SUBMITTED 14 JUL 21 1 REVISIONS REQ. 10 OCT 21; REVISIONS RECD. 12 DEC 21 2 ACCEPTED 12 JAN 22 3 ONLINE-FIRST: FEBRUARY 2022 4 DOI: https://doi.org/10.18295/squmj.2.2022.016 5 6 The association of Human Leukocyte Antigens Complex with Type 1 Diabetes 7 in Omanis 8 Mohammed Al-Balushi,1 Samiya Al-Badi,2 Saif Al-Yaarubi,3 Hamad Al-9 Riyami,4 Azza Al-Shidhani,3 Shaima Al-Hinai,3 Ali Alshirawi,5 Sidgi Hasson,1 10 Elias Said,1 Ali Al-Jabri,1 *Aliya Al Ansari2 11 Departments of 1Microbiology & Immunology, 3Child Health, 4Genetics, 5Medicine, College of 12 Medicine, 2Department of Biology, College of Science, Sultan Qaboos University, Muscat, 13 Oman. 14 *Corresponding Author’s e-mail: alansari@squ.edu.om 15 16 Abstract 17 Background: Identifying the human leukocyte antigens (HLA) high risk alleles, genotypes and 18 haplotypes in different populations is beneficial for understanding their roles in type 1 diabetes 19 (T1D) pathogenesis and intervention practices. Objective: The aim of this study was to identify 20 T1D associated HLA gene alleles in the Omani population. Methods: Our case-control study 21 included 73 diabetic seropositive children (mean age 9.08±3.27 years) and 110 healthy controls. 22 HLA–A, -B, -C, -DRB1, and -DQB1 genes were genotyped using sequence specific primer 23 polymerase chain reaction (SSP-PCR). Results: Two HLA class I alleles (B*08, B*58) and three 24 class II alleles (DQB1*02, DRB1*03 and DRB1*04) were associated with T1D susceptibility, 25 while one class I (B*51) and three class II (DQB1*05, DQB1*06, and DRB1*16) alleles were 26 associated with T1D protection. HLA- DRB1*03 and DQB1*02 alleles showed the strongest risk 27 association among all alleles. Six DRB1 residues (E9, S11, S13, Y30, V70 and K71) were significantly 28 associated with T1D susceptibility. Heterozygous genotypes, HLA-DRB1*03/*04 and 29 DQB1*02/*03 were significantly associated with T1D susceptibility (P=4.29E-07, OR=63.2 and 30 P=0.02, OR=3.6, respectively). Furthermore, we detected a significant combined action of 31 DRB1*03-DQB1*02 haplotype in T1D risk (P=1.76E-05, OR=15), and DRB1*16-DQB1*05 32 haplotype in protection (P=3.12E-2, OR=0.48). Conclusion: Known HLA class II gene alleles are 33 associated with T1D in Omani children. 34 Keywords: Type 1 diabetes; human leukocytes antigens; zygosity; alleles; residues; haplotypes, 35 case-control study; Oman 36 37 Advances in Knowledge 38  HLA class II alleles (DQB1*02, DRB1*03 and DRB1*04) are the major genetic risk 39 factors for T1D in Omanis. 40  Combined action in DRB1*16-DQB1*05 haplotype is associated with T1D protection. 41  Combined action in DRB1*03-DQB1*02 haplotype is associated with T1D risk. 42 Application to Patient Care 43  The associated gene alleles can be used for disease prediction and intervention. 44 45 Introduction 46 Type 1 diabetes (T1D) is a common incurable chronic autoimmune disease of childhood, with an 47 estimated incidence increase o f 9.5% globally.1 It is a complex disease that develops from 48 collective contribution from genetic, epigenetic, and environmental factors.2 49 50 Both the cellular and humoral adaptive immune mechanisms are implicated in T1D. The destruction 51 of β-cells driven by self-reactive CD8+ and CD4+ T cells leads to total insulin deficiency.3 52 Autoantibodies to pancreatic islet β-cell autoantigens are detected prior to disease development 53 and are used as biomarkers for β-cells dysfunction and T1D progression.4 54 55 Determining the associated environmental triggers, autoimmune-mechanisms and predisposing 56 genetic background hold potentials for interventions through prediction, prevention or slowing 57 down the rate of disease progression. 58 59 T1D estimated heritability is high (0.53 to 0.92) and familial and population based genetic studies 60 identified more than 60 genes, responsible for about 80% of the disease heritability.5 Most of the 61 T1D genetic predisposition (60%) is attributed to the human leukocytes antigen (HLA) class I and 62 class II genes, in the major histocompatibility complex (MHC) region, which encode for proteins 63 that present antigenic peptides for CD8+ and CD4+ T cells, respectively.6 64 65 Markedly, 45% of the genetic predisposition is attributed to HLA class II genes7, thus, it is 66 considered as a major genetic risk determinant for T1D. The strongest T1D risk is associated with 67 the DRB1, DQA1, DQB1 gene alleles and there is a cumulative supporting evidence for the role of 68 DRB1 and DQB1 genes in combination as a haplotype.8 In European, more than 95% of T1D 69 cases have DR3 (HLA-DRB1*0301-DQB1*0201) or DR4 (HLA-DRB1*04-DQB1*0302). 7 The 70 same HLA susceptibility and protection gene alleles and haplotypes were reported in Arabs.9 71 72 With the current knowledge about autoantigens, genetic risk alleles and biomarkers, disease 73 interventions are more informed and can be considered at three stages: prior to the development 74 of autoimmunity (primary prevention), after autoimmunity is recognized (secondary prevention) 75 or after diagnosis, if significant numbers of β-cells are left (tertiary prevention). 4 76 77 In a study conducted over two years on Omani children with T1D (9 months -14 years), reported 78 incidence rates of 2.45 and 2.62 per 100, 1000 P-Y in 1993 and 1994, respectively.10 The reported 79 gender-specific incidence rates among boys and girls were 3.23 and 1.99 per 100,000 P-Y in 1993 80 and 2.91 and 1.95 per 100,000 P-Y in 1994, respectively. During the two years, they found higher 81 age-specific incidence rates in the 10–14year old group children compared to the younger age 82 group. Furthermore, a retrospective (June 2006 to May 2013) analysis of 144 T1D Omani children 83 reported that the disease is highly prevalent in the family history of these patients (22%).11 84 85 In Oman, the incidence of T1D is comparatively less than other Arabs, and also, ketoacidosis 86 reported to be less in the Omani cases11. Although the Omani population is genetically related to 87 Mediterranean and West-Asian populations12,13, the high frequency of HLA-DR2 and -DQ1 alleles 88 (DRB1*15 and DRB1*16, and DQB1*05 and DQB1*06, respectively) were suggested as genetic 89 protection factor against T1D in the Omani population.14 However, it remains to be elucidated 90 whether this is true or attributed to low frequency of risk alleles. 91 To identify the potential HLA gene alleles associated with T1D risk and/or protection in Omanis, 92 we genotyped T1D patients, attending the pediatric clinic at Sultan Qaboos University Hospital 93 (SQUH) in Muscat for HLA class I (A, B and C) and class II (DRB1 and DQB1) alleles and 94 compared them to healthy Omani controls. 95 96 Materials and methods 97 Statement on Ethics 98 The study was approved by the Ethics Research Committee in the College of Medicine and Health 99 Science. A written informed consent was obtained from all participants guardians enrolled in the 100 study to use their blood sample for research purpose. 101 102 Cases and Controls 103 One hundred Omani diabetic patients attending the pediatric clinic at SQUH were included based 104 on their medical records (mean age 9.31±3.27 years, 47% male and 53% female). All patients did 105 not have another autoimmune disease or syndrome and the diagnosis of T1D was confirmed by 106 the presence of diabetes autoantibodies to islet cell (ICA) and glutamic acid decarboxylase 107 (GADA). Family history of T1D and T2D in cases was recorded. 108 109 Peripheral venous blood samples (5 ml) were collected in EDTA – anticoagulated vacutainer tubes 110 and stored at -20 ˚C. HLA data for 110 healthy potential bone marrow stem cell donors (mean age 111 10.77±3.36 years, 51% male and 49% female) from the national HLA database was used as the 112 healthy population control. 113 114 DNA was extracted from whole blood samples using QIAamp® DNA Medi Kit according to the 115 manufacturer's instructions (Qiagen, Hilden, Germany). DNA concentration and purity was 116 measured using Nano Drop spectrophotometer (ND 2000; Thermo Scientific, Germany). The 117 extracted DNA (20-35 ng/μl) was HLA genotyped for HLA-A, -B, -C, -DRB1, and -DQB1 loci 118 using a commercial sequence specific primer polymerase chain reaction (SSP-PCR) following the 119 manufacturer’s protocol (Olerup SSP). The generated genotypes data are at low resolution. 120 121 Agarose gel (1.3 %) electrophoresis was used to detect the amplified PCR product. The gel was 122 visualized using the gel documentation system INGENIUS 3 (Syngene) with GeneSys software. 123 The appearance of the internal control bands in all lanes indicated successful amplification of the 124 studied DNA. Negative control wells were checked for contamination. HLA genotypes for each 125 locus were identified using the Olerup SSP score software (version 5.00.72.5T). 126 127 Statistical analysis 128 Hardy-Weinberg equilibrium tests were conducted for each locus using the Basic statistics tool 129 (One locus summary) available at HLA-net (https://hla-net.eu/tools/basic-statistics). Alleles at 130 each locus were considered in Hardy-Weinberg equilibrium if the observed and expected 131 (estimated) frequencies did not differ significantly (P > 0.05). 132 133 Tests for allele associations, zygosity, as well as tests for, independence, difference in association, 134 combined action, interaction, and linkage disequilibrium were conducted using PyHLA.15 135 136 The comparison of allele frequencies was performed using Fisher's exact test. The P value for each 137 test was corrected for multiple comparisons by FDR. Adjusted P values less than 0.05 was 138 considered statistically significant. The strength of the association between HLA antigens and T1D 139 was determined by odds ratio (OR). An OR ≥ 1.5 was associated with susceptibility or ≤ 0.5 with 140 resistance. 141 142 In addition, tests for pockets with significant residues association were conducted using SKDM 143 human leukocyte antigen tool.16 144 145 Results 146 Out of the initially screened 100 T1D patients, 73 (73%, mean age 9.08±3.27 years, 41.1% male 147 and 58.9% female) were included in the study because they were seropositive for GADA and/or 148 ICA autoantibodies. Twenty-six patients (26%) were seronegative (mean age 9.77±3.25 years, 149 61.5% male and 38.5% female), out of which three patients (two males and one female) were 150 heterozygous for mutations in different genes (KLF11, WFS1 and HNF1A). About 23% of the 151 seropositive cases have family history of T1D and 59% of T2D. About 19% of the seronegative 152 cases have family history of T1D and 54% of T2D. One patient was excluded as no antibodies test 153 results were reported. 154 155 All tested loci were in Hardy-Weinberg equilibrium in cases but not in controls (Supplementary 156 data). However, as our single center project is considered as a preliminary study, we did conduct 157 the association tests to detect any potential associations. 158 159 HLA Class I and II Loci are Associated with Risk and Protection of T1D 160 Association test results indicated that the risk and protection of T1D in seropositive cases are 161 associated with alleles belonging to the HLA class I (HLA-B) and class II, (HLA-DRB1 and HLA-162 DQB1) genes [Table 1]. 163 164 The strongest significant susceptibility alleles are the HLA-DRB1*03 (P=9.19E-11, OR= 5) and 165 DQB1*02 (P=9.76E-08, OR=3.5). We also observed that the seropositive cases for GADA 166 (98.6%), ICAs (23.3%) or both autoantibodies (21.9%) have more DRB1*03 or DRB1*04 alleles 167 (95.8%), than the seronegative cases (65.2%) and healthy controls (39%). However, the presence 168 of risk alleles did not correlate with higher GADA autoantibody levels and the presence of 169 protection alleles did not correlate with lower levels. 170 171 Seronegative cases also, showed significant risk association with HLA-DRB1*03 and -DQB1*02 172 alleles but to a lesser extent (P=1.74E-3, OR= 5.6 and P=1.20E-2, OR= 4.4). 173 174 The most significant resistance alleles are HLA-DQB1*06 (P=6.40E-05, OR=0.05) and HLA-175 DQB1*05 (P=9.59E-05, OR=0.4). 176 177 Zygosity at HLA Class II Loci is Associated with Risk and Protection of TID 178 The zygosity tests were performed to investigate homozygous, heterozygous, and zygosity 179 associations based on the genotype frequency differences in cases and controls. The results 180 indicated that HLA-DRB1*03 and DQB1*02 zygosity is associated with disease susceptibility 181 (P=2.3E-05, OR=8.2 and P=6.6E-07, OR=9.4, respectively), i.e., significantly higher frequency 182 of risk allele homozygous genotypes than risk allele absent genotypes in cases compared to in 183 controls [Table 2]. 184 185 Notably, heterozygous genotypes, DRB1*03/04 and DQB1*02/03 are associated with significant 186 T1D risk (P=4.294e-07, OR= 63.2; P=0.02, OR =3.6, respectively). 187 188 However, heterozygosity, i.e., higher frequency of risk alleles (B*08, B*58, DRB1*03 DQB1*02 189 and DRB1*04) heterozygous genotypes than risk allele absent genotypes in cases compared to in 190 controls, is associated with disease protection (P=0.03, OR=0.46; P=1.0E-12, OR=0.08; P=3.5E-191 06, OR=0.17; and P=0.01, OR=0.33, respectively). 192 193 T1D protection is associated with zygosity of protective alleles, DRB1*16 (P=1.3E-3, OR=0.10) 194 and DQB1*05 (P=4.5E-05, OR= 0.11) and susceptibility is associated with DQB1*06 195 heterozygosity (P=4.14E-04, OR=10.77). 196 197 Pocket residues of HLA Class II DRB1 chain are Associated with increased risk of T1D 198 As the HLA genotypes dictate the affinity to the presented peptides, the T1D associated HLA 199 alleles are implicated in the selective presentation of self-peptides. Therefore, we investigated the 200 potentially associated residues in the HLA chains using the pocket test. The results showed that 201 six residues (Glu-9, E9; Ser-11, S11; Ser-13, S13; Tyr-30, Y30; Val-70, V70; and Lys-71, K71) in 202 pockets 4, 6, 7 and 9 of HLA class II DRB1 chain are significantly associated with T1D 203 susceptibility [Table 3] [Figure 1]. 204 205 The zygosity analysis for five associated residues showed that only the heterozygotes are 206 associated with T1D susceptibility (E9 P=1.547E-7, 6.04; S11 P=3.13E-12, 10.43, S13 P=3.13E-12, 207 10.43, V70 P=7.357E-13, 11.68, and K71 P=3.13E-12, 10.43). In contrast, residue Y30 homozygotes 208 (P=1.199E-7, 33.65), heterozygotes (P=0.02305, 6.7) and zygosity (P=8.753E-6, 5.02) are all 209 associated with T1D susceptibility. 210 211 Interactions between T1D associated alleles 212 Since T1D association with HLA alleles reported at the haplotypic context as well as the genotypic 213 context, we also analyzed the associated allele interactions. Two haplotypes found to be associated 214 with risk (HLA-B*08-DRB1*03, P8=1.57E-08, OR=12.71 and HLA-DRB1*03-DQ*02, 215 P8=1.66E-12, OR=14.99) [Table 4] [Figure 2]. However, the interaction analysis indicated that 216 DRB1*03 association with T1D is independent of B*08 (P5=8.23E-04, P6=1.95E-09), while B*08 217 association is dependent (P3=0.64, P4=1) and that both alleles have a combined effect in disease 218 (P8=1.57E -08) [Table 4]. Also, our data indicated that a combined -dependent effect of the HLA-219 DRB1*03-DQ*02 haplotype results in T1D susceptibility, while a combined -dependent effect of 220 the DRB1*16-DQB1*05 haplotype results in protection [Table 4] [Figure 2]. 221 222 Discussion 223 The risk and protection to T1D in Omani are associated with alleles belonging to the HLA-B, HLA-224 DRB1 and HLA-DQB1 genes [Table 1], which were reported in other populations.8 This was 225 expected as the Omani population is genetically related to Arab, Mediterranean and West-Asian 226 populations.12,13,17 227 228 The HLA class I alleles associated with T1D susceptibility are B*08 (P=1.82E-02, OR= 2.51), 229 B*58, P=2.86E-02, OR=2.47) and with protection is B*51 (P=1.82E-02, OR=0.41). These 230 associated were reported in previous studies.18 B*08 association with autoimmune diseases was 231 attributed to its presence in linkage disequilibrium (LD) with DRB1*03,18 which we observed in 232 both cases and controls [Table 4]. Furthermore, results indicated that B*08 association is 233 dependent on DRB1*03. Also, B*58 is part of a significantly associated haplotype in North Indians 234 and Han Chinese and results from both populations suggested that the association is not attributed 235 to the allele itself.19,20 236 237 As predicted by a past study, T1D protection in Omanis was found to be associated with HLA-238 DR2 (DRB1*16) and DQ1 (DQB1*05 and DQB1*06) alleles.14 The highest significant resistance 239 alleles are HLA-DQB1*06 (P=6.40E-05, OR=0.05) and HLA-DQB1*05 (P=9.59E-05, OR=0.4). 240 However, despite the high frequency of the DRB1*16 allele in the Omanis compared to other 241 populations21, its significant association with protection is relatively weaker (P= 0.02, OR=0.5). 242 This is likely due to the presence of different alleles (DRB1*16:01:01, 16:02:01 and 16*64, 243 personal communication) in the Omani population and not all are not protective. 244 245 Notably, about 96% of the seropositive cases have either DRB1*03 or DRB1*04 allele but the 246 presence of these alleles did not associate with higher GADA autoantibody levels. Also, no 247 association was detected between GADA autoantibody levels and risk or protection genotypes. 248 249 The zygosity test showed that the HLA-DRB1*03 and DQB1*02 zygosity are associated with risk, 250 while heterozygosity is associated with protection (P=1.0E-12, OR=0.08 and P=3.5E-06, 251 OR=0.17, respectively), indicating that the risk associated with both alleles is recessive, as 252 suggested by others.7 Also, we detected that heterozygous genotypes, DRB1*03/04 (P=4.294e-07, 253 OR= 63.2) and DQB1*02/03 (P=0.02, OR =3.6), are associated with significant T1D risk. 254 255 In contrast, the protection associated with heterozygosity of the same risk associated alleles may 256 be attributed to the presence of protection alleles in the genotypes. Twenty-seven of the HLA-257 DRB1*03 heterozygous cases (44) have one of the HLA-DR2 protection associated alleles (five 258 cases with DRB1*15 and 22 with DRB1*16) and thirty of the HLA-DQB1*02 heterozygous cases 259 (39) have one of the HLA-DQ1 protection associated alleles (29 cases with DQB1*05 and one 260 with DQB1*06). 261 262 Also, the zygosity test showed that the protection associated with DQB1*05 and DRB1*16 are 263 significant in homozygosity, suggesting that the protection associated with both alleles is 264 recessive. 265 266 The side chains of self-peptide residues interaction with the binding groove pockets, stabilize the 267 peptide–HLA-class II complex and therefore they are known as the anchor residues. The binding 268 grooves of HLA class II chains are characterized by the properties of the P1, P4, P6 and P9 pockets 269 that specificity the anchor residues.22T1D associated residues 9, 11, 13 and 30 are located in the 270 -sheet floor and their side chains are in the peptide-binding groove, while residues 70 and 71 are 271 in the -helix but their side chains are close to residue 13 [Figure 1]. DRB1 S13 is in pocket 4, K71 272 in pockets 4 and 7, V70 in pocket 4, S11 in pocket 6, E9 in pockets 6 and 9 and Y30 in pocket 6. As 273 S13, V70 and K71 were associated with the strongest disease risk based on the P values and OR 274 values, they might be the major contributors from pocket 4. 275 276 S13 and K71 association with T1D susceptibility was reported by others23,24 and they were 277 implicated in joint susceptibility to both T1D and autoimmune thyroid disease.25 S11, S13 and K71 278 residues were also associated with risk to rheumatoid arthritis.26 This suggests common disease 279 mechanisms that operate irrespective of the presented self-peptides. 280 281 Transgenic mice expressing TID human class II susceptibility alleles, showed that MHC class II 282 molecules present specific autoantigenic peptides, such as GAD65 peptides27, which can 283 potentially activate autoreactive CD4+ T cells that is known to assist in targeting  cells by 284 cytotoxic CD8+ and autoantibody producing B cells. 285 286 Interaction tests suggested that the association of HLA-DRB1*03 and -DQB1*02 haplotype with 287 T1D risk is resulting from a combined -dependent effect [Table 4]. Notably, 78% of cases with 288 this haplotype were GADA positive, as reported by others.28 This suggested that both susceptibility 289 HLA alleles and anti GAD are risk factors for T1DM. However, we did not detect an association 290 between risk alleles and higher GADA levels. This may indicate that GADA autoantibody level, 291 which is implicated in the destructive process in the islets, is not genetically driven. 292 293 Also, the analysis indicated that the association with T1D is resulting from a combined -dependent 294 effect of the DRB1*16-DQB1*05 haplotype [Table 4]. This haplotype thought to have a protective 295 role, but its rare occurrence in Caucasians and east -Asians, could not prove its effect in T1D 296 resistance. Furthermore, we also believe that DRB1*16-DQB1*05 haplotype in Omanis could 297 potentially protect autoantibody seropositive first-degree relatives from T1D, like the HLA-298 DRB1*15:01-DQB1*06:02 haplotype in other populations.6 299 300 Although other T1D associated haplotypes were reported in the Omani population, such as 301 DRB1*04-DQB1*03 (7.7%), DRB1*07-DQB1*02 (6.4%) and DRB1*15-DQB1*06 (1%) 12, we 302 did not detect significant LD in the investigated group of cases and controls, which is likely due 303 to small sample size. 304 Notably, the frequency of seronegative cases (26%) is higher than what was reported from other 305 ethnic groups (20%).29 However, a relatively weaker association of T1D with HLA-DRB1 and -306 DQB1 alleles in seronegative cases, may reflect the fact that some of the cases may be positive for 307 other autoantibodies associated with T1D that where not tested for in this study or they may show 308 positive on repeat testing, as reported by Hameed et al. .30 309 310 A major limitation of the study was the sample size, because it was based on a single center. 311 Therefore, we recommend conducting a larger size multi-center study to at least double the cases 312 sample sizes and increase the controls to cases ratio (at least 3:1) to reach acceptable power (≥80%) 313 for verifying our preliminary study results. In addition, sequencing of the associated risk and 314 protection allele should be considered. 315 316 Conclusion 317 The majority of the seropositive T1D cases (71%) have family history of T1D and/or T2D. Despite 318 the study small sample size, we identified DQB1*02, DRB1*03 and DRB1*04 as potential risk 319 alleles in GADA and/or ICA seropositive T1D in Omani children. In addition, we detected an 320 association of the DRB1*16-DQB1*05 haplotype with T1D protection in a combined -dependent 321 manner. 322 323 Acknowledgement 324 We would like to thank all patients and their parents. We also aknowledge the support from Dr Irfan 325 Ullah from the Pediatric Department at SQUH, and Ms Faiza Al-Yahyai and Ms Faiza Al-Ghanami and 326 Ms Iman Al-Hadhili from the Genetic lab. Special thanks to Dr Felix Fan and Dr Abdelhamid 327 Abdesselam for their assistance in utilizing pyHLA. 328 329 Conflict of interest 330 The authors declare that they have no conflict of interest. 331 332 Funding 333 This work was supported by TRC fund, Oman (RClMEDlMICRl14l01). 334 335 Authors’ contributions 336 MA-B, AA-J, SH and ES developed the proposal. MA-B, SA-B, AA-S, SA-H and AA collected 337 the data. MA-B and HA-R ordered the required materials. MA-B and SA-B conducted the 338 laboratory work. SA-Y reviewed the clinical and family histories. SA-B and AA-A analysed the 339 data. AA-A drafted the manuscript. MA-B and SA-Y revised the manuscript. All authors 340 approved the final version of the manuscript. 341 342 References 343 1. Mobasseri M, Shirmohammadi M, Amiri T, Vahed N, Fard HH. Prevalence and incidence 344 of type 1 diabetes in the world : a systematic review and meta-analysis. 2020; 10:98-115. 345 doi: https://doi.org/10.34172/hpp.2020.18. 346 2. 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The influence of type 1 diabetes genetic 421 susceptibility regions, age, sex, and family history on the progression from multiple 422 autoantibodies to type 1 diabetes: A teddy study report. Diabetes. 2017; 66:3122-3129. 423 doi: https://doi.org/10.2337/db17-0261. 424 29. Wang J, Miao D, Babu S, et al. Prevalence of autoantibody-negative diabetes is not rare at 425 all ages and increases with older age and obesity. J Clin Endocrinol Metab. 2007; 92:88-426 92. doi: https://doi.org/10.1210/jc.2006-1494. 427 30. Hameed S, Ellard S, Woodhead HJ, et al. Persistently autoantibody negative (PAN) type 1 428 diabetes mellitus in children. Pediatr Diabetes. 2011; 12:142-149. doi: 429 https://doi.org/10.1111/j.1399-5448.2010.00681.x. 430 431 432 Figure 1. Ribbon model of an HLA-DR molecule peptide-binding groove, showing the position 433 and the side-chain of significantly associated residues. The model was based on 3pdo entry from 434 Protein Data Bank and the figure was prepared using Swiss-PdbViewer (http://spdbv.vital-it.ch/). 435 436 437 438 HLA-B HLA-DRB1 HLA-DQB1 S u sc e p ti b il i ty B*08 DRB1*03 DQB1*02 1.82E-02 1.57E-08 9.19E-11 1.66E-12 9.76E-08 http://spdbv.vital-it.ch/ 2.51 4.97 3.47 R e si st a n c e DRB1*16 DQB1*05 1.65E-02 3.12E-02 9.59E-05 0.53 0.40 Figure 2. A representation of detected combined actions between T1D susceptibility and 439 resistance alleles of HLA genes. 440 Top corrected P values and bottom odds ratios. The lines connecting gene alleles represent 441 combined actions with P values on top. 442 443 Table 1. Distribution of significantly associated HLA alleles in T1D cases and controls 444 Allele Cases % Ctrl % P value OR L95 U95 Adjusted P Susceptibility DRB1*03 49.32 16.36 2.30E-11 4.97 3.07 8.06 9.19E-11 DQB1*02 59.59 29.82 2.44E-08 3.47 2.24 5.39 9.76E-08 DRB1*04 19.86 8.18 1.40E-03 2.78 1.48 5.23 2.70E-03 B*08 19.18 8.64 4.00E-03 2.51 1.34 4.69 1.82E-02 B*58 14.38 6.36 1.72E-02 2.47 1.21 5.04 2.86E-02 Resistance DQB1*06 0.68 11.47 3.20E-05 0.05 0.01 0.40 6.40E-05 DQB1*05 26.03 46.79 7.19E-05 0.40 0.25 0.63 9.59E-05 DRB1*16 20.55 32.73 1.24E-02 0.53 0.33 0.87 1.65E-02 B*51 8.90 19.09 7.30E-03 0.41 0.21 0.80 1.82E-02 DRB1*15 3. 42 8.64 5.38E-02 0.38 0.14 1.03 5.38E-02 Association test was performed using PyHLA program 445 446 Table 2. Zygosity test results for the associated HLA alleles 447 Allele Hom_P Hom_OR Het_P Het_OR Zyg_P Zyg_OR DRB1*03 0.43 0.63 1.05E-12 0.07 2.27E-05 8.22 DQB1*02 0.32 1.60 3.51E-06 0.17 6.59E-07 9.41 DRB1*04 1.00 1.21 0.01 0.35 0.18 3.50 B*08 0.37 2.56 0.04 0.46 0.06 5. 61 B*58 0.63 0.6 0.01 0.33 0.62 1.81 DQB1*06 1.00 1.86 4.14E-04 10.77 0.25 0.17 DQB1*05 0.00 0.19 0.14 1.66 4.51E-05 0.11 DRB1*16 0.00 0.10 1.00 1.01 0.00 0.10 B*51 0.45 0.47 0.22 1.67 0.07 0.27 Abbreviations: Hom, homozygous test (homozygous compared to absent); Het, heterozygous test (heterozygous 448 compared to absent); Zyg, zygosity test (homozygous compared to heterozygous); OR, odds ratio. Zygosity test was 449 performed using PyHLA program. 450 451 Table 3. Significant residue associations in the HLA-DRB1 pockets 452 Position Amino acid Association P value Corrected P Odds Ratio Pocket 4 [13,71,78,70,74,26] 13 S + 2.19E-13 1.69E-11 11.46 71 K + 2.19E-13 1.69E-11 11.46 70 V + 3.41E-13 2.63E-11 11.31 Pocket 6 [9,11,30] 9 E + 1.98E-7 1.37E-5 5.43 11 S + 1.04E-12 7.20E-11 10.43 30 Y + 6.92E-05 4.77E-03 12.29 Pocket 7 [28,61,71,47,67] 71 K + 2.19E-13 1.51E-11 11.46 Pocket 9 [9,60,57,37,38] 9 E + 1.98E-7 1.37E-5 5.43 Residue association test was performed using SKDM program 453 454 Table 4. Significant interaction tests including independent association, Difference, action, and linkage disequilibrium (LD) 455 Alleles A independent of B B independent of A Difference Combined action LD in cases LD in controls Allele A Allele B P3 OR3 P4 OR4 P5 OR5 P6 OR6 P7 OR7 P8 OR8 P9 OR9 P10 OR10 Susceptibility B*08 DRB1*03 0.64 1.29 1 0.81 8.23E- 04 15.67 1.95E- 09 9.86 0.00 0.08 1.57E- 08 12.71 0.02 6.4 0.01 4.03 DQB1*02 DRB1*03 0.59 2.22 0.25 1.91 3.43E- 06 7.83 0.10 6.76 0.24 0.28 1.66E- 12 14.99 1.76E- 05 25.61 1.32E- 08 22.13 Resistance DQB1*05 DRB1*16 0.61 0.52 0.02 0.33 0.50 1.45 1 0.92 0.56 0.36 0.03 0.48 1.76E- 10 47.24 8.83E- 11 29.94 If both P3 and P4 are significant, then A is associated with T1D independently of B. 456 If P5 and P6 are significant, then B is associated with T1D independently of A. 457 If both P3 and P5 are significant, then A and B show interaction in T1D. 458 If P7 is significant, then Difference between A and B is associated with T1D. 459 If P8 is significant, then A and B have combined action. 460 If P9 is significant, then A and B are in LD in cases. 461 If P10 is significant, then A and B are in LD in controls. 462 Interaction tests was performed using PyHLA program 463