Early development of a gonadal tumor in a patient with mixed gonadal dysgenesis

SUMMARY A gonadal tumor was diagnosed in the first months of life in a patient with genital ambiguity, a 45,X/46,XY karyotype, and mixed gonadal dysgenesis. Gonadal biopsies at the age of 3 months revealed dysgenetic testes and a gonadoblastoma on the right testis. Even though gonadal tumors are rare in childhood, this case indicates that prophylactic removal of dysgenetic gonads should be performed as early as possible, especially when the female sex is assigned to a patient with a Y-chromosome sequence.

Early development of a gonadal tumor in a patient with mixed gonadal dysgenesis.

A gonadal tumor was diagnosed in the first months of life in a patient with genital ambiguity, a 45,X/46,XY karyotype, and mixed gonadal dysgenesis. Gonadal biopsies at the age of 3 months revealed dysgenetic testes and a gonadoblastoma on the right testis. Even though gonadal tumors are rare in childhood, this case indicates that prophylactic removal of dysgenetic gonads should be performed as early as possible, especially when the female sex is assigned to a patient with a Y-chromosome sequence.

Functional characterization of five NR5A1 gene mutations found in patients with 46,XY disorders of sex development.

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.

45,X/46,XY ovotesticular disorder of sex development revisited: undifferentiated gonadal tissue may be mistaken as ovarian tissue.

BACKGROUND: The 45,X/46,XY karyotype has been associated with mixed gonadal dysgenesis (MGD) and ovotesticular disorder of sex development (DSD). Our aim was to revise the diagnosis of ovotesticular DSD in two patients in the context of a retrospective study of 45,X/46,XY subjects with genital ambiguity. CASE PRESENTATION: Patient 1 had a left streak gonad; the right one was considered an ovotestis. Patient 2 had a right testis; the left gonad was considered an ovary. Revision of the histological sections was performed. Both the "ovarian" part of the right gonad of patient 1 and the left "ovary" of patient 2 contained ovarian-type stroma with clusters of sex-cordlike structures and rare germ cells, compatible with undifferentiated gonadal tissue (UGT). Misdiagnosis of ovarian tissue in patients with 45,X/46,XY mosaicism or its variants could also be found in six published case reports. CONCLUSIONS: A distinction between 45,X/46,XY ovotesticular DSD and MGD should be made on past and future cases keeping in mind that UGT may be mistaken as ovarian tissue.

408 Cases of Genital Ambiguity Followed by Single Multidisciplinary Team during 23 Years: Etiologic Diagnosis and Sex of Rearing.

Objective. To evaluate diagnosis, age of referral, karyotype, and sex of rearing of cases with disorders of sex development (DSD) with ambiguous genitalia. Methods. Retrospective study during 23 years at outpatient clinic of a referral center. Results. There were 408 cases; 250 (61.3%) were 46,XY and 124 (30.4%) 46,XX and 34 (8.3%) had sex chromosomes abnormalities. 189 (46.3%) had 46,XY testicular DSD, 105 (25.7%) 46,XX ovarian DSD, 95 (23.3%) disorders of gonadal development (DGD), and 19 (4.7%) complex malformations. The main etiology of 46,XX ovarian DSD was salt-wasting 21-hydroxylase deficiency. In 46,XX and 46,XY groups, other malformations were observed. In the DGD group, 46,XY partial gonadal dysgenesis, mixed gonadal dysgenesis, and ovotesticular DSD were more frequent. Low birth weight was observed in 42 cases of idiopathic 46,XY testicular DSD. The average age at diagnosis was 31.7 months. The final sex of rearing was male in 238 cases and female in 170. Only 6.6% (27 cases) needed sex reassignment. Conclusions. In this large DSD sample with ambiguous genitalia, the 46,XY karyotype was the most frequent; in turn, congenital adrenal hyperplasia was the most frequent etiology. Malformations associated with DSD were common in all groups and low birth weight was associated with idiopathic 46,XY testicular DSD.

[The use of FISH on buccal smear to investigate mosaicism with a 45,X cell line: study on healthy men and patients with disorders of sex development]. / Uso da FISH em mucosa oral para investigação de mosaicismo com linhagem 45,X: estudo com homens saudáveis e pacientes com distúrbios da diferenciação do sexo.

OBJECTIVE: To verify whether fluorescence in situ hybridization (FISH) of cells from the buccal epithelium could be employed to detect cryptomosaicism with a 45,X lineage in 46,XY patients. SUBJECTS AND METHODS: Samples of nineteen 46,XY healthy young men and five patients with disorders of sex development (DSD), four 45,X/46,XY and one 46,XY were used. FISH analysis with X and Y specific probes on interphase nuclei from blood lymphocytes and buccal epithelium were analyzed to investigate the proportion of nuclei containing only the signal of the X chromosome. RESULTS: The frequency of nuclei containing only the X signal in the two tissues of healthy men did not differ (p = 0.69). In all patients with DSD this frequency was significantly higher, and there was no difference between the two tissues (p = 0.38), either. CONCLUSIONS: Investigation of mosaicism with a 45,X cell line in patients with 46,XY DSD or sterility can be done by FISH directly using cells from the buccal epithelium.

Uso da FISH em mucosa oral para investigação de mosaicismo com linhagem 45,X: estudo com homens saudáveis e pacientes com distúrbios da diferenciação do sexo / The use of FISH on buccal smear to investigate mosaicism with a 45,X cell line: study on healthy men and patients with disorders of sex development

Objetivo: Verificar se a hibridização in situ por fluorescência (FISH) em células de mucosa oral poderia ser empregada para detectar criptomosaicismo com linhagem 45,X em pacientes 46,XY. Sujeitos e métodos: Amostra de 19 jovens saudáveis 46,XY e cinco pacientes com distúrbios da diferenciação do sexo (DDS), quatro 45,X/46,XY e um 46,XY. FISH com sondas específicas para X e Y em núcleos interfásicos de linfócitos e mucosa oral para investigar a proporção de núcleos contendo apenas o sinal do cromossomo X. Resultados: A frequência de núcleos contendo apenas o sinal do X nos dois tecidos dos homens saudáveis não diferiu (p = 0,69). Em todos os pacientes com DDS essa frequência foi significativamente maior, e também não houve diferença entre os dois tecidos (p = 0,38). Conclusões: A investigação de mosaicismo com linhagem 45,X em pacientes com DDS 46,XY ou esterilidade pode ser feita por FISH diretamente em células de mucosa oral. .

Objective: To verify whether fluorescence in situ hybridization (FISH) of cells from the buccal epithelium could be employed to detect cryptomosaicism with a 45,X lineage in 46,XY patients. Subjects and methods: Samples of nineteen 46,XY healthy young men and five patients with disorders of sex development (DSD), four 45,X/46,XY and one 46,XY were used. FISH analysis with X and Y specific probes on interphase nuclei from blood lymphocytes and buccal epithelium were analyzed to investigate the proportion of nuclei containing only the signal of the X chromosome. Results: The frequency of nuclei containing only the X signal in the two tissues of healthy men did not differ (p = 0.69). In all patients with DSD this frequency was significantly higher, and there was no difference between the two tissues (p = 0.38), either. Conclusions: Investigation of mosaicism with a 45,X cell line in patients with 46,XY DSD or sterility can be done by FISH directly using cells from the buccal epithelium. .

The novel p.Cys65Tyr mutation in NR5A1 gene in three 46,XY siblings with normal testosterone levels and their mother with primary ovarian insufficiency.

BACKGROUND: Disorders of sex development (DSD) is the term used for congenital conditions in which development of chromosomal, gonadal, or phenotypic sex is atypical. Nuclear receptor subfamily 5, group A, member 1 gene (NR5A1) encodes steroidogenic factor 1 (SF1), a transcription factor that is involved in gonadal development and regulates adrenal steroidogenesis. Mutations in the NR5A1 gene may lead to different 46,XX or 46,XY DSD phenotypes with or without adrenal failure. We report a Brazilian family with a novel NR5A1 mutation causing ambiguous genitalia in 46,XY affected individuals without Müllerian derivatives and apparently normal Leydig function after birth and at puberty, respectively. Their mother, who is also heterozygous for the mutation, presents evidence of primary ovarian insufficiency. CASE PRESENTATION: Three siblings with 46,XY DSD, ambiguous genitalia and normal testosterone production were included in the study. Molecular analyses for AR, SRD5A2 genes did not reveal any mutation. However, NR5A2 sequence analysis indicated that all three siblings were heterozygous for the p.Cys65Tyr mutation which was inherited from their mother. In silico analysis was carried out to elucidate the role of the amino acid change on the protein function. After the mutation was identified, all sibs and the mother had been reevaluated. Basal hormone concentrations were normal except that ACTH levels were slightly elevated. After 1 mcg ACTH stimulation test, only the older sib showed subnormal cortisol response. CONCLUSION: The p.Cys65Tyr mutation located within the second zinc finger of DNA binding domain was considered deleterious upon analysis with predictive algorithms. The identification of heterozygous individuals with this novel mutation may bring additional knowledge on structural modifications that may influence NR5A1 DNA-binding ability, and may also contribute to genotype-phenotype correlations in DSD. The slightly elevated ACTH basal levels in all three patients with 46,XY DSD and the subnormal cortisol response after 1 mcg ACTH stimulation in the older sib indicate that a long-term follow-up for adrenal function is important for these patients. Our data reinforce that NR5A1 analysis must also be performed in 46,XY DSD patients with normal testosterone levels without AR mutations.

Screening of Y chromosome microdeletions in 46,XY partial gonadal dysgenesis and in patients with a 45,X/46,XY karyotype or its variants.

BACKGROUND: Partial and mixed gonadal dysgenesis (PGD and MGD) are characterized by genital ambiguity and the finding of either a streak gonad and a dysgenetic testis or two dysgenetic testes. The karyotype in PGD is 46,XY, whereas a 45,X/46,XY mosaicism or its variants (more than two lineages and/or structural abnormalities of the Y chromosome) is generally found in MGD. Such mosaics are also compatible with female phenotype and Turner syndrome, ovotesticular disorder of sex development, and infertility in men with normal external genitalia. During the last few years, evidences of a linkage between Y microdeletions and 45,X mosaicism have been reported. There are also indications that the instability caused by such deletions might be more significant in germ cells. The aim of this work was to investigate the presence of Y chromosome microdeletions in individuals with PGD and in those with 45,X/46,XY mosaicism or its variants and variable phenotypes. METHODS: Our sample comprised 13 individuals with PGD and 15 with mosaicism, most of them with a MGD phenotype (n = 11). Thirty-six sequence tagged sites (STS) spanning the male specific region (MSY) on the Y chromosome (Yp, centromere and Yq) were analyzed by multiplex PCR and some individual reactions. RESULTS: All STS showed positive amplifications in the PGD group. Conversely, in the group with mosaicism, six individuals with MGD had been identified with Yq microdeletions, two of them without structural abnormalities of the Y chromosome by routine cytogenetic analysis. The deleted STSs were located within AZFb and AZFc (Azoospermia Factor) regions, which harbor several genes responsible for spermatogenesis. CONCLUSIONS: Absence of deletions in individuals with PGD does not confirm the hypothesis that instability of the Y chromosome in the gonads could be one of the causes of such condition. However, deletions identified in the second group indicate that mosaicism may be associated with Y chromosome abnormalities detectable only at the molecular level. If patients with mosaicism and Y microdeletions reared as males decide to undergo in vitro fertilization, Y chromosomes which tend to be unstable during cell division may be transmitted to offspring.

The use of fluorescence in situ hybridization in the diagnosis of hidden mosaicism: apropos of three cases of sex chromosome anomalies / O uso da hibridação in situ com fluorescência no diagnóstico de mosaicismo oculto: a propósito de três casos de anomalias de cromossomos sexuais

FISH has been used as a complement to classical cytogenetics in the detection of mosaicism in sex chromosome anomalies. The aim of this study is to describe three cases in which the final diagnosis could only be achieved by FISH. Case 1 was an 8-year-old 46,XY girl with normal female genitalia referred to our service because of short stature. FISH analysis of lymphocytes with probes for the X and Y centromeres identified a 45,X/46,X,idic(Y) constitution, and established the diagnosis of Turner syndrome. Case 2 was a 21-month-old 46,XY boy with genital ambiguity (penile hypospadias, right testis, and left streak gonad). FISH analysis of lymphocytes and buccal smear identified a 45,X/46,XY karyotype, leading to diagnosis of mixed gonadal dysgenesis. Case 3 was a 47,XYY 19-year-old boy with delayed neuromotor development, learning disabilities, psychological problems, tall stature, small testes, elevated gonadotropins, and azoospermia. FISH analysis of lymphocytes and buccal smear identified a 47,XYY/48,XXYY constitution. Cases 1 and 2 illustrate the phenotypic variability of the 45,X/46,XY mosaicism, and the importance of detection of the 45,X cell line for proper management and follow-up. In case 3, abnormal gonadal function could be explained by the 48,XXYY cell line. The use of FISH in clinical practice is particularly relevant when classical cytogenetic analysis yields normal or uncertain results in patients with features of sex chromosome aneuploidy. Arq Bras Endocrinol Metab. 2012;56(8):545-51.

FISH tem sido usado como um complemento para a citogenética clássica na detecção de mosaicismo em anomalias de cromossomos sexuais. O objetivo deste trabalho é descrever três casos nos quais o diagnóstico final só foi obtido por meio de FISH. O caso 1 é uma menina de 8 anos, 46,XY, com genitália feminina normal, encaminhada ao nosso setor devido à baixa estatura. A análise de linfócitos por FISH com sondas centroméricas de X e Y identificou a constituição 45,X/46,X,idic(Y) e estabeleceu o diagnóstico de síndrome de Turner. O caso 2 é um menino de 21 meses, 46,XY, com ambiguidade genital (hipospadia peniana, testículo à direita e gônada disgenética à esquerda). FISH de linfócitos e mucosa oral identificou o cariótipo 45,X/46,XY, levando ao diagnóstico de disgenesia gonadal mista. O caso 3 é um rapaz de 19 anos, 47,XYY, com atraso de desenvolvimento neuromotor, dificuldade de aprendizado, problemas psicológicos, alta estatura, testículos pequenos, gonadotrofinas elevadas e azoospermia. FISH de linfócitos e mucosa oral identificou a constituição 47,XYY/48,XXYY. Os casos 1 e 2 ilustram a variabilidade fenotípica do mosaico 45,X/46,XY e a importância da detecção da linhagem 45,X na avaliação e na condução dos casos. No caso 3, a função gonadal anormal pôde ser explicada pela linhagem 48,XXYY. O uso de FISH na prática clínica é particularmente relevante quando a análise citogenética clássica traz resultados normais ou incertos em pacientes com quadro sugestivo de uma aneuploidia de cromossomos sexuais. Arq Bras Endocrinol Metab. 2012;56(8):545-51.

The use of fluorescence in situ hybridization in the diagnosis of hidden mosaicism: apropos of three cases of sex chromosome anomalies.

FISH has been used as a complement to classical cytogenetics in the detection of mosaicism in sex chromosome anomalies. The aim of this study is to describe three cases in which the final diagnosis could only be achieved by FISH. Case 1 was an 8-year-old 46,XY girl with normal female genitalia referred to our service because of short stature. FISH analysis of lymphocytes with probes for the X and Y centromeres identified a 45,X/46,X,idic(Y) constitution, and established the diagnosis of Turner syndrome. Case 2 was a 21-month-old 46,XY boy with genital ambiguity (penile hypospadias, right testis, and left streak gonad). FISH analysis of lymphocytes and buccal smear identified a 45,X/46,XY karyotype, leading to diagnosis of mixed gonadal dysgenesis. Case 3 was a 47,XYY 19-year-old boy with delayed neuromotor development, learning disabilities, psychological problems, tall stature, small testes, elevated gonadotropins, and azoospermia. FISH analysis of lymphocytes and buccal smear identified a 47,XYY/48,XXYY constitution. Cases 1 and 2 illustrate the phenotypic variability of the 45,X/46,XY mosaicism, and the importance of detection of the 45,X cell line for proper management and follow-up. In case 3, abnormal gonadal function could be explained by the 48,XXYY cell line. The use of FISH in clinical practice is particularly relevant when classical cytogenetic analysis yields normal or uncertain results in patients with features of sex chromosome aneuploidy.

46,XY and 45,X/46,XY testicular dysgenesis: similar gonadal and genital phenotype, different prognosis.

The objective of this study was to describe the change in diagnosis and prognosis of a child with testicular dysgenesis and 46,XY karyotype after detection of a 45,X cell line and to discuss the difficulties caused by the terms mixed gonadal dysgenesis (MGD) and XY partial gonadal dysgenesis (XYPGD). One case was reported including clinical and laboratory findings of a child of 41-day-old infant with 1.3-cm phallus, penoscrotal hypospadias and left prepubertal testis. Karyotype 46,XY (16 cells), normal hormone levels. Right streak gonad, epididymis and müllerian remnants were removed; initial diagnosis was XYPGD. Persistent growth retardation led to further cytogenetic analysis (50 cells) and detection of a 45,X cell line. Detection of a 45,X lineage changed both the diagnosis to MGD and also the prognosis.The number of cells analyzed in karyotyping is critical. Use of MGD and XYPGD to designate both a histological picture and a syndromic diagnosis, results in lack of emphasis on clinical differences between 46,XY and 45,X/46,XY subjects.

46,XY and 45,X/46,XY testicular dysgenesis: similar gonadal and genital phenotype, different prognosis / Disgenesias testiculares 46,XY e 45,X/46,XY: fenótipo genital e gonadal semelhante, prognóstico distinto

The objective of this study was to describe the change in diagnosis and prognosis of a child with testicular dysgenesis and 46,XY karyotype after detection of a 45,X cell line and to discuss the difficulties caused by the terms mixed gonadal dysgenesis (MGD) and XY partial gonadal dysgenesis (XYPGD). One case was reported including clinical and laboratory findings of a child of 41-day-old infant with 1.3-cm phallus, penoscrotal hypospadias and left prepubertal testis. Karyotype 46,XY (16 cells), normal hormone levels. Right streak gonad, epididymis and müllerian remnants were removed; initial diagnosis was XYPGD. Persistent growth retardation led to further cytogenetic analysis (50 cells) and detection of a 45,X cell line. Detection of a 45,X lineage changed both the diagnosis to MGD and also the prognosis.The number of cells analyzed in karyotyping is critical. Use of MGD and XYPGD to designate both a histological picture and a syndromic diagnosis, results in lack of emphasis on clinical differences between 46,XY and 45,X/46,XY subjects.

O objetivo deste trabalho foi descrever a mudança no diagnóstico e no prognóstico de criança com disgenesia testicular e cariótipo 46,XY após detecção de linhagem 45,X e discutir as dificuldades causadas pelas denominações disgenesia gonadal mista (DGM) e disgenesia gonadal parcial XY (DGPXY). Relatou-se um caso incluindo achados clínicos e laboratoriais de uma criança de 41 dias com falo de 1,3 cm, hipospadia penoescrotal e testículo pré-puberal à esquerda. Cariótipo 46,XY (16 células), níveis hormonais normais. Gônada direita (disgenética), epidídimo e remanescentes müllerianos foram removidos; o diagnóstico inicial foi DGPXY. Retardo de crescimento persistente levou à ampliação da análise citogenética (50 células) e à detecção de linhagem 45,X. A detecção de linhagem 45,X modificou o diagnóstico para DGM e também o prognóstico. No cariótipo, o número de células analisadas é crítico. O uso de DGM e DGPXY para designar tanto quadro histológico quanto diagnóstico clínico resulta em falta de ênfase nas diferenças clínicas entre indivíduos 46,XY e 45,X/46,XY.