Andréa Trevas Maciel-Guerra

Mutations in NR5A1 Associated with Ovarian Insufficiency

BACKGROUND The genetic causes of nonsyndromic ovarian insufficiency are largely unknown. A nuclear receptor, NR5A1 (also called steroidogenic factor 1), is a key transcriptional regulator of genes involved in the hypothalamic-pituitary-steroidogenic axis. Mutation of NR5A1 causes 46,XY disorders of sex development, with or without adrenal failure, but growing experimental evidence from studies in mice suggests a key role for this factor in ovarian development and function as well. METHODS To test the hypothesis that mutations in NR5A1 cause disorders of ovarian development and function, we sequenced NR5A1 in four families with histories of both 46,XY disorders of sex development and 46,XX primary ovarian insufficiency and in 25 subjects with sporadic ovarian insufficiency. None of the affected subjects had clinical signs of adrenal insufficiency. RESULTS Members of each of the four families and 2 of the 25 subjects with isolated ovarian insufficiency carried mutations in the NR5A1 gene. In-frame deletions and frameshift and missense mutations were detected. Functional studies indicated that these mutations substantially impaired NR5A1 transactivational activity. Mutations were associated with a range of ovarian anomalies, including 46,XX gonadal dysgenesis and 46,XX primary ovarian insufficiency. We did not observe these mutations in more than 700 control alleles. CONCLUSIONS NR5A1 mutations are associated with 46,XX primary ovarian insufficiency and 46,XY disorders of sex development.

Novel mutations affecting SRY DNA-binding activity: the HMG box N65H associated with 46,XY pure gonadal dysgenesis and the familial non-HMG box R30I associated with variable phenotypes.

Abstract. The SRY gene (sex-determining region of the Y chromosome) initiates the process of male sex differentiation in mammalians. In humans mutations in the SRY gene have been reported to account for 10–15% of the XY sex reversal cases. We describe here two novel missense mutations in the SRY gene after the screening of 17 patients, including 3 siblings, with 46,XY gonadal dysgenesis and 4 true hermaphrodites. One of the mutations, an A to C transversion within the HMG box, causes the N65H substitution and it was found in a patient presenting 46,XY pure gonadal dysgenesis. The Escherichia coli expressed SRYN65H protein did not present DNA-binding activity in vitro. The other mutation, a G to T transversion, causes the R30I substitution. This mutation was found in affected and nonaffected members of a family, including the father, two siblings with partial gonadal dysgenesis, a phenotypic female with pure gonadal dysgenesis, and three nonaffected male siblings. The G to T base change was not found in the SRY sequence of 100 normal males screened by ASO-PCR. The R30I mutation is located upstream to the HMG box, within the 29RRSSS33 phosphorylation site. The E. coli expressed SRYR30I protein was poorly phosphorylated and consequently showed reduced DNA-binding capacity in vitro.

Classical congenital adrenal hyperplasia due to 21-hydroxylase deficiency: a cross-sectional study of factors involved in bone mineral density.

Glucocorticoids are essential in the treatment of patients with congenital adrenal hyperplasia (CAH). The opposite actions of glucocorticoids and androgens in bone mass achievement justify a study of bone mineral density (BMD) in these patients. We evaluated BMD in patients with CAH due to classic 21-hydroxylase (CYP21A2) deficiency and investigated the involvement of clinical and laboratory factors in the BMD. This study assessed the clinical and laboratory factors involved in BMD of 45 patients at the Pediatric Unit of Endocrinology, UNICAMP, who had been diagnosed as having classical CAH due to CYP21A2 deficiency including molecular characterization. The sample consisted of 28 females and 17 males; 23 salt-wasting (SW) and 22 simple virilizing (SV) cases, with average of 9.9 years (ranges, 5.1–16.3 years) when bone densitometry was performed. The DEXA method was used for calculating the areal BMD Z score in L2–L4. The variables were analyzed with reference to the BMD for chronological age (BMD/CA), height age (BMD/HA), and bone age (BMD/BA). The mean Z score for BMD/CA was 0.08 ± 1.21 (−2.55 to 2.64); it was 0.29 ± 1.33 (−2.01 to 4.00) for BMD/HA, and −0.90 ± 1.24 (−3.41 to 1.92) for BMD/BA. The BMD/CA was significantly lower in females and in patients on treatment for a long period and of more advanced chronological age. Weight and body mass index (BMI) Z scores showed a positive correlation with advanced BA. The higher the weight and BMI Z scores, the higher the BMD/HA. The BMD/BA values were significantly higher in the group in which BA was closer to CA. The BMD/BA value was significantly lower when compared to the value obtained with height and chronological ages. Sex, duration of treatment, weight, BMI, and bone age have an effect on areal BMD in patients with CAH due to CYP21A2 deficiency, which may be underestimated when evaluated in relation to CA and HA.

Deafness resulting from mutations in the GJB2 (connexin 26) gene in Brazilian patients

Congenital deafness occurs in approximately 1 in 1000 live births. In developed countries about 60% of hearing loss is genetic. However, in Brazil most cases of hearing loss are due to environmental factors, such as congenital infections (mainly rubella), perinatal anoxia, kernicterus and meningitis. Recently, it has been demonstrated that the GJB2 gene is a major gene underlying congenital sensorial deafness. Mutations in this gene cause 10–20% of all genetic sensory hearing loss. One specific mutation, 35delG, accounts for the majority of mutant alleles. The extent of the hearing impairment varies from mild/moderate to profound, even within the patients homozygous for the common 35delG mutation. There may also be progression with age. Mutation analysis in the GJB2 gene was performed on 36 families (group A) presenting with at least one individual with non‐syndromic deafness (NSD). An unselected series of 26 deaf individuals referred by other services where the environmental factors were not completely excluded was also part of the study (group B).

Bone mineralization in Turner syndrome: a transverse study of the determinant factors in 58 patients

Abstract. Turner syndrome (TS) is characterized by the presence of an X chromosome and total or partial loss of the second sex chromosome, short stature, hypergonadotrophic hypogonadism, and a variable dysmorphic picture. Delayed puberty and estrogen deficiency are some of the determinant factors of osteoporosis in TS, but the whether or not there is an intrinsic bone defect is still obscure. The aim of this study was to evaluate the correlation of the z score of bone mineral density (BMD) with age, weight, height, karyotype, associated diseases, bone age, and estrogen therapy in TS patients. We performed a transverse study with area BMD of L2–L4 with dual-energy X-ray absorptiometry (DEXA) in 58 patients with a cytogenetic diagnosis of TS, whose ages ranged from 5 to 29 years. It was observed that 86% of the patients presented with a BMD z score below −1 SD, and 46.5% with a value below −2.5 SD. There was a significant negative association of BMD with age and height, and a positive association with weight and bone mass index (BMI) z scores. A higher BMD was observed in patients with spontaneous puberty and in those with more than 2 years of hormone replacement. In conclusion, there was a high incidence of reduced bone mass among our patients, which was influenced by weight and BMI, by the use and the time of estrogen replacement, and by the presence of spontaneous puberty.

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

Abstract 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 micro...

Turner's syndrome and thyroid disease: a transverse study of pediatric patients in Brazil.

An increased prevalence of autoimmune thyroid disease (AITD) has been described in Turners syndrome (TS), but the extent of this association is still controversial. Some studies also suggest that AITD is more frequent among patients with X-isochromosome. In order to determine the prevalence of AITD among girls with TS, and to look for an association with age and karyotype, we evaluated 71 patients with a mean age of 11.4 years (range 0-19.9). 15.5% (11/71) were hypothyroid, 17 (23.9%) were positive for thyroid peroxidase (TPO) and/or thyroglobulin (Tg) antibodies, and 24 (33.8%) had thyromegaly. No abnormality was observed before 4 years, and the highest frequencies were observed after 16 years. There were no significant differences concerning thyroid findings among patients with a 45,X karyotype, mosaics, and structural rearrangements. Half of the patients (35/71) exhibited one or more abnormalities, which demonstrates the importance of careful evaluation of thyroid function in all girls with TS.

Complete gonadal dysgenesis in clinical practice: the 46,XY karyotype accounts for more than one third of cases

OBJECTIVE To determine the frequency of XY karyotypes among females with complete gonadal dysgenesis (CGD) and to investigate the frequency of both gonadal tumors and SRY mutations. DESIGN Retrospective study based on data from all patients with CGD seen in our service from 1989 to 2010. SETTING Clinic for disorders of sex development, University Hospital, State University of Campinas. PATIENT(S) Thirty-two patients with hypergonadotropic hypogonadism, streak gonads, internal and external female genitalia, and normal karyotype (46,XX or 46,XY); 31 were index cases and 29 did not have a previously determined karyotype. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) None. RESULT(S) The percentage of XY karyotypes among patients with CGD was 34.5% (10/29). Mean age at diagnosis among XY and XX patients was 17.4 years and 19.9 years, respectively. Gonadal tumors were found in 4 of 9 XY girls, and 7 of 10 had SRY gene mutations. CONCLUSION(S) The previously unreported finding of an elevated frequency of 46,XY karyotype among patients with CGD and the high risk of gonadal neoplasia in such cases indicate that this diagnosis must be kept in mind by clinicians and strengthen the importance of karyotype analysis in females with primary hypogonadism. In addition, the frequency of SRY mutations in XY CGD might be higher than previously considered.

OCT4 immunohistochemistry may be necessary to identify the real risk of gonadal tumors in patients with Turner syndrome and Y chromosome sequences

BACKGROUND The aim of this study was to investigate the frequency of gonadal tumors among patients with Turner syndrome (TS) carrying Y-derivative sequences in their chromosomal constitution. METHODS Six out of 260 patients with TS were selected based on mosaicism of the entire Y chromosome; 10 were included because Y-derivative sequences have been detected by PCR with specific oligonucleotides (sex-determining region on the Y, testis specific-protein, Y and DYZ3) and further confirmed by FISH. The 16 patients were subjected to bilateral gonadectomy at ages varying from 8.7 to 18.2 years. Both histopathological investigation with hematoxylin and eosin (H&E) and immunohistochemical analysis with anti-octamer-binding transcription factor 4 (OCT4) antibody were performed. RESULTS Gonadal neoplasia was not detected in any of the 32 gonads evaluated by H&E; however, four gonads (12%) from three patients (19%) had positive OCT4 staining in 50-80% of nuclei, suggesting the existence of germ cell tumors (gonadoblastoma or in situ carcinoma). CONCLUSIONS Evaluation of the real risk of development of gonadal tumors in TS patients with Y-derivative sequences in their chromosomal constitution may require a specific histopathological study, such as immunohistochemistry with OCT4.

The role of the pediatrician in the management of children with genital ambiguities

OBJECTIVE To present the diagnostic criteria of genital ambiguity, the initial medical management and the attitude expected of pediatricians. SOURCES Review of the scientific literature in the form of articles indexed on MEDLINE, in English and Portuguese, published between 1990 and 2007 and dealing with the pediatric age group. SUMMARY OF THE FINDINGS Pediatricians have a fundamental role to play in the assessment of genital ambiguity, the purpose of which is to arrive at an etiologic diagnosis in the shortest possible time in order to define the patients sex and plan treatment. There are specific diagnostic criteria, but, in general, genitalia are ambiguous whenever there is difficulty in attributing gender to a child. The pediatrician should inform the patients family that assignment of their childs sex will depend upon detailed laboratory investigations, preferably carried out by a multidisciplinary team at a tertiary service. The 46,XX or 46,XY karyotypes are not alone sufficient to define the gender of rearing, although the test is fundamental to guide the investigation. When there are no palpable gonads, the first hypothesis should be congenital adrenal hyperplasia. Other causes included partial androgen insensitivity, 5alpha-reductase deficiency, partial gonadal dysgenesis and hermaphroditism. The family should be provided with support and information throughout the assessment process, and their participation is fundamental in the decision of which gender to rear the child in. CONCLUSIONS Although cases of genital ambiguity are relatively rare for pediatricians, they should be well-informed on the subject and the correct management of these conditions, since they will often be responsible for the initial guidance that families receive and for maintaining contact between them and the multidisciplinary team.