GGC Repeat Expansion of RILPL1 is Associated with Oculopharyngodistal Myopathy.

OBJECTIVE: Oculopharyngodistal myopathy (OPDM) is an adult-onset neuromuscular disease characterized by progressive ptosis, dysarthria, ophthalmoplegia, and distal muscle weakness. Recent studies revealed that GGC repeat expansions in 5'-UTR of LRP12, GIPC1, and NOTCH2NLC are associated with OPDM. Despite these advances, approximately 30% of OPDM patients remain genetically undiagnosed. Herein, we aim to investigate the genetic basis for undiagnosed OPDM patients in two unrelated Chinese Han families. METHODS: Parametric linkage analysis was performed. Long-read sequencing followed by repeat-primed polymerase chain reaction and amplicon length polymerase chain reaction were used to determine the genetic cause. Targeted methylation sequencing was implemented to detect epigenetic changes. The possible pathogenesis mechanism was investigated by quantitative polymerase chain reaction, immunoblotting, RNA fluorescence in situ hybridization, and immunofluorescence staining of muscle biopsy samples. RESULTS: The disease locus was mapped to 12q24.3. Subsequently, GGC repeat expansion in the promoter region of RILPL1 was identified in six OPDM patients from two families, findings consistent with a founder effect, designated as OPDM type 4. Targeted methylation sequencing revealed hypermethylation at the RILPL1 locus in unaffected individuals with ultralong expansion. Analysis of muscle samples showed no significant differences in RILPL1 mRNA or RILPL1 protein levels between patients and controls. Public CAGE-seq data indicated that alternative transcription start sites exist upstream of the RefSeq-annotated RILPL1 transcription start site. Strand-specific RNA-seq data revealed bidirectional transcription from the RILPL1 locus. Finally, fluorescence in situ hybridization/immunofluorescence staining showed that both sense and antisense transcripts formed RNA foci, and were co-localized with hnRNPA2B1 and p62 in the intranuclear inclusions of OPDM type 4 patients. INTERPRETATION: Our findings implicate abnormal GGC repeat expansions in the promoter region of RILPL1 as a novel genetic cause for OPDM, and suggest a methylation mechanism and a potential RNA toxicity mechanism are involved in OPDM type 4 pathogenesis. ANN NEUROL 2022;92:512-526.

Comprehensive Proteomic Analysis of Dysferlinopathy Unveiling Molecular Mechanisms and Biomarkers Linked to Pathological Progression.

AIMS: Previous proteomics studies in dysferlinopathy muscle have been limited in scope, often utilizing 2D-electrophoresis and yielding only a small number of differential expression calls. To address this gap, this study aimed to employ high-resolution proteomics to explore the proteomic landscapes of dysferlinopathy and analyze the correlation between muscle pathological changes and alterations in protein expression in muscle biopsies. METHODS: We conducted a comprehensive approach to investigate the proteomic profile and disease-associated changes in the muscle tissue proteome from 15 patients with dysferlinopathy, exhibiting varying degrees of dystrophic pathology, alongside age-matched controls. Our methodology encompasses tandem mass tag (TMT)-labeled liquid chromatography-mass spectrometry (LC-MS/MS)-based proteomics, protein-protein interaction (PPI) network analysis, weighted gene co-expression network analysis, and differential expression analysis. Subsequently, we examined the correlation between the expression of key proteins and the clinical characteristics of the patients to identify pathogenic targets associated with DYSF mutations in dysferlinopathy. RESULTS: A total of 1600 differentially expressed proteins were identified, with 1321 showing high expression levels and 279 expressed at lower levels. Our investigation yields a molecular profile delineating the altered protein networks in dysferlinopathy-afflicted skeletal muscle, uncovering dysregulation across numerous cellular pathways and molecular processes, including mRNA metabolic processes, regulated exocytosis, immune response, muscle system processes, energy metabolic processes, and calcium transmembrane transport. Moreover, we observe significant associations between the protein expression of ANXA1, ANXA2, ANXA4, ANXA5, LMNA, PYGM, and the extent of histopathologic changes in muscle biopsies from patients with dysferlinopathy, validated through immunoblotting and immunofluorescence assays. CONCLUSIONS: Through the aggregation of expression data from dysferlinopathy-impacted muscles exhibiting a range of pathological alterations, we identified multiple key proteins associated with the dystrophic pathology of patients with dysferlinopathy. These findings provide novel insights into the pathogenesis of dysferlinopathy and propose promising targets for future therapeutic endeavors.

Detection of pTDP-43 via routine muscle biopsy: A promising diagnostic biomarker for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, pathologically characterized by TDP-43 aggregates. Recent evidence has been indicated that phosphorylated TDP-43 (pTDP-43) is present not only in motor neurons but also in muscle tissues. However, it is unclear whether testing pTDP-43 aggregation in muscle tissue would assist in the diagnosis of ALS. We propose three key questions: (i) Is aggregation of pTDP-43 detectable in routine biopsied muscles? (ii) Can detection of pTDP-43 aggregation discriminate between ALS and non-ALS patients? (iii) Can pTDP-43 aggregation be observed in the early stages of ALS? We conducted a diagnostic study comprising 2 groups: an ALS group in which 18 cases underwent muscle biopsy screened from a registered ALS cohort consisting of 802 patients and a non-ALS control group, in which we randomly selected 54 muscle samples from a biospecimen bank of 684 patients. Among the 18 ALS patients, 3 patients carried pathological GGGGCC repeats in the C9ORF72 gene, 2 patients carried SOD1 mutations, and 7 patients were at an early stage with only one body region clinically affected. The pTDP-43 accumulation could be detected in routine biopsied muscles, including biceps brachii, deltoid, tibialis anterior, and quadriceps. Abnormal aggregation of pTDP-43 was present in 94.4% of ALS patients (17/18) compared to 29.6% of non-ALS controls (16/54; p < 0.001). The pTDP-43 aggregates were mainly close to the sarcolemma. Using a semi-quantified pTDP-43 aggregates score, we applied a cut-off value of 3 as a diagnostic biomarker, resulting in a sensitivity of 94.4% and a specificity of 83.3%. Moreover, we observed that accumulation of pTDP-43 occurred in muscle tissues prior to clinical symptoms and electromyographic lesions. Our study provides proof-of-concept for the detection of pTDP-43 accumulation via routine muscle biopsy which may serve as a novel biomarker for diagnosis of ALS.

The phenotypic and genotypic features of Chinese patients with oculopharyngeal muscular dystrophy.

OBJECTIVE: Oculopharyngeal muscular dystrophy (OPMD) is a late-onset inherited neuromuscular disorder, with progressive ptosis and dysphagia as common manifestations. To date, OPMD has rarely been reported among East Asians. The present study summarizes the phenotypic and genotypic features of Chinese patients with OPMD. METHODS: Twenty-one patients with molecularly confirmed OPMD from 9 unrelated families were identified by direct sequencing of the polyadenlyate binding protein nuclear-1 (PABPN1) gene. Immunofluorescence staining of muscle biopsies was conducted to identify the components of protein degradation pathways involved in OPMD. RESULTS: In our cohort, the genetically confirmed OPMD group had a mean age at onset of 50.6 ± 4.2 years (range 45-60 years). Ptosis (42.9%) was the most common initial symptom; patients with ptosis as the first symptom subsequently developed dysphagia within a median time of 5.5 years (range 1-19 years). Evidence of external ophthalmoplegia was found in 38.1% of patients. A total of 33.3% of the patients developed muscle weakness at a median age at onset of 66 years (range 50-70 years), with neck flexor involvement in all patients. Five genotypes were observed in our cohort, including classical (GCG)9-11 repeats in 7 families and non-GCG elongations with additional GCA expansions in 2 families. OPMD muscle biopsies revealed rimmed vacuoles and intranuclear filamentous inclusions. The PABPN1 protein showed substantial accumulation in the nuclei of muscle fiber aggregates and closely colocalized with p62, LC3B and FK2. INTERPRETATION: Our findings indicate wide genetic heterogeneity in OPMD in the Chinese population and demonstrate abnormalities in protein degradation pathways in this disease.

Clinical features, imaging findings and molecular data of limb-girdle muscular dystrophies in a cohort of Chinese patients.

BACKGROUND: Limb-girdle muscular dystrophies (LGMDs) are a group of heterogeneous inherited diseases predominantly characterized by limb-girdle muscle weakness and dystrophic changes on histological analysis. The frequency of LGMD subtypes varies among regions in China and ethnic populations worldwide. Here, we analyzed the prevalence of LGMD subtypes, their corresponding clinical manifestations, and molecular data in a cohort of LGMD patients in Southeast China. METHODS: A total of 81 consecutive patients with clinically suspected LGMDs from 62 unrelated families across Southeast China were recruited for targeted next-generation sequencing and whole-exome sequencing from July 2017 to February 2020. RESULTS: Among 50 patients (41 families) with LGMDs, the most common subtypes were LGMD-R2/LGMD2B (36.6%) and LGMD-R1/LGMD2A (29.3%). Dystroglycanopathies (including LGMD-R9/LGMD2I, LGMD-R11/LGMD2K, LGMD-R14/LGMD2N and LGMD-R20/LGMD2U) were the most common childhood-onset subtypes and were found in 12.2% of the families. A total of 14.6% of the families had the LGMD-R7/LGMD2G subtype, and the mutation c.26_33dupAGGTGTCG in TCAP was the most frequent (83.3%). The only patient with the rare subtype LGMD-R18/LGMD2S had TRAPPC11 mutations; had a later onset than those previously reported, and presented with proximal‒distal muscle weakness, walking aid dependency, fatty liver disease and diabetes at 33 years of age. A total of 22.0% of the patients had cardiac abnormalities, and one patient with LMNA-related muscular dystrophy/LGMD1B experienced sudden cardiac death at 37 years of age. A total of 15.4% of the patients had restrictive respiratory insufficiency. Muscle imaging in patients with LGMD-R1/LGMD2A and LGMD-R2/LGMD2B showed subtle differences, including more severe fatty infiltration of the posterior thigh muscles in those with LGMD-R1/LGMD2A and edema in the lower leg muscles in those with LGMD-R2/LGMD2B. CONCLUSION: We determined the prevalence of different LGMD subtypes in Southeast China, described the detailed clinical manifestations and distinct muscle MRI patterns of these LGMD subtypes and reported the frequent mutations and the cardiorespiratory involvement frequency in our cohort, all of which might facilitate the differential diagnosis of LGMDs, allowing more timely treatment and guiding future clinical trials.