ABCMdb

PMID: 16191501

Chou LS, Lyon E, Wittwer CT
A comparison of high-resolution melting analysis with denaturing high-performance liquid chromatography for mutation scanning: cystic fibrosis transmembrane conductance regulator gene as a model.
Am J Clin Pathol. 2005 Sep;124(3):330-8., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC7 p.Gly542* However, when the absolute temperatures of HRMA were considered, G542X but not F508del homozygotes could be distinguished from wild type. Login to comment 18 ABCC7 p.Ala455Glu ABCC7 p.Arg553* ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Tyr122* ABCC7 p.Arg560Thr ABCC7 p.Met1101Lys ABCC7 p.Phe508Cys Materials and Methods Sample Source and Study Design Eleven commercially genotyped samples were obtained from Coriell Cell Repositories, Coriell Institute for Medical Research, Camden, NJ (Y122X, R334W, R347P, A455E, I507del, F508del, F508C, G542X/G542X, R553X, R560T, and M1101K). Login to comment 22 ABCC7 p.Met1101Lys One homozygous Coriell sample (M1101K) was converted into a heterozygous template by amplifying a 292-base-pair (bp) fragment and mixing (1:1) with wild-type amplicon. Login to comment 31 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Ala455Glu ABCC7 p.Arg553* ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Ile148Thr ABCC7 p.Tyr122* ABCC7 p.Arg560Thr ABCC7 p.Met1101Lys ABCC7 p.Phe508Cys ❚Table 1❚ Mutations Analyzed in the Study Position From 5' Exon (or Intron) Genotype* No. of Samples Nucleotide Change SNP Class† End/Amplicon Size (bp) 3 394delTT 1 Del‡ - 132/234 4 R117H 1 G→A 1 83/270 Y122X 1 T→A 4 99/270 I148T 2 T→C 1 176/270 Intron 4 621+1 2 G→T 2 233/270 7 R334W 1 C→T 1 208/345 R347P 1 G→C 3 248/345 9 A455E 2 C→A 2 155/263 10 I507del 1 Del‡ - 171/292 F508del 3 Del‡ - 174/292 F508del/F508del 1 Del - 174/292 F508C 1 T→G 2 175/292 11 G542X 1 G→T 2 90/175 G542X/G542X 1 G→T 2 90/175 G551D 1 G→A 1 118/175 R553X 2 C→T 1 123/175 R560T 1 G→C 3 145/175 13 2184delA 1 Del‡ - 356/458 17b M1101K 1 T→A 4 196/292 21 N1303K 1 C→G 3 175/250 bp, base pairs; SNP, single nucleotide polymorphism. Login to comment 32 ABCC7 p.Gly542* * All genotypes were heterozygous except homozygous F508del and G542X. Login to comment 39 ABCC7 p.Tyr122* ABCC7 p.Met1101Lys Mutation Scanning by dHPLC Twenty-two of the samples (all except 394delTT, Y122X, 2184delA, and M1101K) also were amplified under conditions optimized for dHPLC with the same primers (Transgenomic, Omaha, NE). Login to comment 49 ABCC7 p.Gly542* Of 20 heterozygous samples, all were detected by melting and 19 by dHPLC for sensitivities of 100% and 95%, respectively. Homozygous mutations F508del and G542X could not be identified by melting analysis or dHPLC when only curve shapes were compared. Login to comment 50 ABCC7 p.Gly542* However, when melting curve position and shape were considered, G542X homozygotes could be identified by high-resolution melting. Login to comment 55 ABCC7 p.Arg117His ABCC7 p.Ile148Thr Mutations altering a wild-type G::C bp (R117H and 621+1) showed greater differences than those that altered a wild-type T::A bp (I148T). Login to comment 57 ABCC7 p.Ile148Thr The sequenced wild-type control sample showed a single peak, whereas the 621+1 and I148T traces had multiple peaks. Login to comment 58 ABCC7 p.Arg117His However, the R117H trace was similar to the control sample, resulting in the single false-negative result for dHPLC in our study (Figure 1D). Login to comment 59 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro Exon 7 was amplified as a longer PCR amplicon (345 bp) and included 2 single base heterozygotes, R334W and R347P. Login to comment 65 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro In contrast, different melting shapes were observed for R334W and R347P in the higher melting domain (Figure 2C), as expected from the location of the mutations. Login to comment 68 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro Under these conditions, R334W and R347P had elution peaks that were different from that for the wild-type and were identified correctly as heterozygotes ❚Figure 2D❚. Login to comment 72 ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Such double peaks usually are interpreted as Temperature (°C) Fluorescence 79 80 81 82 83 84 85 86 100 - 90 - 80 - 70 - 60 - 50 - 40 - 30 - 20 - 10 - 0 - R117H het C::A T::G WT G::C Temperature (°C) Fluorescence 79 80 81 82 83 84 85 86 100 - 90 - 80 - 70 - 60 - 50 - 40 - 30 - 20 - 10 - 0 - 621+1 het C::T A::G WT G::C Temperature (°C) Fluorescence 79 80 81 82 83 84 85 86 100 - 90 - 80 - 70 - 60 - 50 - 40 - 30 - 20 - 10 - 0 - I148T het C::A T::G WT T::A Time (min) Absorbance(mV) 0 1 2 3 20 - 19 - 18 - 17 - 16 - 15 - 14 - 13 - 12 - 11 - 10 - 9 - 8 - 7 - 6 - 5 - 4 - 3 - 2 - 1 - 0 - 621+1 het I148T het R117H het WT A B C D ❚Figure 1❚ High-resolution melting and denaturing high-performance liquid chromatography (dHPLC) analysis of exon 4 of the cystic fibrosis transmembrane conductance regulator gene. Login to comment 73 ABCC7 p.Arg117His ABCC7 p.Ile148Thr Melting data were normalized and temperature shifted as previously described.19 A, Heterozygous (het) R117H and the wild-type (WT) control sample. B, Heterozygous I148T and the WT control sample. C, Intronic heterozygous 621+1 G/T and the WT control sample. D,The dHPLC profile of heterozygous mutations in exon 4. Login to comment 75 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu ABCC7 p.Arg553* ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Arg560Thr ABCC7 p.Phe508Cys Additional mutations in exons 9, 10, 11, and 21 included 7 heterozygous SNPs (A455E, F508C, G542X, G551D, R553X, R560T, and N1303K) and 2 heterozygous 3-base deletions (I507del and F508del). Login to comment 78 ABCC7 p.Gly542* The dHPLC traces of homozygous G542X and F508del mutations were the same as wild-type control samples (Figure 3). Login to comment 80 ABCC7 p.Gly542* G542X but not F508del homozygotes could be discerned from their wild types if curve position (absolute temperature) instead of curve shape was used for comparison. Login to comment 81 ABCC7 p.Arg334Trp ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347Pro ABCC7 p.Gly542* Nearest-neighbor calculations predict that the ∆Tm between wild type and G542X homozygotes is Time (min) Absorbance(mV) 0 1 2 3 4 5 20 - 19 - 18 - 17 - 16 - 15 - 14 - 13 - 12 - 11 - 10 - 9 - 8 - 7 - 6 - 5 - 4 - 3 - 2 - 1 - 0 - R347P het R334W het WT Temperature (°C) Fluorescence 82 83 84 85 86 87 100 - 90 - 80 - 70 - 60 - 50 - 40 - 30 - 20 - 10 - 0 - R334W het C::A T::G R347P het C::C G::G WT G::C Temperature (°C) Fluorescence 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 100 - 90 - 80 - 70 - 60 - 50 - 40 - 30 - 20 - 10 - 0 - WT 0 50 100 150 200 250 300 350 100 90 80 70 60 50 40 30 20 10 Temperature(°C) Base Pairs Temperature (°C) Fluorescence 75 76 77 78 79 80 100 - 90 - 80 - 70 - 60 - 50 - 40 - 30 - 20 - 10 - 0 - A B C D ❚Figure 2❚ High-resolution melting and denaturing high-performance liquid chromatography (dHPLC) analysis of exon 7 of the cystic fibrosis transmembrane conductance regulator gene. Login to comment 84 ABCC7 p.Arg334Trp ABCC7 p.Arg334Trp ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347Pro ABCC7 p.Arg347Pro B, Low melting domain (75°C-80°C) comparison of heterozygous (het) R334W, R347P, and the WT control sample. C, High melting domain (82°C-87°C) comparison of heterozygous R334W, R347P, and the WT control sample. D,The dHPLC profile of the homozygous WT, heterozygous R334W, and heterozygous R347P genotypes. Login to comment 86 ABCC7 p.Gly542* Analysis of 5 different wild types and the single available G542X homozygote revealed a shifted curve position (Figure 4), suggesting that absolute temperature differences may be used to identify homozygous sequence alterations. Login to comment 90 ABCC7 p.Tyr122* ABCC7 p.Met1101Lys Y122X and M1101K are both class 4 SNPs20 (A/T heterozygotes), a class otherwise absent from our study. Login to comment 97 ABCC7 p.Arg117His In our hands, all heterozygous mutations were detected except R117H (exon 4), resulting in a heterozygote detection sensitivity of 95%. Login to comment 98 ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* In comparison, Ravnik-Glavac et al28 reported correct detection of heterozygous Time (min) Absorbance(mV) 0 1 2 3 20 - 19 - 18 - 17 - 16 - 15 - 14 - 13 - 12 - 11 - 10 - 9 - 8 - 7 - 6 - 5 - 4 - 3 - 2 - 1 - 0 - G542X het G542X hom WT Time (min) Absorbance(mV) 0 1 2 3 20 - 19 - 18 - 17 - 16 - 15 - 14 - 13 - 12 - 11 - 10 - 9 - 8 - 7 - 6 - 5 - 4 - 3 - 2 - 1 - 0 - F508del het F508del hom WT A B ❚Figure 3❚ Denaturing high-performance liquid chromatography analysis of mutations G542X (exon 11) and F508del (exon 10) of the cystic fibrosis transmembrane conductance regulator gene. Login to comment 99 ABCC7 p.Gly542* ABCC7 p.Gly542* A, Elution patterns of the wild-type (WT), heterozygous (het) G542X, and homozygous (hom) G542X samples. Login to comment 101 ABCC7 p.Gly542* ABCC7 p.Gly542* Temperature (°C) Fluorescence 77 78 79 80 81 82 83 84 85 86 100 - 90 - 80 - 70 - 60 - 50 - 40 - 30 - 20 - 10 - 0 - G542X het WT G542X hom ❚Figure 4❚ High-resolution melting analysis of exon 11. Login to comment 102 ABCC7 p.Gly542* ABCC7 p.Gly542* Melting curves shown are 5 wild-type (WT) samples, 1 G542X heterozygote (het), and 1 G542X homozygote (hom). Login to comment 104 ABCC7 p.Arg117His 336 DOI: 10.1309/BF3MLJN8J527MWQY (c) American Society for Clinical Pathology Chou et al / CFTR GENE SCANNING BY MELTING R117H with an overall heterozygote detection sensitivity of 90% when a single, predicted column temperature was used for each exon. Login to comment 113 ABCC7 p.Tyr122* ABCC7 p.Met1101Lys To discriminate differences at single base resolution, saturating DNA Temperature (°C) Fluorescence 81 82 83 84 85 86 87 88 100 - 90 - 80 - 70 - 60 - 50 - 40 - 30 - 20 - 10 - 0 - Y122X het A::A T::T WT T::A Temperature (°C) Fluorescence 79 80 81 82 83 84 85 100 - 90 - 80 - 70 - 60 - 50 - 40 - 30 - 20 - 10 - 0 - 2184delA het WT Temperature (°C) Fluorescence 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 100 - 90 - 80 - 70 - 60 - 50 - 40 - 30 - 20 - 10 - 0 - M1101K het A::A T::T WT T::A Temperature (°C) Fluorescence 77 78 79 80 81 82 83 84 100 - 90 - 80 - 70 - 60 - 50 - 40 - 30 - 20 - 10 - 0 - 394delTT het WT A B C D ❚Figure 5❚ High-resolution melting detection of heterozygous A/T single nucleotide polymorphisms and small (1-2 base pair) deletions. Login to comment 114 ABCC7 p.Met1101Lys Melting data were normalized and temperature shifted as previously described.19 A, Heterozygous (het)Y122X (exon 4) and the wild-type (WT) control sample. B, Heterozygous M1101K (exon 17b) and the WT control sample. C, Heterozygous 2184delA (exon 13) and the WT control sample. Login to comment 126 ABCC7 p.Arg334Trp ABCC7 p.Arg334Cys For example, in addition to the R334W mutation studied, 6 more mutations have been reported for this amino acid (R334C, E, H, K, L, Q).35 It would be interesting to compare the melting curves of all 7 R334 mutations to see how many can be distinguished from each other. Login to comment 131 ABCC7 p.Gly542* Homozygous SNP detection has been reported in PCR products up to 544 bp.19 In the present study, dHPLC missed the G542X homozygote, but melting analysis was able to identify the homozygous change using Tm differences. Login to comment