ESPE Abstracts

Background: Congenital Adrenal Hyperplasia (CAH) is an autosomal recessive disorder with more than 90% of cases caused by defects in the steroid-21 hydroxylase (CYP21A2) gene. Such defects are the main cause of 21-hydroxylase enzyme deficiency that affects the biosynthesis of cortisol and aldosterone. The CYP21A2 gene is part of the RCCX module, which is located on chromosome 6p21.3, in the major histocompatibility complex (MHC) class III region. The RCCX module consists of four genes, RP, C4, CYP21, and TNX and can normally be found in two versions in tandem, one consisting of the functional genes RP2, C4B, CYP21A2, and TNXB and the second consisting of the corresponding inactive pseudogenes RP1, CYP21A1P, and TNXA and the functional C4A gene. The genes in the two versions of RCCX module are highly homologous and events such as unequal crossover during meiosis can occur. These events produce non-functional chimeric genes such as CYP21A1P/CYP21A2 and TNXA/TNXB, which are directly related to CAH and steroid overproduction. To date, nine types of CYP21P/CYP21A2 chimeras (CH1-CH9) and three types of TNXA/TNXB chimeras (CAH-X CH1-CH3) have been described.

Methods: Twenty-four DNA samples from patients previously identified with various genetic defects in the CYP21A2 gene were selected and re-analyzed for the presence of chimeric genes, using long-range PCR followed by TaqI restriction enzyme digestion, Sanger sequencing, and multiplex ligation-depended probe amplification.

Results: Of the 24 patients, three patients were heterozygous for the CH1 type of chimera, one patient was heterozygous for the CH9 type of chimera and one patient was a compound heterozygous for the CH1 and CH4 type of chimeras. In addition, one patient was found to be heterozygous for the CAH-X CH1 type of chimera, one patient was homozygous for the CAH-X CH1 type of chimera, and one patient was heterozygous for the CAH-X CH2 type of chimera. Interestingly, one patient was identified as heterozygous for the very rare CH4 chimera that lacks the p.Pro30Leu mutation and a second patient was identified as heterozygous for a new type of chimera.

Conclusions: Using the applied methodologies, we successfully identified and characterized the different types of chimeric genes generated by unequal crossover events. Based on our results, the application of improved and more detailed diagnostic methods will help to identify all genetic abnormalities in CYP21A2 genotyping, therefore, providing more targeted management and genetic counseling for current and future families with CAH.