Congenital Adrenal Hyperplasia (CAH) via the CYP21A2 Gene

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders due to defects in single genes involved in different steps of cortisol biosynthesis (Hannah-Shmouni et al. 2017. PubMed ID: 28476231; Merke and Bornstein. 2005. PubMed ID: 15964450; White and Bachega. 2012. PubMed ID: 23044877). The clinical consequence of deficient cortisol biosynthesis represents a continuous phenotypic spectrum depending on causative gene, genotype, residual activity and age of presentation. Clinical features of CAH include adrenal insufficiency, genital ambiguity or disordered sex development, infertility, short stature, hypertension, and an increased risk of metabolic syndrome during adolescence and adulthood.

Over 95% of CAH cases are caused by 21- hydroxylase deficiency (21-OHD). CAH due to 21-OHD is classified into three subtypes in terms of disease severity: salt wasting (SW), simple virilizing (SV) (these two subtypes together are termed classic CAH), and nonclassic CAH (mild or late onset). The estimated prevalence of classic CAH is ~1/15,000 while nonclassic CAH occurs in ~0.1-0.2% in the general White population, but is more frequent in some populations such as Ashkenazi Jews. A conclusive molecular diagnosis is necessary for better personalized treatment and accurate genetic counselling.

CAH is a group of autosomal recessive disorders. Over 95% of CAH cases are caused by 21- hydroxylase deficiency (21-OHD) due to genetic defects in the enzyme’s encoding gene CYP21A2 (Hannah-Shmouni et al. 2017. PubMed ID: 28476231). Genetic analysis of this gene is complicated by its pseudogene CYP21A1P (approximately 98% identical), and the nature of local surrounding sequence. This region, also known as the RCCX module (RP-C4-CYP21-TNX), in the human leukocyte antigen histocompatibility complex on chromosome 6 is characterized by frequent homologous recombination events due to the existence of highly homologous pseudogenes in tandem.

Overall, pathogenic variants in CYP21A2 can be divided into three types: (1) ~65% are due to gene conversion, where one or more of 12 most common pathogenic variants (P30L, In2G, G110Efs, I172N, I236N, V237E, M239K, V281L, Leu307fs, Q318X, R356W and P453S) from the pseudogene are transferred to the CYP21A2 gene; (2) ~30% are chimeric CYP21A1P/CYP21A2 genes (alternatively called 30kb deletions in the literature) due to unequal crossover at the RCCX module; (3) the remaining ~5% are rare pathogenic variants that are not from the pseudogene. Each chimeric CYP21A1P/CYP21A2 gene contains one to multiple of the 12 most common pathogenic variants depending on the location of junction site (Chen et al. 2012. PubMed ID: 22156666). The classification (determination of junction site) of chimeric CYP21A1P/CYP21A2 genes is clinically relevant. De novo events in CYP21A2 appeared to be extremely rare.

Since our test utilizes an integrative (“one-stop”) strategy via Sanger sequencing to perform a comprehensive evaluation, the detection rate of pathogenic CYP21A2 variants is expected to be equal to what multi-method strategies yield, which is approximately 98% (Krone et al. 2000. PubMed ID: 10720040; Stikkelbroeck et al. 2003. PubMed ID: 12915679; Finkielstain et al. 2011. PubMed ID: 20926536; Krone et al. 2013. PubMed ID: 23337727).

We utilize a long range PCR strategy to specifically amplify the functional CYP21A2 gene next to TNXB at the centromeric tail of the RCCX module. This “one-stop” strategy is able to capture chimeric CYP21A1P/CYP21A2 genes (alternatively called 30kb deletions in the literature) due to unequal crossover at the RCCX module, common variants transferred from the pseudogene via gene conversions and other rare pathogenic variants. We don’t recommend additional testing (such as MLPA for 30kb deletions), except for family follow-up tests.

LIMITATIONS OF THIS TEST: A duplicated CYP21A2 gene or a CYP21A2-like gene next to the pseudogene TNXA at the middle of the RCCX module, which is uncommon, CANNOT be detected via the current strategy (see for example at Koppens et al. 2002. PubMed ID: 12384784; Kleinle et al. 2011. PubMed ID: 19773403; Tsai et al. 2011. PubMed ID: 21324303). Therefore, test results via the current strategy should always be interpreted in context of clinical findings, family history and other laboratory data.