Targeted next generation sequencing of RB1 gene for the molecular diagnosis of Retinoblastoma.
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.
Department of Bioinformatics, Aravind Medical Research Foundation, Madurai, India. bharanid@gmail.com.
Bharanidharan
B
Devarajan
Department of Bioinformatics, Aravind Medical Research Foundation, Madurai, India. loga2719@gmail.com.
Logambiga
L
Prakash
Department of Molecular Genetics, Aravind Medical Research Foundation, Madurai, India. nigira85@gmail.com.
Thirumalai Raj
TR
Kannan
Department of Molecular Genetics, Aravind Medical Research Foundation, Madurai, India. aaloci@gmail.com.
Aloysius A
AA
Abraham
Department of Orbit, Oculoplasty and Oncology, Aravind Eye Hospital, Madurai, India. usha@aravind.org.
Usha
U
Kim
Advisor-Research, Aravind Medical Research Foundation, Madurai, India. muthu@aravind.org.
Veerappan
V
Muthukkaruppan
Department of Molecular Genetics, Aravind Medical Research Foundation, Madurai, India. vanniarajan@aravind.org.
Ayyasamy
A
Vanniarajan
D002648:Child; D002675:Child, Preschool; D019295:Computational Biology; D056915:DNA Copy Number Variations / Q000235:genetics*; D005260:Female; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D009154:Mutation; D057089:Pathology, Molecular; D012175:Retinoblastoma / Q000235:genetics* / Q000473:pathology; D016160:Retinoblastoma Protein / Q000235:genetics*
D016160:Retinoblastoma Protein