WO2004067771A1 - Association of the gys1 genotype with increased risk for diabetes mellitus type 2 - Google Patents

Abstract

The present invention relates to methods, nucleic acid sequences and kits for determining a predisposition for metabolic diseases, including diabetes mellitus. The inventors have identified an association between metabolic diseases and particular mutations/polymorphisms in the region controlling expression of the muscle glycogen synthase 1 gene. The predisposition is determined by assaying a biological sample from an individual for the presence or absence of at least one mutation or polymorphism within the DNA sequence of the region controlling expression of muscle glycogen synthase 1, or by assaying for the presence or absence of at least one mutation or polymorphism which is in linkage disequilibrium with any of the polymorphisms identified by the inventors. The invention also relates to methods for determining the haplotype of mutations/polymorphisms in the muscle glycogen synthase gene.

Description

ASSOCIATION OF THE GYS1 GENOTYPE WITH INCREASED RISK FOR DIABETES MELLITUS TYPE

All patent and non-patent references cited in the present application are hereby incorporated by reference in their entirety.

Field of invention

The present invention relates to methods, nucleic acid sequences and kits for determining a predisposition for metabolic diseases. The invention also relates to methods for determining the haplotype of mutations/polymorphisms in the muscle glycogen synthase gene.

Background of invention

Diabetes.

Diabetes mellitus is a serious metabolic disease that is defined by the presence of chronically elevated levels of blood glucose (hyperglycemia). This state of hyperglycemia is the result of a relative or absolute lack of activity of the peptide hormone, insulin.

Insulin is produced and secreted by the β-cells of the pancreas. Insulin is reported to promote glucose utilisation, protein synthesis, lipogenesis, and the formation and storage of carbohydrate energy as glycogen. Glucose is stored in the body as glycogen, a form of polymerised glucose, which may be converted back into glucose to meet metabolism requirements. Under normal conditions, insulin is secreted at both a basal rale and at enhanced rates following glucose stimulation, all to maintain metabolic homeostasis by glycolysis, gluconeogenesis and lipogenesis.

The term diabetes mellitus encompasses several different hyperglycemic states.

These states include Type 1 (insulin-dependent diabetes mellitus or IDDM) and

Type 2 (non-insulin dependent diabetes mellitus or NIDDM) diabetes. The hyperglycemia present in individuals with Type 1 diabetes is associated with deficient, reduced, or non-existent levels of insulin that are insufficient to maintain blood glucose levels within the physiological range. Conventionally, Type 1 diabetes is treated by administration of replacement doses of insulin, generally by a parenteral route.

Type 2 diabetes is an increasingly prevalent disease of aging and/or changes in lifestyle. It is initially characterised by decreased sensitivity to insulin and a compensatory elevation in circulating insulin concentrations, the latter of which is required to maintain normal blood glucose levels. Increased insulin levels are caused by increased secretion from the pancreatic beta cells, and the resulting hyperinsulinemia is associated with atherosclerotic complications of diabetes. As insulin resistance worsens, the demand on the pancreatic beta cells steadily increases until the pancreas can no longer provide adequate levels of insulin, resulting in elevated levels of glucose in the blood. Ultimately, overt hyperglycemia and hyperlipidemia occur, leading to the devastating long-term complications associated with diabetes, including cardiovascular disease, renal failure and blindness. The exact mechanism(s) causing type 2 diabetes are unknown, but result in impaired glucose transport into skeletal muscle and increased hepatic glucose production, in addition to inadequate insulin response. Dietary modifications are often ineffective, therefore the majority of patients ultimately require pharmaceutical intervention in an effort to prevent and/or slow the progression of the complications of the disease. Many patients can be treated with one or more of the many oral anti- diabetic agents available, to improve insulin sensitivity and increase insulin secretion. Examples of drugs include metformin which suppresses hepatic glucose production; sulfonylurea, which increases the insulin secretion; and glitazones, which improve peripheral insulin-sensitivity. Despite the utility of these agents, 30- 40% of diabetics are not adequately controlled using these medications and require subcutaneous insulin injections. Additionally, each of these therapies has associated side effects. For example, sulfonylureas can cause hypoglycemia and troglitazone can cause anemia, weight gain, and some of them severe hepatoxicity.

As can be understood from the above, it is important to be able to detect diabetes of Type 2 as early as possible in order to initiate the necessary dietary modifications, increase the level of physical activity or administer the required pharmaceutical(s) so that the possible side-effects in the subject can be reduced and/or postponed. Genetic component of diabetes

Although it is generally believed that metabolic diseases, among these diabetes mellitus Type 2, are mainly facilitated by wrong nutrition, lack of physical activity and age, it has also been appreciated that there may be a genetic component in the causes leading to diabetes Type 2. In fact, diabetes is highly heritable. Several studies have identified polymorphisms in genes coding for enzymes and transcription factors that are central in the glucose metabolism.

Maturity onset of diabetes in the Young (MODY) constitutes a subgroup of diabetes mellitus. It is defined by onset before 25 years of age, the patients are usually lean and it has an autosomal dominant inheritance. Contrary to diabetes mellitus type 2 the primary defect is in the glucose-stimulated insulin secretion (Froguel.P, Velho.G: Molecular Genetics of Maturity-onset Diabetes of the Young. Trends Endocrinol.Metab 10:142-146, 1999) (OMIM 125850). At least five subtypes are identified with genetic defects in hepatic nuclear faktor-4α (MODY1), glucokinase (MODY2), hepatic nuclear factor-1α (MODY3), insulin promoter factor- 1 (MODY4), and hepatic nuclear faktor-1 β (MODY5). More than 135 mutations have been detected in these genes. The prevalence is calculated to be 2-5% of all diabetes patients (Ledermann.HM: Maturity-onset diabetes of the young (MODY) at least ten times more common in Europe than previously assumed? Diabetologia 38:1482, 1995). A correlation between mutations in a glucokinase enzyme and Maturity onset of diabetes in the young (MODY2) has been reported (US 5,541 ,060 Bell et al). The physiological background for the correlation is probably that the mutations caused amino acid substitutions in the glucokinase enzyme.

Fenger et al (Diabet. Med 2000, 17:735-40) studied the impact of the intron Xba1- polymorphism in the muscle glycogen synthase gene and concluded that the polymorphism is correlated to insulin resistance and diastolic blood pressure. The reason lying behind the correlation was not determined. Groop et al (New England J

Medicine, 1993, 328:10-14) investigated the same polymorphism and additionally determined the level of activity of glycogen synthase in muscle biopsies and concluded that the level of expression was not affected by the alleles. Nakayama et al (Gene, 1994 150:391-393) have discovered 10 polymorphisms in a region lying 4kb upstream from the transcription initiation site of muscle glycogen synthase. They did not find any difference in allele frequency between type 2 patients and control subjects.

Orho et al (Diabetes 1995, 44:1099-1105) set out to investigate the genetic reasons for impaired glycogen synthase activity in type 2 diabetics. They isolated and sequenced the gene and found 3 polymorphic sites and one mutation. The mutation, which results in a gly/ser exchange in exon 11 was found in 2 of 228 type 2 patients and in 0 of 154 control subjects. These two patients had severe insulin resistance and premature arteriosclerosis.

Bjørbaek et al (Diabetes 1994, 43:976-938) identified a number of polymorphisms in both the coding region and the promoter region of glycogen synthase (GS) and in the insulin-responsive glucose transporter (GLUT4). They concluded that genetic abnormalities in these genes were unlikely to be major contributors to the insulin- resistant glucose utilisation in muscle among Caucasian NIDDM patients.

Shimomura et al (Diabetologica 1997, 40:947-952) have identified two polymorphisms in the coding region of the human muscle glycogen synthase gene.

These two polymorphisms are Met416Val in exon 10 and Pro442Ala in exon 11. There were no statistical differences between NIDDM patients with and without the mutation in terms of glucose effectiveness, age, body mass index, levels of glycated haemoglobin and serum lipids for either of the polymorphisms.

From the above it can be seen that a number of polymorphisms have been identified in the genes which are central in glucose metabolism. However, to date none of the polymorphisms/mutations have turned out to be directly connected to metabolic disorders and none of the polymorphisms account for more than a very small percentage of the subjects suffering from metabolic disorders.

Summary of invention

In a first aspect the invention relates to a method for diagnosing metabolic disorders and/or for predicting an increased risk of a subject for developing metabolic disorders comprising obtaining a biological sample from a subject, assaying for at least one mutation or polymorphism within the DNA sequence of the region controlling expression of muscle glycogen synthase 1 (GYS1), said at least one mutation or polymorphism being located in a target nucleic acid sequence selected from a) the sequence comprising the nucleotides from -950 to -550 of SEQ ID No 1 counted from the transcription start site, or a part thereof, b) the complementary region of the nucleotides from -950 to -550 of SEQ ID No 1 counted from the transcription start site, or a part thereof, c) a sequence being at least 90 % identical with any of a) or b) or part thereof, more preferably at least 95 % identical, more preferably at least 96 % identical, such as at least 97% identical, e.g. at least 98% identical, such as at least 99% identical, e.g. at least 99.5% identical.

The assay can be performed ex vivo or in vivo

The presence of such a polymorphism or mutation is indicative of an increased risk for contracting a metabolic disease and/or indicating the presence of a metabolic disease.

The transcription start site is as described in SEQ ID No 1 of Figure 1. Whenever reference is made to the transcription start site and to SEQ ID No 1 this reference is intended to be to the sequence as disclosed in Figure 1 of the present application.

The inventors of the present invention have determined that mutations in the region defined above are correlated to the occurrence of metabolic disorders, in particular to syndrome X and diabetes. Although a very large part of the GYS1 gene has been scanned for polymorphisms correlated to e.g. diabetes none of the previous workers have been able to identify a polymorphism which accounts for more than a minor fraction of patients. For this reason, GYS1 is not the subject of intense investigation into the genetic background of diabetes. Other genes involved in the activation of muscle glycogen synthase, in particular GSK3, have received far more attention in the prior art. The inventors have discovered that one particular type of polymorphism in the above identified region, namely at position -849 (Poly2) counted from the transcription start site, has a strong correlation to diabetes mellitus, additionally signified by significantly higher level of plasma insulin, a significantly higher insulin secretion index, as well as a higher level of plasma cholesterol and of LDL (low density lipoprotein). These factors are all correlated to metabolic disorders, and more particularly to the diabetes mellitus type 2.

The inventors have furthermore discovered that subjects which carry at least one of a group of three coupled polymorphisms in the above identified region namely at position -916, -618, and -588 (Polyl , Poly3, and Poly4) counted from the transcription start site, have a higher level of cholesterol and LDL. This is in particular the case for subjects being homozygous for the mutant alleles. These factors are indicative of a metabolic disorder such as syndrome X.

It is anticipated that the region encompassing and surrounding the muscle glycogen synthase gene may contain other polymorphisms that are in linkage disequilibrium with either Poly2 or Polyl /Poly3/Poly4. Such other polymorphisms may also serve to diagnose and/or predict an increased risk of a subject for developing the metabolic disorders associated with Poly2 or Polyl /Poly3/Poly4. An example of such other polymorphism is a C to T change at position -740 (Poly5, also indicated in Figure 1).

Thus, in a further aspect, the invention relates to a method for diagnosing metabolic disorders and/or for predicting an increased risk of a subject for developing metabolic disorders comprising assaying for at least one mutation or polymorphism which is in linkage disequilibrium with any one or more of the following alleles: -916:G (Polyl), -849:G (Poly2), -618:C (PolyS), -588:G (Poly4).

The identified subjects carrying the mutant alleles account for 7-10 % of all subjects diagnosed with diabetes mellitus type 2. The importance of the polymorphisms in this respect thus exceeds the importance of any other known polymorphisms. Very importantly the polymorphisms can be detected at a time when the subjects have not developed clinical signs of diabetes, more specifically at a time when they do not have an elevated blood glucose level. Measuring the blood glucose level is still the most important (if not only generally accepted) way of diagnosing diabetes mellitus. However for the type 2 diabetics carrying a polymorphism according to the present invention measuring the blood glucose level is not enough to establish a diabetes diagnosis. The blood glucose level in these subjects is within normal limits. As evidenced by the significantly elevated plasma insulin level and significantly elevated insulin secretion index subjects carrying the polymorphism are indeed type 2 diabetics. The test for polymorphism will also reveal subjects which cannot be classified as diabetics yet. For these subjects development of a true diabetic disorder is merely a matter of time.

Expressed in another way the present inventors have identified a completely new therapeutic group of diabetic patients which may or may not show any of the clinical signs of diabetes mellitus all of which require medical treatment to lower the risk for side effects associated with diabetes.

The diagnostic methods according to the present invention may be described as a method of diagnosis of metabolic diseases but may likewise be regarded as a method of prognosis of metabolic diseases, assessing initiation of therapy and choice of therapeutic or prophylactic means at disposal now and/or by forthcoming developments in the future. Also, the present invention may be described as a method for guiding future research in development of new therapeutic and prophylactic means.

It follows from the above that detecting the absence of the polymorphisms as defined in the present invention (in particular any of Polyl , Poly2, Poly3, Poly4 and any polymorphisms coupled to any of these) amounts to determining a predisposition for not having a metabolic disorder/disease. It also follows that determining the presence of the wild-type allele amounts to determining a predisposition for not having a metabolic disorder/disease.

For all types of metabolic disorders it is of great importance to be able to initiate the correct treatment and/or change of lifestyle as early as possible. For patients with a metabolic disorder carrying a mutation in the above identified region it is of importance to initiate treatment as early as possible, because in these subjects the disorder has a genetic background, and the reason for the disorders cannot be eliminated by a change in lifestyle. Also to prolong the lifetime of the beta-cells in the pancreas it is important to lower the insulin secretion in these subjects. In a further aspect the invention relates to an isolated nucleic acid sequence comprising at least 10 contiguous nucleotides of the region from nucleotide -950 to -550 of SEQ ID NO 1 or the complementary strand.

These isolated oligonucleotides can be used as primers and probes in the detection of the polymorphisms identified by the present inventors in the described region.

In a still further aspect the invention relates to a kit for predicting an increased risk of a subject of developing a metabolic disorder comprising at least one probe comprising a nucleic acid sequence as defined above.

These kits can be used in the detection of polymorphisms in the identified region of the GYS1 promoter.

Furthermore, there is provided a method for detecting the haplotype of the muscle glycogen synthase gene in a subject, said method comprising determining the linkage phase between the nucleotide allele at position -849 of SEQ ID NO 1 (Poly2) counted from the transcription start or the corresponding position in the complementary strand and another polymorphism in said gene or its promoter.

As it is of importance in choosing a medication and in advising on change in lifestyle to know the allele of Poly2 it is also important to know the linkage phase of Poly2 with any other polymorphism which may be located in the GYS1 gene or its promoter (SEQ ID No 2).

Description of Drawings

Fig 1. The human GYS1 promoter. 1524 nucleotides upstream from the translation start site of human muscle glycogen synthase 1. Symbols: Polymorphisms with the symbol B&O are found by C. Bjørbaak et al.,1994. Diabetes and M. Orho et al.,1995.

Diabetes. Polymorphisms with the symbol B are found by C. Bjørbaek et al.,1994.

Diabetes. The transcription start site is marked by (+1). The translation start site is marked by ATG. The four polymorphisms, Poly 1-4, found by the present inventors are highlighted. The positions of the polymorphisms are; Polyl -916, Poly2 -849, Poly3 -618, Poly4 -588, and Polyδ -740. The positions are counted from the transcription start site. Known polymorphisms (Bjørbaek and Orho resp.) are located as follows: -251 G→A, -143 A→G, -43 G→A, -16 T→G, +42 C→T.

Fig 2. The sequence of the human muscle glycogen synthase gene (CHIP bioinformatics database). Upper bold cases are exons, Upper cases are introns, lower cases are the promoter. Nucleotides with an underscore are SNPs. The sequence is referred to in the present invention as SEQ ID No 2.

Fig. 3. Potential transcription factors that bind to the GYS1 promoter. Potential mammalian regulatory sites identified by computer analysis. The upper cases show the consensus sites for the transcription factors, the bold cases show the positions of the polymorphisms.

Detailed description of the invention

Definitions

Detection probe: an oligonucleotide linked to a detectable label Capture probe: an oligonucleotide linked to a solid surface Target nucleotide sequence: a nucleic acid isolated from an individual, comprising at least one polymorphism position identified in the present invention, or comprising a position corresponding to a polymorphism which is in linkage disequilibrium with any of the polymorphism identified herein, as well as further nucleotides upstream or downstream. The target nucleic acid can be used for hybridisation, for sequencing or other analytical purposes. Polymorphisms and mutations are ubiquitous in the genome. The distinction between polymorphisms and mutations lies in the frequency of alleles in a gene, where a mutation is defined if one allele amounts to more than 99% of the alleles, and polymorphisms accounts for all other situations. Both polymorphisms and mutations can be single nucleotide substitutions, deletions, insertions or rearrangements.

Sequence identity: is determined with any of the algorithms GAP, BESTFIT, or FASTA in the Wisconsin Genetics Software Package Release 7.0 or updates, using default gap weights. Potential binding sites for transcription factors in the glycogen synthase promoter

The present inventors have looked for potential core sequences for transcription factors flanking the sequences of the four polymorphisms found in the GYS1 promoter. Possible transcription sites were detected by using Matlnspector V2.2 prediction program, (http://www.transfac.gbf.de/cgi-bin). The consensus sites and the transcription factors identified by computer analysis are shown in figure 3 for Polyl , Poly2, Poly3, and Poly4.

It is important to identify specific binding sites for transcription factors, because a nucleotide substitution can inhibit the binding of transcription factors and thereby repress gene-transcription.

It is found that type 2 diabetic patients have a reduced concentration of GYS1- mRNA and GYS1 -protein in muscle cells. This can be due to a repression of the

GYS1 gene-transcription.

By using the Matlnspector program the present inventors have identified two potential binding sites for the transcription factors; KLF4 (Kruppel like factor 4) and IK2 (Ikaros 4) in the sequence comprising Poly2 (see figure 3).

Ikaros factor 2 (IK2)

Ikaros is a hemolymphopoietic restricted zinc finger transcription factor which is necessary for the ontogeny of lymphocytes.

The Ikaros gene is located on chromosome 7p13-p11.1 in humans, and generates at least eight isoforms through alternative splicing. All Ikaros isoforms have a common C-terminus containing a bipartite transcriptional activation domain and two zinc fingers facilitate dimerization with other Ikaros isoforms. All eight isoforms however differ in their N-terminal domain, which consists of four zinc fingers, three of which are necessary to bind DNA with high affinity. Thus only IK1 , IK2 and IK3 demonstrate high affinity DNA binding, while IK4 through IK8 have little to no DNA binding. The Ikaros homo-or heterodimers recognise the sequence 5^'tGGGA(A/T)^'3, where the core 5'GGGA'3 (3TCCC5) sequence is more important than the flanking nucleotides (Dorsam el al 2002).

Ikaros can also bind to the 5'GGAAA^'3 core motifs in the promoter region of target genes. (Karlsen et al, 2002)

IK2 binding elements can overlap Sp1 binding elements. Therefore IK2 may compete with Sp Ikaros has been shown to both activate and suppress transcription.

IK2 regulates the expression of "vasoactive intestinal peptide receptor 1 (VPAC-1)". The ligand for VPAC1 is "vasoactive intestinal peptide (VIP). VIP is a potent neurotropic factor and activates cytokine production, proliferation and apoptosis. VPAC1 is expressed in the central nervous system, peripheral nervous system, liver, lung, intestines and T-lymphocytes (Dorsam et al, 2002).

Ikaros is also critical for T-cell development and differentiation. It is also suggested that Ikaros has tumor-suppressor activity (Karlsson et al 2002). Mice with heterozygous deletions develop T-cell lymphoma or leukemia.

Ikaros regulates the expression of fibroblast growth factor receptors (FGFR's). FGFR's genes encode a complex family of transmembrane receptor tyrosine kinases. FGFR4 has been reported to be expressed mainly in the brain, nervous system, in adrenal, lung, kidney, pancreas, muscle and spleen. FGFR4 is involved in pituitary tumor development.

Kruppel like factors (SCLF)

Kruppel like factors belong to a family of c-terminal zinc finger transcription factors that exhibit homology to the Drosophilia melanogaster segmentation gene product

Kruppel. The targets of the transcription factor are 5ONCCC3 where N often is a A (Gray et al, 2001), or a GT-box like element (Bieker et al, 2001). Currently there are 12 members of Kruppel like factors which are expressed in different tissues. KLF^'s have different functions, they can be transcriptional activators or repressors.

There are at least 12 different KLF transcription factors which are expressed in different tissue. KLF's have different functions although they primarily bind to the same consensus-site (CNCCC). They primarily regulate differentiation of epithelial cells and help control the development of skeleton and kidneys.

KLF1 activates the β-globin gene and other erythroide cells by binding to the sequence CACCC.

KLF works as a repressor. KLF4 is primarily expressed in the gastrointestinal tract but is also expressed in lungs, testicles, the skin and thymus.

The KLF4 protein can act as both a gene activator and gene suppressor. KLF4 activates the expression of cytochrome P-450, keratin 4, keratin 19, cyclin D1 and p21 (waf/Cip1). KLF4 inhibits cell cycle, CYP1A1 and CD1 (inhibited cyclin D1). KLF4 binds to the sequence CACCC or to the consensus sequence for KLF4, which is 5'(G/A)(G/A)GG(C/T)G(C/T^'3 (Shie et al, 2000).

The gene expression of KLF4 itself is activated by INF-γ, which is a cytokine that is produced by the T-lymphocyte (Chen et al, 2000).

KLF5 can be found in the same places as KLF4. Unlike the suppressing effect of

KLF4 on the cell cycle KLF5 activates the cell cycle and suppresses the expression of KLF4. KLF4 can also suppress the gene transcription of its own gene. As KLF5 and KLF4 bind to the same DNA site (GC box) in the promoter of KLF4 the two Iranscription factors are a kind of antagonists. The transcription factors Cdx2 and APC activate the expression of KLF4 (Dang et al, 2002).

KLF6 activates a collagenous specific molecular chaperone protein (HSP47) that is important for synthesis of collagen. KLF6 binds to the consensus site CACCC but not to CTCCC (Yasuda et al, 2002). Over-expression of HSP47 can lead to development of liver cirrhosis, kidney fibrosis and atherosclerosis. KLF15 is found in adipocytes and myocytes and induces the expression of GLUT4 in muscle and adipose cells (KLF4 does not activate GLUT4 expression but suppresses it). The basal as well as the insulin-stimulated uptake of glucose in adipocyts and myocytes is hereby increased. The expression of GLUT4 depends on the synergy between KLF15 and MEP2A (belongs to the family of MADS-box transcription factors ("Myocyte enhancer factors")) (Gray et al, 2001).

KLF and Sp1 transcription factors contain the same c-terminal zinc finger motive that can bind to GC-boxes. The two factors therefore often recognise the same GC- rich areas. Therefore they can compete for the same DNA binding sites (Shie et al, 2000). Sp1 can also act as co-activator together with KLF.

Detection of the polymorphism The present inventors have discovered that a series of polymorphisms located in the promoter region of the human GYS1 gene are closely linked to an increased risk of a subject for developing metabolic disorders.

Accordingly, in a first main aspect, the invention relates to a method for diagnosing metabolic disorders and/or for predicting an increased risk of a subject for developing metabolic disorders comprising three steps. In the first step, a biological sample is obtained from a subject. For the second step, there are two possible scenarios that solve the same technical problem. In the first scenario, the second step comprises assaying for at least one mutation or polymorphism within the DNA sequence of the region controlling expression of muscle glycogen synthase 1

(GYS1), said at least one mutation or polymorphism being located in a target nucleic acid sequence selected from

- the sequence comprising the nucleotides from -950 to -550 of SEQ ID No 1 counted from the transcription start site, or a part thereof, or - the complementary region of the nucleotides from -950 to -550 of SEQ ID

No 1 counted from the transcription start site, or a part thereof, or

- a sequence being at least 90 % identical with any of a) or b) or a part thereof, more preferably at least 95 % identical, more preferably at least 98 % identical, In a second scenario, the second step comprises assaying for at least one mutation or polymorphism which is in linkage disequilibrium with any one or more of the following alleles: -916:G (Polyl), -849:G (Poly2), -618:C (Poly3), -588:G (Poly4). Both scenarios for the second step are followed by a third step, comprising diagnosing a metabolic disorder and/or predicting an increased risk of a subject for developing metabolic disorder on the basis of the presence or absence of said mutation or polymorphism.

Any polymorphism located in the described region may of course be assessed with the same specificity in the corresponding complementary strand. However it is also expected that these polymorphisms can be found in other animal species and that the consequences of carrying such alleles are much the same for an animal as for a human being. There may also be additional minor variations among human or animal subjects such as SNPs in the region, which have not been discovered yet. Therefore a target nucleic acid sequence for assessing the alleles of the polymorphisms identified herein may differ slightly from the sequence of SEQ ID No

1. This may also be caused by sequencing errors. Consequently, the present inventors contemplate that the polymorphisms can be detected in a sequence having at least 90 % sequence identity, more preferably at least 95 % identical, more preferably at least 98 % identical with SED ID No 1 or its complementary sequence.

It is presently believed that the physiological background of the polymorphisms can be found in defective binding of transcription factors to the promoter region or defective folding of the promoter sequence before and/or during transcription. The typical size of a motif for a transcription factor is in the range of 5-10 nucleotides. As there is an effect on the level of expression of a base change at several discrete positions in the promoter region of the GYS1 gene it is expected that there will also be an effect on the level of expression if any base is changed at least 15 bases from any of the base changes described by the present inventors.

Consequently, in a more preferred embodiment, the target nucleic acid sequence is selected from the sequence comprising the nucleotides from -925 to -575 of SEQ ID No 1 counted from the transcription start site, or a part thereof, the complementary region of the nucleotides from -925 to -575 of SEQ ID No 1 counted from the transcription start site, or a part thereof.

In an even more preferred embodiment, the target nucleic acid sequence is selected from the sequence comprising the nucleotides from -941 to -891 of SEQ ID No 1 counted from the transcription start site, or a part thereof, the complementary region of the nucleotides from -941 to -891 of SEQ ID No 1 counted from the transcription start site, or a part thereof, the sequence comprising the nucleotides from -874 to -824 of SEQ ID No 1 counted from the transcription start site, or a part thereof, the complementary region of the nucleotides from -874 to -824 of SEQ ID No 1 counted from the transcription start site, or a part thereof, the sequence comprising the nucleotides from -643 to -593 of SEQ ID No 1 counted from the transcription start site, or a part thereof, the complementary region of the nucleotides from -643 to -593 of SEQ ID No 1 counted from the transcription start site, or a part thereof, the sequence comprising the nucleotides from -613 to -563 of SEQ ID No 1 counted from the transcription start site, or a part thereof, and the complementary region of the nucleotides from -613 to -563 of SEQ ID No 1 counted from the transcription start site, or a part thereof,

As can be seen from the appended examples (example 2), the C→G change at position -849 of SEQ ID NO 1 counted from the transcription start site or the corresponding mutation in the complementary strand or a mutation linked to said

C→G change is closely linked to the occurrence of diabetes mellitus type 2 in human beings. Furthermore the frequency of this change (7-10%) is relatively high among diabetics with type 2 diabetes. Consequently, this polymorphism and any polymorphism linked to it is the most important of the polymorphisms of the present invention.

Another important group of polymorphisms are those selected from the group of polymorphisms of SEQ ID NO 1 and the corresponding polymorphisms in the complementary strand, said group comprising: T→G at position -916, and/or T→C at position -618, and/or A→G at position -588, and/or a polymorphism/mutation linked to any of the above polymorphisms/mutations, such as C→T at position -740. These polymorphisms are all linked to the occurrence of an elevated plasma cholesterol level (see example 2). In the population which has been investigated by the present inventors these three polymorphisms are tightly linked and only two alleles have been seen, T-T-A and G-C-G respectively. This may differ in other populations. Therefore the present inventors regard these three polymorphisms as potentially independent or they may appear as mutations in other populations.

The metabolic disorder which are correlated with the polymorphisms include syndrome X, metabolic syndrome, dyslipidemia, diabetes, atherosclerosis, and adipositas. This is because the clinical parameters from the patients with the less frequent alleles suffer from increased plasma cholesterol, increased plasma insulin, increased insulin secretion and increased plasma LDL. These clinical parameters are indicative of one or more of the above identified metabolic disorders.

The polymorphism, in particular the polymorphism at -849 of SEQ ID No1 is especially indicative of diabetes mellitus Type 2. The polymorphisms and in particular Poly2 could be classified as a subtype of MODY (Maturity onset of diabetes in the Young) but with the primary defect in the muscles.

The polymorphisms at bases no -916, -618, and -588 are particularly indicative of syndrome X, metabolic syndrome, dyslipidemia, diabetes, atherosclerosis, and adipositas.

Detection methods

It will be apparent to the person skilled in the art that there are a large number of analytical procedures which may be used to detect the presence or absence of one or more of the polymorphisms identified herein. In general, the detection of allelic variation requires a mutation discrimination technique, optionally an amplificati on reaction and a signal generation system. Table 1 lists a number of mutati on detection techniques, some based on the PCR. These may be used in combinati on with a number of signal generation systems, a selection of which is listed in Table 2. Further amplification techniques are listed in Table 3. Many current methods for the detection of allelic variation are reviewed by Nollau et al., Clin. Chem. 43, 1114- 1120, 1997; and in standard textbooks, for example "Laboratory Protocols for Mutation Detection", Ed. by U. Landegren, Oxford University Press, 1996 and "PCR", 2.sup.nd Edition by Newton & Graham, BIOS Scientific Publishers Limited, 1997.

Abbreviations:

ALEX™ Amplification refractory mutation system linear extension APEX Arrayed primer extension

ARMS™ Amplification refractory mutation system

ASH Allele specific hybridisation b-DNA Branched DNA

CMC Chemical mismatch cleavage base pa

COPS Compet tiitive oligonucleotide priming system

DGGE Denaturi ing gradient gel electrophoresis

FRET Fluorescence resonance energy transfer

LCR Ligase chain reaction

MASDA Multiple allele specific diagnostic assay

NASBA Nucleic acid sequence based amplification flt-1 VEGF receptor-1

OLA Oligonucleotide ligation assay

PCR Polymerase chain reaction

PTT Protein truncation test

RFLP Restriction fragment length polymorphism

SERRS Surface enhanced raman resonance spectroscopy

SDA Strand displacement amplification

SNP Single nucleotide polymorphism

SSCP Single-strand conformation polymorphism analysis

SSR Self sustained replication

TGGE Temperature gradient gel electrophoresis Table 1 -Mutation Detection Technigues

General:

DNA sequencing, Sequencing by hybridisation

Scanning: PTT*, SSCP, DGGE, TGGE, Cleavase, Heteroduplex analysis, CMC, Enzymatic mismatch cleavage

* Note: not useful for detection of promoter polymorphisms.

Hybridisation Based

Solid phase hybridisation: Dot blots, MASDA, Reverse dot blots, Oligonucleotide arrays (DNA Chips)

Solution phase hybridisation: Taqman.TM.-U.S. Pat. No. 5,210,015 & U.S. Pat. No.

5,487,972 (Hoffmann-La Roche), Molecular Beacons-Tyagi et al (1996), Nature

Biotechnology, 14, 303; WO 95/13399 (Public Health Inst, New York), ASH

Extension Based: ARMS™-allele specific amplification (as described in European patent No. EP-B-

332435 and U.S. Pat. No. 5,595,890), ALEX™-European Patent No. EP 332435 B1

(Zeneca Limited), COPS-Gibbs et al (1989), Nucleic Acids Research, 17, 2347.

Incorporation Based:

Mini-sequencing, APEX Restriction Enzyme Based:

RFLP, Restriction site generating PCR

Ligation Based:

OLA-Nickerson et al. (1990) P.N.A.S. 87:8923-8927.

Other: Invader assay

Table 2--Signal Generation or Detection Systems

Fluorescence:

FRET, Fluorescence quenching, Fluorescence polarisation—United Kingdom Patent No. 2228998 (Zeneca Limited)

Other:

Chemiluminescence, Electrochemiluminescence, Raman, Radioactivity,

Colorimetric, Hybridisation protection assay, Mass spectrometry, SERRS-WO

97/05280 (University of Strathclyde). Amplification

The DNA may be amplified by one of many methods. One of the best known and widely used amplification methods is the polymerase chain reaction (referred to as PCR) which is described in detail in US 4,683,195, US 4,683,202 and US 4,800,159, however other methods such as LCR (Ligase Chain Reaction, see Genomics (1989)

4:560-569), NASBA (Nucleic Acid Sequence-Based Amplification, see PCR Methods Appl (1995) 4, S177-S184), strand displacement amplification (Current Opinion in Biotechnology (2001) 12:21-27), rolling circle amplification (Current Opinion in Biotechnology (2001) 12:21-27), or non PCR methods such as T7 polymerase amplification can be applied.

If the selected polymorphisms are situated a long distance from each other fairly long fragments of genomic DNA have to be amplified. Several techniques that result in amplification of fairly long fragments of DNA are described in the art. One particular useful procedure for this purpose is the so-called "long range PCR", which allows amplification of very large fragments (Proc. Natl. Acad. Sci. USA. (1994) 91 : 2216-20; Methods-Mol-Biol. (1997) 67: 17-29.). Kits allowing the amplification of templates up to 40,000 base pair long are commercially available, e.g. the TripleMaster™ PCR System (cat. no. 0032 008.208) of Eppendorf AG, Hamburg, Germany.

Amplification may be performed by using the primer pairs, SEQ ID NO 3-10 disclosed below. The upstream and downstream primer may be selected so that the amplified fragment comprises the loci to be assessed. By selecting a primer pair consisting of SEQ ID No 3 and 10 a fragment is amplified which contains all four polymorphic loci.

Table 3-Further Amplification Methods SSR, NASBA, LCR, SDA, b-DNA

The table below discloses primers that can be used for amplification of target polynucleotides which contain the specific polymorphisms, Polyl , Poly2, Poly3, and Poly4 as well as restriction enzymes which can be used for the detection using RFLP. The PCR amplification for Poly2 can be carried out in a volume of 25 μl, containing 200 ng of genomic DNA, 0.2 mM of each dNTP, 1.5 mM MgCI₂, 0.6 μM of each primer, 1 units of HotStarTaq DNA polymerase (Qiagen), 1x HotStar 10xbuffer, 1x HotStar 5x Q-Solution. PCR conditions are as follows: denaturation at 94°C for 15 min, followed by 35 cycles of: denaturation at 94°C for 40 sec; annealing at 59°C for 40 sec; and extension at 72°C for 40 sec with a final extension at 72°C for 10 min. RFLPs are detected after digestion with Mnl\ (New England BioLabs®,USA). The fragments are resolved on a 4% (w/v) SeaKem/Nusive® agarose gel and visualised by staining with GelStar® (BMA, Marine, USA). This amplification protocol can be used for all detection methods which involve amplification.

RFLP

One method is restriction fragment length polymorphism (RFLP) in which a target polynucleotide is cleaved by a restriction endonuclease. The resulting cleaved polynucleotides are separated on a gel or by capillary electrophoresis. If the polymorphism is located in the recognition sequence the specific allele will determine whether the polynucleotide is cleaved in that specific place or not. The result is a difference in length of the cleaved fragments. The table above discloses suitable restriction enzymes for cleaving the amplified fragments. RAPD

One widely used PCR-based approach for detection of polymorphisms involves the use of single short PCR primers of arbitrary sequence called RAPD primers (for random amplified polymorphic DNA; Reiter et al., Proc. Natl. Acad. Sci. USA 89:1477-1481 , 1992; Williams et al., Theoret. Appl. Genet. 82:489-498, 1991).

SSLP

A second category of PCR-based markers are called SSLPs (for simple sequence length polymorphisms). The method employing SSLPs is based on amplification across tandem repeats of one or a few nucleotides known as "microsatellites." Microsatellites occur frequently and randomly in most eukaryotic genomes and display a high degree of polymorphism due to variation in the numbers of repeated units. The technique is not relevant for detection of Polyl , Poly2, Poly3 or Poly4, but it may be relevant for detection of polymorphisms linked to any of these polymorphisms.

AFLP

A third category of PCR-based markers are called AFLPs (for amplified fragment length polymorphisms). In the method employing these markers, DNA from two polymorphic strains is cleaved with one or two restriction endonucleases, and adapters are ligated to the ends of the cleaved fragments (Vos et al., Nucleic Acids Research 23: 4407-4414, 1995). The fragments are then amplified using primers complementary to the adapter(s). The primers contain short stretches of random nucleotides at their 3' ends, which results in limiting the number of amplified fragments generated.

a s spectrometry

Detection of polymorphisms using mass spectrometry combines the technologies of mass spectrometry and polynucleotide hybridisation, amplification, extension and/or ligation techniques. The first step comprises amplification of the target nucleic acid sequence, which amplification may be carried out using the PCR protocol disclosed above. In a second step a difference in molecular weight between the alleles is generated. This can be done by a number of techniques: primer extension, translation of the polymorphic region into a protein sequence, hybridisation with allele specific probes and cleavage of non-hybridised single-stranded DNA. Further details on the numerous approaches to SNP detection with mass spectrometry can be found in Pusch et al, 2002, Pharmacogenomics 3(4):537-548.

Single strand conformation polymorphism Single strand conformational polymorphism (SSCP) is a further technique that can detect SNPs present in an amplified DNA segment (Hayashi, K. Genetic Analysis: Techniques and Applications 9:73-79, 1992). In this method, the double stranded amplified product is denatured. The separated strands assume a specific folded conformation based on intramolecular base pairing during electrophoresis in non- denaturing polyacrylamide gels. The electrophoretic properties of each strand are dependent on the folded conformation. The presence of single nucleotide changes in the sequence can cause a detectable change in the conformation and electrophoretic migration of an amplified sample relative to wild type samples, allowing SNPs to be identified.

Single base extension

Single base extension is a technique that allows the detection of SNPs by hybridising a single strand DNA probe to a captured DNA target (Nikiforov, T. et al. Nucl Acids Res 22:4167-4175). The probe is designed to be adjacent to the polymorphism in question. Once hybridised, the single strand probe is extended by a single base with labelled dideoxynucleotides. This single base corresponds to the allele at the polymorphic site. The labelled, extended products are then detected using calorimetric or fluorescent methodologies or using mass spectrometry. One specific method for single base extension is based on the program SnaPshot for ABI PRISM® 3100 Genetic Analyser (sequencer), from Applied Biosystems.

Sequencing

Sequencing of a target nucleic acid containing the polymorphic locus is also a way to determine the allele of a given locus.

Hybridisation based techniques

One very common and often preferred detection technique comprises hybridising a probe to a target sequence comprising the target nucleic acid sequence. Preferably the detection comprises the use of at least one labelled probe. The oligonucleotides are allowed to hybridise to the DNA under highly stringent conditions. In the scope of the present invention the term "hybridisation" signifies hybridisation under conventional hybridising conditions, preferably under stringent conditions, as described for example in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold

Spring Harbor, N.Y.). The term "stringent" when used in conjunction with hybridisation conditions is as defined in the art, i.e. 15-20°C under the melting point T_m, cf. Sambrook et al, 1989, pages 11.45-11.49. Preferably, the conditions are "highly stringent", i.e. 5-10°C under the melting point T_m. Under highly stringent conditions hybridisation only occurs if the identity between the oligonucleotide sequence and the locus of interest is 100 %, while no hybridisation occurs if there is just one mismatch between oligonucleotide and DNA locus. Such optimised hybridisation results are reached by adjusting the temperature and/or the ionic strength of the hybridisation buffer as described in the art. However equally high specificity may be obtained using high-affinity DNA analogues. One such high- affinity DNA analogues has been termed "locked nucleic acid" (LNA). LNA is a novel class of bicyclic nucleic acid analogues in which the furanose ring conformation is restricted in by a methylene linker that connects the 2'-0 position to the 4'-C position. Common to all of these LNA variants is an affinity toward complementary nucleic acids, which is by far the highest reported for a DNA analogue (ørum et al.

(1999) Clinical Chemistry 45, 1898-1905; WO 99/14226). LNA probes are commercially available from Proligo LLC, Boulder, Colorado, USA. Another high- affinity DNA analogue is the so-called protein nucleic acid (PNA). In PNA compounds, the sugar backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone (Science (1991) 254: 1497-1500).

In the present context, the term "label" means a group which is coupled to the nucleic acid and which can be used for the detection or other subsequent reactions e.g. immobilisation of the nucleic acid. The oligonucleotides may be labelled by a number of methods well known in the art. Conveniently, oligonucleotides may be labelled during their solid-phase synthesis using any of the many commercially available phosphoramidite reagents for 5' labelling. Illustrative examples of oligonucleotide labelling procedures may be found in US Pat 6,255,476. A wide variety of appropriate indicators are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. A fluorescent label is preferred because it provides a very strong signal with low background. It is also optically detectable at high resolution and sensitivity through a quick scanning procedure.

A particular fluorescent label has characteristic excitation and emission spectre which allows the simultaneous detection of several different fluorescent labelled molecules if the labels are selected appropriately.

A large number of different useful fluorescent labels are given in the art and may be selected from the group comprising, but not limited to: Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7 (trademarks for Biological Detection Systems, Inc.), fluorescein, acridin, acridin orange, Hoechst 33258, Rhodamine, Rhodamine Green, Tetramethylrhodamine,

Texas Red, Cascade Blue, Oregon Green, Alexa Fluor (trademarks for Molecular Probes, Inc.), 7-nitrobenzo-2-oxa-1-diazole (NBD), pyrene and Europium, Ruthenium, Samarium, and other rare earth metals.

Other potential labelling moieties include, radioisotopes, chemiluminescent compounds, labelled binding proteins, heavy metal atoms, spectroscopic markers, magnetic labels, and linked enzymes.

In preferred embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known that can be employed to provide a means visible to the human eye or spβctrophotometrically, to identify specific hybridisation with complementary nucleic acid-containing samples.

One preferred embodiment comprises binding of the target nucleic aci d to a solid surface. In both examples the immobilisation is obtained by coupl ng a biotin molecule to the nucleic acid and subsequently immobilising the nude c acid on a streptavidin modified surface. However, the nature of the means for immobilisation and of the support is a matter of choice. Numerous suitable supports and methods of attaching nucleotides to them are well known in the art and widely described in the literature. Thus for example, supports in the form of microtiter wells, tubes, dipsticks, particles, fibres or capillaries may be used, made for example from agarose, cellulose, alginate, teflon, latex or polystyrene. Conveniently, the support may comprise magnetic particles, which permits the ready separation of immobilised material by magnetic aggregation.

The solid support may carry functional groups such as hydroxyl, carboxyl, aldehyde or amino groups for the attachment of nucleotides. These may in general be provided by treating the support to provide a surface coating of a polymer carrying one of such functional groups, e.g. polyurethane together with a polyglycol to provide hydroxyl groups, or a cellulose derivative to provide hydroxyl groups, a polymer or copolymer of acrylic acid or methacrylic acid to provide carboxyl groups or an amino alkylated polymer to provide amino groups. US 4,654,267 describes the introduction of many such surface coatings.

Alternatively, the support may carry one member of an "affinity pair", such as avidin, while the amplified DNA is conjugated to the other member of the affinity pair in casu biotin. Representative specific binding affinity pairs are shown in Table 4.

The streptavidin/biotin binding system is very commonly used in molecular biology, due to the relative ease with which biotin can be incorporated within nucleotide sequences, and indeed the commercial availability of biotin-labelled nucleotides, and thus biotin represents a preferred means for immobilisation.

In a preferred embodiment of the invention, an amplified DNA strand is labelled with a molecule, which is subsequently used to immobilise the labelled DNA strand to a solid surface.

The DNA may be labelled by a number of methods. One convenient method to label DNA is to enclose one labelled amplification primer oligonucleotide in the amplification reaction mixture. During the amplification process the labelled oligonucleotide is built into the DNA fragment which becomes labelled.

During synthesis, oligonucleotides may also be labelled or coupled to chemoreactive groups comprising, but not limited to: sulfhyl, primary amine or phosphate. The subsequent use of such labelled primers in PCR, LCR or similar oligonucleotide dependent amplification methods results in labelled DNA fragments which can be immobilised on specialised surfaces. For instance SH-modified DNA may be immobilised on a gold surface (Steel et al. (2000) Biophys J 79:975-81) likewise 5'- phosphorylated DNA or 5'-aminated DNA may be immobilised by reaction with activated surfaces (Oroskar et al. (1996) Clin Chem 42:1547-55; Sjoroos et al (2001) Clin Chem 47:498-504).

During synthesis, oligonucleotides may also be labelled or coupled to photoreactive groups. Acetophenone, benzophenone, anthraquinone, anthrone or anthrone-like modified DNA can for instance be activated by exposure to UV light and immobilised on a wide range of surfaces as described in European and US patents: EP 0820483, US 6,033,784 and US 5,858,653. Also photoreactive psoralens, coumarins, benzofurans and indols have been used for immobilisation of nucleic acids. An extensive discussion of immobilisation of nucleic acids can be found in WO 85/04674.

Phase specific detection of polymorphisms

The haplotype is the set of alleles borne on one of a pair of homologous chromosomes. Often the particular combination of alleles in a defined region of some chromosome is referred to as the haplotype of that locus. The central dogma of modern molecular genetics teaches that it is the haplotype of the coding part of a gene that determines the amino acid sequence and thus the function of the resulting protein. In connection with studies aimed at establishing an association between the risk of developing a particular disease and the genetic makeup of the patients, information of the haplotype-investigations are superior to conventional genetic investigations of only single loci, because the haplotype provides physiological relevant information from more loci.

The present invention is also directed to the determination of the linkage phase between either Polyl, Poly2, Poly3, and Poly4 and at least one further polymorphism in the muscle glycogen synthase gene. It is also directed to determination of the linkage phase between Poly2 and any of Polyl , Poly3, and

Poly4.

Said other polymorphism may be selected from the group of polymorphisms of SEQ ID No l: C→T at pos tion -740, and/or

G→A at posi tion -251 , and/or A→G at pos ilion —143, and/or G→A at pos: tion -43, and/or T→G at pos ilion -16, and/or C→T at pos ilion +43, and/or a polymorph sm/mutation linked to any of the above polymorphisms/mutations.

Further known polymorphisms include the group consisting of the polymorphisms identified in SEQ ID No 2 in Figure 2. Preferably the known polymorphism is the Xba polymorphism (intron14+377c→t). Methods for detection of haplotypes are known e.g. from PCT/DK02/00552.

In order to obtain phase-specific detection of polymorphisms in the most preferred embodiment, the initial amplification process of this embodiment is an allele specific amplification. An "allele specific amplification" is defined as an amplification process resulting in the amplification of one allele only. In the present context the allele specific amplification results in the amplification of a specific part of only one of the chromosomes forming a chromosome pair. Allele-specific amplification can be accomplished in a number of ways. Allele-specific PCR as described in EP 0332435 is a widely used method. Furthermore, a very efficient method is described in WO 00/56916. The use of LCR for allele-specific amplification is described in WO 89/09835 and also the methods of Nucleic Acid Sequence-Based Amplification (NASBA), strand displacement amplification and rolling circle amplification may in principle be modified to perform an allele specific amplification and thus be used to obtain resolution of the haplotypes.

To detect the haplotype resolved DNA, oligonucleotides, complementary to the remaining loci of interest on the DNA fragment, are made as described. A fluorescent label is preferred because it provides a very strong signal with low background. It is also optically detectable at high resolution and sensitivity through a quick scanning procedure. However other labels may be contemplated.

The linkage phase may be determined by using two allele specific oligonucleotide probes. These two allele specific probes may be either an allele specific detection probe, an allele specific primer, or an allele specific capture probe. These may be combined so that a) one allele specific probe is an allele specific primer and the other allele specific probe is an allele specific detection probe, or b) one allele specific probe is an allele specific primer and the other allele specific probe is an allele specific capture probe, or c) one allele specific probe is an allele specific primer and the other allele specific probe is an allele specific primer, or d) one allele specific probe is an allele specific capture probe and the other allele specific probe is an allele specific detection probe. A further embodiment of phase specific detection comprises amplification of the preselected region. The amplified fragment is hybridised to specially designed probes, which are capable of detecting a multiplicity of polymorphisms. Such "bifunctional" or "multifunctional" probes contain at least 2 stretches of relatively short sequences which are complementary to each of the two studied polymorphisms separated by a region of spacer DNA which will not hybridise with the amplified region under the conditions in the experiment. The length of the spacer sequence is preferably from 8 to 28 nucleotides.

The multifunctional probes may of course span several loci of polymorphism, such as to cover at least 3 polymorphisms, for example at least 4 polymorphisms, such as at least 5 polymorphisms, such as at least 10 polymorphisms. In each case, the sequences hybridising with the target nucleotide sequence are separated by a spacer sequence, which does not hybridise to the target.

To obtain a hybridisation signal from only one chromosome of a chromosome pair, the procedure takes advantage of the fact that intramolecular hybridisation is thermodynamically favourable compared to hybridisation between separate molecules. This difference between intra- and inter-molecular hybridisation is in particular significant when hybridisation is performed at low concentrations of nucleic acid and results in a significant difference even between the hybridisation of the bifunctional probe to one or two amplified fragments, the hybridisation to one fragment being the most favourable.

Under the stringent conditions employed in the hybridisation, only probes which are completely complementary to the sequences, comprising both studied polymorphisms, will form stable hybrids.

Following the stringent hybridisation, the oligonucleotides which have not hybridised to a DNA locus are removed from the amplified DNA (e.g. by precipitation, size fractionation, electrophoreses or by centrifugation). Then the haplotype is determined by detection of fluorescence from the different oligonucleotides hybridised to the amplified DNA. As in the case of the previous embodiments other types of fluorescent or other labels may be applied.

In a further embodiment of the "one-phase system" the detection of the polymorphisms is performed using probes directed against only one polymorphism while the resolution of the haplotypes is performed by allele specific amplification, both procedures being performed in suspension.

Chemometrics According to one embodiment, detection of the presence or absence of a polymorphism comprises recording a spectrum of electromagnetic radiation from a hybrid nucleotide comprising a target nucleotide sequence and a labelled probe and subjecting said spectrum to multivariate analysis, (see also PCT/DK03/00594) It has been determined experimentally that by using the information available from a spectrum combined with multivariate statistics it is possible to distinguish the spectrum recorded from a solution containing a hybrid between one allele and a completely homologous labelled probe from the spectrum recorded from a solution containing the other allele and the labelled probe (which in this case is not completely homologous).

The subjects

The diagnostic methods of the present invention are not restricted to human beings but can be used for any mammal, although preferably the mammal is a human being.

As polymorphisms have been identified in the promoter region of human GYS1 it is expected that similar polymorphisms can be found in animals, in particular in mammals, such as pet animals, such as a dog or a cat, or wherein the mammal is a domestic animal such as a cow, a pig, a horse.

Classification of disorders

Generally the invention relates to diagnosis of subjects for the presence of at least one polymorphism as defined in the present invention. In a narrow sense the disorders may be described as described in the introductory part of the present invention. However the consequences of the polymorphisms identified herein may be involved in a wide variety of diseases. Therefore broadly speaking the invention provides methods for diagnosis of a predisposition of any disease which is related to changes in plasma or blood of glucose and/or lipid levels including LDL and cholesterol levels.

The metabolic disorders of subjects carrying a polymorphism according to the present invention may be classified according to the The International Statistical Classification of Diseases and Related Health Problems, tenth revision, World Health Organisation, 1994.

Generally the invention thus relates to the diagnosis and prognosis of any of the disorders listed below in the subjects carrying a polymorphism according to the present invention. Common for all the diseases classified below is that they may partly or completely be caused by disturbances in glucose and/or lipid and/or cholesterol metabolism and the diseases resulting from this.

Generally the whole group E, Endocrinic and nutrition caused diseases as well as metabolic diseases is encompassed insofar as it relates to disturbances of the glucose metabolism. More specifically, diseases include:

E10: IDDM, insulin dependent Diabetes mellitus.

E11 : NIDDM, non-insulin dependent diabetes mellitus

E12: Diabetes caused by malnutrition.

E13: Other types of diabetes E14: Diabetes without specification

E15: Non-diabetic hypoglychemic coma.

E16: Other diseases in the internal secretion of the pancreas.

E24: Cushing's disease.

E25: Adrenogenital syndrome E20-E35: insofar as they relate to disturbances in the glucose metabolism

E65: Local adipositas

E66: Adipositas.

E67: Other types of hyperalimentation.

E68: After effects of hyperalimentation. E74: Other diseases in the carbohydrate metabolism E75: Diseases in the sphingolipidmetabolism and other lipid deposition diseases E78: Diseases in the lipoprotein metabolism and other lipedemia. E85: Amyloidosis. E89.1 : Diabetes after surgery. G30: Alzheimer. G40: Epilepsia.

G63.2: Diabetic polyneuropathy. H36: Diabetic rhetinopathy. 15.2: Arterial hypertension with endocrine diseases. 20: Angina pectoris 21 : Acute mycardial infarction. 22: Recurrent acute mycardial infarction. 23: Acute implications after acute mycardial infarction. 24: Other types of acute mycardial infarction. 25: Chronic ischaemic heart disease.

42.8 and I42.9: Other types and unspecified types of cardiomyopathy. 43.1 : Cardiomyopathy caused by metabolic diseases. 50.9: Heart incompensation without specification. 52.8: Other types of heart diseases.

61 , I63, 165-66, 168-69, 170-72, I77, I79: insofar as the disease is related to disturbances of the glucose metabolism.

K70-K77: Liver related diseases, insofar as they are related to disturbances of the glucose and/or lipid metabolism.

K85-87: Pancreas related diseases, insofar as they are related to disturbances of the glucose and/or lipid metabolism. N07: Hereditary kidney diseases not classified other places. N08.3: Glomerulonefropathy by diabetes mellitus. N18-N19: Kidney insufficiency, chronic uremia.

010.2: Arterial hypertension during pregnancy caused by hypertensive nephropathy. 010.9: Arterial hypertension during pregnancy caused by unspecified factors. 016: Non-specified arterial hypertension during pregnancy. 024: Diabetes mellitus during pregnancy. 028.1 : Abnormal biochemical parameters during pregnancy. P07: Low birth weight. P70: Transitory disturbances in the carbohydrate metabolism in foetus and neonatals.

R73: Elevated blood glucose. R81 : Glucosuria R83.2: Cerebrospinal liquid with abnormal content of biological substances.

Isolated nucleic acid sequences

In one aspect the invention relates to an isolated nucleic acid sequence comprising at least 10 contiguous nucleotides of the region from nucleotide -950 to -550 of SEQ ID NO 1 or the complementary strand. These isolated nucleic acid sequences can be used as probes or primers for the detection methods described above.

Preferably, said contiguous nucleotides comprise any one or more of the following alleles -916:G (Polyl), -849:G (Poly2), -618:C (Poly3), -588:G (Poly4), -740:T (Polyδ) or their base-pairing counterparts on the complementary strand.

One particularly preferred isolated nucleic acid sequence comprises at least 10 contiguous nucleotides of the region from -879 to -819 of SEQ ID NO 1 or the complementary strand counted from the transcription start site, more preferably from -869 to -829 of SEQ ID NO 1 or the complementary strand. This nucleic acid sequence can be used for diagnosing the Poly2 polymorphism.

A further preferred group of isolated nucleic acid sequence comprises at least 10 contiguous nucleotides selected from the group comprising the region from -946 to -886 of SEQ ID NO 1 or the complementary strand counted from the transcription start site, more preferably from -936 to -896 of SEQ ID NO 1 or the complementary strand, and the region from -648 to -588 of SEQ ID NO 1 or the complementary strand counted from the transcription start site, more preferably from -638 to -598 of SEQ ID NO 1 or the complementary strand, and the region from -618 to -558 of SEQ ID NO 1 or the complementary strand counted from the transcription start site, more preferably from -608 to -568 of SEQ ID NO 1 or the complementary strand. These isolated nucleic acid sequences can be used for diagnosing any of or either of Polyl , Poly3, and Poly4.

A nucleic acid sequence comprising the whole sequence from nucleotide -950 to - 550 of SEQ ID NO 1 or the corresponding complementary strand can be used for phase specific determination of the polymorphisms.

An isolated nucleic acid sequence may be as short as 10 contiguous nucleotides but for some purposes it comprises at least 12 contiguous nucleotides, such as at least 15, e.g. at least 20, such as at least 30, e.g. at least 40, such as at least 50 contiguous nucleotides. Preferably, the isolated nucleic acid is less than 10,000, such as less than 1,000, more preferably less than 100 nucleotides in length.

A special embodiment of this aspect of the invention is a bifunctional probe for detection of the linkage phase between two alleles. This bifunctional probe may for example comprise both an isolated nucleic acid sequence for diagnosing Poly2, and another sequence for diagnosing Polyl , Poly3, or Poly4. These two sequences may be linked by a spacer sequence, which is not capable of hybridising with any nucleotide sequence of SEQ ID NO 1 or the corresponding complementary strand. The hybridisation conditions in this case is preferably high stringency. The length of the spacer is preferably from 8 to 28 nucleotides.

In all of the above cases the mutation/polymorphism may be located in the centre of the nucleic acid sequence, in the 5' end of the nucleic acid sequence, or in the 3' end of the nucleic acid sequence. The mutation/polymorphism may also be located adjacent to the 3' or 5' end of the nucleic acid sequence. Such an isolated nucleic acid sequence can be used for single base extension.

The isolated nucleic acid sequence may be complementary to a sub-sequence of the strand of a target nucleotide sequence comprising a coding strand or complementary to a sub-sequence of a strand of a target sequence comprising a non-coding strand.

The isolated nucleic acid sequences may be made from RNA, DNA, LNA, PNA monomers or chemically modified nucleotides capable of hybridising to a target nucleic acid sequence. Conveniently the sequence is made from DNA but for some purposes where particularly high strong hybridisation is desired the nucleotide sequence comprises at least one LNA monomer.

Kits for diagnosis

Kits-of-parts for the diagnosis of an increased risk of developing a metabolic disorder may comprise at least one nucleic acid sequence comprising at least 10 contiguous nucleotides of the region from nucleotide -950 to -550 of SEQ ID NO 1 , or the complementary strand. Such nucleic acid sequences may be used in the method described herein, i.e. as probes and/or as primers for amplification.

Preferably, the nucleic acid sequence in the kit is one as defined by the present invention. The specific layout of the kit depends on the method used for determining the alleles. The kits may furthermore contain a carrier of information, such as a sheet of paper, containing experimental instructions for the user and/or information relating to the diagnosis and/or prediction of increased risk for metabolic disease that the kit may be used for.

Preferably the kit comprises a probe linked to a detectable label. This may be used for any hybridisation based technique. Preferably the kit also comprises a set of primers for amplifying a region of the muscle glycogen synthase gene comprising at least one of the mutations or polymorphisms, a polymerase, and nucleotide monomers. These primers may be any suitable combination of SEQ ID No 3-10 or other primers capable of amplifying the desired region.

When the detection method includes immobilisation of a target nucleic acid sequence, then one of the primers may be coupled to an entity suitable for a subsequent immobilisation reaction and the kit may also comprise a solid surface to which the entity can be immobilised.

A kit for detection based on RFLP may comprise a restriction enzyme for cleaving an amplified region, such as wherein the restriction enzyme is Mni\, BsiU\, or Ac/I, or other isoschizomers. A kit for detection based on single base extension may further comprise a primer wherein the polymorphic site is adjacent the 3' end, a polymerase and at least one fluorescently labelled nucleotide monomer.

Preferred kits for detection based on hybridisation and detection of a signal comprise a capture probe and a detection probe. These may be allele specific such as wherein either the capture probe and/or the detection probe and/or an amplification primer are allele specific, such as wherein one allele specific probe is specific for a Poly2 allele and the other allele specific probe is specific for a Polyl allele, a Poly3 allele, or a Poly4 allele.

A sequencing based kit may comprise primers for specific amplification of the sequence, a sequencing primer with or without 5' extension with the purpose to use it as a universal sequence primer, mononucleotides, polymerase and/or buffers. Examples of primers for amplification include SEQ ID No 3 to 10. SEQ ID No 3, 5, and 7-10 all include a universal sequence primer.

Examples

Example 1 : Statistical analysis of the twin population

The twin population was stratified according to gender and status of glucose metabolism, that is men and women, and normal glucose tolerance (NGT), impaired glucose tolerance (IGT) and diabetics (DM). Metabolic stratification follows the WHO-recommendations (Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation. Diabet. Med. 1998;15(7):539-53). The number of persons in each group was:

Men Women

NGT 165 170

IGT 53 53

DM 42 35 Analysis of molecular variance (AMOVA) (Weir and Cockerham 1358-

70;Weir;Excoffier, Smouse, and Quattro 479-91) did not reveal any diversities among the subpopulations. All variance was accounted for in the subpopulations.

No sign of selection or lack of neutrality was present.

Pairwise linkage disequilibrium

This was performed by 100000 permutations of the individual genotypes as described (Slatkin and Excoffier 377-83; Slatkin 331-36). The result for the men with

IGT is shown below.

Men IGT

Poly(1 ,2)

Exact P= 0.67078 +/- 0.00144; Chi-square test value= 0.53448 (P = 0.46473, 1 d.f.)

Poly(1 ,3) Exact P= 0.00000 +/- 0.00000 ; Chi-square test value=152.88354 (P = 0.00000, 1 d.f.)

Poly(1 ,4)

Exact P= 0.00000 +/- 0.00000 ; Chi-square test value=152.88354 (P = 0.00000, 1 d.f.) Poly(2,3)

Exact P= 0.64591 +/- 0.00147 ; Chi-square test value= 0.53448 (P = 0.46473, 1 d.f.)

Poly(2,4)

Exact P= 0.64438 +- 0.00148 ; Chi-square test value= 0.53448 (P = 0.46473, 1 d.f.)

Poly(3,4) Exact P= 0.00000 +- 0.00000 ; Chi-square test value=152.88354 (P = 0.00000, 1 d.f.)

In conclusion, Poly 1 , Poly3 and Poly 4 are in complete disequilibrium, while Poly 2 segregates independently of the polymorphisms Polyl , Poly3 and Poly4. Inspection of the genotypes clearly confirmed the linkage of the Poly 1 , Poly 3 and Poly 4. That is, only 2 haplotypes are present for these 3 polymorphisms in contrast to the theoretically expected 8 haplotypes or alleles.

Similar results were found for all the subpopulations.

However Poly2 is only found in one of the haplotypes, namely the TTA-haplotype of Polyl ( (T), Poly3 (T), and Poly4 (A). All subpopulations were in Hardy-Weinberg equilibrium (Guo and Thompson 361- 72).

All calculations were performed using the Arlequin package (Scneider, Roessli, and Excoffier).

Reference List

Excoffier, L., P. E. Smouse, and J. M. Quattro. "Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data." Genetics 131.2 (1992): 479-91.

Guo, S. W. and E. A. Thompson. "Performing the exact test of Hardy-Weinberg proportion for multiple alleles." Biometrics 48.2 (1992): 361-72.

Scneider, S., Roessli, D., and Excoffier, L. Arlequin 2.001 : A software for population genetics data analysis. [2.001]. 2001. Genetics and Biometry Laboratory, University of Geneva, Switzerland, Genetics and Biometry Laboratory,

University of Geneva, Switzerland.

Ref Type: Computer Program

Slatkin, M. "Linkage disequilibrium in growing and stable populations." Genetics 137.1 (1994): 331-36.

Slatkin, M. and L. Excoffier. "Testing for linkage disequilibrium in genotypic data using the Expectation-Maximization algorithm." Heredity 76 ( Pt 4) (1996): 377-83.

Weir, B. S. Genetic Data Analysis II. Sinauer, 1996.

Weir, B. S. and C. C. Cockerham. "Estimating F-statistics for the analysis of population structure." Evolution 38.6 (1984): 1358-70.

Example 2: DNA analysis of variance of the promoter region of the glycogen synthase

1528 bp of the glycogen synthase promoter have been cloned, but only approx. 850 bp upstream of the transcription initiation site have been examined for mutations (Bjørbaek et al. 1994, Marju et al. 1995). No nucleotide variants associated with type 2 diabetes/a diabetic phenotype have been described in the literature.

We have scanned 678 bp of the promoter region of the glycogen synthase for mutations (Figure 1). Four new nucleotide variants were found, (Polyl (T→G), Poly2 (C→G), Poly3 (T→C) and Poly4 (A→G). Three (Polyl, Poly3 and Poly4) out of four nucleotide variants were in complete disequilibrium (p < 0.0001). The four nucleotide variants were tested for association with type 2 diabetes/IGT or other relevant phenotypic variables. The study was based on genotyping of approx. 529 Danish twins (who were normal (glucose tolerant), IGT's or type 2 diabetics, respectively (Table 5). These polymorphisms were detected by RFLP after amplification with PCR. Seven of the Poly-2 mutations were sequenced to confirm the presence of the polymorphism.

Table 5. Baseline data of the studied twin population

Control subjects IGT subjects Type 2 diabetic patients n (men/women) 341 (166/175) 110 (53/57) 79 (44/35)

Mono-/dizygotic twins (n)

Age (years) 65 + (5.1) 67 ± (4.6) 67 ± (4.5)

BMI (kg/m²) 25 ± (4.0) 26 ± (4.5) 28 ± (4.9) f-p-glucose (mmol/l) 5.6 ± (0.5) 5.9 ± (0.6) 9.2 ± (3.4) f-p-insulin (pmol/l) 39 ± (20.2) 55 ± (30.8) 65 ± (41.4)

120 min p-glucose 5.9 ± (1.0) 8.7 ± (0.9) 17.0 ± (6.1)

(pmol/l) f-s-Triglycerid (mmol/l) 1.3 ± (0.5) 1.6 ± (0.8) 1.8 ± (0.8)

Total f-s-cholesterol 6.2 ± (1.1) 6.2 ± (1.2) 6.0 ± (1.1)

(mmol/l) f-s-LDL-cholesterol 4.0 ± (1.0) 4,2 ± (1.1) 4.0 ± (1.1)

(mmol/l) f-sHDL-cholesterol 1.5 ± (0.4) 1.4 ± (0.4) 1.3 ± (0.5)

(mmol/l)

The clinical and physiological characteristics represent means ± standard derivation (SD). n = total number of subjects; f = fasting; p = plasma; s = serum The association study of the three polymorphisms that were linked (Polyl , Poly3 and Poly4) showed no association to the development of type 2 diabetes (Table 6). The genotype/phenotype interaction study of the glucose tolerant twins showed a significant association of the genotype Polyl -GG, Poly3-CC and Poly4-GG with an increased fasting cholesterol value (p = 0.04) and a fasting LDL value (p=0.04) (Table 7). Two glucose tolerant twins (monozygotic) and one homozygotic IGT twin with the genotype Polyl -GG, Poly3-CC and Poly4-GG were identified (Table 8).

Table 6. Prevalence of type 2 diabetes or IGT in 530 Danish twins classified according to genotype of the Polyl , Poly3 and Poly4 polymorphisms

Danish twins Distribution of Polyl , Poly3, and Poly4 genotypes n (%)

TT/TT/AA TG/TC/AG GG/CC/GG p

Subjects with 189 161 (85.2) 27 (14.3) 1 (0.5) 0.80

Type 2 diabetes or IGT Glucose tolerant subjects 314 283 (83.0) 56 (16.4) 2 (0.6)

The genotype distributions were compared with Fisher^'s exact test. If the genotype GG/CC/GG and TG/TC/AG were multiplied and compared with the TT/TT/AA genotype by using Chi-square test the p-value was 0.51 (df = 1)

Table 7. Clinical and antropometrical variables of glucose tolerant control subjects, classified according to GYS1 promoter genotype at position Polyl , Poly3 and Poly4

Clinical and Genotype Polyl , Poly3 and Poly 4 anthropometrical variables

Glucose tolerant TT/TT/AA (CI) TG/TC/AG (CI) GG/CC/GG (CI) p-

3UDJ©G1S value

Number (men/women) 138/145 28/28 0/2

Fasting cholesterol 6.14 6.13 8.48 0.04

(mmol/l) (6.01 - 6.28) (5.84 - 6.42) (8.09 :9.10)

Fasting HDL (mmol/l) 0.74

Fasting LDL (mmol/l) 4.06 4.03 6.22 0.04

(3.93 - 4.18) (3.76 - 4.29) (5.76:6.72) The following parameters were also measured: Age, BMI (Body mass index, kg/m²), Plasma glucose (mmol/L) and plasma insulin (pmol/L) were measured in an oral glucose tolerance test (OGTT) at 0, 30 and 120 minutes, RatO (insulin/glucose ratio) at 0, 30 and 120 minutes of the OGTT, systolic and diastolic blood pressure

(mmHg), insulin resistance index (HOMObeta), insulin secretion index (HOMAres), fasting triglycerides (mmol/L). The results are presented as means ± 95% confidence interval (CI). Log-transformations were used for variables with skewed distributions to normalise the distributions (plasma insulin, rat, HOMAres, triglycerid and HDL). All analysis were done using the SAS statistical package version 8 (SAS

Institute, Gary, N.C., USA). The effect of the polymorphism on quantitative variables was tested with a multivariate analysis of covariance using a mixed linear model. In the analysis age, sex and body mass index (BMI) were included as covariants. Twin pair was included as a random effect parameter. Insulin resistance index (HOMAres) and insulin secretion index (HOMAbeta) was assessed with the homeostasis model assessment (HOMA) (Matthews D.R et al., 1995). A p-value of less than 0.05 was considered statistically significant.

Table 8. Clinical and antropometrical variables of the tree subjects with the genotype Polyl -GG, Poly3-CC and Poly4-GG

Clinical and Subjects with the genotype Polyl -GG, Poly3-CC antropometrical and Poly4-GG variables

Subject 1 2 3

Pair no. 272 272 124

Age 54 54 68

Sex Woman Woman Woman

OϊolUS Normal Normal IGT

Zygoti Monozyg otic Monozygotic Dizygotic

BMI 29.4 28.1 28.1

GlucO 5 5 5.4

GlucSO 7 7.1 9.1

Gluc120 4.8 4.7 7.9

InsO 40 39 34

Ins30 288 410 146 Ins120 144 133 227

Trig lye 2.57 2.28 0.91

Cholesterol 8.09 9.10 5.49

HDL 1.16 1.34 1.58

LDL 5.76 6.72 3.5

GlucO, Gluc30 and Gluc120 are the plasma glucose levels and InsO, Ins30 and Ins120 are the plasma insulin levels at 0, 30 and 120 minutes of an OGTT, Triglyc is the plasma triglyceride level, while HDL and LDL are the fasting plasma levels of high density lipoprotein and low density lipoprotein respectively.

The association study of Poly 2 showed a significant association of heterozygous carriers of the polymorphism to the development of type 2 diabetes/IGT (p = 0.005) (Table 9). By including genotypings of Poly2 from a further 32 twins (who had only stated the clinical status) to the association study, an even higher significant association of heterozygous carriers of Poly2 appeared to the development of type 2 diabetes/IGT (p < 0.0008) (Table 10). The genotype/phenotype interaction study of the glucose tolerant control subjects showed a significantly increased fasting cholesterol value (p = 0.0009), LDL (p = 0.0024), triglycerides (p<0.005), insulin level (p <0.007), and insulin resistance (HOMAres, p < 0.01) with the heterozygous carriers of Poly2 (Table 11). As there were only six heterozygous carriers of Poly2 in the genotype/phenotype interaction study of the glucose tolerant control subjects the same analysis was carried out on the full twin population, where control subjects, IGT's and type 2 diabetics were added up (Table 12). Both the insulin level and insulin resistance was highly significantly increased in carriers of Poly2.

Table 9. Prevalence of type 2 diabetes or IGT in 530 Danish twins classified according to genotype of the Poly2 polymorphism

Danish twins Distribution of Poly2 genotypes n (%)

n GG GC CC p

Subjects with 189 0 13 (6.88) 176 (93.12) 0.005

Type 2 diabetes or IGT

Glucose tolerant 340 0 6 (1.76) 334 (98.24) subjects The genotype distributions were compared with Fisher's exact test.

Table 10. Prevalence of type 2 diabetes or IGT in 562 Danish twins classified according to genotype of the Poly2 polymorphism. Twins without clinical parameters (only status) are included

Danish twins Distribution of Poly2 genotypes n (%)

n GG GC CC P

Subjects with 201 0 15 (7.46) 186 (92.5) <0.0008

Type 2 diabetes or IGT

Glucose tolerant 361 0 6 (1.66) 355 (98.3) subjects

The genotype distributions were compared with chi-square test.

Table 11. Clinical and antropometrical variables of 340 glucose tolerant control subjects, classified according to GYS1 promoter genotype at position Poly2

Clinical and anthropometri- Genotype (Poly2) cal variables

Glucose tolerant subjects CC (CI) CG (CI) p-value

Number (men/women) 334 (163-171) 6 (3/3)

Age (years) 65.6 (65.0-66.3) 65.6 (64.9-66.3) 0.98

BMI (kg/m2) 25.6 (25.1-26.1) 23.6 (20.3-26.8) 0.23

Fasting

Plasma glucose (mmol/i) 5.60 (5.54-5.65) 5.66 (5.29-6.04) 0.72

Plasma insulin (pmol/l) 35.2 (33.4-37.0) 55.1 (40.0-75.9) 0.007

HOMAbeta 62.8 (59.5-66.2) 88.3 (65.6-111). 0.03

HOMAres 1.45 (1.38-1.54) 2.29 (1.63-3.25) 0.01

Fasting triglycerides 1.72 (1.31-2.27) 1.15 (1.11-1.20) 0.005

(mmol/l)

Fasting cholesterol (mmol/l) 6.13 (6.00-6.25) 7.58 (6.74-8.42) 0.0009

SEM (0.06) SEM (0.42)

Fasting HDL (mmol/l) 1.48 (1.43-1.52) 1.43 (1.17-1.77) 0.79

Fasting LDL (mmol/l) 4.03 (3.92-4.15) 5.26 (4.49-6.04) 0.0024

The results are presented as means ± 95% confidence interval (CI). Different transformations were used for variables with skewed distributions to normalise the distributions Logarithmic transformation was performed for plasma insulin, HOMAres, triglyceride and HDL. All analysis were done using the SAS statistical package version 8 (SAS Institute, Cary, N.C., USA). The effect of the polymorphism on quantitative variables was tested with a multivariate analysis of covariance using a mixed linear model. In the analysis age, sex and body mass index (BMI) were included as co-variants. Twin pair was included as a random effect parameter. Insulin resistance index (HOMAres) and insulin secretion index (HOMAbeta) was assessed with the homeostasis model assessment (HOMA) (Matthews D.R et al., 1995). A p-value of less than 0.05 was considered statistically significant. Means are adjusted for age, BMI and sex. Values are back-transformed.

Table 12. Clinical and anthropometrical variables of 529 Danish twins, classified according to GYS1 promoter genotype at position Poly2

Clinical and anthropometrical Genotype (Poly2) variables

Total twin population CC (CI) CG (CI) p-value

Number (men/women) 510 (251/259) 19 (12/7)

Age (years) 66.0 (65.4-66.6) 66.0 (65.4-66.7) 0.97

BMI (kg/m2) 25.8 (25.3-26.3) 25.0 (23.8-27.1) 0.48

Fasting

Plasma glucose (mmol/l) 5.88 (5.99-5.78) 6.25 (6.99-5.92) 0.04

Plasma insulin (pmol/l) 39.3 (37.7-41.3) 58.0 (46.1-73.0) 0.001

HOMAbeta 63.8 (60.6-67.0) 76.5 (60.2-92.8) 0.13

HOMAres 1.93 (1.82-2.04) 3.06 (2.37-3.80) 0.0009

Fasting triglycerides (mmol/l) 1.26 (1.21-1.31) 1.51 (1.23-1.84) 0.08

Fasting cholesterol (mmol/l) 6.20 (6.00-6.20) 6.55 (6.00-7.13) 0.10

Fasting HDL (mmol/l) 1.42 (1.38-1.45) 1.46 (1.27-1.68) 0.61

Fasting LDL (mmol/l) 4,05 (3.95-4.16) 4.32 (3.81-4.84) 0.31

The results are presented as means ± 95% confidence interval (CI). Transformations and statistics as in Table 11.

Claims

Claims

1. A method for diagnosing metabolic disorders and/or for predicting an increased risk of a subject for developing metabolic disorders comprising a) obtaining a biological sample from a subject b) assaying for at least one mutation or polymorphism within the DNA sequence of the region controlling expression of muscle glycogen synthase 1 (GYS1), said at least one mutation or polymorphism being located in a target nucleic acid sequence selected from - the sequence comprising the nucleotides from -950 to -550 of SEQ ID No 1 counted from the transcription start site, or a part thereof,

- the complementary region of the nucleotides from -950 to -550 of SEQ ID No 1 counted from the transcription start site, or a part thereof,

- a sequence being at least 90 % identical with any of a) or b) or a part thereof, more preferably at least 95 % identical, more preferably at least 98

% identical, or assaying for at least one mutation or polymorphism which is in linkage disequilibrium with any one or more of the following alleles: -916:G (Polyl), - 849:G (Poly2), -618:C (Poly3), -588:G (Poly4), c) diagnosing a metabolic disorder and/or predicting an increased risk of a subject for developing metabolic disorder on the basis of the presence or absence of said mutation or polymorphism.

2. The method according to claim 1 , wherein the target nucleic acid sequence is selected from a) the sequence comprising the nucleotides from -950 to -550 of SEQ ID No 1 counted from the transcription start site, or a part thereof, b) the complementary region of the nucleotides from -950 to -550 of SEQ ID No 1 counted from the transcription start site, or a part thereof.

3. The method according to claim 1 , wherein the target nucleic acid sequence is selected from a) the sequence comprising the nucleotides from -925 to -575 of SEQ ID No 1 counted from the transcription start site, or a part thereof, b) the complementary region of the nucleotides from -925 to -575 of SEQ ID No 1 counted from the transcription start site, or a part thereof.

4. The method according to any of the preceding claims, wherein the mutation or polymorphism is a C→G change at position -849 of SEQ ID NO 1 counted from the transcription start site or the corresponding mutation in the complementary strand or a mutation linked to said C→G change in the glycogen synthase gene, its promoter or in genes on the same chromosome.

5. The method according to any of the preceding claims, wherein the mutation or polymorphism is a C→G change at position -849 of SEQ ID NO 1 counted from the transcription start site or the corresponding mutation in the complementary strand.

6. The method according to any of the preceding claims, wherein the mutation or polymorphism is selected from the group of polymorphisms of SEQ ID NO 1 and the corresponding polymorphisms in the complementary strand, said group comprising: T→G at position -916, and/or T→C at position -618, and/or

A→G at position -588, and/or a polymorphism/mutation linked to any of the above polymorphisms/mutations in the glycogen synthase gene, its promoter or in genes on the same chromosome.

7. The method according to any of the preceding claims, wherein the mutation or polymorphism is selected from the group of polymorphisms of SEQ ID NO 1 and the corresponding polymorphisms in the complementary strand, said group comprising: T→G at position -916, and/or T→C at position -618, and/or

A→G at position -588.

8. The method according to any of the preceding claims, wherein the metabolic disorder is selected from the group comprising syndrome X, metabolic syndrome, dyslipidemia, diabetes, atherosclerosis, adipositas.

9. The method according to any of the preceding claims, wherein the metabolic disorder is diabetes mellitus.

10. The method according to claim 9, wherein the diabetes is diabetes mellitus Type

2.

11. The method according to claim 6 or 7, wherein the metabolic disorder is associated with dyslepidemia including increased cholesterol.

12. The method according to any of claims 1 to 7, wherein the metabolic disorder is selected from the group of (according to The International Statistical Classification of Diseases and Related Health Problems, tenth revision, World Health Organisation, 1994): E10: IDDM, insulin dependent diabetes mellitus

E11: NIDDM, non-insulin dependent diabetes mellitus E12: Diabetes caused by malnutrition E13: Other types of diabetes E14: Diabetes without specification E15: Non-diabetic hypoglychemic coma

E16: Other diseases in the internal (secretion) of the pancreas

E24: Cushing's disease

E25: Adrenogenital syndrome

E20-E35: insofar as they relate to disturbances in the glucose metabolism

E65 Local adipositas E66 Adipositas E67 Other types of hyperalimentation

E68 After effects of hyperalimentation E74: Other diseases in the carbohydrate metabolism E75: Diseases in the sphingolipidmetabolism and other lipid deposition diseases E78 Diseases in the lipoproteinmetabolism and other lipedemia E85 Amyloidosis E89 1 : Diabetes after surgery G30 Alzheimer G40 Epilepsia G63.2: Diabetic polyneuropathy H36: Diabetic rhetinopathy 15.2: Arterial hypertension with endocrine diseases 20: Angina pectoris 21 : Acute mycardial infarction 22: Recurrent acute mycardial infarction 23: Acute implications after acute mycardial infarction 24: Other types of acute mycardial infarction 25: Chronic ischaemic heart disease

42.8 and I42.9: Other types and unspecified types of cardiomyopathy 43.1 : Cardiomyopathy caused by metabolic diseases 50.9: Heart incompensation without specification 52.8: Other types of heart diseases

61 , I63, 165-66, 168-69, 170-72, I77, I79: insofar as the disease is related to disturbances of the glucose metabolism

K70-K77: Liver related diseases, insofar as they are related to disturbances of the glucose and/or lipid metabolism

K85-87: Pancreas related diseases, insofar as they are related to disturbances of the glucose and/or lipid metabolism N07: Hereditary kidney diseases not classified other places N08.3: Glomerulonefropathy by diabetes mellitusN18-N19: Kidney insufficiency, chronic uraemia

010.2: Arterial hypertension during pregnancy caused by hypertensive nephropathy

010.9: Arterial hypertension during pregnancy caused by unspecified factors 016: Non-specified arterial hypertension during pregnancy 024: Diabetes mellitus during pregnancy 028.1: Abnormal biochemical parameters during pregnancy P07: Low birth weight

P70: Transitory disturbances in the carbohydrate metabolism in foetus and neonatals

R73: Elevated blood glucose R81 : Glucosuria R83.2: Cerebrospinal liquid with abnormal content of biological substances.

13. The method according to any of the preceding claims, wherein at least one polymorphism is detected using restriction fragment length polymorphism (RFLP).

14. The method according to any of the preceding claims, wherein at least one polymorphism is detected using random amplified polymorphic DNA (RAPD).

15. The method according to any of the preceding claims, wherein at least one polymorphism is detected using mass spectrometry.

16. The method according to any of the preceding claims, wherein at least one polymorphism is detected using single strand conformation polymorphism (SSCP).

17. The method according to any of the preceding claims, wherein at least one polymorphism is detected using single nucleotide extension.

18. The method according to any of the preceding claims, wherein at least one polymorphism is detected using sequencing.

19. The method according to any of the preceding claims, wherein at least one polymorphism is detected by recording a spectrum of electromagnetic radiation from a hybrid nucleotide comprising a target nucleotide sequence and a labelled probe and subjecting said spectrum to multivariate analysis.

20. The method according to any of the preceding claims, wherein detection of at least one polymorphism comprises hybridising a probe to a target nucleic acid sequence.

21. The method according to claim 20, wherein the probe is bound to a detectable label.

22. The method according to claim 21 , wherein the label is a fluorescent reporter group.

23. The method according to claim 22, wherein the fluorescent group is selected from the group consisting of fluorescein, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, acridin, Hoechst 33258, Rhodamine, Rhodamine Green, Tetramethylrhodamine, Texas Red, Cascade Blue, Oregon Green, Alexa Fluor, Tetramethylrhodamine, europium and samarium.

24. The method according to claim 21 , wherein the label is an enzyme tag.

25. The method according to claim 24, wherein the enzyme tag is selected from the group consisting of a beta-Galactosidase, a peroxidase, horseradish peroxidase, a urease, a glycosidase, alkaline phosphatase, chloramphenicol acetyltransferase and a luciferase.

26. The method according to claim 21 , wherein the label is a chemiluminescent group.

27. The method according to claim 26, wherein the chemiluminescent group is selected from the group consisting of hydrazides, such as luminol and oxalate esters.

28. The method according to claim 21 , wherein the label is a radioisotope.

29. The method according to claim 28, wherein the radioisotope is selected from the group consisting of ³²P, ³³P, ³⁵S, ¹²⁵l, ⁴⁵Ca, ¹⁴C and ³H.

30. The method according to claim 20, comprising the use of a capture probe for capturing a target nucleic acid sequence.

31. The method according to any of the preceding claims, comprising amplification of a nucleotide sequence comprising the polymorphism(s) position.

32. The method according to claim 31 , wherein the amplification is performed by an amplification method selected from the group consisting of polymerase chain reaction (PCR), Ligase Chain Reaction (LCR), Nucleic Acid Sequence-Based Amplification (NASBA), strand displacement amplification, rolling circle amplification, and T7-polymerase amplification.

33. The method according to claim 32, wherein amplification comprises use of a primer pair consisting of SEQ ID No 3 and SEQ ID No 10.

34. The method according to claim 32, further comprising: a) the use of a primer coupled to an entity suitable for a subsequent immobilisation reaction, b) performing the amplification reaction, thus obtaining an amplified DNA molecule with said entity coupled to one of the DNA strands, c) dissociating the two strands of the amplified DNA molecule, d) binding only the coupled DNA strand from step b) to a solid surface, e) contacting the bound DNA with a labelled oligonucleotide probe which is capable of detecting a specific polymorphism by nucleic acid hybridisation.

35. The method according to claim 34, wherein said solid surface is selected from the group consisting of a nitrocellulose surface, a cellulose surface, a diazotized surface, a lipid emulsion, a bio membrane, glass, silicate, gelatine, and a nylon surface.

36. The method according to claim 34, wherein the binding comprises the coupling of one member of an affinity pair to the nucleic acid while the other member of said affinity pair is immobilised on the solid surface.

37. The method according to claim 36, wherein said affinity pair is an affinity pair selected from the group consisting of Biotin - streptavidin, Biotin - avidin, Digoxigenin - anti-hapten antibody, fluorescein - anti-hapten antibody, lectins - lectin receptor, Ion - Ion chelators, IgG - protein A, IgG - protein G and magnets - paramagnetic particles.

38. The method according to claim 34, wherein the binding comprises the coupling of the nucleic acid to a photoreactive group.

39. The method according to claim 38, wherein the photoreactive group is a group selected from the group consisting of a quinone, an anthraquinone, a psoralene, a coumarin, a benzofuran, an indole and a photoactivable ketone, including benzophenone and acetophenone.

40. The method according to claim 34, wherein the binding comprises the coupling of the nucleic acid to a chemoreactive group.

41. The method according to claim 40, wherein said chemoreactive group is a group selected from the group consisting of a sulfhydryl, a primary amine and a primary phosphate.

42. The method according to any of the preceding claims, comprising capturing a target nucleic acid with a capture probe linked to a solid surface and detecting the polymorphism by contacting the captured target nucleic acid sequence with a labelled probe.

43. The method according to any of the preceding claims, wherein detection of the presence or absence of a polymorphism is performed by detecting the presence or absence of electromagnetic radiation above a predefined threshold at a predefined wavelength.

44. The method according to any of the preceding claims, wherein detection of the presence or absence of a polymorphism comprises recording a spectrum of electromagnetic radiation from a hybrid nucleotide comprising a target nucleotide sequence and a labelled probe and subjecting said spectrum to multivariate analysis.

45. The method according to any of the preceding claims, further comprising determination of the linkage phase between Poly 2 and at least one polymorphism in the muscle glycogen synthase gene.

46. The method according to claim 45, wherein said other polymorphism is selected from the group of polymorphisms of SEQ ID No 1 : G→A at position -251 , and/or A→G at position -143, and/or G→A at position -43, and/or T→G at position -16, and/or C→T at position +43, and/or a polymorphism/mutation linked to any of the above polymorphisms/mutations.

47. The method according to claim 45, wherein said other polymorphism is selected from the group consisting the polymorphisms identified in SEQ ID No 2 (Fig 2), more preferably the Xba1 polymorphism (intron14+377c→t).

48. The method according to any of the claims 45 to 47, wherein the linkage phase is determined by using two allele-specific oligonucleotide probes.

49. The method according to claim 48, wherein one allele specific probe is an allele specific primer and the other allele specific probe is an allele specific detection probe.

50. The method according to claim 48, wherein one allele specific probe is an allele specific primer and the other allele specific probe is an allele specific capture probe.

51. The method according to claim 48, wherein one allele specific probe is an allele specific primer and the other allele specific probe is an allele specific primer.

52. The method according to claim 48, wherein one allele specific probe is an allele specific capture probe and the other allele specific probe is an allele specific detection probe.

53. The method according to claim 48, wherein the allele specific probes are assembled in a multifunctional detection probe linked by a non-hybridising linker.

54. The method according to any of the preceding claims, wherein the subject is a mammal, preferably a human being.

55. The method according to claim 54, wherein the mammal is a pet animal such as a dog or a cat, or wherein the mammal is a domestic animal such as a cow, a pig, or a horse.

56. A method for detecting the haplotype of the muscle glycogen synthase gene (SEQ ID No 2) in a subject, said method comprising determining the linkage phase between the nucleotide allele at position -849 of SEQ ID NO 1 (Poly2) counted from the transcription start or the corresponding position in the complementary strand and another polymorphism in said gene or its promoter.

57. The method according to claim 56, wherein the haplotype is the haplotype of the region controlling expression of the muscle glycogen synthase gene.

58. The method according to claim 56 or 57, wherein the haplotype is the haplotype of Poly2 and either of Polyl , PolyS or Poly4.

59. An isolated nucleic acid sequence comprising at least 10 contiguous nucleotides of the region from nucleotide -950 to -550 of SEQ ID NO 1 or the complementary strand, said contiguous nucleotides comprising any one or more of the following alleles -916:G (Polyl), -849:G (Poly2), -618:C (Poly3), -588:G

(Poly4), -740:T (Polyδ) or their base-pairing counterparts on the complementary strand.

60. The isolated nucleic acid sequence of claim 59, comprising at least 10 contiguous nucleotides of the region from -879 to -819 of SEQ ID NO 1 or the complementary strand counted from the transcription start site, more preferably from -869 to -829 of SEQ ID NO 1 of the complementary strand.

61. The isolated nucleic acid sequence of claim 59, comprising at least 10 contiguous nucleotides of a) the region from -946 to -886 of SEQ ID NO 1 or the complementary strand counted from the transcription start site, more preferably from -936 to -896 of SEQ ID NO 1 or the complementary strand, or b) the region from -648 to -588 of SEQ ID NO 1 or the complementary strand counted from the transcription start site, more preferably from -638 to -598 of SEQ ID NO 1 or the complementary strand, or c) the region from -618 to -558 of SEQ ID NO 1 or the complementary strand counted from the transcription start site, more preferably from -608 to -568 of

SEQ ID NO 1 or the complementary strand.

62. The isolated nucleic acid sequence according to any of claims 59 to 61 , comprising at least 15 contiguous nucleotides.

63. The isolated nucleic acid sequence according to any of claims 59 to 61 , comprising at least 20 contiguous nucleotides.

64. The isolated nucleic acid sequence according to any of claims 59 to 61 , comprising one sequence according to claim 60 and one sequence according to claim 61 linked by a spacer sequence which is not capable of hybridising with any nucleotide sequence of SEQ ID NO 1 or the corresponding complementary strand.

65. The isolated nucleic acid sequence of claim 64, wherein the length of the spacer is from 8 to 28 nucleotides.

66. The isolated nucleic acid sequence according to any of the claims 59 to 63, wherein the mutation/polymorphism is located in the centre of the nucleic acid sequence.

67. The isolated nucleic acid sequence according to any of the claims 59 to 63, wherein the mutation/polymorphism is located in the 5' end of the nucleic acid sequence.

68. The isolated nucleic acid sequence according to any of the claims 59 to 63, wherein the mutation/polymorphism is located in the 3' end of the nucleic acid sequence.

69. The isolated nucleic acid sequence according to any of the claims 59 to 68, wherein the sequence is complementary to a sub-sequence of the coding strand of a target nucleotide sequence.

70. The isolated nucleic acid sequence according to any of the claims 59 to 68, wherein the sequence is complementary to a sub-sequence to the non-coding strand of a target nucleotide sequence.

71. The isolated nucleic acid sequence according to any of the claims 59 to 70, wherein the nucleotides are selected from RNA, DNA, LNA, PNA monomers or chemically modified nucleotides capable of hybridising to a target nucleic acid sequence, more preferably wherein the nucleotide sequence comprises at least one LNA monomer.

72. A kit-of-parts for predicting an increased risk for a subject of developing a metabolic disorder comprising

- at least one a nucleic acid sequence comprising at least 10 contiguous nucleotides of the region from nucleotide -950 to -550 of SEQ ID NO 1 , or the complementary strand, and

- a carrier of information, such as a sheet of paper, containing experimental instructions for the user and/or information relating to the diagnosis and/or prediction of increased risk for metabolic disease that the kit may be used for.

73. The kit-of-parts according to claim 72, wherein the at least one nucleic acid sequence is a nucleic acid sequence according to any of claim 59 to 71.

74. The kit-of-parts according to claim 72, wherein the nucleic acid sequence comprises at least 10 contiguous nucleotides of the region from -879 to -819 of

SEQ ID NO 1 or the complementary strand counted from the transcription start site, more preferably from -869 to -829 of SEQ ID NO 1 of the complementary strand.

75. The kit-of-parts according to claim 72, wherein the nucleic acid sequence comprises at least 10 contiguous nucleotides selected from the group comprising a) the region from -946 to -886 of SEQ ID NO 1 or the complementary strand counted from the transcription start site, more preferably from -936 to -896 of

SEQ ID NO 1 or the complementary strand, and b) the region from -648 to -588 of SEQ ID NO 1 or the complementary strand counted from the transcription start site, more preferably from -638 to -598 of SEQ ID NO 1 or the complementary strand, and c) the region from -618 to -558 of SEQ ID NO 1 or the complementary strand counted from the transcription start site, more preferably from -608 to -568 of SEQ ID NO 1 or the complementary strand.

76. The kit-of-parts according to any of claims 72 to 75, wherein the nucleic acid sequence comprises at least 15 contiguous nucleotides.

77. The kit-of-parts according to any of claims 72 to 75, wherein the nucleic acid sequence comprises at least 20 contiguous nucleotides.

78. The kit-of-parts according to any of claims 72 to 75, wherein the nucleic acid sequence comprises one sequence according to claim 74 and one sequence according to claim 75 linked by a spacer sequence which is not capable of hybridising with any nucleotide sequence of SEQ ID NO 1 or the corresponding complementary strand.

79. The kit-of-parts according to claim 78, wherein the length of the spacer is from 8 to 28 nucleotides.

80. The kit-of-parts according to any of claims 72 to 77, wherein the mutation/polymorphism is located in the centre of the nucleic acid sequence.

81. The kit-of-parts according to any of claims 72 to 77, wherein the mutation/polymorphism is located in the 5' end of the nucleic acid sequence.

82. The kit-of-parts according to any of claims 72 to 77, wherein the mutation/polymorphism is located in the 3' end of the nucleic acid sequence.

83. The kits-of-parts according to any of claims 72 to 77, wherein the mutation/polymorphism is located adjacent to the 3' or 5' end of the nucleic acid sequence.

84. The kit-of-parts according to any of claims 72 to 82, wherein the sequence is complementary to a sub-sequence of the coding strand of a target nucleotide sequence.

85. The kit-of-parts according to any of claims 72 to 82, wherein the sequence is complementary to a sub-sequence to the non-coding strand of a target nucleotide sequence.

86. The kit-of-parts according to any of claims 72 to 85, wherein the nucleotides are selected from RNA, DNA, LNA, PNA monomers or chemically modified nucleotides capable of hybridising to a target nucleic acid sequence, more preferably wherein the nucleotide sequence comprises at least one LNA monomer.

87. The kit-of-parts according to claim 72, wherein the nucleic acid sequence comprises a sequence according to claim 60.

88. The kit-of-parts according to claim 72 to 87, wherein the nucleic acid sequence is linked to a detectable label

89. The kit-of-parts according to any of claims 72 to 88, further comprising a second primer suitable, together with the first primer, for amplifying a region of the muscle glycogen synthase gene comprising at least one of the mutations or polymorphisms, and - a polymerase, and, optionally, nucleotide monomers.

90. The kit-of-parts according to claim 89, wherein one of the primers is coupled to an entity suitable for a subsequent immobilisation reaction and a solid surface to which the entity can be immobilised.

91. The kit-of-parts according to claim 89, wherein the primer pair comprises at least one allele specific primer.

92. The kit-of-parts according to claim 89, further comprising a restriction enzyme for cleaving an amplified region, such as wherein the restriction enzyme is Mni\, BstU , or Ac/I or other isoschizomers.

93. The kit-of-parts according to claim 89, further comprising a primer wherein the polymorphic site is adjacent to the 3' end, a polymerase and at least one fluorescently labelled nucleotide monomer.

94. The kit-of-parts according to claim 89, further comprising a capture probe and a detection probe.

95. The kit-of-parts according to claim 94, wherein either the capture probe and/or the detection probe and/or an amplification primer is allele-specific, such as wherein one allele-specific probe is specific for a Poly2 allele and the other allele-specific probe is specific for a Polyl allele, a Poly3 allele, or a Poly4 allele.

96. A kit-of-parts for predicting an increased risk of a subject of developing a metabolic disorder by sequencing of part of the GYS1 promoter sequence, said kit comprising at least one primer pair for specific amplification of the promoter sequence, said primer pair comprising a sequence primer with or without 5' extension with the purpose to use it as a universal sequence primer, said kit further comprising mononucleotides, polymerase, and/or buffers, and, preferably, a carrier of information, such as a sheet of paper, containing experimental instructions for the user and/or information relating to the diagnosis and/or prediction of increased risk for metabolic disease that the kit may be used for.

97. The kit-of-parts according to claim 96, wherein the primers for amplification include pairs selected from SEQ ID No 3 to 10.