Research Update on Cerebral Cavernous Malformations

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Research Update on Cerebral Cavernous
Malformations

• Issam A. Awad, MD, MSc, FACS, MA(Hon)
• Professor and Vice Chairman
• Department of Neurosurgery
• Feinberg School of Medicine
• Northwestern University
• Evanston Northwestern Healthcare
• Evanston, Illinois

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Cerebral Vascular Malformations ResearchA
Legacy of Collaborations During Two Decades

• The clinical foundations-- BNI and Cleveland
  Clinic
• (Spetzler, Little, Robinson, Estes,
  DeCorletto, et al.)
• The translational science-- Yale
• (Lifton, Kim, Gunel, Rothbart, Wong, Baev,
  White, Awadallah, et al.)
• Genomics of lesions-- the Colorado team
• (Uranishi, Gault, Shenkar, Lepsch, Hu,
  Sarin, Elliott, et al.)
• A new horizon-- ENH and NWU Integrational
  Neuroscience
• (Shenkar, Meade, Wyrwicz, Rowley, Zao,
  Kohlmeir, Batjer, Edelman, et al.)

Support by NIH (NINDS) R01NS36194 K24NS02153,
Butcher Family Foundation, ENH Research Institute
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Categorization of Disease The Phenotype
Natural History
Clinical-Radiologic-Pathologic Correlations
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Refining the Phenotype

• Structural and molecular dissection
• Angioarchitecture
• Ultrastructure
• Functional pathways -- angiogenesis

Regional venous dysmorphism
Hemorrhagic proliferative dysangiogenesis
(Robinson et al. 1989, 1992, 1993)
Arteriovenous shunting
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Refining the Phenotype

• Structural and molecular dissection
• Angioarchitecture
• Ultrastructure
• Functional pathways -- angiogenesis

CCMs Defective inter-EC junctions and sub-EC
structure (Wong et al. 1998)
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Refining the Phenotype

• Structural and molecular dissection
• Angioarchitecture
• Ultrastructure
• Functional pathways -- angiogenesis

Angiogenesis factor upregulation different
roles in AVMs vs. CCMs ? (Rothbart et al. 1997,
Uranishi et al. 2000)
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Refining the Phenotype
SMA
Myosin
Smoothelin

• Structural and molecular dissection
• Smooth muscle maturation

AVM
Defective SMC maturation in CCMs (Uranishi et al.
2001)
CCM
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The Genetic Substrate of CCMs

• 20-40 familial (multiple lesions)
• Three gene loci (7q, 7p, and 3q) in familial
  cases
• Hispanic Americans of Mexican descent are CCM1
  (Q455X with preserved haplotype)-- founder
  mutation
• CCM1 gene is KRIT1--all CCM1 mutations result in
  premature truncation
• CCM2 gene is MGC4607
• CCM 3 gene is XXXXX

(Gunel et al., Tournier-Lasserve et al., others
1995-2005, Marchuck 2003)
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Mutations in KREV INTERACTION TRAPPED 1 (KRIT1)
cause CCM1
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3UT

















Truncating (73), 15 intron
5UT
Retinal
Cutaneous
Hispanic

Amino Acid
coding
NPXY motif binds icap-1A ( integrin cytoplasnic
domain-assoc protein-1) cell-cell adhesion
Ankyrin Repeats (protein-protein interaction)
FERM domain (links cytoplasmic and transmembrane
proteins)
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Genetic Clues to Molecular Mechanism of Disease
CCM

• KRIT1 is related to cytoskeletal
  proteins--cellular adhesion pathway
• KRIT1 is associated with tube formation during
  angiogenesis
• Working hypothesis-- KRIT1 mediated defective
  inter-EC junctions predispose to CCM lesion
  genesis

Serebriiskii et al 1997 Zhang et al.
2001,2003 Zawistowski et al. 2002, 2003 Gunel et
al. 2002
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Genetic Clues to Molecular Mechanism of Disease
CCM
Angiogenesis Growth Factors/Receptors (VEGF,
Angiopeotin, TGF-?, FGF-2)
Endothelial cells
Cell Adhesion Molecules (CD31)
Krev-1/rap1A (signaling)
Krit1-associated microtubules
ICAP-1?
Extracellular Matrix Proteins ?laminin,
?fibronectin,
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Phenotype, Genotype and Clinical Behavior

• Solitary vs multiple lesions Gr Echo MR (T2) as
  surrogate endophenotype
• Venous angioma
• Previous irradiation
• Penetrance and aggressive behavior
• ( ? gene, mutation, promoter)
• Combined risk

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Sporadic Lesions Hidden Germ Line or Somatic
Mutation ?

• Occult familiality--
• Incomplete penetrance--
• Rare in absence of family history or lesion
  multiplicity (0/gt100) MR with gradient echo
  excellent screen in suspected cases
• Somatic mutations--
• Two CCM cases reported (Kehrer-Sawatzki et al.,
  Gault et al , work in progress (CCM1, CCM2,
  transheterozygous?)
• Sporadic mutation in CVM genes or related
  pathways-- Working hypothesis

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Somatic Mutation in Human CCM Lesion from Patient
with Germ Line Mutation in Second Copy of Same
Gene

• ? How common
• ? Transheterozygous
• ? Other second hits

Gault et al. (Stroke 2005) Somatic mutation in
endothelial cells (laser capture,
unpublished 2005)
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Double Hits and Lesion Genesis Transgenic Mice

• CCM1 mutation alone does not seem to produce CCM
  lesions
• Combined genetic predisposition causes CCM
  lesions (ccm /-, P53 -/-)
• Opportunity to study CCM genesis and progression
  in vivo

T2 (GrEcho) MR of transgenic mouse brain 4.7 T
(collaboration Marchuk Awad, 2005)
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Gene Interactions Differential Gene Expression
mRNA

• CCMs compared to AVMs and STAs
• 42 genes significantly upregulated
• 36 genes significantly downregulated
• AVMs compared to CCMs and STAs
• 48 genes significantly upregulated
• 59 genes significantly downregulated

Shenkar et al. 2003
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Genomics Data Analysis
File containing mean
intensity for each gene
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Scaling (equalize average intensity across chips)
File containing scaled data
Confirmation of
Gene Discovery
Differential Expression
of Known Genes
Matlab
GeneSpring
Genes that vary significantly
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15 genes of interest
Compare groups
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InStat
Genes differentially expressed
Statistical Analysis
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Expression Levels and Significance
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Gene Interactions Differential Gene Expression
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Genes upregulated gt20X in CCM over controls
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Immune Response in CCM ?

• Leaky vessels and antigenic challenge
• Organizing thrombus
• ? Cell type
• ? Monoclonal or oligoclonal

B Cells and Plasma Cells
IGG
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High Field MR of CCM Lesions
1 mm
1 mm
10 mm
1 mm
0.1 mm

• Humans up to 4.5 T
• Animals up to 14 T
• Near histologic resolution

1.5 T
1.5 T
Wyrwicz, Edelman, Awad, Shenkar 2005
3.0 T
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Imaging Cellular Events in CCM Lesions
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Imaging Cellular Events in CCM Lesions
Resolution and Labeling
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Dual Probes For Molecular Imaging of Individual
Cells
Meade et al. 2003-2005
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Building Molecular Markers for MR imaging and
Histologic Validation
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Modern Technical Applications to Microsurgical
Excision of CM

• Functional MR if lesion near eloquent areas
• Intraoperative brain mapping
• Image guided navigation

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Consider and Respect Subcortical Tracts
Tensor Diffusion MR imaging
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Case Study Transcallosal Approach to Thalamic
Lesion
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Case Study Transsylvian Approach to Thalamic
Lesion
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Research Future Integrated Teams for Efficient
Translation
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Future DirectionsAn Evolving Story

• Explaining disease behavior
• Predicting disease behavior
• Modifying disease behavior