Search

Introduction: Ovotesticular disorder of sex development (OT-DSD) is a rare condition defined by concomitance of testicular tissue and ovarian tissue (containing follicles) in the same individual. In SRY-negative 46,XX OT-DSD, the presence of testicular tissue may be due to variations in NR5A1. Our aims were to search for NR5A1 variants in SRY-negative 46,XX OT-DSD patients and to perform a systematic review on the contribution of NR5A1 variations to 46,XX OT-DSD. Methods: Sanger sequencing of NR5A1 was performed in seven SRY-negative 46,XX OT-DSD patients: five simplex cases and two with another sibling with a 46,XX DSD. Systematic review of original studies on NR5A1 sequencing of 46,XX OT-DSD patients was performed according to PRISMA-P guideline. Case reports were selected for analysis of clinical features. Individuals with NR5A1-associated testicular DSD were not included. Results: Sanger sequencing of NR5A1 did not reveal pathogenic variants among our patients. Our cohort was included in this systematic review with seven other articles, totalizing fifty-six 46,XX OT-DSD patients investigated by Sanger or whole-exome sequencing. From them, three NR5A1 pathogenic variants were identified (5% of the cases). Clinical analysis of these 3 cases and 5 case reports revealed: predominance of ovotestis (13/16 gonads) and bilateral OT-DSD (5/8 cases). Conclusion: The etiology of most 46,XX OT-DSD cases remains elusive, highlighting the importance of a deeper molecular investigation.

Introduction: The aim of this retrospective study was to verify the association between the time of diagnosis and initial and final sex assignment in a disorder of sex development (DSD) diagnostic group, looking at the age of the patients at first visit, severity of genital ambiguity, and karyotype. Methods: The time of diagnosis was divided into 3 groups: before 2000, between 2000 and 2006, and after 2006. Data were categorized and analyzed using the χ2 test with α < 0.05. Results: A total of 567 cases were analyzed; 307 were assigned as male, 135 as female, and 125 remained undefined at the first visit. After clinical and laboratory evaluations, 369 patients were male and 198 were female. Neither initial nor final sex assignment proportions changed over time, but there were significant differences in the age at first visit, with referral occurring at an earlier age, as well as more severe genital ambiguity presentations, a higher proportion of sex chromosome aberrations, and a lower frequency of 46,XX DSD cases. This occurred both in the sample as a whole (567 cases) and in the group of 125 patients without definitive sex assignment at the first visit. The results were similar when only 284 patients aged less than 12 months at the first visit were analyzed. Discussion/Conclusion: Over time, there were no changes in sex assignment proportions, but there was an increased awareness of the need for early referral and changes in clinical, cytogenetic, and diagnostic aspects.

Introduction: NR5A1 is an essential transcription factor that regulates several target genes involved in reproduction and endocrine function. Pathogenic variants in this gene are responsible for a wide spectrum of disorders/differences of sex development (DSD). Methods: The molecular study involved Sanger sequencing, in vitro assays, and whole exome sequencing (WES). Results: Four variants were identified within the NR5A1 non-coding region in 3 patients with 46,XY DSD. In vitro analyses showed that promoter activity was affected in all cases. WES revealed variants in SRA1, WWOX, and WDR11 genes. Discussion/Conclusion: Evaluation of clinical and phenotypic significance of variants located in a non-coding region of a gene can be complex, and little is known regarding their association with DSD. Nevertheless, based on the important region for interaction with cofactors essential to promote appropriated sex development and on our in vitro results, it is feasible to say that an impact on gene expression can be expected and that this may be correlated with the DSD pathophysiology presented in our patients. Considering the number of cases that remain elusive after screening for the well-known DSD related genes, we emphasize the importance of a careful molecular analysis of NR5A1 non-coding region which is commonly neglected and might explain some idiopathic DSD cases.

De novo variants in the myelin regulatory factor (MYRF), a transcription factor involved in the differentiation of oligodendrocytes, have been linked recently to the cardiac and urogenital syndrome, while familiar variants are associated with nanophthalmos. Here, we report for the first time on a patient with a de novo stop-gain variant in MYRF (p.Q838*) associated with Scimitar syndrome, 46,XY partial gonadal dysgenesis (GD) and severe hyperopia. Since variants in MYRF have been described in both 46,XX and 46,XY GD, we assumed a role of MYRF in the early development of the bipotential gonad. We used publicly available single cell sequencing data of human testis and ovary from different developmental stages and analysed them for MYRF expression. We identified MYRF expression in the subset of coelomic epithelial cells at stages of gonadal ridge development in 46,XX and 46,XY individuals. Differential gene expression analysis revealed significantly upregulated genes. Within these, we identified CITED2 as a gene containing a MYRF binding site. It has been shown that Cited2−/− mice have gonadal defects in both testis and ovary differentiation, as well as defects in heart development and establishment of the left–right axis. This makes MYRF a potential candidate as an early regulator of gonadal and heart development via upregulation of the transcriptional cofactor CITED2.

Background The Congenital Adrenal Hyperplasia due to 21 hydroxylase deficiency is the most common cause of genital ambiguity in persons with XX sexual chromosomes. Genital ambiguity among persons with XY sexual chromosomes comprises diverse and rare etiologies. The deficiency of 17-beta-hydroxysteroid dehydrogenase type 3 enzyme (HSD17B3) is a rare autosomal recessive disorder due to functionally altered variants of the HSD17B3 gene. In this disorder/difference of sex development, the conversion of androstenedione into testosterone is impaired. The appearance of external genitalia of 46,XY individuals varies from typically male to almost female. Case presentation We report on a child presenting severe ambiguous genitalia. Due to access constraints, specialized care did not start until the child was 10 months old. Parents are consanguineous and were born in an area of high isonymy that is a cluster for rare recessive diseases. A new homozygous missense variant c.785G > T was found in exon 10 of the HSD17B3 gene. Conclusions Researchers-clinicians and researchers-researchers collaborative efforts to elucidate the genetic basis of this disease were critical since this etiologic investigation is not available through the public health system. This case exemplifies the families’ pilgrimage in cases of genital ambiguity due to a rare genetic condition. Recognizing the etiology was the baseline to provide information on prognosis and treatment options, and to shelter family and child doubts and hopes in order to better support their decisions.

In this study, we present 3 cases of Down syndrome (DS) associated with disorders/differences of sex development (DSD) and review the literature on this topic. Case 1: 1-year-old child with male genitalia and DS phenotype, 47,XX,+21 karyotype and testicular DSD. Case 2: 11-month-old child with male genitalia and few DS dysmorphisms, 45,X/47,XY,+21 karyotype, and mixed gonadal dysgenesis. Case 3: 4-month-old child with female genitalia and DS phenotype, 47,XY,+21 karyotype and XY complete gonadal dysgenesis. In the literature, among 188 patients, 107 (57%) had Klinefelter syndrome and 61 (33%) Turner syndrome, 12 (6%) had mixed gonadal dysgenesis, 2 (1%) had partial androgen insensitivity, 2 (1%) ovotesticular DSD, and the others had congenital adrenal hyperplasia, XY partial gonadal dysgenesis, XY complete gonadal dysgenesis, and complete androgen insensitivity (1 case each). A typical DS phenotype was found in all individuals of the revision, with the exception of one case, but DSD features were not always reported. In conclusion, the association of DS with sex chromosome DSD is the most frequently observed, whereas associations with 46,XX and 46,XY DSD is extremely rare.

The aim of this study was to verify the influence of Y chromosome and intrauterine androgens production/action on birth weight and length of children with Disorders/Differences of Sex Development (DSD). This was a cross-sectional and retrospective study. Cases of Turner syndrome (TS), complete (XX and XY), mixed (45,X/46,XY) and partial (XY) gonadal dysgenesis (GD), complete (CAIS) and partial (PAIS) androgen insensitivity syndromes and XX and XY congenital adrenal hyperplasia (CAH) were included. Such conditions were grouped according to karyotype and to intrauterine production/action of androgens. The sample consisted of 293 cases, 50 with TS, 28 mixed GD, 117 CAH (49 XY and 68 XX), 18 CAIS, 10 PAIS, 30 partial GD, 10 XY and 30 XX complete GD. Birth weight and length were lower in TS and mixed GD when compared to XY and XX. In turn, patients with increased androgen production/action (117 cases) had higher birth weight and length when compared to those with absent (108 cases) and decreased (68 cases) production/action. It was observed a negative influence of the 45,X/46,XY karyotype in birth weight and a positive influence of increased androgen production/action. Regarding birth length, there was a negative influence of the TS karyotype and of decreased androgen production/action. In conclusion, in DSD, both karyotype, especially with a 45,X cell line, and intrauterine androgenic production/action influence sex dimorphism of birth weight and length. It can be inferred that in children with normal karyotype and without a DSD, this dimorphism is mainly due to intrauterine androgenic production or action.

NR5A1 is an essential transcription factor that regulates several target genes involved in reproduction and endocrine function. Pathogenic variants in this gene are responsible for a wide spectrum of disorders/differences of sex development (DSD).The molecular study involved Sanger sequencing, in vitro assays, and whole exome sequencing (WES).Four variants were identified within the NR5A1 non-coding region in 3 patients with 46,XY DSD. In vitro analyses showed that promoter activity was affected in all cases. WES revealed variants in SRA1, WWOX, and WDR11 genes.Evaluation of clinical and phenotypic significance of variants located in a non-coding region of a gene can be complex, and little is known regarding their association with DSD. Nevertheless, based on the important region for interaction with cofactors essential to promote appropriated sex development and on our in vitro results, it is feasible to say that an impact on gene expression can be expected and that this may be correlated with the DSD pathophysiology presented in our patients. Considering the number of cases that remain elusive after screening for the well-known DSD related genes, we emphasize the importance of a careful molecular analysis of NR5A1 non-coding region which is commonly neglected and might explain some idiopathic DSD cases.

Objective: To evaluate, in a sample of patients with disorders of sex development (DSD), data related to the age at referral and their correlation with the initial complaints, gender at referral, defined gender after diagnosis and etiological diagnosis. Methods: Retrospective review of the age at the first consultation and the reason for it, initial social gender and gender after the diagnosis, karyotype and etiological diagnosis of all cases treated at a DSD outpatient clinic between 1989 and 2016. Cases that did not involve DSD and DSD diagnoses that do not usually involve ambiguous genitalia, thus not requiring specialized monitoring, were excluded. Results: Of the 1793 treated cases, 1139 were diagnosed with some type of DSD. This study excluded 430 cases (272 with Turner's syndrome, 66 with Klinefelter syndrome, and 92 with pure gonadal dysgenesis), thus a total 709 individuals were included. Of these, 82.9% were referred due to ambiguous genitalia; only one-quarter were still in the first month of life, and 6.6% were referred due to pubertal delay, with most of them aged 10 years or older. Of these patients, 68.6% had a diagnosis of XY DSD, 22.4% of XX DSD, and 9% of sex chromosome abnormalities. Conclusions: This study presents the largest series in the literature of patients with DSD treated in a single center. The time of referral of the majority of patients with ambiguous genitalia fell short of the ideal, and milder cases of ambiguous genitalia and many with pubertal manifestations were referred even later. The results reinforce the importance of continuing education for professionals who will have the first contact with these patients, mainly pediatricians and neonatologists. Resumo Objetivo: Avaliar em uma amostra de pacientes com distúrbios da diferenciação do sexo (DDS), dados relacionados à idade, ao encaminhamento e sua correlação com as queixas iniciais, ao sexo ao encaminhamento e ao sexo final e diagnóstico etiológico. Métodos: Revisão retrospectiva da idade por ocasião da primeira consulta e motivo dela, sexo social inicial e após definição do diagnóstico, cariótipo e diagnóstico etiológico de todos os casos atendidos em um ambulatório especializado em DDS entre 1989 e 2016. Foram excluídos casos que não compreendiam DDS e diagnósticos de DDS que não cursam comumente com ambiguidade genital, não necessitam de acompanhamento especializado. Resultados: Dos 1.793 casos atendidos, 1.139 foram diagnosticados com algum DDS. Excluíram-se 430 (272 síndrome de Turner, 66 síndrome de Klinefelter e 92 disgenesia gonadal pura), totalizando 709. Desses, 82,9% foram encaminhados por ambiguidade genital, somente um quarto ainda no primeiro mês de vida e 6,6% por atraso puberal, a maioria com 10 anos ou mais; 68,6% tiveram diagnóstico de DDS XY; 22,4% DDS XX e 9% de anomalias dos cromossomos sexuais. Conclusões: Este estudo apresenta a maior casuística na literatura de pacientes com DDS atendidos em um único serviço. O momento de encaminhamento da maioria dos pacientes com ambiguidade genital foi aquém do ideal e casos mais leves de ambiguidade e muitos com manifestações puberais foram encaminhados ainda mais tardiamente. Os resultados reforçam a importância do ensino continuado a profissionais que terão o primeiro contato com esses pacientes, principalmente pediatras e neonatologistas.

Historically, the terms partial (PGD) and mixed gonadal dysgenesis (MGD) have been used to describe incomplete testicular differentiation in individuals with 46,XY or 45,X/46,XY karyotypes, respectively. However, it is currently unclear to what extent clinical features actually differ between these individuals. The aim of this study was to compare clinical, laboratory, and histological findings in these 2 groups. Patients with testicular dysgenesis seen in our service between 1989 and 2013 were selected. Sixty-one patients met the inclusion criteria. Individuals with 46,XY and 45,X/46,XY karyotypes were compared regarding genital features, gonadal histology and function, growth, and associated conditions. Twenty-five had mosaicism with a 45,X cell line (MGD), while a 46,XY karyotype (PGD) was found in 36 cases belonging to 32 families. Mutations in NR5A1, WT1, and SRY genes associated with testicular dysgenesis were found in 12 families. There were no significant differences regarding parental consanguinity, degree of external androgenization, gonadal location, histology, and function, and associated conditions. However, in the MGD group, the presence of a uterus, lower birth weight and length, and short stature were more often observed. Therefore, the use of histological features to classify PDG and MGD should be abandoned and replaced by classification based on karyotype.

Ovotesticular disorder of sex development (OT-DSD) is a rare condition characterized by coexistence of ovarian and testicular parenchyma, in separate gonads or in the same gonad (ovotestis), in individuals with variable degrees of genital ambiguity. Karyotype may be 46,XX (60%), 46,XY (10%), or there may be sex chromosomes abnormalities, including mosaicism, chimerism and structural anomalies (30%). A genetic origin may be identified in some subjects with normal XX or XY karyotype, but most cases are of unknown origin. Apparently balanced chromosome rearrangements (translocations, insertions and inversions) may cause truncation, deletion, inactivation or over-expression of specific genes. We report on a case of OT-DSD associated with an X chromosome inversion. Case report: A 3-month old girl was referred due to atypical genitalia. She was born at term to a 42 years old G3P2A1 mother and her 45 years old unrelated husband with normal weight, length and head circumference. She had normal development, no associated health problems, and family history was unremarkable. Physical examination revealed a 3.1-cm phallus with chordee, scrotal hypospadias, partial penoscrotal inversion and a 0.5 cm3 right gonad palpable in the inguinal region; there were no associated dysmorphic features. At 1.5 months there were normal levels of FSH (3.09 IU/L) and LH (3.67 IU/L), and testosterone (155 ng/dL) was in the normal male range. Ultrasound revealed normal uterus and gonads were in the inguinal regions. Urethra-cystoscopy and vaginoscopy at 9 months revealed a urogenital sinus with high vaginal confluence. Laparoscopy and gonadal biopsies were also performed; the left gonad was an ovotestis with multiple ovarian follicles, while the right gonad was a testis. In both gonads the seminiferous tubules had only Sertoli cells. Karyotype revealed a pericentric X chromosome inversion, 46,X,inv(X)(p22.1q26)dn[20]. FISH on peripheral blood and cultured cells from the right gonad with probes for X (DXZ1) and Y (DYZ3) centromeres and SRY (Yp11.3 - 122 Kb) showed only two X chromosome signals. Array GH analysis (Cytoscan 750K, Affymetrix) showed a 1.3 Mb deletion distal to the short arm breakpoint (Xp22.31), which was reported as VOUS, and a 9 Mb region of LOH on chromosome 9. Discussion: Several cases of X pericentric inversion with different breakpoints have been reported; though phenotypes of female heterozygotes are often normal, early menopause, irregular menses, gonadal dysgenesis or sterility have been described. In this case, it is plausible that the genomic rearrangement could have affected long-range regulation of SOX3 (located in Xq27.1) resulting in ectopic expression of this gene in the bipotential gonad. In addition, the features detected in array GH may have a role in the phenotype. Different methods to determine the exact chromosomal breakpoints and copy number variations in this region will be required.

Background: Partial Androgen Insensitivity Syndrome (PAIS) (OMIM # 312300) is one of the causes of Disorders/Differences of Sex Development with 46,XY karyotype and normal or increased testosterone secretion that results in atypical genitalia. In general, it is caused by inactivating mutations on AR gene (Xq12 - OMIM * 313700), therefore it presents an X-linked recessive inheritance. Individuals raised as males have incomplete puberty with micropenis, sparse hairs, gynecomastia and increased LH, testosterone and estradiol serum levels. Therefore, the use of aromatase inhibitor in these cases becomes a logical indication aiming to increase testosterone levels in the tentative of supplanting its peripheral resistance and decreasing estrogen levels. Objective: To present clinical and laboratory data during the first year of use of aromatase inhibitor in puberty of three boys with confirmed molecular diagnosis of PAIS. Results: All subjects used letrozole (2.5 mg daily during 12 months). None reported significant side effects. Cases 1 and 2 are brothers (p.Ala596Tre). Case 1: Onset of treatment at age 12; height changed from 158 cm (z = +1.15) to 166 cm (z = +1.21), Tanner from G2P2T2 to G3P3T1, penis from 4.0 to 6.5 cm, LH from 7.5 to 18.3 IU/L (NR: 1.5 to 9.3 IU/L), testosterone from 361 to 1,347 ng/dL (NR: 165 to 763 ng/dL), estradiol from 35 to

Duplications of dosage sensitive sex-locus Xp21.2 including NR0B1 have been linked to 46,XY gonadal dysgenesis (GD) and their effects are attributed merely to increase gene dosage of NR0B1 (DAX1). Here we present a general mechanism how deletions, duplications, triplications or inversions with or without NR0B1 at Xp21.2 can lead to partial or complete GD by disrupting the cognate topological associated domain (TAD) in the vincinity of NR0B1. Our model is supported by three unrelated patients: two showing a 287kb overlapping duplication at the Xp21.2 locus upstream of NR0B1 containing CXorf21 and GK and one patient having a large new triplication of Xp21.2 as the most likely cause of GD. Whole Genome sequencing uncovered the exact structural rearrangements of the duplications and the triplication. Comparison with a previously published deletion upstream of NR0B1 revealed a common 35kb overlap between the deletion, our newly reported NR0B1 upstream duplications and the triplication as well as all other copy number variations (CNVs) at Xp21.2 reported so far. This overlap contains a strong CCCTC-binding factor (CTCF) binding site representing one boundary of the NR0B1 TAD. All three CNVs at Xp21.2 most likely disrupt this TAD boundary, which isolates NR0B1 from CXorf21 and GK and putatively results in GK and CXorf21 enhancer adoption and ensuing ectopic NR0B1 expression. As a result, the patients’ transcriptomes developed an intermediate expression pattern with both ovarian and testicular features and greatly reduced expression of spermatogenesis-related genes. This model not only allows better diagnosis of GD displaying CNVs at Xp21.2, but also gives deeper insight how spatiotemporal activation of developmental genes can be disrupted by reorganized TADs also in other rare diseases.

A retrospective cross-sectional study was performed in a DSD clinic at a tertiary service (University Hospital) to estimate the frequency of disorders of sex development (DSD) among men who seek medical care because of infertility. The sample included 84 men >20 years of age referred from 2010-2017 due to oligozoospermia or nonobstructive azoospermia of unknown etiology. Twelve cases (14%) were diagnosed as DSD, including Klinefelter Syndrome, 46,XX testicular DSD, and mild androgen insensitivity syndrome. Y chromosome microdeletions were detected in 2 patients. Among the remaining 70 cases there were patients with chromosome abnormalities which are not included in the DSD classification as well as rare NR5A1 variants of uncertain significance and hypergonadotropic hypogonadism and microorchidism in 46,XY subjects. In conclusion, the frequency of DSD in this study was 14%, consisting mainly of sex chromosome abnormalities but also 46,XX and 46,XY DSD. However, this figure may increase as further investigations are conducted in idiopathic cases with signs of primary testicular failure, which may present partial gonadal dysgenesis.

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.

Steroidogenic factor-1 (SF1), encoded by the NR5A1 gene, is a key regulator of steroidogenesis and reproductive development. NR5A1 mutations described in 46,XY patients with disorders of sex development (DSD) can be associated with a range of conditions of phenotypes; however, the genotype-phenotype correlation remains elusive in many cases. In the present study, we describe the impact of five NR5A1 variants (three novel: p.Arg39Cys, p.Ser32Asn, and p.Lys396Argfs*34; and two previously described: p.Cys65Tyr and p.Cys247*) on protein function, identified in seven patients with 46,XY DSD. In vitro functional analyses demonstrate that NR5A1 mutations impair protein functions and result in the DSD phenotype observed in our patients. Missense mutations in the DNA binding domain and the frameshift mutation p.Lys396Argfs*34 lead to both, markedly affected transactivation assays, and loss of DNA binding, whereas the mutation p.Cys247* retained partial transactivation capacity and the ability to bind a consensus SF1 responsive element. SF1 acts in a dose-dependent manner and regulates a cascade of genes involved in the sex determination and steroidogenesis, but in most cases reported so far, still lead to a sufficient adrenal steroidogenesis and function, just like in our cases, in which heterozygous mutations are associated to 46,XY DSD with intact adrenal steroid biosynthesis.