Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) that seems to result from the interaction of genetic (hereditary) and non-genetic (environmental) factors. Therefore, MS could be considered a multi-factorial disease, where more than one gene could be implicated in the risk and progression of the disease (Combella and Martin, 2007; Harbo and Spurkland, 2007; Miller and Leay, 2007). Recent genetic studies on MS have mainly focused on the search for susceptibility genes. Among the candidate loci, the human locus antigen (HLA) located on chromosome 6, seems to be the most important genomic region related to MS (Haines et al., 2002; Kalanie et al., 2000; Amirzargar et al., 1998). In addition to HLA, several genes have been shown to be associated with the risk and progression of MS including genes involved in antigen presentation, cytokine genes and T-cell receptor genes such as CD24 (Jevsek et al., 2006; Bai et al., 2004).
CD24 is a sialoglycoprotein expressed on mature granulocytes in many B cells. The protein is a glycosylphosphatidylinositol (GPI)-anchored protein, that has been shown to be expressed by a variety of cells, most of which are involved in the immune system including neutrophils, B cells and T cells. Recently it was reported that CD24 is expressed by myofiber synaptic nuclei in both embryonic and adult mice. The CD24 gene, which is located in the 6q21 region, might therefore be a good candidate as a marker of MS susceptibility among the genes that have been studied previously (Niino et al., 2007; Bai et al., 2004; Duperray et al., 1990). The CD24 gene harbors a single-nucleotide polymorphism (SNP), which has been reported to be associated with the relative risk and progression of MS in different populations (Liu and Zheng, 2007; Zhou et al., 2003). This region has been described to be in linkage disequilibrium with MS in the population based cohort of central Ohio, USA (Liu and Zheng, 2007; Wang et al., 2007). Moreover, the protein encoded by the CD24 gene has been shown to be an important factor in the induction of experimental autoimmune encephalomyelitis (EAE) in mice.
Previuosly, a population based study, preformed on the prevalence of MS in the central province of Iran, Isfahan, suggested this geographical region to be at medium to high risk for MS (Etemadifar et al., 2006; Ale-Yasin et al., 2002). In the present study, the association of the different genotypes of CD24 SNP with the MS disease in patients residing in the province of Isfahan, Iran was investigated. The results showed that the CD24 V/V genotype may confer susceptibility to risk and development of MS.
MATERIALS AND METHODS
Human subjects, Expanded Disability Status Scale (EDSS) and Progression index (PI): 217 MS patients and 200 healthy individuals (controls) were selected for this study. The patients residing in Isfahan, Iran were referred from the Isfahan MS association; MS disease in all patients was confirmed according to the recommended diagnostic criteria for MS by the international panel on the diagnosis of MS (the McDonald criteria) (McDonald et al., 2001). All the patients were considered as “definite MS” according to the McDonald criteria. Patient’s informed consent was obtained from all participants prior to sample collection. Blood samples from patients and healthy controls were collected in EDTA tubes for eventual DNA extraction and genomic analysis.
The EDSS, a method of determining degree of disability in MS, was used to quantify disability in eight functional systems (FS). EDSS steps 1.0 to 4.5 refer to people with MS who are able to ambulate. EDSS steps 5.0 to 9.5 are defined by the impairment of ambulation. EDSS 10.0 refers to the cases of death due to MS. the PI was measured by dividing the EDSS score by the number of years lapsed since the first symptoms of MS.
Genotyping of the CD24 single nucleotide SNP: The CD24 SNP results in the replacement of C by T at nucleotide 226 in the coding region of exon 2, (Gene Bank accession no. NM-013230). The SNP with the T nucleotide creates a BstXI restriction enzyme site which makes it possible to differentiate the CD24A allele from the CD24V allele by restriction fragment length polymorphism (RFLP) analysis. For this purpose total human genomic DNA from was isolated from nucleated blood cells using standard salting out precipitation method. Allele-specific polymerase chain reaction (PCR) was performed to amplify the DNA region containing the CD24 SNP using specific primers. The forward primer was (5´-TTG TTG CCA CTT GGC ATT TTT GAG GC-3´), and the reverse primer was (5´-GGA TTG GGT TTA GAA GAT GGG GAA A-3´). The PCR condition was as follows: denaturation at 94ºC for 3 minutes, followed by 30 cycles of denaturation at 94ºC for 1 min, annealing at 56.5ºC for 1 min, and extension at 72ºC for 1 min, with a final extension at 72ºC for 3 min. The predicted PCR product was 453 bp in length. Following PCR amplification, the PCR products were digested with BstXI (Fermentas, Germany) for 4h at 55ºC. The digested PCR products were resolved on a 1.5% (w/v) agarose gel. PCR products of the CD24V allele were cut into two small fragments (325 and 129 bp), whereas those of the CD24A were completely resistant to digestion. In heterozygous individuals with two types of alleles A and V (CD24A/V genotype), three fragments of 453, 325 and 129 were expected.
Statistical analysis: Patients and normal controls were examined for any significant difference in their genotype (allele) distribution with respect to CD24 polymorphism at the population level. Pearson’s chi square (χ2) test was used to perform homogeneity assessments for distribution of the genotypes between patients and the control population. The number of individuals falling into each of the three genotypes in the patients and controls were quite high; therefore χ2 test was also used to determine a valid P value.
To assess whether the MS progression was different among patients with different genotypes, the progression index (PI) score was calculated. One-way analysis of variance (ANOVA) was used to compare the PI value of the disease in each genotype between the groups. The difference in progression of the disease between the three genotypes was investigated using the least significant difference (LSD) test.
Genotyping of CD24 SNP in MS patients and controls: Genotyping of CD24 SNP was performed on 217 DNA samples from MS patients, and 200 healthy individuals. As shown in Figure 1, following PCR amplification and BstXI digestion (PCR/RFLP) of total genomic DNA, three different genotypes could be detected, which were CD24A/A, CD24A/V and CD24 V/V. Figure 1 represents a typical PCR/RFLP analysis of samples from six MS patients. Lanes 1, 4 and 6 represent patients with the CD24A/A genotype having the 453 bp fragment, whereas lanes 2 and 5 represent heterozygous samples with the CD24A/V genotype consisting of the 543, 325 and 129 bp fragments. Lane 3 shows a DNA sample with the CD24V/V genotype displaying two bands of 325 and 129 bp.
Upon genotyping of all the patients and controls, the distribution of different genotypes among the population was examined. As represented in Table 1, while there is no significant difference in the frequency of the CD24A/A and CD24A/V genotypes between MS patients and control individuals, the CD24V/V genotype varies significantly between the two populations (P = 0.0193, Odds Ratio 2.4882, 95% confidence interval (CI): 1.416-4.3722). The significant increase in the CD24V/V genotype in individuals with the MS disease may suggest the importance of association of this genotype with the risk and susceptibility of the disease.
The CD24V/V genotype is associated with the progression of MS: To investigate the correlation of the CD24 genotypes and progression of MS, the EDSS and PI of the MS patients with different genotypes were analyzed. The EDSS score and PI were determined for patients for whom more than 3 years had lapsed since the first symptom of MS. Data summarized in Table 2, show that among patients examined, 52 had CD24A/A, 23 had CD24A/V and 29 had the CD24V/V genotypes.
Subsequently, the possibility of differing CD24 genotypes affecting the progression index of the disease was investigated. Table 2 shows the analysis of the progression index for different genotypes. Comparison of genotypes in each group by one-way ANOVA was performed. The results showed a large difference in the progression index of the three different genotypes (F=13.14, P=0.0001).
To investigate the difference in progression between the three genotypes, the LSD test was applied. As shown in Table 3, no significant difference between the average of the progression index of the CD24A/A and CD24A/V genotypes was observed. However, the average progression index in the CD24V/V patients was markedly different from that of the CD24A/V and CD24A/A patients. It should be noted that for the CD24A/A and CD24A/V genotypes, results of the LSD test were not statistically significant. However, in CD24V/V patients, LSD analysis was statistically significant (p < 0.05 and p < 0.01). (see Table 3). Together these data suggest that the progression of MS in patients with the CD24V/V genotype is higher than patients with the CD24A/A and CD24A/V genotypes. Moreover, the progression of the disease is not significantly different in patients with the CD24A/A and /or CD24A/V genotypes.
Iran is considered as a medium to high risk area for MS (Etemadifar et al., 2006). Our previous population based study indicated the prevalence of MS in the central province of Isfahan, Iran, for the period of 2004-2005 as 35.5 per 100,000 [95% CI: 33.6-37.3] in a population of 3,923,255 (Etemadifar et al., 2006). A higher rate in women (54.5) than men (14.9) was detected which is much higher than previously believed (Etemadifar et al., 2006; Carton et al., 1997; Ebers et al., 1986). To date, few studies have examined the molecular pathogenesis of MS in Iranian patients. Those studies carried out have mainly investigated epidemiological and immunological factors related to MS such as HLA antigenic polymorphisms (Kalanie et al., 2000; Haines et al., 1996).
The presence of an SNP in the CD24 gene, which involves a non-conservative amino acid at position 57, has been shown to mediate the association of the CD24 molecule with MS. This SNP involves a replacement of C by T at nucleotide 226, thus leading to the production of two polypeptide chains with different GPI-anchored cell surface efficiencies. Our results support previous reports on the association of the CD24V/V (C/C allele) genotype with risk and development of MS (Otaegui et al., 2006; Zhou et al., 2003). Analysis of the distribution frequencies of different CD24 genotypes among the control healthy individuals and MS patients, indicates a significant difference in the CD24V/V homozygous genotype, but not in the CD24A/A and CD24A/V heterozygous genotypes. This may suggest a recessive role for the CD24 SNP C allele as compared to the T allele, regarding association with MS association. Therefore, in CD24V/V homozygous patients, the CD24 polypeptide chain with amino acid valine at position 57 may function as a risk factor in the development of MS (see Table 1).
Our findings are similar to those reported earlier by Zhou et al. (2003), where a significant difference in the distribution of the CD24V/V genotype, but not the CD24A/A and CD24A/V genotypes was found (P = 0.0193, Odds Ratio 2.4882, 95% CI: 1.416-4.3722). Interestingly, in vivo and in vitro expression analysis of the CD24 alleles by this group indicated a significantly higher expression for the CD24A allele as compared to the CD24V allele. Moreover, peripheral blood lymphocytes (PBLs) with CD24V/V genotypes were found to express higher levels of the CD24 protein compared to the CD24A/V and CD24A/A genotypes. Further investigations on the association of the CD24 SNP genotypes in a Spanish population has also revealed that the CD24V/V genotype is associated with increased risk of developing MS (Otaegui et al., 2006, Kamali-Sarvestani et al., 2006). These data support a significant role for the involvement of the CD24V allele in the molecular pathogenesis of MS, which warrants further investigation. Intriguingly, in a relatively broad population from Belgium and the UK, the association of CD24V/A with susceptibility and progression of MS has not been observed (Goris et al., 2006; Liu et al., 2007). This could further signify the role of the CD24V/V genotype, but not those of the CD24V/A or CD24A/A genotypes in risk and development of MS.
The severity of MS is usually measured according to the EDSS score (McDonald et al., 2001). Analysis of EDSS scores, indicated that the MS patients with CD24V/V genotypes exhibited increased risk of developing MS symptoms as compared to patients with the CD24V/A and CD24A/A genotypes. Moreover, our studies on a limited number of MS patients with family histories show that in comparison with other unaffected siblings, the CD24V allele seems to be preferentially transmitted to the MS offspring (unpublished data). Interestingly, population based studies of MS in relatives of patients and twins suggest a likely concordance pattern of inheritance for the possible MS genes.
Finally, examination of the progression index (PI) of MS patients shows that patients with the CD24V/V genotype display a more rapid progression of the disease (See Table 2). Since the PI was calculated by dividing the EDSS score by the number of years lapsed since the first symptoms of MS, it is feasible that the CD24V/V allele has a positive effect on the progression of the disease in CD24V/V patients. Moreover, the progression of the disease in patients with CD24V/V is much faster than other patients, when examined by ANOVA and the LSD test. However, in CD24V/V patients, LSD analysis was statistically significant (p < 0.05 and p < 0.01).
In summary, these data suggest that patients with the CD24V/V genotype are more prone to MS symptoms, and therefore may require more thorough treatment compared to patients with the CD24V/A and CD24A/A genotypes. However, further molecular verifications are required to fully justify this genotype as an important genetic modifier in the risk and progression of the MS disease.
We would like to thank the patients and their families for participation in this study. We express our gratitude to the personnel of the MS society of Isfahan for their cooperation in collection of samples. This study was supported through a general grant (Pajoohaneh) by the Department of Research of the University of Isfahan, IR Iran.