Endoplasmic Reticulum Stress Induced Senescence in Human Lung Fibroblasts.

Loss of proteostasis and cellular senescence have been previously established as characteristics of aging, however their interaction in the context of lung aging and potential contributions to aging-associated lung remodeling remains understudied. In this study we aimed to characterize endoplasmic reticulum (ER) stress response, cellular senescence, and their interaction in relation to extracellular matrix (ECM) production in lung fibroblasts from young (25-45 years) and old (>60 years) humans. Fibroblasts from young and old patients without significant preexisting lung disease were exposed to vehicle, MG132, etoposide or salubrinal. Afterwards, cells and cell lysates or supernatants were analyzed for ER stress, cellular senescence and ECM changes using protein analysis, proliferation assay and senescence-associated beta galactosidase (SA-ß-Gal) staining. At baseline, fibroblasts from aging individuals showed increased levels of ER stress (ATF6 and PERK), senescence (p21 and McL-1) and ECM marker (COL1A1) compared to those from young individuals. Upon ER stress induction and etoposide exposure, fibroblasts showed an increase in senescence (SA-ß-Gal, p21, Cav-1), ER stress (PERK) and ECM markers (COL1A1 and LUM) compared to vehicle. Additionally, CXCL8 and IL-6 levels were increase in the supernatants of MG132 and etoposide-treated fibroblasts, respectively. Finally, the ER stress inhibitor salubrinal decreased the expression of p21 compared to vehicle and MG132 treatments, however salubrinal inhibited COL1A1 but not p21 expression in MG132-treated fibroblasts. Our study suggests that ER stress response plays an important role in establishment and maintenance of a senescence phenotype in lung fibroblasts and therefore contributes to altered remodeling in the aging lung.

Functional role of glial-derived neurotrophic factor in a mixed allergen murine model of asthma.

Our previous study showed that glial-derived neurotrophic factor (GDNF) expression is upregulated in asthmatic human lungs, and GDNF regulates calcium responses through its receptor GDNF family receptor α1 (GFRα1) and RET receptor in human airway smooth muscle (ASM) cells. In this study, we tested the hypothesis that airway GDNF contributes to airway hyperreactivity (AHR) and remodeling using a mixed allergen mouse model. Adult C57BL/6J mice were intranasally exposed to mixed allergens (ovalbumin, Aspergillus, Alternaria, house dust mite) over 4 wk with concurrent exposure to recombinant GDNF, or extracellular GDNF chelator GFRα1-Fc. Airway resistance and compliance to methacholine were assessed using FlexiVent. Lung expression of GDNF, GFRα1, RET, collagen, and fibronectin was examined by RT-PCR and histology staining. Allergen exposure increased GDNF expression in bronchial airways including ASM and epithelium. Laser capture microdissection of the ASM layer showed increased mRNA for GDNF, GFRα1, and RET in allergen-treated mice. Allergen exposure increased protein expression of GDNF and RET, but not GFRα1, in ASM. Intranasal administration of GDNF enhanced baseline responses to methacholine but did not consistently potentiate allergen effects. GDNF also induced airway thickening, and collagen deposition in bronchial airways. Chelation of GDNF by GFRα1-Fc attenuated allergen-induced AHR and particularly remodeling. These data suggest that locally produced GDNF, potentially derived from epithelium and/or ASM, contributes to AHR and remodeling relevant to asthma.NEW & NOTEWORTHY Local production of growth factors within the airway with autocrine/paracrine effects can promote features of asthma. Here, we show that glial-derived neurotrophic factor (GDNF) is a procontractile and proremodeling factor that contributes to allergen-induced airway hyperreactivity and tissue remodeling in a mouse model of asthma. Blocking GDNF signaling attenuates allergen-induced airway hyperreactivity and remodeling, suggesting a novel approach to alleviating structural and functional changes in the asthmatic airway.

GJA3 Genetic Variation and Autosomal Dominant Congenital Cataracts and Glaucoma Following Cataract Surgery.

Importance: The p.Asp67Tyr genetic variant in the GJA3 gene is responsible for congenital cataracts in a family with a high incidence of glaucoma following cataract surgery. Objective: To describe the clinical features of a family with a strong association between congenital cataracts and glaucoma following cataract surgery secondary to a genetic variant in the GJA3 gene (NM_021954.4:c.199G>T, p.Asp67Tyr). Design, Setting, and Participants: This was a retrospective, observational, case series, genetic association study from the University of Iowa spanning 61 years. Examined were the ophthalmic records from 1961 through 2022 of the family members of a 4-generation pedigree with autosomal dominant congenital cataracts. Main Outcomes and Measures: Frequency of glaucoma following cataract surgery and postoperative complications among family members with congenital cataract due to the p.Asp67Tyr GJA3 genetic variant. Results: Medical records were available from 11 of 12 family members (7 male [63.6%]) with congenital cataract with a mean (SD) follow-up of 30 (21.7) years (range, 0.2-61 years). Eight of 9 patients with congenital cataracts developed glaucoma, and 8 of 8 patients who had cataract surgery at age 2 years or younger developed glaucoma following cataract surgery. The only family member with congenital cataracts who did not develop glaucoma had delayed cataract surgery until 12 and 21 years of age. Five of 11 family members (45.5%) had retinal detachments after cataract extraction and vitrectomy. No patients developed retinal detachments after prophylactic 360-degree endolaser. Conclusions and Relevance: The GJA3 genetic variant, p.Asp67Tyr, was identified in a 4-generation congenital cataract pedigree from Iowa. This report suggests that patients with congenital cataract due to some GJA3 genetic variants may be at especially high risk for glaucoma following cataract surgery. Retinal detachments after cataract extraction in the first 2 years of life were also common in this family, and prophylactic retinal endolaser may be indicated at the time of surgery.

Functional α7 nicotinic receptors in human airway smooth muscle increase intracellular calcium concentration and contractility in asthmatics.

Although nicotinic acetylcholine receptors (nAChRs) are commonly associated with neurons in the brain and periphery, recent data indicate that they are also expressed in non-neuronal tissues. We recently found the alpha7 (α7nAChR) subunit is highly expressed in human airway smooth muscle (hASM) with substantial increase in asthmatics, but their functionality remains unknown. We investigated the location and functional role of α7nAChRs in hASM cells from normal versus mild-moderate asthmatic patients. Immunostaining and protein analyses showed α7nAChR in the plasma membrane including in asthmatics. In asthmatic hASM, patch-clamp recordings revealed significantly higher functional homomeric α7nAChR channels. Real-time fluorescence imaging showed nicotine, via α7nAChR, increases intracellular Ca2+ ([Ca2+]i) independent of ACh effects, particularly in asthmatic hASM, while cellular traction force microscopy showed nicotine-induced contractility including in asthmatics. These results indicate functional homomeric and heteromeric nAChRs that are increased in asthmatic hASM, with pharmacology that likely differ owing to different subunit interfaces that form the orthosteric sites. nAChRs may represent a novel target in alleviating airway hyperresponsiveness in asthma.NEW & NOTEWORTHY Cigarette smoking and vaping exacerbate asthma. Understanding the mechanisms of nicotine effects in asthmatic airways is important. This study demonstrates that functional alpha7 nicotinic acetylcholine receptors (α7nAChRs) are expressed in human airway smooth muscle, including from asthmatics, and enhance intracellular calcium and contractility. Although a7nAChRs are associated with neuronal pathways, α7nAChR in smooth muscle suggests inhaled nicotine (e.g., vaping) can directly influence airway contractility. Targeting α7nAChR may represent a novel approach to alleviating airway hyperresponsiveness in asthma.

Asthmatic lung fibroblasts promote type 2 immune responses via endoplasmic reticulum stress response dependent thymic stromal lymphopoietin secretion.

Lung fibroblasts contribute to asthma pathology partly through modulation of the immune environment in the airway. Tumor necrosis factor-α (TNFα) expression is upregulated in asthmatic lungs. How asthmatic lung fibroblasts respond to TNFα stimulation and subsequently regulate immune responses is not well understood. Endoplasmic reticulum (ER) stress and unfolded protein responses (UPR) play important roles in asthma, but their functional roles are still under investigation. In this study, we investigated TNFα-induced cytokine production in primary lung fibroblasts from asthmatic vs. non-asthmatic human subjects, and downstream effects on type 2 immune responses. TNFα significantly upregulated IL-6, IL-8, C-C motif chemokine ligand 5 (CCL5), and thymic stromal lymphopoietin (TSLP) mRNA expression and protein secretion by lung fibroblasts. Asthmatic lung fibroblasts secreted higher levels of TSLP which promoted IL-33-induced IL-5 and IL-13 production by peripheral blood mononuclear cells. TNFα exposure enhanced expression of ER stress/UPR pathways in both asthmatic and non-asthmatic lung fibroblasts, especially inositol-requiring protein 1α in asthmatics. ER stress/UPR inhibitors decreased IL-6, CCL5, and TSLP protein secretion by asthmatic lung fibroblasts. Our data suggest that TNFα and lung fibroblasts form an important axis in asthmatic lungs to promote asthmatic inflammation that can be attenuated by inhibiting ER stress/UPR pathway.

Calcium-sensing receptor and CPAP-induced neonatal airway hyperreactivity in mice.

BACKGROUND: Continuous positive airway pressure (CPAP) in preterm infants is initially beneficial, but animal models suggest longer term detrimental airway effects towards asthma. We used a neonatal CPAP mouse model and human fetal airway smooth muscle (ASM) to investigate the role of extracellular calcium-sensing receptor (CaSR) in these effects. METHODS: Newborn wild type and smooth muscle-specific CaSR-/- mice were given CPAP for 7 days via a custom device (mimicking CPAP in premature infants), and recovered in normoxia for another 14 days (representing infants at 3-4 years). Airway reactivity was tested using lung slices, and airway CaSR quantified. Role of CaSR was tested using NPS2143 (inhibitor) or siRNA in WT mice. Fetal ASM cells stretched cyclically with/without static stretch mimicking breathing and CPAP were analyzed for intracellular Ca2+ ([Ca2+]i) responses, role of CaSR, and signaling cascades. RESULTS: CPAP increased airway reactivity in WT but not CaSR-/- mice, increasing ASM CaSR. NPS2143 or CaSR siRNA reversed CPAP effects in WT mice. CPAP increased fetal ASM [Ca2+]I, blocked by NPS2143, and increased ERK1/2 and RhoA suggesting two mechanisms by which stretch increases CaSR. CONCLUSIONS: These data implicate CaSR in CPAP effects on airway function with implications for wheezing in former preterm infants. IMPACT: Neonatal CPAP increases airway reactivity to bronchoconstrictor agonist. CPAP increases smooth muscle expression of the extracellular calcium-sensing receptor (CaSR). Inhibition or absence of CaSR blunts CPAP effects on contractility. These data suggest a causal/contributory role for CaSR in stretch effects on the developing airway. These data may impact clinical recognition of the ways that CPAP may contribute to wheezing disorders of former preterm infants.

Prevalence of Open-angle Glaucoma in the Faroese Population.

PURPOSE: The Faroe Islands are home to 50,000 genetically isolated people in the North Atlantic. The prevalence of open-angle glaucoma (OAG) in the Faroese population is unknown. Consequently, we conducted a survey to determine the prevalence of OAG in the Faroese population. We also investigated the role of known glaucoma-causing genes in Faroese OAG. MATERIALS AND METHODS: We conducted a prospective survey of known and newly diagnosed glaucoma patients at the Faroese National Hospital, Landssjukrahusid, Tórshavn between October 1, 2015 to December 31, 2017. In addition we reviewed the only eye care provider in the Faroese Islands by scrutinizing electronic medical records between 2009 and June 15, 2014, October 1, 2015 and the partly overlapping prescriptions for ocular hypotensive medications in 2016 to identify patients with either a diagnosis of glaucoma, a diagnosis of ocular hypertension or a prescription for ocular hypotensive medications. Next, we prospectively confirmed diagnoses with complete eye examinations. Patient DNA samples were tested for variations in known glaucoma-causing genes [myocilin (MYOC), optineurin (OPTN), and TANK binding kinase 1 (TBK1)]. RESULTS: We determined the age-related prevalence of OAG January 1, 2017 in individuals 40 years or older to be 10.7/1000 (1.07%) and highly age-related. A diagnosis of OAG was present in 264 patients, of whom 211 (79.9%) had primary OAG (including normal tension glaucoma), 49 (18.6%) had pseudoexfoliation glaucoma, and 4 (1.5%) had pigmentary glaucoma. Among patients receiving medications for glaucoma, nearly 50% had primary OAG, while the majority of the rest had ocular hypertension or secondary glaucoma. No disease-causing variants were detected in MYOC, OPTN, or TBK1. CONCLUSIONS: The calculated prevalence of OAG in the Faroe Islands was 1.07%. The absence of MYOC, OPTN, or TBK1 disease-causing variants in Faroese primary OAG patients suggests that a different, potentially unique set of genes may be contributing to the pathogenesis of glaucoma in this population.

Aging increases senescence, calcium signaling, and extracellular matrix deposition in human airway smooth muscle.

Lung function declines as people age and their lungs become stiffer. With an increasing elderly population, understanding mechanisms that contribute to these structural and functional changes in the aging lung is important. Part of the aging process is characterized by thicker, more fibrotic airways, and senile emphysema caused by changes in lung parenchyma. There is also senescence, which occurs throughout the body with aging. Here, using human airway smooth muscle (ASM) cells from patients in different age groups, we explored senescence pathways and changes in intracellular calcium signaling and extracellular matrix (ECM) deposition to elucidate potential mechanisms by which aging leads to thicker and stiffer lungs. Senescent markers p21, γH2AX, and ß-gal, and some senescence-associated secretory proteins (SASP) increased with aging, as shown by staining and biochemical analyses. Agonist-induced intracellular Ca2+ responses, measured using fura-2 loaded cells and fluorescence imaging, increased with age. However, biochemical analysis showed that expression of the following markers decreased with age: M3 muscarinic receptor, TRPC3, Orai1, STIM1, SERCA2, MMP2 and MMP9. In contrast, collagen III, and fibronectin deposition increased with age. These data show that senescence increases in the aging airways that is associated with a stiffer but surprisingly greater intracellular calcium signaling as a marker for contractility. ASM senescence may enhance fibrosis in a feed forward loop promoting remodeling and altered calcium storage and buffering.

Glial-derived neurotrophic factor in human airway smooth muscle.

Airway smooth muscle (ASM) cells modulate the local airway milieu via production of inflammatory mediators and growth factors including classical neurotrophins, such as brain-derived neurotrophic factor (BDNF). The glial cell-derived neurotrophic factor (GDNF) family of ligands (GFLs) are nonclassical neurotrophins and their role in the airway is barely understood. The major GFLs, GDNF and Neurturin (NRTN) bind to GDNF family receptor (GFR) α1 and α2 respectively that pair with Ret receptor to accomplish signaling. In this study, we found GDNF is expressed in human lung and increased in adult asthma, while human ASM expresses GDNF and its receptors. Accordingly, we used human ASM cells to test the hypothesis that ASM expression and autocrine signaling by GFLs regulate [Ca2+ ]i . Serum-deprived ASM cells from non-asthmatics were exposed to 10 ng/ml GDNF or NRTN for 15 min (acute) or 24 h (chronic). In fura-2 loaded cells, acute GDNF or NRTN alone induced [Ca2+ ]i responses, and further enhanced responses to 1 µM ACh or 10 µM histamine. Ret inhibitor (SPP86; 10 µM) or specific GDNF chelator GFRα1-Fc (1 µg/ml) showed roles of these receptors in GDNF effects. In contrast, NRTN did not enhance [Ca2+ ]i response to histamine. Furthermore, conditioned media of nonasthmatic and asthmatic ASM cells showed GDNF secretion. SPP86, Ret inhibitor and GFRα1-Fc chelator markedly decreased [Ca2+ ]i response compared with vehicle, highlighting autocrine effects of secreted GDNF. Chronic GDNF treatment increased histamine-induced myosin light chain phosphorylation. These novel data demonstrate GFLs particularly GDNF/GFRα1 influence ASM [Ca2+ ]i and raise the possibility that GFLs are potential targets of airway hyperresponsiveness.

Nicotinic α7 acetylcholine receptor (α7nAChR) in human airway smooth muscle.

Diseases such as asthma are exacerbated by inflammation, cigarette smoke and even nicotine delivery devices such as e-cigarettes. However, there is currently little information on how nicotine affects airways, particularly in humans, and changes in the context of inflammation or asthma. Here, a longstanding assumption is that airway smooth muscle (ASM) that is key to bronchoconstriction has muscarinic receptors while nicotinic receptors (nAChRs) are only on airway neurons. In this study, we tested the hypothesis that human ASM expresses α7nAChR and explored its profile in inflammation and asthma using ASM of non-asthmatics vs. mild-moderate asthmatics. mRNA and western analysis showed the α7 subunit is most expressed in ASM cells and further increased in asthmatics and smokers, or by exposure to nicotine, cigarette smoke or pro-inflammatory cytokines TNFα and IL-13. In these effects, signaling pathways relevant to asthma such as NFκB, AP-1 and CREB are involved. These novel data demonstrate the expression of α7nAChR in human ASM and suggest their potential role in asthma pathophysiology in the context of nicotine exposure.

Neurotrophin Regulation and Signaling in Airway Smooth Muscle.

Structural and functional aspects of bronchial airways are key throughout life and play critical roles in diseases such as asthma. Asthma involves functional changes such as airway irritability and hyperreactivity, as well as structural changes such as enhanced cellular proliferation of airway smooth muscle (ASM), epithelium, and fibroblasts, and altered extracellular matrix (ECM) and fibrosis, all modulated by factors such as inflammation. There is now increasing recognition that disease maintenance following initial triggers involves a prominent role for resident nonimmune airway cells that secrete growth factors with pleiotropic autocrine and paracrine effects. The family of neurotrophins may be particularly relevant in this regard. Long recognized in the nervous system, classical neurotrophins such as brain-derived neurotrophic factor (BDNF) and nonclassical ligands such as glial-derived neurotrophic factor (GDNF) are now known to be expressed and functional in non-neuronal systems including lung. However, the sources, targets, regulation, and downstream effects are still under investigation. In this chapter, we discuss current state of knowledge and future directions regarding BDNF and GDNF in airway physiology and on pathophysiological contributions in asthma.

Calcium-Sensing Receptor Contributes to Hyperoxia Effects on Human Fetal Airway Smooth Muscle.

Supplemental O2 (hyperoxia), necessary for maintenance of oxygenation in premature infants, contributes to neonatal and pediatric airway diseases including asthma. Airway smooth muscle (ASM) is a key resident cell type, responding to hyperoxia with increased contractility and remodeling [proliferation, extracellular matrix (ECM) production], making the mechanisms underlying hyperoxia effects on ASM significant. Recognizing that fetal lungs experience a higher extracellular Ca2+ ([Ca2+]o) environment, we previously reported that the calcium sensing receptor (CaSR) is expressed and functional in human fetal ASM (fASM). In this study, using fASM cells from 18 to 22 week human fetal lungs, we tested the hypothesis that CaSR contributes to hyperoxia effects on developing ASM. Moderate hyperoxia (50% O2) increased fASM CaSR expression. Fluorescence [Ca2+]i imaging showed hyperoxia increased [Ca2+]i responses to histamine that was more sensitive to altered [Ca2+]o, and promoted IP3 induced intracellular Ca2+ release and store-operated Ca2+ entry: effects blunted by the calcilytic NPS2143. Hyperoxia did not significantly increase mitochondrial calcium which was regulated by CaSR irrespective of oxygen levels. Separately, fASM cell proliferation and ECM deposition (collagens but not fibronectin) showed sensitivity to [Ca2+]o that was enhanced by hyperoxia, but blunted by NPS2143. Effects of hyperoxia involved p42/44 ERK via CaSR and HIF1α. These results demonstrate functional CaSR in developing ASM that contributes to hyperoxia-induced contractility and remodeling that may be relevant to perinatal airway disease.

Genetic Association between MMP9 and Choroidal Neovascularization in Age-Related Macular Degeneration.

Purpose: To evaluate the first association specific to exudative age-related macular degeneration (AMD) located near the matrix metalloproteinase 9 (MMP9) gene. Design: Genetic association study. Participants: One thousand seven hundred twelve patients with AMD (672 nonexudative, 1040 exudative) of predominantly northern European descent seeking treatment at the University of Iowa Hospitals and Clinics. Methods: We reanalyzed the International AMD Genetics Consortium (IAMDGC) data to validate the association of polymorphisms near MMP9 with exudative AMD and to identify additional associated single nucleotide polymorphisms (SNPs), especially MMP9 coding sequence SNPs. We genotyped a cohort of 1712 AMD patients from Iowa with 3 SNPs identified with our analysis of the IAMDGC cohort using commercially available real-time quantitative polymerase chain reaction (PCR) assays. Firth regression was used to measure the association between MMP9 SNP genotypes and exudative AMD in our cohort of patients from Iowa. In addition, we developed a PCR-based assay to genotype the Iowa cohort at a short tandem repeat polymorphism (STRP) at the MMP9 locus. Main Outcome Measures: Odds ratios and P values for exudative compared with nonexudative AMD patients in the Iowa cohort for MMP9 SNPs (rs4810482, rs17576, and rs17577) and STRP. Results: We identified 3 SNPs in the MMP9 locus (rs4810482, rs17576, and rs17577) that are highly associated with exudative AMD in patient cohorts of the IAMDGC. These MMP9 SNPs also are associated with exudative AMD in the cohort of 1712 AMD patients from Iowa (rs4810482: odds ratio [OR], 0.82; P = 0.010; rs17576: OR, 0.86; P = 0.046; and rs17577: OR, 0.80; P = 0.041). We also genotyped the cohort of AMD patients from Iowa at rs142450006, another MMP9 polymorphism that previously was associated with exudative AMD. We detected a 4bp STRP, (TTTC)n, at the rs142450006 locus that is highly polymorphic and associated significantly with exudative AMD (OR, 0.78; P = 0.016). Conclusions: This study independently confirms and expands an association between the MMP9 locus and exudative AMD, further implicating a role for extracellular matrix abnormalities in choroidal neovascularization.

Nanophthalmos patient with a THR518MET mutation in MYRF, a case report.

BACKGROUND: Nanophthalmos has a significant genetic background and disease-causing mutations have been recently been reported in the myelin regulatory factor (MYRF) gene. We report clinical features in a patient with nanophthalmos and a Thr518Met MYRF mutation. CASE PRESENTATION: A three-year-old male was discovered to have nanophthalmos after first presenting to the emergency department for a frontal headache, eye pain, emesis, and lethargy. Imaging studies (CT and MRI) were negative except for increased posterior fossa cerebrospinal fluid. Subsequent examinations revealed nanophthalmos (short axial eye lengths 18.1 mm OD and 18.3 mm OS), microcornea, and a large crystalline lens. Peripheral chorioretinal pigment abnormalities were also observed. He experienced episodes of marked ocular hypertension (53 mmHg OD and 60 mmHg) likely due to intermittent angle closure precipitated by nanophthalmos. The ocular hypertension was responsive to topical medicines. Genetic analysis of known nanophthalmos genes MFRP and TMEM98 were negative, while a novel mutation, Thr518Met was detected in MYRF. The Thr518Met mutation was absent from 362 matched normal controls and was extremely rare in a large population database, allele frequency of 0.000024. The Thr518Met mutation altered a highly conserved amino acid in the MYRF protein and three of four algorithms suggested that this mutation is likely pathogenic. Finally, molecular modeling showed that the Thr518Met mutation is damaging to MYRF structure. Together these data suggest that the Thr518Met mutation causes nanophthalmos. CONCLUSIONS: Nanophthalmos may present at an early age with features of angle closure glaucoma and a Thr518Met mutation in MYRF was detected in a patient with nanophthalmos. Prevalence data, homology data, mutation analysis data, and protein modeling data suggest that this variant is pathogenic and may expand the phenotypic range of syndromic nanophthalmos caused by MYRF mutations to include central nervous system abnormalities (increased posterior fossa cerebrospinal fluid).

Heterozygous triplication of upstream regulatory sequences leads to dysregulation of matrix metalloproteinase 19 in patients with cavitary optic disc anomaly.

Patients with a congenital optic nerve disease, cavitary optic disc anomaly (CODA), are born with profound excavation of the optic nerve resembling glaucoma. We previously mapped the gene that causes autosomal-dominant CODA in a large pedigree to a chromosome 12q locus. Using comparative genomic hybridization and quantitative PCR analysis of this pedigree, we report identifying a 6-Kbp heterozygous triplication upstream of the matrix metalloproteinase 19 (MMP19) gene, present in all 17 affected family members and no normal members. Moreover, the triplication was not detected in 78 control subjects or in the Database of Genomic Variants. We further detected the same 6-Kbp triplication in one of 24 unrelated CODA patients and in none of 172 glaucoma patients. Analysis with a Luciferase assay showed that the 6-Kbp sequence has transcription enhancer activity. A 773-bp fragment of the 6-Kbp DNA segment increased downstream gene expression eightfold, suggesting that triplication of this sequence may lead to dysregulation of the downstream gene, MMP19, in CODA patients. Lastly, immunohistochemical analysis of human donor eyes revealed strong expression of MMP19 in optic nerve head. These data strongly suggest that triplication of an enhancer may lead to overexpression of MMP19 in the optic nerve that causes CODA.

Copy number variations of TBK1 in Australian patients with primary open-angle glaucoma.

PURPOSE: To investigate the presence of TBK1 copy number variations in a large, well-characterized Australian cohort of patients with glaucoma comprising both normal-tension glaucoma and high-tension glaucoma cases. DESIGN: A retrospective cohort study. METHODS: DNA samples from patients with normal-tension glaucoma and high-tension glaucoma and unaffected controls were screened for TBK1 copy number variations using real-time quantitative polymerase chain reaction. Samples with additional copies of the TBK1 gene were further tested using custom comparative genomic hybridization arrays. RESULTS: Four out of 334 normal-tension glaucoma cases (1.2%) were found to carry TBK1 copy number variations using quantitative polymerase chain reaction. One extra dose of the TBK1 gene (duplication) was detected in 3 normal-tension glaucoma patients, while 2 extra doses of the gene (triplication) were detected in a fourth normal-tension glaucoma patient. The results were further confirmed by custom comparative genomic hybridization arrays. Further, the TBK1 copy number variation segregated with normal-tension glaucoma in the family members of the probands, showing an autosomal dominant pattern of inheritance. No TBK1 copy number variations were detected in 1045 Australian patients with high-tension glaucoma or in 254 unaffected controls. CONCLUSION: We report the presence of TBK1 copy number variations in our Australian normal-tension glaucoma cohort, including the first example of more than 1 extra copy of this gene in glaucoma patients (gene triplication). These results confirm TBK1 to be an important cause of normal-tension glaucoma, but do not suggest common involvement in high-tension glaucoma.

Ethnic variation in AMD-associated complement factor H polymorphism p.Tyr402His.

Age-related macular degeneration (AMD) is the most common cause of irreversible visual loss in the developed world. Previous studies have demonstrated that the c.1204T>C, p.Tyr402His allelic variant in the complement factor H (CFH) gene is associated with an approximately three-fold increased risk for AMD in Caucasians of predominantly European descent. Both the prevalence as well as the phenotypic spectrum of AMD varies widely among persons of different ethnicities. We hypothesized that populations with a lower prevalence of AMD might also have a lower prevalence of the CFH risk allele. In this study we sought to determine the frequency of this sequence variant in control populations of Caucasians, African Americans, Hispanics, Somalis, and Japanese. Normal control populations were assembled for each ethnic group: Caucasian (n=148), Somali (n=128), African American (n=75), Hispanic (n=81), and Japanese (n=82). Individuals were genotyped using a restriction digest assay and the frequency of the C allele at nucleotide position 1204 of the CFH gene was determined. A bioinformatic approach was used to identify SNPs in linkage disequilibrium with rs1061170 (c.1204T>C, p.Tyr402His) from the human haplotype map project database (HapMap) in order to validate the findings. We found widely discordant frequencies of the risk allele between some of the different ethnic groups: Japanese 0.07+/-0.02, Hispanics 0.17+/-0.03, African-Americans 0.35+/-0.04, Caucasians 0.34+/-0.03, and Somalis 0.34+/-0.03. Allele frequencies generated by analysis of the HapMap database were consistent with these findings. This study suggests that there are other yet unidentified genetic factors important in the pathogenesis of AMD that may mitigate the effects of c.1204T>C, p.Tyr402His variant.

Evaluation of optineurin sequence variations in 1,048 patients with open-angle glaucoma.

PURPOSE: To investigate the association of sequence variations in the optineurin (OPTN) gene in patients with open-angle glaucoma. DESIGN: Prospective case control study. METHODS: The OPTN gene was screened for sequence variations using a combination of single-strand conformational polymorphism analysis and automated DNA sequencing. A total of 1,299 subjects (1048 glaucoma patients and 251 controls) were screened for variations in the four portions of the gene that had been previously associated with glaucoma. A subset of these subjects (376 patients and 176 controls) was screened for variations in the entire coding sequence. Twenty-four percent of the patients and 35% of the controls were Japanese, whereas the remainder were predominantly Caucasian. Allele frequencies were compared with the Fisher exact test. RESULTS: The OPTN sequence variations were not significantly associated with any form of high-tension open-angle glaucoma. One proband with familial normal-tension glaucoma was found to harbor the previously reported Glu50Lys variation. Another previously reported change, Met98Lys, was associated with normal-tension glaucoma in Japanese but not in Caucasian patients. CONCLUSIONS: This study provides some additional evidence for the association of the Glu50Lys OPTN sequence variation with familial normal tension glaucoma. However, because familial normal-tension glaucoma is so rare, this change seems to be responsible for less than 0.1% of all open-angle glaucoma. The Arg545Gln variation is likely to be a nondisease-causing polymorphism. The Met98Lys change may be associated with a fraction of normal-tension glaucoma in patients of Japanese ethnicity.