Table4_How does the age of control individuals hinder the identification of target genes for Huntington’s disease?.DOCX

Several studies have compared the transcriptome across various brain regions in Huntington’s disease (HD) gene-positive and neurologically normal individuals to identify potential differentially expressed genes (DEGs) that could be pharmaceutical or prognostic targets for HD. Despite adhering to technical recommendations for optimal RNA-Seq analysis, none of the genes identified as upregulated in these studies have yet demonstrated success as prognostic or therapeutic targets for HD. Earlier studies included samples from neurologically normal individuals older than the HD gene-positive group. Considering the gradual transcriptional changes induced by aging in the brain, we posited that utilizing samples from older controls could result in the misidentification of DEGs. To validate our hypothesis, we reanalyzed 69 samples from this study, accessible on the SRA database, and employed Propensity Score Matching (PSM) to create a “virtual” control group with a statistically comparable age distribution to the HD gene-positive group. Our study underscores the adverse impact of using neurologically normal individuals over 75 as controls in gene differential expression analysis, resulting in false positives and negatives. We conclusively demonstrate that using such old controls leads to the misidentification of DEGs, detrimentally affecting the discovery of potential pharmaceutical and prognostic markers. This underscores the pivotal role of considering the age of control samples in RNA-Seq analysis and emphasizes its inclusion in evaluating best practices for such investigations. Although our primary focus is HD, our findings suggest that judiciously selecting age-appropriate control samples can significantly improve best practices in differential expression analysis.

已有多项研究对亨廷顿舞蹈病（Huntington’s disease, HD）基因阳性者与神经学健康个体的多个脑区转录组展开比较，以期筛选可作为HD药物研发或预后靶点的潜在差异表达基因（differentially expressed genes, DEGs）。尽管现有研究均遵循了RNA测序（RNA-Seq）最优分析的技术规范，但上述研究中鉴定出的上调基因，目前均未被证实可作为HD的预后或治疗靶点。早期研究纳入的神经学健康个体样本，其年龄普遍高于HD基因阳性组。鉴于大脑衰老会引发渐进性转录组变化，我们推测若使用年龄偏高的对照样本，可能会导致差异表达基因的错误鉴定。为验证这一假说，我们对SRA数据库公开的该研究中69份样本进行了重新分析，并采用倾向得分匹配（Propensity Score Matching, PSM）方法构建了“虚拟”对照组，使其年龄分布与HD基因阳性组在统计学上无显著差异。本研究证实，将75岁以上的神经学健康个体作为对照样本用于基因差异表达分析，会产生假阳性与假阴性结果，带来不良影响。本研究最终证实，使用此类高龄对照样本会导致差异表达基因的错误鉴定，进而阻碍潜在药物靶点与预后标志物的发掘。这凸显了在RNA-Seq分析中考量对照样本年龄的关键作用，并强调需将该因素纳入此类研究的最佳实践评估体系。尽管本研究的核心聚焦于HD，但研究结果表明，审慎选择年龄匹配的对照样本，可显著优化差异表达分析的最佳实践流程。