The first results of genetic screening and exploration of genotype-phenotype correlations in retinoblastoma patients from Belarus

Abstract

Retinoblastoma is an aggressive eye tumor originating from maturing cone precursors in the developing retina and most commonly seen in childhood. In 98 % of patients, retinoblastoma is caused by bi-allelic inactivation of the RB1 tumor suppressor gene. Approximately 40 % of disorders in the RB1 gene are germline. This study aimed to analyze the frequency of germline RB1 variants in a cohort of Belarusian patients with retinoblastoma and to correlate the variants with clinical phenotypes. The study was approved by an Independent Ethics Committee and the Scientific Council of the Belarusian Research Center for Pediatric Oncology, Hematology and Immunology. The study included 20 patients from unrelated families (9 patients with unilateral retinoblastoma, 11 – with bilateral). Two out of eleven patients with bilateral retinoblastoma had a positive family history. Genomic DNA was extracted from peripheral blood mononuclear cells. Using polymerase chain reaction, we obtained fragments including sequences of all exons, regions of splice sites and promoter regions of the RB1 gene. Nucleotide sequences of the obtained amplicons were detected by next-generation sequencing. All clinically significant variants were confirmed by Sanger sequencing. Multiplex ligation-dependent probe amplification (MLPA) or fluorescence in situ hybridization (FISH) were used to detect gross alterations. A genetic analysis of blood relatives was carried out for five probands with detected germline variants. We identified 13 different variants in 14 patients: 38.5 % (n = 5) of them were defects in splice sites; 15.4 % (n = 2) – missense mutations; 15.4 % (n = 2) – small deletions (frameshift); 23% (n = 3) – large deletions; 7.7% (n = 1) – nonsense mutations. Four of these variants had not been previously reported in patients with retinoblastoma from other populations (exon 3: c.350_351delTT, p. Phe117TyrfsTer2; exon 8: c.861+2T>G; exon 24: c.2520+4A>G; Del of exons 16, 17). Germline mutations were detected in 33.3 % (3/9) of patients with unilateral retinoblastoma and in 100% (11/11) of patients with bilateral disease. A genetic screening of relatives showed that three variants were de novo, and two variants were inherited from parents in families with a positive history of retinoblastoma. Here we reported the first results of genetic examination of Belarusian patients with retinoblastoma. Seventy-eight point six per cent (78.6 %) of variants were detected by sequencing, 21.4 % were identified with the help of the MLPA and FISH methods. Among sporadic cases, germline RB1 variants were detected in 66.6 % (12/18) of cases. A full range of screening techniques is required to achieve high sensitivity of detection in retinoblastoma patients. Our study also provides new evidence that will inform patient management and genetic counseling.