Ascertainment of uninterrupted CAG repeat length and disease-modifying variants in fragment-based genetic testing for
Hailey Findlay Black1, Chris Kay1, Jessica Dawson1
1Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, BC, Canada.
Insights
Synonymous variants in Huntington disease (HD) can alter disease onset but are missed in genetic testing. Our new method accurately identifies these variants, ensuring correct diagnostic reporting of HTT CAG repeat length.
Area of Science:
- Genetics
- Neurology
- Molecular Biology
Background:
- Huntington disease (HD) is a neurodegenerative disorder caused by an expanded CAG repeat in the HTT gene.
- Synonymous variants affecting the CAA codon within the HTT CAG repeat can modify HD onset but are currently undetectable by standard genetic testing.
- This leads to inaccurate reporting of uninterrupted CAG repeat length and potential misdiagnosis for individuals near diagnostic cut-offs.
Purpose of the Study:
- To develop and validate a method for identifying synonymous variants within the HTT CAG repeat during genetic testing.
- To ensure accurate diagnostic reporting of uninterrupted CAG repeat length in individuals with Huntington disease.
- To prevent misdiagnosis in patients with alleles close to established diagnostic thresholds.
Main Methods:
- Utilized triplet-primed PCR (TP-PCR) to amplify HTT CAG repeat alleles, including those with canonical and noncanonical interruptions.
- Developed a screening method based on peak height ratio (PHR) analysis of TP-PCR amplification patterns.
- Applied PHR screening to DNA samples from a cohort of symptomatic individuals with diagnostic CAG repeat lengths (40-41).
Main Results:
- TP-PCR successfully amplified HTT CAG repeat alleles with varying interruption patterns.
- The PHR method effectively differentiated between alleles with and without loss or duplication of the CAA interruption.
- Accurate reporting of uninterrupted CAG repeat length was achieved by detecting these previously hidden variants.
Conclusions:
- TP-PCR combined with PHR analysis provides a cost-effective method for detecting disease-modifying noncanonical CAG repeat interruptions.
- This approach enables accurate ascertainment of uninterrupted HTT CAG repeat length.
- The method facilitates precise diagnostic reporting for individuals with Huntington disease, improving diagnostic accuracy.
Purpose:
In Huntington disease (HD), synonymous variants causing loss or duplication of the interrupting CAA codon in the HTT CAG repeat modify disease onset. These variants are undetectable during HD genetic testing, resulting in inaccurate diagnostic reporting of uninterrupted CAG repeat length. Inaccurate reporting of CAG repeat length results in misdiagnosis of individuals with alleles near diagnostic cut-offs. We present a method to identify variant alleles during CAG repeat genotyping, allowing accurate diagnostic reporting of uninterrupted CAG repeat length.
Methods:
We used triplet-primed PCR (TP-PCR) to amplify HTT CAG repeat alleles with canonical or noncanonical repeat interruptions and leveraged differences in peak amplification patterns to develop a screening method based on peak height ratio (PHR). We used PHR to screen blood DNA from a cohort of symptomatic individuals with diagnostic CAG repeat lengths of 40 to 41.
Results:
TP-PCR enables accurate reporting of uninterrupted CAG repeat length in diagnostic testing by detecting HD alleles with loss or duplication of the CAG repeat interruption.
Conclusion:
PHR screening of TP-PCR traces is a cost-effective screening method for detection, ascertainment of uninterrupted HTT CAG repeat length, and accurate diagnostic reporting for individuals with disease-modifying noncanonical CAG repeat interruptions.
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