新型ABCB4基因突变所致的进行性家族性肝内胆汁淤积症3型的临床表型及基因型分析

Clinical phenotype and genotype analysis of progressive familial intrahepatic cholestasis type 3 caused by novel ABCB4 gene mutation

目的探讨ATP结合盒转运蛋白B4(ABCB4)新型剪接变异的分子致病机制及临床特点。方法回顾性总结首都医科大学附属北京儿童医院收治的1例胆汁淤积性肝病患儿的临床资料, 采用全外显子组测序检测致病突变并进行Sanger测序验证, 通过生物信息学预测突变位点的致病性, 通过mRNA异常剪接的Minigene体外验证实验对可能致病突变进行体外功能验证, 并随访患儿出院后的临床转归。结果患儿男, 5岁, 11月龄出现胆汁淤积, 体格检查显示肝脾明显肿大, 肝功能、腹部超声及肝脏病理等检查提示胆汁淤积性肝硬化。基因分析结果显示患儿为ABCB4基因致病性突变c.2860G>A和新发突变c.2065-8T>G的复合杂合子, 突变分别来源于其父母, 对c.2065-8T>G位点进行保守性预测, 显示该区域高度保守并可能影响剪接。Minigene实验结果证实c.2065-8T>G突变导致内含子16滞留7 bp序列在成熟的mRNA中, 在未发生无义介导的mRNA降解的情况下, 氨基酸移码形成截短蛋白(p.Glu689ValfsTer19), 最终确诊为进行性家族性肝内胆汁淤积症3型(PFIC3), 予熊去氧胆酸(UDCA)等药物治疗, 在18个月的随访期内, 患儿的临床症状有所改善。结论 ABCB4基因c.2065-8T>G位点被证实影响剪接过程, 与c.2860G>A构成复合杂合子, 确定为PFIC3的致病原因, 携带此突变的PFIC3患儿以胆汁淤积性肝病为主要表现, 疾病进展相对缓慢, 对UDCA治疗敏感。

Objective To investigate the pathogenic mechanism and clinical characteristics of the novel splicing variant of ATP-binding cassette subfamily B member 4(ABCB4)and provide a basis for subsequent genetic diagnosis.Methods The clinical data of a 5-year-old child with cholestatic liver disease admitted to the Beijing Children′s Hospital of Capital Medical University was retrospectively analyzed.The pathogenic variations were detected by whole exome sequencing and verified by Sanger sequencing,and bioinformatics was used to predict the pathogenicity of the mutation sites.Possible pathogenic variations were verified in vitro by Minigene assay.The clinical outcome was followed after discharge from hospital.Results The 5-year-old boy had developed cholestasis at the age of 11 months.His physical examination showed obvious enlargement of the liver and spleen.Cholestatic cirrhosis was diagnosed by liver function tests,abdominal ultrasonography,liver biopsy and pathology.The results of genetic analysis showed that the patient was a complex heterozygote of the ABCB4 gene,with a pathogenic mutation c.2860G>A and a novel mutation c.2065-8T>G,derived from the mother and father respectively.The conservative prediction of the c.2065-8T>G site showed that this region was highly conserved and may affect splicing.Minigene assay results confirmed that the c.2065-8T>G mutation resulted in a 7 bp retention of intron 16 in the mature mRNA.In the absence of nonsense-mediated mRNA decay,the amino acid frameshift forms a truncated protein,which is represented by p.Glu689ValfsTer19.The patient was diagnosed as progressive familial intrahepatic cholestasis type 3(PFIC3)and treated with ursodeoxycholic acid(UDCA).His clinical symptoms improved during 18 months of follow-up.Conclusions The c.2065-8T>G variant is confirmed to affect the splicing process and exhibits complex heterozygosity with c.2860G>A,which is identified as the cause of the disease.PFIC3 children with this variant showed cholestatic liver disease as the main manifestation with a slow progression and was sensitive to treatment with UDCA.