Phosphorylated neurofilament heavy chain in cerebrospinal fluid and plasma as a Nusinersen treatment response marker in childhood-onset SMA individuals from Serbia.

Introduction: Biomarkers capable of reflecting disease onset and short- and long-term therapeutic effects in individuals with spinal muscular atrophy (SMA) are still an unmet need and phosphorylated neurofilament heavy chain (pNF-H) holds significant promise. Methods: We conducted a longitudinal prospective study to evaluate pNF-H levels in the cerebrospinal fluid (CSF) and plasma of 29 individuals with childhood-onset SMA treated with Nuinersen (SMA type 1: n = 6, 2: n = 17, 3: n = 6). pNF-H levels before and during treatment were compared with the levels of controls (n = 22), patients with Duchenne muscular dystrophy (n = 17), myotonic dystrophy type 1 (n = 11), untreated SMA individuals with chronic type 3 disease (n = 8), and children with presymptomatic SMA (n = 3). Results: SMA type 1 showed the highest mean CSF pNF-H levels before treatment initiation. All Nusinersen-treated individuals (types 1, 2, and 3) showed significantly elevated mean baseline CSF pNF-H compared to controls, which inversely correlated with age at disease onset, age at first dose, disease duration and the initial CHOP INTEND result (SMA type 1 and 2). During 22 months of treatment, CSF pNF-H levels declined during loading doses, stabilizing at reduced levels from the initial maintenance dose in all individuals. Baseline plasma pNF-H levels in type 1 and 2 SMA were significantly increased compared to other cohorts and decreased notably in type 1 after 2 months of treatment and type 2 after 14 months. Conversely, SMA type 3, characterized by lower baseline pNF-H levels, did not show significant fluctuations in plasma pNF-H levels after 14 months of treatment. Conclusion: Our findings suggest that CSF pNF-H levels in untreated SMA individuals are significantly higher than in controls and that monitoring of CSF pNF-H levels may serve as an indicator of rapid short-term treatment response in childhood-onset SMA individuals, irrespective of the subtype of the disease, while also suggesting its potential for assessing long-term suppression of neurodegeneration. Plasma pNF-H may serve as an appropriate outcome measure for disease progression and/or response to treatment in types 1 and 2 but not in type 3. Presymptomatic infants with SMA may show elevated pNF-H levels, confirming early neuronal degeneration.

LTBP4, SPP1, and CD40 Variants: Genetic Modifiers of Duchenne Muscular Dystrophy Analyzed in Serbian Patients.

BACKGROUND: Clinical course variability in Duchenne muscular dystrophy (DMD) is partially explained by the mutation location in the DMD gene and variants in modifier genes. We assessed the effect of the SPP1, CD40, and LTBP4 genes and DMD mutation location on loss of ambulation (LoA). METHODS: SNPs in SPP1-rs28357094, LTBP4-rs2303729, rs1131620, rs1051303, rs10880, and CD40-rs1883832 were genotyped, and their effect was assessed by survival and hierarchical cluster analysis. RESULTS: Patients on glucocorticoid corticosteroid (GC) therapy experienced LoA one year later (p = 0.04). The modifying effect of SPP1 and CD40 variants, as well as LTBP4 haplotypes, was not observed using a log-rank test and multivariant Cox regression analysis. Cluster analysis revealed two subgroups with statistical trends in differences in age at LoA. Almost all patients in the cluster with later LoA had the protective IAAM LTBP4 haplotype and statistically significantly fewer CD40 genotypes with harmful T allele and "distal" DMD mutations. CONCLUSIONS: The modifying effect of SPP1, CD40, and LTBP4 was not replicated in Serbian patients, although our cohort was comparable in terms of its DMD mutation type distribution, SNP allele frequencies, and GC-positive effect with other European cohorts. Cluster analysis may be able to identify patient subgroups carrying a combination of the genetic variants that modify LoA.

Biallelic ADPRHL2 mutations in complex neuropathy affect ADP ribosylation and DNA damage response.

ADP ribosylation is a reversible posttranslational modification mediated by poly(ADP-ribose)transferases (e.g., PARP1) and (ADP-ribosyl)hydrolases (e.g., ARH3 and PARG), ensuring synthesis and removal of mono-ADP-ribose or poly-ADP-ribose chains on protein substrates. Dysregulation of ADP ribosylation signaling has been associated with several neurodegenerative diseases, including Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease. Recessive ADPRHL2/ARH3 mutations are described to cause a stress-induced epileptic ataxia syndrome with developmental delay and axonal neuropathy (CONDSIAS). Here, we present two families with a neuropathy predominant disorder and homozygous mutations in ADPRHL2 We characterized a novel C26F mutation, demonstrating protein instability and reduced protein function. Characterization of the recurrent V335G mutant demonstrated mild loss of expression with retained enzymatic activity. Although the V335G mutation retains its mitochondrial localization, it has altered cytosolic/nuclear localization. This minimally affects basal ADP ribosylation but results in elevated nuclear ADP ribosylation during stress, demonstrating the vital role of ADP ribosylation reversal by ARH3 in DNA damage control.

Glucose and lipid metabolism disorders in children and adolescents with spinal muscular atrophy types 2 and 3.

We aimed to estimate the prevalence of glucose and lipid metabolism disorders in children and adolescents with spinal muscular atrophy (SMA) types 2 and 3. A cross-sectional study was conducted. Medical history, anthropometric measurements, pubertal status, blood chemistry (glucose and insulin levels, lipid profile, aminotransferases, and hemoglobin A1c [HbA1c]), and liver ultrasound were obtained in all patients. Oral glucose tolerance test was performed in those with body mass index (BMI) >25th percentile or glucose or HbA1c levels in the prediabetic range. A total of 37 patients with SMA (22 type 2, 15 type 3) with a median age of 8.5 years (range 2-18.9 years) were included. Eleven patients (29.7%) met the criteria for prediabetes, but none had overt type 2 diabetes. Dyslipidemia was detected in 11 patients (29.7%), and 4 (10.8%) had hepatic steatosis on ultrasound. Sixteen patients (43.2%) had at least one abnormal finding (prediabetes, dyslipidemia, or hepatic steatosis); all but one were non-ambulatory and 12 (75%) had BMI ≥85th percentile. One young child developed fasting hypoglycemia. Our results suggest that non-ambulatory overweight/obese SMA patients are particularly prone to abnormalities in glucose and lipid metabolism. Young underweight patients might develop fasting hypoglycemia.

Cardiac findings in pediatric patients with spinal muscular atrophy types 2 and 3.

BACKGROUND: It is unclear whether the heart is affected in pediatric patients with milder forms of spinal muscular atrophy (SMA). Therefore, we aimed to determine the presence of any cardiac abnormalities in these patients. METHODS: We conducted a cross-sectional study of children and adolescents with SMA types 2 and 3 between July 2018 and July 2019. All patients underwent a comprehensive cardiac evaluation, including history-taking, physical examination, electrocardiography, echocardiography, measurement of cardiac biomarkers (cardiac troponin T [cTnT] and N-terminal pro-brain natriuretic peptide [NT-proBNP]), and 24-hour Holter monitoring. RESULTS: In total, 42 patients were enrolled (27 and 15 with SMA type 2 and 3, respectively). No patient had structural heart disease, except for one with mitral valve prolapse. None had signs of ventricular dysfunction on echocardiography. Both cTnT and NT-proBNP levels were normal in all patients. Electrocardiography showed sinus tachycardia in seven patients (16.7%), and prolonged P-R interval in one (2.4%). Holter monitoring detected benign ventricular arrhythmias in two patients (4.8%), and rare supraventricular premature beats in one. The mean 24-hour heart rate was elevated in six patients (14.3%), whereas both the minimum 24-hour heart rate and the maximum R-R interval were increased in 23 (54.8%). DISCUSSION: The prevalence of cardiac disease in pediatric patients with SMA types 2 and 3 is low; however, these patients may have increased resting heart rates. A complete cardiac history and physical examination are a useful screen. Additional cardiac investigations may be performed as needed.

Phenotypic and genetic spectrum of patients with limb-girdle muscular dystrophy type 2A from Serbia.

Limb-girdle muscular dystrophy (LGMD) type 2A (calpainopathy) is an autosomal recessive disease caused by mutation in the CAPN3 gene. The aim of this study was to examine genetic and phenotypic features of Serbian patients with calpainopathy. The study comprised 19 patients with genetically confirmed calpainopathy diagnosed at the Neurology Clinic, Clinical Center of Serbia and the Clinic for Neurology and Psychiatry for Children and Youth in Belgrade, Serbia during a ten-year period. Eighteen patients in this cohort had c.550delA mutation, with nine of them being homozygous. In majority of the patients, disease started in childhood or early adulthood. The disease affected shoulder girdle - upper arm and pelvic girdle - thigh muscles with similar frequency, with muscles of lower extremities being more severely impaired. Facial and bulbar muscles were spared. All patients in this cohort, except two, remained ambulant. None of the patients had cardiomyopathy, while 21% showed mild conduction defects. Respiratory function was mildly impaired in 21% of patients. Standard muscle histopathology showed myopathic and dystrophic pattern. In conclusion, the majority of Serbian LGMD2A patients have the same mutation and similar phenotype.

Sphingosine 1-phosphate lyase deficiency causes Charcot-Marie-Tooth neuropathy.

OBJECTIVE: To identify the unknown genetic cause in a nuclear family with an axonal form of peripheral neuropathy and atypical disease course. METHODS: Detailed neurologic, electrophysiologic, and neuropathologic examinations of the patients were performed. Whole exome sequencing of both affected individuals was done. The effect of the identified sequence variations was investigated at cDNA and protein level in patient-derived lymphoblasts. The plasma sphingoid base profile was analyzed. Functional consequences of neuron-specific downregulation of the gene were studied in Drosophila. RESULTS: Both patients present an atypical form of axonal peripheral neuropathy, characterized by acute or subacute onset and episodes of recurrent mononeuropathy. We identified compound heterozygous mutations cosegregating with disease and absent in controls in the SGPL1 gene, encoding sphingosine 1-phosphate lyase (SPL). The p.Ser361* mutation triggers nonsense-mediated mRNA decay. The missense p.Ile184Thr mutation causes partial protein degradation. The plasma levels of sphingosine 1-phosphate and sphingosine/sphinganine ratio were increased in the patients. Neuron-specific downregulation of the Drosophila orthologue impaired the morphology of the neuromuscular junction and caused progressive degeneration of the chemosensory neurons innervating the wing margin bristles. CONCLUSIONS: We suggest SPL deficiency as a cause of a distinct form of Charcot-Marie-Tooth disease in humans, thus extending the currently recognized clinical and genetic spectrum of inherited peripheral neuropathies. Our data emphasize the importance of sphingolipid metabolism for neuronal function.

Analysis of PMP22 duplication and deletion using a panel of six dinucleotide tandem repeats.

BACKGROUND: Charcot-Marie-Tooth type 1A (CMT1A) is the most common type of hereditary motor and sensory neuropathies (HMSN), caused by the duplication of the 17p11.2 region that includes the PMP22 gene. Reciprocal deletion of the same region is the main cause of hereditary neuropathy with liability to pressure palsies (HNPP). CMT1A accounts for approximately 50% of HMSN patients. Diagnostics of CMT1A and HNPP are based on quantitative analysis of the affected region or RFLP detection of breakage points. The aim of this study was to improve the sensitivity and efficiency of CMT1A and HNPP genetic diagnostics by introducing analysis of six STR markers (D17S261-D17S122-D17S839-D17S1358-D17S955-D17S921) spanning the duplicated region. METHODS: Forty-six CMT1A and seven HNPP patients, all genetically diagnosed by RFLP analysis, were tested for duplication or deletion using six STR markers. RESULTS: In all CMT1A and HNPP patients, microsatellite analysis comprising six STR markers confirmed the existence of a duplication or deletion. In 89% (41/46) CMT1A patients the confirmation was based on detecting three alleles on at least one locus. In the remaining 11% (5) CMT1A patients, duplication was also confirmed based on two peaks with clear dosage difference for at least two different markers. All HNPP patients (7/7) displayed only one allele for each analyzed locus. CONCLUSIONS: Microsatellite analysis using six selected STR loci showed a high level of sensitivity and specificity for genetic diagnostics of CMT1A and HNPP. The results here strongly suggest STR marker analysis as a method of choice in PMP22 duplication/deletion testing.

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.

An algorithm for genetic testing of Serbian patients with demyelinating Charcot-Marie-Tooth.

Charcot-Marie Tooth (CMT) is a clinically and genetically heterogeneous group of diseases with rough genotype-phenotype correlation, so the final diagnosis requires extensive clinical and electrophysiological examination, family data, and gene mutation analysis. Although there is a common pattern of genetic basis of CMT, there could be some population differences that should be taken into account to facilitate analyses. Here we present the algorithm for genetic testing in Serbian patients with demyelinating CMT, based on their genetic specificities: in cases of no PMP22 duplication, and if -X-linked CMT (CMTX) is not contraindicated by pattern of inheritance (male-to-male transmission), one should test for c.94A>G GJB founder mutation, first. Also, when a patient is of Romani ethnicity, or if there is an autosomal recessive inheritance in a family and unclear ethnicity, c.442C>T mutation in NDRG1 should be tested.

Community-based study of health-related quality of life in spinal cord injury, muscular dystrophy, multiple sclerosis, and cerebral palsy.

PURPOSE: To assess health-related quality of life (HRQOL) in adults with spinal cord injury (SCI), muscular dystrophy (MD), multiple sclerosis (MS), and cerebral palsy (CP). METHODS: This is a multicenter, community-based, cross-sectional study of adults diagnosed with CP (94), MD (99), MS (98), SCI (99), and healthy adults (105). The WHOQOL-BREF and WHOQOL-DIS questionnaire were used. RESULTS: Significant differences in physical functioning between adults with CP and SCI and adults with MS (p = 0.003 and p < 0.001, respectively), as well as between adults with SCI and MD (p = 0.001) were found. Univariate tests revealed significant psychological functioning differences between adults with SCI and MD (p = 0.02) and SCI and MS (p = 0.001). There was a significant difference in physical functioning between controls and adults with SCI (p = 0.049) and a significant difference in psychological functioning between controls and adults with MS (p = 0.039). No statistically significant differences were found between the groups in social and environmental domains. CONCLUSIONS: Physical and physiological functioning were affected to various degrees in the studied neurodisabilities, while all groups reported similar levels of functioning and well-being in social and environmental domains.

Further developments with antisense treatment for myasthenia gravis.

We present further developments in the study of the antisense oligonucleotide EN101. Ongoing in vitro and in vivo studies demonstrate that EN101 is a TLR9-specific ligand that can suppress pro-inflammatory functions and shift nuclear factor kappa B (NF-κB) from the pro-inflammatory canonical pathway to the anti-inflammatory alternative pathway, which results in decreases acetylcholinesterase (AChE) activity. Preliminary results of a double-blinded phase II cross-over study compared 10, 20, and 40 mg EN101 administered to patients with myasthenia gravis. Patients were randomly assigned to one of three treatment groups in weeks 1, 3, and 5 and received their pretreatment dose of pyridostigmine in weeks 2 and 4. Thus far, all doses show a decrease in QMG scores, with a greater response to higher doses.

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.

Childhood cerebral X-linked adrenoleukodystrophy more than 5 years after hematopoietic cell transplantation: the first case from Serbia and southeastern Europe.

We report the clinical course, brain magnetic resonance imaging (MRI), and proton magnetic resonance spectroscopy findings in a boy with childhood cerebral X-linked adrenoleukodystrophy whose neurological disease keeps progressing more than 5 years after conventional hematopoietic cell transplantation with full donor-derived engraftment accomplishment. The described clinical and radiological findings follow all phases of this childhood cerebral X-linked adrenoleukodystrophy: from the clinically asymptomatic pretransplant stage to the present day. This is the first patient not only from Serbia but from the entire area of Southeastern Europe who underwent hematopoietic cell transplantation for childhood cerebral X-linked adrenoleukodystrophy. The presented disease course and the posttransplant outcome in the only case of transplanted adrenoleukodystrophy from Serbia enhances the overwhelming appeal for better X-linked adrenoleukodystrophy screening, earlier disease detection, and contributes to the well-known anticipation of the refined hematopoietic cell transplantation eligibility criteria in future adrenoleukodystrophy treatment.

Mutational analysis of GJB1, MPZ, PMP22, EGR2, and LITAF/SIMPLE in Serbian Charcot-Marie-Tooth patients.

We report the results of mutational analysis in the following genes: GJB1, MPZ, PMP22, EGR2, and LITAF/SIMPLE in 57 Charcot-Marie-Tooth (CMT) patients of Serbian origin without the PMP22 duplication. We found 10 different mutations in 14 CMT patients: 6 mutations in GJB1, 3 in MPZ, and 1 in PMP22. Five of six GJB1 mutations are reported for the first time, and the most frequent one appears to be a founder mutation in the Serbian population. No mutations were found in EGR2 or LITAF. Thus, GJB1 mutation analysis should be done in patients without the PMP22 duplication and male-to-male transmission of CMT.

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.

Arterial ischemic stroke in a child with beta-thalassemia trait and methylentetrahydrofolate reductase mutation.

Genetic and acquired disorders that foster a procoagulable state represent risk factors for stroke in childhood. Although an increased incidence of thromboembolic complications has been reported in patients with thalassemia, severe cerebral thromboembolism has rarely been observed in patients with beta-thalassemia minor. This article describes a case study of a 1-year-old boy who presented with left-sided hemiparesis, seizures, microcytic anemia, and recent infection with reactive thrombocytosis. Ischemic infarction in the territory of the right middle cerebral artery was confirmed by magnetic resonance imaging and magnetic resonance angiography. Genetic tests showed that the patient was heterozygous for the beta(degrees) -thalassemia IVS-I-1 mutation and homozygous for the methylentetrahydrofolate reductase C677T mutation. Based on these findings, it was concluded that the synergistic effects of multiple, genetic, and acquired prothrombotic risk factors brought about the hypercoagulable state that resulted in overt stroke in a thalassemic patient in early childhood.

MFN2 mutation distribution and genotype/phenotype correlation in Charcot-Marie-Tooth type 2.

Mutations in mitofusin 2 (MFN2) have been reported in Charcot-Marie-Tooth type 2 (CMT2) families. To study the distribution of mutations in MFN2 we screened 323 families and isolated patients with distinct CMT phenotypes. In 29 probands, we identified 22 distinct MFN2 mutations, and 14 of these mutations have not been reported before. All mutations were located in the cytoplasmic domains of the MFN2 protein. Patients presented with a classical but rather severe CMT phenotype, since 28% of them were wheelchair-dependent. Some had additional features as optic atrophy. Most patients had an early onset and severe disease status, whereas a smaller group experienced a later onset and milder disease course. Electrophysiological data showed in the majority of patients normal to slightly reduced nerve conduction velocities with often severely reduced amplitudes of the compound motor and sensory nerve action potentials. Examination of sural nerve specimens showed loss of large myelinated fibres and degenerative mitochondrial changes. In patients with a documented family history of CMT2 the frequency of MFN2 mutations was 33% indicating that MFN2 mutations are a major cause in this population.