Pitfalls of PCR-based genotyping in patients with 21-hydroxylase deficiency

Mutation analysis of CYP21A2 gene was performed in seven patients with congenital adrenal hyperplasia (CAH) by combining differential long template polymerase chain reaction (PCR) amplification and amplified created restriction site (ACRS) methods. All mutations were identified, including five alleles of deletions, three alleles of splicing (IVS2-12[C/A] > G), four alleles of Ile172Asn, and two alleles of Arg356Trp. During the course of genetic analysis of CYP21A2, we found that misgenotyping of CAH by PCR-based method is possible if both alleles of a CAH patient were deletion mutations and at least one of them carried a CYP21A1P-CYP21A2 fusion gene. We also found a patient's mother was misgenotyping as IVS2-12[C/A] > G homozygous due to "allele dropout" in the PCR amplification process. We present in this article evidences of mis-genotyping by PCR-based amplification method. Due to the pitfalls observed in this study, we recommend that more methods, including microsatellite linkage analysis and direct sequencing, should be performed with direct amplification of known mutations in prenatal diagnosis of CAH to avoid misdiagnosis.