Identification of an Intronic Single Nucleotide Polymorphism Leading to Allele Dropout During Validation of a CDH1 Sequencing Assay: Implications for Designing Polymerase Chain Reaction-based Assays

Purpose: The CDH1 gene encodes the cell adhesion protein E-cadherin, and CDH1 germline mutations are associated with hereditary diffuse gastric cancer. Identification of individuals at high risk of developing diffuse gastric cancer affords the opportunity for endoscopic screening or elective prophylactic gastrectomy. We set out to develop a CDH1 sequencing assay for clinical use.

Methods: All exons of the CDH1 gene were amplified and sequenced with published and modified primers.

Results: While validating the assay, we encountered a case in which a single nucleotide polymorphism located in intron 15 led to allele dropout and therefore to a false-negative result. The polymorphism leading to allele dropout was located within a primer-binding sequence, five bases away from the 3' end of the primer. A frameshift mutation in exon 15 was detected by an alternative primer that binds away from the polymorphic site. A search of the University of California Santa Cruz single nucleotide polymorphism database revealed other polymorphisms located within primer-binding sites. A total of 12 primers in nine primer sets were modified to minimize allele dropout risk.

Conclusion: The approach of designing primers to avoid known single nucleotide polymorphisms can be generalized to the design of any polymerase chain reaction-based assay and should be employed whenever possible.