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

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+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.

Systematic analysis of variants related to familial hypercholesterolemia in families with premature myocardial infarction

“…This variant is reported to cause FH and is labelled as likely disease-causing in HGMD but the variant may be only pathogenic in combination with another variant in the LDLR gene. 50,51 In our data, we checked for co-segregation with LDLC and observed a positive co-segregation only in one family (family 7421) that also carries an additional co-segregating LDLR variant (c.811G4A (p.(V271I))). The other five families show poor or no evidence that the variant causes FH.…”

Prevalence and functionality of paucimorphic and privateMC4Rmutations in a large, unselected European British population, scanned by meltMADGE

“…A detailed description of the meltMADGE methodology is available [Alharbi et al, 2005;Smith et al, 2006]. MADGE formers and glass plates of the same dimensions were used essentially as previously described [Day and Humphries, 1994;Gaunt et al, 2003], except that meltMADGE gels as used in this work contained 6 M urea.…”

“…Approximately 2 mL of PCR product was loaded from microplates by passive transfer using a 96-slot pin replicator onto the meltMADGE gel. Prior to loading into the gel tank each gel was sealed as previously described [Alharbi et al, 2005;Smith et al, 2006]. Electrophoresis was in 1 Â tris-acetate (TAE) buffer of the same ionic strength and with equivalent ammonium persulfate (APS) concentration as found in the gels to prevent spatial ionic heterogeneity.…”

“…This methodology makes population scanning achievable. In previous tests (including blinded tests), 36 out of 37 different sequence variations in a wide variety of different sequence contexts, including mostly single-base changes but also a few small indels, were recognized [Alharbi et al, 2005]. Here we describe the development of a set of nine meltMADGE mutation scanning assays for the MC4R coding region and promoter and their application in a survey of a cohort of 1,100 anthropometrically characterized subjects.…”

dbSNP in the detail and copy number complexities

“…There may thus be many sequence variants in dbSNP which might, on the basis of the name of the database, be assumed to be SNPs, but which may not validate, either because they represent rare alleles or sequencing artifacts. The 1,000 Genomes Project of complete human genome sequencing will identify many ''private'' sequence variants as well as giving frequency estimates for new polymorphisms and ''paucimorphisms'' [Alharbi et al, 2005]. They defined paucimorphism (pauci meaning few) empirically to cover the minor allele frequency range of 0.05 to 5%, generally falling below the common SNPs represented in HapMap and SNP chips, but such that more than one might be seen in a typical population cohort study.…”