Nemaline Myopathy in Brazilian Patients: Molecular and Clinical Characterization.

Nemaline myopathy (NM), a structural congenital myopathy, presents a significant clinical and genetic heterogeneity. Here, we compiled molecular and clinical data of 30 Brazilian patients from 25 unrelated families. Next-generation sequencing was able to genetically classify all patients: sixteen families (64%) with mutation in NEB, five (20%) in ACTA1, two (8%) in KLHL40, and one in TPM2 (4%) and TPM3 (4%). In the NEB-related families, 25 different variants, 11 of them novel, were identified; splice site (10/25) and frame shift (9/25) mutations were the most common. Mutation c.24579 G>C was recurrent in three unrelated patients from the same region, suggesting a common ancestor. Clinically, the "typical" form was the more frequent and caused by mutations in the different NM genes. Phenotypic heterogeneity was observed among patients with mutations in the same gene. Respiratory involvement was very common and often out of proportion with limb weakness. Muscle MRI patterns showed variability within the forms and genes, which was related to the severity of the weakness. Considering the high frequency of NEB mutations and the complexity of this gene, NGS tools should be combined with CNV identification, especially in patients with a likely non-identified second mutation.

A nebulin super-repeat panel reveals stronger actin binding toward the ends of the super-repeat region.

INTRODUCTION: Nebulin is a giant actin-binding protein in the thin filament of the skeletal muscle sarcomere. Studies of nebulin interactions are limited by the size, complexity, and poor solubility of the protein. We divided the nebulin super-repeat region into a super-repeat panel, and studied nebulin/actin interactions. METHODS: Actin binding was studied using a co-sedimentation assay with filamentous actin and 26 different nebulin super-repeats. RESULTS: The panel revealed notable differences in actin binding between the super-repeats. Both ends of the super-repeat region bound actin significantly more strongly, whereas the central part of the protein bound actin weakly. Thus, the binding between nebulin and actin formed a location-dependent pattern of strong vs. weak binding. DISCUSSION: The nebulin super-repeat panel allowed us to study the actin binding of each super-repeat individually. The panel will be a powerful tool in elucidating nebulin function in health and disease. Muscle Nerve 59:116-121, 2019.

Mutation-specific effects on thin filament length in thin filament myopathy.

OBJECTIVE: Thin filament myopathies are among the most common nondystrophic congenital muscular disorders, and are caused by mutations in genes encoding proteins that are associated with the skeletal muscle thin filament. Mechanisms underlying muscle weakness are poorly understood, but might involve the length of the thin filament, an important determinant of force generation. METHODS: We investigated the sarcomere length-dependence of force, a functional assay that provides insights into the contractile strength of muscle fibers as well as the length of the thin filaments, in muscle fibers from 51 patients with thin filament myopathy caused by mutations in NEB, ACTA1, TPM2, TPM3, TNNT1, KBTBD13, KLHL40, and KLHL41. RESULTS: Lower force generation was observed in muscle fibers from patients of all genotypes. In a subset of patients who harbor mutations in NEB and ACTA1, the lower force was associated with downward shifted force-sarcomere length relations, indicative of shorter thin filaments. Confocal microscopy confirmed shorter thin filaments in muscle fibers of these patients. A conditional Neb knockout mouse model, which recapitulates thin filament myopathy, revealed a compensatory mechanism; the lower force generation that was associated with shorter thin filaments was compensated for by increasing the number of sarcomeres in series. This allowed muscle fibers to operate at a shorter sarcomere length and maintain optimal thin-thick filament overlap. INTERPRETATION: These findings might provide a novel direction for the development of therapeutic strategies for thin filament myopathy patients with shortened thin filament lengths. Ann Neurol 2016;79:959-969.

Mutations in KLHL40 are a frequent cause of severe autosomal-recessive nemaline myopathy.

Nemaline myopathy (NEM) is a common congenital myopathy. At the very severe end of the NEM clinical spectrum are genetically unresolved cases of autosomal-recessive fetal akinesia sequence. We studied a multinational cohort of 143 severe-NEM-affected families lacking genetic diagnosis. We performed whole-exome sequencing of six families and targeted gene sequencing of additional families. We identified 19 mutations in KLHL40 (kelch-like family member 40) in 28 apparently unrelated NEM kindreds of various ethnicities. Accounting for up to 28% of the tested individuals in the Japanese cohort, KLHL40 mutations were found to be the most common cause of this severe form of NEM. Clinical features of affected individuals were severe and distinctive and included fetal akinesia or hypokinesia and contractures, fractures, respiratory failure, and swallowing difficulties at birth. Molecular modeling suggested that the missense substitutions would destabilize the protein. Protein studies showed that KLHL40 is a striated-muscle-specific protein that is absent in KLHL40-associated NEM skeletal muscle. In zebrafish, klhl40a and klhl40b expression is largely confined to the myotome and skeletal muscle, and knockdown of these isoforms results in disruption of muscle structure and loss of movement. We identified KLHL40 mutations as a frequent cause of severe autosomal-recessive NEM and showed that it plays a key role in muscle development and function. Screening of KLHL40 should be a priority in individuals who are affected by autosomal-recessive NEM and who present with prenatal symptoms and/or contractures and in all Japanese individuals with severe NEM.

Other specified and unspecified feeding or eating disorders among women in the community.

OBJECTIVE: To examine the occurrence, course, and clinical picture of the DSM-5 residual categories 'Other Specified Feeding or Eating Disorder' (OSFED) and 'Unspecified Feeding or Eating Disorder' (UFED), to describe potential subtypes, and to evaluate whether the subdivision of the residual category appears meaningful. METHOD: We screened women from the 1975-79 birth cohorts of Finnish twins (N = 2825) for lifetime eating disorders using questionnaires and the SCID interview. This analysis characterizes women who reported clinically significant eating disorder symptoms but did not fulfill diagnostic criteria for DSM-5 anorexia nervosa (AN), bulimia nervosa (BN), or binge eating disorder (BED). RESULTS: Thirty-eight women (21% of those with an eating disorder) fell in the residual OSFED/UFED category. A third of them (N = 14) fulfilled OSFED criteria, whereas two thirds (N = 24) fell in the UFED category. The lifetime prevalence of OSFED/UFED was 1.5% (95% CI 1.1-2.0%), less than half of the prevalence of DSM-IV eating disorder not otherwise specified (EDNOS). The mean age of onset of OSFED/UFED was 18 years, median duration of symptoms was two years, and the 5-year probability of recovery was 60%. Over a third of women with OSFED/UFED suffered from comorbid psychiatric disorders. Both residual categories were clinically heterogeneous and included atypical forms of AN, BN, and BED. CONCLUSIONS: Applying the DSM-5 criteria in a community sample of young women more than halved the occurrence of residual eating disorder diagnoses, but resulted in two instead of one clinically heterogeneous residual categories. Nevertheless, residual eating disorders were associated with considerable clinical severity. © 2016 Wiley Periodicals, Inc. (Int J Eat Disord 2016; 49:1010-1017).

Mutation update: the spectra of nebulin variants and associated myopathies.

A mutation update on the nebulin gene (NEB) is necessary because of recent developments in analysis methodology, the identification of increasing numbers and novel types of variants, and a widening in the spectrum of clinical and histological phenotypes associated with this gigantic, 183 exons containing gene. Recessive pathogenic variants in NEB are the major cause of nemaline myopathy (NM), one of the most common congenital myopathies. Moreover, pathogenic NEB variants have been identified in core-rod myopathy and in distal myopathies. In this update, we present the disease-causing variants in NEB in 159 families, 143 families with NM, and 16 families with NM-related myopathies. Eighty-eight families are presented here for the first time. We summarize 86 previously published and 126 unpublished variants identified in NEB. Furthermore, we have analyzed the NEB variants deposited in the Exome Variant Server (http://evs.gs.washington.edu/EVS/), identifying that pathogenic variants are a minor fraction of all coding variants (∼7%). This indicates that nebulin tolerates substantial changes in its amino acid sequence, providing an explanation as to why variants in such a large gene result in relatively rare disorders. Lastly, we discuss the difficulties of drawing reliable genotype-phenotype correlations in NEB-associated disease.

Mutation update and genotype-phenotype correlations of novel and previously described mutations in TPM2 and TPM3 causing congenital myopathies.

Mutations affecting skeletal muscle isoforms of the tropomyosin genes may cause nemaline myopathy, cap myopathy, core-rod myopathy, congenital fiber-type disproportion, distal arthrogryposes, and Escobar syndrome. We correlate the clinical picture of these diseases with novel (19) and previously reported (31) mutations of the TPM2 and TPM3 genes. Included are altogether 93 families: 53 with TPM2 mutations and 40 with TPM3 mutations. Thirty distinct pathogenic variants of TPM2 and 20 of TPM3 have been published or listed in the Leiden Open Variant Database (http://www.dmd.nl/). Most are heterozygous changes associated with autosomal-dominant disease. Patients with TPM2 mutations tended to present with milder symptoms than those with TPM3 mutations, DA being present only in the TPM2 group. Previous studies have shown that five of the mutations in TPM2 and one in TPM3 cause increased Ca(2+) sensitivity resulting in a hypercontractile molecular phenotype. Patients with hypercontractile phenotype more often had contractures of the limb joints (18/19) and jaw (6/19) than those with nonhypercontractile ones (2/22 and 1/22), whereas patients with the non-hypercontractile molecular phenotype more often (19/22) had axial contractures than the hypercontractile group (7/19). Our in silico predictions show that most mutations affect tropomyosin-actin association or tropomyosin head-to-tail binding.

K7del is a common TPM2 gene mutation associated with nemaline myopathy and raised myofibre calcium sensitivity.

Mutations in the TPM2 gene, which encodes ß-tropomyosin, are an established cause of several congenital skeletal myopathies and distal arthrogryposis. We have identified a TPM2 mutation, p.K7del, in five unrelated families with nemaline myopathy and a consistent distinctive clinical phenotype. Patients develop large joint contractures during childhood, followed by slowly progressive skeletal muscle weakness during adulthood. The TPM2 p.K7del mutation results in the loss of a highly conserved lysine residue near the N-terminus of ß-tropomyosin, which is predicted to disrupt head-to-tail polymerization of tropomyosin. Recombinant K7del-ß-tropomyosin incorporates poorly into sarcomeres in C2C12 myotubes and has a reduced affinity for actin. Two-dimensional gel electrophoresis of patient muscle and primary patient cultured myotubes showed that mutant protein is expressed but incorporates poorly into sarcomeres and likely accumulates in nemaline rods. In vitro studies using recombinant K7del-ß-tropomyosin and force measurements from single dissected patient myofibres showed increased myofilament calcium sensitivity. Together these data indicate that p.K7del is a common recurrent TPM2 mutation associated with mild nemaline myopathy. The p.K7del mutation likely disrupts head-to-tail polymerization of tropomyosin, which impairs incorporation into sarcomeres and also affects the equilibrium of the troponin/tropomyosin-dependent calcium switch of muscle. Joint contractures may stem from chronic muscle hypercontraction due to increased myofibrillar calcium sensitivity while declining strength in adulthood likely arises from other mechanisms, such as myofibre decompensation and fatty infiltration. These results suggest that patients may benefit from therapies that reduce skeletal muscle calcium sensitivity, and we highlight late muscle decompensation as an important cause of morbidity.

Variants in tropomyosins TPM2 and TPM3 causing muscle hypertonia.

Patients with myopathies caused by pathogenic variants in tropomyosin genes TPM2 and TPM3 usually have muscle hypotonia and weakness, their muscle biopsies often showing fibre size disproportion and nemaline bodies. Here, we describe a series of patients with hypercontractile molecular phenotypes, high muscle tone, and mostly non-specific myopathic biopsy findings without nemaline bodies. Three of the patients had trismus, whilst in one patient, the distal joints of her fingers flexed on extension of the wrists. In one biopsy from a patient with a rare TPM3 pathogenic variant, cores and minicores were observed, an unusual finding in TPM3-caused myopathy. The variants alter conserved contact sites between tropomyosin and actin.

Haplotype information of large neuromuscular disease genes provided by linked-read sequencing has a potential to increase diagnostic yield.

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.

A recurrent ACTA1 amino acid change in mosaic form causes milder asymmetric myopathy.

We describe three patients with asymmetric congenital myopathy without definite nemaline bodies and one patient with severe nemaline myopathy. In all four patients, the phenotype had been caused by pathogenic missense variants in ACTA1 leading to the same amino acid change, p.(Gly247Arg). The three patients with milder myopathy were mosaic for their variants. In contrast, in the severely affected patient, the missense variant was present in a de novo, constitutional form. The grade of mosaicism in the three mosaic patients ranged between 20 % and 40 %. We speculate that the milder clinical and histological manifestations of the same ACTA1 variant in the patients with mosaicism reflect the lower abundance of mutant actin in their muscle tissue. Similarly, the asymmetry of body growth and muscle weakness may be a consequence of the affected cells being unevenly distributed. The partial improvement in muscle strength with age in patients with mosaicism might be due to an increased proportion over time of nuclei carrying and expressing two normal alleles.

Self-reported functioning among patients with ultra-rare nemaline myopathy or a related disorder in Finland: a pilot study.

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.

Novel Compound Heterozygous Splice-Site Variants in TPM3 Revealed by RNA Sequencing in a Patient with an Unusual Form of Nemaline Myopathy: A Case Report.

BACKGROUND: Pathogenic variants in the TPM3 gene, encoding slow skeletal muscle α-tropomyosin account for less than 5% of nemaline myopathy cases. Dominantly inherited or de novo missense variants in TPM3 are more common than recessive loss-of-function variants. The recessive variants reported to date seem to affect either the 5' or the 3' end of the skeletal muscle-specific TPM3 transcript. OBJECTIVES: The aim of the study was to identify the disease-causing gene and variants in a Finnish patient with an unusual form of nemaline myopathy. METHODS: The genetic analyses included Sanger sequencing, whole-exome sequencing, targeted array-CGH, and linked-read whole genome sequencing. RNA sequencing was done on total RNA extracted from cultured myoblasts and myotubes of the patient and controls. TPM3 protein expression was assessed by Western blot analysis. The diagnostic muscle biopsy was analyzed by routine histopathological methods. RESULTS: The patient had poor head control and failure to thrive, but no hypomimia, and his upper limbs were clearly weaker than his lower limbs, features which in combination with the histopathology suggested TPM3-caused nemaline myopathy. Muscle histopathology showed increased fiber size variation and numerous nemaline bodies predominantly in small type 1 fibers. The patient was found to be compound heterozygous for two splice-site variants in intron 1a of TPM3: NM_152263.4:c.117+2_5delTAGG, deleting the donor splice site of intron 1a, and NM_152263.4:c.117 + 164 C>T, which activates an acceptor splice site preceding a non-coding exon in intron 1a. RNA sequencing revealed inclusion of intron 1a and the non-coding exon in the transcripts, resulting in early premature stop codons. Western blot using patient myoblasts revealed markedly reduced levels of the TPM3 protein. CONCLUSIONS: Novel biallelic splice-site variants were shown to markedly reduce TPM3 protein expression. The effects of the variants on splicing were readily revealed by RNA sequencing, demonstrating the power of the method.

Disrupted myosin cross-bridge cycling kinetics triggers muscle weakness in nebulin-related myopathy.

Nebulin is a giant protein expressed at high levels in skeletal muscle. Mutations in the nebulin gene (NEB) lead to muscle weakness and various congenital myopathies. Despite increasing clinical and scientific interest, the pathogenesis of weakness remains unknown. The present study, therefore, aims at unraveling the underlying molecular mechanisms. Hence, we recorded and analyzed the mechanics as well as the X-ray diffraction patterns of human membrane-permeabilized single muscle fibers expressing nebulin mutations. Results demonstrated that, during contraction, the cycling rate of myosin heads attaching to actin is dramatically perturbed, causing a reduction in the fraction of myosin-actin interactions in the strong binding state. This phenomenon prevents complete thin-filament activation, more especially proper and full tropomyosin movement, further limiting additional binding of myosin cross-bridges. At the cell level, this reduces the force-generating capacity and, overall, provokes muscle weakness. To reverse such a negative cascade of events, future potential therapeutic interventions should, therefore, focus on the triggering component, the altered myosin cross-bridge cycling kinetics.

Carrier state for the nebulin exon 55 deletion and abnormal prenatal ultrasound findings as potential signs of nemaline myopathy.

OBJECTIVE: To increase awareness to the possibility of nemaline myopathy (NM) when abnormal prenatal ultrasound findings appear together with a carrier state for the common exon 55 deletion in the nebulin gene (NEB) of an Ashkenazi Jewish parent. METHODS: We describe four unrelated pregnancies with abnormal prenatal ultrasound findings resulting in the birth of newborns with NM, where one or both parents were of Ashkenazi Jewish origin. Data was collected retrospectively from the patients' medical files. Molecular analysis of NEB was performed on the DNA from the patients and parents. RESULTS: Prenatal ultrasound findings included polyhydramnios, decreased fetal movements, club feet, and arthrogryposis. A biopsy from two of the newborns was consistent with NM. In all of the newborns, the common NEB exon 55 deletion was detected in the heterozygote state and in three of them, a second novel mutation was found. CONCLUSIONS: Ultrasonographic findings suggestive of a myopathy and a carrier state for the NEB exon 55 deletion in one of the parents should trigger a thorough investigation for NM. The extreme size of NEB imposes great difficulties when searching for a second mutation, especially under the time constraints of an ongoing pregnancy.

Array Comparative Genomic Hybridisation and Droplet Digital PCR Uncover Recurrent Copy Number Variation of the TTN Segmental Duplication Region.

Intragenic segmental duplication regions are potential hotspots for recurrent copy number variation and possible pathogenic aberrations. Two large sarcomeric genes, nebulin and titin, both contain such segmental duplication regions. Using our custom Comparative Genomic Hybridisation array, we have previously shown that a gain or loss of more than one copy of the repeated block of the nebulin triplicate region constitutes a recessive pathogenic mutation. Using targeted array-CGH, similar copy number variants can be detected in the segmental duplication region of titin. Due to the limitations of the array-CGH methodology and the repetitiveness of the region, the exact copy numbers of the blocks could not be determined. Therefore, we developed complementary custom Droplet Digital PCR assays for the titin segmental duplication region to confirm true variation. Our combined methods show that the titin segmental duplication region is subject to recurrent copy number variation. Gains and losses were detected in samples from healthy individuals as well as in samples from patients with different muscle disorders. The copy number variation observed in our cohort is likely benign, but pathogenic copy number variants in the segmental duplication region of titin cannot be excluded. Further investigations are needed, however, this region should no longer be neglected in genetic analyses.

A custom ddPCR method for the detection of copy number variations in the nebulin triplicate region.

The human genome contains repetitive regions, such as segmental duplications, known to be prone to copy number variation. Segmental duplications are highly identical and homologous sequences, posing a specific challenge for most mutation detection methods. The giant nebulin gene is expressed in skeletal muscle. It harbors a large segmental duplication region composed of eight exons repeated three times, the so-called triplicate region. Mutations in nebulin are known to cause nemaline myopathy and other congenital myopathies. Using our custom targeted Comparative Genomic Hybridization arrays, we have previously shown that copy number variations in the nebulin triplicate region are pathogenic when the copy number of the segmental duplication block deviates two or more copies from the normal number, which is three per allele. To complement our Comparative Genomic Hybridization arrays, we have established a custom Droplet Digital PCR method for the detection of copy number variations within the nebulin triplicate region. The custom Droplet Digital PCR assays allow sensitive, rapid, high-throughput, and cost-effective detection of copy number variations within this region and is ready for implementation a screening method for disease-causing copy number variations of the nebulin triplicate region. We suggest that Droplet Digital PCR may also be used in the study and diagnostics of other segmental duplication regions of the genome.

Congenital asymmetric distal myopathy with hemifacial weakness caused by a heterozygous large de novo mosaic deletion in nebulin.

We report the first mosaic mutation, a deletion of exons 11-107, identified in the nebulin gene in a Finnish patient presenting with a predominantly distal congenital myopathy and asymmetric muscle weakness. The female patient is ambulant and currently 26 years old. Muscle biopsies showed myopathic features with type 1 fibre predominance, strikingly hypotrophic type 2 fibres and central nuclei, but no nemaline bodies. The deletion was detected in a copy number variation analysis based on next-generation sequencing data. The parents of the patient did not carry the deletion. Mosaicism was detected using a custom, targeted comparative genomic hybridisation array. Expression of the truncated allele, less than half the size of full-length nebulin, was confirmed by Western blotting. The clinical and histological picture resembled that of a family with a slightly smaller deletion, and that in patients with recessively inherited distal forms of nebulin-caused myopathy. Asymmetry, however, was a novel feature.

Dominantly inherited distal nemaline/cap myopathy caused by a large deletion in the nebulin gene.

We report the first family with a dominantly inherited mutation of the nebulin gene (NEB). This ∼100 kb in-frame deletion encompasses NEB exons 14-89, causing distal nemaline/cap myopathy in a three-generation family. It is the largest deletion characterized in NEB hitherto. The mutated allele was shown to be expressed at the mRNA level and furthermore, for the first time, a deletion was shown to cause the production of a smaller mutant nebulin protein. Thus, we suggest that this novel mutant nebulin protein has a dominant-negative effect, explaining the first documented dominant inheritance of nebulin-caused myopathy. The index patient, a young man, was more severely affected than his mother and grandmother. His first symptom was foot drop at the age of three, followed by distal muscle atrophy, slight hypomimia, high-arched palate, and weakness of the neck and elbow flexors, hands, tibialis anterior and toe extensors. Muscle biopsies showed myopathic features with type 1 fibre predominance in the index patient and nemaline bodies and cap-like structures in biopsies from his mother and grandmother. The muscle biopsy findings constitute a further example of nemaline bodies and cap-like structures being part of the same spectrum of pathological changes.