Transcriptome profiling in collagen VI related myopathies

Muscle Study GroupSeptember 22, 2014

R.J. Butterfield, MD, PhDUniversity of Utah, Salt Lake City UT,

Ullrich congenital muscular dystrophy

Bönnemann CG. The collagen VI-related myopathies Ullrich congenital muscular dystrophy and Bethlem myopathy. Handb Clin Neurol. 2011;101:81-96

Bethlem myopathy

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

ControlCOL6DominantNegativeCOL6Null

PC1

PC2

−30

−20

−10

0

10

20

−30 −20 −10 0 10 20

Significant DE genes

EGR1LENG8CYP26B1RPL22L1EMP1LDB1MAPK8IP3CNTNAP1MCM6FTLNEAT1SFRP1DUSP1PDE4BKREMEN1SLC29A1CSDC2GALNT5

ARMC9KCNJ15FZD2FKBP5DUSP6CDR1HSPB1MCAMCDC6MSMPME1MCM3MTRNR2L10ADCK2PILRBE2F1IDI2MMS22L

WBP2IER2CDC45TNS3ABHD16BCRMP1C1orf192SLC18A2CEMP1STARD10DTLKDM6BNABP1MCM2

Dominant Negative vs Null(718 DE genes)

FAM134BITIH5GGT5TCF7L1SCN9ACDCP1GLI2GPR68IGFBP7LTBP3PVRL3AMIGO2KIAA1644DMDFGD6GPR126PRLRRARRES2

CD24ZDHHC15KIAA1024BCAMCEBPDMMP12LRP5PDE5ASTMN3FLT1GULP1RDH10LDB2CYGBGALNT15HIC1KRT19NXN

SBSNTRIB2NUAK1TNFRSF1BARRDC4MEGF10KIAA1199MUC1CILP2PTCH1COL4A1CAV1ALDH2FEN1

Control vs Null(172 DE genes)

ACECALB2CFDFAM20AFKBP5KIAA1324LAPODGALNT5PTCHD4GALNT15GREM1OLFML2AARMC9NEU1SGMS2SCINDPYDCOL15A1

TMEM132BPTPRNDMDTTYH3PLXNB3LSP1NFIBFTLFESGPRC5CABI3BPGBASPRY1SLC29A1AQP1LDB1IL1RNSERPINF1

TMEM26ADCY3ARHGAP29PDE1CAEBP1PLSCR4NPTXRMLLT11MTUS1GBP5PLOD2TRIB2CD24PLD1

Control vs Dominant Neg(302 DE genes)

Gene Ontologies DMDMolecular FunctionGO:0002162 dystroglycan binding GO:0003779 actin binding GO:0005200 structural constituent of cytoskeleton GO:0005509 calcium ion binding GO:0005515 protein binding

Cellular localizationGO:0005634 nucleus GO:0005829 cytosol GO:0005856 cytoskeleton GO:0005886 plasma membrane GO:0009986 cell surface

Biologic ProcessGO:0001954 positive regulation of cell-matrix adhesion GO:0002027 regulation of heart rate GO:0006355 regulation of transcription, DNA-templated GO:0007517 muscle organ development GO:0007519 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

Downregulation: inflammatory and tissue remodeling genes:•IIL1B,•CXCL3, CXCL5, CXCL6•CCL5•MMP3, MMP10, MMP12, MMP1

Upregulation: extracellular matrix components•COL14A1•COL15A1•COL8A2•COMP

Differential Expression in Dominant Negative (8) vs Control (4) –Network analysis in 5 most significantly GO Categories

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 mutationc.849G>A, incudes glycine substitution

loxP siteto induce deletion ofexons 9-14 withCreexposure

ACEAngiotensin converting enzyme

XLOC_009222

5

10

normal

col6

sample_name

FPKM

quant_status

OK

tracking_id

XLOC_009222

ACEXLOC_009222

5

10

DN

CO

L6null

sample_name

FPKM

quant_status

OK

tracking_id

XLOC_009222

ACE

DN vs control DN vs null

CFDComplement Factor D

TCONS_00020398

0

5

10

15

20

normal

col6

sample_name

FPKM

quant_status

OK

tracking_id

TCONS_00020398

CFD

TCONS_00020398

0

5

10

15

DN

CO

L6null

sample_name

FPKM

quant_status

OK

tracking_id

TCONS_00020398

CFD

DN vs control DN vs null

CXCL5Chemokine (C-X-C motif) ligand 5

TCONS_00032544

0

25

50

75

DN

CO

L6null

sample_name

FPKM

quant_status

OK

tracking_id

TCONS_00032544

CXCL5

DN vs control DN vs null

PI16Protease inhibitor 16

XLOC_018272

0

10

20

normal

col6

sample_name

FPKM

quant_status

OK

tracking_id

XLOC_018272

PI16XLOC_018272

0

10

20

30

DN

COL6null

sample_name

FPKM

quant_status

OK

tracking_id

XLOC_018272

PI16

DN vs control DN vs null

COL15A1Collagen 15

TCONS_00043668

0

100

200

normal

col6

sample_name

FPKM

quant_status

OK

tracking_id

TCONS_00043668

COL15A1TCONS_00043668

0

100

200

300

DN

COL6null

sample_name

FPKM

quant_status

OK

tracking_id

TCONS_00043668

COL15A1

DN vs control DN vs null