Detection of mutations in the dystrophin gene via automated DHPLC screening and direct sequencing

BACKGROUND: Currently molecular diagnostic laboratories focus only on the identification of large deletion and duplication mutations (spanning one exon or more) for Duchenne Muscular Dystrophy (DMD) yielding 65% of causative mutations. These mutations are detected by an existing set of multiplexed polymerase chain reaction (PCR) primer pairs. Due to the large size of the dystrophin gene (79 exons), finding point mutations (substitutions, deletions or insertions of one or several nucleotides) has been prohibitively expensive and laborious. The aim of this project was to develop an effective and convenient method of finding all, or most, mutations in the dystrophin gene with only a moderate increase in cost. RESULTS: Using denaturing high performance liquid chromatography (DHPLC) screening and direct sequencing, 86 PCR amplicons of genomic DNA from the dystrophin gene were screened for mutations in eight patients diagnosed with DMD who had tested negative for large DNA rearragements. Mutations likely to be disease-causative were found in six of the eight patients. All 86 amplicons from the two patients in whom no likely disease-causative mutations were found were completely sequenced and only polymorphisms were found. CONCLUSIONS: We have shown that it is now feasible for clinical laboratories to begin testing for both point mutations and large deletions/duplications in the dystrophin gene. The detection rate will rise from 65% to greater than 92% with only a moderate increase in cost

Compound heterozygosity of predicted loss-of-function DES variants in a family with recessive desminopathy

Background: Variants in the desmin gene (DES) are associated with desminopathy; a myofibrillar myopathy mainly characterized by muscle weakness, conduction block, and dilated cardiomyopathy. To date, only ~50 disease-associated variants have been described, and the majority of these lead to dominant-negative effects. However, the complete genotypic spectrum of desminopathy is not well established. Case presentation: Next-generation sequencing was performed on 51 cardiac disease genes in a proband with profound skeletal myopathy, dilated cardiomyopathy, and respiratory dysfunction. Our analyses revealed compound heterozygous DES variants, both of which are predicted to lead to a loss-of-function. Consistent with recessive inheritance, each variant was identified in an unaffected parent. Conclusions: This case report serves to broaden the variant spectrum of desminopathies and provides insight into the molecular mechanisms of desminopathy, supporting distinct dominant-negative and loss-of-function etiologies

Meta-analyses of deflazacort versus prednisone/prednisolone in patients with nonsense mutation Duchenne muscular dystrophy

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

Electrical impedance myography for reducing sample size in Duchenne muscular dystrophy trials

OBJECTIVE: To evaluate the sensitivity of electrical impedance myography (EIM) to disease progression in both ambulatory and non-ambulatory boys with DMD. METHODS AND PARTICIPANTS: A non-blinded, longitudinal cohort study of 29 ambulatory and 15 non-ambulatory boys with DMD and age-similar healthy boys. Subjects were followed for up to 1 year and assessed using the Myolex((R)) mView(TM) EIM system as part of a multicenter study. RESULTS: In the ambulatory group, EIM 100 kHz resistance values showed significant change compared to the healthy boys. For example, in lower extremity muscles, the average change in EIM 100 kHz resistance values over 12 months led to an estimated effect size of 1.58. Based on these results, 26 DMD patients/arm would be needed for a 12-month clinical trial assuming a 50% treatment effect. In non-ambulatory boys, EIM changes were greater in upper limb muscles. For example, biceps at 100kHz resistance gave an estimated effect size of 1.92 at 12 months. Based on these results, 18 non-ambulatory DMD patients/arm would be needed for a 12-month clinical trial assuming a 50% treatment effect. Longitudinal changes in the 100 kHz resistance values for the ambulatory boys correlated with the longitudinal changes in the timed supine-to-stand test. EIM was well-tolerated throughout the study. INTERPRETATION: This study supports that EIM 100 kHz resistance is sensitive to DMD progression in both ambulatory and non-ambulatory boys. Given the technology\u27s ease of use and broad age range of utility it should be employed as an exploratory endpoint in future clinical therapeutic trials in DMD. TRIAL REGISTRATION: Clincialtrials.gov registration #NCT02340923

Clinical and genetic characterization of AP4B1-associated SPG47.

The hereditary spastic paraplegias (HSPs) are a heterogeneous group of disorders characterized by degeneration of the corticospinal and spinocerebellar tracts leading to progressive spasticity. One subtype, spastic paraplegia type 47 (SPG47 or HSP-AP4B1), is due to bi-allelic loss-of-function mutations in the AP4B1 gene. AP4B1 is a subunit of the adapter protein complex 4 (AP-4), a heterotetrameric protein complex that regulates the transport of membrane proteins. Since 2011, 11 individuals from six families with AP4B1 mutations have been reported, nine of whom had homozygous mutations and were from consanguineous families. Here we report eight patients with AP4B1-associated SPG47, the majority born to non-consanguineous parents and carrying compound heterozygous mutations. Core clinical features in this cohort and previously published patients include neonatal hypotonia that progresses to spasticity, early onset developmental delay with prominent motor delay and severely impaired or absent speech development, episodes of stereotypic laughter, seizures including frequent febrile seizures, thinning of the corpus callosum, and delayed myelination/white matter loss. Given that some of the features of AP-4 deficiency overlap with those of cerebral palsy, and the discovery of the disorder in non-consanguineous populations, we believe that AP-4 deficiency may be more common than previously appreciated

Automated DNA mutation detection using universal conditions direct sequencing: application to ten muscular dystrophy genes

<p>Abstract</p> <p>Background</p> <p>One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive.</p> <p>These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels.</p> <p>The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput.</p> <p>Results</p> <p>An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other patients). Methods and assay sequences are reported in this paper.</p> <p>Conclusion</p> <p>This automated process allows laboratories to discover DNA variations in a short time and at low cost.</p

Multicenter Consensus Approach to Evaluation of Neonatal Hypotonia in the Genomic Era: A Review

IMPORTANCE: Infants with hypotonia can present with a variety of potentially severe clinical signs and symptoms and often require invasive testing and multiple procedures. The wide range of clinical presentations and potential etiologies leaves diagnosis and prognosis uncertain, underscoring the need for rapid elucidation of the underlying genetic cause of disease. OBSERVATIONS: The clinical application of exome sequencing or genome sequencing has dramatically improved the timely yield of diagnostic testing for neonatal hypotonia, with diagnostic rates of greater than 50% in academic neonatal intensive care units (NICUs) across Australia, Canada, the UK, and the US, which compose the International Precision Child Health Partnership (IPCHiP). A total of 74% (17 of 23) of patients had a change in clinical care in response to genetic diagnosis, including 2 patients who received targeted therapy. This narrative review discusses the common causes of neonatal hypotonia, the relative benefits and limitations of available testing modalities used in NICUs, and hypotonia management recommendations. CONCLUSIONS AND RELEVANCE: This narrative review summarizes the causes of neonatal hypotonia and the benefits of prompt genetic diagnosis, including improved prognostication and identification of targeted treatments which can improve the short-term and long-term outcomes. Institutional resources can vary among different NICUs; as a result, consideration should be given to rule out a small number of relatively unique conditions for which rapid targeted genetic testing is available. Nevertheless, the consensus recommendation is to use rapid genome or exome sequencing as a first-line testing option for NICU patients with unexplained hypotonia. As part of the IPCHiP, this diagnostic experience will be collected in a central database with the goal of advancing knowledge of neonatal hypotonia and improving evidence-based practice

Mutations in the Satellite Cell Gene MEGF10 Cause a Recessive Congenital Myopathy with Minicores

We ascertained a nuclear family in which three of four siblings were affected with an unclassified autosomal recessive myopathy characterized by severe weakness, respiratory impairment, scoliosis, joint contractures, and an unusual combination of dystrophic and myopathic features on muscle biopsy. Whole genome sequence from one affected subject was filtered using linkage data and variant databases. A single gene, MEGF10, contained nonsynonymous mutations that co-segregated with the phenotype. Affected subjects were compound heterozygous for missense mutations c.976T > C (p.C326R) and c.2320T > C (p.C774R). Screening the MEGF10 open reading frame in 190 patients with genetically unexplained myopathies revealed a heterozygous mutation, c.211C > T (p.R71W), in one additional subject with a similar clinical and histological presentation as the discovery family. All three mutations were absent from at least 645 genotyped unaffected control subjects. MEGF10 contains 17 atypical epidermal growth factor-like domains, each of which contains eight cysteine residues that likely form disulfide bonds. Both the p.C326R and p.C774R mutations alter one of these residues, which are completely conserved in vertebrates. Previous work showed that murine Megf10 is required for preserving the undifferentiated, proliferative potential of satellite cells, myogenic precursors that regenerate skeletal muscle in response to injury or disease. Here, knockdown of megf10 in zebrafish by four different morpholinos resulted in abnormal phenotypes including unhatched eggs, curved tails, impaired motility, and disorganized muscle tissue, corroborating the pathogenicity of the human mutations. Our data establish the importance of MEGF10 in human skeletal muscle and suggest satellite cell dysfunction as a novel myopathic mechanism

LGMD2I in a North American population

<p>Abstract</p> <p>Background</p> <p>There is a marked variation in clinical phenotypes that have been associated with mutations in <it>FKRP</it>, ranging from severe congenital muscular dystrophies to limb-girdle muscular dystrophy type 2I (LGMD2I).</p> <p>Methods</p> <p>We screened the <it>FKRP </it>gene in two cohorts totaling 87 patients with the LGMD phenotype.</p> <p>Results</p> <p>The c.826C>A, p.L276I mutation was present in six patients and a compound heterozygote mutation in a seventh patient. Six patients had a mild LGMD2I phenotype, which resembles that of Becker muscular dystrophy. The other patient had onset before the age of 3 years, and thus may follow a more severe course.</p> <p>Conclusion</p> <p>These findings suggest that LGMD2I may be common in certain North American populations. This diagnosis should be considered early in the evaluation of LGMD.</p

Pretreatment HLADQA1-HLADRB1 Testing for the Prevention of Azathioprine-Induced Pancreatitis in Inflammatory Bowel Disease: A Prospective Cohort Study

INTRODUCTION:Azathioprine-induced pancreatitis is an idiosyncratic and unpredictable response, occurring in up to 7% of azathioprine-exposed patients with inflammatory bowel disease (IBD). The haplotype HLADQA1-HLADRB1*07:01A\u3eC is strongly associated with azathioprine-induced pancreatitis in IBD. We aimed to evaluate whether pretreatment HLADQA1-HLADRB1*07:01A\u3eC screening will reduce the risk of azathioprine-induced pancreatitis.METHODS:Participants with IBD were screened for HLADQA1-HLADRB1*07:01A\u3eC, and participants with a variant genotype were excluded from azathioprine treatment. Wild-type participants were started on azathioprine and followed for 3 months. The incidence of pancreatitis was compared with unscreened historical controls.RESULTS:HLADQA1-HLADRB1*07:01A\u3eC screening resulted in an 11-fold reduction in the incidence of azathioprine-induced pancreatitis (n = 1/328 or 0.30% vs n = 13/373 or 3.4%). In propensity score-matched cohorts (age and sex), HLA DQA1-HLADRB1*07:01A\u3eC screening was significantly associated with a reduction in the incidence of AZA-induced pancreatitis independent of weight, glucocorticoid exposure, and smoking status (adjusted odds ratio = 0.075, 95% confidence interval = 0.01-0.58, P = 0.01). Up to 45% (n = 271/599) of participants were excluded from azathioprine therapy based on the haplotype in the HLADQA1-HLADRB1*07:01A\u3eC-screened cohort.DISCUSSION:HLADQA1-HLADRB1*07:01A\u3eC screening reduced the risk of azathioprine-induced pancreatitis; however, using this strategy to guide the use of azathioprine therapy in IBD may eliminate a large proportion of patients from being eligible for treatment with azathioprine. In regions where there is access to other IBD therapies, and given the short-term and long-term toxicities associated with azathioprine, HLADQA1-HLADRB1*07:01A\u3eC-screening may be a clinically relevant strategy for enhancing the safe use of azathioprine in IBD. In addition, cost-effectiveness analyses are needed to further solidify the utility of HLADQA1-HLADRB1*07:01A\u3eC screening in IBD populations