The Genetic Basis of Metabolic Disease.

Recent developments in genetics and genomics are providing a detailed and systematic characterization of the genetic underpinnings of common metabolic diseases and traits, highlighting the inherent complexity within systems for homeostatic control and the many ways in which that control can fail. The genetic architecture underlying these common metabolic phenotypes is complex, with each trait influenced by hundreds of loci spanning a range of allele frequencies and effect sizes. Here, we review the growing appreciation of this complexity and how this has fostered the implementation of genome-scale approaches that deliver robust mechanistic inference and unveil new strategies for translational exploitation.

Human Semaphorin 3 Variants Link Melanocortin Circuit Development and Energy Balance.

Hypothalamic melanocortin neurons play a pivotal role in weight regulation. Here, we examined the contribution of Semaphorin 3 (SEMA3) signaling to the development of these circuits. In genetic studies, we found 40 rare variants in SEMA3A-G and their receptors (PLXNA1-4; NRP1-2) in 573 severely obese individuals; variants disrupted secretion and/or signaling through multiple molecular mechanisms. Rare variants in this set of genes were significantly enriched in 982 severely obese cases compared to 4,449 controls. In a zebrafish mutagenesis screen, deletion of 7 genes in this pathway led to increased somatic growth and/or adiposity demonstrating that disruption of Semaphorin 3 signaling perturbs energy homeostasis. In mice, deletion of the Neuropilin-2 receptor in Pro-opiomelanocortin neurons disrupted their projections from the arcuate to the paraventricular nucleus, reduced energy expenditure, and caused weight gain. Cumulatively, these studies demonstrate that SEMA3-mediated signaling drives the development of hypothalamic melanocortin circuits involved in energy homeostasis.

KSR2 mutations are associated with obesity, insulin resistance, and impaired cellular fuel oxidation.

Kinase suppressor of Ras 2 (KSR2) is an intracellular scaffolding protein involved in multiple signaling pathways. Targeted deletion of Ksr2 leads to obesity in mice, suggesting a role in energy homeostasis. We explored the role of KSR2 in humans by sequencing 2,101 individuals with severe early-onset obesity and 1,536 controls. We identified multiple rare variants in KSR2 that disrupt signaling through the Raf-MEKERK pathway and impair cellular fatty acid oxidation and glucose oxidation in transfected cells; effects that can be ameliorated by the commonly prescribed antidiabetic drug, metformin. Mutation carriers exhibit hyperphagia in childhood, low heart rate, reduced basal metabolic rate and severe insulin resistance. These data establish KSR2 as an important regulator of energy intake, energy expenditure, and substrate utilization in humans. Modulation of KSR2-mediated effects may represent a novel therapeutic strategy for obesity and type 2 diabetes.

Author Correction: Genomics of disease risk in globally diverse populations.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genomics of disease risk in globally diverse populations.

Risk of disease is multifactorial and can be shaped by socio-economic, demographic, cultural, environmental and genetic factors. Our understanding of the genetic determinants of disease risk has greatly advanced with the advent of genome-wide association studies (GWAS), which detect associations between genetic variants and complex traits or diseases by comparing populations of cases and controls. However, much of this discovery has occurred through GWAS of individuals of European ancestry, with limited representation of other populations, including from Africa, The Americas, Asia and Oceania. Population demography, genetic drift and adaptation to environments over thousands of years have led globally to the diversification of populations. This global genomic diversity can provide new opportunities for discovery and translation into therapies, as well as a better understanding of population disease risk. Large-scale multi-ethnic and representative biobanks and population health resources provide unprecedented opportunities to understand the genetic determinants of disease on a global scale.

Obesity-Associated GNAS Mutations and the Melanocortin Pathway.

BACKGROUND: GNAS encodes the Gαs (stimulatory G-protein alpha subunit) protein, which mediates G protein-coupled receptor (GPCR) signaling. GNAS mutations cause developmental delay, short stature, and skeletal abnormalities in a syndrome called Albright's hereditary osteodystrophy. Because of imprinting, mutations on the maternal allele also cause obesity and hormone resistance (pseudohypoparathyroidism). METHODS: We performed exome sequencing and targeted resequencing in 2548 children who presented with severe obesity, and we unexpectedly identified 22 GNAS mutation carriers. We investigated whether the effect of GNAS mutations on melanocortin 4 receptor (MC4R) signaling explains the obesity and whether the variable clinical spectrum in patients might be explained by the results of molecular assays. RESULTS: Almost all GNAS mutations impaired MC4R signaling. A total of 6 of 11 patients who were 12 to 18 years of age had reduced growth. In these patients, mutations disrupted growth hormone-releasing hormone receptor signaling, but growth was unaffected in carriers of mutations that did not affect this signaling pathway (mean standard-deviation score for height, -0.90 vs. 0.75, respectively; P = 0.02). Only 1 of 10 patients who reached final height before or during the study had short stature. GNAS mutations that impaired thyrotropin receptor signaling were associated with developmental delay and with higher thyrotropin levels (mean [±SD], 8.4±4.7 mIU per liter) than those in 340 severely obese children who did not have GNAS mutations (3.9±2.6 mIU per liter; P = 0.004). CONCLUSIONS: Because pathogenic mutations may manifest with obesity alone, screening of children with severe obesity for GNAS deficiency may allow early diagnosis, improving clinical outcomes, and melanocortin agonists may aid in weight loss. GNAS mutations that are identified by means of unbiased genetic testing differentially affect GPCR signaling pathways that contribute to clinical heterogeneity. Monogenic diseases are clinically more variable than their classic descriptions suggest. (Funded by Wellcome and others.).

Predicting novel candidate human obesity genes and their site of action by systematic functional screening in Drosophila.

The discovery of human obesity-associated genes can reveal new mechanisms to target for weight loss therapy. Genetic studies of obese individuals and the analysis of rare genetic variants can identify novel obesity-associated genes. However, establishing a functional relationship between these candidate genes and adiposity remains a significant challenge. We uncovered a large number of rare homozygous gene variants by exome sequencing of severely obese children, including those from consanguineous families. By assessing the function of these genes in vivo in Drosophila, we identified 4 genes, not previously linked to human obesity, that regulate adiposity (itpr, dachsous, calpA, and sdk). Dachsous is a transmembrane protein upstream of the Hippo signalling pathway. We found that 3 further members of the Hippo pathway, fat, four-jointed, and hippo, also regulate adiposity and that they act in neurons, rather than in adipose tissue (fat body). Screening Hippo pathway genes in larger human cohorts revealed rare variants in TAOK2 associated with human obesity. Knockdown of Drosophila tao increased adiposity in vivo demonstrating the strength of our approach in predicting novel human obesity genes and signalling pathways and their site of action.

The impact of population-level HbA1c screening on reducing diabetes diagnostic delay in middle-aged adults: a UK Biobank analysis.

AIMS/HYPOTHESIS: Screening programmes can detect cases of undiagnosed diabetes earlier than symptomatic or incidental diagnosis. However, the improvement in time to diagnosis achieved by screening programmes compared with routine clinical care is unclear. We aimed to use the UK Biobank population-based study to provide the first population-based estimate of the reduction in time to diabetes diagnosis that could be achieved by HbA1c-based screening in middle-aged adults. METHODS: We studied UK Biobank participants aged 40-70 years with HbA1c measured at enrolment (but not fed back to participants/clinicians) and linked primary and secondary healthcare data (n=179,923) and identified those with a pre-existing diabetes diagnosis (n=13,077, 7.3%). Among the remaining participants (n=166,846) without a diabetes diagnosis, we used an elevated enrolment HbA1c level (≥48 mmol/mol [≥6.5%]) to identify those with undiagnosed diabetes. For this group, we used Kaplan-Meier analysis to assess the time between enrolment HbA1c measurement and subsequent clinical diabetes diagnosis up to 10 years, and Cox regression to identify clinical factors associated with delayed diabetes diagnosis. RESULTS: In total, 1.0% (1703/166,846) of participants without a diabetes diagnosis had undiagnosed diabetes based on calibrated HbA1c levels at UK Biobank enrolment, with a median HbA1c level of 51.3 mmol/mol (IQR 49.1-57.2) (6.8% [6.6-7.4]). These participants represented an additional 13.0% of diabetes cases in the study population relative to the 13,077 participants with a diabetes diagnosis. The median time to clinical diagnosis for those with undiagnosed diabetes was 2.2 years, with a median HbA1c at clinical diagnosis of 58.2 mmol/mol (IQR 51.0-80.0) (7.5% [6.8-9.5]). Female participants with lower HbA1c and BMI measurements at enrolment experienced the longest delay to clinical diagnosis. CONCLUSIONS/INTERPRETATION: Our population-based study shows that HbA1c screening in adults aged 40-70 years can reduce the time to diabetes diagnosis by a median of 2.2 years compared with routine clinical care. The findings support the use of HbA1c screening to reduce the time for which individuals are living with undiagnosed diabetes.

The influence of rare variants in circulating metabolic biomarkers.

Circulating metabolite levels are biomarkers for cardiovascular disease (CVD). Here we studied, association of rare variants and 226 serum lipoproteins, lipids and amino acids in 7,142 (discovery plus follow-up) healthy participants. We leveraged the information from multiple metabolite measurements on the same participants to improve discovery in rare variant association analyses for gene-based and gene-set tests by incorporating correlated metabolites as covariates in the validation stage. Gene-based analysis corrected for the effective number of tests performed, confirmed established associations at APOB, APOC3, PAH, HAL and PCSK (p<1.32x10-7) and identified novel gene-trait associations at a lower stringency threshold with ACSL1, MYCN, FBXO36 and B4GALNT3 (p<2.5x10-6). Regulation of the pyruvate dehydrogenase (PDH) complex was associated for the first time, in gene-set analyses also corrected for effective number of tests, with IDL and LDL parameters, as well as circulating cholesterol (pMETASKAT<2.41x10-6). In conclusion, using an approach that leverages metabolite measurements obtained in the same participants, we identified novel loci and pathways involved in the regulation of these important metabolic biomarkers. As large-scale biobanks continue to amass sequencing and phenotypic information, analytical approaches such as ours will be useful to fully exploit the copious amounts of biological data generated in these efforts.

Novel loci for childhood body mass index and shared heritability with adult cardiometabolic traits.

The genetic background of childhood body mass index (BMI), and the extent to which the well-known associations of childhood BMI with adult diseases are explained by shared genetic factors, are largely unknown. We performed a genome-wide association study meta-analysis of BMI in 61,111 children aged between 2 and 10 years. Twenty-five independent loci reached genome-wide significance in the combined discovery and replication analyses. Two of these, located near NEDD4L and SLC45A3, have not previously been reported in relation to either childhood or adult BMI. Positive genetic correlations of childhood BMI with birth weight and adult BMI, waist-to-hip ratio, diastolic blood pressure and type 2 diabetes were detected (Rg ranging from 0.11 to 0.76, P-values <0.002). A negative genetic correlation of childhood BMI with age at menarche was observed. Our results suggest that the biological processes underlying childhood BMI largely, but not completely, overlap with those underlying adult BMI. The well-known observational associations of BMI in childhood with cardio-metabolic diseases in adulthood may reflect partial genetic overlap, but in light of previous evidence, it is also likely that they are explained through phenotypic continuity of BMI from childhood into adulthood.

A Specific CNOT1 Mutation Results in a Novel Syndrome of Pancreatic Agenesis and Holoprosencephaly through Impaired Pancreatic and Neurological Development.

We report a recurrent CNOT1 de novo missense mutation, GenBank: NM_016284.4; c.1603C>T (p.Arg535Cys), resulting in a syndrome of pancreatic agenesis and abnormal forebrain development in three individuals and a similar phenotype in mice. CNOT1 is a transcriptional repressor that has been suggested as being critical for maintaining embryonic stem cells in a pluripotent state. These findings suggest that CNOT1 plays a critical role in pancreatic and neurological development and describe a novel genetic syndrome of pancreatic agenesis and holoprosencephaly.

Genetic architecture of human thinness compared to severe obesity.

The variation in weight within a shared environment is largely attributable to genetic factors. Whilst many genes/loci confer susceptibility to obesity, little is known about the genetic architecture of healthy thinness. Here, we characterise the heritability of thinness which we found was comparable to that of severe obesity (h2 = 28.07 vs 32.33% respectively), although with incomplete genetic overlap (r = -0.49, 95% CI [-0.17, -0.82], p = 0.003). In a genome-wide association analysis of thinness (n = 1,471) vs severe obesity (n = 1,456), we identified 10 loci previously associated with obesity, and demonstrate enrichment for established BMI-associated loci (pbinomial = 3.05x10-5). Simulation analyses showed that different association results between the extremes were likely in agreement with additive effects across the BMI distribution, suggesting different effects on thinness and obesity could be due to their different degrees of extremeness. In further analyses, we detected a novel obesity and BMI-associated locus at PKHD1 (rs2784243, obese vs. thin p = 5.99x10-6, obese vs. controls p = 2.13x10-6 pBMI = 2.3x10-13), associations at loci recently discovered with much larger sample sizes (e.g. FAM150B and PRDM6-CEP120), and novel variants driving associations at previously established signals (e.g. rs205262 at the SNRPC/C6orf106 locus and rs112446794 at the PRDM6-CEP120 locus). Our ability to replicate loci found with much larger sample sizes demonstrates the value of clinical extremes and suggest that characterisation of the genetics of thinness may provide a more nuanced understanding of the genetic architecture of body weight regulation and may inform the identification of potential anti-obesity targets.

Controlled Release of Epigenetically-Enhanced Extracellular Vesicles from a GelMA/Nanoclay Composite Hydrogel to Promote Bone Repair.

Extracellular vesicles (EVs) have garnered growing attention as promising acellular tools for bone repair. Although EVs' potential for bone regeneration has been shown, issues associated with their therapeutic potency and short half-life in vivo hinders their clinical utility. Epigenetic reprogramming with the histone deacetylase inhibitor Trichostatin A (TSA) has been reported to promote the osteoinductive potency of osteoblast-derived EVs. Gelatin methacryloyl (GelMA) hydrogels functionalised with the synthetic nanoclay laponite (LAP) have been shown to effectively bind, stabilise, and improve the retention of bioactive factors. This study investigated the potential of utilising a GelMA-LAP hydrogel to improve local retention and control delivery of epigenetically enhanced osteoblast-derived EVs as a novel bone repair strategy. LAP was found to elicit a dose-dependent increase in GelMA compressive modulus and shear-thinning properties. Incorporation of the nanoclay was also found to enhance shape fidelity when 3D printed compared to LAP-free gels. Interestingly, GelMA hydrogels containing LAP displayed increased mineralisation capacity (1.41-fold) (p ≤ 0.01) over 14 days. EV release kinetics from these nanocomposite systems were also strongly influenced by LAP concentration with significantly more vesicles being released from GelMA constructs as detected by a CD63 ELISA (p ≤ 0.001). EVs derived from TSA-treated osteoblasts (TSA-EVs) enhanced proliferation (1.09-fold), migration (1.83-fold), histone acetylation (1.32-fold) and mineralisation (1.87-fold) of human bone marrow stromal cells (hBMSCs) when released from the GelMA-LAP hydrogel compared to the untreated EV gels (p ≤ 0.01). Importantly, the TSA-EV functionalised GelMA-LAP hydrogel significantly promoted encapsulated hBMSCs extracellular matrix collagen production (≥1.3-fold) and mineralisation (≥1.78-fold) in a dose-dependent manner compared to untreated EV constructs (p ≤ 0.001). Taken together, these findings demonstrate the potential of combining epigenetically enhanced osteoblast-derived EVs with a nanocomposite photocurable hydrogel to promote the therapeutic efficacy of acellular vesicle approaches for bone regeneration.

The importance of increasing population diversity in genetic studies of type 2 diabetes and related glycaemic traits.

Type 2 diabetes has a global prevalence, with epidemiological data suggesting that some populations have a higher risk of developing this disease. However, to date, most genetic studies of type 2 diabetes and related glycaemic traits have been performed in individuals of European ancestry. The same is true for most other complex diseases, largely due to use of 'convenience samples'. Rapid genotyping of large population cohorts and case-control studies from existing collections was performed when the genome-wide association study (GWAS) 'revolution' began, back in 2005. Although global representation has increased in the intervening 15 years, further expansion and inclusion of diverse populations in genetic and genomic studies is still needed. In this review, I discuss the progress made in incorporating multi-ancestry participants in genetic analyses of type 2 diabetes and related glycaemic traits, and associated opportunities and challenges. I also discuss how increased representation of global diversity in genetic and genomic studies is required to fulfil the promise of precision medicine for all.

DNA Polymerase Epsilon Deficiency Causes IMAGe Syndrome with Variable Immunodeficiency.

During genome replication, polymerase epsilon (Pol ε) acts as the major leading-strand DNA polymerase. Here we report the identification of biallelic mutations in POLE, encoding the Pol ε catalytic subunit POLE1, in 15 individuals from 12 families. Phenotypically, these individuals had clinical features closely resembling IMAGe syndrome (intrauterine growth restriction [IUGR], metaphyseal dysplasia, adrenal hypoplasia congenita, and genitourinary anomalies in males), a disorder previously associated with gain-of-function mutations in CDKN1C. POLE1-deficient individuals also exhibited distinctive facial features and variable immune dysfunction with evidence of lymphocyte deficiency. All subjects shared the same intronic variant (c.1686+32C>G) as part of a common haplotype, in combination with different loss-of-function variants in trans. The intronic variant alters splicing, and together the biallelic mutations lead to cellular deficiency of Pol ε and delayed S-phase progression. In summary, we establish POLE as a second gene in which mutations cause IMAGe syndrome. These findings add to a growing list of disorders due to mutations in DNA replication genes that manifest growth restriction alongside adrenal dysfunction and/or immunodeficiency, consolidating these as replisome phenotypes and highlighting a need for future studies to understand the tissue-specific development roles of the encoded proteins.

The UK10K project identifies rare variants in health and disease.

The contribution of rare and low-frequency variants to human traits is largely unexplored. Here we describe insights from sequencing whole genomes (low read depth, 7×) or exomes (high read depth, 80×) of nearly 10,000 individuals from population-based and disease collections. In extensively phenotyped cohorts we characterize over 24 million novel sequence variants, generate a highly accurate imputation reference panel and identify novel alleles associated with levels of triglycerides (APOB), adiponectin (ADIPOQ) and low-density lipoprotein cholesterol (LDLR and RGAG1) from single-marker and rare variant aggregation tests. We describe population structure and functional annotation of rare and low-frequency variants, use the data to estimate the benefits of sequencing for association studies, and summarize lessons from disease-specific collections. Finally, we make available an extensive resource, including individual-level genetic and phenotypic data and web-based tools to facilitate the exploration of association results.

ProxECAT: Proxy External Controls Association Test. A new case-control gene region association test using allele frequencies from public controls.

A primary goal of the recent investment in sequencing is to detect novel genetic associations in health and disease improving the development of treatments and playing a critical role in precision medicine. While this investment has resulted in an enormous total number of sequenced genomes, individual studies of complex traits and diseases are often smaller and underpowered to detect rare variant genetic associations. Existing genetic resources such as the Exome Aggregation Consortium (>60,000 exomes) and the Genome Aggregation Database (~140,000 sequenced samples) have the potential to be used as controls in these studies. Fully utilizing these and other existing sequencing resources may increase power and could be especially useful in studies where resources to sequence additional samples are limited. However, to date, these large, publicly available genetic resources remain underutilized, or even misused, in large part due to the lack of statistical methods that can appropriately use this summary level data. Here, we present a new method to incorporate external controls in case-control analysis called ProxECAT (Proxy External Controls Association Test). ProxECAT estimates enrichment of rare variants within a gene region using internally sequenced cases and external controls. We evaluated ProxECAT in simulations and empirical analyses of obesity cases using both low-depth of coverage (7x) whole-genome sequenced controls and ExAC as controls. We find that ProxECAT maintains the expected type I error rate with increased power as the number of external controls increases. With an accompanying R package, ProxECAT enables the use of publicly available allele frequencies as external controls in case-control analysis.

Genome-wide association study of type 2 diabetes in Africa.

AIMS/HYPOTHESIS: Genome-wide association studies (GWAS) for type 2 diabetes have uncovered >400 risk loci, primarily in populations of European and Asian ancestry. Here, we aimed to discover additional type 2 diabetes risk loci (including African-specific variants) and fine-map association signals by performing genetic analysis in African populations. METHODS: We conducted two type 2 diabetes genome-wide association studies in 4347 Africans from South Africa, Nigeria, Ghana and Kenya and meta-analysed both studies together. Likely causal variants were identified using fine-mapping approaches. RESULTS: The most significantly associated variants mapped to the widely replicated type 2 diabetes risk locus near TCF7L2 (p = 5.3 × 10-13). Fine-mapping of the TCF7L2 locus suggested one type 2 diabetes association signal shared between Europeans and Africans (indexed by rs7903146) and a distinct African-specific signal (indexed by rs17746147). We also detected one novel signal, rs73284431, near AGMO (p = 5.2 × 10-9, minor allele frequency [MAF] = 0.095; monomorphic in most non-African populations), distinct from previously reported signals in the region. In analyses focused on 100 published type 2 diabetes risk loci, we identified 21 with shared causal variants in African and non-African populations. CONCLUSIONS/INTERPRETATION: These results demonstrate the value of performing GWAS in Africans, provide a resource to larger consortia for further discovery and fine-mapping and indicate that additional large-scale efforts in Africa are warranted to gain further insight in to the genetic architecture of type 2 diabetes.

Genetic aetiology of glycaemic traits: approaches and insights.

Glycaemic traits such as fasting and post-challenge glucose and insulin measures, as well as glycated haemoglobin (HbA1c), are used to diagnose and monitor diabetes. These traits are risk factors for cardiovascular disease even below the diabetic threshold, and their study can additionally yield insights into the pathophysiology of type 2 diabetes. To date, a diverse set of genetic approaches have led to the discovery of over 97 loci influencing glycaemic traits. In this review, we will focus on recent advances in the genetic aetiology of glycaemic traits, and the resulting biological insights. We will provide a brief overview of results ranging from common, to low- and rare-frequency variant-trait association studies, studies leveraging the diversity across populations, and studies harnessing the power of genetic and genomic approaches to gain insights into the biological underpinnings of these traits.