CCM3/SERPINI1 bidirectional promoter variants in patients with cerebral cavernous malformations: a molecular and functional study
Scimone et al. BMC Medical Genetics
CCM3/SERPINI1 bidirectional promoter variants in patients with cerebral cavernous malformations: a molecular and functional study
Concetta Scimone 0 1
Placido Bramanti 2
Alessia Ruggeri 1
Luigi Donato 0 1
Concetta Alafaci 1
Concetta Crisafulli 1
Massimo Mucciardi 3
Carmela Rinaldi 1
Antonina Sidoti 0 1
Rosalia D'Angelo 1
0 Department of Cutting-Edge Medicine and Therapies, Biomolecular Strategies And Neuroscience, Section of Neuroscience-applied, Molecular Genetics and Predictive Medicine, I.E.ME.S.T , Palermo , Italy
1 Department of Biomedical and Dental Sciences and Morphofunctional Imaging, Division of Medical Biotechnologies and Preventive Medicine, University of Messina , via C. Valeria 1, 98125 Messina , Italy
2 IRCCS Centro Neurolesi “Bonino-Pulejo” , Messina , Italy
3 Department of Economy, University of Messina , Messina , Italy
Background: Cerebral cavernous malformations (CCMs) are vascular anomalies of the nervous system mostly located in the brain presenting sporadically or familial. Causes of familial forms are mutations in CCM1 (Krit1), CCM2 (MGC4607) and CCM3 (PDCD10) genes. Sporadic forms with no affected relative most often have only one lesion and no germ line mutations. However, a number of sporadic cases with multiple lesions have been reported and are indeed genetic cases with a de novo mutation or a mutation inherited from an asymptomatic parent. Methods: Here, we performed an analysis of regulatory region of CCM genes in 60 sporadic patients, negative for mutations in coding region and intron-exon boundaries and large deletion/duplications in CCM genes by direct sequencing and MLPA. Among 5 variants identified in 851-bp region shared by CCM3 and SERPINI1 genes and acting as asymmetric bidirectional promoter, two polymorphisms c.-639 T > C/rs9853967 and c.-591 T > C/rs11714980 were selected. A case-control study was performed to analyze their possible relationships with sporadic CCMs. Promoter haplotypes activities on CCM3/SERPINI1 genes expression were tested by dual-luciferase assay. Results: No variants were identified in CCM1 and CCM2 regulatory regions. In CCM3/SERPINI1 asymmetric bidirectional promoter 5 variants, 2 of them unknown and 3 corresponding to polymorphisms c.-639 T > C/rs9853967, c.-591 T > C/rs11714980 and c.-359G > A/rs9834676 were detected. While rs9853967 and rs11714980 polymorphisms fall in a critical regulatory fragment outside the minimal promoter in intergenic region, other variants had no effects on transcription factor binding according to RegRNA tool. Case-control study performed on 60 patients and 350 healthy controls showed frequencies of the mutated alleles significantly higher in the control group than in patients. Furthermore, the functional assay showed a significant reduction of CCM3 expression for C-C haplotype even more than for T-C and C-T haplotypes. In SERPINI1 direction, the reduction was not statistically significant. Conclusions: Our data indicated that rs9853967 and rs11714980 polymorphisms could be associated with a protective role in CCM disease.
Bidirectional promoter; CCM3/SERPINI1; Polymorphism; Association study; Dual luciferase-assay; CCM genes
Cerebral cavernous malformations (CCMs) are among
the most common cerebral vascular anomalies
characterized by clusters of enlarged blood vessels consisting of
a single layer of endothelium and lacking smooth muscle
and elastic tissue, without intervening brain parenchyma
[1, 2]. CCMs, reported in up to 0.5 % of the population,
are primarily found within the central nervous system
where they result in increased risk for stroke, seizures,
recurrent headaches and focal neurological deficits [1–4].
CCMs are associated with loss-of-function mutations in
any one of the three CCM genes CCM1/KRIT1, CCM2/
MGC4607 or CCM3/PDCD10 and occur in both sporadic
and familial forms , inherited in an autosomal
dominant fashion with a high penetrance.
Familial CCM accounts for only 20 % of cases but
tends to be more severe than sporadic CCM , with
patients exhibiting multiple lesions and increased
More than 150 distinct CCM1, CCM2, and CCM3
germline mutations are known [6–9].
Linkage analyses predicts that 40 % of patients with
familial forms is linked to CCM1 locus, 40 % to CCM3
and, only 20 % to CCM2 [10–13]; however, according
with reported experimental data, these frequencies are
not confirmed by an our recent screening of a cohort of
Italian patients with CCMs . Our data showed that
54 % of patients leads CCM1 mutations, a very lower
percent was observed for CCM3 (6 %) while 18 % leads
mutation at CCM2 locus; the absence of any mutation
in 22 % of patients, leads to consider other possible
elements involving in the development of disease like a
somatic mosaicism of a de novo mutation that occurred
during gestation and is not detectable in DNA extracted
from peripheral blood [5, 6, 14], the possibility of large
deletions or duplications not detected by direct
sequencing or mutations located in regulatory regions of CCM
genes, and finally, the existence of other as yet
No data in the literature exist on CCM genes regulatory
regions. Therefore we focused our attention on molecular
analysis of these regions in a cohort of CCM sporadic
Initially we examined the promoters of CCM1 and
CCM2 genes, located about 4.0 kb (CCM1) and 0.5 Kb
(CCM2) upstream from the ATG start codon, and finally
CCM3 one. This is an evolutionary conserved
bidirectional promoter shared with SERPINI1 gene, coding for
a serine protease inhibitor . Two genes were found
to be closely adjacent to each other in a head-to-head
orientation and separated by an exceptionally short
intergenic region of 851-bp that function as a
bidirectional promoter to regulate transcription of both genes.
A 175-bp fragment from nt1 to 175 in the vicinity of
CCM3 was further determined to function as a minimal
bidirectional promoter. A critical regulatory fragment,
from nt 176–473 outside the minimal promoter in the
intergenic region, was identified to contain a strong
repressive element for SERPINI1 and an enhancer for
CCM3 . In a parallel study, through an in-silico
approach, we investigated the existence of common
pathways to both genes in order to explain the need of
this co-regulation and to detect a possible involvement
of SERPINI1 in CCM pathogenesis (data not shown).
Here, we focused on intergenic region of 851 bp
shared by CCM3 and SERPINI1, where we identified
5 variants, 2 of them unknown (c.-964 G > C and
c.-419G > T) and 3 corresponding to polymorphisms:
c.-639 T > C/rs9853967, c. -591 T > C/rs11714980
and c.-359G > A/rs9834676.
RegRNA analysis showed that the rs9834676
polymorphism and two novel variants do not affect the
transcriptional regulatory motifs. Furthermore,
literature data report rs9853967 and rs11714980
polymorphisms falling in a critical regulatory fragment outside
the minimal promoter in intergenic region .
Therefore, our attention has focused on rs9853967
and rs11714980 polymorphisms and their possible
association with CCM disease. A case-control study was
carried-out and the polymorphisms effects on two genes
expression were determined by a functional assay.
A total of 60 CCM patients (40 % men, 60 % women)
were recruited during 2003–2013.
Detailed clinical and neuroimaging information on
patients and their relatives were collected through direct
interview by review of the medical charts, before CCM
gene molecular analysis and after providing their written
informed consent. On the basis of pedigree analysis and
in the absence of relatives positive to biomolecular
investigation and/or Magnetic Resonance Imaging (MRI)
(standard spin echo and fast turbo spin echo T1- and
T2-weighted axial, coronal, and/or sagittal images) the
patients were considered sporadic. Lesions were single
in 58 patients (97.0 %) and multiple in 2 patients (3.0 %).
The age of symptoms onset, in all patients examined,
coincides with the age at diagnosis and the patient's age
at the time of recruitment. The mean age at onset of
symptoms was 39,5 ± 18,7 years (median, 36.0 years;
range, 10–73 years) (Table 1). A group consisted of 350
unrelated, randomly selected, ethnically matched,
healthy individuals were recruited. Demographic data on
controls are summarized in Table 1. Age at symptoms
onset of male vs female was matched in the CCM
patients, as well as age of male versus female in the
controls. Control individuals were selected to match the
(A) CCM sporadic patients (n = 60)
Male + Female 2 + 2
(B) Healthy controls (n = 350)
204 44,3 ± 17,9
146 42,3 ± 18,3
350 43,5 ± 18,1
p > 0.05 indicates no significant differences. Statistical analysis was tested by
the Student’s t-test. CCM, cerebral cavernous malformation; SD, standard deviation
The age of symptoms onset in all patients (with the exception of
asymptomatic patients) coincides with the age at diagnosis
CCM patients for age. Table 1 indicates no significant
differences (p > 0.05). This study was approved by the
Scientific Ethics Committee of the Azienda Ospedaliera
Universitaria–Policlinico ‘G. Martino’ Messina. Informed
consent was obtained from all patients and controls.
Genetic analysis of the CCM1, CCM2 and CCM3 promoters
DNA was extracted from leukyocytes by using the
standard protocols. The amplification of regulatory regions of
CCM1 and CCM2 genes was performed using the primers
pairs designed according to the published nucleotide
sequence of GenBank (accession no. NG_012964.1 CCM1;
NG_016295.1 CCM2) (Table 2).
Polymerase chain reaction (PCR) was carried out in a
thermal cycler (Gene Amp PCR System 2700; PE Applied
Biosystems, Foster City, CA) under following conditions:
F: 5’ – TTT ACA TTC TAG CTG TGC TA – 3’ 49
R: 5’ - CTG GAC AGG TGC GTT CTC – 3’
CCM3- SERPINI1 F: 5’ - TGAGGCACTGACTTCACTT-3’
R: 5’-CTTAGCTGCTCTCAGGGA-3’ (P1)
F: 5’ - TCCCTGAGAGCAGCTAAG-3’
R: 5’ - GCTCTCGTTCCTGCTTTC-3’ (P2)
aPrimers were designed to amplify a promoter region located about 3.8 kb
(CCM1) and 0.5 kb (CCM2) from the ATG start codon
0.8 μg of genomic DNA was added to 50 μl reaction
mixture containing a 0.2 μm concentration of each primer
and 1 U Taq Gold polymerase (PE Applied Biosystems).
PCR conditions for CCM1 promoter region (655 bp)
were as follows: denaturation at 95 °C for 45 s, annealing
at 58 °C for 35 s and extension at 72 °C for 40 s for
35 cycles, after an initial 10 min denaturation at 95 °C.
For CCM2 promoter region (620 bp) we employed
these conditions: one cycle of denaturation at 95 °C for
10 min, followed by 35 cycles at 94 °C for 40 s, 49 °C for
30 s, and 72 °C for 45 s, before a final extension at 72 °C
for 10 min.
Short intergenic region (851 bp) between two
nonhomologous genes, SERPINI1 and CCM3, was amplified
using two primer sets designed according to published
sequence data (Accession no. NG_ 008217.1 for
SERPINI1; NG_008158.1 for CCM3) and shown in Table 2.
Two fragments of 580 bp (P1) and 383 bp (P2) were
amplified under following conditions: for P1
denaturation at 94 °C for 50 s, annealing at 58 °C for 45 s and
extension at 72 °C for 50 s for 35 cycles, after an initial
10 min denaturation at 95 °C; for P2 denaturation at
94 °C for 40 s, annealing at 56 °C for 35 s and extension
at 72 °C for 45 s for 35 cycles, after an initial 10 min
denaturation at 95 °C.
PCR products were sequenced by direct sequencing.
The nucleotide number relative to polymorphisms
identified in intergenic region of 851 bp was indicated
respect to the transcriptional start sites of the reference
sequence reported by the NCBI database (in direction of
Analysis of data was performed using computer software
Statistical Package for Social Science (SPSS) for Windows
(Version 6.0.1) and Epi Info (version 6.0.4).
Comparisons between means of age of symptoms
onset of male versus female in the CCM group and overall
CCM age at onset versus overall healthy control age
were calculated with the Student’s t-test. For each group
(control and patients), allele frequencies were calculated
by direct gene counting. Estimates of statistical
significance were calculated by standard χ2 analysis for one
degree of freedom. Descriptive analysis included Student’s
t-test of means and the respective standard deviation
(SD) for cases and controls. A two-sided probability
value of < 0.05 was considered to indicate statistical
significance. The results from dual-luciferase assay were
compared using one-way ANOVA for repeated
In silico analysis
, a transcription factor prediction tool, was used to
to evaluate how the identified variants could interfere
with the transcriptional regulatory motifs in CCM3/
The relative position numbers of the nucleotides in this
intergenic region of 851 bp was as follows: 9 (- c.964G > C),
335 (-c.-639 T > C), 383 (-c.-591 T > C), 556 (-
c.419 G > T), 615 (-c.-359G > A).
U373-MG (human, Caucasian, glioblastoma-astrocytoma)
cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10 % Fetal Bovine Serum
(FBS), 100U/ml of penicillin and 1 mg/ml of streptomycin
(Lonza) at 37 °C in a water-satured atmosphere with 5 %
Construction of the reporter gene plasmids
To examine the potential effects of the c.-639 T > C and
c.-591 T > C on the intergenic region of 851 bp
transcription activity, we compared the promoter activity of
four common haplotypes (c.-639 T/c.-591 T) (T-T),
(c.639C/c.-591 T) (C-T), (c.-639 T/c.-591C) (T-C),
(c.639C/c.-591C) (C-C). 851 bp intergenic regions were
amplified by PCR using genomic DNA from donors
carrying each haplotype, using the primers showed in the
Table 3 and under the following conditions: 1 cycle of
95 °C for 5 min; 35 cycles of 94 °C for 40 s, 53 °C for
35 s, and 72 °C for 40 s; and 1 cycle of 72 °C for 10 min.
PCR products were inserted upstream of the luciferase
gene cloned into the pGL4.10 (luc2).
For this purpose, PCR products, as well as pGL4.10
(luc2) were digested with BglII and HindIII (Promega)
and then purified (PureLink™ PCR Purification Kit, Life
Technologies). Vector arms were ligated overnight to
the digested PCR fragment. The novel constructs were
subcloned into E. coli Top 10 cells (Life Technologies)
and single colonies were miniprep. The correct sequence
of all the clones was verified by DNA sequencing.
Transient transfection and promoter assays
Cells were first seeded in 96-well culture plates at a
density of 2 × 104 cells per well. In each well, cells were
then co-transfected with 0.6 μl of FuGENE HD
Transfection Reagent (Promega) and a mixture consisting of
0.2 μg of the pGL4.10 [luc2] promoter constructs and
0.02 μg of the co-reporter vector pGL4.74 [hRluc/TK] in
a serum-free medium and then incubated for 24 h at
37 °C in a humidified atmosphere of 5 % CO2 in air.
After incubation, cells were washed twice with PBS and
lysed by Passive Lysis Buffer (Promega). Luciferase
activity was measured using Dual Luciferase assay kit
(Promega) and GloMax-Luminometer (Promega).
Reporter construct activity was normalized by comparison
with activity from the Renilla luciferase construct.
Luciferase activities are representative of at least three
independent experiments, with each construct tested in
triplicate per experiment.
Genotype and allele frequencies of c.-639 T > C/ rs9853967
and c.-591 T > C/ rs11714980 polymorphisms in patients
In the promoter regions of CCM1 and CCM2 of 60
CCM patients cohort, no variants has been identified.
Conversely, analysis of regulatory intergenic region
shared by CCM3 and SERPINI1 has lead to the
identification of 5 variants, 2 unknown (c.-964 G > C and
c.419G > T) and 3 reported such as polymorphisms in SNP
Sequence?db=core;g=ENSG00000163536;.r=3:167453031167543356: c.-639 T > C/rs9853967, c.-591 T > C/
rs11714980 and c.-359 G > A/rs9834676).
To evaluate the possible influence that these variants
could have on transcriptional regulatory motifs, wild-type
(c.- 964G, c.-639 T, c.-591 T- c.-419 G, c.-359G) and
mutated (c.- 964C, c.-639C, c.-591C, c.-419 T, c.-359A)
sequences of intergenic region shared by CCM3 and
SERPINI1 were analyzed by using RegRNA. In mutated
sequence the loss of transcriptional regulatory motif located
in position 334 ~ 338 (RegRNA ID:R0146) was detected
MotifType=tfbs) respect to wild-type one (http://regrna.
To exclude a possible role of variants c.-964 G > C,
c.-419G > T and c.-359G > A in the loss of this motif
a further analysis was performed: the motif was
absent also in (c.- 964G, c.-639C, c.-591C,
c.-419G,c.-359G) sequence, assigning to c.-639 T > C and
c.591 T > C polymorphisms a role in the loss (http://
Table 3 Primer sequences used to amplify CCM3 promoter region for subsequent cloning
Table 4 Allele and genotype frequencies of CCM3-SERPINI1
controls typed were homozygous for the mutant ‘C’
allele. None was found to be homozygous in the patient
group; 6 heterozygotes (10 %) were found among 60
CCM patients vs the 139/350 (40 %) in control group.
The frequency of the ‘C’ allele was 0.3 for controls and
0.05 for patients (χ2 = 32.7; p = 0.0001).
Same trend was observed for c.-591 T > C/ rs11714980
polymorphism with “C” mutated allele having a
frequency in CCM samples of 0.06 %.
The second step regarded the characterization of the
effects of the c.-639 T > C/ rs9853967 and c.-591 T > C/
For this purpose two constructs of 851 bp were made:
toward the CCM3 and the SERPINI1 direction.
Promoter activity of the four common haplotypes
(T-T, T-C, C-T and C-C) was compared. The T-C and
C-T haplotypes significantly decreased promoter activity
compared with the T-T haplotype; C-C haplotype showed
largest reduction of promoter activity (p < 0.01). In both
cases this reduction was only in CCM3 direction. In
SERPINI1 direction, it was not statistically significant
(p > 0.05) (Fig. 1).
We performed a molecular analysis on CCM genes
regulatory regions in a cohort of CCM sporadic patients
negative for mutations in the coding regions of these
genes as well as for large genomic rearrangements not
detectable to direct sequencing.
Case-control study performed on 60 patients and 350
healthy controls showed a possible association with
CCM disease. Frequencies of the mutated alleles were
significantly higher in the control group than in patients
suggesting a possible protective effect of two
Dual-luciferase assay of the c.-639 T > C/rs9853967
and c.-591 T > C/rs11714980 polymorphisms falling in
&MotifInfo=Transcriptional%20Regulatory%20Motifs&MotifType=tfbs). Furthermore, literature data report
rs9853967 and rs11714980 polymorphisms falling outside
the minimal promoter (from nt 176–473) in a critical
regulatory element identified to contain a strong repressive
element for SERPINI1 and an enhancer for CCM3 .
Because no data about these polymorphisms and CCM
there are in literature and few data about polymorphisms
association and CCM too , we focused our attention
on above mentioned rs9853967 and rs11714980.
First, we analyzed c.-639 T > C/ rs9853967, c.-591 T >
C/rs11714980 polymorphisms frequencies in a group of
CCM sporadic patients vs a healthy control sample.
Genotype distribution is given in Table 4. About the
-639 T > C/rs9853967 polymorphism, 35/350 (10 %)
Fig. 1 Transcription activity analysis of CCM3/SERPINI1 haplotypes of the c.-639 T > C and c.-591 T > C polymorphisms. The transcription activity
was measured using the Dual-Luciferase Reporter Assay System in U373 MG (human, Caucasian, glioblastoma-astrocytoma) cells. Luciferase activities
are representative of at least three independent experiments, with each construct tested in triplicate per experiment. *p < 0.01; **p > 0.05
the critical regulatory fragment of bidirectional promoter
SERPINI1 and CCM3, showed a significant reduction of
CCM3 expression for C-C haplotype even more than for
T-C and C-T haplotypes. In SERPINI1 direction, the
reduction was not statistically significant.
CCM3 reduced expression would seem to disagree
with case-control study data showing a higher frequency
of the mutated alleles in control group than in patients.
This taking into account the only apoptotic role of
CCM3 protein and the association between CCM3
lossof-function mutations and CCM development [18, 19].
Evidences suggest that CCM3 interacts with CCM1
and CCM2 proteins in an intracellular complex sharing
some common signaling pathways [20–23].
Furthermore, it is involved in angiogenesis and
remodeling of cerebral vessels. Thus CCM3 protein acts
as a positive regulator of Ste20-related kinase MST4
 promoting cell growth and transformation via
extracellular signal-regulated kinase (ERK) pathway.
These processes are the basis of pathogenesis,
progression, and oncogenic behavior of human cancers  as well
as of CCM pathogenesis. Furthermore, it was shown that
potent short interfering RNAs (siRNAs) against CCM3 and
MST4 are able to specifically inhibit the expression of
CCM3 and MST4 mRNA, respectively [26–28].
Therefore, based on these observations it is possible to
assume that c.-639 T > C/rs9853967 and c.-591 T > C
polymorphisms in regulatory intergenic region shared by
CCM3 and SERPINI1 genes could have a protective role
against CCM pathogenesis. In fact, two polymorphisms
cause a considerable reduction of CCM3 expression to
dual luciferase-assay, thus justifying the higher
frequencies of mutated alleles found in the control group than
in patients. The reduced expression of CCM3 gene we
observed might be accompanied by reduced ERK activity
and attenuated cell growth. Further studies are needed
to confirm this.
In the present study, for the first time, we found that
c.-639 T > C/rs9853967 and c.-591 T > C/rs11714980
polymorphisms in CCM3/SERPINI1 genes asymmetric
bidirectional promoter were able to determine a sensible
reduction of CCM3 expression. Mutated allelic
frequencies higher in control group than in patients and the
reduction of CCM3 expression especially in C-C haplotype
carriers suggesting a possible protective role of these
polymorphisms in CCM pathogenesis.
CCMs: Cerebral cavernous malformations; DMEM: Dulbecco’s modified
Eagle’s medium; ERK: Extracellular signal-regulated kinase; FBS: Fetal Bovine
Serum; MRI: Magnetic resonance imaging; PCR: Polymerase chain reaction;
SD: Standard deviation; siRNAs: Short Interfering RNAs; SPSS: Statistical
package for social science
CS: carried out the genetic analysis, writing and revision of the manuscript
PB: has been working on recruitment of cases as neurologist AR: carried out
the functional studies LO: carried out the genetic analysis CA: has been
working on recruitment of cases as neurosurgeon and provided clinical
details CC: has been working on statistical analysis MM: has been working on
statistical analysis CR: provided to acquisition of data and sequence
alignment RD: design and conceptualization of the study, drafting of
manuscript AS: design and conceptualization of the study, revising the
manuscript. All authors read and approved the final manuscript.
Consent for publication
All the authors agree to publication.
Ethics approval and consent to partecipate
This study was approved by the Scientific Ethics Committee of the Azienda
Ospedaliera Universitaria- Policlinico ’G. Martino’ Messina. Informed consent
was obtained from all patients and controls.
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