title: Analysis of the allele-specific expression of the mismatch repair gene MLH1 using a simple DHPLC-Based Method.
abstract: 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.
authors.affiliation: INSERM EMI 9906, IFRMP, Faculté de Médecine et Pharmacie, Université de Rouen, Rouen, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Tournier
authors.forename: Grégory
authors.initials: G
authors.lastname: Raux
authors.forename: Fréderic
authors.initials: F
authors.lastname: Di Fiore
authors.forename: Isabelle
authors.initials: I
authors.lastname: Maréchal
authors.forename: Carole
authors.initials: C
authors.lastname: Leclerc
authors.forename: Cosette
authors.initials: C
authors.lastname: Martin
authors.forename: Qing
authors.initials: Q
authors.lastname: Wang
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Buisine
authors.forename: Dominique
authors.initials: D
authors.lastname: Stoppa-Lyonnet
authors.forename: Sylviane
authors.initials: S
authors.lastname: Olschwang
authors.forename: Thierry
authors.initials: T
authors.lastname: Frébourg
authors.forename: Mario
authors.initials: M
authors.lastname: Tosi
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000483:Alleles; D002352:Carrier Proteins; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D000070957:MutL Protein Homolog 1; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins; D009691:Nucleic Acid Denaturation; D020641:Polymorphism, Single Nucleotide; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D000070957:MutL Protein Homolog 1