5 'alfa' - reductase type 2 deficency : importance of hormonal evaluation in the diagnosis, including anti-mullerian hormone levels

Orientador: Gil Guerra JuniorTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias MedicasResumo: As caracteristicas clínicas de pacientes com deficiência da 5a-redutase 2 são fteqüentemente indistinguíveis de outras causas de pseudo-hermaftoditismo masculino, especialmente as síndromes de insensibilidade androgênica; e o diagnóstico diferencial se torna mais dificil na ausência de casos semelhantes na família, quando não permitem estabelecer o modo de herança. O objetivo deste estudo foi avaliar o perfil hormonal, incluindo a dosagem sérica do hormônio anti-MüIleriano (HAM), em pacientes com diagnóstico molecular de deficiência de 5a-redutase 2. Dados de 14 pacientes foram analisados de acordo com a idade e o estádio puberal. Ao exame fisico inicial a ambigüidade genital. Prader grau m foi observada em 11 casos; ambas as gônadas eram palpáveis em todos os casos. Dois pacientes foram gonadectomizados previamente. Foram dosados LH, FSH, testosterona total (T), dihidrotestosterona (DHT) e HAM em todos os pacientes; e T e DHT em 20 controles. A idade variou de 21 dias a 29 anos; 7 pacientes já tinham iniciado a puberdade. Nos pacientes que ainda mantinham as gônadas, LH e FSH foram normais; T/DHT foi elevada em relação aos controles; todos os pacientes pré-púberes apresentaram HAM abaixo de -1 DP para a idade, enquanto que os púberes tiveram HAM compatível com o estádio puberal. Portanto, este estudo mostra a importância da dosagem sérica do HAM para dirigir a pesquisa molecular em pacientes 46,XY com ambigüidade genital e níveis normais ou elevados de T, particularmente nos casos pré-púberes; aqueles com HAM acima da média para a idade devem ter inicialmente avaliado o gene do receptor de andrógenos; se os valores de HAM estiverem abaixo da média, a análise do gene SRD5A2 deve ser realizadaAbstract: The clinical features of 5a-reductase 2 deficiency patients are hardly distinguishable from those of the other causes of pseudohennaphroditism, especially androgen insensitivity syndromes; the diagnosis becomes even more difficult when family history is unremarkable and there are no clues to a pattem of inheritance. The aim of this study was to eValuatethe hormonal profile, including anti-MüIlerian hormone (AMH), in patients with molecular diagnosis of 5a-reductase 2 deficiency. Data from 14 patients have been analyzed according to age and pubertal stage. Sex ambiguity was rated as Prader m in 11 cases; both gonads were palpable in alI but 2 patients, who had been previously submitted to gonadectomy. LH, FSH, testosterone (T), dihydrotestosterone (DHf) and AMH serum levels were measured in alI patients; T and DHf were also measured in 20 controls. The age range was 21 days to 29 years; 7 patients had reached puberty. In the patients who retained the gonads, LH and FSH levels were normal; TIDHf was elevated in alI cases when compared to controls; alI prepubertal patients had AMH levels < -1 SD for age, while pubertal patients had AMH levels compatible with pubertal stage. This study shows the usefulness of AMH to direct molecular analysis in 46,XY patients with genital ambiguity and normal or elevated T levels, particularly in isolated prepubertal cases; those with AMH values above the mean should be screened for mutations in the androgen receptor gene; if values are below the mean, SRD5A2 gene mutations should be investigated.DoutoradoClinica MedicaDoutor em Clínica Médic

Avaliação funcional das celulas de Leydig e de Sertoli em 24 casos de ambiguidade genital com cariotipo 46,XY

Orientador: Gil Guerra JuniorDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: A investigação diagnóstica etiológica da ambigüidade genital é muito complexa. A diferenciação sexual é dependente da ação de dois hormônios testiculares: a testosterona e o hormônio anti-MüIleriano (HAM). Apesar da função testicular ser tradicionalmente avaliada apenas pela capacidade esteroidogênica das células de Leydig e pela espermatogênese, tem sido mostrado recentemente que a determinação sérica do HAM pode ser de grande utilidade para os clínicos, como rnarcador da função das células de Sertoli. O HAM é o responsável pela regressão dos duetos de MüIler no feto, continua a ser secretado após o nascimento pelas células de Sertoli imaturas, e é negativamente regulado pela testosterona na puberdade. O objetivo deste estudo foi avaliar a função das células de Leydig e de Sertoli em pacientes com ambigüidade genital e cariótipo 46,XY para estabelecer parâmetros bioquímicos a fim de auxiliar no diagnóstico etiológico. Foram realizadas as determinações séricas dos andrógenos, do HAM e das gonadotrofinas em 24 pacientes com ambigüidade genital e cariótipo 46,XY. O diagnóstico etiológico destes casos foi: 8 com disgenesia gonadal, 5 com insensibilidade androgênica, 4 com deficiência de 5a-redutase tipo 2, 3 com deficiência da enzima 3p-OH-esteróide desidrogenase, e 4 idiopáticos. Os resultados mostraram que quando a testosterona estava baixa e as gonadotrofinas algumas vezes elevadas no~ pacientes com disgenesia gonadal ou deficiência da enzima 3 p-OH-esteróide desidrogenase, o HAM apresentou-se muito diminuído no primeiro grupo e normal ou elevado no segundo, auxiliando o diagnóstico. Os valores séricos de HAM e gonadotrofinas mostraram-se normais ou elevados em pacientes com deficiência da enzima 3p-OH-esteróide desidrogenase e insensibilidade androgênica, porém nestes casos a testosterona possibilitou o diagnóstico diferencial. Os valores séricos de testosterona e das gonadotrofinas foram normais ou elevados nas insensibilidades androgênicas e nos casos de deficiência de 5a-redutase tipo 2, entretanto, o HAM apresentou-se normal ou elevado nas insensi.bilidades androgênicas, e diminuído nos outros casos, indicando que a testosterona não necessita ser transformada em dehidrotestosterona para inibir o HAM no testículo. Concluiu-se que a avaliação combinada de andrógenos, HAM egonadotrofinas permite a compreensão fisiopatológica testicular e possibilita o estabelecimento do diagnóstico diferencial de pacientes com ambigüidade genitalAbstract: The investigation of the origin of sex ambiguity is a very complex matter. Sex differentiation is dependent upon the action of two testicular hormones: testosterone and anti-Müllerian hormone (AMH). Although testicular function has traditionally been assessed only by examining the steroidogenic capacity of Leydig cells and spermatogenesis, it has recently been shown that the measurement of the serum AMH may also help clinicians, as a marker of Sertoli cell function. AMH is responsible for the regression of Müllerian ducts in the male fetus, continues to be secreted after birth by immature Sertoli cells, and is negatively regulated by testosterone at puberty. The aim of this study was to evaluate both Leydig and Sertoli cell function in 46,XY patients with intersex states in order to establish biochemical patterns that would help to reach an etiologic diagnosis. We measl,lred serum androgens, AMH and gonadotropins in 24 patients with sex ambiguity and XY karyotype. The diagnosis of these cases was: 8 with gonadal dysgenesis, 5 with androgen insensitivity syndrome, 4 with 5a-reductase 2 deficiency, 3 with 3 p-hydroxysteroid dehydrogenase deficiency, and 4 idiopathic. Our results showed that while testosterone was low and gonadotropins sometimes elevated in patients with either gonadal dysgenesis or 3 p-hydroxysteroid dehydrogenase deficiency, AMH was very low in the former and normal or high in the latter, helping to guide the diagnosis. Serum AMH and gonadotropins were normal or high in patients with either 3 p-hydroxysteroid dehydrogenase deficiency or androgen insensitivity syndrome, but testosterone levels made the distinction. Serum testosterone and gonadotropins were normal or high in androgen insensitivity syndrome and 5a-reductase 2 deficiency patients; however, while AMH was normal or elevated in androgen insensitivity syndrome, it was not the case in 5a-reductase 2 deficiency patients, indicating that testosterone does not need to be transformed to dihydrotestosterone to inhibit AMH in the testis. We conclude that the combined measurement of androgens, AMH and gonadotropins enhances the comprehension of testicular pathophysiology and helps to establish the diagnosis in intersex patientsMestradoClinica MedicaMestre em Clinica Medic

Hormônio anti-mülleriano: revisão e contribuição para a investigação das ambigüidades genitais

Etiological investigation of patients with sex ambiguity and XY karyotype is a complex matter. Traditionally, testicular function has been assessed only by examining the steroidogenic capacity of Leydig cells and spermatogenesis. Recently, it has been shown that measurement of the serum antimüllerian hormone (AMH), as a marker of Sertoli cell function, may also help clinicians. The aim of this review is to show historic and physiological aspects of the AMH, and its utility to establish the diagnosis in patients with intersexual states. The authors experience in intersex evaluation combining the measurement of androgens, AMH and gonadotropins is also showed.A investigação etiológica das ambigüidades genitais com cariótipo 46,XY apresenta dificuldades freqüentes. A função testicular tem sido tradicionalmente avaliada pela capacidade esteroidogênica das células de Leydig e pela espermatogênese. Recentemente, demonstrou-se que a avaliação sérica do hormônio anti-mülleriano (HAM) como marcador da função das células de Sertoli pode ser de grande valia nesta investigação. O objetivo desta revisão é apresentar aspectos históricos e fisiológicos do HAM, e sua utilidade na investigação diagnostica de pacientes com intersexo. Também é mostrada a experiência dos autores na avaliação de intersexo com dosagens combinadas de andrógenos, HAM e testosterona.42543

New Mutations, Hotspots, And Founder Effects In Brazilian Patients With Steroid 5alpha-reductase Deficiency Type 2.

Mutations of the steroid 5alpha-reductase type 2 (SRD5A2) gene in 46,XY subjects cause masculinization defects of varying degrees, due to reduced or impaired enzymatic activity. In this study, sequence abnormalities of the SRD5A2 gene were assessed by polymerase chain reaction with specific primers and automated sequencing analysis in DNA samples from 20 patients with suspected steroid 5alpha-reductase type 2 deficiency from 18 Brazilian families. Eleven subjects presented SRD5A2 homozygous single-base mutations (two first cousins and four unrelated patients with G183S, two with R246W, one with del642T, one with G196S, and one with 217_218insC plus the A49T variant in heterozygosis), whereas four were compound heterozygotes (one with Q126R/IVS3+1G>A, one with Q126R/del418T, and two brothers with Q126R/G158R). Three patients were heterozygous for A207D, G196S, and R266W substitutions. The V89L polymorphism was found in heterozygosis in one of them (with A207D) and in one case with an otherwise normal gene sequence. The A49T variant was also detected in heterozygosis in the second case without other sequencing abnormalities. Four patients harbor yet non-described SRD5A2 gene mutations: a single nucleotide deletion (del642T), a G158R amino acid substitution, a splice junction mutation (IVS3+1G>A), and the insertion of a cytosine (217_218insC) occurring at a CCCC motif. This is the first report of a single-nucleotide insertion in the coding sequence of the SRD5A2 gene. In addition to these new mutations, this investigation reveals the prevalence of G183S substitution among a subset of African-Brazilian patients and presents evidences of the recurrence of already known mutations.83569-7

Morphometry and histology of gonads from 13 children with dysgenetic male pseudohermaphroditism

Background.-Dysgenetic male pseudohermaphroditism (DMP) is a sexual differentiation disorder characterized by bilateral dysgenetic testes, persistent mullerian structures, and cryptorchidism in individuals with a 46,XY karyotype. However, the histologic criteria for the diagnosis of DMP are poorly established.Objective.-To determine gonadal histology in children with DMP.Patients and Methods.-Between 1996 and 1998, 13 patients with DMP were evaluated on our service. The clinical diagnosis of DMP was based on a 46,XY karyotype, sex ambiguity, high levels of follicle-stimulating hormone and low levels of antimullerian hormone, a decreased testosterone response to human chorionic gonadotropin stimulation without accumulation of testosterone precursors, and the presence of mullerian structures. Molecular sequencing the HMGbox region of the SRY gene did not reveal any mutations. Biopsies were performed for 22 of 26 gonads (patient age at the time of biopsy, 16 months to 10 years). Conventional microscopy was used to evaluate mean tubular diameter, tubular fertility index, and number of Sertoli cells per tubular profile.Results.-All 26 gonads were located outside of the labioscrotal folds. Their histologic features varied from only a reduction in tubular size to features of a streak gonad. Five of the 22 gonads grossly resembled a streak gonad. The mean tubular diameter was severely reduced (>30% reduction relative to the normal tubular diameter for the patient's age) in 4 gonads, markedly reduced (10%-30%) in 11 gonads, slightly reduced (<10%) in one gonad, and normal in one gonad. The tubular fertility index, expressed as the percentage of tubular profiles containing germ cells, was severely reduced (<30% of normal values) in 9 gonads, markedly reduced (50%-30%) in 2 gonads, and normal in 6 gonads. The number of Sertoli cells per tubular profile was elevated in 16 gonads and normal in one gonad. Thin tubules surrounded by fibrous tissue were occasionally observed.Conclusion.-The histologic findings confirmed the clinical diagnosis of DMP in every patient in the present series. However, gonadal histology was variable, and careful morphometric evaluation may be necessary to establish the diagnosis.Univ Estadual Campinas, Fac Ciencias Med, Grp Study Sex Determinat & Differentiat, BR-13083970 Campinas, SP, BrazilWeb of Scienc

[5alpha-reductase Type 2 Deficiency: Experiences From Campinas (sp) And Salvador (ba)].

To report the experience regarding patients with steroid 5alpha-reductase type 2 deficiency from three different clinical services in Brazil. Twenty five patients with clinical and hormonal features of 5alpha-reductase deficiency from 23 families (15 from Bahia, 7 from São Paulo and 1 from Minas Gerais) were included in this study. Clinical, hormonal and molecular data were evaluated. The molecular analysis of the five exons of the SRD5A2 gene was done by automatic or manual sequencing of PCR products. In ten families, SRD5A2 mutations were found in homozygosis (5 with G183S, 2 with R246W, 1 with G196S, 1 with del642T, 1 with 217_218insC), in three in compound heterozygosis (1 with Q126R/IVS3+1G>A, 1 with Q126R/del418T, 1 with Q126R/G158R) while other three were heterozygous, with only one deleterious mutation (1 with G196S, 1 with A207D, and 1 with R246W). In seven cases, no sequencing abnormalities were detected. The G183S substitution was the most frequently found among miscegenated patients (Afro-Euro-Brazilians) from Bahia. Hormonal and clinical findings did not differ between patients with or without mutations, exception made to a higher frequency of consanguinity and greater severity of genital ambiguity in the first group. Our results reinforce the importance of molecular investigation for the diagnosis of this disease and point out to the finding of a very frequent mutation (G183S) in our series, especially in patients with mixed ethnic background from Bahia, and the description of mutations that have only been reported in Brazilian patients so far.49103-1