Molecular landscape of CAPN3 mutations in limb-girdle muscular dystrophy type R1: from a Chinese multicentre analysis to a worldwide perspective.

BACKGROUND: Limb-girdle muscular dystrophy type R1 (LGMDR1) can be caused by recessive CAPN3 mutations accounting for the majority of LGMD. To date, no systemic evaluation has been performed to analyse the detrimental and normal mutations on CAPN3 and its hotspots. METHODS: CAPN3 variants (n=112) from a total of 124 patients with LGMDR1 recruited in four centres in China were retrospectively analysed. Then external CAPN3 variants (n=2031) from online databases were integrated with our Chinese cohort data to achieve a worldwide perspective on CAPN3 mutations. According to their related phenotypes (LGMDR1 or normal), we analysed consequence, distribution, ethnicity and severity scores of CAPN3 mutations. RESULTS: Two hotspot mutations were identified including c.2120A>G in Chinese population and c.550del in Europe. According to the integrated dataset, 521 mutations were classified as LGMDR1-related and converged on exons 1, 10, 5, 22 and 13 of CAPN3. The remaining 1585 variants were classified as normal-population related. The deleterious ratio of LGMDR1-relevant variants to total variants in each population was 0.26 on average with a maximum of 0.35 in Finns and a minimum of 0.21 in South Asians. Severity evaluation showed that Chinese LGMDR1-related variants exhibited a higher risk (Combined Annotation Dependent Depletion score +1.10) than that from database patients (p<0.001). CONCLUSIONS: This study confirmed two hotspots and LGMDR1-related CAPN3 variants, highlighting the advantages in using a data-based comprehensive analysis to achieve a genetic landscape for patients with LGMDR1.

Compound heterozygous DYSF variants causing limb-girdle muscular dystrophy type 2B in a Chinese family.

BACKGROUND: The dysferlin gene or the DYSF gene encodes the Ca2+ -dependent phospholipid-binding protein dysferlin, which belongs to the ferlin family and is associated with muscle membrane regeneration and repair. Variants in the DYSF gene are responsible for limb-girdle muscular dystrophy type 2B (LGMD2B), also called limb-girdle muscular dystrophy recessive 2 (LGMDR2), a rare subtype of muscular dystrophy involving progressive muscle weakness and atrophy. The present study aimed to identify the variants responsible for the clinical symptoms of a Chinese patient with limb girdle muscular dystrophies (LGMDs) and to explore the genotype-phenotype associations of LGMD2B. METHODS: A series of clinical examinations, including blood tests, magnetic resonance imaging scans for the lower legs, electromyography and muscle biopsy, was performed on the proband diagnosed with muscular dystrophies. Whole exome sequencing was conducted to detect the causative variants, followed by Sanger sequencing to validate these variants. RESULTS: We identified two compound heterozygous variants in the DYSF gene, c.1058 T>C, p.(Leu353Pro) in exon 12 and c.1461C>A/p.Cys487* in exon 16 in this proband, which were inherited from the father and mother, respectively. In silico analysis for these variants revealed deleterious results by PolyPhen-2 (Polymorphism Phenotyping v2; http://genetics.bwh.harvard.edu/pph2), SIFT (Sorting Intolerant From Tolerant; https://sift.bii.a-star.edu.sg), PROVEAN (Protein Variation Effect Analyzer; http://provean.jcvi.org/seq_submit.php) and MutationTaster (http://www.mutationtaster.org). In addition, the two compound heterozygous variants in the proband were absent in 100 control individuals who had an identical ethnic origin and were from the same region, suggesting that these variants may be the pathogenic variants responsible for the LGMD2B phenotypes for this proband. CONCLUSIONS: The present study broadens our understanding of the mutational spectrum of the DYSF gene, which provides a deep insight into the pathogenesis of LGMDs and accelerates the development of a prenatal diagnosis.

Expression, purification, and structural analysis of the full-length human integral membrane protein γ-sarcoglycan.

Mutation of the gene encoding γ-sarcoglycan (SGCG), an integral membrane protein responsible for maintaining the integrity of the muscle cell sarcolemma, results in Limb-Girdle Muscular Dystrophy (LGMD), a congenital disease with no current treatment options. This member of the sarcoglycan glycoprotein family is a vital component of the Dystrophin Complex, which together facilitate normal muscle function. However, very little is known about the structure and dynamics of these proteins, and of membrane glycoproteins in general. This is due to a number of factors, including their complexity, heterogeneity and highly-specific native environments. The expression, purification, and structural study of membrane proteins is further impeded by their hydrophobic nature and consequent propensity to aggregate in aqueous solutions. Here, we report the first successful expression and purification of milligram quantities of full-length recombinant SGCG, utilizing fusion protein-guided overexpression to inclusion bodies in Escherichia coli. Purification of SGCG from the fusion protein, TrpΔLE, was facilitated using chemical cleavage. Cleavage products were then isolated by size-exclusion chromatography. Successful purification of the protein was confirmed using SDS-PAGE and mass spectroscopy. Finally, solution nuclear magnetic resonance spectroscopy of uniformly 15N-labeled SGCG in detergent environments was performed, yielding the first spectra of the full-length membrane glycoprotein, SGCG. These results represent the initial structural studies of SGCG, laying the foundation for further investigation on the interaction and dynamics of other integral membrane proteins. More specifically, this data allows for opportunities in the future for enhanced treatment modalities and cures for LGMD.

Ubiquitin- and ATP-dependent unfoldase activity of P97/VCP•NPLOC4•UFD1L is enhanced by a mutation that causes multisystem proteinopathy.

p97 is a "segregase" that plays a key role in numerous ubiquitin (Ub)-dependent pathways such as ER-associated degradation. It has been hypothesized that p97 extracts proteins from membranes or macromolecular complexes to enable their proteasomal degradation; however, the complex nature of p97 substrates has made it difficult to directly observe the fundamental basis for this activity. To address this issue, we developed a soluble p97 substrate-Ub-GFP modified with K48-linked ubiquitin chains-for in vitro p97 activity assays. We demonstrate that WT p97 can unfold proteins and that this activity is dependent on the p97 adaptor NPLOC4-UFD1L, ATP hydrolysis, and substrate ubiquitination, with branched chains providing maximal stimulation. Furthermore, we show that a p97 mutant that causes inclusion body myopathy, Paget's disease of bone, and frontotemporal dementia in humans unfolds substrate faster, suggesting that excess activity may underlie pathogenesis. This work overcomes a significant barrier in the study of p97 and will allow the future dissection of p97 mechanism at a level of detail previously unattainable.

Lamin-Related Congenital Muscular Dystrophy Alters Mechanical Signaling and Skeletal Muscle Growth.

Laminopathies are a clinically heterogeneous group of disorders caused by mutations in the LMNA gene, which encodes the nuclear envelope proteins lamins A and C. The most frequent diseases associated with LMNA mutations are characterized by skeletal and cardiac involvement, and include autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD), limb-girdle muscular dystrophy type 1B, and LMNA-related congenital muscular dystrophy (LMNA-CMD). Although the exact pathophysiological mechanisms responsible for LMNA-CMD are not yet understood, severe contracture and muscle atrophy suggest that mutations may impair skeletal muscle growth. Using human muscle stem cells (MuSCs) carrying LMNA-CMD mutations, we observe impaired myogenic fusion with disorganized cadherin/ß catenin adhesion complexes. We show that skeletal muscle from Lmna-CMD mice is unable to hypertrophy in response to functional overload, due to defective fusion of activated MuSCs, defective protein synthesis and defective remodeling of the neuromuscular junction. Moreover, stretched myotubes and overloaded muscle fibers with LMNA-CMD mutations display aberrant mechanical regulation of the yes-associated protein (YAP). We also observe defects in MuSC activation and YAP signaling in muscle biopsies from LMNA-CMD patients. These phenotypes are not recapitulated in closely related but less severe EDMD models. In conclusion, combining studies in vitro, in vivo, and patient samples, we find that LMNA-CMD mutations interfere with mechanosignaling pathways in skeletal muscle, implicating A-type lamins in the regulation of skeletal muscle growth.

A limb-girdle myopathy phenotype of RUNX2 mutation in a patient with cleidocranial dysplasia: a case study and literature review.

BACKGROUND: Cleidocranial dysplasia (CCD) is a rare hereditary disorder that arises from heterozygous loss of function mutations in the runt-related transcription factor 2 (RUNX2) gene. As RUNX2 is mainly expressed in osteoblasts, CCD typically affects the skeletal and dental systems. Few studies have investigated RUNX2 mutation effects on non-skeletal systems. Here, we describe limb-girdle myopathy, an uncommon phenotype of CCD, in a patient with a heterozygous missense mutation (p.R225Q) in the RUNX2 gene. CASE PRESENTATION: A 58 year-old man presented with progressive back pain and six months of weakness in the proximal parts of all four limbs. Physical examinations showed that he was short in stature (height, 164.4 cm; weight, 79.1 kg) with a dysmorphic face, including hypertelorism, midface hypoplasia, and chin protrusion. At a young age, he had received orthodontic surgery, due to dental abnormalities. Neurological examinations revealed sloping shoulders, weakness, and atrophy in the proximal areas of the arms, shoulder girdle muscles, and legs. The deep tendon reflex and sensory system were normal. Radiological examinations revealed mild scoliosis, shortened clavicles, and a depressed skull bone, which were consistent with a clinical diagnosis of CCD. Electromyography (EMG) studies showed myogenic polyphasic waves in the deltoid, biceps brachii, and rectus femoris muscles. Instead, the EMG findings were normal in the first dorsal interosseous, tibialis anterior and facial muscles. The EMG findings were compatible with a limb-girdle pattern with facial sparing. The patient's family history showed his father and eldest daughter with similar dysmorphic faces, skeletal disorders and proximal upper extremity weakness. We sequenced the RUNX2 gene and discovered a heterozygous missense mutation (c.G674A, p.R225Q), which altered the C-terminal end of the RUNX2 protein. This mutation was predicted to inactivate the protein and might affect its interactions with other proteins. This mutation co-segregated with the disease phenotypes in the family. CONCLUSIONS: We described limb-girdle myopathy in a patient with CCD that carried a heterozygous RUNX2 missense mutation. This uncommon phenotype expanded the phenotypic spectrum of the RUNX2 p.R225Q mutation. The role of RUNX2 in myogenic development merits future studies. Our findings remind clinicians that myopathic patients with myopathies combined with facial dysmorphism and shortened clavicles should consider the diagnosis of CCD.

Next generation sequencing detection of late onset pompe disease.

INTRODUCTION: We report a patient in whom the diagnosis of a treatable disease was delayed for 30 years. METHODS: Recent discoveries of next generation sequencing (NGS) have allowed us to reconsider the diagnosis of limb girdle muscular dystrophy (LGMD) cases of unknown etiology. RESULTS: A 36-year-old man appeared to have LGMD with onset in shoulder girdle muscles, but all sarcolemmal and cytoskeletal proteins tested by immunoblotting and immunohistochemistry gave normal results. He developed respiratory insufficiency and became dependent on overnight ventilation at age 44. By NGS technology, 2 mutations in the GAA gene (intervening sequence 1 and a missense mutation in exon 11) allowed us to make a definite diagnosis of glycogenosis type II (Pompe disease) and start enzyme replacement therapy at age 71. CONCLUSIONS: Mild nondystrophic features on muscle biopsy and respiratory muscle involvement should suggest late-onset Pompe disease in patients with an unclassified LGMD phenotype. NGS may help make the diagnosis. Muscle Nerve 53: 981-983, 2016.

Dual effects of exercise in dysferlinopathy.

Dysferlinopathy refers to a group of autosomal recessive muscular dystrophies due to mutations in the dysferlin gene causing deficiency of a membrane-bound protein crucially involved in plasma membrane repair. The condition is characterized by marked clinical heterogeneity, the different phenotypes/modes of presentation being unrelated to the genotype. For unknown reasons, patients are often remarkably active before the onset of symptoms. Dysferlin deficiency-related persistence of mechanically induced sarcolemma disruptions causes myofiber damage and necrosis. We postulate that limited myodamage may initially remain hidden with well-preserved resistance to physical strains. By subjecting dysferlin-deficient B6.A/J-Dysf(prmd) mice to long-term swimming exercise, we observed that concentric/isometric strain improved muscle strength and alleviated muscular dystrophy by limiting the accumulation of membrane lesions. By contrast, eccentric strain induced by long-term running in a wheel worsened the dystrophic process. Myofiber damage induced by eccentric strain increased with age, reflecting the accumulation of non-necrotic membrane lesions up to a critical threshold. This phenomenon was modulated by daily spontaneous activity. Transposed to humans, our results may suggest that the past activity profile shapes the clinical phenotype of the myopathy and that patients with dysferlinopathy should likely benefit from concentric exercise-based physiotherapy.

Muscle fatigue, nNOS and muscle fiber atrophy in limb girdle muscular dystrophy.

Muscle fatigability and atrophy are frequent clinical signs in limb girdle muscular dystrophy (LGMD), but their pathogenetic mechanisms are still poorly understood. We review a series of different factors that may be connected in causing fatigue and atrophy, particularly considering the role of neuronal nitric oxide synthase (nNOS) and additional factors such as gender in different forms of LGMD (both recessive and dominant) underlying different pathogenetic mechanisms. In sarcoglycanopathies, the sarcolemmal nNOS reactivity varied from absent to reduced, depending on the residual level of sarcoglycan complex: in cases with complete sarcoglycan complex deficiency (mostly in beta-sarcoglycanopathy), the sarcolemmal nNOS reaction was absent and it was always associated with early severe clinical phenotype and cardiomyopathy. Calpainopathy, dysferlinopathy, and caveolinopathy present gradual onset of fatigability and had normal sarcolemmal nNOS reactivity. Notably, as compared with caveolinopathy and sarcoglycanopathies, calpainopathy and dysferlinopathy showed a higher degree of muscle fiber atrophy. Males with calpainopathy and dysferlinopathy showed significantly higher fiber atrophy than control males, whereas female patients have similar values than female controls, suggesting a gender difference in muscle fiber atrophy with a relative protection in females. In female patients, the smaller initial muscle fiber size associated to endocrine factors and less physical effort might attenuate gender-specific muscle loss and atrophy.

Etiology of limb girdle muscular dystrophy 1D/1E determined by laser capture microdissection proteomics.

Limb girdle muscular dystrophy 1D/1E (OMIM nomenclature LGMD1D, Human Gene Nomenclature Committee LGMD1E), a skeletal and cardiac myopathy, has previously been linked to chromosome 6q23. We used laser capture microdissection to isolate cytoplasmic inclusions from skeletal muscle from a patient with LGMD1D/1E, performed mass spectrometry-based proteomics on these minute inclusions, and identified through bioinformatics desmin as their major constituent. Sequencing in this patient and family members identified the genetic basis of the previously reported 6q23 linked LGMD1D/1E to be due to an intron splice donor site mutation (IVS3+3A>G) of the desmin gene located on chromosome 2q35.

Rare treatable limb girdle muscle disease.

We report two cases of Limb Girdle pattern of muscle weakness caused by hyperparathyroidism due to parathyroid adenoma. It can be easily missed as early symptoms are non specific but once diagnosed it is easily treatable and complete recovery occurs over a period of time.

Dysferlinopathy course and sportive activity: clues for possible treatment.

LGMD2B is a frequent proximo-distal myopathy with rapid evolution after age 20. Exacerbating factors may be physical exercise and inflammation. There is very little information about the effect of sportive activity in LGMD2B, since eccentric exercise frequently results in muscle damage. LGMD2B has often an onset with myalgia and MRI imaging (STIR-sequences) shows myoedema. In a prolonged observational study of a series of 18 MM/LGMD2B patients we have studied the pattern of clinical and radiological evolution. The disease has an abrupt onset in the second decade and most patients perform sports before definite disease onset. On the basis of Gardner-Medwin and Walton scale, grade 4 is reached two years faster in patients who performed sports (over 1000 hours). Other considerations regarding pathogenetic mechanism and response to treatment show a poor response to immunosuppressive treatment of muscle inflammation. Preventing a strenuous physical activity should be recommended in patients with high CK and diagnosed or suspected to have dysferlin deficiency.

Two cases of a rare treatable limb girdle muscle disease.

We report two cases of limb girdle pattern of muscle weakness caused by hyperparathyroidism due to parathyroid adenoma. It can be easily missed as early symptoms are non specific but once diagnosed it is easily treatable and complete recovery occurs over a period of time.

Identifying Plectin Isoform Functions through Animal Models.

Plectin, a high-molecular-weight cytoskeletal linker protein, binds with high affinity to intermediate filaments of all types and connects them to junctional complexes, organelles, and inner membrane systems. In addition, it interacts with actomyosin structures and microtubules. As a multifunctional protein, plectin has been implicated in several multisystemic diseases, the most common of which is epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). A great part of our knowledge about plectin's functional diversity has been gained through the analysis of a unique collection of transgenic mice that includes a full (null) knockout (KO), several tissue-restricted and isoform-specific KOs, three double KOs, and two knock-in lines. The key molecular features and pathological phenotypes of these mice will be discussed in this review. In summary, the analysis of the different genetic models indicated that a functional plectin is required for the proper function of striated and simple epithelia, cardiac and skeletal muscle, the neuromuscular junction, and the vascular endothelium, recapitulating the symptoms of humans carrying plectin mutations. The plectin-null line showed severe skin and muscle phenotypes reflecting the importance of plectin for hemidesmosome and sarcomere integrity; whereas the ablation of individual isoforms caused a specific phenotype in myofibers, basal keratinocytes, or neurons. Tissue-restricted ablation of plectin rendered the targeted cells less resilient to mechanical stress. Studies based on animal models other than the mouse, such as zebrafish and C. elegans, will be discussed as well.

Cervical Hyperextension Deformity After Sagittal Balance Correction in Patient with Congenital Limb-Girdle Myopathy: Surgical Technique and Review of Literature.

BACKGROUND: There is no gold standard surgical treatment for cervical hyperextension deformity, especially in case of muscular dystrophy. Special considerations and caution should be taken as they carry a high risk of early mortality and spinal cord injury. Only a few case reports are available in the literature. CASE DESCRIPTION: We report a case of surgical correction of an iatrogenic cervical hyperextension deformity following sagittal balance correction in a patient with congenital limb-girdle myopathy. The patient was successfully treated by posterior cervical release and fusion after verification of the range of motion, reducibility of the deformity, and absence of any positional spinal cord compression with dynamic radiographic examination and preoperative magnetic resonance imaging in the desired postoperative position. CONCLUSIONS: We suggest posterior cervical release and fusion in case of a radiologically and clinically reducible cervical hyperextension deformity under both motor and sensory spinal evoked potential monitoring. In cases of longstanding, rigid, nonreducible cervical hyperextension, laminectomy and concomitant duroplasty could be considered.

Correction of pseudoexon splicing caused by a novel intronic dysferlin mutation.

OBJECTIVE: Dysferlin is a large transmembrane protein that functions in critical processes of membrane repair and vesicle fusion. Dysferlin-deficiency due to mutations in the dysferlin gene leads to muscular dystrophy (Miyoshi myopathy (MM), limb girdle muscular dystrophy type 2B (LGMD2B), distal myopathy with anterior tibial onset (DMAT)), typically with early adult onset. At least 416 pathogenic dysferlin mutations are known, but for approximately 17% of patients, one or both of their pathogenic variants remain undefined following standard exon sequencing methods that interrogate exons and nearby flanking intronic regions but not the majority of intronic regions. METHODS: We sequenced RNA from myogenic cells to identify a novel dysferlin pathogenic variant in two affected siblings that previously had only one disease-causing variant identified. We designed antisense oligonucleotides (AONs) to bypass the effects of this mutation on RNA splicing. RESULTS: We identified a new pathogenic point mutation deep within dysferlin intron 50i. This intronic variant causes aberrant mRNA splicing and inclusion of an additional pseudoexon (PE, we term PE50.1) within the mature dysferlin mRNA. PE50.1 inclusion alters the protein sequence, causing premature translation termination. We identified this mutation in 23 dysferlinopathy patients (seventeen families), revealing it to be one of the more prevalent dysferlin mutations. We used AON-mediated exon skipping to correct the aberrant PE50.1 splicing events in vitro, which increased normal mRNA production and significantly restored dysferlin protein expression. INTERPRETATION: Deep intronic mutations can be a common underlying cause of dysferlinopathy, and importantly, could be treatable with AON-based exon-skipping strategies.

Avascular necroses inducing limb-girdle syndrome.

Disuse myopathy can be caused by many disorders. However, disuse myopathy with symmetrical weakness of the proximal muscles in the four limbs is rare. Here we report a 45-year-old man who presented with the appearance of myopathy similar to the clinical picture of limb-girdle syndrome with symmetrical weakness of proximal muscles in four limbs. A battery of intensive investigations confirmed that the patient had symmetric disuse myopathy induced by multiple avascular necroses of hip and shoulder joints. After the patient had received bilateral total hip replacement operations he had remarkable improvement in his muscle power in both lower limbs. This report highlights that some myopathies can be reversed if their underlying causes are correctable.

New intronic splicing mutation in the LMNA gene causing progressive cardiac conduction defects and variable myopathy.

BACKGROUND: Most of mutations in the LMNA gene are unique and have been found in only a few unrelated families. The clinical interpretation of new genetic variants, especially beyond the coding area and canonical splice sites, is proving to be difficult and requires advanced investigation. METHODS: This study included patients with progressive cardiac conduction defects with neuromuscular involvement. The clinical evaluation included medical history and 24-h Holter monitoring. The genetic evaluation included mutation screening in the LMNA gene by the Sanger sequence. Sanger sequencing was followed by RT-PCR of the target fragment of cDNA. In silico modeling was performed with CCBulder and Modeller software. RESULTS: The diagnosis of limb-girdle muscular dystrophy type 1B (LGMD1B) was established. The new intronic variant c.513+45T>G was found in the LMNA gene in the proband and affected daughter. The insertion of 45bp was confirmed in the proband's cDNA. The structural and possible functional effects of the aberrant protein were predicted. CONCLUSIONS: Variant c.513+45T>G in the LMNA gene likely translates into the longer lamin A/C proteins with additional 15 amino acids. This variant is thought to be pathogenic. Intronic variants in the LMNA gene located beside canonic splice sites may be responsible for some genotype-negative cases with clinical phenotype of laminopathies.

POPDC1(S201F) causes muscular dystrophy and arrhythmia by affecting protein trafficking.

The Popeye domain-containing 1 (POPDC1) gene encodes a plasma membrane-localized cAMP-binding protein that is abundantly expressed in striated muscle. In animal models, POPDC1 is an essential regulator of structure and function of cardiac and skeletal muscle; however, POPDC1 mutations have not been associated with human cardiac and muscular diseases. Here, we have described a homozygous missense variant (c.602C>T, p.S201F) in POPDC1, identified by whole-exome sequencing, in a family of 4 with cardiac arrhythmia and limb-girdle muscular dystrophy (LGMD). This allele was absent in known databases and segregated with the pathological phenotype in this family. We did not find the allele in a further screen of 104 patients with a similar phenotype, suggesting this mutation to be family specific. Compared with WT protein, POPDC1(S201F) displayed a 50% reduction in cAMP affinity, and in skeletal muscle from patients, both POPDC1(S201F) and WT POPDC2 displayed impaired membrane trafficking. Forced expression of POPDC1(S201F) in a murine cardiac muscle cell line (HL-1) increased hyperpolarization and upstroke velocity of the action potential. In zebrafish, expression of the homologous mutation (popdc1(S191F)) caused heart and skeletal muscle phenotypes that resembled those observed in patients. Our study therefore identifies POPDC1 as a disease gene causing a very rare autosomal recessive cardiac arrhythmia and LGMD, expanding the genetic causes of this heterogeneous group of inherited rare diseases.