A molecular study of first and second RB1 mutational hits in retinoblastoma patients

RBI mutations accountable for biallelic inactivation are crucial events in the development of retinoblastoma because a first mutation (MI) predisposes to retinoblastoma while a second mutation (M2) is required for tumor development. Mutational analyses of this gene showed a wide spectrum of genetic alterations (single base substitutions, insertions, or deletions, as well as small and large deletions). The most frequent second hit in retinoblastoma patients is loss of heterozygosity (LOH) followed by promoter methylation. Molecular analyses of RBI mutations were conducted in 36 patients (20 unilateral and 16 bilateral) using polymerase chain reaction-mediated single-strand conformation polymorphism (SSCP) analysis, sequencing, and LOH analysis. Sixty-four amplified fragments showing abnormal SSCP patterns were sequenced, and mutations were confirmed in five patients (13.89%). Four mutations were located at coding regions, and a fifth one was found at an exon-intron junction. Two mutations were C -> T transitions, two were small-length deletions, and one was a G -> A transition. A total of 47.05% patients showed LOH. In one patient, the parental origin of the mutated allele was detected: the allele retained in the tumor was the paternal one. This work helps to characterize the spectrum of mutations in the Brazilian population, and to confirm that formaldehyde-fixed paraffin tissue can provide valuable information on the RBI status in retinoblastoma patients. (c) 2006 Elsevier Inc. All rights reserved.