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

Mutations involving the SRY-related gene SOX8 are associated with a spectrum of human reproductive anomalies.

© The Author(s) 2018. Published by Oxford University Press. All rights reserved. SOX8 is an HMG-box transcription factor closely related to SRY and SOX9. Deletion of the gene encoding Sox8 in mice causes reproductive dysfunction but the role of SOX8 in humans is unknown. Here, we show that SOX8 is expressed in the somatic cells of the early developing gonad in the human and influences human sex determination. We identified two individuals with 46, XY disorders/differences in sex development (DSD) and chromosomal rearrangements encompassing the SOX8 locus and a third individual with 46, XY DSD and a missense mutation in the HMG-box of SOX8. In vitro functional assays indicate that this mutation alters the biological activity of the protein. As an emerging body of evidence suggests that DSDs and infertility can have common etiologies, we also analysed SOX8 in a cohort of infertile men (n=274) and two independent cohorts of women with primary ovarian insufficiency (POI; n=153 and n=104). SOX8 mutations were found at increased frequency in oligozoospermic men (3.5%; P < 0.05) and POI (5.06%; P=4.5×10 -5 ) as compared with fertile/normospermic control populations (0.74%). The mutant proteins identified altered SOX8 biological activity as compared with the wild-type protein. These data demonstrate that SOX8 plays an important role in human reproduction and SOX8 mutations contribute to a spectrum of phenotypes including 46, XY DSD, male infertility and 46, XX POI.Link_to_subscribed_fulltex

Genetic study of gonadal development in human

Les anomalies du développement sexuel recouvrent un spectre phénotypique large. Les hommes XX présentent dans la majorité des cas un développement testiculaire normal, lié à la présence SRY sur un des chromosomes X. Dans 10% des cas, aucune cause n’est retrouvée. Chez les femmes, l’origine de l’insuffisance ovarienne prématurée (IOP) n’est identifiée que dans 20% des cas. L’objectif de cette thèse a été d’identifier de nouveaux mécanismes moléculaires impliqués dans le développement gonadique, testiculaire et ovarien, ainsi que dans son fonctionnement. L’étude d’une cohorte de patients DSD 46,XX SRY négatifs a mis en évidence une duplication de la région RevSex dans un désert génique en amont de SOX9 chez quatre patients. Ceci a permis de redéfinir la région minimale impliquée dans l'activation de l'expression de SOX9 à une taille maximale de 41.9 kb et de proposer un mécanisme permettant cette expression chez des hommes XX. Le séquençage d’exome chez dix patients de la cohorte n’a pas mis en évidence de mutations dans des gènes d’intérêts. L’ensemble de ces résultats pose la question du rôle des régions régulatrices dans la survenue des DSD.L’analyse d’une cohorte de patientes ayant une IOP a permis d'identifier des délétions incluant le gène CPEB1. Des études précédentes chez la souris ont montré son implication dans le développement folliculaire. Le séquençage du gène CPEB1 dans la cohorte n'a pas mis en évidence de mutation pathogène. Ce travail a permis de montrer que la délétion impliquant le gène CPEB1est une cause rare mais récurrente d'IOP et concerne environ 1% des patientes. Une microdélétion contenant le gène CASP3 un gène de la voie des caspases impliquée dans la régulation du pool folliculaire a également été identifiée chez une patiente. L'ensemble de ces résultats montre l'intérêt de l'étude génétique des patients présentant une anomalie du développement de la gonade ou de son fonctionnement par des techniques d'étude globale du génome.Disorders of Sex Development (DSD) can be identified in new-born and during infancy but also in adults because of infertility. Most 46,XX testicular DSD have a normal testicular development due to the presence of the SRY gene at the tip of one of their X chromosome. However, the genetic causes of 46,XX-SRY negative testicular DSD remain poorly defined. In women, disorders of gonadal development can be responsible for primary ovarian insufficiency (POI) and genetic causes are identify in only 20% of cases. The aim of this thesis was to identify molecular mechanisms involved in gonadal development and in its functioning. The cohort study of 46,XX testicular DSD identified four patients with a duplication in the previously reported RevSex region located about 550 kb upstream of SOX9. One duplication allowed us to refine the minimal region associated with 46,XX-SRY negative DSD to a 40.7–41.9 kb element. Exome sequencing of 10 patients from the cohort did not show any mutation in genes implicated in DSD or in new candidate genes. These results raise questions about the role of the regulatory sequences in the onset of DSD.The cohort study of POI patients identified three patients carrying a microdeletion including CPEB1 a good candidate gene for POI as study in mice showed the implication of CPEB1 in follicular development. Sequencing CPEB1 gene did not identified any mutation. Therefore, heterozygous deletion of CPEB1 gene leading to haploinsufficiency could be responsible for POI in humans. This microdeletion is rare but recurrent and was identified in about 1% of patients with POI. Another microdeletion containing CASP3 gene that belongs to the caspase family, which is implicated in the regulation of the follicular pool, was identified in a patient. Further studies are needed to confirm the role of CASP3 in POI. These results demonstrate the importance of genetic study of patients presenting with DSD or POI using whole genome techniques

Etude des gènes impliqués dans le déterminisme gonadique chez l'homme

Disorders of Sex Development (DSD) can be identified in new-born and during infancy but also in adults because of infertility. Most 46,XX testicular DSD have a normal testicular development due to the presence of the SRY gene at the tip of one of their X chromosome. However, the genetic causes of 46,XX-SRY negative testicular DSD remain poorly defined. In women, disorders of gonadal development can be responsible for primary ovarian insufficiency (POI) and genetic causes are identify in only 20% of cases. The aim of this thesis was to identify molecular mechanisms involved in gonadal development and in its functioning. The cohort study of 46,XX testicular DSD identified four patients with a duplication in the previously reported RevSex region located about 550 kb upstream of SOX9. One duplication allowed us to refine the minimal region associated with 46,XX-SRY negative DSD to a 40.7–41.9 kb element. Exome sequencing of 10 patients from the cohort did not show any mutation in genes implicated in DSD or in new candidate genes. These results raise questions about the role of the regulatory sequences in the onset of DSD.The cohort study of POI patients identified three patients carrying a microdeletion including CPEB1 a good candidate gene for POI as study in mice showed the implication of CPEB1 in follicular development. Sequencing CPEB1 gene did not identified any mutation. Therefore, heterozygous deletion of CPEB1 gene leading to haploinsufficiency could be responsible for POI in humans. This microdeletion is rare but recurrent and was identified in about 1% of patients with POI. Another microdeletion containing CASP3 gene that belongs to the caspase family, which is implicated in the regulation of the follicular pool, was identified in a patient. Further studies are needed to confirm the role of CASP3 in POI. These results demonstrate the importance of genetic study of patients presenting with DSD or POI using whole genome techniques.Les anomalies du développement sexuel recouvrent un spectre phénotypique large. Les hommes XX présentent dans la majorité des cas un développement testiculaire normal, lié à la présence SRY sur un des chromosomes X. Dans 10% des cas, aucune cause n’est retrouvée. Chez les femmes, l’origine de l’insuffisance ovarienne prématurée (IOP) n’est identifiée que dans 20% des cas. L’objectif de cette thèse a été d’identifier de nouveaux mécanismes moléculaires impliqués dans le développement gonadique, testiculaire et ovarien, ainsi que dans son fonctionnement. L’étude d’une cohorte de patients DSD 46,XX SRY négatifs a mis en évidence une duplication de la région RevSex dans un désert génique en amont de SOX9 chez quatre patients. Ceci a permis de redéfinir la région minimale impliquée dans l'activation de l'expression de SOX9 à une taille maximale de 41.9 kb et de proposer un mécanisme permettant cette expression chez des hommes XX. Le séquençage d’exome chez dix patients de la cohorte n’a pas mis en évidence de mutations dans des gènes d’intérêts. L’ensemble de ces résultats pose la question du rôle des régions régulatrices dans la survenue des DSD.L’analyse d’une cohorte de patientes ayant une IOP a permis d'identifier des délétions incluant le gène CPEB1. Des études précédentes chez la souris ont montré son implication dans le développement folliculaire. Le séquençage du gène CPEB1 dans la cohorte n'a pas mis en évidence de mutation pathogène. Ce travail a permis de montrer que la délétion impliquant le gène CPEB1est une cause rare mais récurrente d'IOP et concerne environ 1% des patientes. Une microdélétion contenant le gène CASP3 un gène de la voie des caspases impliquée dans la régulation du pool folliculaire a également été identifiée chez une patiente. L'ensemble de ces résultats montre l'intérêt de l'étude génétique des patients présentant une anomalie du développement de la gonade ou de son fonctionnement par des techniques d'étude globale du génome

Apport de l'analyse chromosomique sur puce à ADN (ACPA) dans le diagnostic étiologique de l'insuffisance ovarienne prématurée

L'insuffisance ovarienne prématurée (IOP) correspond à la survenue d'une aménorrhée primaire ou secondaire avant l'âge de 40 ans. Les causes identifiées sont nombreuses : causes immunologiques, iatrogènes, environnementales et génétiques mais dans la majorité des cas aucune origine n'est trouvée. Afin d'identifier l'étiologie de l'IOP, des études génétiques sont réalisées et en particulier le caryotype. Cet outil de génétique a permis de mettre en évidence de nombreux remaniements chromosomiques et d'identifier de nouvelles causes génétiques d'IOP. Le développement des outils de cytogénétique innovants tels que l'analyse chromosomique sur puce à ADN (ACPA) a permis de mettre en évidence de nouveaux gènes candidats et d'identifier un nouveau syndrome microdélétionnel situé en 15q25.2 et impliquant le gène CPEB1 responsable de près de 1,5 % des cas d'IOP

Recherche de microremaniements chromosomiques par CGH array chez des foetus présentant une hernie diaphragmatique

Les hernies diaphragmatiques congénitales (HCD) surviennent dans environ 1/4000 naissances. L origine génétique semble jouer un rôle important dans le développement de ces HCD. En effet, certains gènes sont connus pour être responsable de HCD chez l Homme et des anomalies chromosomiques récurrentes ont été mises en évidence dans les cas de HCD. Nous avons donc étudié par CGH array 50 foetus présentant une hernie diaphragmatique syndromique ou non dans le but de mettre en évidence de nouveaux remaniements chromosomiques et de nouveaux gènes candidats. Notre étude a révélé la présence de trois remaniements chromosomiques : une délétion 15qter déjà connue comme facteur étiologique, une délétion intragénique du gène PTPRD en 9p23,héritée d un parent sain, qui chez la souris est responsable d anomalies de développement du diaphragme et une duplication 17pter dont l implication dans la survenue de la HCD reste à explorer. Ce travail confirme l intérêt de la CGH array comme outil diagnostic.Congenital diaphragmatic hernia (CDH) is a common major malformation affecting 1/4000 births; Althoug the exact etiology of most cases of CDH remains unknown, it is becoming increasingly clear that genetic factors play an important role. The data supporting genetic etiologies include single gene disorders associated with CDH and the association of CDH with recurring chromosome abnormalities. The aim of this work was to study 50 foetuses with syndromic CDH or isolated CDH array CGH to identify and map new chromosome aberrations in this disease. We identified three different chromosome aberrations : 15q26 deletion which is well known to be implicated in CDH, PTPRD intragenic deletion located on chomosome 9p23 inherited from a healthy parent, responsible for abnormal diaphragmatic development in mouse models and 17 pter duplication which implication requires further exploration.ST QUENTIN EN YVELINES-BU (782972101) / SudocSudocFranceF

First Birth after Sperm Selection through Discontinuous Gradient Centrifugation and Artificial Insemination from a Chromosomal Translocation Carrier

Introduction. Balanced chromosomal carriers, though usually healthy, are confronted with recurrent spontaneous abortions and malformations in the offspring. Those are related to the transmission of an abnormal, chromosomally unbalanced genotype. We evidenced that the proportion of unbalanced spermatozoa can be significantly decreased through a sperm preparation process called discontinuous gradient centrifugation (DGC). We therefore started offering intrauterine inseminations with this procedure to couples with a male translocation carriers. Case Presentation. We report the case of a 37-year-old man carrying a t(3;10)(q25;p13) reciprocal translocation. He and his partner had had trouble conceiving for ten years and had four spontaneous abortions. DGC in this patient decreased the proportion of unbalanced spermatozoa from 63.6% to 52.3%. They were therefore offered intrauterine insemination with DGC, which eventually led to the birth of a healthy female child carrying the paternal translocation. Conclusion. We showed that translocation carriers could be offered intrauterine inseminations with DGC. Before this, the only two options were natural conception with prenatal diagnosis and termination of chromosomally unbalanced fetuses or preimplantation genetic diagnosis, which is a much heavier and costly procedure. We are currently offering this option through a multicentric program in France, and this is the first birth originating from it

A Novel 3q29 Deletion in Association With Developmental Delay and Heart Malformation—Case Report With Literature Review

International audience3q29 deletion syndrome is a rare disorder, causing a complex phenotype. Clinical features are variable and relatively non-specific. Our report aims to present an atypical, de novo deletion in chromosome band 3q29 in a preschool boy, first child of healthy non-consanguineous parents, presenting a particular phenotype (microcephaly, “full moon” face, flattened facial profile, large ears, auricular polyp, and dental dystrophies), motor and cognitive delay, characteristics of autism spectrum disorder and aggressive behavior. He also presented intrauterine growth restriction (birth weight 2,400 g) and a ventricular septal defect. SNP Array revealed a 962 kb copy number loss, on the chromosome 3q29 band (195519857–196482211), consistent with 3q29 microdeletion syndrome. FISH analysis using a RP11-252K11 probe confirmed the deletion in the proband, which was not present in the parents. Although the patient's deletion is relatively small, it partly overlaps the canonical 3q29 deletion (defined between TFRC and DLG1 gene) and extends upstream, associating a different facial phenotype compared to the classic 3q29 deletion, nonetheless showing a similar psychiatric disorder. This deletion is different from the canonical region, as it does not include the PAK2 and DLG1 genes, considered as candidates for causing intellectual disability. Thus, narrowing the genotype-phenotype correlation for the 3q29 band, FBX045 is suggested as a candidate gene for the neuropsychiatric phenotype

Recurrent Intragenic Duplication within the <b><i>NR5A1</i></b> Gene and Severe Proximal Hypospadias

International audienceA heterozygous intragenic duplication within the repeated area (CTGCAGCTG)×2 of the NR5A1 gene was found in a 15-year-old 46,XY DSD (disorders/differences of sex development) patient with micropenis and severe proximal hypospadias. This heterozygous duplication has already been described twice in boys with a similar phenotype, whereas a deletion of 3 amino acids at the same position in the protein SF-1 has been described in a 46,XX patient with primary ovarian failure and short stature. These data suggest that this region within the NR5A1 gene has an important role for SF-1 protein function in gonads and is a hotspot for intragenic rearrangements

SRY ‐negative 46,XX testicular/ovotesticular DSD: Long‐term outcomes and early blockade of gonadotropic axis

International audienceObjective: SRY-negative 46,XX testicular and ovotesticular disorders/differences of sex development (T/OTDSD) represent a very rare and unique DSD condition where testicular tissue develops in the absence of a Y chromosome. To date, very few studies have described the phenotype, clinical and surgical management and long-term outcomes of these patients. Particularly, early blockade of the gonadotropic axis in patients raised in the female gender to minimize postnatal androgenization has never been reported.Design: Retrospective description of sixteen 46,XX T/OTDSD patients.Results: Sixteen 46,XX SRY-negative T/OTDSD were included. Most (12/16) were diagnosed in the neonatal period. Sex of rearing was male for six patients and female for ten, while the clinical presentation varied, with an external masculinization score from 1 to 10. Five patients raised as girl were successfully treated with GnRH analog to avoid virilization during minipuberty. Ovotestes/testes were found bilaterally for 54% of the patients and unilaterally for the others (with a contralateral ovary). Gonadal surgery preserved appropriate tissue in the majority of cases. Spontaneous puberty occurred in two girls and one boy, while two boys required hormonal induction of puberty. One of the girls conceived spontaneously and had an uneventful pregnancy. DNA analyses (SNP-array, next-generation sequencing and whole-exome sequencing) were performed. A heterozygous frameshit mutation in the NR2F2 gene was identified in one patient.Conclusions: This study presents a population of patients with 46,XX SRY-negative T/OTDSD. Early blockade of gonadotropic axis appears efficient to reduce and avoid further androgenization in patients raised as girls