Recent genetic data indicates that around 45,000 to 60,000 years ago, human expansion occurred outside of Africa and spread toward the Middle East branching into Europe and Asia.1 Large number of genetic variations originally existed in African population were gradually lost as particular branched of human were dispersed, leading to genetic diversity among global regions. To this day, the detail of genetic variations comes mainly from Europe and the US, while other developed countries are quickly catching up, developmental countries are still struggling to get sufficient budget, people, and infrastructure to take advantage of the next-generation technologies for genome analysis.2 This is also true in the field of eye genetics. To overcome this situation, Japan/Asian/Global Eye Genetics Consortium (GEGC) was established to cooperatively and effectively collect and pool phenotype-genotype information at global level.
Inherited retinal diseases (IRD) are groups of eye diseases initiated by abnormal retinal development, cellular function or structure in the retina. Patients are often influenced by genetic factor with inherited gene mutation in autosomal dominant, autosomal recessive, X-linked or sporadic forms. IRD is classified to over 36 various eye diseases with a phenotype of retina-specific or with syndromic complications. These eye diseases follow into several groups of characteristic phenotypes when examined by fundus photograph, optical coherence tomography, electroretinography, fluorescein angiography, and other diagnostic methods. A number of therapy for IRD is in clinical trials but so far RPE65 substitution gene therapy is only approved by the US Food and Drug Administration (FDA).3–5 Approximately 300 genes have been reported as disease-causing, mainly identified in patients in Europe and the US. However, the prevalence of these gene mutations differ between ethnic groups. Until 2011, gene mutation analysis for Japanese IRD was performed by direct sequencing. In 2010, we identified novel gene mutations in RP1L1 gene associated with occult macular dystrophy—Miyake Disease, a phenotypically unique retinal disease where fovea response was lost to only be detected by focal electroretinogram.6 Single nucleotide polymorphism linkage analysis and direct sequencing was performed to identify in more than 5 pedigree. This was the first case for a Japanese researcher (Yozo Miyake, who is an ophthalmologist) to identify novel disease–causing gene for retinal disease. This success brought attention to the ophthalmology research community and Ministry of Heath, Labour and Welfare of Japan for further investigation of IRD in Japanese population.
JAPAN EYE GENETICS CONSORTIUM (JEGC)
In 2011, JEGC was established to change the research structure of collecting phenotype-genotype information for Japanese IRD from individual university-based studies to all-Japan. JEGC was funded by the Japanese Ministry of Health, Labour and Welfare, and later by the Japanese Agency for Medical Research and Development (AMED) up to present. The entire board of the Japanese Society for Clinical Electrophysiology of Vision (JSCEV President, Masayuki Horiguchi, Fujita Health University) joined this consortium to set up a system to maintain the quality of diagnosis. JEGC targets 37 ocular diseases including (1) Leber congenital amaurosis, (2) retinitis pigmentosa, (3) enhanced S-cone syndrome, (4) Usher syndrome, (5) Stargardt disease, (6) macular dystrophy (non-Stargardt disease) or cone (cone-rod) dystrophy, (7) occult macular dystrophy, (8) cone/cone-rod dystrophy with normal fundus appearance, (9) North Carolina macular dystrophy, (10) foveal hypoplasia, (11) microphthalmos/nanophthalmos, (12) congenital stationary night blindness, (13) Oguchi disease, (14) flecked retina syndrome, (15) Bietti crystalline corneoretinal dystrophy, (16) choroideremia, (17) achromatopsia, (18) blue cone monochromatism, (19) gyrate atrophy, (20) bradyopsia, (21) retinoschisis, (22) familial drusen, (23) familial age-related macular degeneration, (24) bestrophinopathy, (25) Wnt-signaling retinopathy, (26) Stickler syndrome, (27) Wagner syndrome, (28) dominant optic atrophy, (29) mitochondrial retinopathy, (30) Leber hereditary optic neuropathy, (31) ocular albinism, (32) oculocutaneous albinism, (33) albinism with systemic abnormalities, (34) angioid streaks, (35) retinoblastoma, (36) hereditary optic neuropathy, and (37) hereditary normal tension glaucoma.
Disease Diagnostic, Database, and Sample Collection
The consortium came to an agreement that quality of patient phenotype information was critical for grouping of patients with similar phenotype, natural history study, and for recruitment of patients to clinical trials in the future. To maintain diagnostic quality, “Disease Leader” was appointed from consortium member with experience to specific IRD (Fig. 1). Disease Leaders gather twice a year at Tokyo Medical Center (TMC) to establish diagnostic protocol for each disease and to discuss about patients with difficult phenotype. The accurate IRD diagnosis is also critical for grouping similar diseases to efficiently perform next-generation deoxyribonucleic acid (DNA) sequencing (NGS), such as whole exome/genome sequencing (WES/WGS).
Online JEGC phenotype-genotype database was developed at TMC for all JEGC members to easily access to phenotype text information in Human Phenotype Ontology (HPO) terms, pedigree chart, and phenotype image data such as fundus photo, fluorescein angiogram, autofluorescence, optical coherence tomography, electroretinogram, visual field, etc, retrospectively (Fig. 2). Questions and comments about the diagnosis and the progress of ongoing NGS can be followed by all members without exchange of emails. Significant effort of computer programing and designing of phenotype input menu were spent to minimize the time required for ophthalmologists to input and upload phenotype information. To secure the system, data input and data storage are separated into 2 servers and data are encrypted with daily data backup.
TMC takes full responsibility to centralize the flow of DNA samples and NGS. Sample collection of 7 mL whole blood in ethylenediamine tetraacetic acid (EDTA) tube or 2 mL of saliva collection kit (Oragene DNA, US) followed by outsourced DNA extraction (Genotechs, Japan), while DNA extraction for saliva is done outsourced. All DNA samples were stored at 4°C in Tris-EDTA buffer at fixed concentration or at −20°C in ethanol.
Genotyping of Japanese IRD
Over 10 years, 1538 pedigree (2788 DNA samples) was collected from 38 ophthalmology departments and eye hospitals in Japan. The DNA samples were outsourced for WES (RIKEN, Japan and Macrogen, South Korea). WES was performed by exon capture (Agilent SureSelect Ver. 3-6) and DNA sequencing (Illumina Hiseq. 2000, Hiseq. 2500 and Hiseq. 4000, US) at average of 100 reads. All filtration process from FastQ files were performed at TMC (Fig. 3).
Reads from the FastQ files were mapped to the reference human genome (1000 genomes, phase 2 reference, hs37d5) with the Burrows-Wheeler Alinger software, version 0.7.10. Duplicated reads were then removed by Picard MarkDuplicates module version 1.129, and mapped reads around insertion-deletion polymorphisms (INDELs) were realigned using the Genome Analysis Toolkit (GATK) version 3.3-0. Base-quality scores were recalibrated using GATK. The calling of mutations was performed using the GATK HaplotypeCaller module, and the called single-nucleotide variants and INDELs were annotated with the snpEff software, version 4.1B and the annovar software, version 2015-04-24. 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 HGVD (http://www.genome.med.kyoto-u.ac.jp/SnpDB/index.html). 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 610 people exome data were further analyzed. We also used novel genetic variations, which were not found in the in-house exome data of normal people without ocular disease. Mutations were filtered by hereditary information in the family members.
During 10 years of the study, the diagnostic rate was increased from ~17% to 53%, including 27% of known variants, 18% of novel variants in known gene, 8% of potential novel disease–causing genes (Fig. 4). Rest of the 47% of pedigree is now the target for WGS. To our surprise, around 70% of Japanese patients were affected by novel mutation or by unknown cause. Complete reference is list at JEGC website (https://jegc.org).
Global Eye Genetics Consortium (GEGC)
In 2014, during the World Ophthalmology Congress (WOC) in Tokyo, Japan, a Memorandum of Understanding was signed by former directors Paul Sieving of National Eye Institute (NEI), National Institutes of Health (NIH), and Yozo Miyake of National Institute of Sensory Organs (NISO), TMC, to cooperatively work to study genetic eye diseases in Asia. Gyan Prakash (NEI/NIH) and myself decided to launch the Asian Eye Genetics Consortium (AEGC) during the 2015 ARVO meeting with S. Natarajan (India), Calvin Pang (Hong Kong), Namrata Sharma (India), and others (Fig. 5). The aim of the consortium was to collaboratively explore the possibility of performing eye genetic studies in developing counties in Asia. After 4 years of activity as AEGC, the number of members grew to over 200 from 30 countries including countries outside of Asia. The consortium was renamed Global Eye Genetics Consortium (GEGC) with core organizers Gyan Prakash (NEI/NIH, US), S. Natarajan (Aditya Jyot Eye Hospital, India), Paul N. Baird (University of Melbourne, Australia), Calvin Pang (The Chinese University of Hong Kong, China), and myself. See all members at GEGC website (https://gegc.org).
The aims of GEGC are as follow:
- Share genetic information to isolate common genetic variants associated with genetic eye diseases.
- Establish cost-effective genetic analysis and accurate diagnosis for grouping of genetic eye diseases.
- Develop research-oriented database to collect and catalog genetic eye diseases at global scale.
- Support and foster global collaboration for the advancement of genetic eye research.
- Collaborate with other international or regional organizations with similar goals.
- Organize regional congresses and other educational and scientific activities to promote goals of the consortium.
GEGC Networking, Database, and Publication
The first step of networking was to identify individual working on eye genetics in each developed country, who did not attend international meetings such as ARVO, WOC, AAO or ISER. AEGC/GEGC has organized promotional sessions or presentations at local or international ophthalmology meetings in Australia, China, Hong Kong, India, Japan, Nepal, Singapore, South Korea, Sri Lanka, Taiwan, Thailand, the UK, the US, and Vietnam. AEGC/GEGC session at the Asia Pacific Academy of Ophthalmology (APAO) and South Asian Association for Regional Cooperation (SAARC) Academy of Ophthalmology (SAO) was organized to promote genetic eye research to administrative leaders of each country. AEGC/GEGC annual meeting is organized every year at the ARVO meeting (Fig. 5). Exchange program was set up to invite young scientists or PhD students to laboratory in Japan or the US to experience how genetic lab works. Students will learn about the database, phenotype collection, DNA sampling, genotype filtration, mutant protein experiments, and more. Such programs have helped establish new eye genetics laboratory in India. In 2016, Aditya Jyot Eye Hospital and GEGC jointly opened an Eye Genetics Laboratory in Mumbai, for phenotype-genotype collection for diabetic retinopathy, age-related macular degeneration and IRD (Fig. 5).
In 2020, a phenotype-genotype database GenEye was developed by reprograming JEGC database by Python. GenEye contains 3 diseases including IRD, age-related macular degeneration, and glaucoma. The international standard HPO term is used throughout the database for phenotype description. Further security is added with authentication of user to restrict viewing of their own data to only collaborators before publication. To facilitate the best design of the GEGC database, a series of meetings were held with experts from around the world to garner experience from different data platforms that were being used. These included meeting with Andrew Webster from Moorefields Eye Hospital about the UK IRD database, and meetings with Kerry Goetz and Santa Tumminia from NEI/NIH about the US EyeGene database. GEGC is currently promoting research groups with no database to collect patient data into GenEye. The first phase of GenEye is to accumulate phenotype data from Asia, Africa, Middle East, and South America and plan for the whole genome analysis by funded international collaborations.
Asia, Middle East, Africa, and South America are under-represented in the global knowledge base. However, these regions are important to obtain valuable information to greatly advance our understanding of molecular mechanisms and pathobiology of genetic eye diseases. GenEye will play important role to catalog mutations and variants associated with genetic eye disease across developed countries in Asia, Middle East, Africa, and South America. It may also provide data for artificial intelligence projects to develop future diagnosis in regions without ophthalmologists. Drug companies planning for clinical trial at global scale will also benefit by recruiting patients for specific phenotype/genotype.
AEGC/GEGC activities are published in Advances in Vision Research (editor: Gyan Prakash, Takeshi Iwata), Essentials in Ophthalmology series (series editor: Arun D. Singh) by Springer Nature (Fig. 6). A total of 105 chapters in 3 volumes have been published by GEGC members and the fourth volume is in planning to recruit topics in diagnostic and therapy for genetic eye diseases.
China and India Eye Genetics Consortium (IEGC) and Becoming Member of International Council of Ophthalmology (ICO)
In February 2018, a ceremony was conducted at New Delhi, India, to celebrate the establishment of GEGC India branch, the IEGC, under the leadership of Gyan Prakash (NEI/NIH), Umang Mathur (Dr Shroff’s Charity Eye Hospital, New Delhi), and S. Natarajan (Aditya Jyot Eye Hospital, Mumbai), to divide India to North and South segments to effectively collect phenotype-genotype information to Mumbai and New Delhi. At the meeting, the eye hospitals joined the consortium to discuss target disease for IEGC, sample handling in hot weather, ethic issues, and funding for research. IEGC has the support of the All India Ophthalmology Society (AIOS).
In June of the same year at Chongqing, China, another ceremony was conducted to establish the GEGC China branch, the China Eye Genetics Consortium (CEGC), under the leadership of Zhengqin Yin (Southwest Eye Hospital/Southwest Hospital, Third Military Medical University, Chongqing) and Qingjiong Zhang (Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou) (Fig. 5). The following 2019 meeting in Zhongshan Ophthalmic Center, Guangzhou, organized by Qingjiong Zhang and 2020 virtual CEGC meeting organized by Zibing Jin was held. Both consortia were important to GEGC because it covered approximately half of the global population.
GEGC annual meeting was held on April, 2019, during the ARVO meeting in Vancouver, Canada. Representatives from 18 countries gathered to this meeting. After the introduction of GEGC, former President, Peter Wiedermann, and current President, Neeru Gupta, of ICO welcomed GEGC to be the new member of the ICO. Mrs Pamela Sieving from NEI gave a talk about the scientific activities in Africa and South America based on the bibliometric analysis in these regions. GEGC is now working with ICO to expand to Africa and South America.
IDENTIFICATION OF NOVEL GENES, MECHANISMS OF DISEASE ONSET, AND THERAPEUTICS
WES performed on affected families with retinal disease collected through JEGC/AEGC/GEGC has efficiently identified number of novel disease–causing genes to further improve the detection rate of hereditary eye disorders.
CCT2: Leber congenital amaurosis is a hereditary early-onset retinal dystrophy with severe macular degeneration. In collaboration with Xunlun Sheng (Ningxia Eye Hospital, China), we identified novel compound heterozygous mutations p.T400P/p.R516H in chaperonin-containing TCP-1, subunit 2 (CCT2) gene, which encodes the chaperone protein CCTβ.7 CCTβ protein is a component of 8 subunits (CCT α-θ) forming a 2-ring structure of molecular chaperone. Previous studies have shown zebrafish mutants of CCTβ exhibit abnormal eye phenotype, while its mutation and association with human disease have not been reported.8 The identified novel CCTβ mutants p.T400P/p.R516H are biochemically instable and affinity for adjacent subunit CCTγ was significantly affected 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 (WT) CCTβ expression. However, the expression of T400P and R516H did not exhibit the significant effect. In mouse retina, both CCTβ and CCTγ are expressed in the retinal ganglion cells (RGCs) and connecting cilium of photoreceptor cells. The CCT2 knockdown decreased its major client protein, transducing β1 (Gβ1). Knock-in zebrafish9 and knock-in mouse are now being developed for further pathological study.
LRRTM4: A pedigree in Japan with unique phenotype was identified with autosomal dominant macular dystrophy and atypical absence of ON-type bipolar cell response.10 Whole exome analysis of 4 affected and 4 nonaffected individuals in a 3-generation family identified a novel p.C538Y mutation in a postsynaptic gene, Leucine Rich Repeat Transmembrane Neuronal 4 (LRRTM4).10,11 LRRTM4 belongs to a protein family of synaptic adhesion molecules with roles in synaptic formation and signaling. LRRTM4 transcripts are enriched in rod bipolar cells, which are the secondary neurons in the retina that form synapses with rod photoreceptor.12 These findings by others provide strong evidence for absence of ON-type bipolar cell response in patients.
MCAT: A pedigree with autosomal recessive optic neuropathies was identified by Xunlun Sheng at Ningxia Eye Hospital, China. Previously studies show that 3 disease-causing genes implicated in autosomal recessive forms of optic atrophy that involve progressive degeneration of optic nerve and RGCs encode mitochondrial proteins.13 Using the whole exome analysis, a novel double homozygous mutation p.L81R/p.R212W in malonyl CoA-acyl carrier protein transacylase (MCAT) gene coding, a mitochondrial protein involved in fatty acid biosynthesis was identified responsible for the disease onset.14 MCAT is highly expressed in mitochondria rich RGCs. The disease variants p.L81R/p.R212W lead to structurally unstable MCAT protein with significantly reduced intracellular expression. RGC-specific knockdown of MCAT in mice leads to an attenuated retinal neurofiber layer that resembles the phenotype of optic neuropathy. These results indicate that MCAT plays an essential role in mitochondrial function and maintenance of RGC axons, while novel MCAT p.L81R/p.R212W mutations can lead to autosomal recessive optic neuropathy. Recently, another family with compound heterogynous mutations NM_173467.4/NM_014507.3:c.424-2A>G/NM_173467.4:c.1039G>A (NM_014507.3:c.824G>A) in MCAT was identified in French family, suggesting more families with mutation in MCAT will be identified in the future.15
Patient-Derived iPSCs and Gene-Edited Knock-In Animal Models
The objective of JEGC/GEGC is to identify molecular mechanisms for each disease-causing gene mutation, which will serve as seed information to develop new drugs or to alternatively use drugs currently available for diseases other than eye. The novel candidate mutation requires additional proof of information from in vitro and in vivo experiments of the mutant protein. A number of tools are now available to mimic the patient’s condition in vitro and in vivo. These techniques include the use of patient iPSCs. iPSCs generated from patients and normal individuals within the family can now be differentiated to retinal pigment epithelial cells,16,17 RGCs,18,19 photoreceptor cells,20–22 and glia cells23 to mimic the conditions of the patient in vitro. iPSCs was generated for the first time from glaucoma patients with optineurin (OPTN) E50K mutation and differentiated to neuronal cells, which showed abnormal aggregation in endoplasmic reticulum.24 iPSCs are also differentiated to retinal organoid to replace animal models in the future.
Gene-editing technologies such as zinc finger nuclease,25 transcription activator-like effector nuclease,26,27 and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9)28,29 have been useful tools to efficiently generate knock-out and knock-in mice to observe disease pathology by mutation. These techniques are effective especially when amino acid sequence of protein is highly homologous between human and targeted animal. Zinc finger nuclease was used to generate OPTN E50K knock-in mice30 to develop normal tension glaucoma (NTG) mice model. CRISPR/Cas9 was also used to generate LRRTM4 and CCT2 knock-in mice for periodically examination of fundus photo, optical coherence tomography, and electroretinogram followed by histological analysis. Transcription activator-like effector nuclease is also used to generate larger animal models (Fu, Iwata, Mandai, Yamamoto, 2021 Audacious Goal Initiative, NEI/NIH).
Mechanisms of Disease Onset to Therapeutic Development
Gene responsible for hereditary NTG is well studied with Myocilin, another gene responsible for hereditary glaucoma with high intraocular pressure.31 A Japanese family with OPTN E50K mutation was identified by Kazuhide Kawase at Gifu University, Japan, and patient iPSCs were generated and differentiated to neural cells to observe mutant OPTN protein accumulating in endoplasmic reticulum.24 Immunoprecipitation experiment of E50K mutant and WT OPTN was separately performed to identify interacting proteins by proteomic analysis. This analysis resulted with E50K mutant protein interaction with TANK-binding kinase 1 (TBK1), while WT with other WT OPTN protein. The inhibitor for TBK1, Amlexanox, a FDA-approved and Pharmaceuticals and Medical Devices Agency (PMDA)–approved drug for allergic rhinitis and asthma was later used to successfully disassociate mutant OPTN-TBK1 interaction.30 To test if this mutant OPTN-TBK1 disassociation can lead to improvement of NTG, OPTN E50K knock-in mice was generated by CRISPR/Cas9.30 We administered Amlexanox by oral administration 5 days a week for 1 year, which showed significant neural protection compared with nontreated mice. TBK1 inhibitors are currently prepared for clinical trial in Japan for NTG patients with OPTN mutations.
FUTURE PLANS FOR JEGC AND GEGC
JEGC has provided a boost in IRD research in Japan with over 50 publications on Japanese IRD (see JEGC website, https://jegc.org). JEGC has performed WES for 9 years and is now shifting to whole genome analysis for pedigree with no result in exons by whole exome analysis. Genomic Japan project funded by AMED started in 2020 for rare and intractable diseases to perform short-read and long-read WGS for pedigree with no result by WES. JEGC was selected as one the 12 research groups to work on IRD. A detection of disease-associated mutations in deep intron and promoter/enhancer regulatory region are expected. Novel genes with amino acid substitutions are also in progress.
GEGC has brought a collective thinking from the researchers around the world who have interest in genetic eye research in Asia, Middle East, Africa, and South America. It has created a wide opportunity to establish partnerships among scientists, ophthalmologists, governments, and companies to support research programs for understanding the biology of genetic eye diseases worldwide. However, GEGC still faces challenges to solicit funding to collect DNA samples and to perform NGS in developing countries. GEGC has now grown to over 200 members from 30 countries, covering most of the eye geneticists around the globe. GEGC lost the valuable opportunity to meet in-person with scientists and ophthalmologists at WOC 2020 meeting in Cape Town, South Africa, and ISER 2020 meeting in Buenos Aires, Argentina. We hope to come back to both continents in 2023. Meanwhile, series of GEGC virtual summit is planned for 2022.
The author thanks all the patients, JEGC/GEGC members, and collaborators participated in this study. This work was supported in part by grant to Takeshi Iwata from the Japan Agency for Medical Research and Development, Practical Research Project for Rare/Intractable Diseases (15ek0109072h0003, 16ek0109072h0003, 17ek0109282s0001, 18ek0109282h0002, 19ek0109282h0003), Japanese Ministry of Health, Labour and Welfare (H23-Jitsuyouka(Nannbyo)-Ippan-006, H26-Itaku(Nann)-Ippann-087) and the Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research and Grant-in-Aid for Scientific Research (B).
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