Hans H. Goebel

M2 Polarized Macrophages and Giant Cells Contribute to Myofibrosis in Neuromuscular Sarcoidosis

The etiopathogenesis of sarcoidosis, a systemic granulomatous disease, still remains obscure. A multitude of organs have been described to be affected in systemic sarcoidosis. Skeletal muscles may also be affected, leading to myalgia and weakness. A workup of the specific immune response with emphasis on the macrophage response is provided herein. Affected muscle tissue from seven patients with systemic sarcoidosis was analyzed and compared with that from seven patients with other myopathies containing macrophagocytic infiltration. Monocytes/macrophages and giant cells in granulomas of muscle tissue from patients with sarcoidosis show a status of alternative activation (M2) based on their expression of CD206, CD301, arginase-1, and suppressor of cytokine signaling-1 as a consequence of a functionally type 2 helper T cell (Th2)-biased cytokine profile. Significant fibrosis and up-regulation of CCL18 were associated with the M2 phenotype of macrophages. Conversely, up-regulated Th1 cytokines did not result in significant classical activation of macrophages (M1), with poor inducible nitric oxide synthase and cyclooxygenase-2 production. Giant cell formation was further associated with up-regulated expression of DNAX-activating protein of 12 kDa (DAP12; gene symbol TYROBP). Functionally, alternative activation of macrophages on the basis of a Th2-biased immune response may induce clinical symptoms and chronic evolution of neuromuscular sarcoidosis. This is the first characterization of Th2-mediated immune mechanisms in neuromuscular sarcoidosis suggesting that a specific macrophage activation status leading to myofibrosis may be a key event in the pathogenesis of this disease.

Immune-Mediated Necrotizing Myopathy Is Characterized by a Specific Th1-M1 Polarized Immune Profile

Immune-mediated necrotizing myopathy (IMNM) is considered one of the idiopathic inflammatory myopathies, comprising dermatomyositis, polymyositis, and inclusion body myositis. The heterogeneous group of necrotizing myopathies shows a varying amount of necrotic muscle fibers, myophagocytosis, and a sparse inflammatory infiltrate. The underlying immune response in necrotizing myopathy has not yet been addressed in detail. Affected muscle tissue, obtained from 16 patients with IMNM, was analyzed compared with eight non-IMNM (nIMNM) tissues. Inflammatory cells were characterized by IHC, and immune mediators were assessed by quantitative real-time PCR. We demonstrate that immune- and non-immune-mediated disease can be distinguished by a specific immune profile with significantly more prominent major histocompatibility complex class I expression and complement deposition and a conspicuous inflammatory infiltrate. In addition, patients with IMNM exhibit a strong type 1 helper T cell (T1)/classically activated macrophage M1 response, with detection of elevated interferon-γ, tumor necrosis factor-α, IL-12, and STAT1 levels in the muscle tissue, which may serve as biomarkers and aid in diagnostic decisions. Furthermore, B cells and high expression of the chemoattractant CXCL13 were identified in a subgroup of patients with defined autoantibodies. Taken together, we propose a diagnostic armamentarium that allows for clear differentiation between IMNM and nIMNM. In addition, we have characterized a Th1-driven, M1-mediated immune response in most of the autoimmune necrotizing myopathies, which may guide therapeutic options in the future.

Human NCL Neuropathology

The neuronal ceroid lipofuscinoses (NCL) currently encompass fourteen genetically different forms, CLN1 to CLN14, but are all morphologically marked by loss of nerve cells, particularly in the cerebral and cerebellar cortices, and the cerebral and extracerebral formation of lipopigments. These lipopigments show distinct ultrastructural patterns, i.e., granular, curvilinear/rectilinear and fingerprint profiles. They contain-although to a different degree among the different CLN forms-subunit C of ATP synthase, saposins A and D, and beta-amyloid proteins. Extracerebral pathology, apart from lipopigment formation, which provides diagnostic information, is scant or non-existent. The retina undergoes atrophy in all childhood forms. While many new data and findings have been obtained by immunohistochemistry in mouse and other animal models, similar findings in human NCL are largely missing, thus recommending respective studies of archived brain tissues. The newly described NCL forms, i.e., CLN 10 to CLN 14, also require further studies to provide complete neuropathology. This article is part of a Special Issue entitled: Current Research on the Neuronal Ceroid Lipofuscinoses (Batten Disease).

Enlarging the nosological spectrum of hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS)

Hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS) is an autosomal dominant disease clinically characterized by cognitive decline, personality changes, motor impairment, parkinsonism and seizures. Recently, mutations in the colony‐stimulating factor‐1 receptor (CSF1R) gene have been shown to be associated with HDLS. We report clinical, neuropathological and molecular genetic findings of patients from a new family with a mutation in the CSF1R gene. Disease onset was earlier and disease progression was more rapid compared with previously reported patients. Psychiatric symptoms including personality changes, alcohol abuse and severe depression were the first symptoms in male patients. In the index, female patient, the initial symptom was cognitive decline. Magnetic resonance imaging (MRI) showed bilateral, confluent white matter lesions in the cerebrum. Stereotactic biopsy revealed loss of myelin and microglial activation as well as macrophage infiltration of the parenchyma. Numerous axonal swellings and spheroids were present. Ultrastructural analysis revealed pigment‐containing macrophages. Axonal swellings were detected by electron microscopy not only in the central nervous system (CNS) but also in skin nerves. We identified a heterozygous mutation (c.2330G>A, p.R777Q) in the CSF1R gene. Through this report, we aim to enlarge the nosological spectrum of HDLS, providing new clinical descriptions as well as novel neuropathological findings from the peripheral nervous system.

Mutations in Subunits of the Activating Signal Cointegrator 1 Complex Are Associated with Prenatal Spinal Muscular Atrophy and Congenital Bone Fractures

Transcriptional signal cointegrators associate with transcription factors or nuclear receptors and coregulate tissue-specific gene transcription. We report on recessive loss-of-function mutations in two genes (TRIP4 and ASCC1) that encode subunits of the nuclear activating signal cointegrator 1 (ASC-1) complex. We used autozygosity mapping and whole-exome sequencing to search for pathogenic mutations in four families. Affected individuals presented with prenatal-onset spinal muscular atrophy (SMA), multiple congenital contractures (arthrogryposis multiplex congenita), respiratory distress, and congenital bone fractures. We identified homozygous and compound-heterozygous nonsense and frameshift TRIP4 and ASCC1 mutations that led to a truncation or the entire absence of the respective proteins and cosegregated with the disease phenotype. Trip4 and Ascc1 have identical expression patterns in 17.5-day-old mouse embryos with high expression levels in the spinal cord, brain, paraspinal ganglia, thyroid, and submandibular glands. Antisense morpholino-mediated knockdown of either trip4 or ascc1 in zebrafish disrupted the highly patterned and coordinated process of α-motoneuron outgrowth and formation of myotomes and neuromuscular junctions and led to a swimming defect in the larvae. Immunoprecipitation of the ASC-1 complex consistently copurified cysteine and glycine rich protein 1 (CSRP1), a transcriptional cofactor, which is known to be involved in spinal cord regeneration upon injury in adult zebrafish. ASCC1 mutant fibroblasts downregulated genes associated with neurogenesis, neuronal migration, and pathfinding (SERPINF1, DAB1, SEMA3D, SEMA3A), as well as with bone development (TNFRSF11B, RASSF2, STC1). Our findings indicate that the dysfunction of a transcriptional coactivator complex can result in a clinical syndrome affecting the neuromuscular system.

GNE myopathy in Roma patients homozygous for the p.I618T founder mutation

GNE myopathy is an autosomal-recessive disorder caused by mutations in the GNE gene, encoding the key enzyme in the sialic acid biosynthetic pathway, UDP-N-acetylglucosamine 2-epimerase/N-acetyl mannosamine kinase. We studied 50 Bulgarian Roma patients homozygous for p.I618T, an ancient founder mutation in the kinase domain of the GNE gene, dating before the Gypsy exodus from North West India. The clinical features in the Bulgarian GNE group can be described with disease onset mostly in the third decade, but in individual cases, onset was as early as 10 years of age. The majority of patients had foot drop as the first symptom, but three patients developed hand weakness first. Muscle weakness was early and severe for the tibialis anterior, and minimal or late for quadriceps femoris, and respiratory muscles were only subclinically affected even in the advanced stages of the disease. During a 15-year follow-up period, 32 patients became non-ambulant. The average period between disease onset and loss of ambulation was 10.34u2009±u20094.31 years, ranging from 3 to 20 years. Our analysis of affected sib pairs suggested a possible role of genetic modifying factors, accounting for significant variation in disease severity.

Fetal akinesia caused by a novel actin filament aggregate myopathy skeletal muscle actin gene (ACTA1) mutation

We report a female newborn, diagnosed with fetal akinesia in utero, who died one hour after birth. Post-mortem muscle biopsy demonstrated actin-filament myopathy based on immunolabelling for sarcomeric actin, and large areas of filaments, without rod formation, ultrastructurally. Analysis of DNA extracted from the muscle disclosed a novel de novo heterozygous c.44G>A, GGC>GAC, p.Gly15Asp mutation in the ACTA1 gene. Analysis of the location of the mutated amino-acid in the actin molecule suggests the mutation most likely causes abnormal nucleotide binding, and consequent pathological actin polymerization. This case emphasizes the association of fetal akinesia with actin-filament myopathy.

Ubiquitin ligase defect by DCAF8 mutation causes HMSN2 with giant axons

Objective: To identify the genetic cause of axonal hereditary motor and sensory neuropathy (HMSN2) with infrequent giant axons. Methods: We studied 11 members of a previously described HMSN2 family with infrequent giant axons and variable cardiomyopathy. Whole-exome sequencing (WES) was performed on 2 affected persons and 1 unaffected person. Sanger sequencing was utilized to confirm the identified novel variant tracking with the affected status. Linkage analysis and haplotype mapping were obtained to confirm the causal nature of the identified variant. Cotransfection of HEK293 cells and co-immunoprecipitation assay were performed to assess the impact of the identified mutant protein in the implicated ubiquitin ligase pathway. Results: Giant axons with neurofilament accumulations were found in 3 affected persons who had undergone nerve biopsy evaluations. Six novel variants were identified by WES, but only DCAF8 p.R317C tracked with affected status within the family. Linkage and haplotype analysis using microsatellite markers supported this variant as causal. The mutation is within the DCAF8 WD repeat region critical for its binding to DDB1. Functional analysis shows DCAF8 p.R317C reduces the association of DCAF8 and DDB1, which is important in Cul4-ubiquitin E3 function. Conclusions: Our results indicate that DCAF8 p.R317C mutation is responsible for this specific variety of HMSN2 with infrequent giant axons and mild cardiomyopathy. This mutation results in decreased DDB1–DCAF8 association, leading to an E3 ubiquitin ligase defect that is likely associated with neurofilament degradation.

Myositis non-inflammatory mechanisms: An up-dated review

ABSTRACT Idiopathic inflammatory myopathies (IIM) represent a heterogeneous group of rare muscular diseases, with no clearly known causes. IIM frequently have an incomplete response to treatment due to the difficulty in distinguishing between IIM forms, and due to neglect their non-inflammatory causes. Important data concerning non-immune mechanisms in IIM pathology have been recently accumulated. There is a correlation between inflammatory and non-inflammatory mechanisms, but their involvement in IIM pathogenesis is still unknown. Here we review some of the most important data regarding the non-immune IIM pathology, highlighting possible future therapeutic targets: endoplasmic reticulum stress, ATP metabolism, ROS generation, autophagy, and microRNAs disturbances.

De novo exonic mutation in MYH7 gene leading to exon skipping in a patient with early onset muscular weakness and fiber-type disproportion

Here we report on a case of MYH7-related myopathy in a boy with early onset of muscular weakness and delayed motor development in infancy. His most affected muscles were neck extensors showing a dropped head sign, proximal muscles of lower limbs with positive Gowers sign, and trunk muscles. Brain and spinal cord MRI scans, echocardiography, and laboratory analyses including creatine kinase and lactate did not reveal any abnormalities. Muscle histopathology showed fiber-type disproportion. Whole exome sequencing of the parents-offspring trio revealed a novel de novo c.5655G>A p.(Ala1885=) synonymous substitution of the last nucleotide in exon 38 of the MYH7 gene. Further RNA investigations proved the skipping of exon 38 (p.1854_1885del). This is a first report of an exon-skipping mutation in the MYH7 gene causing myopathy. This report broadens both the phenotypic and genotypic spectra of MYH7-related myopathies.