典型结节性硬化症1例临床表型及基因变异分析

Analysis of clinical phenotype and gene variants in a patient with classic tuberous sclerosis complex

目的收集1例典型结节性硬化症患者的临床资料并检测其致病基因变异。方法收集患者临床资料,应用二代测序法对患者进行致病基因筛查,采用Sanger测序法验证,构建迷你基因质粒转染至人肾上皮细胞系293T细胞,提取RNA进行转录分析。结果患者临床表型包括反复癫痫发作,伴面部血管纤维瘤、甲周纤维瘤、肺淋巴管肌瘤病、肾血管平滑肌脂肪瘤及多发性骨质硬化。二代测序提示患者TSC2基因存在可疑致病变异,经Sanger测序验证,患者TSC2基因第4号外显子存在c.336_336+15delGGTAAGGCCCAGGGCG杂合突变,其父母及100名无关健康对照未检测出该位点变异。该突变位点既往未见报道。迷你基因实验显示,患者TSC2基因mRNA序列发生改变,原4号外显子剪切位点丢失,插入74 bp内含子序列,使剪切位置后移90 bp(r.336delins336+16_336+90)。结论TSC2基因第4号外显子c.336_336+15delGGTAAGGCCCAGGGCG杂合变异可导致异常剪切,可能是该结节性硬化症患者病因。

Objective To analyze clinical phenotypes and pathogenic mutations of a patient with classic tuberous sclerosis complex.Methods Clinical data was collected from a patient with classic tuberous sclerosis complex.Next-generation sequencing was performed to screen pathogenic gene variants,and Sanger sequencing to verify the mutations.Minigene plasmids were constructed and transfected into the human renal epithelial cell line 293T,and RNA was extracted for transcriptional analysis.Results The patient clinically presented with recurrent epileptic seizures,facial angiofibroma,periungual fibroma,pulmonary lymphangioleiomyomatosis,renal angiomyolipoma and multiple osteosclerosis.Next-generation sequencing revealed a suspected pathogenic variant in the TSC2 gene in the patient.Sanger sequencing identified a heterozygous mutation c.336_336+15delGGTAAGGCCCAGGGCG in exon 4 of the TSC2 gene in the patient,but not in his parents or 100 unrelated healthy controls.Moreover,this mutation had not been previously reported.The minigene experiment showed changed mRNA sequence of the TSC2 gene in this patient with loss of the authentic splice site in exon 4 and insertion of a 74-bp intron,which shifted the splice site 90 bp downstream(r.336delins336+16_336+90).Conclusion The novel heterozygous mutation c.336_336+15delGGTAAGGCCCAGGGCG in exon 4 of the TSC2 gene can lead to aberrant splicing,and may contribute to tuberous sclerosis complex in this patient.