Settas Nikolaos, Bittel Adam J, Chen Yi-Wen
Center for Genetic Medicine Research, Children's National Research and Innovation Campus, Washington, DC 20012, USA.
Department of Genomics and Precision Medicine, School of Medicine and Health Science, The George Washington University, Washington, DC 20037, USA.
Int J Mol Sci. 2025 May 28;26(11):5170. doi: 10.3390/ijms26115170.
Facioscapulohumeral muscular dystrophy (FSHD) is caused by the aberrant expression of the double homeobox 4 () gene. In this study, an analysis of human FSHD muscle biopsies revealed differential expressions of six m6A regulators, including writers, readers and eraser proteins. In immortalized human FSHD myoblasts, we found higher levels of mRNA and protein expression of a major m6A regulator, methyltransferase-like protein 3 (), in comparison with myoblasts from unaffected siblings (UASbs). Quantification of the overall RNA m6A levels in the FSHD myoblasts revealed significant elevation compared with their UASb, which was reversed to UASb levels following treatment with an antisense oligonucleotide targeting the . Using Oxford Nanopore direct-RNA sequencing, we mapped m6A across the transcriptome and identified genes harboring differential methylated m6A sites, including several involved in iron homeostasis. Western blot protein quantification showed that FSHD myoblasts had higher levels of ferritin-heavy chain-207 isoform and mitoferrin-1. In addition, our data showed elevation in mitochondrial ferrous iron in FSHD myoblasts. Our findings suggest that m6A RNA modifications play a pivotal role in FSHD pathophysiology and may serve as biomarker for this disease.
面肩肱型肌营养不良症（FSHD）是由双同源盒4（）基因的异常表达引起的。在本研究中，对人类FSHD肌肉活检组织的分析揭示了六种m6A调节因子的差异表达，包括写入蛋白、读取蛋白和擦除蛋白。在永生化的人类FSHD成肌细胞中，我们发现与未受影响的同胞（UASbs）的成肌细胞相比，一种主要的m6A调节因子——甲基转移酶样蛋白3（）的mRNA和蛋白表达水平更高。对FSHD成肌细胞中总体RNA m6A水平的定量分析显示，与它们的UASb相比有显著升高，在用靶向的反义寡核苷酸处理后，其恢复到UASb水平。使用牛津纳米孔直接RNA测序，我们在转录组中绘制了m6A图谱，并鉴定出含有差异甲基化m6A位点的基因，包括几个参与铁稳态的基因。蛋白质免疫印迹定量分析表明，FSHD成肌细胞中重链铁蛋白-207异构体和线粒体铁转运蛋白-1的水平更高。此外，我们的数据显示FSHD成肌细胞中的线粒体亚铁含量升高。我们的研究结果表明，m6A RNA修饰在FSHD病理生理学中起关键作用，可能作为这种疾病的生物标志物。
