Optimising RNA diagnostics for implementation into clinical practice.

Background: Genetic variants that elicit aberrant splicing of pre-messenger RNA (pre-mRNA) are recognised as causative variants in ~30-50% of genetic disorders. However, it is still not possible to predict reliably if and how a variant will impact splicing, limiting the application of in silico splice prediction tools in variant interpretation. Most splicing variants fall outside the essential splice site and, in the absence of RNA testing, remain classed variants of uncertain significance (VUS) according to ACMG-AMP (American College of Medical Genetics and Genomics and Association for Molecular Pathology) guidelines. Sequence analysis of spliced messenger RNA (mRNA) is the only definitive means to determine the precise nature of variant associated mis-splicing. Tissues with limited accessibility, for instance vital organs, present a challenge for RNA testing of genes with tissue specific expression. Fortunately, clinically accessible tissues such as blood and fibroblasts can be used to infer variant associated mis-splicing outcomes in the manifesting tissue. A further challenge arises from the lack of guidance on how functional evidence (PS3/BS3 criteria) from RNA studies should be applied to variant interpretation within the current ACMG-AMP framework. There is an urgent need to establish ACMG-AMP aligned quality standards and guidelines for complex RNA assay data for accurate and consistent variant interpretation between clinical laboratories. Methods: Families were recruited from local area health districts across Australia and New Zealand using inclusion criteria to ascertain putative splicing variants with high clinical suspicion of causality. More than 120 families with diverse monogenic conditions were triaged into PCR-based RNA testing, with comparative RNA-sequencing for 38 cases. Consensus ascertainment criteria, standard practices for PCR-based RNA testing, and RNA assay interpretation rubric were devised through consultation with the clinical and molecular genetics community via surveys, live polls and SpliceACORD consortium (Australasian Consortium for RNA Diagnostics) meetings. Results: Informative RNA assay data was obtained for 96% cases, enabling variant re-classification for 75% of variants. RNA testing reports were used to guide clinical care and genetic counselling, and 75% of diagnosis were clinician-reported to have a positive impact for the family. PCR-based RNA diagnostics has the capacity to analyse 81.3% of clinically significant genes and to allow phasing of RNA splicing events. Variant associated mis-splicing was highly reproducible between affected individuals and heterozygotes, and between different biospecimens. Discussion: We provide a standardised protocol for PCR-based RNA testing and ACMG-AMP aligned recommendations for the interpretation of RNA assay data. Our study demonstrates the significant diagnostic and health benefits of RNA analysis as adjunct testing to extend diagnostic yield from genomic testing

Exoproteome Analysis of the Seaweed Pathogen Nautella italica R11 Reveals Temperature-Dependent Regulation of RTX-Like Proteins

Climate fluctuations have been linked to an increased prevalence of disease in seaweeds, including the red alga Delisea pulchra, which is susceptible to a bleaching disease caused by the bacterium Nautella italica R11 under elevated seawater temperatures. To further investigate the role of temperature in the induction of disease by N. italica R11, we assessed the effect of temperature on the expression of the extracellular proteome (exoproteome) in this bacterium. Label-free quantitative mass spectrometry was used to identify 207 proteins secreted into supernatant fraction, which is equivalent to 5% of the protein coding genes in the N. italica R11 genome. Comparative analysis demonstrated that expression of over 30% of the N. italica R11 exoproteome is affected by temperature. The temperature-dependent proteins include traits that could facilitate the ATP-dependent transport of amino acid and carbohydrate, as well as several uncharacterized proteins. Further, potential virulence determinants, including two RTX-like proteins, exhibited significantly higher expression in the exoproteome at the disease inducing temperature of 24°C relative to non-inducing temperature (16°C). This is the first study to demonstrate that temperature has an influence exoproteome expression in a macroalgal pathogen. The results have revealed several temperature regulated candidate virulence factors that may have a role in macroalgal colonization and invasion at elevated sea-surface temperatures, including novel RTX-like proteins

Cullin-3 Dependent Deregulation of ACTN1 Represents a New Pathogenic Mechanism in Nemaline Myopathy

Nemaline myopathy is a congenital neuromuscular disorder characterized by muscle weakness, fiber atrophy, and presence of nemaline bodies within myofibers. However, understanding of the underlying pathomechanisms is lacking. Recently, mutations in KBTBD13, KLHL40, and KLHL41, three substrate adaptors for the E3 ubiquitin ligase Cullin-3, have been associated with early-onset nemaline myopathies. We hypothesized that deregulation of Cullin-3 and its muscle protein substrates may be responsible for disease development. Using Cullin-3–knockout mice, we identified accumulation of non-muscle α-actinins (ACTN1 and ACTN4) in muscles of these mice, which we also observed in patients with mutations in KBTBD13. Our data reveal that proper regulation of Cullin-3 activity and ACTN1 levels is essential for normal muscle and neuromuscular junction development. While ACTN1 is naturally downregulated during myogenesis, its overexpression in C2C12 myoblasts triggered defects in fusion, myogenesis, and acetylcholine receptor clustering — features that we characterized in Cullin-3–deficient mice. Taken together, our data highlight the importance of Cullin-3–mediated degradation of ACTN1 for muscle development, and indicate what is to our knowledge new pathomechanism for the etiology of myopathies seen in Cullin-3–knockout mice and patients with nemaline myopathy

Loss of calpains-1 and -2 prevents repair of plasma membrane scrape injuries, but not small pores, and induces a severe muscular dystrophy

Copyright © 2020 the American Physiological Society. The ubiquitous calpains, calpain-1 and -2, play important roles in Ca2+-dependent membrane repair. Mechanically active tissues like skeletal muscle are particularly reliant on mechanisms to repair and remodel membrane injury, such as those caused by eccentric damage. We demonstrate that calpain-1 and -2 are master effectors of Ca2+-dependent repair of mechanical plasma membrane scrape injuries, although they are dispensable for repair/removal of small wounds caused by pore-forming agents. Using CRISPR gene-edited human embryonic kidney 293 (HEK293) cell lines, we established that loss of both calpains-1 and -2 (CAPNS1∼/∼) virtually ablates Ca2+-dependent repair of mechanical scrape injuries but does not affect injury or recovery from perforation by streptoly-sin-O or saponin. In contrast, cells with targeted knockout of either calpain-1 (CAPN1∼/∼) or -2 (CAPN2∼/∼) show near-normal repair of mechanical injuries, inferring that both calpain-1 and calpain-2 are equally capable of conducting the cascade of proteolytic cleavage events to reseal a membrane injury, including that of the known membrane repair agent dysferlin. A severe muscular dystrophy in a murine model with skeletal muscle knockout of Capns1 highlights vital roles for calpain-1 and/or -2 for health and viability of skeletal muscles not compensated for by calpain-3 (CAPN3). We propose that the dystrophic phenotype relates to loss of maintenance of plasma membrane/cytoskeletal networks by calpains-1 and -2 in response to directed and dysfunctional Ca2+-signaling, pathways hyperstimulated in the context of membrane injury. With CAPN1 variants associated with spastic paraplegia, a severe dystrophy observed with muscle-specific loss of calpain-1 and -2 activity identifies CAPN2 and CAPNS1 as plausible candidate neuromuscular disease genes

Minimal expression of dysferlin prevents development of dysferlinopathy in dysferlin exon 40a knockout mice

Abstract Dysferlin is a Ca2+-activated lipid binding protein implicated in muscle membrane repair. Recessive variants in DYSF result in dysferlinopathy, a progressive muscular dystrophy. We showed previously that calpain cleavage within a motif encoded by alternatively spliced exon 40a releases a 72 kDa C-terminal minidysferlin recruited to injured sarcolemma. Herein we use CRISPR/Cas9 gene editing to knock out murine Dysf exon 40a, to specifically assess its role in membrane repair and development of dysferlinopathy. We created three Dysf exon 40a knockout (40aKO) mouse lines that each express different levels of dysferlin protein ranging from ~ 90%, ~ 50% and ~ 10–20% levels of wild-type. Histopathological analysis of skeletal muscles from all 12-month-old 40aKO lines showed virtual absence of dystrophic features and normal membrane repair capacity for all three 40aKO lines, as compared with dysferlin-null BLAJ mice. Further, lipidomic and proteomic analyses on 18wk old quadriceps show all three 40aKO lines are spared the profound lipidomic/proteomic imbalance that characterises dysferlin-deficient BLAJ muscles. Collective results indicate that membrane repair does not depend upon calpain cleavage within exon 40a and that ~ 10–20% of WT dysferlin protein expression is sufficient to maintain the muscle lipidome, proteome and membrane repair capacity to crucially prevent development of dysferlinopathy

Recurrent TTN metatranscript-only c.39974-11T>G splice variant associated with autosomal recessive arthrogryposis multiplex congenita and myopathy

We present eight families with arthrogryposis multiplex congenita and myopathy bearing a TTN intron 213 extended splice-site variant (NM_001267550.1:c.39974-11T>G), inherited in trans with a second pathogenic TTN variant. Muscle-derived RNA studies of three individuals confirmed mis-splicing induced by the c.39974-11T>G variant; in-frame exon 214 skipping or use of a cryptic 3 ' splice-site effecting a frameshift. Confounding interpretation of pathogenicity is the absence of exons 213-217 within the described skeletal muscle TTN N2A isoform. However, RNA-sequencing from 365 adult human gastrocnemius samples revealed that 56% specimens predominantly include exons 213-217 in TTN transcripts (inclusion rate >= 66%). Further, RNA-sequencing of five fetal muscle samples confirmed that 4/5 specimens predominantly include exons 213-217 (fifth sample inclusion rate 57%). Contractures improved significantly with age for four individuals, which may be linked to decreased expression of pathogenic fetal transcripts. Our study extends emerging evidence supporting a vital developmental role for TTN isoforms containing metatranscript-only exons.Peer reviewe