ABCMdb

PMID: 15681482

Chou LS, Gedge F, Lyon E
Complete gene scanning by temperature gradient capillary electrophoresis using the cystic fibrosis transmembrane conductance regulator gene as a model.
J Mol Diagn. 2005 Feb;7(1):111-20., [PubMed]

Sentences

No. Mutations Sentence Comment

75 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Ala455Glu ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Arg1162* ABCC7 p.Ile148Thr ABCC7 p.Val520Phe ABCC7 p.Gly85Glu ABCC7 p.Arg560Thr ABCC7 p.Phe508Cys ABCC7 p.Ile506Val Mutation Samples with Known Genotypes Scanned by TGCE* Exon Mutation† Amplicon size (bp) Location of mutation from 5Ј end (bp) Base change Detection‡ 3 G85E 234 124 G to A 1/1 3 394delTT 234 132 del TT 1/1 4 R117H 270 83 G to T 2/2 4 I148T 270 176 T to C 3/3 Intron 4 621 ϩ 1 G/T 270 233 G to T 1/1 5 663delT/663delT 186 75 del T 0/1 Intron 5 711 ϩ 1 G/T 186 124 G to T 1/1 7 R334W 345 208 C to T 1/1 7 R347P 345 248 G to C 1/1 9 A455E 263 155 C to A 2/2 10 I506V 292 168 A to G 1/1 10 ⌬I507 292 171 del ATC 2/2 10 ⌬F508 292 174 del TTT 2/2 10 ⌬F508/⌬F508 292 174 del TTT 0/1 10 F508C 292 175 T to G 1/1 10 V520F 292 210 G to T 1/1 Intron 10 1717-1 G/A 175 50 G to A 1/1 11 G542X 175 90 G to T 2/2 11 G542X/G542X 175 90 G to T 0/1 11 G551D 175 118 G to A 3/3 11 R553X 175 123 C to T 3/3 11 R560T 175 145 G to C 2/2 13 2184delA 834 356 del A 1/1 Intron 14b 2789 ϩ 5G/A 192 102 G to A 1/1 Intron 16 3120 ϩ 1G/A 216 111 G to A 1/1 19 R1162X 322 68 C to T 1/1 19 3659delC 322 111 del C 1/1 20 W1282X 206 154 G to A 1/1 21 N1303K 250 175 C to G 2/2 Total exon/intron Overall accuracy 17 93% *Samples were compared with their respective wild-type control (confirmed by sequencing). Login to comment 76 ABCC7 p.Gly542* ABCC7 p.Gly542* † All genotypes were heterozygous except homozygous sample 663delT/663delT, ⌬F508/ ⌬F508, and G542X/G542X. Login to comment 92 ABCC7 p.Gly551Asp ABCC7 p.Arg117His The best temperature range for both heterozygous R117H (exon 4, 270 bp) (Figure 1; A to C) and the hard-to-discriminate mutation G551D heterozygous (exon 11, 175 bp) (Figure 1; D to F) is 50 to 55°C. Login to comment 99 ABCC7 p.Gly551Asp In this figure, the hard-to-discriminate G551D heterozygous shows better resolution in both 1 to 2 and 1 to 4 dilutions using 1ϫ PCR buffer (Figure 3, A and B). Login to comment 113 ABCC7 p.Arg117His ABCC7 p.Ile148Thr In this 270-bp fragment, heterozygous R117H (G/T) is located 83 bp from the 5Ј end, heterozygous I148T (T/C) is located in the middle of the fragment, and heterozygous 621 ϩ 1 (G/T) is located at the end of the fragment (37 bp from the 3Ј end) (Figure 4, A to D; Table 1). Login to comment 114 ABCC7 p.Arg117His ABCC7 p.Ile148Thr Heterozygous 621 ϩ 1 (G/T) had the least distinct split-peak pattern when compared to heterozygous R117H and I148T (Figure 4; B to D). Login to comment 118 ABCC7 p.Ala455Glu Another example of reduced resolution is heterozygous A455E in exon 9. Login to comment 119 ABCC7 p.Ala455Glu A shoulder instead of a small peak was observed in heterozygous A455E when compared to the wild-type control (Figure 5, A and B). Login to comment 124 ABCC7 p.Arg117His ABCC7 p.Arg117His A: R117H heterozygous (exon 4, 270 bp) without dilution and injected at 5 kV for 30 seconds; B: R117H heterozygous (exon 4) without dilution and injected at 3 kV for 20 seconds. Login to comment 127 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Arg117His A: Wild-type control (exon 4, 270 bp); B: R117H heterozygous (exon 4) with 50 to 55°C gradient; C: R117H heterozygous (exon 4) with 55 to 60°C gradient; D: wild-type control (exon 11, 175 bp); E: G551D heterozygous (exon 11) with 50 to 55°C gradient; F: G551D heterozygous (exon 11) with 55 to 60°C gradient. Login to comment 135 ABCC7 p.Gly542* In our study, homozygous mutants 663 delT (exon 5), ⌬F508 (exon 10), and G542X (exon 11) were not initially detected. Login to comment 142 ABCC7 p.Gly551Asp Effect of dilution with PCR buffers (1ϫ buffer containing 1.5 mmol/L Mgϩ2 ; 10ϫ buffer containing 15 mmol/L Mgϩ2 ) on peak resolution using amplicon G551D heterozygous (exon 11) as an example. Login to comment 146 ABCC7 p.Arg117His ABCC7 p.Ile148Thr A: Wild-type; B: R117H heterozygous (exon 4); C: I148T heterozygous (exon 4); D: 621 ϩ 1 G/T heterozygous sample 1 (intron 4); E: 621 ϩ 1 G/T heterozygous sample 2 (intron 4, from Coriell Repository); F: 621 ϩ 1 G/T heterozygous sample 3 (intron 4, from Coriell Repository). Login to comment 149 ABCC7 p.Ala455Glu A: Wild-type (exon 9 of the CFTR gene); B: A455E heterozygous (exon 9 of the CFTR gene). Login to comment 156 ABCC7 p.Ala455Glu In addition, we have sequenced exon 9 for all 12 samples because the mutation A455E is difficult to be detected in some systems. Login to comment 179 ABCC7 p.Gly551Asp ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Discussion More than 1000 mutations have been reported since the CFTR gene was cloned and characterized in 1989.23,25 Of these mutations, ⌬F508 (a 3-base deletion) is the most frequent mutation and results in a defective cAMP-regulated chloride transport in epithelial cells.26 Other mutations in the CFTR gene such as G542X, G551D, and N1303K occur in greater than 1% in the CF population and are associated with severe pancreatic insufficiency.23,27 Recently, carriers of the I148T mutation have received more attention because I148T has been found in association with the 3199del6 mutation, which may be necessary for the classic CF phenotype.28 Because of the complexity of both the mutations and the phenotypes, a high-throughput mutation scanning method to screen the entire coding region of the CFTR gene may provide valuable clinical information regarding CF genotypes and respective phenotypes. Login to comment 190 ABCC7 p.Pro67Leu ABCC7 p.Gly1287Arg Entire 27 CF Exons of 12 Deidentified DNA Samples Scanned by TGCE Method Exon Amplicon size (bp) Alterations* Confirmation by sequencing Consequence† 1 335 1 125 G/C Sequence variation (5Ј flanking) 2 210 0 3 234 1 332 C/T Change Pro to Leu at 67 4 270 0 5 186 0 6a 248 1 875 ϩ 40 A/G Sequence variation in intron 6a 6b 239 4 IVS6a (GATT)n‡ Sequence variation in intron 6a 7 345 0 8 233 0 9 263 0 10 292 0 11 175 0 12 250 0 13 834 0 14a 248 5 2694 T/G No change (Thr at 854) 14b 192 1 2789 ϩ 2 ins A Suspected deleterious 15 322 1 3030 G/A No change (Thr at 966) 16 216 0 17a 243 0 17b 292 0 18 217 0 19 322 0 20 206 1 3991 G/A Unknown mutation, change Gly to Arg at 1287 21 250 1 4029 A/G No change (Thr at 1299) 22 249 0 23 193 0 24 250 4 4521 G/A No change (Gln at 1463) Total Amplicon analyzed Potential SNP (%)§ 27 324 5% *Number of samples containing potential alterations (n ϭ 12). Login to comment