Kazuki Kuniyoshi

Whole Exome Analysis Identifies Frequent CNGA1 Mutations in Japanese Population with Autosomal Recessive Retinitis Pigmentosa

Objective The purpose of this study was to investigate frequent disease-causing gene mutations in autosomal recessive retinitis pigmentosa (arRP) in the Japanese population. Methods In total, 99 Japanese patients with non-syndromic and unrelated arRP or sporadic RP (spRP) were recruited in this study and ophthalmic examinations were conducted for the diagnosis of RP. Among these patients, whole exome sequencing analysis of 30 RP patients and direct sequencing screening of all CNGA1 exons of the other 69 RP patients were performed. Results Whole exome sequencing of 30 arRP/spRP patients identified disease-causing gene mutations of CNGA1 (four patients), EYS (three patients) and SAG (one patient) in eight patients and potential disease-causing gene variants of USH2A (two patients), EYS (one patient), TULP1 (one patient) and C2orf71 (one patient) in five patients. Screening of an additional 69 arRP/spRP patients for the CNGA1 gene mutation revealed one patient with a homozygous mutation. Conclusions This is the first identification of CNGA1 mutations in arRP Japanese patients. The frequency of CNGA1 gene mutation was 5.1% (5/99 patients). CNGA1 mutations are one of the most frequent arRP-causing mutations in Japanese patients.

Electroretinography of short-wavelength-sensitive cones with a LED built-in electrode and its normal values.

To record electroretinograms (ERG) produced by short-wavelength-sensitive cone mechanisms (SWS-cone ERG), the authors used three kinds (blue, green, and red) of light-emitting diode (LED) which were built into a contact lens electrode assembly. The LEDs were used as both stimulus and background light sources. ERG was recorded using blue LED after 10 min of yellow light adaptation produced by green and red LEDs. Duration of photo-stimulation was either 2 or 100 ms. ERG recorded in normal human subjects showed two positive waves with 2 ms photo-stimulation. Amplitude of the former positive wave (b1-wave) was attenuated when the luminance of yellow background increased, and the latter positive wave (b2-wave) was attenuated when the color of photo-stimulation was green or red. These findings suggest that middle-wavelength-sensitive and long-wavelength-sensitive cone mechanisms generated the former positive wave (b1-wave) and SWS-cone mechanisms generated the latter positive wave (b2-wave). Ratio of b2-wave-amplitude to b1-wave-amplitude with 2 ms photo-stimulation measured on 39 normal subjects ranged from 0.5 to 2.0. It was concluded that this three-colored LED built-in electrode was useful for recording SWS-cone ERG.

Novel RP1L1 Variants and Genotype-Photoreceptor Microstructural Phenotype Associations in Cohort of Japanese Patients With Occult Macular Dystrophy.

PURPOSE To determine the clinical and genetic characteristics of Japanese patients with occult macular dystrophy (OMD) in a nationwide multicenter study. METHODS Twenty-three patients from 21 families with clinically diagnosed OMD were studied at 10 institutions throughout Japan. Ophthalmologic examinations including spectral-domain optic coherence tomography were performed. Patients were classified into two phenotype groups: a classical group having both blurred ellipsoid zone and absence of interdigitation zone of the photoreceptors, and a nonclassical group lacking at least one of these two features. Whole-exome sequencing, direct sequencing, and in silico molecular analysis were performed to detect the pathogenic RP1L1 variants. Statistical associations between the phenotype and genotypes based on the presence of pathogenic RP1L1 variants were investigated. RESULTS There were 12 families with the classical findings and 9 families with the nonclassical findings. Nine pathogenic RP1L1 missense variants were identified in 12 families (57%) including three reported variants, namely, p.R45W, p.S1199C, and p.G1200A, and six novel variants, p.G221R, p.T1194M, p.T1196I, p.G1200D, p.G1200V, and p.V1201G. The pathogenic missense variants in seven families (33%) were located between amino acid numbers 1196 and 1201. A significant association was found between the photoreceptor microstructural phenotypes and molecular genotypes. CONCLUSIONS The spectrum of the morphologic phenotypes and pathogenic RP1L1 variants was documented in a well-characterized Japanese cohort with OMD. A unique motif including six amino acids (1196-1201) downstream of the doublecortin domain could be a hot spot for RP1L1 pathogenic variants. The significant association of the morphologic phenotypes and genotypes indicates that there are two types of pathophysiology underlying the occult macular dysfunction syndrome: a hereditary OMD with the classical phenotype (Miyakes disease), and a nonhereditary OMD-like syndrome with progressive occult maculopathy.

Two siblings with late-onset cone-rod dystrophy and no visible macular degeneration.

Background We report our findings in two siblings with late-onset cone–rod dystrophy (CRD) with no visible macular degeneration. Cases and methods Case 1 was an 82-year-old man who first noticed a decrease in vision and color blindness in his early seventies. His mother and younger sister also had visual disturbances. His decimal visual acuity was 0.3 in the right eye and 0.2 in the left eye. Ophthalmoscopy showed normal fundi, and fluorescein angiography was also normal in both eyes. The photopic single flash and flicker eletroretinograms (ERGs) were severely attenuated and the scotopic ERGs were slightly reduced in both eyes. Case 2 was the 80-year-old younger sister of Case 1. She first noticed a decline in vision and photophobia in both eyes in her early seventies. Her decimal visual acuity was 0.4 in the right eye and 0.2 in the left eye. Ophthalmoscopy showed mottling of the retinal pigment epithelium in the midperiphery with no visible macular degeneration. The photopic single flash and flicker ERGs were severely attenuated, and the scotopic ERGs were slightly reduced in both eyes. Conclusion These siblings are the oldest reported cases of CRD with no visible macular degeneration. Thus, CRD should be considered in patients with reduced visual acuity, color blindness, and photophobia even if they are older than 70 years.

Intravitreal bevacizumab injection and carotid artery stent replacement for neovascular glaucoma in internal carotid artery occlusion

Neovascular glaucoma (NVG) secondary to internal carotid artery (ICA) occlusion is usually resistant to treatment. We report a case of NVG with ICA occlusion improved by intravitreal bevacizumab (IVB) injection and carotid artery stent replacement (CAS), even though we did not perform panretinal photocoagulation. A 67-year-old male with NVG noted visual loss in his left eye. Magnetic resonance angiography showed left ICA occlusion. He was diagnosed with NVG secondary to ICA occlusion. The next day, we carried out IVB injection in his left eye, following which the iris and angle neovascularization regressed, and the intraocular pressure decreased to normal within a day after the injection. CAS was performed on his left ICA at a month post injection. Two months later, we reinjected bevacizumab in his left eye. His condition remained stable with no recurrence over two years. This case indicates that IVB injection and CAS are useful for early-stage NVG secondary to ICA occlusion.

Identification of Novel Mutations in the LRR-Cap Domain of C21orf2 in Japanese Patients With Retinitis Pigmentosa and Cone–Rod Dystrophy

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.

Multifocal electroretinograms in Stargardt's disease/fundus flavimaculatus.

Purpose: To determine whether the characteristics of multifocal electroretinograms (mfERGs) were correlated with the ophthalmic appearance of the fundus in patients with Stargardts disease/fundus flavimaculatus (SFF). Methods: Full-field ERGs, mfERGs, and general ophthalmic examinations were performed on 49 eyes with SFF. Results: The SFF patients were divided into four subtypes according to the classification of Noble and Carr [Arch Ophthalmol 1979;97:1281-1285]. The patients with type 1 SFF had severely reduced mfERGs in the macular area and reduced and delayed responses in the mid-periphery. The type 2 SFF patients had reduced but recordable mfERGs from the center of the macula with more depressed responses in the paramacular area, and the type 3 SFF patients had reduced and delayed mfERGs both in the macula and periphery. The patients with type 4 SFF had normal mfERGs in the macular area and delayed responses in all outer zones. Conclusions: These mfERG findings indicate that each subtype of SFF has unique characteristics corresponding to the abnormal retinal functions.

Novel Mutations in Enhanced S-cone Syndrome

Dear Editor: Enhanced S-cone syndrome (ESCS) is a rare and unique retinal dystrophy with a pattern of autosomal-recessive inheritance. Patients with ESCS show night blindness and high sensitivity to short-wavelength light, because of the 2-fold increased number of short-wavelength-sensitive cones (S cones) with absence of rods in the retina. Since the first discovery of mutations in the NR2E3 gene on chromosome 15q23 in patients with ESCS, 40 mutations have been reported as causes of ESCS and allied diseases (Fig 1, available online at http://aaojournal.org). In this letter, we report novel mutations in the NR2E3 gene that were discovered in 2 cases with ESCS. Cases are 2 Japanese patients who were reported previously. Case 1 was a 31-year-old man whose parents were consanguineous. His vision was 0.7 in the right eye and 0.3 in the left eye. Funduscopy revealed retinal degeneration surrounding the vascular arcade with cystic changes in both maculae (Fig 2, available online at http://aaojournal.org). Perimetry showed ring-shaped scotoma and electrophysiology showed unique responses corresponding to ESCS (Figs 3–6, available online at http://aaojournal.org). During 23-year clinical follow-up, clumped pigmentation has appeared in the retinal degeneration and the cystic changes in the foveal region have become ambiguous (Fig 2). His latest vision was 0.5 in the right eye and 0.3 in the left eye at age 53. Genetic analysis revealed a novel nucleotide substitution (c.151G A) in exon 2 homozygously, resulting in a novel missense mutation (a glycine-to-arginine substitution) at amino acid position 51 (p.G51R; Fig 7; available online at http://aaojournal.org). Case 2 was a 78-year-old woman. Funduscopy showed diffuse mild retinal degeneration with no pigmentation in both eyes (Fig 8, available online at http://aaojournal.org). Optical coherence tomography showed a subtle foveal schisis in the left eye, although the structure of the retina including the outer nuclear layer in the macular area was relatively well maintained (Fig 8). After cataract surgery, her vision improved to 0.3 in the right eye and 0.2 in the left. Genetic analysis revealed compound heterozygous mutations of c.142C T (exon 2) and c.311G A (exon 3), resulting in an arginine-to-cysteine substitution at amino acid position 48 (p.R48C) and an arginine-to-glutamine substitution at amino acid position 104 (p.R104Q; Fig 7). A daughter of case 2 who was asymptomatic and had normal fundus appearance showed only the p.R48C mutation heterozygously, that indicated she was an unaffected carrier relative (Fig 7). NR2E3 protein, a photoreceptor-specific orphan nuclear receptor, plays an important role in the development and differentiation of rods and all cone classes. NR2E3 has 2 functionally important domains, namely, the DNA-binding domain (DBD) and the ligand-binding domain (Fig 1). Mutations within these domains result in serious dysfunction of NR2E3 protein leading to abnormal process of development and differentiation of multipotent progenitor cells to rods and cones. Genetic analysis revealed a homozygous mutation (p.G51R) in case 1 and compound heterozygous mutations of (p.R48C) and (p.R104Q) in case 2. Among these mutations, (p.G51R) and (p.R48C) are novel as causative mutations of ESCS. The mutation (p.G51R) found in case 1 resides in the first zinc finger of the DBD, and the compound heterozygous mutations (p.R48C and p.R104Q), which were found in case 2 reside in the first and second zinc fingers of the DBD. Because the zinc fingers are necessary for maintenance the structure of NR2E3 protein, these mutations in the zinc fingers of NR2E3 result in phenotypes as ESCS (Fig 1). Clinically, case 1 with a homozygous missense mutation (p.G51R) showed typical features as ESCS, whereas case 2 with compound heterozygous mutations (p.R48C and p.R104Q) showed mild retinal degeneration and has kept some level of vision and construction of the macula despite advanced age. In the past, the mutation (p.R104Q) has never been reported except in 1 case, which demonstrated the normal structure and function of the macula with recordable rod electrophysiology. These facts indicate the mutation (p.R104Q) may be correlated with the relatively mild clinical findings as ESCS. We identified 2 novel missense mutations (p.G51R and p.R48C) as causes of ESCS. To our knowledge, the finding of case 1 is the longest-observed clinical case ever reported, and case 2 is the oldest case among all the patients with ESCS so far reported. KAZUKI KUNIYOSHI, MD, TAKAAKI HAYASHI, MD, HIROYUKI SAKURAMOTO, MD, AKIRA NAKAO, MD, TAKASHI SATO, MD, TOMOHIRO UTSUMI, HIROSHI TSUNEOKA, MD, YOSHIKAZU SHIMOMURA, MD Department of Ophthalmology, Kinki University, Faculty of Medicine, Osaka, Japan; Department of Ophthalmology, The Jikei University School of Medicine, Tokyo, Japan

Identification of vitreous proteins in retinopathy of prematurity

Retinopathy of prematurity (ROP) is a disorder of blood vessels in the retina developed in premature infants and the leading cause of the blindness in children. Proteomic analysis was performed to identify vitreous proteins specific to patients with ROP. Vitreous humor samples were obtained from three patients with ROP and two patients with congenital cataract, the latter included as a control group. The vitreous samples were separated by 2D-PAGE and the proteins running as definitive spots were identified by MALDI-TOF MS spectrometry. We identified 13 and 6 proteins in the vitreous from ROP and cataract patients, respectively. Albumin, transferrin, pigment epithelium-derived factor (PEDF) and transthyretin were found in both patient groups. In the samples from ROP patients, PEDF and transthyretin levels were lower than in those from cataract patients, and retinol binding protein 3 and prostaglandin D synthase were not detected. Of the 13 proteins, 9 proteins including α-2-macroglobulin, ceruloplasmin, α-fetoprotein, vitamin D-binding protein, α-1-antitrypsin, α-1-β-glycoprotein, hemopexin, apolipoprotein A-1 and A-lV were found in vitreous samples of only the ROP patients. PEDF has anti-angiogenic and neurotrophic functions. Whether PEDF is increased or decreased in diabetic retinopathy has been controversial but we observed lower PEDF in the ROP samples than in the controls. The proteins specific to or decreased in ROP, if confirmed in future studies, may provide clue to understanding its pathogenesis.

Optical coherence tomographic findings at the fixation point in a case of bilateral congenital macular coloboma

Background Congenital macular coloboma is a rare ocular disease that consists of atrophic lesions in the macula with well-circumscribed borders. We report the findings of spectral domain optical coherence tomography (SD-OCT) at the fixation point in a case of bilateral macular coloboma. Case report The subject is a 4-year-old boy. He visited our hospital at age 1 year and 4 months for the evaluation of strabismus. The fundus examination of both eyes showed round-shaped sharply-demarcated atrophic lesions involving the macula with large choroidal vessels and bared sclera at the base. Immunologic tests including toxoplasmosis, rubella, varicella, herpes virus, and human T-cell leukemia virus were all negative. At age 4 years and 1 month, cycloplegic refraction showed insignificant refractive errors and his best corrected visual acuity was 0.6 bilaterally. The SD-OCT showed a crater-like depression accompanying atrophic neurosensory retina, and the absence of retinal pigment epithelium and choroid. Examination of the fixation behavior by visuscope showed steady fixation with an area 0.5° nasal to the nasal edge of the atrophic lesion bilaterally. The SD-OCT findings at fixation area showed remaining normal retinal structures involving inner segment-outer segment (IS/OS) junction line. Conclusion The findings of SD-OCT have been shown to be useful in the diagnosis of macular coloboma. In the fixation point, the structure of retina and choroid were well preserved.