Limb-girdle Muscular Dystrophy 2L Research Articles

Endoplasmic reticulum maintains ion homeostasis required for plasma membrane repair

Of the many crucial functions of the ER, homeostasis of physiological calcium increase is critical for signaling. Plasma membrane (PM) injury causes a pathological calcium influx. Here, we show that the ER helps clear this surge in cytoplasmic calcium through an ER-resident calcium pump, SERCA, and a calcium-activated ion channel, Anoctamin 5 (ANO5). SERCA imports calcium into the ER, and ANO5 supports this by maintaining electroneutrality of the ER lumen through anion import. Preventing either of these transporter activities causes cytosolic calcium overload and disrupts PM repair (PMR). ANO5 deficit in limb girdle muscular dystrophy 2L (LGMD2L) patient cells compromises their cytosolic and ER calcium homeostasis. By generating a mouse model of LGMD2L, we find that PM injury causes cytosolic calcium overload and compromises the ability of ANO5-deficient myofibers to repair. Addressing calcium overload in ANO5-deficient myofibers enables them to repair, supporting the requirement of the ER in calcium homeostasis in injured cells and facilitating PMR.

P.299Disease progression using quantitative MRI outcome measures in limb girdle muscular dystrophy 2L

Traditionally outcome measures for clinical trials in neuromuscular diseases are based on physical assessments which are dependent on patient effort, and may not be sensitive to progression over short trial periods in slow-progressing diseases. Limb girdle muscular dystrophy type 2L (LGMD2L) is a slowly progressive disease with weakness and atrophy of proximal muscles; elevated creatine kinase levels; and dystrophic findings on muscle biopsy. There is no effective treatment available for LGMD2L, but clinical trials have been conducted over the past years, which call for quantitative and reliable outcome measures to be developed. In this study, we investigated disease progression over 12 months in 16 patients with genetically confirmed LGMD2L, using quantitative outcome measures, including Dixon magnetic resonance imaging (MRI). We used MRI to assess changes in muscle fat content and stationary dynamometry to assess changes in muscle strength. Disease progression was also investigated with handheld dynamometry, handgrip strength and manual muscle testing (MMT). Preliminary results show a tendency of increased muscle fat fraction in the muscles analyzed using the quantitative MRI technique from baseline to 12 months follow up. The muscle fat fraction increased with 2% on average and the intrinsic back muscles showed the largest increase. Stationary dynamometry and handgrip strength decreased slightly, whereas handheld dynamometry and manual muscle testing did not change. The present study demonstrates a slow disease progression in LGMD2L. Dixon MRI detected increases in muscle fat content and is therefore a suitable candidate for an outcome measure in natural history or treatment studies in LGMD2L. The stationary dynamometry also seems to be reliable outcome measures for LMGMD2L.

Dysregulated calcium homeostasis prevents plasma membrane repair in Anoctamin 5/TMEM16E-deficient patient muscle cells

Autosomal recessive mutations in Anoctamin 5 (ANO5/TMEM16E), a member of the transmembrane 16 (TMEM16) family of Ca2+-activated ion channels and phospholipid scramblases, cause adult-onset muscular dystrophies (limb girdle muscular dystrophy 2L (LGMD2L) and Miyoshi Muscular Dystrophy (MMD3). However, the molecular role of ANO5 is unclear and ANO5 knockout mouse models show conflicting requirements of ANO5 in muscle. To study the role of ANO5 in human muscle cells we generated a myoblast line from a MMD3-patient carrying the c.2272C>T mutation, which we find causes the mutant protein to be degraded. The patient myoblasts exhibit normal myogenesis, but are compromised in their plasma membrane repair (PMR) ability. The repair deficit is linked to the poor ability of the endoplasmic reticulum (ER) to clear cytosolic Ca2+ increase caused by focal plasma membrane injury. Expression of wild-type ANO5 or pharmacological prevention of injury-triggered cytosolic Ca2+ overload enable injured patient muscle cells to repair. A homology model of ANO5 shows that several of the known LGMD2L/MMD3 patient mutations line the transmembrane region of the protein implicated in its channel activity. These results point to a role of cytosolic Ca2+ homeostasis in PMR, indicate a role for ANO5 in ER-mediated cytosolic Ca2+ uptake and identify normalization of cytosolic Ca2+ homeostasis as a potential therapeutic approach to treat muscular dystrophies caused by ANO5 deficit.

First familial limb-girdle muscular dystrophy 2L in China

Limb-girdle muscular dystrophy 2L (LGMD2L) is mainly characterized by late adult onset, atrophy of proximal muscles, chronic progressive and asymmetric weakness, accompanied by increased creatine kinase (CK) levels, dystrophic pathological changes and electromyography showing myogenic damage. To date, familial LGMD2L was reported in European countries and had not been reported in China.A careful investigation of the clinical manifestations, muscle performance imaging, biopsy, and target next-generation sequencing (NGS) technology was utilized to identify pathogenic genetic variants in a 4-generation pedigree that includes 6 affected individuals.The results revealed mild-to-moderate hypertrophy of bilateral gastrocnemii and slight weakness and atrophy in the proximal muscles of the lower limbs, with obviously increased serum creatine kinase levels. The symptoms were more serious in the male proband but were also observed in females. Obvious and symmetric atrophy and fat infiltration of posterior segments of the thigh was evident in muscle magnetic resonance imaging (MRI). The pathological changes included a small amount of atrophic and hypertrophic fibers, scattered necrotizing fibers, a small number of increased nuclei, inward migration, mild proliferation of interstitial connective tissue, and no inflammatory cell infiltration. The pathogenic allele was a c.220C > T mutation in the anoctamin 5 (ANO5) gene.The LGMD2L family was characterized by mild chronic myopathy and bilateral gastrocnemius hypertrophy with obviously increased CK levels. Pathological changes included atrophy of fibers with interstitial connective tissues hyperplasia. The pathogenic allele was a c.220C> T mutation in the ANO5 gene.

A novel ANO5 splicing variant in a LGMD2L patient leads to production of a truncated aggregation-prone Ano5 peptide.

Mutations in ANO5 cause several human diseases including gnathodiaphyseal dysplasia 1 (GDD1), limb‐girdle muscular dystrophy 2L (LGMD2L), and Miyoshi myopathy 3 (MMD3). Previous work showed that complete genetic disruption of Ano5 in mice did not recapitulate human muscular dystrophy, while residual expression of mutant Ano5 in a gene trapped mouse developed muscular dystrophy with defective membrane repair. This suggests that truncated Ano5 expression may be pathogenic. Here, we screened a panel of commercial anti‐Ano5 antibodies using a recombinant adenovirus expressing human Ano5 with FLAG and YFP at the N‐ and C‐terminus, respectively. The monoclonal antibody (mAb) N421A/85 was found to specifically detect human Ano5 by immunoblotting and immunofluorescence staining. The antigen epitope was mapped to a region of 28 residues within the N‐terminus. Immunofluorescence staining of muscle cryosections from healthy control subjects showed that Ano5 is localized at the sarcoplasmic reticulum. The muscle biopsy from a LGMD2L patient homozygous for the c.191dupA mutation showed no Ano5 signal, confirming the specificity of the N421A/85 antibody. Surprisingly, strong Ano5 signal was detected in a patient with compound heterozygous mutations (c.191dupA and a novel splice donor site variant c.363 + 4A > G at the exon 6–intron 6 junction). Interestingly, insertion of the mutant intron 6, but not the wild‐type intron 6, into human ANO5 cDNA resulted in a major transcript that carried the first 158‐bp of intron 6. Transfection of the construct encoding the first 121 amino acids into C2C12 cells resulted in protein aggregate formation, suggesting that aggregate‐forming Ano5 peptide may contribute to the pathogenesis of muscular dystrophy.

Gain-of-Function of TMEM16E/ANO5 Scrambling Activity Caused by a Mutation Associated with the Bone Genetic Disease Gnathodiaphyseal Dysplasia

Members of the TMEM16 or anoctamin membrane protein family are involved in a variety of functions that include ion transport, phospholipid scrambling and regulation of other membrane proteins. In particular, TMEM16A, 16B and 16F have a clear plasma membrane localization but while 16A and 16B work as ‘pure’ ion channels, TMEM16F shows both ion channel and scramblase activities. Recent data suggest that also TMEM16E, the closest relative of TMEM16F, may work as a phospholipid scramblase, although a direct confirmation is missing. TMEM16E is highly expressed in bone tissue and skeletal muscle and different mutations in the human TMEM16E (ANO5) gene are associated with the bone disease gnathodiaphyseal dysplasia (GDD) or to different forms of muscular dystrophy (LGMD2L, limb-girdle muscular dystrophy-2l and distal MMD3, miyoshi muscular dystrophy-3). Our data show that human TMEM16E, when overexpressed in mammalian cell lines, displays partial plasma membrane localization and give rise to phospholipid scrambling as well as non-selective ionic currents with slow time-dependent activation at highly depolarized membrane potentials and in presence of high intracellular Ca2+. We address the effect of the GDD-causing mutation T513I, located in the second extracellular loop, on TMEM16E and we show phospholipid scrambling activity and large time-dependent ion currents even at low cytosolic Ca2+ concentrations. Our data provide the first direct demonstration of Ca2+-dependent current and phospholipid scrambling activity for TMEM16E and suggest a gain-of-function phenotype related to a GDD mutation.

Comparing clinical data and muscle imaging of DYSF and ANO5 related muscular dystrophies

In this retrospective cross-sectional study clinical and muscle imaging data of patients with Miyoshi distal myopathy phenotype (MMD1 and MMD3) and limb girdle muscular dystrophy 2L (LGMD2L) were described. MMD1 and MMD3 are genetically heterogenous diseases based on DYSF and ANO5 gene defects. MMD3 and LGMD2L are clinically different diseases caused by an ANO5 gene defect. All groups showed predominant fatty degeneration of the gluteus minimus muscle and of the posterior segments of the thigh and calf muscles with sparing of the gracilis muscle. Muscle atrophy, hypertrophy and asymmetric muscle involvement on muscle imaging did not differ between groups. The pattern of fatty degeneration of muscles and of muscle weakness shows only minor differences between MMD1 (n=6) and MMD3 (n=8) patients with more frequently fatty degeneration of the rectus femoris, anterior tibial, and extensor digitorum muscles and more frequently muscle weakness in the anterior tibial, peroneal and calf muscle in MMD1. In the ANO5 related phenotypes the lateral head of the gastrocnemius muscle was less frequently involved in LGMD2L (n=13) and no differences in the incidence of muscle weakness was found. Therefore, MMD3 and LGMD2L should be considered as part of one spectrum of ANO5 related muscle disease.

Isolated semitendinosus involvement in the initial stages of limb-girdle muscular dystrophy 2L

Unit of Neuromuscular Disorders, Laboratory of Molecular Medicine, Bambino Gesu’ Children’s Research Hospital, Rome, Italy Folkhalsan Institute of Genetics and Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland Pediatric Neurology Unit, Catholic University School of Medicine, Rome, Italy d Institute of Neurology, Catholic University School of Medicine, Rome, Italy Neuromuscular Research Center, University of Tampere and Tampere University Hospital, Tampere, Finland Neurology Department, Vaasa Central Hospital, Vaasa, Finland

P.5.2 A comparison of muscle imaging in DYSF and ANO5 related muscular dystrophies

In this descriptive retrospective and cross-sectional study we compared muscle images of patients with Miyoshi distal myopathy phenotype (MMD1 and MMD3) to limb girdle muscular dystrophy 2L (LGMD2L) patients. MMD1 and MMD3 are genetically heterogenous diseases based on DYSF and ANO5 gene defects. MMD3 and LGMD2L are clinically different diseases caused by an ANO5 gene defect. A total of 35 muscle imaging studies of MMD1, MMD3 and LGMD2L patients was evaluated for fatty degeneration, muscle hypertrophy or atrophy and asymmetric muscle involvement. Eight muscle imaging scans of six MMD1 patients, 11 scans of nine MMD3 patients, and 16 scans of 14 LGMD2L patients were evaluated. Median disease duration was six years in MMD1, 13 years in MMD 3, and five years in LGMD2L. All groups showed predominant involvement of the gluteus minimus muscle and both the posterior segment of the thigh and calf muscles, with sparing of the gracilis muscle. The rectus femoris, anterior tibial, and extensor digitorum muscles were more frequently involved in MMD1 when compared with MMD3. The lateral head of the gastrocnemius muscle was more frequently involved in MMD3 in comparison to LGMD2L. Muscle hypertrophy or atrophy and asymmetric muscle involvement did not differ significantly between the groups. The pattern of fatty degeneration of muscles shows minor differences between MMD3 and MMD1 patients and is similar between the ANO5 related phenotypes. Therefore MMD3 and LGMD2L should be considered as part of one spectrum of ANO5 related muscle disease, equal to the dysferlinopathies, MMD1 and LGMD2B.

ANO5‐muscular dystrophy: clinical, pathological and molecular findings

Anoctamin 5 (ANO5) is a putative intracellular calcium-activated chloride channel. Recessive mutations in ANO5 cause primary skeletal muscle disorders (limb-girdle muscular dystrophy 2L and distal muscular dystrophy), which are phenotypically similar to dysferlinopathy, a muscular dystrophy due to dysferlin-encoding gene (DYSF) mutations. This study reports the phenotype and genotype of seven unrelated patients with ANO5-muscular dystrophy. Three patients had amyloid deposition in muscle and two had cardiac involvement. An additional patient without skeletal muscle amyloidosis had cardiac involvement with septal hypokinesis and supraventricular tachycardia requiring ablation. Amyloid subtyping using laser capture microdissection and mass spectrometry-based proteomic analysis did not identify ANO5 or any fragment of ANO5 in the amyloid deposits, but detected other known amyloidogenic proteins. Three patients had myotonic discharges without clinical myotonia. Four ANO5 mutations are novel, including a heterozygous 0.4Mb deletion involving the entire ANO5 gene. The results of the present study suggest that ANO5 mutations can be associated with amyloid deposition in muscle, but the nature of the amyloid deposits remains indeterminate, as does their relationship with cardiac involvement. ANO5 analysis should be considered in cases of muscle amyloid deposition of indeterminate etiology. Electrical myotonia can accompany ANO5-muscular dystrophy.

Anoctamin 5 Mutations Are a Frequent Cause of Limb Girdle Muscular Dystrophy and of the Miyoshi-Type Muscular Dystrophy in the Netherlands (S15.005)

Objective: To perform Anoctamin 5 (ANO) mutation analysis, neurological and cardiological examination in the genetically undiagnosed remainder of the Dutch limb girdle muscular dystrophy (LGMD) cohort encompassing 105 patients from 68 families and in a proportion of a cohort of 22 patients from 16 families with a Miyoshi-type muscular dystrophy (MMD) in which no dysferlin mutations were found. Background ANO 5 mutations have recently been found to cause LGMD2L, MMD3 and asymptomatic hyperCKemia. Design/Methods: Neurological examination, cardiological investigations (i.e. electrocardiography, Holter monitoring, echocardiography), serum creatine kinase (sCK) activity assessments and ANO 5 mutation analysis were performed in LGMD and MMD patients. Results: 32 LGMD index patients and 12 MMD3 patients from 8 kindreds were examined. ANO 5 mutations were found in 13 sporadic LGMD cases (8 males). Age at onset ranged from 21-57 years, age at examination from 26-69. Symmetrical proximal leg muscle weakness had been the first manifestation. Rhabdomyolysis attacks were mentioned in one patient, and three had cardiological abnormalities, including intraventricular septum thickening (2) and hypertrophic cardiomyopathy (1). SCK was 10-25 times the upper limit of normal (ULN). In 8 patients (all male) from 6 MMD families mutations were identified. Age at onset ranged from 18-51 years and age at examination from 30-67. Uni- or bilateral calf muscle weakness had been the presenting symptom in 7 patients and one had been asymptomatic at time of diagnosis. SCK was 20-40 times ULN. None had associated symptoms and cardiological examination was normal. Exon 5 c.191dupA was the most frequently observed mutation and in addition novel mutations were identified. Conclusions: 1. LGMD 2L is found in 16% of the Dutch LGMD families and MMD3 in 40% of the MMD families.2. In three LGMD2L patients heart involvement was found and in none of the MMD3 patients. Disclosure: Dr. De Visser has received research support from Biogen Idec. Dr. van der Kooi has received research support from Biogen Idec. Dr. Linssen has nothing to disclose. Dr. Ginjaar has nothing to disclose. Dr. Wokke has received research support from Genzyme Corporation. Dr. van Doorn has received personal compensation for activities with Talecris, ZLB, Baxter, and Octapharma. Dr. van Doorn has received research support from Baxter.

165 Novel ANO5 gene mutations, c.989dupT and c.2018A→G causing Limb Girdle Muscular dystrophy 2L

A 41-year-old male presented with difficulty in walking on tiptoes and in standing for long periods and also noticed wasting of both his calves, forearm muscles and thighs for 2...

A founder mutation in Anoctamin 5 is a major cause of limb girdle muscular dystrophy

The limb-girdle muscular dystrophies are a group of disorders with wide genetic and clinical heterogeneity. Recently, mutations in the ANO5 gene, which encodes a putative calcium-activated chloride channel belonging to the Anoctamin family of proteins, were identified in five families with one of two previously identified disorders, limb-girdle muscular dystrophy 2L and non-dysferlin Miyoshi muscular dystrophy. We screened a candidate group of 64 patients from 59 British and German kindreds and found the truncating mutation, c.191dupA in exon 5 of ANO5 in 20 patients, homozygously in 15 and in compound heterozygosity with other ANO5 variants in the rest. An intragenic single nucleotide polymorphism and an extragenic microsatellite marker are in linkage disequilibrium with the mutation, suggesting a founder effect in the Northern European population. We have further defined the clinical phenotype of ANO5-associated muscular dystrophy. Patients show adult onset proximal lower limb weakness with highly raised serum creatine kinase values (average 4500 IU/l) and frequent muscle atrophy and asymmetry of muscle involvement. Onset varies from the early 20 s to 50 s and the weakness is generally slowly progressive, with most patients remaining ambulant for several decades. Distal presentation is much less common but a milder degree of distal lower limb weakness is often observed. Upper limb strength is only mildly affected and cardiac and respiratory function is normal. Females appear less frequently affected. In the North of England population we have identified eight patients with ANO5 mutations, suggesting a minimum prevalence of 0.27/100,000, twice as common as dysferlinopathy. We suggest that mutations in ANO5 represent a relatively common cause of adult onset muscular dystrophy with high serum creatine kinase and that mutation screening, particularly of the common mutation c.191dupA, should be an early step in the diagnostic algorithm of adult limb-girdle muscular dystrophy patients.