Nemaline Myopathy Type Research Articles

A Case Study on Anesthesia for Percutaneous Closure of Large Secundum ASD in a Patient with Nemaline Myopathy Type

A Case Study on Anesthesia for Percutaneous Closure of Large Secundum ASD in a Patient with Nemaline Myopathy Type

Kbtbd13R408C-knockin mouse model reveals impaired relaxation kinetics as novel pathomechanism for NEM6 cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): VENI-Nederlandse organisatie voor Wetenschappelijk Onderzoek (NWO) We identified a novel cardiomyopathy gene: KBTBD13 encoding kelch repeat and BTB (POZ) domain containing 13. The Dutch founder variant KBTBD13R408C not only affects skeletal muscle function resulting in nemaline myopathy type 6 (NEM6), but also affects the heart. In the Dutch NEM6 cohort, various cardiac abnormalities were observed, including systolic dysfunction, diastolic dysfunction, atrial fibrillation, ventricular tachycardia and three patients died of sudden cardiac death. The majority of NEM6 patients harbors the Dutch founder variant, c.1222C>T, p.Arg408Cys (KBTBD13R408C). To provide insight in the mechanism underlying cardiac dysfunction in NEM6, here we assessed cardiac structure and function in Kbtbd13R408C-knockin mice, which closely recapitulate the well characterized skeletal muscle phenotype, i.e. impaired relaxation kinetics and mitochondrial dysfunction. Pressure-volume loop analyses showed that the end-diastolic pressure-volume relation was steeper in Kbtbd13R408C-knockin mice, indicating diastolic dysfunction. Histological evaluation of cardiac structure revealed no increase in fibrosis in Kbtbd13R408C-knockin mice. Also, no alterations in titin isoform composition or myosin heavy chain composition were observed that could account for the increased diastolic stiffness. Next, we studied the contraction and relaxation kinetics in wild type (WT) and Kbtbd13R408C-knockin mice at the intact single cardiomyocyte level by a high-throughput contractility set-up. Our data showed a prolonged relaxation time in Kbtbd13R408C-knockin mice compared to WT mice, indicating impaired relaxation kinetics. No differences in the magnitude of contraction (percentage of shortening) was found between cardiomyocytes of Kbtbd13R408C-knockin and WT mice. In parallel, calcium-handling was assessed by calcium indicator Fura-2AM. These studies reveal that calcium-release is not affected, but calcium-reuptake is slower in Kbtbd13R408C-knockin mice, which might contribute to the impaired relaxation kinetics. Current studies focus on how KBTBD13R408C affects calcium-reuptake kinetics and sarcomere kinetics in NEM6 cardiomyocytes. Hence, our studies provide the first insights in the pathomechanism underlying cardiac dysfunction in NEM6.

Pharmacological Inhibition of Myostatin in a Mouse Model of Typical Nemaline Myopathy Increases Muscle Size and Force.

Nemaline myopathy is one of the most common non-dystrophic congenital myopathies. Individuals affected by this condition experience muscle weakness and muscle smallness, often requiring supportive measures like wheelchairs or respiratory support. A significant proportion of patients, approximately one-third, exhibit compound heterozygous nebulin mutations, which usually give rise to the typical form of the disease. Currently, there are no approved treatments available for nemaline myopathy. Our research explored the modulation of myostatin, a negative regulator of muscle mass, in combating the muscle smallness associated with the disease. To investigate the effect of myostatin inhibition, we employed a mouse model with compound heterozygous nebulin mutations that mimic the typical form of the disease. The mice were treated with mRK35, a myostatin antibody, through weekly intraperitoneal injections of 10 mg/kg mRK35, commencing at two weeks of age and continuing until the mice reached four months of age. The treatment resulted in an increase in body weight and an approximate 20% muscle weight gain across most skeletal muscles, without affecting the heart. The minimum Feret diameter of type IIA and IIB fibers exhibited an increase in compound heterozygous mice, while only type IIB fibers demonstrated an increase in wild-type mice. In vitro mechanical experiments conducted on intact extensor digitorum longus muscle revealed that mRK35 augmented the physiological cross-sectional area of muscle fibers and enhanced absolute tetanic force in both wild-type and compound heterozygous mice. Furthermore, mRK35 administration improved grip strength in treated mice. Collectively, these findings indicate that inhibiting myostatin can mitigate the muscle deficits in nebulin-based typical nemaline myopathy, potentially serving as a much-needed therapeutic option.

P408 Clinical and pathologic characterization of a novel homozygous CFL2 mutation in a patient with nemaline myopathy type 7

P408 Clinical and pathologic characterization of a novel homozygous CFL2 mutation in a patient with nemaline myopathy type 7

P399 Kbtbd13 knock-down prevents and reverts phenotype development and progression in a nemaline myopathy type 6 mouse model

P399 Kbtbd13 knock-down prevents and reverts phenotype development and progression in a nemaline myopathy type 6 mouse model

P402 Nemaline myopathy type 6 caused by variants in the KBTBD13 gene: A cross-sectional study of 24 patients

P402 Nemaline myopathy type 6 caused by variants in the KBTBD13 gene: A cross-sectional study of 24 patients

Detecting impaired muscle relaxation in myopathies with the use of motor cortical stimulation

Impaired muscle relaxation is a notable feature in specific myopathies. Transcranial magnetic stimulation (TMS) of the motor cortex can induce muscle relaxation by abruptly halting corticospinal drive. Our aim was to quantify muscle relaxation using TMS in different myopathies with symptoms of muscle stiffness, contractures/cramps, and myalgia and explore the technique's diagnostic potential. In men, normalized peak relaxation rate was lower in Brody disease (n = 4) (-3.5 ± 1.3 s-1), nemaline myopathy type 6 (NEM6; n = 5) (-7.5 ± 1.0 s-1), and myotonic dystrophy type 2 (DM2; n = 5) (-10.2 ± 2.0 s-1) compared to healthy (n = 14) (-13.7 ± 2.1 s-1; all P ≤ 0.01) and symptomatic controls (n = 9) (-13.7 ± 1.6 s-1; all P ≤ 0.02). In women, NEM6 (n = 5) (-5.7 ± 2.1 s-1) and McArdle patients (n = 4) (-6.6 ± 1.4 s-1) had lower relaxation rate compared to healthy (n = 10) (-11.7 ± 1.6 s-1; both P ≤ 0.002) and symptomatic controls (n = 8) (-11.3 ± 1.8 s-1; both P ≤ 0.008). TMS-induced muscle relaxation achieved a high level of diagnostic accuracy (area under the curve = 0.94 (M) and 0.92 (F)) to differentiate symptomatic controls from myopathy patients. Muscle relaxation assessed using TMS has the potential to serve as a diagnostic tool, an in-vivo functional test to confirm the pathogenicity of unknown variants, an outcome measure in clinical trials, and monitor disease progression.

KBTBD13 is a novel cardiomyopathy gene.

KBTBD13 variants cause nemaline myopathy type 6 (NEM6). The majority of NEM6 patients harbors the Dutch founder variant, c.1222C>T, p.Arg408Cys (KBTBD13 p.R408C). Although KBTBD13 is expressed in cardiac muscle, cardiac involvement in NEM6 is unknown. Here, we constructed pedigrees of three families with the KBTBD13 p.R408C variant. In 65evaluated patients, 12% presented with left ventricle dilatation, 29% with left ventricular ejection fraction< 50%, 8% with atrial fibrillation, 9% with ventricular tachycardia, and 20% with repolarization abnormalities. Five patients received an implantable cardioverter defibrillator, three cases of sudden cardiac death were reported. Linkage analysis confirmed cosegregation of the KBTBD13 p.R408C variant with the cardiac phenotype. Mouse studies revealed that (1) mice harboring the Kbtbd13 p.R408C variant display mild diastolic dysfunction; (2) Kbtbd13-deficient mice have systolic dysfunction. Hence, (1) KBTBD13 is associated with cardiac dysfunction and cardiomyopathy; (2) KBTBD13 should be added to the cardiomyopathy gene panel; (3) NEM6 patients should be referred to the cardiologist.

FP.02 Mutation in KBTBD13 causes stiffening of thin filaments in skeletal muscle

Nemaline myopathy is caused by mutations in genes encoding thin filament proteins. Nemaline myopathy-type 6 (NEM6) is caused by mutations in <i>KBTBD13</i>. NEM6 patients display muscle weakness and slow kinetics of muscle relaxation. Whether KBTBD13 plays a role in thin filament structure/function, and whether such role can explain the NEM6 phenotype, is incompletely understood. Therefore, we developed a KBTBD13R408C–knockin (KI) mouse model. In line with the patients' phenotype, KI mice exhibit nemaline rods and slow relaxation kinetics. We used low-angle x-ray diffraction to study whether structural changes in the myofilaments can explain the contractile phenotype of KI mice. Intact soleus and extensor digitorum longus muscles (EDL) were dissected from wt and KI mice. Low angle x-ray diffraction experiments were conducted at the BioCAT beamline, Argonne National Laboratories. Muscles were mounted between a force transducer and a fixed hook, exposed to the x-ray beam during relaxed and maximal tetanic conditions. Images were recorded with 15ms acquisition time using a Pilatus 3xM1 detector set at 1.26 meter distance from the muscle. Results show that in relaxed soleus muscles the actin monomer spacing (27A) and myosin backbone spacing (28A) are shorter in KI compared to wt mice (2.7329 vs 2.7343nm; 2.8764nm vs 2.8797nm). Upon maximal tetanic activation, which was comparable between KI and wt muscles, the 27A spacing increased less in KI compared to wt muscles, indicating increased stiffness. Upon maximal tetanic activation, the 28A spacing increased in wt muscles, but this increase was comparable to that in KI mice. EDL muscle of KI mice showed no differences in actin monomer or myosin backbone spacings. Results indicate that thin filament stiffness, but not thick filament stiffness, is increased in KI mice, and that this increase is muscle-type specific. Sarcomere modeling shows that the increased stiffness of thin filaments contributes to slow relaxation kinetics.

O.03 Nemaline myopathy type 6: From pathology to therapeutics

Patients harboring variants in <i>KBTBD13</i> (NEM6) display impaired muscle relaxation, which compromises normal muscle function and daily-life activities. Histopathologically, NEM6 is characterized by the presence of myofibers containing nemaline bodies and a predominance of type I fibers. The majority of NEM6 patients harbor the Dutch founder mutation (c.1222C>T, p.Arg408Cys). Recently, we revealed that the R408C variantin <i>KBTBD13</i> slows muscle relaxation through an actin-based mechanism. To date, no therapy is available to treat NEM6. To fill this void, we developed a Kbtbd13^R408C knock-in mouse model. To identify a therapeutic window, we first performed a natural history study to characterize time of disease onset and progression in homozygous Kbtbd13^R408C knock-in at 1, 3, 9 and 18 months of age. Morphological and functional assays of soleus muscle showed that Kbtbd13^R408C knock-in mice closely recapitulate the human NEM6 phenotype: slow relaxation kinetics and muscle weakness was observed, in addition to the presence of nemaline bodies, type I fiber predominance, and hypertrophy of type I and IIa myofibers. This phenotype was absent at 1 month, developed between 1-3 months, and showed little progression after 9 months of age. Our treatment of choice was the knock-down of mutant <i>Kbtbd13</i> transcript. This choice was based on experiments on homozygous Kbtbd13-KO mice, which showed no overt phenotype at 3 and 9 months of age. Thus, Kbtbd13-deficiency is well tolerated. We treated soleus with a single intramuscular injection with an AAV9 containing a short-hairpin RNA for Kbtbd13 (AAV9-shKbtbd13). We hypothesized that a reduction in mutant transcript level would prevent or reverse disease development. Our data demonstrate that a single injection knocked down mutant <i>Kbtbd13</i> transcript levels by >90% in 3 (injected at 1-month, prior to disease development) and 9 month (injected at 7-months, at which an overt disease phenotype was present) old mice. Importantly, in treated mice, the relaxation kinetics were normalized to those of wild-type mice. Additionally, in treated mice, we observed a >70% reduction in the number of nemaline rod positive fibers. In conclusion, our results show the natural history of NEM6 in a novel mouse model and the promise of knocking down mutant transcript to prevent and reverse the NEM6 phenotype.

Respiratory muscle function in patients with nemaline myopathy

In this cross-sectional study, we comprehensively assessed respiratory muscle function in various clinical forms of nemaline myopathy (NM) including non-volitional tests for diaphragm function. Forty-two patients with NM were included (10 males (25-74 y/o); 32 females (11-76 y/o)). The NM forms were typical (n=11), mild (n=7), or childhood-onset with slowness of movements (n=24). Forced vital capacity (FVC) and maximal inspiratory pressure were decreased in typical NM in comparison with childhood-onset NM with slowness (32.0 [29.0-58.5] vs 81.0 [75.0-87.0]%, p<0.01, and 35.0 [24.0-55.0] vs 81.0 [65.0-102.5] cmH2O, p<0.01). Eight patients with childhood-onset NM with slowness had respiratory muscle weakness. There was a low correlation between FVC and Motor Function Measure scores (r=0.48, p<0.01). End-inspiratory diaphragm thickness and twitch mouth pressure were decreased in patients requiring home mechanical ventilation compared to non-ventilated patients with normal lung function (1.8 [1.5-2.4] vs 3.1 [2.0-4.6] mm, p=0.049, and -7.9 [-10.9- -4.0] vs -14.9 [-17.3- -12.6], p=0.04). Our results show that respiratory muscle weakness is present in all NM forms, including childhood-onset NM with slowness, and may be present irrespective of the degree of general motor function impairment. These findings highlight the importance for screening of respiratory function in patients with NM to guide respiratory management.

Generation of two isogenic induced pluripotent stem cell lines from a 10-year-old typical nemaline myopathy patient with a heterozygous dominant c.541G>A (p.Asp179Asn) pathogenic variant in the ACTA1 gene

Nemaline myopathy (NM) is a congenital myopathy typically characterized by skeletal muscle weakness and the presence of nemaline bodies in myofibres. Approximately 25% of NM cases are caused by variants in ACTA1. We generated two induced pluripotent stem cell lines from lymphoblastoid cells of a 10-year-old female with typical NM harbouring a dominant pathogenic variant in ACTA1 (c.541C>A). The isogenic lines displayed typical iPSC morphology, expressed pluripotency markers, and could differentiate into each of the three germ layers. Although the lines have partial or complete X chromosome duplication, they may still prove useful as models of human ACTA1 disease.

NEM6, KBTBD13-Related Congenital Myopathy: Myopathological Analysis in 18 Dutch Patients Reveals Ring Rods Fibers, Cores, Nuclear Clumps, and Granulo-Filamentous Protein Material.

Nemaline myopathy type 6 (NEM6), KBTBD13-related congenital myopathy is caused by mutated KBTBD13 protein that interacts improperly with thin filaments/actin, provoking impaired muscle-relaxation kinetics. We describe muscle morphology in 18 Dutch NEM6 patients and correlate it with clinical phenotype and pathophysiological mechanisms. Rods were found in in 85% of biopsies by light microscopy, and 89% by electron microscopy. A peculiar ring disposition of rods resulting in ring-rods fiber was observed. Cores were found in 79% of NEM6 biopsies by light microscopy, and 83% by electron microscopy. Electron microscopy also disclosed granulofilamentous protein material in 9 biopsies. Fiber type 1 predominance and prominent nuclear internalization were found. Rods were immunoreactive for α-actinin and myotilin. Areas surrounding the rods showed titin overexpression suggesting derangement of the surrounding sarcomeres. NEM6 myopathology hallmarks are prominent cores, rods including ring-rods fibers, nuclear clumps, and granulofilamentous protein material. This material might represent the histopathologic epiphenomenon of altered interaction between mutated KBTBD13 protein and thin filaments. We claim to classify KBTBD13-related congenital myopathy as rod-core myopathy.

CONGENITAL MYOPATHIES 1 – NEMALINE: P.18 KBTBD13, NEM6 congenital myopathy: deep muscle phenotyping reveals ring-rods fibers, nuclear clumps, and protein aggregates as new morphologic markers

Nemaline myopathy type 6 (NEM6) is a rare congenital myopathy characterized by muscle weakness, exercise intolerance and muscle slowness, linked to autosomal dominant KBTBD13 gene mutations. Mutated KBTBD13 interacts improperly with thin filaments/actin provoking impaired muscle-relaxation kinetics. We performed a deep muscle phenotyping including immunohistochemistry and electron microscopy in 18 muscle biopsies of Dutch NEM6 patients in order to correlate muscle morphology with clinical phenotype and pathophysiological mechanisms.

Triggering typical nemaline myopathy with compound heterozygous nebulin mutations reveals myofilament structural changes as pathomechanism

Nebulin is a giant protein that winds around the actin filaments in the skeletal muscle sarcomere. Compound-heterozygous mutations in the nebulin gene (NEB) cause typical nemaline myopathy (NM), a muscle disorder characterized by muscle weakness with limited treatment options. We created a mouse model with a missense mutation p.Ser6366Ile and a deletion of NEB exon 55, the Compound-Het model that resembles typical NM. We show that Compound-Het mice are growth-retarded and have muscle weakness. Muscles have a reduced myofibrillar fractional-area and sarcomeres are disorganized, contain rod bodies, and have longer thin filaments. In contrast to nebulin-based severe NM where haplo-insufficiency is the disease driver, Compound-Het mice express normal amounts of nebulin. X-ray diffraction revealed that the actin filament is twisted with a larger radius, that tropomyosin and troponin behavior is altered, and that the myofilament spacing is increased. The unique disease mechanism of nebulin-based typical NM reveals novel therapeutic targets.

Mutations in the Large Protein Nebulin Trigger Typical Nemaline Myopathy with a Unique Molecular Mechanism

Mutations in the Large Protein Nebulin Trigger Typical Nemaline Myopathy with a Unique Molecular Mechanism

P.151 - ACTA1-related mild or typical nemaline myopathy in a three-generation Finnish family

P.151 - ACTA1-related mild or typical nemaline myopathy in a three-generation Finnish family

Derivation of NEM2 affected human embryonic stem cell line Genea080

The Genea080 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying compound heterozygous mutations in the NEB gene, exon 55 deletion & c.15110dupA, indicative of Nemaline Myopathy Type 2 (NEM2). Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XY and STR analysis demonstrated a male allele pattern. The hESC line had pluripotent cell morphology, 90% of cells expressed Nanog, 95% Oct4, 54% Tra1-60 and 99% SSEA4 and gave a PluriTest Pluripotency score of 32.08, Novelty of 1.3. The cell line was negative for Mycoplasma and visible contamination.

Derivation of NEM2 affected human embryonic stem cell line Genea079

The Genea079 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying compound heterozygous mutations in the NEB gene, exon 55 deletion & c.15110dupA, indicative of Nemaline Myopathy Type 2 (NEM2). Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XY and STR analysis demonstrated a male Allele pattern. The hESC line had pluripotent cell morphology, 86% of cells expressed Nanog, 95% Oct4, 54% Tra1-60 and 98% SSEA4 and gave a PluriTest Pluripotency score of 30.25, Novelty of 1.21. The cell line was negative for Mycoplasma and visible contamination.

Derivation of NEM2 affected human embryonic stem cell line Genea078

The Genea078 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying compound heterozygous mutations in the NEB gene, exon 55 deletion & c.15110dupA, indicative of Nemaline Myopathy Type 2 (NEM2). Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XX and STR analysis demonstrated a female Allele pattern. The hESC line had pluripotent cell morphology, 76% of cells expressed Nanog, 93% Oct4, 67% Tra1–60 and 97% SSEA4 and gave a Pluritest Pluripotency score of 42.18, Novelty of 1.37. The cell line was negative for Mycoplasma and visible contamination.