NR5A1 molecular analysis in 46,XY patients with disorders of sex development

Orientador: Marcilda Palandi de MelloDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O termo Distúrbio da Diferenciação do Sexo (DDS) caracteriza-se pelo desenvolvimento genital ou gonadal incompleto ou desordenado. Os DDS com cariótipo 46,XY são caracterizados por genitália externa ambígua ou feminina, em alguns casos com gônadas disgenéticas, e presença ou ausência de derivados de Müller. Os mais frequentes são a insensibilidade androgênica, deficiência da 5-alfa-redutase tipo 2, disgenesia gonadal e DDS ovário-testicular. Vários são os genes que participam dos processos de determinação e diferenciação do sexo. Alterações no gene NR5A1, que codifica o fator de transcrição SF- 1, é responsável por diferentes fenótipos de DDS. A proteína SF-1 é expressa principalmente em tecidos esteroidogênicos (gônadas, adrenais e placenta), nas células de Sertoli, nas células de Leydig e nos ovários; é o principal regulador do metabolismo do colesterol nas células esteroidogênicas. Além disso, regula a atividade de outros genes, como os CYPs, HSD3B, StAR, SOX9, DAX1, entre outros. Na literatura são descritas alterações no gene NR5A1 associadas à DDS 46,XY, anorquia bilateral, amenorréia primária, falência ovariana precoce, hipospádia, infertilidade masculina, e alguns casos de tumores adrenais e endometrioses. Neste trabalho foi realizada a análise molecular do gene NR5A1 em 86 pacientes com DDS 46,XY, incluindo-se disgenesia gonadal completa (n = 7), disgenesia gonadal parcial (n = 18), DDS 46,XY idiopático (n = 41) e outros (n = 20). Doze alterações foram identificadas neste trabalho, sendo: sete na região codificante (p.Ser32Asn, p.Arg39Cis, p.Lis38*, p.Cis65Tir, p.L80Wfs*8, p.Cis247*, and p.Asp364Trefs*18), uma em sítio de splicing (c.1138+1G>T), duas no exon 1 nãocodificante (c.-133G>A e c.-156_-136ins18pb), três na região 5'UTR (c.-413G>A, c.- 208C>A, e c.-762C>T) e uma na região 3'UTR (c.*1286C>T). As variações aqui descritas, não foram identificadas em controles saudáveis. As análises in silico demonstraram o possível efeito deletério de cada alteração e, suas relações com o fenótipo dos indivíduos. Embora estes resultados demonstrem a importância de cada alteração para o fenótipo, haverá ainda a necessidade de se investigar os efeitos funcionais in vitro. As alterações com potencial deletério foram identificadas em maior frequência nos casos dos distúrbios da diferenciação gonadal (20%) e DDS 46,XY idiopático (22%)Abstract: The term Disorders of Sex Differentiation (DSD) characterize incomplete or disorganized genital or gonadal development. The DSD with 46, XY karyotype may present either ambiguous or female genitalia and also dysgenetic gonads in some cases, with presence or absence of Müllerian derivatives. The most frequent are androgen insensitivity, 5-alpha-reductase type 2 deficiency, gonadal dysgenesis and ovarian-testicular DSD. There are several genes that participate in both sex determination and differentiation processes. Mutations in NR5A1 gene, which encoding SF-1, a transcription factor, are responsible for different phenotypes of DSD. The protein SF-1, which is expressed mainly in steroidogenic tissues (gonads, adrenal glands and placenta), is also express in Sertoli and Leydig cells, in the ovaries, and is the major regulator of cholesterol metabolism in steroidogenic cells. Moreover, it regulates the activity of other genes, such as CYPs, HSD3B, StAR, SOX9, DAX1, among others. The literature describes the association of changes in NR5A1 gene with 46, XY DSD, bilateral anorchia, primary amenorrhea, premature ovarian failure, hypospadias, male infertility, and some cases of adrenal tumors and endometriosis. The present work involved the molecular analysis of NR5A1 gene in 86 patients with 46, XY DSD including complete gonadal dysgenesis (n = 7), partial gonadal dysgenesis (n = 18), idiopathic 46, XY DSD (n = 41) and others (n = 20). Twelve variations had been identified: seven in the coding region (p.Ser32Asn, p.Arg39Cis, p.Lis38*, p.Cis65Tir, p.L80Wfs*8, p.Cis247*, and p.Asp364Trefs*18), one at a splice site (c.1138+1 G>T), two in the noncoding exon 1 (c.-133G>A and c.-156_-136ins18pb), three in the 5'UTR region (c.- 413G>A, c.-208C>A and c.-762C>T) and one in the 3'UTR (c.*1286C>T). The variations herein described, have not been identified in healthy controls. In silico analysis showed possible deleterious effects for each change and its correlations to individual phenotypes. Although those results demonstrate the importance of each change for the phenotype, there in vitro functional effects must be investigated. The potentially deleterious changes were identified more frequently in cases of disorders of gonadal development (20%) and idiopathic 46, XY DSD (22%)MestradoGenetica Animal e EvoluçãoMestra em Genética e Biologia Molecula

Functional analyses of new nucleotide variations in NR5A1 gene in patients 46,XY with disorders of sex development

Orientadores: Maricilda Palandi de Mello, Olaf Hiort, Ralf WernerTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Os Distúrbios da Diferenciação do Sexo (DDS) caracterizam-se pelos desenvolvimentos gonadal e/ou genital incompletos ou desordenados , que levam a uma discordância entre o sexo genético, gonadal e fenotípico do indivíduo afetado. Entre os genes envolvidos na cascata de diferenciação e determinação sexual, encontra-se o NR5A1, que codifica a proteína SF-1, e que quando mutado, pode levar a diferentes fenótipos de DDS, como disgenesia gonadal pura e parcial, DDS ovário testicular 46,XX, DDS 46,XX testicular, anorquia bilateral e hipospádia, além de quadros de amenorréia primária, insuficiência ovariana primária, infertilidade masculina, entre outros ...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digitalAbstract: Disorders of Sex Development (DSD) are defined as congenital conditions in which development of chromosomal, gonadal or anatomical sex is atypical. Mutations in the NR5A1 gene, which encodes the transcription factor SF-1, are responsible for different phenotypes of DSD, such partial and complete gonadal dysgenesis, 46,XX testicular and ovotesticular DSD, bilateral anorchia, hypospadia, and also can be associated with primary amenorrhea, premature ovarian failure, male infertility, adrenal tumors and others...Note: The complete abstract is available with the full electronic documentDoutoradoGenetica Animal e EvoluçãoDoutor em Genetica e Biologia Molecular2013/05603-5, 2013/24333-9FAPES

Mutation update for the NR5A1 gene involved in DSD and infertility

Nuclear receptor subfamily 5 group A member 1 (NR5A1), also named steroidogenic factor 1, is an essential transcription factor that regulates a number of target genes crucial for normal reproductive physiology and endocrine function. It is encoded by NR5A1 gene and is expressed in high doses mainly in steroidogenic tissues, where it controls several steps of adrenal and gonadal development. NR5A1 mutations are associated with a wide phenotypic spectrum of disorders/differences of sex development (DSD), a group of conditions in which development of chromosomal, gonadal, or anatomic sex is atypical. Here, we reviewed 188 NR5A1 mutations from 238 cases reported in literature so far. Additionally, we report the variations p.Ser4*, p.(Cys55Ser), p.(Met78Leu), and p.Met98Glyfs*45, which have not been annotated for NR5A1 before and were identified in some of the 205 46,XY patients of our own cohort. This is the first NR5A1 mutation review which includes both 46,XX and 46,XY karyotype, with the purpose of discussing the complexity of genotype-phenotype correlations among DSD and infertile male patients and also females with primary ovarian failure.4115868CNPQ - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPESP – Fundação de Amparo à Pesquisa Do Estado De São Paulo406605/2018‐12013/05603‐5; 2018/19445‐6; 2015/04763‐

Phenotype-genotype correlation in nr5a1 gene remains elusive: heterozygosis for p.d293n mutation also leads dsd phenotype

We report on a patient with XY partial gonadal dysgenesis (PGD) with a heterozygous mutation that had already been described in homozygous patients from a family with different phenotypes. Methods: Our patient was referred with 14 days of age due to sex ambiguity. He had a 1.5-cm phallus, penoscrotal hypospadias, and both gonads were in the labioscrotal folds. Horm Res Paediatr 2017;88(suppl 1):1–628 Karyotype was 46,XY. At 6 weeks of age he had normal FSH, DHEA and androstenedione, whereas LH, 17-OHprogesterone and testosterone were elevated, slightly elevated and low, respectively. ACTH test resulted normal but there was no response to hCH test performed at 7 months. Antimullerian hormone was also low. He received a male sex assignment and underwent hypospadias repair. He had a right streak gonad and a left dysgenetic testis, leading to the diagnosis of XY PGD. Spontaneous puberty started at 12 years and progressed normally. At 16 years he had high FSH and LH but normal testosterone. Whole exome sequencing was performed to search for patogenic mutations in genes involved in testis differentiation. Results: Whole exome sequencing revealed heterozygosis of the c.877Ggt;A transition within exon 5 of NR5A1. This change leads to p.D293N mutation that had already been described in four homozygous members of a family: one with XY PGD, one with XX complete gonadal dysgenesis (CGD) and two with XY CGD. Heterozygous members of the family were phenotypically normal. The mother of the present patient was born in the same little town where that family originated. Conclusions: To our knowledge, this is the first p.D293N heterozygous individual to present DSD. The recessive inheritance for this mutation concluded on basis of that family pattern and on the relatively high level of residual activity of the mutated SF-1 is no longer appropriated. SF-1 acts in a dose-dependent manner and regulates many other genes involved in sex differentiation process, which could explain the broad spectrum of phenotypes caused by the same mutation. The present case illustrates the difficulty in establishing an appropriate phenotype-genotype correlation and highlights the importance of genetic knowledge for the correlation of NR5A1 mutations with clinical phenotypes881P1 - 151620220

A Novel Look at Dosage-Sensitive Sex Locus Xp21.2 in a Case of 46,XY Partial Gonadal Dysgenesis without <i>NR0B1</i> Duplication

A region of 160 kb at Xp21.2 has been defined as dosage-sensitive sex reversal (DSS) and includes the NR0B1 gene, considered to be the candidate gene involved in XY gonadal dysgenesis if overexpressed. We describe a girl with 46,XY partial gonadal dysgenesis carrying a 297 kb duplication at Xp21.2 upstream of NR0B1 initially detected by chromosomal microarray analysis. Fine mapping of the breakpoints by whole-genome sequencing showed a tandem duplication of TASL (CXorf21), GK and partially TAB3, upstream of NR0B1. This is the first description of an Xp21.2 duplication upstream of NR0B1 associated with 46,XY partial gonadal dysgenesis

Clinical and genetic characteristics of a large international cohort of individuals with rare NR5A1/SF-1 variants of sex developmentResearch in context

Summary: Background: Steroidogenic factor 1 (SF-1/NR5A1) is essential for human sex development. Heterozygous NR5A1/SF-1 variants manifest with a broad range of phenotypes of differences of sex development (DSD), which remain unexplained. Methods: We conducted a retrospective analysis on the so far largest international cohort of individuals with NR5A1/SF-1 variants, identified through the I-DSD registry and a research network. Findings: Among 197 individuals with NR5A1/SF-1 variants, we confirmed diverse phenotypes. Over 70% of 46, XY individuals had a severe DSD phenotype, while 90% of 46, XX individuals had female-typical sex development. Close to 100 different novel and known NR5A1/SF-1 variants were identified, without specific hot spots. Additionally, likely disease-associated variants in other genes were reported in 32 individuals out of 128 tested (25%), particularly in those with severe or opposite sex DSD phenotypes. Interestingly, 48% of these variants were found in known DSD or SF-1 interacting genes, but no frequent gene-clusters were identified. Sex registration at birth varied, with <10% undergoing reassignment. Gonadectomy was performed in 30% and genital surgery in 58%. Associated organ anomalies were observed in 27% of individuals with a DSD, mainly concerning the spleen. Intrafamilial phenotypes also varied considerably. Interpretation: The observed phenotypic variability in individuals and families with NR5A1/SF-1 variants is large and remains unpredictable. It may often not be solely explained by the monogenic pathogenicity of the NR5A1/SF-1 variants but is likely influenced by additional genetic variants and as-yet-unknown factors. Funding: Swiss National Science Foundation (320030-197725) and Boveri Foundation Zürich, Switzerland