Rohlin Anna, Wernersson Josephine, Engwall Yvonne, Wiklund Leif, Björk Jan, Nordling Margareta
Department of Clinical Genetics, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden.
Hum Mutat. 2009 Jun;30(6):1012-20. doi: 10.1002/humu.20980.
We have made an evaluation of mutation detection techniques for their abilities to detect mosaic mutations. In this study, Sanger sequencing, single-strand conformation polymorphism (SSCP)/heteroduplex analysis (HD), protein truncation test (PTT), and denaturating high-performance liquid chromatography (DHPLC) were compared with parallel sequencing. In total DNA samples from nine patients were included in this study. Mosaic mutations were artificially constructed from seven of these samples, which were from heterozygote mutation carriers with the mutant allele present at 50%. The mutations analyzed were as follows: c.646C>T, c.2626C>T, c.2828C>A, c.1817_1818insA, c.2788dupA, c.416_419delAAGA, and c.607delC in the APC gene. The lowest degree of mutant alleles detected with SSCP/HD and DHPLC varied between 5% and 25%, and between 15% and 50% for Sanger sequencing. Three of the mutations were analyzed with PTT with considerable variations in detection levels (from 10 to 100%). Using parallel sequencing a detection frequency down to 1% was reached, but to achieve this high sensitivity sufficient coverage was required. Two patients with natural mosaic mutations were also included in this study. These two mutations had previously been identified with Sanger sequencing (NF2 c.1026_1027delGA) and SSCP/HD (APC c.2700_2701delTC). In conclusion, all the evaluated methods are applicable for mosaic mutation screening even though combinations of the conventional methods should be used to reach an adequate sensitivity. Sanger sequencing alone is not sensitive enough to detect low mosaic levels. Parallel sequencing seems to be the ultimate choice but the possibilities to use this technique is today limited by its complexity, economics, and availability of instruments.
我们对多种突变检测技术检测嵌合突变的能力进行了评估。在本研究中，将桑格测序、单链构象多态性（SSCP）/异源双链分析（HD）、蛋白质截短试验（PTT）和变性高效液相色谱（DHPLC）与平行测序进行了比较。本研究共纳入了9例患者的全DNA样本。其中7个样本人工构建了嵌合突变，这些样本来自杂合子突变携带者，突变等位基因的比例为50%。分析的突变如下：APC基因中的c.646C>T、c.2626C>T、c.2828C>A、c.1817_1818insA、c.2788dupA、c.416_419delAAGA和c.607delC。SSCP/HD和DHPLC检测到的最低突变等位基因程度在5%至25%之间，桑格测序则在15%至50%之间。对其中3种突变进行了PTT分析，检测水平有很大差异（从10%到100%）。使用平行测序可达到低至1%的检测频率，但要实现这种高灵敏度需要足够的覆盖度。本研究还纳入了2例有自然嵌合突变的患者。这两种突变此前分别通过桑格测序（NF2基因c.1026_1027delGA）和SSCP/HD（APC基因c.2700_2701delTC）得以鉴定。总之，所有评估方法都适用于嵌合突变筛查，尽管应采用传统方法的组合以达到足够的灵敏度。单独的桑格测序灵敏度不足以检测低水平的嵌合情况。平行测序似乎是最终选择，但目前该技术的应用受到其复杂性、经济性和仪器可用性的限制。
