Pooled analysis of radiation hybrids identifies loci for growth and drug action in mammalian cells

Khan AH, Lin A, Wang RT, et al.

Genetic screens in mammalian cells commonly focus on loss-of-function methodologies. To evaluate the phenotypic consequences of extra gene copies, we used bulk segregant analysis (BSA) of radiation hybrid (RH) cells. We constructed six pools of RH cells, each consisting of ~2500 independent clones, and placed the pools under selection in media with or without paclitaxel. Low pass sequencing identified 859 growth loci, 38 paclitaxel loci, 62 interaction loci and 3 loci for mitochondrial abundance at genome-wide significance. Resolution was measured as ~30 kb, close to single-gene. Divergent properties were displayed by the RH-BSA growth genes compared to those from loss-of-function screens, refuting the balance hypothesis. In addition, enhanced retention of human centromeres in the RH pools suggests a new approach to functional dissection of these chromosomal elements. Pooled analysis of RH cells showed high power and resolution and should be a useful addition to the mammalian genetic toolkit.

Genome Research, September 2, 2020

A well tolerated core needle muscle biopsy process suitable for children and adults

Barthelemy F, Woods JD, …, Wang RT, et al.

Introduction: Serial muscle biopsies within clinical trials for Duchenne muscular dystrophy (DMD) are critical to document therapeutic responses. Less invasive means of sampling muscle are needed.

Methods: We analyzed a retrospective consecutive case-series cohort of vacuum-assisted core needle muscle biopsy procedures performed on healthy and dystrophic individuals at a single institution assessing for safety and reliability of obtaining sufficient high quality biopsy tissue for histologic assessment in adult and pediatric subjects.

Results: Of 471 muscle cores from 128 biopsy procedures, 377-550mg of total muscle tissue was obtained per procedure with mean core weight of 129mg (SD 25.1mg). All Biopsies were adequate for histological assessment. There were no significant adverse events.

Discussion: This core needle biopsy approach, when combined with improved sample processing, provides a safe means to consistently obtain muscle samples for diagnostic and clinical trial applications.

Muscle & Nerve August 2020

Diagnostic Utility of Transcriptome Sequencing for Rare Mendelian Diseases

Lee H, Huang AY, … Wang, RT, et al.

Purpose: We investigated the value of transcriptome sequencing (RNAseq) in ascertaining the consequence of DNA variants on RNA transcripts to improve the diagnostic rate from exome or genome sequencing for undiagnosed Mendelian diseases spanning a wide spectrum of clinical indications.

Methods: From 234 subjects referred to the Undiagnosed Diseases Network, University of California-Los Angeles clinical site between July 2014 and August 2018, 113 were enrolled for high likelihood of having rare undiagnosed, suspected genetic conditions despite thorough prior clinical evaluation. Exome or genome sequencing and RNAseq were performed, and RNAseq data was integrated with genome sequencing data for DNA variant interpretation genome-wide.

Results: The molecular diagnostic rate by exome or genome sequencing was 31%. Integration of RNAseq with genome sequencing resulted in an additional seven cases with clear diagnosis of a known genetic disease. Thus, the overall molecular diagnostic rate was 38%, and 18% of all genetic diagnoses returned required RNAseq to determine variant causality.

Conclusion: In this rare disease cohort with a wide spectrum of undiagnosed, suspected genetic conditions, RNAseq analysis increased the molecular diagnostic rate above that possible with genome sequencing analysis alone even without availability of the most appropriate tissue type to assess.

Genet Med. October 2019

Delays in diagnosis of Duchenne muscular dystrophy: an evaluation of genotypic and sociodemographic factors.

Counterman, K.J., Furlong, P., Wang, R.T., Martin, A.S. Furlong, P.

Introduction: In this study we investigate associations between genotypic and sociodemographic factors and the age of diagnosis of Duchenne muscular dystrophy (DMD).

Methods: Data were collected from the Duchenne Registry from 2007 to 2019, and then used to assess the impact genotype, race/ethnicity, neighborhood poverty levels, and other sociodemographics factors have on the age of diagnosis of DMD patients without a known family history, using univariate and multivariable linear regression.

Results: The mean age of diagnosis was 4.43 years. Non-Caucasian patients and patients from high-poverty neighborhoods were older at diagnosis (P < .01). Increased year of birth was associated with decreasing age of diagnosis (P < .001). Specific genetic mutation subtypes were associated with later ages of symptom onset and diagnosis (P = .005).

Discussion: After adjusting for genotype and year of birth, the average age of diagnosis was significantly later for traditionally at-risk patients.

Muscle Nerve. October 2019

Targeting RyR activity boosts antisense exon 44 and 45 skipping in human DMD skeletal or cardiac muscle culture models

Barthelemy, F, Wang, RT, et al

Abstract: Systemic delivery of antisense oligonucleotides (AO) for DMD exon skipping has proven effective for reframing DMD mRNA, rescuing dystrophin expression, and slowing disease progression in animal models. In humans with Duchenne muscular dystrophy treated with AOs, low levels of dystrophin have been induced, and modest slowing of disease progression has been observed, highlighting the need for improved efficiency of human skipping drugs. Here, we demonstrate that dantrolene and Rycals S107 and ARM210 potentiate AO-mediated exon skipping of exon 44 or exon 45 in patient-derived myotube cultures with appropriate mutations. Further, dantrolene is shown to boost AO-mediated exon skipping in patient-derived, induced cardiomyocyte cultures. Our findings further validate the ryanodine receptors (RyR) as the likely target responsible for exon skip boosting and demonstrate potential applicability beyond exon 51 skipping. These data provide preclinical support of dantrolene trial as an adjuvant to AO-mediated exon-skipping therapy in humans and identify a novel Rycal, ARM210, for development as a potential exon-skipping booster. Further, they highlight the value of mutation-specific DMD culture models for basic discovery, preclinical drug screening and translation of personalized genetic medicines.

Molecular Therapy: Nucleic Acid. September 2019

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. Top Downloaded Paper 2018-2019.

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.

Genome Med. 2017 Oct 25;9(1):90. doi: 10.1186/s13073-017-0479-0.

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.

Current Opinion in Neurology. 2015 Oct;28(5):535-41. doi: 10.1097/WCO.0000000000000245

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.

PLOS Currents Muscular Dystrophy. 2014 Oct 17. Edition 1. doi: 10.1371/
[pdf] [write up for nonscientists] [PMC]

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.

Science Translation Medicine 2012 Dec 12;4(164)

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.

BMC Genomics. 12:562, November 2011. Highly accessed article.

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 (, 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.