Genetics of Cerebral Cavernous Malformations

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CHAPTER 393 Genetics of Cerebral Cavernous Malformations

Ali Kemal Ozturk, Angeliki Louvi, Murat Günel

Cerebral cavernous malformations (CCMs) are some of the most common types of vascular malformations of the central nervous system (CNS), and they affect nearly 1 in 200 people. CCM lesions are characterized by grossly dilated vascular channels lined by a single layer of endothelium (Fig. 393-1).¹ They typically affect the brain and spinal cord but may also involve the skin and retinal vasculature.² CCMs lack normal vessel wall elements such as smooth muscle and are also devoid of intervening normal parenchyma.³

FIGURE 393-1 Histology of cavernous malformations. These lesions are characterized by grossly dilated vascular channels lined by a single layer of endothelium.

The CCM population prevalence of 0.5% is an estimate based on both magnetic resonance imaging (MRI) and autopsy studies. Although fairly common in the general population, only 20% to 30% of CCMs become symptomatic.⁴^–⁷ Patients are typically seen initially in the third through fifth decades of life because of hemorrhagic stroke, headache, seizures, or focal neurological deficits.⁵ As a result of their rudimentary vessel wall and the low-pressure blood flow through the lesions, CCMs tend to “leak” and cause microhemorrhages as evidenced by a characteristic hemosiderin ring seen on MRI (Fig. 393-2).³ CCM lesions can also lead, albeit rarely, to gross macrohemorrhages causing clinical signs and symptoms reflecting the size and location of the bleeding. Catastrophic hemorrhages are much rarer, again being mainly dependent on the location. Treatment options for CCMs include observation for asymptomatic lesions, antiepileptic medication for seizures, and surgical excision of accessible lesions in patients who suffer from symptomatic hemorrhage or intractable seizures.^8,⁹

FIGURE 393-2 Magnetic resonance imaging of cerebral cavernous malformation (CCM). A and B, Sagittal and coronal images demonstrate a large CCM (arrowhead) with resultant gross hemorrhage (arrows). C and D, Axial images of a patient with the genetic form of CCM demonstrating multiple small lesions with a characteristic hemosiderin ring surrounding them (arrows).

Both sporadic and familial forms of CCM exist. The familial form is usually manifested as multiple lesions in the setting of a strong family history of neurological disease. Sporadic forms almost never involve more than two lesions, and a family history is absent. The heritable nature of CCMs has been well recognized since its original description ¹⁰; however, the limited neuroradiologic data available to early investigators resulted in an incomplete appreciation for affected family members. The advent of MRI has led to the detection of asymptomatic CCMs, and in 1988, Rigamonti and coauthors reported a series of 13 Hispanic American families with CCM, complete with clinical and radiographic features,¹¹ a study complemented in 1998 by the first large series of white, non-Hispanic patients with familial CCM.¹² These and other studies have demonstrated that although only 10% to 20% of affected white individuals have a first-degree relative with the disorder, this figure is as high as 50% in Hispanic Americans, a population in which our group has demonstrated a strong founder mutation.^10,¹³

It is now established that the genetic mode of inheritance of familial CCM is autosomal dominant with a high degree of penetrance, thus rendering it an ideal genetic model for human hemorrhagic stroke. Genetic linkage analyses have mapped three CCM loci to chromosomes 7q (CCM1), 7p (CCM2), and 3q (CCM3).¹⁴^–¹⁹ All three causative genes have now been identified, which has allowed new insight into the pathophysiology of CCMs.

We first review the concept of linkage analysis as a general molecular genetics tool for identification of genes. We then focus on the CCM molecules with an emphasis on their known biologic functions as they apply to the pathogenesis of disease. Finally, we review the clinical implications of these findings and phenotype-genotype correlations.

Molecular Genetic Approaches

A variety of genetic approaches, including association (candidate gene, whole genome association) and linkage (parametric and nonparametric) studies, as well as, more recently, copy number variation (CNV) and whole-exome sequencing approaches, can be used for the analysis of heritable diseases. Linkage analysis is the main approach to identification of rare alleles imparting a major effect on the disease phenotype, especially when a disease with a mendelian form of inheritance is being studied, as is the case for CCM. If the mode of inheritance is known (e.g., autosomal dominant in CCM), parametric, or model-based, linkage analysis is the preferred method.

In parametric linkage analysis, a single extended family pedigree, large enough to support linkage independently, is analyzed. A number of DNA markers, in the form of single nucleotide polymorphisms (SNPs) or short tandem repeats, of known location dispersed throughout the entire human genome are subsequently used to investigate the inheritance of polymorphic regions of the genome by statistically computing recombination frequencies. For each marker, evidence of linkage is sought by using cosegregation of the trait (and presumably the disease-causing gene) and a particular variant of the marker being analyzed. This is possible because the closer the disease and marker loci are located on a chromosome, the less likely it is that a recombination event between them will occur and the more likely that the specific marker variant will be present in all individuals who are affected with the disease. Thus, the genetic distance between the marker locus and the disease-causing gene can be estimated in the genetic unit of morgan (M), with one-one hundredth morgan (1 cM) corresponding to a recombination fraction of 1%.²⁰

Statistically, the logarithm of the odds (LOD) score function is used to test for significance of the linkage ²¹ and is a robust measure of its strength. The LOD score represents the logarithm of the odds that the DNA marker locus is linked to the disease trait and is calculated according to the following formula:

Thus, a LOD score of 3.0 would mean that the probability that the two loci are linked is 1000 times more likely than them being unlinked. However, any two given loci are approximately 50 times more likely to be unlinked than linked because they are most likely on different chromosomes. Correcting for this factor would mean that the two loci with a LOD score of 3.0 are 1000/50 or 20 times more likely to be linked than unlinked. This means that there is 1 in 20 probability that the linkage is due to chance findings, which roughly corresponds to a P value of .05. Therefore, a LOD score of 3.0 is considered to be the threshold for linkage significance.

Using this approach, the area or areas of the genome with the highest LOD score are considered to be the most likely to contain the disease-causing gene or genes. If the linked region is sufficiently small, one may directly proceed to sequence that portion of the DNA to look for allelic variants that may directly explain the disease. If the area in question is comparatively large, fine-mapping techniques to further narrow the linked interval may be necessary.

The availability of new molecular genetic technologies, including high-density SNP arrays, allowed the development of new methodology, such as CNV analysis. Recent studies using the CNV approach have allowed identification of segments of DNA in the genome ranging from kilobases to megabases in length that are lost (deleted) or gained (duplicated).²² Although CNVs are abundantly present and distributed throughout the human genome, thus suggesting that many benign variations exist,²³ it is becoming evident that an increased burden of CNVs is observed in disease states.²⁴^–²⁸ We and others have successfully applied this technology to identify rare disease-causing alleles in CCM.^29,³⁰

Genetics of Cerebral Cavernous Malformations

Familial CCM is an autosomal dominant disease with high, but incomplete penetrance. It should also be kept in mind, however, that mutations in one of the three identified CCM loci are evident in only 96% of familial CCM cases.¹² Thus, the possibility of a fourth, to date unidentified CCM locus should not be excluded. The disease that ensues from mutations in the three loci is clinically and radiographically very similar.

The three CCM genes thus far identified are Krev1 interaction trapped protein-1 (KRIT1) located on chromosome 7q as the CCM1 gene, MGC4607 (or malcavernin) on chromosome 7p as the CCM2 gene, and programmed cell death-10 (PDCD10) on chromosome 3q as the CCM3 gene.³¹^–³⁸ The following sections focus on the molecular biology of these three genes.

CCM1

The CCM1 gene is located on chromosome 7q21 and contains 16 exons that encode a 736–amino acid protein, KRIT1. This is the first CCM gene that was identified and, consequently, has been the most studied in terms of its cellular function. Mutational analysis of this gene has revealed a founder mutation (C742T) that is responsible for more than 90% of familial cases in Hispanic American patients.^13,^32,³³ Numerous studies have now identified in excess of 80 different mutations of this gene leading to clinically evident CCM. Nearly all are nonsense mutations in the form of stop codons or frameshift mutations that result in premature termination of translation.^33,^35,³⁹^–⁴³ Thus, there is strong evidence to suggest that loss of function of the CCM1 protein leads to disease, which probably results from a two-hit mechanism.⁴⁴ In the two-hit model, the first genetic “hit” is inherited, whereas the second is acquired in a subset of cells and rapidly leads to disease afterward because affected cells can no longer synthesize functionally intact proteins.

Krit1 was initially identified in a yeast two-hybrid screen as an interactor of the small Ras family guanosine triphosphatase Krev1/Rap1a.⁴⁵ Even though later studies challenged this finding,^46,⁴⁷ more recent studies have confirmed this interaction, thus suggesting that Rap1a signaling might be important in CCM pathophysiology.^48,⁴⁹

The KRIT1/CCM1 protein contains a number of domains implicated in protein-protein interactions, including a FERM domain at the C-terminal, which is typically critical for binding to the cell membrane.⁵⁰ Recent studies have suggested this domain’s importance in CCM1’s binding cell-cell junctional proteins (see later).^48,⁴⁹ KRIT1 also contains a series of ankyrin repeats, but the proteins that interact with CCM1 through this domain remain unknown.⁴⁵ Finally, CCM1 contains three NPXY domains that are known to interact with phosphotyrosine-binding (PTB) regions. Two of these domains create a binding site for integrin cytoplasmic domain–associated protein-1α (ICAP-1α)^46,⁴⁷ and CCM2 (see later) (Fig. 393-3).⁵¹

FIGURE 393-3 Examination of the Krev1 interaction trapped protein-1 (KRIT1) structure reveals multiple domains. KRIT1 interacts with Rap1a/Krev1 and binds to membranes through its FERM domain. KRIT1 interactions with integrin cytoplasmic domain–associated protein-1α (ICAP-1α) and CCM2 are mediated by two separate NPXY domains, which in turn bind phosphotyrosine-binding (PTB) domains on ICAP-1α and CCM2.

Our group was the first to demonstrate that CCM1 interacts with the plus end of microtubules.⁵² This finding was later confirmed by Beraud-Dufour and colleagues, who also demonstrated that CCM1 can adopt both an open and a closed conformation, depending on the interaction of the N-terminal NPXY motif with its C-terminal.⁴⁸ In vitro, CCM1 interacts with microtubules in its closed conformation, which would in turn help deliver CCM1 to the plasma membrane, where it would bind to ICAP-1α in its open conformation. Even though both the small G-protein Rap1a and ICAP-1α can disrupt CCM1’s association with microtubules, thereby localizing it to endothelial cell-cell junctions, only ICAP-1α appears to be able to unfold CCM1.⁴⁸

ICAP-1α is known to bind to β1 integrin, again through an NPXY/PTB domain interaction.^53,⁵⁴ Integrins, specifically β1 integrin, are important proteins that regulate endothelial cell adhesion and migration throughout angiogenesis.^55,⁵⁶ These observations imply a role for CCM1 in angiogenesis with its involvement in the Rap1a/Krev1 and integrin pathways. More recent studies suggested that after localizing to the cell membrane, CCM1 can also bind to β-catenin and play a role in stabilizing adherens cell junctions and regulating endothelial cell permeability.⁴⁹

In addition, CCM1 binds to both CCM2 and CCM3.^51,⁵⁷ The exact nature of the interaction between the three CCM molecules is currently unknown, but among these three proteins, CCM2 appears to act as a scaffolding protein⁵⁸ and is potentially recruited to cell-cell junctions through its interactions with CCM1.

Even though the molecules involved in the downstream signaling of CCM molecules are unknown, in vitro studies previously suggested that CCM1 translocates to the nucleus upon cellular stimulation,⁵¹ and recent work in Caenorhabditis elegans confirmed this finding by showing interaction of the CCM1 ortholog kri-1 with a transcription factor daf16.⁵⁹ Mutations in kri-1 result in increased life span, mediated though its interactions with daf16. The importance of this aspect of CCM signaling in humans is currently unknown, but dissecting this molecular pathway will have important implications in understanding disease pathophysiology.

In an attempt to gain some insight into CCM pathophysiology, our group focused on the temporal and spatial expression patterns of the three CCM molecules. Our initial studies demonstrated CCM1 to be expressed in many human tissues, including the heart, brain, kidney, liver, pancreas, and spleen.⁶⁰ Furthermore, CCM1 is expressed specifically in the endothelium of capillaries and arterioles, but not in the venous system.⁶¹ CCM1 (but also CCM2 and CCM3; see later) is abundantly expressed in neurons and astrocytic foot processes (Fig. 393-4), an observation potentially related to the finding that CCM lesions almost exclusively form within the CNS.⁶¹

FIGURE 393-4 Immunohistochemical analysis of the CCM1/KRIT1 protein. A, KRIT1 is expressed in arterial endothelium within blood vessels (bv, black arrow) and astrocytic foot processes (fp, open arrow). B, Astrocytic foot processes (fpopen arrow) surrounding blood vessels (bvblack arrow) demonstrate strong KRIT1 expression. C, Large pyramidal neurons also stain strongly for KRIT1.

(From Guzeloglu-Kayisli O, Amankulor NM, Voorhees J, et al. KRIT1/cerebral cavernous malformation 1 protein localizes to vascular endothelium, astrocytes, and pyramidal cells of the adult human cerebral cortex. Neurosurgery. 2004;54:943-949; discussion 949.)

In vivo studies based on mouse models have revealed an essential role for Ccm1 in vascular development: complete loss of Ccm1 results in embryonic lethality at midgestation.⁶² Ccm1^−/− embryos demonstrate vascular defects incompatible with life, namely, dilated precursor brain vessels, an enlarged caudal dorsal aorta (probably caused by increased endothelial proliferation), and variable obstruction of branchial arch arteries and the proximal dorsal aorta.⁶² All these findings suggest a primary defect in arterial morphogenesis. Ccm1^+/− animals are phenotypically normal with no apparent vascular phenotype and do not form CCM lesions. Interestingly, when these Ccm1^+/− animals are crossed with p53^−/− mice, vascular lesions resembling cavernous malformations in the brain develop in approximately 55% of the compound Ccm1^+/−;p53^−/− mutants.⁶³ The mechanism underlying the enhanced phenotype in the absence of p53 is not known but is speculated to be an increased rate of somatic mutations leading to more second hits and thereby resulting in the formation of CCM lesions in the compound animals.

When viewed as a whole, these studies are beginning to shed light on the cellular function of the CCM1 protein and its involvement in angiogenesis. CCM1 probably exerts the majority of its effects at the level of the cerebrovascular unit, where it mediates communication between its components, neural cells, and vascular endothelium of the CNS (Fig. 393-5).

FIGURE 393-5 Schematic diagram of the interaction of cerebral cavernous malformation (CCM) molecules with each other and various regulators. ICAP1α, integrin cytoplasmic domain–associated protein-1α; IRK, insulin receptor kinase; MEKK3, MAP kinase kinase kinase 3; VEGF, vascular endothelial growth factor.

CCM2

In 2003, Liquori and colleagues identified MGC4607 (or malcavernin) as the causative gene in families linked to the CCM2 locus.³⁰ MGC4607 is a 10-exon gene located on chromosome 7p13 that encodes the protein CCM2, or malcavernin.³⁷ Similar to CCM1, almost all mutations identified to date in CCM2 are frameshift mutations or result in stop codons, again implying a loss-of-function mechanism, presumably via a two-hit model.

The CCM2 protein contains a putative PTB domain, also found in ICAP-1α, CCM1’s binding partner. Although binding of CCM1 to both ICAP-1α and CCM2 is mediated via PTB/NPXY domain interactions, the NPXY domains that interact with these two molecules have been shown to be distinct, thus potentially indicating concomitant effects of ICAP-1α and CCM2 on CCM1.⁵¹

Interestingly, the temporal and spatial pattern of CCM2 expression parallels that of CCM1. CCM2, like CCM1, is expressed by arterial endothelium, neurons, and astrocytes (Fig. 393-6).⁶⁴ This expression pattern supported the hypothesis that the two proteins may be involved in a common pathway. It should also be underscored that both proteins are restricted to the arterial endothelium, with exclusion from the venous circulation. Given that histologically CCMs are abnormally dilated microvessels, this finding is somewhat unexpected but has now been confirmed by in vivo studies as well.^62,⁶⁵

FIGURE 393-6 Immunohistochemical analysis of the CCM2/malcavernin protein. A, Low magnification of cerebral tissue demonstrates CCM2 expression by arterial endothelium (black arrow) and pyramidal neurons (white arrows). B and C, High magnification of cerebral arteriolar endothelial cells further demonstrates CCM2 expression (arrow). D, CCM2 expression in the intima of microvessels (black arrows) and the astrocytic foot processes surrounding these vessels (white arrows). E, CCM2 expression in astrocytes and astrocytic foot processes (white arrows) encapsulating the capillary endothelium (black arrows). F, Cross section of the cerebral cortex revealing cytoplasmic expression of CCM2 by both pyramidal neurons (arrows) and glial cells. G, Higher magnification of pyramidal neurons shows immunopositive basal dendrites and apical dendritic extensions. E and F, Axial T1-weighted images with gadolinium in a patient with CCM demonstrates a cerebellar DVA. The two entities frequently co-exist.

(From Seker A, Pricola KL, Guclu B, et al. CCM2 expression parallels that of CCM1. Stroke. 2006;37:518-523.)

Considerable homology exists between CCM2 and the rodent protein OSM, which is involved in osmosensing and mechanosensing of the extracellular matrix.⁵⁸ Like OSM, CCM2 has been shown to function as a scaffolding protein and signals through p38, a pathway that is involved in environmental stress signaling and plays a major role during angiogenesis.^58,⁶⁶ More recent in vitro assays have further elucidated this pathway and showed that CCM2, when overexpressed, has the potential to sequester CCM1 and form a complex with MAP kinase kinase kinase 3 (MEKK3).⁵¹ These results are the beginning of our understanding of CCM pathophysiology and imply that CCM2, as well as possibly CCM1 through interactions with CCM2, may assert its influence via the p38 pathway and could lead to disease by perturbing CNS angiogenesis.

In vivo studies in zebrafish and mice have demonstrated further parallels between Ccm1 and Ccm2. In mice, similar to Ccm1, complete loss of Ccm2 is embryonically lethal as a result of vascular defects.⁶⁵ Vascular lesions reminiscent of small cavernous malformations develop in approximately 10% of Ccm2^+/− animals.⁶⁵ Ccm2 inactivation in the endothelium causes cardiovascular pathology; however, both neural and smooth musclespecific conditional mutants appear normal.⁶⁷^–⁶⁹ In zebrafish, recessive mutations of either the Ccm1 or Ccm2 orthologs santa and valentine result in a lethal cardiac phenotype with massive dilation of the cardiac chamber, apparently secondary to failure to generate appropriate concentric thickness of the myocardial wall.⁷⁰

CCM3

The CCM3 gene was discovered in 2005 by Bergametti and colleagues to be PDCD10.^34,³⁶ Because it is the most recently discovered, this gene and its encoded protein are the least studied and understood. PDCD10 is a seven-exon gene located on 3q26 that encodes a 212–amino acid protein. Similar to CCM1 and CCM2, identified mutations of the CCM3 gene lead to stop codons and frameshifts and result in truncated protein products. In silico analyses have failed to demonstrate any known functional domains for CCM3.

Our group investigated the expression pattern of CCM3 in embryonic and postnatal mouse brain and in various human organs. Similar to CCM1 and CCM2, the CCM3 protein is expressed strongly in neurons, astrocytes, and arterial endothelium.⁷¹ CCM3 is either weakly present in the venous circulation or completely absent, thus underscoring the fact that CCM lesions are a result of arterial pathology (Fig. 393-7).

FIGURE 393-7 Immunohistochemical analysis of the CCM3/PDCD10 protein. A, Cortical arterial endothelium demonstrates strong expression of CCM3. B, CCM3 expression is observed in the cytoplasm of large pyramidal neurons (arrows) but is notably absent from the cortical venous endothelium (arrowhead). C and D, Similar staining pattern in human cerebellum with expression noted in neurons (arrowheads) and vessels (arrow).

(From Tanriover G, Boylan AJ, Diluna ML, et al. PDCD10, the gene mutated in cerebral cavernous malformation 3, is expressed in the neurovascular unit. Neurosurgery. 2008;62:930-938; discussion 938.)

Little is known regarding the function of CCM3. It was first identified as being upregulated on deprivation of growth factor and induction of apoptosis in the TF-1 premyeloid cell line, as well as in fibroblast cell lines, which suggested a role for the protein in apoptotic pathways.⁷² In vitro, CCM3 appears to be both necessary and sufficient to induce apoptosis: overexpression of CCM3 (but not of mutated, biologically inactive forms of the protein identified in CCM patients) elicits activation of caspase 3 and cell death. Moreover, in the presence of proapoptotic stimuli, CCM3 levels are increased concomitantly with levels of activated caspase 3 and p38.⁷¹

An important discovery came from Voss and colleagues, who showed a potential link between the CCM3 and CCM2 proteins: in a yeast two-hybrid system, CCM3 binds to and is phosphorylated by serine/threonine kinase 25 (STK25), a molecule that also forms a complex with CCM2. Furthermore, CCM3 colocalizes and coprecipitates with CCM2. Interestingly, in the presence of CCM1, the amount of CCM3 immunoprecipitated with CCM2 increased.⁵⁷ Hilder and coworkers hypothesized that the three CCM proteins are scaffold- or adaptor-type proteins that organize a large macromolecular complex and, with the use of a proteomics approach, demonstrated that the three proteins participate in a larger signaling complex.⁶⁶ Taken together, these results pointed for the first time to an interaction among the three different CCM proteins by showing that CCM3 may be a part of the CCM1/CCM2 protein complex via its interactions with CCM2. This complex would have the potential to participate and influence integrin-mediated signaling and the p38 pathway and could significantly alter endothelial cell adhesion and migration during angiogenesis with resulting cavernous malformations. Global or endothelial deletion of Ccm3 causes embryonic lethality associated with defects in vascular development and VEGFR2 signaling,⁷³ and in zebrafish, CCM3-mediated signaling through Ste20-like kinases is involved in cardiovascular development.⁷⁴

Our group examined the role of Ccm3 in neurovascular development and disease and showed that loss of Ccm3 in neural cells has cell autonomous effects that cause activation of astrocytes, in addition to cell nonautonomous effects in the vasculature, including a diffusely dilated and simplified vascular tree and formation of lesions that resemble typical human cavernomas (Louvi and Gunel, unpublished).

Clinical Genetics

Despite multiple similarities between the CCM1, CCM2, and CCM3 families and their disease manifestations, closer inspection does reveal some intrafamily and interfamily differences. Studies that attempt to understand and predict phenotypic behavior based on genotypic makeup are called genotype-phenotype correlation studies. These studies have potentially important clinical applications and may have repercussions for patient prognosis and genetic counseling.

All three CCM genes have varying degrees of clinical penetrance. Studies estimate this penetrance to be approximately 70% for CCM1, 40% for CCM2, and 60% for CCM3.⁷⁵ Thus, clinical disease will develop in different proportions of patients with mutations for each gene.

Among these patients, types of clinical manifestations might also vary. For example, hemorrhage rates might be lowest in the CCM1 group ⁷⁶ and highest in the CCM3 patients, particularly during childhood.⁷⁵ Even though CCM1 patients may have a more benign course, there is evidence to support the fact that more lesions develop as they age.⁷⁵ These early studies are beginning to demonstrate differences in the clinical behavior and radiographic features of the three CCM groups. To further complicate the issue, it is also known that the same mutation that segregates within a large family can lead to different disease manifestations, with some members of the family suffering from progressive bleeding and others remaining asymptomatic all their lives. These observations prove the complexity of the genetics of CCM and suggest that other modifier genes or yet unidentified environmental risk factors (or both) can modify progression of the disease. Further studies are currently aimed at understanding these genetic and nongenetic risk factors. Such studies may ultimately reveal certain subgroups of patients who harbor risk factors that render their lesions more prone to rupture and therefore may need to be treated more aggressively.

Conclusion and Future Directions

Because the families that harbor mutations in the three CCM genes are clinically and radiographically virtually indistinguishable, we had previously hypothesized that all three genes encode proteins that are involved in common or parallel pathways. In support of this hypothesis, our group has demonstrated that CCM1, CCM2, and CCM3 are very similar in their temporal and spatial expression pattern, specifically, in neurons, astrocytes, and arterial endothelium, with little, if any involvement of the venous circulation.^61,^64,⁷¹ Support of this theory also comes from in vivo studies in mice and zebrafish, which have shown that defects in Ccm1 and Ccm2 lead to similar lethal problems involving early angiogenesis.^62,^65,⁷⁰

In addition, in vitro analyses have revealed CCM1 to bind to microtubules and travel between the cell membrane, where it interacts with molecules involved in cell adhesion ^48,⁵² and the nucleus. CCM1 also binds to CCM2, which acts as a scaffolding protein, and signals through the p38 mitogen-activated protein kinase signaling pathway, known to be important in diverse cellular processes, including the stress response, proliferation, apoptosis, and differentiation. More recent evidence adds CCM3 to the CCM1/CCM2 complex, thus lending further support to the hypothesis that all three CCM proteins are involved in the same pathway.⁵⁷ Initial insight into CCM3 function suggests that its signaling is important within the neurovascular unit of the CNS and that it potentially affects cellular proliferation and apoptosis.^51,⁶⁶

With the identification of all three genes that lead to familial CCM, major steps have been taken toward understanding the pathophysiology of CCM at a molecular level. Although our understanding of the involved genes and molecules is constantly evolving, there is still much that needs to be investigated, and many areas of disease formation are still a mystery. Phenotype-genotype correlation studies may help predict disease severity and the extent of disease, as well as prognosis. Why do certain lesions bleed whereas others are clinically silent? Are there modifier genes that may account for this variation and the clinically and radiographically incomplete penetrance? Is there a fourth CCM locus that may account for the familial cases without any evident mutation in the three CCM genes?

Although many questions remain unanswered, CCM is a prime example of how investigation of a familial form of a disease may help reveal its pathophysiology through genetic studies followed by molecular biology. This understanding will provide important insight into both familial and sporadic forms of disease by dissecting the involved molecules, which are oftentimes shared. Ultimately, the involved molecules and pathways will be the substrate for novel therapeutic strategies and possibly disease prevention.