Endocrine Abstracts

Background: Congenital adrenal hyperplasia (CAH) is a group of monogenic disorders, with autosomal recessive inheritance which is classified as a sexual development disorder due to androgen excess. It results from a defect in one of the steroidogenic enzymes which leads to the accumulation of intermediate metabolites affecting the production of Glucocorticoids, Mineralocorticoids, and/or sex steroids, resulting in a wide spectrum of clinical presentations. CAH resulting from 11β-hydroxylase (11β-OHD) deficiency is caused by pathogenic variants occuring in the CYP11B1 gene, located on the long arm of chromosome 8 (8q24.3). This gene encodes for 11β-OHD, a mitochondrial hemoprotein formed by a 503 amino acid chain and expressed mainly at the adrenal gland. In the present study, we identified a novel homozygous intronic variant c.1200+5G>C located in intron 7 of CYP11B1 gene in a Tunisian family. The aim of this study was to characterize the functional consequences of this variant on the CYP11B1 function.

Methods: In the way to characterize the functional implications of this variation on mRNA splicing, we conducted, as a first intention, an in-silico prediction and modeling study to determine if it can activate or inhibit acceptor or donor canonical splicing sites or if it is involved only in the activation of other cryptic splice sites. In the second step, to confirm and assess the outcome of the in-silico analyses, we performed an in vitro functional study using the “Exon Trapping” approach.

Results: The in-silico analysis suggested that the c.1200+5G>C variant would result in retention of intron 7 leading to a frameshift and the appearance of a premature stop codon which would alter the conformation as well as the function of the mutated protein. However, in vitro functional studies revealed that the variation would rather result in the excision of exon 7 located just upstream site. In essence, our in vitro assays confirmed the pathogenicity role of the c.1200+5G>C variant as well as its association with a severe clinical phenotype.

Conclusions: The divergence between the results of the in-silico prediction and the in vitro functional analysis highlights the complementarity and limitations of bioinformatic tools, which although predictive, must systematically be validated by experimental approaches.