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Massively parallel sequencing for early molecular diagnosis in Leber congenital amaurosis

Frauke Coppieters UGent, BRAM DE WILDE UGent, Steve Lefever UGent, Ellen De Meester UGent, Nina De Rocker UGent, Caroline Van Cauwenbergh UGent, Filip Pattyn UGent, Françoise Meire, Bart Leroy UGent and JAN HELLEMANS UGent, et al. (2012) GENETICS IN MEDICINE. 14(6). p.576-585
abstract
Purpose: Leber congenital amaurosis (LCA) is a rare congenital retinal dystrophy associated with 16 genes. Recent breakthroughs in LCA gene therapy offer the first prospect of treating inherited blindness, which requires an unequivocal and early molecular diagnosis. While present genetic tests do not address this due to a tremendous genetic heterogeneity, massively parallel sequencing (MPS) strategies might bring a solution. Here, we developed a comprehensive molecular test for LCA based on targeted MPS of all exons of 16 known LCA genes. Methods: We designed a unique and flexible workflow for targeted resequencing of all 236 exons from 16 LCA genes based on quantitative PCR (qPCR) amplicon ligation, shearing, and parallel sequencing of multiple patients on a single lane of a short-read sequencer. Twenty-two prescreened LCA patients were included, five of whom had a known molecular cause. Results: Validation of 107 variations was performed as proof of concept. In addition, the causal genetic defect and a single heterozygous mutation were identified in 3 and 5, respectively, of 17 patients without previously identified mutations. Conclusion: We propose a novel targeted MPS-based approach that is suitable for accurate, fast, and cost-effective early molecular testing in LCA, and easily applicable in other genetic disorders.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
MUTATIONS, DNA-SEQUENCE, VISUAL FUNCTION, MODIFIER ALLELES, RETINAL DYSTROPHIES, GENOTYPING MICROARRAY, GENE-THERAPY, qPCR, molecular diagnosis, massively parallel sequencing, Leber congenital amaurosis, MODEL, PLATFORMS, CAPTURE
journal title
GENETICS IN MEDICINE
Genet. Med.
volume
14
issue
6
pages
576 - 585
Web of Science type
Article
Web of Science id
000305005000002
JCR category
GENETICS & HEREDITY
JCR impact factor
5.56 (2012)
JCR rank
20/161 (2012)
JCR quartile
1 (2012)
ISSN
1098-3600
DOI
10.1038/gim.2011.51
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
2134021
handle
http://hdl.handle.net/1854/LU-2134021
date created
2012-06-06 11:08:16
date last changed
2012-10-03 16:00:57
@article{2134021,
  abstract     = {Purpose: Leber congenital amaurosis (LCA) is a rare congenital retinal dystrophy associated with 16 genes. Recent breakthroughs in LCA gene therapy offer the first prospect of treating inherited blindness, which requires an unequivocal and early molecular diagnosis. While present genetic tests do not address this due to a tremendous genetic heterogeneity, massively parallel sequencing (MPS) strategies might bring a solution. Here, we developed a comprehensive molecular test for LCA based on targeted MPS of all exons of 16 known LCA genes. 
Methods: We designed a unique and flexible workflow for targeted resequencing of all 236 exons from 16 LCA genes based on quantitative PCR (qPCR) amplicon ligation, shearing, and parallel sequencing of multiple patients on a single lane of a short-read sequencer. Twenty-two prescreened LCA patients were included, five of whom had a known molecular cause. 
Results: Validation of 107 variations was performed as proof of concept. In addition, the causal genetic defect and a single heterozygous mutation were identified in 3 and 5, respectively, of 17 patients without previously identified mutations. 
Conclusion: We propose a novel targeted MPS-based approach that is suitable for accurate, fast, and cost-effective early molecular testing in LCA, and easily applicable in other genetic disorders.},
  author       = {Coppieters, Frauke and DE WILDE, BRAM and Lefever, Steve and De Meester, Ellen and De Rocker, Nina and Van Cauwenbergh, Caroline and Pattyn, Filip and Meire, Fran\c{c}oise  and Leroy, Bart and HELLEMANS, JAN and Vandesompele, Jo and De Baere, Elfride},
  issn         = {1098-3600},
  journal      = {GENETICS IN MEDICINE},
  keyword      = {MUTATIONS,DNA-SEQUENCE,VISUAL FUNCTION,MODIFIER ALLELES,RETINAL DYSTROPHIES,GENOTYPING MICROARRAY,GENE-THERAPY,qPCR,molecular diagnosis,massively parallel sequencing,Leber congenital amaurosis,MODEL,PLATFORMS,CAPTURE},
  language     = {eng},
  number       = {6},
  pages        = {576--585},
  title        = {Massively parallel sequencing for early molecular diagnosis in Leber congenital amaurosis},
  url          = {http://dx.doi.org/10.1038/gim.2011.51},
  volume       = {14},
  year         = {2012},
}

Chicago
Coppieters, Frauke, BRAM DE WILDE, Steve Lefever, Ellen De Meester, Nina De Rocker, Caroline Van Cauwenbergh, Filip Pattyn, et al. 2012. “Massively Parallel Sequencing for Early Molecular Diagnosis in Leber Congenital Amaurosis.” Genetics in Medicine 14 (6): 576–585.
APA
Coppieters, F., DE WILDE, B., Lefever, S., De Meester, E., De Rocker, N., Van Cauwenbergh, C., Pattyn, F., et al. (2012). Massively parallel sequencing for early molecular diagnosis in Leber congenital amaurosis. GENETICS IN MEDICINE, 14(6), 576–585.
Vancouver
1.
Coppieters F, DE WILDE B, Lefever S, De Meester E, De Rocker N, Van Cauwenbergh C, et al. Massively parallel sequencing for early molecular diagnosis in Leber congenital amaurosis. GENETICS IN MEDICINE. 2012;14(6):576–85.
MLA
Coppieters, Frauke, BRAM DE WILDE, Steve Lefever, et al. “Massively Parallel Sequencing for Early Molecular Diagnosis in Leber Congenital Amaurosis.” GENETICS IN MEDICINE 14.6 (2012): 576–585. Print.