Vaccine breakthrough infection and onward transmission of SARS-CoV-2 Beta (B.1.351) variant, Bavaria, Germany, February to March 2021.

A breakthrough infection occurred in a fully Comirnaty (BNT162b2) vaccinated healthcare worker with high levels of neutralising antibodies with the SARS-CoV-2 B.1.351 (Beta) variant in February 2021. The infection was subsequently transmitted to their unvaccinated spouse. Sequencing revealed an identical virus in both spouses, with a match of all nine single nucleotide polymorphisms typical for B.1.351. To the best of our knowledge, no transmission of any variant of SARS-CoV-2 from a fully vaccinated person has been described before.

Bioenergetic Impairment in Congenital Muscular Dystrophy Type 1A and Leigh Syndrome Muscle Cells.

Skeletal muscle has high energy requirement and alterations in metabolism are associated with pathological conditions causing muscle wasting and impaired regeneration. Congenital muscular dystrophy type 1A (MDC1A) is a severe muscle disorder caused by mutations in the LAMA2 gene. Leigh syndrome (LS) is a neurometabolic disease caused by mutations in genes related to mitochondrial function. Skeletal muscle is severely affected in both diseases and a common feature is muscle weakness that leads to hypotonia and respiratory problems. Here, we have investigated the bioenergetic profile in myogenic cells from MDC1A and LS patients. We found dysregulated expression of genes related to energy production, apoptosis and proteasome in myoblasts and myotubes. Moreover, impaired mitochondrial function and a compensatory upregulation of glycolysis were observed when monitored in real-time. Also, alterations in cell cycle populations in myoblasts and enhanced caspase-3 activity in myotubes were observed. Thus, we have for the first time demonstrated an impairment of the bioenergetic status in human MDC1A and LS muscle cells, which could contribute to cell cycle disturbance and increased apoptosis. Our findings suggest that skeletal muscle metabolism might be a promising pharmacological target in order to improve muscle function, energy efficiency and tissue maintenance of MDC1A and LS patients.

Lifetime exercise intolerance with lactic acidosis as key manifestation of novel compound heterozygous ACAD9 mutations causing complex I deficiency.

We report a 36-year-old female having lifetime exercise intolerance and lactic acidosis with nausea associated with novel compound heterozygous Acyl-CoA dehydrogenase 9 gene (ACAD9) mutations (p.Ala390Thr and p.Arg518Cys). ACAD9 is an assembly factor for the mitochondrial respiratory chain complex I. ACAD9 mutations are recognized as frequent causes of complex I deficiency. Our patient presented with exercise intolerance, rapid fatigue, and nausea since early childhood. Mild physical workload provoked the occurrence of nausea and vomiting repeatedly. Her neurological examination, laboratory findings and muscle biopsy demonstrated no abnormalities. A bicycle spiroergometry provoked significant lactic acidosis during and following exercise pointing towards a mitochondrial disorder. Subsequently, the analysis of respiratory chain enzyme activities in muscle revealed severe isolated complex I deficiency. Candidate gene sequencing revealed two novel heterozygous ACAD9 mutations. This patient report expands the mutational and phenotypic spectrum of diseases associated with mutations in ACAD9.

Generation of a human iPSC line from a patient with a defect of intergenomic communication.

Human iPSC line PG64SV.2 was generated from fibroblasts of a patient with a defect of intergenomic communication. This patient harbored a homozygous mutation (c.2243G>C; p.Trp748Ser) in the gene encoding the catalytic subunit of the mitochondrial DNA polymerase gamma gene (POLG). Reprogramming factors Oct3/4, Sox2, Klf4, and cMyc were delivered using a non integrative methodology that involves the use of Sendai virus.

Abnormal proliferation and spontaneous differentiation of myoblasts from a symptomatic female carrier of X-linked Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy (EDMD) is a neuromuscular disease characterized by early contractures, slowly progressive muscular weakness and life-threatening cardiac arrhythmia that can develop into cardiomyopathy. In X-linked EDMD (EDMD1), female carriers are usually unaffected. Here we present a clinical description and in vitro characterization of a mildly affected EDMD1 female carrying the heterozygous EMD mutation c.174_175delTT; p.Y59* that yields loss of protein. Muscle tissue sections and cultured patient myoblasts exhibited a mixed population of emerin-positive and -negative cells; thus uneven X-inactivation was excluded as causative. Patient blood cells were predominantly emerin-positive, but considerable nuclear lobulation was observed in non-granulocyte cells - a novel phenotype in EDMD. Both emerin-positive and emerin-negative myoblasts exhibited spontaneous differentiation in tissue culture, though emerin-negative myoblasts were more proliferative than emerin-positive cells. The preferential proliferation of emerin-negative myoblasts together with the high rate of spontaneous differentiation in both populations suggests that loss of functional satellite cells might be one underlying mechanism for disease pathology. This could also account for the slowly developing muscle phenotype.

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.

Immortalized myogenic cells from congenital muscular dystrophy type1A patients recapitulate aberrant caspase activation in pathogenesis: a new tool for MDC1A research.

BACKGROUND: Congenital muscular dystrophy Type 1A (MDC1A) is a severe, recessive disease of childhood onset that is caused by mutations in the LAMA2 gene encoding laminin-α2. Studies with both mouse models and primary cultures of human MDC1A myogenic cells suggest that aberrant activation of cell death is a significant contributor to pathogenesis in laminin-α2-deficiency. METHODS: To overcome the limited population doublings of primary cultures, we generated immortalized, clonal lines of human MDC1A myogenic cells via overexpression of both CDK4 and the telomerase catalytic component (human telomerase reverse transcriptase (hTERT)). RESULTS: The immortalized MDC1A myogenic cells proliferated indefinitely when cultured at low density in high serum growth medium, but retained the capacity to form multinucleate myotubes and express muscle-specific proteins when switched to low serum medium. When cultured in the absence of laminin, myotubes formed from immortalized MDC1A myoblasts, but not those formed from immortalized healthy or disease control human myoblasts, showed significantly increased activation of caspase-3. This pattern of aberrant caspase-3 activation in the immortalized cultures was similar to that found previously in primary MDC1A cultures and laminin-α2-deficient mice. CONCLUSIONS: Immortalized MDC1A myogenic cells provide a new resource for studies of pathogenetic mechanisms and for screening possible therapeutic approaches in laminin-α2-deficiency.

Facioscapulohumeral muscular dystrophy and Charcot-Marie-Tooth neuropathy 1A - evidence for "double trouble" overlapping syndromes.

BACKGROUND: We report on a patient with genetically confirmed overlapping diagnoses of CMT1A and FSHD. This case adds to the increasing number of unique patients presenting with atypical phenotypes, particularly in FSHD. Even if a mutation in one disease gene has been found, further genetic testing might be warranted in cases with unusual clinical presentation. CASE PRESENTATION: The reported 53 years old male patient suffered from walking difficulties and foot deformities first noticed at age 20. Later on, he developed scapuloperoneal and truncal muscle weakness, along with atrophy of the intrinsic hand and foot muscles, pes cavus, claw toes and a distal symmetric hypoesthesia. Motor nerve conduction velocities were reduced to 20 m/s in the upper extremities, and not educible in the lower extremities, sensory nerve conduction velocities were not attainable. Electromyography showed both, myopathic and neurogenic changes. A muscle biopsy taken from the tibialis anterior muscle showed a mild myopathy with some neurogenic findings and hypertrophic type 1 fibers. Whole-body muscle MRI revealed severe changes in the lower leg muscles, tibialis anterior and gastrocnemius muscles were highly replaced by fatty tissue. Additionally, fatty degeneration of shoulder girdle and straight back muscles, and atrophy of dorsal upper leg muscles were seen. Taken together, the presenting features suggested both, a neuropathy and a myopathy. Patient's family history suggested an autosomal dominant inheritance.Molecular testing revealed both, a hereditary motor and sensory neuropathy type 1A (HMSN1A, also called Charcot-Marie-Tooth neuropathy 1A, CMT1A) due to a PMP22 gene duplication and facioscapulohumeral muscular dystrophy (FSHD) due to a partial deletion of the D4Z4 locus (19 kb). CONCLUSION: Molecular testing in hereditary neuromuscular disorders has led to the identification of an increasing number of atypical phenotypes. Nevertheless, finding the right diagnosis is crucial for the patient in order to obtain adequate medical care and appropriate genetic counseling, especially in the background of arising curative therapies.

Flow cytometry for the analysis of α-dystroglycan glycosylation in fibroblasts from patients with dystroglycanopathies.

α-dystroglycan (α-DG) is a peripheral membrane protein that is an integral component of the dystrophin-glycoprotein complex. In an inherited subset of muscular dystrophies known as dystroglycanopathies, α-DG has reduced glycosylation which results in lower affinity binding to several extracellular matrix proteins including laminins. The glycosylation status of α-DG is normally assessed by the binding of the α-DG antibody IIH6 to a specific glycan epitope on α-DG involved in laminin binding. Immunocytochemistry and immunoblotting are two of the most widely used methods to detect the amount of α-DG glycosylation in muscle. While the interpretation of the presence or absence of the epitope on muscle using these techniques is straightforward, the assessment of a mild defect can be challenging. In this study, flow cytometry was used to compare the amount of IIH6-reactive glycans in fibroblasts from dystroglycanopathy patients with defects in genes known to cause α-DG hypoglycosylation to the amount in fibroblasts from healthy and pathological control subjects. A total of twenty one dystroglycanopathy patient fibroblasts were assessed, as well as fibroblasts from three healthy controls and seven pathological controls. Control fibroblasts have clearly detectable amounts of IIH6-reactive glycans, and there is a significant difference in the amount of this glycosylation, as measured by the mean fluorescence intensity of an antibody recognising the epitope and the percentage of cells positive for the epitope, between these controls and dystroglycanopathy patient fibroblasts (p<0.0001 for both). Our results indicate that the amount of α-DG glycosylation in patient fibroblasts is comparable to that in patient skeletal muscle. This method could complement existing immunohistochemical assays in skeletal muscle as it is quantitative and simple to perform, and could be used when a muscle biopsy is not available. This test could also be used to assess the pathogenicity of variants of unknown significance in genes involved in dystroglycanopathies.

miR-411 is up-regulated in FSHD myoblasts and suppresses myogenic factors.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscle disorder, which is linked to the contraction of the D4Z4 array at chromosome 4q35. Recent studies suggest that this shortening of the D4Z4 array leads to aberrant expression of double homeobox protein 4 (DUX4) and causes FSHD. In addition, misregulation of microRNAs (miRNAs) has been reported in muscular dystrophies including FSHD. In this study, we identified a miRNA that is differentially expressed in FSHD myoblasts and investigated its function. METHODS: To identify misregulated miRNAs and their potential targets in FSHD myoblasts, we performed expression profiling of both miRNA and mRNA using TaqMan Human MicroRNA Arrays and Affymetrix Human Genome U133A plus 2.0 microarrays, respectively. In addition, we over-expressed miR-411 in C2C12 cells to determine the effect of miR-411 on myogenic markers. RESULTS: Using miRNA and mRNA expression profiling, we identified 8 miRNAs and 1,502 transcripts that were differentially expressed in FSHD myoblasts during cell proliferation. One of the 8 differentially expressed miRNAs, miR-411, was validated by quantitative RT-PCR in both primary (2.1 fold, p<0.01) and immortalized (2.7 fold, p<0.01) myoblasts. In situ hybridization showed cytoplasmic localization of miR-411 in FSHD myoblasts. By analyzing both miRNA and mRNA data using Partek Genomics Suite, we identified 4 mRNAs potentially regulated by miR-411 including YY1 associated factor 2 (YAF2). The down-regulation of YAF2 in immortalized myoblasts was validated by immunoblotting (-3.7 fold, p<0.01). C2C12 cells were transfected with miR-411 to determine whether miR-411 affects YAF2 expression in myoblasts. The results showed that over-expression of miR-411 reduced YAF2 mRNA expression. In addition, expression of myogenic markers including Myod, myogenin, and myosin heavy chain 1 (Myh1) were suppressed by miR-411. CONCLUSIONS: The study demonstrated that miR-411 was differentially expressed in FSHD myoblasts and may play a role in regulating myogenesis.

Transplantation of genetically corrected human iPSC-derived progenitors in mice with limb-girdle muscular dystrophy.

Mesoangioblasts are stem/progenitor cells derived from a subset of pericytes found in muscle that express alkaline phosphatase. They have been shown to ameliorate the disease phenotypes of different animal models of muscular dystrophy and are now undergoing clinical testing in children affected by Duchenne's muscular dystrophy. Here, we show that patients with a related disease, limb-girdle muscular dystrophy 2D (LGMD2D), which is caused by mutations in the gene encoding α-sarcoglycan, have reduced numbers of this pericyte subset and thus produce too few mesoangioblasts for use in autologous cell therapy. Hence, we reprogrammed fibroblasts and myoblasts from LGMD2D patients to generate human induced pluripotent stem cells (iPSCs) and developed a protocol for the derivation of mesoangioblast-like cells from these iPSCs. The iPSC-derived mesoangioblasts were expanded and genetically corrected in vitro with a lentiviral vector carrying the gene encoding human α-sarcoglycan and a promoter that would ensure expression only in striated muscle. When these genetically corrected human iPSC-derived mesoangioblasts were transplanted into α-sarcoglycan-null immunodeficient mice, they generated muscle fibers that expressed α-sarcoglycan. Finally, transplantation of mouse iPSC-derived mesoangioblasts into α-sarcoglycan-null immunodeficient mice resulted in functional amelioration of the dystrophic phenotype and restoration of the depleted progenitors. These findings suggest that transplantation of genetically corrected mesoangioblast-like cells generated from iPSCs from LGMD2D patients may be useful for treating this type of muscular dystrophy and perhaps other forms of muscular dystrophy as well.

A novel mutation in the myotilin gene (MYOT) causes a severe form of limb girdle muscular dystrophy 1A (LGMD1A).

Here we describe a patient with limb girdle muscular dystrophy 1A (LGMD1A) due to a novel myotilin gene (MYOT) mutation with late onset, rapid progression, loss of ambulation and respiratory failure. The onset of weakness in proximal muscles and muscle MRI findings are clearly different from the pattern identified in myofibrillar myopathies (MFM) related to MYOT mutations. Moreover, there was very limited evidence of myofibrillar pathology in several muscle biopsies obtained during the disease course. We conclude, that MYOT mutations need to be considered as a rare cause of adult-onset, dominant LGMD without clear-cut MFM pathology.

Biobanking in rare disorders.

Biobanks are collections of biomaterials with associated data. Biobanking is an essential tool to provide access to high quality human biomaterial for fundamental and translational research. Research for rare disorders benefits from the provision of human biomaterials through biobanks, and each human sample from a person with a rare disorder has a high value as it may hold the key to answer an important research question. Transnational cooperation in biobanking is an important catalyst to share limited resources and achieve optimal outcomes as in other areas of rare disorder research. Networks of biobanks aim to assure common practices and quality standards, and facilitate access to rare disorder biomaterials for the scientific community.

Late-onset ptosis and myopathy in a patient with a heterozygous insertion in POLG2.

Polymerase gamma 1 (POLG) mutations are a frequent cause of both autosomal dominant and recessive complex neurological phenotypes. In contrast, only a single pathogenic mutation in one patient was reported in POLG2 so far. Here we describe a 62-year-old woman, carrying a novel heterozygous sequence variant in the POLG2 gene. She developed bilateral ptosis at 30 years of age, followed by exercise intolerance, muscle weakness and mild CK increase in her late forties. Muscle histology and respiratory chain activities were normal. Southern blot and long range PCR detected multiple mtDNA deletions, but no depletion in muscle DNA. Sequencing of POLG, PEO1, ANT1, OPA1 and RRM2B showed normal results. A novel heteroallelic 24 bp insertion (c.1207_1208ins24) was detected in POLG2. This 24 bp insertion into exon 7 causes missplicing and loss of exon 7 in myoblast cDNA. We did not detect POLG2 mutations in 62 patients with multiple mtDNA deletions in muscle DNA, suggesting that POLG2 mutations may represent a rare cause of autosomal dominant PEO.

Facioscapulohumeral muscular dystrophy presenting with unusual phenotypes and atypical morphological features of vacuolar myopathy.

Facioscapulohumeral muscular dystrophy (FSHD) is the third most common muscular dystrophy and usually follows an autosomal dominant trait. Clinically, FSHD affects facial muscles and proximal upper limb and girdle muscles, but may present with variable clinical phenotypes even within the same family. Most genetically confirmed FSHD patients exhibit unspecific morphological signs of a degenerative myopathy. We report on five unrelated patients who carried the pathogenic FSHD mutation on chromosome 4q35. Muscle biopsies revealed numerous rimmed vacuoles and filamentous cytoplasmic inclusions in all cases. Clinically, the patients suffered from weakness and atrophy predominantly of the lower limb muscles. In conclusion, we suggest considering FSHD in the differential diagnosis of adult-onset distal myopathies with rimmed vacuoles.

Heteroplasmic mutation in the anticodon-stem of mitochondrial tRNA(Val) causing MNGIE-like gastrointestinal dysmotility and cachexia.

While mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is typically associated with mutations in the nuclear gene encoding for thymidine phosphorylase (ECGF1, TYMP), a similar clinical phenotype was described in patients carrying mutations in the nuclear-encoded polymerase gamma (POLG1) as well as a few mitochondrial tRNA genes. Here we report a novel mutation in the mitochondrial tRNA(Val) (MTTV) gene in a girl presenting with clinical symptoms of MNGIE-like gastrointestinal dysmotility and cachexia. Clinical, histological, biochemical and single cell investigations were performed. The heteroplasmic m.1630A>G mutation was detected in the mitochondrial tRNA(Val) (MTTV) gene in the patient's muscle, blood leukocytes and myoblasts, as well as in blood DNA of the unaffected mother. We provide clinical, biochemical, histological, and molecular genetic evidence on the single cell level for the pathogenicity of this mutation. Our finding adds to the genetic heterogeneity of MNGIE-like gastrointestinal symptoms and highlights the importance of a thorough genetic workup in case of suspected mitochondrial disease.

The myopathic form of coenzyme Q10 deficiency is caused by mutations in the electron-transferring-flavoprotein dehydrogenase (ETFDH) gene.

Coenzyme Q10 (CoQ10) deficiency is an autosomal recessive disorder with heterogenous phenotypic manifestations and genetic background. We describe seven patients from five independent families with an isolated myopathic phenotype of CoQ10 deficiency. The clinical, histological and biochemical presentation of our patients was very homogenous. All patients presented with exercise intolerance, fatigue, proximal myopathy and high serum CK. Muscle histology showed lipid accumulation and subtle signs of mitochondrial myopathy. Biochemical measurement of muscle homogenates showed severely decreased activities of respiratory chain complexes I and II + III, while complex IV (COX) was moderately decreased. CoQ10 was significantly decreased in the skeletal muscle of all patients. Tandem mass spectrometry detected multiple acyl-CoA deficiency, leading to the analysis of the electron-transferring-flavoprotein dehydrogenase (ETFDH) gene, previously shown to result in another metabolic disorder, glutaric aciduria type II (GAII). All of our patients carried autosomal recessive mutations in ETFDH, suggesting that ETFDH deficiency leads to a secondary CoQ10 deficiency. Our results indicate that the late-onset form of GAII and the myopathic form of CoQ10 deficiency are allelic diseases. Since this condition is treatable, correct diagnosis is of the utmost importance and should be considered both in children and in adults. We suggest to give patients both CoQ10 and riboflavin supplementation, especially for long-term treatment.

Functional analysis of apoptosis induction in acute myeloid leukaemia-relevance of karyotype and clinical treatment response.

Deficiencies or structural defects of the apoptotic machinery have been postulated as a potential mechanism for a broad resistance of acute myeloid leukaemia (AML) blasts towards cytotoxic therapy comprising chemotherapeutic agents with diverse pharmacodynamic principles but also cell-mediated cytotoxicity of the graft-versus-leukaemia effect, for example, in the setting of allogeneic transplantation. This hypothesis was systematically tested by functionally analysing the early, intermediate and late events of the apoptotic process in primary AML (n = 31) blasts following activation of the intrinsic and extrinsic pathway of apoptosis (etoposide and cytarabine as DNA damaging agents, FAS-ligand as an activator of the death receptor pathway). Activation of the extrinsic pathway by FAS-ligand did not induce apoptosis in primary AML, instead the proapoptotic signal was shown to 'fade', even in the early phase of the apoptotic sequence. However, activation of the intrinsic pathway induced severe cytotoxicity in all samples that showed the characteristic features of typical apoptosis, with a prominent apoptotic volume decrease (blebbing) in the early phase, significant increases in caspase 3 activity (intermediate or effector phase) and breakdown of cellular energy production in the late phase of apoptosis. These characteristics did not differ between prognostically favourable versus unfavourable AML karyotypes or between clinically responding versus refractory AML--indicating that a functional apoptotic apparatus is present even in the unfavourable AML subgroups. Our data indicate that the mechanism for a broad clinical resistance is not a dysfunctional apparatus per se but rather the consequence of anti-apoptotic regulation impeding otherwise functional apoptotic machinery.