The Power of Proficiency Testing: Unraveling Single-Nucleotide Polymorphism Interference, With Potential Impact on...

The College of American Pathologists proficiency testing program has been instrumental in identifying problems in clinical testing.

To describe how this program was used to identify a single-nucleotide polymorphism that affects clinical testing for spinocerebellar ataxia type 3.

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.

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 3' 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' end being complementary to the single-nucleotide polymorphism, showed the expected heterozygous pattern. The possibility of DNA polymerase 3'-5' 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'-5' exonuclease activity yielded both homozygous and expected heterozygous results with the published forward primer.

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.