Abstract
Cerebral cavernous malformations (CCM) are CNS vascular anomalies associated with seizures, headaches, and hemorrhagic strokes. The CCM1 gene was screened in 35 sporadic cases with either single or multiple CCM. It was found that 29% of the individuals with multiple CCM have a CCM1 mutation, whereas cases with only one malformation have none. Sporadic cases with multiple malformations warrant the same approach as individuals who have a familial history of CCM.
Cerebral cavernous malformation (CCM; MIM 116860) is a common disorder characterized by abnormally enlarged capillary cavities in the brain without intervening normal parenchyma.1 It is found in 0.1 to 0.5% of the population and represents 10 to 20% of the cerebral vascular lesions.1 CCM most often occurs sporadically but may also be inherited dominantly with incomplete penetrance. Patients may have single or multiple malformations that can lead to focal neurologic signs, hemorrhagic strokes, seizures, or sometimes death.1 Patients can be either managed conservatively or treated with surgical resection when lesions cause recurrent hemorrhage or seizures.
Mutations in the gene CCM1, located on chromosome 7q21.2, account for nearly all Hispanic and approximately 40%2 of non-Hispanic familial CCM cases. All CCM1 mutations found to date are predicted to result in a truncated protein.3,4⇓ Two other loci were found by linkage analysis: CCM2 on 7p13-p15 and CCM3 on 3q25.2-q27, calculated to account for 20 and 40%, respectively, of all remaining familial cases.2
We assessed the incidence of mutation in the CCM1 gene in individuals harboring single or multiple cavernous malformations and who have no family history of the disease. For this purpose, individuals were screened for mutation in the CCM1 gene by denaturing high-performance liquid chromatography (dHPLC).
Methods.
Patients.
DNA was collected from 21 sporadic cases with one single malformation and 14 sporadic cases with multiple malformations. All individuals participated in the International Familial Cavernous Angioma Study and were located in Switzerland and Germany. In all cases, DNA was collected with informed consent, and the study was approved by the Committee for the Protection of Human Subjects at Dartmouth College (Hanover, NH). Criteria for inclusion in the study were 1) neuroradiologic diagnosis of either single or multiple cavernous malformations by MRI (including T1, T2, and gradient echo sequences); 2) histologic verification of at least one neuroradiologically diagnosed cavernous malformation; 3) no familial history for typical clinical manifestations of cavernous malformations such as seizures, hemorrhage of the CNS, focal neurologic deficits, and headache; and 4) no unexplained death of a family member at an early age (as putative indicator for a fatal hemorrhage of a cavernous malformation).
Mutation detection by dHPLC and sequencing.
For dHPLC of the gene CCM1, patient DNA was extracted from peripheral blood lymphocytes by standard methods. The CCM1 gene contains 16 coding and 3 noncoding exons.5 Each of the 16 coding exons was amplified by PCR with intronic primers.6 PCR-amplified products were denatured by heating to 95 °C for 5 minutes, followed by cooling to room temperature over a 45-minute period to enhance heteroduplex formation. The sequences of these fragments were analyzed using Wavemaker software package (Transgenomic, Omaha, NE). DHPLC detects 96% of all single-base substitutions as well as insertions and deletions one to several basepairs in length compared with single-strand conformational polymorphism (SSCP), which has a power of mutation detection of 85%.7 Each variant found by the dHPLC was reamplified by PCR and sequenced on an ABI 3700 sequencer, according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA).
Results.
A total of 35 sporadic cases were screened for mutation in the CCM1 gene. Of the 14 cases with multiple malformations, 4 (29%) have a CCM1 mutation leading to truncation of the protein (table). Subject 3 has a nonsense mutation in exon 9 (Trp271Stop),3 created by a G-to-C transition at nucleotide position 813, predicted to lead to a protein missing most of its domains. Subject 14 has an invariant splice donor site mutation in exon 16 (IVS16+2T→A), and Subject 34 has an invariant splice acceptor site mutation in exon 8 (IVS8-2A→G). Subject 32 has a nonsense mutation in exon 15 (Glu567Stop), created by a G-to-T transversion at nucleotide position 1,699, predicted to lead to a truncated protein with half of its FERM (band 4.1, ezrin, radixin, moesin) domain missing.
Table Mutations in sporadic cases with cerebral cavernous malformations
In contrast, only a missense mutation was found in 1 of the 21 individuals with a single malformation. Subject 7 has a Lys479Thr missense in exon 14 due to an A-to-C transversion at nucleotide position 1,436. The lysine, which is found in the FERM domain, is conserved within the mouse protein. The variation could not be found in 96 control subjects of European descent. Yet, the only missense mutations ever reported in familial cases actually led to splicing errors.4 Unfortunately, the effect of this missense on splicing was not tested, as blood was no longer available from this subject. However, this variation does not seem to be in a conserved sequence for splicing. It is likely to be a rare polymorphism, but further molecular testing would be required to be certain.
Discussion.
The gene CCM1 encodes for a 736-amino acid protein called the Krev interaction trapped 1 (Krit1) protein. Krit1 contains three ankyrin repeats, a FERM domain, and an NPXY (Asn-Pro-X-Tyr) motif. Molecular studies show that the NPXY motif seems to modulate a strong interaction with the integrin cytoplasmic domain-associated protein 1 (icap1α), a protein involved with β1-dependent angiogenesis.8 In addition, Krit1 has been shown to be a microtubule-associated protein that may help determine endothelial cell shape and function in response to cell-cell and cell-matrix interactions by guiding cytoskeletal structure.9
The chance of finding a deleterious mutation in an individual with multiple malformations (4 in 14) compared with an individual with only a single malformation (0 in 21) is significant (p = 0.004, Student t-test); 29% (what we found) and 40% (the expected value) are not statistically different (p = 0.361, Student t-test). Hence, our results show that sporadic cases with multiple malformations seem to harbor CCM1 mutations in approximately the same proportion that familial cases harbor CCM1 mutations. By extension, mutations in the candidate genes CCM2 and CCM3 may account for the cases where no CCM1 mutation was found. The CCM1 mutations found in these sporadic individuals are either germline mutations or have been inherited from an asymptomatic parent. Clinically, these individuals may be considered as CCM familial cases that have a higher recurrence risk of malformation formation and a 50% chance of offspring transmission. MRI or other CCM screening method of the patient’s family may also be appropriate for early detection diagnosis. In contrast, sporadic cases with only one malformation do not seem to have any CCM1 mutation, and their malformation may have been caused by a one-time random mutational event in the CCM1 gene at the site of the malformation or by mutation in other genes. These cases would have a much lower risk of malformation formation recurrence and little chance of offspring transmission.
A similar study10 that included 64 sporadic cases with a single malformation, 6 sporadic cases with multiple malformations, and 2 individuals from a CCM family for a total of 72 patients found that none of the cases had a CCM1 mutation. Their results for patients with only one malformation seem to be concordant with our data, suggesting that an inherited CCM1 mutation is not the cause of the disease. However, the fact that they did not find any mutation in their cases with multiple malformations does not contradict our study but could simply suggest a small sample size or missed mutation due to the limitation of their mutation detection technique (SSCP).7
Although there are many new molecular findings, the pathologic mechanism of CCM1 still remains unknown. There are at least two possible mechanisms hypothesized for malformation development. The first mechanism is Knudson’s second hit hypothesis in which the normal allele is deactivated, and the second one is a haploinsufficiency model. Further studies will need to confirm either one of these theories.
Acknowledgments
D.J. Verlaan is supported by an FRSQ-FCAR scholarship.
The authors thank the patients and the physicians who referred them for their participation in this study.
- Received June 3, 2003.
- Accepted in final form December 1, 2003.
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