Journal of Neuroimmunology 104 (2000) 174–178
www.elsevier.com / locate / jneuroin
Association study of a new polymorphism in the PECAM-1 gene in
multiple sclerosis
Francesca L. Sciacca a , Cinzia Ferri a , Sandra D’Alfonso b , Elisabetta Bolognesi b ,
Filippo Martinelli Boneschi c , Barbara Cuzzilla d , Bruno Colombo c , Giancarlo Comi c , Nicola Canal c ,
Luigi M.E. Grimaldi a,c,e , *
a
Neuroimmunology Unit, San Raffaele Scientific Institute, Milano, Italy
Medical Sciences Department, University of Eastern Piedmont, Novara, Italy
c
Neurological Clinic, Department of Neuroscience, San Raffaele Scientific Institute, Milano, Italy
d
Tissue Typing Laboratory, Department of Hematology, San Raffaele Scientific Institute, Milano, Italy
e
IRCCS Oasi Maria Santissima, Troina, Italy
b
Received 5 October 1999; received in revised form 30 November 1999; accepted 1 December 1999
Abstract
Genetic polymorphisms of immunorelevant genes may modulate occurrence or clinical features of multifactorial diseases. PECAM-1 is
an adhesion molecule crucial for transmigration of cells from blood to tissues, but its genetic contribution to multifactorial diseases has
never been investigated. We have identified and characterized a tetranucleotide repeat polymorphism within the third intron of PECAM-1.
In a cohort of healthy controls (HC), we found 10 alleles. An assessment of the association of this polymorphism with multiple sclerosis
(MS) showed similar allele and genotype frequencies in HC and MS patients as well as in MS patients differing for the gravity of their
disease course. We conclude that although potentially able to affect organ-specific autoimmune diseases, this new PECAM-1
polymorphism, does not seem to contribute to the genetic background of MS. 2000 Elsevier Science B.V. All rights reserved.
Keywords: PECAM-1 ; Multiple sclerosis; Polymorphism; Tetranucleotide repeats
1. Introduction
Susceptibility to MS is likely to be under a polygenic
control, but the majority of the genes involved is yet to be
identified. Of the many association studies of polymorphic
immunorelevant molecule genes so far reported for MS
(HLA antigens, T cell receptor subunits, several cytokines), only few have, in fact, shown positive results
(Hillert and Olerup, 1993; Vandenbroeck et al., 1997;
Mycko et al., 1998; Sciacca et al., 1999). Additional loci
involved in MS susceptibility and in the modification of its
clinical course remain to be characterized.
The platelet endothelial cell adhesion molecule-1
(PECAM-1 ) is a member of the Ig superfamily (Newman
et al., 1990) expressed, among others, in human endotheli-
*Corresponding author. Tel.: 139-02-2643-4867; fax: 139-02-26434855.
E-mail address: luigi.grimaldi@hsr.it (L.M.E. Grimaldi)
al cells (EC) and T-lymphocyte subsets (Muller et al.,
1989; Ashman and Aylett, 1991). PECAM-1 is involved in
EC–EC interaction, contributing to vessel integrity (Albelda et al., 1990), and in EC–leukocyte interaction,
regulating leukocyte diapedesis into tissues (Muller et al.,
1993; Ferrero et al., 1998; Yong et al., 1998). The
PECAM-1 region more relevant in trans-endothelial migration seems to be the first Ig-like domain, coded by
PECAM-1 exon 3 (Liao et al., 1997).
Due to its strategic role in cell–cell interaction, genetic
variations in PECAM-1 may have profound biological
effects in inflammatory or organ–specific autoimmune
processes requiring extravasation of pathogenetic cells into
target organs, as in the case of multiple sclerosis (MS)
(Al-Omaishi et al., 1999). Besides its functional relevance,
PECAM-1 is an attractive candidate gene in MS susceptibility because it maps in 17q13, about 3cM from the
D17S807 microsatellite marker which showed positive
scores in three indipendent MS linkage studies (Sawcer et
al., 1996; Kuokkanen et al., 1997; D’Alfonso et al., 1999).
0165-5728 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved.
PII: S0165-5728( 99 )00274-X
F.L. Sciacca et al. / Journal of Neuroimmunology 104 (2000) 174 – 178
For these reasons we looked for polymorphisms in the
PECAM-1 gene and performed an association study of a
newly identified polymorphism in MS patients compared
to age- and ethnicity-matched healthy controls (HC).
175
tion or, when necessary, an internal primer (59CCTGGCATTTTCCTTACC).
2.3. Data analyses
2. Materials and methods
2.1. MS patients and controls
Two hundred and five random healthy blood donors
(mean age 35.2610.1), seen at the Tissue Typing Laboratory of San Raffaele Scientific Institute of Milano (Italy),
were analyzed as healthy controls (HC). The MS cohort
included 204 patients (mean age 39.5611.5), affected by
relapsing–remitting (RR; Poser et al., 1983) MS, seen at
the MS Center of San Raffaele Scientific Institute of
Milano, (Italy). All subjects were from peninsular regions
of Italy. We selected a subgroup of benign MS patients,
defined as having a stabilized expanded disability status
scale (EDSS) score #3 after at least 10 years from disease
onset (Weinshenker et al., 1995) and compared them with
non-benign MS patients, defined as having EDSS score .3
within 10 years from disease onset (Sciacca et al., 1999).
2.2. Genetic analysis
Genomic DNA was extracted from buffycoats of healthy
blood donors and from venous blood of MS patients. Two
hundred microliters of buffycoat or blood were used for
DNA extraction (QiAmp blood kit; Qiagen, Chatsworth,
CA, USA). A region encompassing the third exon and the
third intron of PECAM-1 was amplified by PCR using 0.5
mM of the following primers: forward 59CTGTGATTGTGAACAACAAA-39 and backward 59ACAGAGTGAGACTCCATC-39 (Perkin–Elmer Cetus,
Norwalk, CT, USA); 100–250 ng DNA, heated at 958C for
109, was amplified by 30 cycles PCR at 958C / 558C / 728C
in 50 ml reaction containing 0.016 U / ml AmpliTaq Gold, 2
mM MgCl 2 , 10 mM Tris–HCl pH 8.3, 50 mM KCl, 200
mM each dNTP (all reagents from Perkin–Elmer Cetus).
PCR amplified products were run on ethidium bromide
stained 5% agarose or silver stained acrylamide gels. For
large population screenings, PCR and fluorescence-based
technique were used to discriminate allele sizes (Huang et
al., 1998). The 59-end of the forward primers was labelled
with TET or FAM dye and PCR performed with previously described conditions. Amplified products were run in an
ABI 373 sequencer (Perkin–Elmer Cetus) and data were
analyzed with a GeneScan 672 and the Genotyper software
(Perkin–Elmer Cetus).
Nineteen PCR-amplified fragments were extracted from
agarose gels (Amicon–Millipore Corporation, Bedford,
MA, USA) and sequenced by the ABI 373 sequencer
using the same primers employed for the PCR amplifica-
Allele frequencies (AF) and genotype frequencies (GF)
were compared by 23N contingency tables in MS patients
vs. controls, and in benign vs. non-benign MS patients.
Alleles or genotypes having frequencies #5% in at least
one of the two cohorts were gathered into a single group.
Statistical significance was evaluated by x 2 test with the
appropriate degrees of freedom (df55 in all but in GF
comparison of benign vs. non-benign MS, where df56).
The frequencies of each single allele were compared with
232 contingency tables in MS patients vs. controls, and in
benign vs. non-benign MS patients. No correction for the
number of comparisons was applied.
3. Results
3.1. Identification and characterization of a new
polymorphism
PCR amplification of a region encompassing the third
exon and intron of PECAM-1 was initially performed on
DNA extracted from 25 HC, revealing one or two amplified fragments for each subject and suggesting the
existence of allelic variants of the amplified region.
Amplification products were heterogeneous in size, being
of about 300 (named A1) or about 270 (named A2) bp;
Fig. 1 shows the amplification products of 7 representative
individuals.
Nineteen fragments were extracted from gels and sequenced. We observed 7 allelic variants whose size reflected the number of repeats of a CCTT tetranucleotide.
While A2 had 6 CCTT repeats, in A1 the number of
tetranucleotides was variable (14, 15, 17, 18, 19 and 21)
and is indicated by the number following the A1 code
(e.g., A1.14514 CCTT repeats).
Fig. 1. PCR amplification products of DNA from 7 individuals (sample
numbers are indicated at the top of each lane): larger alleles, having
similar lengths, were named ‘‘A1’’ while the shorter allele was called
‘‘A2’’.
F.L. Sciacca et al. / Journal of Neuroimmunology 104 (2000) 174 – 178
176
Table 1
Allele number (n) and frequency (AF) of PECAM-1 polymorphism in
healthy controls (HC), in the entire cohort of patients with multiple
sclerosis (MS) and in those MS patients having a benign or non-benign
course of disease
Allele
A2
A1.13
A1.14
A1.15
A1.16
A1.17
A1.18
A1.19
A1.20
A1.21
Total
HC
MS
All
n (AF)
All
n (AF)
Benign
n (AF)
Non-benign
n (AF)
205 (0.500)
0
8 (0.020)
58 (0.141)
27 (0.066)
43 (0.105)
47 (0.115)
9 (0.022)
12 (0.029)
1 (0.002)
195 (0.478)
3 (0.007)
14 (0.034)
57 (0.140)
31 (0.076)
41 (0.100)
43 (0.105)
18 (0.044)
6 (0.015)
0
64 (0.533)
0
2 (0.017)
18 (0.150)
10 (0.083)
11 (0.092)
9 (0.075)
5 (0.042)
1 (0.008)
0
72
2
6
24
11
19
13
4
3
410
408
120
3.2. Distribution of PECAM-1 polymorphism and
association study with MS
We studied the distribution of this polymorphism in a
large cohort of Italian HC using fluorescent primers to
detect allele size. We observed additional A1 alleles
indicating that the number of CCTT repeats ranged from
13 to 21. Allele and genotype frequencies of HC are
reported in Tables 1 and 2. The distribution of CCTT
alleles and genotypes was similar between men and
women (data not shown). Both HC and MS populations
were in Hardy Weinberg equilibrium for this locus.
The overall distribution of CCTT alleles and genotypes
was not significantly different between MS patients and
HC (AF: 632x 2 52.5, P50.8; GF: 632x 2 55.7, P50.3).
Finally, we studied among MS patients the possible
association of this polymorphism with different disease
courses. The overall distribution of CCTT alleles and
genotypes did not differ between benign and non-benign
(0.468)
(0.013)
(0.039)
(0.156)
(0.071)
(0.123)
(0.084)
(0.026)
(0.019)
0
154
Table 2
Genotype number (n) and frequency (GF) of PECAM-1 polymorphism in healthy controls (HC), in the entire cohort of patients with multiple sclerosis
(MS) and in those MS patients having a benign or non-benign course of disease
Genotypes
A2 /A2
A2 /A1.13
A2 /A1.14
A2 /A1.15
A2 /A1.16
A2 /A1.17
A2 /A1.18
A2 /A1.19
A2 /A1.20
A2 /A1.21
A1.14 /A1.15
A1.14 /A1.16
A1.14 /A1.17
A1.14 /A1.18
A1.14 /A1.20
A1.15 /A1.15
A1.15 /A1.16
A1.15 /A1.17
A1.15 /A1.18
A1.15 /A1.19
A1.15 /A1.20
A1.16 /A1.16
A1.16 /A1.17
A1.16 /A1.18
A1.16 /A1.19
A1.16 /A1.20
A1.17 /A1.17
A1.17 /A1.18
A1.17 /A1.19
A1.18 /A1.18
A1.18 /A1.19
A1.19 /A1.19
Total
HC
MS
All
n (GF)
All
n (GF)
Benign
n (GF)
Non-benign
n (GF)
55 (0.268)
0
3 (0.015)
26 (0.127)
12 (0.059)
22 (0.107)
21 (0.102)
3 (0.015)
7 (0.034)
1 (0.005)
1 (0.005)
2 (0.010)
0
1 (0.005)
1 (0.005)
6 (0.029)
4 (0.020)
8 (0.039)
4 (0.020)
0
3 (0.015)
1 (0.005)
0
4 (0.020)
2 (0.010)
1 (0.005)
1 (0.005)
9 (0.044)
2 (0.010)
3 (0.015)
2 (0.010)
0
48
3
9
18
21
18
19
7
4
15 (0.250)
0
2 (0.03)
5 (0.083)
10 (0.167)
5 (0.083)
8 (0.133)
3 (0.050)
1 (0.017)
0
0
0
0
0
0
3 (0.050)
0
5 (0.083)
0
2 (0.033)
0
0
0
0
0
0
0
1 (0.017)
0
0
0
0
16
2
3
11
6
10
6
1
1
205
1
2
1
1
8
1
9
8
3
1
1
2
3
6
5
3
1
1
(0.235)
(0.0150)
(0.044)
(0.088)
(0.103)
(0.088)
(0.093)
(0.0340)
(0.020)
0
(0.005)
(0.010)
(0.005)
0
(0.005)
(0.039)
(0.005)
(0.044)
(0.039)
(0.015)
(0.005)
(0.005)
(0.010)
(0.015)
0
0
0
(0.029)
(0.025)
(0.015)
(0.005)
(0.005)
204
60
1
1
1
2
1
4
2
1
1
1
1
2
2
1
(0.208)
(0.026)
(0.039)
(0.143)
(0.078)
(0.130)
(0.078)
(0.013)
(0.013)
0
0
(0.013)
(0.013)
0
(0.013)
(0.026)
(0.013)
(0.052)
(0.026)
(0.013)
(0.013)
(0.013)
0
(0.013)
0
0
0
(0.026)
(0.026)
(0.013)
0
0
77
F.L. Sciacca et al. / Journal of Neuroimmunology 104 (2000) 174 – 178
MS patients (AF: 632x 2 52.2, P50.8; GF: 732x 2 57.3,
P50.3). The comparison of the frequencies of each single
allele between groups (MS patients vs. HC and benign vs.
non-benign MS) showed no significant difference.
4. Discussion
Multifactorial diseases are determined by a discrete
number of genetic and environmental factors. Familial
linkage analysis or population association studies using
polymorphic markers could unravel in these diseases new
genetic loci potentially fostering susceptibility or clinical
variability. Irrespective of their functional significance,
polymorphisms could also be markers of disease, when
they are in linkage dysequilibrium with a locus associatied
with it.
To assess the role of PECAM-1 in MS using an
association analysis approach, we initially searched for
previously unreported polymorphisms and found an interesting microsatellite characterized by variable number (6
or 13–21) of CCTT tetranucleotide repeats in the third
intron of PECAM-1. Although we can not exclude the
existence of alleles with 7–12 CCTT repeats, it is possible
that the longer alleles (A1) were generated by duplication
of the 6 A2 tetranucleotides, conferring instability and
further expansion to the repeat region.
The pathogenic potential of such repeat expansions is
increasingly recognized in human pathology. Excessive
expansions of polymorphic triplet repeats have been
associated with several neurodegenerative diseases (e.g., a
GAA expansion in the X25 gene with Friedreich ataxia; a
CGG expansion in the FMR1 gene promoter with the
fragile X syndrome; a CAG expansion in Huntington’s
disease, spinocerebellar ataxia types I–VII and Kennedy’s
disease). When located in a non-coding (intronic) region,
such polymorphisms can influence the transcription process either by forming DNA secondary structures or
conferring instability to the primary transcript, thus
modifying the production or the viability of the mature
molecule (Weitzmann et al., 1997).
When the possible association of this polymorphism
with MS was finally assessed, however, we observed a
similar distribution of alleles and genotypes in HC and MS
patients, as well as in MS subgroups of patients with very
different disease courses. Accordingly, this polymorphism
does not seem to contribute to the genetic background of
MS, at least in Italian individuals. Additional studies might
reveal pathogenic potentials of this polymorphism in other
human diseases.
Acknowledgements
This work was supported by the Armenise–Harvard
Foundation and partially by MURST 60%.
177
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