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Nemaline myopathy (NM) is a congenital skeletal muscle disorder that typically results in muscle weakness and the presence of rod-like structures (nemaline bodies) in the sarcoplasma and/or in the nuclei of myofibres. Two induced pluripotent stem cell (iPSC) lines were generated from the lymphoblastoid cells of a 1-month-old male with severe NM caused by a homozygous recessive mutation in the ACTA1 gene (c.121C T, p.Arg39Ter). The iPSC lines demonstrated typical morphology, expressed pluripotency markers, exhibited trilineage differentiation potential and displayed a normal karyotype. These isogenic lines represent a potential resource to investigate and model recessive ACTA1 disease in a human context.

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 Regulators of Complement Activation (RCA) gene cluster comprises several tandemly arranged genes with shared functions within the immune system. RCA members, such as complement receptor 2 (CR2), are well-established susceptibility genes in complex autoimmune diseases. Altered expression of RCA genes has been demonstrated at both the functional and genetic level, but the mechanisms underlying their regulation are not fully characterised. We aimed to investigate the structural organisation of the RCA gene cluster to identify key regulatory elements that influence the expression of CR2 and other genes in this immunomodulatory region. Using 4C, we captured extensive CTCF-mediated chromatin looping across the RCA gene cluster in B cells and showed these were organised into two topologically associated domains (TADs). Interestingly, an inter-TAD boundary was located within the CR1 gene at a well-characterised segmental duplication. Additionally, we mapped numerous gene-gene and gene-enhancer interactions across the region, revealing extensive co-regulation. Importantly, we identified an intergenic enhancer and functionally demonstrated this element upregulates two RCA members (CR2 and CD55) in B cells. We have uncovered novel, long-range mechanisms whereby autoimmune disease susceptibility may be influenced by genetic variants, thus highlighting the important contribution of chromatin topology to gene regulation and complex genetic disease., This work was supported by the National Institutes of Health [R01 AI24717 to JH], the Australian Government Research Training Program Scholarship at the University of Western Australia [to JC and JSC], the Spanish Government [BFU2016-74961-P to JG-S] and an institutional grant Unidad de Excelencia María de Maeztu [MDM-206-0687 to the Department of Gene Regulation and Morphogenesis, Centro Andaluz de Biología del Desarrol].

International audience; Nemaline myopathy (NM) is a congenital myopathy typically characterized by skeletal muscle weakness and the presence of nemaline bodies in myofibres. Approximately 25% of NM cases are caused by variants in ACTA1. We generated two induced pluripotent stem cell lines from lymphoblastoid cells of a 10-year-old female with typical NM harbouring a dominant pathogenic variant in ACTA1 (c.541C>A). The isogenic lines displayed typical iPSC morphology, expressed pluripotency markers, and could differentiate into each of the three germ layers. Although the lines have partial or complete X chromosome duplication, they may still prove useful as models of human ACTA1 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.

International audience; AbstractNemaline myopathy (NM) is a congenital myopathy typically characterized by skeletal muscle weakness and the presence of abnormal thread- or rod-like structures (nemaline bodies) in myofibres. Pathogenic variants in the skeletal muscle alpha actin gene, ACTA1, cause approximately 25% of all NM cases. We generated two induced pluripotent stem cell lines from lymphoblastoid cells of a 4-month-old female with severe NM harbouring a dominant variant in ACTA1 (c.553C > A). The isogenic lines displayed characteristic iPSC morphology, expressed pluripotency markers, differentiated into cells of all three germ layers, and possessed normal karyotypes. These lines could be useful models of human ACTA1 disease.

Ovine congenital progressive muscular dystrophy (OCPMD) was first described in Merino sheep flocks in Queensland and Western Australia in the 1960s and 1970s. The most prominent feature of the disease is a distinctive gait with stiffness of the hind limbs that can be seen as early as 3 weeks after birth. The disease is progressive. Histopathological examination had revealed dystrophic changes specifically in type I (slow) myofibres, while electron microscopy had demonstrated abundant nemaline bodies. Therefore, it was never certain whether the disease was a dystrophy or a congenital myopathy with dystrophic features. In this study, we performed whole genome sequencing of OCPMD sheep and identified a single base deletion at the splice donor site (+ 1) of intron 13 in the type I myofibre-specific TNNT1 gene (KT218690 c.614 + 1delG). All affected sheep were homozygous for this variant. Examination of TNNT1 splicing by RT-PCR showed intron retention and premature termination, which disrupts the highly conserved 14 amino acid C-terminus. The variant did not reduce TNNT1 protein levels or affect its localization but impaired its ability to modulate muscle contraction in response to Ca2+ levels. Identification of the causative variant in TNNT1 finally clarifies that the OCPMD sheep is in fact a large animal model of TNNT1 congenital myopathy. This model could now be used for testing molecular or gene therapies. Electronic supplementary material The online version of this article (10.1186/s40478-020-01017-1) contains supplementary material, which is available to authorized users.

[Aims] Recessive variants in CAPN3 gene are the cause of the commonest form of autosomal recessive limb girdle muscle dystrophy. However, two distinct in-frame deletions in CAPN3 (NM_000070.3:c.643_663del21 and c.598_621del15) and more recently, Gly445Arg and Arg572Pro substitutions have been linked to autosomal dominant (AD) forms of calpainopathy. We report 21 affected individuals from seven unrelated families presenting with an autosomal dominant form of muscular dystrophy associated with five different heterozygous missense variants in CAPN., [Methods] We have used massively parallel gene sequencing (MPS) to determine the genetic basis of a dominant form of limb girdle muscular dystrophy in affected individuals from seven unrelated families., [Results] The c.700G> A, [p.(Gly234Arg)], c.1327T> C [p.(Ser443Pro], c.1333G> A [p.(Gly445Arg)], c.1661A> C [p.(Tyr554Ser)] and c.1706T> C [p.(Phe569Ser)] CAPN3 variants were identified. Affected individuals presented in young adulthood with progressive proximal and axial weakness, waddling walking and scapular winging or with isolated hyperCKaemia. Muscle imaging showed fatty replacement of paraspinal muscles, variable degrees of involvement of the gluteal muscles, and the posterior compartment of the thigh and minor changes at the mid-leg level. Muscle biopsies revealed mild myopathic changes. Western blot analysis revealed a clear reduction in calpain 3 in skeletal muscle relative to controls. Protein modelling of these variants on the predicted structure of calpain 3 revealed that all variants are located in proximity to the calmodulin-binding site and are predicted to interfere with proteolytic activation., [Conclusions] We expand the genotypic spectrum of CAPN3-associated muscular dystrophy due to autosomal dominant missense variants., This study was funded in part by Instituto de Salud Carlos III through the project PI14/00738 to M. O. (co-funded by European Regional Development Fund. ERDF, a way to build Europe). We thank CERCA Programme / Generalitat de Catalunya for institutional support NGL (APP1117510) and GR (APP1122952) are supported by the Australian National Health and Medical Research Council (NHMRC). This work is also funded by an NHMRC Project Grant (APP1080587).

Nemaline myopathy (NM) caused by mutations in the gene encoding nebulin (NEB) accounts for at least 50% of all NM cases worldwide, representing a significant disease burden. Most NEB-NM patients have autosomal recessive disease due to a compound heterozygous genotype. Of the few murine models developed for NEB-NM, most are Neb knockout models rather than harbouring Neb mutations. Additionally, some models have a very severe phenotype that limits their application for evaluating disease progression and potential therapies. No existing murine models possess compound heterozygous Neb mutations that reflect the genotype and resulting phenotype present in most patients. We aimed to develop a murine model that more closely matched the underlying genetics of NEB-NM, which could assist elucidation of the pathogenetic mechanisms underlying the disease. Here, we have characterised a mouse strain with compound heterozygous Neb mutations; one missense (p.Tyr2303His), affecting a conserved actin-binding site and one nonsense mutation (p.Tyr935*), introducing a premature stop codon early in the protein. Our studies reveal that this compound heterozygous model, NebY2303H, Y935X, has striking skeletal muscle pathology including nemaline bodies. In vitro whole muscle and single myofibre physiology studies also demonstrate functional perturbations. However, no reduction in lifespan was noted. Therefore, NebY2303H,Y935X mice recapitulate human NEB-NM and are a much needed addition to the NEB-NM mouse model collection. The moderate phenotype also makes this an appropriate model for studying NEB-NM pathogenesis, and could potentially be suitable for testing therapeutic applications.

Ovine congenital progressive muscular dystrophy (OCPMD) was first described in Merino sheep flocks in Queensland and Western Australia (WA) in the 1960s and 70s. The most prominent feature of the disease is a distinctive gait with stiffness of the hind limbs that can be seen as early as three weeks after birth. The disease is progressive. Histopathological examination had revealed dystrophic changes specifically in slow myofibres, while electron microscopy had demonstrated abundant nemaline bodies. Therefore, it was never certain whether the disease was a dystrophy or a nemaline myopathy with dystrophic features. In this study, we performed whole genome sequencing of WA OCPMD sheep and identified a single base deletion at the splice donor site (+1) of intron 13 of the slow myofibre-specific TNNT1 gene (KT218690 c.614+1delG). All affected sheep were homozygous for this variant. Examination of TNNT1 splicing by RT-PCR showed intron retention and premature termination, which disrupts the highly conserved 14 amino acid C-terminus. The variant did not reduce TNNT1 protein levels or affect its localization but impaired its ability to modulate muscle contraction in response to Ca2+ levels. Identification of the causative variant in TNNT1 finally clarifies that the WA OCPMD sheep is in fact a large animal model of nemaline myopathy with dystrophic features. This model could now be used for testing molecular or gene therapies.

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.

A probabilistic method is proposed to quantify peak component acceleration (PCA) demands for nonstructural components attached to elastic and inelastic structures. Incremental dynamic analyses and site-specific ground-motion hazard information are used to estimate PCA hazard curves and component uniform hazard spectra (CUHS) based on various structural and nonstructural parameters. For a given structural system, the primary parameters of interest are the location of the component within the structure, the ratio of the period of the component to the modal periods of the primary structure, and the component damping ratio. Representative results for shear wall structures illustrate the value of applying the method to acceleration-sensitive components, as the quantification of CUHS facilitates the implementation of performance-based design and evaluation approaches. The variability in the component responses presented highlights the need for a robust probabilistic seismic demand estimation methodology for nonstructural components in which the major sources of variability are incorporated.

The yes-associated protein (YAP) is a key effector of the mammalian Hippo signaling pathway. YAP has eight known alternately spliced isoforms and these are widely expressed across multiple tissues. Variable effects have been ascribed to different YAP isoforms by inducing their expression in cells, but whether these differences are due to variability in the transcriptional potency of individual YAP isoforms has not been addressed. Indeed a systematic comparison of the transcriptional potencies of YAP isoforms has not been done. To address this, using overexpression and transcriptional reporter analyses we investigated the transcriptional activities of several human YAP isoforms and determined the effects of the splice variant insertions within the transactivation domain on its transcriptional potency. Utilising full-length coding sequence constructs we determined that the number of WW domains and disruption of the leucine zipper motif within YAP’s transactivation domain both contribute to transcriptional activity. Notably, disruption of YAP’s leucine zipper had a greater effect on transcriptional activity than the absence of the second WW domain. Using GAL4-YAP transcriptional activation domain fusion proteins we found that disruption of the leucine zipper significantly decreased YAP’s transcriptional activity in several cell lines. Our data indicates that expression of different YAP isoforms with varying transcriptional potencies may enable fine control of Hippo pathway signaling. Furthermore the specific isoform being utilised should be taken into consideration when interpreting published data or when designing experiments to ascribe YAP’s function., Highlights • Transcriptional activities of yes-associated protein (YAP) isoforms were compared. • YAP’s WW domains and leucine zipper motif both contribute to transcriptional activity. • Absence of YAP’s second WW domain weakens transcriptional potency. • Disruption of YAP’s leucine zipper weakens the transactivation domain (TAD). • Potency of the TAD from YAP α, β, γ, δ isoforms is cell-context dependent.

This paper addresses the quantification of peak component acceleration demands for acceleration-sensitive nonstructural components attached to or suspended from inelastic structural wall and moment-resisting frame structures. Under certain conditions, for a given ground motion hazard level, inelasticity of the primary structures increases the peak component acceleration demands when compared to the accelerations experienced by the component if the primary structure behaves in the linear elastic range. These conditions include nonstructural components located near the bottom of the structure with periods of vibration that are between the higher modal periods of the supporting structure. This amplification of peak component acceleration demands increases with a decrease in the damping ratio of the component and is more pronounced for structural systems that exhibit a concentration of inelastic behavior near the bottom of the structure. A short discussion on the relevance of these results in the context of current seismic strength demand estimation for nonstructural components is included.