Candidate gene region 2q33 in European families with coeliac disease.

Chromosome region 2q33 harbours a cluster of genes, CTLA-4, CD28, ICOS and closely located PD-1, all related to immune activation and considered as promising candidate genes for susceptibility to coeliac disease (CD). We present here the results of a genetic linkage and association analysis of nine markers located in this gene region in a large combined European material of 796 families with CD from Finland, Sweden, Norway, UK, France and Italy. The joint analysis supports earlier findings that this susceptibility locus, assigned as CELIAC3, merits further studies. Nominally significant linkage to CD was found in 314 families including affected sib pairs. Each of the five populations showed weak associations to several marker alleles, but the analysis revealed, however, no conclusive evidence for a primary functional gene or gene variant present in the total set of families. The results suggest that the CD risk due to 2q33 gene region is complex and may involve more than one susceptibility allele, which possibly differ from other autoimmune diseases.

Genetic association of coeliac disease susceptibility to polymorphisms in the ICOS gene on chromosome 2q33.

An interesting candidate gene region for coeliac disease (CD), a common multifactorial disease, is a segment on 2q33-37 harbouring the genes for the CD28, cytotoxic T-lymphocyte-associated antigen-4 (CTLA4), inducible costimulator (ICOS), and programmed death-1 (PD-1), all receptors that regulate lymphocyte activation. Several studies have suggested a role for this locus in immune-mediated diseases. To study further our previous finding of genetic linkage of this region to CD, we studied 25 polymorphic markers to identify the putative disease-associated polymorphism. Transmission/disequilibrium test in 106 Finnish families with CD indicated that only four polymorphisms, all located in the ICOS gene, showed evidence for genetic association. Strong linkage disequilibrium (LD), based on the analysis of 424 haplotypes, encompassed not only the associated ICOS markers but also many polymorphisms in the CTLA4 gene. Our results demonstrate that due to LD, it appears not easy to identify the genuine susceptibility factor in this region without larger multipopulation studies. Furthermore, the results did not support the evidence that polymorphisms in CTLA4 were the major susceptibility locus for CD.

Genetic dissection between coeliac disease and dermatitis herpetiformis in sib pairs.

Gluten sensitive enteropathy has various manifestations, of which the two major forms are classical coeliac disease (cCD) and dermatitis herpetiformis (DH). In cCD predominantly the small intestine is affected, whereas in DH also the skin is affected showing typical rash and IgA deposits. The symptoms in both forms are dependent on gluten intake. The factors diversifying these two clinical outcomes are unknown. In the present report we evaluated the role of the major genetic susceptibility locus, HLA DQ, in 25 families, in which both forms of the disease, cCD and DH, occurred in siblings. By using the family-based approach it can be assumed that within each family variation in environmental factors is substantially lower than in the standard case-control setting, and also the problems related to population stratification can be avoided. Results from the Finnish family material with 25 discordant and 85 concordant sib pairs, and from additional case-control material comprising 71 unrelated Hungarian DH and 68 cCD patients, together indicated that the HLA DQ locus did not differ between the two major outcomes of gluten sensitive enteropathy. The non-HLA DR;DQ factors are critical for the different clinical manifestations of gluten sensitivity.

CD80 (B7-1) and CD86 (B7-2) genes and genetic susceptibility to coeliac disease.

The role of the costimulatory molecules B7-1 (CD80) and B7-2 (CD86) in T-cell activation makes them good candidates for coeliac disease susceptibility genes. We conducted a genetic linkage study of the CD80/86 gene region in the general Finnish population and in a local subisolate. No linkage was found in either population.

Not all HLA DR3 DQ2 haplotypes confer equal susceptibility to coeliac disease: transmission analysis in families.

BACKGROUND: HLA-DQ is the only established susceptibility factor for coeliac disease. We tested whether all HLA haplotypes with the known risk marker, HLA-DQ2, confer equal susceptibility to coeliac disease, i.e. whether haplotype transmission from DQ2 homozygous parents to patients is random. The random transmission would strengthen the importance of DQ2 as the only risk factor within the HLA region. METHODS: Inheritance of DQ2-positive haplotypes from parent to patients was investigated in 14 of 127 Finnish coeliac families who had an HLA-DQ2 homozygous parent. HLA alleles and 18 HLA-linked microsatellite markers were used to determine the haplotypes, which were divided into those transmitted and those non-transmitted from DQ2-homozygous parents to patients: RESULTS: Transmitted haplotypes differed clearly from those not transmitted. The alleles in the transmitted haplotypes were strongly conserved and predominantly consisted of the well-known HLA-A*01, B*08, DRB1*03, DQ2, DPB1*0101 haplotype. The non-transmitted haplotypes, on the other hand, were significantly more heterogeneous; in particular, markers near HLA-A and -B genes differed from the transmitted haplotypes. CONCLUSIONS: The results suggest that DQ2 is not the only HLA-linked genetic risk factor for coeliac disease but the conserved haplotype harbours at least one other risk gene.

Coeliac disease among healthy members of multiple case coeliac disease families.

BACKGROUND: The main objective of the study was to assess the frequency of undetected coeliac disease among the first-degree relatives of families with two or more previously diagnosed coeliac disease patients. The value of the serum endomysial antibody test as a single means of detecting clinically silent coeliac disease was evaluated. The correlation of endomysial and tissue transglutaminase antibodies and the correlation of endomysial antibodies to the HLA typical for coeliac disease was determined. METHODS: A total of 137 multiple-case coeliac disease families with 872 family members were recruited; 466 healthy family members were simultaneously screened for gliadin and endomysial antibodies and thereafter for tissue tranglutaminase antibodies. Antibody-positive persons were typed for HLA-DQ2 and DQ8. The diagnosis of coeliac disease was based on the typical mucosal lesion on small-bowel biopsies. RESULTS: Forty-four (9.4%) of the healthy family members were positive for endomysial and 48 (10.3%) for gliadin antibodies; 42 biopsies revealed 29 new coeliac disease patients (6.2% of healthy individuals). Endomysial antibodies detected 97% and gliadin antibodies 52% of the new cases. All 44 endomysial-antibody-positive and 35 of 48 gliadin-antibody-positive individuals were positive for DQ2. Tissue transglutaminase antibodies corresponded well with endomysial antibodies. CONCLUSIONS: Undetected coeliac disease is common even among healthy first-degree relatives of multiple case families. The findings emphasize the value of serum endomysial antibodies in the detection of clinically silent coeliac disease. Endomysial-antibody-positive individuals, unlike gliadin-antibody-positive ones, share the coeliac disease-type HLA-DQ.

Genetic dissection between silent and clinically diagnosed symptomatic forms of coeliac disease in multiplex families.

BACKGROUND: Coeliac disease has a large variation in clinical outcome. In addition to the classical disease with malabsorption, many individuals have a silent form, in which subject symptoms are missing but autoantibodies and mucosa lesions are identical to the symptomatic disease. AIM: To investigate whether differences in HLA DR-DQ genes explain the variation in outcome. MATERIALS AND METHODS: HLA DQ alleles were determined in 28 multiplex families with sibling pairs in which one had the symptomatic disease but the other had the silent form. RESULTS: No differences in the distribution of HLA DR-DQ haplotypes could be observed. The clinically diagnosed coeliac disease seemed to have earlier onset than silent coeliac disease. CONCLUSIONS: Results indicate that the major genetic susceptibility locus, HLA DQ, does not determine the exact clinical outcome of coeliac disease.

Technical note: linkage disequilibrium and disease-associated CTLA4 gene polymorphisms.

CTLA4 and CD28 are important regulators of T lymphocyte activation. Gene region 2q33 carrying genes for both CTLA4 and CD28 has been shown to be linked to many autoimmune diseases. Disease associations with particular CTLA4 gene polymorphisms have been reported. Recently, first lines of evidence emerged for functional effects of CTLA4 gene polymorphisms. Two independent studies reported a reduced inhibitory function of CTLA4 in individuals with certain CTLA4 genotypes: those with a high number of microsatellite repeats in one study and those with allele +49*G in exon 1 in the other one. We analyzed the strength of linkage disequilibrium between the three known CTLA4 polymorphisms among 577 independent chromosomes. Our results show that the polymorphisms previously suggested to be the functional risk factors nearly always occur together in a very frequent haplotype. Due to this strong linkage disequilibrium, we conclude that the previous reports studying merely a single polymorphism could not distinguish which variation actually caused the functional difference. Hence, either mutagenesis approaches or studies with data on all linked polymorphisms are still needed to determine the genuine functional risk polymorphism in this gene region.

Candidate gene region 15q26 and genetic susceptibility to coeliac disease in Finnish families.

BACKGROUND: Studies in the Irish and British populations have indicated that chromosome region 15q26 could include a novel non-HLA-linked locus conferring genetic susceptibility to coeliac disease. The locus is of particular interest, since a type I diabetes risk locus, IDDM3, maps to the same position. It was tested whether this locus shows evidence for genetic linkage to coeliac disease in Finland. METHODS: Ninety-nine Finnish families with at least one affected sibpair were studied. Five microsatellite markers mapped within approximately 20 cM region on chromosome 15q26 were typed. Non-parametric linkage (NPL) scores and allelic transmission (TDT) were studied. RESULTS: No evidence for genetic linkage could be obtained by the NPL scores calculated by the Genehunter program. However, transmission/disequilibrium analysis (TDT) revealed that haplotype D15S107*1-D15S120*6 was statistically significantly more frequently transmitted to affected than expected by chance (TDT chi2 9.0; P = 0.003). The subgroup of families having this haplotype, however, did not differ from the others, regarding to disease manifestation, HLA status, or geographical origin. CONCLUSION: The 15q26 region appears not to be a major non-HLA susceptibility locus for gluten sensitivity in Finland, but a particular haplotype which may harbour a susceptibility gene was identified.

Candidate gene regions and genetic heterogeneity in gluten sensitivity.

BACKGROUND: Gluten sensitivity is a common multifactorial disorder, manifested in the small intestine or on the skin as typical coeliac disease or dermatitis herpetiformis, respectively. The only established genetic risk factor is HLA DQ2. AIMS: We tested genetic linkage of previously reported chromosomal loci 5q and 11q in Finnish families with gluten sensitivity. We also tested if genetic linkage to candidate loci on 5q, 11q, 2q33, and HLA DQ differed with respect to clinical manifestations or sex. SUBJECTS: We studied 102 Finnish families with affected sibpairs. For heterogeneity analysis, families were divided into subgroups according to sex and the presence of dermatitis herpetiformis, the skin manifestation of gluten sensitivity. METHODS: Non-parametric linkage between microsatellite markers and disease was tested. Linkage heterogeneity between subgroups was tested using the M test. The transmission/disequilibrium test and association analysis were performed. RESULTS: Evidence of linkage to 11q (MLS 1.37), but not to 5q, was found in the entire dataset of 102 families. Heterogeneity between subgroups was suggested: families with only the intestinal disease showed linkage mainly to 2q33 whereas families with dermatitis herpetiformis showed linkage to 11q and 5q, but not to 2q33. Linkage in all three non-HLA loci was strongest in families with predominantly male patients. HLA DQ2 conferred much stronger susceptibility to females than males. CONCLUSIONS: Independent evidence for the suggested genetic linkage between 11q and gluten sensitivity was obtained. The possible linkage heterogeneity suggests genetic differences between intestinal and skin manifestations, and the gender dependent effect of HLA DQ2.

Use of closely related affected individuals for the genetic study of complex diseases in founder populations.

We propose a method, the maximum identity length contrast (MILC) statistic, to locate genetic risk factors for complex diseases in founder populations. The MILC approach compares the identity length of parental haplotypes that are transmitted to affected offspring with the identity length of those that are not transmitted to affected offspring. Initially, the statistical properties of the method were assessed using randomly selected affected individuals with unknown relationship. Because both nuclear families with multiple affected sibs and large pedigrees are often available in founder populations, we performed simulations to investigate the properties of the MILC statistic in the presence of closely related affected individuals. The simulation showed that the use of closely related affected individuals greatly enhances the power of the statistic. For a given sample size and type I error, the use of affected sib pairs, instead of affected individuals randomly selected from the population, could increase the power by a factor of two. This increase was related to an increase of kinship-coefficient contrast between haplotype groups when closely related individuals were considered. The MILC approach allows the simultaneous use of affected individuals from a founder population and affected individuals with any kind of relationship, close or remote. We used the MILC approach to analyze the role of HLA in celiac disease and showed that the effect of HLA may be detected with the MILC approach by typing only 11 affected individuals, who were part of a single large Finnish pedigree.

Concordance of dermatitis herpetiformis and celiac disease in monozygous twins.

Celiac disease can be defined as the classical manifestation of gluten sensitivity, which primarily affects the small intestine. Gluten sensitivity has also a skin manifestation, i.e., dermatitis herpetiformis. Both diseases have a strong genetic association with HLA DQ on chromosome 6. In this study we tried to estimate how much different clinical expressions of gluten sensitivity are determined by genetic factors, and hence how feasible they are for genetic mapping; therefore, we studied all six monozygous twin pairs found among 1292 prospectively collected patients of dermatitis herpetiformis in Finland. Three of the six twin pairs were concordant for dermatitis herpetiformis and for simultaneous enteropathy, celiac disease. Two other twin pairs were partially discordant, one of each pair had dermatitis herpetiformis and celiac disease, whereas the other had solely the gut manifestation of gluten sensitivity, i.e., celiac disease. Only one pair was found to be discordant for gluten sensitivity. All the pairs had typical risk alleles for gluten sensitivity, i.e., either HLA DQ2 or DQ8. These results demonstrate that the genetic component in gluten sensitivity as broadly defined is very strong (5/6 concordant). Genetically identical individuals can have clearly distinguished phenotypes, either dermatitis herpetiformis or celiac disease, suggesting that environmental factors determine the exact phenotype of this multifactorial disease. These findings are of importance in genetic linkage analyses, which focus to only certain phenotypic properties of a complex trait.

Single founder mutation (W380G) in type II protein C deficiency in Finland.

The present study investigated the genetic basis for type II protein C deficiency in Finland, where this form has an unusually high incidence. We demonstrated that, first, a single novel mutation W380G in the protein C gene (PROC) explained 25/26 index patients, estimated to represent two thirds of all families with type II deficiency in Finland. Second, extended chromosomal conservation, i.e. a specific haplotype, around the W380G mutation was indicated in unrelated patients. Third, a local geographical origin for the W380G mutation was suggested by genealogical data. These results are in contrast to the heterogeneity in type II protein C deficiency elsewhere, but closely parallel disorders of the Finnish disease heritage. The high frequency of the type II disease can be explained by founder effect and subsequent enrichment of a single mutation in Finland. The present study also provided a simple means for genetic diagnosis of this disease and the genetic test can be included in the routine screenings in this population.

Major histocompatibility complex (MHC)-linked microsatellite markers in a founder population.

The Finnish population is genetically relatively homogeneous and has a narrow gene pool as a result of founder effect followed by rapid population growth. We here demonstrate that microsatellite markers are highly informative tools for major histocompatibility complex (MHC) analysis in this population. First, no variation in 12 MHC-linked microsatellites could be observed in certain CYP21-deficient chromosomes, which as a result of founder effect most likely derived from common ancestors. Second, amongst 131 Finnish chromosomes, some, but not all, apparently HLA-identical chromosomes also carried identical microsatellites, suggesting that these loci could be applied for identification of haplotypes which have a relatively recent shared origins. Finally, when the microsatellites were studied between ethnically more distant individuals (Finnish vs. non-Finnish), who were matched for the HLA alleles, much more differences were observed. This showed that the similarity in microsatellites was population specific. The microsatellite typing can therefore be informative in fine mapping MHC-linked susceptibility genes and can help in matching bone marrow transplants in isolated populations. Linkage disequilibrium was found to be much higher in the MHC than in another region (5q31) of similar size, indicating that there may be particular mechanisms keeping the MHC haplotypes conserved.

CD28/CTLA4 gene region on chromosome 2q33 confers genetic susceptibility to celiac disease. A linkage and family-based association study.

Celiac disease (CD) is a common small intestinal injury caused by sensitivity to gliadin in genetically-predisposed individuals. The only susceptibility locus established is the HLA-DQ. We tested whether the chromosomal region of the CD28/CTLA4 genes on 2q33 is linked to CD. These genes encode receptors regulating the T-lymphocyte activation. Recently, this gene region was reported to be linked to the susceptibility to many autoimmune diseases, including insulin-dependent diabetes (IDDM12locus). It is thus an obvious candidate locus also for CD, since the intestinal injury is mediated by the immune system. Genetic linkage between seven marker loci in this gene region and CD was studied in 69 Finnish families. In the multipoint linkage analysis, the highest non-pararametric linkage score (NPL) was 1.75 (P=0.04) for D2S116, suggesting weak linkage for this candidate locus. To evaluate this finding, an additional 31 families were typed for all markers. In the combined set of 100 families the NPL score for marker D2S116 was 2.55 (P=0.006) and for other markers 1.90-2.47 (P=0.029-0.007), supporting genuine linkage at this region. Significantly, locus D2S116 also showed a clear allelic association in these 100 families (P=0.0001). The transmission/disequilibrium test (TDT) for locus D2S116 gave preliminary evidence for preferential maternal non-transmission of allele *136 to patients (TDTmax=8.3; P<0.05). No paternal deviation was found suggesting that the effect of the locus might be mediated by a sex-dependent factor protective against CD. Our results indicate that the CD28/CTLA4 gene region can contain a novel susceptibility locus for CD and support the hypothesis that CD has an immune system-mediated component. Like the HLA, the CD28/CTLA4 genes appear to be associated with genetic susceptibility to various autoimmune diseases.