Type 1 diabetes mellitus is a chronic autoimmune disease characterized by progressive beta cell destruction and subsequent insulin deficiency. Onset typically occurs in childhood or adolescence, concomitant with the presence of autoantibodies such as glutamic acid decarboxylase-65 (GADA) and insulinoma-associated antigen 2 (IA-2A) (1,2). First-degree relatives of individuals with autoimmune diabetes are more likely to express these autoantibodies and are at increased risk for developing type 1 diabetes, which indicates a role for genetic factors in the etiology of the disease (3,4).

Key genetic determinants of the autoimmune component of type 1 diabetes have been identified in the major histocompatibility class II regions of chromosome 6, such as Human Leukocyte Antigen (HLA) DRB1 and DQB1 (5–11). Although susceptibility and resistance to type 1 diabetes have mapped to these loci, studies reporting these associations were conducted primarily in Caucasian populations (6,7,9,10). Few studies have investigated the race/ethnicity-specific distribution of alleles at these HLA loci in black (5,12) or Hispanic populations (11–13). Moreover, the joint effect of these loci on specific clinical characteristics of onset and severity, especially in these minority populations, is not well understood.

SEARCH for Diabetes in Youth (SEARCH) is a large multi-center observational study among a racial/ethnically diverse population of children and adolescents with diabetes. We describe the distribution of DRB1-DQB1 haplotypes in non-Hispanic white (NHW), non-Hispanic black (NHB), and Hispanic youth with autoantibody-positive type 1 diabetes, and examine the associations between these haplotypes and clinical characteristics of diabetes, such as age at diagnosis, presence of diabetic ketoacidosis (DKA) at onset, multiple autoantibody positivity, and fasting C-peptide (FCP) levels.

The study sample consisted of 1,662 youth with autoimmune diabetes (49% female), ranging in age from 3 to 21 years, of whom 77% were NHW, 10% NHB and 13% Hispanic (Table 1). The majority of NHW youth (99.3%) reported NHW as their only race/ethnicity. Among NHB youth, 83.7% self-identified as black or African American only, 15.7% as both black and NHW, and 0.6% as both black and Asian/Pacific Islander. Most Hispanic youth self-identified as Mexican or Mexican American (71.1%); others reported Puerto Rican (4.7%), Cuban (1.0%), Central American (3.8%), South American (3.0%), European Spanish (5.7%), or “Latino” or “Hispanic” only (10.7%).

Although mean age at diagnosis and mean age at study visit were similar among all racial/ethnic groups, NHB and Hispanic youth had a slightly longer duration of diabetes at the time of the visit. NHB youth were also more likely to test positive for GADA than either NHW or Hispanic youth. NHW and Hispanics were more likely to test positive for IA-2A, and among all youth who tested positive for IA-2A, had significantly higher mean IA-2A titers than NHB. NHB and Hispanic youth were more likely to present with DKA at diabetes onset than NHW youth. Although both NHB and Hispanics had a higher prevalence of overweight and obesity than NHW, they had similar FCP levels at the study visit.

Overall, 85% of youth in the study were carriers of at least one DRB1*03, DRB1*04 or DRB1*09 haplotype for susceptibility to type 1 diabetes. However, the distribution of many specific DRB1-DQB1 haplotypes varied by race/ethnicity (Table 2). While the DRB1*0301-DQB1*0201 susceptibility haplotype did not differ in frequency among the racial/ethnic groups, the frequency of the DRB1*0401-DQB1*0302 susceptibility haplotype was substantially higher in NHW than NHB or Hispanic youth. DRB1*0405-DQB1*0302 was more frequent in NHB and Hispanic than NHW youth, while DRB1*0407-DQB1*0302 was more frequent in Hispanic youth only. The frequencies of susceptibility DRB1*0901-DQB1*0201 and DRB1*0901-DQB1*0202 haplotypes were significantly higher in NHB than NHW and Hispanic youth. In general, frequencies of the neutral haplotypes either did not differ among racial/ethnic groups, or were higher in NHW compared to NHB and Hispanic youth (e.g. DRB1*0101-DQB1*0501). As expected, haplotypes shown to be protective for type 1 diabetes were of low frequency among all groups. In fact, the most protective haplotypes including DRB1*11, DRB1*14, DRB1*15 and DRB1*16 alleles typically had frequencies ≤ 1% in all racial/ethnic groups (Supplementary Table 1). The protective haplotype DRB1*0401-DQB1*0301 was more frequent in NHW, while frequency of modestly protective DRB1*0701-DQB1*0202 was higher among NHB.

While the frequency of susceptibility haplotypes DRB1*03-DQB1*XX were similar among all racial/ethnic groups, associations with characteristics of diabetes onset were not observed across all race/ethnicities. DRB1*03-DQB1*XX haplotypes were significantly associated with positive titers for both GADA and IA-2A in Hispanic youth only (Table 3); Hispanic youth carrying haplotypes with a DRB1*03 allele had 82% greater odds of testing positive for both GADA and IA-2A than non-carriers (OR: 1.82, 95% CI: 1.05–3.15). NHW youth carrying haplotypes with this allele had significantly younger mean age of diabetes onset compared to non-carriers (9.9 ± 0.2 vs. 10.5 ± 0.2 years; p=0.011) (Supplemental Figure 1). The susceptibility haplotype DRB1*04-DQB1*0302 was also significantly associated with positive titers for multiple autoantibodies in Hispanic youth; carriers of this haplotype were more than twice as likely to test positive for both GADA and IA-2A as non-carriers (OR: 2.13, 95% CI: 1.15–3.94) (Table 3). Hispanic carriers of this haplotype were also more than twice as likely to have had DKA at onset (OR: 2.38, 95% CI: 1.07–5.31) (Table 3) and significantly lower mean FCP at the study visit (0.45 ± 0.03 vs. 0.67 ± 0.08 ng/ml, p=0.007) compared to non-carriers (Supplemental Figure 2).

While neutral haplotypes did not appear to significantly alter risk for multiple autoantibody positivity, they appeared to have race/ethnicity-specific effects on other characteristics of diabetes onset. NHB youth with haplotypes containing a DRB1*01 allele were more than three times as likely to have had DKA at onset (OR= 3.62, 95% CI: 1.18–11.06) (Table 3), while NHW youth carrying this allele had significantly older age of onset (10.7 ± 0.2 vs. 10.1 ± 0.2 years; p=0.037) compared to non-carriers (Supplemental Figure 1). Interestingly, the modestly protective haplotypes, DRB1*04-DQB1*0301 and DRB1*0701-DQB1*XX were associated with increased odds of positive titers for multiple autoantibodies in NHW (OR= 1.98, 95% CI: 1.32–2.97 and OR=1.51, 95% CI: 1.05–2.17, respectively) (Table 3). DRB1*04-DQB1*0301 was also associated with older age of onset in NHW youth (11.0 ± 0.3 vs. 10.1 ± 0.1 years; p=0.017) (Supplemental Figure 1). Protective and neutral haplotypes containing the DRB1*13 allele were generally more frequent in NHB and NHW youth, and appeared to have a protective effect for multiple autoantibody positivity and DKA at onset. NHB youth carrying a DRB1*13 allele were 57% less likely to test positive for both GADA and IA-2A compared to non-carriers (OR= 0.43, 95% CI: 0.19–0.95). DRB1*13 may also confer protection from DKA at onset in NHB youth (OR= 0.40, 95% CI: 0.13–1.19) (Table 3).

In this diverse sample of 1,662 youth with diabetes, we found the distribution of HLA DRB1-DQB1 haplotypes to vary by race/ethnicity. DRB1-DQB1 associations with characteristics of diabetes onset and severity also varied by race/ethnicity, although not always in accordance with race/ethnicity-specific haplotype frequencies. Moreover, a few haplotypes previously shown to be “neutral” or “protective” with respect to risk for type 1 diabetes were associated with diabetes-related characteristics in some groups. As such, these findings may explain some of the variation in clinical presentation of autoimmune diabetes by race/ethnicity.

Of the susceptibility haplotypes, the combined frequency of those containing at least one DRB1*03 allele ranged from 27.5% in Hispanic youth, to 28.8% and 28.9% in NHW and NHB youth, respectively. The frequency of susceptibility haplotypes DRB1*03-DQB1*0201 and DRB1*04-DQB1*0302 (28.4% and 34.5%, respectively) in our NHW sample were slightly lower than those reported by the Type 1 Diabetes Genetics Consortium (T1DGC) (34.1% and 39.4%, respectively) and other Caucasian samples (6,9,20). Additionally, the frequency of neutral DRB1*01-DQB1*XX and protective DRB1*04-DQB1*0301, DRB1*0701-DQB1*XX and DRB1*13-DQB1*XX haplotypes (8.9%, 4.8%, 5.5%, and 6.6%, respectively) were slightly higher than those previously reported in the T1DGC (7.5%, 1.7%, 3.6%, and 3.1%, respectively) (9). This variation may be due to differences in the geographic location of proband ascertainment, and/or enrichment of family members with type 1 diabetes in the T1DGC. Scant data exist on the frequency of these haplotypes in Hispanic or black individuals with diabetes for comparison, although others have noted higher frequencies of DQB1*0302 among NHW and Hispanic youth and DQB1*02XX among black youth with type 1 diabetes, which are consistent with our findings (12).

Few studies have examined associations between DRB1-DQB1 haplotypes and clinical characteristics of diabetes onset or severity, especially in black or Hispanic populations. While the frequency of haplotypes containing a DRB1*03 allele was similar among all racial/ethnic groups, we found the presence of these haplotypes to be significantly associated with multiple autoantibody positivity among Hispanic youth only. DRB1*04-DQB1*0302 haplotypes, which were more frequent in NHW and Hispanics, were associated with multiple autoantibody positivity in both groups. DRB1*04-DQB1*0302 was also significantly associated with increased risk for DKA in Hispanic youth, which may indicate a role for this particular set of haplotypes in severity of onset in Hispanics. Interestingly, despite their “protective” designations, we also found DRB1*04-DQB1*0301 and DRB1*0701-DQB1*XX to be associated with multiple autoantibody positivity in NHW youth. This finding is supported by a study conducted in northern European children and adolescents with type 1 diabetes, which reported both DRB1*04 and DRB1*07 associations with IA-2A positivity (21). Moreover, Noble et al. recently reported that the effect of DRB1*0701-DQB1*0201, which was the highest frequency DRB1*07 haplotype in our sample, particularly among NHB youth, varies by DQA1 allele (22). The presence of European-derived DQA1*0201 on the haplotype confers protection, while the presence of DQA1*0301, linked to African ancestry, confers susceptibility to type 1 diabetes (22). Although DQA1 was not typed in our sample, it is possible that the association observed with increased autoantibody positivity is due to a greater presence of DQA1*0301 than DQA1*0201.

We acknowledge the limitations of our investigation. GADA and IA-2A were the only autoantibodies measured from blood samples drawn at the SEARCH study visit. Therefore, in restricting our analyses to youth who tested positive for at least one of the two autoantibodies measured, we excluded those who may have been positive for other diabetes-related autoantibodies in the absence of GADA or IA-2A. Self-reported race/ethnicity precluded us from accounting for racial/ethnic admixture in the estimation of haplotype frequencies and their effects on diabetes-related characteristics. Moreover, the modest sample sizes for NHB and Hispanic youth precluded us from examining DRB1-DQB1 haplogenotypes in more detail, and limited our power to detect association with some traits; most associations observed at a nominal significance level (p<0.05) would not survive correction for multiple testing. Despite these limitations, our population-based approach combined with the multi-ethnic composition of this cohort allowed for exploration of HLA-associated phenotypes in major racial/ethnic groups largely understudied. SEARCH participants are also well characterized in terms of traits associated with diabetes onset.

In conclusion, the frequencies of DRB1-DQB1 haplotypes and their association with clinical features of autoimmune diabetes vary across major racial/ethnic groups in the United States. Our findings also suggest a role for “neutral” and “protective” haplotypes in the severity of diabetes onset that has not been previously explored, particularly in racial/ethnic minority groups. Taken together, these findings may explain, in part, the variation in clinical presentation of autoimmune diabetes within and across racial/ethnic groups.