Detection Of Frameshift Mutations Research Articles

Nature and mRNA effect of 282 differentNF1point mutations: focus on splicing alterations

Neurofibromatosis type 1 (NF1) is a common autosomal dominant genetic disorder caused by mutations in the NF1 gene. In this paper we report our experience using the cDNA-SSCP/HD analysis as a mutational screening approach and the double characterization of all mutations at the DNA and RNA levels. Two hundred and eighty-two different mutations (in 374 independent patients) were identified, 140 of which were novel in our population. Most of these mutations are unique and distributed along the gene. However, we also detected 37 recurrent mutations. Our approach is limited with respect to the detection of single base substitutions, but it is highly effective in the detection of frameshift mutations and mutations that affect the correct splicing. Due to this bias we focus here in the characterization of these two types of mutations. Forty-seven percent of mutations found were frameshift mutations, with small deletions being 2.3 times more common than small insertions. At the mRNA level, 44% of mutations affected the correct splicing, 80% of them located in the consensus sequences, with the donor site being much more frequently involved. The remaining 20% consisted of mutations located in deep intronic sites and mutations located in the coding region. In general the latter group produces different types of mutated transcripts with specific proportions for each mutation. The double characterization of mutations at the DNA and RNA levels enables to detect a broader spectrum of mutations than any single level approach, and provides a greater understanding of their molecular pathogenesis.

Detection of frameshift mutations of the transforming growth factor β receptor II in gastric cancers with microsatellite instability

OBJECTIVE To study the relationship among microsatellite instability (MSI), frameshift mutations (FM) of the transforming growth factor β receptor II (TGFβR II), methylation state of the hMLH1 promoter and hMLH1 protein expression level in gastric cancers, and to explore their relationship to gastric carcinogenesis.

Gene expression of galectin-9/ecalectin, a potent eosinophil chemoattractant, and/or the insertional isoform in human colorectal carcinoma cell lines and detection of frame-shift mutations for protein sequence truncations in the second functional lectin domain.

The family of Ca2+-independent galactoside-binding lectins with the beta-strand topology of the jelly-roll, referred to as galectins, is known to mediate and modulate a variety of cellular activities. Their functional versatility explains the current interest in monitoring their expression in cancer research, so far primarily focused on galectin-1 and -3. Tandem-repeat-type galectin-9 and its (most probably) allelic variant ecalectin, a potent eosinophil chemoattractant, are known to be human leukocyte products. We show by RT-PCR with primers specific for both that their mRNA is expressed in 17 of 21 human colorectal cancer lines. As also indicated by restriction analysis, in addition to the expected transcript of 571 bp an otherwise identical isoform coding for a 32-amino acid extension of the link peptide was detected. Positive cell lines differentially expressed either one (7 lines) or both transcripts (10 lines). Sequence analysis of RT-PCR products, performed in four cases, allowed to assign the standard transcript to ecalectin in the case of SW480 cells and detected two point mutations in the insert of the link peptide-coding sequence in WiDr and Colo205. Furthermore, this analysis identified the insertion of a single nucleotide into the coding sequence generating a frame-shift mutation, an event which has so far not been reported for any galectin. This alteration encountered in both transcripts of the WiDr line and the isoform transcript of Colo205 cells will most likely truncate the protein part within the second (C-terminal) carbohydrate recognition domain. Our results thus reveal the presence of mRNA for a galectin-9-isoform or a potent eosinophil chemoattractant (ecalectin) or a truncated version thereof with preserved N-terminal carbohydrate recognition domain in established human colon cancer cell lines.

The early detection of frameshift mutations induced by a food-borne carcinogen in rats: a new tool for molecular epidemiology.

The accumulation of genetic changes is considered as the main factor that determines the development of cancer. Recent progresses in genetics and molecular biology led to the discovery of many new molecular markers and to the development of techniques able to monitor these markers. As a consequence, molecular epidemiology has emerged as a powerful approach to study the ternary relationship between the environment, the behaviour and the genetic predisposition of each individual. Susceptibility to cancer is determined at different levels such as the genetic polymorphism of enzymes involved in the activation and detoxification of carcinogens, the polymorphism of genes that maintains the genome stability, like those involved in DNA repair or recombination processes, and finally the polymorphism in oncogenes or tumour suppressor genes. Consequently, the full assessment of each individual's genetic predisposition is a long and difficult task. As the accumulation of mutations in somatic cells integrates all these parameters, its measurement would facilitate the evaluation of the individual predisposition status, provided that a marker common to a large spectrum of carcinogens could be found. Our current studies on the molecular mechanisms of carcinogen-induced mutagenesis has revealed that G-rich repetitive sequences are mutational hot spots for several major classes of environmental genotoxins such as aromatic and heterocyclic amines, polycyclic hydrocarbons and oxidative agents. We thus consider the possibility that these sequences form a new class of biomarkers for carcinogen exposure. In order to validate this hypothesis, we designed a sensitive PCR-based assay able to detect specific mutations induced by a common food-borne carcinogen in the colon epithelium of rats exposed for a short period to this carcinogen. This assay is sensitive enough to allow early detection of induced mutations and therefore allows to differentiate between unexposed animal and those exposed for a period as short as 1 week.