Overestimated frequency of a possible emphysema-susceptibility allele whenmicrosomal epoxide hydrolase is genotyped by the conventional polymerase chain reaction-based method

A recent association study suggested that the His113 variant of microsomal epoxide hydrolase (mEPHX) may confer a risk for development of emphysema, p resumably by increasing susceptibility to smoking injury. Before considerin g a possible role of this enzyme in pulmonary disease, we attempted to char acterize the genetic polymorphism further. The Tyr/His113 polymorphism with in exon 3 of mEPHX was initially examined in 62 healthy individuals by conv entional methods involving polymerase chain reaction (PCR)-based determinat ion of a restriction fragment length polymorphism (RFLP). Genomic nucleotid e sequences, including the polymorphic site and the downstream primer seque nce, were further analyzed in 95 unrelated, healthy Japanese volunteers by single-stranded conformation polymorphism (SSCP analysis and direct sequenc ing. Genotyping by the first method (PCR-RFLP) revealed that the allelic di stribution in our test population apparently deviated from Hardy-Weinberg e quilibrium. Sequence analysis showed that a synonymous nucleotide substitut ion, AAG to AAA (Lys119), was located just within the published primer site . The AAA at codon 119 was present only in alleles with Tyr113, and its fre quency reached 0.31 in our panel of 190 Japanese alleles. This substitution potentially hampered PCR amplification because of the nucleotide mismatch, with the result that the frequency of the Tyr113 variation was underestima ted. The frequency of His113, a possible emphysema susceptibility allele of the mEPHX gene, was thus overestimated when human DNA samples were genotyp ed in the conventional way. Depending on the population(s) tested, this ano maly could represent a pitfall for PCR-based association studies.