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Rapid genomic diagnosis programs are transforming rare disease diagnosis in acute pediatrics. A ventilated newborn with cerebellar hypoplasia underwent rapid exome sequencing (75 h), identifying a novel homozygous ASNS splice-site variant (NM_133436.3:c.1476+1G>A) of uncertain significance. Rapid ASNS splicing studies using blood-derived messenger RNA from the family trio confirmed a consistent pattern of abnormal splicing induced by the variant (cryptic 5' splice-site or exon 12 skipping) with absence of normal ASNS splicing in the proband. Splicing studies reported within 10 days led to reclassification of c.1476+1G>A as pathogenic at age 27 days. Intensive care was redirected toward palliation. Cost analyses for the neonate and his undiagnosed, similarly affected deceased sibling, demonstrate that early diagnosis reduced hospitalization costs by AU$100,828. We highlight the diagnostic benefits of adjunct RNA testing to confirm the pathogenicity of splicing variants identified via rapid genomic testing pipelines for precision and preventative medicine.Copyright © 2020 Wiley Periodicals LLC

Contains fulltext : 196367.pdf (Publisher’s version ) (Open Access), OBJECTIVE: Comprehensive clinical characterization of congenital titinopathy to facilitate diagnosis and management of this important emerging disorder. METHODS: Using massively parallel sequencing we identified 30 patients from 27 families with 2 pathogenic nonsense, frameshift and/or splice site TTN mutations in trans. We then undertook a detailed analysis of the clinical, histopathological and imaging features of these patients. RESULTS: All patients had prenatal or early onset hypotonia and/or congenital contractures. None had ophthalmoplegia. Scoliosis and respiratory insufficiency typically developed early and progressed rapidly, whereas limb weakness was often slowly progressive, and usually did not prevent independent walking. Cardiac involvement was present in 46% of patients. Relatives of 2 patients had dilated cardiomyopathy. Creatine kinase levels were normal to moderately elevated. Increased fiber size variation, internalized nuclei and cores were common histopathological abnormalities. Cap-like regions, whorled or ring fibers, and mitochondrial accumulations were also observed. Muscle magnetic resonance imaging showed gluteal, hamstring and calf muscle involvement. Western blot analysis showed a near-normal sized titin protein in all samples. The presence of 2 mutations predicted to impact both N2BA and N2B cardiac isoforms appeared to be associated with greatest risk of cardiac involvement. One-third of patients had 1 mutation predicted to impact exons present in fetal skeletal muscle, but not included within the mature skeletal muscle isoform transcript. This strongly suggests developmental isoforms are involved in the pathogenesis of this congenital/early onset disorder. INTERPRETATION: This detailed clinical reference dataset will greatly facilitate diagnostic confirmation and management of patients, and has provided important insights into disease pathogenesis. Ann Neurol 2018;83:1105-1124.

Item does not contain fulltext, OBJECTIVE: To describe the clinical and genetic characteristics of presynaptic congenital myasthenic syndrome secondary to biallelic variants in SLC18A3. METHODS: Individuals from 2 families were identified with biallelic variants in SLC18A3, the gene encoding the vesicular acetylcholine transporter (VAChT), through whole-exome sequencing. RESULTS: The patients demonstrated features seen in presynaptic congenital myasthenic syndrome, including ptosis, ophthalmoplegia, fatigable weakness, apneic crises, and deterioration of symptoms in cold water for patient 1. Both patients demonstrated moderate clinical improvement on pyridostigmine. Patient 1 had a broader phenotype, including learning difficulties and left ventricular dysfunction. Electrophysiologic studies were typical for a presynaptic defect. Both patients showed profound electrodecrement on low-frequency repetitive stimulation followed by a prolonged period of postactivation exhaustion. In patient 1, this was unmasked only after isometric contraction, a recognized feature of presynaptic disease, emphasizing the importance of activation procedures. CONCLUSIONS: VAChT is responsible for uptake of acetylcholine into presynaptic vesicles. The clinical and electrographic characteristics of the patients described are consistent with previously reported mouse models of VAChT deficiency. These findings make it very likely that defects in VAChT due to variants in SLC18A3 are a cause of congenital myasthenic syndrome in humans.

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 s

LAMP2 variants cause Danon disease, an X-Linked dominant lysosomal disorder (OMIM #300257) characterized by hypertrophic cardiomyopathy (HCM), rhythm disturbance, mild intellectual disability and skeletal myopathy. ACMG classification guidelines struggle with splice variants of uncertain significance. RNA splicing studies are new in cardiac genomics but can be critical in confirming pathogenicity. Studies demonstrating cost-effectiveness will further support its introduction clinically. Case Report: A 6-month-old boy was found incidentally to have severe concentric HCM with preserved systolic function following review for patent ductus arteriosus. Initial investigations to determine the aetiology were nondiagnostic. By 12 months he had developed subtle skeletal myopathy with raised CK (1152 H) and developmental delay. Result(s): Genomic testing identified a LAMP2 splicing-variant of uncertain significance (VUS) (ChrX GRCh37: g.119576451T>A, NM-013995.2: c.928+3A>T), according to ACMG guidelines. The variant was absent from a large population database (gnoMAD v2.1) and had not been previously reported. It segregated with disease in an affected older sibling. The mother was negative for the LAMP2 c.928+3A>T variant, suggesting gonadal mosaicism. Functional testing of LAMP2 pre-mRNA splicing using blood from both siblings established that c.928+3A>T variant induced exon-7 skipping (r.865-928del) with no evidence of residual normal splicing. Exon 7 skipping induces a frameshift (p.Lys289Phefs*26) with clear, deleterious consequences for the encoded protein, enabling re-classification to class 5 (pathogenic). Conclusion(s): RNA diagnostics are increasingly used clinically to clarify pathogenicity. Increasing availability of adjunct supportive studies will lead to improved diagnostic rates and patient outcomes from genomic testing.

LAMP2 variants cause Danon disease, an X-Linked dominant lysosomal disorder (OMIM #300257) characterized by hypertrophic cardiomyopathy (HCM), rhythm disturbance, mild intellectual disability and skeletal myopathy. ACMG classification guidelines struggle with splice variants of uncertain significance. RNA splicing studies are new in cardiac genomics but can be critical in confirming pathogenicity. Studies demonstrating cost-effectiveness will further support its introduction clinically. Case Report: A 6-month-old boy was found incidentally to have severe concentric HCM with preserved systolic function following review for patent ductus arteriosus. Initial investigations to determine the aetiology were nondiagnostic. By 12 months he had developed subtle skeletal myopathy with raised CK (1152 H) and developmental delay. Result(s): Genomic testing identified a LAMP2 splicing-variant of uncertain significance (VUS) (ChrX GRCh37: g.119576451T>A, NM-013995.2: c.928+3A>T), according to ACMG guidelines. The variant was absent from a large population database (gnoMAD v2.1) and had not been previously reported. It segregated with disease in an affected older sibling. The mother was negative for the LAMP2 c.928+3A>T variant, suggesting gonadal mosaicism. Functional testing of LAMP2 pre-mRNA splicing using blood from both siblings established that c.928+3A>T variant induced exon-7 skipping (r.865-928del) with no evidence of residual normal splicing. Exon 7 skipping induces a frameshift (p.Lys289Phefs*26) with clear, deleterious consequences for the encoded protein, enabling re-classification to class 5 (pathogenic). Conclusion(s): RNA diagnostics are increasingly used clinically to clarify pathogenicity. Increasing availability of adjunct supportive studies will lead to improved diagnostic rates and patient outcomes from genomic testing.