166 results on '"Wieben, ED"'
Search Results
2. Abstract P3-07-51: Regulation of DNA methyltransferases via TRAF6 determines breast cancer response to decitabine
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Yu, J, primary, Qin, B, additional, Boughey, JC, additional, Moyer, AM, additional, Visscher, DW, additional, Sinnwell, JP, additional, Yin, P, additional, Thompson, KJ, additional, Docter, TJ, additional, Kalari, KR, additional, Suman, VJ, additional, Wieben, ED, additional, Felten, SJ, additional, Conners, AL, additional, Jones, KN, additional, McLaughlin, SA, additional, Copland JA, III, additional, Moreno Aspitia, A, additional, Northfelt, DW, additional, Gray, RJ, additional, Ingle, JN, additional, Lou, Z, additional, Weinshilboum, R, additional, Goetz, MP, additional, and Wang, L, additional
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- 2016
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3. Abstract P3-07-29: Role of germline BRCA status and tumor homologous recombination (HR) deficiency in response to neoadjuvant weekly paclitaxel followed by anthracycline-based chemotherapy
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Boughey, JC, primary, Kalari, KR, additional, Suman, VJ, additional, McLaughlin, SA, additional, Moreno Aspitia, A, additional, Moyer, AM, additional, Northfelt, DW, additional, Gray, RJ, additional, Vedell, PT, additional, Tang, X, additional, Dockter, TJ, additional, Jones, KN, additional, Felten, SJ, additional, Conners, AL, additional, Hart, SN, additional, Visscher, DW, additional, Wieben, ED, additional, Ingle, JN, additional, Hartman, A-R, additional, Timms, K, additional, Elkin, E, additional, Jones, J, additional, Wang, L, additional, Weinshilboum, RW, additional, and Goetz, MP, additional
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- 2016
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4. A comprehensive assessment of RNA-seq accuracy, reproducibility and information content by the Sequencing Quality Control Consortium
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Su, Z, Labaj, PP, Li, S, Thierry-Mieg, J, Thierry-Mieg, D, Shi, W, Wang, C, Schroth, GP, Setterquist, RA, Thompson, JF, Jones, WD, Xiao, W, Xu, W, Jensen, RV, Kelly, R, Xu, J, Conesa, A, Furlanello, C, Gao, H, Hong, H, Jafari, N, Letovsky, S, Liao, Y, Lu, F, Oakeley, EJ, Peng, Z, Praul, CA, Santoyo-Lopez, J, Scherer, A, Shi, T, Smyth, GK, Staedtler, F, Sykacek, P, Tan, X-X, Thompson, EA, Vandesompele, J, Wang, MD, Wang, J, Wolfinger, RD, Zavadil, J, Auerbach, SS, Bao, W, Binder, H, Blomquist, T, Brilliant, MH, Bushel, PR, Cain, W, Catalano, JG, Chang, C-W, Chen, T, Chen, G, Chen, R, Chierici, M, Chu, T-M, Clevert, D-A, Deng, Y, Derti, A, Devanarayan, V, Dong, Z, Dopazo, J, Du, T, Fang, H, Fang, Y, Fasold, M, Fernandez, A, Fischer, M, Furio-Tari, P, Fuscoe, JC, Caiment, F, Gaj, S, Gandara, J, Ge, W, Gondo, Y, Gong, B, Gong, M, Gong, Z, Green, B, Guo, C, Guo, L, Guo, L-W, Hadfield, J, Hellemans, J, Hochreiter, S, Jia, M, Jian, M, Johnson, CD, Kay, S, Kleinjans, J, Lababidi, S, Levy, S, Li, Q-Z, Li, L, Li, P, Li, Y, Li, H, Li, J, Lin, SM, Lopez, FJ, Lu, X, Luo, H, Ma, X, Meehan, J, Megherbi, DB, Mei, N, Mu, B, Ning, B, Pandey, A, Perez-Florido, J, Perkins, RG, Peters, R, Phan, JH, Pirooznia, M, Qian, F, Qing, T, Rainbow, L, Rocca-Serra, P, Sambourg, L, Sansone, S-A, Schwartz, S, Shah, R, Shen, J, Smith, TM, Stegle, O, Stralis-Pavese, N, Stupka, E, Suzuki, Y, Szkotnicki, LT, Tinning, M, Tu, B, van Deft, J, Vela-Boza, A, Venturini, E, Walker, SJ, Wan, L, Wang, W, Wieben, ED, Willey, JC, Wu, P-Y, Xuan, J, Yang, Y, Ye, Z, Yin, Y, Yu, Y, Yuan, Y-C, Zhang, J, Zhang, KK, Zhang, W, Zhang, Y, Zhao, C, Zheng, Y, Zhou, Y, Zumbo, P, Tong, W, Kreil, DP, Mason, CE, Shi, L, Su, Z, Labaj, PP, Li, S, Thierry-Mieg, J, Thierry-Mieg, D, Shi, W, Wang, C, Schroth, GP, Setterquist, RA, Thompson, JF, Jones, WD, Xiao, W, Xu, W, Jensen, RV, Kelly, R, Xu, J, Conesa, A, Furlanello, C, Gao, H, Hong, H, Jafari, N, Letovsky, S, Liao, Y, Lu, F, Oakeley, EJ, Peng, Z, Praul, CA, Santoyo-Lopez, J, Scherer, A, Shi, T, Smyth, GK, Staedtler, F, Sykacek, P, Tan, X-X, Thompson, EA, Vandesompele, J, Wang, MD, Wang, J, Wolfinger, RD, Zavadil, J, Auerbach, SS, Bao, W, Binder, H, Blomquist, T, Brilliant, MH, Bushel, PR, Cain, W, Catalano, JG, Chang, C-W, Chen, T, Chen, G, Chen, R, Chierici, M, Chu, T-M, Clevert, D-A, Deng, Y, Derti, A, Devanarayan, V, Dong, Z, Dopazo, J, Du, T, Fang, H, Fang, Y, Fasold, M, Fernandez, A, Fischer, M, Furio-Tari, P, Fuscoe, JC, Caiment, F, Gaj, S, Gandara, J, Ge, W, Gondo, Y, Gong, B, Gong, M, Gong, Z, Green, B, Guo, C, Guo, L, Guo, L-W, Hadfield, J, Hellemans, J, Hochreiter, S, Jia, M, Jian, M, Johnson, CD, Kay, S, Kleinjans, J, Lababidi, S, Levy, S, Li, Q-Z, Li, L, Li, P, Li, Y, Li, H, Li, J, Lin, SM, Lopez, FJ, Lu, X, Luo, H, Ma, X, Meehan, J, Megherbi, DB, Mei, N, Mu, B, Ning, B, Pandey, A, Perez-Florido, J, Perkins, RG, Peters, R, Phan, JH, Pirooznia, M, Qian, F, Qing, T, Rainbow, L, Rocca-Serra, P, Sambourg, L, Sansone, S-A, Schwartz, S, Shah, R, Shen, J, Smith, TM, Stegle, O, Stralis-Pavese, N, Stupka, E, Suzuki, Y, Szkotnicki, LT, Tinning, M, Tu, B, van Deft, J, Vela-Boza, A, Venturini, E, Walker, SJ, Wan, L, Wang, W, Wieben, ED, Willey, JC, Wu, P-Y, Xuan, J, Yang, Y, Ye, Z, Yin, Y, Yu, Y, Yuan, Y-C, Zhang, J, Zhang, KK, Zhang, W, Zhang, Y, Zhao, C, Zheng, Y, Zhou, Y, Zumbo, P, Tong, W, Kreil, DP, Mason, CE, and Shi, L
- Abstract
We present primary results from the Sequencing Quality Control (SEQC) project, coordinated by the US Food and Drug Administration. Examining Illumina HiSeq, Life Technologies SOLiD and Roche 454 platforms at multiple laboratory sites using reference RNA samples with built-in controls, we assess RNA sequencing (RNA-seq) performance for junction discovery and differential expression profiling and compare it to microarray and quantitative PCR (qPCR) data using complementary metrics. At all sequencing depths, we discover unannotated exon-exon junctions, with >80% validated by qPCR. We find that measurements of relative expression are accurate and reproducible across sites and platforms if specific filters are used. In contrast, RNA-seq and microarrays do not provide accurate absolute measurements, and gene-specific biases are observed for all examined platforms, including qPCR. Measurement performance depends on the platform and data analysis pipeline, and variation is large for transcript-level profiling. The complete SEQC data sets, comprising >100 billion reads (10Tb), provide unique resources for evaluating RNA-seq analyses for clinical and regulatory settings.
- Published
- 2014
5. Abstract P1-08-10: Integration of next generation sequencing (NGS) and patient derived xenografts (PDX) to identify novel markers of paclitaxel (T) response in the breast cancer genome guided therapy study (BEAUTY)
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Goetz, MP, primary, Boughey, JC, additional, Kalari, KR, additional, Eckel-Passow, J, additional, Suman, VJ, additional, Sicotte, H, additional, Hart, SN, additional, Moyer, AM, additional, Visscher, DW, additional, Yu, J, additional, Gao, B, additional, Sinnwell, JP, additional, Mahoney, DW, additional, Barman, P, additional, Vedell, P, additional, Tang, X, additional, Thompson, K, additional, Dockter, TJ, additional, Jones, KN, additional, Conners, AL, additional, McLaughlin, SA, additional, Moreno-Aspitia, A, additional, Northfelt, DW, additional, Gray, RJ, additional, Wieben, ED, additional, Farrugia, G, additional, Schultz, C, additional, Ingle, JN, additional, Wang, L, additional, and Weinshilboum, RW, additional
- Published
- 2013
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6. Pharmacogenetics of the mycophenolic acid targets inosine monophosphate dehydrogenases IMPDH1 and IMPDH2: gene sequence variation and functional genomics
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Wu, T-Y, primary, Peng, Y, additional, Pelleymounter, LL, additional, Moon, I, additional, Eckloff, BW, additional, Wieben, ED, additional, Yee, VC, additional, and Weinshilboum, RM, additional
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- 2010
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7. Alpha1-antitrypsin and neutrophil elastase imbalance and lung cancer risk.
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Yang P, Bamlet WR, Sun Z, Ebbert JO, Aubry M, Taylor WR, Marks RS, Deschamps C, Swensen SJ, Wieben ED, Cunningham JM, Melton LJ, and de Andrade M
- Abstract
OBJECTIVE: Imbalance between alpha(1)-antitrypsin and neutrophil elastase is an underlying cause of lung tissue damage that may create a favorable host environment for carcinogenesis. We conducted a case-control study to investigate whether genetic variations indicative of alpha(1)-antitrypsin deficiency (A1ATD) or an excess of neutrophil elastase modify lung cancer risk DESIGN: The case patients were 305 consecutively identified primary lung cancer patients, and the control subjects were 338 community residents. Protease inhibitor-1 (PI1), encoding alpha(1)-antitrypsin, was typed by an isoelectric focusing assay. Neutrophil elastase-2 (ELA2), encoding neutrophil elastase, was typed by two single-nucleotide polymorphism sites. Multivariable logistic regression models tested the independent and interactive effects of PI1, ELA2, tobacco smoke exposure, COPD, and family history of lung cancer RESULTS: Sex and ethnicity were comparable between case patients and control subjects, but case patients were more likely to be smokers, and to have a history of COPD, environmental tobacco smoke exposure, and a positive family history of lung cancer. Haplotype analysis indicated an overall strong association between the two ELA2 markers and lung cancer risk. Our best-fitting model showed significant and independent effects of the PI1-deficient allele (odds ratio [OR], 2.0; 95% confidence interval [CI], 1.4 to 3.0) and the ELA2 T-G haplotype (OR, 4.1; 95% CI, 1.9 to 8.9) on lung cancer risk, and an increased risk (OR, 2.6; 95% CI, 2.4 to 2.8) for individuals carrying both a PI1-deficient allele and a G-G haplotype CONCLUSIONS: Genotypes indicative of A1ATD and/or an excess of neutrophil elastase are significantly associated with lung cancer risk. Our findings may provide opportunities to better understand the mechanisms of lung cancer development and risk reduction. [ABSTRACT FROM AUTHOR]
- Published
- 2005
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8. Human cytosolic sulfotransferase database mining: identification of seven novel genes and pseudogenes.
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Freimuth, RR, Wiepert, M, Chute, CG, Wieben, ED, and Weinshilboum, RM
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GENES ,SOMATOMEDIN ,DATABASES ,DATA mining - Abstract
A total of 10 SULT genes are presently known to be expressed in human tissues. We performed a comprehensive genome-wide search for novel SULT genes using two different but complementary approaches, and developed a novel graphical display to aid in the annotation of the hits. Seven novel human SULT genes were identified, five of which were predicted to be pseudogenes, including two processed pseudogenes and three pseudogenes that contained introns. Those five pseudogenes represent the first unambiguous SULT pseudogenes described in any species. Expression-profiling studies were conducted for one novel gene, SULT6B1, and a series of alternatively spliced transcripts were identified in the human testis. SULT6B1 was also present in chimpanzee and gorilla, differing at only seven encoded amino-acid residues among the three species. The results of these database mining studies will aid in studies of the regulation of these SULT genes, provide insights into the evolution of this gene family in humans, and serve as a starting point for comparative genomic studies of SULT genes.The Pharmacogenomics Journal (2004) 4, 54-65. doi:10.1038/sj.tpj.6500223 Published online 16 December 2003 [ABSTRACT FROM AUTHOR]
- Published
- 2004
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9. Primer on medical genomics. Part VII: The evolving concept of the gene.
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Wieben ED and Wieben, Eric D
- Abstract
The draft sequence of the human genome was reported 2 years ago, and the task of filling gaps and polishing the sequence is nearing completion. However, despite this remarkable achievement, there is still no definitive assessment of the number of genes contained in the human genome. In part, this uncertainty reflects our growing understanding of the complexity and diversity of gene structure. Examples of complex gene structure are considered in the context of a discussion about the evolution of our understanding of gene structure and function. [ABSTRACT FROM AUTHOR]
- Published
- 2003
10. Single-cell multiomics reveal divergent effects of DNMT3A- and TET2-mutant clonal hematopoiesis in inflammatory response.
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Mohammed Ismail W, Fernandez JA, Binder M, Lasho TL, Kim M, Geyer SM, Mazzone A, Finke CM, Mangaonkar AA, Lee JH, Wang L, Kim KH, Simon VA, Rakhshan Rohakthar F, Munankarmy A, Byeon SK, Schwager SM, Harrington JJ, Snyder MR, Robertson KD, Pandey A, Wieben ED, Chia N, Gaspar-Maia A, and Patnaik MM
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- Humans, Inflammation genetics, SARS-CoV-2, Respiratory Distress Syndrome genetics, Male, Female, Multiomics, DNA Methyltransferase 3A, COVID-19 genetics, DNA (Cytosine-5-)-Methyltransferases genetics, DNA (Cytosine-5-)-Methyltransferases metabolism, Single-Cell Analysis, Dioxygenases, Mutation, Clonal Hematopoiesis genetics, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins metabolism
- Abstract
Abstract: DNMT3A and TET2 are epigenetic regulator genes commonly mutated in age-related clonal hematopoiesis (CH). Despite having opposed epigenetic functions, these mutations are associated with increased all-cause mortality and a low risk for progression to hematologic neoplasms. Although individual impacts on the epigenome have been described using different model systems, the phenotypic complexity in humans remains to be elucidated. Here, we make use of a natural inflammatory response occurring during coronavirus disease 2019 (COVID-19), to understand the association of these mutations with inflammatory morbidity (acute respiratory distress syndrome [ARDS]) and mortality. We demonstrate the age-independent, negative impact of DNMT3A mutant (DNMT3Amt) CH on COVID-19-related ARDS and mortality. Using single-cell proteogenomics we show that DNMT3A mutations involve myeloid and lymphoid lineage cells. Using single-cell multiomics sequencing, we identify cell-specific gene expression changes associated with DNMT3A mutations, along with significant epigenomic deregulation affecting enhancer accessibility, resulting in overexpression of interleukin-32 (IL-32), a proinflammatory cytokine that can result in inflammasome activation in monocytes and macrophages. Finally, we show with single-cell resolution that the loss of function of DNMT3A is directly associated with increased chromatin accessibility in mutant cells. Hence, we demonstrate the negative prognostic impact of DNMT3Amt CH on COVID-19-related ARDS and mortality. DNMT3Amt CH in the context of COVID-19, was associated with inflammatory transcriptional priming, resulting in overexpression of IL32. This overexpression was secondary to increased chromatic accessibility, specific to DNMT3Amt CH cells. DNMT3Amt CH can thus serve as a potential biomarker for adverse outcomes in COVID-19., (© 2025 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)
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- 2025
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11. Genomic epidemiology reveals the dominance of Hennepin County in the transmission of SARS-CoV-2 in Minnesota from 2020 to 2022.
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Scotch M, Lauer K, Wieben ED, Cherukuri Y, Cunningham JM, Klee EW, Harrington JJ, Lau JS, McDonough SJ, Mutawe M, O'Horo JC, Rentmeester CE, Schlicher NR, White VT, Schneider SK, Vedell PT, Wang X, Yao JD, Pritt BS, and Norgan AP
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- Humans, Minnesota epidemiology, Pandemics, Communicable Disease Control, Genomics, SARS-CoV-2 genetics, COVID-19 epidemiology
- Abstract
Importance: We analyzed over 22,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes of patient samples tested at Mayo Clinic Laboratories during a 2-year period in the COVID-19 pandemic, which included Alpha, Delta, and Omicron variants of concern to examine the roles and relationships of Minnesota virus transmission. We found that Hennepin County, the most populous county, drove the transmission of SARS-CoV-2 viruses in the state after including the formation of earlier clades including 20A, 20C, and 20G, as well as variants of concern Alpha and Delta. We also found that Hennepin County was the source for most of the county-to-county introductions after an initial predicted introduction with the virus in early 2020 from an international source, while other counties acted as transmission "sinks." In addition, major policies, such as the end of the lockdown period in 2020 or the end of all restrictions in 2021, did not appear to have an impact on virus diversity across individual counties., Competing Interests: The authors declare no conflict of interest.
- Published
- 2023
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12. εγ-Thalassemia, a New Hemoglobinopathy Category.
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Oliveira JL, Thompson CH, Saravanaperumal SA, Koganti T, Jenkinson G, Hein MS, Kohorst MA, Hasadsri L, Nguyen PL, Matern D, Kipp BR, Klee EW, Wieben ED, Hoyer JD, and Rangan A
- Subjects
- Humans, Female, Fetal Hemoglobin genetics, Multiplex Polymerase Chain Reaction, Thalassemia genetics, Hemoglobinopathies, beta-Thalassemia diagnosis, beta-Thalassemia genetics
- Abstract
Background: Large β-globin gene cluster deletions (hereditary persistence of fetal hemoglobin [Hb] or β-, δβ-, γδβ-, and ϵγδβ-thalassemia), are associated with widely disparate phenotypes, including variable degrees of microcytic anemia and Hb F levels. When present, increased Hb A2 is used as a surrogate marker for β-thalassemia. Notably, ϵγδβ-thalassemias lack the essential regulatory locus control region (LCR) and cause severe transient perinatal anemia but normal newborn screen (NBS) results and Hb A2 levels. Herein, we report a novel deletion of the ϵ, Aγ, Gγ, and ψβ loci with intact LCR, δ-, and β-regions in 2 women and newborn twins., Methods: Capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), DNA sequencing, multiplex ligation-dependent probe amplification (MLPA), gap-polymerase chain reaction (gap-PCR), and long-read sequencing (LRS) were performed., Results: NBS showed an Hb A > Hb F pattern for both twins. At 20 months, Hb A2 was increased similarly to that in the mother and an unrelated woman. Unexplained microcytosis was absent and the twins lacked severe neonatal anemia. MLPA, LRS, and gap-PCR confirmed a 32 599 base pair deletion of ϵ (HBE1) through ψβ (HBBP1) loci., Conclusions: This deletion represents a hemoglobinopathy category with a distinct phenotype that has not been previously described, an ϵγ-thalassemia. Both the NBS Hb A > F pattern and the subsequent increased Hb A2 without microcytosis are unusual. A similar deletion should be considered when this pattern is encountered and appropriate test methods selected for detection. Knowledge of the clinical impact of this new category will improve genetic counselling, with distinction from the severe transient anemia associated with ϵγδβ-thalassemia., (© American Association for Clinical Chemistry 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
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- 2023
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13. Genomic epidemiology reveals the dominance of Hennepin County in transmission of SARS-CoV-2 in Minnesota from 2020-2022.
- Author
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Scotch M, Lauer K, Wieben ED, Cherukuri Y, Cunningham JM, Klee EW, Harrington JJ, Lau JS, McDonough SJ, Mutawe M, O'Horo JC, Rentmeester CE, Schlicher NR, White VT, Schneider SK, Vedell PT, Wang X, Yao JD, Pritt BS, and Norgan AP
- Abstract
SARS-CoV-2 has had an unprecedented impact on human health and highlights the need for genomic epidemiology studies to increase our understanding of virus evolution and spread, and to inform policy decisions. We sequenced viral genomes from over 22,000 patient samples tested at Mayo Clinic Laboratories between 2020-2022 and use Bayesian phylodynamics to describe county and regional spread in Minnesota. The earliest introduction into Minnesota was to Hennepin County from a domestic source around January 22, 2020; six weeks before the first confirmed case in the state. This led to the virus spreading to Northern Minnesota, and eventually, the rest of the state. International introductions were most abundant in Hennepin (home to the Minneapolis/St. Paul International (MSP) airport) totaling 45 (out of 107) over the two-year period. Southern Minnesota counties were most common for domestic introductions with 19 (out of 64), potentially driven by bordering states such as Iowa and Wisconsin as well as Illinois which is nearby. Hennepin also was, by far, the most dominant source of in-state transmissions to other Minnesota locations (n=772) over the two-year period. We also analyzed the diversity of the location source of SARS-CoV-2 viruses in each county and noted the timing of state-wide policies as well as trends in clinical cases. Neither the number of clinical cases or major policy decisions, such as the end of the lockdown period in 2020 or the end of all restrictions in 2021, appeared to have impact on virus diversity across each individual county., Competing Interests: Competing interests The authors declare no competing interests.
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- 2023
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14. Comparison of TCF4 repeat expansion length in corneal endothelium and leukocytes of patients with Fuchs endothelial corneal dystrophy.
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Wieben ED, Aleff RA, Rinkoski TA, Baratz KH, Basu S, Patel SV, Maguire LJ, and Fautsch MP
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- Aged, Aged, 80 and over, Blotting, Southern, Child, DNA analysis, Endothelium, Corneal metabolism, Female, Fuchs' Endothelial Dystrophy epidemiology, Fuchs' Endothelial Dystrophy genetics, Genetic Predisposition to Disease, Genotype, Humans, Leukocytes metabolism, Male, Middle Aged, Polymerase Chain Reaction, DNA genetics, Endothelium, Corneal pathology, Fuchs' Endothelial Dystrophy pathology, Leukocytes pathology, Transcription Factor 4 genetics, Trinucleotide Repeat Expansion
- Abstract
Expansion of CTG trinucleotide repeats (TNR) in the transcription factor 4 (TCF4) gene is highly associated with Fuchs Endothelial Corneal Dystrophy (FECD). Due to limitations in the availability of DNA from diseased corneal endothelium, sizing of CTG repeats in FECD patients has typically been determined using DNA samples isolated from peripheral blood leukocytes. However, it is non-feasible to extract enough DNA from surgically isolated FECD corneal endothelial tissue to determine repeat length based on current technology. To circumvent this issue, total RNA was isolated from FECD corneal endothelium and sequenced using long-read sequencing. Southern blotting of DNA samples isolated from primary cultures of corneal endothelium from these same affected individuals was also assessed. Both long read sequencing and Southern blot analysis showed significantly longer CTG TNR expansion (>1000 repeats) in the corneal endothelium from FECD patients than those characterized in leukocytes from the same individuals (<90 repeats). Our findings suggest that the TCF4 CTG repeat expansions in the FECD corneal endothelium are much longer than those found in leukocytes., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Michael P. Fautsch, PhD is a consultant for Qlaris Bio, Inc; Sanjay V. Patel, MD is a consultant for Senju Pharmaceutical Co., Ltd, GlaxoSmithKline PLC, Santen Pharmaceutical Co., Ltd, and Emmecell. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
- Published
- 2021
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15. Relationship of Body Mass Index With Fuchs Endothelial Corneal Dystrophy Severity and TCF4 CTG18.1 Trinucleotide Repeat Expansion.
- Author
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Kinariwala BB, Xu TT, Baratz KH, Aleff RA, Patel SV, Maguire LJ, Fautsch MP, Wieben ED, Millen AE, and Patel SP
- Subjects
- Aged, Alleles, Body Mass Index, Female, Follow-Up Studies, Fuchs' Endothelial Dystrophy diagnosis, Fuchs' Endothelial Dystrophy metabolism, Genotype, Humans, Male, Patient Acuity, Retrospective Studies, Slit Lamp Microscopy, Transcription Factor 4 metabolism, Trinucleotide Repeat Expansion, DNA genetics, Endothelium, Corneal pathology, Fuchs' Endothelial Dystrophy genetics, Genetic Predisposition to Disease, Transcription Factor 4 genetics
- Abstract
Purpose: To investigate the association of body mass index (BMI) with Fuchs endothelial corneal dystrophy (FECD) severity and TCF4 CTG18.1 expansion., Methods: A total of 343 patients with FECD were enrolled from the Mayo Clinic. FECD severity was graded by slit-lamp biomicroscopy. BMI values were obtained from the electronic medical records. DNA extracted from leukocytes was analyzed for CTG18.1 expansion length, with ≥40 repeats considered expanded. Wilcoxon signed-rank tests were used to compare FECD grade and CTG18.1 expansion length in patients by BMI (<25, ≥25 to <30, and ≥30 kg/m2). FECD grade was regressed on age, sex, BMI, and CTG18.1 expansion and, separately, BMI on CTG18.1 expansion. Models were investigated for effect modification by age and sex with an interaction term of P < 0.05 considered statistically significant., Results: When examining the association between BMI and FECD, there was a significant interaction between BMI and sex (P for interaction = 0.004). When controlling for age and CTG18.1 expansion, a positive association was observed between BMI and FECD grade in women, but not in men. In addition, BMI was not associated with CTG18.1 expansion when controlling for age and sex., Conclusions: BMI was positively associated with FECD severity among women but not men. There was no significant association between BMI and CTG18.1 expansion. These findings suggest that increased BMI is potentially a modifiable risk factor for FECD disease progression among women., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)
- Published
- 2021
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16. Improved Characterization of Complex β-Globin Gene Cluster Structural Variants Using Long-Read Sequencing.
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Rangan A, Hein MS, Jenkinson WG, Koganti T, Aleff RA, Hilker CA, Blommel JH, Porter TR, Swanson KC, Lundquist P, Nguyen PL, Shi M, He R, Viswanatha DS, Jen J, Klee EW, Kipp BR, Hoyer JD, Wieben ED, and Oliveira JL
- Subjects
- Anemia, Sickle Cell genetics, Female, Gene Duplication, Heterozygote, Humans, India, Infant, Infant, Newborn, Male, Middle Aged, Multigene Family, beta-Globins analysis, Sequence Analysis, DNA methods, Thalassemia genetics, beta-Globins genetics
- Abstract
Complex insertion-deletion (indel) events in the globin genes manifest in widely variable clinical phenotypes. Many are incompletely characterized because of a historic lack of efficient methods. A more complete assessment enables improved prediction of clinical impact, which guides emerging therapeutic choices. Current methods have limited capacity for breakpoint assignment and accurate assessment of mutation extent, especially in cases containing duplications or multiple deletions and insertions. Technology, such as long-read sequencing, holds promise for significant impact in the characterization of indel events because of read lengths that span large regions, resulting in improved resolution. Four known complex β-globin gene cluster indel types were assessed using single-molecule, real-time sequencing technology and showed high correlation with previous reports, including the Caribbean locus control deletion (g.5,305,478_5,310,336del), a large β-gene duplication containing the Hb S mutation (g.4,640,335_5,290,171dup with g.5,248,232T>A, c.20A>T; variant allele fraction, 64%), and two nested variants (double deletions with intervening inversion): the Indian
G γ(A γδβ)0 -thalassemia (g.5,246,804-5,254,275del, g.5,254,276_5,269,600inv, and g.5,269,601_5,270,442del) and the Turkish/Macedonian (δβ)0 thalassemia (g.5,235,064_5,236,652del, g.5,236,653_5,244,280inv, and g.5,244,281_5,255,766del). Our data confirm long-read sequencing as an efficient and accurate method to identify these clinically significant complex events. Limitations include high-complexity sample preparation requirements, which hinder routine use in clinical laboratories. Continued improvements in sample and data workflow processes are needed to accommodate volumes in a tertiary clinical laboratory., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)- Published
- 2021
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17. Characterization of a dual media system for culturing primary normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells.
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Rinkoski TA, Bahler CK, Pacheco JM, Khanna ML, Holmes DM, Roy Chowdhury U, Baratz KH, Patel SV, Maguire LJ, Wieben ED, and Fautsch MP
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- Cell Proliferation, Culture Media, Endothelium, Corneal, Humans, Cell Culture Techniques, Endothelial Cells pathology, Fuchs' Endothelial Dystrophy pathology
- Abstract
Primary cultures of human corneal endothelial cells (HCECs) are an important model system for studying the pathophysiology of corneal endothelium. The purpose of this study was to identify and validate an optimal primary culture model of normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells by comparing cell morphology and marker expression under different media conditions to in vivo donor tissues. Primary and immortalized HCECs, isolated from normal and FECD donors, were cultured in proliferation media (Joyce, M4, Bartakova) alone or sequentially with maturation media (F99, Stabilization 1, M5). CD56, CD73 and CD166 expressions were quantified in confluent and matured cell lines by flow cytometry. HCECs that were allowed to proliferate in Joyce's medium followed by maturation in low-mitogen containing media yielded cells with similar morphology to corneal endothelial tissues. Elevated expression of CD56 and CD166 and low expression of CD73 correlated with regular, hexagonal-like HCEC morphology. CD56:CD73 > 2.5 was most consistent with normal HCEC morphology and mimicked corneal endothelial tissue. Immortalization of normal HCECs by hTERT transduction showed morphology and CD56:CD73 ratios similar to parental cell lines. HCECs established from FECD donors showed reduced CD56:CD73 ratios compared to normal HCECs which coincided with reduced uniformity and regularity of cell monolayers. Overall, a dual media system with Joyce's medium for proliferation and a low-mitogen media for maturation, provided normal cultures with regular, hexagonal-like cell morphologies consistent with corneal endothelial cells in vivo. CD56:CD73 expression ratio >2.5 was predictive of in vivo-like cellular morphology., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Sanjay V. Patel is a consultant for Senju Pharmaceutical Co., Ltd., GlaxoKlineSmith plc, Santen Pharmaceutical Co., Ltd., and Emmecell. Michael P. Fautsch is a consultant for Qlaris Bio. Inc.
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- 2021
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18. Long-read targeted sequencing uncovers clinicopathological associations for C9orf72-linked diseases.
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DeJesus-Hernandez M, Aleff RA, Jackson JL, Finch NA, Baker MC, Gendron TF, Murray ME, McLaughlin IJ, Harting JR, Graff-Radford NR, Oskarsson B, Knopman DS, Josephs KA, Boeve BF, Petersen RC, Fryer JD, Petrucelli L, Dickson DW, Rademakers R, Ebbert MTW, Wieben ED, and van Blitterswijk M
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- Aged, Cerebellum metabolism, Cross-Sectional Studies, DNA Repeat Expansion genetics, Female, Humans, Male, Middle Aged, C9orf72 Protein genetics, Neurodegenerative Diseases genetics, Sequence Analysis, DNA methods
- Abstract
To examine the length of a hexanucleotide expansion in C9orf72, which represents the most frequent genetic cause of frontotemporal lobar degeneration and motor neuron disease, we employed a targeted amplification-free long-read sequencing technology: No-Amp sequencing. In our cross-sectional study, we assessed cerebellar tissue from 28 well-characterized C9orf72 expansion carriers. We obtained 3507 on-target circular consensus sequencing reads, of which 814 bridged the C9orf72 repeat expansion (23%). Importantly, we observed a significant correlation between expansion sizes obtained using No-Amp sequencing and Southern blotting (P = 5.0 × 10-4). Interestingly, we also detected a significant survival advantage for individuals with smaller expansions (P = 0.004). Additionally, we uncovered that smaller expansions were significantly associated with higher levels of C9orf72 transcripts containing intron 1b (P = 0.003), poly(GP) proteins (P = 1.3 × 10- 5), and poly(GA) proteins (P = 0.005). Thorough examination of the composition of the expansion revealed that its GC content was extremely high (median: 100%) and that it was mainly composed of GGGGCC repeats (median: 96%), suggesting that expanded C9orf72 repeats are quite pure. Taken together, our findings demonstrate that No-Amp sequencing is a powerful tool that enables the discovery of relevant clinicopathological associations, highlighting the important role played by the cerebellar size of the expanded repeat in C9orf72-linked diseases., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain.)
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- 2021
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19. TCF4-mediated Fuchs endothelial corneal dystrophy: Insights into a common trinucleotide repeat-associated disease.
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Fautsch MP, Wieben ED, Baratz KH, Bhattacharyya N, Sadan AN, Hafford-Tear NJ, Tuft SJ, and Davidson AE
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- Fuchs' Endothelial Dystrophy pathology, Gene Expression Regulation, Genetic Predisposition to Disease, Genotype, Humans, Polymorphism, Genetic, Fuchs' Endothelial Dystrophy genetics, Transcription Factor 4 genetics, Trinucleotide Repeat Expansion genetics
- Abstract
Fuchs endothelial corneal dystrophy (FECD) is a common cause for heritable visual loss in the elderly. Since the first description of an association between FECD and common polymorphisms situated within the transcription factor 4 (TCF4) gene, genetic and molecular studies have implicated an intronic CTG trinucleotide repeat (CTG18.1) expansion as a causal variant in the majority of FECD patients. To date, several non-mutually exclusive mechanisms have been proposed that drive and/or exacerbate the onset of disease. These mechanisms include (i) TCF4 dysregulation; (ii) toxic gain-of-function from TCF4 repeat-containing RNA; (iii) toxic gain-of-function from repeat-associated non-AUG dependent (RAN) translation; and (iv) somatic instability of CTG18.1. However, the relative contribution of these proposed mechanisms in disease pathogenesis is currently unknown. In this review, we summarise research implicating the repeat expansion in disease pathogenesis, define the phenotype-genotype correlations between FECD and CTG18.1 expansion, and provide an update on research tools that are available to study FECD as a trinucleotide repeat expansion disease. Furthermore, ongoing international research efforts to develop novel CTG18.1 expansion-mediated FECD therapeutics are highlighted and we provide a forward-thinking perspective on key unanswered questions that remain in the field., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)
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- 2021
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20. Identification of Possible Risk Variants of Familial Strabismus Using Exome Sequencing Analysis.
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An JY, Jung JH, Choi L, Wieben ED, and Mohney BG
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- Cadherins genetics, Case-Control Studies, Child, ERG1 Potassium Channel genetics, Epidermal Growth Factor genetics, Female, Humans, Male, Membrane Proteins genetics, Prospective Studies, Strabismus diagnosis, Genetic Predisposition to Disease, Genetic Testing methods, Strabismus genetics, Exome Sequencing
- Abstract
Purpose: To investigate candidate genes associated with familial strabismus and propose a theory of their interaction in familial strabismus associated with early neurodevelopment., Methods: Eighteen families, including 53 patients diagnosed with strabismus and 34 unaffected family members, were analyzed. All patients with strabismus and available unaffected family members were evaluated using whole exome sequencing. The primary outcome was to identify rare occurring variants among affected individuals and investigate the evidence of their genetic heterogeneity. These results were compared with exome sequencing analysis to build a comprehensive genetic profile of the study families., Results: We observed 60 variants from 58 genes in 53 patients diagnosed with strabismus. We prioritized the most credible risk variants, which showed clear segregation in family members affected by strabismus. As a result, we found risk variants in four genes ( FAT3, KCNH2 , CELSR1 , and TTYH1 ) in five families, suggesting their role in development of familial strabismus. In other families, there were several rare genetic variants in affected cases, but we did not find clear segregation pattern across family members., Conclusion: Genomic sequencing holds great promise in elucidating the genetic causes of strabismus; further research with larger cohorts or other related approaches are warranted.
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- 2021
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21. Disease Expression and Familial Transmission of Fuchs Endothelial Corneal Dystrophy With and Without CTG18.1 Expansion.
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Xu TT, Li YJ, Afshari NA, Aleff RA, Rinkoski TA, Patel SV, Maguire LJ, Edwards AO, Brown WL, Fautsch MP, Wieben ED, and Baratz KH
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- Adult, Aged, Aged, 80 and over, Blotting, Southern, DNA genetics, Female, Genetic Predisposition to Disease, Genotype, Humans, Inheritance Patterns, Male, Microsatellite Repeats, Middle Aged, Pedigree, Penetrance, Polymerase Chain Reaction, Polymorphism, Genetic, Prospective Studies, Young Adult, Fuchs' Endothelial Dystrophy genetics, Transcription Factor 4 genetics, Trinucleotide Repeat Expansion genetics
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Purpose: To characterize inheritance, penetrance, and trinucleotide repeat expansion stability in Fuchs endothelial corneal dystrophy (FECD)., Methods: One thousand unrelated and related subjects with and without FECD were prospectively recruited. CTG18.1 repeat length (CTG18.1L) was determined via short tandem repeat assay and Southern blotting of leukocyte DNA. Multivariable logistic regression and generalized estimating equation models were employed., Results: There were 546 unrelated FECD cases (67.6% female; 70 ± 10 years) and 235 controls (63.8% female; 73 ± 8 years; all ≥ 50 years). CTG18.1 expansion (CTG18.1exp+) was observed in 424 (77.7%) cases and 18 (7.7%) controls (P = 2.48 × 10-44). CTG18.1 expansion was associated with FECD severity (P = 5.62 × 10-7). The family arm of the study included 331 members from 112 FECD-affected families; 87 families were CTG18.1exp+. Autosomal dominant inheritance with variable expression of FECD was observed, regardless of expansion status. FECD penetrance of CTG18.1 expansion increased with age, ranging from 44.4% in the youngest (19-46 years) to 86.2% in the oldest (64-91 years) age quartiles. Among 62 parent-offspring transmissions of CTG18.1exp+, 48 (77.4%) had a change in CTG18.1L ≤ 10 repeats, and eight (12.9%) were ≥50 repeats, including five large expansions (∼1000-2000 repeats) that contracted. Among 44 offspring who did not inherit the CTG18.1exp+ allele, eight (18.2%) exhibited FECD., Conclusions: CTG18.1 expansion was highly associated with FECD but demonstrated incomplete penetrance. CTG18.1L instability occurred in a minority of parent-offspring transmissions, with large expansions exhibiting contraction. The observation of FECD without CTG18.1 expansion among family members in CTG18.1exp+ families highlights the complexity of the relationship between the FECD phenotype and CTG18.1 expansion.
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- 2021
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22. Transplant chimerism in porcine structural vascularized bone allotransplants.
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Houben RH, Aleff RA, Friedrich PF, Shin AY, Wieben ED, van Wijnen AJ, and Bishop AT
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- Animals, DNA genetics, Female, Liver metabolism, Male, Osteogenesis, RNA genetics, RNA metabolism, Spleen metabolism, Swine, Transplantation, Homologous, Bone Transplantation, Chimerism, Neovascularization, Physiologic
- Abstract
Background: Bone allotransplant viability can be maintained long-term by implanting arteriovenous (AV) bundles and creating an autogenous neoangiogenic circulation. Only short-term immunosuppression is required. This study investigates the origin of viable osteocytes observed in areas of active bone remodeling in orthotopically transplanted tibiae in a Yucatan mini-pig model., Methods: Segmental tibial defects created in female Yucatan minipigs (N = 14) were reconstructed with a matched vascularized composite allotransplant from a male donor. The circulation was microsurgically restored, with simultaneous autogenous AV-bundle implantation in group 1 (N = 7). A ligated AV-bundle was implanted as a no-angiogenesis control in group 2 (N = 7). After 20-weeks, repopulation of the allotransplant was assessed by real-time qPCR measurement of relative copy numbers of a Y chromosome-specific gene (SRY) and an autosomal housekeeping gene, ribosomal protein L4 (RPL4). A lower SRY/RPL4 ratio demonstrates replacement of male allogeneic cells with female, autogenous cells in the sample. Genomic DNA was extracted from cross-sections of the allotransplant, liver and spleen. Additionally, areas of new bone formation within the allotransplant were sampled by laser capture microdissection. A comparison was made between groups as well as male control samples. RNA was extracted from bone as well, as a measure of metabolically active cells., Results: Laser-captured areas of new bone formation in animals with both normal and ligated AV-bundles were found to have significantly lower relative copy numbers of SRY (p = 0.03) than control specimens from male bone, indicating replacement by female (autogenous) bone-forming cells. Analysis of an entire segment of the allotransplant from Group 1 was similarly reduced (p = 0.04), unlike that from Group 2. RNA expression of SRY was observed in both groups. No chimerism could be found in non-bone tissues (liver and spleen)., Conclusion: We observed a significant level of transplant chimerism in areas of new bone formation sampled by laser capture microdissection. The migration of autogenous cells including osteocytes was seen in both groups. Survival of some allogeneic (male) cells was also demonstrable. No microchimerism was found in liver and spleen., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)
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- 2020
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23. Correction to: Recommendations for performance optimizations when using GATK3.8 and GATK4.
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Heldenbrand JR, Baheti S, Bockol MA, Drucker TM, Hart SN, Hudson ME, Iyer RK, Kalmbach MT, Kendig KI, Klee EW, Mattson NR, Wieben ED, Wiepert M, Wildman DE, and Mainzer LS
- Abstract
Following publication of the original article [1], the author explained that Table 2 is displayed incorrectly. The correct Table 2 is given below. The original article has been corrected.
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- 2019
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24. Recommendations for performance optimizations when using GATK3.8 and GATK4.
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Heldenbrand JR, Baheti S, Bockol MA, Drucker TM, Hart SN, Hudson ME, Iyer RK, Kalmbach MT, Kendig KI, Klee EW, Mattson NR, Wieben ED, Wiepert M, Wildman DE, and Mainzer LS
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- Algorithms, Chromosomes, Human genetics, Genome, Human, Haplotypes genetics, High-Throughput Nucleotide Sequencing, Humans, Genomics methods, Software
- Abstract
Background: Use of the Genome Analysis Toolkit (GATK) continues to be the standard practice in genomic variant calling in both research and the clinic. Recently the toolkit has been rapidly evolving. Significant computational performance improvements have been introduced in GATK3.8 through collaboration with Intel in 2017. The first release of GATK4 in early 2018 revealed rewrites in the code base, as the stepping stone toward a Spark implementation. As the software continues to be a moving target for optimal deployment in highly productive environments, we present a detailed analysis of these improvements, to help the community stay abreast with changes in performance., Results: We re-evaluated multiple options, such as threading, parallel garbage collection, I/O options and data-level parallelization. Additionally, we considered the trade-offs of using GATK3.8 and GATK4. We found optimized parameter values that reduce the time of executing the best practices variant calling procedure by 29.3% for GATK3.8 and 16.9% for GATK4. Further speedups can be accomplished by splitting data for parallel analysis, resulting in run time of only a few hours on whole human genome sequenced to the depth of 20X, for both versions of GATK. Nonetheless, GATK4 is already much more cost-effective than GATK3.8. Thanks to significant rewrites of the algorithms, the same analysis can be run largely in a single-threaded fashion, allowing users to process multiple samples on the same CPU., Conclusions: In time-sensitive situations, when a patient has a critical or rapidly developing condition, it is useful to minimize the time to process a single sample. In such cases we recommend using GATK3.8 by splitting the sample into chunks and computing across multiple nodes. The resultant walltime will be nnn.4 hours at the cost of $41.60 on 4 c5.18xlarge instances of Amazon Cloud. For cost-effectiveness of routine analyses or for large population studies, it is useful to maximize the number of samples processed per unit time. Thus we recommend GATK4, running multiple samples on one node. The total walltime will be ∼34.1 hours on 40 samples, with 1.18 samples processed per hour at the cost of $2.60 per sample on c5.18xlarge instance of Amazon Cloud.
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- 2019
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25. Sentieon DNASeq Variant Calling Workflow Demonstrates Strong Computational Performance and Accuracy.
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Kendig KI, Baheti S, Bockol MA, Drucker TM, Hart SN, Heldenbrand JR, Hernaez M, Hudson ME, Kalmbach MT, Klee EW, Mattson NR, Ross CA, Taschuk M, Wieben ED, Wiepert M, Wildman DE, and Mainzer LS
- Abstract
As reliable, efficient genome sequencing becomes ubiquitous, the need for similarly reliable and efficient variant calling becomes increasingly important. The Genome Analysis Toolkit (GATK), maintained by the Broad Institute, is currently the widely accepted standard for variant calling software. However, alternative solutions may provide faster variant calling without sacrificing accuracy. One such alternative is Sentieon DNASeq, a toolkit analogous to GATK but built on a highly optimized backend. We conducted an independent evaluation of the DNASeq single-sample variant calling pipeline in comparison to that of GATK. Our results support the near-identical accuracy of the two software packages, showcase optimal scalability and great speed from Sentieon, and describe computational performance considerations for the deployment of DNASeq.
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- 2019
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26. Gene Expression and Missplicing in the Corneal Endothelium of Patients With a TCF4 Trinucleotide Repeat Expansion Without Fuchs' Endothelial Corneal Dystrophy.
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Wieben ED, Baratz KH, Aleff RA, Kalari KR, Tang X, Maguire LJ, Patel SV, and Fautsch MP
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- Aged, Aged, 80 and over, Female, Genotype, Humans, Male, Trinucleotide Repeat Expansion genetics, Exome Sequencing, A Kinase Anchor Proteins genetics, Alternative Splicing, Endothelium, Corneal metabolism, Fuchs' Endothelial Dystrophy genetics, Gene Expression Regulation physiology, Kinesins genetics, Minor Histocompatibility Antigens genetics, Proto-Oncogene Proteins genetics, RNA-Binding Proteins genetics, Transcription Factor 4 genetics
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Purpose: CTG trinucleotide repeat (TNR) expansion in an intron of the TCF4 gene is the most common genetic variant associated with Fuchs' endothelial corneal dystrophy (FECD). Although several mechanisms have been implicated in the disease process, their exact pathophysiologic importance is unclear. To understand events leading from TCF4 TNR expansion to disease phenotype, we characterized splicing, gene expression, and exon sequence changes in a rare cohort of patients with TNR expansions but no phenotypic FECD (RE+/FECD-)., Methods: Corneal endothelium and blood were collected from patients undergoing endothelial keratoplasty for non-FECD corneal edema. Total RNA was isolated from corneal endothelial tissue (n = 3) and used for RNASeq. Gene splicing and expression was assessed by Mixture of Isoforms (MISO) and MAP-RSeq software. Genomic DNA was isolated from blood mononuclear cells and used for whole genome exome sequencing. Base calling was performed using Illumina's Real-Time Analysis., Results: Three genes (MBNL1, KIF13A, AKAP13) that were previously identified as misspliced in patients with a CTG TNR expansion and FECD disease (RE+/FECD+) were found normally spliced in RE+/FECD- samples. Gene expression differences in pathways associated with the innate immune response, cell signaling (e.g., TGFβ, WNT), and cell senescence markers were also identified between RE+/FECD- and RE+/FECD+ groups. No consistent genetic variants were identified in RE+/FECD- patient exomes., Conclusions: Identification of novel splicing patterns and differential gene expression in RE+/FECD- samples provides new insights and more relevant gene targets that may be protective against FECD disease in vulnerable patients with TCF4 CTG TNR expansions.
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- 2019
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27. Amplification-free long-read sequencing of TCF4 expanded trinucleotide repeats in Fuchs Endothelial Corneal Dystrophy.
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Wieben ED, Aleff RA, Basu S, Sarangi V, Bowman B, McLaughlin IJ, Mills JR, Butz ML, Highsmith EW, Ida CM, Ekholm JM, Baratz KH, and Fautsch MP
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- Alleles, Computational Biology methods, Fuchs' Endothelial Dystrophy diagnosis, Gene Editing, Genetic Association Studies, Genomics methods, Genotype, Humans, Phenotype, Trinucleotide Repeats, RNA, Guide, CRISPR-Cas Systems, Fuchs' Endothelial Dystrophy genetics, Gene Amplification, Genetic Predisposition to Disease, Transcription Factor 4 genetics, Trinucleotide Repeat Expansion
- Abstract
Amplification of a CAG trinucleotide motif (CTG18.1) within the TCF4 gene has been strongly associated with Fuchs Endothelial Corneal Dystrophy (FECD). Nevertheless, a small minority of clinically unaffected elderly patients who have expanded CTG18.1 sequences have been identified. To test the hypothesis that the CAG expansions in these patients are protected from FECD because they have interruptions within the CAG repeats, we utilized a combination of an amplification-free, long-read sequencing method and a new target-enrichment sequence analysis tool developed by Pacific Biosciences to interrogate the sequence structure of expanded repeats. The sequencing was successful in identifying a previously described interruption within an unexpanded allele and provided sequence data on expanded alleles greater than 2000 bases in length. The data revealed considerable heterogeneity in the size distribution of expanded repeats within each patient. Detailed analysis of the long sequence reads did not reveal any instances of interruptions to the expanded CAG repeats, but did reveal novel variants within the AGG repeats that flank the CAG repeats in two of the five samples from clinically unaffected patients with expansions. This first examination of the sequence structure of CAG repeats in CTG18.1 suggests that factors other than interruptions to the repeat structure account for the absence of disease in some elderly patients with repeat expansions in the TCF4 gene., Competing Interests: B.B., I.J.M and J.M.E. are employees of Pacific Biosciences. All other authors declare that no competing interests exist.
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- 2019
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28. Association of rs613872 and Trinucleotide Repeat Expansion in the TCF4 Gene of German Patients With Fuchs Endothelial Corneal Dystrophy.
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Okumura N, Hayashi R, Nakano M, Tashiro K, Yoshii K, Aleff R, Butz M, Highsmith EW, Wieben ED, Fautsch MP, Baratz KH, Komori Y, Ueda E, Nakahara M, Weller J, Tourtas T, Schlötzer-Schrehardt U, Kruse F, and Koizumi N
- Subjects
- Adolescent, Adult, Aged, Aged, 80 and over, Alleles, Female, Genotype, Humans, Male, Middle Aged, Polymorphism, Single Nucleotide, Young Adult, Fuchs' Endothelial Dystrophy genetics, Genetic Predisposition to Disease, Transcription Factor 4 genetics, Trinucleotide Repeat Expansion
- Abstract
Purpose: To investigate single nucleotide polymorphisms (SNPs) and trinucleotide repeat (TNR) expansion in the transcription factor 4 (TCF4) gene in a large cohort of German patients with Fuchs endothelial corneal dystrophy (FECD)., Methods: Genomic DNA was obtained from 398 patients with FECD and from 58 non-FECD controls. Thirty-seven previously reported SNPs were evaluated by genotyping. The 398 FECD samples were analyzed for TNR expansions by short tandem repeat assays and Southern blotting. The possible associations between the TNR length and clinical parameters (age, sex, visual acuity, and central corneal thickness) were analyzed in 132 patients., Results: The SNPs in COL8A2, TCF8, LOXHD1, and AGBL1 showed no heterogeneity in 36 cases, although SLCA411 showed 3 nonsense mutations. SNPs were detected for TCF4 (rs613872, rs2123392, rs17089887, rs1452787, and rs1348047), but only rs613872 showed a significant association with FECD (P = 9.93 × 10). Overall, 315/398 (79%) patients harbored TNR lengths >50, whereas no non-FECD controls harbored TNR lengths >50. The TCF4 SNP rs613872 genotype was TT: 39 (67%), TG: 18 (31%), and GG: 1 (2%) in non-FECD controls; TT: 39 (47%), TG: 38 (46%), and GG: 6 (7%) in FECD cases harboring TNR <50; and TT: 23 (8%), TG: 224 (79%), and GG: 38 (13%) in FECD cases harboring TNR >50 (P = 2.93 × 10). No significant association was detected between the TNR length and clinical parameters., Conclusions: Our large German cohort demonstrated a significant association between the risk allele G in rs613872 and FECD, irrespective of TNR expansion, although this risk allele was more frequent in FECD cases with TNR expansion than without.
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- 2019
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29. Effect of Trinucleotide Repeat Expansion on the Expression of TCF4 mRNA in Fuchs' Endothelial Corneal Dystrophy.
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Okumura N, Hayashi R, Nakano M, Yoshii K, Tashiro K, Sato T, Blake DJ, Aleff R, Butz M, Highsmith EW, Wieben ED, Fautsch MP, Baratz KH, Komori Y, Nakahara M, Tourtas T, Schlötzer-Schrehardt U, Kruse F, and Koizumi N
- Subjects
- Adult, Aged, Aged, 80 and over, Blotting, Southern, Female, Genotyping Techniques, Humans, Male, Microsatellite Repeats, Middle Aged, Real-Time Polymerase Chain Reaction, Young Adult, Fuchs' Endothelial Dystrophy genetics, Gene Expression Regulation physiology, RNA, Messenger genetics, Transcription Factor 4 genetics, Trinucleotide Repeat Expansion
- Abstract
Purpose: CTG trinucleotide repeat (TNR) expansion is frequently found in transcription factor 4 (TCF4) in Fuchs' endothelial corneal dystrophy (FECD), though the effect of TNR expansion on FECD pathophysiology remains unclear. The purpose of this study was to evaluate the effect of TNR expansion on TCF4 expression in corneal endothelium of patients with FECD., Methods: Peripheral blood DNA and Descemet membrane with corneal endothelium were obtained from 203 German patients with FECD. The CTG TNR repeat length in TCF4 was determined by short tandem repeat (STR) assays and Southern blotting using genomic DNA. Genotyping of rs613872 in TCF4 was performed by PCR. TCF4 mRNA levels in corneal endothelium were evaluated by quantitative PCR using three different probes. Control corneal endothelial samples were obtained from 35 non-FECD subjects., Results: The STR assay and Southern blotting showed that 162 of the 203 patients with FECD (80%) harbored CTG trinucleotide repeat lengths larger than 50. Quantitative PCR using all three probes demonstrated that TCF4 mRNA is significantly upregulated in the corneal endothelium of patients with FECD, regardless of the presence of TNR expansion. However, the length of the TNR tended to show a positive correlation with TCF4 expression level. No correlation was shown between the genotype of TCF4 SNP, rs613872, and the level of TCF4 expression., Conclusions: Our findings showed that TCF4 mRNA is upregulated in the corneal endothelium of patients with FECD. Further studies on the effects of TCF4 upregulation on corneal endothelial cell function will aid in understanding the pathophysiology of FECD.
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- 2019
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30. CDKN2A Germline Rare Coding Variants and Risk of Pancreatic Cancer in Minority Populations.
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McWilliams RR, Wieben ED, Chaffee KG, Antwi SO, Raskin L, Olopade OI, Li D, Highsmith WE Jr, Colon-Otero G, Khanna LG, Permuth JB, Olson JE, Frucht H, Genkinger J, Zheng W, Blot WJ, Wu L, Almada LL, Fernandez-Zapico ME, Sicotte H, Pedersen KS, and Petersen GM
- Subjects
- Adult, Aged, Aged, 80 and over, Female, Germ-Line Mutation, Humans, Male, Middle Aged, Minority Groups, Pancreatic Neoplasms, Cyclin-Dependent Kinase Inhibitor p16 genetics
- Abstract
Background: Pathogenic germline mutations in the CDKN2A tumor suppressor gene are rare and associated with highly penetrant familial melanoma and pancreatic cancer in non-Hispanic whites (NHW). To date, the prevalence and impact of CDKN2A rare coding variants (RCV) in racial minority groups remain poorly characterized. We examined the role of CDKN2A RCVs on the risk of pancreatic cancer among minority subjects. Methods: We sequenced CDKN2A in 220 African American (AA) pancreatic cancer cases, 900 noncancer AA controls, and 183 Nigerian controls. RCV frequencies were determined for each group and compared with that of 1,537 NHW patients with pancreatic cancer. Odds ratios (OR) and 95% confidence intervals (CI) were calculated for both a case-case comparison of RCV frequencies in AAs versus NHWs, and case-control comparison between AA cases versus noncancer AA controls plus Nigerian controls. Smaller sets of Hispanic and Native American cases and controls also were sequenced. Results: One novel missense RCV and one novel frameshift RCV were found among AA patients: 400G>A and 258_278del. RCV carrier status was associated with increased risk of pancreatic cancer among AA cases (11/220; OR, 3.3; 95% CI, 1.5-7.1; P = 0.004) compared with AA and Nigerian controls (17/1,083). Further, AA cases had higher frequency of RCVs: 5.0% (OR, 13.4; 95% CI, 4.9-36.7; P < 0.001) compared with NHW cases (0.4%). Conclusions: CDKN2A RCVs are more common in AA than in NHW patients with pancreatic cancer and associated with moderately increased pancreatic cancer risk among AAs. Impact: RCVs in CDKN2A are frequent in AAs and are associated with risk for pancreatic cancer. Cancer Epidemiol Biomarkers Prev; 27(11); 1364-70. ©2018 AACR., (©2018 American Association for Cancer Research.)
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- 2018
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31. Gene expression in the corneal endothelium of Fuchs endothelial corneal dystrophy patients with and without expansion of a trinucleotide repeat in TCF4.
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Wieben ED, Aleff RA, Tang X, Kalari KR, Maguire LJ, Patel SV, Baratz KH, and Fautsch MP
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- Aged, Aged, 80 and over, Alternative Splicing, Female, Genetic Variation, Humans, Male, Middle Aged, Endothelium, Corneal metabolism, Fuchs' Endothelial Dystrophy genetics, Gene Expression Profiling, Transcription Factor 4 genetics, Trinucleotide Repeat Expansion
- Abstract
Fuchs Endothelial Corneal Dystrophy (FECD) is a late onset, autosomal dominant eye disease that can lead to loss of vision. Expansion of a CTG trinucleotide repeat in the third intron of the transcription factor 4 (TCF4) gene is highly associated with FECD. However, only about 75% of FECD patients in the northern European population possess an expansion of this repeat. The remaining FECD cases appear to be associated with variants in other genes. To better understand the pathophysiology of this disease, we compared gene expression profiles of corneal endothelium from FECD patients with an expanded trinucleotide repeat (RE+) to those that do not have a repeat expansion (RE-). Comparative analysis of these two cohorts showed widespread RNA mis-splicing in RE+, but not in RE- samples. Quantitatively, we identified 39 genes in which expression was significantly different between RE+ and RE- samples. Examination of the mutation profiles in the RE- samples did not find any mutations in genes previously associated with FECD, but did reveal one sample with a rare variant of laminin subunit gamma 1 (LAMC1) and three samples with rare variants in the gene coding for the mitochondrial protein peripheral-type benzodiazepine receptor-associated protein 1 (TSPOAP1)., Competing Interests: The authors have declared that no competing interests exist.
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- 2018
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32. Fuchs' Endothelial Corneal Dystrophy in Patients With Myotonic Dystrophy, Type 1.
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Winkler NS, Milone M, Martinez-Thompson JM, Raja H, Aleff RA, Patel SV, Fautsch MP, Wieben ED, and Baratz KH
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- Adult, Aged, Endothelium, Corneal metabolism, Female, Fuchs' Endothelial Dystrophy genetics, Fuchs' Endothelial Dystrophy pathology, Genetic Predisposition to Disease, Humans, Male, Middle Aged, Myotonic Dystrophy genetics, Myotonic Dystrophy pathology, Pedigree, Phenotype, Polymerase Chain Reaction, Prospective Studies, Slit Lamp Microscopy, Young Adult, Fuchs' Endothelial Dystrophy complications, Myotonic Dystrophy complications, Myotonin-Protein Kinase genetics, Transcription Factor 4 genetics, Trinucleotide Repeat Expansion genetics
- Abstract
Purpose: RNA toxicity from CTG trinucleotide repeat (TNR) expansion within noncoding DNA of the transcription factor 4 (TCF4) and DM1 protein kinase (DMPK) genes has been described in Fuchs' endothelial corneal dystrophy (FECD) and myotonic dystrophy, type 1 (DM1), respectively. We prospectively evaluated DM1 patients and their families for phenotypic FECD and report the analysis of CTG expansion in the TCF4 gene and DMPK expression in corneal endothelium., Methods: FECD grade was evaluated by slit lamp biomicroscopy in 26 participants from 14 families with DM1. CTG TNR length in TCF4 and DMPK was determined by a combination of Gene Scan and Southern blotting of peripheral blood leukocyte DNA., Results: FECD grade was 2 or higher in 5 (36%) of 14 probands, significantly greater than the general population (5%) (P < 0.001). FECD segregated with DM1; six of eight members of the largest family had both FECD and DM1, while the other two family members had neither disease. All DNA samples from 24 subjects, including four FECD-affected probands, were bi-allelic for nonexpanded TNR length in TCF4 (<40 repeats). Considering a 75% prevalence of TCF4 TNR expansion in FECD, the probability of four FECD probands lacking TNR expansion was 0.4%. Neither severity of DM1 nor DMPK TNR length predicted the presence of FECD in DM1 patients., Conclusions: FECD was common in DM1 families, and the diseases cosegregated. TCF4 TNR expansion was lacking in DM1 families. These findings support a hypothesis that DMPK TNR expansion contributes to clinical FECD.
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- 2018
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33. Autosomal dominant calpainopathy due to heterozygous CAPN3 C.643_663del21.
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Martinez-Thompson JM, Niu Z, Tracy JA, Moore SA, Swenson A, Wieben ED, and Milone M
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- Adult, Aged, Calpain metabolism, Creatine Kinase metabolism, DNA Mutational Analysis, Electromyography, Female, Heterozygote, High-Throughput Nucleotide Sequencing, Humans, Male, Middle Aged, Muscle Proteins metabolism, Muscle Weakness etiology, Muscular Atrophy etiology, Muscular Atrophy pathology, Muscular Dystrophies, Limb-Girdle complications, Muscular Dystrophies, Limb-Girdle metabolism, Muscular Dystrophies, Limb-Girdle physiopathology, Mutation, Pedigree, Sequence Analysis, DNA, Sequence Deletion, Calpain genetics, Muscle Proteins genetics, Muscle Weakness physiopathology, Muscular Atrophy diagnostic imaging, Muscular Dystrophies, Limb-Girdle genetics, Paraspinal Muscles diagnostic imaging
- Abstract
Introduction: A calpain-3 (CAPN3) gene heterozygous deletion (c.643_663del21) was recently linked to autosomal dominant (AD) limb-girdle muscular dystrophy. However, the possibility of digenic disease was raised. We describe 3 families with AD calpainopathy carrying this isolated mutation., Methods: Probands heterozygous for CAPN3 c.643_663del21 were identified by targeted next generation or whole exome sequencing. Clinical findings were collected for probands and families. Calpain-3 muscle Western blots were performed in 3 unrelated individuals., Results: Probands reported variable weakness in their 40s or 50s, with myalgia, back pain, or hyperlordosis. Pelvic girdle muscles were affected with adductor and hamstring sparing. Creatine kinase was normal to 1,800 U/L, independent of weakness severity. Imaging demonstrated lumbar paraspinal muscle atrophy. Electromyographic findings and muscle biopsies were normal to mildly myopathic. Muscle calpain-3 expression was reduced., Discussion: This study provides further evidence for AD calpainopathy associated with CAPN3 c.643_663del21. No pathogenic variants in other genes known to cause myopathy were detected. Muscle Nerve 57: 679-683, 2018., (© 2017 Wiley Periodicals, Inc.)
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- 2018
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34. Utility of DNA, RNA, Protein, and Functional Approaches to Solve Cryptic Immunodeficiencies.
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Cousin MA, Smith MJ, Sigafoos AN, Jin JJ, Murphree MI, Boczek NJ, Blackburn PR, Oliver GR, Aleff RA, Clark KJ, Wieben ED, Joshi AY, Pichurin PN, Abraham RS, and Klee EW
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- Biomarkers, Computational Biology methods, DNA, Female, Gene Expression Profiling, Genetic Variation, Humans, Immunologic Deficiency Syndromes diagnosis, Immunophenotyping, Infant, Proteins, RNA, Exome Sequencing, Genomics methods, Immunologic Deficiency Syndromes etiology, Immunologic Deficiency Syndromes metabolism, Proteomics methods
- Abstract
Purpose: We report a female infant identified by newborn screening for severe combined immunodeficiencies (NBS SCID) with T cell lymphopenia (TCL). The patient had persistently elevated alpha-fetoprotein (AFP) with IgA deficiency, and elevated IgM. Gene sequencing for a SCID panel was uninformative. We sought to determine the cause of the immunodeficiency in this infant., Methods: We performed whole-exome sequencing (WES) on the patient and parents to identify a genetic diagnosis. Based on the WES result, we developed a novel flow cytometric panel for rapid assessment of DNA repair defects using blood samples. We also performed whole transcriptome sequencing (WTS) on fibroblast RNA from the patient and father for abnormal transcript analysis., Results: WES revealed a pathogenic paternally inherited indel in ATM. We used the flow panel to assess several proteins in the DNA repair pathway in lymphocyte subsets. The patient had absent phosphorylation of ATM, resulting in absent or aberrant phosphorylation of downstream proteins, including γH2AX. However, ataxia-telangiectasia (AT) is an autosomal recessive condition, and the abnormal functional data did not correspond with a single ATM variant. WTS revealed in-frame reciprocal fusion transcripts involving ATM and SLC35F2 indicating a chromosome 11 inversion within 11q22.3, of maternal origin. Inversion breakpoints were identified within ATM intron 16 and SLC35F2 intron 7., Conclusions: We identified a novel ATM-breaking chromosome 11 inversion in trans with a pathogenic indel (compound heterozygote) resulting in non-functional ATM protein, consistent with a diagnosis of AT. Utilization of several molecular and functional assays allowed successful resolution of this case.
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- 2018
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35. Repeat-Associated Non-ATG (RAN) Translation in Fuchs' Endothelial Corneal Dystrophy.
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Soragni E, Petrosyan L, Rinkoski TA, Wieben ED, Baratz KH, Fautsch MP, and Gottesfeld JM
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- Blotting, Western, Cell Proliferation, Cells, Cultured, Endothelium, Corneal metabolism, Fibroblasts metabolism, Fluorescent Antibody Technique, Indirect, Gene Expression physiology, Humans, Introns, Real-Time Polymerase Chain Reaction, Transfection, Fuchs' Endothelial Dystrophy genetics, Protein Biosynthesis, Transcription Factor 4 genetics, Trinucleotide Repeat Expansion genetics
- Abstract
Purpose: The strongest genetic association with Fuchs' endothelial corneal dystrophy (FECD) is the presence of an intronic (CTG·CAG)n trinucleotide repeat (TNR) expansion in the transcription factor 4 (TCF4) gene. Repeat-associated non-ATG (RAN) translation, an unconventional protein translation mechanism that does not require an initiating ATG, has been described in many TNR expansion diseases, including myotonic dystrophy type 1 (DM1). Given the similarities between DM1 and FECD, we wished to determine whether RAN translation occurs in FECD., Methods: Antibodies against peptides in the C-terminus of putative RAN translation products from TCF4 were raised and validated by Western blotting and immunofluorescence (IF). CTG·CAG repeats of various lengths in the context of the TCF4 gene were cloned in frame with a 3× FLAG tag and transfected in human cells. IF with antipeptide and anti-FLAG antibodies, as well as cytotoxicity and cell proliferation assays, were performed in these transfected cells. Corneal endothelium derived from patients with FECD was probed with validated antibodies by IF., Results: CTG·CAG repeats in the context of the TCF4 gene are transcribed and translated via non-ATG initiation in transfected cells and confer toxicity to an immortalized corneal endothelial cell line. An antipeptide antibody raised against the C-terminus of the TCF4 poly-cysteine frame recognized RAN translation products by IF in cells transfected with CTG·CAG repeats and in FECD corneal endothelium., Conclusions: Expanded CTG·CAG repeats in the context of the third intron of TCF4 are transcribed and translated via non-ATG initiation, providing evidence for RAN translation in corneal endothelium of patients with FECD.
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- 2018
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36. A prospective genome-wide study of prostate cancer metastases reveals association of wnt pathway activation and increased cell cycle proliferation with primary resistance to abiraterone acetate-prednisone.
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Wang L, Dehm SM, Hillman DW, Sicotte H, Tan W, Gormley M, Bhargava V, Jimenez R, Xie F, Yin P, Qin S, Quevedo F, Costello BA, Pitot HC, Ho T, Bryce AH, Ye Z, Li Y, Eiken P, Vedell PT, Barman P, McMenomy BP, Atwell TD, Carlson RE, Ellingson M, Eckloff BW, Qin R, Ou F, Hart SN, Huang H, Jen J, Wieben ED, Kalari KR, Weinshilboum RM, Wang L, and Kohli M
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- Aged, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Cell Cycle, Cell Proliferation, Genome-Wide Association Study, Humans, Male, Middle Aged, Neoplasm Metastasis drug therapy, Neoplasm Metastasis genetics, Prospective Studies, Prostatic Neoplasms, Castration-Resistant drug therapy, Abiraterone Acetate administration & dosage, Drug Resistance, Neoplasm genetics, Prednisone administration & dosage, Prostatic Neoplasms, Castration-Resistant genetics, Wnt Signaling Pathway genetics
- Abstract
Background: Genomic aberrations have been identified in metastatic castration-resistant prostate cancer (mCRPC), but molecular predictors of resistance to abiraterone acetate/prednisone (AA/P) treatment are not known., Patients and Methods: In a prospective clinical trial, mCRPC patients underwent whole-exome sequencing (n = 82) and RNA sequencing (n = 75) of metastatic biopsies before initiating AA/P with the objective of identifying genomic alterations associated with resistance to AA/P. Primary resistance was determined at 12 weeks of treatment using criteria for progression that included serum prostate-specific antigen measurement, bone and computerized tomography imaging and symptom assessments. Acquired resistance was determined using the end point of time to treatment change (TTTC), defined as time from enrollment until change in treatment from progressive disease. Associations of genomic and transcriptomic alterations with primary resistance were determined using logistic regression, Fisher's exact test, single and multivariate analyses. Cox regression models were utilized for determining association of genomic and transcriptomic alterations with TTTC., Results: At 12 weeks, 32 patients in the cohort had progressed (nonresponders). Median study follow-up was 32.1 months by which time 58 patients had switched treatments due to progression. Median TTTC was 10.1 months (interquartile range: 4.4-24.1). Genes in the Wnt/β-catenin pathway were more frequently mutated and negative regulators of Wnt/β-catenin signaling were more frequently deleted or displayed reduced mRNA expression in nonresponders. Additionally, mRNA expression of cell cycle regulatory genes was increased in nonresponders. In multivariate models, increased cell cycle proliferation scores (≥ 50) were associated with shorter TTTC (hazard ratio = 2.11, 95% confidence interval: 1.17-3.80; P = 0.01)., Conclusions: Wnt/β-catenin pathway activation and increased cell cycle progression scores can serve as molecular markers for predicting resistance to AA/P therapy., (© The Author 2017. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)
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- 2018
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37. Androgen Receptor Variant AR-V9 Is Coexpressed with AR-V7 in Prostate Cancer Metastases and Predicts Abiraterone Resistance.
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Kohli M, Ho Y, Hillman DW, Van Etten JL, Henzler C, Yang R, Sperger JM, Li Y, Tseng E, Hon T, Clark T, Tan W, Carlson RE, Wang L, Sicotte H, Thai H, Jimenez R, Huang H, Vedell PT, Eckloff BW, Quevedo JF, Pitot HC, Costello BA, Jen J, Wieben ED, Silverstein KAT, Lang JM, Wang L, and Dehm SM
- Subjects
- Cell Line, Tumor, Cell Proliferation genetics, Disease-Free Survival, Drug Resistance, Neoplasm genetics, Humans, Male, Neoplasm Metastasis, Prospective Studies, Prostatic Neoplasms, Castration-Resistant genetics, Prostatic Neoplasms, Castration-Resistant metabolism, Protein Isoforms genetics, Protein Isoforms metabolism, RNA Interference, Receptors, Androgen metabolism, Androstenes therapeutic use, Gene Expression Regulation, Neoplastic drug effects, Genetic Variation, Prostatic Neoplasms, Castration-Resistant drug therapy, Receptors, Androgen genetics
- Abstract
Purpose: Androgen receptor (AR) variant AR-V7 is a ligand-independent transcription factor that promotes prostate cancer resistance to AR-targeted therapies. Accordingly, efforts are under way to develop strategies for monitoring and inhibiting AR-V7 in castration-resistant prostate cancer (CRPC). The purpose of this study was to understand whether other AR variants may be coexpressed with AR-V7 and promote resistance to AR-targeted therapies. Experimental Design: We utilized complementary short- and long-read sequencing of intact AR mRNA isoforms to characterize AR expression in CRPC models. Coexpression of AR-V7 and AR-V9 mRNA in CRPC metastases and circulating tumor cells was assessed by RNA-seq and RT-PCR, respectively. Expression of AR-V9 protein in CRPC models was evaluated with polyclonal antisera. Multivariate analysis was performed to test whether AR variant mRNA expression in metastatic tissues was associated with a 12-week progression-free survival endpoint in a prospective clinical trial of 78 CRPC-stage patients initiating therapy with the androgen synthesis inhibitor, abiraterone acetate. Results: AR-V9 was frequently coexpressed with AR-V7. Both AR variant species were found to share a common 3' terminal cryptic exon, which rendered AR-V9 susceptible to experimental manipulations that were previously thought to target AR-V7 uniquely. AR-V9 promoted ligand-independent growth of prostate cancer cells. High AR-V9 mRNA expression in CRPC metastases was predictive of primary resistance to abiraterone acetate (HR = 4.0; 95% confidence interval, 1.31-12.2; P = 0.02). Conclusions: AR-V9 may be an important component of therapeutic resistance in CRPC. Clin Cancer Res; 23(16); 4704-15. ©2017 AACR ., (©2017 American Association for Cancer Research.)
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- 2017
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38. Tumor Sequencing and Patient-Derived Xenografts in the Neoadjuvant Treatment of Breast Cancer.
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Goetz MP, Kalari KR, Suman VJ, Moyer AM, Yu J, Visscher DW, Dockter TJ, Vedell PT, Sinnwell JP, Tang X, Thompson KJ, McLaughlin SA, Moreno-Aspitia A, Copland JA, Northfelt DW, Gray RJ, Hunt K, Conners A, Sicotte H, Eckel-Passow JE, Kocher JP, Ingle JN, Ellingson MS, McDonough M, Wieben ED, Weinshilboum R, Wang L, and Boughey JC
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- Adult, Aged, Animals, Breast Neoplasms genetics, Breast Neoplasms metabolism, Chemotherapy, Adjuvant, Exome genetics, Female, Humans, Mice, Inbred NOD, Mice, Knockout, Mice, SCID, Middle Aged, Mutation, Neoadjuvant Therapy, Prospective Studies, Sequence Analysis, DNA methods, Treatment Outcome, Triple Negative Breast Neoplasms genetics, Triple Negative Breast Neoplasms metabolism, Tumor Suppressor Protein p53 genetics, Antineoplastic Agents therapeutic use, Breast Neoplasms drug therapy, Triple Negative Breast Neoplasms drug therapy, Xenograft Model Antitumor Assays methods
- Abstract
Background: Breast cancer patients with residual disease after neoadjuvant chemotherapy (NAC) have increased recurrence risk. Molecular characterization, knowledge of NAC response, and simultaneous generation of patient-derived xenografts (PDXs) may accelerate drug development. However, the feasibility of this approach is unknown., Methods: We conducted a prospective study of 140 breast cancer patients treated with NAC and performed tumor and germline sequencing and generated patient-derived xenografts (PDXs) using core needle biopsies. Chemotherapy response was assessed at surgery., Results: Recurrent "targetable" alterations were not enriched in patients without pathologic complete response (pCR); however, upregulation of steroid receptor signaling and lower pCR rates (16.7%, 1/6) were observed in triple-negative breast cancer (TNBC) patients with luminal androgen receptor (LAR) vs basal subtypes (60.0%, 21/35). Within TNBC, TP53 mutation frequency (75.6%, 31/41) did not differ comparing basal (74.3%, 26/35) and LAR (83.3%, 5/6); however, TP53 stop-gain mutations were more common in basal (22.9%, 8/35) vs LAR (0.0%, 0/6), which was confirmed in The Cancer Genome Atlas and British Columbia data sets. In luminal B tumors, Ki-67 responses were observed in tumors that harbored mutations conferring endocrine resistance ( p53, AKT, and IKBKE ). PDX take rate (27.4%, 31/113) varied according to tumor subtype, and in a patient with progression on NAC, sequencing data informed drug selection (olaparib) with in vivo antitumor activity observed in the primary and resistant (postchemotherapy) PDXs., Conclusions: In this study, we demonstrate the feasibility of tumor sequencing and PDX generation in the NAC setting. "Targetable" alterations were not enriched in chemotherapy-resistant tumors; however, prioritization of drug testing based on sequence data may accelerate drug development., (© The Author, 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com)
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- 2017
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39. Retinoic acid receptor alpha drives cell cycle progression and is associated with increased sensitivity to retinoids in T-cell lymphoma.
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Wang X, Dasari S, Nowakowski GS, Lazaridis KN, Wieben ED, Kadin ME, Feldman AL, and Boddicker RL
- Subjects
- Cell Line, Tumor, Cyclin-Dependent Kinases genetics, Cyclin-Dependent Kinases metabolism, Gene Expression Regulation, Neoplastic, Humans, Lymphoma, T-Cell metabolism, Mutation, Proteolysis, RNA Interference, RNA, Small Interfering genetics, Retinoic Acid Receptor alpha metabolism, Cell Cycle genetics, Drug Resistance, Neoplasm genetics, Lymphoma, T-Cell genetics, Retinoic Acid Receptor alpha genetics, Retinoids pharmacology
- Abstract
Peripheral T-cell lymphomas (PTCLs) are aggressive non-Hodgkin lymphomas with generally poor outcomes following standard therapy. Few candidate therapeutic targets have been identified to date. Retinoic acid receptor alpha (RARA) is a transcription factor that modulates cell growth and differentiation in response to retinoids. While retinoids have been used to treat some cutaneous T-cell lymphomas (CTCLs), their mechanism of action and the role of RARA in CTCL and other mature T-cell lymphomas remain poorly understood. After identifying a PTCL with a RARAR394Q mutation, we sought to characterize the role of RARA in T-cell lymphoma cells. Overexpressing wild-type RARA or RARAR394Q significantly increased cell growth in RARAlow cell lines, while RARA knockdown induced G1 arrest and decreased expression of cyclin-dependent kinases CDK2/4/6 in RARAhigh cells. The retinoids, AM80 (tamibarotene) and all-trans retinoic acid, caused dose-dependent growth inhibition, G1 arrest, and CDK2/4/6 down-regulation. Genes down-regulated in transcriptome data were enriched for cell cycle and G1-S transition. Finally, RARA overexpression augmented chemosensitivity to retinoids. In conclusion, RARA drives cyclin-dependent kinase expression, G1-S transition, and cell growth in T-cell lymphoma. Synthetic retinoids inhibit these functions in a dose-dependent fashion and are most effective in cells with high RARA expression, indicating RARA may represent a therapeutic target in some PTCLs.
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- 2017
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40. Erratum to: Conserved recurrent gene mutations correlate with pathway deregulation and clinical outcomes of lung adenocarcinoma in never-smokers.
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Sun Z, Wang L, Eckloff BW, Deng B, Wang Y, Wampfler JA, Jang J, Wieben ED, Jen J, You M, and Yang P
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- 2017
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41. Trinucleotide Repeat Expansion in the Transcription Factor 4 (TCF4) Gene Leads to Widespread mRNA Splicing Changes in Fuchs' Endothelial Corneal Dystrophy.
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Wieben ED, Aleff RA, Tang X, Butz ML, Kalari KR, Highsmith EW, Jen J, Vasmatzis G, Patel SV, Maguire LJ, Baratz KH, and Fautsch MP
- Subjects
- Adult, Aged, Aged, 80 and over, Alleles, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors metabolism, Blotting, Southern, Endothelium, Corneal metabolism, Endothelium, Corneal pathology, Female, Fuchs' Endothelial Dystrophy diagnosis, Fuchs' Endothelial Dystrophy metabolism, Genotype, Humans, Male, Middle Aged, Polymerase Chain Reaction, Transcription Factor 4, Transcription Factors metabolism, Trinucleotide Repeat Expansion, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors genetics, Fuchs' Endothelial Dystrophy genetics, Gene Expression Regulation, Genetic Predisposition to Disease, RNA Splicing genetics, RNA, Messenger genetics, Transcription Factors genetics
- Abstract
Purpose: To identify RNA missplicing events in human corneal endothelial tissue isolated from Fuchs' endothelial corneal dystrophy (FECD)., Methods: Total RNA was isolated and sequenced from corneal endothelial tissue obtained during keratoplasty from 12 patients with FECD and 4 patients undergoing keratoplasty or enucleation for other indications. The length of the trinucleotide repeat (TNR) CTG in the transcription factor 4 (TCF4) gene was determined using leukocyte-derived DNA analyzed by a combination of Southern blotting and Genescan analysis. Commercial statistical software was used to quantify expression of alternatively spliced genes. Validation of selected alternative splicing events was performed by using RT-PCR. Gene sets identified were analyzed for overrepresentation using Web-based analysis system., Results: Corneal endothelial tissue from FECD patients containing a CTG TNR expansion sequence in the TCF4 gene revealed widespread changes in mRNA splicing, including a novel splicing event involving FGFR2. Differential splicing of NUMA1, PPFIBP1, MBNL1, and MBNL2 transcripts were identified in all FECD samples containing a TNR expansion. The differentially spliced genes were enriched for products that localize to the cell cortex and bind cytoskeletal and cell adhesion proteins., Conclusions: Corneal endothelium from FECD patients harbors a unique signature of mis-splicing events due to CTG TNR expansion in the TCF4 gene, consistent with the hypothesis that RNA toxicity contributes to the pathogenesis of FECD. Changes to the endothelial barrier function, a known event in the development of FECD, was identified as a key biological process influenced by the missplicing events.
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- 2017
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42. Molecular Modeling and Functional Analysis of Exome Sequencing-Derived Variants of Unknown Significance Identify a Novel, Constitutively Active FGFR2 Mutant in Cholangiocarcinoma.
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Egan JB, Marks DL, Hogenson TL, Vrabel AM, Sigafoos AN, Tolosa EJ, Carr RM, Safgren SL, Hesles EE, Almada LL, Romecin-Duran PA, Iguchi E, Ala'Aldeen A, Kocher JA, Oliver GR, Prodduturi N, Mead DW, Hossain A, Huneke NE, Tagtow CM, Ailawadhi S, Ansell SM, Banck MS, Bryce AH, Carballido EM, Chanan-Khan AA, Curtis KK, Resnik E, Gawryletz CD, Go RS, Halfdanarson TR, Ho TH, Joseph RW, Kapoor P, Mansfield AS, Meurice N, Nageswara Rao AA, Nowakowski GS, Pardanani A, Parikh SA, Cheville JC, Feldman AL, Ramanathan RK, Robinson SI, Tibes R, Finnes HD, McCormick JB, McWilliams RR, Jatoi A, Patnaik MM, Silva AC, Wieben ED, McAllister TM, Rumilla KM, Kerr SE, Lazaridis KN, Farrugia G, Stewart AK, Clark KJ, Kennedy EJ, Klee EW, Borad MJ, and Fernandez-Zapico ME
- Abstract
Purpose: Genomic testing has increased the quantity of information available to oncologists. Unfortunately, many identified sequence alterations are variants of unknown significance (VUSs), which thus limit the clinician's ability to use these findings to inform treatment. We applied a combination of in silico prediction and molecular modeling tools and laboratory techniques to rapidly define actionable VUSs., Materials and Methods: Exome sequencing was conducted on 308 tumors from various origins. Most single nucleotide alterations within gene coding regions were VUSs. These VUSs were filtered to identify a subset of therapeutically targetable genes that were predicted with in silico tools to be altered in function by their variant sequence. A subset of receptor tyrosine kinase VUSs was characterized by laboratory comparison of each VUS versus its wild-type counterpart in terms of expression and signaling activity., Results: The study identified 4,327 point mutations of which 3,833 were VUSs. Filtering for mutations in genes that were therapeutically targetable and predicted to affect protein function reduced these to 522VUSs of interest, including a large number of kinases. Ten receptortyrosine kinase VUSs were selected to explore in the laboratory. Of these, seven were found to be functionally altered. Three VUSs (FGFR2 F276C, FGFR4 R78H, and KDR G539R) showed increased basal or ligand-stimulated ERK phosphorylation compared with their wild-type counterparts, which suggests that they support transformation. Treatment of a patient who carried FGFR2 F276C with an FGFR inhibitor resulted in significant and sustained tumor response with clinical benefit., Conclusion: The findings demonstrate the feasibility of rapid identification of the biologic relevance of somatic mutations, which thus advances clinicians' ability to make informed treatment decisions., Competing Interests: AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relation-ships may not relate to the subject matter of this manuscript. For more information about ASCO’s conflict of interest policy, please refer to www.asco.org/rwc or po.ascopubs.org/site/ifc. Jan B. Egan No relationship to disclose David L. Marks Stock and Other Ownership Interests: WebMD, Cardinal Health (I), Prothena (I), Perrigo (I) Tara L. Hogenson No relationship to disclose Anne M. Vrabel No relationship to disclose Ashley N. Sigafoos No relationship to disclose Ezequiel J. Tolosa No relationship to disclose Ryan M. Carr No relationship to disclose Stephanie L. Safgren No relationship to disclose Elisa Enriquez Hesles No relationship to disclose Luciana L. Almada No relationship to disclose Paola A. Romecin-Duran No relationship to disclose Eriko Iguchi No relationship to disclose Aryan Ala’Aldeen No relationship to disclose Jean-Pierre A. Kocher No relationship to disclose Gavin R. Oliver No relationship to disclose Naresh Prodduturi No relationship to disclose David W. Mead No relationship to disclose Asif Hossain No relationship to disclose Norine E. Huneke No relationship to disclose Colleen M. Tagtow No relationship to disclose Sikander Ailawadhi Consulting or Advisory Role: Amgen, Takeda Pharmaceuticals, Novartis Research Funding: Pharmacyclics (Inst) Travel, Accommodations, Expenses: Amgen, Takeda Pharmaceuticals, Novartis Stephen M. Ansell Honoraria: WebMD, Research To Practice Research Funding: Bristol-Myers Squibb (Inst), Celldex Therapeutics (Inst), Seattle Genetics (Inst), Merck (Inst), Affimed (Inst), Trillium Therapeutics (Inst) Michaela S. Banck No relationship to disclose Alan H. BryceNo relationship to disclose Estrella M. Carballido No relationship to disclose Asher A. Chanan-Khan No relationship to disclose Kelly K. Curtis Employment: INC Research Stock and Other Ownership Interests: INC Research Travel, Accommodations, Expenses: INC Research Ernesto ResnikNo relationship to disclose Chelsea D. Gawryletz No relationship to disclose Ronald S. Go Speakers’ Bureau: OnLive, Takeda Pharmaceuticals Thorvardur R. Halfdanarson Consulting or Advisory Role: Lexicon (Inst), Ipsen (Inst), Merrimack (Inst) Research Funding: Esanex (Inst), Ipsen (Inst), Boston Biomedical (Inst) Thai H. Ho No relationship to disclose Richard W. Joseph Consulting or Advisory Role: Bristol-Myers Squibb, Nektar, Genoptix, Eisai, Bristol-Myers Squibb Research Funding: Merck, Bristol-Meyers Squibb, Roche (Inst), Genentech (Inst), X4P Pharmaceuticals (Inst), Amgen (Inst) Prashant Kapoor Consulting or Advisory Role: Sanofi (Inst) Research Funding: Amgen (Inst), Takeda Pharmaceuticals (Inst) Aaron S. Mansfield Consulting or Advisory Role: TrovaGene (Inst), Genentech (Inst), Bristol-Myers Squibb (Inst) Travel, Accommodations, Expenses: Bristol-Myers Squibb Nathalie Meurice No relationship to disclose Amulya A. Nageswara Rao No relationship to disclose Grzegorz S. Nowakowski Consulting or Advisory Role: Celgene (Inst), MorphoSys (Inst), Genentech (Inst) Research Funding: Celgene (Inst) Animesh Pardanani No relationship to disclose Sameer A. Parikh Research Funding: Pharmacyclics (Inst) John C. Cheville No relationship to disclose Andrew L. Feldman Consulting or Advisory Role: Infinity Pharmaceuticals Patents, Royalties, Other Intellectual Property: Inventor of technology for which the Mayo Clinic holds an unlicensed patent or has submitted a patent application (Inst) Ramesh K. Ramanathan Honoraria: Taiho Pharmaceutical, Cerulean Pharma, Pharmacyclics, Insys Therapeutics, Novocure Consulting or Advisory Role: Cerulean Pharma, Novocure, INSYS Therapeutics, Pharmacyclics Research Funding: AbbVie (Inst), Celgene (Inst), Merck (Inst), Schering Plough (Inst), Merrimack (Inst), Boston Biomedical (Inst), BERG (Inst), Superlab Far East (Inst) Steven I. Robinson Travel, Accommodations, Expenses: Tricon Pharmaceuticals Raoul Tibes Research Funding: Novartis (Inst), Merck (Inst), Seattle Genetics (Inst), Boehringer Ingelheim (Inst), Astellas Pharma (Inst), Astex Pharmaceuticals (Inst)Heidi D. Finnes Jennifer B. McCormick No relationship to disclose Robert R. McWilliams Consulting or Advisory Role: Merrimack (Inst) Research Funding: PRISM BioLab (Inst), Genentech (Inst), Novartis (Inst), NewLink Genetics (Inst), Eli Lilly (Inst), Aduro Biotech (Inst), Pfizer (Inst), Sanofi (Inst) Travel, Accommodations, Expenses: AstraZeneca Aminah Jatoi Research Funding: Entera Health, Boston Biologics Mrinal M. Patnaik No relationship to disclose Alvin C. Silva No relationship to disclose Eric D. Wieben Patents, Royalties, Other Intellectual Property: Champions Oncology–licensed IP-targeted therapy with abiraterone acetate ($0 received), WuXi AppTec–licensed IP-breast cancer xenografts ($12,737 to Mayo Clinic; Inst), Affymetrix-licensed IP on several variants in drug processing enzymes ($0 in past 2 years), Life Technologies–licensed IP on several variants in drug processing enzymes ($0 in past 2 years) Tammy M. McAllister No relationship to disclose Kandelaria M. Rumilla No relationship to disclose Sarah E. Kerr Research Funding: Abbott Molecular Konstantinos N. Lazaridis No relationship to disclose Gianrico Farrugia No relationship to disclose A. Keith Stewart Consulting or Advisory Role: Celgene, Bristol-Myers Squibb, Janssen Pharmaceuticals, Roche, Amgen Karl J. Clark Patents, Royalties, Other Intellectual Property: Inventor on patents associated with the Sleeping Beauty transposon system Eileen J. Kennedy Patents, Royalties, Other Intellectual Property: Patent on unrelated work (US patent 9,458,189) for “ligation of stapled polypeptides” issued October 4, 2016 Eric W. Klee Patents, Royalties, Other Intellectual Property: Royalties on software development Mitesh J. Borad Stock and Other Ownership Interests: GlaxoSmithKline, Gilead Sciences Consulting or Advisory Role: G1 Therapeutics, TD2, Fujifilm (Inst), Agios (Inst), INSYS Therapeutics (Inst), Novartis (Inst), ArQule (Inst), Celegene (Inst), Inspyr Therapeutics, Halozyme Therapeutics (Inst) Research Funding: Boston Biomedical (Inst), miRNA Therapeutics (Inst), Senhwa Biosciences (Inst), Medimmune (Inst), Bioline (Inst), Agios (Inst), Halozyme Therapeutics (Inst), Celgene (Inst), Threshold Pharmaceuticals (Inst), Toray (Inst), Dicerna Pharmaceuticals (Inst), Sillajen (Inst), Eisai (Inst), Taiho Pharmaceuticals (Inst), EMD Serono (Inst), Isis Pharmaceuticals (Inst), Incyte (Inst), Sun BioPharma (Inst), ARIAD Pharmaceuticals (Inst), ImClone Systems (Inst) Travel, Accommodations, Expenses: ArQule, Celgene, AstraZeneca Martin E. Fernandez-Zapico No relationship to disclose
- Published
- 2017
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43. Outcome of Whole Exome Sequencing for Diagnostic Odyssey Cases of an Individualized Medicine Clinic: The Mayo Clinic Experience.
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Lazaridis KN, Schahl KA, Cousin MA, Babovic-Vuksanovic D, Riegert-Johnson DL, Gavrilova RH, McAllister TM, Lindor NM, Abraham RS, Ackerman MJ, Pichurin PN, Deyle DR, Gavrilov DK, Hand JL, Klee EW, Stephens MC, Wick MJ, Atkinson EJ, Linden DR, Ferber MJ, Wieben ED, and Farrugia G
- Subjects
- Adolescent, Adult, Aged, Child, Child, Preschool, Female, Genetic Predisposition to Disease, Humans, Infant, Male, Middle Aged, Minnesota, Young Adult, Exome, Genetic Testing economics, Genetic Testing methods, Genomics, High-Throughput Nucleotide Sequencing, Precision Medicine methods
- Abstract
Objective: To describe the experience and outcome of performing whole-exome sequencing (WES) for resolution of patients on a diagnostic odyssey in the first 18 months of an individualized medicine clinic (IMC)., Patients and Methods: The IMC offered WES to physicians of Mayo Clinic practice for patients with suspected genetic disease. DNA specimens of the proband and relatives were submitted to WES laboratories. We developed the Genomic Odyssey Board with multidisciplinary expertise to determine the appropriateness for IMC services, review WES reports, and make the final decision about whether the exome findings explain the disease. This study took place from September 30, 2012, to March 30, 2014., Results: In the first 18 consecutive months, the IMC received 82 consultation requests for patients on a diagnostic odyssey. The Genomic Odyssey Board deferred 7 cases and approved 75 cases to proceed with WES. Seventy-one patients met with an IMC genomic counselor. Fifty-one patients submitted specimens for WES testing, and the results have been received for all. There were 15 cases in which a diagnosis was made on the basis of WES findings; thus, the positive diagnostic yield of this practice was 29%. The mean cost per patient for this service was approximately $8000. Medicaid supported 27% of the patients, and 38% of patients received complete or partial insurance coverage., Conclusion: The significant diagnostic yield, moderate cost, and notable health marketplace acceptance for WES compared with conventional genetic testing make the former method a rational diagnostic approach for patients on a diagnostic odyssey., (Copyright © 2016 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.)
- Published
- 2016
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44. Whole Exome Sequencing and Heterologous Cellular Electrophysiology Studies Elucidate a Novel Loss-of-Function Mutation in the CACNA1A-Encoded Neuronal P/Q-Type Calcium Channel in a Child With Congenital Hypotonia and Developmental Delay.
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Weyhrauch DL, Ye D, Boczek NJ, Tester DJ, Gavrilova RH, Patterson MC, Wieben ED, and Ackerman MJ
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- Child, Preschool, Humans, Male, Muscle Hypotonia congenital, Mutation, Missense, Calcium Channels genetics, Developmental Disabilities genetics, Exome genetics, Muscle Hypotonia genetics, Patch-Clamp Techniques
- Abstract
Background: A 4-year-old boy born at 37 weeks' gestation with intrauterine growth retardation presented with developmental delay with pronounced language and gross motor delay, axial hypotonia, and dynamic hypertonia of the extremities. Investigations including the Minnesota Newborn Screen, thyroid stimulating hormone/thyroxin, and inborn errors of metabolism screening were negative. Cerebral magnetic resonance imaging and spectroscopy were normal. Genetic testing was negative for coagulopathy, Smith-Lemli-Opitz, fragile X, and Prader-Willi/Angelman syndromes. Whole genome array analysis was unremarkable., Methods: Whole exome sequencing was performed through a commercial testing laboratory to elucidate the underlying etiology for the child's presentation. A de novo mutation was hypothesized. In attempt to establish pathogenicity of our candidate variant, cellular electrophysiologic functional analysis of the putative de novo mutation was performed using patch-clamp technology., Results: Whole exome sequencing revealed a p.P1353L variant in the CACNA1A gene, which encodes for the α1-subunit of the brain-specific P/Q-type calcium channel (CaV2.1). This presynaptic high-voltage-gated channel couples neuronal excitation to the vesicular release of neurotransmitter and is implicated in several neurologic disorders. DNA Sanger sequencing confirmed that the de novo mutation was absent in both parents and present in the child only. Electrophysiologic analysis of P1353L-CACNA1A demonstrated near complete loss of function, with a 95% reduction in peak current density., Conclusions: Whole exome sequencing coupled with cellular electrophysiologic functional analysis of a de novoCACNA1A missense mutation has elucidated the probable underlying pathophysiologic mechanism responsible for the child's phenotype. Genetic testing of CACNA1A in patients with congenital hypotonia and developmental delay may be warranted., (Copyright © 2016. Published by Elsevier Inc.)
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- 2016
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45. Mutational Landscapes of Sequential Prostate Metastases and Matched Patient Derived Xenografts during Enzalutamide Therapy.
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Kohli M, Wang L, Xie F, Sicotte H, Yin P, Dehm SM, Hart SN, Vedell PT, Barman P, Qin R, Mahoney DW, Carlson RE, Eckel-Passow JE, Atwell TD, Eiken PW, McMenomy BP, Wieben ED, Jha G, Jimenez RE, Weinshilboum R, and Wang L
- Subjects
- Animals, Benzamides, Heterografts, Humans, Male, Mice, Neoplasm Metastasis, Neoplasm Transplantation, Nitriles, Phenylthiohydantoin pharmacology, Gene Expression Regulation, Neoplastic drug effects, Mutation, Phenylthiohydantoin analogs & derivatives, Prostatic Neoplasms drug therapy, Prostatic Neoplasms genetics, Prostatic Neoplasms metabolism, Prostatic Neoplasms pathology, Xenograft Model Antitumor Assays methods
- Abstract
Developing patient derived models from individual tumors that capture the biological heterogeneity and mutation landscape in advanced prostate cancer is challenging, but essential for understanding tumor progression and delivery of personalized therapy in metastatic castrate resistant prostate cancer stage. To demonstrate the feasibility of developing patient derived xenograft models in this stage, we present a case study wherein xenografts were derived from cancer metastases in a patient progressing on androgen deprivation therapy and prior to initiating pre-chemotherapy enzalutamide treatment. Tissue biopsies from a metastatic rib lesion were obtained for sequencing before and after initiating enzalutamide treatment over a twelve-week period and also implanted subcutaneously as well as under the renal capsule in immuno-deficient mice. The genome and transcriptome landscapes of xenografts and the original patient tumor tissues were compared by performing whole exome and transcriptome sequencing of the metastatic tumor tissues and the xenografts at both time points. After comparing the somatic mutations, copy number variations, gene fusions and gene expression we found that the patient's genomic and transcriptomic alterations were preserved in the patient derived xenografts with high fidelity. These xenograft models provide an opportunity for predicting efficacy of existing and potentially novel drugs that is based on individual metastatic tumor expression signature and molecular pharmacology for delivery of precision medicine.
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- 2015
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46. Whole-Exome Sequencing of 10 Scientists: Evaluation of the Process and Outcomes.
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Lindor NM, Schahl KA, Johnson KJ, Hunt KS, Mensink KA, Wieben ED, Klee E, Black JL, Highsmith WE Jr, Thibodeau SN, Ferber MJ, Aypar U, Ji Y, Graham RP, Fiksdal AS, Sarangi V, Ormond KE, Riegert-Johnson DL, McAllister TM, Farrugia G, and McCormick JB
- Subjects
- Adult, Female, Healthy Volunteers, Humans, Male, Outcome and Process Assessment, Health Care, Patient Selection, Research Design, Sequence Analysis, DNA methods, Attitude of Health Personnel, Exome, Genetic Counseling methods, Genetic Testing methods, Genome-Wide Association Study methods
- Abstract
Objective: To understand motivations, educational needs, and concerns of individuals contemplating whole-exome sequencing (WES) and determine what amount of genetic information might be obtained by sequencing a generally healthy cohort so as to more effectively counsel future patients., Patients and Methods: From 2012 to 2014, 40 medically educated, generally healthy scientists at Mayo Clinic were invited to have WES conducted on a research basis; 26 agreed to be in a drawing from which 10 participants were selected. The study involved pre- and posttest genetic counseling and completion of 4 surveys related to the experience and outcomes. Whole-exome sequencing was conducted on DNA from blood from each person., Results: Most variants (76,305 per person; range, 74,505-77,387) were known benign allelic variants, variants in genes of unknown function, or variants of uncertain significance in genes of known function. The results of suspected pathogenic/pathogenic variants in Mendelian disorders and pharmacogenomic variants were disclosed. The mean number of suspected pathogenic/pathogenic variants was 2.2 per person (range, 1-4). Four pharmacogenomic genes were included for reporting; variants were found in 9 of 10 participants., Conclusion: This study provides data that may be useful in establishing reality-based patient expectations, outlines specific points to cover during counseling, and increases confidence in the feasibility of providing adequate preparation and counseling for WES in generally healthy individuals., (Copyright © 2015 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.)
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- 2015
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47. Exome sequencing reveals frequent deleterious germline variants in cancer susceptibility genes in women with invasive breast cancer undergoing neoadjuvant chemotherapy.
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Ellingson MS, Hart SN, Kalari KR, Suman V, Schahl KA, Dockter TJ, Felten SJ, Sinnwell JP, Thompson KJ, Tang X, Vedell PT, Barman P, Sicotte H, Eckel-Passow JE, Northfelt DW, Gray RJ, McLaughlin SA, Moreno-Aspitia A, Ingle JN, Moyer AM, Visscher DW, Jones K, Conners A, McDonough M, Wieben ED, Wang L, Weinshilboum R, Boughey JC, and Goetz MP
- Subjects
- Adult, Aged, Aged, 80 and over, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Biomarkers, Tumor, Breast Neoplasms drug therapy, Databases, Genetic, Female, Gene Frequency, Genes, BRCA1, Genes, BRCA2, Genes, p53, Humans, Middle Aged, Neoadjuvant Therapy, Neoplasm Invasiveness, Neoplasm Staging, Young Adult, Breast Neoplasms genetics, Breast Neoplasms pathology, Exome, Genetic Predisposition to Disease, Germ-Line Mutation, High-Throughput Nucleotide Sequencing
- Abstract
When sequencing blood and tumor samples to identify targetable somatic variants for cancer therapy, clinically relevant germline variants may be uncovered. We evaluated the prevalence of deleterious germline variants in cancer susceptibility genes in women with breast cancer referred for neoadjuvant chemotherapy and returned clinically actionable results to patients. Exome sequencing was performed on blood samples from women with invasive breast cancer referred for neoadjuvant chemotherapy. Germline variants within 142 hereditary cancer susceptibility genes were filtered and reviewed for pathogenicity. Return of results was offered to patients with deleterious variants in actionable genes if they were not aware of their result through clinical testing. 124 patients were enrolled (median age 51) with the following subtypes: triple negative (n = 43, 34.7%), HER2+ (n = 37, 29.8%), luminal B (n = 31, 25%), and luminal A (n = 13, 10.5%). Twenty-eight deleterious variants were identified in 26/124 (21.0%) patients in the following genes: ATM (n = 3), BLM (n = 1), BRCA1 (n = 4), BRCA2 (n = 8), CHEK2 (n = 2), FANCA (n = 1), FANCI (n = 1), FANCL (n = 1), FANCM (n = 1), FH (n = 1), MLH3 (n = 1), MUTYH (n = 2), PALB2 (n = 1), and WRN (n = 1). 121/124 (97.6%) patients consented to return of research results. Thirteen (10.5%) had actionable variants, including four that were returned to patients and led to changes in medical management. Deleterious variants in cancer susceptibility genes are highly prevalent in patients with invasive breast cancer referred for neoadjuvant chemotherapy undergoing exome sequencing. Detection of these variants impacts medical management.
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- 2015
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48. Recessive MYH6 Mutations in Hypoplastic Left Heart With Reduced Ejection Fraction.
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Theis JL, Zimmermann MT, Evans JM, Eckloff BW, Wieben ED, Qureshi MY, O'Leary PW, and Olson TM
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- Cardiac Myosins chemistry, Echocardiography, Family Health, Female, Gene Frequency, Genes, Recessive, Genome, Human genetics, Heterozygote, Humans, Hypoplastic Left Heart Syndrome physiopathology, Male, Models, Molecular, Myosin Heavy Chains chemistry, Pedigree, Protein Structure, Tertiary, Sequence Analysis, DNA methods, Stroke Volume physiology, Cardiac Myosins genetics, Genetic Predisposition to Disease genetics, Hypoplastic Left Heart Syndrome genetics, Mutation, Myosin Heavy Chains genetics, Stroke Volume genetics
- Abstract
Background: The molecular underpinnings of hypoplastic left heart are poorly understood. Staged surgical palliation has dramatically improved survival, yet eventual failure of the systemic right ventricle necessitates cardiac transplantation in a subset of patients. We sought to identify genetic determinants of hypoplastic left heart with latent right ventricular dysfunction in individuals with a Fontan circulation., Methods and Results: Evaluation of cardiac structure and function by echocardiography in patients with hypoplastic left heart and their first-degree relatives identified 5 individuals with right ventricular ejection fraction ≤40% after Fontan operation. Whole genome sequencing was performed on DNA from 21 family members, filtering for genetic variants with allele frequency <1% predicted to alter protein structure or expression. Secondary family-based filtering for de novo and recessive variants revealed rare inherited missense mutations on both paternal and maternal alleles of MYH6, encoding myosin heavy chain 6, in 2 patients who developed right ventricular dysfunction 3 to 11 years postoperatively. Parents and siblings who were heterozygous carriers had normal echocardiograms. Protein modeling of the 4 highly conserved amino acid substitutions, residing in both head and tail domains, predicted perturbation of protein structure and function., Conclusions: In contrast to dominant MYH6 mutations with variable penetrance identified in other congenital heart defects and dilated cardiomyopathy, this study reveals compound heterozygosity for recessive MYH6 mutations in patients with hypoplastic left heart and reduced systemic right ventricular ejection fraction. These findings implicate a shared molecular basis for the developmental arrest and latent myopathy of left and right ventricles, respectively., (© 2015 American Heart Association, Inc.)
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- 2015
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49. How well do whole exome sequencing results correlate with medical findings? A study of 89 Mayo Clinic Biobank samples.
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Middha S, Lindor NM, McDonnell SK, Olson JE, Johnson KJ, Wieben ED, Farrugia G, Cerhan JR, and Thibodeau SN
- Abstract
Whole exome sequencing (WES) is increasingly being used for diagnosis without adequate information on predictive characteristics of reportable variants typically found on any given individual and correlation with clinical phenotype. In this study, we performed WES on 89 deceased individuals (mean age at death 74 years, range 28-93) from the Mayo Clinic Biobank. Significant clinical diagnoses were abstracted from electronic medical record via chart review. Variants [Single Nucleotide Variant (SNV) and insertion/deletion] were filtered based on quality (accuracy >99%, read-depth >20, alternate-allele read-depth >5, minor-allele-frequency <0.1) and available HGMD/OMIM phenotype information. Variants were defined as Tier-1 (nonsense, splice or frame-shifting) and Tier-2 (missense, predicted-damaging) and evaluated in 56 ACMG-reportable genes, 57 cancer-predisposition genes, along with examining overall genotype-phenotype correlations. Following variant filtering, 7046 total variants were identified (~79/person, 644 Tier-1, 6402 Tier-2), 161 among 56 ACMG-reportable genes (~1.8/person, 13 Tier-1, 148 Tier-2), and 115 among 57 cancer-predisposition genes (~1.3/person, 3 Tier-1, 112 Tier-2). The number of variants across 57 cancer-predisposition genes did not differentiate individuals with/without invasive cancer history (P > 0.19). Evaluating genotype-phenotype correlations across the exome, 202(3%) of 7046 filtered variants had some evidence for phenotypic correlation in medical records, while 3710(53%) variants had no phenotypic correlation. The phenotype associated with the remaining 44% could not be assessed from a typical medical record review. These data highlight significant continued challenges in the ability to extract medically meaningful predictive results from WES.
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- 2015
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50. Trinucleotide Repeat Expansion in the TCF4 Gene in Fuchs' Endothelial Corneal Dystrophy in Japanese.
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Nakano M, Okumura N, Nakagawa H, Koizumi N, Ikeda Y, Ueno M, Yoshii K, Adachi H, Aleff RA, Butz ML, Highsmith WE, Tashiro K, Wieben ED, Kinoshita S, and Baratz KH
- Subjects
- Aged, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors metabolism, Blotting, Southern, Female, Fuchs' Endothelial Dystrophy metabolism, Genotype, Humans, Introns, Japan, Male, Microsatellite Repeats, Polymerase Chain Reaction, Retrospective Studies, Transcription Factor 4, Transcription Factors metabolism, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors genetics, DNA genetics, Fuchs' Endothelial Dystrophy genetics, Genetic Predisposition to Disease, Transcription Factors genetics, Trinucleotide Repeat Expansion
- Abstract
Purpose: The purpose of this study was to evaluate the association between the intronic expansion of a trinucleotide repeat (TNR) in the TCF4 gene and Fuchs' endothelial corneal dystrophy (FECD) in a Japanese population., Methods: Forty-seven Japanese FECD patients and 96 age-matched controls were recruited. FECD patients and controls were examined by slit-lamp and noncontact specular microscopy. The repeat length was determined by direct sequencing and short tandem repeat assay of PCR-amplified DNA and Southern blotting of unamplified DNA., Results: A TNR expansion, defined as >50 CTG repeats in the TCF4 gene was identified in 12 of 47 FECD cases (26%) and 0 of 96 controls (0%; P < 0.001). Sensitivity and specificity in this study were 26% and 100%, respectively. The clinical characteristics of FECD patients with TNR expansion were not distinct from those without TNR expansion., Conclusions: These findings show for the first time in a Japanese population the association of the TNR expansion in TCF4 with FECD. In contrast to Caucasian cohorts in whom the TNR expansion is present in most patients with FECD, a CTG expansion is present in a minority of Japanese subjects, indicating other genetic variants as common causes of phenotypically identical disease in this population.
- Published
- 2015
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