Table1_Evidence of survival bias in the association between APOE-Є4 and age at ischemic stroke onset.DOCX

IntroductionLarge genome-wide association studies (GWASs) using case–control study designs have now identified tens of loci associated with ischemic stroke (IS). As a complement to these studies, we performed GWAS in a case-only design to identify loci influencing the age at onset (AAO) of ischemic stroke.
MethodsAnalyses were conducted in a discovery cohort of 10,857 ischemic stroke cases using a linear regression framework. We meta-analyzed all SNPs with p-value <1 x 10−5 in a sexcombined or sex-stratified analysis using summary data from two additional replication cohorts.
ResultsIn the women-only meta-analysis, we detected significant evidence for the association of AAO with rs429358, an exonic variant in apolipoprotein E (APOE) that encodes for the APOE-Є4 allele. Each copy of the rs429358:T>C allele was associated with a 1.29-year earlier stroke AAO (meta p-value = 2.48 x 10−11). This APOE variant has previously been associated with increased mortality and ischemic stroke AAO. We hypothesized that the association with AAO may reflect a survival bias attributable to an age-related decrease in mortality among APOE-Є4 carriers and have no association to stroke AAO per se. A simulation study showed that a variant associated with overall mortality might indeed be detected with an AAO analysis. A variant with a 2-fold increase in mortality risk would lead to an observed effect of AAO that is comparable to what we found.
DiscussionIn conclusion, we detected a robust association of the APOE locus with stroke AAO and provided simulations to suggest that this association may be unrelated to ischemic stroke per se but related to a general survival bias.

引言
采用病例对照研究设计的大型全基因组关联研究（genome-wide association studies, GWAS）迄今已鉴定出数十个与缺血性脑卒中（ischemic stroke, IS）相关的基因座。作为此类研究的补充，本研究采用仅病例研究设计开展全基因组关联研究，以鉴定影响缺血性脑卒中发病年龄（age at onset, AAO）的基因座。

方法
本研究以10857例缺血性脑卒中病例作为发现队列，采用线性回归框架开展分析。本研究利用另外两个独立验证队列的汇总数据，针对P值<1×10^−5的所有单核苷酸多态性（single nucleotide polymorphism, SNPs）开展不分性别或性别分层的meta分析。

结果
在仅针对女性的meta分析中，我们检测到rs429358与缺血性脑卒中发病年龄存在显著关联；rs429358是载脂蛋白E（apolipoprotein E, APOE）的外显子变异，编码APOE-Є4等位基因。每携带1份rs429358:T>C等位基因，脑卒中发病年龄可提前1.29年（meta分析P值=2.48×10^−11）。该APOE变异此前已被证实与死亡率升高及缺血性脑卒中发病年龄相关。我们推测，其与发病年龄的关联可能反映了APOE-Є4携带者中年龄相关死亡率下降所导致的存活偏倚，而非与缺血性脑卒中发病年龄本身存在直接关联。一项模拟研究证实，与总体死亡率相关的变异确实可通过发病年龄分析被检测到。若某变异使死亡率风险升高2倍，其对应的发病年龄观测效应与本研究发现的结果相当。

讨论
综上，本研究证实APOE基因座与脑卒中发病年龄存在显著关联，并通过模拟研究提示，该关联可能并非与缺血性脑卒中本身相关，而是与普遍的存活偏倚有关。