FBN1基因c.4336G>A突变导致35号外显子缺失可能引发马方综合征

Exon 35 skipping of FBN1 caused by c.4336G>A mutation may lead to Marfan syndrome

目的探究1例马方综合征家系的遗传学病因及检出突变c.4336G>A对原纤维蛋白-1(FBN1)基因剪接效应的影响。方法对2019年8月就诊于空军军医大学西京医院心血管外科的1例胸主动脉夹层动脉瘤患者,采用多重PCR联合二代测序技术对其进行遗传性主动脉疾病相关的15个基因的组合检测,应用Sanger测序技术对检出位点进行验证并对部分家庭成员进行同一位点的检测。通过剪接预测软件预测该突变位点对剪接的影响,并提取正常人及患者新鲜外周血中RNA,通过逆转录PCR及Sanger测序,验证突变对剪接过程的影响。根据美国医学遗传学与基因组学学会(ACMG)遗传变异分类标准与指南分析检出位点的致病性。结果基因组合检测结果显示先证者携带有FBN1基因杂合变异c.4336G>A,Sanger测序结果与二代测序结果一致。Sanger测序对部分家庭成员检测发现,该家系中还存在4名携带者,其中3例已出现胸主动脉瘤或夹层的表现;dbscSNV_ada_score和dbscSNV_rf_score预测检出位点可能影响剪接,患者及正常人RNA经逆转录PCR并对PCR产物进行Sanger测序后,结果表明该位点导致患者FBN1基因35号外显子缺失;根据ACMG遗传变异分类标准与指南判读该位点致病等级为“致病”。结论FBN1基因c.4336G>A位点被证实影响剪接过程,导致FBN1基因35号外显子缺失,这可能是该家系中多名马方综合征患者的致病原因。

Objective To investigate the genetic etiology of a Marfan syndrome pedigree,and the impact of c.4336G>A variant on the splicing process of FBN1 gene.Methods The proband was admitted to the Department of Cardiovascular Surgery of Xijing Hospital due to thoracic aortic aneurysm and dissection in August 2019.Multiplex PCR and next generation sequencing technology were used to detect 15 genes associated with hereditary aortic diseases in the proband.Then the pathogenic sites were further verified by Sanger sequencing,and above examinations were also performed among the family members of the proband.The effect of the mutation on mRNA splicing was predicted by splicing prediction software.RNAs from peripheral blood cells of the proband and the healthy person were extracted,and the effect of the mutation on mRNA splicing was verified by reverse transcription PCR and Sanger sequencing.The pathogenicity was analyzed by the recommendations from the American College of Medical Genetics(ACMG).Results The gene panel detected a missense mutation of FBN1 gene(c.4336G>A)in the proband.Sanger sequencing results were consistent with that of panel.Sanger sequencing results showed that 4 family members were carriers of the same variant,and 3 out of the 4 family members presented signs of thoracic aortic aneurysm and dissection.The dbscSNV_ada_score and dbscSNV_rf_score software predicted that this mutation would lead to the occurrence of abnormal splicing of mRNA.The skipping of exon 35 was verified in the subsequent examinations by reverse transcription PCR and Sanger sequencing.The variant was classified as"pathogenic"according to ACMG guideline.Conclusion FBN1 c.4336G>A mutation can cause the skipping of exon 35,and this might be the genetic mechanistic of severe cardiovascular abnormalities observed in this Marfan syndrome pedigree.