Significance of HLA in Graves’ disease and Graves’ orbitopathy in Asian and Caucasian populations – a systematic review

Introduction Graves’ disease (GD) and Graves’ orbitopathy (GO) development were suspected to be HLA-related in both Asian and Caucasian populations. However, most studies were performed with application of serological methods or low resolution genetic typing, which led to inconsistent results even among the same population. The present review is intended to summarize the state-of-art knowledge on the HLA significance in GD and GO in Asians and Caucasians, as well as to find the most significant alleles for each of the populations. Methods PubMed was searched for relevant articles using the following search terms: HLA plus thyroid-associated ophthalmopathy or Graves’ disease or Graves’ orbitopathy or thyroid eye disease or thyroid-associated orbitopathy. Results In Asian population GD was found to be associated mostly with B*46:01, DPB1*05:01, DRB1*08:02/03, DRB1*16:02, DRB1*14:03, DRB1*04:05, DQB1*05:02 and DQB1*03:03, while DRB1*07:01, DRB1*01:01, DRB1*13:02, DRB1*12:02 are potentially protective. HLA-B*38:02, DRB1*16:02, DQA1*01:02, DQB1*05:02 can be considered associated with increased risk of GO in Asians, while HLA-B*54:01 may play protective role. In Caucasians, C*07:01, DQA1*05:01, DRB1*03, DQB1*02:01 are associated with GD risk while DRB1*07:01, DQA1*02:01 may be protective. Significance of HLA in the course of GD and novel aspects of HLA amino acid variants and potential HLA-based treatment modalities were also discussed.


Introduction
Graves' disease (GD) is an autoimmune thyroid disorder caused by production of antibodies against thyrotropin (TSH) receptor.TSH-receptor antibodies (TRAb) usually stimulate thyroid hormone production, but they can also block TSH-receptor (TSHR) or have ambivalent character with no impact on thyroid function (1).Additionally, insulin-like growth factor-1 (IGF-1) receptor (IGF-1R) was demonstrated to play an important role in GD development (2).An activation of IGF-1R on orbital fibroblasts may result from the binding of stimulatory IGF-1R antibodies (IGF-1R-Ab) to IGF-1R.Synergistic crosstalk between TSHR and IGF-1R after binding of stimulatory TRAb to TSHR may also be a mechanism leading to IGF-1R activation (3).The prevalence of GD in general population is about 0.5-2.0%(1,4).Like many other autoimmune diseases, GD is typically induced by environmental factors in genetically predisposed individuals (5,6).Graves' orbitopathy (GO) is the most frequent extrathyroidal manifestation of GD, with the estimated incidence of 2.67-3.3cases/100 000/year in women and 0.54-0.9cases/100 000/year in men (7).GO significantly deteriorates patients' quality of life (QoL) and may even lead to a sight-threatening conditions (7).
Therefore, the knowledge on risk factors associated with GD and GO development seems to be crucial for the prevention and management of GO.Many environmental agents were demonstrated to be potential GD-triggering factors, including viral infections, vaccines (8), drugs (9), stress (5), reactive oxygen species (ROS) overproduction (10).Smoking, high serum TRAb levels, severe or unstable hyperthyroidism as well as hypercholesterolemia are already known risk factors of GO development and progression (7,11).
However, as not all exposed individuals are affected andon the other hand -GD and GO are more frequent among family members, it seems obvious that environmental factors always act on a genetic susceptibility which is crucial for the disease development.The strength of genetic susceptibility was proven in environmental, family and twin studies, which suggested that 70% of the risk of GD can be attributed to genetic factors (12).Among genes associated with the autoimmune response, human leukocyte antigen (HLA) genes seem to play a prominent role as a molecular background of GD and GO (13-15).Many different HLA alleles were postulated as GD and/or GO risk factors in Caucasian and Asian populations.The role of other genes such as TSHR, cytotoxic T lymphocyte-associated factor 4 (CTLA-4), protein tyrosine phosphatase non-receptor type 22 (PTPN22), Fc receptor like 3 (FCEL3), interleukin 2 receptor A (IL2RA), thyroglobulin (TG) or cluster of differentiation 40 (CD40) genes was also postulated in GD development (16)(17)(18)(19).Similarly, the risk of GO occurrence in GD patients is associated with genetic susceptibility.Except for HLA, several other genes were suggested as GO-related, including TSHR, CTLA4, tumor necrosis factor (TNF), interferon g (IFN g), interleukin 1A (IL1A) and its receptor (IL1AR), protein tyrosine phosphatase non-receptor type 12 (PTPN12), peroxisome proliferator-activated receptor g (PPAR g) or intracellular adhesion molecule 1 (ICAM-1) genes (4, [20][21][22][23][24].There is evidence that the risk of GD and/or GO is not related to a single gene, but is a result of a complex interaction between genetic factors (4).Nevertheless, considering the importance of the major histocompatibility complex (MHC) for autoimmune responses and taking into account high polymorphism of HLA region, HLA seems to play a prominent role as a molecular background of GD and GO (4, 14,15).
However, results of studies regarding either Caucasian or Asian population were not coherent.The major reasons of this inconsistency are: different sizes of study groups and different methods used by the researchers, including low resolution or serological methods (13).Serological methods detect antigens only, so their accuracy is very low and their application is currently not recommended.More precise results can be obtained with DNA typing methods for HLA analysis.These include i.a.: polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), PCR sequence-specific oligonucleotide probe (PCR-SSOP), PCR sequence-specific primer (PCR-SSP), PCR single-strand conformation polymorphism (PCR-SSCP), and sequence-based typing (SBT).These methods, except for the SBT, require more probes and primers to maintain the acceptable (low or intermediate) resolution, and are unable to detect new polymorphism (25).On the other hand, SBT method can identify all sequence motifs and is capable of detecting new undefined alleles (25).Low resolution methods provide results for the entire allelic group, but not for a particular allele.Serological methods or low resolution DNAbased methods of HLA typing provide results only at antigen or allelic group level.The quality of the results was significantly improved by high-resolution DNA-based typing.In order to further improve this quality, next-generation sequencing (NGS) methods which are based on deep-sequencing of the entire HLA gene followed by bioinformatic processing, have been introduced to provide typing results at the allelic level (25,26).The use of serological or low resolution methods may lead to erroneous conclusions and inconsistency in the study results depending on the applied method.More and more studies which demonstrated method-dependent error in HLA analysis are available.Among a strictly controlled cohort in whom HLA typing was performed for bone marrow transplantation, discrepancies exceeding 29% were reported between less sensitive methods and NGS method (27).Another important example of the risk related to low resolution methods is HLA-B*27 test, which is commonly used to confirm a diagnosis of ankylosing spondylitis.However, it has been recently demonstrated that alleles HLA-B*27:06 and HLA-B*27:09 are probably not associated with the disease, so the results based on less precise methods may lead to a wrong diagnosis (28).Therefore, the significance of applied HLA-typing method seems crucial to unequivocally determine HLA-related background of autoimmune diseases, including GD.
The purpose of this review is to summarize the current knowledge on the HLA significance in GD and GO in Asians and Caucasians, as well as to indicate the most reliable sets of high riskrelated or protective alleles for each of the populations.Identification of an actual sets of such alleles can constitute a reliable tool for the individual risk assessment, and would play an important role in a development of personalized medicine.Additionally, other aspects of HLA significance in GD/GO were also summarized.

Methods
PubMed was searched for relevant articles using the following search terms: HLA plus one of the following: thyroid-associated ophthalmopathy or Graves' disease or Graves' orbitopathy or thyroid eye disease or thyroid-associated orbitopathy.Studies which analyzed HLA-associated susceptibility to GD were included only if comparison with healthy control group was performed.Results based exclusively on comparison with patients with other autoimmune diseases, without a control group, were excluded.In the case of HLA-related background of GO, only studies which provided comparison between GD patients with GO and without GO were included.A total number of 1197 studies were found through initial database searching.Subsequently, after thorough screening of titles and abstracts, 368 studies were selected after exclusion of duplicates, articles not available in English, or in full text, and irrelevant papers.These studies were reviewed in detail.Two hundred seventy two of these were excluded as they did not meet the inclusion criteria or due to data overlapping, insufficient data, or for other reasons.Study selection flowchart is available as a Supplementary Material.A total number of 96 papers were included and, on the basis of the studied population and analyzed factors, these papers were divided into the following groups of papers 1. Results on HLA-related GD risk in Asians, 2. Results on HLArelated GO risk in Asians, 3. Results on HLA-related GD risk in Caucasians, 4. Results on HLA-related GO risk in Caucasians, 5. Results on other correlations between HLA and GD/GO (recurrence risk, clinical course, amino acid variants significance etc.).For further analysis of Group 1 and Group 3, only studies performed with genetic (not serological) methods were included, and only alleles which were reported as related to increased or decreased risk in more than one study were presented as summary results.

Population-dependent differences and comparison difficulties
An importance of significant ethnic differences was indicated in many studies on HLA-related susceptibility to GD (5,6,(29)(30)(31)(32)(33)(34).Furthermore, the results already published for either Asian or Caucasian populations are inconsistent (6,(29)(30)(31)(32)(33)(34).Additionally, the symbols of alleles used for the previous methods differ from the ones currently used.Application of high-resolution methods changed the obtained results significantly, because antigens previously denoted by a given symbol have been separated into many different alleles (13).This fact obviously highly influenced the accuracy and coherence of the already published results.

HLA and GD development
Many studies on the potential significance of HLA for GD in Asians were performed.Since 1978, several of them have postulated the impact of HLA-B*46 in the development of GD (31)(32)(33).However, with application of serological methods, in some studies, HLA*Bw46 significance was demonstrated in males only, with lack of correlation in females (34)(35)(36).Similar gender-limited relationship was observed between GD and HLA-DR9 and DQB1*03:03 (36).A metanalysis of 14 studies performed by Li et al. in 2013 demonstrated association between HLA-B*46 and GD in Asians (31).Although, most of the authors of the included studies applied serological methods, more recent reports confirmed this correlation (37-40).The overview of the published results on HLA significance in GD are demonstrated in Table 1.It is clearly visible that the obtained results are not consistent and potential high risk alleles or protective ones are different even in similar populations (i.e. in the Chinese, the Japanese, the Korean etc.).Several antigens other than Bw46 were postulated by authors who used serological methods, including, for example, HLA-DRw8, -DQw4, -B5, Dw12 and -A11 antigens (44)(45)(46).At the end of the 20th century, polymerase chain reaction (PCR) sequence specific oligonucleotide probe (SSOP) method was introduced and many authors applied it at least for MHC class II analysis.Similarly to the serology-based reports, the studies with combined serological and SSOP methods revealed inconsistent results.Chan et al. reported that the risk of GD was higher in patients with HLA-A*2, Cw1, DRB1*16:02, DRB1*03:01, DRB1*14:05, DRB5*02, DQB1*05:02, while the presence of HLA-DRB1*15:01 and -DQB1*03:01 played a protective role (35).On the other hand, Japanese authors presented that the most important factor was the presence of HLA-DPB1*05:01 and/or HLA-A*2, with the risk being the highest in carriers of both of them (46).In one Taiwanese study, HLA-A*02:07 was found a GD risk factor (37), while other Taiwanese authors showed correlation between GD and HLA-B*46:01, DPB1*05:01, DQB1*03:02, DRB1*15:01 and DRB1*16:02, with the strongest relationship existing with HLA-DPB1*05:01 (38).
There are discrepancies in the obtained results, not only between methods applied by the authors or between subpopulations based on patients' nationality, but also between studies performed with the same method and among the same subpopulation (40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57).Additionally, many studies included MHC class II alleles only, or even one group mostly HLA-DRB1 (48)(49)(50)(51)(52). Therefore, direct comparison of the results, or any attempt to include the studies into metanalysis, would be subjected to high risk of error.The most reliable results obtained with NGS method are scarce.Katahira et al. did not compare their GD patients to a control group but only to patients with Hashimoto thyroiditis.Therefore, they are not suitable for comparison with others.Ueda et al. presented results as a list of alleles associated with high risk of GD and a list of 3 haplotypes which showed significant protective effects against the development of GD in Japanese population Taking into account the difficulties in direct comparison of the results, we decided to analyze a number of studies which revealed a given allele as the high risk one or the protective one.In order to obtain the most reliable results we included all studies which used methods which   2).On the other hand, the following alleles were reported as potentially protective: DRB1*07/DRB1*07:01 (4 studies), DRB1*01:01 (3 studies), DRB1*13:02 (3 studies), DRB1*12:02 (3 studies) (Table 2).
It is worth indicating, that among the alleles found as related to high risk of GD on the basis of our present review, HLA-DRB1*16:02 is in linkage disequilibrium with DQB1*05:02 (58), so they cannot be considered independent risk predictors.Additionally, among alleles demonstrated as related to lower risk HLA-DRB1*01:01 is in linkage disequilibrium with DQB1*05:01,

HLA and GO development
Similarly to GD, the results concerning HLA associations with GO development are inconsistent.The overview of the published results is presented in Table 3. Inoue et al. used serological method in Japanese patients and demonstrated that only DQw3 seemed to be a GO risk factor (59).However, in a study published a year later, Inoue et al. found that the risk of GO was associated with the following sets of HLA antigens: HLA-DQw4 without presence of -A31, HLA-A11 without presence of -DPw2, and with a copresence of HLA-B5 and -Dw12 (45).Other authors applied serological methods, in another group of patients from Japan, and obtained entirely different results (60).They reported that predisposition to severe GO can be related to a presence of HLA-DR14 and DQ1 antigens, while HLA-B35, B54, DR4, and DQ4 were postulated to play a protective role (60).
In Iranian cohort, no differences in allele distribution between patients with and without GO was postulated (61).On the contrary, in a Korean study, HLA-C*03:03 allele had higher frequency of occurrence in GO as compared to non-GO patients (14).In a Chinese cohort described by Huang et al., the HLA-B*38:02, DRB1*16:02, DQA1*01:02 and DQB1*05:02 were postulated as GO high risk alleles (62).No consistent results were found (Table 3), therefore, no allele can be unequivocally selected as actually GO related.Taking into account the significance of the applied method and the size of groups, the results presented by Huang et al. in 2021 can be considered the most reliable, as they were performed by NGS method in 82 GO patients, 272 GD patients and 411 healthy controls (Table 3) (62).Therefore the alleles HLA-B*38:02, DRB1*16:02, DQA1*01:02 and DQB1*05:02 can be considered associated with increased risk of GO in Asians, although such relationship was demonstrated in one study only and requires further confirmation.Moreover, HLA-DRB1*16:02 is in linkage disequilibrium with DQA1*01:02 and DQB1*05:02 (58), so their associations with GO cannot be considered independent.On the other hand, the significance of HLA-B*38:02 seems to be entirely independent.In this study, no potentially protective alleles were selected, however there was a concordance in two previous studies in regard to HLA-B*54/HLA-B*54:01 which was demonstrated to play a protective role (14,60).Therefore HLA-B*54:01 can be considered as associated with lower GO risk in Asian population.
Many other alleles were postulated, but hardly any consistency was found between the results.Vita 13).Unfortunately, it was the first study which applied NGS in Caucasian patients, therefore, no comparison with other NGS-based studies is possible.The overview of studies focused on associations between HLA and GD in Caucasian population is presented in Table 4.
Similarly to Asian population, there were discrepancies in the applied methods, group size and analyzed MHC classes (13, 63-91).Most of the studies included only one MCH class, mainly class 2, while some studies included even only one gene, predominantly HLA-DRB1 (Table 4).Therefore, due to difficulties in direct comparison of the results, we analyzed numbers of studies which revealed a given allele as a high risk one or a protective one.In order to obtain the most reliable results we included studies which used methods which allowed to obtain allelic specificity, or studies which reported allele group concordant with results in which a specific allele was provided (e.g.DRB1*03 and DRB1*03:01/04).We did not include any of the results obtained by serological methods.
The significance of HLA-DRB1*03:01 as GD high risk allele is the only one which is common for both Asian and Caucasian populations.The alleles HLA-DRB1*03:01, DQA1*05:01 and DQB1*02:01 are in strong linkage disequilibrium in Caucasian population (58).Therefore, their significance cannot be considered entirely independent, however, the single presence of any of them can be correlated with the risk of GD.Moreover, some studies demonstrated a common association of HLA-B*08:01, which was also reported as GD high risk allele, with DRB1*03:01 and DQB1*02:01 (6,13,29,30,77).In our recent study, the combination of these three alleles occurred in 22% of patients with GD, while it was found only in 5.87% of the healthy control group (13).Interestingly, in patients in whom HLA-B*08:01 was present, it occurred with alleles other than the ones described above only in 1.9% of GD patients (13).HLA-B*08:01 is in linkage disequilibrium with HLA-C*07:01another high risk allele, whose significance in GD was also demonstrated (92, 93).
On the other hand, HLA-DRB1*07:01 was the allele most commonly demonstrated to be a protective one.Interestingly, this observation is the only one consistent with results of studies in Asians in regard to the protective alleles.It should be indicated that HLA-DRB1*07:01 is in linkage disequilibrium with two other potentially protective alleles -HLA-DQB1*02:02 and DQA1*02:01 (58).HLA-C*03:04, which is in no linkage disequilibrium with other discussed alleles, seems to be the only entirely independent protective allele (34,92).Therefore, the alleles HLA-C*03:04, DRB1*07:01, DQB1*02:02 and DQA1*02:01 can be considered a group of GD protective alleles.
Some differences in the results between Caucasian and Asian populations seem unexpected.Among the alleles, which are in linkage disequilibrium with HLA-DQA1*05:01, only DRB1*03:01 was proved to be GD-related in both Asian and Caucasian populations (13, 30, 35, 54, 91).However, these linkage disequilibrium-based correlations are not exclusive for the Caucasian population, but are common in all analyzed populations, including Asians (58).Therefore, it remains unexplained why these correlations found in Caucasians are absent in Asians, in whom completely different alleles were reported as high risk of GD development.Furthermore, on the basis of the present review, a phenomenon of opposite roles of HLA-DQB1*02:01 can be observed.This allele was demonstrated as high risk in Caucasians (6, 13), but it was also reported as protective in Asian pediatric studies (48,49).This population-dependent opposite correlation indicates the potential significance of other factors influencing GD risk in either population.Further studies with application of high resolution methods are required to solve this puzzle.

HLA and GO
Similarly to the current state of art in Asians, the results concerning HLA associations with GO development in Caucasians are inconsistent (Table 6).From the first study in 1980, DR3 antigen was being demonstrated in serological studies to be not only associated with GD high risk, but also with GO development (66).However, several studies, using different methods, both serological and genetic, did not confirmed such association (36,72,97) and the results were highly divergent (80,(94)(95)(96)(97)(98)(99).Even a quite recent study performed by Yin et al. did not confirm the existence of HLA-related susceptibility to GO in the group of patients with GD and postulated the importance of environmental or epigenetic factors only (99).However, the authors of that study, similarly to some other earlier ones, focused only on the frequency of HLA-DR3, without assessing the frequencies of other alleles, and applied low resolution method.Recently, the significance of HLA-DRB1*03:01 was demonstrated by  (15).Unfortunately, it was the first study which applied NGS in Caucasian patients, and thereforesimilarly to the studies on GD described aboveno comparison with other NGS-based studies is possible.The overview of studies focused on associations between HLA and GO in Caucasian is presented in Table 6.
In our study, based on NGS method, HLA-DQB1*02:01 was also found to be a high risk allele (15).Such phenomenon could be expected as this allele is in linkage disequilibrium with HLA-DRB1*03:01 (58).Interestingly, another allele demonstrated to be GO-related in our studyi.e.HLA-DRB1*14:01, was previously observed to be GO-related in Japanese patients (60) (Table 1).This similarity is the only one between our results in Caucasian population and the already published data for Asian population in regard to GO.Nevertheless, such a lack of coherence between Asians and Caucasians could be expected, because HLA-related susceptibility for many autoimmune diseases differs between these two populations.In our NGS-based study, the highest risk of GO was associated with the presence of HLA-C*08:02 (OR 6.9) and -B*37:01 (OR 4.5) as well as DRB1*14:01 (OR 6.2) (15).There is no linkage disequilibrium between these three alleles (58, 92, 93), so the presence of any of them constitutes independent high risk factor.
Interestingly, in the same study, we demonstrated a protective effect of HLA-C*04:01 allele, which was previously reported as related to increased risk of subacute thyroiditis (SAT) (100,101).This coincidence can be considered an explanation of the fact that  SAT and GO actually occur together extremally rarely in clinical practice.It is worth noting that HLA alleles had already been suggested to be related with the course of SAT and GD in patients with simultaneous presence of both these diseases (102).(40).
Some studies suggested that the age of GD onset is HLAdependent.As it was indicated above, it was postulated that HLA-Bw46 is associated with younger age of GD onset (32, 34), while B5 was suggested as related to older age of onset (32).Few years later, Onuma et al. published entirely opposite results and stated that HLA-B46 is related to late onset of GD, while DPB1*05:01 is associated with early onset (47).In Japanese and Chinese studies, which included children only, DQB1*03:03 was demonstrated to be GD-related (48,49).However, in a recent study performed in Korean children, previously postulated significance of HLA-B*46:01 was confirmed, andadditionally -HLA-C*01:02, DPB1*02:02, DPB1*05:01, occurred to be significantly associated with GD (14).
In a recent study by Azizi et al. with application of SSP method, DQB1*05 HLA polymorphism was demonstrated to be related to GD relapse (103).The authors analyzed relapse rates in patient during 48 months period after methimazole withdrawal (103).Unfortunately, no other similar study in Asians is available for comparison.

HLA and GD recurrence and course
In Caucasian population, HLA-DQA1*05 variant was demonstrated to have ability to predict relapse.Surprisingly, combinations with other HLA risk genes forming the risk haplotype DRB1*03-DQA1*05-DQB1*02 did not improve the predictive value (104).Several years earlier, no significance of HLA DQA1*05:01 in GD recurrence was reported by Badenhoop et al. (95) However, a study in which patients were observed for two years after antithyroid drug withdrawal revealed that HLA-DRB1*03, DQA1*05, and DQB1*02 polymorphisms are strong predictors for recurrence after antithyroid drug therapy (105).There is a strong linkage disequilibrium between the them and their significance in GD relapse in Caucasians should be considered.
Most of the studies focused on GD recurrence used serological methods.In a study performed by Shifferdecker et al. in German population, HLA DR5 was associated with relapse of GD, whereas HLA DR7 and B12 were negatively correlated with relapse (75).A role of B8 was postulated by Irvine et al, who observed significantly more common relapses in B8 positive patients (106).Another study demonstrated significance of HLA-DR3 haplotype and lack of association with HLA-B8 haplotype and GD relapse (107).Some studies underlined the role of HLA-DR3 (65, 108), while other did not confirmed this association (109).Similarly, de Bruin et al. found no significance of HLA-DR3, but demonstrated that HLA-Cw7 was associated with relapse, while HLA-DR4 was protective against recurrence (110).In a study by Young et al., neither HLA-B8 nor HLA-DR3 conferred increased likelihood of relapse (111).Significant association of GD relapse and DQA2 genotype was found with application of RFLP method (112).Most of the authors did not find any correlation between HLA and GD recurrence (113-117).
Few studies analyzed the severity of GD in regard to HLA.Preus et al. demonstrated that patients with severe course were characterized by a high frequency of HLA-A1 andHLA-B8, while mild course patients showed a higher frequency of HLA-B12 (118).Some studies focused directly on the HLA associations with GD onset.In a study by Lavard et al.DRB1*03 and DRB3*01:01 were associated with juvenile GD onset (<20 years of age), whereas DRB3*02:02 was associated with adult onset of the disease (85).Other clinical course-related associations demonstrated that HLA-DR3 positive GD patients were also found to be more resistant to radioiodine therapy than patients negative for these antigens (66).Additionally, patients with GD and haplotype HLA-DRB1*03/ DRB1*11 were found to have higher FT4/TT3 ratio and antithyroglobulin antibody levels (91).
Unfortunately, most of studies which analyzed GD recurrence or the disease course were performed decades ago and applied serological methods.Therefore, the described discrepancies in the obtained results were not unexpected, and currently, no clear correlation in regard to HLA and GD severity or age of the disease onset can be confirmed.Further studies using high resolution methods may provide conclusive results.

HLA and non-genetic risk factors of GO
Increased level of total cholesterol (TC) and/orof lowdensity lipoprotein cholesterol (LDL) is generally known to increase the risk of GO.Very recently, our research team demonstrated a significant correlation between the higher TC/ LDL levels (11) and the occurrence of GO-related high-risk alleles (HLA-B*37:01 and C*03:02).Moreover, the presence of alleles associated with GD without GO (HLA-C*17:01 and B*08:01), as well as alleles which are in linkage disequilibrium with B*08:01 (i.e., HLA-DRB1*03:01 and DQB1*02:01), was shown to be correlated with lower TC levels (11).These results seem important as they provide evidence that correlations between TC/LDL and GO can be HLA-dependent and confirm the relevance of TC/LDL lowering therapy in the cases where the risk of the development of GO is significant.

Significance of HLA amino acid variants in Asians and Caucasians
The significance of amino acid variants of HLA molecules were also postulated in the context of risk of GD development.In a study by Shin et al., both Leu35 (OR = 23.38,P = 0.0002) and Glu55 (OR = 23.38,P = 0.0002) of HLA-DPB1 were strongly associated with GD (14).These authors concluded that amino-acid signatures of the HLA-DP b chain, might contribute to the molecular pathogenesis of GD (14).However, in Japanese population, amino acid variants in HLA-DRB1 allotypes were examined, and critical significance of position 9 amino acid variants for GD development was found (57).Glu-9 variant, which was reported as related to GD high risk, is encoded by HLA-DRB1*03:01, DRB1*04:05 and -DRB1*14:03 alleles, which confer susceptibility to GD in several studies.On the other hand, Cys-9 variant, which was reported as protective against GD, is encoded by HLA-DRB1*07:01, DRB1*01:01 and DRB1*15:02 alleles, which were demonstrated to be protective against GD (57).It is speculated that changes in the charge and polarity of amino acids at these positions modify the three-dimensional structure of the DR peptide-binding pocket, which leads to susceptibility to or protection against GD (57, 119).Negatively charged Glu-9 was demonstrated to confer susceptibility to GD, while uncharged Cys-9 conferred protection against GD (57).
It was postulated that HLA-DR3 (HLA-DRB1*03) Arg-74 is the critical amino acid for the development of GD (88, 120).The group HLA-DR3 includes more than 30 alleles.A HLA-DRb (beta chain of HLA-DR3) pocket variant with arginine at position 74 (HLA-DRb-Arg74) is associated with an increased risk of GD, while glutamine (HLA-DRb-Gln74) variant was protective (88, 119, 120).Three-dimensional computer modeling of the HLA-DR pocket allowed proving that electrostatic potential of HLA-DRb-Arg74 generates a more positively charged P4 HLA-DR pocket than HLA-DRb-Gln74.Therefore, the susceptibility to GD may be related to electrostatic potential of HLA-DR pocket and positive charge can be a high risk factor.The significance of this phenomenon can be confirmed by the finding that, among DRB1 molecules, DRB1*03 contains a positively charged Arg at b74, and DRB1*07 contains a non-charged Gln at b74 position (88).This positive charge difference is believed to facilitate auto-antigen presentation and T-cell activation because of its increased binding affinity of pathogenic self-peptides to the HLA-DR pocket (121).Candidate pathogenic peptides in GD include peptides derived from TSHR and Tg.This interaction between peptides of thyroidal origin and the HLA-DRb-Arg74 pocket was considered as potential therapeutic target (121).Such an antigen-specific immunotherapy may constitute a crucial progress in the GD therapy, as current treatment modalities do not reverse the autoimmune process.It is well known that TSHR is the major autoantigen in GD.A TSHR peptide, designated TSHR.132, was found to be a dominant TSHR peptide in GD (122-124).TSHR.132 was demonstrated to bind with high affinity to recombinant HLA-DRb-Arg74 and to cells that express HLA-DRb-Arg74 (121).Cepharanthine is a small molecule compound which was demonstrated to inhibit T-cell activation by TSHR.132 ex vivo in splenocytes isolated from humanized mice induced with EAGD (experimental autoimmune Graves' disease) (123,124).
Cepharantine directly interacts with Arg74 and blocks the HLA-DR3 pocket variant associated with AITD including GD. Due to the significant role of HLA-DR3 in many autoimmune diseases, cepharantine may be a potentially efficient causative therapy for GD and other autoimmune disorders such as Addison's disease, Hashimoto's thyroiditis, myasthenia gravis or SLE (121,123,124).
Additional various HLA polymorphisms were also described as associated with GD.In two independent Caucasian populations, an intronic variant (rs3094228) in HLA complex P5 (HCP5) was demonstrated to be associated with GD susceptibility and age of onset, which indicates a potential role of long non-coding ribonucleic acids, including HCP5, in GD pathogenesis (125,126).HCP5 gene polymorphism (rs3094228) was associated with an earlier age of GD onset and patients with higher number of the HCP5 risk alleles tend to have a significantly earlier onset of GD (126).

TABLE 1
Associations between HLA and Graves' disease in Asian population.

TABLE 2
Alleles reported as Graves' disease (GD)-related in more than one study in Asian population.

TABLE 3
Associations between HLA and Graves' orbitopathy in Asian population.

TABLE 4
Associations between HLA and Graves' disease in Caucasian population.

TABLE 5
Alleles reported as Graves' disease (GD)-related in more than one study in Caucasian population.

TABLE 6
Associations between HLA and Graves' orbitopathy in Caucasian population.