A functional genomic framework to elucidate novel causal non-alcoholic fatty liver disease genes.

Background & Aims: Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver pathology in western countries, with serious public health consequences. Efforts to identify causal genes for NAFLD have been hampered by the relative paucity of human data from gold-standard magnetic resonance quantification of hepatic fat. To overcome insufficient sample size, genome-wide association studies using NAFLD surrogate phenotypes have been used, but only a small number of loci have been identified to date. In this study, we combined GWAS of NAFLD composite surrogate phenotypes with genetic colocalization studies followed by functional in vitro screens to identify bona fide causal genes for NAFLD. Approach & Results: We used the UK Biobank to explore the associations of our novel NAFLD score, and genetic colocalization to prioritize putative causal genes for in vitro validation. We created a functional genomic framework to study NAFLD genes in vitro using CRISPRi. Our data identify VKORC1, TNKS, LYPLAL1 and GPAM as regulators of lipid accumulation in hepatocytes and suggest the involvement of VKORC1 in the lipid storage related to the development of NAFLD. Conclusions: Complementary genetic and genomic approaches are useful for the identification of NAFLD genes. Our data supports VKORC1 as a bona fide NAFLD gene. We have established a functional genomic framework to study at scale putative novel NAFLD genes from human genetic association studies.

Distilling causality between physical activity traits and obesity via Mendelian randomization.

BACKGROUND: Whether obesity is a cause or consequence of low physical activity levels and more sedentary time has not yet been fully elucidated. Better instrumental variables and a more thorough consideration of potential confounding variables that may influence the causal inference between physical activity and obesity are needed. METHODS: Leveraging results from our recent genome-wide association study for leisure time moderate-to-vigorous intensity (MV) physical activity and screen time, we here disentangle the causal relationships between physical activity, sedentary behavior, education-defined by years of schooling-and body mass index (BMI), using multiple univariable and multivariable Mendelian Randomization (MR) approaches. RESULTS: Univariable MR analyses suggest bidirectional causal effects of physical activity and sedentary behavior with BMI. However, multivariable MR analyses that take years of schooling into account suggest that more MV physical activity causes a lower BMI, and a higher BMI causes more screen time, but not vice versa. In addition, more years of schooling causes higher levels of MV physical activity, less screen time, and lower BMI. CONCLUSIONS: In conclusion, our results highlight the beneficial effect of education on improved health and suggest that a more physically active lifestyle leads to lower BMI, while sedentary behavior is a consequence of higher BMI.

It remains unclear exactly how physical activity, sedentary behavior (usually time spent sitting or lying, often in front of a screen), and obesity influence each other, and what role education plays in this relationship. Here, we use genetic information to study this relationship. We show that if you're more physically active, you're likely to be thinner. If your weight is higher, you tend to spend more time in front of the TV or computer. Additionally, getting more years of education leads to more physical activity, less screen time, and a lower weight later in life. The take-home messages are that being more physically active can prevent obesity; watching more TV is a result but not the cause of obesity; and education stimulates a healthier lifestyle later in life. These findings may help to guide public health messaging around healthy lifestyles.

GWAS of random glucose in 476,326 individuals provide insights into diabetes pathophysiology, complications and treatment stratification.

Conventional measurements of fasting and postprandial blood glucose levels investigated in genome-wide association studies (GWAS) cannot capture the effects of DNA variability on 'around the clock' glucoregulatory processes. Here we show that GWAS meta-analysis of glucose measurements under nonstandardized conditions (random glucose (RG)) in 476,326 individuals of diverse ancestries and without diabetes enables locus discovery and innovative pathophysiological observations. We discovered 120 RG loci represented by 150 distinct signals, including 13 with sex-dimorphic effects, two cross-ancestry and seven rare frequency signals. Of these, 44 loci are new for glycemic traits. Regulatory, glycosylation and metagenomic annotations highlight ileum and colon tissues, indicating an underappreciated role of the gastrointestinal tract in controlling blood glucose. Functional follow-up and molecular dynamics simulations of lower frequency coding variants in glucagon-like peptide-1 receptor (GLP1R), a type 2 diabetes treatment target, reveal that optimal selection of GLP-1R agonist therapy will benefit from tailored genetic stratification. We also provide evidence from Mendelian randomization that lung function is modulated by blood glucose and that pulmonary dysfunction is a diabetes complication. Our investigation yields new insights into the biology of glucose regulation, diabetes complications and pathways for treatment stratification.

Loss of RREB1 in pancreatic beta cells reduces cellular insulin content and affects endocrine cell gene expression.

AIMS/HYPOTHESIS: Genome-wide studies have uncovered multiple independent signals at the RREB1 locus associated with altered type 2 diabetes risk and related glycaemic traits. However, little is known about the function of the zinc finger transcription factor Ras-responsive element binding protein 1 (RREB1) in glucose homeostasis or how changes in its expression and/or function influence diabetes risk. METHODS: A zebrafish model lacking rreb1a and rreb1b was used to study the effect of RREB1 loss in vivo. Using transcriptomic and cellular phenotyping of a human beta cell model (EndoC-ßH1) and human induced pluripotent stem cell (hiPSC)-derived beta-like cells, we investigated how loss of RREB1 expression and activity affects pancreatic endocrine cell development and function. Ex vivo measurements of human islet function were performed in donor islets from carriers of RREB1 type 2 diabetes risk alleles. RESULTS: CRISPR/Cas9-mediated loss of rreb1a and rreb1b function in zebrafish supports an in vivo role for the transcription factor in beta cell mass, beta cell insulin expression and glucose levels. Loss of RREB1 also reduced insulin gene expression and cellular insulin content in EndoC-ßH1 cells and impaired insulin secretion under prolonged stimulation. Transcriptomic analysis of RREB1 knockdown and knockout EndoC-ßH1 cells supports RREB1 as a novel regulator of genes involved in insulin secretion. In vitro differentiation of RREB1KO/KO hiPSCs revealed dysregulation of pro-endocrine cell genes, including RFX family members, suggesting that RREB1 also regulates genes involved in endocrine cell development. Human donor islets from carriers of type 2 diabetes risk alleles in RREB1 have altered glucose-stimulated insulin secretion ex vivo, consistent with a role for RREB1 in regulating islet cell function. CONCLUSIONS/INTERPRETATION: Together, our results indicate that RREB1 regulates beta cell function by transcriptionally regulating the expression of genes involved in beta cell development and function.

CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations.

CRISPR-Cas9 genome editing has potential to cure diseases without current treatments, but therapies must be safe. Here we show that CRISPR-Cas9 editing can introduce unintended mutations in vivo, which are passed on to the next generation. By editing fertilized zebrafish eggs using four guide RNAs selected for off-target activity in vitro, followed by long-read sequencing of DNA from >1100 larvae, juvenile and adult fish across two generations, we find that structural variants (SVs), i.e., insertions and deletions ≥50 bp, represent 6% of editing outcomes in founder larvae. These SVs occur both at on-target and off-target sites. Our results also illustrate that adult founder zebrafish are mosaic in their germ cells, and that 26% of their offspring carries an off-target mutation and 9% an SV. Hence, pre-testing for off-target activity and SVs using patient material is advisable in clinical applications, to reduce the risk of unanticipated effects with potentially large implications.

Identification of 371 genetic variants for age at first sex and birth linked to externalising behaviour.

Age at first sexual intercourse and age at first birth have implications for health and evolutionary fitness. In this genome-wide association study (age at first sexual intercourse, N = 387,338; age at first birth, N = 542,901), we identify 371 single-nucleotide polymorphisms, 11 sex-specific, with a 5-6% polygenic score prediction. Heritability of age at first birth shifted from 9% [CI = 4-14%] for women born in 1940 to 22% [CI = 19-25%] for those born in 1965. Signals are driven by the genetics of reproductive biology and externalising behaviour, with key genes related to follicle stimulating hormone (FSHB), implantation (ESR1), infertility and spermatid differentiation. Our findings suggest that polycystic ovarian syndrome may lead to later age at first birth, linking with infertility. Late age at first birth is associated with parental longevity and reduced incidence of type 2 diabetes and cardiovascular disease. Higher childhood socioeconomic circumstances and those in the highest polygenic score decile (90%+) experience markedly later reproductive onset. Results are relevant for improving teenage and late-life health, understanding longevity and guiding experimentation into mechanisms of infertility.

Genome-wide discovery of genetic loci that uncouple excess adiposity from its comorbidities.

Obesity is a major risk factor for cardiometabolic diseases. Nevertheless, a substantial proportion of individuals with obesity do not suffer cardiometabolic comorbidities. The mechanisms that uncouple adiposity from its cardiometabolic complications are not fully understood. Here, we identify 62 loci of which the same allele is significantly associated with both higher adiposity and lower cardiometabolic risk. Functional analyses show that the 62 loci are enriched for genes expressed in adipose tissue, and for regulatory variants that influence nearby genes that affect adipocyte differentiation. Genes prioritized in each locus support a key role of fat distribution (FAM13A, IRS1 and PPARG) and adipocyte function (ALDH2, CCDC92, DNAH10, ESR1, FAM13A, MTOR, PIK3R1 and VEGFB). Several additional mechanisms are involved as well, such as insulin-glucose signalling (ADCY5, ARAP1, CREBBP, FAM13A, MTOR, PEPD, RAC1 and SH2B3), energy expenditure and fatty acid oxidation (IGF2BP2), browning of white adipose tissue (CSK, VEGFA, VEGFB and SLC22A3) and inflammation (SH2B3, DAGLB and ADCY9). Some of these genes may represent therapeutic targets to reduce cardiometabolic risk linked to excess adiposity.

Amplification-free long-read sequencing reveals unforeseen CRISPR-Cas9 off-target activity.

BACKGROUND: One ongoing concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target activity is challenging. Here, we present SMRT-OTS and Nano-OTS, two novel, amplification-free, long-read sequencing protocols for detection of gRNA-driven digestion of genomic DNA by Cas9 in vitro. RESULTS: The methods are assessed using the human cell line HEK293, re-sequenced at 18x coverage using highly accurate HiFi SMRT reads. SMRT-OTS and Nano-OTS are first applied to three different gRNAs targeting HEK293 genomic DNA, resulting in a set of 55 high-confidence gRNA cleavage sites identified by both methods. Twenty-five of these sites are not reported by off-target prediction software, either because they contain four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. Additional experiments reveal that 85% of Cas9 cleavage sites are also found by other in vitro-based methods and that on- and off-target sites are detectable in gene bodies where short-reads fail to uniquely align. Even though SMRT-OTS and Nano-OTS identify several sites with previously validated off-target editing activity in cells, our own CRISPR-Cas9 editing experiments in human fibroblasts do not give rise to detectable off-target mutations at the in vitro-predicted sites. However, indel and structural variation events are enriched at the on-target sites. CONCLUSIONS: Amplification-free long-read sequencing reveals Cas9 cleavage sites in vitro that would have been difficult to predict using computational tools, including in dark genomic regions inaccessible by short-read sequencing.

Translating GWAS-identified loci for cardiac rhythm and rate using an in vivo image- and CRISPR/Cas9-based approach.

A meta-analysis of genome-wide association studies (GWAS) identified eight loci that are associated with heart rate variability (HRV), but candidate genes in these loci remain uncharacterized. We developed an image- and CRISPR/Cas9-based pipeline to systematically characterize candidate genes for HRV in live zebrafish embryos. Nine zebrafish orthologues of six human candidate genes were targeted simultaneously in eggs from fish that transgenically express GFP on smooth muscle cells (Tg[acta2:GFP]), to visualize the beating heart. An automated analysis of repeated 30 s recordings of beating atria in 381 live, intact zebrafish embryos at 2 and 5 days post-fertilization highlighted genes that influence HRV (hcn4 and si:dkey-65j6.2 [KIAA1755]); heart rate (rgs6 and hcn4); and the risk of sinoatrial pauses and arrests (hcn4). Exposure to 10 or 25 µM ivabradine-an open channel blocker of HCNs-for 24 h resulted in a dose-dependent higher HRV and lower heart rate at 5 days post-fertilization. Hence, our screen confirmed the role of established genes for heart rate and rhythm (RGS6 and HCN4); showed that ivabradine reduces heart rate and increases HRV in zebrafish embryos, as it does in humans; and highlighted a novel gene that plays a role in HRV (KIAA1755).

Publisher Correction: Multiancestry genome-wide association study of 520,000 subjects identifies 32 loci associated with stroke and stroke subtypes.

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

Test-retest variability of VO2max using total-capture indirect calorimetry reveals linear relationship of VO2 and Power.

This study aimed to analyze the intra-individual variation in VO2max of human subjects using total-capture and free-flow indirect calorimetry. Twenty-seven men (27 ± 5 year; VO2max 49-79 mL•kg-1 •min-1 ) performed two maximal exertion tests (CPETs) on a cycle ergometer, separated by a 7 ± 2 day interval. VO2 and VCO2 were assessed using an indirect calorimeter (Omnical) with total capture of exhalation in a free-flow airstream. Thirteen subjects performed a third maximal exertion test using a breath-by-breath calorimeter (Oxycon Pro). On-site validation was deemed a requirement. For the Omnical, the mean within-subject CV for VO2max was 1.2 ± 0.9% (0.0%-4.4%) and for ergometer workload P max 1.3 ± 1.3% (0%-4.6%). VO2max values with the Oxycon Pro were significantly lower in comparison with Omnical (P < 0.001; t test) with mean 3570 vs 4061 and difference SD 361 mL•min-1 . Validation results for the Omnical with methanol combustion were -0.05 ± 0.70% (mean ± SD; n = 31) at the 225 mL•min-1 VO2 level and -0.23 ± 0.80% (n = 31) at the 150 mL•min-1 VCO2 level. Results using gas infusion were 0.04 ± 0.75% (n = 34) and -0.99 ± 1.05% (n = 24) over the respective 500-6000 mL•min-1 VO2 and VCO2 ranges. Validation results for the Oxycon Pro in breath-by-breath mode were - 2.2 ± 1.6% (n = 12) for VO2 and 5.7 ± 3.3% (n = 12) for VCO2 over the 1000-4000 mL•min-1 range. On a Visual analog scale, participants reported improved breathing using the free-flow indirect calorimetry (score 7.6 ± 1.2 vs 5.1 ± 2.7, P = 0.008). We conclude that total capturing free-flow indirect calorimetry is suitable for measuring VO2 even with the highest range. VO2max was linear with the incline in P max over the full range.

PR interval genome-wide association meta-analysis identifies 50 loci associated with atrial and atrioventricular electrical activity.

Electrocardiographic PR interval measures atrio-ventricular depolarization and conduction, and abnormal PR interval is a risk factor for atrial fibrillation and heart block. Our genome-wide association study of over 92,000 European-descent individuals identifies 44 PR interval loci (34 novel). Examination of these loci reveals known and previously not-yet-reported biological processes involved in cardiac atrial electrical activity. Genes in these loci are over-represented in cardiac disease processes including heart block and atrial fibrillation. Variants in over half of the 44 loci were associated with atrial or blood transcript expression levels, or were in high linkage disequilibrium with missense variants. Six additional loci were identified either by meta-analysis of ~105,000 African and European-descent individuals and/or by pleiotropic analyses combining PR interval with heart rate, QRS interval, and atrial fibrillation. These findings implicate developmental pathways, and identify transcription factors, ion-channel genes, and cell-junction/cell-signaling proteins in atrio-ventricular conduction, identifying potential targets for drug development.

Multiancestry genome-wide association study of 520,000 subjects identifies 32 loci associated with stroke and stroke subtypes.

Stroke has multiple etiologies, but the underlying genes and pathways are largely unknown. We conducted a multiancestry genome-wide-association meta-analysis in 521,612 individuals (67,162 cases and 454,450 controls) and discovered 22 new stroke risk loci, bringing the total to 32. We further found shared genetic variation with related vascular traits, including blood pressure, cardiac traits, and venous thromboembolism, at individual loci (n = 18), and using genetic risk scores and linkage-disequilibrium-score regression. Several loci exhibited distinct association and pleiotropy patterns for etiological stroke subtypes. Eleven new susceptibility loci indicate mechanisms not previously implicated in stroke pathophysiology, with prioritization of risk variants and genes accomplished through bioinformatics analyses using extensive functional datasets. Stroke risk loci were significantly enriched in drug targets for antithrombotic therapy.

Biological/Genetic Regulation of Physical Activity Level: Consensus from GenBioPAC.

PURPOSE: Physical activity unquestionably maintains and improves health; however, physical activity levels globally are low and not rising despite all the resources devoted to this goal. Attention in both the research literature and the public policy domain has focused on social-behavioral factors; however, a growing body of literature suggests that biological determinants play a significant role in regulating physical activity levels. For instance, physical activity level, measured in various manners, has a genetic component in both humans and nonhuman animal models. This consensus article, developed as a result of an American College of Sports Medicine-sponsored round table, provides a brief review of the theoretical concepts and existing literature that supports a significant role of genetic and other biological factors in the regulation of physical activity. CONCLUSIONS: Future research on physical activity regulation should incorporate genetics and other biological determinants of physical activity instead of a sole reliance on social and other environmental determinants.

Maternal diabetes and incidence of childhood cancer - a nationwide cohort study and exploratory genetic analysis.

BACKGROUND: The etiology of childhood cancer is not well understood, but may be linked to prenatal and perinatal factors, such as maternal diabetes. However, this association has not been examined in depth. We aimed to determine if maternal diabetes is associated with risk of childhood brain tumor (CBT), leukemia (all types combined and acute lymphoblastic leukemia [ALL] separately), and lymphoma. METHODS: All children born in Sweden between 1973 and 2014 (n=4,239,965) were followed from birth until first cancer diagnosis, age 15 years, or December 31, 2015. Data on maternal diabetes, childhood cancer, and covariates were obtained from nationwide health registers. Incidence rate ratios (IRRs) and 95% confidence intervals (CIs) were calculated using Cox regression adjusted for potential confounders/mediators. Additionally, we performed an exploratory analysis using results from published genome-wide association studies and functional annotation. RESULTS: Maternal diabetes was associated with lower risk of CBT (adjusted IRR [95% CI]: 0.56 [0.35-0.91]) and higher risk of leukemia (adjusted IRR: 1.47 [1.13-1.92] for all leukemia combined and 1.64 [1.23-2.18] for ALL). These associations were similar for both maternal type 1 diabetes and gestational diabetes. Associations of five previously identified genetic loci were compatible with a causal effect of diabetes traits on neuroblastoma and common Hodgkin's lymphoma. CONCLUSION: Children whose mother had diabetes had lower risk of CBT and higher risk of leukemia, compared with children whose mother did not have diabetes. Our results are compatible with a role of prenatal and perinatal glycemic environment in childhood cancer etiology.

Correction: Genome-wide physical activity interactions in adiposity - A meta-analysis of 200,452 adults.

[This corrects the article DOI: 10.1371/journal.pgen.1006528.].

Genome-wide physical activity interactions in adiposity - A meta-analysis of 200,452 adults.

Physical activity (PA) may modify the genetic effects that give rise to increased risk of obesity. To identify adiposity loci whose effects are modified by PA, we performed genome-wide interaction meta-analyses of BMI and BMI-adjusted waist circumference and waist-hip ratio from up to 200,452 adults of European (n = 180,423) or other ancestry (n = 20,029). We standardized PA by categorizing it into a dichotomous variable where, on average, 23% of participants were categorized as inactive and 77% as physically active. While we replicate the interaction with PA for the strongest known obesity-risk locus in the FTO gene, of which the effect is attenuated by ~30% in physically active individuals compared to inactive individuals, we do not identify additional loci that are sensitive to PA. In additional genome-wide meta-analyses adjusting for PA and interaction with PA, we identify 11 novel adiposity loci, suggesting that accounting for PA or other environmental factors that contribute to variation in adiposity may facilitate gene discovery.

Extensive Type II Muscle Fiber Atrophy in Elderly Female Hip Fracture Patients.

BACKGROUND: Sarcopenia, or the loss of muscle mass and strength, is known to increase the risk for falls and (hip) fractures in older people. The objective of this study was to assess the skeletal muscle fiber characteristics in elderly female hip fracture patients. METHOD: Percutaneous needle biopsies were collected from the vastus lateralis muscle in 15 healthy young women (20 ± 0.4 years), 15 healthy elderly women (79 ± 1.7 years), and 15 elderly women with a fall-related hip fracture (82 ± 1.5 years). Immunohistochemical analyses were performed to assess Type I and Type II muscle fiber size, and myonuclear and satellite cell content. RESULTS: Type II muscle fiber size was significantly different between all groups (p < .05), with smaller Type II muscle fibers in the hip fracture patients (2,609 ± 185 µm2) compared with healthy elderly group (3,723 ± 322 µm2) and the largest Type II muscle fibers in the healthy young group (4,755 ± 335 µm2). Furthermore, Type I muscle fiber size was significantly lower in the hip fracture patients (4,684 ± 211 µm2) compared with the healthy elderly group (5,842 ± 316 µm2, p = .02). The number of myonuclei per Type II muscle fiber was significantly lower in the healthy elderly and hip fracture group compared with the healthy young group (p = .011 and p = .002, respectively). Muscle fiber satellite cell content did not differ between groups. CONCLUSION: Elderly female hip fracture patients show extensive Type II muscle fiber atrophy when compared with healthy young or age-matched healthy elderly controls. Type II muscle fiber atrophy is an important hallmark of sarcopenia and may predispose to falls and (hip) fractures in the older population.