Reply: a bias in genotyping of the ERBB2 (HER2) Ile655Val variant

Patrick R. Benusiglio *, Craig Luccarini, Oluseun Ajai, Bruce A.J. Ponder and Paul D. Pharoah

Department of Oncology, University of Cambridge, Strangeways Research Laboratory, Wort's Causeway, Cambridge CB1 8RN, UK

* To whom correspondence should be addressed. Email: pbenusiglio{at}yahoo.com

Dear Sir,

In a recent study of the neighbouring ERBB2 single nucleotide polymorphisms (SNPs) Ile654Val and Ile655Val, Frank et al. concluded that TaqMan genotyping of Ile655Val provided inaccurate results (1). In their assay, a valine at position 654 interfered with the correct binding of the Ile655Val probes. As a result, the individuals with the Ile654-Ile655/Val654-Val655 genotype combination were called Ile655 homozygotes. Because of this bias, the authors questioned the validity of our results on Ile655Val and breast cancer risk (2).

We share the concerns expressed by Frank et al. regarding the reliability of TaqMan genotyping when there is a second SNP, known or yet to be identified, close to the SNP under study. The TaqMan assay is a measure of allele dose, and position on the allelic discrimination plot varies with the number of alleles (zero, one or two) bound by the probes. If the binding of the TaqMan probe for Val655 is inhibited by a Val at position 654, individuals with the Ile654-Ile655/Val654-Val655 combination would appear as Ile655 hemizygotes, and not Ile655 homozygotes. Indeed, this is what we have observed.

We have genotyped Ile654Val in the 4449 individuals previously studied for Ile655Val using a TaqMan assay. Ile654Val genotypes were not affected by the proximity of Ile655Val since probes were complementary to a 16 base pair region not including Ile655Val. We observed 85 heterozygotes for Ile654Val. If the bias of the Ile655Val assay was as extreme as that described by Frank et al. none of these 85 would have been called Ile655Val heterozygotes. However, we called 19 of these 85 as heterozygotes for Ile655Val, as well as one Ile655 homozygote and 12 Val655 homozygotes. There were also 53 individuals with undetermined Ile655Val genotype. Of these, 32 (13 cases and 19 controls) were in a small cluster close to the Ile655Val heterozygote cluster on the allelic discrimination plot (Figure 1). These 32 individuals are probably Ile655Val heterozygotes appearing as hemizygotes because of the altered binding capacity of the probe. We have re-analysed our case-control data treating these samples as Ile655Val heterozygotes; the results were not affected (data not shown).



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Fig. 1. Ile655Val TaqMan allelic discrimination plot. Seven samples hemizygous for Ile655 (circled) sit close to the heterozygote cluster (light grey).

 
The reliability of TaqMan genotyping can indeed be altered by a second SNP located in the vicinity of the SNP under study. If the second SNP is known, probes must be designed so that they are complementary to a target sequence excluding it. If the second SNP is yet to be identified, TaqMan discrimination plots will exhibit the characteristic pattern similar to the one shown in Figure 1, in which case we recommend investigation of the region surrounding the SNP under study by direct sequencing.Conflict of Interest Statement: None declared.

References

  1. Frank,B., Hemminki,K., and Burwinkel,B. (2005) A bias in genotyping the ERBB2 (Her2) Ile655Val variant. Carcinogenesis, 26, 1649.[Free Full Text]
  2. Benusiglio,P.R., Lesueur,F., Luccarini,C., Conroy,D.M., Shah,M., Easton,D.F., Day,N.E., Dunning,A.M., Pharoah,P.D. and Ponder,B.A. (2005) Common ERBB2 polymorphisms and risk of breast cancer in a white British population: a case-control study. Breast Cancer Res., 7, R204–R209.[CrossRef][ISI][Medline]
Received June 22, 2005; accepted July 4, 2005.





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