A Study On The Mutation Analysis Of Rb1 Gene And The Screening Of Differential Proteins In Retinoblastoma

Retinoblastoma (RB) is a primary malignant intraocular neoplasm that arises from immature retinoblasts within the developing retina. It is the most common primary intraocular malignancy of childhood-it occurs in about 1:21000 individuals. The neoplasm has strong tendencies to invade the brain via the optic nerve and to metastasize widely. The median age at the diagnosis is approximately 12 months in children who have bilateral retinoblastoma versus 24 months in those who suffer unilateral disease. Approximately 60~70% of retinoblastoma cases are unilateral while the remaining 30~40% are bilateral. In unilateral cases, usually only a single tumor is present in the affected eye. In bilateral cases, multifocal tumors in both eyes are the rule. Most of children who have the sporadic form of retinoblastoma are affected unilaterally. In contrast, a small number of patients have a prior family history of retinoblastoma. The affected child in hereditary cases usually, but not always, has multiple tumors in both eyes. Because the mutations arise in both coding region and splicing sile of RNA in Rb1 gene, it results in the change of RB protein structurally and functionally. The biological effect the mutation gave will be the tumorgenesis of RB by the elimination of the negative regulation of the cell circle by Rb1 gene. In this study, with the PCR-SSCP combined sequencing, we analysized the mutations of Rb1 gene in 40 tumor samples of retinoblastom. All 27 exons of Rb1 gene had been gone through for the mutation analysis, including the promoter region. 19 mutations were totally found from 18 samples while 2 mutations were existed in the same tumor sample. 9 mutations (about 50% of whole mutations) out of 19 incidents were deletion ranged from 1 to 27 base pairs. 4 of them were existed in the coding region of Rb1 gene while the other 4 were localized in the splicing sites of mRNA. Only one mutation of deletion was found in the intron of Rb1 gene. Three insertion mutations were presented in Rb1 gene with the numbe of base pair 2 to 8. Two of them were existed among the sequence of ORF of Rb1 gene while one mutation affected the region of mRNA splice site. Most of the point mutation found in Rb1 gene were C to T mutation. We found 7 point mutations totally. All the bases, except one, which were replaced were C and all of them were affecting the coding region of Rb1 gene. The mutations found in the Rb1 gene will finally interfere the normal structure of RB protein and the performance of the protein. They will finally cause the tumorigenesis of retinoblastoma. By this study, we found that the techniques of PCR-SSCP combined sequencing can be used in the mutation analysis of the Rb1 gene. And we have shown that the technique has potential as a repid and efficient aid to screen and offer genetic counseling of familial members in the hereditary retinoblastoma.