Person: Gorham, Joshua
Loading...
Email Address
AA Acceptance Date
Birth Date
Research Projects
Organizational Units
Job Title
Last Name
Gorham
First Name
Joshua
Name
Gorham, Joshua
8 results
Search Results
Now showing 1 - 8 of 8
Publication ViroFind: A novel target-enrichment deep-sequencing platform reveals a complex JC virus population in the brain of PML patients(Public Library of Science, 2018) Chalkias, Spyros; Gorham, Joshua; Mazaika, Erica; Parfenov, Michael; Dang, Xin; DePalma, Steve; McKean, David; Seidman, Christine; Seidman, Jonathan; Koralnik, Igor J.Deep nucleotide sequencing enables the unbiased, broad-spectrum detection of viruses in clinical samples without requiring an a priori hypothesis for the source of infection. However, its use in clinical research applications is limited by low cost-effectiveness given that most of the sequencing information from clinical samples is related to the human genome, which renders the analysis of viral genomes challenging. To overcome this limitation we developed ViroFind, an in-solution target-enrichment platform for virus detection and discovery in clinical samples. ViroFind comprises 165,433 viral probes that cover the genomes of 535 selected DNA and RNA viruses that infect humans or could cause zoonosis. The ViroFind probes are used in a hybridization reaction to enrich viral sequences and therefore enhance the detection of viral genomes via deep sequencing. We used ViroFind to detect and analyze all viral populations in the brain of 5 patients with progressive multifocal leukoencephalopathy (PML) and of 18 control subjects with no known neurological disease. Compared to direct deep sequencing, by using ViroFind we enriched viral sequences present in the clinical samples up to 127-fold. We discovered highly complex polyoma virus JC populations in the PML brain samples with a remarkable degree of genetic divergence among the JC virus variants of each PML brain sample. Specifically for the viral capsid protein VP1 gene, we identified 24 single nucleotide substitutions, 12 of which were associated with amino acid changes. The most frequent (4 of 5 samples, 80%) amino acid change was D66H, which is associated with enhanced tissue tropism, and hence likely a viral fitness advantage, compared to other variants. Lastly, we also detected sparse JC virus sequences in 10 of 18 (55.5%) of control samples and sparse human herpes virus 6B (HHV6B) sequences in the brain of 11 of 18 (61.1%) control subjects. In sum, ViroFind enabled the in-depth analysis of all viral genomes in PML and control brain samples and allowed us to demonstrate a high degree of JC virus genetic divergence in vivo that has been previously underappreciated. ViroFind can be used to investigate the structure of the virome with unprecedented depth in health and disease state.Publication Titin mutations in iPS cells define sarcomere insufficiency as a cause of dilated cardiomyopathy(American Association for the Advancement of Science (AAAS), 2015) Hinson, John Travis; Chopra, Anant; Nafissi, N.; Polacheck, William J.; Benson, Craig Carlyle; Swist, S.; Gorham, Joshua; Yang, Luhan; Schafer, S.; Sheng, Calvin Chen; Haghighi, Alireza; Homsy, Jason; Hubner, N.; Church, George; Cook, S. A.; Linke, Wolfgang; Chen, Christopher; Seidman, Jonathan; Seidman, ChristineHuman mutations that truncate the massive sarcomere protein titin [TTN-truncating variants (TTNtvs)] are the most common genetic cause for dilated cardiomyopathy (DCM), a major cause of heart failure and premature death. Here we show that cardiac microtissues engineered from human induced pluripotent stem (iPS) cells are a powerful system for evaluating the pathogenicity of titin gene variants. We found that certain missense mutations, like TTNtvs, diminish contractile performance and are pathogenic. By combining functional analyses with RNA sequencing, we explain why truncations in the A-band domain of TTN cause DCM, whereas truncations in the I band are better tolerated. Finally, we demonstrate that mutant titin protein in iPS cell–derived cardiomyocytes results in sarcomere insufficiency, impaired responses to mechanical and β-adrenergic stress, and attenuated growth factor and cell signaling activation. Our findings indicate that titin mutations cause DCM by disrupting critical linkages between sarcomerogenesis and adaptive remodeling.Publication Cardiovascular homeostasis dependence on MICU2, a regulatory subunit of the mitochondrial calcium uniporter(National Academy of Sciences, 2017) Bick, Alexander; Wakimoto, Hiroko; Kamer, Kimberli; Sancak, Yasemin; Goldberger, Olga; Axelsson, Anna; DeLaughter, Daniel; Gorham, Joshua; Mootha, Vamsi; Seidman, J. G.; Seidman, ChristineComparative analyses of transcriptional profiles from humans and mice with cardiovascular pathologies revealed consistently elevated expression of MICU2, a regulatory subunit of the mitochondrial calcium uniporter complex. To determine if MICU2 expression was cardioprotective, we produced and characterized Micu2−/− mice. Mutant mice had left atrial enlargement and Micu2−/− cardiomyocytes had delayed sarcomere relaxation and cytosolic calcium reuptake kinetics, indicating diastolic dysfunction. RNA sequencing (RNA-seq) of Micu2−/− ventricular tissues revealed markedly reduced transcripts encoding the apelin receptor (Micu2−/− vs. wild type, P = 7.8 × 10−40), which suppresses angiotensin II receptor signaling via allosteric transinhibition. We found that Micu2−/− and wild-type mice had comparable basal blood pressures and elevated responses to angiotensin II infusion, but that Micu2−/− mice exhibited systolic dysfunction and 30% lethality from abdominal aortic rupture. Aneurysms and rupture did not occur with norepinephrine-induced hypertension. Aortic tissue from Micu2−/− mice had increased expression of extracellular matrix remodeling genes, while single-cell RNA-seq analyses showed increased expression of genes related to reactive oxygen species, inflammation, and proliferation in fibroblast and smooth muscle cells. We concluded that Micu2−/− mice recapitulate features of diastolic heart disease and define previously unappreciated roles for Micu2 in regulating angiotensin II-mediated hypertensive responses that are critical in protecting the abdominal aorta from injury.Publication Loss of RNA expression and allele-specific expression associated with congenital heart disease(Nature Publishing Group, 2016) McKean, David; Homsy, Jason; Wakimoto, Hiroko; Patel, Neil; Gorham, Joshua; DePalma, Steven; Ware, James S.; Zaidi, Samir; Ma, Wenji; Patel, Nihir; Lifton, Richard P.; Chung, Wendy K.; Kim, Richard; Shen, Yufeng; Brueckner, Martina; Goldmuntz, Elizabeth; Sharp, Andrew J.; Seidman, Christine; Gelb, Bruce D.; Seidman, J. G.Congenital heart disease (CHD), a prevalent birth defect occurring in 1% of newborns, likely results from aberrant expression of cardiac developmental genes. Mutations in a variety of cardiac transcription factors, developmental signalling molecules and molecules that modify chromatin cause at least 20% of disease, but most CHD remains unexplained. We employ RNAseq analyses to assess allele-specific expression (ASE) and biallelic loss-of-expression (LOE) in 172 tissue samples from 144 surgically repaired CHD subjects. Here we show that only 5% of known imprinted genes with paternal allele silencing are monoallelic versus 56% with paternal allele expression—this cardiac-specific phenomenon seems unrelated to CHD. Further, compared with control subjects, CHD subjects have a significant burden of both LOE genes and ASE events associated with altered gene expression. These studies identify FGFBP2, LBH, RBFOX2, SGSM1 and ZBTB16 as candidate CHD genes because of significantly altered transcriptional expression.Publication Stable Gene Targeting in Human Cells Using Single-Strand Oligonucleotides with Modified Bases(Public Library of Science, 2012) Rios, Xavier; Briggs, Adrian; Christodoulou, Danos; Gorham, Joshua; Seidman, Jonathan; Church, GeorgeRecent advances allow multiplexed genome engineering in E. coli, employing easily designed oligonucleotides to edit multiple loci simultaneously. A similar technology in human cells would greatly expedite functional genomics, both by enhancing our ability to test how individual variants such as single nucleotide polymorphisms (SNPs) are related to specific phenotypes, and potentially allowing simultaneous mutation of multiple loci. However, oligo-mediated targeting of human cells is currently limited by low targeting efficiencies and low survival of modified cells. Using a HeLa-based EGFP-rescue reporter system we show that use of modified base analogs can increase targeting efficiency, in part by avoiding the mismatch repair machinery. We investigate the effects of oligonucleotide toxicity and find a strong correlation between the number of phosphorothioate bonds and toxicity. Stably EGFP-corrected cells were generated at a frequency of \(\sim\)0.05% with an optimized oligonucleotide design combining modified bases and reduced number of phosphorothioate bonds. We provide evidence from comparative RNA-seq analysis suggesting cellular immunity induced by the oligonucleotides might contribute to the low viability of oligo-corrected cells. Further optimization of this method should allow rapid and scalable genome engineering in human cells.Publication Dissecting Spatio-Temporal Protein Networks Driving Human Heart Development and Related Disorders(Nature Publishing Group, 2010) Lage, Kasper; Møllgård, Kjeld; Greenway, Steven; Workman, Christopher T; Bendsen, Eske; Hansen, Niclas T; Rigina, Olga; Roque, Francisco S; Wiese, Cornelia; Christoffels, Vincent M; Tommerup, Niels; Brunak, Søren; Larsen, Lars A; Wakimoto, Hiroko; Gorham, Joshua; Roberts, Amy; Smoot, Leslie; Pu, William; Donahoe, Patricia; Seidman, Christine; Seidman, JonathanAberrant organ development is associated with a wide spectrum of disorders, from schizophrenia to congenital heart disease, but systems-level insight into the underlying processes is very limited. Using heart morphogenesis as general model for dissecting the functional architecture of organ development, we combined detailed phenotype information from deleterious mutations in 255 genes with high-confidence experimental interactome data, and coupled the results to thorough experimental validation. Hereby, we made the first systematic analysis of spatio-temporal protein networks driving many stages of a developing organ identifying several novel signaling modules. Our results show that organ development relies on surprisingly few, extensively recycled, protein modules that integrate into complex higher-order networks. This design allows the formation of a complicated organ using simple building blocks, and suggests how mutations in the same genes can lead to diverse phenotypes. We observe a striking temporal correlation between organ complexity and the number of discrete functional modules coordinating morphogenesis. Our analysis elucidates the organization and composition of spatio-temporal protein networks that drive the formation of organs, which in the future may lay the foundation of novel approaches in treatments, diagnostics, and regenerative medicine.Publication Stable Gene Targeting in Human Cells Using Single-Strand Oligonucleotides with Modified Bases(Public Library of Science (PLoS), 2012-05-14) Rios, Xavier; Briggs, Adrian W.; Christodoulou, Danos; Gorham, Joshua; Seidman, Jonathan; Church, GeorgeRecent advances allow multiplexed genome engineering in E. coli, employing easily designed oligonucleotides to edit multiple loci simultaneously. A similar technology in human cells would greatly expedite functional genomics, both by enhancing our ability to test how individual variants such as single nucleotide polymorphisms (SNPs) are related to specific phenotypes, and potentially allowing simultaneous mutation of multiple loci. However, oligo-mediated targeting of human cells is currently limited by low targeting efficiencies and low survival of modified cells. Using a HeLa-based EGFP-rescue reporter system we show that use of modified base analogs can increase targeting efficiency, in part by avoiding the mismatch repair machinery. We investigate the effects of oligonucleotide toxicity and find a strong correlation between the number of phosphorothioate bonds and toxicity. Stably EGFP-corrected cells were generated at a frequency of ~0.05% with an optimized oligonucleotide design combining modified bases and reduced number of phosphorothioate bonds. We provide evidence from comparative RNA-seq analysis suggesting cellular immunity induced by the oligonucleotides might contribute to the low viability of oligo-corrected cells. Further optimization of this method should allow rapid and scalable genome engineering in human cells.Publication Small-Molecule Screen Identifies De Novo Nucleotide Synthesis as a Vulnerability of Cells Lacking SIRT3(2018) Gonzalez Herrera, Karina; Zaganjor, Elma; Ishikawa, Yoshinori; Spinelli, Jessica; Yoon, Haejin; Lin, Jia-Ren; Satterstrom, F. Kyle; Ringel, Alison; Mulei, Stacy; Souza, Amanda; Gorham, Joshua; Benson, Craig C.; Seidman, Jonathan; Sorger, Peter; Clish, Clary B.; Haigis, MarciaSummary Sirtuin 3 (SIRT3) is a NAD+-dependent deacetylase downregulated in aging and age-associated diseases such as cancer and neurodegeneration and in high-fat diet (HFD)-induced metabolic disorders. Here, we performed a small-molecule screen and identified an unexpected metabolic vulnerability associated with SIRT3 loss. Azaserine, a glutamine analog, was the top compound that inhibited growth and proliferation of cells lacking SIRT3. Using stable isotope tracing of glutamine, we observed its increased incorporation into de novo nucleotide synthesis in SIRT3 knockout (KO) cells. Furthermore, we found that SIRT3 KO cells upregulated the diversion of glutamine into de novo nucleotide synthesis through hyperactive mTORC1 signaling. Overexpression of SIRT3 suppressed mTORC1 and growth in vivo in a xenograft tumor model of breast cancer. Thus, we have uncovered a metabolic vulnerability of cells with SIRT3 loss by using an unbiased small-molecule screen.