Co-Occurrence of Myotonic Dystrophy Type 1 and Limb-Girdle Muscular Dystrophy Type 2B: A Case Report.

Introduction: Myotonic dystrophy type 1 (DM1) is an autosomal dominant neuromuscular disease whose pattern of weakness is predominantly distal. Limb-girdle muscular dystrophy type 2B/R2-dysferlin-related (LGMD2B/R2) is another neuromuscular disease, which presents an autosomal recessive inheritance and is marked by proximal muscle weakness. Even if uncommon, comorbid inherited pathologies must be considered in cases of atypical presentations, especially in those with family history of consanguinity. Case Presentation: Herein, we report the unique case of a patient diagnosed with both DM1 and LGMD2B/R2: a 38-year-old woman in follow-up of DM1 in a neuromuscular disease service presenting prominent proximal weakness. The patient's parents were consanguineous, and creatine kinase levels were elevated. A multi-gene panel test was performed and revealed the diagnosis of LGMD2B/R2. Conclusion: Genetic diseases with atypical presentations should raise the possibility of a second disorder, prompting an appropriate investigation. Overlooking a second diagnosis can implicate in not offering adequate genetic counseling, support, or specific treatment.

Limb-Girdle Muscular Dystrophy Type 2B (LGMD2B) caused by Pathogenic Splice and Missense Variants of DYSF Gene among Iranians with Muscular Dystrophy.

Background: The phenotypic range of limb-girdle muscular dystrophies (LGMDs) varies significantly because of genetic heterogeneity ranging from very mild to severe forms. Molecular analysis of the DYSF gene is challenging due to the wide range of mutations and associated complications in interpretations of novel DYSF variants with uncertain significance. Thus, in the current study, we performed the NGS analysis and its results are confirmed with Sanger sequencing to find the plausible disease-causing variants in patients with muscular dystrophy and their relatives via segregation analysis. Materials and Methods: Nine patients with LGMD type 2B (LGMD2B) characteristics were screened for putative mutations by the whole-exome sequencing (WES) test. Either the patients themselves or their parents and first relatives were investigated in the segregation analysis through Sanger sequencing. The majority of variants were classified as pathogenic through American College of Medical Genetics and Genomics (ACMG) guidelines, segregation results, and in silico predictions. Results: Results revealed eight variants in DYSF gene, including three splicing (c.1149+4A>G, c.2864+1G>A, and c.5785-7G>A), two nonsense (p.Gln112Ter and p.Trp2084Ter), two missense (p.Thr1546Pro and p.Tyr1032Cys), and one frameshift (p.Asp1067Ilefs), among nine Iranian families. One of the eight identified variants was novel, including p.Asp1067Ilefs, which was predicted to be likely pathogenic based on the ACMG guidelines. Notably, prediction tools suggested the damaging effects of studied variants on dysferlin structure. Conclusion: Conclusively, the current report introduced eight variants including a novel frameshift in DYSF gene with noticeable pathogenic effects. This study significantly can broaden the diagnostic spectrum of LGMD2B in combination with previous reports about DYSF mutations and may pave the way for a rapidly high-ranked identification of the accurate type of dysferlinopathy.

Analysis of Dysferlin Direct Interactions with Putative Repair Proteins Links Apoptotic Signaling to Ca2+ Elevation via PDCD6 and FKBP8.

Quantitative surface plasmon resonance (SPR) was utilized to determine binding strength and calcium dependence of direct interactions between dysferlin and proteins likely to mediate skeletal muscle repair, interrupted in limb girdle muscular dystrophy type 2B/R2. Dysferlin canonical C2A (cC2A) and C2F/G domains directly interacted with annexin A1, calpain-3, caveolin-3, affixin, AHNAK1, syntaxin-4, and mitsugumin-53, with cC2A the primary target and C2F lesser involved, overall demonstrating positive calcium dependence. Dysferlin C2 pairings alone showed negative calcium dependence in almost all cases. Like otoferlin, dysferlin directly interacted via its carboxy terminus with FKBP8, an anti-apoptotic outer mitochondrial membrane protein, and via its C2DE domain with apoptosis-linked gene (ALG-2/PDCD6), linking anti-apoptosis with apoptosis. Confocal Z-stack immunofluorescence confirmed co-compartmentalization of PDCD6 and FKBP8 at the sarcolemmal membrane. Our evidence supports the hypothesis that prior to injury, dysferlin C2 domains self-interact and give rise to a folded, compact structure as indicated for otoferlin. With elevation of intracellular Ca2+ in injury, dysferlin would unfold and expose the cC2A domain for interaction with annexin A1, calpain-3, mitsugumin 53, affixin, and caveolin-3, and dysferlin would realign from its interactions with PDCD6 at basal calcium levels to interact strongly with FKBP8, an intramolecular rearrangement facilitating membrane repair.

Recurrent, non-traumatic, non-exertional rhabdomyolysis after immunologic stimuli in a healthy adolescent female: a case report.

BACKGROUND: Dysferlinopathy refers to a heterogenous group of autosomal recessive disorders that affect a skeletal muscle protein called dysferlin. These mutations are associated with limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, asymptomatic hyperCKemia, and distal myopathy with anterior tibial onset. CASE PRESENTATION: A 16 year old female presented with myalgia, weakness and dark urine one week after her second BNT162b2 mRNA (Pfizer) vaccine. Initial serum creatine kinase (CK) was measured at 153,000 IU/L, eventually up-trending to over 200,000 IU/L. However, stable renal function precluded hemodialysis allowing discharge after 10 days of intravenous (IV) hydration and alkaline diuresis. Just two years prior to the current presentation, the patient was hospitalized following Group A Streptococcal pharyngitis infection complicated by rhabdomyolysis. She presented with fatigue, lower extremity weakness, and dark oliguria with CK measuring 984,800 IU/L. IV hydration was attempted however hemodialysis was ultimately required throughout her 24-day hospital stay. Her episode was presumed to be idiopathic and no further work-up was performed at that time. During the patient's current hospitalization, she reported similar symptomology (myalgias and weakness) following her first quadrivalent Gardasil vaccine at age 11. No hospitalization was required at that time. A comprehensive workup was now initiated while the patient was being treated for her suspected second or third non-exertional, non-traumatic rhabdomyolysis. Rheumatologic, metabolic, infectious, and endocrinologic workup were all unremarkable. Patient eventually had whole exome sequencing performed which revealed a heterozygous pathogenic variant in the DYSF gene (DYSF c.2643 + 1G > A) encoding dysferlin. No clinically significant sequelae occurred thus far. CONCLUSIONS: While there have been reports of symptomatic heterozygote carriers of dysferlinopathies, to our knowledge none have been associated with recurrent rhabdomyolysis after immunogenic stimuli. This unique case presentation highlights the importance of a multi-disciplinary care team, the utility of modern whole-exome gene sequencing, and the future challenges of balancing vaccine risk vs benefit.

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.

The genetic profile of dysferlinopathy in a cohort of 209 cases: Genotype-phenotype relationship and a hotspot on the inner DysF domain.

Dysferlinopathy is a group of autosomal recessive muscular dystrophies caused by variants in the dysferlin gene (DYSF), with variable proximal and distal muscle involvement. We performed DYSF gene analyses of 200 cases suspected of having dysferlinopathy (Cohort 1), and identified diagnostic variants in 129/200 cases, including 19 novel variants. To achieve a comprehensive genetic profile of dysferlinopathy, we analyzed the variant data from 209 affected cases from unrelated 209 families, including 80 previously diagnosed and 129 newly diagnosed cases (Cohort 2). Among the 90 types of variants identified in 209 cases, the NM_003494.3: c.2997G>T; p.Trp999Cys, was the most frequent (96/420; 22.9%), followed by c.1566C>G; p.Tyr522* (45/420; 10.7%) on an allele base. p.Trp999Cys was found in 70/209 cases (33.5%), including 20/104 cases (19.2%) with the Miyoshi muscular phenotype and 43/82 cases (52.4%) with the limb-girdle phenotype. In the analysis of missense variants, p.Trp992Arg, p.Trp999Arg, p.Trp999Cys, p.Ser1000Phe, p.Arg1040Trp, and p.Arg1046His were located in the inner DysF domain, representing in 113/160 missense variants (70.6%). This large cohort highlighted the frequent missense variants located in the inner DysF domain as a hotspot for missense variants among our cohort of 209 cases (>95%, Japanese) and hinted at their potential as targets for future therapeutic strategies.

Functions of Vertebrate Ferlins.

Ferlins are multiple-C2-domain proteins involved in Ca2+-triggered membrane dynamics within the secretory, endocytic and lysosomal pathways. In bony vertebrates there are six ferlin genes encoding, in humans, dysferlin, otoferlin, myoferlin, Fer1L5 and 6 and the long noncoding RNA Fer1L4. Mutations in DYSF (dysferlin) can cause a range of muscle diseases with various clinical manifestations collectively known as dysferlinopathies, including limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. A mutation in MYOF (myoferlin) was linked to a muscular dystrophy accompanied by cardiomyopathy. Mutations in OTOF (otoferlin) can be the cause of nonsyndromic deafness DFNB9. Dysregulated expression of any human ferlin may be associated with development of cancer. This review provides a detailed description of functions of the vertebrate ferlins with a focus on muscle ferlins and discusses the mechanisms leading to disease development.

Effects of Prednisone on a Patient with Dysferlinopathy Assessed by Maximal Voluntary Isometric Contraction: Alternate-Day Low-Dose Administration for a 17-Year Period.

Glucocorticoids are candidates for the pharmacological treatment of dysferlinopathy. Deflazacort, however, showed a worse effect on muscle strength than placebo. Alternate-day low-dose prednisone may have beneficial effects with fewer adverse effects. The outcomes for a female patient with dysferlinopathy (limb-girdle muscular dystrophy type 2B) were assessed by maximal voluntary isometric contraction (MVIC) using a newly devised chair and arm table with push-pull type strain gauges. Grip strength was also measured isometrically. Prednisone 15 mg was started orally at the age of 24 years and was taken every other day in the morning until 41 years of age. The MVIC of flexion of the knees and elbows increased gradually and significantly. The MVIC of extension of the knees and elbows increased to a lesser extent. Isometric grip strength showed no remarkable increase, but strength was sustained over 10 years. Muscle fiber types account for these differences. The beneficial effects of alternate-day prednisone treatment on dysferlinopathy are reported.

Identification of Novel Antisense-Mediated Exon Skipping Targets in DYSF for Therapeutic Treatment of Dysferlinopathy.

Dysferlinopathy is a progressive myopathy caused by mutations in the dysferlin (DYSF) gene. Dysferlin protein plays a major role in plasma-membrane resealing. Some patients with DYSF deletion mutations exhibit mild symptoms, suggesting some regions of DYSF can be removed without significantly impacting protein function. Antisense-mediated exon-skipping therapy uses synthetic molecules called antisense oligonucleotides to modulate splicing, allowing exons harboring or near genetic mutations to be removed and the open reading frame corrected. Previous studies have focused on DYSF exon 32 skipping as a potential therapeutic approach, based on the association of a mild phenotype with the in-frame deletion of exon 32. To date, no other DYSF exon-skipping targets have been identified, and the relationship between DYSF exon deletion pattern and protein function remains largely uncharacterized. In this study, we utilized a membrane-wounding assay to evaluate the ability of plasmid constructs carrying mutant DYSF, as well as antisense oligonucleotides, to rescue membrane resealing in patient cells. We report that multi-exon skipping of DYSF exons 26-27 and 28-29 rescues plasma-membrane resealing. Successful translation of these findings into the development of clinical antisense drugs would establish new therapeutic approaches that would be applicable to âˆ¼5%-7% (exons 26-27 skipping) and ∼8% (exons 28-29 skipping) of dysferlinopathy patients worldwide.

Limb-girdle muscular dystrophy type 2B: An unusual cause of proximal muscular weakness in Saudi Arabia.

Dysferlinopathies encompass a group of neuromuscular diseases characterized by the absence of dysferlin in skeletal muscle. It is a genetic disorder caused by a mutation in the dysferlin gene (DYSF) with an autosomal recessive mode of inheritance. In this article, we report a case of Limb-girdle muscular dystrophy type 2B with a rare homozygous duplication c.164dupA, p.(Ile57Hisfs*8) (rs863225020) in DYSF in a Saudi patient. To the best of our knowledge, this is the first case from Saudi Arabia with complete clinical data, pathology findings, radiology findings, and genetic analysis. Although there is no curative treatment for this disease, an accurate diagnosis is important to avoid using steroids and immunosuppressive medications, which are not effective and may have several side effects. Further studies are needed to explore potential therapies for this rare condition.

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.

Heterogeneous characteristics of Korean patients with dysferlinopathy.

Dysferlinopathy is caused by mutations in the DYSF gene. To characterize the clinical spectrum, we investigated the characteristics of 31 Korean dysferlinopathy patients confirmed by immunohistochemistry. The mean age of symptom onset was 22.23 ± 7.34 yr. The serum creatine kinase (CK) was highly increased (4- to 101-fold above normal). The pathological findings of muscle specimens showed nonspecific dystrophic features and frequent inflammatory cell infiltration. Muscle imaging studies showed fatty atrophic changes dominantly in the posterolateral muscles of the lower limb. The patients with dysferlinopathy were classified by initial muscle weakness: fifteen patients with Miyoshi myopathy phenotype (MM), thirteen patients with limb girdle muscular dystrophy 2B phenotype (LGMD2B), two patients with proximodistal phenotype, and one asymptomatic patient. There were no differences between LGMD2B and MM groups in terms of onset age, serum CK levels and pathological findings. Dysferlinopathy patients usually have young adult onset and high serum CK levels. However, heterogeneity of clinical presentations and pathologic findings upon routine staining makes it difficult to diagnose dysferlinopathy. These limitations make immunohistochemistry currently the most important method for the diagnosis of dysferlinopathy.

Heterogeneous Characteristics of Korean Patients with Dysferlinopathy

Dysferlinopathy is caused by mutations in the DYSF gene. To characterize the clinical spectrum, we investigated the characteristics of 31 Korean dysferlinopathy patients confirmed by immunohistochemistry. The mean age of symptom onset was 22.23 +/- 7.34 yr. The serum creatine kinase (CK) was highly increased (4- to 101-fold above normal). The pathological findings of muscle specimens showed nonspecific dystrophic features and frequent inflammatory cell infiltration. Muscle imaging studies showed fatty atrophic changes dominantly in the posterolateral muscles of the lower limb. The patients with dysferlinopathy were classified by initial muscle weakness: fifteen patients with Miyoshi myopathy phenotype (MM), thirteen patients with limb girdle muscular dystrophy 2B phenotype (LGMD2B), two patients with proximodistal phenotype, and one asymptomatic patient. There were no differences between LGMD2B and MM groups in terms of onset age, serum CK levels and pathological findings. Dysferlinopathy patients usually have young adult onset and high serum CK levels. However, heterogeneity of clinical presentations and pathologic findings upon routine staining makes it difficult to diagnose dysferlinopathy. These limitations make immunohistochemistry currently the most important method for the diagnosis of dysferlinopathy.

Mutation analysis of dysferlin gene in a Chinese family with Miyoshi myopathy / 临床检验杂志

Objective Two autosomal recessive forms of muscular dystrophy:LGMD2B and Miyoshi myopathy may be indused by dysferlin gene mutation.The purpose of this study was to define molecular defects in dysferlin gene in a family with Miyoshi myopathy.Methods mRNA from peripheral blood in a Chinese Miyoshi myopathy pedigree was amplified by RT-PCR and the mutation was determined by sequencing the amplified products.Results The results of sequencing revealed a novel homozygous mutation,a 6429delG,on exon 53 of the dysferlin gene for the patients.Conclusion The 6429delG mutation in the dysferlin gene of patients creates a frameshift mutation which induces a stop codon at 2035 on exon 54 and the premature dysferlin contributes to the Miyoshi myopathy in the Chinese pedigree.

Clinical and pathological features of limb-girdle muscular dystrophy type 2B / 临床神经病学杂志

Objective To investigate the clinical and pathological features of limb-girdle muscular dystrophy type 2B(LGMD2B).Methods The clinical data and the pathological result of skeletal muscle in five patients with LGMD2B were analyzed retrospectively.Results Five patients presented muscle atrophy and weakness(four limbs in three patients and both lower limbs in two patients),and the chronic onset,and the progressive deterioration.The pathological examination showed that the muscle fibers degenerating,necrotic and regenerating in different extents were observed,and the infiltration of inflammatory cells were appeared on all of cases.Immunohistochemical stains showed that the expression of anti-dysferlin monoclonal antibody was negative,anti-Dystrophy,Sarcoglycan and dystroglycan monoclonal antibodies were positive normaly,anti-CD8+T cell monoclonal antibody was negative,and anti-MHC-1 monoclonal antibody was up-regulated in five patients.Conclusions The clinical characters of LGMD2B are chronic onset,progressive muscle atrophy and weakness in four limbs or both lower limbs.The pathological characters of LGMD2B are similar to the changes of polymyositis.