COL4A5基因剪接变异致Alport综合征患者1例的遗传学分析及体外验证

Genetic analysis and in vitro validation of a case of Alport syndrome due to a splicing variant of COL4A5 gene

目的探讨1例Alport综合征(AS)患者的遗传学特征并验证其存在的剪接变异。方法选取2021年1月8日因"体检发现尿蛋白3+、尿潜血3+"就诊于内蒙古医科大学附属医院肾脏内科的1例AS患者作为研究对象,收集患者临床资料。抽取患者及其父母的外周血样本,提取基因组DNA,利用全外显子组测序及Sanger测序确定基因变异位点,应用mRNA异常剪接体外验证实验研究变异对转录产物的影响。应用生物学软件分析变异位点的氨基酸保守性并模拟胶原蛋白Ⅳ模型,分析变异蛋白的三维结果。对患者肾组织进行免疫荧光与免疫组织化学检查,确认是否存在AS肾脏损伤。结果患者为21岁男性,门诊尿常规检查显示24 h尿蛋白3.53 g/24 h(大量蛋白尿),血尿酸491μmol/L(高尿酸血症)。基因测序结果显示患者COL4A5基因存在剪接变异c.835-9T>A,患者父母未见该变异。体外实验结果显示c.835-9T>A(p.279_297del)导致COL4A5基因mRNA的第15外显子缺失57 bp,造成第279~297位氨基酸残基缺失,影响COL4A5基因编码的α5链的二级结构的稳定性。受损的氨基酸在不同物种中高度进化保守。同源建模显示突变的α5链参与的Col-Ⅳ三聚化可以完成,但三维结构变形严重。免疫荧光结果诊断为AS肾脏损伤。根据美国医学遗传学与基因组学学会相关变异评级指南,c.835-9T>A评级为可能致病性变异(PVS1_Moderate+PS3_Moderate+PM2_Supporting+PS2+PP3+PP4)。结论COL4A5:c.835-9T>A考虑是AS患者致病的原因,体外实验证实为剪接变异。通过肾脏组织病理学检查为该变异的致病性提供了体内证据,并拓展了COL4A5基因的变异谱。

Objective To explore the genetic basis for a patient with Alport syndrome(AS)and confirm the existence of a splicing variant.Methods An AS patient diagnosed at the Affiliated Hospital of Inner Mongolia Medical University on January 8,2021 for significant proteinuria and occult hematuria was selected as the study subject.Clinical data was collected.Peripheral blood samples were collected for the extraction of genomic DNA.Whole exome sequencing and Sanger sequencing were carried out to identify potential genetic variants.An in vitro experiment was also conducted to verify the abnormal mRNA splicing.Bioinformatic software was used to analyze the conservation of amino acids of the variant sites and simulate the 3D structure of the variant collagen IV protein.Immunofluorescence and immunohistochemistry were carried out on renal tissues from the patient to confirm the presence of AS kidney injury.Results The patient,a 21-year-old male,had a 24-hour urine protein of 3.53 g/24 h,which fulfilled the diagnostic criteria for proteinuria.His blood uric acid has also increased to 491μmol/L.DNA sequencing revealed that he has harbored a c.835-9T>A splice variant of the COL4A5 gene,which was not found in either of his parents.In vitro experiment confirmed that the variant has removed 57 bp from the exon 15 of the mRNA of the COL4A5 gene.The deletion may cause loss of amino acid residues from positions 279 to 297,which in turn may affect the stability of the secondary structure of theα5 chain encoded by the COL4A5 gene.The amino acids are conserved across various species.The result of homology modeling indicated that the trimerization of Col-IV with the mutatedα5 chain could be achieved,however,the 3D structure was severely distorted.The AS kidney damage was confirmed through immunofluorescence assays.Based on the guidelines from the American College of Medical Genetics and Genomics,the c.835-9T>A variant was classified as likely pathogenic(PVS1_Moderate+PS3_Moderate+PM2_Supporting+PS2+PP3+PP4).Conclusion The c.835-9T>A variant of the COL4A5 gene probably underlay the AS in this patient.In vitro experiment has confirmed the abnormal splicing caused by the variant.Histopathological examination of the kidney tissue has provided in vivo evidence for its pathogenicity.Above finding has expanded the mutational spectrum of the COL4A5 gene.