Mutations in SRY and WT1 genes required for gonadal development are not responsible for XY partial gonadal dysgenesis

The WT1 transcription factor regulates SRY expression during the initial steps of the sex determination process in humans, activating a gene cascade leading to testis differentiation. In addition to causing Wilms' tumor, mutations in WT1 are often responsible for urogenital defects in men, while SRY mutations are mainly related to 46,XY pure gonadal dysgenesis. In order to evaluate their role in abnormal testicular organogenesis, we screened for SRY and WT1 gene mutations in 10 children with XY partial gonadal dysgenesis, 2 of whom with a history of Wilms' tumor. The open reading frame and 360 bp of the 5' flanking sequence of the SRY gene, and the ten exons and intron boundaries of the WT1 gene were amplified by PCR of genomic DNA. Single-strand conformation polymorphism was initially used for WT1 mutation screening. Since shifts in fragment migration were only observed for intron/exon 4, the ten WT1 exons from all patients were sequenced manually. No mutations were detected in the SRY 5' untranslated region or within SRY open-reading frame sequences. WT1 sequencing revealed one missense mutation (D396N) in the ninth exon of a patient who also had Wilms' tumor. In addition, two silent point mutations were found in the first exon including one described here for the first time. Some non-coding sequence variations were detected, representing one new (IVS4+85A>G) and two already described (-7ATG T>G, IVS9-49 T>C) single nucleotide polymorphisms. Therefore, mutations in two major genes required for gonadal development, SRY and WT1, are not responsible for XY partial gonadal dysgenesis.172

An Illustrative Case Of Léri-weill Dyschondrosteosis

We report on a girl presenting Léri-Weill dyschondrosteosis (LWD) due to deletion of the SHOX gene. Her family included individuals with short stature alone or with both short stature and mesomelia or Madelung's deformity. The deletion was demonstrated through detection of hemizygosity for microsatellite markdrs SHOX-CA repeat, DXYS10092, DXYS10093 and DXYS10091 localized around the SHOX gene, with retention of paternal alleles in the proband and three of her sisters who had short stature as the only clinical feature. Hemizygosity for these loci was also observed in their mother, who had short stature too. The deletion in the proband was however larger, including locus DXY 10083. The proband's only sister with normal height did not carry the deletion. Family history suggests transmission of the deletion from the proband's maternal great-grandfather to her grandfather via the Y chromosome, and from the grandfather to the proband's mother via the X chromosome after crossing-over in the pseudoautosomal region proximal to the SHOX gene. Copyright © 2008, Sociedade Brasileira de Genética.314839842Belin, V., Cusin, V., Viot, G., Girlich, D., Toutain, A., Moncla, A., Vekemans, M., Cormier-Daire, V., SHOX mutations in dyschondrosteosis (Léri-Weill syndrome) (1998) Nat Genet, 19, pp. 67-69Benito-Sanz, S., del Blanco, D.G., Aza-Carmona, M., Magano, L.F., Lapunzina, P., Argente, J., Campos-Barros, A., Heath, K.E., PAR1 deletions downstream of SHOX are the most frequent defect in a Spanish cohort of Léri-Weill dyschondrosteosis (LWD) probands (2006) Hum Mutat, 27, p. 1062Benito-Sanz, S., del Blanco, D.G., Huber, C., Thomas, N.S., Aza-Carmona, M., Bunyan, B., Maloney, V., Campos-Barros, A., Characterization of SHOX deletions in Léri-Weill dyschondrosteosis (LWD) reveals genetic heterogeneity and no recombination hotspots (2006) Am J Hum Genet, 79, pp. 409-412Benito-Sanz, S., Thomas, N.S., Huber, C., Gorbenko del Blanco, D., Aza-Carmona, M., Crolla, J.A., Maloney, V., Campos-Barros, A., A novel class of pseudoautosomal region 1 deletions downstream of SHOX Is associated with Léri-Weill dyschondrosteosis (2005) Am J Hum Genet, 77, pp. 533-544Clement-Jones, M., Schiller, S., Rao, E., Blaschke, R.J., Zuniga, A., Zeller, R., Robson, S.C., Strachan, T., The short stature homeobox gene SHOX is involved in skeletal abnormalities in Turner syndrome (2000) Hum Mol Genet, 9, pp. 695-702Ellison, J.W., Wardak, Z., Young, M.F., Gehron Robey, P., Laig-Webster, M., Chiong, W., PHOG, a candidate gene for involvement in the short stature of Turner syndrome (1997) Hum Mol Genet, 6, pp. 1341-1347Filatov, D.A., Gerrard, D.T., High mutation rates in human and ape pseudoautosomal genes (2003) Gene, 317, pp. 67-77Fukami, M., Kato, F., Tajima, T., Yokoya, S., Ogata, T., Transactivation function of an approximately 800-bp evolutionarily conserved sequence at the SHOX 3′ region: Implication for the downstream enhancer (2006) Am J Hum Genet, 78, pp. 167-170Henry, A., Thorburn, M.J., Madelung's deformity. A clinical and cytogenetic study (1967) J Bone Joint Surg, 49 B, pp. 66-73Jorge, A.A., Souza, S.C., Nishi, M.Y., Billerbeck, A.E., Liborio, D.C., Kim, C.A., Arnhold, I.J., Mendonca, B.B., SHOX mutations in idiopathic short stature and Leri-Weill dyschondrosteosis: Frequency and phenotypic variability (2007) Clin Endocrinol, 66, pp. 130-135Lien, S., Szyda, J., Schechinger, B., Rappold, G., Arnheim, N., Evidence for heterogeneity in recombination in the human pseudoautosomal region: High resolution analysis by sperm typing and radiation-hybrid mapping (2000) Am J Hum Genet, 66, pp. 557-566Rao, E., Weiss, B., Fukami, M., Rump, A., Niesler, B., Mertz, A., Muroya, K., Winkelmann, M., Pseudoautosomal deletions encompassing a novel homeobox gene cause growth failure in idiopathic short stature and Turner syndrome (1997) Nat Genet, 16, pp. 54-63Rappold, G., Blum, W.F., Shavrikova, E.P., Crowe, B.J., Roeth, R., Quigley, C.A., Ross, J.L., Niesler, B., Genotypes and phenotypes in children with short stature: Clinical indicators of SHOX haploinsufficiency (2007) J Med Genet, 44, pp. 306-313Rappold, G.A., Fukami, M., Niesler, B., Schiller, S., Zumkeller, W., Bettendorf, M., Heinrich, U., Onigata, K., Deletions of the homeobox gene SHOX(short stature homeobox) are an important cause of growth failure in children with short stature (2002) J Clin Endocrinol Metab, 87, pp. 1402-1406Schneider, K.U., Sabherwal, N., Jantz, K., Röth, R., Muncke, N., Blum, W.F., Cutler Jr, G.B., Rappold, G., Identification of major raombinant hotspot in patients with short stature and SHOX deficiency (2005) Am J Hum Genet, 77, pp. 89-96Shears, D.J., Vassal, H.J., Goodman, F.R., Palmer, R.W., Reardon, W., Superti-Furga, A., Scambler, P.J., Winter, R.M., Mutation and deletion of the pseudoautosomal gene SHOX cause Léri-Weill dyschondrosteosis (1998) Nat Genet, 19, pp. 70-72Zebala, L.P., Manske, P.R., Goldfarb, C.A., (200't) Madelung's deformity: A spectrum of presentation.. The J Hand Surg, 32 A, pp. 1393-1401Zinn, A.R., Ramos, P., Ross, J., (1006) A second recombination hotspot associated with SHOX deletions Am J Hum Genet, 78, pp. 523-52

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

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOObjective: 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. © ABE&M todos os direitos reservados.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 epithelium584328334FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO2011/50189-7Verificar 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 aproporção 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 tambémnão 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 FISHdiretamente em células de mucosa ora

Spontaneous Puberty In Girls With Early Diagnosis Of Turner Syndrome [puberdade Espontânea Em Meninas Com Diagnóstico Precoce De Síndrome De Turner]

Objective: To verify if the frequency of spontaneous pubertal development among girls with Turner syndrome (TS) diagnosed in infancy and childhood is greater than that of patients diagnosed later. Subjects and methods: Thirty three girls aged 13 years diagnosed at the same service. Results: Sixteen of 32 informative patients had signs of spontaneous puberty, a frequency greater than that of patients diagnosed later. In six patients, there was no progression of puberty; menarche occurred in six, and one became pregnant, but the fetus was a stillborn. Spontaneous puberty was absent in all cases with 45,X karyotype. Conclusions: The greater prevalence of spontaneous puberty in girls whose diagnosis was not based on pubertal delay suggests that, among those diagnosed later, there is a bias towards patients with hypogonadism. © ABE&M todos os direitos reservados.569653657Bondy, C.A., Turner syndrome study group. Care of girls and women with Turner syndrome: A guideline of the Turner Syndrome Study Group (2007) J Clin Endocrinol Metab., 92 (1), pp. 10-25Reynaud, K., Cortvrindt, R., Verlinde, F., de Schepper, J., Bourgain, C., Smitz, J., Number of ovarian follicles in human fetuses with the 45, X karyotype (2004) Fertil Steril., 81 (4), pp. 1112-1119Conte, F.A., Grumbach, M.M., Kaplan, S.L., A diphasic pattern of gonadotropin secretion in patients with the syndrome of gonadal dysgenesis (1975) J Clin Endocrinol Metab., 40 (4), pp. 670-674Ropelato, M.G., Escobar, M.E., Gottlieb, S., Bergada, C., Gonadotropin secretion in prepubertal normal and agonadal children evaluated by ultrasensitive time-resolved immunofluorometric assays (1997) Horm Res., 48 (4), pp. 164-172Chrysis, D., Spiliotis, B.E., Stene, M., Cacciari, E., Davenport, M.L., Gonadotropin secretion in girls with Turner syndrome measured by an ultrasensitive immunochemiluminometric assay (2006) Horm Res., 65 (5), pp. 261-266Turner, H.H., A syndrome of infantilism, congenital webbed neck and cubitus valgus (1938) Endocrinology, 23, pp. 566-574Lippe, B., Westra, S.J., Boechat, M.I., Ovarian function in Turner syndrome: Recognizing the spectrum (1993) Basic and clinical approach to Turner syndrome, pp. 117-122. , In: Hibi I, Takano K, editors, Amsterdam, NL: Elsevier Science PublishersPrice, D.A., Albertsson-Wikland, K., Demography, auxology and response to recombinant human growth hormone treatment in girls with Turner's syndrome in the Kabi Pharmacia International growth study (1993) Acta Paediatr., 82 (s391), pp. 69-74Lippe, B., Turner syndrome (1996) Pediatric Endocrinology, pp. 387-422. , In: Sperling MA, editor, Philadelphia, USA: WB SaundersPasquino, A.M., Passeri, F., Pucarelli, I., Segni, M., Municchi, G., Italian's Study Group for Turner's syndrome. Spontaneous pubertal development in Turner's syndrome (1997) J Clin Endocrinol Metab., 82 (6), pp. 1810-1813Hjerrild, B.E., Mortensen, K.H., Gravholt, C.H., Turner syndrome and clinical treatment (2008) Br Med Bull., 86, pp. 77-93Hadnott, T.N., Gould, H.N., Gharib, A.M., Bondy, C.A., Outcomes of spontaneous and assisted pregnancies in Turner syndrome: The U.S. National Institutes of Health experience (2011) Fertil Steril., 95 (7), pp. 2251-2256Carvalho, A.B., Guerra Jr., G., Baptista, M.T.M., Marques-de-Faria, A.P., Lemos-Marini, S.H., Maciel-Guerra, A.T., Turner syndrome: A pediatric diagnosis frequently made by non-pediatricians (2010) J Pediatr (Rio J), 86 (2), pp. 121-125Sutton, E.J., McInerney-Leo, A., Bondy, C.A., Gollust, S.E., King, D., Biesecker, B., Turner syndrome: Four challenges across the lifespan (2005) Am J Med Genet A., 139 A (2), pp. 57-66Hagen, C.P., Main, K.M., Kjaergaard, S., Juul, A., FSH, LH, inhibin B and estradiol levels in Turner syndrome depend on age and karyotype: Longitudinal study of 70 Turner girls with or without spontaneous puberty (2010) Hum Reprod., 25 (12), pp. 3134-3141Fechner, P.Y., Davenport, M.L., Qualy, R.L., Ross, J.L., Gunther, D.F., Eugster, E.A., Differences in follicle-stimulating hormone secretion between 45, X monosomy Turner syndrome and 45, X/46, XX mosaicism are evident at an early age (2006) J Clin Endocrinol Metab., 91 (12), pp. 4896-4902Hook, E.B., Exclusion of chromosome mosaicism: Tables of 90 percent, 95 percent and 99 percent confidence limits and comments on use (1977) Am J Hum Genet., 29, pp. 94-97Massa, G., Verlinde, F., de Schepper, J., Thomas, M., Bourguignon, J.P., Craen, M., Trends in age at diagnosis of Turner syndrome (2005) Arch Dis Child, 90 (3), pp. 267-268Hagen, C.P., Aksglaede, L., Sørensen, K., Main, K.M., Boas, M., Cleemann, L., Serum levels of anti-Müllerian hormone as a marker of ovarian function in 926 healthy females from birth to adulthood and in 172 Turner syndrome patients (2012) J Clin Endocrinol Metab., 95 (11), pp. 5003-501

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 müllerian 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 antimüllerian hormone, a decreased testosterone response to human chorionic gonadotropin stimulation without accumulation of testosterone precursors, and the presence of müllerian 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 fertililty 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.1255652656Robboy, S.J., Miller, T., Donahoe, P.K., Dysgenesis of testicular and streak gonads in the syndrome of mixed gonadal dysgenesis: Perspective derived from clinicopathologic analysis of twenty-one cases (1982) Hum Pathol, 13, pp. 700-716Troche, V., Hernandez, E., Neoplasia arising in dysgenetic gonads (1986) Obstet Gynecol Surv, 41, pp. 74-79Krasna, I.H., Lee, M.L., Smilow, P., Sciorra, L., Eierman, L., Risk of malignancy in bilateral streak gonads: The role of the Y chromosome (1992) J Pediatr Surg, 27, pp. 1376-1380Rohatgi, M., Gupta, D.K., Menon, P.S., Verma, I.C., Mathur, M., Mixed gonadal dysgenesis and dysgenetic male pseudohermaphroditism - A critical analysis (1992) Indian J Pediatr, 59, pp. 487-500Rey, R.A., Belville, C., Nhou-Fékété, C., Evaluation of gonadal function in 107 intersex patients by means of serum antimüllerian hormone measurement (1999) J Clin Endocrinol Metab, 84, pp. 627-631Stuchi-Perez, E.G., Lukas-Croisier, C., Castro, M., Evaluation of the tubular and interstitial functions of the testis in 46,XY patients with ambiguous genitalia (2000) J Pediatr Endocrinol Metab, 13, pp. 605-612Chang, H.J., Clark, R.D., Bachman, H., The phenotype of 45,X/46,XY mosaicism: An analysis of 92 prenatally diagnosed cases (1990) Am J Hum Genet, 46, pp. 156-167Rajfer, J., Walsh, P.C., Mixed gonadal dysgenesis: Dysgenetic male pseudoher-maphroditism (1981) The Intersex Child: Pediatric and Adolescent Endocrinology, pp. 103-115. , Josso N, ed. Basel, Switzerland: S. KargerBorer, J.G., Nitti, V.W., Glassberg, K.I., Mixed gonadal dysgenesis and dysgenetic male pseudohermaphroditism (1995) J Urol, 153, pp. 1267-1273Donahoe, P.K., Crawford, J.D., Hendren, W.H., Mixed gonadal dysgenesis: Pathogenesis and management (1979) J Pediatr Surg, 14, pp. 287-300Pelletier, J., Bruening, W., Kashtan, C.E., Germline mutations in the Wilms' tumor supressor gene are associated with abnormal urogenital development in Denys-Drash syndrome (1991) Cell, 67, pp. 437-1147Carré-Éusebe, D., Imbeaud, S., Harbison, M., New, M.I., Josso, N., Picard, J.Y., Variants of the anti-Müllerian hormone gene in a compound heterozygote with the persistent Müllerian duct syndrome and his family (1992) Hum Genet, 90, pp. 389-394Nistal, M., Paniagua, R., Non-neoplastic diseases of the testis (1996) Urologic Surgical Pathology, pp. 458-565. , Bostiwick DG, Eble JN, eds. St Louis, Mo: MosbyLennox, B., Ahmad, K.M., Mack, W.S., A method for determining the relative total length of the tubules in the testis (1970) J Pathol, 102, pp. 229-238Jimenez, R., Sanchez, A., Burgos, M., Dias De La Guardia, R.C., Puzzling out the genetics of mammalian sex determination (1996) Trends Genet, 12, pp. 164-166Müller, J., Skakkebaek, N.F., Quantification of germ cells and seminiferous tubules by stereological examination of testicles from 50 boys who suffered from sudden death (1983) Int J Androl, 6, pp. 143-156Cortes, D., Müller, J., Skakkebaek, N.E., Proliferation of Sertoli cells during development of the human testis assessed by stereological methods (1987) Int J Androl, 10, pp. 589-596Nistal, M., Abaurrea, M.A., Paniagua, R., Morphological and histometric study on the human Sertoli cell from birth to the onset of puberty (1982) J Anat, 14, pp. 351-363Van Niekerk, W.A., Retief, A.E., The gonads of human true hermaphrodites (1981) Hum Genet, 58, pp. 117-122Guerra Jr., G., De Mello, M.P., Assumpção, J.G., True hermaphrodites in the southeastern region of Brazil: A different cytogenetic and gonadal profile (1998) J Pediatr Endocrinol Metab, 11, pp. 519-524Quigley, C.A., De Bellis, A., Marschke, K.B., El-Awady, M.K., Wilson, E.M., French, F.S., Androgen receptor defects: Historical, clinical and molecular perspectives (1995) Endocr Rev, 16, pp. 271-32

Mutations of androgen receptor gene in Brazilian patients with male pseudohermaphroditism

We describe the identification of point mutations in the androgen receptor gene in five Brazilian patients with female assignment and behavior. The eight exons of the gene were amplified by the polymerase chain reaction (PCR) and analyzed for single-strand conformation polymorphism (SSCP) to detect the mutations. Direct sequencing of the mutant PCR products demonstrated single transitions in three of these cases: G<FONT FACE="Symbol">®</font>A in case 1, within exon C, changing codon 615 from Arg to His; G<FONT FACE="Symbol">®</font>A in case 2, within exon E, changing codon 752 from Arg to Gln, and C<FONT FACE="Symbol">®</font>T in case 3, within exon B, but without amino acid change