ABCMdb

PMID: 19959634

Aguirre-Lamban J, Riveiro-Alvarez R, Garcia-Hoyos M, Cantalapiedra D, Avila-Fernandez A, Villaverde-Montero C, Trujillo-Tiebas MJ, Ramos C, Ayuso C
Comparison of high-resolution melting analysis with denaturing high-performance liquid chromatography for mutation scanning in the ABCA4 gene.
Invest Ophthalmol Vis Sci. 2010 May;51(5):2615-9. Epub 2009 Dec 3., [PubMed]

Sentences

No. Mutations Sentence Comment

12 ABCA4 p.Arg187His ABCA4 p.Cys2137Tyr ABCA4 p.Trp1724Cys One homozygous mutation was not detected by dHPLC; however, the p.Cys2137Tyr homozygote was distinguished from the wild-type by HRM technique. In the same way, one novel change in exon 5 (p.Arg187His) was found only by means of the HRM technique. In addition, dHPLC identified the mutation p.Trp1724Cys in one sample; however, HRM detected the mutation in two samples. Login to comment 61 ABCA4 p.Arg187His ABCA4 p.Arg187His ABCA4 p.Tyr954Ser ABCA4 p.Tyr954Ser Except p.Arg187His and p.Tyr954Ser variants, the rest of changes were previously reported as disease-associated allele.14 The p.Arg187His and p.Tyr954Ser variants were not found in 100 ethnically matched control chromosomes. Login to comment 63 ABCA4 p.Cys2137Tyr Homozygous sequence alteration (p.Cys2137Tyr) could be identified from wild-type by HRM analyses (Fig. 1). Login to comment 64 ABCA4 p.Arg187His ABCA4 p.Arg187His ABCA4 p.Tyr954Ser ABCA4 p.Tyr954Ser Except p.Arg187His and p.Tyr954Ser variants, the rest of changes were previously reported as disease-associated allele.14 The p.Arg187His and p.Tyr954Ser variants were not found in 100 ethnically matched control chromosomes. Login to comment 65 ABCA4 p.Arg187His The exon 5 included one novel heterozygous single-nucleotide substitution (p.Arg187His) identified in two STGD patients. Login to comment 66 ABCA4 p.Cys2137Tyr Homozygous sequence alteration (p.Cys2137Tyr) could be identified from wild-type by HRM analyses (Fig. 1). Login to comment 68 ABCA4 p.Arg187His The exon 5 included one novel heterozygous single-nucleotide substitution (p.Arg187His) identified in two STGD patients. Login to comment 70 ABCA4 p.Arg187His The p.Arg187His trace was similar to the control sample, resulting in a false-negative result for dHPLC (Fig. 2B). Login to comment 71 ABCA4 p.Trp1724Cys Exon 36 included the p.Trp1724Cys mutation detected in two STGD patients. Login to comment 73 ABCA4 p.Arg187His The p.Arg187His trace was similar to the control sample, resulting in a false-negative result for dHPLC (Fig. 2B). Login to comment 74 ABCA4 p.Trp1724Cys Exon 36 included the p.Trp1724Cys mutation detected in two STGD patients. Login to comment 78 ABCA4 p.Tyr954Ser The p.Tyr954Ser variant had not been reported and was not found in 100 ethnically matched control chromosomes. Login to comment 81 ABCA4 p.Tyr954Ser The p.Tyr954Ser variant had not been reported and was not found in 100 ethnically matched control chromosomes. Login to comment 87 ABCA4 p.Arg187His ABCA4 p.Arg187His ABCA4 p.Arg187His ABCA4 p.Trp1724Cys ABCA4 p.Trp1724Cys ABCA4 p.Tyr954Ser ABCA4 p.Ala762Glu Mutations Analyzed Family Exon Genotype Mutation Detected by dHPLC Mutation Detected by HRM Nucleotide Change Amino Acid Change ARDM-167 5 c.560G>A p.Arg187His No Yes ARDM-257 5 c.560G>A p.Arg187His No Yes ARDM-164 6 c.700CϾT p.Gln234X Yes Yes ARDM-135 8 c.1029_1030insT p.Asn344fsX Yes No ARDM-240 15 c.2285CϾA p.Ala762Glu Yes Yes ARDM-248 19 c.2861A>C p.Tyr954Ser Yes Yes ARDM-90 - IVS21-2AϾT - Yes Yes ARDM-40 27 c.3943CϾT p.Gln1315X Yes Yes ARDM-158 30 c.4537delC p.Gln1513fsX1525 Yes Yes ARDM-38 33 c.4739delT p.Leu1580fs Yes Yes ARDM-163 36 c.5172GϾT p.Trp1724Cys Not Yes ARDM-197 36 c.5172GϾT p.Trp1724Cys Yes Yes ARDM-181 - IVS38ϩ5GϾA - Yes Yes ARDM-125 40 - p.KNLFA1876dup Yes Yes ARDM-183 43 c.5929GϾA(False-) p.Gly1977Ser(False-) Yes Yes ARDM-146 44 c.6140TϾA p.lle2047Asn Yes Yes ARDM-174 - IVS44ϩ2TϾA - Yes Yes ARDM-247 47 c.6410GϾA p.Cys2137Tyr* Yes Yes ARDM-84 47 c.6410GϾA p.Cys2137Tyr† No Yes ARDM-225 48 c.6559CϾT p.Gln2187X Yes Yes Previously unreported mutations are shown in bold. Login to comment 90 ABCA4 p.Arg187His ABCA4 p.Cys2137Tyr ABCA4 p.Cys2137Tyr ABCA4 p.Cys2137Tyr ABCA4 p.Cys2137Tyr Normalized and Temp-Shifted Difference Plot T empera ture (°C) 89.5 89 88.5 88 87.5 87 86.5 86 85.5 85 84.5 R elative S ignal Difference 16.539 15.039 13.539 12.039 10.539 9.039 7.539 6.039 4.539 3.039 1.539 0.039 -1.461 A B p.Cys2137Tyr het p.Cys2137Tyr hom WT p.Cys2137Tyr het p.Cys2137Tyr hom WT FIGURE 1. Login to comment 92 ABCA4 p.Cys2137Tyr ABCA4 p.Cys2137Tyr ABCA4 p.Trp1724Cys (A) Melting curves for two wild-type (WT), one p.Cys2137Tyr heterozygous (het), and one p.Cys2137Tyr homozygous (hom) sample. Login to comment 93 ABCA4 p.Cys2137Tyr ABCA4 p.Cys2137Tyr (B) dHPLC profile of the heterozygous (het) p.Cys2137Tyr, the homozygous (hom) p.Cys2137Tyr, and the WT control sample. Login to comment 95 ABCA4 p.Tyr954Ser The p.Tyr954Ser variant was not found in 100 ethnically matched control chromosomes, suggesting the presence of a disease-associated allele. Login to comment 97 ABCA4 p.Cys2137Tyr In contrast, dHPLC depends on a specific temperature that requires optimization.19 It generally is accepted that routine dHPLC scanning does not detect homozygous sequence alterations, but the situation for melting analyses is less clear.20 In this study, a homozygous mutation (p.Cys2137Tyr) was detected by HRM. Login to comment 101 ABCA4 p.Arg187His ABCA4 p.Arg187His ABCA4 p.Arg187His In a previous study, all the errors were made with PCR products 400 bp or larger, suggesting a dependence on product length.17 In exon 5, HRM analysis detected a previously unreported mutation (p.Arg187His) in two STGD patients. Login to comment 103 ABCA4 p.Arg187His (A) Melting data normalized and temperature shifted shown 2 p.Arg187His heterozygote (het), and 1 wild-type (WT) sample. Login to comment 104 ABCA4 p.Arg187His ABCA4 p.Arg187His Previous analysis by dHPLC of the exon 5 did not allow the detection of the p.Arg187His mutation. Login to comment 106 ABCA4 p.Trp1724Cys The p.Trp1724Cys mutation was identified in two STGD patients. Login to comment 109 ABCA4 p.Tyr954Ser The p.Tyr954Ser variant was not found in 100 ethnically matched control chromosomes, suggesting the presence of a disease-associated allele. Login to comment 111 ABCA4 p.Cys2137Tyr In contrast, dHPLC depends on a specific temperature that requires optimization.19 It generally is accepted that routine dHPLC scanning does not detect homozygous sequence alterations, but the situation for melting analyses is less clear.20 In this study, a homozygous mutation (p.Cys2137Tyr) was detected by HRM. Login to comment 115 ABCA4 p.Arg187His ABCA4 p.Arg187His Therefore, the HRM analysis had a Normalized and Shifted Melting Curves T empera ture (°C) 89 88 87 86 85 84 83 82 81 80 79 78 77 76 75 74 73 72 71 R elative S ignal (% ) 100.000 80.000 60.000 40.000 20.000 0.000 A B WT p.Arg187His WT p.Arg187His FIGURE 2. Login to comment 117 ABCA4 p.Arg187His (A) Melting data normalized and temperature shifted shown 2 p.Arg187His heterozygote (het), and 1 wild-type (WT) sample. Login to comment 118 ABCA4 p.Arg187His (B) The dHPLC profile of the heterozygous (het) p.Arg187His and the WT control sample. Login to comment