University Program in Genetics and Genomics, Tuesday Seminar Series, Duke University

Talk I gave at Duke, UPGG, Tuesday Seminar Series

Transcript

1. Systems Genetics of Complex Skeletal Phenotypes Charles R. Farber, Ph.D. Center for Public Health Genomics, University of Virginia @cr_farber crf2s@virginia.edu farberlab.com University Program in Genetics and Genomics, Tuesday Seminar, Duke, 11/16/15 Slides on speakerdeck.com
2. • Condition of reduced bone strength • Common - 12 million cases in U.S. • Costly - ~$18 billion Osteoporosis http://www.neuros.net/en/osteoporosis.php
4. Variants, Genes and Gene Networks
5. Variation in Bone Strength and Related Traits Variants, Genes and Gene Networks
6. Systems Genetics
7. Adjacency Matrix TOM = Topological Overlap Measure Weighted Gene Co- Expression Network Analysis (WGCNA) Zhang and Horvath. 2005. Statistical Applications in Genetics and Molecular Biology 4: Article17. Samples Expression
8. Co-Expression Networks
9. Co-Expression Networks Gene (Node) Edge
10. Co-Expression Networks Gene (Node) Edge
11. Co-Expression Networks Gene (Node) Edge
12. Co-Expression Networks • Modular • Small number of highly connected “hub” genes (scale-free) Gene (Node) Edge
13. Co-Expression Network Cell Cycle P=4.0 x 10-30 Mitochondria P=1.1 x 10-64 Extracellular Matrix P=1.9 x 10-16 Extracellular Matrix P=4.4 x 10-20 Ribosome P=5.1 x 10-14
14. http://www.maxi-file.com/wp-content/uploads/2012/02/carousel-filing-system-lateral-filing-shelves-Maxi-File-850x409.jpg
15. http://www.maxi-file.com/wp-content/uploads/2012/02/carousel-filing-system-lateral-filing-shelves-Maxi-File-850x409.jpg Osteoblast Genes Osteoblast Differentiation Cell Adhesion
16. Hybrid Mouse Diversity Panel – 
 A Resource for Systems Genetics 100 Mouse Inbred Strains Whole Genome Gene Expression Profiles SNP Genotypes DNA RNA Hybrid Mouse Diversity Panel (HMDP) Bennett, B. J., Farber, C. R., Orozco, L., Kang, H. M., et al. (2010). A high-resolution association mapping panel for the dissection of complex traits in mice. Genome Research, 20(2), 281–290.
17. HMDP Bone Co- expression Network Predicting Function Mesner, L. D., et al. (2014). The Journal of Clinical Investigation, 124(6), 2736–2749. Farber, C. R., et al. (2011). PLoS Genetics, 7(4), e1002038. Gene Discovery Farber, C. R., et al. (2014). Journal of Bone and Mineral Research. Jun;29(6):1402-11. Calabrese, G., et al. (2012). PLoS Genetics, 8(12), e1003150. Informing GWAS Unpublished 10,968 Genes 21 Co-expression Modules
18. Bicaudal C1 homolog (BICC1) is a genetic determinant of Bone Mineral Density (BMD)
19. Bicaudal C1 homolog (BICC1) is a genetic determinant of Bone Mineral Density (BMD)
20. 0 2 4 6 2 4 6 8 10 12 −log10(BICC1 eQTL P-values) GEFOSII FN-BMD MESA L5 BMD BICC1 expression in skeletal muscle −log10(BMD P-values) 59.9 60.0 60.1 60.2 Chromosome 10 (Mbp; hg18) BICC1 rs7096154, P=3.1x10-4 rs12247559, P=3.7x10-4 rs10740740, P=3.9x10-4 rs10740740, P=8.6x10-13 Bicaudal C1 homolog (BICC1) is a genetic determinant of Bone Mineral Density (BMD)
21. HMDP Bone Co-expression Network
22. HMDP Bone Co-expression Network Where is Bicc1 located in the network? What genes share connections with Bicc1?
23. HMDP Bone Co-expression Network Where is Bicc1 located in the network? What genes share connections with Bicc1? Bicc1
24. Bicc1 is a member of Module 6
25. Bicc1 is a member of Module 6 Connectivity shared with Bicc1 (TOM) Random genes M6 genes M6 OB genes 0.000 0.005 0.010 0.015 0.020 0.025 0.030 *
26. Bicc1 is positively correlated with the expression of module 6 genes Mef2c Smad3 Wwtr1 Acvr2b Ski Bmp4 Zfp521 Ptk2 Igfbp5 Twsg1 Shox2 Gli3 Acvr1 Twist1 Chrd Smad5 Smad1 Gli2 Wnt10b Zhx3 Fgfr1 Akp2 Shox2 Pthr1 Pard6g Maged1 Gja1 Sp7 Cd276 Tmem119 Snai2 Lrp5 Bmp7 Fbn2 Relative Expression (%) 0 50 100 150 200 * Bicc1 low expressing strains (N=15) Bicc1 high expressing strains (N=15) * Bicc1 low expressing strains (N=15) Bicc1 high expressing strains (N=15) Module 6 Module 9
27. * SC Bicc1_1 Bicc1_2 Bicc1 Relative Expression (%) 0 20 40 60 80 100 120 * * SC Bicc1_1 Bicc1_2 ng AKP/ug Protein 0.0 0.1 0.2 0.3 0.4 0.5 * * cc1+/+ cc1+/jcpk Runx2 Sp7 Col1a1 Bglap Ibsp Relative Expression (%) 0 50 100 150 200 SC Bicc1_1 Bicc1_2 ** ** ** * * SC Bicc1_1 Bicc1_2 Alizarin Red (OD405nm) 0 1 2 3 4 * * SC Bicc1_1 Bicc1_2 Primary calvarial osteoblasts Bicc1 siRNAs Differentiate Quantify Mineralization
28. Bicc1 Network 0.083 0.095 0.097 0.079 0.074 0.070 0.068 0.068 0.066 0.065 0.064 0.063 Megf8 Nfib Bicc1 Sobp Dcn Itga11 Pkd2 Kctd17 Spg20 Nfatc4 Arhgef5 Dlg3 Ankrd25 Hoxc8 Lrig3 Tshz2 0.069 0.062 0.061 10.0 10.5 9.5 10.0 10.5 Bicc1 expression Pkd2 expression r=0.75 ; p=0 11.0
29. Bicc1 Network 0.083 0.095 0.097 0.079 0.074 0.070 0.068 0.068 0.066 0.065 0.064 0.063 Megf8 Nfib Bicc1 Sobp Dcn Itga11 Pkd2 Kctd17 Spg20 Nfatc4 Arhgef5 Dlg3 Ankrd25 Hoxc8 Lrig3 Tshz2 0.069 0.062 0.061 10.0 10.5 9.5 10.0 10.5 Bicc1 expression Pkd2 expression r=0.75 ; p=0 11.0
30. Polycystins, Primary Cilia and Bone Pazour GJ. JASN. 2004
31. Polycystins, Primary Cilia and Bone Pazour GJ. JASN. 2004
32. Polycystins, Primary Cilia and Bone Pazour GJ. JASN. 2004
33. Polycystins, Primary Cilia and Bone Pazour GJ. JASN. 2004 Xiao ZS. JBC. 2006; Xiao ZS. JBC. 2010.
34. Pkd2 knockdown decreases osteoblast proliferation and differentiation SC Pkd2_1 Pkd2_2 Pkd2 Relative Expression (%) 0 20 40 60 80 100 120 * * SC Pkd2_1 Pkd2_2 ng AKP/ug Protein 0.0 0.1 0.2 0.3 0.4 0.5 0.6 * * Runx2 Sp7 Col1a1 Ocn Ibsp Relative Expression (%) 0 50 100 150 200 SC Pkd2_1 siRNA Pkd2_2 siRNA # * # # * * ** * * SC Pkd2_1 Pkd2_2 SC Pkd2_1 Pkd2_2 Alizarin Red (OD405nm) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 * * SC Pkd2_1 Pkd2_2 Nodule Count 0 50 100 150 200 250 * *
35. Pkd2 rescues Bicc1 deficiency Alizarin Red (OD405nm) No. of Nodules Percent of Bicc1+/+ Bicc1+/+ Bicc1+/+ , Pkd2 Bicc1jcpk/jcpk , Pkd2 0 50 100 150 200 * * * * * Bicc1jcpk/jcpk
36. • Genome-Wide Association Studies (GWAS) have identified thousands of associations 1. Gene(s) 2. Function
37. 1. Genes that influence a disease are often functionally related (Goh, K.-I. et al. 2007. PNAS) 2. Functionally related genes are often co-expressed (Nayak, R. R. et al. 2009. Genome Research) Observations
38. Gene X Gene Y Gene Z Gene A Gene B Gene C Non-Causal Causal GWAS loci
39. Gene X Gene Y Gene Z Gene A Gene B Gene C Non-Causal Causal GWAS loci Functionally related and co- expressed
40. Gene X Gene Y Gene Z Gene A Gene B Gene C Non-Causal Causal GWAS loci Functionally related and co- expressed Genes located within GWAS loci
41. Enriched co-expression modules Gene X Gene Y Gene Z Gene A Gene B Gene C Non-Causal Causal GWAS loci Functionally related and co- expressed Genes located within GWAS loci
42. Enriched co-expression modules Gene X Gene Y Gene Z Gene A Gene B Gene C Non-Causal Causal GWAS loci Functionally related and co- expressed Genes located within GWAS loci Gene X Gene Y Gene Z Prioritize Genes Predict Function
43. 64 SNPs P<5.0 x 10-8 234 Genes Bone transcriptomic profiles from male mice of 96 inbred mouse strains (Calabrese et al. 2012. PLoS Genetics) Genome-wide co-expression network GEFOSII BMD GWAS (N~80K) (Estrada, K., et al. 2012. Nature Genetics)
44. GWAS gene enrichment in modules 6 and 9 0 1 2 3 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Module −log10(Enrichment P-value) 6 9
45. GWAS gene enrichment in modules 6 and 9 0 1 2 3 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Module −log10(Enrichment P-value) 6 9 • Enriched for genes with well- known roles in osteoblasts • Preferentially expressed in osteoblast-lineage cells • Module 6 and 9 genes, as a group, were co- expressed
46. Osteoblast Functional Module (OFM) GWAS SNP Chr Mbp Gene rs1053051 12 107.4 TMEM263 rs4233949 2 54.7 SPTBN1 rs4790881 17 2.1 SMG6 rs3736228 11 68.2 PPP6R3 rs4790881 17 2.1 HIC1 rs13245690 7 120.8 CPED1 rs7071206 10 79.4 DLG5 rs7851693 9 133.5 FUBP3 rs3801387 7 121.0 FAM3C rs11623869 14 103.9 MARK3 rs7071206 10 79.4 KCNMA1 GWAS SNP Chr Mbp Gene rs11755164 6 44.6 RUNX2 rs2016266 12 53.7 SP7 rs2062377 8 120.0 TNFRSF11B rs7932354 11 46.7 LRP4 rs3736228 11 68.2 LRP5 rs4792909 17 41.8 SOST rs1373004 10 54.4 DKK1 rs2887571 12 1.6 WNT5B rs7521902 1 22.5 WNT4 rs13245690 7 120.8 WNT16 rs12407028 1 68.6 WLS rs227584 17 42.2 HDAC5 GWAS SNP Chr Mbp Gene rs6532023 4 88.8 MEPE rs4792909 17 41.8 MEOX1 rs1366594 5 88.4 MEF2C rs7932354 11 46.7 MDK rs1566045 16 51.0 CYLD rs7017914 8 71.6 EYA1 rs479336 1 172.2 DNM3OS rs1346004 2 166.6 GALNT3 rs7108738 11 15.7 INSC rs736825 12 54.4 HOXC8 rs736825 12 54.4 HOXC6 1. Module 6 and 9 genes 2. Genes whose expression was correlated (P<0.001) with the eigengene of either module
47. DNM3OS WNT4 WLS WLS KCNMA1 INSC PPP6R3 WNT5B SP7 HOXC6 MARK3 SMG6 SOST GALNT3 SPTBN1 MEPE MEF2C CPED1 WNT16 TNFRSF11B FUBP3 ZBTB40 MBL2 ARHGAP1 MTERF2 SALL1SALL1 C17orf53 CDC5L XKR9 WNT4 DKK1 DLG5 LRP4 MDK LRP5 TMEM263 HOXC8 CYLD CYLD HIC1 MEOX1 HDAC5 RUNX2 WNT16 CPED1 FAM3C EYA1 C1orf105 DNM3 PIGC ZBTB40 DIRAS3 RPE65 DIRAS3 RPE65 SOX6 AMBRA1 ATG13 CKAP5 DGKZ F2 ZNF408 C11orf24 RIC8B ERC1 AAAS ESPL1 SP1 HOXC4 HOXC5 CKB EIF5 TRMT61A SRR TSR1 ASB16 ATXN7L3 TMUB2 CSRNP3 C2orf73SPP1 SUPT3H ING3 COLEC10 LACTB2 TRAM1 ASS1 PRDM12 5 10 15 20 rs479336 rs7521902 rs17482952 rs12407028 rs7071206 rs7108738 rs3736228 rs2887571 rs2016266 rs736825 rs11623869 rs4790881 rs4792909 rs1346004 rs4233949 rs6532023 rs1366594 rs13245690 rs3801387 rs2062377 rs7851693 rs6426749 rs1373004 rs7932354 rs1053051 rs1564981 rs1566045 rs227584 rs11755164 rs7017914 Distance to TSS (log2(bp)) OFM OFM_NONOFM 0 10 20 30 40 50 OFM OFM_NONOFM Genes (%) P=0.02 N=9 of 50 N=21 of 39 OFM genes are in close proximity to lead GWAS SNP P=0.056
48. OFM SNPs overlap active regulatory elements ENCODE Project Consortium. (2014). Nature. SNP Group OFM Lead SNPs (N=30) NOFM Lead SNPs (N=34) OFM matched random SNP set 0 10 20 30 40 50 H3K27ac H3K4me1 H3K4me2 H3K4me3 H3K79me2 H3K36me3 H3K9me3 H3K27me3 Overlap (%) P=0.02 P=0.02 P<0.001 P<0.001 P<0.001 Histone Modifications Primary Human Osteoblasts
49. OFM histone overlap is specific to osteoblasts ADRENAL BLOOD BONE BRAIN BREAST CERVIX ESC ESC_DERIVED FAT GI_COLON GI_DUODENUM GI_ESOPHAGUS GI_INTESTINE GI_RECTUM GI_STOMACH HEART IPSC LIVER LUNG MUSCLE MUSCLE_LEG OVARY PANCREAS PLACENTA SKIN SPLEEN STROMAL_CONNECTIVE THYMUS VASCULAR OSTEOBLASTS CHONDROCYTES MESENCHYMAL STEM CELLS FIBROBLASTS 10 20 30 40 −0.1 0.0 0.1 0.2 0.3 Difference (% OFM overlap - % NOFM overlap) Overlap (%) H3K27ac Roadmap Epigenomics Consortium. (2015). Nature. OFM Overlap (%)
50. Osteoblast Functional Module (OFM) GWAS SNP Chr Mbp Gene rs1053051 12 107.4 TMEM263 rs4233949 2 54.7 SPTBN1 rs4790881 17 2.1 SMG6 rs3736228 11 68.2 PPP6R3 rs4790881 17 2.1 HIC1 rs13245690 7 120.8 CPED1 rs7071206 10 79.4 DLG5 rs7851693 9 133.5 FUBP3 rs3801387 7 121.0 FAM3C rs11623869 14 103.9 MARK3 rs7071206 10 79.4 KCNMA1 GWAS SNP Chr Mbp Gene rs11755164 6 44.6 RUNX2 rs2016266 12 53.7 SP7 rs2062377 8 120.0 TNFRSF11B rs7932354 11 46.7 LRP4 rs3736228 11 68.2 LRP5 rs4792909 17 41.8 SOST rs1373004 10 54.4 DKK1 rs2887571 12 1.6 WNT5B rs7521902 1 22.5 WNT4 rs13245690 7 120.8 WNT16 rs12407028 1 68.6 WLS rs227584 17 42.2 HDAC5 GWAS SNP Chr Mbp Gene rs6532023 4 88.8 MEPE rs4792909 17 41.8 MEOX1 rs1366594 5 88.4 MEF2C rs7932354 11 46.7 MDK rs1566045 16 51.0 CYLD rs7017914 8 71.6 EYA1 rs479336 1 172.2 DNM3OS rs1346004 2 166.6 GALNT3 rs7108738 11 15.7 INSC rs736825 12 54.4 HOXC8 rs736825 12 54.4 HOXC6
51. Chr14q32.32 GWAS locus Estrada, K. et al. (2012). Nature Genetics. 0 1 2 3 4 5 6 7 rs11623869 • • •• • • • • • • • • • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • • •• • • • • • • • • • • • • • 0.2 0.4 0.6 0.8 r2 GEFOSII Femoral Neck BMD EIF5 SNORA28 MARK3 CKB TRMT61A BAG5 APOPT1 103.75 103.8 103.85 103.9 103.95 104 Position on chr14 (Mb) -log10(P-value)
52. Chr14q32.32 GWAS locus Estrada, K. et al. (2012). Nature Genetics. 0 1 2 3 4 5 6 7 rs11623869 • • •• • • • • • • • • • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • • •• • • • • • • • • • • • • • 0.2 0.4 0.6 0.8 r2 GEFOSII Femoral Neck BMD EIF5 SNORA28 MARK3 CKB TRMT61A BAG5 APOPT1 103.75 103.8 103.85 103.9 103.95 104 Position on chr14 (Mb) -log10(P-value) 9.00 9.25 9.50 9.75 10.00 0.06 0.07 0.08 0.09 0.10 Femoral BMD (g/cm2) Bone Mark3 Expression (log 2 (INT)) r=-0.25, P=0.036
53. Chr14q32.32 GWAS locus Estrada, K. et al. (2012). Nature Genetics. 0 1 2 3 4 5 6 7 rs11623869 • • •• • • • • • • • • • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • • •• • • • • • • • • • • • • • 0.2 0.4 0.6 0.8 r2 GEFOSII Femoral Neck BMD EIF5 SNORA28 MARK3 CKB TRMT61A BAG5 APOPT1 103.75 103.8 103.85 103.9 103.95 104 Position on chr14 (Mb) -log10(P-value) 9.00 9.25 9.50 9.75 10.00 0.06 0.07 0.08 0.09 0.10 Femoral BMD (g/cm2) Bone Mark3 Expression (log 2 (INT)) r=-0.25, P=0.036 Mark3 Osteoblast activity & BMD
54. Primary calvarial osteoblasts Mark3 siRNAs Differentiate Quantify Mineralization SC Mark3_1 Mark3_2 0.09 0.12 0.15 0.18 0.21 Alizarin Red (mM) SC Mark3 1 Mark3 2 P<0.05 0.00 0.25 0.50 0.75 1.00 SC Mark3 1 Mark3 2 Mark3 Relative Expression P<0.01
55. Increased Femoral BMD in Mark3 Gene Trap
56. Increased Femoral BMD in Mark3 Gene Trap Mark3 KOMP Gene Trap (tm1a) targeted ES Cells +/tm1a 45% reduction in Mark3 expression
57. Increased Femoral BMD in Mark3 Gene Trap Femoral BMD 12 week Males Genotype Adjusted BMD (g/cm2) 0.066 0.068 0.070 0.072 0.074 0.076 +/+ +/tm1a N=13 N=17 P=0.01 Mark3 KOMP Gene Trap (tm1a) targeted ES Cells +/tm1a 45% reduction in Mark3 expression
58. http://www.gtexportal.org/; GTEx Consortium. (2015). Science. 0 5 10 15 rs11623869 • • • • • • • • • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • • • • • • • 0.2 0.4 0.6 0.8 r2 EIF5 SNORA28 MARK3 CKB TRMT61A BAG5 APOPT1 103.75 103.8 103.85 103.9 103.95 104 Position on chr14 (Mb) GTEX MARK3 THYROID eQTL -log10(P-value) MARK3 local eQTL
59. http://www.gtexportal.org/; GTEx Consortium. (2015). Science. 0 5 10 15 rs11623869 • • • • • • • • • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • • • • • • • 0.2 0.4 0.6 0.8 r2 EIF5 SNORA28 MARK3 CKB TRMT61A BAG5 APOPT1 103.75 103.8 103.85 103.9 103.95 104 Position on chr14 (Mb) GTEX MARK3 THYROID eQTL Prostate Thyroid Adrenal Gland Artery Coronary Colon Transverse Pancreas Skin Sun Exposed Stomach Adipose Subcutaneous Muscle Skeletal Lung BMD 0.00 0.25 0.50 0.75 Effect of the rs11623869 “T” allele 4.6 x 10-2 5.2 x 10-16 7.4 x 10-3 4.2 x 10-2 1.1 x 10-2 9.9 x 10-3 3.6 x 10-2 2.9 x 10-2 1.7 x 10-2 1.4 x 10-2 1.6 x 10-13 1.1 x 10-2 -log10(P-value) MARK3 local eQTL
60. Farber Lab • Larry Mesner, Ph.D. • Gina Calabrese • Brianne Ray, Ph.D. • Eric Lum • Achilleas Pitsillides, Ph.D. University of Virginia • Stephen Rich, Ph.D. • Ani Manichaikul, Ph.D. UCLA • Jake Lusis, Ph.D. • Brian Bennett, Ph.D. • Luz Orozco, Ph.D. • Calvin Pan • Anatole Ghazalpour, Ph.D. • Hyun Min Kang, Ph.D. • Eleazar Eskin, Ph.D. University of Rochester • Cheryl Ackert-Bicknell, Ph.D. • Dana Godfrey Maine Medical Center Research Institute • Cliff Rosen, M.D. Johns Hopkins University • Tom Clemens, Ph.D. University of Missouri • Elizabeth Bryda, Ph.D. Vanderbilt University • Guanqing Wu, Ph.D. Harvard • Yi-Hsiang Hsu, Ph.D. USCD • Micheal H. Criqui, M.D. • Matthew Allison, M.D. Harbor-UCLA • Matthew Budoff, M.D. University of Namur, Belgium • Olivier De Backer, Ph.D. • Carlos Dombret, Ph.D. R01-AR057759 R56-AR064790 R01-AR064790
61. Farber lab and CPHG/UVa are hiring! Two open rank tenure-track computational biology faculty positions in the Center for Public Health Genomics Three open rank tenure-track faculty positions in biomedical data sciences at UVa crf2s@virginia.edu Two positions (Postdoc/Analyst) in genomic data science in the Farber lab