Volumetric brain reductions in adult patients with phenylketonuria and their relationship with blood phenylalanine levels.

BACKGROUND: Continued dietary treatment since early diagnosis through newborn screening programs usually prevents brain-related complications in phenylketonuria (PKU). However, subtle neurocognitive and brain alterations may be observed in some adult patients despite early treatment. Nevertheless, neuropsychological and neuroimaging studies in the field remain scarce. OBJECTIVES: This work aimed to determine possible neuropsychological and structural brain alterations in treated adult patients with PKU. METHODS: Thirty-five patients with PKU and 22 healthy controls (HC) underwent neuropsychological assessment and T1-weighted magnetic resonance imaging on a 3 T scanner. FreeSurfer (v.7.1) was used to obtain volumetric measures and SPSS (v27.0.1.0) was used to analyze sociodemographic, neuropsychological, volumetric, and clinical data (p < 0.05). RESULTS: Adult patients with PKU showed significantly lower performance than HC in Full Scale IQ (t = 2.67; p = .010) from the WAIS-IV. The PKU group also showed significantly lower volumes than HC in the pallidum (U = 224.000; p = .008), hippocampus (U = 243.000; p = .020), amygdala (U = 200.000; p = .002), and brainstem (t = 3.17; p = .006) as well as in total cerebral white matter volume (U = 175.000; p = .001). Blood phenylalanine (Phe) levels in PKU patients were negatively correlated with the pallidum (r = -0.417; p = .013) and brainstem (r = -0.455, p = .006) volumes. CONCLUSIONS: Adult patients with early-treated PKU showed significantly lower global intelligence than HC. Moreover, these patients showed reduced global white matter volume as well as reductions in the volume of several subcortical grey matter structures, which might be related to the existence of underlying neurodevelopmental alterations. Higher blood Phe levels were also negatively correlated with pallidum and brainstem, suggesting a higher vulnerability of these structures to Phe toxicity.

Preparing Enteral Formulas for Adult Patients with Phenylketonuria: A Minor Necessity but Major Challenge-A Case Report.

Phenylketonuria (PKU) is the most frequent of the congenital errors of amino acid (AA) metabolism worldwide. It leads to the accumulation of the essential AA phenylalanine (Phe) and it is associated with severe neurological defects. The early diagnosis and treatment of this rare disease, achieved through newborn screening and low-Phe diet, has profoundly changed its clinical spectrum, resulting in normal cognitive development. We face the first generation of PKU patients perinatally diagnosed and treated who have reached adulthood, whose special needs must be addressed, including feeding through enteral nutrition (EN). However, recommendations regarding EN in PKU constitute a gap in the literature. Although protein substitutes for patients with PKU are offered in multiple forms (Phe-free L-amino acid or casein glycomacropeptide supplements), none of these commercial formulas ensures the whole provision of daily total energy and protein requirements, including a safe amount of Phe. Consequently, the combination of different products becomes necessary when artificial nutrition via tube feeding is required. Importantly, the composition of these specific formulas may result in physicochemical interactions when they are mixed with standard EN products, leading to enteral feeding tubes clogging, and also gastrointestinal concerns due to hyperosmolality. Herein, we present the first reported case of EN use in an adult patient with PKU, where the separate administration of protein substitutes and the other EN products avoided physicochemical interactions.

Identification of Unique microRNA Profiles in Different Types of Idiopathic Inflammatory Myopathy.

Dermatomyositis (DM), antisynthetase syndrome (AS), immune-mediated necrotizing myopathy (IMNM), and inclusion body myositis (IBM) are four major types of idiopathic inflammatory myopathy (IIM). Muscle biopsies from each type of IIM have unique transcriptomic profiles. MicroRNAs (miRNAs) target messenger RNAs (mRNAs), thereby regulating their expression and modulating transcriptomic profiles. In this study, 18 DM, 12 IMNM, 6 AS, 6 IBM, and 6 histologically normal muscle biopsies underwent miRNA profiling using the NanoString nCounter system. Eleven miRNAs were exclusively differentially expressed in DM compared to controls, seven miRNAs were only differentially expressed in AS, and nine miRNAs were specifically upregulated in IBM. No differentially expressed miRNAs were identified in IMNM. We also analyzed miRNA-mRNA associations to identify putative targets of differentially expressed miRNAs. In DM and AS, these were predominantly related to inflammation and cell cycle progression. Moreover, our analysis showed an association between miR-30a-3p, miR-30e-3p, and miR-199b-5p downregulation in DM and the upregulation of target genes induced by type I interferon. In conclusion, we show that muscle biopsies from DM, AS, and IBM patients have unique miRNA signatures and that these miRNAs might play a role in regulating the expression of genes known to be involved in IIM pathogenesis.

Screening for late-onset Pompe disease in Internal Medicine departments in Spain.

BACKGROUND: The screening of high-risk populations using dried blood spots (DBS) has allowed the rapid identification of patients with Pompe disease, mostly in Neurology departments. The aim of the study was to determine the prevalence of late-onset Pompe disease (LOPD) among patients not previously diagnosed or tested for this entity despite presenting possible signs or symptoms of the disease in Internal Medicine departments in Spain. METHODS: This epidemiological, observational, cross-sectional, multicenter study included a single cohort of individuals with clinical suspicion of LOPD seen at Internal Medicine departments in Spain. The diagnosis of LOPD was initially established on the basis of the result of DBS. If decreased enzyme acid-alpha-1,4-glucosidase (GAA) activity was detected in DBS, additional confirmatory diagnostic measurements were conducted, including GAA activity in lymphocytes, fibroblasts, or muscle and/or genetic testing. RESULTS: The diagnosis of LOPD was confirmed in 2 out of 322 patients (0.6%). Reasons for suspecting LOPD diagnosis were polymyositis or any type of myopathy of unknown etiology (in one patient), and asymptomatic or pauci-symptomatic hyperCKemia (in the other). The time between symptom onset and LOPD diagnosis was 2.0 and 0.0 years. Both patients were asymptomatic, with no muscle weakness. Additionally, 19.7% of the non-LOPD cases received an alternative diagnosis. CONCLUSIONS: Our study highlights the existence of a hidden population of LOPD patients in Internal Medicine departments who might benefit from early diagnosis and early initiation of potential treatments.

Integrated Multi-Omics Analysis for Inferring Molecular Players in Inclusion Body Myositis.

Inclusion body myositis (IBM) is an acquired inflammatory myopathy affecting proximal and distal muscles that leads to weakness in patients over 50. It is diagnosed based on clinical and histological findings in muscle related to inflammation, degeneration, and mitochondria. In relation to IBM, a shortage of validated disease models and a lack of biomarkers and effective treatments constitute an unmet medical need. To overcome these hurdles, we performed an omics analysis of multiple samples from IBM patients (saliva, fibroblasts, urine, plasma, and muscle) to gain insight into the pathophysiology of IBM. Degeneration was evident due to the presence of amyloid ß peptide 1-42 (Aß1-42) in the saliva of the analyzed IBM patients. The presence of metabolic disarrangements in IBM was indicated by an imbalanced organic acid profile in fibroblasts and urine. Specifically, abnormal levels of L-pyroglutamic and orotic acid were supported by the abnormal expression of related metabolites in plasma and urine (glutathione and pyrimidines) and the aberrant expression of upstream gene regulators (L2HGDH, IDH2, OPLAH, and ASL) in muscle. Combined levels of L-pyroglutamic and orotic acid displayed an outstanding biomarker signature in urine with 100% sensitivity and specificity. The confirmation of systemic metabolic disarrangements in IBM and the identification of novel biomarkers reported herein unveil novel insights that require validation in larger cohorts.

Transcriptional derepression of CHD4/NuRD-regulated genes in the muscle of patients with dermatomyositis and anti-Mi2 autoantibodies.

OBJECTIVES: Myositis is a heterogeneous family of diseases including dermatomyositis (DM), immune-mediated necrotising myopathy (IMNM), antisynthetase syndrome (AS) and inclusion body myositis (IBM). Myositis-specific autoantibodies define different subtypes of myositis. For example, patients with anti-Mi2 autoantibodies targeting the chromodomain helicase DNA-binding protein 4 (CHD4)/NuRD complex (a transcriptional repressor) have more severe muscle disease than other DM patients. This study aimed to define the transcriptional profile of muscle biopsies from anti-Mi2-positive DM patients. METHODS: RNA sequencing was performed on muscle biopsies (n=171) from patients with anti-Mi2-positive DM (n=18), DM without anti-Mi2 autoantibodies (n=32), AS (n=18), IMNM (n=54) and IBM (n=16) as well as 33 normal muscle biopsies. Genes specifically upregulated in anti-Mi2-positive DM were identified. Muscle biopsies were stained for human immunoglobulin and protein products corresponding to genes specifically upregulated in anti-Mi2-positive muscle biopsies. RESULTS: A set of 135 genes, including SCRT1 and MADCAM1, was specifically overexpressed in anti-Mi2-positive DM muscle. This set was enriched for CHD4/NuRD-regulated genes and included genes that are not otherwise expressed in skeletal muscle. The expression levels of these genes correlated with anti-Mi2 autoantibody titres, markers of disease activity and with the other members of the gene set. In anti-Mi2-positive muscle biopsies, immunoglobulin was localised to the myonuclei, MAdCAM-1 protein was present in the cytoplasm of perifascicular fibres, and SCRT1 protein was localised to myofibre nuclei. CONCLUSIONS: Based on these findings, we hypothesise that anti-Mi2 autoantibodies could exert a pathogenic effect by entering damaged myofibres, inhibiting the CHD4/NuRD complex, and subsequently derepressing the unique set of genes defined in this study.

Unravelling inclusion body myositis using a patient-derived fibroblast model.

BACKGROUND: Inclusion body myositis (IBM) is an inflammatory myopathy clinically characterized by proximal and distal muscle weakness, with inflammatory infiltrates, rimmed vacuoles and mitochondrial changes in muscle histopathology. There is scarce knowledge on IBM aetiology, and non-established biomarkers or effective treatments are available, partly due to the lack of validated disease models. METHODS: We have performed transcriptomics and functional validation of IBM muscle pathological hallmarks in fibroblasts from IBM patients (n = 14) and healthy controls (n = 12), paired by age and sex. The results comprise an mRNA-seq, together with functional inflammatory, autophagy, mitochondrial and metabolic changes between patients and controls. RESULTS: Gene expression profile of IBM vs control fibroblasts revealed 778 differentially expressed genes (P-value adj < 0.05) related to inflammation, mitochondria, cell cycle regulation and metabolism. Functionally, an increased inflammatory profile was observed in IBM fibroblasts with higher supernatant cytokine secretion (three-fold increase). Autophagy was reduced considering basal protein mediators (18.4% reduced), time-course autophagosome formation (LC3BII 39% reduced, P-value < 0.05), and autophagosome microscopic evaluation. Mitochondria displayed reduced genetic content (by 33.9%, P-value < 0.05) and function (30.2%-decrease in respiration, 45.6%-decline in enzymatic activity (P-value < 0.001), 14.3%-higher oxidative stress, 135.2%-increased antioxidant defence (P-value < 0.05), 11.6%-reduced mitochondrial membrane potential (P-value < 0.05) and 42.8%-reduced mitochondrial elongation (P-value < 0.05)). In accordance, at the metabolite level, organic acid showed a 1.8-fold change increase, with conserved amino acid profile. Correlating to disease evolution, oxidative stress and inflammation emerge as potential markers of prognosis. CONCLUSIONS: These findings confirm the presence of molecular disturbances in peripheral tissues from IBM patients and prompt patients' derived fibroblasts as a promising disease model, which may eventually be exported to other neuromuscular disorders. We additionally identify new molecular players in IBM associated with disease progression, setting the path to deepen in disease aetiology, in the identification of novel biomarkers or in the standardization of biomimetic platforms to assay new therapeutic strategies for preclinical studies.

Variants in DTNA cause a mild, dominantly inherited muscular dystrophy.

DTNA encodes α-dystrobrevin, a component of the macromolecular dystrophin-glycoprotein complex (DGC) that binds to dystrophin/utrophin and α-syntrophin. Mice lacking α-dystrobrevin have a muscular dystrophy phenotype, but variants in DTNA have not previously been associated with human skeletal muscle disease. We present 12 individuals from four unrelated families with two different monoallelic DTNA variants affecting the coiled-coil domain of α-dystrobrevin. The five affected individuals from family A harbor a c.1585G > A; p.Glu529Lys variant, while the recurrent c.1567_1587del; p.Gln523_Glu529del DTNA variant was identified in the other three families (family B: four affected individuals, family C: one affected individual, and family D: two affected individuals). Myalgia and exercise intolerance, with variable ages of onset, were reported in 10 of 12 affected individuals. Proximal lower limb weakness with onset in the first decade of life was noted in three individuals. Persistent elevations of serum creatine kinase (CK) levels were detected in 11 of 12 affected individuals, 1 of whom had an episode of rhabdomyolysis at 20 years of age. Autism spectrum disorder or learning disabilities were reported in four individuals with the c.1567_1587 deletion. Muscle biopsies in eight affected individuals showed mixed myopathic and dystrophic findings, characterized by fiber size variability, internalized nuclei, and slightly increased extracellular connective tissue and inflammation. Immunofluorescence analysis of biopsies from five affected individuals showed reduced α-dystrobrevin immunoreactivity and variably reduced immunoreactivity of other DGC proteins: dystrophin, α, ß, δ and γ-sarcoglycans, and α and ß-dystroglycans. The DTNA deletion disrupted an interaction between α-dystrobrevin and syntrophin. Specific variants in the coiled-coil domain of DTNA cause skeletal muscle disease with variable penetrance. Affected individuals show a spectrum of clinical manifestations, with severity ranging from hyperCKemia, myalgias, and exercise intolerance to childhood-onset proximal muscle weakness. Our findings expand the molecular etiologies of both muscular dystrophy and paucisymptomatic hyperCKemia, to now include monoallelic DTNA variants as a novel cause of skeletal muscle disease in humans.

Transcriptomic profiling reveals distinct subsets of immune checkpoint inhibitor induced myositis.

OBJECTIVES: Inflammatory myopathy or myositis is a heterogeneous family of immune-mediated diseases including dermatomyositis (DM), antisynthetase syndrome (AS), immune-mediated necrotising myopathy (IMNM) and inclusion body myositis (IBM). Immune checkpoint inhibitors (ICIs) can also cause myositis (ICI-myositis). This study was designed to define gene expression patterns in muscle biopsies from patients with ICI-myositis. METHODS: Bulk RNA sequencing was performed on 200 muscle biopsies (35 ICI-myositis, 44 DM, 18 AS, 54 IMNM, 16 IBM and 33 normal muscle biopsies) and single nuclei RNA sequencing was performed on 22 muscle biopsies (seven ICI-myositis, four DM, three AS, six IMNM and two IBM). RESULTS: Unsupervised clustering defined three distinct transcriptomic subsets of ICI-myositis: ICI-DM, ICI-MYO1 and ICI-MYO2. ICI-DM included patients with DM and anti-TIF1γ autoantibodies who, like DM patients, overexpressed type 1 interferon-inducible genes. ICI-MYO1 patients had highly inflammatory muscle biopsies and included all patients that developed coexisting myocarditis. ICI-MYO2 was composed of patients with predominant necrotising pathology and low levels of muscle inflammation. The type 2 interferon pathway was activated both in ICI-DM and ICI-MYO1. Unlike the other types of myositis, all three subsets of ICI-myositis patients overexpressed genes involved in the IL6 pathway. CONCLUSIONS: We identified three distinct types of ICI-myositis based on transcriptomic analyses. The IL6 pathway was overexpressed in all groups, the type I interferon pathway activation was specific for ICI-DM, the type 2 IFN pathway was overexpressed in both ICI-DM and ICI-MYO1 and only ICI-MYO1 patients developed myocarditis.

The pattern of MHC class I expression in muscle biopsies from patients with myositis and other neuromuscular disorders.

OBJECTIVE: Diagnostic muscle biopsies are routinely immunostained for major histocompatibility complex class I (MHC-I) protein. In this study we analysed the prevalence and patterns of MHC-I immunostaining in biopsies from patients with different types of myopathies and neurogenic disorders. METHODS: All 357 diagnostic muscle biopsies processed at the Johns Hopkins Neuromuscular Pathology Laboratory from August 2013 to January 2017 were immunostained for MHC-I. The prevalence and patterns of MHC-I immunostaining were compared between patients with histologically normal muscle biopsies (n = 31), idiopathic inflammatory myopathies (IIMs; n = 170), non-inflammatory myopathies (n = 60) and neurogenic disorders (n = 96). RESULTS: MHC-I immunostaining was abnormal in most patients with DM (98%), sporadic IBM (sIBM; 100%), immune-mediated necrotizing myopathy (IMNM; 100%) and polymyositis (77%). In contrast, MHC-I immunostaining was less frequently present in non-inflammatory myopathies (32%) or neurogenic disorders (30%). Overall, abnormal MHC-I immunostaining had a sensitivity of 0.95 and a specificity of 0.82 for diagnosing IIMs. A focal MHC-I staining pattern was associated with IMNM, whereas a global pattern was more prevalent in sIBM and a perifascicular pattern was significantly more common in dermatomyositis. Among 18 DM biopsies without perifascicular atrophy, 50% had a perifascicular MHC-I staining pattern. Sarcoplasmic upregulation staining was more common than sarcolemmal staining across all groups. CONCLUSION: MHC-I immunostaining was useful to distinguish IIMs from non-inflammatory myopathies or neurogenic disorders. Of note, a perifascicular MHC-I staining pattern was present only in those with DM, including half of those without perifascicular atrophy; many of these biopsies may not otherwise have been diagnostic for DM.

Clinico-pathological phenotypes of systemic sclerosis-associated myopathy: analysis of a large multicentre cohort.

OBJECTIVE: The objective of this study was to analyse the clinico-serological and histological phenotypes of patients with SSc with associated myopathy. METHODS: From November 2002 to September 2020, 52 patients with SSc underwent a muscle biopsy for suspected myopathy. We established two subgroups according to the histological findings based on the presence of isolated fibrosis or fibrosis together with significant inflammation. These patterns were designated as fibrosing and inflammatory, respectively. Clinical data, antibody profile, electrophysiologic studies, muscle biopsy findings and data regarding treatment, mortality and survival were compared between the two groups. RESULTS: Fourteen biopsies had a fibrosing pattern, whereas 26 showed an inflammatory pattern that could be classified (according to the predominant pattern) into DM (n = 7), necrotizing myopathy (n = 4) and non-specific myositis (n = 15). Additionally, 12 muscle biopsies were reported as neurogenic atrophy (n = 2), or normal muscle or minimal changes (n = 10). Compared with the inflammatory group, SSc patients with the fibrosing pattern presented a higher prevalence of ischaemic heart disease (38.5% vs 3.8%, P = 0.011), conduction abnormalities or arrhythmias (61.5% vs 26.9%, P = 0.036), anti-topo I antibodies (42.9% vs 11.5%, P = 0.044), greater median ESR (53.5 mm/h vs 32.5 mm/h, P = 0.013), with poor response to treatment and a higher mortality (42.9% vs 3.8%, P = 0.004) and lower cumulative survival (P = 0.035). CONCLUSIONS: Patients with SSc-associated myopathy require a comprehensive approach that encompasses clinical, serological and histopathological aspects, given their outcome predictive capacity. At least two different phenotypes can be drawn, considering clinico-pathological features. Significant differences are delineated between both a fibrotic and an inflammatory phenotype.

Coexisting autoantibodies against transcription factor Sp4 are associated with decreased cancer risk in patients with dermatomyositis with anti-TIF1γ autoantibodies.

OBJECTIVES: In dermatomyositis (DM), autoantibodies are associated with unique clinical phenotypes. For example, anti-TIF1γ autoantibodies are associated with an increased risk of cancer. The purpose of this study was to discover novel DM autoantibodies. METHODS: Phage ImmunoPrecipitation Sequencing using sera from 43 patients with DM suggested that transcription factor Sp4 is a novel autoantigen; this was confirmed by showing that patient sera immunoprecipitated full-length Sp4 protein. Sera from 371 Johns Hopkins patients with myositis (255 with DM, 28 with antisynthetase syndrome, 40 with immune-mediated necrotising myopathy, 29 with inclusion body myositis and 19 with polymyositis), 80 rheumatological disease controls (25 with Sjogren's syndrome, 25 with systemic lupus erythematosus and 30 with rheumatoid arthritis (RA)) and 200 healthy comparators were screened for anti-SP4 autoantibodies by ELISA. A validation cohort of 46 anti-TIF1γ-positive patient sera from the University of Pittsburgh was also screened for anti-Sp4 autoantibodies. RESULTS: Anti-Sp4 autoantibodies were present in 27 (10.5%) patients with DM and 1 (3.3%) patient with RA but not in other clinical groups. In patients with DM, 96.3% of anti-Sp4 autoantibodies were detected in those with anti-TIF1γ autoantibodies. Among 26 TIF1γ-positive patients with anti-Sp4 autoantibodies, none (0%) had cancer. In contrast, among 35 TIF1γ-positive patients without anti-Sp4 autoantibodies, 5 (14%, p=0.04) had cancer. In the validation cohort, among 15 TIF1γ-positive patients with anti-Sp4 autoantibodies, 2 (13.3%) had cancer. By comparison, among 31 TIF1γ-positive patients without anti-Sp4 autoantibodies, 21 (67.7%, p<0.001) had cancer. CONCLUSIONS: Anti-Sp4 autoantibodies appear to identify a subgroup of anti-TIF1γ-positive DM patients with lower cancer risk.

Neuronal induction and bioenergetics characterization of human forearm adipose stem cells from Parkinson's disease patients and healthy controls.

Neurodegenerative diseases, such as Parkinson's disease, are heterogeneous disorders with a multifactorial nature involving impaired bioenergetics. Stem-regenerative medicine and bioenergetics have been proposed as promising therapeutic targets in the neurologic field. The rationale of the present study was to assess the potential of human-derived adipose stem cells (hASCs) to transdifferentiate into neuronal-like cells (NhASCs and neurospheres) and explore the hASC bioenergetic profile. hASC neuronal transdifferentiation was performed through neurobasal media and differentiation factor exposure. High resolution respirometry was assessed. Increased MAP-2 neuronal marker protein expression upon neuronal induction (p<0.05 undifferentiated hASCs vs. 28-36 days of differentiation) and increased bIII-tubulin neuronal marker protein expression upon neuronal induction (p<0.05 undifferentiated hASCs vs. 6-28-36 days of differentiation) were found. The bioenergetic profile was detectable through high-resolution respirometry approaches in hASCs but did not lead to differential oxidative capacity rates in healthy or clinically diagnosed PD-hASCs. We confirmed the capability of transdifferentiation to the neuronal-like profile of hASCs derived from the forearms of human subjects and characterized the bioenergetic profile. Suboptimal maximal respiratory capacity trends in PD were found. Neuronal induction leading to positive neuronal protein expression markers is a relevant issue that encourages the suitability of NhASC models in neurodegeneration.

Differential diagnosis of necrotizing myopathy.

PURPOSE OF REVIEW: Necrotizing myopathy is a broad term. It includes patients with the recently described immune-mediated necrotizing myopathies (IMNM) who have specific antibodies, such as anti-hydroxy-3-methylglutaryl-CoA reductase or anti-signal recognition particle, seronegative phenotypes that can be associated with cancer, and other types of myositis and connective tissue diseases involving necrotic muscle fibers as a characteristic pathologic feature. Necrotizing myopathies that are not immune-mediated, such as those caused by drugs, dystrophies, infections, or even hypothyroidism are also included. The purpose of this review is to address the differential diagnosis of these disorders. RECENT FINDINGS: New IMNM have been described over the last few years, some of them related with checkpoint inhibitors, drugs that are being increasingly used in cancer treatment. Necrotizing myopathy has also been reported in association with specific phenotypes and autoantibodies (e.g. anti-Mi2 dermatomyositis, antisynthetase syndrome, and myositis associated with antimitochondrial antibodies). Rarer cases associated with graft-versus-host disease and severe acute respiratory syndrome coronavirus 2 infection are also emerging. SUMMARY: Differentiation between patients with IMNM and those without the superimposed autoimmune phenomena helps clinicians determine the best individualized approach to use and the appropriate immunosuppressive therapy, whenever needed.

Circulating Cell-Free Mitochondrial DNA in Cerebrospinal Fluid as a Biomarker for Mitochondrial Diseases.

BACKGROUND: Mitochondrial diseases (MD) are genetic metabolic disorders that impair normal mitochondrial structure or function. The aim of this study was to investigate the status of circulating cell-free mitochondrial DNA (ccfmtDNA) in cerebrospinal fluid (CSF), together with other biomarkers (growth differentiation factor-15 [GDF-15], alanine, and lactate), in a cohort of 25 patients with a molecular diagnosis of MD. METHODS: Measurement of ccfmtDNA was performed by using droplet digital PCR. RESULTS: The mean copy number of ccfmtDNA was approximately 6 times higher in the MD cohort compared to the control group; patients with mitochondrial deletion and depletion syndromes (MDD) had the higher levels. We also detected the presence of both wild-type mtDNA and mtDNA deletions in CSF samples of patients with single deletions. Patients with MDD with single deletions had significantly higher concentrations of GDF-15 in CSF than controls, whereas patients with point mutations in mitochondrial DNA presented no statistically significant differences. Additionally, we found a significant positive correlation between ccfmtDNA levels and GDF-15 concentrations (r = 0.59, P = 0.016). CONCLUSION: CSF ccfmtDNA levels are significantly higher in patients with MD in comparison to controls and, thus, they can be used as a novel biomarker for MD research. Our results could also be valuable to support the clinical outcome assessment of MD patients.

The Impact of Mitochondrial Deficiencies in Neuromuscular Diseases.

Neuromuscular diseases (NMDs) are a heterogeneous group of acquired or inherited rare disorders caused by injury or dysfunction of the anterior horn cells of the spinal cord (lower motor neurons), peripheral nerves, neuromuscular junctions, or skeletal muscles leading to muscle weakness and waste. Unfortunately, most of them entail serious or even fatal consequences. The prevalence rates among NMDs range between 1 and 10 per 100,000 population, but their rarity and diversity pose difficulties for healthcare and research. Some molecular hallmarks are being explored to elucidate the mechanisms triggering disease, to set the path for further advances. In fact, in the present review we outline the metabolic alterations of NMDs, mainly focusing on the role of mitochondria. The aim of the review is to discuss the mechanisms underlying energy production, oxidative stress generation, cell signaling, autophagy, and inflammation triggered or conditioned by the mitochondria. Briefly, increased levels of inflammation have been linked to reactive oxygen species (ROS) accumulation, which is key in mitochondrial genomic instability and mitochondrial respiratory chain (MRC) dysfunction. ROS burst, impaired autophagy, and increased inflammation are observed in many NMDs. Increasing knowledge of the etiology of NMDs will help to develop better diagnosis and treatments, eventually reducing the health and economic burden of NMDs for patients and healthcare systems.

Machine learning algorithms reveal unique gene expression profiles in muscle biopsies from patients with different types of myositis.

OBJECTIVES: Myositis is a heterogeneous family of diseases that includes dermatomyositis (DM), antisynthetase syndrome (AS), immune-mediated necrotising myopathy (IMNM), inclusion body myositis (IBM), polymyositis and overlap myositis. Additional subtypes of myositis can be defined by the presence of myositis-specific autoantibodies (MSAs). The purpose of this study was to define unique gene expression profiles in muscle biopsies from patients with MSA-positive DM, AS and IMNM as well as IBM. METHODS: RNA-seq was performed on muscle biopsies from 119 myositis patients with IBM or defined MSAs and 20 controls. Machine learning algorithms were trained on transcriptomic data and recursive feature elimination was used to determine which genes were most useful for classifying muscle biopsies into each type and MSA-defined subtype of myositis. RESULTS: The support vector machine learning algorithm classified the muscle biopsies with >90% accuracy. Recursive feature elimination identified genes that are most useful to the machine learning algorithm and that are only overexpressed in one type of myositis. For example, CAMK1G (calcium/calmodulin-dependent protein kinase IG), EGR4 (early growth response protein 4) and CXCL8 (interleukin 8) are highly expressed in AS but not in DM or other types of myositis. Using the same computational approach, we also identified genes that are uniquely overexpressed in different MSA-defined subtypes. These included apolipoprotein A4 (APOA4), which is only expressed in anti-3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) myopathy, and MADCAM1 (mucosal vascular addressin cell adhesion molecule 1), which is only expressed in anti-Mi2-positive DM. CONCLUSIONS: Unique gene expression profiles in muscle biopsies from patients with MSA-defined subtypes of myositis and IBM suggest that different pathological mechanisms underly muscle damage in each of these diseases.

Mitochondrial Dysfunction: A Common Hallmark Underlying Comorbidity between sIBM and Other Degenerative and Age-Related Diseases.

Sporadic inclusion body myositis (sIBM) is an inflammatory myopathy associated, among others, with mitochondrial dysfunction. Similar molecular features are found in Alzheimer's disease (AD) and Type 2 Diabetes Mellitus (T2DM), underlying potential comorbidity. This study aims to evaluate common clinical and molecular hallmarks among sIBM, AD, and T2DM. Comorbidity with AD was assessed in n = 14 sIBM patients by performing neuropsychological and cognitive tests, cranial magnetic resonance imaging, AD cerebrospinal fluid biomarkers (levels of amyloid beta, total tau, and phosphorylated tau at threonine-181), and genetic apolipoprotein E genotyping. In the same sIBM cohort, comorbidity with T2DM was assessed by collecting anthropometric measures and performing an oral glucose tolerance test and insulin determinations. Results were compared to the standard population and other myositis (n = 7 dermatomyositis and n = 7 polymyositis). Mitochondrial contribution into disease was tested by measurement of oxidative/anaerobic and oxidant/antioxidant balances, respiration fluxes, and enzymatic activities in sIBM fibroblasts subjected to different glucose levels. Comorbidity of sIBM with AD was not detected. Clinically, sIBM patients showed signs of misbalanced glucose homeostasis, similar to other myositis. Such misbalance was further confirmed at the molecular level by the metabolic inability of sIBM fibroblasts to adapt to different glucose conditions. Under the standard condition, sIBM fibroblasts showed decreased respiration (0.71 ± 0.08 vs. 1.06 ± 0.04 nmols O2/min; p = 0.024) and increased anaerobic metabolism (5.76 ± 0.52 vs. 3.79 ± 0.35 mM lactate; p = 0.052). Moreover, when glucose conditions were changed, sIBM fibroblasts presented decreased fold change in mitochondrial enzymatic activities (-12.13 ± 21.86 vs. 199.22 ± 62.52 cytochrome c oxidase/citrate synthase ratio; p = 0.017) and increased oxidative stress per mitochondrial activity (203.76 ± 82.77 vs. -69.55 ± 21.00; p = 0.047), underlying scarce metabolic plasticity. These findings do not demonstrate higher prevalence of AD in sIBM patients, but evidences of prediabetogenic conditions were found. Glucose deregulation in myositis suggests the contribution of lifestyle conditions, such as restricted mobility. Additionally, molecular evidences from sIBM fibroblasts confirm that mitochondrial dysfunction may play a role. Monitoring T2DM development and mitochondrial contribution to disease in myositis patients could set a path for novel therapeutic options.

Neutrophil dysregulation is pathogenic in idiopathic inflammatory myopathies.

Idiopathic inflammatory myopathies (IIM) are characterized by muscle inflammation and weakness, myositis-specific autoantibodies (MSAs), and extramuscular organ damage. The role of neutrophil dysregulation and neutrophil extracellular traps (NETs) in IIM is unclear. We assessed whether pathogenic neutrophil subsets (low-density granulocytes [LDGs]) and NETs were elevated in IIM, associated with clinical presentation and MSAs, and their effect on skeletal myoblasts and myotubes. Circulating NETs and LDGs were quantified and correlated with clinical measures. Specific MSAs were tested for their ability to induce NETs. NETs and neutrophil gene expression were measured in IIM biopsies. Whether NETs damage skeletal myoblasts and myotubes was tested. Circulating LDGs and NETs were increased in IIM. IIM LDGs had an enhanced ability to form NETs. LDGs and NETs correlated with IIM disease activity and muscle damage. The serum MSA anti-MDA5 correlated with circulating and tissue NETs and directly enhanced NET formation. An enhanced neutrophil gene signature was present in IIM muscle and associated with muscle injury and tissue IFN gene signatures. IIM NETs decreased the viability of myotubes in a citrullinated histone-dependent manner. Dysregulated neutrophil pathways may play pathogenic roles in IIM through their ability to directly injure muscle cells and other affected tissues.