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Background: Current carrier screening methods do not identify a proportion of carriers that may have children affected by spinal muscular atrophy (SMA). Additional genetic data is essential to inform accurate risk assessment and genetic counselling of SMA carriers. This study aims to quantify the various genotypes among parents of children with SMA. Method: A retrospective cohort study was undertaken at Sydney Children’s Hospital Network, the major SMA referral centre for New South Wales, Australia. Participants included children with genetically confirmed SMA born between 2005 and 2021. Data was collected on parent genotype inclusive of copy number of SMN1 exons 7 and 8. The number of SMN2 exon 7 copies were recorded for the affected children. Descriptive statistics were used to determine the proportion of carriers of 2+0 genotype classified as silent carriers. Chi-square test was used to correlate the association between parents with a heterozygous SMN1 exon 7 deletion and two copies of exon 8 and ≥3 SMN2 copy number in the proband. Results: SMA carrier testing was performed in 118/154 (76.6%) parents, incorporating 59 probands with homozygous SMN1 deletions and one proband with compound heterozygote pathogenic variants. Among parents with a child with SMA, 7.6% had two copies of SMN1 exon 7. When only probands with a homozygous SMN1 exon 7 deletion were included, 6.9% of parents had two copies of SMN1 exon 7. An association was observed between heterozygous deletion of SMN1 exon 7 with two copies of exon 8 in a parent and ≥3 SMN2 copy number in the affected proband (p = 0.07). Conclusions: This study confirmed a small but substantial proportion of silent carriers not identified by conventional screening within an Australian context. Accordingly, the effectiveness of carrier screening for SMA is linked with genetic counselling to enable health literacy regarding high and low risk results and is complemented by new-born screening and maintaining clinical awareness for SMA. Gene conversion events may underpin the associations between parent carrier status and proband SMN2 copy number.

Paediatric hyperCKaemia without weakness presents a clinical conundrum. Invasive investigations with low diagnostic yields, including muscle biopsy, may be considered unjustifiable. Improved access to genome-wide genetic testing has shifted first-line investigations towards genetic studies in neuromuscular disease. This research aims to provide an evidence-based diagnostic approach to paediatric hyperCKaemia without weakness, a current gap in the literature. We identified 47 individuals (10-months to 16-years-old; 34 males, 13 females) from 43 families presenting with hyperCKaemia on two or more occasions, without weakness, from The Children's Hospital at Westmead Neuromuscular Clinic Database. Clinical features, investigations and outcomes were analysed via retrospective chart review. Genetic testing has been performed in 34/43. Genetic variants explaining hyperCKaemia were identified in 25/34 (74%) using multiplex ligation-dependent probe amplification, massive parallel sequencing, single gene testing and exome sequencing. Pathogenic/likely pathogenic variants were identified in 19 neuromuscular disease genes and six metabolic myopathy genes. Individuals with metabolic diagnoses had higher peak creatine kinase levels that sometimes normalized. Conversely, creatine kinase levels remained persistently elevated those with neuromuscular diagnoses. In summary, a genetic cause is found in most paediatric patients with hyperCKaemia without weakness informing clinical management and counselling. Thus, we propose a diagnostic algorithm for this cohort.

BACKGROUND & AIMS: Constitutional mismatch repair deficiency (CMMRD) is a rare recessive childhood cancer predisposition syndrome caused by germline mismatch repair variants. Constitutional microsatellite instability (cMSI) is a CMMRD diagnostic hallmark and may associate with cancer risk. We quantified cMSI in a large CMMRD patient cohort to explore genotype-phenotype correlations using novel MSI markers selected for instability in blood.METHODS: Three CMMRD, 1 Lynch syndrome, and 2 control blood samples were genome sequenced to >120× depth. A pilot cohort of 8 CMMRD and 38 control blood samples and a blinded cohort of 56 CMMRD, 8 suspected CMMRD, 40 Lynch syndrome, and 43 control blood samples were amplicon sequenced to 5000× depth. Sample cMSI score was calculated using a published method comparing microsatellite reference allele frequencies with 80 controls.RESULTS: Thirty-two mononucleotide repeats were selected from blood genome and pilot amplicon sequencing data. cMSI scoring using these MSI markers achieved 100% sensitivity (95% CI, 93.6%-100.0%) and specificity (95% CI 97.9%-100.0%), was reproducible, and was superior to an established tumor MSI marker panel. Lower cMSI scores were found in patients with CMMRD with MSH6 deficiency and patients with at least 1 mismatch repair missense variant, and patients with biallelic truncating/copy number variants had higher scores. cMSI score did not correlate with age at first tumor.CONCLUSIONS: We present an inexpensive and scalable cMSI assay that enhances CMMRD detection relative to existing methods. cMSI score is associated with mismatch repair genotype but not phenotype, suggesting it is not a useful predictor of cancer risk.

usc Refereed/Peer-reviewed Purpose: Genetic variants causing aberrant premessenger RNA splicing are increasingly being recognized as causal variants in genetic disorders. In this study, we devise standardized practices for polymerase chain reaction (PCR)-based RNA diagnostics using clinically accessible specimens (blood, fibroblasts, urothelia, biopsy). Methods: A total of 74 families with diverse monogenic conditions (31% prenatal-congenital onset, 47% early childhood, and 22% teenage-adult onset) were triaged into PCR-based RNA testing, with comparative RNA sequencing for 19 cases. Results: Informative RNA assay data were obtained for 96% of cases, enabling variant reclassification for 75% variants that can be used for genetic counseling (71%), to inform clinical care (32%) and prenatal counseling (41%). Variant-associated mis-splicing was highly reproducible for 28 cases with samples from ≥2 affected individuals or heterozygotes and 10 cases with ≥2 biospecimens. PCR amplicons encompassing another segregated heterozygous variant was vital for clinical interpretation of 22 of 79 variants to phase RNA splicing events and discern complete from partial mis-splicing. Conclusion: RNA diagnostics enabled provision of a genetic diagnosis for 64% of recruited cases. PCR-based RNA diagnostics has capacity to analyze 81.3% of clinically significant genes, with long amplicons providing an advantage over RNA sequencing to phase RNA splicing events. The Australasian Consortium for RNA Diagnostics (SpliceACORD) provide clinically-endorsed, standardized protocols and recommendations for interpreting RNA assay data.

A precise genetic diagnosis of a dystrophinopathy has far-reaching implications for affected boys and their families. We present three boys with DMD single nucleotide variants associated with Becker muscular dystrophy presenting with myalgia, reduced exercise capacity, neurodevelopmental symptoms and elevated creatine kinase. The DMD variants were difficult to classify: AIII:1 a synonymous variant in exon 13 c.1602G>A, p.Lys534Lys; BIII:1 an essential splice-site variant in intron 33 c.4674+1G>A, and CII:1 a missense mutation within the cysteine-rich domain, exon 66 c.9619T>C, p.Cys3207Arg. Complementary DNA (cDNA) analysis using muscle-derived mRNA established splice-altering effects of variants for AIII:1 and BIII:1, and normal splicing in CII:1. Western blot analysis demonstrated mildly to moderately reduced dystrophin levels (17.6 - 36.1% the levels of controls), supporting dystrophinopathy as a probable diagnosis. These three cases highlight the diagnostic utility of muscle biopsy for mRNA studies and western blot to investigate DMD variants of uncertain pathogenicity, by exploring effects on splicing and dystrophin protein levels.

Aim: Compare efficacies of deflazacort and prednisone/prednisolone in providing clinically meaningful delays in loss of physical milestones in patients with nonsense mutation Duchenne muscular dystrophy. Materials & methods: Placebo data from Phase IIb (ClinicalTrials.gov Identifier: NCT00592553) and ACT DMD (ClinicalTrials.gov Identifier: NCT01826487) ataluren nonsense mutation Duchenne muscular dystrophy clinical trials were retrospectively combined in meta-analyses (intent-to-treat population; for change from baseline to week 48 in 6-min walk distance [6MWD] and timed function tests). Results: Significant improvements in change in 6-min walk distance with deflazacort versus prednisone/prednisolone (least-squares mean difference 39.54 m [95% CI: 13.799, 65.286; p = 0.0026]). Significant and clinically meaningful improvements in 4-stair climb and 4-stair descend for deflazacort versus prednisone/prednisolone. Conclusion: Deflazacort provides clinically meaningful delays in loss of physical milestones over 48 weeks compared with prednisone/prednisolone for patients with nonsense mutation Duchenne muscular dystrophy.

Reduced muscle tone, muscle weakness, and physical fatigue can impact considerably on quality of life for children with neurofibromatosis type 1 (NF1). Human muscle biopsies and mouse models of NF1 deficiency in muscle show intramyocellular lipid accumulation, and preclinical data have indicated that L-carnitine supplementation can ameliorate this phenotype. The aim of this study is to examine whether daily L-carnitine supplementation is safe and feasible, and will improve muscle strength and reduce fatigue in children with NF1. A 12-week Phase 2a trial was conducted using 1000 mg daily oral levocarnitine tartrate supplementation. Recruited children were between 8 and 12 years old with a clinical diagnosis of NF1, history of muscle weakness and fatigue, and naive to L-carnitine. Primary outcomes were safety (self-reporting, biochemical testing) and compliance. Secondary outcomes included plasma acylcarnitine profiles, functional measures (muscle strength, long jump, handwriting speed, 6-minute-walk test [6MWT]), and parent-reported questionnaires (PedsQL™, CBCL/6-18). Six children completed the trial with no self-reported adverse events. Biochemical tests for kidney and liver function were normal, and the average compliance was 95%. Plasma acylcarnitine levels were low, but within a range not clinically linked to carnitine deficiency. For strength measures, there was a mean 53% increase in dorsiflexion strength (95% confidence interval [CI] 8.89-60.75; p = 0.02) and mean 66% increase in plantarflexion strength (95% CI 12.99-134.1; p = 0.03). In terms of muscle performance, there was a mean 10% increase in long jump distance (95% CI 2.97-16.03; p = 0.01) and 6MWT distance (95% CI 5.88-75.45; p = 0.03). Comparison with the 1000 Norms Project data showed a significant improvement in Z-score for all of these measures. Parent reports showed no negative impact on quality of life, and the perceived benefits led to the majority of individuals remaining on L-carnitine after the study. Twelve weeks of L-carnitine supplementation is safe and feasible in children with NF1, and a Phase 3 trial should confirm the efficacy of treatment.

BackgroundThe activating signal cointegrator 1 (ASC-1) complex acts as a transcriptional coactivator for a variety of transcription factors and consists of four subunits: ASCC1, ASCC2, ASCC3 and TRIP4. A single homozygous mutation in ASCC1 has recently been reported in two families with a severe muscle and bone disorder.ObjectiveWe aim to contribute to a better understanding of the ASCC1-related disorder.MethodsHere, we provide a clinical, histological and genetic description of three additional ASCC1 families.ResultsAll patients presented with severe prenatal-onset muscle weakness, neonatal hypotonia and arthrogryposis, and congenital bone fractures. The muscle biopsies from the affected infants revealed intense oxidative rims beneath the sarcolemma and scattered remnants of sarcomeres with enlarged Z-bands, potentially representing a histopathological hallmark of the disorder. Sequencing identified recessive nonsense or frameshift mutations in ASCC1, including two novel mutations.ConclusionOverall, this work expands the ASCC1 mutation spectrum, sheds light on the muscle histology of the disorder and emphasises the physiological importance of the ASC-1 complex in fetal muscle and bone development.

PURPOSE: To investigate the effect of PLXNA1 variants on the phenotype of patients with autosomal dominant and recessive inheritance patterns and to functionally characterize the zebrafish homologs plxna1a and plxna1b during development. METHODS: We assembled ten patients from seven families with biallelic or de novo PLXNA1 variants. We describe genotype-phenotype correlations, investigated the variants by structural modeling, and used Morpholino knockdown experiments in zebrafish to characterize the embryonic role of plxna1a and plxna1b. RESULTS: Shared phenotypic features among patients include global developmental delay (9/10), brain anomalies (6/10), and eye anomalies (7/10). Notably, seizures were predominantly reported in patients with monoallelic variants. Structural modeling of missense variants in PLXNA1 suggests distortion in the native protein. Our zebrafish studies enforce an embryonic role of plxna1a and plxna1b in the development of the central nervous system and the eye. CONCLUSION: We propose that different biallelic and monoallelic variants in PLXNA1 result in a novel neurodevelopmental syndrome mainly comprising developmental delay, brain, and eye anomalies. We hypothesize that biallelic variants in the extracellular Plexin-A1 domains lead to impaired dimerization or lack of receptor molecules, whereas monoallelic variants in the intracellular Plexin-A1 domains might impair downstream signaling through a dominant-negative effect.

Aim: Assess the totality of efficacy evidence for ataluren in patients with nonsense mutation Duchenne muscular dystrophy (nmDMD). Materials & methods: Data from the two completed randomized controlled trials (ClinicalTrials.gov: NCT00592553; NCT01826487) of ataluren in nmDMD were combined to examine the intent-to-treat (ITT) populations and two patient subgroups (baseline 6-min walk distance [6MWD] ≥300–Results: Statistically significant differences in 6MWD change with ataluren versus placebo were observed across all three meta-analyses. Least-squares mean difference (95% CI): ITT (n = 342), +17.2 (0.2–34.1) m, p = 0.0473; ≥300–Conclusion: These meta-analyses support previous evidence for ataluren in slowing disease progression versus placebo in patients with nmDMD over 48 weeks. Treatment benefit was most evident in patients with a baseline 6MWD ≥300–

Multiple studies have shown that genomic testing has a high diagnostic yield and an impact on clinical management for patients with suspected genetic conditions. Therefore, there has been a push worldwide to apply rapid genomic sequencing in critically ill neonatal and pediatric patients. The goal of this study was to investigate the practicality of applying ultrarapid genomic testing for critically ill neonatal and pediatric patients with suspected monogenic conditions in Australia. The study recruited a total of 108 patients prospectively from March 2018 to February 2019, and data were collected until May 2019. Of the 12 hospitals that acted as collaborating sites, 5 were women's hospitals, 3 women's and children's hospitals, and 4 children's hospitals. Eligible patients included those who were admitted to a neonatal or pediatric intensive care unit (NICU or PICU) and were referred to clinical genetics for a possible monogenic condition. Chromosomal microarray was a requirement before enrollment if there was a suspected chromosomal condition. Chromosomal microarrays were performed concurrently with ultrarapid exome sequencing when the probability of a positive result on microarray was low. Additionally, rapid mitochondrial genome sequencing was performed alongside ultrarapid exome sequencing if a mitochondrial condition was suspected. When possible, ultrarapid exome sequencing was performed in both parents as well as the child (trio). The primary outcome of this study measured the time from the last sample received to the ultrarapid exome sequencing report finalized. Other outcomes measured were the diagnostic yield, change in clinical management after the report was finalized, number of reports returned before hospital discharge, and time from admission to the report being finalized. Of the 108 patients enrolled, the median age was 28 days, with a range of 0 days to 17 years. Overall, 34% were female, 57% were NICU admissions, 33% were PICU admissions, and 9% were from other hospital wards. The mean time it took from the last sample received to the report being issued was 3.3 days (95% confidence interval [CI], 3.2-3.5 days). The mean time from hospital admission to the sequencing report being finalized was 17.5 days (95% CI, 14.6-21.1 days). The mean time from hospital admission to clinical genetics referral was 8.1 days (95% CI, 6.0-10.8 days). Overall, 94% of reports were issued within the goal of 5 calendar days, and 56 molecular diagnoses were made for 55 patients (51%). Among the 62 patients in the NICU, 35 had a clear molecular diagnosis from ultrarapid exome sequencing (56%), whereas in the 36 PICU patients, 17 had a clear molecular diagnosis (47%), and in the 10 patients from other hospital wards, 3 had a clear molecular diagnosis (30%). After the ultrarapid exome sequencing report was finalized, clinical management changed for 48 of the 108 patients enrolled (44%). Overall, the ultrarapid sequencing reports that revealed a clear molecular diagnosis were regarded as “very useful” or “useful” by referring providers in 52 of 55 cases (95%) and “neutral” in 3 of 55 cases (5%). Negative reports were regarded as “very useful” or “useful” in 31 of 53 cases (58%), “neutral” in 20 of 53 cases, and “not useful” in 2 of 53 cases (4%). Altogether, this study demonstrates the utility of ultrarapid exome sequencing in critically ill pediatric patients with a suspected monogenic condition in Australia. Refereed/Peer-reviewed

Great progress has been made in the clinical translation of several therapeutic strategies for spinal muscular atrophy (SMA), including measures to selectively address Survival Motor Neuron (SMN) protein deficiency with SMN1 gene replacement or modulation of SMN2 encoded protein levels, as well as neuroprotective approaches and supporting muscle strength and function. This review highlights these novel therapies. This is particularly vital with the advent of the first disease modifying therapy, which has brought to the fore an array of questions surrounding who, how and when to treat, and stimulated challenges in resource limited healthcare systems to streamline access for those eligible for drug therapy. The overhaul of the landscape for all those involved in SMA extends to the design of further drug trials and the necessity of multidisciplinary supportive care to potentiate the effects of disease modifying medications. The impact of respiratory complications in SMA is central to management in the current era of emerging novel therapies. These fundamental changes in our knowledge and management approach to those with SMA are explored further in this review.

Background Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder with a population frequency of approximately 1 in 10,000. The most common epigenetic defect in BWS is a loss of methylation (LOM) at the 11p15.5 imprinting centre, KCNQ1OT1 TSS-DMR, and affects 50% of cases. We hypothesised that genetic factors linked to folate metabolism may play a role in BWS predisposition via effects on methylation maintenance at KCNQ1OT1 TSS-DMR. Results Single nucleotide variants (SNVs) in the folate pathway affecting methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR), 5-methyltetrahydrofolate-homocysteine S-methyltransferase (MTR), cystathionine beta-synthase (CBS) and methionine adenosyltransferase (MAT1A) were examined in 55 BWS patients with KCNQ1OT1 TSS-DMR LOM and in 100 unaffected cases. MTHFR rs1801133: C>T was more prevalent in BWS with KCNQ1OT1 TSS-DMR LOM (p T, rs150331990: A>G and rs757460628: G>A encoding NP_001124295 p.Arg136Cys, p.His1118Arg and p.Arg1223His, respectively. These variants have population frequencies of less than 1 in 1000 and were absent from 100 control cases. Functional characterization using a hemimethylated DNA trapping assay revealed a reduced methyltransferase activity relative to wild-type DNMT1 for each variant ranging from 40 to 70% reduction in activity. Conclusions This study is the first to examine folate pathway genetics in BWS and to identify rare DNMT1 missense variants in affected individuals. Our data suggests that reduced DNMT1 activity could affect maintenance of methylation at KCNQ1OT1 TSS-DMR in some cases of BWS, possibly via a maternal effect in the early embryo. Larger cohort studies are warranted to further interrogate the relationship between impaired MTHFR enzymatic activity attributable to MTHFR rs1801133: C>T, dietary folate intake and BWS. Electronic supplementary material The online version of this article (10.1186/s13148-018-0546-4) contains supplementary material, which is available to authorized users.

Monitoring optic nerve sheath meningiomas (ONSM) in Neurofibromatosis type 2 (NF2) patients remains difficult. Other ocular manifestations of NF2 may obscure ophthalmic assessment of optic nerve function in these patients. Serial magnetic resonance imaging (MRI) used to assess the optic nerve is not without limitations, being expensive and often requiring general anaesthetic in children, with associated risks. This study was undertaken to describe the use of multifocal visual evoked potentials (multifocal VEP, mfVEP) in the regular monitoring of NF2 patients with ONSM. This study involved three NF2 patients with ONSM who undertook mfVEP testing at an academic ophthalmic centre. Same day mfVEP and routine ophthalmic testing were undertaken. Topographical function of the optic nerve was assessed, utilising tools such as asymmetry deviation and accumap severity index. Results were assessed alongside MRI and visual acuity (VA). From the three patients, five eyes had ONSMs, of which two caused unilateral blindness. The remaining three affected eyes had initial VAs 6/6, 6/24, and 6/18. Over follow up, ranging from 5 to 12 years, all tumours progressed, and VA declined for all patients. Multifocal VEP detected optic nerve functional loss corresponding with visual decline. This case series suggests mfVEP is effective in the objective topographic monitoring of optic nerve function in NF2 patients with ONSM. Due also to its safety in a paediatric population, the test may be considered in the routine monitoring of these patients, to be used to assist regular ophthalmic review and MRI scans.

A male neonate presented with severe weakness, hypotonia, contractures and congenital scoliosis. Skeletal muscle specimens showed marked atrophy and degeneration of fast fibres with striking nemaline rods and hypertrophy of slow fibres that were ultrastructurally normal. A neuromuscular gene panel identified a homozygous essential splice variant in TNNT3 (chr11:1956150G>A, NM_006757.3:c.681+1G>A). TNNT3 encodes skeletal troponin-T(fast) and is associated with autosomal dominant distal arthrogryposis. TNNT3 has not previously been associated with nemaline myopathy, a rare congenital myopathy linked to defects in proteins associated with thin filament structure and regulation. cDNA studies confirmed pathogenic consequences of the splice variant, eliciting exon-skipping and intron retention events leading to a frameshift. Western blot showed deficiency of troponin-T(fast) protein with secondary loss of troponin-I(fast). We establish a homozygous splice variant in TNNT3 as the likely cause of severe congenital nemaline myopathy with distal arthrogryposis (NM-DA), characterised by specific involvement of Type-2 fibres and deficiency of troponin-T(fast).

PURPOSE: Neurofibromatosis type 1 (NF1) is characterized by a highly variable clinical presentation, but almost all NF1-affected adults present with cutaneous and/or subcutaneous neurofibromas. Exceptions are individuals heterozygous for the NF1 in-frame deletion, c.2970_2972del (p.Met992del), associated with a mild phenotype without any externally visible tumors. METHODS: A total of 135 individuals from 103 unrelated families, all carrying the constitutional NF1 p.Met992del pathogenic variant and clinically assessed using the same standardized phenotypic checklist form, were included in this study. RESULTS: None of the individuals had externally visible plexiform or histopathologically confirmed cutaneous or subcutaneous neurofibromas. We did not identify any complications, such as symptomatic optic pathway gliomas (OPGs) or symptomatic spinal neurofibromas; however, 4.8% of individuals had nonoptic brain tumors, mostly low-grade and asymptomatic, and 38.8% had cognitive impairment/learning disabilities. In an individual with the NF1 constitutional c.2970_2972del and three astrocytomas, we provided proof that all were NF1-associated tumors given loss of heterozygosity at three intragenic NF1 microsatellite markers and c.2970_2972del. CONCLUSION: We demonstrate that individuals with the NF1 p.Met992del pathogenic variant have a mild NF1 phenotype lacking clinically suspected plexiform, cutaneous, or subcutaneous neurofibromas. However, learning difficulties are clearly part of the phenotypic presentation in these individuals and will require specialized care. ispartof: GENETICS IN MEDICINE vol:21 issue:4 pages:867-876 ispartof: location:United States status: published

ObjectiveTo describe the diagnostic utility of whole-genome sequencing and RNA studies in boys with suspected dystrophinopathy, for whom multiplex ligation-dependent probe amplification and exomic parallel sequencing failed to yield a genetic diagnosis, and to use remnant normal DMD splicing in 3 families to define critical levels of wild-type dystrophin bridging clinical spectrums of Duchenne to myalgia.MethodsExome, genome, and/or muscle RNA sequencing was performed for 7 males with elevated creatine kinase. PCR of muscle-derived complementary DNA (cDNA) studied consequences for DMD premessenger RNA (pre-mRNA) splicing. Quantitative Western blot was used to determine levels of dystrophin, relative to control muscle.ResultsSplice-altering intronic single nucleotide variants or structural rearrangements in DMD were identified in all 7 families. Four individuals, with abnormal splicing causing a premature stop codon and nonsense-mediated decay, expressed remnant levels of normally spliced DMD mRNA. Quantitative Western blot enabled correlation of wild-type dystrophin and clinical severity, with 0%–5% dystrophin conferring a Duchenne phenotype, 10% ± 2% a Becker phenotype, and 15% ± 2% dystrophin associated with myalgia without manifesting weakness.ConclusionsWhole-genome sequencing relied heavily on RNA studies to identify DMD splice-altering variants. Short-read RNA sequencing was regularly confounded by the effectiveness of nonsense-mediated mRNA decay and low read depth of the giant DMD mRNA. PCR of muscle cDNA provided a simple, yet informative approach. Highly relevant to genetic therapies for dystrophinopathies, our data align strongly with previous studies of mutant dystrophin in Becker muscular dystrophy, with the collective conclusion that a fractional increase in levels of normal dystrophin between 5% and 20% is clinically significant.

IMPORTANCE: Widespread adoption of rapid genomic testing in pediatric critical care requires robust clinical and laboratory pathways that provide equitable and consistent service across health care systems.OBJECTIVE: To prospectively evaluate the performance of a multicenter network for ultra-rapid genomic diagnosis in a public health care system.DESIGN, SETTING, AND PARTICIPANTS: Descriptive feasibility study of critically ill pediatric patients with suspected monogenic conditions treated at 12 Australian hospitals between March 2018 and February 2019, with data collected to May 2019. A formal implementation strategy emphasizing communication and feedback, standardized processes, coordination,distributed leadership, and collective learning was used to facilitate adoption.EXPOSURES Ultra-rapid exome sequencing.MAIN OUTCOMES AND MEASURES: The primary outcome was time from sample receipt to ultra-rapid exome sequencing report. The secondary outcomes were the molecular diagnostic yield, the change in clinical management after the ultra-rapid exome sequencing report, the time from hospital admission to the laboratory report, and the proportion of laboratory reports returned prior to death or hospital discharge RESULTS: The study population included 108 patients with a median age of 28 days (range, 0 days to 17 years); 34% were female; and 57% were from neonatal intensive care units, 33% were from pediatric intensive care units, and 9% were from other hospital wards. The meantime from sample receipt to ultra-rapid exome sequencing report was 3.3 days (95% CI,3.2-3.5 days) and the median time was 3 days (range, 2-7 days). The mean time from hospital admission to ultra-rapid exome sequencing report was 17.5 days (95% CI, 14.6-21.1 days) and 93 reports (86%) were issued prior to death or hospital discharge. A molecular diagnosis was established in 55 patients (51%). Eleven diagnoses (20%) resulted from using the following approaches to augment standard exome sequencing analysis: mitochondrial genome sequencing analysis, exome sequencing–based copy number analysis, use of international databases to identify novel gene–disease associations, and additional phenotyping and RNA analysis. In 42 of 55 patients (76%) with a molecular diagnosis and 6 of 53 patients (11%)without a molecular diagnosis, the ultra-rapid exome sequencing result was considered as having influenced clinical management. Targeted treatments were initiated in 12 patients(11%), treatment was redirected toward palliative care in 14 patients (13%), and surveillance for specific complications was initiated in 19 patients (18%).CONCLUSIONS AND RELEVANCE: This study suggests feasibility of ultra-rapid genomic testing in critically ill pediatric patients with suspected monogenic conditions in the Australian public health care system. However, further research is needed to understand the clinical value of such testing, and the generalizability of the findings to other health care settings. Refereed/Peer-reviewed

BACKGROUND: Cough augmentation techniques are taught by health-care providers to improve secretion clearance and to help prevent respiratory infections in children with neuromuscular disease. There is some evidence of the effectiveness of a manually assisted cough when applied by health-care providers. However, it is unknown whether parents and caregivers may also be effective in applying manually assisted cough. The aim of this study was to evaluate whether parents and caregivers are effective at applying a manually assisted cough to a child with neuromuscular disease after being taught by a health-care provider. METHODS: For this prospective cohort study, children and their parents or caregivers were recruited from neuromuscular clinics in the Sydney Children's Hospitals Network. Cough peak flow was the outcome measure for the strength of the child's cough. Children were eligible to participate if their unassisted cough peak flow at baseline was

Background and objectiveDysferlinopathies are a group of muscle disorders caused by mutations in the DYSF gene. Previous muscle imaging studies describe a selective pattern of muscle involvement in smaller patient cohorts, but a large imaging study across the entire spectrum of the dysferlinopathies had not been performed and previous imaging findings were not correlated with functional tests.MethodsWe present cross-sectional T1-weighted muscle MRI data from 182 patients with genetically confirmed dysferlinopathies. We have analysed the pattern of muscles involved in the disease using hierarchical analysis and presented it as heatmaps. Results of the MRI scans have been correlated with relevant functional tests for each region of the body analysed.ResultsIn 181 of the 182 patients scanned, we observed muscle pathology on T1-weighted images, with the gastrocnemius medialis and the soleus being the most commonly affected muscles. A similar pattern of involvement was identified in most patients regardless of their clinical presentation. Increased muscle pathology on MRI correlated positively with disease duration and functional impairment.ConclusionsThe information generated by this study is of high diagnostic value and important for clinical trial development. We have been able to describe a pattern that can be considered as characteristic of dysferlinopathy. We have defined the natural history of the disease from a radiological point of view. These results enabled the identification of the most relevant regions of interest for quantitative MRI in longitudinal studies, such as clinical trials.Clinical trial registrationNCT01676077.

Neurofibromatosis type 1 (NF1), one of the most common genetic disorders with an estimated prevalence of 1:3000 live births, is characterized by a highly variable clinical presentation. To date, only two clinically relevant intragenic genotype-phenotype correlations have been reported for NF1 missense mutations affecting p.Arg1809 and an in-frame 1-amino acid deletion p.Met922del. Both variants predispose to a distinct mild NF1 phenotype with neither externally visible cutaneous/plexiform neurofibromas nor other tumors. Here, we report 162 patients (129 unrelated probands and 33 affected relatives) carrying a constitutional missense mutation affecting one of five neighboring NF1 codons Leu844, Cys845, Ala846, Leu847 and Gly848, located in the Cysteine-Serine-Rich Domain (CSRD). These recurrent missense mutations affect ~0.8% of unrelated NF1 mutation-positive probands in the UAB cohort. A substantial fraction of these patients presented with a severe phenotype, including plexiform and/or spinal neurofibromas, symptomatic optic pathway gliomas, malignant neoplasms or osseous lesions. Major superficial plexiform neurofibromas and symptomatic spinal neurofibromas were more prevalent compared with classic NF1 cohorts (both p

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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.

BackgroundSpinal muscular atrophy (SMA) is a devastating motor neuron disorder causing progressive muscle weakness and respiratory insufficiency. We present the initial Australian experiences implementing the expanded access programme (EAP) to enable preapproval access to nusinersen, the first disease-modifying therapy, for SMA type 1.MethodsAn Australian multicentre, open-label EAP for nusinersen enrolled patients with infantile-onset SMA type 1 from November 2016 to September 2017. Standard-of-care medical therapy and treatment with intrathecal nusinersen were provided to all patients. Clinical and diagnostic characteristics, molecular genetics, treatment administered, and functional motor outcomes were assessed.ResultsA total of 20 patients with SMA type 1 met the inclusion criteria, of whom 16 consented and received nusinersen treatment. Median time to diagnosis from symptom onset was 5.0 months and was correlated with age of onset (r=0.54, PSMN2 copies.ConclusionsThe nusinersen EAP highlights difficulties in achieving early diagnosis and/or prevention, the evolution of optimal clinical care in a time of uncertain prognostication, resource implications and ethical issues in clinical practice for SMA type 1. These challenges are broadly relevant to the realisation of all novel therapeutics in neurological disorders.

Neuromuscular disorders in children are a heterogeneous group of conditions with a variable age of presentation and overlapping clinical manifestations, many of which have progressive respiratory morbidity. Respiratory insufficiency occurs as a consequence of an imbalance between demands on the respiratory system and respiratory muscle capacity. Daytime measures of pulmonary function are used routinely in these children to assess respiratory status and monitor the consequences of the progression of muscle weakness. This review describes the current evidence for daytime pulmonary function tests and their ability to predict imminent respiratory morbidity.

Introduction: Dystrophinopathy is a rare, severe muscle disorder, and nonsense mutations are found in 13% of cases. Ataluren was developed to enable ribosomal readthrough of premature stop codons in nonsense mutation (nm) genetic disorders. Methods: Randomized, double-blind, placebo-controlled study; males ≥5 years with nm-dystrophinopathy received study drug orally 3 times daily, ataluren 10, 10, 20 mg/kg (N = 57); ataluren 20, 20, 40 mg/kg (N = 60); or placebo (N = 57) for 48 weeks. The primary endpoint was change in 6-Minute Walk Distance (6MWD) at Week 48. Results: Ataluren was generally well tolerated. The primary endpoint favored ataluren 10, 10, 20 mg/kg versus placebo; the week 48 6MWD Δ = 31.3 meters, post hoc P = 0.056. Secondary endpoints (timed function tests) showed meaningful differences between ataluren 10, 10, 20 mg/kg, and placebo. Conclusions: As the first investigational new drug targeting the underlying cause of nm-dystrophinopathy, ataluren offers promise as a treatment for this orphan genetic disorder with high unmet medical need. Muscle Nerve 50: 477–487, 2014

BACKGROUND: Duchenne muscular dystrophy (DMD) is a severe, progressive, and rare neuromuscular, X-linked recessive disease. Dystrophin deficiency is the underlying cause of disease; therefore, mutation-specific therapies aimed at restoring dystrophin protein production are being explored. We aimed to assess the efficacy and safety of ataluren in ambulatory boys with nonsense mutation DMD. METHODS: We did this multicentre, randomised, double-blind, placebo-controlled, phase 3 trial at 54 sites in 18 countries located in North America, Europe, the Asia-Pacific region, and Latin America. Boys aged 7-16 years with nonsense mutation DMD and a baseline 6-minute walk distance (6MWD) of 150 m or more and 80% or less of the predicted normal value for age and height were randomly assigned (1:1), via permuted block randomisation (block size of four) using an interactive voice-response or web-response system, to receive ataluren orally three times daily (40 mg/kg per day) or matching placebo. Randomisation was stratified by age (

Objective: To describe the baseline clinical and functional characteristics of an international cohort of 193 patients with dysferlinopathy. Methods: The Clinical Outcome Study for dysferlinopathy (COS) is an international multicenter study of this disease, evaluating patients with genetically confirmed dysferlinopathy over 3 years. We present a cross-sectional analysis of 193 patients derived from their baseline clinical and functional assessments. Results: There is a high degree of variability in disease onset, pattern of weakness, and rate of progression. No factor, such as mutation class, protein expression, or age at onset, accounted for this variability. Among patients with clinical diagnoses of Miyoshi myopathy or limb-girdle muscular dystrophy, clinical presentation and examination was not strikingly different. Respiratory impairment and cardiac dysfunction were observed in a minority of patients. A substantial delay in diagnosis was previously common but has been steadily reducing, suggesting increasing awareness of dysferlinopathies. Conclusions: These findings highlight crucial issues to be addressed for both optimizing clinical care and planning therapeutic trials in dysferlinopathy. This ongoing longitudinal study will provide an opportunity to further understand patterns and variability in disease progression and form the basis for trial design.

Published research exploring confidence and perceptions of effectiveness in performing a manually assisted cough on children with neuromuscular disease is not available. This descriptive study aimed to describe confidence and perceived effectiveness of parents, children, and therapists in parent performed manually assisted cough. A total of 28 children with neuromuscular disease, one of their parents, and physiotherapist participated. Overall, 40% of parents, 52% of children, and 46% of therapists were very confident in parents’ ability to perform effective manually assisted coughs. Parents, children, and therapists largely perceived the parental manually assisted coughs as somewhat effective (68%, 60%, and 57%, respectively). Approximately, half of parents (48%), children (52%), and therapists (50%) felt very confident in parents’ ability to retain the technique between clinic visits. Interestingly, percentage agreement statistics indicate that a reasonable proportion (30%) of pairs of respondents did not agree in their ratings. Overall, high percentages of favourable ratings were noted for all questions.