BackgroundThe genetic profile of retinitis pigmentosa (RP) in East Asian populations has not been well characterised. Therefore, we conducted a large-scale sequencing study to investigate the genes and variants causing RP in a Japanese population.MethodsA total of 1209 Japanese patients diagnosed with typical RP were enrolled. We performed deep resequencing of 83 known causative genes of RP using next-generation sequencing. We defined pathogenic variants as those that were putatively deleterious or registered as pathogenic in the Human Gene Mutation Database or ClinVar database and had a minor allele frequency in any ethnic population of ≤0.5% for recessive genes or ≤0.01% for dominant genes as determined using population-based databases.ResultsWe successfully sequenced 1204 patients with RP and determined 200 pathogenic variants in 38 genes as the cause of RP in 356 patients (29.6%). Variants in six genes (EYS, USH2A, RP1L1, RHO, RP1 and RPGR) caused RP in 65.4% (233/356) of those patients. Among autosomal recessive genes, two known founder variants in EYS [p.(Ser1653fs) and p.(Tyr2935*)] and four East Asian-specific variants [p.(Gly2752Arg) in USH2A, p.(Arg658*) in RP1L1, p.(Gly2186Glu) in EYS and p.(Ile535Asn) in PDE6B] and p.(Cys934Trp) in USH2A were found in ≥10 patients. Among autosomal dominant genes, four pathogenic variants [p.(Pro347Leu) in RHO, p.(Arg872fs) in RP1, p.(Arg41Trp) in CRX and p.(Gly381fs) in PRPF31] were found in ≥4 patients, while these variants were unreported or extremely rare in both East Asian and non-East Asian population-based databases.ConclusionsEast Asian-specific variants in causative genes were the major causes of RP in the Japanese population.

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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.

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 Leber's 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.

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.

X-linked retinitis pigmentosa (XLRP) is a type of severe retinal dystrophy, and female carriers of XLRP demonstrate markedly variable clinical severity. In this study, we aimed to elucidate the clinical findings of male patients with and female carriers of XLRP in a Japanese cohort and demonstrate the genetic contribution. Twelve unrelated families (13 male patients, 15 female carriers) harboring pathogenic mutations in RPGR or RP2 were included, and comprehensive ophthalmic examinations were performed. To identify potential pathogenic mutations, targeted next-generation sequencing was employed. Consequently, we identified 11 pathogenic mutations, of which five were novel. Six and five mutations were detected in RPGR and RP2, respectively. Only one mutation was detected in ORF15. Affected male patients with RP2 mutations tended to have lower visual function than those with RPGR mutations. Female carriers demonstrated varying visual acuities and visual fields. Among the female carriers, 92% had electroretinographical abnormalities and 63% had a radial autofluorescent pattern, and the carriers who had higher myopia showed worse visual acuity and more severe retinal degeneration. Our results expand the knowledge of the clinical phenotypes of male patients with and female carriers of XLRP and suggest the possibility that RP2 mutations are relatively highly prevalent in Japan.

Leber congenital amaurosis (LCA) is a genetically and clinically heterogeneous disease, and represents the most severe form of inherited retinal dystrophy (IRD). The present study reports the mutation spectra and frequency of known LCA and IRD-associated genes in 34 Japanese families with LCA (including three families that were previously reported). A total of 74 LCA- and IRD-associated genes were analysed via targeted-next generation sequencing (TS), while recently discovered LCA-associated genes, as well as known variants not able to be screened using this approach, were evaluated via additional Sanger sequencing, long-range polymerase chain reaction, and/or copy number variation analyses. The results of these analyses revealed 30 potential pathogenic variants in 12 (nine LCA-associated and three other IRD-associated) genes among 19 of the 34 analysed families. The most frequently mutated genes were CRB1, NMNAT1, and RPGRIP1. The results also showed the mutation spectra and frequencies identified in the analysed Japanese population to be distinctly different from those previously identified for other ethnic backgrounds. Finally, the present study, which is the first to conduct a NGS-based molecular diagnosis of a large Japanese LCA cohort, achieved a detection rate of approximately 56%, indicating that TS is a valuable method for molecular diagnosis of LCA cases in the Japanese population.

This study aimed to evaluate retinal structure in the early stage of Leber’s congenital amaurosis (LCA) caused by RPGRIP1 mutations. Four patients from two families were included. Case 1 was a 13-year-old girl, cases 2 and 3 were 7-year-old monozygotic twin brothers of case 1, and case 4 was a 17-year-old boy. Comprehensive ophthalmic examinations were performed, including visual acuity measurements, perimetry, electroretinography (ERG), and optical coherence tomography (OCT). To identify potential pathogenic mutations, 74 genes known to cause retinitis pigmentosa or LCA were assessed using targeted next-generation sequencing. OCT showed photoreceptor outer nuclear layer (ONL) thinning in all patients. The lamellar structure was retained in all patients, whereas the ellipsoid zone was extinguished in cases 1, 2, and 3. In case 4, the ellipsoid zone was maintained at 9 years of age but became blurred at 17 years of age. In case 1, OCT indicated slight photoreceptor ONL thinning during the period between 7 and 11 years of age. Mutation analysis revealed RPGRIP1 mutations as the cause for autosomal recessive LCA in all patients. Photoreceptor ONL on OCT is relatively well preserved in the early stage of LCA caused by RPGRIP1 mutations.