Exome-Wide Association Study on Alanine Aminotransferase Identifies Sequence Variants in the GPAM and APOE Associated With Fatty Liver Disease.

BACKGROUND & AIMS: Fatty liver disease (FLD) is a growing epidemic that is expected to be the leading cause of end-stage liver disease within the next decade. Both environmental and genetic factors contribute to the susceptibility of FLD. Several genetic variants contributing to FLD have been identified in exome-wide association studies. However, there is still a missing hereditability indicating that other genetic variants are yet to be discovered. METHODS: To find genes involved in FLD, we first examined the association of missense and nonsense variants with alanine aminotransferase at an exome-wide level in 425,671 participants from the UK Biobank. We then validated genetic variants with liver fat content in 8930 participants in whom liver fat measurement was available, and replicated 2 genetic variants in 3 independent cohorts comprising 2621 individuals with available liver biopsy. RESULTS: We identified 190 genetic variants independently associated with alanine aminotransferase after correcting for multiple testing with Bonferroni method. The majority of these variants were not previously associated with this trait. Among those associated, there was a striking enrichment of genetic variants influencing lipid metabolism. We identified the variants rs2792751 in GPAM/GPAT1, the gene encoding glycerol-3-phosphate acyltransferase, mitochondrial, and rs429358 in APOE, the gene encoding apolipoprotein E, as robustly associated with liver fat content and liver disease after adjusting for multiple testing. Both genes affect lipid metabolism in the liver. CONCLUSIONS: We identified 2 novel genetic variants in GPAM and APOE that are robustly associated with steatosis and liver damage. These findings may help to better elucidate the genetic susceptibility to FLD onset and progression.

Hepatic Molecular Signatures Highlight the Sexual Dimorphism of Nonalcoholic Steatohepatitis (NASH).

BACKGROUND AND AIMS: Nonalcoholic steatohepatitis (NASH) is considered as a pivotal stage in nonalcoholic fatty liver disease (NAFLD) progression, given that it paves the way for severe liver injuries such as fibrosis and cirrhosis. The etiology of human NASH is multifactorial, and identifying reliable molecular players and/or biomarkers has proven difficult. Together with the inappropriate consideration of risk factors revealed by epidemiological studies (altered glucose homeostasis, obesity, ethnicity, sex, etc.), the limited availability of representative NASH cohorts with associated liver biopsies, the gold standard for NASH diagnosis, probably explains the poor overlap between published "omics"-defined NASH signatures. APPROACH AND RESULTS: Here, we have explored transcriptomic profiles of livers starting from a 910-obese-patient cohort, which was further stratified based on stringent histological characterization, to define "NoNASH" and "NASH" patients. Sex was identified as the main factor for data heterogeneity in this cohort. Using powerful bootstrapping and random forest (RF) approaches, we identified reliably differentially expressed genes participating in distinct biological processes in NASH as a function of sex. RF-calculated gene signatures identified NASH patients in independent cohorts with high accuracy. CONCLUSIONS: This large-scale analysis of transcriptomic profiles from human livers emphasized the sexually dimorphic nature of NASH and its link with fibrosis, calling for the integration of sex as a major determinant of liver responses to NASH progression and responses to drugs.

Transancestral fine-mapping of four type 2 diabetes susceptibility loci highlights potential causal regulatory mechanisms.

To gain insight into potential regulatory mechanisms through which the effects of variants at four established type 2 diabetes (T2D) susceptibility loci (CDKAL1, CDKN2A-B, IGF2BP2 and KCNQ1) are mediated, we undertook transancestral fine-mapping in 22 086 cases and 42 539 controls of East Asian, European, South Asian, African American and Mexican American descent. Through high-density imputation and conditional analyses, we identified seven distinct association signals at these four loci, each with allelic effects on T2D susceptibility that were homogenous across ancestry groups. By leveraging differences in the structure of linkage disequilibrium between diverse populations, and increased sample size, we localised the variants most likely to drive each distinct association signal. We demonstrated that integration of these genetic fine-mapping data with genomic annotation can highlight potential causal regulatory elements in T2D-relevant tissues. These analyses provide insight into the mechanisms through which T2D association signals are mediated, and suggest future routes to understanding the biology of specific disease susceptibility loci.

Incidence and Predictive Factors of Postprandial Hyperinsulinemic Hypoglycemia After Roux-en-Y Gastric Bypass: A Five year Longitudinal Study.

BACKGROUND: Postprandial hyperinsulinemic hypoglycemia (PHH) is often reported after Roux-en-Y gastric bypass (RYGB). In the absence of a prospective study, the clinical and biological determinants of PHH remain unclear. OBJECTIVE: To determine the incidence and predictive factors of PHH after RYGB. METHODS: Participants were 957 RYGB patients enrolled in an ongoing longitudinal cohort study. We analyzed the results of an oral glucose tolerance test (OGTT) routinely performed before surgery and 1 and/or 5 years after. PHH was defined as blood glucose < 50 mg/dL AND plasma insulin > 3 mU/L at 120 minutes post glucose challenge. Validated indices of insulin sensitivity (Matsuda index), beta-cell function (Insulinogenic index), and beta-cell mass (fasting C-peptide: glucose ratio) were calculated, from glucose, insulin, and c-peptide values measured during OGTT. RESULTS: OGTT results were available in all patients at baseline, in 85.6% at 12 months and 52.8% at 60 months. The incidence of PHH was 0.5% at baseline, 9.1% * and 7.9%* at 12 months and 60 months following RYGB (*: P < 0.001). In multivariate logistic regression analysis, PHH after RYGB was independently associated with lower age (P = 0.005), greater weight loss (P = 0.031), as well as higher beta-cell function (P = 0.002) and insulin sensitivity (P < 0.001), but not with beta-cell mass (P = 0.381). A preoperative elevated beta-cell function was an independent predictor of PHH after RYGB (receiver operating characteristics curve area under the curve 0.68, P = 0.04). CONCLUSIONS:: The incidence of PHH significantly increased after RYGB but remained stable between 1 and 5 years. The estimation of beta-cell function with an OGTT before surgery can identify patients at risk for developing PHH after RYGB.

A Federated Database for Obesity Research: An IMI-SOPHIA Study.

Obesity is considered by many as a lifestyle choice rather than a chronic progressive disease. The Innovative Medicines Initiative (IMI) SOPHIA (Stratification of Obesity Phenotypes to Optimize Future Obesity Therapy) project is part of a momentum shift aiming to provide better tools for the stratification of people with obesity according to disease risk and treatment response. One of the challenges to achieving these goals is that many clinical cohorts are siloed, limiting the potential of combined data for biomarker discovery. In SOPHIA, we have addressed this challenge by setting up a federated database building on open-source DataSHIELD technology. The database currently federates 16 cohorts that are accessible via a central gateway. The database is multi-modal, including research studies, clinical trials, and routine health data, and is accessed using the R statistical programming environment where statistical and machine learning analyses can be performed at a distance without any disclosure of patient-level data. We demonstrate the use of the database by providing a proof-of-concept analysis, performing a federated linear model of BMI and systolic blood pressure, pooling all data from 16 studies virtually without any analyst seeing individual patient-level data. This analysis provided similar point estimates compared to a meta-analysis of the 16 individual studies. Our approach provides a benchmark for reproducible, safe federated analyses across multiple study types provided by multiple stakeholders.

A comparison of changes in bone turnover markers after gastric bypass and sleeve gastrectomy, and their association with markers of interest.

BACKGROUND: It is still debated whether differences in bone turnover markers (BTMs) exist between the 2 most popular bariatric surgery procedures (Roux-en-Y gastric bypass [RYGB] and sleeve gastrectomy [SG]). OBJECTIVES: To compare changes in BTMs after RYGB and SG, and to investigate their association with predefined markers of interest. SETTING: University hospital, Lille, France. METHODS: An ancillary investigation of a prospective cohort was conducted. SG patients with severe obesity ≥40 years were matched one-to-one to RYGB patients for age, sex, body mass index (BMI), and menopausal status. BTMs, as well as predefined markers of interest, were measured at baseline, 12, and 24 months after bariatric surgery. RESULTS: Sixty-four patients (66% women) had a mean (standard deviation [SD]) age of 49.6 years (5.1) and a mean (SD) BMI of 45.0 kg/m2 (6.0). From baseline to 12 months, a significant increase in BTMs was observed in both groups (P < .001). Moreover, RYGB was associated with a greater increase in C-terminal telopeptide (ß-CTX) and procollagen type 1 N-terminal propeptide (PINP) compared with SG (P < .0001). From 12 to 24 months, a significant decrease in BTMs was observed in both groups, but no significant differences were found between RYGB and SG. However, BTMs did not return to baseline levels. The changes in PINP and ß-CTX at 12 months were independently associated with the type of surgical procedure, after adjusting for weight or each predefined marker of interest (all P < .0001). CONCLUSION: RYGB was associated with a greater increase in BTMs than SG at 12 and 24 months.

PSD3 downregulation confers protection against fatty liver disease.

Fatty liver disease (FLD) is a growing health issue with burdening unmet clinical needs. FLD has a genetic component but, despite the common variants already identified, there is still a missing heritability component. Using a candidate gene approach, we identify a locus (rs71519934) at the Pleckstrin and Sec7 domain-containing 3 (PSD3) gene resulting in a leucine to threonine substitution at position 186 of the protein (L186T) that reduces susceptibility to the entire spectrum of FLD in individuals at risk. PSD3 downregulation by short interfering RNA reduces intracellular lipid content in primary human hepatocytes cultured in two and three dimensions, and in human and rodent hepatoma cells. Consistent with this, Psd3 downregulation by antisense oligonucleotides in vivo protects against FLD in mice fed a non-alcoholic steatohepatitis-inducing diet. Thus, translating these results to humans, PSD3 downregulation might be a future therapeutic option for treating FLD.

NASH-related increases in plasma bile acid levels depend on insulin resistance.

BACKGROUND & AIMS: Plasma bile acids (BAs) have been extensively studied as pathophysiological actors in non-alcoholic steatohepatitis (NASH). However, results from clinical studies are often complicated by the association of NASH with type 2 diabetes (T2D), obesity, and insulin resistance (IR). Here, we sought to dissect the relationship between NASH, T2D, and plasma BA levels in a large patient cohort. METHODS: Four groups of patients from the Biological Atlas of Severe Obesity (ABOS) cohort (Clinical Trials number NCT01129297) were included based on the presence or absence of histologically evaluated NASH with or without coincident T2D. Patients were matched for BMI, homeostatic model assessment 2 (HOMA2)-assessed IR, glycated haemoglobin, age, and gender. To study the effect of IR and BMI on the association of plasma BA and NASH, patients from the HEPADIP study were included. In both cohorts, fasting plasma BA concentrations were measured. RESULTS: Plasma BA concentrations were higher in NASH compared with No-NASH patients both in T2D and NoT2D patients from the ABOS cohort. As we previously reported that plasma BA levels were unaltered in NASH patients of the HEPADIP cohort, we assessed the impact of BMI and IR on the association of NASH and BA on the combined BA datasets. Our results revealed that NASH-associated increases in plasma total cholic acid (CA) concentrations depend on the degree of HOMA2-assessed systemic IR, but not on ß-cell function nor on BMI. CONCLUSIONS: Plasma BA concentrations are elevated only in those NASH patients exhibiting pronounced IR. LAY SUMMARY: Non-alcoholic steatohepatitis (NASH) is a progressive liver disease that frequently occurs in patients with obesity and type 2 diabetes. Reliable markers for the diagnosis of NASH are needed. Plasma bile acids have been proposed as NASH biomarkers. Herein, we found that plasma bile acids are only elevated in patients with NASH when significant insulin resistance is present, limiting their utility as NASH markers.

Hepatic transcriptomic signatures of statin treatment are associated with impaired glucose homeostasis in severely obese patients.

BACKGROUND: Clinical data identified an association between the use of HMG-CoA reductase inhibitors (statins) and incident diabetes in patients with underlying diabetes risk factors such as obesity, hypertension and dyslipidemia. The molecular mechanisms however are unknown. METHODS: An observational cross-sectional study included 910 severely obese patients, mean (SD) body mass index (BMI) 46.7 (8.7), treated with or without statins (ABOS cohort: a biological atlas of severe obesity). Data and sample collection took place in France between 2006 and 2016. Transcriptomic signatures of statin treatment in human liver obtained from genome-wide transcriptomic profiling of five different statin drugs using microarrays were correlated to clinico-biological phenotypes and also assigned to biological pathways and mechanisms. Patients from the non-statin-users group were matched to patients in the statin users group by propensity score analysis to minimize confounding effects from age, gender, parental familial history of diabetes, BMI, waist circumference, systolic and diastolic blood pressure and use of anti-hypertensive drugs as pre-specified covariates. RESULTS: We determined the hepatic, statin-related gene signature from genome-wide transcriptomic profiling in severely obese patients with varying degrees of glucose tolerance and cardio-metabolic comorbidities. One hundred and fifty seven patients on statin treatment in the matched cohort showed higher diabetes prevalence (OR = 2.67; 95%CI, 1.60-4.45; P = 0.0002) and impairment of glucose homeostasis. This phenotype was associated with molecular signatures of increased hepatic de novo lipogenesis (DNL) via activation of sterol regulatory element-binding protein 1 (SREBP1) and concomitant upregulation of the expression of key genes in both fatty acid and triglyceride metabolism. CONCLUSIONS: A DNL gene activation profile in response to statins is associated with insulin resistance and the diabetic status of the patients. Identified molecular signatures thus suggest that statin treatment increases the risk for diabetes in humans at least in part via induction of DNL. TRIAL REGISTRATION: NCT01129297 . Registered May 242,010 (retrospectively registered).

CoDE-seq, an augmented whole-exome sequencing, enables the accurate detection of CNVs and mutations in Mendelian obesity and intellectual disability.

OBJECTIVE: The molecular diagnosis of extreme forms of obesity, in which accurate detection of both copy number variations (CNVs) and point mutations, is crucial for an optimal care of the patients and genetic counseling for their families. Whole-exome sequencing (WES) has benefited considerably this molecular diagnosis, but its poor ability to detect CNVs remains a major limitation. We aimed to develop a method (CoDE-seq) enabling the accurate detection of both CNVs and point mutations in one step. METHODS: CoDE-seq is based on an augmented WES method, using probes distributed uniformly throughout the genome. CoDE-seq was validated in 40 patients for whom chromosomal DNA microarray was available. CNVs and mutations were assessed in 82 children/young adults with suspected Mendelian obesity and/or intellectual disability and in their parents when available (ntotal = 145). RESULTS: CoDE-seq not only detected all of the 97 CNVs identified by chromosomal DNA microarrays but also found 84 additional CNVs, due to a better resolution. When compared to CoDE-seq and chromosomal DNA microarrays, WES failed to detect 37% and 14% of CNVs, respectively. In the 82 patients, a likely molecular diagnosis was achieved in >30% of the patients. Half of the genetic diagnoses were explained by CNVs while the other half by mutations. CONCLUSIONS: CoDE-seq has proven cost-efficient and highly effective as it avoids the sequential genetic screening approaches currently used in clinical practice for the accurate detection of CNVs and point mutations.

An Expanded Genome-Wide Association Study of Type 2 Diabetes in Europeans.

To characterize type 2 diabetes (T2D)-associated variation across the allele frequency spectrum, we conducted a meta-analysis of genome-wide association data from 26,676 T2D case and 132,532 control subjects of European ancestry after imputation using the 1000 Genomes multiethnic reference panel. Promising association signals were followed up in additional data sets (of 14,545 or 7,397 T2D case and 38,994 or 71,604 control subjects). We identified 13 novel T2D-associated loci (P < 5 × 10-8), including variants near the GLP2R, GIP, and HLA-DQA1 genes. Our analysis brought the total number of independent T2D associations to 128 distinct signals at 113 loci. Despite substantially increased sample size and more complete coverage of low-frequency variation, all novel associations were driven by common single nucleotide variants. Credible sets of potentially causal variants were generally larger than those based on imputation with earlier reference panels, consistent with resolution of causal signals to common risk haplotypes. Stratification of T2D-associated loci based on T2D-related quantitative trait associations revealed tissue-specific enrichment of regulatory annotations in pancreatic islet enhancers for loci influencing insulin secretion and in adipocytes, monocytes, and hepatocytes for insulin action-associated loci. These findings highlight the predominant role played by common variants of modest effect and the diversity of biological mechanisms influencing T2D pathophysiology.

Eating Behavior, Low-Frequency Functional Mutations in the Melanocortin-4 Receptor (MC4R) Gene, and Outcomes of Bariatric Operations: A 6-Year Prospective Study.

OBJECTIVE: Data on the effects of eating behavior and genetics on outcomes of gastrointestinal surgery for diabesity have been sparse, often flawed, and controversial. We aimed to assess long-term outcomes of bariatric operations in patients characterized for eating behavior and rare mutations in the melanocortin-4 receptor (MC4R) gene, which is strongly implicated in energy balance. RESEARCH DESIGN AND METHODS: Between 1996 and 2005, 1,264 severely obese Swiss patients underwent current laparoscopic adjustable gastric banding, gastroduodenal bypass, or a hybrid operation. Of these, 872 patients were followed for a minimum of 6 years and were screened for MC4R mutations. Using regression models, we studied relationships between eating behavior and MC4R mutations and postoperative weight loss, complications, and reoperations after 6 years. RESULTS: At baseline, rare functional MC4R mutation carriers exhibited a significantly higher prevalence of binge eating disorder (BED) or loss-of-control eating independent of age, sex, and BMI. Six years after bariatric surgery, the mutation carriers had more major complications than wild-type subjects independent of age, baseline BMI, sex, operation type, and weight loss. Furthermore, high baseline BMI, male sex, BED, and functional MC4R mutations were independent predictors of higher reoperation rates. CONCLUSIONS: Sequencing of MC4R and eating typology, combined with stratification for sex and baseline BMI, might significantly improve patient allocation to banding or bypass operations for diabesity as well as reduce both complication and reoperation rates.

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.

Association between large detectable clonal mosaicism and type 2 diabetes with vascular complications.

Large chromosomal clonal mosaic events (CMEs) have been suggested to be linked to aging and to predict cancer. Type 2 diabetes (T2D) has been conceptualized as an accelerated-aging disease and is associated with higher prevalence of cancers. Here we aimed to assess the association between T2D and CME occurrence in blood. We evaluated the presence of CMEs in 7,659 individuals (including 2,208 with T2D) using DNA arrays. A significant association between CME occurrence and T2D was found (odds ratio (OR) = 5.3; P = 5.1 × 10(-5)) and was stronger when we only considered non-obese individuals with T2D (OR = 5.6; P = 4.9 × 10(-5)). Notably, CME carriers with T2D had higher prevalence of vascular complications than non-carriers with T2D (71.4% versus 37.1%, respectively; P = 7.7 × 10(-4)). In CME carriers, we found an increase in the percentage of abnormal cells over 6 years (P = 8.60 × 10(-3)). In conclusion, given the increased risk of cancer in CME carriers, our results may have profound clinical implications in patients with severe T2D.