Congenital adrenal hyperplasia is an autosomal recessive inborn error of metabolism due to impaired activity of one enzyme required for cortisol biosynthesis. Steroid 21-hydroxylase (21OH) deficiency is the cause in more than 90% of the patients. The 21OH is encoded by the CYP21A2 gene located on the chromosome 6 in the region known as the RCCX module. Due to the high homology and tandem-repeat organization of the RCCX module, this region of the genome is subjected to unequal crossover events leading to large rearrangements. Different clinical presentations are classified in classical form that can be salt-wasting (01% of enzyme activity) or simple-virilizing (15% of enzyme activity) and late-onset non-classical form (1560% of enzyme activity). This work aims to standardization of fast and cheap molecular techniques to detect CYP21A2 gene polymorphisms in Brazilian newborns from public health network of Rio Grande do Sul state. First of all DNA extraction was carried by salting out methodology from peripheral blood of patients with high dosage of 17-hydroxyprogesterone (21OH enzyme substrate) on newborn screening, followed by quantified of total DNA. CYP21A2 gene was amplified as Krone et al, 2002. Primers were designed on Primer 3 software to nested PCR multiplex reaction for amplified six regions, followed by SNaPshot reaction to analyze the 13 SNPs most frequently in Brazilian population. The standardization of these techniques was validated by sequencing techniques. For detection large rearrangements was used the commercial MLPA kit. Blood sample from 235 patients was collected and standardized the CYP21A2 gene amplification and Nested PCR multiplex. Commercial MLPA kit was already validated. Standardization process of SNaPshot is still in development. Therefore, with this work we will be able to do a population study relating the polymorphisms more present in this population with their phenotype.
10 - 12 Sep 2016
European Society for Paediatric Endocrinology