RB1 gene mutations in Turkish retinoblastoma patients.
Authors: Kebudi Rejin , Akdeniz Demet , Bayramova Jamila , Erciyas Seda Kilic , Turkcan Gozde Kuru , Tuncer Bugra Seref , Sukruoglu Ozge , Celik Betul , Gultaslar Busra Kurt , Buyukkapu Sema Bay , Tuncer Samuray , Yazici Hulya 
Published: June 2022

e22007 Background: Retinoblastoma (RB) is the most common intraocular malignancy in children, caused by mutations in the tumor suppressor RB1 gene. Patients with bilateral/trilateral/familial RB are considered to have hereditary RB (1 germline, 1 somatic mutation); although most unilateral RB are nonhereditary (2 somatic mutations), some have germline RB1 mutation. Identification of RB1 mutations and genetic counseling is essential to assess the risk of developing RB in the patients´ relatives and to prevent the disease. The aim of this study is to assess the frequency/type of RB1 gene mutations in a large cohort of Turkish RB cases. Methods: RB1 gene mutation screening was performed in peripheral blood samples of 219 individuals (122 children with RB/14 family members with RB/83 healthy family members of 47 probands with RB1 mutations) followed in the Istanbul University, Oncology Institute. All 27 exons and close intronic regions of the RB1 gene were sequenced for small deletions and insertions using the Sanger sequencing (2013-2018) or Next Generation Sequencing (2019-2021); large deletions and duplications were investigated both by Multiplex Ligation Probe Amplification (MLPA) and copy number variation (CNV). Correlation with demographic and clinical data were evaluated. Results: After RB1 mutation screening, mutations were detected in 57 (41.9%) of 136 patients. RB1 mutations was observed in 23/84 (27.4%) patients with unilateral RB, in 30/47 (63.8%) bilateral RB, in 3/3 trilateral RB, and1/2 unilateral retinoma. Of these mutations, 45 (78.9%) were small genetic rearrangements and 12 (21.1%) large genetic rearrangements. Frameshift mutations were found in 11, nonsense in 18, splice error in 11, and missense mutation in 1, synonymous substitution in 2, upstream substitution in 2 patients. Ten novel mutations were found. Three of 83 healthy family members also had germline RB1 mutation. The disease was hereditary in 13 (22.8%); and de novo (77.2 %) in 44 of the 57 patients with mutations. Three siblings were found to have RB1 mutation, while their parents did not. Patients diagnosed as infants (RB1 mutation found in 63.2% of 70 infants vs in 36.8% of older ones, p:0.021); those with bilateral/trilateral RB (66 vs 27.4%, p:0.0001); those with light (green-blue) iris color vs dark color (71.4% vs 36.5%. p:0.003) had higher frequency of RB1 gene mutation. There was no significant correlation regarding gender or ICRB stage and RB1 mutation frequency. Conclusions: RB1 gene mutation was found in 41.9% of Turkish children with RB, in 63.8% of bilateral and 27.4% of unilateral RB. 10 novel mutations of the RB1 gene were found according to the Leiden Open Variation Database and the Human Gene Mutation Database.The mutation frequency was significantly higher in patients with bilateral/trilateral RB, in infants, in those with light iris color. In bilateral RB with no RB1 mutation, other genes/RB1 gene methylation and expression status may play a role in the pathogenesis.