Biology subjects

Stojkovic, T.

Publications and source records attributed to Stojkovic, T..

5 recordsLinked to original sources

bioRxiv2025

DAB2 as a biomarker and mechanistic link between lipid dysregulation and disease progression in LGMD R2

Limb-girdle muscular dystrophy R2 (LGMD R2) is an autosomal recessive disorder caused by dysferlin deficiency, leading to progressive muscle weakness and wasting. Despite advances in understanding the mechanisms linking dysferlin loss to membrane fragility and muscle degeneration, the lack of robust clinical biomarkers has limited disease monitoring and therapeutic evaluation. Here, we identify Disabled-2 (DAB2) as a molecular and clinical biomarker for LGMD R2. Transcriptomic profiling revealed a significant upregulation of DAB2 in induced pluripotent stem cell (iPSC)-derived myotubes from patients with LGMD R2. Its expression correlated with disease severity in muscle biopsies from a cohort of 14 dysferlin-deficient individuals and in dysferlin knockout Bla/J mice, where levels increased with disease progression. Crucially, we demonstrate that DAB2 upregulation in muscle is normalized following treatment with AAV gene therapy expressing full-length dysferlin, positioning DAB2 as a dynamic biomarker for both disease monitoring and therapeutic response. Based on the role of DAB2 in lipid trafficking and the reported pathological lipid accumulation in LGMD R2, we then investigated its contribution to disease-associated lipid dysregulation. Consistent with this hypothesis, we show that high DAB2 levels paralleled lipid deposition in affected patients, iPSC-derived myotubes and mouse muscles, while siRNA- mediated DAB2 knockdown reduced lipid accumulation in LGMD R2 myotubes. Together, our findings establish DAB2 as a mechanistic link between disease severity and lipid dysregulation, and highlight its potential as a key prognostic marker, opening new avenues for precision medicine approaches in LGMD R2 and other related muscular dystrophies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/675589v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@152c1f1org.highwire.dtl.DTLVardef@3af57eorg.highwire.dtl.DTLVardef@cb4630org.highwire.dtl.DTLVardef@16d384b_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Comparative analysis of CRISPR/Cas9-targeted nanopore sequencing approaches in repeat expansion disorders

More than 50 repeat expansion disorders have been identified, with long-read sequencing marking a new milestone in the diagnosis of these disorders. Despite these major achievements, the comprehensive characterization of short tandem repeats in a pathological context remains challenging, primarily due to their inherent characteristics such as motif complexity, high GC content, and variable length. In this study, our aim was to thoroughly characterize repeat expansions in two neuromuscular diseases: myotonic dystrophy type 1 (DM1) and oculopharyngodistal myopathy (OPDM) using CRISPR/Cas9- targeted long-read sequencing (Oxford Nanopore Technologies, ONT). We conducted precise analyses of the DM1 and OPDM loci, determining repeat size, repeat length distribution, expansion architecture and DNA methylation, using three different basecalling strategies (MinKnow software, Dorado and Bonito). We demonstrated the importance of the basecalling strategy in repeat expansion characterization. We proposed guidelines to perform CRISPR-Cas9 targeted long-read sequencing (no longer supported by ONT), from library preparation to bioinformatical analyses. Finally, we showed, for the first time, somatic mosaicism, hypermethylation of LRP12 loci in OPDM symptomatic patients and changes in the repeat tract structure of these patients. We propose a strategy based on CRISPR/Cas9-enrichment long-read sequencing for repeat expansion diseases, which could be readily applicable in research but also in diagnostic settings.

bioinformatics↗

bioRxiv2024

CCG interruptions are unstable and hypermethylated in DM1 patients

Myotonic dystrophy type 1 (DM1) exhibits highly heterogeneous clinical manifestations caused by an unstable CTG repeat expansion reaching up to 4,000 CTG. The dynamics of CTG repeats and clinical variability depends on the CTG repeat number, CNG repeat interruptions, DNA methylation, somatic mosaicism, but also gene modifiers. Around 10% of the DM1 population carries triplet repeat interruptions (CCG, CGG, CTC, CAG), which differ in number and nature between DM1 families. CCG interruptions have been associated with stabilization of the CTG repeat expansions and a milder phenotype in the DM1 population. However, the dynamics and precise role of interruptions in CTG repeat instability remain relatively underexplored due to the complexities of analyzing them. In this study, we showed that the number of CCG interruptions within the expansion varies within tissues and between generations using single-molecule real-time long-read sequencing. Although the interrupted expanded alleles showed global stabilization, the CCG interruptions themselves displayed variability across generations and within somatic tissues. Importantly, our findings reveal, for the first time, CCG hypermethylation within the CTG expansion, which is linked to downstream hypermethylation of the repeat. These results support the hypothesis that methylation of CCG interruptions within the expanded DMPK alleles may contribute to the stabilization of trinucleotide repeats.

molecular biology↗

bioRxiv2022

Identification of a CCG-enriched expanded allele in DM1 patients using Amplification-free long-read sequencing

Myotonic dystrophy type 1 (DM1) exhibits highly heterogeneous clinical manifestations caused by an unstable CTG repeat expansion reaching up to 4,000 CTG. The clinical variability depends on CTG repeat number, CNG repeat interruptions and somatic mosaicism. Currently, none of these factors are simultaneously and accurately determined due to the limitations of gold standard methods used in clinical and research laboratories. An amplicon method for targeting DM1 locus using Single-Molecule Real-Time sequencing was recently developed to accurately analyze expanded alleles. However, amplicon-based sequencing still depends on PCR and the inherent bias towards preferential amplification of smaller repeats can be problematic in DM1. Thus, an amplification-free long-read sequencing method was developed using the CRISPR/Cas9 technology in DM1. This method was used to sequence the DM1 locus in patients with CTG repeat expansion ranging from 130 to > 1000 CTG. We showed that elimination of PCR amplification improves the accuracy of measurement of inherited repeat number and somatic repeat variations, two important key factors in the DM1 severity and age at onset. For the first time, an expansion composed of over 85% CCG repeats was identified using this innovative method in a DM1 family with an atypical clinical profile. No-Amplification targeted sequencing represents a promising method that can overcome research and diagnosis shortcomings, with translational implications for clinical and genetic counseling in DM1.

molecular biology↗

bioRxiv2019

Metformin rescues muscle function in BAG3 myofibrillar myopathy models

Dominant de novo mutations in the co-chaperone BAG3 cause a severe form of myofibrillar myopathy, exhibiting progressive muscle weakness, muscle structural failure, and protein aggregation.\n\nTo identify therapies we generated two zebrafish models, one conditionally expressing BAG3P209L and one with a nonsense mutation in bag3. Whilst transgenic BAG3P209L expressing fish display protein aggregation, modelling the early phase of the disease, bag3-/- fish demonstrate impaired autophagic activity, exercise dependent fibre disintegration, and reduced swimming activity, consistent with later stages.\n\nWe confirmed the presence of impaired autophagy in patient samples and screened autophagy promoting compounds for their effectiveness at removing protein aggregates, identifying nine including Metformin. Further evaluation demonstrated Metformin is not only able to remove the protein aggregates in zebrafish and human myoblasts but is also able to rescue the fibre disintegration and swimming deficit observed in the bag3-/- fish. Therefore, repurposing Metformin provides a promising therapy for BAG3 myopathy.

genetics↗