Data_Sheet_1_The repeat length of C9orf72 is associated with the survival of amyotrophic lateral sclerosis patients without C9orf72 pathological expansions.pdf

ObjectiveTo explore whether the repeat lengths of the chromosome 9 open reading frame 72 (C9orf72) gene and the ataxin-2 (ATXN2) gene in amyotrophic lateral sclerosis (ALS) patients without C9orf72 repeat expansions confer a risk of ALS or survival disadvantages in ALS.
MethodsWe screened a hospital-based cohort of Chinese patients with sporadic ALS without C9orf72 repeat expansions and neurologically healthy controls for C9orf72 GGGGCC and AXTN2 CAG repeat length to compare the frequency of possible detrimental length alleles using several thresholds. Furthermore, the clinical features of ALS were compared between patients with ALS subgroups using different length thresholds of maximum C9orf72 and ATXN2 repeat alleles, such as sex, age of onset, diagnostic delay, and survival.
ResultsOverall, 879 sporadic patients with ALS and 535 controls were included and the repeat lengths of the C9orf72 and ATXN2 were both detected. We found significant survival differences in patients using a series of C9orf72 repeat length thresholds from 2 to 5, among which the most significant difference was at the cutoff value of 2 (repeats 2 vs. >2: median survival 67 vs. 55 months, log-rank p = 0.032). Furthermore, Cox regression analysis revealed the role of age of onset [hazard ratio (HR) 1.04, 95% CI 1.03–1.05, p < 0.001], diagnostic delay (0.95, 0.94–0.96, p < 0.001), and carrying C9orf72 repeat length of 2 (0.72, 0.59–0.89, p = 0.002) in the survival of patients without C9orf72 repeat expansions. In addition, bulbar onset was associated with poorer survival when the patients carried the maximum C9orf72 repeat allele over 2 (1.81, 1.32–2.48, p < 0.001). However, no survival difference was found when applying a series of continuous cutoff values of ATXN2 or stratified by C9orf72 repeats of 2.
ConclusionThe length of 2 in the maximum C9orf72 repeat allele was identified to be associated with favorable survival in ALS patients without C9orf72 repeat expansions. Our findings from the clinical setting implicated the possible cutoff definition of detrimental C9orf72 repeats, which should be helpful in the understanding of genetics in ALS and in clinical genetic counseling.

**研究目的**：旨在探讨无C9orf72基因重复扩增的肌萎缩侧索硬化症（amyotrophic lateral sclerosis, ALS）患者中，9号染色体开放阅读框72（C9orf72）与共济失调蛋白2（ATXN2）基因的重复序列长度，是否会增加ALS发病风险或对ALS患者的生存产生不利影响。
**研究方法**：本研究纳入基于医院队列的中国散发性ALS患者（无C9orf72基因重复扩增）及神经系统健康对照，对C9orf72基因GGGGCC重复序列与ATXN2基因CAG重复序列长度进行检测，通过多种阈值标准比较可能具有致病风险的等位基因频率。此外，依据最大C9orf72与ATXN2重复等位基因的不同长度阈值对ALS患者进行亚组分组，比较不同亚组间ALS临床特征，包括性别、起病年龄、诊断延迟时间及生存情况。
**研究结果**：本研究共纳入879例散发性ALS患者与535例对照，均完成了C9orf72与ATXN2基因重复序列长度检测。结果显示，采用2至5的一系列C9orf72重复序列长度阈值进行分析时，患者的生存情况存在显著差异；其中以阈值2时差异最为显著（重复数=2 vs. >2：中位生存期分别为67个月与55个月，对数秩检验p=0.032）。进一步Cox回归分析显示，在无C9orf72重复扩增的ALS患者中，起病年龄[风险比（hazard ratio, HR）=1.04，95%置信区间（95% CI）=1.03~1.05，p<0.001]、诊断延迟时间（HR=0.95，95% CI=0.94~0.96，p<0.001）以及携带C9orf72重复数为2的等位基因（HR=0.72，95% CI=0.59~0.89，p=0.002）均与患者生存相关。此外，当患者携带的最大C9orf72重复等位基因数超过2时，球部起病与较差的生存预后相关（HR=1.81，95% CI=1.32~2.48，p<0.001）。然而，采用一系列连续阈值分析ATXN2基因重复序列，或依据C9orf72重复数为2进行分层分析时，均未发现生存情况存在显著差异。
**研究结论**：在无C9orf72基因重复扩增的ALS患者中，最大C9orf72重复等位基因的重复数为2，与患者良好的生存预后相关。本研究基于临床队列的结果，明确了具有致病风险的C9orf72基因重复序列的临界阈值，这将有助于加深对ALS遗传学机制的理解，并为临床遗传咨询提供参考依据。