V. Milic Rasic

Intellectual Ability in the Duchenne Muscular Dystrophy and Dystrophin Gene Mutation Location

Abstract Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy during childhood. Mutations in dystrophin (DMD) gene are also recognized as a cause of cognitive impairment. We aimed to determine the association between intelligence level and mutation location in DMD genes in Serbian patients with DMD. Forty-one male patients with DMD, aged 3 to 16 years, were recruited at the Clinic for Neurology and Psychiatry for Children and Youth in Belgrade, Serbia. All patients had defined DMD gene deletions or duplications [multiplex ligation- dependent probe amplification (MLPA), polymerase chain reaction (PCR)] and cognitive status assessment (Wechsler Intelligence Scale for Children, Brunet-Lezine scale, Vineland-Doll scale). In 37 patients with an estimated full scale intelligence quotient (FSIQ), six (16.22%) had borderline intelligence (70<FSIQ ≤85), while seven (18.92%) were intellectually impaired (FSIQ <70). The FSIQ was not associated with proximal and distal mutations when boundaries were set at exons 30 and 45. However, FSIQ was statistically significantly associated with mutation location when we assumed their functional consequence on dystrophin isoforms and when mutations in the 5’-untranslated region (5’UTR) of Dp140 (exons 45-50) were assigned to affect only Dp427 and Dp260. Mutations affecting Dp140 and Dp71/Dp40 have been associated with more frequent and more severe cognitive impairment. Finally, the same classification of mutations explained the greater proportion of FSIQ variability associated with cumulative loss of dystrophin isoforms. In conclusion, cumulative loss of dystrophin isoforms increases the risk of intellectual impairment in DMD and characterizing the genotype can define necessity of early cognitive interventions in DMD patients.

Epidemiology of Charcot-Marie-Tooth Disease in the Population of Belgrade, Serbia

Background: The aim of this study was to determine prevalence and 15-year survival in Charcot-Marie-Tooth disease (CMT). Methods: The study covers the period from 1 January 1988 to 31 December 2007 in the territory of Belgrade. Data on a number of CMT-affected persons and their basic demographic characteristics as well as data on the disease were collected from medical records. Data on the course and outcome of the disease were obtained through direct contact with patients, their families and their physicians. Results: We registered 161 patients with CMT in the population of Belgrade. The most frequent type was CMT1. The crude prevalence of CMT disease in the Belgrade population on 31 December 2007 was 9.7/100,000 for all subtypes, 7.1/100,000 for CMT1, and 2.3/100,000 for CMT2. Gender-specific prevalence was 11.2/100,000 for males and 8.3/100,000 for females. The highest age-specific prevalence was registered in the oldest age group (75+ years; 19.1/100,000), and the lowest one in patients aged 5–14 years (5.0/100,000). The cumulative probability of 15-year survival for CMT patients in Belgrade was 85.6 ± 7.8% (44.9 ± 31.8% for males and 98.2 ± 1.8% for females). Conclusions: The prevalence of CMT found in Belgrade is similar to the prevalence registered in Southern European countries.

PP12.16 – 2973: Congenital cerebellar ataxias – Clinical, radiological and genetic considerations

Objective Congenital ataxias, according to Hardings classification, represent a subgroup of early onset ataxias. The aim of this study was to evaluate initial clinical presentations, course of the disease and MRI characteristics in order to prioritize future investigations, including appropriate genetic testing. Methods The patients were selected from clinical ataxia registry at the tertiary center (n=51) after excluding patients with cerebellar malformations, later ataxia onset and known underlying disease. We focused on the following points: 1) congenital onset&clinical course 2) associated features 3) occurrence of peripheral neuropathy 4) neuroimaging results. The statistical analysis has done using T test,: version SPSS 15.0. Results There were 14 subjects (27.5%) from 10 kindred presented with congenital onset – 3 families with 2 or more affected members. Clinical characteristics: 86% subjects initially presented with mild to severe generalized hypotonia and global development delay – the mean age of independent walking was 3.9±2.8 and speech 2.5±0.7. Early generalized ataxia is noticed in all subjects, accompanied with nystagmus in 57% and other ocular abnormalities in 64%. Tendon reflexes were present in 79%. The accompanying findings (n=5) were orthopedic – 3, dystonia – 1 and optic atrophy – 1 and axonal motor neuropathy in one. Laboratory and metabolic workup were negative in all subjects. 86% are functioning within normal or low intellectual level. Global cerebellar hypoplasia was the most common MRI finding – in 93% and correlated significantly with clinical findings. Genetic confirmation was obtained for one family – missense mutation in ITPR1 gene. Conclusion The selected group of patient, presented as non-progressive cerebellar ataxia, showed rather uniform clinical characteristics – mostly isolated ataxia “improving” with aging (SARA, t test 0.015). The presence of cerebellar hypoplasia on MRI mostly correlated with phenotypes, suggesting genetic basis of the disease. We assume that new DNA sequencing technologies would help delineate genetic and pathophysiological mechanism of congenital ataxias.

PP09.9 – 2907: Is it easy to recognize HINT1 neuropathy

Objective Autosomal recessive axonal neuropathy with neuromyotonia and mutation in HINT1 gene form a new disease entity among the myotonic syndromes (Zimon M et al, 2012) and presents one of the most frequent genotype within CMT2/HMN (Lassuthova et al, 2014). Purpose: To present clinical and neurophysiology problems in diagnostic procedure of HINT1 neuropathy. Methods Conventional ENMG technique was applied (Premier, Medelec apparatus) according to standard protocol. Molecular genetics studies were done in VIB Department of Molecular Genetics, Antwerpen and in Neurogenetic lab. in Belgrade. Results 18 HINT1 (founder R37P) patients from 11 families expressed unique CMT phenotype, sometimes with clinically hardly recognizable myotonia. ENMG was typical for axonal neuropathies with neuromyotonia (NT), but in some patients NT was detectable only in non-distal limb muscles. Multigeneration inheritance was found in two families. Conclusion Our patients expressed founder mutation in HINT1 gene and possible pseudodominant transmission which could stimulate AD-CMT2 diagnosis. ENG is enough for differentiating axonal from demyelinating neuropathies, but needle EMG for distal and proximal muscles is necessary for detecting NT.

5. Molecular genetics of myotonic disorders in Serbian patients

Purpose: To present genetic findings in myotonic dystrophy type 1 (DM1) and autosomal recessive axonal neuropathy with neuromyotonia (ARAN-NM) in Serbia. Methods: Various DNA analyses were used to characterize DMPK CTG repeats, which expansions cause DM1. Linkage analyses and next generation sequencing were used to identify gene associated with ARAN-NM. Results: In a cohort of 35 unrelated DM1 patients we found association between expansion-biased somatic instability of mutant alleles over time and progression of neuromuscular symptoms, negative linear correlation of age at onset and average expansion size in patients with progenitor allele less than 245 repeats, and lack of correlation between expansion size and cardiac involvement and polyneuropathy. The large sized normal alleles (>18 repeats), from which expanded alleles originate, had frequency of 8.72% in 235 healthy controls, indicating similar DM1 incidence in Serbia as in Europe (∼1/8000), which was subsequently confirmed by molecular epidemiological study. Loss-of-function mutations in the HINT1 gene were identified as a major cause of ARAN-NM and all Serbian patients were p.R37P homozygote with confirmed founder effect. Conclusion: The main cause of myotonic disorders is the DMPK CTG and a new gene associated with ARAN-NM is the HINT1 , indicating different genetic basis from other genetically characterized non-dystrofic myotonic syndromes.

4. Neurophysiologic and clinical characteristic of neuromyotonia in new hereditary neuromuscular disorder

Purpose: To present clinical and neurophysiologic findings in patients with hereditary axonal neuropathies and myotonia. Methods: Conventional ENMG technique was applied (Premier, Medelec apparatus) on patients with distal motor and sensitive deficits and arm myotonia. Molecular genetics studies were done in the PCR Laboratory, Belgrade and VIB Department of Molecular Genetics, Antwerpen. Results: All 14 patients from 8 families expressed very severe neuropathy, sporadic in two cases and autosomal recessive in others. ENMG was typical for axonal neuropathies (small or no detectable SNAP and CMAP, especially on leg’s nerves and normal or mildly decreased sensitive and motor velocity). Spontaneous activity was not myotonic, it was neuromyotonic, showing excess in excitability of the peripheral motor axons rather than muscles. Homozygote mutation (p.R37P) in new HINT1 gene was detected. Still, the exact mechanism through which HINT1 mutations cause peripheral neuropathy with neuromyotonia remains unclear. Conclusion: HINT1 neuropathies or autosomal recessive axonal neuropathy with neuromyotonia form a new disease entity among the myotonic syndromes and have to be differentiated clinically, neurophysiologically and genetically from both myotonic dystrophy and the various channelopathies causing non-dystrophic forms of myotonia.

31.Familial autoimmune myasthenia gravis

myography with nerve conduction studies was found in two thirds of patients, a finding similar to the analysis of root conduction time. However, in order to detect subclinical CSM we have found prolongation of central motor conduction time in 29% of patients, while application of dynamic tests increased abnormalities to 43%. Within the same group od patients abnormal median SSEP was observed for 38% of patients with cervical spondyloarthrosis. Conclusions: Routine application of extended neurophysiological protocol aimed to evaluate function of central and peripheral motor neurons, including CMCT measurement in different head positions, provide us better chances to recognize the earliest stages of CSM.

4.Electromyography test as diagnostic procedure in recessive cerebellar ataxias

cerebellar ataxias—V. Milic Rasic , S. Todorovic , V. Brankovic , I. Novakovic , J. Baets , S. Vermeer d ( Clinic for Neurology and Psychiatry for Children and Youth, University of Belgrade, Serbia, b Institute of Biology and Human Genetics, Medical Faculty, University of Belgrade, Serbia, c VIB Department of Molecular Genetics, University of Antwerp, Belgium, d Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands)

P25.7 Rehabilitation possibility depends on electromyography and genotype in hereditary neuropathy

Results: We observed a progressive enhancement of MEPs from the first to the third session, exclusively for the motor imagery task, but only when TMS was delivered over the right M1 (i.e., the non-dominant hemisphere). No learning effects emerged for the left M1 stimulation during imagery. Action observation tasks did not change tonically the corticospinal output in both hemispheres. Conclusions: These findings suggest that motor imagery is sustained by a cortical network susceptible to learning only for the non-dominant hemisphere. On the other hand, the absence of any effects of observation tasks may depend of the more automatic nature of these processes.

P5.18 Electromyography findings in family with cerebellar ataxia and new genotype

Introduction: F-waves are late responses to motor nerve stimulation with relevant applications in the electrodiagnostic evaluation of the proximal tracts of the peripheral nervous system such as the brachial or lumbosacral plexi and the spinal roots. In our clinical experience, F-waves from stimulation of the radial nerve recorded from the anconeus muscle can be useful in the evaluation of C7 radiculopathies and of posterior cord and middle trunk plexopathies. Objectives: To report normal values of anconeus muscle F-waves (AFWs). Methods: We tested 25 healthy adults (15 F, 10 M; aged 18 54 years; height: 158 178 cm) on both arms. We recorded the anconeus muscle on the dorsal surface of the forearm (bipolar montage with G1 4 cm distal to the olecranon and G2 placed 2 3 cm further distally). The radial nerve was stimulated 4 cm proximal to the olecranon with 0.2 ms square waves at intensity of 120% of the one required for a maximal M response. Analysis was performed on one limb per subject selected randomly. The measurements consisted of (1) F-wave persistence (PERS); (2) minimum (FMIN), (3) mean (FMEAN) and (4) maximum (FMAX) F-wave latencies and the inter-side difference of persistence (D-PERS) and of FMIN (D-FMIN). Additionally we evaluated the distal motor latency (DLM) and amplitude (M-AMPL) of M-responses from the anconeus muscle. Results: All parameters had normal distributions (Shapiro-Wilk’s W test). Asymmetry and kurtosis were negligible. FMIN, FMEAN and FMAX values correlated significantly with the subjects’ height (r = 0.75, r = 0.76 and r = 0.76 respectively), showing an increase of around 0.1 ms per every cm of height. We report hereafter the mean±SD of all parameters. PERS: 39.4±23.0 ms. FMIN: 17.8±2.1 ms. FMEAN: 18.7±1.9 ms. FMAX: 19.8±1.7 ms. D-PERS: 11.5±8.7 ms. D-FMIN: 0.9±0.9 ms. DLM: 3.0±0.4. M-AMPL: 6.5±1.8. Conclusions: AFWs are a reliable and repeatable electrodiagnostic technique of considerable potential clinical use.