February 2012
Volume 53, Issue 2
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Retina  |   February 2012
High Prevalence of Mutations in the EYS Gene in Japanese Patients with Autosomal Recessive Retinitis Pigmentosa
Author Affiliations & Notes
  • Masaki Iwanami
    From the Department of Ophthalmology, Hospital, and
    Department of Rehabilitation Engineering, Research Institute, National Rehabilitation Center for Persons with Disabilities, Tokorozawa, Japan.
  • Mio Oshikawa
    Department of Rehabilitation Engineering, Research Institute, National Rehabilitation Center for Persons with Disabilities, Tokorozawa, Japan.
  • Tomomi Nishida
    From the Department of Ophthalmology, Hospital, and
  • Satoshi Nakadomari
    From the Department of Ophthalmology, Hospital, and
  • Seishi Kato
    Department of Rehabilitation Engineering, Research Institute, National Rehabilitation Center for Persons with Disabilities, Tokorozawa, Japan.
  • Corresponding author: Masaki Iwanami, Department of Ophthalmology, Hospital, National Rehabilitation Center for Persons with Disabilities, 4-1 Namiki, Tokorozawa, Saitama 359-8555, Japan; miwanami1@gmail.com
Investigative Ophthalmology & Visual Science February 2012, Vol.53, 1033-1040. doi:10.1167/iovs.11-9048
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      Masaki Iwanami, Mio Oshikawa, Tomomi Nishida, Satoshi Nakadomari, Seishi Kato; High Prevalence of Mutations in the EYS Gene in Japanese Patients with Autosomal Recessive Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2012;53(2):1033-1040. doi: 10.1167/iovs.11-9048.

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

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Abstract

Purpose.: To screen for disease-causing mutations in the Eyes shut homolog (EYS) gene in Japanese patients with retinitis pigmentosa (RP).

Methods.: Blood samples were obtained from 68 RP patients and 68 controls. Genomic DNA was extracted from the blood samples and used for screening of mutations in the coding exons by direct sequencing. Each patient underwent a detailed clinical examination.

Results.: Nine nucleotide sequence variations causing amino acid changes were observed in homozygous or heterozygous alleles in 26 patients but not in 68 controls. Seven truncating mutations were found in 21 (32.8%) of 64 patients with nonsyndromic RP composed of 23 autosomal recessive RP (arRP) and 41 sporadic cases. The most abundant mutation was p.S1653Kfs*2, which was generated by a single adenine insertion into exon 26 (c.4957dupA) and was carried by 15 patients. The mutation p.Y2935*, produced by a single nucleotide substitution (c.8805C>A) in the last exon, was carried by five patients. These two truncating mutations were probably founder mutations because each was carried by the particular haplotype. The patients with homozygous or compound heterozygous truncating mutations showed a severe decline in visual acuity, whereas those with a single truncating mutation showed a mild decline.

Conclusions.: One-third of Japanese patients with nonsyndromic arRP carried probable pathogenic mutations in the EYS gene, including two founder mutations. Because the genotype was correlated with the phenotype, genotyping in the EYS gene could be a valuable tool for predicting long-term prognoses of Japanese patients with arRP and thus could be useful for genetic counseling and future gene therapy.

Retinitis pigmentosa (RP) is the most common genetic disorder in inherited retinal dystrophy. It has a prevalence of approximately 1 in 4000 persons worldwide. 1 In Japan, RP was the major cause of visual impairment among persons who entered 29 rehabilitation centers during the past 20 years, accounting for 25.0% (2001) and 15.8% (2006) of these cases. 2 RP is characterized by progressive visual loss caused by degenerative abnormalities of retinal photoreceptors. In the early stages, rod photoreceptor function is predominantly impaired, leading to defective dark adaptation, night blindness, and constriction of the visual field, followed by impairment of visual acuity from the loss of cone photoreceptor cells and eventually complete blindness. 1  
RP is genetically heterogeneous; it is transmitted as an autosomal dominant (ad), autosomal recessive (ar), or X-linked recessive disorder. To date more than 42 causative genes and seven loci have been identified (RetNet [http://www.sph.uth.tmc.edu/RetNet]), which is estimated to account for approximately 50% of all cases. 3 Thirty-two genes have been identified for the autosomal recessive form of RP (arRP). Mutation in each gene is observed in 1% to 2% of arRP cases, 4 with the exceptions of ATP-binding cassette, sub-family A, member 4 (ABCA4) and Usher syndrome type2A (USH2A) genes, mutations of which are observed in 5.6% and 7.0% of arRP patients, respectively. 5,6 Recent reports have suggested a global involvement of EYS, with the prevalence in different populations of persons with arRP (e.g., Spanish, French, Israeli) ranging from approximately 5% to 15%. 7 13 In Japan, large-scale mutation screening of ABCA4, 14 rhodopsin, 15 and 30 RP-causing genes 16 have been performed on arRP patients, but no major gene responsible for RP has been identified. 
EYS (Online Mendelian Inheritance in Man no. 612424) is the largest gene known to be expressed in the human eye, spanning more than 2 Mb within the RP25 locus (6q12). 7,8 The human EYS protein is a homolog of the Drosophila eyes shut/spacemaker (eys) protein, which is an extracellular matrix protein essential for photoreceptor development and morphology of the insect eye. 17,18 The longest isoform of EYS encodes a protein of 3165 amino acids, containing a signal peptide, 28 EGF-like domains, and 5 laminin A G-like domains (LamG). 7,8 All types of mutations have been reported in EYS: insertion, deletion, nonsense or missense substitution, and splice site mutation. 7 13 Most are truncating mutations, leading to premature truncation codons (PTCs). Some missense mutations were also identified by segregation studies and found in combination with a truncating mutation as probable causative mutations. 9 13,19 EYS is expressed in the retina, and the EYS protein is localized to outer segments of photoreceptor cells. Considering the evolutionary data and the function of the Drosophila homolog eys, EYS is likely to play a role in the modeling of retinal architecture. 17,18  
In this study, we screened 68 Japanese RP patients who visited the low-vision clinic at our center to search for RP-associated mutations in the EYS gene. As a result, we found that EYS is a major causative gene of nonsyndromic arRP in the Japanese population. The genotype-phenotype correlation was examined to serve as a prognostic indicator. 
Methods
Subjects
We studied 68 unrelated RP patients who visited the low-vision clinic at our center, as previously reported. 15,20 RP subjects were selected on the basis of clinical findings, patient history, and family history. Of these 68 patients, 4 were diagnosed as having adRP and 64 as having nonsyndromic arRP; the latter group consisted of 23 pedigrees with a recessive mode of inheritance (arRP) and 41 sporadic cases. Sixty-eight controls were recruited from among students at a college affiliated with our center and members of our staff of the center, all of whom declared having neither a personal history nor a family history of night blindness or unexplained visual loss. The study was approved by the institutional review board of the National Rehabilitation Center for Persons with Disabilities and was conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from all subjects in this study. 
Genetic Analysis
Genomic DNA was isolated from venous blood of the subjects using a DNA purification kit (Puregene; Gentra Systems, Minneapolis, MN). An additional 100 control DNA samples isolated from B-lymphoblast cell lines derived from unrelated healthy Japanese volunteers were obtained from the Health Science Research Resources Bank (Osaka, Japan). The genomic sequence of the EYS locus (NT_007299.13) and mRNA sequence (NM_001142800.1) were retrieved from the National Center for Biotechnology Information. Nucleotide A of the initiation codon of EYS was defined as position 1. The intronic primer sequences of 40 coding exons of the EYS gene for polymerase chain reaction (PCR) amplification are listed in Supplementary Table S1. Two hundred nanograms of genomic DNA were amplified with Taq polymerase (TaKaRa PrimeSTAR; Takara Bio Inc., Shiga, Japan), and mutation analysis was performed by direct sequencing of purified PCR products (BigDye Terminator Cycle Sequencing Kit; Applied Biosystems, Foster City, CA). The sequence primers are listed in Supplementary Table S2. Sequencing reaction products were run on an automated capillary sequencer (3130xl Genetic Analyzer; Applied Biosystems). The sequence variations were designated in accordance with the Human Genome Variation Society recommendations (http://www.hgvs.org/). 
Haplotype Analysis
Haplotypes were estimated from unphased genotypes using Clark's algorithm 21 and the expectation-maximization (EM) algorithm. 22 The Arlequin program (Schneider, Roessli, Excoffier, Arlequin version 2.000: a software package for population genetics data analysis; Geneva, Switzerland) was used for the analysis using the EM algorithm. The haplotype block in the EYS locus was estimated on the basis of LD plot analysis using HapMap (http://hapmap.ncbi.nlm.nih.gov/cgi-perl/gbrowse/hapmap27_B36/). 
Clinical Evaluation
A complete ophthalmic examination was performed, including refraction, visual acuity, visual field, biomicroscopic slit lamp examination, electroretinography (ERG), and funduscopy. Best-corrected visual acuity was measured in each eye with a Landolt chart, and the decimal values were converted to the logarithm of the minimal angle of resolution (logMAR) units. The visual field was assessed by Goldman kinetic perimetry (V-4e, I-4e, and I-2e targets). The clinical diagnosis was based on visual acuity, visual field, fundus photographs, and ERG findings. ERG recordings were performed in accordance with the guidelines provided by the International Society for Clinical Electrophysiology of Vision, using a monopolar contact lens electrode. Basic retinal imaging was performed using color fundus photographs, and an optical coherence tomography (OCT) device (Spectralis; Heidelberg Engineering, Heidelberg, Germany) was also used in some patients. 
Results
Sequence Variations
Supplementary Table S3 shows the nucleotide sequence variations in the EYS gene found in a total of 136 chromosomes from 68 RP patients, of whom 64 had nonsyndromic arRP (23 arRP and 41 sporadic) and 4 had adRP. We identified 83 nucleotide sequence variations that correspond to markers SV01 to SV83 in Supplementary Table S3, consisted of 66 single nucleotide substitutions and 16 insertion/deletion variations; 16 of these variations were novel. The 10 variations causing amino acid changes are listed in Table 1; they consisted of 3 missense variations (1 was novel) and 7 truncating mutations (6 were novel). SV25 (p.G843E) was included in the list in spite of a probable rare single nucleotide polymorphism (SNP) found in controls because it was unexpectedly abundant in RP patients compared with controls. Truncating mutations were produced by substitution (SV80, SV83), insertion (SV04, SV59), replacement (SV09, SV77), or deletion (SV81). Interestingly, SV59 (p.S1653Kfs*2) and SV83 (p.Y2935*) were found in multiple patients, as was p.G843E. 
Table 1.
 
Mutations Causing Amino Acid Changes Found in the EYS Gene of Japanese Patients with Retinitis Pigmentosa
Table 1.
 
Mutations Causing Amino Acid Changes Found in the EYS Gene of Japanese Patients with Retinitis Pigmentosa
Marker Exon Nucleotide Change Amino Acid Change Domain* Type SNP ID RP† Control‡ Reference
SV04 8 c.1211dupA p.Asn404Lysfs*3 EGF Frameshift 1 0 11
SV09 10 c.1485_1493delGGTTATTGAinsCGAAAAG p.Val495Glufs*13 EGF Frameshift 1 0 Novel
SV25 16 c.2528G>A p.Gly843Glu EGF Missense rs74419361 11 2 dbSNP§
SV38 23 c.3489T>A p.Asn1163Lys EGF Missense rs150951106 1 0 9,12
SV42 25 c.3809T>G p.Val1270Gly EGF Missense 1 0 Novel
SV59 26 c.4957dupA p.Ser1653Lysfs*2 Close to coiled-coil Frameshift 22 0 Novel
SV77 35 c.7028_7029delTGinsATCGT p.Leu2343Hisfs*105 EGF Frameshift 1 0 Novel
SV80 37 c.7283C>A p.Ser2428* LamG Nonsense 1 0 Novel
SV81 39 c.7665_7666delCA p.Tyr2555* LamG Nonsense 1 0 Novel, 10‖
SV83 43 c.8805C>A p.Tyr2935* EGF Nonsense 6 0 Novel
The amino acid sequence changes were observed in homozygous or heterozygous alleles in 26 of the RP patients, including 11 arRP and 14 sporadic cases, as shown in Table 2, but not in the 68 controls, with the exception of p.G843E, which was carried by 2 controls. Seven truncating mutations were found in 21 (32.8%) of the 64 patients with nonsyndromic arRP. Three missense variations were observed in 12 patients. The patients were classified into 5 groups on the basis of allele types: group A consisted of patients with homozygous truncating mutations, group B of patients with probable compound heterozygous truncating mutations, group C of patients with heterozygous truncating and missense mutations, group D of patients with single heterozygous truncating mutations, and group E of patients with missense mutations. 
Table 2.
 
Retinitis Pigmentosa Patients Carrying EYS Mutations
Table 2.
 
Retinitis Pigmentosa Patients Carrying EYS Mutations
No. Patient Sex Age (y) Inheritance Mutation 1 Mutation 2
Group A, homozygous truncating mutations
1 RP37* F 43 ad p.S1653Kfs*2 p.S1653Kfs*2
2 RP63† M 35 ar p.S1653Kfs*2 p.S1653Kfs*2
3 RP04 F 53 s p.S1653Kfs*2 p.S1653Kfs*2
4 RP12 F 49 s p.S1653Kfs*2 p.S1653Kfs*2
5 RP38 F 57 s p.S1653Kfs*2 p.S1653Kfs*2
6 RP57 M 52 s p.S1653Kfs*2 p.S1653Kfs*2
7 RP49‡ F 45 ar p.Y2935* p.Y2935*
Group B, probable compound heterozygous truncating mutations
8 RP16 F 57 ar p.S1653Kfs*2 p.N404Kfs*3
9 RP44‡ F 47 ar p.S1653Kfs*2 p.Y2935*
10 RP54 M 56 s p.V495Efs*13 p.L2343Hfs*105
11 RP29 M 54 s p.Y2935* p.Y2555*
Group C, heterozygous truncating and missense mutations
12 RP43 F 69 ar p.S1653Kfs*2 p.G843E
13 RP62 M 63 ar p.S1653Kfs*2 p.G843E
14 RP08 F 67 s p.S1653Kfs*2 p.G843E
15 RP28 M 43 s p.S1653Kfs*2 p.G843E
16 RP55 M 51 s p.S1653Kfs*2 p.G843E
17 RP50 M 58 s p.S2428* p.G843E
Group D, single heterozygous truncating mutation
18 RP03† F 56 ar p.S1653Kfs*2
19 RP61 F 62 s p.S1653Kfs*2
20 RP68 M 54 s p.S1653Kfs*2
21 RP26 F 28 ar p.Y2935*
22 RP45 M 64 ar p.Y2935*
Group E, missense mutation
7 RP49‡ F 45 ar p.G843E p.G843E
23 RP21 M 33 ar p.G843E
9 RP44‡ F 47 ar p.G843E
24 RP66 M 71 s p.G843E
25 RP10 F 50 ar p.N1163K
26 RP14 F 54 s p.V1270G
Truncating Mutations
Seven truncating mutations were found in 22 out of 68 RP patients (Table 2). The most abundant one was p.S1653Kfs*2, which was generated by a single adenine insertion into exon 26 (c.4957dupA), and was carried by 15 (15/64 = 23.4%) of the 64 non-syndromic arRP patients and 1 adRP patient (RP37). As shown in Table 2, 7 patients were homozygous (group A), 4 were probable compound heterozygous (group B), and 11 were heterozygous (group C and D). Probable compound heterozygous mutations in combination with p.S1653Kfs*2 were identified: p.N404Kfs*3 for RP16, p.Y2935* for RP44, and p.G843E for 5 patients. RP37 in the adRP panel had homozygous p.S1653Kfs*2 alleles, suggesting the possibility of arRP. The other 3 adRP patients had no mutation in the EYS gene. 
Mutation p.Y2935* in the last exon was produced by a single nucleotide substitution (c.8805C>A), which was carried by 5 patients. RP49 was homozygous for this mutation. RP44 and RP29 were probable compound heterozygous in combination with p.S1653Kfs*2 and p.Y2555*, respectively. RP26 and RP45 were single heterozygous. 
RP54 carried double heterozygous mutations, both of which were produced by replacement: c.1485_1493 delGGTTATTGAinsCGAAAAG (p.V495Efs*13) and c.7028_7029delTGinsATCGT (p.L2343Hfs*105). The remaining single truncating mutations (p.N404Kfs*3, p.S2428*, p.Y2555*) were combined with one of three abundant mutations as described above. The mutation p.N404Kfs*3 (C.1211dupA) was previously reported in a Moroccan Jewish patient. 11 p.Y2555* was reported in Caucasian patient, 10 but the genotype (c.7665C>G) was different from that (c.7665_7666delCA) observed in our case. 
Missense Mutations
Of 3 missense mutations, p.G843E in exon 16 was carried by 10 patients, and the allele frequency was 8.1% in RP patients. This substitution has already been annotated as SNP (c.2528G>A, rs74419361) with an allele frequency of 1.3% in the 1000 genomes (http://www.1000genomes.org/). In fact, this substitution was detected in 2 of the 68 controls and 1 of the samples from another 100 Japanese controls. RP49 was homozygous for p.G843E as well as for p.Y2935* and thus carried 2 different homozygous mutations. RP44 carrying combined truncating mutations also had heterozygous p.G843E. Six patients carrying p.G843E were heterozygous in combination with 2 truncating mutations (group C), and 2 were single heterozygous (group E). The single heterozygous mutations p.N1163K and p.V1270G were detected in RP10 and RP14, respectively. 
Haplotype Analysis
The truncating mutations p.S1653Kfs*2 and p.Y2935* were carried by multiple patients (16 and 5, respectively), suggesting that these mutations are probably founder mutations rather than recurrent mutations. If the mutation is attributed to a founder effect, the mutation should exist in a particular haplotype. To examine this possibility, the haplotype structures around these mutations were estimated using SNPs obtained from 68 patients and 68 controls. First, the region of the haplotype block containing each mutation was estimated on the basis of LD plot analysis using HapMap. The RP patient-derived SNPs found within each haplotype block were used to estimate the haplotype of this region. Tables 3 and 4 show the haplotype structures, suggesting that each mutation exists in a particular haplotype: HBA03 for SV59 (p.S1653Kfs*2) and HBB01 for SV83 (p.Y2935*). HBA03 is a minor haplotype with a frequency of only 8.1% in the controls, while HBB01 is a major haplotype with a frequency of 57.4% in the controls. The results strongly support that these two mutations are probable founder mutations. In addition, the haplotype structure around SV25 (p.G843E) was estimated, and this variation was confirmed to be a SNP characterizing a haplotype HBC06 as shown in Table 5
Table 3.
 
Haplotypes of the Region around SV59 (p.S1653Kfs*2)
Table 3.
 
Haplotypes of the Region around SV59 (p.S1653Kfs*2)
Haplotype Name Sequence Variation Marker Patients Controls
SV46 SV48 SV49 SV50 SV51 SV52 SV53 SV54 SV55 SV56 SV57 SV59 SV60 SV61 Chromosomes Frequency Chromosomes Frequency
HBA01 C C A C C A C T C A G A C 54 0.397 82 0.603
HBA02 C C A C C A T T C A G A C 41 0.301 34 0.250
HBA03 G T G G T G C C T G A A G 12 0.088 11 0.081
HBA03m G T G G T G C C T G A insA A G 22 0.162 0 0.000
HBA04 C C A C C A C T C A G C C 7 0.052 7 0.051
HBA05 C C A C C A C C T G A A G 0 0.000 2 0.015
Total 136 1 136 1
Table 4.
 
Haplotypes of the Region around SV81 (p.Tyr2555*) and SV83 (p.Tyr2935*)
Table 4.
 
Haplotypes of the Region around SV81 (p.Tyr2555*) and SV83 (p.Tyr2935*)
Haplotype Name Sequence Variation Marker Patients Controls
SV79 SV82 (SV81) SV83 Chromosomes Frequency Chromosomes Frequency
HBB01 A A C 52 0.382 78 0.574
HBB01m A A A 7 0.052 0 0
HBB02 G A C 42 0.309 31 0.228
HBB02m G del C 1 0.007 0 0
HBB03 A T C 34 0.25 27 0.198
Total 136 1 136 1
Table 5.
 
Haplotypes of the Region around SV25 (p.G843E)
Table 5.
 
Haplotypes of the Region around SV25 (p.G843E)
Haplotype Name Sequence Variation Marker Patients Controls
SV23 SV24 SV25 SV26 SV27 SV28 Chromosomes Frequency Chromosomes Frequency
HBC01 T C G T A A 50 0.368 57 0.419
HBC02 T G G C A C 43 0.316 44 0.324
HBC03 C C G T A A 26 0.191 21 0.154
HBC04 T G G C G C 6 0.044 7 0.051
HBC05 C G G C A C 0 0.000 5 0.037
HBC06 T G A C A C 11 0.081 2 0.015
Total 136 1 136 1
Clinical Features
Clinical and functional findings in 26 patients harboring the EYS mutations are summarized in Supplementary Table S4. The patients' age ranged from 28 to 71 years (average 52) when genomic testing was performed. These patients showed typical RP symptoms such as night blindness and progressive constriction of visual fields. Both myopic and hyperopic refractive errors were present. Cataracts were seen at a relatively young age. Fifteen patients had a cataract surgery, and their ages at the time of surgery ranged from 31 to 67 (average 50). Fundus appearance was typical for RP, including attenuation of the retinal arteries and bone spicule pigment deposits in the mid-periphery of the retina. Pallor of the optic discs was observed in patients at the end stage. Electroretinography responses were non-recordable in all patients with the EYS mutations in this study. 
Segregation Analysis
A segregation study was performed for the family of RP38 with the probable founder mutation p.S1653Kfs*2, and the results showed that the disease phenotype segregated the subjects (Fig. 1). The OCT images of a horizontal section including the macular and peripheral region demonstrated a reduction of retinal thickness in RP38 (II-1) due to the loss of sensory retinal tissue (Fig. 1C). The mother (I-2, age 91) and the sibling (II-4, age 58) were carriers of p.S1653Kfs*2 without manifestation of the RP phenotype. The fundus images of the sibling and mother were normal (Figs. 1D, 1E), although the mother had the cataract. The OCT image of the sibling clearly showed the normal retinal structure (Fig. 1D). 
Figure 1.
 
Segregation study for the family of RP38. (A) Pedigree of the family of RP38, who carried p.S1653Kfs*2 (M1). Arrow: Proband. M1/M1 represents a homozygous mutant, whereas M1/+ indicates a heterozygous carrier. (B) Mutation found in the RP38 family. c.4957dupA causes p.S1653Kfs*2. The proband (II-1) has homozygous mutations, and the unaffected sibling (II-4) has a heterozygous allele. Clinical features of the proband II-1 (C), the affected sibling II-4 (D), and the unaffected mother I-2 (E) were shown. FP, fundus photograph; OCT, optical coherence tomography; VF, visual field; L, left; R, right. The number in each panel indicates the age of the patient when the examination was performed. In the OCT panel, an arrow indicates the junction between the inner and outer segments of photoreceptors, and an arrowhead indicates an RPE complex.
Figure 1.
 
Segregation study for the family of RP38. (A) Pedigree of the family of RP38, who carried p.S1653Kfs*2 (M1). Arrow: Proband. M1/M1 represents a homozygous mutant, whereas M1/+ indicates a heterozygous carrier. (B) Mutation found in the RP38 family. c.4957dupA causes p.S1653Kfs*2. The proband (II-1) has homozygous mutations, and the unaffected sibling (II-4) has a heterozygous allele. Clinical features of the proband II-1 (C), the affected sibling II-4 (D), and the unaffected mother I-2 (E) were shown. FP, fundus photograph; OCT, optical coherence tomography; VF, visual field; L, left; R, right. The number in each panel indicates the age of the patient when the examination was performed. In the OCT panel, an arrow indicates the junction between the inner and outer segments of photoreceptors, and an arrowhead indicates an RPE complex.
Genotype-Phenotype Correlation
To characterize the genotype-phenotype correlation, the clinical features were compared among the different groups (groups A-D). Fifteen patients revisited our center for clinical follow-up, so that the time course data of the visual acuity and visual field were available. To examine the relationship between these clinical features and genotype, the time course of the visual acuity of the patients was plotted in Figure 2 using the data available for 5 patients from group A, 3 from group B, 5 from group C, and 2 from group D. During the clinical follow-up period (2–12 years; average, 7.3 years), patients in groups A and B, who carried homozygous or compound heterozygous truncating mutations, showed a severe decline in visual acuity, except RP12 and RP49 (Fig. 2A). Similarly, a severe decline was observed in the visual field of all these patients except RP37 (Supplementary Fig. S1A). In contrast, patients in groups C and D, who carried a heterozygous truncating mutation, showed a mild decline in visual acuity and visual field (Fig. 2B; Supplementary Fig. S1B). RP50 showed severe loss of visual acuity and visual field in his 50s, whereas RP08 and RP43 showed residual functions in central vision and visual field at ages 69 and 76, respectively. 
Figure 2.
 
The time course of visual acuity score in the right eye. (A) Patients carrying homozygous truncating mutations or probable compound heterozygous truncating mutations. (B) Patients carrying a heterozygous truncating mutation. Visual acuity was measured with a Landolt chart, and the decimal values were converted to the logMAR units. 2.7 logMAR and 2.8 logMAR correspond to hand movement and light perception, respectively.
Figure 2.
 
The time course of visual acuity score in the right eye. (A) Patients carrying homozygous truncating mutations or probable compound heterozygous truncating mutations. (B) Patients carrying a heterozygous truncating mutation. Visual acuity was measured with a Landolt chart, and the decimal values were converted to the logMAR units. 2.7 logMAR and 2.8 logMAR correspond to hand movement and light perception, respectively.
The phenotypes characterized by the time course of the visual acuity and visual field were confirmed by the fundus and OCT images of patients in each group. For example, RP04, with a severe decline of visual field in group A, showed the end stage of severe retinal thinning and foveal atrophy at age 60, with clumping structures that seemed to be composed of the proliferating retinal pigment epithelial cells (Fig. 3A). RP49, with a moderate decline of visual field in group A, showed moderate loss of sensory retinal tissue (Fig. 3B). RP43 in group C and RP68 in group D showed preserved sensory retinal tissue with the junction line between the inner and outer segments of photoreceptor cells in the macular region (Figs. 3C, 3D), suggesting the weaker pathogenicity of a single heterozygous truncating mutation. 
Figure 3.
 
Clinical features of patients carrying the truncating mutations. (A) RP04 carrying the homozygous p.S1653Kfs*2 mutation (M1). (B) RP49 carrying the homozygous p.Y2935* mutation (M2) and the homozygous p.G843E mutation (M3). (C) RP43 carrying the heterozygous p.S1653Kfs*2 mutation (M1) and p.G843E mutation (M3). (D) RP68 carrying the heterozygous p.S1653Kfs*2 mutation (M1) and unknown mutation (M*). FP, fundus photograph; OCT, optical coherence tomography; VF, visual field; L, left; R, right. The number in each panel indicates the age of the patient when the examination was performed. In the OCT panel, an arrow indicates the junction between the inner and outer segments of photoreceptors, and an arrowhead indicates an RPE complex.
Figure 3.
 
Clinical features of patients carrying the truncating mutations. (A) RP04 carrying the homozygous p.S1653Kfs*2 mutation (M1). (B) RP49 carrying the homozygous p.Y2935* mutation (M2) and the homozygous p.G843E mutation (M3). (C) RP43 carrying the heterozygous p.S1653Kfs*2 mutation (M1) and p.G843E mutation (M3). (D) RP68 carrying the heterozygous p.S1653Kfs*2 mutation (M1) and unknown mutation (M*). FP, fundus photograph; OCT, optical coherence tomography; VF, visual field; L, left; R, right. The number in each panel indicates the age of the patient when the examination was performed. In the OCT panel, an arrow indicates the junction between the inner and outer segments of photoreceptors, and an arrowhead indicates an RPE complex.
Discussion
We found that one-third of Japanese patients with nonsyndromic arRP carried probable pathogenic mutations in the EYS gene. This prevalence is higher than the prevalences reported in other populations: 11% in British and Chinese, 10 12% in French, 9 5% in Dutch and Canadian, 12 7% in Israeli, 11 and 15.9% in Spanish. 13 This high prevalence can be attributed to the existence of two major variants, p.S1653Kfs*2 and p.Y2935*, which are probably founder mutations because each was carried by the particular haplotype. The discovery of these founder mutations will greatly simplify diagnostic genetic screening in Japanese arRP patients, and these mutations will become a potential target for gene therapy. Similar founder mutations in the EYS gene have been reported in other ethnic populations: p.T135Lfs*26 in Moroccan Jewish, 11 p.H2740Yfs*28 in Iraqi Jewish, 11 and p.Y3156* in Dutch. 8 Further examination is necessary to reveal the origin of these population-specific founder mutations. 
The genotypes of group A (homozygous truncating mutations) and group B (probable compound heterozygous truncating mutations), shown in Table 2, meet the requirement for an autosomal recessive mode of inheritance, suggesting that all truncating mutations in these groups are pathogenic. In fact, most patients in these groups showed a severe decline in visual acuity. Since the mRNA transcribed from the EYS gene containing these truncating mutations has a PTC, the transcripts should be degraded by the nonsense-mediated decay (NMD) mechanism, 23 leading to the loss of the EYS protein. Alternatively, the truncating mutations may produce a truncated EYS protein lacking the C-terminal region, which has no or little of the function of EYS. The patients in groups C and D had a single truncating mutation (p.S1653Kfs*2 or p.Y2935*). Most of these patients showed a mild decline in visual acuity. Thus, we could predict the prognosis of the patient by examining whether the patient had homozygous truncating mutations or a single heterozygous truncating mutation. Considering the non-adRP mode of inheritance, each patient with a single truncating mutation should have had another mutation allele. There are several possibilities: the large deletion of an allele that cannot be detected by direct sequencing 24 ; a second pathogenic mutation located in the promoter region, the 5′-UTR region, the regulatory element in the intron, an unknown exon, or the 3′-UTR region 13 ; a mutation in the second gene involved in the maintenance of photoreceptor cells in association with EYS. The second mutation in another allele remains to be sought. 
The combination of a truncating mutation and p.G843E is intriguing because c.2528G>A generating p.G843E is one of the SNPs with approximately 1% of minor allele frequency. The glycine at position 843 is not only highly conserved among the EGF domains in the EYS protein, it is evolutionarily conserved among EYS homologs from various species (orangutan, marmoset, horse, dog, opossum, platypus, chicken, fly). 13 Thus, p.G843E is possibly pathogenic because of the reduction of EYS function. Alternatively, there may be a p.G843E-linked mutation that causes the reduction of EYS function. If a patient carrying homozygous p.G843E alleles were to be found, the pathogenicity of p.G843E or the p.G843E-linked mutation could be confirmed. RP49 had homozygous p.G843E alleles but also had homozygous p.Y2935* alleles that seem to have been responsible primarily for the manifestation of RP. Because p.Y2935* is located in the EGF domain encoded by the last exon, the transcript may escape degradation by NMD, 23 resulting in the production of a truncated EYS protein lacking the C-terminal EGF and LamG domains. RP49 showed a mild decline in visual acuity compared with the other patients, suggesting that the protein with p.Y2935* and p.G843E may have weak EYS activity. The mild symptoms of patients carrying both p.G843E and a truncation mutation can also be explained by assuming the weak activity of the mutant EYS protein with p.G843E or a reduction of EYS due to the p.G843E-linked mutation. On the other hand, RP50, who carried p.S2428* and p.G843E, completely lost visual acuity, implying that this patient has further mutations in the EYS gene or in another gene responsible for maintaining the photoreceptor cells. 
Two patients had a single missense change, p.N1163K (c.3489T>A) and p.V1270G (c.3809T>G). p.V1270G is located close to the EGF domain in the N-terminal half region, and a valine at position 1270 is conserved among different species. The pathogenicity of these missense changes remains unclear. p.N1163K concerns a residue in a calcium-binding EGF domain, which is conserved among other EGF domains and among different species. p.N1163K has been reported in two patients of different origins. 9,12 It is possible that these missense changes could be involved in the manifestation of RP in combination with an unknown mutation in another allele. 
Although the function of the EYS protein must be completely or almost completely lost in the eyes of patients carrying two alleles with truncating mutations due to degradation of truncated mRNA by NMD or to the loss of the C-terminal region, the patients had normal visual acuity until adolescence, indicating that EYS may be dispensable for the development of photoreceptor cells and that another factor may compensate for the loss of EYS to maintain the function of photoreceptor cells. If this is the case, the severity of symptoms may depend on the amount, activity, or both of this compensating factor. From this perspective, RP12 is interesting because this patient had mild symptoms despite carrying double truncating mutations. This case can be explained by the higher activity of the compensating factor. One candidate for the factor compensating the impaired function of EYS is a variant of EYS. Recently, we showed that human retinoblastoma Y79 expressed three variants of the EYS gene, including a long full-length cDNA clone with 7989 bp encoding a short form of EYS with 594 amino acids, but were not able to obtain the full-length cDNA clone encoding the longest form of EYS. 25 Thus, there seem to be many forms of EYS variants, including unknown ones. Given that the short EYS variants contain the N-terminal EGF domains identical to those of the long form, these variants may partially compensate for the loss of the long form of EYS. In addition, the existence of other compensating factors is expected from the fact that EYS orthologs are disrupted or absent from rodent genomes. 7  
Eys was first identified as a gene involved in the morphogenesis of the inter-rhabdomere space of Drosophila eyes 17,18 and was also shown to be expressed in Drosophila mechanosensory organs in which eys provides mechanical support to the mechanoreceptor cell. 26 In the vertebrate, the porcine EYS was shown to localize in the outer segment of the photoreceptor cell layer. 7 These findings lead us to postulate that human EYS may contribute to maintain the structure of the photoreceptor layer by providing rigidity to the outer segment of photoreceptor cells. To understand the disease mechanism caused by mutations observed in the EYS gene of arRP patients, it is necessary to reveal the molecular and biochemical characteristics of the EYS protein. 
In conclusion, we found that the main cause of nonsyndromic arRP in a Japanese population was mutation in the EYS gene and that one-third of the patients with nonsyndromic arRP had founder mutations. In addition, we found that the type of mutation was related to the severity of the symptoms. Thus, genotyping in the EYS gene could be a valuable tool for the diagnosis and prediction of the long-term prognosis of Japanese patients with arRP and thus could be useful for genetic counseling and future gene therapy. 
Supplementary Materials
Figure sf01, TIF - Figure sf01, TIF 
Table st1, XLS - Table st1, XLS 
Table st2, XLS - Table st2, XLS 
Table st3, XLS - Table st3, XLS 
Table st4, XLS - Table st4, XLS 
Footnotes
 Disclosure: M. Iwanami, None; M. Oshikawa, None; T. Nishida, None; S. Nakadomari, None; S. Kato, None
The authors thank Akiko Nakajima and Yuuki Noshiro for their technical assistance and all the participating RP patients and control subjects. 
References
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Figure 1.
 
Segregation study for the family of RP38. (A) Pedigree of the family of RP38, who carried p.S1653Kfs*2 (M1). Arrow: Proband. M1/M1 represents a homozygous mutant, whereas M1/+ indicates a heterozygous carrier. (B) Mutation found in the RP38 family. c.4957dupA causes p.S1653Kfs*2. The proband (II-1) has homozygous mutations, and the unaffected sibling (II-4) has a heterozygous allele. Clinical features of the proband II-1 (C), the affected sibling II-4 (D), and the unaffected mother I-2 (E) were shown. FP, fundus photograph; OCT, optical coherence tomography; VF, visual field; L, left; R, right. The number in each panel indicates the age of the patient when the examination was performed. In the OCT panel, an arrow indicates the junction between the inner and outer segments of photoreceptors, and an arrowhead indicates an RPE complex.
Figure 1.
 
Segregation study for the family of RP38. (A) Pedigree of the family of RP38, who carried p.S1653Kfs*2 (M1). Arrow: Proband. M1/M1 represents a homozygous mutant, whereas M1/+ indicates a heterozygous carrier. (B) Mutation found in the RP38 family. c.4957dupA causes p.S1653Kfs*2. The proband (II-1) has homozygous mutations, and the unaffected sibling (II-4) has a heterozygous allele. Clinical features of the proband II-1 (C), the affected sibling II-4 (D), and the unaffected mother I-2 (E) were shown. FP, fundus photograph; OCT, optical coherence tomography; VF, visual field; L, left; R, right. The number in each panel indicates the age of the patient when the examination was performed. In the OCT panel, an arrow indicates the junction between the inner and outer segments of photoreceptors, and an arrowhead indicates an RPE complex.
Figure 2.
 
The time course of visual acuity score in the right eye. (A) Patients carrying homozygous truncating mutations or probable compound heterozygous truncating mutations. (B) Patients carrying a heterozygous truncating mutation. Visual acuity was measured with a Landolt chart, and the decimal values were converted to the logMAR units. 2.7 logMAR and 2.8 logMAR correspond to hand movement and light perception, respectively.
Figure 2.
 
The time course of visual acuity score in the right eye. (A) Patients carrying homozygous truncating mutations or probable compound heterozygous truncating mutations. (B) Patients carrying a heterozygous truncating mutation. Visual acuity was measured with a Landolt chart, and the decimal values were converted to the logMAR units. 2.7 logMAR and 2.8 logMAR correspond to hand movement and light perception, respectively.
Figure 3.
 
Clinical features of patients carrying the truncating mutations. (A) RP04 carrying the homozygous p.S1653Kfs*2 mutation (M1). (B) RP49 carrying the homozygous p.Y2935* mutation (M2) and the homozygous p.G843E mutation (M3). (C) RP43 carrying the heterozygous p.S1653Kfs*2 mutation (M1) and p.G843E mutation (M3). (D) RP68 carrying the heterozygous p.S1653Kfs*2 mutation (M1) and unknown mutation (M*). FP, fundus photograph; OCT, optical coherence tomography; VF, visual field; L, left; R, right. The number in each panel indicates the age of the patient when the examination was performed. In the OCT panel, an arrow indicates the junction between the inner and outer segments of photoreceptors, and an arrowhead indicates an RPE complex.
Figure 3.
 
Clinical features of patients carrying the truncating mutations. (A) RP04 carrying the homozygous p.S1653Kfs*2 mutation (M1). (B) RP49 carrying the homozygous p.Y2935* mutation (M2) and the homozygous p.G843E mutation (M3). (C) RP43 carrying the heterozygous p.S1653Kfs*2 mutation (M1) and p.G843E mutation (M3). (D) RP68 carrying the heterozygous p.S1653Kfs*2 mutation (M1) and unknown mutation (M*). FP, fundus photograph; OCT, optical coherence tomography; VF, visual field; L, left; R, right. The number in each panel indicates the age of the patient when the examination was performed. In the OCT panel, an arrow indicates the junction between the inner and outer segments of photoreceptors, and an arrowhead indicates an RPE complex.
Table 1.
 
Mutations Causing Amino Acid Changes Found in the EYS Gene of Japanese Patients with Retinitis Pigmentosa
Table 1.
 
Mutations Causing Amino Acid Changes Found in the EYS Gene of Japanese Patients with Retinitis Pigmentosa
Marker Exon Nucleotide Change Amino Acid Change Domain* Type SNP ID RP† Control‡ Reference
SV04 8 c.1211dupA p.Asn404Lysfs*3 EGF Frameshift 1 0 11
SV09 10 c.1485_1493delGGTTATTGAinsCGAAAAG p.Val495Glufs*13 EGF Frameshift 1 0 Novel
SV25 16 c.2528G>A p.Gly843Glu EGF Missense rs74419361 11 2 dbSNP§
SV38 23 c.3489T>A p.Asn1163Lys EGF Missense rs150951106 1 0 9,12
SV42 25 c.3809T>G p.Val1270Gly EGF Missense 1 0 Novel
SV59 26 c.4957dupA p.Ser1653Lysfs*2 Close to coiled-coil Frameshift 22 0 Novel
SV77 35 c.7028_7029delTGinsATCGT p.Leu2343Hisfs*105 EGF Frameshift 1 0 Novel
SV80 37 c.7283C>A p.Ser2428* LamG Nonsense 1 0 Novel
SV81 39 c.7665_7666delCA p.Tyr2555* LamG Nonsense 1 0 Novel, 10‖
SV83 43 c.8805C>A p.Tyr2935* EGF Nonsense 6 0 Novel
Table 2.
 
Retinitis Pigmentosa Patients Carrying EYS Mutations
Table 2.
 
Retinitis Pigmentosa Patients Carrying EYS Mutations
No. Patient Sex Age (y) Inheritance Mutation 1 Mutation 2
Group A, homozygous truncating mutations
1 RP37* F 43 ad p.S1653Kfs*2 p.S1653Kfs*2
2 RP63† M 35 ar p.S1653Kfs*2 p.S1653Kfs*2
3 RP04 F 53 s p.S1653Kfs*2 p.S1653Kfs*2
4 RP12 F 49 s p.S1653Kfs*2 p.S1653Kfs*2
5 RP38 F 57 s p.S1653Kfs*2 p.S1653Kfs*2
6 RP57 M 52 s p.S1653Kfs*2 p.S1653Kfs*2
7 RP49‡ F 45 ar p.Y2935* p.Y2935*
Group B, probable compound heterozygous truncating mutations
8 RP16 F 57 ar p.S1653Kfs*2 p.N404Kfs*3
9 RP44‡ F 47 ar p.S1653Kfs*2 p.Y2935*
10 RP54 M 56 s p.V495Efs*13 p.L2343Hfs*105
11 RP29 M 54 s p.Y2935* p.Y2555*
Group C, heterozygous truncating and missense mutations
12 RP43 F 69 ar p.S1653Kfs*2 p.G843E
13 RP62 M 63 ar p.S1653Kfs*2 p.G843E
14 RP08 F 67 s p.S1653Kfs*2 p.G843E
15 RP28 M 43 s p.S1653Kfs*2 p.G843E
16 RP55 M 51 s p.S1653Kfs*2 p.G843E
17 RP50 M 58 s p.S2428* p.G843E
Group D, single heterozygous truncating mutation
18 RP03† F 56 ar p.S1653Kfs*2
19 RP61 F 62 s p.S1653Kfs*2
20 RP68 M 54 s p.S1653Kfs*2
21 RP26 F 28 ar p.Y2935*
22 RP45 M 64 ar p.Y2935*
Group E, missense mutation
7 RP49‡ F 45 ar p.G843E p.G843E
23 RP21 M 33 ar p.G843E
9 RP44‡ F 47 ar p.G843E
24 RP66 M 71 s p.G843E
25 RP10 F 50 ar p.N1163K
26 RP14 F 54 s p.V1270G
Table 3.
 
Haplotypes of the Region around SV59 (p.S1653Kfs*2)
Table 3.
 
Haplotypes of the Region around SV59 (p.S1653Kfs*2)
Haplotype Name Sequence Variation Marker Patients Controls
SV46 SV48 SV49 SV50 SV51 SV52 SV53 SV54 SV55 SV56 SV57 SV59 SV60 SV61 Chromosomes Frequency Chromosomes Frequency
HBA01 C C A C C A C T C A G A C 54 0.397 82 0.603
HBA02 C C A C C A T T C A G A C 41 0.301 34 0.250
HBA03 G T G G T G C C T G A A G 12 0.088 11 0.081
HBA03m G T G G T G C C T G A insA A G 22 0.162 0 0.000
HBA04 C C A C C A C T C A G C C 7 0.052 7 0.051
HBA05 C C A C C A C C T G A A G 0 0.000 2 0.015
Total 136 1 136 1
Table 4.
 
Haplotypes of the Region around SV81 (p.Tyr2555*) and SV83 (p.Tyr2935*)
Table 4.
 
Haplotypes of the Region around SV81 (p.Tyr2555*) and SV83 (p.Tyr2935*)
Haplotype Name Sequence Variation Marker Patients Controls
SV79 SV82 (SV81) SV83 Chromosomes Frequency Chromosomes Frequency
HBB01 A A C 52 0.382 78 0.574
HBB01m A A A 7 0.052 0 0
HBB02 G A C 42 0.309 31 0.228
HBB02m G del C 1 0.007 0 0
HBB03 A T C 34 0.25 27 0.198
Total 136 1 136 1
Table 5.
 
Haplotypes of the Region around SV25 (p.G843E)
Table 5.
 
Haplotypes of the Region around SV25 (p.G843E)
Haplotype Name Sequence Variation Marker Patients Controls
SV23 SV24 SV25 SV26 SV27 SV28 Chromosomes Frequency Chromosomes Frequency
HBC01 T C G T A A 50 0.368 57 0.419
HBC02 T G G C A C 43 0.316 44 0.324
HBC03 C C G T A A 26 0.191 21 0.154
HBC04 T G G C G C 6 0.044 7 0.051
HBC05 C G G C A C 0 0.000 5 0.037
HBC06 T G A C A C 11 0.081 2 0.015
Total 136 1 136 1
Figure sf01, TIF
Table st1, XLS
Table st2, XLS
Table st3, XLS
Table st4, XLS
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