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Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids
Published on: June 8, 2019
Multiplex PCR screening detects small p53 deletions and insertions in human ovarian cancer cell lines
I B Runnebaum1, X W Tong, V Moebus
1Molekularbiologie-Labor, Universitäts-Frauenklinik, Ulm, Germany.
Abstract:
Mutations at the p53 tumor suppressor gene locus are a frequent genetic alteration associated with human ovarian carcinoma. Little information exists regarding whether mutational events occur other than point mutations and large deletions, causing loss of heterozygosity. Small intragenic deletions and insertions in the p53 gene have been observed in various human neoplasias. We developed a multiplex polymerase chain reaction (MPCR) screening assay to amplify the complete p53 coding region from genomic DNA in a single step. Deletions and/or insertions were found in six out of 11 newly established ovarian carcinoma cell lines. MPCR detected deletions as small as 2 bp, as confirmed by nucleotide sequence analysis. Most of the observed alterations (6/7) were homozygous or hemizygous. Structural aberrations of the p53 gene possibly leading to loss of p53 cell cycle control may be a consequence of a slipped-mispairing mechanism in rapid DNA replication during repetitious ovulation and wound repair of ovarian epithelial cells. MPCR may be a valuable tool for screening for possible p53 deletion and insertion mutations not only in ovarian cancer but also in other malignancies.
Insights
Multiplex PCR identified small deletions and insertions in the p53 gene of ovarian cancer cell lines. This method efficiently detects p53 mutations, crucial for understanding ovarian carcinoma development.
Area of Science:
- Molecular Biology
- Genetics
- Oncology
Background:
- p53 tumor suppressor gene mutations are common in ovarian carcinoma.
- Limited data exists on non-point mutation/deletion events like small intragenic alterations in p53.
- Such alterations can lead to loss of heterozygosity and impact tumor suppressor function.
Purpose of the Study:
- To develop and validate a multiplex polymerase chain reaction (MPCR) assay for detecting small deletions and insertions in the p53 gene.
- To screen newly established ovarian carcinoma cell lines for these p53 alterations.
- To investigate the potential mechanisms and implications of p53 gene structural aberrations in ovarian cancer.
Main Methods:
- Development of a single-step MPCR assay to amplify the entire p53 coding region from genomic DNA.
- Screening of 11 ovarian carcinoma cell lines using the MPCR assay.
- Confirmation of MPCR findings, including small deletions, via nucleotide sequence analysis.
Main Results:
- The MPCR assay successfully amplified the complete p53 coding region.
- Deletions and/or insertions were detected in 6 out of 11 ovarian carcinoma cell lines.
- MPCR identified deletions as small as 2 base pairs; most alterations were homozygous or hemizygous.
- Nucleotide sequence analysis confirmed the MPCR-detected mutations.
Conclusions:
- MPCR is a sensitive and effective tool for detecting small p53 intragenic deletions and insertions.
- Structural aberrations in the p53 gene are present in a significant proportion of ovarian carcinoma cell lines.
- These p53 alterations may arise from DNA replication errors during ovulation/repair and contribute to ovarian cancer progression by disrupting cell cycle control.
