Patricia Momigliano-Richiardi

Concordance, disease progression, and heritability of coeliac disease in Italian twins

Background and aims: We adopted the twin method to disentangle the genetic and environmental components of susceptibility to coeliac disease (CD). We estimated disease concordance rate by zygosity and HLA genotypes, discordance times, progression rates to disease, and heritability. Methods: We crosslinked the Italian Twin Registry with the membership lists of the Italian Coeliac Disease Association and recruited 23 monozygotic (MZ) and 50 dizygotic (DZ) twin pairs with at least one affected member. Zygosity was assigned by DNA fingerprinting, and HLA-DQ and DR alleles were genotyped. Disease status was ascertained by antiendomysial, anti-human tissue transglutaminase antibodies, and bowel biopsy. Results: Concordance was significantly higher in MZ (83.3% probandwise, 71.4% pairwise) than in DZ (16.7% probandwise, 9.1% pairwise) pairs. Concordance was not affected by sex or HLA genotype of the co-twin and being MZ was significantly associated with the occurrence of CD (Cox adjusted hazard ratio 14.3 (95% confidence interval 4.0–50.3)). In 90% of concordant pairs the discordance time was ⩽2 years. MZ and DZ co-twins had 70% and 9% cumulative probability of having symptomatic or silent forms of CD, respectively, within five years. Under ACE (additive genetic, common, and unshared environmental factors) models, with CD population prevalences of 1/91 and 1/1000, heritability estimates were 87% and 57%, respectively. Conclusion: MZ pairs have a high probability of being concordant, regardless of sex or HLA genotype. Most of the affected co-twins receive a diagnosis within two years. A remarkable proportion of phenotypic variance is due to genetic factors.

Systemic lupus erythematosus candidate genes in the Italian population: evidence for a significant association with interleukin-10.

OBJECTIVE To determine whether 7 candidate genes, including tumor necrosis factor receptor II, bcl-2, CTLA-4, interleukin-10 (IL-10), CD19, Fcy receptor type IIA (CD32), and IL-1 receptor antagonist, may contribute to susceptibility to systemic lupus erythematosus (SLE) in the Italian population. METHODS The association with SLE of intragenic markers for each candidate gene, including either microsatellites or dimorphisms, was analyzed. Gene frequencies of these gene markers were compared for patients and ethnically matched controls. Significance was tested by chi-square test on 2 x 2 tables and by Monte Carlo simulation on 2 x N tables. RESULTS A significant increase was found in SLE patients (0.170 versus 0.095; chi2y = 4.11, P = 0.0425) in the frequency of the 140-basepair allele of the IL10.G microsatellite located in the promoter region of the IL-10 gene. This finding was confirmed in a second independent panel where, again, the frequency of the 140-bp allele was found to be significantly increased in SLE patients versus controls (0.176 versus 0.086; chi2y = 3.95, P = 0.0470). Considering the 2 panels together, the relative risk conferred by the presence of the 140-bp allele was 1.78 (95% confidence interval 1.19-2.66). Conversely, no significant association was detected for the remaining 6 candidate genes, even when the patients were stratified according to the presence of different clinical and immunologic features according to the presence of the associated HLA-DR or IL-10 alleles. CONCLUSION Of the 7 candidate genes tested, only IL-10 was significantly associated with SLE in Italian patients. This genetic marker represents, apart from HLA, the only genetic susceptibility factor for SLE found so far in the Italian population.

New polymorphisms in the IL-10 promoter region.

Interleukin-10 (IL-10) is an important immunoregulatory cytokine. We searched for new sequence variations in the 5′ flanking region of the IL-10 gene by denaturing high performance liquid chromatography. A 3996 bp region spanning position −3934 to +61 was amplified in 12 polymerase chain reaction (PCR) fragments and each fragment was screened for variations in 23 Italian individuals. The following eight sequence variations all consisting of single base pair substitutions were identified: −3533A/T, −2769A/G, −2739A/G, −2013A/G, −1349A/G, −1255C/T, −851A/G, −657A/G. The new polymorphisms were analysed in an additional panel of random Italian individuals. The same samples were also tested for the IL10.G and IL10.R microsatellites, and for the two previously described single nucleotide polymorphisms (SNPs) at positions −1082 and −592. Highly significant pairwise linkage disequilibria were observed between alleles at most SNPs. Three major haplotypic combinations of alleles at multiple SNP sites were observed.

HLA-multiple sclerosis association in Continental Italy and correlation with disease prevalence in Europe

The association with HLA-DRB1 alleles was tested in 609 Continental Italian MS patients and 836 controls. The phenotype frequency of DRB1*15 in the patients was significantly higher (0.316 vs. 0.112; p(c)<10(-6); Odds Ratio (OR)=3.64) with no dose effect. DRB1*10 was also significantly increased (OR=2.19; p(c)=0.047) and DRB1*07 decreased (OR=0.60; p(c)=1.3 x 10(-3)) independently of DR15 and of each other. We did not detect an influence of the DR phenotype on disease course, age at onset/diagnosis, gender or familiarity. No association with Class I was detected in a random subset of patients and controls. A comparison of the HLA association data in Northern and Southern European populations shows a parallel between disease prevalence and DR15 frequency.

Osteopontin gene haplotypes correlate with multiple sclerosis development and progression

Osteopontin (OPN) is an inflammatory cytokine highly expressed in multiple sclerosis (MS) plaques. In a previous work, we showed that four OPN polymorphisms form three haplotypes (A, B, and C) and that homozygotes for haplotype-A display lower OPN levels than non-AA subjects. In this work, we evaluated the distribution of these OPN haplotypes in 425 MS patients and 688 controls. Haplotype-A homozygotes had about 1.5 lower risk of developing MS than non-AA subjects. Clinical analysis of 288 patients showed that AA patients displayed slower switching from a relapsing remitting to a secondary progressive form and milder disease with slower evolution of disability. MS patients displayed increased OPN serum levels, which were partly due to the increased frequency of non-AA subjects. Moreover in AA patients, OPN levels were higher than in AA controls and similar to those found in both non-AA patients and controls, which suggests a role of the activated immune response. These data suggest that OPN genotypes may influence MS development and progression due to their influence on OPN levels.

Determination of SNP allele frequencies in pooled DNAs by primer extension genotyping and denaturing high-performance liquid chromatography.

By testing DNA pools rather than single samples the number of tests for a case-control association study can be decreased to only two for each marker: one on the patient and one on the control pool. A fundamental requirement is that each pool represents the frequency of the markers in the corresponding population beyond the influence of experimental errors. Consequently the latter must be carefully determined. To this aim, we prepared pools of different size (49-402 individuals) with accurately quantified DNAs, estimated the allelic frequencies in the pools of two SNPs by primer extension genotyping followed by DHPLC analysis and compared them with the real frequencies determined in the single samples. Our data show that (1) the method is highly reproducible: the standard deviation of repeated determinations was +/-0.014; (2) the experimental error (i.e., the discrepancy between the estimated and real frequencies) was +/-0.013 (95% C.I.: 0.0098-0.0165). The magnitude of this error was not correlated to the pool size or to the type of SNP. The effect of the observed experimental error on the power of the association test was evaluated. We conclude that this method constitutes an efficient tool for high-throughput association screenings provided that the experimental error is low. We therefore recommend that before a pool is used for extensive association studies, its quality, i.e., the experimental error, is verified by determining the difference between estimated and real frequencies for at least one marker.

Genetics of multiple sclerosis: linkage and association studies.

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system caused by an interplay of environmental and genetic factors. The only genetic region that has been clearly demonstrated by linkage and association studies to contribute to MS genetic susceptibility is the human leukocyte antigen (HLA) system. The majority of HLA population studies in MS have focused on Caucasians of Northern European descent, where the predisposition to disease has been consistently associated with the class II DRBl*1501-DQAl*0102-DQBl*0602 haplotype. Apositive association with DR4 was detected in Sardinians and in other Mediterranean populations. Moreover DR1, DR7, DR11 have been found to be protective in several populations.Systematic searches aimed at identifying non-HLA susceptibility genes were undertaken in several populations by means of linkage studies with microsatellite markers distributed across the whole genome. The conclusion of these studies was that there is no major MS locus, and genetic susceptibility to the disease is most likely explained by the presence of different genes each conferring a small contribution to the overall familial aggregation.The involvement of several candidate genes was tested by association studies, utilizing either a population-based (case control) or a family-based (transmission disequilibrium test) approach. Candidate genes were selected mainly on the basis of their involvement in the autoimmune pathogenesis and include immunorelevant molecules such as cytokines, cytokine receptors, immunoglobulin, T cell receptor subunits and myelin antigens. With the notable exception of HLA, association studies met only modest success. This failure may result from the small size of the tested samples and the small number of markers considered for each gene. New tools for large scale screening are needed to identify genetic determinants with a low phenotypic effect. Large collaborative studies are planned to screen several thousands of patients with MS with several thousands of genetic markers. The tests are increasingly based on the DNA pooling procedure.

HLA-class I markers and multiple sclerosis susceptibility in the Italian population.

Previous studies reported an association with multiple sclerosis (MS) of distinct HLA-class I markers, namely HLA-A*02, HLA-Cw*05 and MOG-142L. In this work, we tested the association with MS of A*02 and Cw*05 in 1273 Italian MS patients and 1075 matched controls, which were previously analyzed for MOG-142, and explored the relationship among these three markers in modulating MS risk. HLA-A*02 conferred a statistically robust MS protection (odds ratio, OR=0.61; 95% confidence intervals, CI=0.51–0.72, P<10−9), which was independent of DRB1*15 and of any other DRB1* allele and remained similar after accounting for the other two analyzed class I markers. Conversely, the protective effect we previously observed for MOG-142L was secondary to its linkage disequilibrium with A*02. Cw*05 was not associated considering the whole sample, but its presence significantly enhanced the protection in the HLA-A*02-positive group, independently of DRB1: the OR conferred by A*02 in Cw*05-positive individuals (0.22, 95% CI=0.13–0.38) was significantly lower than in Cw*05-negative individuals (0.69, 95% CI=0.58–0.83) with a significant (P=4.94 × 10−5) multiplicative interaction between the two markers. In the absence of A*02, Cw*05 behaved as a risk factor, particularly in combination with DRB1*03 (OR=3.89, P=0.0006), indicating that Cw*05 might be a marker of protective or risk haplotypes, respectively.

Genetic interaction of CTLA-4 with HLA-DR15 in multiple sclerosis patients

Multiple sclerosis is a chronic inflammatory disease of the central nervous system with a genetic component. Until now, the more consistent association with the disease is found with the major histocompatibility complex, especially HLA‐DRB1*1501‐DQB1*0602 haplotype. In this report, we demonstrate the interaction of Cytotoxic T Lymphocyte‐associated antigen 4 (CTLA‐4 [CD152]) gene with DRB1*15 haplotype in multiple sclerosis genetic susceptibility. Our data were obtained from two European independent family‐based studies including 610 multiple sclerosis family trios. Ann Neurol 2003;54:119–122

Association tests with systemic lupus erythematosus (SLE) of IL10 markers indicate a direct involvement of a CA repeat in the 5′ regulatory region

Many lines of evidence suggest that IL10 is a strong candidate gene for systemic lupus erythematosus (SLE) susceptibility. In our previously reported study an allele (IL10.G-140bp) of the microsatellite IL10.G located at position −1100 was significantly increased in Italian SLE patients in comparison with controls. Starting from this observation, we tested if sequence variations in the vicinity of IL10.G were more strongly associated with SLE. We performed a comprehensive association study including 26 SNPs (of which four were newly identified in the present study by DHPLC analysis) spanning 8.5 Kb of the 5′ flanking and the transcribed region of the IL10 gene. The association study was performed by the DNA pool method on an extended panel of Italian patients (205) and controls (631). Haplotypic associations were studied by individual typing of seven selected markers surrounding IL10.G. Gene, genotype and haplotype frequencies were not significantly different in patients and controls. Thus the IL10.G microsatellite remains to date the only IL10 marker associated with SLE in our population. A meta-analysis of all published results indicates a possible direct role of the IL10.G repeat number in SLE susceptibiliy.