Genome and RNA sequencing boost neuromuscular diagnoses to 62% from 34% with exome sequencing alone.

OBJECTIVE: Most families with heritable neuromuscular disorders do not receive a molecular diagnosis. Here we evaluate diagnostic utility of exome, genome, RNA sequencing, and protein studies and provide evidence-based recommendations for their integration into practice. METHODS: In total, 247 families with suspected monogenic neuromuscular disorders who remained without a genetic diagnosis after standard diagnostic investigations underwent research-led massively parallel sequencing: neuromuscular disorder gene panel, exome, genome, and/or RNA sequencing to identify causal variants. Protein and RNA studies were also deployed when required. RESULTS: Integration of exome sequencing and auxiliary genome, RNA and/or protein studies identified causal or likely causal variants in 62% (152 out of 247) of families. Exome sequencing alone informed 55% (83 out of 152) of diagnoses, with remaining diagnoses (45%; 69 out of 152) requiring genome sequencing, RNA and/or protein studies to identify variants and/or support pathogenicity. Arrestingly, novel disease genes accounted for <4% (6 out of 152) of diagnoses while 36.2% of solved families (55 out of 152) harbored at least one splice-altering or structural variant in a known neuromuscular disorder gene. We posit that contemporary neuromuscular disorder gene-panel sequencing could likely provide 66% (100 out of 152) of our diagnoses today. INTERPRETATION: Our results emphasize thorough clinical phenotyping to enable deep scrutiny of all rare genetic variation in phenotypically consistent genes. Post-exome auxiliary investigations extended our diagnostic yield by 81% overall (34-62%). We present a diagnostic algorithm that details deployment of genomic and auxiliary investigations to obtain these diagnoses today most effectively. We hope this provides a practical guide for clinicians as they gain greater access to clinical genome and transcriptome sequencing.

Vibration Therapy as an Early Intervention for Children Aged 2-4 Years with Cerebral Palsy: A Feasibility Study.

Aims: To evaluate the feasibility and acceptability of vibration therapy (VT) in preschool children with cerebral palsy (CP) and obtain preliminary data on its potential effectiveness.Methods: Nine children aged 2.5-4.8 years (4 boys) with CP GMFCS levels I-III participated in a single-group feasibility study, undergoing a 12-week control period without intervention, followed by 12 weeks of home-based VT (four times/week, 9 min/day, frequency 20 Hz). We assessed adherence to VT protocol, adverse events, and family acceptability of VT. Clinical assessments included motor function (GMFM-66), body composition (DXA), mobility (10-meter walk/run test), and health-related quality of life (PedsQL).Results: VT was well tolerated and acceptable to families, with high adherence levels reported (mean = 93%). There were no observed between-period differences (ΔControl vs ΔVT) except for an improvement in the PedsQL "Movement & Balance" dimension with VT (p = 0.044). Nonetheless, changes after the VT but not the Control period were suggestive of potential treatment benefits for mobility, gross motor function, and body composition (lean mass and legs bone mineral density).Conclusion: Home-based VT is feasible and acceptable for preschool children with CP. Our preliminary data suggest potential health benefits from VT for these children, supporting larger randomized trials to assess its effectiveness properly. Clinical trial registration number: Australian New Zealand Clinical Trials Registry (ACTRN12618002027291).

Compound heterozygous splicing variants expand the genotypic spectrum of EMC1-related disorders.

EMC1 encodes subunit 1 of the endoplasmic reticulum (ER) membrane protein complex (EMC), a transmembrane domain insertase involved in membrane protein biosynthesis. Variants in EMC1 are described as a cause of global developmental delay, hypotonia, cortical visual impairment, and commonly, cerebral atrophy on MRI scan. We report an individual with severe global developmental delay and progressive cerebellar atrophy in whom exome sequencing identified a heterozygous essential splice-site variant in intron-3 of EMC1 (NM_015047.3:c.287-1G>A). Whole genome sequencing (WGS) identified a deep intronic variant in intron-20 of EMC1 (NM_015047.3:c.2588-771C>G) that was poorly predicted by in silico programs to disrupt pre-mRNA splicing. Reverse Transcription-PCR (RT-PCR) revealed stochastic activation of a pseudo-exon associated with the c.2588-771C>G variant and mis-splicing arising from the c.287-1G>A variant. This case highlights the utility of WGS and RNA studies to identify and assess likely pathogenicity of deep intronic variants and expands the genotypic and phenotypic spectrum of EMC1-related disorders.

WGS and RNA Studies Diagnose Noncoding DMD Variants in Males With High Creatine Kinase.

OBJECTIVE: To describe the diagnostic utility of whole-genome sequencing and RNA studies in boys with suspected dystrophinopathy, for whom multiplex ligation-dependent probe amplification and exomic parallel sequencing failed to yield a genetic diagnosis, and to use remnant normal DMD splicing in 3 families to define critical levels of wild-type dystrophin bridging clinical spectrums of Duchenne to myalgia. METHODS: Exome, genome, and/or muscle RNA sequencing was performed for 7 males with elevated creatine kinase. PCR of muscle-derived complementary DNA (cDNA) studied consequences for DMD premessenger RNA (pre-mRNA) splicing. Quantitative Western blot was used to determine levels of dystrophin, relative to control muscle. RESULTS: Splice-altering intronic single nucleotide variants or structural rearrangements in DMD were identified in all 7 families. Four individuals, with abnormal splicing causing a premature stop codon and nonsense-mediated decay, expressed remnant levels of normally spliced DMD mRNA. Quantitative Western blot enabled correlation of wild-type dystrophin and clinical severity, with 0%-5% dystrophin conferring a Duchenne phenotype, 10% ± 2% a Becker phenotype, and 15% ± 2% dystrophin associated with myalgia without manifesting weakness. CONCLUSIONS: Whole-genome sequencing relied heavily on RNA studies to identify DMD splice-altering variants. Short-read RNA sequencing was regularly confounded by the effectiveness of nonsense-mediated mRNA decay and low read depth of the giant DMD mRNA. PCR of muscle cDNA provided a simple, yet informative approach. Highly relevant to genetic therapies for dystrophinopathies, our data align strongly with previous studies of mutant dystrophin in Becker muscular dystrophy, with the collective conclusion that a fractional increase in levels of normal dystrophin between 5% and 20% is clinically significant.

Exercise Cardiac Magnetic Resonance Imaging in Boys With Duchenne Muscular Dystrophy Without Cardiac Disease.

BACKGROUND: Duchenne muscular dystrophy is caused by mutations in the DMD gene, resulting in cardiomyopathy in all affected children by 18 years. Although cardiomyopathy is now the leading cause of mortality in these children, there is ongoing debate regarding timely diagnosis, secondary prevention, and treatment of this condition. The purpose of this study was to use exercise cardiac magnetic resonance imaging in asymptomatic young boys with Duchenne muscular dystrophy to describe their heart function and compare this with healthy controls. METHODS: We studied 11 boys with Duchenne muscular dystrophy aged 8.6 to 13.9 years and 11 healthy age- and sex-matched controls. RESULTS: Compared with the controls, boys with Duchenne muscular dystrophy had lower ejection fraction at rest (57% versus 63%; P = 0.004). During submaximal exercise, they reached similar peak tachycardia but increased their heart rate and cardiac output only half as much as controls (P = 0.003 and P = 0.014, respectively). End-systolic volume remained higher in boys with Duchenne muscular dystrophy both at rest and during exercise. When transthoracic echocardiography was compared with cardiac magnetic resonance imaging, 45% of the echocardiograms had suboptimal or poor views in the Duchenne muscular dystrophy group. CONCLUSIONS: Boys with Duchenne muscular dystrophy had abnormalities in left ventricular systolic function that were exaggerated by exercise stress. Exercise cardiac magnetic resonance imaging is feasible in a select population of children with Duchenne muscular dystrophy, and it has the potential to unmask early signs of cardiomyopathy.

Exercise Training as Part of Musculoskeletal Management for Congenital Myopathy: Where Are We Now?

Congenital myopathy is a heterogeneous group of muscle disorders characterized by muscle weakness and hypotonia. This condition is associated with a range of skeletal, respiratory, and ophthalmologic complications and requires a multidisciplinary therapeutic approach aimed at maximizing the function and independence of patients. One promising direction for therapeutic intervention is physical exercise rehabilitation, given its demonstrated ability to promote muscle and bone health of patients with a variety of neuromuscular conditions. However, there are few data to assist health care professionals identify the optimal physical activity levels and exercise type, including the intensity, frequency, and duration. This lack of empirical evidence is particularly problematic given the fact that inappropriate exercise modes can potentially cause muscle damage in patients with congenital myopathy. In this article, we discuss the rationale behind the incorporation of two types of physical exercises, strength and aerobic training, into the clinical care of patients with congenital myopathy. Given the paucity of literature on the management of congenital myopathy, we review the results of published research on the treatment of both congenital myopathy and other neuromuscular diseases that could provide helpful insights into the physical rehabilitation of patients with congenital myopathy. We also discuss the potential benefits of vibration therapy, which has been studied in patients with other neuromuscular disorders over the last two decades. We conclude by proposing directions for future research on physical rehabilitation of patients with congenital myopathy.

Isolated seizures during the first episode of relapsing myelin oligodendrocyte glycoprotein antibody-associated demyelination in children.

Myelin oligodendrocyte glycoprotein (MOG) antibodies have a strong association with acute disseminated encephalomyelitis (ADEM) in children, and bilateral and recurrent optic neuritis in children and adults. Recent reports suggest that seizures and encephalopathy may occur in children and adults with MOG antibody-associated disease. We describe the clinical, laboratory, and radiological course of four MOG antibody-positive children who first presented with isolated seizures without fulfilling clinical or radiological criteria for ADEM or other central nervous system demyelination syndromes, who months to years later developed more typical demyelination. This case series highlights a novel observation that isolated seizures in the absence of ADEM may be the index presentation for MOG antibody-associated disease, which should therefore be considered a form of autoimmune epilepsy. It would be reasonable to test for MOG antibodies in children with seizures accompanied by subtle inflammatory changes on magnetic resonance imaging or cerebrospinal fluid analyses, particularly if followed by demyelination, given the clinical and therapeutic implications of an expedited diagnosis in minimizing long-term disability. WHAT THIS PAPER ADDS: Isolated seizures in the absence of acute disseminated encephalomyelitis may be the index presentation for myelin oligodendrocyte glycoprotein antibody-associated demyelination.

CONVULSIONES AISLADAS DURANTE UNA PRIMERA RECAÍDA DE UN EVENTO DESMIELINIZANTE ASOCIADA A ANTICUERPOS CONTRA LA GLICOPROTEINA DE LOS OLIGODENDROCITOS DE LA MIELINA: Los anticuerpos contra la Glicoproteina de los Oligodendrocitos de la Mielina (MOG) están fuertemente asociados con la Encefalomielitis Aguda Diseminada (EMAD) en niños, y con la Neuritis Óptica bilateral recurrente en niños y adultos. Recientes reportes sugieren que convulsiones y encefalopatía pueden presentarse con enfermedad asociada a anticuerpos MOG. Describimos la evolución clínica, serologica y radiológica de cuatro niños con anticuerpos positivos para MOG cuya primera presentación fueron convulsiones aisladas sin cumplir todos los criterios cínicos ni radiológicos para EMAD u otro síndrome desmielinizante del sistema nervioso central; y, que luego de meses a años, desarrollaron una desmielinización típica. Esta serie de casos resalta una nueva observación, en la cual, convulsiones aisladas en ausencia de EMAD puede ser la presentación de enfermedad asociada a anticuerpos para MOG; y, por lo tanto, debe ser considerada una forma de epilepsia autoinmune. Sería razonable buscar anticuerpos para MOG en aquellos niños con convulsiones que se acompañan de cambios inflamatorios sutiles en la resonancia magnética y análisis de líquido cefalorraquídeo, en particular, en aquellos que posteriormente desarrollan desmielinización teniendo en cuenta las implicancias clínicas y terapéuticas de realizar un diagnóstico rápido con el fin de minimizar la discapacidad a largo plazo.

CONVULSÕES ISOLADAS DURANTE O PRIMEIRO EPISÓDIO DE DESMIELINIZAÇÃO ASSOCIADA AO ANTICORPO DE GLICOPROTEÍNA DE OLIGODENDRÓCITO DA MIELINA EM CRIANÇAS: Anticorpos de glicoproteína de oligodendrócito da mielina (GOM) têm forte associação com encefalomielite aguda disseminada (EMAD) em crianças, e com neurite óptica bilateral e recorrente em crianças e adultos. Relatos recentes sugerem que convulsões e encefalopatia podem ocorrer em adultos e crianças com doença associada aos anticorpos GOM. Descrevemos o curso clínico, laboratorial, e radiológico de crianças com anticorpos GOM positivos que apresentaram convulsões isoladas pela primeira vez, sem preencher os critérios clínicos e radiológicos para EMAD ou para outras síndromes desmielinizantes do sistema nervoso central, que nos meses ou anos seguintes desenvolveram desmielinização mais típica. Esta série de casos realça uma nova observação de que convulsões isoladas na ausência de EMAD podem ser sinal de doença relacionada ao anticorpo GOM, e deve portanto ser considerada uma forma de epilepsia auto-imune. Seria razoável testar anticorpos GOM em crianças com convulsões acompanhada de doenças inflamatórias sutis à ressonância magnética ou na análise do fluido cérebro-espinhal, particularmente se seguida por desmielinização, dadas as implicações clínicas e terapêuticas do diagnóstico rápido em minimizar incapacidades no longo prazo.

Imaging the heart to detect cardiomyopathy in Duchenne muscular dystrophy: A review.

Duchenne Muscular Dystrophy is the most common paediatric neuromuscular disorder. Mutations in the DMD gene on the X-chromosome result in progressive skeletal muscle weakness as the main clinical manifestation. However, cardiac muscle is also affected, with cardiomyopathy becoming an increasingly recognised cause of morbidity, and now the leading cause of mortality in this group. The diagnosis of cardiomyopathy has often been made late due to technical limitations in transthoracic echocardiograms and delayed symptomatology in less mobile patients. Increasingly, evidence supports earlier pharmacological intervention in cardiomyopathy to improve outcomes. However, the optimal timing of initiation remains uncertain, and the benefits of prophylactic therapy are unproven. Current treatment guidelines suggest initiation of therapy once cardiac dysfunction is detected. This review focuses on new and evolving techniques for earlier detection of Duchenne muscular dystrophy-associated cardiomyopathy. Transthoracic echocardiography or cardiac magnetic resonance imaging performed under physiological stress (dobutamine or exercise), can unmask early cardiac dysfunction. Cardiac magnetic resonance imaging can define cardiac function with greater accuracy and reliability than an echocardiogram, and is not limited by body habitus. Improved imaging techniques, used in a timely fashion, offer the potential for early detection of cardiomyopathy and improved long-term outcomes.

Congenital Titinopathy: Comprehensive characterization and pathogenic insights.

OBJECTIVE: Comprehensive clinical characterization of congenital titinopathy to facilitate diagnosis and management of this important emerging disorder. METHODS: Using massively parallel sequencing we identified 30 patients from 27 families with 2 pathogenic nonsense, frameshift and/or splice site TTN mutations in trans. We then undertook a detailed analysis of the clinical, histopathological and imaging features of these patients. RESULTS: All patients had prenatal or early onset hypotonia and/or congenital contractures. None had ophthalmoplegia. Scoliosis and respiratory insufficiency typically developed early and progressed rapidly, whereas limb weakness was often slowly progressive, and usually did not prevent independent walking. Cardiac involvement was present in 46% of patients. Relatives of 2 patients had dilated cardiomyopathy. Creatine kinase levels were normal to moderately elevated. Increased fiber size variation, internalized nuclei and cores were common histopathological abnormalities. Cap-like regions, whorled or ring fibers, and mitochondrial accumulations were also observed. Muscle magnetic resonance imaging showed gluteal, hamstring and calf muscle involvement. Western blot analysis showed a near-normal sized titin protein in all samples. The presence of 2 mutations predicted to impact both N2BA and N2B cardiac isoforms appeared to be associated with greatest risk of cardiac involvement. One-third of patients had 1 mutation predicted to impact exons present in fetal skeletal muscle, but not included within the mature skeletal muscle isoform transcript. This strongly suggests developmental isoforms are involved in the pathogenesis of this congenital/early onset disorder. INTERPRETATION: This detailed clinical reference dataset will greatly facilitate diagnostic confirmation and management of patients, and has provided important insights into disease pathogenesis. Ann Neurol 2018;83:1105-1124.

Cost-effectiveness of massively parallel sequencing for diagnosis of paediatric muscle diseases.

Childhood-onset muscle disorders are genetically heterogeneous. Diagnostic workup has traditionally included muscle biopsy, protein-based studies of muscle specimens, and candidate gene sequencing. High throughput or massively parallel sequencing is transforming the approach to diagnosis of rare diseases; however, evidence for cost-effectiveness is lacking. Patients presenting with suspected congenital muscular dystrophy or nemaline myopathy were ascertained over a 15-year period. Patients were investigated using traditional diagnostic approaches. Undiagnosed patients were investigated using either massively parallel sequencing of a panel of neuromuscular disease genes panel, or whole exome sequencing. Cost data were collected for all diagnostic investigations. The diagnostic yield and cost effectiveness of a molecular approach to diagnosis, prior to muscle biopsy, were compared with the traditional approach. Fifty-six patients were analysed. Compared with the traditional invasive muscle biopsy approach, both the neuromuscular disease panel and whole exome sequencing had significantly increased diagnostic yields (from 46 to 75% for the neuromuscular disease panel, and 79% for whole exome sequencing), and reduced the cost per diagnosis from USD$16,495 (95% CI: $12,413-$22,994) to USD$3706 (95% CI: $3086-$4453) for the neuromuscular disease panel and USD $5646 (95% CI: $4501-$7078) for whole exome sequencing. The neuromuscular disease panel was the most cost-effective, saving USD$17,075 (95% CI: $10,654-$30,064) per additional diagnosis, over the traditional diagnostic pathway. Whole exome sequencing saved USD$10,024 (95% CI: $5795-$17,135) per additional diagnosis. This study demonstrates the cost-effectiveness of investigation using massively parallel sequencing technologies in paediatric muscle disease. The findings emphasise the value of implementing these technologies in clinical practice, with particular application for diagnosis of Mendelian diseases, and provide evidence crucial for government subsidy and equitable access.

The New Zealand Neuromuscular Disease Patient Registry; Five Years and a Thousand Patients.

The New Zealand Neuromuscular Disease Patient Registry has been recruiting for five years. Its primary aim is to enable people with neuromuscular disease to participate in research including clinical trials. It has contributed data to large anonymised cohort studies and many feasibility studies, and has provided practical information and advice to researchers wanting to work with people with neuromuscular conditions. 1019 people have enrolled since the Registry's launch in August 2011 with over 70 different diagnoses. Of these; 8 patients have been involved in clinical trials, 134 in other disease-specific research and 757 have contributed anonymised data to cohort studies. As a result the Registry is now effectively facilitating almost all neuromuscular research currently taking place in New Zealand.

Improving genetic diagnosis in Mendelian disease with transcriptome sequencing.

Exome and whole-genome sequencing are becoming increasingly routine approaches in Mendelian disease diagnosis. Despite their success, the current diagnostic rate for genomic analyses across a variety of rare diseases is approximately 25 to 50%. We explore the utility of transcriptome sequencing [RNA sequencing (RNA-seq)] as a complementary diagnostic tool in a cohort of 50 patients with genetically undiagnosed rare muscle disorders. We describe an integrated approach to analyze patient muscle RNA-seq, leveraging an analysis framework focused on the detection of transcript-level changes that are unique to the patient compared to more than 180 control skeletal muscle samples. We demonstrate the power of RNA-seq to validate candidate splice-disrupting mutations and to identify splice-altering variants in both exonic and deep intronic regions, yielding an overall diagnosis rate of 35%. We also report the discovery of a highly recurrent de novo intronic mutation in COL6A1 that results in a dominantly acting splice-gain event, disrupting the critical glycine repeat motif of the triple helical domain. We identify this pathogenic variant in a total of 27 genetically unsolved patients in an external collagen VI-like dystrophy cohort, thus explaining approximately 25% of patients clinically suggestive of having collagen VI dystrophy in whom prior genetic analysis is negative. Overall, this study represents a large systematic application of transcriptome sequencing to rare disease diagnosis and highlights its utility for the detection and interpretation of variants missed by current standard diagnostic approaches.

Targeted Re-Sequencing Emulsion PCR Panel for Myopathies: Results in 94 Cases.

BACKGROUND: Molecular diagnostics in the genetic myopathies often requires testing of the largest and most complex transcript units in the human genome (DMD, TTN, NEB). Iteratively targeting single genes for sequencing has traditionally entailed high costs and long turnaround times. Exome sequencing has begun to supplant single targeted genes, but there are concerns regarding coverage and needed depth of the very large and complex genes that frequently cause myopathies. OBJECTIVE: To evaluate efficiency of next-generation sequencing technologies to provide molecular diagnostics for patients with previously undiagnosed myopathies. METHODS: We tested a targeted re-sequencing approach, using a 45 gene emulsion PCR myopathy panel, with subsequent sequencing on the Illumina platform in 94 undiagnosed patients. We compared the targeted re-sequencing approach to exome sequencing for 10 of these patients studied. RESULTS: We detected likely pathogenic mutations in 33 out of 94 patients with a molecular diagnostic rate of approximately 35%. The remaining patients showed variants of unknown significance (35/94 patients) or no mutations detected in the 45 genes tested (26/94 patients). Mutation detection rates for targeted re-sequencing vs. whole exome were similar in both methods; however exome sequencing showed better distribution of reads and fewer exon dropouts. CONCLUSIONS: Given that costs of highly parallel re-sequencing and whole exome sequencing are similar, and that exome sequencing now takes considerably less laboratory processing time than targeted re-sequencing, we recommend exome sequencing as the standard approach for molecular diagnostics of myopathies.

Variants in the Oxidoreductase PYROXD1 Cause Early-Onset Myopathy with Internalized Nuclei and Myofibrillar Disorganization.

This study establishes PYROXD1 variants as a cause of early-onset myopathy and uses biospecimens and cell lines, yeast, and zebrafish models to elucidate the fundamental role of PYROXD1 in skeletal muscle. Exome sequencing identified recessive variants in PYROXD1 in nine probands from five families. Affected individuals presented in infancy or childhood with slowly progressive proximal and distal weakness, facial weakness, nasal speech, swallowing difficulties, and normal to moderately elevated creatine kinase. Distinctive histopathology showed abundant internalized nuclei, myofibrillar disorganization, desmin-positive inclusions, and thickened Z-bands. PYROXD1 is a nuclear-cytoplasmic pyridine nucleotide-disulphide reductase (PNDR). PNDRs are flavoproteins (FAD-binding) and catalyze pyridine-nucleotide-dependent (NAD/NADH) reduction of thiol residues in other proteins. Complementation experiments in yeast lacking glutathione reductase glr1 show that human PYROXD1 has reductase activity that is strongly impaired by the disease-associated missense mutations. Immunolocalization studies in human muscle and zebrafish myofibers demonstrate that PYROXD1 localizes to the nucleus and to striated sarcomeric compartments. Zebrafish with ryroxD1 knock-down recapitulate features of PYROXD1 myopathy with sarcomeric disorganization, myofibrillar aggregates, and marked swimming defect. We characterize variants in the oxidoreductase PYROXD1 as a cause of early-onset myopathy with distinctive histopathology and introduce altered redox regulation as a primary cause of congenital muscle disease.

Variants in SLC18A3, vesicular acetylcholine transporter, cause congenital myasthenic syndrome.

OBJECTIVE: To describe the clinical and genetic characteristics of presynaptic congenital myasthenic syndrome secondary to biallelic variants in SLC18A3. METHODS: Individuals from 2 families were identified with biallelic variants in SLC18A3, the gene encoding the vesicular acetylcholine transporter (VAChT), through whole-exome sequencing. RESULTS: The patients demonstrated features seen in presynaptic congenital myasthenic syndrome, including ptosis, ophthalmoplegia, fatigable weakness, apneic crises, and deterioration of symptoms in cold water for patient 1. Both patients demonstrated moderate clinical improvement on pyridostigmine. Patient 1 had a broader phenotype, including learning difficulties and left ventricular dysfunction. Electrophysiologic studies were typical for a presynaptic defect. Both patients showed profound electrodecrement on low-frequency repetitive stimulation followed by a prolonged period of postactivation exhaustion. In patient 1, this was unmasked only after isometric contraction, a recognized feature of presynaptic disease, emphasizing the importance of activation procedures. CONCLUSIONS: VAChT is responsible for uptake of acetylcholine into presynaptic vesicles. The clinical and electrographic characteristics of the patients described are consistent with previously reported mouse models of VAChT deficiency. These findings make it very likely that defects in VAChT due to variants in SLC18A3 are a cause of congenital myasthenic syndrome in humans.

Diagnosis and etiology of congenital muscular dystrophy: We are halfway there.

OBJECTIVE: To evaluate the diagnostic outcomes in a large cohort of congenital muscular dystrophy (CMD) patients using traditional and next generation sequencing (NGS) technologies. METHODS: A total of 123 CMD patients were investigated using the traditional approaches of histology, immunohistochemical analysis of muscle biopsy, and candidate gene sequencing. Undiagnosed patients available for further testing were investigated using NGS. RESULTS: Muscle biopsy and immunohistochemical analysis found deficiencies of laminin α2, α-dystroglycan, or collagen VI in 50% of patients. Candidate gene sequencing and chromosomal microarray established a genetic diagnosis in 32% (39 of 123). Of 85 patients presenting in the past 20 years, 28 of 51 who lacked a confirmed genetic diagnosis (55%) consented to NGS studies, leading to confirmed diagnoses in a further 11 patients. Using the combination of approaches, a confirmed genetic diagnosis was achieved in 51% (43 of 85). The diagnoses within the cohort were heterogeneous. Forty-five of 59 probands with confirmed or probable diagnoses had variants in genes known to cause CMD (76%), and 11 of 59 (19%) had variants in genes associated with congenital myopathies, reflecting overlapping features of these conditions. One patient had a congenital myasthenic syndrome, and 2 had microdeletions. Within the cohort, 5 patients had variants in novel (PIGY and GMPPB) or recently published genes (GFPT1 and MICU1), and 7 had variants in TTN or RYR1, large genes that are technically difficult to Sanger sequence. INTERPRETATION: These data support NGS as a first-line tool for genetic evaluation of patients with a clinical phenotype suggestive of CMD, with muscle biopsy reserved as a second-tier investigation. Ann Neurol 2016;80:101-111.

Prominent scapulae mimicking an inherited myopathy expands the phenotype of CHD7-related disease.

CHD7 variants are a well-established cause of CHARGE syndrome, a disabling multi-system malformation disorder that is often associated with deafness, visual impairment and intellectual disability. Less severe forms of CHD7-related disease are known to exist, but the full spectrum of phenotypes remains uncertain. We identified a de novo missense variant in CHD7 in a family presenting with musculoskeletal abnormalities as the main manifestation of CHD7-related disease, representing a new phenotype. The proband presented with prominent scapulae, mild shoulder girdle weakness and only subtle dysmorphic features. Investigation revealed hypoplasia of the trapezius and sternocleidomastoid muscles and semicircular canal defects, but he did not fulfill diagnostic criteria for CHARGE syndrome. Although the shoulders are often sloping and anteverted in CHARGE syndrome, the underlying neuromuscular cause has never been investigated. This report expands the phenotypes associated with CHD7 mutations to include a musculoskeletal presentation, with hypoplasia of the shoulder and neck muscles. CHD7 should be considered in patients presenting in childhood with stable scapular winging, particularly if accompanied by dysmorphic features and balance difficulties.

Mutations in PIGY: expanding the phenotype of inherited glycosylphosphatidylinositol deficiencies.

Glycosylphosphatidylinositol (GPI)-anchored proteins are ubiquitously expressed in the human body and are important for various functions at the cell surface. Mutations in many GPI biosynthesis genes have been described to date in patients with multi-system disease and together these constitute a subtype of congenital disorders of glycosylation. We used whole exome sequencing in two families to investigate the genetic basis of disease and used RNA and cellular studies to investigate the functional consequences of sequence variants in the PIGY gene. Two families with different phenotypes had homozygous recessive sequence variants in the GPI biosynthesis gene PIGY. Two sisters with c.137T>C (p.Leu46Pro) PIGY variants had multi-system disease including dysmorphism, seizures, severe developmental delay, cataracts and early death. There were significantly reduced levels of GPI-anchored proteins (CD55 and CD59) on the surface of patient-derived skin fibroblasts (∼20-50% compared with controls). In a second, consanguineous family, two siblings had moderate development delay and microcephaly. A homozygous PIGY promoter variant (c.-540G>A) was detected within a 7.7 Mb region of autozygosity. This variant was predicted to disrupt a SP1 consensus binding site and was shown to be associated with reduced gene expression. Mutations in PIGY can occur in coding and non-coding regions of the gene and cause variable phenotypes. This article contributes to understanding of the range of disease phenotypes and disease genes associated with deficiencies of the GPI-anchor biosynthesis pathway and also serves to highlight the potential importance of analysing variants detected in 5'-UTR regions despite their typically low coverage in exome data.

Recessive ACTA1 variant causes congenital muscular dystrophy with rigid spine.

Variants in ACTA1, which encodes α-skeletal actin, cause several congenital myopathies, most commonly nemaline myopathy. Autosomal recessive variants comprise approximately 10% of ACTA1 myopathy. All recessive variants reported to date have resulted in loss of skeletal α-actin expression from muscle and severe weakness from birth. Targeted next-generation sequencing in two brothers with congenital muscular dystrophy with rigid spine revealed homozygous missense variants in ACTA1. Skeletal α-actin expression was preserved in these patients. This report expands the clinical and histological phenotype of ACTA1 disease to include congenital muscular dystrophy with rigid spine and dystrophic features on muscle biopsy. This represents a new class of recessive ACTA1 variants, which do not abolish protein expression.