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Identification of germline and somatic mutations affecting the retinoblastoma gene
J M Dunn1, R A Phillips, A J Becker
1Hospital for Sick Children, Research Institute, Toronto, Canada.
Abstract:
Retinoblastoma (RB) is a malignant tumor of developing retina that arises when abnormalities resulting in loss of function affect both alleles of the gene at the retinoblastoma locus (RB1) on chromosome 13q. The majority of RB tumors do not show gross alterations in a 4.7-kb fragment (4.7R), which is a candidate RB1 gene. To search for more subtle mutations, the ribonuclease protection method was used to analyze 4.7R messenger RNA from RB tumors. Five of 11 RB tumors, which exhibit normal 4.7R DNA and normal-sized RNA transcripts, showed abnormal ribonuclease cleavage patterns. Three of the five mutations affected the same region of the messenger RNA, consistent with an effect on splicing involving an as yet unidentified 5' exon. The high frequency of mutations in 4.7R supports the identification of 4.7R as the RB1 gene. However, the unusual nature of some of the abnormalities of 4.7 R alleles indicates that the accepted sequence of genetic events involved in the genesis of RB may require reevaluation.
Insights
Subtle mutations in the RB1 gene
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Retinoblastoma (RB) is a malignant retinal tumor caused by biallelic loss-of-function mutations in the RB1 gene.
- The 4.7R fragment is a candidate for the RB1 gene, but gross DNA alterations are infrequent in RB tumors.
- Subtle genetic alterations may contribute to retinoblastoma development.
Purpose of the Study:
- To investigate subtle mutations in the 4.7R candidate RB1 gene in retinoblastoma tumors.
- To analyze messenger RNA (mRNA) transcripts of the 4.7R fragment for abnormalities.
- To determine if mutations in 4.7R are frequent and if they affect splicing.
Main Methods:
- Ribonuclease protection assay was used to analyze 4.7R mRNA from 11 retinoblastoma tumors.
- Tumors with normal 4.7R DNA and normal-sized RNA transcripts were specifically examined.
- Abnormal ribonuclease cleavage patterns were identified as indicative of mutations.
Main Results:
- Five of 11 retinoblastoma tumors exhibited abnormal ribonuclease cleavage patterns in 4.7R mRNA.
- These mutations were detected despite normal 4.7R DNA and normal-sized RNA transcripts.
- Three mutations affected a specific region, suggesting splicing alterations involving an unidentified 5' exon.
Conclusions:
- The high frequency of mutations in 4.7R supports its role as the RB1 gene.
- Unusual mutations in 4.7R alleles suggest a need to reevaluate the genetic events in retinoblastoma genesis.
- Splicing defects may play a significant role in the development of retinoblastoma.