Transcriptome profiling in collagen VI related myopathies Muscle Study Group September 22, 2014 R.J. Butterfield, MD, PhD University of Utah, Salt Lake City UT, Ullrich congenital muscular dystrophy Bethlem myopathy Bönnemann CG. The collagen VI-related myopathies Ullrich congenital muscular dystrophy and Bethlem myopathy. Handb Clin Neurol. 2011;101:81-96 COL6 Structure and Genetics • Most mutations in triple helix domain and disrupt Gly-X-Y repeat – Function as dominant negative – Abnormal COL6 in the matrix • Null mutation produce no COL6 in the matrix – < 5% of patients – Current mouse model is homozygous nonsense mutation in Col6a2 • Phenotype variable even with identical mutation Problem • COL6 in muscle is expressed primarily by interstitial fibroblasts not by myogenic cells – Interaction between the fibroblast, myofiber, and ECM results in disease • The factors underlying this interaction are largely unknown Hypothesis 1. Identification of interactions between myofiber, fibroblast, and matrix will lead to better understanding of disease pathogenesis and provide targets for therapy 2. Differences between dominant negative and null mutants highlight the role of abnormal COL6 in the matrix Transcriptome Analysis • RNA-seq – Sequence all expressed transcripts from poly-A purified RNA – 18 billion bp sequenced in 50bp reads • Map to 23,159 RefSeq genes • 12,629 genes with sufficient depth to test differential expression • Analysis – Differential expression (DE) • Cufflinks, DEseq, EdgeR – Identification of critical genes and pathways in pathogenesis from DE genes • Gene Ontology analysis • Pathway analysis Samples for RNA-seq • Total RNA from cultured dermal fibroblast • Patients with UCMD or Intermediate phenotype – 9 Control – 10 Dominant negative • 6 Glycine substitution in triple helix • 4 Exon skipping – 5 Null Overall sample relatedness Principal components based on expression levels Control COL6 Dominant Negative COL6 Null 20 PC2 10 0 −10 −20 −30 −30 −20 −10 0 PC1 10 20 Significant DE genes Control vs Dominant Neg (302 DE genes) ACE CALB2 CFD FAM20A FKBP5 KIAA1324L APOD GALNT5 PTCHD4 GALNT15 GREM1 OLFML2A ARMC9 NEU1 SGMS2 SCIN DPYD COL15A1 TMEM132B PTPRN DMD TTYH3 PLXNB3 LSP1 NFIB FTL FES GPRC5C ABI3BP GBA SPRY1 SLC29A1 AQP1 LDB1 IL1RN SERPINF1 TMEM26 ADCY3 ARHGAP29 PDE1C AEBP1 PLSCR4 NPTXR MLLT11 MTUS1 GBP5 PLOD2 TRIB2 CD24 PLD1 Control vs Null (172 DE genes) FAM134B ITIH5 GGT5 TCF7L1 SCN9A CDCP1 GLI2 GPR68 IGFBP7 LTBP3 PVRL3 AMIGO2 KIAA1644 DMD FGD6 GPR126 PRLR RARRES2 CD24 ZDHHC15 KIAA1024 BCAM CEBPD MMP12 LRP5 PDE5A STMN3 FLT1 GULP1 RDH10 LDB2 CYGB GALNT15 HIC1 KRT19 NXN SBSN TRIB2 NUAK1 TNFRSF1B ARRDC4 MEGF10 KIAA1199 MUC1 CILP2 PTCH1 COL4A1 CAV1 ALDH2 FEN1 Dominant Negative vs Null (718 DE genes) EGR1 LENG8 CYP26B1 RPL22L1 EMP1 LDB1 MAPK8IP3 CNTNAP1 MCM6 FTL NEAT1 SFRP1 DUSP1 PDE4B KREMEN1 SLC29A1 CSDC2 GALNT5 ARMC9 KCNJ15 FZD2 FKBP5 DUSP6 CDR1 HSPB1 MCAM CDC6 MSMP ME1 MCM3 MTRNR2L10 ADCK2 PILRB E2F1 IDI2 MMS22L WBP2 IER2 CDC45 TNS3 ABHD16B CRMP1 C1orf192 SLC18A2 CEMP1 STARD10 DTL KDM6B NABP1 MCM2 Gene Ontologies DMD Molecular Function GO:0002162 GO:0003779 GO:0005200 GO:0005509 GO:0005515 dystroglycan binding actin binding structural constituent of cytoskeleton calcium ion binding protein binding Cellular localization GO:0005634 GO:0005829 GO:0005856 GO:0005886 GO:0009986 nucleus cytosol cytoskeleton plasma membrane cell surface Biologic Process GO:0001954 GO:0002027 GO:0006355 GO:0007517 GO:0007519 positive regulation of cell-matrix adhesion regulation of heart rate regulation of transcription, DNA-templated muscle organ development skeletal muscle tissue development Enrichment of Gene Ontologies among DE Genes Control vs Dominant Negative Enrichment of Gene Ontologies among DE Genes Control vs Null Differential Expression in Dominant Negative (8) vs Control (4) –Network analysis in 5 most significantly GO Categories Downregulation: inflammatory and tissue remodeling genes: •IIL1B, •CXCL3, CXCL5, CXCL6 •CCL5 •MMP3, MMP10, MMP12, MMP1 Upregulation: extracellular matrix components •COL14A1 •COL15A1 •COL8A2 •COMP Conclusions • Transcriptome profiles are different depending on mutation type (presence or absence of abnormal collagen VI in matrix) – Genes controlling cell cycle, regeneration are important in Null mutants – Genes controlling production of extracellular matrix and matrix remodeling are important in Dominant Negative mutants • Current mouse model (Null) may not reflect extent of molecular pathogenesis Future Direction • Identify molecular pathways among DE genes important in pathogenesis – Identify regulators of interactions between fibroblast, myofiber and matrix • Identify biomarkers and targets for therapy • Mouse model (pending K08) with dominant negative mutation inducible to null (via Cre exposure) – Allows temporal and spatial control of the expression of the mutation Acknowledgement • Bob Weiss—Utah – Diane Dunn • Carsten Bonnemann—NIH, NINDS – Ying Hu – Yaqun Zou – Funding • CureCMD • Primary Children’s Hospital Foundation Gene Ontology Enrichment Dominant Negative vs Null 1kB Col6a1-mouse chromosome 10 Missense mutation c.849G>A, incudes glycine substitution loxP site to induce deletion of exons 9-14 with Cre exposure ACE Angiotensin converting enzyme DN vs control DN vs null ACE ACE XLOC_009222 XLOC_009222 10 quant_status 10 quant_status OK FPKM FPKM OK tracking_id tracking_id XLOC_009222 XLOC_009222 5 5 COL6null DN col6 normal sample_name sample_name CFD Complement Factor D DN vs control DN vs null CFD CFD TCONS_00020398 TCONS_00020398 15 20 quant_status 10 OK quant_status OK FPKM FPKM 15 10 tracking_id tracking_id TCONS_00020398 TCONS_00020398 5 5 0 0 COL6null DN col6 normal sample_name sample_name CXCL5 Chemokine (C-X-C motif) ligand 5 DN vs control DN vs null CXCL5 TCONS_00032544 75 FPKM quant_status OK 50 tracking_id TCONS_00032544 25 0 COL6null DN sample_name PI16 Protease inhibitor 16 DN vs control DN vs null PI16 PI16 XLOC_018272 XLOC_018272 30 20 FPKM FPKM 20 quant_status quant_status OK OK tracking_id tracking_id 10 XLOC_018272 XLOC_018272 10 0 0 COL6null DN col6 normal sample_name sample_name COL15A1 Collagen 15 DN vs control DN vs null COL15A1 COL15A1 TCONS_00043668 TCONS_00043668 300 200 quant_status FPKM FPKM quant_status 200 OK OK tracking_id tracking_id TCONS_00043668 100 TCONS_00043668 100 0 0 COL6null DN col6 normal sample_name sample_name