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BACKGROUND: There are currently no specific biomarkers for multiple sclerosis (MS). Identifying robust biomarkers for MS is crucial to improve disease diagnosis and management. METHODS: This study first used six Mendelian randomisation methods to assess causal relationship of 174 metabolites with MS, incorporating data from European-ancestry metabolomics (n=8569-86 507) and MS (n=14 802 MS cases, 26 703 controls) genomewide association studies. Genetic scores for identified causal metabolite(s) were then computed to predict MS disability progression in an independent longitudinal cohort (AusLong study) of 203 MS cases with up to 15-year follow-up. RESULTS: We found a novel genetic causal effect of serine on MS onset (OR=1.67, 95% CI 1.51 to 1.84, p=1.73×10-20), such that individuals whose serine level is 1 SD above the population mean will have 1.67 times the risk of developing MS. This is robust across all sensitivity methods (OR ranges from 1.49 to 1.67). In an independent longitudinal MS cohort, we then constructed time-dynamic and time-fixed genetic scores based on serine genetic instrument single-nucleotide polymorphisms, where higher scores for raised serum serine level were associated with increased risk of disability worsening, especially in the time-dynamic model (RR=1.25, 95% CI 1.10 to 1.42, p=7.52×10-4). CONCLUSIONS: These findings support investigating serine as an important candidate biomarker for MS onset and disability progression.

Intraocular anti-vascular endothelial growth factor (VEGF) therapies are the front-line treatment for diabetic macular edema (DME); however, treatment response varies widely. This study aimed to identify genetic determinants associated with anti-VEGF treatment response in DME. We performed a genome-wide association study on 220 Australian patients with DME treated with anti-VEGF therapy, genotyped on the Illumina Global Screening Array, and imputed to the Haplotype Reference Consortium panel. The primary outcome measures were changes in central macular thickness (CMT in microns) and best-corrected visual acuity (BCVA in ETDRS letters) after 12 months. Association between single nucleotide polymorphism (SNP) genotypes and DME outcomes were evaluated by linear regression, adjusting for the first three principal components, age, baseline CMT/BCVA, duration of diabetic retinopathy, and HbA1c. Two loci reached genome-wide significance (p < 5 × 10−8) for association with increased CMT: a single SNP on chromosome 6 near CASC15 (rs78466540, p = 1.16 × 10−9) and a locus on chromosome 12 near RP11-116D17.1 (top SNP rs11614480, p = 2.69 × 10−8). Four loci were significantly associated with reduction in BCVA: two loci on chromosome 11, downstream of NTM (top SNP rs148980760, p = 5.30 × 10−9) and intronic in RP11-744N12.3 (top SNP rs57801753, p = 1.71 × 10−8); one near PGAM1P1 on chromosome 5 (rs187876551, p = 1.52 × 10−8); and one near TBC1D32 on chromosome 6 (rs118074968, p = 4.94 × 10−8). In silico investigations of each locus identified multiple expression quantitative trait loci and potentially relevant candidate genes warranting further analysis. Thus, we identified multiple genetic loci predicting treatment outcomes for anti-VEGF therapies in DME. This work may potentially lead to managing DME using personalized treatment approaches.

OBJECTIVES: To assess whether insulin therapy impacts the effectiveness of anti-vascular endothelial growth factor (anti-VEGF) injection for the treatment of diabetic macular edema (DME) in type 2 diabetes mellitus. METHODS: This was a retrospective multi-center analysis. The best-corrected visual acuity (BCVA) at 12 months, BCVA change, central macular thickness (CMT), CMT change, and cumulative injection number were compared between the insulin and the oral hypoglycemic agent (OHA) groups. RESULTS: The mean final BCVA and CMT improved in both the insulin (N = 137; p < 0.001; p < 0.001, respectively) and the OHA group (N = 61; p = 0.199; p < 0.001, respectively). The two treatment groups were comparable for final BCVA (p = 0.263), BCVA change (p = 0.184), final CMT (p = 0.741), CMT change (p = 0.458), and the cumulative injections received (p = 0.594). The results were comparable between the two groups when stratified by baseline vision (p > 0.05) and baseline HbA1c (p > 0.05). CONCLUSION: Insulin therapy does not alter treatment outcomes for anti-VEGF therapy in DME.

Advances in genome sequencing technologies have unlocked new possibilities in identifying disease-associated and causative genetic markers, which may in turn enhance disease diagnosis and improve prognostication and management strategies. With the capability of examining genetic variations ranging from single-nucleotide mutations to large structural variants, whole-genome sequencing (WGS) is an increasingly adopted approach to dissect the complex genetic architecture of neurologic diseases. There is emerging evidence for different structural variants and their roles in major neurologic and neurodevelopmental diseases. This review first describes different structural variants and their implicated roles in major neurologic and neurodevelopmental diseases, and then discusses the clinical relevance of WGS applications in neurology. Notably, WGS-based detection of structural variants has shown promising potential in enhancing diagnostic power of genetic tests in clinical settings. Ongoing WGS-based research in structural variations and quantifying mutational constraints can also yield clinical benefits by improving variant interpretation and disease diagnosis, while supporting biomarker discovery and therapeutic development. As a result, wider integration of WGS technologies into health care will likely increase diagnostic yields in difficult-to-diagnose conditions and define potential therapeutic targets or intervention points for genome-editing strategies.

OBJECTIVE: Paediatric (childhood or congenital) cataract is an opacification of the normally clear lens of the eye and has a genetic basis in at least 18% of cases in Australia. This study aimed to replicate clinical gene screening to identify variants likely to be causative of disease in an Australian patient cohort. METHODS AND ANALYSIS: Sixty-three reported isolated cataract genes were screened for rare coding variants in 37 Australian families using genome sequencing. RESULTS: Disease-causing variants were confirmed in eight families with variant classification as 'likely pathogenic'. This included novel variants PITX3 p.(Ter303LeuextTer100), BFSP1 p.(Glu375GlyfsTer2), and GJA8 p.(Pro189Ser), as well as, previously described variants identified in genes GJA3, GJA8, CRYAA, BFSP1, PITX3, COL4A1 and HSF4. Additionally, eight variants of uncertain significance with evidence towards pathogenicity were identified in genes: GJA3, GJA8, LEMD2, PRX, CRYBB1, BFSP2, and MIP. CONCLUSION: These findings expand the genotype-phenotype correlations of both pathogenic and benign variation in cataract-associated genes. They further emphasise the need to develop additional evidence such as functional assays and variant classification criteria specific to paediatric cataract genes to improve interpretation of variants and molecular diagnosis in patients.

Corneal dystrophies describe a clinically and genetically heterogeneous group of inherited disorders. The International Classification of Corneal Dystrophies (IC3D) lists 22 types of corneal dystrophy, 17 of which have been demonstrated to result from pathogenic variants in 19 identified genes. In this study, we investigated the diagnostic yield of genetic testing in a well-characterised cohort of 58 individuals from 44 families with different types of corneal dystrophy. Individuals diagnosed solely with Fuchs endothelial corneal dystrophy were excluded. Clinical details were obtained from the treating ophthalmologist. Participants and their family members were tested using a gene candidate and exome sequencing approach. We identified a likely molecular diagnosis in 70.5% families (31/44). The detection rate was significantly higher among probands with a family history of corneal dystrophy (15/16, 93.8%) than those without (16/28, 57.1%, p = .015), and among those who had undergone corneal graft surgery (9/9, 100.0%) compared to those who had not (22/35, 62.9%, p = .041). We identified eight novel variants in five genes and identified five families with syndromes associated with corneal dystrophies. Our findings highlight the genetic heterogeneity of corneal dystrophies and the clinical utility of genetic testing in reaching an accurate clinical diagnosis.

PURPOSE. Over a third of patients with Acanthamoeba keratitis (AK) experience severe inflammatory complications (SICs). This study aimed to determine if some contact lens (CL) wearers with AK were predisposed to SICs due to variations in key immune genes. METHODS. CL wearers with AK who attended Moorfields Eye Hospital were recruited prospectively between April 2013 and October 2014. SICs were defined as scleritis and/or stromal ring infiltrate. Genomic DNA was processed with an Illumina Low Input Custom Amplicon assay of 58 single nucleotide polymorphism (SNP) targets across 18 genes and tested for association in PLINK. RESULTS. Genomic DNA was obtained and analyzed for 105 cases of AK, 40 (38%) of whom experienced SICs. SNPs in the CXCL8 gene encoding IL-8 was significantly associated with protection from SICs (chr4: Rs1126647, odds ratio [OR] = 0.3, P = 0.005, rs2227543, OR = 0.4, P = 0.007, and rs2227307, OR = 0.4, P = 0.02) after adjusting for age, sex, steroids prediagnosis, and herpes simplex keratitis (HSK) misdiagnosis. Two TLR-4 SNPs were associated with increased risk of SICs (chr9: Rs4986791 and rs4986790, both OR = 6.9, P = 0.01). Th-17 associated SNPs (chr1: IL-23R rs11209026, chr2: IL-1β rs16944, and chr12: IL-22 rs1179251) were also associated with SICs. CONCLUSIONS. The current study identifies biologically relevant genetic variants in patients with AK with SICs; IL-8 is associated with a strong neutrophil response in the cornea in AK, TLR-4 is important in early AK disease, and Th-17 genes are associated with adaptive immune responses to AK in animal models. Genetic screening of patients with AK to predict severity is viable and this would be expected to assist disease management.

Keratoconus is characterised by reduced rigidity of the cornea with distortion and focal thinning that causes blurred vision, however, the pathogenetic mechanisms are unknown. It can lead to severe visual morbidity in children and young adults and is a common indication for corneal transplantation worldwide. Here we report the first large scale genome-wide association study of keratoconus including 4,669 cases and 116,547 controls. We have identified significant association with 36 genomic loci that, for the first time, implicate both dysregulation of corneal collagen matrix integrity and cell differentiation pathways as primary disease-causing mechanisms. The results also suggest pleiotropy, with some disease mechanisms shared with other corneal diseases, such as Fuchs endothelial corneal dystrophy. The common variants associated with keratoconus explain 12.5% of the genetic variance, which shows potential for the future development of a diagnostic test to detect susceptibility to disease.

Inherited paediatric cataract is a rare Mendelian disease that results in visual impairment or blindness due to a clouding of the eye's crystalline lens. Here we report an Australian family with isolated paediatric cataract, which we had previously mapped to Xq24. Linkage at Xq24-25 (LOD = 2.53) was confirmed, and the region refined with a denser marker map. In addition, two autosomal regions with suggestive evidence of linkage were observed. A segregating 127 kb deletion (chrX:g.118373226_118500408del) in the Xq24-25 linkage region was identified from whole-genome sequencing data. This deletion completely removed a commonly deleted long non-coding RNA gene LOC101928336 and truncated the protein coding progesterone receptor membrane component 1 (PGRMC1) gene following exon 1. A literature search revealed a report of two unrelated males with non-syndromic intellectual disability, as well as congenital cataract, who had contiguous gene deletions that accounted for their intellectual disability but also disrupted the PGRMC1 gene. A morpholino-induced pgrmc1 knockdown in a zebrafish model produced significant cataract formation, supporting a role for PGRMC1 in lens development and cataract formation. We hypothesise that the loss of PGRMC1 causes cataract through disrupted PGRMC1-CYP51A1 protein-protein interactions and altered cholesterol biosynthesis. The cause of paediatric cataract in this family is the truncating deletion of PGRMC1, which we report as a novel cataract gene.

PURPOSE: Developmental abnormalities of the ocular anterior segment in some cases can lead to ocular hypertension and glaucoma. CPAMD8 is a gene of unknown function recently associated with ocular anterior segment dysgenesis, myopia, and ectopia lentis. We sought to assess the contribution of biallelic CPAMD8 variants to childhood and juvenile open-angle glaucoma. DESIGN: Retrospective, multicenter case series. PARTICIPANTS: A total of 268 probands and their relatives with a diagnosis of childhood or juvenile open-angle glaucoma. PURPOSE: Developmental abnormalities of the ocular anterior segment in some cases can lead to ocular hypertension and glaucoma. CPAMD8 is a gene of unknown function recently associated with ocular anterior segment dysgenesis, myopia, and ectopia lentis. We sought to assess the contribution of biallelic CPAMD8 variants to childhood and juvenile open-angle glaucoma. METHODS: Patients underwent a comprehensive ophthalmic assessment, with DNA from patients and their relatives subjected to genome, exome, or capillary sequencing. CPAMD8 RNA expression analysis was performed on tissues dissected from cadaveric human eyes. MAIN OUTCOME MEASURES: Diagnostic yield within a cohort of childhood and juvenile open-angle glaucoma, prevalence and risk of ophthalmic phenotypes, and relative expression of CPAMD8 in the human eye. RESULTS: We identified rare (allele frequency < 4×10-5) biallelic CPAMD8 variants in 5.7% (5/88) of probands with childhood glaucoma and 2.1% (2/96) of probands with juvenile open-angle glaucoma. When including family members, we identified 11 individuals with biallelic variants in CPAMD8 from 7 unrelated families. Nine of these individuals were diagnosed with glaucoma (9/11, 81.8%), with a mean age at diagnosis of 9.22±14.89 years, and all individuals with glaucoma required 1 or more incisional procedures to control high intraocular pressure. Iris abnormalities were observed in 9 of 11 individuals, cataract was observed in 8 of 11

Nuclear cataract is the most common type of age-related cataract and a leading cause of blindness worldwide. Age-related nuclear cataract is heritable (h2 = 0.48), but little is known about specific genetic factors underlying this condition. Here we report findings from the largest to date multi-ethnic meta-analysis of genome-wide association studies (discovery cohort N = 14,151 and replication N = 5299) of the International Cataract Genetics Consortium. We confirmed the known genetic association of CRYAA (rs7278468, P = 2.8 × 10-16) with nuclear cataract and identified five new loci associated with this disease: SOX2-OT (rs9842371, P = 1.7 × 10-19), TMPRSS5 (rs4936279, P = 2.5 × 10-10), LINC01412 (rs16823886, P = 1.3 × 10-9), GLTSCR1 (rs1005911, P = 9.8 × 10-9), and COMMD1 (rs62149908, P = 1.2 × 10-8). The results suggest a strong link of age-related nuclear cataract with congenital cataract and eye development genes, and the importance of common genetic variants in maintaining crystalline lens integrity in the aging eye.

Glaucoma, a disease characterized by progressive optic nerve degeneration, can be prevented through timely diagnosis and treatment. We characterize optic nerve photographs of 67,040 UK Biobank participants and use a multitrait genetic model to identify risk loci for glaucoma. A glaucoma polygenic risk score (PRS) enables effective risk stratification in unselected glaucoma cases and modifies penetrance of the MYOC variant encoding p.Gln368Ter, the most common glaucoma-associated myocilin variant. In the unselected glaucoma population, individuals in the top PRS decile reach an absolute risk for glaucoma 10 years earlier than the bottom decile and are at 15-fold increased risk of developing advanced glaucoma (top 10% versus remaining 90%, odds ratio = 4.20). The PRS predicts glaucoma progression in prospectively monitored, early manifest glaucoma cases (P = 0.004) and surgical intervention in advanced disease (P = 3.6 × 10-6). This glaucoma PRS will facilitate the development of a personalized approach for earlier treatment of high-risk individuals, with less intensive monitoring and treatment being possible for lower-risk groups.

Nanophthalmos and posterior microphthalmos are ocular abnormalities in which both eyes are abnormally small, and typically associated with extreme hyperopia. We recruited 40 individuals from 13 kindreds with nanophthalmos or posterior microphthalmos, with 12 probands subjected to exome sequencing. Nine probands (69.2%) were assigned a genetic diagnosis, with variants in MYRF, TMEM98, MFRP, and PRSS56. Two of four PRSS56 families harbored the previously described c.1066dupC variant implicated in over half of all reported PRSS56 kindreds, with different surrounding haplotypes in each family suggesting a mutational hotspot. Individuals with a genetic diagnosis had shorter mean axial lengths and higher hyperopia than those without, with recessive forms associated with the most extreme phenotypes. These findings detail the genetic architecture of nanophthalmos and posterior microphthalmos in a cohort of predominantly European ancestry, their relative clinical phenotypes, and highlight the shared genetic architecture of rare and common disorders of refractive error.

The original version of this Article contained errors in the spelling of the authors Fan Liu and M. Arfan Ikram, which were incorrectly given as Fan Lui and Arfan M. Ikram. In addition, the original version of this Article also contained errors in the author affiliations which are detailed in the associated Publisher Correction.

The knowledge on the genetic background of refractive error and myopia has expanded dramatically in the past few years. This white paper aims to provide a concise summary of current genetic findings and defines the direction where development is needed. We performed an extensive literature search and conducted informal discussions with key stakeholders. Specific topics reviewed included common refractive error, any and high myopia, and myopia related to syndromes. To date, almost 200 genetic loci have been identified for refractive error and myopia, and risk variants mostly carry low risk but are highly prevalent in the general population. Several genes for secondary syndromic myopia overlap with those for common myopia. Polygenic risk scores show overrepresentation of high myopia in the higher deciles of risk. Annotated genes have a wide variety of functions, and all retinal layers appear to be sites of expression. The current genetic findings offer a world of new molecules involved in myopiagenesis. As the missing heritability is still large, further genetic advances are needed. This Committee recommends expanding large-scale, in-depth genetic studies using complementary big data analytics, consideration of gene-environment effects by thorough measurement of environmental exposures, and focus on subgroups with extreme phenotypes and high familial occurrence. Functional characterization of associated variants is simultaneously needed to bridge the knowledge gap between sequence variance and consequence for eye growth.

A new avenue of mining published genome-wide association studies includes the joint analysis of related traits. The power of this approach depends on the genetic correlation of traits, which reflects the number of pleiotropic loci, i.e. genetic loci influencing multiple traits. Here, we applied new meta-analyses of optic nerve head (ONH) related traits implicated in primary open-angle glaucoma (POAG); intraocular pressure and central corneal thickness using Haplotype reference consortium imputations. We performed a multi-trait analysis of ONH parameters cup area, disc area and vertical cup-disc ratio. We uncover new variants; rs11158547 in PPP1R36-PLEKHG3 and rs1028727 near SERPINE3 at genome-wide significance that replicate in independent Asian cohorts imputed to 1000 Genomes. At this point, validation of these variants in POAG cohorts is hampered by the high degree of heterogeneity. Our results show that multi-trait analysis is a valid approach to identify novel pleiotropic variants for ONH.

PURPOSE: Age-related macular degeneration (AMD) is the worldwide leading cause of blindness among the elderly. Although genome-wide association studies (GWAS) have identified AMD risk variants, their roles in disease etiology are not well-characterized, and they only explain a portion of AMD heritability. METHODS: We performed pathway analyses using summary statistics from the International AMD Genomics Consortium's 2016 GWAS and multiple pathway databases to identify biological pathways wherein genetic association signals for AMD may be aggregating. We determined which genes contributed most to significant pathway signals across the databases. We characterized these genes by constructing protein-protein interaction networks and performing motif analysis. RESULTS: We determined that eight genes (C2, C3, LIPC, MICA, NOTCH4, PLCG2, PPARA, and RAD51B) "drive" the statistical signals observed across pathways curated in the Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome, and Gene Ontology (GO) databases. We further refined our definition of statistical driver gene to identify PLCG2 as a candidate gene for AMD due to its significant gene-level signals (P < 0.0001) across KEGG, Reactome, GO, and NetPath pathways. CONCLUSIONS: We performed pathway analyses on the largest available collection of advanced AMD cases and controls in the world. Eight genes strongly contributed to significant pathways from the three larger databases, and one gene (PLCG2) was central to significant pathways from all four databases. This is, to our knowledge, the first study to identify PLCG2 as a candidate gene for AMD based solely on genetic burden. Our findings reinforce the utility of integrating in silico genetic and biological pathway data to investigate the genetic architecture of AMD.

BACKGROUND: Fuchs endothelial corneal dystrophy (FECD) is a progressive and potentially a sight threatening disease, and a common indication for corneal grafting in the elderly. Aberrant thickening of Descemet's membrane, formation of microscopic excrescences (guttae) and gradual loss of corneal endothelial cells are the hallmarks of the disease. The aim of this study was to identify differentially abundant proteins between FECD-affected and unaffected Descemet's membrane. METHODS: Label-free quantitative proteomics using nanoscale ultra-performance liquid chromatography-mass spectrometry (nUPLC-MSE ) was employed on affected and unaffected Descemet's membrane extracts, and interesting findings were further investigated using quantitative reverse transcription-polymerase chain reaction and immunohistochemical techniques. RESULTS: Quantitative proteomics revealed significantly lower abundance of apolipoprotein E (APOE) and immunoglobulin heavy constant gamma 1 protein (IGHG1) in affected Descemet's membrane. The difference in the distribution of APOE between affected and unaffected Descemet's membrane and of IGHG1 detected by immunohistochemistry support their down-regulation in the disease. Comparative gene expression analysis showed significantly lower APOE mRNA levels in FECD-affected than unaffected corneal endothelium. IGHG1 gene is expressed at extremely low levels in the corneal endothelium, precluding relative expression analysis. CONCLUSIONS: This is the first study to report comparative proteomics of Descemet's membrane tissue, and implicates dysregulation of APOE and IGHG1 proteins in the pathogenesis of Fuchs endothelial corneal dystrophy.

Mitochondrial haplogroups H1, H2 and UK have previously been reported to be associated with proliferative diabetic retinopathy (PDR) in Caucasian patients with diabetes. We aimed to replicate this finding with a larger sample and expand the analysis to include different severities of DR, and diabetic macular edema (DME). Caucasian participants (n = 2935) with either type 1 or type 2 diabetes from the Australian Registry of Advanced Diabetic Retinopathy were enrolled in this study. Twenty-two mitochondrial single nucleotide polymorphisms were genotyped by MassArray and haplogroups reconstructed using Haplogrep. Chi square tests and logistic regressions were used to test associations between haplogroup and DR phenotypes including any DR, non-proliferative DR (NPDR), proliferative DR (PDR) and DME. After stratifying the samples in type 1 and type 2 diabetes groups, and adjusting for sex, age, diabetes duration, concurrent HbA1c and hypertension, neither haplogroups H1, H2, UK, K or JT were associated with any DR, NPDR, PDR or DME.

Emmanuelle Souzeau, who contributed to analysis of data, was inadvertently omitted from the author list in the originally published version of this Article. This has now been corrected in both the PDF and HTML versions of the Article.

Many genes have been suggested as candidate genes for keratoconus based on their function, their proximity to associated polymorphisms or due to the identification of putative causative variants within the gene. However, very few of these genes have been assessed for rare variation in keratoconus more broadly. In contrast, VSX1 and SOD1 have been widely assessed, however, the vast majority of studies have been small and the findings conflicting. In a cohort of Australians of European descent, consisting of 385 keratoconus cases and 396 controls, we screened 21 keratoconus candidate genes: BANP, CAST, COL4A3, COL4A4, COL5A1, FOXO1, FNDC3B, HGF, IL1A, IL1B, ILRN, IMMP2L, MPDZ, NFIB, RAB3GAP1, RAD51, RXRA, SLC4A11, SOD1, TF and VSX1. The candidate genes were sequenced in these individuals by either whole exome sequencing or targeted gene sequencing. Variants were filtered to identify rare (minor allele frequency <1%), potentially pathogenic variants. A total of 164 such variants were identified across the two groups with no variants fulfilling these criteria in cases in IL1RN, BANP, IL1B, RAD51 or SOD1. The frequency of variants was compared between cases and controls using chi-square or Fishers' Exact tests for each gene with at least one rare potentially pathogenic variant identified in the case cohort. The number of rare potentially pathogenic variants per gene ranged from three (RXRA) to 102 (MPDZ), however for all genes, there was no difference in the frequency between the cases and controls. We conclude that rare potentially pathogenic variation in the 21 candidate genes assessed do not play a major role in keratoconus susceptibility and pathogenesis.

BACKGROUND: Pediatric cataract is an important cause of blindness and visual impairment in children. A large proportion of pediatric cataracts are inherited, and many genes have been described for this heterogeneous Mendelian disease. Surveys of schools for the blind in Bhutan, Cambodia, and Sri Lanka have identified many children with this condition and we aimed to identify the genetic causes of inherited cataract in these populations. METHODS: We screened, in parallel, 51 causative genes for inherited cataracts in 33 probands by Ampliseq enrichment and sequencing on an Ion Torrent PGM. Rare novel protein coding variants were assessed for segregation in family members, where possible, by Sanger sequencing. RESULTS: We identified 24 rare (frequency <1% in public databases) or novel protein coding variants in 12 probands and confirmed segregation of variants with disease in the extended family where possible. Of these, six are predicted to be the cause of disease in the patient, with four other variants also highly likely to be pathogenic. CONCLUSION: This study found that 20%-30% of patients in these countries have a mutation in a known cataract causing gene, which is considerably lower than the 60%-70% reported in Caucasian cohorts. This suggests that additional cataract genes remain to be discovered in this cohort of Asian pediatric cataract patients.

PURPOSE: Sex hormones may be associated with primary open-angle glaucoma (POAG), although the mechanisms are unclear. We previously observed that gene variants involved with estrogen metabolism were collectively associated with POAG in women but not men; here we assessed gene variants related to testosterone metabolism collectively and POAG risk. METHODS: We used two datasets: one from the United States (3853 cases and 33,480 controls) and another from Australia (1155 cases and 1992 controls). Both datasets contained densely called genotypes imputed to the 1000 Genomes reference panel. We used pathway- and gene-based approaches with Pathway Analysis by Randomization Incorporating Structure (PARIS) software to assess the overall association between a panel of single nucleotide polymorphisms (SNPs) in testosterone metabolism genes and POAG. In sex-stratified analyses, we evaluated POAG overall and POAG subtypes defined by maximum IOP (high-tension [HTG] or normal tension glaucoma [NTG]). RESULTS: In the US dataset, the SNP panel was not associated with POAG (permuted P = 0.77), although there was an association in the Australian sample (permuted P = 0.018). In both datasets, the SNP panel was associated with POAG in men (permuted P ≤ 0.033) and not women (permuted P ≥ 0.42), but in gene-based analyses, there was no consistency on the main genes responsible for these findings. In both datasets, the testosterone pathway association with HTG was significant (permuted P ≤ 0.011), but again, gene-based analyses showed no consistent driver gene associations. CONCLUSIONS: Collectively, testosterone metabolism pathway SNPs were consistently associated with the high-tension subtype of POAG in two datasets.

BACKGROUND: Diabetic macular edema (DME) and proliferative diabetic retinopathy (PDR) are sight-threatening complications of diabetes mellitus and leading causes of adult-onset blindness worldwide. Genetic risk factors for diabetic retinopathy (DR) have been described previously, but have been difficult to replicate between studies, which have often used composite phenotypes and been conducted in different populations. This study aims to identify genetic risk factors for DME and PDR as separate complications in Australians of European descent with type 2 diabetes. METHODS: Caucasian Australians with type 2 diabetes were evaluated in a genome-wide association study (GWAS) to compare 270 DME cases and 176 PDR cases with 435 non-retinopathy controls. All participants were genotyped by SNP array and after data cleaning, cases were compared to controls using logistic regression adjusting for relevant covariates. RESULTS: The top ranked SNP for DME was rs1990145 (p = 4.10 × 10- 6, OR = 2.02 95%CI [1.50, 2.72]) on chromosome 2. The top-ranked SNP for PDR was rs918519 (p = 3.87 × 10- 6, OR = 0.35 95%CI [0.22, 0.54]) on chromosome 5. A trend towards association was also detected at two SNPs reported in the only other reported GWAS of DR in Caucasians; rs12267418 near MALRD1 (p = 0.008) in the DME cohort and rs16999051 in the diabetes gene PCSK2 (p = 0.007) in the PDR cohort. CONCLUSION: This study has identified loci of interest for DME and PDR, two common ocular complications of diabetes. These findings require replication in other Caucasian cohorts with type 2 diabetes and larger cohorts will be required to identify genetic loci with statistical confidence. There is considerable overlap in the patient cohorts with each retinopathy subtype, complicating the search for genes that contribute to PDR and DME biology.

Central corneal thickness (CCT) is a highly heritable trait associated with complex eye diseases such as keratoconus and glaucoma. We perform a genome-wide association meta-analysis of CCT and identify 19 novel regions. In addition to adding support for known connective tissue-related pathways, pathway analyses uncover previously unreported gene sets. Remarkably, >20% of the CCT-loci are near or within Mendelian disorder genes. These included FBN1, ADAMTS2 and TGFB2 which associate with connective tissue disorders (Marfan, Ehlers-Danlos and Loeys-Dietz syndromes), and the LUM-DCN-KERA gene complex involved in myopia, corneal dystrophies and cornea plana. Using index CCT-increasing variants, we find a significant inverse correlation in effect sizes between CCT and keratoconus (r = -0.62, P = 5.30 × 10-5) but not between CCT and primary open-angle glaucoma (r = -0.17, P = 0.2). Our findings provide evidence for shared genetic influences between CCT and keratoconus, and implicate candidate genes acting in collagen and extracellular matrix regulation.

PURPOSE: Diabetic retinopathy is the most common eye complication in patients with diabetes. The purpose of this study is to identify genetic factors contributing to severe diabetic retinopathy. METHODS: A genome-wide association approach was applied. In the Genetics of Diabetes Audit and Research in Tayside Scotland (GoDARTS) datasets, cases of severe diabetic retinopathy were defined as type 2 diabetic patients who were ever graded as having severe background retinopathy (Level R3) or proliferative retinopathy (Level R4) in at least one eye according to the Scottish Diabetic Retinopathy Grading Scheme or who were once treated by laser photocoagulation. Controls were diabetic individuals whose longitudinal retinopathy screening records were either normal (Level R0) or only with mild background retinopathy (Level R1) in both eyes. Significant Single Nucleotide Polymorphisms (SNPs) were taken forward for meta-analysis using multiple Caucasian cohorts. RESULTS: Five hundred and sixty cases of type 2 diabetes with severe diabetic retinopathy and 4,106 controls were identified in the GoDARTS cohort. We revealed that rs3913535 in the NADPH Oxidase 4 (NOX4) gene reached a p value of 4.05 × 10-9 . Two nearby SNPs, rs10765219 and rs11018670 also showed promising p values (p values = 7.41 × 10-8 and 1.23 × 10-8 , respectively). In the meta-analysis using multiple Caucasian cohorts (excluding GoDARTS), rs10765219 and rs11018670 showed associations for diabetic retinopathy (p = 0.003 and 0.007, respectively), while the p value of rs3913535 was not significant (p = 0.429). CONCLUSION: This genome-wide association study of severe diabetic retinopathy suggests new evidence for the involvement of the NOX4 gene.

Open-angle glaucoma (OAG) is a major cause of blindness worldwide. To identify new risk loci for OAG, we performed a genome-wide association study in 3,071 OAG cases and 6,750 unscreened controls, and meta-analysed the results with GWAS data for intraocular pressure (IOP) and optic disc parameters (the overall meta-analysis sample size varying between 32,000 to 48,000 participants), which are glaucoma-related traits. We identified and independently validated four novel genome-wide significant associations within or near MYOF and CYP26A1, LINC02052 and CRYGS, LMX1B, and LMO7 using single variant tests, one additional locus (C9) using gene-based tests, and two genetic pathways - "response to fluid shear stress" and "abnormal retina morphology" - in pathway-based tests. Interestingly, some of the new risk loci contribute to risk of other genetically-correlated eye diseases including myopia and age-related macular degeneration. To our knowledge, this study is the first integrative study to combine genetic data from OAG and its correlated traits to identify new risk variants and genetic pathways, highlighting the future potential of combining genetic data from genetically-correlated eye traits for the purpose of gene discovery and mapping.

BACKGROUND: Cataract is a major cause of severe visual impairment in childhood. The purpose of this study was to determine the genetic cause of syndromic congenital cataract in an Australian mother and son. METHOD: Fifty-one genes associated with congenital cataract were sequenced in the proband using a custom Ampliseq library on the Ion Torrent Personal Genome Machine (PGM). Reads were aligned against the human genome (hg19) and variants were annotated. Variants were prioritised for validation by Sanger sequencing if they were novel, rare or previously reported to be associated with paediatric cataract and were predicted to be protein changing. Variants were assessed for segregation with the phenotype in the affected mother. RESULT: A novel likely pathogenic variant was identified in the transactivation domain of the MAF gene (c.176C > G, p.(Pro59Arg)) in the proband and his affected mother., but was absent in 326 unrelated controls and absent from public variant databases. CONCLUSION: The MAF variant is the likely cause of the congenital cataract, Asperger syndrome, seizures, hearing loss and facial characteristics in the proband, providinga diagnosis of Aymé-Gripp syndrome for the family.

The structure of the cornea is vital to its transparency, and dystrophies that disrupt corneal organization are highly heritable. To understand the genetic aetiology of Fuchs endothelial corneal dystrophy (FECD), the most prevalent corneal disorder requiring transplantation, we conducted a genome-wide association study (GWAS) on 1,404 FECD cases and 2,564 controls of European ancestry, followed by replication and meta-analysis, for a total of 2,075 cases and 3,342 controls. We identify three novel loci meeting genome-wide significance (P<5 × 10-8): KANK4 rs79742895, LAMC1 rs3768617 and LINC00970/ATP1B1 rs1200114. We also observe an overwhelming effect of the established TCF4 locus. Interestingly, we detect differential sex-specific association at LAMC1, with greater risk in women, and TCF4, with greater risk in men. Combining GWAS results with biological evidence we expand the knowledge of common FECD loci from one to four, and provide a deeper understanding of the underlying pathogenic basis of FECD.

Pediatric cataract is a leading cause of childhood blindness. This study aimed to determine the genetic cause of pediatric cataract in Australian families by screening known disease-associated genes using massively parallel sequencing technology. We sequenced 51 previously reported pediatric cataract genes in 33 affected individuals with a family history (cases with previously known or published mutations were excluded) using the Ion Torrent Personal Genome Machine. Variants were prioritized for validation if they were predicted to alter the protein sequence and were absent or rare with minor allele frequency <1% in public databases. Confirmed mutations were assessed for segregation with the phenotype in all available family members. All identified novel or previously reported cataract-causing mutations were screened in 326 unrelated Australian controls. We detected 11 novel mutations in GJA3, GJA8, CRYAA, CRYBB2, CRYGS, CRYGA, GCNT2, CRYGA, and MIP; and three previously reported cataract-causing mutations in GJA8, CRYAA, and CRYBB2 The most commonly mutated genes were those coding for gap junctions and crystallin proteins. Including previous reports of pediatric cataract-associated mutations in our Australian cohort, known genes account for >60% of familial pediatric cataract in Australia, indicating that still more causative genes remain to be identified.

Variation in FOXC1 and PITX2 is associated with Axenfeld-Rieger syndrome, characterised by structural defects of the anterior chamber of the eye and a range of systemic features. Approximately half of all affected individuals will develop glaucoma, but the age at diagnosis and the phenotypic spectrum have not been well defined. As phenotypic heterogeneity is common, we aimed to delineate the age-related penetrance and the full phenotypic spectrum of glaucoma in FOXC1 or PITX2 carriers recruited through a national disease registry. All coding exons of FOXC1 and PITX2 were directly sequenced and multiplex ligation-dependent probe amplification was performed to detect copy number variation. The cohort included 53 individuals from 24 families with disease-associated FOXC1 or PITX2 variants, including one individual diagnosed with primary congenital glaucoma and five with primary open-angle glaucoma. The overall prevalence of glaucoma was 58.5% and was similar for both genes (53.3% for FOXC1 vs 60.9% for PITX2, P=0.59), however, the median age at glaucoma diagnosis was significantly lower in FOXC1 (6.0±13.0 years) compared with PITX2 carriers (18.0±10.6 years, P=0.04). The penetrance at 10 years old was significantly lower in PITX2 than FOXC1 carriers (13.0% vs 42.9%, P=0.03) but became comparable at 25 years old (71.4% vs 57.7%, P=0.38). These findings have important implications for the genetic counselling of families affected by Axenfeld-Rieger syndrome, and also suggest that FOXC1 and PITX2 contribute to the genetic architecture of primary glaucoma subtypes.

PURPOSE: To identify biological processes associated with POAG and its subtypes, high-tension (HTG) and normal-tension glaucoma (NTG), by analyzing rare potentially damaging genetic variants. METHODS: A total of 122 and 65 unrelated HTG and NTG participants, respectively, with early onset advanced POAG, 103 non-glaucoma controls and 993 unscreened ethnicity-matched controls were included in this study. Study participants without myocilin disease-causing variants and non-glaucoma controls were subjected to whole exome sequencing on an Illumina HiSeq2000. Exomes of participants were sequenced on an Illumina HiSeq2000. Qualifying variants were rare in the general population (MAF < 0.001) and potentially functionally damaging (nonsense, frameshift, splice or predicted pathogenic using SIFT or Polyphen2 software). Genes showing enrichment of qualifying variants in cases were selected for pathway and network analysis using InnateDB. RESULTS: POAG cases showed enrichment of rare variants in camera-type eye development genes (p = 1.40×10-7, corrected p = 3.28×10-4). Implicated eye development genes were related to neuronal or retinal development. HTG cases were significantly enriched for key regulators in the unfolded protein response (UPR) (p = 7.72×10-5, corrected p = 0.013). The UPR is known to be involved in myocilin-related glaucoma; our results suggest the UPR has a role in non-myocilin causes of HTG. NTG cases showed enrichment in ion channel transport processes (p = 1.05×10-4, corrected p = 0.027) including calcium, chloride and phospholipid transporters involved in plasma membrane homeostasis. Network analysis also revealed enrichment of the MHC Class I antigen presentation pathway in HTG, and the EGFR1 and cell-cycle pathways in both HTG and NTG. CONCLUSION: This study suggests that mutations in eye development genes are enriched in POAG. HTG can result from aberrant responses to protein misfolding which may be amenable to molecular chaperone therapy. NTG is assoc

Fuchs' endothelial corneal dystrophy (FECD) is a progressive, vision impairing disease. Common single nucleotide polymorphisms (SNPs) and a trinucleotide repeat polymorphism, thymine-guanine-cytosine (TGC), in the TCF4 gene have been associated with the risk of FECD in some populations. We previously reported association of SNPs in TCF4 with FECD risk in the Australian population. The aim of this study was to determine whether TGC repeat polymorphism in TCF4 is associated with FECD in the Australian population. In 189 unrelated Australian cases with advanced late-onset FECD and 183 matched controls, the TGC repeat polymorphism located in intron 3 of TCF4 was genotyped using a short tandem repeat (STR) assay. The repeat length was verified by direct sequencing in selected homozygous carriers. We found significant association between the expanded TGC repeat (≥ 40 repeats) in TCF4 and advanced FECD (P = 2.58 × 10-22; OR = 15.66 (95% CI: 7.79-31.49)). Genotypic analysis showed that 51% of cases (97) compared to 5% of controls (9) were heterozygous or homozygous for the expanded repeat allele. Furthermore, the repeat expansion showed stronger association than the most significantly associated SNP, rs613872, in TCF4, with the disease in the Australian cohort. This and haplotype analysis of both the polymorphisms suggest that considering both the polymorphisms together rather than either of the two alone would better predict susceptibility to FECD in the Australian population. This is the first study to report association of the TGC trinucleotide repeat expansion in TCF4 with advanced FECD in the Australian population.

Genome editing using clustered regularly interspersed short palindromic repeats (CRISPR) and CRISPR-associated proteins offers the potential to facilitate safe and effective treatment of genetic diseases refractory to other types of intervention. Here, we identify some of the major challenges for clinicians, regulators, and human research ethics committees in the clinical translation of CRISPR-mediated somatic cell therapy.

BACKGROUND: Cataract is a major cause of childhood blindness worldwide. The purpose of this study was to determine the genetic cause of paediatric cataract in a South Australian family with a bilateral lamellar paediatric cataract displaying variable phenotypes. CASE PRESENTATION: Fifty-one genes implicated in congenital cataract in human or mouse were sequenced in an affected individual from an Australian (Caucasian) family using a custom Ampliseq library on the Ion Torrent Personal Genome Machine. Reads were mapped against the human genome (hg19) and variants called with the Torrent Suite software. Variants were annotated to dbSNP 137 using Ion Reporter (IR 1.6.2) and were prioritised for validation if they were novel or rare and were predicted to be protein changing. We identified a previously reported oligomerization disrupting mutation, c.62G > A (p.R21Q), in the Crystallin alpha A (CRYAA) gene segregating in this three generation family. No other novel or rare coding mutations were detected in the known cataract genes sequenced. Microsatellite markers were used to compare the haplotypes between the family reported here and a previously published family with the same segregating mutation. Haplotype analysis indicated a potential common ancestry between the two South Australian families with this mutation. The work strengthens the genotype-phenotype correlations between this functional mutation in the crystallin alpha A (CRYAA) gene and paediatric cataract. CONCLUSION: The p.R21Q mutation is the most likely cause of paediatric cataract in this family. The recurrence of this mutation in paediatric cataract families is likely due to a familial relationship.

BACKGROUND: Genome-wide association studies (GWAS) have identified association of common alleles with primary open-angle glaucoma (POAG) and its quantitative endophenotypes near numerous genes. This study aims to determine whether rare pathogenic variants in these disease-associated genes contribute to POAG. METHODS: Participants fulfilled strict inclusion criteria of advanced POAG at a young age of diagnosis. Myocilin mutation carriers were excluded using direct sequencing. Whole exome sequencing was performed on 187 glaucoma cases and 103 local screened nonglaucoma controls then joint-called with exomes of 993 previously sequenced Australian controls. GWAS-associated genes were assessed for enrichment of rare predicted pathogenic variants in POAG. Significantly enriched genes were compared against Exome Aggregation Consortium (ExAC) public control. RESULTS: Eighty-six GWAS disease or trait-associated glaucoma genes were captured and sequenced. CARD10 showed enrichment after Bonferroni correction for rare variants in glaucoma cases (OR = 13.2, P = 6.94 × 10-5) with mutations identified in 4.28% of our POAG cohort compared to 0.27% in controls. CARD10 was significantly associated with optic disc parameters in previous GWAS. The whole GWAS gene set showed no enrichment in POAG overall (OR = 1.12, P = 0.51). CONCLUSION: We report here an enrichment of rare predicted pathogenic coding variants within a GWAS-associated locus in POAG (CARD10). These findings indicate that both common and rare pathogenic coding variants in CARD10 may contribute to POAG pathogenesis.

BACKGROUND: Glaucoma is a leading cause of irreversible blindness. Pathogenic variants in the Myocilin gene (MYOC) cause juvenile open angle glaucoma (JOAG) in 8-36% of cases, and display an autosomal dominant inheritance with high penetrance. Molecular diagnosis is important for early identification as therapies are effective in minimizing vision loss and MYOC variants can be associated to severe glaucoma. MYOC variants are usually inherited, however a fifth of carriers do not report a family history. The occurrence of de novo MYOC variants is currently unknown. CASE PRESENTATION: In this study we investigated a 14 year old male Caucasian patient diagnosed with JOAG, and no family history of glaucoma. A novel probably deleterious MYOC:p.(Pro254Leu) variant was identified in the index case. This variant was not present in the parents or the siblings. CONCLUSION: This is the second report of a de novo MYOC variant in a sporadic case of JOAG and it is currently unknown if this mechanism occurs more frequently. This finding emphasizes the importance of screening individuals with JOAG for MYOC mutations irrespective of a negative family history.

Whole genome and whole exome sequencing technologies are being increasingly used in research. However, they have the potential to identify incidental findings (IF), findings not related to the indication of the test, raising questions regarding researchers' responsibilities toward the return of this information to participants. In this study we discuss the ethical considerations related to the return of IF to research participants, emphasizing that the type of the study matters and describing the current practice standards. There are currently no legal obligations for researchers to return IF to participants, but some viewpoints consider that researchers might have an ethical one to return IF of clinical validity and clinical utility and that are actionable. The reality is that most IF are complex to interpret, especially since they were not the indication of the test. The clinical utility often depends on the participants' preferences, which can be challenging to conciliate and relies on participants' understanding. In summary, in the context of a lack of clear guidance, researchers need to have a clear plan for the disclosure or nondisclosure of IF from genomic research, balancing their research goals and resources with the participants' rights and their duty not to harm.

Pooled DNA based GWAS to determine genetic association of SNPs with visual acuity (VA) outcome in anti-vascular endothelial growth factor (anti-VEGF) treated neovascular age-related macular degeneration (nAMD) patients. We performed pooled DNA based GWAS on 285 anti-VEGF treated nAMD patients using high density Illumina 4.3 M array. Primary outcome was change in VA in Early Treatment Diabetic Retinopathy Study (ETDRS) letters after 6 months of anti-VEGF treatment (patients who lost ≥5 ETDRS letters classified as non-responders and all remaining classified as responders). GWAS analysis identified 44 SNPs of interest: 37 with strong evidence of association (p < 9 × 10-8), 2 in drug resistance genes (p < 5 × 10-6) and 5 nonsynonymous changes (p < 1 × 10-4). In the validation phase, individual genotyping of 44 variants showed three SNPs (rs4910623 p = 5.6 × 10-5, rs323085 p = 6.5 × 10-4 and rs10198937 p = 1.30 × 10-3) remained associated with VA response at 6 months. SNP rs4910623 also associated with treatment response at 3 months (p = 1.5 × 10-3). Replication of these three SNPs in 376 patients revealed association of rs4910623 with poor VA response after 3 and 6 months of treatment (p = 2.4 × 10-3 and p = 3.5 × 10-2, respectively). Meta-analysis of both cohorts (673 samples) confirmed association of rs4910623 with poor VA response after 3 months (p = 1.2 × 10-5) and 6 months (p = 9.3 × 10-6) of treatment in nAMD patients.