A bias in genotyping the ERBB2 (HER2) Ile655Val variant.

Dear Sir, As part of association studies on cancer risk ERBB2 Ile655Val is a frequently analysed polymorphism. Recent publications have analysed the association of the common ERBB2 single nucleotide polymorphism (SNP) Ile655Val with breast cancer risk using TaqMan allelic discrimination (1,2). However, this genotyping method appears to be inappropriate regarding Ile655Val as the adjacent variant Ile654Val interferes with the correct binding of the TaqMan probe. Frank et al. performed their case-control study by directly sequencing both Ile654Val and Ile655Val (3). As the Ile654Val variant is rare, only the heterozygous genotype (Ile/Val) was detected in the cases or the controls. In addition, Val654 (1960G) was observed to be in complete linkage with the more common Val655 (1963G). Our primary outcome of Ile655Val genotyping using TaqMan allelic discrimination (Assay-on-Demand no. C_7452451_1 by Applied Biosystems, ABI, Foster City, CA) were not concordant with our sequencing results (Table I). Obviously labelled TaqMan probes were not capable of binding the Val654 allele, thereby providing false homozygous results concerning Ile655Val. (The genotype constellation Val654-Val655/Ile654-Ile655 resulted in false homozygous Ile655 instead of heterozygous Ile655/ Val655 calls.) The same is expected to apply to self-designed probes (e.g. using the ABI Assay-by-Design service), which consequently comprise both SNP sites. Due to the rareness of Ile654Val, the introduced genotyping bias seems negligible. Considering our data (3), 1.1% of the controls are heterozygous carriers for Ile654Val. However, the error rate regarding the heterozygous carriers of Ile655Val (39.3%) would add up to ~2.5%. It is noteworthy that the introduced bias for the breast cancer cases—comprising 2.9% heterozygotes for Ile654Val—would rise to ~7.5%. Given these facts, we would like to point out that the use of alternative genotyping methods, such as direct sequencing, restriction or primer extension, is indispensable for further studies investigating ERBB2 Ile655Val (3,4). In general, this example demonstrates the importance of verifying a portion of genotyping results using an independent method like sequencing. Additionally, sequence polymorphisms in the vicinity of the SNP to be analysed should be carefully considered when designing PCR/sequencing primers or TaqMan probes.