Investigation of Chimeric Transcripts Derived from LINE-1 and Alu Retrotransposons in Cerebellar Tissues of Individuals with Autism Spectrum Disorder (ASD)

LINE-1 and Alu retrotransposons are components of the human genome and have been implicated in many human diseases. These elements can influence human transcriptome plasticity in various mechanisms. Chimeric transcripts derived from LINE-1 and Alu can also impact the human transcriptome, such as exonization and post-transcriptional modification. However, its specific role in ASD neuropathology remains unclear, particularly in the cerebellum tissues. We performed RNA-sequencing of post-mortem cerebellum tissues from ASD and unaffected individuals for transposable elements profiling and chimeric transcript identification. The majority of free transcripts of transposable elements were not changed in the cerebellum tissues of ASD compared with unaffected individuals. Nevertheless, we observed that chimeric transcripts derived from LINE-1 and Alu were embedded in the transcripts of differentially expressed genes in the cerebellum of ASD, and these genes were related to developments and abnormalities of the cerebellum. In addition, the expression levels of these genes were correlated with the significantly decreased thickness of the molecular layer in the cerebellum of ASD. We also found that global methylation and expression of LINE-1 and Alu elements were not changed in ASD, but observed in the ASD sub-phenotypes. Our findings showed associations between transposable elements and cerebellar abnormalities in ASD, particularly in distinct phenotypic subgroups. Further investigations using appropriate models are warranted to elucidate the structural and functional implications of LINE-1 and Alu elements in ASD neuropathology. Overall design: Post-mortem cerebellum tissues from ASD and unaffected individuals were originally obtained from the Harvard Brain Tissue Resource Center. The snap-frozen post-mortem brain tissues used in this study consist of 13 samples from cerebellum tissues of ASD (n=8) and unaffected controls (n=5). The snap-frozen post-mortem brain tissues were used for RNA isolations before molecular analysis. Total RNA was sent to BGI Hong Kong for RNA-sequencing analysis using the MGI DNBSEQ technology platform. Briefly, total RNA was used to prepare stranded mRNA library construction by mRNA enrichment and was then sequenced by the DNBSEQ-G400 (150 bp paired-end).

LINE-1与Alu反转录转座子（retrotransposons）是人类基因组的组成部分，且与多种人类疾病密切相关。这类元件可通过多种机制影响人类转录组可塑性。由LINE-1和Alu衍生的嵌合转录本（chimeric transcripts）同样可对人类转录组产生影响，例如外显子化与转录后修饰。然而，其在自闭症谱系障碍（ASD, Autism Spectrum Disorder）神经病理学中的具体作用仍不明确，针对小脑组织的相关研究尤为匮乏。
本研究对自闭症患者与健康对照者的死后小脑组织开展RNA测序，以进行转座元件（transposable elements）谱分析与嵌合转录本鉴定。与健康对照相比，自闭症患者小脑组织中大多数游离的转座元件转录本未出现表达变化。
尽管如此，我们观察到，源自LINE-1与Alu的嵌合转录本嵌入了自闭症患者小脑内差异表达基因的转录本序列中，且这些基因与小脑发育及发育异常密切相关。此外，上述基因的表达水平与自闭症患者小脑分子层厚度的显著降低呈相关性。
我们还发现，自闭症患者整体的LINE-1与Alu元件的全局甲基化水平及表达量未发生显著改变，但该变化在自闭症亚表型（sub-phenotypes）中有所体现。本研究结果揭示了转座元件与自闭症患者小脑异常之间的关联，尤其是在特定表型亚组中。后续需采用合适的模型开展深入研究，以阐明LINE-1与Alu元件在自闭症神经病理学中的结构与功能意义。
总体实验设计：本研究使用的自闭症患者与健康对照者的死后小脑组织最初取自哈佛脑组织资源中心（Harvard Brain Tissue Resource Center）。本次研究使用的快速冷冻死后脑组织样本共13份，其中自闭症患者小脑组织样本8份（n=8），健康对照样本5份（n=5）。在开展分子分析前，先从快速冷冻的死后脑组织中提取总RNA。总RNA被送往BGI香港分部，采用MGI DNBSEQ技术平台开展RNA测序分析。简要而言，以总RNA为起始材料，通过mRNA富集制备链特异性mRNA文库，随后使用DNBSEQ-G400测序平台开展150 bp双端测序。