High density mapping
GWAS do not inevitably lead to identification of a gene or genes in a given locus associated with disease.The most strongly associated SNPs are often only markers for the functional variant responsible for the observed genetic effect and most associated regions harbor several genes.Therefore, additional fine mapping of the loci in even larger sample sets is often necessary. To do this cost-efficiently a Cardio-Metabochip has been developed for metabolic/cardiovascular gene mapping. This custom-design Illumina Infinium genotyping chip contains ∼200,000 polymorphisms selected to cover association signals from a wide range of metabolic disorders (T2DM, lipid disorders, obesity, and cardiovascular disease), was designed to perform both deep replication of major disease signals and fine mapping of established loci. Meta-analysis of previous GWAS with an additional 22,669 T2DM cases and 58,119 controls genotyped using the Cardio-Metabochip has recently added another eight new loci associated with T2DM in the European population .
Next-generation sequencing will provide even denser coverage of genetic variation. Scientists in Iceland recently identified four new rare and low-frequency variants (minor allele frequency 0–5%) associated with T2DM through whole-genome sequencing of 2630 Icelanders and imputation of genotypes in more than 290,000 closely related individuals . The GoT2D consortium aims to map lower frequency variants via low-coverage whole-genome sequencing, deep exome sequencing, and next-generation 2.5M SNP chip genotyping of 1,325 cases and 1,325 controls selected in the phenotype extremes of T2DM. A custom-designed Exome Chip containing rare variants that have been seen at least three times in different studies has also been designed and is currently used to try to identify rare variants associated with T2DM.
Functions of associated genes
Most identified diabetes loci have not been mechanistically tied to the disease. While loci are commonly referred to by the names of candidate genes located close to them, only a few are close to strong biologic candidates, including the melatonin receptor (MTNR1B) and the insulin receptor substrate-1 (IRS1). For others, like TCF7L2 and GIPR, the evidence is quite strong that an intronic SNP is the causal SNP. Melatonin receptor 1B (MTNR1B) has been found to be associated with both fasting glucose and T2DM risk [48–50]. Melatonin works as a chronobiotic factor, adjusting the timing of the biologic clock. Its receptors are present in the pancreas and melatonin is proposed to contribute to the nocturnal lowering of insulin in humans. The MTNR1B risk genotype is associated with impaired early insulin release to both oral and intravenous glucose and insulin secretion deteriorates over time in the risk allele carriers . The proposed mechanism by which MTNR1B polymorphism could predispose to T2DM involves altered expression of MTNR1B in pancreatic β-cells leading to decreased cAMP/cGMP concentrations via G proteins and, thereby, impaired insulin secretion.
The insulin receptor substrate 1 (IRS1) gene encodes a protein that mediates insulin’s control of various cellular processes by transmitting signals from the insulin receptor to intracellular signaling pathways. The C allele of rs2943641 has been shown to be associated with insulin resistance and increased risk of diabetes.The genetic variant causes reduced basal levels of IRS1 protein and decreased insulin induction of IRS1-associated phosphatidylinositol-3-hydroxykinase activity in human skeletal muscle biopsies .
TCF7L2 is a transcription factor playing an important role in the Wnt signaling pathway. The risk allele is associated with decreased insulinogenic index and lower disposition index, suggesting a reduced capacity for insulin secretion in relation to insulin sensitivity. Since it was identified as a diabetes gene it has been shown to be important for several vital functions in the pancreatic islet, including pancreas development, determination of β-cell mass, and maintenance of the secretory function of mature β-cells.
The incretin hormone GIP (glucose-dependent insulinotropic polypeptide) promotes pancreatic β-cell function by potentiating insulin secretion and β-cell proliferation. The GIP receptor (GIPR] locus showed association to postprandial insulin levels in a meta-analysis performed by the MAGIC consortium but was surprisingly not associated with risk of diabetes in the DIAGRAM+ study [13,52]. The reason seems to be that the same variant results in decreased BMI, which neutralizes the effect of the SNP on risk of T2DM. GIP influences expression of the inflammatory cytokine OPN in islets and fat, which in turn, has protective effects on β-cell proliferation and potentially apoptosis, but detrimental effects on insulin sensitivity [53,54].
Many of the other recently identified loci can be subgrouped based on their association with other phenotypes with a key role in T2DM etiology. Exploration of the effects of T2DM-associated variants on glucose and insulin traits in nondiabetic populations has shown that most of the known loci act through an effect on insulin secretion rather than insulin resistance (Table 26.1) [13,24,55,56].
Fasting glucose-raising alleles of the MADD, GIPR, GCK, FADS, DGKB, PROX1, TCF7L2, SLC30A8, HHEX/IDE, CDKAL1, CDKN2A/2B, and C2CD4B loci have all been associated with either abnormal insulin processing or secretion whereas GCKR and IGF1 are associated with OGTT-based disposition indices and β-cell function [55,57]. The DIAGRAM+ consortium observed that three loci (TCF7L2, ARAP1, and CDKAL1) were associated with reduced fasting insulin also suggestive of β-cell dysfunction, whereas the T2DM risk alleles at PPARG, FTO, IRS1, GCKR, and KLF14 were associated with higher fasting insulin, indicating a primary effect on insulin action [13,57].