Poster abstracts

Poster number 16 submitted by Reeva Gupta

Missense or silent mutations in p53 exon 7 ESE result in exon skipping

Reeva Gupta (The Center for Childhood Cancer, Columbus Childrens Research Institute and the Department of Pediatrics, The Ohio State University School of Medicine and Public Health, Columbus, Ohio), Jessica Fleming (The Center for Childhood Cancer, Columbus Childrens Research Institute, Columbus, Ohio), Jill Molfenter (The Ohio State University School of Medicine and Public Health, Columbus, Ohio), Dawn S. Chandler (The Center for Childhood Cancer, Columbus Childrens Research Institute and the Department of Pediatrics, The Ohio State University School of Medicine and Public Health, Columbus, Ohio)

Abstract:
Germ line mutations in TP53 gene have been observed in Li-Fraumeni Syndrome, a familial cancer syndrome. This syndrome is characterized by a diverse set of early-onset malignancies including breast carcinoma, sarcomas and brain tumors. Germ line analysis for p53 has reported mutations between exon 5 and 9.

The most commonly observed missense mutations are within codon 248. The changes in exon 7 results in amino acid alterations from an arginine (R) to either trypothan (W) or glutamine (Q) which affect protein function. Incidentally silent mutations have been identified at the same location. ESE finder database predicted that 248 codon lies within the two overlapping SF2/ASF predicted binding sites. Interestingly, the four reported 248 missense and silent mutations were predicted to disrupt one or both the binding sites. We hypothesize that these mutations disrupt p53 normal splicing, resulting in truncated p53 protein with altered function that ultimately contributes to the cancer phenotype.

To confirm the candidate ESE function for codon 248 in exon 7 we are using minigene splicing assays. The exon 7 ESE was cloned in the SXN-13 minigene exon 2 to determine the skipping or inclusion of the exon 2. The ESE was also altered to include the four reported exon 7 mutations.

Our results indicate that the wild type p53 ESE promotes proper exon recognition while mutant ESEs give rise to transcripts in which the exon has been skipped. The most deleterious splicing mutations in the exon 7 ESE are CGG248CGT (silent), CGG248CGA (silent), CGG248TGG(R > W), CGG248CAG(R > Q).

We are currently analyzing patient cell lines which harbor these p53 ESE mutations for the presence of the altered p53 transcripts. The identification of silent p53 mutation that affect p53 splicing will help in determination of the functional role of these new spliced isoforms in the initiation of cancer.

Keywords: Exon splicing enhancers, TP53, silent or missense mutations