Endocrine Abstracts

Congenital adrenal hyperplasia (CAH) is usually caused by the mutations of steroid 21-hydroxylase gene (CYP21A2). CYP21A2 resides in RCCX copy number variation (CNV), and the genomic structure of RCCX CNV creates difficulties in the genetic testing of CAH. An RCCX CNV allele on one chromosome can carry CYP21A2 in various numbers. Homozygous deletion of complete CYP21A2 results in most severe form of CAH, whereas an additional and intact CYP21A2 copy can replace the lost function of the other CYP21A2 on the same chromosome. Therefore, the copy number determination of CYP21A2 has got a profound impact in genetic testing of CAH.

The clinical chemistry validation of duplex real-time quantitative polymerase chain reaction (RT-qPCR) based on hydrolysis probes for the determination of CYP21A2 copy number was performed on 18 genomic DNA samples from European reference population of HapMap project (CEU), 19 samples with good DNA quality and 12 samples with bad DNA quality. The validation of RT-qPCR for steroid 21-hydrosylase pseudogene and the verifications of RT-qPCRs for complement component genes, HERV-K(C4) CNV alleles and RCCX CNV breakpoint region were also achieved. Estimated limit of detection for CYP21A2 RT-qPCR was lower than measurement concentration by two order of magnitude, and nonspecific PCR product was not detected by melting curve analysis and microcapillary electrophoresis. PCR efficiency was 1.019 ± 0.077, and linearity was R² = 0.988 ± 0.008. Repeatability was 0.35 CV% and reproducibility was 0.57 CV%. Accuracy was 7.23 ± 4.28% of the CYP21A2 copy number, however it was 15.15 ± 7.53% in samples with bad DNA quality. Both clinical sensitivity and specificity were 100%, however, a relatively small number of samples was examined (n = 49). Robustness was not influenced by the smaller changes of PCR conditions, but different instrument and inadequate RT-qPCR reagent could diminish it. The validation and verification of RT-qPCR for the copy number of other genetic elements of RCCX CNV had got some flaws, however, their performance were reasonable.

The validation of RT-qPCR assay for the copy number determination of CYP21A2 is fitted for the genetic testing of CAH. In case of need, ambiguous finding in CYP21A2 copy number can be solved by the consideration of the copy numbers of other RCCX CNV elements.