Next-Generation Sequencing Revealed L-Carnitine Rescues Congenital Myopathy in Transgenic Zebrafish with Tropomyosin 3 Novel Mutation. Next-Generation Sequencing Revealed L-Carnitine Rescues Congenital Myopathy in Transgenic Zebrafish with Tropomyosin 3 Novel Mutation

Purpose: Congenital myopathies (CM) are a group of clinically and genetically heterogeneous muscle disorders, characterized by muscle weakness and hypotonia from birth. Currently, no definite treatment exists for CM. A novel and de novo mutation in tropomyosin 3 [TPM3(E151G)] was identified from a 7-year-old boy with clinically diagnosed CM. TPM3(E151A) mutation was previously reported as causing CM. In this study, we created transgenic zebrafish to investigate the pathogenicity and find potential treatment. The goals of this study are to describe the RNA contents in muscle specimens derived from TPM3(WT), TPM3(E151A), TPM3(E152G) at embryos and adult stages and to compare the differences TPM3(E151A), TPM3(E151G) mutants and TPM3(WT). The biological function of L-carnitine treatment TPM3(E151G) versus without treatment are also predicted and evaluated. Methods: Muscle mRNA profiles of 6 months adult zebrafish from TPM3(WT), TPM3(E151A) and TPM3(E151G) were extracted and performed deep sequencing, in duplicate, using Illumina Novaseq6000. We also obtained the muscle mRNA profiles of 2 day's zebrafish embryos from TPM3(WT), TPM3(E151A), TPM3(E151G) and TPM3(E151G) treated with L-carnitine. RNA profiles of adult and embryos were generated by deep sequencing, using Illumina NovaSeq 6000. Results: After acquiring of clean reads, we mapped about 40 million sequence reads per sample to the zebrafish genome (GRCz11). Saturation rate analysis was then performed evaluating the gene coverage rate along with read depth increase. The transcripts mapping to difference functional region of the genome was then analyzed. Circos software was used to map the consistency of samples with zebrafish genome on a chromosomal level. The quantification of transcript intensity was analyzed using Fragments Per Kilobase of exon modelper Million mapped fragments (FPKM). Conclusions: Our study represents the first detailed analysis of zebrafish muscle transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles between TPM3(WT), TPM3(E151A) and TPM3(E151G) and L-carnitine treatment. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. Overall design: Next-generation sequencing experiment was performed by NHRI Genomics Core Facility. Sequencing libraries 1 µg total RNA were generated using RNA HyperPrep kit with RiboErase (KAPA Biosystems, Pleasanton, CA, USA), and sequenced on NovaSeq 6000 instrument (Illumina, San Diego, CA, USA). All RNA sample preparation procedures were carried out according to the Illumina's official protocol.

研究目的：先天性肌病（Congenital myopathies, CM）是一类兼具临床与遗传异质性的肌肉疾病，以出生后即出现肌无力与肌张力低下为典型特征。目前针对CM尚无确切有效的治疗方案。本研究从1名临床确诊为CM的7岁男性患儿体内，鉴定出原肌球蛋白3（tropomyosin 3, TPM3）的新发突变[TPM3(E151G)]；此前已有TPM3(E151A)突变引发CM的报道。本研究构建转基因斑马鱼模型，旨在探究该类突变的致病机制并筛选潜在治疗策略。本研究的目标包括：分析TPM3野生型（TPM3(WT)）、TPM3(E151A)及TPM3(E152G)突变型斑马鱼在胚胎期与成年阶段肌肉样本的RNA表达谱，对比TPM3(E151A)、TPM3(E151G)突变体与TPM3(WT)之间的表达差异；同时评估左旋肉碱（L-carnitine）处理与未处理的TPM3(E151G)突变体的生物学功能差异。

研究方法：本研究提取了6月龄TPM3(WT)、TPM3(E151A)及TPM3(E151G)成年斑马鱼的肌肉组织mRNA，设置生物学重复进行双份深度测序，测序平台为Illumina NovaSeq6000。此外，我们还获取了TPM3(WT)、TPM3(E151A)、TPM3(E151G)以及经左旋肉碱处理的TPM3(E151G)2日龄斑马鱼胚胎的肌肉mRNA表达谱，同样通过Illumina NovaSeq 6000平台完成深度测序。

研究结果：获取洁净读段（clean reads）后，我们将每个样本约4000万条序列读段比对至斑马鱼参考基因组（GRCz11）。随后开展饱和度分析，评估随测序读长增加的基因覆盖度变化。对比对至基因组不同功能区域的转录本进行注释与分析。使用Circos软件在染色体水平上绘制样本与斑马鱼基因组的比对一致性图谱。采用每百万映射读段的外显子模型每千碱基片段数（Fragments Per Kilobase of exon model per Million mapped fragments, FPKM）对转录本表达强度进行定量分析。

研究结论：本研究首次借助RNA测序技术，结合生物学重复，对斑马鱼肌肉转录组开展了全面细致的分析。本研究中优化的数据分析流程，可为TPM3(WT)、TPM3(E151A)、TPM3(E151G)突变体及左旋肉碱处理组之间的表达谱比较研究提供标准化框架。研究结果证实，下一代测序（next-generation sequencing, NGS）可对细胞或组织内的mRNA进行全面且更精准的定量与定性评估。我们认为，基于RNA-seq的转录组表征技术可加速遗传网络分析进程，并助力解析复杂的生物学功能。

实验整体设计：本研究的下一代测序实验由NHRI基因组核心设施完成。使用RNA HyperPrep试剂盒搭配RiboErase（KAPA Biosystems，美国加利福尼亚州普莱森顿）构建测序文库，总RNA投入量为1 μg，随后在NovaSeq 6000测序仪（Illumina，美国加利福尼亚州圣迭戈）上完成测序。所有RNA样本制备流程均严格遵循Illumina官方实验方案。