薄基底膜肾病并发局灶节段性肾小球硬化症一家系的COL4A3和COL4A4基因突变分析

Mutation analysis of COL4A3/COL4A4 genes in a family with thin basement membrane nephropathy and focal segmental glomerulosclerosis

目的探讨薄基底膜肾病（TBMN）合并局灶节段性肾小球硬化症（FSGS）的遗传学机制。方法对一病理学诊断为TBMN合并FSGS患者及其家系的COL4A3和COL4A4基因突变，应用与COL4A3和COL4A4基因连锁的微卫星标记连锁分析方法进行分析。PCR扩增COL4A3和COL4A4全部98个外显子后，直接测序筛查突变。同时测序排除已为公认的FSGS相关基因NPHS1、NPHS2、WT1、TRPC6、ACTN4、CD2AP突变导致FSGS的可能。结果微卫星标记连锁分析显示此家系与COL4A3和COL4A4基因连锁。直接测序在此家系中发现疾病患者COL4A4基因1214位的鸟嘌呤突变为腺嘌呤，导致Ⅳ型胶原仅4链第405位甘氨酸突变为谷氨酸，并且发现COL4A3基因一多态性IVS1．4C〉T。此多态性随疾病分布，可能与致病相关。未发现FSGS相关基因的突变。结论此家系是在TBMN的基础上发生FSGS。Ⅳ型胶原d4链突变及随疾病分布的基因多态性是否导致TBMN合并FSGS或使其易感性增加尚待更多家系进一步研究。

Objective To elucidate whether focal segmental glomerulosclerosis （FSGS） is a secondary development of the COL4-1inked thin basement membrane nephropathy （TBMN） or the primary FSGS produces thin glomerular basement membrane （GBM）. Methods The family members presented microscopic hematuria, increasing proteinuria with the years and a dual pathological diagnosis of FSGS and TBMN was made in the proband. DNA linkage analysis at locus 2q36-37 that contains the COL4A3/COL4A4 genes was performed with polymorphic microsatellite markers D2S434, D2S279, D2S1370, D2S256 and D2S427. Haplotypes were constructed at the COL4A3/COL4A4 loci for affected and unaffected family members. All exons of COL4A3 and COL4A4 genes were screened for mutations in the proband. Mutation screening was also performed for NPHS1, NPHS2, CD2AP, WT1, TRPC6 and ACTN4 to exclude familial FSGS. Mutation or polymorphism found in the family were examined in 50 healthy controls. Results In this family hematuria segregated with the 55224 haplotype at the COL4A3/COL4A4 locus. G to A substitution at nucleotide 1214 resulting in an glyeine being replaced by glutamate （G405E） was demonstrated for the first time in exon 20 of COL4A4 gene. G405E was present in all four members of the family with hematuria but not in the seven unaffected family members nor in 50 healthy controls. A novel polymorphism segregating with hematuria （IVS1-4C 〉T in exon 2 of COL4A3） was also found which was only present in all four affected family members but not in the seven unaffected family members. No mutations were demonstrated in FSGS associated genes, however, a novel SNP （R268Q）, which distributed with the disease incompletely, was described in the NPHS1 gene coding nephrin, the podocyte slit diaphragm protein. Conclusions In this family, FSGS occurres on the basis of TBMN. Maybe the particular COL4A3/COL4A4 mutation and polymorphism work together to develop proteinuria and eventually leading to FSGS. But whether the mutation and the polymorphism segregating with the disease predispose to develop FSGS in TBMN patients is required further study.