Multiple sclerosis and HLA genotypes: A possible influence on brain atrophy.

BACKGROUND: The strongest genetic determinant for multiple sclerosis (MS) is located at the human leukocyte antigen (HLA) class II DRB1 and DQB1 loci. OBJECTIVES: To investigate the possible role of predisposing HLA genotypes in determining brain atrophy. METHODS: HLA genotypes were categorized as high risk (two predisposing haplotypes) or medium/low risk (one or no predisposing haplotypes). Patients underwent a brain magnetic resonance imaging (MRI) study and volumes of white matter (WM), gray matter (GM), and whole brain (WB) were estimated with SIENAX. Longitudinal atrophy was also assessed with SIENA. RESULTS: The study included 240 MS patients. In 51/240 (21%) subjects, a high-risk HLA genotype was observed, while medium- and low-risk HLA genotypes were 109/240 (45%) and 80/240 (34%), respectively. Multiple regression analysis found that the high-risk HLA genotype was associated with significant reduction in WB ( p = 0.02) and GM ( p = 0.03) volumes compared with the medium-/low-risk HLA genotypes, independently from MS clinical features. The longitudinal study included 60 patients and showed a brain volume loss of -0.79% in high-risk HLA genotype group versus -0.56% in low-risk HLA genotype. CONCLUSION: Our results suggest an influence of HLA genotype on WB and GM atrophy. Further investigations are necessary to confirm these findings.

Immune and Epstein-Barr virus gene expression in cerebrospinal fluid and peripheral blood mononuclear cells from patients with relapsing-remitting multiple sclerosis.

BACKGROUND: Gene expression analyses in paired cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMC) from patients with multiple sclerosis (MS) are restrained by the low RNA amounts from CSF cells and low expression levels of certain genes. Here, we applied a Taqman-based pre-amplification real-time reverse-transcription polymerase chain reaction (RT-PCR) (PreAmp RT-PCR) to cDNA from CSF cells and PBMC of MS patients and analyzed multiple genes related to immune system function and genes expressed by Epstein-Barr virus (EBV), a herpesvirus showing strong association with MS. Using this enhanced RT-PCR method, we aimed at the following: (1) identifying gene signatures potentially useful for patient stratification, (2) understanding whether EBV infection is perturbed in CSF and/or blood, and (3) finding a link between immune and EBV infection status. METHODS: Thirty-one therapy-free patients with relapsing-remitting MS were included in the study. Paired CSF cells and PBMC were collected and expression of 41 immune-related cellular genes and 7 EBV genes associated with latent or lytic viral infection were determined by PreAmp RT-PCR. Clinical, radiological, CSF, and gene expression data were analyzed using univariate and multivariate (cluster analysis, factor analysis) statistical approaches. RESULTS: Several immune-related genes were differentially expressed between CSF cells and PBMC from the whole MS cohort. By univariate analysis, no or only minor differences in gene expression were found associated with sex, clinical, or radiological condition. Cluster analysis on CSF gene expression data grouped patients into three clusters; clusters 1 and 2 differed by expression of genes that are related mainly to innate immunity, irrespective of sex and disease characteristics. By factor analysis, two factors grouping genes involved in antiviral immunity and immune regulation, respectively, accurately discriminated cluster 1 and cluster 2 patients. Despite the use of an enhanced RT-PCR method, EBV transcripts were detected in a minority of patients (5 of 31), with evidence of viral latency activation in CSF cells or PBMC and of lytic infection in one patient with active disease only. CONCLUSIONS: Analysis of multiple cellular and EBV genes in paired CSF cell and PBMC samples using PreAmp RT-PCR may yield new information on the complex interplay between biological processes underlying MS and help in biomarker identification.

Inter-Laboratory Concordance of Cerebrospinal Fluid and Serum Kappa Free Light Chain Measurements.

The kappa index (K-Index), calculated by dividing the cerebrospinal fluid (CSF)/serum kappa free light chain (KFLC) ratio by the CSF/serum albumin ratio, is gaining increasing interest as a marker of intrathecal immunoglobulin synthesis. However, data on inter-laboratory agreement of these measures is lacking. The aim was to assess the concordance of CSF and serum KFLC measurements, and of K-index values, across different laboratories. KFLC and albumin of 15 paired CSF and serum samples were analyzed by eight participating laboratories. Four centers used Binding Site instruments and assays (B), three used Siemens instruments and assays (S), and one center used a Siemens instrument with a Binding Site assay (mixed). Absolute individual agreement was calculated using a two-way mixed effects intraclass correlation coefficient (ICC). Cohen's kappa coefficient (k) was used to measure agreement on positive (≥5.8) K-index values. There was an excellent agreement in CSF KFLC measurements across all laboratories (ICC (95% confidence interval): 0.93 (0.87-0.97)) and of serum KFLC across B and S laboratories (ICC: 0.91 (0.73-0.97)), while ICC decreased (to 0.81 (0.53-0.93)) when including the mixed laboratory in the analysis. Concordance for a positive K-Index was substantial across all laboratories (k = 0.77) and within S laboratories (k = 0.71), and very good (k = 0.89) within B laboratories, meaning that patients rarely get discordant results on K-index positivity notwithstanding the testing in different laboratories and the use of different platforms/assays.

Genetic loci linked to type 1 diabetes and multiple sclerosis families in Sardinia.

BACKGROUND: The Mediterranean island of Sardinia has a strikingly high incidence of the autoimmune disorders Type 1 Diabetes (T1D) and Multiple Sclerosis (MS). Furthermore, the two diseases tend to be co-inherited in the same individuals and in the same families. These observations suggest that some unknown autoimmunity variant with relevant effect size could be fairly common in this founder population and could be detected using linkage analysis. METHODS: To search for T1D and MS loci as well as any that predispose to both diseases, we performed a whole genome linkage scan, sequentially genotyping 593 microsatellite marker loci in 954 individuals distributed in 175 Sardinian families. In total, 413 patients were studied; 285 with T1D, 116 with MS and 12 with both disorders. Model-free linkage analysis was performed on the genotyped samples using the Kong and Cox logarithm of odds (LOD) score statistic. RESULTS: In T1D, aside from the HLA locus, we found four regions showing a lod-score > or =1; 1p31.1, 6q26, 10q21.2 and 22q11.22. In MS we found three regions showing a lod-score > or =1; 1q42.2, 18p11.21 and 20p12.3. In the combined T1D-MS scan for shared autoimmunity loci, four regions showed a LOD >1, including 6q26, 10q21.2, 20p12.3 and 22q11.22. When we typed more markers in these intervals we obtained suggestive evidence of linkage in the T1D scan at 10q21.2 (LOD = 2.1), in the MS scan at 1q42.2 (LOD = 2.5) and at 18p11.22 (LOD = 2.6). When all T1D and MS families were analysed jointly we obtained suggestive evidence in two regions: at 10q21.1 (LOD score = 2.3) and at 20p12.3 (LOD score = 2.5). CONCLUSION: This suggestive evidence of linkage with T1D, MS and both diseases indicates critical chromosome intervals to be followed up in downstream association studies.

Intrathecal oligoclonal bands synthesis in multiple sclerosis: is it always a prognostic factor?

BACKGROUND: Oligoclonal IgM (OCMB) and IgG (OCGB) bands were found to be associated with poor multiple sclerosis (MS) prognosis. OBJECTIVE: We aimed to evaluate the prognostic value of OCMB/OCGB in a cohort of Sardinian MS patients. MATERIALS AND METHODS: We recruited patients from the University of Cagliari. They underwent lumbar puncture for diagnostic purposes. Demographic and the following clinical data were recorded: clinical course; time to reach EDSS 3 and 6; EDSS at last follow-up; and MS treatments. The influence of gender, clinical course, age at onset, treatments, and OCGB/OCMB on reaching EDSS 3 was analysed using Cox regression. Kaplan-Meier curves were used to study the time to reach EDSS 3 considering OCMB/OCGB and therapies. RESULTS: The enrolled number of subjects was 503. The variables influencing the achievement of EDSS 3.0 were: male gender (p = 0.005); progressive course (p = 0.001); age at onset (p < 0.001); and disease-modifying drugs (p < 0.001). The OCGB/OCMB status was not significant. Kaplan-Meier analysis showed no difference in time to reach EDSS 3 for patients with and without OCGB or OCMB in both treated and non-treated groups. CONCLUSION: Our study did not confirm the poor prognostic value of OCMB/OCGB. These results may be influenced by the peculiar genetic background associated with the risk of MS in Sardinians.

Variation of the myelin oligodendrocyte glycoprotein gene is not primarily associated with multiple sclerosis in the Sardinian population.

BACKGROUND: Multiple sclerosis (MS) is consistently associated with particular HLA-DRB1-DQB1 haplotypes. However, existing evidence suggests that variation at these loci does not entirely explain association of the HLA region with the disease. The MOG locus is a prime positional and functional candidate for such additional predisposing effects but the analysis is complicated by the strong, albeit labyrinthine pattern of linkage disequilibrium in the region. Here we have assessed the association of MOG variation with MS in the Sardinian population to see if it represents an independent contributor to MS predisposition. RESULTS: After re-sequencing the MOG gene in 21 healthy parents of MS patients we detected 134 variants, 33 of which were novel. A set of 40 informative SNPs was then selected and assessed for disease association together with 1 intragenic microsatellite in an initial data set of 239 MS families. This microsatellite and 11 SNPs were found to be positively associated with MS, using the transmission disequilibrium test, and were followed up in an additional 158 families (total families analysed = 397). While in these 397 families, 8 markers showed significant association with MS, through conditional tests we determined that these MOG variants were not associated with MS independently of the main DRB1-DQB1 disease associations. CONCLUSION: These results indicate that variation within the MOG gene is not an important independent determinant of MS-inherited risk in the Sardinian population.

PTPRC (CD45) C77G mutation does not contribute to multiple sclerosis susceptibility in Sardinian patients.

A linkage and association of the CD45 (protein-tyrosine phosphatase, receptor-type C) C77G polymorphism and multiple sclerosis (MS) has been found in some studies but not in others. We analysed the C77G polymorphism in MS patients from the genetically homogeneous population of Sardinia. Using the transmission disequilibrium test, the mutation has been sought in 241 patients and 217 healthy sibs (HS) from singleton MS families and it was found in 5 (2.07 %) affected and 3 (1.38%) HS from 7 heterozygous parents (1.45 %). Transmission of the G77 allele was 71.4 % (TDT = 1.3, P = 0.26) in patients and 50% (TDT = 0, P = 1) in HS. Stratifying families according to carriage of MS-predisposing (DR+) or not-predisposing (DR-) HLA-DR-DQ genotype in patients, percentage of G77 transmission to DR+ patients was 33 (TDT = 0.33, P = 0.56, Pc = 1.12), while it was 100 (TDT = 4, P = 0.045, Pc = 0.09) in the DR-patients. We concluded that, despite the presence of CD45 G77 polymorphism in a few patients who did not carry the HLADR- DQ MS-predisposing molecules, CD45 did not contribute to development of the disease in Sardinian MS.

Vitamin D responsive elements within the HLA-DRB1 promoter region in Sardinian multiple sclerosis associated alleles.

Vitamin D response elements (VDREs) have been found in the promoter region of the MS-associated allele HLA-DRB1*15:01, suggesting that with low vitamin D availability VDREs are incapable of inducing *15:01 expression allowing in early life autoreactive T-cells to escape central thymic deletion. The Italian island of Sardinia exhibits a very high frequency of MS and high solar radiation exposure. We test the contribution of VDREs analysing the promoter region of the MS-associated DRB1 *04:05, *03:01, *13:01 and *15:01 and non-MS-associated *16:01, *01, *11, *07:01 alleles in a cohort of Sardinians (44 MS patients and 112 healthy subjects). Sequencing of the DRB1 promoter region revealed a homozygous canonical VDRE in all *15:01, *16:01, *11 and in 45/73 *03:01 and in heterozygous state in 28/73 *03:01 and all *01 alleles. A new mutated homozygous VDRE was found in all *13:03, *04:05 and *07:01 alleles. Functionality of mutated and canonical VDREs was assessed for its potential to modulate levels of DRB1 gene expression using an in vitro transactivation assay after stimulation with active vitamin D metabolite. Vitamin D failed to increase promoter activity of the *04:05 and *03:01 alleles carrying the new mutated VDRE, while the *16:01 and *03:01 alleles carrying the canonical VDRE sequence showed significantly increased transcriptional activity. The ability of VDR to bind the mutant VDRE in the DRB1 promoter was evaluated by EMSA. Efficient binding of VDR to the VDRE sequence found in the *16:01 and in the *15:01 allele reduced electrophoretic mobility when either an anti-VDR or an anti-RXR monoclonal antibody was added. Conversely, the Sardinian mutated VDRE sample showed very low affinity for the RXR/VDR heterodimer. These data seem to exclude a role of VDREs in the promoter region of the DRB1 gene in susceptibility to MS carried by DRB1* alleles in Sardinian patients.