Novel DMRT1 3'UTR+11insT mutation associated to XY partial gonadal dysgenesis

The Y-chromosome-located SRY gene encodes a small testis-specific protein containing a DNA-binding motif known as the HMG (high mobility group) box. However, mutations in SRY are not frequent especially in cases of 46,XY partial gonadal dysgenesis. Several sex-determining genes direct the fate of the bipotential gonad to either testis or ovary. In addition, heterozygous small deletions in 9p can cause complete and partial XY gonadal dysgenesis without other symptoms. Human DMRT1 gene, which is located at 9p24.3, is expressed in testis and ovary and has been considered, among others, a candidate autosomal gene responsible for gonadal dysgenesis. In this report we describe a nucleotide insertion in DMRT1 3'UTR in a patient of XY partial gonadal dygenesis. The 3'UTR+11insT is located within a conserved motif important for mRNA stabilization.

Serum androgen levels in elite female athletes.

OBJECTIVE: Prior to the implementation of the blood steroidal module of the Athlete Biological Passport, we measured the serum androgen levels among a large population of high-level female athletes as well as the prevalence of biochemical hyperandrogenism and some disorders of sex development (DSD). METHODS AND RESULTS: In 849 elite female athletes, serum T, dehydroepiandrosterone sulphate, androstenedione, SHBG, and gonadotrophins were measured by liquid chromatography-mass spectrometry high resolution or immunoassay. Free T was calculated. The sampling hour, age, and type of athletic event only had a small influence on T concentration, whereas ethnicity had not. Among the 85.5% that did not use oral contraceptives, 168 of 717 athletes were oligo- or amenorrhoic. The oral contraceptive users showed the lowest serum androgen and gonadotrophin and the highest SHBG concentrations. After having removed five doped athletes and five DSD women from our population, median T and free T values were close to those reported in sedentary young women. The 99th percentile for T concentration was calculated at 3.08 nmol/L, which is below the 10 nmol/L threshold used for competition eligibility of hyperandrogenic women with normal androgen sensitivity. Prevalence of hyperandrogenic 46 XY DSD in our athletic population is approximately 7 per 1000, which is 140 times higher than expected in the general population. CONCLUSION: This is the first study to establish normative serum androgens values in elite female athletes, while taking into account the possible influence of menstrual status, oral contraceptive use, type of athletic event, and ethnicity. These findings should help to develop the blood steroidal module of the Athlete Biological Passport and to refine more evidence-based fair policies and recommendations concerning hyperandrogenism in female athletes.

Amenorréia primária e cariótipo XY: identificando pacientes em risco

OBJETIVO: verificar a prevalência e as características clínicas de pacientes com amenorréia primária e cariótipo XY avaliadas em nosso Serviço com o intuito de identificar achados que possam auxiliar em seu reconhecimento. MÉTODOS: no período de Janeiro de 1975 a Novembro de 2007, foram avaliadas 104 pacientes com amenorréia primária. Para todos os casos foi realizada a análise pelo cariótipo por bandas GTG. Destas, 21 (20,2%) apresentavam uma constituição 46,XY. Contudo, duas foram excluídas do estudo por terem prontuários incompletos. Das 19 pacientes que compuseram a amostra, a maior parte veio encaminhada pela ginecologia (63,2%). Suas idades variaram entre 16 e 41 anos (média de 22,1 anos). Realizou-se uma coleta de dados sobre sua história familiar e pregressa, exame físico e resultados de exames complementares. Para determinação dos seus diagnósticos levaram-se em consideração essas informações. RESULTADOS: a síndrome de resistência aos androgênios foi o diagnóstico predominante (n=12; 63,2%). Cinco pacientes (26,3%) apresentavam disgenesia gonadal pura XY (DGP XY), uma (5,3%) deficiência de 17-alfa hidroxilase e uma (5,3%) deficiência de 5-alfa redutase. Achados clínicos freqüentemente observados nessas pacientes incluíram desenvolvimento anormal dos caracteres sexuais secundários (n=19), agenesia uterina com vagina em fundo de saco (n=14), história familiar de amenorréia (n=8) e gônadas palpáveis no canal inguinal (n=5). Duas delas apresentavam história de hérnia inguinal. Hipertensão arterial sistêmica foi diagnosticada somente na paciente com deficiência de 17-alfa hidroxilase, e malignização gonadal, naquela com DGP XY. CONCLUSÕES: a freqüência de pacientes com cariótipo XY (20%) foi superior à usualmente descrita na literatura (3 a 11%). Acreditamos que isso tenha relação com a forma de encaminhamento das pacientes ao Serviço. Certos achados da história clínica e do exame físico deveriam ser rotineiramente avaliados em indivíduos com amenorréia primária. Dessa forma, haveria um reconhecimento mais precoce das pacientes 46,XY e, conseqüentemente, um adequado manejo clínico das mesmas.

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.

DSD Due to 5 alpha-Reductase 2 Deficiency - from Diagnosis to Long Term Outcome

Most of the patients with 5 alpha-RD 2 deficiency are reared in the female social sex due to their severely undervirilized external genitalia but similar to 60% who have not been submitted to orchiectomy in childhood undergo male social sex change at puberty. In our cohort of 30 cases from 18 families, all subjects were registered in the female social sex except for two children-one who had an affected uncle and the other who was diagnosed before being registered. The majority of the patients were satisfied with the long-term results of their treatment and surprisingly, penile length was not associated with satisfactory or unsatisfactory sexual activity. Steroid 5 alpha-RD2 deficiency should be included in the differential diagnosis of all newborns with 46,XY DSD with normal testosterone production before gender assignment or any surgical intervention because these patients should be considered males at birth.

Preserved Fertility In A Patient With Gynecomastia Associated With The P.pro695ser Mutation In The Androgen Receptor.

The androgen insensitivity syndrome (AIS) is described as a dysfunction of the androgen receptor (AR) in 46,XY individuals, which can be associated with mutations in the AR gene or can be due to unknown mechanisms. Different mutations in AIS generally cause variable phenotypes that range from a complete hormone resistance to a mild form usually associated with male infertility. The purpose of this study was to search for mutations in the AR gene in a fertile man with gynecomastia and to evaluate the influence of the mutation on the AR transactivation ability. Sequencing of the AR gene revealed the p.Pro695Ser mutation. It is located within the AR ligand-binding domain. Bioinformatics analysis indicated a deleterious role, which was verified after testing transactivation activity and N-/C-terminal (N/C) interaction by in vitro expression of a reporter gene and 2-hybrid assays. p.Pro695Ser showed low levels of both transactivation activity and N/C interaction at low dihydrotestosterone (DHT) conditions. As the ligand concentration increased, both transactivation activity and N/C interaction also increased and reached normal levels. Therefore, this study provides functional insights for the p.Pro695Ser mutation described here for the first time in a patient with mild AIS. The expression profile of p.Pro695Ser not only correlates to the patient's phenotype, but also suggests that a high-dose DHT therapy may overcome the functional deficit of the mutant AR.

Six new cases confirm the clinical molecular profile of complete combined 17α-hydroxylase/ 17,20-lyase deficiency in Brazil

In 2004, Costa-Santos and cols. reported 24 patients from 19 Brazilian families with 17α-hydroxylase deficiency and showed that p.W406R and p.R362C corresponded to 50% and 32% of CYP17A1 mutant alleles, respectively. The present report describes clinical and molecular data of six patients from three inbred Brazilian families with 17α-hydroxlyse deficiency. All patients had hypogonadism, amenorrhea and hypertension at diagnosis. Two sisters were found to be 46,XY with both gonads palpable in the inguinal region. All patients presented hypergonadotrophic hypogonadism, with high levels of ACTH (> 104 ng/mL), suppressed plasmatic renin activity, low levels of potassium (< 2.8 mEq/L) and elevated progesterone levels (> 4.4 ng/mL). Three of them, including two sisters, were homozygous for p.W406R mutation and the other three (two sisters and one cousin) were homozygous for p.R362C. The finding of p.W406R and p.R362C in the CYP17A1 gene here reported in additional families, confirms them as the most frequent mutations causing complete combined 17α-hydroxylase/17,20-lyase deficiency in Brazilian patients.

Structural aspects of the p.P222Q homozygous mutation of HSD3B2 gene in a patient with congenital adrenal hyperplasia

Type II 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase (3β-HSD2), encoded by the HSD3B2 gene, is a key enzyme involved in the biosynthesis of all the classes of steroid hormones. Deleterious mutations in the HSD3B2 gene cause the classical deficiency of 3β-HSD2, which is a rare autosomal recessive disease that leads to congenital adrenal hyperplasia (CAH). CAH is the most frequent cause of ambiguous genitalia and adrenal insufficiency in newborn infants with variable degrees of salt losing. Here we report the molecular and structural analysis of the HSD3B2 gene in a 46,XY child, who was born from consanguineous parents, and presented with ambiguous genitalia and salt losing. The patient carries a homozygous nucleotide c.665C>A change in exon 4 that putatively substitutes the proline at codon 222 for glutamine. Molecular homology modeling of normal and mutant 3β-HSD2 enzymes emphasizes codon 222 as an important residue for the folding pattern of the enzyme and validates a suitable model for analysis of new mutations.

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. Arq Bras Endocrinol Metab. 2012;56(8):545-51

A proteome analysis of conditioned media from human neonatal fibroblasts used in the maintenance of human embryonic stem cells

The pathways involved in the maintenance of human embryonic stem (hES) cells remain largely unknown, although some signaling pathways have been identified in mouse embryonic stem (mES) cells. Fibroblast feeder layers are used to maintain the undifferentiated growth of hES cells and an examination of the conditioned media (CM) of human neonatal fibroblasts (HNFs) could provide insights into the maintenance of hES cells. The neonatal foreskin fibroblast line (HNF02) used in this study was shown to have a normal 2n = 46, XY chromosomal complement and to support the undifferentiated growth of the Embryonic Stem Cell International Pte. Ltd.-hES3 cell line. The CM of HNF02 was examined using two-dimensional liquid chromatography-tandem mass spectrometry (2-D LCMS) and two-dimensional electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (2-DE/MALDI). A total of 102 proteins were identified, 19 by 2-DE/MALDI, 53 by 2-D LCMS and 30 by both techniques. These proteins were classified into 15 functional groups. Proteins identified in the extracellular matrix and differentiation and growth factor functional categories were considered most likely to be involved in the maintenance of hES cell growth, differentiation and pluripotency as these groups contained proteins involved in a variety of events including cell adhesion, cell proliferation and inhibition of cell proliferation, Writ signaling and inhibition of bone morphogenetic proteins.

Tetrasomy 8 in a patient with chronic lymphocytic leukemia

We report a case of a 47-year-old man diagnosed with chronic lymphocytic leukemia (CLL) with two extra copies of chromosome 8. Classical cytogenetic analysis by the immunostimulatory combination of DSP30 and interleukin 2 showed tetrasomy of chromosome 8 in 60% of the metaphase cells (48,XY,+8,+8[12]/46,XY[8]). Spectral karyotype analysis confirmed the abnormality previously seen by G banding. Additionally, interphase fluorescence in situ hybridization using an LSI CEP 8 probe performed on peripheral blood cells without any stimulant agent showed tetrasomy of chromosome 8 in 54% of analyzed cells (108 of 200). To our knowledge, tetrasomy 8 as the sole chromosomal abnormality in CLL has not been previously described. The prognostic significance of tetrasomy 8 in CLL remains to be elucidated. However, the patient has been followed up in the outpatient hospital since 2004 without any therapeutic intervention and has so far remained stable. (C) 2010 Elsevier Inc. All rights reserved.

Clonal Complex Chromosome Aberration in Non-Ossifying Fibroma

Cytogenetic information of non-ossifying fibromas (NOFs) is exceptionally limited. This fact relies, in part, on their benign nature but mainly because most cases evolve undetected or there is no need for surgical intervention. We report the case of a NOF arising in the left tibia of a 14-year-old male with an invariable clonal translocation. The karyotype was denoted as 42-46,XY,t(11;3;14)(q23;p21;p11). There are only two previous reported cases of clonally aberrant NOF. Records from additional cases will be essential to assess whether consistent karyotypic aberrations define this lesion. Pediatr Blood Cancer 2010;54:764 767. (C) 2010 Wiley-Liss, Inc.

Amenorréia e anormalidades do cromossomo X

OBJETIVO: correlacionar as manifestações clínicas de pacientes com amenorréia e anormalidades do cromossomo X. MÉTODOS: realizou-se uma análise retrospectiva dos achados clínicos e laboratoriais das pacientes com amenorréia e anormalidades do cromossomo X, atendidas entre janeiro de 1975 e novembro de 2007. Suas medidas antropométricas foram avaliadas através de tabelas de crescimento padrão, sendo que, quando presentes, dismorfias menores e maiores foram anotadas. O estudo dos cromossomos foi realizado através do cariótipo com bandamento GTG. RESULTADOS: do total de 141 pacientes com amenorréia, 16% apresentavam anormalidades numéricas e 13% estruturais do cromossomo X. Destas pacientes com anormalidade do X (n=41), 35 possuíam descrição clínica completa. Todas elas apresentavam hipogonadismo hipergonadotrófico. Amenorréia primária foi observada em 24 pacientes, das quais 91,7% com fenótipo de síndrome de Turner. Com exceção de um caso com deleção Xq22-q28, todas as demais pacientes com este fenótipo apresentavam alterações envolvendo Xp (uma com uma linhagem 46,XY associada). Os dois casos restantes com apenas amenorréia primária possuíam deleções proximais de Xq. Entre as 11 pacientes com amenorréia secundária, 54,5% apresentavam fenótipo de Turner (todas com monossomia do X isolada ou em mosaico). Entre aquelas com fenótipo de falência ovariana isolada observaram-se somente deleções Xq e trissomia do X. CONCLUSÕES: a análise cromossômica deve sempre ser realizada em mulheres com falência ovariana de causa não conhecida, mesmo na ausência de achados dismórficos. Esta também é de extrema importância em pacientes sindrômicas, pois, além de confirmar o diagnóstico, é capaz de identificar pacientes em risco, como nos casos com uma linhagem 46,XY.

Anormalidades cromossômicas em casais com história de aborto recorrente

OBJETIVO: verificar a prevalência e as características clínicas de casais com história de abortos de repetição e anormalidade cromossômica atendidos em nosso serviço. MÉTODOS: foram avaliados retrospectivamente todos os casais encaminhados de janeiro de 1975 a junho de 2008 por história de abortos de repetição. Foram incluídos no estudo somente aqueles casais, em que a análise cromossômica feita com o cariótipo por bandas GTG foi realizada com sucesso. Foram coletados dados clínicos referentes às suas idades, bem como o número de abortamentos, natimortos, crianças polimalformadas, nativivos por casal e resultado do exame de cariótipo. Para comparação da frequência das alterações cromossômicas encontradas em nosso estudo com as da literatura, bem como entre os diferentes subgrupos de nossa amostra, foi utilizado o teste exato de Fisher (p<0,05). RESULTADOS: a amostra foi composta de 108 casais. As idades variaram de 21 a 58 anos entre os homens (média de 31,4 anos) e de 19 a 43 anos entre as mulheres (média de 29,9 anos). O número de abortos oscilou de dois a nove (média de 3,2). Alterações cromossômicas foram observadas em um dos parceiros em dez casais (9,3%) e corresponderam, respectivamente, a três casos (30%) de translocação recíproca [dois de t(5;6) e um de t(2;13)], dois (20%) de translocação Robertsoniana [um de der(13;14) e um de der(13;15)], cinco (50%) de mosaicismo (mos) [dois casos de mos 45,X/46,XX, um de mos 46,XX/47,XXX, um de mos 46,XY/47,XXY e um de mos 46,XY/47,XYY] e um (10%) de inversão cromossômica [inv(10)]. Em um dos casais, a parceira apresentava duas alterações concomitantes: t(2;13) e der(13;14). Anormalidades cromossômicas foram verificadas em 5% dos casais com história de dois abortamentos, em 10,3% com três abortos e 14,3% com quatro ou mais abortos. CONCLUSÕES: a frequência de anormalidades cromossômicas verificada neste estudo (9,3%) foi similar à da maior parte dos trabalhos realizados nos últimos 20 anos, a qual variou de 4,8 a 10,8%. Contudo, nos chamou a atenção o alto percentual de pacientes com mosaicismo. Acreditamos que este achado possa estar associado ao maior número de metáfases rotineiramente analisadas no presente serviço.

Mutations in the SRY, DAX1, SF1 and WNT4 genes in Brazilian sex-reversed patients

In most mammals, male development is triggered by the transient expression of the SRY gene, which initiates a cascade of gene interactions ultimately leading to the formation of a testis from the indifferent fetal gonad. Mutation studies have identified several genes essential for early gonadal development. We report here a molecular study of the SRY, DAX1, SF1 and WNT4 genes, mainly involved in sexual determination, in Brazilian 46,XX and 46,XY sex-reversed patients. The group of 46,XX sex-reversed patients consisted of thirteen 46,XX true hermaphrodites and four 46,XX males, and was examined for the presence of the SRY gene and for the loss of function (inactivating mutations and deletions) of DAX1 and WNT4 genes. In the second group consisting of thirty-three 46,XY sex-reversed patients we investigated the presence of inactivating mutations in the SRY and SF1 genes as well as the overexpression (duplication) of the DAX1 and WNT4 genes. The SRY gene was present in two 46,XX male patients and in none of the true hermaphrodites. Only one mutation, located outside homeobox domain of the 5' region of the HMG box of SRY (S18N), was identified in a patient with 46,XY sex reversal. A novel 8-bp microdeletion of the SF1 gene was identified in a 46,XY sex-reversed patient without adrenal insufficiency. The dosage of DAX1 and WNT4 was normal in the sex-reversed patients studied. We conclude that these genes are rarely involved in the etiology of male gonadal development in sex-reversed patients, a fact suggesting the presence of other genes in the sex determination cascade.