Loss of function variants in DNAJB4 cause a myopathy with early respiratory failure.

DNAJ/HSP40 co-chaperones are integral to the chaperone network, bind client proteins and recruit them to HSP70 for folding. We performed exome sequencing on patients with a presumed hereditary muscle disease and no genetic diagnosis. This identified four individuals from three unrelated families carrying an unreported homozygous stop gain (c.856A > T; p.Lys286Ter), or homozygous missense variants (c.74G > A; p.Arg25Gln and c.785 T > C; p.Leu262Ser) in DNAJB4. Affected patients presented with axial rigidity and early respiratory failure requiring ventilator support between the 1st and 4th decade of life. Selective involvement of the semitendinosus and biceps femoris muscles was seen on MRI scans of the thigh. On biopsy, muscle was myopathic with angular fibers, protein inclusions and occasional rimmed vacuoles. DNAJB4 normally localizes to the Z-disc and was absent from muscle and fibroblasts of affected patients supporting a loss of function. Functional studies confirmed that the p.Lys286Ter and p.Leu262Ser mutant proteins are rapidly degraded in cells. In contrast, the p.Arg25Gln mutant protein is stable but failed to complement for DNAJB function in yeast, disaggregate client proteins or protect from heat shock-induced cell death consistent with its loss of function. DNAJB4 knockout mice had muscle weakness and fiber atrophy with prominent diaphragm involvement and kyphosis. DNAJB4 knockout muscle and myotubes had myofibrillar disorganization and accumulated Z-disc proteins and protein chaperones. These data demonstrate a novel chaperonopathy associated with DNAJB4 causing a myopathy with early respiratory failure. DNAJB4 loss of function variants may lead to the accumulation of DNAJB4 client proteins resulting in muscle dysfunction and degeneration.

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.

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 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.

Cerebellar ataxia, neuropathy, vestibular areflexia syndrome due to RFC1 repeat expansion.

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.

Genetics of neuromuscular fetal akinesia in the genomics era.

Fetal hypokinesia or akinesia encompasses a broad spectrum of disorders, united by impaired movement in utero. Often, the underlying aetiology is genetic in origin, affecting part of the neuromuscular system. The affordable and high-throughput nature of next-generation DNA sequencing has led to an explosion in disease gene discovery across rare diseases, including fetal akinesias. A genetic diagnosis has clinical utility as it may affect management and prognosis and informs recurrence risk, facilitating family planning decisions. More broadly, knowledge of disease genes increasingly allows population-based preconception carrier screening, which has reduced the incidence of recessive diseases in several populations. Despite gains in knowledge of the genetics of fetal akinesia, many families lack a genetic diagnosis. In this review, we describe the developments in Mendelian genetics of neuromuscular fetal akinesia in the genomics era. We examine genetic diagnoses with neuromuscular causes, specifically including the lower motor neuron, peripheral nerve, neuromuscular junction and muscle.

Hecatomb: an integrated software platform for viral metagenomics.

BACKGROUND: Modern sequencing technologies offer extraordinary opportunities for virus discovery and virome analysis. Annotation of viral sequences from metagenomic data requires a complex series of steps to ensure accurate annotation of individual reads and assembled contigs. In addition, varying study designs will require project-specific statistical analyses. FINDINGS: Here we introduce Hecatomb, a bioinformatic platform coordinating commonly used tasks required for virome analysis. Hecatomb means "a great sacrifice." In this setting, Hecatomb is "sacrificing" false-positive viral annotations using extensive quality control and tiered-database searches. Hecatomb processes metagenomic data obtained from both short- and long-read sequencing technologies, providing annotations to individual sequences and assembled contigs. Results are provided in commonly used data formats useful for downstream analysis. Here we demonstrate the functionality of Hecatomb through the reanalysis of a primate enteric and a novel coral reef virome. CONCLUSION: Hecatomb provides an integrated platform to manage many commonly used steps for virome characterization, including rigorous quality control, host removal, and both read- and contig-based analysis. Each step is managed using the Snakemake workflow manager with dependency management using Conda. Hecatomb outputs several tables properly formatted for immediate use within popular data analysis and visualization tools, enabling effective data interpretation for a variety of study designs. Hecatomb is hosted on GitHub (github.com/shandley/hecatomb) and is available for installation from Bioconda and PyPI.

Heterozygous loss-of-function variants in SPTAN1 cause a novel early childhood onset distal myopathy with chronic neurogenic features.

Background: Neurogenetic disorders caused by pathogenic variants in four genes encoding non-erythrocytic spectrins ( SPTAN1, SPTBN1, SPTBN2, SPTBN4) range from peripheral and central nervous system involvement to complex syndromic presentations. Heterozygous pathogenic variants in SPTAN1 are exemplary for this diversity with phenotypes spanning almost the entire spectrum. Methods: Through international collaboration we identified 14 families with genetically unsolved distal weakness and unreported heterozygous SPTAN1 loss-of-function variants including frameshift, nonsense and splice-acceptor variants. Clinical data, electrophysiology, muscle CT or MRI and muscle biopsy findings were collected and standardized. SPTAN1 protein, mRNA expression analysis and cDNA sequencing was performed on muscle tissue from two patients. Results: All 20 patients presented with early childhood onset distal weakness. The severity varied both within families and between different families. Foot abnormalities ranged from hammer toes and pes cavus to distal arthrogryposis. Electrophysiology showed mixed myogenic and neurogenic features. Muscle MRI or CT in 10 patients showed fatty infiltration of the distal lower limb anterior compartment and/or selective involvement of the extensor hallucis longus muscle. Muscle biopsy revealed myopathic changes with mild dystrophic and chronic neurogenic changes in 7 patients. Finally, we provide proof for nonsense mediated decay in tissues derived from two patients. Conclusions: We provide evidence for the association of SPTAN1 loss-of-function variants with childhood onset distal myopathy in 14 families. This finding extends the phenotypic spectrum of SPTAN1 loss-of-function variants ranging from intellectual disability to distal weakness with a predominant myogenic cause. KEY MESSAGES: SPTAN1 loss-of-function variants, including frameshift, nonsense and splice site variants cause a novel childhood onset distal weakness syndrome with primarily skeletal muscle involvement. Hereditary motor neuropathies and distal myopathic disorders present a well-known diagnostic challenge as they demonstrate substantial clinical and genetic overlap. The emergence of SPTAN1 loss-of-function variants serves as a noteworthy example, highlighting a growing convergence in the spectrum of genotypes linked to both hereditary motor neuropathies and distal myopathies.

A CCG expansion in ABCD3 causes oculopharyngodistal myopathy in individuals of European ancestry.

Oculopharyngodistal myopathy (OPDM) is an inherited myopathy manifesting with ptosis, dysphagia and distal weakness. Pathologically it is characterised by rimmed vacuoles and intranuclear inclusions on muscle biopsy. In recent years CGG • CCG repeat expansion in four different genes were identified in OPDM individuals in Asian populations. None of these have been found in affected individuals of non-Asian ancestry. In this study we describe the identification of CCG expansions in ABCD3, ranging from 118 to 694 repeats, in 35 affected individuals across eight unrelated OPDM families of European ancestry. ABCD3 transcript appears upregulated in fibroblasts and skeletal muscle from OPDM individuals, suggesting a potential role of over-expression of CCG repeat containing ABCD3 transcript in progressive skeletal muscle degeneration. The study provides further evidence of the role of non-coding repeat expansions in unsolved neuromuscular diseases and strengthens the association between the CGG • CCG repeat motif and a specific pattern of muscle weakness.

Introme accurately predicts the impact of coding and noncoding variants on gene splicing, with clinical applications.

Predicting the impact of coding and noncoding variants on splicing is challenging, particularly in non-canonical splice sites, leading to missed diagnoses in patients. Existing splice prediction tools are complementary but knowing which to use for each splicing context remains difficult. Here, we describe Introme, which uses machine learning to integrate predictions from several splice detection tools, additional splicing rules, and gene architecture features to comprehensively evaluate the likelihood of a variant impacting splicing. Through extensive benchmarking across 21,000 splice-altering variants, Introme outperformed all tools (auPRC: 0.98) for the detection of clinically significant splice variants. Introme is available at https://github.com/CCICB/introme .

STRling: a k-mer counting approach that detects short tandem repeat expansions at known and novel loci.

Expansions of short tandem repeats (STRs) cause many rare diseases. Expansion detection is challenging with short-read DNA sequencing data since supporting reads are often mapped incorrectly. Detection is particularly difficult for "novel" STRs, which include new motifs at known loci or STRs absent from the reference genome. We developed STRling to efficiently count k-mers to recover informative reads and call expansions at known and novel STR loci. STRling is sensitive to known STR disease loci, has a low false discovery rate, and resolves novel STR expansions to base-pair position accuracy. It is fast, scalable, open-source, and available at: github.com/quinlan-lab/STRling .

The Impact of Next-Generation Sequencing on the Diagnosis, Treatment, and Prevention of Hereditary Neuromuscular Disorders.

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.

Cylindrical spirals in two families: Clinical and genetic investigations.

Cylindrical spirals are a rare ultrastructural finding on muscle biopsy, with fewer than 20 reported cases since its first description in 1979. These structures are sometimes observed with tubular aggregates and are thought to comprise longitudinal sarcoplasmic reticulum. While mutations in genes encoding key components of Ca2+ handling (ORAI1 and STIM1) underlie tubular aggregate myopathy, no causative genes have been associated with cylindrical spirals. Here we describe two families with cylindrical spirals on muscle biopsy with a suspected genetic cause. In one family we identified a known truncating variant in EBF3, previously associated with a neurodevelopmental disorder. The affected individuals in this family present with clinical features overlapping with those described for EBF3 disease. An isolated proband in the second family harbours bi-allelic truncating variants in TTN and her clinical course and other features on biopsy are highly concordant for titinopathy. From experimental studies, EBF3 is known to be involved in Ca2+ regulation in muscle, thus EBF3 dysregulation may represent a novel mechanism of impaired Ca2+ handling leading to cylindrical spirals. Additional cases of EBF3 disease or titinopathy with cylindrical spirals need to be identified to support the involvement of these genes in the pathogenesis of cylindrical spirals.

Targeted gene panel use in 2249 neuromuscular patients: the Australasian referral center experience.

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.

Recessive MYH7-related myopathy in two families.

Myopathies due to recessive MYH7 mutations are exceedingly rare, reported in only two families to date. We describe three patients from two families (from Australia and the UK) with a myopathy caused by recessive mutations in MYH7. The Australian family was homozygous for a c.5134C > T, p.Arg1712Trp mutation, whilst the UK patient was compound heterozygous for a truncating (c.4699C > T; p.Gln1567*) and a missense variant (c.4664A > G; p.Glu1555Gly). All three patients shared key clinical features, including infancy/childhood onset, pronounced axial/proximal weakness, spinal rigidity, severe scoliosis, and normal cardiac function. There was progressive respiratory impairment necessitating non-invasive ventilation despite preserved ambulation, a combination of features often seen in SEPN1- or NEB-related myopathies. On biopsy, the Australian proband showed classical myosin storage myopathy features, while the UK patient showed multi-minicore like areas. To establish pathogenicity of the Arg1712Trp mutation, we expressed mutant MYH7 protein in COS-7 cells, observing abnormal mutant myosin aggregation compared to wild-type. We describe skinned myofiber studies of patient muscle and hypertrophy of type II myofibers, which may be a compensatory mechanism. In summary, we have expanded the phenotype of ultra-rare recessive MYH7 disease, and provide novel insights into associated changes in muscle physiology.

Lethal multiple pterygium syndrome: A severe phenotype associated with a novel mutation in the nebulin gene.

Fetal akinesia deformation sequence is a clinically and genetically heterogeneous disorder characterized by a variable combination of fetal akinesia, intrauterine growth restriction, developmental abnormalities such as cystic hygroma, hydrops fetalis, pulmonary hypoplasia, occasional arthrogryposis, and pterygia. The pathogenetic mechanisms of fetal akinesia deformation sequence include neuropathy, muscular disorders, neuromuscular junction disorders, maternal myasthenia gravis, restrictive dermopathy and others. We here report an Egyptian family presenting with recurrent lethal multiple pterygium syndrome. The diagnosis was based on antenatal sonographic demonstration of complete fetal akinesia and a large cystic hygroma with severe limb contractures evident on postmortem examination. Next generation sequencing performed on the second affected fetus identified a novel homozygous essential splice-site variant in the nebulin gene. In conclusion, our report adds further evidence for the involvement of the nebulin gene in the etiology of fetal akinesia deformation sequence/lethal multiple pterygium syndrome.

Expanding the phenotypic spectrum associated with mutations of DYNC1H1.

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.