Ashavaid Tester F, Kondkar Altaf A, Dherai Alpa J, Raghavan Rani, Udani Soonu V, Udwadia Zarir F, Desai Devendra
Research Laboratories, P.D. Hinduja National Hospital and Medical Research Centre, Mahim, Mumbai, India.
Mol Diagn. 2005;9(2):59-66. doi: 10.1007/BF03260073.
Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CFTR gene. The most severe, DeltaF508, mutation accounts for nearly 70% of CF cases worldwide. Besides DeltaF508, there are other point mutations, namely G542X, G551D, R553X, N1303K, and 621+1(G-->T), which are common among Caucasians. Additionally, a polyT polymorphism in intron 8 is also involved in the pathogenesis of CF. However, neither the prevalence nor the types of mutations causing CF in India are known. In this study, we aimed at estimating the frequency of the above common mutations and polymorphism in clinically suspected CF cases. We also carried out partial analysis of the CFTR gene, limited to exons 10 and 11, to identify other variations in these exons.
The multiplex amplification refractory mutation system (ARMS) test was applied for rapid simultaneous analysis of six most common CF mutations, in 100 normal and 39 elevated sweat chloride cases. The scanning of exons 10 and 11 was carried out by single-stranded conformation polymorphism/heteroduplex (SSCP/HD) analysis, followed by DNA sequencing in 50 normal and 37 elevated sweat chloride cases. A single ARMS-polymerase chain reaction assay was used to distinguish the 5T, 7T, and 9T alleles in 100 normal and 33 elevated sweat chloride cases.
The multiplex ARMS analysis identified the DeltaF508 mutation at an allele frequency of 24% in Indian CF cases. However, the other predominant CF mutations were found to be absent. The 7T polyT variant was observed to be the most common allele, followed by the 9T, and 5T, which was the lowest. The DeltaF508 mutation was observed in all instances with the 9T variant. The SSCP/HD and DNA sequencing additionally revealed a known polymorphism (M470V, exon 10) and a known mutation [1525-1(G-->A), intron 9]. The 1525-1(G-->A) mutation, observed in a single 4-year-old male, is predicted to code for a class II defective CFTR protein.
The findings of this study suggest a difference in relative frequencies and spectrum of CFTR mutations in Indian CF cases. A larger screening study of the entire CFTR gene in clinically well defined CF cases is required to delineate common mutations in the CFTR gene and enable molecular diagnosis of CF in India.
囊性纤维化（CF）是一种由CFTR基因突变引起的常染色体隐性疾病。最严重的ΔF508突变占全球CF病例的近70%。除了ΔF508，还有其他点突变，即G542X、G551D、R553X、N1303K和621 + 1（G→T），这些在白种人中很常见。此外，内含子8中的多聚T多态性也参与了CF的发病机制。然而，在印度导致CF的突变的患病率和类型均不清楚。在本研究中，我们旨在估计临床疑似CF病例中上述常见突变和多态性的频率。我们还对CFTR基因进行了部分分析，限于外显子10和11，以确定这些外显子中的其他变异。
采用多重扩增不应性突变系统（ARMS）检测对100例正常人和39例汗液氯化物升高的病例进行六种最常见CF突变的快速同步分析。通过单链构象多态性/异源双链（SSCP/HD）分析对外显子10和11进行扫描，随后对50例正常人和37例汗液氯化物升高的病例进行DNA测序。使用单一ARMS聚合酶链反应测定法区分100例正常人和33例汗液氯化物升高的病例中的5T、7T和9T等位基因。
多重ARMS分析在印度CF病例中以24%的等位基因频率鉴定出ΔF508突变。然而，未发现其他主要的CF突变。观察到7T多聚T变体是最常见的等位基因，其次是9T，而5T是最低的。在所有9T变体的病例中均观察到ΔF508突变。SSCP/HD和DNA测序还揭示了一个已知的多态性（M470V，外显子10）和一个已知的突变[1525 - 1（G→A），内含子9]。在一名4岁男性中观察到的1525 - 1（G→A）突变预计编码一种II类缺陷型CFTR蛋白。
本研究结果表明印度CF病例中CFTR突变的相对频率和谱存在差异。需要对临床明确的CF病例进行更大规模的全CFTR基因筛查研究，以确定CFTR基因中的常见突变，并实现印度CF的分子诊断。
