New molecular findings in congenital myopathies due to selenoprotein N gene mutations

Selenoprotein N-related myopathy (SEPN1-RM) is an early-onset muscle disorder that can manifest clinically as congenital muscular dystrophy with spinal rigidity and can result in specific pathological entities such as multiminicore disease, desmin-related myopathy with Mallory body-like inclusions, and congenital fiber-type disproportion. Here we describe the clinical, histopathological, muscle magnetic resonance imaging (MRI) and genetic findings of three Italian SEPN1-RM families. Proband 1 is a 31-year-old female who was floppy at birth and developed axial and mild lower limb-girdle weakness. The second proband is a 13-year-old boy with RSMD1. Probands 3 and 4 were brothers showing clinical phenotype of congenital myopathy. Muscle MRI demonstrated selective involvement of sartorius, gluteal muscles and distal gastrocnemius and sparing of rectus femoris and gracilis. Muscle histopathology showed in proband 1 myopathic changes with mild connective tissue increase and some fibres lacking the Z-line, while probands 2 and 3 had multiminicores. SEPN1 gene analysis revealed five mutations, three of which are novel. Proband 1 was a compound heterozygote for a 92-bp (exon 1) and a 1-bp deletion (exon 9); proband 2 had a 99-bp deletion and a 10-bp duplication in exon 1, and proband 3 presented a novel homozygous mutation in intron 10 acceptor splice site.     

Two patients with 'Dropped head syndrome' due to mutations in LMNA or SEPN1 genes

Dropped head syndrome is characterized by severe weakness of neck extensor muscles with sparing of the flexors. It is a prominent sign in several neuromuscular conditions, but it may also be an isolated feature with uncertain aetiology. We report two children in whom prominent weakness of neck extensor muscles is associated with mutations in lamin A/C (LMNA) and selenoprotein N1 (SEPN1) genes, respectively. This report expands the underlying causes of the dropped head syndrome which may be the presenting feature of a congenital muscular dystrophy.     

Differential effects of PINK1 nonsense and missense mutations on mitochondrial function and morphology

Mutations of the PINK1 gene are a cause of autosomal recessive Parkinson's disease (PD). PINK1 encodes a mitochondrial kinase of unknown function which is widely expressed in both neuronal and non-neuronal cells. We have studied fibroblast cultures from four family members harbouring the homozygous p.Q456X mutation in PINK1, three of their wild-type relatives, one individual with the homozygous p.V170G mutation and five independent controls. Results showed bioenergetic abnormalities involving decreased activities of complexes I and IV along with increased activities of complexes II and III in the missense p.V170G mutant. There were increased basal levels of mitochondrial superoxide dismutase in these cells and an exaggerated increase of reduced glutathione in response to paraquat-induced free radical formation. Furthermore, swollen and enlarged mitochondria were observed in this sample. In the p.Q456X nonsense mutants, the respiratory chain enzymes were unaffected, but ATP levels were significantly decreased. These results confirm that mutations of PINK1 cause abnormal mitochondrial morphology, bioenergetic function and oxidative metabolism in human tissues but suggest that the biochemical consequences may vary between mutations.     

Desmin-related myopathy with Mallory body-like inclusions is caused by mutations of the selenoprotein N gene

Desmin-related myopathies (DRMs) are a heterogeneous group of muscle disorders, morphologically defined by intrasarcoplasmic aggregates of desmin. Mutations in the desmin and the alpha-B crystallin genes account for approximately one third of the DRM cases. The genetic basis of the other forms remain unknown, including the early-onset, recessive form with Mallory body-like inclusions (MB-DRMs), first described in five related German patients. Recently, we identified the selenoprotein N gene (SEPN1) as responsible for SEPN-related myopathy (SEPN-RM), a unique early-onset myopathy formerly divided in two different nosological categories: rigid spine muscular dystrophy and the severe form of classical multiminicore disease. The finding of Mallory body-like inclusions in two cases of genetically documented SEPN-RM led us to suspect a relationship between MB-DRM and SEPN1. In the original MB-DRM German family, we demonstrated a linkage of the disease to the SEPN1 locus (1p36), and subsequently a homozygous SEPN1 deletion (del 92 nucleotide -19/+73) in the affected patients. A comparative reevaluation showed that MB-DRM and SEPN-RM share identical clinical features. Therefore, we propose that MB-DRM should be categorized as SEPN-RM. These findings substantiate the molecular heterogeneity of DRM, expand the morphological spectrum of SEPN-RM, and implicate a necessary reassessment of the nosological boundaries in early-onset myopathies.     

A novel 3-bp deletion in the PANK2 gene of Dutch patients with pantothenate kinase-associated neurodegeneration: evidence for a founder effect

Mutation analysis was performed in four apparently unrelated Dutch families with pantothenate kinase-associated neurodegeneration, formerly known as Hallervorden-Spatz syndrome. A novel 3-bp deletion encompassing the nucleotides GAG at positions 1,142 to 1,144 of exon 5 of the PANK2 gene was found in all patients. One patient was compound heterozygous; she also carried a novel nonsense mutation (Ser68Stop). The other patients were homozygous for the 1142_1144delGAG mutation. The 1142_1144delGAG mutation was also found in a German patient of unknown descent. We used polymorphic microsatellite markers flanking the PANK2 gene (spanning a region of approximately 8 cM) for haplotype analyses in all these families. A conserved haplotype of 1.5 cM was found for the 1142_1144delGAG mutation carriers. All the Dutch families originated from the same geographical region within the Netherlands. The results indicate a founder effect and suggest that the 1142_1144delGAG mutation probably originated from one common ancestor. It was estimated that this mutation arose at the beginning of the ninth century, approximately 38 generations ago.     

Early onset myopathy with a novel mutation in the Selenoprotein N gene (SEPN1)

Mutations in SEPN1 have been associated with three autosomal recessive congenital myopathies, including rigid spine muscular dystrophy, multiminicore disease and desmin-related myopathy with Mallory body-like inclusions. These disorders constitute the SEPN1 related myopathies (SEPN-RM). On the basis of clinical and laboratory features compatible with SEPN-RM, we performed mutation analysis of SEPN1 in 11 unrelated patients and found one case with pathogenic mutations. He showed early onset axial muscle weakness and developed scoliosis with respiratory insufficiency. Muscle biopsy showed increased variability of fiber size and slight, focal increase of connective tissue. A few fibers showed mini-core changes. SEPN1 mutation analysis revealed that the patient was a compound heterozygote: a previously described insertion (713-714 insA), and a novel nonsense mutation (R439stop).     

A study of FHL1, BAG3, MATR3, PTRF and TCAP in Australian muscular dystrophy patients

FHL1, BAG3, MATR3 and PTRF are recently identified myopathy genes associated with phenotypes that overlap muscular dystrophy. TCAP is a rare reported cause of muscular dystrophy not routinely screened in most centres. We hypothesised that these genes may account for patients with undiagnosed forms of muscular dystrophy in Australia. We screened a large cohort of muscular dystrophy patients for abnormalities in FHL1 (n = 102) and TCAP (n = 100) and selected patients whose clinical features overlapped the phenotypes previously described for BAG3 (n = 9), MATR3 (n = 15) and PTRF (n = 7). We found one FHL1 mutation (c.311G>A, p.C104Y) in a boy with rapidly progressive muscle weakness and reducing body myopathy who was initially diagnosed with muscular dystrophy. We identified no pathogenic mutations in BAG3, MATR3, PTRF or TCAP. In conclusion, we have excluded these five genes as common causes of muscular dystrophy in Australia. Patients with reducing body myopathy may be initially diagnosed as muscular dystrophy.     

New FKRP mutations causing congenital muscular dystrophy associated with mental retardation and central nervous system abnormalities. Identification of a founder mutation in Tunisian families

The congenital muscular dystrophies (CMD) constitute a clinically and genetically heterogeneous group of autosomal recessive myopathies. Patients show congenital hypotonia, muscle weakness, and dystrophic changes on muscle biopsy. Mutations in four genes (FKT1, POMGnT1, POMT1, FKRP) encoding putative glycosyltransferases have been identified in a subset of patients characterized by a deficient glycosylation of -dystroglycan on muscle biopsy. FKRP mutations account for a broad spectrum of patients with muscular dystrophy, from a severe congenital form with or without mental retardation (MDC1C) to a much milder limb-girdle muscular dystrophy (LGMD2I). We identified two novel homozygous missense FKRP mutations, one, A455D, in six unrelated Tunisian patients and the other, V405L, in an Algerian boy. The patients, between the ages of 3 and 12 years, presented with a severe form of MDC1C with calf hypertrophy and high serum creatine kinase levels. None had ever walked. Two had cardiac dysfunction and one strabismus. They all had mental retardation, microcephaly, cerebellar cysts, and hypoplasia of the vermis. White matter abnormalities were found in five, mostly when cranial magnetic resonance imaging was performed at a young age. These abnormalities were shown to regress in one patient, as has been observed in patients with Fukuyama CMD. Identification of a new microsatellite close to the FKRP gene allowed us to confirm the founder origin of the Tunisian mutation. These results strongly suggest that particular FKRP mutations in the homozygous state induce structural and clinical neurological lesions in addition to muscular dystrophy. They also relate MDC1C to other CMD with abnormal protein glycosylation and disordered brain function.     

Homozygous mutations in VAMP1 cause a presynaptic congenital myasthenic syndrome

We report 2 families with undiagnosed recessive presynaptic congenital myasthenic syndrome (CMS). Whole exome or genome sequencing identified segregating homozygous variants in VAMP1: c.51_64delAGGTGGGGGTCCCC in a Kuwaiti family and c.146G>C in an Israeli family. VAMP1 is crucial for vesicle fusion at presynaptic neuromuscular junction (NMJ). Electrodiagnostic examination showed severely low compound muscle action potentials and presynaptic impairment. We assessed the effect of the nonsense mutation on mRNA levels and evaluated the NMJ transmission in VAMP1^lew/lew mice, observing neurophysiological features of presynaptic impairment, similar to the patients. Taken together, our findings highlight VAMP1 homozygous mutations as a cause of presynaptic CMS. Ann Neurol 2017;81:597–603     

Study of Survival of Motor Neuron (SMN) and Neuronal Apoptosis Inhibitory Protein (NAIP) gene deletions in SMA patients

In view of the paucity of deletion studies of survival of motor neuron (SMN) and neuronal apoptosis inhibitor protein (NAIP) genes in Indian SMA patients, this study has been undertaken to determine the status of SMN1, SMN2 and NAIP gene deletions in Indian SMA patients. Clinically and neurophysiologically diagnosed SMA patients were included in the study. A gene deletion study was carried out in 45 proximal SMA patients and 50 controls of the same ethnic group. Both SMN1 and NAIP genes showed homozygous absence in 76 % and 31 % respectively in proximal SMA patients. It is proposed that the lower deletion frequency of SMN1 gene in Indian patients may be due to mutations present in other genes or population variation, which need further study.     

Novel mutations and repeated findings of mutations in familial Alzheimer disease

Twenty-one unrelated patients with a history of suspected familial Alzheimer disease (FAD) were screened for mutations in PSEN1, PSEN2, and APP, the known FAD genes encoding the presenilins (PS1 and PS2) and the amyloid precursor protein (APP). The mutation detection rate was 57%. Of the nine pathogenic mutations found in 12 cases, three were in APP, one in PSEN2, and five in PSEN1, including two novel Greek mutations (L113Q and N135S). Whereas our findings suggest the possibility of single founders for the majority of mutations, we found evidence of recurrence of the APP mutations V717L and V717I.     

Next generation sequencing for molecular diagnosis of neuromuscular diseases

Inherited neuromuscular disorders (NMD) are chronic genetic diseases posing a significant burden on patients and the health care system. Despite tremendous research and clinical efforts, the molecular causes remain unknown for nearly half of the patients, due to genetic heterogeneity and conventional molecular diagnosis based on a gene-by-gene approach. We aimed to test next generation sequencing (NGS) as an efficient and cost-effective strategy to accelerate patient diagnosis. We designed a capture library to target the coding and splice site sequences of all known NMD genes and used NGS and DNA multiplexing to retrieve the pathogenic mutations in patients with heterogeneous NMD with or without known mutations. We retrieved all known mutations, including point mutations and small indels, intronic and exonic mutations, and a large deletion in a patient with Duchenne muscular dystrophy, validating the sensitivity and reproducibility of this strategy on a heterogeneous subset of NMD with different genetic inheritance. Most pathogenic mutations were ranked on top in our blind bioinformatic pipeline. Following the same strategy, we characterized probable TTN, RYR1 and COL6A3 mutations in several patients without previous molecular diagnosis. The cost was less than conventional testing for a single large gene. With appropriate adaptations, this strategy could be implemented into a routine genetic diagnosis set-up as a first screening approach to detect most kind of mutations, potentially before the need of more invasive and specific clinical investigations. An earlier genetic diagnosis should provide improved disease management and higher quality genetic counseling, and ease access to therapy or inclusion into therapeutic trials.     

Phenotypic spectrum of Fukutinopathy: most severe phenotype of Fukutinopathy

Fukuyama-type congenital muscular dystrophy (FCMD), Walker-Warburg syndrome (WWS), and muscle-eye-brain (MEB) disease are clinically similar autosomal recessive disorders characterized by congenital muscular dystrophy, cobblestone lissencephaly, and eye anomalies. Among them, WWS is the most severe syndrome. Causative genes for FCMD (Fukutin), WWS (POMT1), and MEB (POMGnT1) have been identified. The vast majority of Japanese FCMD patients carry at least one copy of an ancestral founder insertion mutation. Patients homozygous for this insertion show a milder phenotype than do compound heterozygotes, carrying the insertion in combination with a missense or nonsense mutation on the other allele. No Japanese FCMD patients have been identified with nonfounder mutations on both alleles. A Turkish boy with characteristics of WWS was detected to have a homozygous nonsense mutation in exon 5 of Fukutin. This is the first case worldwide in which a Fukutin mutation has been found outside the Japanese population. Later, another Turkish boy with WWS phenotype was found to have a homozygous nonsense mutation in exon 4 of Fukutin. These two Turkish boys represent the most severe end of the phenotypic spectrum of Fukutin mutations. The Japanese FCMD patients carrying at least one copy of a founder mutation in the noncoding region may produce a lower level of mature Fukutin than normal and generate a relatively mild FCMD phenotype. The homozygous nonsense mutations within the coding region identified in Turkish patients are predicted to cause a total loss of fukutin activity and are likely to produce a more severe phenotype which closely resembles WWS.     

Spinal muscular atrophy genotyping by gene dosage using multiple ligation-dependent probe amplification

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by degeneration of the anterior horn cells of the spinal cord, causing symmetric proximal muscle weakness. SMA is classified in three clinical types, SMA I, SMA II, and SMA III, based on the severity of the symptoms and the age of onset. About 95% of SMA cases are caused by homozygous deletion of the survival motor neuron 1 (SMN1) gene (5q13), or its conversion to SMN2. The molecular diagnosis of this disease is usually carried out by a polymerase chain reaction–restriction fragment length polymorphism approach able to evidence the absence of both SMN1 copies. However, this approach is not able to identify heterozygous healthy carriers, which show a very high frequency in general population (1:50). We used the multiple ligation-dependent probe amplification (MLPA) approach for the molecular diagnosis of SMA in 19 affected patient and in 57 individuals at risk to become healthy carriers. This analysis detected the absence of the homozygous SMN1 in all the investigated cases, and allowed to discriminate between SMN1 deletion and conversion to SMN2 on the basis of the size showed by the peaks specific for the different genes mapped within the SMA critical region. Moreover, MLPA analysis evidenced a condition of the absence of the heterozygous SMN1 in 33 out of the 57 relatives of the affected patients, demonstrating the usefulness of this approach in the identification of healthy carriers. Thus, the MLPA technique represents an easy, low cost, and high throughput system in the molecular diagnosis of SMA, both in affected patients and in healthy carriers.     

SEPN1: associated with congenital fiber-type disproportion and insulin resistance

OBJECTIVE : Our first objective was to determine whether SEPN1 gene mutations are a cause of congenital fiber-type disproportion (CFTD), a rare form of congenital myopathy in which relative hypotrophy of type 1 (slow twitch) muscle fibers is the principal abnormality on histology. Second, we investigated an association between SEPN1-related myopathy and insulin resistance. METHODS: We sequenced SEPN1 in five unrelated CFTD patients with scoliosis and respiratory muscle weakness and screened an additional 22 CFTD patients for abnormalities in SEPN1 by Western blotting and restriction digest for the 943G-->A mutation. We performed oral glucose tolerance tests (OGTTs) in eight SEPN1-related myopathy patients. RESULTS: Two sisters with CFTD were homozygous for the 943G-->A SEPN1 mutation and had clinical features typical of previously reported patients with SEPN1-related myopathy. Five of eight SEPN1-related myopathy patients had abnormalities on OGTT suggestive of insulin resistance. INTERPRETATION: SEPN1 is the second genetic cause of CFTD and the first cause of autosomal recessive CFTD to be identified to our knowledge. CFTD is the fourth clinicopathological presentation that can be associated with mutations in SEPN1. Insulin resistance may be a specific, previously unrecognized aspect of SEPN1-related myopathy.     

Core myopathies

The core myopathies, Central Core Disease and Multiminicore Disease, are heterogeneous congenital myopathies with the common defining histopathological feature of focally reduced oxidative enzyme activity (central cores, multiminicores). Mutations in the gene encoding for the skeletal muscle ryanodine (RyR1) receptor are the most common cause. Mutations in the selenoprotein N (SEPN1) gene cause a less common variant. Pathogenic mechanisms underlying dominant RYR1 mutations have been extensively characterized, whereas those associated with recessive RYR1 and SEPN1 mutations are emerging. Identifying a specific genetic defect from the histopathological diagnosis of a core myopathy is complex and ought to be informed by a combined appraisal of histopathological, clinical, and, increasingly, muscle magnetic resonance imaging data. The present review aims at giving an overview of the main genetic and clinicopathological findings, with a major emphasis on features likely to inform the diagnostic process, as well as current treatments and perspectives for future research.     

A novel mutation at the N-terminal of SMN Tudor domain inhibits its interaction with target proteins

Abstract Although most patients with spinal muscular atrophy (SMA) are homozygous for deletion of the SMN1 gene, some patients bear one SMN1 copy with a subtle mutation. Detection of such an intragenic mutation may be helpful not only in confirming diagnosis but also in elucidating functional domains of the SMN protein. In this study, we identified a novel mutation in SMN1 of two Japanese patients with type I SMA. DHPLC and sequencing analysis revealed that they harbored a point mutation in SMN1 exon 3, 275G > C, leading to tryptophan-to-serine substitution at amino acid 92 (W92S) at the Nterminal of SMN Tudor domain. In-vitro protein binding assays showed that the mutation severely reduced interaction of the domain with SmB protein and fibrillarin, suggesting that it impairs the critical function of SMN. In conclusion, we reported here that a novel mutation, W92S, in the Tudor domain affects the interaction of SMN with the target proteins.