A hMLH1 genomic mutation and associated novel mRNA defects in a hereditary non-polyposis colorectal cancer family

Q. Tanko, B. Franklin, Henry T. Lynch, J. Knezetic

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Hereditary non-polyposis colorectal cancer (HNPCC), or Lynch syndrome I, is responsible for as high as 10% of all colorectal cancers (CRCs) newly diagnosed in any given year. This disorder has an autosomal dominant inheritance pattern and is almost fully penetrant (>85%). It occurs when there is a mutation in any one of six mismatch repair genes: hMLH1, hMSH2, hPMS1, hPMS2, hMSH3 and hMSH6. Mutations in these genes allow mistakes in tumor suppressor genes and oncogenes to accumulate which eventually leads to cancer. The founder of an HNPCC family in the Creighton University Hereditary Cancer Institute database was known to produce truncated hMLH1 protein, a product of one of the aforementioned mismatch repair genes. Lymphoblasts were isolated from ten members of this HNPCC family (six affected and four unaffected) and two persons from outside this family (both unaffected controls). RNA and DNA were purified from these lymphoblasts which had been transformed by the Epstein-Barr virus (EBV). The hypothesis was that a mutation in the hMLH1 gene perpetuated defects in its mRNA and functional protein. hMLH1 RNA transcripts were detected in reverse transcriptase polymerase chain reactions (RT-PCR) whereby total poly A+ RNA was converted to a complementary DNA (cDNA), amplified using hMLH1 specific primers, purified and cycle sequenced. Likewise, DNA was employed as template for PCR amplification of hMLH1 exons; PCR products were then directly cycle sequenced. Affected family members were found to produce hMLH1 mRNA lacking exons 6 and 7 (and wild-type mRNA). A splicing mutation at 546 -2 (two bases 5′ to exon 7) was located in the genomic DNA samples from the six family members with the HNPCC phenotype. This mutation caused deletion of exon 7 from the mRNA. None of the four unaffected family members or the two unaffected persons outside of this family had the above defects in their hMLH1 mRNA and DNA.

Original languageEnglish
Pages (from-to)37-42
Number of pages6
JournalMutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Volume503
Issue number1-2
DOIs
StatePublished - Jun 19 2002

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Colorectal Neoplasms
Messenger RNA
Mutation
Exons
DNA Mismatch Repair
DNA
Genes
RNA
Hereditary Nonpolyposis Colorectal Neoplasms
Inheritance Patterns
Polymerase Chain Reaction
Sequence Deletion
Tumor Suppressor Genes
Reverse Transcriptase Polymerase Chain Reaction
Human Herpesvirus 4
Oncogenes
Neoplasms
Proteins
Complementary DNA
Databases

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Health, Toxicology and Mutagenesis

Cite this

A hMLH1 genomic mutation and associated novel mRNA defects in a hereditary non-polyposis colorectal cancer family. / Tanko, Q.; Franklin, B.; Lynch, Henry T.; Knezetic, J.

In: Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, Vol. 503, No. 1-2, 19.06.2002, p. 37-42.

Research output: Contribution to journalArticle

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