Autosomal Recessive Dystrophy Research Articles

Dysferlin mediates membrane tubulation and links T-tubule biogenesis to muscular dystrophy.

The multi-C2 domain protein dysferlin localizes to the plasma membrane and the T-tubule system in skeletal muscle; however, its physiological mode of action is unknown. Mutations in the DYSF gene lead to autosomal recessive limb-girdle muscular dystrophy type 2B and Miyoshi myopathy. Here, we show that dysferlin has membrane tubulating capacity and that it shapes the T-tubule system. Dysferlin tubulates liposomes, generates a T-tubule-like membrane system in non-muscle cells, and links the recruitment of phosphatidylinositol 4,5-bisphosphate to the biogenesis of the T-tubule system. Pathogenic mutant forms interfere with all of these functions, indicating that muscular wasting and dystrophy are caused by the dysferlin mutants' inability to form a functional T-tubule membrane system.

A POGLUT1 mutation causes a muscular dystrophy with reduced Notch signaling and satellite cell loss.

Skeletal muscle regeneration by muscle satellite cells is a physiological mechanism activated upon muscle damage and regulated by Notch signaling. In a family with autosomal recessive limb‐girdle muscular dystrophy, we identified a missense mutation in POGLUT1 (protein O‐glucosyltransferase 1), an enzyme involved in Notch posttranslational modification and function. In vitro and in vivo experiments demonstrated that the mutation reduces O‐glucosyltransferase activity on Notch and impairs muscle development. Muscles from patients revealed decreased Notch signaling, dramatic reduction in satellite cell pool and a muscle‐specific α‐dystroglycan hypoglycosylation not present in patients' fibroblasts. Primary myoblasts from patients showed slow proliferation, facilitated differentiation, and a decreased pool of quiescent PAX7+ cells. A robust rescue of the myogenesis was demonstrated by increasing Notch signaling. None of these alterations were found in muscles from secondary dystroglycanopathy patients. These data suggest that a key pathomechanism for this novel form of muscular dystrophy is Notch‐dependent loss of satellite cells.

A.O.7 - Research for the pathogenesis and therapy of dysferlinopathy using proteomics approach

Dysferlinopathies are a group of autosomal recessive muscular dystrophies caused by mutations in dysferlin gene (DYSF). There are two main phenotypes including Miyoshi muscular dystrophy and limb-girdle muscular dystrophy type 2B. DYSF is composed of 55 exons coding for 2,080 amino acid residues. DYSF is expressed in the plasma membrane of skeletal muscles and is involved in calcium-mediated membrane fusion events and membrane repair. We previously identified over 50 different mutations across the entire DYSF in approximately 60% of the patients. Our research theme is focusing on these DYSF and DYSF-related proteins. Recent advances have identified some components of the cell membrane repair process, particularly those involved in a putative pathway specific to striated muscles. Two important members are DYSF and Mitsugumin 53 (MG53). Compared to DYSF, molecular size of MG53 is rather small and can be expressed and work as membrane repair molecules. To find the binding partner of DYSF, which can be working as the similar functional role, would be a good clue to find the therapeutic strategy of dysferlinopathy. Using recombinant proteins and affinity column binding methods combined with LC/MS/MS, we identified novel DYSF-binding proteins. Protein X, one of the novel proteins, is considered to be an essential component of the membrane repair process. To directly evaluate the membrane repair capacity of the X knockdown C2C12 cells, we measured FM fluorescent dye entry after laser-induced damage to cells. The wild-type cells could effectively reseal sarcolemmal membranes, as they showed only minimal FM dye entry after laser damage. In contrast, significant entry of FM dye into the X knockdown cells was observed after laser-induced damage, suggesting insufficient membrane repair in X-deficient cells. To elucidate the molecular function of protein X in dysferlinopathy condition, we will also use several kinds of myoblasts originated from dysferlinopathy patients.

A novel mutation in alpha sarcoglycan gene in an Iranian family with limb girdle muscular dystrophy 2D

Objective and importance: The sarcoglycanopathies (SGPs) are a subgroup of autosomal recessive limb girdle muscular dystrophies. They are caused by mutations in gamma, alpha, beta, and delta sarcoglycans (SGs) genes. Alpha-SGPs are the most frequent form of SGPs. Muscle biopsy studies in patients with SGPs have indicated that loss of one SG subunit leads to instability of whole SG complex. Autozygosity mapping is a powerful gene mapping approach for rare recessive inherited disorders in consanguineous families.Clinical presentation: In the present study, proband was a 9 year old girl from consanguineous parents. She was diagnosed at the age of 5 when she had problems climbing stairs. Her creatine kinase level was 16428 U/L. Proximal weakness and ankle contracture were also observed in the patient.Techniques: Autozygosity mapping, using short tandem repeat (STR) markers linked to the SG genes, showed co-segregation of the phenotype with STR markers linked to the SGCA (Alpha-sarcoglycan) gene. Her muscle biopsy also suggested alpha sarcoglycanopathy. Mutation analyses revealed a novel homozygous deletion of 11 base pairs in exon 4 of this gene. This deletion introduces a premature termination codon after the 4th amino acid. This will eliminate the expression of the downstream part of the extracellular domain of the protein. This domain has a critical role by associating with other molecules of dystrophin–glycoprotein complexes.Conclusion: IHC (Immunohistochemistry) studies combined with autozygosity mapping and mutation screening is an efficient diagnostic method in the SGPs.

A rare form of limb girdle muscular dystrophy (type 2E) seen in an Iranian family detected by autozygosity mapping

Sarcoglycanopathies (SGPs) constitute a subgroup of autosomal recessive limb girdle muscular dystrophies (LGMDs) which are caused by mutations in sarcoglycan (SGs) genes. SG proteins form a core complex consisting of α, β, γ and δ sarcoglycans which are encoded by SGCA, SGCB, SGCG and SGCD genes, respectively. Genetic defect, in any of these SG proteins, results in instability of the whole complex. This effect can be helpful in interpreting muscle biopsy results. Autozygosity mapping is a gene mapping approach which can be applied in large consanguineous families for tracking the defective gene in most autosomal recessive disorders. In the present study, we used autozygosity mapping, to find the gene responsible for muscular dystrophy. Proband was a 10-year-old boy referred to our center for ruling out DMD (Duchenne muscular dystrophy). According to the pedigree and clinical reports, we assessed him for SGPs. Haplotyping, using the four short tandem repeat (STR) markers for each of the SG genes, showed that the phenotype may segregate with SGCB gene; and observing two crossing overs which occurred within the gene suggested that the mutation might be in the first two exons of SGCB gene. Mutation analysis showed a 26 bp duplication (10 bp before the initiation codon till 13 bp after the ATG start codon). This will cause a frameshift in protein synthesis.

Limb-girdle muscular dystrophy type 2A in Brazilian children.

Calpainopathy is an autosomal recessive limb girdle muscular dystrophy (LGMD2A) caused by mutations in CAPN3 gene. To present clinical and histological findings in six children with a molecular diagnosis of LGMD2A and additionally the MRI findings in two of them. We retrospectively assessed medical records of 6 patients with mutation on CAPN3 gene. All patients were female (three to 12 years). The mean of age of disease onset was 9 years. All of them showed progressive weakness with predominance in lower limbs. Other findings were scapular winging, joint contractures and calf hypertrophy. One female had a more severe phenotype than her dizygotic twin sister that was confirmed by muscle MRI. Muscle biopsies showed a dystrophic pattern in all patients. In this cohort of children with LGMD2A, the clinical aspects were similar to adults with the same disorder.

Diagnosis of cobblestone lissencephaly and associated Chiari malformation using fetal MRI

Walker-Warburg Syndrome (WWS) is a rare and severe form of autosomal recessive congenital muscular dystrophy affecting the brain, eyes, and muscles. We report a case of an infant diagnosed with cobblestone lissencephaly as well as a Chiari malformation, consistent with likely WWS based on fetal anomalies noted on prenatal ultrasound and confirmed with magnetic resonance imaging (MRI) at 35 week. Although multiple posterior fossa abnormalities have been reported in the setting of WWS, to our knowledge, the co-existence of a Chiari malformation has never before been reported. We describe the fetal MRI and pathological findings of WWS and discuss the role of MRI as an antenatal imaging tool.

Inherited progressive limb-girdle muscular dystrophy type 2A (calpainopathy): a review of literature

The Russian literature gives inadequate attention to the problem of one of the most common form of autosomal recessive progressive limb-girdle muscular dystrophies type 2A (LGMD2A) (calpainopathy). This paper highlights current views on the physiological role of calpain 3 protein, possible pathogenetic mechanisms for the development of myodystrophy, diagnostic criteria, and therapeutic approaches. Clinical neurologists» awareness of LGMD2A will be able to reduce the time to a correct diagnosis, to take measures to slow down the rate of disease progression, to make medical and genetic counselling, a follow-up, and monitoring, as well as to use measures for the physical and social adaptation of a patient. Key words: progressive limb-girdle muscular dystrophy, calpainopathy, calpain 3, inherited myopathy, creatinine phosphokinase, electromyography, muscle magnetic resonance imaging, genetic analysis, muscle biopsy, differential diagnosis.

P88 – 2855: Deficient alpha-dystroglycan presenting with rhabdomyolysis

Objective To describe a case of deficient alpha-dystroglycan revealed by acute rhabdomyolysis. Rhabdomyolysis frequently occurs in metabolic myopathies induced by exercise or triggered by intercurrent disease. Furthermore, rhabdomyolysis has been described in muscular dystrophies, particularly Duchenne and Becker dystrophies. It has only rarely been reported in limb girdle muscular dystrophies (LGMD), even though it may be the first and unique presenting feature of some type of LGMD. Case description and results A 12 year old boy complained about fatigue after summer holidays with more intensive exercise than usually. As laboratory investigations showed mild elevation of serum AST (264 IU/l, N Conclusion Limb girdle muscular dystrophy type 2I (LGMD2I) is one of the most common autosomal recessive limb girdle muscular dystrophies presenting in childhood. It may present with acute rhabdomyolysis as the first sign. It should be included in the diagnostic work-up of rhabdomyolysis and persistent high CK levels should prompt muscle biopsy and/or molecular diagnosis.

Homozygous Nonsense Mutations in RBP3 Gene Cause Early-Onset Retinal Dystrophies Associated With High Myopia.

The very detailed report by Arno et al. 1 of early-onset autosomal recessive dystrophies associated with high myopia occurring in young children carrying homozygous nonsense mutations of the RBP3 gene highlights the power of the combination of meticulous phenotyping and whole-exome sequencing analysis in the field of inherited retinal dystrophies. A homozygous missense mutation of the RBP3 gene was previously associated with autosomal recessive retinitis pigmentosa (RP) and high myopia in adult patients. 2 The recent findings emphasize the importance of longitudinal phenotypic studies based on the addition of the most modern methods of investigation, such as fundus autofluorescence imaging and spectral-domain optical coherence tomography (OCT), to the classical methods used for the diagnosis of retinal dystrophies. The authors not only have discovered novel phenotypes of retinal dystrophies caused by different mutations in the same gene RBP3, but also show unequivocally that the loss of the inner segment ellipsoid band over peripheral macular areas is a key marker of the novel dystrophies found. Whether these novel phenotypes will evolve toward RP remains to be demonstrated by rigorous follow-up studies of each affected patient. This report illustrates the necessity of increasing our knowledge on the unknown and probably diverse functions of the interphotoreceptor retinoid binding protein (IRBP). 3 The roles of IRBP in the normal development of human eye and retina are far from being fully understood. The exploration of the role of IRBP in the pathophysiology of nonsyndromic myopia requires the urgent implementation of cleverly designed innovative experiments. Finally, this important study points toward the possibility of a window of opportunity for gene therapy or pharmacological therapies in some patients affected by RBP3 homozygous mutations.

Targeted next-generation sequencing for the genetic diagnosis of dysferlinopathy

Dysferlinopathy comprises a group of autosomal recessive muscular dystrophies caused by mutations in the DYSF gene. Due to the large size of the gene and its lack of mutational hot spots, analysis of the DYSF gene is time-consuming and laborious using conventional sequencing methods. By next-generation sequencing (NGS), DYSF gene analysis has previously been validated through its incorporation in multi-gene panels or exome analyses. However, individual validation of NGS approaches for DYSF gene has not been performed. Here, we established and validated a hybridization capture-based target-enrichment followed by next-generation sequencing to detect mutations in patients with dysferlinopathy. With this approach, mean depth of coverage was approximately 450 fold and almost all (99.3%) of the targeted region had sequence coverage greater than 20 fold. When this approach was tested on samples from patients with known DYSF mutations, all known mutations were correctly retrieved. Using this method on 32 consecutive patient samples with dysferlinopathy, at least two pathogenic variants were detected in 28 (87.5%) samples and at least one pathogenic variant was identified in all samples. Our results suggested that the NGS-based screening method could facilitate efficient and accurate genetic diagnosis of dysferlinopathy.

A prospective study on the immunophenotypic characterization of limb girdle muscular dystrophies 2 in India.

In this prospective study conducted over 2 years, 300 nonconsecutive cases of autosomal recessive limb girdle muscular dystrophies (AR-LGMD) were characterized, based on phenotypic features, biochemical findings, electrophysiological studies, muscle immunohistochemistry (IHC), and western blot (WB) analysis. Muscle biopsy was performed in 280 index cases. 226 biopsies were subjected for IHC, and, 176 of these for WB analysis. A total of 246 patients were finally analyzed. This figure included 20 affected siblings. LGMD2B was the most common form and comprised of 33.3% (n = 82) of the entire cohort. This was followed by alpha-dystroglycanopathies with 61 (24.79%) patients (LGMD2I in 15, 2K in 10 and combined deficiency of both in the remaining). LGMD 2C-F was present in 35 (14.23%) cases and LGMD2A in 22 (10.2%) cases, and were identified by routine WB, densitometry method and autocatalytic assay. LGMD2G was present in 8 patients (3.25%), and LGMD2H and 2J in 2 cases each, respectively. For the first time, we have identified patients with LGMD2G, 2H, 2I, and 2K by the WB technique. These may be the common forms of autosomal recessive (AR)-LGMD's among Indian patients and need identification for prognostication and appropriate counseling. Although not a nationwide study, our data is sufficient to provide information about the relative proportions of various LGMD2 subtypes in India. Diagnosing LGMD2 based on classical clinical features, IHC and WB is fairly sensitive and specific; however, further genetic studies are required to confirm the diagnosis.

Diagnostic clues and manifesting carriers in fukutin-related protein (FKRP) limb-girdle muscular dystrophy

Mutations in the fukutin-related protein (FKRP) gene are a known cause of autosomal recessive limb-girdle muscular dystrophy. Clinically, patients resemble Becker's muscular dystrophy and generally present in the first two decades of life with a mild, progressive phenotype. Cardiac involvement is variable. Heterozygous carriers are usually clinically unaffected. We report a patient presenting later in life with life-threatening cardiac failure and we describe for the first time clinically manifesting carriers in the family.

LAMA2-related congenital muscular dystrophy complicated by West syndrome

BackgroundMutations in the LAMA2 gene cause autosomal recessive laminin α2 related congenital muscular dystrophy. In patients with partial laminin α2 deficiency the phenotype is usually milder than in those with absent protein. Apart from the typical white matter abnormalities, there is an increased risk of cerebral complications such as epilepsy and mental retardation, despite a structurally normal brain. Methods/resultsWe present a patient with primary partial laminin α2 deficiency due to a homozygous novel LAMA2 missense mutation who developed West syndrome in his first year of life. To our knowledge, this combination has not previously been reported. A 5 year-old boy exhibited global hypotonia with generalized muscle weakness from birth. At 8 months of age he presented infantile spasms and an EEG finding of hypsarrhythmia. Seizures were controlled in a few weeks with intramuscular synthetic ACTH, followed by valproic acid. Two years later antiepileptic medication was withdrawn. He achieved unsupported walking at the age of 4, but his cognitive status corresponded to a 2 year-old child. Epilepsy has not recurred and brain MRI showed the typical white matter abnormalities without associated neuronal migration defects. ConclusionThis report widens the clinical spectrum of cerebral manifestations related with mutations in LAMA2. The beginning of a severe epileptic encephalopathy modifies the natural history of the disease.

Limb girdle muscular dystrophy type 2G with myopathic-neurogenic motor unit potentials and a novel muscle image pattern

BackgroundLimb girdle muscular dystrophy type 2G (LGMD2G) is a subtype of autosomal recessive muscular dystrophy caused by mutations in the telethonin gene. There are few LGMD2G patients worldwide reported, and this is the first description associated with early tibialis anterior sparing on muscle image and myopathic-neurogenic motor unit potentials.Case presentationHere we report a 31 years old caucasian male patient with progressive gait disturbance, and severe lower limb proximal weakness since the age of 20 years, associated with subtle facial muscle weakness. Computed tomography demonstrated soleus, medial gastrocnemius, and diffuse thigh muscles involvement with tibialis anterior sparing. Electromyography disclosed both neurogenic and myopathic motor unit potentials. Muscle biopsy demonstrated large groups of atrophic and hypertrophic fibers, frequent fibers with intracytoplasmic rimmed vacuoles full of autophagic membrane and sarcoplasmic debris, and a total deficiency of telethonin. Molecular investigation identified the common homozygous c.157C > T in the TCAP gene.ConclusionThis report expands the phenotypic variability of telethoninopathy/ LGMD2G, including: 1) mixed neurogenic and myopathic motor unit potentials, 2) facial weakness, and 3) tibialis anterior sparing. Appropriate diagnosis in these cases is important for genetic counseling and prognosis.

Clinical aspects of patients with sarcoglycanopathies under steroids therapy.

Patients with sarcoglycanopathies, which comprise four subtypes of autosomal recessive limb-girdle muscular dystrophies, usually present with progressive weakness leading to early loss of ambulation and premature death, and no effective treatment is currently available. To present clinical aspects and outcomes of six children with sarcoglycanopathies treated with steroids for at least one year. Patient files were retrospectively analyzed for steroid use. Stabilization of muscle strength was noted in one patient, a slight improvement in two, and a slight worsening in three. In addition, variable responses of forced vital capacity and cardiac function were observed. No overt clinical improvement was observed in patients with sarcoglycanopathies under steroid therapy. Prospective controlled studies including a larger number of patients are necessary to determine the effects of steroids for sarcoglycanopathies.

L.I.2: The ABC of autosomal recessive limb girdle muscular dystrophy

Autosomal recessive limb girdle muscular dystrophy (LGMD2) is a group of heterogeneous muscle diseases that share several common denominators. Beside the same mode of inheritance, patients develop progressive weakness and wasting of their shoulder and pelvic girdle muscles whilst muscle biopsies generally show dystrophic histological features. Coming to a specific diagnosis in a patient with LGMD2 can be quite challenging. Diagnostic clues for the clinician can be the age of onset, the ethnic background, the involvement of respiratory muscles, the heart or other organ systems, the selective pattern of muscle pathology and immunoanalysis of the muscle biopsy. Next generation sequencing strategies, especially gene panel and exome sequencing approaches, are gradually replacing the often cumbersome gene by gene mutation analysis and should help to provide all patients with LGMD2 with a precise diagnosis over the next few years. The identification of the underlying genetic defect in almost 20 different forms of LGMD2 so far has helped to generate animal models and to study disease pathogenesis. Nevertheless there is currently no licensed drug for LGMD2 and treatment strategies focus on the improvement of symptoms. One of the biggest challenges is the low number of identified LGMD2 patients. Whereas in some forms of LGMD2 several hundred patients have been diagnosed, in other forms only very few families have been characterized. Over the coming years it will be important to develop a successful translational research pathway in LGMD2 by creating patient registries, developing standards of care and establishing validated outcome measures and disease biomarkers. These objectives can only be achieved through a close partnership between scientists, healthcare professionals, patient organizations, the pharmaceutical industry and regulators. The presentation will focus on the current stage of translational research approaches in LGMD2 and on future perspectives.

G.P.287: Genetic and epigenetic determinants of low dysferlin expression in monocytes

Dysferlinopathies are autosomal recessive inherited muscular dystrophies caused by mutations in the gene DYSF. Dysferlin is primarily expressed in skeletal muscle, cardiac muscle and peripheral blood monocytes. Dysferlin expression in skeletal muscle and monocytes strongly correlate in the healthy and disease states. We evaluated the efficiency of the monocyte assay to detect carriers and to determine the carrier frequency of dysferlinopathies in the general population. We enrolled 149 healthy volunteers and collected peripheral blood samples for protein analysis. While eighteen of these individuals with protein levels in the range of 40–64% were predicted to be carriers by the monocyte assay, subsequent DYSF sequencing analysis in 14 of these 18 volunteers detected missense variants in only four. Analysis of DNA methylation patterns at DYSF locus showed no changes in DNA methylation levels at CpG island and shores between samples. Our results suggest that: (1) dysferlin expression can also be regulated by factors outside of the dysferlin gene, but not related to DNA methylation; (2) carrier frequency and therefore the number of affected individuals could be higher than previously estimated; and (3) although reliable for evaluating dysferlinopathies, results of the monocyte assay can not be used when determining carrier status and molecular analysis of DYSF must be performed.

G.P.315: Cases of normal to mildly elevated creatine kinase in muscle-eye-brain disease patients and delay in diagnosis

The dystroglycanopathies are a genetically heterogeneous group of autosomal recessive muscular dystrophies that have in common abnormal glycosylation of alpha-dystroglycan. The spectrum of clinical features is wide and includes muscle-eye-brain disease (MEB), which typically encompass congenital muscular dystrophy, cobblestone lissencephaly and eye abnormalities. Serum creatine kinase (CK) is often elevated greater than fivefold. We describe three cases of MEB in whom the combination of low or normal serum CK and predominant upper motor neurone features led to delayed diagnosis. Case 1: 4-year-old boy presented antenatally with ventriculomegaly. MRI revealed cobblestone lissencephaly and cerebellar polymicrogyria. He was myopic. CK was 173 IU/L at 4 days. He appeared to have an evolving CP with central hypotonia, limb spasticity. His CK was 563 IU/L and compound heterozygous mutations in POMGnT1, c.511C>T and c.1895+1G>T was found at 2 years. Case 2: 4-year-old girl presented at 5 days with hypotonia. CK was 228 IU/L at 2 weeks and 248 IU/L by 3 months. MRI showed lissencephaly and widespread polymicrogyria with cerebellar cysts. She was myopic. She is bottom shuffling with preserved strength but evolving CP-like picture. At 2 years compound heterozygous mutations in POMGnT1, c.385C>T and c.1460delG were found. Case 3: 14 year-old male presented at 3 months with myopia. MRI was consistent with a neuronal migration defect. CK was 386 IU/L at 11 months. Presentation was that of an evolving motor disorder. He was diagnosed age 8 with a compound heterozygous mutations in LARGE. These cases highlight that patients with dystroglycanopathies with POMGnT1 and LARGE mutations can have normal or only mildly elevated CK levels, even beyond the first 6 months. The presentation suggests CP with preservation of muscle strength. It is important to suspect dystroglycanopathies with typical MRI changes and eye involvement, even in the light of normal CK without weakness.

Common recessive limb girdle muscular dystrophies differential diagnosis: why and how?

Limb girdle muscular dystrophies are heterogeneous autosomal hereditary neuromuscular disorders. They produce dystrophic changes on muscle biopsy and they are associated with mutations in several genes involved in muscular structure and function. Detailed clinical, laboratorial, imaging, diagnostic flowchart, photographs, tables, and illustrated diagrams are presented for the differential diagnosis of common autosomal recessive limb girdle muscular dystrophy subtypes diagnosed nowadays at one reference center in Brazil. Preoperative image studies guide muscle biopsy site selection. Muscle involvement image pattern differs depending on the limb girdle muscular dystrophy subtype. Muscle involvement is conspicuous at the posterior thigh in calpainopathy and fukutin-related proteinopathy; anterior thigh in sarcoglycanopathy; whole thigh in dysferlinopathy, and telethoninopathy. The precise differential diagnosis of limb girdle muscular dystrophies is important for genetic counseling, prognostic orientation, cardiac and respiratory management. Besides that, it may probably, in the future, provide specific genetic therapies for each subtype.