Importância funcional de novas variações nucleotídicas no gene CYP21A2

Congenital Adrenal Hyperplasia (CAH), one of the most frequent autosome recessive disorders, is caused by defects in steroidogenic enzymes involved in the cortisol biosynthesis. One consequence is the deviation of the synthesis towards the production of androgenic hormones. In the CAH classical form, intrauterine prenatal exposure to androgen excess in critical stages of sexual differentiation leads to external genitalia virilization of the 46,XX fetus resulting in ambiguous genitalia at birth, pseudopuberty in both sexes and salt wasting crisis when CYP21A2 residual activity is less than 1%. Signs of androgen excess in the non-classic form can be acne, hirsutism and menstrual disorders in young women but they can manifest early in development as precocious pubarche, accelerated linear growth and advanced skeletal maturation. However, it is well known that some individuals with non-classic CAH can remain asymptomatic. The objectives of this project were to analyze the functional role of ten novel CYP21A2 missense mutations (p.Leu12Met; p.Arg16Cys; p.Ser101Asn; p.Ser202Gly; p.Pro267Leu; p.Val358Ile; p.Arg369Gln; p.Asp377Tyr; p.Thr450Met; and p.Leu461Pro), one novel in frame deletion (p.Gln398_Ala391del), two mutations that has previously been identified but lacking functional studies (p.Ser113Phe and p.Thr450Pro) and three combined alleles (p.Ile172Asn+p.Val358Ile; p.Val281Leu+p.Arg369Gln; and p.Asp377Tyr+p.Leu461Pro); and establish a genotype-phenotype correlation for the mutations investigated in vitro. Besides all classic methods used in molecular studies, the methodology included the expression of normal and mutant CYP21A2 proteins for enzymatic activity assays and enzymatic kinetics as well as the verification of protein expression by Western blot. In vitro studies showed that one mutation is a normal variant of the CYP21A2 gene (p.Leu12Met) showing residual activity close to 100%; nine (p.Arg16Cys; p.Ser101Asn; p.Ser202Gly; p.Pro267Leu; p.Val358Ile; p.Arg369Gln; p.Asp377Tyr; p.Thr450Met; and p.Leu461Pro) result in mild non-classic CAH with residual activities varying between 35% and 95% and three result in classic CAH (p.Ser113Phe; p.Gln398_Ala391del and p.Thr450Pro) with residual activities below 4%. Alleles harboring two mutations in cis result in a synergistic effect on the enzyme residual activity. This study allowed the correlation between phenotype and genotype (AU)