Roles of Aging, Circular RNAs, and RNA Editing in the Pathogenesis of Amyotrophic Lateral Sclerosis: Potential Biomarkers and Therapeutic Targets.

Amyotrophic lateral sclerosis (ALS) is an incurable motor neuron disease caused by upper and lower motor neuron death. Despite advances in our understanding of ALS pathogenesis, effective treatment for this fatal disease remains elusive. As aging is a major risk factor for ALS, age-related molecular changes may provide clues for the development of new therapeutic strategies. Dysregulation of age-dependent RNA metabolism plays a pivotal role in the pathogenesis of ALS. In addition, failure of RNA editing at the glutamine/arginine (Q/R) site of GluA2 mRNA causes excitotoxicity due to excessive Ca2+ influx through Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, which is recognized as an underlying mechanism of motor neuron death in ALS. Circular RNAs (circRNAs), a circular form of cognate RNA generated by back-splicing, are abundant in the brain and accumulate with age. Hence, they are assumed to play a role in neurodegeneration. Emerging evidence has demonstrated that age-related dysregulation of RNA editing and changes in circRNA expression are involved in ALS pathogenesis. Herein, we review the potential associations between age-dependent changes in circRNAs and RNA editing, and discuss the possibility of developing new therapies and biomarkers for ALS based on age-related changes in circRNAs and dysregulation of RNA editing.

The RNA editor ADAR2 promotes immune cell trafficking by enhancing endothelial responses to interleukin-6 during sterile inflammation.

Immune cell trafficking constitutes a fundamental component of immunological response to tissue injury, but the contribution of intrinsic RNA nucleotide modifications to this response remains elusive. We report that RNA editor ADAR2 exerts a tissue- and stress-specific regulation of endothelial responses to interleukin-6 (IL-6), which tightly controls leukocyte trafficking in IL-6-inflamed and ischemic tissues. Genetic ablation of ADAR2 from vascular endothelial cells diminished myeloid cell rolling and adhesion on vascular walls and reduced immune cell infiltration within ischemic tissues. ADAR2 was required in the endothelium for the expression of the IL-6 receptor subunit, IL-6 signal transducer (IL6ST; gp130), and subsequently, for IL-6 trans-signaling responses. ADAR2-induced adenosine-to-inosine RNA editing suppressed the Drosha-dependent primary microRNA processing, thereby overwriting the default endothelial transcriptional program to safeguard gp130 expression. This work demonstrates a role for ADAR2 epitranscriptional activity as a checkpoint in IL-6 trans-signaling and immune cell trafficking to sites of tissue injury.

Effect of Serum Perampanel Concentration on Sporadic Amyotrophic Lateral Sclerosis Progression.

BACKGROUND AND PURPOSE: To clarify the effect of perampanel (PER) on sporadic amyotrophic lateral sclerosis (sALS) progression, the relationship between the changes in Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) scores and serum PER concentrations was investigated. METHODS: 12 patients with sALS from our hospital who agreed to participate and completed the PER for sALS randomized phase 2 study were included. After completing the study, we retrospectively obtained serum PER concentration data from the patients. Based on their mean PER concentrations, we divided the patients who had been taking PER into two groups: four patients with a mean PER concentration of ≥400 ng/mL were assigned to the H group, and three with a mean PER concentration of <400 ng/mL were assigned to the L group. The control group consisted of five patients who had been taking a placebo. We obtained the ALSFRS-R scores of each patient at 36 and 48 weeks after randomization. The differences in ALSFRS-R scores at baseline (0 weeks) and each subsequent week were used in the analysis. RESULTS: At 48 weeks, there were no differences in the degree of deterioration of the bulbar, upper and lower limb, and respiratory ALSFRS-R subscores and total ALSFRS-R score. However, at 36 weeks, the bulbar subscore was significantly lower in the H group than in the control group (p=0.032). CONCLUSIONS: Because high PER concentrations may exacerbate bulbar symptoms in patients with sALS, serum PER measurements may be beneficial when patients with sALS are taking PER.

Pathological features of glial cells and motor neurons in the anterior horn of the spinal cord in sporadic ALS using ADAR2 conditional knockout mice.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective degeneration of motor neurons (MNs). In the MNs of patients with ALS, adenosine deaminase acting on RNA 2 (ADAR2)-mediated RNA editing of GluA2 mRNA at the Q/R site is profoundly deficient. In genetically modified mice (ADAR2flox/flox/VAChT-Cre.Fast; AR2), the selective knockout of ADAR2 in cholinergic neurons induced progressive loss of lower MNs. MNs exhibiting an age-related increase in abnormal TDP-43 localization and reduced ADAR2 immunoreactivity are localized in the lateral areas of the anterior horns (AHs) in aged wild-type mice. However, the patterns in the AHs of AR2 mice remain unknown. In this study, we investigated whether similar degeneration is observed in AR2 mice. We compared the number of astrocytes and MNs in the lateral and medial AHs of the lumbar spinal cord of 12-month-old AR2 mice with age-matched wild-type mice. The number of MNs significantly decreased in both the lateral and medial areas in AR2 mice AHs, particularly in the former. The number of reactive astrocytes increased significantly in the lateral areas of the AHs of AR2 mice. In conclusion, stronger activation of astrocytes with reduction of MNs in the ADAR2 deficiency-related lateral area increases in AR2 mice AHs. Fast fatigable MNs are expected to be present in the lateral area of the AHs. We found that MN death is more common in the lateral area of AHs associated with FF MNs due to differences in vulnerability to MN under ADAR2 deficiency.

Prolyl Isomerase Pin1 Expression in the Spinal Motor Neurons of Patients With Sporadic Amyotrophic Lateral Sclerosis.

BACKGROUND AND PURPOSE: Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease. Selective deficiency of edited adenosine deaminase acting on RNA 2 (ADAR2), a key molecule in the acquisition of Ca2+ resistance in motor neurons, has been reported in sporadic ALS (sALS) spinal motor neurons. Since ADAR2 activity is positively regulated by prolyl isomerase Protein never in mitosis gene A interacting-1 (Pin1), a known phosphorylation-dependent peptidyl-prolyl cis/trans isomerase, we investigated Pin1 expression in spinal motor neurons in sALS. METHODS: Specimens of the spinal cord were obtained from the lumbar region in eight sALS patients and age-matched five controls after postmortem examinations. The specimens were double stained with anti-Pin1 and anti-TAR DNA-binding protein of 43 kDa (TDP-43) antibodies, and examined under a fluorescence microscope. RESULTS: This study analyzed 254 and 422 spinal motor neurons from 8 sALS patients and 5 control subjects, respectively. The frequency of motor neurons with high cytoplasmic Pin1 expression from the spinal cord did not differ significantly between sALS specimens without cytoplasmic TDP-43 inclusions and control specimens. However, in sALS specimens, neurons for which the Pin1 immunoluminescence intensity in the cytoplasm was at least twice that in the background were more common in specimens with cytoplasmic TDP-43 inclusions (p<0.05 in χ² test). CONCLUSIONS: In sALS, neurons with higher expression levels of Pin1 levels had more TDP-43 inclusions. Despite the feedback mechanism between Pin1 and ADAR2 being unclear, since Pin1 positively regulates ADAR2, our results suggest that higher Pin1 expression levels in motor neurons with TDP-43 pathology from sALS patients represent a compensatory mechanism.

Testing of the therapeutic efficacy and safety of AMPA receptor RNA aptamers in an ALS mouse model.

In motor neurons of sporadic amyotrophic lateral sclerosis (ALS) patients, the RNA editing at the glutamine/arginine site of the GluA2 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors is defective or incomplete. As a result, AMPA receptors containing the abnormally expressed, unedited isoform of GluA2 are highly Ca2+-permeable, and are responsible for mediating abnormal Ca2+ influx, thereby triggering motor neuron degeneration and cell death. Thus, blocking the AMPA receptor-mediated, abnormal Ca2+ influx is a potential therapeutic strategy for treatment of sporadic ALS. Here, we report a study of the efficacy and safety of two RNA aptamers targeting AMPA receptors on the ALS phenotype of AR2 mice. A 12-wk continuous, intracerebroventricular infusion of aptamers to AR2 mice reduced the progression of motor dysfunction, normalized TDP-43 mislocalization, and prevented death of motor neurons. Our results demonstrate that the use of AMPA receptor aptamers as a novel class of AMPA receptor antagonists is a promising strategy for developing an ALS treatment approach.

Randomized phase 2 study of perampanel for sporadic amyotrophic lateral sclerosis.

OBJECTIVE: To evaluate the efficacy and safety of perampanel in patients with sporadic amyotrophic lateral sclerosis (SALS). METHODS: This randomized, double-blind, placebo-controlled, multicenter, phase 2 clinical study was conducted at 12 sites. Patients with probable or definite ALS as defined by revised El Escorial criteria were enrolled. Sixty-six patients were randomly assigned (1:1:1) to receive placebo, 4 mg perampanel, or 8 mg perampanel daily for 48 weeks. Adverse events (AEs) were recorded throughout the trial period. The primary efficacy outcome was the change in Amyotrophic Lateral Sclerosis Rating Scale-Revised (ALSFRS-R) score after 48 weeks of treatment. RESULTS: One patient withdrew before starting the treatment. Of 65 patients included, 18 of 22 patients randomized to placebo (82%), 14 of 22 patients randomized to 4 mg perampanel (64%), and 7 of 21 patients randomized to 8 mg perampanel (33%) completed the trial. There was a significant difference in the change of ALSFRS-R scores [- 8.4 (95% CI - 13.9 to - 2.9); p = 0.015] between the placebo and the perampanel 8 mg group, primarily due to worsening of the bulbar subscore in the perampanel 8 mg group. Serious AEs were more frequent in the perampanel 8 mg group than in the placebo group (p = 0.0483). CONCLUSIONS: Perampanel was associated with a significant decline in ALSFRS-R score and was linked to worsening of the bulbar subscore in the 8 mg group.

RNA Editing: A New Therapeutic Target in Amyotrophic Lateral Sclerosis and Other Neurological Diseases.

The conversion of adenosine to inosine in RNA editing (A-to-I RNA editing) is recognized as a critical post-transcriptional modification of RNA by adenosine deaminases acting on RNAs (ADARs). A-to-I RNA editing occurs predominantly in mammalian and human central nervous systems and can alter the function of translated proteins, including neurotransmitter receptors and ion channels; therefore, the role of dysregulated RNA editing in the pathogenesis of neurological diseases has been speculated. Specifically, the failure of A-to-I RNA editing at the glutamine/arginine (Q/R) site of the GluA2 subunit causes excessive permeability of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors to Ca2+, inducing fatal status epilepticus and the neurodegeneration of motor neurons in mice. Therefore, an RNA editing deficiency at the Q/R site in GluA2 due to the downregulation of ADAR2 in the motor neurons of sporadic amyotrophic lateral sclerosis (ALS) patients suggests that Ca2+-permeable AMPA receptors and the dysregulation of RNA editing are suitable therapeutic targets for ALS. Gene therapy has recently emerged as a new therapeutic opportunity for many heretofore incurable diseases, and RNA editing dysregulation can be a target for gene therapy; therefore, we reviewed neurological diseases associated with dysregulated RNA editing and a new therapeutic approach targeting dysregulated RNA editing, especially one that is effective in ALS.

[Perampanel for Sporadic Amyotrophic Lateral Sclerosis].

Disease-specific and site-selective deficiency of an RNA editing enzyme, adenosine deaminase acting on RNA 2 (ADAR2), has been demonstrated in sporadic amyotrophic lateral sclerosis (sALS) motor neurons. ADAR2 regulates Ca2+ influx through α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors via adenosine-to-inosine conversion at the glutamine/arginine site of GluA2 mRNA, which makes ADAR2 a key factor in acquired Ca2+ resistance in motor neurons. Deficient ADAR2 of sALS motor neurons is supposed to lead to excessive Ca2+ influx through AMPA receptors, resulting in TDP-43 pathology and nuclear pore complex pathology, and eventually motor neuronal death. We considered that AMPA receptor antagonists could strongly prevent excessive Ca2+ influx through AMPA receptors and block motor neuronal degeneration in sALS. Perampanel, a selective non-competitive AMPA receptor antagonist, has been reported to prevent deterioration in a mouse model for sALS, in which ADAR2 is conditionally knocked out in motor neurons. Because of the therapeutic potency of perampanel for sporadic ALS, we have performed a multicenter randomized, double-blinded, placebo-controlled, parallel-group phase 2 clinical trial. The primary outcome measure is the change in ALS functional rating scale-revised after 48 weeks of treatment. The results of this study will be available in early 2020.

Extracellular RNAs as Biomarkers of Sporadic Amyotrophic Lateral Sclerosis and Other Neurodegenerative Diseases.

Recent progress in the research for underlying mechanisms in neurodegenerative diseases, including Alzheimer disease (AD), Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS) has led to the development of potentially effective treatment, and hence increased the need for useful biomarkers that may enable early diagnosis and therapeutic monitoring. The deposition of abnormal proteins is a pathological hallmark of neurodegenerative diseases, including ß-amyloid in AD, α-synuclein in PD, and the transactive response DNA/RNA binding protein of 43kDa (TDP-43) in ALS. Furthermore, progression of the disease process accompanies the spreading of abnormal proteins. Extracellular proteins and RNAs, including mRNA, micro RNA, and circular RNA, which are present as a composite of exosomes or other forms, play a role in cell-cell communication, and the role of extracellular molecules in the cell-to-cell spreading of pathological processes in neurodegenerative diseases is now in the spotlight. Therefore, extracellular proteins and RNAs are considered potential biomarkers of neurodegenerative diseases, in particular ALS, in which RNA dysregulation has been shown to be involved in the pathogenesis. Here, we review extracellular proteins and RNAs that have been scrutinized as potential biomarkers of neurodegenerative diseases, and discuss the possibility of extracellular RNAs as diagnostic and therapeutic monitoring biomarkers of sporadic ALS.

Impaired Nucleoporins Are Present in Sporadic Amyotrophic Lateral Sclerosis Motor Neurons that Exhibit Mislocalization of the 43-kDa TAR DNA-Binding Protein.

BACKGROUND AND PURPOSE: Disruption of nucleoporins has been reported in the motor neurons of patients with sporadic amyotrophic lateral sclerosis (sALS). However, the precise changes in the morphology of nucleoporins associated with the pathology of the 43-kDa TAR DNA-binding protein (TDP-43) in the disease process remain unknown. We investigated the expression of nucleoporins that constitute the nuclear pore complex (NPC) in spinal motor neurons that exhibit sALS in relation to TDP-43 pathology, which is a reliable neuropathological hallmark of sALS. METHODS: Paraffin-embedded sections of the lumbar spinal cord were obtained for immunofluorescence analysis from seven control subjects and six sALS patients. Anti-TDP-43 antibody, anti-nucleoporin p62 (NUP62) antibody, and anti-karyopherin beta 1 (KPNB1) antibody were applied as primary antibodies, and then visualized using appropriate secondary antibodies. The sections were then examined under a fluorescence microscope. RESULTS: NUP62 and KPNB1 immunoreactivity appeared as a smooth round rim bordering the nuclear margin in normal spinal motor neurons that exhibited nuclear TDP-43 immunoreactivity. sALS spinal motor neurons with apparent TDP-43 mislocalization demonstrated irregular, disrupted nuclear staining for NUP62 or KPNB1. Some atrophic sALS spinal motor neurons with TDP-43 mislocalization presented no NUP62 immunoreactivity. CONCLUSIONS: Our findings suggest a close relationship between NPC alterations and TDP-43 pathology in the degenerative process of the motor neurons of sALS patients.

ADAR2-dependent A-to-I RNA editing in the extracellular linear and circular RNAs.

Currently, no reliable biomarkers of amyotrophic lateral sclerosis (ALS) exist. In sporadic ALS, RNA editing at the glutamine/arginine site of GluA2 mRNA is specifically reduced in the motor neurons due to the downregulation of adenosine deaminase acting on RNA 2 (ADAR2). Furthermore, TDP-43 pathology, the pathological hallmark of ALS, is observed in the ADAR2-lacking motor neurons in ALS patients and conditional ADAR2 knockout mice, suggesting a pivotal role of ADAR2 downregulation in the ALS pathogenesis. Extracellular RNAs were shown to represent potential disease biomarkers and the editing efficiencies at their ADAR2-dependent sites may reflect cellular ADAR2 activity, suggesting that these RNAs isolated from the body fluids may represent the biomarkers of ALS. We searched for ADAR2-dependent sites in the mouse motor neurons and human-derived cultured cells and found 10 sites in five host RNAs expressed in SH-SY5Y cells and their culture medium. Of these, the arginine/glycine site of SON mRNA was newly identified as an ADAR2-dependent site. Furthermore, we detected a circular RNA with an ADAR2-dependent site in the SH-SY5Y cells and their culture medium. Therefore, the changes in the editing efficiencies at the identified host RNA sites isolated from the body fluids may represent potential biomarkers of ALS.

Cell death cascade and molecular therapy in ADAR2-deficient motor neurons of ALS.

TAR DNA-binding protein (TDP-43) pathology in the motor neurons is the most reliable pathological hallmark of amyotrophic lateral sclerosis (ALS), and motor neurons bearing TDP-43 pathology invariably exhibit failure in RNA editing at the GluA2 glutamine/arginine (Q/R) site due to down-regulation of adenosine deaminase acting on RNA 2 (ADAR2). Conditional ADAR2 knockout (AR2) mice display ALS-like phenotype, including progressive motor dysfunction due to loss of motor neurons. Motor neurons devoid of ADAR2 express Q/R site-unedited GluA2, and AMPA receptors with unedited GluA2 in their subunit assembly are abnormally permeable to Ca2+, which results in progressive neuronal death. Moreover, analysis of AR2 mice has demonstrated that exaggerated Ca2+ influx through the abnormal AMPA receptors overactivates calpain, a Ca2+-dependent protease, that cleaves TDP-43 into aggregation-prone fragments, which serve as seeds for TDP-43 pathology. Activated calpain also disrupts nucleo-cytoplasmic transport and gene expression by cleaving molecules involved in nucleocytoplasmic transport, including nucleoporins. These lines of evidence prompted us to develop molecular targeting therapy for ALS by normalization of disrupted intracellular environment due to ADAR2 down-regulation. In this review, we have summarized the work from our group on the cell death cascade in sporadic ALS and discussed a potential therapeutic strategy for ALS.

Ethanol Preference and Drinking Behavior Are Controlled by RNA Editing in the Nucleus Accumbens.

RNA editing plays critical roles in normal brain function, and alteration of its activity causes various disorders. We previously found that chronic consumption of ethanol was associated with increased levels of RNA editing of serotonin 2C receptor in the nucleus accumbens (NAc). However, it remains unknown whether RNA editing in the NAc modulates alcohol addiction through the brain reward system. To investigate the involvement of NAc RNA editing in alcohol addiction, we generated NAc-specific knockout mice of the double-stranded RNA-specific adenosine deaminase ADAR2 using AAV-GFP/Cre and conducted a battery of behavioral tests including anxiety- and depression-like behaviors. In addition, NAc-specific ADAR2 knockout mice were exposed to ethanol vapor for 20 days, followed by ethanol-drinking and conditioned place preference (CPP) tests. NAc-specific ADAR2 knockout mice showed a significant decrease in locomotor activity in the open field test although they did not develop anxiety- and depression-like behaviors. In addition, the enhancements of ethanol intake and ethanol preference that are usually observed after chronic ethanol vapor exposure were significantly reduced in these mice. These results suggest that ADAR2-mediated RNA editing in the NAc is involved in determination of alcohol preference after chronic alcohol consumption.

Ca2+ -permeable AMPA receptors associated with epileptogenesis of hypothalamic hamartoma.

Hypothalamic hamartoma (HH), composed of neurons and glia without apparent cytologic abnormalities, is a rare developmental malformation in humans. Patients with HH often have characteristic medically refractory gelastic seizures, and intrinsic epileptogenesis within the lesions has been speculated. Herein we provide evidence to suggest that in HH neurons, Ca2+ permeability through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors is aberrantly elevated. In needle biopsy specimens of HH tissue, field potential recordings demonstrated spontaneous epileptiform activities similar to those observed in other etiologically distinct epileptogenic tissues. In HH, however, these activities were clearly abolished by application of Joro Spider Toxin (JSTX), a specific inhibitor of the Ca2+ -permeable AMPA receptor. Consistent with these physiologic findings, the neuronal nuclei showed disappearance of adenosine deaminase acting on RNA 2 (ADAR2) immunoreactivity. Furthermore, examination of glutamate receptor 2 (GluA2) messenger RNA (mRNA) revealed that editing efficiency at the glutamine/arginine site was significantly low. These results suggest that neurons in HH may bear Ca2+ -permeable AMPA receptors due to dislocation of ADAR2.

Altered Intracellular Milieu of ADAR2-Deficient Motor Neurons in Amyotrophic Lateral Sclerosis.

Transactive response DNA-binding protein (TDP-43) pathology, and failure of A-to-I conversion (RNA editing) at the glutamine/arginine (Q/R) site of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit GluA2, are etiology-linked molecular abnormalities that concomitantly occur in the motor neurons of most patients with amyotrophic lateral sclerosis (ALS). Adenosine deaminase acting on RNA 2 (ADAR2) specifically catalyzes GluA2 Q/R site-RNA editing. Furthermore, conditional ADAR2 knockout mice (AR2) exhibit a progressive ALS phenotype with TDP-43 pathology in the motor neurons, which is the most reliable pathological marker of ALS. Therefore, the evidence indicates that ADAR2 downregulation is a causative factor in ALS, and AR2 mice exhibit causative molecular changes that occur in ALS. We discuss the contributors to ADAR2 downregulation and TDP-43 pathology in AR2 mouse motor neurons. We describe mechanisms of exaggerated Ca2+ influx amelioration via AMPA receptors, which is neuroprotective in ADAR2-deficient motor neurons with normalization of TDP-43 pathology in AR2 mice. Development of drugs to treat diseases requires appropriate animal models and a sensitive method of evaluating efficacy. Therefore, normalization of disrupted intracellular environments resulting from ADAR2 downregulation may be a therapeutic target for ALS. We discuss the development of targeted therapy for ALS using the AR2 mouse model.

Calpain-dependent disruption of nucleo-cytoplasmic transport in ALS motor neurons.

Nuclear dysfunction in motor neurons has been hypothesized to be a principal cause of amyotrophic lateral sclerosis (ALS) pathogenesis. Here, we investigated the mechanism by which the nuclear pore complex (NPC) is disrupted in dying motor neurons in a mechanistic ALS mouse model (adenosine deaminase acting on RNA 2 (ADAR2) conditional knockout (AR2) mice) and in ALS patients. We showed that nucleoporins (Nups) that constituted the NPC were cleaved by activated calpain via a Ca2+-permeable AMPA receptor-mediated mechanism in dying motor neurons lacking ADAR2 expression in AR2 mice. In these neurons, nucleo-cytoplasmic transport was disrupted, and the level of the transcript elongation enzyme RNA polymerase II phosphorylated at Ser2 was significantly decreased. Analogous changes were observed in motor neurons lacking ADAR2 immunoreactivity in sporadic ALS patients. Therefore, calpain-dependent NPC disruption may participate in ALS pathogenesis, and inhibiting Ca2+-mediated cell death signals may be a therapeutic strategy for ALS.

Cancer association as a risk factor for anti-HMGCR antibody-positive myopathy.

OBJECTIVE: To show cancer association is a risk factor other than statin exposure for anti-3-hydroxy-3-methylglutaryl coenzyme A reductase autoantibody-positive (anti-HMGCR Ab+) myopathy. METHODS: We analyzed the clinical features and courses of 33 patients (23 female and 10 male) with anti-HMGCR Ab+ myopathy among 621 consecutive patients with idiopathic inflammatory myopathies. RESULTS: Among the 33 patients, 7 (21%) were statin-exposed and 26 were statin-naive. In relation with cancer, there were 12 patients (statin-exposed, n = 4) with cancers detected within 3 years of myopathy diagnosis (cancer association), 3 patients (all statin-naive) with cancers detected more than 3 years before myopathy diagnosis (cancer history), 10 cancer-free patients followed up for more than 3 years (all statin-naive), and 8 patients without cancer detection but followed up for less than 3 years (statin-exposed, n = 3). Therefore, 12 patients with cancer association (36%) formed a larger group than that of 7 statin-exposed patients (21%). Among 12 patients with cancer association, 92% had cancer detection within 1 year of myopathy diagnosis (after 1.3 years in the remaining patient), 83% had advanced cancers, and 75% died of cancers within 2.7 years. Of interest, 1 patient with cancer history had sustained increase in creatine kinase level over 12 years from cancer removal to the development of weakness. CONCLUSIONS: Patients with cancer association formed a large group with poor prognosis in our series of patients with anti-HMGCR Ab+ myopathy. The close synchronous occurrence of cancers and myopathies suggested that cancer association is one of the risk factors for developing anti-HMGCR Ab+ myopathy.