WO2010072608A1

WO2010072608A1 - Pcsk1 single nucleotide polymorphism in type 2 diabetes

Info

Publication number: WO2010072608A1
Application number: PCT/EP2009/067064
Authority: WO
Inventors: Soren Germer; Mitchell Lee Martin
Original assignee: F. Hoffmann-La Roche Ag
Priority date: 2008-12-22
Filing date: 2009-12-14
Publication date: 2010-07-01

Abstract

The present invention provides a polymorphism in the PCSK1 gene which is indicative for a susceptibility to type 2 diabetes.

Description

PCSKl SINGLE NUCLEOTIDE POLYMORPHISM IN TYPE 2 DIABETES

Type 2 diabetes (T2D) is an increasingly prevalent complex disease in both developed and developing societies characterized by defective insulin secretion in the pancreatic β cells and insulin resistance in the liver, muscle and adipose tissues. The molecular pathogenic mechanisms are not fully understood. This study hypothesized that common polymorphisms in the genes en- coding known insulin-producing and -responsive pathways influence susceptibility to T2D.

T2D has been shown to have a strong familial transmission based on studies of concordance rates of monozygotic twin pairs with T2D. The high prevalence of the disease and increasing population affected shows an unmet medical need to define other genetic factors involved in T2D and to more precisely define the associated risk factors. Also needed are diagnostic assays to identify the propensity to develop T2D and therapeutic agents for prevention and treatment of the disease.

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

SNPs are the most common form of genetic variation responsible for differences in disease susceptibility and drug response. SNPs can directly contribute to or, more commonly, serve as markers for many phenotypic endpoints such as disease risk or the drug response differences between patients. Identification of these genetic factors can lead to diagnostic methods, reagents and reagent kits for the identification of individuals who have a propensity to develop certain diseases.

Summary of the Invention

The instant invention concerns the identification of genetic factors in the PCSKl gene that predisposes individuals to diabetes, with a focus specifically on nucleic acid fragments of genes having single nucleotide polymorphisms ("SNPs") which are amenable to diagnostic and therapeutic intervention.

In a first object, the present invention provides a method for determining a susceptibility of a human subject to type 2 diabetes comprising: providing a nucleic acid sample of the subject, detecting the nucleotide present at position 782 of a PCSKl gene as defined by the positions in Seq. Id. No. 1, wherein the presence of a guanine at position 782 indicates a susceptibility to type 2 diabetes.

In a second object, the present invention provides a method of estimating the responsiveness of a subject to compounds whose action depends on PCSKl activity comprising providing a nucleic acid sample of the subject, detecting the nucleotide present at position 782 of a PCSKl gene as defined by the positions in Seq. Id. No. 1, wherein the presence of a guanine at position 782 indicates that the subject is statistically more likely to have an altered than average response to the compound than an individual lacking the guanine at position 782 of a PCSKl gene as defined by the positions in Seq. Id. No. 1.

In a preferred embodiment of the method for estimating the responsiveness of a subject to compounds whose action depends on PCSKl activity, the compound is selected from the group consisting of metformin, DPPIV inhibitors, GLP-I analogues, SGLT2 inhibitors, HbHSDl inhibitors, statins, PPAR-gammas, PPAR-alfa and fenofibrates, insulin, orlistat and sulfonylureas.

Exemplary SGLT2 inhibitors are dapagliflozin, remogliflozin, sergliflozin. Exemplary DPPIV inhibitors are vildagliptin, sitagliptin, saxagliptin, linagliptin and alogliptin.

In another preferred embodiment of the methods of the present invention, the polymorphism is detected by sequence determination.

In a further preferred embodiment of the methods of the present invention, the polymorphism is detected by gel electrophoresis. In yet a further preferred embodiment of the methods of the present invention, the polymorphism is detected by a single strand conformational polymorphism (SSCP) method.

In a third object, the present invention provides a kit for determining a susceptibility of a human subject to type 2 diabetes comprising: a PCR primer pair allowing amplification of a nucleic acid sequence including position 782 of a PCSKl gene as defined by the positions in Seq. Id. No. 1 and reagents for performing the PCR reaction., wherein the presence of a guanine at position 782 indicates a susceptibility to type 2 diabetes. In accordance with the invention, genotyping of polymorphic variants can be carried out using any suitable methodology known in the art and it is to be understood that the invention is in no way limited by the precise technique used to carry out the genotyping.

Known techniques which may be used for genotyping single nucleotide polymorphisms in- elude ligation detection reaction (LDR; Day, D. J., Speiser, P. W., White, P. C. & Barany, F. Genomics 29, 152-62 (1995)), mass spectrometry, particularly matrix-assisted laser desorp- tion/ionization time-of- flight mass spectrometry (MALDI-TOF-MS), single nucleotide primer extension and DNA chips or microarrays (see review by Schafer, A. J. and Hawkins, J. R. in Nature Biotechnology, VoI 16, pp 33-39 (1998)). The use of DNA chips or microarrays may enable simultaneous genotyping at many different polymorphic loci in a single individual or the simultaneous genotyping of a single polymorphic locus in multiple individuals. SNPs may also be scored by DNA sequencing.

In addition to the above, SNPs are commonly scored using PCR-based techniques, such as PCR-SSP using allele-specifϊc primers (described by Bunce M, et al, Tissue Antigens, 1995; 50: 23-31). This method generally involves performing DNA amplification reactions using genomic DNA as the template and two different primer pairs, the first primer pair comprising an allele- specifϊc primer which under appropriate conditions is capable of hybridising selectively to the wild type allele and a non allele-specifϊc primer which binds to a complementary sequence elsewhere within the gene in question, the second primer pair comprising an allele-specifϊc primer which under appropriate conditions is capable of hybridising selectively to the variant allele and the same non allele-specifϊc primer. Further suitable techniques for scoring SNPs include PCR ELISA and denaturing high performance liquid chromatography (DHPLC).

If the SNP results in the abolition or creation of a restriction site, genotyping can be carried out by performing PCR using non-allele specific primers spanning the polymorphic site and di- gesting the resultant PCR product using the appropriate restriction enzyme (also known as PCR- RFLP). Restriction fragment length polymorphisms, including those resulting from the presence of a single nucleotide polymorphism, may be scored by digesting genomic DNA with an appropriate enzyme then performing a Southern blot using a labeled probe corresponding to the polymorphic region (see Molecular Cloning: A Laboratory Manual, Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

The known techniques for genotyping polymorphisms are of general applicability and it will be readily apparent to persons skilled in the art that many of the known techniques may be adapted for the scoring of single nucleotide polymorphisms in the PCSKl gene.

Genotyping is preferably carried out in vitro, and is most preferably performed on an iso- lated sample containing genomic DNA prepared from a suitable tissue sample obtained from the subject under test. Most commonly, genomic DNA is prepared from a sample of whole blood according to standard procedures which are well known in the art. If genomic sequence data for the individual under test in the region containing the SNP is available, for example in a genomic sequence database as a result of a prior genomic sequencing exercise, then genotyping of the SNP may be accomplished by searching the available sequence data.

In the case of genetic variants which have a detectable effect on the mRNA transcripts transcribed from a given gene, for example variants which cause altered splicing or which affect transcript termination or which affect the level or mRNA expression, then as an alternative to detecting the presence of the variant at the genomic DNA level, the presence of the variant may be inferred by evaluating the mRNA expression pattern using any suitable technique. Similarly, in the case of genetic variants which have a detectable effect on the protein products encoded by a gene, for example variants which cause a change in primary amino acid sequence, structure or properties of the encoded protein, the presence of the variant may be inferred by evaluating the sequence, structure or properties of the protein using any convenient technique. The above- described screening methods may be used prognostically to identify individuals pre-disposed to or protected from type 2 diabetes by virtue of their genetic make-up. The "susceptibility to type 2 diabetes" may be manifest as an increased risk of developing disease as compared to individuals who do not possess a variant allele (i.e. subjects who are homozygous for the corresponding normal allele(s) having adenine at position 782 as defined by the positions in Seq. Id. No. 1). In a particular embodiment the method may be used to screen asymptomatic individuals (i.e. individuals who do not exhibit significant symptoms of type 2 diabetes according to standard diagnostic criteria) in order to identify those "at risk" of developing type 2 diabetes. In particular embodiments, the screens may be used to assess risk of developing type 2 diabetes in human subjects. The results of such screens may facilitate early intervention with therapeutic treatments, particularly prophylactic treatments aimed at preventing, reducing or delaying the clinical symptoms of type 2 diabetes.

Experimental Part

Forty- five candidate genes from the insulin secretion and insulin action pathways were selected respectively. A one-stage case-control association study in 493 confirmed diabetic cases and 506 confirmed controls of European origin was conducted. Tests for association between genetic polymorphisms and T2D under allelic and genotypic models were performed. The key findings of this study included that a polymorphism in PCSKl was significantly associated with protection from T2D under the allelic test.

Study Population A total of 999 patients were included in the study to investigate the variation in the presence of SNPs between control and Type 2 diabetes populations. From the case-control study, the phenotype was simply "diabetes". Other sub-phenotypes could be included in the analysis including age, gender and waist-to-hip ratio. The samples were collected as described in Zee et al, Atherosclerosis 197:931-936 (2008). The population contained roughly equal numbers of males and females (513 males and 486 females). Samples were also well matched with identical numbers of males (288 cases and 225 controls) and females (205 cases and 281 controls) in the diabetic and unaffected groups.

In any population based study, it is important to match the cases and controls in order to avoid spurious results based on unknown, confounding factors. In the context of genetics studies, this means that case and control populations should be genetically similar across the genome, with the exception of regions containing genes that predispose to the phenotype being studied. That is, a random set of markers should show broadly similar allele frequencies in the case and control populations. Population stratification was unlikely to be present in this study as the pa- tients and controls were not only matched for gender but for self-reported ethnicity as well.

SNP Selection

The SNPs chosen to assess the association of PCSKl to Type 2 diabetes were selected from the publically available human HapMap, Phase II (www.hapmap.org) according to the RefSeq gene mapping per UCSC genome build 35/36. Common SNPs (>5% minor allele fre- quency (MAF)) within and immediately flanking (+/- 2.5kb) the PCSKl gene were selected using the LD Select method (CS. Carlson et al., Am J Hum Genet (2004) 74:106-120) to interrogate the largest proportion of genetic diversity of PCSKl with the fewest numbers of SNPs.

SNP Genotyping

The GC-clamp assay (see Wang et al., (6):885-93 (2005)) was used to genotype the SNPs. This is a melting curve assay that relies on allele specific PCR, without labeled oligonucleotides. Depending on the sample genotype either one or the other, or both, allele-specific primer(s) amplify. With tails of unequal length attached to allele-specific primers, the PCR product has a distinct melting temperature that depends largely on product length and GC content. Depending on which of the two primers is responsible for the amplification, genotypes can be determined by inspection of a melting curve on a real-time PCR instrument.

Summary of Allelic Association Results

Marker-trait association was examined using contingency table analyses and Fisher's Exact test for empirical p-values. A summary of the results from the allelic chi-square association test (2x2, 1 d.f.), followed by correction for multiple testing using False Discovery Rate are shown for the PCSKl SNP found to be significant at q<0.05:

Chi-sq allelic Chi-sq genotypic Allelic

SNP p £ V- a MAF Odds Ratio [95CI] gene rs6232 0.00043 0.0337 0.00144 0.083 0.05 0.46 [0.29-0.72] PCSKl

Summary of the Genotypic Association Results

A genotypic chi-square association test (2x3, 2 d.f.) for SNPs in PCSKl were not found to be significant at q<0.05 following False Discovery Rate correction for multiple testing.

While there are shown and described presently preferred embodiments of the invention, it is to be distinctly understood that the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims.

Claims

1. A method for determining a susceptibility of a human subject to type 2 diabetes comprising: providing a nucleic acid sample of the subject, detecting the nucleotide present at position 782 of a PCSKl gene as defined by the positions in Seq. Id. No. 1, wherein the presence of a guanine at position 782 indicates a susceptibility to type 2 diabetes.

2. A method of estimating the responsiveness of a subject to compounds whose action depends on PCSKl activity comprising providing a nucleic acid sample of the subject, detecting the nucleotide present at position 782 of a PCSKl gene as defined by the positions in Seq. Id. No. 1, wherein the presence of a guanine at position 782 indicates that the subject is statistically more likely to have an altered than average response to the compound than an individual lacking the guanine at position 782 of a PCSKl gene as defined by the positions in Seq. Id. No. 1.

3. The method of claim 2, wherein the compound whose action depends on PCKSl activity is selected from the group consisting of metformin, DPPIV inhibitors, GLP-I analogues, SGLT2 inhibitors, HbHSDl inhibitors, statins, PPAR-gammas, PPAR-alfa and fenofϊbrates, in- sulin, orlistat and sulfonylureas.

4. The method of claims 1 to 3, wherein the polymorphism is detected by sequence determination.

5. The method of claim 1 to 3, wherein the polymorphism is detected by gel electrophoresis.

6. The method of claim 1 to 3, wherein the polymorphism is detected by a single strand conformational polymorphism (SSCP) method.

7. A kit for determining a susceptibility of a human subject to type 2 diabetes comprising: a PCR primer pair allowing amplification of a nucleic acid sequence including position 782 of a PCSKl gene as defined by the positions in Seq. Id. No. 1 and reagents for performing the PCR reaction., wherein the presence of a guanine at position 782 indicates a susceptibility to type 2 diabetes.

8. The methods and kits substantially as described herein before.