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Published on: November 5, 2019
Epigenetic and copy number variation analysis in retinoblastoma by MS-MLPA
Gabriella Livide1, Maria Carmela Epistolato, Mariangela Amenduni
1Department of Biotechnology, University of Siena, Siena, Italy.
Abstract:
Retinoblastoma is the most common primary intraocular malignancy in children. Two step inactivation of RB1 (M1-M2) represents the key event in the pathogenesis of retinoblastoma but additional genetic and epigenetic events (M3-Mn) are required for tumor development. In the present study, we employed Methylation Specific Multiplex Ligation Probe Assay to investigate methylation status and copy number changes of 25 and 39 oncosuppressor genes, respectively. This technique was applied to analyse 12 retinoblastomas (5 bilateral and 7 unilateral) and results were compared to corresponding normal retina. We identified hypermethylation in seven new genes: MSH6 (50%), CD44 (42%), PAX5 (42%), GATA5 (25%), TP53 (8%), VHL (8%) and GSTP1 (8%) and we confirmed the previously reported hypermethylation of MGMT (58%), RB1 (17%) and CDKN2 (8%). These genes belong to key pathways including DNA repair, pRB and p53 signalling, transcriptional regulation, protein degradation, cell-cell interaction, cellular adhesion and migration. In the same group of retinoblastomas, a total of 29 copy number changes (19 duplications and 10 deletions) have been identified. Interestingly, we found deletions of the following oncosuppressor genes that might contribute to drive retinoblastoma tumorigenesis: TP53, CDH13, GATA5, CHFR, TP73 and IGSF4. The present data highlight the importance of epigenetic changes in retinoblastoma and indicate seven hypermethylated oncosuppressors never associated before to retinoblastoma pathogenesis. This study also confirms the presence of copy number variations in retinoblastoma, expecially in unilateral cases (mean 3 ± 1.3) where these changes were found more frequently respect to bilateral cases (mean 1.4 ± 1.1).
Insights
This study reveals new epigenetic alterations in retinoblastoma, a childhood eye cancer. Researchers found hypermethylation in seven previously unlinked genes and copy number variations, particularly in unilateral cases, advancing understanding of tumor development.
Area of Science:
- Oncology
- Genetics
- Epigenetics
Background:
- Retinoblastoma is the most common pediatric intraocular malignancy.
- Tumorigenesis involves RB1 gene inactivation plus additional genetic/epigenetic events.
- Understanding these events is crucial for targeted therapies.
Purpose of the Study:
- To investigate methylation status and copy number changes of 25 and 39 oncosuppressor genes in retinoblastoma.
- To identify novel genes epigenetically altered in retinoblastoma pathogenesis.
- To compare genetic alterations between unilateral and bilateral retinoblastoma cases.
Main Methods:
- Methylation Specific Multiplex Ligation Probe Assay (MS-MLPA) was used.
- Analyzed 12 retinoblastoma samples (5 bilateral, 7 unilateral) and matched normal retina.
- Assessed methylation and copy number variations in key oncosuppressor genes.
Main Results:
- Identified hypermethylation in seven novel genes: MSH6, CD44, PAX5, GATA5, TP53, VHL, and GSTP1.
- Confirmed hypermethylation of previously reported genes: MGMT, RB1, and CDKN2.
- Detected 29 copy number changes, including deletions in TP53, CDH13, GATA5, CHFR, TP73, and IGSF4.
- Found copy number variations more frequent in unilateral (mean 3) than bilateral (mean 1.4) retinoblastoma.
Conclusions:
- Epigenetic changes, including novel hypermethylated oncosuppressors, play a significant role in retinoblastoma development.
- Copy number variations are prevalent and potentially drive tumorigenesis, especially in unilateral cases.
- Findings expand the understanding of retinoblastoma's genetic landscape and identify potential therapeutic targets.
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