SEVERAL STUDIES HAVE reported an increased frequency

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1 X/01/$03.00/0 Vol. 86, No. 2 The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A. Copyright 2001 by The Endocrine Society Possible Human Leukocyte Antigen-Mediated Genetic Interaction between Type 1 and Type 2 Diabetes* HAIYAN LI, EERO LINDHOLM, PETER ALMGREN, ÅSA GUSTAFSSON, CAROL FORSBLOM, LEIF GROOP, AND TIINAMAIJA TUOMI Diabetes and Endocrine Research Laboratory (H.L., E.L., P.A., Å.G., L.G., T.T.), Department of Endocrinology, Lund University, S Malmö, Sweden; and Department of Internal Medicine (C.F., T.T.), Helsinki University Central Hospital, FIN Helsinki, Finland ABSTRACT We assessed the prevalence of families with both type 1 and type 2 diabetes in Finland; and we studied, in patients with type 2 diabetes, the association between a family history of type 1 diabetes, glutamic acid decarboxylase (GAD) antibodies (GADab), and type 1 diabetesassociated human leukocyte antigen (HLA) DQB1-genotypes. Further, in mixed type 1/type 2 diabetes families, we investigated whether sharing an HLA haplotype with a family member with type 1 diabetes influenced the manifestation of type 2 diabetes. Among 695 families ascertained through the presence of more than 1 patient with type 2 diabetes, 100 (14%) also had members with type 1 diabetes. Type 2 diabetic patients from the mixed families had, more often, GADab (18% vs. 8%, P ) and DQB1*0302/X genotype (25% vs. 12%, P 0.005) than patients from families with only type 2 diabetes; Received April 19, Revision received October 6, Accepted October 16, Address all correspondence and requests for reprints to: Dr. Tiinamaija Tuomi, Wallenberg Laboratory, Department of Endocrinology, Lund University, S Malmö, Sweden. * This study was financially supported by the Sigrid Juselius Foundation, the Påhlsson Foundation, the Medical Faculty of the Lund University, the Malmö University Hospital, the Swedish National Board of Health and Welfare, the Swedish Medical Doctors Association, the Crafoord Foundation, the Jalmari and Rauha Ahokas Foundation, the Novo Nordisk Foundation, the Anna Lisa and Sven-Eric Lundgren Foundation, and the Ernhold Lundström Foundation. The Botnia study is supported by the Sigrid Juselius Foundation, JDF Wallenberg, EC (BM4-CT ), Swedish Medical Research Foundation, Academy of Finland, Finnish Diabetes Research Society, Swedish Diabetes Association, and Novo Nordisk Foundation. but they had a lower frequency of DQB1*02/0302 genotype, compared with adult-onset type 1 patients (4% vs. 27%, P ). In the mixed families, the insulin response to oral glucose load was impaired in patients who had HLA class II risk haplotypes, either DR3(17)- DQA1*0501-DQB1*02 or DR4*0401/4-DQA1*0301-DQB1*0302, compared with patients without such haplotypes (P 0.016). This finding was independent of the presence of GADab. We conclude that type 1 and type 2 diabetes cluster in the same families. A shared genetic background with a patient with type 1 diabetes predisposes type 2 diabetic patients both to autoantibody positivity and, irrespective of antibody positivity, to impaired insulin secretion. The findings support a possible genetic interaction between type 1 and type 2 diabetes mediated by the HLA locus. (J Clin Endocrinol Metab 86: , 2001) SEVERAL STUDIES HAVE reported an increased frequency of type 2 diabetes (noninsulin-dependent diabetes mellitus) in families with type 1 diabetes (insulindependent diabetes mellitus) (1 4). In Sweden, 32% of patients with type 1 diabetes, compared with 12.5% of a nondiabetic reference group, reported a family history of type 2 diabetes (1). On the other hand, frequent occurrence of type 1 diabetes in relatives of patients with type 2 diabetes has been observed (5 7). A parental history of type 2 diabetes is associated with an increased risk of type 1 diabetes in siblings of type 1 diabetic patients (8, 9) and with an increased risk for nephropathy in type 1 diabetic patients (10). The consequence of such genetic admixture for type 2 diabetes is not known. In populations of European origin, 10% of patients diagnosed with type 2 diabetes have antibodies to glutamic acid decarboxylase (GADab), which is associated with subsequent development of insulin deficiency (11 15). However, the interaction between type 1 and type 2 diabetes may not be restricted to GADab-positive patients. In populations with a high prevalence of type 1 diabetes, a large proportion of patients with type 2 diabetes should have inherited susceptibility genes for both types of diabetes. Thus, type 1 diabetes susceptibility genes could contribute to the polygenic etiology of type 2 diabetes and modify its clinical manifestation. Indeed, a family history of type 1 diabetes was associated with lower fasting C-peptide concentration and a lower frequency of cardiovascular disease in patients with type 2 diabetes (16). Some studies have reported an increased frequency of human leukocyte antigen (HLA)-DR4 or HLA-DR3/DR4 (17 20) in patients with type 2 diabetes, but this increase was mainly restricted to patients with relative insulin deficiency or islet cell antibodies (ICA) and/or GAD (15, 17, 20). In addition, excess transmission of DR4-linked haplotypes, from parents with type 2 diabetes to offspring with type 1 diabetes, has been reported (21). Taken together, these data point to a genetic interaction between type 1 and type 2 diabetes that could be mediated by the HLA locus. To further elucidate this interaction, we investigated, in families with both type 1 and type 2 diabetes, whether family history of type 1 diabetes and/or sharing a risk HLA haplotype with a member with type 1 diabetes influenced the manifestation of type 2 diabetes. Subjects Materials and Methods The Botnia study is a population-based study aiming at the identification of genes that increase susceptibility to type 2 diabetes (22). Since 574

2 HLA AND TYPE 1 AND TYPE 2 DIABETES , we have recruited 850 families with at least 1 member with type 2 diabetes, from Finland (16). Of them, 695 (82%) had more than 1 type 2 diabetic patient and were included in the present study. The mean family size was 8, including (on average) 2.4 type 2 diabetic patients per family (n 1658). All available family members were invited to participate in the study, and an oral glucose tolerance test (OGTT) was performed on more than 90% of the subjects studied. Diabetes was diagnosed according to the new World Health Organization criteria (23). Diagnosis of type 1 diabetes was based on initiation of insulin treatment within 6 months after diagnosis and/or fasting C-peptide concentrations less than 0.2 nmol/l. Patients who did not fulfill these criteria were considered to have type 2 diabetes. However, all type 2 diabetic patients in the present study had been treated with diet and/or oral hypoglycemic agents for at least 1 yr before commencing insulin treatment. Informed consent was obtained from all subjects, and the study was approved by the local ethics committee. Families with genetically confirmed maturity-onset diabetes of the young (MODY1, MODY2, MODY3) (24) were excluded. Families with only type 2 diabetes were called common type 2 families. Families in which type 1 diabetes occurred among the first-, second-, or third-degree relatives of type 2 diabetic patients were called mixed type 1/2 families. GADab were analyzed in 1451 of the 1658 (88%) patients diagnosed with type 2 diabetes. Study design To compare the HLA-DQB1 genotype frequencies, we randomly selected one patient from each mixed type 1/2 family (n 93) and compared them with randomly selected patients from 195 common type 2 families. In addition, we compared the genotype frequencies with those of: 1) 172 GADab-negative control subjects from the Botnia study [nondiabetic spouses, 77 males (M)/95 females (F); mean age, yr]; and 2) 126 patients with adult-onset type 1 diabetes from the Diabetes 2000 Registry in Southern Sweden (73 M/53 F; age at diagnosis, 20 yr; mean, yr). These adult-onset type 1 diabetic patients were of similar age, at diagnosis of diabetes, and had a similar frequency of DQB1 genotypes as those Finnish adult-onset type 1 diabetic patients participating in the Botnia study (n 32) (15). Among the mixed type 1/2 families, we compared the glucose and insulin responses during OGTT between family members who had (HLA ) or did not have (HLA ) a type 1 diabetes susceptibility HLA haplotype (either DR3(17)-DQA1*0501-DQB1*02 or DR4*0401/4- DQA1*0301-DQB1*0302) (25, 26). We studied 36 families with, on average, 9 members (range, 4 40). The comparisons were restricted to those family members not treated with insulin who would share, at most, 25% of the chromosomes, i.e. second-degree or more distant relatives of the type 1 diabetic patients. These included 68 type 2 diabetic (42 HLA and 26 HLA ) and 122 nondiabetic (75 HLA and 47 HLA ) relatives. To test whether differences in glucose and insulin responses between the HLA and HLA family members were attributable to the presence of susceptibility class II HLA DRB1-DQB1 haplotypes per se or whether actual sharing of the chromosomal region with a patient with type 1 diabetes was needed, we established HLA-sharing identity by descent (IBD) by following the haplotype of the type 1 diabetic proband in the families. In families with more than one patient with type 1 diabetes, the patient diagnosed first was regarded as the type 1 proband, whose HLA haplotypes were followed. Among the 36 families included, 27 (64%) diabetic and 33 (44%) nondiabetic HLA subjects shared the class II haplotype IBD (HLA-IBD ) with the type 1 proband of the family. As with the mixed type 1/2 family members, we also compared the glucose and insulin response to OGTT, according to the presence of risk DRB1-DQB1 haplotypes, in a group of unrelated GADab-negative (122 diabetic and 158 nondiabetic) subjects without a family history of type 1 diabetes. Subjects with one risk and one protective (DR2*15- DQB1*0602/3) haplotype were excluded from the analysis. HLA genotyping The second exon of the HLA DQA1 and DQB1 genes was amplified by PCR, followed by dot-blot hybridization with sequence-specific oligonucleotide probes labeled with digoxigenin (DIG Oligonucleotide 3 -End Labeling Kit, Roche Molecular Biochemicals, Mannheim, Germany) (15). Nine DQB1 probes were used to distinguish DQB1 alleles 0201 or 0202 (02), 0301, 0302, 0303, 0501, and either 0602 or 0603 (0602/3), and 0401 or 0402 (0401/2). Ten DQA1 probes were used to distinguish DQA1 alleles 0101, 0102, 0103, 0301, 0302, 0201, 0501, and either 0401 or 0601 (0401/0601). The probe sequences originated from the 11th International Histocompatibility Workshop (27). HLA DRB1 was typed by a group-specific PCR, employing seven primer-pairs to amplify DR1, DR2, DR3/5/6/8, DR4, DR7, DR9, and DR10 (28). Using restriction fragment length polymorphism analysis, the DR1 group was further subdivided into DRB1*0101, *0102, and *0103 alleles; the DR2 group into DR2(15) and DR2(16) alleles; and the DR3/ 5/6/8 group into DR3(17) and DR3(18), DR5(11) and DR5(12), DR6(13) and DR6(14), and DRB1*0801/3 and DRB1*0802/4 alleles. The DR4 group was similarly divided into six subgroups: DRB1*0401/4/8 (*0401, *0404, or *0408); DRB1*0402; DRB1*0403/7 (*0403 or *0407); DRB1*0405/ 9/10 (*0405, *0409, or *0410); DRB1*0406; and DRB1*0411. A further subtyping for the DR4 subgroups was performed by dot-blotting to identify the *0403 and *0401/4 alleles (29). In pedigrees where IBD segregation of the class II HLA-haplotypes could not unambiguously be deduced, we typed three additional microsatellite markers (D6S291, D6S1691, and D6S299) located 5 cm centromeric, 3 cm telomeric, and 5 cm telomeric to the HLA class II locus, respectively (data not shown). The primer sequences, allele sizes, and genetic marker order were obtained from the Genome Database. The markers were typed by fluorescence-based PCR (30). The extended HLA haplotypes were constructed both manually and with the GENE- HUNTER linkage program (31). Metabolic measurements An OGTT was performed for all subjects more than 15 yr old, with fasting blood glucose less than 10 mmol/l, and not treated with insulin. After 12 h of overnight fast, the subjects ingested 75 g glucose in a vol of 300 ml. Samples for measurements of blood glucose and serum insulin were drawn at 10, 0, 30, 60, and 120 min; and incremental glucose or insulin areas under the curve were calculated. Blood glucose was measured with a hexokinase method with a coefficient of variation (CV) less than 1% (Roche Molecular Biochemicals). Serum insulin concentrations were measured by RIA (Pharmacia Biotech, Uppsala, Sweden) with an interassay CV of 5%. Fasting serum-c-peptide concentrations were measured in duplicate by an RIA with an interassay CV of 9% (Human C-peptide RIA Kit, Linco Research, Inc., St. Charles, MO). Hemoglobin A 1c (HbA 1c ) concentration was measured by a high-pressure liquid chromatography with a reference range of 5 7%. GADab were measured by a radioimmunoprecipitation method employing 35 S- labeled recombinant human GAD 65 produced by in vitro transcription/ translation (15). Statistical analysis Data are presented as mean sd for normally distributed variables, as median (interquartile range) for nonnormally distributed variables, and as percentages for categorical variables. The statistical analysis was performed with either Biomedical Data Processing (BMDP; Los Angeles, CA) or Number Cruncher Statistical Systems (NCSS; Kaysville, UT) statistical software. The group frequencies were compared using the -squared or Fisher s exact tests, and group means using the Mann- Whitney or the Kruskall-Wallis tests. Results Characteristics of families with both type 1 and type 2 diabetes Among 695 families, ascertained through the presence of more than 1 patient with type 2 diabetes, patients with type 1 diabetes were observed in 100 families (14%). Of them, 30% had more than one member with type 1 diabetes. Thirtyseven (5.3%) of 695 randomly selected probands with type 2 diabetes had a first-degree relative with type 1 diabetes. Compared with patients from families with only type 2 diabetes, the type 2 diabetic patients from the mixed type 1/2

3 576 LI ET AL. JCE&M 2001 Vol. 86 No. 2 families had a lower body mass index (BMI) (P 0.004) and lower fasting C-peptide concentrations (P 0.031) (Table 1). They were also more often treated with insulin (all patients: 42% vs. 32%; P 0.003; GADab patients: 55% vs. 59%, P NS; GADab patients: 38% vs. 30%, P 0.050). However, when compared with the adult-onset type 1 diabetic patients, the mixed patients were older at diagnosis (P ) and had higher BMI (P ) and higher fasting C-peptide concentrations (P ) (Table 1). Role of autoimmunity and HLA genotype GADab were found more often in type 2 diabetic patients from the mixed type 1/2 families (18%) than in those from the common type 2 families (8%, P ) (Table 1). Fortytwo of the 139 (30%) GADab-positive patients with type 2 diabetes had a family history of type 1 diabetes, compared with 198 of 1312 GADab-negative patients (15%) (P ). Of the 126 patients with adult onset type 1 diabetes, 82 (65%) had GADab after a median diabetes duration of 17.5 (18.2) yr. The frequency of GADab among the nondiabetic relatives was not significantly different from the frequency in the general population (4.4%, 17 of 383), either in the mixed type 1/2 (4.3%, 13 of 297) or in the common type 2 diabetes (3.1%, 28 of 905) families. Figure 1 shows the DQB1 genotype frequencies stratified according to GADab positivity and family history of diabetes. The type 2 diabetic patients from the mixed 1/2 families had, nearly as often, the 0302/X genotype conferring an increased risk for type 1 diabetes (25%) as the adult-onset type 1 diabetic patients (32%). The frequency was twice as high as that seen in patients from the common type 2 families (12%, P 0.005) or in the nondiabetic control subjects (12%, P 0.008). Of note, although the GADab patients, in general, had an increased frequency of the 0302/X genotype, it was higher in both GADab and GADab patients from mixed, compared with those from the common type 2 families (Fig. 1). On the contrary, the frequency of the 02/0302 genotype conferring the highest risk for type 1 diabetes did not differ among the patients from mixed families (4%), common type 2 families (3%), or the nondiabetic control subjects (4%), whereas it was significantly lower in all these groups, compared with the adult-onset type 1 patients (27%, P 0.001) (Fig. 1). The presence of the protective genotype 0602(3)/X showed antibody-dependent variation between the groups. Although its frequency among the GADab-negative patients from the mixed families (20%) did not differ from patients with the common form of type 2 diabetes (28%) or the control subjects (30%), it was virtually absent from both the GADabpositive mixed patients (0%) and the adult-onset type 1 patients (2%) (Fig. 1). The genotype frequencies of the nondiabetic relatives from the mixed type 1/2 families were 6% for 02/0302, 17% for 0302/X, 23% for 0201/X, and 17% for 0602(3)/X. Thus, they did not significantly differ from the type 2 diabetic patients from the same families (4%, 25%, 25%, and 16%, respectively). HLA risk-haplotypes and insulin response among subjects from mixed type 1/2 families Diabetic relatives. Despite similar age and BMI, the fasting glucose concentration (P 0.005) and the incremental glucose area during OGTT (P 0.004) were significantly higher in diabetic HLA patients than in their diabetic HLA relatives (Table 2). In addition, insulin response during OGTT [insulin area: 3666 (3514) vs (5437), P 0.044] and the ratio of insulin to glucose area [5.9 (11.3) vs (17.9), P 0.005] were significantly lower in the HLA patients than in the HLA patients. These differences were unaltered when only GADab patients were considered [Fig. 2, glucose area: 601 (343) vs. 393 (241) mmol/l; P 0.004; insulin area: 3706 (3803) vs (5692) mu/l, P The ratio of insulin to glucose area: 6.1 (12.5) vs (17.2), P 0.002]. Also, similar results were obtained when the subjects were stratified according to the DQB1-genotype instead of the risk-haplotype [patients with DQB1*02/0302 or 0302/X genotype vs. others: insulin area, 3503 (3070) vs (5103), P 0.049; the ratio of insulin to glucose area, 4.4 (4.9) vs.11.7 (14.6), P 0.004]. We also analyzed the subjects according to whether they TABLE 1. Clinical characteristics of adult-onset type 1 diabetic patients and type 2 diabetic patients from mixed type 1/2 or common type 2 diabetes families Type 1 diabetes P value a Mixed Type 1/2 P value b Common Type 2 Type 2 diabetes families families Number Sex (M/F) 73/53 113/ /766 Age (yr) Age at diagnosis (yr) Duration of diabetes (yr) 17.5 (18.2) (12.2) 8.5 (11.3) BMI (kg/m 2 ) Fasting blood glucose (mmol/l) Fasting serum insulin (mu/l) NA 12.1 (13.7) 12.9 (11.9) Fasting serum C-peptide (nmol/l) 0.00 (0.00) (0.48) c 0.57 (0.50) HbA 1c (%) GADab positivity 65% % (42/240) % (97/1211) Values are mean SD or median (interquartile range). NA, Not applicable. a P values are given for the comparison between adult-onset type 1 diabetic patients and type 2 patients from mixed type 1/2 diabetes families. b P values are given for the comparison between type 2 patients from mixed type 1/2 or common type 2 diabetes families. c Adjusted for BMI.

4 HLA AND TYPE 1 AND TYPE 2 DIABETES 577 FIG. 1. The frequency (%) of type 1 diabetes-associated HLA DQB1 genotypes (0302/X, 02/0302, 0602(3)/X,and 02/X) in adult-onset type 1 diabetic patients (n 126), type 2 patients from mixed type 1/2 families (n 93) or common type 2 families (n 195), and in nondiabetic control subjects (n 172). f, GADab subjects;, GADab subjects. TABLE 2. Clinical characteristics of patients with type 2 diabetes and nondiabetic subjects from the mixed type 1/2 families according to the presence of HLA risk (HLA ) haplotypes a Type 2 diabetic subjects Nondiabetic subjects HLA P values b HLA HLA HLA Number Sex (M/F) 16/26 10/16 37/38 21/26 Age (yr) 66.2 (19.3) 70.3 (25.1) 45.0 (24.6) 42.4 (22.3) Age at diagnosis (yr) 57.0 (18.0) (28.0) NA NA Duration of diabetes (yr) 5.3 (11.5) 3.6 (7.6) NA NA BMI (kg/m 2 ) 27.2 (5.7) 26.2 (5.2) 25.0 (5.9) 25.1 (5.6) Fasting blood glucose (mmol/l) 7.1 (2.3) (1.6) 4.7 (0.7) 4.7 (0.6) Glucose area (mmol/l 2 h) 626 (327) (249) 217 (188) 156 (157) Fasting serum insulin (mu/l) 10.5 (7.2) 10.0 (11.9) 7.3 (5.1) 6.5 (3.9) Insulin area (mu/l 2 h) 3666 (3514) (5437) 3763 (2644) 3589 (3286) HbA 1c (%) 6.4 (1.1) (1.3) 5.2 (0.8) 5.3 (0.7) Values are median (75% 25% interquartile range). a Either DR3(17)-DQA1*0501-DQB1*02 or DR4*0401/4-DQA1*0301-DQB1*0302 haplotype. b P values are given for the comparison between the HLA and the HLA Type 2 diabetic subjects. actually shared the HLA risk haplotype with their type 1 diabetic relative. When only the patients sharing HLA haplotypes IBD with the type 1 diabetic proband were included, the difference in glucose area [682 (286) vs. 442 (306) mmol/l; P 0.002], as well as the ratio of insulin to glucose area [4.8 (4.7) vs (15.3)] and the early-phase insulin secretion [30-min insulin area, 320 (278) vs. 572 (594) mu/l, P 0.015] were maintained between the HLA-IBD (n 27) and HLA- IBD (n 41) groups. Again, the differences in glucose and insulin responses were not attributable to the presence of GADab [GADab, glucose area: 682 (324) vs. 433 (300) mmol/l, P 0.004; 30-min insulin area: 321 (191) vs. 620 (696) mu/l, P 0.041; the ratio of insulin to glucose area: 5.9 (3.5) vs (15.2), P 0.020; see Fig. 4]. Nondiabetic relatives. Among the nondiabetic relatives, the glucose and insulin responses did not significantly differ between the HLA and the HLA subjects (Table 2). However, when only the 33 (44%) nondiabetic subjects sharing HLA haplotypes IBD with the type 1 diabetic proband were considered, they had greater glucose area [249 (261) vs. 172 (161) mmol/l, P 0.003] and lower incremental insulin-to-

5 578 LI ET AL. JCE&M 2001 Vol. 86 No. 2 FIG. 2. Blood glucose and serum insulin concentrations (median SEM) during an OGTT in GADab type 2 diabetic relatives from the mixed type 1/2 families. The patients are grouped according to whether they have (F, HLA, n 35, solid line) or do not have (E, HLA, n 23, dashed line) a risk HLA-haplotype. P for the difference in glucose area and P for insulin area between HLA and HLA patients. glucose ratio at 30 min [6.6 (5.5) vs. 9.8 (8.6) mu/mmol, P 0.017] or 2 h [12.4 (9.5) vs (13.9), P 0.007] than the 89 HLA-IBD subjects. Once again, these differences did not change when the GADab subjects were excluded (Figs. 3 and 4). HLA risk-haplotypes and insulin response among unrelated subjects without type 1 diabetes family history To further test whether high-risk HLA haplotypes predisposing to type 1 diabetes per se explained the above findings or whether actual sharing of the chromosome IBD with the type 1 diabetic relative was essential, we also studied GADab-negative unrelated type 2 diabetic (n 122) and nondiabetic (n 158) subjects without a family history of type 1 diabetes. We compared the insulin and glucose responses during OGTT between those with (HLA )- and without (HLA )-risk HLA haplotypes (see Subjects and Methods). The proportion of HLA class II haplotypes between the diabetic and nondiabetic subjects was similar (34% vs. 34%). Different from the relatives in the mixed type 1/2 families (see above), there was no difference between the HLA (n 41) and the HLA (n 81) diabetic subjects, with respect to either glucose [548 (294) vs. 573 (224) mmol/l 2h]or insulin [4243 (6029) vs (5711) mu/l 2 h] areas during FIG. 3. Blood glucose and serum insulin concentrations (median SEM) during an OGTT in GADab nondiabetic relatives from the mixed type 1/2 families. The subjects are grouped according to whether they share (f, HLA-IBD,n 30, solid line) or do not share (, HLA IBD,n 87, dashed line) a risk HLA-haplotype IBD with a type 1 diabetic relative. P for the difference in glucose area between HLA-IBD and HLA-IBD nondiabetic subjects. OGTT (Table 3 and Fig. 4). Similarly, among the nondiabetic subjects, no difference was observed between the HLA (n 53) and the HLA (n 105) nondiabetic subjects, with respect to insulin [4397 (3985) vs (4104) mu/l 2h]and glucose [204 (206) vs. 204 (148) mmol/l 2 h] areas during OGTT (Table 3 and Fig. 4). Discussion We have previously shown that type 2 diabetic patients with a family history of type 1 diabetes have lower fasting C-peptide concentrations and a lower frequency of cardiovascular disease than patients with family history of type 2 diabetes only (16). Because they were also more often positive for GADab and had type 1 diabetes associated HLAtypes, the present study was undertaken to try to evaluate the relationship of these components and their contribution to the manifestation of diabetes. In keeping with some earlier data on clustering of the two diabetes types in the same families (1 3, 6, 8, 9, 16, 21), our data show a 2-fold increased prevalence of type 1 diabetes in families with type 2 diabetes, compared with the prevalence in the background population (32). Thus, altogether, 14% of our families with more than one type 2 diabetic patient had also members with type 1 diabetes, and 5% of the patients had a first-degree relative with type 1 diabetes. Of note, this clustering of both forms of

6 HLA AND TYPE 1 AND TYPE 2 DIABETES 579 FIG. 4. Insulin-to-glucose ratio (Ins/Glu; median SEM) during an OGTT in GADab subjects who are: 1) type 2 diabetic (upper left) or nondiabetic (lower left) relatives from the mixed type 1/2 families; or 2) type 2 diabetic (upper right) or nondiabetic (lower right) unrelated subjects without family history of type 1 diabetes. The subjects from the mixed type 1/2 families are grouped according to whether they share (f, HLA-IBD, solid line) or do not share (, HLA-IBD, dashed line) a risk HLA-haplotype IBD with a type 1 diabetic relative. The unrelated subjects are grouped according to whether they have (F, HLA, solid line) or do not have (E, HLA, dashed line) a risk HLA-haplotype. For the difference in the ratio of insulin to glucose area, P in GADab type 2 diabetic relatives and P in GADab nondiabetic relatives. TABLE 3. Clinical characteristics of unrelated GADab-negative type 2 diabetic and nondiabetic subjects without family history of type 1 diabetes according to the presence of class II HLA risk (HLA ) haplotypes Diabetic subjects Nondiabetic subjects HLA a HLA b HLA HLA Number Sex (M/F) 16/25 39/42 29/24 43/62 Age (yr) 66.5 (14.0) 67.5 (16.6) 57.6 (19.0) c 50.4 (26.1) Age at diagnosis (yr) 61.0 (13.2) 62.0 (12.0) NA NA Duration of diabetes (yr) 3.8 (6.6) 5.5 (8.8) NA NA BMI (kg/m 2 ) 28.0 (5.9) 27.9 (5.2) 27.4 (5.1) d 25.0 (4.5) Fasting blood glucose (mmol/l) 6.9 (1.9) 6.9 (3.0) 5.1 (0.6) 5.0 (0.6) Glucose area (mmol/l 2 h) 548 (294) 573 (224) 204 (206) 204 (148) Fasting serum insulin (mu/l) 11.0 (9.7) 12.5 (10.8) 7.4 (5.7) 6.5 (4.6) Insulin area (mu/l 2 h) 4243 (6029) 4604 (5711) 4397 (3985) 4519 (4104) HbA 1c (%) 6.6 (1.7) 6.2 (1.5) 5.4 (0.5) 5.4 (0.6) Values are median (75% 25% interquartile range). a Subjects with either DR3(17)-DQB1*02 and/or DR4(0401/4)-DQB1*0302 and without DR2*15-DQB1*0603/2 haplotype; b Subjects with protective and/or neutral class II HLA haplotypes. c P and d P for the comparison between the HLA and the HLA nondiabetic subjects. diabetes does not seem to be restricted to Scandinavian populations (33, 34). The data further support the view that a shared genetic background with a type 1 diabetic family member influences the phenotype of the type 2 diabetic patient, i.e. they were less obese, had earlier onset of diabetes and lower C-peptide concentrations, and were more often treated with insulin than type 2 diabetic patients without such family history. As

7 580 LI ET AL. JCE&M 2001 Vol. 86 No. 2 reported earlier, GADab positivity was also more common in mixed, than in common type 2, patients (17.5% vs. 8%; P ) (16). The prevalence of GADab (9%) in the whole type 2 diabetes population is in accordance with figures reported from other countries (14, 35). The type 2 diabetic patients from the mixed type 1/2 families shared an increase in the moderate-risk HLA- DQB1*0302/X genotype (36) with the adult-onset patients with type 1 diabetes. Because they were relatives of type 1 diabetic patients, this was per se not unexpected. However, similar sharing of the genotype conferring the highest risk, 02/0302, or absence of the genotype conferring protection, 0602(3)/X, was not observed except for the GADab-positive subgroup of patients from the mixed type 1/2 families. It is of interest that, if we compare GADab-positive patients from mixed type 1/2 families and the common type 2 families, only the patients from mixed families share the 02/0302 and 0602(3) association with type 1 diabetic patients, whereas all GADab-positive patients share the 0302/X association. This finding suggests that part of the observed heterogeneity among the GADab-positive type 2 diabetic patients (i.e. patients with latent autoimmune diabetes) (11, 13 15) could be ascribed to type 1 family history. The high frequency of GADab in mixed patients could reflect autoimmune insulitis, and one could argue that the differences were attributable to misclassification of the patients, i.e. adult-onset type 1 diabetes masquerading as type 2 diabetes. Because of the cross-sectional nature of the study, we cannot totally refute that interpretation, but we consider it unlikely for the following reasons. An increase in the 0302/X genotype frequency was seen also in the GADabnegative patients from the mixed type 1/2 families, and sharing of class II HLA risk haplotypes with a family member with type 1 diabetes was associated with a reduced insulin response to OGTT independent of the presence of GADab. Also, all type 2 diabetic patients were noninsulin-treated for at least 1 yr after diagnosis. On the other hand, a considerable proportion of the patients would fulfill the criteria for latent autoimmune diabetes in adults. It is possible that some of the patients diagnosed with type 2 diabetes in the mixed type 1/2 families could have been positive for GADab or other autoantibodies at onset but not at the time of testing. However, all available data for GADab show that they are stable over the years (37 40). As to the other antibodies, no data exist on patients with newly-diagnosed adult-onset type 1 diabetes at the age of over 40 yr, which would be the relevant age-group for our study. In the study by Gorus et al., 85 88% of patients between 20 and 40 yr at diagnosis were positive for GADab, which represented 92 95% of those positive for any diabetes-associated autoantibody (41); 1.4% were positive for IA2-ab, and 4% for ICA, though negative for GADab. In addition, 12% of Swedish patients who were yr old at diagnosis of diabetes of unclear type had ICA in the absence of GADab (42). However, among patients clinically diagnosed with type 2 diabetes, as our patients were, only % of the 1538 UKPDS patients who were yr old at diagnosis were ICA in the absence of GADab. Accordingly, our cross-sectional data on the Botnia Study showed 0.5% of 517 GADab type 2 diabetic patients to have IA2-ab and 0.6% to have ICA (15). In addition, IA2ab were analyzed in 75% of the GADab relatives included in the analysis of insulin response to OGTT, and none of them had IA2ab (data not shown). Thus, we do not think that the results can be explained by misclassification of patients. Could it be that 0201/0302 is associated with a more aggressive insulitis (and thus, susceptibility to type 1 diabetes), whereas 0302/X confers risk to both types of diabetes, e.g. through subclinical insulitis? An increase in HLA-DR4, which is in linkage-disequilibrium with the DQB1*0302 allele and in type 1 diabetes-associated HLA-haplotypes, has previously been reported in patients with type 2 diabetes (18, 19, 21, 43). More recently, it was shown that type 2 patients with DR4 had a lower cardiovascular mortality rate than that of DR4 negative patients (44). This is compatible with our data on lower BMI, lower C-peptide concentrations, and lower frequency of coronary artery disease in mixed patients compared with common type 2 patients (16), because both obesity and insulin resistance are considered risk factors for cardiovascular disease (45). The nature of such an autoimmune process remains obscure but could involve two possibilities. A genetically determined autoimmune attack could have reduced the pancreatic -cell capacity of the HLA-sharing relatives early in life. Only histological analysis of the pancreatic islets could confirm this. Another possible explanation is that the HLAsharing relatives are in the process of developing type 1 diabetes or have subclinical autoimmune insulitis reducing their -cell capacity. It is well established that 6 10% of first-degree relatives of patients with type 1 diabetes develop type 1 diabetes (1, 46 49). First-degree relatives with ICA (26, 48, 50, 51) or relatives of young probands (21, 52) seem to have a higher risk of developing type 1 diabetes, but the effect of probands age is controversial (47). For siblings, HLA-identity with the proband is associated with a 10 30% risk, and HLA-haploidentity with a 4 9% risk, of developing type 1 diabetes, whereas the risk for HLA-nonidentical siblings does not differ from that for the general population (26, 48, 53, 54). It is not known what the risk of developing type 1 diabetes is for more distant relatives who are haploidentical for the HLA-type. However, based on the risk estimates for the first-degree relatives, it is unlikely that a major proportion of the second-degree or more distant relatives that we have been studying would develop type 1 diabetes. In contrast to the findings in family members, type 2 diabetic patients without type 1 family history who had highor moderate-risk class II HLA alleles did not demonstrate any deterioration of glucose tolerance or impairment in insulin secretion. The same applied for the nondiabetic relatives of type 1 diabetic subjects, unless they shared the HLA haplotypes IBD with the type 1 diabetic relative. This would suggest that some other gene than the HLA-DQB1 (or combination of alleles) in this region is required to manifest the metabolic effects. The current study was not designed to identify these genetic factors, but there are several candidates. Although the class II HLA locus accounts for most of the increased susceptibility to type 1 diabetes ( s 3.1), at least 15 other chromosomal loci have been shown to increase susceptibility to type 1 diabetes. Some of them are located near the HLA region on the short arm of chromosome 6 (55, 56). Linkage disequilibrium mapping, using a dense SNP

8 HLA AND TYPE 1 AND TYPE 2 DIABETES 581 map of the region in the mixed families, could provide some clues to the nature of these genetic factors. We conclude that genetic susceptibility to type 1 diabetes conferred by the HLA locus predisposes to impaired -cell function in patients with type 2 diabetes. The findings have important clinical implications by emphasizing the profound effect of a family history of type 1 diabetes on the phenotype of patients with type 2 diabetes. Further studies are required to precisely define the alleles or combination of alleles increasing susceptibility to impaired -cell function in patients with type 2 diabetes. Acknowledgments Anita Nilsson and Britt Bruveris-Svenburg are acknowledged for technical assistance; and Monica Gullström, Maja Häggblom, Sonja Paulaharju, Susanne Salmela, Leena Sarelin, Mikko Lehtovirta, Auli Hyrkkö, and the rest of the Botnia Research Group for recruiting and clinically studying the subjects. References 1. 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