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A nonsense mutation in MLH1 causes exon skipping in three unrelated HNPCC families
1University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.
Abstract:
Germline mutations in the DNA mismatch repair genes MSH2 and MLH1 are responsible for the majority of hereditary nonpolyposis colorectal cancer (HNPCC) families. A common mutation mechanism is to disrupt MLH1 and MSH2 mRNA splicing. The disruption creates aberrant mRNAs lacking specific coding exons (exon skipping). Here, we report a novel skipping of MLH1 exon 12 caused by an AAG to TAG nonsense mutation at codon 461 in three HNPCC families of North American origins. The nonsense codon was found in a conserved haplotype in the three unrelated families and seems to represent a founder mutation. The skipping created an aberrant MLH1 mRNA transcript lacking exon 12. The effect of the codon 461 nonsense mutation on exon 12 skipping is evident even though it was placed in a minigene construct containing entirely different coding sequences. Notably, the effect of the nonsense mutation on exon skipping is incomplete. Accordingly, a second aberrant MLH1 transcript encompassing the nonsense codon is also produced. Whereas the latter transcript is unstable, presumably because of nonsense-mediated mRNA decay, neither of the aberrant transcripts seems to affect the stability of wild-type MLH1 mRNA. This study demonstrates that the germ-line nonsense mutation at codon 461 of MLH1 disrupts normal MLH1 mRNA processing, and that exon skipping underlies pathogenesis in these HNPCC families.
Insights
A novel nonsense mutation in the MLH1 gene causes exon skipping, leading to hereditary nonpolyposis colorectal cancer (HNPCC). This genetic defect disrupts MLH1 mRNA processing and underlies disease development in affected families.
Area of Science:
- Genetics
- Molecular Biology
- Oncology
Background:
- Hereditary nonpolyposis colorectal cancer (HNPCC) is primarily caused by germline mutations in DNA mismatch repair genes MLH1 and MSH2.
- A common mechanism involves mutations disrupting mRNA splicing, leading to exon skipping.
Purpose of the Study:
- To identify and characterize a novel MLH1 mutation causing exon skipping in HNPCC families.
- To investigate the molecular mechanism by which this mutation affects MLH1 mRNA processing and contributes to HNPCC pathogenesis.
Main Methods:
- Analysis of MLH1 gene in HNPCC families.
- Minigene construct to assess the effect of the mutation on mRNA splicing.
- mRNA stability studies, including nonsense-mediated decay analysis.
Main Results:
- A novel AAG to TAG nonsense mutation at codon 461 in MLH1 was identified in three unrelated North American HNPCC families, suggesting a founder effect.
- This mutation causes skipping of MLH1 exon 12, resulting in an aberrant mRNA transcript.
- The mutation's effect on exon skipping was confirmed using a minigene construct.
- Incomplete exon skipping resulted in a second aberrant transcript, which was unstable due to nonsense-mediated decay.
- Neither aberrant transcript affected wild-type MLH1 mRNA stability.
Conclusions:
- The germline nonsense mutation at MLH1 codon 461 disrupts normal MLH1 mRNA processing through exon skipping.
- This aberrant splicing mechanism is a key factor in the pathogenesis of HNPCC in these families.