The Location of Disease-Causing DES Variants Determines the Severity of Phenotype and the Morphology of Sarcoplasmic Aggregates.

Desmin (DES) is the main intermediate muscle filament that connects myofibrils individually and with the nucleus, sarcolemma, and organelles. Pathogenic variants of DES cause desminopathy, a disorder affecting the heart and skeletal muscles. We aimed to analyze the clinical features, morphology, and distribution of desmin aggregates in skeletal muscle biopsies of patients with desminopathy and to correlate these findings with the type and location of disease-causing DES variants. This retrospective study included 30 patients from 20 families with molecularly confirmed desminopathy from 2 neuromuscular referral centers. We identified 2 distinct patterns of desmin aggregates: well-demarcated subsarcolemmal aggregates and diffuse aggregates with poorly delimited borders. Pathogenic variants located in the 1B segment and the tail domain of the desmin molecule are more likely to present with early-onset cardiomyopathy compared to patients with variants in other segments. All patients with mutations in the 1B segment had well-demarcated subsarcolemmal aggregates, but none of the patients with variants in other desmin segments showed such histological features. We suggest that variants located in the 1B segment lead to well-shaped subsarcolemmal desmin aggregation and cause disease with more frequent cardiac manifestations. These findings will facilitate early identification of patients with potentially severe cardiac syndromes.

Genetic fitness and selection intensity in a population affected with high-incidence spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 (SCA1) is the major and likely the only type of autosomal dominant cerebellar ataxia in the Sakha (Yakut) people of Eastern Siberia. The prevalence rate of SCA1 has doubled over the past 21 years peaking at 46 cases per 100,000 rural population. The age at death correlates closely with the number of CAG triplet repeats in the mutant ATXN1 gene (r = -0.81); most patients with low-medium (39-55) repeat numbers survived until the end of reproductive age. The number of CAG repeats expands in meiosis, particularly in paternal transmissions; the average total increase in intergenerational transmissions in our cohort was estimated at 1.6 CAG repeats. The fertility rates of heterozygous carriers of 39-55 CAG repeats in women were no different from those of the general Sakha population. Overall, the survival of mutation carriers through reproductive age, unaltered fertility rates, low childhood mortality in SCA1-affected families, and intergenerational transmission of increasing numbers of CAG repeats in the ATXN1 gene indicate that SCA1 in the Sakha population will be maintained at high prevalence levels. The low (0.19) Crow's index of total selection intensity in our SCA1 cohort implies that this mutation is unlikely to be eliminated through natural selection alone.

Adult-onset autosomal dominant spastic paraplegia linked to a GTPase-effector domain mutation of dynamin 2.

BACKGROUND: Hereditary Spastic Paraplegia (HSP) represents a large group of clinically and genetically heterogeneous disorders linked to over 70 different loci and more than 60 recognized disease-causing genes. A heightened vulnerability to disruption of various cellular processes inherent to the unique function and morphology of corticospinal neurons may account, at least in part, for the genetic heterogeneity. METHODS: Whole exome sequencing was utilized to identify candidate genetic variants in a four-generation Siberian kindred that includes nine individuals showing clinical features of HSP. Segregation of candidate variants within the family yielded a disease-associated mutation. Functional as well as in-silico structural analyses confirmed the selected candidate variant to be causative. RESULTS: Nine known patients had young-adult onset of bilateral slowly progressive lower-limb spasticity, weakness and hyperreflexia progressing over two-to-three decades to wheel-chair dependency. In the advanced stage of the disease, some patients also had distal wasting of lower leg muscles, pes cavus, mildly decreased vibratory sense in the ankles, and urinary urgency along with electrophysiological evidence of a mild distal motor/sensory axonopathy. Molecular analyses uncovered a missense c.2155C > T, p.R719W mutation in the highly conserved GTP-effector domain of dynamin 2. The mutant DNM2 co-segregated with HSP and affected endocytosis when expressed in HeLa cells. In-silico modeling indicated that this HSP-associated dynamin 2 mutation is located in a highly conserved bundle-signaling element of the protein while dynamin 2 mutations associated with other disorders are located in the stalk and PH domains; p.R719W potentially disrupts dynamin 2 assembly. CONCLUSION: This is the first report linking a mutation in dynamin 2 to a HSP phenotype. Dynamin 2 mutations have previously been associated with other phenotypes including two forms of Charcot-Marie-Tooth neuropathy and centronuclear myopathy. These strikingly different pathogenic effects may depend on structural relationships the mutations disrupt. Awareness of this distinct association between HSP and c.2155C > T, p.R719W mutation will facilitate ascertainment of additional DNM2 HSP families and will direct future research toward better understanding of cell biological processes involved in these partly overlapping clinical syndromes.

A new disease allele for the p.C30071R mutation in titin causing hereditary myopathy with early respiratory failure.

Hereditary myopathy with early respiratory failure is an autosomal dominant myopathy caused by mutations in the 119th fibronectin-3 domain of titin. To date all reported patients with the most common mutation in this domain (p.C30071R) appear to share ancestral disease alleles. We undertook this study of two families with the p.C30071R mutation to determine whether they share the same haplotype as previously reported British families or whether the mutation arose as a de novo event. We sequenced the 119th fibronectin-3 domain in these two probands and flanking polymorphisms associated with the British haplotype in hereditary myopathy with early respiratory failure. A family of Indian descent had a haplotype that was not compatible with the British shared haplotype. Cloning of the 119th fibronectin-3 domain in this patient demonstrated polymorphisms rs191484894 and novel noncoding variant c.90225C>T on the same allele as the mutation, which is distinct from previously reported British families. This proves that the p.C30071R mutation itself (rather than the haplotype containing this mutation) causes hereditary myopathy with early respiratory failure and suggests its independent origin in different ethnic groups.

Myofibrillar myopathies: new developments.

PURPOSE OF REVIEW: Myofibrillar myopathies (MFMs) are a heterogeneous group of skeletal and cardiac muscle diseases. In this review, we highlight recent discoveries of new genes and disease mechanisms involved in this group of disorders. RECENT FINDINGS: The advent of next-generation sequencing technology, laser microdissection and mass spectrometry-based proteomics has facilitated the discovery of new MFM causative genes and pathomechanisms. New mutations have also been discovered in 'older' genes, helping to find a classification niche for MFM-linked disorders showing variant phenotypes. Cell transfection experiments using primary cultured myoblasts and newer animal models provide insights into the pathogenesis of MFMs. SUMMARY: An increasing number of genes are involved in the causation of variant subtypes of MFM. The application of modern technologies in combination with classical histopathological and ultrastructural studies is helping to establish the molecular diagnosis and reach a better understanding of the pathogenic mechanisms of each MFM subtype, thus putting an emphasis on the development of specific means for prevention and therapy of these incapacitating and frequently fatal diseases.

Exome sequencing identifies titin mutations causing hereditary myopathy with early respiratory failure (HMERF) in families of diverse ethnic origins.

BACKGROUND: Hereditary myopathy with early respiratory failure (HMERF) was described in several North European families and recently linked to a titin gene (TTN) mutation. We independently studied HMERF-like diseases with the purpose to identify the cause, refine diagnostic criteria, and estimate the frequency of this disease among myopathy patients of various ethnic origins. METHODS: Whole exome sequencing analysis was carried out in a large U.S. family that included seven members suffering from skeletal muscle weakness and respiratory failure. Subsequent mutation screening was performed in further 45 unrelated probands with similar phenotypes. Studies included muscle strength evaluation, nerve conduction studies and concentric needle EMG, respiratory function test, cardiologic examination, and muscle biopsy. RESULTS: A novel TTN p.Gly30150Asp mutation was identified in the highly conserved A-band of titin that co-segregated with the disease in the U.S. family. Screening of 45 probands initially diagnosed as myofibrillar myopathy (MFM) but excluded based on molecular screening for the known MFM genes led to the identification of a previously reported TTN p.Cys30071Arg mutation in one patient. This same mutation was also identified in a patient with suspected HMERF. The p.Gly30150Asp and p.Cys30071Arg mutations are localized to a side chain of fibronectin type III element A150 of the 10th C-zone super-repeat of titin. CONCLUSIONS: Missense mutations in TTN are the cause of HMERF in families of diverse origins. A comparison of phenotypic features of HMERF caused by the three known TTN mutations in various populations allowed to emphasize distinct clinical/pathological features that can serve as the basis for diagnosis. The newly identified p.Gly30150Asp and the p.Cys30071Arg mutation are localized to a side chain of fibronectin type III element A150 of the 10th C-zone super-repeat of titin.

Impairment of protein degradation in myofibrillar myopathy caused by FLNC/filamin C mutations.

Myofibrillar myopathy caused by FLNC/filamin C mutations is characterized by disintegration of myofibrils and a massive formation of protein aggregates within skeletal muscle fibers. We performed immunofluorescence studies in skeletal muscle sections from filaminopathy patients to detect disturbances of protein quality control mechanisms. Our analyses revealed altered expression of chaperone proteins and components of proteasomal and autophagic degradation pathways in abnormal muscle fibers that harbor protein deposits but not in neighboring muscle fibers without pathological protein aggregation. These findings suggest a dysfunction of protein stabilizing and degrading mechanisms that leads to a pathological accumulation of protein aggregates in abnormal fibers. Accordingly, a pharmacological modulation of chaperone activity may be a promising therapeutic strategy to prevent protein aggregation and to reduce disease progression. Newly established filaminopathy cell culture models provide a suitable basis for testing such pharmacological approaches.

Filamin C-related myopathies: pathology and mechanisms.

The term filaminopathy was introduced after a truncating mutation in the dimerization domain of filamin C (FLNc) was shown to be responsible for a devastating muscle disease. Subsequently, the same mutation was found in patients from diverse ethnical origins, indicating that this specific alteration is a mutational hot spot. Patients initially present with proximal muscle weakness, while distal and respiratory muscles become affected with disease progression. Muscle biopsies of these patients show typical signs of myofibrillar myopathy, including disintegration of myofibrils and aggregation of several proteins into distinct intracellular deposits. Highly similar phenotypes were observed in patients with other mutations in Ig-like domains of FLNc that result in expression of a noxious protein. Biochemical and biophysical studies showed that the mutated domains acquire an abnormal structure causing decreased stability and eventually becoming a seed for abnormal aggregation with other proteins. The disease usually presents only after the fourth decade of life possibly as a result of ageing-related impairments in the machinery that is responsible for disposal of damaged proteins. This is confirmed by mutations in components of this machinery that cause a highly similar phenotype. Transfection studies of cultured muscle cells reflect the events observed in patient muscles and, therefore, may provide a helpful model for testing future dedicated therapeutic strategies. More recently, FLNC mutations were also found in families with a distal myopathy phenotype, caused either by mutations in the actin-binding domain of FLNc that result in increased actin-binding and non-specific myopathic abnormalities without myofibrillar myopathy pathology, or a nonsense mutation in the rod domain that leads to RNA instability, haploinsufficiency with decreased expression levels of FLNc in the muscle fibers and myofibrillar abnormalities, but not to the formation of desmin-positive protein aggregates required for the diagnosis of myofibrillar myopathy.

Clinical and myopathological characteristics of desminopathy caused by a mutation in desmin tail domain.

BACKGROUND: Most of the previously described pathogenic mutations in desmin are located in highly conserved α-helical domains that play an important role in intermediate filament assembly. The role of the C-terminus non-α-helical 'tail' domain is much less investigated and until recently mutations in this domain have been implicated in only a few patients. The majority of reported desminopathy cases caused by the tail mutations were sporadic, creating a representation bias regarding the disease frequency and phenotypic characteristics. METHODS: We performed detailed genotype-phenotype analysis of autosomal dominant desminopathy associated with tail domain mutations in a four-generation autosomal dominant family with 16 members affected by a progressive cardiac and/or skeletal myopathy caused by a c.1346A>C (p.Lys449Thr) mutation located in the tail domain of desmin. RESULTS: Phenotypic features in patients with tail domain mutations are similar to those in patients with mutations localized in the 1B and 2B α-helical domains. CONCLUSION: We recommend that the tail domain is searched for mutations as intensely as desmin coil domains which until recently were considered to be more 'functional'.

Pathophysiology of protein aggregation and extended phenotyping in filaminopathy.

Mutations in FLNC cause two distinct types of myopathy. Disease associated with mutations in filamin C rod domain leading to expression of a toxic protein presents with progressive proximal muscle weakness and shows focal destructive lesions of polymorphous aggregates containing desmin, myotilin and other proteins in the affected myofibres; these features correspond to the profile of myofibrillar myopathy. The second variant associated with mutations in the actin-binding domain of filamin C is characterized by weakness of distal muscles and morphologically by non-specific myopathic features. A frameshift mutation in the filamin C rod domain causing haploinsufficiency was also found responsible for distal myopathy with some myofibrillar changes but no protein aggregation typical of myofibrillar myopathies. Controversial data accumulating in the literature require re-evaluation and comparative analysis of phenotypes associated with the position of the FLNC mutation and investigation of the underlying disease mechanisms. This is relevant and necessary for the refinement of diagnostic criteria and developing therapeutic approaches. We identified a p.W2710X mutation in families originating from ethnically diverse populations and re-evaluated a family with a p.V930_T933del mutation. Analysis of the expanded database allows us to refine clinical and myopathological characteristics of myofibrillar myopathy caused by mutations in the rod domain of filamin C. Biophysical and biochemical studies indicate that certain pathogenic mutations in FLNC cause protein misfolding, which triggers aggregation of the mutant filamin C protein and subsequently involves several other proteins. Immunofluorescence analyses using markers for the ubiquitin-proteasome system and autophagy reveal that the affected muscle fibres react to protein aggregate formation with a highly increased expression of chaperones and proteins involved in proteasomal protein degradation and autophagy. However, there is a noticeably diminished efficiency of both the ubiquitin-proteasome system and autophagy that impairs the muscle capacity to prevent the formation or mediate the degradation of aggregates. Transfection studies of cultured muscle cells imitate events observed in the patient's affected muscle and therefore provide a helpful model for testing future therapeutic strategies.

Novel FLNC mutation in a patient with myofibrillar myopathy in combination with late-onset cerebellar ataxia.

INTRODUCTION: Mutations in the gene that encodes filamin C, FLNC, represent a rare cause of a distinctive type of myofibrillar myopathy (MFM). METHODS: We investigated an Italian patient by means of muscle biopsy, muscle and brain imaging and molecular analysis of MFM genes. RESULTS: The patient harbored a novel 7256C>T, p.Thr2419Met mutation in exon 44 of FLNC. Clinical, pathological and muscle MRI findings were similar to the previously described filaminopathy cases. This patient had, in addition, cerebellar ataxia with atrophy of cerebellum and vermis evident on brain MRI scan. Extensive screening failed to establish a cause of cerebellar atrophy. CONCLUSIONS: We report an Italian filaminopathy patient, with a novel mutation in a highly conserved region. This case raises the possibility that the disease spectrum caused by FLNC may include cerebellar dysfunction.

Kuru: genes, cannibals and neuropathology.

Kuru was the first human transmissible spongiform encephalopathy (TSE) or prion disease identified, occurring in the Fore linguistic group of Papua New Guinea. Kuru was a uniformly fatal cerebellar ataxic syndrome, usually followed by choreiform and athetoid movements. Kuru imposed a strong balancing selection on the Fore population, with individuals homozygous for the 129 Met allele of the gene (PRNP) encoding for prion protein (PrP) being the most susceptible. The decline in the incidence of kuru in the Fore has been attributed to the exhaustion of the susceptible genotype and ultimately by discontinuation of exposure via cannibalism. Neuropathologically, kuru-affected brains were characterized by widespread degeneration of neurons, astroglial and microglial proliferation, and the presence of amyloid plaques. These early findings have been confirmed and extended by recent immunohistochemical studies for the detection of the TSE-specific PrP (PrP). Confocal laser microscopy also showed the concentration of glial fibrillary acidic protein-positive astrocytic processes at the plaque periphery. The fine structure of plaques corresponds to that described earlier by light microscopy. The successful experimental transmission of kuru led to the awareness of its similarity to Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker disease and formed a background against which the recent epidemics of iatrogenic and variant Creutzfeldt-Jakob disease could be studied.

Genetic variants in potassium channels are associated with type 2 diabetes in a Mongolian population.

BACKGROUND: Recent genome-wide association studies (GWAS) have identified more than 40 common sequence variants associated with type 2 diabetes (T2D). However, the results are not always the same in populations with differing genetic backgrounds. In the present study, we evaluated a hypothesis that a North Asian population living in a geographic area with unusually harsh environmental conditions would develop unique genetic risks. METHODS: A population-based association study was performed with 21 single-nucleotide polymorphisms (SNPs) in nine genes selected according to the results of GWAS conducted in other populations. The study participants included 393 full-heritage Mongolian individuals (177 diagnosed with T2D and 216 matched controls). Genotyping was performed by TaqMan methodology. RESULTS: The strongest association was detected with SNPs located within the potassium channel-coding genes KCNQ1 (highest odds ratio [OR] = 1.92; P = 3.4 × 10(-5) ) and ABCC8 (OR = 1.79; P = 5 × 10(-4) ). Genetic variants identified as strongly influencing the risk of T2D in other populations (e.g. KCNJ11 or TCF7L2) did not show significant association in Mongolia. CONCLUSIONS: The strongest T2D risk-associated SNPs in Mongolians are located within two of three tested potassium channel-coding genes. Accumulated variations in these genes may be related to the exposure to harsh environmental conditions.

Clinical and myopathological evaluation of early- and late-onset subtypes of myofibrillar myopathy.

Myofibrillar myopathies (MFM) are a group of disorders associated with mutations in DES, CRYAB, MYOT, ZASP, FLNC, or BAG3 genes and characterized by disintegration of myofibrils and accumulation of degradation products into intracellular inclusions. We retrospectively evaluated 53 MFM patients from 35 Spanish families. Studies included neurologic exam, muscle imaging, light and electron microscopic analysis of muscle biopsy, respiratory function testing and cardiologic work-up. Search for pathogenic mutations was accomplished by sequencing of coding regions of the six genes known to cause MFM. Mutations in MYOT were the predominant cause of MFM in Spain affecting 18 of 35 families, followed by DES in 11 and ZASP in 3; in 3 families the cause of MFM remains undetermined. Comparative analysis of DES, MYOT and ZASP associated phenotypes demonstrates substantial phenotypic distinctions that should be considered in studies of disease pathogenesis, for optimization of subtype-specific treatments and management, and directing molecular analysis.

Nemaline myopathy type 6: clinical and myopathological features.

Nemaline myopathy (NEM) is one of the most common congenital myopathies. A unique subtype, NEM6, maps to chromosome 15q21-q23 in two pedigrees, but the causative gene has not been determined. We conducted clinical examination and myopathological studies in a new NEM family. Genotyping and gene screening were accomplished by searching known and 18 new candidate genes. The disease started in childhood by affecting proximal and distal muscles and causing slowness of movements. Muscle biopsies showed numerous nemaline rods and core-like formations. Suggestive linkage to chromosome 15q22-q23 was established. Genes known to be mutated in NEM or core-rod myopathy were screened and excluded. No pathogenic mutations were identified in other candidate genes. The disease in this Spanish family was classified as NEM6. It is phenotypically similar and probably allelic to the two previously reported NEM6 pedigrees. Further studies of these families will lead to the identification of the NEM6 gene.

Dominant mutations in KBTBD13, a member of the BTB/Kelch family, cause nemaline myopathy with cores.

We identified a member of the BTB/Kelch protein family that is mutated in nemaline myopathy type 6 (NEM6), an autosomal-dominant neuromuscular disorder characterized by the presence of nemaline rods and core lesions in the skeletal myofibers. Analysis of affected families allowed narrowing of the candidate region on chromosome 15q22.31, and mutation screening led to the identification of a previously uncharacterized gene, KBTBD13, coding for a hypothetical protein and containing missense mutations that perfectly cosegregate with nemaline myopathy in the studied families. KBTBD13 contains a BTB/POZ domain and five Kelch repeats and is expressed primarily in skeletal and cardiac muscle. The identified disease-associated mutations, C.742C>A (p.Arg248Ser), c.1170G>C (p.Lys390Asn), and c.1222C>T (p.Arg408Cys), located in conserved domains of Kelch repeats, are predicted to disrupt the molecule's beta-propeller blades. Previously identified BTB/POZ/Kelch-domain-containing proteins have been implicated in a broad variety of biological processes, including cytoskeleton modulation, regulation of gene transcription, ubiquitination, and myofibril assembly. The functional role of KBTBD13 in skeletal muscle and the pathogenesis of NEM6 are subjects for further studies.

Inheritance patterns and phenotypic features of myofibrillar myopathy associated with a BAG3 mutation.

Myofibrillar myopathies are a heterogeneous group of neuromuscular disorders characterized by disintegration of myofibrils. The inheritance pattern is commonly autosomal dominant, but there has been a striking absence of secondary cases noted in a BAG3-associated subtype. We studied three families with BAG3 p.Pro209Leu mutation showing a severe phenotype of myofibrillar myopathy and axonal neuropathy with giant axons. In one family, transmission to a pair of siblings has occurred from their asymptomatic father who showed somatic mosaicism. In two other families, neither of the parents was affected or showed detectable level of somatic mosaicism. These observations suggest that the BAG3 variant of myofibrillar myopathy may result from a spontaneous mutation at an early point of embryonic development and that transmission from a mosaic parent may occur more than once. The study underlines the importance of parental evaluation as it may have implications for genetic counseling.

DNA sequencing errors in molecular diagnostics of filamin myopathy.

BACKGROUND: Filamin myopathy is a neuromuscular disorder manifesting with predominantly limb-girdle muscle weakness and in many patients with diaphragm paralysis and cardiomyopathy, caused by mutations in the filamin C (FLNC) gene. Molecular diagnosis of filamin myopathy based on direct DNA sequencing of coding exons is compromised by the presence of a high homology pseudogene (pseFLNC) located approximately 53.6 kb downstream of the functional FLNC gene on chromosome 7q. METHODS: Molecular cloning, RT-PCR and real-time PCR methods were used to detect sequence differences between the FLNC and pseFLNC that are implicated in known or potential molecular diagnostic errors. Overall, 50 patients with a phenotype resembling filamin myopathy have been screened for mutations in FLNC. RESULTS: FLNC sequence inconsistencies caused by the interference from pseFLNC were identified and diagnostic errors involving, in particular, the detection of the most frequent disease-causing FLNC p.W2710X mutation resolved. Mismatches between the FLNC and pseFLNC sequences were tabulated for future use. CONCLUSIONS: We devise a strategy that allows one to discern mutations occurring in the functional FLNC from those harbored in pseFLNC, thus preventing possible complications in future research and patient genetic testing.

Epidemiology of Viliuisk encephalomyelitis in Eastern Siberia.

BACKGROUND: Viliuisk encephalomyelitis is a disorder that starts, in most cases, as an acute meningoencephalitis. Survivors of the acute phase develop a slowly progressing neurologic syndrome characterized by dementia, dysarthria, and spasticity. An epidemic of this disease has been spreading throughout the Yakut Republic of the Russian Federation. Although clinical, neuropathologic, and epidemiologic data suggest infectious etiology, multiple attempts at pathogen isolation have been unsuccessful. METHODS: Detailed clinical, pathologic, laboratory, and epidemiologic studies have identified 414 patients with definite Viliuisk encephalomyelitis in 15 of 33 administrative regions of the Yakut Republic between 1940 and 1999. All data are documented in a Registry. RESULTS: The average annual Viliuisk encephalomyelitis incidence rate at the height of the epidemic reached 8.8 per 100,000 population and affected predominantly young adults. The initial outbreak occurred in a remote isolated area of the middle reaches of Viliui River; the disease spread to adjacent areas and further in the direction of more densely populated regions. The results suggest that intensified human migration from endemic villages led to the emergence of this disease in new communities. Recent social and demographic changes have presumably contributed to a subsequent decline in disease incidence. CONCLUSIONS: Based on the largest known set of diagnostically verified Viliuisk encephalomyelitis cases, we demonstrate how a previously little-known disease that was endemic in a small indigenous population subsequently reached densely populated areas and produced an epidemic involving hundreds of persons.