Cerebral cavernous malformations (CCMs) is a common form of vascular anomaly with abnormally enlarged capillary cavities that are prone to rupture causing hemorrhagic stroke, accounting for 5%-15% of all vascular malformations in the central nervous system.1–3 Cases can occur sporadically or be inherited in an autosomal dominant fashion with incomplete penetrance.4 Familial CCMs have been due to mutations at 3 loci identified by linkage analysis and positional cloning: CCM1 (OMIM 116860) on 7q21 that is more responsible for CCMs,5–7CCM2 (OMIM 603284) on 7p13, and CCM3 (OMIM 603285) on 3q26,8 which are less commonly associated with this disorder.9,10 So far, the reported familial CCMs have been mainly described in the Mexican/Hispanic population, demonstrating founder effect in this community.11–13 However, there are a few Chinese families with CCMs having been referred during recent decade.14–16 We here report a Chinese family with CCMs due to a novel mutation in exon 14 of CCM1 gene. Our study expands the spectrum of mutations in CCM1 linked to CCMs and implies the potential implications in the prenatal counseling or genetic diagnosis for this family in the future.
We studied a 25-member Chinese family with CCMs, involving seven patients (five male and two female). The index person was a 50-year-old man with hemorrhagic stroke in the left basal ganglia. After acquiring informed consent following the protocols approved by the local ethics committee, all of the participants underwent detailed medical history, clinical evaluation highlighting results of neurological, dermatological, and ophthalmological examinations, and mutation analysis.
Brain magnetic resonance imaging (MRI) acquisition
Participants completed a clinical brain MRI on 1.5 Tesla GE Signa and Genesis systems (Siemens, Germany), including axial and coronal T1-weighted images, axial and coronal T2-weighted images, fluid-attenuated inversion recovery, diffusion-weighted images, spin echo sequence and gradient echo sequence. The MRI slice/gap thickness was 6.0/0.5 mm, and the matrix size was 224 × 256 for all sequences. Experienced neuroradiologists reviewed the brain MRI images.
Brain histopathological and ultrastructural examinations
Two patients with serious hemiplegia had surgical treatment and histopathologic and ultrastructural examinations were performed on the specimens.
Blood samples were obtained from the 25 family members with their informed consent and were added to ethylene diamine tetraacetic acid (EDTA). Genomic DNA was extracted from peripheral blood.
PCR was performed to amplify all of the coding exons of the three CCM genes (CCM1, CCM2 and CCM3). The primers were designed to encompass each exon and its flanking exon-intronic splice sites. Primers sequences were shown in Table 1. PCR was conducted under the following conditions: pre-denaturation at 94°C for 5 minutes, 35 cycles of denaturation at 95°C for 30 seconds, annealing at suitable temperature (Table 1) for 30 seconds, extension at 72°C for 30 seconds, and final extension at 72°C for 5 minutes. PCR amplified products underwent 1.5% agarose gel electrophoresis and were then analyzed by direct sequencing on ABI sequencer (Applied Biosystems Inc., USA). One of the amplification primers was used as a sequencing primer, and the sequencing results were analyzed by a BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi). DNA fragments with mutations were cloned into a PMD-18T vector (Takara, Japan) and transformed into E.coli bacterium. The identified variation in the proband was examined in other affected and unaffected family members as well as 100 healthy controls from China.
The family pedigree is shown in Figure 1. The index person is a 50-year-old man with sudden onset of right limbs weakness and slurred speech. Neurological examination showed sensory and movement disturbance in right limbs. Dermatological examination demonstrated a 3 cm × 2 cm irregular bluish violet mass with protruding vessels in the lateral malleolus of his right foot (Figure 2).
The proband's father died of hemorrhagic stroke at the age of 76 years and could be considered as a patient with CCMs. All 25 remaining living family members took part in the investigation. There was a wide range in clinical manifestations as well as the age of onset within the family. Three patients presented with recurrent headache, three with hemiplegia, one with seizure, one with difficulty in swallowing or dysdipsia and one with hemianopia. Four patients with skin cavernous hemangioma had CCMs. Ophthalmological examination showed no abnormalities.
Brain MRI or CT
The index patient's brain CT (Figure 3A) showed a hemorrhagic and calcified lesion in the left basal ganglia. Further brain MRI (Figure 3B-F) revealed multiple CCMs in the cerebral hemisphere, cerebellar and brainstem. MRI is the most sensitive modality for the diagnosis of CCMs. With T2-weighted sequences, the lesions are typically characterized by an area of mixed signal intensity. Gradient-echo (GRE) sequence MRI could find microcavernous hemangiomas with a central reticulated core and a peripheral rim of decreased signal intensity related to deposition of hemosiderin.
Other than the proband, six patients were identified by brain MRI (four male and two female), aged from 8–79-years old (mean age of 35.8 years). All of them displayed multiple CCMs. The number of intracranial lesions ranged from 18 to 49, and size from 0.2–5.0 cm.
Brain histopathological and ultrastructural features
Two patients' pathological examinations of the excised tissues confirmed CCMs (Figure 4). The lesions are berry-like hemangiomas, with typically discrete sublobes, owing to hemorrhage in various stages during illness evolution. Cavernous angiomas are composed of abnormally enlarged sinusoids. Hemorrhagic residua within the lesions are commonly seen (Figure 4A). Under an ultramicroscope, it was found that the basement membranes of sinusoids become thicker and looser. Parts of them are layered. And many disorganized collagen bundles exist in it. Sinusoids are formed by a monolayer endothelial cell and a large number of red blood cells are seen in the cavities (Figure 4B).
Screening of the 16 coding exons (exons 5–20) and flanking sequences of CCM1 gene revealed a heterozygous T deletion in exon 14 (c.1396delT) of the index patient (Figure 5A and 5B). All of the patients but no unaffected family members or unrelated healthy controls carried the same mutation (Figure 5C). This frameshift mutation was predicted to cause a premature translational termination signal at nucleotide (NT) 1481 to 1483, with a truncated Krev interaction trapped-1 (KRIT1) protein of 493 amino acids.
The most common presenting symptoms of CCMs include hemorrhagic stroke, seizure, recurrent headache, and focal neurological deficits.17–19 Patients' symptoms are clinical heterogeneity within or between families affected with CCMs. Many asymptomatic family members had obvious intracranial lesions revealed by brain MRI, but were clinically silent. It could be due to incomplete penetrance.20 Apart from the diversity of symptoms, the age of onset and severity, also varied to a large extent.21 The reasons for this variability are unclear, but there is a strong correlation between the age of patients and the number of lesions: the younger, the less lesions.22 In the family, it is also confirmed by the youngest 8-year-old boy with the least lesions comparing with the other six patients.
MRI is the most sensitive modality for the diagnosis of CCMs.23 GRE sequence could find micro-cavernous hemangiomas with a central reticulated core and a peripheral rim of decreased signal intensity related to the deposition of hemosiderin. The excised intracranial lesions of two patients have been performed on pathological examinations. CCMs are typically discrete multi-sublobes berry-like lesions that contain hemorrhage in various stages of illness evolution. The resected hemangiomas show abnormal ultrastructural pathological features. The recurrent embolization of CCMs has resulted in endothelial cells denudation. The thin walls of abnormal vessels, lacking significant subendothelial support, along with the rare tight junctions between endothelial cells, may contribute to the known propensity of CCMs for recurrent micro-hemorrhage.
Cerebral cavernous malformations can be both sporadic and familial. Almost half of CCMs cases are heriditory in nature and follow autosomal dominant trait with variable penetrance.24 However, approximately 60% of the sporadic cases of CCMs are demonstrated to be familial ones with identified familial mutations actually.25 Familial CCMs are more susceptible to have multiple intracranial lesions and is mainly distributed in Hispanic populations of Mexican descent. In our study, all the patients in this Chinese family had multiple intracranial lesions. Four patients with skin cavernous hemangioma all had CCMs. Gunel et al26 performed a linkage analysis in a Hispanicfamily and mapped the first gene CCM1 responsible for CCMs to 7q11.2-q21. And then they identified a strong founder effect that the identical haplotypes over a short segment of chromosome 7q existed in several unrelated Hispanic kindreds with FCCMs.11 However, this disorder is genetically heterogeneous and two additional genes have also been mapped on 7p15–13 (CCM2) and 3q25.2–27 (CCM3), respectively, which are less commonly associated with CCMs.8
CCM1 includes 16 coding exons and encodes Krev interaction trapped-1 (KRIT1) protein, a 736-amino acid microtubule-associated protein, containing two functional motifs: three putative ankyrin repeats and a C-terminal FERM domain. The protein plays an important regulatory role in angiogenesis by involvement in Rap1A (also known as Krev1)/Ras signal transduction pathways as well as in tumor suppression processes.27 In addition, KRIT1 is reported to interact with intergrin cytoplasmic domain-associated protein-1 (ICAP-1), which regulates cell adhesion processes and extracellular matrix (ECM) interactions by binding to the N-terminal region of KRIT1.28
To date, CCM1 mutations account for a large proportion of identified pathogenetic mutations of CCMs. Among the 135 CCM1 mutations, 43 are small deletions, 23 small insertions and 38 missense/nonsense mutations, 24 splicing mutations, three small indels, three gross deletions and one gross insertion/duplication. Most of these mutations were significantly more often clustered in the second half of the CCM1 gene.
Though familial CCMs are more common in the Mexican/ Hispanic population, there are a few studies performed in Chinese community in recent decade. Interestingly, all the reported CCMs mutations in Chinese families are in the CCM1 gene, including two deletion mutations,15,16 two missense mutations14,29 and one splicing mutation.30 Chen et al14 and Xu et al29 reported two point mutations in exon 19 (c.2835CT, p.Q698X) and exon14 (c.1298CG, p.S430X) of CCM1 gene, respectively, in three Chinese families with CCMs. Mao et al16 first found an AT deletion mutation in exon 13 (c.1292delAT), resulting in a truncated KRIT1 protein. In 2011, another deletion mutation in exon 12 of CCM1 gene (c.1197delCAAA) was also identified.15 Till now, there is only one splicing mutation of a GTA deletion at the acceptor splicing site of intron 9/exon 10 in CCM1, causing a truncated protein by creating a premature termination code at the 23rd amino acid downstream from the sequence alteration.30 It appears that CCM1, rather than CCM2 or CCM3, is likely to be the dominant pathogenic cause of familial CCMs in Chinese population. All of these mutations are located within the second half of CCM1 gene and predicted to produce a truncated KRIT1 protein, which is consistent with the international reports.6 We hypothesize a loss of function due to mutated gene which may produce unstable mRNA or proteins without function is the most likely pathogenetic mechanism of CCMs.
Our study here reports a novel mutation, deletion of a T in exon 14 (c.1396delT) of CCM1 gene, which also could result in a premature stop codon at the second half of CCM1, with a 493-amino acid truncated protein. The C-terminal mutations may remove ankyrin repeats and lose the recognition site Rap1A which interacts with KRIT1. However, these inferences are required to be validated in more Chinese familial CCMs or by further molecular biological research.
We thank the patients and their families for their participation.
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