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 References Albelda, S.M., Oliver, P.D., Romer, L.H., Buck, C.A., 1990. 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