Association of Selective HLA Class II Susceptibility-Conferring and Protective Haplotypes with Type 2 Diabetes in Patients from Bahrain and Lebanon

Type 2 (non-insulin-dependent) diabetes mellitus (T2DM) is the most common diabetes form (19), and susceptibility to it is determined by environmental and genetic factors (9, 19), the latter being complex and poorly defined (5, 19). While the association of HLA class II genes in type 1 diabetes pathogenesis was reported for several ethnicities (1, 14), studies on HLA class II association with T2DM provided inconsistent results, since an association (16), no association (7), or a weak link between HLA class II and T2DM has been reported. A number of studies focused on the association of HLA with T2DM morbidity and mortality (17), highlighted by increased frequency of HLA-DR3 and -DR4 in islet cell autoantibody (ICA)-positive patients refractory to oral anti-diabetic drugs as reported by some (11) but not others (20), and association of HLA-DRB1*1502 with T2DM in anti-glutamic acid decarboxylase (GAD)-positive patients (10).

We previously reported on the distribution of HLA class II alleles and haplotypes among T2DM patients in the Bahraini population (16), an Arab Peninsula population with a high prevalence (24% of the adult population) of T2DM (2). In view of the heterogeneity of Arabs with distinct ethnic backgrounds and racial origins (3), this study addresses the association of HLA-DRB1 and HLA-DQB1 haplotypes with T2DM in Bahraini and Lebanese Arabs.

T2DM patients comprised 115 Lebanese (59 males and 56 females; mean age, 55.2 ± 13.5 years) and 110 Bahraini (59 males and 51 females; mean age, 52.3 ± 9.6 years) unrelated patients. Exclusion criteria included other types of diabetes, autoimmune diseases, and positive anti-GAD, anti-protein tyrosine phosphatase-related protein (IA-2), or ICA autoantibody responses. Family history of diabetes (80/110 Bahraini patients and 77/115 Lebanese patients) or body mass index did not influence selection of subjects. The control group included 121 Lebanese (57 males and 64 females; mean age, 54.5 ± 13.8 years) and 154 Bahraini (79 males and 75 females; mean age, 52.9 ± 9.0 years) subjects with normal fasting/random glucose levels and no known personal or family history of diabetes. Demographic details, which included duration, first-degree family history, complications, and treatment for diabetes, were recorded. The Arabian Gulf University Ethics Committee approved the study (which was done according to Helsinki guidelines), and informed consent was obtained from all participants.

Total genomic DNA was extracted from EDTA-anticoagulated venous blood by the phenol-chloroform method. HLA-DRB1 and HLA-DQB1 gene alleles were analyzed using the PCR sequence-specific priming (SSP) technique, using an SSP2L HLA class II genotyping kit according to the manufacturer's specifications (One Lambda, Thousand Oaks, CA). Allele frequencies were determined by the gene counting method, and haplotype frequencies were determined by the maximum likelihood method, using the Arlequin (version 2.000) population analysis software. P values were corrected for the number of alleles tested (Pc) using Bonferroni's inequality method; significance was determined at a P value of <0.05.

Significant DRB1 and DQB1 allelic differences were seen between Lebanese and Bahraini T2DM patients and controls. When Bonferroni's correction was applied, DRB1*070101 was significantly more common in both patient groups. While DQB1*0201 was significantly more common among Lebanese patients, none of the DQB1 loci were found to be significantly different between Bahraini patients and controls (Table 1). DRB1-DQB1 haplotype analysis showed that the frequency of DRB1*110101-DQB1*0201 was higher, while the frequencies of DRB1*040101-DQB1*050101, DRB1*110101-DQB1*030101, DRB1*130101-DQB1*060101, and DRB1*140101-DQB1*050101 were lower in Lebanese patients than in controls (Table 2). The frequencies of DRB1*040101-DQB1*0302 and DRB1*070101-DQB1*0201 were higher, while the frequency of DRB1*110101-DQB1*0201 was lower in Bahraini patients than in controls (Table 2). Logistic regression analysis demonstrated, after controlling for confounding variables, that DRB1*070101-DQB1*0201 and DRB1*110101-DQB1*0201 served a dominant role among Bahraini and Lebanese subjects, respectively (Table 3). Other haplotypes were rejected according to the model employed.

Unique susceptibility-conferring and protective HLA class II haplotypes were seen among Bahraini and Lebanese T2DM patients, with DR7- and DR4-containing (Bahraini) as well as DR11-containing (Lebanese) haplotypes being positively associated with T2DM. Whereas DR4-containing haplotypes conferred susceptibility among Bahraini patients, they were protective among their Lebanese counterparts. DRB1*110101-DQB1*0201 served a susceptibility-conferring role among Lebanese patients but was largely protective among Bahraini patients. The susceptibility conferred by DRB1*040101-DQB1*0302 and DRB1*070101-DQB1*0201 haplotypes among Bahraini subjects confirmed our previous finding (16), with key differences between the two studies, as DRB1*160101-DQB1*050101 was reported earlier to be protective and DRB1*150101-DQB1*060101 and DRB1*070101-DQB1*050101 were reported to confer susceptibility (16). This was largely due to the larger sample analyzed here (110 patients versus 89 patients).

DRB1*040101-DQB1*0302 conferred disease susceptibility in Bahraini patients, in agreement with previous studies on Caucasian (6) and non-Caucasian (8, 12) patients, but in disagreement with others which suggested that DR4 was protective (17). This may be due to ethnic background, patient selection, and sample size. In contrast to the case for Bahrainis, DRB1*040101-DQB1*0302 was neutral among Lebanese patients due to the high prevalence of DRB1*04 among healthy Lebanese (3) and other Mediterranean populations (15). DRB1*110101 was positively or negatively associated with T2DM when present with DQB1*0201 or DQB1*030101, respectively, suggesting that DRB1*110101 did not influence the disease susceptibility, which apparently resided in the DQB1 allele it associated with (18).

While DRB1*070101 was the susceptibility allele in both Lebanese and Bahraini patients, DQB1*0201 was present at higher frequency among Lebanese patients than among Bahraini patients due to the high prevalence of DQB1*0201 among healthy Bahrainis (3). Regression analysis confirmed that DRB1*070101-DQB1*0201 (Bahrain) or DRB1*110101-DQB1*0201 (Lebanon) was a risk factor for T2DM, indicating that HLA class II genotype influenced T2DM pathogenesis in these communities. While the association of specific HLA genotypes with T2DM reportedly disappeared after controlling for GAD or ICA levels (13), this does not appear to be the case here, as all patients were GAD and ICA negative.

While the mechanisms underlying these associations remain to be seen, it is possible that the association of DRB1-DQB1 haplotypes with T2DM and its complications may reside in differential affinity to antigenic fragments presented by each haplotype (4). Susceptibility-conferring haplotypes may bind to and present specific antigens, thereby precipitating hypoglycemia, while “protective” haplotypes may have reduced or no affinity for such antigens. A larger study which addresses class II genotype distribution among T2DM from other communities with a high T2DM prevalence, together with functional association with T2DM complications and response to therapy, are needed to confirm the association of HLA class II with T2DM.

• American Society for Microbiology