原发性局灶节段性肾小球硬化患者CD2AP基因突变的研究

CD2AP mutation in patients with focal segmental glomerulosclerosis

目的了解原发性局灶节段性肾小球硬化(FSGS)患者CD2AP基因突变特点。方法研究对象为2001年至2004年我院收治的82例病理确诊为FSGS患者,年龄12-76岁,男性43例,女性39例,临床诊断为肾病综合征(NS)者55例,非NS 27例;60例健康正常人为对照组。外周血基因组DNA PCR扩增后直接测序。冰冻切片免疫荧光双染色,激光共聚焦显微镜采集图像检测突变患者肾组织中CD2AP和podocin蛋白的表达。结果 (1)发现2个CD2AP外显子突变,1个为2号外显子160G>A杂合突变,造成第54位氨基酸由缬氨酸变为异亮氨酸 (V54I),该患者为非NS患者,已出现肾功能不全。另1个为4号外显子358A>G杂合突变,造成第120位氨基酸由异亮氨酸变为缬氨酸(1120V),该患者为NS患者,曾复发2次,目前肾功能尚正常。正常对照120条染色体中未发现同样突变。查阅文献和基因库,未发现相同突变报道。 (2)CD2AP外显子突变患者肾小球内CD2AP表达明显减低,同时伴有podocin表达的降低。(3) 发现1个启动子区突变、2个内含子突变和8个SNP位点,其中一个单核苷酸多态性(SNP)位点以往未见报道。结论CD2AP突变可能是原发性FSGS的致病原因之一。CD2AP外显子突变可导致CD2AP蛋白表达减少,并影响podocin的表达。

Objective To investigate a possible relationship between CD2AP mutation and focal segmental glomendosclerosis （FSGS）. Methods Genomic DNA from peripheral blood cells of FSGS patients were extracted, and CD2AP mutation was analyzed by polymerase chain reaction （PCR） and direct sequencing. Immunofluorescence staining, confocal laser scanning microscopy and LSM-510 graphic system were used to detect the expression and distribution of protein, including both CD2AP and podocin, in patients with CD2AP mutation. Eighty-two Chinese patients with idiopathic FSGS, including 43 males and 39 females whose age ranged from 12 to 76 years old were enrolled in this study. Of these, 55 had nephrotic syndrome （NS）. Sixty genomic DNA from 60 healthy volunteers were selected as normal control group. Results （1） Two CD2AP heterozygous mutations were detected in exons. One was 160G 〉 A in exon 2, which caused the 54th amino acid changed from valine to isoleucine, and occurred in one non-NS patient with renal failure. Another was 358A 〉 G in exon 4, which caused the 120th amino acid changed from isoleucine to valine, and occurred in one NS patient with normal renal function and relapse twice. The same mutations were not found in the control group of 60 healthy people. （2） A decreased expression was observed in glomendi stained with CD2AP antibody, accompanied with decreasing of podocin, in the patients with CD2AP mutation.（3） Moreover, 2 mutations in introns, 1 in promoter region and 1 SNP were first reported. Conclusions The mutations in CD2AP may cause FSGS in both NS and non-NS patient. The decreasing expression of CD2AP resulting from CD2AP gene mutation may affect the expression of poclocin.