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N. Ahmad, T. Romanowicz, H. Pace, C. Shields, J. Shields; Molecular Genetics of 25 year old recurrence of RB . Invest. Ophthalmol. Vis. Sci. 2004;45(13):5194.
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Purpose: Recurrence of RB beyond 4 years following radiotherapy is extremely rare. We investigated the molecular genetics underlying an extremely unusual case of RB, 25 years after therapy. . Shields et al (2002) reported that the patient was diagnosed and treated for bilateral RB at 12 months of age, with enucleation of the left eye and external beam radiation therapy (EBRT) to the right eye. She presented with a mushroom shaped choroidal recurrence of RB 25 years later. The right eye was enucleated and histopathologic examination confirmed that the tumor was poorly differentiated, mitotically active RB. Methods: We screened the entire genomic RB1 gene of this patient by Conformation Sensitive Gel Electrophoresis that revealed an anomalous migration of the PCR product containing exon 2, which was directly sequenced to characterize the variation. DNA isolated from paraffin blocks of tumor from each eye was screened for this variation. Polymorphism sites in intron 1, intron 17 and intron 26 were investigated to evaluate loss of heterozygosity Results:We detected a heterozygous single base deletion of ‘C’ at nucleotide 5450, 2nd position of codon 55 leading to a premature stop codon downstream at exon 64 in the genomic DNA. This mutation was present in the RB1 gene of both tumor samples as homozygous mutations. This is the first report of this mutation at this particular site in the RB1 gene. Comparative genotyping of polymorphic loci in DNA from blood and tumour was performed to investigate loss of heterozygosity (LOH). Polymorphisms at intron 1 and 17 were uninformative since the genomic DNA was homozygous. However, genomic DNA was heterozygous for the polymorphism in intron 26 as was DNA of the tumor from the right eye. The tumor from the left eye was homozygous. Conclusions: These results indicate this new RB mutation is a germ–line mutation and subsequent offspring will be at risk. These results underscore the current clinical practice to avoid EBRT since it has been noted that patients with the familial form of the disease who undergo EBRT have about a 30% chance of developing another malignant cancer in or out of the field of radiation by 30 years of age. The LOH results indicate that the tumor from the right eye shows LOH at one polymorphic locus (at least at the mutant loci) but retained alleles at another suggesting mitotic recombination as the second mutation. These results which indicate that the second event in each tumor was different, may also explain the lag in tumor formation in the right eye.
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