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Enhanced Sensitivity for Detection of Low‐Level Germline Mosaic RB 1 Mutations in Sporadic Retinoblastoma Cases Using Deep Semiconductor Sequencing
Author(s) -
Chen Zhao,
Moran Kimberly,
RichardsYutz Jennifer,
Toorens Erik,
Gerhart Daniel,
Ganguly Tapan,
Shields Carol L.,
Ganguly Arupa
Publication year - 2014
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.22488
Subject(s) - sanger sequencing , biology , ion semiconductor sequencing , genetics , dna sequencing , deep sequencing , germline mutation , mutation , retinoblastoma , exon , gene , microbiology and biotechnology , genome
Sporadic retinoblastoma ( RB ) is caused by de novo mutations in the RB 1 gene. Often, these mutations are present as mosaic mutations that cannot be detected by S anger sequencing. Next‐generation deep sequencing allows unambiguous detection of the mosaic mutations in lymphocyte DNA . Deep sequencing of the RB 1 gene on lymphocyte DNA from 20 bilateral and 70 unilateral RB cases was performed, where S anger sequencing excluded the presence of mutations. The individual exons of the RB 1 gene from each sample were amplified, pooled, ligated to barcoded adapters, and sequenced using semiconductor sequencing on an I on T orrent P ersonal G enome M achine. Six low‐level mosaic mutations were identified in bilateral RB and four in unilateral RB cases. The incidence of low‐level mosaic mutation was estimated to be 30% and 6%, respectively, in sporadic bilateral and unilateral RB cases, previously classified as mutation negative. The frequency of point mutations detectable in lymphocyte DNA increased from 96% to 97% for bilateral RB and from 13% to 18% for unilateral RB . The use of deep sequencing technology increased the sensitivity of the detection of low‐level germline mosaic mutations in the RB 1 gene. This finding has significant implications for improved clinical diagnosis, genetic counseling, surveillance, and management of RB .