Whole Exome Sequencing analysis of constitutional DNA in argentinean Retinoblastoma patients

Abstract

Whole Exome Sequencing analysis of constitutional DNA in argentinean Retinoblastoma patientsParma, Diana; Luce, Leonela; Carcione Micaela; Mazzanti Chiara; Ferrer, Marcela; Giliberto Florencia; Szijan Irena.Retinoblastoma (RB) is a pediatric tumor of the developing retina, caused by mutations in RB1 tumor suppressor gene. RB1 molecular alterations are small mutations in 80% of cases and gross deletions/duplications in 20%. In hereditary RB, identification of mutations is essential for genetic assessment. The purpose of this work was to detect small mutations in RB1 by Whole Exome Sequencing (WES) in 7 argentinean RB patients. WES was performed in leukocytes DNA from 2 familial and 1 sporadic bilateral and 4 early unilateral RB cases. The pathogenicity of the identified variants was determined according to: its presence in RB1 mutation database; its absence in sequence consortiums (ExAC and GnomAD); and predictive softwares. All pathogenic mutations were corroborated by Sanger Sequencing. Gross mutations were screened by MLPA. We have found an average of 13 sequence variants in RB1, 5 of them were present in all patients. We identified the disease causing mutations in 2/7 cases. In one bilateral familial case, a nonsense mutation (g.70286C>A) in exon 12 was detected, the patient´s affected father showed the same mutation. The sporadic bilateral RB presented a substitution (g.5550G>A) in the last nucleotide of exon 2, according to previous reports this variant leads to aberrant splicing. In an unilateral RB patient, a likely benign inframe deletion was observed. The 4 unilateral and the other familial bilateral RB patients did not show pathogenic small mutations nor gross molecular alterations in RB1. Thus, screening was enlarged to genes associated with RB1 pathways and other tumors, but no deleterious variant was identified. We can conclude that WES was able to find constitutional mutations in RB1 gene, allowing confirmation of diagnosis and genetic assessment. Further studies must be performed in patients with no causative mutation found. Finally, the importance of this work relies on the fact that is the first one applying WES for RB molecular diagnosis in Argentina.