Violeta Mihaylova

Congenital myasthenic syndromes: spotlight on genetic defects of neuromuscular transmission

The neuromuscular junction (NMJ) is a complex structure that efficiently communicates the electrical impulse from the motor neuron to the skeletal muscle to induce muscle contraction. Genetic and autoimmune disorders known to compromise neuromuscular transmission are providing further insights into the complexities of NMJ function. Congenital myasthenic syndromes (CMSs) are a genetically and phenotypically heterogeneous group of rare hereditary disorders affecting neuromuscular transmission. The understanding of the molecular basis of the different types of CMSs has evolved rapidly in recent years. Mutations were first identified in the subunits of the nicotinic acetylcholine receptor (AChR), but now mutations in ten different genes - encoding post-, pre- or synaptic proteins - are known to cause CMSs. Pathogenic mechanisms leading to an impaired neuromuscular transmission modify AChRs or endplate structure or lead to decreased acetylcholine synthesis and release. However, the genetic background of many CMS forms is still unresolved. A precise molecular classification of CMS type is of paramount importance for the diagnosis, counselling and therapy of a patient, as different drugs may be beneficial or deleterious depending on the molecular background of the particular CMS.

Congenital myasthenic syndromes: achievements and limitations of phenotype-guided gene-after-gene sequencing in diagnostic practice: a study of 680 patients.

Congenital myasthenic syndromes (CMSs) are clinically and genetically heterogeneous disorders characterized by a neuromuscular transmission defect. Even though CMSs are genetic disorders, they are highly treatable, and the appropriate drug treatment depends on the underlying genetic defect. This highlights the importance of genetic testing in CMS. In recent years, the molecular basis of CMS has constantly broadened and disease‐associated mutations have been identified in 14 genes encoding proteins of the neuromuscular junction. In the dawn of novel sequencing strategies, we report on our 14‐year experience in traditional Sanger‐based mutation screening of a large cohort of 680 independent patients with suspected CMS. In total, we identified disease‐causing mutations in 299 patients (44%) of patients in various known CMS genes, confirming the high degree of genetic heterogeneity associated with the disease. Apart from four known founder mutations, and a few additional recurrent mutations, the majority of variants are private, found in single families. The impact of previously reported genotype–phenotype correlations on efficiency of genetic testing was analyzed in our population. Taking our experiment into account, we present our algorithm for genetic testing in CMS. Hum Mutat 33:1474–1484, 2012.

REFINEMENT OF THE CLINICAL PHENOTYPE IN MUSK-RELATED CONGENITAL MYASTHENIC SYNDROMES

Congenital myasthenic syndromes (CMS) are a heterogeneous group of inherited disorders caused by genetic defects that affect transmission at the neuromuscular junction.1 To date, 10 genes are known to cause CMS if mutated.2 Mutations in the muscle specific kinase ( MUSK ) gene have been published in a single family worldwide.3 Two siblings of this family were reported carrying heteroallelic MUSK mutations. ### Case reports. We report on CMS caused by a novel homozygous missense mutation in MUSK in 5 affected sibs (patients 1–5) from a consanguineous Sudanese family. The father and the maternal grandmother are first-degree cousins. The patients were followed up for 5 years (ages at the end of follow-up: 9–19.5 years). All studies were carried out with informed consent of the patients’ parents and approved by the institutional ethics review board. All affected individuals demonstrated ptosis at age 1–3 years and fatigability, when walking for a long distance, more pronounced in the evening. At the first examination they had exercise-induced weakness of the deltoid muscle. Four of them had partial ophthalmoparesis (except patient 3). Patients 2 and 3 showed modified Gowers sign and waddling gait or pronounced lordosis, respectively. Treatment with pyridostigmine (30–60 mg/day) led to slight benefit. Increased doses were reported to result in the “feeling of muscle stiffness” and the medication was discontinued. Five years later (table), there was some progression, i.e., ophthalmoparesis in all, involving additional directions of gaze in patients 2 and 5 and mild facial weakness. Patient …

Distal myopathy with upper limb predominance caused by filamin C haploinsufficiency.

Objective: In this study, we investigated the detailed clinical findings and underlying genetic defect in 3 presumably related Bulgarian families displaying dominantly transmitted adult onset distal myopathy with upper limb predominance. Methods: We performed neurologic, electrophysiologic, radiologic, and histopathologic analyses of 13 patients and 13 at-risk but asymptomatic individuals from 3 generations. Genome-wide parametric linkage analysis was followed by bidirectional sequencing of the filamin C (FLNC) gene. We characterized the identified nonsense mutation at cDNA and protein level. Results: Based on clinical findings, no known myopathy subtype was implicated in our distal myopathy patients. Light microscopic analysis of affected muscle tissue showed no specific hallmarks; however, the electron microscopy revealed changes compatible with myofibrillar myopathy. Linkage studies delineated a 9.76 Mb region on chromosome 7q22.1-q35 containing filamin C (FLNC), a gene previously associated with myofibrillar myopathy. Mutation analysis revealed a novel c.5160delC frameshift deletion in all patients of the 3 families. The mutation results in a premature stop codon (p.Phe1720LeufsX63) that triggers nonsense-mediated mRNA decay. FLNC transcript levels were reduced in muscle and lymphoblast cells from affected subjects and partial loss of FLNC in muscle tissue was confirmed by protein analysis. Conclusions: The FLNC mutation that we identified is distinct in terms of the associated phenotype, muscle morphology, and underlying molecular mechanism, thus extending the currently recognized clinical and genetic spectrum of filaminopathies. We conclude that filamin C is a dosage-sensitive gene and that FLNC haploinsufficiency can cause a specific type of myopathy in humans.

Molecular characterisation of congenital myasthenic syndromes in Southern Brazil

Objective To perform genetic testing of patients with congenital myasthenic syndromes (CMS) from the Southern Brazilian state of Parana. Patients and methods Twenty-five CMS patients from 18 independent families were included in the study. Known CMS genes were sequenced and restriction digest for the mutation RAPSN p.N88K was performed in all patients. Results We identified recessive mutations of CHRNE in ten families, mutations in DOK7 in three families and mutations in COLQ, CHRNA1 and CHRNB1 in one family each. The mutation CHRNE c.70insG was found in six families. We have repeatedly identified this mutation in patients from Spain and Portugal and haplotype studies indicate that CHRNE c.70insG derives from a common ancestor. Conclusions Recessive mutations in CHRNE are the major cause of CMS in Southern Brazil with a common mutation introduced by Hispanic settlers. The second most common cause is mutations in DOK7. The minimum prevalence of CMS in Parana is 0.18/100 000.

Genetic heterogeneity and minor CYP1B1 involvement in the molecular basis of primary congenital glaucoma in Gypsies

Primary congenital glaucoma (PCG) is a genetically heterogeneous disorder of autosomal recessive inheritance, with mutations in the cytochrome P450 1B1 (CYP1B1) gene detected in an average of ∼50% of cases worldwide. The Roma/Gypsies are considered to be a rare example of a single founder CYP1B1 mutation, E387K (identified in the Slovak Roma), accounting for 100% of disease alleles. Contrary to this concept, unusual genetic heterogeneity was revealed in this study of 21 Gypsy PCG patients from Bulgaria and 715 controls from the general Gypsy population. In our small sample of affected subjects, we identified five different CYP1B1 mutations – four known (E229K, R368H, E387K and R390C) and one novel and potentially pathogenic (F445I), which together accounted for ∼30% of disease alleles. E387K was rare in both the patient and the control group, indicating that its high frequency in the Slovak Roma is the product of local founder effect not representative of the overall molecular pattern of PCG in the Gypsy population. Data on other Mendelian disorders and on the population genetics of the Gypsies suggest that a true founder mutation is likely to exist and has remained undetected. Our analysis of another candidate gene, MYOC, and the GLC3B and GLC3C loci did not provide support for their involvement. The molecular basis of PCG in the Gypsies is thus unresolved, and diagnostic analyses should be extended beyond the E387K mutation.

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.

GNE myopathy in Roma patients homozygous for the p.I618T founder mutation

GNE myopathy is an autosomal-recessive disorder caused by mutations in the GNE gene, encoding the key enzyme in the sialic acid biosynthetic pathway, UDP-N-acetylglucosamine 2-epimerase/N-acetyl mannosamine kinase. We studied 50 Bulgarian Roma patients homozygous for p.I618T, an ancient founder mutation in the kinase domain of the GNE gene, dating before the Gypsy exodus from North West India. The clinical features in the Bulgarian GNE group can be described with disease onset mostly in the third decade, but in individual cases, onset was as early as 10 years of age. The majority of patients had foot drop as the first symptom, but three patients developed hand weakness first. Muscle weakness was early and severe for the tibialis anterior, and minimal or late for quadriceps femoris, and respiratory muscles were only subclinically affected even in the advanced stages of the disease. During a 15-year follow-up period, 32 patients became non-ambulant. The average period between disease onset and loss of ambulation was 10.34 ± 4.31 years, ranging from 3 to 20 years. Our analysis of affected sib pairs suggested a possible role of genetic modifying factors, accounting for significant variation in disease severity.

Wilson's disease in two consecutive generations in a Bulgarian Roma family.

Sirs: Wilson’s disease (WD) is a relatively rare inherited autosomal recessive disorder of copper metabolism that results in accumulation of copper in the liver and subsequently in other organs, mainly the central nervous system [1, 2]. The diagnosis is determined by the presence of the characteristic symptoms in conjunction with laboratory testing indicating impaired copper metabolism [2–4]. WD can be quite effectively treated; thus, early recognition and diagnosis are crucial [1, 2, 4]. We report on a family with WD in two consecutive generations belonging to the Roma sub-isolate “feredjelli”. The proband was a 24-year-old man presenting with a 2-year history of intention tremor developed first in the right hand followed by the left hand. Family history disclosed a deceased sister with liver cirrhosis, tremor and laboratory findings suggesting WD, but genetic testing was not performed. On neurological examination masked face, rigidity in the arms, festinating gait, postural tremor in both hands, dysmetria and bilateral intention tremor on knee-to-heel and finger-to-nose testing, dysdiadochokinesis and scanning dysarthria were found. Physical examination was unremarkable. Routine hematological and biochemical investigations were normal. The serum ceruloplasmin level was low: 0.08 g/l (normal > 0.2 g/l). The basal 24-hour urinary copper excretion was increased (14.6 μmol/24 h, normal < 1.1 μmol/24). On slit-lamp examination Kayser-Fleischer rings were present. The diagnosis of WD was made and treatment with d-penicillamine started. The patient married an unrelated woman from the same ethnic group and had 2 children. The proband’s son was first brought to medical attention at the age of 10 years because of acute enterocolitis. On physical examination hepatomegaly was found. The neurological examination was normal. There was elevation of aspartate transaminase (AST) 58 U/l (normal ≤ 37 U/l) and alanine transaminase (ALT) 50 U/l (normal ≤ 41 U/l). The ceruloplasmin was 0.14 g/l, the basal 24-h urinary copper excretion was 4.74 μmol/24 h. After oral administration of 1000 mg/d d-penicillamine for two days and 24-h urine collection during the second day, the urinary copper excretion was 41.58 μmol/24 h (d-penicillamine challenge test, values > 25 μmoles/24 h considered diagnostic for WD) [4]. The slitlamp examination failed to demonstrate Kayser-Fleischer rings. At 6 years of age the proband’s daughter was free of symptoms but on examination hepatomegaly was found. The neurological examination was unremarkable. There was elevation of transaminase activities (AST 120 U/l, ALT 189 U/l) and alkaline phosphatase 1054 U/l (normal < 910 U/l). The ceruloplasmin was 0.24 g/l, the basal 24-h urinary copper excretion was 1.86 μmol/24 h with a significant increase after d-penicillamine challenge test (33.3 μmol/24 h). No Kayser-Fleischer rings were found on slit-lamp examination. After obtaining informed consent from the proband and his wife, blood samples from the propositus and his children were collected and genomic DNA isolated. Molecular genetic studies using SSCP analysis and subsequent direct sequencing detected homozygosity for H1069Q in the proband and his children and wild type/H1069Q heterozygosity in the proband’s wife. The presented case is another family with WD in two consecutive generations [5] and the first of Roma ethnicity (Fig. 1). Although the worldwide heterozygous carrier rate is considered to be 1:100 [3], in the endogamous population of the LETTER TO THE EDITORS

Unique PABPN1 gene mutation in a large Bulgarian family with OPMD

JO N 2769 The study included six affected individuals from two generations. The diagnosis was established based on the dominant inheritance (Fig. 1), late onset and typical clinical features (Table 1). Limb weakness was only encountered in subject 4 showing not only proximal weakness and wasting but also marked weakness and atrophy of thenar and tibialis anterior muscles (MRC 3). No sensory abnormalities were detected in any of the subjects. After obtaining informed consent, blood samples were drawn for DNA extraction from all affected and 4 unaffected individuals. The part of exon 1 of the PABPN1 gene containing the GCG/GCA elongation was sequenced as described [6]. We found three additional GCA repeats heterozygously in all affected individuals, which were not found in the four unaffected family members and in 100 healthy controls and 35 other patients with OPMD. To our knowledge, this is the first OPMD family with a (GCG)6(GCA)3(GCA)3GCG genotype. In contrast to other populations, where the majority of OPMD patients carry classical GCG repeat expansions, our single Bulgarian family demonstrated a non-GCG elongation, further supporting the genetic heterogeneity and probably the existence of multiple founders of OPMD [3, 4, 6, 9]. Similar to the other reported OPMD mutations, additional (GCA)3 repeats can be the result of a duplication of the wildtype (GCA)3 due to unequal crossingover of two PABPN1 alleles [6, 7, 9– 11]. Despite the unique genotype, our patients presented with the classical symptoms. The disease onset was in the fifth or sixth decade of life. All but one reported ptosis and dysphagia. However, V. Mihaylova T. Müller I. Petrova I. Tournev S. Cherninkova M. C. Walter M. Deschauer