Over 50 single nucleotide polymorphisms (SNPs) have been identified by genome wide association studies (GWAS) to be associated with susceptibility to type 2 diabetes (T2D); however the causal gene in most cases is not known. In this study we sought to identify which may be the most likely causal genes at five T2D GWAS loci by measuring their expression in control and T2D islets, as well as observing their regulation by glucose. We measured the expression of ten genes at five loci (CDKN2A/2B, CDC123/CAMK-1D, HHEX/IDE, TSPAN8/LGR5, and DGKB/TMEM 195), in control and human pancreatic islets by real-time PCR. We then measured the expression of these genes in the rodent pancreatic beta cell line INS-1 exposed to 5.6 mmol/l, 11 mmol/l and 28 mmol/l glucose for 48 hours. We found differential expression of the longest isoform of CDKN2B specifically between control and T2D human islets, whereas the shortest isoform of this gene had no expression in islets. Tmem195 was the only gene to show differential expression in response to increasing glycemia in INS-1 cells under the conditions described. Our study is an example of how the differential expression of genes in loci spanning more than one gene can aid identification of the more likely causal gene.
Genome wide association studies (GWAS) have identified more than 50 single nucleotide polymorphisms (SNPs) associated with susceptibility to type 2 diabetes (T2D) in human populations of different ethnicities [1-3]. Variants associated with T2D risk predictors, such as fasting glucose and fasting insulin have also been identified [
Identification of precisely which gene may be affected by the variant is not straightforward; most candidates are predicted by the distance from the index SNP, their location in open chromatin and by biological plausibility. The recent characterization of the genome by ENCODE [6-11] highlights many previously uncharacterized regulatory elements which may also inform on the identity of the gene affected, but in many cases, the index locus still will encompass several possible candidates. An important first step in identifying which genes play a role in the etiology of diabetes is to determine which of those genes are expressed in human islet samples: genes not expressed either in control or type 2 diabetic beta cells may be less likely to be causative genes. Whether or not candidate genes are expressed in relevant tissues is complicated by the fact that their expression may also be affected by the diabetic microenvironment [
In this study, we aimed to use gene expression profiling in human islet samples to provide evidence to aid definition of the causal genes at 5 loci which were amongst the first to be associated with T2D susceptibility by GWAS and had more than one candidate gene at each locus [1,2]. In each case the most significantly associated SNP was positioned at a locus spanning two genes where both are equally plausible candidates in terms of proximity to the index SNP [1,2]. The 5 loci were located at chromosome 10p13 (CAMK1D/CDC123), 9p21.3 (CDK N2A/CDKN2B), 7p21.2 (DGKB/TMEM195), 10q23.33 (HHEX/IDE) 12q21.1 (TSPAN8/LGR5). Moreover, all but the TSPAN8/LGR5 locus have been associated with glucose and insulin traits [18-20], indicating that they may have their effect through altered beta cell function. We analyzed the expression of target genes in normal human islet samples (n = 5) and to establish whether the expression of candidate genes could be responsive to the diabetic microenvironment, we analyzed the expression of the same set of genes in islet samples from donors with T2D (n = 3). We also analyzed the expression of the 10 genes in the rodent pancreatic beta cell line INS-1 treated with exogenous glucose.
Islet samples from control and T2D donors were procured from ProCell Biotech (Newport Beach, USA). Islet purity and viability measurements were conducted by the donor company and were determined by dithizone and fluorescein diacetate/propidium iodide staining, respectively. The control and T2D islet donor cohort information is shown in
RNA was extracted from the cells and human isletpreparations using the MirVana RNA Isolation pro-
cedure, according to the manufacturer’s instructions (Life Technologies, Foster City, USA). RNA from the INS-1 cells (3 biological replicates), and from diabetic and non-diabetic human islets was reverse transcribed using Superscript III enzyme (Life Technologies, Foster City, USA), after DNA was removed from the RNA samples using TURBO DNA-free (Life Technologies, Foster City, California). Loci were chosen on the basis that they have been associated with T2D susceptibility or fasting glucose by GWAS and include 2 equally likely candidate genes based on proximity to the index SNP [1,2]. Expression of the 10 candidate genes was analyzed on custom designed cards using TaqMan Low Density Array (TLDA) on the ABI Prism 7900HT platform (Life Technologies, Foster City, USA). Expression of each target was measured by relative quantification, and normalized back to the global ΔCt in order to allow comparison of expression levels across genes and between tissues.
The rodent pancreatic beta cell line INS-1 was cultured in RPMI 1640 medium (Life Technologies, Foster City, USA) at an ambient glucose concentration of 11 mmol/l, with 10% fetal calf serum and 1% penicillin/ streptomycin. Cells were incubated under these conditions in a humidified atmosphere at 37˚C for 72 hours. After 72 hours, the INS-1 cells were then seeded at a density of 1.4 ´ 104 cells/cm2 in 25 cm2 flasks. Cells were cultured at one of three glucose concentrations for 48 hours: 5.6 mmol/l (low glucose), 11 mmol/l (ambient glucose), and 28 mmol/l (high glucose) (
Significant changes in gene expression between control and T2D human islets were identified using the Mann-Whitney U test and the most stable endogenous controls (B2M, GUSB) were found using the GeNorm algorithm [
Dozens of loci have been associated with T2D susceptibility by GWAS, although the causal gene or genes in most cases remain elusive. We have analyzed the expression of ten genes at five loci in control and T2D human
islets in the hopes of identifying which gene at each of these loci is more likely to be the causal gene. All but one (TSPAN8/LGR5) of these loci have been associated with glucose and insulin traits [18-20], indicating that they may have their effect through altered beta cell function.
Results of our screen are given in
The CDKN2B gene is in a locus spanning two potential candidate genes (CDKN2A/2B). This particular susceptibility locus is also associated with impaired GSIS, with individuals with the risk allele for rs10811661 showing decreased insulin release after an oral or IV glucose challenge [
proliferation and develop diabetes and Cdkn2a transgenic mice models show differences in islet cell proliferation [
We observed an up-regulation of Tmem195 in response to increasing glycemia in INS-1 cells, although we found little evidence for expression of this gene or the other candidate gene at this locus, DGKB, in human islets (
estimates of beta cell function and decreased GSIS [
At the remaining two loci (CAMK1D/CDC123 and HHEX/IDE), all 4 genes showed evidence of expression in human islets, but we found no evidence of significant gene expression differences between control and T2D islets. Cdc123, Hhex and Ide (Camk1d is not present in rat) did not show differential regulation in INS-1 cells in response to increasing glycemia, providing no evidence for a role of these genes in T2D progression. These results could indicate that altered gene expression may not mediate the effects of these genes at these loci, although isoform-specific expression differences for CAMK1D and IDE, which encode more than one transcript variant, cannot be ruled out. Both loci have been associated with aspects of beta cell function, indicating that they have their effect through altered beta cell function [18,20,32].
In conclusion, we have demonstrated that expression profiling of human islets could be a useful tool in indicating causality at T2D susceptibility loci. Our data suggest that CDKN2B, rather than CDKN2A, and TSPAN8 rather than LGR5, may be better candidates at their respective loci, based on their expression profiles in nondiabetic and diabetic human islets, and in beta cells treated with glucose in vitro.
This study was funded by the Wellcome Trust Project Grant WT081278MA to L.W. Harries and by a charitable donation from the Mendip Golf Club.