M.P. de Mello

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.

Inhibition of CYP21A2 Enzyme Activity Caused by Novel Missense Mutations Identified in Brazilian and Scandinavian Patients

BACKGROUND Most patients with 21-hydroxylase deficiency carry CYP21A1P-derived mutations, but an increasing number of novel and rare mutations have been reported in disease-causing alleles. OBJECTIVE Functional effects of three novel (p.G56R, p.L107R, p.L142P) and one recurrent (p.R408C) CYP21A2 mutations were investigated. The degree of enzyme impairment caused by p.H62L alone or combined to p.P453S was also analyzed. DESIGN The study included 10 Brazilian and two Scandinavian patients. To determine the deleterious role of each mutant protein, in vitro assays were performed in transiently transfected COS-1 cells. For a correct genotype-phenotype correlation, the enzymatic activities were evaluated toward the two natural substrates, 17-hydroxyprogesterone and progesterone. RESULTS Low levels of residual activities obtained for p.G56R, p.L107R, p.L142P, and p.R408C mutants classified them as classical congenital adrenal hyperplasia mutations, whereas the p.H62L showed an activity within the range of nonclassical mutations. Apparent kinetic constants for p.H62L confirmed the nonclassical classification as the substrate binding capacity was within the same magnitude for mutant and normal enzymes. A synergistic effect was observed for the allele bearing the p.H62L+p.P453S combination because it caused a significant reduction in the enzymatic activity. CONCLUSIONS We describe the functional analysis of five rare missense mutations identified in Brazilian and Scandinavian patients. The p.G56R, p.L107R, and p.L142P are reported for the first time. Most probably these novel mutations are closer to null than the p.I172N, but for the p.G56R, that might not be the case, and the p.H62L is definitely a nonclassical mutation.

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 effect of fetal androgen metabolism-related gene variants on external genitalia virilization in congenital adrenal hyperplasia

The 21‐hydroxylase deficiency (21OHD) is caused by CYP21A2 mutations resulting in severe or moderate enzymatic impairments. 21OHD females carrying similar genotypes present different degrees of external genitalia virilization, suggesting the influence of other genetic factors. Single nucleotide variants (SNVs) in the CYP3A7 gene and in its transcription factors, related to fetal 19‐carbon steroid metabolism, could modulate the genital phenotype. To evaluate the influence of the 21OHD genotypes and the CYP3A7, PXR and CAR SNVs on the genital phenotype in 21OHD females. Prader scores were evaluated in 183 patients. The CYP3A7, PXR and CAR SNVs were screened and the 21OHD genotypes were classified according to their severity: severe and moderate groups. Patients with severe genotype showed higher degree of genital virilization (Prader median III, IQR III‐IV) than those with moderate genotype (III, IQR II‐III) (p < 0.001). However, a great overlap was observed between genotype groups. Among all the SNVs tested, only the CAR rs2307424 variant correlated with Prader scores (r2 = 0.253; p = 0.023). The CYP21A2 genotypes influence the severity of genital virilization in 21OHD females. We also suggest that the CAR variant, which results in a poor metabolizer phenotype, could account for a higher degree of external genitalia virilization.

GAPO syndrome: a new syndromic cause of premature ovarian insufficiency

Abstract Premature ovarian insufficiency has the following causes: genetic, autoimmune, metabolic, infectious, and iatrogenic dysfunctions (including radiotherapy, chemotherapy and surgery). However, premature ovarian insufficiency remains without a definite cause in a substantial number of cases. This article describes GAPO syndrome in association with premature ovarian insufficiency, as well as a novel ANTXR1 gene mutation. Histopathological study of the ovaries of a woman with hypergonadotropic hypogonadism revealed extensive deposition of hyaline extracellular material, with bilateral parenchymal atrophy and follicular depletion. Molecular study revealed a novel ANTXR1 gene mutation. The homozygous c.378 + 3A > G transition at the consensus donor splice site of intron 4 was identified. Our results support the involvement of ANTRX1 gene mutations in deregulated extracellular matrix. In addition, our study identified a novel ANTXR1 mutation causing GAPO syndrome, indicating it as a new cause of early loss of ovarian function.

Impairment in Anthropometric Parameters and Body Composition in Females with Classical 21-Hydroxylase Deficiency

The aim of this study was to evaluate the physical measurements and body composition of female patients with the classic form of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. Twenty-eight girls with CAH were classified according to both hormonal control (well or not well controlled) and the clinical form of the disease (simple virilizing or salt-wasting). In the control group, 112 healthy individuals were included, divided into two subgroups (male and female). Both patients and controls were subdivided by age into three groups according to pubertal stage: < or =10 years (prepubertal), 11-15 years (pubertal), and > or =15 years (postpubertal). Anthropometrical evaluations and bioelectrical impedance were used to obtain the physical measurement and body composition data. The patients with the simple virilizing form presented higher values for BMI, waist, arm fat area, and fat mass percentage. The not well controlled group presented shorter leg length. Values obtained for BMI as well as for arm fat area, brachial circumference, waist, hip, bi-iliac diameter and fat mass percentage were significantly higher in the patients than in the controls, whereas leg length, hand size and the percentages of water and lean mass were lower. Alterations in body composition were observed in all age groups, mainly by increase of fat mass with age. After puberty, impairments in limb measurements (leg, hand and foot) were more evident. Patients with CAH presented differences in anthropometric parameters but mainly in body composition. Hence, more comprehensive and careful anthropometric evaluation during monitoring of patients with CAH is recommended.

Two Novel Mutations in the Thyroid Hormone Receptor β in Patients with Resistance to Thyroid Hormone (RTH β): Clinical, Biochemical, and Molecular Data.

The syndrome of resistance to thyroid hormone (RTH β) is an inherited disorder characterized by variable tissue hyposensitivity to 3,5,30-L-triiodothyronine (T(3)), with persistent elevation of free-circulating T(3) (FT(3)) and free thyroxine (FT(4)) levels in association with nonsuppressed serum thyrotropin (TSH). Clinical presentation is variable and the molecular analysis of THRB gene provides a short cut diagnosis. Here, we describe 2 cases in which RTH β was suspected on the basis of laboratory findings. The diagnosis was confirmed by direct THRB sequencing that revealed 2 novel mutations: the heterozygous p.Ala317Ser in subject 1 and the heterozygous p.Arg438Pro in subject 2. Both mutations were shown to be deleterious by SIFT, PolyPhen, and Align GV-GD predictive methods.

Genetic variation and relationships at six VNTR loci in two distinct sample populations in Brazil

Background: The Brazilian population has been the focus of intensive genetic study due to admixture characteristics whereas there are few reports on the variability of VNTR loci in Brazil. Primary objective: The aim of this study was to analyse genetic parameters in sample populations from two geographically distant regions: São Luís City, in Maranhão State and Campinas City, in São Paulo State. We investigated if distinct colonization influences could produce detectable differences in genetic background. Subject and methods: DNA samples from peripheral drained blood were obtained from unrelated individuals who underwent paternity testing. Allelic variation in six VNTR loci (D2S44, D4S139, D5S110, D8S358, D10S28 and D17S79) was evaluated. The results were compared to reference databases available for general Latin-derived European and African–American populations as well as for other Brazilian groups. Results: This study reveals that forensic population parameters did not show differences among regions, although we detected admixture values varying between the south-east and north-east of Brazil. Conclusions: Differences between the two samples are probably due to different admixture proportions of European- and African-derived alleles in each region; both populations are in Hardy–Weinberg equilibrium. In addition, the allelic frequency for all loci, in both populations, can be used as database for forensic purposes.

Two Novel Mutations In The Thyroid Hormone Receptor β In Patients With Resistance To Thyroid Hormone (rth β): Clinical, Biochemical, And Molecular Data

The syndrome of resistance to thyroid hormone (RTH β) is an inherited disorder characterized by variable tissue hyposensitivity to 3,5,30-L-triiodothyronine (T(3)), with persistent elevation of free-circulating T(3) (FT(3)) and free thyroxine (FT(4)) levels in association with nonsuppressed serum thyrotropin (TSH). Clinical presentation is variable and the molecular analysis of THRB gene provides a short cut diagnosis. Here, we describe 2 cases in which RTH β was suspected on the basis of laboratory findings. The diagnosis was confirmed by direct THRB sequencing that revealed 2 novel mutations: the heterozygous p.Ala317Ser in subject 1 and the heterozygous p.Arg438Pro in subject 2. Both mutations were shown to be deleterious by SIFT, PolyPhen, and Align GV-GD predictive methods.

Two Novel Mutations In The Thyroid Hormone Receptor β In Patients With Resistance To Thyroid Hormone (rth β): Clinical, Biochemical, And Molecular Data.

The syndrome of resistance to thyroid hormone (RTH β) is an inherited disorder characterized by variable tissue hyposensitivity to 3,5,30-L-triiodothyronine (T(3)), with persistent elevation of free-circulating T(3) (FT(3)) and free thyroxine (FT(4)) levels in association with nonsuppressed serum thyrotropin (TSH). Clinical presentation is variable and the molecular analysis of THRB gene provides a short cut diagnosis. Here, we describe 2 cases in which RTH β was suspected on the basis of laboratory findings. The diagnosis was confirmed by direct THRB sequencing that revealed 2 novel mutations: the heterozygous p.Ala317Ser in subject 1 and the heterozygous p.Arg438Pro in subject 2. Both mutations were shown to be deleterious by SIFT, PolyPhen, and Align GV-GD predictive methods.