Targeted next generation sequencing of RB1 gene for the molecular diagnosis of Retinoblastoma

Abstract

Background: The spectrum of RB1gene mutations in Retinoblastoma (RB) patients and the necessity of multiple traditional methods for complete variant analysis make the molecular diagnosis a cumbersome, labor-intensive and time-consuming process. Here, we have used targeted next generation sequencing (NGS) approach with in-house analysis pipeline to explore its potential for the molecular diagnosis of RB.

Methods: Thirty-three patients with RB and their family members were selected randomly. DNA from patient blood and/or tumor was used for RB1 gene targeted sequencing. The raw reads were obtained from Illumina Miseq. An in-house bioinformatics pipeline was developed to detect both single nucleotide variants (SNVs) and small insertions/deletions (InDels) and to distinguish between somatic and germline mutations. In addition, ExomeCNV and Cn. MOPS were used to detect copy number variations (CNVs). The pathogenic variants were identified with stringent criteria, and were further confirmed by conventional methods and cosegregation in families.

Results: Using our approach, an array of pathogenic variants including SNVs, InDels and CNVs were detected in 85% of patients. Among the variants detected, 63% were germline and 37% were somatic. Interestingly, nine novel pathogenic variants (33%) were also detected in our study.

Conclusions: We demonstrated for the first time that targeted NGS is an efficient approach for the identification of wide spectrum of pathogenic variants in RB patients. This study is helpful for the molecular diagnosis of RB in a comprehensive and time-efficient manner.

Figure 1

Analysis pipeline to identify pathogenic variants in tumor and blood samples from retinoblastoma patients.

Figure 2

Confirmation of novel pathogenic splice variants. (A) Cosegregation of variants in the family was confirmed by Sanger sequencing of blood samples of Patient RB4, his mother and sibling, who had heterozygous c.265-9 T > A variant that created a new splice site acceptor. (B) Patient RB24 had a de novo heterozygous in-frame deletion of three bases identified at the start site of exon 20. Red arrows denote the variant.

Figure 3

Agarose gel electrophoresis for the confirmation of small deletions. (A) 17 bp deletion in the Promoter region was observed in blood samples of patient RB1 and his father. (B) 29 bp deletion in Exon 1 was observed in blood samples of patient RB11, her mother and siblings. The size of actual and deleted product is indicated by straight and dotted arrows respectively in both gels.

Figure 4

Confirmation of copy number variations (CNVs) by MLPA. (A) Patient RB7 had an affected father and both of them showed deletion of exons 10-12. (B) Patient RB32 had a somatic deletion of exons 7-27 which was not detected in blood. The deletions were denoted by the red spots below the deletion cut-off line (red) in the ratio chart.