Treatment-resistant ophthalmoplegia in Myasthenia gravis: extraocular muscle pathology, the role of TGFβ1 and the derivation of induced pluripotency towards 'disease-in-a-dish' modeling

Myasthenia gravis (MG) is an autoimmune disease in which pathogenic antibodies target specific neuromuscular junction proteins, most frequently acetylcholine receptors (AChR). Among those without detectable AChR-antibodies, a subgroup of patients has antibodies directed against muscle-specific tyrosine kinase (MuSK). In MG the pathogenic antibodies result in failure of neuromuscular transmission with consequent fatiguable skeletal muscle weakness. MG frequently affects the extraocular muscles (EOMs) early in the course of the disease, resulting in diplopia and ptosis, which is usually reversible with treatment. A treatment-resistant ophthalmoplegia and ptosis occurs as a complication of MG in a distinct subset of cases referred to as OP-MG. The EOMs are highly specialised muscle tissue with a unique physiological and immunological microenvironment with a large satellite cell niche, a distinct muscle fibroblast population, different transcriptional and cellular signaling pathways and fewer intrinsic complement regulatory proteins to protect them against antibody- activated complement-mediated damage. We hypothesised that in OP-MG, there is a differential response of the EOMs to the underlying MG disease process(es) on a genetic and molecular level, resulting in abnormal myofibre homeostasis. We aimed to report descriptive clinical-pathological data pertaining to EOM function and histopathological and ultrastructural EOM tissue analysis of a patient with OP- MG versus that of a non-MG control (both consented to EOM donation at ocular realignment surgery). EOM tissue from an OP-MG individual with AChR- and MuSK- antibody negative MG, demonstrated predominantly myopathic pathology and ultrastructural evidence of mitochondrial stress. The OP-MG EOM findings differ from the control EOM, which showed normal muscle histopathology in a patient undergoing strabismus surgery for a sensory exotropia in a non-seeing eye (loss of retinal stimulus for fusion) and a similar duration of deviation. These OP-MG findings appear to better correlate with previously reported histology/ultrastructure in limb muscle in MuSK-positive MG rather than AChR-positive MG. We next focussed on transforming growth factor beta-1 (TGFβ1) as a critical cytokine involved in muscle repair. An auto-induction pathway in muscle allows TGFβ1 expression to influence the transdifferentiation of satellite cells into myofibroblasts or myoblasts. In orbital fibroblasts, TGFβ1 has also been shown to upregulate decay accelerating factor (DAF), a complement regulatory protein expressed at lower levels in EOMs than other muscles, which should protect against complement-mediated injury. We established OP-MG and control-MG phenotype-specific dermal fibroblast cell lines and performed immunoblotting to evaluate TGFβ1-induced Smad3 phosphorylation and Daf expression in mouse myotubes. We demonstrated repression of phosphorylated-Smad3, a marker of the canonical TGFβ1 pathway, in OP-MG versus control MG fibroblasts after treatment with TGFβ1. We also demonstrated that TGFβ1 significantly upregulates Daf expression levels in mouse myoblasts. Taken together, these results suggest that OP-MG fibroblasts (and possibly myofibroblasts) are likely to be more susceptible to complement-mediated damage and abnormal myofibrogenesis due to their altered response to TGFβ1 stimulation and secondary DAF upregulation. Finally we investigated the feasability of establishing an in vitro disease model for MG or OP-MG by reprogramming dermal fibroblasts into disease phenotype-specific induced pluripotent stem (iPS) cells. We successfully generated and characterised iPS cells for one individual. However, this process was very labour-intensive, cost-inefficient and time-consuming, taking approximately four months to establish pluripotency in a single patient and thereby limited its further application(s). In conclusion, the EOM ultrastructural findings of an OP-MG case are novel and show similar findings to those described in limb skeletal muscle of MuSK-positive MG patients. The TGFβ1 pathway appears to be differentially regulated in OP-MG compared to control-MG cases and this may impact DAF upregulation in the EOMs in MG patients. Finally, our group is exploring an alternative method of establishing a 'disease-in-a-dish' model that is more cost-effective and practically feasible than the iPS cell route

Extraocular Muscle Findings in Myasthenia Gravis Associated Treatment-Resistant Ophthalmoplegia

We report the histopathological and ultrastructural tissue analysis of extraocular muscle (EOM) obtained from a patient with seronegative myasthenia gravis (MG) with treatment-resistant ophthalmoplegia for 3.5 years. The EOM demonstrated predominantly myopathic features and ultrastructural evidence of mitochondrial dysfunction, but the most striking features were increased endomysial collagen and adipocyte replacement of muscle fibers. By contrast, control EOM from a patient undergoing strabismus surgery for a sensory exotropia in a nonseeing eye and a similar duration of deviation, showed normal muscle histology. Although the histopathological and ultrastructural findings largely resemble those of limb muscle in MG, the abundant endomysial collagen may be nonspecific and secondary to poor force generation as a result of chronic ophthalmoplegia

Genetic variants and cellular stressors associated with exfoliation syndrome modulate promoter activity of a lncRNA within the LOXL1 locus

Exfoliation syndrome (XFS) is a common, age-related, systemic fibrillinopathy. It greatly increases risk of exfoliation glaucoma (XFG), a major worldwide cause of irreversible blindness. Coding variants in the lysyl oxidase-like 1 (LOXL1) gene are strongly associated with XFS in all studied populations, but a functional role for these variants has not been established. To identify additional candidate functional variants, we sequenced the entire LOXL1 genomic locus (∼40 kb) in 50 indigenous, black South African XFS cases and 50 matched controls. The variants with the strongest evidence of association were located in a well-defined 7-kb region bounded by the 3'-end of exon 1 and the adjacent region of intron 1 of LOXL1. We replicated this finding in US Caucasian (91 cases/1031 controls), German (771 cases/1365 controls) and Japanese (1484 cases/1188 controls) populations. The region of peak association lies upstream of LOXL1-AS1, a long non-coding RNA (lncRNA) encoded on the opposite strand of LOXL1. We show that this region contains a promoter and, importantly, that the strongly associated XFS risk alleles in the South African population are functional variants that significantly modulate the activity of this promoter. LOXL1-AS1 expression is also significantly altered in response to oxidative stress in human lens epithelial cells and in response to cyclic mechanical stress in human Schlemm's canal endothelial cells. Taken together, these findings support a functional role for the LOXL1-AS1 lncRNA in cellular stress response and suggest that dysregulation of its expression by genetic risk variants plays a key role in XFS pathogenesis

A common variant mapping to CACNA1A is associated with susceptibility to exfoliation syndrome

Exfoliation syndrome (XFS) is the most common recognizable cause of open-angle glaucoma worldwide. To better understand the etiology of XFS, we conducted a genome-wide association study (GWAS) of 1,484 cases and 1,188 controls from Japan and followed up the most significant findings in a further 6,901 cases and 20,727 controls from 17 countries across 6 continents. We discovered a genome-wide significant association between a new locus (CACNA1A rs4926244) and increased susceptibility to XFS (odds ratio (OR) = 1.16, P = 3.36 × 10(-11)). Although we also confirmed overwhelming association at the LOXL1 locus, the key SNP marker (LOXL1 rs4886776) demonstrated allelic reversal depending on the ancestry group (Japanese: OR(A allele) = 9.87, P = 2.13 × 10(-217); non-Japanese: OR(A allele) = 0.49, P = 2.35 × 10(-31)). Our findings represent the first genetic locus outside of LOXL1 surpassing genome-wide significance for XFS and provide insight into the biology and pathogenesis of the disease

A common variant mapping to <i>CACNA1A </i>is associated with susceptibility to exfoliation syndrome

Author manuscript available from PMC http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4605818/Exfoliation syndrome (XFS) is the most common recognizable cause of open-angle glaucoma worldwide. To better understand the etiology of XFS, we conducted a genome-wide association study (GWAS) of 1,484 cases and 1,188 controls from Japan and followed up the most significant findings in a further 6,901 cases and 20,727 controls from 17 countries across 6 continents. We discovered a genome-wide significant association between a new locus (CACNA1A rs4926244) and increased susceptibility to XFS (odds ratio (OR) = 1.16, P = 3.36 × 10−11). Although we also confirmed overwhelming association at the LOXL1 locus, the key SNP marker (LOXL1 rs4886776) demonstrated allelic reversal depending on the ancestry group (Japanese: ORA allele = 9.87, P = 2.13 × 10−217; non-Japanese: ORA allele = 0.49, P = 2.35 × 10−31). Our findings represent the first genetic locus outside of LOXL1 surpassing genome-wide significance for XFS and provide insight into the biology and pathogenesis of the disease

Genetic Association Study Of Exfoliation Syndrome Identifies A Protective Rare Variant At Loxl1 And Five New Susceptibility Loci

Exfoliation syndrome (XFS) is the most common known risk factor for secondary glaucoma and a major cause of blindness worldwide. Variants in two genes, LOXL1 and CACNA1A, have previously been associated with XFS. To further elucidate the genetic basis of XFS, we collected a global sample of XFS cases to refine the association at LOXL1, which previously showed inconsistent results across populations, and to identify new variants associated with XFS. We identified a rare protective allele at LOXL1 (p.Phe407, odds ratio (OR) = 25, P = 2.9 x 10(-14)) through deep resequencing of XFS cases and controls from nine countries. A genome-wide association study (GWAS) of XFS cases and controls from 24 countries followed by replication in 18 countries identified seven genome-wide significant loci (P < 5 x 10(-8)). We identified association signals at 13q12 (POMP), 11q23.3 (TMEM136), 6p21 (AGPAT1), 3p24 (RBMS3) and 5q23 (near SEMA6A). These findings provide biological insights into the pathology of XFS and highlight a potential role for naturally occurring rare LOXL1 variants in disease biology.Wo