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Clinical, pathological, magnetic resonance imaging features of a Chinese family with cerebral cavernous malformation induced by a novel CCM1 gene mutation ※ WANG Xue, LIU Xue-wu , Nora Lee, LIU Qi-ji, LI Wen-na, HAN Tao, WEI Kun-kun, QIAO Shan and CHI Zhao-fu Department of Neurology, Qilu Hospital, Shandong University, Jinan, Shandong 250012, China (WANG Xue, LIU Xue-wu, LI Wen-na, HAN Tao, WEI Kun-kun, QIAO Shan and CHI Zhao-fu) Department of Neurology, Hartford Hospital, Hartford, Connecticut 06106, USA (Nora Lee) Department of Medical Genetics, Key Laboratory for Experimental Teratology of the Ministry of Education, Shandong University School of Medicine, Jinan, Shandong 250012, China( LIU Qi-ji) ※Correspondence to : LIU Xue-wu , Department of Neurology, Qilu Hospital, Shandong University, Jinan, Shandong 250012, China (Tel: +8682166166, Fax: 0531-6927944 , E-Mail: [email protected]) This study was supported by grants from the National Natural Science Foundation of China (81171071) and the Natural Science Foundation of Shandong province (No. ZR2010HM052). Conflict of interest: none. Keywords: cavernous malformation; pathology; magnetic resonance imaging (MRI); CCM1; gene mutation; Chinese family Background Familial cerebral cavernous malformations (CCMs), characterized by hemorrhagic stroke, recurrent headache and epilepsy, are congenital vascular anomalies of the central nervous system. Family CCMs is an autosomal dominant inherited disorder and three CCM genes have been identified. We report a Chinese family with CCMs and intend to explore clinical, pathological, magnetic resonance imaging (MRI) features and pathogenic gene mutation of this family. Methods 25 family members underwent brain MRI examination and clinical check. Two patients with surgical indications had surgical treatment and the specimens were performed histopathological and microstructural examination. In addition, polymerase chain reaction (PCR) and direct sequencing were performed with genomic DNA extracted from 25 family members’ blood samples for mutation detection. Results Brain MRI identified abnormal results in 7 family members. All of them had multiple intracranial lesions and four cases had skin cavernous hemangioma. T2-weighted sequence showed the lesions were typically characterized by an area of mixed signal intensity. Gradient-echo (GRE) sequence was more sensitive to find micro-cavernous hemangiomas. There was a wide range in the clinical manifestations as well as the age of onset in the family. The youngest patient was a 8-year-old boy with least intracranial lesions. Histopathological and microstructural examination showed the CCMs were typically discrete multi-sublobes berry-like lesions, with hemorrhage in various stages of illness evolution. They were formed by abnormally enlarged sinusoids and the thin basement membranes. A novel T deletion mutation in exon 14 of CCM1 gene was identified by mutation detection in the 7 patients. But unaffected members and healthy controls did not carry this mutation. Conclusions The clinical manifestations were heterogeneity within this family. The results of brain MRI and pathological examination conformed to the diagnosis of CCMs. We identified a novel mutation (c.1396delT) was the disease-causing mutation for this family and extended the mutational spectrum of CCMs. Cerebral cavernous malformations(CCMs) is one common form of vascular anomalies with abnormally enlarged capillary cavities that is 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 inherit 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 5-7 , CCM2 (OMIM 603284) on 7p13 and 8 CCM3 (OMIM 603285) on 3q26 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 CCMs having been referred during recent decade 14-16 . However, there are a few Chinese families with . 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. METHODS Participants We studied a 25-member Chinese family with CCMs, involving 7 patients (5 male, 2 female). The index person was a fifty years 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 on neurological, dermatological, and ophthalmological examinations, and mutation analysis. Brain 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 histopathologic and ultrastructural examinations Two patients with serious hemiplegia had surgical treatment and the specimens were performed histopathologic and ultrastructural examinations. Blood samples Blood samples were obtained from 25 family individuals with their informed consent and added to ethylene diamine tetraacetic acid (EDTA). Genomic DNA was extracted from peripheral blood. Mutation detection PCR was performed to amplify all of the coding exons of 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 with following conditions: pre-denaturation at 94℃ for 5 min, 35 cycles of denaturation at 95℃ for 30 s, annealing at suitable temperature (Table 1) for 30 s, extension at 72℃ for 30 s, and final extension at 72℃ for 5 min. PCR amplified products underwent 1.5%agarose gel electrophoresis and 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, China) 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. RESULTS Clinical features The family pedigree was shown in Fig.1. The index person was 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 3cm*2cm irregular bluish violet mass with protruding vessels in the lateral malleolus of his right foot (Fig.2). The proband’s father died of hemorrhagic stroke at the age of 76 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 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 (Fig.3-1) showed a hemorrhagic and calcification lesion in the left basal ganglia. Further brain MRI (Fig.3-2,3,4,5,6) 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 micro-cavernous hemangiomas with a central reticulated core and a peripheral rim of decreased signal intensity related to deposition of hemosiderin. Other than the proband, 6 patients were identified by brain MRI (four male and two female), aged from 8-79 years old (mean age 35.8 years old). 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 histopathologic and ultrastructural features Two patients’ pathological examinations of the excised tissues confirmed CCMs (Fig.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 abnormal enlarged sinusoids. Hemorrhagic residua within the lesions are commonly seen (Fig.4-1). Under a ultramicroscope, it is found 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 (Fig.4-2). Genetic analysis Screening of the 16 coding exons (exon5-20) and flanking sequences of CCM1 gene revealed a heterozygous T deletion in exon 14 (c.1396delT) of the index patient (Fig. 5-1). All of the patients but no unaffected family members or unrelated healthy controls carried the same mutation (Fig.5-2, 3). 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. DISCUSSION The most common presenting symptoms of CCMs include hemorrhagic stroke, seizure, recurrent . Patients’ symptoms are clinical heterogeneity within or 17-19 headache and focal neurological deficits between families affected with CCMs. Many asymptomatic family members had obvious intracranial lesions revealed by brain MRI, but clinically silent. It could be due to incomplete penetrance 20 . Apart 21 from the diversity symptoms, the age of onset and severity, have varied to a large extent . 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 22. In the family, it is also confirmed by a youngest 8-year-old boy with the least lesions comparing with other 6 patients. MRI is the most sensitive modality for the diagnosis of CCMs 23. Gradient-echo (GRE) sequence could find micro-cavernous hemangiomas with a central reticulated core and a peripheral rim of decreased signal intensity related to 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 inherited 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 mainly distribute 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 al. performed a linkage analysis in a Hispanic-family and mapped the first gene CCM1 responsible for CCMs to 7q11.2-q21 26 . 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 singal 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 KRIT128. 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, 3 small indels, 3 gross deletions and one gross insertions/duplications. 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 2 deletion mutations 15, 16 , 2 missense mutations 14,29 and one splicing mutation 30. Chen et al. and Xu et al. reported two point mutations in exon 19 (c.2835C >T, p.Q698X) and exon14 (c.1298C>G, p.S430X) of CCM1 gene respectively in three Chinese families with CCMs 14, 29 . Mao et al. first found a AT deletion mutation in exon 13 (c.1292delAT), resulting in a truncated KRIT1 protein 16. In 2011, another deletion mutation in exon12 of CCM1 gene (c.1197delCAAA) was also identified 15. To now, there is only one splicing mutation of a GTA deletion at the acceptor splicing site of intron9/exon10 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 are consistent with the international reports 6. We hypothesize a loss of function that mutated gene may product unstable mRNA or proteins without function is the most likely pathogenetic mechanism of CCMs. Our study here report 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 interacts with KRIT1. However, these inferences are required to be validated in more Chinese familial CCMs or by further molecular biological research. Acknowledgments Thank the patients and their families for their participation. Statement: The abstract of this paper had been published in the form of a poster at the American neurology annual meeting. REFERENCES 1. Robinson JR, Awad IA, Little JR. Natural history of the cavernous angioma. J Neurosurg 1991; 75: 709-714. 2. Cavalcanti DD, Kalani MY, Martirosyan NL, Eales J, Spetzler RF, Preul MC. Cerebral cavernous malformations: from genes to proteins to disease. J Neurosurg 2012; 116: 122-132. 3. Moriarity JL, Wetzel M, Clatterbuck RE, Javedan S, Sheppard JM, Hoenig-Rigamonti K, et al. The natural history of cavernous malformations: a prospective study of 68 patients. Neurosurgery 1999; 44: 1166-1171; discussion 1172-1163. 4. 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Treatment of epileptogenic cavernomas: surgery versus radiosurgery. Cerebrovasc Dis 2007; 24: 116-120; discussion 121. 18. Labauge P, Denier C, Bergametti F, Tournier-Lasserve E. Genetics of cavernous angiomas. Lancet Neurol 2007; 6: 237-244. 19. Rabinstein AA, Tisch SH, McClelland RL, Wijdicks EF. Cause is the main predictor of outcome in patients with pontine hemorrhage. Cerebrovasc Dis 2004; 17: 66-71. 20. Gianfrancesco F, Cannella M, Martino T, Maglione V, Esposito T, Innocenzi G , et al. Highly variable penetrance in subjects affected with cavernous cerebral angiomas (CCM) carrying novel CCM1 and CCM2 mutations. Am J Med Genet B Neuropsychiatr Genet 2007; 144B: 691-695. 21. Belousova OB, Konovalov AN, Okishev DN, Sazonova OB, Shamov A. Hereditary cerebral cavernous malformations: analysis of 12 families. Zh Vopr Neirokhir Im N N Burdenko 2011; 75: 34-45; discussion 45-37. 22. Labauge P, Laberge S, Brunereau L, Levy C, Tournier-Lasserve E. 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Table 1 Primer pairs for CCM1、CCM2 and CCM3 genes Gene and exon Primer sequence(5’-3’) CCM1 exon5F GGTTTTGAGTAGTTGAACAGTA CCM1 exon5R ACAAGATATAATAAGGCTTTAT CCM1 exon6F GCTTATTGTAGTATTCTAGGGC CCM1 exon6R TTTTGCAAGTTTGTTAAATGTA CCM1 exon7F ACCAGGACCCTAAATCTACAAG CCM1 exon7R ACTTCCTCAATAGACCTTTACT CCM1 exon8F TGAGTGGACAAGGTCACAGAGC CCM1 exon8R CCAGGACAACCTTATCTTCGGG CCM1 exon9F ATTCACAAGTGTCATATATCAG CCM1 exon9R AATTACACTTGAGATAAAACGT CCM1 exon10-11F ATGGGTTTTTGTTATTGTTTTC CCM1 exon10-11R CAATGCCTAAGGACACTAACAG CCM1 exon12F TAATAATCCTTTGCTCTCTGTC CCM1 exon12R GATGAACTCTCAAACATACAAT CCM1 exon13F CTAAACATAATACATACCAAGT CCM1 exon13R GTAAAGAAGCCATCTAATCGTC CCM1 exon14F GTTGAAGGAAAGTATCTTGTTA CCM1 exon14R CACTCCCAAAAAGGAATAATGA CCM1 exon15F AGAAGTGCAGACAGTTTAATAC CCM1 exon15R TAAGTAGATCAACTGAAACTCC CCM1 exon16F TTTCCCATATTAAGTTGTTCAT CCM1 exon16R ATTCCAAACCAATGTAATGTAT CCM1 exon17F TGATTACTATACTTTAGCTACT CCM1 exon17R ATAACACTAACAAAGTTTCAAC CCM1 exon18F TCTATTTGGATTATCAATGGTA CCM1 exon18R TTAGTGTCCCCCTTCCTCTTAT CCM1 exon19F TAGTAAGAGAAAATAGGTGAAG CCM1 exon19R CTAACACAATAGTTTATGAAGT CCM1 exon20F ATAGATAGGGAACTGCCCAATG CCM1 exon20R CACACCAAACATAAGTACAGAT CCM2 exon1F CCTGAAAGTGAGGATGAGCGAC CCM2 exon1R CCGCCGACCACAGCAGTAGGAC CCM2 exon2F TTGGCTACTTCTGTTTGTTA CCM2 exon2R TTTATCAGCATAGACAAGAC CCM2 exon3F TCTGTAAGAACCCAGGACTC CCM2 exon3R CTCATTCCTCACAGCCACGA CCM2 exon4F GTATTTCTGATGCCCTGTGG CCM2 exon4R AAACAGCAGCACCCAACACG CCM2 exon5F CCCTTCACCTGAGTCGTTCT CCM2 exon5R GGCAATGGCAGAGGAAGTAG CCM2 exon6F TGTTTATTGAGCATCTGGGC CCM2 exon6R TGAAACCCTATGGGCTAATG PCR-product size (bp) Annealing temperature (℃) 412 56 354 58 367 56 487 58 397 54 725 54 397 56 368 54 286 52 392 54 350 56 215 52 424 54 226 54 622 58 319 54 409 56 352 56 322 54 412 58 402 56 Gene and exon Primer sequence(5’-3’) CCM2 exon7F TTGATGAAGGACAGCAGGGT CCM2 exon7R ACAACACCAGACAACGCATT CCM2 exon8F GAAGCCACCCGCTCACATAC CCM2 exon8R CCAGGAAAAGGCAGTGATGT CCM2 exon9F GTTAGTGGCTGTGGCAAGGT CCM2 exon9R GTTCAAATCTCCCATCCCTG CCM2 exon10F TGATTTCAACCAGGATGCTA CCM2 exon10R AAAGAGCCTTCCACCCCGCA CCM3 exon1F CTACGAGTCCCCATAAGCCT CCM3 exon1R GAGACTTCCTGGAGGCGTTC CCM3 exon2F GAGGTGAGGCTGCTGTTTTC CCM3 exon2R TAATCCCTCGGTTTCCTCAT CCM3 exon3F AAAAGGTAAGTTTCTTGCCA CCM3 exon3R TTGCCCTGATACAATGCCTA CCM3 exon4F ATGGAATCTCAGAAATGTGC CCM3 exon4R TTTATCTTTTGGAAGTGTTT CCM3 exon5F GCTCCCAACAGATTCTAAAC CCM3 exon5R AAACAGTAGGGAAGGAAGAT CCM3 exon6F ACCTGCCTTTGACCAACATT CCM3 exon6R CTCTGAAACCAAACGCCATA CCM3 exon7-8F CTAAAATCCCCACTCCAACC CCM3 exon7-8R TGCCTTCATTATCAGTTGCC CCM3 exon9F AATAATACTAACTTGTCTTT CCM3 exon9R GTCAGAAACAAACACCAATA PCR-product size(bp) Annealing temperature(℃) 255 54 312 56 351 52 654 56 388 54 320 56 517 55 287 58 307 56 385 58 779 56 520 57 Fig.1. The pedigree of the Chinese family with CCMs. The arrow indicates the index patient (Patient II1) and all affected members are indicated by filled symbols. Filled circle: affected female member, filled square: affected male member, unfilled circle: unaffected female member, unfilled square: unaffected male member. Filled square with a crossed line mean that the individual has died. Fig.2. a 3cm*2cm irregular bluish violet mass with protruding vessels in the lateral malleolus of right foot of the index patient(left).His daughter left foot had similar lesion(right). 1 4 2 5 3 6 Fig.3. Brain CT (1) of the index patient shows an irregular mixed-density lesion in the left basal ganglia, hyperdensity region represents calcification with bleeding. Axial T1、T2-weighted MRI(3、4) of the proband ,T2- fluid-attenuated inversion recovery MRI(2),as well as gradient echo sequence (GRE)(5,6) , reveal multiple CCM lesions in the cerebral hemisphere, cerebellar and brainstem. All lesions share a feature: mixed-signal regions surrounded by a hypointense ring of hemosiderin. 1 2 Fig.4. Photomicrograph of CCMs from the index patient(Ⅱ1) shows the blood vessels with different sizes and malformations. The sinusoids cavities are enlarged abnormally. And the basement membrane, together with the endothelial cells in its surface, highlight to the sinusoids cavities (Fig.4-1). HE staining. Original magnification×200. Under ultrastructure sinusoids are formed by a monolayer endothelial cell and a large number of red blood cells are seen in the cavities. The basement membranes become thicker, looser and layered. And many disorganized collagen bundles exist in it.(Fig.4-2).Double fixed (glutaraldehyde, OsO4), double staining (uranyl acetate - citric acid lead method) ×4000. Fig.5. Part sequencing result of 14 exon in CCM1 (from 5’to 3’): (1) a heterozygous T deletion at NT1396 in the index patient(Ⅱ1); and (2) sequencing result of bacilli PCR (patientⅡ1): a T deletion at NT1396, replaced by a G; and (3) the normal genome DNA sequence: a T at NT1396.