Abstract
Since individuals with Addison’s disease (AD) present considerable co-occurrence of additional autoimmune conditions, clustering of autoimmunity was also predicted among their relatives. The study was aimed to assess circulating autoantibodies in first-degree relatives of patients with AD and to correlate them with the established genetic risk factors (PTPN22 rs2476601, CTLA4 rs231775, and BACH2 rs3757247). Antibodies were evaluated using validated commercial assays, and genotyping was performed using TaqMan chemistry. The studied cohort comprised 112 female and 75 male relatives. Circulating autoantibodies were found in 69 relatives (36.9%). Thyroid autoantibodies, that is antibodies to thyroid peroxidase (aTPO) and thyroglobulin (aTg), were detectable in 25.1 and 17.1% relatives, respectively. Antibodies to 21-hydroxylase (a21OH) were found in 5.8% individuals, and beta cell-specific antibodies to ZnT8, GAD, and IA2 were found in 7.5, 8.0, and 2.7%, respectively. The prevalence of a21OH (P = 0.0075; odds ratio (OR) 7.68; 95% CI 1.903–36.0), aTPO (P < 0.0001; OR 3.85; 95% CI 1.873–7.495), and aTg (P < 0.0001; OR 7.73; 95% CI 3.112–19.65), as well as aGAD (P = 0.0303; OR 3.38; 95% CI 1.180–9.123) and aZnT8 (P = 0.032; OR 6.40; 95% CI 1.846–21.91), was significantly increased in carriers of rs2476601 T allele. Moreover, T allele appeared to be a risk factor for multiple circulating autoantibody specificities (P = 0.0009; OR 5.79; 95% CI 1.962–15.81). None of the studied autoantibodies demonstrated significant association with rs231775 in CTLA4 (P > 0.05), and only weak association was detected between BACH2 rs3757247 and circulating aTPO (P = 0.0336; OR 2.12; 95%CI 1.019–4.228). In conclusion, first-degree relatives of patients with AD, carriers of the PTPN22 rs2476601 T allele, are at particular risk of developing autoantibodies to endocrine antigens.
Introduction
Numerous genes associated with susceptibility to autoimmune diseases have been identified to date. Many of these genetic variants are shared by several disorders, including not only organ-specific but also systemic conditions. Furthermore, familial clustering of autoimmunity was observed among the relatives of patients suffering from systemic lupus erythematosus (SLE), multiple sclerosis, Graves’ disease, and type 1 diabetes (T1D) (1, 2). Recently, we and others have also confirmed increased risk for autoimmune disorders in families of patients with autoimmune adrenal insufficiency, that is Addison’s disease (AD) (3, 4). Patients suffering from AD display extreme predisposition to other autoimmune disorders, and more than half of them present with additional autoimmune diseases (5, 6, 7). This particular trait suggests an inborn immune defect, which potentially may be transmitted within their families. However, most of the autoimmune endocrine diseases, including the majority of AD cases, display complex inheritance patterns. Furthermore, although less than 10% of AD patients have a relative with the same condition, various other endocrine diseases emerge in their family members (4, 5, 8). Our former study revealed that nearly 40% first-degree relatives of the AD patients displayed circulating serum autoantibodies to the endocrine gland-specific antigens compared to just 8.4% subjects with no family history of autoimmunity (9). Familial incidence of the autoimmune disease might be partially due to similar environmental exposures, but high concordance rates in monozygotic twins support substantial role of the genetic factors associated with the incidence of T1D, AD, and autoimmune thyroid disease (AITD) (10, 11, 12). Numerous analyses confirmed that these disorders share much of their molecular background, with the most prominent effect of the class II HLA haplotypes, especially DR3/DQ2 and DR4/DQ8 (13, 14). Polymorphic variants of other genes implicated in the immune function, such as PTPN22, CTLA4, and BACH2, are equally associated with endocrine autoimmunity (15, 16, 17, 18). PTPN22 encodes a negative regulator of T cell receptor signaling, which additionally may promote inflammation through Toll-like receptor-induced type 1 interferon production (15). Furthermore, its p.620W variant (rs2476601) may affect the development of Treg cells (19). CTLA4 is an inhibitory checkpoint molecule, which downregulates T cell responses; hence, its functional polymorphisms may impair protein action and lower the threshold for T cell activation (20, 21). Finally, BACH2 is a transcription factor essential for B cell differentiation and class switch recombination, also involved in shifting the T cell balance toward the development of Treg cells (22). Carriers of the risk alleles at PTPN22 1858T, CTLA4 CT60, and BACH2 rs3757247 seem more prone to develop multiple autoimmune conditions (23, 24).
Therefore, the aim of our study was to evaluate the association of the established genetic markers of the autoimmunity risk, i.e. polymorphisms in PTPN22, CTLA4, and BACH2 genes, with the presence of serum autoantibodies against endocrine-specific antigens in first-degree relatives of patients suffering from autoimmune AD.
Patients and methods
In this study, relatives of 78 individuals with autoimmune AD were evaluated. The original patients’ cohort comprised 56 affected females and 22 males. Their mean age was 50.4 ± 12.5 years, and the mean age at AD onset was 37.0 ± 11.9 years. Only 15 (19.2%) of them suffered from isolated AD, while the remaining 63 (80.8%) displayed autoimmune comorbidities: Hashimoto’s thyroiditis in 46 (59.0%), Graves’ disease in 14 (17.9%), chronic atrophic gastritis in 11 (14.1%), T1D in 10 (12.8%), vitiligo in 7 (9.0%), hypergonadotropic hypogonadism in 6 (7.7%), alopecia in 1 (1.3%), celiac disease in 1 (1.3%), and Sjögren syndrome in 1 (1.3%) person with AD.
Eventually, our cross-sectional study comprised 187 first-degree relatives (33 parents, 58 siblings, and 96 children) of these AD patients. The participants were recruited through their affected relatives followed at the regional referral outpatient clinic for the endocrine diseases. Only family members of patients with confirmed autoimmune origin (positive serum autoantibodies to 21-hydroxylase (a21OH)) of the primary adrenocortical failure were enrolled. Families with monogenic autoimmune polyglandular syndrome type 1 were not recruited. Relatives were considered eligible if they were at least 12 years old. The study protocol complied with the Declaration of Helsinki and was approved by the local ethical board at Poznan University of Medical Sciences (decisions 539/17 and 39/18). All adult participants and legal representatives of the minors gave their informed written consent. Individuals aged from 16 to 18 years old had to formally agree for their participation as well.
Blood samples were taken in the morning after overnight fast, centrifuged to separate serum, and stored at –20°C until analyzed. Circulating antibodies to autoantigens are as follows: steroid a21OH, thyroid peroxidase (aTPO), thyroglobulin (aTg), glutamic acid decarboxylase (aGAD), zinc transporter-8 (aZnT8), and tyrosine phosphatase (aIA2) were detected by RIA- and ELISA-based commercial assays: 21-Hydroxylase Autoantibody ELISA Kit (RSR Ltd. Cardiff, UK), Anti-TPOn RIA kit, ThermoScientific (BRAHMS GmbH, Hennigsdorf, Germany), Anti-TGn RIA kit, ThermoScientific (BRAHMS GmbH), GAD65 Ab RIA kit (DRG International Inc., Marburg, Germany), Zinc Transporter 8 Autoantibody ELISA Kit (RSR Ltd), and IA2 Ab RIA kit (DRG International Inc.), respectively. RIA results were read on scintillation gamma counter Wallac 1470 Wizard (Perkin Elmer, Turku, Finland), while the absorbance from ELISA assays was analyzed on ELx808 Absorbance Reader (BioTek) using Gen5 Microplate Reader Software.
Polymorphisms rs2476601 in PTPN22, rs231775 in CTLA4, and rs3757247 in BACH2 gene were investigated. Genomic DNA was extracted from the peripheral blood using Gentra Puregene Blood Kit (Qiagen). Genotyping was performed in Bio-Rad CFX96 Real-Time Detection System, using validated commercial TaqMan SNP Genotyping assays (C_16021387_20 for rs2476601, C_2415786_20 for rs231775, and C_27475051_10 for rs3757247, respectively) following the conditions recommended by the manufacturer (Applied Biosystems by Thermo Fisher Scientific). Genotypes were confirmed in 10% samples by direct DNA sequencing with BigDye® Terminator Cycle Sequencing Ready Reaction Kit and 3730xl Genetic Analyzer (Thermo Fisher Scientific). Moreover, a subset of 32 randomly selected samples was genotyped twice for accuracy. Genotypes were checked for Hardy–Weinberg equilibrium (threshold P > 0.05) using an online calculator available at the GeneCalc website (https://gene-calc.pl/hardy-weinberg-page).
The control cohort consisted of 393 healthy blood donors (245 females and 148 males) with negative history of autoimmunity, and no clinical signs of the autoimmune disorders. Moreover, given that Hashimoto’s thyroiditis, which may develop insidiously (25), is one of the most common autoimmune conditions in the population, each candidate for healthy control underwent evaluation of serum aTPO, and only subjects with negative result were enrolled. Their mean age was 40.1 ± 16.4 years.
Statistical analyses were performed using GraphPad Prism 6.0c (GraphPad Software). The chi-squaretest was used for association analyses on 2 × 2 and 2 × 3 contingency tables. Two-tailed P values <0.05 were considered statistically significant. The power of the study was evaluated by means of the online PS power and sample size calculator v.2.1.30 (Vanderbilt University, TN, USA). Assuming an allelic odds ratio (OR) of 1.5 which is close to that reported in former studies of rs2476601, rs231775, and rs3757247 (25, 26, 27) and given the minor allele frequencies as observed in the control group, our analysis showed 63, 89, and 90% power to detect an effect of the studied polymorphisms in PTPN22, CTLA4, and BACH2, respectively (at P = 0.05).
Results
The investigated cohort of the first-degree relatives of patients with AD comprised 112 (59.9%) females and 75 males. Their mean age was 36.4 ± 16.7 years – 155 participants were adults, while 32 (17.1%) subjects were 12–18 years old. Serologic analysis revealed the presence of circulating autoantibodies in 69 relatives (36.9%), with just one of the studied antibodies found in 35 (18.7%) individuals, and more than one autoantibody detectable in the remaining 34 (18.2%) relatives (Table 1). Thyroid autoantibodies, aTPO and aTg, were the most frequent, found in 25.1 and 17.1% relatives, respectively. Moreover, aTPO and aTg were the most commonly coexisting antibodies, detected together in 18 (9.6%) relatives, whereas other antibody combinations were observed in just one to three subjects. a21OH were detectable in 5.8% individuals. Finally, beta cell-specific antibodies to ZnT8, GAD, and IA2 were discovered in 7.5, 8.0 and 2.7% of the relatives, respectively. Sex-related differences in the prevalence of the studied autoantibodies were only found for aTPO, which appeared more frequent in female relatives compared to males (31.3% vs 16.0%, P = 0.0185) (data not shown), yielding an OR of 2.386 (95% CI 1.176–4.924). The frequencies of the circulating autoantibodies among parents, siblings, and children of the AD patients were equally explored (Supplementary Table 1, see section on supplementary materials given at the end of this article) but did not reveal significant differences between various family members (all P-values >0.05).
Serological characteristics of the first-degree relatives of patients with Addison’s disease.
| Autoantibody | Relatives |
|---|---|
| a21OH positivity | 11 (5.8%) |
| aTPO positivity | 47 (25.1%) |
| aTg positivity | 32 (17.1%) |
| aZnT8 positivity | 14 (7.5%) |
| aGAD positivity | 15 (8.0%) |
| aIA2 positivity | 5 (2.7%) |
| Any autoantibody positive | 69 (36.9%) |
| One positive autoantibody | 35 (18.7%) |
| Two positive autoantibodies | 19 (10.2%) |
| Three positive autoantibodies | 10 (5.3%) |
| Four positive autoantibodies | 4 (2.1%) |
| Five positive autoantibodies | 1 (0.5%) |
Serum autoantibodies against: a21OH, 21-hydroxylase; aTPO, thyroid peroxidase; aGAD, glutamic acid decarboxylase; aIA2, tyrosine phosphatase; aZnT8, zinc transporter-8.
Similar to our previous study led in a smaller cohort of 113 relatives (9), we evaluated the association of the AD patient’s own features with the risk of autoimmunity in his/her family. We could confirm that circulating autoantibodies were found more frequently in relatives of the affected males (54.7%) than in relatives of AD females (29.9%), producing an OR of 2.840 (95% CI 1.463–5.336, P = 0.0015). Family members of the AD patients suffering from polyendocrine autoimmunity equally appeared at significantly higher risk of autoimmunity vs relatives of individuals with isolated AD (41.1% vs 19.4%, OR 2.886 (95% CI 1.217–7.478), P=0.0157).
Genotype frequencies of the three analyzed polymorphisms remained in Hardy–Weinberg equilibrium in both studied cohorts (P values for rs2476601 were 0.142 in relatives and 0.718 in controls, for rs231775 they were 0.926 in relatives and 0.612 in controls, and for rs3757247 they were 0.348 in relatives and 0.775 in controls, respectively).
The frequencies of genotypes and alleles of all studied polymorphisms were significantly different in AD relatives and controls (Table 2). PTPN22 rs2476601 minor T allele was found in 21.1% relatives’ alleles compared to its 11.6% prevalence among subjects with no history of autoimmunity (OR 2.045 (95% CI 1.469–2.847), P < 0.0001). rs231775 variant in the CTLA4 gene was also significantly more frequent in family members of the AD patients (48.1% vs 37.5%, OR 1.544 (95%CI 1.204–1.981), P = 0.0006). Finally, BACH2 rs3757247 minor T allele was found more often in AD relatives (51.6%) than in controls (43.1%) (OR 1.408 (95%CI 1.099–1.803), P = 0.0066). In line with this, genotype distributions differed between the relatives and controls, although in the case of rs3757247, the significance did not survive correction for multiple tests (corrected P-value = 0.0083).
Genotype and allele distribution of the three studied polymorphisms in first-degree relatives (RELAT) of patients suffering from Addison’s disease compared to control subjects (CON).
| rs2476601 PTPN22 | RELAT (%) | CON (%) | rs231775 CTLA4 | RELAT (%) | CON (%) | rs3757247 BACH2 | RELAT (%) | CON (%) |
|---|---|---|---|---|---|---|---|---|
| CC | 113 (60.4) | 308 (78.4) | AA | 50 (26.7) | 151 (38.4) | CC | 47 (25.1) | 128 (32.7) |
| CT | 69 (36.9) | 79 (20.1) | AG | 94 (50.3) | 189 (48.1) | CT | 87 (46.5) | 189 (48.3) |
| TT | 5 (2.7) | 6 (1.5) | GG | 43 (23.0) | 53 (13.5) | TT | 53 (28.4) | 74 (19.0) |
| P-value | <0.0001 | 0.0024 | 0.0223 | |||||
| C | 295 (78.9) | 695 (88.4) | A | 194 (51.9) | 491 (62.5) | C | 181 (48.4) | 445 (56.9) |
| T | 79 (21.1) | 91 (11.6) | G | 180 (48.1) | 295 (37.5) | T | 193 (51.6) | 337 (43.1) |
| P-value | <0.0001 | 0.0006 | 0.0066 | |||||
| OR (95% CI) | 2.045 (1.469–2.847) | OR (95% CI) | 1.544 (1.204–1.981) | OR (95% CI) | 1.408 (1.099–1.803) | |||
In the next step, an attempt was made to determine whether polymorphisms in PTPN22, CTLA4, and BACH2 genes might be associated with circulating autoantibodies in the first-degree relatives of patients suffering from AD. Due to limited numbers of carriers of particular genotypes, the subgroups were merged for analyses. In case of PTPN22 rs2476601 and CTLA4 rs231775, for which the mutant alleles are autoimmunity risk factors, the analyses were performed according to the dominant model, by comparing the frequency of antibodies in the wild-type homozygotes vs risk allele carriers (combined heterozygotes and homozygotes). On the contrary, rs3757247 BACH2 was evaluated based on the recessive model, as our former study demonstrated significant excess of the mutant TT homozygotes at this locus in patients with type 2 autoimmune polyendocrine syndrome (25). The current analysis revealed significantly increased prevalence of a21OH (P = 0.0075; OR 7.68; 95% CI 1, 903–36.0), thyroid antibodies aTPO (P < 0.0001; OR 3.85; 95% CI 1.873–7.495) and aTg (P < 0.0001; OR 7.73; 95% CI 3.112–19.65), as well as aGAD (P = 0.0303; OR 3.38; 95% CI 1.180–9.123) and aZnT8 (P = 0.032; OR 6.40; 95% CI 1.846–21.91) in carriers of PTPN22 rs2476601 T allele (Table 3). Among the evaluated autoantibodies, only aIA2 did not display significant association with rs2476601. None of the autoantibodies found in first-degree relatives demonstrated significant association with rs231775 in CTLA4 (all P-values >0.05), and only weak association was detected between BACH2 rs3757247 and circulating aTPO (P = 0.0336; OR 2.12; 95%CI 1.019–4.228). Overall, carriers of the rs2476601 T allele revealed nearly fourfold increased risk of detection of any serum autoantibody compared to the wild-type homozygous family members (P < 0.0001; OR 3.77; 95% CI 1.982–7.142). Moreover, the T allele appeared to be a risk factor for multiple circulating autoantibody specificities (P = 0.0009; OR 5.79; 95% CI 1.962–15.81). On the contrary, these relationships were not observed in case of two other studied polymorphisms, in CTLA4 and BACH2 genes (P-values >0.05).
Prevalence of specific serum autoantibodies in first-degree relatives of patients suffering from Addison’s disease with regard to their PTPN22, CTLA4, and BACH2 genotypes.
| Polymorphism | rs2476601 PTPN22 | rs231775 CTLA4 | rs3757247 BACH2 | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Positive antibody | CC 113 (%) | CT+TT 74 (%) | P-value | AA 50 (%) | AG+GG 137 (%) | P-value | CC+CT 134 (%) | TT 53 (%) | P-value |
| a21OH | 2 (1.8) | 9 (12.2) | 0.0075 | 1 (2.0) | 10 (7.3) | 0.2932 | 8 (6.0) | 3 (5.7) | 0.9999 |
| aZnT8 | 3 (2.7) | 11 (14.9) | 0.0032 | 3 (6.0) | 11 (8.0) | 0.7629 | 10 (7.5) | 4 (7.5) | 0.9999 |
| aGAD | 5 (4.4) | 10 (13.5) | 0.0303 | 2 (4.0) | 13 (9.5) | 0.3614 | 11 (8.2) | 4 (7.5) | 0.9999 |
| aIA2 | 3 (2.7) | 2 (2.7) | 0.9999 | 1 (2.0) | 4 (2.9) | 0.9999 | 2 (1.5) | 3 (5.7) | 0.1389 |
| aTPO | 17 (15.0) | 30 (40.5) | <0.0001 | 8 (16.0) | 39 (28.5) | 0.0819 | 28 (20.1) | 19 (35.8) | 0.0336 |
| aTg | 7 (6.2) | 25 (33.8) | <0.0001 | 5 (10.0) | 27 (19.7) | 0.1187 | 24 (17.9) | 8 (15.1) | 0.6449 |
| Any antibody | 28 (24.8) | 41 (55.4) | <0.0001 | 17 (34.0) | 52 (38.0) | 0.6197 | 50 (37.3) | 19 (35.8) | 0.8516 |
| One antibody | 21 (75.0) | 14 (34.1) | 0.0009 | 7 (58.3) | 28 (49.1) | 0.5619 | 24 (52.2) | 11 (47.8) | 0.7335 |
| >1 antibody | 7 (25.0) | 27 (67.9) | 5 (41.7) | 29 (50.9) | 22 (47.8) | 12 (52.2) | |||
serum autoantibodies against: a21OH, 21-hydroxylase; aTg, thyroglobulin; aTPO, thyroid peroxidase; aGAD, glutamic acid decarboxylase; aIA2, tyrosine phosphatase; aZnT8, zinc transporter-8.
Discussion
In the current study, both genetic and serologic autoimmunity markers were explored in the first-degree relatives of patients suffering from the autoimmune AD. More than one-third of the studied relatives presented with positive serum autoantibody, a proportion that largely exceeds overall autoimmunity estimates in the general population, ranging between 3.2 and 9.4% (28). As expected, thyroid-specific autoantibodies, aTPO and aTg, were the most common, often detectable in combination. Serologic markers of beta cell-targeted autoimmunity were also frequent, found in 2.7% up to 8.0% of the studied individuals, and autoantibodies to 21-hydroxylase were detected in 5.8% relatives. Therefore, various endocrine glands were targeted in relatives of the AD patients, indicating a generalized autoimmune problem in those families, rather than susceptibility to any particular disease. In line, our former survey revealed a wide range of self-reported autoimmune conditions in AD families (4). Moreover, several (18.2%) subjects in our current investigation displayed multiple circulating autoantibodies. Apart from aTPO, which was more prevalent in females, no sex-related differences nor predisposition connected with relationship to the patient (parents, siblings, or offspring) were noted. However, as in previous analysis, relatives of the male AD patients, and of those suffering from polyendocrine autoimmunity, appeared most prone to develop autoantibodies (9).
AD features considerable genetic susceptibility with several well-established risk factors. Family members are supposed to share much of the genetic constitution with their affected relatives and, therefore, share predisposition to autoimmunity. This observation has been very well corroborated in respect of the HLA system – specific class II alleles are associated with endocrine autoimmunity in patients’ relatives (29, 30). Current analysis was focused on polymorphic variants of three genes cardinal for the immune function. Their polymorphisms are strongly associated with AD, as demonstrated in Norwegian, Swedish, UK, Italian, German, and Polish cohorts (18, 26, 27, 31, 32, 33). Furthermore, they are recognized risk factors for T1D, AITD, and other autoimmune conditions, including rheumatoid arthritis, SLE, systemic sclerosis, and vitiligo (34, 35, 36). Based on that, it seemed worthwhile to analyze the relationship between these polymorphisms and the presence of endocrine tissue-specific autoantibodies in first-degree relatives of patients with autoimmune AD. As might have been presumed, all three studied polymorphisms were significantly more frequent in families of the AD patients compared to controls (Table 2). When examined with regard to autoantibody prevalence, rs2476601 PTPN22 T allele appeared to be a considerable risk factor for serum a21OH, aTPO, aTg, and ZnT8 in AD relatives. Slightly less convincing association was found for aGAD, which displayed borderline statistical significance (P = 0.030). However, it seems probable that with increased sample size, aGAD results would reach unequivocal level. On the contrary, aIA2 did not display association with rs2476601, but its prevalence remained low, in line with former reports from T1D adults and their relatives (37).
Rs2476601 leads to substitution of arginine with tryptophan (R620W) in the P1 domain of the lymphoid protein tyrosine phosphatase, critical for downstream control of T cell receptor signaling (15). Despite its major role in T cell function, PTPN22 seems also implicated in B cell biology, comprising signal transduction, proliferation, and resistance to apoptosis (38). Hitherto analyses of the association between PTPN22 polymorphism and the presence of specific autoantibodies were mainly focused on T1D. Rs2476601 was significantly associated with aGAD in populations from Denmark, Italy, and Brazil, including patients with newly diagnosed and long-lasting diabetes (39, 40, 41). Moreover, in healthy Finnish subjects followed prospectively, an association was found between rs2476601 and appearance of serum aGAD, especially in people with a family history of T1D (42). On the contrary, this association was not replicated in British and German patients (43, 44). As for aZnT8, a small analysis from a multiethnic Brazilian population has shown its association with rs2476601 in T1D patients and their first-degree relatives (45). Earlier studies in British and German patients with diabetes did not confirm the relationship of PTPN22 with thyroid autoimmunity (43, 46). However, according to more robust data, PTPN22 variant is significantly associated with the occurrence of aTPO and a21OH in the muti-ethnic T1D groups (44, 47). To date, the polymorphism of the PTPN22 gene has not been assessed in terms of its coincidence with autoantibodies in patients with AD, let alone in their relatives. Our study is the first observation of this type, further supported by the association of rs2476601 with multiple circulating autoantibodies.
In contrast, polymorphic variant of another prominent immune regulator, CTLA4, failed to reveal any association with the circulating autoantibodies in our relatives’ cohort. rs231775 used to be associated with endocrine autoimmunity, and functional studies revealed that it might alter CTLA4 expression in CD4 Treg cells (32, 48, 49). rs231775 was associated with increased rates of aIA2 and aGAD among T1D patients from Asia, whereas European cohort did not confirm this relationship (49, 50). Several papers support its association with Graves’ disease and circulating antibodies to TSH receptor (16, 51, 52). A small study in Polish children with AITD revealed increased proportion of aTPO and aTg in rs231775 variant carriers (52). On the contrary, no effect of rs231775 on circulating a21OH in Italian AD patients was detected (32). Likewise, in our analysis, although the mutated rs231775 G allele revealed increased frequency in first-degree relatives of patients with AD, no association with any of the studied autoantibodies was found. In fact, the genuine causal variant responsible for the susceptibility to autoimmune reactions may be located elsewhere within the 2q33.2 locus (53). Moreover, autoimmunity-predisposing genes may possibly operate thorough various mechanisms, not necessarily via autoantibody synthesis.
Finally, only marginal association was detected between BACH2 rs3757247 and serum aTPO (P = 0.0336), while all other studied autoantibodies did not display relationship with this variant. BACH2 was first identified as a critical player in differentiation of the mature B cells into antibody-secreting plasma cells (22). Therefore, it seemed plausible that its genetic variants might affect the autoantibody synthesis. Former analyses supported its association not only with autoimmune conditions, T1D, AD, or Graves’ disease, but also with the presence of specific autoantibodies (17, 18, 27, 44, 54). Several studies confirmed the relationship between BACH2 polymorphisms and circulating aTPO in patients with AITD and those suffering from autoimmune diabetes (44, 45, 55). Furthermore, rs3757247 was correlated with increased risk of appearance of aGAD in prospectively followed children with HLA-DR high-risk genotypes (56). On the other hand, although BACH2 polymorphism has been pinpointed through extended exome sequencing as AD risk factor, this association appeared independent of aTPO presence [18]. Other studies were focused on various BACH2 polymorphisms, most often rs11755527 and rs619192, which remain in tight linkage disequilibrium with intronic rs3757247 investigated in our cohort. A haplotype block which covers the 5′ portion of BACH2 precludes precise dissection of a single causative polymorphism implicated in autoimmunity (27).
We need to acknowledge that there are several limitations to our current research. First, we only focused on markers specific for the endocrine autoimmunity, whereas other autoimmune conditions, fairly common in the population, may emerge in members of the autoimmunity-prone families. For instance, chronic atrophic gastritis frequently coincides with AD and AITD; therefore, parietal cell antibodies could have been evaluated as well (7, 57). Next, our relatives’ cohort was of limited size; however, it was composed of carefully selected individuals, with well-documented family history of AD. We hoped that evaluation of the studied polymorphisms would allow to generate a preliminary immunogenetic screening profile, capable of predicting autoimmunity prior to clinical symptoms of the disease. These analyses would be particularly useful in family members of individuals with adrenal autoimmunity, especially relatives of males with AD suffering from polyglandular autoimmunity, who seem particularly prone to autoimmune reactions. Since genetic associations in our study appeared less common than expected, we could not achieve that aim. The only autoantibody which seemed related with more than one polymorphism, was aTPO, but its association with BACH2 remained borderline. Therefore, further analyses, preferably prospective and involving larger cohorts, are warranted to fully evaluate clinical utility of studying susceptibility alleles as autoimmunity predictors in AD families. Nonetheless, based on our data, we conclude that among the first-degree relatives of individuals with AD, carriers of the PTPN22 rs2476601 T allele are at considerable risk of developing autoantibodies to the endocrine antigens. This preselected population may benefit from regular clinical and serological screening toward the autoimmune conditions.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/EC-23-0008.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector.
Acknowledgements
The authors would like to thank all our patients with Addison’s disease and their relatives for their participation and comprehension.
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