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Mutation scanning by meltMADGE: validations using BRCA1 and LDLR, and demonstration of the potential to identify severe, moderate, silent, rare, and paucimorphic mutations in the general population

Mutation scanning by meltMADGE: validations using BRCA1 and LDLR, and demonstration of the potential to identify severe, moderate, silent, rare, and paucimorphic mutations in the general population
Mutation scanning by meltMADGE: validations using BRCA1 and LDLR, and demonstration of the potential to identify severe, moderate, silent, rare, and paucimorphic mutations in the general population
We have developed a mutation-scanning approach suitable for whole population screening for unknown mutations. The method, meltMADGE, combines thermal ramp electrophoresis with MADGE to achieve suitable cost efficiency and throughput. The sensitivity was tested in blind trials using 54 amplicons representing the BRCA1 coding region and a panel of 94 unrelated family breast cancer risk consultands previously screened in a clinical diagnostic laboratory. All 10 common polymorphisms, 15/15 previously identified disease-causing mutations, and three previously untested single base changes were identified. Assays of LDLR exons 3 and 8 were validated in 460 familial hypercholesteremics and detected 8/9 known variants. We then applied the exon 3 assay in several DNA banks representing 8000 subjects with known cholesterol values and applied both assays in one DNA bank (n = 3600). In exon 3 we identified one previously reported moderate mutation, P84S (n = 1), also associated with moderate hypercholesteremia in this subject; an unreported silent variant, N76N (n = 1); and known severe hypercholesteremia splice mutation 313+1GA (n = 2). Around exon 8 we identified a paucimorphism (n = 35) at the splice site 1061–8TC (known to be in complete linkage disequilibrium with T705I) and unreported sequence variants 1186+11GA (n = 1) and D335N GA (n = 1). The cholesterol value for D335N was on the 96.2 percentile and for T705I, 2/35 carriers were above the 99th percentile. Thus, variants with predicted severe, moderate, and no effect were identified at the population level. In contrast with case collections, CpG mutations predominated. MeltMADGE will enable definition of the full population spectrum of rare, paucimorphic, severe, moderate (forme fruste), and silent mutations and effects.
1088-9051
967-977
Alharbi, Khalid K.
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Aldahmesh, Mohammed A.
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Spanakis, Emmanuel
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Haddad, Lema
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Whittall, Roslyn A.
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Chen, Xiao-he
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Rassoulian, Hamid
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Smith, Matt J.
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Sillibourne, Julie
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Ball, Nicola J.
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Graham, Nikki J.
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Briggs, Patricia J.
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Simpson, Iain A.
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Phillips, David I.W.
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Lawlor, Deborah A.
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Ye, Shu
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Humphries, Stephen E.
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Cooper, Cyrus
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Smith, George Davey
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Ebrahim, Shah
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Eccles, Diana M.
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Day, Ian N.M.
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Alharbi, Khalid K.
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Aldahmesh, Mohammed A.
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Spanakis, Emmanuel
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Haddad, Lema
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Whittall, Roslyn A.
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Chen, Xiao-he
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Rassoulian, Hamid
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Smith, Matt J.
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Sillibourne, Julie
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Ball, Nicola J.
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Graham, Nikki J.
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Briggs, Patricia J.
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Simpson, Iain A.
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Phillips, David I.W.
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Lawlor, Deborah A.
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Ye, Shu
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Humphries, Stephen E.
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Cooper, Cyrus
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Smith, George Davey
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Ebrahim, Shah
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Eccles, Diana M.
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Day, Ian N.M.
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Alharbi, Khalid K., Aldahmesh, Mohammed A., Spanakis, Emmanuel, Haddad, Lema, Whittall, Roslyn A., Chen, Xiao-he, Rassoulian, Hamid, Smith, Matt J., Sillibourne, Julie, Ball, Nicola J., Graham, Nikki J., Briggs, Patricia J., Simpson, Iain A., Phillips, David I.W., Lawlor, Deborah A., Ye, Shu, Humphries, Stephen E., Cooper, Cyrus, Smith, George Davey, Ebrahim, Shah, Eccles, Diana M. and Day, Ian N.M. (2005) Mutation scanning by meltMADGE: validations using BRCA1 and LDLR, and demonstration of the potential to identify severe, moderate, silent, rare, and paucimorphic mutations in the general population. Genome Research, 15 (7), 967-977. (doi:10.1101/gr.3313405).

Record type: Article

Abstract

We have developed a mutation-scanning approach suitable for whole population screening for unknown mutations. The method, meltMADGE, combines thermal ramp electrophoresis with MADGE to achieve suitable cost efficiency and throughput. The sensitivity was tested in blind trials using 54 amplicons representing the BRCA1 coding region and a panel of 94 unrelated family breast cancer risk consultands previously screened in a clinical diagnostic laboratory. All 10 common polymorphisms, 15/15 previously identified disease-causing mutations, and three previously untested single base changes were identified. Assays of LDLR exons 3 and 8 were validated in 460 familial hypercholesteremics and detected 8/9 known variants. We then applied the exon 3 assay in several DNA banks representing 8000 subjects with known cholesterol values and applied both assays in one DNA bank (n = 3600). In exon 3 we identified one previously reported moderate mutation, P84S (n = 1), also associated with moderate hypercholesteremia in this subject; an unreported silent variant, N76N (n = 1); and known severe hypercholesteremia splice mutation 313+1GA (n = 2). Around exon 8 we identified a paucimorphism (n = 35) at the splice site 1061–8TC (known to be in complete linkage disequilibrium with T705I) and unreported sequence variants 1186+11GA (n = 1) and D335N GA (n = 1). The cholesterol value for D335N was on the 96.2 percentile and for T705I, 2/35 carriers were above the 99th percentile. Thus, variants with predicted severe, moderate, and no effect were identified at the population level. In contrast with case collections, CpG mutations predominated. MeltMADGE will enable definition of the full population spectrum of rare, paucimorphic, severe, moderate (forme fruste), and silent mutations and effects.

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Published date: 2005

Identifiers

Local EPrints ID: 25195
URI: http://eprints.soton.ac.uk/id/eprint/25195
ISSN: 1088-9051
PURE UUID: 35d0769c-119d-4f26-888d-23e966b1e752
ORCID for Cyrus Cooper: ORCID iD orcid.org/0000-0003-3510-0709

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Date deposited: 07 Apr 2006
Last modified: 03 Dec 2019 01:58

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Contributors

Author: Khalid K. Alharbi
Author: Mohammed A. Aldahmesh
Author: Emmanuel Spanakis
Author: Lema Haddad
Author: Roslyn A. Whittall
Author: Xiao-he Chen
Author: Hamid Rassoulian
Author: Matt J. Smith
Author: Julie Sillibourne
Author: Nicola J. Ball
Author: Nikki J. Graham
Author: Patricia J. Briggs
Author: Iain A. Simpson
Author: David I.W. Phillips
Author: Deborah A. Lawlor
Author: Shu Ye
Author: Stephen E. Humphries
Author: Cyrus Cooper ORCID iD
Author: George Davey Smith
Author: Shah Ebrahim
Author: Diana M. Eccles
Author: Ian N.M. Day

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