Transcripts with splicings of exons 15 and 16 of the hMLH1 gene in normal lymphocytes: implications in RNA-based mutation screening of hereditary non-polyposis colorectal cancer

Palmirotta, Raffaele; Verı̀, Maria Concetta; Curia, Maria Cristina; Aceto, Gitana María; D'Amico, Franca; Esposito, Diana L.; Arcuri, Paola; Mariani-Costantini, Renato; Messerini, Luca; Mori, Sabrina; Cama, Alessandro; Battista, Pasquale

doi:10.1016/s0959-8049(98)00031-8

Cited by 11 publications

(10 citation statements)

References 16 publications

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“…fs, Frame Shift with paremature nonsense codon created. the detection of constitutional MLH1 and MSH2 mutations by sequencing of cDNA, nested PCR was performed to produce the template [Kohonen-Corish et al, 1996;Herfarth et al, 1997;Palmirotta et al, 1998]. Here we were able to show that efficient detection of mutations can be achieved with 42 cycles of PCR amplification if specific primers are used for RT and that non-specific products can be minimized.…”

Section: Discussionmentioning

confidence: 69%

“…The limitations of cost and sensitivity may be partially overcome by RNA-based sequencing, where the entire coding region can be amplified in just a few overlapping fragments and any abnormalities localized outside of the exonic structure identified. Difficulties in the detection of changes can still be encountered such as alternative splicing [Hori et al, 1992;Arakawa et al, 1994;Charbonnier et al, 1995;Kohonen-Corish et al, 1996;Xia et al, 1996;Mori et al, 1997;Xu et al, 1997;Genuardi et al, 1998;Palmirotta et al, 1998] and low expression of mutant alleles [Lim et al, 1992;Maquat, 1995;Liu et al, 1996;Nyström-Lahti et al, 1996;Andreutti-Zaugg et al, 1997;Jäger et al, 1997;Culbertson, 1999;Wang et al, 1999]. It has been suggested that it is difficult to reliably detect abnormal transcripts of MSH2 or MLH1 in peripheral blood lymphocytes without additional stimulation of their proliferation in tissue culture.…”

Section: Introductionmentioning

confidence: 99%

“…Currently mRNA changes identified by nucleotide sequencing have been performed using nested-PCR in order to increase the efficiency of amplification of cDNA specific for MSH2, MLH1 [Kohonen-Corish et al, 1996;Herfarth et al, 1997;Palmirotta et al, 1998]. Nested-PCR creates experimental conditions that favor the amplification of shorter sequences such as alternative transcripts and the generation of large numbers of abnormal transcripts [Charbonnier et al, 1995;Kohonen-Corish et al, 1996;Xia et al, 1996;Mori et al, 1997;Viel et al, 1997;Genuardi et al, 1998;Palmirotta et al, 1998]. The second goal of this study was to establish if simple (not nested) PCR which only use a slightly increased number of amplification cycles could improve mutation detection in MSH2 and MLH1.…”

Section: Introductionmentioning

confidence: 99%

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Optimization of experimental conditions for RNA-based sequencing of MLH1 and MSH2 genes

et al. 2000

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The most sensitive technique for the detection of germline mutations is exon by exon sequencing of the gene under investigation using genomic DNA as a template for analysis. This approach, however, has cost and sensitivity limitations that can, at least in part, be overcome by RNA‐based analysis. Germline mutations of MLH1 and MSH2 are the most frequent cause of the inherited susceptibility to colorectal and other epithelial cancers known as hereditary non‐polyposis colorectal cancer (HNPCC). We compared the analysis of the MLH1 and MSH2 genes using mRNA and genomic DNA as starting material from 21 HNPCC patients. All samples were investigated by RT‐PCR, sequencing of cDNA and simultaneous sequencing of genomic DNA. The cDNA was generated using specific primers complementary to the ends of MLH1 and MSH2 genes, respectively. Mutations in MLH1 and MSH2 were detected in 11 out of 21 unrelated patients. In 10 out of 11 cases, mutations were detected independently of the type of primers used for reverse transcription (RT). One novel missense mutation (K751R) in MLH1 was detected using this method. One nonsense mutation (E205X) in MSH2 was only detectable when RT was performed using MSH2 gene‐specific primers. Shorter PCR products indicative of alternatively spliced transcripts were not observed when MLH1 or MSH2 specific cDNA RT primers were employed to generate template, except in one case where exon skipping was observed for exons 9 and 10. In this report we demonstrate that primers specific for RT of MLH1 and MSH2 are crucial for increasing the sensitivity of cDNA analysis. DNA sequencing using RNA as a basis for template construction may be a valuable and economical alternative to genomic DNA sequencing. Hum Mutat 17:52–60, 2001. © 2001 Wiley‐Liss, Inc.

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Section: Discussionmentioning

confidence: 69%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Optimization of experimental conditions for RNA-based sequencing of MLH1 and MSH2 genes

et al. 2000

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“…The genomic transition described here was reported for a different HNPCC case, where the authors found no alteration on cDNA derived from lymphoblastoid cell lines (9). Furthermore, one group reported a transcript lacking exon 15 as a common isoform (19). Common transcripts lacking one or several exons were reported for exons 9/10, 9-11 and 10/11 of hMLH1 (7), and for exon 13 of hMSH2 (8,10).…”

Section: Discussionmentioning

confidence: 99%

Quantitative Differences between Aberrant Transcripts Which Occur as Common Isoforms and due to Mutation-based Exon Skipping of the Mismatch Repair Gene hMLH1

Plaschke

Bulitta²,

Saeger³

et al. 1999

CCLM

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About one-third of hereditary non-polyposis colorectal cancer-related mutations in the mismatch repair gene hMLH1 result in the loss of entire exons from the wild type transcripts. Here we describe quantitative differences of hMLH1 transcripts without exon 15, exon 16 or exon 17 in several members of a family with hereditary non-polyposis colorectal cancer. The transcript lacking exon 15 is caused by a G to A transition affecting the last nucleotide of the respective exon and results in a truncated protein. The transcripts lacking exon 16 or exon 17, which are in-frame deletions, were also found in all tested samples of a normal population and represent common isoforms. Reverse transcription-polymerase chain reaction-based relative quantification revealed about 50 % signal intensity for the mutation-based transcript, but less than 10% for the common isoforms, if compared to the wild type. All aberrant transcripts were detected from blood-derived cDNAs but not from samples of normal colon epithelium. Although the biological significance of the common isoforms is unknown, they might lead to false risk assessment in hereditary non-polyposis colorectal cancer cases.

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“…Finding mutation in hMLH1 poses a particularly complicated problem because alternative splicing is common in this gene, which may generate transcripts that code for truncated protein (Charbonnier et al, 1995;Kohonen-Corish et al, 1996;Palmirotta et al, 1998). Some of the commonly observed splice variants include those with skipping of exons 10 and 11, exons 9 and 10, exons 9 ± 11, exons 15 and 16.…”

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confidence: 99%

A novel germline 1.8-kb deletion of hMLH1 mimicking alternative splicing: a founder mutation in the Chinese population

Chan

Yuen

et al. 2001

Oncogene

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We have previously reported that there is a high incidence of microsatellite instability (MSI) and germline mismatch repair gene mutation in colorectal cancer arising from young Hong Kong Chinese. Most of the germline mutations involve hMSH2, which is di erent from the mutation spectrum in the Western population. It is well known that alternative splicing is common in hMLH1, which complicates RNA based mutation detection methods. In contrast, large deletions in hMLH1, commonly observed in some ethnic groups, tend to escape detection by exon-by-exon direct DNA sequencing. Here we report the detection of a novel germline 1.8 kb deletion involving exon 11 of hMLH1 in a local hereditary non-polyposis colorectal cancer family. This mutation generates a mRNA transcript with deletion of exons 10 ± 11, which is indistinguishable from one of the most common and predominant hMLH1 splice variants. A diagnostic test based on PCR of the breakpoint region led to the identi®cation of an additional young colorectal cancer patient with this mutation. Haplotype analysis suggests that they may share a common ancestral mutation. Our results caution investigators in the interpretation of alternative splicing and have important implications for the design of hMLH1 mutation detection strategy in the Chinese population. Oncogene (2001) 20, 2976 ± 2981.

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Transcripts with splicings of exons 15 and 16 of the hMLH1 gene in normal lymphocytes: implications in RNA-based mutation screening of hereditary non-polyposis colorectal cancer

Cited by 11 publications

References 16 publications

Optimization of experimental conditions for RNA-based sequencing of MLH1 and MSH2 genes

Optimization of experimental conditions for RNA-based sequencing of MLH1 and MSH2 genes

Quantitative Differences between Aberrant Transcripts Which Occur as Common Isoforms and due to Mutation-based Exon Skipping of the Mismatch Repair Gene hMLH1

A novel germline 1.8-kb deletion of hMLH1 mimicking alternative splicing: a founder mutation in the Chinese population

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