Birgit Köhler

Five novel mutations in steroidogenic factor 1 (SF1, NR5A1) in 46,XY patients with severe underandrogenization but without adrenal insufficiency

Steroidogenic factor 1 (SF1, NR5A1) is a nuclear receptor that regulates multiple genes involved in adrenal and gonadal development, steroidogenesis, and the reproductive axis. Human mutations in SF1 were initially found in two 46,XY female patients with severe gonadal dysgenesis and primary adrenal failure. However, more recent case reports have suggested that heterozygous mutations in SF1 may also be found in patients with 46,XY partial gonadal dysgenesis and underandrogenization but normal adrenal function. We have analyzed the gene encoding SF1 (NR5A1) in a cohort of 27 patients with 46,XY disorders of sex development (DSD) from the German network of DSD. Heterozygous SF1 mutations were found in 5 out of 27 (18.5%) of cases. Four patients with SF1 mutations presented with the similar phenotype of mild gonadal dysgenesis, severe underandrogenization, and absent Müllerian structures. Of these, two patients harbored missense mutations within the DNA‐binding region of SF1 (p.C33S, p.R84H), one patient had a nonsense mutation (p.Y138X) and one patient had a frameshift mutation (c.1277dupT) predicted to disrupt RNA stability or protein function. One additional patient ([c.424_427dupCCCA]+[p.G146A]) displayed a more marked phenotype of severe gonadal dysgenesis, normal female external genitalia, and Müllerian structures. Functional studies of the missense mutants (p.C33S, p.R84H) and of one nonsense mutant (p.Y138X) revealed impaired transcriptional activation of SF1‐responsive target genes. To date, adrenal insufficiency has not occurred in any of the patients. Thus, SF1 mutations are a relatively frequent cause of 46,XY DSD in humans. Hum Mutat 29(1), 59–64, 2008.

Mutations of Genes Involved in the Innate Immune System as Predictors of Sepsis in Very Low Birth Weight Infants

Mutations of genes involved in the innate immune system have been reported to be associated with an increased sepsis rate in adults. We determined the −159T mutation of the CD14 gene, the 896G mutation of the toll-like receptor 4 gene, the 3020insC mutation of the NOD2 gene (NOD2-3020insC), the IL-6 174G/C promoter polymorphism (IL6-174G/C), and the mannose-binding lectin genotype and their association to the subsequent development of neonatal sepsis in a large cohort of very low birth weight (VLBW) infants. Fifty (14%) of 356 VLBW infants developed blood culture–proven sepsis during their stay in the hospital. VLBW infants carrying the NOD2-3020insC allele (n =15) and the IL6-174G allele (n =121) had a significantly higher rate of blood culture–proven sepsis (33% and 19.8%, respectively) than VLBW infants without these genotypes (p = 0.046 and 0.035, respectively). In a multivariate logistic regression analysis, gestational age less than 28 wk (odds ratio, 3.2; 95% confidence interval, 1.7–6.0; p < 0.001) and the homozygous IL6-174G allele (odds ratio, 1.9; 95% confidence interval, 1.0–3.9; p = 0.039) were predictive for the development of sepsis, whereas the NOD2-3020insC allele was only of borderline significance (odds ratio, 3.2; 95% confidence interval, 1.0–10.4; p = 0.052). VLBW infants with repeated episodes of sepsis had higher frequencies of the NOD2-3020insC and IL6-174G allele. The increased sepsis rate of homozygous IL6-174G carriers was especially related to an increase in Gram-positive infections, and was not observed in VLBW infants who received prophylaxis with teicoplanin (frequency of Gram-positive sepsis in homozygous IL6-174G carriers without prophylaxis 16.5%versus 2.4% in homozygous IL6-174G carriers with prophylaxis; p = 0.033).

The spectrum of phenotypes associated with mutations in steroidogenic factor 1 (SF-1, NR5A1, Ad4BP) includes severe penoscrotal hypospadias in 46,XY males without adrenal insufficiency

Objective Hypospadias is a frequent congenital anomaly but in most cases an underlying cause is not found. Steroidogenic factor 1 (SF-1, NR5A1, Ad4BP) is a key regulator of human sex development and an increasing number of SF-1 (NR5A1) mutations are reported in 46,XY disorders of sex development (DSD). We hypothesized that NR5A1 mutations could be identified in boys with hypospadias. Design and methods Mutational analysis of NR5A1 in 60 individuals with varying degrees of hypospadias from the German DSD network. Results Heterozygous NR5A1 mutations were found in three out of 60 cases. These three individuals represented the most severe end of the spectrum studied as they presented with penoscrotal hypospadias, variable androgenization of the phallus and undescended testes (three out of 20 cases (15%) with this phenotype). Testosterone was low in all three patients and inhibin B/anti-Müllerian hormone (AMH) were low in two patients. Two patients had a clear male gender assignment. Gender re-assignment to male occurred in the third case. Two patients harbored heterozygous nonsense mutations (p.Q107X/WT, p.E11X/WT). One patient had a heterozygous splice site mutation in intron 2 (c.103-3A/WT) predicted to disrupt the main DNA-binding motif. Functional studies of the nonsense mutants showed impaired transcriptional activation of an SF-1-responsive promoter (Cyp11a). To date, adrenal insufficiency has not occurred in any of the patients. Conclusions SF-1 (NR5A1) mutations should be considered in 46,XY individuals with severe (penoscrotal) hypospadias, especially if undescended testes, low testosterone, or low inhibin B/AMH levels are present. SF-1 mutations in milder forms of idiopathic hypospadias are unlikely to be common.

Genotype-phenotype analysis in congenital adrenal hyperplasia due to P450 oxidoreductase deficiency

Context: P450 oxidoreductase deficiency (PORD) is a unique congenital adrenal hyperplasia variant that manifests with glucocorticoid deficiency, disordered sex development (DSD), and skeletal malformations. No comprehensive data on genotype-phenotype correlations in Caucasian patients are available. Objective: The objective of the study was to establish genotype-phenotype correlations in a large PORD cohort. Design: The design of the study was the clinical, biochemical, and genetic assessment including multiplex ligation-dependent probe amplification (MLPA) in 30 PORD patients from 11 countries. Results: We identified 23 P450 oxidoreductase (POR) mutations (14 novel) including an exonic deletion and a partial duplication detected by MLPA. Only 22% of unrelated patients carried homozygous POR mutations. p.A287P was the most common mutation (43% of unrelated alleles); no other hot spot was identified. Urinary steroid profiling showed characteristic PORD metabolomes with variable impairment of 17α-hydroxylase and 21-hydroxylase. Short cosyntropin testing revealed adrenal insufficiency in 89%. DSD was present in 15 of 18 46,XX and seven of 12 46,XY individuals. Homozygosity for p.A287P was invariably associated with 46,XX DSD but normal genitalia in 46,XY individuals. The majority of patients with mild to moderate skeletal malformations, assessed by a novel scoring system, were compound heterozygous for missense mutations, whereas nearly all patients with severe malformations carried a major loss-of-function defect on one of the affected alleles. Conclusions: We report clinical, biochemical, and genetic findings in a large PORD cohort and show that MLPA is a useful addition to POR mutation analysis. Homozygosity for the most frequent mutation in Caucasians, p.A287P, allows for prediction of genital phenotype and moderate malformations. Adrenal insufficiency is frequent, easily overlooked, but readily detected by cosyntropin testing.

Psychosexual Development in Adolescents and Adults with Disorders of Sex Development—Results from the German Clinical Evaluation Study

INTRODUCTION Both biological and psychosocial factors influence psychosexual development. High levels of pre- and postnatal androgens lead to more male-typical behavior. So far, the influence of androgens on gender identity and sexual orientation is unclear. Disorders of sex development (DSDs) are heterogeneous genetic conditions with different levels of prenatal androgens resulting in variations of genital development. Through DSD, the role of the different factors, especially androgen exposure, on psychosexual development can be evaluated. AIM The purpose of the study was to assess psychosexual development in adolescents and adults with different forms of DSD. METHODS For the examination of psychosexual development of 66 adolescents and 110 adults with DSD, the authors used the Utrecht Gender Dysphoria Scale for adolescents, the Questionnaire of Gender Identity for adults, and a condition-specific DSD study questionnaire. Individuals were analyzed in four subgroups reflecting the karyotype, absence/presence of androgen effects, and gender of rearing. MAIN OUTCOME MEASURES Main outcome measures used were gender identity, friendships, love and sexual relationships, and sexual orientation in adolescents and adults with DSD. RESULTS Individuals with DSD did not show increased gender dysphoria. However, partnership and sexuality were identified to be difficult areas of life. Both adolescents and adults with DSD reported fewer experiences regarding love or sexual relationships compared with unaffected individuals. Especially men with DSD and undervirilization and women with DSD and androgen effects less often had a love relationship. Adult women with DSD and androgen effects more frequently engaged in love and sexual relationships with individuals of the same gender compared with women without DSD. CONCLUSION Individuals with DSD experience atypical hormonal influences (higher levels of androgens in girls/women and lower levels in androgens in boys/men); however, they did not show increased gender dysphoria in this study. However, partnership and sexual relationships are difficult areas of life for adolescents and adults with DSD. We recommend that individuals with DSD should get support from a multiprofessional team with competency in assessing and counseling issues regarding relationships and sexuality. Contact to other individuals with DSD can be helpful for nonprofessional support and exchange of experiences.

Pubertal Presentation in Seven Patients with Congenital Adrenal Hyperplasia due to P450 Oxidoreductase Deficiency

Adolescents with oxidoreductase deficiency present with impaired pubertal development, manifesting in girls with hypergonadotropic hypogonadism and ovarian cysts while boys may show delayed but spontaneous pubertal progression.

Bilateral Wilms Tumor in a Boy with Severe Hypospadias and Cryptorchidism Due to a Heterozygous Mutation in the WT1 Gene

Mutations in the WT1 gene causing Wilms tumors were first reported in WAGR syndrome (Wilms tumor, Aniridia, Genitourinary malformation, mental Retardation) and Denys Drash syndrome (pseudohermaphroditism, Wilms tumor, nephropathy), but only in a few patients with hypospadias and cryptorchidism without other signs of Denys Drash (DDS) or WAGR syndrome WT1 mutations were identified. We report a boy, who was born in 1989 with hypospadias and bilateral cryptorchidism. Previous karyotyping and endocrine studies had ruled out any known cause of male pseudohermaphroditism. Subsequently, he developed a bilateral Wilms tumor, which was detected by palpation at the age of 15 months during a routine visit by the general pediatrician. Because of its extensive size, surgery and chemotherapy were needed for treatment. Analysis of the WT1 gene was performed 5 y after diagnosis and revealed a C to T transition in one allele generating a stop codon at codon 362 and subsequently leading to a truncated protein with loss of its ability to bind to DNA. No signs of DDS or WAGR syndrome are present in the boy. The work up of this patient and the so far known few comparable cases from the literature lead to the conclusion that in newborns with severe urogenital malformations not due to known chromosomal or endocrine disorders mutational screening of the WT1 gene should be performed, to evaluate the high risk of developing a Wilms tumor. We favor mutational screening in these patients as an easy tool for investigation, because in the future it will probably decrease the necessity of frequent control visits in patients without a WT1 mutation.

Complete androgen insensitivity syndrome is frequently due to premature stop codons in exon 1 of the androgen receptor gene: an international collaborative report of 13 new mutations.

OBJECTIVE To confirm the clinical diagnosis of complete androgen insensitivity syndrome (CAIS) by molecular genetic analysis and to determine the prevalence of exon 1 mutations in the androgen receptor (AR) transactivation defects of a large series of CAIS patients. DESIGN International retrospective study. SETTING University Hospital of Montpellier, Department of Hormonology. PATIENT(S) 105 patients with normal female external genitalia, bilateral intra-abdominal or inguinal testis, normal breast development, absent or sparse pubic hair, normal or high endogenous testosterone production, hypoplastic or absent wolffian structures, and 46,XY karyotype. INTERVENTION(S) Sequencing of the AR gene. MAIN OUTCOME MEASURE(S) Prevalence of AR exon 1 mutations. RESULT(S) Over a 10-year period (1997 to 2007), we identified 78 AR gene mutations in 105 patients with CAIS; 21 of them were located in exon 1, and 13 of these were new mutations. We report 13 new mutations in the AR gene. All but one were stop codons, and the last was a splicing abnormality. CONCLUSION(S) The finding that 27% of the mutations in our cohort were localized in exon 1 versus 15% in previous works justifies the sequencing of this exon in patients with CAIS.

Testosterone production during puberty in two 46,XY patients with disorders of sex development and novel NR5A1 (SF-1) mutations

Background Steroidogenic factor 1 (SF-1, NR5A1) is a key transcriptional regulator of many genes involved in the hypothalamic–pituitary–gonadal axis and mutations in NR5A1 can result in 46,XY disorders of sex development (DSD). Patients with this condition typically present with ambiguous genitalia, partial gonadal dysgenesis, and absent/rudimentary Müllerian structures. In these cases, testosterone is usually low in early infancy, indicating significantly impaired androgen synthesis. Further, Sertoli cell dysfunction is seen (low inhibin B, anti-Müllerian hormone). However, gonadal function at puberty in patients with NR5A1 mutations is unknown. Subjects and methods Clinical assessment, endocrine evaluation, and genetic analysis were performed in one female and one male with 46,XY DSD who showed spontaneous virilization during puberty. The female patient presented at adolescence with clitoral hypertrophy, whereas the male patient presented at birth with severe hypospadias and entered puberty spontaneously. Molecular analysis of NR5A1 was performed followed by in vitro functional analysis of the two novel mutations detected. Results Testosterone levels were normal during puberty in both patients. Analysis of NR5A1 revealed two novel heterozygous missense mutations in the ligand-binding domain of SF-1 (patient 1: p.L376F; patient 2: p.G328V). The mutant proteins showed reduced transactivation of the CYP11A promoter in vitro. Conclusion Patients with 46,XY DSD and NR5A1 mutations can produce sufficient testosterone for spontaneous virilization during puberty. Phenotypic females (46,XY) with NR5A1 mutations can present with clitoromegaly at puberty, a phenotype similar to other partial defects of androgen synthesis or action. Testosterone production in 46,XY males with NR5A1 mutations can be sufficient for virilization at puberty. As progressive gonadal dysgenesis is likely, gonadal function should be monitored in adolescence and adulthood, and early sperm cryopreservation considered in male patients if possible.

Utilization of Health Care Services and Satisfaction with Care in Adults Affected by Disorders of Sex Development (DSD)

ABSTRACTBACKGROUNDDisorders of sex development (DSD) are a heterogeneous group of rare genetic disorders of sex determination or differentiation. Evidence-based guidelines concerning gender assignment and surgical and hormonal treatment are limited for many DSD entities, and health care is highly fragmented across various sub-specialties and settings. A lack of informed consent, secrecy about the condition, shame, and impaired sexual and psychosocial functioning may affect satisfaction with care.OBJECTIVESThe main goal of this study was to describe satisfaction with care in individuals with DSD and to identify factors associated with low satisfaction with care.METHODS / MAIN MEASURESUsing both biological (chromosomes) and social categories (sex of rearing), we classified participants according to the nomenclature of the European Society for Pediatric Endocrinology/Lawson Wilkins Pediatric Endocrine Society (ESPE/LWPES) consensus statement. We used standardized measures to assess satisfaction with care (CSQ-8), health-related quality of life (SF-36), psychological symptoms (BSI), and gender identity (FGI), in addition to self-constructed questionnaires probing experiences with health care and access to self-help groups.PARTICIPANTSA total of 110 adults were recruited between January 2005 and December 2007 in four study centers in Germany, Austria, and German-speaking Switzerland.RESULTSReports of half the participants scored below the cut-off indicating low quality of care. Women with XX DSD conditions and virilization (i.e., congenital adrenal hyperplasia) reported the highest scores for satisfaction with care, and women with XY DSD conditions and complete lack of androgen effects reported the lowest scores. Satisfaction with care was positively associated with indicators of psychological well-being.CONCLUSIONSSatisfaction with care is lowest among participants with the rarest conditions, highlighting the lack of evidence-based recommendations and the lack of coordination of care. Associations of satisfaction and well-being indicate the need to ensure access to mental health services.