ABCMdb

PMID: 19372188

Bickmann JK, Kamin W, Wiebel M, Hauser F, Wenzel JJ, Neukirch C, Stuhrmann M, Lackner KJ, Rossmann H
A novel approach to CFTR mutation testing by pyrosequencing-based assay panels adapted to ethnicities.
Clin Chem. 2009 Jun;55(6):1083-91. Epub 2009 Apr 16., [PubMed]

Sentences

No. Mutations Sentence Comment

42 ABCC7 p.Asn1303Lys Except for the N1303K assay (exon 21), the nested PCR assay for the analysis of 1342-6 (T)n (5T/ 7T/9T) [intervening sequence 8 (IVS 8)], and the 1342-12 (TG)n-5T (IVS 8) reflex test, all PCRs proceeded under standard amplification conditions. Login to comment 62 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg553* ABCC7 p.Arg347Pro ABCC7 p.Asn1303Lys ABCC7 p.Gly542* We had initially focused on CF mutations potentially prevalent in our local, ethnically mixed, but mainly German population: F508del, I507del, 1677delTA, R347P, G542X, G551D, R553X, N1303K, 1717-1GϾA, 3849ϩ10kb CϾT, CFTRdele2,3 (21 kb), R117H, 1342-6 (T)n (5T/7T/9T) (reported by our laboratory only if a R117H allele was present, unless genetic analysis served to investigate a case of CBAVD or atypical mild CF), and the (TG)n region starting at base position 1342-12 of IVS 8 (exclusively tested in the case of a 5T allele) (Fig. 1, boldface text), with an expected sensitivity of 85% among German patients. Login to comment 86 ABCC7 p.Ile148Thr The resulting PSQ assays targeted 44 CF/CBAVD mutations (potential detection of adjacent mutations not included) and 2 variations [I148T and 1342-6 (T)n (5T/7T/9T)]. Login to comment 88 ABCC7 p.Arg117His ABCC7 p.Arg117His A PSQ-adapted long-range PCR [modification of method of Pont-Kingdon et al. (25)] for haplotyping of 5T and R117H will complement the panel if an R117H-5T (CF haplotype) control becomes available. Login to comment 100 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Arg347Pro ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Ser549Asn ABCC7 p.Gln552* Diagnostic evaluation of the PSQ-based first-level testing of a predominantly German CF population.a Panethnic population Clinical diagnosis All patients Sweat test-confirmed CF Suspected atypical CF Carrier screening Chromosomes, n 310 184 96 30 PSQ screen 168 (54.2%) 158 (85.9%) 5 (5.2%) 5 (33.3%) Conventional sequencing 25 (8.1%) 25 (13.6%) 0 (0%) 0 (0%) Total detected alleles 193 (62.3%) 183 (99.5%) 5 (5.2%) 5 (33.3%) German ethnicity Other ethnicities Clinical diagnosis Sweat test-confirmed CF Sweat test-confirmed CF Chromosomes, n 146 38 PSQ screen F508del 106 (72.6%) 14 (36.8%) I507del 1 (0.7%) 1 (2.6%) 1677delTA 0 (0%) 2 (5.3%) G551D 6 (4.1%) 0 (0%) R553X 2 (1.4%) 0 (0%) Q552X 1 (0.7%) 0 (0%) G542X 2 (1.4%) 1 (2.6%) S549N 0 (0%) 2 (5.3%) W1282X 1 (0.7%) 3 (7.9%) R117H 1 (0.7%) 0 (0%) 1342-12 (TG)11-5T 0 (0%) 0 (0%) R347P 2 (1.4%) 1 (2.6%) 3849ϩ10kb CϾT 2 (1.4%) 0 (0%) N1303K 3 (2.1%) 3 (7.9%) 1717-1 GϾA 1 (0.7%) 0 (0%) CFTRdele2,3 (21 kb) 2 (1.4%) 1 (2.6%) Sum 130 (89.0%) 28 (73.7%) Conventional sequencing 16 (11.0%) 9 (23.7%) Total detected alleles 146 (100%) 37 (97.4%) a Data are presented as the number of chromosomes (percent). Login to comment 108 ABCC7 p.Gly551Asp ABCC7 p.Arg553* A second patient was genotyped as F508del/ I507delinourlaboratorybuthadpreviouslybeencharac- terized as homozygous for F508del, and a third patient was F508del/G551D in our laboratory and F508del/ R553X in the other laboratory. Login to comment 110 ABCC7 p.Gly551Asp ABCC7 p.Arg553* Furthermore, the analysis of control samples clearly demonstrated that PSQ reliably distinguished G551D and R553X (Fig. 1 in the online DataSupplement),aswellasF508delandI507del(Fig.3). Login to comment 111 ABCC7 p.Arg553* BecauseformermethodsoftenusedtomistakeG551Dfor R553X, and I507del for the more common mutation F508del, and because both samples had been genotyped several years before, an error during precharacterization seemed the most probable explanation for the discrepancy. Login to comment 113 ABCC7 p.Ile506Val The clinically irrelevant variants I506V, I507V,andF508C,whicharelocatedincloseproximityto these mutations, change the expected pyrograms dramatically (involving all peaks 3Ј of the base exchange), thereby virtually eliminating any chance of misdiagnosis. Login to comment 118 ABCC7 p.Gly542* ABCC7 p.Ser549Asn An example of the integration of a mutation that was not primarily targeted (S549N) into a PSQ assay for G542X. Login to comment 119 ABCC7 p.Gly542* ABCC7 p.Gly544Ser ABCC7 p.Gly544Val The G542X (1756 GϾT) assay had originally been designed to include adjacent but rare mutations G544S (1762 GϾA) and G544V (1763 GϾT), with the sequence to analyze reaching as far as base 1783. Login to comment 121 ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn Conventional sequencing of exon 11 confirmed the homozygous state of S549N, and MLPA analysis excluded the combination of S549N with a gross deletion. Login to comment 122 ABCC7 p.Ser549Asn By changing the order of base injections, we obtained pyrograms optimized to detect S549N, facilitating automated genotype calling of this mutation in the future (extended assay, right panel). Login to comment 123 ABCC7 p.Gly542* ABCC7 p.Ser549Asn See Fig. 1 in the online Data Supplement for example pyrograms of a wild-type sample and heterozygous samples for G542X and S549N. Login to comment 129 ABCC7 p.Gly551Asp ABCC7 p.Arg553* ABCC7 p.Phe508Cys ABCC7 p.Ile506Val ABCC7 p.Ile507Val Furthermore, the PSQ-based first-level test avoids common pitfalls, as do the most recent assays: It correctly discriminates G551D and R553X, as well as I507del and F508del (Fig. 3; see Fig. 1 in the online Data Supplement), thus obviating reflex testing for benign sequence variations such as I506V, I507V, and F508C. Login to comment 130 ABCC7 p.Arg117His With respect to haplotyping of R117H-5T, we suggest an assay based on a long-range PCR described by Pont-Kingdon et al. (25) that we have adapted to PSQ but have not yet been able to validate because of a lack of positive-control samples. Login to comment 148 ABCC7 p.Phe508Cys ABCC7 p.Ile506Val ABCC7 p.Ile507Val Fig. 2 in the online Data Supplement illustrates the capability of the assay to discriminate I507del, F508del, 1677delTA, and the interfering benign variants I506V, I507V, and F508C. Login to comment 153 ABCC7 p.Gly551Asp ABCC7 p.Arg553* ABCC7 p.Gly542* ABCC7 p.Ser549Asn ABCC7 p.Gln552* The fact that simultaneously detecting some mutations (e.g.: F508del, 1677delTA, and I507del; G542X and S549N; or G551D, R553X, and Q552X) within a single assay improves the sensitivity of each PSQ run underlines even further the advantages that arise from detecting neighboring mutations as well as the target mutation within one assay (Table 2). 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