFS or PPP1R3AFS which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal The term "immunogenic fragment" also includes any polypeptide or oligopeptide fragment of PPARγFS or PPP1R3AFS which is useful in any of the antibody production methods disclosed herein or known in the art.
"Labeling moiety" refers to any reporter molecule including radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, substrates, cofactors, inhibitors, or magnetic particles than can be attached to or incorporated into a polynucleotide, protein, or antibody. A wide variety conjugation techniques are known in the art and include both direct synthesis and chemical conjugation, particularly to amines, thiols and other side groups which may be present. Visible labels and dyes include but are not limited to anthocyanins, β glucuronidase, biotin, BIODIPY, Coomassie blue, Cy3 and Cy5, 4,6-diamidino- 2-phenylindole (DAPI), digoxigenin, fluorescein, FTTC, gold, green fluorescent protein (GFP), nssamine, luciferase, phycoerythrin, rhodamine, spyro red, silver, streptavidin, and the like. Radioactive markers include radioactive forms of hydrogen, iodine, phosphorous, sulfur, and the like.
"Ligand" refers to any agent, molecule, or compound which will bind specifically to a polynucleotide or to an epitope of a protein. Such ligands stabilize or modulate the activity of polynucleotides or proteins and may be composed of inorganic and/or organic substances including minerals, cofactors, nucleic acids, proteins, carbohydrates, fats, and lipids. The term "microarray" refers to an arrangement of a plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microarray. The term "modulate" refers to a change in the activity of PPARγ or PPP1R3A. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of PPARγ or PPP1R3A.
A "multispecific molecule" can bind with at least two different binding specificities to at least two different molecules or two different sites on a molecule. Antibodies can perform as multispecific molecules in that they can bind to both a target protein and a pharmaceutical agent.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material. "Oligonucleotide" refers a single-stranded molecule from about 18 to about 60 nucleotides in length which may be used in hybridization or amplification technologies or in regulation of replication, transcription or translation. Equivalent terms are amplicon, amplimer, primer, and oligomer.
"Operably linked" refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
A "pharmaceutical agent" may be an antibody, an antisense molecule, a multispecific molecule, a peptide, a protein, a radionuclide, a small drug molecule, a cytospecific or cytotoxic drug such as abrin, actinomyosin D, cisplatin, crotin, doxorubicin, 5-fluorouracil, methotrexate, ricin, vincristine, vinblastine, or any combination of these elements.
"Post-translational modification" of a protein can involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and the like. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cellular location, cell type, pH, enzymatic milieu, and the like.
"Probe" refers to polynucleotides encoding PPARγFS or PPP1R3AFS, their complements, or fragments thereof, which are used to detect identical, allelic or related polynucleotides. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. "Primers" are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR).
"Protein" refers to a polypeptide or any portion thereof. A "portion" of a protein refers to that length of amino acid sequence which would retain at least one biological activity, a domain identified by PFAM or PRINTS analysis or an antigenic deteπninant of the protein identified using Kyte-Doolittle algorithms of the PROTEAN program (DNASTAR, Madison WI). An "oligopeptide" is an amino acid sequence from about five residues to about 15 residues that is used as part of a fusion protein to produce an antibody.
A "recombinant nucleic acid" is a nucleic acid that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook and Russell (supra). The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell. Alternatively, such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
An "RNA equivalent," in reference to a DNA molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
"Sample" is used in its broadest sense as containing nucleic acids, proteins, and antibodies. A sample may comprise a bodily fluid such as ascites, blood, cerebrospinal fluid, lymph, semen, sputum, urine and the like; the soluble fraction of a cell preparation, or an aliquot of media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue, a tissue biopsy, or a tissue print; buccal cells, skin, hair, a hair follicle; and the like.
The terms "specific binding" and "specifically binding" refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A," the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody. The term "substantially purified" refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably at least about 75% free, and most preferably at least about 90% free from other components with which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any rigid or semi-rigid support to which polynucleotides, proteins, or antibodies are bound and includes magnetic or nonmagnetic beads, capillaries or other tubing, chips, fibers, filters, gels, membranes, plates, polymers, slides, wafers, and micro particles with a variety of surface forms including channels, columns, pins, pores, trenches, and wells. "Transformation" describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term "transformed cells" includes stable transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating phasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time. A "transgenic organism," as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by micro injection or by infection with a recombinant virus. In another embodiment, the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002) Science 295:868-872). The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook and Russell (supra).
"Variant" refers to molecules that are recognized variations of a protein or the polynucleotides that encode it. Splice variants may be determined by BLAST score, wherein the score is at least 100, and most preferably at least 400. Allelic variants have a high percent identity to the cDNAs and may differ by about three bases per hundred bases. "Single nucleotide polymorphism" (SNP) refers to a change in a single base as a result of a substitution, insertion or deletion. The change may be conservative (purine for purine) or non-conservative (purine to pyrimidine) and may or may not result in a change in an encoded amino acid or its secondary, tertiary, or quaternary structure. THE INVENTION
Using mutational screening (Example I) a heterozygous frameshift was identified resulting in a premature stop mutation in PPARγ (Figure lA;PPARγFS;SEQ ID NO:l) that was present in a Europid pedigree (Family A; Figure 2). (For purposes of comparison, the nucleic acid and protein sequences of the normal PPARγ are given in the Sequence Listing as SEQ ID NO:5 and SEQ ID NO:7, respectively, and for the normal PPP1R3A as SEQ ED NO:6 and SEQ ID NO:8, respectively). The grandparents (individuals Ii and Iii) had typical late-onset type 2 diabetes with no clinical features of severe insulin resistance. Three of their six children and two of their grandchildren had acanthosis nigricans, a dermatological marker of extreme insulin resistance. All five individuals with acanthosis nigricans had markedly elevated fasting plasma insulin levels, indicative of severe insulin resistance. The PPARγFS mutation (P) was found in the grandfather (Ii), all five relatives with severe insulin resistance, and one other relative with normal insulin levels (Ilvi). Further candidate-gene studies revealed a heterozygous frameshift/premature stop mutation in PPP1R3A (Figure 1B;PPP1R3AFS;SEQ ID NO:2) that was also present in this family. In this case, the mutation (R3) was present in the grandmother (Iii), in all five individuals with severe insulin resistance and in one other relative (Elii). Thus, all five family members with severe insulin resistance, and no other family members, were doubly heterozygous with respect to the two frameshift mutations of these two unlinked genes. The doubly heterozygous individuals were variably affected by additional features of syndrome X, a condition characterized by obesity, insulin resistance, dyslipidemia, hypertriglyceridemia, and high LDL (low-density lipoprotein)/low HDL (Hauler, H. (2002) Eur J Clin Nut 56:S25-S29). Dyslipidemia is defined by triglycerides > 2mmol/L and high- density lipoprotein (HDL) < lmmol/L. Figure 3 shows that fasting insulin levels in the singly heterozygous and wildtype family members were within the normal range. By contrast, the double heterozygotes showed extreme hyperinsulinemia and, to a variable extent, diabetes, hyperlipidemia and hypertension (Figure 2 and Table 1). As diabetes, hypertension or dyslipidemia were also present in some other members of the kindred, these phenotypes do not seem to require mutations in both PPARγ and PPP1R3A.
A cohort of prebends with syndromes of severe insulin resistance (n = 129) was screened for the PPARγ and PPP1R3A frameshift mutations. The PPARγFS frameshift mutation was not detected in any other individuals, whereas one Europid individual carried the same heterozygous frameshift mutation of PPP1R3A that was found in family A. This individual (iii, family B; Figure 4) presented with acanthosis nigricans at age 20 years. He had a body mass index (BMI) of 36.5 kg m2 and a fasting insulin level of 437 pmol i1 (normal <80 ρmol/1). He inherited the mutation from his moderately obese father (Ii; BMI 30 kg m2), who also has marked hyperinsulinemia (fasting insulin 178 pmol l 1; Figure 4). The two other family members who did not carry these mutations were clinically and biochemically normal Notably, subject HI (family B) subsequently lost 40 kg and reduced his BMI to 27 kg m2. By that time, his fasting insulin level had fallen to 93 pmol l 1. These results suggest that carriers of the PPP1R3A frameshift mutation develop fasting hyperinsulinemia when obese.
Figure 5 shows the results of a DNA binding assay for wild type PPARγl and PPARγ2 and the corresponding mutant isoforms when complete with RXR using an electrophoretic mobility shift assay. Unlike their wildtype counterparts (WTγl and WTγ2), neither mutant PPARγ isoforms (FSγl or FSγ2) formed heterodimeric complexes when co-incubated with a radio labeled probe encoding the acyl-CoA. oxidase, PPARE (Figure 5A). Accordingly, and in contrast to wildtype receptors, neither mutant receptor mediated transactivation when cotransfected with a reporter gene containing PPARE and increasing concentrations of the thiazolidinedione, rosiglitazone (Figure 6). Moreover, unlike the previously reported naturally occurring missense PPARγ mutations (Barrios et al., supra) the truncated PPARγ mutant proteins, FSγl and FSγ2, do not show dominant-negative activity when co-expressed with the wildtype receptor (Figure 7). Transcriptional responses to lOOng or 200ng WT receptor were identical (data not shown). The results of these studies show that the loss of the DBD domain of PPARγ in the truncated PPARγFS protein results in the loss of transcriptional function of the protein.
Figure 8A shows the presence of the truncated PPP1R3A mutant protein in CHO cells transfected with the FS mutation and compared with cells likewise transfected with the wildtype gene. The mutant gene produces a protein of the expected size, approximately 83 kDa. The wildtype PPP1R3A undergoes rapid proteolysis to produce proteolytic fragments of a similar size. Figure 8B demonstrates that when the cell lysate from either PPP1R3A (WT) or PPP1R3AFS (FS) transfected cells is immunoprecipitated with anti-HA and then western blotted with anti-PPIC antibody, the truncated PPP1R3AFS protein interacts with PP1C with an efficiency similar to PPP1R3A. Figure 9 shows a comparison of the intracellular distribution for wildtype PPP1R3A and wildtype PPP1R3 AFS. The results show a significant fraction of wildtype PPP1R3 A localized to intracellular membranes, as expected. However, the mutant PPP1R3 AFS protein is almost exclusively cytosolic.
As a result of 1) the a priori knowledge that both the PPARγ and PPP1R3A genes are intimately involved in insulin action 2) the fact that the mutations found resulted in truncated proteins with clear abnormalities in their function (PPARγFS) or localization ( PPP1R3AFS) and 3) the observation that only the five doubly heterozygous members of family A and not seven other members had unequivocal severe insulin resistance, it appears highly likely that the extreme insulin resistant phenotype seen in this family is the result of an interaction between the two mutations. This likelihood is consistent with previously known evidence of cooperativity between muscle and adipose tissues in insulin regulation.
It is further evident that the mutation in PPP1R3 A alters the function of the gene, e.g. , loss of the SRBD binding domain (Figure IB). Furthermore, as the truncated PPP1R3A mutant protein can still interact with the catalytic subunit, PP1C, it may actively interfere with the normal function of that subunit. Therefore, modulation of the activity of the PPP1R3A gene by any means, such as a small molecule drug, in particular an agonist of PPP1R3 A, could be an effective means of treating diabetes.
Additional evidence indicates that the PPP1R3AFS mutation is prevalent at a higher frequency in Type 2 diabetics than control populations, and therefore may be diagnostic of a predisposition for the development of diabetes. Cambridgeshire Case/Control Study a. The Cambridge shire Case Control population consists of a collection of 517 type 2 diabetics and 517 matched controls. The cases were a random sample of Europid men and women with type 2 diabetes, aged 47-75 years from a population-based diabetes register in a geographically defined region of Cambridge shire, UK. In the control subjects, diabetes mellitus was excluded by a medical record search and it they had glycated hemoglobin (HbAlc) levels >6.0%. b. The PPP1R3 AFS mutation was present in 16 of 517 (3.1 %) type 2 diabetic and 3 of 517 (0.6%) age and sex-matched non-diabetic control subjects. (One of the controls was homozygous; all others were heterozygous). c. These results give an odds ratio of 5.5 with exact 95% confidence intervals of 1.55-29.4, and a significant probability value of p = 0.003. This means that in this population, individuals with the PPP1R3AFS mutation are 5.5 times more likely to develop type 2 diabetes than those without the mutation. A p value of 0.05 or lower is considered statistically significant. d. One additional type 2 diabetic subject had a novel nonsense mutation (protein STOP mutation) predicted to truncate the protein at position 662, which is adjacent to where the PPP1R3AFS mutation occurs. This mutation was not found among control subjects.
EPIC-Norfolk Cohort Study a. The EPIC-Norfolk study is a population-based cohort study involving 25,000 individuals recruited from general practices throughout Norfolk, UK. Prevalent and incident cases of diabetes were included in the analysis. Cases were ascertained from multiple sources including primary care registers, hospital clinics, self-report and reported medication. Controls were selected from the same cohort study and were individually matched for age, sex and General Practice. Controls were excluded if they had a HbAlc >6.0%. b. 512 cases were considered with 516 controls. The frequency of the PPP1R3AFS mutation was lower than in the previous study, and similar in type 2 diabetics (0.78%) and controls (0.97%). It is noteworthy that although the change in this population appears to be more common in control subjects than in the previous study, more controls than diabetics were tested in this study. Combined Results a. The combined data set consists of a total of 1029 patients with type 2 diabetes and 1033 controls. b. Analysis shows that the Mantel-Hansel weighted odds for the frameshift mutation and type 2 diabetes to be 2.53 (95% confidence limits of 1.06-6.70), p = 0.03. This means that in this population, individuals with the PPP1R3AFS mutation are 2.53 times more likely to develop type 2 diabetes than those without the mutation. The p value shows that the result is statistically significant. c. Combining the results of the two matched case-control studies shows that the PPP1R3AFS mutation is important in predisposing a subject to type 2 diabetes. Characterization and Use of the Invention Sequencing
Methods for sequencing nucleic acids are well known in the art and may be used to practice any of the embodiments of the invention. These methods employ enzymes such as the Klenow fragment of DNA polymerase I, SEQUENASE, Taq DNA polymerase and thermostable T7 DNA polymerase (Amersham Biosciences (APB), Piscataway NJ), or combinations of polymerases and proofreading exonucleases (Invitrogen, Carlsbad CA). Sequence preparation is automated with machines such as the MICROLAB 2200 system (Hamilton, Reno NV) and the DNA ENGINE thermal cycler (MJ Research, Watertown MA) and sequencing, with the PRISM 3700, 377 or 373 DNA sequencing systems (ABI) or the MEGABACE 1000 DNA sequencing system (APB). The nucleic acid sequences of the cDNAs presented in the Sequence Listing were prepared by such automated methods and may contain occasional sequencing errors and unidentified nucleotides, designated with an N, that reflect state-of-the-art technology at the time the cDNA was sequenced. Vector, linker, and polyA sequences were masked using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis. Ns and SNPs can be verified either by resequencing the cDNA or using algorithms to compare multiple sequences that overlap the area in which the Ns or SNP occur. Both of these techniques are well known to and used by those skilled in the art. The sequences may be analyzed using a variety of algorithms described in Ausubel et al. (1997; Short Protocols in Molecular Biology, John Wiley & Sons, New York NY, unit 1.1) and in Meyers (1995; Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853). Shotgun sequencing may also be used to complete the sequence of a particular cloned insert of interest. Shotgun strategy involves randomly breaking the original insert into segments of various sizes and cloning these fragments into vectors. The fragments are sequenced and reassembled using overlapping ends until the entire sequence of the original insert is known. Shotgun sequencing methods are well known in the art and use thermostable DNA polymerases, heat-labile DNA polymerases, and primers chosen from representative regions flanking the cDNAs of interest. Incomplete assembled sequences are inspected for identity using various algorithms or programs such as CONSED (Gordon (1998) Genome Res 8:195-202) which are well known in the art. Contaminating sequences, including vector or chimeric sequences, can be removed, and deleted sequences can be restored to complete the assembled, finished sequences. Extension of a Nucleic Acid Sequence
The sequences of the invention may be extended using various PCR-based methods known in the art. For example, the XL-PCR kit (ABI), nested primers, and cDNA or genomic DNA libraries may be used to extend the nucleic acid sequence. For all PCR-based methods, primers may be designed using software, such as LOLIGO primer analysis software (Molecular Biology Insights, Cascade CO) to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to a target molecule at temperatures from about 55C to about 68C. When extending a sequence to recover regulatory elements, genomic, rather than cDNA libraries are used. Hybridization The polynucleotide and fragments thereof can be used in hybridization technologies for various purposes. A probe may be designed or derived from unique regions such as the 5' regulatory region or from a nonconserved region (i.e., 5 ' or 3 'of the nucleotides encoding the conserved catalytic domain of the protein) and used in protocols to identify naturally occurring molecules encoding the PPP1R3AFS, allelic variants, or related molecules. The probe may be DNA or RNA, may be single-stranded, and should have at least 50% sequence identity to any of the nucleic acid sequences, SEQ ID NOs:3 or 4. Hybridization probes may be produced using oligolabeling, nick-translation, end-labeling, or PCR amplification in the presence of a reporter molecule. A vector containing the nucleic acid or a fragment thereof may be used to produce an mRNA probe in vitro by addition of an RNA polymerase and labeled nucleotides. These procedures may be conducted using kits such as those provided by APB. The stringency of hybridization is determined by G+C content of the probe, salt concentration, and temperature. In particular, stringency can be increased by reducing the concentration of salt or raising the hybridization temperature. Hybridization can be performed at low stringency with buffers, such as 5xSSC with 1% sodium dodecyl sulfate (SDS) at 60C, which permits the formation of a hybridization complex between nucleic acid sequences that contain some mismatches. Subsequent washes are performed at higher stringency with buffers such as 0.2xSSC with 0.1% SDS at either 45C (medium stringency) or 68C (high stringency). At high stringency, hybridization complexes will remain stable only where the nucleic acids are completely complementary. In some membrane-based hybridizations, from about 35% to about 50% formalize can be added to the hybridization solution to reduce the temperature at which hybridization is performed. Background signals can be reduced by the use of detergents such as Sarkosyl or TRITON X-100 (Sigma- Aldrich) and a blocking agent such as denatured salmon sperm DNA. Selection of components and conditions for hybridization are well known to those skilled in the art and are reviewed in Isabel (supra) and Sambrook et al (1989) Molecular Cloning, A Laboratory Manual Cold Spring Harbor Press, Plain view NY.
Arrays may be prepared and analyzed using methods well known in the art. Oligonucleotides or cDNAs may be used as hybridization probes or targets to monitor the expression level of large numbers of genes simultaneously or to identify genetic variants, mutations, and single nucleotide polymorphisms. Arrays may be used to determine gene function; to understand the genetic basis of a condition, disease, or disorder; to diagnose a condition, disease, or disorder; and to develop and monitor the activities of therapeutic agents. (See, e.g., USN 5,474,796; Schena et al. (1996) Proc Natl Acad Sci 93:10614-10619; Heller et al. (1997) Proc Natl Acad Sci 94:2150-2155; USN 5,605,662.)
Hybridization probes are also useful in mapping the naturally occurring genomic sequence. The probes may be hybridized to a particular chromosome, a specific region of a chromosome, or an artificial chromosome construction. Such constructions include human artificial chromosomes , yeast artificial chromosomes, bacterial artificial chromosomes, bacterial PI constructions, or the cDNAs of libraries made from single chromosomes. OPCR
QPCR is a method for quantifying a nucleic acid molecule based on detection of a fluorescent signal produced during PCR amplification (Gibson et al. (1996) Genome Res 6:995-1001; Heid et al. (1996) Genome Res 6:986-994). Amplification is carried out on machines such as the PRISM 7700 detection system (ABI) which consists of a 96-well thermal cycler connected to a laser and charge-coupled device (CCD) optics system. To perform QPCR, a PCR reaction is carried out in the presence of a doubly labeled probe. The probe, which is designed to anneal between the standard forward and reverse PCR primers, is labeled at the 5' end by a flourogenic reporter dye such as 6-carboxyfluorescein (6-FAM) and at the 3 'end by a quencher molecule such as
6-carboxy-tetrame yl-rhodamine (TAMRA). As long as the probe is intact, the 3 'quencher extinguishes fluorescence by the 5' reporter. However, during each primer extension cycle, the annealed probe is degraded as a result of the intrinsic 5 ' to 3' nuclease activity of Taq polymerase (Holland et al (1991) Proc Natl Acad Sci 88:7276-7280). This degradation separates the reporter from the quencher, and fluorescence is detected every few seconds by the CCD. The higjier the starting copy number of the nucleic acid, the sooner an increase in fluorescence is observed. A cycle threshold (C- ) value, representing the cycle number at which the PCR product crosses a fixed threshold of detection is determined by the instrument software. The Cτ is inversely proportional to the copy number of the template and can therefore be used to calculate either the relative or absolute initial concentration of the nucleic acid molecule in the sample. The relative concentration of two different molecules can be calculated by determining their respective . values (comparative Cτ method). Alternatively, the absolute concentration of the nucleic acid molecule can be calculated by constructing a standard curve using a housekeeping molecule of known concentration. The process of calculating O- values, preparing a standard curve, and determining starting copy number is performed using SEQUENCE DETECTOR 1.7 software (ABI). Expression
Any one of a multitude of polynucleotides encoding PPARγFS or PPP1R3 AFS may be cloned into a vector and used to express the protein, or portions thereof, in host cells. The nucleic acid sequence can be engineered by such methods as DNA shuffling (USPN 5,830,721) and site-directed mutagenesis to create new restriction sites, alter glycosylation patterns, change codon preference to increase expression in a particular host, produce splice variants, extend half-life, and the like. The expression vector may contain transcriptional and translational control elements (promoters, enhancers, specific initiation signals, and polyadenylated 3' sequence) from various sources which have been selected for their efficiency in a particular host. The vector, cDNA, and regulatory elements are combined using in vitro recombinant DNA techniques, synthetic techniques, and/or in vivo genetic recombination techniques well known in the art and described in Sambrook (supra, ch. 4, 8, 16 and 17).
A variety of host systems may be transformed with an expression vector. These include, but are not limited to, bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems transformed with baculovirus expression vectors or plant cell systems transformed with expression vectors containing viral and/or bacterial elements (Ausubel supra, unit 16). In mammalian cell systems, an adenovirus transcriptional/ translational complex may be utilized. After sequences are ligated into the El or E3 region of the viral genome, the infective virus is used to transform and express the protein in host cells. The Rous sarcoma virus enhancer or SV40 or EBV-based vectors may also be used for high-level protein expression.
Routine cloning, subcloning, and propagation of nucleic acid sequences can be achieved using the multifunctional pBLUESCRIPT vector (Stratagene, La Jolla CA) or pSPORTl plasmid (Invitrogen). Introduction of a nucleic acid sequence into the multiple cloning site of these vectors disrupts the lacZ gene and allows colorimetric screening for transformed bacteria. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
For long term production of recombinant proteins, the vector can be stably transformed into cell lines along with a selectable or visible marker gene on the same or on a separate vector. After transformation, cells are allowed to grow for about 1 to 2 days in enriched media and then are transferred to selective media. Selectable markers, antimetabolite, antibiotic, or herbicide resistance genes, confer resistance to the relevant selective agent and allow growth and recovery of cells which successfully express the introduced sequences. Resistant clones identified either by survival on selective media or by the expression of visible markers may be propagated using culture techniques. Visible markers are also used to estimate the amount of protein expressed by the introduced genes. Verification that the host cell contains the desired poynucleotide is based on DNA-DNA or DNA-RNA hybridizations or PCR amplification. The host cell may be chosen for its ability to modify a recombinant protein in a desired fashion. Such modifications include acetylation, carboxylation, glycosylation, phosphorylation, lipidation, acylation and the like. Post-translational processing which cleaves a "prepro" form may also be used to specify protein targeting, folding, and/or activity. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities may be chosen to ensure the correct modification and processing of the recombinant protein. Recovery of Proteins from Cell Culture
Heterologous moieties engineered into a vector for ease of purification include glutathione S- transferals (GST), 6xHis, FLAG, MRC, and the like. GST and 6-His are purified using affinity matrices such as immobilized glutathione and metal-chelate resins, respectively. FLAG and MRC are purified using monoclonal and polyclonal antibodies. For ease of separation following purification, a sequence encoding a proteolytic cleavage site may be part of the vector located between the protein and the heterologous moiety. Methods for recombinant protein expression and purification are discussed in Isabel (supra, unit 16). Protein Identification
Several techniques have been developed which permit rapid identification of proteins using HPLC and MS. Beginning with a sample containing proteins, the method is: 1) proteins are separated using two-dimensional gel electrophoresis (2-DE), 2) selected proteins are excised from the gel and digested with a protease to produce a set of peptides; and 3) the peptides are subjected to mass spectral analysis to derive peptide ion mass and spectral pattern information. The MS information is used to identify the protein by comparing it with information in a protein database (Shevenko et al. (1996) Proc Natl Acad Sci 93:14440-14445). Proteins are separated by 2DE employing isoelectric focusing (EEF) in the first dimension followed by SDS-PAGE in the second dimension. For IEF, an immobilized pH gradient strip is useful to increase reproducibility and resolution of the separation. Alternative techniques may be used to improve resolution of very basic, hydrophobic, or high molecular weight proteins. The separated proteins are detected using a stain or dye such as silver stain, Coomassie blue, or spyro red (Molecular Probes, Eugene OR) that is compatible with MS. Gels may be blotted onto a PVDF membrane for western analysis and optically scanned using a STORM scanner (APB) to produce a computer-readable output which is analyzed by pattern recognition software such as MELANIE (GeneBio, Geneva, Switzerland). The software annotates individual spots by assigning a unique identifier and calculating their respective x,y coordinates, molecular masses, isoelectric points, and signal intensity. Individual spots of interest, such as those representing differentially expressed proteins, are excised and proteolytically digested with a site- specific protease such as trypsin or chymotrypsin, singly or in combination, to generate a set of small peptides, preferably in the range of 1-2 kDa. Prior to digestion, samples may be treated with reducing and alkylatmg agents, and following digestion, the peptides are then separated by liquid chromatography or capillary electrophoresis and analyzed using MS.
MS converts components of a sample into gaseous ions, separates the ions based on their mass-to-charge ratio, and determines relative abundance. For peptide mass fingerprinting analysis, a MA -DI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight), ESI (Electrospray
Ionization), and TOF-TOF (Time of Flight Time of Flight) machines are used to determine a set of highly accurate peptide masses. Using analytical programs, such as TURBOSEQUEST software (Finnigan, San Jose CA), the MS data is compared against a database of theoretical MS data derived from known or predicted proteins. A rninimum match of three peptide masses is used for reliable protein identification. If additional information is needed for identification, Tandem-MS may be used to derive information about individual peptides. In tandem-MS, a first stage of MS is performed to determine individual peptide masses. Then selected peptide ions are subjected to fragmentation using a technique such as collision induced dissociation (CID) to produce an ion series. The resulting fragmentation ions are analyzed in a second round of MS, and their spectral pattern may be used to determine a short stretch of amino acid sequence (Dancik et al (1999) J Comput Biol 6:327-342).
Assuming the protein is represented in the database, a combination of peptide mass and fragmentation data, together with the calculated MW and pi of the protein, will usually yield an unambiguous identification. If no match is found, protein sequence can be obtained using direct chemical sequencing procedures well known in the art (cf. Creighton (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York NY). Chemical Synthesis of Peptides
Proteins or portions thereof may be produced not only by recombinant methods, but also by using chemical methods well known in the art. Solid phase peptide synthesis may be carried out in a batchwise or continuous flow process which sequentially adds α-amino- and side chain-protected amino acid residues to an insoluble polymeric support via a linker group. A linker group such as me ylamine- derivatized polyethylene glycol is attached to poly(styrene-co-divinylbenzene) to form the support resin. The amino acid residues are N-α-protected by acid labile Boc (t-butyloxycarbonyl) or base-labile Fmoc (9-fluorenylmethoxycarbonyl). The carboxyl group of the protected amino acid is coupled to the amine of the linker group to anchor the residue to the solid phase support resin. Trifluoroacetic acid or piperidine are used to remove the protecting group in the case of Boc or Fmoc, respectively. Each additional amino acid is added to the anchored residue using a coupling agent or pre-activated amino acid derivative, and the resin is washed. The full length peptide is synthesized by sequential deprotection, coupling of derivitized amino acids, and washing with dichloromethane and/or N, N-dimethylformamide. The peptide is cleaved between the peptide carboxy terminus and the linker group to yield a peptide acid or amide. (Novabiochem 1997/98 Catalog and Peptide Synthesis Handbook, San Diego CA pp. S1-S20). Automated synthesis may also be carried out on machines such as the 431 A peptide synthesizer (ABI). A protein or portion thereof may be purified by preparative HPLC and its composition confirmed by -unino acid analysis or by sequencing (Creighton, supra). Antibodies
Antibodies, or immunoglobulins (Ig), are components of immune response expressed on the surface of or secreted into the circulation by B cells. The prototypical antibody is a tetramer composed of two identical heavy polypeptide chains (H-chains) and two identical light polypeptide chains (L-chains) interlinked by disulfide bonds which binds and neutralizes foreign antigens. Based on their H-chain, antibodies are classified as IgA, IgD, IgE, IgG or IgM. The most common class, IgG, is tetrameric while other classes are variants or multimers of the basic structure.
Antibodies are described in terms of their two functional domains. Antigen recognition is mediated by the Fab (antigen binding fragment) region of the antibody, while effector functions are mediated by the Fc (crystallizable fragment) region. The binding of antibody to antigen triggers destruction of the antigen by phagocytic white blood cells such as macrophages and neutrophils. These cells express surface Fc receptors that specifically bind to the Fc region of the antibody and allow the phagocytic cells to destroy antibody-bound antigen. Fc receptors are single-pass transmembrane glycoproteins containing about 350 amino acids whose extracellular portion typically contains two or three Ig domains (Sears et al. (1990) J Immunol 144:371-378). Preparation and Screening of Antibodies
Various hosts including mice, rats, rabbits, goats, llamas, camels, and human cell lines may be immunized by injection with an antigenic determinant. Adjuvants such as Freund's, mineral gels, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemacyanin (KLH; Sigma- Aldrich), and dinitrophenol may be used to increase immunological response. In humans, BCG (bacilli Calmette-Guerin) and Corvnebacterium parvum increase response. The antigenic determinant may be an oligopeptide, peptide, or protein. When the amount of antigenic determinant allows immunization to be repeated, specific polyclonal antibody with high affinity can be obtained (Klinman and Press (1975) Transplant Rev 24:41-83). Oligopeptides which may contain between about five and about fifteen amino acids identical to a portion of the endogenous protem may be fused with proteins such as KLH in order to produce antibodies to the chimeric molecule. Monoclonal antibodies may be prepared using any technique which provides for the production of antibodies by continuous cell lines in culture. These include the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al (1975) Nature 256:495-497; Kozbor et al (1985) J Immunol Methods 81:31-42; Cote et al. (1983) Proc Natl Acad Sci 80:2026-2030; and Cole et al. (1984) Mol Cell Biol 62:109-120).
Chimeric antibodies may be produced by techniques such as splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity (Morrison et al. (1984) Proc Natl Acad Sci 81:6851-6855; Neuberger et al. (1984) Nature 312:604-608; and Takeda et al. (1985) Nature 314:452-454). Alternatively, techniques described for antibody production may be adapted, using methods known in the art, to produce specific, single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton (1991) Proc Natl Acad Sci 88:10134-10137). Antibody fragments which contain specific binding sites for an antigenic determinant may also be produced. For example, such fragments include, but are not limited to, F(abτ)2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries maybe constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al. (1989) Science 246:1275-1281).
Antibodies may also be produced by inducing production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al. (1989; Proc Natl Acad Sci 86:3833-3837) or Winter et al. (1991; Nature 349:293-299). A protein may be used in screening assays of phagemid or B-lymphocyte immunoglobulin libraries to identify antibodies having a desired specificity. Numerous protocols for competitive binding or immunoassays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Antibody Specificity
Various methods such as Scatchard analysis combined with radioimmunoassay techniques may be used to assess the affinity of particular antibodies for a protein. Affinity is expressed as an association constant, Ka, which is defined as the molar concentration of protein-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions. The Ka determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple antigenic determinants, represents the average affinity, or avidity, of the antibodies. The Ka determined for a preparation of monoclonal antibodies, which are specific for a particular antigenic determinant, represents a true measure of affinity. High-affinity antibody preparations with Ka ranging from about 109 to 1012 IVmole are commonly used in immunoassays in which the protein-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with K., ranging from about 106 to 107 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of the protein, preferably in active form, from the antibody (Catty (1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington DC; Eiddell and Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations maybe further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing about 5-10 mg specific antibody/ml, is generally employed in procedures requiring precipitation of protein-antibody complexes. Procedures for making antibodies, evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are discussed in Catty (supra) and Ausubel (supra-) pp. 11.1-11.31. DIAGNOSTICS The presence of the mutations for PPP1R3 AFS and PPARγFS in DNA as detected by genotyping of DNA may be used to diagnose diabetes, including type 2 diabetes. In addition, the presence of the PPP1R3 AFS mutation in DNA as determined by genotyping may be used to diagnose a predisposition to, or a risk factor for, insulin resistance and diabetes.
The co-expression of PPP1R3AFS and PPARγFS, may also be detected using PPP1R3AFS and PPARγFS, a polynucleotide encoding PPP1R3AFS or PPARγFS, or an antibody that specifically binds PPP1R3AFS or PPARγFS, and at least one of the assays described below to diagnose severe insulin resistance and diabetes, including type 2 diabetes. In addition, the expression of PPP1R3AFS detected using any of the compositions for PPP1R3AFS noted above, and at least one of the assays described below can be used to diagnose a predisposition, or a risk factor for the development of diabetes.
Labeling of Molecules for Assay
A wide variety of reporter molecules and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid, amino acid, and antibody assays. Synthesis of labeled molecules may be achieved using kits such as those supplied by Promega (Madison WI) or APB for incorporation of a labeled nucleotide such as 32P-dCTP (APB), Cy3-dCTP or Cy5-dCTP (Qiagen-
Operon, Alameda CA), or amino acid such as 35S-methionine (APB). Nucleotides and amino acids may be directly labeled with a variety of substances including fluorescent, chemiluminescent, or chromogenic agents, and the like, by chemical conjugation to amines, thiols and other groups present in the molecules using reagents such as BIODIPY or FTTC (Molecular Probes). Nucleic Acid Assays
The polynucleotides, cDNAs, fragments, oligonucleotides, complementary PNAs, and peptide nucleic acids (PNA) may be used to detect and quantify differential gene expression for diagnosis of a disorder. Disorders associated with such differential expression of the nucleic acids of the invention include diabetes. The diagnostic assay may use hybridization or amplification technology to compare gene expression in a biological sample from a patient to standard samples in order to detect differential gene expression. Qualitative or quantitative methods for this comparison are well known in the art. Expression Profiles
An expression profile comprises the expression of a plurality of polynucleotides or protein as measured using standard assays with a sample. The polynucleotides, proteins or antibodies of the invention may be used as elements on a array to produce an expression profile. In one embodiment, the array is used to diagnose or monitor the progression of disease.
An expression profile comprises the expression of a plurality of polynucleotides or proteins as measured using standard assays with a sample. The polynucleotides, proteins or antibodies of the invention may be used as elements in the assay to produce the expression profile. In one embodiment, an array upon which the elements are immobilized is used to diagnose, stage or monitor the progression or treatment of a disorder.
For example, the polynucleotides, proteins or antibodies may be labeled using standard methods and added to a biological sample from a patient under conditions for the complex formation. After an incubation period, the sample is washed, and the amount of label (or signal) associated with each complexes is quantified and compared with a standard value. If the amount of complex formation in the patient sample is altered in comparison to normal or disease standards, then complex formation can be used to indicate the presence of a disorder.
In order to provide standards for establishing differential expression, normal and disease profiles are established. This is accomplished by combining a sample taken from a normal subject, either animal or human, with a polynucleotide under conditions for complex formation to occur. Standard complex formation may be quantified by comparing the values obtained using samples from normal subjects with values from an experiment in which a known amount of a purified control is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who were diagnosed with a particular condition, disease, or disorder. Deviation from standard values toward those associated with a particular disorder is used to diagnose or stage that disorder.
By analyzing changes in patterns of gene expression, a disorder can be diagnosed earlier, sometimes even before the patient is symptomatic. The invention can be used to formulate a prognosis and to design a treatment regimen. The invention can also be used to monitor the efficacy of treatment or to establish a dosage that causes a change in the expression profile indicative of successful treatment. For treatments with known side effects, the expression profile is employed to improve the treatment regimen so that expression patterns associated with the onset of undesirable side effects are avoided. This approach may be more sensitive and rapid than waiting for the patient to show inadequate improvement, or to manifest side effects, before altering the course of treatment. In another embodiment, animal models which mimic a human disease can be used to characterize expression profiles associated with a particular condition, disease, or disorder; or treatment of the condition, disease, or disorder. Novel treatment regimens may be tested in these animal models using an expression profile over time. In addition, an expression profile may be used with cell cultures or tissues removed from animal models to rapidly screen large numbers of candidate drug molecules, looking for ones that produce an expression profile similar to those of known therapeutic drugs, with the expectation that molecules with the same expression profile will likely have similar therapeutic effects. Thus, the invention provides the means to rapidly determine the molecular mode of action of a drug.
Such expression profiles may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies or in clinical trials or to monitor the treatment of an individual patient. Once the presence of a condition is established and a treatment protocol is initiated, expression may be analyzed on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in a normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to years. Protein and Antibody Assays
Antibodies which specifically bind PPP1R3AFS or PPARγFS maybe used to candidate those proteins. Immunological methods for detecting and measuring complex formation as a measure of protein expression using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include antibody or protein arrays, ELISA, FACS, spatial immobilization such as 2D- PAGE and SC, HPLC or MS, RLAs and western analysis. Such immunoassays typically involve the measurement of complex formation between the protein and its specific antibody. These assays and their quantitation against purified, labeled standards are well known in the art (Ausubel, supra, unit 10.1-10.6). A two-site, monoclonal-based immunoassay utilizing antibodies reactive to two non-interfering epitopes is preferred, but a competitive binding assay may be employed (Pound (1998) Immunochemical Protocols, Humana Press, Totowa NJ).
These methods are also useful for diagnosing diseases that show differential protein expression. Normal or standard values for protein expression are estabhshed by combining body fluids or cell extracts taken from a normal mammalian or human subject with specific antibodies to a protein under conditions for complex formation. Standard values for complex formation in normal and diseased tissues are established by various methods, often photometric means. Then complex formation as it is expressed in a subject sample is compared with the standard values. Deviation from the normal standard and toward the diseased standard provides parameters for disease diagnosis or prognosis while deviation away from the diseased and toward the normal standard may be used to evaluate treatment efficacy. Antibody arrays allow the development of techniques for high-throughput screening of recombinant antibodies. Such methods use robots to pick and grid bacteria containing antibody genes, and a filter-based ELIZA to screen and identify clones that express antibody fragments. Because liquid handling is eliminated and the clones are arrayed from master stocks, the same antibodies can be spotted multiple times and screened against multiple antigens simultaneously. Antibody arrays are highly useful in the identification of differentially expressed proteins. See de Wild et al. (2000) Nature Biotechnol 18:989-94. THERAPEUTICS
The evidence presented in Figures 1 - 9 and in Table 1 clearly demonstrates that the extreme insulin resistant phenotype and the presence of type 2 diabetes in family A is associated with the presence of two mutations in PPARγ and PPP1R3A, PPARγ FS and PPP1R3AFS, and further that the extreme insulin resistance and diabetes in family A is a result of the combined effect of the two mutations. It is furthermore evident that the truncation in the PPP1R3 AFS mutant protein abrogates normal protein function thus leading to a diminished activity of the protein relative to normal PPP1R3 A. Therefore, a treatment for diabetes may employ an agonist of PPP1R3A or other means of increasing the activity of the protein.
In the treatment of diabetes in which it is desirable to increase expression or activity of PPP1R3A, a pharmaceutical agent such as an agonist, transcription factor or a small drug molecule that specifically binds the protein and increases its expression or activity may be administered to a subject in need of such treatment. In another embodiment, a pharmaceutical composition comprising an agonist, transcription factor or a small drug molecule and a pharmaceutical carrier may be administered to a subject to treat decreased expression or activity associated with the endogenous protein. In one aspect, an antibody that specifically binds the protein can act as a carrier to effect delivery. In an additional embodiment, a vector expressing the encoding polynucleotide, or fragments thereof, may be administered to a subject to treat the disorder.
Any of the polynucleotides, complementary molecules, or fragments thereof, proteins or portions thereof, vectors delivering these nucleic acid molecules or expressing the proteins, therapeutic antibodies, and ligands binding the polynucleotide or protein may be administered in combination with other therapeutic agents. Selection of the agents for use in combination therapy may be made by one of ordinary skill in the art according to conventional pharmaceutical principles. A combination of therapeutic agents may act synergistically to affect treatment of a particular disorder at a lower dosage of each agent. Modification of Gene Expression Using Nucleic Acids Gene expression may be modified by designing complementary or antisense molecules (DNA, RNA, or PNA) to the control, 5', 3', or other regulatory regions of the gene encoding PPP1R3AFS or PPARγFS. Oligonucleotides designed to inhibit transcription initiation are preferred. Similarly, inhibition can be achieved using triple helix base-pairing which inhibits the binding of polymerase, transcription factors, or regulatory molecules (Gee et al. In: Huber and Carr (1994) Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-177). A complementary molecule may also be designed to block translation by preventing binding between ribosomes and mRNA. In one alternative, a library or plurality of polynucleotides may be screened to identify those which specifically bind a regulatory, nontranslated sequence. Ribosomes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of
RNA. The mechanism of ribosome action involves sequence-specific hybridization of the ribosome molecule to complementary target RNA followed by endonucleolytic cleavage at sites such as GUN, Glu, and GUN. Once such sites are identified, an oligonucleotide with the same sequence may be evaluated for secondary structural features which would render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing their hybridization with complementary oligonucleotides using ribonuclease protection assays.
Complementary nucleic acids and ribosomes of the invention may be prepared via recombinant expression, in vitro or in vivo, or using solid phase phosphoramidite chemical synthesis. In addition, RNA molecules may be modified to increase intracellular stability and half-life by addition of flanking sequences at the 5' and/or 3' ends of the molecule or by the use of phosphorothioate or 2' O-methyl rather than phosphodiesterases linkages within the backbone of the molecule. Modification is inherent in the production of PNAs and can be extended to other nucleic acid molecules. Either the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, or the modification of adenine, cytidine, guanine, thymine, and uridine with acetyl-, methyl-, thio- groups renders the molecule more resistant to endogenous endonucleases. RNA Interference
RNA interference (RNAi), also known as double-stranded RNA (dsRNA)-induced gene silencing, is a method of interfering with the transcription of specific rnRNAs through the production of small RNAs (siRNAs) and short hairpin RNAs (shRNAs). These RNAs are naturally formed in a multicomponent nuclease complex (RISC) in the presence of an RNAse HI family nuclease (Dicer), and they serve as a guide to identify and destroy complementary transcripts. Transient infection of cells with RNAs capable of interference can bypass the need for Dicer and result in silencing of a gene for the lifespan of the introduced RNAs, usually from about 2 to about 7 days. See McManus and Sharp (2002) Nature Reviews 3:737-747. The RANi pathway is believed to have evolved in early eucaryote as a cell-based immunity against viral and genetic parasites. It is considered, however, to have great potential as a method for identifying gene function, particularly in signal transduction disorders (such as insulin resistance and type 2 diabetes), and for providing a highly specific means for treating such disorders. Gene Therapy
The polynucleotides of the invention can be used in gene therapy, polynucleotides can be delivered ex vivo to target cells, such as cells of bone marrow. Once stable integration and transcription and or translation are confirmed, the bone marrow maybe reintroduced into the subject. Expression of the protein encoded by the polynucleotide may correct a disorder associated with mutation of a normal sequence, reduction or loss of an endogenous target protein, or overepression of an endogenous or mutant protein. Alternatively, polynucleotides may be delivered in vivo using vectors such as retro virus, adenovirus, Aden-associated virus, herpes simplex virus, and bacterial plastics. Non- viral methods of gene delivery include cationic liposomes, polylysine conjugates, artificial viral envelopes, and direct injection of DNA (Anderson (1998) Nature 392:25-30; Dachs et al. (1997) Oncol Res 9:313-325; Chu et a]. (1998) J Mol Med 76(3-4):184-192; Weiss et al. (1999) CeU Mol Life Sci 55(3):334-358; Agrawal (1996) Antisense Therapeutics. Humana Press, Totowa NJ; and August et al. (1997) Gene Therapy (Advances in Pharmacology, Vol. 40), Academic Press, San Diego CA). Screening and Purification Assays
The polynucleotide encoding PPP1R3AFS or PPARγFS may be used to screen a library or a plurality of molecules or compounds for specific binding affinity. The libraries maybe antisense molecules, artificial chromosome constructions, branched nucleic acid molecules, DNA molecules, peptides, peptide nucleic acid, proteins such as transcription factors, enhancers, or repressions, RNA molecules, ribosomes, and other ligands which regulate the activity, replication, transcription, or translation of the endogenous gene. The assay involves combining a polynucleotide with a library or plurality of molecules or compounds under conditions allowing specific binding, and detecting specific binding to identify at least one molecule which specifically binds the polynucleotide.
In one embodiment, the polynucleotide of the invention may be incubated with a plurality of purified molecules or compounds and binding activity determined by methods well known in the art, e.g., a gel-retardation assay (USN 6,010,849) or a reticulocyte lysate transcriptional assay. In another embodiment, the polynucleotide may be incubated with nuclear extracts from biopsies and/or cultured cells and tissues. Specific binding between the polynucleotide and a molecule or compound in the nuclear extract is initially determined by gel shift assay and may be later confirmed by recovering and raising antibodies against that molecule or compound. When these antibodies are added into the assay, they cause a supershift in the gel-retardation assay. In another embodiment, the polynucleotide may be used to purify a molecule or compound using affinity chromatography methods well known in the art. In one embodiment, the polynucleotide is chemically reacted with cyanogen bromide groups on a polymeric resin or gel. Then a sample is passed over and reacts with or binds to the polynucleotide. The molecule or compound which is bound to the polynucleotide may be released from the polynucleotide by increasing the salt concentration of the flow- through medium and collected.
In a further embodiment, the protein or a portion thereof may be used to purify a ligand from a sample. A method for using a protein to purify a ligand would involve combining the protein with a sample under conditions to allow specific binding, detecting specific binding between the protein and ligand, recovering the bound protein, and using a chaotropic agent to separate the protein from the purified ligand.
In a further embodiment, PPP1R3 A may be used to screen a plurality of molecules or compounds in any of a variety of screening assays. The portion of the protein employed in such screening may be free in solution, affixed to an biotic or biotic substrate (e.g. borne on a cell surface), or located infra cellularly. For example, in one method, viable or fixed prokaryotic host cells that are stable transformed with recombinant nucleic acids that have expressed and positioned a peptide on their cell surface can be used in screening assays. The cells are screened against a plurality or libraries of ligands, and the specificity of binding or formation of complexes between the expressed protein and the ligand can be measured. Depending on the particular kind of molecules or compounds being screened, the assay may be used to identify agonists, antagonists, antibodies, DNA molecules, small drug molecules, immunoglobulins, inhibitors, mimetics, peptides, peptide nucleic acids, proteins, and RNA molecules or any other ligand, which specifically binds the protein.
In one aspect, this invention contemplates a method for high throughput screening using very small assay volumes and very small amounts of test compound as described in USN 5,876,946, incorporated herein by reference. This method is used to screen large numbers of molecules and compounds via specific binding. In another aspect, this invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding the protein specifically compete with a test compound capable of binding to the protein. Molecules or compounds identified by screening may be used in a mammalian model system to evaluate their toxicity or therapeutic potential. Pharmaceutical Compositions
Pharmaceutical compositions may be formulated and administered, to a subject in need of such treatment, to attain a therapeutic effect. Such compositions contain the instant protein, agonists, antagonists, small drug molecules, immunoglobulins, inhibitors, mimetics, multispecific molecules, peptides, peptide nucleic acids, pharmaceutical agent, proteins, and RNA molecules. Compositions may be manufactured by conventional means such as mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing. The composition may be provided as a salt, formed with acids such as hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic, or as a lyophilized powder which may be combined with a sterile buffer such as saline, dextrose, or water. These compositions may include auxiliaries or excipients which facilitate processing of the active compounds. Auxiliaries and excipients may include coatings, fillers or binders including sugars such as lactose, sucrose, mannitol, glycerol, or sorbitol; starches from corn, wheat, rice, or potato; proteins such as albumin, gelatin and collagen; cellulose in the form of hydroxypropylmethyl-cellulose, methyl cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; lubricants such as magnesium stearate or talc; disintegrating or solubilizing agents such as the, agar, alginic acid, sodium alginate or cross-linked polyvinyl pyrrolidone; stabilizers such as carbopol gel, polyethylene glycol, or titanium dioxide; and dyestuffs or pigments added for identify the product or to characterize the quantity of active compound or dosage.
These compositions may be administered by any number of routes including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal.
The route of administration and dosage will determine formulation; for example, oral administration maybe accomplished using tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, or suspensions; parenteral administration may be formulated in aqueous, physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Suspensions for injection may be aqueous, containing viscous additives such as sodium carboxymethyl cellulose or dextran to increase the viscosity, or oily, containing lipophilic solvents such as sesame oil or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Penetrants well known in the art are used for topical or nasal administration. Toxicity and Therapeutic Efficacy
A therapeutically effective dose refers to the amount of active ingredient which ameliorates symptoms or condition. For any compound, a therapeutically effective dose can be estimated from cell culture assays using normal and neoplastic cells or in animal models. Therapeutic efficacy, toxicity, concentration range, and route of administration maybe determined by standard pharmaceutical procedures using experimental animals.
The therapeutic index is the dose ratio between therapeutic and toxic effects--LD50 (the dose lethal to 50% of the population)/ED50 (the dose therapeutically effective in 50% of the population)— and large therapeutic indices are preferred. Dosage is within a range of circulating concentrations, includes an ED50 with little or no toxicity, and varies depending upon the composition, method of delivery, sensitivity of the patient, and route of administration. Exact dosage will be determined by the practitioner in light of factors related to the subject in need of the treatment.
Dosage and administration are adjusted to provide active moiety that maintains therapeutic effect. Factors for adjustment include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular composition.
Normal dosage amounts may vary from 0.1 μg, up to a total dose of about 1 g, depending upon the route of administration. The dosage of a particular composition maybe lower when administered to a patient in combination with other agents, drugs, or hormones. Guidance as to particular dosages and methods of delivery is provided in the pharmaceutical literature. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Mack Publishing, Easton PA). Model Systems
Animal models may be used as bioassays where they exhibit a phenotypic response similar to that of humans and where exposure conditions are relevant to human exposures. Mammals are the most common models, and most infectious agent, cancer, drug, and toxicity studies are performed on rodents such as rats or mice because of low cost, availability, lifespan, gestation period, numbers of progeny, and abundant reference literature. Inbred and outbred rodent strains provide a convenient model for investigation of the physiological consequences of under- or over-expression of genes of interest and for the development of methods for diagnosis and treatment of diseases. A mammal inbred to over-express a particular gene (for example, secreted in milk) may also serve as a convenient source of the protein expressed by that gene. Toxicology
Toxicology is the study of the effects of agents on living systems. The majority of toxicity studies are performed on rats or mice. Observation of qualitative and quantitative changes in physiology, behavior, hemostatic processes, and lethality in the rats or mice are used to generate a toxicity profile and to assess consequences on human health following exposure to the agent. Genetic toxicology identifies and analyzes the effect of an agent on the rate of endogenous, spontaneous, and induced genetic mutations. Genotoxic agents usually have common chemical or physical properties that facilitate interaction with nucleic acids and are most harmful when chromosomal aberrations are transmitted to progeny. Toxicological studies may identify agents that increase the frequency of structural or functional abnormalities in the tissues of the progeny if administered to either parent before conception, to the mother during pregnancy, or to the developing organism. Mice and rats are most frequently used in these tests because their short reproductive cycle allows the production of the numbers of organisms needed to satisfy statistical requirements.
Acute toxicity tests are based on a single administration of an agent to the subject to determine the symptom ology or lethality of the agent. Three experiments are conducted: 1) an initial dose-range- finding experiment, 2) an experiment to narrow the range of effective doses, and 3) a final experiment for establishing the dose-response curve.
Subchronic toxicity tests are based on the repeated administration of an agent. Rat and dog are commonly used in these studies to provide data from species in different families. With the exception of carcinogenesis, there is considerable evidence that daily administration of an agent at high-dose concentrations for periods of three to four months will reveal most forms of toxicity in adult animals.
Chronic toxicity tests, with a duration of a year or more, are used to test whether long term administration may elicit toxicity, teratogenesis, or carcinogenesis. When studies are conducted on rats, a minimum of three test groups plus one control group are used, and animals are examined and monitored at the outset and at intervals throughout the experiment. Transgenic Animal Models
Transgenic rodents that over-express or under-express a gene of interest may be inbred and used to model human diseases or to test therapeutic or toxic agents. (See, e.g., USN 5,175,383 and USN 5,767,337.) In some cases, the introduced gene may be activated at a specific time in a specific tissue type during fetal or postnatal development. Expression of the transcend is monitored by analysis of phenotype, of tissue-specific mRNA expression, or of serum and tissue protein levels in transgenic animals before, during, and after challenge with experimental drug therapies. Embryonic Stem Cells
Embryonic (ES) stem cells isolated from rodent embryos retain the ability to form embryonic tissues. When ES cells are placed inside a carrier embryo, they resume normal development and contribute to tissues of the live-born animal. ES cells are the preferred cells used in the creation of experimental knockout and knocking rodent strains. Mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and are grown under culture conditions well known in the art. Vectors used to produce a transgenic strain contain a disease gene candidate and a marker gene, the latter serves to identify the presence of the introduced disease gene. The vector is transformed into ES cells by methods well known in the art, and transformed ES cells are identified and micro injected into mouse cell blastocyst such as those from the C57BL/6 mouse strain. The blastocyst are surgically transferred to pseudo pregnant dams, and the resulting chimeric progeny are genotype and bred to produce heterozygous or homozygous strains. ES cells derived from human blastocyst may be manipulated in vitro to differentiate into at least eight separate cell lineages. These lineages are used to study the differentiation of various cell types and tissues in vitro, and they include endoderm, mesoderm, and ectodermal cell types which differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes. Knockout Analysis
In gene knockout analysis, a region of a gene is enzymatically modified to include a non- mammalian gene such as the neomycin phosphotransferase gene (neo; Capecchi (1989) Science 244:1288-1292). The modified gene is transformed into cultured ES cells and integrates into the endogenous genome by homologous recombination. The inserted sequence disrupts transcription and translation of the endogenous gene. Transformed cells are injected into rodent blastalae, and the blastalae are implanted into pseudo pregnant dams. Transgenic progeny are crossbred to obtain homozygous inbred lines which lack a functional copy of the mammalian gene. In one example, the mammalian gene is a human gene. Knockin Analysis ES cells can be used to create knocking humanized animals (pigs) or transgenic animal models
(mice or rats) of human diseases. With knocking technology, a region of a human gene is injected into animal ES cells, and the human sequence integrates into the animal cell genome. Transformed cells are injected into blastalae and the blastalae are implanted as described above. Transgenic progeny or inbred lines are studied and treated with pharmaceutical agents to obtain information on treatment of the analogous human condition. These methods have been used to model several human diseases. Non-Human Primate Model
The field of animal testing deals with data and methodology from basic sciences such as physiology, genetics, chemistry, pharmacology and statistics. These data are paramount in evaluating the effects of therapeutic agents on non-human primates as they can be related to human health. Monkeys are used as human surrogates in vaccine and drag evaluations, and their responses are relevant to human exposures under similar conditions. Cynomolgus and Rhesus monkeys (Macaca fascicularis and Macaca mulatta, respectively) and Common Marmosets (Callithrix Bacchus) are the most common non-human primates (NHPs) used in these investigations. Since great cost is associated with developing and maintaining a colony of NHPs, early research and toxicological studies are usually carried out in rodent models. In studies using behavioral measures such as drug addiction, NHPs are the first choice test animal. In addition, NHPs and individual humans exhibit differential sensitivities to many drugs and toxins and can be classified as a range of phenotypes from "extensive metabolites" to "poor metabolites" of these agents. In additional embodiments, the polynucleotides which encode the protein maybe used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of polynucleotides that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
EXAMPLES
I Mutational Screening
Genomic DNA from subjects was randomly pre-amplified in a primer extension pre-amplification (PEP) reaction (Zhang et al (1992) Proc Natl Acad Sci USA 89:547-5851). All coding eons and splice junctions of PPARγ transcripts and PPP1R3 A were amplified by PCR from PEP DNA with gene-specific primers (primer sequences available upon request). PCR products were studied using single-stranded conformation polymorphism analysis and direct sequencing of all abnormal conformers (Thorpe et al.(1999) Immunogenetics 49:256-265). The PPP1R3A frameshift mutation was screened in participants in two independent, population-based, case-control studies in East Anglia, UK. The Cambridgeshire Case Control population consists of a collection of 517 type 2 diabetics and 517 matched controls. The cases were a random sample of Europid men and women with type 2 diabetes, aged 47-75 years from a population-based diabetes register in a geographically defined region of Cambridgeshire, UK. The presence of type 2 diabetes was assumed if the onset of diabetes was after the age of 30 y and insulin therapy was not used in the first year after diagnosis. The EPIC-Norfolk study is a population-based cohort study involving 25,000 individuals recruited from general practices throughout Norfolk, UK. Prevalent and incident cases of diabetes were included in the analysis. Cases were ascertained from multiple sources including primary care registers, hospital clinics, self-report and reported medication. Estimated ascertainment using the capture-recapture method was 9%. Controls were individually age- and gender-matched to each of the cases. Controls were excluded that had glycated hemoglobin (HbAlc) levels >6.0%. The PPARγ frameshift mutation was not detected in any individuals from the first population-based cohort.
II DNA-Binding Assays
Receptor binding of proteins to DNA was assessed in electrophoretic mobility supershift assays as described in Collingwood et al (1994 Mol Endocrinol 8:1262-1277) using 35S-labeled, in vitro translated receptors quantified by SDS-PAGE analysis and a 32P-labeled oligonucleotide duplex corresponding to the PPARE derived from the acyl-CoA. oxidase gene (Zamia et al. (1995) Genes Dev 11:835-846).
III Transactivation Assays
Transfections of 293 EBNA cells were carried out in 24-well plates using 500 ng of (PPARE)3TKLUC and 100 ng of receptor expression vector (wildtype, frameshift mutant, or empty vector, pcDNA3) (Invitrogen Corp, Carlsbad, CA) Using the calcium phosphate method (Collingwood et al, supra). Luciferase values were normalized to β-galactosidase activity from the internal control phasmid, Bosβgal (Collingwood et al., supra), and represent the mean +/- sem. of at least three independent experiments each performed in triplicate. IV Immunofluorescence Microscopy
CHO cells were transiently transfected (EUGENE; Roche Diagnostics, Ltd, East Sussex, UK) with N-terminal, HA-tagged expression vectors containing wildtype or mutated PPP1R3A. PACCMV.pKpA-HA-PPPlR3A was a gift from P. Cohen (Rasmussen, SK (2000) Diabetologia 43:718- 722). Cells were fixed in 3% paraformaldehyde/0.05% gluteraldehyde in 100 mM potassium HEPES/3 mM MgCls buffer (pH 7.5) for 15 min., treated with 0.5% borohydride/PBS for 10 min., and then blocked and permeabilized in 1% BSA/0.1% saponin for 20 min. When permeabilizing cells before fixation, the cells were incubated for 5 min. in 0.5% saponin in 80 mm potassium PIPES/5 mM EGTA/1 mM MgCl2 (pH 6.8) at room temperature. Cells were labeled with a rat anti-HA (1:100; Santa Cruz Biotechnology) followed by Texas Red goat anti-rat (1:200; Molecular Probes, Inc., Eugene, OR). Confocal images were collected using a LEICA TCS SP system (Leica Microsystems, Inc., Eaton, PA) and processed them using Adobe Photoshop software (Adobe Systems, Inc., San Jose, CA).
V Clinical Studies
EMCL determinations were made as previously described (Rico-Saenz, J. (1999) J Appl Physiol 85:1778-1785) and plasma Leptin concentrations were determined using the assay described below. Leptin Assay
This micro titre-plate format assay is based on the use of two commercially available monoclonal antibodies (MAN 398 and BAM 398 obtained from R&D Systems Europe, Ltd). As one of these antibodies has been biotinylated, incubating with Europium-labelled streptavidin allows the use of time resolved fluorometry as the detection system. Calibration is with recombinant human Leptin (Cat No. 398-LP, R&D Systems). The comparison with an established radioimmunoassay gave the following statistical data using Deeming calculations: N= 343; Range 0.5-121.7 NG/ml; Intercept = -1.0339; Slope = 1.0491; r = 0.9547. The within batch coefficient of variation (CV) was ≤ 4.4 % within the range 1.8 - 63.4 ng/ml. Between run (n=30) CV's were 7.1% at 2.1 ng/ml, 3.9% at 14.9 ng/ml, and 5.7 % at 54.9 ng/ml respectively. The detection limit is 0.1 NG/ml (3 standard deviations from zero). The assay was automated using the AutoDELFIA immunoassay system (Pekin Elmer Life Sciences).
VI Chromosome Mapping
Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIRG), and Genethon are used to determine if any of the polynucleotides presented in the Sequence Listing have been mapped. Any of the fragments of the polynucleotide encoding PPP1R3AFS or PPARγFS that have been mapped result in the assignment of all related regulatory and coding sequences to the same location. The genetic map locations are described as ranges, or intervals, of human chromosomes. The map position of an interval, in cm (which is roughly equivalent to 1 megabase of human DNA), is measured relative to the
5 teπninus of the chromosomal p-arm.
VII Hybridization and Application Technologies and Analyses Immobilization of polynucleotides on a Substrate
The polynucleotides are applied to a substrate by one of the following methods. A mixture of polynucleotides is fractionated by gel electrophoresis and transferred to a nylon membrane by capillary
10 transfer. Alternatively, the polynucleotides are individually ligated to a vector and inserted into bacterial host cells to form a library. The polynucleotides are then arranged on a substrate by one of the following methods. In the first method, bacterial cells containing individual clones are robotically picked and arranged on a nylon membrane. The membrane is placed on LB agar containing selective agent (carbenicillin, kanamycin, ampicillin, or chloramphenicol depending on the vector used) and incubated at
15 37C for 16 hr. The membrane is removed from the agar and consecutively placed colony side up in 10% SDS, denaturing solution (1.5 M NaCl, 0.5 M NaOH. ), neutralizing solution (1.5 M NaCl, 1 M Tris, pH 8.0), and twice in 2xSSC for 10 min each. The membrane is then UV irradiated in a STRATALENKER UN-cross linker (Stratagene).
In the second method, polynucleotides are amplified from bacterial vectors by thirty cycles of
20 PCR using primers complementary to vector sequences flanking the insert. PCR amplification increases a starting concentration of 1-2 ng nucleic acid to a final quantity greater than 5 μg. Amplified nucleic acids from about 400 bp to about 5000 bp in length are purified using SEPHACRYL-400 beads (APB). Purified nucleic acids are arranged on a nylon membrane manually or using a dot/slot blotting manifold and suction device and are immobilized by denataration, neutralization, and UV irradiation as described
25 above. Purified nucleic acids are robotically arranged and immobilized on polymer-coated glass slides using the procedure described in USΝ 5,807,522. Polymer-coated slides are prepared by cleaning glass microscope slides (Corning Life Sciences) by ultrasound in 0.1% SDS and acetone, etching in 4% hydrofluoric acid (VWR. Scientific Products, West Chester PA), coating with 0.05% aminopropyl silane (Sigma Aldrich) in 95% ethanol, and curing in a HOC oven. The slides are washed extensively with
30 distilled water between and after treatments. The nucleic acids are arranged on the slide and then immobilized by exposing the array to UV irradiation using a STRATA LINKER UV-cross linker (Stratagene). Arrays are then washed at room temperature in 0.2% SDS and rinsed three times in distilled water. Non-specific binding sites are blocked by incubation of arrays in 0.2% casein in phosphate buffered saline (PBS; Tropix, Bedford MA) for 30 min at 60C; then the arrays are washed in 0.2% SDS and rinsed in distilled water as before. Probe Preparation for Membrane Hybridization
Hybridization probes derived from the polynucleotides of the Sequence Listing are employed for screening cDNAs, mRNAs, or genomic DNA in membrane-based hybridizations. Probes are prepared by diluting the polynucleotides to a concentration of 40-50 ng in 45 μl TE buffer, denaturing by heating to 100C for five min, and briefly centrifuging. The denatured polynucleotide is then added to a REDEPRIME tube (APB), gently mixed until blue color is evenly distributed, and briefly centrifuged. Five μl of [32P]dCTP is added to the tube, and the contents are incubated at 37C for 10 min. The labeling reaction is stopped by adding 5 μl of 0.2M EDTA, and probe is purified from unincorporated nucleotides using a PROBEQUANT G-50 microcolumn (APB). The purified probe is heated to 100C for five min, snap cooled for two min on ice, and used in membrane-based hybridizations as described below. Probe Preparation for Polymer Coated Slide Hybridization
Hybridization probes derived from mRNA isolated from samples are employed for screening polynucleotides of the Sequence Listing in array-based hybridizations. Probe is prepared using the
GEMbright kit (Incyte Genomics) by diluting mRNA to a concentration of 200 ng in 9 μl TE buffer and adding 5 μl 5x buffer, 1 μl 0.1 M DTT, 3 μl Cy3 or Cy5 labeling mix, 1 μl RNAse inhibitor, 1 μl reverse transcriptase, and 5 μl lx yeast control mRNAs. Yeast control mRNAs are synthesized by in vitro transcription from noncoding yeast genomic DNA (W. Lei, unpublished). As quantitative controls, one set of control mRNAs at 0.002 ng, 0.02 ng, 0.2 ng, and 2 ng are diluted into reverse transcription reaction mixture at ratios of 1:100,000, 1:10,000, 1:1000, and 1:100 (w/w) to sample mRNA respectively. To examine mRNA differential expression patterns, a second set of control mRNAs are diluted into reverse transcription reaction mixture at ratios of 1:3, 3:1, 1:10, 10:1, 1:25, and 25:1 (w/w). The reaction mixture is mixed and incubated at 37C for two hr. The reaction mixtare is then incubated for 20 min at 85C, and probes are purified using two successive CHROMA SPEN+TE 30 columns (Clontech, Palo Alto CA). Purified probe is ethanol precipitated by diluting probe to 90 μl in DEPC-treated water, adding 2 μl lmg/ml glycogen, 60 μl 5 M sodium acetate, and 300 μl 100% ethanol. The probe is centrifuged for 20 min at 20,800xg, and the pellet is resuspended in 12 μl resuspension buffer, heated to 65C for five min, and mixed thoroughly. The probe is heated and mixed as before and then stored on ice. Probe is used in high density array-based hybridizations as described below. Membrane-based Hybridization
Membranes are pre-hybridized in hybridization solution containing 1% Sarkosyl and lx high phosphate buffer (0.5 M NaCl, 0.1 M Na2HP04, 5 mM EDTA, pH 7) at 55C for two hr. The probe, diluted in 15 ml fresh hybridization solution, is then added to the membrane. The membrane is hybridized with the probe at 55C for 16 hr. Following hybridization, the membrane is washed for 15 min at 25C in ImM Tris (pH 8.0), 1% Sarkosyl, and four times for 15 min each at 25C in ImM Tris (pH 8.0). To detect hybridization complexes, XOMAT-AR film (Eastman Kodak, Rochester NY) is exposed to the membrane overnight at -70C, developed, and examined visually. Polymer Coated Slide-based Hybridization
Probe is heated to 65C for five min, centrifuged five min at 9400 rpm in a 5415C microcentrifuge (Eppendorf Scientific, Westbury NY), and then 18 μl is aliquoted onto the array surface and covered with a coverslip. The arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 μl of 5xSSC in a corner of the chamber. The chamber containing the arrays is incubated for about 6.5 hr at 60C. The arrays are washed for 10 min at 45C in lxSSC, 0.1% SDS, and three times for 10 min each at 45C in O.lxSSC, and dried.
Hybridization reactions are performed in absolute or differential hybridization formats. In the absolute hybridization format, probe from one sample is hybridized to array elements, and signals are detected after hybridization complexes form. Signal strength correlates with probe mRNA levels in the sample. In the differential hybridization format, differential expression of a set of genes in two biological samples is analyzed. Probes from the two samples are prepared and labeled with different labeling moieties. A mixture of the two labeled probes is hybridized to the array elements, and signals are examined under conditions in which the emissions from the two different labels are individually detectable. Elements on the array that are hybridized to equal numbers of probes derived from both biological samples give a distinct combined fluorescence (Shalon WO95/35505).
Hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5. The excitation laser light is focused on the array using a 20X microscope objective (Nikon, Melville NY). The slide containing the array is placed on a computer- controled X-Y stage on the microscope and raster-scanned past the objective with a resolution of 20 micrometers. In the differential hybridization format, the two fluorophores are sequentially excited by the laser. Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Filters positioned between the array and the photomultiplier tabes are used to separate the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5. The sensitivity of the scans is calibrated using the signal intensity generated by the yeast control mRNAs added to the probe mix. A specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000. The output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (A D) conversion board (Analog Devices, Norwood MA) installed in an IBM-compatible PC computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using the emission spectrum for each fluorophore. A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS program (Incyte Genomics). VIII Complementary Molecules
Antisense molecules complementary to the cDNA, from about 5 bp to about 5000 bp in length, are used to detect or inhibit gene expression. Detection is described in Example VH. To inhibit transcription by preventing promoter binding, the complementary molecule is designed to bind to the most unique 5' sequence and includes nucleotides of the 5' UTR upstream of the initiation codon of the open reading frame. Complementary molecules include genomic sequences (such as enhancers or introns) and are used in triple helix base pairing to compromise the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. To inhibit translation, a complementary molecule is designed to prevent ribosomal binding to the mRNA encoding the protein. Complementary molecules are placed in expression vectors and used to transform a cell line to test efficacy; into an organ, tumor, synovial cavity, or the vascular system for transient or short term therapy; or into a stem cell, zygote, or other reproducing lineage for long term or stable gene therapy. Transient expression lasts for a month or more with a non-replicating vector and for three months or more if elements for inducing vector replication are used in the transformation/expression system. Stable transformation of dividing cells with a vector encoding the complementary molecule produces a transgenic cell line, tissue, or organism (USPN 4,736,866). Those cells that assimilate and replicate sufficient quantities of the vector to allow stable integration also produce enough complementary molecules to compromise or entirely eliminate activity of the cDNA encoding the protein. IX Antibody Arrays Protein:protein interactions
In an alternative to yeast two hybrid system analysis of proteins, an antibody array can be used to study protein-protein interactions and phosphorylation. A variety of protein ligands are immobilized on a membrane using methods well known in the art. The array is incubated in the presence of cell lysate until proteimantibody complexes are formed. Proteins of interest are identified by exposing the membrane to an antibody specific to the protein of interest. In the alternative, a protein of interest is labeled with digoxigenin (DIG) and exposed to the membrane; then the membrane is exposed to anti-DIG antibody which reveals where the protein of interest forms a complex. The identity of the proteins with which the protein of interest interacts is determined by the position of the protein of interest on the membrane. Proteomic Profiles
Antibody arrays can also be used for high-throughput screening of recombinant antibodies. Bacteria containing antibody genes are robotically-picked and griddled at high density (up to 18,342 different double-spotted clones) on a filter. Up to 15 antigens at a time are used to screen for clones to identify those that express binding antibody fragments. These antibody arrays can also be used to identify proteins which are differentially expressed in samples (de Wildt, supra)
X Screening Molecules for Specific Binding with the polynucleotide or Protein
The polynucleotide, protein, or antibody is labeled with a nucleotide such as 32P-dCTP, Cy3- dCTP, or Cy5-dCTP, an amino acid such as 35S-methionine, or reagents such as BIODEPY or E-TTC (Molecular Probes, Eugene OR). Kits for direct synthesis or chemical conjugation are supplied by companies such as APB, Invitrogen, Promega, or Qiagen. Libraries of candidate molecules ors compounds previously arranged on a substrate are incubated in the presence of labeled polynucleotide or protein. After incubation under conditions for either a nucleic acid or amino acid sequence, the substrate is washed, and any position on the substrate retaining label, which indicates specific binding or complex formation, is assayed, and the ligand is identified. Data obtained using different concentrations of the nucleic acid or protein are used to calculate affinity between the labeled nucleic acid or protein and the bound molecule.
XI Two-Hybrid Screen
A yeast two-hybrid system, MATCHMAKER LexA Two-Hybrid system (Clontech Laboratories), is used to screen for peptides that bind the protein of the invention. A polynucleotide encoding the protein is inserted into the multiple cloning site of a pLexA vector, ligated, and transformed into E. coh. cDNA, prepared from mRNA, is inserted into the multiple cloning site of a pB42AD vector, ligated, and transformed into E. coli to construct a cDNA library. The pLexA plasmid and pB42AD- cDNA library constructs are isolated from E. coli and used in a 2:1 ratio to co-transform competent yeast EGY48[p8op-lacZ] cells using a polyethylene glycol/lithium acetate protocol Transformed yeast cells are plated on synthetic dropout (SD) media lacking histidine (-His), tryptophan (-Trp), and uracil (-Ura), and incubated at 30C until the colonies have grown up and are counted. The colonies are pooled in a minimal volume of lx TE (pH 7.5), replated on SD/-His/-I-eu/-Trp/-Ura media supplemented with 2% galactose (Gal), 1% raffinose (Raf), and 80 mg/ml 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-Gal), and subsequently examined for growth of blue colonies. Interaction between expressed protein and cDNA fusion proteins activates expression of a LEU2 reporter gene in EGY48 and produces colony growth on media lacking leucine (-Leu). Interaction also activates expression of β-galactosidase from the p8op-lacZ reporter construct that produces blue color in colonies grown on X-Gal Positive interactions between expressed protein and cDNA fusion proteins are verified by isolating individual positive colonies and growing them in SD/-Trp/-Ura liquid medium for 1 to 2 days at 30C. A sample of the culture is plated on SD/-Trp/-Ura media and incubated at 30C until colonies appear. The sample is replica-plated on SD/-Trp/-Ura and SD/-His/-Trp/-Ura plates. Colonies that grow on SD cont-uning histidine but not on media lacking histidine have lost the pLexA plasmid. Histidine- requiring colonies are grown on SD/Gal/Raf/X-Gal/-Trp/-Ura, and white colonies are isolated and propagated. The pB42AD-cDNA plasmid, which contains a cDNA encoding a protein that physically interacts with the protein, is isolated from the yeast cells and characterized.
All patents and publications mentioned in the specification are incorporated by reference herein. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.
TABLE 1
Family A Fami ly B
Doubly PPARv FS PPP1R3AFS Reference heterozygou. mutant mutant values subjects heterozygotes heterozygotes
Figl I Ii i IIiv Him Il v Ii Ilvi In lllπ Hi h reference
Age 9 47 41 25 21 71 32 71 20 20 65
Gender F F F F F M M F M M M
BMI (kg/m2) 268 26 28 314 29 242 258 329 189 365 30
Blood 190/ 140/ 130/ 130/ 150/ 170/ 125/ 170/ 105/ 135/ 172/ pressure 110 80* 84 70 110 90* 90 105* 69 82* 93*
Measured 843 635 837 468 794 n/a 752 n/a n/a n/a n/a 100% body fat as percentage of predicted body fat ||
Glucose S6 64 44 92* 39 12φ 46 45 52 44 62 35-63 mmolL
Insulin 195 359 197 411 346 61 46 56 31 437 178 <80 pmolL
% insulin 27 15 28 14 20 87 115 95 168 13 30 100% sensitivity
(HOMA) 1
Tπglyceπdes 61 2 It 34 346 101 66 15 11 07 15 24 Desirable < mmol/L
HDL 082 063 081 052 104 071 102 184 136 07 091 Desirable > 9mmol/L
NEFA 1442 202t 526 2532 867 1219 584 933 n/a n/a n/a 280 - 920 umol/L
Uπc acid 031 024 023 023 028 035 031 023 032 017 044 015-035 mmolL
Leptin 121 44 82 173 124 12 09 132 06 146 198 ug/L
IMCL 198 191 255 283 449 n/a 283 n/a n/a n/a n/a 136 creatine ±66§ ratio (soleus)
All samples were obtained following an overnight fast. *, Measurements affected by anti-hypertensive therapy;
|| , Body fat was quantified by magnetic resonance imaging (MRl) as described previously^!. Predicted body fat32: for women = (1.48*BMI ( g/m2)) - 7.00, for men = (1.281*BMI (kg/m2)) - 1013 t, Measurements affected by lipid lowering therapy, φ, Abnormalities detected at the time of screening;
1, HOMA (homeostasis model assessment)33 is likely to be influenced by the diabetic status of some individuals
§ IMCL reference values represent mean and SD of measurements from 76 control subjects (unpublished observations EL Thomas and JD Bell)

Claims

What is claimed is:
1. An isolated polynucleotide, or the complete complement thereof, encoding a protein comprising an amino acid sequence of SEQ ID NO:l or SEQ ID NO:2.
2. An isolated polynucleotide comprising a nucleic acid sequence of SEQ ID NO:3 or SEQ ED
NO:4, or the complement of the nucleic acid sequence.
3. A recombinant polynucleotide comprising a promoter operably linked to a polynucleotide of claim 1.
4. A cell transformed with a recombinant polynucleotide of claim 3.
5. A method of producing a polypeptide of claim 1 , the method comprising: a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed.
6. An isolated polypeptide encoded by a nucleic acid molecule of claim 2.
7. An isolated polypeptide produced by the method of claim 5.
8. A method for detecting expression of a protein having the amino acid sequence of SEQ ED NO:l or SEQ ID NO:2. in a sample, the method comprising: a) performing an assay to determine the amount of the protein of claim 6 in a sample; and b) comparing the amount of protein to a standard, thereby detecting expression of the protein in the sample.
9. The method of claim 8 wherein the assay is two-dimensional polyacrylamide electrophoresis, western analysis, mass spectrophotomefry, enzyme-linked immunosorbent assay , radioimmunoassays, fluorescence activated cell sorting, or array technology.
10. The method of claim 8, wherein the expression of SEQ ID NO:2 is diagnostic of a predisposition to the development of diabetes.
11. The method of claim 8, wherein the co-expression of SEQ ID NO:l and SEQ ID NO:2 is diagnostic of the presence of type 2 diabetes.
12. A method for detecting the presence of type 2 diabetes in a subject, the method comprising: a) obtaining a sample of DNA from a subject; b) submitting the sample to a mutational screening assay; and c) detecting the presence of the mutations for PPARγFS and PPP1R3AFS in the subject DNA sample, wherein the presence of the mutations indicates the presence of type 2 diabetes in the subject.
13. A method for diagnosing a predisposition to the development of diabetes in a subject, the method comprising: a) obtaining a sample of DNA from a subject; b) submitting the sample to a mutational screening assay; and c) detecting the presence of the mutation for PPP1R3 AFS in the subject DNA sample, wherein the presence of the mutation indicates a predisposition to the development of diabetes in the subject.
14. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 2, the method comprising: a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof.
15. The method of claim 14 further comprising amplifying the nucleic acids of the sample prior to hybridization.
16. The method of claim 14 wherein the presence of SEQ ED NO:3 and SEQ ID NO:4 is diagnostic of type 2 diabetes.
17. The method of claim 14 wherein the presence of SEQ ID NO:4 is diagnostic of a predisposition to the development of diabetes.
18. An isolated polynucleotide comprising at least 20 contiguous nucleotide of SEQ ID NO:3 which includes nucleotide T554 of SEQ ID NO:3, or comprising at least 20 contiguous nucleotides of SEQ ED NO:4 which includes nucleotide G1985 of SEQ ID NO:4.
19. An isolated polypeptide encoded by the polynucleotide of claim 18.
20. A method of treating diabetes comprising administering to a subject in need of such treatment an effective amount of an agonist of PPP1R3A.
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