WO2008096009A2 - Polymorphism in the slc30a8 gene - Google Patents

Abstract

The present invention relates to a single nucleotide polymorphisme (SNP) identified in the slc30a8 gene, leading to a specific amino acid sequence of the coded protein, associated with susceptibility to diabetes. The invention is also relative to a transgenic mammalian model comprising a protein encoded bythe slc30a8 gene, having a specific polypeptidic sequence, which can be useful for elucidating the onset mechanism of diabetes, and for the screening of therapeutically active drugs for the treatment of diabetes, especially type II diabetes, and metabolic disorders, including syndrome X and obesity. The mammalian animal considered can be a mouse (preferably), a rat, a dog, a macaque, a chimp or a pig.

Description

MAMMALIAN ANIMAL MODEL COMPRISING A POLYMORPHISM IN

THE SLC30A8 GENE

The present invention relates to a single nucleotide polymorphism (SNP) identified in the slc30a8 gene, leading to a specific amino acid sequence of the coded protein, associated with susceptibility to diabetes. The invention is also relative to a transgenic mammalian model comprising a protein encoded by the slc30a8 gene, having a specific polypeptidic sequence, which can be useful for elucidating the onset mechanism of diabetes, and for the screening of therapeutically active drugs for the treatment of diabetes, especially type II diabetes, and metabolic disorders, including syndrome X and obesity.

The mammalian animal considered can be a mouse (preferably), a rat, a dog, a macaque, a chimp or a pig.

FIELD OF THE INVENTION The present invention relates generally to the fields of genetics and medicine. The present invention more particularly relates to a human diabetes susceptibility gene, slc30a8. The invention more specifically relates to a certain allele of the slc30a8 gene on chromosome 8 related to susceptibility to diabetes and representing a novel target for therapeutic intervention. The present invention more specifically relates to a transgenic animal model comprising a particular SNP (single nucleotide polymorphism) in the slc30a8 gene, which can be used for research and for the screening of therapeutically active drugs for the treatment of diabetes, especially type II diabetes, and metabolic disorders, including syndrome X and obesity.

BACKGROUND OF THE INVENTION

The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated herein by reference, and for convenience, are referenced by author and date in the following text and respectively grouped in the appended List of References. Diabetes mellitus, a metabolic disease in which carbohydrate utilization is reduced and lipid and protein utilization is enhanced, is caused by an absolute or relative deficiency of insulin.

Diabetes mellitus is among the most common of all metabolic disorders, affecting up to 11% of the population by age 70. Type I diabetes (insulin dependent diabetes mellitus or IDDM) represents about 5 to 10% of this group and is the result of a progressive autoimmune destruction of the pancreatic β-cells with subsequent insulin deficiency. IDDM is characterized by a partial or complete inability to produce insulin. Patients with IDDM would die without daily insulin injections to control their disease. Little advancement in resolving the pathogenesis of diabetes was made until the mid-1970s when evidence began to accumulate to suggest that IDDM had an autoimmune etiopathogenesis. It is now generally accepted that IDDM results from the chronic autoimmune destruction of the insulin producing pancreatic β-cells. Lymphocytes and other inflammatory cells have been observed within the islets of Langerhans in newly diagnosed IDDM patients and have been found preferentially in regenerating islets composed of β-cells rather than those of other cell types. This active immunological process is associated with a variety of autoantibodies to β-cells cytoplasmic and membrane antigens, insulin, and insulin receptors. Although the general mechanism by which IDDM occurs is known, IDDM becomes clinically evident only after the vast majority of pancreatic β-cells have been irrevocably destroyed and the individual becomes dependent upon exogenous insulin.

In both humans and diabetes-prone non-obese diabetic (NOD) mice, genes mapping to the major histocompatibility complex have been associated with susceptibility to IDDM and shown to be very important in the disease process (Todd, 1990). Studies of NOD mice have mapped at least 12 other susceptibility genes to specific chromosomal locations (Prochazaka et al, 1987; Todd et al, 1991; De Gouyon et al, 1993; Morahan et al, 1994; Serreze et al., 1994; Comall et al., 1991; Garchon et al., 1991). In humans, markers near the insulin/insulin- like growth factor loci have also been associated with IDDM (Bell et al., 1984).

Epidemiological and molecular genetic studies indicate that only 40% of the genetic susceptibility is due to alleles of the MHC class II genes, suggesting additional non-MHC genes are involved (Todd et al., 1988; Buzzetti. et al., 1998). Genome-wide searches and analyses of specific genes have identified more at least 17 loci that contribute to the disease (Buzzetti et al., 1998; Todd, 1995; Concannon et al., 1998; Hashimoto et al., 1994; Davies et al., 1994; Mein et al., 1998; Verge et al., 1998). Significant evidence for linkage was reported for a ~-7 cM region on chromosome lq42-43 (Concannon et al., 1998). This region of chromosome 1 contains the angiotensinogen (AGT) gene.

Non-insulin dependent diabetes mellitus (NIDDM), or type II diabetes, is a heterogenous disorder characterized by chronic hyperglycemia leading to progressive micro- and macrovascular lesions in the cardiovascular, renal and visual systems as well as diabetic neuropathy.

The health implications of Type II diabetes are enormous. In 1995, there were 135 million adults with diabetes worldwide. It is estimated that close to 300 million will have diabetes in the year 2025. (King H., etal, Diabetes Care, 21(9): 1414-1431 (1998)).

The causes of the fasting hyperglycemia and/or glucose intolerance associated with this form of diabetes are not well understood. Unfortunately, the disease is associated with early morbidity and mortality. Subtypes of the disease can be identified based at least to some degree on the time of onset of the symptoms. The principal type of NIDDM occurs at a later time of onset, typically at midlife. Early-onset NIDDM or maturity-onset diabetes of the young (MODY) shares many features with the more common form(s) of NIDDM but onset occurs in early life. Although most forms of NIDDM do not exhibit simple Mendelian inheritance, the contribution of heredity is well recognized (Rotter et al., 1990). The genetic basis of a few rare monogenic syndromes of NIDDM have been elucidated, but together, these syndromes account for a very small minority of cases (Taylor et al., 1992; Froguel et al., 1993; Steiner et al., 1990; Kadowaki et al., 1994). It is likely that the common forms of NIDDM are complex and heterogenous, and result when a pool of mutant genes, each of which contributes modestly and in a subtle way, interact with each other and with environmental, aging and behavioral influences to lead to the expression of the disease. This pool of genes may vary between populations and among individuals within a population, despite the illusion of a clinically homogenous phenotype. Certain loci have been linked to rare early-onset forms of Type II diabetes that is associated with chronic hyperglycemia and monogenic inheritance (i.e. maturity onset diabetes of the young (MODY) loci) (Bell et al., 1991; Froguel et al., 1992; Hattersley et al., 1992; Vaxillaire et al., 1995). The defects in the glucokinase (GCK) gene on human chromosome 7 have been found to be responsible for the relatively rare M0DY2 phenotype. (Froguel et al., 1992).

The genes responsible for MODYl and M0DY3 have been identified to be transcription factors hepatocyte nuclear factor 4 (HNF4) and hepatocyte nuclear factor 1 FINF 1, repsectively. (Yamagata et al., 1996a; Yamagata et al. 1996b). The diabetes gene HNF4/M0DY1 regulates expression of the AGT gene (Yanai et al., 1999). Mutations in FINF4/MODY1 have been demonstrated to cause predisposition to maturity onset diabetes of the young. (MODY) a genetically heterogenouse monogenic form of non-insulin- dependent diabetes mellitus (NIDDM) (Yamagata et al., 1996b; Lindner et al., 1997).

Another locus has been identified for a rare early-onset form with mitochondrial inheritance (Van den Ouwenland et al., 1992). In addition, Harris et al. (1996) identified a locus of NIDDMl on chromosome 2 that appears to play a role in Mexican American diabetes. Further, Mahtani et al. (1996) report evidence of the existence of a gene on human chromosome 12, NIDDM2, that causes NIDDM associated with low insulin secretion. The paper suggests that NIDDM2 and M0DY3 represent different alleles of the same gene with severe mutations causing M0DY3 and milder mutations giving rise to later-onset NIDDM characterized by low insulin secretion.

In view of the heterogeneity of genes associated with IDDM or NIDDM, it is desired to identify additional genes associated with IDDM or NIDDM for diagnostic and therapeutic purposes. The high prevalence of the disease and increasing population affected shows an unmet medical need to define other genetic factors involved in Type II diabetes and to more precisely define the associated risk factors. Also needed are diagnostic assays to identify the propensity to develop Type II diabetes and therapeutic agents for prevention and treatment of the disease. The role of slc30a8 gene on chromosome 8 related to susceptibility to diabetes and its use for research and for the screening of therapeutically active drugs for the treatment of diabetes, especially type II diabetes, and metabolic disorders, including syndrome X and obesity is disclosed in WO 2004/046355.

A nucleic acid sequence at which more than one sequence is possible in a population (either a natural population or a synthetic population, e.g., a library of synthetic molecules) is referred to herein as a "polymorphic site." Polymorphic sites can allow for differences in sequences based on substitutions, insertions, or deletions. Such substitutions, insertions, or deletions can result in frame shifts, the generation of premature stop codons, the deletion or addition of one or more amino acids encoded by a polynucleotide, alter splice sites, and affect the stability or transport of mRNA. Where a polymorphic site is a single nucleotide in length, the site is referred to as a single nucleotide polymorphism ("SNP").

SNPs are the most common form of genetic variation responsible for differences in disease susceptibility and drug response. It is generally admitted that specific SNPs exist at a frequence of less than 1% in a whole population. SNPs can directly contribute to or, more commonly, are associated and serve as markers for many phenotypic endpoints such as disease risk or the drug response differences between patients.

Identification of these genetic factors can lead to the design of transgenic models that can be used in diabetes research and drug screening.

SUMMARY OF THE INVENTION

The invention relates to transgenic mammalian animals containing a polynucleotide comprising an alteration in the slc30a8 gene locus, which can be used for research in the field of diabetes and related disorders, as well as for the screening of therapeutically active drugs in diabetes.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

The term "gene" shall be construed to include any type of coding nucleic acid, including genomic DNA (gDNA), complementary DNA (cDNA), synthetic or semisynthetic DNA, as well as any form of corresponding RINA. The term gene particularly includes recombinant nucleic acids encoding slc30a8, i.e., any non naturally occurring nucleic acid molecule created artificially, e.g., by assembling, cuffing, ligating or amplifying sequences.

Within the context of this invention, the slc30a8 gene locus designates all slc30a8 sequences or products in a cell or organism, including Slc30a8 coding sequences, Slc30a8 non-coding sequences (e.g., introns), Slc30a8 regulatory sequences controlling transcription and/or translation (e.g., promoter, enhancer, terminator, etc.), as well as all corresponding expression products, such as Slc30a8 RNAs (e.g., mRNAs) and Slc30a8 polypeptides (e.g., a pre-protein and a mature protein). The Slc30a8 gene locus also comprises surrounding sequences of the Slc30a8 gene which include SNPs that are in linkage disequilibrium with SNPs located in the Slc30a8 gene.

The term "SNP" refers to a single nucleotide polymorphism at a particular position in the human genome that varies among a population of individuals. SNPs exist at a low frequence in a population, generally in less than 1% of the individuals. As used herein, a SNP may be identified by its name or by location within a particular sequence. SNPs may be silent or non-silent. When non-silent, translation of the SNP leads to an identified mutation in the polypeptide corresponding to translation of the said polynucleotide.

SNPs positions, when non-silent, can also be identified on the gene product, such as a protein encoded by the corresponding nucleotide sequence. The specific amino acid sequence related to the SNP, such as identified in the polypeptides sequences herein presented in SEQ ID N°l-8, are indicated in bold.

As used herein, the polypeptides sequences disclosed by the SEQ. ID. N°.l-8 of the present invention encompasses the complements of nucleotide sequences coding for said polypeptides. In addition, as used herein, the term "SNP" encompasses any allele among a set of alleles having a specific SNP encoding for the specific amino acid sequence such as presented in SEQ ID N° 1 to 8.

The term "allele" refers to a specific nucleotide sequence among a selection of nucleotides defining a SNP. The term "minor allele" refers to an allele of a SNP that occurs less frequently within a population of individuals than the major allele.

The term "major allele" refers to an allele of a SNP that occurs more frequently within a population of individuals than the minor allele.

The term "at-risk allele" refers to an allele that is associated with Type II diabetes. The term "haplotype" refers to a combination of particular alleles from two or more

SNPs.

The term "at-risk haplotype" refers to a haplotype that is associated with Type II diabetes. The term "polynucleotide" refers to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/ or their analogs. Polynucleotides may have any three-dimensional structure including single- stranded, double-stranded and triple helical molecular structures, and may perform any function, known or unknown. The following are non-limiting embodiments of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, short interfering nucleic acid molecules (siNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated KNA of any sequence, nucleic acid probes, and primers. A polynucleotide may also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs.

A "substantially isolated" or "isolated" polynucleotide is one that is substantially free of the sequences with which it is associated in nature. By substantially free is meant at least 50%, at least 70%, at least 80%, or at least 90% free of the materials with which it is associated in nature. An "isolated polynucleotide" also includes recombinant polynucleotides, which, by virtue of origin or manipulation: (1) are not associated with all or a portion of a polynucleotide with which it is associated in nature, (2) are linked to a polynucleotide other that to which it is linked in nature, or (3) does not occur in nature.

The term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y (1989), 6.3.1- 6.3.6. A non-limiting example of stringent hybridization conditions are hybridization in 6x sodium chloride/sodium citrate (SSC) at about 45<0>C, followed by one or more washes in 0.2. x SSC, 0.1% SDS at 50-65OC.

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

A "host cell" includes an individual cell or cell culture which can be or has been a recipient for vector (s) or for incorporation of nucleic acid molecules and/or proteins. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent due to natural, accidental, or deliberate mutation. A host cell includes cells transfected with the polynucleotides of the present invention. An "isolated host cell" is one which has been physically dissociated from the organism from which it was derived.

The terms "individual," "host," and "subject" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. The terms "transformation," "transfection," and "genetic transformation" are used interchangeably herein to refer to the insertion or introduction of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion, for example, lipofection, transduction, infection, electroporation, CaPO4 precipitation, DEAE- dextran, particle bombardment, etc. The exogenous polynucleotide may be maintained as a non- integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome. The genetic transformation maybe transient or stable.

A slc30a8 gene is typically double-stranded, although other forms may be contemplated, such as single-stranded. slc30a8 genes may be obtained from various sources and according to various techniques known in the art, such as by screening DNA libraries or by amplification from various natural sources. Recombinant nucleic acids may be prepared by conventional techniques, including chemical synthesis, genetic engineering, enzymatic techniques, or a combination thereof. Suitable slc30a8 gene sequences may be found on gene banks, such as Unigene Cluster Hs.532270 and Unigene Representative Sequences AYl 17411. In particular, the human gene is accessible on Genbank under accession number

AYl 17411; the mouse gene is accessible under number NM_172816; other sequences are available on the database "Ensembl": rat gene under number ENSRNOT 6410; dog gene under number ENSCAFT 1287; chimp gene under number ENSPTRT 37978; ENSMMUT_7003; and pig gene under number ENSCPOT_3107. Typical stringent hybridisation conditions include temperatures above 300° C, preferably above 350C, more preferably in excess of 420C, 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 slc30a8 polypeptide designates any protein or polypeptide encoded by a slc30a8 gene as disclosed above. The term "polypeptide" refers to any molecule comprising a stretch of amino acids. This term includes molecules of various length, 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 slc30a8 polypeptide comprises all or part of SEQ ID Nos: 1-8 or a variant thereof.

The terms "response to a treatment" refer to treatment efficacy, including but not limited to ability to metabolize a therapeutic compound, to the ability to convert a pro-drug to an active drug, and to the pharmacokinetics (absorption, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual.

The terms "adverse effects to a treatment" refer to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors. "Side effects to a treatment" include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, generalized urticaria, bronchoconstriction, hypotension, and shock. The present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

As used herein, the singular form of any term can alternatively encompass the plural form and vice versa. Main publications and references cited herein are incorporated by reference in their entirety for any purpose.

Polymorphisms in the slc30a8 gene which are associated with diabetes or an associated metabolic disorder, or protection/resistance against diabetes, may be identified by comparing the sequences of the slc30a8 gene from patients presenting diabetes or an associated metabolic disorder or protection/resistance against diabetes and "control" individuals.

SEQUENCES OF ENCODED PROTEINS SEQ ID NOl - Slc30a8 human MEFLERTYLV NDKAAKMHAF TLESVELQQK PVNKDQCPRE RPEELESGGM YHCHSGSKPT EKGANEYAYA KWKLCSASAI CFIFMIAEVV GGHIAGSLAV VTDAAHLLID LTSFLLSLFS LWLSSKPPSK RLTFGWHRAE ILGALLSILC IWVVTGVLVY LACERLLYPD YQIQATVMII VSSCAVAANI VLTVVLHQRC LGHNHKEVQA NASVRAAFVH ALGDLFQSIS VLISALIIYF KPEYKIADPI CTFIFSILVL ASTITILKDF SILLMEGVPK SLNYSGVKEL ILAVDGVLSV HSLHIWSLTM NQVILSAHVA TAASRDSQVV RREIAKALSK SFTMHSLTIQ

MESPVDQDPD CLFCEDPCD

SEQ ID NO2 - Slc30a8 human with the specific sequence R325W MEFLERTYLV NDKAAKMHAF TLESVELQQK PVNKDQCPRE RPEELESGGM YHCHSGSKPT EKGANEYAYA KWKLCSASAI CFIFMIAEVV GGHIAGSLAV VTDAAHLLID LTSFLLSLFS LWLSSKPPSK RLTFGWHRAE ILGALLSILC IWVVTGVLVY LACERLLYPD YQIQATVMII VSSCAVAANI VLTVVLHQRC LGHNHKEVQA NASVRAAFVH ALGDLFQSIS VLISALIIYF KPEYKIADPI CTFIFSILVL ASTITILKDF SILLMEGVPK SLNYSGVKEL ILAVDGVLSV HSLHIWSLTM NQVILSAHVA TAASWDSQVV RREIAKALSK SFTMHSLTIQ

MESPVDQDPD CLFCEDPCD

SEQ ID NO3 - SLc30a8 mouse

MEFLERTYLV NDQATKMYAF PLDRELRQKP VNKDQCPGDR PEHPEAGGIY HCHNSAKATG NRSSKQAHAK WRLCAASAIC FIFMVAEVVG GHVAGSLAIL TDAAHLLIDL TSFLLSLFSL WLSSRPPSKR LTFGWYRAEI LGALLSVLCI WVVTGVLLYL ACERLLYPDY QIQAGIMITV SGCAVAANIV LTMILHQRNF GYNHKDVQAN ASVRAAFVHA LGDVFQSISV LISALIIYFK PDYKIADPVC TFIFSILVLA STVMILKDFS ILLMEGVPKG LSYNSVKEII LAVDGVISVH SLHIWSLTVN QVILSVHVAT AASQDSQSVR TGIAQALSSF DLHSLTIQIE SAADQDPSCL LCEDPQD

SEQ ID NO4 - SLc30a8 Rat

MEFLERTYLV NDQATKMYAF TSDRERGQKP VNKDQCPGDG PERPEAGAIY HCHNSFKATG NRSSKQVHAK WRLCAASAIC FFFMVAEVVG GHVAGSLAVL TDAAHLLIDL TSFLLSLFSL WLSSRPPSKR LTFGWYRAEI LGALLSVLCI WVVTGVLVYL ACERLLYPDY QIQAGIMITV SGCAVAANIV LTLILHQRHL GHNHKDAQAN ASVRAAFVHA LGDVFQSTSV LISALIIYFK PDYKMADPVC TFISSVLALA STVMILKDFS ILLMEGVPKG LSYNSVKELL LTVDGVISVH NLHIWSLTVN QVILSVHVAT AASQDSQSVR TGIACALSSS FDLHSLTIQI ESAADQDPSC LLCEDPQD

SEQ ID NO5 - SLc30a8 Canis

MEFLERTYLV NDRATKMYAF NLDSVELQQK SLNKDQCPGE KPEELESGAI YHCHSNSKAT ENRANEQVYA KWKLYAASGV CFIFMIAEVV GGHIAGSLAV ITDAAHLLID LTSFLLSLFS LWLSSKPPSK QLTFGWHRAE ILGALLSILC VWVVTGVLVY LACERLLYPD YQIQGTVMIL VSGCAVAANI MLSVILHQKH PGHNHKEVQA NASVRAAFVH ALGDLFQSIS VLTSALI IYF KPDYKMADPI CTFVFSILVL ASTITVLKDF SILLMEGVPK NLNYSDVKEL ILAVDGVVSV HSLHIWSLAM NQVILSAHVA AAASRDSQVV RREIVKALSN SYTVHSLTIQ MESPADQDPN CFFCEDPRD

SEQ ID NO6 - SLc30a8 Chimp

MEFLERTYLV NDKAAKMYAF TLESVELQQK PVNKDQCPRE RPEELESGGM YHCHSGSKPT EKGANEYAYA KWKLCSASAI CFIFMIAEVV GGHIAGSLAV VTDAAHLLID LTSFLLSLVS MWLSSKPPSK RLTFGWHRAE ILGALLSILC IWVVTGVLVY LACERLLYPD YQIQATVMII VSSCAVAANI VLTVVLHQRC LGHNHKEVQG NASVRAAFVH ALGDLFQSIS VLISALIIYF KPEYKIADPI CTFIFSILVL ASTITILKDF SILLMEGVPK SLNYSGVKEL ILAVDGVVSV HSLHIWSLTV NQVILSAHVA TAASRDSQVV RREIAKALSK SFTMHSLTIQ

MESPVDQDPD CLFCEDPCD

SEQ ID NO7 - SLc30a8 Macaque

MEFLERTYLV NDKAAKMYAF TLESVELQQK PVNKDHCPRE RPEELESGGV YHCHSGSQAT ENRANEHAYA KWKLCAASAI CFIFMIAEVV GGHFAGSLAV

VTDAAHLLID LTSFLLSLFS LWLSSKPPSK RLTFGWHRAE ILGALLSILC

IWVVTGVLVY LACERLLYPD YQIQATVMII VSSCAVAANI ILTVVLHQRR

LGHNHKEVQA NASVRAAFVH ALGDLFQSIS VLISALI IYF KPEYKLADPI

CTFIFSILVL ASTITILKDF SILLMEGVPK SLNYNGVKEL ILAVDGVVSV HSLHIWSLTM NQVILSAHVA TAASRESQVV RREIARALSK SFTVYSLTIQ

MESPVDQDPD CLFCEDPRD

SEQ ID NO8 - SLc30a8 mouse with the specific sequence Q324W

MEFLERTYLV NDQATKMYAF PLDRELRQKP VNKDQCPGDR PEHPEAGGIY HCHNSAKATG NRSSKQAHAK WRLCAASAIC FIFMVAEVVG GHVAGSLAIL

TDAAHLLIDL TSFLLSLFSL WLSSRPPSKR LTFGWYRAEI LGALLSVLCI

WVVTGVLLYL ACERLLYPDY QIQAGIMITV SGCAVAANIV LTMILHQRNF

GYNHKDVQAN ASVRAAFVHA LGDVFQSISV LISALIIYFK PDYKIADPVC

TFIFSILVLA STVMILKDFS ILLMEGVPKG LSYNSVKEII LAVDGVISVH SLHIWSLTVN QVILSVHVAT AASWDSQSVR TGIAQALSSF DLHSLTIQIE

SAADQDPSCL LCEDPQD

TRANSGENIC ANIMALS

The present invention concerns SNPs identified in the slc30a8 gene, more particularly SNPs associated with a susceptibility to diabetes or an associated metabolic disorder or protection/resistance against diabetes. More particularly, the SNPs are non silent.

Prefered SNPs according to the invention are located in a codon of the polynucleotide sequence encoding for a strech of aminoacids of the slc30a8 protein AASXX' SQV, wherein X is selected among R and, Q, and X' is selected among D and E.

More preferred SNP is located in a codon of the polynucleotide sequence encoding for the above sequence where X is not R and Q. Most preferably, the SNP in the slc30a8 gene is non- silent and, when translated, comprises the following strech of aminoacids: AASWX' SQV wherein X' is selected among D and E.

In a most preferred embodiment, the SNP is in a codon of the gene coding for the mouse slc30a8 gene, leading to a mutation Q324W. The corresponding slc30a8 protein comprising the said mutation is represented on SEQ ID NO 8.

The invention concerns a transgenic animal expressing a polynucleotide comprising a SNP located in the polynucleotide sequence encoding for the protein having the sequence identified by SEQ. ID. NO. 8.

The invention also concerns a transgenic animal containing a polynucleotide comprising a SNP encoding for the proteins having the sequences identified by SEQ. ID. NO. 3-7.

The present invention concerns a transgenic non-human animal, more particularly a mammal preferably selected among rat, mouse, dog, chimp and macaque, wherein said transgenic animal expresses an heterologous slc30a8 gene comprising a SNP as defined above. In a most preferred embodiment, a transgenic non-human animal is a mouse expressing the human slc30a8 gene comprising a SNP encoding for the polypeptidic sequence identified by SEQ ID NO. 1-2.

Expression of said heterologous slc30a8 gene comprising a SNP can be obtained by introducing in said animal genome a gene comprising a polynucleotide sequence coding for the corresponding slc30a8 protein under control of regulatory elements functional in said animal. Such regulatory elements comprising promoters, terminator sequences, introns, enhancers, dunctional in mammal cells are well known in the art.

Expression of said heterologous slc30a8 gene comprising a SNP can be also be obtained by introducing the identified sequence into the native gene in the genome of said animal. The slc30a8 protein comprising the corresponding amino acid is then expressed by a modified sequence of the native allele, under control of the regulatory elements of the said native gene.

Introduction of exogenous sequences in the genome of animals is well known in the art, including homologous recombination technologies such as disclosed in Hammer & al. (Nature. 1985 Jun 20-26;315(6021):680-3. Production of transgenic rabbits, sheep and pigs by microinjection), Thompson & al. (Cell. 1989 Jan 27;56(2):313-21. Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells), Baribault & Kemler (MoI Biol Med. 1989 Dec;6(6):481-92.

Embryonic stem cell culture and gene targeting in transgenic mice) and Tojo & al. (Cytotechnology. 1995-1996; 19(2): 161-5. Establishment of a novel embryonic stem cell line by a modified procedure).

The present invention relates to a transgenic non-human mammal into which a similar identified sequence at the position encoding for aminoacid 325 in the human protein sequence is introduced in the slc30a8 gene, in particular a transgenic non-human mammal into which a tryptophane replaces the normal aminoacid at the equivalent position of aminoacid 325 in the human sequence, is introduced in the slc30a8 gene, and more particularly a transgenic mouse in which the Slc30a8/Q324W gene sequence (SEQ ID N°8 is expressed instead of the slc30a8 gene (SEQ ID N°7). The present invention relates also to a transgenic non- human mammal into which a human slc30a8 gene encoding for a protein having either R or W polymorphisms at position 325 (SEQ ID N°l-2) is expressed instead of the endogenous slc30a8 gene (SEQ ID N°7).

Technologies for the preparation of transgenic animals, including introducing a single mutation in a specified target gene are well known in the art, including Robertson EJ

(Biol Reprod. 1991 Feb;44(2):238-45. Using embryonic stem cells to introduce mutations into the mouse germ line) and Aarts & al. (Nucleic Acids Res. 2006;34(21):el47.

Generation of a mouse mutant by oligonucleotide-mediated gene modification in ES cells).

A transgenic mouse according to the invention can be obtained by a process comprising the steps of: a transgene is prepared by incorporating a mouse Slc30a8/Q324W gene (SEQ ID N°8 - example), which have been separated by a screening of an imprinted gene that exhibits maternal expression, into a vector; the transgene is microinjected into a male pronucleus of a fertilized egg and is cultured; subsequently the transgene is brought back into an oviduct of a pseudopregnant mouse. The inventors have found that the diabetes onset is triggered by the expression of a human Slc30a8/R325W gene (SEQ ID N°l) in humans. The Slc30a8/Q324W gene (encoding for SEQ ID N°8) incorporation in mouse corresponds to the intention of validating the link to type II diabetes susceptibility observed with the expression of the human Slc30a8/R325W gene in humans.

The following oligonucleotide may be used for the introduction of the said specific polymorphism in the mouse genome by usual techniques: Target sequence (SEQ ID NO9):

TGCTACAGCTGCCAGC CAG GACAGCCAGTCTGTGC Mutated oligonucleotide (SEQ ID NOlO):

TGCTACAGCTGCCAGC TGG GACAGCCAGTCTGTGC The present invention also relates to a method for the preparation of a transgenic mouse characterized in comprising the steps of: a transgene containing cDNA that encodes a human slc30a8 protein (SEQ ID N°l) or preferably a human slc30a8 protein mutated at position 325, and more preferably a human slc30a8/R325W protein (SEQ ID N°2); subsequently the transgene is microinjected into a male proneucleus of a mouse fertilized egg; thus obtained egg cell is cultured and then transplanted into an oviduct of a pseudopregnant female mouse; after rearing up the recipient animal, baby mice that have the above-mentioned cDNA are selected from the mice born from the recipient animal. DRUG SCREENING

An embodiment of the invention is its use to predict an individual's response to a particular drug to treat Type II diabetes. It is a well-known phenomenon that in general, patients do not respond equally to the same drug. Much of the differences in drug response to a given drug is thought to be based on genetic and protein differences among individuals in certain genes and their corresponding pathways. The present invention defines particular SNPs, haplotypes, and genes that are associated with Type II diabetes. Some current or future therapeutic agents maybe able to affect pathways that are related to such SNPs, haplotypes, and/ or genes directly or indirectly and therefore, be effective in those patients whose Type II diabetes risk is in part determined by such SNPs, haplotypes, and/or genes. On the other hand, those same drugs may be less effective or ineffective in those patients who do not have particular alleles of said SNPs and/or haplotypes. Therefore, the SNPs and/or haplotypes of the present invention maybe used to guide choice of therapeutic agent in a given individual. In one embodiment, a method for monitoring the effectiveness of a drug in the disclosed transgenic non-human mammal to design a treatment for Type II diabetes comprises, monitoring the levels of fasting blood glucose and insulin secretion after treatment with said drug, and comparing these levels before said treatment and after said treatment, monitoring the beta cell mass in pancreatic islets and comparing these masses before said treatment and after said treatment, monitoring diabetic complications (retinopathy, micro-angiopathy, vascular complications) and comparing these complications before said treatment and after said treatment.

With a transgenic mouse as an example of the transgenic non-human mammal, the generating method of a transgenic mouse is explained more specifically as follows: a transgene containing cDNA that encodes a mouse Slc30a8/Q324W gene (see example with SEQ ID N°8) or a human slc30a8/R325W gene (SEQ ID N°2), which have been separated by a screening of an imprinted gene that exhibits maternal expression, into a vector; the transgene is microinjected into a male pronucleus of a fertilized egg and is cultured; subsequently the transgene is brought back into an oviduct of a pseudopregnant female mouse; after rearing up the recipient animal, baby mice that have the above-mentioned cDNA are selected from the mice born from the recipient animal. As the above-mentioned fertilized egg of a mouse, any fertilized egg obtained through a mating of mice derived from 129/sv, C57BL/6, BALB/c, C3H, SJL/Wt or the like can be used, however, it is preferable to use a fertilized egg from B6C3H mice being obtainable by mating a C57BL/6 (B6) mouse with a C3H mouse because it is possible to distinguish the independence of male and female pronuclei in cytoplasm at a pronuclear stage. Further, the appropriate number of transgenes to be introduced is 100 to 3000 molecules per fertilized egg. Still further, the baby mice having cDNA can be selected by dot hybridization method wherein a mouse Slc30a8/Q324W gene or a human Slc30a8/R325W gene is being introduced with crude DNA extracted from a tail of a mouse or the like is used as a probe, PCR method using a specific primer, or other such methods.

For the screening method of a remedy for diabetes of the present invention using the transgenic non-human mammal of the present invention, a diabetic-prone transgenic non- human mammal is mainly used. Specifically, by administering a subject material to a diabetic-prone transgenic non-human mammal and then by measuring the glucose level in urine, or blood collected from the foot of an eye ball, a tail or the like, of the transgenic non-human mammal, or by considering the survival rate etc, the therapeutic effect of the subject material against diabetes is evaluated.

In the screening method of the invention, the subject material (the material tto be studied for its potential effect in the treatment of diabetes) is administered to the transgenic non-human mammal according to the invention and the pancreatic beta cell mass is assessed. The present invention makes it possible to conduct a screening of a novel remedy for diabetes by using a mouse expressing the gene from mouse Slc30a8/Q324W or the gene from human Slc30a8/R325W. Further, the transgenic non-human mammal such as the mouse expressing mouse Slc30a8/Q324W or human Slc30a8/R325W genes of the present invention can be used as model animal for diabetes researches and is useful for elucidating the onset mechanism and for the development of a novel remedy for diabetes. The present invention is also related to a method for detecting the susceptibility of an individual to diabetes or a similar metabolic disease, comprising a step of identification of a single nucleotide polymorphism such as described previously, wherein the presence of said SNP in an individual is associated to a risk to develop said disease by the individual, said risk being more important than if the SNP is absent, i.e. non detected.

REFERENCES

- Aarts M et al. (2006) Nucleic Acids Res. 34(21):el47.

- Bell, G. et al. (1984). Diabetes 33:176. - Baribault H, Kemler R. (1989) MoI Biol Med. Dec;6(6):481-92.

- Comall, R. J. et al. (1991) Nature 353:262.

- Davies, J. L. et al. (1994). Nature 371 :130-136.

- De Gouyon, B. et al. (1993). Proc. Nat. Acad. Sci. USA 90:1877.

- Froguel, P. et al. (1992). Nature 356:162-164. - Froguel, P. et al. (1993). N. Engl. J. Med. 328:697-702.

- Garchon, H. J. et al. (1991). Nature 353:260.

- Hammer RE et al. (1985) Nature. Jun 20-26;315(6021):680-3.

- Harris, C. L. et al. (1996). Nature Genet. 13:161-166.

- Harris, M. L, et al. (1992) Diabetes Care 15:815-819 - Hattersley, A. T. et al. (1992). Lancet 339:1307-1310.

- Kadowaki et al. (1994). N. Engl. J. Med. 330:962-968.

- King, H. and Zimmer, P. (1988). WId Hlth. Statist. Quart. 41 : 190- 196;

- Lindner, T. et al. (1997). J. Clin. Invest. 100:1400-1405.

- Mahtani, M. M. et al. (1996). Nature Genetics 14:90-94. - Morahan, G. et al. (1994). Proc. Nat. Acad. Sci. USA 91 :5998

- Prochazka, M. et al. (1987). Science 237:286.

- Robertson EJ (1991) Biol Reprod. Feb;44(2):238-45.

- Rotter et al. (1990). Diabetes Mellitus: Theory and Practice, 378-413.

- Serreze, D. et al. (1994). J. Exp. Med. 180:1553. - Steiner et al. (1990). Diabetes Care 13:600-609.

- Taylor et al. (1992). Endocrine Rev. 13:566-595.

- Thompson S et al. (1989) Cell. Jan 27;56(2):313-21.

- Todd, J. A. (1990). Immunol. Today 11 :122.

- Todd, J. A., et al. (1991). Nature 351 :542. - Tojo H et al. (1995-1996) Cytotechnology.19(2): 161-5.

- Van den Ouwenland, J. M. W. et al. (1992). Nature Genet. 1 :368-371.

- Verge, C. F. et al. (1998). J. Clin. Invest. 102:1569-1575.

- Yamagata, K. et al. (1996). Nature 384:458-460.

- Yanai, K. et al. (1999). J. Biol. Chem. 274:34605-34612.

Claims

I . A human protein having the sequence such as shown in SEQ ID N°2, with a W at position 325. 2. A mouse protein having the sequence such as shown in SEQ ID N°8, with a

W at position 324.

3. Polynucleotides encoding for proteins having sequences such as shown in SEQ ID N°2 or SEQ ID N°8.

4. SNPs present in the Slc30a8 gene, located in a strech of codons encoding for the aminoacids sequence AASXX'SQV in the slc30a8 protein, wherein X is selected among R and Q, and X' is selected among D and E.

5. SNP of claim 4 where X is not R and Q.

6. Non-silent SNP present in the Slc30a8 gene, located in a strech of codons encoding for the aminoacids sequence AASWX' SQV wherein X' is selected among D and E.

7. SNP of one of claims 4 to 7, wherein a specific codon is modified in the gene coding for the mouse slc30a8 gene, leading to the aminoacids replacement Q324W in the corresponding protein.

8. A transgenic non human mammal containing a polynucleotide wherein said polynucleotide encodes for a protein having anyone of the sequences shown in SEQ. ID.

NO. 3-7.

9. A transgenic non-human mammal preferably selected among rat, mouse, dog, chimp and macaque, wherein said transgenic animal expresses an heterologous slc30a8 gene comprising a SNP as defined in one of claims 4 to 7. 10. The transgenic animal of claim 9, wherein expression of the heterologous slc30a8 gene comprising a SNP is obtained by introducing into said animal genome a gene comprising a polynucleotide sequence coding for the corresponding slc30a8 protein under control of regulatory elements functional in said animal.

I I. The transgenic animal of claim 9, wherein expression of said heterologous slc30a8 gene comprising a SNP is obtained by introducing the polymorphism into the native gene in the genome of said animal.

12. A transgenic non-human mammal, including rat, mouse, dog, chimp, macaque, into which a similar polymorphism at the equivalent position of aminoacid 325 in the human sequence is introduced in the slc30a8 gene having a sequence such as shown in SEQ ID N°3-7.

13. A transgenic non-human mammal into which a human Slc30a8/R325W gene is introduced, wherein said gene encodes for a protein having the sequence shown in SEQ ID N°2.

14. The transgenic non-human mammal according to claim 12, wherein the transgenic non-human mammal is a mouse.

15. A method for generating a transgenic mouse comprising the steps of:

- obtaining a transgene containing cDNA that encodes a mouse Slc30a8/Q324W protein or a human slc30a8/R325W protein,

- microinjecting said transgene into a male proneucleus of a mouse fertilized egg,

- culturing said obtained egg cell,

- transplanting said egg into an oviduct of a pseudopregnant female mouse,

- selecting baby mice that express the above-mentioned cDNA from the mice born from the recipient animal.

16 A screening method of a remedy for diabetes characterized in using a similar gene polymorphism encoding for a specific aminoacid at the position 325 in the human sequence, wherein the diabetic prone transgenic non-human mammal according to claims 8 to 14 is used. 17. The screening method of a remedy for diabetes according to claim 16, wherein a subject material is administered to a transgenic non- human mammal and the level of glucose in urine and/or blood obtained from the transgenic non-human mammal is measured.

18. The screening method of a remedy for diabetes according to claim 16, wherein the transgenic non-human mammal is a mouse.

19. The screening method of claim 16, wherein a subject material is administered to the transgenic non-human mammal and the pancreatic beta cell mass is assessed.

20. A method for detecting the susceptibility of an individual to diabetes or a similar metabolic disease, comprising a step of identification of a single nucleotide polymorphism such as described in claims 4 to 7, wherein the presence of said SNP in an individual is associated to a risk to develop said disease by the individual, said risk being more important than if the SNP is absent.