Free
Biochemistry and Molecular Biology  |   April 2014
Whole Exome Sequencing in Thai Patients With Retinitis Pigmentosa Reveals Novel Mutations in Six Genes
Author Affiliations & Notes
  • Worapoj Jinda
    Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
  • Todd D. Taylor
    Laboratory for Integrated Bioinformatics, Core for Precise Measuring and Modeling, RIKEN Center for Integrative Medical Sciences, Tsurumi-ku, Yokohama, Kanagawa, Japan
  • Yutaka Suzuki
    Department of Medical Genome Sciences, The University of Tokyo, Kashiwa, Chiba, Japan
  • Wanna Thongnoppakhun
    Division of Molecular Genetics, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
  • Chanin Limwongse
    Division of Molecular Genetics, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
    Division of Medical Genetics, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
  • Patcharee Lertrit
    Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
  • Prapat Suriyaphol
    Bioinformatics and Data Management for Research Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
  • Adisak Trinavarat
    Department of Ophthalmology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
  • La-ongsri Atchaneeyasakul
    Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
    Department of Ophthalmology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
  • Correspondence: La-ongsri Atchaneeyasakul, Department of Ophthalmology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand; atchanee@hotmail.com
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 2259-2268. doi:10.1167/iovs.13-13567
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Worapoj Jinda, Todd D. Taylor, Yutaka Suzuki, Wanna Thongnoppakhun, Chanin Limwongse, Patcharee Lertrit, Prapat Suriyaphol, Adisak Trinavarat, La-ongsri Atchaneeyasakul; Whole Exome Sequencing in Thai Patients With Retinitis Pigmentosa Reveals Novel Mutations in Six Genes. Invest. Ophthalmol. Vis. Sci. 2014;55(4):2259-2268. doi: 10.1167/iovs.13-13567.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose.: To identify disease-causing mutations and describe genotype–phenotype correlations in Thai patients with nonsyndromic retinitis pigmentosa (RP).

Methods.: Whole exome sequencing was performed in 20 unrelated patients. Eighty-six genes associated with RP, Leber congenital amaurosis, and cone-rod dystrophy were analyzed for variant detection.

Results.: Seventeen variants (13 novel and 4 known) in 13 genes were identified in 11 patients. These variants include 10 missense substitutions, 2 nonsense mutations, 3 deletions, 1 insertion, and 1 splice site change. Nine patients with identified inheritance patterns carried a total of 10 potentially pathogenic mutations located in genes CRB1, C8orf37, EYS, PROM1, RP2, and USH2A. Three of the nine patients also demonstrated additional heterozygous variants in genes ABCA4, GUCY2D, RD3, ROM1, and TULP1. In addition, two patients carried variants of uncertain significance in genes FSCN2 and NR2E3. The RP phenotypes of our patients were consistent with previous reports.

Conclusions.: This is the first report of mutations in Thai RP patients. These findings are useful for genotype–phenotype comparisons among different ethnic groups.

Introduction
Retinitis pigmentosa (RP) is the most commonly inherited retinal disease and is both clinically and genetically heterogeneous. The worldwide prevalence of RP is approximately 1 in 3500 to 5000. 1 The severity and age of onset of RP can vary among patients. Night blindness is usually the first symptom during adolescence with subsequent peripheral visual field loss occurring in young adulthood. In the later stages of disease, patients start to lose their central vision, usually between the ages of 40 to 60. 2 Retinitis pigmentosa presents different modes of inheritance including autosomal recessive (arRP), autosomal dominant (adRP), X-linked recessive (xlRP), and at least two rare forms, digenic and mitochondrial trait. 1  
Mutations in at least 57 associated genes distributed among all RP modes of inheritance have been identified (The Retinal Information Network [RetNet]). The proteins encoded by these genes are involved in a variety of functions, including the phototransduction cascade, vitamin A metabolism, photoreceptor structural and cytoskeletal formation, cell-to-cell signaling or synaptic interaction, RNA intron-splicing factors, intracellular protein trafficking, pH regulation, and phagocytosis. 2  
There are many factors that contribute to the complexity of RP. Different mutations in the same RP-associated gene can cause different diseases. Furthermore, clinical variability, which may be due to modifier genes, has been observed in different affected individuals both in inter- and intrafamilial cases with the same mutation. 3,4 Although many RP-associated genes have been reported, mutation screening of these genes fails to detect the disease-causing mutations in approximately 40% of RP patients. 1 Due to genetic heterogeneity and the fact that no clear genotype-phenotype correlations have been described, sequence analysis of the entire coding region for each candidate gene is the most commonly used molecular genetic test for RP. However, this method is laborious, time-consuming, and expensive. Recent developments in next-generation sequencing (NGS) represent an efficient, time-saving, and cost-effective tool for variant detection in affected individuals. 5 To date, NGS has been successfully applied to identify mutations in genes known to be associated with RP 616 and has also led to the discovery of several novel candidate RP genes. 8,1113  
In this study, we present the first mutation analysis of 20 unrelated Thai patients with nonsyndromic RP using whole exome sequencing (WES). Seventeen different variants in 13 genes were identified in 11 patients. We also describe the genotype–phenotype correlation of each patient. 
Methods
Clinical Data and Sample Collection
Twenty unrelated Thai patients with nonsyndromic RP were evaluated at the Division of Ophthalmology, Siriraj Hospital, Bangkok. The ophthalmological examination included best-corrected visual acuity (BCVA) using Snellen chart, slit-lamp biomicroscopy, dilated fundus examination, optical coherence tomography (OCT, when available), full-field electroretinogram (ERG), and visual evoked potential (VEP). All patients were of Thai ancestry ranging in age from 5 to 69 years. This study was approved by the Institutional Review Board of Siriraj Hospital Mahidol University, being adherent to the Declaration of Helsinki. Peripheral blood samples were collected from all participants after informed consent was obtained. Deoxyribonucleic acid was extracted from leukocytes according to the salting-out method. 17 Ethnically matched, unrelated healthy individuals were used for normal control screening. 
Whole Exome Sequencing (WES)
Exome capture was performed using solution hybrid selection with a commercial enrichment kit (SureSelectXT Human All Exon Automated Target Enrichment Kit; Agilent Technologies, Santa Clara, CA, USA) for Illumina paired-end multiplexed sequencing. Exome capture libraries of 2 × 100 bp paired-end reads were sequenced on an array scanner (Illumina HiSequation 2000; Illumina, Inc., San Diego, CA, USA) at the Department of Medical Genome Sciences, The University of Tokyo. Three DNA samples were pooled on each lane of the flow cell for sequencing. 
Sequence Data Alignment, Variant Calling and Identification
The Illumina paired-end DNA sequence data were mapped and aligned to the reference human genome NCBI Build 37 (hg19), using the Burrows-Wheeler Aligner (BWA) program. A set of utilities (Sequence Alignment/Map [SAMtools], a tab-delimited text file that contains sequence alignment data; Wellcome Trust Sanger Institute, Hinxton, UK) was used to convert the alignments from SAM to BAM files (the binary version of a SAM file), sort and merge the alignments, remove PCR duplicates, and index the alignment data. A software package (The Genome Analysis Toolkit [GATK]; Broad Institute, Cambridge, MA, USA) was used for single nucleotide polymorphism (SNP) and indel calling. Visualization and filtration of the sequence variations were performed using a high-performance viewer (Integrative Genomics Viewer [IGV]; Broad Institute) in comparison with SNP information (common, flagged, and multiple-location mapped SNPs) from the dbSNP137 database. 
Identification of Variants
Due to the overlap of clinical features and RP-associated genes with other inherited retinal diseases, 86 genes associated with RP, Leber congenital amaurosis (LCA), and cone-rod dystrophy (CRD) were analyzed for variant detection (Supplementary Fig. S1). 
Filtration and Prioritization of Variants
To distinguish between pathogenic and nonpathogenic variants, the annotated sequencing data were filtered and prioritized based on the following criteria: (1) variant located in coding sequence; (2) nonsynonymous variant; (3) nonsense variant; (4) short insertion or deletion variant (indel) in coding sequence; (5) variant located at splice donor or acceptor site, and (6) variant reported in flagged SNPs but not in common or multiple-location mapped SNPs of the dbSNP137 database. 
In Silico Prediction of Variants
Six computational tools were used to predict the possible effects of the identified amino acid substitutions on protein function: PolyPhen-2, (SIFT), VarioWatch, MutationTaster, Prediction of Pathological Mutations (PMut), and Screening for Non-Acceptable Polymorphisms (SNAP). Only the following outputs were considered as deleterious variants: PolyPhen-2 (probably or possibly damaging); SIFT (damaging); VarioWatch (very high or high); MutationTaster (disease causing); PMut (pathological); and SNAP (non-neutral). For estimating evolutionary conservation, multiple sequence alignments of the affected amino acids in different vertebrate species were performed using sequence alignment program (ClustalW; The Biology Workbench, San Diego, CA, USA). 
Variant Validation
The workflow for variant validation is shown in Supplementary Figure S2. Variants were selected for confirmation by Sanger sequencing when three or more of the prediction outputs from the six computational prediction tools suggested that a variant was deleterious. The primer sequencing program (Primer3; University of Massachusetts, Boston, MA, USA) was used for designing amplicon-specific primers to amplify the regions containing each variant. The PCR products were treated with a reagent (ExoSAP-IT; Affymetrix, Santa Clara, CA, USA) before processing for direct sequencing. Sequencing was performed using a cycle-sequencing kits (ABI BigDye Terminator v3.1; Applied Biosystems, Grand Island, NY, USA) on an analyzer (ABI 3130xl Genetic Analyzer; Applied Biosystems). To verify the pathogenicity of novel variants identified in this study, denaturing HPLC (Transgenomic, Inc., San Jose, CA, USA) analysis was performed for detecting each variant on at least 110 DNA samples (220 chromosomes) from unrelated healthy individuals. 
Segregation Analysis
To assess variant segregation within each patient's family, Sanger sequencing was also performed on the family members from whom DNA was available. 
Protein Structure and Functional Prediction
The protein structures and functions resulting from missense variants identified in this study were predicted using the freely available web service HOPE (Have Your Protein Explained). 18  
Results
Twenty unrelated Thai patients were recruited for this pilot study. Most patients experienced poor night vision. The BCVA ranged from hand motion (HM) to 6/6. The OCT was performed in two patients (RP023 and RP069), which demonstrated no evidence of cystoid macular edema. A summary of clinical information and family pedigrees are shown in Table 1 and the Figure, respectively. The average read depth of WES observed in the 20 samples was approximately 150. The putative deleterious variants were detected in 11 out of 20 patients (55%). Nine of them carried a total of 10 potentially pathogenic mutations and five additional heterozygous variants, while the other two having two variants of uncertain significance (VUS). 
Figure
 
Pedigrees and genotypes of the RP families. Genotype data are presented below the patient and members, where applicable, of their family. Filled symbols with an arrow indicate the probands. Squares: males. Circles: females. Slashed: decreased family members. Double line: consanguineous marriage. Filled black circle: obligate carrier. Normal alleles are indicated by ‘+'. Potentially pathogenic mutations, additional variants, and variants of uncertain significance, are indicated by “M,” “V,” and “VUS,” respectively.
Figure
 
Pedigrees and genotypes of the RP families. Genotype data are presented below the patient and members, where applicable, of their family. Filled symbols with an arrow indicate the probands. Squares: males. Circles: females. Slashed: decreased family members. Double line: consanguineous marriage. Filled black circle: obligate carrier. Normal alleles are indicated by ‘+'. Potentially pathogenic mutations, additional variants, and variants of uncertain significance, are indicated by “M,” “V,” and “VUS,” respectively.
Table 1
 
Clinical Information for Patients in this Study
Table 1
 
Clinical Information for Patients in this Study
Sample Sex Age Presenting Symptoms BCVA ERG VEP Fundus Examination
Onset At Exam RE LE RE LE RE LE
RP009 M 27 30 Poor night vision; flashing 6/6 6/9 NR NR Normal Normal Pink optic disc; generalized RPE changes without macular involvement; moderate bone spicules
RP010 F 60 69 Progressive visual loss 6/24 NLP NR NR Normal NR Pale optic disc LE > RE; heavy bone spicules 360 degrees
RP011 F 19 29 Poor night vision 6/9 6/9 NR NR Normal Normal Mildly pale optic disc; generalized RPE changes without macular involvement; mild bone spicules
RP016 M 26 36 Progressive visual loss; poor night vision CF CF NR NR Moderately decreased amplitude Moderately decreased amplitude Pale optic disc; generalized RPE changes; macular hyperpigmentation; bone spicules in macula only
RP019 F 47 49 Progressive visual loss; poor night vision; poor color discrimination 6/24 6/18 NR NR Normal Normal Pale optic disc; moderate bone spicules in mid periphery
RP022 F 11 11 Blurred vision since birth; photophobia; poor night vision 6/60 6/60 NR NR Normal Normal Pale optic disc; heavy bone spicules 360 degree; macular RPE changes
RP023* M 25 33 Progressive visual loss; poor night vision; tearing; photosensitivity 6/60 6/18 NR NR Moderately decreased amplitude Moderately decreased amplitude Pale optic disc; heavy bone spicules 360 degrees; macular RPE changes
RP027 M 50 51 Poor night vision 6/36 6/24 NR NR Moderately decreased amplitude Moderately decreased amplitude Pink optic disc; RPE changes in midperiphery without macular involvement; no bone spicules
RP038 M 18 48 Progressive visual loss; flashing HM CF NR NR Markedly decreased amplitude Markedly decreased amplitude Pale optic disc; generalized RPE changes with macular involvement; minimal bone spicules
RP069* M 19 23 Progressive visual loss; poor night vision HM HM NR NR Markedly decreased amplitude Markedly decreased amplitude Pale optic disc; generalized RPE changes with macular sheen; no bone spicules
RP087/1* M 3 5 Blurred distant vision; poor night vision; high myopia 6/36 6/60 NR NR Normal Normal Pink disc; generalized RPE changes without macular involvement; no bone spicules
RP087/2* M 5 10 Blurred distant vision; poor night vision; mild myopia with high astigmatism 6/60 6/60 NR NR Normal Normal Pink disc; generalized RPE changes without macular involvement; no bone spicules
Potentially Pathogenic Mutations
Ten different putative, disease-causing mutations, eight novel and two known, were identified in nine patients (Table 2, Supplementary Table S1). These include five autosomal recessive genes (CRB1, C8orf37, EYS, PROM1, and USH2A) and one X-linked recessive gene (RP2). These mutations include five missense substitutions, two nonsense mutations, two deletions, and one splice site change. None of the novel missense variants were detected in at least 220 alleles from the unrelated normal controls. 
Table 2
 
Variants Identified in the RP Patients
Table 2
 
Variants Identified in the RP Patients
Sample MOI in Family Gene MOI of Gene Variant Detected Reference
Chr Exon Nucleotide Change Amino Acid Change State Domain
Potentially pathogenic mutations
 RP009 Isolated RP USH2A arRP  1  4 c.773A>C p.Gln258Pro Het Novel
USH2A  1 22 c.4732C>T p.Arg1578Cys Het Laminin G-like 1 Reported19
 RP011 Isolated RP EYS arRP  6  8 c.1260_1260delG p.Asn421Metfs*8 Het Novel
EYS  6 31 c.6416G>A p.Cys2139Tyr Het EGF-like 21 Reported31
 RP019 Isolated RP EYS arRP  6 31 c.6416G>A p.Cys2139Tyr Hom EGF-like 21 Reported31
 RP022 Isolated RP CRB1 arRP and arLCA  1  9 c.3442T>C p.Cys1148Arg Hom EGF-like 15 Novel
 RP023 arRP CRB1 arRP and arLCA  1  7 c.2539T>A p.Phe847Ile Hom Laminin G-like 2 Novel
 RP027 Isolated RP EYS arRP  6 42 c.8107G>T p.Glu2703Ter Hom Novel
 RP038 Isolated RP C8orf37 arRP and arCRD  8 Intron 2 c.243+2T>C splice site change Hom Novel
 RP069 arRP PROM1 arRP and adCRD  4  4 c.442A>T p.Lys148Ter Hom Cytoplasmic Novel
 RP087 xlRP RP2 xlRP X  2 c.395_419delCCACTCAA p.Thr133Glnfs*15 Hem C-CAP/cofactor C-like Novel
CCCATCATTGAGTCTTC
Additional heterozygous variants
 RP009 Isolated RP ABCA4 arRP and arCRD  1  2 c.71G>A p.Arg24His Het Transmembrane Reported20
RD3 arLCA  1  2 c.94_95delG p.Glu32Serfs*2 Het Coiled coil Novel
 RP022 Isolated RP GUCY2D adCRD and arLCA 17  4 c.1138C>T p.Arg380Cys Het Extracellular Novel
 RP038 Isolated RP ROM1 adRP 11  1 c.339_340insG p.Leu114Alafs*18 Het Transmembrane Reported22–24
TULP1 arRP and arLCA  6  1 c.43G>A p.Asp15Asn Het Novel
Variants of uncertain significance
 RP010 Isolated RP NR2E3 arRP and adRP 15  4 c.424C>T p.Arg142Trp Het Novel
 RP016 Isolated RP FSCN2 adRP 17  4 c.1264C>T p.Arg422Trp Het Fascin-2 Novel
Additional Heterozygous Variants
Among the nine patients with potentially pathogenic mutations, three (RP009, RP022, and RP038 probands) also carried a heterozygous variant in one of five genes (ABCA4, GUCY2D, RD3, ROM1, and TULP1; Fig., Table 2). 
The RP009 proband carried four variants in three genes (USH2A, ABCA4, and RD3) (Fig., Tables 2, 3, Supplementary Fig. S3). A compound heterozygous mutation of a known variant, c.4732C>T (p.Arg1578Cys), and a novel variant, c.773A>C (p.Gln258Pro), were identified in USH2A. The variant p.Arg1578Cys was previously reported as a disease-causing mutation in a patient with Usher syndrome type 2 (USH2), 19 while the p.Gln258Pro variant was not detected in the screening of 224 normal chromosomes. For the ABCA4 gene, a single known heterozygous mutation reported in a patient with Stargardt disease type 1, 20 c.71G>A (p.Arg24His), was detected. Finally, a novel heterozygous frameshift variant, c.94_95delG (p.Glu32Serfs*2), was found in the RD3 gene. 
Table 3
 
Summary of Variant Prediction Using Six Different Tools
Table 3
 
Summary of Variant Prediction Using Six Different Tools
Sample Gene Amino Acid Change Prediction Tool* Familial Alleles in Control Vertebrate
PolyPhen-2 SIFT VarioWatch MutationTaster PMut SNAP Segregation Chromosomes Conservation
Potentially pathogenic mutations
 RP009 USH2A p.Gln258Pro Probably damaging Damaging High Disease causing Neutral Non-neutral NA 0/224 Yes
USH2A p.Arg1578Cys Possibly damaging Damaging High Disease causing Neutral Non-neutral NA NA§ Yes
 RP011 EYS p.Asn421Metfs*8 Disease causing Yes NA Yes
EYS p.Cys2139Tyr Probably damaging Damaging High Polymorphism Pathological Non-neutral Op NA§ Yes
 RP019 EYS p.Cys2139Tyr Probably damaging Damaging High Polymorphism Pathological Non-neutral Yes NA§ Yes
 RP022 CRB1 p.Cys1148Arg Probably damaging Damaging High Disease causing Pathological Non-neutral Yes 0/242 Yes
 RP023 CRB1 p.Phe847Ile Possibly damaging Tolerated High Polymorphism Neutral Non-neutral NA 0/224 Yes
 RP027 EYS p.Glu2703Ter Very high Disease causing NA NA Yes
 RP038 C8orf37 Splice site change Disease causing Yes NA
 RP069 PROM1 p.Lys148Ter Very high Disease causing Yes NA Yes
 RP087 RP2 p.Thr133Glnfs*15 Disease causing Yes NA Yes
Additional heterozygous variants
 RP009 ABCA4 p.Arg24His Probably damaging Tolerated High Disease causing Pathological Non-neutral NA§ Yes
RD3 p.Glu32Serfs*2 Disease causing NA Yes
 RP022 GUCY2D p.Arg380Cys Probably damaging Damaging High Disease causing Neutral Neutral Opo 0/220 Yes
 RP038 ROM1 p.Leu114Alafs*18 Disease causing Op 2/240 Yes
TULP1 p.Asp15Asn Probably damaging Tolerated High Polymorphism Neutral Non-neutral Opo 0/232 Yes
Variants of uncertain significance
 RP010 NR2E3 p.Arg142Trp Possibly damaging NA NA Disease causing Pathological Non-neutral NA 0/236 Yes
 RP016 FSCN2 p.Arg422Trp Possibly damaging Tolerated High Polymorphism Pathological Non-neutral NA 0/224 No
In addition, the RP022 proband carried two variants, but three alleles, in genes CRB1 and GUCY2D (Fig.). In the CRB1 gene, a novel homozygous mutation, c.3442T>C (p.Cys1148Arg), was detected. The occurrence of this mutation at a highly conserved cysteine (Supplementary Fig. S3), its absence in the 242 normal chromosomes screened, and the results of the six prediction tools support its true pathogenicity (Table 3). In addition, HOPE prediction showed that the substitution from cysteine 1148 to arginine changed a highly conserved cysteine in the EGF-like 15 domain and caused a disruption of the disulfide bridge with Cys1163, which in turn could lead to misfolding and thereby affect the binding properties of CRB1 product (crumbs homolog 1). Both asymptomatic parents and a brother of the patient were found to be heterozygous carriers of p.Cys1148Arg. Another novel heterozygous variant in the GUCY2D gene, c.1138C>T (p.Arg380Cys), was detected in the patient and his unaffected mother. 
Variants of Uncertain Significance (VUS)
Single novel heterozygous variants predicted to be deleterious were detected in genes FSCN2 and NR2E3 in two isolated RP patients (Fig., Table 2). These include a c.1264C>T (p.Arg422Trp) variant in FSCN2 and a c.424C>T (p.Arg142Trp) variant in the NR2E3 identified in the RP016 and RP010 probands, respectively. 
Discussion
We present here the first mutation report in Thai patients with nonsyndromic RP using WES. Eighty-six genes associated with RP, LCA, and CRD were analyzed to identify potentially pathogenic variants. Our study in 20 unrelated patients uncovered 10 potentially pathogenic mutations and two VUSs in nine and two patients, respectively. We also describe genotype–phenotype correlations of the identified genetic variants (Tables 1, 2). 
Potentially Pathogenic Mutations With and Without the Additional Variants
The RP038 proband carries three variants, but four alleles in three genes, C8orf37, ROM1, and TULP1. A novel homozygous splice-donor-site mutation, c.243+2T>C, identified in C8orf37 is most likely to be the cause of arRP in this family. This change is predicted to introduce a premature termination codon (PTC) that could lead to activation of the nonsense-mediated mRNA decay (NMD) pathway. 21 The proband's phenotype was accordant with a previous report in which all affected members demonstrated early impairment of the macula with age of onset ranging from infancy to late teenage years. 13 Furthermore, two additional variants were identified in the ROM1 and TULP1 genes. A frameshift insertion in ROM1, c.331_332insG (dbSNP rs137955062), was previously described in monogenic adRP 22,23 with disease severity ranging from asymptomatic to severe, and in digenic RP when coinherited with a p.Leu185Pro mutation in the PRPH2 gene. 24 This variant was also detected in two out of 240 normal chromosomes, indicating that the c.331_332insG variant is not the causative mutation for the RP038 proband. A novel heterozygous variant, c.43G>A, in TULP1 was identified in the RP038 proband and his unaffected elder brother. Previous reports have described TULP1 variants as the causes of both arRP and arLCA (RetNet). These data imply that the change in TULP1 in the RP038 proband does not correlate with the disease. 
In our study, two CRB1 gene mutations in exons 9 (RP022 proband) and 7 (RP023 proband) were identified. This is in accordance with previous publications that showed mutations mainly in exons 9 (41%) and 7 (27%). 25 These findings confirm the importance of the domains included in these two exons, exon 7: laminin G-like 2, exon 9: laminin G-like 3 and EGF-like 15 through 17. Previous reports of CRB1 gene mutations in retinal dystrophies showed variable age of onset and BCVA. 25,26 In the RP022 proband, who showed onset of blurred vision since birth, a homozygous mutation (p.Cys1148Arg) in CRB1 and a heterozygous variant (p.Arg380Cys) in GUCY2D were detected, whereas a homozygous mutation (p.Phe847Ile) in CRB1 was identified in the RP023 proband with onset of RP occurring at age 25 years. Given the fact that various GUCY2D gene mutations have been reported to cause both adCRD and arLCA (RetNet), and that the p.Arg380Cys variant was predicted to be deleterious, GUCY2D cannot be excluded from acting as a possible modifier gene. Coinheritance of variants in both CRB1 and GUCY2D might explain the earlier onset of the disease in the RP022 proband compared with the RP023 proband. 
In a previous report, a compound heterozygous and a homozygous mutation identified in the USH2A gene were associated with USH2 and RP without hearing loss, respectively. 27 Here, four variants in three different genes (USH2A, ABCA4, and RD3) were detected in the RP009 proband. Among the identified variants, a compound heterozygous mutation (p.Arg1578Cys and p.Gln258Pro) detected in USH2A is likely the cause of the disease. While the p.Arg1578Cys mutation was previously reported in a patient with USH2, 19 the RP009 proband has no hearing loss at age 30 years. Previous reports showed a wide range of BCVA among patients with USH2A mutations. 28,29 The BCVA and the macula of the RP009 proband were both normal. Although single heterozygous ABCA4 gene variants have been reported to cause age-related macular degeneration, 4,30 this finding was not observed in the RP009 proband at the time of examination. For RD3, all causative mutations were reported only in arLCA. Due to the lack of cosegregation analysis in family members, it is difficult to describe the ABCA4 and RD3 gene variants as possible modifier genes or as possible causes of phenotypic variation within the patient's family. 
Reported EYS mutations are distributed along the length of the gene. 31,32 In this study, a reported homozygous missense mutation (p.Cys2139Tyr) in exon 31 31 and a novel homozygous nonsense mutation (p.Glu2703Ter) in exon 42 of EYS were detected in the RP019 and RP027 probands, respectively. Normally, the Cys residue at position 2139 forms a disulfide bridge with Cys2130 (provided in the public domain by The Universal Protein Resource [UniProt], http://www.uniprot.org/uniprot/Q5T1H1/). Substitution of p.Cys2139Tyr may affect the conformation of the EYS protein. In the RP011 proband, a compound heterozygous mutation of a novel frameshift mutation (p.Asn421Metfs*8) in exon 8 and the same missense mutation as in the RP019 proband were observed (Table 2). The p.Glu2703Ter mutation is located near the C-terminal, leading to the loss of 441 amino acids in the EYS protein, while the p.Asn421Metfs*8 mutation introduces eight new amino acids. Both null alleles are predicted to induce a PTC that can lead to activation of the NMD pathway. 21 The RP011 proband developed poor night vision at age 19. This data supports the previous report in a French family with a compound heterozygous mutation of p.Cys2139Tyr and c.2847-1G>T with RP onset at age 21 years. 31 A homozygous nonsense mutation (p.Glu1836Ter) in exon 26 reported in a Chinese arRP patient was associated with onset of night blindness at age 15 years, 33 while the RP027 proband with the mutation in exon 42 developed poor night vision at age 50 years. The severity of protein truncation may determine the difference in age of onset. 
In the RP069 proband, a novel homozygous nonsense mutation (p.Lys148Ter) in the PROM1 gene leads to a truncation of 717 C-terminal amino acids from the full-length 865 residues and could activate the NMD pathway. 21 Homozygous truncation mutations in PROM1 have been identified in CRD and RP patients with early onset and severe visual impairment (6/120 to HM). 3437 Our patient developed progressive visual loss since age 19 years, which was later than those reported previously. However, the BCVA rapidly deteriorated to HM at age 23 years. The severity of visual impairment can be explained by an in vivo study that showed expression of PROM1 by rod and cone photoreceptor cells. The absence of PROM1 product (prominin-1 protein) specifically impaired photoreceptor outer segment and disk morphogenesis. 38  
A novel 25-bp deletion in exon 2 of the RP2 gene, identified in the RP087 family, is located in the C-CAP/cofactor C-like domain. This frameshift deletion leads to a truncated XRP2 protein missing 217 C-terminal amino acids from the full-length 350 residues possibly resulting in activation of the NMD pathway. 21 Exon 2 is the most common site of mutations reported in HGMD (provided in the public domain by The Human Gene Mutation Database, http://www.hgmd.cf.ac.uk/ac/index.php), suggesting the importance of this domain on XRP2 protein function. The proband RP087/1 and his affected elder brother, RP087/2, developed visual impairment at the ages of three and five, respectively. High myopia and high astigmatism were demonstrated. This information supports the genotype–phenotype correlation of mutations in RP2 with early onset of the disease and a high degree of refractive error. 39,40  
Variants of Uncertain Significance
A novel missense variant (p.Arg422Trp) in the FSCN2 gene identified in the RP016 proband is associated with disease onset at age 26 and severe visual impairment. Only one reported mutation, 208delG, has been identified in Japanese families with adRP and autosomal dominant macular dystrophy (adMD). 41,42 However, this mutation was also identified in both affected and unaffected members of Chinese families with RP, CRD, LCA, and normal controls, implying that 208delG was not the causative mutation of these diseases. 43 Different variants in FSCN2 were detected in Spanish patients with adRP and adMD, but none of these variants cosegregated in the families. 44 The FSCN2 gene product (retinal fascin 2) was proposed to play a role in photoreceptor disk morphogenesis. 45 Although the arginine residue 422 of retinal fascin 2 is not evolutionarily conserved, the results from HOPE prediction showed that Arg422 is located on the surface of this protein. The hydrophobicity difference between the arginine and tryptophan residues may affect the hydrogen-bond formation leading to the loss of protein interactions with other molecules. In fact, no tryptophan homologues of FSCN2 were found during evolutionary analysis as assessed by multiple alignments among distant species. This residue is normally glutamine in other vertebrates (Supplementary Fig. S3). A lack of DNA samples from additional family members makes it impossible to describe the segregation of p.Arg422Trp in this family. Cosegregation analysis and further in-depth functional study of this variant is needed for understanding the disease mechanism. 
A novel heterozygous variant (p.Arg142Trp) in the NR2E3 gene was identified in the RP010 proband. A previous study showed 33 disease-causing NR2E3 mutations with only one mutation causing adRP and two mutations causing arRP. 46 The RP010 proband demonstrated a relatively good prognosis with late disease onset and 6/24 vision in her right eye. Her left eye had no light perception due to anterior ischemic optic neuropathy. Although the identified variant (p.Arg142Trp) was predicted to be deleterious, it would require the family's DNA samples to confirm the significance of this missense variant. 
In summary, WES is a powerful approach for the identification of pathogenic genes in RP. Further analysis will reveal other known or novel genes contributing to the disease. 
Web Resources
The URLs for data presented herein are provided in the public domain as follows: 
Supplementary Materials
Acknowledgments
The authors thank the patients and their families for participation in this study. We also thank Fusano Todokoro and the technical staff at the Department of Medical Genome Sciences, The University of Tokyo, for their technical support. 
Supported by the operational expenditure fund of RIKEN (TDT); MEXT KAKENHI Grant 221S0002; the Royal Golden Jubilee PhD Program, Grant PHD/0102/2552 (WJ); the Siriraj Foundation (L-OA); and the “Chalermphrakiat” Grant, Faculty of Medicine Siriraj Hospital, Mahidol University (L-OA, WT, CL, PL, PS). 
Disclosure: W. Jinda, None; T.D. Taylor, None; Y. Suzuki, None; W. Thongnoppakhun, None; C. Limwongse, None; P. Lertrit, None; P. Suriyaphol, None; A. Trinavarat, None; L.-O. Atchaneeyasakul, None 
References
Anasagasti A Irigoyen C Barandika O Current mutation discovery approaches in retinitis pigmentosa. Vision Res . 2012; 75: 117–129. [CrossRef] [PubMed]
Hartong DT Berson EL Dryja TP. Retinitis pigmentosa. Lancet . 2006; 368: 1795–1809. [CrossRef] [PubMed]
Venturini G Rose AM Shah AZ CNOT3 is a modifier of PRPF31 mutations in retinitis pigmentosa with incomplete penetrance. PLoS Genet . 2012; 8: e1003040. [CrossRef] [PubMed]
Poloschek CM Bach M Lagreze WA ABCA4 and ROM1: implications for modification of the PRPH2-associated macular dystrophy phenotype. Invest Ophthalmol Vis Sci . 2010; 51: 4253–4265. [CrossRef] [PubMed]
Ng SB Turner EH Robertson PD Targeted capture and massively parallel sequencing of 12 human exomes. Nature . 2009; 461: 272–276. [CrossRef] [PubMed]
Rio Frio T McGee TL Wade NM A single-base substitution within an intronic repetitive element causes dominant retinitis pigmentosa with reduced penetrance. Hum Mutat . 2009; 30: 1340–1347. [CrossRef] [PubMed]
Bowne SJ Sullivan LS Koboldt DC Identification of disease-causing mutations in autosomal dominant retinitis pigmentosa (adRP) using next-generation DNA sequencing. Invest Ophthalmol Vis Sci . 2011; 52: 494–503. [CrossRef] [PubMed]
Zuchner S Dallman J Wen R Whole-exome sequencing links a variant in DHDDS to retinitis pigmentosa. Am J Hum Genet . 2011; 88: 201–206. [CrossRef] [PubMed]
Benaglio P McGee TL Capelli LP Next generation sequencing of pooled samples reveals new SNRNP200 mutations associated with retinitis pigmentosa. Hum Mutat . 2011; 32: E2246–E2258. [CrossRef] [PubMed]
Bowne SJ Humphries MM Sullivan LS A dominant mutation in RPE65 identified by whole-exome sequencing causes retinitis pigmentosa with choroidal involvement. Eur J Hum Genet . 2011; 19: 1074–1081. [CrossRef] [PubMed]
Tucker BA Scheetz TE Mullins RF Exome sequencing and analysis of induced pluripotent stem cells identify the cilia-related gene male germ cell-associated kinase (MAK) as a cause of retinitis pigmentosa. Proc Natl Acad Sci U S A . 2011; 108: E569–E576. [CrossRef] [PubMed]
Ozgul RK Siemiatkowska AM Yucel D Exome sequencing and cis-regulatory mapping identify mutations in MAK, a gene encoding a regulator of ciliary length, as a cause of retinitis pigmentosa. Am J Hum Genet . 2011; 89: 253–264. [CrossRef] [PubMed]
Estrada-Cuzcano A Neveling K Kohl S Mutations in C8orf37, encoding a ciliary protein, are associated with autosomal-recessive retinal dystrophies with early macular involvement. Am J Hum Genet . 2012; 90: 102–109. [CrossRef] [PubMed]
Neveling K Collin RW Gilissen C Next-generation genetic testing for retinitis pigmentosa. Hum Mutat . 2012; 33: 963–972. [CrossRef] [PubMed]
Liu T Jin X Zhang X A novel missense SNRNP200 mutation associated with autosomal dominant retinitis pigmentosa in a Chinese family. PLoS One . 2012; 7: e45464. [CrossRef] [PubMed]
Fu Q Wang F Wang H Next generation sequencing based molecular diagnosis of a chinese patient cohort with autosomal recessive retinitis pigmentosa. Invest Ophthalmol Vis Sci . 2013; 54: 4158–4166. [CrossRef] [PubMed]
Miller SA Dykes DD Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res . 1988; 16: 1215. [CrossRef] [PubMed]
Venselaar H Te Beek TA Kuipers RK Protein structure analysis of mutations causing inheritable diseases. An e-Science approach with life scientist friendly interfaces. BMC Bioinformatics . 2010; 11: 548. [CrossRef] [PubMed]
Le Quesne Stabej P Saihan Z Rangesh N Comprehensive sequence analysis of nine Usher syndrome genes in the UK National Collaborative Usher Study. J Med Genet . 2012; 49: 27–36. [CrossRef] [PubMed]
Lewis RA Shroyer NF Singh N Genotype/phenotype analysis of a photoreceptor-specific ATP-binding cassette transporter gene, ABCR, in Stargardt disease. Am J Hum Genet . 1999; 64: 422–434. [CrossRef] [PubMed]
Zhang J Sun X Qian Y At least one intron is required for the nonsense-mediated decay of triosephosphate isomerase mRNA: a possible link between nuclear splicing and cytoplasmic translation. Mol Cell Biol . 1998; 18: 5272–5283. [PubMed]
Sakuma H Inana G Murakami A A heterozygous putative null mutation in ROM1 without a mutation in peripherin/RDS in a family with retinitis pigmentosa. Genomics . 1995; 27: 384–386. [CrossRef] [PubMed]
Bascom RA Liu L Heckenlively JR Mutation analysis of the ROM1 gene in retinitis pigmentosa. Hum Mol Genet . 1995; 4: 1895–1902. [CrossRef] [PubMed]
Kajiwara K Berson EL Dryja TP. Digenic retinitis pigmentosa due to mutations at the unlinked peripherin/RDS and ROM1 loci. Science . 1994; 264: 1604–1608. [CrossRef] [PubMed]
Bujakowska K Audo I Mohand-Said S CRB1 mutations in inherited retinal dystrophies. Hum Mutat . 2012; 33: 306–315. [CrossRef] [PubMed]
Henderson RH Mackay DS Li Z Phenotypic variability in patients with retinal dystrophies due to mutations in CRB1. Br J Ophthalmol . 2011; 95: 811–817. [CrossRef] [PubMed]
Liu X Tang Z Li C Novel USH2A compound heterozygous mutations cause RP/USH2 in a Chinese family. Mol Vis . 2010; 16: 454–461. [CrossRef] [PubMed]
Garcia-Garcia G Aparisi MJ Jaijo T Mutational screening of the USH2A gene in Spanish USH patients reveals 23 novel pathogenic mutations. Orphanet J Rare Dis . 2011; 6: 65. [CrossRef] [PubMed]
Xu W Dai H Lu T Seven novel mutations in the long isoform of the USH2A gene in Chinese families with nonsyndromic retinitis pigmentosa and Usher syndrome Type II. Mol Vis . 2011; 17: 1537–1552. [PubMed]
Souied EH Ducroq D Gerber S Age-related macular degeneration in grandparents of patients with Stargardt disease: genetic study. Am J Ophthalmol . 1999; 128: 173–178. [CrossRef] [PubMed]
Audo I Sahel JA Mohand-Said S EYS is a major gene for rod-cone dystrophies in France. Hum Mutat . 2010; 31: E1406–E1435. [CrossRef] [PubMed]
Barragan I Borrego S Pieras JI Mutation spectrum of EYS in Spanish patients with autosomal recessive retinitis pigmentosa. Hum Mutat . 2010; 31: E1772–E1800. [CrossRef] [PubMed]
Huang Y Zhang J Li C Identification of a novel homozygous nonsense mutation in EYS in a Chinese family with autosomal recessive retinitis pigmentosa. BMC Med Genet . 2010; 11: 121. [CrossRef] [PubMed]
Maw MA Corbeil D Koch J A frameshift mutation in prominin (mouse)-like 1 causes human retinal degeneration. Hum Mol Genet . 2000; 9: 27–34. [CrossRef] [PubMed]
Zhang Q Zulfiqar F Xiao X Severe retinitis pigmentosa mapped to 4p15 and associated with a novel mutation in the PROM1 gene. Hum Genet . 2007; 122: 293–299. [CrossRef] [PubMed]
Pras E Abu A Rotenstreich Y Cone-rod dystrophy and a frameshift mutation in the PROM1 gene. Mol Vis . 2009; 15: 1709–1716. [PubMed]
Permanyer J Navarro R Friedman J Autosomal recessive retinitis pigmentosa with early macular affectation caused by premature truncation in PROM1. Invest Ophthalmol Vis Sci . 2010; 51: 2656–2663. [CrossRef] [PubMed]
Zacchigna S Oh H Wilsch-Brauninger M Loss of the cholesterol-binding protein prominin-1/CD133 causes disk dysmorphogenesis and photoreceptor degeneration. J Neurosci . 2009; 29: 2297–2308. [CrossRef] [PubMed]
Jayasundera T Branham KE Othman M RP2 phenotype and pathogenetic correlations in X-linked retinitis pigmentosa. Arch Ophthalmol . 2010; 128: 915–923. [CrossRef] [PubMed]
Ji Y Wang J Xiao X Mutations in RPGR and RP2 of Chinese patients with X-linked retinitis pigmentosa. Curr Eye Res . 2010; 35: 73–79. [CrossRef] [PubMed]
Wada Y Abe T Takeshita T Mutation of human retinal fascin gene (FSCN2) causes autosomal dominant retinitis pigmentosa. Invest Ophthalmol Vis Sci . 2001; 42: 2395–2400. [PubMed]
Wada Y Abe T Itabashi T Autosomal dominant macular degeneration associated with 208delG mutation in the FSCN2 gene. Arch Ophthalmol . 2003; 121: 1613–1620. [CrossRef] [PubMed]
Zhang Q Li S Xiao X The 208delG mutation in FSCN2 does not associate with retinal degeneration in Chinese individuals. Invest Ophthalmol Vis Sci . 2007; 48: 530–533. [CrossRef] [PubMed]
Gamundi MJ Hernan I Maseras M Sequence variations in the retinal fascin FSCN2 gene in a Spanish population with autosomal dominant retinitis pigmentosa or macular degeneration. Mol Vis . 2005; 11: 922–928. [PubMed]
Tubb BE Bardien-Kruger S Kashork CD Characterization of human retinal fascin gene (FSCN2) at 17q25: close physical linkage of fascin and cytoplasmic actin genes. Genomics . 2000; 65: 146–156. [CrossRef] [PubMed]
Schorderet DF Escher P. NR2E3 mutations in enhanced S-cone sensitivity syndrome (ESCS), Goldmann-Favre syndrome (GFS), clumped pigmentary retinal degeneration (CPRD), and retinitis pigmentosa (RP). Hum Mutat . 2009; 30: 1475–1485. [CrossRef] [PubMed]
Figure
 
Pedigrees and genotypes of the RP families. Genotype data are presented below the patient and members, where applicable, of their family. Filled symbols with an arrow indicate the probands. Squares: males. Circles: females. Slashed: decreased family members. Double line: consanguineous marriage. Filled black circle: obligate carrier. Normal alleles are indicated by ‘+'. Potentially pathogenic mutations, additional variants, and variants of uncertain significance, are indicated by “M,” “V,” and “VUS,” respectively.
Figure
 
Pedigrees and genotypes of the RP families. Genotype data are presented below the patient and members, where applicable, of their family. Filled symbols with an arrow indicate the probands. Squares: males. Circles: females. Slashed: decreased family members. Double line: consanguineous marriage. Filled black circle: obligate carrier. Normal alleles are indicated by ‘+'. Potentially pathogenic mutations, additional variants, and variants of uncertain significance, are indicated by “M,” “V,” and “VUS,” respectively.
Table 1
 
Clinical Information for Patients in this Study
Table 1
 
Clinical Information for Patients in this Study
Sample Sex Age Presenting Symptoms BCVA ERG VEP Fundus Examination
Onset At Exam RE LE RE LE RE LE
RP009 M 27 30 Poor night vision; flashing 6/6 6/9 NR NR Normal Normal Pink optic disc; generalized RPE changes without macular involvement; moderate bone spicules
RP010 F 60 69 Progressive visual loss 6/24 NLP NR NR Normal NR Pale optic disc LE > RE; heavy bone spicules 360 degrees
RP011 F 19 29 Poor night vision 6/9 6/9 NR NR Normal Normal Mildly pale optic disc; generalized RPE changes without macular involvement; mild bone spicules
RP016 M 26 36 Progressive visual loss; poor night vision CF CF NR NR Moderately decreased amplitude Moderately decreased amplitude Pale optic disc; generalized RPE changes; macular hyperpigmentation; bone spicules in macula only
RP019 F 47 49 Progressive visual loss; poor night vision; poor color discrimination 6/24 6/18 NR NR Normal Normal Pale optic disc; moderate bone spicules in mid periphery
RP022 F 11 11 Blurred vision since birth; photophobia; poor night vision 6/60 6/60 NR NR Normal Normal Pale optic disc; heavy bone spicules 360 degree; macular RPE changes
RP023* M 25 33 Progressive visual loss; poor night vision; tearing; photosensitivity 6/60 6/18 NR NR Moderately decreased amplitude Moderately decreased amplitude Pale optic disc; heavy bone spicules 360 degrees; macular RPE changes
RP027 M 50 51 Poor night vision 6/36 6/24 NR NR Moderately decreased amplitude Moderately decreased amplitude Pink optic disc; RPE changes in midperiphery without macular involvement; no bone spicules
RP038 M 18 48 Progressive visual loss; flashing HM CF NR NR Markedly decreased amplitude Markedly decreased amplitude Pale optic disc; generalized RPE changes with macular involvement; minimal bone spicules
RP069* M 19 23 Progressive visual loss; poor night vision HM HM NR NR Markedly decreased amplitude Markedly decreased amplitude Pale optic disc; generalized RPE changes with macular sheen; no bone spicules
RP087/1* M 3 5 Blurred distant vision; poor night vision; high myopia 6/36 6/60 NR NR Normal Normal Pink disc; generalized RPE changes without macular involvement; no bone spicules
RP087/2* M 5 10 Blurred distant vision; poor night vision; mild myopia with high astigmatism 6/60 6/60 NR NR Normal Normal Pink disc; generalized RPE changes without macular involvement; no bone spicules
Table 2
 
Variants Identified in the RP Patients
Table 2
 
Variants Identified in the RP Patients
Sample MOI in Family Gene MOI of Gene Variant Detected Reference
Chr Exon Nucleotide Change Amino Acid Change State Domain
Potentially pathogenic mutations
 RP009 Isolated RP USH2A arRP  1  4 c.773A>C p.Gln258Pro Het Novel
USH2A  1 22 c.4732C>T p.Arg1578Cys Het Laminin G-like 1 Reported19
 RP011 Isolated RP EYS arRP  6  8 c.1260_1260delG p.Asn421Metfs*8 Het Novel
EYS  6 31 c.6416G>A p.Cys2139Tyr Het EGF-like 21 Reported31
 RP019 Isolated RP EYS arRP  6 31 c.6416G>A p.Cys2139Tyr Hom EGF-like 21 Reported31
 RP022 Isolated RP CRB1 arRP and arLCA  1  9 c.3442T>C p.Cys1148Arg Hom EGF-like 15 Novel
 RP023 arRP CRB1 arRP and arLCA  1  7 c.2539T>A p.Phe847Ile Hom Laminin G-like 2 Novel
 RP027 Isolated RP EYS arRP  6 42 c.8107G>T p.Glu2703Ter Hom Novel
 RP038 Isolated RP C8orf37 arRP and arCRD  8 Intron 2 c.243+2T>C splice site change Hom Novel
 RP069 arRP PROM1 arRP and adCRD  4  4 c.442A>T p.Lys148Ter Hom Cytoplasmic Novel
 RP087 xlRP RP2 xlRP X  2 c.395_419delCCACTCAA p.Thr133Glnfs*15 Hem C-CAP/cofactor C-like Novel
CCCATCATTGAGTCTTC
Additional heterozygous variants
 RP009 Isolated RP ABCA4 arRP and arCRD  1  2 c.71G>A p.Arg24His Het Transmembrane Reported20
RD3 arLCA  1  2 c.94_95delG p.Glu32Serfs*2 Het Coiled coil Novel
 RP022 Isolated RP GUCY2D adCRD and arLCA 17  4 c.1138C>T p.Arg380Cys Het Extracellular Novel
 RP038 Isolated RP ROM1 adRP 11  1 c.339_340insG p.Leu114Alafs*18 Het Transmembrane Reported22–24
TULP1 arRP and arLCA  6  1 c.43G>A p.Asp15Asn Het Novel
Variants of uncertain significance
 RP010 Isolated RP NR2E3 arRP and adRP 15  4 c.424C>T p.Arg142Trp Het Novel
 RP016 Isolated RP FSCN2 adRP 17  4 c.1264C>T p.Arg422Trp Het Fascin-2 Novel
Table 3
 
Summary of Variant Prediction Using Six Different Tools
Table 3
 
Summary of Variant Prediction Using Six Different Tools
Sample Gene Amino Acid Change Prediction Tool* Familial Alleles in Control Vertebrate
PolyPhen-2 SIFT VarioWatch MutationTaster PMut SNAP Segregation Chromosomes Conservation
Potentially pathogenic mutations
 RP009 USH2A p.Gln258Pro Probably damaging Damaging High Disease causing Neutral Non-neutral NA 0/224 Yes
USH2A p.Arg1578Cys Possibly damaging Damaging High Disease causing Neutral Non-neutral NA NA§ Yes
 RP011 EYS p.Asn421Metfs*8 Disease causing Yes NA Yes
EYS p.Cys2139Tyr Probably damaging Damaging High Polymorphism Pathological Non-neutral Op NA§ Yes
 RP019 EYS p.Cys2139Tyr Probably damaging Damaging High Polymorphism Pathological Non-neutral Yes NA§ Yes
 RP022 CRB1 p.Cys1148Arg Probably damaging Damaging High Disease causing Pathological Non-neutral Yes 0/242 Yes
 RP023 CRB1 p.Phe847Ile Possibly damaging Tolerated High Polymorphism Neutral Non-neutral NA 0/224 Yes
 RP027 EYS p.Glu2703Ter Very high Disease causing NA NA Yes
 RP038 C8orf37 Splice site change Disease causing Yes NA
 RP069 PROM1 p.Lys148Ter Very high Disease causing Yes NA Yes
 RP087 RP2 p.Thr133Glnfs*15 Disease causing Yes NA Yes
Additional heterozygous variants
 RP009 ABCA4 p.Arg24His Probably damaging Tolerated High Disease causing Pathological Non-neutral NA§ Yes
RD3 p.Glu32Serfs*2 Disease causing NA Yes
 RP022 GUCY2D p.Arg380Cys Probably damaging Damaging High Disease causing Neutral Neutral Opo 0/220 Yes
 RP038 ROM1 p.Leu114Alafs*18 Disease causing Op 2/240 Yes
TULP1 p.Asp15Asn Probably damaging Tolerated High Polymorphism Neutral Non-neutral Opo 0/232 Yes
Variants of uncertain significance
 RP010 NR2E3 p.Arg142Trp Possibly damaging NA NA Disease causing Pathological Non-neutral NA 0/236 Yes
 RP016 FSCN2 p.Arg422Trp Possibly damaging Tolerated High Polymorphism Pathological Non-neutral NA 0/224 No
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×