Triplet repeat primed PCR simplifies testing for Huntington disease

Diagnostic and predictive testing for Huntington disease (HD) requires an accurate determination of the number of CAG repeats in the Huntingtin (HHT) gene. Currently, when a sample appears to be homozygous for a normal allele, additional testing is required to confirm amplification from both alleles. If the sample still appears homozygous, Southern blot analysis is performed to rule out an undetected expanded HTT allele. Southern blot analysis is expensive, time-consuming, and labor intensive and requires high concentrations of DNA. We have developed a chimeric PCR process to help streamline workflow; true homozygous alleles are easily distinguished by this simplified method, and only very large expanded alleles still require Southern blot analysis. Two hundred forty-six HD samples, previously run with a different fragment analysis method, were analyzed with our new method. All samples were correctly genotyped, resulting in 100% concordance between the methods. The chimeric PCR assay was able to identify expanded alleles up to >150 CAG repeats. This method offers a simple strategy to differentiate normal from expanded CAG alleles, thereby reducing the number of samples reflexed to Southern blot analysis. It also provides assurance that expanded alleles are not routinely missed because of allele dropout.