Association between serotonin 2C receptor gene (HTR2C) polymorphisms and psychopathological symptoms in children and adolescents

Serotonin 2C receptors (5HT2C) are involved in serotonin-driven dynamic equilibrium adjustments responsible for homeostatic stability in brain structures that modulate behavior and emotions. Single nucleotide polymorphisms (SNPs) from the serotonin 2C receptor gene (HTR2C) have been associated with several neurological and mental disorders, including abnormalities in cognitive and emotional processes. The aim of this study was to evaluate the association between the rs6318 SNP of the HTR2C gene and behavioral characteristics exhibited by children and adolescents based on the Child Behavior Checklist (CBCL/6-18) inventory. Eighty-five psychiatric outpatients between 8 and 18 years of age underwent genotyping of the rs6318 SNP. The CBCL/6-18 scale was administered to their caregivers. The chi-squared test was used to assess differences in the frequency of C and G alleles of the rs6318 SNP relative to the grouped CBCL/6-18 scores; significance level was 5%. The presence of the G allele of rs6318 was found to be associated with characteristics of aggressive behavior and social problems, and aggressive behavior was found to be associated with heterozygosis in females. These findings contribute to the identification of mental and behavioral phenotypes associated with gene expression.

The novel p.E89K mutation in the SRY gene inhibits DNA binding and causes the 46,XY disorder of sex development

Male sex determination in humans is controlled by the SRY gene, which encodes a transcriptional regulator containing a conserved high mobility group box domain (HMG-box) required for DNA binding. Mutations in the SRY HMG-box affect protein function, causing sex reversal phenotypes. In the present study, we describe a 19-year-old female presenting 46,XY karyotype with hypogonadism and primary amenorrhea that led to the diagnosis of 46,XY complete gonadal dysgenesis. The novel p.E89K missense mutation in the SRY HMG-box was identified as a de novo mutation. Electrophoretic mobility shift assays showed that p.E89K almost completely abolished SRY DNA-binding activity, suggesting that it is the cause of SRY function impairment. In addition, we report the occurrence of the p.G95R mutation in a 46,XY female with complete gonadal dysgenesis. According to the three-dimensional structure of the human SRY HMG-box, the substitution of the conserved glutamic acid residue by the basic lysine at position 89 introduces an extra positive charge adjacent to and between the positively charged residues R86 and K92, important for stabilizing the HMG-box helix 2 with DNA. Thus, we propose that an electrostatic repulsion caused by the proximity of these positive charges could destabilize the tip of helix 2, abrogating DNA interaction.

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.

Phenotype and genotype correlation of the microconversion from the CYP21A1P to the CYP21A2 gene in congenital adrenal hyperplasia

Deficiency of 21-hydroxylase is the most common form of congenital adrenal hyperplasia (CAH-21OH). We determined by allele-specific PCR the frequency of microconversion in the CYP21A2 gene in 50 Brazilian patients with the classical (salt wasting: SW and simple virilizing: SV) forms and nonclassical (NC) form of CAH-21OH and correlated genotype with phenotype. Genotypes were classified into three mutation groups (A, B, and C) based on the amount of enzymatic activity in in vitro studies using adrenal cells. In 94 unrelated alleles, we diagnosed 76 percent of the affected alleles after screening for 7 microconversions. The most frequent point mutations observed in this series were I172N (19 percent), V281L (18 percent), and IVS2,A/C>G,-12 (15 percent). In the SW form, the most frequent mutation was IVS2,A/C>G,-12 (38 percent), in the SV form it was I172N (53 percent), and in the NC form it was V281L (57.7 percent). We observed a good correlation between genotype and phenotype. Discordance between genotype and phenotype was found in one SV patient with a mild mutation in one of the alleles (R356W/V281L). However, we cannot rule out the presence of an additional mutation in these alleles. We also observed a good correlation of genotype with 17alpha-hydroxyprogesterone, testosterone, and androstenedione levels. The severity of external genitalia virilization correlated with the severity of mutation. In conclusion, the frequencies described in the present study did not differ from worldwide studies, including the Brazilian population. The few differences observed may reflect individual sample variations. This new Brazilian cohort study suggests the presence of new mutations in Brazilian patients with different forms of CAH-21OH

Molecular analysis of CYP21 and C4 genes in Brazilian families with the classical form of steroid 21-hydroxylase deficiency

The most common enzymatic defect of steroid synthesis is deficiency of the adrenal steroid 21-hydroxylase. Inhibition of the formation of cortisol results in an increased pituitary release of ACTH which in turn drives the adrenal cortex to overproduce androgens. This hormonal setting affects the development of genetic females by misdirecting the differentiation of external genitalia towards the male type. Since the isolation of the gene encoding 21-hydroxylase enzyme in 1984, gene deletions, large gene conversions, and microconversions have been reported to be responsible for the disease. In this paper, we report a study of this genetic defect in 22 families with one or more affected offspring diagnosed as having the classical form of congenital adrenal hyperplasia. The DNA from 30 patients was analyzed with three restriction enzymes. Hybridization with a 21-hydroxylase cDNA probe and the 5' end of a C4 genomic probe disclosed gene deletion in 7.3 percent (3/41) of the disease-related chromosomes. The rate of large gene conversion was 17.1 percent (7/41), and no abnormality in the hybridization pattern was observed in 75.6 percent (31/41) of the disease alleles. Densitometry of the autoradiographs was used to determine the ratio of the copy-number of the 2 1-hydroxylase gene (CYP21B) to the copy-number of its pseudogene (CYP21A). Differences in phenotype, the low frequency of gene deletion, and the high frequency of gene conversion compared with other studies in different populations indicated that 21-hydroxylase deficiency in the Brazilian population may involve different molecular mutations.

Use of y-chromosome-specific DNA probes to evaluate an XX male

The function of a Y human chromosomal DNA sequence was evaluated. The Y-5 probe was isolated from a flow-sorted chromosome library and detects Y-specific sequences. The Y-5 probe and other T-specific probes were used to analyze an XX male patient without ambiguous genitalia. DNA sequences from the short arm of the chromosome Y that were detected with pDP1007 and DP105 in the patient's genome explain the testis differentation observed in this case. Failure of the patient's DNA to hybridize to the Y-5 probe shows that the primitive gonads can differentiate into testes even in the absence of this chromosome region. In contrast, a gene controlling spermatogenesis may exist in this region because the patient azoospermic