Homozygous p.Val89Leu plays an important pathogenic role in 5α-reductase type 2 deficiency patients with homozygous p.Arg246Gln in SRD5A2.

OBJECTIVE: To evaluate the pathogenic role of a few benign variants and hypomorphic pathogenic variants in SRD5A2. DESIGN AND METHODS: We retrospectively analyzed phenotypes and genotypes in 23 Indian patients with genetically proven steroid 5α-reductase 2 (SRD5A2) deficiency. The interactions of the SRD5A2 enzymes resulting due to the most common benign variant (p.Val89Leu), the most common (hypomorphic) pathogenic variant (p.Arg246Gln) and the double variants (p.Val89Leu and p.Arg246Gln) in SRD5A2 were compared with that of the wild type (WT) enzyme by molecular dynamics (MD) simulation. RESULTS: The majority (n = 19, 82.61%) of patients presented for atypical genitalia and had male gender identity (16/20). Including the two novel ones (p.Leu83Pro, p.Ala28Leufs*103), a total of nine different pathogenic variants were observed. p.Arg246Gln was the most common pathogenic variant (n = 12). Homozygous p.Arg246Gln (n = 9) variant was associated with milder undervirilization (Sinnecker score of ≤3a: 8/9 vs 6/14, P = 0.04) and had concurrent homozygous p.Val89Leu in all patients. Interestingly, asymptomatic fathers of two index patients were homozygous for p.Arg246Gln which questioned the pathogenicity of the variation as a sole factor. Unlike all symptomatic homozygous p.Arg246Gln patients who were also homozygous for p.Val89Leu, asymptomatic homozygous p.Arg246Gln fathers were heterozygous for p.Val89Leu. On MD simulation SRD5A2 p.Val89Leu-Testeosterone (T) and SRD5A2 p.Arg246Gln-T complexes, but not SRD5A2 p.Val89Leu and p.Arg246Gln-T complex, demonstrated close interaction between NADPH and T as that of SRD5A2 WT-T. CONCLUSIONS: p.Arg246Gln may not be pathogenic as a sole variation even in the homozygous state; additional contribution of homozygous p.Val89Leu variant may be essential for the pathogenicity of p.Arg246Gln in SRD5A2.

5α-Reductase-2 deficiency: is gender assignment recommended in infancy? Two case-reports and review of the literature.

PURPOSE: Gender assignment represents one of the most controversial aspects of the clinical management of individuals with Differences of Sex Development, including 5α-Reductase-2 deficiency (SRD5A2). Given the predominant female appearance of external genitalia in individuals with SRD5A2 deficiency, most of them were assigned to the female sex at birth. However, in the last years the high rate of gender role shift from female to male led to recommend a male gender assignment. METHODS: We here describe two cases of subjects with SRD5A2 deficiency assigned as females at birth, reporting their clinical histories and psychometric evaluations (Body Uneasiness Test, Utrecht Gender Dysphoria Scale, Bem Sex-Role Inventory, Female Sexual Distress Scale Revised, visual analogue scale for gender identity and sexual orientation) performed at the time of referral at the Florence Gender Clinic. RESULTS: Both patients underwent early surgical interventions without being included in the decision-making process. They had to conform to a binary feminine gender role because of social/familiar pressure, with a significant impact on their psychological well-being. Psychometric evaluations identified clinically significant body uneasiness and gender incongruence in both subjects. No sexually related distress and undifferentiated gender role resulted in the first subject and sexually related distress and androgynous gender role resulted in the second subject. CONCLUSIONS: The reported cases suggest the possibility to consider a new approach for gender assignment in these individuals, involving them directly in the decision-making process and allowing them to explore their gender identity, also with the help of GnRH analogues to delay pubertal modifications.

CYP21A2 genetic profile in 14 Egyptian children with suspected congenital adrenal hyperplasia: a diagnostic challenge.

CYP21A2 genotyping remains an important element in the diagnosis and management of congenital adrenal hyperplasia, and establishing accurate genotype-phenotype correlations has facillitated adequate genetic counseling and prenatal management for at-risk families. Despite extensive efforts to establish a clear genotype-phenotype correlation, some discordance remains. Establishing a diagnosis of congenital adrenal hyperplasia on the basis of biochemical and clinical data is occasionally challenging, and the identification of CYP21A2 mutations may help confirm the diagnosis. We review the diagnostic challenges despite an extensive genetic evaluation for 14 patients with a suspected clinical and biochemical diagnosis of congenital adrenal hyperplasia. Other diagnostic entities should be considered in the absence of convincing genetic data.

5α-Reductase-2 Deficiency: Clinical Findings, Endocrine Pitfalls, and Genetic Features in a Large Italian Cohort.

Clinical records (n = 24) with an established diagnosis of 5α-reductase-2 deficiency were reviewed. A previous misdiagnosis was present in about 70% (period from first observation to definitive diagnosis: 9.1 ± 10.8 years), and in 8 children gonadal removal was performed before certain diagnosis. Initial sex assignment was female in 16/24 (67%) and male in 8/24 (33%) cases. After diagnosis, sex re-assignment was performed in 5 babies (4 girls to male sex; 1 boy to female sex). Baseline testosterone/DHT ratio was diagnostic in 6/12 subjects (first months of life n = 4; puberty n = 2), while post-hCG testosterone/DHT ratio was diagnostic in all tested individuals (choosing both the cut-off value 15 or 10). Eighteen different mutations in the steroid-5α-reductase-2 (SRD5A2) gene were identified, 5 of which have never been reported. In conclusion, a time lag exists before the diagnosis of 5α-reductase-2 deficiency is established; sex assignment and gonadal removal may be performed before certain diagnosis. Sex re-assignment is usually female to male, but the contrary may occur. A large variability in clinical phenotypes and genetic mutations was present in this cohort. Accurate endocrine evaluation is recommended in babies possibly affected by 5α-reductase-2 deficiency, since the use of appropriate cut-off values of testosterone/DHT ratio after hCG stimulation may permit to select individuals for SRD5A2 gene analysis. A genotype-phenotype correlation was not found in this study.

Two different patterns of mini-puberty in two 46,XY newborns with 17ß-hydroxysteroid dehydrogenase type 3 deficiency.

We report two newborns with female external genitalia and bilateral inguinal swelling who were diagnosed with 17ß-hydroxysteroid dehydrogenase type 3 deficiency, a rare cause of 46,XY disorder of sexual development. The first case had normal clitoral size and vaginal and urethral openings, palpable gonads in the inguinal region, low testosterone, and low levels of basal and GNRH-stimulated gonadotropin. The second case had similar external genitalia, low testosterone but borderline basal and normal stimulated gonadotropin levels. Low testosterone/androstenedione ratios (0.22 and 0.24, respectively; normal, >0.8) after human chorionic gonadotropin stimulation indicated 17ß-hydroxysteroid dehydrogenase type 3 deficiency. HSD17B3 sequencing revealed a homozygous novel mutation (c.464A>C, p.H155P) in exon 6 in the first case and homozygous c.239G>A (p.R80Q) in exon 3 in the second.

17ßHSD-3 enzyme deficiency due to novel mutations in the HSD17B3 gene diagnosed in a neonate.

17-ß-Hydroxysteroid dehydrogenase type 3 (17ßHSD-3) is present almost exclusively in the testes, and converts androstenedione (A) to testosterone (T). 17ßHSD-3 deficiency is rare. The diagnosis can be missed in early childhood as the clinical presentation may be subtle. The most frequent presentation of 17 HSD-3 deficiency is a 46,XY individual with female external genitalia, labial fusion and a blind ending vagina, with or without clitoromegaly. A low testosterone/androstenedion (T/A) ratio is suggestive of 17ßHSD-3 deficiency, and such diagnosis can be confirmed with molecular genetic studies. A 12-day newborn was referred to our hospital because of palpable gonads in the labia majora. On physical examination, the baby had female external genitalia and palpable gonads in the labia majora. T/A ratio was 0.26 and the diagnosis was 17ßHSD-3 deficiency, which was confirmed by the evidence of compound heterozygousity novel frameshift mutations in exon 9 and 10 of HSD17B3 gene.

5-Alpha reductase deficiency: a 40-year retrospective review.

PURPOSE OF REVIEW: 5-Alpha reductase is an enzyme responsible for the conversion of testosterone to dihydrotestosterone. This key enzyme is responsible for triggering masculinization of the male external genitalia. Discovery of 5-alpha reductase deficiency as a syndrome of disordered male sexual development led to our molecular understanding of the role that this key enzyme plays in male sexual differentiation. This article will review the clinical and molecular history behind the discovery of 5-alpha reductase deficiency. RECENT FINDINGS: Three different genes encoding for 5-alpha reductase have been identified, with 5-alpha reductase type 2 being implicated in disordered male sexual development. SUMMARY: The discovery of 5-alpha reductase deficiency has not only shed light on the crucial role of 5-alpha reductase, testosterone, and dihydrotestosterone in male sexual differentiation but it also has facilitated the discovery of novel therapeutic applications of 5-alpha reductase inhibitors in clinical practice.

Transgenic expression of Map3k4 rescues T-associated sex reversal (Tas) in mice.

Disorders of sex development in the human population range in severity from mild genital defects to gonadal sex reversal. XY female development has been associated with heterozygous mutations in several genes, including SOX9, WT1 and MAP3K1. In contrast, XY sex reversal in mice usually requires complete absence of testis-determining gene products. One exception to this involves T-associated sex reversal (Tas), a phenomenon characterized by the formation of ovotestes or ovaries in XY mice hemizygous for the hairpin-tail (T(hp)) or T-Orleans (T(Orl)) deletions on proximal mouse chromosome 17. We recently reported that mice heterozygous for a null allele of Map3k4, which resides in the T(hp) deletion, exhibit XY ovotestis development and occasional gonadal sex reversal on the sensitized C57BL/6J-Y(AKR) (B6-Y(AKR)) genetic background, reminiscent of the Tas phenotype. However, these experiments did not exclude the possibility that loss of other loci in the T(hp) deletion, or other effects of the deletion itself, might contribute to Tas. Here, we show that disruption to Sry expression underlies XY gonadal defects in B6-Y(AKR) embryos harbouring the T(hp) deletion and that a functional Map3k4 bacterial artificial chromosome rescues these abnormalities by re-establishing a normal Sry expression profile. These data demonstrate that Map3k4 haploinsufficiency is the cause of T-associated sex reversal and that levels of this signalling molecule are a major determinant of the expression profile of Sry.

Role of aldo-keto reductase family 1 (AKR1) enzymes in human steroid metabolism.

Human aldo-keto reductases AKR1C1-AKR1C4 and AKR1D1 play essential roles in the metabolism of all steroid hormones, the biosynthesis of neurosteroids and bile acids, the metabolism of conjugated steroids, and synthetic therapeutic steroids. These enzymes catalyze NADPH dependent reductions at the C3, C5, C17 and C20 positions on the steroid nucleus and side-chain. AKR1C1-AKR1C4 act as 3-keto, 17-keto and 20-ketosteroid reductases to varying extents, while AKR1D1 acts as the sole Δ(4)-3-ketosteroid-5ß-reductase (steroid 5ß-reductase) in humans. AKR1 enzymes control the concentrations of active ligands for nuclear receptors and control their ligand occupancy and trans-activation, they also regulate the amount of neurosteroids that can modulate the activity of GABAA and NMDA receptors. As such they are involved in the pre-receptor regulation of nuclear and membrane bound receptors. Altered expression of individual AKR1C genes is related to development of prostate, breast, and endometrial cancer. Mutations in AKR1C1 and AKR1C4 are responsible for sexual development dysgenesis and mutations in AKR1D1 are causative in bile-acid deficiency.

Clinical and molecular spectrum of patients with 17β-hydroxysteroid dehydrogenase type 3 (17-β-HSD3) deficiency / Espectro clínico e molecular de pacientes com deficiência de 17β-hidroxiesteroide desidrogenase tipo 2 (17-β-HSD3)

The enzyme 17β-hydroxysteroid dehydrogenase type 3 (17-β-HSD3) catalyzes the conversion of androstenedione to testosterone in the testes, and its deficiency is a rare disorder of sex development in 46,XY individuals. It can lead to a wide range of phenotypic features, with variable hormonal profiles. We report four patients with the 46,XY karyotype and 17-β-HSD3 deficiency, showing different degrees of genital ambiguity, increased androstenedione and decreased testosterone levels, and testosterone to androstenedione ratio < 0.8. In three of the patients, diagnosis was only determined due to the presence of signs of virilization at puberty. All patients had been raised as females, and female gender identity was maintained in all of them. Compound heterozygosis for c.277+2T>G novel mutation, and c.277+4A>T mutation, both located within the intron 3 splice donor site of the HSD17B3 gene, were identified in case 3. In addition, homozygosis for the missense p.Ala203Val, p.Gly289Ser, p.Arg80Gln mutations were found upon HSD17B3 gene sequencing in cases 1, 2, and 4, respectively. Arq Bras Endocrinol Metab. 2012;56(8):533-9.

A enzima 17β-hidroxiesteroide desidrogenase tipo 3 (17-β-HSD3) catalisa a conversão de androstenediona a testosterona nos testículos, e sua deficiência é uma forma rara de distúrbio do desenvolvimento do sexo em indivíduos 46,XY. A desordem apresenta um amplo espectro de características fenotípicas e de resultados de dosagens laboratoriais. Neste trabalho, são relatados quatro casos de deficiência da 17-β-HSD3 com cariótipo 46,XY, ambiguidade genital em diversos graus, androstenediona aumentada, testosterona diminuída, e relação testosterona e androstenediona < 0,8. Em três das pacientes, o diagnóstico foi suspeitado devido à presença de sinais de virilização na puberdade. Todos os pacientes foram criados como mulheres, e a identidade de gênero feminino foi mantida em todas elas. A heterozigose composta da mutação nova c.277+2T>G e da mutação c.277+4A>T, ambas localizadas no sítio doador de splicing do íntron 3 do gene HSD17B3, foi identificada no caso 3. Além dessas, as mutações missense p.Ala203Val, p.Gly289Ser, p.Arg80Gln foram identificadas em homozigose pelo sequenciamento do gene HSD17B3 dos casos 1, 2 e 4, respectivamente. Arq Bras Endocrinol Metab. 2012;56(8):533-9.

Clinical and molecular spectrum of patients with 17ß-hydroxysteroid dehydrogenase type 3 (17-ß-HSD3) deficiency.

The enzyme 17ß-hydroxysteroid dehydrogenase type 3 (17-ß-HSD3) catalyzes the conversion of androstenedione to testosterone in the testes, and its deficiency is a rare disorder of sex development in 46,XY individuals. It can lead to a wide range of phenotypic features, with variable hormonal profiles. We report four patients with the 46,XY karyotype and 17-ß-HSD3 deficiency, showing different degrees of genital ambiguity, increased androstenedione and decreased testosterone levels, and testosterone to androstenedione ratio < 0.8. In three of the patients, diagnosis was only determined due to the presence of signs of virilization at puberty. All patients had been raised as females, and female gender identity was maintained in all of them. Compound heterozygosis for c.277+2T>G novel mutation, and c.277+4A>T mutation, both located within the intron 3 splice donor site of the HSD17B3 gene, were identified in case 3. In addition, homozygosis for the missense p.Ala203Val, p.Gly289Ser, p.Arg80Gln mutations were found upon HSD17B3 gene sequencing in cases 1, 2, and 4, respectively.

Consequences of POR mutations and polymorphisms.

P450 oxidoreductase (POR) transports electrons from NADPH to all microsomal cytochrome P450 enzymes, including steroidogenic P450c17, P450c21 and P450aro. Severe POR mutations A287P (in Europeans) and R457H (in Japanese) cause the Antley-Bixler skeletal malformation syndrome (ABS) plus impaired steroidogenesis (causing genital anomalies), but the basis of ABS is unclear. We have characterized the activities of ∼40 POR variants, showing that assays based on P450c17 activities, but not cytochrome c assays, correlate with the clinical phenotype. The human POR gene is highly polymorphic: the A503V sequence variant, which decreases P450c17 activities to ∼60%, is found on ∼28% of human alleles. A promoter polymorphism (∼8% of Asians and ∼13% of Caucasians) at -152 reduces transcriptional activity by half. Screening of 35 POR variants showed that most mutants lacking activity with P450c17 or cytochrome c also lacked activity to support CYP1A2 and CYP2C19 metabolism of EOMCC (a fluorogenic non-drug substrate), although there were some remarkable differences: Q153R causes ABS and has ∼30% of wild-type activity with P450c17 but had 144% of WT activity with CYP1A2 and 284% with CYP2C19. The effects of POR variants on CYP3A4, which metabolizes nearly 50% of clinically used drugs, was examined with multiple, clinically relevant drug substrates, showing that A287P and R457H dramatically reduce drug metabolism, and that A503V variably impairs drug metabolism. The degree of activity can vary with the drug substrate assayed, as the drugs can influence the conformation of the P450. POR is probably an important contributor to genetic variation in both steroidogenesis and drug metabolism.

SRD5A2 gene mutations--a population-based review.

Knowledge of steroid 5 alpha-reductase type 2 (SRD5A2) gene mutations is expanding, and its role has been implicated in various disease susceptibilities concerning reproductive health. Extensive research has revealed the tendency for specific SRD5A2 gene mutations to be passed along certain racial, ethnic and geographically isolated groups, which suggests population specificity of these mutations. The review provides evidence of variation in the mutational spectrum of the SRD5A2 gene leading to population-specific high prevalence of characteristic disease or phenotypic expression.

Gender identity of children and young adults with 5alpha-reductase deficiency.

Male pseudohermaphroditism (46,XY DSD) due to 5alpha-reductase deficiency has been recognized for the last few decades. There is scant literature on this entity in India. We compiled data on five patients with this disorder. Four of our five patients were reared as females. Our assessment of these children reveals that they had male gender identity from childhood. Three of the four reared as females chose to change gender role at adolescence, while the fourth is still prepubertal. We conclude that all these patients had male gender identity from early childhood. The parents took note of this only after the appearance of male secondary sexual characteristics at puberty, thereby giving an impression of change in gender identity and gender role.

Novel compound heterozygous mutations in the SRD5A2 gene from 46,XY infants with ambiguous external genitalia.

Dihydrotestosterone is crucial for normal development of external genitalia and prostate in the male embryo. Autosomal recessive mutations in the 5 alpha-reductase type 2 (SRD5A2) gene disrupt the synthesis of dihydrotestosterone in the urogenital tract and give rise to genetic males with undervirilized external genitalia that may be female-like or ambiguous. In this study, three unrelated 46,XY children (0.5, 3, and 8 years old) who presented severe undermasculinization at birth were examined for genetic abnormalities in the SRD5A2 gene. Coding sequence abnormalities were ascertained by exon-specific polymerase chain reaction (PCR), single-stranded conformational polymorphism (SSCP), and sequencing analysis. Functional properties of the mutant alleles were investigated by means of site-directed mutagenesis assays. DNA molecular studies showed that all three patients were compound heterozygotes for SRD5A2 mutations. Patient 1 had a point mutation 547G --> A in exon 3 (G183S) and a novel dinucleotidic mutation 634,635CC --> TG in exon 4 (P212X). This double change results in premature termination signal (TGA) at codon 212, which predicts the expression of a truncated 211-amino acid protein. Patient 2 was the carrier of mutations G115D in exon 3 and S210F in exon 4. Patient 3 had two substitution mutations in exon 1, including a novel G --> C transversion at nucleotide 169 (E57Q) and a G --> A transition at nucleotide 254 (G85D). In transitory transfection assays, the recombinant cDNAs harboring mutations E57Q and G85D showed residual 5 alpha-reductase activity, whereas those with mutations G115D, S210F, and P212X were devoid of activity. In contrast, the G183S substitution affected the catalytic activity of the enzyme by decreasing its affinity for testosterone substrate. We describe six different mutations of the SRD5A2 gene detected in three children with genital ambiguity. These genotypes are consistent with the clinical phenotype of steroid 5 alpha-reductase 2 deficiency. Our data suggest that the combined gene variants (E57Q/G85D, G115D/S210F, and G183S/P212X) result in subfunctional or nonfunctional enzymes, causing masculinization defects in these patients. This further underscores that exon 4 of SRD5A2 may be a site prone to inactivating mutations.

Identification of mutations in the SRD5A2 gene in Thai patients with male pseudohermaphroditism.

OBJECTIVE: To describe two unrelated Thai patients with suspected 5alpha-reductase type 2 deficiency and perform mutation analysis of the SRD5A2 gene. DESIGN: Case report. SETTING: A pediatric endocrinology clinic at a university hospital. PATIENT(S): Two unrelated patients with 46,XY karyotype, born with ambiguous genitalia, were studied. One was reared as a boy and the other was reared as a girl. INTERVENTION(S): The entire coding regions of the SRD5A2 gene were assessed by polymerase chain reaction (PCR) and sequencing analysis. MAIN OUTCOME MEASURE(S): Molecular characterization of the SRD5A2 gene. RESULT(S): Four different pathogenic mutations (three missense and one nonsense) were identified. These were located at exon 1 (p.Q6X and p.L20P), exon 3 (p.G183S), and exon 4 (p.G203S). The T>C transition (c.59T>C) resulting in a leucine-to-proline substitution at codon 20 (p.L20P) has not been previously described and was not detected in 100 unaffected, ethnic-matched control chromosomes. In addition, p.G183S, previously identified only among patients from mixed African-European ancestry and in the Dominican Republic, was also detected in a Thai patient. CONCLUSION(S): This study demonstrates that the SRD5A2 gene is responsible for 5alpha-reductase type 2 deficiency across different populations and emphasizes the important role of genetic testing for the definite diagnosis and genetic counseling before gender assignment or any surgical intervention.

17beta-hydroxysteroid dehydrogenase 3 deficiency in a male pseudohermaphrodite.

OBJECTIVE: To present the clinical, biochemical, and genetic features of a male pseudohermaphrodite whose condition was caused by 17beta-hydroxysteroid dehydrogenase 3 (17beta-HSD3) deficiency. DESIGN: Case report. SETTING: Gynecology practice in a university teaching hospital. PATIENT(S): A 15-year-old black American male pseudohermaphrodite with 17beta-HSD3 deficiency. INTERVENTION(S): Laboratory evaluation, genetic mutation analysis, bilateral gonadectomy, and hormone replacement. MAIN OUTCOME MEASURE(S): Endocrinologic evaluation and genetic analysis. RESULT(S): A diagnosis of 17beta-HSD3 deficiency made on the basis of hormone evaluation was confirmed through genetic mutation analysis of the HSD17B3 gene. Female phenotype was attained after gonadectomy, passive vaginal dilatation, and hormone therapy. CONCLUSION(S): Deficiency of 17beta-HSD3 was diagnosed in this patient on the basis of endocrinologic evaluation and was confirmed with genetic mutation analysis. The patient was able to retain her female sexual identity after surgical and medical treatment.