CLEC3B is a novel causative gene for macular-retinal dystrophy.

PURPOSE: Macular degeneration is the leading cause of blindness worldwide. In this study, we aimed to define a new subtype of macular-retinal dystrophy and its genetic predisposition in 5 families. METHODS: Exome sequencing was performed to determine the putative disease-causing genes in patients with inherited macular disorders confirmed through comprehensive ophthalmic examinations. To validate its functional consequence, adeno-associated virus-mediated mutant gene was delivered into the murine retina, and both structural and functional tests were performed to investigate its pathological effects in vivo. RESULTS: In total, 5 multigenerational families diagnosed with autosomal dominant maculoretinopathy were found to carry a pathogenic variant in a new gene, CLEC3B, which encodes tetranectin, a plasminogen kringle-4 binding protein. Consistent with the disease phenotypes of patients, mice that received subretinal injections with the CLEC3B variant displayed multiple subretinal hyperreflective deposits, reduced retinal thickness, and decreased electroretinographic responses. Moreover, the optokinetic tracking response indicated that spatial frequency was significantly lower (P < .05), implying impaired visual function in these mice. CONCLUSION: We have presented a new subtype of macular-retinal dystrophy in 5 families as well as a new pathogenic gene, CLEC3B, providing new insights into maculoretinopathy etiology.

Mutation spectrum and genotype-phenotype correlation of inherited retinal dystrophy in Taiwan.

BACKGROUND: Inherited retinal dystrophy (IRD) is a group of irreversible retinal degenerative disorders with significant genotypic and phenotypic heterogeneity, which cause difficulty in making a precise clinical diagnosis. Furthermore, the mutation spectrum of IRD in Taiwan remains unknown. Therefore, our study focused on investigating the spectrum of mutations among Taiwanese families with IRD using targeted exome sequencing (TES) technology. METHODS: We recruited a total of 60 unrelated Taiwanese families with IRD; most of them were retinitis pigmentosa. We employed TES to investigate 284 candidate genes. Bioinformatics analysis, Sanger sequencing-based co-segregation testing, and computational assessment were performed to validate each mutation and its pathogenicity. The genotype-phenotype correlation was analysed in all patients with mutations defined in the guidelines provided by the American College of Medical Genetics. RESULTS: We successfully identified genetic causes in 32 families (detection rate of 53.3%). Among them, 16 had a sporadic inheritance (16/36, 44.4%); eight had an autosomal recessive inheritance (8/14, 57.1%); four had an autosomal dominant inheritance (4/5, 80%); four had an X-linked inheritance (4/5, 80%). Among 38 pathological mutations in 19 known genes, 20 mutations are reported here for the first time. Novel mutation spectrum and genotype-phenotype correlations were revealed as well. CONCLUSION: Here we achieved a detection rate of 53.3% and elucidated the mutation spectrum in Taiwanese families with IRD for the first time. The results indicated that CYP4V2 and USH2A might be the most common pathogenic genes in IRD patients in Taiwan.

Functional characterization of CEP250 variant identified in nonsyndromic retinitis pigmentosa.

Retinitis pigmentosa (RP) is the most common manifestation of inherited retinal diseases with high degree of genetic, allelic, and phenotypic heterogeneity. CEP250 encodes the C-Nap1 protein and has been associated with various retinal phenotypes. Here, we report the identification of a mutation (c.562C>T, p.R188*) in the CEP250 in a consanguineous family with nonsyndromic RP. To gain insights into the molecular pathomechanism underlying CEP250 defects and the functional relevance of CEP250 variants in humans, we conducted a functional characterization of CEP250 variant using a novel Cep250 knockin mouse line. Remarkably, the disruption of Cep250 resulted in severe impairment of retinal function and significant retinal morphological alterations. The homozygous knockin mice showed significantly reduced retinal thickness and ERG responses. This study not only broadens the spectrum of phenotypes associated with CEP250 mutations, but also, for the first time, elucidates the function of CEP250 in photoreceptors using a newly established animal model.

Genetic control of DNA methylation is largely shared across European and East Asian populations.

DNA methylation is an ideal trait to study the extent of the shared genetic control across ancestries, effectively providing hundreds of thousands of model molecular traits with large QTL effect sizes. We investigate cis DNAm QTLs in three European (n = 3701) and two East Asian (n = 2099) cohorts to quantify the similarities and differences in the genetic architecture across populations. We observe 80,394 associated mQTLs (62.2% of DNAm probes with significant mQTL) to be significant in both ancestries, while 28,925 mQTLs (22.4%) are identified in only a single ancestry. mQTL effect sizes are highly conserved across populations, with differences in mQTL discovery likely due to differences in allele frequency of associated variants and differing linkage disequilibrium between causal variants and assayed SNPs. This study highlights the overall similarity of genetic control across ancestries and the value of ancestral diversity in increasing the power to detect associations and enhancing fine mapping resolution.

Rationale and protocol paper for the Asia Pacific Network for inherited eye diseases.

PURPOSE: There are major gaps in our knowledge of hereditary ocular conditions in the Asia-Pacific population, which comprises approximately 60% of the world's population. Therefore, a concerted regional effort is urgently needed to close this critical knowledge gap and apply precision medicine technology to improve the quality of lives of these patients in the Asia-Pacific region. DESIGN: Multi-national, multi-center collaborative network. METHODS: The Research Standing Committee of the Asia-Pacific Academy of Ophthalmology and the Asia-Pacific Society of Eye Genetics fostered this research collaboration, which brings together renowned institutions and experts for inherited eye diseases in the Asia-Pacific region. The immediate priority of the network will be inherited retinal diseases (IRDs), where there is a lack of detailed characterization of these conditions and in the number of established registries. RESULTS: The network comprises 55 members from 35 centers, spanning 12 countries and regions, including Australia, China, India, Indonesia, Japan, South Korea, Malaysia, Nepal, Philippines, Singapore, Taiwan, and Thailand. The steering committee comprises ophthalmologists with experience in consortia for eye diseases in the Asia-Pacific region, leading ophthalmologists and vision scientists in the field of IRDs internationally, and ophthalmic geneticists. CONCLUSIONS: The Asia Pacific Inherited Eye Disease (APIED) network aims to (1) improve genotyping capabilities and expertise to increase early and accurate genetic diagnosis of IRDs, (2) harmonise deep phenotyping practices and utilization of ontological terms, and (3) establish high-quality, multi-user, federated disease registries that will facilitate patient care, genetic counseling, and research of IRDs regionally and internationally.

Exome sequencing in retinal dystrophy patients reveals a novel candidate gene ER membrane protein complex subunit 3.

Inherited retinal dystrophies (IRDs) are a heterogeneous group of visual disorders caused by different pathogenic mutations in genes and regulatory sequences. The endoplasmic reticulum (ER) membrane protein complex (EMC) subunit 3 (EMC3) is the core unit of the EMC insertase that integrates the transmembrane peptides into lipid bilayers, and the function of its cytoplasmic carboxyl terminus remains to be elucidated. In this study, an insertional mutation c.768insT in the C-terminal coding region of EMC3 was identified and associated with dominant IRDs in a five-generation family. This mutation caused a frameshift in the coding sequence and a gain of an additional 16 amino acid residues (p.L256F-fs-ext21) to form a helix structure in the C-terminus of the EMC3 protein. The mutation is heterozygous with an incomplete penetrance, and cosegregates in all patients examined. This finding indicates that the C-terminus of EMC3 is essential for EMC functions and that EMC3 may be a novel candidate gene for retinal degenerative diseases.

Global spectrum of USH2A mutation in inherited retinal dystrophies: Prompt message for development of base editing therapy.

Purpose: Mutation in the USH2A gene is the most common cause of inherited retinal dystrophy (IRD), including non-syndromic retinitis pigmentosa (RP) and Usher syndrome II (USH2). Gene editing and therapy targeting USH2A, especially the hotspot region, would benefit a large proportion of IRD patients. In this study, we comprehensively analyzed the genetic spectrum of the USH2A gene, aiming to identify global hot spot mutations in USH2A-related IRDs and differences in hot spot regions across continents. Materials and methods: A retrospective USH2A-related IRD study was conducted, including our IRD cohort, and reported USH2A studies worldwide. Results: A total of 3,972 mutated USH2A alleles of approximately 1,935 patients were collected from 33 cohort studies worldwide, containing 102 alleles of 51 patients in our IRD cohort. Mutations in exon 13 were the most common, reaching 18.4% globally and a higher frequency of 22% in America, 19.2% in Europe, and a lower 12% in East Asia. Pathogenic mutations that affected 10 of the 72 exons of USH2A, exon 2, exon 13, exon 41-43, exon 50, exon 54, exon 57, exon 61, and exon 63 in total were responsible for half of global USH2A mutant alleles. With base editors including adenine base editor (ABE), cytidine base editor (CBE), and glycosylase base editor (GBE), 76.3% of single nucleotide variations (SNVs) and 58% of all mutations in USH2A are correctable. Meantime, four novel pathogenic mutations were revealed in our IRD cohort, p. (Val1130Cysfs*72), p. (Ala2139fs*14), p. (Gly4139Arg), and p. (Val4166Cysfs*7). Conclusion: In this study, we revealed four novel mutations, expanding the spectrum of USH2A mutations, and importantly presented global hotspot exons and mutations of USH2A as well as the proportion of SNVs that can be restored by different base editors, providing a perspective for exploring high-efficiency and broader-reaching gene editing and gene therapies.

Second hit impels oncogenesis of retinoblastoma in patient-induced pluripotent stem cell-derived retinal organoids: direct evidence for Knudson's theory.

Retinoblastoma (Rb) is a type of malignant tumor due to abnormal retinogenesis with biallelic mutations of the RB1 gene. Its pathogenesis has been proposed as a "two-mutation hypothesis" by Knudson since 1971; however, there remain some debates on disease onset sufficiency of the biallelic RB1 mutations. To obtain straightforward evidence for this hypothesis, we investigated whether two-hit mutations of the RB1 gene drive tumorigenesis in patient-induced pluripotent stem cell (hiPSC)-derived human retinal organoids (hROs) and whether single allelic mutation hiPSC-derived hROs exhibit molecular and cellular defects. We generated hiPSCs with a heterozygous germline mutation (RB1m1/ wt ) from a Rb patient. A second-allele RB1 gene mutation was knocked in to produce compound heterozygous mutations (RB1m1/m2 ) in the hiPSCs. These two hiPSC lines were independently developed into hROs through a stepwise differentiation. The hiPSC-RB1m1/m2 derived organoids demonstrated tumorigenesis in dishes, consistent with Rb profiles in spatiotemporal transcriptomes, in which developmentally photoreceptor fate-determining markers, CRX and OTX2, were highly expressed in hiPSC-RB1m1/m2 derived hROs. Additionally, ARR3+ maturing cone precursors were co-labeled with proliferative markers Ki67 or PCNA, in agreement with the consensus that human Rb is originated from maturing cone precursors. Finally, we demonstrated that retinal cells of hROs with monoallelic RB1 mutation were abnormal in molecular aspects due to its haploinsufficiency. In conclusion, this study provides straightforward supporting evidence in a way of reverse genetics for "two-hit hypothesis" in the Rb tumorigenesis and opens new avenues for development of early intervention and treatment of Rb.

Mutation of SLC7A14 causes auditory neuropathy and retinitis pigmentosa mediated by lysosomal dysfunction.

Lysosomes contribute to cellular homeostasis via processes including macromolecule degradation, nutrient sensing, and autophagy. Defective proteins related to lysosomal macromolecule catabolism are known to cause a range of lysosomal storage diseases; however, it is unclear whether mutations in proteins involved in homeostatic nutrient sensing mechanisms cause syndromic sensory disease. Here, we show that SLC7A14, a transporter protein mediating lysosomal uptake of cationic amino acids, is evolutionarily conserved in vertebrate mechanosensory hair cells and highly expressed in lysosomes of mammalian cochlear inner hair cells (IHCs) and retinal photoreceptors. Autosomal recessive mutation of SLC7A14 caused loss of IHCs and photoreceptors, leading to presynaptic auditory neuropathy and retinitis pigmentosa in mice and humans. Loss-of-function mutation altered protein trafficking and increased basal autophagy, leading to progressive cell degeneration. This study implicates autophagy-lysosomal dysfunction in syndromic hearing and vision loss in mice and humans.

Consanguinity-based analysis of exome sequencing yields likely genetic causes in patients with inherited retinal dystrophy.

BACKGROUND: Consanguineous families have a relatively high prevalence of genetic disorders caused by bi-allelic mutations in recessive genes. This study aims to evaluate the effectiveness and efficiency of a consanguinity-based exome sequencing approach to capturing genetic mutations in inherited retinal dystrophy families with consanguineous marriages. METHODS: Ten unrelated consanguineous families with a proband affected by inherited retinal dystrophy were recruited in this study. All participants underwent comprehensive ophthalmic examinations. Whole exome sequencing was performed, followed by a homozygote-prior strategy to rapidly filter disease-causing mutations. Bioinformatic prediction of pathogenicity, Sanger sequencing and co-segregation analysis were carried out for further validation. RESULTS: In ten consanguineous families, a total of 10 homozygous mutations in 8 IRD genes were identified, including 2 novel mutations, c.1654_1655delAG (p. R552Afs*5) in gene FAM161A in a patient diagnosed with retinitis pigmentosa, and c.830T > C (p.L277P) in gene CEP78 in a patient diagnosed with cone and rod dystrophy. CONCLUSION: The genetic etiology in consanguineous families with IRD were successfully identified using consanguinity-based analysis of exome sequencing data, suggesting that this approach could provide complementary insights into genetic diagnoses in consanguineous families with variant genetic disorders.

Whole-Exome Sequencing in a Cohort of High Myopia Patients in Northwest China.

High myopia (HM) is one of the leading causes of visual impairment worldwide. In order to expand the myopia gene spectrum in the Chinese population, we investigated genetic mutations in a cohort of 27 families with HM from Northwest China by using whole-exome sequencing (WES). Genetic variations were filtered using bioinformatics tools and cosegregation analysis. A total of 201 candidate mutations were detected, and 139 were cosegregated with the disease in the families. Multistep analysis revealed four missense variants in four unrelated families, including c.904C>T (p.R302C) in CSMD1, c.860G>A (p.R287H) in PARP8, c.G848A (p.G283D) in ADAMTSL1, and c.686A>G (p.H229R) in FNDC3B. These mutations were rare or absent in the Exome Aggregation Consortium (ExAC), 1000 Genomes Project, and Genome Aggregation Database (gnomAD), indicating that they are new candidate disease-causing genes. Our findings not only expand the myopia gene spectrum but also provide reference information for further genetic study of heritable HM.

Genotype Profile of Global EYS-Associated Inherited Retinal Dystrophy and Clinical Findings in a Large Chinese Cohort.

PURPOSE: The aim of this study was to probe the global profile of the EYS-associated genotype-phenotype trait in the worldwide reported IRD cases and to build a model for predicting disease progression as a reference for clinical consultation. METHODS: This retrospective study of 420 well-documented IRD cases with mutations in the EYS gene included 39 patients from a genotype-phenotype study of inherited retinal dystrophy (IRD) conducted at the Beijing Institute of Ophthalmology and 381 cases retrieved from global reports. All patients underwent ophthalmic evaluation. Mutations were revealed using next-generation sequencing, followed by Sanger DNA sequencing and real-time quantitative PCR analysis. Multiple regression models and statistical analysis were used to assess the genotype and phenotype characteristics and traits in this large cohort. RESULTS: A total of 420 well-defined patients with 841 identified mutations in the EYS gene were successfully obtained. The most common pathogenic variant was a frameshift c.4957dupA (p.S1653Kfs∗2) in exon 26, with an allele frequency of 12.7% (107/841), followed by c.8805C > A (p.Y2935X) in exon 43, with an allele frequency of 5.9% (50/841). Two new hot spots were identified in the Chinese cohort, c.1750G > T (p.E584X) and c.7492G > C (p.A2498P). Several EYS mutation types were identified, with CNV being relatively common. The mean age of onset was 20.54 ± 11.33 (4-46) years. Clinical examinations revealed a typical progression of RPE atrophy from the peripheral area to the macula. CONCLUSION: This large global cohort of 420 IRD cases, with 262 distinct variants, identified genotype-phenotype correlations and mutation spectra with hotspots in the EYS gene.

Genetic Screening Revealed Latent Keratoconus in Asymptomatic Individuals.

PURPOSE: To adopt molecular screening in asymptomatic individuals at high risk of developing keratoconus as a combinative approach to prevent subclinical patients from post-refractive surgery progressive corneal ectasia. METHODS: In this study, 79 Chinese and nine Greek families with keratoconus were recruited, including 91 patients with clinically diagnosed keratoconus as well as their asymptomatic but assumptive high-risk first-degree relatives based on underlying genetic factor. Mutational screening of VSX1, TGFBI, and ZEB1 genes and full clinical assessment including Pentacam Scheimpflug tomography were carried out in these individuals. RESULTS: Five variants in VSX1 and TGFBI genes were identified in three Chinese families and one Greek family, and four of them were novel ones. Surprisingly, ultra-early corneal changes in Belin/Ambrosio Enhanced Ectasia Display of Pentacam corneal topography together with co-segregated variants were revealed in the relatives who had no self-reported symptoms. CONCLUSIONS: Variants of VSX1 and TGFBI genes identified in both the clinically diagnosed and subclinical patients may cause the keratoconus through an autosomal dominant inheritance pattern, with different variable expressivity. Combining genetic with Belin/AmbrosioEnhanced Ectasia Display can be used to identify patients with latent keratoconus. This study indicates that genetic testing may play an important supplementary role in re-classifying the disease manifestation and evaluating the preoperative examination of refractive surgery.

Genotype-Phenotype Analysis and Mutation Spectrum in a Cohort of Chinese Patients With Congenital Nystagmus.

Purpose: Congenital nystagmus (CN) is a genetically and clinically heterogeneous ocular disorder that manifests as involuntary, periodic oscillations of the eyes. To date, only FRMD7 and GPR143 have been reported to be responsible for causing CN. Here, we aimed to identify the disease-causing mutations and describe the clinical features in the affected members in our study. Methods: All the subjects underwent a detailed ophthalmic examination. Direct sequencing of all coding exons and splice site regions in FRMD7 and GPR143 and a mutation assessment were performed in each patient. Results: We found 14 mutations in 14/37 (37.8%) probands, including nine mutations in the FRMD7 gene and five mutations in the GPR143 gene, seven of which are novel, including c.284G>A(R95K), c.964C>T(P322S), c.284+10T>G, c.901T>C (Y301H), and c.2014_2023delTCACCCATGG(S672Pfs*12) in FRMD7, and c.250+1G>C, and c.485G>A (W162*) in GPR143. The mutation detection rate was 87.5% (7/8) of familial vs. 24.1% (7/29) of sporadic cases. Ten mutations in 24 (41.7%) non-syndromic subjects and 4 mutations in 13(30.8%) syndromic subjects were detected. A total of 77.8% (7/9) of mutations in FRMD7 were concentrated within the FERM and FA domains, while all mutations in GPR143 were located in exons 1, 2, 4 and 6. We observed that visual acuity tended to be worse in the GPR143 group than in the FRMD7 group, and no obvious difference in other clinical manifestations was found through comparisons in different groups of patients. Conclusions: This study identified 14 mutations (seven novel and seven known) in eight familial and 29 sporadic patients with congenital nystagmus, expanding the mutational spectrum and validating FRMD7 and GPR143 as mutation hotspots. These findings also revealed a significant difference in the screening rate between different groups of participants, providing new insights for the strategy of genetic screening and early clinical diagnosis of CN.

Nonhuman Primate Model of Oculocutaneous Albinism with TYR and OCA2 Mutations.

Human visual acuity is anatomically determined by the retinal fovea. The ontogenetic development of the fovea can be seriously hindered by oculocutaneous albinism (OCA), which is characterized by a disorder of melanin synthesis. Although people of all ethnic backgrounds can be affected, no efficient treatments for OCA have been developed thus far, due partly to the lack of effective animal models. Rhesus macaques are genetically homologous to humans and, most importantly, exhibit structures of the macula and fovea that are similar to those of humans; thus, rhesus macaques present special advantages in the modeling and study of human macular and foveal diseases. In this study, we identified rhesus macaque models with clinical characteristics consistent with those of OCA patients according to observations of ocular behavior, fundus examination, and optical coherence tomography. Genomic sequencing revealed a biallelic p.L312I mutation in TYR and a homozygous p.S788L mutation in OCA2, both of which were further confirmed to affect melanin biosynthesis via in vitro assays. These rhesus macaque models of OCA will be useful animal resources for studying foveal development and for preclinical trials of new therapies for OCA.

Relationship Between Cone Loss and Microvasculature Change in Retinitis Pigmentosa.

Purpose: To objectively quantify cone density (CD) and microvascular density (MVD) in normal subjects and patients with moderate or severe retinitis pigmentosa (RP) by adaptive optics (AO) and optical coherence tomography angiography (OCTA) and to evaluate the changes in the parafoveal regions. Method: Thirty-seven eyes from 20 RP patients and 54 eyes from 29 age-matched healthy participants underwent AO fundus and OCTA imaging. AO images covering a 3-mm-diameter area centered on the fovea were subdivided into 5 equidistant concentric rings (C1-C5). An automated algorithm was used to quantify the mean cone density (mCD; cells/mm2). Macular MVDs (%) in the superficial capillary plexus (SCP) and deep capillary plexus (DCP) were assessed by OCTA. Results: In the moderate RP group, CDs in C2 and C3 were each significantly lower than in the normal group (both P < 0.05). In the severe RP group, CDs were significantly lower than in normal eyes in each concentric ring (all P < 0.001; C1-C5). In both RP groups, MVDs were significantly lower than in normal eyes for both the SCP and DCP (both P < 0.05). The mean CD was significantly correlated with the MVD in the DCP (r = 0.43; P = 0.028) but not in the SCP (r = -0.19, P = 0.323). Conclusions: Decreased CD was present in the moderate and severe RP groups. This was accompanied by a decreased MVD in the DCP. Direct assessment of photoreceptors in RP patients by high-resolution imaging technologies is crucial for the future development of RP therapeutics.

Slc7a14 Is Indispensable in Zebrafish Retinas.

Previous study has identified SLC7A14 as a new causative gene of retinitis pigmentosa (RP). However, the role of SLC7A14 has not been fully characterized. The goal of this study was to investigate the biological features of slc7a14 in zebrafish. To determine the expression of slc7a14 in developing zebrafish, we performed in situ hybridization (ISH) and quantitative real-time PCR. Morpholino knockdown and overexpression experiments were performed to study the role of slc7a14 in zebrafish retinas. Immunostaining was carried out to observe structural changes. Visual motor responses (VMR) and optokinetic responses (OKR) were analyzed to assess visual behaviors. Terminal deoxynucleotidyl transferase (dUTP) nick-end labeling (TUNEL) staining was performed to survey apoptotic retinal cells. We found that slc7a14 was highly expressed in neuronal tissues, including the brain, spinal cord and retina, and that the expression levels increased during early embryogenesis. Consistently, ISH showed a similar expression pattern. Knockdown of slc7a14 led to dose-dependent microphthalmia that was reversed by overexpression. The immunostaining results revealed that the rod-specific protein zpr-3 and the retinal pigment epithelium-specific protein zpr-2 (decreased to 44.48%) were significantly suppressed in the slc7a14-silenced morphants. Notably, visual behaviors (the VMR and the OKR) were severely impaired in the slc7a14-deficient morphant, especially the VMR OFF response. In addition, apoptotic cells were observed in the retina at 3 days post fertilization (dpf) and 5 dpf by TUNEL assay. Our results demonstrated that slc7a14 is essential for visually mediated behaviors in zebrafish. Temporary silencing of slc7a14 in larvae led to severe visual impairments, consistent with the manifestations observed in RP patients. Our findings provide further insights into the genetic mechanisms of RP predisposition caused by SLC7A14 mutations.