NEB Mutations Research Articles

Genetic and clinical landscape of ER + /PR- breast cancer in China

BackgroundEstrogen receptor-positive and progesterone receptor-negative (ER + /PR-) breast cancer comprise a special type. More than 10% breast cancer patients belonged to ER + /PR-.MethodsIn order to better understand this patient population, we utilized a unique dataset from China, examining the clinicopathological features and genomic profiles of ER + /PR- breast cancers. Our study involved three cohorts: Cohort 1 included 2120 unselected ER-positive female patients with re-evaluated clinicopathological and survival data; Cohort 2 comprised 442 ER-positive females who underwent genetic testing; and Cohort 3 consisted of 77 ER-positive/HER2-negative females tested with MammaPrint and BluePrint.ResultsPatients were stratified into four categories based on the PR/ER ratio. Clinically, ER + /PR- tumors (PR/ER ratio = 0) showed the lowest proportion of T1 tumors (10.88%) and highest proportion of HER2-positive tumors (28.36%) than did other ER + /PR + tumors groups. The ER + /PR- group contained a higher number of underweight patients (20.20%). Independently of HER2 status, ER + /PR- patients demonstrated the poorest prognosis. Genomically, the most prevalent mutations were PIK3CA (50%) in ER + /PR + tumors and TP53 (65%) in ER + /PR- tumors. ER + /PR- tumors presented more frequent mutations in TP53, ERBB2, CDK12, SPEN, and NEB, with mutation rates of 65%, 42%, 27%, 13%, and 10%, respectively. Additionally, the Tumor Mutational Burden (TMB) was higher in the ER + /PR- group compared to the ER + /PR + group. The MammaPrint score for the ER + /PR-/HER2- group was significantly lower than that of other groups. In the BluePrint analysis, only four patients were classified as Basal-Type, all of whom were ER + /PR-/HER2-.ConclusionsIn this study, we identified the clinical and genetic characteristics of ER + /PR- breast cancer patients in China. Distinct PR statuses indicated different biological processes of ER + breast cancer and survival outcomes. Future treatment strategies may need to be tailored for ER + /PR- patients.

P403 The replacement kinetics of the giant muscle protein nebulin are slow and further reduced by a frequently observed mutation in Neb

P403 The replacement kinetics of the giant muscle protein nebulin are slow and further reduced by a frequently observed mutation in Neb

NEB mutations disrupt the super-relaxed state of myosin and remodel the muscle metabolic proteome in nemaline myopathy

Nemaline myopathy (NM) is one of the most common non-dystrophic genetic muscle disorders. NM is often associated with mutations in the NEB gene. Even though the exact NEB-NM pathophysiological mechanisms remain unclear, histological analyses of patients’ muscle biopsies often reveal unexplained accumulation of glycogen and abnormally shaped mitochondria. Hence, the aim of the present study was to define the exact molecular and cellular cascade of events that would lead to potential changes in muscle energetics in NEB-NM. For that, we applied a wide range of biophysical and cell biology assays on skeletal muscle fibres from NM patients as well as untargeted proteomics analyses on isolated myofibres from a muscle-specific nebulin‐deficient mouse model. Unexpectedly, we found that the myosin stabilizing conformational state, known as super-relaxed state, was significantly impaired, inducing an increase in the energy (ATP) consumption of resting muscle fibres from NEB-NM patients when compared with controls or with other forms of genetic/rare, acquired NM. This destabilization of the myosin super-relaxed state had dynamic consequences as we observed a remodeling of the metabolic proteome in muscle fibres from nebulin‐deficient mice. Altogether, our findings explain some of the hitherto obscure hallmarks of NM, including the appearance of abnormal energy proteins and suggest potential beneficial effects of drugs targeting myosin activity/conformations for NEB-NM.

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.

O.01 Correlating molecular genetic testing for rare genetic variants with a broad clinicopathologic spectrum of congenital myopathies

Congenital myopathies (CMs) are a group of clinically heterogeneous disorders presenting with a range of overlapping findings, making diagnosis challenging. Despite advances in genetic testing, many cases remain undiagnosed. The Beggs' laboratory CM cohort, which currently totals over 1,100 individuals, constitutes a robust dataset of existing cases. Subjects were ascertained based on clinical presentation and pathologic diagnoses suggestive of CM. We report that over 65% have been solved by a combination of single gene, panel, or genomic sequencing, and/or RNA-Seq. Of 727 solved cases, 610 had mutations in either ACTA1, DNM2, MTM1, NEB, RYR1, SELENON, TPM2, TPM3, or TTN. All these genes are well-known causes of congenital myopathy. While all patients shared a myopathic pattern of weakness and hypotonia, a wide spectrum of clinicopathologic features were appreciated among the cases. The remaining 117 cases had mutations in one of 52 different genes, including a number of rare or novel genes, as well as some more commonly mutated non-CM disease genes in patients who presented atypically for those associated conditions. Among a heterogeneous group of 96 probands with congenital myopathy with nonspecific or no pathology, 31 (32%) had SELENON mutations; 9 (9.4%) had RYR1 mutations and 6 (6.25%) had TTN mutations. This survey of the Beggs' laboratory cohort cases, accumulated over roughly 20 years, emphasizes the crucial and growing role of WES/WGS in establishing a molecular genetic diagnosis of CM, as opposed to disease-based gene panels or single-gene testing, which may miss cases where the presentation does not fit the classical description of the disorder, or where multiple genes may present with overlapping clinicopathologic diagnoses. Moreover, certain syndromes not known to present predominantly with myopathic symptoms may go undiagnosed if genetic testing is done in a more targeted fashion based on specific clinical symptoms or pathologic diagnosis alone.

P.03 Myosin dysregulation in nemaline myopathy

Nemaline myopathy (NM) is one of the most common congenital myopathies. NM is often related to pathogenic variants in the <i>NEB, ACTA1, TPM2</i> and <i>TPM3</i> genes. Even though the exact disease mechanisms remain to be elucidated, histological analyses of NM patients' muscle biopsies may reveal unexplained accumulation of glycogen and abnormal mitochondria. Thus, the aim of the present work was to identify what could cause such change in muscle energetics. For that, we applied mant-ATP chase experiments on skeletal muscle fibres from patients diagnosed with NM. We observed that the myosin-stabilising conformational state, known as the super-relaxed state, was significantly impaired, inducing an increase in the energy (ATP) consumption of resting patient muscle fibres expressing NEB and ACTA1 mutations. Such alteration was linked to hyper-phosphorylation of myosin-binding protein C, a protein linking myosin molecules to the thin filaments. Hence, our results may indicate a contribution of myosin to some of the histopathological aberrations related to NM.

A Cross-Sectional Study of Nemaline Myopathy.

ObjectiveNemaline myopathy (NM) is a rare neuromuscular condition with clinical and genetic heterogeneity. To establish disease natural history, we performed a cross-sectional study of NM, complemented by longitudinal assessment and exploration of pilot outcome measures.MethodsFifty-seven individuals with NM were recruited at 2 family workshops, including 16 examined at both time points. Participants were evaluated by clinical history and physical examination. Functional outcome measures included the Motor Function Measure (MFM), pulmonary function tests (PFTs), myometry, goniometry, and bulbar assessments.ResultsThe most common clinical classification was typical congenital (54%), whereas 42% had more severe presentations. Fifty-eight percent of individuals needed mechanical support, with 26% requiring wheelchair, tracheostomy, and feeding tube. The MFM scale was performed in 44 of 57 participants and showed reduced scores in most with little floor/ceiling effect. Of the 27 individuals completing PFTs, abnormal values were observed in 65%. Last, bulbar function was abnormal in all patients examined, as determined with a novel outcome measure. Genotypes included mutations in ACTA1 (18), NEB (20), and TPM2 (2). Seventeen individuals were genetically unresolved. Patients with pathogenic ACTA1 and NEB variants were largely similar in clinical phenotype. Patients without genetic resolution had more severe disease.ConclusionWe present a comprehensive cross-sectional study of NM. Our data identify significant disabilities and support a relatively stable disease course. We identify a need for further diagnostic investigation for the genetically unresolved group. MFM, PFTs, and the slurp test were identified as promising outcome measures for future clinical trials.

Progression or Not - A Small Natural History Study of Genetical Confirmed Congenital Myopathies.

Clinical characteristics of patients with congenital myopathies (CM) are well known but there is a lack of knowledge about the natural history and course of disease of the different genetic subtypes. In 2010 we assessed the national cohort of Danish patients with CM to decide genetic diagnosing and describe genotype- phenotype relationships.AIM of this follow-up study was to evaluate the course of disease since the initial study and to evaluate the applicability of standard assessment methods to reflect change over time and patients own opinion on the course of disease. All available genetically diagnosed patients studied by us in 2010 (n = 41) were invited to the follow-up study; assessment of motor function (MFM-32), muscle strength (MRC %)and respiratory function (FVC %) and prime assessor were the same as in the initial study. Patients were asked whether the course of disease had progresses, was stable or had improved. 23 patients (15-61 y) accepted the invitation. Mean follow-up time was 7.7 years. Loss of muscle strength was more prominent in patients with mutations in DNM2, RYR1 and TPM2/3 genes and deterioration in FVC % was more evident in patients carrying NEB and ACTA1 gene mutations. MFM-sum score was less sensitive to change compared to MRC-sum score. In general, agreement between the patient's own opinion of the course of disease and results of assessments was good. The number of patients in the study is too small to be conclusive, but the results indicate that CM can be stable or slowly progressive depending on the genetic subtype.

A novel mutation in NEB causing foetal nemaline myopathy with arthrogryposis during early gestation

Nemaline myopathies are a clinically and genetically heterogeneous group of congenital myopathies, mainly characterized by muscle weakness, hypotonia and respiratory insufficiency. Here, we report a male foetus of consanguineous parents with a severe congenital syndrome characterized by arthrogryposis detected at 13 weeks of gestation. We describe severe complex dysmorphic facial and musculoskeletal features by post mortem fetal examination confirming the prenatal diagnosis. Histomorphological and ultrastructural studies of skeletal muscle reveal mini-rods in myotubes caused by a novel homozygous splice-site mutation in NEB (NM_001164508, chr2:g.152,417,623C>A GRCh37.p11 | c.19,102–1G>T ENST00000397345.3). No rods were seen in the myocardium. We discuss the relevance of this mutation in the context of nemaline myopathies associated with early developmental musculoskeletal disorders.

Nebulin nemaline myopathy recapitulated in a compound heterozygous mouse model with both a missense and a nonsense mutation in Neb

Nemaline myopathy (NM) caused by mutations in the gene encoding nebulin (NEB) accounts for at least 50% of all NM cases worldwide, representing a significant disease burden. Most NEB-NM patients have autosomal recessive disease due to a compound heterozygous genotype. Of the few murine models developed for NEB-NM, most are Neb knockout models rather than harbouring Neb mutations. Additionally, some models have a very severe phenotype that limits their application for evaluating disease progression and potential therapies. No existing murine models possess compound heterozygous Neb mutations that reflect the genotype and resulting phenotype present in most patients. We aimed to develop a murine model that more closely matched the underlying genetics of NEB-NM, which could assist elucidation of the pathogenetic mechanisms underlying the disease. Here, we have characterised a mouse strain with compound heterozygous Neb mutations; one missense (p.Tyr2303His), affecting a conserved actin-binding site and one nonsense mutation (p.Tyr935*), introducing a premature stop codon early in the protein. Our studies reveal that this compound heterozygous model, NebY2303H, Y935X, has striking skeletal muscle pathology including nemaline bodies. In vitro whole muscle and single myofibre physiology studies also demonstrate functional perturbations. However, no reduction in lifespan was noted. Therefore, NebY2303H,Y935X mice recapitulate human NEB-NM and are a much needed addition to the NEB-NM mouse model collection. The moderate phenotype also makes this an appropriate model for studying NEB-NM pathogenesis, and could potentially be suitable for testing therapeutic applications.

Nebulin: big protein with big responsibilities

Nebulin, encoded by NEB, is a giant skeletal muscle protein of about 6669 amino acids which forms an integral part of the sarcomeric thin filament. In recent years, the nebula around this protein has been largely lifted resulting in the discovery that nebulin is critical for a number of tasks in skeletal muscle. In this review, we firstly discussed nebulin’s role as a structural component of the thin filament and the Z-disk, regulating the length and the mechanical properties of the thin filament as well as providing stability to myofibrils by interacting with structural proteins within the Z-disk. Secondly, we reviewed nebulin’s involvement in the regulation of muscle contraction, cross-bridge cycling kinetics, Ca2+-homeostasis and excitation contraction (EC) coupling. While its role in Ca2+-homeostasis and EC coupling is still poorly understood, a large number of studies have helped to improve our knowledge on how nebulin affects skeletal muscle contractile mechanics. These studies suggest that nebulin affects the number of force generating actin-myosin cross-bridges and may also affect the force that each cross-bridge produces. It may exert this effect by interacting directly with actin and myosin and/or indirectly by potentially changing the localisation and function of the regulatory complex (troponin and tropomyosin). Besides unravelling the biology of nebulin, these studies are particularly helpful in understanding the patho-mechanism of myopathies caused by NEB mutations, providing knowledge which constitutes the critical first step towards the development of therapeutic interventions. Currently, effective treatments are not available, although a number of therapeutic strategies are being investigated.

P.160The spectrum of disease-causing and normal variation in the nebulin gene

The nebulin gene (NEB) with 183 exons is one of the largest genes in human. More than 240 different disease-causing variants in NEB have been identified in some 220 families. Around 50% of nemaline myopathies (NM) are caused by mutations in NEB. Mutations in NEB also cause core-rod myopathy, distal forms of NM, distal myopathy without nemaline rods, and multiple pterygium syndrome. The majority (95%) of the disease-causing variants in NEB are recessive point mutations, mainly nonsense and splice site mutations, and small indels, causing frameshifts. The remaining 5% of mutations are large copy number variations (CNVs). In addition to the recurrent CNVs in the triplicate region and the founder mutation with world-wide occurrence, deletion of exon 55, 11 different large pathogenic duplications and deletions have been identified in NEB. Until recently, all identified mutations in NEB have been recessive. Now it is known that large in-frame deletions in NEB, leading to the expression of substantially smaller proteins, can have a dominant-negative effect, and cause dominant distal forms of NM. The genome aggregation database (gnomAD) contains exome and genome data from 141,456 unrelated individuals, excluding patients with severe pediatric disorders. The total number of different NEB variants listed in gnomAD is 10,634. Most of the variants (96%) are very rare, only present in a few heterozygotes. Intronic variants comprise 43%, missense variants 40%, synonymous variants 14%, loss-of-function variants 3%, and UTR variants 0.2% of the total variation. It is noteworthy that 12.5% of the intronic variants are in the splice regions (excluding splice donor and acceptor sites), and may affect splicing. Rare variants are the most common variant type in NEB, complicating the interpretation of pathogenicity. Therefore, the pathogenicity of especially missense and splice site variants in NEB requires verification by functional analyses at the protein and mRNA level.

P.164Expression of alternative nebulin isoforms containing super repeat S21a or S21b in skeletal muscle

Nebulin is a giant sarcomeric protein, binding to actin along the length of the thin filament. Mutations in NEB are the most common cause of autosomal recessive nemaline myopathy (NM). NEB has 183 exons, and undergoes extensive alternative splicing, giving rise to numerous different protein isoforms. The protein structure is modular, with 32-35 amino-acid actin-binding simple repeats forming 26 different super repeats spanning most of the protein. Two alternative isoforms of super repeat S21, S21a and S21b, are encoded by two mutually exclusive exons, exon 143 and exon 144, respectively. S21a and S21b differ in both charge and hydrophobicity. The amino-acid sequence encoded by exon 143 shows complete homology between mouse, rat and human, indicating a central, conserved role for this region of the nebulin protein. To study the expression and function of these developmentally and clinically intriguing alternatively spliced exons, we have developed specific monoclonal antibodies, NEB-143 and NEB-144, against the isoforms containing S21a or S21b, respectively. We have previously shown that nebulin containing exon 144 is the first isoform expressed in myogenesis in vitro, while regulated expression of nebulin containing exon 143 occurs at later stages of muscle development. In this study, we investigated the expression of the S21a (exon 143) nebulin isoform further, in several adult muscles and myofibre types. Our results revealed an interesting pattern of alternating correlation between the expression of S21a and different types of MyHC myofibres, both slow and fast, with preferential expression in fast, MyHC IIX fibres.

New mutations found by Next-Generation Sequencing screening of Spanish patients with Nemaline Myopathy.

Nemaline Myopathy (NM) is a rare genetic disorder that encompasses a large spectrum of myopathies characterized by hypotonia and generalized muscle weakness. To date, mutations in thirteen different genes have been associated with NM. The most frequently responsible genes are NEB (50% of cases) and ACTA1 (15–25% of cases). In this report all known NM related genes were screened by Next Generation Sequencing in five Spanish patients in order to genetically confirm the clinical and histological diagnosis of NM. Four mutations in NEB (c.17779_17780delTA, c.11086A>C, c.21076C>T and c.2310+5G>A) and one mutation in ACTA1 (c.871A>T) were found in four patients. Three of the four mutations in NEB were novel. A cDNA sequencing assay of the novel variants c.17779_17780delTA, c.11086A>C and c.2310+5G>A revealed that the intronic variant c.2310+5G>A affected the splicing process. Mutations reported here could help clinicians and geneticists in NM diagnosis.

CONGENITAL MYOPATHIES: NEMALINE AND TITINOPATHIES: P.244A ddPCR method for the analysis of copy number variation in the segmental duplication regions of the sarcomeric giants nebulin and titin

The sarcomeric giants nebulin and titin (encoded by the NEB and TTN genes, respectively) both harbour large segmental duplication (SD) regions known to be expressed. In NEB, this region contains eight exons repeated three times. In TTN, nine exons are repeated threefold, after which the two first exons of the block reappear alone a fourth time. Mutations in NEB are known to cause nemaline myopathy (NM), and mutations in TTN can cause different myopathies, including tibial myopathy and cardiomyopathies. Using our targeted NM Comparative Genomic Hybridization array (NM-CGH-array), we have previously shown that copy number variations (CNV) in the NEB SD are potentially pathogenic. Recent studies using our CGH-array for neuromuscular disorders (NMD-CGH-array) revealed recurrent and potentially pathogenic CNVs in the TTN SD region also. To validate these findings, we have established a custom Digital Droplet PCR (ddPCR) assay for the analysis of the SD regions of NEB and TTN.

NEXT GENERATION SEQUENCING AND EXPERIMENTAL MYOLOGY: P.372Next generation sequencing: new phenotype-genotype correlations

Congenital myopathies and muscular dystrophies are clinically and genetically heterogeneous diseases with broadly overlapping features. Next generation sequencing (NGS) techniques represent a powerful tool to identify genetic variants in these disorders in contrast to classic time consuming gene by gene approach. We describe the results obtained in an extensive study of 150 genetically undiagnosed patients. The patients were classified, according to their clinical phenotype, as affected by congenital myopathy (CM), congenital muscular dystrophy (CMD), limb girdle muscle dystrophy (LGMD), distal myopathies (DM) and exercise intolerance. All the patients were followed at the French South-West Reference Center of neuromuscular disorders. The causing gene mutations could be identified in 46% of the patients. In most of the cases the variants were localized in genes classically implicated in each phenotype group (RYR1, ACTA1, NEB mutations in congenital myopathy group for example). However, one key finding from our study was the identification of new phenotype-genotype correlations in 10% of the cases. Two FLNC gene mutations in patients with congenital myopathy and severe cardiopathy; NEB variants in three patients with adult onset pure distal myopathy; ACTA1 neomutation in a patient with asymptomatic elevation of creatin kinase; 11 GCG repeats expansions in PABPN1 in a patient with predominant axial myopathy without ptosis. Of special interest is the emerging role of TTN gene mutations in muscular disease. We found clear pathogenic variants (compound heterozygous or homozygous stop codon, frameshift or splicing) in seven patients with quite heterogeneous clinical picture from congenital centronuclear myopathy to congenital distal arthrogryposis or limb girdle muscular weakness with cardiopathy The aim of the work is to present the global results of the study and more particularly the new phenotype-genotype correlations.

Characterization of congenital myopathies at a Korean neuromuscular center.

Congenital myopathies are muscle diseases characterized by specific histopathologic features, generalized hypotonia from birth, and perinatal complications, although some cases develop during childhood or, rarely, in adulthood. We undertook this study to characterize congenital myopathies among patients registered at our institution. Clinical, histopathologic, and genetic features were evaluated in 34 patients recruited for this study. The majority of patients experienced a childhood onset, and no disease-related mortality was recorded during follow-up. Functional outcomes were no better for those with late-onset disease, indicating later disease progression can be significant. Nemaline myopathy was the most frequent pathology, followed by central core disease and centronuclear myopathy. Among the 18 (54.5%) genetically confirmed patients, NEB and RYR1 mutations were the most common, followed by DNM2 mutations. This study shows features not previously reported and suggests that congenital myopathy should be considered an important issue among adult patients. Muscle Nerve 58: 235-244, 2018.

Phenotypes, genotypes, and prevalence of congenital myopathies older than 5 years in Denmark.

Objective:Congenital myopathy as a nosologic entity has long been recognized, but knowledge of overall and subtype prevalence and phenotype-genotype relationship is scarce, especially in the adult population.Methods:A national cohort of 107 patients ≥5 years diagnosed with congenital myopathy were prospectively assessed clinically, histologically, and genetically.Results:Twenty-five patients were excluded because of atypical features or alternative etiologies. The remaining 82 were on average 28 years old. Histologic examination revealed 14 (17%) with core disease, 15 (18%) centronuclear myopathy, 12 (15%) nemaline rods, 27 (33%) congenital fiber-type disproportion or type I predominance, and 14 (17%) nonspecific myopathic changes. Genetic etiology was identified in 46 patients (56.1%); 22.0% were heterozygous or compound heterozygous for mutations in RYR1, 7.3% had DNM2 mutations, and 7.3% NEB mutations. Less than 5% had mutations in ACTA1, TPM2/3, MTM1, TTN, SEPN1, or SC4NA. A genetic cause was established in 83% with specific histology (cores/rods/centronuclear myopathy) vs 29% with unspecific histology. The detailed clinical examination found gene-dependent discrepancies in the pattern of muscle affection and walking ability. Although walking ability was delayed in patients with ACTA1, TPM2/3, and RYR1 mutations, it was within normal limits in patients with NEB and DNM2 mutations.Conclusions:We found that overall, genetic and histologic prevalence of congenital myopathy in Denmark differs from previous retrospective reports. Less RYR1 and more DNM2 and NEB mutations and less core histology were present in our cohort. These differences may be explained by our prospective design, the older cohort of patients, and by differences in genetic background.

Exome sequencing reveals a nebulin nonsense mutation in a dog model of nemaline myopathy.

Nemaline myopathy (NM) is a congenital muscle disorder associated with muscle weakness, hypotonia, and rod bodies in the skeletal muscle fibers. Mutations in 10 genes have been implicated in human NM, but spontaneous cases in dogs have not been genetically characterized. We identified a novel recessive myopathy in a family of line-bred American bulldogs (ABDs); rod bodies in muscle biopsies established this as NM. Using SNP profiles from the nuclear family, we evaluated inheritance patterns at candidate loci and prioritized TNNT1 and NEB for further investigation. Whole exome sequencing of the dam, two affected littermates, and an unaffected littermate revealed a nonsense mutation in NEB (g.52734272 C>A, S8042X). Whole tissue gel electrophoresis and western blots confirmed a lack of full-length NEB in affected tissues, suggesting nonsense-mediated decay. The pathogenic variant was absent from 120 dogs of 24 other breeds and 100 unrelated ABDs, suggesting that it occurred recently and may be private to the family. This study presents the first molecularly characterized large animal model of NM, which could provide new opportunities for therapeutic approaches.

Mutation‐specific effects on thin filament length in thin filament myopathy

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. 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. 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. 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.