Our aim was to present the experience of systematic, routine use of next generation sequencing (NGS) in clinical diagnostics of myopathies.
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.
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.
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.
We aimed to detect the causative gene in five unrelated families with recessive inheritance pattern neurological disorders involving the central nervous system, and the potential function of the
NEMF
...gene in the central nervous system. Exome sequencing (ES) was applied to all families and linkage analysis was performed on family 1. A minigene assay was used to validate the splicing effect of the relevant discovered variants. Immunofluorescence (IF) experiment was performed to investigate the role of the causative gene in neuron development. The large consanguineous family confirms the phenotype-causative relationship with homozygous frameshift variant (NM_004713.6:c.2618del) as revealed by ES. Linkage analysis of the family showed a significant single-point LOD of 4.5 locus. Through collaboration in GeneMatcher, four additional unrelated families’ likely pathogenic NEMF variants for a spectrum of central neurological disorders, two homozygous splice-site variants (NM_004713.6:c.574+1G>T and NM_004713.6:c.807-2A>C) and a homozygous frameshift variant (NM_004713.6: c.1234_1235insC) were subsequently identified and segregated with all affected individuals. We further revealed that knockdown (KD) of
Nemf
leads to impairment of axonal outgrowth and synapse development in cultured mouse primary cortical neurons. Our study demonstrates that disease-causing biallelic
NEMF
variants result in central nervous system impairment and other variable features.
NEMF
is an important player in mammalian neuron development.
The challenging anatomic predispositions in adult patients with spinal muscular atrophy (SMA) preclude the conventional lumbar punctures. Consequently, an introduction of alternative method for ...intrathecal delivery of nusinersen is required. Cone-beam CT (CBCT) allows volumetric display of the area of interest, pre-procedural planning and real time needle guidance which results in accurate anatomic navigation. The aim of the study was to evaluate technical success, safety, and feasibility of CBCT lumbar intrathecal delivery of nusinersen in the adult SMA patients with challenging anatomical access.
Thirty-eight adult SMA patients were treated in our institution. Patients with challenging access were selected by multidisciplinary board for image guided administration of nusinersen either due to implantation of the posterior fusion instrumentation, severe scoliosis defined as Cobb's angle > 40º or body mass index over 35. Technical success, radiation exposure and occurrence of adverse events were assessed.
Twenty patients were selected, and 108 CBCT-guided procedures were performed. Each patient underwent at least 4 administrations. Transforaminal approach was performed in 82% of patients. The technical success was 100%, with primary success of 93.5%. The median radiation effective dose of the administrations was 5 mSv, the mean value equalled 10 mSv. Only mild adverse events were reported in the study.
CBCT-guided lumbar intrathecal administrations of nusinersen in an adult SMA population with challenging access was feasible and safe image guided method.
ABSTRACT
Introduction: The aim of this study was to create reference values for jitter measured with concentric needle electrodes. Methods: Operators worldwide contributed recordings from orbicularis ...oculi (OO), frontalis (FR), and extensor digitorum (ED) muscles in healthy controls. Criteria for acceptable signal quality were agreed upon in advance. Fifteen or 20 recordings of acceptable quality from each muscle were required for voluntary and electrical stimulation recordings, respectively. Results: Recordings from 59 to 92 subjects were obtained for each muscle and activation type. Outlier limits for mean consecutive difference and individual jitter data for voluntary activation were: OO, 31 and 45 µs; FR, 28 and 38 µs; ED, 30 and 43 µs; and for electrical stimulation they were: OO, 27 and 36 µs; FR, 21 and 28 µs; ED, 24 and 35 µs. Conclusion: Reference jitter values from concentric needle electrode recordings were developed from signals of defined quality while seeking to avoid creating supernormal values. Muscle Nerve 53: 351–362, 2016
Hereditary sensory neuropathy type I (HSN I) is an axonal form of autosomal-dominant hereditary motor and sensory neuropathy distinguished by prominent sensory loss that leads to painless injuries. ...Unrecognized, these can result in delayed wound healing and osteomyelitis, necessitating distal amputations. To elucidate the genetic basis of an HSN I subtype in a family in which mutations in the few known HSN I genes had been excluded, we employed massive parallel exon sequencing of the 14.3 Mb disease interval on chromosome 14q. We detected a missense mutation (c.1065C>A, p.Asn355Lys) in atlastin-1 (ATL1), a gene that is known to be mutated in early-onset hereditary spastic paraplegia SPG3A and that encodes the large dynamin-related GTPase atlastin-1. The mutant protein exhibited reduced GTPase activity and prominently disrupted ER network morphology when expressed in COS7 cells, strongly supporting pathogenicity. An expanded screen in 115 additional HSN I patients identified two further dominant ATL1 mutations (c.196G>C p.Glu66Gln and c.976 delG p.Val326TrpfsX8). This study highlights an unexpected major role for atlastin-1 in the function of sensory neurons and identifies HSN I and SPG3A as allelic disorders.
Facioscapulohumeral muscular dystrophy (FSHD) is the third most common hereditary muscular dystrophy, caused by the contraction of the D4Z4 repeats on the permissive 4qA haplotype on chromosome 4, ...resulting in the faulty expression of the
gene. Traditional diagnostics are based on Southern blotting, a time- and effort-intensive method that can be affected by single nucleotide variants (SNV) and copy number variants (CNV), as well as by the similarity of the D4Z4 repeats located on chromosome 10. We aimed to evaluate optical genome mapping (OGM) as an alternative molecular diagnostic method for the detection of FSHD. We first performed optical genome mapping with EnFocus™ FSHD analysis using DLE-1 labeling and the Saphyr instrument in patients with inconclusive diagnostic Southern blot results, negative FSHD2 results, and clinically evident FSHD. Second, we performed OGM in parallel with the classical Southern blot analysis for our prospectively collected new FSHD cases. Finally, panel exome sequencing was performed to confirm the presence of FSHD2. In two patients with diagnostically inconclusive Southern blot results, OGM was able to identify shortened D4Z4 repeats on the permissive 4qA alleles, consistent with the clinical presentation. The results of the prospectively collected patients tested in parallel using Southern blotting and OGM showed full concordance, indicating that OGM is a useful alternative to the classical Southern blotting method for detecting FSHD1. In a patient showing clinical FSHD but no shortened D4Z4 repeats in the 4qA allele using OGM or Southern blotting, a likely pathogenic variant in SMCHD1 was detected using exome sequencing, confirming FSHD2. OGM and panel exome sequencing can be used consecutively to detect FSHD2.
Genetic studies have managed to explain many cases of familial amyotrophic lateral sclerosis (ALS) through mutations in several genes. However, the cause of a majority of sporadic cases remains ...unknown. Recently, epigenetics, especially miRNA studies, show some promising aspects. We aimed to evaluate the differential expression of 10 miRNAs, including miR-9, miR-338, miR-638, miR-663a, miR-124a, miR-143, miR-451a, miR-132, miR-206, and let-7b, for which some connection to ALS was shown previously in ALS culture cells, animal models or patients, and in three miRNA host genes, including
(miR-9),
(miR-338), and
(miR-638), in leukocyte samples of 84 patients with sporadic ALS. We observed significant aberrant dysregulation across our patient cohort for miR-124a, miR-206, miR-9, let-7b, and miR-638. Since we did not use neurological controls we cannot rule out that the revealed differences in expression of investigated miRNAs are specific for ALS. Nevertheless, the group of these five miRNAs is worth of additional research in leukocytes of larger cohorts from different populations in order to verify their potential association to ALS disease. We also detected a significant up-regulation of the
gene and down-regulation of the
gene, and thus, for the first time, we connected these with sporadic ALS cases. These findings open up new research toward miRNAs as diagnostic biomarkers and epigenetic processes involved in ALS. The detected significant deregulation of
and
in sporadic ALS also represents an interesting finding. The
gene was previously found to be mutated in Charcot-Marie-Tooth neuropathy-type CMT2M and centronuclear myopathy (CNM). In addition, as recent studies connected
and frontotemporal dementia (FTD) and
and hereditary spastic paraplegia (HSP), these two genes together with our results genetically connect, at least in part, five diseases, including FTD, HSP, Charcot-Marie-Tooth (type CMT2M), CNM, and ALS, thus opening future research toward a better understanding of the cell biology involved in these partly overlapping pathologies.
Ataxia, causing imbalance, dizziness and falls, is a leading cause of neurological disability. We have recently identified a biallelic intronic AAGGG repeat expansion in replication factor complex ...subunit 1 (RFC1) as the cause of cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and a major cause of late onset ataxia. Here we describe the full spectrum of the disease phenotype in our first 100 genetically confirmed carriers of biallelic repeat expansions in RFC1 and identify the sensory neuropathy as a common feature in all cases to date. All patients were Caucasian and half were sporadic. Patients typically reported progressive unsteadiness starting in the sixth decade. A dry spasmodic cough was also frequently associated and often preceded by decades the onset of walking difficulty. Sensory symptoms, oscillopsia, dysautonomia and dysarthria were also variably associated. The disease seems to follow a pattern of spatial progression from the early involvement of sensory neurons, to the later appearance of vestibular and cerebellar dysfunction. Half of the patients needed walking aids after 10 years of disease duration and a quarter were wheelchair dependent after 15 years. Overall, two-thirds of cases had full CANVAS. Sensory neuropathy was the only manifestation in 15 patients. Sixteen patients additionally showed cerebellar involvement, and six showed vestibular involvement. The disease is very likely to be underdiagnosed. Repeat expansion in RFC1 should be considered in all cases of sensory ataxic neuropathy, particularly, but not only, if cerebellar dysfunction, vestibular involvement and cough coexist.
Abstract Amyotrophic lateral sclerosis (ALS) is a complex fatal neurodegenerative disease characterized by progressive degeneration and loss of upper motor neurons in the cerebral cortex and lower ...motor neurons in brainstem and spinal cord. We established the frequencies of mutations in 4 major ALS-associated genes, SOD1 , TARDBP , FUS , and C9ORF72 in a representative cohort of 85 Slovenian patients with sporadic form of ALS. Pathogenic massive hexanucleotide repeat expansion mutation in C9ORF72 was detected in 5.9% of patients and was the most common cause of the disease. In the remaining 3 genes, we identified 4 changes in 3 patients, p.Val14Met in SOD1 , silent mutation p.Arg522Arg in FUS , and p.Gly93Cys in SOD1 together with a novel synonymous variant c.990A>G (p.Leu330Leu) in TARDBP gene, respectively. This study represents the first genetic screening of major causative genes for ALS in a cohort of sporadic ALS patients from Slovenia and is according to our knowledge the first such study in Slavic population. Overall, we genetically characterized 8.2% sporadic ALS patients.
The aim of this study was to evaluate the quantitative motor unit potential (MUP) analysis in the diagnosis of myopathy and neuropathy of the diaphragm.
Diaphragm template-operated quantitative EMG ...were performed in 30 patients with myotonic dystrophy type 1 (DM1), 17 with myotonic dystrophy type 2 (DM2) and 40 with amyotrophic lateral sclerosis (ALS).
Low MUP amplitude precluded MUP analysis in 21% of DM1 patients. Only a single DM1 patient had EMG findings consistent with myopathy. In this patient, and another 4 DM1 and 3 DM2 patients, findings were consistent with neuropathy. Neuropathic MUP changes were found in 92% of ALS patients, but due to motor neuron cell loss in only 60% MUPs analyses could be done. Phrenic nerve conduction studies correlated with respiratory function tests, whereas MUP parameters did not.
Quantitative MUP analysis was not able to adequately sample diaphragm MUPs in patients with chronic myopathy or motor neuronopathy.
L’objectif de cette étude était d’évaluer l’intérêt de l’analyse quantifiée automatisée des potentiels d’unité motrice (PUM) dans le diagnostic d’anomalies myopathiques ou neuropathiques au niveau du diaphragme.
Des enregistrements EMG diaphragmatiques ont été réalisés chez 30 patients atteints de dystrophie myotonique de type 1 (DM1), 17 avec une dystrophie myotonique de type 2 (DM2) et 40 avec une sclérose latérale amyotrophique (SLA).
La faible amplitude des PUM a empêché l’analyse quantifiée dans 21 % des patients DM1. Seul un seul patient DM1 avait des résultats EMG compatibles avec une myopathie. Chez ce patient, ainsi que chez 4 autres patients DM1 et 3 patients DM2, les résultats étaient en revanche compatibles avec une neuropathie. Des anomalies neuropathiques des PUM ont été observées dans 92 % des patients atteints de SLA, mais en raison de la perte neuronale motrice, l’analyse des PUM n’a pu être réalisée que dans 60 % des cas. Les paramètres de conduction du nerf phrénique étaient corrélés avec les tests de la fonction respiratoire, alors que les paramètres des PUM ne l’étaient pas.
L’analyse quantifiée automatique des PUM n’a pas permis d’échantillonner de manière adéquate les PUM au niveau du diaphragme chez des patients présentant une myopathie chronique ou une neuronopathie motrice.
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