Loss of ciliary zonule protein hydroxylation and lens stability as a predicted consequence of biallelic ASPH variation

This author accepted manuscript is made available following 12 month embargo from date of publication (January 2019) in accordance with the publisher’s archiving policy “This is an Accepted Manuscript of an article published by Taylor & Francis in Ophthalmic Genetics on 2 January 2019, available online: http://www.tandfonline.com/10.1080/13816810.2018.1561904”Purpose: Stability of the crystalline lens requires formation of microfibril bundles and their higher-order structures of ciliary zonules. Trauma, malformation, or degeneration of the ciliary zonules can lead to dislocation or displacement of the lens, which in turn can cause transient or permanent loss of visual acuity. The purpose of this study was to identify the predicted substrates of ASPH, a 2-oxoglutarate- and Fe2+-dependent hydroxylase, which may account for the lens instability phenotype of ASPH-associated syndromes. Methods: A single proband of European ancestry with spherophakia and high myopia was subjected to exome sequencing. Proteins containing the ASPH hydroxylation motif were identified within the SwissProt protein database. Results: We identified 105 putative substrates of ASPH-mediated hydroxylation in the human proteome, of which two (FBN1 and LTBP2) are associated with inherited ectopia lentis syndromes, and are essential for microfibril and ciliary zonule development. Conclusion: Our results implicate ASPH-mediated hydroxylation in the formation of FBN1/LTBP2 microfibril bundles and competent ciliary zonules

A forward genetic screen reveals roles for Nfkbid, Zeb1, and Ruvbl2 in humoral immunity

Using chemical germ-line mutagenesis, we screened mice for defects in the humoral immune response to a type II T-independent immunogen and an experimental alphavirus vector. A total of 26 mutations that impair humoral immunity were recovered, and 19 of these mutations have been positionally cloned. Among the phenovariants were bumble, cellophane, and Worker ascribed to mutations in Nfkbid, Zeb1, and Ruvbl2, respectively. We show that IκBNS, the nuclear IκB-like protein encoded by Nfkbid, is required for the development of marginal zone and peritoneal B-1 B cells and additionally required for extrafollicular antibody responses to T-independent and -dependent immunogens. Zeb1 is also required for marginal zone and peritoneal B-1 B-cell development as well as T-cell development, germinal center formation, and memory B-cell responses. Finally, Ruvbl2 is required for T-cell development and maximal T-dependent antibody responses. Collectively, the mutations that we identified give us insight into the points at which disruption of an antibody response can occur. All of the mutations identified to date directly affect lymphocyte development or function; none have an exclusive effect on cells of the innate immune system

Expression Pattern of Immunoproteasome Subunits in Human Thymus

The expression pattern of immunoproteasomes in human thymus has not been analyzed but may have important consequences during thymic selection. Here we examined the expression patterns of immunoproteasome subunits in fetal and adult thymic tissues by immunohistochemistry and found that all three subunits are expressed in both cortical and medullary stromal cells. These data suggest that thymic selection in human can be affected by peptide repertoires generated by immunoproteasomes

Risk stratification and clinical utility of polygenic risk scores in ophthalmology

Combining genetic and clinical data into an informative risk prediction profile has been an important ambition of personalized medicine. Single-nucleotide polymorphisms are commonly found throughout the genome and account for the majority of interindividual genetic variation. To date, genome-wide association studies have led to the discovery of thousands of disease-associated loci, including across dozens of ophthalmic diseases and traits. However, compared with the clinical utility of identifying rare Mendelian variants, the translation of these results to clinical practice has so far been limited because such variants are found commonly in the population, and individually account for a very small risk. Recently, combining large numbers of these genetic variants into polygenic risk scores (PRS) has shown clinically meaningful risk prediction across several common diseases. PRS have the potential to translate the discovery of common risk variants into individualized disease risk prediction, prognostication, and may enable targeted treatments. In this context, we review the clinical utility of PRS in three common, genetically complex ophthalmic conditions: primary open angle glaucoma, age-related macular degeneration, and myopia. Translational Relevance: Common genetic variants can be used to effectively stratify the risk of disease development and progression and may be used to guide screening, triaging, monitoring, or treatment thresholds.Ayub Qassim, Emmanuelle Souzeau, Georgie Hollitt, Mark M. Hassall, Owen M. Siggs, and Jamie E. Crai

A hypomorphic allele of ZAP-70 reveals a distinct thymic threshold for autoimmune disease versus autoimmune reactivity

ZAP-70 is critical for T cell receptor (TCR) signaling. Tyrosine to phenylalanine mutations of Y315 and Y319 in ZAP-70 suggest these residues function to recruit downstream effector molecules, but mutagenesis and crystallization studies reveal that these residues also play an important role in autoinhibition ZAP-70. To address the importance of the scaffolding function, we generated a zap70 mutant mouse (YYAA mouse) with Y315 and Y319 both mutated to alanines. These YYAA mice reveal that the scaffolding function is important for normal development and function. Moreover, the YYAA mice have many similarities to a previously identified ZAP-70 mutant mouse, SKG, which harbors a distinct hypomorphic mutation. Both YYAA and SKG mice have impaired T cell development and hyporesponsiveness to TCR stimulation, markedly reduced numbers of thymic T regulatory cells and defective positive and negative selection. YYAA mice, like SKG mice, develop rheumatoid factor antibodies, but fail to develop autoimmune arthritis. Signaling differences that result from ZAP-70 mutations appear to skew the TCR repertoire in ways that differentially influence propensity to autoimmunity versus autoimmune disease susceptibility. By uncoupling the relative contribution from T regulatory cells and TCR repertoire during thymic selection, our data help to identify events that may be important, but alone are insufficient, for the development of autoimmune disease

Rare variants in optic disc area gene CARD10 enriched in primary open-angle glaucoma

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.Tiger Zhou, Emmanuelle Souzeau, Shiwani Sharma, Owen M. Siggs, Ivan Goldberg, Paul R. Healey, Stuart Graham, Alex W. Hewitt, David A. Mackey, Robert J. Casson, John Landers, Richard Mills, Jonathan Ellis, Paul Leo, Matthew A. Brown, Stuart MacGregor, Kathryn P. Burdon and Jamie E. Crai

Biallelic CPAMD8 variants are a frequent cause of childhood and 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. Design: Retrospective, multicenter case series. Participants: A total of 268 probands and their relatives with a diagnosis of childhood or 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 -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 individuals (72.7%), and retinal detachment was observed in 3 of 11 individuals (27.3%). CPAMD8 expression was highest in neural crest-derived tissues of the adult anterior segment, suggesting that CPAMD8 variation may cause malformation or obstruction of key drainage structures. Conclusions: Biallelic CPAMD8 variation was associated with a highly heterogeneous phenotype and in our cohorts was the second most common inherited cause of childhood glaucoma after CYP1B1 and juvenile open-angle glaucoma after MYOC. CPAMD8 sequencing should be considered in the investigation of both childhood and juvenile open-angle glaucoma, particularly when associated with iris abnormalities, cataract, or retinal detachment

Biallelic CPAMD8 Variants Are a Frequent Cause of Childhood and Juvenile Open-Angle Glaucoma

© 2020 by the American Academy of Ophthalmology. This is an open access article under the CC BY-NC-ND licensePurpose 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 individuals (72.7%), and retinal detachment was observed in 3 of 11 individuals (27.3%). CPAMD8 expression was highest in neural crest–derived tissues of the adult anterior segment, suggesting that CPAMD8 variation may cause malformation or obstruction of key drainage structures. Conclusions Biallelic CPAMD8 variation was associated with a highly heterogeneous phenotype and in our cohorts was the second most common inherited cause of childhood glaucoma after CYP1B1 and juvenile open-angle glaucoma after MYOC. CPAMD8 sequencing should be considered in the investigation of both childhood and juvenile open-angle glaucoma, particularly when associated with iris abnormalities, cataract, or retinal detachment