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
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DOI：
10.1101/gr.3313405
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发表时间：
2005-07-01
期刊：
影响因子：
7
通讯作者：
Day, INM
中科院分区：
文献类型：
--
作者：
Alharbi, KK;Aldahmesh, MA;Day, INM
我们开发了一种适用于全人群筛选未知突变的突变扫描方法。这种名为MeltMADGE的方法结合了热斜坡电泳法和MADGE法，以获得适当的成本效益和产量。这种敏感性是在盲法试验中测试的，使用了54个代表BRCA1编码区的扩增片段和一个由94名先前在临床诊断实验室筛选的无关家族乳腺癌风险顾问组成的小组。所有10个常见的多态、15/15个以前发现的致病突变和3个以前未检测到的单碱基变化都得到了鉴定。LDLR外显子3和8的检测在460名家族性高胆固醇患者中得到验证，并检测到8/9已知的变异。然后，我们在几个DNA库中应用了外显子3的分析，这些DNA库代表了大约8000名胆固醇水平已知的受试者，并在一个DNA库中同时应用了两种分析方法(n=3600)。在外显子3中，我们发现了一个先前报道的中度突变，P84S(n=1)，也与中度高胆固醇血症相关；一个未报道的沉默变异，N76N(n=1)；以及已知的严重高胆固醇血症剪接突变313+1G->；A(n=2)。在外显子8附近，我们在剪接点1061-8T->；C处发现了35个稀缺多态(n=35)(已知与T7051处于完全连锁不平衡状态)，以及未报道的序列变体1186+11G->；A(n=1)和D335NG->；A(n=1)。D335N携带者胆固醇值在96.2百分位数，T7051携带者2/35携带者在第99百分位数以上。因此，在人群水平上确定了预测为严重、中度和无影响的变异。与病例集相比，CpG突变占主导地位。MeltMADGE将能够定义罕见、稀有、严重、中度(形成突变)和沉默突变和影响的全部种群谱。
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 similar to 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 T7051) 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 T7051, 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.