Utah's ARUP Laboratories has replaced its previous approach to PCR-based Huntington's disease testing with a triple repeat primed PCR method developed by ARUP's' Elaine Lyon.
Lyon and colleagues described the new approach in a study published this week in the Journal of Molecular Diagnostics. The team originally developed the method to detect the presence or absence of expanded alleles in the X chromosome's FMR1 gene. The repeat alleles are associated with fragile X syndrome and other developmental and degenerative disorders (PCR Insider, 5/6/2010)
After developing that assay in collaboration with Celera, the ARUP researchers then decided to try to apply the approach to Huntington's disease by measuring the number of CAG repeats in the HHT gene. The group hoped the method would simplify its testing algorithm by removing the need to reflex to additional PCR and Southern blot analyses.
"We wanted to try the triplet repeat primed PCR approach that worked for Fragile X to simplify our testing process for Huntington's disease and be confident of the results from a single PCR," Lyon told PCR Insider.
"In a typical PCR … amplifying over the repeat region with its high GC content can be challenging, with a chance that a large expansion may not amplify," she explained. "So because of the concern of missing an expanded allele, we did further testing using a second PCR and Southern blots." All in all, she said, the process could easily take several weeks to yield a final result.
With triplet repeat primed PCR, Lyon said, "one primer is designed outside the region and the other is partially in the repeat region. This allows primers to anneal and amplify from each repeat, resulting in PCR products that differ by one repeat."
Separating the products by capillary electrophoresis creates a characteristic stutter pattern for expanded alleles that researchers can then use to distinguish between true homozygous samples and highly expanded alleles — up to more than 100 CAG repeats in the group's JMD study.
In the study, Lyon and colleagues compared their previous testing method — relying on additional PCR steps and Southern blot to confirm apparent homozygous results — to a triplet repeat primed approach based on what they developed in their earlier work with Fragile X.
According to Lyon, designing primers for the assay was a little more complicated than with Fragile X because the researchers needed to design "around known benign variants in the Huntington's gene, which could interfere with the assay and result in an allele dropout."
To evaluate the assay's accuracy, the group looked at 246 samples including 14 from a Coriell Institute for Medical Research repository, three samples from the College of American Pathology, and 229 blinded samples that had been analyzed using ARUP's standard previous workflow.
According to the study authors, the triplet repeat primed PCR approach showed "perfect concordance," characterizing all 246 samples within one CAG repeat of the results established for the repository samples and obtained using ARUP's previous testing paradigm.
The group was able to accurately size repeat numbers ranging from 14 to 101. For one sample with greater than 200 repeats, the assay was able to detect the allele, but the primers failed to amplify the full-length product, according to the authors. Thus, the group set 101 repeats as the assay's upper limit for accurate sizing of expanded alleles.
According to Lyon, ARUP is now offering a laboratory-developed triplet repeat primed PCR test for Huntington's through its CLIA lab. "Since Huntington's disease isn’t as prevalent as Fragile X we haven’t collaborated with other companies or discussed commercialization other than offering it [ourselves,]" she said. The group's FMR1 test is currently offered commercially by Abbott.
Lyon said, the new Huntington's disease assay completely removes the lab's need for Southern blot when analyzing adult samples. In children, however, CAG expansions can often be much larger — sometimes over 200 repeats.
Although the group was able to detect greater-than-100-fold expanded alleles in its study, the authors wrote that homozygous samples from symptomatic kids should still be reflexed to Southern blot just to be safe.
"Southern blot may still be useful to confirm or size very large repeats that are likely in children affected with Huntington disease. We have been able to detect repeats over 100 but there remains a question [of whether] we could potentially miss one," Lyon said.
According to Lyon, the team is now interested in exploring using triplet repeat primed PCR to test for other triplet-repeat disorders, such as Friedreich ataxia and spinocerebellar ataxia.