Iandry RABEARIVELO

CoDE-seq, an augmented whole-exome sequencing, enables the accurate detection of CNVs and mutations in Mendelian obesity and intellectual disability. Montagne & al. - Molecular Metabolism - May 2018

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.

Loss-of-function mutations in ADCY3 cause monogenic severe obesity. Saeed & al. - Nature Genetics - January 2018

Study of monogenic forms of obesity has demonstrated the pivotal role of the central leptin-melanocortin pathway in controlling energy balance, appetite and body weight 1 . The majority of loss-of-function mutations (mostly recessive or co-dominant) have been identified in genes that are directly involved in leptin-melanocortin signaling. These genes, however, only explain obesity in <5% of cases, predominantly from outbred populations 2 . We previously showed that, in a consanguineous population in Pakistan, recessive mutations in known obesity-related genes explain ~30% of cases with severe obesity3-5. These data suggested that new monogenic forms of obesity could also be identified in this population. Here we identify and functionally characterize homozygous mutations in the ADCY3 gene encoding adenylate cyclase 3 in children with severe obesity from consanguineous Pakistani families, as well as compound heterozygous mutations in a severely obese child of European-American descent. These findings highlight ADCY3 as an important mediator of energy homeostasis and an attractive pharmacological target in the treatment of obesity.

Increased hepatic PDGF-AA signaling mediates liver insulin resistance in obesity associated type 2 diabetes. Abderrahmani & al. - December 2017

Type 2 diabetes (T2D) is closely linked with non-alcoholic fatty liver disease (NAFLD) and hepatic insulin resistance, but the involved mechanisms are still elusive. Using DNA methylome and transcriptome analyses of livers from obese individuals, we found that both hypomethylation at a CpG site in PDGFA (encoding platelet derived growth factor alpha) and PDGFA overexpression are associated with increased T2D risk, hyperinsulinemia, increased insulin resistance and increased steatohepatitis risk. Both genetic risk score studies and human cell modeling pointed to a causative impact of high insulin levels on PDGFA CpG site hypomethylation, PDGFA overexpression, and increased PDGF-AA secretion from liver. We found that PDGF-AA secretion further stimulates its own expression through protein kinase C activity and contributes to insulin resistance through decreased expression of both insulin receptor substrate 1 and of insulin receptor. Importantly, hepatocyte insulin sensitivity can be restored by PDGF-AA blocking antibodies, PDGF receptor inhibitors and by metformin opening therapeutic avenues. Conclusion: Therefore, in the liver of obese patients with T2D, the increased PDGF-AA signaling contributes to insulin resistance, opening new therapeutic avenues against T2D and NAFLD.

Cdkn2a deficiency promotes adipose tissue browning. Rabhi & al. - Molecular Metabolism - December 2017

Objectives: Genome-wide association studies have reported that DNA polymorphisms at the CDKN2A locus modulate fasting glucose in human and contribute to type 2 diabetes (T2D) risk. Yet the causal relationship between this gene and defective energy homeostasis remains elusive. Here we sought to understand the contribution of Cdkn2a to metabolic homeostasis.
Methods: We first analyzed glucose and energy homeostasis from Cdkn2a-deficient mice subjected to normal or high fat diets. Subsequently Cdkn2a-deficient primary adipose cells and human-induced pluripotent stem differentiated into adipocytes were further characterized for their capacity to promote browning of adipose tissue. Finally CDKN2A levels were studied in adipocytes from lean and obese patients.
Results: We report that Cdkn2a deficiency protects mice against high fat diet-induced obesity, increases energy expenditure and modulates adaptive thermogenesis, in addition to improving insulin sensitivity. Disruption of Cdkn2a associates with increased expression of brown-like/beige fat markers in inguinal adipose tissue and enhances respiration in primary adipose cells. Kinase activity profiling and RNA-sequencing analysis of primary adipose cells further demonstrate that Cdkn2a modulates gene networks involved in energy production and lipid metabolism, through the activation of the Protein Kinase A (PKA), PKG, PPARGC1A and PRDM16 signaling pathways, key regulators of adipocyte beiging. Importantly, CDKN2A expression is increased in adipocytes from obese compared to lean subjects. Moreover silencing CDKN2A expression during human-induced pluripotent stem cells adipogenic differentiation promoted UCP1 expression.
Conclusion: Our results offer novel insight into brown/beige adipocyte functions, which has recently emerged as an attractive therapeutic strategy for obesity and T2D. Modulating Cdkn2a-regulated signaling cascades may be of interest for the treatment of metabolic disorders.

High Prevalence of Rare Monogenic Forms of Obesity in Obese Guadeloupean Afro-Caribbean Children. Foucan & al. - The Journal of Clinical Endocrinology & Metabolism - December 2017

Context: The population from Guadeloupe Island exhibits high prevalence of obesity.
Objective: We aimed to investigate whether rare genetic mutations in genes involved in monogenic obesity (or diabetes) might be causal in this population of Afro-Caribbean ancestry.
Design and setting: This was a secondary analysis of a study on obesity conducted in schoolchildren from Guadeloupe in 2013, which aimed to assess the changes in children profiles after a lifestyle intervention program. Through next-generation sequencing, we sequenced coding regions of 59 genes involved in monogenic obesity or diabetes, in participants from this study. Participants and interventions A total of 25 obese schoolchildren from Guadeloupe were screened for rare mutations (non-synonymous, splice-site, or insertion/deletion) in 59 genes. Main outcome measures Correlation between phenotypes and mutations of interest.
Results: We detected five rare heterozygous mutations in five different obese children: MC4R p.Ile301Thr and SIM1 p.Val326Thrfs*43 mutations which were pathogenic; SIM1 p.Ser343Pro and SH2B1 p.Pro90His mutations which were likely pathogenic; and NTRK2 p.Leu140Phe that was of uncertain significance. In parallel, we identified seven carriers of mutation in ABCC8 (p.Lys1521Asn and p.Ala625Val) or KCNJ11 (p.Val13Met and p.Val151Met), which were of uncertain significance.
Conclusions: We were able to detect pathogenic or likely pathogenic mutations linked to severe obesity in >15% of this population, which is much higher than what we observed in Europeans (~5%).

Expression and functional assessment of candidate type 2 diabetes susceptibility genes identify four new genes contributing to human insulin secretion. Ndiaye & al. - Molecular Metabolism - April 2017

Genome-wide association studies (GWAS) have identified >100 loci independently contributing to type 2 diabetes (T2D) risk. However, translational implications for precision medicine and for the development of novel treatments have been disappointing, due to poor knowledge of how these loci impact T2D pathophysiology. Here, we aimed to measure the expression of genes located nearby T2D associated signals and to assess their effect on insulin secretion from pancreatic beta cells.

What is the best NGS enrichment method for the molecular diagnosis of monogenic diabetes and obesity? Philippe & al. - PLOS One - November 2015

Molecular diagnosis of monogenic diabetes and obesity is of paramount importance for both the patient and society, as it can result in personalized medicine associated with a better life and it eventually saves health care spending. Genetic clinical laboratories are currently switching from Sanger sequencing to next-generation sequencing (NGS) approaches but choosing the optimal protocols is not easy. Here, we compared the sequencing coverage of 43 genes involved in monogenic forms of diabetes and obesity, and variant detection rates, resulting from four enrichment methods based on the sonication of DNA (Agilent SureSelect, RainDance technologies), or using enzymes for DNA fragmentation (Illumina Nextera, Agilent HaloPlex). We analyzed coding exons and untranslated regions of the 43 genes involved in monogenic diabetes and obesity. We found that none of the methods achieves yet full sequencing of the gene targets. Nonetheless, the RainDance, SureSelect and HaloPlex enrichment methods led to the best sequencing coverage of the targets; while the Nextera method resulted in the poorest sequencing coverage. Although the sequencing coverage was high, we unexpectedly found that the HaloPlex method missed 20% of variants detected by the three other methods and Nextera missed 10%. The question of which NGS technique for genetic diagnosis yields the highest diagnosis rate is frequently discussed in the literature and the response is still unclear. Here, we showed that the RainDance enrichment method as well as SureSelect, which are both based on the sonication of DNA, resulted in a good sequencing quality and variant detection, while the use of enzymes to fragment DNA (HaloPlex or Nextera) might not be the best strategy to get an accurate sequencing.

Highly Sensitive Diagnosis of 43 Monogenic Forms of Diabetes or Obesity Through One-Step PCR-Based Enrichment in Combination With Next-Generation Sequencing. Bonnefond & al. - Diabetes Care - February 2014

Objective: Accurate etiological diagnosis of monogenic forms of diabetes and obesity is useful as it can lead to marked improvements in patient care and genetic counseling. Currently, molecular diagnosis based on Sanger sequencing is restricted to only a few genes, as this technology is expensive, time-consuming, and labor-intensive. High-throughput next-generation sequencing (NGS) provides an opportunity to develop innovative cost-efficient methods for sensitive diabetes and obesity multigene screening.
Research Design and Methods: We assessed a new method based on PCR enrichment in microdroplets (RainDance Technologies) and NGS using the Illumina HiSeq2000 for the molecular diagnosis of 43 forms of monogenic diabetes or obesity. Forty patients carrying a known causal mutation for those subtypes according to diagnostic laboratories were blindly reanalyzed.
Results: Except for one variant, we reidentified all causal mutations in each patient associated with an almost-perfect sequencing of the targets (mean of 98.6%). We failed to call one highly complex indel, although we identified a dramatic drop of coverage at this locus. In three patients, we detected other mutations with a putatively deleterious effect in addition to those reported by the genetic diagnostic laboratories.
Conclusions: Our NGS approach provides an efficient means of highly sensitive screening for mutations in genes associated with monogenic forms of diabetes and obesity. As cost and time to deliver results have been key barriers to uncovering a molecular cause in the many undiagnosed cases likely to exist, the present methodology should be considered in patients displaying features of monogenic diabetes or obesity.

A pipeLine Dedicated to Oligonucleotides Design (ALDOv1.2). Rabearivelo & al. - July 2012

Poster presented at "Journées Ouvertes en Biologie, Informatique et Mathématiques" JOBIM 2012 in Rennes.
Abstract: Design of primers and probes is an important step in the conception of molecular detection reagents. Bioinformatics is very helpful both for the design of these oligonucleotides and to validate candidates in silico before in vitro testing, which is an expensive and timeconsuming task. ALDOv1.2 is a bioinformatics pipeline dedicated to the conception of primers and probes for qPCR applications . Moreover, a part of the pipeline may be used for monitoring assay performance to cope with the emergence of new variants of targeted alleles. To our knowledge, this tool is the first one able to automatically adapt primers and probe sequences in response to new evolving target alleles.

A pipeLine dedicated to Oligonucleotides Design (ALDO) : A Workflow for Molecular Diagnostics Assay Design. Rabearivelo & al. - July 2011

Poster presented at "Journées Ouvertes en Biologie, Informatique et Mathématiques" JOBIM 2011 in Paris.
Abstract: Primers and probes design is an important step of molecular diagnostics (MDx) reagents conception. Bioinformatics is very helpful for the design of these oligonucleotides, but also to validate candidates in silico before in vitro tests, an expensive and time-consuming task. ALDO is a bioinformatics pipeline dedicated to the conception of MDx qPCR primers and probes. ALDO was applied to Influenza primers and probes design and validation.