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Oculopharyngodistal myopathy (OPDM) is an adult-onset neuromuscular disease characterized by progressive ocular, facial, pharyngeal and distal limb muscle involvement. Trinucleotide repeat expansions in LRP12 or GIPC1 were recently reported to be associated with OPDM. However, a significant portion of OPDM patients have unknown genetic causes. In this study long-read whole-genome sequencing and repeat-primed polymerase chain reaction were performed and we identified GGC repeat expansions in the NOTCH2NLC gene in 16.7% (4/24) of a cohort of Chinese OPDM patients, designated as OPDM type 3 (OPDM3). Methylation analysis indicated that methylation levels of the NOTCH2NLC gene were unaltered in OPDM3 patients, but increased significantly in asymptomatic carriers. Quantitative real-time PCR analysis indicated that NOTCH2NLC mRNA levels were increased in muscle but not in blood of OPDM3 patients. Immunofluorescence on OPDM muscle samples and expressing mutant NOTCH2NLC with (GGC)69 repeat expansions in HEK293 cells indicated that mutant NOTCH2NLC-polyGlycine protein might be a major component of intranuclear inclusions, and contribute to toxicity in cultured cells. In addition, two RNA-binding proteins, hnRNP A/B and MBNL1, were both co-localized with p62 in intranuclear inclusions in OPDM muscle samples. These results indicated that a toxic protein gain-of-function mechanism and RNA gain-of-function mechanism may both play a vital role in the pathogenic processes of OPDM3. This study extended the spectrum of NOTCH2NLC repeat expansion related diseases to a predominant myopathy phenotype presenting as OPDM, and provided evidence for possible pathogenesis of these diseases.

Background Erythrocytes are a major source of peripheral α-synuclein (α-Syn). The goal of the current investigation is to evaluate erythrocytic total, oligomeric/aggregated, and phosphorylated α-Syn species as biomarkers of Parkinson’s disease (PD). PD and healthy control blood samples were collected along with extensive clinical history to determine whether total, phosphorylated, or aggregated α-Syn derived from erythrocytes (the major source of blood α-Syn) are more promising and consistent biomarkers for PD than are free α-Syn species in serum or plasma. Methods Using newly developed electrochemiluminescence assays, concentrations of erythrocytic total, aggregated and phosphorylated at Ser129 (pS129) α-Syn, separated into membrane and cytosolic components, were measured in 225 PD patients and 133 healthy controls and analyzed with extensive clinical measures. Results The total and aggregated α-Syn levels were significantly higher in the membrane fraction of PD patients compared to healthy controls, but without alterations in the cytosolic component. The pS129 level was remarkably higher in PD subjects than in controls in the cytosolic fraction, and to a lesser extent, higher in the membrane fraction. Combining age, erythrocytic membrane aggregated α-Syn, and cytosolic pS129 levels, a model generated by using logistic regression analysis was able to discriminate patients with PD from neurologically normal controls, with a sensitivity and a specificity of 72 and 68%, respectively. Conclusions These results suggest that total, aggregated and phosphorylated α-Syn levels are altered in PD erythrocytes and peripheral erythrocytic α-Syn is a potential PD biomarker that needs further validation. Electronic supplementary material The online version of this article (10.1186/s40035-019-0155-y) contains supplementary material, which is available to authorized users.

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ObjectiveTo investigate the diagnostic value of implementing a stepwise genetic testing strategy (SGTS) in genetically unsolved cases with dystrophinopathies.MethodsAfter routine genetic testing in 872 male patients with highly suspected dystrophinopathies, we identified 715 patients with a pathogenic DMD variant. Of the 157 patients who had no pathogenic DMD variants and underwent a muscle biopsy, 142 patients were confirmed to have other myopathies, and 15 suspected dystrophinopathies remained genetically undiagnosed. These 15 patients underwent a more comprehensive evaluation as part of the SGTS pipeline, which included the stepwise analysis of dystrophin mRNA, short-read whole-gene DMD sequencing, long-read whole-gene DMD sequencing and in silico bioinformatic analyses.ResultsSGTS successfully yielded a molecular diagnosis of dystrophinopathy in 11 of the 15 genetically unsolved cases. We identified 8 intronic and 2 complex structural variants (SVs) leading to aberrant splicing in 10 of 11 patients, of which 9 variants were novel. In one case, a molecular defect was detected on mRNA and protein level only. Aberrant splicing mechanisms included 6 pseudoexon inclusions and 4 alterations of splice sites and splicing regulatory elements. We showed for the first time the exonisation of a MER48 element as a novel pathogenic mechanism in dystrophinopathies.ConclusionOur study highlights the high diagnostic utility of implementing a SGTS pipeline in dystrophinopathies with intronic variants and complex SVs.

Parkinson's disease is a neurodegenerative disorder characterized by the transmission and accumulation of toxic species of α-synuclein (α-syn). Extracellular vesicles (EVs) are believed to play a vital role in the spread of toxic α-syn species. Recently, peripheral α-syn pathology has been investigated, but little attention has been devoted to erythrocytes, which contain abundant α-syn. In this study, we first demonstrated that erythrocyte-derived EVs isolated from Parkinson's disease patients carried elevated levels of oligomeric α-syn, compared to those from healthy controls. Moreover, human erythrocyte-derived EVs, when injected into peripheral blood in a mouse model of Parkinson's disease, were found to readily cross the blood-brain barrier (BBB). These EVs accumulated in astrocyte endfeet, a component of the BBB, where they impaired glutamate uptake, likely via interaction between excitatory amino acid transporter 2 (EAAT2) and oligomeric α-syn. These data suggest that erythrocyte-derived EVs and the oligomeric α-syn carried in them may play critical roles in the progression or even initiation of Parkinson's disease. Additionally, the mechanisms involved are attributable at least in part to dysfunction of astrocytes induced by these EVs. These observations provide new insight into the understanding of the mechanisms involved in Parkinson's disease.

Background Sarcoglycanopathies comprise four subtypes of autosomal recessive limb-girdle muscular dystrophy (LGMD2C, LGMD2D, LGMD2E, and LGMD2F) that are caused, respectively, by mutations in the SGCG , SGCA , SGCB , and SGCD genes. Knowledge about the clinical and genetic features of sarcoglycanopathies in Chinese patients is limited. The aims of this study were to investigate in detail the clinical manifestations, sarcoglycan expression, and gene mutations in Chinese patients with sarcoglycanopathies and to identify possible correlations between them. Results Of 3638 patients for suspected neuromuscular diseases (1733 with inherited myopathies, 1557 with acquired myopathies, and 348 unknown), 756 patients had next-generation sequencing (NGS) diagnostic panel. Twenty-five patients with sarcoglycanopathies (11.5%) were identified from 218 confirmed LGMDs, comprising 18 with LGMD2D, 6 with LGMD2E, and one with LGMD2C. One patient with LGMD2D also had Charcot-Marie-Tooth 1A. The clinical phenotypes of the patients with LGMD2D or LGMD2E were markedly heterogeneous. Muscle biopsy showed a dystrophic pattern in 19 patients and mild myopathic changes in 6. The percentage of correct prediction of genotype based on expression of sarcoglycan was 36.0% (4 LGMD2D, 4 LGMD2E, and one LGMD2C). There was a statistically significant positive correlation between reduction of α-sarcoglycan level and disease severity in LGMD2D. Thirty-five mutations were identified in SGCA , SGCB , SGCG , and PMP22 , 16 of which were novel. Exon 3 of SGCA was a hotspot region for mutations in LGMD2D. The missense mutation c.662G > A (p.R221H) was the most common mutation in SGCA . Missense mutations in both alleles of SGCA were associated with a relative benign disease course. No obvious clinical, sarcoglycan expression, and genetic correlation was found in LGMD2E. Conclusions This study expands the clinical and genetic spectrum of sarcoglycanopathies in Chinese patients and provides evidence that disease severity of LGMD2D may be predicted by α-sarcoglycan expression and SGCA mutation. Electronic supplementary material The online version of this article (10.1186/s13023-019-1021-9) contains supplementary material, which is available to authorized users.

The study aimed to evaluate the body composition of patients with mitochondrial diseases (MD) and correlate it with disease severity. Overall, 89 patients (age ≥ 18 years) with MD were recruited, includin g 49 with chronic progressive external ophthalmoplegia (CPEO) and 40 with mitochondrial encephalomyopathy with lactate acidosis and stroke-like episodes (MELAS). Body composition, including fat mass index (FMI), fat-free mass index (FFMI), skeletal muscle mass index (SMI), and appendicular skeletal muscle mass index (ASMI), were examined using multifrequency bioelectric impedance analysis. Clinical assessments, including muscle strength, usual gait speed, and disease severity determined by the Newcastle Mitochondrial Disease Adult Scale score (NMDAS), were performed. The comparisons between patients group and age- and gender-matched healthy controls, as well as the correlations between anthropometric measurements, body composition, and disease severity were analyzed. Height, weight, body mass index (BMI), FFMI, SMI, and ASMI were significantly lower in patients with MD than in healthy controls. Notably, low muscle mass was noted in 69.7% (62/89) of MD patients, with 22 patients also presenting with compromised physical performance as indicated by decreased gait speed, resulting in 24.7% satisfied the sarcopenia diagnostic criteria. Disease severity was more negatively correlated with ASMI than it was with height, weight, and BMI. Subgroup analysis showed that in the MELAS subgroup, disease severity was negatively correlated with height, weight, and ASMI; whereas in the CPEO subgroup, it was only negatively correlated with ASMI and SMI. Additionally, ASMI was positively associated with muscle strength. Altogether, compared with BMI, ASMI is a more sensitive biomarker predicting disease severity of MD, both in MELAS and CPEO patients.