Hemang Parikh

Genome-Wide Association Analysis Identifies Loci for Type 2 Diabetes and Triglyceride Levels

New strategies for prevention and treatment of type 2 diabetes (T2D) require improved insight into disease etiology. We analyzed 386,731 common single-nucleotide polymorphisms (SNPs) in 1464 patients with T2D and 1467 matched controls, each characterized for measures of glucose metabolism, lipids, obesity, and blood pressure. With collaborators (FUSION and WTCCC/UKT2D), we identified and confirmed three loci associated with T2D—in a noncoding region near CDKN2A and CDKN2B, in an intron of IGF2BP2, and an intron of CDKAL1—and replicated associations near HHEX and in SLC30A8 found by a recent whole-genome association study. We identified and confirmed association of a SNP in an intron of glucokinase regulatory protein (GCKR) with serum triglycerides. The discovery of associated variants in unsuspected genes and outside coding regions illustrates the ability of genome-wide association studies to provide potentially important clues to the pathogenesis of common diseases.

A systems genetics approach identifies genes and pathways for type 2 diabetes in human islets.

Close to 50 genetic loci have been associated with type 2 diabetes (T2D), but they explain only 15% of the heritability. In an attempt to identify additional T2D genes, we analyzed global gene expression in human islets from 63 donors. Using 48 genes located near T2D risk variants, we identified gene coexpression and protein-protein interaction networks that were strongly associated with islet insulin secretion and HbA(1c). We integrated our data to form a rank list of putative T2D genes, of which CHL1, LRFN2, RASGRP1, and PPM1K were validated in INS-1 cells to influence insulin secretion, whereas GPR120 affected apoptosis in islets. Expression variation of the top 20 genes explained 24% of the variance in HbA(1c) with no claim of the direction. The data present a global map of genes associated with islet dysfunction and demonstrate the value of systems genetics for the identification of genes potentially involved in T2D.

Impact of an Exercise Intervention on DNA Methylation in Skeletal Muscle From First-Degree Relatives of Patients With Type 2 Diabetes

To identify epigenetic patterns, which may predispose to type 2 diabetes (T2D) due to a family history (FH) of the disease, we analyzed DNA methylation genome-wide in skeletal muscle from individuals with (FH+) or without (FH−) an FH of T2D. We found differential DNA methylation of genes in biological pathways including mitogen-activated protein kinase (MAPK), insulin, and calcium signaling (P ≤ 0.007) and of individual genes with known function in muscle, including MAPK1, MYO18B, HOXC6, and the AMP-activated protein kinase subunit PRKAB1 in skeletal muscle of FH+ compared with FH− men. We further validated our findings from FH+ men in monozygotic twin pairs discordant for T2D, and 40% of 65 analyzed genes exhibited differential DNA methylation in muscle of both FH+ men and diabetic twins. We further examined if a 6-month exercise intervention modifies the genome-wide DNA methylation pattern in skeletal muscle of the FH+ and FH− individuals. DNA methylation of genes in retinol metabolism and calcium signaling pathways (P < 3 × 10−6) and with known functions in muscle and T2D including MEF2A, RUNX1, NDUFC2, and THADA decreased after exercise. Methylation of these human promoter regions suppressed reporter gene expression in vitro. In addition, both expression and methylation of several genes, i.e., ADIPOR1, BDKRB2, and TRIB1, changed after exercise. These findings provide new insights into how genetic background and environment can alter the human epigenome.

Candidate genes for type 2 diabetes.

About 150 million people suffer from type 2 diabetes in the world today and it has been predicted that this number will be doubled within 15 years [1]. It is no longer a disease of the elderly, age at onset has decreased with the epidemic increase in prevalence. There are large ethnic and geographic variations in the prevalence of type 2 diabetes. In Scandinavia, where type 1 diabetes is common, type 2 diabetes accounts for about 85% of all cases with diabetes. However, the distinction between type 1 and type 2 diabetes may not be as clear as hitherto thought. In addition, the disease seems to be quite heterogeneous [2]. There is plenty of evidence that type 2 diabetes is inherited, the life time risk in offspring of one diabetic parent is about 40% and the relative risk (λs) for a sibling to a patient with type 2 diabetes is about 3. It is though unlikely that the cause of the explosion of diabetes is found in our genes. It is rather the rapid change of the environment with an affluent westernized society which triggers the epidemic in genetically predisposed individuals. What then makes up this genetic predisposition? In general, the chronic hyperglycemia characteristic of type 2 diabetes require both insulin resistance in muscle and liver and impaired insulin secretion in pancreatic β-cells. Insulin resistance by itself cannot cause diabetes. As long as the β-cell can compensate for the degree of insulin resistance, glucose tolerance remains normal. Accumulation of abdominal fat often predates the manifestation of these defects. It has therefore been suggested that the normal function of the subcutaneous adipose tissue is to serve as a buffer for excess intake of fat. When this buffer capacity is exceeded the body starts to deposit fat in places where it normally does not belong, e.g. muscle, liver and β-cells. According to this hypothesis a common defect could explain all defects. There is, however, no proof for this and it is likely that the genetic predisposition involves several pathways in different organs. Type 2 diabetes is considered a paradigm for a multifactorial polygenic disease where common variations in several genes interact (epistasis) to cause the disease when exposed to the affluent environment of too much food and too little exercise. In the following we will discuss potential candidates which could contribute to this genetic predisposition to type 2 diabetes.

A variant in the transcription factor 7-like 2 (TCF7L2) gene is associated with an increased risk of gestational diabetes mellitus

Aims/hypothesisGenetic and epidemiological studies suggest an association between gestational diabetes mellitus and type 2 diabetes. Both are polygenic multifactorial disorders characterised by beta cell dysfunction and insulin resistance. Our aim was to investigate whether common genetic variants that have previously been associated with type 2 diabetes or related phenotypes would also confer risk for gestational diabetes mellitus.Materials and methodsIn 1,881 unrelated pregnant Scandinavian women (649 women with gestational diabetes mellitus, 1,232 non-diabetic control subjects) we genotyped the transcription factor 7-like 2 (TCF7L2 rs7903146), adiponectin (ADIPOQ +276G > T), peroxisome-proliferator activated receptor, gamma 2 (PPARG Pro12Ala), PPARG-coactivator, 1 alpha (PPARGC1A Gly482Ser), forkhead box C2 (FOXC2 −512C > T) and β3-adrenergic receptor (ADRB3 Trp64Arg) polymorphisms using TaqMan allelic discrimination assay or RFLP.ResultsThe CC, CT and TT genotype frequencies of the TCF7L2 rs7903146 variant differed significantly between women with gestational diabetes mellitus and control women (46.3, 43.6 and 10.1% vs 58.5, 35.3 and 6.2%, p = 3.7 × 10−6, corrected p value [Pc] for multiple testing Pc = 2.2 × 10−5). The T-allele was associated with an increased risk of gestational diabetes mellitus (odds ratio 1.49 [95% CI 1.28–1.75], p = 4.9 × 10−7 [Pc = 2.8 × 10−6]). Compared with wild-type CC-genotype carriers, heterozygous (CT-genotype) and homozygous (TT-genotype) carriers had a 1.6-fold (95% CI 1.26–1.93, p = 3.7 × 10−5 [Pc = 0.0002]) and a 2.1-fold (95% CI 1.41–2.99, p = 0.0001 [Pc = 0.0008]) increased risk of gestational diabetes mellitus, respectively. The other polymorphisms studied were not significantly associated with gestational diabetes mellitus (ADIPOQ +276G > T: 1.17 [1.01–1.36], p = 0.039 [Pc = 0.23]; PPARG Pro12Ala: 1.06 [0.87–1.29], p = 0.53; PPARGC1A Gly482Ser: 0.96 [0.83–1.10], p = 0.54; FOXC2 −512C > T: 1.01 [0.87–1.16], p = 0.94; and ADRB3 Trp64Arg: 1.22 [0.95–1.56], p = 0.12).Conclusions/interpretationThe TCF7L2 rs7903146 variant is associated with an increased risk of gestational diabetes mellitus in Scandinavian women.

Differential gene expression in adipose tissue from obese human subjects during weight loss and weight maintenance.

BACKGROUND Differential gene expression in adipose tissue during diet-induced weight loss followed by a weight stability period is poorly characterized. Markers of these processes may provide a deeper understanding of underlying mechanisms. OBJECTIVE We aimed to identify differentially expressed genes in human adipose tissue during weight loss and weight maintenance after weight loss. DESIGN RNA from subcutaneous abdominal adipose tissue from 9 obese subjects was analyzed by using a complementary DNA microarray at baseline after weight loss on a low-calorie diet and after weight maintenance. RESULTS Subjects lost 18.8 ± 1.8% of weight and maintained this loss during weight maintenance (1.1 ± 2.1%; range: -9.3 to 10.6%). Most differentially expressed genes exhibited a reciprocal regulation and returned to baseline after weight loss (2163 genes) and weight maintenance (3175 genes). CETP and ABCG1, both of which participate in the HDL-mediated reverse cholesterol transport (RCT), were among the most upregulated of the 750 genes that were differentially expressed after both processes. Several genes involved in inflammation were downregulated. The use of real-time polymerase chain reaction confirmed or partially confirmed the previously implicated genes TNMD and MMP9 (both downregulated), PNPLA3 (upregulated), and CIDEA and SCD (both reciprocally regulated). CONCLUSIONS The beneficial effects of weight loss should be investigated after long-term weight maintenance. The processes of weight loss and weight maintenance should be viewed as biologically distinct. CETP and ABCG1 may be important mediators of these effects through HDL-mediated RCT.

Common variants in MODY genes increase the risk of gestational diabetes mellitus.

Aims/hypothesisImpaired beta cell function is the hallmark of gestational diabetes mellitus (GDM) and MODY. In addition, women with MODY gene mutations often present with GDM, but it is not known whether common variants in MODY genes contribute to GDM.Subjects and methodsWe genotyped five common variants in the glucokinase (GCK, commonly known as MODY2), hepatocyte nuclear factor 1-α (HNF1A, commonly known as MODY3) and 4-α (HNF4A commonly known as MODY1) genes in 1,880 Scandinavian women (648 women with GDM and 1,232 pregnant non-diabetic control women).ResultsThe A allele of the GCK −30G→A polymorphism was more common in GDM women than in control subjects (odds ratio [OR] 1.28 [95% CI 1.06−1.53], p=0.008, corrected p value, p=0.035). Under a recessive model [AA vs GA+GG], the OR increased further to 2.12 (95% CI 1.21−3.72, p=0.009). The frequency of the L allele of the HNF1A I27L polymorphism was slightly higher in GDM than in controls (1.16 [95% CI 1.001−1.34], p=0.048, corrected p value, p=0.17). However, the OR increased under a dominant model (LL+IL vs II; 1.31 [95% CI 1.08−1.60], p=0.007). The rs2144908, rs2425637 and rs1885088 variants, which are located downstream of the primary beta cell promoter (P2) of HNF4A, were not associated with GDM.Conclusions/interpretationThe −30G→A polymorphism of the beta-cell-specific promoter of GCK and the I27L polymorphism of HNF1A seem to increase the risk of GDM in Scandinavian women.

Prioritizing genes for follow-up from genome wide association studies using information on gene expression in tissues relevant for type 2 diabetes mellitus

BackgroundGenome-wide association studies (GWAS) have emerged as a powerful approach for identifying susceptibility loci associated with polygenetic diseases such as type 2 diabetes mellitus (T2DM). However, it is still a daunting task to prioritize single nucleotide polymorphisms (SNPs) from GWAS for further replication in different population. Several recent studies have shown that genetic variation often affects gene-expression at proximal (cis) as well as distal (trans) genomic locations by different mechanisms such as altering rate of transcription or splicing or transcript stability.MethodsTo prioritize SNPs from GWAS, we combined results from two GWAS related to T2DM, the Diabetes Genetics Initiative (DGI) and the Wellcome Trust Case Control Consortium (WTCCC), with genome-wide expression data from pancreas, adipose tissue, liver and skeletal muscle of individuals with or without T2DM or animal models thereof to identify T2DM susceptibility loci.ResultsWe identified 1,170 SNPs associated with T2DM with P < 0.05 in both GWAS and 243 genes that were located in the vicinity of these SNPs. Out of these 243 genes, we identified 115 differentially expressed in publicly available gene expression profiling data. Notably five of them, IGF2BP2, KCNJ11, NOTCH2, TCF7L2 and TSPAN8, have subsequently been shown to be associated with T2DM in different populations. To provide further validation of our approach, we reversed the approach and started with 26 known SNPs associated with T2DM and related traits. We could show that 12 (57%) (HHEX, HNF1B, IGF2BP2, IRS1, KCNJ11, KCNQ1, NOTCH2, PPARG, TCF7L2, THADA, TSPAN8 and WFS1) out of 21 genes located in vicinity of these SNPs were showing aberrant expression in T2DM from the gene expression profiling studies.ConclusionsUtilizing of gene expression profiling data from different tissues of individuals with or without T2DM or animal models thereof is a powerful tool for prioritizing SNPs from WGAS for further replication studies.

CLPTM1L Promotes Growth and Enhances Aneuploidy in Pancreatic Cancer Cells

Genome-wide association studies (GWAS) of 10 different cancers have identified pleiotropic cancer predisposition loci across a region of chromosome 5p15.33 that includes the TERT and CLPTM1L genes. Of these, susceptibility alleles for pancreatic cancer have mapped to the CLPTM1L gene, thus prompting an investigation of the function of CLPTM1L in the pancreas. Immunofluorescence analysis indicated that CLPTM1L localized to the endoplasmic reticulum where it is likely embedded in the membrane, in accord with multiple predicted transmembrane domains. Overexpression of CLPTM1L enhanced growth of pancreatic cancer cells in vitro (1.3-1.5-fold; PDAY7 < 0.003) and in vivo (3.46-fold; PDAY68 = 0.039), suggesting a role in tumor growth; this effect was abrogated by deletion of two hydrophilic domains. Affinity purification followed by mass spectrometry identified an interaction between CLPTM1L and non-muscle myosin II (NMM-II), a protein involved in maintaining cell shape, migration, and cytokinesis. The two proteins colocalized in the cytoplasm and, after treatment with a DNA-damaging agent, at the centrosomes. Overexpression of CLPTM1L and depletion of NMM-II induced aneuploidy, indicating that CLPTM1L may interfere with normal NMM-II function in regulating cytokinesis. Immunohistochemical analysis revealed enhanced staining of CLPTM1L in human pancreatic ductal adenocarcinoma (n = 378) as compared with normal pancreatic tissue samples (n = 17; P = 1.7 × 10(-4)). Our results suggest that CLPTM1L functions as a growth-promoting gene in the pancreas and that overexpression may lead to an abrogation of normal cytokinesis, indicating that it should be considered as a plausible candidate gene that could explain the effect of pancreatic cancer susceptibility alleles on chr5p15.33.

An integrated transcriptome and epigenome analysis identifies a novel candidate gene for pancreatic cancer

BackgroundPancreatic cancer is a highly lethal cancer with limited diagnostic and therapeutic modalities.MethodsTo begin to explore the genomic landscape of pancreatic cancer, we used massively parallel sequencing to catalog and compare transcribed regions and potential regulatory elements in two human cell lines derived from normal and cancerous pancreas.ResultsBy RNA-sequencing, we identified 2,146 differentially expressed genes in these cell lines that were enriched in cancer related pathways and biological processes that include cell adhesion, growth factor and receptor activity, signaling, transcription and differentiation. Our high throughput Chromatin immunoprecipitation (ChIP) sequence analysis furthermore identified over 100,000 regions enriched in epigenetic marks, showing either positive (H3K4me1, H3K4me3, RNA Pol II) or negative (H3K27me3) correlation with gene expression. Notably, an overall enrichment of RNA Pol II binding and depletion of H3K27me3 binding were seen in the cancer derived cell line as compared to the normal derived cell line. By selecting genes for further assessment based on this difference, we confirmed enhanced expression of aldehyde dehydrogenase 1A3 (ALDH1A3) in two larger sets of pancreatic cancer cell lines and in tumor tissues as compared to normal derived tissues.ConclusionsAs aldehyde dehydrogenase (ALDH) activity is a key feature of cancer stem cells, our results indicate that a member of the ALDH superfamily, ALDH1A3, may be upregulated in pancreatic cancer, where it could mark pancreatic cancer stem cells.