JP2008522597A - Single nucleotide polymorphism (SNP) associated with type II diabetes - Google Patents

Abstract

Disclose the association of type 2 diabetes with single nucleotide polymorphisms and haplotypes. Also disclosed are diagnostic applications in identifying persons with type 2 diabetes or those at risk of developing type 2 diabetes, and the discovery of therapeutic agents and methods.

Description

  Diabetes, a metabolic disease with reduced carbohydrate utilization and increased lipid and protein utilization, is caused by complete or relative deficiency of insulin. In more severe cases, diabetes is characterized by chronic hyperglycemia, diabetes, loss of water and electrolytes, ketoacidosis, and coma. Long-term complications include the occurrence of neuropathy, retinopathy, nephropathy, degenerative changes in generalized large and small blood vessels, and increased susceptibility to infection. The most common form of diabetes is type 2, non-insulin dependent diabetes, characterized by hyperglycemia resulting from impaired insulin secretion and insulin resistance in the target tissue. Both genetic and environmental factors contribute to the disease. For example, obesity plays a major role in the development of this disease. Type 2 diabetes is often mild onset of mild form of diabetes.

  The health significance of type 2 diabetes is enormous. In 1995, there were 135 million diabetic adults worldwide. In 2025, it is estimated that nearly 300 million people have diabetes. (King H., et al., Diabetes Care, 21 (9): 1414-1431 (1998)).

  Type 2 diabetes has been shown to have strong familial transmission: 40% of monozygotic twin pairs with type 2 diabetes also have a first-degree relative with one or several diabetes . Barnett et al. 20 Diabetologia 87-93 (1981). In Pima Indians, the relative risk of becoming diabetic increases by a factor of 2 for children born from one diabetic parent and by a factor of 6 if both parents are affected. Knowler, W. C., et al. Genetic Susceptibility to Environmental Factors. A Challenge for Public Intervention 67-74 (Almquist & Wiksele International: Stockholm, 1988). It has been observed that the agreement of identical twins for type 2 diabetes exceeds 90% compared to about 50% for identical twins affected by type 1 diabetes. Barnett, A. H., et al. 20 (2) Diabetologia 87-93 (1981). Non-diabetic twins of type 2 diabetic patients have been shown to have decreased insulin secretion and decreased glucose tolerance after an oral glucose tolerance test. Barnett, A.H., et al. 282 Brit. Med. J. 1656-1658 (1981).

  The high prevalence of the disease and the increasing affected population define the other genetic factors involved in type 2 diabetes and the unmet medical issues that define the associated risk factors more clearly Indicates the need. There is also a need for diagnostic assays for identifying a propensity to develop type 2 diabetes and therapeutic agents for the prevention and treatment of this disease.

  A nucleic acid sequence that can have multiple sequences in a population (either a natural population or a synthetic population such as, for example, a library of synthetic molecules) is referred to herein as a “polymorphic site”. Polymorphic sites can take into account sequence differences based on substitutions, insertions, or deletions. Such substitutions, insertions or deletions result in frameshifts, premature stop codon generation, deletion or addition of one or more amino acids encoded by the polynucleotide, altering the splice site, and mRNA May affect the stability or transportation. If the polymorphic site is 1 nucleotide long, this site is referred to as a single nucleotide polymorphism (“SNP”).

  SNP is the most common form of genetic change responsible for differences in disease susceptibility and drug response. SNPs can directly contribute to markers for many phenotypic endpoints such as disease risk or drug response differences between patients or, more generally, disease risk or drug response differences between patients Can serve as a marker for many phenotypic endpoints.

  Identification of such genetic factors can lead to diagnostic methods, reagents, and reagent kits for identification of individuals who are prone to develop certain diseases.

  The present invention focuses on candidate genes having single nucleotide polymorphisms (“SNPs”) that facilitate diagnostic and therapeutic interventions, and specifically, individuals with diabetes, focusing on nucleic acid fragments of the genes. It relates to the identification of genetic factors that are predisposed to morbidity.

  In certain embodiments, the invention provides sequences identified by sequence identification numbers (“SEQ ID NOs”) 1-92 as well as vectors, recombinant host cells, transgenic animals, and compositions comprising such polynucleotides. And an isolated polynucleotide comprising an SNP located within a sequence selected from the group consisting of the complement of the sequences identified by SEQ ID NOs: 1-92. The present invention also provides a method of diagnosing susceptibility to type 2 diabetes in an individual by detecting one or more at-risk alleles of SNPs associated with type 2 diabetes. Furthermore, the present invention provides a method of diagnosing susceptibility to type 2 diabetes in an individual by detecting one or more haplotypes associated with type 2 diabetes.

  In addition to the drugs themselves and pharmaceutical compositions containing these drugs, methods of identifying drugs that can alter the course of the disease are also contemplated by the present invention.

  Single nucleotide polymorphisms, the most frequent DNA sequence changes in the human genome, are gaining increasing importance for a wide range of biological and biochemical applications. SNPs have been used to explore the evolutionary history of human populations and to analyze forensic samples. SNP also plays a major role in genetic analysis. In addition, pharmacogenetics uses these DNA changes to elucidate the genetic factors underlying different drug efficacy or side effects. Finally, SNPs are thought to help identify genes involved in complex diseases.

  The present invention relates to the identification of specific loci or single nucleotide polymorphisms (SNPs) that are specifically identified as having a phenotype associated with type 2 diabetes. As a result, such individuals can be instructed to intervene, such as dietary changes, exercise, and / or medication, before signs of disease appear. The identification of genes involved in the type 2 diabetes locus opens the way to a better understanding of the disease process, which in turn may lead to improved diagnosis and treatment.

  To identify SNPs, we analyzed genes that may be related to type 2 diabetes. Subsequently, nucleic acid sequences containing SNPs were genotyped in diabetic cases and matched controls. A statistical analysis was then performed to find an association with type 2 diabetes in the analysis of the control and diabetic populations. After analysis of 1,769 SNPs in 186 genes, it was found that one SNP was statistically associated with type 2 diabetes (p ≦ 0.05).

  The term “SNP” refers to a single nucleotide polymorphism at a specific location in the human genome that varies between populations of individuals. As used herein, a SNP may be identified by its name or may be identified by location within a particular sequence. SNPs identified in the SEQ ID NO in FIG. 1 are shown in parentheses. For example, the SNP “[G / A]” of SEQ ID NO: 1 in FIG. 1 indicates that the nucleotide base (or allele) at that position in the sequence can be either guanine or adenine. Alleles associated with type 2 diabetes in FIG. 1 (eg, guanine of SEQ ID NO: 1) are shown in separate columns. The nucleotide adjacent to the SNP of each SEQ ID NO: 1 is the flanking sequence used to identify the location of the SNP in the genome.

  As used herein, a nucleotide sequence disclosed by a SEQ ID NO of the present invention encompasses the complement of the nucleotide sequence. Further, as used herein, the term “SNP” encompasses any allele in a set of alleles.

  The term “allele” refers to a specific nucleotide among selected nucleotides that define a SNP.

  The term “minor allele” refers to an allele of an SNP that occurs less frequently in a population of individuals than a major allele.

  The term “major allele” refers to an allele of an SNP that occurs more frequently in a population of individuals than a minor allele.

  The term “at risk allele” refers to an allele associated with type 2 diabetes. Figures 1 and 3-5 illustrate a number of at-risk alleles of the present invention.

  The term “haplotype” refers to a combination of specific alleles from two or more SNPs.

  The term “at-risk haplotype” refers to a haplotype associated with type 2 diabetes. FIG. 2 shows a number of at-risk haplotypes of the present invention.

  The term “polynucleotide” refers to a polymeric form of nucleotides of any length. A polynucleotide may comprise deoxyribonucleotides, ribonucleotides, and / or analogs thereof. A polynucleotide may have any three-dimensional structure, including single-stranded, double-stranded, and triple-helix molecular structures, and may perform any function known or unknown. The following are non-limiting embodiments of a polynucleotide: gene or gene fragment, exon, intron, mRNA, tRNA, rRNA, short interfering nucleic acid molecule (siNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide , Plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may also be modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs.

  A “substantially isolated” or “isolated” polynucleotide is a polynucleotide that is substantially free of naturally associated sequences. By substantially free, it is meant that it is free of at least 50%, at least 70%, at least 80%, or at least 90% of the originally relevant substance. An “isolated polynucleotide” also depends on origin or manipulation: (1) a polynucleotide that is not associated with all or part of the associated polynucleotide, and (2) a polynucleotide other than the originally linked polynucleotide. Or (3) a recombinant polynucleotide that does not naturally occur.

  The term “hybridizes under stringent conditions” typically refers to nucleotide sequences that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to each other. It is intended to describe the conditions for hybridization and washing that remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1.-6.3.6. Non-limiting examples of stringent hybridization conditions include hybridization at about 45 ° C. in 6 × sodium chloride / sodium citrate (SSC) followed by 0.2 × SSC, 0.1% SDS. One or more washes at 50-65 ° C.

  The term “vector” refers to a DNA molecule that carries the inserted DNA and can be persisted in a host cell. Vectors are also known as cloning vectors, cloning vehicles, or vehicles. The term “vector” includes vectors that function primarily for insertion of nucleic acid molecules into cells, replication vectors that function primarily for replication of nucleic acids, and primarily for transcription and / or translation of DNA or RNA. A functional expression vector is included. Also included are vectors that provide more than one of the above functions.

  A “host cell” is an individual cell or cell that can be a recipient of a vector or a recipient of nucleic acid molecule and / or protein integration, or a recipient of a vector recipient or nucleic acid molecule and / or protein. Culture is included. A host cell contains the progeny of one host cell, and the progeny is not necessarily completely (formally or in terms of total DNA content) the original parent due to natural, accidental or intentional mutation. May not be the same. Host cells include cells transfected with a polynucleotide of the invention. An “isolated host cell” is a cell that is physically separated from the organism from which it was derived.

  “Individual”, “host”, and “subject” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human.

“Transformation”, “transfection”, and “genetic transformation” are methods used for insertion, eg, lipofection, transduction, infection, electroporation, CaPO ₄ precipitation, DEAE-dextran, particle bombardment, etc. Regardless of, it is used interchangeably herein to refer to the insertion or introduction of an exogenous polynucleotide into a host cell. The exogenous polynucleotide may be maintained as a non-integrated vector, such as a plasmid, or alternatively may be integrated into the host cell genome. Genetic transformation may be transient or stable.

  The present invention utilizes conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology to the extent possible to those skilled in the art unless otherwise indicated.

  As used herein, the singular form of any term may instead include the plural and vice versa.

  All publications and references cited herein are hereby incorporated by reference in their entirety for any purpose.

  The present invention provides a sequence that can be used to identify patients with type 2 diabetes, where the presence of a particular allele of SNP (a particular nucleotide base) is prone to developing type 2 diabetes. Provided is an isolated polynucleotide comprising a SNP located within a sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 . In certain embodiments, the polynucleotide is selected from the group consisting of the sequence identified by SEQ ID NOs: 1-92 and the complement of the sequence identified by SEQ ID NOs: 1-92. In another embodiment, the polynucleotide comprises at least a portion of a sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92.

  The invention also hybridizes to a nucleotide sequence present in a test sample and is complementary or partially complementary, the sequence identified by SEQ ID NO: 1-92 and SEQ ID NO: 1-92 An isolated polynucleotide comprising an SNP located within a sequence selected from the group consisting of the complement of sequences identified by. In certain embodiments, the isolated polynucleotide hybridizes to a nucleotide sequence present in a test sample, is complementary, or is partially complementary, a sequence identified by SEQ ID NOs: 1-92 And selected from the group consisting of the complement of the sequences identified by SEQ ID NOs: 1-92. In a further embodiment, the isolated polynucleotide hybridizes to a nucleotide sequence present in the test sample, is complementary, or is partially complementary, a sequence identified by SEQ ID NOs: 1-92 And at least part of a sequence selected from the group consisting of the complement of the sequences identified by SEQ ID NOs: 1-92.

  The present invention also provides an isolated polynucleotide comprising one or more haplotypes selected from the group consisting of the haplotypes identified in FIG. 2 that exhibit a tendency to develop type 2 diabetes.

  Furthermore, using standard molecular biology techniques and the sequence information provided herein, the polynucleotides of the invention can be isolated. Using all or part of a sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 (see, eg, Sambrook et al. , Ed., Molecular Cloning: A laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y, 1989) Cloning techniques can be used to isolate a polynucleotide.

  Polynucleotides can be amplified by standard PCR amplification techniques using cDNA, mRNA, or genomic DNA as a template and appropriate oligonucleotide primers. The polynucleotide so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to all or part of a polynucleotide can be prepared using standard synthetic techniques, such as, for example, using an automated DNA synthesizer.

  Probes based on the sequence of the polynucleotides of the invention can be used to detect transcripts or genomic sequences. The probe may include a labeling group attached thereto, such as a radioisotope, a fluorescent compound, an enzyme, an enzyme cofactor, and the like. Measuring the level of a nucleic acid molecule encoding a protein in a sample of cells from a subject, eg, detecting mRNA levels, or whether the gene encoding the protein is mutated or deleted Such probes can be used as part of a diagnostic test kit to identify cells or tissues that fail to express the protein, such as by determining.

  In certain embodiments, the present invention also provides a SNP located within a sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92. A polypeptide encoded by the polynucleotide is provided. In certain embodiments, the polypeptide is encoded by a polynucleotide selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92. In another embodiment, the polypeptide comprises at least a portion of a sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92. Encoded by a polynucleotide. Antibodies that bind such polypeptides are also contemplated.

  The invention also provides a polypeptide encoded by a polynucleotide comprising a haplotype selected from the group consisting of the haplotypes identified in FIG.

  In certain embodiments, the invention is also identified by the haplotype identified in FIG. 2, or the sequence identified by SEQ ID NOs: 1-92 and SEQ ID NOs: 1-92, operably linked to regulatory sequences. A vector comprising an SNP located within a sequence selected from the group consisting of the complement of sequences is provided. In certain embodiments, the vector is selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 operably linked to a regulatory sequence. Contains an array. In another embodiment, the vector is selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 operably linked to a regulatory sequence. At least part of the sequence.

  In certain embodiments, the present invention also provides a recombinant host cell comprising such a vector. In certain embodiments, the present invention is also selected from the group consisting of the haplotype identified in FIG. 2, or the sequence identified by SEQ ID NOs: 1-92 and the complement of the sequence identified by SEQ ID NOs: 1-92 A method for producing a polypeptide encoded by a polynucleotide comprising an SNP located within a sequence comprising culturing a recombinant host cell comprising such a polynucleotide under conditions suitable for expression A method comprising: In certain embodiments, under conditions for expression, comprising a sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92, Polypeptides are produced by culturing recombinant host cells containing nucleotides. In another embodiment, under conditions for expression, a portion of a sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 The polypeptide is produced by culturing a recombinant host cell comprising the polynucleotide comprising

  A haplotype identified in FIG. 2, or a SNP located within a sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 Also contemplated by the present invention are transgenic animals comprising the polynucleotide comprising. In certain embodiments, the transgenic animal comprises a polynucleotide comprising a sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92. In another embodiment, the transgenic animal comprises at least a portion of a sequence selected from the group consisting of the sequence identified by SEQ ID NOs: 1-92 and the complement of the sequence identified by SEQ ID NOs: 1-92. Including polynucleotides.

  In another aspect, compositions and kits comprising the polynucleotides, proteins, antibodies, vectors, and / or host cells of the invention are contemplated.

  One application of the present invention includes the prediction of those at higher risk of developing type 2 diabetes. To clarify the individual's risk to the population, diagnostic tests that identify genetic factors contributing to type 2 diabetes are used in conjunction with, or separate from, known clinical risk factors. May be. Means for identifying individuals at risk for type 2 diabetes should result in better prevention and treatment plans, including more aggressive management of current clinical risk factors. In certain embodiments, the present invention is a method of diagnosing susceptibility to type 2 diabetes in an individual, wherein the nucleic acid of a particular gene or gene fragment indicates that the presence of a polymorphism in the nucleic acid indicates susceptibility to type 2 diabetes A method comprising detecting a polymorphism in

  In certain embodiments, the present invention is a method of diagnosing type 2 diabetes or susceptibility to type 2 diabetes in an individual comprising the specific alleles of the SNPs included in SEQ ID NOs: 1-92 and shown in FIG. Including a method comprising determining the presence or absence. In one aspect of the invention, the method comprises screening one of the at-risk alleles associated with type 2 diabetes shown in FIG. Accordingly, in certain embodiments, the present invention provides a method of diagnosing or determining susceptibility to type 2 diabetes in an individual or a method of screening individuals susceptible to type 2 diabetes, comprising SEQ ID NOs: 1-92 Detecting an at risk allele of a SNP associated with type 2 diabetes located within a sequence selected from the group consisting of the sequence identified and the complement of the sequence identified by SEQ ID NOs: 1-92 Provide a method. In certain embodiments, the SNP is located within SEQ ID NO: 23 and the complement of SEQ ID NO: 23. In certain embodiments, the SNP is located within the complement of SEQ ID NO: 32 and SEQ ID NO: 32. In certain embodiments, the SNP is located within SEQ ID NO: 35 and the complement of SEQ ID NO: 35. In certain embodiments, the SNP is located within SEQ ID NO: 40 and the complement of SEQ ID NO: 40. In certain embodiments, the SNP is located within SEQ ID NO: 41 and the complement of SEQ ID NO: 41. In certain embodiments, the SNP is located within SEQ ID NO: 17 and the complement of SEQ ID NO: 17. In certain embodiments, the SNP is located within SEQ ID NO: 36 and the complement of SEQ ID NO: 36. In certain embodiments, the SNP is located within SEQ ID NO: 74 and the complement of SEQ ID NO: 74. In certain embodiments, the SNP is located within SEQ ID NO: 62 and the complement of SEQ ID NO: 62. In certain embodiments, the SNP is located within SEQ ID NO: 10 and the complement of SEQ ID NO: 10. In certain embodiments, the SNP is located within the SEQ ID NO: 11 and SEQ ID NO: 11 complements. In certain embodiments, the SNP is located within SEQ ID NO: 8 and the complement of SEQ ID NO: 8. In certain embodiments, the SNP is located within SEQ ID NO: 54 and the complement of SEQ ID NO: 54. In certain embodiments, the SNP is located within SEQ ID NO: 65 and the complement of SEQ ID NO: 65. In certain embodiments, the SNP is located within SEQ ID NO: 9 and the complement of SEQ ID NO: 9. In certain embodiments, the SNP is located within SEQ ID NO: 67 and the complement of SEQ ID NO: 67. In certain embodiments, the SNP is located within the complement of SEQ ID NO: 4 and SEQ ID NO: 4. In certain embodiments, the SNP is located within SEQ ID NO: 91 and the complement of SEQ ID NO: 91. In certain embodiments, the SNP is located within SEQ ID NO: 92 and the complement of SEQ ID NO: 92. In certain embodiments, the SNP is located within the complement of SEQ ID NO: 68 and SEQ ID NO: 68. In certain embodiments, the SNP is located within SEQ ID NO: 69 and the complement of SEQ ID NO: 69. In certain embodiments, the SNP is located within SEQ ID NO: 7 and the complement of SEQ ID NO: 7. In certain embodiments, the SNP is located within SEQ ID NO: 20 and the complement of SEQ ID NO: 20. In certain embodiments, the SNP is located within the complement of SEQ ID NO: 16 and SEQ ID NO: 16. In certain embodiments, the SNP is located within the complement of SEQ ID NO: 31 and SEQ ID NO: 31. In certain embodiments, the SNP is located within SEQ ID NO: 42 and the complement of SEQ ID NO: 42. In certain embodiments, the SNP is located within SEQ ID NO: 39 and the complement of SEQ ID NO: 39. In certain embodiments, the SNP is located within SEQ ID NO: 53 and the complement of SEQ ID NO: 53. In certain embodiments, the SNP is located within SEQ ID NO: 38 and the complement of SEQ ID NO: 38.

  In certain embodiments, the present invention is selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 under conditions suitable for hybridization Contacting the sample with an isolated polynucleotide comprising a sequence (or a portion of the sequence), and including certain alleles of SNPs associated with (or not associated with) type 2 diabetes if hybridization occurs The sequence and sequence identified by SEQ ID NOs: 1-92, comprising assessing whether hybridization has occurred between a polynucleotide in the sample and the isolated polynucleotide, wherein a polynucleotide is present in the sample Numbers in the sample containing SNPs located within the sequence selected from the group consisting of the complements of the sequences identified by numbers 1 to 92 A method for detecting the presence of a renucleotide is provided. In certain embodiments of the above methods, the isolated polynucleotide is completely complementary to the polynucleotide present in the sample. In another embodiment of the above method, the isolated polynucleotide is partially complementary to the polynucleotide present in the sample. In another embodiment, the isolated polynucleotide is at least 80% identical to the polynucleotide present in the sample and can selectively hybridize to the polynucleotide. If desired, amplification of the polynucleotide present in the sample can be performed using methods known in the art.

  The present invention further includes a second polynucleotide comprising a sequence selected from the group consisting of a sequence identified by SEQ ID NO: 1-92 and a complement of the sequence identified by SEQ ID NO: 1-92 and at least partially A method for assaying a sample for the presence of a first polynucleotide that is complementary to: (a) contacting the sample with a second polynucleotide under conditions suitable for hybridization; and (b ) Comprising assessing whether hybridization has occurred between the first polynucleotide and the second polynucleotide, and providing a method wherein the first polynucleotide is present in the sample if hybridization has occurred . In certain embodiments of the methods previously described herein, the presence of the first polynucleotide indicates a tendency to develop type 2 diabetes or type 2 diabetes. In a further embodiment of the method, the second polynucleotide is completely complementary to a portion of the sequence of the first polynucleotide. In another embodiment, the method further comprises amplification of at least a portion of the first polynucleotide. In further embodiments, the second polynucleotide is 99 or less nucleotides in length and: (a) is at least 80% identical to the sequence of consecutive nucleotides in the first polynucleotide, or (b) Either of which can selectively hybridize to the first polynucleotide.

  Includes an allele of SNP associated with type 2 diabetes located within a sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 A method of assaying a sample for the presence of a polypeptide associated with type 2 diabetes encoded by the polynucleotide, the method comprising contacting the sample with an antibody that specifically binds to the polypeptide is also included in the present invention. Is intended by. In certain embodiments, by contacting a sample with an antibody that specifically binds to a polypeptide (consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 The presence of a polypeptide associated with type 2 diabetes in the sample encoded by the polynucleotide (including a sequence selected from the group) is assayed. In another embodiment, by contacting the sample with an antibody that specifically binds to the polypeptide (from the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92) The presence of a polypeptide associated with type 2 diabetes in a sample encoded by the polynucleotide (including at least a portion of a sequence selected from the group) is assayed.

  The present invention also includes a first SNP comprising an SNP located within a sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 A reagent for assaying a sample for the presence of a polynucleotide comprising a second polynucleotide comprising a contiguous nucleotide sequence that is at least partially complementary to a portion of the first polynucleotide. In certain embodiments of the reagent, the second polynucleotide is completely complementary to a portion of the first polynucleotide.

  The present invention also includes a first SNP comprising an SNP located within a sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 A reagent kit for assaying a sample for the presence of a polynucleotide, in separate containers: (a) the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 One or more labeled second polynucleotides comprising a sequence selected from the group consisting of a body; and (b) a reagent kit comprising a reagent for detection of the label.

  In another embodiment, associated with type 2 diabetes, located within a sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 A kit comprising a polynucleotide that can be used to assay a sample for the presence of a polynucleotide comprising a particular allele of a SNP that is (or is not) associated with is contemplated. An antibody that can be used to assay a sample for the presence of a protein associated with (or not associated with) type 2 diabetes encoded by a polynucleotide comprising an allele of an SNP associated with (or not associated with) type 2 diabetes A kit containing is also contemplated.

  Another method of diagnosing susceptibility to type 2 diabetes in an individual is type 2, wherein the presence of a change in the expression or composition of a polypeptide in a test sample compared to a control sample indicates susceptibility to type 2 diabetes Determining the expression or composition of a polypeptide in a control sample encoded by a polynucleotide containing an allele of SNP not associated with diabetes, and the same except that it comprises an allele of SNP associated with type 2 diabetes Comparing the expression or composition of the polypeptide in the test sample encoded by the polynucleotide.

  In certain embodiments, the present invention also relates to a method of diagnosing type 2 diabetes or susceptibility to type 2 diabetes in an individual comprising determining the presence or absence of a haplotype in the individual. In one aspect of the invention, the method includes screening one of the at-risk haplotypes shown in FIG. Therefore, the present invention is a method for diagnosing susceptibility to type 2 diabetes in an individual or a method for screening individuals having susceptibility to type 2 diabetes, comprising the group consisting of the haplotypes shown in FIG. A method comprising detecting a haplotype associated with a more selected type 2 diabetes.

  The presence or absence of haplotypes may be determined by various methods including, for example, using enzymatic amplification of nucleic acids from an individual, electrophoretic analysis, restriction fragment length polymorphism analysis, and / or sequence analysis. .

  A method for diagnosing susceptibility to type 2 diabetes in an individual, or a method for screening an individual for susceptibility to type 2 diabetes: (a) obtaining a polynucleotide sample from the individual; and (b) Also included is a method comprising analyzing a polynucleotide sample for the presence or absence of a haplotype, including the haplotype shown in FIG. 2, wherein the presence of the haplotype corresponds to susceptibility to type 2 diabetes.

  In certain embodiments, a method is provided for determining or diagnosing susceptibility to type 2 diabetes in an individual comprising detecting a number of SNPs identified in FIG. 1 or FIG. In certain embodiments, the method of determining susceptibility to type 2 diabetes in an individual comprises detecting a number of SNPs identified in SEQ ID NOs: 23, 32, 35, 40, 41, 17, and / or 36. Including. In another embodiment, the method of determining susceptibility to type 2 diabetes in an individual comprises detecting a number of SNPs identified in SEQ ID NOs: 74, 62, 10, 11, 8, and / or 54. . In another embodiment, the method of determining susceptibility to type 2 diabetes in an individual detects a number of SNPs identified in SEQ ID NOs: 65, 9, 67, 4, 91, 92, 68, and / or 69. The process of carrying out is included. In another embodiment, the method of determining susceptibility to type 2 diabetes in an individual detects a number of SNPs identified in SEQ ID NOs: 7, 20, 16, 31, 42, 39, 53, and / or 38 The process of carrying out. In certain embodiments, the sample is assayed for the presence in the sample of the first polynucleotide comprising one or more at-risk alleles of FIG. 1 by contacting the sample with a probe polynucleotide that is complementary to the first polynucleotide. .

  In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 23 and SEQ ID NO: 23. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 32 and SEQ ID NO: 32. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 35 and SEQ ID NO: 35. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 40 and SEQ ID NO: 40. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 41 and SEQ ID NO: 41. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 17 and SEQ ID NO: 17. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 36 and SEQ ID NO: 36. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 74 and SEQ ID NO: 74. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 62 and SEQ ID NO: 62. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 10 and SEQ ID NO: 10. In certain embodiments, at least one SNP is located within the SEQ ID NO: 11 and SEQ ID NO: 11 complements. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 8 and SEQ ID NO: 8. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 54 and SEQ ID NO: 54. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 65 and SEQ ID NO: 65. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 9 and SEQ ID NO: 9. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 67 and SEQ ID NO: 67. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 4 and SEQ ID NO: 4. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 91 and SEQ ID NO: 91. In certain embodiments, at least one SNP is located within SEQ ID NO: 92 and the complement of SEQ ID NO: 92. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 68 and SEQ ID NO: 68. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 69 and SEQ ID NO: 69. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 7 and SEQ ID NO: 7. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 20 and SEQ ID NO: 20. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 16 and SEQ ID NO: 16. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 31 and SEQ ID NO: 31. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 42 and SEQ ID NO: 42. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 39 and SEQ ID NO: 39. In certain embodiments, at least one SNP is located within SEQ ID NO: 53 and the complement of SEQ ID NO: 53. In certain embodiments, at least one SNP is located within the complement of SEQ ID NO: 38 and SEQ ID NO: 38.

  In certain methods of the invention, a therapeutic agent for type 2 diabetes is contemplated. Type 2 diabetes therapeutic agent comprises polypeptide activity and / or the haplotype identified in FIG. 2 or the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 It can be an agent that alters (eg, enhances or inhibits) the expression of a polynucleotide comprising a SNP located within a more selected sequence. Such agents are encoded by a polynucleotide, polypeptide, receptor, binding agent, peptidomimetic, fusion protein, prodrug, antibody, agent that alters polynucleotide expression, a gene or polynucleotide of the invention. Agents that alter the activity of a polypeptide, agents that alter the post-transcriptional processing of a polypeptide encoded by a gene or polynucleotide of the invention, agents, genes or agents that alter the interaction of a polypeptide with a binding agent or receptor Included are agents that alter transcription of a splice variant encoded by a polynucleotide, and ribosomes. In certain embodiments, the present invention also relates to pharmaceutical compositions comprising at least one type 2 diabetes therapeutic agent as described herein.

  Type 2 diabetes therapeutic agents are, for example, by altering the transcription of a splicing variant by altering post-translational processing of the polypeptide, eg, by up-regulating transcription or translation of the polynucleotide encoding the polypeptide. Or by interfering with polypeptide activity (eg, by binding to a polypeptide or by binding to another polypeptide that interacts with a polypeptide of interest). Polypeptide activity or expression of a polynucleotide can be altered by various means, such as by down-regulating expression, transcription, or translation, or by altering the interaction of a polypeptide of interest with a polypeptide binding agent. Can be changed wear.

  In certain embodiments, the present invention also relates to a method of treating an individual suffering from type 2 diabetes by administering to the individual a therapeutically effective amount of a type 2 diabetes therapeutic agent. In certain embodiments, the therapeutic agent for type 2 diabetes is an agonist, and in another embodiment, the therapeutic agent for type 2 diabetes is an antagonist. In certain embodiments, the present invention further relates to the use of a therapeutic agent for type 2 diabetes for the manufacture of a medicament for use in the treatment of type 2 diabetes.

  The therapeutic agents as described herein can be delivered in the form of a composition or alone. They can be administered systemically or can be targeted to specific tissues. Therapeutic agents are produced by a variety of means, including chemical synthesis; recombinant production and in vivo production (eg, transgenic animals, see US Pat. No. 4,873,316 to Meade et al., Incorporated herein by reference in its entirety). As well as can be isolated using standard methods known in the art. In addition, any combination of the above therapies (eg, administration of a polypeptide in combination with antisense therapy targeting mRNA; first splicing mutation in combination with antisense therapy targeting a second splicing variant) Body administration) can also be used.

  In certain embodiments, the present invention also includes a method of monitoring the effectiveness of the therapeutic agents of the present invention against type 2 diabetes using methods known in the art. Another application of the present invention is its use to predict an individual's response to a particular therapeutic agent. For example, SNPs or haplotypes may be used as pharmacogenomic diagnostics to predict drug response and guide therapeutic agent selection in a given individual.

  In another embodiment, the invention provides a method of identifying an agent that alters the expression of a polynucleotide comprising an allele of an SNP associated with type 2 diabetes, comprising: (a) a sequence under conditions for expression (1) An allele of a SNP associated with type 2 diabetes located within a sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92, and ( 2) contacting a polynucleotide comprising a promoter region operably linked to a reporter gene with an agent to be tested; (b) assessing the level of expression of the reporter gene in the presence of the agent; ) Assessing the level of expression of the reporter gene in the absence of the drug; and (d) the level of expression and step in step (b) for differences indicating that expression has been altered by the drug. Said method comprising comparing the level of expression in c).

  In another embodiment, the present invention is a method of identifying a suitable agent for treating type 2 diabetes, comprising: (a) the haplotype identified in FIG. 2 or the sequence identified by SEQ ID NOs: 1-92 And contacting the polynucleotide with the agent to be tested, comprising a SNP located within a sequence selected from the group consisting of the complement of the sequences identified by SEQ ID NOs: 1-92; and (b) type 2 It relates to a method comprising determining whether an agent binds to a polynucleotide, alters a polynucleotide, or affects a polynucleotide in a manner believed to be useful in the treatment of diabetes.

  In certain embodiments, the expression of the polynucleotide in the presence of the agent is one of one or more splicing variants that differs in type or amount from the expression of the one or more splicing variants in the absence of the agent. Including expression.

  In another embodiment, the present invention is a method of identifying a suitable agent for treating type 2 diabetes, comprising: (a) the haplotype identified in FIG. 2 or the sequence identified by SEQ ID NOs: 1-92 And contacting the polypeptide with an agent to be tested, encoded by a polynucleotide comprising a SNP located within a sequence selected from the group consisting of the complement of the sequences identified by SEQ ID NOs: 1-92; And (b) a method believed to be useful in the treatment of type 2 diabetes, comprising the step of determining whether the agent binds to the polypeptide, alters the polypeptide, or affects the polypeptide . In addition to agents identified by the above methods, pharmaceutical compositions containing such agents are also contemplated.

  In certain embodiments, located within a sequence selected from the group consisting of the haplotype identified in FIG. 2 or the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 The polynucleotide comprising the SNP is used in “antisense” therapy where the polynucleotide is administered or generated in situ and specifically hybridizes to mRNA and / or genomic DNA. An antisense polynucleotide that specifically hybridizes to mRNA and / or DNA inhibits expression of the polypeptide encoded by the mRNA and / or DNA, for example, by inhibiting translation and / or transcription. The binding of the antisense polynucleotide can be by conventional base-pair complementarity or via specific interactions in the main groove of the double helix, for example when binding to a DNA duplex.

  The antisense construct can be delivered, for example, as an expression plasmid. When a plasmid is transcribed in a cell, it produces RNA that is complementary to a portion of the mRNA and / or DNA encoding the polypeptide. Alternatively, an antisense construct can be a polynucleotide probe that is generated ex vivo and introduced into a cell, after which it is expressed by hybridizing to mRNA and / or genomic DNA encoding the polypeptide. Inhibit. In certain embodiments, the polynucleotide probe is a modified oligonucleotide that is resistant to endogenous nucleases, such as, for example, exonucleases and / or endonucleases, thereby providing in vivo stability. Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoamidate, phosphothioate, and methylphosphonate analogs of DNA (US Pat. No. 5,176,996, all of which are hereby incorporated by reference in their entirety). No. 5,264,564 and 5,256,775). In addition, general approaches to making oligomers useful in antisense therapy are also described, for example, by Van der Krol et al. (Bio Techniques 6: 958-976 (1988), both of which are incorporated herein by reference in their entirety. ); And Stein et al. (Cancer Res. 48: 2659-2668 (1988)). For antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site may be used.

  To perform antisense therapy, an oligonucleotide is designed that is complementary to the mRNA encoding the polypeptide. Antisense oligonucleotides bind to mRNA transcripts and prevent translation. Full complementarity is not required as long as the oligonucleotide has sufficient complementarity to be able to hybridize with RNA and form a stable duplex. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense oligonucleotide. In general, the longer the oligonucleotides that hybridize, the more they may contain and the more base mismatches with RNA that still form stable duplexes (or even triplets). One skilled in the art can ascertain the degree of mismatch that can be tolerated by using standard methods.

  Oligonucleotides used in antisense therapy can be DNA, RNA, or chimeric mixtures or derivatives or modifications thereof, single-stranded or double-stranded. Oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve molecular stability, hybridization, and the like. Oligonucleotides can be other adducts such as peptides (eg to target host cell receptors in vivo) or cell membranes (eg Letsinger et al., Proc. Natl. Acad. Sci. USA 86: 6553 -6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci USA 84: 648-652 (1987); PCT International Publication Number: see WO 88/09810) or blood brain barrier (eg PCT International Publication Number) : Agents that promote transport beyond WO 89/10134), or cleavage agents that induce hybridization (see, eg, Krol et al., Bio Techniques 6: 958-976 (1988)), or intercalating agents. Can be included. (See, for example, Zon, Pharm. Res. 5: 539-549 (1988)). For this purpose, the oligonucleotide may be conjugated to another molecule (eg, a peptide, a crosslinker that induces hybridization, a transport agent, a cleavage agent that induces hybridization).

  In certain embodiments, the antisense molecule is delivered to cells that express a polypeptide associated with type 2 diabetes in vivo. Many methods can be used to deliver antisense DNA or RNA to cells; for example, antisense molecules can be injected directly into a tissue site or targeted to a desired cell Designed, modified antisense molecules (eg, antisense linked to peptides or antibodies that specifically bind to receptors or antigens expressed on the surface of target cells) can be administered systemically. Alternatively, if the antisense oligonucleotide is placed under the control of a strong promoter (eg, pol III or pol II), a recombinant DNA construct is utilized. By using such a construct to transfect target cells in a patient, a sufficient amount is expected to form a base pair complementary to the endogenous transcript and thereby prevent translation of the mRNA. Transcription of single-stranded RNA is caused. For example, the vector can be introduced in vivo such that it is taken up by the cell and directs the transcription of the antisense RNA. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA techniques and methods standard in the art. For example, a plasmid vector, cosmid vector, or viral vector can be used to prepare a recombinant DNA construct that can be introduced directly into a tissue site. Alternatively, viral vectors that selectively infect the desired tissue can be used, in which case administration may be accomplished by another route (eg, systemically). In another embodiment of the invention, small double-stranded interfering RNA (RNA interference (RNAi)) can be used. RNAi is a post-transcriptional process in which double-stranded RNA is introduced and sequence-specific gene silencing results through catalytic degradation of the targeted mRNA. For example, all of its teachings are incorporated herein by reference in their entirety; Elbashir, SM et. Al., Nature 411: 494-498 (2001); Lee, NS, Nature Biotech. 19: 500-505 (2002) ); Lee, SK. Et al., Nature Medicine 8 (7): 681-686 (2002).

  In certain embodiments, the invention provides a sequence selected from the group consisting of the haplotype identified in FIG. 2 or a sequence identified by SEQ ID NOs: 1-92 and a complement of the sequence identified by SEQ ID NOs: 1-92 A short interfering nucleic acid (“siNA”) molecule comprising a double-stranded RNA polynucleotide that down-regulates expression of the polynucleotide, including the SNP identified in. In certain embodiments, the present invention provides a gene comprising a SNP identified by a sequence selected from the group consisting of the sequence identified by SEQ ID NOs: 1-92 and the complement of the sequence identified by SEQ ID NOs: 1-92 In US Patent Publication Nos. US 2004/0192626 A1, US 2004/0203145 A1, and US 2004/0198682 A1 (all of which are hereby incorporated by reference in their entirety). Polynucleotides, compositions, and methods used in RNA interference (such as).

  Target homologous recombination can also be used to reduce endogenous expression of a gene product by inactivating or “knocking out” the gene or its promoter. For example, selectable using a non-functional gene (or a completely unrelated DNA sequence) in which the DNA complementary to the endogenous gene (either the coding or regulatory region of the gene) is flanked by changes A gene can be transfected into cells expressing in vivo with or without markers and / or negative selectable markers. Insertion of a DNA construct via targeted homologous recombination results in gene inactivation. A suitable vector, such as described above, can be used to administer or target the recombinant DNA construct directly to the required in vivo site. Alternatively, similar methods can be used to increase the expression of an unaltered gene: target homologous recombination can be used to replace the gene in the cell, as described above, without being altered. A DNA construct comprising a functional gene, or its complement, or part thereof can be inserted. In another embodiment, target homologous recombination can be used to insert a DNA construct comprising a polynucleotide that encodes a polypeptide variant that differs from the polypeptide present in the cell.

  Alternatively, the internalization of the gene product can be achieved by targeting a deoxyribonucleotide sequence complementary to the regulatory region (ie, promoter and / or enhancer) to form a triple helix structure that prevents transcription of the gene in the target cell in the body. Sexual expression can be reduced. (Generally, Helene, C., Anticancer Drug Des., 6 (6): 569-84 (1991); Helene, C., et al., Ann. NY Acad. Sci. 660: 27-36 ( 1992); and Maher, LJ, Bioassays 14 (12): 807-15 (1992)), all of which are hereby incorporated by reference in their entirety. Similarly, the antisense constructs described herein are used in the manipulation of tissue by blocking tissue differentiation in both normal biological activity of the gene product, eg, in vivo tissue culture and ex vivo tissue culture. be able to. In addition, using antisense technology (eg, microinjection of antisense molecules, or transfection using plasmids whose transcripts are antisense with respect to RNA or nucleic acid sequences) are involved in the development of type 2 diabetes and related conditions The role of one or more genes involved in the pathway can be investigated. Such techniques can be used for cell culture, but can also be used to create transgenic animals.

  The polynucleotides, proteins, and / or therapeutic agents of the invention described herein can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the polynucleotide, protein, and / or therapeutic agent, and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” refers to any and all solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic, which are compatible with pharmaceutical administration. It is intended to include agents and absorption delaying agents, and similar agents. The use of such solvents and agents for pharmaceutically active substances is well known in the art. Except where any conventional solvent or agent is incompatible with the active compound, its use in the composition is contemplated. Supplementary active compounds can also be incorporated into the compositions.

  A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include, for example, parenteral administration such as intravenous administration, intradermal administration, subcutaneous administration, oral administration (eg inhalation), transdermal administration (topical administration), transmucosal administration, and rectal administration It is. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain the following components: sterile diluents such as water for injection, saline, fixed oils, polyethylene glycols, Glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; acetic acid, citric acid, or phosphoric acid Buffers and agents for adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

  Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or sterile injectable dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL ™ (BASF; Parsippany, N. J.) or phosphate buffered saline (PBS). The composition may be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersant containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, magnesium monostearate and gelatin.

  Incorporate the active compound (eg, polynucleotide, polypeptide, or antibody) in the required amount in an appropriate solvent, along with one or a combination of the components listed above, followed by filter sterilization if necessary. By doing so, sterile injectable solutions can be prepared. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersant and the other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, some methods of preparation include vacuum drying and lyophilization to produce a powder of the active ingredient and the desired additional ingredients from that solution that have been filter sterilized so far. There is.

  Oral compositions usually include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. An oral composition is prepared using a flowable carrier for use as a mouthwash, wherein the compound in the flowable carrier is applied orally and is swallowed and exhaled or swallowed You can also Pharmaceutically acceptable binding agents, and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, and the like can include any of the following ingredients or compounds of similar nature: binders such as microcrystalline cellulose, gum tragacanth, or gelatin; starch or lactose Excipients such as alginic acid or corn starch; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or peppermint, methyl salicylate, or Fragrance additive such as orange fragrance.

  For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

  Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid. Transmucosal administration can also be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated in ointments, salves, gels, or creams as known in the art.

  The compounds can also be prepared in the form of suppositories for rectal delivery (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas.

  In certain embodiments, the active compounds are prepared with carriers that will protect the compound against rapid removal from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for the preparation of such formulations will be apparent to those skilled in the art. Materials can also be obtained commercially from Alza and Nova Pharmaceuticals. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811.

  It is advantageous to formulate oral and parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to a physically discrete unit suitable as a unit dosage for the subject being treated; each unit is as desired in connection with the required pharmaceutical carrier. Contains a predetermined amount of active compound calculated to produce a therapeutic effect. The specifications for the dosage unit forms of the present invention are determined by the unique characteristics of the active compound and the particular therapeutic effect achieved, as well as limitations inherent in the art for formulating such active compounds for the treatment of individuals. And the specific characteristics of the active compound and the specific therapeutic effect on what has been achieved, as well as the limitations inherent in the art of formulating such active compounds for the treatment of individuals.

  The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. For example, gene therapy by intravenous injection, local administration (see US Pat. No. 5,328,470) or by stereotaxic injection (see, eg, Chen et al. (1994) Proc. Natl Acad. Sci. USA 91: 3054-3057) The vector can be delivered to the subject. The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent or can include a sustained release matrix in which the gene delivery vehicle is embedded. Alternatively, where a complete gene delivery vector can be produced intact from a recombinant cell, eg, a retroviral vector, the pharmaceutical preparation includes one or more cells that produce the gene delivery system. Can do.

  The pharmaceutical composition can be included in a container, pack, or dispenser along with instructions for administration. Kits comprising the therapeutic agents of the present invention are contemplated.

  Another aspect of the present invention is its use to predict an individual's response to a particular drug for treating type 2 diabetes. In general, it is a well-known phenomenon that patients do not respond to the same drug as well. Many of the differences in drug response to a given drug are believed to be based on genetic and protein differences between individuals in certain genes and the pathways to which they correspond. The present invention defines specific SNPs, haplotypes, and genes associated with type 2 diabetes. Some current or future therapeutic agents can affect pathways that are directly or indirectly related to such SNPs, haplotypes, and / or genes, and thus type 2 diabetes There may be one that can be effective in patients whose risk is determined in part by such SNPs, haplotypes, and / or genes. On the other hand, those same drugs may be less effective or ineffective in patients who do not have a specific allele of the SNP and / or haplotype. Thus, the SNPs and / or haplotypes of the present invention can be used as pharmacogenomic diagnostics to predict drug response in a given individual and guide therapeutic agent selection.

  In certain embodiments, the method for monitoring the effectiveness of a drug for the treatment of type 2 diabetes is one or more selected from the group of SNPs consisting of the SNPs identified in FIG. 1 prior to treatment with the drug. Compares the level of expression of genes associated with type 2 diabetes, including SNP, the level of expression of genes after treatment with drugs, and the level of expression of genes before and after treatment The process of carrying out.

  In another embodiment, the method for predicting the efficacy of a given therapeutic agent in the treatment of type 2 diabetes is the sequence identified by SEQ ID NO: 1-92 and the sequence identified by SEQ ID NO: 1-92 Screening for the presence or absence of one or more SNPs located within a sequence selected from the group consisting of the complements of:

  In another embodiment, the method for predicting the effectiveness of a given therapeutic agent in the treatment of type 2 diabetes comprises screening for the presence or absence of one or more haplotypes identified in FIG.

  Another application of the present invention is the specific identification of rate limiting pathways involved in type 2 diabetes. Disease genes with genetic mutations that are significantly more common in diabetic patients compared to controls represent a specifically demonstrated causal stage in the development of type 2 diabetes. That is, the uncertainty of whether the gene is responsible or simply reactive to the disease process is eliminated. The protein encoded by the disease gene defines the rate-limiting molecule involved in the biological process of type 2 diabetes predisposition. The protein encoded by such a type 2 gene or its interacting protein in its molecular pathway represents a drug target that can be selectively regulated by small molecule therapy, protein therapy, antibody therapy, or nucleic acid therapy. As the population suffering from type 2 diabetes is increasing, such specific information is highly needed.

（表1）2型糖尿病と関係があることが発見された遺伝子 Genes that have not been previously known to be related to type 2 diabetes by SNP-based associations but have been found to be related to type 2 diabetes by SNP-based associations in the present invention include the following: Is included.

(Table 1) Genes found to be associated with type 2 diabetes

  In certain embodiments, the present invention provides a method for identifying a gene associated with type 2 diabetes, comprising: (a) a sequence identified by SEQ ID NO: 1-92 and a sequence identified by SEQ ID NO: 1-92 Identifying a gene comprising a SNP located within a sequence selected from the group consisting of complements; and (b) for an individual having an at-risk allele for differences indicating that the gene is associated with type 2 diabetes It relates to a method comprising the step of comparing the expression of the gene with the expression of the gene in an individual without an at-risk allele.

  In another embodiment, the present invention is a method for identifying a gene associated with type 2 diabetes, comprising: (a) identifying a gene comprising the at-risk haplotype identified in FIG. 2; and (b) the gene is For a difference indicating that it is associated with type 2 diabetes, the present invention relates to a method comprising comparing the expression of a gene in an individual having an at-risk haplotype with the expression of a gene in an individual not having an at-risk haplotype.

（表2）この研究で用いた症例試料および対照試料のまとめ Example
Example 1: A total of 600 patients were included in the study to investigate mutations in the presence of SNPs and SNP haplotypes as between the study population control population and the type 2 diabetes population. One Swedish cohort was used for SNP discovery, and another Polish diabetic cohort was used in related studies (see Table 2). Samples from an additional 300 matched controls were analyzed for use in related studies.

(Table 2) Summary of case and control samples used in this study

  From the case-control study, the phenotype was simply “diabetes”. Other subphenotypes could be included in the analysis, including BMI, hemoglobin AIC, heart disease (such as MI), nephropathy, etc. The sample is a protocol detailed in Ardlie et al., Testing for population subdivision and association in four case-control populations, Am J Hum Genet. 71: 304-311, 2002, which is incorporated herein by reference in its entirety. Collected by Genomics Collaborative (CGI).

  Finally, the population included roughly the same number of men and women (274 men and 326 women). The samples were also well matched in the same number of men (163 cases and 163 controls) and the same number of women (137 cases and 137 controls) in the diabetic and unaffected populations.

  In studies based on any population, it is important to match cases and controls to avoid false results based on unknown confounders. In the context of genetic studies, this means that the case population and the control population should be genetically identical across the genome, except for regions containing genes that give the phenotypic tendency being studied. That is, a random set of markers should show widely similar allele frequencies in the case population and the control population. Because patients and controls were not only matched for gender and ethnicity but were selected from the same country (Poland), population stratification was unlikely to exist in this study. However, to test for population stratification, data were analyzed using the software program STRUCTURE 2.0 (March 2002) by Falush et al; all of which are incorporated herein by reference; Pritchard et al, See also Inference of population structure: Extensions to linked loci and correlated allele frequencies; GENETICS; forthcoming (2003); Pritchard et al., Inference of Population Structure Using Multilocus Genotype Data; GENETICS 155: 945-959 (2000a). STRUCTURE is incorporated herein by reference in its entirety; a model as described in Pritchard et al., Association mapping in structured population, AM. J. HUM. GENET. 671: 170-81 (2000). A clustering method based on is executed. The program was allowed to classify the data into a predefined number of clusters without any intervention. The data set consisted of 150 markers selected based on three criteria. First, minor alleles had to have a frequency> 5% across the population. Second, at least 80% of individuals are required to have a genotype, and third, the marker should not be closer than 100 kb to any other marker in the set.

  Data stratification analysis showed no clear clustering. A variety of factors indicated that there was no structure, including the following: The proportion of the genome of individuals from each of the clusters was the same for cases and controls, and all individuals were mixed. That is, they were considered to have genes from all clusters and did not improve the potential by adding more parameters (ie fitting more clusters).

  In summary, there was no consistent genetic bias between cases and controls. The best fit cluster created by STRUCTURE appeared to be independent of the phenotype of the individual samples.

Example 2: Sample collection and SNP discovery in diabetic population candidate genes
Three hundred (300) samples from diabetic patients were analyzed for the SNP discovery process. A total of 186 genes (identified to be associated with diabetes genes) were utilized for SNP discovery. Of these genes, 62 were analyzed using ParAllele's mismatch repair detection system (MRD), and 341 SNPs were detected. The other 1659 SNPs were identified by de novo sequencing and identified from the public database (National Center for Biotechnology Information).

  Of the 186 genes analyzed for SNP discovery, 62 were analyzed through the mismatch repair detection platform (MRD), and 341 SNPs were detected. The purpose of the analysis was to detect among these targets SNPs with a frequency of 2% or higher in the study population including the diabetic and control populations. Information about all gene exons was obtained from the Ensembl (The Sanger Centre, Cambridge, UK) Build 33 database. Since the sequence immediately upstream of the transcript is enriched in regulatory sequences, the first 350 bp upstream was encoded as exon 0. A total of 990 regions were identified, including not only exon 0 but also each exon and human mouse homology. 175 of these regions were human mouse homologous and 815 were exons (including exon 0). The number of exons per gene ranged from 2 (including exon 0) to 58 (NOS2A) as in PPPIR3C. Several large exons were divided into two or more targets, and pairs of small exons in close proximity were combined to form a single target. In total, primers were designed to generate 1011 targets and amplify 999 amplicons.

Example 3: Mismatch repair detection (MRD)
MRD uses the Escherichia coli mismatch repair system to detect mutations or SNPs. Modrich, P., Mechanisms and biological effects of mismatch repair, ANN REV, GENET, 25: 2259-53 (1991), which is incorporated herein by reference in its entirety. Specific strains are created artificially to classify the pool of transformed fragments into two pools: a pool with mutations and a pool without mutations. MRD has previously been described as a method for multiple mutation scanning. Faham. M., et al., Mismatch repair detection (MDRD): high-throughput scanning for DNA variations, HUM MOL. GENET, 10 (16); p1657-64 (2001), which is incorporated herein by reference in its entirety. ). Use MRD with standard dideoxy terminator sequence analysis to find common mutant alleles in two different populations. A PCR reaction using pooled genomic DNA from a population as a template is mixed with PCR fragments from a single haploid individual. Sanger sequencing analysis, where pooled populations are sequenced directly, is not sensitive enough to detect rare alleles from genomic pools. Instead, many PCR reactions are pooled and a single MRD reaction is performed to generate a pool of colonies that are enriched for mutant alleles relative to haploid standards. A single amplification reaction from the pool enriched for mutants is performed on each amplicon, followed by a sequence reaction to identify common and rare mutations in the studied population. See Fakhrai-Rad et al., SNP Discovery in Pooled Samples With Mismatch Repair Detection, COLD SPRING HARBOR LABORATORY PRESS, 14: 1404-1412 (2004), which is incorporated herein by reference in its entirety.

  The net result of this process is that the need to amplify and sequence many individuals is replaced by a pool enrichment process performed on thousands of amplicons in a multiplexed manner. The sequence analysis process is therefore summarized in the work of sequencing haploid standards and the results of MRD enriched pools. To reduce the false positive SNP discovery rate, amplicon sequencing is typically performed in both the forward and reverse directions.

  The sensitivity of MRD-based SNP discovery is limited by the background generated by MRD enrichment of nongenomic DNA mismatches. These can occur in two ways: oligonucleotide mutations and PCR errors. Both oligo errors and PCR errors in the PCR primer introduce a set of fragments containing the mutation in the absence of any actual DNA mutation. These fragments are thought to be enriched with the actual mutations, meaning that it is impossible to enrich for mutations that occur less frequently than the background level. To minimize these problems, PCR using oligos with a low percentage of mutations and high fidelity polymerases is used. A control experiment was conducted using patients with mutations in the BRCA1 gene. These patients were sequenced to identify mutations in the BRCA1 exon. These DNAs were then pooled to create samples in which individual SNPs were found at varying frequencies. These pools were then concentrated and sequenced. MRD is very sensitive to mutations as low as 1%, with complete rediscovery if the amplicon shows SNP at a frequency> 10%.

  In the human population, there are other practical issues that impose other limitations on the sensitivity of MRD-based SNP discovery. The first of these is that multiple SNPs can occur on a particular sequence analysis fragment. If this occurs with two SNPs with very different frequencies, it appears that there is a tendency to dominate in the pool with the higher frequency SNPs enriched, and the signal of the rarer SNPs is suppressed. This effect can be mitigated in several ways. The first is to use fairly small PCR fragments to minimize the chance of multiple SNPs occurring within a single fragment (typically using a ~ 300 bp fragment). Next, if it is known that common SNPs are generated, PCR primers can be designed to remove these SNPs. These limitations will be compared with the very high cost of sequence analysis and analysis of many individuals with typical techniques. By introducing Poisson noise into the selection of a small population, coverage is reduced by reducing the number of individuals that are sequenced by classical methods.

Example 4: SNP Genotyping Using Molecular Inversion Probes After the end of the SNP discovery phase using samples from diabetic patients, a set of 300 samples from a second diabetic cohort and 300 from non-diabetic controls Another set of samples, including a set of samples, was utilized for the genotyping phase of the project.

  A molecular inversion probe (MIP) was used for SNP genotyping. The sequence for 1739 SNPs was analyzed. A total of 1591 of these SNPs were unique in the NCB1 database (Build 33). The 40 bp sequence flanking 327 of the SNPs was unique. 82 validated SNPs out of 102 from public databases (which were not detected by SNP discovery) were unique in the genome. This gave just 2,000 SNPs for which the MIP probe was designed. Of these 2,000 probes, 1,769 (88.4%) produced validated assays. These genotyping was then performed in 300 diabetic cases and 300 ethnic and gender matched controls.

  These SNPs were chosen to provide information on 186 genes that may play a role in susceptibility to diabetes. These genes are at least one gene from all chromosomes except chromosome 21 and Y, and are located throughout the genome. Genes vary in size from 0 to 992 kb. The length of a gene is measured by the size of the region between the most widely spaced SNPs in each gene, so that a gene with only one SNP is recorded as having a size of 0 kb. Please keep in mind.

  The oligonucleotide probe in this process undergoes a single molecule rearrangement from a non-amplifiable molecule to an amplifiable molecule. This rearrangement is mediated by an enzymatic “gap filling” process that occurs in genomic DNA and allele-specific procedures. The gap filling process results in an important intermediate state where the probe is circularized. This state allows selection for unimolecular interactions through exonuclease treatment that is believed to degrade all cross-reactive and unreacted probes. After inversion, the probe is amplified using common fluorescently labeled PCR primers. See Hardenbol et al., Multiplexed genotyping with sequence tagged molecular inversion probes, 21 NAT. BIOTECHNOL. (6): 673-78 (June 2003), which is incorporated herein by reference in its entirety.

  To identify alleles, 4 identical reactions are used for SNPs. By using a single nucleotide species (A, C, G, or T), each of the four multiplexing reactions yields a different SNP allele. After inversion, PCR is performed using a common primer pair so that all probes that have undergone inversion are amplified in each reaction. Inferring the SNP allele by identifying which label is present on the MIP probe amplicon resulting from four separate reactions by using different fluorescent labels in each of these four reactions Can do.

  After amplification, the 4 reactions are hybridized to a universal oligonucleotide array. Relative base incorporation is measured by the fluorescent signal at the corresponding complementary tag site on the DNA array. Make four intensity values for each probe. To determine if the SNP is homozygous or heterozygous for a given individual, compare the two values for the expected allele base, and as a quality control check, for the probe To measure signal versus noise, two non-allelic bases are compared to the allelic base.

Example 5: Summary of Allele Association Results Marker-trait associations were examined using contingency table analysis on empirical p-values and Fisher's exact test. A summary of the results from an allele chi-square association test (2 × 2, 1 df) from one particular study is shown in FIG. 3, where the number of SNPs was found to be significant at p ≦ 0.05.

Example 6: Summary of genotype relevance results Summary of results from the chi-square relevance test (2 × 3, 2 df) of genotypes of a particular study, significant for S ≦ p 0.05 This is shown in FIG.

Example 7: Chi-square test for recessive effect If the underlying genetic susceptibility to disease follows an additive genetic model, both allele and genotype tests are most appropriate, Includes tests for dominance. However, both genotype and allele tests do not address the recessive allele effect, and if present, they are likely to be overlooked. To address this issue, we performed another series of chi-square tests in which each SNP minor allele was modeled as a recessive effect (2 × 2, 1 df). Some SNPs were significant in the recessive test (see FIG. 5), some of which were already associated with the allele test. FIG. 6 provides a summary of SNPs found to be associated with type 2 diabetes using an allele association test, a genotype association test, and a chi-square test for recessive effects.

Example 8: Evaluation for at-risk haplotypes The haplotypes described herein have been found to be more frequent in individuals with type 2 diabetes than in individuals without type 2 diabetes. Thus, these haplotypes have predictive value regarding type 2 diabetes or susceptibility to type 2 diabetes in individuals. In certain embodiments described herein, an individual at risk for type 2 diabetes is an individual for whom an at-risk haplotype is identified.

  In certain embodiments, an at-risk haplotype is a haplotype that confers a significant risk of type 2 diabetes. In certain embodiments, significance associated with a haplotype is measured by an odds ratio. In a further embodiment, significance is measured as a percentage. In certain embodiments, significant risk is measured as an odds ratio of at least about 1.2, including but not limited to: 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. In a further embodiment, an odds ratio of at least about 1.2 is significant. In a further embodiment, an odds ratio of at least about 1.5 is significant. In a further embodiment, a significant increase in risk of at least about 1.7 is significant. In a further embodiment, the significant increase in risk is about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 95%, and 98%, at least about 20%. In a further embodiment, the significant increase in risk is at least about 50%. However, it is understood that identifying whether a risk is medically significant may depend on a variety of factors, including specific diseases, haplotypes, and often environmental factors.

  For genotyping the presence of SNPs, such as fluorescence-based techniques (Chen, et al., Genome Res. 9, 492 (1999)), PCR, LCR, nested PCR, and other techniques for nucleic acid amplification Standard techniques can be used. In certain embodiments, the method comprises an SNP of an SNP that is excessive or more frequent compared to a healthy control population, indicating that the individual has type 2 diabetes or is susceptible to type 2 diabetes. Assessing presence or frequency in the individual. The presence of two or more SNPs may indicate the presence of an at-risk haplotype that can be used to screen individuals. For example, the at-risk haplotype can include the haplotype identified in FIG. 2, the combination of SNPs identified in FIG. 1, or the combination of SNPs identified in FIG. 1 or FIG. The presence of at-risk haplotypes indicates susceptibility to type 2 diabetes and thus individuals that fall within the target population for the treatment methods described herein.

Example 9: SNP for peripheral vascular disease
The association with a clear phenotype of peripheral vascular disease (“PVD”) was also investigated among diabetic patients. The strongest association detected for PVD is the SNP in SEQ ID NO: 44 (rs1491238) and SEQ ID NO: 45 (rs2306985), located in the MTP (microsomal triglyceride transfer protein) gene on chromosome 4. G ”allele. For rs1491238 in SEQ ID NO: 44, the chi-square is χ ² = 18.0 (p ≦ 0.0001), and for rs2306985 in SEQ ID NO: 45, the chi-square is χ ² = 18.8 (p = 0.001) Met. The allele odds ratio for the “G” allele was less than 1; for rs1491238, OR = 0.49 (0.35-0.68), and for rs2306985, OR = 0.48 (0.35-0.67). The population contribution risk for both SNPs is 23%, and a quarter of the population with at least one “G” allele in either of these SNPs is protected from PVD.

FIGS. 1A-1F (collectively referred to herein as “FIG. 1”) show SEQ ID NOs: 1-92 with SNPs shown in parentheses within each sequence. Each SNP allele associated with type 2 diabetes is shown in a separate column. 2A-2KK (collectively referred to herein as “FIG. 2”) show haplotypes associated with type 2 diabetes. 3A-3B (collectively referred to herein as “FIG. 3”) show how much the at-risk allele identified in FIG. 1 for each SNP is compared to type 2 diabetes based on the allele chi-square association test. Indicates whether relevant (significance at p ≦ 0.05). Figure 1 shows how the at-risk allele identified in Figure 1 for each SNP is associated with type 2 diabetes based on a chi-square test for recessive effects (significance at p <0.05). Figure 1 shows how at-risk alleles identified in Figure 1 for each SNP are associated with type 2 diabetes based on the genotype chi-square association test (significance at p <0.05). 6A-6B (collectively referred to herein as “FIG. 6”) is associated with type 2 diabetes using an allele association test, genotype association test, and / or chi-square test for recessive effects Provides a summary of SNPs found.

Claims (62)

  A method for determining susceptibility to type 2 diabetes in an individual comprising a sequence identified by SEQ ID NO: 1-92 and a complement of the sequence identified by SEQ ID NO: 1-92 Detecting a SNP at-risk allele associated with type 2 diabetes located within the selected sequence.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 23 or the complement of SEQ ID NO: 23.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 32 or the complement of SEQ ID NO: 32.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 35 or the complement of SEQ ID NO: 35.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 40 or the complement of SEQ ID NO: 40.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 41 or the complement of SEQ ID NO: 41.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 17 or the complement of SEQ ID NO: 17.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 36 or the complement of SEQ ID NO: 36.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 74 or the complement of SEQ ID NO: 74.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 62 or the complement of SEQ ID NO: 62.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 10 or the complement of SEQ ID NO: 10.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 11 or the complement of SEQ ID NO: 11.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 8 or the complement of SEQ ID NO: 8.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 54 or the complement of SEQ ID NO: 54.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 65 or the complement of SEQ ID NO: 65.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 9 or the complement of SEQ ID NO: 9.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 67 or the complement of SEQ ID NO: 67.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 4 or the complement of SEQ ID NO: 4.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 91 or the complement of SEQ ID NO: 91.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 92 or the complement of SEQ ID NO: 92.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 68 or the complement of SEQ ID NO: 68.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 69 or the complement of SEQ ID NO: 69.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 7 or the complement of SEQ ID NO: 7.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 20 or the complement of SEQ ID NO: 20.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 16 or the complement of SEQ ID NO: 16.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 31 or the complement of SEQ ID NO: 31.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 42 or the complement of SEQ ID NO: 42.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 39 or the complement of SEQ ID NO: 39.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 53 or the complement of SEQ ID NO: 53.   2. The method of claim 1, wherein the SNP is located within SEQ ID NO: 38 or the complement of SEQ ID NO: 38.   An isolated polynucleotide comprising a SNP located within a sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92.   A method for assaying for the presence of a first polynucleotide having a SNP associated with type 2 diabetes or a tendency to develop type 2 diabetes in a sample, the sequences and sequences identified by SEQ ID NOs: 1-92 Contacting a sample with a second polynucleotide comprising a nucleotide sequence selected from the group consisting of the complement of the sequences identified by Nos. 1-92 and hybridizing to the first polynucleotide under stringent conditions The method including the process to make.   SNP located within a sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 operably linked to a regulatory sequence A vector comprising an isolated polynucleotide comprising   34. A recombinant host cell comprising the vector of claim 33.   Coded by an isolated polynucleotide having a SNP located within a sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 35. A method for producing a polypeptide to be produced; comprising culturing the recombinant host cell of claim 34 under conditions suitable for expression of said polynucleotide.   An isolated poly having an SNP located within a sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 in the sample A method of assaying for the presence of a polypeptide encoded by a nucleotide, comprising contacting a sample with an antibody that specifically binds to the encoded polypeptide.   A transgenic animal comprising a polynucleotide having a SNP located within a sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92. A method of identifying an agent that alters the expression of a polynucleotide comprising a SNP associated with type 2 diabetes, comprising the following steps:
(A) Inside a sequence selected from the group consisting of (1) a sequence identified by SEQ ID NO: 1-92 and a complement of the sequence identified by SEQ ID NO: 1-92 under conditions for expression (2) contacting a polynucleotide comprising a promoter region operably linked to a reporter gene with an agent to be tested;
(B) evaluating the level of reporter gene expression in the presence of the drug;
(C) evaluating the level of expression of the reporter gene in the absence of the drug; and (d) determining the level of expression in step (b) for the difference indicating that the expression has been altered by the drug, step (c) Comparing the level of expression in. At least partially complementary to a portion of a second polynucleotide comprising a sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 A method for assaying a sample for the presence of a first polynucleotide of interest comprising the following steps:
a) contacting the sample with a second polynucleotide under conditions suitable for hybridization; and
b) assessing whether hybridization has occurred between the first polynucleotide and the second polynucleotide;
The method wherein the first polynucleotide is present in the sample when hybridization occurs.   40. The method of claim 39, wherein the presence of the first polynucleotide indicates a tendency to develop type 2 diabetes or type 2 diabetes.   41. The method of claim 39 or 40, wherein the second polynucleotide is completely complementary to a portion of the sequence of the first polynucleotide.   42. The method of any one of claims 39-41, further comprising amplification of at least a portion of the first polynucleotide.   The second polynucleotide is 99 or less nucleotides in length and: (a) at least 80% identical to the sequence of consecutive nucleotides in the first polynucleotide; or (b) the first 43. The method of any one of claims 39 to 42, wherein the method is either capable of selectively hybridizing to a polynucleotide.   Regarding the presence of the first polynucleotide comprising SNP located within the sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 A reagent for assaying a sample, comprising a second polynucleotide comprising a contiguous nucleotide sequence that is at least partially complementary to a portion of the first polynucleotide.   45. The reagent of claim 44, wherein the second polynucleotide is completely complementary to a portion of the first polynucleotide. Regarding the presence of the first polynucleotide comprising SNP located within the sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 Reagent kit for assaying a sample comprising the following in separate containers:
a) one or more labeled second polys comprising a sequence selected from the group consisting of the sequence identified by SEQ ID NO: 1-92 and the complement of the sequence identified by SEQ ID NO: 1-92 Nucleotides; and
b) Reagents for label detection.   A method for diagnosing susceptibility to type 2 diabetes in an individual, comprising the step of detecting a haplotype associated with type 2 diabetes selected from the group consisting of the haplotypes shown in FIG.   48. The method of claim 47, wherein detecting the presence of a haplotype comprises enzymatic amplification of nucleic acid from the individual.   48. The method of claim 47, further comprising electrophoretic analysis in detecting the presence of a haplotype.   48. The method of claim 47, further comprising restriction fragment length polymorphism analysis in detecting the presence of a haplotype.   48. The method of claim 47, further comprising sequence analysis in detecting the presence of a haplotype. A method of diagnosing susceptibility to type 2 diabetes in an individual comprising the following steps:
a) obtaining a polynucleotide sample from the individual; and
b) analyzing the polynucleotide sample for the presence or absence of the haplotype, including the haplotype shown in FIG. 2, wherein the presence of the haplotype corresponds to susceptibility to type 2 diabetes.   A method for identifying a gene associated with type 2 diabetes, comprising: (a) selected from the group consisting of a sequence identified by SEQ ID NO: 1-92 and a complement of the sequence identified by SEQ ID NO: 1-92 Identifying a gene containing an SNP located within the sequence to be identified; and (b) expressing the expression of the gene in an individual having an at-risk allele for at-risk for differences indicating that the gene is associated with type 2 diabetes. Comparing the expression of said gene in an individual without an allele.   A method for identifying a suitable agent for treating type 2 diabetes comprising: (a) a sequence identified by SEQ ID NO: 1-92 and a complement of the sequence identified by SEQ ID NO: 1-92 Contacting the polynucleotide with an agent to be tested, comprising an SNP located within a sequence selected from the group; and (b) an approach that is believed to be useful for treating type 2 diabetes, Determining whether it binds to, alters or affects a polynucleotide.   A method for identifying a suitable agent for treating type 2 diabetes comprising: (a) a sequence identified by SEQ ID NO: 1-92 and a complement of the sequence identified by SEQ ID NO: 1-92 Contacting the polypeptide with an agent to be tested, encoded by a polynucleotide comprising an SNP located within a sequence selected from the group; and (b) considered useful for treating type 2 diabetes Determining whether the agent binds to the polypeptide, alters the polypeptide, or affects the polypeptide in such a manner.   56. An agent identified by the method of claim 55.   57. A pharmaceutical composition comprising the agent according to claim 56.   58. The pharmaceutical composition of claim 57, comprising a therapeutically effective amount of the drug.   39. An agent identified by the method of claim 38.   60. A pharmaceutical composition comprising the agent of claim 59.   61. The pharmaceutical composition of claim 60, comprising a therapeutically effective amount of the drug.   Methods, polynucleotides, vectors, recombinant host cells, transgenic animals, reagents, agents, and pharmaceutical compositions as hereinbefore described, particularly with reference to the examples.

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