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The factors driving initiation of pathological expansion of tandem repeats remain largely unknown. Here, we assessed theFGF14-SCA27B (GAA)•(TTC) repeat locus in 2,530 individuals by long-read and Sanger sequencing and identified a 5’-flanking 17-bp deletion-insertion in 70.34% of alleles (3,463/4,923). This common sequence variation was present nearly exclusively on alleles with fewer than 30 GAA-pure repeats and was associated with enhanced meiotic stability of the repeat locus.

The late-onset cerebellar ataxias (LOCAs) have largely resisted molecular diagnosis.We sequenced the genomes of six persons with autosomal dominant LOCA who were members of three French Canadian families and identified a candidate pathogenic repeat expansion. We then tested for association between the repeat expansion and disease in two independent case-control series - one French Canadian (66 patients and 209 controls) and the other German (228 patients and 199 controls). We also genotyped the repeat in 20 Australian and 31 Indian index patients. We assayed gene and protein expression in two postmortem cerebellum specimens and two induced pluripotent stem-cell (iPSC)-derived motor-neuron cell lines.In the six French Canadian patients, we identified a GAA repeat expansion deep in the first intron ofA dominantly inherited deep intronic GAA repeat expansion in

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) is a progressive late-onset, neurological disease. Recently, a pentanucleotide expansion in intron 2 of RFC1 was identified as the genetic cause of CANVAS. We screened an Asian-Pacific cohort for CANVAS and identified a novel RFC1 repeat expansion motif, (ACAGG)exp, in three affected individuals. This motif was associated with additional clinical features including fasciculations and elevated serum creatine kinase. These features have not previously been described in individuals with genetically-confirmed CANVAS. Haplotype analysis showed our patients shared the same core haplotype as previously published, supporting the possibility of a single origin of the RFC1 disease allele. We analysed data from >26 000 genetically diverse individuals in gnomAD to show enrichment of (ACAGG) in non-European populations.

The impact of high-throughput sequencing in genetic neuromuscular disorders cannot be overstated. The ability to rapidly and affordably sequence multiple genes simultaneously has enabled a second golden age of Mendelian disease gene discovery, with flow-on impacts for rapid genetic diagnosis, evidence-based treatment, tailored therapy development, carrier-screening, and prevention of disease recurrence in families. However, there are likely many more neuromuscular disease genes and mechanisms to be discovered. Many patients and families remain without a molecular diagnosis following targeted panel sequencing, clinical exome sequencing, or even genome sequencing. Here we review how massively parallel, or next-generation, sequencing has changed the field of genetic neuromuscular disorders, and anticipate future benefits of recent technological innovations such as RNA-seq implementation and detection of tandem repeat expansions from short-read sequencing.

Neuregulin 1 signals are essential for the development and function of Schwann cells, which form the myelin sheath on peripheral axons. Disruption of myelin in the peripheral nervous system can lead to peripheral neuropathy, which is characterized by reduced axonal conduction velocity and sensorimotor deficits. Charcot-Marie-Tooth disease is a group of heritable peripheral neuropathies that may be caused by variants in nearly 100 genes. Despite the evidence that Neuregulin 1 is essential for many aspects of Schwann cell development, previous studies have not reported variants in the neuregulin 1 gene (NRG1) in patients with peripheral neuropathy. We have identified a rare missense variant in NRG1 that is homozygous in a patient with sensory and motor deficits consistent with mixed axonal and de-myelinating peripheral neuropathy. Our in vivo functional studies in zebrafish indicate that the patient variant partially reduces NRG1 function. This study tentatively suggests that variants at the NRG1 locus may cause peripheral neuropathy and that NRG1 should be investigated in families with peripheral neuropathy of unknown cause.

Expansions of short tandem repeats (STRs) cause dozens of rare Mendelian diseases. However, STR expansions, especially those arising from repeats not present in the reference genome, are challenging to detect from short-read sequencing data. Such “novel” STRs include new repeat units occurring at known STR loci, or entirely new STR loci where the sequence is absent from the reference genome. A primary cause of difficulty detecting STR expansions is that reads arising from STR expansions are frequently mismapped or unmapped. To address this challenge, we have developed STRling, a new STR detection algorithm that counts k-mers (short DNA sequences of length k) in DNA sequencing reads, to efficiently recover reads that inform the presence and size of STR expansions. As a result, STRling can call expansions at both known and novel STR loci. STRling has a sensitivity of 83% for 14 known STR disease loci, including the novel STRs that cause CANVAS and DBQD2. It is the first method to resolve the position of novel STR expansions to base pair accuracy. Such accuracy is essential to interpreting the consequence of each expansion. STRling has an estimated 0.078 false discovery rate for known pathogenic loci in unaffected individuals and a 0.20 false discovery rate for genome-wide loci in unaffected individuals when using variants called from long-read data as truth. STRling is fast, scalable on cloud computing, open-source, and freely available at https://github.com/quinlan-lab/STRling.

The impact of high-throughput sequencing in genetic neuromuscular disorders cannot be overstated. The ability to rapidly and affordably sequence multiple genes simultaneously has enabled a second golden age of Mendelian disease gene discovery, with flow-on impacts for rapid genetic diagnosis, evidence-based treatment, tailored therapy development, carrier-screening, and prevention of disease recurrence in families. However, there are likely many more neuromuscular disease genes and mechanisms to be discovered. Many patients and families remain without a molecular diagnosis following targeted panel sequencing, clinical exome sequencing, or even genome sequencing. Here we review how massively parallel, or next-generation, sequencing has changed the field of genetic neuromuscular disorders, and anticipate future benefits of recent technological innovations such as RNA-seq implementation and detection of tandem repeat expansions from short-read sequencing.

Although mutations in more than 90 genes are known to cause CMT, the underlying genetic cause of CMT remains unknown in more than 50% of affected individuals. The discovery of additional genes that harbor CMT2-causing mutations increasingly depends on sharing sequence data on a global level. In this way—by combining data from seven countries on four continents—we were able to define mutations in ATP1A1, which encodes the alpha1 subunit of the Na+,K+-ATPase, as a cause of autosomal-dominant CMT2. Seven missense changes were identified that segregated within individual pedigrees: c.143T>G (p.Leu48Arg), c.1775T>C (p.Ile592Thr), c.1789G>A (p.Ala597Thr), c.1801_1802delinsTT (p.Asp601Phe), c.1798C>G (p.Pro600Ala), c.1798C>A (p.Pro600Thr), and c.2432A>C (p.Asp811Ala). Immunostaining peripheral nerve axons localized ATP1A1 to the axolemma of myelinated sensory and motor axons and to Schmidt-Lanterman incisures of myelin sheaths. Two-electrode voltage clamp measurements on Xenopus oocytes demonstrated significant reduction in Na+ current activity in some, but not all, ouabain-insensitive ATP1A1 mutants, suggesting a loss-of-function defect of the Na+,K+ pump. Five mutants fall into a remarkably narrow motif within the helical linker region that couples the nucleotide-binding and phosphorylation domains. These findings identify a CMT pathway and a potential target for therapy development in degenerative diseases of peripheral nerve axons.

Aim: To explore the relationship between balance deficit, falls, and activities of daily living in a cohort with Charcot-Marie-Tooth disease. Method: A cohort survey of individuals with Charcot-Marie-Tooth disease attending a neurogenetic clinic was carried out. Surveys were sent to individuals on a database of a state-wide neurogenetic service located in Perth, Australia. Results: Out of 71 surveys, 40 were returned (56%). The cohort were predominantly male (68%), the average age was 55.5 years (range 18–84) and average age at symptom onset was 23 years (range 5–65). Excluding skeletal foot changes, balance deficits (87.5%) and loss of balance confidence (90%) were the most frequently rated symptoms and those most severely affecting physical functioning (71% and 64% respectively). Ankle splinting was frequently reported in this population (65%). The majority of participants (69%) had fallen at least once in the past year and 40% of fallers reported having never seen a physiotherapist. Conclusions: Despite balance deficits and loss of balance confidence being frequently reported to impact daily activities, and falls and near-falls being common in this cohort with Charcot-Marie-Tooth disease, more than a third had not seen a physiotherapist. Future management to reduce the disease burden should investigate the effects of a timely multidisciplinary approach to manage balance dysfunction, and strategies to reduce falls in this population.

Nephropathic cystinosis is an autosomal recessive lysosomal disease in which cystine cannot exit the lysosome to complete its degradation in the cytoplasm, thus accumulating in tissues. Some patients develop a distal myopathy involving mainly hand muscles. Myopathology descriptions from only 5 patients are available in the literature. We present a comprehensive clinical, pathological and genetic description of 3 patients from 2 families with nephropathic cystinosis. Intrafamiliar variability was detected in one family in which one sibling developed a severe distal myopathy while the other sibling did not show any signs of skeletal muscle involvement. One of the patients was on treatment with Cysteamine for over 12 years but still developed the usual complications of nephropathic cystinosis in his twenties. Novel pathological findings consisting in sarcoplasmic deposits reactive for slow myosin were identified. Three previously known and one novel mutation are reported. Nephropathic cystinosis should be included in the differential diagnosis of distal myopathies in those with early renal failure. Novel clinical and pathological features are reported here contributing to the characterization of the muscle involvement in nephropathic cystinosis.

Autosomal dominant mutations of DYNC1H1 cause a range of neurogenetic diseases, including mental retardation with cortical malformations, hereditary spastic paraplegia and spinal muscular atrophy. Using SNP array, linkage analysis and next generation sequencing, we identified two families and one isolated proband sharing a known spinal muscular atrophy, lower extremity predominant (SMALED) causing mutation DYNC1H1 c.1792C>T, p.Arg598Cys, and another family harbouring a c.2327C>T, p.Pro776Leu mutation. Here, we present a detailed clinical and pathological examination of these patients, and show that patients with DYNC1H1 mutations may present with a phenotype mimicking a congenital myopathy. We also highlight features that increase the phenotypic overlap with BICD2, which causes SMALED2. Serial muscle biopsies were available for several patients, spanning from infancy and early childhood to middle age. These provide a unique insight into the developmental and pathological origins of SMALED, suggesting in utero denervation with reinnervation by surrounding intact motor neurons and segmental anterior horn cell deficits. We characterise biopsy features that may make diagnosis of this condition easier in the future.

Early-onset muscle disease includes three major entities that present generally at or before birth: congenital myopathies, congenital muscular dystrophies and congenital myasthenic syndromes. Almost exclusively there is weakness and hypotonia, although cases manifesting hypertonia are increasingly being recognised. These diseases display a wide phenotypic and genetic heterogeneity, with the uptake of next generation sequencing resulting in an unparalleled extension of the phenotype-genotype correlations and "diagnosis by sequencing" due to unbiased sequencing. Perhaps now more than ever, detailed clinical evaluations are necessary to guide the genetic diagnosis; with arrival at a molecular diagnosis frequently occurring following dialogue between the molecular geneticist, the referring clinician and the pathologist. There is an ever-increasing blurring of the boundaries between the congenital myopathies, dystrophies and myasthenic syndromes. In addition, many novel disease genes have been described and new insights have been gained into skeletal muscle development and function. Despite the advances made, a significant percentage of patients remain without a molecular diagnosis, suggesting that there are many more human disease genes and mechanisms to identify. It is now technically- and clinically-feasible to perform next generation sequencing for severe diseases on a population-wide scale, such that preconception-carrier screening can occur. Newborn screening for selected early-onset muscle diseases is also technically and ethically-achievable, with benefits to the patient and family from early management of these diseases and should also be implemented. The need for world-wide Reference Centres to meticulously curate polymorphisms and mutations within a particular gene is becoming increasingly apparent, particularly for interpretation of variants in the large genes which cause early-onset myopathies: NEB, RYR1 and TTN. Functional validation of candidate disease variants is crucial for accurate interpretation of next generation sequencing and appropriate genetic counseling. Many published "pathogenic" variants are too frequent in control populations and are thus likely rare polymorphisms. Mechanisms need to be put in place to systematically update the classification of variants such that accurate interpretation of variants occurs. In this review, we highlight the recent advances made and the challenges ahead for the molecular diagnosis of early-onset muscle diseases.

[Objective] To develop, test, and iterate a comprehensive neuromuscular targeted gene panel in a national referral center., [Methods] We designed two iterations of a comprehensive targeted gene panel for neuromuscular disorders. Version 1 included 336 genes, which was increased to 464 genes in Version 2. Both panels used TargetSeqTM probe‐based hybridization for target enrichment followed by Ion Torrent sequencing. Targeted high‐coverage sequencing and analysis was performed on 2249 neurology patients from Australia and New Zealand (1054 Version 1, 1195 Version 2) from 2012 to 2015. No selection criteria were used other than referral from a suitable medical specialist (e.g., neurologist or clinical geneticist). Patients were classified into 15 clinical categories based on the clinical diagnosis from the referring clinician., [Results] Six hundred and sixty‐five patients received a genetic diagnosis (30%). Diagnosed patients were significantly younger that undiagnosed patients (26.4 and 32.5 years, respectively; P = 4.6326E‐9). The diagnostic success varied markedly between disease categories. Pathogenic variants in 10 genes explained 38% of the disease burden. Unexpected phenotypic expansions were discovered in multiple cases. Triage of unsolved cases for research exome testing led to the discovery of six new disease genes., [Interpretation] A comprehensive targeted diagnostic panel was an effective method for neuromuscular disease diagnosis within the context of an Australasian referral center. Use of smaller disease‐specific panels would have precluded diagnosis in many patients and increased cost. Analysis through a centralized laboratory facilitated detection of recurrent, but under‐recognized pathogenic variants., Research funding: The Fred Liuzzi Foundation, Australian Postgraduate Award, Australian Genomics Health Alliance. Grant Number: GNT1113531, Fundación Alfonso Martín Escudero, Junta de Andalucía‐Consejería de Salud. Grant Number: B‐0005‐2017, Australian National Health and Medical Research Council. Grant Numbers: APP1117510, APP1122952, APP1080587, Western Australian Department of Health Future Health’s WA Merit Award.

See Paisán-Ruiz and Jen (doi:10.1093/brain/awaa015) for a scientific commentary on this article. Cortese et al. describe the full disease phenotype, including progression of ataxia, in 100 confirmed carriers of RFC1 repeat expansions. RFC1 repeat expansion should be considered in all cases of sensory ataxic neuropathy, particularly if cerebellar dysfunction, vestibular involvement and cough coexist., Ataxia, causing imbalance, dizziness and falls, is a leading cause of neurological disability. We have recently identified a biallelic intronic AAGGG repeat expansion in replication factor complex subunit 1 (RFC1) as the cause of cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and a major cause of late onset ataxia. Here we describe the full spectrum of the disease phenotype in our first 100 genetically confirmed carriers of biallelic repeat expansions in RFC1 and identify the sensory neuropathy as a common feature in all cases to date. All patients were Caucasian and half were sporadic. Patients typically reported progressive unsteadiness starting in the sixth decade. A dry spasmodic cough was also frequently associated and often preceded by decades the onset of walking difficulty. Sensory symptoms, oscillopsia, dysautonomia and dysarthria were also variably associated. The disease seems to follow a pattern of spatial progression from the early involvement of sensory neurons, to the later appearance of vestibular and cerebellar dysfunction. Half of the patients needed walking aids after 10 years of disease duration and a quarter were wheelchair dependent after 15 years. Overall, two-thirds of cases had full CANVAS. Sensory neuropathy was the only manifestation in 15 patients. Sixteen patients additionally showed cerebellar involvement, and six showed vestibular involvement. The disease is very likely to be underdiagnosed. Repeat expansion in RFC1 should be considered in all cases of sensory ataxic neuropathy, particularly, but not only, if cerebellar dysfunction, vestibular involvement and cough coexist.

IntroductionEmerging evidence indicates that rehabilitation can improve ataxia, mobility and independence in everyday activities in individuals with hereditary cerebellar ataxia. However, with the rarity of the genetic ataxias and known recruitment challenges in rehabilitation trials, most studies have been underpowered, non-randomised or non-controlled. This study will be the first, appropriately powered randomised controlled trial to examine the efficacy of an outpatient and home-based rehabilitation programme on improving motor function for individuals with hereditary cerebellar ataxia.Methods and analysisThis randomised, single-blind, parallel group trial will compare a 30-week rehabilitation programme to standard care in individuals with hereditary cerebellar ataxia. Eighty individuals with a hereditary cerebellar ataxia, aged 15 years and above, will be recruited. The rehabilitation programme will include 6 weeks of outpatient land and aquatic physiotherapy followed immediately by a 24- week home exercise programme supported with fortnightly physiotherapy sessions. Participants in the standard care group will be asked to continue their usual physical activity. The primary outcome will be the motor domain of the Functional Independence Measure. Secondary outcomes will measure the motor impairment related to ataxia, balance, quality of life and cost-effectiveness. Outcomes will be administered at baseline, 7 weeks, 18 weeks and 30 weeks by a physiotherapist blinded to group allocation. A repeated measures mixed-effects linear regression model will be used to analyse the effect of the treatment group for each of the dependent continuous variables. The primary efficacy analysis will follow the intention-to-treat principle.Ethics and disseminationThe study has been approved by the Monash Health Human Research Ethics Committee (HREC/18/MonH/418) and the Human Research Ethics Committee of the Northern Territory Department of Health and Menzies School of Health Research (2019/3503). Results will be published in peer-reviewed journals, presented at national and/or international conferences and disseminated to Australian ataxia support groups.Trial registration numberACTRN12618000908235.

Leigh syndrome (LS) is a progressive mitochondrial neurodegenerative disorder, whose symptoms most commonly include psychomotor delay with regression, lactic acidosis and a failure to thrive. Here we describe three siblings with LS, but with additional manifestations including hypertrophic cardiomyopathy, hepatosplenomegaly, cholestatic hepatitis, and seizures. All three affected siblings were found to be homoplasmic for an m. 5559A>G mutation in the T stem of the mitochondrial DNA-encoded MT-TW by next generation sequencing. The m.5559A>G mutation causes a reduction in the steady state levels of tRNA(Trp) and this decrease likely affects the stability of other mitochondrial RNAs in the patient fibroblasts. We observe accumulation of an unprocessed transcript containing tRNA(Trp), decreased de novo protein synthesis and consequently lowered steady state levels of mitochondrial DNA-encoded proteins that compromise mitochondrial respiration. Our results show that the m.5559A>G mutation at homoplasmic levels causes LS in association with severe multi-organ disease (LS-plus) as a consequence of dysfunctional mitochondrial RNA metabolism.

An MYH2 mutation p.(Glu706Lys) was originally described in a family with autosomal dominant inheritance, where the affected family members presented with multiple congenital contractures and ophthalmoplegia, progressing to a proximal myopathy in adulthood. Another patient with a dominant mutation p.(Leu1870Pro) was described, presenting as a congenital myopathy with ophthalmoplegia. Here, we present a patient with symptoms beginning at age 16 years, of prominent distal but also proximal weakness, bulbar involvement and ophthalmoplegia. Initially, clinically classified as oculopharyngodistal myopathy, the patient was found to carry a novel, de novo MYH2 mutation c.5630T>C p.(Leu1877Pro). This expands the phenotype of dominant MYH2 myopathies with the clinical phenotype overlapping the oculopharyngodistal myopathy spectrum.

BACKGROUND: Primary chronic intestinal pseudo-obstruction (CIPO) is a rare, potentially life-threatening disorder characterized by severely impaired gastrointestinal motility. The objective of this study was to examine the contribution of ACTG2, LMOD1, MYH11, and MYLK mutations in an Australasian cohort of patients with a diagnosis of primary CIPO associated with visceral myopathy. METHODS: Pediatric and adult patients with primary CIPO and suspected visceral myopathy were recruited from across Australia and New Zealand. Sanger sequencing of the genes encoding enteric gamma-actin (ACTG2) and smooth muscle leiomodin (LMOD1) was performed on DNA from patients, and their relatives, where available. MYH11 and MYLK were screened by next-generation sequencing. KEY RESULTS: We identified heterozygous missense variants in ACTG2 in 7 of 17 families (~41%) diagnosed with CIPO and its associated conditions. We also identified a previously unpublished missense mutation (c.443C>T, p.Arg148Leu) in one family. One case presented with megacystis-microcolon-intestinal hypoperistalsis syndrome in utero with subsequent termination of pregnancy at 28 weeks' gestation. All of the substitutions identified occurred at arginine residues. No likely pathogenic variants in LMOD1, MYH11, or MYLK were identified within our cohort. CONCLUSIONS AND INFERENCES: ACTG2 mutations represent a significant underlying cause of primary CIPO with visceral myopathy and associated phenotypes in Australasian patients. Thus, ACTG2 sequencing should be considered in cases presenting with hypoperistalsis phenotypes with suspected visceral myopathy. It is likely that variants in other genes encoding enteric smooth muscle contractile proteins will contribute further to the genetic heterogeneity of hypoperistalsis phenotypes.

Short tandem repeat (STR) expansions have been identified as the causal DNA mutation in dozens of Mendelian diseases. Historically, pathogenic STR expansions could only be detected by single locus techniques, such as PCR and electrophoresis. The ability to use short read sequencing data to screen for STR expansions has the potential to reduce both the time and cost to reaching diagnosis and enable the discovery of new causal STR loci. Most existing tools detect STR variation within the read length, and so are unable to detect the majority of pathogenic expansions. Those tools that can detect large expansions are limited to a set of known disease loci and as yet no new disease causing STR expansions have been identified with high-throughput sequencing technologies.Here we address this by presenting STRetch, a new genome-wide method to detect STR expansions at all loci across the human genome. We demonstrate the use of STRetch for detecting pathogenic STR expansions in short-read whole genome sequencing data with a very low false discovery rate. We further demonstrate the application of STRetch to solve cases of patients with undiagnosed disease and apply STRetch to the analysis of 97 whole genomes to reveal variation at STR loci. STRetch assesses expansions at all STR loci in the genome and allows screening for novel disease-causing STRs.STRetch is open source software, available fromgithub.com/Oshlack/STRetch.

Introduction: Mutations in the choline kinase beta (CHKB) gene are associated with a congenital muscular dystrophy with giant mitochondria at the periphery of muscle fibers. Methods: We describe a patient of Italian origin in whom whole-exome sequencing revealed a novel homozygous nonsense mutation, c.648C>A, p.(Tyr216*), in exon 5 of CHKB. Results: The patient presented with limb-girdle weakness and hypotonia from birth with mental retardation, and had sudden and transient deteriorations of muscle strength with acute intercurrent illnesses. Previously undescribed sarcolemmal overexpression of utrophin was noted in the muscle biopsy. Conclusions: Pathological features broaden the description of the entity and provide new insight in the pathogenic mechanisms. This case highlights the usefulness of next-generation sequencing in the diagnosis of rare and incompletely understood conditions. Muscle Nerve 51: 140–143, 2015

Contains fulltext : 88858.pdf (Publisher’s version ) (Closed access) We identified a member of the BTB/Kelch protein family that is mutated in nemaline myopathy type 6 (NEM6), an autosomal-dominant neuromuscular disorder characterized by the presence of nemaline rods and core lesions in the skeletal myofibers. Analysis of affected families allowed narrowing of the candidate region on chromosome 15q22.31, and mutation screening led to the identification of a previously uncharacterized gene, KBTBD13, coding for a hypothetical protein and containing missense mutations that perfectly cosegregate with nemaline myopathy in the studied families. KBTBD13 contains a BTB/POZ domain and five Kelch repeats and is expressed primarily in skeletal and cardiac muscle. The identified disease-associated mutations, C.742C>A (p.Arg248Ser), c.1170G>C (p.Lys390Asn), and c.1222C>T (p.Arg408Cys), located in conserved domains of Kelch repeats, are predicted to disrupt the molecule's beta-propeller blades. Previously identified BTB/POZ/Kelch-domain-containing proteins have been implicated in a broad variety of biological processes, including cytoskeleton modulation, regulation of gene transcription, ubiquitination, and myofibril assembly. The functional role of KBTBD13 in skeletal muscle and the pathogenesis of NEM6 are subjects for further studies.

Background: Laing early onset distal myopathy (MPD1) is an autosomal dominant myopathy caused by mutations within the slow skeletal muscle fibre myosin heavy chain gene, MYH7. It is allelic with myosin storage myopathy, with the commonest form of familial hypertrophic cardiomyopathy, and with one form of dilated cardiomyopathy. However, the clinical picture of MPD1 is distinct from these three conditions. Objective: To collate and discuss the histological features reported in the muscle biopsies of MPD1 patients and to outline the clinical features. Results: The phenotype of MPD1 was consistent, with initial weakness of great toe/ankle dorsiflexion, and later development of weakness of finger extension and neck flexion. Age of onset was the only variable, being from birth up to the 20s, but progression was always very slow. The pathological features were variable. In this retrospective series, there were no pathognomonic diagnostic features, although atrophic type I fibres were found in half the families. Rimmed vacuoles are consistently seen in all other distal myopathies with the exception of Myoshi distal myopathy. However, they were found in a minority of patients with MPD1, and were not prominent when present. Immunohistochemical staining for slow and fast myosin showed co-expression of slow and fast myosin in some type I fibres, possibly indicating a switch to type II status. This may be a useful aid to diagnosis. Conclusions: The pathological findings in MPD1 are variable and appear to be affected by factors such as the specific muscle biopsied, the age of the patient at biopsy, and the duration of disease manifestations.

The distal myopathies are a heterogeneous group of disorders that pose a challenge to both the clinician and geneticist. This article summarizes the findings of recent clinical, genetic and molecular studies and the current diagnostic approach to this group of patients.Publications over the past 5 years describe a number of new clinical phenotypes and genetic loci and further emphasize the overlap in clinical phenotype between a number of these disorders and between the distal and limb girdle myopathies and hereditary inclusion body myopathies. Recent studies have led to the identification of the genes and mutations responsible for early onset (Laing) myopathy and tibial (Udd) myopathy, and for distal myopathy with rimmed vacuoles (Nonaka), which has been shown to be allelic with quadriceps sparing hereditary inclusion body myopathy (IBM2), and have elucidated the underlying pathogenetic mechanisms in these conditions. New diagnostic approaches using magnetic resonance imaging, and a blood-based assay for dysferlin deficiency, have also been reported.These findings have important implications for future genetic linkage and gene expression studies and for the diagnostic approach to patients with a distal myopathy phenotype. They also hold promise for the eventual development of therapies for this group of disorders.

We previously linked Laing-type early-onset autosomal dominant distal myopathy (MPD1) to a 22-cM region of chromosome 14. One candidate gene in the region, MYH7, which is mutated in cardiomyopathy and myosin storage myopathy, codes for the myosin heavy chain of type I skeletal muscle fibers and cardiac ventricles. We have identified five novel heterozygous mutations - Arg1500Pro, Lys1617del, Ala1663Pro, Leu1706Pro, and Lys1729del in exons 32, 34, 35, and 36 of MYH7 - in six families with early-onset distal myopathy. All five mutations are predicted, by in silico analysis, to locally disrupt the ability of the myosin tail to form the coiled coil, which is its normal structure. These findings demonstrate that heterozygous mutations toward the 3' end of MYH7 cause Laing-type early-onset distal myopathy. MYH7 is the fourth distal-myopathy gene to have been identified

The congenital myopathies are a group of disorders characterised by the predominance of specific histological features observed in biopsied muscle. Central core disease and nemaline myopathy are examples of congenital myopathies that have specific histological characteristics but significantly overlapping clinical pictures. Central core disease is an autosomal dominant disorder with variable penetrance which has been linked principally to the gene for the skeletal muscle calcium release channel (RYR1). Two recent reports have identified the 3' transmembrane domain of this gene as a common site for mutations. Two other studies have reported single families that have features of both central core disease and nemaline myopathy (core/rod disease) caused by mutations in RYR1. Screening of the 3' region (exons 93-105) of the RYR1 gene for mutations in 27 apparently unrelated patients with either central core disease or core/rod disease by single strand conformation polymorphism analysis and DNA sequencing identified three described and nine novel mutations in 15 patients.

Dystroglycanopathies are a heterogeneous group of diseases with a broad phenotypic spectrum ranging from severe disorders with congenital muscle weakness, eye and brain structural abnormalities and intellectual delay to adult-onset limb-girdle muscular dystrophies without mental retardation. Most frequently the disease onset is congenital or during childhood. The exception is FKRP mutations, in which adult onset is a common presentation. Here we report eight patients from five non-consanguineous families where next generation sequencing identified mutations in the GMPPB gene. Six patients presented as an adult or adolescent-onset limb-girdle muscular dystrophy, one presented with isolated episodes of rhabdomyolysis, and one as a congenital muscular dystrophy. This report expands the phenotypic spectrum of GMPPB mutations to include limb-girdle muscular dystrophies with adult onset with or without intellectual disability, or isolated rhabdomyolysis.

SummaryA novel heteroplasmic 7587T→C mutation in the mitochondrial genome which changes the initiation codon of the gene encoding cytochrome c oxidase subunit II (COX II), was found in a family with mitochondrial disease. This T→C transition is predicted to change the initiating methionine to threonine. The mutation load was present at 67% in muscle from the index case and at 91% in muscle from the patient's clinically affected son. Muscle biopsy samples revealed isolated COX deficiency and mitochondrial proliferation. Single-muscle-fiber analysis revealed that the 7587C copy was at much higher load in COX-negative fibers than in COX-positive fibers. After microphotometric enzyme analysis, the mutation was shown to cause a decrease in COX activity when the mutant load was >55%–65%. In fibroblasts from one family member, which contained >95% mutated mtDNA, there was no detectable synthesis or any steady-state level of COX II. This new mutation constitutes a new mechanism by which mtDNA mutations can cause disease-defective initiation of translation.

Brain-lung-thyroid syndrome (BLTS) characterized by congenital hypothyroidism, respiratory distress syndrome, and benign hereditary chorea is caused by thyroid transcription factor 1 (NKX2-1/TTF1) mutations. We report the clinical and molecular characteristics of four cases presenting with primary hypothyroidism, respiratory distress, and neurological disorder. Two of the four patients presenting with the triad of BLTS had NKX2-1 mutations, and one of these NKX2-1 [c.890_896del (p.Ala327Glyfs(star)52)] is a novel variant. The third patient without any identified NKX2-1 mutations was a carrier of mitochondrial mutation; this raises the possibility of mitochondrial mutations contributing to thyroid dysgenesis. Although rare, the triad of congenital hypothyroidism, neurological, and respiratory signs is highly suggestive of NKX2-1 anomalies. Screening for NKX2-1 mutations in patients with thyroid, lung, and neurological abnormalities will enable a unifying diagnosis and genetic counseling for the affected families. In addition, identification of an NKX2-1 defect would be helpful in allaying the concerns about inadequate thyroxine supplementation as the cause of neurological defects observed in some children with congenital hypothyroidism

Central Core Disease (CCD) and Multi-minicore Disease (MmD) (the "core myopathies") have been mainly associated with mutations in the skeletal muscle ryanodine receptor (RYR1) and the selenoprotein N (SEPN1) gene. A proportion of cases remain unresolved. Mutations in MYH7 encoding the beta myosin heavy chain protein have been implicated in cardiac and, less frequently, skeletal muscle disorders. Here we report four patients from two families with a histopathological diagnosis of MmD, presenting in childhood with slowly progressive muscle weakness, more proximal in Family 1 and more distal in Family 2, and variable degrees of cardiorespiratory impairment evolving later in life. There was also a strong family history of sudden death in the first family. Muscle biopsies obtained in early childhood showed multiple minicores as the most prominent feature. Sequencing of the MYH7 gene revealed heterozygous missense mutations, c.4399C>G; p.Leu1467Val (exon 32) in Family 1 and c.4763G>C; p.Arg1588Pro (exon 34) in Family 2. These findings suggest MYH7 mutations as another cause of a myopathy with multiple cores, in particular if associated with dominant inheritance and cardiac involvement. However, clinical features previously associated with this genetic background, namely a more distal distribution of weakness and an associated cardiomyopathy, may only evolve over time.

Defects of the mitochondrial respiratory chain are associated with a diverse spectrum of clinical phenotypes, and may be caused by mutations in either the nuclear or the mitochondrial genome (mitochondrial DNA (mtDNA)). Isolated complex I deficiency is the most common enzyme defect in mitochondrial disorders, particularly in children in whom family history is often consistent with sporadic or autosomal recessive inheritance, implicating a nuclear genetic cause. In contrast, although a number of recurrent, pathogenic mtDNA mutations have been described, historically, these have been perceived as rare causes of paediatric complex I deficiency. We reviewed the clinical and genetic findings in a large cohort of 109 paediatric patients with isolated complex I deficiency from 101 families. Pathogenic mtDNA mutations were found in 29 of 101 probands (29%), 21 in MTND subunit genes and 8 in mtDNA tRNA genes. Nuclear gene defects were inferred in 38 of 101 (38%) probands based on cell hybrid studies, mtDNA sequencing or mutation analysis (nuclear gene mutations were identified in 22 probands). Leigh or Leigh-like disease was the most common clinical presentation in both mtDNA and nuclear genetic defects. The median age at onset was higher in mtDNA patients (12 months) than in patients with a nuclear gene defect (3 months). However, considerable overlap existed, with onset varying from 0 to >60 months in both groups. Our findings confirm that pathogenic mtDNA mutations are a significant cause of complex I deficiency in children. In the absence of parental consanguinity, we recommend whole mitochondrial genome sequencing as a key approach to elucidate the underlying molecular genetic abnormality.

Oculopharyngodistal (OPD) myopathy (OMIM 164310) is a clinically and genetically distinct entity for which a molecular defect has not yet been found. Mutations in MYH2 have been described in patients with ophthalmoplegia presenting with multiple contractures at birth, later developing a proximal myopathy in the adulthood. We present a patient clinically classified as OPD myopathy who was found to carry a MYH2 de novo mutation. An Australian male of Italian background presented at 18 years old with predominantly distal but also proximal limb weakness, ophthalmoplegia, dysphagia and facial weakness. He later developed respiratory involvement. Parents were not affected. The course of the disease was progressive, being wheelchair bound by the age of 50. He had 4 muscle biopsies done from age 20 to 46, none of which showed rimmed vacuoles. Dystrophic features, type I predominance, and abundant lobulated fibres were consistently found. Ultrastructure showed foci of Z band streaming. In one biopsy, total absence of type II fibres was seen. However, unlike previously reported cases, the structural changes did not seem to be more severe at an older age. A panel of 277 neuromuscular disease-causing genes were sequenced using next generation sequencing methods. A missense variation in exon 39 of MYH2: c.T5630C (p.L1877P) not previously seen in normal populations, was found. The mutation introduces a proline in the tail of myosin, presumably interfering with the assembly of thick filaments, as in MYH7 mutations causing Laing distal myopathy. Segregation studies proved it to be a de novo mutation. This case expands the phenotype of mutations in MYH2 to include predominantly distal limb weakness. We suggest that mutations in MYH2 should be considered in patients with distal weakness and ophthalmoplegia.

Two problems for molecular diagnosis of neurogenetic disorders are high levels of genetic heterogeneity and involvement of large genes, e.g. titin. These problems result in many patients not being diagnosed by current Sanger-sequencing based diagnostics. To offset these problems we have developed a TargetSeq (Life Technologies) capture panel consisting of 335 disease genes with mutations detectable by next generation sequencing (NGS). The genes were 276 neurogenetic and 59 cardiomyopathy genes, targeted together because of clinical overlap. The 335 panel genes were captured from pools of 16 bar-coded patient DNA samples and sequenced 16 patients at a time using Ion Proton™ (Life Technologies) sequencing. Variant analysis was by either custom Annovar or Cartagenia (Cartagenia, Inc.) based pipelines. Testing the panel identified 88% of the known small-scale mutations in 28 positive controls. Known mutations deliberately chosen in regions not sequenced well by NGS were not detected. Using the FishingCNV package, we identified 90% of CNV controls, including 100% of CMT1A/HNPP duplication/deletion controls. Although one single exon CNV was detected, not all small CNVs were detected. We have screened >300 molecularly undiagnosed probands, identifying mutations in 59 disease genes, many of which were not previously analysed by the diagnostic laboratory due to Sanger sequencing costs. Using the targeted panel is not ethically different to traditional sequential Sanger sequencing diagnostics. The panel needs updating regularly with new disease gene discoveries. We now receive samples from all round Australasia for analysis of the 335-gene panel, which is cheaper than Sanger.

Muscle mass and strength are variable traits in humans. Many French Canadians (FC) became international celebrities because of their exceptional strength. Though muscle hypertrophy has been associated in many mammals with myostatin mutations, to date only a single pediatric case has been reported in humans. Dominant pathological mutations in the CLCN1, SCN4A, CAV3, ATP2A1, LPIN1, LMNA and RYR1 gene have been known to lead to muscle hypertrophy in humans, in particular selective muscle hypertrophy in certain myopathies. These conditions are associated with either weakness or increased strength. We recruited a cohort of more than 115 cases belonging to more than 65 families of a heterogeneous dominant condition that we refer to as: “Strongman syndrome” (SM). In all families, the most affected case suffers from incapacitating myalgias with variable degree of muscle cramps, and they also have a personal and familial history of above average strength. On examination these patients have large well defined muscles, above average strength, non-electric prolonged muscle contractions and progressive weakness on repeated contractions. Exome sequencing of a large FC family identified a single segregating variant predicted to be damaging in the DCST2 gene shared also by two other FC families. The DCST2 gene is expressed in skeletal muscle and is predicted to code for a six transmembrane domains channel-like protein. To better characterize the herculean strength we assesses different biometric variables in a subset of cases with and without DCST2 mutations and investigated if myofiber hypertrophy is present. Results of quantifiable methods to evaluate the clinical and pathological phenotype of strongman cases will be presented, as well as functional data about DCST2. The uncovering of the function of DCST2 and other strongman genes will contribute to a better understanding of pathways important for muscle hypertrophy and strength in health and disease.

The collagen VI muscular dystrophies, Bethlem myopathy and Ullrich congenital muscular dystrophy, form a continuum of clinical phenotypes. Glycine mutations in the triple helix have been identified in both Bethlem and Ullrich congenital muscular dystrophy, but it is not known why they cause these different phenotypes.We studied eight new patients who presented with a spectrum of clinical severity, screened the three collagen VI messenger RNA for mutations, and examined collagen VI biosynthesis and the assembly pathway.All eight patients had heterozygous glycine mutations toward the N-terminal end of the triple helix. The mutations produced two assembly phenotypes. In the first patient group, collagen VI dimers accumulated in the cell but not the medium, microfibril formation in the medium was moderately reduced, and the amount of collagen VI in the extracellular matrix was not significantly altered. The second group had more severe assembly defects: some secreted collagen VI tetramers were not disulfide bonded, microfibril formation in the medium was severely compromised, and collagen VI in the extracellular matrix was reduced.These data indicate that collagen VI glycine mutations impair the assembly pathway in different ways and disease severity correlates with the assembly abnormality. In mildly affected patients, normal amounts of collagen VI were deposited in the fibroblast matrix, whereas in patients with moderate-to-severe disability, assembly defects led to a reduced collagen VI fibroblast matrix. This study thus provides an explanation for how different glycine mutations produce a spectrum of clinical severity.

Hereditary myosin myopathies are a newly emerged group of diseases caused by mutations in skeletal muscle myosin heavy chain (MyHC) genes. The phenotypes of these diseases are varied, ranging from prenatal nonprogressive arthrogrypotic syndromes to adult-onset progressive muscle weakness. They are caused by mutations in skeletal muscle myosin heavy chain (MyHC) genes. Mutations have been reported in two of three MyHC isoforms expressed in adult limb skeletal muscle: type I (slow/β-cardiac MyHC; MYH7) and type IIa (MYH2). Most of the mutations described in MYH7 are associated with hypertrophic/dilated cardiomyopathy, with no skeletal muscle involvement. However, some mutations are associated with two distinct skeletal myopathies, namely Laing distal myopathy and myosin storage myopathy. Although initially thought not to have associated cardiac involvement, recent reports have indicated co-existent cardiac and skeletal muscle disease can occur in both. A myopathy associated with a specific mutation in MYH2 is associated with congenital joint contractures and external ophthalmoplegia. Mutations in embryonic MyHC (MYH3) and perinatal MyHC (MYH8) are associated with distal arthrogryposis syndromes with no or minor muscle weakness. This may be expected in myosin isoforms expressed predominantly during muscle development. Clinical findings, muscle morphology and molecular genetics in hereditary myosin myopathies are summarized in this chapter.

Three infants are described who had nemaline rods on muscle biopsy and isolated deficiency of complex I of the respiratory chain on biochemical analysis. They all manifested failure to thrive from birth, and hypotonia and muscle weakness within the first three months of life. Different genetic defects leading to isolated complex I deficiency have been described associated with a variety of morphological changes on muscle biopsy, but rods have not been described. Nemaline rods have been secondary phenomena in a number of conditions, as well as being the primary abnormality in nemaline myopathy. However, the combination of nemaline rods and complex I deficiency is an association not previously reported.

Several pedigrees have recently been reported in which dominantly inherited familial Alzheimer's disease is associated in some family members with spastic paraparesis and non-neuritic 'cotton wool' plaques. Here we report clinical, genetic and neuropathological findings in two further large pedigrees in which this combination of phenotypes is associated with a deletion of exon 9 of the presenilin-1 (PS-1) gene caused by mutations at the splice acceptor site. In both pedigrees, individuals with paraparesis at presentation had a later than average age at onset of symptoms. In addition, one subject with paraparesis had a much less prominent dementia syndrome than his dementia-affected siblings. As PS-1 mutations are almost always associated with a particularly aggressive form of presenile dementia, these findings suggest the existence of a protective or delaying factor in individuals with spastic paraparesis.

Item does not contain fulltext Nemaline myopathy is a congenital neuromuscular disorder characterized by muscle weakness and the presence of nemaline rods. Five genes have now been associated with nemaline myopathy: alpha-tropomyosin-3 (TPM3), alpha-actin (ACTA1), nebulin (NEB), beta-tropomysin (TPM2) and troponin T (TNNT1). In addition, mutations in the ryanodine receptor gene (RYR1) have been associated with core-rod myopathy. Here we report linkage in two unrelated families, with a variant of nemaline myopathy, with associated core-like lesions. The clinical phenotype consists of muscle weakness in addition to a peculiar kind of muscle slowness. A genome-wide scan revealed a locus for nemaline myopathy with core-like lesions on chromosome 15q21-q23 for both families. Combining the two families gave a two-point LOD score of 10.65 for D15S993. The alpha-tropomyosin-1 gene (TPM1) located within this region is the strongest candidate gene. However, no mutations were found in the protein-coding region of TPM1, although small deletions or mutations in an intron cannot be excluded. The critical region contains few other candidate genes coding for muscle proteins and several genes of unknown function, and has not yet been sequenced completely. The novel phenotype of nemaline myopathy in the two presented families corresponds to an also novel, as yet uncharacterized, genotype.

The autosomal recessive spinocerebellar ataxias are a clinically and genetically heterogeneous group of neurodegenerative disorders associated with 10 disease genes and seven other loci. Because of the genetic and phenotypic heterogeneity of this disease, a large percentage of cases go without a genetic diagnosis. In the family under investigation, two out of eight siblings are affected, and the parents are second cousins, suggesting a recessive disease. The two affected brothers presented at 55 and 54 years respectively with weakness about the ankles, poor balance and chronic cough. There were cerebellar signs with saccadic interruption of eye movements and a wide-based gait, and later on dysarthria. Creatine kinase was elevated, to between 580 and 1020 (N

Charcot–Marie–Tooth disease (CMT) is a common inherited peripheral neuropathy associated with mutations in 54 known genes. CMT is characterized by distal wasting of the legs and later hands. Skeletal deformations are frequent including pes cavus and hammer toes. CMT is subdivided into three subtypes: CMT1 is demyelinating, CMT2 axonal, and intermediate CMT mixed. The clinical and molecular heterogeneity of this disease means many families remain without a molecular diagnosis. This proof of principal study aimed to investigate the use of whole exome sequencing (WES), allied with linkage analysis, to detect known or novel mutations in CMT families in which CMT1A had been excluded. We looked at three families, two dominant and one consanguineous. All other known CMT regions were excluded by linkage analysis in one dominant family; all CMT regions except MPZ, by homozygosity mapping in the consanguineous family. In both these families, WES excluded all known CMT genes including MPZ in the consanguineous family. This suggests the CMT genes in these two families are novel. The second dominant family presented with pes cavus and hammer toes in the first decade followed by difficulty in participation in sports in the second decade. Nerve conduction studies showed a severe sensorimotor axonal peripheral neuropathy. Linkage analysis excluded all known CMT genes except a 65 Mbp region on chromosome 16 including three known CMT genes. One of these, AARS (encoding alanyl-tRNA synthetase), fitted with the autosomal dominant inheritance in the family. WES of the proband identified a heterozygous variant, c.G986A (p.R329H) in AARS, which is a known pathogenic mutation. The variant was confirmed by Sanger sequencing in the proband and all other affected family members available, indicating the mutation is the cause of the disease in the family. Our study highlights the potential of WES in diagnostics and that more CMT genes remain to be found.

Mutations in over 20 genes are associated with distal myopathies. Yet, many patients remain unresolved. To genetically characterize a cohort of distal myopathies we recruited patients with distal weakness and normal motor nerve conduction studies. We used next generation sequencing methods to study a panel of 277 genes that have been associated with muscle and nerve diseases plus Sanger sequencing of not well covered target regions. Sixteen patients were included. Five were characterized as Oculopharyngo-dystal myopathies (OPD) and those were studied separately. In the general group all were sporadic cases. The average age of onset was 34 years old. Three patients had facial weakness, 8 had proximal weakness, in 4 the anterior compartment of lower limbs was more severely affected and 3 the posterior compartment was more affected. Muscle biopsy showed necrotic and regenerating fibres in 5 patients, vacuoles in 3, denervative features in 2, inflammation in 1 and eosinophilic aggregates in 1. Gastrocnemius was the most commonly affected muscle in the MRI. Genetic analysis showed in four patients: a known LDB3 mutation, a novel mutation in FLNC, a possible splice site mutation in FIG4 and a known mutation in SOD1. Retrospective review of this last patient confirmed a clinical diagnosis of ALS. In the OPD group, 2 patients were related. The average age of onset was 24 years old. Three patients had facial involvement, 4 had ophthalmoparesis, 4 had ptosis, 3 had proximal weakness, in 2 the posterior compartment of lower legs was predominantly involved. Two patients had muscle biopsies both having rimmed vacuoles. Expansions in PABPN1 were excluded in all. One patient was found to have a mutation in MYH2, and 1 in POLG2. The other 3 remain unresolved. Genetic causes of clinically diagnosed distal myopathies are diverse, with others yet to be identified. Even in patients with no neuropathic signs neurogenic diseases are still to be considered.