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Am J Hum Genet. Dec 2001; 69(6): 1301–1313.
Published online Oct 11, 2001.
PMCID: PMC1235542

A Genomewide Scan for Type 1–Diabetes Susceptibility in Scandinavian Families: Identification of New Loci with Evidence of Interactions

European Consortium for IDDM Genome Studies1

Abstract

Type 1 diabetes mellitus (TIDM) has a multifactorial etiology, with major genetic-susceptibility determinants located in the HLA and insulin-gene (INS) regions. Linkage data implicating other disease-susceptibility loci are conflicting. This is likely due to (1) the limited power for detection of contributions of additional susceptibility loci, given the limited number of informative families available for study, (2) factors such as genetic heterogeneity between populations, and (3) potential gene-gene and gene-environment interactions. To circumvent some of these problems, we have conducted a genomewide linkage analysis for T1DM-susceptibility loci in 408 multiplex families from Scandinavia, a population expected to be homogeneous for genetic and environmental factors. In addition to verifying the HLA and INS susceptibility loci, the study provides confirmation of IDDM15 on chromosome 6q21. Suggestive evidence of additional susceptibility loci was found on chromosomes 2p, 5q, and 16p. For some loci, the support for linkage increased substantially when families were stratified on the basis of HLA or INS genotypes, with statistically significant heterogeneity between the stratified subgroups. Our data support both the existence of non-HLA genes of significance for T1DM and interaction between HLA and non-HLA loci in the determination of the T1DM phenotype.

Introduction

Type 1 diabetes mellitus (T1DM [MIM 222100]) develops in susceptible individuals because of a gradual autoimmune destruction of the insulin-producing β-cells of the endocrine pancreas, with a consequent dependence on daily insulin injections for survival. Increased concordance for T1DM in MZ twins (30%–50%), compared to that in DZ twins (4.8%–27%) and in siblings (4.4%–12.5%), suggests that susceptibility is determined partly by genetic factors (Degnbøl and Green 1978; Tillil and Köbberling 1987; Olmos et al. 1988; Kaprio et al. 1992; Kumar et al. 1993; Lorenzen et al. 1994; Kyvik et al. 1995; Wagner et al. 1995; Davis 1996). This is also supported by familial clustering, as illustrated by an estimated recurrence risk in siblings (λS) of 15 (Risch 1987).

Monogenic or oligogenic forms of T1DM have been described, and genetic studies in families affected with it have allowed mapping of the corresponding genes to chromosomes 10q25 and 2p12 (Verge et al. 1998; Delépine et al. 2000). In the chromosome 2 region, disease-causing mutations in the gene for eukaryotic translation-initiation factor–2 alpha kinase–3 gene (EIF2AK3) have been identified in patients with Wolcott-Rallison syndrome (neonatal insulin-dependent diabetes and epiphyseal dysplasia) (Delépine et al. 2000).

The major histocompatibility complex (MHC) on chromosome 6p21.3 is a major susceptibility locus (referred to as “IDDM1”) for the common multifactorial from of T1DM (Nerup et al. 1974; Pociot 1996), estimated to account for 40%–50% of λS (Risch 1987). The insulin-gene locus (IDDM2 [MIM 125852]) has also been shown to contribute to disease susceptibility (Bell et al. 1984; Julier et al. 1991). In addition, environmental factors are also important in T1DM, as illustrated by <100% concordance in MZ twins and by the increase in incidence, over time, in certain age groups, particularly in the Scandinavian population (Diabetes Epidemiology Research International Group 1990; Tuomiletho et al. 1999; Dahlquist et al. 2000).

During the past decade, a large number of genetic studies have been devoted to the search for additional T1DM-susceptibility loci, either by linkage or by association with candidate genes. The major findings of these studies are summarized in table 1.

Table 1
Summary of Previous Linkage Results and Exclusion Data from Present Study

Both the relatively minor risk contribution of each of the non-HLA loci and the possible genetic and/or environmental heterogeneity within and between studies are likely to contribute to difficulties in replication of some of these linkage results. Many studies have had low power because of small sample sizes. In particular, data stratification based on HLA genotype or other criteria, which may be necessary for detection of the effects of some of the susceptibility loci, further increases the need for large sample sizes. In this context, it is probable that identification of T1DM-susceptibility loci will require the combination of data from several large linkage studies of multiple populations. Many of the previous investigations have been performed in partially overlapping groups of patients (Davies et al. 1994; Hashimoto et al. 1994; Concannon et al. 1998; Mein et al. 1998). Consequently, the aim of this investigation was to perform a genomewide search for T1DM-susceptibility genes in an independent collection of patients, consisting of 424 families (table 2) from Denmark, Norway, and Sweden—a relatively homogeneous population, in terms of its ethnic background and environmental exposures.

Table 2
Families of Patients Included in Genomewide Linkage Analysis

Subjects and Methods

Patients and Families

DNA from families with at least two affected individuals other than parent-child pairs was collected in Denmark, Norway, and Sweden (table 2). Individuals were considered affected (1) if they had been diagnosed with diabetes at age <15 years, required insulin treatment at the time of onset, and had remained on insulin treatment subsequently or (2) if diabetes with either ketoacidosis or requirement of insulin therapy from time of onset had been diagnosed at age >15 years. The median age at onset was 13 years (range 0–71 years). It previously had been shown that these criteria are consistent with the diagnosis of T1DM (Mølbak et al. 1994). To exclude families with maturity-onset diabetes of the young (MODY), the Danish families with diabetes reported in three consecutive generations were screened for MODY mutations. The Swedish families were identified through the two nationwide incidence registries: the Swedish Childhood Registry (Dahlquist et al. 1982, 1985; Nyström et al. 1990; Dahlquist and Mustonen 1994) and the Diabetes Incidence Study in Sweden (Östman et al. 1986; Blohmé et al. 1992). The Danish families were identified through the Danish Study Group of Diabetes in Childhood (Pociot et al. 1993), and the Danish IDDM Epidemiology and Genetics Group (Lorenzen et al. 1998). The Norwegian families were recruited during 1993–95, through advertisements, in the journal of the Norwegian Diabetes Association, for families with both (a) two sibs with T1DM and (b) parents without the disease, and the recruitment was followed by phone calls to include all family members. In most cases, blood samples were obtained, by mail, from local physicians. The diabetic siblings were asked to complete a form to confirm T1DM; in cases in which these sibs were uncertain of their T1DM status, their physicians were contacted. During 1996–97, recruitment was extended by letters to diabetes nurses (n=480) at all pediatric and internal medicine departments in Norway. In all, 408 multiplex families from Scandinavia, comprising 464 affected full-sib pairs, were studied (table 2); when available, nonaffected siblings (216 nonaffected sib pairs) were also included in the scan.

Complementary Families: French and U.S. Families

The French- and U.S.-family panels are identical to those described elsewhere (Delépine et al. 1997). In all, 126 families with 158 affected sib pairs were available from France, and 255 families with 310 affected sib pairs were available from the United States. The latter were obtained principally from the HBDI family collection.

DNA Extraction

DNA was extracted by standard procedures, by either phenol/chloroform extraction or a salting-out method (Miller et al. 1988).

Microsatellite Genotyping

The genomewide linkage study was undertaken by use of the Medical Research Council (MRC) microsatellite panel (Reed et al. 1997). Markers that did not pass our quality-control procedures (see below) were replaced by new markers selected from the Généthon map. Additional markers were added both to regions that are sparsely covered by the MRC panel and in three regions of specific interest: HLA, on 6p21; IDDM15 (MIM 601666), on 6q21; and the EIF2AK3 locus, on 2p12.

Microsatellite markers were characterized by PCR amplification with fluorescence-labeled primers on PTC-225 thermocyclers (MJ Research). PCR was performed in a final volume of 15 μl (Buffer 1× [ Perkin-Elmer], 0.2 mM dNTP, 1.5–3.0 mM MgCl2, 0.4 U Taq Gold, 0.2 mM of each primer, and 5–15 ng DNA). The samples were amplified for 35 cycles, each consisting of 30 s at 94 C, 30 s at the primer-specific annealing temperature (45°C–61°C), and 30 s at 72°C. Initial denaturation was performed at 94°C for 10 min, and a final elongation step was performed at 72°C for 1 min. PCR products from 10–21 different primer pairs were pooled prior to separation on either an ABI373 or an ABI377 (Applied Biosystems), with either 12-cm or 36-cm well-to-read, denaturing 6% polyacrylamide gels, followed by analysis using either version 1.2 or version 3.1 of GENESCAN (Applied Biosystems). Single-strand size markers GS-350 or GS-500 (Applied Biosystems) were used. The allele assignments were made by version 2.0 of GENOTYPER (Applied Biosystems). Each gel contained an internal DNA control (individual 2 in CEPH family 1347; Coriell Institute), which was used for calibration of the allele sizes. In all, 324 markers were genotyped and included in the scan before quality control was performed.

Family structures were reassessed and corrected on the basis of the genomewide-scan data, by the SIBERROR program, with markers separated by >30 cM (Ehm and Wagner 1998). In particular, half-sibs and MZ twins were identified, and one member in each MZ pair was excluded from further analysis.

The following quality-control tests were routinely performed for each marker (and genotypes were recharacterized or removed as necessary): heterogeneity, in allele frequencies, between gels (possibly due to differences in allele sizing); Mendelian consistency, by the PEDCHECK program (O'Connell and Weeks 1998); and excess of homozygosity (generally reflecting either missed amplification of some alleles or, in heterozygotes, preferential amplification of one allele). The observed number of homozygotes was compared with the number expected on the basis of the observed allele frequencies, by a χ2 test. A cutoff of P<.001 was used. Special care was also given to ensure consistency in allele sizing across all the families. These tests led to the exclusion of six microsatellite markers, which displayed excess of homozygosity that could not be corrected by reinterpretation of the gel data. Finally, we obtained consistent and corrected genotypes for a total of 318 microsatellite markers, which were used for analysis. The average success rate for genotypes after the quality-control tests were performed was 84.9%. The average heterozygosity of the markers was .79 (range .56–.91).

HLA and INS Genotyping

The HLA-DRB1 (MIM 142857) genotype was determined by allele-specific PCR for the DR3 and DR4 alleles (Olerup and Zetterquist 1992, 1993). To determine the susceptibility status at the INS (IDDM2) locus, the INS polymorphism INS-23/HphI was genotyped (Lucassen et al. 1993).

Genetic Map

The genetic map was assessed by both the CRIMAP program and the ASPEX program (see the The ASPEX Package: Affected Sib Pair Exclusion Mapping web site) (Lander and Green 1987; Hauser et al. 1996), which gave results that, in terms of marker order and distances, were similar to those for the publicly available linkage maps (e.g., that of the Center for Medical Genetics, Marshfield Medical Research Foundation). To avoid possible distortions of the map in families with diabetes due to adjacent susceptibility loci, we used the Marshfield map distances in the multilocus linkage analyses, with complementary estimates from other publicly available linkage maps, extrapolations from physical maps, and estimates from the present T1DM-Scandinavian map, where necessary. The average spacing between the 318 markers was 11.3 cM. In all, 23 intermarker distances were >20 cM.

Linkage Analysis

Linkage was assessed by nonparametric linkage methods. First, the data were scanned by programs including only nuclear families. Single-locus linkage analysis was performed by the ANALYZE package. Multilocus linkage analysis and exclusion mapping were performed by the SIBIBD program from ASPEX (see the The ASPEX Package: Affected Sib Pair Exclusion Mapping web site) (provided by D. Hinds and N. Risch), under a multiplicative model, as described elsewhere (Risch et al. 1999). Allele frequencies in all family members were estimated by the DOWNFREQ program. Additional single-locus and multilocus linkage analyses were performed by ALLEGRO (Gudbjartsson et al. 2000), with similar results. For chromosome X, MAPMAKER/SIBS (Kruglyak and Lander 1995) was also used for linkage calculations, as suggested by Nyholt (2000), resulting in similar results. Loci reaching a LOD score [gt-or-equal, slanted]2.0 in either the single-locus analysis or the multilocus analysis were further investigated by single-locus and multilocus linkage analyses by ALLEGRO, which allowed the inclusion of all affected family members (Gudbjartsson et al. 2000). A linear model with equal weighting of affected relative pairs was used. No correction was made for number of affected individuals in each family. Locus-specific λS was estimated as described by Risch (1987).

Transmission/Disequilibrium Test (TDT) Analysis

TDT analysis was performed by the ANALYZE program, with all affected siblings in each family (Terwilliger 1995).

Linkage Analysis for IDDM15

For IDDM15 on chromosome 6q, analysis was performed, as described elsewhere (Delépine et al. 1997), to correct for linkage to the HLA region. The analysis was restricted to families in which the parental origin of HLA and IDDM15 haplotypes could be determined. HLA haplotypes were determined on the basis of HLA-DRB1 and the nearby microsatellite markers D6S276, D6S273, and TNFA. Marker D6S283 was chosen as the marker locus for the IDDM15 locus (Delépine et al. 1997).

Stratification Based on Population of Origin, HLA, and INS

Secondary statistical analyses were performed, with the data subdivided by either population of origin, HLA type, or INS genotype. For stratification based on HLA type, families were divided into four independent groups: (1) DR3/4, comprising families in which all the affected siblings were HLA DR3/4 (132 families and 146 affected sib pairs); (2) DR4, comprising families in which all affected siblings carried HLA-DR4, excluding families in category 1 (164 families and 206 affected sib pairs); (3) DR3, comprising families in which all affected siblings carried HLA-DR3, excluding those in category 1 (61 families and 69 affected sib pairs); and (4) other, comprising families in which all affected siblings are neither DR3 nor DR4 (36 families and 53 affected sib pairs).

For stratification based on INS, positive susceptibility (“+”) was defined in terms of homozygosity (+/+) and nonsusceptibility (“−”) was defined in terms of other genotypes (+/− and −/−), where “+” denotes the T1DM-associated allele at the INS-23/HphI site, which is in strong linkage disequilibrium with the small-size class at the INS-5′VNTR polymorphism (Julier et al. 1994). Families were subdivided into three independent groups: (1) INS+, comprising families in which all affected sibs carried the +/+ genotype (239 families and 279 affected sib pairs); (2) INS−, comprising families in which all affected siblings carried the −/− genotype (56 families and 60 affected sib pairs); and (3) INS+/INS−, comprising families in which affected siblings were discordant, or +/− (98 families and 135 affected sib pairs). Results from the secondary statistical analyses are reported only when the results of tests for differences in linkage are significant at P<.05 (heterogeneity test).

Results

Primary Linkage Analysis

Genotypes for 318 marker loci were obtained in 408 multiplex families (464 affected sib pairs). Multilocus-LOD-score curves, plotted for the entire genome, together with the exclusion data (for effects of λS[gt-or-equal, slanted]1.5), are shown in figure 1. In addition to HLA, 14 regions were detected with a LOD score of 1.0 in multilocus analysis, 8 of which also were detected at this significance level in single-locus analyses (table 3).

Figure  1
Multipoint linkage analysis using ASPEX multipoint linkage analysis (solid line) and exclusion mapping (dashed line) using ASPEX in all affected sib pairs. Genetic distances (in Kosambi Morgans) are given along the X-axis.
Table 3
Regions with LOD Score 1.0: All Families

In multilocus analysis, a LOD score of 4.8 was observed in the IDDM15 region (markers D6S300–D6S283), corresponding to a secondary peak in the broad HLA-linked region. LOD scores >2 were also observed on chromosome 2, at D2S113 near EIF2AK3 (LOD score 2.1), on chromosome 5, near marker D5S407 (LOD score 2.2), and on chromosome 16p, in a wide region from D16S407 to D16S287 (LOD score 2.8). LOD scores of 1.0 in single- or multilocus analyses were also observed on chromosomes 4, 9, 10, 12, 13 (two regions), 19 (two regions), 21, and X.

Susceptibility loci with λS=1.5 were excluded from 61% of the genome (LOD score <−2.0). For λS=2.0, 89% of the genome was excluded. Apart from IDDM15, none of the 17 previously reported non-HLA putative susceptibility loci achieved a LOD score >0.8. Effects of λS[gt-or-equal, slanted]1.5 could be excluded at eight of the loci, effects of λS[gt-or-equal, slanted]2.0 at the other five (table 1). However, linkage in the presence of association was confirmed at the IDDM2 locus (INS) by TDT, with an excess of transmission of INS+ from heterozygous parents to diabetic children (261 vs. 164; P=10-6); there was no distortion in the transmission of INS alleles to nonaffected children (84 vs. 89).

The IDDM15 region on chromosome 6q is linked to HLA, with an estimated recombination rate (θ) of .32 in male meioses and .47 in female meioses (Delépine et al. 1997). Therefore, statistical analysis was performed to evaluate the significance of cosegregation of IDDM15 with the disease, with the contribution from HLA being taken into account. This yielded suggestive evidence of linkage in the Scandinavian families, with P=.002 at marker D6S283, the marker that, in a previous analysis (Delépine et al. 1997), had shown the strongest evidence of linkage (table 4). We then combined the data from the Scandinavian families with data from the French and U.S. multiplex families. There was no evidence, at this locus, of heterogeneity between the Scandinavian families and the French and U.S. families. Taking into account the 95% confidence interval for the estimate of the distance between HLA and D6S283, an estimate obtained from data on the 59 large reference families from the Centre d’Étude du Polymorphisme Humain (Delépine et al. 1997), we estimated that the P value associated with this statistic in the combined family set would be 3×10-5–5×10-8 (7×10-7 for the recombination rates given above).

Table 4
Results of IDDM15 Linkage Analysis, Adjusted for HLA Linkage, for Marker D6S283

Secondary Linkage Analyses

Multilocus linkage analysis was also undertaken with the data subdivided according to HLA subtype, INS susceptibility status, or population of origin (see the “Stratification Based on Population of Origin, HLA, and INS” subsection of Subjects and Methods), in regions with a LOD score >1 in the primary analysis. This allowed for testing of either heterogeneity of particular genetic effects due to interaction with specific HLA- or INS-risk haplotypes or population-of-origin heterogeneity for T1DM.

Table 5 shows the results when the test of heterogeneity between categories (HLA, INS, or population of origin) was significant at P<.05. A chromosome region near D16S405 on chromosome 16p displayed weak but significant evidence of heterogeneity in the stratification based on HLA (P=.012), with increased evidence of linkage in the DR3/4 group (LOD score 3.6; 65% IBD) (table 5). In addition, a region near marker D16S3113 on chromosome 16p demonstrates significant evidence of heterogeneity in the stratification based on INS (P=.0005), with increased evidence of linkage in the INS− group (LOD score 3.4; 71% IBD) (table 5). Weak evidence of heterogeneity in the stratification based on population of origin was found in the extended chromosome 16p region near marker D16S3131 (P=.037), with increased evidence of linkage in the Danish population (LOD score 2.8; 61% IBD) (table 5). Interestingly, the three peaks observed in the HLA-DR3/4, INS−, and Danish groups have distinct positions within the extended chromosome 16p region and cover a 30-cM region (fig. 2). In addition, the region on chromosome 2p near marker D2S113 showed increased evidence of linkage in the DR3/4 group (LOD score 2.7; 65% IBD), although the test for heterogeneity in the stratification based on HLA did not reach statistical significance (P>.05; data not shown). Evidence of linkage under stratification based on either HLA, INS, or population of origin was not increased in the chromosome 5q region compared with the complete data set (data not shown). There was also some evidence of heterogeneity in linkage to HLA when the data were stratified on the basis of INS (P=.012), with increased evidence of linkage in the INS− group (85% IBD) compared both with the INS+ group (71% IBD) and with the intermediate IBD value in the INS+/INS− group (76%) (table 5).

Figure  2
Multipoint linkage analysis using ALLEGRO, in HLA DR3/4 families (thinner black hashed line), non-DR3/4 families (thinner black solid line), INS+ families (gray hashed line), INS− families (solid gray line), and all families (thicker black solid ...
Table 5
Analysis of Heterogeneity in IBD Sharing and Stratification

Two additional markers, D4S412 and D13S153, located near markers detected with a LOD score >1.0 in the complete data set (21 cM from D4S403 and 12.5 cM from D13S153) showed evidence of heterogeneity when stratified on the basis of either HLA (P=.031 and P=.018, respectively). The LOD was 2.6 in the other-DR group, for D4S412, and was 3.2 in the DR4 group, for D13S153.

Linkage Analysis Using All Family Members

To include all affected individuals, linkage was calculated, by the ALLEGRO program, for regions reaching a LOD score of 2 in the primary linkage analysis. These data are displayed in figure 2. On chromosome 2, the LOD score was 2.6 among patients who also carried DR3/4. On chromosome 5, the LOD score was 2.2 in the complete data set, whereas it was 3.7 on chromosome 16 among patients who also carried DR3/4 and was 3.5 in the INS− group (these peaks were 29 cM apart).

Discussion

In the present genomewide linkage analysis in a large collection of Scandinavian families, we confirmed linkage to the disease-susceptibility loci IDDM1 (HLA region), IDDM2 (INS), and IDDM15. We also found suggestive evidence of linkage (LOD score >2) in three additional regions, on chromosomes 2p, 5p11-q13, and 16p. In the first and third of these three regions, the evidence of linkage was increased in secondary analyses when the data were stratified on the basis of either HLA or INS. HLA was confirmed as a major susceptibility locus for T1DM (multilocus LOD score 42.6), with an observed locus-specific λS of ~3.6. TDT analysis performed with an INS variant associated with T1DM susceptibility provided confirmation of linkage to IDDM2 (P=10-6). The IDDM1 and IDDM2 loci showed marginally significant evidence of interaction (P=.012). Linkage to the INS region was not significant, as expected, because of the limited power of this approach, given the low λS attributed to the locus because of the high carrier frequency of the susceptibility allele in the population (Julier et al. 1991; Spielman et al. 1993).

The pattern of linkage on 6q was similar to that observed in French and U.S. families (Delépine et al. 1997), with the LOD-score curve's maxima occurring at IDDM1 (HLA) and IDDM15 and being separated by >30 cM. After adjusting for linkage to IDDM1, we obtained P=.002 in the test for linkage to IDDM15 in the Scandinavian families. However, 147 of the Danish families were included in both the present study and the original report of linkage to IDDM15 (Delépine et al. 1997). When the new data were combined with those previously published on Danish, French, and U.S. families, the significance of the HLA-adjusted test statistic was P=7×10-7. Linkage at this locus has also been reported, in combined U.S. and U.K. families, by Concannon et al. (1998), although the U.S. families in the latter study overlap partially with both those in the report by Delépine et al. (1997) and those in the present study. Thus, the combined evidence in favor of IDDM15 is the most compelling that has been reported thus far for any of the putative T1DM-susceptibility loci, except for IDDM1 and IDDM2.

We selected additional markers on chromosome 2p12, to cover the region of EIF2AK3, which is mutated in patients with Wolcott-Rallison syndrome (Delépine et al. 2000). Although this syndrome is not known to involve an autoimmune process, patients develop permanent insulin-dependent diabetes shortly after birth, which leads to the proposition that this gene is a candidate for T1DM (Delépine et al. 2000). The evidence of linkage at this locus was increased in the HLA DR3/4+ sib pairs (multilocus LOD score 2.6), compared with that observed in the complete data set. This suggests that EIF2AK3—or another gene located in this region—may contribute to T1DM susceptibility. The EIF2AK3 region is distinct from the previously reported putative susceptibility loci—IDDM7 (MIM 600321), IDDM12 (MIM 601388), and IDDM13 (MIM 601318)—on chromosome 2q (Cordell et al. 1995; Luo et al. 1995; Owerbach and Gabbay 1995; Concannon et al. 1998; Mein et al. 1998). Significant linkage to the IDDM7–IDDM12 region has been reported, with replication, in a partly independent data set (table 1). Suggestive linkage to IDDM13 has been reported (table 1). CTLA4, which maps to the IDDM12 region, has been shown to be associated with T1DM (Nisticò et al. 1996; Marron et al. 1997; Larsen et al. 1999). Since we have excluded linkage to IDDM7–IDDM12 for λS[gt-or-equal, slanted]2, we cannot exclude the possibility that this region contains a gene with a relatively small effect on the risk for development of T1DM. The IDDM13 region was excluded at λS[gt-or-equal, slanted]1.5, suggesting that this region either does not contain a susceptibility gene in this population or contains one with a very small effect on the risk for T1DM.

The region of linkage on chromosome 16p extends over ~50 cM between D16S405 and D16S207 (fig. 2). Furthermore, increased evidence of linkage with heterogeneity was obtained in groups defined on the basis of HLA, INS, and population of origin, with peaks in the three groups extending over a 30-cM region. The 1-LOD support interval for the HLA-DR3/4 and INS− groups did not overlap (being located at 0–22 and 26–45 cM respectively; see fig. 2). In addition, single-point data in the INS− group reached its maximum for D16S3113 and D16S3131 (LOD score 2.9 and 3.0, respectively) whereas in the DR3/4 group the LOD score for these markers was 0.2 and 0.1, respectively. These observations suggest that this extended region contains more than one T1DM-susceptibility locus. However, simulation studies have shown that linkage analysis of multifactorial diseases may lead to linkage peaks some distance from the causative genetic effect (Roberts et al. 1999). The region partly overlaps that reported at D16S420–D16S261 (Mein et al. 1998), for which a LOD score of 1.2 was found in the UK263-family subset. In the U.K. study, the LOD score was also greater in HLA-DR3/4 sib pairs (LOD score 2.2). However, this region is distinct from a second region, on 16q22-q24, that has been reported to be linked to T1DM in U.K. families (Mein et al. 1998).

Suggestive evidence of linkage on chromosome 5p11-q13 also was found. This region had not previously been linked to T1DM; however, in two independent studies of multiple sclerosis, evidence of linkage had been found in this region. In one study, a LOD >3 was reached with a dominant model of inheritance (Kuokkanen et al. 1996; Sawcer et al. 1996; Oturai et al. 1999). This region is different both from the recently reported IDDM18 (MIM 605598) locus (IL12B) at 5q31.1-q33.1 (Morahan et al. 2001) and from other chromosome 5 T1DM loci reported in U.K. families (Mein et al. 1998).

Eleven other chromosome regions were identified that have nominal evidence of linkage (.05>P>.00074; table 3). Although some of these overlap putative regions with T1DM-susceptibility loci from the literature, the data presently available are not sufficient to determine which, if any, of these regions contain disease-susceptibility loci. Effects of λS[gt-or-equal, slanted]1.5 could be excluded at previously reported T1DM loci on chromosome 1, 6, 11, 14, 16, and 18 (table 1 and fig. 1), showing that these loci per se do not play a detectable role in T1DM susceptibility in the Scandinavian population.

We have observed significant heterogeneity in IBD sharing, for the chromosome 16 region, when sib pairs were grouped on the basis of shared HLA alleles (table 5). Similar results also were observed, for both the chromosome 16 region and the HLA region, when sib pairs were grouped on the basis of shared INS genotype (table 5). This suggests that there is both an interaction between the chromosome 16 region and both the HLA and INS and, similarly, an interaction between the INS and HLA loci. The LOD-score differences between different HLA and INS groups, presented in figure 2, are exploratory in nature and do not prove an interaction, for example, between the chromosome 2 region and the HLA region.

The results of this study demonstrate highly significant evidence of linkage to IDDM15 at 6q, when data from the present study are combined with those from previous investigations. Evidence of interaction between susceptibility loci for T1DM has been obtained in a number of studies (Davies et al. 1994; Cordell et al. 1995; Mein et al. 1998). This concept is supported in the present study, in particular by the results obtained, after stratification based on either HLA or INS, on linkage to chromosome 16p (table 5). Gene-gene interaction is clearly an important feature of genetic predisposition, and studies addressing this are very much in demand to explain the molecular basis of disease and to develop new intervention modalities. Extensive investigations of populations, such as the Scandinavian families studied here, and the combination of data from multiple sources are required to obtain the requisite power to detect interacting loci with relatively small effects on familial recurrence risk of the disease.

Acknowledgments

Drs. Dag Undlien, Jesper Johannesen and Ole P. Kristiansen are acknowledged for their efforts in the identification and collection of parts of the Norwegian and Danish families; the Norwegian Diabetes Association and the ISID group (diabetes nurses) are acknowledged for help in collection of Norwegian multiplex families; the Diabetes Incidence Study in Sweden is acknowledged for help in collection of Swedish multiplex families; and the Danish Study Group for Diabetes in Childhood and the Danish IDDM Epidemiology and Genetics Group are acknowledged for their help in collection of the Danish families. We thank Sivonne Arvidsson, Ingrid Delin, Chris Groves, Marianne Olsson, Susanne Palm, Liv Paltiel, Line Rooas, and Berit Rydlander for excellent technical assistance. I.K. was a Swedish medical research council–Wellcome Trust visiting fellow and was supported by an AMF Jubilee Fund stipend. This work was supported by European Union BioMed program grant BMH4-CT97-2311; Juvenile Diabetes Research Foundation International grants DK-96-012, 1-1998-168, and 2-2000-570; The Danish Medical Research Council grant 9502027; The Danish National Science Research Council grant 9601730; the Sehested-Hansen Foundation; the Swedish Strategic Funds; Swedish Medical Research Council grant K98-19X-12674-01A; TryggHansa; Arbetsmarknadens Försäkrings Aktiebolag; Novo Nordisk Fonden; Svenska Diabetesförbundet; Barndiabetes Fonden; Smedbergs Stiftelse; Sven Järring Fonden; Torsten och Ragnar Söderbergs Stiftelser; Stiftelsen för vetenskapligt arbete inom diabetologin; Första Maj blommans riksförbund; The Health Research Council in the South-East of Sweden; and The Scientific Committee of Jönköping County Council.

Electronic-Database Information

Accession numbers and URLs for data in this article are as follows:

ASPEX Package, The: Affected Sib Pair Exclusion Mapping, ftp://lahmed.stanford.edu/pub/aspex/index.html
Center for Medical Genetics, Marshfield Medical Research Foundation, http://research.marshfieldclinic.org/genetics/
Online Mendelian Inheritance of Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim (for T1DM [MIM 222100], HLA-DRB1 [MIM 142857], IDDM2 [MIM 125852], IDDM7 [MIM 600321], IDDM12 [MIM 601388], IDDM13 [MIM 601318], IDDM15 [MIM 601666], and IDDM18 [MIM 605598])

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