A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

Use an avian cell culture model to analyze effects of endocrine disruptors on the testicular tissue

International audienceIn recent decades, many tests in vivo or in vitro models mainly based on mammalian (rat-mouse) and fish were used to assess the risks raised by contact or ingestion of molecules with an pharmaceutical, agricultural or natural origin. But, none or few in vitro tests using bird models have been explored despite their advantages: the embryonic gonads of birds have a high plasticity of development, and sperm production is nearly two times faster than in rodents. Thus, birds present one gonad in female and two gonads in male. In the presence of estrogenic substances or antagonist of estrogen production, during the first days of the embryonic life, the sex gonadic phenotype could change. Hence, as part of a national project (post-Grenelle Environment Forum), we have established the in vitro culture of germ cells and somatic cells from chicken testis and we have evaluated the sensitivity against two molecules known to be endocrine disruptors: phtalates (mono-(2-ethylhexyl) phthalate, MEHP) and ethinyl estradiol (EE2) in comparison to previous studies using rodent and human system. After 96 h of culture in presence of 10μM MEHP, cultures present a structural alteration, a reduction in cell proliferation and in germ cells population. These results are in accordance with previous studies using rat, mice or humans culture of testicular cells and with similar sensitivity levels or even better sensitivity for some 'end-points' (biological parameters). Moreover, we observed an increase in proliferative germ cells and a decrease in apoptosis or oxydative stress starting at the lower EE2 dosis (0.03 ng/ml, output value of wastewater) to 300 ng/ml. These cells preparations were also able to be frozen and thawed for a latter use, because VASA-positive germ cells were detected after 96h of culture. In conclusion, the establishment of this testicular cells culture could be considered as an in vitro alternative method, complementary to mammals model, limiting the number of animals used and with an elevated sensitivity at least for these two endocrine disruptors

Développement d'outils de diagnostic en reprotoxicologie aviaire

National audienc

Goal oriented random forest (GORF)

International audienc

Goal oriented random forest (GORF)

International audienc

Goal oriented random forest (GORF)

International audienc

Effects of mono-(2-ethylhexyl) phthalate (MEHP) on chicken germ cells cultured in vitro

In recent decades, many toxicological tests based on in vivo or in vitro models, mainly from mammalian (rat-mouse) and fish species, were used to assess the risks raised by contact or ingestion of molecules of pharmaceutical, agricultural, or natural origin. But no, or few, in vitro tests using other non-mammalian models such as bird have been explored despite their advantages: the embryonic gonads of birds have a high plasticity of development sensitive to estrogen, and sperm production is nearly two times faster than in rodents. Hence, we have established an in vitro culture of germ cells and somatic cells from chicken post-natal testis, and we have evaluated the sensitivity against the endocrine disruptor compound mono-(2-ethylhexyl) phthalate (MEHP) in comparison to previous studies using rodent and human models. After 96 h of exposure in presence of 10 μM MEHP, chicken seminiferous tubules cultures present a structural alteration, a reduction in cell proliferation and in germ cells population. Apoptosis of germ and somatic cells increases in presence of 1 μM MEHP. Furthermore, MEHP does not affect inhibin B and lactate production by Sertoli cells. These results are in accordance with previous studies using rat, mice, or human culture of testicular cells and in similar range of exposures or even better sensitivity for some "end-points" (biological parameters). In conclusion, the establishment of this postnatal testicular cells culture could be considered as an alternative method to in vivo experiments frequently used for evaluating the impact on the terrestrial wildlife species. This method could be also complementary to mammal model due to the limiting number of animals used and its elevated sensitivity

Chromosomal microarray analysis in fetuses with an isolated congenital heart defect: A retrospective, nationwide, multicenter study in France

International audienceObjectives Congenital heart defects (CHDs) may be isolated or associated with other malformations. The use of chromosome microarray (CMA) can increase the genetic diagnostic yield for CHDs by between 4% and 10%. The objective of this study was to evaluate the value of CMA after the prenatal diagnosis of an isolated CHD. Methods In a retrospective, nationwide study performed in France, we collected data on all cases of isolated CHD that had been explored using CMAs in 2015. Results A total of 239 fetuses were included and 33 copy number variations (CNVs) were reported; 19 were considered to be pathogenic, six were variants of unknown significance, and eight were benign variants. The anomaly detection rate was 10.4% overall but ranged from 0% to 16.7% as a function of the isolated CHD in question. The known CNVs were 22q11.21 deletions (n = 10), 22q11.21 duplications (n = 2), 8p23 deletions (n = 2), an Alagille syndrome (n = 1), and a Kleefstra syndrome (n = 1). Conclusion The additional diagnostic yield was clinically significant (3.1%), even when anomalies in the 22q11.21 region were not taken into account. Hence, patients with a suspected isolated CHD and a normal karyotype must be screened for chromosome anomalies other than 22q11.21 duplications and deletions

Spectrum of mutations in the renin-angiotensin system genes in autosomal recessive renal tubular dysgenesis

Autosomal recessive renal tubular dysgenesis (RTD) is a severe disorder of renal tubular development characterized by early onset and persistent fetal anuria leading to oligohydramnios and the Potter sequence, associated with skull ossification defects. Early death occurs in most cases from anuria, pulmonary hypoplasia, and refractory arterial hypotension. The disease is linked to mutations in the genes encoding several components of the renin–angiotensin system (RAS): AGT (angiotensinogen), REN (renin), ACE (angiotensin-converting enzyme), and AGTR1 (angiotensin II receptor type 1). Here, we review the series of 54 distinct mutations identified in 48 unrelated families. Most of them are novel and ACE mutations are the most frequent, observed in two-thirds of families (64.6%). The severity of the clinical course was similar whatever the mutated gene, which underlines the importance of a functional RAS in the maintenance of blood pressure and renal blood flow during the life of a human fetus. Renal hypoperfusion, whether genetic or secondary to a variety of diseases, precludes the normal development/ differentiation of proximal tubules. The identification of the disease on the basis of precise clinical and histological analyses and the characterization of the genetic defects allow genetic counseling and early prenatal diagnosis