DMD genotype correlations from the Duchenne Registry: Endogenous exon skipping is a factor in prolonged ambulation for individuals with a defined mutation sub-type
Abstract: Antisense oligonucleotide (AON)‐mediated exon skipping is an emerging therapeutic for individuals with Duchenne muscular dystrophy (DMD). Skipping of exons adjacent to common exon deletions in DMD using AONs can produce in‐frame transcripts and functional protein. Targeted skipping of DMD exons 8, 44, 45, 50, 51, 52, 53, and 55 is predicted to benefit 47% of affected individuals. We observed a correlation between mutation subgroups and age at loss of ambulation in the Duchenne Registry, a large database of phenotypic and genetic data for DMD (N = 765). Males amenable to exon 44 (N = 74) and exon 8 skipping (N = 18) showed prolonged ambulation compared to other exon skip groups and nonsense mutations (P = 0.035 and P < 0.01, respectively). In particular, exon 45 deletions were associated with prolonged age at loss of ambulation relative to the rest of the exon 44 skip amenable cohort and other DMD mutations. Exon 3–7 deletions also showed prolonged ambulation relative to all other exon 8 skippable mutations. Cultured myotubes from DMD patients with deletions of exons 3–7 or exon 45 showed higher endogenous skipping than other mutations, providing a potential biological rationale for our observations. These results highlight the utility of aggregating phenotypic and genotypic data for rare pediatric diseases to reveal progression differences, identify potentially confounding factors, and probe molecular mechanisms that may affect disease severity.
Wang RT, Barthelemy F, Martin AS, Douine ED, Eskin A, Lucas A, Lavigne J, Peay H, Khanlou N, Sweeney L, Cantor RM, Miceli MC, Nelson SF.
Human Mutation August 31, 2018 Jun 16. doi: 10.1002/humu.23561. Editor’s Choice Award
Next-generation mapping: a novel approach for detection of pathogenic structural variants with a potential utility in clinical diagnosis
Abstract: Background: Massively parallel DNA sequencing, such as exome sequencing, has become a routine clinical procedure to identify pathogenic variants responsible for a patient’s phenotype. Exome sequencing has the capability of reliably identifying inherited and de novo single-nucleotide variants, small insertions, and deletions. However, due to the use of 100–300-bp fragment reads, this platform is not well powered to sensitively identify moderate to large structural variants (SV), such as insertions, deletions, inversions, and translocations. Methods: To overcome these limitations, we used next-generation mapping (NGM) to image high molecular weight double-stranded DNA molecules (megabase size) with fluorescent tags in nanochannel arrays for de novo genome assembly. We investigated the capacity of this NGM platform to identify pathogenic SV in a series of patients diagnosed with Duchenne muscular dystrophy (DMD), due to large deletions, insertion, and inversion involving the DMD gene. Results: We identified deletion, duplication, and inversion breakpoints within DMD. The sizes of deletions were in the range of 45–250 Kbp, whereas the one identified insertion was approximately 13 Kbp in size. This method refined the location of the break points within introns for cases with deletions compared to current polymerase chain reaction (PCR)-based clinical techniques. Heterozygous SV were detected in the known carrier mothers of the DMD patients, demonstrating the ability of the method to ascertain carrier status for large SV. The method was also able to identify a 5.1-Mbp inversion involving the DMD gene, previously identified by RNA sequencing. Conclusions
We showed the ability of NGM technology to detect pathogenic structural variants otherwise missed by PCR-based techniques or chromosomal microarrays. NGM is poised to become a new tool in the clinical genetic diagnostic strategy and research due to its ability to sensitively identify large genomic variations.
Barseghyan H, Tang W, Wang RT, Almalvez M, Segura E, Bramble MS, Lipson A, Douine ED, Lee H, Délot EC, Nelson SF, Vilain E.
What Can DuchenneConnect Teach Us About Treating Duchenne Muscular Dystrophy?
PURPOSE OF REVIEW: This review aims to describe the benefits and limitations of using the Duchenne Connect patient registry to provide information particularly in regard to active treatment choices in Duchenne muscular dystrophy and their impact on disease progression.
RECENT FINDINGS: Clinical trials and natural history studies are difficult for rare diseases like Duchenne muscular dystrophy. Using an online patient self-report survey model, Duchenne Connect provides relevant data that are difficult to gather in other ways. Validation of the overall dataset is supported by comparable mutational spectrum relative to other cohorts and demonstrated beneficial effect of corticosteroid use in prolonging ambulation. These types of analyses are provocative and allow multivariate analyses across the breadth of patient and physician medication and supplement practices. Because the data are self-reported and online, the barrier to participation is low and great potential exists for novel directions of further research in a highly participatory forum.
SUMMARY: Patient registries for Duchenne and Becker muscular dystrophy (DBMD) are powerful tools for monitoring patient outcomes, comparing treatment options, and relating information between patients, researchers, and clinicians. Duchenne Connect is an online patient self-report registry for individuals with DBMD that facilitates aggregation of treatment modalities, outcomes, and genotype data and has played a vital role in furthering DBMD research, particularly in the USA, in a highly participatory and low-cost manner.
Wang, R.T., Nelson, S.F.
Online Self-Report Data for Duchenne Muscular Dystrophy Confirms Natural History and Can Be Used to Assess for Therapeutic Benefits
Abstract: To assess the utility of online patient self-report outcomes in a rare disease, we attempted to observe the effects of corticosteroids in delaying age at fulltime wheelchair use in Duchenne muscular dystrophy (DMD) using data from 1,057 males from DuchenneConnect, an online registry. Data collected were compared to prior natural history data in regard to age at diagnosis, mutation spectrum, and age at loss of ambulation. Because registrants reported differences in steroid and other medication usage, as well as age and ambulation status, we could explore these data for correlations with age at loss of ambulation. Using multivariate analysis, current steroid usage was the most significant and largest independent predictor of improved wheelchair-free survival. Thus, these online self-report data were sufficient to retrospectively observe that current steroid use by patients with DMD is associated with a delay in loss of ambulation. Comparing commonly used steroid drugs, deflazacort prolonged ambulation longer than prednisone (median 14 years and 13 years, respectively). Further, use of Vitamin D and Coenzyme Q10, insurance status, and age at diagnosis after 4 years were also significant, but smaller, independent predictors of longer wheelchair-free survival. Nine other common supplements were also individually tested but had lower study power. This study demonstrates the utility of DuchenneConnect data to observe therapeutic differences, and highlights needs for improvement in quality and quantity of patient-report data, which may allow exploration of drug/therapeutic practice combinations impractical to study in clinical trial settings. Further, with the low barrier to participation, we anticipate substantial growth in the dataset in the coming years.
Wang RT, Silverstein Fadlon CA, Ulm JW, Jankovic I, Eskin A, Lu A, Rangel Miller V, Cantor RM, Li N, Elashoff R, Martin AS, Peay HL, Halnon N, Nelson SF.
This was the first and largest ever study showing the superiority of deflazacort over prednisone and predates the positive results of the Marathon Pharmaceuticals/PTC Therapeutics study.
Dantrolene enhances antisense-mediated exon skipping in human and mouse models of Duchenne muscular dystrophy
Abstract: Duchenne muscular dystrophy (DMD) causes profound and progressive muscle weakness and loss, resulting in early death. DMD is usually caused by frameshifting deletions in the gene DMD, which leads to absence of dystrophin protein. Dystrophin binds to F-actin and components of the dystrophin-associated glycoprotein complex and protects the sarcolemma from contraction-induced injury. Antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach aimed at restoring the DMD reading frame and allowing expression of an intact dystrophin glycoprotein complex. To date, low levels of dystrophin protein have been produced in humans by this method. We performed a small-molecule screen to identify existing drugs that enhance antisense-directed exon skipping. We found that dantrolene, currently used to treat malignant hyperthermia, potentiates antisense oligomer-guided exon skipping to increase exon skipping to restore the mRNA reading frame, the sarcolemmal dystrophin protein, and the dystrophin glycoprotein complex in skeletal muscles of mdx mice when delivered intramuscularly or intravenously. Further, dantrolene synergized with multiple weekly injections of antisense to increase muscle strength and reduce serum creatine kinase in mdx mice. Dantrolene similarly promoted antisense-mediated exon skipping in reprogrammed myotubes from DMD patients. Ryanodine and Rycal S107, which, like dantrolene, targets the ryanodine receptor, also promoted antisense-driven exon skipping, implicating the ryanodine receptor as the critical molecular target.
Kendall, G.C., Mokhonova, E.I., Moran, M., Sejbuk, N.E., Wang, D.W., Silva, O., Wang, R.T., Martinez, L., Lu, Q.L., Damoiseaux, R., Spencer, M.J., Nelson, S.F., Miceli, M.C.
Effects of genome-wide copy number variation on expression in mammalian cells
BACKGROUND: There is only a limited understanding of the relation between copy number and expression for mammalian genes. We fine mapped cis and trans regulatory loci due to copy number change for essentially all genes using a human-hamster radiation hybrid (RH) panel. These loci are called copy number expression quantitative trait loci (ceQTLs). RESULTS: Unexpected findings from a previous study of a mouse-hamster RH panel were replicated. These findings included decreased expression as a result of increased copy number for 30% of genes and an attenuated relationship between expression and copy number on the X chromosome suggesting an Xist independent form of dosage compensation. In a separate glioblastoma dataset, we found conservation of genes in which dosage was negatively correlated with gene expression. These genes were enriched in signaling and receptor activities. The observation of attenuated X-linked gene expression in response to increased gene number was also replicated in the glioblastoma dataset. Of 523 gene deserts of size > 600 kb in the human RH panel, 325 contained trans ceQTLs with -log10 P > 4.1. Recently discovered genes, ultra conserved regions, noncoding RNAs and microRNAs explained only a small fraction of the results, suggesting a substantial portion of gene deserts harbor as yet unidentified functional elements. CONCLUSION: Radiation hybrids are a useful tool for high resolution mapping of cis and trans loci capable of affecting gene expression due to copy number change. Analysis of two independent radiation hybrid panels show agreement in their findings and may serve as a discovery source for novel regulatory loci in noncoding regions of the genome.
Wang, R.T., Ahn, S., Park, C.P., Khan, A., Lange, K., Smith, D.J.
A genome-scale map of the human interactome constructed from radiation hybrid genotyping data
Lin, A., Wang, R.T., Ahn, S., Park, C., Smith, D.J.
Genome Research. 20: 1122-1132, August 2010. Highlighted in “Epistasis: A network of interactors” by T. Casci in Nature Reviews Genetics, 11: 531, Aug. 2010.
Directed Gene Regulatory Networks in Mammals Using Expression and Comparative Genomic Hybridization Microarray Data from Radiation Hybrids
Ahn, S., Wang, R.T., Park, C.P., Lin, A., Leahy, R.M., Lange, K., Smith, D.J.
PLoS Computational Biology. 5(6): e1000407, June 2009. Highlighted in “This Week in PLoS” in GenomeWeb (http://www.genomeweb.com), Jun. 15, 2009.
Fine mapping of regulatory loci for mammalian gene expression using radiation hybrids
Park, C.P., Ahn, S., Bloom, J.S., Lin, A., Wang, R.T., Wu, T., Sekar, A., Khan, A.H., Farr, C.J., Lusis, A.J., Leahy, R.M., Lange, K., Smith, D.J.
Nature Genetics, 40(4):421-429, April 2008.
Achieving Effective Treatment of Patients With Chronic Psychotic Illness and Comorbid Substance Dependence.
Ho, A.P., Tsuang, J.T., Liberman, R.P., Wang, R., Wilkins, J.N., Eckman, T.A., Shaner, A.L.
Am J Psychiatry, 156: 1765 – 1770. November 1999.