HLA-DRB1*1501 and Spinal Cord Magnetic Resonance Imaging Lesions in Multiple SclerosisHLA-DRB1*1501 and Spinal Cord MR Lesions in MS FREE

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system characterized by inflammation, demyelination, and axonal loss in the brain and spinal cord. The current hypothesis is that MS is caused by a complex interplay of genetic and environmental factors. The genetic influence is characterized by the interaction of multiple genes that exert modest effects. The most striking among these is the association of the major histocompatibility complex (MHC) with MS susceptibility, which has consistently been reported over the past decades.¹

Clinically, MS is a heterogeneous disease with a diverse spectrum of neurologic deficits and variable outcome. Some studies^{1 - 8} have focused on genetic predictors of disease phenotypes (such as disease severity, disease subtypes, magnetic resonance [MR] imaging characteristics, and response to treatment), showing some effect of different genes.

Besides clinical heterogeneity, considerable variation exists between patients regarding type and anatomic location of the lesions.^{9 - 10} Varying degrees of involvement of the cerebrum, brainstem, cerebellum, and spinal cord have been described in postmortem and MR imaging studies.^{11 - 12} This variability is unexplained, and much effort is being put in unraveling it. Several arguments point toward a genetic role in the regional distribution of lesions in the brain and spinal cord in MS. First, differences in pathologic manifestation of MS have been observed between Asian and Western populations. Asian-type MS is characterized by predominant involvement of the optic nerve and spinal cord, whereas Western-type MS predominantly involves the brain. Genetic differences within the HLA region between these populations could underlie this variability.^{3 ,13 - 16} Second, recent studies^{17 - 18} confirm a tendency of patients to develop relapses in the same locations, including spinal cord lesions. This tendency for localized exacerbations could be genetically predetermined. Third, involvement of a genetic factor has already been demonstrated in lesion distribution in experimental autoimmune encephalomyelitis (an animal model that shows many similarities to MS); Butterfield et al¹⁹ showed that in mouse experimental autoimmune encephalomyelitis the lesions in the brain and spinal cord were controlled by different quantitative trait loci.

Many researchers have demonstrated the clinical relevance of spinal cord lesions in diagnosing MS and disability accumulation. However, we are unaware of any studies that included spinal cord MR imaging variables in genetic analyses searching for genotype-phenotype correlations.

In this study, we used a DNA chip containing a set of single-nucleotide polymorphisms (SNPs) in candidate genes to assess the genetic effect on regional lesion distribution in the brain and spinal cord as seen on MR imaging. The SNPs on this chip were selected based on published associations with MS pathogenesis, prognosis, or response to treatment.

Unrelated Dutch Caucasian patients were selected retrospectively from natural history studies conducted in the Department of Neurology and Radiology, Vrije Universiteit Medical Center, Amsterdam, the Netherlands. Patients were selected for the availability of DNA material, as well as brain and spinal cord MR images that fulfilled certain standardized requirements (described herein) and were obtained less than 2 years apart. The study was performed with the approval of the medical ethics committee of the VU University Medical Center, and informed consent was obtained from all participants. Patients were diagnosed as having MS as ascertained by Poser et al²⁰ or revised McDonald criteria.²¹ For patients included in the analyses, clinical data were collected, including age, sex, type of disease, age at onset, disease course, and duration of disease. Disability status was determined for all subjects using Kurtzke's Expanded Disability Status Scale²² and, whenever available, the Multiple Sclerosis Functional Composite Scale.²³

Single-nucleotide polymorphisms were selected based on involvement in MS pathogenesis, prognosis, or response to treatment according to the literature published before July 2007. The polymorphisms were confirmed and associated with an identifier using dbSNP database (http://www.ncbi.nlm.nih.gov/SNP). Nucleotide sequences for the design of allele-specific probes and polymerase chain reaction primers were retrieved in the SNPper database (http://snpper.chip.org/bio). Sequence-specific probes and primers were designed using freely available Primer3 software (http://frodo.wi.mit.edu/).

If a polymorphism was not present in the database, position and sequences were established by performing a BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using the data available in the literature.

Genomic DNA was isolated from anticoagulated blood using DNAzol reagent (Molecular Research Center, Inc, Cincinnati, Ohio). Genotyping was performed using a newly developed low-density DNA microarray based on allele-specific probes. The design, fabrication, validation, and analysis of the arrays were performed using the procedure described by Tejedor et al²⁴ with minor modifications.

Scans were acquired on 1.0-T or 1.5-T scanners (Siemens AG, Erlangen, Germany) with standard head coils using 2-dimensional conventional or fast spin-echo proton density–weighted and T2-weighted images (repetition time, 2200-3000 milliseconds; echo times, 20-30 and 80-100 milliseconds with section thicknesses of 3 to 5 mm, a maximum gap between sections of 0.5 mm, and an in-plane resolution of 1 × 1 mm². Lesions were identified by an expert reader and then outlined on the corresponding proton density–weighted image using home-developed semiautomated seed-growing software based on a local thresholding technique (Show_images).²⁵ Lesion areas were multiplied by the intersection distance to obtain total T2-weighted brain lesion volume for each patient.

Spinal cord scanning included a cardiac-triggered sagittal proton density–weighted and T2-weighted dual-echo spin-echo sequence with a section thickness of 3 mm covering the whole spinal cord (repetition time, 2500-3000 milliseconds; echo times, 20-30 and 80-100 milliseconds), with a gap between sections of 0.3 mm, and an in-plane resolution of 1 × 1 mm². From this sequence, the number of focal lesions and the presence of diffuse abnormalities were scored by an experienced reader (C.L.). Diffuse abnormalities were defined as poorly delineated areas with increased signal intensity compared with the signal intensity of spinal cerebrospinal fluid best seen on proton density–weighted images.²⁶ Lesion volume was assessed in the spinal cord as the total number of focal pathologic segments involved (total extension of all spinal cord lesions over several corresponding vertebral segments).

First, the associations between the brain variable (T2-weighted lesion load) and the spinal cord variables (the number of focal lesions and the presence of diffuse abnormalities) were tested per SNP and per clinical variable. We used the nonparametric Spearman ρ rank correlation test, Kruskal-Wallis test, Mann-Whitney test, and χ² test as appropriate, applying the false discovery rate according to Benjamini and Hochberg²⁷ to correct for multiple testing. The corrected number represents the expected proportion of false discoveries for a given P value cutoff. We used the cutoff point of P < .05 after false discovery rate correction. Spearman rank correlation coefficient was used to test the correlations between 2 scaled variables.

Second, multivariate analysis by general linear models was performed on the associated SNPs (P < .10 significance after correction for multiple testing). We used log-transformed brain T2-weighted lesion loads and the square root–transformed number of focal abnormalities in the spinal cord to correct for significant clinical variables and the type of MR imaging system (1.0 T vs 1.5 T).

All analyses were performed using commercially available software packages. These included SPSS (version 15; SPSS Inc, Chicago, Illinois), Excel 2003 (Microsoft Corporation, Redmond, Washington), and HelixTree (Golden Helix, Inc, Bozeman, Montana).

One hundred fifty patients were included in the analysis. Our patient group reflects a representative population with MS, with 36.7% being male and 20.0% having primary progressive MS (Table 1). Most patients (132 of 150) demonstrated abnormalities (mostly focal lesions) on spinal cord MR imaging, while all patients had abnormalities on brain MR imaging.

In total, 80 SNPs in 44 genes were studied on the DNA chip. Twelve SNPs were excluded from further analysis (5 SNPs were monomorphic, and 7 SNPs had a minor allele frequency <5% [eTable]). Hardy-Weinberg equilibrium was calculated for all SNPs.

In the univariate analyses of T2-weighted lesion load in the brain vs all SNPs on the DNA chip, the only suggestive correlation was rs2107538 (CCL5) (OMIM 187011) (Table 2). Two clinical covariates were significant and were included in the general linear model for brain T2-weighted lesion load, namely, disease duration and MS subtype. The type of MR imaging system (1.0 T vs 1.5 T) was not associated with T2-weighted lesion load in the brain. After inclusion of rs2107538 (CCL5) in the model that contained the clinical covariates, this SNP showed significant association with brain T2-weighted lesion load (P = .03).

Several MHC SNPs were found to be related to the number of focal spinal cord abnormalities (Table 2). The most significant is SNP rs3135388. Carrier status of the A allele (surrogate marker for HLA-DRB1*1501) was associated with significantly more lesions in the spinal cord (Figure). The median number of focal abnormalities in carriers of HLA-DRB1*1501 (n = 80) was 4 lesions and in noncarriers (n = 70) was 2 lesions (P < .001, Mann-Whitney test).

When corrected for multiple testing, 5 SNPs within the MHC class II region (rs3135388, rs2395182, rs2239802, rs2227139, and rs2213584) remained significant, and 1 SNP within the C2TA gene (class II transactivator) showed a suggestive result. The 5 HLA SNPs are in strong linkage disequilibrium.

In addition, the aforementioned MHC class II SNPs were also found to be correlated with a higher lesion volume in the spinal cord. Specifically, carriership of HLA-DRB1*1501 was associated with more segments of the spinal cord affected by focal lesions (P = .01, Mann-Whitney test).

No clinical covariate (disease duration, age of patients, or MS subtype) was significantly correlated with the number of focal lesions in the spinal cord. No general linear model that included the 5 significantly associated MHC class II SNPs could be produced because of the high level of collinearity owing to the high linkage disequilibrium (r² range, 0.33-0.99). Only rs3087456 (C2TA) (OMIM 600005) showed a marginally significant P value (P = .05) in the general linear model that included rs3135388 (P = .002). No association was observed between the presence of diffuse abnormalities and the evaluated SNPs.

In the present candidate gene study, we observed an association between several SNPs within the MHC class II region and the number of focal abnormalities in the spinal cord. The most significant results were found for rs3135388. This SNP is a surrogate marker for the HLA-DRB1*1501 allele (r² = 0.97).²⁸

After correction for multiple testing, no genes were significantly related to T2-weighted lesion load in the brain. The MHC class II SNPs that were associated with more lesions in the spinal cord and with a higher lesion volume in the spinal cord do not seem to affect the presence of T2-weighted lesions in the brain.

Studies^{6 ,29 - 30} have unambiguously shown that HLA-DRB1*1501 strongly influences MS susceptibility. There is some evidence that this haplotype might also be associated with a more severe disease course.^{1 ,4 ,6 ,31} Recent findings show a correlation between carriership of HLA-DRB1*1501 and Expanded Disability Status Scale scores using an extremes of outcome strategy (comparing patients with severe and mild disability only).⁴ In our study, we could not demonstrate a correlation between carriership of HLA-DRB1*1501 and Expanded Disability Status Scale scores. This might be because of the impossibility of performing the extremes of outcome analysis owing to a small sample size. However, findings from this study suggest that spinal cord lesions might be an additional explanation for the described relationship between HLA-DRB1*1501 and MS disease severity.

Abnormalities in the spinal cord correlate with the degree of disability and with the date of diagnosis.^{32 - 33} In our study, we also noticed an association between Expanded Disability Status Scale scores and the number of focal abnormalities in the spinal cord (P = .02, ρ = 0.185). Future studies should include additional MR imaging variables of the spinal cord such as atrophy (cross-sectional area), as this variable might correlate better with clinical disability.³⁴

In a recent article, Okuda et al³⁵ showed in their patients that HLA-DRB1*1501 affects disease severity as measured by clinical variables and by brain MR imaging variables. In our patients, we could not demonstrate this effect on T2-weighted lesion load in the brain or on cognitive function (using PASAT [Paced Auditory Serial Addition Task]²³ scores). Post hoc power analysis detected a power of less than 25% to detect a T2-weighted lesion load difference of 1 mL, suggesting that a type II error cannot be ruled out. This also warrants careful interpretation of the suggestive positive finding of rs2107538 (CCL5) in association with brain T2-weighted lesion load, although previous findings have shown an influence of this gene on other MR imaging variables.³⁶

Previous data were sometimes conflicting about the effect of HLA haplotypes on brain MR imaging features. Some studies^{31 ,35} found a relationship between HLA genotypes and brain MR imaging quantitative markers, while other studies^{31 ,37} did not observe this effect of HLA-DRB1*1501. In addition, no effect of HLA-DRB1*1501 was shown on lesion distribution within the brain using T1-weighted and T2-weighted lesion maps.³⁸ To date, we are unaware of any study that specifically relates HLA-DRB1*1501 to spinal cord abnormalities on MR imaging in a Caucasian population.

The molecular basis of the association with greater involvement of the spinal cord in patients carrying the HLA-DRB1*1501 allele remains unknown. Major histocompatibility complex class II genes are involved in self vs nonself immune recognition.³⁹ These genes encode for polymorphic surface glycoproteins. Variability in this region may determine individual differences in T-cell responses. It is postulated that MHC class II may present variable central nervous system antigens to T cells, possibly producing different lesion distribution in animals and in humans.⁴⁰

Moreover, Stromnes et al⁴¹ reported different lesion distribution in the central nervous system (spinal cord vs brain parenchyma) in 2 mouse experimental autoimmune encephalomyelitis models with different MHC strains. This was found to be mediated by variable preferential MOG (myelin oligodendrocyte glycoprotein) epitope presentation and ultimately by an alternative ratio of T-helper type 17 to T-helper type 1. The present study indicates a different mechanism of lesion formation in the brain vs the spinal cord, with a possible indirect role of the MHC class II genes. The role of the genes on this mechanism is unclear. The translation of these findings from animal models to the human situation warrants further studies.

Because of the complexity of the MHC class II region (epistatic effects and high linkage disequilibrium), future studies using high-density HLA mapping are warranted to unravel the genetic influence on lesion distribution and to determine the responsible HLA allele.

In conclusion, this study demonstrates more focal lesions in the spinal cord among carriers of the HLA-DRB1*1501 allele. If confirmed in independent samples, these observations may provide important insight in MS disease heterogeneity and its underlying mechanisms.

Correspondence: Madeleine H. Sombekke, MD, Department of Neurology, VU University Medical Center, Vrije Universiteit Amsterdam, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands (m.sombekke@vumc.nl).

Accepted for Publication: June 12, 2009.

Author Contributions:Study concept and design: Sombekke, Lukas, Crusius, Tejedor, Killestein, Arteta, Martínez, Uitdehaag, Peña, Vrenken, and Polman. Acquisition of data: Sombekke, Lukas, Crusius, and Barkhof. Analysis and interpretation of data: Sombekke, Lukas, Crusius, Tejedor, Killestein, Arteta, Martínez, Uitdehaag, Knol, Geurts, De Jager, Barkhof, Vrenken, and Polman. Drafting of the manuscript: Sombekke, Crusius, Tejedor, Arteta, Martínez, and Vrenken. Critical revision of the manuscript for important intellectual content: Crusius, Tejedor, Killestein, Arteta, Martínez, Uitdehaag, Knol, Peña, Geurts, De Jager, Barkhof, Vrenken, and Polman. Statistical analysis: Sombekke, Uitdehaag, Knol, and De Jager. Obtained funding: Tejedor, Arteta, Martínez, and Polman. Administrative, technical, and material support: Crusius, Tejedor, Arteta, Martínez, and Vrenken. Study supervision: Crusius, Uitdehaag, Barkhof, Vrenken, and Polman.

Financial Disclosure: Dr Lukas received a research grant from Bayer Schering Germany. Drs Tejedor, Arteta, and Martínez are employees of the corporate sponsor. Dr Killestein has been involved in clinical trials of companies that market drugs for multiple sclerosis (Bayer Schering Germany, AG, Biogen Idec, Merck Serono, and Teva Neuroscience) and some companies that have development programs for future drugs to treat multiple sclerosis. Dr Uitdehaag received honoraria for consultancy from Novartis and Merck Serono. Dr De Jager received speaking and advisory board fees from Biogen Idec, honoraria for serving on the advisory board of Teva Neuroscience, and research support from Affymetrix Inc and Source MDx Inc. Dr Vrenken received program grant 05-358c and project grants 05-566MS and 08-633MS from the Dutch Multiple Sclerosis Research Foundation and project grant EADNI-06-10004 from the Alzheimer's Association. Dr Polman received consulting fees or speaking fees from Actelion, Biogen Idec, Bayer Schering Germany, Teva Neuroscience, Merck Serono, Novartis, GlaxoSmithKline, UCB, Roche, and Antisense Therapeutics, as well as grant support from Biogen Idec, Bayer Schering Germany, GlaxoSmithKline, Novartis, UCB, Merck Serono, and Teva Neuroscience.

Funding/Support: This study was supported by Progenika Biopharma, SA. The MS Center Amsterdam is supported by program grant 05-358 from the Dutch Multiple Sclerosis Research Foundation. Dr De Jager is a Harry Weaver Neuroscience Scholar of the National Multiple Sclerosis Society.

Role of the Sponsor: The corporate sponsor participated in the design and conduct of the study (including selection of single-nucleotide polymorphisms for the DNA chip, which is intellectually protected by the corporate sponsor), the genetic analysis, and the review and approval of the final manuscript.

Additional Contributions: M. van de Wiel, PhD, provided statistical advice. We thank all the patients for their participation.