Protocol for a Prospective, Observational Cost-effectiveness Analysis of Returning Secondary Findings of Genome Sequencing for Unexplained Suspected Genetic Conditions.

PURPOSE: Although costly, genome-wide sequencing (GWS) detects an extensive range of variants, enhancing our ability to diagnose and assess risk for an increasing number of diseases. In addition to detecting variants related to the indication for testing, GWS can detect secondary variants in BRCA1, BRCA2, and other genes for which early intervention may improve health. As the list of secondary findings grows, there is increased demand for surveillance and management by multiple specialists, adding pressure to constrained health care budgets. Secondary finding testing is actively debated because some consider it opportunistic screening for future health risks that may not manifest. Given the economic implications of secondary finding testing and follow-up and its unproven clinical utility, the objective is to assess the incremental cost-effectiveness of secondary finding ascertainment per case detected and per unit of improved clinical utility in families of children with unexplained suspected genetic conditions undergoing clinical GWS. METHODS: Those undergoing trio genome or exome sequencing are eligible for the study. Positive secondary finding index cases will be matched to negative controls (1:2) based on age group, primary result(s) type, and clinical indication. During the 2-year study, 71 cases and 142 matched controls are expected. Health service use will be collected in patients and 1 adult family member every 6 months. The per-child and per-dyad total cost will be determined by multiplying use of each resource by a corresponding unit price and summing all cost items. Costs will be estimated from the public and societal payer perspectives. The mean cost per child and per dyad for secondary finding-positive and secondary finding-negative groups will be compared statistically. If important demographic differences are observed between groups, ordinary least-squares regression, log transformation, or other nonparametric technique will be used to compare adjusted mean costs. The ratio of the difference in mean cost to the secondary finding yield will be used to estimate incremental cost-effectiveness. In secondary analyses, effectiveness will be estimated using the number of clinical management changes due to secondary findings or the Clinician-Reported Genetic Testing Utility Index (C-GUIDE) score, a validated measure of clinical utility. Sensitivity analysis will be undertaken to assess the robustness of the findings to variation in key parameters. IMPLICATIONS: This study generates key evidence to inform clinical practice and funding allocation related to secondary finding testing. The inclusion of family members and a new measure of clinical utility represent important advancements in economic evaluation in genomics.

Reverse effects of Streptococcus mutans physiological states on neutrophil extracellular traps formation as a strategy to escape neutrophil killing.

Bacteria in nature are present in different lifestyles with distinct characteristics. Streptococcus mutans is the etiologic pathogen of dental caries and could easily gain access into the bloodstream after oral surgery and adopt a biofilm lifestyle, resulting in infective endocarditis. A growing amount of evidence have revealed that the large web-like structure composed of extracellular DNA and antimicrobial proteins released by neutrophils, named Neutrophil Extracellular Traps (NETs), play an active role in the defense against bacterial invasion. The present study demonstrated that NETs formation was discriminatively affected by S. mutans biofilm and its planktonic counterpart. The free-floating planktonic S. mutans exhibited an active NETs response, whereas the biofilm community exhibited a reverse negative NETs response. Besides, impaired biofilm killing correlated with the decrease in NETs production. Unlike planktonic cells, biofilm avoided the burst of reactive oxygen species (ROS) when co-culture with neutrophils, and the NADPH-oxidase pathway was partially involved. A mice infection model also supported the distinguishing response of neutrophils challenged by different lifestyles of S. mutans. In conclusion, different bacterial physiological states can affect the distinct response of the host-microbe interaction, thus contributing to the anti-pathogen immune response activation and immune surveillance survival.

A novel gingipain regulatory gene in Porphyromonas gingivalis mediates host cell detachment and inhibition of wound closure.

The black pigmentation-related genes in Porphyromonas gingivalis are primarily involved in regulating gingipain functions. In this study, we identified a pigmentation-related gene, designated as pgn_0361. To characterize the role of pgn_0361 in regulating P. gingivalis-mediated epithelial cell detachment and inhibition of wound closure, PgΔ0361, an isogenic pgn_0361-defective mutant strain, and PgΔ0361C, a complementation strain, were constructed using P. gingivalis ATCC 33277. The gingipain and hemagglutination activities, as well as biofilm formation, were examined in all three strains. The effect of P. gingivalis strains on epithelial cell detachment was investigated using the HO-1-N-1 and Ca9-22 epithelial cell lines. The inhibition of wound closure by heat-killed P. gingivalis cells and culture supernatant was analyzed using an in vitro wound closure assay. Compared to the wild-type strain, the PgΔ0361 strain did not exhibit gingipain or hemagglutination activity but exhibited enhanced biofilm formation. Additionally, the PgΔ0361 strain exhibited attenuated ability to detach the epithelial cells and to inhibit wound closure in vitro. Contrastingly, the culture supernatant of PgΔ0361 exhibited high gingipain activity and strong inhibition of wound closure. The characteristics of PgΔ0361C and wild-type strains were comparable. In conclusion, the pgn_0361 gene is involved in regulating gingipains. The PGN_0361-defective strain exhibited reduced virulence in terms of epithelial cell detachment and inhibition of wound closure. The culture supernatant of the mutant strain highly inhibited wound closure, which may be due to high gingipain activity.

Myotonic Myopathy With Secondary Joint and Skeletal Anomalies From the c.2386C>G, p.L769V Mutation in SCN4A.

The phenotypic spectrum associated with the skeletal muscle voltage-gated sodium channel gene (SCN4A) has expanded with advancements in genetic testing. Autosomal dominant SCN4A mutations were first linked to hyperkalemic periodic paralysis, then subsequently included paramyotonia congenita, several variants of myotonia, and finally hypokalemic periodic paralysis. Biallelic recessive mutations were later identified in myasthenic myopathy and in infants showing a severe congenital myopathy with hypotonia. We report a patient with a pathogenic de novo SCN4A variant, c.2386C>G p.L769V at a highly conserved leucine. The phenotype was manifest at birth with arthrogryposis multiplex congenita, severe episodes of bronchospasm that responded immediately to carbamazepine therapy, and electromyographic evidence of widespread myotonia. Another de novo case of p.L769V has been reported with hip dysplasia, scoliosis, myopathy, and later paramyotonia. Expression studies of L796V mutant channels showed predominantly gain-of-function changes, that included defects of slow inactivation. Computer simulations of muscle excitability reveal a strong predisposition to myotonia with exceptionally prolonged bursts of discharges, when the L796V defects are included. We propose L769V is a pathogenic variant, that along with other cases in the literature, defines a new dominant SCN4A disorder of myotonic myopathy with secondary congenital joint and skeletal involvement.

Biallelic Mutations in LRRC56, Encoding a Protein Associated with Intraflagellar Transport, Cause Mucociliary Clearance and Laterality Defects.

Primary defects in motile cilia result in dysfunction of the apparatus responsible for generating fluid flows. Defects in these mechanisms underlie disorders characterized by poor mucus clearance, resulting in susceptibility to chronic recurrent respiratory infections, often associated with infertility; laterality defects occur in about 50% of such individuals. Here we report biallelic variants in LRRC56 (known as oda8 in Chlamydomonas) identified in three unrelated families. The phenotype comprises laterality defects and chronic pulmonary infections. High-speed video microscopy of cultured epithelial cells from an affected individual showed severely dyskinetic cilia but no obvious ultra-structural abnormalities on routine transmission electron microscopy (TEM). Further investigation revealed that LRRC56 interacts with the intraflagellar transport (IFT) protein IFT88. The link with IFT was interrogated in Trypanosoma brucei. In this protist, LRRC56 is recruited to the cilium during axoneme construction, where it co-localizes with IFT trains and is required for the addition of dynein arms to the distal end of the flagellum. In T. brucei carrying LRRC56-null mutations, or a variant resulting in the p.Leu259Pro substitution corresponding to the p.Leu140Pro variant seen in one of the affected families, we observed abnormal ciliary beat patterns and an absence of outer dynein arms restricted to the distal portion of the axoneme. Together, our findings confirm that deleterious variants in LRRC56 result in a human disease and suggest that this protein has a likely role in dynein transport during cilia assembly that is evolutionarily important for cilia motility.

Effects of bacterial physiological states and bacterial species on host-microbe interactions.

This study investigated how the physiological states of Aggregatibacter actinomycetemcomitans (Aa) and Streptococcus mitis affect their intracellular invasion capabilities and the resulting host cell responses. The physiological states included two forms of planktonic states, floating or sedimented (by centrifugation) and the biofilm state (with centrifugation). Confluent epithelial Ca9-22 cells were challenged with floating or sedimented planktonic cultures, or with 24-h biofilms for 3 h. The results show that intracellular invasion efficiencies were clearly affected by the bacterial physiological states. For both bacterial species, the sedimented-cells displayed 2-10 times higher invasion efficiency than the floating-cells (p < 0.05). The invasion efficiency of Aa biofilms was three fold lower than sedimented cells, whereas those of S. mitis biofilms were similar to sedimented cells. Unlike invasion, the metabolic activities of Ca9-22 were unaffected by different bacterial physiological states. However, Aa biofilms induced higher IL-1ß expression than planktonic cultures. In conclusion, different bacterial physiological states can affect the outcomes of (in vitro) host-microbe interaction in different ways.

A novel multisystem disease associated with recessive mutations in the tyrosyl-tRNA synthetase (YARS) gene.

Aminoacyl-tRNA synthetases (ARSs) are a group of ubiquitously expressed enzymes that are best known for their function in the first step of protein translation but have been increasingly associated with secondary functions including transcription and translation control and extracellular signaling. Mutations in numerous ARSs have been linked to a growing number of both autosomal dominant and autosomal recessive human diseases. The tyrosyl-tRNA synthetase (YARS) links the amino acid tyrosine to its cognate tRNA. We report two siblings who presented with failure to thrive (FTT), hypertriglyceridemia, developmental delay, liver dysfunction, lung cysts, and abnormal subcortical white matter. Using exome sequencing the siblings were found to harbor bi-allelic pathogenic-appearing variants within the YARS gene (NM_003680.3):c.638C>T p.(Pro213Leu) and c.1573G>A p.(Gly525Arg). These YARS variants occur in the catalytic domain and the C-terminal domain, respectively. Mutations in YARS have been previously associated with an autosomal dominant form of Charcot-Marie-Tooth (CMT); our findings suggest the disease spectrum associated with YARS dysregulation is broader than peripheral neuropathy. © 2016 Wiley Periodicals, Inc.

Epithelial cell detachment by Porphyromonas gingivalis biofilm and planktonic cultures.

Porphyromonas gingivalis is present as a biofilm at the sites of periodontal infections. The detachment of gingival epithelial cells induced by P. gingivalis biofilms was examined using planktonic cultures as a comparison. Exponentially grown planktonic cultures or 40-h biofilms were co-incubated with epithelial cells in a 24-well plate for 4 h. Epithelial cell detachment was assessed using imaging. The activity of arginine-gingipain (Rgp) and gene expression profiles of P. gingivalis cultures were examined using a gingipain assay and quantitative PCR, respectively. P. gingivalis biofilms induced significantly higher cell detachment and displayed higher Rgp activity compared to the planktonic cultures. The genes involved in gingipain post-translational modification, but not rgp genes, were significantly up-regulated in P. gingivalis biofilms. The results underline the importance of including biofilms in the study of bacterial and host cell interactions.

Mandibulofacial Dysostosis with Microcephaly: Mutation and Database Update.

Mandibulofacial dysostosis with microcephaly (MFDM) is a multiple malformation syndrome comprising microcephaly, craniofacial anomalies, hearing loss, dysmorphic features, and, in some cases, esophageal atresia. Haploinsufficiency of a spliceosomal GTPase, U5-116 kDa/EFTUD2, is responsible. Here, we review the molecular basis of MFDM in the 69 individuals described to date, and report mutations in 38 new individuals, bringing the total number of reported individuals to 107 individuals from 94 kindreds. Pathogenic EFTUD2 variants comprise 76 distinct mutations and seven microdeletions. Among point mutations, missense substitutions are infrequent (14 out of 76; 18%) relative to stop-gain (29 out of 76; 38%), and splicing (33 out of 76; 43%) mutations. Where known, mutation origin was de novo in 48 out of 64 individuals (75%), dominantly inherited in 12 out of 64 (19%), and due to proven germline mosaicism in four out of 64 (6%). Highly penetrant clinical features include, microcephaly, first and second arch craniofacial malformations, and hearing loss; esophageal atresia is present in an estimated ∼27%. Microcephaly is virtually universal in childhood, with some adults exhibiting late "catch-up" growth and normocephaly at maturity. Occasionally reported anomalies, include vestibular and ossicular malformations, reduced mouth opening, atrophy of cerebral white matter, structural brain malformations, and epibulbar dermoid. All reported EFTUD2 mutations can be found in the EFTUD2 mutation database (http://databases.lovd.nl/shared/genes/EFTUD2).

Gold nanorods as a theranostic platform for in vitro and in vivo imaging and photothermal therapy of inflammatory macrophages.

Inflammatory macrophages play pivotal roles in the development of atherosclerosis. Theranostics, a promising approach for local imaging and photothermal therapy of inflammatory macrophages, has drawn increasing attention in biomedical research. In this study, gold nanorods (Au NRs) were synthesized, and their in vitro photothermal effects on the macrophage cell line (Ana-1 cells) under 808 nm near infrared reflection (NIR) were investigated by the CCK8 assay, calcein AM/PI staining, flow cytometry, transmission electron microscopy (TEM), silver staining and in vitro micro-computed tomography (CT) imaging. These Au NRs were then applied to an apolipoprotein E knockout (Apo E) mouse model to evaluate their effects on in vivo CT imaging and their effectiveness as for the subsequent photothermal therapy of macrophages in femoral artery restenosis under 808 nm laser irradiation. In vitro photothermal ablation treatment using Au NRs exhibited a significant cell-killing efficacy of macrophages, even at relatively low concentrations of Au NRs and low NIR powers. In addition, the in vivo results demonstrated that the Au NRs are effective for in vivo imaging and photothermal therapy of inflammatory macrophages in femoral artery restenosis. This study shows that Au nanorods are a promising theranostic platform for the diagnosis and photothermal therapy of inflammation-associated diseases.

An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes.

Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.

Periostin enhances adipose-derived stem cell adhesion, migration, and therapeutic efficiency in Apo E deficient mice with hind limb ischemia.

INTRODUCTION: Therapeutic angiogenesis by transplantation of autologous/allogeneic adipose-derived stem cells (ADSCs) is a potential approach for severe ischemic diseases. However, poor viability, adhesion, migration and differentiation limit the therapeutic efficiency after the cells were transplanted into the targeted area. Periostin, an extracellular matrix protein, exhibits a critical role in wound repair as well as promotes cell adhesion, survival, and angiogenesis. METHOD: ADSCs were obtained and genetically engineered with periostin gene (P-ADSCs). The viability, proliferation, migration, and apoptosis of P-ADSCs under hypoxia were analyzed. Moreover, P-ADSCs were implanted into Apo E deficient mice with hind limb ischemia. The Laser Doppler perfusion index, immunofluorescence, and histological pathology assay were tested to evaluate the therapeutic effects. The associated molecular mechanism of periostin on the proliferation, adhesion, migration, and differentiation of ADSCs was also analyzed. RESULTS: The in vitro studies have shown that periostin-transfected ADSCs (P-ADSCs) promoted viability, proliferation, and migration of ADSCs. Apoptosis of ADSCs was inhibited under hypoxic conditions. The Laser Doppler perfusion index was significantly higher in the P-ADSCs group compared with that in the ADSC and control groups after 4 weeks. Immunofluorescence and histological pathology assay showed that the P-ADSCs were in and around the ischemic sites, and some cells differentiated into capillaries and endothelium. Microvessel densities were significantly improved in P-ADSCs group compared with those in the control group. The molecular mechanisms that provide the beneficial effects of periostin were connected with the upregulated expression of integrinß1/FAK/PI3K/Akt/eNOS signal pathway and the increased secretion of growth factors. CONCLUSION: Overexpression of periostin by gene transfection on ADSCs promotes survival, migration, and therapeutic efficiency, which will bring new insights into the treatment of critical limb ischemia.

An effective approach to reduce inflammation and stenosis in carotid artery: polypyrrole nanoparticle-based photothermal therapy.

Photothermal therapy (PTT), as a promising treatment for tumours, has rarely been reported for application in artery restenosis, which is a common complication of endovascular management due to enduring chronic inflammation and abnormal cell proliferation. In our study, biodegradable polypyrrole nanoparticles (PPy-NPs) were synthesized and characterized, including their size distribution, UV-vis-NIR absorbance, molar extinction coefficients, and photothermal properties. We then verified that PPy-NP incubation followed by 915 nm near-infrared (NIR) laser irradiation could effectively ablate inflammatory macrophages in vitro, leading to significant cell apoptosis and cell death. Further, it was found that a combination of local PPy-NP injection with 915 nm NIR laser irradiation could significantly alleviate arterial inflammation by eliminating infiltrating macrophages and further ameliorating artery stenosis in an ApoE(-/-) mouse model, without showing any obvious toxic side effects. Thus, we propose that PTT based on PPy-NPs as photothermal agents and a 915 nm NIR laser as a power source can serve as a new effective treatment for reducing inflammation and stenosis formation in inflamed arteries after endovascular management.

LIMS2 mutations are associated with a novel muscular dystrophy, severe cardiomyopathy and triangular tongues.

Limb girdle muscular dystrophy (LGMD) is a heterogeneous group of genetic disorders leading to progressive muscle degeneration and often associated with cardiac complications. We present two adult siblings with childhood-onset of weakness progressing to a severe quadriparesis with the additional features of triangular tongues and biventricular cardiac dysfunction. Whole exome sequencing identified compound heterozygous missense mutations that are predicted to be pathogenic in LIMS2. Biopsy of skeletal muscle demonstrated disrupted immunostaining of LIMS2. This is the first report of mutations in LIMS2 and resulting disruption of the integrin linked kinase (ILK)-LIMS-parvin complex associated with LGMD.

Noninvasive detection of macrophages in atherosclerotic lesions by computed tomography enhanced with PEGylated gold nanoparticles.

Macrophages are becoming increasingly significant in the progression of atherosclerosis (AS). Molecular imaging of macrophages may improve the detection and characterization of AS. In this study, dendrimer-entrapped gold nanoparticles (Au DENPs) with polyethylene glycol (PEG) and fluorescein isothiocyanate (FI) coatings were designed, tested, and applied as contrast agents for the enhanced computed tomography (CT) imaging of macrophages in atherosclerotic lesions. Cell counting kit-8 assay, fluorescence microscopy, silver staining, and transmission electron microscopy revealed that the FI-functionalized Au DENPs are noncytotoxic at high concentrations (3.0 µM) and can be efficiently taken up by murine macrophages in vitro. These nanoparticles were administered to apolipoprotein E knockout mice as AS models, which demonstrated that the macrophage burden in atherosclerotic areas can be tracked noninvasively and dynamically three-dimensionally in live animals using micro-CT. Our findings suggest that the designed PEGylated gold nanoparticles are promising biocompatible nanoprobes for the CT imaging of macrophages in atherosclerotic lesions and will provide new insights into the pathophysiology of AS and other concerned inflammatory diseases.

An in vitro study on the effect of free amino acids alone or in combination with nisin on biofilms as well as on planktonic bacteria of Streptococcus mutans.

Free D-amino acids (D-AAs) are one of the most striking features of the peptidoglycan composition in bacteria and play a key role in regulating and disassembling bacterial biofilms. Previous studies have indicated that the antimicrobial peptide nisin can inhibit the growth of the cariogenic bacteria Streptococcus mutans. The present study investigated the effect of free amino acids either alone or in combination with nisin on biofilm and on planktonic S. mutans bacteria. The results of the MIC and MBC analyses showed that D-cysteine (Cys), D- or L-aspartic acid (Asp), and D- or L-glutamic acid (Glu) significantly improve the antibacterial activity of nisin against S. mutans and that the mixture of D-Cys, D-Asp, and D-Glu (3D-AAs) and the mixture of L-Cys, L-Asp, and L-Glu (3L-AAs) at a concentration of 40 mM can prevent S. mutans growth. Crystal violet staining showed that the D- or L-enantiomers of Cys, Asp, and Glu at a concentration of 40 mM can inhibit the formation of S. mutans biofilms, and their mixture generated a stronger inhibition than the components alone. Furthermore, the mixture of the three D-AAs or L-AAs may improve the antibacterial activity of nisin against S. mutans biofilms. This study underscores the potential of free amino acids for the enhancement of the antibacterial activity of nisin and the inhibition of the cariogenic bacteria S. mutans and biofilms.

Identification of novel mutations confirms PDE4D as a major gene causing acrodysostosis.

Acrodysostosis is characterized by nasal hypoplasia, peripheral dysostosis, variable short stature, and intellectual impairment. Recently, mutations in PRKAR1A were reported in patients with acrodysostosis and hormone resistance. Subsequently, mutations in a phosphodiesterase gene (PDE4D) were identified in seven sporadic cases. We sequenced PDE4D in seven acrodysostosis patients from five families. Missense mutations were identified in all cases. Families showed de novo inheritance except one family with three affected children whose father was subsequently found to have subtle features of acrodysostosis. There were no recurrent mutations. Short stature and endocrine resistance are rare in this series; however, cognitive involvement and obesity were frequent. This last finding is relevant given PDE4D is insulin responsive and potentially involved in lipolysis. PDE4D encodes a cyclic AMP regulator and places PDE4D-related acrodysostosis within the same family of diseases as pseudohypoparathyroidism, pseudopseudohypoparathyroidism, PRKAR1A-related acrodysostosis and brachydactyly-mental retardation syndrome; all characterized by cognitive impairment and short distal extremities.

TMEM237 is mutated in individuals with a Joubert syndrome related disorder and expands the role of the TMEM family at the ciliary transition zone.

Joubert syndrome related disorders (JSRDs) have broad but variable phenotypic overlap with other ciliopathies. The molecular etiology of this overlap is unclear but probably arises from disrupting common functional module components within primary cilia. To identify additional module elements associated with JSRDs, we performed homozygosity mapping followed by next-generation sequencing (NGS) and uncovered mutations in TMEM237 (previously known as ALS2CR4). We show that loss of the mammalian TMEM237, which localizes to the ciliary transition zone (TZ), results in defective ciliogenesis and deregulation of Wnt signaling. Furthermore, disruption of Danio rerio (zebrafish) tmem237 expression produces gastrulation defects consistent with ciliary dysfunction, and Caenorhabditis elegans jbts-14 genetically interacts with nphp-4, encoding another TZ protein, to control basal body-TZ anchoring to the membrane and ciliogenesis. Both mammalian and C. elegans TMEM237/JBTS-14 require RPGRIP1L/MKS5 for proper TZ localization, and we demonstrate additional functional interactions between C. elegans JBTS-14 and MKS-2/TMEM216, MKSR-1/B9D1, and MKSR-2/B9D2. Collectively, our findings integrate TMEM237/JBTS-14 in a complex interaction network of TZ-associated proteins and reveal a growing contribution of a TZ functional module to the spectrum of ciliopathy phenotypes.

A complex regulatory network of transcription factors critical for ocular development and disease.

The PITX2 'homeobox' and FOXC1 and FOXC2 'forkhead box' transcription factors are critical for eye development and cause human ocular diseases when mutated. We have identified biochemical and genetic links between these transcription factors and a transcriptional regulator protein PRKC apoptosis Wilms' tumor 1 regulator (PAWR) that we propose to functionally connect all these proteins in a common pathway critically involved in eye development. We discovered all binary physical interactions between FOXC1, PITX2, FOXC2 and PAWR. Importantly, PAWR modulates the abilities of PITX2, FOXC1 and FOXC2 to activate their genetic targets. Together with either FOXC1 or FOXC2, PAWR increases PITX2 activity. PAWR reduces PITX2 activity in the absence of FOXC1 or FOXC2. At the same time, PAWR also exerts different regulatory effects on different FOXC target sites. Furthermore, morpholino knockdown of pitx2, foxc1 and pawr in zebrafish indicate that PAWR, FOXC1 and PITX2 genetically interact, and are in the same developmental pathway. These data for the first time tie PITX2, FOXC1, FOXC2 and PAWR into a common regulatory pathway. We have therefore identified a functional link between three transcription factors, modulated by PAWR, which we propose underlies the similar ocular phenotypes and glaucoma pathology caused by mutations of these genes.