Rebello George, Ramesar Rajkumar, Vorster Alvera, Roberts Lisa, Ehrenreich Liezle, Oppon Ekow, Gama Dumisani, Bardien Soraya, Greenberg Jacquie, Bonapace Giuseppe, Waheed Abdul, Shah Gul N, Sly William S
Medical Research Council Human Genetics Research Unit, Division of Human Genetics, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Observatory, Cape Town 7925, South Africa.
Proc Natl Acad Sci U S A. 2004 Apr 27;101(17):6617-22. doi: 10.1073/pnas.0401529101. Epub 2004 Apr 16.
Genetic and physical mapping of the RP17 locus on 17q identified a 3.6-megabase candidate region that includes the gene encoding carbonic anhydrase IV (CA4), a glycosylphosphatidylinositol-anchored protein that is highly expressed in the choriocapillaris of the human eye. By sequencing candidate genes in this region, we identified a mutation that causes replacement of an arginine with a tryptophan (R14W) in the signal sequence of the CA4 gene at position -5 relative to the signal sequence cleavage site. This mutation was found to cosegregate with the disease phenotype in two large families and was not found in 36 unaffected family members or 100 controls. Expression of the mutant cDNA in COS-7 cells produced several findings, suggesting a mechanism by which the mutation can explain the autosomal dominant disease. In transfected COS-7 cells, the R14W mutation (i) reduced the steady-state level of carbonic anhydrase IV activity expressed by 28% due to a combination of decreased synthesis and accelerated turnover; (ii) led to up-regulation of immunoglobulin-binding protein, double-stranded RNA-regulated protein kinase-like ER kinase, and CCAAT/enhancer-binding protein homologous protein, markers of the unfolded protein response and endoplasmic reticulum stress; and (iii) induced apoptosis, as evidenced by annexin V binding and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining, in most cells expressing the mutant, but not the WT, protein. We suggest that a high level of expression of the mutant allele in the endothelial cells of the choriocapillaris leads to apoptosis, leading in turn to ischemia in the overlying retina and producing autosomal dominant retinitis pigmentosa.
对17号染色体长臂上RP17基因座进行的遗传和物理图谱分析确定了一个3.6兆碱基的候选区域，该区域包含编码碳酸酐酶IV（CA4）的基因，CA4是一种糖基磷脂酰肌醇锚定蛋白，在人眼脉络膜毛细血管中高度表达。通过对该区域的候选基因进行测序，我们在CA4基因信号序列中相对于信号序列切割位点的-5位置发现了一个导致精氨酸被色氨酸取代（R14W）的突变。在两个大家族中发现该突变与疾病表型共分离，而在36名未受影响的家庭成员或100名对照中未发现。在COS-7细胞中表达突变型cDNA产生了几个结果，提示了该突变可解释常染色体显性疾病的机制。在转染的COS-7细胞中，R14W突变：（i）由于合成减少和周转加速，使碳酸酐酶IV活性的稳态水平降低了28%；（ii）导致免疫球蛋白结合蛋白、双链RNA调节蛋白激酶样内质网激酶和CCAAT/增强子结合蛋白同源蛋白上调，这些是未折叠蛋白反应和内质网应激的标志物；（iii）在大多数表达突变蛋白而非野生型蛋白的细胞中诱导凋亡，这通过膜联蛋白V结合和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记染色得以证实。我们认为，脉络膜毛细血管内皮细胞中突变等位基因的高表达导致凋亡，进而导致上方视网膜缺血，并产生常染色体显性视网膜色素变性。
