常染色体显性遗传Alport综合征致病基因COL4A4杂合剪接突变的致病性分析及基因型-表型关联分析

Deciphering the pathogenicity of COL4A4 heterozygous splicing mutations and the genotype-phenotype correlation in autosomal dominant Alport syndrome

目的探讨常染色体显性遗传Alport综合征(autosomal dominant Alport syndrome, ADAS)致病基因COL4A4杂合剪接突变的致病机制及基因型与表型的关联, 以加深对COL4A4剪接突变的认识以及对ADAS表型异质性的理解。方法本研究为病例系列分析。从3家医院收集5个ADAS家系先证者及家系成员的临床资料。对从先证者中经全外显子组测序(whole exome sequencing, WES)发现的COL4A4杂合剪接变异, 通过RNA体内剪接或Minigene体外实验分析其对mRNA正常剪接的影响。结果在此5个ADAS家系患者中经WES发现了4个COL4A4杂合剪接变异位点。家系1、家系2、家系3和家系4中多数患者呈孤立性镜下血尿或合并微量蛋白尿, 个别患者合并显性蛋白尿、进入老年期后出现肾功能轻度减退。家系5中4例患者均呈快速肾功能进展, 于28~41岁进展至终末期肾病。家系1、家系2患者携带的c.735+3A>G和家系3患者携带的c.694-1G>C均可引起COL4A4的第12号外显子跳跃导致42 bp核苷酸框内缺失(c.694735del), 家系4中发现的c.2056+3A>G可引起COL4A4的第26号外显子跳跃导致69 bp核苷酸框内缺失(c.19882056del)。家系5中发现的c.2716+5G>T可引起包括第29号外显子3’端100 bp缺失、第30号外显子跳跃和第31号外显子5’端89 bp缺失的360 bp核苷酸大片段框内缺失(c.24462805del)。结论 COL4A4杂合剪接突变导致不同大小核苷酸框内缺失与ADAS表型存在一定关联。通过体内或体外实验明确COL4A4剪接突变影响mRNA剪接的具体方式可能有助于分析ADAS基因型对表型的影响。

Objective Through the investigation of the pathogenicity of COL4A4 heterozygous splicing mutations and the genotype-phenotype correlation in autosomal dominant Alport syndrome(ADAS),to better understand the impact of COL4A4 heterozygous splicing mutations on ADAS.Methods The study was a case series analysis.Patients from 5 ADAS families with COL4A4 heterozygous splicing mutations detected by whole exome sequencing were recruited by three hospitals.In vivo transcriptional analysis and/or in vitro minigene splicing assay were conducted to determine the splicing patterns and assess the pathogenicity of COL4A4 heterozygous splicing mutations.Results In the five ADAS pedigrees carrying COL4A4 heterozygous splicing mutations,four novel ADAS splicing patterns were described.In pedigree 1-4,most patients presented with continuous hematuria or/and microalbuminuria.Otherwise,the proband in pedigree 4 presented with macroalbuminuria and the proband in pedigree 1 had progressed to chronic kidney disease stage 2 at the age of 70 years old.In pedigree 5,all patients developed end-stage renal disease between 28 and 41 years old.c.735+3A>G detected in pedigree 1 and pedigree 2 and c.694-1G>C detected in pedigree 3 both led to exon 12 skipping in COL4A4,resulting in 42 nucleotides in-frame deletion(c.694_735del).c.2056+3A>G detected in pedigree 4 led to COL4A4 exon 26 skipping,which caused in-frame deletion of 69 nucleotides(c.1988_2056del).c.2716+5G>T detected in pedigree 5 led to a 360 nucleotides large in-frame deletion,including 100 bp sequence at the 3'end of exon 29,the whole sequence of exon 30 and 89 bp sequence at the 5'end of exon 31(c.2446_2805del).Conclusions Renal prognosis differs significantly for patients with small in-frame deletions versus large in-frame deletion splicing abnormalities.Determination of the pathogenicity and the splicing patterns of COL4A4 heterozygous splicing mutations using in vivo and in vitro transcriptional analysis may provide renal prognostic information.