The power of proficiency testing: Unraveling single-nucleotide polymorphism interference, with potential impact on clinical testing of spinocerebellar ataxia type 3

Context.-The College of American Pathologists proficiency testing program has been instrumental in identifying problems in clinical testing. Objective.-To describe how this program was used to identify a single-nucleotide polymorphism that affects clinical testing for spinocerebellar ataxia type 3. Design.-A proficiency testing sample with discordant results for spinocerebellar ataxia type 3 analysis was further evaluated by targeted Sanger sequencing and genotype polymerase chain reaction using multiple DNA polymerases. Results.-Of 28 laboratories responding in the spinocerebellar ataxia type 3 Proficiency Survey, 18 reported an incorrect homozygous result and 10 reported the expected heterozygous result. A heterozygous single-nucleotide polymorphism complementary to the 30 end of a published forward primer was identified in the proficiency testing sample, which may have led to allele dropout. However, this primer was used by only 3 of 18 laboratories (16%) reporting a homozygous result. A new forward primer of identical sequence, except for the 3 ⁰ end being complementary to the single-nucleotide polymorphism, showed the expected heterozygous pattern. The possibility of DNA polymerase 3 ⁰ -5 ⁰ exonuclease activity contributing to allele dropout was investigated by testing 9 additional polymerases with and without exonuclease activity. No clear pattern emerged, but enzymes with and without 3 ⁰ -5 ⁰ exonuclease activity yielded both homozygous and expected heterozygous results with the published forward primer. Conclusions.-Proactive systematic primer sequence checking is recommended because single-nucleotide polymorphism interference may result in allele dropout and impact clinical testing. Allele dropout is also influenced by other factors, including DNA polymerase exonuclease activity.