MOLECULAR CHARACTERIZATION OF FOUR FREQUENT POLYMORPHISMS IN THE CFTR GENE: ASSESSING ITS ROLE IN CYSTIC FIBROSIS DISEASE
Abstract
To date, more than 200 sequence polymorphisms have been identified in the CFTR gene thus far and only a minority has been characterized at the cellular localization and the protein levels. In previous studies, molecular genetic analysis of the entire coding region of the CFTR gene in patients identified four common polymorphisms: c.1540A>G is located in Exon 10 of CFTR; c.2694T>G in Exon 14a, whereas c.4404C>T and c.4521G>A are located in Exon 24. The aim of this study was therefore to examine the possible effects of these polymorphisms on subcellular localization and CFTR processing in different constructs to disclose their impact on the clinical phenotype. The subcellular localization using confocal microscopy of coding single-nucleotide polymorphisms c.1540A>G, c.2694T>G, c.4404C>T and c.4521G>A have shown correct membrane localization and normal effect on maturation of CFTR protein. Although maturation patterns were not affected, total amounts of mature CFTR protein were reduced for c.2694T>G and c.4521G>A polymorphisms. The double c.[2694T>G; 4521G>A], triple c.[1540A>G;2694T> G;4521G>A] and quadruple c.[1540A>G; 2694T>G;4404C>T; 4521G>A] polymorphisms have shown to be exclusively cytoplasmic consistent with an endoplasmic reticulum localization. Western blot analysis of CFTR protein indicated that the double and triple mutants had effects on the maturation of CFTR protein. So, more severe effect on CFTR protein was present when these polymorphisms were combined in complex alleles in cis, supporting the influence of these frequent polymorphisms on the clinical features of CF patients. These findings suggest that SNPs may be responsible for variation in the phenotypic expression of CFTR mutations.References
Bombieri, C., S. Giorgi, S. Carles, R. de Cid, F. Belpinati, C. Tandoi, N. Pallares-Ruiz, C. Lazaro, B. M. Ciminelli, M. C. Romey, T. Casals, F. Pompei, G. Gandini, M. Claustres, X. Estivill, P. F. Pignatti and G. Modiano (2000). A new approach for identifying non-pathogenic mutations: an analysis of the cystic fibrosis transmembrane regulator gene in normal individuals. Hum. Genet., 106: 172-178.
Boyle, M. P. (2003). Nonclassic cystic fibrosis and CFTR-related diseases. Current Opinion in Pulmonary Medicine, 9: 498-503.
Chang, M. C., Y. T. Chang, S. C. Wei, Y. W. Tien, P. C. Liang, I. S. Jan, Y. N. Su and J. M. Wong (2007). Spectrum of mutations and variants/haplotypes of CFTR and genotype-phenotype correlation in idiopathic chronic pancreatitis and controls in Chinese by complete analysis. Clin. Genet., 71: 530-539.
Chu, C. S., B. C. Trapnell, S. Curritin and G. R. Cutting (1993). Genetic basis of variable exon 9 skipping in cystic fibrosis transmembrane conductance regulator mRNA. Nat. Genet., 3: 151-156.
Cuppens, H., W. Lin, M. Jaspers, B. Costes, H. Teng, A. Vankeer-berghen, M. Jorissen, G. Droogmans, I. Reynaert, M. Goossens, B. Nilius and J. J. Cassiman (1998). Polyvariant mutant cystic fibrosis transmembrane conductance regulator genes. The polymorphic (Tg)m locus explains the partial penetrance of the T5 polymorphism as a disease mutation. J. Clin. Invest., 101: 487-496.
Cuppens, H., P. Marynen, C. De Boeck and J. J. Cassiman (1993). Detection of 98.5% of the mutations in 200 Belgian cystic fibrosis alleles by reverse dotblot and sequencing of the complete coding region and exon/intron junctions of the CFTR gene. Genomics, 18: 693-697.
Davies, J. C., U. Griesenbach and E. Alton (2005). Modifier Genes in Cystic Fibrosis. Pediatric Pulmonology, 39: 383-391.
De Cid, R., M. D. Ramos, L. Aparisi, C. García, J. Mora, X. Estivill and T. Casals (2010). Independent contribution of common CFTR variants to chronic pancreatitis. Pancreas, 39: 209-215.
George Priya Doss, C., R. Rajasekaran, C. Sudandiradoss, K. Ramanathan, R. Purohit and Sethumadhavan R. (2008). A novel computational and structural analysis of nsSNPs in CFTR gene. Genomic Med., 2: 23-32.
Huang, Q., W. Ding and M. X. Wei (2008). Comparative analysis of common CFTR polymorphisms poly-T, TG-repeats and M470V in a healthy Chinese population. World J. Gastroenterol., 14: 1925-1930.
Laëmmli, U. K. and M. Favre (1973). Maturation of the head of bacteriophage T4. I. DNA packaging events. J. Mol. Biol., 80: 575-599.
Lee, J. H., J. H. Choi, W. Namkung, J. W. Hanrahan, J. Chang, S. Y. Song, S. W. Park, D. S. Kim, J. H. Yoon, Y. Suh, I. J. Jang, J. H. Nam, S. J. Kim, M. O. Cho, J. E. Lee, K. H. Kim and M. G. Lee (2003). A haplotypebased molecular analysis of CFTR mutations associated with respiratory and pancreatic diseases. Hum. Mol. Genet., 12: 2321-2332.
Nikolic, A., A. Divac, M. Stankovic, J. Dinic, B. Tomic and M. Ljujic (2006). Analysis of common CFTR polymorphisms 5 T, M470 V and R75Q in healthy Serbian population. Genetika, 42: 996-998.
Noone, P. G., C. A. Pue, Z. Zhou, K. J. Friedman, E. L. Wakeling, M. Ganeshananthan, R. H. Simon, L. M. Silverman and M. R. Knowles (2000). Lung disease associated with the IVS8 5T allele of the CFTR gene. Am. J. Respir. Crit. Care Med., 162: 1919-24.
Qiao, D., L. Yi, L. Hua, Z. Xu, Y. Ding, D. Shi, L. Ni, N. Song, Y. Wang and H. Wu (2008). Cystic fibrosis transmembrane conductance regulator (CFTR) gene 5T allele may protect against prostate cancer: A case-control study in Chinese Han population. Journal of Cystic Fi-brosis, 7: 3210-214.
Riordan, J. R. (1999). Cystic fibrosis as a disease of misprocessing of the cystic fibrosis transmembrane conductance regulator glycoprotein. Am. J. Hum. Genet., 64: 1499-1504.
Steiner, B., K. Truninger, J. Sanz, A. Schaller and S. Gallati (2004). The role of common single-nucleotide polymorphisms on exon 9 and exon 12 skipping in nonmutated CFTR alleles. Hum. Mutat., 24: 120-129.
Wang, D. and W. Sadée (2006). Searching for polymorphisms that affect gene expression and mRNA processing: example ABCB1 (MDR1). American Association of Pharmaceutical Scientists (AAPS) Journal, 8: 515-520.
Yarden, J., D. Radojkovic, K. De Boeck, M. Jr Macek, D. Zemkova, V. Vavrova, R. Vlietinck, J. J. Cassiman and H. Cuppens (2004). Polymorphisms in the mannose binding lectin gene affect the cystic fibrosis pulmonary phenotype. J. Med. Genet., 41: 629-633.
Yarden, J., D. Radojkovic, K. De Boeck, M. Jr Macek, D. Zemkova, V. Vavrova, R. Vlietinck, J. J. Cassiman and H. Cuppens (2005). Association of tumour necrosis factor alpha variants with the CF pulmonary phenotype. Thorax, 60: 320-325.
