Ethnicity-related DMD Genotype Landscapes in European and Non-European Countries.

OBJECTIVE: Genetic diagnosis and mutation identification are now compulsory for Duchenne (DMD) and Becker muscular dystrophies (BMD), which are due to dystrophin (DMD) gene mutations, either for disease prevention or personalized therapies. To evaluate the ethnic-related genetic assortments of DMD mutations, which may impact on DMD genetic diagnosis pipelines, we studied 328 patients with DMD and BMD from non-European countries. METHODS: We performed a full DMD mutation detection in 328 patients from 10 Eastern European countries (Poland, Hungary, Lithuania, Romania, Serbia, Croatia, Bosnia, Bulgaria, Ukraine, and Russia) and 2 non-European countries (Cyprus and Algeria). We used both conventional methods (multiplex ligation-dependent probe amplification [MLPA] followed by gene-specific sequencing) and whole-exome sequencing (WES) as a pivotal study ran in 28 patients where DMD mutations were already identified by standard techniques. WES output was also interrogated for DMD gene modifiers. RESULTS: We identified DMD gene mutations in 222 male patients. We identified a remarkable allele heterogeneity among different populations with a mutation landscape often country specific. We also showed that WES is effective for picking up all DMD deletions and small mutations and its adoption could allow a detection rate close to 90% of all occurring mutations. Gene modifiers haplotypes were identified with some ethnic-specific configurations. CONCLUSIONS: Our data provide unreported mutation landscapes in different countries, suggesting that ethnicity may orient genetic diagnosis flowchart, which can be adjusted depending on the mutation type frequency, with impact in drug eligibility.

SCN1A mutation spectrum in a cohort of Bulgarian patients with GEFS+ phenotype.

BACKGROUND: Dravet syndrome (DS) is the most severe form of Generalized Epilepsy with Febrile Seizures plus (GEFS+) syndrome with a clear genetic component in 85% of the cases. It is characterized by fever-provoked seizure onset around six months of age and subsequent developmental deterioration later in life. METHODS: In the current study, 60 patients with fever-provoked seizures and suspicion either of GEFS+ (50 patients) or of DS (10 patients) were referred for SCN1A gene sequence analysis. RESULTS: SCN1A gene sequencing revealed clinically significant variants in 11 patients (18.3%); seven pathogenic (11.7%) and four likely pathogenic (6.7%). Five of these variants have not been reported previously. Among the preselected group of ten DS patients, five had pathogenic SCN1A variants which confirmed diagnosis of DS. In four patients with preliminary diagnosis GEFS+, the detected SCN1A variant enabled us to specify the diagnosis of DS in these patients. Thus, SCN1A sequencing led to confirmation of the genetic diagnosis in 50% (5/10) of DS patients, as well as clarification of the diagnosis of DS in 8% of GEFS+ patients (4/50). In this study, four patients with truncating mutations had refractory seizures and additional psychomotor abnormalities. Additionally, pathogenic missense mutations were detected in three children with comparable phenotypes, which support the observations that missense mutations in critical channel function regions can cause a devastating epileptic condition. CONCLUSIONS: This is the first systematic screening of SCN1A gene in our country, which expands the spectrum of SCN1A variants with five novel variants from Bulgaria and demonstrates the clinical utility of confirmatory SCN1A testing, which helps clinicians make early and precise diagnoses. It is important for a better followup, choice of proper treatment, avoidance of development of refractory seizures and neuropsychological complications. Identification of pathogenic variants in SCN1A in the milder GEFS+ and severe DS cases, will help to offer adequate prenatal diagnosis and improve the genetic counselling provided to affected families.

Clinical Spectrum and Genetic Variability in Bulgarian Patients with Niemann-Pick Disease Type C.

BACKGROUND: Niemann-Pick disease type C (NP-C) is a rare autosomal-recessive lysosomal storage disorder caused by mutations in either the NPC1 (in 95% of cases) or the NPC2 gene. METHODS: In a prospective, observational cohort study, all Bulgarian patients diagnosed with NP-C to date (since 2010) underwent detailed neurological examination and neuro-ophthalmological, neuropsychological and psychiatric evaluations, as well as brain MRI, abdominal ultrasound and hearing tests. Plasma chitotriosidase was also measured, when possible. RESULTS: The Bulgarian national NP-C cohort comprised 11 patients who were diagnosed based on molecular genetic analysis (n = 9) and/or filipin staining of skin fibroblasts (n = 3). The mean age at onset was 14.4 (SD 8.3). Diagnoses were achieved 1-23 years after initial clinical presentation. All patients who underwent genetic mutation analysis were compound heterozygotes: a total of 12 NPC1 mutations were recorded, 5 of which were novel. Two patients had late-infantile onset, 4 had juvenile onset, and the remaining 5 had the adult-onset form of NP-C. Initial symptoms were neurological in 9 patients, visceral in one, and predominantly psychiatric in another. Vertical gaze palsy was present in all patients. Dysarthria, pyramidal involvement, cognitive impairment, and organomegaly with varied severity were observed in 10 of them. Ataxia was present in 9 and dystonia in 7. Four patients had epileptic seizures, and gelastic cataplexy was reported in 5. Brain MRI revealed hyperintense white matter lesions in 5 patients and cortical and/or cerebellar atrophy in 4. CONCLUSIONS: This Bulgarian NP-C cohort showed wide variability in terms of NPC1 mutations and predominant forms of neurological involvement. Diagnosing NP-C is challenging, and it was often delayed in this cohort due to the heterogeneity of patients' clinical signs and symptoms.

Cerebellar Degeneration as a Rare Paraneoplastic Syndrome in a Child With Hodgkin Lymphoma.

We report a rare case of cerebellar degeneration as a paraneoplastic syndrome in an 8-year-old boy with Hodgkin lymphoma that presented during first-line treatment. Antibodies against Purkinje cells (anti-Tr antibodies) were detected in the serum of the patient. After successful treatment of the lymphoma, the cerebellar symptoms resolved partially. Childhood presentation of paraneoplastic cerebellar degeneration is extremely rare, with only a few reports in the literature. For this reason, the description of all such cases contributes to the enrichment of the medical knowledge and will improve the diagnosis and the treatment of this complication.

15q13.3 microdeletions in a prospectively recruited cohort of patients with idiopathic generalized epilepsy in Bulgaria.

PURPOSE: The chromosome 15q13.3 region is a genomic rearrangement hotspot linked to idiopathic generalized epilepsies (IGEs) and such rearrangements remain the strongest risk factor for IGE known to date. Increasing evidence suggests that genetic variations can be highly population-specific. Therefore, we aimed to assess the frequency of 15q13.3 microdeletions in IGE patients from Bulgaria. METHODS: A cohort of 100 patients with various IGE syndromes was screened for large deletions/duplications by MLPA. All deletions and duplications were confirmed by array CGH analysis as previously described. RESULTS: In 100 prospectively recruited Bulgarian patients with IGE, we found one case with a microdeletion, which amounted to 1% frequency for this copy number variant. CONCLUSION: We confirm the frequency of 1% for the 15q13.3 microdeletion in a prospectively recruited cohort of Bulgarian epilepsy patients, demonstrating that this variation represents a significant risk factor for IGE for various populations and that it is retrospectively detected frequency is not due to selection bias.

Autosomal-recessive congenital cerebellar ataxia is caused by mutations in metabotropic glutamate receptor 1.

Autosomal-recessive congenital cerebellar ataxia was identified in Roma patients originating from a small subisolate with a known strong founder effect. Patients presented with global developmental delay, moderate to severe stance and gait ataxia, dysarthria, mild dysdiadochokinesia, dysmetria and tremors, intellectual deficit, and mild pyramidal signs. Brain imaging revealed progressive generalized cerebellar atrophy, and inferior vermian hypoplasia and/or a constitutionally small brain were observed in some patients. Exome sequencing, used for linkage analysis on extracted SNP genotypes and for mutation detection, identified two novel (i.e., not found in any database) variants located 7 bp apart within a unique 6q24 linkage region. Both mutations cosegregated with the disease in five affected families, in which all ten patients were homozygous. The mutated gene, GRM1, encodes metabotropic glutamate receptor mGluR1, which is highly expressed in cerebellar Purkinje cells and plays an important role in cerebellar development and synaptic plasticity. The two mutations affect a gene region critical for alternative splicing and the generation of receptor isoforms; they are a 3 bp exon 8 deletion and an intron 8 splicing mutation (c.2652_2654del and c.2660+2T>G, respectively [RefSeq accession number NM_000838.3]). The functional impact of the deletion is unclear and is overshadowed by the splicing defect. Although ataxia lymphoblastoid cell lines expressed GRM1 at levels comparable to those of control cells, the aberrant transcripts skipped exon 8 or ended in intron 8 and encoded various species of nonfunctional receptors either lacking the transmembrane domain and containing abnormal intracellular tails or completely missing the tail. The study implicates mGluR1 in human hereditary ataxia. It also illustrates the potential of the Roma founder populations for mutation identification by exome sequencing.

Spontaneous recurrent mutations and a complex rearrangement in the MECP2 gene in the light of current models of mutagenesis.

Mutations in the methyl-CpG-binding protein 2 (MECP2) gene are associated with Rett syndrome (RTT). The MECP2 gene has some unique characteristics: (1) it is mainly affected by de novo mutations, due to recurrent independent mutational events in a defined "hot spot" regions or positions; (2) complex mutational events along a single allele are frequently found in this gene; (3) most mutations arise on paternal X chromosome. The recurrent point mutations involve mainly CpG dinucleotides, where C>T transitions are explained by methylation-mediated deamination. The complex mutational events might be explained by the genomic architecture of the region involving the MECP2 gene. The finding that most spontaneous mutations arise on paternal X-chromosome supports the higher contribution of replication-mediated mechanism of mutagenesis. We present 9 types of mutations in the MECP2 gene, detected in a group of 22 Bulgarian and 6 Romanian classical RTT patients. Thirteen patients were clarified on molecular level (46.4%). The point mutations in our sample account for 61.5%. One intraexonic deletion was detected in the present study (7.7%). One novel insertion c.321_322insGAAG, p.(Lys107_Leu108insGluAlafs2*) was found (7.7%). Large deletions and complex mutations account for 23%. A novel complex mutational event c.[584_624del41insTT; 638delTinsCA] was detected in a Romanian patient. We discuss different types of the MECP2 mutations detected in our sample in the light of the possible mechanisms of mutagenesis. Complex gene rearrangements involving a combination of deletions and insertions have always been most difficult to detect, to specify precisely and hence to explain in terms of their underlying mutational mechanisms.

New mutations in the ATM gene and clinical data of 25 AT patients.

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by cerebellar degeneration, immunodeficiency, oculocutaneous telangiectasias, chromosomal instability, radiosensitivity, and cancer predisposition. The gene mutated in the patients, ATM, encodes a member of the phosphatidylinositol 3-kinase family proteins. The ATM protein has a key role in the cellular response to DNA damage. Truncating and splice site mutations in ATM have been found in most patients with the classical AT phenotype. Here we report of our extensive ATM mutation screening on 25 AT patients from 19 families of different ethnic origin. Previously unknown mutations were identified in six patients including a new homozygous missense mutation, c.8110T>C (p.Cys2704Arg), in a severely affected patient. Comprehensive clinical data are presented for all patients described here along with data on ATM function generated by analysis of cell lines established from a subset of the patients.

Mosaicism of a missense SCN1A mutation and Dravet syndrome in a Roma/Gypsy family.

SCN1A mutations account for a large proportion of Dravet syndrome patients, and are reported in other cases of epilepsy, such as some families with genetic epilepsy with febrile seizures plus (GEFS+). While most Dravet syndrome cases are caused by de novo mutations, 5% inherit a mutation from a mildly affected or symptom-free parent. Parental mosaicism has been identified, with documented cases involving truncating mutations or gene rearrangements. We describe a Roma/Gypsy family, where a missense mutation in SCN1A, p.D194N, is transmitted from a mosaic GEFS+ father to a child with Dravet syndrome. Mosaicism may be more common than assumed and should be considered regardless of the nature of the mutation.

Generalized epilepsy with febrile seizures plus: novel SCN1A mutation.

Genetic generalized epilepsy with febrile seizures plus (GEFS+) is an idiopathic generalized epileptic syndrome of heterogeneous phenotype. The cases described here are of two brothers, one with severe myoclonic epilepsy of infancy (Dravet syndrome) and the other myoclonic-astatic epilepsy. Their father experienced one simple febrile seizure in infancy and two generalized tonic-clonic seizures after head trauma in adulthood, and had generalized epileptiform activity in the electroencephalogram. He died in a severe sport accident before genetic testing could be performed. In both siblings, but not in their healthy mother, DNA analysis identified an unreported point mutation (c.3925 C>T) in exon 20 of the SCN1A gene. The missense mutation was therefore assumed to be inherited from the father, who had a very mild clinical picture, with a single febrile seizure and only occasional generalized tonic-clonic seizures. The offspring have GEFS+ phenotypes with opposite severity, an illustration of the broad intrafamilial variability of SCN1A gene mutations.

Transient mutism and pathologic laughter in the course of cerebellitis.

The phenomenon of cerebellar mutism with subsequent dysarthria is most commonly described as a part of posterior fossa syndrome after surgery for neoplasms in childhood. Pathologic laughter, on the other hand, is observed primarily in various neurologic diseases in adults. In the present case, a child manifested transient mutism and pathologic laughter during a severe cerebellitis. Headache, vertigo, and impaired consciousness developed during an acute respiratory infection. Thereafter, severe ataxia, mutism, and involuntary laughter became the main clinical features, as well as pyramidal signs. Magnetic resonance imaging revealed cerebellar swelling and T(2) hyperintensity. During steroid treatment, a gradual vanishing of the pathologic laughter and improvement of the motor and speech functions occurred. Recovery was slow and incomplete, and follow-up magnetic resonance imaging showed cerebellar atrophy. This case confirms that mutism is a rare, but possible, manifestation in acute parainfectious cerebellitis and provides a novel example of pathologic laughter during this disease in childhood.

Partial epilepsy syndrome in a Gypsy family linked to 5q31.3-q32.

PURPOSE: The restricted genetic diversity and homogeneous molecular basis of Mendelian disorders in isolated founder populations have rarely been explored in epilepsy research. Our long-term goal is to explore the genetic basis of epilepsies in one such population, the Gypsies. The aim of this report is the clinical and genetic characterization of a Gypsy family with a partial epilepsy syndrome. METHODS: Clinical information was collected using semistructured interviews with affected subjects and informants. At least one interictal electroencephalography (EEG) recording was performed for each patient and previous data obtained from records. Neuroimaging included structural magnetic resonance imaging (MRI). Linkage and haplotype analysis was performed using the Illumina IVb Linkage Panel, supplemented with highly informative microsatellites in linked regions and Affymetrix SNP 5.0 array data. RESULTS: We observed an early-onset partial epilepsy syndrome with seizure semiology strongly suggestive of temporal lobe epilepsy (TLE), with mild intellectual deficit co-occurring in a large proportion of the patients. Psychiatric morbidity was common in the extended pedigree but did not cosegregate with epilepsy. Linkage analysis definitively excluded previously reported loci, and identified a novel locus on 5q31.3-q32 with an logarithm of the odds (LOD) score of 3 corresponding to the expected maximum in this family. DISCUSSION: The syndrome can be classified as familial temporal lobe epilepsy (FTLE) or possibly a new syndrome with mild intellectual deficit. The linked 5q region does not contain any ion channel-encoding genes and is thus likely to contribute new knowledge about epilepsy pathogenesis. Identification of the mutation in this family and in additional patients will define the full phenotypic spectrum.

Early clinical and electrophysiologic features of the two most common autosomal recessive forms of Charcot-Marie-Tooth disease in the Roma (Gypsies).

Our recent studies of the genetic epidemiology of neuromuscular disorders in Gypsies in Bulgaria have revealed that two private disorders, hereditary motor and sensory neuropathy type Lom and hereditary motor and sensory neuropathy type Russe, account for most cases of Charcot-Marie-Tooth disease in this population. In this study, we examined the clinical and electrophysiologic manifestations of the two disorders in childhood, aiming to identify the distinctive features that allow early differential diagnosis. The study included 13 patients, aged between 2 and 15 years. The childhood clinical manifestations of both neuropathies were similar, although they tended to be more severe in hereditary motor and sensory neuropathy type Lom. The nerve conduction velocities in hereditary motor and sensory neuropathy type Lom were lower than in hereditary motor and sensory neuropathy type Russe. Brainstem auditory evoked potentials were abnormal in hereditary motor and sensory neuropathy type Lom, even at an early age, and normal in hereditary motor and sensory neuropathy type Russe. Although electrophysiologic data provide a more reliable differentiation than clinical data, the definitive diagnosis should rely on genetic testing. (J Child Neurol 2006;21:20-25).

Acute reversible parkinsonism in Epstein-Barr virus-related encephalitis lethargica-like illness.

A 10-year-old boy presented with fever, headache, vomiting, and hypersomnolence. An akinetic-rigid syndrome with tremor, dysphagia, dysphonia, and sialorrhea, as well as pyramidal signs, developed. Slightly elevated protein content was found in the cerebrospinal fluid and serological investigations were suggestive of a primary Epstein-Barr virus infection. Magnetic resonance imaging (MRI) showed massive bilateral hyperintense striatal and punctiform periventricular lesions. After 2-month treatment with steroids and antiparkinsonian drugs, all features resolved without sequelae. Control MRI demonstrated only minimal residual lesions in both putamina. Strongly resembling the encephalitis lethargica-like syndrome, this case is an unusual presentation of parainfectious acute disseminated encephalitis.

Case of subacute sclerosing panencephalitis with atypical absences and myoclonic-atonic seizures as a first symptom.

The 2-year history of a 10-year-old boy with subacute sclerosing panencephalitis is presented. After 6 months of epilepsy manifested by atypical absences and myoclonic-atonic seizures with an electroencephalographic (EEG) correlation of generalized spike-and polyspike-wave discharges, the child developed chorioretinitis and demonstrated a dramatic intellectual decline during corticosteroid treatment. Neuroimaging investigations did not demonstrate any pathologic changes, including at the time of fully developed disease, with neurologic deficits, periodic spasms, polymorphic epileptic seizures, and dementia. The typical generalized periodic complexes and general slowing replaced the epileptic abnormalities in the EEG. The initial anticonvulsant treatment was temporarily effective, and the purposeful isoprinosine therapy had no significant beneficial effect. The subacute sclerosing panencephalitis rapidly developed to stages III to IV, and only during the vegetative state did computed tomography show marked brain atrophy. This case is among the few described in the literature with atypical absences and myoclonic-atonic seizures as the first manifestation of subacute sclerosing panencephalitis.

Subacute sclerosing panencephalitis in Bulgaria (1978-2002).

The epidemiology of subacute sclerosing panencephalitis (SSPE) has changed substantially since the introduction of measles vaccine. We studied the incidence of SSPE in Bulgaria based on cases admitted to the Child Neurology Clinic, University Hospital of Neurology and Psychiatry, Sofia, for a 25-year period (1978-2002). The SSPE incidence prior to and during the period of routine measles immunization was analyzed. SSPE was diagnosed in 40 children (29 males and 11 females, mean age 8.5 years), 28 from 1978 to 1984 (average 4 patients/year), and 12 from 1995 to 2002 (average 1.7 patients/year). Thirty-eight cases (95%) were non-immunized and had early measles infection. Age at onset of SSPE ranged from 8 to 11 years (52.5%) with a mean latent period of 7 years following measles infection. The increase in SSPE incidence (1995-2002) following a 10-year disease-free period (1985-1994) appears to be related to early measles infection (mean age 11 months) during the measles epidemic of 1991-1992. During the period 1995-2002, children had earlier measles infection (average 11 months) and earlier onset of SSPE (mean age 8.4 years) than in the period 1978-1984 (mean age at measles infection 18 months, and of SSPE onset 11.2 years). The SSPE incidence in Bulgaria during the 25-year period from 1978 to 2002 confirms the importance of early measles infection as a risk factor for SSPE, and the role of routine measles immunization in SSPE prevention.

Clinical and epidemiological characteristics of subacute sclerosing panencephalitis in Bulgaria during the past 25 years (1978-2002).

The aims of the present study are to establish the subacute sclerosing panencephalitis (SSPE) incidence in Bulgaria for the 25-year period 1978-2002; to analyze the SSPE incidence prior to, and in the period of, routine measles immunization; and, to analyze the clinical characteristics of SSPE. SSPE was diagnosed in a total of 40 children; 28 of were diagnosed between 1978 and 1984, and 12 between 1995 and 2002. Thirty-eight cases (95%) were non-immunized and have had an early measles infection (mean age 16 months). The SSPE onset occurred primarily between 8 and 11 years of age (52.5%) with a mean latent period of about 7 years after the measles infection. After the 10-year disease-free period (1985-1994), the SSPE incidence increased between 1995 and 2002 because of the 1991-1992 measles epidemic. During the period 1995-2002 children with earlier measles infection and earlier SSPE onset predominated, compared to the period 1978-1984. The initial clinical manifestations included intellectual deterioration in 35%, extrapyramidal hyperkinesias in 29%, epileptic seizures in 15%, hemiparesis in 10%, and visual disturbances in 10% of the cases. Nine children (22.5%) demonstrated an atypical onset. A rapidly progressive course was observed in 4 children (10%) and a chronic progressive course with pseudoremissions over 2 years-in 8 cases (20%). Our analysis of the SSPE incidence in Bulgaria for the 25-year period (1978-2002) supports the importance of early measles infection as a crucial risk factor for this persistent neuroinfection. Moreover, it confirms the role of routine measles immunization in SSPE prevention.