EP2102368A1 - Gène btbd9 de susceptibilité au diabète humain - Google Patents

Gène btbd9 de susceptibilité au diabète humain

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Publication number
EP2102368A1
EP2102368A1 EP08701566A EP08701566A EP2102368A1 EP 2102368 A1 EP2102368 A1 EP 2102368A1 EP 08701566 A EP08701566 A EP 08701566A EP 08701566 A EP08701566 A EP 08701566A EP 2102368 A1 EP2102368 A1 EP 2102368A1
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EP
European Patent Office
Prior art keywords
diabetes
btbd9
gene
type
subject
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP08701566A
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German (de)
English (en)
Inventor
Anne Philippi
Jörg Hager
Francis Rousseau
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IntegraGen SA
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IntegraGen SA
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Publication date
Application filed by IntegraGen SA filed Critical IntegraGen SA
Publication of EP2102368A1 publication Critical patent/EP2102368A1/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
    • C12Q2600/158—Expression markers
    • 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
    • C12Q2600/172—Haplotypes

Definitions

  • the present invention relates to a method for determining a predisposition to diabetes in patients, preferably in patients with obesity.
  • type 1 diabetes a malignant neoplasm originating from type 2 diabetes
  • type 2 diabetes other specific types
  • gestational diabetes mellitus ADA, 2003.
  • type 2 diabetes a malignant neoplasm originating from type 2 diabetes
  • ADA gestational diabetes mellitus
  • over 80% of cases of Diabetes are due to type 2 diabetes, 5 to 10 % to type 1 diabetes, and the remainder to other specific causes.
  • Type 1 diabetes formerly known as insulin-dependent
  • the pancreas fails to produce the insulin which is essential for survival. This form develops most frequently in children and adolescents, but is being increasingly diagnosed later in life.
  • Type 2 diabetes mellitus formerly known as non-insulin dependent diabetes mellitus (NIDDM), or adult onset Diabetes, is the most common form of diabetes, accounting for approximately 90-95% of all diabetes cases.
  • Type 2 diabetes is characterized by insulin resistance of peripheral tissues, especially muscle and liver, and primary or secondary insufficiency of insulin secretion from pancreatic beta-cells.
  • Type 2 diabetes is defined by abnormally increased blood glucose levels and diagnosed if the fasting blood glucose level is superior to 126 mg/dl (7.0 mmol/1) or blood glucose levels are superior to 200 mg/dl (11.0 mmol/1) 2 hours after an oral glucose uptake of 75g (oral glucose tolerance test, OGTT).
  • Pre-diabetic states with already abnormal glucose values are defined as fasting hyperglycemia (FH) >6.1 mmol/1 and ⁇ 7.0 mmol/1 or impaired glucose tolerance (IGT) >7.75 mmol/1 and ⁇ 11.0 mmol/1 2 hours after an OGTT.
  • FH fasting hyperglycemia
  • ITT impaired glucose tolerance
  • Table 1 Classification of Type 2 diabetes (WHO, 2006)
  • the number of people with type 2 diabetes will expectedly more than double over the next 25 years, to reach a total of 366 million by 2030 (WHO/IDF, 2006). Most of this increase will occur as a result of a 150% rise in developing countries. In the US 7% of the general population are considered diabetic (over 15 million diabetics and an estimated 15 million people with impaired glucose tolerance).
  • diabetes forms associated with monogenetic defects in beta cell function are frequently characterized by onset of hyperglycemia at an early age (generally before age 25 years). They are referred to as maturity-onset diabetes of the Young (MODY) and are characterized by impaired insulin secretion with minimal or no defects in insulin action (Herman WH et al, 1994; Clement K et all, 1996; Byrne MM et all, 1996).
  • HNF hepatocyte nuclear factor
  • Glucokinase converts glucose to glucose-6-phosphase, the metabolism of which, in turn, stimulates insulin secretion by the beta cell. Because of defects in the glucokinase gene, increased plasma levels of glucose are necessary to elicit normal levels of insulin secretion.
  • a third form is associated with a mutation in the HnfMa gene on chromosome 2Oq (Bell GI et all, 1991; Yamagata K et all, 1996).
  • HNF-4 ⁇ is a transcription factor involved in the regulation of the expression of HNF-4 ⁇ .
  • Point mutations in mitochondrial DNA can cause diabetes mellitus primarily by impairing pancreatic beta cell function (Reardon W et all, 1992; VanDen Ouwenland JMW et all, 1992; Kadowaki T et all, 1994). There are unusual causes of diabetes that result from genetically determined abnormalities of insulin action.
  • the metabolic abnormalities associated with mutation of the insulin receptor may range from hyperinsulinemia and modest hyperglycemia to severe diabetes (Kahn CR et all, 1976; Taylor SI, 1992).
  • Type 2 diabetes is a major risk factor for serious micro- and macro-vascular complications. The two major diabetic complications are cardiovascular disease, culminating in myocardial infarction.
  • Diabetic retinopathy is an important cause of blindness, and occurs as a result of long-term accumulated damage to the small blood vessels in the retina. After 15 years of diabetes, approximately 2% of people become blind, and about 10% develop severe visual impairment. Diabetic neuropathy is damage to the nerves as a result of diabetes, and affects up to 50% of all diabetics. Although many different problems can occur as a result of diabetic neuropathy, common symptoms are tingling, pain, numbness, or weakness in the feet and hands. Combined with reduced blood flow, neuropathy in the feet increases the risk of foot ulcers and eventual limb amputation.
  • Obesity is associated with insulin resistance and therefore a major risk factor for the development of type 2 diabetes.
  • Obesity is defined as a condition of abnormal or excessive accumulation of adipose tissue, to the extent that health may be impaired.
  • the body mass index (BMI; kg/m 2 ) provides the most useful, albeit crude, population-level measure of obesity.
  • Obesity has also been defined using the WHO classification of the different weight classes for adults.
  • Table 2 Classification of overweight in adults according to BMI (WHO, 2006)
  • Type 2 diabetes is treated either by oral application of anti-glycemic molecules or insulin injection.
  • the oral antidiabetics either increase insulin secretion from the pancreatic beta- cells or that reduce the effects of the peripheral insulin resistance. Multiple rounds of differing treatments before an efficient treatment is found significantly decreases the compliance rates in diabetic patients.
  • the present invention now discloses the identification of a diabetes susceptibility gene.
  • the invention thus provides a diagnostic method of determining whether a subject is at risk of developing type 2 diabetes, which method comprises detecting the presence of an alteration in the BTBD9 gene locus in a biological sample of said subject.
  • the invention pertains to single nucleotide polymorphisms in the BTBD9 gene on chromosome 6 associated with type 2 diabetes and body weight.
  • the subject to test is affected with obesity.
  • the subject shows a body mass index (BMI; kg/m 2 ) of at least 27, preferably of at least 30.
  • Figure 1 High density mapping using Genomic Hybrid Identity Profiling (GenomeHIP). Graphical presentation of the linkage peak on chromosome 6p21.32-p21.1. The curve depict the linkage results for the GenomeHip procedure in the region. A total of 8 Bac clones on human chromosome 6 ranging from position p-ter-33315824 to 43294973-cen were tested for linkage using GenomeHip. Each point on the x-axis corresponds to a clone. Significant evidence for linkage was calculated for clone BACA23ZF11 (p-value 2.8E-08). The whole linkage region encompasses a region from 36 721 951 base pairs to 41255967 base pairs on human chromosome 6. The p-value less to 2x10 " corresponding to the significance level for significant linkage was used as a significance level for whole genome screens as proposed by Lander and Kruglyak (1995).
  • the present invention discloses the identification of BTBD9 as a diabetes susceptibility gene in individuals with type 2 diabetes. More specifically the invention pertains to individuals with both type 2 diabetes and a BMI > 27 kg/m .
  • Various nucleic acid samples from diabetes families were submitted to a particular GenomeHIP process. This process led to the identification of particular identical-by-descent (IBD) fragments in said populations that are altered in diabetic subjects with a BMI > 27 kg/m .
  • IBD identical-by-descent
  • Type 2 diabetes is characterized by chronic hyperglycemia caused by pancreatic insulin secretion deficiency and/or insulin resistance of peripheral insulin sensitive tissues (e.g. muscle, liver). Long term hyperglycemia has been shown to lead to serious damage to various tissue including nerves tissue and blood vessels.
  • Type 2 diabetes accounts for 90 % all diabetes mellitus cases around the world (10% being type 1 diabetes characterized by the auto-immune destruction of the insulin producing pancreatic beta-cells). About 70-80% of type 2 diabetics are considered overweight or obese and the excess in body weight significantly contributes to the peripheral insulin resistance and constitutes a major independent risk factor for the development of type 2 diabetes. Obesity is generally assessed by calculating the body mass index (BMI; kg/m 2 ), as described above.
  • BMI body mass index
  • the invention described here pertains to a genetic risk factor for individuals to develop type 2 diabetes.
  • the invention describes increased risk for overweight individuals (BMI > 27 kg/m 2 ). More preferably the invention describes increased risk for overweight individuals (BMI > 30 kg/m 2 ).
  • the BTBD9 gene locus designates all BTBD9 sequences or products in a cell or organism, including BTBD9 coding sequences, BTBD9 non-coding sequences (e.g., introns), BTBD9 regulatory sequences controlling transcription and/or translation (e.g., promoter, enhancer, terminator, etc.), as well as all corresponding expression products, such as BTBD9 RNAs (e.g., mRNAs) and BTBD9 polypeptides (e.g., a pre -protein and a mature protein).
  • the BTBD9 gene locus also comprise surrounding sequences of the BTBD9 gene which include SNPs that are in linkage disequilibrium with SNPs located in the BTBD9 gene.
  • BBD9 gene designates the gene BTB (POZ) domain containing 9, as well as variants or fragments thereof, including alleles thereof (e.g., germline mutations) which are related to susceptibility to TYPE 2 DIABETES.
  • BTBD9 gene may also be referred to as RP3-322I12.1, FLJ32945, KIAA1880, MGC 120517, MGC 120519, MGC 120520, dJ322I12.1. It is located on chromosome 6 at position 6p21.2.
  • the cDNA sequence is shown as SEQ ID NO:1, and the protein as SEQ ID NO:2 (GenBank Source: BC101356 - BC101354; Unigene: Hs.l 16233).
  • the BTB (for BR-C, ttk and bab) or POZ (for Pox virus and Zinc finger) domain is present near the N-terminus of a fraction of zinc finger (pfam00096) proteins and in proteins that contain the pfamO1344 motif such as Kelch and a family of pox virus proteins.
  • the BTB/POZ domain mediates homomeric dimerisation and in some instances heteromeric dimerisation.
  • the structure of the dimerised PLZF BTB/POZ domain has been solved and consists of a tightly intertwined homodimer.
  • the central scaffolding of the protein is made up of a cluster of alpha-helices flanked by short beta-sheets at both the top and bottom of the molecule.
  • POZ domains from several zinc finger proteins have been shown to mediate transcriptional repression and to interact with components of histone deacetylase co- repressor complexes including N-CoR and SMRT.
  • the POZ or BTB domain is also known as BR-C/Ttk or ZiN.
  • gene shall be construed to include any type of coding nucleic acid, including genomic DNA (gDNA), complementary DNA (cDNA), synthetic or semi-synthetic DNA, as well as any form of corresponding RNA.
  • genomic DNA gDNA
  • cDNA complementary DNA
  • synthetic or semi-synthetic DNA as well as any form of corresponding RNA.
  • the BTBD9 variants include, for instance, naturally-occurring variants due to allelic variations between individuals (e.g., polymorphisms), mutated alleles related to diabetes, alternative splicing forms, etc.
  • the term variant also includes BTBD9 gene sequences from other sources or organisms.
  • Variants are preferably substantially homologous to SEQ ID No 1, i.e., exhibit a nucleotide sequence identity of at least about 65%, typically at least about 75%, preferably at least about 85%, more preferably at least about 95% with SEQ ID No 1.
  • Variants of a BTBD9 gene also include nucleic acid sequences, which hybridize to a sequence as defined above (or a complementary strand thereof) under stringent hybridization conditions.
  • Typical stringent hybridisation conditions include temperatures above 30° C, preferably above 35°C, more preferably in excess of 42°C, and/or salinity of less than about 500 mM, preferably less than 200 mM.
  • Hybridization conditions may be adjusted by the skilled person by modifying the temperature, salinity and/or the concentration of other reagents such as SDS, SSC, etc.
  • a fragment of a BTBD9 gene designates any portion of at least about 8 consecutive nucleotides of a sequence as disclosed above, preferably at least about 15, more preferably at least about 20 nucleotides, further preferably of at least 30 nucleotides. Fragments include all possible nucleotide lengths between 8 and 100 nucleotides, preferably between 15 and 100, more preferably between 20 and 100.
  • a BTBD9 polypeptide designates any protein or polypeptide encoded by a BTBD9 gene as disclosed above.
  • polypeptide refers to any molecule comprising a stretch of amino acids. This term includes molecules of various lengths, such as peptides and proteins.
  • the polypeptide may be modified, such as by glycosylations and/or acetylations and/or chemical reaction or coupling, and may contain one or several non-natural or synthetic amino acids.
  • a specific example of a BTBD9 polypeptide comprises all or part of SEQ ID No: 2.
  • the invention now provides diagnosis methods based on a monitoring of the BTBD9 gene locus in a subject.
  • diagnosis includes the detection, monitoring, dosing, comparison, etc., at various stages, including early, pre- symptomatic stages, and late stages, in adults or children.
  • Diagnosis typically includes the prognosis, the assessment of a predisposition or risk of development, the characterization of a subject to define most appropriate treatment (pharmacogenetics), etc.
  • the present invention provides diagnostic methods to determine whether a subject, more particularly an obese subject, is at risk of developing type 2 diabetes resulting from a mutation or a polymorphism in the BTBD9 gene locus. It is therefore provided a method of detecting the presence of or predisposition to type 2 diabetes in a subject, more particularly a subject with obesity, the method comprising detecting in a biological sample from the subject the presence of an alteration in the BTBD9 gene locus in said sample. The presence of said alteration is indicative of the presence or predisposition to type 2 diabetes.
  • said method comprises a preliminary step of providing a sample from a subject.
  • the presence of an alteration in the BTBD9 gene locus in said sample is detected through the genotyping of a sample.
  • said alteration is one or several SNP(s) or a haplotype of SNPs associated with type 2 diabetes. More preferably, said SNP associated with type 2 diabetes is as shown in Table 3A, i.e. said SNP is selected from the group consisting of SNP87, SNP88, SNP92, and SNP94. Other SNP(s), as listed in Table 3B, may be informative too.
  • Table3A - SNPs on BTBD9 gene associated with type 2 diabetes:
  • Table 3B - Additional SNPs on BTBD9 gene:
  • the SNP is allele T of SNP87.
  • said haplotype comprises or consists of several SNPs selected from the group consisting of SNP87, SNP88, SNP92, SNP94, more particularly the following haplotype: 2 - 1 - 1 - 1 (i.e. SNP87 is T, SNP88 is T, SNP92 is C and SNP94 is G).
  • the invention further provides a method for preventing type 2 diabetes in a subject, more particularly a subject with obesity, comprising detecting the presence of an alteration in the BTBD9 gene locus in a sample from the subject, the presence of said alteration being indicative of the predisposition to type 2 diabetes, and administering a prophylactic treatment against type 2 diabetes.
  • the alteration may be determined at the level of the BTBD9 gDNA, RNA or polypeptide.
  • the detection is performed by sequencing all or part of the BTBD9 gene or by selective hybridisation or amplification of all or part of the BTBD9 gene. More preferably a BTBD9 gene specific amplification is carried out before the alteration identification step.
  • An alteration in the BTBD9 gene locus may be any form of mutation(s), deletion(s), rearrangement(s) and/or insertion(s) in the coding and/or non-coding region of the locus, alone or in various combination(s). Mutations more specifically include point mutations. Deletions may encompass any region of two or more residues in a coding or non-coding portion of the gene locus, such as from two residues up to the entire gene or locus. Typical deletions affect smaller regions, such as domains (introns) or repeated sequences or fragments of less than about 50 consecutive base pairs, although larger deletions may occur as well. Insertions may encompass the addition of one or several residues in a coding or non-coding portion of the gene locus.
  • Insertions may typically comprise an addition of between 1 and 50 base pairs in the gene locus. Rearrangement includes inversion of sequences.
  • the BTBD9 gene locus alteration may result in the creation of stop codons, frameshift mutations, amino acid substitutions, particular RNA splicing or processing, product instability, truncated polypeptide production, etc.
  • the alteration may result in the production of a BTBD9 polypeptide with altered function, stability, targeting or structure.
  • the alteration may also cause a reduction in protein expression or, alternatively, an increase in said production.
  • the alteration in the BTBD9 gene locus is selected from a point mutation, a deletion and an insertion in the BTBD9 gene or corresponding expression product, more preferably a point mutation and a deletion.
  • one or several SNP in the BTBD9 gene and certain haplotypes comprising SNP in the BTBD9 gene can be used in combination with other SNP or haplotype associated with type 2 diabetes and located in other gene(s).
  • the method comprises detecting the presence of an altered BTBD9 RNA expression.
  • Altered RNA expression includes the presence of an altered RNA sequence, the presence of an altered RNA splicing or processing, the presence of an altered quantity of RNA, etc. These may be detected by various techniques known in the art, including by sequencing all or part of the BTBD9 RNA or by selective hybridisation or selective amplification of all or part of said RNA, for instance.
  • the method comprises detecting the presence of an altered BTBD9 polypeptide expression.
  • Altered BTBD9 polypeptide expression includes the presence of an altered polypeptide sequence, the presence of an altered quantity of BTBD9 polypeptide, the presence of an altered tissue distribution, etc. These may be detected by various techniques known in the art, including by sequencing and/or binding to specific ligands (such as antibodies), for instance.
  • BTBD9 gene or RNA expression or sequence
  • suitable methods include allele-specific oligonucleotide (ASO), allele-specific amplification, Southern blot (for DNAs), Northern blot (for RNAs), single-stranded conformation analysis (SSCA), PFGE, fluorescent in situ hybridization (FISH), gel migration, clamped denaturing gel electrophoresis, heteroduplex analysis, RNase protection, chemical mismatch cleavage, ELISA, radio-immunoassays (RIA) and immuno-enzymatic assays (IEMA).
  • ASO allele-specific oligonucleotide
  • Southern blot for DNAs
  • Northern blot for RNAs
  • SSCA single-stranded conformation analysis
  • FISH fluorescent in situ hybridization
  • gel migration clamped denaturing gel electrophoresis
  • heteroduplex analysis RNase protection
  • ELISA radio-immunoassays
  • IEMA immuno-enzymatic as
  • Some of these approaches are based on a change in electrophoretic mobility of the nucleic acids, as a result of the presence of an altered sequence. According to these techniques, the altered sequence is visualized by a shift in mobility on gels. The fragments may then be sequenced to confirm the alteration.
  • Some others are based on specific hybridisation between nucleic acids from the subject and a probe specific for wild type or altered BTBD9 gene or RNA.
  • the probe may be in suspension or immobilized on a substrate.
  • the probe is typically labeled to facilitate detection of hybrids.
  • Some of these approaches are particularly suited for assessing a polypeptide sequence or expression level, such as Northern blot, ELISA and RIA. These latter require the use of a ligand specific for the polypeptide, more preferably of a specific antibody.
  • the method comprises detecting the presence of an altered BTBD9 gene expression profile in a sample from the subject. As indicated above, this can be accomplished more preferably by sequencing, selective hybridisation and/or selective amplification of nucleic acids present in said sample.
  • Sequencing Sequencing can be carried out using techniques well known in the art, using automatic sequencers.
  • the sequencing may be performed on the complete BTBD9 gene or, more preferably, on specific domains thereof, typically those known or suspected to carry deleterious mutations or other alterations.
  • Amplification Amplification is based on the formation of specific hybrids between complementary nucleic acid sequences that serve to initiate nucleic acid reproduction.
  • Amplification may be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA). These techniques can be performed using commercially available reagents and protocols. Preferred techniques use allele-specific PCR or PCR-SSCP. Amplification usually requires the use of specific nucleic acid primers, to initiate the reaction.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence based amplification
  • Nucleic acid primers useful for amplifying sequences from the BTBD9 gene or locus are able to specifically hybridize with a portion of the BTBD9 gene locus that flank a target region of said locus, said target region being altered in certain subjects having type 2 diabetes. Examples of such target regions are provided in Table 3 A or Table 3B.
  • Primers that can be used to amplify BTBD9 target region comprising SNPs as identified in Tables 3A or 3B may be designed based on the sequence of SEQ ID No 1 or on the genomic sequence of BTBD9. In a particular embodiment, primers may be designed based on the sequence of SEQ ID Nos 3-22.
  • Typical primers of this invention are single-stranded nucleic acid molecules of about 5 to 60 nucleotides in length, more preferably of about 8 to about 25 nucleotides in length.
  • the sequence can be derived directly from the sequence of the BTBD9 gene locus. Perfect complementarity is preferred, to ensure high specificity. However, certain mismatch may be tolerated.
  • the invention also concerns the use of a nucleic acid primer or a pair of nucleic acid primers as described above in a method of detecting the presence of or predisposition to type 2 diabetes in a subject, in particular in a subject with obesity.
  • Selective hybridization Hybridization detection methods are based on the formation of specific hybrids between complementary nucleic acid sequences that serve to detect nucleic acid sequence alteration(s).
  • a particular detection technique involves the use of a nucleic acid probe specific for wild type or altered BTBD9 gene or RNA, followed by the detection of the presence of a hybrid.
  • the probe may be in suspension or immobilized on a substrate or support (as in nucleic acid array or chips technologies).
  • the probe is typically labeled to facilitate detection of hybrids.
  • a particular embodiment of this invention comprises contacting the sample from the subject with a nucleic acid probe specific for an altered BTBD9 gene locus, and assessing the formation of an hybrid.
  • the method comprises contacting simultaneously the sample with a set of probes that are specific, respectively, for wild type BTBD9 gene locus and for various altered forms thereof.
  • various samples from various subjects may be treated in parallel.
  • a probe refers to a polynucleotide sequence which is complementary to and capable of specific hybridisation with a (target portion of a) BTBD9 gene or RNA, and which is suitable for detecting polynucleotide polymorphisms associated with BTBD9 alleles which predispose to or are associated with obesity or an associated disorder.
  • Probes are preferably perfectly complementary to the BTBD9 gene, RNA, or target portion thereof. Probes typically comprise single-stranded nucleic acids of between 8 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. It should be understood that longer probes may be used as well.
  • a preferred probe of this invention is a single stranded nucleic acid molecule of between 8 to 500 nucleotides in length, which can specifically hybridise to a region of a BTBD9 gene or RNA that carries an alteration.
  • a specific embodiment of this invention is a nucleic acid probe specific for an altered (e.g., a mutated) BTBD9 gene or RNA, i.e., a nucleic acid probe that specifically hybridises to said altered BTBD9 gene or RNA and essentially does not hybridise to a BTBD9 gene or RNA lacking said alteration. Specificity indicates that hybridisation to the target sequence generates a specific signal which can be distinguished from the signal generated through non-specific hybridisation. Perfectly complementary sequences are preferred to design probes according to this invention. It should be understood, however, that a certain degree of mismatch may be tolerated, as long as the specific signal may be distinguished from non-specific hybridisation.
  • probes are nucleic acid sequences complementary to a target portion of the genomic region including the BTBD9 gene or RNA carrying a point mutation as listed in Table 3A or Table 3B above. More particularly, the probes can comprise a sequence selected from the group consisting of SEQ ID Nos 3-22 or a fragment thereof comprising the SNP or a complementary sequence thereof.
  • the sequence of the probes can be derived from the sequences of the BTBD9 gene and RNA as provided in the present application. Nucleotide substitutions may be performed, as well as chemical modifications of the probe. Such chemical modifications may be accomplished to increase the stability of hybrids (e.g., intercalating groups) or to label the probe. Typical examples of labels include, without limitation, radioactivity, fluorescence, luminescence, enzymatic labeling, etc.
  • the invention also concerns the use of a nucleic acid probe as described above in a method of detecting the presence of or predisposition to type 2 diabetes in a subject or in a method of assessing the response of a subject to a treatment of type 2 diabetes or an associated disorder.
  • alteration in the BTBD9 gene locus may also be detected by screening for alteration(s) in BTBD9 polypeptide sequence or expression levels.
  • a specific embodiment of this invention comprises contacting the sample with a ligand specific for a BTBD9 polypeptide and determining the formation of a complex.
  • ligands may be used, such as specific antibodies.
  • the sample is contacted with an antibody specific for a BTBD9 polypeptide and the formation of an immune complex is determined.
  • Various methods for detecting an immune complex can be used, such as ELISA, radioimmunoassays (RIA) and immuno- enzymatic assays (IEMA).
  • an antibody designates a polyclonal antibody, a monoclonal antibody, as well as fragments or derivatives thereof having substantially the same antigen specificity. Fragments include Fab, Fab'2, CDR regions, etc. Derivatives include single-chain antibodies, humanized antibodies, poly-functional antibodies, etc.
  • An antibody specific for a BTBD9 polypeptide designates an antibody that selectively binds a BTBD9 polypeptide, namely, an antibody raised against a BTBD9 polypeptide or an epitope-containing fragment thereof. Although non-specific binding towards other antigens may occur, binding to the target BTBD9 polypeptide occurs with a higher affinity and can be reliably discriminated from non-specific binding.
  • the method comprises contacting a sample from the subject with (a support coated with) an antibody specific for an altered form of a BTBD9 polypeptide, and determining the presence of an immune complex.
  • the sample may be contacted simultaneously, or in parallel, or sequentially, with various (supports coated with) antibodies specific for different forms of a BTBD9 polypeptide, such as a wild type and various altered forms thereof.
  • the invention also concerns the use of a ligand, preferably an antibody, a fragment or a derivative thereof as described above, in a method of detecting the presence of or predisposition to type 2 diabetes in a subject, in particular in a subject with obesity.
  • diagnostic kits comprising products and reagents for detecting in a sample from a subject the presence of an alteration in the BTBD9 gene or polypeptide, in the BTBD9 gene or polypeptide expression, and/or in BTBD9 activity.
  • Said diagnostic kit comprises any primer, any pair of primers, any nucleic acid probe and/or any ligand, preferably antibody, described in the present invention.
  • Said diagnostic kit can further comprise reagents and/or protocols for performing a hybridization, amplification or antigen-antibody immune reaction.
  • the diagnosis methods can be performed in vitro, ex vivo or in vivo, preferably in vitro or ex vivo. They use a sample from the subject, to assess the status of the BTBD9 gene locus.
  • the sample may be any biological sample derived from a subject, which contains nucleic acids or polypeptides.
  • samples include fluids, tissues, cell samples, organs, biopsies, etc. Most preferred samples are blood, plasma, saliva, urine, seminal fluid, etc.
  • the sample may be collected according to conventional techniques and used directly for diagnosis or stored.
  • the sample may be treated prior to performing the method, in order to render or improve availability of nucleic acids or polypeptides for testing. Treatments include, for instant, lysis (e.g., mechanical, physical, chemical, etc.), centrifugation, etc.
  • nucleic acids and/or polypeptides may be pre -purified or enriched by conventional techniques, and/or reduced in complexity. Nucleic acids and polypeptides may also be treated with enzymes or other chemical or physical treatments to produce fragments thereof. Considering the high sensitivity of the claimed methods, very few amounts of sample are sufficient to perform the assay.
  • the sample is preferably contacted with reagents such as probes, primers or ligands in order to assess the presence of an altered BTBD9 gene locus.
  • Contacting may be performed in any suitable device, such as a plate, tube, well, glass, etc.
  • the contacting is performed on a substrate coated with the reagent, such as a nucleic acid array or a specific ligand array.
  • the substrate may be a solid or semi-solid substrate such as any support comprising glass, plastic, nylon, paper, metal, polymers and the like.
  • the substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc.
  • the contacting may be made under any condition suitable for a complex to be formed between the reagent and the nucleic acids or polypeptides of the sample.
  • the finding of an altered BTBD9 polypeptide, RNA or DNA in the sample is indicative of the presence of an altered BTBD9 gene locus in the subject, which can be correlated to the presence, predisposition or stage of progression of type 2 diabetes.
  • an individual having a germ line BTBD9 mutation has an increased risk of developing type 2 diabetes.
  • the determination of the presence of an altered BTBD9 gene locus in a subject also allows the design of appropriate therapeutic intervention, which is more effective and customized.
  • BTBD9 gene locus the practitioner of ordinary skill in the art can easily identify additional SNPs in linkage disequilibrium with this first SNP. Indeed, any SNP in linkage disequilibrium with a first SNP associated with type 2 diabetes will be associated with this trait. Therefore, once the association has been demonstrated between a given SNP and type 2 diabetes, the discovery of additional SNPs associated with this trait can be of great interest in order to increase the density of SNPs in this particular region.
  • Identification of additional SNPs in linkage disequilibrium with a given SNP involves: (a) amplifying a fragment from the genomic region comprising or surrounding a first SNP from a plurality of individuals; (b) identifying of second SNPs in the genomic region harboring or surrounding said first SNP; (c) conducting a linkage disequilibrium analysis between said first SNP and second SNPs; and (d) selecting said second SNPs as being in linkage disequilibrium with said first marker. Subcombinations comprising steps (b) and (c) are also contemplated.
  • SNPs in linkage disequilibrium can also be used in the methods according to the present invention, and more particularly in the diagnosic methods according to the present invention.
  • a linkage locus of Crohn's disease has been mapped to a large region spanning 18cM on chromosome 5q31 (Rioux et al., 2000 and 2001).
  • LD linkage disequilibrium
  • the authors developed an ultra-high-density SNP map and studied a denser collection of markers selected from this map.
  • Multilocus analyses defined a single common risk haplotype characterised by multiple SNPs that were each independently associated using TDT. These SNPs were unique to the risk haplotype and essentially identical in their information content by virtue of being in nearly complete LD with one another. The equivalent properties of these SNPs make it impossible to identify the causal mutation within this region on the basis of genetic evidence alone.
  • Mutations in the BTBD9 gene which are responsible for type 2 diabetes may be identified by comparing the sequences of the BTBD9 gene from patients presenting type 2 diabetes and control individuals. Based on the identified association of SNPs of BTBD9 and type 2 diabetes, the identified locus can be scanned for mutations. In a preferred embodiment, functional regions such as exons and splice sites, promoters and other regulatory regions of the BTBD9 gene are scanned for mutations.
  • patients presenting type 2 diabetes carry the mutation shown to be associated with type 2 diabetes and controls individuals do not carry the mutation or allele associated with type 2 diabetes or an associated disorder. It might also be possible that patients presenting type 2 diabetes carry the mutation shown to be associated with type 2 diabetes with a higher frequency than controls individuals.
  • the method used to detect such mutations generally comprises the following steps: amplification of a region of the BTBD9 gene comprising a SNP or a group of SNPs associated with type 2 diabetes from DNA samples of the BTBD9 gene from patients presenting type 2 diabetes and control individuals; sequencing of the amplified region; comparison of DNA sequences of the BTBD9 gene from patients presenting type 2 diabetes and control individuals; determination of mutations specific to patients presenting type 2 diabetes.
  • identification of a causal mutation in the BTBD9 gene can be carried out by the skilled person without undue experimentation by using well-known methods.
  • causal mutations have been identified in the following examples by using routine methods.
  • Hugot et al. (2001) applied a positional cloning strategy to identify gene variants with susceptibly to Crohn's disease in a region of chromosome 16 previously found to be linked to susceptibility to Crohn's disease.
  • microsatellite markers were genotyped and tested for association to Crohn's disease using the transmission disequilibrium test.
  • a borderline significant association was found between one allele of the microsatellite marker D 16Sl 36.
  • Eleven additional SNPs were selected from surrounding regions and several SNPs showed significant association. SNP5-8 from this region were found to be present in a single exon of the NOD2/CARD15 gene and shown to be non-synonymous variants.
  • the present invention demonstrates the correlation between type 2 diabetes and the BTBD9 gene locus.
  • the invention thus provides a novel target of therapeutic intervention.
  • Various approaches can be contemplated to restore or modulate the BTBD9 activity or function in a subject, particularly those carrying an altered BTBD9 gene locus.
  • Supplying wild-type function to such subjects is expected to suppress phenotypic expression of type 2 diabetes in a pathological cell or organism.
  • the supply of such function can be accomplished through gene or protein therapy, or by administering compounds that modulate or mimic BTBD9 polypeptide activity (e.g., agonists as identified in the above screening assays).
  • BTBD9 activity e.g., peptides, drugs, BTBD9 agonists, or organic compounds
  • a subject or to suppress the deleterious phenotype in a cell may also be used to restore functional BTBD9 activity in a subject or to suppress the deleterious phenotype in a cell.
  • Restoration of functional BTBD9 gene function in a cell may be used to prevent the development of type 2 diabetes or to reduce progression of said diseases.
  • Such a treatment may suppress the type 2 diabetes -associated phenotype of a cell, particularly those cells carrying a deleterious allele.
  • the GenomeHIP platform was applied to allow rapid identification of a TYPE 2 DIABETES susceptibility gene.
  • the technology consists of forming pairs from the DNA of related individuals. Each DNA is marked with a specific label allowing its identification. Hybrids are then formed between the two DNAs. A particular process (WO00/53802) is then applied that selects all fragments identical-by-descent (IBD) from the two DNAs in a multi step procedure. The remaining IBD enriched DNA is then scored against a BAC clone derived DNA microarray that allows the positioning of the IBD fraction on a chromosome.
  • IBD identical-by-descent
  • the inventors By screening the aforementioned 4.5 Megabases in the linked chromosomal region, the inventors identified the BTBD9 gene as a candidate for type 2 diabetes. This gene is indeed present in the critical interval, with evidence for linkage delimited by the clones outlined above.
  • Table 4 Linkage results for chromosome 6 in the BTBD9 locus: Indicated is the region correspondent to BAC clones with evidence for linkage. The start and stop positions of the clones correspond to their genomic location based on NCBI Build 36 sequence respective to the start of the chromosome (p-ter).
  • the method is based on likelihood ratio tests in a logistic model:
  • Iog( ⁇ 3 ⁇ ) mu + ⁇ beta, .X 1
  • X 1 are variables which represent the allele or haplotypes in some way depending upon the particular test, and mu and beta; are coefficients to be estimated.
  • Reference for this application of log-linear models is Cordell & Clayton, AJHG (2002) In cases of uncertain haplotype, the method for case-control sample is a standard unconditional logistic regression identical to the model-free method T5 of EHPLUS (Zhao et al Hum Hered (2000) and the log-linear modelling of Mander.
  • the beta ! are log odds ratios for the haplotypes.
  • the EM algorithm is used to obtain maximum likelihood frequency estimates.
  • genotype A A genotype A a and genotype a a where a represented the associate allele of the SNP with TYPE 2 DIABETES.
  • Dominant transmission model for associated allele (a) were tested by counting A a and a a genotype together. The statistic test was carried out using the standard Chi-square independence test with 1 df (genotype distribution, 2x2 table). Recessive transmission model for associated allele (a) were tested by counting A A and A a genotype together. The statistic test was carried out using the standard Chi-square independence test with 1 df (genotype distribution, 2x2 table). Additive transmission model for associated allele (a) were tested using the standard Chi-square independence test with 2 df (genotype distribution, 2x3 table). 0
  • Nonsense mutation in the glucokinase gene causes early-onset non- insulin-dependent diabetes mellitus. Nature 356:721-22

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Abstract

Procédé de diagnostic, permettant de déterminer si un sujet, de préférence obèse, risque de développer un diabète de type 2, qui consiste à détecter la présence d'une altération dans le locus du gène BTBD9 à l'intérieur d'un échantillon biologique du sujet.
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