Introduction

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system characterized by an autoimmune response against myelin proteins, which results in progressive neurological dysfunction.¹ In Caucasian populations it is the most common cause of acquired neurological dysfunction arising during early and mid-adulthood, affecting more than 1 million people in North America and Western Europe. The course of the disease may consist of recurrent attacks each followed by a variable degree of recovery (relapsing-remitting) or, in a minority of patients, of a progressive course from onset (primary-progressive). Intermediate phenotypes are also common. A large body of research supports a multifactorial etiology, with an underlying genetic susceptibility likely acting in concert with undefined environmental exposures.² In a recent report, transcriptional profiling using large-scale sequencing of expressed tagged sequences (ESTs) from MS lesions has identified a number of genes that are potentially involved in disease pathogenesis.³ One of the genes showing maximal differential expression between patient samples and controls was osteopontin (OPN). Microarray analysis of spinal cord RNA from rats with experimental allergic encephalomyelitis (EAE) also revealed increased OPN transcripts. In addition, OPN-deficient mice were resistant to MOG-induced progressive EAE and had frequent remissions³ suggesting a significant role for this gene in determining disease course.

OPN maps to 4q21–q25 and codes for a 60 kDa secreted phosphoprotein with pleiotropic functions, including roles in tissue remodeling, cell survival and cellular immunity.^4,5 OPN costimulates T-cell proliferation,⁴ and is classified as a T helper cell-1 (Thl) cytokine, upregulating interferon-gamma and interleukin-12 production, while downregulating that of interleukin-10.⁵ Here we investigated whether four single-nucleotide polymorphisms (SNPs) in the OPN gene were correlated with susceptibility to MS or any of several clinical end points in a well-characterized cohort of 821 patients.

Results and discussion

The data set studied consisted of 184 multicase families including 434 MS patients (total n=1312 genotyped individuals). In addition, we studied a second, independent, data set (n=387) composed primarily of patients with no family history of MS, giving a total of 821 clinically definite MS patients. This group was comprised of 617 females and 204 males (sex ratio=3.0 : l), with an overall mean age of onset of 30.0 (8.7) years and mean disease duration of 14.1 (10.1) years. All patients were non-Hispanic Caucasian and of European descent. Diagnostic criteria, ascertainment protocols, and other clinical and demographic characteristics of MS patients and families have been summarized elsewhere.⁶ A strong association to the HLA-DR locus overall (P=1.9 10^-6), and specifically with the HLA-DR2 haplotype (DRB1*1501-DQB1*0602) (P=1.6 10^-7) was observed (data not shown), as previously reported in a subset of this population.⁶

To test the OPN polymorphisms for association with MS, two complimentary analytical approaches were used considering the diversity in pedigree structure of the studied data set. The multicase families were analyzed using the pedigree disequilibrium test (PDT)^7,8 for both allelic and genotypic effects (Table 1). Alleles and haplotypes were also examined using the likelihood-ratio test (Table 2) implemented in TRANSMIT.⁹ No significant associations were observed for OPN allele or genotype in all families or subgroups stratified by HLA-DR2 status.

Table 1 - Global PDT P-values for allele and genotype results for OPN polymorphisms in multicase MS families.

Full table

Table 2 - Global TRANSMIT P-values for OPN polymorphisms in multicase MS families.

Full table

TRANSMIT can consider transmission of multiple marker haplotypes even in the presence of phase uncertainty and missing parental genotypes. Haplotype assignments revealed strong associations between alleles at the different SNP sites within OPN, resulting in two major haplotypes: one comprised of all wild-type SNP alleles or '1111' with a frequency of 72.0%, and a second comprised of all rare alleles or '2222' with a frequency of 22.0%. All other OPN haplotypes were present at frequencies 5.0%. The four SNP haplotypes as well as all pairwise haplotype combinations were analyzed, and no significant results were observed. When analyzed separately, all possible nuclear families from the multicase data set (n=292; total affecteds=459), as well as only one nuclear family selected from each multicase family (n=184), yielded comparable results (data not shown).

We also carried out case–control testing using randomly selected familial index cases and a control group, which did not reveal significant OPN allelic associations (Table 3). OPN SNP allele distributions for controls, patients and unaffected founders were similar.

Table 3 - Allele frequencies for OPN SNPs in MS index patients and Caucasian controls.

Full table

Given the striking phenotypic effect of OPN-deficient mice,³ we hypothesize that genomic variations in OPN are responsible, at least in part, for the clinical heterogeneity in MS. Therefore, we examined the effect of OPN genotypes on four well-defined clinical phenotypes such as disease course and severity in the combined MS patient data set using logistic models estimated by generalized estimating equations (GEE), which take into account any correlation between family members (Table 4). As a result of the strong disequilibrium observed between SNPs within the OPN locus, only two of the four original OPN polymorphisms were selected to study potential genotype–phenotype correlations, 1284A/C and 327T/C. Patients were coded by genotype (wild-type homozygous '11', heterozygous '12' and homozygous '22'), and gender, age of onset, HLA-DR2 status, and disease duration (when appropriate) were included as covariates in all analyses. Patients were also categorized by disease course (see Table 4). Disability was also assessed at entry with the Expanded Disability Status Scale (EDSS).¹⁰ Mild (benign) and severe disease patient classifications based upon EDSS scores maintained over or achieved within designated time intervals were also used in this study.⁶

Table 4 - OPN genotype–clinical phenotype correlations in MS patients.

Full table

A modest but interesting trend was observed in the data set. Patients carrying at least one wild-type OPN SNP 1284A allele were less likely to have a mild course (OR=0.3, P=0.08), and exhibited an increased risk for a secondary-progressive clinical course (OR=2.4, P=0.05). Similar results were also observed for OPN SNP 327T/C genotypes. The results, however, require careful interpretation because of multiple comparison effect.

Overall, we observed no evidence of genetic association between the OPN polymorphisms and MS susceptibility. However, the paucity of patients with mild disease carrying at least one wild-type A allele in the 1284A/C SNP suggests that there may be an effect of OPN in disease pathogenesis that, conceivably, may be regulated through downstream molecules in the OPN-mediated immunological cascade. These findings will require confirmation in an independent population.

Notes

Electronic-database information

URLs for data in this article are as follows:

Center for Human Genetics, http://wwwchg.mc.duke.edu/software/pdt.html for Pedigree Disequilibrium Test computer program. A beta-version of the geno-PDT program is available upon request (emartin@chg.mc.duke.edu).

References

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Acknowledgements

We thank the MS patients and their families for making this study possible. The collection of subjects and all experiments were performed under the approval of the Committee of Human Research at UC San Francisco. This work was funded by the National Multiple Sclerosis Society (NMSS) grants RG2542 (SLH) and RG2901 (JRO), and NIH grants NS26799 (SLH, JRO). We thank S. Toth, J. Mueller and D. Litman from Pyrosequencing for assisting in development of the genotyping assays.