Progress 09/01/13 to 08/31/18
Outputs
Target Audience:Academic and industry scientists working on genomics and genomic selection, especially with respect to disease resistance. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided training and experience in working with poultry, genomics and computational biology, and critical analytical thinking and writing. How have the results been disseminated to communities of interest?Besides what was report previously, the following national and international presentations were made this past year: • Poultry workshop, Plant and Animal Genome XXVI, January 13-17, 2018, San Diego, CA. • 12th International Symposium on Marek's Disease and Avian Herpesviruses, July 29 to August 2, 2018, Yangzhou, China. • International Symposium on Animal Health and Inheritance of Disease Resistance, August 3-5, 2018, Beijing, China. ? What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Significant accomplishments were made for both goals. With respect to Goal #1, the major findings were: • Like all virulent Marek's disease viruses (MDV), all Marek's disease (MD) vaccines viruses (HVT, SB-1, and Rispens) can integrate into the chicken host genome with similar kinetics. However, compare to virulent MDVs, the integration frequency is much lower. This result has significant implications on how MD vaccines might work to protect chickens. • Virulent MDVs show a cellular phenotype that we define as integrated only, which is the presence of the viral genome integrated into the chicken genome (almost always in or next to the telomeres) and no evidence of viral replication. Thus, no MD vaccines are the recombinant MDV lacking Meq, the viral oncogene, shown the integrated only phenotype. We interpret this result that only virulent MDVs are selected to continue to replicate, which may eventually form gross tumors. • The genetic resistance to the chicken can have a dramatic influence on the MDV integration profile. Specifically, MD resistant birds compared to susceptible one exhibited much lower levels of MDV integration. Thus, host genetic status is likely to influence the degree that virulent MDVs can replicate and integrate in chicken cells, which impact the ability for future transformation by the virus. With respect to Goal #2, the major findings were: • In MD tumors, there are ~600 somatic variants including 1-3 genes with nonsynonymous mutations. This very small number is similar to what is observed in pediatric cancers, which is in agreement with our expectation given that gross MD tumors arise as soon as 4 weeks after viral infection. • Ikaros, the master regulator of B and T cell development, is the first MD driver gene identified. Somatic variants are typically found in the zinc-finger DNA binding domains, which is a similar pattern to what has been observed for human acute lymphoblastic leukemias (ALL). • MD tumors result from at least "two hits." The first being expression of MDV Meq, the viral oncogene, which likely blocks apoptosis. The second hit is somatic mutations in Ikaros and potential other driver genes that lead to unregulated cellular proliferation. In conclusion, these results dramatically increase our fundamental knowledge of MDV-induced pathogenesis, especially with respect to MD tumors. Furthermore, these results can be further leveraged for the development of improved MD vaccines and the selection of MD resistant chickens. ?
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2018
Citation:
1. McPherson, M.C., H.H Cheng, J.M. Smith, and M.E. Delany. Vaccination and host Mareks disease-resistance genotype significantly reduce oncogenic Gallid alphaherpesvirus 2 telomere integration in host birds. In press. Cytogenetics and Genome Research.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
2. McPherson, M.C., H.H Cheng, and M.E. Delany. 2016. Mareks disease herpesvirus vaccines integrate into chicken host chromosomes yet lack a virus-host phenotype associated with oncogenic transformation. Vaccine 34:5554-5561.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
3. McPherson, M.C., and M.E. Delany. 2016. Virus and host genomic, molecular and cellular interactions during Mareks disease pathogenesis and oncogenesis. Poultry Science 95:412-429.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
4. Schmid, M., J. Smith, D.W. Burt, B.L. Aken, P.B. Antin, A.L. Archibald, C. Ashwell, P.J. Blackshear, C. Boschiero, C.T. Brown, S.C. Burgess, H.H. Cheng, W. Chow, Coble D.J., A. Cooksey, R.P.M.A. Crooijmans, J. Damas, R.V.N. Davis, D.-J. de Koning D., Delany M.E., T. Derrien, T.T. Desta, I.C. Dunn, M. Dunn, H. Ellegren, L. E�ry, I.k. Erb, M. Farr�, M. Fasold, D. Fleming, P. Flicek, K.E. Fowler, L. Fr�sard, D.P. Froman, V. Garceau, P.P. Gardner, A.A. Gheyas, D.K. Griffin, M.A.M. Groenen, T. Haaf, O. Hanotte, A. Hart, J. H�sler, S.B. Hedges S.B., J. Hertel, K. Howe, A. Hubbard, D.A. Hume D.A., P. Kaiser, D. Kedra, S.J. Kemp, C. Klopp, K.E. Kniel K.E., R. Kuo, S. Lagarrigue, S.J. Lamont S.J., D.M. Larkin, R.A. Lawal, S.M. Markland, F. McCarthy, H.A. McCormack, M.C. McPherson, A. Motegi, S.A. Muljo, A. M�nsterberg, R. Nag, I. Nanda, M. Neuberger, A. Nitsche, C. Notredame, H. Noyes, R. O'Connor, E.A. O'Hare, A.J. Oler, S.C. Ommeh, H. Pais, M. Persia, F. Pitel, L. Preeyanon, B.P. Prieto, P. Barja, E. M. Pritchett, D.D. Rhoads, C. Robinson, M.N. Romanov, M. Rothschild, P.-F. Roux, C.J. Schmidt, A.-S. Schneider, M.G. Schwartz, S.M. Searle, M.S. Skinner, C.A. Smith, P.F. Stadler, T.E. Steeves, C. Steinlein, L. Sun, M. Takata, I. Ulitsky, W. Wang, Y. Wang, W.C. Warren, J.M.D. Wood, D. Wragg, and H. Zhou. 2015. Third report on chicken genes and chromosomes 2015. Cytogenetics and Genome Research. 145(2):78-179.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
5. Robinson, C.M., H.H. Cheng, and M.E. Delany. 2014. Mareks disease virus and chicken host genome interactions. Viral genome integration occurs early post-infection and over a timeframe associated with latency, yet integration alone is not sufficient for cellular transformation. Cytogenetic and Genome Research 144:142-154.
|
Progress 09/01/16 to 08/31/17
Outputs
Target Audience:Academic and industry scientists working on genomics and improved vaccines, especially with respect to disease resistance. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided training and experience in working with poultry, genomics and computational biology, and critical analytical thinking and writing. How have the results been disseminated to communities of interest?National and international presentations have been made: • Poultry workshop, Plant and Animal Genome XXIV, January 14-18, 2017, San Diego, CA • National Breeders Roundtable, May 4-5, 2017, St. Louis, MO • ASV 36th Annual Meeting Veterinary Vaccine Club Workshop, June 24, 2017, Madison, WI What do you plan to do during the next reporting period to accomplish the goals?Continue as planned and then write report results in scientific publication.
Impacts
What was accomplished under these goals?
Progress under Objective 2 (Identify and characterize somatic alterations in MD transformed cells) was ongoing this year and is 90% complete. All the data have been generated and final analyses are being conducted. Thus far, 26 MD tumors from 22 birds and their corresponding controls (for DNA seq, normal tissue from the same bird; for RNA seq, CD4 T cells from uninfected birds) have been characterized with whole genome sequencing and transcriptome profiling. To identify various classes of somatic mutations, several software packages shown below were implemented: • single nucleotide variants (SNVs) - MuSE, MuTect, JointSNVMix2, SomaticSniper, VarDict, and VarScan 2 were utilized and further filtered using SomaticSeq (Fang et al., 2015). An average of ~2,500 SNVs were found per tumor. Considering only non-synonymous SNVs, ~56.5 were identified (range of 25 to 76). This figure is in the range found for adult solid tumors but higher than pediatric cancers, which was originally anticipated class based on the very early onset of tumors. C to T is the most common substitution, and is most similar to COSMIC signatures 7 and 11. • insertions and deletions (indels) - Indelocator, VarDict, VarScan 2, and LoFreq were used. The final results are currently being confirmed. • somatic copy number alterations (SCNAs) and loss of heterozygosity (LOH) - Both Control-FREEC and copyCat were used and identified 253 and 542 SCNAs, respectively. However, only 9 SCNAs were in common suggesting a high degree of false positives. With respect to LOH events, Control-FREEC identified 2,779 of which 381 were also found with HD-CNV with a median size of 163 Kb and a range of 10 Kb to 11.4 Mb. • structural variants (SVs) - BreakDancer, Delly, and novoBreak were used, which identified 917, 1,451, and 163 SVs, respectively. Of these, 39 (28 deletions and 11 inversions) were identified with at least 2 algorithms. • To identify a high-confidence list of candidate driver genes from the datasets generated above, MuSiC, OncodriveCLUST, and MUFFINN were employed to identify significantly mutated genes and pathways among our tumors samples. In total, 54 genes were identified by at least two of the methods. Our top driver gene is IKAROS Family Zinc Finger 1 (IKZF1), a tumor suppressor gene that encodes a zinc finger protein. IKZF1 has been shown to be associated with T-cell development, specifically in determining whether a thymocyte will lineate towards a CD4 or CD8 cell type; MD tumors are predominantly transformed CD4 cells. This gene has been shown to be frequently mutated in Acute Lymphoblastic Leukemia (ALL) in the N terminus, which disables the ability of the protein to attach to DNA, thereby losing its function In both ALL and in our experiment, we see significant mutations across samples (about 62% in our cases). More importantly, we also see clustering of highly deleterious mutations specifically in specific zinc finger binding domain, which is also observed in humans. We also see hemizygous mutations in one allele suggesting a dominant negative mutation, which has also been observed in humans. Adding to our interest is the report that IKZF1 is also a major factor in the maintenance of viral latency in Epstein-Barr virus (EBV), a gammaherpesvirus. IKZF1 does this indirectly by altering the expression of Oct-2, Bcl-6, and other transcription factors that directly EBV reactivation and plasma differentiation. Expression of IKZF1 may help explain the role of EBV as a human tumor-associated virus since viral gene EBNA-1 is necessary for B cell transformation and is required for replication and maintenance of EBV episomes during latency. As cancer is considered a "pathway disease" and to aid in our identification of MD driver genes, transcriptome analysis was also performed. 1,394 and 361 genes were significantly up-and down-regulated in MD tumors compared to control CD4 T cells. Of particular note is that IKZF1 showed reduced expression in 6 of the 26 samples. Interestingly, none of these 6 samples showed detectable somatic mutations in the exons suggesting possible variants in regulatory regions. Gene enrichment analysis of the differentially-expressed genes identified the following pathways: multicellular organismal signaling, bicellular tight junction and extracellular matrix component, leukocyte proliferation, leukocyte differentiation, leukocyte activation, hemopoiesis, cell activation, and positive regulation of immune system process. It is hoped that our ongoing analyses, refinements in computational pipelines (which is challenging as most need to be heavily modified for use outside of human and mouse), and integration with the other datasets should enable the identification of key driving genes for MD as well as information on somatic mutation frequency, somatic mutation signatures, and significantly mutated regions. ?
Publications
|
Progress 09/01/15 to 08/31/16
Outputs
Target Audience:Academic and industry scientists working on genomics and improved vaccines, especially with respect to disease resistance. Changes/Problems:Continue as planned and then write report results in scientific publication. What opportunities for training and professional development has the project provided?This project provided training and experience in working with poultry, genomics and computational biology, and critical analytical thinking and writing. Individuals with significant training include Alec Steep (graduate student, Genetics Program, Michigan State U.) and Charmaine Robinson and Marla McPherson (graduate students, Integrative Genetics and Genomics Graduate Group, UC Davis) How have the results been disseminated to communities of interest?National and international presentations have been made: • Poultry workshop, Plant and Animal Genome XXIV, January 9-13, 2016, San Diego, CA • 11th International Symposium on Marek's Disease and Avian Herpesviruses, July 6-9, 2016, Tours, France • 35th International Society of Animal Genetics Conference, July 23-27, 2016, Salt Lake City Utah ? What do you plan to do during the next reporting period to accomplish the goals?Continue as planned and then write report results in scientific publication.
Impacts
What was accomplished under these goals?
Progress under Objective 1 (Characterize MDV integration profiles as a function of genetic background, tissue, time after infection, and/or vaccine status) with 80% completed. We find that all tested MD vaccine strains can integrate at the chicken host telomeres as early as 1 day after vaccination in lymphoid tissue similar to the oncogenic MDV strains, but do not establish the specific, transformation-associated viral phenotype observed for oncogenic viruses during early infection. Based on these results, physical integration of the MDV genome into host telomeres along with a cease of viral replication appears to have a cooperative implication in MDV's ability to induce oncogenic transformation. This result contributes to an updated vaccine model with the following activities and impacts: MD vaccines cause an infection and host immune response which primes the system to react against MDV exposure; vaccine strains can integrate into host chromosomes, however, unlike the disease-virus cell harboring integrations do not transition to a latent-cell population and thus vaccines do not contribute to cancer-growth. Progress under Objective 2 (Identify and characterize somatic alterations in MD transformed cells) with 75% completed. All the data have been generated and analysis is underway, which has required substantial modification and updates to existing software as most are designed for biomedical efforts. Analysis of DNA sequencing is the most advanced thus far with 7 different somatic single nucleotide variant callers having co-identified ~2,500 SNPs per tumor. Interestingly, a higher proportion of SNPs were found in promoter regions, which suggests regulatory regions and gene expression may be important for tumor development. Examination of SNPs in coding regions identified an average of ~40 non-synonymous SNPs per tumors. This low number of variants may increase our chances of finding causative driver genes. If we further refine genes with somatic non-synonymous SNPs to those that are significantly mutated or show clustering of mutations across multiple samples, are also associated with cancer based on various databases (e.g., The Cancer Genome Atlas), and the change in coding sequence is predicted to have a functional impact (e.g., stop codon gain or loss), then a number of interesting candidates can be found. One example is IKAROS Family Zinc Finger 1 (IKZF1), a tumor suppressor gene that encodes a zing finger protein. IKZF1 has been shown to be associated with T-cell development, specifically in determining whether a thymocyte will lineate towards a CD4 or CD8 cell type. This gene has been shown to be frequently mutated in Acute Lymphoblastic Leukemia (ALL) in the N terminus, which disables its ability to attach to DNA, thereby losing its function. In both ALL and in our experiment, we see significant mutations across samples (about 19% in our case). More importantly, we also see clustering of highly deleterious mutations specifically in a zinc finger binding domain. We also see hemizygous mutations in one allele suggesting a dominant negative mutation, when has been observed in humans. It is hoped that our ongoing analyses, refinements in computational pipelines (which is challenging as most need to be heavily modified for use outside of human and mouse), and integration with the other datasets should enable the identification of key driving genes for MD as well as information on somatic mutation frequency, somatic mutation signatures, and significantly mutated regions. ?
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Robinson, C.M., Cheng, H.H., and Delany, M.E. 2014. Temporal kinetics of Marek's disease herpesvirus: integration occurs early after infection in both B and T cells. Cytogenet. Genome Res. 144:142-54.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Schmid, M., J. Smith, D.W. Burt, B.L. Aken, P.B. Antin, A.L. Archibald, C. Ashwell, P.J. Blackshear, C. Boschiero, C.T. Brown, S.C. Burgess, H.H. Cheng, W. Chow, Coble D.J., A. Cooksey, R.P.M.A. Crooijmans, J. Damas, R.V.N. Davis, D.-J. de Koning D., Delany M.E., T. Derrien, T.T. Desta, I.C. Dunn, M. Dunn, H. Ellegren, L. E�ry, I.k. Erb, M. Farr�, M. Fasold, D. Fleming, P. Flicek, K.E. Fowler, L. Fr�sard, D.P. Froman, V. Garceau, P.P. Gardner, A.A. Gheyas, D.K. Griffin, M.A.M. Groenen, T. Haaf, O. Hanotte, A. Hart, J. H�sler, S.B. Hedges S.B., J. Hertel, K. Howe, A. Hubbard, D.A. Hume D.A., P. Kaiser, D. Kedra, S.J. Kemp, C. Klopp, K.E. Kniel K.E., R. Kuo, S. Lagarrigue, S.J. Lamont S.J., D.M. Larkin, R.A. Lawal, S.M. Markland, F. McCarthy, H.A. McCormack, M.C. McPherson, A. Motegi, S.A. Muljo, A. M�nsterberg, R. Nag, I. Nanda, M. Neuberger, A. Nitsche, C. Notredame, H. Noyes, R. O'Connor, E.A. O'Hare, A.J. Oler, S.C. Ommeh, H. Pais, M. Persia, F. Pitel, L. Preeyanon, B.P. Prieto, P. Barja, E. M. Pritchett, D.D. Rhoads, C. Robinson, M.N. Romanov, M. Rothschild, P.-F. Roux, C.J. Schmidt, A.-S. Schneider, M.G. Schwartz, S.M. Searle, M.S. Skinner, C.A. Smith, P.F. Stadler, T.E. Steeves, C. Steinlein, L. Sun, M. Takata, I. Ulitsky, W. Wang, Y. Wang, W.C. Warren, J.M.D. Wood, D. Wragg, and H. Zhou. 2015. Third report on chicken genes and chromosomes 2015. Cytogenet. Genome Res. 145(2):78-179.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
McPherson, M.C., and Delany, M.E. 2016. Virus and host genomic, molecular, and cellular interactions during Marek's disease pathogenesis and oncogenesis. Poult. Sci. 95:412-29.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
McPherson, M.C., Cheng, H.H., and Delany, M.E. 2016. Mareks disease herpesvirus vaccines integrate into chicken host chromosomes yet lack a virus-host phenotype associated with oncogenic transformation. Vaccine 34:5554-5561.
|
Progress 09/01/14 to 08/31/15
Outputs
Target Audience:Academic and industry scientists working on genomics and improved vaccines, especially with respect to disease resistance. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided training and experience in working with poultry, genomics and computational biology, and critical analytical thinking and writing. Individuals with significant training include Alec Steep (graduate student, Genetics Program, Michigan State U.) and Charmaine Robinson and Marla McPherson (graduate students, Integrative Genetics and Genomics Graduate Group, UC Davis) How have the results been disseminated to communities of interest?National and international presentations have been made: Poultry workshop, Plant and Animal Genome XXIV, January 9-13, 2016, San Diego, CA 11th International Symposium on Marek's Disease and Avian Herpesviruses, July 6-9, 2016, Tours, France 35th International Society of Animal Genetics Conference, July 23-27, 2016, Salt Lake City Utah What do you plan to do during the next reporting period to accomplish the goals?Continue as planned and then write report results in scientific publication.
Impacts
What was accomplished under these goals?
Progress under Objective 1 (Characterize MDV integration profiles as a function of genetic background, tissue, time after infection, and/or vaccine status) with 80% completed. We find that all tested MD vaccine strains can integrate at the chicken host telomeres as early as 1 day after vaccination in lymphoid tissue similar to the oncogenic MDV strains, but do not establish the specific, transformation-associated viral phenotype observed for oncogenic viruses during early infection. Based on these results, physical integration of the MDV genome into host telomeres along with a cease of viral replication appears to have a cooperative implication in MDV's ability to induce oncogenic transformation. This result contributes to an updated vaccine model with the following activities and impacts: MD vaccines cause an infection and host immune response which primes the system to react against MDV exposure; vaccine strains can integrate into host chromosomes, however, unlike the disease-virus cell harboring integrations do not transition to a latent-cell population and thus vaccines do not contribute to cancer-growth. Progress under Objective 2 (Identify and characterize somatic alterations in MD transformed cells) with 75% completed. All the data have been generated and analysis is underway, which has required substantial modification and updates to existing software as most are designed for biomedical efforts. Analysis of DNA sequencing is the most advanced thus far with 7 different somatic single nucleotide variant callers having co-identified ~2,500 SNPs per tumor. Interestingly, a higher proportion of SNPs were found in promoter regions, which suggests regulatory regions and gene expression may be important for tumor development. Examination of SNPs in coding regions identified an average of ~40 non-synonymous SNPs per tumors. This low number of variants may increase our chances of finding causative driver genes. If we further refine genes with somatic non-synonymous SNPs to those that are significantly mutated or show clustering of mutations across multiple samples, are also associated with cancer based on various databases (e.g., The Cancer Genome Atlas), and the change in coding sequence is predicted to have a functional impact (e.g., stop codon gain or loss), then a number of interesting candidates can be found. One example is IKAROS Family Zinc Finger 1 (IKZF1), a tumor suppressor gene that encodes a zing finger protein. IKZF1 has been shown to be associated with T-cell development, specifically in determining whether a thymocyte will lineate towards a CD4 or CD8 cell type. This gene has been shown to be frequently mutated in Acute Lymphoblastic Leukemia (ALL) in the N terminus, which disables its ability to attach to DNA, thereby losing its function. In both ALL and in our experiment, we see significant mutations across samples (about 19% in our case). More importantly, we also see clustering of highly deleterious mutations specifically in a zinc finger binding domain. We also see hemizygous mutations in one allele suggesting a dominant negative mutation, when has been observed in humans. It is hoped that our ongoing analyses, refinements in computational pipelines (which is challenging as most need to be heavily modified for use outside of human and mouse), and integration with the other datasets should enable the identification of key driving genes for MD as well as information on somatic mutation frequency, somatic mutation signatures, and significantly mutated regions.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
Robinson, C.M., Cheng, H.H., and Delany, M.E. 2014. Temporal kinetics of Marek's disease herpesvirus: integration occurs early after infection in both B and T cells. Cytogenet. Genome Res. 144:142-54.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2016
Citation:
McPherson, M.C., and Delany, M.E. 2016. Virus and host genomic, molecular, and cellular interactions during Marek's disease pathogenesis and oncogenesis. Poult. Sci. 95:412-29.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2016
Citation:
McPherson, M.C., Cheng, H.H., and Delany, M.E. 2016. Mareks disease herpesvirus vaccines integrate into chicken host chromosomes yet lack a virus-host phenotype associated with oncogenic transformation. Vaccine 34:5554-5561.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Schmid, M., J. Smith, D.W. Burt, B.L. Aken, P.B. Antin, A.L. Archibald, C. Ashwell, P.J. Blackshear, C. Boschiero, C.T. Brown, S.C. Burgess, H.H. Cheng, W. Chow, Coble D.J., A. Cooksey, R.P.M.A. Crooijmans, J. Damas, R.V.N. Davis, D.-J. de Koning D., Delany M.E., T. Derrien, T.T. Desta, I.C. Dunn, M. Dunn, H. Ellegren, L. E�ry, I.k. Erb, M. Farr�, M. Fasold, D. Fleming, P. Flicek, K.E. Fowler, L. Fr�sard, D.P. Froman, V. Garceau, P.P. Gardner, A.A. Gheyas, D.K. Griffin, M.A.M. Groenen, T. Haaf, O. Hanotte, A. Hart, J. H�sler, S.B. Hedges S.B., J. Hertel, K. Howe, A. Hubbard, D.A. Hume D.A., P. Kaiser, D. Kedra, S.J. Kemp, C. Klopp, K.E. Kniel K.E., R. Kuo, S. Lagarrigue, S.J. Lamont S.J., D.M. Larkin, R.A. Lawal, S.M. Markland, F. McCarthy, H.A. McCormack, M.C. McPherson, A. Motegi, S.A. Muljo, A. M�nsterberg, R. Nag, I. Nanda, M. Neuberger, A. Nitsche, C. Notredame, H. Noyes, R. O'Connor, E.A. O'Hare, A.J. Oler, S.C. Ommeh, H. Pais, M. Persia, F. Pitel, L. Preeyanon, B.P. Prieto, P. Barja, E. M. Pritchett, D.D. Rhoads, C. Robinson, M.N. Romanov, M. Rothschild, P.-F. Roux, C.J. Schmidt, A.-S. Schneider, M.G. Schwartz, S.M. Searle, M.S. Skinner, C.A. Smith, P.F. Stadler, T.E. Steeves, C. Steinlein, L. Sun, M. Takata, I. Ulitsky, W. Wang, Y. Wang, W.C. Warren, J.M.D. Wood, D. Wragg, and H. Zhou. 2015. Third report on chicken genes and chromosomes 2015. Cytogenet. Genome Res. 145(2):78-179.
|
Progress 09/01/13 to 08/31/14
Outputs
Target Audience:Academic and industry scientists working on genomics and improved vaccines, especially with respect to disease resistance. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided training and experience in working with poultry, genomics and computational biology, and critical analytical thinking and writing. How have the results been disseminated to communities of interest?National and international presentations have been made. What do you plan to do during the next reporting period to accomplish the goals?Continue as planned and then write report results in scientific publication.
Impacts
What was accomplished under these goals?
Marek's disease (MD), a lymphoproliferative disease of chickens caused by the highly pathogenic Marek's disease virus (MDV) is the most serious chronic disease problem that costs the worldwide poultry industry $1-2 billion per year. Despite control measures including biosecurity and MD vaccines, new and more virulent MDV strains have and are predicted to arise in the future resulting in disease outbreaks. Thus, there is a critical need to improve existing measures or provide alternatives. This year, with respect to Objective 1 (MDV integration profiles), efforts has primarily focused on variation of MDV integration profiles for commonly used MD vaccine strains (i.e., HVT, SB-1, and Rispens). We find that all tested MD vaccine strains can integrate at the chicken host telomeres as early as 1 day after vaccination in lymphoid tissue similar to the oncogenic MDV strains, but do not establish the specific, transformation-associated viral phenotype observed for oncogenic viruses during early infection. Based on these results, physical integration of the MDV genome into host telomeres along with a cease of viral replication appears to have a cooperative implication in MDV's ability to induce oncogenic transformation. With respect to Objective 1 (MD driver mutations), Approximately 200 line 6 x 7 F1 chicks were challenged at hatch with 1,000 pfu JM/102W strain MDV; this strain induces large tumors. At 4-8 weeks after infection, ~100 birds that developed one or more tumors were collected; normal (control) tissues were also collected from each individual bird. To date, genomic screens (and samples) include: Whole genome sequencing (26 tumors and matched controls) to identify somatic single nucleotide polymorphisms (SNPs), short insertions and deletions (indels), copy number alterations (CNAs), and structural variants (SVs). Whole transcriptome sequencing (26 tumors and unmatched CD4 T cells from uninfected birds at 2, 4, 6, and 8 weeks of age) were collected to identify genes and pathways associated with MD tumors, as well as to validate somatic SNPs in coding regions. 15K SNP genotyping (72 tumors and various controls) was used to identify somatic CNAs and loss of heterozygosity (LOH). Cytogenetic screens for MDV integration sites (72 tumors and uninfected controls) was used to identify SVs and MDV integration profiles Analysis of this data is ongoing, but initially focusing on DNA sequencing in item #1, 7 different somatic single nucleotide variant callers have co-identified ~2,500 SNPs per tumor. Interestingly, a higher proportion of SNPs were found in promoter regions, which suggests regulatory regions and gene expression may be important for tumor development. Examination of SNPs in coding regions identified an average of ~40 non-synonymous SNPs per tumors. This low number of variants may increase our chances of finding causative driver genes. If we further refine genes with somatic non-synonymous SNPs to those that are significantly mutated or show clustering of mutations across multiple samples, are also associated with cancer based on various databases (e.g., The Cancer Genome Atlas), and the change in coding sequence is predicted to have a functional impact (e.g., stop codon gain or loss), then a number of interesting candidates can be found. One example is IKAROS Family Zinc Finger 1 (IKZF1), a tumor suppressor gene that encodes a zing finger protein. IKZF1 has been shown to be associated with T-cell development, specifically in determining whether a thymocyte will lineate towards a CD4 or CD8 cell type. This gene has been shown to be frequently mutated in Acute Lymphoblastic Leukemia (ALL) in the N terminus, which disables its ability to attach to DNA, thereby losing its function. In both ALL and in our experiment, we see significant mutations across samples (about 19% in our case). More importantly, we also see clustering of highly deleterious mutations specifically in a zinc finger binding domain. We also see hemizygous mutations in one allele suggesting a dominant negative mutation, when has been observed in humans. It is hoped that our ongoing analyses, refinements in computational pipelines (which is challenging as most need to be heavily modified for use outside of human and mouse), and integration with the other datasets should enable the identification of key driving genes for MD as well as information on somatic mutation frequency, somatic mutation signatures, and significantly mutated regions.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Robinson, C.M., Cheng, H.H., and Delany, M.E. 2014. Temporal kinetics of Marek's disease herpesvirus: integration occurs early after infection in both B and T cells. Cytogenet. Genome Res. 144:142-54.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
McPherson, M.C., and Delany, M.E. 2016. Virus and host genomic, molecular, and cellular interactions during Marek's disease pathogenesis and oncogenesis. Poult. Sci. 95:412-29.
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