Rare intronic mutation between Exon 62 and 63 (c.9225-285A>G) of the dystrophin gene associated with atypical BMD phenotype.

Dystrophinopathies are predominantly caused by deletions, duplications and point mutations in the coding regions of the dystrophin gene with less than 1% of all pathogenic mutations identified within intronic sequences. We describe a 17-year-old male with a Becker muscular dystrophy diagnosis and mental disability due to an intron mutation that led to aberrant splicing and formation of an additional exon. Histopathological analysis of muscle tissue revealed signs of muscular dystrophy and reduced signal for dystrophin, alpha-sarcoglycan, and alpha-dystroglycan. Multiplex ligation-dependent probe amplification screening and total sequencing of the dystrophin gene did not identify a mutation in the coding regions. However, next generation sequencing revealed an intron mutation between exons 62 and 63 of the dystrophin gene known for pseudoexon formation and disruption of the reading frame. We report a functional consequence of this mutation as an increased intracellular-weighted sodium signal (assessed by 23Na-magnetic resonance imaging) in leg muscles.

[Congenital myasthenic syndromes in adulthood : Challenging, rare but treatable]. / Kongenitale myasthene Syndrome im Erwachsenenalter : Diagnostisch herausfordernd, selten, aber behandelbar.

The congenital myasthenic syndromes (CMS) represent a heterogeneous group of diseases with a broad spectrum of phenotypes. The common characteristic is an inherited genetic defect of the neuromuscular junction. Although in some patients the specific gene defect remains to be detected, the increasing identification of causative genes in recent years has already provided unique insights into the functionality of structural proteins at the neuromuscular junction. Neonatal and early childhood onset is observed in most CMS subtypes; however, late onset in adolescence or adulthood also occurs and establishing the diagnosis at these stages imposes particular challenges. To enable appropriate therapeutic interventions for an at least in principle treatable condition, determining the genetic cause is warranted. In this overview, the critical clinical and diagnostic features of the different CMS subtypes are presented and illustrated using typical cases. Furthermore, specific diagnostic clues are outlined. Finally, the overlap between CMS and muscular dystrophies is discussed. Illustrating characteristic patient examples, the essential clinical and additional diagnostic findings of various CMS subtypes and special diagnostic indications are presented.

Extension of the phenotype of biallelic loss-of-function mutations in SLC25A46 to the severe form of pontocerebellar hypoplasia type I.

Biallelic mutations in SLC25A46, encoding a modified solute transporter involved in mitochondrial dynamics, have been identified in a wide range of conditions such as hereditary motor and sensory neuropathy with optic atrophy type VIB (OMIM: *610826) and congenital lethal pontocerebellar hypoplasia (PCH). To date, 18 patients from 13 families have been reported, presenting with the key clinical features of optic atrophy, peripheral neuropathy, and cerebellar atrophy. The course of the disease was highly variable ranging from severe muscular hypotonia at birth and early death to first manifestations in late childhood and survival into the fifties. Here we report on 4 patients from 2 families diagnosed with PCH who died within the first month of life from respiratory insufficiency. Patients from 1 family had pathoanatomically proven spinal motor neuron degeneration (PCH1). Using exome sequencing, we identified biallelic disease-segregating loss-of-function mutations in SLC25A46 in both families. Our study adds to the definition of the SLC25A46-associated phenotypic spectrum that includes neonatal fatalities due to PCH as the severe extreme.

Molecular characterization of congenital myasthenic syndromes in Spain.

Congenital myasthenic syndromes (CMS) are a heterogeneous group of genetic disorders, all of which impair neuromuscular transmission. Epidemiological data and frequencies of gene mutations are scarce in the literature. Here we describe the molecular genetic and clinical findings of sixty-four genetically confirmed CMS patients from Spain. Thirty-six mutations in the CHRNE, RAPSN, COLQ, GFPT1, DOK7, CHRNG, GMPPB, CHAT, CHRNA1, and CHRNB1 genes were identified in our patients, with five of them not reported so far. These data provide an overview on the relative frequencies of the different CMS subtypes in a large Spanish population. CHRNE mutations are the most common cause of CMS in Spain, accounting for 27% of the total. The second most common are RAPSN mutations. We found a higher rate of GFPT1 mutations in comparison with other populations. Remarkably, several founder mutations made a large contribution to CMS in Spain: RAPSN c.264C > A (p.Asn88Lys), CHRNE c.130insG (Glu44Glyfs*3), CHRNE c.1353insG (p.Asn542Gluf*4), DOK7 c.1124_1127dup (p.Ala378Serfs*30), and particularly frequent in Spain in comparison with other populations, COLQ c.1289A > C (p.Tyr430Ser). Furthermore, we describe phenotypes and distinguishing clinical signs associated with the various CMS genes which might help to identify specific CMS subtypes to guide diagnosis and management.

Novel homozygous RARS2 mutation in two siblings without pontocerebellar hypoplasia - further expansion of the phenotypic spectrum.

BACKGROUND: Pontocerebellar hypoplasia type 6 (PCH6) is a mitochondrial disease caused by mutations in the RARS2 gene. RARS2 encodes mitochondrial arginyl transfer RNA synthetase, an enzyme involved in mitochondrial protein translation. A total of 27 patients from 14 families have been reported so far. Characteristic clinical features comprise neonatal lactic acidosis, severe encephalopathy, intractable seizures, feeding problems and profound developmental delay. Most patients show typical neuroradiologic abnormalities including cerebellar hypoplasia and progressive pontocerebellar atrophy. METHODS: We describe the clinical, biochemical and molecular features of 2 siblings with a novel homozygous mutation in RARS2. Both patients presented neonatally with lactic acidosis. While the older sibling had severe neurological symptoms with microcephaly, seizures and developmental delay, the younger patient was still neurologically asymptomatic at the age of 2 months. RESULTS: MRI studies in both children lacked pontocerebellar involvement. The expression of the OXPHOS complex proteins was decreased in both patients, whereas oxygen consumption was increased. CONCLUSIONS: Characteristic neuroradiological abnormalities of PCH6 such as vermis and cerebellar hypoplasia and progressive pontocerebellar atrophy may be missing in patients with RARS2 mutations. RARS2 testing should therefore also be performed in patients without pontocerebellar hypoplasia but otherwise typical clinical symptoms.

Long-term follow-up in patients with congenital myasthenic syndrome due to RAPSN mutations.

Rapsyn (RAPSN) mutations are a common cause of postsynaptic congenital myasthenic syndromes. We present a comprehensive description of the clinical and molecular findings of ten patients with CMS due to mutations in RAPSN, mostly with a long-term follow-up. Two patients were homozygous and eight were heterozygous for the common p.Asn88Lys mutation. In three of the heterozygous patients we have identified three novel mutations (c.869T > C; p.Leu290Pro, c.1185delG; p.Thr396Profs*12, and c.358delC; p.Gln120Serfs*8). In our cohort, the RAPSN mutations lead to a relatively homogeneous phenotype, characterized by fluctuating ptosis, occasional bulbar symptoms, neck muscle weakness, and mild proximal muscle weakness with exacerbations precipitated by minor infections. Interestingly, episodic exacerbations continue to occur during adulthood. These were characterized by proximal limb girdle weakness and ptosis, and not so much by respiratory insufficiency after age 6. All patients presented during neonatal period and responded to cholinergic agonists. In most of the affected patients, additional use of 3,4-diaminopyridine resulted in significant clinical benefit. The disease course is stable except for intermittent worsening.

KLHL40-related nemaline myopathy with a sustained, positive response to treatment with acetylcholinesterase inhibitors.

Congenital myopathies are a group of inherited muscle disorders characterized by hypotonia, weakness and a non-dystrophic muscle biopsy with the presence of one or more characteristic histological features. Neuromuscular transmission defects have recently been reported in several patients with congenital myopathies (CM). Mutations in KLHL40 are among the most common causes of severe forms of nemaline myopathy. Clinical features of affected individuals include fetal akinesia or hypokinesia, respiratory failure, and swallowing difficulties at birth. Muscle weakness is usually severe and nearly half of the individuals have no spontaneous antigravity movement. The average age of death has been reported to be 5 months in a recent case series. Herein we present a case of a patient with a nemaline myopathy due to KLHL40 mutations (c.604delG, p.Ala202Argfs*56 and c.1513G>C, p.Ala505Pro) with an impressive and prolonged beneficial response to treatment with high-dose pyridostigmine. Myasthenic features or response to ACEI have not previously been reported as a characteristic of nemaline myopathy or KLHL40-related myopathy.

Novel TPM3 mutation in a family with cap myopathy and review of the literature.

Cap myopathy is a rare congenital myopathy characterized by the presence of caps within muscle fibres and caused by mutations in ACTA1, TPM2 or TPM3. Thus far, only three cases with TPM3-related cap myopathy have been described. Here, we report on the first autosomal dominant family with cap myopathy in three-generations, caused by a novel heterozygous mutation in the alpha-tropomyosin-slow-encoding gene (TPM3; exon 4; c.445C>A; p.Leu149Ile). The three patients experienced first symptoms of muscle weakness in childhood and followed a slowly progressive course. They presented generalized hypotrophy and mild muscle weakness, elongated face, high arched palate, micrognathia, scoliosis and respiratory involvement. Intrafamilial variability of skeletal deformities, respiratory involvement and mild cardiac abnormalities was noted. Muscle MRI revealed a recognizable pattern of fatty muscle infiltration and masseter muscle hypertrophy. Subsarcolemmal caps were present in 6-10% of the fibres and immunoreactive with anti-tropomyosin antibodies. We conclude that the MRI-pattern of muscle involvement and the presence of masseter muscle hypertrophy in cap myopathy may guide molecular genetic diagnosis towards a mutation in TPM3. Regular respiratory examinations are important, even if patients have no anamnestic clues. We compare our findings to all cases of cap myopathy with identified mutations (n=11), thus far reported in the literature.

[Neuromuscular signal transmission in adulthood. Current facets of acquired and hereditary disorders]. / Neuromuskuläre Signalübertragung im Erwachsenenalter. Aktuelle Facetten erworbener und angeborener Störungen.

The availability of early diagnosis and modern effective therapies has reduced mortality and disability linked to late-onset acquired or hereditary neuromuscular transmission disorders. Nevertheless, identification of the pathogenesis of these diseases remains a challenge. In addition to non-specific and fluctuating presenting symptoms current diagnostic work-up strategies include electrophysiology, antibody measurements and less frequently molecular genetics. For differential diagnostic purposes there is an increasing demand for improving awareness concerning late-onset congenital myasthenic syndromes (CMS) which are rare but nevertheless symptomatically treatable diseases. Especially in seronegative myasthenic syndromes, molecular genetic analyses of CMS genes should be integrated into the differential diagnostic work-up. Therefore, some facets of neuromuscular synaptogenesis in the context of seronegative acquired myasthenic syndromes and recently uncovered congenital myasthenic syndromes are reviewed.

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.

Severe Myoclonic Epilepsy in Infancy - Adult Phenotype with Bradykinesia, Hypomimia, and Perseverative Behavior: Report of Five Cases.

Dravet syndrome or severe myoclonic epilepsy in infancy (SMEI) is an epileptic syndrome characterised by refractory epilepsy and intellectual disability, typically presenting with febrile and afebrile generalised and unilateral clonic/tonic-clonic seizures in the first year of life and other types of seizures appearing later in the course of the disease. Five adult patients with SMEI and SCN1A mutations are reported, in which motor and behavioural abnormalities were outstanding symptoms. Bradykinesia, responding with latency, slow speaking with a thin voice, midface hypomimia and perseveration were distinctive features in all cases. These symptoms may be fit to define the adult phenotype of SMEI beyond seizure/epilepsy criteria. The motor and behavioural symptoms are discussed in the context of a possibly underlying frontal lobe/mesofrontal and cerebellar dysfunction.

Ephedrine therapy in eight patients with congenital myasthenic syndrome due to DOK7 mutations.

In congenital myasthenic syndrome with DOK7 mutations ephedrine was reported to be beneficial in single patients. We carried out a small, open and prospective cohort study in eight European patients manifesting from birth to 12 years. Five patients showed limb-girdle and facial weakness, three a floppy infant syndrome with bulbar symptoms and/or respiratory distress. Ephedrine was started with 25 mg/day and slowly increased to 75-100 mg/day. Within weeks after starting therapy an improvement was observed in all patients and clinical follow-up disclosed positive effects more pronounced on proximal muscle weakness and strength using MRC scale. Effects on facial weakness were less pronounced. Vital capacity measurements and repetitive stimulation tests did not improve in the same way as clinical symptoms did. These investigations are appropriate to confirm the diagnosis in case of pathological results, but they might not be appropriate means to monitor patients under ephedrine therapy.

Impaired receptor clustering in congenital myasthenic syndrome with novel RAPSN mutations.

OBJECTIVE: Congenital myasthenic syndromes (CMS) with underlying RAPSN mutations turned out to be of high clinical relevance due to their worldwide frequency. To date, all reported patients with CMS with sequence variations in the translated region of RAPSN carry the mutation N88K on at least one allele. The authors report two patients lacking the common N88K allele but harboring differing novel mutations of the RAPSN gene on both alleles: one patient is homozygous for a missense mutation (R164C); the second patient is compound heterozygous for a splice (IVS1-15C>A) and another missense mutation (L283P). METHODS: The authors analyzed the RAPSN gene for sequence variations and carried out in vitro studies in order to delineate the potential pathogenicity of the three novel RAPSN mutations. RESULTS: For the putative splice mutation (IVS1-15C>A), the authors constructed wild-type and mutated RAPSN minigenes for transfection and subsequent RNA analysis. The mutation generates a novel acceptor splice site leading to retention of 13 nucleotides of intron 1 in the mature mRNA and subsequently to a frameshift transcript. Cotransfection of wild-type AChR subunits with RAPSN-constructs carrying R164C and L283P indicate that both mutations diminish coclustering of AChR with rapsyn. CONCLUSIONS: Screening for the common mutation RAPSN N88K facilitates targeted genetic analysis in congenital myasthenic syndromes. However, absence of a N88K allele does not exclude underlying RAPSN mutations as cause of the congenital myasthenic syndromes. Sequencing of the entire gene may be considered in patients with joint contractures and respiratory problems even in the absence of the mutation N88K.

Long-term improvement of slow-channel congenital myasthenic syndrome with fluoxetine.

We report on a 15-year-old patient who was diagnosed with congenital myasthenic syndrome (CMS) at the age of 7 months. At initial diagnosis, the CMS was not further characterized. The patient was treated for several years with the anticholinesterase drug (Mestinon), without clinical benefit. The patient deteriorated progressively and became dependent on home nocturnal ventilatory support, being unable to take part in daily life activities at age of 12 years. At age 14, the slow-channel syndrome mutation CHRNE L269F (805C>T) was detected and acetylcholinesterase inhibitor therapy was immediately stopped. Fluoxetine therapy was started and gradually increased over 2 months. The boy improved dramatically in strength and endurance and was taken off ventilatory support 1 month after the fluoxetine therapy was initiated. The clinical improvement was confirmed by functional respiratory and electrophysiological tests.

Leigh syndrome caused by mutations in the flavoprotein (Fp) subunit of succinate dehydrogenase (SDHA).

Detailed clinical, neuroradiological, histological, biochemical, and genetic investigations were undertaken in a child suffering from Leigh syndrome. The clinical symptoms started at age five months and led to a severe progressive neurodegenerative disorder causing epilepsy, psychomotor retardation, and tetraspasticity. Biochemical measurement of skeletal muscle showed a severe decrease in mitochondrial complex II. Sequencing of SDHA revealed compound heterozygosity for a nonsense mutation in exon 4 (W119X) and a missense mutation in exon 3 (A83V), both absent in normal controls. In six additional patients--five with Leigh or Leigh-like syndrome and one with neuropathy and ataxia associated with isolated deficiency of complex II--mutations in SDHA were not detected, indicating genetic heterogeneity.

Facing the genetic heterogeneity in neuromuscular disorders: linkage analysis as an economic diagnostic approach towards the molecular diagnosis.

The identification of an ever increasing number of gene defects in patients with neuromuscular disorders has disclosed both marked phenotype and genotype variability and considerable disease overlap. In order to offer an economic strategy to characterise the molecular defect in patients with unclassified neuromuscular disorders, we designed DNA marker sets for linkage analysis of 62 distinct neuromuscular disorders gene loci, including all known muscular dystrophies, congenital myopathies, congenital myasthenic syndromes and myotonias. Genotyping of marker loci of 140 clinically well-characterised families with unclassified neuromuscular disorders reduced the number of candidates to one or two genes in 49 % of the families. Subsequent mutation analysis and genome-wide scans enabled the determination of the genetic defect in 31 % of the families including the identification of a new gene and a new mutation in an unexpected candidate gene. This highlights the effective application of this approach both for diagnostic strategies as well as for the identification of new loci and genes.

An intronic base alteration of the CHRNE gene leading to a congenital myasthenic syndrome.

Reported is a patient with a congenital myasthenic syndrome due to two compound heterozygous mutations of the CHRNE gene. The molecular consequences of a novel intronic base alteration (CHRNE IVS5-16GA) remote from the splice acceptor site were investigated in vivo and in vitro. In conclusion, RNA analysis may be necessary to reveal unexpected splicing aberrations due to intronic mutations that are not part of the consensus splice site.