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.

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.

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.

Employees with mental disorders seeking support from the workers compensation system - experiences from Denmark.

BACKGROUND: In Europe it is commonly accepted that psychosocial hazards may influence the mental health of employees. However, mental disorders such as depression are generally not acknowledged as an occupational disease covered by the workers compensation system. Studies indicate that workers compensation claim processes may affect employee's health negatively due to a demanding case process. If filing a workers' compensation claim can harm the employees' health, it is highly relevant to pay attention to employees with mental health claims, as they are most likely vulnerable and face a very low chance of compensation. OBJECTIVE: This study investigates how employees with work-related mental disorders experience the process of seeking workers compensation from the Danish Workers' Compensation System. METHOD: Interview (N = 13) and questionnaire (N = 436) data from claimants were analysed. RESULTS: Analysis showed that even though many employees wished for the claim to influence the conditions at the workplace, there seemed to be a lack of preventive health and safety initiatives in the workplaces. Central stakeholders such as health and safety representatives were often not involved. Management involvement was often experienced negatively, and the Danish Working Environment Authority rarely conducted workplace inspections. Employees experienced inadequate information about the workers' compensation process and experienced a lack of coordination between stakeholders. CONCLUSION: A more supportive and coordinated approach in the Workers' Compensation System is recommendable. The processes in the system could be evaluated using the Social Insurance Literacy concept, to ensure sufficient support of the claimants and reduce potential harmful aspects of the process.

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.

Type 2 Diabetes risk alleles in Peptidyl-glycine Alpha-amidating Monooxygenase influence GLP-1 levels and response to GLP-1 Receptor Agonists.

Patients with type 2 diabetes vary in their response to currently available therapeutic agents (including GLP-1 receptor agonists) leading to suboptimal glycemic control and increased risk of complications. We show that human carriers of hypomorphic T2D-risk alleles in the gene encoding peptidyl-glycine alpha-amidating monooxygenase (PAM), as well as Pam-knockout mice, display increased resistance to GLP-1 in vivo. Pam inactivation in mice leads to reduced gastric GLP-1R expression and faster gastric emptying: this persists during GLP-1R agonist treatment and is rescued when GLP-1R activity is antagonized, indicating resistance to GLP-1's gastric slowing properties. Meta-analysis of human data from studies examining GLP-1R agonist response (including RCTs) reveals a relative loss of 44% and 20% of glucose lowering (measured by glycated hemoglobin) in individuals with hypomorphic PAM alleles p.S539W and p.D536G treated with GLP-1R agonist. Genetic variation in PAM has effects on incretin signaling that alters response to medication used commonly for treatment of T2D.

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.

Oxygen minimum zone cryptic sulfur cycling sustained by offshore transport of key sulfur oxidizing bacteria.

Members of the gammaproteobacterial clade SUP05 couple water column sulfide oxidation to nitrate reduction in sulfidic oxygen minimum zones (OMZs). Their abundance in offshore OMZ waters devoid of detectable sulfide has led to the suggestion that local sulfate reduction fuels SUP05-mediated sulfide oxidation in a so-called "cryptic sulfur cycle". We examined the distribution and metabolic capacity of SUP05 in Peru Upwelling waters, using a combination of oceanographic, molecular, biogeochemical and single-cell techniques. A single SUP05 species, U Thioglobus perditus, was found to be abundant and active in both sulfidic shelf and sulfide-free offshore OMZ waters. Our combined data indicated that mesoscale eddy-driven transport led to the dispersal of U T. perditus and elemental sulfur from the sulfidic shelf waters into the offshore OMZ region. This offshore transport of shelf waters provides an alternative explanation for the abundance and activity of sulfide-oxidizing denitrifying bacteria in sulfide-poor offshore OMZ waters.

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.

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.

The fungal community changes over time in developing wheat heads.

Under normal conditions, wheat is colonized by a multitude of fungi that can have beneficial or adverse effects on plant growth and yield. To study the effect of spraying wheat heads with fungicides on the fungal community from emergence to harvest we applied an amplicon sequencing approach on single wheat heads. The climatic data showed that the spring of 2014 was very dry and without precipitation in the two weeks around flowering. An initial quantitative PCR showed that the total amount of fungal DNA increased during the entire period, without significant difference between sprayed and control wheat heads. Amplicon sequencing of the internal transcribed spacer 2 (ITS2) region showed that operational taxonomic units (OTUs) identified as Sporobolomyces roseus dominated in the first weeks, whereas Alternaria infectoria OTUs dominated in the last weeks before harvest. The only observed significant difference was that the control wheat heads contained more of the powdery mildew causing Blumeria graminis f. sp. tritici OTUs compared with the sprayed wheat heads. The dry conditions around flowering most likely also had an effect on Fusarium head blight infection as Fusarium OTUs were only sporadically encountered. Analyses of secondary metabolites produced by Fusarium and Alternaria in the wheat heads confirmed the observations from the amplicon sequencing. Enniatin B was the most frequent contaminant present in four sprayed (49-538 ng/g) and three control (56-355 ng/g) wheat heads. The A. infectoria secondary metabolites infectopyrone and 4Z-infectopyrone were however consistently observed in all samples collected the last five weeks before harvest.

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.

Intra-articular glucocorticoid, bupivacaine and morphine reduces pain, inflammatory response and convalescence after arthroscopic meniscectomy.

Convalescence after arthroscopic meniscectomy is dependent on pain and the inflammatory response. The aim of the study was therefore to investigate the effect of intra-articular bupivacaine + morphine + methylprednisolone versus bupivacaine + morphine or saline on postmeniscectomy pain, mobilisation and convalescence. In a double-blind randomized study 60 patients undergoing arthroscopic meniscectomy were allocated to intra-articular saline, intra-articular bupivacaine 150 mg + morphine 4 mg or the same dose of bupivacaine + morphine + intra-articular methylprednisolone 40 mg. All patients were instructed to resume normal activities immediately after operation. Pain during movement and walking, leg muscle force and joint effusion, use of crutches and duration of sick leave were assessed. Combined bupivacaine and morphine significantly reduced pain, time of immobilisation and duration of convalescence. Addition of methylprednisolone further reduced pain, use of additional analgesics, joint swelling and convalescence, improved muscle function and prevented the inflammatory response (acute phase protein) (P < 0.05). A multimodal analgesic and anti-inflammatory treatment may enhance post-arthroscopic convalescence, which depends on the trauma induced inflammatory response and pain.

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.

Combined intra-articular glucocorticoid, bupivacaine and morphine reduces pain and convalescence after diagnostic knee arthroscopy.

We studied the effect of intra-articullar saline vs. bupivacaine + morphine or bupivacaine morphine + methylprednisolone after diagnostic knee arthroscopy. In a double-blind randomized study, 60 patients undergoing diagnostic knee arthroscopy without a therapeutic procedure were allocated to groups receiving intra-articular saline, intra-articular bupivacaine 150 mg + morphine 4 mg or the same dose of bupivacaine + morphine + intra-articular methylprednisolone 40 mg at the end of arthroscopy during general anesthesia. All patients were instructed to resume normal activities immediately after the procedure. Pain during movement and walking, leg muscle force and joint effusion, use of crutches and duration of sick leave were assessed. A combination of bupivacaine and morphine reduced pain, duration of immobilization and of convalescence. The addition of methylprednisolone further reduced pain, use of more analgesics, joint swelling and convalescence.