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. 2024 Aug 2:2:101882.
doi: 10.1016/j.gimo.2024.101882. eCollection 2024.

Ascertainment of uninterrupted CAG repeat length and disease-modifying variants in fragment-based genetic testing for Huntington Disease

Hailey Findlay Black  1 , Chris Kay  1 , Jessica Dawson  1 , Stephanie Bortnick  1 , Kyla Javier  1 , Qingwen Xia  1 , Cheuk Hin Chau  1 , Tess Leavitt  1 , Larissa Arning  2 , Huu Phuc Nguyen  2 , Michael R Hayden  1
Affiliations

Ascertainment of uninterrupted CAG repeat length and disease-modifying variants in fragment-based genetic testing for Huntington Disease

Hailey Findlay Black et al. Genet Med Open. .

Abstract

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.

Keywords: Genetic modifiers; Huntington disease; Loss of interruption; Triplet-primed PCR; Variant screening.

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Conflict of interest statement

Michael R. Hayden is the CEO of Prilenia Therapeutics, a private company, and serves on the public boards of Ionis Pharmaceuticals, Oxford Biomedica, AbCellera and 89bio. All other authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Triplet-primed PCR and peak height ratio screening methods. A. HTT canonical CAG and CCG repeat structure, indicating locations of interrupting codons and primer binding sites for triplet-primed PCR (TP-PCR) forward and reverse primers. B. TP-PCR traces of canonical, CAG duplication of interruption (DOI), CAG-CCG loss of interruption (LOI), CAG LOI, and CCG LOI alleles with uninterrupted CAG repeat lengths of 41; bold numbers show diagnostic CAG repeat lengths, and red numbers indicate misestimation of uninterrupted CAG repeat length. C. Peak height ratio (PHR) and secondary peak height ratio (2° PHR) calculations, with TP-PCR trace indicating peak naming notation. D. Effect of diagnostic CAG repeat length and genotype on PHR in TP-PCR sizing of blood DNA samples. Dashed line indicates logistic fit for canonical samples; grey shaded area indicates PHRs <80% of predicted value. E. 2° PHR enables CAG-length-dependent separation of DOI from LOI variants. Dashed line indicates proposed threshold for differentiation of LOI and DOI variants.
Figure 2
Figure 2
Peak height ratio screening of (CAG)40-41 blood DNA. A. Peak height ratio (PHR) values of (CAG)40-41 alleles with known canonical, known noncanonical, or unknown presumed-canonical genotypes. P values indicate results of Wilcoxon rank-sum tests. B. Secondary peak height ratio (2° PHR) values of (CAG)40-41 alleles with low PHR. Dashed line indicates the previous proposed threshold for differentiation of loss of interruption (LOI) from duplication of interruption (DOI) variants. C. Proposed screening workflow for identification and size-correction of mis-sized variant alleles, including optional 2° PHR screening of suspected variants.
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References

    1. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. The Huntington’s Disease Collaborative Research Group. Cell. 1993;72(6):971–983. doi: 10.1016/0092-8674(93)90585-e. - DOI - PubMed
    1. Bean L., Bayrak-Toydemir P., ACMG Laboratory Quality Assurance Committee Addendum: American College of Medical Genetics and Genomics Standards and Guidelines for Clinical Genetics Laboratories, 2014 edition: technical standards and guidelines for Huntington disease. Genet Med. 2021;23(12):2461. doi: 10.1038/s41436-020-0893-3. - DOI - PubMed
    1. Caron N.S., Wright G.E.B., Hayden M.R. In: GeneReviews®. Adam M.P., Feldman J., Mirzaa G.M., et al., editors. University of Washington; Seattle: 1998. Huntington disease; pp. 1–34.https://www.ncbi.nlm.nih.gov/books/NBK1305/
    1. Squitieri F., Jankovic J. Huntington’s disease: how intermediate are intermediate repeat lengths? Mov Disord. 2012;27(14):1714–1717. doi: 10.1002/mds.25172. - DOI - PubMed
    1. Bao Y.F., Li X.Y., Dong Y., Wu Z.Y. Loss of CAA interruption and intergenerational CAG instability in Chinese patients with Huntington’s disease. J Mol Med (Berl) 2023;101(7):869–876. doi: 10.1007/s00109-023-02329-0. - DOI - PubMed

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