Age-dependent somatic expansion of the ATXN3 CAG repeat in the blood and buccal cell DNA of individuals with spinocerebellar ataxia type 3

Spinocerebellar ataxia type 3 (SCA3), a currently untreatable disorder, is caused by the expansion of a genetically unstable polyglutamine-encoding complex CAG repeat in the ATXN3 gene. Longer alleles are generally associated with earlier onset and frequent intergenerational expansions mediate the anticipation observed in this disorder. Somatic expansion of the repeat has also been implicated in disease progression and slowing the rate of somatic expansion in patients has recently been proposed as a therapeutic strategy. Here, we utilised high-throughput ultra-deep MiSeq amplicon sequencing of the ATXN3 repeat to precisely define the number of repeats, the exact sequence structure, the phased genotype of an adjacent single nucleotide polymorphism and to accurately quantify somatic expansion in blood and buccal cell DNA samples of a cohort of individuals with SCA3 from the Azores islands (Portugal). We revealed systematic mis-sizing of the ATXN3 repeat and high levels of inaccuracy of the traditional fragment length analysis approach that have important implications for attempts to identify modifiers of clinical and molecular phenotypes, including genetic instability. Quantification of somatic expansion in blood DNA revealed the expected effects of age and CAG repeat length, although the effect of repeat length was surprisingly modest with much stronger associations with age at sampling. We also observed an association of the downstream rs12895357 single nucleotide polymorphism with the rate of somatic expansion, and a higher level of somatic expansion in buccal cell DNA compared to blood. Although the levels of somatic expansion are much lower per repeat unit, the average level of somatic expansion at the ATXN3 locus in SCA3 patients is much higher than is typically observed at the HTT locus in Huntington disease patients. These data suggest that the ATXN3 locus in SCA3 patients in blood or buccal cell DNA might serve as a good biomarker for clinical trials testing suppressors of somatic expansion with peripheral exposure. ### Competing Interest Statement Within the last 36 months Professor Monckton has been a scientific consultant and/or received an honoraria/grants from AMO Pharma, Dyne, F. Hoffman-La Roche, LoQus23, MOMA Therapeutics, Novartis, Ono Pharmaceuticals, Pfizer Pharmaceuticals, Rgenta Therapeutics, Sanofi, Sarepta Therapeutics Inc, Script Biosciences, Triplet Therapeutics, and Vertex Pharmaceuticals. Professor Monckton also had research contracts with AMO Pharma and Vertex Pharmaceuticals. The other authors have declared no competing interests. ### Funding Statement The study was funded by a Newton Mosharafa PhD Scholarship award to AMS from the British Council and the Egyptian Ministry of Higher Education and Scientific Research. MR is supported by FCT (https://doi.org/10.54499/CEECIND/03018/2018/CP1556/CT0009). The Fundo Regional para a Ciencia e Tecnologia (FRCT, Governo Regional dos Acores) has supported this work, under the PRO-SCIENTIA program. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Ethical Board of the University of the Azores (Parecer 1/2020) gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.