Disrupting the Repeat Domain of Premelanosome Protein (PMEL) Produces Dysamyloidosis and Dystrophic Ocular Pigment Reflective of Pigmentary Glaucoma.

Pigmentary glaucoma has recently been associated with missense mutations in PMEL that are dominantly inherited and enriched in the protein's fascinating repeat domain. PMEL pathobiology is intriguing because PMEL forms functional amyloid in healthy eyes, and this PMEL amyloid acts to scaffold melanin deposition. This is an informative contradistinction to prominent neurodegenerative diseases where amyloid formation is neurotoxic and mutations cause a toxic gain of function called "amyloidosis". Preclinical animal models have failed to model this PMEL "dysamyloidosis" pathomechanism and instead cause recessively inherited ocular pigment defects via PMEL loss of function; they have not addressed the consequences of disrupting PMEL's repetitive region. Here, we use CRISPR to engineer a small in-frame mutation in the zebrafish homolog of PMEL that is predicted to subtly disrupt the protein's repetitive region. Homozygous mutant larvae displayed pigmentation phenotypes and altered eye morphogenesis similar to presumptive null larvae. Heterozygous mutants had disrupted eye morphogenesis and disrupted pigment deposition in their retinal melanosomes. The deficits in the pigment deposition of these young adult fish were not accompanied by any detectable glaucomatous changes in intraocular pressure or retinal morphology. Overall, the data provide important in vivo validation that subtle PMEL mutations can cause a dominantly inherited pigment pathology that aligns with the inheritance of pigmentary glaucoma patient pedigrees. These in vivo observations help to resolve controversy regarding the necessity of PMEL's repeat domain in pigmentation. The data foster an ongoing interest in an antithetical dysamyloidosis mechanism that, akin to the amyloidosis of devastating dementias, manifests as a slow progressive neurodegenerative disease.

Non-Synonymous variants in premelanosome protein (PMEL) cause ocular pigment dispersion and pigmentary glaucoma.

Pigmentary glaucoma (PG) is a common glaucoma subtype that results from release of pigment from the iris, called pigment dispersion syndrome (PDS), and its deposition throughout the anterior chamber of the eye. Although PG has a substantial heritable component, no causative genes have yet been identified. We used whole exome sequencing of two independent pedigrees to identify two premelanosome protein (PMEL) variants associated with heritable PDS/PG. PMEL encodes a key component of the melanosome, the organelle essential for melanin synthesis, storage and transport. Targeted screening of PMEL in three independent cohorts (n = 394) identified seven additional PDS/PG-associated non-synonymous variants. Five of the nine variants exhibited defective processing of the PMEL protein. In addition, analysis of PDS/PG-associated PMEL variants expressed in HeLa cells revealed structural changes to pseudomelanosomes indicating altered amyloid fibril formation in five of the nine variants. Introduction of 11-base pair deletions to the homologous pmela in zebrafish by the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 method caused profound pigmentation defects and enlarged anterior segments in the eye, further supporting PMEL's role in ocular pigmentation and function. Taken together, these data support a model in which missense PMEL variants represent dominant negative mutations that impair the ability of PMEL to form functional amyloid fibrils. While PMEL mutations have previously been shown to cause pigmentation and ocular defects in animals, this research is the first report of mutations in PMEL causing human disease.

FOXF2 is required for cochlear development in humans and mice.

Molecular mechanisms governing the development of the human cochlea remain largely unknown. Through genome sequencing, we identified a homozygous FOXF2 variant c.325A>T (p.I109F) in a child with profound sensorineural hearing loss (SNHL) associated with incomplete partition type I anomaly of the cochlea. This variant is not found in public databases or in over 1000 ethnicity-matched control individuals. I109 is a highly conserved residue in the forkhead box (Fox) domain of FOXF2, a member of the Fox protein family of transcription factors that regulate the expression of genes involved in embryogenic development as well as adult life. Our in vitro studies show that the half-life of mutant FOXF2 is reduced compared to that of wild type. Foxf2 is expressed in the cochlea of developing and adult mice. The mouse knockout of Foxf2 shows shortened and malformed cochleae, in addition to altered shape of hair cells with innervation and planar cell polarity defects. Expressions of Eya1 and Pax3, genes essential for cochlear development, are reduced in the cochleae of Foxf2 knockout mice. We conclude that FOXF2 plays a major role in cochlear development and its dysfunction leads to SNHL and developmental anomalies of the cochlea in humans and mice.

Functional Domains and Evolutionary History of the PMEL and GPNMB Family Proteins.

The ancient paralogs premelanosome protein (PMEL) and glycoprotein nonmetastatic melanoma protein B (GPNMB) have independently emerged as intriguing disease loci in recent years. Both proteins possess common functional domains and variants that cause a shared spectrum of overlapping phenotypes and disease associations: melanin-based pigmentation, cancer, neurodegenerative disease and glaucoma. Surprisingly, these proteins have yet to be shown to physically or genetically interact within the same cellular pathway. This juxtaposition inspired us to compare and contrast this family across a breadth of species to better understand the divergent evolutionary trajectories of two related, but distinct, genes. In this study, we investigated the evolutionary history of PMEL and GPNMB in clade-representative species and identified TMEM130 as the most ancient paralog of the family. By curating the functional domains in each paralog, we identified many commonalities dating back to the emergence of the gene family in basal metazoans. PMEL and GPNMB have gained functional domains since their divergence from TMEM130, including the core amyloid fragment (CAF) that is critical for the amyloid potential of PMEL. Additionally, the PMEL gene has acquired the enigmatic repeat domain (RPT), composed of a variable number of imperfect tandem repeats; this domain acts in an accessory role to control amyloid formation. Our analyses revealed the vast variability in sequence, length and repeat number in homologous RPT domains between craniates, even within the same taxonomic class. We hope that these analyses inspire further investigation into a gene family that is remarkable from the evolutionary, pathological and cell biology perspectives.

Mutations of conserved non-coding elements of PITX2 in patients with ocular dysgenesis and developmental glaucoma.

Mutations in FOXC1 and PITX2 constitute the most common causes of ocular anterior segment dysgenesis (ASD), and confer a high risk for secondary glaucoma. The genetic causes underlying ASD in approximately half of patients remain unknown, despite many of them being screened by whole exome sequencing. Here, we performed whole genome sequencing on DNA from two affected individuals from a family with dominantly inherited ASD and glaucoma to identify a 748-kb deletion in a gene desert that contains conserved putative PITX2 regulatory elements. We used CRISPR/Cas9 to delete the orthologous region in zebrafish in order to test the pathogenicity of this structural variant. Deletion in zebrafish reduced pitx2 expression during development and resulted in shallow anterior chambers. We screened additional patients for copy number variation of the putative regulatory elements and found an overlapping deletion in a second family and in a potentially-ancestrally-related index patient with ASD and glaucoma. These data suggest that mutations affecting conserved non-coding elements of PITX2 may constitute an important class of mutations in patients with ASD for whom the molecular cause of their disease have not yet been identified. Improved functional annotation of the human genome and transition to sequencing of patient genomes instead of exomes will be required before the magnitude of this class of mutations is fully understood.

Comparison of Bioinformatics Prediction, Molecular Modeling, and Functional Analyses of FOXC1 Mutations in Patients with Axenfeld-Rieger Syndrome.

Mutations in the forkhead box C1 gene (FOXC1) cause Axenfeld-Rieger syndrome (ARS). Here, we investigated the effect of four ARS missense variants on FOXC1 structure and function, and examined the predictive value of four in silico programs for all 31 FOXC1 missense variants identified to date. Molecular modeling of the FOXC1 forkhead domain predicts that c.402G> A (p.C135Y) alters FOXC1's structure. In contrast, c.378A> G (p.H128R) and c.481A> G (p.M161V) are not predicted to change FOXC1's structure. Functional analysis indicates that p.H128R reduced DNA binding, transactivation, nuclear localization, and has a longer protein half-life than normal. p.C135Y significantly disrupts FOXC1's DNA binding, transactivation, and nuclear localization. p.M161V reduces transactivation capacity without affecting other FOXC1 functions. C.1103C> A (p.T368N) is indistinguishable from wild-type FOXC1 in all tests, consistent with being a rare benign variant. Comparison of these four variants, plus 18 previously characterized FOXC1 missense variants, with predictions from four commonly used in silico bioinformatics programs indicated that sorting intolerant from tolerant (SIFT), polymorphism phenotyping (PolyPhen-2), and MutPred can sensitively identify as pathogenic only FOXC1 mutations with significant functional defects. This information was used to predict, as disease-causing, nine additional FOXC1 missense variations. Importantly, our results indicate SIFT, PolyPhen-2, and MutPred can reliably be used to predict missense variant pathogenicity for forkhead transcription factors.

Fractalkine enhances oligodendrocyte regeneration and remyelination in a demyelination mouse model.

Demyelinating disorders of the central nervous system (CNS) occur when myelin and oligodendrocytes are damaged or lost. Remyelination and regeneration of oligodendrocytes can be achieved from endogenous oligodendrocyte precursor cells (OPCs) that reside in the adult CNS tissue. Using a cuprizone mouse model of demyelination, we show that infusion of fractalkine (CX3CL1) into the demyelinated murine brain increases de novo oligodendrocyte formation and enhances remyelination in the corpus callosum and cortical gray matter. This is achieved by increased OPC proliferation in the cortical gray matter as well as OPC differentiation and attenuation of microglia/macrophage activation both in corpus callosum and cortical gray matter. Finally, we show that activated OPCs and microglia/macrophages express fractalkine receptor CX3CR1 in vivo, and that in OPC-microglia co-cultures fractalkine increases in vitro oligodendrocyte differentiation by modulating both OPC and microglia biology. Our results demonstrate a novel pro-regenerative role of fractalkine in a demyelinating mouse model.

Mutation of the bone morphogenetic protein GDF3 causes ocular and skeletal anomalies.

Ocular mal-development results in heterogeneous and frequently visually disabling phenotypes that include coloboma and microphthalmia. Due to the contribution of bone morphogenetic proteins to such processes, the function of the paralogue Growth Differentiation Factor 3 was investigated. Multiple mis-sense variants were identified in patients with ocular and/or skeletal (Klippel-Feil) anomalies including one individual with heterozygous alterations in GDF3 and GDF6. These variants were characterized, individually and in combination, through integrated biochemical and zebrafish model organism analyses, demonstrating appreciable effects with western blot analyses, luciferase based reporter assays and antisense morpholino inhibition. Notably, inhibition of the zebrafish co-orthologue of GDF3 accurately recapitulates patient phenotypes. By demonstrating the pleiotropic effects of GDF3 mutation, these results extend the contribution of perturbed BMP signaling to human disease and potentially implicate multi-allelic inheritance of BMP variants in developmental disorders.

Yeast two-hybrid analysis of a human trabecular meshwork cDNA library identified EFEMP2 as a novel PITX2 interacting protein.

PURPOSE: Mutations in the homeobox transcription factor paired-like homeodomain transcription factor 2 (PITX2) cause Axenfeld-Reiger syndrome (ARS), which is associated with anterior segment dysgenesis (ASD) and glaucoma. To understand ARS pathogenesis, it is essential to know the normal functions of PITX2 and the proteins with which PITX2 interacts in the eye. Therefore, we used a unique cDNA library that we created from human trabecular meshwork (TM) primary cells to discover PITX2-interacting proteins (PIPs). METHODS: A human TM cDNA library was created from primary cells in the ProQuest Two-Hybrid prey vector: pEXP-AD502. Human PITX2A and PITX2C isoforms were used independently as "bait" to identify novel PIPs. A total of 1.25×106 clones were screened by yeast two-hybrid (Y2H) analyses. PIPs obtained from each Y2H experiment were confirmed by yeast retransformation and mammalian co-immunoprecipitation assays. RESULTS: EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) was identified by both PITX2A and PITX2C isoforms as a novel PIP from Y2H analyses. EFEMP2 is 443 amino acids long with six epidermal growth factor (EGF)-like modules and one fibulin-like module. The PITX2-interaction domain in EFEMP2 lies between the second EGF-like module and the COOH-terminal fibulin-like module. Co-immunoprecipitation assays in COS-7 cells confirmed the interaction between PITX2 and EFEMP2. CONCLUSIONS: We discovered EFEMP2 as a novel PITX2-interacting protein. Further, our cDNA library made from human TM primary cells is a unique and effective resource to identify novel interacting proteins for glaucoma and ASD candidates. This resource could be used both for discovery and validation of interactomes identified from in silico analysis.

Hepatoma Derived Growth Factor Enhances Oligodendrocyte Genesis from Subventricular Zone Precursor Cells.

Oligodendrocytes, the myelinating cells of the central nervous system (CNS), perform vital functions in neural protection and communication, as well as cognition. Enhanced production of oligodendrocytes has been identified as a therapeutic approach for neurodegenerative and neurodevelopmental disorders. In the postnatal brain, oligodendrocytes are generated from the neural stem and precursor cells (NPCs) in the subventricular zone (SVZ) and parenchymal oligodendrocyte precursor cells (OPCs). Here, we demonstrate exogenous Hepatoma Derived Growth Factor (HDGF) enhances oligodendrocyte genesis from murine postnatal SVZ NPCs in vitro without affecting neurogenesis or astrogliogenesis. We further show that this is achieved by increasing proliferation of both NPCs and OPCs, as well as OPC differentiation into oligodendrocytes. In vivo results demonstrate that intracerebroventricular infusion of HDGF leads to increased oligodendrocyte genesis from SVZ NPCs, as well as OPC proliferation. Our results demonstrate a novel role for HDGF in regulating SVZ precursor cell proliferation and oligodendrocyte differentiation.

Agathisflavone Modifies Microglial Activation State and Myelination in Organotypic Cerebellar Slices Culture.

Oligodendrocytes produce the myelin that is critical for rapid neuronal transmission in the central nervous system (CNS). Disruption of myelin has devastating effects on CNS function, as in the demyelinating disease multiple sclerosis (MS). Microglia are the endogenous immune cells of the CNS and play a central role in demyelination and repair. There is a need for new potential therapies that regulate myelination and microglia to promote repair. Agathisflavone (FAB) is a non-toxic flavonoid that is known for its anti-inflammatory and neuroprotective properties. Here, we examined the effects of FAB (5-50 µM) on myelination and microglia in organotypic cerebellar slices prepared from P10-P12 Sox10-EGFP and Plp1-DsRed transgenic mice. Immunofluorescence labeling for myelin basic protein (MBP) and neurofilament (NF) demonstrates that FAB significantly increased the proportion of MBP + /NF + axons but did not affect the overall number of oligodendroglia or axons, or the expression of oligodendroglial proteins CNPase and MBP. FAB is known to be a phytoestrogen, but blockade of α- or ß- estrogen receptors (ER) indicated the myelination promoting effects of FAB were not mediated by ER. Examination of microglial responses by Iba1 immunohistochemistry demonstrated that FAB markedly altered microglial morphology, characterized by smaller somata and reduced branching of their processes, consistent with a decreased state of activation, and increased Iba1 protein expression. The results provide evidence that FAB increases the extent of axonal coverage by MBP immunopositive oligodendroglial processes and has a modulatory effect upon microglial cells, which are important therapeutic strategies in multiple neuropathologies.

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.

FOXQ1 is Differentially Expressed Across Breast Cancer Subtypes with Low Expression Associated with Poor Overall Survival.

PURPOSE: Forkhead box Q1 (FOXQ1) has been shown to contribute to the development and progression of cancers, including ovarian and breast cancer (BC). However, research exploring FOXQ1 expression, copy number variation (CNV), and prognostic value across different BC subtypes is limited. Our purpose was to evaluate FOXQ1 mRNA expression, CNV, and prognostic value across BC subtypes. MATERIALS AND METHODS: We determined FOXQ1 expression and CNV in BC patient tumors using RT-qPCR and qPCR, respectively. We also analyzed FOXQ1 expression and CNV in BC cell lines in the CCLE database using K-means clustering. The prognostic value of FOXQ1 expression in the TCGA-BRCA database was assessed using univariate and multivariate Cox's regression analysis as well as using the online tools OncoLnc, GEPIA, and UALCAN. RESULTS: Our analyses reveal that FOXQ1 mRNA is differentially expressed between different subtypes of BC and is significantly decreased in luminal BC and HER2 patients when compared to normal breast tissue samples. Furthermore, analysis of BC cell lines showed that FOXQ1 mRNA expression was independent of CNV. Moreover, patients with low FOXQ1 mRNA expression had significantly poorer overall survival compared to those with high FOXQ1 mRNA expression. Finally, low FOXQ1 expression had a critical impact on the prognostic values of BC patients and was an independent predictor of overall survival when it was adjusted for BC subtypes and to two other FOX genes, FOXF2 and FOXM1. CONCLUSION: Our study reveals for the first time that FOXQ1 is differentially expressed across BC subtypes and that low expression of FOXQ1 is indicative of poor prognosis in patients with BC.

Fractalkine signaling regulates oligodendroglial cell genesis from SVZ precursor cells.

Neural and oligodendrocyte precursor cells (NPCs and OPCs) in the subventricular zone (SVZ) of the brain contribute to oligodendrogenesis throughout life, in part due to direct regulation by chemokines. The role of the chemokine fractalkine is well established in microglia; however, the effect of fractalkine on SVZ precursor cells is unknown. We show that murine SVZ NPCs and OPCs express the fractalkine receptor (CX3CR1) and bind fractalkine. Exogenous fractalkine directly enhances OPC and oligodendrocyte genesis from SVZ NPCs in vitro. Infusion of fractalkine into the lateral ventricle of adult NPC lineage-tracing mice leads to increased newborn OPC and oligodendrocyte formation in vivo. We also show that OPCs secrete fractalkine and that inhibition of endogenous fractalkine signaling reduces oligodendrocyte formation in vitro. Finally, we show that fractalkine signaling regulates oligodendrogenesis in cerebellar slices ex vivo. In summary, we demonstrate a novel role for fractalkine signaling in regulating oligodendrocyte genesis from postnatal CNS precursor cells.

The Chromatin Regulator Ankrd11 Controls Palate and Cranial Bone Development.

Epigenetic and chromatin regulation of craniofacial development remains poorly understood. Ankyrin Repeat Domain 11 (ANKRD11) is a chromatin regulator that has previously been shown to control neural stem cell fates via modulation of histone acetylation. ANKRD11 gene variants, or microdeletions of the 16q24.3 chromosomal region encompassing the ANKRD11 gene, cause KBG syndrome, a rare autosomal dominant congenital disorder with variable neurodevelopmental and craniofacial involvement. Craniofacial abnormalities include a distinct facial gestalt, delayed bone age, tooth abnormalities, delayed fontanelle closure, and frequently cleft or submucosal palate. Despite this, the dramatic phenotype and precise role of ANKRD11 in embryonic craniofacial development remain unexplored. Quantitative analysis of 3D images of KBG syndromic subjects shows an overall reduction in the size of the middle and lower face. Here, we report that mice with heterozygous deletion of Ankrd11 in neural crest cells (Ankrd11nchet) display a mild midfacial hypoplasia including reduced midfacial width and a persistent open fontanelle, both of which mirror KBG syndrome patient facial phenotypes. Mice with a homozygous Ankrd11 deletion in neural crest cells (Ankrd11ncko) die at birth. They show increased severity of several clinical manifestations described for KBG syndrome, such as cleft palate, retrognathia, midfacial hypoplasia, and reduced calvarial growth. At E14.5, Ankrd11 expression in the craniofacial complex is closely associated with developing bony structures, while expression at birth is markedly decreased. Conditional deletion of Ankrd11 leads to a reduction in ossification of midfacial bones, with several ossification centers failing to expand and/or fuse. Intramembranous bones show features of delayed maturation, with bone remodeling severely curtailed at birth. Palatal shelves remain hypoplastic at all developmental stages, with a local reduction in proliferation at E13.5. Our study identifies Ankrd11 as a critical regulator of intramembranous ossification and palate development and suggests that Ankrd11nchet and Ankrd11ncko mice may serve as pre-clinical models for KBG syndrome in humans.

Regulation of CNS precursor function by neuronal chemokines.

Oligodendrocyte and neural precursor cells (OPCs and NPCs, respectively) in the central nervous system (CNS) have diverse roles in development and homeostasis. During development, precursors build the CNS. In adulthood, they maintain their ability to proliferate and generate differentiated progeny, indicating their tremendous potential to regenerate and repair injured or degenerated CNS. How can we utilize this capability? Cross-talk between neurons and OPCs may hold some clues. Neurons communicate with OPCs via two mechanisms: 1) paracrine secretion of ligands, and 2) neuronal activity and bona fide synapses with OPCs. Intriguingly, OPCs express receptors for chemokines, which are small signalling molecules produced by various cells, including neurons. In addition to inducing chemotaxis, chemokines also regulate cell proliferation, survival and differentiation. In this review, we will summarize the roles of neuronally secreted chemokines and their documented ability to directly regulate the diverse functions of OPCs and NPCs in the developing as well as adult normal and injured CNS. We will focus on the following neuronal chemokines: CCL2, CCL3, CCL20, CCL21, CXCL1, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12 and CX3CL1. We will discuss the implications for neuronal chemokine signalling in OPCs and NPCs not only in developmental myelination and adult CNS regeneration, but also in cognition, behavior, neuroinflammation and neuronal function.

A Novel PITX2c Gain-of-Function Mutation, p.Met207Val, in Patients With Familial Atrial Fibrillation.

Genome-wide studies have associated several genetic variants upstream of PITX2 on chromosome 4q25 with atrial fibrillation (AF), suggesting a potential role of PITX2 in AF. Our study aimed at identifying rare coding variants in PITX2 predisposing to AF. The Polymerase chain reaction sequencing of PITX2c was performed in 60 unrelated patients with idiopathic AF. The p.Met207Val variant was identified in 1 of 60 French patients with early onset AF and in none of 389 French referents. This variant, located in the inhibitory domain 1 distal to the homeodomain, was evaluated by the software MutationTaster as a disease-causing mutation with a probability of 0.999. Reporter gene assays demonstrated that p.Met207Val caused a 3.1-fold increase in transactivation activity of PITX2c in HeLa cells in comparison with its wild-type counterpart. When the variant was coexpressed with wild-type PITX2c in the HL-1 immortalized mouse atrial cell line, this gain-of-function caused an increase in the mRNA level of KCNH2 (2.6-fold), SCN1B (1.9-fold), GJA5 (3.1-fold), GJA1 (2.1-fold), and KCNQ1 in the homozygous form (1.8-fold). These genes encode for the IKr channel α subunit, the ß-1 Na+ channel subunit, connexin 40, connexin 43 and the IKs channel α subunit, respectively. These conditions may contribute to the propensity to AF found in patients carrying the p.Met207Val variant. In conclusion, the present report is the first to associate a gain-of-function mutation of PITX2c with increased vulnerability to AF, therefore, restoration of normal PITX2c function may be a potential therapeutic target in AF patients.

FOXC1 Regulates Expression of Prostaglandin Receptors Leading to an Attenuated Response to Latanoprost.

Purpose: This study examines the effect of FOXC1 on the prostaglandin pathway in order to explore FOXC1's role in the prostaglandin-resistant glaucoma phenotype commonly seen in Axenfeld-Rieger syndrome. Methods: Binding and transcriptional activity of FOXC1 to the gene coding for the EP3 prostaglandin receptor (PTGER3) were evaluated through ChIP-qPCR and luciferase-based assays. Immortalized trabecular meshwork cells (TM1) and HeLa cells had FOXC1 mRNA reduced via siRNA interference. qPCR and Western blot experiments were conducted to examine the changes in prostaglandin receptor expression brought about by lowered FOXC1. TM1 cells were then treated with 10 µM latanoprost acid and/or an siRNA for FOXC1. The expression of fibronectin and matrix metalloproteinase 9 were evaluated via qPCR in each treatment condition. Results: ChIP-qPCR and luciferase experiments confirmed that FOXC1 binds to and activates transcription of the EP3 gene prostaglandin receptor. qPCR and Western experiments in HeLa and TM1 cells showed that FOXC1 siRNA knockdown results in significantly lowered EP3 levels (protein and RNA). In addition, RNA levels of the other prostaglandin receptor genes EP1 (PTGER1), EP2 (PTGER2), EP4 (PTGER4), and FP (PTGFR) were altered when FOXC1 was knocked down in TM1 and HeLa cells. Analysis of fibronectin expression in TM1 cells after treatment with 10 µM latanoprost acid showed a statistically significant increase in expression; this increase was abrogated by cotreatment with a siRNA for FOXC1. Conclusions: We show the abrogation of latanoprost signalling when FOXC1 is knocked down via siRNA in a trabecular meshwork cell line. We propose that the lower levels of active FOXC1 in Axenfeld-Rieger syndrome patients with glaucoma account for the lack of response to prostaglandin-based medications.

Analyses of a novel L130F missense mutation in FOXC1.

OBJECTIVE: To understand how the novel L130F mutation, found in 2 patients with Axenfeld-Rieger syndrome, disrupts function of the forkhead box C1 protein (FOXC1). METHODS: Sequencing DNA from patients with Axenfeld-Rieger syndrome identified a novel missense mutation that results in an L130F substitution in the FOXC1 gene. Site-directed mutagenesis was used to introduce the L130F mutation into the FOXC1 complementary DNA. The level of L130F protein expression was determined by means of immunoblotting. We determined the mutant protein's ability to localize to the nucleus, bind DNA, and transactivate a reporter construct. RESULTS: The FOXC1 L130F mutant protein is expressed at levels similar to those of wild-type FOXC1. The L130F protein, however, migrated at an apparent reduced molecular weight compared with the wild-type protein, suggesting that the mutant and wild-type proteins may be differentially phosphorylated. The L130F protein also had a significantly impaired capacity to localize to the nucleus, bind DNA, and transactivate reporter genes. CONCLUSIONS: The disease-causing L130F mutation further demonstrates that helix 3 of the forkhead domain is important for the FOXC1 protein to properly localize to the nucleus, bind DNA, and activate gene expression. CLINICAL RELEVANCE: The inability of FOXC1 to function owing to the L130F mutation provides further insight into how disruptions in the FOXC1 gene lead to human Axenfeld-Rieger syndrome.