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Multiple sclerosis is an autoimmune and inflammatory disease of the CNS characterised by recurrent destruction of myelin which results in heterogeneous and unpredictable clinical manifestations. Although the factors able to foster the initial changes leading to CNS demyelination in patients with multiple sclerosis are still unknown, a discrete number of genes are likely to predispose to the occurrence of multiple sclerosis and to influence its clinical variables.
Due to the inflammatory nature of the pathogenic mechanisms mediating CNS damage during multiple sclerosis, association studies have been focused on candidate genes coding for immunorelevant molecules. The interleukin (IL)-1 gene cluster (includingIL-1A, IL-1B,IL-1RN), located on the long arm of chromosome 2 (2q12–22; OMIM Database of National Center for Biotechnology Information; www3.ncbi.nlm.nih.gov:80/Omim) and polymorphic in different sites, represents a good target for association studies in multiple sclerosis. IL-1, in fact, has been detected within multiple sclerosis lesions.1 Moreover, lipopolysaccharide stimulated peripheral blood monocytes of patients with multiple sclerosis produce more IL-1α and IL-1β than controls.2 Due to the potential involvement of IL-1α and IL-1β in multiple sclerosis, a genetically determined polymorphic variation in their production may contribute to the occurrence of multiple sclerosis, or modulate its clinical features. Furthermore, we have recently reported the association between the variable number of tandem repeat polymorphism (VNTR) of IL-1RNwith multiple sclerosis susceptibility and prognosis.3 IL-1A -889 andIL-1B -551 polymorphic loci are located 375 and 320 kb respectively from IL-1RN VNTR. To determine whether a particular allele or genotype ofIL-1A or IL-1Baffects occurrence or clinical variables of multiple sclerosis, we performed a case-control association study in a large cohort of patients with multiple sclerosis and age and ethnicity matched healthy controls.
Patients were clinically diagnosed at the multiple sclerosis centre of the San Raffaele Hospital in Milano as affected by clinically definite multiple sclerosis according to Poser's criteria. All patients had a relapsing-remitting course. Genotype frequencies of theIL-1A -889 polymorphism were calculated in a cohort of 399 patients with sporadic relapsing-remitting multiple sclerosis (247 women, 152 men; age 37.3 (SD 12); mean age at disease onset 27.3 (SD 9.3)) and 439 healthy controls (151 women, 288 men; mean age 37.6 (SD 10.6)). Gene frequencies of theIL-1B -511 polymorphism were calculated in a cohort of 321 patients with sporadic relapsing-remitting multiple sclerosis (203 women, 118 men; mean age 38 (SD 12); mean age at disease onset 27.4 (SD 9.2)) and 403 HC (146 women, 257 men; mean age 38 (SD 10.9)).
IL-1A -889 andIL-1B -511 C/T polymorphisms were characterised by polymerase chain reaction (95°C 30 seconds, 55°C 30 seconds, and 72°C 40 seconds; 40 cycles) using genomic DNA and following primers: IL-1α FW 5′-AAGCTTGTTCTACCACCT GAACTAGGC-3′, IL-1α RV 5′-TTAA TATGAGCCTTCCATG-3′; IL-1β FW 5′-TGGCATTGATCTGGTTCATC-3′, IL-1β RV 5′-GTTTAGGAATCTTCCCACTT-3′). Amplified products were digested with restriction endonuclease (NcoI and AvaI).
Statistical analyses were performed with the SAS statistical package (SAS Institute Inc, Cary, NC, USA). The p values were considered not significant (NS) when greater than 0.05. Gene frequencies were compared by χ2 test with two degrees of freedom. The homogeneity of Kaplan-Meyer disease free curves over strata was tested using the rank statistics (log-rank and Wilcoxon tests).
The IL-1A andIL-1B alleles were in Hardy-Weinberg equilibrium in all populations and their distributions were not affected by sex or age (data not shown). We found a similar gene free distribution between patients with multiple sclerosis and healthy controls for both IL-1A andIL-1B polymorphisms (table) (IL-1A: multiple sclerosisv healthy controls, χ2=0.5, NS; IL-1B: multiple sclerosisv healthy controls, χ2=1.3, NS).
The possible association between a givenIL-1A or IL-1Bgenotype and accumulation of clinical burden over time was assessed by comparing two groups of patients with multiple sclerosis differing for their disease outcome and classified as “benign” (patients with a stabilised expanded disability status scale (EDSS) score⩽3 after at least 10 years from disease onset) and “non-benign” (patients with an EDSS score>3 within 10 years from disease onset, table).3 In the case of theIL-1A polymorphism, the gene frequency of 91 benign was not statistically different from those of the 107 non-benign patients with multiple sclerosis (χ2=5.9, NS). As for theIL-1B polymorphism, the gene frequency of the 73 benign patients was also similar to those of the 82 non-benign patients with multiple sclerosis (χ2=4.0, NS).
To assess whether IL-1A and/orIL-1B genotypes had an influence on the age at onset of multiple sclerosis, Kaplan-Meier disease free survival curves were calculated, plotting the age at disease onset of each patient versus the respective IL-1 genotype (data not shown). Again, neither IL-1A (log rank χ2=3.9, NS) nor IL-1B (log rank χ2= 0.72, NS) genotypes seemed to affect the distribution of the age at multiple sclerosis onset.
A handful of inflammation associated polymorphisms have been shown to positively associate with the occurrence of multiple sclerosis or to influence its clinical variables, including polymorphisms located within or nearby the HLA locus (important for multiple sclerosis susceptibility), or the IL-1RN,IL-4 and IL-1Bgenes3 (possibly important in modulating the course of disease).
Our analysis could not demonstrate any association between these two previously untested IL-1A andIL-1B promoter polymorphisms and the occurrence of multiple sclerosis, the age at disease onset, or the accumulation of clinical burden over time in affected patients.
Given the inflammatory nature of multiple sclerosis pathological lesions and the positive association of the relatedIL-1RN VNTR with some of the clinical features of multiple sclerosis, these results are somewhat unexpected and contrast with data coming from similar genetic studies in Alzheimer's disease, a degenerative disease of the CNS where the role of inflammatory mechanisms is not as established as in multiple sclerosis.4
These data contribute to the debate over the role of inflammation in multiple sclerosis, especially as recent reports have surprisingly suggested that inflammation may even be protective (rather than having a pathogenic role) in experimental animal models of multiple sclerosis.5
This work was supported by a grant from the Armenise-Harvard Foundation.
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