背景:TPM3基因的致病变异,编码慢骨骼肌α-原肌球蛋白占不到5%的线虫性肌病病例。TPM3中显性遗传或从头错义变体比隐性功能丧失变体更常见。迄今为止报道的隐性变体似乎影响骨骼肌特异性TPM3转录物的5'或3'末端。
目的:本研究的目的是在一名患有不寻常形式的线虫性肌病的芬兰患者中鉴定致病基因和变异体。
方法:遗传分析包括桑格测序,全外显子组测序,靶向阵列-CGH,和连锁阅读全基因组测序。对从患者和对照的培养的成肌细胞和肌管提取的总RNA进行RNA测序。通过Western印迹分析评估TPM3蛋白表达。通过常规组织病理学方法分析诊断性肌肉活检。
结果:患者头部控制不佳,未能茁壮成长,但没有低omimia,他的上肢明显比他的下肢弱,与组织病理学结合的特征提示TPM3引起的线虫性肌病。肌肉组织病理学显示纤维大小变化增加,主要在1型小纤维中存在许多线虫体。发现该患者是TPM3内含子1a中两个剪接位点变体的复合杂合:NM_152263.4:c.117_5delTAGG,删除内含子1a的供体剪接位点,和NM_152263.4:c.117+64C>T,它激活内含子1a非编码外显子之前的受体剪接位点。RNA测序显示转录物中包含内含子1a和非编码外显子,导致早期过早终止密码子。使用患者成肌细胞的Western印迹显示TPM3蛋白的水平显着降低。
结论:新型双等位基因剪接位点变异体显示显著降低TPM3蛋白表达。变异体对剪接的影响很容易通过RNA测序揭示,展示了该方法的力量。
BACKGROUND: Pathogenic variants in the TPM3 gene, encoding slow skeletal muscle α-
tropomyosin account for less than 5% of nemaline myopathy cases. Dominantly inherited or de novo missense variants in TPM3 are more common than recessive loss-of-function variants. The recessive variants reported to date seem to affect either the 5\' or the 3\' end of the skeletal muscle-specific TPM3 transcript.
OBJECTIVE: The aim of the study was to identify the disease-causing gene and variants in a Finnish patient with an unusual form of nemaline myopathy.
METHODS: The genetic analyses included Sanger sequencing, whole-exome sequencing, targeted array-CGH, and linked-read whole genome sequencing. RNA sequencing was done on total RNA extracted from cultured myoblasts and myotubes of the patient and controls. TPM3 protein expression was assessed by Western blot analysis. The diagnostic muscle biopsy was analyzed by routine histopathological methods.
RESULTS: The patient had poor head control and failure to thrive, but no hypomimia, and his upper limbs were clearly weaker than his lower limbs, features which in combination with the histopathology suggested TPM3-caused nemaline myopathy. Muscle histopathology showed increased fiber size variation and numerous nemaline bodies predominantly in small type 1 fibers. The patient was found to be compound heterozygous for two splice-site variants in intron 1a of TPM3: NM_152263.4:c.117+2_5delTAGG, deleting the donor splice site of intron 1a, and NM_152263.4:c.117 + 164 C>T, which activates an acceptor splice site preceding a non-coding exon in intron 1a. RNA sequencing revealed inclusion of intron 1a and the non-coding exon in the transcripts, resulting in early premature stop codons. Western blot using patient myoblasts revealed markedly reduced levels of the TPM3 protein.
CONCLUSIONS: Novel biallelic splice-site variants were shown to markedly reduce TPM3 protein expression. The effects of the variants on splicing were readily revealed by RNA sequencing, demonstrating the power of the method.