Search Results (8)

Pathogenic variants localized in the gene coding for the Fukutin-Related Protein (FKRP) are responsible for Limb-Girdle Muscular Dystrophy type 9 (LGMDR9), Congenital Muscular Dystrophies type 1C (MDC1C), Walker–Warburg Syndrome (WWS), and Muscle–Eye–Brain diseases (MEBs). LGMDR9 is the fourth most common hereditary Limb Girdle Muscular Dystrophy in Italy. LGMDR9 patients with severe disease show an overlapping Duchenne/Becker phenotype and may have secondary dystrophin reduction on muscle biopsy. We conducted a molecular analysis of the FKRP gene by direct sequencing in 153 patients from Southern Italy (Calabria) with Duchenne/Becker-like phenotypes without confirmed genetic diagnosis. Mutational screening of the patients (112 men and 41 women, aged between 5 and 84 years), revealed pathogenic variants in 16 subjects. The most frequent variants identified were c.427C > A, p.R143S, and c.826C > A, p.L276I (NM_024301.5). The results obtained show that the Duchenne/Becker-like phenotype is frequently determined by mutations in the FKRP gene in our cohort and highlight the importance of considering LGMDR9 in the differential diagnosis of dystrophinopathies in Calabria. Finally, this study, which, to our knowledge, is the first conducted on Calabrian subjects, will contribute to the rapid identification and management of LGMDR9 patients. Full article

Rat animal models are widely used owing to their relatively superior cognitive abilities and higher similarity compared with mouse models to human physiological characteristics. However, their use is limited because of difficulties in establishing embryonic stem cells and performing genetic modifications, and insufficient embryological research. In this study, we established optimal superovulation and fertilized–egg transfer conditions, including optimal hormone injection concentration (≥150 IU/kg of PMSG and hCG) and culture medium (mR1ECM), to obtain high-quality zygotes and establish in vitro fertilization conditions for rats. Next, sgRNA with optimal targeting activity was selected by performing PCR analysis and the T7E1 assay, and the CRISPR/Cas9 system was used to construct a rat model for muscular dystrophy by inducing a deficiency in the fukutin gene without any off-target effect detected. The production of fukutin knockout rats was phenotypically confirmed by observing a drop-in body weight to one-third of that of the control group. In summary, we succeeded in constructing the first muscular dystrophy disease rat model using the CRISPR/CAS9 system for increasing future prospects of producing various animal disease models and encouraging disease research using rats. Full article

The diaphragm muscle is essential for breathing, and its dysfunctions can be fatal. Many disorders affect the diaphragm, including muscular dystrophies. Despite the clinical relevance of targeting the diaphragm, there have been few studies evaluating diaphragm function following a given experimental treatment, with most of these involving anti-inflammatory drugs or gene therapy. Cell-based therapeutic approaches have shown success promoting muscle regeneration in several mouse models of muscular dystrophy, but these have focused mainly on limb muscles. Here we show that transplantation of as few as 5000 satellite cells directly into the diaphragm results in consistent and robust myofiber engraftment in dystrophin- and fukutin-related protein-mutant dystrophic mice. Transplanted cells also seed the stem cell reservoir, as shown by the presence of donor-derived satellite cells. Force measurements showed enhanced diaphragm strength in engrafted muscles. These findings demonstrate the feasibility of cell transplantation to target the diseased diaphragm and improve its contractility. Full article

Limb-Girdle Muscular Dystrophy R9 (LGMDR9) is a dystroglycanopathy caused by Fukutin-related protein (FKRP) defects leading to the deficiency of α-DG glycosylation, essential to membrane integrity. Recombinant adeno-associated viral vector (rAAV) gene therapy offers great therapeutic promise for such neuromuscular disorders. Pre-clinical studies have paved the way for a phase 1/2 clinical trial aiming to evaluate the safety and efficacy of FKRP gene therapy in LGMDR9 patients. To demonstrate product activity, quality, and consistency throughout product and clinical development, regulatory authorities request several quality controls, including a potency assay aiming to demonstrate and quantify the intended biological effect of the gene therapy product. In the present study, we generated FKRP knock-out (KO) cells fully depleted of α-DG glycosylation using CRISPR-Cas9 to assess the functional activity of a rAAV-FKRP gene therapy. We then developed a high-throughput On-Cell-Western methodology to evaluate the restoration of α-DG glycosylation in KO-FKRP cells and determine the biological activity of the FKRP transgene. The determination of the half maximal effective concentration (EC₅₀) provides a method to compare the rAAV-FKRP batch using a reference standard. The generation of KO-FKRP muscle cells associated with the high-throughput On-Cell-Western technique may serve as a cell-based potency assay to assess rAAV-FKRP gene therapy products. Full article

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease that mainly affects boys due to X-linked recessive inheritance. In most affected individuals, MLPA or sequencing-based techniques detect deletions, duplications, or point mutations in the dystrophin-encoding DMD gene. However, in a small subset of patients clinically diagnosed with DMD, the molecular cause is not identified with these routine methods. Evaluation of the 60 DMD patients in our center revealed three cases without a known genetic cause. DNA samples of these patients were analyzed using whole-exome sequencing (WES) and, if unconclusive, optical genome mapping (OGM). WES led to a diagnosis in two cases: one patient was found to carry a splice mutation in the DMD gene that had not been identified during previous Sanger sequencing. In the second patient, we detected two variants in the fukutin gene (FKTN) that were presumed to be disease-causing. In the third patient, WES was unremarkable, but OGM identified an inversion disrupting the DMD gene (~1.28 Mb) that was subsequently confirmed with long-read sequencing. These results highlight the importance of reanalyzing unsolved cases using WES and demonstrate that OGM is a useful method for identifying large structural variants in cases with unremarkable exome sequencing. Full article

Fukutin encoded by FKTN is a ribitol 5-phosphate transferase involved in glycosylation of α-dystroglycan. It is known that mutations in FKTN affect the glycosylation of α-dystroglycan, leading to a dystroglycanopathy. Dystroglycanopathies are a group of syndromes with a broad clinical spectrum including dilated cardiomyopathy and muscular dystrophy. In this study, we reported the case of a patient with muscular dystrophy, early onset dilated cardiomyopathy, and elevated creatine kinase levels who was a carrier of the compound heterozygous variants p.Ser299Arg and p.Asn442Ser in FKTN. Our work showed that compound heterozygous mutations in FKTN lead to a loss of fully glycosylated α-dystroglycan and result in cardiomyopathy and end-stage heart failure at a young age. Full article

The causative gene of Fukuyama congenital muscular dystrophy (fukutin) is involved in formation of the basement membrane through glycosylation of alpha-dystroglycan. However, there are other proposed functions that have not been fully understood. Using cultured astrocytes (1321N1), we found nuclear localization of fukutin and a positive relationship between fukutin expression and cell proliferation. Among potential proteins regulating cell proliferation, we focused on cyclin D1, by reverse-transcription polymerase chain reaction, Western blotting, immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), and sandwich ELISA. Expression of cyclin D1 was significantly downregulated by fukutin knockdown and significantly upregulated by fukutin overexpression. Moreover, fukutin was proven to bind to the activator protein-1 (AP-1) binding site of cyclin D1 promoter, as well as the AP-1 component c-Jun. The c-Jun phosphorylation status was not significantly influenced by knockdown or overexpression of fukutin. The present results provide in vitro evidence for a novel function of fukutin, which participates in cell proliferation by enhancing cyclin D1 expression through forming a complex with AP-1. It is likely that fukutin is a potential cofactor of AP-1. Full article

Atrial fibrillation (AF) is the most common cardiac arrhythmia, and it is associated with an increased risk of heart failure, stroke, dementia, and death. Recently, titin-truncating variants (TTNtv), which are predominantly associated with dilated cardiomyopathy (DCM), were associated with early-onset AF. Furthermore, genome-wide association studies (GWAS) associated AF with other structural genes. In this study, we investigated whether early-onset AF was associated with loss-of-function variants in DCM-associated genes encoding cytoskeletal proteins. Using targeted sequencing, we examined a cohort of 527 Scandinavian individuals with early-onset AF and a control group of individuals free of AF (n = 383). The patients had onset of AF before 50 years of age, normal echocardiogram, and no other cardiovascular disease at onset of AF. We identified six individuals with rare loss-of-function variants in three different genes (dystrophin (DMD), actin-associated LIM protein (PDLIM3), and fukutin (FKTN)), of which two variants were novel. Loss-of-function variants in cytoskeletal genes were significantly associated with early-onset AF when patients were compared with controls (p = 0.044). Using publicly available GWAS data, we performed genetic correlation analyses between AF and 13 other traits, e.g., showing genetic correlation between AF and non-ischemic cardiomyopathy (p = 0.0003). Our data suggest that rare loss-of-function variants in cytoskeletal genes previously associated with DCM may have a role in early-onset AF, perhaps through the development of an atrial cardiomyopathy. Full article