Rastreamento de alterações patogênicas por PCR-AE e padronização do sequenciamento do gene CYP21A2

Abstract

Congenital adrenal hyperplasia (CAH) is a disorder of sex differentiation (DSD) characterized by deficiency of adrenal steroid biosynthesis. It comprises a set of autosomal recessive disorders, with approximately 95% of cases being due to a deficiency of the enzyme 21-hydroxylase (21OHD). In its classical form, the accumulation of cortisol and aldosterone precursors are converted into androgens leading to progressive virilization, rapid somatic growth with accelerated skeletal maturation and concomitant salt loss in the most severe form of the disease which, if left untreated, can lead to adrenal crisis, shock, and death. This work continues the research line of the LGMH/HUPAA/UFAL, where molecular diagnosis of HAC-21OHD by allele-specific polymerase chain reaction (PCR-AE) is performed and standardized diagnosis by Sanger sequencing of the CY21A2 gene for unresolved cases. Through the PCR-AE technique and/or by restriction enzyme digestion, eight frequent alterations derived from the CYP21A1P pseudogene and one rare alteration, not derived from it, were investigated in 19 families (16 individuals diagnosed with HAC-21OHD and 19 relatives). Using this technique, it was possible to elucidate the diagnosis of 56.25% of our series (9 affected individuals), and 43.75% (7 individuals) proceeded to investigation by techniques of complete sequencing of the gene and by MLPA (Multiplex ligation-dependent Probe Amplification). Through these techniques, the genotype of 2 of these cases was elucidated at the partner institution. Moreover, the complete sequencing of the CYP21A2 gene was standardized at the LGMH, and therefore, 5 cases from this series are still under investigation in our research group. During the standardization of the complete sequencing technique of the CYP21A2 gene by the Sanger method, we elucidated the molecular diagnosis of a family referred for genetic counseling where both parents have the genotype p.Gln319*+p.Arg484fs*5. The molecular diagnosis of most cases of HAC21OHD can be elucidated quickly and inexpensively by screening for frequent alterations. For cases not elucidated, the complete sequencing of the CYP21A2 gene is valuable since more than 400 pathogenic alterations have been related to HAC-21OHD, thus, the standardization of this technique, in the present work, enriches even more the molecular diagnosis of HAC performed in the state by the LGMH/HUPAA/UFAL.