Case Report: Longitudinal follow-up and testicular sperm extraction in a patient with a pathogenic NR5A1 (SF-1) frameshift variant: p.(Phe70Serfs*5)

BackgroundSteroidogenic factor 1 (SF-1), encoded by the nuclear receptor subfamily 5 group A member 1 (NR5A1) gene, is a transcriptional factor crucial for adrenal and gonadal organogenesis. Pathogenic variants of NR5A1 are responsible for a wide spectrum of phenotypes with autosomal dominant inheritance including disorders of sex development and oligospermia–azoospermia in 46,XY adults. Preservation of fertility remains challenging in these patients.ObjectiveThe aim was to offer fertility preservation at the end of puberty in an NR5A1 mutated patient.Case reportThe patient was born of non-consanguineous parents, with a disorder of sex development, a small genital bud, perineal hypospadias, and gonads in the left labioscrotal fold and the right inguinal region. Neither uterus nor vagina was detected. The karyotype was 46,XY. Anti-Müllerian hormone (AMH) and testosterone levels were low, indicating testicular dysgenesis. The child was raised as a boy. At 9 years old, he presented with precocious puberty treated by triptorelin. At puberty, follicle-stimulating hormone (FSH), luteinising hormone (LH), and testosterone levels increased, whereas AMH, inhibin B, and testicular volume were low, suggesting an impaired Sertoli cell function and a partially preserved Leydig cell function. A genetic study performed at almost 15 years old identified the new frameshift variant NM_004959.5: c.207del p.(Phe70Serfs*5) at a heterozygous state. He was thus addressed for fertility preservation. No sperm cells could be retrieved from three semen collections between the ages of 16 years 4 months and 16 years 10 months. A conventional bilateral testicular biopsy and testicular sperm extraction were performed at 17 years 10 months of age, but no sperm cells were found. Histological analysis revealed an aspect of mosaicism with seminiferous tubules that were either atrophic, with Sertoli cells only, or presenting an arrest of spermatogenesis at the spermatocyte stage.ConclusionWe report a case with a new NR5A1 variant. The fertility preservation protocol proposed at the end of puberty did not allow any sperm retrieval for future parenthood

Aberrant Splicing Is the Pathogenicity Mechanism of the p.Glu314Lys Variant in CYP11A1 Gene

Context: The cholesterol side chain cleavage enzyme (CYP11A1) catalyzes the conversion of cholesterol to pregnenolone, the first rate-limiting step of steroidogenesis. CYP11A1 mutations are associated with primary adrenal insufficiency (PAI) as well as disorders of sex development (DSD) in 46,XY patients.Objective: To define the pathogenicity mechanism for the p.Glu314Lys variant, previously reported, and found in four additional patients with CYP11A1 deficiency.Subjects and Methods: DNA of four patients presenting with delayed PAI and/or 46,XY DSD were studied by Sanger or Massively Parallel sequencing. Three CYP11A1 mutations were characterized in vitro and in silico, and one by mRNA analysis on testicular tissue.Results: All patients were compound heterozygous for the previously described p.Glu314Lys variant. In silico studies predicted this mutation as benign with no effect on splicing but mRNA analysis found that it led to incomplete exon 5 skipping. This mechanism was confirmed by minigene experiment. The protein carrying this mutation without exon skipping should conserve almost normal activity, according to in vitro studies. Two other mutations found in trans, the p.Arg120Gln and p.Arg465Trp, had similar activity compared to negative control, consistent with the in silico studies.Conclusions: We provide biological proof that the p. Glu314Lys variant is pathogenic due to its impact on splicing and seems responsible for the moderate phenotype of the four patients reported herein. The present study highlights the importance of considering the potential effect of a missense variant on splicing when it is not predicted to be disease causing

Loss of LGR4/GPR48 causes severe neonatal salt-wasting due to disrupted WNT signaling altering adrenal zonation.

Disorders of isolated mineralocorticoid deficiency causing potentially life-threatening salt-wasting crisis early in life have been associated with gene variants of aldosterone biosynthesis or resistance, but in some patients no such variants are found. WNT/β-catenin signaling is crucial for differentiation and maintenance of the aldosterone producing adrenal zona glomerulosa (zG). We describe a highly consanguineous family with multiple perinatal deaths or infants presenting at birth with failure to thrive, severe salt-wasting crises associated with isolated hypoaldosteronism, nail anomalies, short stature, and deafness. Whole exome sequencing revealed a homozygous splice variant in the R-SPONDIN receptor LGR4 gene (c.618-1G>C) regulating WNT signaling. The resulting transcripts affected protein function and stability, and resulted in loss of Wnt/β-catenin signaling in vitro. The impact of LGR4 inactivation was analyzed by adrenal cortex specific ablation of Lgr4, using Lgr4Flox/Flox mated with Sf1:Cre mice. Inactivation of Lgr4 within the adrenal cortex in the mouse model caused decreased WNT signaling, aberrant zonation with deficient zG and reduced aldosterone production. Thus, human LGR4 mutations establish a direct link between LGR4 inactivation and decreased canonical WNT signaling with abnormal zG differentiation and endocrine function. Therefore, variants in WNT signaling and its regulators should systematically be considered in familial hyperreninemic hypoaldosteronism

MICRODELETIONS DU CHROMOSOME Y (METHODES DE DEPISTAGE, RELATION PHENOTYPE-GENOTYPE)

LYON1-BU Santé (693882101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Critères diagnostiques du syndrome des ovaires polykystiques et importance du dosage de l'hormone anti-mullérienne

LYON1-BU Santé (693882101) / SudocSudocFranceF

Implication de facteurs sertoliens dans la physiologie et la physiopathologie de la spermatogenèse (étude des ARNm de la clusterine et du stem cell factor)

La cellule de Sertoli a un rôle fondamental dans le contrôle des différentes étapes de la spermatogenèse. Le stem cell factor et la clusterine sont produits par la cellule de Sertoli. Nous avons mesuré par RT-PCR quantitative leurs ARNm au cours du développement et après avoir induit un trouble de la spermatogenèse chez le rat et dans des biopsies testiculaires de patients présentant une azoospermie soit obstructive (groupe contrôle) soit par trouble de la spermatogenèse. Les résultats suggèrent que la différenciation des cellules de Sertoli est altérée dans les troubles de la spermatogenèse constitutifs ou idiopathiques chez l'homme, contrairement aux troubles de la spermatogenèse acquis chez l'homme ou induit chez le rat adulteThe sertoli cell is essential in the control of the different stages in the spermatogenisis. Stem cell factor and clusterin are product by the Sertoli Cell. We mesured by quantitative RT PCR their mRNA during the developpement and after induction of spermatogenisis failure in the rat and in the testicular biopsies from men with either obstructive azoospermia (control group) or spermatogenic failure. The results suggest that Sertoli cell differentiation is altered in cases of constitutive or idiopathic spermatogenic failure in human, in opposite of acquired spermatogenesic failure in human or induced spermatogenic failure in adult ratLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Transmissible microdeletion of the Y-chromosome encompassing two DAZ copies, four RBMY1 copies, and both PRY copies

International audienceObjective: To study a transmissible partial AZFb and -c microdeletion. Design: Case report. Setting: Service of Reproductive Medicine, Molecular Biology, CHU Lyon, France. Patient(s): A case of oligoasthenospermia with partial spermatogenic failure. Screening for Yq microdeletions revealed the absence of sY143, suggesting an AZFb microdeletion. Intervention(s): Sequence-tagged site mapping indicated that the deletion encompassed a portion of the AZFb and -c region. Genomic DNA from the patient's father gave the same pattern. During the course of these investigations, a pregnancy occurred. On the 46,XY amniocyte and cord blood DNA, the same microdeletion was found. Main Outcome Measure(s): Study of the fine structure of the Y-chromosome and the gene copy number. Result(s): The three males of this family have a rearrangement including a deletion encompassing r3 and r4, the palindrome P3, and its boundary regions: u3 and u1 in its distal part. This induced a reduction in DAZ and RBMY1 copy number and complete loss of PRY. Conclusion(s): PRY is not indispensable to complete spermatogenesis; and with two RBMY1 and two DAZ copies, complete spermatogenesis can be conserved. (Fertil Steril (R) 2010;94:2770.e11-e16. (C) 2010 by American Society for Reproductive Medicine.

Hyperandrogenic states in women: pitfalls in laboratory diagnosis

International audienceMeasuring total testosterone level is the first-line approach in assessing androgen excess in women. The main pitfalls in measuring testosterone relate to its low concentration and to the structural similarity between circulating androgens and testosterone, requiring accurate techniques with high specificity and sensitivity. These goals can be achieved by immunoassay using a specific anti-testosterone monoclonal antibody, ideally after an extraction step. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)will be commonly used for measuring testosterone, providing optimal accuracy with a low limit of detection. Yet, the pitfalls of these two techniques are well identified and must be recognized and systematically addressed. In general, laboratories using direct testosterone immunoassay and mass spectrometry need to operate within a quality framework and be actively engaged in external quality control processes and standardization, so as to ensure appropriate interpretation irrespective of the particular laboratory. Circulating testosterone is strongly bound to sex-hormone-binding globulin (SHBG), and SHBG levels are typically low in overweight hyperandrogenic patients. Thus, low SHBG may decrease circulating testosterone to normal values, which will mask androgen excess status. One way to avoid this pitfall, awaiting direct free testosterone assays that are yet to be developed, is to measure SHBG and calculate free testosterone. A few other pitfalls will be discussed in this review, including those of adrenal androgen exploration, with the aim of helping clinicians to better handle laboratory investigation of androgen excess disorders in women

Involvement of chemerin and CMKLR1 in the progesterone decrease by PCOS granulosa cells

International audiencePolycystic ovarian syndrome (PCOS) is the main cause of infertility in women. It is frequently associated with reduced progesterone production by human luteinised granulosa cells (hlGCs). However, the molecular mechanisms involved in these steroidogenesis alterations in PCOS patients are unclear. In a dihydrotestosterone-induced PCOS mouse model, steroid production is maintained in the setting of chemokine-like receptor 1 ( Cmklr1 ) knockout. Thus, chemerin and chemerin receptors in terms of expression and progesterone regulation could be different in control and PCOS hlGCs. We first confirmed that progesterone levels in both plasma ( P < 0.0001) and follicular fluid (FF) ( P < 0.0001) were significantly reduced in PCOS normal weight women compared to control women. These data were associated with a lower STAR mRNA expression in both in vivo ( P < 0.0001) and in vitro ( P < 0.0001) hlGCs from PCOS women. Secondly, chemerin FF levels ( P < 0.0001) and RARRES2 ( P < 0.05) and CMKLR1 ( P < 0.0001) mRNA levels in GCs were higher in PCOS normal weight patients. Thirdly, treatment of hlGCs with a specific nanobody (the VHH CA4910) targeting the human receptor for CMKLR1 leading to its inactivation abolished chemerin-induced progesterone inhibition, suggesting the involvement of CMKLR1 in this process. Furthermore, the inhibition of progesterone secretion induced by chemerin was two-fold higher in PCOS hlGCs ( P < 0.05). Moreover, the VHH CA4910 reinstated a normal progesterone secretion with lower concentrations in PCOS hlGCs, suggesting a different chemerin sensitivity between PCOS and control hlGCs. Thus, chemerin, through CMKLR1, could be involved in the steroidogenesis alterations in PCOS hlGCs