背景:Dysferlinopathy是一种由DYSF基因突变引起的常染色体隐性遗传疾病。这项研究报道了DYSF基因中的两个纯合相邻错义突变,临床上表现为双侧下肢无力和小腿肿胀。DYSF基因中的两个纯合子相邻错义突变可能与异常蛋白病的发展有关,但是确切的机制需要进一步研究。方法:回顾性分析一个受病患儿家庭的临床资料。从该家族成员收集外周血样品用于全外显子组测序(WES)和拷贝数变异分析。采用Sanger测序来确认潜在的致病变体。人类拼接搜索器,SpliceAI,和varSEAK数据库用于预测突变对剪接功能的影响。通过体内剪接测定和体外小基因测定,确定了由于DYSF基因中两个纯合相邻错义突变而导致的异常剪接在异常酶病中的致病机制。结果:先证者为42岁女性,表现为下肢无力2年,小腿水肿。两个纯合DYSF变体,c.5628C>Ap.D1876E和c.5633A>Tp.Y1878F,在先证者中被确认。生物信息学数据库提示DYSF的突变c.5628C>A对剪接信号没有显著影响。人类拼接Finder版本2.4.1表明DYSF突变的c.5633A>T引起辅助序列的改变和ESE/ESS基序比的显著改变。VarSEAK和SpleeAI提示DYSF突变的c.5633A>T没有剪接作用。体内剪接测定和体外小基因测定均显示两个相邻的突变:DYSF基因中的c.5628C>Ap。D1876E和c.5633A>Tp。Y1878F导致Exon50跳跃,导致蛋白质内32个氨基酸缺失。DYSF基因中的点突变c.5628C>Ap.D1876E影响了体外剪接,而DYSF基因中c.5633A>Tp.Y1878F的点突变不影响剪接功能。结论:本研究首次证实了DYSF的两个纯合突变与异常铁蛋白病的发生有关。DYSF中c.5628C>Ap.D1876E突变影响了剪接功能,可能是致病因素之一。
Background: Dysferlinopathy is an autosomal recessive disorder caused by mutations in the DYSF gene. This study reported two
homozygous adjacent missense mutations in the DYSF gene, presenting clinically with bilateral lower limb weakness and calf swelling. Two
homozygous adjacent missense mutations in the DYSF gene may be associated with the development of dysferlinopathy, but the exact mechanism needs further investigation. Methods: A retrospective analysis of clinical data from a dysferlinopathy-affected family was conducted. Peripheral blood samples were collected from members of this family for whole-exome sequencing (WES) and copy number variation analysis. Sanger sequencing was employed to confirm potential pathogenic variants. The Human Splicing Finder, SpliceAI, and varSEAK database were used to predict the effect of mutations on splicing function. The pathogenic mechanism of aberrant splicing in dysferlinopathy due to two
homozygous adjacent missense mutations in the DYSF gene was determined by an in vivo splicing assay and an in vitro minigene assay. Results: The proband was a 42-year-old woman who presented with weakness of the lower limbs for 2 years and edema of the lower leg. Two
homozygous DYSF variants, c.5628C>A p. D1876E and c.5633A>T p. Y1878F, were identified in the proband. Bioinformatics databases suggested that the mutation c.5628C>A of DYSF had no significant impact on splicing signals. Human Splicing Finder Version 2.4.1 suggested that the c.5633A>T of DYSF mutation caused alteration of auxiliary sequences and significant alteration of the ESE/ESS motif ratio. VarSEAK and SpliceAI suggested that the c.5633A>T of DYSF mutation had no splicing effect. Both an in vivo splicing assay and an in vitro minigene assay showed two adjacent mutations: c.5628C>A p. D1876E and c.5633A>T p. Y1878F in the DYSF gene leading to an Exon50 jump that resulted in a 32-aa amino acid deletion within the protein. Point mutation c.5628C>A p. D1876E in the DYSF gene affected splicing in vitro, while point mutation c.5633A>T p. Y1878F in the DYSF gene did not affect splicing function. Conclusion: This study confirmed for the first time that two
homozygous mutations of DYSF were associated with the occurrence of dysferlinopathy. The c.5628C>A p. D1876E mutation in DYSF affected the splicing function and may be one of the contributing factors to the pathogenicity.