Utilities for high throughput use of the single strand conformational polymorphism method: screening of 791 patients with familial hypercholesterolaemia for mutations in exon 3 of the low density lipoprotein receptor gene.

Whittall, Ros; Gudnason, Vilmundur; Weavind, G.P.; Day, Lorna; Humphries, Steve E.; Day, Ian N.M.

doi:10.1136/jmg.32.7.509

Cited by 36 publications

(7 citation statements)

References 26 publications

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“…To examine the sensitivity of melt-MADGE, 460 DNA samples from the Simon Broom Familial Hypercholesterolemia (SBFH) register (Betteridge et al 1999;Neil et al 2004), previously screened for mutations of LDLR using the SSCP technique (Whittall et al 1995), were used. MeltMADGE mutation scanning was undertaken by author K.K.A.…”

Section: Wwwgenomeorgmentioning

confidence: 99%

Mutation scanning by meltMADGE: Validations usingBRCA1andLDLR, and demonstration of the potential to identify severe, moderate, silent, rare, and paucimorphic mutations in the general population

Alharbi¹,

Aldahmesh²,

Spanakis³

et al. 2005

Genome Res.

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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+1G→A (n = 2). Around exon 8 we identified a paucimorphism (n = 35) at the splice site 1061-8T→C (known to be in complete linkage disequilibrium with T705I) and unreported sequence variants 1186+11G→A (n = 1) and D335N G→A (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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Section: Wwwgenomeorgmentioning

confidence: 99%

Mutation scanning by meltMADGE: Validations usingBRCA1andLDLR, and demonstration of the potential to identify severe, moderate, silent, rare, and paucimorphic mutations in the general population

Alharbi¹,

Aldahmesh²,

Spanakis³

et al. 2005

Genome Res.

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“…34,35 LDLR was screened by SSCP analysis. 36 Potentially FH-causing SSCP band shifts were subsequently sequenced using dRhodamine Bigdye fluorescent terminator sequencing, according to Perkin Elmer Applied Biosystems on an ABI DNA 377 Sequencer. LDLR major rearrangements were screened by analysing exons 3, 5, 8, 14, 17 by universal primer quantitative fluorescent multiplex PCR (UPQFM-PCR).…”

Section: Molecular Analysismentioning

confidence: 99%

A molecular genetic service for diagnosing individuals with familial hypercholesterolaemia (FH) in the United Kingdom

et al. 2001

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A genetic diagnostic service for familial hypercholesterolaemia (FH) has been established over the last 4 years in the Clinical Molecular Genetics Laboratory at Great Ormond Street Hospital for Children NHS Trust (GOSH), London. In total there have been 368 referrals; 227 probands and 141 family members, which have come from a number of lipid clinics and from general practitioners. FH is caused by mutations in the lowdensity lipoprotein receptor gene (LDLR) and these are analysed by SSCP, DNA sequencing and direct assays. The clinically indistinguishable disorder, familial defective apolipoprotein B100 (FDB) is caused by one of three mutations in the apolipoprotein B100 gene (APOB) which are analysed by direct assays. Mutations predicted to be pathogenic were found in 76 probands, 67 in LDLR (23 previously undescribed) and nine in APOB. The mutation detection rate was 53% in paediatric probands, 32% in adults with a`definite' FH diagnosis (tendon xanthoma positive) and 14% in adults with a`possible' FH diagnosis (tendon xanthoma negative). The predicted loss of sensitivity that would result from reducing the number of exons tested has been assessed, and a molecular screening strategy suitable for UK patients is proposed. A similar strategy may be useful for other countries where genetic heterogeneity results in a wide mutation spectrum for FH.

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“… Mutation analyses of the LDLR and APOB genes in a family with FH are as described [4,5]. Filled symbols represent subjects with hyperlipidaemia.…”

Section: Resultsmentioning

confidence: 99%

“…DNA tests for relative tracing are feasible and useful [2], especially in paediatric cases where there is an overlap between cholesterol levels in FH and normal subjects [3]. We have established a clinical genetic diagnostic service for FH [2] and have been using single strand conformation polymorphism (SSCP) for rapid mutation screening of the LDLR [4]. The method gives a detection rate in adult FH probands of ∼40% [5] and in child FH probands of ∼70% (Heath and Humphries, unpublished data).…”

mentioning

confidence: 99%

Rapid detection of polymorphisms in exons 10, 11 and 12 of the low density lipoprotein receptor gene (LDLR) and their use in a clinical genetic diagnostic setting

Heath¹,

Humphries²

1999

Clinical Genetics

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Utilities for high throughput use of the single strand conformational polymorphism method: screening of 791 patients with familial hypercholesterolaemia for mutations in exon 3 of the low density lipoprotein receptor gene.

Cited by 36 publications

References 26 publications

Mutation scanning by meltMADGE: Validations usingBRCA1andLDLR, and demonstration of the potential to identify severe, moderate, silent, rare, and paucimorphic mutations in the general population

Mutation scanning by meltMADGE: Validations usingBRCA1andLDLR, and demonstration of the potential to identify severe, moderate, silent, rare, and paucimorphic mutations in the general population

A molecular genetic service for diagnosing individuals with familial hypercholesterolaemia (FH) in the United Kingdom

Rapid detection of polymorphisms in exons 10, 11 and 12 of the low density lipoprotein receptor gene (LDLR) and their use in a clinical genetic diagnostic setting

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