Exome sequencing identifies mutations in the gene TTC7A in French-Canadian cases with hereditary multiple intestinal atresia.

BACKGROUND: Congenital multiple intestinal atresia (MIA) is a severe, fatal neonatal disorder, involving the occurrence of obstructions in the small and large intestines ultimately leading to organ failure. Surgical interventions are palliative but do not provide long-term survival. Severe immunodeficiency may be associated with the phenotype. A genetic basis for MIA is likely. We had previously ascertained a cohort of patients of French-Canadian origin, most of whom were deceased as infants or in utero. The goal of the study was to identify the molecular basis for the disease in the patients of this cohort. METHODS: We performed whole exome sequencing on samples from five patients of four families. Validation of mutations and familial segregation was performed using standard Sanger sequencing in these and three additional families with deceased cases. Exon skipping was assessed by reverse transcription-PCR and Sanger sequencing. RESULTS: Five patients from four different families were each homozygous for a four base intronic deletion in the gene TTC7A, immediately adjacent to a consensus GT splice donor site. The deletion was demonstrated to have deleterious effects on splicing causing the skipping of the attendant upstream coding exon, thereby leading to a predicted severe protein truncation. Parents were heterozygous carriers of the deletion in these families and in two additional families segregating affected cases. In a seventh family, an affected case was compound heterozygous for the same 4bp deletion and a second missense mutation p.L823P, also predicted as pathogenic. No other sequenced genes possessed deleterious variants explanatory for all patients in the cohort. Neither mutation was seen in a large set of control chromosomes. CONCLUSIONS: Based on our genetic results, TTC7A is the likely causal gene for MIA.

A pragmatic, randomized, controlled study evaluating the impact of access to smoking cessation pharmacotherapy coverage on the proportion of successful quitters in a Canadian population of smokers motivated to quit (ACCESSATION).

BACKGROUND: Many smokers find the cost of smoking cessation medications a barrier. Financial coverage for these medications increases utilization of pharmacotherapies. This study assesses whether financial coverage increases the proportion of successful quitters. METHODS: A pragmatic, open-label, randomized, controlled trial was conducted in 58 Canadian sites between March 2009 and September 2010. Smokers (≥10 cigarettes/day) without insurance coverage who were motivated to quit within 14 days were randomized (1:1) in a blinded manner to receive either full coverage eligibility for 26 weeks or no coverage. Pharmacotherapies covered were varenicline, bupropion, or nicotine patches/gum. Investigators/subjects were unblinded to study group assignment after randomization and prior to choosing a smoking cessation method(s). All subjects received brief smoking cessation counseling. The primary outcome measure was self-reported 7-day point prevalence of abstinence (PPA) at week 26. RESULTS: Of the 1380 randomized subjects (coverage, 696; no coverage, 684), 682 (98.0%) and 435 (63.6%), respectively, were dispensed at least one smoking cessation medication dose. The 7-day PPA at week 26 was higher in the full coverage versus no coverage group: 20.8% (n = 145) and 13.9% (n = 95), respectively; odds ratio (OR) = 1.64, 95% confidence interval (CI) 1.23-2.18; p = 0.001. Urine cotinine-confirmed 7-day PPA at week 26 was 15.7% (n = 109) and 10.1% (n = 69), respectively; OR = 1.68, 95% CI 1.21-2.33; p = 0.002. After pharmacotherapy, coverage eligibility was withdrawn from the full coverage group, continuous abstinence between weeks 26 and 52 was 6.6% (n = 46) and 5.6% (n = 38), in the full coverage and no coverage groups, respectively; OR = 1.19, 95% CI 0.76-1.87; p = 0.439. CONCLUSIONS: In this study, the adoption of a smoking cessation medication coverage drug policy was an effective intervention to improve 26-week quit rates in Canada. The advantages were lost once coverage was discontinued. Further study is required on the duration of coverage to prevent relapse to smoking. (clinicaltrials.gov identifier: NCT00818207; the study was sponsored by Pfizer Inc.).

Effects of dexamethasone on human trabecular meshwork cells in vitro.

PURPOSE: To study the effects of dexamethasone sodium phosphate (Dex) on human trabecular meshwork (HTM) cells in vitro. METHODS: HTM cells were treated with Dex 2 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.1 mg/ml, or 0.05 mg/ml for 24 h. Cell viability was measured by a trypan blue exclusion test. Caspase-3/7, -8, -9 and -12 activities were measured by fluorochrome assays as mean signal intensity (msi) to assess apoptosis. Mitochondrial dehydrogenase activity was determined by a WST assay to quantify mitochondrial damage. RESULTS: Mean cell viabilities of HTM cells exposed to Dex at the higher doses of 2 mg/ml, 1 mg/ml, and 0.5 mg/ml were reduced: 11.9 % ± 3.5 (P < 0.001), 31.2 % ± 3.2 (P < 0.001), and 76.6 % ± 4.4 (P < 0.01). At the lower doses of 0.25 mg/ml, 0.1 mg/ml or 0.05 mg/ml, no significant cell viability reductions were seen: 96.3 % ± 0.7 (P > 0.05), 95.3 % ± 2.5 (P > 0.05) and 93.8 % ± 2.3 (P > 0.05), respectively compared to untreated HTM cells (97.0 % ± 1.9). Caspase-3/7 activity (msi) of HTM cells exposed to Dex 2, 1 or 0.5 mg/ml was 21068 ± 2498 (P < 0.001), 26994 ± 3104 (P < 0.001) and 20416 ± 1150 (P < 0.001) compared to untreated HTM cells 1148 ± 803. Caspase-9 activity (msi) of HTM cells after exposure to Dex 2, 1 or 0.5 mg/ml was 14188 ± 1203 (P < 0.001), 13256 ± 1564 (P < 0.001) and 15041 ± 1584 (P < 0.001) compared to untreated HTM cells 1748 ± 524. The lower doses of Dex did not significantly increase caspase-3/7 or -9 activities. There were no increases for caspase-8 or -12 activities at any of the tested Dex doses. The WST assay showed mitochondrial dehydrogenase activities of 14.3 ± 0.7 (P < 0.001), 9.6 ± 0.3 (P < 0.001) and 56.0 ± 7.6 (P < 0.001) at 2 mg/ml, 1 mg/ml and 0.5 mg/ml Dex compared to untreated HTM cells (186.1 ± 15.0). CONCLUSIONS: Dex at 0.25, 0.1 and 0.05 mg/ml clinical dose did not cause significant reduction in cell viability, increased apoptosis, or mitochondrial dysfunction of HTM cells in vitro. At high doses (2, 1 or 0.5 mg/ml) Dex caused apoptosis via mitochondrial pathways.

How pragmatic or explanatory is the randomized, controlled trial? The application and enhancement of the PRECIS tool to the evaluation of a smoking cessation trial.

BACKGROUND: Numerous explanatory randomized trials support the efficacy of chronic disease interventions, including smoking cessation treatments. However, there is often inadequate adoption of these interventions for various reasons, one being the limitation of generalizability of the explanatory studies in real-world settings. Randomized controlled trials can be rated as more explanatory versus pragmatic along 10 dimensions. Pragmatic randomized clinical trials generate more realistic estimates of effectiveness with greater relevance to clinical practice and for health resource allocation decisions. However, there is no clear method to scale each dimension during the trial design phase to ensure that the design matches the intended purpose of the study. METHODS: We designed a pragmatic, randomized, controlled study to maximize external validity by addressing several barriers to smoking cessation therapy in ambulatory care. We analyzed our design and methods using the recently published 'Pragmatic-Explanatory Continuum Indicatory Summary (PRECIS)' tool, a qualitative method to assess trial design across 10 domains. We added a 20-point numerical rating scale and a modified Delphi process to improve consensus in rating these domains. RESULTS: After two rounds of review, there was consensus on all 10 domains of study design. No single domain was scored as either fully pragmatic or fully explanatory; but overall, the study scored high on pragmatism. CONCLUSIONS: This addition to the PRECIS tool may assist other trial designers working with interdisciplinary co-investigators to rate their study design while building consensus.

Economic impact of delays in listing decisions by provincial drug plans after a positive Common Drug Review recommendation: the case of a smoking-cessation treatment.

Although varenicline (Champix), a smoking-cessation treatment, was recommended for listing by the Common Drug Review (CDR) in 2007, only one CDR-participating drug insurance plan listed it in March 2011 (Saskatchewan). This study estimated the economic impact of delays in the public listing of varenicline in Canada. Using statistical data and peer-reviewed research, social costs and benefits of reimbursing varenicline were estimated. Flows of attempted and successful quitters were projected over a five-year period for three scenarios: immediate listing (2007), one- to four-year listing delays, and no reimbursement. Benefits of public reimbursement of varenicline would have been greatest in the first year ($271 million) and then decreased due to the erosion in smoking prevalence. The current three-year listing delay prevented a projected 17,729 current smokers from quitting, translating into a projected additional lifetime social burden of $700 million. The sizeable opportunity cost of delaying varenicline reimbursement implies broader economic issues for policy makers.

Glaucoma-associated WDR36 variants encode functional defects in a yeast model system.

Primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide. POAG is associated with a characteristic progression of changes to ocular morphology and degeneration at the optic nerve head with the loss of visual fields. Physical mapping efforts identified genomic loci in which to search for causative POAG gene mutations. WDR36, at locus GLC1G, was initially identified as a gene with a low frequency of non-synonymous sequence variations that were exclusive to adult-onset POAG patients. It has since been shown that rare WDR36 sequence variants are also present in the normal population at similarly low frequencies. The lack of a consistent genotype:phenotype correlation prompted us to investigate the functional consequences of WDR36 sequence variations. WDR36 is involved in rRNA processing, a critical step in ribosome biogenesis, and is very similar to yeast Utp21p which is a member of the small subunit (SSU) processome complex responsible for maturation of 18S rRNA. We, therefore, developed a yeast model system to test the functional and phenotypic consequences of POAG-associated sequence variants introduced into UTP21. Alone, the POAG variants did not produce any significant defects in cell viability or rRNA processing. However, when combined with disruption of STI1 (which synthetically interacts with UTP21), 5 of the 11 tested variants had increased or decreased cell viability which corresponded to reduced or elevated levels of pre-rRNA, respectively. These results demonstrate that, in the correct genetic background, WDR36 sequence variants can lead to an altered cellular phenotype, supporting the theory that WDR36 participates in polygenic forms of glaucoma.

Co-variation of STI1 and WDR36/UTP21 alters cell proliferation in a glaucoma model.

PURPOSE: To investigate the role of multigenic variation in primary open-angle glaucoma (POAG) involving the rRNA processing gene WD repeat domain 36 (WDR36). METHODS: We examined the heat shock protein 70/90 (HSP70/90)-organizing co-chaperone stress-induced-phosphoprotein 1 (STI1) as a potential co-modifying gene in glaucoma patients found to harbor WDR36 amino acid variation. The STI1 gene was sequenced and its POAG-associated amino acid variant K434R, as well as the single nucleotide polymorphism (SNP) P173T, were tested for functional defects in a yeast model system previously used to characterize WDR36 variants (using the homologous yeast gene U3 protein 21 [UTP21]). RESULTS: A POAG patient heterozygous for the WDR36 variant L25P was discovered to also carry the STI1 variant K434R in a heterozygous state. Variant K434R, located at an evolutionarily-conserved site, was not found in a pool of clinically-examined individuals lacking WDR36 variation which included 55 normal controls and 20 patients with normal tension glaucoma (NTG). STI1 (K434R) and the homologous yeast variant K470R were able to rescue yeast growth-inhibition by the HSP90-inhibitor radicicol. Double mutant haploid strains expressing human STI1 (K434R) and recombinant yeast UTP21 variants did not have significantly different levels of 18S rRNA from the corresponding hSTI1 (WT) strains. However, specific double mutant K434R strains exhibited significantly slower culture growth at 37 °C. Double mutant P173T strains also displayed altered growth rates at 37 °C. CONCLUSIONS: STI1 variation does not play a significant direct role in the genetics of POAG. However, as previously found for the STI1 null allele, non-synonymous variants of human STI1 confer growth dysregulation in the context of specific yeast UTP21 mutations and heat stress. Based on the genetic association of two co-heterozygous STI1 and WDR36 variants in a POAG patient and the functional analyses performed in a model system for basic eukaryotic cellular processes, these experiments point to a conserved molecular pathway involving STI1 and WDR36.

FOXC1 is required for cell viability and resistance to oxidative stress in the eye through the transcriptional regulation of FOXO1A.

Mutations in the human FOXC1 transcription factor gene underlie Axenfeld-Rieger (AR) syndrome, a disorder characterized by anterior segment malformations in the eye and glaucoma. Through the use of an inducible FOXC1 protein, along with an intermediate protein synthesis blocker, we have determined direct targets of FOXC1 transcriptional regulation. FOXC1 regulates the expression of FOXO1A and binds to a conserved element in the FOXO1A promoter in vivo. The zebrafish foxO1a orthologs exhibit a robust expression pattern in the periocular mesenchyme. Furthermore, FOXO1A expression is reduced in cultured human trabecular meshwork (TM) cells and in the zebrafish developing eye when FOXC1 expression is knocked down by siRNAs and morpholino antisense oliognucleotides, respectively. We also demonstrate that reduced FOXC1 expression increases cell death in cultured TM cells in response to oxidative stress, and increases cell death in the developing zebrafish eye. These studies have uncovered a novel role for FOXC1 as an essential mediator of cellular homeostasis in the eye and indicate that a decreased resistance to oxidative stress may underlie AR-glaucoma pathogenesis. Given that FOXO1A influences cellular homeostasis when positively or negatively regulated; the dysregulation of FOXO1A activities in the eye through FOXC1 loss of function mutations and FOXC1 gene duplications provides an explanation into how seemingly similar human disorders can arise from both increases and decreases in FOXC1 gene dose.

The novel Y371D myocilin mutation causes an aggressive form of juvenile open-angle glaucoma in a Caucasian family from the Middle-East.

PURPOSE: To search for the genetic cause of juvenile open-angle glaucoma (JOAG) in a Caucasian family and to perform genotype/phenotype correlation studies in the kindred. METHODS: Six members of a three-generation family originating from Uzbekistan and now living in the Middle East were recruited from one large clinic in Israel. Ophthalmologic investigations comprised of visual field assessments, intraocular pressure measurements, optic disc evaluation, and gonioscopy. Medical charts were obtained to date the onset of glaucoma and to evaluate aggressivity of the trait. We screened the myocilin gene (MYOC, OMIM 601652) by direct genomic sequencing of its three exons in all family members. RESULTS: JOAG segregated as an autosomal dominant trait in four members of the family. The proband, a 14-year-old girl, had been diagnosed with juvenile open-angle glaucoma at 12 years old. Her mother, maternal aunt, and maternal grandfather all had JOAG that started at an early age. The disorder progressed rapidly even under optimal medical treatment, and all four patients had to undergo trabeculectomy. One missense mutation, Y371D (1111t-->g, Tyr [Y] 371 Asp [D]), was identified. This mutation cosegregated with the disorder in all affected members and was absent in 200 Caucasian controls. The Y371D MYOC mutation has not been reported before. One cousin of the proband was a silent heterozygotic carrier of the mutation and was still asymptomatic at nine years of age. CONCLUSIONS: We identified a novel mutation (Y371D) in MYOC from a Caucasian family who presented with an aggressive form of JOAG that required early trabeculectomy. Genetic screening of the MYOC mutation was beneficial in predicting one asymptomatic heterozygotic carrier.

FOXC1 modulates MYOC secretion through regulation of the exocytic proteins RAB3GAP1, RAB3GAP2 and SNAP25.

The neurodegenerative disease glaucoma is one of the leading causes of blindness in the world. Glaucoma is characterized by progressive visual field loss caused by retinal ganglion cell (RGC) death. Both surgical glaucoma treatments and medications are available, however, they only halt glaucoma progression and are unable to reverse damage. Furthermore, many patients do not respond well to treatments. It is therefore important to better understand the mechanisms involved in glaucoma pathogenesis. Patients with Axenfeld-Rieger syndrome (ARS) offer important insight into glaucoma progression. ARS patients are at 50% risk of developing early onset glaucoma and respond poorly to treatments, even when surgical treatments are combined with medications. Mutations in the transcription factor FOXC1 cause ARS. Alterations in FOXC1 levels cause ocular malformations and disrupt stress response in ocular tissues, thereby contributing to glaucoma progression. In this study, using biochemical and molecular techniques, we show that FOXC1 regulates the expression of RAB3GAP1, RAB3GAP2 and SNAP25, three genes with central roles in both exocytosis and endocytosis, responsible for extracellular trafficking. FOXC1 positively regulates RAB3GAP1 and RAB3GAP2, while either increase or decrease in FOXC1 levels beyond its normal range results in decreased SNAP25. In addition, we found that FOXC1 regulation of RAB3GAP1, RAB3GAP2 and SNAP25 affects secretion of Myocilin (MYOC), a protein associated with juvenile onset glaucoma and steroid-induced glaucoma. The present work reveals that FOXC1 is an important regulator of exocytosis and establishes a new link between FOXC1 and MYOC-associated glaucoma.

A genome-wide scan maps a novel juvenile-onset primary open angle glaucoma locus to chromosome 5q.

PURPOSE: To map the disease-associated locus of a family with autosomal dominant juvenile-onset primary open angle glaucoma (JOAG) and to screen the novel glaucoma gene WD repeat domain 36 (WDR36). METHODS: Complete ophthalmic examination and genomic DNA were obtained from 27 family members, in which nine were confirmed JOAG patients. Myocilin (MYOC), optineurin (OPTN), and WDR36 were screened for mutations by polymerase chain reaction and direct sequencing. Genome-wide scanning was carried out using the ABI PRISM Linkage Mapping Set MD-10. Two-point and multipoint linkage analyses were performed with the MLINK, ILINK, and LINKMAP programs. For fine mapping, additional markers flanking the most promising region on chromosome 5q were also analyzed. The significance of LOD scores was tested with simulation analyses using FASTLINK. Haplotypes were constructed using Simwalk2. RESULTS: MYOC or OPTN mutations were excluded in all family members. A maximum LOD score value of 4.82 at theta=0.00 was obtained for the marker D5S2011. Markers D5S2065, D5S1384, D5S471, D5S503, D5S2098, and D5S638 had LOD score values over 4.0 at theta=0.00. Haplotype analysis and recombination mapping further confined this region to 5q22.1-q32 within a region of 36 Mb flanked by D5S2051 and D5S2090. Screening of the novel WDR36 glaucoma-associated gene, which lies centromeric to the disease interval, revealed no mutations within any of the 23 coding exons or splicing junctions. CONCLUSIONS: Our results provided the mapping of a novel locus for JOAG at 5q and excluded coding or splicing junctions mutations within the WDR36 gene.

Novel myocilin mutation in a Chinese family with juvenile-onset open-angle glaucoma.

OBJECTIVE: To search for the genetic cause of juvenile-onset open-angle glaucoma (JOAG) in a Chinese family. METHODS: In a 3-generation glaucoma family affected with JOAG or ocular hypertension, we screened myocilin (MYOC) and optineurin (OPTN) for mutations and investigated apolipoprotein E (APOE) polymorphisms in 6 family members, 2 of them patients with JOAG, 2 patients with ocular hypertension, and 2 patients who were asymptomatic. Normal controls included 200 unrelated Chinese subjects. The COS-7 cell line was transfected with expression vectors encoding wild-type or mutated MYOC complementary DNA. Cellular and secreted MYOC proteins were characterized by Western blotting. RESULTS: One missense MYOC mutation, 734G>A: Cys245Tyr, was identified. It occurred in all 4 family members afflicted with JOAG or ocular hypertension but not in asymptomatic family members. No OPTN variations were observed. APOE polymorphism frequencies were similar to those for controls. The Cys245Tyr MYOC mutation cosegregated with the disorder within the family. It was absent in the 200 control subjects. The Cys245Tyr mutant MYOC protein formed homomultimeric complexes that migrated at molecular weights larger than their wild-type counterparts. These mutant complexes remained sequestered intracellularly in COS-7 cells. CONCLUSIONS: The Cys245Tyr MYOC mutation was the genetic cause of JOAG in this Chinese family. This mutation may alter covalent bonds that formed between MYOC cysteines. Clinical Relevance Genetic tests of MYOC mutations may be beneficial to predict new cases of the disease in families with JOAG.

Clinical and economic benefits of extended treatment with apixaban for the treatment and prevention of recurrent venous thromboembolism in Canada.

Background and objective Venous thromboembolism (VTE) is associated with long-term clinical and economic burden. Clinical guidelines generally recommend at least 3 months of anticoagulation, but, in clinical practice, concerns over bleeding risk often limit extended treatment. Apixaban was studied for extended VTE treatment in the AMPLIFY-EXT trial, demonstrating superiority to placebo in VTE reduction without increasing risk of major bleeding. This study assessed the long-term clinical and economic benefits of extending treatment with apixaban when clinical equipoise exists compared to standard of care with enoxaparin/warfarin and other novel oral anti-coagulants (NOACs) for the treatment and prevention of recurrent VTE in Canada. Methods A Markov model was developed to follow patients with VTE over their lifetimes. Efficacy and safety for apixaban and enoxaparin/warfarin were based on AMPLIFY and AMPLIFY-EXT, while relative efficacy to other NOACs was synthesized by network meta-analysis (NMA). Dosages for NOACs and enoxaparin/warfarin were based on their respective trials and were given up to 18 months and up to 6 months, followed by no treatment, respectively. Patient quality adjusted life years (QALYs) were based on published studies, and costs for resource utilization were from a Ministry of Health perspective, expressed as 2014 CAD ($). Results Extended treatment with apixaban compared to enoxaparin/warfarin resulted in fewer recurrent VTEs, VTE-related deaths, and bleeding events, but at slightly increased cost. The incremental cost-effectiveness ratio was $4828 per QALY gained. Compared to other NOACs, apixaban had the fewest bleeding events, similar recurrent VTE events, and the lowest overall cost, which was driven by the strong bleeding profile. In scenario analyses of acute and lifetime treatments, apixaban was cost-effective against all strategies. Conclusions Extended treatment with apixaban can offer substantial clinical benefits and is a cost-effective alternative to enoxaparin/warfarin and other NOACs.

A common disease haplotype for the Q368STOP mutation of the myocilin gene in Australian and Canadian glaucoma families.

PURPOSE: To ascertain whether there is a common disease haplotype for the Q368STOP mutation of the myocilin gene in Australian and Canadian families with primary open-angle glaucoma (POAG). DESIGN: Family pedigree study. METHODS: A disease haplotype for the Q368STOP mutation of the myocilin gene has previously been identified in 15 Tasmanian families with POAG. The four microsatellite markers that constitute this 0.14-megabase (Mb) disease haplotype were genotyped in individuals from a large French Canadian family with POAG (family CT) and two unrelated French Canadian individuals with ocular hypertension. RESULTS: The Tasmanian Q368STOP disease haplotype was identified in affected individuals from family CT, and the same alleles were shared at the four microsatellite markers in the two unrelated French Canadian individuals. CONCLUSION: The same disease haplotype for the Q368STOP mutation of the myocilin gene was found in both the Tasmanian and French Canadian populations, supporting the view that this mutation arose from a common Caucasian founder.

Gene expression profile of the human trabecular meshwork: NEIBank sequence tag analysis.

PURPOSE: To characterize the gene expression pattern in the human trabecular meshwork (TM) and identify candidate genes for glaucoma by expressed sequence tag (EST) analysis as part of the NEIBank project. METHODS: RNA was extracted from dissected human TM and used to construct unamplified, un-normalized cDNA libraries in the pSPORT1 vector. More than 4000 clones were sequenced from the 5' end. Clones were clustered and identified using GRIST software. In addition, the expression patterns of genes encoding olfactomedin-domain proteins were analyzed by RT-PCR. RESULTS: After non-mRNA contaminants were removed, 3459 independent TM-expressed clones were obtained. These were grouped in 1888 clusters, potentially representing individual expressed genes. Transcripts for the myocilin gene, a locus for inherited glaucoma, formed the third most abundant cluster in the TM collection, and several other genes implicated in glaucoma (PITX2, CYP1B1, and optineurin) were also represented. One abundant TM transcript was from the gene for the angiopoietin-like factor CTD6, which is located at on the long arm of chromosome 1, area 36.2-36.1 in the region of the glaucoma locus GLC3B, whereas other transcripts were from genes close to known glaucoma loci. The TM collection contains cDNAs for genes that are preferentially expressed in the lymphatic endothelium (matrix Gla protein, apolipoprotein D precursor, and selenoprotein P precursor). In addition to EST profiling, RT- PCR was used to detect transcripts of the olfactomedin-domain proteins latrotoxin receptor Lec3 and optimedin in the TM. CONCLUSIONS: The TM libraries are a good source of molecular markers for TM and candidate genes for glaucoma. The abundance of myocilin cDNAs corresponds to the critical role of this gene in glaucoma and contrasts with libraries derived from cultured tissue. The expression profile raises the possibility that cells of the TM and Schlemm's canal may be more similar to lymphatic, rather than blood vascular endothelium.

Intracellular sequestration of hetero-oligomers formed by wild-type and glaucoma-causing myocilin mutants.

PURPOSE: To investigate mechanism(s) by which mutations in the olfactomedin domain of myocilin (MYOC), also known as the trabecular meshwork-induced glucocorticoid response (TIGR) gene, cause autosomal dominant open-angle glaucoma, the structure and properties of wild-type (WT) MYOC protein were examined, when expressed alone or simultaneously with the Q368X or K423E disease-associated polypeptides. METHODS: Myocilin was analyzed in human aqueous humor and human trabecular meshwork (HTM) tissues. COS-7 and immortalized human trabecular meshwork (iHTM) cell lines were transfected with expression vectors encoding WT MYOC, mutated, and/or epitope-tagged cDNAs. MYOC proteins were characterized by double-epitope tagging procedures and/or Western blot analysis. RESULTS: MYOC polypeptides formed highly similar oligomers in aqueous humor, HTM tissues, transfected COS-7, and iHTM cell lines. These complexes ranged in size from 116 kDa to more than 200 kDa. The smallest complex, approximately 116 kDa, resulted from dimerization between two MYOC monomers. Expression of a 150-kDa complex was strongest in aqueous humor. Cotransfections of the WT construct with either the Q368X or K423E cDNA produced MYOC(WT)/MYOC(mutant) heterodimers and higher molecular weight hetero-oligomeric complexes. WT homo-oligomeric complexes were secreted in the extracellular media of both cell lines whereas the Q368X and K423E mutant/mutant homomultimers and heteromeric WT/mutant oligomers remained sequestered intracellularly. CONCLUSIONS: Formation of heteromeric WT/mutant complexes may provide a critical mechanism by which mutant myocilin polypeptides produce autosomal dominant open-angle glaucoma. The intracellular sequestration of abnormal WT/mutant complexes could lead to the malfunction of MYOC-expressing cells and to POAG potentially involving a dominant negative effect.

Axenfeld-Rieger anomaly: a novel mutation in the forkhead box C1 (FOXC1) gene in a 4-generation family.

OBJECTIVE: To characterize DNA mutations in a pedigree of Axenfeld-Rieger anomaly (ARA) (Online Mendelian Inheritance of Man 601631), a clinically and genetically heterogeneous, autosomal dominantly inherited disorder associated with anterior chamber abnormalities and glaucoma. DESIGN: Observational case-control and DNA linkage and screening studies. PARTICIPANTS: Affected (10 cases) and unaffected (5 controls) members of a family with ARA. METHODS: Clinical characteristics of ARA were documented by history or physical examination of symptomatic individuals. With their informed consent, a blood sample was collected from each of 10 affected and 5 unaffected family members. DNA was tested for linkage to the IRID1 locus at chromosome 6p25, a known locus for ARA/Rieger syndrome. A candidate gene previously mapped at this locus, FOXC1, was screened for mutations in cases and controls. Main Outcome Measure Linkage of the ARA phenotype at the 6p25 locus and mutation detected in FOXC1. RESULTS: Direct sequencing of FOXC1 detected a new mutation, T272C, that segregated with the ARA phenotype in this family and was not detected in DNA from family members without ARA. This mutation, a T-->C transition, is predicted to result in a change of isoleucine to threonine (Ile9lThr) in a highly conserved location within the first helix of the forkhead domain. CONCLUSION: Characterization of the FOXC1 mutation in family members with ARA furthers our understanding of the molecular origin of developmental glaucoma and other anterior segment disorders.

Effects of triamcinolone acetonide on human trabecular meshwork cells in vitro.

AIM: To study the effects of triamcinolone acetonide (TA) on cultured human trabecular meshwork (HTM) cells. MATERIALS AND METHODS: HTM cells were cultured and treated with 125, 250, 500 and 1000 µg/mL concentration of TA for 24 h. The cells were treated with both crystalline TA (TA-C) (commercial preparation) and solubilized TA (TA-S). Cell viability was measured by a trypan blue dye exclusion test. The activity of caspse-3/7 was measured by a fluorescence caspase kit and DNA laddering was evaluated by electrophoresis on 3% agarose gel. Levels of lactate dehydrogenase (LDH) were assessed with LDH cytotoxicity assay kit-II. RESULTS: Mean cell viabilities of HTM cells after 24 h exposure to TA-C 125, 250, 500, and 1000 µg/mL were 75.4 ±2.45% (P < 0.0001), 49.43 ± 1.85% (P < 0.0001), 17.07 ± 2.39% (P < 0.0001), and 3.7 ± 0.9% (P < 0.0001), respectively, compared with the untreated HTM cells 92.49 ± 1.21%. The mean cell viabilities with 125, 250, 500, and 1000 µg/mL of TA-S were 94.47 ± 1.60% (P > 0.05), 90.13 ± 0.40% (P < 0.01), 85.57 ± 0.47% (P < 0.001), and 71.67 ± 3.30% (P < 0.0001), respectively, compared to DMSO-equivalent cultures. Untreated HTM control had a cell viability of 96.57 ± 1.98%. DMSO-treated controls of 125, 250, 500, and 1000 µg/mL had a cell viability of 94.73 ± 0.57%, 96.97 ± 1.08%, 93.97 ± 1.85%, and 97.27 ± 1.15%, respectively. There was no increase of caspase-3/7 activity in cultures treated with either TA-C or TA-S. DNA laddering showed no bands in the TA-C or TA-S treated cultures. There were significantly higher LDH release rates at all concentrations of TA-C compared to TA-S. CONCLUSIONS: Results show that the effect of TA-C and TA-S on HTM cells is due to cell death by necrosis at all concentrations except 125 µg/mL of TA-S. Elevated levels of LDH confirmed necrotic cell death. Our study also infers the relative safety of TA-S over TA-C.

A Canadian study of the cost-effectiveness of apixaban compared with enoxaparin for post-surgical venous thromboembolism prevention.

BACKGROUND: Occurrence of a venous thromboembolism (VTE) in patients undergoing major orthopedic surgery who are not given thromboprophylactic therapy presents considerable danger to patient medical outcomes and a significant economic burden to the health care system at large. Apixaban is a direct factor Xa inhibitor that has been shown in clinical trial use to safely reduce the composite of VTE and mortality rates in patients undergoing total hip arthroplasty (THA) and total knee arthroplasty (TKA); however, the cost-effectiveness of apixaban treatment in Canadian settings has not been studied. Our study evaluated the cost-effectiveness of apixaban compared with enoxaparin as VTE preventive therapy in patients undergoing elective THA or TKA in Canada. METHODS: An economic model, including both a decision-tree component and a Markov model, was created. The decision tree considered VTE, bleeding, and mortality incidence that occurred in patients within 90 days post-surgery using data from the Apixaban Versus Enoxaparin for Thromboprophylaxis After Knee or Hip Replacement (ADVANCE) trials, which compared apixaban therapy with 30-mg twice daily and 40-mg daily enoxaparin treatment. The Markov model provided the option to simulate events that may occur over the long term, such as recurrent VTE and post-thrombotic syndrome. Outcomes during the short-term phase directly impact the risk of events occurring during the long-term phase (5 years post-surgery). RESULTS: The results of our analysis indicated that apixaban is dominant (ie, more effective and less expensive) than enoxaparin in treating patients undergoing THA and TKA. There were fewer occurrences of VTEs, bleeding events, recurrent VTEs, and post-thrombotic syndrome events in the TKA population with apixaban therapy. Similar results were seen in patients undergoing THA, with the exception of bleeding events, which were more common with apixaban treatment. Savings of $180 to $270 per patient are expected with apixaban treatment compared with enoxaparin treatment, and health outcomes in general are better with apixaban use. Sensitivity analyses yielded consistent results across the THA and TKA populations. CONCLUSION: : This is the first economic evaluation of apixaban use for VTE thromboprophylaxis in the Canadian setting, and our study results show apixaban to be a cost-effective treatment alternative to preventive treatment with enoxaparin.

PITX2 is involved in stress response in cultured human trabecular meshwork cells through regulation of SLC13A3.

PURPOSE: Mutations of the PITX2 gene cause Axenfeld-Rieger syndrome (ARS) and glaucoma. In this study, the authors investigated genes directly regulated by the PITX2 transcription factor to gain insight into the mechanisms underlying these disorders. METHODS: RNA from nonpigmented ciliary epithelium cells transfected with hormone-inducible PITX2 and activated by mifepristone was subjected to microarray analyses. Data were analyzed using dCHIP algorithms to detect significant differences in expression. Genes with significantly altered expression in multiple microarray experiments in the presence of activated PITX2 were subjected to in silico and biochemical analyses to validate them as direct regulatory targets. One target gene was further characterized by studying the effect of its knockdown in a cell model of oxidative stress, and its expression in zebrafish embryos was analyzed by in situ hybridization. RESULTS: Solute carrier family 13 sodium-dependent dicarboxylate transporter member 3 (SLC13A3) was identified as 1 of 47 potential PITX2 target genes in ocular cells. PITX2 directly regulates SLC13A3 expression, as demonstrated by luciferase reporter and chromatin immunoprecipitation assays. Reduction of PITX2 or SLC13A3 levels by small interfering RNA (siRNA)-mediated knockdown augmented the death of transformed human trabecular meshwork cells exposed to hydrogen peroxide. Zebrafish slc13a3 is expressed in anterior ocular regions in a pattern similar to that of pitx2. CONCLUSIONS: The results indicate that SLC13A3 is a direct downstream target of PITX2 transcriptional regulation and that levels of PITX2 and SLC13A3 modulate responses to oxidative stress in ocular cells.