Genetic loci linked to Type 1 Diabetes and Multiple Sclerosis families in Sardinia

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>In T1D, aside from the HLA locus, we found four regions showing a lod-score ≥1; 1p31.1, 6q26, 10q21.2 and 22q11.22. In MS we found three regions showing a lod-score ≥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).</p> <p>Conclusion</p> <p>This suggestive evidence of linkage with T1D, MS and both diseases indicates critical chromosome intervals to be followed up in downstream association studies.</p

The Distribution of HLA class II haplotypes reveals that the Sardinian population is genetically differentiated from the other Caucasian populations

In this study we have established the frequencies of the DRB1-DQA1-DQB1 haplotypes in a large cohort of Sardinian new-borns and found that the most frequent haplotypes were detected at frequencies unique to the Sardinians. Other haplotypes, common in other Caucasian populations, are rare or absent across the island. Next, the DRB1-DQA1-DQB1 haplotype frequencies obtained in Sardinians and those reported in other human populations were used to compute genetic distances and construct phylogenetic trees. A clear-cut pattern appeared with a split between the three major human groups: Caucasians, Asians and Blacks. Among the Caucasians there were three major clusters: a group representing the North-Africans, a group including most of the European-derived populations and a group encompassing Bulgaria, Greece and Sardinia. When we increased the resolution of the tree using the genetic distances calculated from both DRB1-DQA1-DQB1 haplotypes and class I HLA A, B, C allelic frequencies, the Sardinians clearly emerged as the major outlier among the various European populations considered in this study. These results indicate that the genetic structure of the present Sardinian population is the result of a fixation of haplotypes, which are very rare elsewhere, and are most likely to have originated from a relatively large group of founders

Confirmation of the <i>DRB1-DQB1 loci</i> as the major component of <i>IDDM1</i> in the isolated founder population of Sardinia

There is considerable uncertainty and debate concerning the application of linkage disequilibrium (LD) mapping in common multifactorial diseases, including the choice of population and the density of the marker map. Previously, it has been shown that, in the large cosmopolitan population of the UK, the established type 1 diabetes IDDM1 locus in the HLA region could be mapped with high resolution by LD. The LD curve peaked at marker D6S2444, 85 kb from the HLA class II gene DQB1, which is known to be a major determinant of IDDM1. However, given the many unknown parameters underlying LD, a validation of the approach in a genetically distinct population is necessary. In the present report we have achieved this by the LD mapping of IDDM1 in the isolated founder population of Sardinia. Using a dense map of microsatellite markers, we determined the peak of LD to be located at marker D6S2447, which is only 6.5 kb from DQB1. Next, we typed a large number of SNPs defining allelic variation at functional candidate genes within the critical region. The association curve, with both classes of marker, peaked at the loci DRB1-DQB1. These results, while representing conclusive evidence that the class II loci DRB1-DQB1 dominate the association of the HLA region to type 1 diabetes, provide empirical support for LD mapping

A Gene dosage effect of the DQA1*0501/DQB1*0201 allelic combination influences the clinical heterogeneity of celiac disease

This study reports the HLA-DR and DQ molecular characterization of 62 CD patients of Sardinian descent. Patients were divided in two groups (36 in group I and 26 in group II) according to the clinical features at the disease onset. Among the patients of group I, having the fully expressed form of CD and a mean age of 3 years at disease onset, a significant increase of DRB1*0301, DQA1*0501. DQB1*0201 homozygotes, encoding in cis two DQ (α1*0501,β1*0201) susceptibility heterodimers, was observed when compared either with the patients of group II (pIII &lt; 0.012) or with healthy individuals (pI &lt; 10-6). On the other hand, in the patients of group II, presenting oligosymptomatic forms and a mean age of 5.7 years at the disease onset, the haplotype combinations encoding in cis or in trans only one DQ (α1*0501,β1*0201) heterodimer were significantly increased in comparison either with the patients of group I (pIII &lt; 0.026) or with controls (pII &lt; 10-6). These findings suggest that a double dose of DQA1*0501, DQB1*0201 genes may predispose a person to an earlier onset and to more severe disease manifestations. Genotype analysis showed that only three patients (all in group I) failed to form in trans or in cis the DQ (α1*0501,β1*0201) heterodimer and carried the DQA1*0101,DQB1*0501 haplotype, suggesting its possible role in CD susceptibility. In addition, a significant increment of DQB1*0501 gene (pc &lt; 0.0065) was found comparing the frequency of DQB1 alleles in CD patients and healthy controls, after exclusion of DQB1*0201 chromosomes. In conclusion, this study suggests that the clinical picture of CD may be influenced by the DQA1*0501/DQB1*0201 susceptibility genes with a gene dosage effect and that Sardinian patients who are DQ (α1*0501,β1*0201) negative may carry another DQ heterodimer (i.e., DQ [α1*0101,β1*0501]) able to confer CD susceptibility in addition to the primary DQA1*0501/DQB1*0201 gene associations

Anti-Actin IgA Antibodies Identify Celiac Disease Patients with a Marsh 3 Intestinal Damage among Subjects with Moderate Anti-TG2 Levels

A new diagnostic tool (algorithm-1) for coeliac disease (CD) permitting the diagnosis without performing the duodenal biopsy has been recently proposed by the European Society for Paediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN). It combines symptoms associated with CD, high anti-transglutaminase type 2 antibody (anti-TG2) levels, anti-endomysium-IgA antibodies (EMA), and at-risk HLA. Our aims were (i) to evaluate retrospectively in 227 individuals (149 CD patients and 78 controls) the algorithm-1, (ii) to reduce the number of duodenal biopsies among CD patients for whom algorithm-1 is not applicable through the addition of antiactin IgA antibodies (AAA-IgA), and (iii) to evaluate prospectively algorithm-1 and AAA-IgA in 50 patients with suspected CD. Algorithm-1 identified 70 out of 149 CD patients with Marsh 3 lesions. Adding AAA-IgA to the remaining patients with anti-TG2 levels comprised between 4 and 10 times upper limit of normal (ULN) allowed the detection of further 20 patients with a Marsh 3 damage. In our prospective study, algorithm-1 identified 23 out of 50 patients, whilst further 7 were recognized adding AAA-IgA. We confirm that algorithm-1 may avoid the duodenal biopsy in many CD patients and underscores the usefulness of AAA-IgA in reducing the number of duodenal biopsies in patients with moderate anti-TG2 levels

HLA-DQB1*0305 and −DQB1*0304 alleles among Sardinians: evolutionary and practical implications for oligotyping

This study, performed in individuals of Sardiaian descent, reports an epidemiologic and molecular analysis of the recently identified DQB1*0304 and DQB1*0305 alleles*. These two alleles having a gene frequency of 0.017 and 0.005, respectively, are not uncommon in Sardinia and are distributed fairly uniformly on the island. The analysis of DQB1 second and third exons of the two alleles revealed that although they have always been found included within the same DRB1*0403-DQA1*03 haplotype, they had a different origin. The sequence pattern of DQB1*0305 confirmed that it originated from the DQB1*0302 “recipient” gene by the insertion of a DQB1*0402 nucleotide stretch, within its β-sheet region, while that of DQB1*0304 suggested that it originated from the DQB1*0301 gene, either by a single point mutation at codon 57 (GCC instead of GAC) or, alternatively, by a segmental transfer of a DQB1*0302 motif, including codon 57, within its α-helic region. Independently from the mechanism of generation, the fact that DQB1*0304 originated from DQB1*0301 allele was intriguing considering that, in over 1500 HLA class II Sardinian haplotypes examined, neither the putative parental DRB1*0403-DQA1*03-DQB1*0301 nor any DRB1*04-DQA1*03-DQB1*0301 haplotypes were found. Finally, since the assignment of DQB1*0305 may be inaccurate with the traditional panel of probes commonly used for DQB1 oligotyping, the use of an additional oligonucleotide probe is recommended

The Co-inheritance of type 1 diabetes and multiple sclerosis in Sardinia cannot be explained by genotype variation in the HLA region alone

compared with the surrounding southern European populations. Surprisingly, a 5-fold increased prevalence of T1D has also been observed in Sardinian MS patients. Susceptibility to both disorders is associated with common variants of the HLA-DRB1 and -DQB1 loci. In this study, we determined the relative contribution of genotype variation of these loci to the co-occurrence of the two disorders in Sardinia. We genotyped 1052 T1D patients and 1049 MS patients (31 of whom also had T1D) together with 1917 ethnically matched controls. On the basis of the absolute risks for T1D of the HLA-DRB1-DQB1 genotypes, we established that these loci would only contribute to a 2-fold increase in T1D prevalence in MS patients. From this evidence, we conclude that shared disease associations due to the HLA-DRB1-DQB1 loci provide only a partial explanation for the observed increased prevalence of T1D in Sardinian MS patients. The data suggest that variation at other non-HLA class II loci, and/or unknown environmental factors contribute significantly to the co-occurrence of these two traits

Combinations of specific DRB1, DQA1, DQB1 haplotypes are associated with insulin-dependent diabetes mellitus in Sardinia

The Sardinian population has an extremely high incidence of IDDM (30.2 of 100.000 in the age group of 0–14 years). This study reports the molecular characterization of HLA class II genes in 120 IDDM sporadic patients and 89 healthy subjects of Sardinian origin. Compared with other Caucasians, both Sardinian patients and controls had an unusual distribution of haplotypes and genotypes. In particular, there was a high gene frequency of the DRB1*0301, DQA1*0501, DQB1*0201 susceptibility haplotype both in patients (0.58) and controls (0.23) while a reduction of the BRB1*1501, DQA1*0102, DQB1*0602 protective haplotype (0.03) was observed in the healthy population. This distribution may partially explain the high incidence of IDDM reported in Sardinia. The analysis of the DQβ57 and DQα52 residues showed that the absence of Asp 57 and the presence of Arg 52 were associated with IDDM in a dose-response manner. On the other hand, we found that (a) a very similar distribution of these residues was found when comparing Sardinians with another healthy Caucasian population from the same latitude but with a lower rate of IDDM incidence; (b) several genotypes encoding the identical DQα52/DQβ57 phenotype carried very differnt relative risks; and (c) the DRB1*0403, DQA1*0301, DQB1*0304 haplotype (DQβ57 Asp-neg and DQα52 Argpos) was found in 40% of the DR4-positive controls but not in patients (p = 0.00034), while the DRB1*0405, DQA1*0301, and DQB1*0302 haplotype carrying the same residues at the same positions was found in 70% of the DR4-positive patients and in only one control (p = 0.00003). These findings suggest that S0IDDM susceptibility cannot be completely explained by the model in which only DQα52 and DQβ57 residues are taken into account

Conditional linkage disequilibrium analysis of a complex disease superlocus, <i>IDDM1</i> in the HLA region, reveals the presence of independent modifying gene effects influencing the type 1 diabetes risk encoded by the major <i>HLA-DBQ1</i>, -<i>DRB1</i> disease <i>loci</i>

Type 1 diabetes mellitus is a common disease with a complex mode of inheritance. Its aetiology is underpinned by a major locus, insulin-dependent diabetes mellitus 1 (IDDM1) in the human leukocyte antigen (HLA) region of chromosome 6p21, and an unknown number of loci of lesser individual effect. In linkage analyses IDDM1 is a single peak, but it is evident that the linkage is caused by allelic variation of three adjacent genes in a 75 kb region, namely the class II genes, HLA-DRB1, -DQA1 and -DQB1. However, even these three genes may not explain all of the HLA association. We investigated, in the founder population of Sardinia, whether non-DQ/DR polymorphic markers within a 9.452 Mb region encompassing the whole HLA complex further influence the disease risk, after taking into account linkage disequilibrium with the disease loci HLA-DQB1, -DQA1 and -DRB1. We generalized the conditional association test, the haplotype method, to detect marker associations that are independent of the main DR/DQ disease associations. Three regions were identified as risk modifiers. These associations were not only independent of the polymorphic exon 2 sequences of HLA-DQB1, -DQA1 and -DRB1, but also independent of each other. The individual contributions of these risk modifiers were relatively modest but their combined impact was highly significant. Together, alleles of single nucleotide polymorphisms at the DMB and DOB genes, and the microsatellite locus TNFc, identified ∼40% of Sardinian DR3 haplotypes as non-predisposing. This conditional analysis approach can be applied to any chromosome region involved in the predisposition to complex traits

Major factors influencing linkage disequilibrium by analysis of different chromosome regions in distinct populations: demography, chromosome recombination frequency and selection

Linkage disequilibrium (LD) mapping of disease genes is complicated by population- and chromosome-region-specific factors. We have analysed demographic factors by contrasting intermarker LD results obtained in a large cosmopolitan population (UK), a large genetic isolate (Sardinia) and a subisolate (village of Gavoi) for two regions of the X chromosome. A dramatic increase of LD was found in the subisolate. Demographic history of populations therefore influences LD. Chromosome-region-specific effects, namely the pattern and frequency of homologous recombination, were next delineated by the analysis of chromosome 6p21, including the HLA region. Patterns of global LD in this region were very similar in the UK and Sardinian populations despite their entirely distinct demographies, and correlate well with the pattern of recombinations. Nevertheless, haplotypes extend across recombination hot spots indicative of selection of certain haplotypes. Subisolate aside, chromosome-region-specific differences in LD patterns appear to be more important than the differences in intermarker LD between distinct populations