Molekulargenetische Untersuchung metabolischer Myopathien

Die Arbeit fasst molekulargenetische Untersuchungen zu den wichtigsten metabolischen Myopathien (mitochondriale Enzephalomyopathien, Myophosphorylase-Mangel und muskulärer CPT II-Mangel) zusammen. Bei 21 Patienten mit mitochondrialen Enzephalomyopathien fand sich die Mutation A3243G der mtDNA, die eine große phänotypische Variabilität zeigte. Das häufigste Symptom war eine Hörstörung, die bei fast alle Patienten klinisch oder subklinisch vorlag. Schmerzhafte Muskelsteife wurde als neuer Phänotyp erkannt. Die Identifikation einer neuen Mutation C11777A der mtDNA bei einem Patienten mit Erstmanifestation einer Enzephalopathie im 67. Lebensjahr zeigte, daß auch bei älteren Patienten an eine Mitochondriopathie gedacht werden muß. Diese Mutation betrifft das gleiche Kodon wie die Mutation G11778A, die mit der Leber’schen Optikusneuropathie assoziiert ist, zeigte jedoch bemerkenswerterweise einen sehr differenten Phänotyp. Deletionen der mtDNA, die in der Regel mit einer CPEO assoziiert waren, fanden sich bei 43 Index-Patienten im Southern-Blot. Dabei zeigte sich, daß singuläre Deletionen häufiger sind und mit sporadischem Auftreten assoziiert sind, im Vergleich zu multiplen Deletionen, die sowohl autosomal vererbt als auch sporadisch auftreten können. Bei einem Patienten mit autosomal dominant vererbter CPEO wurde eine neue Mutation (K319T) im nukleären Twinkle-Gen identifiziert. Bei diesem Patienten zeigten sich die für diese Erkrankung wegweisenden multiplen Deletionen der mtDNA nur mittels Long-PCR und Real-time-PCR, nicht jedoch mittels Southern-Blot-Technik, die bislang als Methode der Wahl galt, was die diagnostischen Schwierigkeiten illustriert. Bei sechs Patienten mit Myophosphorylase-Mangel wurden drei neue Mutationen (Y84X, R93W und R269X) identifiziert und so die Kenntnis der genetische Variabilität dieser Erkrankung erweitert. Bei 37 Patienten mit muskulärem CPT II-Mangel, wurden fünf neuen Mutationen (M214T, Y479F, 413delAG-F448L, 515del4 und IVS3+5G->A) identifiziert. Erstmals wurde bei muskulärem CPT II-Mangel eine Mutation in einer Splice-Site nachgewiesen. Eine klare Genotyp-Phänotyp-Korrelation fand sich nicht, jedoch war Fasten als Auslöser der Attacken nur bei Patienten mit einer trunkierenden Mutation relevant. Eine Allelhäufigkeit von jeweils 8% für die Mutationen P50H and Q413fs-F448L legte nahe, dass bei der molekularen Routine-Diagnostik neben der Untersuchung der häufigen Mutation S113L auch die Suche nach diesen beiden Mutationen sinnvoll ist.Molecular genetic investigations in patients with common metabolic myopathies (mitochondrial encephalomyopathies, myophosphorylase deficiency, muscle CPT II deficiency) are summarized. The A3243G mutation of mtDNA was identified in 21 patients with mitochondrial encephalomyopathies revealing a marked phenotypic variability. The most common symptom was hearing loss that was present in nearly all patients clinically or subclinically. Painful muscle stiffness was described as a new phenotype. Identification of a novel mutation C11777A of mtDNA in a patient with encephalopathy manifesting at age 67 years showed, that even in older patients the diagnosis of mitochondrial disorders has to be considered. This mutation affects the same codon as the G11778A mutation, that is associated with Leber hereditary optic neuropathy. However, it is remarkable that it results in a totally different phenotype. Deletions of mtDNA were found in 43 index patients by Southern blot analysis and were associated with CPEO. Single deletions, that occurred sporadically, were more frequent than multiple deletions, that were inherited autosomally or occurred sporadically. In one family with autosomal dominant CPEO a new mutation (K319T) was identified in the nuclear Twinkle gene. Multiple mtDNA deletions were evident in the index case by both long-range and real-time PCR but not by conventional Southern blot analysis, highlighting the diagnostic difficulties associated with characterising patients with multiple mtDNA deletions. In six patients with myophosphorylase deficiency three novel mutations (Y84X, R93W, R269X) were identified thus expanding the genetic heterogenity in patients with McArdle disease. In 37 patients with muscle CPT II deficiency five novel mutations (M214T, Y479F, 413delAG-F448L, 515del4, and IVS3+5G->A) were identified. The latter mutation was the first splice junction mutation to be described in muscle CPT II deficiency. No clear genotype-phenotype correlation could be observed in the patients with CPT II deficiency. However, triggering of attacks by fasting was relevant only in patients with a truncating mutation. The P50H and Q413fs-F448L mutation were identified in 8% of the mutant alleles respectively indicating that these two mutations should be included in routine screening of patients with suspected CPT II deficiency additionally to the common S113L mutation.von Marcus Deschaue

McArdle Disease : clinical, biochemical, histological and molecular genetic analysis of 60 patients

A clinical, biochemical, histological and molecular genetic analysis of 60 McArdle patients (33 males and 27 females; mean age at diagnosis: 37 years) was performed. The objective of this study was to identify a possible genotype–phenotype correlation in McArdle disease. All patients complained of exercise-induced myalgia and fatigue; permanent weakness was present in 47% of the patients. Five percent of patients conveyed of masticatory muscle weakness. Age of onset was <15 years in 92% patients. Serum creatine kinase was elevated 5 to13-fold. Forearm ischemic test showed decreased lactate production but excessively increased ammonia upon exercise (n = 16). Muscle biopsies revealed highly reduced or missing myophosphorylase activity (n = 20) (mean: 0.17 ± 0.35 U/g tissue; normal: 12–61) and histologically, sub-sarcolemmal glycogen accumulation (n = 9). Molecular genetic analysis revealed the common p.Arg50Ter mutation in 68% of the patients. Other rather frequent mutations were p.Arg270Ter (allele frequency: 5%) followed by c.2262delA and p.Met1Val (allele frequencies: 3%). Twenty-four other rare mutations were also identified. No genotype–phenotype correlation was observed. The analysis highlights that testing of the p.Arg50Ter mutation could be performed first in molecular genetic testing of patients with exercise intolerance possibly due to McArdle disease. However, there is enormous mutation heterogeneity in McArdle disease thus sequencing of the myophosphorylase gene is needed in patients highly suspicious of McArdle diseasePublikationsfond ML

Combined Treatment With Pembrolizumab and Allogenic BK Virus-Specific T Cells in Progressive Multifocal Leukoencephalopathy A Case Report

Objective We report a combination of BK virus-specific T cells and pembrolizumab as a treatment option in progressive multifocal leukoencephalopathy (PML). Results A 57-year-old male patient diagnosed with PML presented a fast-progressing right hemiparesis, aphasia, and cognitive deficits. Brain MRI showed a severe leukoencephalopathy with diffusion restriction. The patient was treated with 10 doses of pembrolizumab (2 mg/kg body weight) in differing intervals and 2 partially human leukocyte antigen-matched allogenic BK virus-specific T cell transfusions after the fifth pembrolizumab treatment. Although pembrolizumab alone decreased the viral load but failed to control the virus, BK-specific T cell transfer further enhanced the decline of JC virus copies in the CSF. Moreover, the regression of leukoencephalopathy and disappearance of diffusion restriction in subsequent brain MRI were observed. The combined treatment resulted in a clinical stabilization with improvements of the cognitive and speech deficits. Discussion This case supports the hypothesis that pembrolizumab is more efficient in the presence of an appropriate number of functional antigen-specific T cells. Thus, the combined treatment of pembrolizumab and virus-specific T cells should be further evaluated as a treatment option for PML in future clinical trials

Uncovering genetic mimics in multiple sclerosis

Background : Multiple sclerosis (MS) shares clinical/radiological features with several monogenic diseases that can mimic MS. Objective : We aimed to determine if exome sequencing can identify monogenic diseases in patients diagnosed with MS according to the McDonald criteria thus uncovering them as being misdiagnosed. Methods : We performed whole exome sequencing in a cohort of 278 patients with MS, clinically or radiologically isolated syndrome without cerebrospinal fluid-specific oligoclonal bands (CSF-OCBs) (n = 228), a positive family history of MS (n = 44), or both (n = 6), thereby focusing on individuals potentially more likely to have underlying monogenic conditions mimicking MS. We prioritized 495 genes associated with monogenic diseases sharing features with MS. Results : A disease-causing variant in NOTCH3 was identified in one patient without CSF-OCBs, no spinal lesions, with non-response to immunotherapy, and a family history of dementia, thereby converting the diagnosis to cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Moreover, 18 patients (6.5% of total) carried variants of unclear significance. Conclusion : Monogenic diseases being misdiagnosed as MS seem rare in patients diagnosed with MS according to the McDonald criteria, even in CSF-OCB negative cases. The detected pathogenic NOTCH3 variant emphasizes CADASIL as a rare differential diagnosis and highlights the relevance of genetic testing in selected MS cases with atypical presentations

T2-Weighted Dixon Turbo Spin Echo for Accelerated Simultaneous Grading of Whole-Body Skeletal Muscle Fat Infiltration and Edema in Patients With Neuromuscular Diseases

Objective The assessment of fatty infiltration and edema in the musculature of patients with neuromuscular diseases (NMDs) typically requires the separate performance of T-1-weighted and fat-suppressed T-2-weighted sequences. T-2-weighted Dixon turbo spin echo (TSE) enables the generation of T-2-weighted fat- and water-separated images, which can be used to assess both pathologies simultaneously. The present study examines the diagnostic performance of T-2-weighted Dixon TSE compared with the standard sequences in 10 patients with NMDs and 10 healthy subjects. Methods Whole-body magnetic resonance imaging was performed including T-1-weighted Dixon fast field echo, T-2-weighted short-tau inversion recovery, and T-2-weighted Dixon TSE. Fatty infiltration and intramuscular edema were rated by 2 radiologists using visual semiquantitative rating scales. To assess intermethod and interrater agreement, weighted Cohen's coefficients were calculated. Results The ratings of fatty infiltration showed high intermethod and high interrater agreement (T-1-weighted Dixon fast field echo vs T-2-weighted Dixon TSE fat image). The evaluation of edematous changes showed high intermethod and good interrater agreement (T-2-weighted short-tau inversion recovery vs T-2-weighted Dixon TSE water image). Conclusions T-2-weighted Dixon TSE imaging is an alternative for accelerated simultaneous grading of whole-body skeletal muscle fat infiltration and edema in patients with NMDs

Clonal hematopoiesis as a pitfall in germline variant interpretation in the context of Mendelian disorders

Clonal hematopoiesis because of somatic mutations in hematopoietic stem/progenitor cells is an age-related phenomenon and commonly observed when sequencing blood DNA in elderly individuals. Several genes that are implicated in clonal hematopoiesis are also associated with Mendelian disorders when mutated in the germline, potentially leading to variant misinterpretation. We performed a literature search to identify genes associated with age-related clonal hematopoiesis followed by an OMIM query to identify the subset of genes in which germline variants are associated with Mendelian disorders. We retrospectively screened for diagnostic cases in which the presence of age-related clonal hematopoiesis confounded exome sequencing data interpretation. We found 58 genes in which somatic mutations are implicated in clonal hematopoiesis, while germline variants in the same genes are associated with Mendelian (mostly neurodevelopmental) disorders. Using five selected cases of individuals with suspected monogenic disorders, we illustrate how clonal hematopoiesis in either variant databases or exome sequencing datasets poses a pitfall, potentially leading to variant misclassification and erroneous conclusions regarding gene-disease associations

Brain Iron and Metabolic Abnormalities in C19orf12 Mutation Carriers: A 7.0 Tesla MRI Study in Mitochondrial Membrane Protein–Associated Neurodegeneration

Background Mitochondrial membrane protein‐associated neurodegeneration is an autosomal‐recessive disorder caused by C19orf12 mutations and characterized by iron deposits in the basal ganglia. Objectives The aim of this study was to quantify iron concentrations in deep gray matter structures using quantitative susceptibility mapping MRI and to characterize metabolic abnormalities in the pyramidal pathway using 1H MR spectroscopy in clinically manifesting membrane protein‐associated neurodegeneration patients and asymptomatic C19orf12 gene mutation heterozygous carriers. Methods We present data of 4 clinically affected membrane protein‐associated neurodegeneration patients (mean age: 21.0 ± 2.9 years) and 9 heterozygous gene mutation carriers (mean age: 50.4 ± 9.8 years), compared to age‐matched healthy controls. MRI assessments were performed on a 7.0 Tesla whole‐body system, consisting of whole‐brain gradient‐echo scans and short echo time, single‐volume MR spectroscopy in the white matter of the precentral/postcentral gyrus. Quantitative susceptibility mapping, a surrogate marker for iron concentration, was performed using a state‐of‐the‐art multiscale dipole inversion approach with focus on the globus pallidus, thalamus, putamen, caudate nucleus, and SN. Results and Conclusion In membrane protein‐associated neurodegeneration patients, magnetic susceptibilities were 2 to 3 times higher in the globus pallidus (P = 0.02) and SN (P = 0.02) compared to controls. In addition, significantly higher magnetic susceptibility was observed in the caudate nucleus (P = 0.02). Non‐manifesting heterozygous mutation carriers exhibited significantly increased magnetic susceptibility (relative to controls) in the putamen (P = 0.003) and caudate nucleus (P = 0.001), which may be an endophenotypic marker of genetic heterozygosity. MR spectroscopy revealed significantly increased levels of glutamate, taurine, and the combined concentration of glutamate and glutamine in membrane protein‐associated neurodegeneration, which may be a correlate of corticospinal pathway dysfunction frequently observed in membrane protein‐associated neurodegeneration patients

Unusual multisystemic involvement and a novel BAG3 mutation revealed by NGS screening in a large cohort of myofibrillar myopathies

Myofibrillar myopathies (MFM) are a group of phenotypically and genetically heterogeneous neuromuscular disorders, which are characterized by protein aggregations in muscle fibres and can be associated with multisystemic involvement.Methods We screened a large cohort of 38 index patients with MFM for mutations in the nine thus far known causative genes using Sanger and next generation sequencing (NGS). We studied the clinical and histopathological characteristics in 38 index patients and five additional relatives (n = 43) and particularly focused on the associated multisystemic symptoms.Results We identified 14 heterozygous mutations (diagnostic yield of 37%), among them the novel p.Pro209Gln mutation in the BAG3 gene, which was associated with onset in adulthood, a mild phenotype and an axonal sensorimotor polyneuropathy, in the absence of giant axons at the nerve biopsy. We revealed several novel clinical phenotypes and unusual multisystemic presentations with previously described mutations: hearing impairment with a FLNC mutation, dysphonia with a mutation in DES and the first patient with a FLNC mutation presenting respiratory insufficiency as the initial symptom. Moreover, we described for the first time respiratory insufficiency occurring in a patient with the p.Gly154Ser mutation in CRYAB. Interestingly, we detected a polyneuropathy in 28% of the MFM patients, including a BAG3 and a MYOT case, and hearing impairment in 13%, including one patient with a FLNC mutation and two with mutations in the DES gene. In four index patients with a mutation in one of the MFM genes, typical histological findings were only identified at the ultrastructural level (29%).Conclusions We conclude that extraskeletal symptoms frequently occur in MFM, particularly cardiac and respiratory involvement, polyneuropathy and/or deafness. BAG3 mutations should be considered even in cases with a mild phenotype or an adult onset. We identified a genetic defect in one of the known genes in less than half of the MFM patients, indicating that more causative genes are still to be found. Next generation sequencing techniques should be helpful in achieving this aim

Informal Caregiving in Amyotrophic Lateral Sclerosis (ALS): A High Caregiver Burden and Drastic Consequences on Caregivers’ Lives

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive autonomy loss and need for care. This does not only affect patients themselves, but also the patients’ informal caregivers (CGs) in their health, personal and professional lives. The big efforts of this multi-center study were not only to evaluate the caregivers’ burden and to identify its predictors, but it also should provide a specific understanding of the needs of ALS patients’ CGs and fill the gap of knowledge on their personal and work lives. Using standardized questionnaires, primary data from patients and their main informal CGs (n = 249) were collected. Patients’ functional status and disease severity were evaluated using the Barthel Index, the revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) and the King’s Stages for ALS. The caregivers’ burden was recorded by the Zarit Burden Interview (ZBI). Comorbid anxiety and depression of caregivers were assessed by the Hospital Anxiety and Depression Scale. Additionally, the EuroQol Five Dimension Five Level Scale evaluated their health-related quality of life. The caregivers’ burden was high (mean ZBI = 26/88, 0 = no burden, ≥24 = highly burdened) and correlated with patients’ functional status (rp = −0.555, p < 0.001, n = 242). It was influenced by the CGs’ own mental health issues due to caregiving (+11.36, 95% CI [6.84; 15.87], p < 0.001), patients’ wheelchair dependency (+9.30, 95% CI [5.94; 12.66], p < 0.001) and was interrelated with the CGs’ depression (rp = 0.627, p < 0.001, n = 234), anxiety (rp = 0.550, p < 0.001, n = 234), and poorer physical condition (rp = −0.362, p < 0.001, n = 237). Moreover, female CGs showed symptoms of anxiety more often, which also correlated with the patients’ impairment in daily routine (rs = −0.280, p < 0.001, n = 169). As increasing disease severity, along with decreasing autonomy, was the main predictor of caregiver burden and showed to create relevant (negative) implications on CGs’ lives, patient care and supportive therapies should address this issue. Moreover, in order to preserve the mental and physical health of the CGs, new concepts of care have to focus on both, on not only patients but also their CGs and gender-associated specific issues. As caregiving in ALS also significantly influences the socioeconomic status by restrictions in CGs’ work lives and income, and the main reported needs being lack of psychological support and a high bureaucracy, the situation of CGs needs more attention. Apart from their own multi-disciplinary medical and psychological care, more support in care and patient management issues is required