WO2014134970A1 - Nouveau biomarqueur pour le diabète de type 2 - Google Patents

Nouveau biomarqueur pour le diabète de type 2 Download PDF

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Publication number
WO2014134970A1
WO2014134970A1 PCT/CN2014/000226 CN2014000226W WO2014134970A1 WO 2014134970 A1 WO2014134970 A1 WO 2014134970A1 CN 2014000226 W CN2014000226 W CN 2014000226W WO 2014134970 A1 WO2014134970 A1 WO 2014134970A1
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Prior art keywords
diabetes
sequence
type
subject
risk
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PCT/CN2014/000226
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English (en)
Inventor
Ronald Ching-Wan MA
Wing-Yee So
Juliana Chung-Ngor CHAN
Cheng Hu
Rong Zhang
Weiping JIA
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The Chinese University Of Hong Kong
Hospital Authority
Shanghai Jiaotong University Affiliated Sixth People's Hospital
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Publication of WO2014134970A1 publication Critical patent/WO2014134970A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • Type 2 diabetes is a common complex disease characterised by deficient insulin secretion and decreased insulin sensitivity.
  • T2D Type 2 diabetes
  • 285 million people worldwide were affected by type 2 diabetes (Shaw et al, (2010) Diabetes Res Clin Pract 87: 4-14), with 60% of them located in Asia (Chan et al. (2009) JAMA 301 : 2129-2140; Ramachandran et al. (2010) Lancet 375: 408-418).
  • the present invention provides a method for assessing the presence or risk of type 2 diabetes (T2D) or cardiovascular disease in a subject.
  • the method includes these steps: (a) performing an assay that determines nucleotide sequence of at least a portion of the PAX4-SND1 genomic sequence that is present in a biological sample taken from the subject, and (b) comparing the sequence determined in step (a) with a standard sequence of the corresponding PAX4-SND1 genomic sequence, wherein a variation in the sequence determined in step (a) when compared with the standard sequence indicates the presence or risk of type 2 diabetes or cardiovascular disease in the subject.
  • the sample is a blood or saliva sample.
  • the subject is of Asian descent, such as a Chinese, Korean, Japanese, especially a Han Chinese.
  • the subject has a family history of type 2 diabetes but has not been diagnosed of type 2 diabetes, while in other cases, the subject has no family history and has not been diagnosed of type 2 diabetes.
  • the method is particular effective in detecting or assessing the risk of developing cardiovascular disease in a subject, who has already been diagnosed with type 2 diabetes. After performing steps (a) and (b) as described above, a sequence variation indicates the presence or risk of developing cardiovascular disease in the subject.
  • the assay in step (a) may comprise an amplification reaction, such as a polymerase chain reaction (PCR); or the assay in step (a) may comprise mass spectrometry.
  • amplification reaction such as a polymerase chain reaction (PCR)
  • mass spectrometry a polymerase chain reaction
  • sequence variants include a polymorphism rs7801 1 1 , rs806187, rsl 40971 , rs806179, rs806178, rs7781 189, or rs806176.
  • the method of this invention is not limited to be used with just one sequence variation. In some cases, especially after a sequence variation is detected in the portion of PAX4-SND1 genomic sequence after steps (a) and (b), a further step may be taken to detect a second sequence variation in a second genomic sequence, e.g. , one that is different from the PAX4-SND1 genomic sequence, or one is in another portion of the PAX4-SND1 genomic sequence.
  • Two or more such additional sequence variants can be used for this purpose.
  • the diagnosis of presence or risk of type 2 diabetes or cardiovascular disease is further supported.
  • One example of the second sequence variation is a polymorphism rs2737250, located near TRPS1. Additional examples can be found in Tables 2, 6, 7, and 8.
  • one or more treatment steps should be taken.
  • a physician may prescribe administering to the subject a cholesterol lowering drug or a blood glucose lowering drug.
  • the subject once indicated as at risk of developing type 2 diabetes or cardiovascular disease according to the methods described above, may receive one or more further steps of monitoring for any of these conditions on a regular basis, utilizing physical examination tools, laboratory tests and application of various scanning and/or scoping technologies to image high risk anatomical areas. Preventive steps may also be taken such as changing dietary habits, increasing physical activity level, etc.
  • the present invention provides a kit for assessing the presence or risk of T2D or cardiovascular disease in a subject.
  • the kit includes two oligonucleotide primers for specifically amplifying: (1) at least a segment of the PAX4-SND1 genomic sequence; or (2) complement of (1), in an amplification reaction.
  • Such an amplification reaction may be a polymerase chain reaction (PCR), such as RT-PCR.
  • the kit may include an agent that can differentially indicate a sequence variation within the genomic sequence following its amplification, e.g., an oligonucleotide probe that specifically binds to one version of the genomic sequence but not to other versions.
  • the kit typically further includes an instruction manual. BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 Summary of study design. CHB, Han Chinese in Beijing, China; JPT, Japanese in Tokyo, Japan.
  • Figure 2 Manhattan plot of combined genome-wide association results from the Hong Kong 1, Hong Kong 2 and Shanghai studies based on the random effect models.
  • the j-axis represents the -logio p value
  • the x-axis represents the 2,925,090 analysed SNPs.
  • the dotted line indicates the threshold of significance p ⁇ x 10 5 .
  • There are 44 points with p ⁇ x 10 ⁇ 5 and the arrows and labels localise the susceptibility loci to type 2 diabetes uncovered in the present study.
  • Figure 3 Regional plots for the identified variant rsl0229583, including results for both genotyped and imputed SNPs in the Chinese population.
  • the top positioned circle and diamond represent the sentinel SNP in meta-analysis of three GWAS in the stage 1 and the East Asian meta-analysis in stages 1+2+3, respectively.
  • Other SNPs are coloured according to their level of LD, which is measured by r 2 , with the sentinel SNP.
  • the recombination rates estimated from the 1000 Genomes project JPT+CHB data are shown. CHB, Han Chinese in Beijing, China; JPT, Japanese in Tokyo, Japan.
  • Figure 4 Forest plot for meta-analysis of the association between type 2 diabetes and rs 10229583 for all populations in the present study. ORs and 95% CIs were reported with respect to the type 2 diabetes-related risk alleles (G).
  • FIG. 6 Multidimensional scaling analysis (MDS) plot showing the first two principal components, based on genotype data of 1 1 populations from HapMap (African ancestry in Southwest USA (ASW), Utah residents with Northern and Western European ancestry from the CEPH collection (CEU), Han Chinese in Beijing, China (CHB), Chinese in Metropolitan Denver, Colorado (CHD), kanni Indians in Houston, Texas (GIH), Japanese in Tokyo, Japan (JPT), Luhya in Webuye, Kenya (LWK), Mexican ancestry in Los Angeles, California (MEX), Maasai in Kinyawa, Kenya (MKK), Samsung in Italy (TSI) and Yoruban in Ibadan, Nigeria (YRI)), as well as the 3 case-controls cohorts (Hong Kong GWAS 1 (HK1), Hong Kong GWAS 2 (HK2) and Shanghai GWAS (SH)) in the stage 1 genome scan of the present study.
  • FIG. 7 Multidimensional scaling analysis (MDS) plot shows the first two principal components, based on genotype data of 3 case-controls cohorts (Hong Kong GWAS 1 , Hong Kong GWAS 2 and Shanghai GWAS) in the stage 1 genome scan of the present study without HapMap scaling.
  • MDS Multidimensional scaling analysis
  • Figure 8 Q-Q plot for combined genome-wide association results in a total of 684 T2D patients and 955 controls based on the 2,925,090 analyzed SNPs.
  • the curvy lines above and below the straight diagonal lines represent the upper and lower boundaries of the 95% confidence bands.
  • Figure 9 Distribution of ENCODE open chromatin sites around the identified gene region (NCBI Build 36.1/hgl8 CHR7:127033000-127084685) annotated in the UCSC human genome browser on human (website: genome.ucsc.edu/).
  • the distribution of open chromatin across the identified gene region in pancreatic islets (depicted as peaks) is highlighted inside the box (labeled Panlsle FAIRE FD and highlighted by red arrow).
  • the position of rs 10229583 and other tagging SNPs in high LD to this lead SNP are marked by the black arrows at the top of the figure.
  • Figure 10 Comparison of varLD scores within 100Kb region centred on index SNP rsl0229583 between pairs of populations using HapMap phase III CHB, JPT, CEU and YRI data.
  • Figure 11 Linkage disequilibrium for SNPs within the region near PAX4 on chromosome 7 between 126.95 Mb and 127.06 Mb (Build 36). Pairwise r 2 among SNPs for HapMap CEU and CHB are indicated in upper and lower block, respectively. Shades of grey represent the strength of pairwise r 2 . Rsl0229583 and rs6467136 are the SNPs showing significant association with T2D in the present study and the study conducted by the East Asian Consortium, respectively. DEFINITIONS
  • Type 2 diabetes refers to a metabolic disorder that is characterized by high blood glucose in the context of varying combinations of insulin resistance and insulin deficiency.
  • Type 2 diabetes may be caused by a combination of lifestyle and genetic factors. Diabetes can be caused by distinct clinical entities such as endocrine disorders (e.g., Cushing's syndrome) and chronic pancreatitis.
  • type 2 diabetes often include polyuria (frequent urination), polydipsia (increased thirst), polyphagia (increased hunger), fatigue, and weight loss.
  • the abnormal neurohormonal and metabolic milieu characterized by hyperglycemia, dyslipidemia and low grade inflammation can trigger a cascade of signaling pathways, which can lead to cell death and dysregulated cell growth, giving rise to multiple morbidities including heart disease, strokes, limb amputation, visual loss, kidney failure, cancers, and cognitive impairment.
  • cardiovascular disease refers to a broad class of diseases that involve the heart or blood vessels (arteries and veins) and affect the cardiovascular system, such as conditions related to atherosclerosis (arterial disease). These include but not limited to stroke, coronary heart disease and peripheral vascular disease.
  • Known risk factors for cardiovascular diseases include unhealthy eating, lack of exercise, obesity, suboptimally managed diabetes, abnormal blood lipids, high blood pressure, excessive consumption of alcohol, use of tobacco, as well as genetic background.
  • diabetic cardiovascular disease specifically refers to a cardiovascular disease that is associated with or secondary to diabetes.
  • a BMI of 20 to 25 kg/m 2 is considered optimal weight; a BMI lower than 20 kg/m 2 suggests the person is underweight whereas a BMI above 25 kg/m 2 may indicate the person is overweight; a BMI above 30 kg/m suggests the person is obese; and a BMI over 40 kg/m indicates the person to be morbidly obese.
  • Asians have more body fat for the same degree of BMI and waist circumference.
  • Asians are defined as ⁇ 23 kg/m and >25 kg/m respectively. While high BMI may predict risk for diabetes or prediabetes, people with low BMI, which correlates with beta cell function, are also at high risk, especially if these subjects develop central obesity, which tends to be associated with insulin resistance or reduced insulin sensitivity.
  • biological sample includes any section of tissue or bodily fluid taken from a test subject such as a biopsy and autopsy sample, and frozen section taken for histologic purposes, or processed forms of any of such samples.
  • Biological samples include blood and blood fractions or products (e.g., serum, plasma, platelets, white blood cells, red blood cells, and the like), sputum or saliva, lymph and tongue tissue, cultured cells, e.g. , primary cultures, explants, and transformed cells, stool, urine, stomach biopsy tissue etc.
  • a biological sample is typically obtained from a eukaryotic organism, which may be a mammal, may be a primate and may be a human subject.
  • biopsy refers to the process of removing a tissue sample for diagnostic or prognostic evaluation, and to the tissue specimen itself. Any biopsy technique known in the art can be applied to the methods of the present invention. The biopsy technique applied will depend on the tissue type to be evaluated (e.g., tongue, colon, prostate, kidney, bladder, lymph node, liver, bone marrow, blood cell, stomach tissue, etc.) among other factors. Representative biopsy techniques include, but are not limited to, excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy, and bone marrow biopsy and may comprise endoscopy such as colonoscopy. A wide range of biopsy techniques are well known to those skilled in the art who will choose between them and implement them with minimal experimentation.
  • isolated nucleic acid molecule means a nucleic acid molecule that is separated from other nucleic acid molecules that are usually associated with the isolated nucleic acid molecule.
  • an "isolated" nucleic acid molecule includes, without limitation, a nucleic acid molecule that is free of nucleotide sequences that naturally flank one or both ends of the nucleic acid in the genome of the organism from which the isolated nucleic acid is derived (e.g., a cDNA or genomic DNA fragment produced by a polymerase chain reaction or restriction endonuclease digestion).
  • an isolated nucleic acid molecule is generally introduced into a vector (e.g., a cloning vector or an expression vector) for convenience of manipulation or to generate a fusion nucleic acid molecule.
  • an isolated nucleic acid molecule can include an engineered nucleic acid molecule such as a recombinant or a synthetic nucleic acid molecule.
  • nucleic acid or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form.
  • nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide
  • SNPs polymorphisms
  • complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et ah, Nucleic Acid Res. 19:5081 (1991);
  • nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • gene means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) involved in the transcription and/or translation of the gene product and the regulation of the transcription and/or translation, as well as intervening sequences (introns) between individual coding segments (exons).
  • polypeptide polypeptide
  • peptide protein
  • protein protein
  • amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may include those having non-naturally occurring D-chirality, as disclosed in WO01/12654, which may improve the stability (e.g., half-life), bioavailability, and other characteristics of a polypeptide comprising one or more of such D-amino acids. In some cases, one or more, and potentially all of the amino acids of a therapeutic polypeptide have D-chirality.
  • Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical
  • immunoglobulin or "antibody” (used interchangeably herein) refers to an antigen-binding protein having a basic four-polypeptide chain structure consisting of two heavy and two light chains, said chains being stabilized, for example, by interchain disulfide bonds, which has the ability to specifically bind antigen. Both heavy and light chains are folded into domains.
  • antibody also refers to antigen- and epitope-binding fragments of antibodies, e.g., Fab fragments, that can be used in immunological affinity assays.
  • Fab fragments antigen- and epitope-binding fragments of antibodies
  • pepsin digests an antibody C-terminal to the disulfide linkages in the hinge region to produce F(ab)' 2 , a dimer of Fab which itself is a light chain joined to V R -C R I by a disulfide bond.
  • the F(ab)' 2 can be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab') 2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially a Fab with part of the hinge region (see, e.g., Fundamental Immunology, Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that fragments can be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody also includes antibody fragments either produced by the modification of whole antibodies or synthesized using recombinant DNA methodologies.
  • the specified binding agent e.g., an antibody
  • Specific binding of an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein or a protein but not its similar "sister" proteins.
  • immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein or in a particular form.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immuno reactivity).
  • a specific or selective binding reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
  • the term “specifically bind” when used in the context of referring to a polynucleotide sequence forming a double-stranded complex with another polynucleotide sequence describes "polynucleotide hybridization” based on the Watson-Crick base-pairing, as provided in the definition for the term “polynucleotide hybridization method.”
  • a "polynucleotide hybridization method" as used herein refers to a method for detecting the presence and/or quantity of a pre-determined polynucleotide sequence based on its ability to form Watson-Crick base-pairing, under appropriate hybridization conditions, with a polynucleotide probe of a known sequence. Examples of such hybridization methods include Southern blot, Northern blot, and in situ hybridization.
  • Primers refer to oligonucleotides that can be used in an amplification method, such as a polymerase chain reaction (PCR), to amplify a nucleotide sequence based on the polynucleotide sequence corresponding to a gene of interest, e.g., the cDNA or human genomic sequence PAX4-SND1 or a portion thereof.
  • PCR polymerase chain reaction
  • at least one of the PCR primers for amplification of a polynucleotide sequence is sequence-specific for that polynucleotide sequence. The exact length of the primer will depend upon many factors, including temperature, source of the primer, and the method used.
  • the oligonucleotide primer typically contains at least 10, or 15, or 20, or 25 or more nucleotides, although it may contain fewer nucleotides or more nucleotides.
  • the factors involved in determining the appropriate length of primer are readily known to one of ordinary skill in the art.
  • primer pair means a pair of primers that hybridize to opposite strands a target DNA molecule or to regions of the target DNA which flank a nucleotide sequence to be amplified.
  • the term "primer site” means the area of the target DNA or other nucleic acid to which a primer hybridizes.
  • a "label,” “detectable label,” or “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes ⁇ e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins that can be made detectable, e.g., by incorporating a radioactive component into the peptide or used to detect antibodies specifically reactive with the peptide.
  • a detectable label is attached to a probe or a molecule with defined binding characteristics ⁇ e.g., a polypeptide with a known binding specificity or a polynucleotide), so as to allow the presence of the probe (and therefore its binding target) to be readily detectable.
  • binding characteristics e.g., a polypeptide with a known binding specificity or a polynucleotide
  • a "standard sequence” as used herein refers to the polynucleotide sequence of a predetermined genomic DNA segment, e.g., a defined portion or the entire length of a human genomic sequence of a given range and location, such as the human PAX4-SND1 genomic sequence, including 2 kb upstream and 2 kb downstream flanking sequences, that is present in a publically accessible database, e.g., the University of California Santa Cruz database (hgl 8), as the standard human genomic sequence for these particular genes.
  • a genomic DNA sequence determined from a test sample is compared with a "standard sequence,” the test sequence is aligned with the "standard sequence” at the corresponding nucleotide bases of the genomic sequence to reveal any sequence variation.
  • the standard genomic sequences for human PAX4 and SND1 genes are provided as below:
  • PAX4 (paired box 4, chr7: 127250346-127255982, hgl9) is the closest gene to the SNP rsl0229583 (chr7: 127246903, hgl9).
  • the PAX4 gene encodes 4 protein-coding isoforms (Tablel). Because the PAX4 gene is at the reverse (-) strand, rsl0229583 is about 3.4 kb downstream to the most 3 ' exon of the PAX4 gene.
  • PAX4-001 ENST00000341640. 2010 ENSP00000339906, 343 9 127250346 127255780
  • PAX4-002 ENST00000463946 ⁇ 613 ENSP00000451923 341 8 127250992 127255724
  • PAX4-004 ENST00000378740 I 08S ENSP00000368014 34S ⁇ 127250865 127255982
  • the term "amount” as used in this application refers to the quantity of a polynucleotide of interest or a polypeptide of interest present in a sample. Such quantity may be expressed in the absolute terms, i.e. , the total quantity of the polynucleotide or polypeptide in the sample, or in the relative terms, i.e. , the concentration of the polynucleotide or polypeptide in the sample.
  • an effective amount of a cholesterol lowering drug or a blood glucose lowering drug is the amount of said drug to achieve a decreased level of cholesterol or blood glucose, respectively, in a patient who has been given the drug for therapeutic purposes.
  • An amount adequate to accomplish this is defined as the "therapeutically effective dose.”
  • the dosing range varies with the nature of the therapeutic agent being administered and other factors such as the route of administration and the severity of a patient's condition.
  • subject or “subject in need of treatment,” as used herein, includes individuals who seek medical attention due to risk of, or actual suffering from type 2 diabetes or cardiovascular/renal disease associated with diabetes. Subjects also include individuals currently undergoing therapy that seek manipulation of the therapeutic regimen. Subjects or individuals in need of treatment include those that demonstrate symptoms of type 2 diabetes or related cardiovascular/renal disease, or are at risk of suffering from type 2 diabetes or diabetic cardiovascular/renal disease or related symptoms.
  • a subject in need of treatment includes individuals with a genetic predisposition or family history for type 2 diabetes or diabetic cardiovascular/renal disease, those who have suffered relevant symptoms in the past, those who have been exposed to a triggering substance or event, as well as those suffering from chronic or acute symptoms of the condition.
  • a "subject in need of treatment” may be at any age of life.
  • the present inventors performed studies to identify new type 2 diabetes susceptibility loci in Southern Han Chinese individuals. A meta-analysis was performed of three GWAS comprising 684 patients with type 2 diabetes and 955 controls, and analysed 2.9 million
  • SNPs single-nucleotide polymorphisms
  • Putatively associated SNPs (p ⁇ l > ⁇ 10 ⁇ 5 ) were genotyped de novo in two independent Southern Han Chinese cohorts (10,383 cases and 6,974 controls), and SNPs reaching a genome- wide significance of /? ⁇ 5x 10 " were replicated in silico in five East Asian and three non-East Asian populations for a total of 31,541 cases and 60,344 controls.
  • the inventors discovered for the first time the correlation between genomic sequence variation in the human PAX4-SND1 genomic sequence and medical conditions such as type 2 diabetes and diabetic cardiovascular and renal diseases in human subjects.
  • This discovery allows medical professionals to identify subjects at risk cardiovascular or renal disease in a patient with type 2 diabetes or assess the risk of developing type 2 diabetes and diabetic cardiovascular and/or renal disease in a subject at risk by studying the subject's PAX4-SND1 genomic sequence and then comparing the subject's sequence with a standard PAX4-SND1 genomic sequence that has been determined as a part of the standard human genome. Detection of such sequence variation(s) indicates the presence or elevated risk of developing type 2 diabetes or diabetic cardiovascular and/or renal disease in the subject, as well as the early onset of these conditions.
  • the detection of pertinent genomic sequence variation(s) can further guide physicians to devise or modify treatment plans for a subject in both prevention and therapeutic measures.
  • type 2 diabetes patients have an increased risk of coronary heart disease (CHD) when they possess the genomic sequence variation in the human PAX4-SND1 genomic sequence at 7q32.
  • CHD coronary heart disease
  • a recent genome -wide association study in the Chinese populations identified association between a novel variant at 7q32 near paired box 4 (PAX4) and T2D, which was confirmed in other East Asian populations. This study aimed to investigate the association of this novel 7q32 variant and CHD risk in an 8- year prospective cohort of Chinese patients with T2D.
  • nucleic acids sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences.
  • kb kilobases
  • proteins sizes are given in kilodaltons (kDa) or amino acid residue numbers. Protein sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.
  • Oligonucleotides that are not commercially available can be chemically synthesized, e.g. , according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Lett.
  • oligonucleotides are synthesized using any art-recognized strategy, e.g., native acrylamide gel electrophoresis or anion-exchange high performance liquid chromatography (HPLC) as described in Pearson and Reanier, J. Chrom. 255: 137-149 (1983).
  • HPLC high performance liquid chromatography
  • sequence of interest used in this invention e.g., the polynucleotide sequence of the human PAX4 and SND1 genes, and synthetic oligonucleotides (e.g., primers) can be verified using, e.g., the chain termination method for sequencing double-stranded templates of Wallace et al., Gene 16: 21-26 (1981).
  • sequence of interest used in this invention e.g., the polynucleotide sequence of the human PAX4 and SND1 genes, and synthetic oligonucleotides (e.g., primers) can be verified using, e.g., the chain termination method for sequencing double-stranded templates of Wallace et al., Gene 16: 21-26 (1981).
  • the present invention relates to determining at least a portion of the genomic sequence of a pertinent region, such as the human PAX4-SND1 segment and/or its transcript(s), found in a biological sample taken from a person being tested, as a means to detect the presence and/or to assess the risk of developing type 2 diabetes, or cardiovascular/renal diseases in that person.
  • the first steps of practicing this invention are to obtain a biological sample (e.g., tissue or bodily fluid sample) from a test subject and extract genomic DNA or RNA from the sample.
  • a biological sample is obtained from a person to be tested or assessed for risk of developing type 2 diabetes or associated cardiovascular or renal disease using a method of the present invention. Collection of a tissue or fluid sample from an individual is performed in accordance with the standard protocol laboratories, hospitals or clinics generally follow, such as during a biopsy, blood drawing, saliva collection, or oral swab. An appropriate amount of sample is collected and may be stored according to standard procedures prior to further preparation.
  • genomic DNA found in a subject's sample may be performed using essentially any tissue or bodily fluid, so long as genomic DNA is expected to be present in such sample.
  • the methods for preparing tissue or fluid samples for nucleic acid extraction are well known among those of skill in the art. For example, a subject's epithelial tissue sample should be first treated to disrupt cellular membrane so as to release nucleic acids contained within the cells.
  • Possible sequence variation within a segment of a pertinent genomic sequence is investigated to provide indication as to whether a test subject is suffering from type 2 diabetes and associated cardiovascular or renal disease, or whether the subject is at risk of developing type 2 diabetes and associated complications including cardiovascular or renal disease in the future.
  • a segment of the genomic sequence of an appropriate length is selected for sequencing analysis.
  • the segment may be chosen from the genomic sequence of a pertinent gene or genes defined by the same boundaries defining the gene's genomic sequence, plus about 2,000 base pairs upstream and downstream from the boundaries.
  • the human PAX4- SND1 genomic sequence will encompass the upstream boundary of the upstream gene, PAX4, to the downstream boundary of the downstream gene, SNDl, plus 2000 bp upstream and downstream from the upstream and downstream boundaries, respectively.
  • the PAX4-SND1 genomic sequence may be as long as: 2000 bp genomic sequence immediately upstream from the PAX4 genomic sequence + the PAX4 genomic sequence + the genomic sequence between the PAX4 and SNDl genomic sequences + the SNDl genomic sequence + 2000 bp genomic sequence immediately downstream from the SNDl genomic sequence.
  • the length of the genomic sequence being analyzed may be a segment of the above and is usually at least 15 or 20 contiguous nucleotides, and may be longer with at least 25, 30, 50, 100, 200, 300, 400, or more contiguous nucleotides.
  • RNA contamination should be eliminated to avoid interference with DNA analysis.
  • other components such as proteins and lipids may be removed from the biological sample prior to further analysis of the genomic DNA.
  • RNA RNA sequence-based analysis such that the genomic sequence of one or more of the pertinent genes, or one or more of its transcripts, found in a test subject may be determined and then compared with a standard sequence to detect any possible sequence variation.
  • An amplification reaction is optional prior to the sequence analysis.
  • a variety of polynucleotide amplification methods are well established and frequently used in research. For instance, the general methods of polymerase chain reaction (PCR) for polynucleotide sequence amplification are well known in the art and are thus not described in detail herein.
  • PCR reagents and protocols are also available from commercial vendors, such as Roche Molecular Systems.
  • PCR amplification is typically used in practicing the present invention, one of skill in the art will recognize that amplification of the relevant genomic sequence may be accomplished by any known method, such as the ligase chain reaction (LCR), transcription- mediated amplification, and self-sustained sequence replication or nucleic acid sequence-based amplification ( ASBA), each of which provides sufficient amplification.
  • LCR ligase chain reaction
  • ASBA nucleic acid sequence-based amplification
  • Additional means suitable for determining the polynucleotide sequence of a genomic DNA include but are not limited to mass spectrometry, primer extension, polynucleotide hybridization, real-time PCR, melting curve analysis, high resolution melting analysis, heteroduplex analysis, pyrosequencing, and electrophoresis.
  • genomic DNA sequence variations may also be detected by way of analyzing RNA sequences transcribed from the pertinent DNA sequences, which may include portion of the coding sequence or non-coding sequence of a genomic locus of interest (e.g., the PAX4-SND1 genomic sequence).
  • Methods for RNA extraction from a biological sample, sequence analysis of RNA or DNA molecules, optionally involving amplification techniques such as reverse transcription based amplification processes, e.g., RT-PCR, are well known in the art.
  • Suitable samples for RNA sequence analysis may include peripheral blood monocytes (PBMC) and specific tissue samples such as fat and muscles.
  • PBMC peripheral blood monocytes
  • specific tissue samples such as fat and muscles.
  • the standard genomic sequence(s) for one or more pertinent genes such as the human PAX4 and SND1 genes and their isoforms, will be chosen before the comparison with a test subject's genomic sequence of the corresponding gene at the corresponding location may be performed.
  • genomic sequence variation in one or more of the specific genes named above and the presence or heightened risk of developing type 2 diabetes or cardiovascular and renal diseases among subjects having such variation, especially those fitting certain profiles, such as those of Asian descent, in particular Han Chinese
  • the present inventors have provided a valuable tool for clinicians to determine, often in combination with other information and diagnostic or predictive or screening test results, how a subject having certain genomic sequence variation(s) should be monitored and/or treated for type 2 diabetes and diabetic cardiovascular or renal disease such that the symptoms of these conditions may be prevented, eliminated, ameliorated, reduced in severity and/or frequency, or delayed in their onset.
  • a physician may arrange for regular monitoring of various symptoms of type 2 diabetes or diabetic cardiovascular and renal diseases in a subject who has been deemed by the method of the present invention to have an elevated risk of developing type 2 diabetes.
  • the physician may also prescribe both pharmacological and non-pharmacological treatments such as lifestyle modification ⁇ e.g., reduce body weight by 5%, high fiber diet, walking for at least 150 minutes weekly) and medicines known to reduce risk of onset of diabetes (e.g., metformin, alpha glucosidase inhibitors, lipase inhibitors) to a subject who has been deemed by the method of the present invention to have an elevated risk of developing type 2 diabetes.
  • lifestyle modification e.g., reduce body weight by 5%, high fiber diet, walking for at least 150 minutes weekly
  • medicines known to reduce risk of onset of diabetes e.g., metformin, alpha glucosidase inhibitors, lipase inhibitors
  • the attending physician may prescribe medications to control risk factors such as high levels of blood cholesterol and triglycerol (e.g., statins and fibrates) and reduce angiotensin II activity (e.g., Angiotensin converting enzyme inhibitor (ACEI) and angiotensin II receptor blocker (ARB)), as well as place the subject under regular testing and monitoring of coronary artery condition and kidney function.
  • risk factors such as high levels of blood cholesterol and triglycerol (e.g., statins and fibrates) and reduce angiotensin II activity (e.g., Angiotensin converting enzyme inhibitor (ACEI) and angiotensin II receptor blocker (ARB)
  • ACEI Angiotensin converting enzyme inhibitor
  • ARB angiotensin II receptor blocker
  • the present invention provides compositions and kits for practicing the methods described herein to detect possible genomic sequence variation of certain gene(s) and the transcripts thereof in a subject, which can be used for various purposes such as detecting or diagnosing the presence of type 2 diabetes and diabetic cardiovascular or renal disease in a subject, determining the risk of developing type 2 diabetes and diabetic cardiovascular or renal disease in a subject, and guiding the treatment plan for these conditions in the subject.
  • Kits for carrying out assays for determining the nucleotide sequence of a relevant genomic sequence typically include at least one oligonucleotide useful for specific hybridization with a predetermined segment of a pertinent genomic sequence (e.g., human PAX4-SND1 genomic sequence).
  • this oligonucleotide is labeled with a detectable moiety.
  • the oligonucleotide specifically hybridizes with the standard sequence only but not with any of the variant sequences.
  • the oligonucleotide specifically hybridizes with one particular version of the variant sequence but not with other versions, nor with the standard sequence.
  • kits may include at least two oligonucleotide primers that can be used in the amplification of at least one segment of a pertient genomic sequence (such as the human PAX4-SND1 genomic sequence) or transcripts thereof by PCR.
  • at least one of the oligonucleotide primers is designed to anneal only to the standard sequence or only to a particular version of the variant sequences, for example, the G allele of rs 10229583.
  • kits of this invention may provide instruction manuals ⁇ e.g., internet- based decision support tools) to guide users in analyzing test samples and assessing the presence or future risk of type 2 diabetes and diabetic cardiovascular or renal disease in a test subject.
  • instruction manuals e.g., internet- based decision support tools
  • the present invention can also be embodied in a device or a system comprising one or more such devices, which is capable of carrying out all or some of the method steps described herein.
  • the device or system performs the following steps upon receiving a biological sample taken from a subject being tested for detecting type 2 diabetes or diabetic cardiovascular or renal disease, assessing the risk of developing type 2 diabetes or diabetic cardiovascular or renal disease, or guiding treatment of a subject having or at risk of developing any one of these conditions: (a) determining in the sample the nucleotide sequence of a pertinent genomic DNA segment or its transcript; (b) comparing the sequence determined from the sample with a corresponding standard sequence; and (c) providing an output indicating whether type 2 diabetes or diabetic cardiovascular/renal disease is present in the subject or whether the subject is at risk of developing type 2 diabetes or diabetic
  • the device or system of the invention performs the task of steps (b) and (c), after step (a) has been performed and the genomic sequence determined from (a) has been entered into the device.
  • the device or system is partially or fully automated.
  • GWAS Genome -wide association studies
  • the present inventors performed a meta-analysis of three GWAS comprising 684 patients with type 2 diabetes and 955 controls of Southern Han Chinese descent.
  • the inventors followed up the top signals in two independent Southern Han Chinese cohorts (totalling 10,383 cases and 6,974 controls) and performed in silico replication in multiple populations. They identified CDKN2A/B and four novel type 2 diabetes association signals with p ⁇ 1 x 10 "5 from the meta-analysis. Thirteen loci within these four loci were followed up in two independent Chinese cohorts, and rs 10229583 at 7q32 was found to be associated with type 2 diabetes in a combined analysis of 1 1,067 cases and 7,929 controls l0 "8; OR [95% CI] 1.18 [1.11 , 1.25]). In silico replication revealed consistent associations across multiethnic groups, including five East
  • the rs 10229583 risk variant was associated with elevated fasting plasma glucose, impaired beta cell function in controls, and an earlier age at diagnosis for the cases.
  • the novel variant lies within an islet-selective cluster of open regulatory elements. There was significant heterogeneity of effect between Han Chinese and individuals of European descent, Malaysians and Indians. rs 10229583 near PAX4 is identified as a novel locus for type 2 diabetes in Chinese and other populations and provides new insights into the pathogenesis of type 2 diabetes.
  • stage 1 Participants In the first-stage discovery cohort (stage 1), genome-wide scanning was performed in three different case-control samples: 198 Hong Kong Chinese individuals (99 patients with type 2 diabetes and 99 healthy controls) in Hong Kong GWAS 1 , 1 ,047 Hong Kong Chinese individuals (388 with type 2 diabetes and 659 controls) in Hong Kong GWAS 2 and 394 Shanghai Chinese (197 patients with type 2 diabetes and 197 normal controls) in the Shanghai GWAS.
  • Individuals included in the stage 2 replication included 5,366 with type 2 diabetes and 2,474 controls from Hong Kong, and 4,035 cases and 3,964 controls from Shanghai. 325 cases and 368 controls from 178 Hong Kong families were also included, as well as 657 cases and 168 controls from 248 Shanghai families.
  • Table 4 shows the quality control for the participants in stage 1.
  • SNPs were excluded from further analysis if: (1) /? ⁇ l x l0 ⁇ 4 for HWE; (2) minor allele frequency (MAF) was ⁇ 1%; (3) call rate was ⁇ 95%; in particular, SNPs with MAF>1 % but ⁇ 5% were excluded if their call rate was ⁇ 99%; or (4) the SNPs showed a significant difference in MAF (p ⁇ x 1(T 4 ) between the Hong Kong control cohorts with other conditions (450 with epilepsy cases, 1 10 with eczema and 99 non-hypertensive individuals). Only SNPs that passed the quality control criteria for both cases and controls were used for further analysis. Table 5 shows the quality control of the genotyping results in stage 1. Genotypes were imputed for autosomal SNPs according to the 1000 Genomes reference panel. See the ESM Methods for further details.
  • MassARRAY platform (Sequenom; San Diego, CA, USA). Family samples were genotyped using TaqMan SNP Genotyping Assays (Applied Biosystems, Foster City, CA, USA) or by direct sequencing.
  • GWAMA software website: well.ox.ac.uk/gwama/
  • Magi and Morris BMC Bioinformatics 2010, 1 1 :288 was used to calculate the combined estimates of the ORs (95% CIs) from multiple groups by weighting the natural log-transformed ORs of each study using the inverse of their variance under the random effect model (DerSimonian and Laird (1986) Control Clin Trials 7: 177-188).
  • the random effect model SNPs with some degree of heterogeneity between studies were excluded, which helped to attenuate the number of false-positive findings in this study.
  • ALR alternating logistic regressions
  • bioinformatics and czs-expression quantitative trait loci (eQTL) analysis was performed for functional implication of the identified SNP. See the ESM Methods for additional information on methods, including adjustment for genomic control and the gene network analysis.
  • Meta-analysis of patients with Chinese ancestry A summary of the study design and the clinical characteristics of the participants in all stages are shown in Fig. 1 and Table 1.
  • stage 1 684 patients with type 2 diabetes and 955 controls were genotyped. No population stratification were detected between case and control individuals in multidimensional scaling analysis for all GWAS (Fig. 7).
  • Meta-analysis was implemented to combine the individual association results for 2,925,090 imputed and genotyped SNPs (under additive genetic models) available in all three GWAS using the inverse-variance approach for random effect models.
  • stage 1 meta-analysis of three Chinese GWAS 44 SNPs within five loci were prioritised for follow-up (Fig. 2 and Table 8). No substantial change was observed in the stage 1 results after adjusting either for ⁇ . s (1.01— 1.04 in individual cohorts) or the first principle component in the meta-analysis, reflecting that the results were not likely to be due to population stratification (Fig. 8 and Table 9).
  • CDKN2A/B has previously been reported to be strongly associated with type 2 diabetes.
  • two SNPs in CDKN2A/B showing strong signals for type 2 diabetes in the present study were in high LD (r 2 ⁇ 0.8) with rs 1081 1661 , which is well-replicated in most populations.
  • 13 top and proxy SNPs among the remaining 42 SNPs in four regions were taked forward to stage 2, de novo replication, in two independent Chinese case-control cohorts (Table 6).
  • Genotypes were successfully obtained for 1 1 SNPs in Hong Kong replication 1 cohort with 5,366 cases and 2,474 controls, and Shanghai replication 1 cohort with 4,035 cases and 3,964 controls to proceed for subsequent analysis (Table 7).
  • rsl0229583 and rs2737250 located on chromosomes 7 and 8, respectively, gave p ⁇ 4.5x l0 " (threshold of significance after Bonferroni correction) with the same directions of association as the original signals (Table 2).
  • These two SNPs were genotyped in 1 ,518 additional samples from 426 families of Han Chinese descent (325 cases and 368 controls from 178 Hong Kong families, and 657 cases and 168 controls from 248 Shanghai families).
  • FPG fasting plasma glucose
  • variant and its tagging SNPs lie within an area near PAX4 and SND1 , which is enriched with DNase I hypersenstitive sites, histone H3 lysine modifications and CCCTC factor binding in human islets (Fig. 9) (Stitzel et al. (2010) Cell Metab 12: 443-455).
  • eQTL data were only available for PAX4 in adipose tissue, but not LCLs or skin, for which no expression data were available from MuTHER. There was a nominal association (p ⁇ 0.05) between the variant and expression of C7orf54 and ARF5 in LCLs, and C7orf68 in adipose tissue.
  • the r 2 between the GWAS SNP and the peak eQTL SNPs ranged between 0.56 and 1.
  • Type 2 diabetes in Asians is characterised by an earlier AAD, strong family history and evidence of impaired beta cell function (Chan et al. (2009) JAMA 301 : 2129-2140; and
  • the novel locus for type 2 diabetes identified, rsl 0229583, is located downstream of the ARF5 and PAX4 genes in 7q32, and upstream of SND1.
  • PAX4 a member of the paired box family of transcription factors, plays a critical role in pancreatic beta cell formation during fetal development (Bran et al. (2004) J Cell Biol 167: 1 123-1 135; Li et al. (2006) Leuk Res 30: 1547- 1553) and is therefore a very strong candidate for the implicated gene.
  • PAX4 is expressed in early pancreatic endocrine cells, but expression is later restricted to beta cells and it is not expressed in mature pancreas (Habener et al. (2005) Endocrinology 146: 1025-1034). In pancreatic endocrine cells, PAX4 represses ghrelin and glucagon expression, and can induce the expression of PDX1, a key transcription factor for islet development. Targeted disruption of PAX4 in mice was found to lead to reduced beta cell mass at birth (Wang et al. (2004) Dev Biol 266: 178-189).
  • a risk variant at HNF4a has been found to be associated with increased risk of type 2 diabetes, and carriers of the risk allele have impaired beta cell function (Silander et al. (2004) Diabetes 53: 1 141-1 149).
  • the MAF of the R121W PAX4 mutation was 1 % in Asians, and the mutation is in low LD with rs 10229583. It is possible that both rare mutations and common variation within the same gene confer risk towards type 2 diabetes independently.
  • the common variant here identified, rsl0229583 may be associated with altered gene expression, while the other rare non-synonymous mutations lead to impaired gene function.
  • the recent East Asian meta-analysis comprising eight type 2 diabetes GWAS identified a locus on chromosome 7 near GRIP and GCC1-PAX4 to be associated with type 2 diabetes.
  • the protein encoded by GCC1 may play a role in transmembrane transport (Luke et al. (2005) Biochem J 388: 835-841).
  • the variant identified from the East Asian study, rs6467136, appears to be independent of our signal, with r 2 0.044 in our Chinese samples (Fig. 1 1).
  • ARF5 belongs to a family of guanine nucleotide -binding proteins that have been shown to play a role in vesicular trafficking and as activators of phospholipase D (Lebeda and Haun (1999) Gene 237: 209-214). Islet expression of ARF5 was found to be induced threefold in rats receiving a high-carbohydrate diet (Song et al. (2001) Diabetes 50: 2053-2060). The nearby SND1 gene, also known as the plOO transcription co- activator, is a member of the micronuclease family and plays a key role in transcription and splicing. The pi 00 transcriptional co-activator is present in endocrine cells and tissues, including the pancreas of cattle (Broadhurst et al. (2005) Biochim Biophys Acta 1681 : 126-133).
  • PAX4 mutations were first identified in Asian MODY probands (Shimajiri et al. (2001) Diabetes 50: 2864-2869; Tokuyama et al. (2006) Metabolism 55: 213- 216), but seldom found in those of European descent (Dupont et al. (1999) Diabetologia 42: 480- 484; Dusatkova et al. (2010) Diabet Med 27: 1459-1460). This suggests that PAX4, like KCNQl, may be particularly relevant for the pathogenesis of type 2 diabetes in East Asians individuals.
  • rs 10229583 is also in strong LD with a region spanning the neighbouring SND1 gene (Fig. 3). Further resequencing and transethnic mapping should help to identify the causal gene variant for type 2 diabetes within this region.
  • rs 10229583 near PAX4 has been identified as a novel locus for type 2 diabetes in Chinese and other populations, providing new insights into the pathogenesis of type 2 diabetes.
  • Example 2 Sequence Variation Near PAX4 Linked to Early Onset of Coronary Heart Disease Among Type 2 Diabetes Patients
  • Type 2 diabetes (T2D) patients have a 2-4 fold increased risk of coronary heart disease (CHD) compared with the general population.
  • CHD coronary heart disease
  • a recent genome-wide association study conducted by the present inventors in the Chinese populations identified association between a sequence variant at 7q32 near paired box 4 (PAX4) and T2D, which was confirmed in other East Asian populations. See details in Example 1.
  • Type 2 diabetes was diagnosed according to the 1998 World Health Organization (WHO) criteria. Patients with classic type 1 diabetes with acute ketotic presentation or continuous requirement of insulin within 1 year of diagnosis were excluded. Written informed consent was obtained from all participants. This study was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong.
  • WHO World Health Organization
  • stage 1 genome -wide scanning was performed in 202 Hong Kong Chinese individuals (102 type 2 diabetes patients and 100 healthy controls) (Hong Kong GWAS 1 cohort).
  • 102 type 2 diabetes cases were selected with young-onset diabetes diagnosed at age ⁇ 40 years, positive family history and overweight, and 100 controls were selected using the criteria of 1) no past diagnostic history of type 2 diabetes, impaired fasting glucose (IFG) or impaired glucose tolerance (IGT); 2) without family history of type 2 diabetes; and 3) with BMI ⁇ 25 kg/m 2 and waist circumference ⁇ 90 cm and 80 cm for men and women, respectively.
  • IGF impaired fasting glucose
  • ITT impaired glucose tolerance
  • Hong Kong Chinese individuals were genome-scanned (400 type 2 diabetes patients from the Hong Kong Diabetes Registry and 668 non-diabetic controls) (Hong Kong GWAS 2 cohort).
  • the 668 diseased controls were individuals aged > 16 years old with diseases other than type 2 diabetes that included 457 epilepsy cases, 11 1 eczema cases and 100 healthy individuals without hypertension (recruited from the control arm of a hypertension study).
  • the genome -wide scan was performed in 394 samples, including 197 type 2 diabetes patients and 197 normal glucose regulation controls.
  • the type 2 diabetes patients were probands of diabetic pedigrees with fasting plasma glucose > 7.0 mmol/L and/or 2-h post plasma glucose > 1 1.1 mmol/L who were diagnosed before 40 years old.
  • Type 1 diabetes and mitochondrial diabetes were excluded based on clinical, immunological and genetic criteria.
  • the controls were individuals with normal glucose regulation with fasting plasma glucose ⁇ 6.1 mmol/L and 2-h plasma glucose ⁇ 7.8 mmol/L as assessed by standard 75g OGTTs, negative diabetic family history, aged over 50 years old and with a BMI below 23kg/m 2 .
  • HOMA-IR homeostasis model assessment of beta-cell function
  • ⁇ - ⁇ homeostasis model assessment of beta-cell function
  • Stumvoll indices for beta-cell function were calculated for Shanghai controls which underwent OGTT with measurement of insulin levels (Stumvoll et al. (2000) Diabetes Care 23: 295-301).
  • the case cohort consisted of 5,366 unrelated type 2 diabetes patients (mean age 56.7 ⁇ 13.4 years, 45.1% male, mean duration of T2D 6.6 ⁇ 6.9 years) selected from the Hong Kong Diabetes Registry (HKDR).
  • the control cohort consisted of 2474 individuals ascertained from 3 sources: a) 985 adolescents from a community-based school survey of cardiovascular risk factors (mean age 15.5 ⁇ 1.9 years, 44.2% male) (Ng et al. (2010) J Clin Endocrinol Metab 95: 2418- 2425); b) 513 hospital staff and adult volunteers participating in a community-based health screening program (mean age 42.0 ⁇ 10.4 years, 47% male) (Ng et al.
  • SHDS I and II Shanghai Diabetes Study (SHDS) I and II, which are community-based surveys of diabetes performed in 1998-2001 (SHDS I) and 2007-2008 (SHDS II).
  • the controls had fasting plasma glucose ⁇ 6.1 mmol/L and 2-h plasma glucose ⁇ 7.8 mmol/L as assessed by standard 75g OGTTs, and had no family history of diabetes mellitus.
  • Type 2 diabetes cases were selected from individuals registered as having type 2 diabetes. Diabetes was originally diagnosed according to the World Health Organization (WHO) criteria, type 2 diabetes was clinically defined as disease with a gradual adult onset. Individuals who tested positive for antibodies to glutamic acid decarboxylase (GAD) and those diagnosed with a mitochondrial disease or MODY were not included in the case group.
  • WHO World Health Organization
  • Controls were individuals registered as individuals not having type 2 diabetes but with diseases other than type 2 diabetes, comprised of 13 distinct diseases, or healthy volunteers. Individuals who had been analyzed in the previous report (Yamauchi et al., 2010 Nat Genet 42(10):864-868) were excluded from the present study. Altogether, 4,878 individuals with type 2 diabetes (case 1 , age, 65.8 ⁇ 10.0 years; BMI, 24.1 ⁇ 3.8 kg/m2; (all values are expressed as mean ⁇ s.d.)) and
  • 3,345controls (control 1 , age, 52.5 ⁇ 15.2 years; BMI, 22.5 ⁇ 3.8 kg/m2; (all values are expressed as mean ⁇ s.d.)) were genotyped. A total of 7,541 individuals belonging to the Hondo cluster (4,470 cases and 3,071 controls) were selected. Samples were directly genotyped using Illurnina HurnanHap610-Quad (type 2 diabetes patients) and 550K BeadChip (controls).
  • nondiabetic control individuals were as follows: (1) no history of diabetes and (2) fasting plasma glucose ⁇ 5.6 mmol/L and plasma glucose 2-h after ingestion of 75gm oral glucose load ⁇ 7.8 mmol/L at both baseline and follow up studies.
  • the Singapore case-control study contained individuals from three sources: 1) 1998 Singapore National Health Survey (NHS98); 2) Singapore Malay Eye Study (SiMES); and 3) Singapore Diabetes Cohorts Study (SDCS) (Tan et ah (2010) J Clin Endocrinol Metab 95: 390- 397).
  • NHS98 United States National Health Survey
  • SiMES Singapore Malay Eye Study
  • SDCS Singapore Diabetes Cohorts Study
  • FPG fasting plasma glucose
  • 2HPG 2 hour post-challenge glucose
  • Type 2 diabetic cases were identified as those with previously diagnosed type 2 diabetes and current use of antidiabetic treatment or who meet the following criteria: 1) 30 ⁇ age ⁇ 70, 2) fasting plasma glucose >7.0 mmol/1, 3) 2-h postprandial plasma glucose >1 1.1 mmol/1 in a standard 75 g oral glucose tolerance test (OGTT) or plasma HbAlc >6.5%.
  • the nondiabetic controls were selected according to the following criteria: 1) age >30, 2) no past history of diagnosis of diabetes and no family history of diabetes, 3) fasting glucose ⁇ 5.6 mmol/1, 4) 2-h OGTT ⁇ 7.8 mmol/1 and/or HbAl c content ⁇ 5.6%.
  • the studies were approved by local ethnic committees of each participating institution, and written informed consents were obtained from all participants.
  • the DNA samples were genotyped using the Illumina Human660W-Quad BeadChip (Illumina, Inc., San Diego, CA, USA), and the genotypes were called using the Illumina GenCall algorithm. Some of samples were excluded if their genotype call rates ⁇ 97%, excessive heterozygosity, gender mismatches between the reported and genetically inferred gender or duplicates among other samples. Principle component analysis was used to assess population structure of the samples and detected outliers along the first two eigenvectors which were excluded from further analyses. SNPs with genotype call rate ⁇ 95%, MAF ⁇ 0.5% or deviation from Hardy- Weinberg equilibrium (p ⁇ 10 "6 ) in control groups were also excluded.
  • DIAGRAM+ study comprised 8,130 type 2 diabetes cases and 38,987 controls from eight type 2 diabetes GWAS of European descent, including the Wellcome Trust Case Control Consortium (WTCCC), Diabetes Genetics Initiative (DGI) and Finland-US Investigation of NIDDM genetics (FUSION) scans (the individuals of a previous joint analysis), with those from scans performed by deCODE genetics, the Diabetes Gene Discovery Group, the Cooperative Health Research in the Region of Augsburg group (KORAgen), the Rotterdam study and the European Special Population Research Network (EUROSPAN) (for details of sample characteristics, see Supplementary Table 2 in Voight et ah (2010) Nat Genet 42: 579-589). Additional information on methods
  • the SNP ID (rs number) was standardized according to dbSNP build 129, and their physical positions were standardized according to build 36. SNPs were further excluded sequentially if: 1) their polymorphisms were A T or C/G; 2) absent from dbSNP build 129; 3) genotyped in only case or only control cohorts; 4) absent from the 1000 Genomes reference panel for CHB+JPT (March 2010 release of pilot project 1). For each sample set in stage 1 , all SNPs were aligned to the positive strand and imputed (via the MLE approach) using the MACH 1.0 software (Li et al. (2010) Genet Epidemiol 34: 816-834). Genotypes were imputed for autosomal SNPs that were present in the March 2010 release of phased 1000
  • Genomes genotype data from 60 CHB+JPT founders (Nature 467: 1061-1073, 2010), but were not present in the genome -wide chip or did not pass direct genotyping QC. Cases and controls were merged into a single cohort for imputation based on 440,194, 435,953 and 274,752 quality autosomal SNPs in Hong Kong GWAS 1, Hong Kong GWAS 2 and Shanghai GWAS case- control cohorts, respectively.
  • For the Hong Kong GWAS 1 cohort one-step imputation was applied.
  • two-step imputation was used to improve imputation efficiency, by randomly selecting 100 cases and 100 controls for model parameter estimation first before imputation.
  • Genomic control was applied to correct for relatedness of the individuals and adjust for potential population stratification (Devlin and Roeder (1999) Biometrics 55: 997- 1004).
  • the inflation factor ⁇ was estimated by taking the median of the distribution of the ⁇ 2 statistic from all quality SNPs in association test, and then divide by the median of the expected ⁇ 2 distribution.
  • the p values were calculated corrected for genomic control by dividing the observed ⁇ statistic by ⁇ . In this study, they were adjusted for GC in two levels. Firstly, each individual study was corrected for ⁇ separately in directly genotyped and imputed SNPs. Then they were further adjusted for GC on the meta-analysis results. eQTL analysis
  • MuTHER resource website: muther.ac.uk
  • Log 2 transformed expression signals were normalized separately per tissue as follows: quantile normalization was performed across technical replicates of each individual followed by quantile normalization across all individuals.
  • Genotyping was done with a combination of Illumina arrays (HumanHap300, HumanHap610Q, lMDuo and 1.2MDuo). Untyped HapMap2 SNPs were imputed using the IMPUTE software package (v2). The number of adipose samples with genotypes and expression values is per tissue was 778 for LCLs, 667 in skin and 776 in adipose.
  • the eQTL data were also examined for SNPs which showed nominal association (p ⁇ 0.05) with the expression of nearby genes in the different tissues from the MuTHER dataset.
  • the LD between the MuTHER eQTL peaks within the dataset and rsl 0229583 were then examined.
  • GenCord project is the study of association analysis for eQTL to nearby SNPs in three cell types (primary fibroblasts, lymphoblastoid cells and T cells) from the umbilical cords of 75 individuals (Dimas et al. (2009) Science 325: 1246- 1250).
  • the Java based application is the study of association analysis for eQTL to nearby SNPs in three cell types (primary fibroblasts, lymphoblastoid cells and T cells) from the umbilical cords of 75 individuals (Dimas et al. (2009) Science 325: 1246- 1250).
  • Genevar website: sanger.ac.uk/resources/software/genevar/
  • Genevar was used to retrieve the relevant association results of our variant and expression of genes within 1MB of the SNP from the GenCord project.
  • the LD between the variant and the eQTL peak within the dataset was also examined.
  • SNAP website: broadinstitute.org/mpg/snap/ldsearch.php. Selection criteria were as follows: (1) 1000 Genome Pilot 1 ; (2) r 2 limit > 0.8; (3) Population Panel: CHBJPT; and (4) Distance
  • GeneMania (Warde-Farley et ah (2010) Nucleic Acids Res 38: W214-220). Data was updated by GeneMania as of Feb 2012. The weight on each edge, as represented by the thickness of the edge, was computed by GeneMania and reflects the degree of confidence of the relationships between any gene pair within the network. Genes that are identified from the MuTHER eQTL analysis above were entered as prior knowledge, and were used to guide the gene-network building. Pathway information was obtained from Pathway Commons (Cerami et al. (201 1) Nucleic Acids Res 39: D685-690) and genetic interactions were obtained from BioGrid (Stark et al. (201 1) Nucleic Acids Res 39: D698-704).
  • T2D patient 99 (40.4) 40.6 ⁇ 8.8 31.8 ⁇ 7.7 8.0 ⁇ 8.3 30.9 ⁇ 4.4 -
  • HK2 Diseased control 659 (48.7) 37.1 ⁇ 17.0 - - 23.3 ⁇ 3.7 -
  • T2D patient 388 (49.5) 60.6 ⁇ 10.8 51.1 ⁇ 12.1 9.5 ⁇ 7.0 25.0 ⁇ 3.8 -
  • T2D patient 197 (57.9) 41.6 ⁇ 10.4 34.5 ⁇ 4.8 7.3 ⁇ 8.5 23.8 ⁇ 4.1 -
  • T2D patient 4035 (52.0) 61.2 ⁇ 12.1 54.2 ⁇ 11.3 7.2 ⁇ 6.9 24.5 ⁇ 3.5 -
  • T2D patient 325 (40.6) 48.0 ⁇ 14.4 41.7 ⁇ 13.1 6.3 ⁇ 7.6 25.9 ⁇ 4.4 -
  • T2D patient 657 (43.7) 54.6 ⁇ 15.6 50.0 ⁇ 14.2 4.9 ⁇ 7.3 23.9 ⁇ 3.5 -
  • T2D patient 4465 (68.0) 65.8 ⁇ 10.0 56.5 ⁇ 11.4 9.4 ⁇ 8.4 24.1 ⁇ 3.8
  • T2D patient 1042 (51.7) 56.4 ⁇ 8.6 25.5 ⁇ 3.3 7.0 ⁇ 2.6 Korean 2 Control 1305 (54.5) 65.2 ⁇ 2.6 23.9 ⁇ 3.0 5.0 ⁇ 0.5
  • T2D patient 1082 (37.2) 65.1 ⁇ 9.7 55.7 ⁇ 12.0 - 25.3 ⁇ 3.9 -
  • T2D patient 928 (64.9) 63.7 ⁇ 10.8 52.2 ⁇ 14.4 25.4 ⁇ 3.8
  • T2D patient 794 (51.0) 62.3 ⁇ 9.90 54.4 ⁇ 11.2 - 27.8 ⁇ 4.9 -
  • T2D type 2 diabetes
  • P, Pmeta and p het represent p values from logistic regression without any adjustment under the additive genetic model, meta-analysis under a fixed effect model
  • Asians Korean replication None 1,042 2,943 0.894 0.878 1.17 (0.99, 1.38) 0.0577
  • Phet refers to the p value obtained from the heterogeneity test
  • Step 1 Monomorphic SNPs 58,440 57,680 0 53,422 194 51 ,746 25,397 24,237
  • Step 2 SNPs with 100% missing genotype 78 78 0 112 0 0 2 0
  • Step 3 SNPs with MAF ⁇ 5% and their call rate
  • Step 4 SNPs with MAF > 5% and their call rate
  • Step 5 SNPs with overall MAF ⁇ 1% 9,656 9,967 81 26,447 1 ,311 27,618 236 214
  • Step 6 SNPs without HWE (P ⁇ 1 10 ⁇ 4 ) 463 458 54 598 19 575 12,281 12,486
  • Step 7 SNPs with different MAF between
  • HKl 99 T2D vs 99 controls
  • HK2 388 T2D vs 659 controls
  • SHGWA 197 T2D vs 197 controls
  • HK1 99 T2D vs 99 controls
  • HK2 388 T2D vs 659 controls
  • SHGWA 197 T2D vs 197 controls
  • Non- gene(s) risk (95% CI) (95% CI) (95% CI) (95% CI) (uncorrect)
  • P unadjusted and P adjusted represent P values calculated from linear regression with and without adjustmen for sex, BMI and Hb a i c (where appropriate) under the additive genetic model.
  • P values were obtained from met analysis of two Chinese T2D case cohorts (Hong Kong and Shanghai) under a fixed effect model.
  • Q test P and I 2 refer to the statistical significance and quantified index of heterogeneity test of OR between Chinese and other populations, respectively.
  • Monte Carlo and ⁇ test P refer to the P values testing for the regional LD variation and allelic difference between CHB and other populations, respectively.
  • CHB Han Chinese in Beijing, China
  • JPT Japanese in Tokyo, Japan
  • CEU Utah residents with Northern and Western European ancestry from the CEPH collection
  • YRI Yoruban in Ibadan, Nigeria.

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Abstract

La présente invention concerne un procédé pour estimer la présence et le risque de développer le diabète de type 2 et une maladie cardiovasculaire chez un sujet par la détection d'une variation de séquence dans la séquence génomique localisée entre les gènes PAX4 et SND1 dans 7q32. L'invention concerne également une trousse et un dispositif utiles pour un tel procédé. De plus, la présente invention concerne une méthode de traitement du diabète de type 2 chez des patients qui ont été testés et montrés avoir la ou les variations génétiques pertinentes.
PCT/CN2014/000226 2013-03-08 2014-03-10 Nouveau biomarqueur pour le diabète de type 2 WO2014134970A1 (fr)

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WO2019237209A1 (fr) * 2018-06-15 2019-12-19 Opti-Thera Inc. Scores de risque polygéniques pour prédire des complications de maladie et/ou une réponse à une thérapie
CN111584080A (zh) * 2020-04-18 2020-08-25 中国医学科学院北京协和医院 一种区分mody与t1d和t2d的诊断模型

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019237209A1 (fr) * 2018-06-15 2019-12-19 Opti-Thera Inc. Scores de risque polygéniques pour prédire des complications de maladie et/ou une réponse à une thérapie
CN112513295A (zh) * 2018-06-15 2021-03-16 Opti 西拉公司 用于预测疾病并发症和/或对治疗的反应的多基因风险评分
EP3807883A4 (fr) * 2018-06-15 2022-03-23 Opti-Thera Inc. Scores de risque polygéniques pour prédire des complications de maladie et/ou une réponse à une thérapie
CN111584080A (zh) * 2020-04-18 2020-08-25 中国医学科学院北京协和医院 一种区分mody与t1d和t2d的诊断模型
CN111584080B (zh) * 2020-04-18 2022-07-26 中国医学科学院北京协和医院 一种区分mody与t1d和t2d的诊断试剂盒和诊断系统

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