MACF1 links Rapsyn to microtubule- and actin-binding proteins to maintain neuromuscular synapses.

Complex mechanisms are required to form neuromuscular synapses, direct their subsequent maturation, and maintain the synapse throughout life. Transcriptional and post-translational pathways play important roles in synaptic differentiation and direct the accumulation of the neurotransmitter receptors, acetylcholine receptors (AChRs), to the postsynaptic membrane, ensuring for reliable synaptic transmission. Rapsyn, an intracellular peripheral membrane protein that binds AChRs, is essential for synaptic differentiation, but how Rapsyn acts is poorly understood. We screened for proteins that coisolate with AChRs in a Rapsyn-dependent manner and show that microtubule actin cross linking factor 1 (MACF1), a scaffolding protein with binding sites for microtubules (MT) and actin, is concentrated at neuromuscular synapses, where it binds Rapsyn and serves as a synaptic organizer for MT-associated proteins, EB1 and MAP1b, and the actin-associated protein, Vinculin. MACF1 plays an important role in maintaining synaptic differentiation and efficient synaptic transmission in mice, and variants in MACF1 are associated with congenital myasthenia in humans.

Joint effect of the SMN2 and SERF1A genes on childhood-onset types of spinal muscular atrophy in Serbian patients.

Spinal muscular atrophy (SMA) is caused by functional loss of the survival of motor neuron 1 (SMN1) gene. Despite genetic homogeneity, phenotypic variability indicates the involvement of disease modifiers. SMN1 is located in 5q13.2 segmental duplication, enriched in genes and prone to unequal rearrangements, which results in copy number polymorphism (CNP). We examined the influence of CNP of 5q13.2 genes and their joint effect on childhood-onset SMA phenotype. Multiplex ligation-dependent probe amplification (MLPA) was used to construct 5q13.2 alleles and assess copy number of the SMN2, small EDRK-rich factor 1A (SERF1A) and NLR family apoptosis inhibitory protein (NAIP) genes in 99 Serbian patients with SMN1 homozygous absence (23-type I, 37-type II and 39-mild type III) and 122 patients' parents. Spearman rank test was performed to test correlation of individual genes and SMA type. Generalized linear models and backward selection were performed to obtain a model explaining phenotypic variation with the smallest set of variables. 5q13.2 alleles most commonly associated with type I harbored large-scale deletions, while those detected in types II and III originated from conversion of SMN1 to SMN2. Inverse correlation was observed between SMN2, SERF1A and NAIP CNP and SMA type (P=2.2e-16, P=4.264e-10, P=2.722e-8, respectively). The best minimal model describing phenotypic variability included SMN2 (P<2e-16), SERF1A (P<2e-16) and their interaction (P=0.02628). SMN2 and SERF1A have a joint modifying effect on childhood-onset SMA phenotype.

Loss-of-function mutations in HINT1 cause axonal neuropathy with neuromyotonia.

Inherited peripheral neuropathies are frequent neuromuscular disorders known for their clinical and genetic heterogeneity. In 33 families, we identified 8 mutations in HINT1 (encoding histidine triad nucleotide-binding protein 1) by combining linkage analyses with next-generation sequencing and subsequent cohort screening of affected individuals. Our study provides evidence that loss of functional HINT1 protein results in a distinct phenotype of autosomal recessive axonal neuropathy with neuromyotonia.

Targeted next-generation sequencing of a 12.5 Mb homozygous region reveals ANO10 mutations in patients with autosomal-recessive cerebellar ataxia.

Autosomal-recessive cerebellar ataxias comprise a clinically and genetically heterogeneous group of neurodegenerative disorders. In contrast to their dominant counterparts, unraveling the molecular background of these ataxias has proven to be more complicated and the currently known mutations provide incomplete coverage for genotyping of patients. By combining SNP array-based linkage analysis and targeted resequencing of relevant sequences in the linkage interval with the use of next-generation sequencing technology, we identified a mutation in a gene and have shown its association with autosomal-recessive cerebellar ataxia. In a Dutch consanguineous family with three affected siblings a homozygous 12.5 Mb region on chromosome 3 was targeted by array-based sequence capture. Prioritization of all detected sequence variants led to four candidate genes, one of which contained a variant with a high base pair conservation score (phyloP score: 5.26). This variant was a leucine-to-arginine substitution in the DUF 590 domain of a 16K transmembrane protein, a putative calcium-activated chloride channel encoded by anoctamin 10 (ANO10). The analysis of ANO10 by Sanger sequencing revealed three additional mutations: a homozygous mutation (c.1150_1151del [p.Leu384fs]) in a Serbian family and a compound-heterozygous splice-site mutation (c.1476+1G>T) and a frameshift mutation (c.1604del [p.Leu535X]) in a French family. This illustrates the power of using initial homozygosity mapping with next-generation sequencing technology to identify genes involved in autosomal-recessive diseases. Moreover, identifying a putative calcium-dependent chloride channel involved in cerebellar ataxia adds another pathway to the list of pathophysiological mechanisms that may cause cerebellar ataxia.

Eosinophilic myositis as presenting symptom in gamma-sarcoglycanopathy.

The patient reported here presented with first symptoms at the age of 10 showing an abnormal gait, calf hypertrophy and winged scapulae. She was diagnosed with eosinophilic myositis after muscle biopsy. A second muscle biopsy at the age of 20 and subsequent genetic testing, however, revealed the underlying condition of a primary gamma-sarcoglycanopathy, or LGMD2C. To our knowledge, this is the first LGMD2C patient reported who initially presented with eosinophilic myositis. Eosinophilia has been reported previously in patients with Calpainopathy and Becker Muscular Dystrophy and might be an early, but transient feature of a wider range of muscular dystrophies.

Clinical case report atypical myopathy in a young girl with 91 CTG repeats in DM1 locus and a positive DM1 family history.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant inheritable disease associated with an expansion of CTG repeats in the 3' UTR of the DMPK gene. The subject is an 11-year-old girl with atypical myopathy. Because the proband's family has a positive DM1 history, a molecular-genetic analysis for DM1 was performed. This study showed that proband had a small DMPK expansion (91 CTG repeats) although the observed myopathy would not normally be associated with DM1. These results show how the phenotypic manifestation of DM1 can have unusual symptoms with a completely unexpected relationship to genotype.

Epidemiology of myotonic dystrophy type 1 (Steinert disease) in Belgrade (Serbia).

The aim of this study was to estimate the incidence and prevalence of myotonic dystrophy type 1 (DM1) in Belgrade during the period 1983-2002. The patients who had DM1 were ascertained through hospital records from all neurological departments in Belgrade during 1983-2002. The molecular genetic analysis was performed in all patents included in the study. We identified 101 DM1 patients (52 males and 49 females). The average annual incidence rate of DM1 in Belgrade for the period observed was 2.0/1,000,000 (95% confidence interval (CI), 0.3-8.3), 2.1/1,000,000 (95% CI, 0.3-8.3) for males and 2.0/1,000,000 (95% CI, 0.3-8.3) for females. The highest age-specific DM1 incidence was registered in the age group 20-49: 3.4/1,000,000 (95% CI, 0.5-7.6), 4.0/1,000,000 (95% CI, 1.1-10.2) in males and 2.5/1,000,000 (95% CI, 0.5-7.6) in females. In the population of Belgrade, a cumulative probability of acquiring DM1 was 1 per 8621 for men and 1 per 9259 for women (1 per 8940 of the population for both sexes). The prevalence of DM1 in Belgrade on 31 December 2002 was 5.3/100,000 (95% CI, 4.2-6.6).

Proximal dystrophin gene deletions and protein alterations in becker muscular dystrophy.

Alterations in production of cytoskeletal protein dystrophin caused by in-frame gene mutations lead to the Becker muscular dystrophy. In this study we analyzed genotype-phenotype correlation in a group of Becker muscular dystrophy patients with deletions affecting the proximal part of dystrophin gene, encompassing exons 3-13. Four patients with deletions affecting N terminal dystrophin domain had early onset and faster progression of the disease, while three patients with deletions in the proximal part of dystrophin's rod domain had a more benign disease course. Our study suggests that proximal gene deletions in Becker muscular dystrophy have various phenotypic effects depending on the affected domain of protein dystrophin.

[Epidemiology of myotonic dystrophy type 1 in the population of central Serbia].

The objective of this epidemiological survey was to estimate the frequency and distribution of Myotonic dystrophy type 1 (MD1) (Steinert's disease) in central Serbia, during the period 1983-2002. The data on the number of diagnosed MD1 patients were obtained using the analysis of hospital records, which were examined in all the relevant neurological institutions in central Serbia in the mentioned period. Incidence rate and prevalence were used for the data analysis. In the study period in central Serbia, 154 patients (78 males and 76 females) with MD1 were identified. The average annual incidence rate of MD1 was 1.3 (95% CI-confidence interval 0.1-7.2) per 1,000,000 population, 1.4/1,000,000 (95% CI 0.1-7.2) for males, 1.3/1,000,000 (95% CI 0.1-7.2) for females. The trend of MD1 incidence rates in the observed period in central Serbia had a tendency of the statistically significant decrease, according to the linear model, in both male (y = 0.205 - 0.0066x, p = 0.021) and female populations (y = 0.1788 - 0.0048x, p = 0.032). The prevalence of MD1 on December 31, 2002 in central Serbia was 3.8/100,000 (95% IP 3.2-4.6), 3.7/100,000 (95% IP 3.3 - 4.8) for males, 3.3/100,000 (95% IP 3.0 - 4.4) for females.

The recurrence risk of ischemic stroke in childhood.

OBJECTIVE: To determine the risk of recurrence of ischemic stroke in children and to evaluate the influence of etiological factors and underlying mechanisms on recurrence rate. SUBJECTS AND METHODS: Thirty-six children (21 boys and 15 girls) with clinically and radiographically proven ischemic cerebral infarction were prospectively followed up over a period of 1-9 years (median 5 years 5 months). The median age of onset of stroke was 8.4 years (1-16 years). Patients with hemorrhagic stroke, neonatal infarction and sinovenous thrombosis were not included. The patients were analyzed according to the mechanisms and etiology of the initial and recurrent stroke event. RESULTS: For the initial stroke, cardioembolic (33.3%) and arteriopathic processes (36.1%) were identified as the most probable mechanisms of arterial ischemic stroke. Prothrombotic abnormalities were found in 4 children (11.1%). Underlying pathology in the remaining 7 (19.4%) was not known. Recurrent ischemic infarction was diagnosed in 5 children (13.9%) within 5 days to 18 months (median 6 months) after the first stroke manifestation. In 3 of them stroke recurrence was due to cardiac or transcardiac embolism. Cardiac abnormality prior to the first stroke was detected in 1 child. Clinically silent multiple cerebral infarcts disclosed by MRI preceded the overt stroke episode in 2 patients. CONCLUSION: Congenital and acquired heart diseases were the most common cause of repeated stroke in our study. The risk of recurrence appeared to be fivefold higher in children with cardiac disease irrespective of the coexistence of other risk factors. The risk factors of stroke in children were multiple and overlapping. Consequently, recognition of the major one and its underlying mechanism is crucial for both effective therapeutic approach and the prevention of recurrence.

[Clinical course and prognosis in hypoxic-ischemic encephalopathy]. / Klinicki tok i prognoza hipoksijsko-ishemijske encefalopatije.

BACKGROUND: Establishing the value of neurological examination, and additional diagnostic methods (ultrasonography and magnetic resonance imaging of the brain) in the diagnosis and prognosis of hypoxic-ischemic encephalopathy and its treatment, tracking the clinical course, and making the prognosis of neurological development in newborn infants with hypoxic-ischemic encephalopathy. METHODS: The group of 40 term newborn infants with suspected intrauterine asphyxia was examined. All the infants were prospectively followed until the 3rd year of age at the Clinic for Neurology and Psychiatry for Children and Youth in order to estimate their neurological development and to diagnose the occurrence of persistent neurological disorders. All the infants were analyzed by their gestational age and Apgar score in the 1st and the 5th minute of life. They were all examined neurologically and by ultrasonography in the first week of life and, repeatedly, at the age of 1, 3, 6, 9, 12, 18, as well as in the 24th month of life. They were treated by the standard methods for this disease. Finally, all the infants were examined neurologically and by magnetic resonance imaging of the brain in their 3rd year of age. On the basis of neurological finding infants were divided into 3 groups: infants with normal neurological finding, infants with mild neurological symptomatology, and infants with severe neurological disorders. RESULTS: It was shown that neurological finding, ultrasonography and magnetic resonance imaging of the brain positively correlated with the later neurological development of the infants with hypoxic-ischemic encephalopathy. CONCLUSION: Only the combined use of these techniques had full diagnostic and prognostic significance, emphasizing that the integrative approach was very important in the diagnosis of brain lesions in infants.

[Duchenne's and Becker's muscular dystrophy: analysis of phenotype-genotype correlation in 28 patients] ]. / Disenova i Bekerova misicna distrofija: analiza korelacije fenotip-genotip kod 28 bolesnika.

Duchenne's and Becker's muscular dystrophy (DMD & BMD) is a X linked disease caused by mutations in the dystrophic gene. DMD is the malign form of the disease, which significantly shortens the lifetime of the patient, while BMD has late onset with slow progression. Sixty five percent of DMD and BMD cases are caused by deletion of one or more exons in the dystrophic gene, while duplications cause these diseases in 6 to 7% of the cases. There are two hot spots for deletions and duplications. These are exons in the proximal part of the gene (3rd to 18th) and exons of a distal part of the gene (45th to 52nd). The remaining 30% of DMD and BMD cases are caused by point mutations, small deletions or inversions in the dystrophic gene. The correlation between the severity of the disease and the position of deletion shows that most of the out of frame deletions cause DMD phenotype, while in frame deletions result in BMD phenotype. We report on the results of 28 non-related DMD and BMD patients. In 57% of cases deletions were detected and all were found in the distal hot spot of the gene. These results suggest that in most of the cases, out of frame deletions produce DMD phenotype while in frame deletions result in BMD phenotype. This is in compliance with data from literature.

250 CTG repeats in DMPK is a threshold for correlation of expansion size and age at onset of juvenile-adult DM1.

Myotonic dystrophy type 1 (DM1) is associated with an expansion of CTG repeats in the 3'UTR of the DMPK gene. It is accepted, as in other trinucleotide diseases, that the number of the repeats is correlated with age at onset and severity of the disease. However, assessment of genotype-phenotype correlation in DM1 is complicated with the expansion-biased somatic instability of mutant alleles over time and difficulties in precise assessment of the number of repeats by standard Southern blot hybridization. In order to clarify this issue we defined DM1 expansion size in lymphocytes by three parameters: size of progenitor, average, and largest allele, using a more precise small-pool/long-range PCR technique. We found a negative linear correlation of age at onset and average expansion size in juvenile-adult DM1 patients (35 out of 46) whose progenitor allele is less than 245 repeats long. Our result favors the hypothesis of the existence of a threshold in the progenitor allele size beyond which number of CTG repeats does not influence age at onset. Potential clinical significance is that the average allele size could be a useful indicator for the age at onset in juvenile-adult DM1 patients with relatively short progenitor allele. To test whether somatic instability of mutant alleles influences the progression of DM1, patients were divided in three phenotypic classes according to the severity of neuromuscular symptoms. We showed that the largest expansion in each DM1 phenotypic class reflects somatic instability of mutant allele over time independently of progenitor allele size and patient's age at sampling. The mean of the largest expansion was significantly different between phenotypic classes, implying the possible association between expansion-biased somatic instability of mutant alleles over time and progression of neuromuscular symptoms.