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

BMC Cancer

Bharanidharan Devarajan²  Logambiga Prakash²  Thirumalai Raj Kannan¹  Aloysius A Abraham¹  Usha Kim⁴  Veerappan Muthukkaruppan³  Ayyasamy Vanniarajan¹ 

[1] Department of Molecular Genetics, Aravind Medical Research Foundation, Madurai, India

[2] Department of Bioinformatics, Aravind Medical Research Foundation, Madurai, India

[3] Advisor-Research, Aravind Medical Research Foundation, Madurai, India

[4] Department of Orbit, Oculoplasty and Oncology, Aravind Eye Hospital, Madurai, India

关键词: Molecular diagnosis;    Targeted next generation sequencing;    Retinoblastoma;   

Others  :  1177488
DOI  :  10.1186/s12885-015-1340-8

 received in 2014-11-21, accepted in 2015-04-22,  发布年份 2015

【 摘 要 】

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.

【 授权许可】

   
2015 Devarajan et al.; licensee BioMed Central.

【 图 表 】

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