Analysis of modulatory factors involved in the phenotype of external genitalia virilization in females with classical form of 21-hydroxylase deficiency

Author(s)

Laura Cesar Kaupert

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
• Defense date: 2012-10-04

Examining board members

Tânia Aparecida Sartori Sanchez Bachega; Maria Lucia Cardillo Correa Giannella; Gil Guerra Júnior; Cláudio Elias Kater; Carlos Alberto Longui

• Advisor: Tânia Aparecida Sartori Sanchez Bachega

Abstract

Congenital Adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OH) is an autosomic recessive disorder characterized by an impairment in the cortisol and/or aldosterone synthesis, being the most frequent cause of 46,XX disorder of sex development. The disease presents a wide phenotypic variability resulting from different CYP21A2 gene mutations and a strong correlation has been observed among genotypes, clinical forms and basal hormone levels. However, this correlation is not observed regarding the degree of prenatal external genitalia virilization in females and an interindividual variability in the synthesis, metabolism and/or peripheral action of androgens could corroborate for these findings. Objectives: to evaluate if allelic variants in genes related to the androgen synthesis, metabolism and peripheral action could modulate the genital phenotype in CAH females bearing similar CYP21A2 mutations. Differences in the HSD17B5, SRD5A1, SRD5A2 and RA gene expression in genital skin were evaluated among patients with different degrees of external genital virilization. Patients: were selected 187 CAH females and clinical and hormonal data were retrospectively evaluated. The degree of external genitalia virilization was classified according to Prader (P) scores and patients were divided into 4 groups: P I+II, P III, P IV and P V. Patients were also grouped according to 21OH genotypes: group A bearing mutations predicting < 2% of residual enzymatic activity (n= 122) and group B between 3 to 7% (n= 58). Methodology: the exonic flanking regions of CYP3A7, PXR e CAR genes were PCR amplified and sequenced. The exons 12-13 of POR and the promotor region of HSD17B5 were sequenced to screen the functional polymorphisms. The V89L and A49T SRD5A2 alleles were screened by PCR-RFLP and the CAG polymorphic tract of AR gene by capillary electrophoresis and GeneScan analysis. The differential gene expression in genital skin was evaluated by real time PCR and the CYC, PGK1 e B2M housekeeping genes were used. The t-test, Mann-Whitney, Kruskal-Wallis, Fisher and uni- and multiple linear regression tests were used in statistical analysis. Results: Prader score in group A varied from II to V (III: III - IV) and in group B from I to V (III: II - III) (P< 0,001). The CYP3A7*1C allele was identified in 2.5% of alleles, CYP3A7*2 in 24%, rs2307424 CAR in 31%, A503V POR in 25%, -71G HSD17B5 in 33%, rs2518047 HSD17B5 in 17%, V89L SRD5A2 em 31% and A49T SRD5A2 in 1% of alleles. The rs2307424 CAR (P= 0.023; r2= 0.253) and rs2518047 HSD17B5 variants (P= 0.006; r2= 0.144) were associated with the degree of external genitalia virilization, and they were found in a higher frequency in more virilized patients. The remaining variants were not associated with Prader scores (P> 0.05). The HSD17B5, SRD5A1, SRD5A2 and RA gene expressions did not significantly differ between patients presenting Prader score III and IV (P> 0.05); however, 4/7 samples from patients with Prader IV and just 1/5 patients with Prader III presented an increased SRD5A2 expression. Conclusion: In this multicentric study the 21OH genotypes were correlated with the degree of external genitalia virilization in CAH females. The rs2307424 CAR and the rs2518047 HSD17B5 variants, related to the prenatal androgen metabolism and synthesis, respectively, explained some of the interindividual variability of genital phenotype in CAH females bearing similar CYP21A2 mutations. Differences in the expression of genes involved in the peripheral androgen action did not corroborate for the variability of genital phenotype in CAH (AU)