Download Clinical, pathological, magnetic resonance imaging features of a

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.
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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.