The importance of heteroduplexes in interpreting the results of PCR-RED diagnostic assays: Application to the analysis of mutations in the steroid 21-hydroxylase gene in a case of congenital adrenal hyperplasia

Background: Congenital adrenal hyperplasia (CAH) due to deficiency of steroid 21-hydroxylase (CYP21) is an autosomal recessive disease that is a major cause of ambiguous genitalia at birth in females. The milder late-onset form of the disease can cause mild virilization in women including hirsutism, infertility, and acne. Characterization of the causative mutations in a patient requires finding mutations on both chromosomes. Methods and Results: We use polymerase chain reaction (PCR) followed by re striction enzyme digestion (RED), among several other methods, to detect mutations in the CYP21 gene. The authors found two different point mutations in a patient with CAH, V281L and Q318X. Did we find two mutations in trans which would account for the disease, or two mutations in cis with a still unknown mutation(s) on the other chromosome? We devised a method to determine the cis/trans nature of the mutations using PCR amplification, digestion with a restriction enzyme (ApaL I) diagnostic for V281L, gel purification of the mutant band which lacks the cutting site, digestion with a second restriction enzyme (Pst I) which detects Q318X, and gel electrophoretic analysis of the products. Although the results initially supported a cis orientation, consideration of the impact of heteroduplex formation during the PCR on the products of RED showed that the orientation was actually trans. Conclusions: When DNA that is heterozygous for a mutation is amplified using the PCR, four double-stranded DNA products call be formed: the two homoduplexes and two heteroduplexes. The heteroduplex DNA must be considered to ensure correct interpretation of the results of PCR-RED analysis.