Sevim Erdem-Ozdamar

A novel desmin mutation leading to autosomal recessive limb-girdle muscular dystrophy: distinct histopathological outcomes compared with desminopathies

Background Autosomal recessive limb girdle muscular dystrophy (LGMD2) is a heterogeneous group of myopathies characterised by progressive muscle weakness involving proximal muscles of the shoulder and pelvic girdles including at least 17 different genetic entities. Additional loci have yet to be identified as there are families which are unlinked to any of the known loci. Here we have investigated a consanguineous family with LGMD2 with two affected individuals in order to identify the causative gene defect. Methods and results We performed genome wide homozygosity mapping and mapped the LGMD2 phenotype to chromosome 2q35–q36.3. DNA sequence analysis of the highly relevant candidate gene DES revealed a homozygous splice site mutation c.1289-2A>G in the two affected family members. Immunofluorescent staining and western blot analysis showed that the expression and the cytoskeletal network formation of mutant desmin were well preserved in skeletal muscle fibres. Unlike autosomal dominant desminopathies, ultrastructural alterations such as disruption of myofibrillar organisation, formation of myofibrillar degradation products and dislocation/aggregation of membranous organelles were not present. This novel splice site mutation results in addition of 16 amino acids within the tail domain of desmin, which has been suggested to interact with lamin B protein. We also detected a specific disruption of desmin-lamin B interaction in the skeletal muscle of the patient by confocal laser scanning microscopy. Conclusions Our study reveals that autosomal recessive mutations in DES cause LGMD2 phenotype without features of myofibrillar myopathy.

Remission with fingolimod in a case of demyelinating polyneuropathy

D. Facial onset sensory and motor neuronopathy (FOSMN syndrome): a novel syndrome in neurology. Brain 2006;129:3384–3390. 2. Isoardo G, Troni W. Sporadic bulbospinal muscle atrophy with facialonset sensory neuropathy. Muscle Nerve 2008;37:659–662. 3. Hokonohara T, Shigeto H, Kawano Y, Ohyagi Y, Uehara M, Kira J. Facial onset sensory and motor neuronopathy (FOSMN) syndrome responding to immunotherapies. J Neurol Sci 2008;275:157–158. 4. Fluchere F, Verschueren A, Cintas P, Franques J, Serratrice J, Weiller PJ, et al. Clinical features and follow-up of four new cases of facial-onset sensory and motor neuronopathy. Muscle Nerve 2011;43:136–140. 5. Vucic S, Stein TD, Hedley-Whyte ET, Reddel SR, Tisch S, Kotschet K, Cros D, et al. FOSMN syndrome: novel insight into disease pathophysiology. Neurology 2012;79:73–79. 6. Dobrev D, Barohn RJ, Anderson NE, Kilfoyle D, Khan S, McVey AL, et al. Facial onset sensorimotor neuronopathy syndrome: a case series. J Clin Neuromuscul Dis 2012;14:7–10. 7. Barca E, Russo M, Mazzeo A, Terranova C, Toscano A, Girlanda P. Facial onset sensory motor neuronopathy: not always a slowly progressive disorder. J Neurol 2013;260:1415–1416. 8. Knopp M, Vaghela NN, Shanmugam SV, Rajabally YA. Facial onset sensory motor neuronopathy: an immunoglobulin-responsive case. J Clin Neuromuscul Dis 2013;14:176–179. 9. Sonoda K, Sasaki K, Tateishi T, Yamasaki R, Hayashi S, Sakae N, et al. TAR DNA-binding protein 43 pathology in a case clinically diagnosed with facial-onset sensory and motor neuronopathy syndrome: an autopsied case report and a review of the literature. J Neurol Sci 2013;332:148–153. 10. Ziso B, Williams T, Walters J, Jaiser S, Wieshmann U, Jacob A. Fosmn: facial onset sensory motor neuronopathy. A “benign” differential diagnosis to bulbar onset motor neuron disease. The first cohort from the UK. J Neurol Neurosurg Psychiatry 2013;84:e2. 11. Dalla Bella E, Rigamonti A, Mantero V, Morbin M, Saccucci S, Gellera C, et al. Heterozygous D90A-SOD1 mutation in a patient with facial onset sensory motor neuronopathy (FOSMN) syndrome: a bridge to amyotrophic lateral sclerosis. J Neurol Neurosurg Psychiatry 2014 doi:10.1136/jnnp-2013–307416. [Epub ahead of print].

Three Turkish families with different transthyretin mutations

Transthyretin (TTR)-related hereditary amyloidosis, also called familial amyloid polyneuropathy (FAP), is a rare autosomal dominant systemic disorder that presents with progressive axonal sensory, autonomic and/or motor neuropathies. The present report describes three families with three different TTR mutations who were followed from 1995 to 2014. Only one of these families expressed the Val30Met mutation, which is the most common mutation in endemic regions; all members of this family had late disease onset but varied severities and clinical presentations of the disease. The second family expressed the Thr49Ser mutation, which has not been well documented previously. Our limited experience obtained from these patients indicates that this mutation presents with autonomic neuropathy but a greater degree of cardiac involvement, especially fatal heart failure. The third mutation, Glu54Lys, has been identified as a cause of severe familial amyloid polyneuropathy; the family members with this mutation exhibited severe motor and autonomic neuropathy, early vitreous opacity, and fatal heart failure. Three of the patients with the Val30Met mutation were treated with tafamidis for longer than one year and cessation of the polyneuropathy resulted. However, a short trial of tafamidis in two patients with the Glu54Lys mutation, who showed severe systemic and neurological involvement, did not gain any clinical benefits.

Overexpression of amyloid beta precursor protein enhances expression and secretion of ST6Gal1 in C2C12 myogenic cell line

The Abeta (amyloid‐beta) peptide is derived from the sequential cleavage of AbetaPP (amyloid‐beta precursor protein) by two enzymes, the β‐ and γ‐secretases. The major β‐secretase, identified as the novel transmembrane aspartic protease BACE1 (beta site APP‐cleaving enzyme 1), mediates the primary amyloidogenic cleavage of AbetaPP and initiates the production of Abeta. It has been implicated in the proteolytic processing of another substrate, namely ST6Gal1 (β galactoside α2,6‐sialyltransferase 1), which is the major α2,6‐sialyltransferase responsible for the broad synthesis of glycoproteins and glycolipids. The present study investigated the effect of overexpression of AbetaPP on expression and secretion of ST6Gal1 in skeletal muscle cells by inducing overexpression of wild‐type full‐length 751‐AbetaPP in the mouse myogenic cell line C2C12. Expression and secretion of the ST6Gal1 enzyme were analysed by Western blot and/or immunofluorescence staining. The results of our study demonstrated that AbetaPP overexpression in C2C12 cells increased the expression and the secretion of ST6Gal1 enzyme in vitro.

Transcript levels of plastin 3 and neuritin 1 modifier genes in spinal muscular atrophy siblings

In single gene disorders, patients with the same genotype may have variations in severity. One of the main factors affecting disease severity is modifier genes. Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by degeneration of alpha motor neurons. Plastin 3 (PLS3) is a phenotypic modifier of SMA, and neuritin 1 (NRN1) has also been suggested as a possible modifier gene. The aim of the present study was therefore to analyze PLS3 and NRN1 expression in SMA siblings in four families.

Myophosphorylase (PYGM) mutations determined by next generation sequencing in a cohort from turkey with McArdle disease

This study aimed to identify PYGM mutations in patients with McArdle disease from Turkey by next generation sequencing (NGS). Genomic DNA was extracted from the blood of the McArdle patients (n = 67) and unrelated healthy volunteers (n = 53). The PYGM gene was sequenced with NGS and the observed mutations were validated by direct Sanger sequencing. A diagnostic algorithm was developed for patients with suspected McArdle disease. A total of 16 deleterious PYGM mutations were identified, of which 5 were novel, including 1 splice-site donor, 1 frame-shift, and 3 non-synonymous variants. The p.Met1Val (27-patients/11-families) was the most common PYGM mutation, followed by p.Arg576* (6/4), c.1827+7A>G (5/4), c.772+2_3delTG (5/3), p.Phe710del (4/2), p.Lys754Asnfs (2/1), and p.Arg50* (1/1). A molecular diagnostic flowchart is proposed for the McArdle patients in Turkey, covering the 6 most common PYGM mutations found in Turkey as well as the most common mutation in Europe. The diagnostic algorithm may alleviate the need for muscle biopsies in 77.6% of future patients. A prevalence of any of the mutations to a geographical region in Turkey was not identified. Furthermore, the NGS approach to sequence the entire PYGM gene was successful in detecting a common missense mutation and discovering novel mutations in this population study.

Spinal muscular atrophy type III: Molecular genetic characterization of Turkish patients.

Spinal Muscular Atrophy (SMA) is a neurodegenerative disease with autosomal recessive inheritance. Homozygous loss of exon 7 of the Survival of motor neuron 1 (SMN1) gene is the main cause of SMA. Although progressive muscle weakness and atrophy are common symptoms, disease severity varies from severe to mild. Type III is one of the milder and less frequent forms of SMA. In this study, we report molecular genetic characteristics of 24 Turkish type III SMA patients. Homozygous loss of SMN1 exon 7 and 8 was analysed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and multiplex ligation dependent probe amplification (MLPA). SMN2, homologue of SMN1, and Neuronal apoptosis inhibitory protein (NAIP) genes were also evaluated considering their influence on disease severity. We determined that male patients who were born in consanguineous families were predominant in our cohort and these patients mostly carry the homozygous loss of SMN1 exon 7 and 8 and four copies of SMN2 gene without NAIP deletions.

Recent therapeutic developments in spinal muscular atrophy

Proximal spinal muscular atrophy (SMA) is an inherited neurodegenerative disease with a heterogeneous clinical phenotype. Although there is no cure for SMA, several strategies are currently being developed. In this review, we summarize the ongoing clinical trials and molecular mechanisms of successful approaches to SMA treatment.

Establishment of primary myoblast cell cultures from cryopreserved skeletal muscle biopsies to serve as a tool in related research & development studies

BACKGROUND Primary myoblast cell cultures display the phenotypic characteristics and genetic defects of the donor tissue and represent an in vitro model system reflecting the disease pathology. They have been generated only from freshly harvested tissue biopsies. Here, we describe a novel technique to establish myoblast cell cultures from cryopreserved skeletal muscle biopsy tissues that are useful for diagnostic and research purposes. METHODS AND RESULTS This protocol was performed on seven gradually frozen muscle biopsy specimens from various neuromuscular disorders that were stored in dimethylsulfoxide (DMSO)-supplemented freezing media at -80 °C for up to one year. After storage for varying periods of time, primary myoblast cultures were successfully established from all cryopreserved biopsy tissues without any chromosomal abnormality. Desmin immunoreactivity confirmed that the cell cultures contained >90% pure myoblasts. The myoblasts differentiated into multinucleated myotubes successfully. Furthermore, there were no statistically significant differences in cell viability, metabolic activity, population doubling time, and myocyte enhancer factor 2 (MEF2C) expression between cell cultures established from freshly harvested and one year-stored frozen tissue specimens. CONCLUSIONS This protocol opens up new horizons for basic research and the pre-clinical studies of novel therapies by using cryopreserved skeletal muscle biopsies stored under suitable conditions in tissue banks.

Ocular surface alterations and in vivo confocal microscopic characteristics of corneas in patients with myasthenia gravis

Purpose: To evaluate ocular surface alterations and characteristics of corneal basal epithelium and subbasal nerves in patients with myasthenia gravis. Materials and methods: Myasthenia gravis patients (n = 21) and healthy controls (n = 20) were enrolled. All participants underwent ocular surface testing in the following order: tear break-up time, lissamine green staining, Schirmer I test with anesthesia, and Ocular Surface Disease Index questionnaire. The Cochet-Bonnet esthesiometer was used to measure corneal sensitivity. Basal epithelial cells and subbasal nerves were evaluated using in vivo confocal microscopy. Results: Myasthenia gravis patients had higher Ocular Surface Disease Index score (13.9 ± 15.0 vs 1.4 ± 2.2, p < 0.001) and lissamine green staining score (0.6 ± 0.4 vs 0.2 ± 0.4, p = 0.007). Break-up time score (9.3 ± 3.0 vs 9.9 ± 1.9, p = 0.481) and Schirmer I test score (16.5 ± 9.2 vs 19.3 ± 8.4, p = 0.323) did not differ significantly. Corneal sensation was 0.4 g/mm2 in all eyes. Patients with myasthenia gravis had lower basal epithelial cell density (3775.7 ± 938.1 vs 4983.1 ± 608.5, p < 0.001) and total nerve density (1956.1 ± 373.3 vs 2277.9 ± 405.0, p = 0.012) and higher subbasal nerve tortuosity (1.9 ± 0.8 vs 1.6 ± 0.7, p = 0.007) than controls. A significant increase in Ocular Surface Disease Index scores was found with decreasing basal epithelial cell density (rho = −0.518, p = 0.001). There was a significantly moderate negative correlation between the duration of myasthenia gravis and the number of corneal nerves (rho = −0.497, p = 0.022). Conclusion: Significant alterations of basal epithelial cells and subbasal nerves were demonstrated in myasthenia gravis patients although there was no difference of corneal sensitivity between myasthenia gravis patients and healthy controls. Thus, it should be borne in mind that myasthenia gravis patients deserve further evaluation with regard to ocular surface disease.