Deep Intronic FGF14 GAA Repeat Expansion in Late-Onset Cerebellar Ataxia.

BACKGROUND: The late-onset cerebellar ataxias (LOCAs) have largely resisted molecular diagnosis. METHODS: 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. RESULTS: In the six French Canadian patients, we identified a GAA repeat expansion deep in the first intron of FGF14, which encodes fibroblast growth factor 14. Cosegregation of the repeat expansion with disease in the families supported a pathogenic threshold of at least 250 GAA repeats ([GAA]≥250). There was significant association between FGF14 (GAA)≥250 expansions and LOCA in the French Canadian series (odds ratio, 105.60; 95% confidence interval [CI], 31.09 to 334.20; P<0.001) and in the German series (odds ratio, 8.76; 95% CI, 3.45 to 20.84; P<0.001). The repeat expansion was present in 61%, 18%, 15%, and 10% of French Canadian, German, Australian, and Indian index patients, respectively. In total, we identified 128 patients with LOCA who carried an FGF14 (GAA)≥250 expansion. Postmortem cerebellum specimens and iPSC-derived motor neurons from patients showed reduced expression of FGF14 RNA and protein. CONCLUSIONS: A dominantly inherited deep intronic GAA repeat expansion in FGF14 was found to be associated with LOCA. (Funded by Fondation Groupe Monaco and others.).

A KLHL40 3' UTR splice-altering variant causes milder NEM8, an under-appreciated disease mechanism.

Nemaline myopathy 8 (NEM8) is typically a severe autosomal recessive disorder associated with variants in the kelch-like family member 40 gene (KLHL40). Common features include fetal akinesia, fractures, contractures, dysphagia, respiratory failure and neonatal death. Here, we describe a 26-year-old man with relatively mild NEM8. He presented with hypotonia and bilateral femur fractures at birth, later developing bilateral Achilles' contractures, scoliosis, and elbow and knee contractures. He had walking difficulties throughout childhood and became wheelchair bound from age 13 after prolonged immobilization. Muscle magnetic resonance imaging at age 13 indicated prominent fat replacement in his pelvic girdle, posterior compartments of thighs and vastus intermedius. Muscle biopsy revealed nemaline bodies and intranuclear rods. RNA sequencing and western blotting of patient skeletal muscle indicated significant reduction in KLHL40 mRNA and protein, respectively. Using gene panel screening, exome sequencing and RNA sequencing, we identified compound heterozygous variants in KLHL40; a truncating 10.9 kb deletion in trans with a likely pathogenic variant (c.*152G > T) in the 3' untranslated region (UTR). Computational tools SpliceAI and Introme predicted the c.*152G > T variant created a cryptic donor splice site. RNA-seq and in vitro analyses indicated that the c.*152G > T variant induces multiple de novo splicing events that likely provoke nonsense mediated decay of KLHL40 mRNA explaining the loss of mRNA expression and protein abundance in the patient. Analysis of 3' UTR variants in ClinVar suggests variants that introduce aberrant 3' UTR splicing may be underrecognized in Mendelian disease. We encourage consideration of this mechanism during variant curation.

Genetics of inherited peripheral neuropathies and the next frontier: looking backwards to progress forwards.

Inherited peripheral neuropathies (IPNs) encompass a clinically and genetically heterogeneous group of disorders causing length-dependent degeneration of peripheral autonomic, motor and/or sensory nerves. Despite gold-standard diagnostic testing for pathogenic variants in over 100 known associated genes, many patients with IPN remain genetically unsolved. Providing patients with a diagnosis is critical for reducing their 'diagnostic odyssey', improving clinical care, and for informed genetic counselling. The last decade of massively parallel sequencing technologies has seen a rapid increase in the number of newly described IPN-associated gene variants contributing to IPN pathogenesis. However, the scarcity of additional families and functional data supporting variants in potential novel genes is prolonging patient diagnostic uncertainty and contributing to the missing heritability of IPNs. We review the last decade of IPN disease gene discovery to highlight novel genes, structural variation and short tandem repeat expansions contributing to IPN pathogenesis. From the lessons learnt, we provide our vision for IPN research as we anticipate the future, providing examples of emerging technologies, resources and tools that we propose that will expedite the genetic diagnosis of unsolved IPN families.

Ablation of the carboxy-terminal end of MAMDC2 causes a distinct muscular dystrophy.

The extracellular matrix (ECM) has an important role in the development and maintenance of skeletal muscle, and several muscle diseases are associated with the dysfunction of ECM elements. MAMDC2 is a putative ECM protein and its role in cell proliferation has been investigated in certain cancer types. However, its participation in skeletal muscle physiology has not been previously studied. We describe 17 individuals with an autosomal dominant muscular dystrophy belonging to two unrelated families in which different heterozygous truncating variants in the last exon of MAMDC2 co-segregate correctly with the disease. The radiological aspect of muscle involvement resembles that of COL6 myopathies with fat replacement at the peripheral rim of vastii muscles. In this cohort, a subfascial and peri-tendinous pattern is observed in upper and lower limb muscles. Here we show that MAMDC2 is expressed in adult skeletal muscle and differentiating muscle cells, where it appears to localize to the sarcoplasm and myonuclei. In addition, we show it is secreted by myoblasts and differentiating myotubes into to the extracellular compartment. The last exon encodes a disordered region with a polar residue compositional bias loss of which likely induces a toxic effect of the mutant protein. The precise mechanisms by which the altered MAMDC2 proteins cause disease remains to be determined. MAMDC2 is a skeletal muscle disease-associated protein. Its role in muscle development and ECM-muscle communication remains to be fully elucidated. Screening of the last exon of MAMDC2 should be considered in patients presenting with autosomal dominant muscular dystrophy, particularly in those with a subfascial radiological pattern of muscle involvement.

Bi-allelic loss-of-function OBSCN variants predispose individuals to severe recurrent rhabdomyolysis.

Rhabdomyolysis is the acute breakdown of skeletal myofibres in response to an initiating factor, most commonly toxins and over exertion. A variety of genetic disorders predispose to rhabdomyolysis through different pathogenic mechanisms, particularly in patients with recurrent episodes. However, most cases remain without a genetic diagnosis. Here we present six patients who presented with severe and recurrent rhabdomyolysis, usually with onset in the teenage years; other features included a history of myalgia and muscle cramps. We identified 10 bi-allelic loss-of-function variants in the gene encoding obscurin (OBSCN) predisposing individuals to recurrent rhabdomyolysis. We show reduced expression of OBSCN and loss of obscurin protein in patient muscle. Obscurin is proposed to be involved in sarcoplasmic reticulum function and Ca2+ handling. Patient cultured myoblasts appear more susceptible to starvation as evidenced by a greater decreased in sarcoplasmic reticulum Ca2+ content compared to control myoblasts. This likely reflects a lower efficiency when pumping Ca2+ back into the sarcoplasmic reticulum and/or a decrease in Ca2+ sarcoplasmic reticulum storage ability when metabolism is diminished. OSBCN variants have previously been associated with cardiomyopathies. None of the patients presented with a cardiomyopathy and cardiac examinations were normal in all cases in which cardiac function was assessed. There was also no history of cardiomyopathy in first degree relatives, in particular in any of the carrier parents. This cohort is relatively young, thus follow-up studies and the identification of additional cases with bi-allelic null OBSCN variants will further delineate OBSCN-related disease and the clinical course of disease.

Partial loss-of-function variant in neuregulin 1 identified in family with heritable peripheral neuropathy.

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.

Systemic AAV8-mediated delivery of a functional copy of muscle glycogen phosphorylase (Pygm) ameliorates disease in a murine model of McArdle disease.

McArdle disease is a disorder of carbohydrate metabolism that causes painful skeletal muscle cramps and skeletal muscle damage leading to transient myoglobinuria and increased risk of kidney failure. McArdle disease is caused by recessive mutations in the muscle glycogen phosphorylase (PYGM) gene leading to absence of PYGM enzyme in skeletal muscle and preventing access to energy from muscle glycogen stores. There is currently no cure for McArdle disease. Using a preclinical animal model, we aimed to identify a clinically translatable and relevant therapy for McArdle disease. We evaluated the safety and efficacy of recombinant adeno-associated virus serotype 8 (rAAV8) to treat a murine model of McArdle disease via delivery of a functional copy of the disease-causing gene, Pygm. Intraperitoneal injection of rAAV8-Pygm at post-natal day 1-3 resulted in Pygm expression at 8 weeks of age, accompanied by improved skeletal muscle architecture, reduced accumulation of glycogen and restoration of voluntary running wheel activity to wild-type levels. We did not observe any adverse reaction to the treatment at 8 weeks post-injection. Thus, we have investigated a highly promising gene therapy for McArdle disease with a clear path to the ovine large animal model endemic to Western Australia and subsequently to patients.

Pathogenic Abnormal Splicing Due to Intronic Deletions that Induce Biophysical Space Constraint for Spliceosome Assembly.

A precise genetic diagnosis is the single most important step for families with genetic disorders to enable personalized and preventative medicine. In addition to genetic variants in coding regions (exons) that can change a protein sequence, abnormal pre-mRNA splicing can be devastating for the encoded protein, inducing a frameshift or in-frame deletion/insertion of multiple residues. Non-coding variants that disrupt splicing are extremely challenging to identify. Stemming from an initial clinical discovery in two index Australian families, we define 25 families with genetic disorders caused by a class of pathogenic non-coding splice variant due to intronic deletions. These pathogenic intronic deletions spare all consensus splice motifs, though they critically shorten the minimal distance between the 5' splice-site (5'SS) and branchpoint. The mechanistic basis for abnormal splicing is due to biophysical constraint precluding U1/U2 spliceosome assembly, which stalls in A-complexes (that bridge the 5'SS and branchpoint). Substitution of deleted nucleotides with non-specific sequences restores spliceosome assembly and normal splicing, arguing against loss of an intronic element as the primary causal basis. Incremental lengthening of 5'SS-branchpoint length in our index EMD case subject defines 45-47 nt as the critical elongation enabling (inefficient) spliceosome assembly for EMD intron 5. The 5'SS-branchpoint space constraint mechanism, not currently factored by genomic informatics pipelines, is relevant to diagnosis and precision medicine across the breadth of Mendelian disorders and cancer genomics.

The relationship between beta-ureidopropionase deficiency due to UPB1 variants and human phenotypes is uncertain.

BACKGROUND: Beta-ureidopropionase deficiency, caused by variants in UPB1, has been reported in association with various neurodevelopmental phenotypes including intellectual disability, seizures and autism. AIM: We aimed to reassess the relationship between variants in UPB1 and a clinical phenotype. METHODS: Literature review, calculation of carrier frequencies from population databases, long-term follow-up of a previously published case and reporting of additional cases. RESULTS: Fifty-three published cases were identified, and two additional cases are reported here. Of these, 14 were asymptomatic and four had transient neurological features; clinical features in the remainder were variable and included non-neurological presentations. Several of the variants previously reported as pathogenic are present in population databases at frequencies higher than expected for a rare condition. In particular, the variant most frequently reported as pathogenic, p.Arg326Gln, is very common among East Asians, with a carrier frequency of 1 in 19 and 1 in 907 being homozygous for the variant in gnomAD v2.1.1. CONCLUSION: Pending the availability of further evidence, UPB1 should be considered a 'gene of uncertain clinical significance'. Caution should be used in ascribing clinical significance to biochemical features of beta-ureidopropionase deficiency and/or UPB1 variants in patients with neurodevelopmental phenotypes. UPB1 is not currently suitable for inclusion in gene panels for reproductive genetic carrier screening. SYNOPSIS: The relationship between beta-ureidopropionase deficiency due to UPB1 variants and clinical phenotypes is uncertain.

A TOR1AIP1 variant segregating with an early onset limb girdle myasthenia-Support for the role of LAP1 in NMJ function and disease.

Rare pathogenic variants in TOR1AIP1 (OMIM 614512), coding the inner nuclear membrane protein lamin-associated protein 1 (LAP1), have been associated with a spectrum of disorders including limb girdle muscular dystrophy with cardiac involvement and a severe multisystem phenotype. Recently, Cossins et al reported two siblings with limb girdle muscular dystrophy and impaired transmission of the neuromuscular synapse, demonstrating that defective LAP1 may lead to a congenital myasthenic syndrome. Herein, we describe the association of TOR1AIP1 deficiency with congenital myasthenic syndrome in three siblings.

Identification of a novel heterozygous DYSF variant in a large family with a dominantly-inherited dysferlinopathy.

AIMS: Dysferlinopathy is an autosomal recessive muscular dystrophy, caused by bi-allelic variants in the gene encoding dysferlin (DYSF). Onset typically occurs in the second to third decade and is characterised by slowly progressive skeletal muscle weakness and atrophy of the proximal and/or distal muscles of the four limbs. There are rare cases of symptomatic DYSF variant carriers. Here, we report a large family with a dominantly inherited hyperCKaemia and late-onset muscular dystrophy. METHODS AND RESULTS: Genetic analysis identified a co-segregating novel DYSF variant [NM_003494.4:c.6207del p.(Tyr2070Metfs*4)]. No secondary variants in DYSF or other dystrophy-related genes were identified on whole genome sequencing and analysis of the proband's DNA. Skeletal muscle involvement was milder and later onset than typical dysferlinopathy presentations; these clinical signs manifested in four individuals, all between the fourth and sixth decades of life. All individuals heterozygous for the c.6207del variant had hyperCKaemia. Histological analysis of skeletal muscle biopsies across three generations showed clear dystrophic signs, including inflammatory infiltrates, regenerating myofibres, increased variability in myofibre size and internal nuclei. Muscle magnetic resonance imaging revealed fatty replacement of muscle in two individuals. Western blot and immunohistochemical analysis of muscle biopsy demonstrated consistent reduction of dysferlin staining. Allele-specific quantitative PCR analysis of DYSF mRNA from patient muscle found that the variant, localised to the extreme C-terminus of dysferlin, does not activate post-transcriptional mRNA decay. CONCLUSIONS: We propose that this inheritance pattern may be underappreciated and that other late-onset muscular dystrophy cases with mono-allelic DYSF variants, particularly C-terminal premature truncation variants, may represent dominant forms of disease.

Neurogenetic fetal akinesia and arthrogryposis: genetics, expanding genotype-phenotypes and functional genomics.

BACKGROUND: Fetal akinesia and arthrogryposis are clinically and genetically heterogeneous and have traditionally been refractive to genetic diagnosis. The widespread availability of affordable genome-wide sequencing has facilitated accurate genetic diagnosis and gene discovery in these conditions. METHODS: We performed next generation sequencing (NGS) in 190 probands with a diagnosis of arthrogryposis multiplex congenita, distal arthrogryposis, fetal akinesia deformation sequence or multiple pterygium syndrome. This sequencing was a combination of bespoke neurogenetic disease gene panels and whole exome sequencing. Only class 4 and 5 variants were reported, except for two cases where the identified variants of unknown significance (VUS) are most likely to be causative for the observed phenotype. Co-segregation studies and confirmation of variants identified by NGS were performed where possible. Functional genomics was performed as required. RESULTS: Of the 190 probands, 81 received an accurate genetic diagnosis. All except two of these cases harboured class 4 and/or 5 variants based on the American College of Medical Genetics and Genomics guidelines. We identified phenotypic expansions associated with CACNA1S, CHRNB1, GMPPB and STAC3. We describe a total of 50 novel variants, including a novel missense variant in the recently identified gene for arthrogryposis with brain malformations-SMPD4. CONCLUSIONS: Comprehensive gene panels give a diagnosis for a substantial proportion (42%) of fetal akinesia and arthrogryposis cases, even in an unselected cohort. Recently identified genes account for a relatively large proportion, 32%, of the diagnoses. Diagnostic-research collaboration was critical to the diagnosis and variant interpretation in many cases, facilitated genotype-phenotype expansions and reclassified VUS through functional genomics.

Nemaline Myopathy in Brazilian Patients: Molecular and Clinical Characterization.

Nemaline myopathy (NM), a structural congenital myopathy, presents a significant clinical and genetic heterogeneity. Here, we compiled molecular and clinical data of 30 Brazilian patients from 25 unrelated families. Next-generation sequencing was able to genetically classify all patients: sixteen families (64%) with mutation in NEB, five (20%) in ACTA1, two (8%) in KLHL40, and one in TPM2 (4%) and TPM3 (4%). In the NEB-related families, 25 different variants, 11 of them novel, were identified; splice site (10/25) and frame shift (9/25) mutations were the most common. Mutation c.24579 G>C was recurrent in three unrelated patients from the same region, suggesting a common ancestor. Clinically, the "typical" form was the more frequent and caused by mutations in the different NM genes. Phenotypic heterogeneity was observed among patients with mutations in the same gene. Respiratory involvement was very common and often out of proportion with limb weakness. Muscle MRI patterns showed variability within the forms and genes, which was related to the severity of the weakness. Considering the high frequency of NEB mutations and the complexity of this gene, NGS tools should be combined with CNV identification, especially in patients with a likely non-identified second mutation.

Biallelic hypomorphic variants in ALDH1A2 cause a novel lethal human multiple congenital anomaly syndrome encompassing diaphragmatic, pulmonary, and cardiovascular defects.

This study shows a causal association between ALDH1A2 variants and a novel, severe multiple congenital anomaly syndrome in humans that is neonatally lethal due to associated pulmonary hypoplasia and respiratory failure. In two families, exome sequencing identified compound heterozygous missense variants in ALDH1A2. ALDH1A2 is involved in the conversion of retinol (vitamin A) into retinoic acid (RA), which is an essential regulator of diaphragm and cardiovascular formation during embryogenesis. Reduced RA causes cardiovascular, diaphragmatic, and associated pulmonary defects in several animal models, matching the phenotype observed in our patients. In silico protein modeling showed probable impairment of ALDH1A2 for three of the four substitutions. In vitro studies show a reduction of RA. Few pathogenic variants in genes encoding components of the retinoic signaling pathway have been described to date, likely due to embryonic lethality. Thus, this study contributes significantly to knowledge of the role of this pathway in human diaphragm and cardiovascular development and disease. Some clinical features in our patients are also observed in Fryns syndrome (MIM# 229850), syndromic microphthalmia 9 (MIM# 601186), and DiGeorge syndrome (MIM# 188400). Patients with similar clinical features who are genetically undiagnosed should be tested for recessive ALDH1A2-deficient malformation syndrome.

Mutations in ATP1A1 Cause Dominant Charcot-Marie-Tooth Type 2.

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.

A novel RFC1 repeat motif (ACAGG) in two Asia-Pacific CANVAS families.

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.

A Maori specific RFC1 pathogenic repeat configuration in CANVAS, likely due to a founder allele.

Cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS) is a recently recognized neurodegenerative disease with onset in mid- to late adulthood. The genetic basis for a large proportion of Caucasian patients was recently shown to be the biallelic expansion of a pentanucleotide (AAGGG)n repeat in RFC1. Here, we describe the first instance of CANVAS genetic testing in New Zealand Maori and Cook Island Maori individuals. We show a novel, possibly population-specific CANVAS configuration (AAAGG)10-25(AAGGG)exp, which was the cause of CANVAS in all patients. There were no apparent phenotypic differences compared with European CANVAS patients. Presence of a common disease haplotype among this cohort suggests this novel repeat expansion configuration is a founder effect in this population, which may indicate that CANVAS will be especially prevalent in this group. Haplotype dating estimated the most recent common ancestor at ∼1430 ce. We also show the same core haplotype as previously described, supporting a single origin of the CANVAS mutation.

A homozygous UBA5 pathogenic variant causes a fatal congenital neuropathy.

BACKGROUND: UBA5 is the activating enzyme of UFM1 in the ufmylation post-translational modification system. Different neurological phenotypes have been associated with UBA5 pathogenic variants including epilepsy, intellectual disability, movement disorders and ataxia. METHODS AND RESULTS: We describe a large multigenerational consanguineous family presenting with a severe congenital neuropathy causing early death in infancy. Whole exome sequencing and linkage analysis identified a novel homozygous UBA5 NM_024818.3 c.31C>T (p.Arg11Trp) mutation. Protein expression assays in mouse tissue showed similar levels of UBA5 in peripheral nerves to the central nervous system. CRISPR-Cas9 edited HEK (human embrionic kidney) cells homozygous for the UBA5 p.Arg11Trp mutation showed reduced levels of UBA5 protein compared with the wild-type. The mutant p.Arg11Trp UBA5 protein shows reduced ability to activate UFM1. CONCLUSION: This report expands the phenotypical spectrum of UBA5 mutations to include fatal peripheral neuropathy.

Biallelic mutations in nucleoporin NUP88 cause lethal fetal akinesia deformation sequence.

Nucleoporins build the nuclear pore complex (NPC), which, as sole gate for nuclear-cytoplasmic exchange, is of outmost importance for normal cell function. Defects in the process of nucleocytoplasmic transport or in its machinery have been frequently described in human diseases, such as cancer and neurodegenerative disorders, but only in a few cases of developmental disorders. Here we report biallelic mutations in the nucleoporin NUP88 as a novel cause of lethal fetal akinesia deformation sequence (FADS) in two families. FADS comprises a spectrum of clinically and genetically heterogeneous disorders with congenital malformations related to impaired fetal movement. We show that genetic disruption of nup88 in zebrafish results in pleiotropic developmental defects reminiscent of those seen in affected human fetuses, including locomotor defects as well as defects at neuromuscular junctions. Phenotypic alterations become visible at distinct developmental stages, both in affected human fetuses and in zebrafish, whereas early stages of development are apparently normal. The zebrafish phenotypes caused by nup88 deficiency are rescued by expressing wild-type Nup88 but not the disease-linked mutant forms of Nup88. Furthermore, using human and mouse cell lines as well as immunohistochemistry on fetal muscle tissue, we demonstrate that NUP88 depletion affects rapsyn, a key regulator of the muscle nicotinic acetylcholine receptor at the neuromuscular junction. Together, our studies provide the first characterization of NUP88 in vertebrate development, expand our understanding of the molecular events causing FADS, and suggest that variants in NUP88 should be investigated in cases of FADS.

Bi-allelic mutations in MYL1 cause a severe congenital myopathy.

Congenital myopathies are typically characterised by early onset hypotonia, weakness and hallmark features on biopsy. Despite the rapid pace of gene discovery, ∼50% of patients with a congenital myopathy remain without a genetic diagnosis following screening of known disease genes. We performed exome sequencing on two consanguineous probands diagnosed with a congenital myopathy and muscle biopsy showing selective atrophy/hypotrophy or absence of type II myofibres. We identified variants in the gene (MYL1) encoding the skeletal muscle fast-twitch specific myosin essential light chain (ELC) in both probands. A homozygous essential splice acceptor variant (c.479-2A > G, predicted to result in skipping of exon 5 was identified in Proband 1, and a homozygous missense substitution (c.488T>G, p.(Met163Arg)) was identified in Proband 2. Protein modelling of the p.(Met163Arg) substitution predicted it might impede intermolecular interactions that facilitate binding to the IQ domain of myosin heavy chain, thus likely impacting on the structure and functioning of the myosin motor. MYL1 was markedly reduced in skeletal muscle from both probands, suggesting that the missense substitution likely results in an unstable protein. Knock down of myl1 in zebrafish resulted in abnormal morphology, disrupted muscle structure and impaired touch-evoked escape responses, thus confirming that skeletal muscle fast-twitch specific myosin ELC is critical for myofibre development and function. Our data implicate MYL1 as a crucial protein for adequate skeletal muscle function and that MYL1 deficiency is associated with severe congenital myopathy.