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- byO 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-

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©2011 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 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 retinal 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 recessive 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 sequences 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 these 7 variants against the 1000 Ge- (http://samtools.sourceforge.net/); nonsynonymous variants nomes Project Consortium database15 identified 3 com- were labeled using ANNOVAR (http://www.openbioinformatics mon polymorphisms previously observed in normal ge- .org/annovar/). Further filtering was performed (1) with a nomes; this fact led to the elimination of a fourth variant prioritization of genes known to associate with retinal disor- based on a recessive model (eTable 2). The list of can- ders (a list of 177 genes from RetNet, http://www.sph.uth.tmc .edu/retnet/), (2) by the PolyPhen-2 Web resource didate genes was thus reduced to 2: CNGA3 with 2 het- (http://genetics.bwh.harvard.edu/pph2/)14 based on high like- erozygous candidate mutations and TLR3 with 1 homo- lihood of damage (pph2 prob Ͼ0.9) to protein function caused zygous candidate mutation. by a variant, (3) by applying a recessive model, ie, either 2 Literature review indicated that TLR3 (toll-like recep- heterozygous mutations occurring in the same gene or a tor 3, also known as CD283) encodes a member of the

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Reads Covering Reads/ Mutation Mutation, No. Variant Orientation, No. Orientation First amplified library 38 19 C 5 ϩ 14 − c.829CϾT 19 T 3 ϩ 16 − 34 5T 2 ϩ 3− c.1580TϾG 29 G 21 ϩ 8− Second amplified library 28 6C 3 ϩ 3− c.829CϾT 22 T 10 ϩ 12 − 42 31 T 24 ϩ 7− c.1580TϾG 11 G 5 ϩ 6−

Wild-type Wild-type

G A G G T T C A A C C G C C T A C T G A A G A G G G C A A G C T G G C C G T G G T G

c.829C > T p.Arg277Cys c.1580T > G p.Leu527Arg

G A G G T T C A A C N G C C T A C T G A A G A G G G C A A G C N G G C C G T G G T G

Figure 1. Identification of 2 heterozygous mutations in CNGA3 in a patient diagnosed with autosomal recessive achromatopsia determined by direct sequencing of polymerase chain reaction amplicons. Wild-type sequence compared with heterozygous mutant electropherogram sections of exon 7 of CNGA3. Arrows identify the mutation sites.

toll-like receptor family, which plays a fundamental role Table. All of the reads containing the 2 changes map to in pathogen recognition and activation of innate immu- only 1 site. nity. It was identified by association study as a candidate gene whose L412F polymorphism might be protec- CASE HISTORY AND CLINICAL FINDINGS tive in patients with geographic atrophy related to age- related macular degeneration; on this basis, it was included The proband (II:1; Figure 3) presented shortly after birth in RetNet.17-19 Therefore, in this context, TLR3 is not a with nystagmus. It was noted that she was fascinated by retinal disease gene. In contrast, CNGA3 is known to as- bright lights but performed poorly in a bright environ- sociate with achromatopsia, and one of the candidate mu- ment and appeared to see better in the dark. There was no tations, c.829CϾT p.R277C, has previously been re- family history of ophthalmic disease, and the parents were ported.16 Therefore, knowledge about the 2 candidate not related. Examination at 9 months of age revealed ab- genes overwhelmingly supported the hypothesis that the sence of fixation, and she could not recognize faces in the retinal disorder in question is achromatopsia and the pro- light. A small-amplitude, rapid, pendular nystagmus was band carries 2 heterozygous mutations: c.829CϾT present and there was a paradoxical pupillary response. p.R277C and c.1580TϾG p.L527R, a novel CNGA3 mu- Findings of anterior segment, ocular media, and fundus ex- tation (Table, Figure 1, and Figure 2). Sequencing amination were normal. The cycloplegic refraction was coverage at c.829CϾT and c.1580TϾG are shown in the ϩ8.00 dioptrically sphere OU. A clinical diagnosis of achro-

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©2011 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 A 227 I:1 I:2 I R277C/+ L527R/+ NP 001289.1 265KVGTNYPEV-RFNRLLKFSRLFEFFDR 290 XP 001156943.1 265KVGTNYPEV-RFNRLLKFSRLFEFFDR 290 XP 538462.2 250KLGVNYPEL-RFNRLLKFSRLFEFFDR 275 NP 776704.1 281KLGMNYPEL-RFNRLLKLARLFEFFDR 306 II:1 II:2 II:3 NP 034048.1 203KLGVNYPEL-RFNRLLKFSRLFEFFDR 228 R277C/L527R R277C/L527R R277C/+ NP 445947.1 183KLGMNYPEL-RFNRLLRFSRLFEFFDR 208 NP 990552.1 304KLGLNYPEL-RFNRLLRIARLFEFFDR 329 XP 695626.3 276KVGFNNPEL-RFNRLFKMARLFEFFDR 301 Figure 3. Family ACHM-GAWa01. The exome of patient II:1 (proband) was XP 683934.3 293KVGYNNPEL-RFNRLFKMARLFEYFDR 318 massively parallel sequenced. Both II:1 and II:2 are affected and carried the 2 NP 477116.1 208SLYLPCPVIVRLNRLLRINRLWEWFDR 234 mutations, p.R277C and p.L527R, compound-heterozygously. Heterozygous XP 319802.4 203ATRLPCPIIVRINRLLRLPRMWEWFDR 229 carriers of either the R277C allele or the L527R allele were unaffected. Accordingly, 100% concordance of genotype and phenotype was observed.

B 527 her 2 younger brothers (II:2; Figure 3) also has achro- I matopsia. NP 001289.1 514DIGKEMYIINEGKLAVVADDGVTQFVV 540 XP 001156943.1 514DIGKEMYIINEGKLAVVADDGVTQFVV 540 XP 538462.2 502DIGREMYIIKEGKLAVVADDG ITQFVV 528 MUTATION ANALYSIS NP 776704.1 533DIGREMYIIKEGKLAVVAEDGITQFVV 559 AND SEGREGATION ANALYSIS NP 034048.1 455DIGREMYIIKEGKLAVVADDGVTQFVV 481 NP 445947.1 435DIGREMYIIKEGKLAVVADDGVTQFVV 461 NP 990552.1 556DIGREMYIIKEGKLAVVADDGITQFVV 582 The mutations (Table and Figure 1) affect evolution- XP 695626.3 528DIGREMYIIKEGKLAVVADDGITQFVV 554 arily highly conserved amino acid residues in the CNGA3 XP 683934.3 545DIGREMYIIKEGKLAVVADDGVTQFVV 571 protein (Figure 2). In addition, the mutations were ex- NP 477116.1 461DVGKEMYIVKRGKLSVVGDDGITVLAT 487 cluded in 100 healthy European control samples (200 chromosomes) by direct DNA sequencing (data not C shown). Segregation analysis (Figure 3) proved the pres- R277C Pore Extracellular ence of 2 mutant alleles that were inherited indepen- dently and compound-heterozygously. Both the pro-

S 1 S 2 S 3 S 4 S 5 S 6 band (II:1, Figure 3) and her affected brother (II:2, Figure 3) carried the 2 mutations p.R277C and p.L527R, while the unaffected brother (II:3, Figure 3) was a het- Intracellular erozygous carrier of the R277C allele, which was inherited with the paternal allele. The mother was heterozy- L527R COOH NH2 gous for the L527R mutation.

cGMP-binding site COMMENT

Figure 2. Multiple sequence alignment of CNGA3 polypeptides of different species showing the conserved amino acid residues arginine 277 (A) and Achromatopsia (rod monochromacy or total color blind- leucine 527 (B) and their neighboring amino acids, respectively (http://www ness) is a congenital or early-onset retinal disorder with cone .ncbi.nlm.nih.gov/homologene). The mutations affect evolutionarily highly photoreceptor function loss. It is estimated to affect 1 in conserved amino acid residues in CNGA3. The polypeptide sequences are 21 NP_001289.1 CNGA3, Homo sapiens; XP_001156943.1 CNGA3, Pan troglo- 30 000 individuals worldwide. Clinically it is character- dytes; XP_538462.2 CNGA3, Canis lupus familiaris; NP_776704.1 CNGA3, ized by reduced visual acuity, pendular nystagmus, pho- Bos taurus; NP_034048.1 CNGA3, Mus musculus; NP_445947.1 CNGA3, tophobia, small central scotomas, eccentric fixation, and Rattus norvegicus; NP_990552.1 CNGA3, Gallus gallus; XP_695626.3 reduced or complete loss of color discrimination. In ERG LOC567242, Danio rerio; XP_683934.3 LOC556112, Danio rerio; NP_477116.1 Cng, Drosophila melanogaster; and XP_319802.4 recordings, photopic (cone) responses are absent or mark- AgaP_AGAP009050, Anopheles gambiae. C, A schematic of CNGA3 channel edly diminished, whereas scotopic (rod) responses are es- protein (adapted from Zagotta and Siegelbaum20) and the mutation sites are sentially normal and remain stable. Genetically, achroma- shown. Cylinders denote helical structures in the protein; S1 through S6 rep- resent transmembrane helices. The mutations affect amino acid residues topsia is a heterogeneous condition inherited as an located in functionally important domains. autosomal recessive trait. To date, mutations in 4 genes are known to associate with this disorder. The primary biological mechanism underlying achromatopsia was made. This was electrodiagnostically con- matopsia is a defective cone phototransduction cascade firmed with an electroretinogram (ERG) under sedation (photopigment → G-protein transducin → phosphodi- at 18 months of age. Scotopic ERG findings were normal esterase/cGMP → CNG channel; for review, see the ar- but cone responses to flicker were extinguished and a re- ticle by Lamb and Pugh22). The 4 genes known to asso- duced B1 wave amplitude of 45 µV on photopic ERG was ciate with achromatopsia are essential components of this observed. At 10 years of age, visual acuity was 20/200 OD cascade, CNGA313 and CNGB3,23,24 encoding the channel- and 20/200 OS, 20/160 with both eyes open, and 6/60 with forming ␣ and the modulatory ␤ subunit of the cone- both eyes open for a near target. specific cyclic nucleotide (cGMP)–gated cation channel The proband’s parents (both of European descent) had (CNG), respectively; GNAT2 is the gene for the cone- normal findings on ophthalmic examination, while 1 of specific ␣ subunit of the G-protein transducin,25-27 while

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©2011 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 PDE6C encodes the ␣Ј subunit of the cone-specific phos- informatics filtering scheme. Furthermore, the prin- phodiesterase.28,29 In a homologous CNGA3 knockout ciples outlined here apply equally well to retinal disor- mouse model, the number of cone photoreceptors is de- ders, corneal diseases, or other ocular disease subgroups. creased and the remaining cones showed abnormal mor- Selecting variants according to the high likelihood of phology and degenerated; physiologically, measurable damage to protein function has been used successfully in cone function was absent.30 Taken together, results ob- several studies.4 However, there is a graded scale of poten- tained from both animal model and human genotype- tial damage; where to draw the line in the high- phenotype correlations strongly support the notion that throughput screening is not always clear-cut. The same dif- normal CNGA3 function is critical for cone phototrans- ficulty affects filtering by mutation frequency.2,6 No doubt, duction; mutations in the CNGA3 gene that damage better quantitative models are being developed to address CNGA3 protein function lead to achromatopsia. these issues. In the meantime, successful identification of The mutations identified in this study are most likely causative mutations for mendelian disorders depends largely causative because of the expected damage to CNGA3 pro- on the circumstance of each case; furthermore, different tein function and hence disruption of the cone photo- filtering procedures and parameters can lead to the same transduction cascade. Arginine 277 is located in the highly correct mutation identification. conserved S4 transmembrane helix (Figure 2); the R277C From the outset, it is clear that exome sequencing would mutation has been shown by in vitro expression studies fail to identify some disease-causing mutations (eAppen- to result in disrupted channel biogenesis and stability.31 dix). Nevertheless, the odds are in favor of success, espe- The R277C mutation is observed recurrently in patients cially when viewed in the context of a large number of at- with achromatopsia and their families; it has been sug- tempts. When this more restrictive but cost-effective gested that it might be owing to a founder mutation in approach fails, the default would be whole-genome se- patients of European descent.16 Similarly, the novel mu- quencing. Several rare diseases have been analyzed suc- tation L527R affects a highly conserved leucine residue cessfully by this exhaustive and more expensive method, in the cGMP binding domain of the protein (Figure 2), eg, Miller syndrome,32 metachondromatosis,33 severe and an apparent clustering of achromatopsia-causing mu- hypercholesterolemia,34 and Charcot-Marie-Tooth dis- tations in amino acid positions 510 to 529 has been ob- ease.35 Therefore, the new DNA sequencing technology has served.16 Furthermore, another causative mutation that made it feasible, for the first time, to identify the genetic affects L527 and segregates within an unrelated achro- cause of the rarest forms of disorders, with only single or matopsia family has been observed recently (S.K.). very few affected individuals. In summary, (1) clinical examination of the family, (2) normal scotopic ERG but severely compromised phot- Submitted for Publication: December 13, 2010; final re- opic ERG of the proband, (3) mutations deemed to dis- vision received March 29, 2011; accepted April 4, 2011. rupt CNGA3 protein function, and (4) 100% concor- Correspondence: Fulton Wong, PhD, MPP, Depart- dance of genotype and phenotype in this family provide ments of Ophthalmology and Neurobiology, Duke Uni- strong evidence that the 2 compound heterozygous mu- versity School of Medicine, Durham, NC 27710 (fulton tations, p.R277C and p.L527R, are the achromatopsia- [email protected]). causing mutations in this family. Financial Disclosure: None reported. An effective filter used in the present study was limit- Funding/Support: This study was supported by Deutsche ing the search to genes known to associate with retinal dis- Forschungsgesellschaft grant KFO134-Ko2176/1-1 (Dr orders; it streamlined the process to the correct end point. Kohl). For mutations that occur in genes not already known to Online-Only Material: The eAppendix, eTables, and eFig- be associated with retinal disorders, the same filter would ure are available at http://www.archophthalmol.com. have misguided the process away from the causative mu- Additional Contributions: Britta Baumann provided ex- tations. An alternative strategy would be selection of vari- cellent technical assistance and Huixin Siew partici- ants that are private to the family, ie, variants observed in pated in bioinformatics analysis. the proband’s immediate family but perhaps not in the general population. 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Correction

Error in Abstract. In the Ophthalmic Molecular Genet- ics article titled “Prolonged Pursuit by Optokinetic Drum Testing in Asymptomatic Female Carriers of Novel FRMD7 Splice Mutation c.1050 ϩ5GϾA” by Khan et al, published in the July issue of the Archives (2011; 129[7]:936-940), there is an error in the “Results” section of the abstract. The sentence, “A brother and the father—all 3 of whom were asymptomatic—had unremarkable examination findings” should have read, “A brother and the father—both of whom were asymptomatic—had unremarkable examination findings.” This article was corrected online.

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