Pseudoautosomal region 1 overdosage affects the global transcriptome in iPSCs from patients with Klinefelter syndrome and high-grade X chromosome aneuploidies

Klinefelter syndrome (KS) is the most prevalent aneuploidy in males and is characterized by one or more supernumerary X chromosomes. Here, using a paradigmatic cohort of KS-inducible pluripotent stem cells (iPSCs) carrying 49-XXXXY, 48-XXXY, and 47-XXY karyotypes, we identified genes within the pseudoautosomal region 1 (PAR1) region as the most susceptible to dosage-dependent transcriptional dysregulation and therefore potentially responsible for the progressively worsening phenotype in higher grade X aneuploidies. By contrast, biallelically expressed non-PAR escape genes displayed high interclonal and interpatient variability in iPSCs and differentiated derivatives, suggesting that these genes could be associated with variable KS traits. By interrogating KS-iPSCs at single-cell resolution, we showed that PAR1 and non-PAR escape genes were not only resilient to X-inactive specific transcript (XIST)-mediated inactivation but also that their transcriptional regulation was disjointed from the absolute XIST expression level. Finally, we explored the transcriptional effects of X chromosome overdosage on autosomes and identified the transcription factor nuclear respiratory factor 1 as the master regulator of zinc finger protein X-linked. Our findings provided the first evidence of an X-dosage-sensitive autosomal transcription factor regulating an X-linked gene in low- and high-grade X aneuploidies. Overall design: Bulk transcriptomic profiles of iPSC derived from Klinefelter syndrome patients and controls. The cohort includes low and high grade KS clones. For each patient two or three independent clones have been generated and for each clone two or three independent RNA-Seq libraries have been processed

克莱费尔特综合征（Klinefelter syndrome, KS）是男性群体中最常见的染色体非整倍体疾病，以携带一条或多条额外X染色体为典型特征。本研究依托包含49-XXXXY、48-XXXY及47-XXY核型的克莱费尔特综合征诱导多能干细胞（inducible pluripotent stem cells, iPSCs）典型队列，鉴定出假常染色体区域1（pseudoautosomal region 1, PAR1）内的基因最易受剂量依赖性转录失调影响，推测其可能与更高等级X染色体非整倍体的进行性加重表型密切相关。

与之相对，双等位基因表达的非PAR逃逸基因在iPSCs及分化子代细胞中呈现显著的克隆间与患者间变异，提示这类基因或与克莱费尔特综合征的可变表型相关联。

通过对克莱费尔特综合征iPSCs开展单细胞分辨率分析，本研究证实PAR1基因与非PAR逃逸基因不仅可抵御X失活特异性转录本（X-inactive specific transcript, XIST）介导的X染色体失活，且其转录调控模式与XIST的绝对表达水平相互脱节。

最后，本研究探究了X染色体过量对常染色体的转录调控效应，并鉴定出转录因子核呼吸因子1（nuclear respiratory factor 1）为X连锁锌指蛋白的核心调控因子。

本研究首次提供证据表明，在低、高等级X染色体非整倍体中，存在受X剂量敏感调控的常染色体转录因子，其可靶向调控X连锁基因。

整体实验设计：克莱费尔特综合征患者与健康对照来源的iPSCs批量转录组谱。该队列涵盖低、高等级克莱费尔特综合征克隆；每位患者可构建2~3个独立克隆，每个克隆可制备2~3个独立RNA测序（RNA-Seq）文库。