Combined use of clinical and MRI Keywords angiogenesis; CCM1; CCM2; CCM3; cerebral cavernous malformations; cerebral hemorrhage; KRIT1; PDCD10; stroke; vascular malformations Corresponde
Trang 1Recent insights into cerebral cavernous malformations: the molecular genetics of CCM
Florence Riant1,2, Francoise Bergametti2, Xavier Ayrignac2, Gwenola Boulday2and Elisabeth
Tournier-Lasserve1,2
1 AP-HP, Hoˆpital Lariboisie`re, Laboratoire de Ge´ne´tique, Paris, France
2 INSERM UMR-S 740, Universite´ Paris 7 Diderot, France
Introduction
Cerebral cavernous malformations (CCM⁄ OMIM
116860) are vascular lesions histologically
character-ized by abnormally enlarged capillary cavities
with-out intervening brain parenchyma From large series
based on necropsy and⁄ or magnetic resonance
imag-ing (MRI) studies, their prevalence in the general
population has been estimated to be close to 0.1–
0.5% Most CCMs are located within the central
nervous system but they sometimes affect either the
retina or the skin [1]
CCM occur both sporadically and in a familial
context The proportion of familial cases has been
esti-mated to be as high as 50% in Hispano-American
CCM patients [2] and close to 10–40% in Caucasian
patients [1] The CCM pattern of inheritance is
autosomal dominant with incomplete clinical and neuroradiological penetrance The presence of multiple lesions on cerebral MRI is one of the main features of familial CCM which is an evolutive condition as assessed by the strong correlation between patient age and the number of lesions (Fig 1) [1–3] The average age-of-onset is around 30 years but symptoms can start in early infancy or in old age The main symptoms include seizures and cerebral hemorrhages Sporadic cases most often have a single lesion on MRI, are not inherited and do not carry a CCM gene germline mutation However, some CCM patients who have multiple MRI lesions do not have any known clinically affected relative and therefore present as sporadic cases Combined use of clinical and MRI
Keywords
angiogenesis; CCM1; CCM2; CCM3;
cerebral cavernous malformations; cerebral
hemorrhage; KRIT1; PDCD10; stroke;
vascular malformations
Correspondence
E Tournier-Lasserve, INSERM UMR-S 740;
Universite´ Paris7 Diderot, 10 Avenue du
Verdun, 75010 Paris, France
Fax: +33 157278594
Tel: +33 157278593
E-mail: tournier-lasserve@univ-paris-diderot.fr
(Received 1 August 2009, revised 4
November 2009, accepted 4 December
2009)
doi:10.1111/j.1742-4658.2009.07535.x
Cerebral cavernous malformations (CCM) are vascular lesions which can occur as a sporadic (80% of the cases) or familial autosomal dominant form (20%) Three CCM genes have been identified: CCM1⁄ KRIT1, CCM2⁄ MGC4607 and CCM3 ⁄ PDCD10 Almost 80% of CCM patients affected with a genetic form of the disease harbor a heterozygous germline mutation in one of these three genes Recent work has shown that a two-hit mechanism is involved in CCM pathogenesis which is caused by a com-plete loss of any of the three CCM proteins within endothelial cells lining the cavernous capillary cavities These data were an important step towards the elucidation of the mechanisms of this condition
Abbreviations
CCM, cerebral cavernous malformations; MRI, magnetic resonance imaging; PDCD10, Programmed Cell Death 10.
Trang 2screening with molecular testing has helped to clarify
what might have first seemed confusing [1]
Three CCM genes have been mapped and identified
in the recent years These molecular genetics data have
provided useful information for clinical care of the
patients and were an important step towards the
understanding of the mechanisms of this disorder This
minireview summarizes the advances in CCM
molecu-lar genetics and the remaining gaps in this field In
addition to the identification of CCM genes, a number
of recent biochemical in vitro studies and in vivo CCM
animal model studies have helped to unravel the
func-tional roles of these proteins and are be the focus of
the two accompanying minireviews by Faurobert and
Albiges-Rizo [4] and Chan et al [5]
CCM genes germline mutations Genetic linkage analyses mapped three CCM loci to chromosome 7q (CCM1), 7p (CCM2) and 3q (CCM3) [6,7] A strong founder effect has been oberved in His-pano-American CCM patients with most families linked to the CCM1 locus [8] In Caucasian families, the proportions of families linked to each CCM locus were 40% (CCM1), 20% (CCM2) and 40% (CCM3) [7] The three genes located at these loci have now been identified (Fig 2) [9–13]
The CCM1 gene contains 16 coding exons which encode for Krit1, a 736-amino acid protein containing three ankyrin domains and one band 4.1 ezrin radixin moesin (FERM) domain CCM2, a 10-exon gene, encodes for the MGC4607 protein, also called malc-avernin, which contains a phosphotyrosin binding domain CCM3 includes seven exons which encode for Programmed Cell Death 10 (PDCD10), a protein with-out any known conserved functional domain, which may be involved in apoptosis Considerable progress has been made recently in understanding the biochemi-cal pathways in which those proteins might be involved (see Faurobert and Albiges-Rizo [4])
More than 150 distinct CCM1⁄ CCM2 ⁄ CCM3 germ-line mutations have been published to date [9–23] Those mutations were highly stereotyped because almost all led to a premature termination codon through different mechanisms including nonsense, splice-site and frameshift mutational events, as well as large genomic rearrangements These data strongly suggest that a loss of function, through mRNA decay
of the mutated allele, is the most likely pathophysio-logical mechanism involved in CCM patients Only four ‘missense’ mutations within CCM1 have been reported to date; interestingly, all of them actually activated cryptic splice sites and led to an aberrant splicing of CCM1 mRNA and a frameshift with a
Fig 1 Cerebral magnetic resonance imaging of a 4-year-old familial
CCM patient Multiple CCM lesions are shown (arrowheads) as
well as a cerebral hemorrhage (arrow).
Fig 2 CCM loci and genes Three CCM
genes have been mapped and identified to
date, CCM1 ⁄ KRIT1, CCM2 ⁄ MGC4607 and
CCM3 ⁄ PDCD10 In 22% of CCM cases
with multiple lesions, no mutation is
detected in these three genes using
currently available technologies.
Trang 3premature stop codon [10,24] The only known
mis-sense mutation which did not affect splicing has been
located within the C-terminal part of the
phosphotyro-sin binding domain of CCM2 [12] This mutation has
been shown to abolish the interaction of CCM2 and
CCM1, strongly suggesting its causality [25] Only four
inframe deletions have been reported: two affect
ex-ons 17 and 18 of CCM1, one deletes exon 2 of CCM2
and one deletes exon 5 of CCM3 The last two
dele-tions have been used to map potential relevant
interac-tion domains of CCM2 and CCM3 [23,26] However,
it is not known if these putative truncated proteins
were indeed produced and stable in vivo
Sequencing of all coding exons of the three CCM
genes and search for genomic rearrangements using
cDNA and⁄ or quantitative multiplex PCR such as
multiplex ligation-dependent probe amplification in
Caucasian non-Hispano-American CCM multiplex
families led to the identification of the causative
muta-tion in 95% of the families [23,27] Approximately
72% of multiplex families harbored a mutation in
CCM1, 18% in CCM2 and 10% in CCM3 The
CCM3 proportion was much lower than expected,
based on previous linkage data which suggested that
40% of CCM families were linked to the CCM3 locus
The mutation detection rate was lower in sporadic
cases with multiple lesions, ranging from 45% to 67%
[22,23,27] Most of these sporadic cases with multiple
lesions had either inherited their mutation from one of
their asymptomatic parents because of incomplete
pen-etrance or had a de novo mutation Sporadic cases with
multiple lesions in whom no mutation was detected are
nevertheless most likely affected by a genetic form of
the disease Several hypotheses may be raised to
explain the absence of any detected mutation,
includ-ing a somatic mosaicism of a de novo mutation which
occured during gestation and is not detectable in DNA
extracted from peripheral blood cells It will be
impor-tant to solve this in the future because it is of interest
for genetic counseling [1] With regard to sporadic
CCM cases with a unique lesion on cerebral MRI, no
mutation was detected in reported series [16,17]
Com-bination of these data with those obtained in familial
CCM strongly suggests that sporadic cases with a
unique lesion who would harbor a germline mutation
are most likely very rare
Haplotyping data strongly suggested a founder effect
in the Hispano-American CCM population; this was
confirmed by the detection of a Q455X stop codon
mutation in CCM1 in most families with this ethnic
background [10] Recurrent mutations have also been
identified in a few additional populations [21,28]
How-ever, in most cases, despite their highly stereotyped
consequences, germline CCM mutations are ‘private’ mutations present in only one or very few families
Biallelic somatic and germline mutations in CCM lesions Based on the autosomal dominant pattern of inheri-tance of CCM and the presence of multiple lesions in familial CCM, contrasting with the detection of a sin-gle lesion in nonhereditary cavernous angiomas, it has been proposed that a second hit affecting the wild-type allele might be involved in CCM lesions pathophysiol-ogy, as reported previously in retinoblastoma or other vascular malformations [29,30] According to this hypothesis, CCM formation would be caused by a complete loss, within affected cells, of the two alleles
of a given CCM gene Loss of one of the alleles (first hit) would be the result of a germline mutation and loss of the second allele (second hit) will occur somati-cally
This hypothesis is not easy to test because of the heterogeneous cellular nature of CCM lesions and the very limited number of endothelial cells lining the cap-illary cavities Indeed, direct sequencing of the DNA extracted from a heterogeneous lesion may not detect the mutation depending of the proportion of the cells which harbor this mutation within the lesion This approach was initially used to screen CCM lesions from both sporadic and a few familial patients and did not detect any somatic mutation except in one spo-radic case [31] In this latter case, two CCM1 missense mutations, F97S and K569E, were detected in the CCM lesion and were shown to be absent in the blood
of the patient However, the data were difficult to interprete because of the nature of the mutations which were not truncating mutations (a possible aber-rant splicing effect of these two mutations was not investigated) and the fact that the biallelism of these mutations was not explored
In 2005, Gault et al reported the first biallelic CCM1 germline and somatic truncating mutation in a CCM lesion, strongly supporting this ‘two-hit’ mecha-nism in the formation of lesions, at least in CCM1 patients; they demonstrated recently that this second hit occurred within the endothelial cells [32,33]
Biallelic somatic and germline mutations in each of the three CCM genes were recently reported by Akers
et al [34] These authors amplified and sequenced a large number of clones from 10 CCM lesions resected from patients harboring a heterozygous germline muta-tion in either CCM1 (two patients), CCM2 (five patients) and CCM3 (two patients) One CCM lesion was analyzed for each patient They were able to
Trang 4convincingly establish the presence of a biallelic
somatic and germline deleterious mutation in four of
these lesions from two CCM1 patients, one CCM2
patient and one CCM3 patient The proportion of
amplicons carrying the somatic mutation ranged from
4% to 16% None of these mutations was detected
through direct sequencing of lesion DNA, emphasizing
the lack of sensitivity of direct sequencing of lesion
DNA These data established the existence of biallelic
somatic and germline mutations, whatever the nature
of the CCM gene involved, at least in some lesions
No mutation was detected in the six remaining lesions
Several hypotheses may be raised to explain this
absence of mutation including the incomplete
sensitiv-ity of this type of approach which would miss a second
hit consisting in either large genomic deletions and⁄ or
epigenetic silencing mechanisms
Interestingly, the authors showed, using laser
cap-ture, that the somatic mutation occurred in endothelial
cells and not in the intervening neural tissue The
pro-portion of endothelial cells which harbor the somatic
mutation was estimated in one lesion and shown to be
close to 30%, suggesting the mosaicism of this somatic
mutation These data are in agreement with those
obtained very recently with an
immunohistochemistry-based approach which showed a mosaic loss of
expres-sion of CCM proteins in endothelial cells lining CCM
caverns [35] This question would, however, require
additional investigations It would also be important
to analyze several lesions from a given patient to test
for the presence of the same mutation in multiple
lesions A unique somatic mutation has indeed been
detected in multifocal lesions in another hereditary
vascular condition suggesting a common origin for
abnormal endothelial cells lying in distant sites [36]
Altogether these data strongly suggest that CCM, as
several other hereditary vascular conditions, show a
paradominant inheritance It remains to determine
when do occur the somatic, second hit, events
Are there additional CCM genes?
Previous linkage data obtained on 20 large North
American families suggested that the three CCM loci
on 7p, 7q and 3q would most likely account for all
CCM families [7] However, despite extensive screening
of exonic sequences for point mutations and deletions,
no mutation was detected in 5% of familial CCM
cases and a larger proportion of sporadic cases wth
multiple lesions [27] In addition, the proportion of
families showing a mutation within PDCD10 (10%) at
the CCM3 locus on chromosome 3q25, was much
lower than expected based on linkage data (40%)
Several mutually nonexclusive hypotheses may explain these data such as: (a) the existence of muta-tions affecting cis-regulatory elements located at long distances from known CCM transcription units; (b) epigenetic silencing of these three genes; and (c) the existence of additional nonidentified CCM genes, one
of which is possibly located close to PDCD10
Recently, an additional gene, Zona Pellucida-like Domain containing 1(ZPLD1), has been reported to be disrupted in a CCM patient harboring a balanced translocation between chromosome X and chromo-some 3q [37] ZPLD1 is located on chromochromo-some three centromeric to PDCD10 The expression of the mRNA
in lymphoblastoid cell lines of the patient was shown
to be significantly decreased suggesting that the inter-ruption of this gene may be causal However, the same authors screened this gene in 20 additional CCM patients without any mutation in CCM1⁄ CCM2 ⁄ CCM3 and did not detect any mutation These data suggest that either this gene is involved in very rare CCM patients or its interruption does not cause CCM but that the translocation present in this patient deregulated the expression of a gene unidentified yet Additional work currently conducted in several teams should help in the next future to identify the molecular anomalies of CCM patients ‘without’ mutations
Conclusions and future The recent identification of the three CCM genes is an important step towards the elucidation of the mecha-nisms of this condition It helped to clarify several fea-tures of this condition including its incomplete clinical and MRI penetrance as well as the molecular basis of sporadic cases with multiple lesions Additional large series studies are needed to evaluate genotype–pheno-type correlations (particularly the prognosis) depending
of the nature of the mutated gene Several additional questions, however, have to be adressed What is the nature of the molecular anomaly in familial CCM cases in whom no mutation has been detected? Are sporadic cases CCM patients with multiple lesions showing a mosaicism for a germline mutation? Are there modifying genes that may explain the intrafamil-ial clinical variability? In addition to these questions, one main challenge is to understand the mechanisms
of this condition The recent identification of several of the biochemical pathways involving CCM proteins as well as the analysis of several fish and mouse CCM animal models has already provided a number of clues
to this goal (see Faurobert and Albiges-Rizo [4] and Chan et al [5])
Trang 51 Labauge P, Denier C, Bergametti F &
Tournier-Las-serve E (2007) Genetics of cavernous angiomas Lancet
Neurol Mar 6(3), 237–244
2 Rigamonti D, Drayer BP, Johnson PC, Hadley MN,
Zabramski J & Spetzler RF (1988) Cerebral cavernous
malformations Incidence and familial occurence
N Engl J Med 319, 343–347
3 Labauge P, Laberge S, Brunereau L, Le´vy C &
Tour-nier-Lasserve E (1998) Hereditary cerebral cavernous
angiomas: clinical and genetic features in 57 French
families Lancet 352, 1892–1897
4 Faurobert E & Albiges-Rizo C (2010) Recent insights
into cerebral cavernous malformations: a complex
jigsaw puzzle under construction FEBS J 277, 1084–
1096
5 Chan AC, Li DY, Berg MJ & Whitehead KJ (2010)
Recent insights into cerebral cavernous malformations:
animal models of CCM and the human phenotype
FEBS J 277, 1076–1083
6 Dubovsky J, Zabramski JM, Kurth J, Spetzler RF,
Rich SS, Orr HT & Weber JL (1995) A gene
responsi-ble for cavernous malformations of the brain maps to
chromosome 7q Hum Mol Genet 4, 453–458
7 Craig HD, Gu¨nel M, Cepeda O, Johnson EW, Ptacek
L, Steinberg GK, Ogilvy CS, Berg MJ, Crawford SC,
Scott RM et al (1998) Multilocus linkage identifies two
new loci for a mendelian form of stroke, cerebral
caver-nous malformation, at 7p15-13 and 3q25.2-27 Hum
Mol Genet 7, 1851–1855
8 Guˆnel M, Awad IA, Finberg K, Anson JA, Steinberg
GK, Batjer HH, Kopitnik TA, Morrison L, Giannotta
SL, Nelson-Williams C et al (1996) A founder mutation
as a cause of cerebral cavernous malformation in
hispa-nic americans N Engl J Med 334, 946–951
9 Laberge-le Couteulx S, Jung HH, Labauge P,
Houtte-ville JP, Lescoat C, Cecillon M, Marechal E, Joutel A,
Bach JF & Tournier-Lasserve E (1999) Mutations in
CCM1, encoding KRIT1, cause hereditary cavernous
angiomas Nat Genet 23, 189–193
10 Sahoo T, Johnson EW, Thomas JW, Kuehl PM, Jones
TL, Dokken CG, Touchman JW, Gallione CJ, Lee-Lin
SQ, Kosofsky B et al (1999) Mutations in the gene
encoding KRIT1, a Krev-1⁄ rap1a binding protein,
cause cerebral cavernous malformations (CCM1) Hum
Mol Genet 8, 2325–2333
11 Liquori CL, Berg MJ, Siegel AM, Huang E,
Zawis-towski JS, Stoffer T, Verlaan D, Balogun F, Hughes L,
Leedom TP et al (2003) Mutations in a gene encoding
a novel protein containing a phosphotyrosine-binding
domain cause type 2 cerebral cavernous malformations
Am J Hum Genet 73(6), 1459–1464
12 Denier C, Goutagny S, Labauge P, Krivosic V, Arnoult
M, Cousin A, Benabid AL, Comoy J, Frerebeau P,
Gilbert B et al (2004) Mutations within the MGC4607 gene cause cerebral cavernous malformations Am J Hum Genet 74, 326–337
13 Bergametti F, Denier C, Labauge P, Arnoult M, Boetto
S, Clanet M, Coubes P, Echenne B, Ibrahim R, Irthum
B et al (2005) Mutations within the programmed cell death 10 gene cause cerebral cavernous malformations
Am J Hum Genet 76, 42–51
14 Cave´-Riant F, Denier C, Labauge P, Ce´cillon M, Maciazek J, Joutel A, Laberge-Le Couteulx S & Tour-nier-Lasserve E (2002) Spectrum and expression analysis
of KRIT1 mutations in 121 consecutive and unrelated patients with Cerebral Cavernous Malformations Eur J Hum Genet 10, 733–740
15 Laurans MS, DiLuna ML, Shin D, Niazi F, Voorhees
JR, Nelson-Williams C, Johnson EW, Siegel AM, Stein-berg GK, Berg MJ et al (2003) Mutational analysis of
206 families with cavernous malformations J Neurosurg
99, 38–43
16 Verlaan DJ, Laurent SB, Sure U, Bertalanffy H, Ander-mann E, AnderAnder-mann F, Rouleau GA & Siegel AM (2004) CCM1 mutation screen of sporadic cases with cerebral cavernous malformations Neurology 62, 1213– 1215
17 Verlaan DJ, Laurent SB, Rouleau GA & Siegel AM (2004) No CCM2 mutations in a cohort of 31 sporadic cases Neurology 63, 1979
18 Liquori CL, Berg MJ, Squitieri F, Ottenbacher M, Sorlie M, Leedom TP, Cannella M, Maglione V, Ptacek
L, Johnson EW et al (2006) Low frequency of PDCD10 mutations in a panel of CCM3 probands: potential for a fourth CCM locus Hum Mutat 27, 118
19 Verlaan DJ, Roussel J, Laurent SB, Elger CE, Siegel
AM & Rouleau GA (2005) CCM3 mutations are uncommon in cerebral cavernous malformations Neurology 65, 1982–1983
20 Guclu B, Ozturk AK, Pricola KL, Bilguvar K, Shin D, O’Roak BJ & Gunel M (2005) Mutations in apoptosis-related gene, PDCD10, cause cerebral cavernous mal-formation 3 Neurosurgery 57, 1008–1013
21 Liquori CL, Berg MJ, Squitieri F, Leedom TP, Ptacek
L, Johnson EW & Marchuk DA (2007) Deletions in CCM2 are a common cause of cerebral cavernous mal-formations Am J Hum Genet 80, 69–75
22 Liquori CL, Penco S, Gault J, Leedom TP, Tassi L, Esposito T, Awad IA, Frati L, Johnson EW, Squitieri
F et al (2008) Different spectra of genomic deletions within the CCM genes between Italian and
American CCM patient cohorts Neurogenetics Feb 9, 25–31
23 Stahl S, Gaetzner S, Voss K, Brackertz B, Schleider E, Su¨ru¨cu¨ O, Kunze E, Netzer C, Korenke C, Finckh U
et al.(2008) Novel CCM1, CCM2, and CCM3 mutations in patients with cerebral cavernous malformations: in-frame deletion in CCM2 prevents
Trang 6formation of a CCM1⁄ CCM2 ⁄ CCM3 protein complex.
Hum Mutat May, 29, 709–717
24 Verlaan DJ, Siegel AM & Rouleau GA (2002) Krit1
missense mutations lead to splicing errors in cerebral
cavernous malformations Am J Hum Genet 70, 1564–
1567
25 Zawistowski JS, Stalheim L, Uhlik MT, Abell AN,
Ancrile BB, Johnson GL & Marchuk DA (2005) CCM1
and CCM2 protein interactions in cell signaling:
impli-cations for cerebral cavernous malformations
pathogen-esis Hum Mol Genet 14, 2521–2531
26 Voss K, Stahl S, Hogan BM, Reinders J, Schleider E,
Schulte-Merker S & Felbor U (2009) Functional
ana-lyses of human and zebrafish 18-amino acid in-frame
deletion pave the way for domain mapping of the
cere-bral cavernous malformation 3 protein Hum Mutat Jun
30, 1003–1011
27 Denier C, Labauge P, Bergametti F, Marchelli F, Riant
F, Arnoult M, Maciazek J, Vicaut E, Brunereau L &
Tournier-Lasserve E (2006) Genotype-phenotype
corre-lations in cerebral cavernous malformations patients
Ann Neurol Nov 60, 550–6
28 Ortiz L, Costa AF, Bellido ML, Solano F,
Garcı´a-Mor-eno JM, Gamero MA, Izquierdo G, Chadli A, Falcao
F, Ferro J et al (2007) Study of cerebral cavernous
malformation in Spain and Portugal: high prevalence of
a 14 bp deletion in exon 5 of MGC4607 (CCM2 gene)
J Neurol Mar 254, 3
29 Knudson AG (1971) Mutation and cancer: statistical
study of retinoblastoma Proc Natl Acad Sci USA 68,
820–823
30 Limaye N, Boon LM & Vikkula M (2009) From
germ-line towards somatic mutations in the pathophysiology
of vascular anomalies Hum Mol Genet Apr 15, 18
1, R65–74
31 Kehrer-Sawatzki H et al (2002) Mutation and expres-sion analysis of the KRIT1 gene associated with cere-bral cavernous malformations Acta Neuropathol 104, 231–240
32 Gault J, Shenkar R, Recksiek P & Awad IA (2005) Biallelic Somatic and Germ Line CCM1 Truncating Mutations in a Cerebral Cavernous Malformation Lesion Stroke 36, 872
33 Gault J, Awad IA, Recksiek P, Shenkar R, Breeze R, Handler M & Kleinschmidt-DeMasters BK (2009) Cerebral cavernous malformations: somatic mutations
in vascular endothelial cells Neurosurgery Jul 65, 138–144
34 Akers AL, Johnson E, Steinberg GK, Zabramski JM & Marchuk DA (2009) Biallelic somatic and germline mutations in cerebral cavernous malformations (CCMs): evidence for a two-hit mechanism of CCM pathogenesis Hum Mol Genet Mar 1, 18, 919–930
35 Pagenstecher A, Stahl S, Sure U & Felbor U (2009) A two-hit mechanism causes cerebral cavernous malforma-tions: complete inactivation of CCM1, CCM2 or CCM3 in affected endothelial cells Hum Mol Genet Mar 1, 18, 911–918
36 Limaye N, Wouters V, Uebelhoer M, Tuominen M, Wirkkala R, Mulliken JB, Eklund L, Boon LM & Vikkula M (2009) Somatic mutations in angiopoietin receptor gene TEK cause solitary and multiple spora-dic venous malformations Nat Genet Jan, 41, 118– 124
37 Gianfrancesco F, Esposito T, Penco S, Maglione V, Liquori CL, Patrosso MC, Zuffardi O, Ciccodicola A, Marchuk DA & Squitieri F (2008) ZPLD1 gene is disrupted in a patient with balanced translocation that exhibits cerebral cavernous malformations Neuro-science, 155, 345–349