Background: Retinoblastoma (RB) is a rare childhood malignant disorder caused by the biallelic inactivation of RB1 gene. However, the mechanisms that enable RB cells to acquire the additional hallmarks of cancer are still unknown. Hence, it is postulated that other genetic defects like focal amplification of MYCN, BCOR, TNFα and OTX2 may be present that drives tumor progression in RB.
Methods: Tumor and blood samples from 7 non-familial unilateral and 4 bilateral (2 non-familial and 1 familial) RB patients were investigated for underlying RB1 mutational spectrum by Sanger Sequencing and Next Generation Sequencing (NGS). They were further investigated for TNFα and KIF13A gene amplification and MGMT hypermethylation. Functional consequence of all mutations was analyzed in-silico (PROVEAN, Mutation Taster, SIFT and CADD) and compared against population databases (1,000 genomes, EXAC and ESP) and gene locus specific mutation database such as rb1-lsdb and HGMD.
Results: We identified 10 novel RB1 mutations (3 germline and 7 somatic) which arose sporadically. Family screening showed that the parents did not carry the respective mutations found in probands. The chi square test for independence for novel variants vs. known variants; germline vs. somatic variants as well as variants present in bilateral vs. unilateral cases did not show any statistical significance. In addition to RB1 mutations, four tumors had both TNFα and KIF13A amplifications, while one tumor had TNFα amplification concurrently with promoter hypermethylation of MGMT.
Conclusions: Our study adds to the genetic spectrum of RB1 mutations, which is important as one of the basis for attributing disease pathogenicity when the same variants are subsequently found in other unrelated patients with similar phenotype. In addition, our findings indicate that other genes may play a potentially supportive role in multistep process of tumorigenesis and disease progression.
