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.

中文翻译：

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3 型脊髓小脑共济失调患者血液和口腔细胞 DNA 中 ATXN3 CAG 重复序列的年龄依赖性体细胞扩增

脊髓小脑性共济失调 3 型 (SCA3) 是一种目前无法治疗的疾病，是由 ATXN3 基因中遗传不稳定的多聚谷氨酰胺编码复合物 CAG 重复序列的扩增引起的。较长的等位基因通常与较早发病有关，并且频繁的代际扩展介导了在这种疾病中观察到的预期。重复的体细胞扩张也与疾病进展有关，并且最近提出减缓患者体细胞扩张的速率作为一种治疗策略。在这里，我们利用 ATXN3 重复序列的高通量超深 MiSeq 扩增子测序来精确定义重复次数、确切的序列结构、相邻单核苷酸多态性的定相基因型，并准确量化血液和口腔细胞中的体细胞扩张来自亚速尔群岛（葡萄牙）的一组 SCA3 患者的 DNA 样本。我们揭示了 ATXN3 重复序列的系统性错误大小以及传统片段长度分析方法的高度不准确性，这对于尝试识别临床和分子表型修饰因素（包括遗传不稳定性）具有重要意义。血液 DNA 中体细胞扩增的量化揭示了年龄和 CAG 重复长度的预期影响，尽管重复长度的影响令人惊讶地温和，与采样时的年龄有更强的关联。我们还观察到下游 rs12895357 单核苷酸多态性与体细胞扩增速率的关联，以及与血液相比，口腔细胞 DNA 中更高水平的体细胞扩增。尽管每个重复单元的体细胞扩张水平要低得多，但 SCA3 患者中 ATXN3 基因座的体细胞扩张平均水平远高于亨廷顿病患者中 HTT 基因座通常观察到的体细胞扩张水平。这些数据表明，SCA3 患者血液或口腔细胞 DNA 中的 ATXN3 基因座可能作为临床试验测试外周暴露体细胞扩张抑制因子的良好生物标志物。