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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. A genome-wide CRISPR knockout screen in the human EndoC-βH1 pancreatic beta cell line identifies 580 regulators of intracellular insulin content. Loss of CALCOCO2 perturbs insulin granule homeostasis in pancreatic beta cells.

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 β-like cells, we establish that the p.Arg138* allele results in reduced SLC30A8 expression due to haploinsufficiency. In human β cells, loss of SLC30A8 leads to increased glucose responsiveness and reduced K ATP 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. The rare loss-of-function allele p.Arg138* in SLC30A8 (encoding ZnT8) mediates protection against type 2 diabetes (T2D) through promoting better insulin secretion and enhanced glucose responsiveness, suggesting ZnT8 as a target for T2D treatment.

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.

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 β-cell model and primary islets from cadaveric donors. Thus, our results demonstrate a role for PAM in β-cell function, and establish molecular mechanisms for T2D risk alleles at this locus. Coding variants in peptidylglycine α-amidating monooxygenase ( PAM ) associated with type 2 diabetes risk negatively impact overall PAM activity via defects in expression and catalytic function, resulting in reduced insulin content and altered dynamics of insulin secretion.

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. Summary 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.

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The molecular mechanisms underpinning susceptibility loci for type 2 diabetes (T2D) remain poorly understood. Coding variants in peptidylglycine [alpha]-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 [beta]-cell model and primary islets from cadaveric donors. Thus, our results demonstrate a role for PAM in [beta]-cell function, and establish molecular mechanisms for T2D risk alleles at this locus.

Genome-wide association studies (GWAS) have identified more than 150 loci associated with type 2 diabetes (T2D) risk. Most association signals are located in noncoding regions of the genome, and uncertainty remains over the causal gene in the vast majority of cases. Motivated by the overall aim of accelerating the translation of genetic discoveries into molecular mechanisms for disease pathogenesis, the work presented in this thesis sought to identify candidate causal genes, and to study their roles in human beta cell function. The causal mechanism for non-coding variants at the CDKN2A/B locus has remained elusive, though animal models have implicated Cdkn2a in beta cell function. To determine the effect of CDKN2A haploinsufficiency on glucose homeostasis in humans, I analysed data from oral and intravenous glucose tolerance tests in individuals carrying rare CDKN2A loss-offunction mutations. Compared with controls, carriers displayed increased insulin secretion, impaired insulin sensitivity, and reduced hepatic insulin clearance. Follow-up studies in the human beta cell line, EndoC-Î2H1, demonstrated cell-cycle independent effects of CDKN2A on insulin secretion. At the PAM/PPIP5K2 locus, two non-synonymous variants in PAM are associated with both T2D risk and insulinogenic index. To elucidate a causal mechanism, I investigated possible local and extra-pancreatic roles of PAM in beta cell function. I demonstrated that PAM deficiency results in reduced insulin content and altered dynamics of insulin secretion in EndoC-Î2H1 and primary beta cells. I also identified the amidated granular packaging protein Chromogranin A as an endogenous PAM substrate, establishing a direct link to granulogenesis in human beta cells. Lastly, to enable systematic, large-scale prioritization of causal genes for T2D risk, I performed a high-throughput RNAi screen for beta cell dysfunction. Among 300 genes selected from 75 risk loci, I identified significant hits at half of these regions. The hits were highly enriched for known monogenic diabetes genes, but also revealed the poorly characterised transcription factor ZMIZ1 to be one of the strongest regulators of insulin secretion. Silencing of ZMIZ1 in primary human islets subsequently revealed a core beta cell network to be negatively affected, highlighting a plausible causal mechanism. Overall, this thesis has addressed a significant outstanding challenge in the translation of genetic signals into disease mechanisms. Using a range of different strategies, including genetic data, candidate gene biology, and functional genetic screening, the work has successfully identified several candidate causal mechanisms for beta cell dysfunction at T2D risk loci. These results provide a deeper understanding of the fundamental mechanisms underpinning disease predisposition, and thus highlight potential new therapeutic applications.

Molecular mechanisms underpinning the genetic risk for type 2 diabetes (T2D) remain poorly understood, hindering translation into new therapies. Recently, genome-wide studies identified two coding variants in Peptidylglycine Alpha-amidating Monooxygenase (PAM) associated with T2D risk and measures of beta cell dysfunction. Here, we demonstrate that both risk alleles impact negatively on overall PAM activity, but via distinct effects on expression and catalytic function. In a human beta cell model, PAM silencing caused decreased insulin content and altered dynamics of granule exocytosis. Analysis of primary human beta cells from cadaveric donors confirmed an effect on exocytosis in carriers of the p.D563G T2D-risk allele. Finally, we show that the granular packaging protein Chromogranin A is a PAM substrate and a strong candidate for mediating downstream effects on insulin secretion. Taken together, our results establish a role for PAM in beta cell function, and uncover a novel mechanism for T2D-associated PAM alleles.

A rare loss-of-function variant p.Arg138* in SLC30A8 encoding the zinc transporter 8 (ZnT8) 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, especially compared with individuals matched for the genotype of a common T2D risk variant in SLC30A8, p.Arg325. In genome-edited human IPS-derived -like cells, we establish that the p.Arg138* variant results in reduced SLC30A8 expression due to haploinsufficiency. In human -cells loss of SLC30A8 leads to increased glucose responsiveness and reduced KATP channel function, which was also seen in isolated islets from carriers of the T2D-protective allele p.Trp325. These data position ZnT8 as an appealing target for treatment aiming at maintaining insulin secretion capacity in T2D.