Identification of novel genes by targeted exome sequencing in Retinoblastoma

ABSTRACT

Background

Retinoblastoma (RB) is initiated by mutation in both alleles of RB1 gene. However, few cases may occur even in the absence of RB1 mutation suggesting the role of genes other than RB1.

Methodology

The current study was planned to utilize targeted exome sequencing in Indian RB patients affected with unilateral non-familial RB. 75 unilateral RB patients below 5 years of age were enrolled. Genomic DNA was extracted from blood and tumor tissue. From peripheral blood DNA, all coding and exon/intron regions were amplified using PCR and direct sequencing. Cases which did not harbor pathogenic variants in peripheral blood DNA were further screened for mutations in their tumor tissue DNA using targeted exome sequencing. Three pathogenicity prediction tools (Mutation Taster, SIFT, and PolyPhen-2) were used to determine the pathogenicity of non-synonymous variations. An in-house bioinformatics pipeline was devised for the mutation screening by targeted exome sequencing. Protein modeling studies were also done to predict the effect of the mutations on the protein structure and function.

Results

Using the mentioned approach, we found two novel variants (g.69673_69674insT and g.48373314C>A) in RB1 gene in peripheral blood DNA. We also found novel variants in eight genes (RB1, ACAD11, GPR151, KCNA1, OTOR, SOX30, ARL11, and MYCT1) that may be associated with RB pathogenesis.

Conclusion

The present study expands our current knowledge regarding the genomic landscape of RB and also highlights the importance of NGS technologies to detect genes and novel variants that may play an important role in cancer initiation, progression, and prognosis.

KEYWORDS:

• Retinoblastoma
• next generation sequencing
• sporadic
• pathophysiology
• non-synonymous variations

Acknowledgments

We are indebted to the patients and families who took part in this study.

Author contributions

Conceptualization, Shilpa Bisht, Bhavna Chawla and Rima Dada; Data curation, Rima Dada; Formal analysis, Shilpa Bisht; Funding acquisition, Rima Dada; Investigation, Rima Dada; Methodology, Shilpa Bisht, Bhavna Chawla and Rima Dada; Project administration, Rima Dada; Resources, Rima Dada; Software, Amit Kumar, Viswanathan Vijayan, Manoj Kumar and Pradeep Sharma; Supervision, Bhavna Chawla and Rima Dada; Visualization, Shilpa Bisht, Bhavna Chawla and Rima Dada; Writing—original draft, Shilpa Bisht, Bhavna Chawla and Rima Dada; Writing—review & editing, Shilpa Bisht, Bhavna Chawla, Amit Kumar, Viswanathan Vijayan, Manoj Kumar, Pradeep Sharma and Rima Dada.

Institutional review board statement

This study was conducted according to the guidelines of the Declaration of Helsinki. Ethical approval for this work was obtained from the Institute’s Ethics Committee (Ref. No. IESC/T-319/23.06.2015).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/13816810.2022.2106497

Additional information

Funding

This research was funded by Indian Council of Medical Research, New Delhi, India (5/10/FR/15/2019-RBMCH) to R.D. S.B. is funded by Council of Scientific & Industrial Research (Human Resource Development Group), New Delhi, India, in the form of a Senior Research Fellowship. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.