COL6 RNA-seq

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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
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