Genetic characterization of 1,210 Japanese pedigrees with inherited retinal diseases by whole-exome sequencing

Inherited retinal diseases (IRDs) comprise a phenotypically and genetically heterogeneous group of ocular disorders that cause visual loss via progressive retinal degeneration. Here we report the genetic characterization of 1,210 IRD pedigrees enrolled through the Japan Eye Genetic Consortium and analyzed by whole exome sequencing. The most common phenotype was retinitis pigmentosa (RP, 43%), followed by macular dystrophy/cone- or cone-rod dystrophy (MD/CORD, 13%). In total, 67 causal genes were identified in 37% (448/1210) of the pedigrees. The first and second most frequently mutated genes were EYS and RP1, associated primarily with autosomal recessive (ar) RP, and RP and arMD/CORD, respectively. Examinations of variant frequency in total and by phenotype showed high accountability of a frequent EYS missense variant (c.2528G>A). In addition to the two known EYS founder mutations (c.4957dupA and c.8805C>G) of arRP, we observed a frequent RP1 variant (c.5797C>T) in patients with arMD/CORD. This article is protected by copyright. All rights reserved.

Suga A, Yoshitake K, Minematsu N, Tsunoda K, Fujinami K, Miyake Y, Kuniyoshi K, Hayashi T, Mizobuchi K, Ueno S, Terasaki H, Kominami T, Nao-I N, Mawatari G, Mizota A, Shinoda K, Kondo M, Kato K, Sekiryu T, Nakamura M, Kusuhara S, Yamamoto H, Yamamoto S, Mochizuki K, Kondo H, Matsushita I, Kameya S, Fukuchi T, Hatase T, Horiguchi M, Shimada Y, Tanikawa A, Yamamoto S, Miura G, Ito N, Murakami A, Fujimaki T, Hotta Y, Tanaka K, Iwata T.
Hum Mutat. 2022 Oct 25. doi: 10.1002/humu.24492. Online ahead of print.PMID: 36284460

The macula is a unique structure in higher primates, where cone and rod photoreceptors show highest density in the fovea and the surrounding area, respectively. The hereditary macular dystrophies represent a heterozygous group of rare disorders characterized by central visual loss and atrophy of the macula and surrounding retina. Here we report an atypical absence of ON-type bipolar cell response in a Japanese patient with autosomal dominant macular dystrophy (adMD). To identify a causal genetic mutation for the adMD, we performed whole-exome sequencing (WES) on four affected and four-non affected members of the family for three generations, and identified a novel p.C538Y mutation in a post-synaptic gene, LRRTM4. WES analysis revealed seven rare genetic variations in patients. We further referred to our in-house WES data from 1360 families with inherited retinal diseases, and found that only p.C538Y mutation in LRRTM4 was associated with adMD-affected patients. Combinatorial filtration using public database of single-nucleotide polymorphism frequency and genotype-phenotype annotated database identified novel mutation in atypical adMD.

Kawamura Y, Suga A, Fujimaki T, Yoshitake K, Tsunoda K, Murakami A, Iwata T.
J Hum Genet. 2018 Aug;63(8):893-900. doi: 10.1038/s10038-018-0465-4. Epub 2018 May 14. PMID: 29760528

Leber congenital amaurosis (LCA) is a hereditary early-onset retinal dystrophy that is accompanied by severe macular degeneration. In this study, novel compound heterozygous mutations were identified as LCA-causative in chaperonin-containing TCP-1, subunit 2 (CCT2), a gene that encodes the molecular chaperone protein, CCTβ. The zebrafish mutants of CCTβ are known to exhibit the eye phenotype while its mutation and association with human disease have been unknown. The CCT proteins (CCT α-θ) forms ring complex for its chaperon function. The LCA mutants of CCTβ, T400P and R516H, are biochemically instable and the affinity for the adjacent subunit, CCTγ, was affected distinctly in both mutants. The patient-derived induced pluripotent stem cells (iPSCs), carrying these CCTβ mutants, were less proliferative than the control iPSCs. Decreased proliferation under Cct2 knockdown in 661W cells was significantly rescued by wild-type CCTβ expression. However, the expression of T400P and R516H didn't exhibit the significant effect. In mouse retina, both CCTβ and CCTγ are expressed in the retinal ganglion cells and connecting cilium of photoreceptor cells. The Cct2 knockdown decreased its major client protein, transducing β1 (Gβ1). Here we report the novel LCA mutations in CCTβ and the impact of chaperon disability by these mutations in cellular biology.

Minegishi Y, Sheng X, Yoshitake K, Sergeev Y, Iejima D, Shibagaki Y, Monma N, Ikeo K, Furuno M, Zhuang W, Liu Y, Rong W, Hattori S, Iwata T.
Sci Rep. 2016 Sep 20;6:33742. doi: 10.1038/srep33742. PMID: 27645772

Cytoplasmic localization of Flag-tagged C21orf2 wild type and mutant proteins. Insert shows overlapping Flag (green) and Tubulin (red) immunostaining.Inherited retinal dystrophy is a rare disease, but has a critical impact on the patient’s quality of life, in that fundamental medical treatments are almost not available until recently. Latest advances of gene therapy technologies and stem cell-based drug screenings are changing the situation, yet, it is still under investigation how the variety of genetic mutations causes the disease, and, further, causal genes/mutations are not identified in up to 30% of the cases.

To identify novel causal genes and mutations of inherited retinal diseases in Japanese population, we have been collecting 872 families with patient(s) and their healthy siblings and/or parents, and working on Whole Exome Sequencing (WES) analysis on 594 families.

One of the novel mutations found in this study, we reported mutations in a ciliary gene, C21orf2, from the patients affected by autosomal recessive retinitis pigmentosa (arRP) and autosomal recessive cone-rod dystrophy (arCRD), respectively.

RP is the most common inherited retinal dystrophy with the prevalence of 1 in 4,000 worldwide. The disease onset and progression of RP is highly variable. However, many patients fall into night blindness followed by the loss of mid-peripheral visual field, which are caused by the rod photoreceptor degeneration. CRD is a further rare disease with the prevalence of 1 in 40,000. Patients typically have photoaversion, decreased visual acuity and loss of central visual field, reflecting the degeneration of cone photoreceptors. RP and CRD are genetically highly variable: RP and CRD can be inherited in any type of hereditary manner; autosomal dominant, autosomal recessive, and X-linked. Further, RP and CRD have at least 80 and 30 causal genes, respectively, with those numbers increasing. In addition, RP and CRD can appear as an associating symptom of syndromic diseases, such as Jubert syndrome, Bardet-Biedl syndrome, Jeune syndrome, and Usher syndrome type 2.

Localization of C21orf2 protein (green) in the photoreceptor connecting cilium indicated by the acetylated Tubulin (red). Mutations in C21orf2 were identified by family-based genetic segregations. The arRP patients had compound heterozygous mutation (p. Y107H, p. V111M), and the arCRD patients had homozygous mutation (p. Y107C). All these mutated amino acids were conserved from human to a unicellular organism, chlamydomonas, suggesting their importance in the protein function. In fact, in vitro studies showed that the mutated C21orf2 proteins expressed at lower levels, and diffusely distributed in the cytoplasm compared to the wild type C21orf2 protein co-localizing to the Tubulin. C21orf2 protein localized to the primary cilia of rodent and primate photoreceptors in vivo. 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. C21orf2 was also identified in atypical retinal dystrophy patients and also in the patients with syndromic skeletal dystrophy. Different effects of each mutation and genetic backgrounds may result in the symptom isolated to the eye or syndromic diseases.

Suga A, Mizota A, Kato M, Kuniyoshi K, Yoshitake K, Sultan W, Yamazaki M, Shimomura Y, Ikeo K, Tsunoda K, Iwata T.
Invest Ophthalmol Vis Sci 2016;57:4255-4263