Tournier Isabelle, Raux Grégory, Di Fiore Fréderic, Maréchal Isabelle, Leclerc Carole, Martin Cosette, Wang Qing, Buisine Marie-Pierre, Stoppa-Lyonnet Dominique, Olschwang Sylviane, Frébourg Thierry, Tosi Mario
INSERM EMI 9906, IFRMP, Faculté de Médecine et Pharmacie, Université de Rouen, Rouen, France.
Hum Mutat. 2004 Apr;23(4):379-84. doi: 10.1002/humu.20008.
Quantitative measures of allele-specific gene expression allow the indirect detection of mutations or sequence variants in regulatory elements or in other non-coding regions that may result in significant physiological or pathological changes of gene expression and may contribute to Mendelian or multifactorial disorders. We have devised a simple method, based on RT-PCR and single nucleotide primer extension (SNuPE) with unlabelled dideoxynucleotides, followed by DHPLC (denaturing high performance liquid chromatography). We established optimal conditions for separation of the extended products corresponding to the alleles of the c.655A>G (p.Ile219Val) SNP, which is the most frequent exonic polymorphism of MLH1. We then genotyped 99 unrelated control subjects and measured the allele-specific MLH1 expression in the 40 heterozygous controls found in this group. This method allowed us to define a narrow range of normal biallelic expression of MLH1, each allele contributing between 44.7% and 55.3% of the total expression. We then measured the allele-specific expression in hereditary nonpolyposis colorectal cancer (HNPCC) patients with MLH1 mRNAs bearing different stop-codon or frame-shift mutations, or in-frame deletions, in order to detect the effects of nonsense-mediated mRNA decay (NMD). Defects that induce mRNA instability were identified unambiguously and the data were consistent with current models of NMD. This study provides a sensitive tool to identify indirectly MLH1 defects that may escape detection in genomic DNA screenings but result in a quantitative change at the mRNA level.
等位基因特异性基因表达的定量测量能够间接检测调控元件或其他非编码区域中的突变或序列变异，这些变异可能导致基因表达发生显著的生理或病理变化，并可能引发孟德尔疾病或多因素疾病。我们设计了一种简单的方法，该方法基于逆转录聚合酶链反应（RT-PCR）和使用未标记双脱氧核苷酸的单核苷酸引物延伸（SNuPE），随后进行变性高效液相色谱（DHPLC）分析。我们确定了分离与c.655A>G（p.Ile219Val）单核苷酸多态性（SNP）等位基因相对应的延伸产物的最佳条件，该SNP是错配修复蛋白1（MLH1）最常见的外显子多态性。然后，我们对99名无亲缘关系的对照受试者进行基因分型，并测量了该组中40名杂合对照受试者的MLH1等位基因特异性表达。该方法使我们能够确定MLH1正常双等位基因表达的狭窄范围，每个等位基因在总表达中所占比例为44.7%至55.3%。接着，我们测量了携带不同终止密码子或移码突变、框内缺失的MLH1信使核糖核酸（mRNA）的遗传性非息肉病性结直肠癌（HNPCC）患者的等位基因特异性表达，以检测无义介导的mRNA降解（NMD）的影响。明确鉴定出了诱导mRNA不稳定的缺陷，且数据与当前的NMD模型一致。本研究提供了一种灵敏的工具，可间接识别在基因组DNA筛查中可能未被检测到但在mRNA水平导致定量变化的MLH1缺陷。
