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Identification of Variants in CNGA3 As Cause for Achromatopsia by Exome Sequencing of a Single Patient

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Identification of Variants in CNGA3 As Cause for Achromatopsia by Exome Sequencing of a Single Patient OPHTHALMIC MOLECULAR GENETICS SECTION EDITOR: JANEY L. WIGGS, MD, PhD Identification of Variants in CNGA3 as Cause for Achromatopsia by Exome Sequencing of a Single Patient Kevin Lam, MSc; Haiyan Guo, PhD; Graham A. Wilson, MBChB; Susanne Kohl, PhD; Fulton Wong, PhD, MPP Objective: To report disease-causing mutations in the Results: Analysis yielded a molecular diagnosis of achro- cyclic nucleotide-gated channel ␣ 3 gene (CNGA3) iden- matopsia. Two compound heterozygous mutations were tified by exome sequencing and bioinformatics filtering identified in CNGA3 of this patient, c.829CϾT p.R277C in a single patient. and c.1580TϾG p.L527R; they were not observed in the normal population and cosegregated with the pheno- Methods: The entire protein-coding sequence of a pa- type of achromatopsia in the patient’s family. tient with a retinal disease was enriched by in-solution targeted capture and massively parallel sequenced at 50- Conclusion: These mutations are the cause of achro- fold coverage. The assembled sequence was compared matopsia in this family. with databases of normal genomic sequences to identify Clinical Relevance: The key advantages of massively nonsynonymous variants, which were further filtered (1) parallel sequencing over linkage mapping and cloning with a prioritization of genes associated with retinal dis- are highlighted by (1) the small sample size required for eases, (2) according to the likelihood of variant damage successful analysis and (2) the rapid and high- to protein function, (3) following the predictions of a re- throughput manner in which the mutations are identi- cessive model, and (4) against common polymorphisms fied. This new tool will likely have major effects on the observed in normal genomes. Clinical evaluation and seg- management and research of rare genetic eye diseases in regation analysis of the mutant alleles in the patient’s fam- the new era of personalized genomic medicine. ily were performed; mutations were excluded in healthy controls. Arch Ophthalmol. 2011;129(9):1212-1217 VER THE LAST QUARTER within the human genome for sequenc- century, advances in ing with the same depth of coverage as ophthalmic molecular whole-genome sequencing and at a much genetics have been lower cost.3,4 The rationale for this ap- driven to a large extent proach is that, while the protein-coding re- Oby new DNA technology. Technological gions compose only 1% of the genome, it advances in DNA sequencing and widely is in this portion that 85% of disease- Author Affiliations: AITBiotech accessible databases of annotated human causing mutations occur.5 Thus, mas- Pte Ltd, Singapore (Mr Lam and genomic sequences are creating a new sively parallel sequencing of this 1% of hu- Dr Guo); Department of paradigm for the study of rare ocular ge- man DNA (approximately 30 million bases Ophthalmology, Dunedin School of Medicine, University netic diseases. The transforming technol- in length) is a cost-effective and efficient of Otago, Dunedin, New ogy is massively parallel sequencing of the way to identify pathogenic mutations of Zealand (Dr Wilson); Molecular entire genome of individual persons, which mendelian diseases. Several recent ar- Genetics Laboratory, Institute is also known as next- or second- ticles have described the successful appli- for Ophthalmic Research, generation sequencing.1 While the high cation of this method, using for analysis Department of Ophthalmology, cost of this new technology is preventing DNA from only a few subjects.3,5-12 University Tuebingen, full realization of its effects at this time, a To apply whole-exome sequencing and Tuebingen, Germany (Dr Kohl); slightly restrictive application is making bioinformatics filtering to the study of rare and Departments of Ophthalmology and substantial headway in the analysis of men- genetic eye disorders, we sequenced the 2 Neurobiology, Duke University delian disorders. The new method tar- exome of a single patient. As expected,ap- School of Medicine, Durham, gets and captures the 180 000 exons (col- proximately15 000 genetic variants were North Carolina (Dr Wong). lectively called the exome) dispersed identified in this individual’s exome se- ARCH OPHTHALMOL / VOL 129 (NO. 9), SEP 2011 WWW.ARCHOPHTHALMOL.COM 1212 ©2011 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 quence. By applying a few general screening criteria (fil- homozygous mutation, and (4) by removing common poly- ters) in the subsequent bioinformatics analysis to sepa- morphisms observed in a pool of normal genomes rate the background polymorphisms from disease- (http://browser.1000genomes.org/index.html).15 Reads gener- causing mutations, we aimed to make an accurate ated from the second amplified library were assembled in the molecular diagnosis. The only clinical information used same manner as described, and the coverage at the 2 mutation sites was determined. for bioinformatics filtering was that the patient has a reti- nal disorder. In our study, this general filtering scheme quickly identified 2 heterozygous mutations in the CNGA3 MUTATION AND SEGREGATION ANALYSES gene, which is known to associate with autosomal re- cessive achromatopsia.13 Exon 7 and flanking intronic sequences of the CNGA3 gene were amplified by polymerase chain reaction with genomic DNA as tem- Ј METHODS plates (primer sequences: forward 5 -TCAGAGTGCATTTCCT- GTAGT-3Ј and reverse 5Ј-GCTTTCAAAGGGTGAGTAGA-3Ј).16 Amplicons were sequenced using the ABI PRISM Big Dye Ter- FRAGMENT LIBRARY CONSTRUCTION minator Cycle Sequencing Ready Reaction Kit (PerkinElmer Ap- plied Biosystems, Foster City, California) and separated on an ABI Genomic DNA was isolated from the blood sample using the PRISM 3100 Genetic Analyzer (Applied Biosystems). Sequenc- Gentra Puregene blood kit (Qiagen, Valencia, California). A frag- ing primers were the following: for c.829CϾT p.R277C 5Ј- ment library of genomic DNA was prepared by using the SOLiD GCATACTGTGTAGCCGTGAGG-3Ј and for c.1580TϾG fragment library construction kit (Life Technologies, Carls- p.L527R 5Ј-GTGGGCAATGTGGGCTCC-3Ј. Editing, se- bad, California). In brief, 3 µg of genomic DNA was frag- quence alignment, and mutation detection were performed ap- mented into lengths ranging from 100 to 150 base pairs (bp) plying the Lasergene Software package (DNASTAR; Lasergene, using a Covaris S2 System (Covaris, Woburn, Massachusetts). London, England). After end repair, the DNA was ligated with P1 and P2 adaptors Segregation analysis by DNA sequencing for the presence from the SureSelect AB adaptor kit (Agilent, Santa Clara, Cali- and independent inheritance of 2 mutant alleles was per- fornia). Size selection of 150- to 200-bp DNA fragments was formed in all family members. Mutations were excluded in 100 performed on a SOLiD library size selection gel (Life Tech- healthy (European descent) control samples (200 chromo- nologies), followed by nick translation and 12-cycle poly- somes) by direct DNA sequencing. merase chain reaction amplification. The quantity and quality of the amplified fragment library was assessed by an Agilent 2100 bioanalyzer before enrichment. RESULTS TARGETED CAPTURE AND EXOME SEQUENCING BIOINFORMATICS ANALYSIS From the prepared fragment library, 500 ng was enriched for From the captured library, 91 346 458 fifty-bp SOLiD exons by using the SureSelect human all-exon kit (version 1; reads were generated. Of these, 57 330 833 reads (62.7%) Agilent). The kit was designed to enrich for all the coding se- quences covering a total of 38 megabases in length. In brief, had at least 1 reported alignment to human genome hg18; the prepared DNA was in-solution hybridized with SureSelect 56.19% were on target, and 84.30% mapped to only 1 biotinylated RNA baits for 24 hours at 65°C, and the captured site. The mapped readings provided 49.77-fold cover- DNA-RNA hybrid library was purified by streptavidin-coated age (range, 0-5705ϫ) for the targeted exon sequences; magnetic beads. After digestion of the RNA baits, the captured 80.81% of bases were covered at more than 7-fold depth DNA library was further amplified and used for emulsion poly- (eFigure; http://www.archophthalmol.com). Using SAM- merase chain reaction according to the manufacturer’s instruc- tools and a cutoff equal to or greater than 20 of the Phred- tions (Life Technologies), based on a library concentration of like quality score, 62 518 variants were called; 15 292 were 0.5pM. The amplified library was then sequenced as single- exonic variants. Of these, ANNOVAR identified 7680 non- end 50-bp reads on a SOLiD 3 plus system (Life Technolo- synonymous variants; 3533 were not in dbSNP130. The gies). The sample was run on 1 quad of a SOLiD sequencing slide (Life Technologies). A second amplified library was ob- types of variants identified, including indels and splice- tained from the same captured library and sequenced indepen- site variants, are summarized in eTable 1. Further filter- dently. ing with a prioritization of genes known to associate with retinal disorders reduced the list to 102 variants associ- BIOINFORMATICS ANALYSIS ated with 51 genes. Selecting variants on the basis of pre- dicted damage to protein function by PolyPhen-2 re- The 50-bp SOLiD reads were mapped to human genome hg18 sulted in a list of 13 genes and 15 variants. By applying a in color space using Bowtie version 0.12.3 (http://bowtie-bio recessive model, this list was shortened to 5 genes and 7 .sourceforge.net). Genetic variants were called using SAMtools variants. Screening
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