Aceto Gitana M, De Lellis Laura, Catalano Teresa, Veschi Serena, Radice Paolo, Di Iorio Angelo, Mariani-Costantini Renato, Cama Alessandro, Curia Maria Cristina
Department of Human Movement Sciences, University G d'Annunzio, Chieti, Italy.
Clin Chem. 2009 Sep;55(9):1711-8. doi: 10.1373/clinchem.2009.126300. Epub 2009 Jul 23.
Altered germline expression of genes may represent a powerful marker of genetic or epigenetic predisposition to cancer or other diseases.
We developed and validated a method of nonfluorescent primer extension that uses a single dideoxynucleotide and denaturing HPLC (DHPLC) to analyze the relative allele expression. We devised 5 independent assays for measuring allele-specific expression (ASE) to exploit different markers of mismatch repair genes MLH1 [mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli)] and MSH2 [mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli)]. We initially confirmed method reproducibility with genomic DNA (gDNA) from individuals heterozygous for a frequent single-nucleotide polymorphism in the MLH1 gene. After this preliminary validation with gDNA, we confirmed assay reproducibility with cDNA templates from control individuals. Relative allele expression was estimated by comparing the heights of the peaks corresponding to the 2 alleles. Results obtained with gDNA templates were used to normalize cDNA results.
With these DHPLC-based primer-extension assays, we detected and confirmed a 5-fold imbalance in MLH1 allele expression in a mutation-negative patient with hereditary nonpolyposis colorectal cancer and in another patient with a modest degree of imbalance in MLH1 expression. Among control individuals, the relative expression of MLH1 alleles displayed a narrow range of variation.
Independent DHPLC-based primer-extension assays for measuring and confirming ASE can be developed for different sequence variants of interest. This DHPLC application provides a cost-effective method for detecting ASE in cases for which conventional screening fails to detect pathogenic mutations in candidate genes and may be applicable for confirming ASE revealed by other methods, such as those used for transcriptome-wide analyses. .
基因种系表达的改变可能是癌症或其他疾病遗传或表观遗传易感性的有力标志物。
我们开发并验证了一种非荧光引物延伸方法，该方法使用单个双脱氧核苷酸和变性高效液相色谱（DHPLC）来分析相对等位基因表达。我们设计了5种独立的测定方法来测量等位基因特异性表达（ASE），以利用错配修复基因MLH1[mutL同源物1，结肠癌，非息肉病2型（大肠杆菌）]和MSH2[mutS同源物2，结肠癌，非息肉病1型（大肠杆菌）]的不同标志物。我们最初用来自MLH1基因常见单核苷酸多态性杂合个体的基因组DNA（gDNA）确认了方法的可重复性。在用gDNA进行初步验证后，我们用对照个体的cDNA模板确认了测定的可重复性。通过比较对应于两个等位基因的峰高来估计相对等位基因表达。用gDNA模板获得的结果用于标准化cDNA结果。
通过这些基于DHPLC的引物延伸测定，我们在一名遗传性非息肉病性结直肠癌的突变阴性患者和另一名MLH1表达存在适度失衡的患者中检测并确认了MLH1等位基因表达存在5倍的失衡。在对照个体中，MLH1等位基因的相对表达显示出狭窄的变异范围。
可以针对感兴趣的不同序列变体开发基于DHPLC的独立引物延伸测定来测量和确认ASE。这种DHPLC应用提供了一种经济有效的方法，用于在常规筛查未能检测到候选基因中的致病突变的情况下检测ASE，并且可能适用于确认其他方法（如用于全转录组分析的方法）所揭示的ASE。
