Katsuhiro Hosono

Two novel mutations in the EYS gene are possible major causes of autosomal recessive retinitis pigmentosa in the Japanese population.

Retinitis pigmentosa (RP) is a highly heterogeneous genetic disease including autosomal recessive (ar), autosomal dominant (ad), and X-linked inheritance. Recently, arRP has been associated with mutations in EYS (Eyes shut homolog), which is a major causative gene for this disease. This study was conducted to determine the spectrum and frequency of EYS mutations in 100 Japanese arRP patients. To determine the prevalence of EYS mutations, all EYS exons were screened for mutations by polymerase chain reaction amplification, and sequence analysis was performed. We detected 67 sequence alterations in EYS, of which 21 were novel. Of these, 7 were very likely pathogenic mutations, 6 were possible pathogenic mutations, and 54 were predicted non-pathogenic sequence alterations. The minimum observed prevalence of distinct EYS mutations in our study was 18% (18/100, comprising 9 patients with 2 very likely pathogenic mutations and the remaining 9 with only one such mutation). Among these mutations, 2 novel truncating mutations, c.4957_4958insA (p.S1653KfsX2) and c.8868C>A (p.Y2956X), were identified in 16 patients and accounted for 57.1% (20/35 alleles) of the mutated alleles. Although these 2 truncating mutations were not detected in Japanese patients with adRP or Lebers congenital amaurosis, we detected them in Korean arRP patients. Similar to Japanese arRP results, the c.4957_4958insA mutation was more frequently detected than the c.8868C>A mutation. The 18% estimated prevalence of very likely pathogenic mutations in our study suggests a major involvement of EYS in the pathogenesis of arRP in the Japanese population. Mutation spectrum of EYS in 100 Japanese patients, including 13 distinct very likely and possible pathogenic mutations, was largely different from the previously reported spectrum in patients from non-Asian populations. Screening for c.4957_4958insA and c.8868C>A mutations in the EYS gene may therefore be very effective for the genetic testing and counseling of RP patients in Japan.

YPEL5 protein of the YPEL gene family is involved in the cell cycle progression by interacting with two distinct proteins RanBPM and RanBP10

YPEL5 is a member of the YPEL gene family that is highly conserved in the eukaryotic species and apparently involved in a certain cell division-related function. In this study, we examined the functional and phylogenetic aspects of YPEL5 protein in more detail. During cell cycle, YPEL5 protein was detected at different subcellular localizations; at interphase, it was located in the nucleus and centrosome, then it changed location sequentially to spindle poles, mitotic spindle, and spindle midzone during mitosis, and finally transferred to midbody at cytokinesis. Knockdown of YPEL5 function by siRNA or anti-sense morpholino oligonucleotide inhibited the growth of cultured COS-7 cells and early development of medaka fish embryos, indicating its involvement in cell cycle progression. Interestingly, RanBPM (Ran Binding Protein in the Microtubule organizing center, encoded by RANBP9) was identified as a YPEL5-binding protein by yeast two-hybrid method. A paralog of RanBPM, namely RanBP10 (encoded by RANBP10), was found to be another YPEL5-binding protein, and these two protein genes are highly conserved each other. Comparative genomic analysis allowed us to define a new gene family consisting of RanBPM and RanBP10, named Scorpin, providing a basis to better understand how they interact with YPEL5.

Mutation analysis of the MYO7A and CDH23 genes in Japanese patients with Usher syndrome type 1

Usher syndrome (USH) is an autosomal recessive disorder characterized by retinitis pigmentosa and hearing loss. USH type 1 (USH1), the second common type of USH, is frequently caused by MYO7A and CDH23 mutations, accounting for 70–80% of the cases among various ethnicities, including Caucasians, Africans and Asians. However, there have been no reports of mutation analysis for any responsible genes for USH1 in Japanese patients. This study describes the first mutation analysis of MYO7A and CDH23 in Japanese USH1 patients. Five mutations (three in MYO7A and two in CDH23) were identified in four of five unrelated patients. Of these mutations, two were novel. One of them, p.Tyr1942SerfsX23 in CDH23, was a large deletion causing the loss of 3 exons. This is the first large deletion to be found in CDH23. The incidence of the MYO7A and CDH23 mutations in the study population was 80%, which is consistent with previous findings. Therefore, mutation screening for these genes is expected to be a highly sensitive method for diagnosing USH1 among the Japanese.

Genotype–phenotype correlation of PAX6 gene mutations in aniridia

The objective of this study was to investigate the genotype–phenotype correlation of the PAX6 gene in aniridia. We clinically examined 5 families and 16 sporadic patients with aniridia. We performed chromosomal analysis and PCR analysis of the PAX6 gene using patient genomic DNA. Chromosomal analysis demonstrated deletions at 11p13 in one allele in four sporadic patients. Seven nonsense mutations, two frameshifts (two insertions), four splice junction errors and two missense mutations were found, and all were heterozygous. The iris phenotype ranged from total to normal in each patient, and the characteristic phenotypes, including cataract, glaucoma or optic nerve hypoplasia, varied widely even among members of the same family. Foveal hypoplasia was detected in all patients except for one. No obvious genotype–phenotype correlation was identified; however, the aniridia phenotype between the two eyes in each patient was quite similar in all patients. Because PAX6 regulates numerous downstream genes and its expression is regulated by several factors during eye development, the aniridia phenotype may be complex even in family members. However, because PAX6 regulation, resulting from both paternal and maternal alleles associated with PAX6, is considered to be roughly similar in both eyes of each patient, the aniridia phenotype may be similar in both eyes of each patient.

Clinical Phenotype in Ten Unrelated Japanese Patients with Mutations in the EYS Gene

ABSTRACT Background: To characterize the clinical phenotypes associated with previously-reported mutations of the eyes shut homolog (EYS) gene, including a truncating mutation, c.4957_4958insA, which is a major causative mutation for retinitis pigmentosa (RP) in Japan. Materials and Methods: The study population comprised ten unrelated RP subjects with very likely pathogenic mutations in both alleles, four of them with a homozygous c.4957_4958insA mutation. The phenotype analysis was based on ophthalmic examination, Goldmann perimetry, and digital fundus photography. Results: The study population included six men and four women aged 34–74 years. The average age at first visit was 31 years (range, 14–44 years), and the patients typically presented with night blindness as the initial symptom and subsequently developed progressive constriction of the visual field. Myopia was noted in 9/20 affected eyes. For most patients, central visual acuity was preserved relatively well up to their thirties, after which it deteriorated rapidly over the next two decades. The visual acuity of patients homozygous for the c.4957_4958insA mutation was uniform. Visual fields were constricted symmetrically, and the extent of constriction seemed to be better correlated with age than visual acuity. The fundus displayed bone spicules, which increased in density with age, and attenuated retinal vessels. Conclusions: Although additional studies with more patients with mutations of the EYS gene are required, it appears that patients share a relatively uniform phenotype with near-normal central visual function up to their twenties. The patients homozygous for the c.4957_4958insA mutation showed a uniform course of visual acuity changes.

Novel GUCY2D Gene Mutations in Japanese Male Twins with Leber Congenital Amaurosis

Purpose. Leber congenital amaurosis (LCA), a genetically and clinically heterogeneous disease, is the earliest onset retinitis pigmentosa (RP) and is the most severe of hereditary retinal dystrophies. This study was conducted to investigate genetic and clinical features of LCA in a set of Japanese male twins with LCA. Methods. To identify causative mutations, 74 genes known to cause RP or LCA were examined by targeted-next generation sequencing (NGS). Targeted-NGS was performed using a custom designed Agilent HaloPlex target enrichment kit with Illumina Miseq sequencer. Identified potential pathogenic mutations were confirmed using Sanger sequencing. Clinical analyses were based on ophthalmic examination, fundus photography, and electroretinography (ERG). Results. Compound heterozygous GUCY2D mutations of novel splicing mutation c.2113+2_2113+3insT and novel missense mutation p.L905P were detected in both twins. Their father and mother were heterozygous for c.2113+2_2113+3insT and p.L905P, respectively. The twins had phenotypic features similar to those previously reported in patients with GUCY2D mutations. This included early childhood onset of visual loss, nystagmus, unrecordable ERG, photophobia, and hyperopia. Conclusions. To the best of our knowledge, this is the first report of genetic and clinical features of Japanese LCA twins with GUCY2D mutation, which were detected using targeted-NGS.

The first USH2A mutation analysis of Japanese autosomal recessive retinitis pigmentosa patients: a totally different mutation profile with the lack of frequent mutations found in Caucasian patients.

Retinitis pigmentosa (RP) is a highly heterogeneous genetic disease. The USH2A gene, which accounts for approximately 74–90% of Usher syndrome type 2 (USH2) cases, is also one of the major autosomal recessive RP (arRP) causative genes among Caucasian populations. To identify disease-causing USH2A gene mutations in Japanese RP patients, all 73 exons were screened for mutations by direct sequencing. In total, 100 unrelated Japanese RP patients with no systemic manifestations were identified, excluding families with obvious autosomal dominant inheritance. Of these 100 patients, 82 were included in this present study after 18 RP patients with very likely pathogenic EYS (eyes shut homolog) mutations were excluded. The mutation analysis of the USH2A revealed five very likely pathogenic mutations in four patients. A patient had only one very likely pathogenic mutation and the others had two of them. Caucasian frequent mutations p.C759F in arRP and p.E767fs in USH2 were not found. All the four patients exhibited typical clinical features of RP. The observed prevalence of USH2A gene mutations was approximately 4% among Japanese arRP patients, and the profile of the USH2A gene mutations differed largely between Japanese patients and previously reported Caucasian populations.

Novel HPS6 mutations identified by whole-exome sequencing in two Japanese sisters with suspected ocular albinism.

Hermansky–Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by oculocutaneous albinism, platelet dysfunction and ceroid deposition. We report suspected ocular albinism in two Japanese sisters, caused by mutations in the HPS6 (Hermansky–Pudlak syndrome 6) gene. Trio-based whole-exome sequencing (WES) identified novel compound heterozygous mutations in HPS6 (c.1898delC: mother origin and c.2038C>T: father origin) in the two sisters. To date, 10 associated mutations have been detected in HPS6. Although we detected no general manifestations, including platelet dysfunction, in the sisters, even in long-term follow-up, we established a diagnosis of HPS type 6 based on the HPS6 mutations and absence of dense bodies in the platelets, indicating that WES can identify cases of HPS type 6. To the best of our knowledge, this is the first report of HPS6 mutations in Japanese patients.

Visual Outcomes in Japanese Patients with Retinitis Pigmentosa and Usher Syndrome Caused by USH2A Mutations

ABSTRACT Purpose: EYS and USH2A are the most common causative genes for retinitis pigmentosa (RP) in Japan. We determined the clinical outcomes for USH2A-related non-syndromic RP or Usher syndrome type II (USH2). Methods: Two non-syndromic RP and 11 USH2 patients with previously identified USH2A mutations were included. Their complete history and medical records were collected using standard procedures. Visual fields and acuity were compared with those of patients with EYS mutations. Clinical analyses were based on ophthalmic and otolaryngologic examinations. Results: In all patients, the fundus displayed changes typical of RP. Most patients showed relatively well-preserved visual acuity in their thirties or forties, with rapid deterioration in their fifties. Concentric constriction started in the twenties or thirties, and no effective residual visual field was observed after the fifties. Conclusions: The visual outcome for non-syndromic RP or USH2 patients with USH2A mutations is consistent with that for RP patients with EYS mutations.

Interaction between optineurin and the bZIP transcription factor NRL.

Although the gene encoding optineurin (OPTN) is a causative gene for glaucoma and amyotrophic lateral sclerosis, it is ubiquitously expressed in all body tissues, including the retina. To study the function of OPTN in retinal ganglion cells as well as the whole retina, we previously isolated OPTN‐interacting proteins and identified the gene encoding the bZIP transcription factor neural retina leucine zipper (NRL), which is a causative gene for retinitis pigmentosa. Herein, we investigated the binding between OPTN and NRL proteins in HeLaS3 cells. Co‐expression of HA‐tagged NRL and FLAG‐tagged OPTN in HeLaS3 cells followed by immunoprecipitation and Western blotting with anti‐tag antibodies demonstrated the binding of these proteins in HeLaS3 cells, which was confirmed by proximity ligation assay. NRL is the first OPTN‐binding protein to show eye‐specific expression. A series of partial‐deletion OPTN plasmids demonstrated that the tail region (423–577 amino acids [aa]) of OPTN was necessary for binding with NRL. Immunostaining showed that Optn (rat homologue of OPTN) was expressed in rat photoreceptors and localised in the cytoplasm of photoreceptor cells. This is a novel demonstration of Optn expression in photoreceptor cells. OPTN was not detected in photoreceptor nuclei under our experimental conditions. Further analyses are necessary to elucidate the function of OPTN and the significance of its possible binding with NRL in photoreceptor cells.