Next generation sequencing in research and diagnostics of ocular birth defects
Introduction
The goal of our study was to evaluate the capacity of array based sequence capture target enrichment (SCE) and massively parallel, next generation sequencing (NGS) to successfully identify mutations in candidate genes for the developmental ocular birth defects anophthalmia, microphthalmia and coloboma.
The advent of NGS technologies is expected to transform the practice of medical genetics [1], [2], [3]. With the high throughput and decreased sequencing costs achieved by NGS, it is no longer impossible to sequence hundreds or even thousands of exons and other genomic sequences in an individual with a suspected genetic disease. It is predicted that in the near future NGS might replace array based techniques and Sanger sequencing in their current clinical applications for the detection of mutations [1], [3]. Additionally, NGS provides entirely new research and diagnostic capabilities, including whole genome screening for novel mutations and sequencing biological specimens for the genomic signature of novel infectious agents [4], [5].
NGS could be particularly advantageous in research and testing for genetically heterogeneous hereditary conditions. Common disorders evaluated by clinical geneticists are caused by heterogeneous Mendelian loci and lend themselves to enrichment strategies followed by NGS. Examples include intellectual disability [6], deafness [7], familial cardiomyopathy [8] and retinitis pigmentosa [9]. In these conditions there are often very subtle phenotypic differences between affected patients to guide molecular diagnostics by indicating which gene is likely to be mutated in a particular individual. Current diagnostic evaluation proceeds by sequencing a series of genes, individually or in small sets, based on the relative frequency of the mutations and the sensitivity of available assays. If there is no predominant mutation(s) causing the disease, the pathogenic change often remains unknown even after very extensive and expensive molecular testing. With enrichment strategies followed by NGS, sequencing of all genes implicated in a particular genetic disorder could be performed simultaneously, efficiently and at low cost.
While clearly superior to traditional Sanger sequencing, NGS has had little impact on clinical testing to date. There are very few examples of successful application of NGS in translational research and diagnostics. Clinical testing using NGS is currently offered for Hypertrophic Cardiomyopathy (HCM), Dilated Cardiomyopathy (DCM) and Long QT syndrome (http://www.genedx.com/). NGS has also been explored as a method to perform rapid human leukocyte antigen (HLA) typing for high resolution allele identification [10], [11], and to develop assays for Neurofibromatosis Type 1 [12], autosomal recessive ataxia [13] and mitochondrial disorders caused by mutations in the mitochondrial genome and 362 nuclear genes controlling mitochondrial function [14]. Ng et al. applied targeted sequencing of all coding regions (“exome”) to show the presence of causative mutations in four unrelated individuals with a rare dominantly inherited disorder, Freeman–Sheldon syndrome (FSS) [15] and to discover the gene for a rare recessive disorder of previously unknown cause, Miller disease [16]. Exciting applications have also been described in cancer research, where NGS has been applied in discovering new candidate genes for acute myeloid leukemia [17], [18], glioblastoma multiforme [19] and other malignancies [20].
We describe the first study which showed the feasibility of using genomic enrichment by sequence capture followed by NGS to investigate genetic causes of the ocular birth defects anophthalmia, microphthalmia and coloboma. These eye anomalies are among the most prevalent causes of childhood blindness, affecting annually ∼2 per 10,000 newborns worldwide [21]. Although they can be of different origins, the majority are caused by defects in genes which regulate normal eye development [22], [23], [24], [25]. There is increased evidence that mutations in large numbers (possibly hundreds) of different genes can cause congenital eye malformations, but no single gene is responsible for a high percentage of cases [23], [24], [25]. Anophthalmia, microphthalmia and coloboma therefore represent disorders where simultaneous sequencing of large numbers of candidate genes by NGS is an ideal approach to study genetic causes and demonstrate the feasibility of NGS for clinical diagnostics.
We showed that the combination of array based SCE with re-sequencing on the GS FLX instrument using Titanium chemistry allows concurrent sequencing of more than 100 candidate genes for anophthalmia, microphthalmia and coloboma. However, improvements will be necessary in several areas including accuracy, speed, ease of data analysis and cost to allow successful diagnostic implementation of NGS for simultaneous mutation testing in hundreds of genes in genetically heterogeneous human diseases.
Section snippets
Materials and methods
We tested whether two known sequence changes in the renal-coloboma syndrome (a.k.a. Papillorenal syndrome, OMIM #120330) associated gene PAX2, which were previously characterized by Sanger sequencing, can reliably be detected by NGS. The first variant was a missense change in exon 5, c.527G > C, which resulted in serine to threonine amino-acid change S176T. This base substitution was identified in one of our previous studies in a father of a proband with ocular birth defects, but not in his
Results
We performed target enrichment by array based SCE and sequencing using GS FLX instrument on two DNA samples with known mutations in the PAX2 gene. The sequencing was performed simultaneously for 112 candidate genes for ocular birth defects which were selected based on extensive literature search. A custom SCE array designed for target enrichment contained probes for all coding regions (total of 1017 exons) of the selected 112 genes. The size of the entire target region was 373,083 bp. Since
Discussion
SCE and GS FLX sequencing allowed us to simultaneously sequence 112 candidate genes for ocular birth defects in two independent DNA samples, and to detect two known mutations in the PAX2 gene.
Although whole genome sequencing represents the most comprehensive approach to identifying disease causing mutations in patients with hereditary disorders, such sequencing and analysis is not yet possible [39]. We therefore focused our studies on genes which are known to be associated with ocular birth
Acknowledgments
We gratefully acknowledge Einat Snir and Jennifer Bair from The University of Iowa DNA Facility for technical assistance with GS FLX sequencing and manuscript review, and Eric Cabot from the University of Wisconsin-Madison Biotechnology Center for help with reviewing the sequencing data.
References (45)
- et al.
Keeping up with the next generation: massively parallel sequencing in clinical diagnostics
J. Mol. Diagn.
(2008) - et al.
Massively parallel sequencing: the next big thing in genetic medicine
Am. J. Hum. Genet.
(2009) Genetics of intellectual disability
Curr. Opin. Genet. Dev.
(2008)- et al.
Rapid high-throughput human leukocyte antigen typing by massively parallel pyrosequencing for high-resolution allele identification
Hum. Immunol.
(2009) - et al.
Congenital malformations of the eye and orbit
Otolaryngol. Clin. North Am.
(2007) - et al.
Developmental eye disorders
Curr. Opin. Genet. Dev.
(2005) - et al.
Identification of two single nucleotide polymorphisms in exon 8 of PAX2
Mol. Genet. Metab.
(1999) Enabling technologies of genomic-scale sequence enrichment for targeted high-throughput sequencing
Genomics
(2009)- et al.
Next-generation sequencing: from basic research to diagnostics
Clin. Chem.
(2009) Next-generation sequencing transforms today’s biology
Nat. Methods
(2008)
Next-generation sequencing: applications beyond genomes
Biochem. Soc. Trans.
The genetics of deafness
Ment. Retard. Dev. Disabil. Res. Rev.
Genetic determinants of cardiac hypertrophy
Curr. Opin. Cardiol.
Hereditary retinal disease
Curr. Opin. Ophthalmol.
High-resolution, high-throughput HLA genotyping by next-generation sequencing
Tissue Antigens
DNA sequence capture and enrichment by microarray followed by next-generation sequencing for targeted resequencing: neurofibromatosis type 1 gene as a model
Clin. Chem.
Next generation sequence analysis for mitochondrial disorders
Genome Med.
Targeted capture and massively parallel sequencing of 12 human exomes
Nature
Exome sequencing identifies the cause of a Mendelian disorder
Nat. Genet.
DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome
Nature
Recurring mutations found by sequencing an acute myeloid leukemia genome
N. Engl. J. Med.
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