The HLA class I A locus affects susceptibility to type 1 diabetes

Human leukocyte antigen A (HLA-A) genotypes were determined for samples from 283 multiplex, Caucasian, type 1 diabetes families from the Human Biological Data Interchange (HBDI) using an immobilized probe assay. Distribution of HLA-A alleles transmitted to patients was significantly different from that in affected family-based controls (AFBAC) (p = 0.004). Transmission disequilibrium test (TDT) analysis revealed differential transmission of several HLA-A alleles from parents to affected offspring. HLA class II DRB1 and DQB1 loci were also typed, allowing assignment of HLA-A alleles to haplotypes and calculation of linkage disequilibrium values. Some of the apparent effects of HLA-A alleles on type 1 diabetes susceptibility were attributable to linkage disequilibrium with DR and DQ alleles, although others were not. The differences in frequencies between patients and controls of alleles A*0101, A*2402, and A*3002 could not be explained by linkage disequilibrium alone. Our results suggest an important role for class I antigens in modulating susceptibility to type 1 diabetes.     

Preferential transmission of maternal allele with DQA1*0301-DQB1*0302 haplotype to affected offspring in families with type 1 diabetes

To approach the possible involvement of an epigenetic mechanism in the pathogenesis of type 1 diabetes, we investigate here a parent-of-origin effect in transmission of the susceptible alleles at HLA-DQ loci by the trans-mission disequilibrium test. When we examined alleles of affected offspring of Japanese origin in 28 nuclear families, the maternal alleles were significantly different from the paternal alleles. Furthermore, the maternal alleles with the susceptible DQA1^*0301-DQB1^*0302 haplotype showed strong transmission disequilibrium with antiglutamic acid decarboxylase antibody-positive type 1 diabetes, while the paternal alleles with the same haplotype did not. This differential transmission disequilibrium of the susceptible allele was confirmed by the contingency table analysis for transmitted or nontransmitted alleles of both parental origin. The unique transmission of the susceptible allele observed supports the hypothesis that an epigenetic mechanism including genomic imprinting at the HLA-DQ region is involved in the pathogenesis and the genetic complexity of type 1 diabetes. Received: April 5, 1999 / Accepted: May 29, 1999     

Heterogeneity of vitamin D receptor gene association with celiac disease and type 1 diabetes mellitus

OBJECTIVE: Vitamin D has been shown to exert multiple immunomodulatory effects and is known to suppress T-cell activation by binding to the vitamin D receptor (VDR). To determine whether VDR gene polymorphisms are related to the susceptibility to celiac disease, we investigated its implication as a candidate gene in the Basque population. Because celiac disease and type 1 diabetes share common susceptibility loci, we also analyzed families with type 1 diabetes mellitus. METHODS: A total of 37 families with celiac disease and 64 type 1 diabetic families of Basque origin with at least one affected offspring were genotyped for four VDR restriction-site polymorphisms (Fok I, Bsm I, Apa I and Taq I). The AFBAC approach was used to test for association. RESULTS: Comparison of VDR genotypes of the patients with those of 88 healthy individuals identified "ff" as a risk genotype for celiac disease [p = 0.01; OR = 3.45 (1.12-10.79)]. On the other hand, a significantly higher frequency of haplotype "fBAt" was observed in the type 1 diabetic group [p(c) = 0.02; OR = 4.4 (1.5-15.3)]. CONCLUSION: Our findings suggest that polymorphisms within the vitamin D receptor gene are markers of susceptibility to or protection from autoimmune diseases, although, at least in the Basque population, association of VDR variants with celiac disease and type 1 diabetes seems to be heterogeneous.     

Evidence by allelic association-dependent methods for a type 1 diabetes polygene (IDDM6) on chromosome 18q21

Type 1 diabetes is a common polygenic disease. Fine mapping of polygenes by affected sibpair linkage analysis is not practical and allelic association or linkage disequilibrium mapping will have to be employed to attempt to detect founder chromosomes. Given prior evidence of linkage of the Jk-D18S64 region of chromosome 18q12-q21 to type 1 diabetes, we evaluated the 12 informative microsatellite markers in the region for linkage with disease by the transmission disequilibrium test (TDT) in a UK data set of type 1 diabetic families (n = 195). Increased transmission of allele 4 of marker D18S487 to affected children was detected (P = 0.02). Support for this was extended in a total of 1067 families from four different countries by isolating, and evaluating by the TDT, two novel microsatellites within 70 kb of D18S487. Evidence for linkage and association was P = 5 x 10(-5) and 3 x 10(-4), respectively. There was no evidence for increased transmission of associated alleles to nonaffected siblings. Analysis of an additional 390 families by the TDT did not extend the evidence further, and reduced support in the total 1457 families to P = 0.001 for linkage and P = 0.003 for association. However, evidence for linkage by affected sibpair allele sharing was strong (P = 3.2 x 10(-5)) in the second data set. Heterogeneity in TDT results between data sets was, in part, accounted for by the presence of more than one common disease- associated haplotype (allelic heterogeneity) which confounds the analysis of individual alleles by the TDT. Guidelines for strategies for the mapping of polygenes are suggested with the emphasis on collections of large numbers of families from multiple populations that should be as genetically homogeneous as possible.      

Insulin-like growth factor 1 promoter polymorphism influences insulin gene variable number of tandem repeat-associated risk for juvenile onset type 1 diabetes

Insulin-like growth factor 1 (IGF1) plays an important role in the development and function of pancreatic beta-cells and contributes to infant growth, which we recently reported to be associated with type 1 diabetes (T1D). Here, we studied an IGF1 microsatellite in 206 families with T1D and its interaction with the polymorphism near the insulin (INS) gene variable number of tandem repeats. The IGF1 microsatellite was associated with T1D (P = 0.045), which was mainly caused by a protective effect of the 194 bp allele (36% transmission to affected offspring). Interestingly, co-segregation of this IGF1 194 bp allele affected the risk of INS alleles. These results provide the first evidence for an association of IGF1 with T1D and imply that co-inheritance of these functional genetic variants of IGF1 and insulin predispose to T1D.     

Transmission of haplotypes of microsatellite markers rather than single marker alleles in the mapping of a putative type 1 diabetes susceptibility gene (IDDM6)

Allelic association methods based on increased transmission of marker alleles will have to be employed for the mapping of complex disease susceptibility genes. However, because the extent of association of single marker alleles with disease is a function of the relative frequency of the allele on disease-associated chromosomes versus non disease-predisposing chromosomes, the most associated marker allele in a region will not necessarily be closest to the disease locus. To overcome this problem we describe a haplotype-based approach developed for mapping of the putative type 1 diabetes susceptibility gene IDDM6. Ten microsatellite markers spanning a 550 kb segment of chromosome 18q21 in the putative IDDM6 region were genotyped in 1708 type 1 diabetic Caucasian families from seven countries. The most likely ancestral diabetogenic chromosome was reconstructed in a stepwise fashion by analysing linkage disequilibrium between a previously defined haplotype of three adjacent markers and the next marker along the chromosome. A plot of transmission from heterozygous parents to affected offspring of single marker alleles present on the ancestral chromosome versus the physical distance between them, was compared with a plot of transmission of haplotypes of groups of three adjacent markers. Analysing transmission of haplotypes largely negated apparent decreases in transmission of single marker alleles. Peak support for association of the D18S487 region with IDDM6 is P = 0.0002 (corrected P = 0.01). The results also demonstrate the utility of polymorphic microsatellite markers to trace and delineate extended and presumably ancient haplotypes in the analysis of common disease and in the search for identical-by-descent chromosome regions that carry an aetiological variant.      

The TAF5L gene on chromosome 1q42 is associated with type 1 diabetes in Russian affected patients

Type 1 diabetes (T1D) is a multifactorial autoimmune disease, with strong genetic component. Several susceptibility loci contribute to genetic predisposition to T1D. One of these loci have been mapped to chromosome 1q42 in UK and US joined affected family data sets but needs to be replicated in other populations. In this study, we evaluated sixteen microsatellites located on 1q42 for linkage with T1D in 97 Russian affected sibling pairs. A 2.7-cm region of suggestive linkage to T1D between markers D1S1644 and D1S225 was found by multipoint linkage analysis. The peak of linkage was shown for D1S2847 (P = 0.0005). Transmission disequilibrium test showed significant undertransmission of the 156-bp allele of D1S2847 from parents to diabetic children (28 transmissions vs. 68 nontransmissions, P = 0.043) in Russian affected families. A preferential transmission from parents to diabetic offspring was also shown for the T(-25) and T1362 alleles of the C/T(-25) and C/T1362 dimorphisms, both located at the TAF5L gene, which is situated 103 kb from D1S2847. Together with the A/C744 TAF5L SNP, these markers share common T(-25)/A744/T1362 and C(-25)/C744/T1362 haplotypes associated with higher and lower risk of diabetes (Odds Ratio = 2.15 and 0.62, respectively). Our results suggest that the TAF5L gene, encoding TAF5L-like RNA polymerase II p300/CBP associated factor (PCAF)-associated factor, could represent the susceptibility gene for T1D on chromosome 1q42 in Russian affected patients.     

CTLA-4/CD 28 region polymorphisms in children from families with autoimmune hepatitis

Susceptibility to autoimmune hepatitis is associated with particular human leucocyte antigen class II alleles. However, non-HLA genetic factors are likely to be required for development of the disease. Among the candidate genes, the cytotoxic T-lymphocyte antigen 4 (CTLA-4) and CD28 genes, located on chromosome 2q33 in humans, encode a cell surface molecule playing a dominant role in the regulation of T-cell activation. The CTLA-4 and CD28 polymorphisms were investigated in children from 32 families with autoimmune hepatitis (AIH). The transmission/disequilibrium test revealed increased transmission of the (AT)8 (dinucleotide repeat) and A (exon 1) alleles of CTLA-4 gene from heterozygous parents to affected offspring (87.5% and 83.5%) with type 1 AIH, compared with unaffected offspring (50.0% for both, p = 0.009 and 0.02, respectively). In contrast, no deviation in transmission for CTLA-4 polymorphisms was found between type 2 AIH patients and unaffected offspring. No evidence for association was found between CD28 gene polymorphism or D2S72 genetic marker and both types of AIH. This study identified the CTLA-4 gene polymorphism as a non-HLA determinant that predisposes to AIH type 1 in children. The genetic heterogeneity seen in the present study provides a new argument in favor of pathogenic differences between type 1 and type 2 AIH.     

IDDM12 (CTLA4) on 2q33 and IDDM13 on 2q34 in genetic susceptibility to type 1 diabetes (insulin-dependent)

Type 1 diabetes (insulin-dependent) is a multifactorial disease with polygenic susceptibility. The major genetic component (IDDM1) resides within the HLA region, but several non-HLA loci have been implicated in the genetic susceptibility. In the present study, we have analysed two such loci, IDDM12 (CTLA4) on 2q33 and IDDM13 on 2q34, in Danish (n = 254) and Spanish (n = 39) type 1 diabetic multiplex families. No significant evidence of linkage of IDDM12 was observed in any of the two studied data sets. However, when the present data were combined with previously published data, they strengthened the evidence of linkage at this locus, p = 0.00002. For the IDDM13 region, we found some positive evidence of linkage of the D2S137-D2S164-D2S1471 markers (p-values 0.007, 0.02, and 0.007, respectively) using transmission disequilibrium testing (TDT) and the Tsp version of the TDT. Importantly, random transmission of all tested alleles was observed in unaffected offspring (p > 0.3). Stratification for HLA (high risk and non-high risk genotypes) in the Danish families did not reveal heterogeneity at IDDM12 or IDDM13. In conclusion, our data on an entirely new family data set did not support the existence of IDDM12 as a type 1 diabetes susceptibility locus in the Danish population. In addition, we found support for evidence of linkage and association of the IDDM13/D2S137-D2S1471 region (approximately 3.5 cM) to type 1 diabetes, however, further studies are needed to substantiate this observation.     

The IL12B 3' untranslated region DNA polymorphism is not associated with early-onset type 1 diabetes

A recent study employing Australian and UK type 1 diabetes families has demonstrated significant transmission bias to affected offspring of a polymorphism (1188A allele; termed allele 1) in the 3' untranslated region (3'UTR) of the interleukin 12B (IL12B) gene which encodes the IL-12p40 subunit of the pro-inflammatory cytokine IL-12. However, results from replication studies in other populations have been controversial. We performed both case-control (n=120 cases; n=330 controls) and family-based (n=307 families) association studies, using the transmission disequilibrium test, to investigate if allele 1 is associated with early-onset type 1 diabetes in Northern Ireland. No association was observed between allele 1 and type 1 diabetes in either case-control (80.8% vs 80.8%; P=0.98) or family-based (49.7% transmissions; P=0.94) studies. Our results do not support earlier reports of an association between allele 1 in the 3'UTR of the IL12B gene and type 1 diabetes.     

Genetic variants of RANTES are associated with serum RANTES level and protection for type 1 diabetes

RANTES (regulated on activation, normal T-cell expressed and secreted) is a T-helper type 1 (Th1) chemokine that promotes T-cell activation and proliferation. RANTES is genetically associated with asthma, sarcoidosis and multiple sclerosis. The concentration of RANTES is increased at inflammation sites in different autoimmune diseases. Type 1 diabetes (T1D) is a Th1-mediated disease with complex genetic predisposition. We tested RANTES as a candidate gene for association with T1D using three single-nucleotide polymorphism (SNP) variants (rs4251719, rs2306630 and rs2107538) to capture haplotype information. The minor alleles of all SNPs were transmitted less frequently to T1D offspring (transmission rates 37.3% (P=0.002), 38.7% (P=0.007) and 41.0% (P=0.01)) and were less frequently present in patients compared to controls (P=0.009, 0.03 and 0.04, respectively). A similar protective effect was observed for the haplotype carrying three minor alleles (transmission disequilibrium test (TDT): P=0.003; odds ratio (OR)=0.55; confidence interval (CI): 0.37-0.83; case/control: P=0.03; OR=0.74; CI: 0.55-0.98). Both patients and controls carrying the protective haplotype express significantly lower serum levels of RANTES compared to non-carriers. Subsequently, we tested a cohort of 310 celiac disease patients, but failed to detect association. RANTES SNPs are significantly associated with RANTES serum concentration and development of T1D. The rs4251719*A-rs2306630*A-rs2107538*A haplotype associated with low RANTES production confers protection from T1D. Our data imply that RANTES is associated with T1D both genetically and functionally, and contributes to diabetes-prone Th1 cytokine profile.     

No linkage of P187S polymorphism in NAD(P)H: Quinone oxidoreductase (NQO1/DIA4) and type 1 diabetes in the Danish population

Recent genome screening studies have identified novel regions of possible interest for susceptibility to type 1 diabetes. One of these is a 30‐35 cM region mapping to 16q22‐q24 (D16S515‐D16S520), where also the gene encoding NAD(P)H: quinone oxidoreductase (NQO1) maps. Data has suggested association of a polymorphism (P187S) in the NQO1 gene and type 1 diabetes. NQO1 is involved in protection against oxidative stress, which is likely to be involved in β‐cell destruction. By use of the transmission disequilibrium test (TDT), we analyzed the P187S polymorphism for association to type 1 diabetes in a population‐based sample of 247 Danish nuclear type 1 diabetic families. Random transmission patterns were observed to all affected offspring (p_tdt = 0.82), to index cases (p_tdt = 0.77), as well as to unaffected offspring (p_tdt = 0.93). Hence, the NQO1 polymorphism is not likely to be an etiological mutation underlying the reported linkage of the 16q22‐q24 region. Hum Mutat 14:67–70, 1999. © 1999 Wiley‐Liss, Inc.     

A functional variant of IRS1 is associated with type 1 diabetes in families from the US and UK

A collection of 767 multiplex type 1 diabetes families from the US and UK were tested for linkage to the IRS1 gene and for allelic association with a specific variant of IRS1, G972R. Pedigree disequilibrium testing revealed preferential transmission of the 972R allele to affected offspring in these families (P = 0.02). Linkage analyses conditioning on status at IRS1 position 972 suggest the possibility of interaction with an unidentified locus on chromosome 8.     

Intercellular adhesion molecule-1 (ICAM-1) K469E polymorphism: no association with type 1 diabetes among Finns

The intercellular adhesion molecule-1 (ICAM-1) has an important role in the process of lymphocyte migration and activation, and is supposed to be involved in the pathogenesis of type 1 diabetes. We studied A/G (K469E) polymorphism of the ICAM-1 gene in 218 type 1 diabetes patients and 212 controls from Finland and found no association. We then studied transmission of the ICAM-1 alleles in 102 Finnish families using a transmission disequilibrium test (TDT). Alleles A and G were transmitted to the affected offspring in 50% each. Stratification by the HLA-DQB1-DQA1 genotypes, sex and age at onset did not reveal association. Our data demonstrate that in the Finnish population K469E polymorphism of the ICAM-1 gene is not associated with type 1 diabetes.     

Evidence for a type 1 diabetes susceptibility locus (IDDM10) on human chromosome 10p11-q11

A region of linkage to type 1 diabetes has been defined on human chromosome 10p11-q11 (IDDM10; P = 0.0007) using 236 UK and 76 US affected sibpairs and a 1 cM resolution microsatellite marker map. Analysis by the transmission disequilibrium test (TDT) in 1159 families with at least one diabetic child, from the UK, the US, Norway, Sardinia and Italy provided additional support for linkage at D10S193 (P = 0.006, Pc = 0.17). Notably, 5.1 cM distal to D10S193, marker D10S588 also provided positive TDT results (P = 0.009, Pc = 0.25) but the allele under analysis was also preferentially transmitted to nonaffected siblings (P = 0.0008, Pc = 0.02). This allele was positively associated in an independent UK case control study and, importantly, was neutrally transmitted in control CEPH families. These results suggest a type 1 diabetes susceptibility locus on chromosome 10p11-q11 (provisionally designated IDDM10) and demonstrate the necessity of analysis of non affected siblings in disease families, as well as analysis of control families.      

DAT1, DRD4, and DRD5 polymorphisms are not associated with ADHD in Dutch families.

Recent meta-analyses have indicated that the dopamine transporter gene (DAT1) and the dopamine receptor genes D4 (DRD4) and D5 (DRD5) are associated with attention-deficit hyperactivity disorder (ADHD), although single studies frequently failed to show significant association. In a family-based sample of 236 Dutch children with ADHD, we have investigated the previously described variable number of tandem repeat (VNTR) polymorphisms and two additional microsatellites at the DAT1 and DRD4 loci. DRD5 was investigated using the microsatellite that was previously found to be associated. Transmission disequilibrium tests (TDTs) did not show preferential transmission of alleles or two-marker haplotypes to affected offspring. These data suggest that DAT1, DRD4, and DRD5 do not contribute substantially to ADHD in the Dutch population.     

Functional genetic polymorphisms in cytokines and metabolic genes as additional genetic markers for susceptibility to develop type 1 diabetes

Genetic association with type 1 diabetes (T1D) has been established for two chromosomal regions: HLA DQ/DR (IDDM1) and INS VNTR (IDDM2). To identify additional genetic markers, we tested polymorphisms in regulatory regions of several cytokine and important metabolic genes. These polymorphisms exhibit functional consequences for expression and function. Functional genetic polymorphisms of proinflammatory (T-helper-1: IL-2, IL-12 and IFN-gamma), anti-inflammatory (T-helper-2: IL-4, IL-6 and IL-10) and metabolic (IGF-I, VDR and INS) genes were determined in 206 Dutch simplex families with juvenile onset T1D and the results were analysed using the transmission disequilibrium test. Significantly increased transmission to T1D probands was observed for the loci IDDM1, IDDM2 and the vitamin D receptor. Although none of the other individual polymorphisms was associated with disease individually, the combination of T-helper-2 and metabolic/growth alleles IL-10(*)R2, IL-4(*)C, VDR(*)C and IGF-I(*)wt was found to be transmitted more frequently than expected (67%, P(c)=0.015). We conclude that additional genetic predisposition to T1D is defined by combinations of markers (eg Th2 and metabolic) rather than by a single marker. The consequences of the increased transmission of a low Th2 expressing genotypes together with a normal Th1 profile may result in a net proinflammatory cytokine expression pattern.     

A linkage analysis of the CTLA4 gene region in Finnish patients with type 1 diabetes.

The cytotoxic T-lymphocyte antigen 4 (CTLA4) region on 2q33 has been shown to be linked to, and associated with, type 1 diabetes (T1D) and suggested to be one of the loci contributing to diabetes aetiology. The polymorphisms responsible for the effect are yet not defined, and the findings reported for the known markers have been discrepant in various populations. We analysed 15 markers around the CTLA4 gene in 138 Finnish affected sib-pair families. A maximum multipoint LOD score (MMLS) of 0.83 at the CTLA4-(AT)n microsatellite was obtained for the whole data set. When stratified, the MMLS increased to 2.61 in the IBS2 (identical by state 2) dataset. In a transmission/disequilibrium test (TDT), some sex-specific effects were observed in transmissions of alleles of CTLA4-(AT)n and D2S105 in siblings. The transmission of the CTLA4 +49 A/G single nucleotide polymorphism (SNP) did not deviate from the expected frequency in this study. In conclusion, our study confirms the linkage of the CTLA4 region to T1D in the Finnish population. In addition, the observations suggest that the polymorphism actually involved in the disease is not the CTLA4 +49 A/G SNP but a polymorphism in linkage disequilibrium with CTLA4 markers and probably closer to CTLA4-(AT)n than to the CTLA4 +49 A/G SNP.     

An extended transmission/disequilibrium test (TDT) for multi-allele marker loci

The transmission/disequilibrium test (TDT) was recently introduced by Spielman et al. (1993) as a test for linkage and linkage disequilibrium. The test is based on the unequal probability of transmission of two different marker alleles from parents to affected offspring, when the marker locus and the hypothetical disease locus are linked and are in linkage disequilibrium. The probabilities of marker allele transmission to affected offspring conditional on parental genotype have been derived by Ott (1989) for a biallelic marker and a recessive disorder with no phenocopies. Here, we derive the transmission probabilities for a multi-allele marker locus and a generalized single locus disease model in a random sample of affected individuals from a randomly mating population. The form of these transmission probabilities suggests an extension of the TDT to multi-allele marker loci, in which the alternative hypothesis is restricted to take account of the likely pattern of unequal transmission when the recombination fraction is near 0. We show how our extended TDT can be implemented by standard software for logistic regression, although we have also written our own program which is available on request. We have evaluated the approximate power of the test under a range of realistic assumptions, and it appears that the test will often have good power when linkage disequilibrium is strong and if the disease is recessive.     

MICA is associated with type 1 diabetes in the Belgian population, independent of HLA-DQ

To ascertain association of MICA with type 1 diabetes (T1D) in the Belgian population, well-characterized antibody-positive patients were analyzed for MICA transmembrane gene polymorphism in both an association study and a nuclear family study. The frequency of MICA5 was significantly increased in the T1D patient group (18%) compared with the control population (12%, OR=1.6, pc<10(-3)), whereas MICA9 was decreased (11% versus 16%, OR=0.7, pc<0.01). A p value<10(-3) for the association of MICA conditional on HLA class II and p=0.01 for the conditional extended transmission disequilibrium test were obtained, indicating that MICA is associated with type 1 diabetes, independent of HLA-DQ. Analysis of estimated extended HLA-DQ-MICA haplotypes revealed individual effects of MICA alleles. The most significant effect was seen for MICA5 on the HLA-DQA1*03-DQB1*0302-MICA haplotype (OR=2.5, p<10(-3)). A significant protective effect was seen for the combination of DQA1*01-DQB1*0602/3 and MICA5.1 (OR=0.3, p<10(-3)). However, patients stratified according to the presence or absence of the different MICA alleles did not differ in terms of age at onset, sex, or other diabetes-related clinical and epidemiological data. In conclusion, MICA is associated with type 1 diabetes in the Belgian population and the observed association does not result from the HLA-DQ associated risk.