A truncating variant of RAD51B associated with primary ovarian insufficiency provides insights into its meiotic and somatic functions

Primary ovarian insufficiency (POI) causes female infertility by abolishing normal ovarian function. Although its genetic etiology has been extensively investigated, most POI cases remain unexplained. Using whole-exome sequencing, we identified a homozygous variant in RAD51B –(c.92delT) in two sisters with POI. In vitro studies revealed that this variant leads to translation reinitiation at methionine 64. Here, we show that this is a pathogenic hypomorphic variant in a mouse model. Rad51bc.92delT/c.92delT mice exhibited meiotic DNA repair defects due to RAD51 and HSF2BP/BMRE1 accumulation in the chromosome axes leading to a reduction in the number of crossovers. Interestingly, the interaction of RAD51B-c.92delT with RAD51C and with its newly identified interactors RAD51 and HELQ was abrogated or diminished. Repair of mitomycin-C-induced chromosomal aberrations was impaired in RAD51B/Rad51b-c.92delT human and mouse somatic cells in vitro and in explanted mouse bone marrow cells. Accordingly, Rad51b-c.92delT variant reduced replication fork progression of patient-derived lymphoblastoid cell lines and pluripotent reprogramming efficiency of primary mouse embryonic fibroblasts. Finally, Rad51bc.92delT/c.92delT mice displayed increased incidence of pituitary gland hyperplasia. These results provide new mechanistic insights into the role of RAD51B not only in meiosis but in the maintenance of somatic genome stability.This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Grant 2014/14231-0 (to MMF); FAPESP Grant 2013/02162-8, Nucleo de Estudos e Terapia Celular e Molecular (NETCEM), Conselho Nacional de Desenvolvimento Científico e Tecnológico Grant 303002/2016- 6 (to BBM); and FAPESP Grant 2014/50137-5 (to SELA). This work was supported by MINECO (PID2020-120326RB-I00) and by Junta de Castilla y León (CSI239P18 and CSI148P20). NFM, FSS, and MRMH are supported by European Social Fund/JCyLe grants (EDU/310/2015, EDU/556/2019 and EDU/1992/2020). YBC and RSU are funded by a grant from MINECO (BS-2015–073993 and BFU2017-89408-R). Experiments performed at CNIO were supported by grant PID2019-106707-RB to JM, co-sponsored by EU ERDF funds. SM was supported by an international postdoctoral contract “CNIO Friends”. The proteomic analysis was performed in the Proteomics Facility of Centro de Investigación del Cáncer, Salamanca, Grant PRB3(IPT17/0019 -ISCIII-SGEFI/ERDF). CIC-IBMCC is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia cofunded by the Castilla–León autonomous government and the European Regional Development Fund (CLC–2017–01). Veitia’s Lab is supported by the University of Paris and the Centre National de la Recherche Scientifique

Loss of Function of the Nuclear Receptor NR2F2, Encoding COUP-TF2, Causes Testis Development and Cardiac Defects in 46,XX Children

Emerging evidence from murine studies suggests that mammalian sex determination is the outcome of an imbalance between mutually antagonistic male and female regulatory networks that canalize development down one pathway while actively repressing the other. However, in contrast to testis formation, the gene regulatory pathways governing mammalian ovary development have remained elusive. We performed exome or Sanger sequencing on 79 46,XX SRY-negative individuals with either unexplained virilization or with testicular/ovotesticular disorders/differences of sex development (TDSD/OTDSD). We identified heterozygous frameshift mutations in NR2F2, encoding COUP-TF2, in three children. One carried a c.103_109delGGCGCCC (p.Gly35Argfs( *)75) mutation, while two others carried a c.97_103delCCGCCCG (p.Pro33Alafs( *)77) mutation. In two of three children the mutation was de novo. All three children presented with congenital heart disease (CHD), one child with congenital diaphragmatic hernia (CDH), and two children with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES). The three children had androgen production, virilization of external genitalia, and biochemical or histological evidence of testicular tissue. We demonstrate a highly significant association between the NR2F2 loss-of-function mutations and this syndromic form of DSD (p = 2.44 x 10(-8)). We show that COUP-TF2 is highly abundant in a FOXL2-negative stromal cell population of the fetal human ovary. In contrast to the mouse, these data establish COUP-TF2 as a human "pro-ovary" and "anti-testis" sex-determining factor in female gonads. Furthermore, the data presented here provide additional evidence of the emerging importance of nuclear receptors in establishing human ovarian identity and indicate that nuclear receptors may have divergent functions in mouse and human biology

Genetics of 46,XY gonadal dysgenesis

International audienceIn 46,XY men, testis is determined by a genetic network(s) that both promotes testis formation and represses ovarian development. Disruption of this process results in a lack of testis-determination and affected individuals present with 46,XY gonadal dysgenesis (GD), a part of the spectrum of Disorders/Differences of Sex Development/Determination (DSD). A minority of all cases of GD are associated with pathogenic variants in key players of testis-determination, SRY, SOX9, MAP3K1 and NR5A1. However, most of the cases remain unexplained. Recently, unbiased exome sequencing approaches have revealed new genes and loci that may cause 46,XY GD. We critically evaluate the evidence to support causality of these factors and describe how functional studies are continuing to improve our understanding of genotype-phenotype relationships in genes that are established causes of GD. As genomic data continues to be generated from DSD cohorts, we propose several recommendations to help interpret the data and establish causality

The genetic make-up of ovarian development and function: the focus on the transcription factor FOXL2.

International audienceIn a 46 XY individual, the presence of the Y chromosome harboring the testis-determining factor (SRY) triggers testis determination and differentiation. In a 46 XX individual, the absence of SRY and the activation of genes associated with the female pathway lead to ovarian development. The latter process has long been considered as a default pathway. However, recent studies have cast doubts on this dogma. Here, after a brief overview of the main steps of ovarian development, we focus on three genes WNT4, RSPO1 and FOXL2 that are essential for ovarian determination, differentiation and/or maintenance. Special attention is paid to FOXL2 whose mutations are responsible for the blepharophimosis syndrome, often associated with female infertility, and for cancer. We highlight the cooperation of WNT4, RSPO1 and FOXL2 within a regulatory network and the need for further research to better understand their role in defining and maintaining ovarian identity

Fibroblast growth factor 23 in children with or without heart failure: a prospective study

Background Elevated fibroblast growth factor 23 (FGF23) levels have been associated with mortality in adults with heart failure (HF), but data on FGF23 levels in paediatric HF are limited. In this prospective cohort study, we aimed to assess the prognostic value of FGF23 in children with chronic HF.Methods We prospectively enrolled 40 children with chronic HF and 20 matched healthy controls. In each patient, a complete diagnostic workup was performed, including transthoracic echocardiography to evaluate cardiac systolic and diastolic functions. Serum FGF23, renal function tests, parathyroid hormone, serum calcium and phosphate were measured in patients and controls. N-terminal probrain natriuretic peptide (NT-proBNP) was measured in patients. The severity of symptoms was assessed using the modified Ross HF classification for children. Patients were followed for 1 year, and clinical worsening events such as death and HF hospitalisation were recorded.Results Patients with HF had significantly higher FGF23 levels compared with controls (355.68±97.27 pg/mL and 60.20±11.04 pg/mL, respectively; p<0.001). Three patients died and 11 were admitted with HF. In comparison with patients without clinical worsening events, these 14 patients exhibited significantly higher FGF23 levels (320.04±89.56 pg/mL and 421.86±75.50 pg/mL, respectively; p<0.001). FGF23 was positively correlated with NT-proBNP and left ventricular end-diastolic diameter and negatively correlated with ejection fraction and fractional shortening. The ability of FGF23 to predict clinical worsening events in patients was analysed using a receiver operating characteristic curve. The optimal cut-off point was 375 pg/mL, with 85.71% sensitivity, 84.62% specificity, positive predictive value of 75.0, negative predictive value of 91.7 and area under the curve (AUC) of 0.878. Multivariable regression analysis revealed that FGF23 is the only independent predictor of clinical worsening events in children with chronic HF.Conclusion FGF23 levels were elevated in children with chronic HF and increased significantly as Ross score class increased. FGF23 levels increased in patients who experienced clinical worsening events

A homozygous donor splice-site mutation in the meiotic gene MSH4 causes primary ovarian insufficiency

Premature ovarian insufficiency (POI) is a frequent pathology that affects women under 40 years of age, characterized by an early cessation of menses and high FSH levels. Despite recent progresses in molecular diagnosis, the etiology of POI remains idiopathic in most cases. Whole-exome sequencing of members of a Colombian family affected by POI allowed us to identify a novel homozygous donor splice-site mutation in the meiotic gene MSH4 (MutS Homolog 4). The variant followed a strict mendelian segregation within the family and was absent in a cohort of 135 women over 50 years of age without history of infertility, from the same geographical region as the affected family. Exon trapping experiments showed that the splice-site mutation induced skipping of exon 17. At the protein level, the mutation p.Ile743_Lys785del is predicted to lead to the ablation of the highly conserved Walker B motif of the ATP-binding domain, thus inactivating MSH4. Our study describes the first MSH4 mutation associated with POI and increases the number of meiotic/DNA repair genes formally implicated as being responsible for this condition. © The Author 2017. Published by Oxford University Press. All rights reserved

A homozygous donor splice-site mutation in the meiotic gene MSH4 causes primary ovarian insufficiency

Premature ovarian insufficiency (POI) is a frequent pathology that affects women under 40 years of age, characterized by an early cessation of menses and high FSH levels. Despite recent progresses in molecular diagnosis, the etiology of POI remains idiopathic in most cases. Whole-exome sequencing of members of a Colombian family affected by POI allowed us to identify a novel homozygous donor splice-site mutation in the meiotic gene MSH4 (MutS Homolog 4). The variant followed a strict mendelian segregation within the family and was absent in a cohort of 135 women over 50 years of age without history of infertility, from the same geographical region as the affected family. Exon trapping experiments showed that the splice-site mutation induced skipping of exon 17. At the protein level, the mutation p.Ile743_Lys785del is predicted to lead to the ablation of the highly conserved Walker B motif of the ATP-binding domain, thus inactivating MSH4. Our study describes the first MSH4 mutation associated with POI and increases the number of meiotic/DNA repair genes formally implicated as being responsible for this condition. © The Author 2017. Published by Oxford University Press. All rights reserved

DHH pathogenic variants involved in 46,XY disorders of sex development differentially impact protein self-cleavage and structural conformation

International audienceIn humans, pathogenic variants in the DHH gene underlie cases of 46,XY gonadal dysgenesis. DHH is part of the Hedgehog family of proteins, which require extensive processing, including self-cleavage of the precursor for efficient signalling. In our work, we have assessed the effect of several human DHH pathogenic variants involved in recessive complete or partial gonadal dysgenesis, on protein processing and sub-cellular localization. We found that a subset of variants was unable to perform self-cleavage, which correlated albeit not perfectly with an altered subcellular localization of the resulting proteins. For the processing-proficient variants, we used structural modelling tools and molecular dynamic (MD) simulations to predict the potential impact of the variants on protein conformation and/or interaction with partners. Our study contributes to a better understanding of the molecular mechanisms involved in DHH dysfunction leading to 46,XY disorders of sex development