Nebulin, a critical protein of the skeletal muscle thin filament, plays important roles in physiological processes such as regulating thin filament length (TFL), cross-bridge cycling, and myofibril alignment. Pathogenic variants in the nebulin gene (NEB) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking curative therapies. In this study, we examined a cohort of ten NEM2 patients, each with unique pathogenic variants, aiming to understand their impact on mRNA, protein, and functional levels. Results show that pathogenic truncation variants affect NEB mRNA stability and lead to nonsense-mediated decay of the mutated transcript. Moreover, a high incidence of cryptic splice site activation was found in patients with pathogenic splicing variants that are expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with either relatively normal or markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, or reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a pathogenic duplication variant in nebulin that resulted in a four-copy gain in the triplicate region of NEB and a much larger nebulin protein and longer TFL. Additionally, we investigated the effect of Omecamtiv mecarbil (OM), a small-molecule activator of cardiac myosin, on force production of type 1 muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87 to 318%, with the largest effects in patients with the lowest level of nebulin. In summary, this study indicates that post-transcriptional or post-translational mechanisms regulate nebulin expression. Moreover, we propose that the pathomechanism of NEM2 involves not only shortened but also elongated thin filaments, along with the disruption of actin-binding sites resulting from pathogenic splicing variants. Significantly, our findings highlight the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with large reductions in nebulin levels.

Nemaline myopathy (NM) is a rare congenital neuromuscular disorder characterized by muscle weakness and hypotonia, slow gross motor development, and decreased respiratory function. Mutations in at least twelve genes, all of each encode proteins that are either components of the muscle thin filament or regulate its length and stability, have been associated with NM. Mutations in Nebulin (NEB), a giant filamentous protein localized in the sarcomere, account for more than 50% of NM cases. At present, there remains a lack of understanding of whether NEB genotype influences nebulin function and NM-patient phenotypes. In addition, there is a lack of therapeutically tractable models that can enable drug discovery and address the current unmet treatment needs of patients. To begin to address these gaps, here we have characterized five new zebrafish models of NEB-related NM. These mutants recapitulate most aspects of NEB-based NM, showing drastically reduced survival, defective muscle structure, reduced contraction force, shorter thin filaments, presence of electron-dense structures in myofibers, and thickening of the Z-disks. This study represents the first extensive investigation of an allelic series of nebulin mutants, and thus provides an initial examination in pre-clinical models of potential genotype-phenotype correlations in human NEB patients. It also represents the first utilization of a set of comprehensive outcome measures in zebrafish, including correlation between molecular analyses, structural and biophysical investigations, and phenotypic outcomes. Therefore, it provides a rich source of data for future studies exploring the NM pathomechanisms, and an ideal springboard for therapy identification and development for NEB-related NM.

HIV-associated myopathies include HIV-associated polymyositis, inclusion body myositis, diffuse infiltrative lymphocytosis syndrome and sporadic late-onset nemaline myopathy (HIV-NM). HIV-NM typically manifests as a painless, progressive proximal and axial muscle weakness with characteristic histological findings of intracytoplasmic rods, or nemaline bodies, seen in atrophic muscle fibres. HIV-NM presents prior to or shortly after initiation of antiretroviral therapy (ART) and is treated with intravenous immunoglobulin, glucocorticoids or immunosuppression. We present a case of HIV-NM in a patient with well-controlled HIV on decades-long ART with progressive bent spine syndrome, or camptocormia. This case highlights the importance of considering HIV-associated myopathies such as HIV-NM in patients with HIV who present with musculoskeletal complaints.

Mutations in the KLHL40 gene are a common cause of severe or even lethal nemaline myopathy. Some cases with mild forms have been described, although the cases are still anecdotal. The aim of this paper was to systematically review the cases described in the literature and to describe a 12-year clinical and imaging follow-up in an Italian patient with KLHL40- related myopathy in order to suggest possible follow-up measurements.
Having searched through three electronic databases (PubMed, Scopus, and EBSCO), 18 articles describing 65 patients with homozygous or compound heterozygous KLHL40 mutations were selected. A patient with a KLHL40 homozygous mutation (c.1582G>A/p.E528K) was added and clinical and genetic data were collected.
The most common mutation identified in our systematic review was the (c.1516A>C) followed by the (c.1582G>A). In our review, 60% percent of the patients died within the first 4 years of life. Clinical features were similar across the sample. Unfortunately, however, there is no record of the natural history data in the surviving patients. The 12-year follow-up of our patient revealed a slow improvement in her clinical course, identifying muscle MRI as the only possible marker of disease progression.
Due to its clinical and genotype homogeneity, KLHL40-related myopathy may be a condition that would greatly benefit from the development of new gene therapies; muscle MRI could be a good biomarker to monitor disease progression.

Rare or novel missense variants in large genes such as TTN and NEB are frequent in the general population, which hampers the interpretation of putative disease-causing biallelic variants in patients with sporadic neuromuscular disorders. Often, when the first initial genetic analysis is performed, the reconstructed haplotype, i.e. phasing information of the variants is missing. Segregation analysis increases the diagnostic turnaround time and is not always possible if samples from family members are lacking. To overcome this difficulty, we investigated how well the linked-read technology succeeded to phase variants in these large genes, and whether it improved the identification of structural variants. Linked-read sequencing data of nemaline myopathy, distal myopathy, and proximal myopathy patients were analyzed for phasing, single nucleotide variants, and structural variants. Variant phasing was successful in the large muscle genes studied. The longest continuous phase blocks were gained using high-quality DNA samples with long DNA fragments. Homozygosity increased the number of phase blocks, especially in exome sequencing samples lacking intronic variation. In our cohort, linked-read sequencing added more information about the structural variation but did not lead to a molecular genetic diagnosis. The linked-read technology can support the clinical diagnosis of neuromuscular and other genetic disorders.

Nemaline myopathies are the most common form of congenital myopathies. Variants in ACTA1 (NEM3) comprise 15-25% of all nemaline myopathy cases. Patients harboring variants in ACTA1 present with a heterogeneous disease course characterized by stable or progressive muscle weakness and, in severe cases, respiratory failure and death. To date, no specific treatments are available. Since NEM3 is an actin-based thin filament disease, we tested the ability of tirasemtiv, a fast skeletal muscle troponin activator, to improve skeletal muscle function in a mouse model of NEM3, harboring the patient-based p.Asp286Gly variant in Acta1. Acute and long-term tirasemtiv treatment significantly increased muscle contractile capacity at submaximal stimulation frequencies in both fast-twitch extensor digitorum longus and gastrocnemius muscle, and intermediate-twitch diaphragm muscle in vitro and in vivo. Additionally, long-term tirasemtiv treatment in NEM3 mice resulted in a decreased respiratory rate with preserved minute volume, suggesting more efficient respiration. Altogether, our data support the therapeutic potential of fast skeletal muscle troponin activators in alleviating skeletal muscle weakness in a mouse model of NEM3 caused by the Acta1:p.Asp286Gly variant.

During striated muscle development the first periodically repeated units appear in the premyofibrils, consisting of immature sarcomeres that must undergo a substantial growth both in length and width, to reach their final size. Here we report that, beyond its well established role in sarcomere elongation, the Sarcomere length short (SALS) protein is involved in Z-disc formation and peripheral growth of the sarcomeres. Our protein localization data and loss-of-function studies in the Drosophila indirect flight muscle strongly suggest that radial growth of the sarcomeres is initiated at the Z-disc. As to thin filament elongation, we used a powerful nanoscopy approach to reveal that SALS is subject to a major conformational change during sarcomere development, which might be critical to stop pointed end elongation in the adult muscles. In addition, we demonstrate that the roles of SALS in sarcomere elongation and radial growth are both dependent on formin type of actin assembly factors. Unexpectedly, when SALS is present in excess amounts, it promotes the formation of actin aggregates highly resembling the ones described in nemaline myopathy patients. Collectively, these findings helped to shed light on the complex mechanisms of SALS during the coordinated elongation and thickening of the sarcomeres, and resulted in the discovery of a potential nemaline myopathy model, suitable for the identification of genetic and small molecule inhibitors.

BACKGROUND: Nemaline myopathy (NM) and related disorders (NMr) form a heterogenous group of ultra-rare (1:50,000 live births or less) congenital muscle disorders. To elucidate the self-reported physical, psychological, and social functioning in the daily lives of adult persons with congenital muscle disorders, we designed a survey using items primarily from the Patient Reported Outcomes Measurement Information System, PROMIS®, and conducted a pilot study in patients with NM and NMr in Finland. The items were linked to International Classification of Functioning, Disability and Health (ICF) categories.
RESULTS: In total, 20 (62.5%) out of 32 invited persons resident in Finland participated in the study; 12 had NM and 8 NMr, 15 were women and 5 men aged 19-75 years. Sixteen (80%) were ambulatory and 4 (20%) NM patients used wheelchairs. The results from the PROMIS measuring system and ICF categories both indicated that non-ambulatory patients of this study faced more challenges in all areas of functioning than ambulatory ones, but the differences were smaller in the domains measuring psychological and social functioning than in physical functioning. In addition, the COVID-19 pandemic adversely affected the functioning of non-ambulatory patients more than that of ambulatory patients. The interindividual differences were, however, noticeable.
CONCLUSIONS: To our knowledge, this pilot study is the first comprehensive survey-based study of the physical, psychological, and social functioning of adult persons with nemaline myopathy or related disorders. The results indicate vulnerability of non-ambulatory patients being at higher risk to a decrease in general functioning during global or national exceptional periods. The responses also gave directions for modifying and improving the survey for future studies.

A novel variant of unknown significance c.8A > G (p.Glu3Gly) in TPM3 was detected in two unrelated families. TPM3 encodes the transcript variant Tpm3.12 (NM_152263.4), the tropomyosin isoform specifically expressed in slow skeletal muscle fibers. The patients presented with slowly progressive muscle weakness associated with Achilles tendon contractures of early childhood onset. Histopathology revealed features consistent with a nemaline rod myopathy. Biochemical in vitro assays performed with reconstituted thin filaments revealed defects in the assembly of the thin filament and regulation of actin-myosin interactions. The substitution p.Glu3Gly increased polymerization of Tpm3.12, but did not significantly change its affinity to actin alone. Affinity of Tpm3.12 to actin in the presence of troponin ± Ca2+ was decreased by the mutation, which was due to reduced interactions with troponin. Altered molecular interactions affected Ca2+-dependent regulation of the thin filament interactions with myosin, resulting in increased Ca2+ sensitivity and decreased relaxation of the actin-activated myosin ATPase activity. The hypercontractile molecular phenotype probably explains the distal joint contractions observed in the patients, but additional research is needed to explain the relatively mild severity of the contractures. The slowly progressive muscle weakness is most likely caused by the lack of relaxation and prolonged contractions which cause muscle wasting. This work provides evidence for the pathogenicity of the TPM3 c.8A > G variant, which allows for its classification as (likely) pathogenic.

BACKGROUND: Nemaline myopathy (NM) is a congenital disease characterized by nonprogressive or slowly progressing muscle weakness and may increase the risk of anesthesia in case of respiratory muscle or cardiac involvement. Care should be taken to prevent respiratory failure after surgery.
METHODS: A 35-year-old man with NM, who had difficult airway, restrictive ventilatory pattern, and pulmonary hypertension, required general anesthesia for surgery because of limited mouth opening.
METHODS: The patient was diagnosed with NM (ACTA1 mutation) and coronoid hyperplasia.
METHODS: Awake fiberoptic nasal intubation was performed following preparations for analgesia. General anesthesia was maintained using inhalational anesthetics and opioids without using neuromuscular blocking agents.
RESULTS: General anesthesia remained well maintained during surgery, with no movement or spontaneous breathing of the patient and he recovered from anesthesia uneventfully without complications.
CONCLUSIONS: This report highlights the safe performance of anesthesia induction and recovery in a case where anesthesia management is necessary for surgery in a patient of NM at a high risk of anesthesia-related complications.