In vitro and molecular modeling analysis of two mutant desert hedgehog proteins associated with 46,XY gonadal dysgenesis.

Mutations of Desert hedgehog (DHH) have been associated to 46,XY pure gonadal dysgenesis (PGD) and to mixed gonadal dysgenesis (MGD); however, there have been no functional studies of mutations described in DHH. To determine if mutations p.L162P and Δ1086delG yield functional impairment, we performed in vitro and in silico analysis of both DHH mutants. In complementary DNA of DHH, we performed site-directed mutagenesis, which was confirmed by DNA sequencing. Protein extracts were obtained from HEK293cells transfected with different constructs and analyzed by Western blot; besides, densitometric analysis of chemiluminescent signals was performed. In addition, the structure of the wt-DHH and its two mutant proteins was inferred using in silico protein molecular modeling. In the Western blot analysis, we observed the absence of signal for p.L162P in DHH-N and a diminished signal for Δ1086delG in DHH-C, when compared to wt-DHH. Protein modeling showed notable conformational changes for the side chains of p.L162P, while the secondary structure was drastically modified in Δ1086delG, when compared to wt-DHH. To our knowledge, this is the first study focused to determine by in vitro studies, the effect of two specific mutations in DHH associated with 46,XY PGD and MGD. Our results suggest that both mutations have a deleterious effect on the expression of the DHH mutant proteins.

High incidence of Y-chromosome microdeletions in gonadal tissues from patients with 45,X/46,XY gonadal dysgenesis.

A higher incidence of Y-chromosome microdeletions was found on gonadal DNA than on peripheral blood lymphocyte DNA and on streak gonads than on dysgenetic testis in 11 patients with 45,X/46,XY gonadal dysgenesis. It is probable that an association between Y-chromosome microdeletions and severity of the phenotype in 45,X/46,XY patients exists.

Testosterone deficiency in young women with 46,XX spontaneous premature ovarian failure.

OBJECTIVE: To determine whether women with 46,XX spontaneous premature ovarian failure have lower serum free-T levels than do control women. DESIGN: Cross-sectional. SETTING: National Institutes of Health Clinical Research Center. PATIENT(S): Women with 46,XX spontaneous premature ovarian failure (n = 130). INTERVENTION(S): Evaluation while off any estrogen therapy and then again after receiving a standardized hormone regimen. Regularly menstruating control women (n = 65) were sampled during the midfollicular phase. MAIN OUTCOME MEASURE(S): Serum total T by RIA after extraction and column chromatography, free T by equilibrium dialysis, and sex hormone-binding globulin by immunoradiometric assay. RESULT(S): While off estrogen therapy patients had a median serum free-T concentration that was statistically significantly lower than controls (2.2 vs. 3.3 pg/mL). This dropped significantly lower to 1.9 pg/mL while the patients were on physiologic transdermal E(2) therapy. This is despite the fact that sex hormone-binding globulin levels did not change. While on E(2) therapy, 13% of women (95% confidence interval, 7.9%-20.3%) had serum free-T levels below the lower limit of normal (<1.1 pg/mL). CONCLUSION(S): As a group, young women with 46,XX spontaneous premature ovarian failure have reduced circulating free-T levels, both during an interval off of estrogen therapy and while on physiologic transdermal E(2) therapy.

Molecular evaluation of the SRY gene for gonads of patients with mixed gonadal dysgenesis.

AIM: To determine whether the SRY gene is present in the gonads of patients with mixed gonadal dysgenesis (MGD). METHODS: Molecular analysis was performed in three patients with MGD. Polymerase chain reactions were used to test for the presence of the SRY gene in the peripheral lymphocytes, testes and streak gonads. RESULTS: Chromosome analysis revealed 45,XO/46,XY in two patients, and 46,XY in the third patient. In the peripheral lymphocytes and testicular tissue, the SRY sequences were positive in all cases. However, the SRY sequence was detected in the streak gonad in only two of the three patients. CONCLUSIONS: It is interesting that we identified both SRY-positive and SRY-negative streak gonads. Although the SRY gene has a very important role in testicular differentiation, genes other than the SRY gene might also influence the development of the indifferent gonad in MGD.

Mixed gonadal dysgenesis: endogenous hormonal effects in the endometrium and histogenesis of germinoma.

The clinicopathologic features of two patients with mixed gonadal dysgenesis are presented, with specific reference to the relationship between endogenous sex hormones and the endometrium and the development of neoplastic disease. One patient, whose immature gonad contained granulosa cells and theca cells, had elevated serum estrogen levels and an endometrium with frequent ciliated metaplasia and squamous metaplasia. Another patient had elevated serum testosterone levels and atrophic endometrium. Both had gonadal tumors, more specifically, germinomas, which contained many calcified nodules within the tumor. These findings suggest that these germinomas arose from a gonadoblastoma.

Correlation of the testicular determinant factor sequence zinc finger Y with varying gonadal phenotypes in a series of 13 subjects with gonadal dysgenesis due to Y aneuploidy.

Deoxyribonucleic acid samples from a series of 13 subjects with 45,X/46,X,altered Y, and varying gonadal phenotypes (streak-streak, n = 9; streak-testis, n = 2; testis-testis, n = 2) were analyzed for the presence of the candidate testicular determinant factor sequence zinc finger Y. The Y-specific probes Y97 mapped to Y centromere, pDP105 A,B mapped to Yp and distal Yq11, respectively, hybridized with the deoxyribonucleic acid from all the 13 study subjects. The same deoxyribonucleic acid samples were analyzed for the presence of the zinc finger Y sequence. Eleven of the 13 subjects were positive for the zinc finger Y sequence. Four zinc finger Y-positive subjects had unilateral (n = 2) or bilateral (n = 2) testicular differentiation. Among the nine subjects with bilateral streak gonads, seven showed the presence of this sequence. The lack of testicular differentiation in the presence of quantitatively normal or almost normal zinc finger Y bands could not be explained by mosaicism alone. Mutations not detectable by analysis with the method of Southern with pDP1007, may occur in the testicular determinant factor gene vitiating testicular development.

Mixed gonadal dysgenesis. A review of 15 patients reporting single cases of malignant intratubular germ cell neoplasia of the testis, endometrial adenocarcinoma, and a complex vascular anomaly.

The clinicopathologic features of 15 patients with mixed gonadal dysgenesis are presented with special regard to cardiovascular and neoplastic disease. Seven (47%) cases, all phenotypic females, had gonadal tumors: gonadoblastoma (5), germinoma (4), malignant intratubular germ cell neoplasia (1), and a unique gonadal stromal tumor (1). Gonadoblastoma was found in 4 of 10 testes and 4 of 17 streak gonads, and associated with germinoma in 4 cases. One patient developed grade 1 endometrial adenocarcinoma after estrogen therapy. Cardiovascular diseases (ie, bicuspid aortic valve, and a unique right aortic arch with a retroesophageal arch segment, aberrant left subclavian artery, coarctation, and dissection) are documented in our series. At the time of diagnosis of mixed gonadal dysgenesis, removal of streak gonads or testes will prevent further gonadal tumor development Cardiovascular examination may identify treatable and potentially lethal disease.