Abstract
Objective: Cerebral cavernous malformations (CCM) occur in familial and sporadic forms that cannot be distinguished by phenotype. Mutations in Krit1, a gene located at the CCM1 locus on chromosome 7q21, account for the majority of familial CCM cases. The authors investigated the role that mutations at the CCM1 locus play in sporadic cavernomas and the prevalence of occult familial forms among symptomatic cavernomas.
Methods: The authors screened the DNA of cavernomas and adjacent normal brain tissue of 72 consecutive patients treated at the Neurosurgical Department/Ludwig-Maximilian University for mutations in Krit1. Eight of the patients had been suspected to have a mutation at CCM1, as they showed multiple cavernomas or clinically familial forms.
Results: None of the patients showed a mutation at the CCM1 site, either in cavernomas or in normal brain tissue.
Conclusion: Mutations in Krit1 are seldom a cause of sporadic cavernomas.
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Cerebral cavernomas are present in about 0.5% of the general population.1-4⇓⇓⇓ Cavernomas may increase in size and number, and they often become symptomatic in patients aged 20 to 40 years. These patients usually present with seizures, acute cerebral hemorrhage, and headache. MRI is used to establish the diagnosis of cerebral cavernoma, especially by hemosiderin-sensitive sequences.3-6⇓⇓⇓ Cerebral cavernomas are classified into two different forms: sporadic or familial. The familial form is strongly suspected when multiple cavernomas are present, autosomal dominant inheritance is very likely according to the family history, or typical MRI patterns occur in several family members.3,7-9⇓⇓⇓
Three different gene loci—CCM1 (7q21–22), CCM2 (7p13–15), and CCM3 (3q25.2–27)—have been described so far.10-13⇓⇓⇓ In Hispanic Americans, virtually all familial cavernomas can be attributed to a founder mutation at the CCM1 locus.11,14-17⇓⇓⇓⇓ Cases involving non-Hispanic families have also exhibited mutations at the CCM1 locus (figure).7,13,16-25⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓ Mutations at the CCM2 and CCM3 locus are reported less frequently.13
Figure. Schematic genomic structure of the CCM1 gene encoding Krit1, depicting the published mutation sites and types, and the nomenclature used in the literature (1 through 20) and in the current study (4a through 12). Exons at sites of published mutations are indicated by different symbols according to the type of mutation they harbor (# = splice site mutations, * = frameshift mutations, • = nonsense mutations). A total of 44 different mutations have been identified to date.16-24,29⇓⇓⇓⇓⇓⇓⇓⇓⇓
We investigated whether CCM1 mutations can be found in the tissue of sporadic cavernomas, for we suspected that familial forms might be more frequent among the general population than reported. If CCM1 mutations were involved in the development of sporadic cavernomas, the same mutation might be detected in the adjacent normal brain tissue. This would clarify whether the underlying genetic event is a germline or a sporadic mutation at the CCM1 locus.
Materials and methods.
Patients.
Seventy-two patients with cerebral cavernomas (43 men and 29 women) who have been consecutively treated at the Neurosurgical Department of the Ludwig-Maximilians University Munich since 1990 were included in the study (demographic data are presented in the table). Initial clinical symptoms included epileptic seizures, focal neurologic deficits, and signs of increased intracranial pressure such as headache or nausea.
Table 1 Demographic data
Treatment protocol.
Microsurgical tumor resection was the treatment of choice.
Screening for CCM1 mutations.
Single-strand conformational polymorphism (SSCP) analysis and direct DNA sequencing for CCM1 mutations.
Serial 4-μm-thick paraffin sections were cut from the tissue blocks. Typical cavernous vessel formations as well as adherent normal brain tissue were identified, marked separately on hematoxylin-eosin stained slides, and copied on the adjacent unstained sections. The marked areas were then scraped from the slides and placed at the bottom of sterile 1.5 mL tubes. The DNA was extracted by a xylol-ethanol-acetone procedure and digested with proteinase K, as described elsewhere.26,27⇓ Mutations were prescreened by PCR-SSCP analysis of the original exons 1 to 12 on the CCM1 gene as well as the four additional encoding exons recently identified17,28,29⇓⇓ and defined in this study as 4a, 5a, 6a, and 7a. The primers were designed according to previous studies (see the supplementary table at www.neurology.org).19,22⇓ Normal control DNA was obtained from the temporal lobe of a patient who underwent epilepsy surgery for hippocampal sclerosis. Every slightly abnormal migration pattern was considered a questionable mobility shift and analyzed by direct DNA sequencing. PCR was preformed with 2 μL DNA solution, 3 pmol of each primer, 50 μM of dNTP, 1 μCi of [α-33P]-dCTP (ICN, Costa Mesa, CA; specific activity, 3,000 Ci/mmol), 10 mM Tris (pH 8.8), 50 mM KCl, 1 mM MgCl2, and 0.2 U Taq polymerase (Sigma, Deisenhofen, Germany) in a final volume of 10 μL. The PCR mixture for the original exons 1 to 12 and for exon 7a underwent an initial step of denaturation at 95 °C for 5 minutes, then 35 cycles of denaturation (94 °C) for 30 seconds, annealing (56 °C) for 60 seconds, and extension (72 °C) for 60 seconds, with a final step at 72 °C for 6 minutes using a Robocycler 96 gradient temperature cycler (Stratagene GmbH, Heidelberg, Germany). The annealing temperature for exons 4a and 6a was changed to 60 °C, for exon 5a to 65 °C. Twenty-five microliters of stop solution (USB, Cleveland, OH) were added to the 10-μL PCR reaction mixture. All samples except for the undenatured control were heated at 99 °C for 5 minutes, and 4 μL were immediately loaded onto a 6% polyacrylamide nondenaturing gel containing 6% glycerol. Gels were run at 40 W for 3 hours with fan cooling at room temperature, dried at 80 °C, and autoradiographed for 24 to 48 hours.
The samples showing abnormal migration patterns were further analyzed by direct DNA sequencing. Sequencing was performed in both directions with an ABI377 automated sequencer using the ABI dRhodamine terminator cycle kit according to the recommendations of the manufacturer (Applied Biosystems Perkin Elmer Cooperation ABI377, Weiterstadt, Germany). In brief, PCR was performed as previously described in a final volume of 40 μL. Sixty microliters of distilled water as well as 500 μL of binding buffer were added in a centrifugation column. The mixture was twice washed with washing buffer and finally elution buffer and centrifuged at 13,000 revolutions per minute. NaOH and iced 100% ethanol were added to the pellet at the bottom of the 1.5 mL Eppendorf tube, and the tube was frozen at 80 °C for 30 minutes and then recentrifuged for 30 minutes. The pellet was then dried and dissolved in 30 to 50 μL of distilled water depending on the amount of PCR product verified on agarose gel. A new PCR with 4 μL of DNA, 1.4 μL of the primer (3 pmol), and 2 μL of the previous PCR product in a total volume of 10 μL was performed with 25 cycles of denaturation (96 °C) for 10 seconds, annealing (50 °C) for 5 seconds, and extension (60 °C) for 4 minutes using a Robocycler 96 gradient temperature cycler (Stratagene GmbH). The new PCR product was resuspended with 10 μL distilled water. Two microliters of NaOH and 55 μL of 100% ethanol were added to each tube and centrifuged for 30 minutes at 13,000 Upm. A second washing step was performed with 70% ethanol. After removal of the liquid phase 3 μL of loading buffer, consisting of 50 mmol EDTA (ph8) and 100 mg/mL dextran blue and formamid, was added to the pellet. The solutions were loaded on a 6% polyacrylamide/7M urea gel in an automated sequencer (Applied Biosystems Perkin Elmer Cooperation, ABI377).
Results.
DNA extracted from cavernoma sinusoids and normal brain tissue of all 72 patients was screened for mutations using the SSCP method on the original exons 1 to 12 of CCM1 and the four recently described additional coding exons 4a through 7a. Fifteen samples showed a questionable mobility shift in the SSCP analysis (see supplementary figure e2 at www.neurology.org). Additional direct DNA sequencing in both directions was performed on these samples as well as 86 other samples with slightly suspicious SSCP patterns. We did not detect a mutation in any of the samples. All sequencing data generated showed a regular wild-type DNA sequence (see supplementary figure e3, A and B, at www.neurology.org).
Cavernomas and normal brain tissue did not harbor a mutation at the CCM1 locus in any of the 72 patients in our series, including the six patients with multiple cavernomas and the two patients who were brother and sister.
Discussion.
CCM1 is the site of mutation in almost all Hispanic American kindreds and in an estimated 35 to 40% of all white families with cerebral cavernomas.7,13-15⇓⇓⇓ A founder mutation in Hispanic Americans has been identified on Krit1, a gene on chromosome 7q 21–22 at the CCM1 locus.11 This substitution of C by T at nucleotide 742 on exon 6 of Krit1 leads to a premature stop codon.16,17⇓ Further investigations revealed several other mutations within the Krit1 gene in white families with familial cavernous malformation (see the figure). The majority of the published mutations cause a premature termination codon, which leads to a loss of function or an underexpression of Krit1.16-25⇓⇓⇓⇓⇓⇓⇓⇓⇓ The Krit1 protein is thought to interact with rap1a, a Ras-family GTPase that is hypothesized to function as a tumor suppressor gene.30,31⇓ Another possible function of Krit1 is to mediate protein-protein interaction.32 Recently, specific interactions of Krit1 with the integrin cytoplasmic domain-associated protein 1 (icap1a), a protein involved in β1-dependent angiogenesis, could be demonstrated.33 The expression profiles of Krit1 and KREV-1/rap1a show that they are not restricted to vascular or cerebrovascular tissue but are also expressed in varying amounts in heart, kidney, lung, liver, skeletal muscle, placenta, and pancreas.16,17,19⇓⇓ It is unknown why cavernous malformations develop mainly in the cerebral vessel tissue.
The working model—namely, that cerebral cavernous malformations are benign vascular tumors that develop due to an alteration in an important growth control pathway involving Krit1 and its interaction with Krev-1/rap1a—has been suggested before.16 The focal nature of cerebral cavernomas is thought to be either due to loss of the wild-type Krit1 allele in the developing cerebral vasculature or a result of mechanical or environmental triggers in the cerebral vasculature, coupled with the underlying genetic defect in Krit1 as a double hit model.16,20⇓ Krit1 mutation in a sporadic case18 as well as a de novo germline mutation of CCM1 in a patient with multiple cerebral cavernomas have already been reported.21 The ultimate role of Krit1 mutations in the development of cerebral cavernomas, however, remains unclear. No candidate genes have been identified so far at the two additional loci for mutational sites in cerebral cavernomas, characterized by multilocus linkage as CCM2 at 7p13–15 and CCM3 at 3q25.2–27.13
We also investigated mutations of the well-characterized CCM1 locus in sporadic cavernoma tissue of white families as the main site of mutations in cerebral cavernomas. One working model was to identify whether DNA of sporadic cavernoma cells shows mutations at the CCM1 locus, which would lead to potential growth regulation disturbances. However, DNA of adjacent normal brain tissue cells does not show any mutation. We found that none of the patients had a mutation at the CCM1 locus, either in cavernoma tissue or in normal brain tissue. Thus, CCM1 mutations seem to be less frequent than estimated.
We also considered whether the prevalence of familial forms of cerebral cavernomas is much higher than reported. This has also been proposed in the literature.7,8⇓ Owing to the absence of mutations of the 16 exons of CCM1, we could not identify a familial form or a de novo germline mutation of cerebral cavernomas in our series, despite having six cases of multiple cavernomas and a brother and a sister who were both affected. Reports of patients with multiple or apparent de novo cavernomas, which are classified as sporadic cavernomas after negative MRI screening results in close relatives, support the finding that even multiple cavernomas do not necessarily show a genetic alteration at the CCM1 locus.34 Because there might be an estimated mutation rate of 20% at the CCM2 and CCM3 loci, a familial background cannot be excluded on the basis of the absence of a mutation at the CCM1 locus. The prevalence of mutations at these loci may be more frequent than estimated so far, and should be considered more often in genetic investigations of white kindreds. Further studies on the pathogenetic effect of CCM1 mutations on the development of cerebral cavernomas and the identification of potential gene candidates at the CCM2 and CCM3 loci are needed to increase our understanding of the development of sporadic single or multiple cavernomas.
Acknowledgments
Supported by a grant from the “Curt-Bohnewand-Fond,” Medical Faculty of the Ludwig-Maximilians University, Munich, Germany.
Acknowledgment
The authors thank J. Benson for copyediting the manuscript. They also thank G.A. Rouleau and D.J. Verlaan for their helpful advice and discussion.
- Received March 6, 2002.
- Accepted December 18, 2002.
References
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