Sensitivity of the denaturing gradient gel electrophoresis technique in detection of known mutations and novel Asian mutations in the CFTR gene

Abstract

More than 500 mutations have been identified in the CFTR gene, making it an excellent system for testing mutation scanning techniques. To assess the sensitivity of denaturing gradient gel electrophoresis (DGGE), we collected a representative group of 202 CFTR mutations. All mutations analyzed were detected by scanning methods other than the DGGE approach evaluated in this study. DGGE analysis was performed on 24 of the 27 exons and their flanking splice site sequences. After optimization, 201 of the 202 control samples produced an altered migration pattern in the region in which an alteration occurred. The remaining sample was sequenced and found not to have the reported mutation. The ability of DGGE to identify novel mutations was evaluated in three Asian CF patients with four unknown CF alleles. Three novel Asian mutations were detected-K166E, L568X, and 3121-2 A-->G (in homozygosity)-accounting for all CF alleles. These results indicate that an optimized DGGE scanning strategy is highly sensitive and specific and can detect 100% of mutations.

Absence of mutations in the interspecies conserved regions of the CFTR promoter region in cystic fibrosis (CF) and CF related patients.

“…In this study, DNA of the CF or CBAVD patients had been previously thoroughly studied for mutations throughout the whole coding sequence of the CFTR gene. We have previously shown that DGGE scanning can detect up to 100% of the mutations in a PCR fragment.2' 22 The fact that we have been unable to find any abnormalities reinforces the idea that the promoter region in the CFTR is not frequently mutated. However, our approach could have missed some important sites involved in the regulation of the human CFTR gene because, firstly, the consensus regulatory sequences are usually relatively short and they may vary by one or two nucleotides between species and, secondly, species specific regulatory elements seem to exist.…”

Improved detection of cystic fibrosis mutations in the heterogeneous U.S. population using an expanded, pan-ethnic mutation panel

“…8 Our data show that a panel of CF mutations that increases detection rates in U.S. Hispanics, Caucasians, and African Americans does not provide a similar increase in Asians.…”

“…Since all individuals are seldom tested for the same mutations, overall sensitivity data are often extrapolated or assumed to be additive across studies. In small, recently published studies of U.S. ethnic groups, detection rates were 75% in African Americans, 6 58% in Hispanics, 7 33% in Asians, 8 and 4% in Native Americans. 9 In a large study of U.S. Caucasians in 1994, the detection rate was 78.6% (derived from ref.…”

Assessing high-resolution melt curve analysis for accurate detection of gene variants in complex DNA fragments

“…Therefore, we aimed to determine (if any) potential advantages and/or limitations of the HRM method of particular note for clinical laboratories planning to adopt this new technique. Several studies have reported on the high sensitivity, ease of data interpretation, and the low cost associated with using DGGE for detecting DNA sequence variation [Hayes, 2003;Macek et al, 1997;Petersen et al, 2002;van der Hout et al, 2006]. Depending on the enzyme used for amplification, the cost of reagents to perform DGGE screening is a half to three-quarter that required for HRM using these platforms and dyes.…”