The Role of Collagen VIII in the Aging Mouse Kidney.

The gradual loss of kidney function due to increasing age is accompanied by structural changes such as fibrosis of the tissue. The underlying molecular mechanisms are complex, but not yet fully understood. Non-fibrillar collagen type VIII (COL8) could be a potential factor in the fibrosis processes of the aging kidney. A pathophysiological significance of COL8 has already been demonstrated in the context of diabetic kidney disease, with studies showing that it directly influences both the development and progression of renal fibrosis occurring. The aim of this study was to investigate whether COL8 impacts age-related micro-anatomical and functional changes in a mouse model. The kidneys of wild-type (Col8-wt) and COL8-knockout (Col8-ko) mice of different age and sex were characterized with regard to the expression of molecular fibrosis markers, the development of nephrosclerosis and renal function. The age-dependent regulation of COL8 mRNA expression in the wild-type revealed sex-dependent effects that were not observed with collagen IV (COL4). Histochemical staining and protein analysis of profibrotic cytokines TGF-ß1 (transforming growth factor) and CTGF (connective tissue growth factor) in mouse kidneys showed significant age effects as well as interactions of the factors age, sex and Col8 genotype. There were also significant age and Col8 genotype effects in the renal function data analyzed by urinary cystatin C. In summary, the present study shows, for the first time, that COL8 is regulated in an age- and sex-dependent manner in the mouse kidney and that the expression of COL8 influences the severity of age-induced renal fibrosis and function.

Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy.

A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early-onset Fuchs' endothelial corneal dystrophy (FECD), which progressively impairs vision through the loss of corneal endothelial cells. We demonstrate that CRISPR/Cas9-based postnatal gene editing achieves structural and functional rescue in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA) efficiently knocked down mutant COL8A2 expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. There were no adverse sequelae on histology or electroretinography. Col8a2 start codon disruption represents a non-surgical strategy to prevent vision loss in early-onset FECD. As this demonstrates the ability of Ad-Cas9-gRNA to restore the phenotype in adult post-mitotic cells, this method may be widely applicable to adult-onset diseases, even in tissues affected with disorders of non-reproducing cells.

Variable phenotype of Knobloch syndrome due to biallelic COL18A1 mutations in children.

PURPOSE: Knobloch syndrome is a rare, recessively inherited disorder classically characterized by high myopia, retinal detachment, and occipital encephalocele. Our aim is to report the clinical and genetic findings of four Israeli children affected by Knobloch syndrome. METHODS: Retrospective study of four patients diagnosed with Knobloch syndrome, who underwent full ophthalmic examination, electroretinography, and neuroradiologic imaging. Genetic analysis included whole exome sequencing (WES) and Sanger sequencing. RESULTS: The four patients included in this study had high myopia and nystagmus at presentation. Ocular findings included vitreous syneresis, macular atrophy, macular coloboma, and retinal detachment. One child had iris transillumination defects and an albinotic fundus, initially leading to an erroneous clinical diagnosis of albinism. Electroretinography revealed a marked cone-rod pattern of dysfunction in all four children. Brain imaging demonstrated none to severe occipital pathology. Cutaneous scalp changes were present in three patients. WES analysis, confirmed by Sanger sequencing revealed COL18A1 biallelic null mutations in all affected individuals, consistent with autosomal recessive inheritance. CONCLUSIONS: This report describes variable features in patients with Knobloch syndrome, including marked lack of eye pigment similar to albinism in one child, macular coloboma in two children as well as advanced cone-rod dysfunction in all children. One patient had normal neuroradiologic findings, emphasizing that some affected individuals have isolated ocular disease. Awareness of this syndrome, with its variable phenotype may aid early diagnosis, monitoring for potential complications, and providing appropriate genetic counseling.

COL8A2 Regulates the Fate of Corneal Endothelial Cells.

Purpose: To investigate the effect of COL8A2 repression on corneal endothelial cells (CECs) in vitro and in vivo. Methods: Cultured human CECs (hCECs) were transfected with COL8A2 siRNA (siCOL8A2), and the cell viability and proliferation rate were measured. The expression of cell proliferation-associated molecules was evaluated by Western blotting and real-time reverse transcription PCR. Cell shape, Wingless-INT (WNT) signaling, and mitochondrial oxidative stress were also measured. For in vivo experiments, siCOL8A2 was transfected into rat CECs (rCECs), and corneal opacity and corneal endothelium were evaluated. Results: After transfection with siCOL8A2, COL8A2 expression was reduced (80%). Cell viability, cell proliferation rate, cyclin D1 expression, and the number of cells in the S-phase were reduced in siCOL8A2-treated cells. The cell attained a fibroblast-like shape, and SNAI1, pSMAD2, and ß-catenin expression, along with mitochondrial mass and oxidative stress levels, were altered. Corneal opacity increased, and the CECs were changed in rats in the siCOL8A2 group. Conclusions: COL8A2 is required to maintain normal wound healing and CEC function.

Four Loci Are Associated with Cardiorespiratory Fitness and Endurance Performance in Young Chinese Females.

Cardiorespiratory fitness (CRF) and endurance performance are characterized by a complex genetic trait with high heritability. Although research has identified many physiological and environmental correlates with CRF, the genetic architecture contributing to CRF remains unclear, especially in non-athlete population. A total of 762 Chinese young female participants were recruited and an endurance run test was used to determine CRF. We used a fixed model of genome-wide association studies (GWAS) for CRF. Genotyping was performed using the Affymetrix Axiom and illumina 1 M arrays. After quality control and imputation, a linear regression-based association analysis was conducted using a total of 5,149,327 variants. Four loci associated with CRF were identified to reach genome-wide significance (P < 5.0 × 10-8), which located in 15q21.3 (rs17240160, P = 1.73 × 10-9, GCOM1), 3q25.31 (rs819865, P = 8.56 × 10-9, GMPS), 21q22.3 (rs117828698, P = 9.59 × 10-9, COL18A1), and 17q24.2 (rs79806428, P = 3.85 × 10-8, PRKCA). These loci (GCOM1, GMPS, COL18A1 and PRKCA) associated with cardiorespiratory fitness and endurance performance in Chinese non-athlete young females. Our results suggest that these gene polymorphisms provide further genetic evidence for the polygenetic nature of cardiorespiratory endurance and be used as genetic biomarkers for future research.

Identification of proliferative diabetic retinopathy-associated genes on the protein-protein interaction network by using heat diffusion algorithm.

Diabetic retinopathy is a common complication of diabetes mellitus that causes pathogenic damage to the retina. Particularly, the proliferative diabetic retinopathy (PDR) state can cause abnormal angiogenesis in the retina tissues and trigger the retina destruction in advanced stage. In the clinic, the symptoms during the initiation and progression of PDR are relatively unrecognizable. Therefore, various studies have focused on the pathogenesis of PDR. According to published literature, genetic contributions play an irreplaceable role in the initiation and progression of PDR. Although many computational methods, such as shortest path- and random walk with restart-based methods, have been applied in screening the potential pathogenic factors of PDR, advanced computational methods, which may provide essential supplements for previous ones, are still widely needed. In this study, a novel computational method was presented to infer novel PDR-associated genes. Different from previous methods, the method used in this work employed a different network algorithm, that is, the Laplacian heat diffusion algorithm. This algorithm was applied on the protein-protein interaction network reported in the STRING database. Three screening tests were performed to filter the most likely inferred genes. A total of 26 genes were accessed using the proposed method. Compared with the two previous predictions, most of the identified genes were novel, and only one gene was shared. Several inferred genes, such as CSF3, COL18A1, CXCR2, CCR1, FGF23, CXCL11, and IL13, were related to the pathogenesis of PDR.

RNAseq-Based Prioritization Revealed COL6A5, COL8A1, COL10A1 and MIR146A as Common and Differential Susceptibility Biomarkers for Psoriasis and Psoriatic Arthritis: Confirmation from Genotyping Analysis of 1417 Italian Subjects.

Psoriasis (Ps) and Psoriatic Arthritis (PsA) are characterized by a multifactorial etiology, involving genetic and environmental factors. The present study aimed to investigate polymorphisms (SNPs) within genes involved in extracellular matrix and cell homeostasis and microRNA genes as susceptibility biomarkers for Ps and PsA. Bioinformatic analysis on public RNA-seq data allowed for selection of rs12488457 (A/C, COL6A5), rs13081855 (G/T, COL8A1), rs3812111 (A/T, COL10A1) and rs2910164 (C/G, MIR146A) as candidate biomarkers. These polymorphisms were analyzed by Real-Time PCR in a cohort of 1417 Italian patients (393 Ps, 424 PsA, 600 controls). Statistical and bioinformatic tools were utilized for assessing the genetic association and predicting the effects of the selected SNPs. rs12488457, rs13081855 and rs2910164 were significantly associated with both Ps (p = 1.39 × 10-8, p = 4.52 × 10-4, p = 0.04, respectively) and PsA (p = 5.12 × 10-5, p = 1.19 × 10-6, p = 0.01, respectively). rs3812111, instead, was associated only with PsA (p = 0.005). Bioinformatic analysis revealed common and differential biological pathways involved in Ps and PsA. COL6A5 and COL8A1 take part in the proliferation and angiogenic pathways which are altered in Ps/PsA and contribute to inflammation together with MIR146A. On the other hand, the exclusive association of COL10A1 with PsA highlighted the specific involvement of bone metabolism in PsA.

Genetic polymorphisms in collagen-related genes are associated with pelvic organ prolapse.

OBJECTIVE: Pelvic organ prolapse (POP) is a common health issue that has a profound negative influence on women's quality of life. Genetic susceptibility to POP has been increasingly investigated. In this study, we assessed the single-nucleotide polymorphisms (SNPs) of six collagen-related genes (COL14A1, COL5A1, COL4A2, COL3A1, COL1A1, and COL18A1) and the genetic association with POP in Chinese women. METHODS: We performed a candidate gene association study of case women (n = 48) with stage III and IV prolapse and control women (n = 48) without prolapse. A target region sequencing approach was used to identify the SNPs in collagen-related genes. The association between SNPs and POP was examined by Fisher exact tests for unadjusted model and logistic regression analysis adjusted for delivery and pregnancy. RESULTS: There was a significant association between COL14A1 SNPs (rs4870723, rs2305600, and rs2305598; P = 0.013, 0.019, and 0.028, respectively), a COL5A1 SNP (rs3827852; P = 0.016), and COL4A2 SNPs (rs76425569, rs388222, and rs2281968; P = 0.049 for the three, and rs445348, P = 0.040) and POP, respectively. Although there was no significant association between the COL3A1 SNP and POP, there was a trend toward significance for COL14A1 SNP (rs2305603), COL4A2 SNP (rs74941798), two COL1A1 SNPs (rs2586488 and rs2249492) and three COL18A1 SNPs (rs1050351, rs56335679, and rs55690336), and POP. CONCLUSION: We are the first to evaluate the relationship between COL14A1, COL5A1, and COL4A2 polymorphisms and POP, besides COL3A1, COL1A1, and COL18A1, which have been reported previously. We found several candidate SNPs that were significantly associated with prolapse in Chinese women. Our results provide new evidence for further investigation of the involvement of these potential genes in the etiology of POP.

Stem cells from human cardiac adipose tissue depots show different gene expression and functional capacities.

BACKGROUND: The composition and function of the adipose tissue covering the heart are poorly known. In this study, we have investigated the epicardial adipose tissue (EAT) covering the cardiac ventricular muscle and the EAT covering the left anterior descending artery (LAD) on the human heart, to identify their resident stem cell functional activity. METHODS: EAT covering the cardiac ventricular muscle was isolated from the apex (avoiding areas irrigated by major vessels) of the heart (ventricular myocardium adipose tissue (VMAT)) and from the area covering the epicardial arterial sulcus of the LAD (PVAT) in human hearts excised during heart transplant surgery. Adipose stem cells (ASCs) from both adipose tissue depots were immediately isolated and phenotypically characterized by flow cytometry. The different behavior of these ASCs and their released secretome microvesicles (MVs) were investigated by molecular and cellular analysis. RESULTS: ASCs from both VMAT (mASCs) and the PVAT (pASCs) were characterized by the expression of CD105, CD44, CD29, CD90, and CD73. The angiogenic-related genes VEGFA, COL18A1, and TF, as well as the miRNA126-3p and miRNA145-5p, were analyzed in both ASC types. Both ASCs were functionally able to form tube-like structures in three-dimensional basement membrane substrates. Interestingly, pASCs showed a higher level of expression of VEGFA and reduced level of COL18A1 than mASCs. Furthermore, MVs released by mASCs significantly induced human microvascular endothelial cell migration. CONCLUSION: Our study indicates for the first time that the resident ASCs in human epicardial adipose tissue display a depot-specific angiogenic function. Additionally, we have demonstrated that resident stem cells are able to regulate microvascular endothelial cell function by the release of MVs.

Type XVIII Collagen Modulates Keratohyalin Granule Formation and Keratinization in Oral Mucosa.

Epithelial keratinization involves complex cellular modifications that provide protection against pathogens and chemical and mechanical injuries. In the oral cavity, keratinized mucosa is also crucial to maintain healthy periodontal or peri-implant tissues. In this study, we investigated the roles of type XVIII collagen, a collagen-glycosaminoglycan featuring an extracellular matrix component present in the basement membrane, in oral mucosal keratinization. Histological analysis of keratinized and non-keratinized oral mucosa showed that type XVIII collagen was highly expressed in keratinized mucosa. Additionally, a 3D culture system using human squamous carcinoma cells (TR146) was used to evaluate and correlate the changes in the expression of type XVIII collagen gene, COL18A1, and epithelial keratinization-related markers, e.g., keratin 1 (KRT1) and 10 (KRT10). The results showed that the increase in COL18A1 expression followed the increase in KRT1 and KRT10 mRNA levels. Additionally, loss-of-function analyses using silencing RNA targeting COL18A1 mRNA and a Col18-knockout (KO) mouse revealed that the absence of type XVIII collagen induces a dramatic decrease in KRT10 expression as well as in the number and size of keratohyalin granules. Together, the results of this study demonstrate the importance of type XVIII collagen in oral mucosal keratinization.

Genetic factors for idiopathic choroidal neovascularization.

Objective: The aim of this study was to investigate genetic factors associated with idiopathic choroidal neovascularization (ICNV). Methods: We conducted a case-control study including 69 cases with ICNV and 114 controls who underwent cataract surgery. Single nucleotide polymorphisms (SNPs) from genes reported to be related to AMD, CNV and uveitis were selected for this study. Results: In an univariate analysis, the rs669676 SNP located in the COL8A1 gene was associated with the proportion of people who has idiopathic CNV ( X2 = 9.3453, corrected p-value = 0.1). For the rs669676 SNP, minor allele homozygotes, in the dominant model of genotype analysis (GG versus AA-GA), it showed significant differences in the ICNV group vs controls (p = .01, OR = 1.219 (95%CI: 1.04-1.429)). Conclusions: The rs669676 SNP located in the COL8A1 gene may contribute to a genetic susceptibility for ICNV.

Atopic Eczema: Genetic Analysis of COL6A5, COL8A1, and COL10A1 in Mediterranean Populations.

To date, the genes associated with susceptibility to Atopic Eczema (AE) are mainly implicated in immunity, inflammation, and maintenance of skin barrier. Little is known about the possible relationship between genes modulating Extra-Cellular Matrix (ECM) and AE etiopathogenesis. In this regard, the primary objective of the present study has been the investigation of susceptibility biomarkers localized within genes encoding collagen proteins. Several studies have shown that polymorphisms within the genes encoding such proteins may generate abnormal connective tissues, making them more susceptible to mechanical stress, loss of epidermal integrity, and aging. We therefore decided to investigate three polymorphisms located in COL6A5, COL8A1, and COL10A1 as potential susceptibility biomarkers for AE in a cohort of 1470 subjects of Mediterranean origin. The genes of interest have been selected considering that the ECM and immune/inflammatory response are strongly dysregulated in AE and other complex disorders. The study confirmed that the susceptibility to AE depends on a complex interaction between latitude, geographical localization, and the differential distribution of genetic variants among populations exposed to similar environmental factors.

RAD51B (rs8017304 and rs2588809), TRIB1 (rs6987702, rs4351379, and rs4351376), COL8A1 (rs13095226), and COL10A1 (rs1064583) Gene Variants with Predisposition to Age-Related Macular Degeneration.

BACKGROUND: Age-related macular degeneration (AMD) is a progressive neurodegenerative disease of a central part of the neural retina (macula) and a leading cause of blindness in elderly people. While it is known that the AMD is a multifactorial disease, genetic factors involved in lipid metabolism, inflammation, and neovascularization are currently being widely studied in genome-wide association studies (GWAS). The aim of our study was to evaluate the impact of new single nucleotide polymorphisms (SNPs) in RAD51B, TRIB1, COL8A1, and COL10A1 genes on AMD development. METHODS: Case-control study involved 254 patients diagnosed with early AMD, 244 patients with exudative AMD, and 942 control subjects. The genotyping of RAD51B (rs8017304 and rs2588809), TRIB1 (rs6987702, rs4351379, and rs4351376), COL8A1 (rs13095226), and COL10A1 (rs1064583) was carried out using TaqMan assays by a real-time polymerase chain reaction (RT-PCR) method. RESULTS: Statistically significant difference was found in genotype (TT, TC, and CC) distribution of COL8A1 rs13095226 between exudative AMD and control groups (60.2%, 33.6%, and 6.1% vs. 64.9%, 32.3%, and 2.9%, respectively, p = 0.036). Also, comparing with TT+TC, rs13095226 CC genotype was associated with 3.5-fold increased odds of exudative AMD development (OR = 3.540; 95% CI: 1.415-8.856; p = 0.007). CONCLUSION: Our study revealed a strong association between a variant in COL8A1 (rs13095226) and exudative AMD development.

TCF4 and COL8A2 Gene Polymorphism Screening in a Greek Population of Late-onset Fuchs Endothelial Corneal Dystrophy.

BACKGROUND/AIM: Fuchs' endothelial corneal dystrophy (FECD) is a hereditary, progressive, bilateral, and irreversible disorder of the corneal endothelium. The purpose of this study was to develop a novel, accurate and high-throughput real-time polymerase chain reaction (PCR) method and melting-curve analysis in order to genotype the rs613872 polymorphism in the transcription factor 4 (TCF4) gene and to implement it on a well-ascertained sample of 22 Greek FECD patients and 58 healthy individuals, age- and sex-matched. PATIENTS AND METHODS: DNA was extracted from blood samples, which were screened with the DNA sequencing method in order to detect the g.31753T>G/p.L450W (rs8035192) and g.31767C>A/p.Q455K (rs8035191) mutations in a COL8A2 genomic region. RESULTS: TCF4 risk G allele frequency increased to 48% in FECD patients compared to 17% in healthy-subjects [OR=4.82 (95% CI=1.98-11.73)]. No p.L450W and p.Q455K COL8A2 gene mutations were detected. CONCLUSION: We confirmed that rs613872 in the TCF4 gene is strongly and statistically associated with late-onset FECD in a Greek population.

Biomechanical changes to Descemet's membrane precede endothelial cell loss in an early-onset murine model of Fuchs endothelial corneal dystrophy.

Early-onset Fuchs endothelial corneal dystrophy (FECD) has been associated with nonsynonymous mutations in collagen VIII α2 (COL8A2), a key extracellular matrix (ECM) protein in Descemet's membrane (DM). Two knock-in strains of mice have been generated to each express a mutant COL8A2 protein (Col8a2L450W/L450W and Col8a2Q455K/Q455K) that recapitulate the clinical phenotype of early-onset FECD including endothelial cell loss, cellular polymegathism and pleomorphism, and guttae. Due to abnormalities in ECM protein composition and structure in FECD, the stiffness of DM in Col8a2 knock-in mice and wildtype (WT) controls was measured using atomic force microscopy at 5 and 10 months of age, coinciding with the onset of FECD phenotypic abnormalities. At 5 months, only sporadic guttae were identified via in vivo confocal microscopy (IVCM) in Col8a2Q455K/Q455K mice, otherwise both strains of Col8a2 transgenic mice were indistinguishable from WT controls in terms of endothelial cell density and size. By 10 months of age, Col8a2L450W/L450W and Col8a2Q455K/Q455K mice developed reduced corneal endothelial density, increased endothelial cell area and guttae, with the Col8a2Q455K/Q455K strain exhibiting a more severe phenotype. However, at 5 months of age, prior to the development endothelial cell abnormalities, Col8a2L450W/L450W and Col8a2Q455K/Q455K mice knock-in mice had reduced tissue stiffness of DM that was statistically significant in the Col8a2Q455K/Q455K mice when compared with wildtype controls. These data indicate that alterations in the tissue compliance of DM precede phenotypic changes in endothelial cell count and morphology, and may play a role in onset and progression of FECD.

Comparative gene array analyses of severe elastic fiber defects in late embryonic and newborn mouse aorta.

Elastic fibers provide reversible elasticity to the large arteries and are assembled during development when hemodynamic forces are increasing. Mutations in elastic fiber genes are associated with cardiovascular disease. Mice lacking expression of the elastic fiber genes elastin ( Eln-/-), fibulin-4 ( Efemp2-/-), or lysyl oxidase ( Lox-/-) die at birth with severe cardiovascular malformations. All three genetic knockout models have elastic fiber defects, aortic wall thickening, and arterial tortuosity. However, Eln-/- mice develop arterial stenoses, while Efemp2-/- and Lox-/- mice develop ascending aortic aneurysms. We performed comparative gene array analyses of these three genetic models for two vascular locations and developmental stages to determine differentially expressed genes and pathways that may explain the common and divergent phenotypes. We first examined arterial morphology and wall structure in newborn mice to confirm that the lack of elastin, fibulin-4, or lysyl oxidase expression provided the expected phenotypes. We then compared gene expression levels for each genetic model by three-way ANOVA for genotype, vascular location, and developmental stage. We found three genes upregulated by genotype in all three models, Col8a1, Igfbp2, and Thbs1, indicative of a common response to severe elastic fiber defects in developing mouse aorta. Genes that are differentially regulated by vascular location or developmental stage in all three models suggest mechanisms for location or stage-specific disease pathology. Comparison of signaling pathways enriched in all three models shows upregulation of integrins and matrix proteins involved in early wound healing, but not of mature matrix molecules such as elastic fiber proteins or fibrillar collagens.

Membrane associated collagen XIII promotes cancer metastasis and enhances anoikis resistance.

BACKGROUND: Increased collagen expression and deposition are associated with cancer progression and poor prognosis in breast cancer patients. However, function and regulation of membrane-associated collagen in breast cancer have not been determined. Collagen XIII is a type II transmembrane protein within the collagen superfamily. Experiments in tissue culture and knockout mouse models show that collagen XIII is involved in cell adhesion and differentiation of certain cell types. In the present study, we determined roles of collagen XIII in breast cancer progression and metastasis. METHODS: We analyzed the association of collagen XIII expression with breast cancer development and metastasis using published gene expression profiles generated from human breast cancer tissues. Utilizing gain- and loss- of function approaches and 3D culture assays, we investigated roles of collagen XIII in regulating invasive tumor growth. Using the tumorsphere/mammosphere formation assay and the detachment cell culture assay, we determined whether collagen XIII enhances cancer cell stemness and induces anoikis resistance. We also inhibited collagen XIII signaling with ß1 integrin function-blocking antibody. Finally, using the lung colonization assay and the orthotopic mammary tumor model, we investigated roles of collagen XIII in regulating breast cancer colonization and metastasis. Cox proportional hazard (log-rank) test, two-sided Student's t-test (two groups) and one-way ANOVA (three or more groups) analyses were used in this study. RESULTS: Collagen XIII expression is significantly higher in human breast cancer tissue compared with normal mammary gland. Increased collagen XIII mRNA levels in breast cancer tissue correlated with short distant recurrence free survival. We showed that collagen XIII expression promoted invasive tumor growth in 3D culture, enhanced cancer cell stemness, and induced anoikis resistance. Collagen XIII expression induced ß1 integrin activation. Blocking ß1 integrin activation significantly reduced collagen XIII-induced invasion and mammosphere formation. Importantly, silencing collagen XIII in MDA-MB-231 cells reduced lung colonization and metastasis. CONCLUSIONS: Our results demonstrate a novel function of collagen XIII in promoting cancer metastasis, cell invasion, and anoikis resistance.

Activation of JNK signaling in osteoblasts is inversely correlated with collagen synthesis in age-related osteoporosis.

The age-related reduction in the function of osteoblasts plays a central role in the pathogenesis of bone loss and osteoporosis. Collagen synthesis is a primary function of differentiated osteoblasts, however, the mechanisms for age-related changes in collagen synthesis in human osteoblasts remain elusive. We use Gene Ontology (GO) analysis and Gene Set Enrichment Analysis (GSEA) analysis to exploit the transcriptional profiles of osteoblasts from young and old donors. A panel of collagen members was downregulated in aged osteoblasts, including COL12A1, COL5A1, COL5A3, COL8A1 and COL8A2. Co-expression analysis followed by GO analysis revealed that oxidoreductase activity and kinase activity were inversely correlated with collagen synthesis in osteoblasts. GESA analysis further showed that JNK signaling was upregulated in aged osteoblasts. Consistently, MAP3K4 and MAP4K2, upstream of JNK, were also increased in aged osteoblasts. Moreover, expression levels of MAP3K4 were significantly inversely correlated with levels of the collagen genes. Those transcriptomic results were further verified by examining clinical specimens of osteoporosis by immunohistochemistry. These results provide transcriptomic evidence that deregulated JNK signaling may impair collagen synthesis in osteoblasts and imply a therapeutic value of JNK inhibitors for treating osteoporosis and preventing skeletal aging by counteracting the age-related reduction in the function of osteoblasts.

COL18A1 is a candidate eye iridocorneal angle-closure gene in humans.

Primary angle-closure glaucoma (PACG) is a common form of glaucoma in the Far East. Its defining feature is iridocorneal angle closure. In addition to PACG, indications of angle closure are included in the diagnostic criteria of related conditions primary angle-closure suspect (PACS) and primary angle closure (PAC). To the best of our knowledge, a causative gene for iridocorneal angle closure in humans has not been identified. This study aimed to identify the genetic cause of iridocorneal angle closure in a pedigree with at least 10 individuals diagnosed with PACS, PAC or PACG. Results of linkage analysis, segregation analysis of 44 novel variations, whole exome sequencing of 10 individuals, screenings of controls and bioinformatics predictions identified a mutation in COL18A1 that encodes collagen type XVIII as the most likely cause of angle closure in the pedigree. The role of COL18A1 in the etiology of Knobloch syndrome (KS) that is consistently accompanied by optic anomalies, available functional data on the encoded protein and the recognized role of collagens and the extracellular matrix in glaucoma pathogenesis supported the proposed role of the COL18A1 mutation in the pedigree. Subsequent identification of other COL18A1 mutations in PACS affected individuals of two unrelated families further supported that COL18A1 may affect angle closure. These PACS individuals were parents and grandparents of KS-affected children. In conclusion, a gene that affects angle closure in humans, a critical feature of PACG, has been identified. The findings also reinforce the importance of collagens in eye features and functions.

Whole-Exome Sequencing in Age-Related Macular Degeneration Identifies Rare Variants in COL8A1, a Component of Bruch's Membrane.

PURPOSE: Genome-wide association studies and targeted sequencing studies of candidate genes have identified common and rare variants that are associated with age-related macular degeneration (AMD). Whole-exome sequencing (WES) studies allow a more comprehensive analysis of rare coding variants across all genes of the genome and will contribute to a better understanding of the underlying disease mechanisms. To date, the number of WES studies in AMD case-control cohorts remains scarce and sample sizes are limited. To scrutinize the role of rare protein-altering variants in AMD cause, we performed the largest WES study in AMD to date in a large European cohort consisting of 1125 AMD patients and 1361 control participants. DESIGN: Genome-wide case-control association study of WES data. PARTICIPANTS: One thousand one hundred twenty-five AMD patients and 1361 control participants. METHODS: A single variant association test of WES data was performed to detect variants that are associated individually with AMD. The cumulative effect of multiple rare variants with 1 gene was analyzed using a gene-based CMC burden test. Immunohistochemistry was performed to determine the localization of the Col8a1 protein in mouse eyes. MAIN OUTCOME MEASURES: Genetic variants associated with AMD. RESULTS: We detected significantly more rare protein-altering variants in the COL8A1 gene in patients (22/2250 alleles [1.0%]) than in control participants (11/2722 alleles [0.4%]; P = 7.07×10-5). The association of rare variants in the COL8A1 gene is independent of the common intergenic variant (rs140647181) near the COL8A1 gene previously associated with AMD. We demonstrated that the Col8a1 protein localizes at Bruch's membrane. CONCLUSIONS: This study supported a role for protein-altering variants in the COL8A1 gene in AMD pathogenesis. We demonstrated the presence of Col8a1 in Bruch's membrane, further supporting the role of COL8A1 variants in AMD pathogenesis. Protein-altering variants in COL8A1 may alter the integrity of Bruch's membrane, contributing to the accumulation of drusen and the development of AMD.