The heterogeneous cellular nature of the CCM lesions may have inhibited previous attempts to detect somatic mutations within the vascular tissue. Direct sequencing of an amplified product effectively identifies heterozygous germline mutations. However, if a somatic mutation is present in substantially <50% of the alleles, it may not be easily detected. Our strategy for somatic mutation analysis involving sequencing of individual PCR amplicons provided the sensitivity to detect mutations that are not present in an entire cellular population and that might otherwise remain undetected by direct sequencing of the amplified product. The overall sensitivity of our approach is approximately similar to that used by Gault et al.
for the same purpose (30
). Denaturing high-pressure liquid chromatography also enabled screening of all the CCM1
exons, such that a biallelic somatic mutation could be identified in a CCM lesion despite its presence in only a fraction of the cells.
We have shown that somatic mutations are present in all forms of inherited CCMs, although we were unable to detect somatic mutations in each sample examined. We suggest that the architecture and microanatomy of the CCM lesions can affect the ability to detect somatic mutations. Tissue heterogeneity can cause problems with somatic mutation detection for solid tumors (33
) and also pose similar problems with the CCM lesion, which comprised multiple cell and tissue types. In addition to vascular components, the bulk lesion contains intervening connective tissue. Additionally, as the lesion is itself a vascular structure, it can be difficult to distinguish the vasculature of the lesion proper from feeding and draining vessels. Any given section through the surgically resected lesion may contain different fractions of cells harboring the somatic mutation. Some sections might contain more interstitial tissues, fewer caverns, more blood or a part of the lesion consisting primarily of feeding or draining vessels. This problem is especially relevant for cases in which histology slides are the only source of tissue available, as the slides represent a single slice through the lesion. In support of this contention, we have observed that different DNA preparations isolated from different regions of the same CCM lesion result in an apparent difference in the frequency of the somatic mutation. In extreme cases, the fraction of clones exhibiting a somatic mutation might fall below the threshold for initial detection.
We chose to sequence 48 clones for each exon as two exons could be completed in a 96-well microtiter plate. Using this number of clones, power calculations showed that we have 95% power to observe a mutation in more than one clone if 20% of the alleles harbored the mutation. However, the power to detect a mutation decreases markedly as the number of alleles harboring the mutation falls below the 20% level. Although in one case we have identified a somatic mutation that was present in only 6% of the alleles sampled, it is more likely that we would miss mutations present at such low levels in the section of the lesion that was analyzed.
We have also noted that the quality of the tissue sample can have profound effects on the ability to identify a somatic mutation. Surgically resected tissue that was immediately frozen provided genomic DNA that amplified very well and, in general, showed the fewest singleton changes and other PCR-induced errors. In contrast, formalin-fixed paraffin-embedded tissue samples contained DNA that was more difficult to amplify and usually showed a higher frequency of random nucleotide changes. Both singletons and even multiple clone changes were detected in formalin-fixed tissue, none of which was validated through multiple rounds of amplification, cloning and sequencing. The fixation process can destroy the integrity of the DNA and result in sequence changes that are indistinguishable from PCR error.
Finally, our somatic mutation identification strategy was specifically designed to identify mutations that are readily apparent by DNA sequencing: single nucleotide changes and small insertions or deletions. Our approach would miss altogether other types of second hits. A classic mechanism for a second genetic hit is LOH, resulting from large deletions, unbalanced translocations or mitotic recombination. The identification of this type of second hit requires the ability to observe the loss of one allele of a linked heterozygous marker. In a relatively homogeneous lesion sample, the loss of one allele is readily apparent, either as complete loss of one allele (LOH) or, more commonly, as a statistically significant imbalance in the ratio of the two alleles. However, it becomes increasingly difficult to observe LOH with increasing amounts of cellular heterogeneity in the lesion tissue (33
). Given the complex microanatomy and mixed cellular composition of CCM lesions, LOH would be very difficult if not impossible to detect. Epigenetic effects such as promoter methylation may also represent a mechanism by which the wild-type allele may be silenced. Again, these types of mutations are not readily identifiable by our strategy. Samples that did not exhibit somatic mutations as simple sequence changes might be explained by any of the previously described technical caveats or by different classes of second hit mutations.
Another potential two-hit mechanism would be the development of a somatic mutation in a gene other than that which harbors the germline mutation. For CCMs, one of the other CCM genes might make attractive candidates for a second hit in samples, where we did not identify a somatic mutation. The concept of trans-heterozygosity as a two-hit mechanism has precedence in autosomal-dominant polycystic kidney disease (PKD). PKD has been shown to follow the two-hit mechanism for pathogenesis. PKD cysts may arise due to inheritance of a mutated copy of PKD1 or PKD2, followed by a second hit to the germline mutation (34
). The cysts sometimes instead show trans-heterozygous germline and somatic mutations, one in each of the two PKD
). However, our mouse models of CCM argue against trans-heterozygosity. We have crossed the Ccm1
lines to create mice that are doubly heterozygous for both germline mutations. These animals are trans-heterozygous in all cells and tissues and yet they do not develop CCM lesions. In light of this observation, we do not favor trans-heterozygosity as a mechanism for the second genetic hit for CCMs.
The two-hit mechanism for disease pathogenesis was originally described for tumor suppressor genes and inherited cancers. However, in recent years, the two-hit mechanism underlying disease pathogenesis has also emerged for non-malignant diseases. PKD is a clear example of a non-malignant phenotype that follows the two-hit mechanism (34
). The two-hit genetic mechanism has been implicated as the underlying mechanism for the development of venous and glomuvenous malformations (36
). Somatic mutations have been identified in a population of non-proliferating T cells in autoimmune lymphoproliferative syndrome (ALPS). This rare population of cells is believed to promote the proliferation of non-mutated lymphocytes and to indirectly cause the onset of ALPS (38
). CCM may follow a similar mechanism of pathogenesis. The second hit of the wild-type allele might initiate both the proliferative and remodeling capabilities of neighboring endothelial cells to result in the genesis of multicavernous CCM lesions.
All three CCM genes have been shown by in situ
and immunohistochemical studies to be expressed in a variety of cell types within the brain, including neurons, astrocytes and vascular endothelium. Although these studies have shown some inconsistencies in the expression pattern, all agree that all three CCM transcripts/proteins are robustly expressed in the neurons (26
). These collective observations suggested that CCM pathogenesis might be primarily due to a neural defect. However, we have shown here that the somatic mutations in the CCM tissue are present only in the endothelial cells lining the caverns of the lesion. Regardless of the role of these proteins in other cell types, this observation strongly supports an important role of the CCM proteins in the vascular endothelium for the maintenance of normal vessel integrity. A critical role for the CCM proteins in the vascular endothelium is also supported by biochemical studies, which demonstrate that KRIT1 is localized to endothelial cell junctions and is responsible for maintenance and stabilization of the integrity of tight junctions (14
). Additionally, a significant role of the CCM1 and CCM2 gene products in the endothelial cell is supported by developmental studies of the null phenotypes in mice (CCM1) (27
) and zebrafish (santa and valentine, CCM1 and CCM2, respectively) (45
), which exhibit primarily vascular (or cardiovascular) phenotypes.
Our data also support existing clinical and histological data, showing that CCM lesions exhibit characteristics of both vascular tumors and vascular malformations. These lesions appear to grow as a result of both remodeling of the existing vasculature and proliferation of the lesion endothelial cells. The three forms of CCM follow the two-hit mechanism of pathogenesis, suggesting that the CCM genes also show characteristics of tumor suppressor genes. Loss of both copies of the gene may explain some of the proliferative capacity in the EC component (47
). However, loss of both copies also appears to initiate other pathogenic mechanisms, including vascular remodeling (20
) and changes in vascular permeability due to loss of tight junctions (3
). Further analysis of these genes and gene products in normal vascular growth will shed further light on the distinct roles of the CCM genes in vascular biology and in the pathogenesis of CCM.