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The major advances in genetic neuromuscular disorders in the last 30 years have been: (a) identification of the genetic basis for hundreds of these disorders, (b) through knowing the genes, understanding their pathobiology and (c) subsequent implementation of evidence-based treatments for some of the disorders. New genomic technologies are providing precision diagnosis, mode of inheritance and likely prognosis for more patients than ever before. Parents of children with a genetic diagnosis can then use preimplantation or prenatal diagnosis to avoid having further affected children if they wish. But is this the best we can do for genetic neuromuscular disorders? Since the 1980s, it has been argued it would be better to identify Duchenne muscular dystrophy carrier mothers, rather than diagnose their affected sons. Carrier screening for recessive disorders can identify couples with a high chance of having affected children. It allows couples reproductive choice and can prevent infant morbidity and mortality and significant distress for families. Professional bodies in many countries now recommend prospective parents should be informed about carrier screening. Implementing and funding expensive therapies increases the cost-effectiveness of carrier screening, increasing its attractiveness to governments. Best practice for genetic neuromuscular disorders should include equitable access to carrier screening.

Reproductive genetic carrier screening (RGCS) provides people with information about their chance of having children with autosomal recessive or X-linked genetic conditions, enabling informed reproductive decision-making. RGCS is recommended to be offered to all couples during preconception or in early pregnancy. However, cost and a lack of awareness may prevent access. To address this, the Australian Government funded Mackenzie’s Mission—the Australian Reproductive Genetic Carrier Screening Project. Mackenzie’s Mission aims to assess the acceptability and feasibility of an easily accessible RGCS program, provided free of charge to the participant. In study Phase 1, implementation needs were mapped, and key study elements were developed. In Phase 2, RGCS is being offered by healthcare providers educated by the study team. Reproductive couples who provide consent are screened for over 1200 genes associated with >750 serious, childhood-onset genetic conditions. Those with an increased chance result are provided comprehensive genetic counseling support. Reproductive couples, recruiting healthcare providers, and study team members are also invited to complete surveys and/or interviews. In Phase 3, a mixed-methods analysis will be undertaken to assess the program outcomes, psychosocial implications and implementation considerations alongside an ongoing bioethical analysis and a health economic evaluation. Findings will inform the implementation of an ethically robust RGCS program.

Eleven percent of Australian infants have confirmed food allergy. We hypothesized earlier introduction may lead to higher rates of infant anaphylaxis, irrespective of whether the overall rate of food allergy in the population was ultimately reduced.To determine whether a public health campaign, targeting earlier introduction of allergenic foods, affected rates of infant anaphylaxis.Data were obtained from St John Ambulance (SJA) Western Australia and Western Australian emergency departments (ED) on infant (≤12 months) anaphylaxis over a 5-year period (July 1, 2015 to June 30, 2020). Adrenaline administration data were collected in the SJA dataset. Poisson regression was undertaken to assess trends in anaphylaxis over time. Segmented regression analysis was undertaken to assess differences in anaphylaxis rates before and after intervention.The SJA and ED datasets included 172 and 294 events, respectively, coded as infant anaphylaxis. Rates of infant anaphylaxis increased over time for both SJA and ED datasets, with a 1-year increase rate ratio of 1.21 (95% confidence interval, 1.09-1.35; P value.01) and 1.11 (95% confidence interval, 1.02-1.20; P = .01), respectively. Segmented regression indicated no significant difference in rates after intervention. Adrenaline was not coded as being administered in 109 of the 172 anaphylaxis events.Rates of infant anaphylaxis increased over the 5-year reporting period; however, there was no clear increase related to the timing of the public health campaign implementation. Reported adrenaline use was suboptimal. Assessing rates of food allergy in all age groups is required to determine whether there has been an overall reduction in food allergy owing to the intervention.

Reproductive genetic carrier screening aims to offer couples information about their chance of having children with certain autosomal recessive and X-linked genetic conditions. We developed a gene list for use in “Mackenzie’s Mission”, a research project in which 10,000 couples will undergo screening. Criteria for selecting genes were: the condition should be life-limiting or disabling, with childhood onset, such that couples would be likely to take steps to avoid having an affected child; and/or be one for which early diagnosis and intervention would substantially change outcome. Strong evidence for gene-phenotype relationship was required. Candidate genes were identified from OMIM and via review of 23 commercial and published gene lists. Genes were reviewed by 16 clinical geneticists using a standard operating procedure, in a process overseen by a multidisciplinary committee which included clinical geneticists, genetic counselors, an ethicist, a parent of a child with a genetic condition and scientists from diagnostic and research backgrounds. 1300 genes met criteria. Genes associated with non-syndromic deafness and non-syndromic differences of sex development were not included. Our experience has highlighted that gene selection for a carrier screening panel needs to be a dynamic process with ongoing review and refinement.

ObjectiveTo test the hypothesis that monogenic neuropathies such as Charcot-Marie-Tooth disease (CMT) contribute to frequent but often unexplained neuropathies in the elderly, we performed genetic analysis of 230 patients with unexplained axonal neuropathies and disease onset ≥35 years.MethodsWe recruited patients, collected clinical data, and conducted whole-exome sequencing (WES; n = 126) and MME single-gene sequencing (n = 104). We further queried WES repositories for MME variants and measured blood levels of the MME-encoded protein neprilysin.ResultsIn the WES cohort, the overall detection rate for assumed disease-causing variants in genes for CMT or other conditions associated with neuropathies was 18.3% (familial cases 26.4%, apparently sporadic cases 12.3%). MME was most frequently involved and accounted for 34.8% of genetically solved cases. The relevance of MME for late-onset neuropathies was further supported by detection of a comparable proportion of cases in an independent patient sample, preponderance of MME variants among patients compared to population frequencies, retrieval of additional late-onset neuropathy patients with MME variants from WES repositories, and low neprilysin levels in patients' blood samples. Transmission of MME variants was often consistent with an incompletely penetrant autosomal-dominant trait and less frequently with autosomal-recessive inheritance.ConclusionsA detectable fraction of unexplained late-onset axonal neuropathies is genetically determined, by variants in either CMT genes or genes involved in other conditions that affect the peripheral nerves and can mimic a CMT phenotype. MME variants can act as completely penetrant recessive alleles but also confer dominantly inherited susceptibility to axonal neuropathies in an aging population.

[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., Research funding: The Fred Liuzzi Foundation, Australian Postgraduate Award, Australian Genomics Health Alliance. Grant Number: GNT1113531, Fundación Alfonso Martín Escudero, Junta de Andalucía‐Consejería de Salud. Grant Number: B‐0005‐2017, Australian National Health and Medical Research Council. Grant Numbers: APP1117510, APP1122952, APP1080587, Western Australian Department of Health Future Health’s WA Merit Award.

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.

IntroductionPreconception carrier screening (PCS) identifies couples at risk of having children with recessive genetic conditions. New technologies have enabled affordable sequencing for multiple disorders simultaneously, including identifying carrier status for many recessive diseases. The aim of the study was to identify the most effective way of delivering PCS in Western Australia (WA) through the public health system.Methods and analysisThis is a multicentre cohort pilot study of 250 couples who have used PCS, conducted at three sites: (1) Genetic Services of Western Australia, (2) a private genetic counselling practice in Perth and (3) participating general practice group practices in the Busselton region of WA. The primary outcome of the pilot study was to evaluate the feasibility of implementing the comprehensive PCS programme in the WA healthcare system. Secondary outcome measures included evaluation of the psychosocial impact of couples, such as reproductive autonomy; identification of areas within the health system that had difficulties in implementing the programme and evaluation of tools developed during the study.Ethics and disseminationApproval was provided by the Women and Newborn Health Service Human Research Ethics Committee (HREC) at King Edward Memorial Hospital for Women (RGS0000000946) and the University of Western Australia (UWA) HREC (RA/4/20/4258). Participants may choose to withdraw at any time. Withdrawal will in no way affect participating couples' medical care. Study couples will be redirected to another participating health professional for consultation or counselling in the event of a health professional withdrawing. All evaluation data will be deidentified and stored in a password-protected database in UWA. In addition, all hard copy data collected will be kept in a locked cabinet within a secure building. All electronic data will be stored in a password-protected, backed-up location in the UWA Institutional Research Data Store. All evaluative results will be published as separate manuscripts, and selected results will be presented at conferences.

OBJECTIVE: Congenital myopathies are typically characterised by early onset hypotonia, weakness and hallmark features on biopsy. Despite the rapid pace of gene discovery, approximately 50% of patients with a congenital myopathy remain without a genetic diagnosis following screening of known disease genes. METHODS: 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. RESULTS: We identified variants in the gene (MYL1) encoding the skeletal muscle fast-twitch specific myosin essential light chain 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 modeling 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 essential light chain is critical for myofibre development and function. INTERPRETATION: Our data implicate MYL1 as a crucial protein for adequate skeletal muscle function and that MYL1 deficiency is associated with a severe congenital myopathy.

BackgroundPreconception carrier screening (PCS) provides the potential to empower couples to make reproductive choices before having an affected child. An important question is what factors influence the decision to use or not use PCS.MethodsWe analysed the relationship between knowledge, attitudes and intentions to participate in PCS using logistic regression in 832 participants in Western Australia.ResultsTwo-thirds of participants said they would take the test, with 92% of these supporting screening for diseases reducing the lifespan of children and infants. Those who had good genetic knowledge were seven times more likely to intend to use PCS (p≤0.001), while those with high genetic knowledge were four times more likely to (p=0.002) and raised concerns such as insurance and confidentiality.Decreasing genetic knowledge correlated positively with religiosity and apprehension (p≤0.001), which correlated negatively with intention to use PCS (p≤0.001). Increasing genetic knowledge correlated positively with factors representing positive attitudes (p≤0.001), which correlated positively with intention to use PCS (p≤0.001). Many participants with good genetic knowledge nevertheless answered questions that tested understanding incorrectly.80% of participants stated they would prefer to access the test through their general practitioners and 30% would pay up to $A200.ConclusionsKnowledge is instrumental in influencing participation. Having good genetic knowledge may not be enough to understand core concepts of PCS and may impact informed decision-making. This study recommends that continuous education of health professionals and thus the community, in PCS is crucial to reduce misconceptions.

BACKGROUND: Primary chronic intestinal pseudo-obstruction (CIPO) is a rare, potentially life-threatening disorder characterized by severely impaired gastrointestinal motility. The objective of this study was to examine the contribution of ACTG2, LMOD1, MYH11, and MYLK mutations in an Australasian cohort of patients with a diagnosis of primary CIPO associated with visceral myopathy. METHODS: Pediatric and adult patients with primary CIPO and suspected visceral myopathy were recruited from across Australia and New Zealand. Sanger sequencing of the genes encoding enteric gamma-actin (ACTG2) and smooth muscle leiomodin (LMOD1) was performed on DNA from patients, and their relatives, where available. MYH11 and MYLK were screened by next-generation sequencing. KEY RESULTS: We identified heterozygous missense variants in ACTG2 in 7 of 17 families (~41%) diagnosed with CIPO and its associated conditions. We also identified a previously unpublished missense mutation (c.443C>T, p.Arg148Leu) in one family. One case presented with megacystis-microcolon-intestinal hypoperistalsis syndrome in utero with subsequent termination of pregnancy at 28 weeks' gestation. All of the substitutions identified occurred at arginine residues. No likely pathogenic variants in LMOD1, MYH11, or MYLK were identified within our cohort. CONCLUSIONS AND INFERENCES: ACTG2 mutations represent a significant underlying cause of primary CIPO with visceral myopathy and associated phenotypes in Australasian patients. Thus, ACTG2 sequencing should be considered in cases presenting with hypoperistalsis phenotypes with suspected visceral myopathy. It is likely that variants in other genes encoding enteric smooth muscle contractile proteins will contribute further to the genetic heterogeneity of hypoperistalsis phenotypes.

Patients with ‘stiff child’ syndrome usually have mutations at the interface of actin and tropomyosin that could affect the equilibrium of the Ca2+-dependent switch of muscle. ACTA1 K326N was previously reported and we now report two de novo TPM3 (Tpm3.12 protein) mutations, ΔE218 and ΔE224, resulting in a significant hypercontractile phenotype with marked congenital muscle stiffness associated with ventilatory failure in one case.The atomic resolution structure of tropomyosin bound to actin in the ‘switched off’ state shows that tropomyosin makes contact with actin at only two points: one of which is a cluster of basic amino acids: actin K326, K328 and R147. We have demonstrated that two Tpm2.2 mutations, ΔE139 and E181K, and the actin K326N mutation destabilize this actin-tropomyosin interface. We predicted that equivalent charge loss mutations at Tpm3.12 EE 218-219, EE 224-224, or ED 257-258 would also destabilise the interaction with actin leading to a partial switch-on of the muscle. The two newly discovered stiff patient mutation are located at two of the three predicted gain of function sites.We used the quantitative in vitro motility assay and skeletal muscle thin filaments containing recombinant mutant Tpm3.12 expressed in a Baculovirus/sf9 system. ΔE218 led to a 2.5-fold increase in Ca2+-sensitivity (EC50 ratio ΔE218/WT = 0.40 ± 0.07, ). ΔE224 also showed an increase in Ca2+-sensitivity by 2.2-fold (EC50 ratio ΔE224/WT for = 0.46 ± 0.09). It has been previously shown that there was a 2.5 fold increase in Ca2+ sensitivity for ACTC K326N mutation (EC50 ratio K326N/WT = 0.4 ± 0.05, p=0.07).The increased Ca2+-sensitivity indicates that both mutations cause a gain of function that was predicted from the structural analysis and that can account for the stiff patient syndrome.

Background Fetal akinesia/hypokinesia, arthrogryposis and severe congenital myopathies are heterogeneous conditions usually presenting before or at birth. Although numerous causative genes have been identified for each of these disease groups, in many cases a specific genetic diagnosis remains elusive. Due to the emergence of next generation sequencing, virtually the entire coding region of an individual’s DNA can now be analysed through “whole” exome sequencing, enabling almost all known and novel disease genes to be investigated for disorders such as these. Methods Genomic DNA samples from 45 patients with fetal akinesia/hypokinesia, arthrogryposis or severe congenital myopathies from 38 unrelated families were subjected to next generation sequencing. Clinical features and diagnoses for each patient were supplied by referring clinicians. Genomic DNA was used for either whole exome sequencing or a custom-designed neuromuscular sub-exomic supercapture array containing 277 genes responsible for various neuromuscular diseases. Candidate disease-causing variants were investigated and confirmed using Sanger sequencing. Some of the cases within this cohort study have been published previously as separate studies. Results A conclusive genetic diagnosis was achieved for 18 of the 38 families. Within this cohort, mutations were found in eight previously known neuromuscular disease genes (CHRND, CHNRG, ECEL1, GBE1, MTM1, MYH3, NEB and RYR1) and four novel neuromuscular disease genes were identified and have been published as separate reports (GPR126, KLHL40, KLHL41 and SPEG). In addition, novel mutations were identified in CHRND, KLHL40, NEB and RYR1. Autosomal dominant, autosomal recessive, X-linked, and de novo modes of inheritance were observed. Conclusions By using next generation sequencing on a cohort of 38 unrelated families with fetal akinesia/hypokinesia, arthrogryposis, or severe congenital myopathy we therefore obtained a genetic diagnosis for 47 % of families. This study highlights the power and capacity of next generation sequencing (i) to determine the aetiology of genetically heterogeneous neuromuscular diseases, (ii) to identify novel disease genes in small pedigrees or isolated cases and (iii) to refine the interplay between genetic diagnosis and clinical evaluation and management. Electronic supplementary material The online version of this article (doi:10.1186/s13023-015-0364-0) contains supplementary material, which is available to authorized users.

Arthrogryposis multiplex congenita is defined by the presence of contractures across two or more major joints and results from reduced or absent fetal movement. Here, we present three consanguineous families affected by lethal arthrogryposis multiplex congenita. By whole-exome or targeted exome sequencing, it was shown that the probands each harbored a different homozygous mutation (one missense, one nonsense, and one frameshift mutation) in GPR126. GPR126 encodes G-protein-coupled receptor 126, which has been shown to be essential for myelination of axons in the peripheral nervous system in fish and mice. A previous study reported that Gpr126(-/-) mice have a lethal arthrogryposis phenotype. We have shown that the peripheral nerves in affected individuals from one family lack myelin basic protein, suggesting that this disease in affected individuals is due to defective myelination of the peripheral axons during fetal development. Previous work has suggested that autoproteolytic cleavage is important for activating GPR126 signaling, and our biochemical assays indicated that the missense substitution (p.Val769Glu [c.2306TA]) impairs autoproteolytic cleavage of GPR126. Our data indicate that GPR126 is critical for myelination of peripheral nerves in humans. This study adds to the literature implicating defective axoglial function as a key cause of severe arthrogryposis multiplex congenita and suggests that GPR126 mutations should be investigated in individuals affected by this disorder.

For each family the affected individuals sequenced are shown as well as the type of NGS that was performed for each patient. The disease group and consanguinity for each family is also indicated as are the coverage statistics for each sample and NGS. (DOCX 21Â kb)

Nemaline myopathy is one of the major subtypes of congenital myopathy and is known to be caused by mutations in nine genes including cofilin-2 ( CFL2 ).1 Typical nemaline myopathy presentations include proximal weakness, hypotonia, respiratory difficulties and facial weakness.1 This study was approved by The University of Western Australia Human Research Ethics Committee with written consent from patients. After an uneventful birth the proband was admitted to the intensive care unit (ICU) with respiratory distress, although he did not require mechanical ventilation and was discharged after 5 days. A few weeks later he was readmitted following recurrent episodes of apnoea lasting seconds, associated with cyanosis. Most episodes presented after feeding and were aborted by stimulation. Upon admission to ICU, hypotension and respiratory acidosis were detected and orotracheal intubation and mechanical ventilation initiated. After correcting metabolic disturbances, the patient remained hypotonic, though fully awake and alert when taken off sedation. Extraocular movements were estimated to be normal and deep tendon reflexes were present. Head circumference was on the third centile. Extubation failed and the patient required 24-h continuous ventilation support before passing away at 12 months. Nerve conduction studies, MRI, EEG and echocardiography were normal. An electromyograph (EMG) showed apparent myotonic discharges and serum creatine kinase (CK) was mildly elevated. The proband was Saudi Arabian and his parents were first cousins (figure 1A). Two cousins of the proband (figure 1A III:3 and III.4) had a similar disease, both requiring 24-h mechanical ventilation. One cousin (III:3) passed away at 14 months, while the 10-month-old brother (III:4) is still on 24-h ventilation. Due to the severity of the disease in the proband, diagnostic mutation analysis of SMN1 and ACTA1 was performed, but both were excluded. Figure 1 Genetic, histological and immunohistochemical analysis of the CFL2 variant identified through whole exome sequencing. (A) Partial pedigree …

The congenital myopathies include a wide spectrum of clinically, histologically and genetically variable neuromuscular disorders many of which are caused by mutations in genes for sarcomeric proteins. Some congenital myopathy patients have a hypercontractile phenotype. Recent functional studies demonstrated that ACTA1 K326N and TPM2 ΔK7 mutations were associated with hypercontractility that could be explained by increased myofibrillar Ca(2+) sensitivity. A recent structure of the complex of actin and tropomyosin in the relaxed state showed that both these mutations are located in the actin-tropomyosin interface. Tropomyosin is an elongated molecule with a 7-fold repeated motif of around 40 amino acids corresponding to the 7 actin monomers it interacts with. Actin binds to tropomyosin electrostatically at two points, through Asp25 and through a cluster of amino acids that includes Lys326, mutated in the gain-of-function mutation. Asp25 interacts with tropomyosin K6, next to K7 that was mutated in the other gain-of-function mutation. We identified four tropomyosin motifs interacting with Asp25 (K6-K7, K48-K49, R90-R91 and R167-K168) and three E-E/D-K/R motifs interacting with Lys326 (E139, E181 and E218), and we predicted that the known skeletal myopathy mutations ΔK7, ΔK49, R91G, ΔE139, K168E and E181K would cause a gain of function. Tests by an in vitro motility assay confirmed that these mutations increased Ca(2+) sensitivity, while mutations not in these motifs (R167H, R244G) decreased Ca(2+) sensitivity. The work reported here explains the molecular mechanism for 6 out of 49 known disease-causing mutations in the TPM2 and TPM3 genes, derived from structural data of the actin-tropomyosin interface.

Importance To our knowledge, the efficacy of transferring next-generation sequencing from a research setting to neuromuscular clinics has never been evaluated. Objective To translate whole-exome sequencing (WES) to clinical practice for the genetic diagnosis of a large cohort of patients with limb-girdle muscular dystrophy (LGMD) for whom protein-based analyses and targeted Sanger sequencing failed to identify the genetic cause of their disorder. Design, Setting, and Participants We performed WES on 60 families with LGMDs (100 exomes). Data analysis was performed between January 6 and December 19, 2014, using the xBrowse bioinformatics interface (Broad Institute). Patients with LGMD were ascertained retrospectively through the Institute for Neuroscience and Muscle Research Biospecimen Bank between 2006 and 2014. Enrolled patients had been extensively investigated via protein studies and candidate gene sequencing and remained undiagnosed. Patients presented with more than 2 years of muscle weakness and with dystrophic or myopathic changes present in muscle biopsy specimens. Main Outcomes and Measures The diagnostic rate of LGMD in Australia and the relative frequencies of the different LGMD subtypes. Our central goals were to improve the genetic diagnosis of LGMD, investigate whether the WES platform provides adequate coverage of known LGMD-related genes, and identify new LGMD-related genes. Results With WES, we identified likely pathogenic mutations in known myopathy genes for 27 of 60 families. Twelve families had mutations in known LGMD-related genes. However, 15 families had variants in disease-related genes not typically associated with LGMD, highlighting the clinical overlap between LGMD and other myopathies. Common causes of phenotypic overlap were due to mutations in congenital muscular dystrophy–related genes (4 families) and collagen myopathy–related genes (4 families). Less common myopathies included metabolic myopathy (2 families), congenital myasthenic syndrome ( DOK7 ), congenital myopathy ( ACTA1 ), tubular aggregate myopathy ( STIM1 ), myofibrillar myopathy ( FLNC ), and mutation of CHD7 , usually associated with the CHARGE syndrome. Inclusion of family members increased the diagnostic efficacy of WES, with a diagnostic rate of 60% for “trios” (an affected proband with both parents) vs 40% for single probands. A follow-up screening of patients whose conditions were undiagnosed on a targeted neuromuscular disease–related gene panel did not improve our diagnostic yield. Conclusions and Relevance With WES, we achieved a diagnostic success rate of 45.0% in our difficult-to-diagnose cohort of patients with LGMD. We expand the clinical phenotypes associated with known myopathy genes, and we stress the importance of accurate clinical examination and histopathological results for interpretation of WES, with many diagnoses requiring follow-up review and ancillary investigations of biopsy specimens or serum samples.

Cardiac actin ( ACTC ) is the fetal homologue of skeletal muscle actin ( ACTA1 ), and is switched off at birth in skeletal muscle but remains highly expressed in adult heart and in regenerating muscle. ACTC is 99% identical to ACTA1 protein, differing at only 4 amino acids. We previously showed an ACTC transgene could functionally replace ACTA1 in postnatal skeletal muscle of Acta1 knockout mice that normally die within a few days of birth, indicating that ACTC could have a therapeutic role in skeletal muscle actin diseases. We harnessed the power of two different recombinant inbred (RI) mouse models including BXD and the more recently developed Collaborative Cross (CC), to identify genetic elements controlling Actc expression. RI mice are genetic reference populations consisting of large numbers of inbred mouse strains descended from either 2 (BXD) or 8 (CC) founder strains. Characterization of these strains for a trait of interest allows for the mapping of gene(s) mediating that trait to genomic intervals called quantitative trait loci (QTL). We have identified in both RI sets a highly significant QTL regulating Actc expression in skeletal muscle. This QTL also regulates Actc expression in particular non-skeletal muscle tissues (eye and lung). Identification of the regulatory genetic element may provide a target for modifying expression of cardiac actin by therapeutic intervention.

First Notes: Why Chemical MagicA" Foreword Acknowledgments About the Authors Presentation: Performing Tips Understanding the Audience and the Environment Routines Suggested Routines Recovering from Blunders Demonstration: Safety Guidelines Glossary of Chemical Safety Symbols Terminology I Terminology II Basic Demonstrations Intermediate Demonstrations Advanced Demonstrations Index Last Notes: Start of a Catalytic Journey The 10 Secrets of Chemical Magic.

ophthalmoplegia. The OPA1 protein is essential for normal mitochondrial fusion. In mouse DOA (such as OPA1Q285STOP), autophagy (recycling of spent cellular components) is dysregulated in retinal ganglion cells (RGCs), and mitophagy (mitochondrial recycling) has been implicated. Quantifying mitophagy is technically demanding. The hallmarks of mitophagy are organelles called autophagosomesis, co-localisating with, and then engulfing mitochondria. We validated and used two high throughput imaging systems [Imagestream (Amnis) and InCell analyser (GE)] to quantify mitophagy in fibroblasts. Co-localisation of mitochondria and autophagosomes was increased in fibroblasts in five patients from four families with severe OPA1 phenotypes indicating increased mitophagy. ImageStream also showed that basal mitophagy was increased when control cultures were depleted of Opa1 by siRNA. Western blotting confirmed increased basal autophagy and autophagic flux in the presence of activators. Increased mitochondrial fragmentation, mitophagy and failure of mitochondrial transport may together cause local depletion of mitochondria in critical regions of the affected neurons, such as axons or synapses. To investigate mitophagy in a DOA model we crossed the OPA1Q285STOP mouse with our RedMIT/GFP-LC3 mouse harbouring red fluorescent mitochondria and green fluorescent autophagosomes. We showed increased co-localisation between mitochondria and autophagosomes in both cell types investigated, confirming increased mitophagy in this model. The role of mitophagy in mitochondrial disease remains controversial, but appears to be excessive and poorly selective in OPA1 mutants. We intend to use our model to identify drug modulators of mitophagy for treating OPA1 and other mitochondrial patients.

Charcot–Marie–Tooth disease (CMT) is a common inherited peripheral neuropathy associated with mutations in 54 known genes. CMT is characterized by distal wasting of the legs and later hands. Skeletal deformations are frequent including pes cavus and hammer toes. CMT is subdivided into three subtypes: CMT1 is demyelinating, CMT2 axonal, and intermediate CMT mixed. The clinical and molecular heterogeneity of this disease means many families remain without a molecular diagnosis. This proof of principal study aimed to investigate the use of whole exome sequencing (WES), allied with linkage analysis, to detect known or novel mutations in CMT families in which CMT1A had been excluded. We looked at three families, two dominant and one consanguineous. All other known CMT regions were excluded by linkage analysis in one dominant family; all CMT regions except MPZ, by homozygosity mapping in the consanguineous family. In both these families, WES excluded all known CMT genes including MPZ in the consanguineous family. This suggests the CMT genes in these two families are novel. The second dominant family presented with pes cavus and hammer toes in the first decade followed by difficulty in participation in sports in the second decade. Nerve conduction studies showed a severe sensorimotor axonal peripheral neuropathy. Linkage analysis excluded all known CMT genes except a 65 Mbp region on chromosome 16 including three known CMT genes. One of these, AARS (encoding alanyl-tRNA synthetase), fitted with the autosomal dominant inheritance in the family. WES of the proband identified a heterozygous variant, c.G986A (p.R329H) in AARS, which is a known pathogenic mutation. The variant was confirmed by Sanger sequencing in the proband and all other affected family members available, indicating the mutation is the cause of the disease in the family. Our study highlights the potential of WES in diagnostics and that more CMT genes remain to be found.