A genome-wide CRISPR screen identifies CALCOCO2 as a regulator of beta cell function influencing type 2 diabetes risk.

Identification of the genes and processes mediating genetic association signals for complex diseases represents a major challenge. As many of the genetic signals for type 2 diabetes (T2D) exert their effects through pancreatic islet-cell dysfunction, we performed a genome-wide pooled CRISPR loss-of-function screen in a human pancreatic beta cell line. We assessed the regulation of insulin content as a disease-relevant readout of beta cell function and identified 580 genes influencing this phenotype. Integration with genetic and genomic data provided experimental support for 20 candidate T2D effector transcripts including the autophagy receptor CALCOCO2. Loss of CALCOCO2 was associated with distorted mitochondria, less proinsulin-containing immature granules and accumulation of autophagosomes upon inhibition of late-stage autophagy. Carriers of T2D-associated variants at the CALCOCO2 locus further displayed altered insulin secretion. Our study highlights how cellular screens can augment existing multi-omic efforts to support mechanistic understanding and provide evidence for causal effects at genome-wide association studies loci.

Loss of ZnT8 function protects against diabetes by enhanced insulin secretion.

A rare loss-of-function allele p.Arg138* in SLC30A8 encoding the zinc transporter 8 (ZnT8), which is enriched in Western Finland, protects against type 2 diabetes (T2D). We recruited relatives of the identified carriers and showed that protection was associated with better insulin secretion due to enhanced glucose responsiveness and proinsulin conversion, particularly when compared with individuals matched for the genotype of a common T2D-risk allele in SLC30A8, p.Arg325. In genome-edited human induced pluripotent stem cell (iPSC)-derived ß-like cells, we establish that the p.Arg138* allele results in reduced SLC30A8 expression due to haploinsufficiency. In human ß cells, loss of SLC30A8 leads to increased glucose responsiveness and reduced KATP channel function similar to isolated islets from carriers of the T2D-protective allele p.Trp325. These data position ZnT8 as an appealing target for treatment aimed at maintaining insulin secretion capacity in T2D.

Type 2 diabetes risk alleles in PAM impact insulin release from human pancreatic ß-cells.

The molecular mechanisms underpinning susceptibility loci for type 2 diabetes (T2D) remain poorly understood. Coding variants in peptidylglycine α-amidating monooxygenase (PAM) are associated with both T2D risk and insulinogenic index. Here, we demonstrate that the T2D risk alleles impact negatively on overall PAM activity via defects in expression and catalytic function. PAM deficiency results in reduced insulin content and altered dynamics of insulin secretion in a human ß-cell model and primary islets from cadaveric donors. Thus, our results demonstrate a role for PAM in ß-cell function, and establish molecular mechanisms for T2D risk alleles at this locus.

Prioritising Causal Genes at Type 2 Diabetes Risk Loci.

PURPOSE OF REVIEW: Genome-wide association studies (GWAS) for type 2 diabetes (T2D) risk have identified a large number of genetic loci associated with disease susceptibility. However, progress moving from association signals through causal genes to functional understanding has so far been slow, hindering clinical translation. This review discusses the benefits and limitations of emerging, unbiased approaches for prioritising causal genes at T2D risk loci. RECENT FINDINGS: Candidate causal genes can be identified by a number of different strategies that rely on genetic data, genomic annotations, and functional screening of selected genes. To overcome the limitations of each particular method, integration of multiple data sets is proving essential for establishing confidence in the prioritised genes. Previous studies have also highlighted the need to support these efforts through identification of causal variants and disease-relevant tissues. Prioritisation of causal genes at T2D risk loci by integrating complementary lines of evidence promises to accelerate our understanding of disease pathology and promote translation into new therapeutics.

Human genetics as a model for target validation: finding new therapies for diabetes.

Type 2 diabetes is a global epidemic with major effects on healthcare expenditure and quality of life. Currently available treatments are inadequate for the prevention of comorbidities, yet progress towards new therapies remains slow. A major barrier is the insufficiency of traditional preclinical models for predicting drug efficacy and safety. Human genetics offers a complementary model to assess causal mechanisms for target validation. Genetic perturbations are 'experiments of nature' that provide a uniquely relevant window into the long-term effects of modulating specific targets. Here, we show that genetic discoveries over the past decades have accurately predicted (now known) therapeutic mechanisms for type 2 diabetes. These findings highlight the potential for use of human genetic variation for prospective target validation, and establish a framework for future applications. Studies into rare, monogenic forms of diabetes have also provided proof-of-principle for precision medicine, and the applicability of this paradigm to complex disease is discussed. Finally, we highlight some of the limitations that are relevant to the use of genome-wide association studies (GWAS) in the search for new therapies for diabetes. A key outstanding challenge is the translation of GWAS signals into disease biology and we outline possible solutions for tackling this experimental bottleneck.

Systematic Functional Characterization of Candidate Causal Genes for Type 2 Diabetes Risk Variants.

Most genetic association signals for type 2 diabetes risk are located in noncoding regions of the genome, hindering translation into molecular mechanisms. Physiological studies have shown a majority of disease-associated variants to exert their effects through pancreatic islet dysfunction. Systematically characterizing the role of regional transcripts in ß-cell function could identify the underlying disease-causing genes, but large-scale studies in human cellular models have previously been impractical. We developed a robust and scalable strategy based on arrayed gene silencing in the human ß-cell line EndoC-ßH1. In a screen of 300 positional candidates selected from 75 type 2 diabetes regions, each gene was assayed for effects on multiple disease-relevant phenotypes, including insulin secretion and cellular proliferation. We identified a total of 45 genes involved in ß-cell function, pointing to possible causal mechanisms at 37 disease-associated loci. The results showed a strong enrichment for genes implicated in monogenic diabetes. Selected effects were validated in a follow-up study, including several genes (ARL15, ZMIZ1, and THADA) with previously unknown or poorly described roles in ß-cell biology. We have demonstrated the feasibility of systematic functional screening in a human ß-cell model and successfully prioritized plausible disease-causing genes at more than half of the regions investigated.

Loss-of-Function Mutations in the Cell-Cycle Control Gene CDKN2A Impact on Glucose Homeostasis in Humans.

At the CDKN2A/B locus, three independent signals for type 2 diabetes risk are located in a noncoding region near CDKN2A. The disease-associated alleles have been implicated in reduced ß-cell function, but the underlying mechanism remains elusive. In mice, ß-cell-specific loss of Cdkn2a causes hyperplasia, while overexpression leads to diabetes, highlighting CDKN2A as a candidate effector transcript. Rare CDKN2A loss-of-function mutations are a cause of familial melanoma and offer the opportunity to determine the impact of CDKN2A haploinsufficiency on glucose homeostasis in humans. To test the hypothesis that such individuals have improved ß-cell function, we performed oral and intravenous glucose tolerance tests on mutation carriers and matched control subjects. Compared with control subjects, carriers displayed increased insulin secretion, impaired insulin sensitivity, and reduced hepatic insulin clearance. These results are consistent with a model whereby CDKN2A loss affects a range of different tissues, including pancreatic ß-cells and liver. To test for direct effects of CDKN2A-loss on ß-cell function, we performed knockdown in a human ß-cell line, EndoC-bH1. This revealed increased insulin secretion independent of proliferation. Overall, we demonstrated that CDKN2A is an important regulator of glucose homeostasis in humans, thus supporting its candidacy as an effector transcript for type 2 diabetes-associated alleles in the region.

Genetic fine mapping and genomic annotation defines causal mechanisms at type 2 diabetes susceptibility loci.

We performed fine mapping of 39 established type 2 diabetes (T2D) loci in 27,206 cases and 57,574 controls of European ancestry. We identified 49 distinct association signals at these loci, including five mapping in or near KCNQ1. 'Credible sets' of the variants most likely to drive each distinct signal mapped predominantly to noncoding sequence, implying that association with T2D is mediated through gene regulation. Credible set variants were enriched for overlap with FOXA2 chromatin immunoprecipitation binding sites in human islet and liver cells, including at MTNR1B, where fine mapping implicated rs10830963 as driving T2D association. We confirmed that the T2D risk allele for this SNP increases FOXA2-bound enhancer activity in islet- and liver-derived cells. We observed allele-specific differences in NEUROD1 binding in islet-derived cells, consistent with evidence that the T2D risk allele increases islet MTNR1B expression. Our study demonstrates how integration of genetic and genomic information can define molecular mechanisms through which variants underlying association signals exert their effects on disease.

The pancreatic ß cell: recent insights from human genetics.

Diabetes mellitus is a metabolic disease characterised by relative or absolute pancreatic ß cell dysfunction. Genetic variants implicated in disease risk can be identified by studying affected individuals. To understand the mechanisms driving genetic associations, variants must be translated through causative transcripts to biological insights. Studies into the genetic basis of Mendelian forms of diabetes have successfully identified genes involved in both ß cell function and pancreatic development. For type 2 diabetes (T2D), genome-wide association studies (GWASs) are uncovering an ever-increasing number of susceptibility variants that exert their effect through ß cell dysfunction, but translation to mechanistic understanding has in most cases been slow. Improved annotations of the islet genome and advances in whole-genome and -exome sequencing (WHS and WES) have facilitated recent progress.

Phenotypic severity of homozygous GCK mutations causing neonatal or childhood-onset diabetes is primarily mediated through effects on protein stability.

Mutations in glucokinase (GCK) cause a spectrum of glycemic disorders. Heterozygous loss-of-function mutations cause mild fasting hyperglycemia irrespective of mutation severity due to compensation from the unaffected allele. Conversely, homozygous loss-of-function mutations cause permanent neonatal diabetes requiring lifelong insulin treatment. This study aimed to determine the relationship between in vitro mutation severity and clinical phenotype in a large international case series of patients with homozygous GCK mutations. Clinical characteristics for 30 patients with diabetes due to homozygous GCK mutations (19 unique mutations, including 16 missense) were compiled and assigned a clinical severity grade (CSG) based on birth weight and age at diagnosis. The majority (28 of 30) of subjects were diagnosed before 9 months, with the remaining two at 9 and 15 years. These are the first two cases of a homozygous GCK mutation diagnosed outside infancy. Recombinant mutant GCK proteins were analyzed for kinetic and thermostability characteristics and assigned a relative activity index (RAI) or relative stability index (RSI) value. Six of 16 missense mutations exhibited severe kinetic defects (RAI ≤ 0.01). There was no correlation between CSG and RAI (r(2) = 0.05, P = 0.39), indicating that kinetics alone did not explain the phenotype. Eighty percent of the remaining mutations showed reduced thermostability, the exceptions being the two later-onset mutations which exhibited increased thermostability. Comparison of CSG with RSI detected a highly significant correlation (r(2) = 0.74, P = 0.002). We report the largest case series of homozygous GCK mutations to date and demonstrate that they can cause childhood-onset diabetes, with protein instability being the major determinant of mutation severity.