Table2_How does the age of control individuals hinder the identification of target genes for Huntington’s disease?.XLSX

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 146 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.

已有多项研究对比了亨廷顿病（Huntington’s disease, HD）基因阳性患者与神经功能正常个体的多脑区转录组，旨在发掘可作为HD治疗或预后靶点的潜在差异表达基因（differentially expressed genes, DEGs）。尽管这些研究均遵循了优化RNA测序（RNA-Seq）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功能正常个体样本，其年龄高于HD基因阳性患者组。鉴于大脑衰老会引发渐进性转录组改变，我们提出假设：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。为验证该假设，我们对SRA数据库中公开的该研究的146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基因阳性患者组在统计学上无显著差异。本研究明确了在基因差异表达分析中，使用年龄超过75岁的神经功能正常个体作为对照所带来的不良影响，该做法会导致假阳性与假阴性结果的产生。我们最终证实，使用此类高龄对照样本会导致差异表达基因的错误鉴定，进而对潜在治疗与预后标志物的发掘产生不利影响。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。