A haplotype-based molecular analysis of CFTR mutations associated with respiratory and pancreatic diseases

Abstract

Aberrant membrane transport caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is associated with a wide spectrum of respiratory and digestive diseases as well as cystic fibrosis. Using a gene scanning method, we found 11 polymorphisms and mutations of the CFTR gene in the Korean population. Individual variants at these sites were analyzed by conventional DNA screening in 117 control and 75 patients having bronchiectasis or chronic pancreatitis. In a haplotype determination based on a Bayesian algorithm, 15 haplotypes were assembled in the 192 individuals tested. Several haplotypes, especially with Q1352H, IVS8 T5, and E217G, were found to have disease associations in a case-control study. Notably, a common polymorphism of M470V appears to affect the intensity of the disease association. Among the two haplotypes having IVS8 T5, the T5-V470 haplotype showed higher disease association than the T5-M470 haplotype. In addition, a Q1352H mutation found in a V470 background showed the strongest disease association. The physiological significances of the identified mutations were rigorously analyzed. Non-synonymous E217G and Q1352H mutations in the M470 background caused a 60-80% reduction in CFTR-dependent Cl(-) currents and HCO3(-) -transport activities. Surprisingly, the additional M470V polymorphic variant with the Q1352H mutation completely abolished CFTR-dependent anion transport activities. These findings provide the first evidence on the importance of CFTR mutations in the Asian population. Importantly, the results also reveal that interactions between multiple genetic variants in cis affect the final function of the gene products.

Long-Range (17.7 kb) Allele-Specific Polymerase Chain Reaction Method for Direct Haplotyping of R117H and IVS-8 Mutations of the Cystic Fibrosis Transmembrane Regulator Gene

“…In contrast, detection of two or more mutations on the same gene and determination of their phase (or haplotype) is more challenging. Advances in the field of human genome mapping, 1 the search for complex disease determinants, 2 pharmacogenomics, 3 and accumulation of data from mutation screening programs 4 underline the need to develop additional molecular haplotyping methods. Current technologies require physical separation of chromosomes or cloning or are limited to analysis of single nucleotide polymorphism (SNPs) present in the range of 1 kilobase (kb).…”

Targeted mutation screening panels expose systematic population bias in detection of cystic fibrosis risk

“…Others have drawn attention to the low clinical utility of current targeted mutation panels outside of European populations, but, to our knowledge, this report is the first systematic, quantitative demonstration of population bias with a large, genetically diverse data set. 5,[27][28][29] Our classification system and method of discovery have general utility for other recessive disease genes. The presence of an analytical bias in identifying CFTR mutations on carrier screening panels calls into question whether this bias is present among all disease-associated genes tested by current platforms, especially those not as well annotated as CFTR.…”

“…Based on the static European definition of classic CF, the disease was thought to be exceedingly rare in South and East Asia. 27,29 Many mutations that cause classic CF were identified in Europeans after the discovery of the CFTR gene in 1989 and the ensuing analysis of patient DNA sequences. At the same time, additional protein-changing CFTR variants were discovered in association with diseases that had been thought to have etiologies distinct from CF.…”

“…1 Different populations have particular CFTR allele spectra that are associated with distinct manifestations of disease. 4,27,29 A system for scoring CFTR disease liability needs to focus on the degree to which a defined mutation increases morbidity and mortality in certain ancestral backgrounds, irrespective of a narrow presentation of disease symptoms. Ours is not the first application of computational analysis to novel variants in the CFTR gene.…”

Genetically Characterized Positive Control Cell Lines Derived from Residual Clinical Blood Samples

“…Two cell lines (GM07441 and GM13591), obtained from the NIGMS collection, are compound heterozygotes for mutations included in the screening panel and are therefore particularly valuable as positive controls. GM13591 was also shown by 2 testing laboratories to be heterozygous for the variant M470V, which may be associated with disease in individuals with particular haplotype backgrounds (19,20 ). Nine cell lines derived from residual clinical samples carry 4 mutations included the 2001 ACMG panel that were not available from public sources at the time this study was initiated: 1898 ϩ 1GϾA is carried by 4 cell lines, I148T by 3, and 2184delA and 1078delT by 1 each.…”