Complete occipitalization of the atlas with bilateral external auditory canal atresia.

Fusion of the atlas with the occipital bone is a rare congenital dysplasia known as occipitalization of the atlas, occipitocervical synostosis, assimilation of the atlas, or atlanto-occipital fusion. It is a component of the paraxial mesodermal maldevelopment and commonly associated with other dysplasias of the craniovertebral junction. External auditory canal atresia or external aural atresia is a rare congenital absence of the external auditory canal. It occurs as the consequence of the maldevelopment of the first pharyngeal cleft due to defects of cranial neural crest cells migration and/or differentiation. It is commonly associated with the dysplasias of the structures derived from the first and second pharyngeal arches including microtia. We present the coexistence of the occipitalization of the atlas and congenital aural atresia, an uncommon combination of the paraxial mesodermal maldevelopment, and defects of cranial neural crest cells. The association is most probably syndromic as minimal diagnostic criteria for the oculoariculovertebral spectrum are fulfilled. From the clinical point of view, it is important to be aware that patients with microtia must obtain also appropriate diagnostic imaging studies of the craniovetebral junction due to eventual concomitant occipitalization of the atlas and frequently associated C1-C2 instability.

Transcriptomic profiling of TK2 deficient human skeletal muscle suggests a role for the p53 signalling pathway and identifies growth and differentiation factor-15 as a potential novel biomarker for mitochondrial myopathies.

BACKGROUND: Mutations in the gene encoding thymidine kinase 2 (TK2) result in the myopathic form of mitochondrial DNA depletion syndrome which is a mitochondrial encephalomyopathy presenting in children. In order to unveil some of the mechanisms involved in this pathology and to identify potential biomarkers and therapeutic targets we have investigated the gene expression profile of human skeletal muscle deficient for TK2 using cDNA microarrays. RESULTS: We have analysed the whole transcriptome of skeletal muscle from patients with TK2 mutations and compared it to normal muscle and to muscle from patients with other mitochondrial myopathies. We have identified a set of over 700 genes which are differentially expressed in TK2 deficient muscle. Bioinformatics analysis reveals important changes in muscle metabolism, in particular, in glucose and glycogen utilisation, and activation of the starvation response which affects aminoacid and lipid metabolism. We have identified those transcriptional regulators which are likely to be responsible for the observed changes in gene expression. CONCLUSION: Our data point towards the tumor suppressor p53 as the regulator at the centre of a network of genes which are responsible for a coordinated response to TK2 mutations which involves inflammation, activation of muscle cell death by apoptosis and induction of growth and differentiation factor 15 (GDF-15) in muscle and serum. We propose that GDF-15 may represent a potential novel biomarker for mitochondrial dysfunction although further studies are required.

Isolation, characterization and differentiation of cells expressing pluripotent/multipotent markers from adult human ovaries.

Pluripotent stem cells are still generally accepted not to exist in adult human ovaries, although increasing studies confirm the presence of pluripotent/multipotent stem cells in adult mammalian ovaries, including those of humans. The aim of this study is to isolate, characterize and differentiate in vitro stem cells that originate from the adult human ovarian cortex and that express markers of pluripotency/multipotency. After enzymatic degradation of small ovarian cortex biopsies retrieved from 18 women, ovarian cell cultures were successfully established from 17 and the formation of cell colonies was observed. The presence of cells/colonies expressing some markers of pluripotency (alkaline phosphatase, surface antigen SSEA-4, OCT4, SOX-2, NANOG, LIN28, STELLA), germinal lineage (DDX4/VASA) and multipotency (M-CAM/CD146, Thy-1/CD90, STRO-1) was confirmed by various methods. Stem cells from the cultures, including small round SSEA-4-positive cells with diameters of up to 4 µm, showed a relatively high degree of plasticity. We were able to differentiate them in vitro into various types of somatic cells of all three germ layers. However, these cells did not form teratoma when injected into immunodeficient mice. Our results thus show that ovarian tissue is a potential source of stem cells with a pluripotent/multipotent character for safe application in regenerative medicine.

Diagnostic yield of exome sequencing in myopathies: Experience of a Slovenian tertiary centre.

BACKGROUND: Our aim was to present the experience of systematic, routine use of next generation sequencing (NGS) in clinical diagnostics of myopathies. METHODS: Exome sequencing was performed on patients with high risk for inherited myopathy, which were selected based on the history of the disease, family history, clinical presentation, and diagnostic workup. Exome target capture was performed, followed by sequencing on HiSeq 2500 or MiSeq platforms. Data analysis was performed using internally developed bioinformatic pipeline. RESULTS: The study comprised 86 patients, including 22 paediatric cases (26%). The largest group were patients referred with an unspecified myopathy (47%), due to non-specific or incomplete clinical and laboratory findings, followed by congenital myopathies (22%) and muscular dystrophies (22%), congenital myotonias (6%), and mitochondrial myopathies (3%). Altogether, a diagnostic yield was 52%; a high diagnostic rate was present in paediatric patients (64%), while in patients with unspecified myopathies the rate was 35%. We found 51 pathogenic/likely pathogenic variants in 23 genes and two pathogenic copy number variations. CONCLUSION: Our results provide evidence that phenotype driven exome analysis diagnostic approach facilitates the diagnostic rate of complex, heterogeneous disorders, such as myopathies, particularly in paediatric patients and patients with unspecified myopathies.

Innervation and electrical pulse stimulation - in vitro effects on human skeletal muscle cells.

Contraction-induced adaptations in skeletal muscles are well characterized in vivo, but the underlying cellular mechanisms are still not completely understood. Cultured human myotubes represent an essential model system for human skeletal muscle that can be modulated ex vivo, but they are quiescent and do not contract unless being stimulated. Stimulation can be achieved by innervation of human myotubes in vitro by co-culturing with embryonic rat spinal cord, or by replacing motor neuron activation by electrical pulse stimulation (EPS). Effects of these two in vitro approaches, innervation and EPS, were characterized with respects to the expression of myosin heavy chains (MyHCs) and metabolism of glucose and oleic acid in cultured human myotubes. Adherent human myotubes were either innervated with rat spinal cord segments or exposed to EPS. The expression pattern of MyHCs was assessed by quantitative polymerase chain reaction, immunoblotting, and immunofluorescence, while the metabolism of glucose and oleic acid were studied using radiolabelled substrates. Innervation and EPS promoted differentiation towards different fiber types in human myotubes. Expression of the slow MyHC-1 isoform was reduced in innervated myotubes, whereas it remained unaltered in EPS-treated cells. Expression of both fast isoforms (MyHC-2A and MyHC-2X) tended to decrease in EPS-treated cells. Both approaches induced a more oxidative phenotype, reflected in increased CO2 production from both glucose and oleic acid. Novelty: Innervation and EPS favour differentiation into different fiber types in human myotubes. Both innervation and EPS promote a metabolically more oxidative phenotype in human myotubes.

Case Report: SATB2-Associated Syndrome Overlapping With Clinical Mitochondrial Disease Presentation: Report of Two Cases.

SATB2-associated syndrome (SAS) is an autosomal dominant neurogenetic multisystemic disorder. We describe two individuals with global developmental delay and hypotonia who underwent an extensive evaluation to rule out an underlying mitochondrial disorder before their eventual diagnosis of SAS. Although the strict application of the clinical mitochondrial disease score only led to the designation of "possible" mitochondrial disorder for these two individuals, other documented abnormalities included nonspecific neuroimaging findings on magnetic resonance imaging and magnetic resonance spectroscopy, decreased complex I activity on muscle biopsy for patient 2, and variation in the size and relative proportion of types of muscle fibers in the muscle biopsies that were aligned with mitochondrial diseases. SAS should be in the differential diagnoses of mitochondrial disorders, and broad-spectrum diagnostic tests such as exome sequencing need to be considered early in the evaluation process of undiagnosed neurodevelopmental disorders.

Characterisation of flexor digitorum profundus, flexor digitorum superficialis and extensor digitorum communis by electrophoresis and immunohistochemical analysis of myosin heavy chain isoforms in older men.

INTRODUCTION: Few data exist on the fiber type composition of the extrinsic finger muscles. The aim of the present study was to describe myosin heavy chain (MyHC) composition of flexor digitorum profundus (FDP), flexor digitorum superficialis (FDS) and extensor digitorum communis (EDC). MyHC composition is relevant for whole muscle contractile performance and several studies on single muscle fibers demonstrated that fibers expressing only slow MyHC-1 develop less specific force than fibers expressing fast MyHCs. Since contraction force of finger extensors is smaller than of finger flexors a hypothesis was posited that the content of MyHC-1 is higher in EDC than in extrinsic finger flexors. METHODS: Autopsy samples of FDP, FDS, and EDC in 27 healthy older men were analyzed and compared with each other and with biceps brachii (BB). MyHC isoforms were quantified on silver-stained 6% SDS-PAGE. Muscle fibers were classified immunohistochemically according to the expression of adult MyHC isoforms and their morphometric parameters were determined. RESULTS: EDC stood out for its higher proportion of slow MyHC-1 (50%) compared to FDP (37%), FDS (38%) and BB (35%) (p<0.001 in all), and its lower proportion of fast MyHC-2x (13%) compared to FDP (26%, p=0.001), FDS (22%, p=0.028) and BB (29%, p<0.001) detected on SDS-PAGE. Immunohistochemically, EDC had a higher area proportion of pure slow type-1 fibers (63%) than FDP (47%, p=0.002), FDS (49%, p=0.007) and BB (47%, p=0.002), and lower area proportion of pure fast type-2x fibers (2%) than FDP (12%, p=0.014), FDS (8%, p=0.256) and BB (14%, p=0.002). All muscles contained a similar area proportion of pure type-2a fibers and hybrid type-2a/2x fibers. CONCLUSIONS: The study results support the hypothesis that the content of MyHC-1 is higher in EDC than in extrinsic finger flexors, which is in agreement with the lower contraction force of finger extensors compared to finger flexors.

Selective screening of late-onset Pompe disease (LOPD) in patients with non-diagnostic muscle biopsies.

AIMS: As of 2016, there were five patients with Pompe in Slovenia (two infantile, one childhood and two adult onset) with a prevalence of 1:400 000; however, the prevalence of late-onset Pompe disease (LOPD) in some other countries means this ratio could be an underestimate. Since an LOPD muscle biopsy could be unspecific or even normal, the purpose of this study is to assess the prevalence of LOPD in patients with non-diagnostic muscle biopsies. METHODS: Six hundred biopsies were recorded at the Neuromuscular Tissue Bank of the University of Ljubljana for the period 2004-2014. All adult patients with non-diagnostic muscle biopsies were invited to the National Slovenian Neuromuscular Centre for dried blood spot testing for LOPD. RESULTS: A total of 90 patients (56% of those invited) responded. No patient with LOPD was found. A total of 49 patients (54%) had fixed muscle weakness, 31 (34%) had mild symptoms and no weakness and 10 (11%) had asymptomatic hyperCKemia. Ventilatory insufficiency associated with proximal muscle weakness was found in two patients (2%). No patients exhibited vacuolar myopathy, globular accumulations of glycogen or regions of increased acid phosphatase activity within the sarcoplasm. CONCLUSIONS: The study results do not support the hypothesis that LOPD is underestimated in Slovenian patients with non-diagnostic muscle biopsies; this could be consistent with the fact that LOPD is of low prevalence in Slovenia, as is the case in the populations of Finland, French-speaking Belgium, west Sweden and west Denmark.

Size and Proportions of Slow-Twitch and Fast-Twitch Muscle Fibers in Human Costal Diaphragm.

Smaller diaphragmatic motor unit potentials (MUPs) compared to MUPs of limb muscles lead to the hypothesis that diaphragmatic muscle fibers, being the generators of MUPs, might be also smaller. We compared autopsy samples of costal diaphragm and vastus lateralis of healthy men with respect to fibers' size and expression of slow myosin heavy chain isoform (MyHC-1) and fast 2A isoform (MyHC-2A). Diaphragmatic fibers were smaller than fibers in vastus lateralis with regard to the mean minimal fiber diameter of slow-twitch (46.8 versus 72.2 µm, p < 0.001), fast-twitch (45.1 versus 62.4 µm, p < 0.001), and hybrid fibers (47.3 versus 65.0 µm, p < 0.01) as well as to the mean fiber cross-sectional areas of slow-twitch (2376.0 versus 5455.9 µm2, p < 0.001), fast-twitch (2258.7 versus 4189.7 µm2, p < 0.001), and hybrid fibers (2404.4 versus 4776.3 µm2, p < 0.01). The numerical proportion of slow-twitch fibers was higher (50.2 versus 36.3%, p < 0.01) in costal diaphragm and the numerical proportion of fast-twitch fibers (47.2 versus 58.7%, p < 0.01) was lower. The numerical proportion of hybrid fibers did not differ. Muscle fibers of costal diaphragm have specific characteristics which support increased resistance of diaphragm to fatigue.

GDF-15 Is Elevated in Children with Mitochondrial Diseases and Is Induced by Mitochondrial Dysfunction.

BACKGROUND: We previously described increased levels of growth and differentiation factor 15 (GDF-15) in skeletal muscle and serum of patients with mitochondrial diseases. Here we evaluated GDF-15 as a biomarker for mitochondrial diseases affecting children and compared it to fibroblast-growth factor 21 (FGF-21). To investigate the mechanism of GDF-15 induction in these pathologies we measured its expression and secretion in response to mitochondrial dysfunction. METHODS: We analysed 59 serum samples from 48 children with mitochondrial disease, 19 samples from children with other neuromuscular diseases and 33 samples from aged-matched healthy children. GDF-15 and FGF-21 circulating levels were determined by ELISA. RESULTS: Our results showed that in children with mitochondrial diseases GDF-15 levels were on average increased by 11-fold (mean 4046pg/ml, 1492 SEM) relative to healthy (350, 21) and myopathic (350, 32) controls. The area under the curve for the receiver-operating-characteristic curve for GDF-15 was 0.82 indicating that it has a good discriminatory power. The overall sensitivity and specificity of GDF-15 for a cut-off value of 550pg/mL was 67.8% (54.4%-79.4%) and 92.3% (81.5%-97.9%), respectively. We found that elevated levels of GDF-15 and or FGF-21 correctly identified a larger proportion of patients than elevated levels of GDF-15 or FGF-21 alone. GDF-15, as well as FGF-21, mRNA expression and protein secretion, were significantly induced after treatment of myotubes with oligomycin and that levels of expression of both factors significantly correlated. CONCLUSIONS: Our data indicate that GDF-15 is a valuable serum quantitative biomarker for the diagnosis of mitochondrial diseases in children and that measurement of both GDF-15 and FGF-21 improves the disease detection ability of either factor separately. Finally, we demonstrate for the first time that GDF-15 is produced by skeletal muscle cells in response to mitochondrial dysfunction and that its levels correlate in vitro with FGF-21 levels.

The EuroBioBank Network: 10 years of hands-on experience of collaborative, transnational biobanking for rare diseases.

The EuroBioBank (EBB) network (www.eurobiobank.org) is the first operating network of biobanks in Europe to provide human DNA, cell and tissue samples as a service to the scientific community conducting research on rare diseases (RDs). The EBB was established in 2001 to facilitate access to RD biospecimens and associated data; it obtained funding from the European Commission in 2002 (5th framework programme) and started operation in 2003. The set-up phase, during the EC funding period 2003-2006, established the basis for running the network; the following consolidation phase has seen the growth of the network through the joining of new partners, better network cohesion, improved coordination of activities, and the development of a quality-control system. During this phase the network participated in the EC-funded TREAT-NMD programme and was involved in planning of the European Biobanking and Biomolecular Resources Research Infrastructure. Recently, EBB became a partner of RD-Connect, an FP7 EU programme aimed at linking RD biobanks, registries, and bioinformatics data. Within RD-Connect, EBB contributes expertise, promotes high professional standards, and best practices in RD biobanking, is implementing integration with RD patient registries and 'omics' data, thus challenging the fragmentation of international cooperation on the field.

Functional outcome of children with mitochondrial diseases.

We evaluated the functional outcome in a cohort of 22 children with mitochondrial diseases. The Pediatric Evaluation of Disability Inventory was administered and scaled scores determined. A large variability in functional skills was seen in all 3 assessed domains: Self-Care (46.4 ± 25.6 S.D.; median 42.4; range 11.8-100), Mobility (47.1 ± 30.4 S.D.; median 46.7; range 6.1-100), and Social Function (49.7 ± 22.3 S.D.; median 45.6; range 21.6-100). There was also a large variability in caregiver assistance, ranging from independent functioning to total dependence on assistance for the following: Self-Care (29.0 ± 33.7 S.D.; median 10.1; range 0-100), Mobility (36.6 ± 38.5 S.D.; median 26.1; range 0-100), and Social Function (42.9 ± 32.4 S.D.; median 44.4; range 0-100). The Pediatric Evaluation of Disability Inventory scores describe in depth different, but strongly related, aspects of everyday functioning in children with mitochondrial diseases. The importance of caregiver assistance in helping these children should not be underestimated. The use of assistive devices was estimated, which were found to be used by few patients.