We aimed to investigate the epidemiology and natural history of FKRP-related limb-girdle muscular dystrophy R9 (LGMDR9) in Norway.We identified 153 genetically confirmed subjects making the overall prevalence 2.84/10 0,0 0 0, the highest reported figure worldwide.Of the 153 subjects, 134 (88 %) were homozygous for FKRP c.826C > A giving a carrier frequency for this variant of 1/101 in Norway.Clinical questionnaires and patient notes from 101 subjects, including 88 c.826C > A homozygotes, were reviewed, and 43/101 subjects examined clinically.Age of onset in c.826C > A homozygotes demonstrated a bimodal distribution.Female subjects showed an increased cumulative probability of wheelchair dependency and need for ventilatory support.Across the cohort, the need for ventilatory support preceded wheelchair dependency in one third of the cases, usually due to sleep apnea.In c.826C > A homozygotes, occurrence of cardiomyopathy correlated positively with male gender but not with age or disease stage.This study highlights novel gender differences in both loss of ambulation, need for ventilatory support and the development of cardiomyopathy.Our results confirm the need for vigilance in order to detect respiratory insufficiency and cardiac involvement, but indicate that these events affect males and females differently.

We describe five families from different regions in Norway with a late-onset autosomal-dominant hereditary polyneuropathy sharing a heterozygous variant in the SLC12A6 gene. Mutations in the same gene have previously been described in infants with autosomal-recessive hereditary motor and sensory neuropathy with corpus callosum agenesis and mental retardation (Andermann syndrome), and in a few case reports describing dominantly acting de novo mutations, most of them with onset in childhood. The phenotypes in our families demonstrated heterogeneity. Some of our patients only had subtle to moderate symptoms and some individuals even no complaints. None had CNS manifestations. Clinical and neurophysiological evaluations revealed a predominant sensory axonal polyneuropathy with slight to moderate motor components. In all 10 patients the identical SLC12A6 missense variant, NM_001365088.1 c.1655G>A p.(Gly552Asp), was identified. For functional characterization, the mutant potassium chloride cotransporter 3 was modelled in Xenopus oocytes. This revealed a significant reduction in potassium influx for the p.(Gly552Asp) substitution. Our findings further expand the spectrum of SLC12A6 disease, from biallelic hereditary motor and sensory neuropathy with corpus callosum agenesis and mental retardation and monoallelic early-onset hereditary motor and sensory neuropathy caused by de novo mutations, to late-onset autosomal-dominant axonal neuropathy with predominant sensory deficits.

We present a retrospective 21-year follow-up of two sisters with X-linked biallelic CAG expansions in the androgen receptor (AR) gene causing Kennedy disease. Two sisters inherited CAG expansions from their mother who was a carrier and their father who had Kennedy disease. Genetic testing revealed alleles comprising 43/45, and 43/43 CAG repeats in the younger and older sister, respectively. They were referred to a neurologist for further evaluation. Both reported similar symptoms with chronic backache, pain and cramps in upper- and lower extremities, and fasciculations in their faces and extremities. Neurological examination demonstrated postural hand tremor in both and EMG revealed chronic neurogenic changes. Reevaluation of the patients at ages 74 and 83 showed slight progression of clinical manifestations. As opposed to male patients, these two females showed minimal disease progression and have maintained normal level of function into old age.

The SRY-related HMG box gene 10 (SOX10), located on 22q13.1, encodes a member of the SOX family of transcription factors involved in the regulation of embryonic development and in the determination of cell fate and differentiation. SOX10 is one of the six causal genes for Waardenburg syndrome, which is a dominantly inherited auditory-pigmentary disorder characterized by sensorineural hearing impairment and abnormal pigmentation of the hair, skin and iris. Waardenburg syndrome is categorized into four subtypes based on clinical features (WS1-WS4). Here we present eight families (eleven patients) harboring pathogenic variants in SOX10. The patients displayed both allelic and clinical variability: bilateral profound hearing impairment (11/11), malformations of the semicircular canals (5/11), motor skill developmental delay (5/11), pigmentary defects (3/ 11) and Hirschsprung's disease (3/11) were some of the clinical manifestations observed. The patients demonstrate a spectrum of pathogenic SOX10 variants, of which six were novel (c.267del, c.299_300insA, c.335T >C, c.366_376del, c.1160_1179dup, and exon 3-4 deletion), and two were previously reported (c.336G>A and c.422T>C). Six of the variants occurred de novo whereas two were dominantly inherited. The pathogenic SOX10 variants presented here add novel information to the allelic variability of Waardenburg syndrome and illustrate the considerable clinical heterogeneity.

Aims Limb-girdle muscular dystrophy R9 (LGMDR9) is an autosomal recessive disorder caused by mutations in the fukutin-related protein gene (FKRP), encoding a glycosyltransferase involved in alpha-dystroglycan modification. Muscle atrophy, a significant feature of LGMDR9, occurs by a change in the normal balance between protein synthesis and protein degradation. The ubiquitin-proteasome system (UPS) and autophagy-lysosomal system play a key role in protein degradation in skeletal muscle cells, but their involvement in the pathology of LGMDR9 is still largely unknown. We have aimed at clarifying whether proteolysis through the UPS and the autophagy-lysosomal pathway is dysregulated in LGMDR9 patients. Methods Vastus lateralis biopsies from 8 normal controls and 12 LGMDR9 patients harbouring the c.826C>A/c.826C>A FKRP genotype were assessed for protein markers related to UPS, the autophagy-lysosomal system and endoplasmic reticulum (ER) stress/unfolded protein response (UPR), followed by ultrastructural analysis by transmission electron microscopy (TEM). Results Protein levels of E3 ubiquitin ligases Atrogin-1 and MuRF1 showed a pattern similar to normal controls. Elevation of the autophagy markers Atg7, LC3B-II, decreased level of p62 as well as downregulation of the negative autophagy regulator mTORC1, indicated an activation of autophagy in LGMDR9. Mitophagy markers Bnip3 and Parkin were decreased. TEM analysis demonstrated accumulation of autophagosome-like structures in LGMDR9 muscle. There was also an increase in the expression of ER stress/UPR markers PDI, peIF2 alpha and CHOP and a decrease in IRE1 alpha. However, GRP94, Bip and Calnexin remained unchanged. Conclusion Our findings indicate that autophagy and ER stress are induced in LGMDR9 muscle.

Neuromuscular disorders are both clinically and genetically a very heterogeneous group of disorders. Accordingly, the ability to interrogate a broad array of genetic alterations, by next generation sequencing, has provided an accurate molecular diagnosis for a larger group of patients. We will present the results of three years of panel based sequencing including 400 patients. We original used a neuromuscular associated gene panel containing 256 genes, which was eventually expanded to 328 genes. We used Illumina TruSight rapid capture technology to prepare Illumina TruSight one targeted sequencing libraries which were sequenced using an Illumina NextSeq 500 desktop sequencer. Data were analysed using Illumina BaseSpace BWA Enrichment Workflow. Variants were annotated and filtered with cartagenia bench lab NGS software applying different criteria. Variants were curated using cartagenia bench and alamut visual (Interactive Biosoftware). We investigated the diagnostic yield of panel-based sequencing of 400 patients who had already received standard of care genetic evaluation/diagnostic testing. We identified a molecular cause in approximately 25% in this cohort and in 10% we found variants of uncertain clinical significance. The detection rate was restricted due to the inclusion of patients presenting with atypical phenotypes. Technically, the choice of gene panel itself, the limited coverage of exon 1 in most genes and the inability to detect copy number variation, equally limited the finding percentage. In conclusion, the use of next generation sequencing targeted gene panels is very helpful in the diagnosis of patients with neuromuscular disorders. It improves our understanding of the relative prevalence of the different neuromuscular disorders, their respective genetic etiology, their inheritance modes and mechanisms, it will allow for improved treatment, management, and enrolment of molecularly diagnosed individuals in clinical trials.

Limb girdle muscular dystrophy type 2I (LGMD2I) is a progressive disorder caused by mutations in the FuKutin-Related Protein gene (FKRP). LGMD2I displays clinical heterogeneity with onset of severe symptoms in early childhood to mild calf and thigh hypertrophy in the second or third decade. Patients homozygous for the common FKRP mutation c.826C>A (p.Leu276Ile) show phenotypes within the milder end of the clinical spectrum. However, this group also manifests substantial clinical variability. FKRP deficiency causes hypoglycosylation of α-dystroglycan; a component of the dystrophin associated glycoprotein complex. α-Dystroglycan hypoglycosylation is associated with loss of interaction with laminin α2, which in turn results in laminin α2 depletion. Here, we have attempted to clarify if the clinical variability seen in patients homozygous for c.826C>A is related to alterations in muscle fibre pathology, α-DG glycosylation levels, levels of laminin α2 as well as the capacity of α-DG to bind to laminin. We have assessed vastus lateralis muscle biopsies from 25 LGMD2I patients harbouring the c.826C>A/c.826C>A genotype by histological examination, immunohistochemistry and immunoblotting. No clear correlation was found between clinical severity, as determined by self-reported walking function, and the above features, suggesting that more complex molecular processes are contributing to the progression of disease.

BackgroundUsher syndrome (USH) is a genetically heterogeneous deafness-blindness syndrome, divided into three clinical subtypes: USH1, USH2 and USH3.MethodsMutations in 21 out of 26 investigated Danish unrelated individuals with USH were identified, using a combination of molecular diagnostic methods.ResultsBefore Next Generation Sequencing (NGS) became available mutations in nine individuals (1 USH1, 7 USH2, 1 USH3) were identified by Sanger sequencing of USH1C, USH2A or CLRN1 or by Arrayed Primer EXtension (APEX) method. Mutations in 12 individuals (7 USH1, 5 USH2) were found by targeted NGS of ten known USH genes. Five novel pathogenic variants were identified. We combined our data with previously published, and obtained an overview of the USH mutation spectrum in Denmark, including 100 unrelated individuals; 32 with USH1, 67 with USH2, and 1 with USH3. Macular edema was observed in 44 of 117 individuals. Olfactory function was tested in 12 individuals and found to be within normal range in all.ConclusionMutations that lead to USH1 were predominantly identified in MYO7A (75%), whereas all mutations in USH2 cases were identified in USH2A. The MYO7A mutation c.93C> A, p.(Cys31*) accounted for 33% of all USH1 mutations and the USH2A c.2299delG, p.(Glu767Serfs*21) variant accounted for 45% of all USH2 mutations in the Danish cohort.

α-Mannosidosis is an autosomal recessive lysosomal storage disorder caused by mutations in the MAN2B1 gene, encoding lysosomal α-mannosidase. The disorder is characterized by a range of clinical phenotypes of which the major manifestations are mental impairment, hearing impairment, skeletal changes, and immunodeficiency. Here, we report an α-mannosidosis mutation database, amamutdb.no, which has been constructed as a publicly accessible online resource for recording and analyzing MAN2B1 variants (http://amamutdb.no). Our aim has been to offer structured and relational information on MAN2B1 mutations and genotypes along with associated clinical phenotypes. Classifying missense mutations, as pathogenic or benign, is a challenge. Therefore, they have been given special attention as we have compiled all available data that relate to their biochemical, functional, and structural properties. The α-mannosidosis mutation database is comprehensive and relational in the sense that information can be retrieved and compiled across datasets; hence, it will facilitate diagnostics and increase our understanding of the clinical and molecular aspects of α-mannosidosis. We believe that the amamutdb.no structure and architecture will be applicable for the development of databases for any monogenic disorder.

BACKGROUND:Alpha-mannosidosis is caused by mutations in MAN2B1, leading to loss of lysosomal alpha-mannosidase activity. Symptoms include intellectual disabilities, hearing impairment, motor function disturbances, facial coarsening and musculoskeletal abnormalities.METHODS:To study the genotype-phenotype relationship for alpha-mannosidosis 66 patients were included. Based on the predicted effect of the mutations and the subcellular localisation of mutant MAN2B1 in cultured cells, the patients were divided into three subgroups. Clinical and biochemical data were collected. Correlation analyses between each of the three subgroups of genotype/subcellular localisation and the clinical and biochemical data were done to investigate the potential relationship between genotype and phenotype in alpha-mannosidosis. Statistical analyses were performed using the SPSS software. Analyses of covariance were performed to describe the genotype-phenotype correlations. The phenotype parameters were modelled by the mutation group and age as a covariate. P values of <0.05 were considered as statistically significant.RESULTS:Complete MAN2B1 genotypes were established for all patients. We found significantly higher scores in the Leiter-R test, lower concentrations of CSF-oligosaccharides, higher point scores in the Bruininks-Oseretsky Test of Motor Proficiency subtests (BOT-2); Upper limb coordination and Balance, and a higher FVC% in patients in subgroup 3, harbouring at least one variant that allows localisation of the mutant MAN2B1 protein to the lysosomes compared to subgrou 2 and/or subgroup 1 with no lysosomal localization of the mutant MAN2B1 protein.CONCLUSION:Our results indicate a correlation between the MAN2B1 genotypes and the cognitive function, upper limb coordination, balance, FVC% and the storage of oligosaccharides in CSF. This correlation depends on the subcellular localisation of the mutant MAN2B1 protein.

Background: Cowpox virus (CPXV), a rodent-borne Orthopoxvirus (OPV) that is indigenous to Eurasia can infect humans, cattle, felidae and other animals. Molecular characterization of CPXVs isolated from different geographic locations is important for the understanding of their biology, geographic distribution, classification and evolution. Our aim was to characterize CPXVs isolated from Fennoscandia on the basis of A-type inclusion (ATI) phenotype, restriction fragment length polymorphism (RFLP) profiles of atip gene fragment amplicon, and phylogenetic tree topology in conjunction with the patristic and genetic distances based on full length DNA sequence of the atip and p4c genes.Methods: ATI phenotypes were determined by transmission electron microcopy and RFLP profiles were obtained by restriction enzyme digestion of the atip gene fragment PCR product. A 6.2 kbp region spanning the entire atip and p4c genes of Fennoscandian CPXV isolates was amplified and sequenced. The phylogenetic affinity of Fennoscandian CPXV isolates to OPVs isolated from other geographic regions was determined on the basis of the atip and p4c genes.Results: Fennoscandian CPXV isolates encoded full length atip and p4c genes. They produce wild type V+ ATI except for CPXV-No-H2. CPXVs were resolved into six and seven species clusters based on the phylogeny of the atip and p4c genes respectively. The CPXVs isolated from Fennoscandia were grouped into three distinct clusters that corresponded to isolates from Norway, Sweden and Finland.Conclusion: CPXV is a polyphyletic assemblage of six or seven distinct clusters and the current classification in which CPXVs are united as one single species should be re-considered. Our results are of significance to the classification and evolution of OPVs.

To investigate if there is a correlation between clinical expression (duration of disease, age, walking function) and morphological alterations in affected muscle (structural changes, immunohistochemical and the level of alpha-DG glycosylation) among 25 patients with Limb Girdle Muscular Dystrophy type 2I (LGMD2I) all with the common C.826C>A mutation ). Muscle biopsies were obtained from 25 patients. Quantitative evaluation of morphological alterations in muscle sections, and immunohistochemistry (IHC) with antibodies directed against the alpha-dystroglycan glycan epitope, was performed by light microscopy. A semi-quantitative assessment of changes was recorded, point-graded and summarized as morphological sum-score for each biopsy.. Western blot (WB) analysis on muscle biopsy homogenates were carried with antibodies directed towards the core alpha-DG as well as the alpha-DG-glycan epitope. Laminin overlay was included. Muscle biopsies from 25 patients with LGMD2I presented large variation in morphological features. All biopsies presented reduced molecular weight of alpha-DG as detected with alpha-DG core antibody on WB analysis. Immunohistochemistry and WB analysis directed towards the alpha-DG-glycan epitope, as well as laminin overlay, demonstrated large variation in signal intensity among the LGMD2I patients. Results from IHC and WB/laminin overlay correlated poorly. There was no obvious correlation between ages at onset or duration of disease at biopsy versus the level of alpha-DG glycosylation. Likewise, there was no apparent association between severities of disease and the sum-scores of structural changes in the muscle biopsies. The clinical and morphological variability seen among LGMD2I patients with identical FKRP genotype must therefore be explained by other genetic or environmental mechanisms. To investigate if there is a correlation between clinical expression (duration of disease, age, walking function) and morphological alterations in affected muscle (structural changes, immunohistochemical and the level of alpha-DG glycosylation) among 25 patients with Limb Girdle Muscular Dystrophy type 2I (LGMD2I) all with the common C.826C>A mutation ). Muscle biopsies were obtained from 25 patients. Quantitative evaluation of morphological alterations in muscle sections, and immunohistochemistry (IHC) with antibodies directed against the alpha-dystroglycan glycan epitope, was performed by light microscopy. A semi-quantitative assessment of changes was recorded, point-graded and summarized as morphological sum-score for each biopsy.. Western blot (WB) analysis on muscle biopsy homogenates were carried with antibodies directed towards the core alpha-DG as well as the alpha-DG-glycan epitope. Laminin overlay was included. Muscle biopsies from 25 patients with LGMD2I presented large variation in morphological features. All biopsies presented reduced molecular weight of alpha-DG as detected with alpha-DG core antibody on WB analysis. Immunohistochemistry and WB analysis directed towards the alpha-DG-glycan epitope, as well as laminin overlay, demonstrated large variation in signal intensity among the LGMD2I patients. Results from IHC and WB/laminin overlay correlated poorly. There was no obvious correlation between ages at onset or duration of disease at biopsy versus the level of alpha-DG glycosylation. Likewise, there was no apparent association between severities of disease and the sum-scores of structural changes in the muscle biopsies. The clinical and morphological variability seen among LGMD2I patients with identical FKRP genotype must therefore be explained by other genetic or environmental mechanisms.