Sanger Sequencing of Exon 14 of RB1 Gene Among North Indian Children with Retinoblastoma

Abstract

Introduction: The RB1 gene has been studied for decades. A large number of genetic analytical studies on this gene are published in the Western world, but very few studies from India are available online. RB1 is the most critical gene involved in the causation of retinoblastoma, and Exon 14 is one of the highly mutated exons of this gene. Aim: To discover the genetic variants in Exon 14 of the RB1 gene in North Indian retinoblastoma patients. Materials and Methods: This was an inter- and intra-institutional cross-sectional observational study. The Department of Anatomy, Ophthalmology, and Paediatrics at King George’s Medical University UP, Lucknow, collaborated with the Department of Biochemistry, ERA University, Lucknow. After obtaining written informed consent from their parents or legal guardians, 40 clinically and radiologically diagnosed retinoblastoma patients were included as study participants. The age range was 12-84 months. 2 ml of peripheral venous blood was withdrawn from 29 patients, and we obtained tumour tissue from 14 patients. We isolated the genomic DNA, which was amplified by PCR, and the amplified products were sent for Sanger sequencing. Chromatograms were analyzed using the BioEdit software. Results: Most patients (92.5%) were below five years of age. Male preponderance was seen, as there were 37.5% females and 62.5% males. In the majority of cases (72.5%), the tumor was seen unilaterally with predominant involvement of the right eye; while in 27.5% of cases, both eyes were seen affected. We did not observe any variations in the sequences of exon 14. Conclusions: As the RB1 gene has 27 exons, therefore screening for mutations in all exons is required to reach a concrete conclusion. Genetic variants might be located in the intronic regions, which may affect the proper functioning of the RB1 gene; therefore, sequencing of the entire gene will be more helpful. Next Generation Sequencing (NGS) is preferred because of the large size of the gene. KEYWORDS: Retinoblastoma, DNA, PCR, RB1 gene, sequencing.