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Gene. Author manuscript; available in PMC 2010 October 28.
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Published in final edited form as:
Gene. 2006 July 5; 376(1): 95–101. doi:10.1016/j.gene.2006.02.016.
The alleles of PECAM-1
Melanie S. Novinskaa, Bradley C. Pietzb, Thomas M. Ellisc, Debra K. Newmana,d, and Peter
J. Newmana,e,f,g,*
a Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI 53201 USA
b
Product Development Laboratory, BloodCenter of Wisconsin, Milwaukee, WI 53201 USA
c
Laboratory of Histocompatibility and Immunogenetics, BloodCenter of Wisconsin, Milwaukee, WI
53201 USA
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d
Department of Microbiology, Medical College of Wisconsin, Milwaukee, WI 53226 USA
e
Department of Pharmacology, Medical College of Wisconsin, Milwaukee, WI 53226 USA
f
Department of Cellular Biology, Medical College of Wisconsin, Milwaukee, WI 53226 USA
g
Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, WI 53226 USA
Abstract
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Previous studies have reported the existence of eleven different single nucleotide polymorphisms
(SNPs) within human PECAM-1 mRNA, several of which have recently been associated with
disease. Though SNPs in the PECAM-1 gene have been known for some time, the genetic background
on which they exist, and their association into distinct allelic isoforms has not yet been established.
To identify the major allelic isoforms of PECAM-1, we determined the nucleotide sequence of
individual full-length cloned cDNAs derived from anonymous, unrelated volunteer individuals.
Initial sequence analysis of 34 alleles from 17 individuals confirmed the presence of two distinct
human PECAM-1 alleles (L98S536R643 and V98N536G643) within the human population. Each of
these were found, upon more detailed analysis, to be superimposed on a previously unreported a2479g
nucleotide polymorphism within the 3′ untranslated region (3′UTR) that occurred on both allelic
isoforms - yielding a total of four major alleles. Multiplex Luminex bead analysis of an additional
259 individuals allowed identification of 117 individuals homozygous for either the L98S536 or
V98N536 allele, and sequence analysis around the R643G and a2479g polymorphic sites permitted
accurate determination of significant differences in the gene frequencies of LSRa, LSRg, VNGa, and
VNGg among Caucasian individuals. Identification of these PECAM-1 allelic isoforms should
facilitate future detailed examination of PECAM-1-related disease associations, and may help resolve
previously disparate results.
Keywords
single nucleotide polymorphism; disease association; CD31; PECAM-1
*
Address correspondence to: Dr. Peter J. Newman, Blood Research Institute, BloodCenter of Wisconsin, P.O. Box 2178, 638 N. 18th
Street, Milwaukee, WI 53201, Phone: (414) 937-6237, Fax: (414) 937-6284, peter.newman@bcw.edu.
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Novinska et al.
Page 2
1. Introduction
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PECAM-1 (CD31) is a vascular cell adhesion and signaling receptor that is expressed on the
surfaces of platelets, leukocytes, and endothelial cells (Newman et al 1990), and is encoded
by a ~70 kb gene near the end of the long arm of chromosome 17 (17q23) (Gumina et al
1996). PECAM-1 exists in mature form as a 130 kDa Type I transmembrane glycoprotein
comprised of a 574 amino acid extracellular domain containing six Ig-like homology domains,
a 19 amino acid transmembrane domain, and a cytoplasmic tail of varying length due to
alternative splicing (Kirschbaum et al 1994). Ig-domain 1 mediates homophilic binding (Liao
et al 1997; Newton et al 1997; Sun et al 1996), while Ig-domain 6 binds calcium (Jackson et
al 1997a) and has been suggested to participate in cis interactions with integrin αvβ3 within the
plane of the plasma membrane (Wong et al 2000). The cytoplasmic tail of PECAM-1 possesses
two Immunoreceptor Tyrosine Inhibitory Motifs (ITIMs) (Newman 1999) which, upon
tyrosine phosphorylation, recruit and activate the protein tyrosine phosphatase, SHP-2
(Jackson et al 1997b; Masuda et al 1997; Sagawa et al 1997). PECAM-1 has been demonstrated
to participate in a variety of physiological events, including leukocyte adhesion and migration,
angiogenesis, apoptosis, and modulation of Immunoreceptor Tyrosine Activating Motif
(ITAM)-mediated cellular activation (for recent reviews, see (Ilan and Madri 2003; Newman
1997; Newman and Newman 2003)).
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Like most genes, variations within the nucleotide sequence of the PECAM-1 gene have been
reported, with individual polymorphic residues identified within the 5′UTR, the extracellular
and cytoplasmic domains, and the 3′UTR (summarized in Table 1). While the effects of these
polymorphisms on PECAM-1-mediated adhesion or signaling have not yet been determined,
mismatches at PECAM-1 amino acid residues 98, 536, or 643 have often (Balduini et al
1999;Behar et al 1996;Cavanagh et al 2005;Grumet et al 2001;Maruya et al 1998), though not
universally (Nichols et al 1996), been associated with an increased incidence of acute graftversus-host disease (GVHD), and these and other PECAM-1 polymorphisms have been linked
with early onset of atherosclerosis (Elrayess et al 2003), increased risk of cardiovascular disease
(Elrayess et al 2004;Fang et al 2005;Listi et al 2004;Sasaoka et al 2001;Song et al 2003;Wei
et al 2004), and susceptibility to malarial infection (Kikuchi et al 2001), though the latter is
also controversial (Casals-Pascual et al 2001).
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While the frequencies of individual PECAM-1 polymorphisms have been determined in a
limited number of population studies, these polymorphisms have not, to date, been linked into
distinct PECAM-1 alleles, hampering efforts to more definitively establish PECAM-1-related
disease associations. The purpose of the present investigation, therefore, was to determine the
major alleles bearing each of the most commonly reported SNPs within the PECAM-1 gene.
This information should not only permit more precise bio-epidemiological associations to be
made amongst different human populations, but also enable biochemical and cell biological
studies to be performed to investigate whether functional differences between PECAM-1 allelic
isoforms might be causally linked to the reported disease associations of PECAM-1 SNPs.
2. Materials and Methods
2.1 Preparation of genomic and cDNA from human whole blood
Human whole blood was obtained from anonymous volunteer blood donors. RNA was isolated
using a QIAamp® RNA Blood Mini Kit according to manufacturer’s instructions (Qiagen,
Valencia, CA). cDNA was then prepared from human RNA using the SuperScript™ FirstStrand Synthesis for RT-PCR kit (Invitrogen, Carlsbad CA). Following cDNA synthesis,
RNase (1 μl) (Invitrogen) was added, tubes incubated at 37°C for 20 minutes, and then put on
ice or frozen at −20°C for later use. Genomic DNA was isolated from whole blood using a
DNA Blood Mini Kit (Qiagen) following lysis of red blood cells with ammonium chloride.
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Novinska et al.
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2.2 PCR amplification, cloning, and sequencing
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Primers used to generate nested PCR products were designed to amplify a 2520 bp region
encompassing all known individual polymorphisms within the human PECAM-1 transcript
(including the 5′- and 3′UTRs, and were manufactured by Integrated DNA Technologies, Inc.
(Coralville, IA). Primary PCR reactions were carried out using PfuTurbo® DNA Polymerase
(Stratagene, La Jolla, CA) using primers corresponding to human PECAM-1 18–35 (sense) 5′gccatggctgccattacc-3′ and 2573–2554 (antisense) 5′-taagaaccggcagcttagcc-3′. Amplification
was performed for 35 cycles (95°C for 30 seconds, 59°C for 30 seconds with a ramping speed
of 3°C/second and a 10°C gradient, 72°C for 3 minutes) in an Eppendorf Mastercycler Gradient
Thermal Cycler (Brinkmann Instruments, Westbury, NY). Nested PCR reactions were
performed for 25 cycles using identical amplification conditions using internally nested primers
corresponding to PECAM-1 26–43 (sense) 5′-tgccattacctgaccagc-3′ and 2545–2528
(antisense) 5′-tgctgtgttctgtgggag-3′.
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PCR products were separated from primers on a 1.5% agarose gel and then purified using the
QIAquick PCR Purification kit (Qiagen) according to manufacturer’s instructions. They were
then ligated into the pPCR-Script Amp SK(+) cloning vector and transformed into XL10-Gold
Kan ultracompetent cells (Stratagene). Plasmids were isolated using the QIAprep Miniprep kit
(Qiagen), analyzed by selective restriction enzyme digestion, and their PECAM-1 cDNA
inserts fully sequenced in a 96-well plate using an Applied Biosystems Model 3100 Capillary
Sequencer using the following primers: 355–372 (sense) 5′-aagaacctgaccctgcag-3′, 411–392
(antisense) 5′-aggcttgacgtgagaggtgg-3′, 1177-1194 (sense) 5′-ttttccaagcccgaactg-3′, 1588–
1607 (sense) 5′-gcggtattcaaagacaaccc -3′, 2030–2047 (sense) 5′-tcggagtgatcattgctc-3′, and
2545–2528 (antisense) 5′-tgctgtgtctgtgggag-3′. Sequencing reactions were performed for 25
cycles (96°C for 30 seconds, 50°C for 15 seconds, and 60°C for 4 minutes). Sequence analysis
of smaller defined regions surrounding the R643G and a2479g polymorphisms was performed
by PCR amplification using 5′ctagaatttcccttgtcactcaccc-3′ (sense) and 5′gctaccttcattgacacatcggct-3′ (antisense), and 5′-gggcaatcttcaatcttgag-3′ (sense) and 5′tgggagcagggcaggttcataaat-3′ (antisense) primers, respectively, and sequenced using (sense)
5′-ctagaatttcccttgtcactcaccc-3′, (antisense) 5′-gctaccttcattgacacatcggct-3′, (sense) 5′gggcaatcttcaatcttgag-3′.
2.3 Genotyping using Luminex Beads
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Large-scale population genotyping for the L98V and S536N polymorphisms was performed
using a MultiCode-PLx (EraGen Biosciences, Madison, WI) multiplex assay. Fluorescently
labeled allele-specific elongation products were hybridized to Luminex™ (Austin, TX)
microspheres and detected on the Luminx100 instrument using methods recently described in
detail (Pietz et al 2005). Primers used for both locus-specific amplification and allele-specific
extension reactions were synthesized by EraGen Biosciences (Madison, WI, see
http://www.eragen.com/diagnostics/technology.html). The following primers (‘x’ denotes the
IsoBase residue, isoC) were used in multiplex allele-independent amplification of PECAM-1
around the L98V polymorphism (sense) 5′-xatctatgactcagggac-3′, (antisense) 5′gtgctcagttccaag-3′, and around the S536N polymorphic site (sense) 5′ttctatcaaatgacctcaaat-3′, (antisense) 5′-xaggctgtgcagtaat-3′. Allele-specific elongation
reactions were performed with primers specific to the L98V polymorphism 5′caaggactcaccttccaccaacag-3′ and 5′-caaggactcaccttccaccaacac-3′, and primers specific to the
S536N polymorphic site 5′-ttggaccaagcagaaggctag-3′ and 5′-tttggaccaagcagaaggctaa-3′.
2.4 Statistical analysis
Hardy-Weinberg equilibrium analysis was performed, with all deviations from HardyWeinberg equilibrium analyzed using a Fisher’s Exact Test contingency table. Statistically
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significant differences in the gene frequencies of PECAM-1 allelic isoforms were determined
by Chi-square analysis.
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3. Results and Discussion
3.1 Identification of the primary alleles of human PECAM-1
Single nucleotide polymorphisms within the PECAM-1 gene, including several that result in
amino acid substitutions, were originally identified by comparing the sequences of PECAM-1
cDNAs cloned from different laboratories (Newman et al 1990; Simmons et al 1990;
Stockinger et al 1990; Zehnder et al 1992), as well as the single sequence derived thus far from
PECAM-1 genomic DNA (Kirschbaum et al 1994). The frequencies for several individual
SNPs have since been determined in several small studies, and the results obtained from these
are summarized in Table 1, which additionally shows the location of the SNPs relative to the
exon in which they are encoded.
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Because PECAM-1 SNPs have recently been implicated as risk factors for graft rejection,
malarial infection, and cardiovascular disease (Table 2), we undertook identification of linked
polymorphisms that might more precisely define PECAM-1 allelic isoforms. In preliminary
studies, RNA was isolated from small blood samples derived from seventeen unrelated,
anonymous volunteer blood donors, converted to cDNA, and nested PCR was performed to
amplify full-length PECAM-1 transcripts (shown schematically in Figures 1A & 1B). The final
2500 bp amplified product (Figure 1C), encompassing all known PECAM-1 polymorphisms,
including those within the 5′ and 3′UTRs, was subcloned into a plasmid vector, and five clones
from each donor were sequenced in their entirety to determine the linkage of SNPs carried on
each of the two PECAM-1 alleles present in that individual. Sequence analysis of 34 alleles
from 17 individuals revealed the presence of two distinct human PECAM-1 alleles
(L98S536R643 and V98N536G643), each of which was superimposed on an additional a2479g
nucleotide polymorphism within the 3′UTR that occurred on both allelic isoforms – yielding
a total of four major alleles, depicted in Figure 2. None of the other reported polymorphisms
noted in Table 1 were found in any of the 34 alleles examined, indicating that they are likely
to be rare variants of one or more of the four primary alleles. The genetic background on which
they occur remains to be established.
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To determine the gene frequencies of the four major PECAM-1 alleles - LSRa, LSRg, VNGa,
and VNGg, genomic DNA from 259 healthy Caucasian blood donors was subjected to
Luminex-based MultiCode-PLx bead hybridization analysis that detected fluorescently labeled
allele-specific elongation products hybridized to Luminex™ microspheres in multiplex (Pietz
et al 2005). Of these, 49 individuals were found to be L98S536 homozygotes and 68 people
were V98N536 homozygotes. PCR amplification and sequence analysis of the regions
surrounding the polymorphisms at R643G and a2479g was performed on these samples, and
from them the gene frequencies for LSRa, LSRg, VNGa, and VNGg were calculated, and found
to be 0.14, 0.28, 0.27, and 0.31, respectively. Chi-square analysis revealed significant
differences in the frequencies of the LSR versus VNG allelic isoforms (p<0.025), and also
between the LSRa and LSRg alleles (p<0.01). None of the other gene frequencies (e.g. between
VNGa and VNGg) were found to be significantly different.
3.2 The N536S and G643R polymorphisms within exons 8 and 12 travel together as a
haplotype block
Haplotype blocks are 10–100 kb regions of the human genome that contain SNPs sufficiently
close to each other as to be nearly always inherited together - i.e. in strong linkage
disequilibrium (Daly et al 2001; Gabriel et al 2002; Shifman et al 2003). Thus, though few or
no recombination events occur within the “blocks” themselves, intervals between haplotype
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Novinska et al.
Page 5
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blocks are commonly sites of recombination, leading to the loss of linkage disequilibrium
between blocks. The observation that two major human PECAM-1 alleles, L98S536R643 and
V98N536G643, are each superimposed upon an additional a2479g nucleotide polymorphism
within the 3′UTR, can be explained by the fact that exons 12–16 within the PECAM-1 gene
are widely dispersed amongst large introns (more than 26,000 base pairs exist between the
R643G polymorphism in exon 12 and the a2479g nucleotide polymorphism within exon 16),
and that the middle third of the PECAM-1 gene (e.g. exons 8–12) travels together as a haplotype
block. A crossing-over event somewhere between exons 12 and 16 (depicted schematically in
Figure 3) was likely responsible for generating the four major alleles of PECAM that we
observed in this study. Moreover, though the S536N (exon 8) and R643G (exon 12)
polymorphisms have consistently been found in nearly complete linkage disequilibrium
((Elrayess et al 2004; Maruya et al 1998; Sasaoka et al 2001; Wei et al 2004), and this study),
evidence exists that the L98V polymorphism within exon 3 can be inherited independently
(Cavanagh et al 2005; Listi et al 2004; Maruya et al 1998; Sasaoka et al 2001), and may not
be as tightly-linked to the centrally-located haplotype block within the PECAM-1 gene. Thus,
a second crossing-over event (depicted with dotted lines in Figure 3) appears to be responsible
for forming the less common VSR and LNG allelic isoforms. Future studies will be required
to determine whether the frequencies of PECAM-1 alleles might vary within different ethnic
groups.
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3.3 Potential consequences of the a2479g polymorphism within the 3′UTR of PECAM-1 mRNA
Though no functional consequences of, or disease associations with, the a2479g nucleotide
polymorphism have yet been reported, 3′UTRs can influence both mRNA stability (Rousseau
et al 2003) and translation efficiency, and have also been associated with disease states. For
example, a polymorphism in the 3′UTR of the interleukin-12B gene has been found to associate
with late onset of type 1 diabetes mellitus (Windsor et al 2004), while another study
demonstrated that a polymorphism within the 3′UTR of the cyclooxygenase-2 gene contributes
to lung cancer susceptibility within the Chinese population (Hu et al 2005). Interestingly, the
“prioritization” of synthesis of certain mRNAs involved in the synthesis of seleniumcontaining proteins have been shown to be influenced by sequences within 3′UTRs, and
polymorphisms within this region may also regulate expression (Hesketh 2004). Based upon
these findings, it may be of future interest to examine whether the PECAM-1 3′UTR
polymorphism affects PECAM-1 mRNA and protein expression levels, and thereby
PECAM-1-mediated adhesion and/or signaling.
3.4 Disease Associations with PECAM-1 Allelic Isoforms
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Our identification of four major allelic isoforms for PECAM-1 within the Caucasian population
may permit more detailed and accurate examination of PECAM-1-related disease associations
(Table 2). Thus, although Sasaoka and colleagues (Sasaoka et al 2001) found a disproportionate
increase in the frequencies of the L98, S536, and R643 forms of PECAM-1 in patients admitted
for myocardial infarction (MI), especially males <60 yrs old, Listi et al. (Listi et al 2004) found
an increased frequency (38.6% in patients compared with 24.6% in healthy individuals) of the
R643 polymorphism in Sicilian MI patients in the absence of significant differences in the
frequencies of L98 or S536. Similarly, Song and colleagues (Song et al 2003) found a significant
increase in the frequencies of V98 and N536 polymorphisms in patients presenting with early
onset of coronary artery disease (CAD), while two other studies (Fang et al 2005;Wei et al
2004) found only V98 associated with CAD. Finally, PECAM-1 serves as an endothelial cell
receptor for certain strains of Plasmodium falciparum-infected erythrocytes, and there are two
reports regarding susceptibility of individuals carrying the Leu98 polymorphism to malarial
infection: one showing an association (Kikuchi et al 2001) and one not showing such a
connection (Casals-Pascual et al 2001). It is possible that re-genotyping these populations for
PECAM-1 alleles, including the newly described 3′UTR polymorphism at nucleotide 2479
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Novinska et al.
Page 6
(this manuscript), rather than for individual SNPs, might help resolve the seemingly disparate
results obtained in each of these studies.
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Finally, although the consequences for allogeneic bone marrow transplantation of mismatching
individual PECAM-1 polymorphisms has been controversial (Balduini et al 1999; Behar et al
1996; Grumet et al 2001; Maruya et al 1998; Nichols et al 1996), Cavanagh et al.(Cavanagh
et al 2005), recently suggested, based on the results of a small study, that genotyping for
linked PECAM-1 polymorphisms might be a better predictor of both acute graft-versus-host
disease (P=0.004) and overall patient survival.
3.5 Conclusions
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The major finding of the present study is that, rather than existing as a single invariant species,
PECAM-1 is actually represented by distinct molecular isoforms that differ in functionally
important regions within both the extracellular and cytoplasmic domains. In addition, a
polymorphism within the 3′ untranslated region further distinguishes these two protein
isoforms, yielding four primary human PECAM-1 alleles: LSRa, LSRg, VNGa, and VNGg,
with frequencies 0.14, 0.28, 0.27, 0.31, respectively, within the Caucasian population. The
middle third of the PECAM-1 gene displays strong linkage disequilibrium, and is inherited as
a haplotype block. A crossing over event within the 26 kb region lying between the
polymorphism in exon 12 and the polymorphism within exon 16 has led to the generation of
these four major PECAM-1 alleles, while another recombination event within the first third of
the gene led to the generation of at least two additional alleles that are relatively rare, at least
in Caucasians of European descent. Because PECAM-1 polymorphisms affect residues within
domains of the protein that are involved in adhesion and signal transduction, it may be
important in the future to determine whether differences exist in the biochemical and cell
biological properties of these distinct isoforms. Finally, identification of the primary alleles of
PECAM-1 should provide both investigators and clinicians with the ability to more accurately
establish correlation of these alleles with diseases in which PECAM-1 polymorphisms have
been implicated, including CAD, MI, and GVHD.
Acknowledgments
The authors thank Daniel B. Rowe, Ph.D., Division of Biostatistics, Medical College of Wisconsin, for his assistance
in statistical analysis. This study was funded by grants HL-40126 and Training Grant HL-07209 from the Heart, Lung,
and Blood Institute of the National Institutes of Health grant.
Abbreviations
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PECAM-1
platelet endothelial cell adhesion molecule-1
SNP
single nucleotide polymorphism
Ig
immunoglobulin
ITIM
immunoreceptor tyrosine inhibitory motif
ITAM
immunoreceptor tyrosine activating motif
GVHD
graft-versus-host disease
PCR
polymerase chain reaction
MI
myocardial infarction
CAD
coronary artery disease
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Page 7
References
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Balduini CL, Noris P, Giorgiani G, Martinetti M, Klersy C, Spedini P, Belletti S, MacCario R, Gusberti
L, Locatelli F. Incompatibility for CD31 and human platelet antigens and acute graft-versus-host
disease after bone marrow transplantation. Br J Haematol 1999;106:723–729. [PubMed: 10468865]
Behar E, Chao NJ, Hirake DD, Krishnaswamy S, Brown BW, Zehnder JL, Grumet FC. Polymorphism
of adhesion molecule CD31 and its role in acute graft-versus-host disease. N Engl J Med
1996;334:286–291. [PubMed: 8532023]
Casals-Pascual C, Allen S, Allen A, Kai O, Lowe B, Pain A, Roberts DJ. Short report: codon 125
polymorphism of CD31 and susceptibility to malaria. Am J Trop Med Hyg 2001;65:736–737.
[PubMed: 11791967]
Cavanagh G, Chapman CE, Carter V, Dickinson AM, Middleton PG. Donor CD31 genotype impacts on
transplant complications after human leukocyte antigen-matched sibling allogeneic bone marrow
transplantation. Transplantation 2005;79:602–605. [PubMed: 15753851]
Daly MJ, Rioux JD, Schaffner SF, Hudson TJ, Lander ES. High-resolution haplotype structure in the
human genome. Nat Genet 2001;29:229–232. [PubMed: 11586305]
Elrayess MA, Webb KE, Bellingan GJ, Whittall RA, Kabir J, Hawe E, Syvanne M, Taskinen MR, Frick
MH, Nieminen MS, Kesaniemi YA, Pasternack A, Miller GJ, Humphries SE. R643G polymorphism
in PECAM-1 influences transendothelial migration of monocytes and is associated with progression
of CHD and CHD events. Atherosclerosis 2004;177:127–135. [PubMed: 15488875]
Elrayess MA, Webb KE, Flavell DM, Syvanne M, Taskinen MR, Frick MH, Nieminen MS, Kesaniemi
YA, Pasternack A, Jukema JW, Kastelein JJ, Zwinderman AH, Humphries SE. A novel functional
polymorphism in the PECAM-1 gene (53G>A) is associated with progression of atherosclerosis in the
LOCAT and REGRESS studies. Atherosclerosis 2003;168:131–138. [PubMed: 12732396]
Fang L, Wei H, Chowdhury SH, Gong N, Song J, Heng CK, Sethi S, Koh TH, Chatterjee S. Association
of Leu125Val polymorphism of platelet endothelial cell adhesion molecule-1 (PECAM-1) gene &
soluble level of PECAM-1 with coronary artery disease in Asian Indians. Indian J Med Res
2005;121:92–99. [PubMed: 15756041]
Gabriel SB, Schaffner SF, Nguyen H, Moore JM, Roy J, Blumenstiel B, Higgins J, DeFelice M, Lochner
A, Faggart M, Liu-Cordero SN, Rotimi C, Adeyemo A, Cooper R, Ward R, Lander ES, Daly MJ,
Altshuler D. The structure of haplotype blocks in the human genome. Science 2002;296:2225–2229.
[PubMed: 12029063]
Grumet FC, Hiraki DD, Brown BWM, Zehnder JL, Zacks ES, Draksharapu A, Parnes J, Negrin RS.
CD31 mismatching affects marrow transplantation outcome. Biol Blood Marrow Transplant
2001;7:503–512. [PubMed: 11669217]
Gumina RJ, Kirschbaum N, Rao PN, vanTuinen P, Newman PJ. The human PECAM1 gene maps to
17q23. Genomics 1996;34:229–232. [PubMed: 8661055]
Gumina RJ, Kirschbaum NE, Piotrowski K, Newman PJ. Characterization of the human Platelet/
Endothelial Cell Adhesion Molecule-1 promoter: Identification of a GATA-2 binding element
required for optimal transcriptional activity. Blood 1997;89:1260–1269. [PubMed: 9028949]
Hesketh J. 3′-Untranslated regions are important in mRNA localization and translation: lessons from
selenium and metallothionein. Biochem Soc Trans 2004;32:990–993. [PubMed: 15506944]
Hu Z, Miao X, Ma H, Wang X, Tan W, Wei Q, Lin D, Shen H. A common polymorphism in the 3′UTR
of cyclooxygenase 2/prostaglandin synthase 2 gene and risk of lung cancer in a Chinese population.
Lung Cancer 2005;48:11–17. [PubMed: 15777967]
Ilan N, Madri JA. PECAM-1: old friend, new partners. Curr Opin Cell Biol 2003;15:515–524. [PubMed:
14519385]
Jackson DE, Loo RO, Holyst MT, Newman PJ. Identification and characterization of functional cation
coordination sites in Platelet Endothelial Cell Adhesion Molecule-1. Biochemistry 1997a;36:9395–
9404. [PubMed: 9235983]
Jackson DE, Ward CM, Wang R, Newman PJ. The protein-tyrosine phosphatase SHP-2 binds PECAM-1
and forms a distinct signaling complex during platelet aggregation. Evidence for a mechanistic link
between PECAM-1 and integrin-mediated cellular signaling. J Biol Chem 1997b;272:6986–6993.
[PubMed: 9054388]
Gene. Author manuscript; available in PMC 2010 October 28.
Novinska et al.
Page 8
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Kikuchi M, Looareesuwan S, Ubalee R, Tasanor O, Suzuki F, Wattanagoon Y, Na-Bangchang K, Kimura
A, Aikawa M, Hirayama K. Association of adhesion molecule PECAM-1/CD31 polymorphism with
susceptibility to cerebral malaria in Thais. Parasitol Int 2001;50:235–239. [PubMed: 11719109]
Kirschbaum NE, Gumina RJ, Newman PJ. Organization of the gene for human Platelet/Endothelial Cell
Adhesion Molecule-1 (PECAM-1) reveals alternatively spliced isoforms and a functionally complex
cytoplasmic domain. Blood 1994;84:4028–4037. [PubMed: 7994021]
Liao F, Ali J, Greene T, Muller WA. Soluble domain 1 of platelet-endothelial cell adhesion molecule
(PECAM) is sufficient to block transendothelial migration in vitro and in vivo. J Exp Med
1997;185:1349–1357. [PubMed: 9104821]
Listi F, Candore G, Lio D, Cavallone L, Colonna-Romano G, Caruso M, Hoffmann E, Caruso C.
Association between platelet endothelial cellular adhesion molecule 1 (PECAM-1/CD31)
polymorphisms and acute myocardial infarction: a study in patients from Sicily. Eur J Immunogenet
2004;31:175–178. [PubMed: 15265022]
Maruya E, Saji H, Seki S, Fujii Y, Kato K, Kai S, Hiraoka A, Kawa K, Hoshi Y, Ito K, Yokoyama S,
Juji T. Evidence that CD31, CD49b, and CD62L are immunodominant minor histocompatibility
antigens in HLA identical sibling bone marrow transplants. Blood 1998;92:2169–2176. [PubMed:
9731077]
Masuda M, Osawa M, Shigematsu H, Harada N, Fujiwara K. Platelet endothelial cell adhesion molecule-1
is a major SH-PTP2 binding protein in vascular endothelial cells. Febs Lett 1997;408:331–336.
[PubMed: 9188788]
Newman PJ. The biology of PECAM-1. J Clin Invest 1997;99:3–8. [PubMed: 9011572]
Newman PJ. Switched at birth: a new family for PECAM-1. J Clin Invest 1999;103:5–9. [PubMed:
9884328]
Newman PJ, Berndt MC, Gorski J, White GC, Lyman S, Paddock C, Muller WA. PECAM-1 (CD31)
cloning and relation to adhesion molecules of the immunoglobulin gene superfamily. Science
1990;247:1219–1222. [PubMed: 1690453]
Newman PJ, Newman DK. Signal transduction pathways mediated by PECAM-1. New roles for an old
molecule in platelet and vascular cell biology. Arterioscler Thromb Vasc Biol 2003;23:953–964.
[PubMed: 12689916]
Newton JP, Buckley CD, Jones EY, Simmons DL. Residues on both faces of the first immunoglobulin
fold contribute to homophilic binding sites on PECAM-1/CD31. J Biol Chem 1997;272:20555–
20563. [PubMed: 9252369]
Nichols WC, Antin JH, Lunetta KL, Terry VH, Hertel CE, Wheatley MA, Arnold ND, Siemieniak DR,
Boehnke M, Ginsburg D. Polymorphism of adhesion molecule CD31 is not a significant risk factor
for graft-versus-host disease. Blood 1996;88:4429–44344. [PubMed: 8977234]
Pietz BC, Warden MB, DuChateau BK, Ellis TM. Multiplex genotyping of human minor
histocompatibility antigens. Human Immunology. 2006 (in press).
Rousseau P, Le DM, Mouillot G, Marcou C, Carosella ED, Moreau P. The 14 bp deletion-insertion
polymorphism in the 3′ UT region of the HLA-G gene influences HLA-G mRNA stability. Hum
Immunol 2003;64:1005–1010. [PubMed: 14602228]
Sagawa K, Kimura T, Swieter M, Siraganian RP. The protein-tyrosine phosphatase SHP-2 associates
with tyrosine-phosphorylated adhesion molecule PECAM-1 (CD31). J Biol Chem 1997;272:31086–
31091. [PubMed: 9388260]
Sasaoka T, Kimura A, Hohta SA, Fukuda N, Kurosawa T, Izumi T. Polymorphisms in the plateletendothelial cell adhesion molecule-1 (PECAM-1) gene, Asn563Ser and Gly670Arg, associated with
myocardial infarction in the Japanese. Ann N Y Acad Sci 2001;947:259–269. [PubMed: 11795274]
Shifman S, Kuypers J, Kokoris M, Yakir B, Darvasi A. Linkage disequilibrium patterns of the human
genome across populations. Hum Mol Genet 2003;12:771–776. [PubMed: 12651872]
Simmons DL, Walker C, Power C, Pigott R. Molecular cloning of CD31, a putative intercellular adhesion
molecule closely related to carcinoembryonic antigen. J Exp Med 1990;171:2147–2152. [PubMed:
2351935]
Song FC, Chen AH, Tang XM, Zhang WX, Qian XX, Li JQ, Lu Q. Association of platelet endothelial
cell adhesion molecule-1 gene polymorphism with coronary heart disease. Di Yi Jun Yi Da Xue Xue
Bao 2003;23:156–158. [PubMed: 12581968]
Gene. Author manuscript; available in PMC 2010 October 28.
Novinska et al.
Page 9
NIH-PA Author Manuscript
Stockinger H, Gadd SJ, Eher R, Majdic O, Schreiber W, Kasinrerk W, Strass B, Schnabl E, Knapp W.
Molecular characterization and functional analysis of the leukocyte surface protein CD31. J Immunol
1990;145:3889–3897. [PubMed: 1700999]
Sun QH, DeLisser HM, Zukowski MM, Paddock C, Albelda SM, Newman PJ. Individually distinct Ig
homology domains in PECAM-1 regulate homophilic binding and modulate receptor affinity. J Biol
Chem 1996;271:11090–11098. [PubMed: 8626652]
Wei H, Fang L, Chowdhury SH, Gong N, Xiong Z, Song J, Mak KH, Wu S, Koay E, Sethi S, Lim YL,
Chatterjee S. Platelet-endothelial cell adhesion molecule-1 gene polymorphism and its soluble level
are associated with severe coronary artery stenosis in Chinese Singaporean. Clin Biochem
2004;37:1091–1097. [PubMed: 15589815]
Windsor L, Morahan G, Huang D, McCann V, Jones T, James I, Christiansen FT, Price P. Alleles of the
IL12B 3′UTR associate with late onset of type 1 diabetes. Hum Immunol 2004;65:1432–1436.
[PubMed: 15603869]
Wong CW, Wiedle G, Ballestrem C, Wehrle-Haller B, Etteldorf S, Bruckner M, Engelhardt B, Gisler
RH, Imhof BA. PECAM-1/CD31 trans-homophilic binding at the intercellular junctions is
independent of its cytoplasmic domain; evidence for heterophilic interaction with integrin αvβ3 in
cis. Mol Biol Cell 2000;11:3109–3121. [PubMed: 10982404]
Zehnder JL, Hirai K, Shatsky M, McGregor JL, Levitt LJ, Leung LLK. The cell adhesion molecule CD31
is phosphorylated after cell activation. Down-regulation of CD31 in activated T lymphocytes. J Biol
Chem 1992;267:5243–5249. [PubMed: 1544907]
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Figure 1. Protocol for determining PECAM-1 alleles
A. Flow diagram of methods used to determine PECAM-1 alleles. B. Schematic of primary
and nested PCR reactions. C. Representative agarose gel of final PCR products.
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Figure 2. The four major allelic isoforms of human PECAM-1
1,25′UTR and 3′UTR polymorphisms show nucleotide number based on the transcription start
site of PECAM cDNA. 3gf = gene frequency. Frequencies were derived from genotyping 234
alleles from 117 unrelated volunteer blood donors, and were consistent with Hardy-Weinberg
equilibrium analysis. ¶Chi-square analysis revealed that significant differences exist in both
the frequencies of the combined LSR versus VNG isoforms (p<0.025), and also between LSRa
and LSRg (p<0.01).
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Figure 3. Likely crossing-over events yielding human PECAM-1 alleles
The 16 exons of PECAM-1 are widely dispersed amongst introns, with more than 26 kb
between the R643G polymorphism in exon 12 and the a2479g nucleotide polymorphism within
exon 16. A crossing-over event is likely to have taken place within this region, resulting in the
generation of the four major alleles of PECAM-1. Dotted lines illustrate what was likely a
second, more recent crossing-over event that has yielded two additional less common
PECAM-1 allelic isoforms, previously observed by (Cavanagh et al 2005; Maruya et al
1998). Exon/Intron spacing is drawn to scale.
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Table 1
Exon
1 Nucleotide base substitution
2 Amino acid substitution
3 Amino acid substitution
Frequency
References
1
g53a in 5′ UTR
N/A
N/A
G 0.98, A 0.02
Elrayess (a)
3
G442A
V53M
V80M
V 0.99, M 0.01
Maruya
3
A548T
N88I
N115I
Unknown
*
Gene. Author manuscript; available in PMC 2010 October 28.
3
C577G
L98V
L125V
L ~0.5, V ~0.5
Fang, Grumet, Listi, Maruya, Sasaoka, Wei
4
A656G
N124D
N151D
Unknown
*
6
A1329G
I348M
I375M
Unknown
*
6
A1376T
D364V
D391V
Unknown
*
7
T1656C
H457H
H484H
Unknown
*
8
G1892A
S536N
S563N
S ~0.5, N ~0.5
Fang, Grumet, Listi, Maruya, Sasaoka, Wei
12
A2212G
R643G
R670G
R ~0.5, G ~0.5
Elrayess (b), Grumet, Listi, Maruya, Sasaoka,
16
a2479g in 3′ UTR
N/A
N/A
Unknown
This study
Novinska et al.
Single nucleotide polymorphisms within PECAM-1 exons
1
Numbering based on PECAM-1 transcription start site (Gumina et al 1997).
2
Numbering based on the mature protein sequence.
3
Numbering based on protein sequence including the 27 amino acid signal peptide.
N/A: not applicable – base change is within an untranslated region
(a)
(b)
Elrayess MA, et al. 2003
Elrayess MA, et al. 2004
*
Polymorphisms deduced from differences in the originally-reported PECAM-1 cDNA sequences (Newman et al 1990; Simmons et al 1990; Stockinger et al 1990; Zehnder et al 1992)
Page 13
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Table 2
Diseases associated with PECAM-1 SNPs
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¶ Amino acid
Disease association
Reference
g53 (5′UTR)
atherosclerosis
Elrayess (a)
Mismatch at L98V
GVHD
Balduini, Behar, Cavanagh, Grumet
V98
CHD or CAD
Song, Wei, Fang
N536
CHD or CAD
Song
V98 + N536
Cerebral malaria
Kikuchi
S536
MI
Sasaoka
Mismatch at S536N
GVHD
Cavanagh, Grumet, Maruya
R643
MI
Sasaoka, Listì, Elrayess (b)
G643
CHD or CAD
Elrayess (b)
Mismatch at R643G
GVHD
Cavanagh, Grumet, Maruya
¶
Numbering based on mature amino acid sequence
NIH-PA Author Manuscript
(a)
(b)
Elrayess MA, et al 2003
Elrayess MA, et al 2004
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Gene. Author manuscript; available in PMC 2010 October 28.