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V. L. Parsam, C. Kannabiran, J. Mohd, S. G. Honavar, G. K. Vemuganti; Transcript Analysis of Constitutional RB1 Gene Mutations in Retinoblastoma Patients Reveals Different Patterns of Missplicing. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1577.
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To analyze the effects of mutations in splice sites, as well as substitutions and deletions in the coding sequence of the RB1 gene on the mRNA and to use transcript analysis to detect mutations in cases where mutations were not known.
Total RNA from fresh blood of 16 patients and available family members was isolated using Trizol reagent and first strand cDNA synthesis was carried out using oligo dT. The complete RB1 cDNA was amplified in 5 overlapping fragments. Amplified cDNA was analyzed by gel electrophoresis to check for sizes of RB1 transcripts. Abnormal transcripts were characterized by gel elution and sequencing.
Transcript analysis of 2 splice site mutations, IVS22+5 G>C and IVS11-1 G>A identified in genomic DNA of 2 patients, revealed single exon skipping in both cases. A missense substitution of (c.652T>G) p.Leu218Val in exon 7 found in a proband with bilateral Rb resulted in two abnormal transcripts- one transcript had deletion of a part of exon 7 and a second transcript had a part of exon 7 as well as exon 8 deleted. Out of 7 probands with no mutations detected in genomic DNA, RNA analysis was informative in 3 patients. In 2 of these cases, exonic deletions were detected- one patient had a deletion of exon 6 and the 2nd patient had more than one aberrant transcript involving exons 21 and 22. A deletion of exons 23-25 identified by quantitative PCR of genomic DNA in a case of familial Rb was confirmed by RNA analysis of the proband and affected family members.
RNA analysis revealed the effects of splice mutations, as well the splicing defect due to a missense substitution. In cases with no identifiable mutations in genomic DNA, the detection of gross splicing defects suggests the utility of mRNA screening in detection of mutations. Analysis of RB1 mRNA can be used as a first line of genetic testing for retinoblastoma patients.
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