Loss of supervillin causes myopathy with myofibrillar disorganization and autophagic vacuoles

The muscle specific isoform of the supervillin protein (SV2), encoded by the SVIL gene, is a large sarcolemmal myosin II- and F-actin-binding protein. Supervillin (SV2) binds and co-localizes with costameric dystrophin and binds nebulin, potentially attaching the sarcolemma to myofibrillar Z-lines. Despite its important role in muscle cell physiology suggested by various in vitro studies, there are so far no reports of any human disease caused by SVIL mutations. We here report four patients from two unrelated, consanguineous families with a childhood/adolescence onset of a myopathy associated with homozygous loss-of-function mutations in SVIL. Wide neck, anteverted shoulders and prominent trapezius muscles together with variable contractures were characteristic features. All patients showed increased levels of serum creatine kinase but no or minor muscle weakness. Mild cardiac manifestations were observed. Muscle biopsies showed complete loss of large supervillin isoforms in muscle fibres by western blot and immunohistochemical analyses. Light and electron microscopic investigations revealed a structural myopathy with numerous lobulated muscle fibres and considerable myofibrillar alterations with a coarse and irregular intermyofibrillar network. Autophagic vacuoles, as well as frequent and extensive deposits of lipoproteins, including immature lipofuscin, were observed. Several sarcolemma-associated proteins, including dystrophin and sarcoglycans, were partially mis-localized. The results demonstrate the importance of the supervillin (SV2) protein for the structural integrity of muscle fibres in humans and show that recessive loss-of-function mutations in SVIL cause a distinctive and novel myopathy

Evaluating the association of biallelic OGDHL variants with significant phenotypic heterogeneity

BACKGROUND: Biallelic variants in OGDHL, encoding part of the α-ketoglutarate dehydrogenase complex, have been associated with highly heterogeneous neurological and neurodevelopmental disorders. However, the validity of this association remains to be confirmed. A second OGDHL patient cohort was recruited to carefully assess the gene-disease relationship. METHODS: Using an unbiased genotype-first approach, we screened large, multiethnic aggregated sequencing datasets worldwide for biallelic OGDHL variants. We used CRISPR/Cas9 to generate zebrafish knockouts of ogdhl, ogdh paralogs, and dhtkd1 to investigate functional relationships and impact during development. Functional complementation with patient variant transcripts was conducted to systematically assess protein functionality as a readout for pathogenicity. RESULTS: A cohort of 14 individuals from 12 unrelated families exhibited highly variable clinical phenotypes, with the majority of them presenting at least one additional variant, potentially accounting for a blended phenotype and complicating phenotypic understanding. We also uncovered extreme clinical heterogeneity and high allele frequencies, occasionally incompatible with a fully penetrant recessive disorder. Human cDNA of previously described and new variants were tested in an ogdhl zebrafish knockout model, adding functional evidence for variant reclassification. We disclosed evidence of hypomorphic alleles as well as a loss-of-function variant without deleterious effects in zebrafish variant testing also showing discordant familial segregation, challenging the relationship of OGDHL as a conventional Mendelian gene. Going further, we uncovered evidence for a complex compensatory relationship among OGDH, OGDHL, and DHTKD1 isoenzymes that are associated with neurodevelopmental disorders and exhibit complex transcriptional compensation patterns with partial functional redundancy. CONCLUSIONS: Based on the results of genetic, clinical, and functional studies, we formed three hypotheses in which to frame observations: biallelic OGDHL variants lead to a highly variable monogenic disorder, variants in OGDHL are following a complex pattern of inheritance, or they may not be causative at all. Our study further highlights the continuing challenges of assessing the validity of reported disease-gene associations and effects of variants identified in these genes. This is particularly more complicated in making genetic diagnoses based on identification of variants in genes presenting a highly heterogenous phenotype such as "OGDHL-related disorders"

Quasi-simultaneous magnetic particle imaging and navigation of nanomag/synomag-D particles in bifurcation flow experiments

Magnetic Particle Imaging (MPI) is used to visualize the distribution of superparamagnetic nanoparticles within 3D volumes with high sensitivity in real time. Recently, MPI is utilized to navigate micron-sized particles and micron-sized swimmers, since the magnetic field topology of the MPI scanner is well suited to apply magnetic forces. In this work, we analyze the magnetic mobility and imaging performance of nanomag/synomag-D for Magnetic Particle Imaging/Navigation (MPIN). With MPIN the focus fields are constantly switching between imaging and magnetic force mode, thus enabling quasi-simultaneous navigation and imaging of particles. In flow bifurcation experiment with a 100 % stenosis on one branch, we determine the limiting flow velocity of 1.36 mL/s, which allows all particles to flow only through one branch towards the stenosis. During this experiment, we image the accumulation of the particles within the stenosis. In combination with therapeutic substances, this approach has high potential for targeted drug delivery.Deutsche Forschungsgemeinschaft (DFG)Bundesministerium für Bildung und Forschung (BMBF

Characterization of the Synomag®-D-PEG-OMe nanoparticles for the encapsulation in human and murine red blood cells

It was shown that the encapsulation of SPIO-based contrast agents in the red blood cells (RBCs) increases the circulation time in blood of these nanomaterials. Not all iron oxide particles are eligible for the entrapment into RBCs, depending on several factors and synthesis protocol. We have recently identified some type of nanoparticles that can be loaded with our method into RBCs to produce biocompatible SPIO-RBCs carriers that could be used as new intravascular tracers for biomedical applications, such as Magnetic Particle Imaging (MPI). Here, we report the first in vitro results obtained by using the Synomag®-D-PEG-OMe nanoparticles with both human and murine RBCs. MPS analysis showed that human Synomag®-D-PEG-OMe-loaded RBCs produced a signal that is weaker respect to the remarkable signal obtained with ferucarbotran loaded-RBCs prepared at the same condition, but it is to be noted that the encapsulation efficiency of Synomag®-D-PEG-OMe into cells is lower compared to ferucarbotran nanoparticles

Encapsulation of new MPI tracer nanoparticles in the human red blood cells

Although Magnetic Particle Imaging (MPI) is not yet in clinical use, it is highly promising for several medical ap-plications, and especially for applications in diagnostic vascular in vivo imaging and imaging-guided vascular interventions. Furthermore, in the last years, different superparamagnetic iron oxide (SPIO) based contrast agents have been developed and approved for niche clinical applications in Magnetic Resonance Imaging (MRI) as alterna-tive to Gadolinium-based contrast agents (GBCAs) due to the risk for patients suffering from kidney dysfunction or nephrogenic systemic fibrosis (NSF). Recently, the potential of RBCs loaded with different SPIO nanoparticles as blood-pool tracer agents with longer blood retention time for MRI and MPI has been investigated. Here, we report the first in vitro results with the highly efficient dextran-based MPI tracer particles perimag® and synomag®-D to study their eligibility to be encapsulated into human RBCs and the potential of these new SPIO-RBC constructs as tracer material for MPI

Encapsulation in human and murine erythrocytes of the Synomag®-D-PEG-OMe tracer for MPI application

Recently, the potential of red blood cells (RBCs) loaded with superparamagnetic iron oxide (SPIO)-based nanoparticles as new blood-pool tracer material for the Magnetic Particle Imaging (MPI) has been investigated. It was shown that the encapsulation of SPIO-based contrast agents in the RBCs increase the circulation time in blood of these nanomaterials. However, not all iron oxide nanoparticles are eligible to the encapsulation into RBCs, depending on several factors such as dispersant agent nature, nanoparticle size and synthesis protocol. Therefore, we have recently started a program to identify those nanoparticles that can be potentially loaded with our method into RBCs. The goal is to produce biocompatible SPIO-RBCs carriers that can be used as new intravascular magnetic susceptible agents in biomedical applications, such as MRI and MPI. Here, we report the in vitro results obtained by using the Synomag®-D-PEG-OMe nanoparticle suspension (micromod Partikeltechnologie GmbH) with both human and murine red blood cells. MPS analysis showed that human Synomag®-D-PEG-OMe-loaded RBCs produced a signal that is weaker respect to the remarkable signal of ferucarbotran loaded-RBCs prepared at the same condition, but it is to be noted that the encapsulation efficiency of Synomag®-D-PEG-OMe into cells is lower compared to ferucarbotran nanoparticles