Heterozygous pathogenic variants in POMC are not responsible for monogenic obesity: Implication for MC4R agonist use.

PURPOSE: Recessive deficiency of proopiomelanocortin (POMC) causes childhood-onset severe obesity. Cases can now benefit from the melanocortin 4 receptor agonist setmelanotide. Furthermore, a phase 3 clinical trial is evaluating setmelanotide in heterozygotes for POMC. We performed a large-scale genetic analysis to assess the effect of heterozygous, pathogenic POMC variants on obesity. METHODS: A genetic analysis was performed in a family including 2 cousins with childhood-onset obesity. We analyzed the obesity status of heterozygotes for pathogenic POMC variants in the Human Gene Mutation Database. The association between heterozygous pathogenic POMC variants and obesity risk was assessed using 190,000 exome samples from UK Biobank. RESULTS: The 2 cousins carried a compound heterozygous pathogenic variant in POMC. Six siblings were heterozygotes; only 1 of them had obesity. In Human Gene Mutation Database, we identified 60 heterozygotes for pathogenic POMC variants, of whom 14 had obesity. In UK Biobank, heterozygous pathogenic POMC variants were not associated with obesity risk, but they modestly increased body mass index levels. CONCLUSION: Heterozygous pathogenic POMC variants do not contribute to monogenic obesity, but they slightly increase body mass index. Setmelanotide use in patients with obesity, which would only be based on the presence of a heterozygous POMC variant, can be questioned.

Dominant negative mutation in oxalate transporter SLC26A6 associated with enteric hyperoxaluria and nephrolithiasis.

BACKGROUND: Nephrolithiasis (NL) is a complex multifactorial disease affecting up to 10%-20% of the human population and causing a significant burden on public health systems worldwide. It results from a combination of environmental and genetic factors. Hyperoxaluria is a major risk factor for NL. METHODS: We used a whole exome-based approach in a patient with calcium oxalate NL. The effects of the mutation were characterised using cell culture and in silico analyses. RESULTS: We identified a rare heterozygous missense mutation (c.1519C>T/p.R507W) in the SLC26A6 gene that encodes a secretory oxalate transporter. This mutation cosegregated with hyperoxaluria in the family. In vitro characterisation of mutant SLC26A6 demonstrated that Cl--dependent oxalate transport was dramatically reduced because the mutation affects both SLC26A6 transport activity and membrane surface expression. Cotransfection studies demonstrated strong dominant-negative effects of the mutant on the wild-type protein indicating that the phenotype of patients heterozygous for this mutation may be more severe than predicted by haploinsufficiency alone. CONCLUSION: Our study is in line with previous observations made in the mouse showing that SLC26A6 inactivation can cause inherited enteric hyperoxaluria with calcium oxalate NL. Consistent with an enteric form of hyperoxaluria, we observed a beneficial effect of increasing calcium in the patient's diet to reduce urinary oxalate excretion.

Contribution of heterozygous PCSK1 variants to obesity and implications for precision medicine: a case-control study.

BACKGROUND: Rare biallelic pathogenic mutations in PCSK1 (encoding proprotein convertase subtilisin/kexin type 1 [PC1/3]) cause early-onset obesity associated with various endocrinopathies. Setmelanotide has been approved for carriers of these biallelic mutations in the past 3 years. We aimed to perform a large-scale functional genomic study focusing on rare heterozygous variants of PCSK1 to decipher their putative impact on obesity risk. METHODS: This case-control study included all participants with overweight and obesity (ie, cases) or healthy weight (ie, controls) from the RaDiO study of three community-based and one hospital-based cohort in France recruited between Jan 1, 1995, and Dec 31, 2000. In adults older than 18 years, healthy weight was defined as BMI of less than 25·0 kg/m2, overweight as 25·0-29·9 kg/m2, and obesity as 30·0 kg/m2 or higher. Participants with type 2 diabetes had fasting glucose of 7·0 mmol/L or higher or used treatment for hyperglycaemia (or both) and were negative for islet or insulin autoantibodies. Functional assessment of rare missense variants of PCSK1 was performed. Pathogenicity clusters of variants were determined with machine learning. The effect of each cluster of PCSK1 variants on obesity was assessed using the adjusted mixed-effects score test. FINDINGS: All 13 coding exons of PCSK1 were sequenced in 9320 participants (including 7260 adults and 2060 children and adolescents) recruited from the RaDiO study. We detected 65 rare heterozygous PCSK1 variants, including four null variants and 61 missense variants that were analysed in vitro and clustered into five groups (A-E), according to enzymatic activity. Compared with the wild-type, 15 missense variants led to complete PC1/3 loss of function (group A; reference) and rare exome variant ensemble learner (REVEL) led to 15 (25%) false positives and four (7%) false negatives. Carrying complete loss-of-function or null PCSK1 variants was significantly associated with obesity (six [86%] of seven carriers vs 1518 [35%] of 4395 non-carriers; OR 9·3 [95% CI 1·5-177·4]; p=0·014) and higher BMI (32·0 kg/m2 [SD 9·3] in carriers vs 27·3 kg/m2 [6·5] in non-carriers; mean effect π 6·94 [SE 1·95]; p=0·00029). Clusters of PCSK1 variants with partial or neutral effect on PC1/3 activity did not have an effect on obesity or overweight and on BMI. INTERPRETATION: Only carriers of heterozygous, null, or complete loss-of-function PCSK1 variants cause monogenic obesity and, therefore, might be eligible for setmelanotide. In silico tests were unable to accurately detect these variants, which suggests that in vitro assays are necessary to determine the variant pathogenicity for genetic diagnosis and precision medicine purposes. FUNDING: Agence Nationale de la Recherche, European Research Council, National Center for Precision Diabetic Medicine, European Regional Development Fund, Hauts-de-France Regional Council, and the European Metropolis of Lille.

Pharmacological HDAC inhibition impairs pancreatic ß-cell function through an epigenome-wide reprogramming.

Histone deacetylases enzymes (HDACs) are chromatin modifiers that regulate gene expression through deacetylation of lysine residues within specific histone and non-histone proteins. A cell-specific gene expression pattern defines the identity of insulin-producing pancreatic ß cells, yet molecular networks driving this transcriptional specificity are not fully understood. Here, we investigated the HDAC-dependent molecular mechanisms controlling pancreatic ß-cell identity and function using the pan-HDAC inhibitor trichostatin A through chromatin immunoprecipitation assays and RNA sequencing experiments. We observed that TSA alters insulin secretion associated with ß-cell specific transcriptome programming in both mouse and human ß-cell lines, as well as on human pancreatic islets. We also demonstrated that this alternative ß-cell transcriptional program in response to HDAC inhibition is related to an epigenome-wide remodeling at both promoters and enhancers. Our data indicate that HDAC activity could be required to protect against loss of ß-cell identity with unsuitable expression of genes associated with alternative cell fates.

Biallelic Mutations in P4HTM Cause Syndromic Obesity.

We previously demonstrated that 50% of children with obesity from consanguineous families from Pakistan carry pathogenic variants in known monogenic obesity genes. Here, we have discovered a novel monogenetic recessive form of severe childhood obesity using an in-house computational staged approach. The analysis included whole-exome sequencing data of 366 children with severe obesity, 1,000 individuals of the Pakistan Risk of Myocardial Infarction Study (PROMIS) study, and 200,000 participants of the UK Biobank to prioritize genes harboring rare homozygous variants with putative effect on human obesity. We identified five rare or novel homozygous missense mutations predicted deleterious in five consanguineous families in P4HTM encoding prolyl 4-hydroxylase transmembrane (P4H-TM). We further found two additional homozygous missense mutations in children with severe obesity of Indian and Moroccan origin. Molecular dynamics simulation suggested that these mutations destabilized the active conformation of the substrate binding domain. Most carriers also presented with hypotonia, cognitive impairment, and/or developmental delay. Three of the five probands died of pneumonia during the first 2 years of the follow-up. P4HTM deficiency is a novel form of syndromic obesity, affecting 1.5% of our children with obesity associated with high mortality. P4H-TM is a hypoxia-inducible factor that is necessary for survival and adaptation under oxygen deprivation, but the role of this pathway in energy homeostasis and obesity pathophysiology remains to be elucidated.

Rare Variant Analysis of Obesity-Associated Genes in Young Adults With Severe Obesity From a Consanguineous Population of Pakistan.

Recent advances in genetic analysis have significantly helped in progressively attenuating the heritability gap of obesity and have brought into focus monogenic variants that disrupt the melanocortin signaling. In a previous study, next-generation sequencing revealed a monogenic etiology in ∼50% of the children with severe obesity from a consanguineous population in Pakistan. Here we assess rare variants in obesity-causing genes in young adults with severe obesity from the same region. Genomic DNA from 126 randomly selected young adult obese subjects (BMI 37.2 ± 0.3 kg/m2; age 18.4 ± 0.3 years) was screened by conventional or augmented whole-exome analysis for point mutations and copy number variants (CNVs). Leptin, insulin, and cortisol levels were measured by ELISA. We identified 13 subjects carrying 13 different pathogenic or likely pathogenic variants in LEPR, PCSK1, MC4R, NTRK2, POMC, SH2B1, and SIM1. We also identified for the first time in the human, two homozygous stop-gain mutations in ASNSD1 and IFI16 genes. Inactivation of these genes in mouse models has been shown to result in obesity. Additionally, we describe nine homozygous mutations (seven missense, one stop-gain, and one stop-loss) and four copy-loss CNVs in genes or genomic regions previously linked to obesity-associated traits by genome-wide association studies. Unexpectedly, in contrast to obese children, pathogenic mutations in LEP and LEPR were either absent or rare in this cohort of young adults. High morbidity and mortality risks and social disadvantage of children with LEP or LEPR deficiency may in part explain this difference between the two cohorts.

Compound genetic etiology in a patient with a syndrome including diabetes, intellectual deficiency and distichiasis.

BACKGROUND: We studied a young woman with atypical diabetes associated with mild intellectual disability, lymphedema distichiasis syndrome (LDS) and polymalformative syndrome including distichiasis. We used different genetic tools to identify causative pathogenic mutations and/or copy number variations. RESULTS: Although proband's, diabetes mellitus occurred during childhood, type 1 diabetes was unlikely due to the absence of detectable autoimmunity. DNA microarray analysis first identified a de novo, heterozygous deletion at the chr16q24.2 locus. Previously, thirty-three pathogenic or likely pathogenic deletions encompassing this locus have been reported in patients presenting with intellectual deficiency, obesity and/or lymphedema but not with diabetes. Of note, the deletion encompassed two topological association domains, whose one included FOXC2 that is known to be linked with LDS. Via whole-exome sequencing, we found a heterozygous, likely pathogenic variant in WFS1 (encoding wolframin endoplasmic reticulum [ER] transmembrane glycoprotein) which was inherited from her father who also had diabetes. WFS1 is known to be involved in monogenic diabetes. We also found a likely pathogenic variant in USP9X (encoding ubiquitin specific peptidase 9 X-linked) that is involved in X-linked intellectual disability, which was inherited from her mother who had dyscalculia and dyspraxia. CONCLUSIONS: Our comprehensive genetic analysis suggested that the peculiar phenotypes of our patient were possibly due to the combination of multiple genetic causes including chr16q24.2 deletion, and two likely pathogenic variants in WFS1 and USP9X.

Genetic Causes of Severe Childhood Obesity: A Remarkably High Prevalence in an Inbred Population of Pakistan.

Monogenic forms of obesity have been identified in ≤10% of severely obese European patients. However, the overall spectrum of deleterious variants (point mutations and structural variants) responsible for childhood severe obesity remains elusive. In this study, we genetically screened 225 severely obese children from consanguineous Pakistani families through a combination of techniques, including an in-house-developed augmented whole-exome sequencing method (CoDE-seq) that enables simultaneous detection of whole-exome copy number variations (CNVs) and point mutations in coding regions. We identified 110 (49%) probands carrying 55 different pathogenic point mutations and CNVs in 13 genes/loci responsible for nonsyndromic and syndromic monofactorial obesity. CoDE-seq also identified 28 rare or novel CNVs associated with intellectual disability in 22 additional obese subjects (10%). Additionally, we highlight variants in candidate genes for obesity warranting further investigation. Altogether, 59% of cases in the studied cohort are likely to have a discrete genetic cause, with 13% of these as a result of CNVs, demonstrating a remarkably higher prevalence of monofactorial obesity than hitherto reported and a plausible overlapping of obesity and intellectual disabilities in several cases. Finally, inbred populations with a high prevalence of obesity provide unique, genetically enriched material in the quest of new genes/variants influencing energy balance.