August 2016
Volume 57, Issue 10
Open Access
Genetics  |   August 2016
Identification of Novel Mutations in the LRR-Cap Domain of C21orf2 in Japanese Patients With Retinitis Pigmentosa and Cone–Rod Dystrophy
Author Affiliations & Notes
  • Akiko Suga
    National Institute of Sensory Organs Tokyo Medical Center, National Hospital Organization, Tokyo, Japan
  • Atsushi Mizota
    Department of Ophthalmology, Teikyo University School of Medicine, Tokyo, Japan
  • Mitsuhiro Kato
    Department of Pediatrics, Showa University School of Medicine, Tokyo, Japan
  • Kazuki Kuniyoshi
    Department of Ophthalmology, Kinki University Faculty of Medicine, Osakasayama City, Osaka, Japan
  • Kazutoshi Yoshitake
    Japan Software Management, Yokohama, Japan
  • William Sultan
    National Institute of Sensory Organs Tokyo Medical Center, National Hospital Organization, Tokyo, Japan
  • Masashi Yamazaki
    Department of Orthopaedic Surgery, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan
  • Yoshikazu Shimomura
    Department of Ophthalmology, Kinki University Faculty of Medicine, Osakasayama City, Osaka, Japan
  • Kazuho Ikeo
    CIB-DDBJ, National Institute of Genetics, Mishima, Shizuoka, Japan
  • Kazushige Tsunoda
    National Institute of Sensory Organs Tokyo Medical Center, National Hospital Organization, Tokyo, Japan
  • Takeshi Iwata
    National Institute of Sensory Organs Tokyo Medical Center, National Hospital Organization, Tokyo, Japan
  • Correspondence: Takeshi Iwata, National Institute of Sensory Organs, Tokyo Medical Center, National Hospital Organization, 2-5-1 Higashigaoka, Meguro-ku, Tokyo 152-8902, Japan; takeshi.iwata@kankakuki.go.jp
Investigative Ophthalmology & Visual Science August 2016, Vol.57, 4255-4263. doi:10.1167/iovs.16-19450
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      Akiko Suga, Atsushi Mizota, Mitsuhiro Kato, Kazuki Kuniyoshi, Kazutoshi Yoshitake, William Sultan, Masashi Yamazaki, Yoshikazu Shimomura, Kazuho Ikeo, Kazushige Tsunoda, Takeshi Iwata; Identification of Novel Mutations in the LRR-Cap Domain of C21orf2 in Japanese Patients With Retinitis Pigmentosa and Cone–Rod Dystrophy. Invest. Ophthalmol. Vis. Sci. 2016;57(10):4255-4263. doi: 10.1167/iovs.16-19450.

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

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Abstract

Purpose: C21orf2 encodes a ciliary protein related to syndromic and nonsyndromic retinal degeneration. The purpose of this study was to identify novel mutations of C21orf2 associated with syndromic autosomal recessive retinitis pigmentosa (arRP) and autosomal recessive cone–rod dystrophy (arCRD) by using whole exome sequencing of a Japanese cohort.

Methods: Whole exome sequencing was performed on DNA from affected and healthy members from 147 families with retinal degenerations. Identified nonsense and missense mutations were further restricted by using the reported single nucleotide variation frequencies and inherited patterns. The effect of the mutations was examined by in vitro assays.

Results: Novel mutations in C21orf2 were found in Japanese patients with arRP with skeletal defects or arCRD. Compound heterozygous mutations, from one family (p.V111M and p.Y107H), and a homozygous mutation, from another family (p.Y107C), were all located in the leucine-rich repeat C-terminal domain required for protein stabilization. C21orf2 was expressed in the retina through the developing to the mature stage, and the protein localized to the photoreceptor cilia in the adult retina. In vitro expression showed reduced levels and affected localizations of mutated protein products compared to the wild type.

Conclusions: The identified C21orf2 mutations decreased protein stability and affected cytoplasmic localization of C21orf2. Since C21orf2 was required for ciliogenesis, our data suggested that reduced levels of functional C21orf2 induced photoreceptor degradation through abnormal cilia formation, leading to arRP or arCRD in the retina.

Ciliopathies are genetic diseases caused by the abnormal formation and/or maintenance of the primary cilia. Because the primary cilium is almost ubiquitous in vertebrate cells and plays a variety of roles, such as intraflagellar transport, mechanosensing, and signal transduction, ciliopathies are expressed as combinations of abnormalities in multiple organs including the retina, ear, brain, kidney, liver, bone, and gonads.1 
The retina is one of the primarily affected tissues in syndromic ciliopathies. Joubert syndrome (JBTS, OMIM No. 213300), Bardet–Biedl syndrome (BBS, OMIM No. 209900), Senior–Løken syndrome (SLS, OMIM No. 266900), Usher syndrome type 2 (USH2A, OMIM No. 276901), and Jeune's asphyxiating thoracic dystrophy (JATD, OMIM No. 208500) include retinal degeneration as one of the associated phenotypes.1 On the other hand, the abnormalities of ciliary proteins can also result in isolated retinal dystrophy caused by photoreceptor degeneration.25 Mutations of the ciliary genes were reported in Leber congenital amaurosis (LCA, OMIM No. 204000), macular dystrophy (MD), cone–rod dystrophy (CRD), and retinitis pigmentosa (RP, OMIM No. 268000). Interestingly, the causal genes for syndromic ciliopathies and isolated retinal ciliopathies are overlapping in part. For example, mutations of CEP290 cause several syndromic ciliopathies, such as JATD, BBS, JBTS, Meckel syndrome, and SLS, but also the retinal ciliopathy, LCA.6 The mechanism by which the same causal gene results in syndromic or nonsyndromic symptom is considered to be due to the different impacts of each mutation, and also due to the individual genetic background, though the relationships are not clearly demonstrated in most cases. 
To study the clinical and genetic characteristics of inherited retinal diseases in the Japanese population, 808 subjects from 496 Japanese families with inherited retinal diseases were selected for the Japan Whole Exome Project.7 That cohort included patients with RP, CRD, LCA, MD, Stargardt's disease, congenital stationary night blindness, and other retinal diseases. Whole exome sequencing (WES) was performed with 147 families. This study identified new putative disease-related genes and a number of novel mutations of previously reported genes.8,9 Among the novel mutations, missense mutations of a ciliary gene, C21orf2, occurred in Japanese patients with autosomal recessive retinitis pigmentosa (arRP) and autosomal recessive cone–rod dystrophy (arCRD). The mutations were located in the short leucine-rich repeat C-terminal (LRRCT) domain, which is required for the stability and structural integrity of leucine-rich repeat (LRR) proteins.10 C21orf2 protein localized to the primary cilium of the photoreceptors. In vitro expression assays indicated that these mutations affected protein stability and localization. These data suggested that the identified mutations reduced the expression of functional C21orf2 protein, which led to the degeneration of photoreceptors by disrupting ciliogenesis and ciliary function. 
Materials and Methods
Patient Recruitment and Diagnostic
Individuals 1-1 and 1-2 were referred to Teikyo University Hospital. Individuals 2-1 and 2-3 were referred to Kinki University Hospital. Clinical diagnoses and evaluations for RP and CRD were based on the clinical history, decimal best-corrected visual acuity (BCVA), fundus examination, visual field test, and ERG findings. The research protocols were approved by the Ethics Review Board of Teikyo University Faculty of Medicine and the Ethics Review Board of Kinki University Faculty of Medicine (approval No. 22-132). The research followed the tenets of the Declaration of Helsinki. Informed consent was obtained from all the participants. 
Whole Exome Analysis
Genomic DNA was extracted by using ORAgene (DNA Genotek, Ottawa, ON, Canada). Whole exome sequencing was performed by Macrogen Japan (Kyoto, Japan). We applied WES using Sureselect All Exon V4 (Agilent Technologies, Santa Clara, CA, USA), and Hiseq2000 (Illumina, San Diego, CA, USA). Reads were mapped to the reference human genome (1000 genomes, phase 2 reference, hs37d5) with the Burrows-Wheeler Alinger software, version 0.7.10.11 Duplicate reads were then removed by Picard MarkDuplicates module version 1.129, and mapped reads around insertion–deletion polymorphisms (INDELs) were realigned by using the Genome Analysis Toolkit (GATK) version 3.3-0.12 Base-quality scores were recalibrated by using GATK. The averaged coverages were above 48, and >93% of the bases were covered with more than five reads (Supplementary Table S1). The calling of mutations was performed by using the GATK HaplotypeCaller module, and the called single-nucleotide variants and INDELs were annotated with the SnpEff software, version 4.1B13 and the ANNOVAR software, version 2015-04-24.14 The mutations were annotated with the SnpEff score (“HIGH,” “MODERATE,” or “LOW”) and with the allele frequency in the 1000 genomes database, Exome Aggregation Consortium (ExAC) database, and human genetic variation database (HGVD) (http://www.genome.med.kyoto-u.ac.jp/SnpDB/index.html; provided in the public domain by Kyoto University, Kyoto, Japan; accessed December 7, 2013). The mutations were then filtered so that only those with “HIGH” or “MODERATE” SnpEff scores, indicating that the amino acid sequence would be functionally affected, and with frequency < 0.1% in the 1000 genomes database, ExAC database, HGVD, and the in-house database of exome data from 610 patients, were further analyzed. We also used new variations that were not found in the in-house exome data of 14 healthy controls without ocular diseases. Mutations were filtered by hereditary information in the family members. 
Animals and Tissue Preparations
Animal experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and approved by the Tokyo Medical Center Experimental Animal Committee. To examine the mRNA and protein expression, mice were killed at the indicated age and the eyes were enucleated. After the removal of the lens, the retina was separated from other tissue such as the sclera and retinal pigment epithelium (RPE). In the embryonic day 16 (E16) mice, the retina and the RPE were not separated. 
Reverse Transcription–PCR
The isolated retina was lysed with TRIzoL Reagent (Ambion, Thermo Scientific, Waltham, MA, USA) for total RNA extraction. Complementary DNA (cDNA) was synthesized with SuperScript III First-Strand Synthesis System (Life Technologies, Thermo Scientific) following the manufacturer's instructions. Primers used were as follows: C21orf2 variant 1 and 4 (forward: 5′-CCACCATGAAGCTGACACGA-3′; reverse: 5′-CAGCTCTTCCTCGGTCACAG-3′), C21orf2 variant 1 and 3 (forward: 5′-AGCTCTGCAACTGAGACCAG-3′; reverse: 5′-CCCGCAGCAGTAGCAATATG-3′), Gapdh (forward: 5′-GGGGAGCCAAAAGGGTCATCATCT-3′; reverse: 5′-CGACGCCTGCTTCACCACCTTCTT-3′). 
Western Blot
The antibodies used in this study are summarized in Table 1. The isolated retina was lysed with RIPA (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40), supplemented with complete Protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) and 1 mM PMSF (ALEXIS Biochemicals, ENZO Life Science, Farmingdale, NY, USA). Retinal lysates were denatured and separated by 12% SDS-PAGE, and transferred to polyviniylidine fluoride membranes by using Trans-Blot Turbo (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 3% bovine serum albumin and incubated with primary antibodies at 4°C overnight. After washing with PBS containing 0.1% Tween-20 (PBST), membranes were incubated with secondary antibodies at room temperature for 30 minutes. After washing with PBST, membranes were incubated with SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Thermo Scientific), and the signals were detected by using ChemiDoc XRS (Bio-Rad Laboratories). 
Table 1
 
Antibodies Used in This Study
Table 1
 
Antibodies Used in This Study
Immunostaining
Mouse eyes were fixed in 4% paraformaldehyde at 4°C overnight and cryopreserved with O.C.T compound (SAKURA, Tokyo, Japan). Monkey eyes were provided by the National Institute of Biomedical Innovation (Tsukuba, Japan), fixed in 4% paraformaldehyde at 4°C overnight, and paraffin embedded. Cryosections were treated with antibody retrieval solution (Dako, Agilent Technologies), washed with PBS with 0.3% Triton X-100, incubated with Protein Block (Dako), and incubated with primary antibodies at 4°C overnight. After washing with PBST, cryosections were incubated with secondary antibodies at room temperature for 30 minutes. Paraffin sections were deparaffinized and immunostained by using the same protocol. Images were taken with an LSM 700 confocal microscope (ZEISS, Oberkochen, Germany). 
Plasmid Construction
Human C21orf2 cDNA was provided by RIKEN BRC (Tsukuba, Japan) through the National Bio-Resource Project of the MEXT, Japan.1518 The coding sequence was cloned into pEF1a-FLAG-N1 (constructed by Yuichi Kawamura, in preparation). Point mutations were induced by using the KOD-Plus-Mutagenesis Kit (TOYOBO, Osaka, Japan) with the following primers: V111M (forward: 5′-CTGCGCACCCTGCCGCG-3′; reverse: 5′-CATGGTCATGCGGTAGCGGTGG-3′), Y107H (forward: 5′-CACCGCATGACCGTGCTGCG-3′, reverse: 5′-GCGGTGGGGGCTGGTGC-3′). 
Cell Culture
HEK 293T cells were maintained in Dulbecco's modified Eagle's medium (Sigma-Aldrich Corp., St. Louis, MO, USA) supplemented with 10% FBS and penicillin–streptomycin (Gibco, Thermo Scientific). For protein expression assays, plasmids were transfected into HEK293T cell lines with TransIT-PRO Transfection Kit (Mirus Bio, Madison, WI, USA). For proteasome inhibitory assay, HEK293T cells were treated with or without 50 μg/mL cycloheximide and 10 μM MG132 (Calbiochem, Merck Millipore, Darmstadt, Hesse, Germany) for 6 hours, and lysed with RIPA. Sodium dodecyl phosphate–PAGE and Western blot were performed as described above. 
Results
Clinical Information
Clinical information of all the patients is summarized in Table 2. Patients 1-1 and 1-2 (Fig. 1A) have been previously diagnosed with early-onset RP with osteochondrodysplasia.19 The patients were followed up for 14 years with regular ophthalmic tests and additional screening. As reported previously,19 the patients have short stature (<mean ± 3 standard deviations) and narrowed thoraxes (Fig. 1C, upper panel; Table 2). Detailed examination revealed that patient 1-1 had shortening of the metacarpals in both hands (Fig. 1C, lower panel; Table 2). The parents of both patients noticed night blindness in their children from 3 years of age. Dilated funduscopy of patient 1-1 showed a salt-and-pepper appearance and narrowed retinal vessels at the age of 20 years (Fig. 1D), with minor difference from the funduscopy at age 9 years.19 Electroretinogram of the combined rod and cone response (Flash) was nonrecordable (Fig. 1E). A visual field test showed loss of middle peripheral area and decreased sensitivity in the central area in both eyes (Fig. 1F). Further, optical coherence tomography (OCT) showed thinning and disorganization of the photoreceptor layer outside the macular area (Fig. 1G). The symptoms of patient 1-2, indicated by the fundus image, the OCT, and the ERG, were similar to those of patient 1-1 (Supplementary Fig. S1, data not shown). Short stature, narrow thorax, and pneumonia appeared at almost the same age in patients 1-1 and 1-2 (Table 2). Hearing impairment and renal anomalies were not detected in either patient. Their parents showed no abnormalities related to body development, stature, or ocular history. 
Table 2
 
Summary of Clinical Information of Each Patient
Table 2
 
Summary of Clinical Information of Each Patient
Figure 1
 
Clinical characteristics of patients with mutations in C21orf2. (A) Pedigree of family 1. Each mutated allele was inherited by two siblings. (B) Pedigree of family 2. A homozygous mutation was found in the two affected siblings. Affected individuals are indicated in black. Subjects included in the whole exome sequence are indicated by asterisks. (C) X-ray photographs showing the narrowed thorax of patient 1-2 (upper panel) and showing the shortening of the fifth metacarpal of right hand and the third and fifth metacarpals of left hand of patient 1-1 (lower panel). (DG) Ophthalmic symptoms of patient 1-1. (D) Fundus image showing salt-and-pepper appearance in peripheral retina and the narrowing of retinal vessels. (E) Response was not recordable by combined rod and cone ERG (Flash). (F) Goldmann kinetic visual fields show narrowing of the visual fields. (G) Optical coherence tomography through the macular area shows thinning of photoreceptor layer outside the macula and disappearance of ellipsoid zone out of macular area. (HK) Clinical data from patient 2-1. (H) Fundus images. (I) Electroretinograms from both eyes. (J) Goldmann kinetic visual fields showing a large scotopic area in the macular area. (K) Optical coherence tomography showing retinal degeneration and disappearance of ellipsoid zone.
Figure 1
 
Clinical characteristics of patients with mutations in C21orf2. (A) Pedigree of family 1. Each mutated allele was inherited by two siblings. (B) Pedigree of family 2. A homozygous mutation was found in the two affected siblings. Affected individuals are indicated in black. Subjects included in the whole exome sequence are indicated by asterisks. (C) X-ray photographs showing the narrowed thorax of patient 1-2 (upper panel) and showing the shortening of the fifth metacarpal of right hand and the third and fifth metacarpals of left hand of patient 1-1 (lower panel). (DG) Ophthalmic symptoms of patient 1-1. (D) Fundus image showing salt-and-pepper appearance in peripheral retina and the narrowing of retinal vessels. (E) Response was not recordable by combined rod and cone ERG (Flash). (F) Goldmann kinetic visual fields show narrowing of the visual fields. (G) Optical coherence tomography through the macular area shows thinning of photoreceptor layer outside the macula and disappearance of ellipsoid zone out of macular area. (HK) Clinical data from patient 2-1. (H) Fundus images. (I) Electroretinograms from both eyes. (J) Goldmann kinetic visual fields showing a large scotopic area in the macular area. (K) Optical coherence tomography showing retinal degeneration and disappearance of ellipsoid zone.
Patients 2-1 and 2-3 were Japanese siblings whose parents had no known consanguinity, although both of them were from a small rural area of Osaka prefecture (Fig. 1B). Patients 2-1 and 2-3 did not exhibit short stature or any syndromic disorders including renal, neural, skeletal, or thoracic abnormalities. They did not have any hearing difficulties. Patient 2-1 was 48 years old when she was referred to Kinki University Hospital for the first time with declining vision that was noticed over the previous several years. She reported photoaversion, rather than night blindness (Table 2). She was emmetropic and her decimal BCVA was 0.1 in either eye. Anterior segments and media were normal in both eyes. Dilated funduscopy revealed bull's eye maculopathy and annular degeneration of the retina and RPE within the vascular arcades (Fig. 1H). The far inferior retina was also degenerative with some small, round pigments. The retinal vessels were mildly attenuated. These findings were symmetrical in both eyes, except for round pigments, which were only present in the left eye. The rod and flash ERGs were reduced, and the cone and flicker ERGs were nonrecordable, indicating CRD (Fig. 1I). Visual field test showed large central scotoma in both eyes (Fig. 1J) and OCT revealed abnormally laminated retina and extinguished ellipsoid zone (Fig. 1K). During 21 years of clinical observation, the retinal degeneration had gradually progressed and a few small clumped pigments had appeared in the retinal degeneration. The patient's BCVA at age 69 years was 0.02 in the right eye and 0.04 in the left eye, although she could walk by herself using her peripheral vision. 
Patient 2-3 was the older brother of patient 2-1. He noticed night blindness when he was in high school and visual field constriction in his twenties (Table 2). He had been diagnosed with RP in the 1970s while he was in his late twenties, which progressed into legal blindness at 40 years of age. He lost light perception at 60 years of age. When he first visited our clinic at 64 years of age, his fundi were found to be diffusely degenerated (Supplementary Fig. S1C). He had nuclear cataracts in both eyes, which progressed to mature cataracts in his late sixties, and made it difficult to determine whether he had late-stage RP or CRD. Electroretinogram was nonrecordable in both eyes (Supplementary Fig. S1D). Their nonsymptomatic, generally healthy older sister (2-2) had normal fundi, ERG findings, and OCT images except for a retinal vein occlusion in the right eye (data not shown). 
Identification of Candidate Disease-Causative Mutations in Family 1 and Family 2
In family 1, comparison of patient whole exome sequences with the reference human genome (hs37d5) detected 65,593 and 65,085 variants in patients 1-1 and 1-2, respectively. The patients' whole exome analyses were focused on nucleotide variations causing amino acid changes and occurring at low frequency in the 1000 genome database, HGVD, and our in-house database (Materials and Methods). A total of 109 and 103 variants met these criteria in patients 1-1 (Supplementary Table S2) and 1-2 (Supplementary Table S3), respectively. These mutations were filtered by using the pattern of inheritance (homozygous recessive, heterozygous recessive, or de novo mutation) using parental DNA. C21orf2 was the only identified disease-causing gene with compound heterozygous mutations p.V111M (c.G331A) and p.Y107H (c.T319C) (RefSeq ID: NM_004928). 
In family 2, a total of 70,837 and 71,175 variants were identified in patients 2-1 and 2-3, respectively. A total of 83 and 110 variants were identified as candidates in patients 2-1 (Supplementary Table S4) and 2-3 (Supplementary Table S5), respectively. We filtered the remaining variants by using the pattern of inheritance with sibling DNA and identified C21orf2 as the only putative disease-causing gene. Genetic investigation revealed C21orf2 was a candidate gene with homozygous mutations p.Y107C (c.A320G) (RefSeq ID: NM_004928). Those mutations in C21orf2 were not found in another patient as collected for the Japan Whole Exome Project. 
C21orf2 encodes a short LRR protein with 256 amino acid residues. C21orf2 has very recently been reported as a causal gene for JATD20 and retinal dystrophy with macular staphyloma.21 The previously reported mutations are located in the LRRs and in the C-terminal region (Fig. 2A, lower panel, blue arrowheads). All of the missense mutations identified in this study were located in exon 4 (Fig. 2A, upper panel), corresponding to the LRRCT domain (Fig. 2A, lower panel, black arrowheads). The LRRCT is a capping motif downstream of the last LRR in a subfamily of LRR proteins.22 The mutated amino acid residues Y107 and V111 were involved in the consensus sequence of LRRCT (YRxxΦxxxΦPxΦxxLD) (Fig. 2B). Comparison of the amino acid sequences of the LRRCT between the human C21orf2 and its homologous proteins from monkey, mouse, zebrafish, and Chlamydomonas organisms showed that Y107 and V111 were highly conserved (Fig. 1G, magenta), suggesting their importance for protein function. 
Figure 2
 
Mutations in the LRRCT of C21orf2. (A) Schematic images of C21orf2 mRNA (NM_004928, upper panel) and protein (NP_004919, lower panel). Exon numbers are shown below. The mutations identified in this study are shown in black, and the previously reported mutations are in blue. (B) Comparison of the amino acid sequences of LRRCT from human, cynomolgus monkey (XP_005548606), mouse (NP_080707), zebrafish (XP_003199580), and Chlamydomonas (XP_001692815) C21orf2 homologues. The consensus sequence of LRRCT is shown below the alignment (YRxxΦxxxΦPxΦxxLD). Φ represents a hydrophobic residue, x represents any residue. Mutated amino acid residues V111 and Y107 are shown in magenta.
Figure 2
 
Mutations in the LRRCT of C21orf2. (A) Schematic images of C21orf2 mRNA (NM_004928, upper panel) and protein (NP_004919, lower panel). Exon numbers are shown below. The mutations identified in this study are shown in black, and the previously reported mutations are in blue. (B) Comparison of the amino acid sequences of LRRCT from human, cynomolgus monkey (XP_005548606), mouse (NP_080707), zebrafish (XP_003199580), and Chlamydomonas (XP_001692815) C21orf2 homologues. The consensus sequence of LRRCT is shown below the alignment (YRxxΦxxxΦPxΦxxLD). Φ represents a hydrophobic residue, x represents any residue. Mutated amino acid residues V111 and Y107 are shown in magenta.
C21orf2 Is Expressed in the Photoreceptor Outer Segment
The small interfering RNA (siRNA)-based in vitro knockdown assays indicated that C21orf2 was required for the formation of primary cilia in mammalian cell lines.23 To assess whether the mutations in C21orf2 could lead to retinal degeneration, we first examined the expression patterns of C21orf2 in the retina. Several splice variants were predicted for C21orf2. Since all of the protein isoforms covered the identified mutation, we tested whether a shorter isoform (NM_001271442 for mRNA, NP_001258371 for protein) that starts from the third LRR,24 and a longer isoform (NM_00127144, NP_001258370) that had a 120 amino acid insert in the C-terminal region, were expressed in the retina in addition to isoform 1 (NM_004928, NP_004919). Comparison of the mRNA expression levels between variant 1 and variant 4 (Fig. 3A, upper panel), and between variant 1 and variant 3 (Fig. 3A, lower panel), indicated that variant 1 was the main mRNA expressed in the developing (E16, postnatal day 7 [P7]) and mature retina (5 weeks [5w]) (Fig. 3A, black arrowheads). 
Figure 3
 
C21orf2 is expressed in the photoreceptor outer segment. (A) Reverse transcription–PCR of mouse retina isolated from indicated stages. Amplified product from each splicing variant was indicated as C21orf2_v1, C21orf2_v3, and C21orf2_v4. Size of DNA is indicated to the right. (B) Western blot analysis of mouse and monkey retinal lysates applying anti-C21orf2 antibody. C21orf2 protein was expressed in the mouse retina at the indicated stages (arrowhead). Molecular mass is indicated to the right. (CF) Immunostaining of the mouse and monkey retinal sections with anti-C21orf2 antibody. C21orf2 (green) was detected in the connecting cilium and the outer segment indicated by acTUBA (red) in the mouse (C) and monkey (E) retina. Magnified images are shown in (D) and (F), respectively. White arrows indicate colocalization of C21orf2 and acTUBA. (F) C21orf2 signals were also detected in the inner segments in the monkey retina (arrowheads). Scale bars: 50 μm in the upper panels, 20 μm in the lower panel (C). acTUBA, acetylated tubulin.
Figure 3
 
C21orf2 is expressed in the photoreceptor outer segment. (A) Reverse transcription–PCR of mouse retina isolated from indicated stages. Amplified product from each splicing variant was indicated as C21orf2_v1, C21orf2_v3, and C21orf2_v4. Size of DNA is indicated to the right. (B) Western blot analysis of mouse and monkey retinal lysates applying anti-C21orf2 antibody. C21orf2 protein was expressed in the mouse retina at the indicated stages (arrowhead). Molecular mass is indicated to the right. (CF) Immunostaining of the mouse and monkey retinal sections with anti-C21orf2 antibody. C21orf2 (green) was detected in the connecting cilium and the outer segment indicated by acTUBA (red) in the mouse (C) and monkey (E) retina. Magnified images are shown in (D) and (F), respectively. White arrows indicate colocalization of C21orf2 and acTUBA. (F) C21orf2 signals were also detected in the inner segments in the monkey retina (arrowheads). Scale bars: 50 μm in the upper panels, 20 μm in the lower panel (C). acTUBA, acetylated tubulin.
Protein expression levels were also examined in the mouse and the monkey retina. A band corresponding to the size of isoform 1 was detected in the developing and mature retina (Fig. 3B, black arrowhead). C21orf2 protein was also detected in the monkey retina (Fig. 3B), which appeared heavier than the mouse C21orf2, corresponding to the longer predicted amino acid (aa) length (318 aa for cynomolgus monkey, 249 aa for mouse). C21orf2 was detected in the connecting cilium of the photoreceptor cells in the mouse and monkey retina (Figs. 3C–F, green) and was associated with acetylated tubulin (Figs. 3D, 3F, red). In the monkey retina, C21orf2 was also detected in the inner segments (Fig. 3F, arrowheads). Negative controls are shown in Supplementary Figure S2
Mutations in the LRRCT Affected the Amount and Localization of C21orf2 Protein
The LRRCT was required for the proper folding of LRR protein,9 and mutations in the LRRCT were expected to affect the stability of C21orf2. Thus, we next compared the expression levels of transfected wild-type C21orf2 (WT), Y107H mutant, or V111M mutant proteins in vitro (Fig. 4A). Expression levels of Y107H (0.66 ± 0.13, mean ± standard error) and V111M (0.59 ± 0.12) were significantly reduced compared to the WT (1.00 ± 0.1). Mutant proteins were preferentially degraded by the proteasome.25 Thus, we tested whether treatment with a proteasome inhibitor could rescue the expression levels of mutated proteins (Supplementary Fig. S3). The protein expression of the Y107H mutant increased to levels similar to those of the WT protein (CHX/MG132: Y107H: 0.93 ± 0.22, WT: 0.87 ± 0.29) after treatment with the proteasome inhibitor MG132, indicating that Y107H was degraded by the proteasome. Because C21orf2 located to the ciliary region of the photoreceptors in association with acetylated tubulin (Figs. 3C, 3D), we also tested if the mutations affected protein localization of C21orf2 in vitro (Fig. 4B). Flag-tagged WT protein made fiber-like structures in the NIH3T3 cells; however, Y107H protein looked sparse and dotted in the cytoplasm, and V111M showed intermediate phenotype. These data suggested that the Y107H or V111M mutations enhanced C21orf2 protein degradation and affected protein localization. 
Figure 4
 
Mutated C21orf2 protein was degraded. (A) Western blot of the FLAG-tagged WT, Y107H mutant, and V111M mutant of C21orf2 protein expressed in the HEK293T cells. Relative expression levels normalized to actin (ACTB) were shown in the right panel. *: i < 0.05. (B) Cytoplasmic localization of FLAG-tagged WT, Y107H mutant, and V111M mutant proteins (green). Magnified images of dotted rectangles are inserted. Scale bars: 20 μm.
Figure 4
 
Mutated C21orf2 protein was degraded. (A) Western blot of the FLAG-tagged WT, Y107H mutant, and V111M mutant of C21orf2 protein expressed in the HEK293T cells. Relative expression levels normalized to actin (ACTB) were shown in the right panel. *: i < 0.05. (B) Cytoplasmic localization of FLAG-tagged WT, Y107H mutant, and V111M mutant proteins (green). Magnified images of dotted rectangles are inserted. Scale bars: 20 μm.
Discussion
The WES in two pedigrees revealed novel missense mutations in the C21orf2 gene in Japanese patients with RP and CRD. The C21orf2 protein localized to the cilium of retinal photoreceptor cells. Reduced expression and mislocalization of mutated proteins were detected as compared to wild type in vitro, suggesting that a decrease in functional protein led to the phenotypes of recessive retinal ciliopathies. 
In recent reports, C21orf2 has been reported as a causal gene for JATD,20 a syndromic ciliopathy with retinal degeneration, and for retinal dystrophy with macular staphyloma.21 Those reports suggest that mutations in C21orf2 could cause several types of retinal ciliopathy in a syndromic and nonsyndromic manner, which was also supported by this study showing that mutations in C21orf2 were associated with RP with skeletal defects and also with nonsyndromic CRD. Thus far, C21orf2 mutations have been primarily associated with photoreceptor degenerations, and they also cause skeletal anormality in some cases, diagnosed as JATD. The genotype–phenotype correlations are not clear yet, though whether the symptoms are isolated to the retina or syndromic seem to be dependent on the individual genetic background. For example, compound heterozygous mutations (p.L224P, p.R73P) have been associated with two JATD patients with CRD and also with a nonsyndromic CRD patient.20,26 In addition, another group21 has described that only one of two patients carrying the same mutation exhibits short stature. In our study, the phenotypic variation could be due to the different impacts of compound heterozygous mutations and homozygous mutations, and also due to the individual genetic background. 
All the identified missense mutations in this study were located in the highly-conserved LRRCT region of the C21orf2 protein. Further, two unrelated families had mutations in the first tyrosine residue of the LRRCT consensus sequence. It was of great interest to us that a kinetic study27 has shown that LRRCT is an essential core domain required for proper folding of the LRR protein PP32. Since the replacement of the first tyrosine or the fifth valine of the LRRCT consensus sequence affects the folding of LRR protein,10,27 missense mutations in these amino acids were expected to decrease the levels of functional protein. We preferentially studied the compound heterozygous mutations to confirm the mutation impacts on both Y107 and V111. Our protein expression assays showed lower amounts and different cytoplasmic localizations of Y107H and V111M mutant proteins as compared to wild type. In particular, the Y107H mutation was expected to change the local charge by replacing the hydrophilic uncharged tyrosine with hydrophilic basic histidine. The other mutation, Y107C, was also expected to affect the C21orf2 protein folding, since the mutation removed the hydroxyl bond between the tyrosine and aspartic acid, similar to the replacement of the tyrosine residue with the hydrophobic phenylalanine.10 In vitro knockdown assays indicate that C21orf2 is necessary for ciliogenesis,20,23 and thus, reduced expression of C21orf2 protein may have had a significant impact on ciliogenesis or the maintenance of ciliary function in photoreceptor cells. Disease-related mutations in C21orf2 have been only recently reported, and we did not find additional patients having mutations in the LRRCT of C21orf2 in our cohort. Further genetic studies will reveal whether the mutations in LRRCT occur frequently in Japanese and global populations. 
In this report, we identified novel mutations in C21orf2 in Japanese patients with autosomal recessive RP or CRD. The mutations reduced the amount of C21orf2 protein, which led to photoreceptor degeneration, and in some cases, skeletal defects through abnormal cilia formation and ciliary function. 
Acknowledgments
Supported in part by a grant to TI from the Japan Agency for Medical Research and Development, Practical Research Project for Rare/Intractable Diseases, 15ek0109072h0002. Computations were partially performed on the supercomputer at National Institute of Genetics, Research Organization of Information and Systems. 
Disclosure: A. Suga, None; A. Mizota, None; M. Kato, None; K. Kuniyoshi, None; K. Yoshitake, None; W. Sultan, None; M. Yamazaki, None; Y. Shimomura, None; K. Ikeo, None; K. Tsunoda, None; T. Iwata, None 
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Figure 1
 
Clinical characteristics of patients with mutations in C21orf2. (A) Pedigree of family 1. Each mutated allele was inherited by two siblings. (B) Pedigree of family 2. A homozygous mutation was found in the two affected siblings. Affected individuals are indicated in black. Subjects included in the whole exome sequence are indicated by asterisks. (C) X-ray photographs showing the narrowed thorax of patient 1-2 (upper panel) and showing the shortening of the fifth metacarpal of right hand and the third and fifth metacarpals of left hand of patient 1-1 (lower panel). (DG) Ophthalmic symptoms of patient 1-1. (D) Fundus image showing salt-and-pepper appearance in peripheral retina and the narrowing of retinal vessels. (E) Response was not recordable by combined rod and cone ERG (Flash). (F) Goldmann kinetic visual fields show narrowing of the visual fields. (G) Optical coherence tomography through the macular area shows thinning of photoreceptor layer outside the macula and disappearance of ellipsoid zone out of macular area. (HK) Clinical data from patient 2-1. (H) Fundus images. (I) Electroretinograms from both eyes. (J) Goldmann kinetic visual fields showing a large scotopic area in the macular area. (K) Optical coherence tomography showing retinal degeneration and disappearance of ellipsoid zone.
Figure 1
 
Clinical characteristics of patients with mutations in C21orf2. (A) Pedigree of family 1. Each mutated allele was inherited by two siblings. (B) Pedigree of family 2. A homozygous mutation was found in the two affected siblings. Affected individuals are indicated in black. Subjects included in the whole exome sequence are indicated by asterisks. (C) X-ray photographs showing the narrowed thorax of patient 1-2 (upper panel) and showing the shortening of the fifth metacarpal of right hand and the third and fifth metacarpals of left hand of patient 1-1 (lower panel). (DG) Ophthalmic symptoms of patient 1-1. (D) Fundus image showing salt-and-pepper appearance in peripheral retina and the narrowing of retinal vessels. (E) Response was not recordable by combined rod and cone ERG (Flash). (F) Goldmann kinetic visual fields show narrowing of the visual fields. (G) Optical coherence tomography through the macular area shows thinning of photoreceptor layer outside the macula and disappearance of ellipsoid zone out of macular area. (HK) Clinical data from patient 2-1. (H) Fundus images. (I) Electroretinograms from both eyes. (J) Goldmann kinetic visual fields showing a large scotopic area in the macular area. (K) Optical coherence tomography showing retinal degeneration and disappearance of ellipsoid zone.
Figure 2
 
Mutations in the LRRCT of C21orf2. (A) Schematic images of C21orf2 mRNA (NM_004928, upper panel) and protein (NP_004919, lower panel). Exon numbers are shown below. The mutations identified in this study are shown in black, and the previously reported mutations are in blue. (B) Comparison of the amino acid sequences of LRRCT from human, cynomolgus monkey (XP_005548606), mouse (NP_080707), zebrafish (XP_003199580), and Chlamydomonas (XP_001692815) C21orf2 homologues. The consensus sequence of LRRCT is shown below the alignment (YRxxΦxxxΦPxΦxxLD). Φ represents a hydrophobic residue, x represents any residue. Mutated amino acid residues V111 and Y107 are shown in magenta.
Figure 2
 
Mutations in the LRRCT of C21orf2. (A) Schematic images of C21orf2 mRNA (NM_004928, upper panel) and protein (NP_004919, lower panel). Exon numbers are shown below. The mutations identified in this study are shown in black, and the previously reported mutations are in blue. (B) Comparison of the amino acid sequences of LRRCT from human, cynomolgus monkey (XP_005548606), mouse (NP_080707), zebrafish (XP_003199580), and Chlamydomonas (XP_001692815) C21orf2 homologues. The consensus sequence of LRRCT is shown below the alignment (YRxxΦxxxΦPxΦxxLD). Φ represents a hydrophobic residue, x represents any residue. Mutated amino acid residues V111 and Y107 are shown in magenta.
Figure 3
 
C21orf2 is expressed in the photoreceptor outer segment. (A) Reverse transcription–PCR of mouse retina isolated from indicated stages. Amplified product from each splicing variant was indicated as C21orf2_v1, C21orf2_v3, and C21orf2_v4. Size of DNA is indicated to the right. (B) Western blot analysis of mouse and monkey retinal lysates applying anti-C21orf2 antibody. C21orf2 protein was expressed in the mouse retina at the indicated stages (arrowhead). Molecular mass is indicated to the right. (CF) Immunostaining of the mouse and monkey retinal sections with anti-C21orf2 antibody. C21orf2 (green) was detected in the connecting cilium and the outer segment indicated by acTUBA (red) in the mouse (C) and monkey (E) retina. Magnified images are shown in (D) and (F), respectively. White arrows indicate colocalization of C21orf2 and acTUBA. (F) C21orf2 signals were also detected in the inner segments in the monkey retina (arrowheads). Scale bars: 50 μm in the upper panels, 20 μm in the lower panel (C). acTUBA, acetylated tubulin.
Figure 3
 
C21orf2 is expressed in the photoreceptor outer segment. (A) Reverse transcription–PCR of mouse retina isolated from indicated stages. Amplified product from each splicing variant was indicated as C21orf2_v1, C21orf2_v3, and C21orf2_v4. Size of DNA is indicated to the right. (B) Western blot analysis of mouse and monkey retinal lysates applying anti-C21orf2 antibody. C21orf2 protein was expressed in the mouse retina at the indicated stages (arrowhead). Molecular mass is indicated to the right. (CF) Immunostaining of the mouse and monkey retinal sections with anti-C21orf2 antibody. C21orf2 (green) was detected in the connecting cilium and the outer segment indicated by acTUBA (red) in the mouse (C) and monkey (E) retina. Magnified images are shown in (D) and (F), respectively. White arrows indicate colocalization of C21orf2 and acTUBA. (F) C21orf2 signals were also detected in the inner segments in the monkey retina (arrowheads). Scale bars: 50 μm in the upper panels, 20 μm in the lower panel (C). acTUBA, acetylated tubulin.
Figure 4
 
Mutated C21orf2 protein was degraded. (A) Western blot of the FLAG-tagged WT, Y107H mutant, and V111M mutant of C21orf2 protein expressed in the HEK293T cells. Relative expression levels normalized to actin (ACTB) were shown in the right panel. *: i < 0.05. (B) Cytoplasmic localization of FLAG-tagged WT, Y107H mutant, and V111M mutant proteins (green). Magnified images of dotted rectangles are inserted. Scale bars: 20 μm.
Figure 4
 
Mutated C21orf2 protein was degraded. (A) Western blot of the FLAG-tagged WT, Y107H mutant, and V111M mutant of C21orf2 protein expressed in the HEK293T cells. Relative expression levels normalized to actin (ACTB) were shown in the right panel. *: i < 0.05. (B) Cytoplasmic localization of FLAG-tagged WT, Y107H mutant, and V111M mutant proteins (green). Magnified images of dotted rectangles are inserted. Scale bars: 20 μm.
Table 1
 
Antibodies Used in This Study
Table 1
 
Antibodies Used in This Study
Table 2
 
Summary of Clinical Information of Each Patient
Table 2
 
Summary of Clinical Information of Each Patient
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