Accuracy of RNAseq based SNP discovery and genotyping in Populusnigra.

BACKGROUD: Populus nigra is a major tree species of ecological and economic importance for which several initiatives have been set up to create genomic resources. In order to access the large number of Single Nucleotide Polymorphisms (SNPs) typically needed to carry out a genome scan, the present study aimed at evaluating RNA sequencing as a tool to discover and type SNPs in genes within natural populations of P. nigra. RESULTS: We have devised a bioinformatics pipeline to call and type SNPs from RNAseq reads and applied it to P. nigra transcriptomic data. The accuracy of the resulting RNAseq-based SNP calling and typing has been evaluated by (i) comparing their position and alleles to those previously reported in candidate genes, (ii) assessing their genotyping accuracy with respect to a previously available SNP chip and (iii) evaluating their inter-annual repeatability. We found that a combination of several callers yields a good compromise between the number of variants type and the accuracy of genotyping. We further used the resulting genotypic data to carry out basic genetic analyses whose results confirm the quality of the RNAseq-based SNP dataset. CONCLUSIONS: We demonstrated the potential and accuracy of RNAseq as an efficient way to genotype SNPs in P. nigra. These results open prospects towards the use of this technology for quantitative and population genomics studies.

TOGGLE: toolbox for generic NGS analyses.

BACKGROUND: The explosion of NGS (Next Generation Sequencing) sequence data requires a huge effort in Bioinformatics methods and analyses. The creation of dedicated, robust and reliable pipelines able to handle dozens of samples from raw FASTQ data to relevant biological data is a time-consuming task in all projects relying on NGS. To address this, we created a generic and modular toolbox for developing such pipelines. RESULTS: TOGGLE (TOolbox for Generic nGs anaLysEs) is a suite of tools able to design pipelines that manage large sets of NGS softwares and utilities. Moreover, TOGGLE offers an easy way to manipulate the various options of the different softwares through the pipelines in using a single basic configuration file, which can be changed for each assay without having to change the code itself. We also describe one implementation of TOGGLE in a complete analysis pipeline designed for SNP discovery for large sets of genomic data, ready to use in different environments (from a single machine to HPC clusters). CONCLUSION: TOGGLE speeds up the creation of robust pipelines with reliable log tracking and data flow, for a large range of analyses. Moreover, it enables Biologists to concentrate on the biological relevance of results, and change the experimental conditions easily. The whole code and test data are available at https://github.com/SouthGreenPlatform/TOGGLE .

Dominant PDX1 deficiency causes highly penetrant diabetes at different ages, associated with obesity and exocrine pancreatic deficiency: Lessons for precision medicine.

OBJECTIVE: Heterozygous pathogenic or likely pathogenic (P/LP) PDX1 variants cause monogenic diabetes. We comprehensively examined the phenotypes of carriers of P/LP PDX1 variants, and delineated potential treatments that could be efficient in an objective of precision medicine. METHODS: The study primarily involved a family harboring a novel P/LP PDX1 variant. We then conducted an analysis of documented carriers of P/LP PDX1 variants, from the Human Gene Mutation Database (HGMD), RaDiO study, and Type 2 Diabetes Knowledge Portal (T2DKP) including 87 K participants. RESULTS: Within the family, we identified a P/LP PDX1 variant encoding p.G232S in four relatives. All of them exhibited diabetes, albeit with very different ages of onset (10-40 years), along with caudal pancreatic agenesis and childhood-onset obesity. In the HGMD, 79 % of carriers of a P/LP PDX1 variant displayed diabetes (with differing ages of onset from eight days of life to 67 years), 63 % exhibited pancreatic insufficiency and surprisingly 40 % had obesity. The impact of P/LP PDX1 variants on increased risk of type 2 diabetes mellitus was confirmed in the T2DKP. Dipeptidyl peptidase 4 inhibitor (DPP4i) and glucagon-like peptide-1 receptor agonist (GLP1-RA), enabled good glucose control without hypoglycemia and weight management. CONCLUSIONS: This study reveals diverse clinical presentations among the carriers of a P/LP PDX1 variant, highlighting strong variations in diabetes onset, and unexpectedly high prevalence of obesity and pancreatic development abnormalities. Clinical data suggest that DPP4i and GLP1-RA may be the best effective treatments to manage both glucose and weight controls, opening new avenue in precision diabetic medicine.

Exploring the role of purinergic receptor P2RY1 in type 2 diabetes risk and pathophysiology: Insights from human functional genomics.

OBJECTIVE: Human functional genomics has proven powerful in discovering drug targets for common metabolic disorders. Through this approach, we investigated the involvement of the purinergic receptor P2RY1 in type 2 diabetes (T2D). METHODS: P2RY1 was sequenced in 9,266 participants including 4,177 patients with T2D. In vitro analyses were then performed to assess the functional effect of each variant. Expression quantitative trait loci (eQTL) analysis was performed in pancreatic islets from 103 pancreatectomized individuals. The effect of P2RY1 on glucose-stimulated insulin secretion was finally assessed in human pancreatic beta cells (EndoCßH5), and RNA sequencing was performed on these cells. RESULTS: Sequencing P2YR1 in 9,266 participants revealed 22 rare variants, seven of which were loss-of-function according to our in vitro analyses. Carriers, except one, exhibited impaired glucose control. Our eQTL analysis of human islets identified P2RY1 variants, in a beta-cell enhancer, linked to increased P2RY1 expression and reduced T2D risk, contrasting with variants located in a silent region associated with decreased P2RY1 expression and increased T2D risk. Additionally, a P2RY1-specific agonist increased insulin secretion upon glucose stimulation, while the antagonist led to decreased insulin secretion. RNA-seq highlighted TXNIP as one of the main transcriptomic markers of insulin secretion triggered by P2RY1 agonist. CONCLUSION: Our findings suggest that P2RY1 inherited or acquired dysfunction increases T2D risk and that P2RY1 activation stimulates insulin secretion. Selective P2RY1 agonists, impermeable to the blood-brain barrier, could serve as potential insulin secretagogues.

Knocking Down CDKN2A in 3D hiPSC-Derived Brown Adipose Progenitors Potentiates Differentiation, Oxidative Metabolism and Browning Process.

Human induced pluripotent stem cells (hiPSCs) have the potential to be differentiated into any cell type, making them a relevant tool for therapeutic purposes such as cell-based therapies. In particular, they show great promise for obesity treatment as they represent an unlimited source of brown/beige adipose progenitors (hiPSC-BAPs). However, the low brown/beige adipocyte differentiation potential in 2D cultures represents a strong limitation for clinical use. In adipose tissue, besides its cell cycle regulator functions, the cyclin-dependent kinase inhibitor 2A (CDKN2A) locus modulates the commitment of stem cells to the brown-like type fate, mature adipocyte energy metabolism and the browning of adipose tissue. Here, using a new method of hiPSC-BAPs 3D culture, via the formation of an organoid-like structure, we silenced CDKN2A expression during hiPSC-BAP adipogenic differentiation and observed that knocking down CDKN2A potentiates adipogenesis, oxidative metabolism and the browning process, resulting in brown-like adipocytes by promoting UCP1 expression and beiging markers. Our results suggest that modulating CDKN2A levels could be relevant for hiPSC-BAPs cell-based therapies.

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.

ß-Cell-Specific E2f1 Deficiency Impairs Glucose Homeostasis, ß-Cell Identity, and Insulin Secretion.

The loss of pancreatic ß-cell identity has emerged as an important feature of type 2 diabetes development, but the molecular mechanisms are still elusive. Here, we explore the cell-autonomous role of the cell-cycle regulator and transcription factor E2F1 in the maintenance of ß-cell identity, insulin secretion, and glucose homeostasis. We show that the ß-cell-specific loss of E2f1 function in mice triggers glucose intolerance associated with defective insulin secretion, altered endocrine cell mass, downregulation of many ß-cell genes, and concomitant increase of non-ß-cell markers. Mechanistically, epigenomic profiling of the promoters of these non-ß-cell upregulated genes identified an enrichment of bivalent H3K4me3/H3K27me3 or H3K27me3 marks. Conversely, promoters of downregulated genes were enriched in active chromatin H3K4me3 and H3K27ac histone marks. We find that specific E2f1 transcriptional, cistromic, and epigenomic signatures are associated with these ß-cell dysfunctions, with E2F1 directly regulating several ß-cell genes at the chromatin level. Finally, the pharmacological inhibition of E2F transcriptional activity in human islets also impairs insulin secretion and the expression of ß-cell identity genes. Our data suggest that E2F1 is critical for maintaining ß-cell identity and function through sustained control of ß-cell and non-ß-cell transcriptional programs. ARTICLE HIGHLIGHTS: ß-Cell-specific E2f1 deficiency in mice impairs glucose tolerance. Loss of E2f1 function alters the ratio of α- to ß-cells but does not trigger ß-cell conversion into α-cells. Pharmacological inhibition of E2F activity inhibits glucose-stimulated insulin secretion and alters ß- and α-cell gene expression in human islets. E2F1 maintains ß-cell function and identity through control of transcriptomic and epigenetic programs.

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.

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.