European Journal of Endocrinology (2012) 166 341–349 ISSN 0804-4643 CASE REPORT Pubertal androgenization and gonadal histology in two 46,XY adolescents with NR5A1 mutations and predominantly female phenotype at birth M Cools1, P Hoebeke2, K P Wolffenbuttel3, H Stoop4, R Hersmus4, M Barbaro5, A Wedell5, H Bru¨ggenwirth6, L H J Looijenga4 and S L S Drop7 1Division of Pediatric Endocrinology, Department of Pediatrics, Division of Pediatric Urology, Department of Urology, University Hospital Ghent, Ghent University, Building 3K12D, De Pintelaan 185, 9000 Ghent, Belgium, 2University Hospital Ghent, Ghent University, Ghent, Belgium, 3Division of Pediatric Urology, Department of Urology, Erasmus Medical Center, Sophia Children’s Hospital, Rotterdam, The Netherlands, 4Department of Pathology, Josephine Nefkens Institute, Daniel Den Hoed Cancer Center,Erasmus Medical Center,Rotterdam, The Netherlands, 5Department of Molecular Medicine and Surgery, Karolinska Institutet, Center for Inherited Metabolic Diseases (CMMS), Karolinska University Hospital, Stockholm, Sweden, 6Department of Clinical Genetics and 7Division of Pediatric Endocrinology, Department of Pediatrics, Erasmus Medical Center, Sophia Children’s Hospital, Rotterdam, The Netherlands (Correspondence should be addressed to M Cools; Email: [email protected]) Abstract Objective: Most patients with NR5A1 (SF-1) mutations and poor virilization at birth are sex-assigned female and receive early gonadectomy. Although studies in pituitary-specific Sf-1 knockout mice suggest hypogonadotropic hypogonadism, little is known about endocrine function at puberty and on germ cell tumor risk in patients with SF-1 mutations. This study reports on the natural course during puberty and on gonadal histology in two adolescents with SF-1 mutations and predominantly female phenotype at birth. Design and methods: Clinical and hormonal data and histopathological studies are reported in one male and one female adolescent with, respectively, a nonsense mutation (c.9TOA, p.Tyr3X) and a deletion of the first two coding exons (NCBI36/hg18 Chr9:g.(126306276-126307705)_(126303229- 126302828)del) of NR5A1, both predicted to fully disrupt gene function. Results: LH and testosterone concentrations were in the normal male range, virilization was disproportionate to the neonatal phenotype. In the girl, gonadectomy at 13 years revealed incomplete spermatogenesis and bilateral precursor lesions of testicular carcinoma in situ. In the boy, at the age of 12, numerous germ cells without signs of malignancy were present in bilateral testicular biopsy specimen. Conclusions:InSF-1 mutations, the neonatal phenotype poorly predicts virilization at puberty. Even in poorly virilized cases at birth, male gender assignment may allow spontaneous puberty without signs of hypogonadotropic hypogonadism, and possibly fertility. Patients with SF-1 mutations are at increased risk for malignant germ cell tumors. In case of preserved gonads, early orchidopexy and germ cell tumor screening is warranted. The finding of premalignant and/or malignant changes should prompt gonadectomy or possibly irradiation. European Journal of Endocrinology 166 341–349 Introduction 46,XY individuals from phenotypically females with complete gonadal dysgenesis with or without adrenal Steroidogenic factor 1 (SF-1), encoded by the NR5A1 failure (5–10) to males with penoscrotal hypospadias gene, is known to be involved in adrenal and gonadal (8, 11, 12) or bilateral anorchia (13). In 46,XX women, development and differentiation. In addition, in both NR5A1 mutations have been associated with premature males and females, SF-1 is a key promoter of gonadal ovarian failure (3). The clinical and genetic charac- hormone production, by regulating the transcription teristics of these patients have been reviewed elsewhere of several genes involved in steroidogenesis (1–3). SF-1 (14–16). mainly acts through direct binding to the promoter The observed Sf-1 expression in the ventromedial regions of its target genes, however, indirect effects have hypothalamus and gonadotrophs and impaired gonado- also been studied (1, 2, 4). trophin production in Sf-1 knockout mice suggest an The clinical presentation of patients affected additional role for Sf-1 in the normal regulation of by NR5A1 mutations is very diverse, ranging in puberty, reproduction, and metabolism (14, 17). In line q 2012 European Society of Endocrinology DOI: 10.1530/EJE-11-0392 Online version via www.eje-online.org Downloaded from Bioscientifica.com at 10/01/2021 11:34:16PM via free access 342 M Cools and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2012) 166 with this hypothesis, pituitary-specific Sf-1 knockout The PCR products were separated by capillary electro- mice develop hypogonadotropic hypogonadism (18). phoresis on an ABI 3100 Genetic Analyzer (Applied Since most of the initially reported 46,XY patients, on Biosystems, Warrington, UK). Trace data were analyzed the basis of absent or poor virilization at birth, were sex- using the Gene Mapper v3.7 software and exported to assigned female and received gonadectomy at a young an Excel spreadsheet (Microsoft). Analysis, using peak age, little is known about the natural course of puberty heights, was performed as described previously (20). in 46,XY patients with NR5A1 mutations. In addition, the risk for the development of germ cell tumors in older Sequencing DNA was extracted from peripheral blood individuals remains an important but unexplored issue. cells using standard procedures. The coding exons and In this study, we present detailed clinical, hormonal, exon/intron boundaries (exons 2–7) were amplified, and histopathological investigations at the onset of using the primers summarized in Tables 1 and 2. All puberty in two patients with NR5A1 haploinsufficiency, PCRs were performed by TaKaRa LA Taq polymerase. and with a predominantly female phenotype at birth. Subsequently, the fragments obtained were analyzed One of them was reassigned male at the age of 2 years by direct sequence analysis on an ABI 3730XL DNA after the finding of a palpable labioscrotal gonad and his sequencer. Data analysis was carried out using Seqscape testis function has been followed through puberty. software (Applied Biosystems). The sequence variant was confirmed by a second experiment, including PCR, on the same DNA sample. Material and methods Hormonal investigations Pathological studies Testosterone random values were obtained by coat-a- Gonadal tissue was fixed in 10% formalin for 24 h. Slices count RIA (Siemens, Los Angeles, CA, USA) (case 1) and of 3 mm thickness were prepared. Experimental con- liquid chromatography/tandem mass spectrometry ditions for immunohistochemistry are summarized in (UPLC Waters quattro premier, Waters Corporation, Tables 1 and 2.StainingwasperformedbyDAB/H2O2 Milford, MA, USA) (case 2). LH and FSH random values (OCT3/4 (POU5A1), SOX9, FOXL2, and SF-1 (NR5A1)) were obtained by electrochemoluminescence assay or New Fuchsin/Sodium nitrite Naphtol ASMX phosphate (Roche Diagnostics E170 Modular). Inhibin B (Inhibin (TSPYand AMH), and counterstaining with hematoxylin. B, Oxford Bio-Innovation Ltd, Oxford, UK) and AMH (Beckman Coulter Company, Suarle´e, Belgium) were Ethics measured by ELISA. Human chorionic gonadotropin (HCG) and ACTH tests were performed according to our Informed consent to publish results was obtained from local protocols. Values for testosterone were obtained at both patients and their parents. baseline and 72 h after i.m. injection of Pregnyl (Organon, Merck, NJ, USA), 1500 U. Values for cortisol were obtained at baseline and 60 min after i.v. injection Results of Synacthen (Novartis Pharma), 250 mg. Case 1 is a girl from consanguineous parents, who first came to medical attention at the age of 13 years, Gene analyses because of progressive virilization in the past year (facial hair growth, deepening of the voice, and muscular Multiplex ligation-dependent probe amplification body) and lack of breast development. Her younger (MLPA) An in-house-designed synthetic probe set for sister and brother are healthy. Discrete clitoromegaly MLPA was used to detect single exon deletions/duplica- had been noticed at birth but was judged insufficient to tions on the NR5A1 gene (19). MLPA reactions were prompt medical investigations. At presentation, she had performed by the reagents and the recommendations of pubic and axillary hair but no breast development the EK1 MLPA reagent kit (MRC-Holland, Amsterdam, (Tanner B1P3A2), clitoral length was 40 mm. On The Netherlands), starting from 100 ng genomic DNA. questionnaires on gender role behavior and gender Table 1 Summary of primers used for sequence analysis of the NR5A1 gene. Amplicon Forward primer Reverse primer NR5A1SQE0203 50-gggcacagagaggggattac-30 50-gttctcttgcagcgactgg-30 NR5A1SQE04-1 50-ggattgcctgaaaagctgag-30 50-agggctctgggtagccgta-30 NR5A1SQE04-2 50-gcttcaagctggagacagg-30 50-caaaggacagtcgggctaag-30 NR5A1SQE05 50-gaaggggtgagaggaaggtc-30 50-gtggggaaagggctgataat-30 NR5A1SQE06 50-cccatctgacctgcacct-30 50-aggatgggagcctggaataa-30 NR5A1SQE07 50-atgcccatgtctttgatggt-30 50-tcccaaacacacagtgtcaga-30 www.eje-online.org Downloaded from Bioscientifica.com at 10/01/2021 11:34:16PM via free access EUROPEAN JOURNAL OF ENDOCRINOLOGY (2012) 166 Puberty and gonadal histology in SF-1 mutations 343 Table 2 Experimental conditions for the pathological studies. Incubation Primary AB Supplied by Dilution Pretreatment AG retrieval time Secondary AB 0 OCT3/4 Santa Cruz Biotechnology, 1/1000 3% H2O2 for 5 pH 6,