Genetic risk factors for pathogenic IgG4 autoantibodies: a systematic review and meta-analysis of HLA class II associations in patients with IgG4 autoimmune diseases

Autoimmune diseases caused by pathogenic IgG4 subclass autoantibodies (IgG4-AID) include diseases like MuSK myasthenia gravis, pemphigus vulgaris or thrombotic thrombocytopenic purpura. Their etiology is still unknown. Polymorphisms in the human leukocyte antigen (HLA) gene locus, particularly in HLA-DRB1, are known genetic susceptibility factors for autoimmune diseases. We hypothesized a similar role for HLA polymorphisms in IgG4-AID and conducted a systematic review and meta-analysis with case-control studies on IgG4-AID based on MOOSE/ HuGENet guidelines. Genotype (G) and (A) and 95% and the HLA-DRB1*14-DQB1*05 haplotype / OR 95% were increased while HLA-DRB1*13 OR 95% CI p < 95% CI p < 0.00001) was decreased in IgG4-AID patients. and the HLA-DRB1*13 allele may protect from IgG4 autoimmunity.

Whether IgG4-AID have distinct genetic risk factors that may predispose for the production of pathogenic IgG4 is unknown. A major contributor to genetic susceptibility to autoimmunity are the highly polymorphic human leucocyte antigen (HLA) genes on chromosome 6p21.3 that encode the major histocompatibility complex (MHC) (16,17). HLA-DR, HLA-DQ, and HLA-DP encode the MHC II molecules on antigen-presenting cells and thymic epithelial cells that present self-and foreign antigen peptides to CD4+ T helper cells, which is essential for T-cell activation or the development and maintenance of tolerance (18,19). HLA-DR has been linked to aberrant presentation of self-peptide to autoreactive T helper cells in the thymus (20), and a recent study showed that distinct HLA-DR variants may directly influence the immune response towards autoimmunity or tolerance (21).
Furthermore, genetic polymorphisms in the HLA-DRB1 gene are associated with a range of autoimmune diseases, such as rheumatoid arthritis, diabetes mellitus type I or systemic lupus erythematosus (22).
GWAS data suggests that HLA class II gene polymorphisms may also play a role for susceptibility to several different IgG4-AID (3), and specifically the HLA-DRB1 and DQB1 loci were associated with individual diseases (23,24), but systematic data are lacking.
Therefore, we wanted to investigate whether genetic susceptibility to develop pathogenic IgG4 autoantibodies may be linked to distinct HLA class II alleles. To this end, we conducted a systematic review and meta-analysis of case-control studies reporting HLA class II associations in individual IgG4-AID.
We found that patients with IgG4-AID had significantly increased frequencies of the HLA-DQB1*05 and HLA-DRB1*14 alleles and the HLA-DRB1*14-DQB1*05 haplotype, and a significant negative association with HLA-DRB1* 13. Notably, HLA-DQB1*05 is not positively associated with classical autoimmunity and could be a genetic risk factor for the production of IgG4 subclass autoantibodies.

Methods
The systematic review was based on recommendations by the HuGENet™ HuGE Review Handbook, version 1.0 (released by the EQUATOR network, 2015, (25)) and MOOSE guidelines for Meta-Analyses and Systematic Reviews of Observational Studies (26

Number and characteristics of included studies
After search and screening, 52 full-text articles with a total of 64 datasets (table 1, table S2) were included in the qualitative synthesis and 51 full-text articles with 62 datasets in the quantitative synthesis (figure 1). The following number of studies was identified: 36 on pemphigus, seven on TTP, five on MuSK MG, three on CIDP. Allele, genotype or haplotype frequencies were extracted and analyzed separately.
Due to lack of data on HLA-DP, only polymorphisms in the HLA-DR and HLA-DQ genes were extracted. The following studies and datasets were included in the qualitative synthesis but excluded from the meta-analysis as they did not fit all selection criteria: the study by Joly et al., 2020 (marked with ( †)), and one dataset from the Delgado study (1997). Studies that did not differentiate between disease subgroups of pemphigus or CIDP were marked with ( ‡).
Data from pemphigus studies using the same control for pemphigus foliaceus and pemphigus vulgaris were pooled and marked with (**). Studies marked with (*) were included after discussion with W.B..    In summary, accounting for the predominance of pemphigus studies, we observed a positive association with HLA-DRB1*14 and HLA-DQB1*05 alleles and the DRB1*14-DQB1*05 haplotype and a negative association with HLA-DRB1*13.

Analysis of higher resolution data
We were interested to know whether the association was due to specific alleles, but high resolution data was only available for a fraction of studies as most studies only reported onefield resolution data (table S3 and S4). We analyzed the available datasets with higher resolution data, which were mostly from pemphigus studies. Data of the available variants (figure S13-S15) was analyzed and positive associations with HLA-DRB1*14:01, HLA-DRB1*14:04, HLA-DRB1*04:02 and HLA-DQB1*05:03 were observed.

Within ancestry analysis
To study the potential effect of ancestry, we conducted a within-ancestry analysis from the three countries with the highest number of datasets (Brazil: 6 studies, Turkey and Japan: each 5 studies) separately (figure S16-S18). A trend for similar outcomes could be observed in all three populations where enough data was available, but there was variation in the strength of the association, e.g. the OR for HLA-DRB1*14 was higher in Japan than in Brazil or Turkey.
An across-ancestry analysis was not considered feasible with the available data.

Evaluation of heterogeneity and publication bias
The heterogeneity was assessed by Tau 2 , X 2 and I 2 tests (table S5), whereas potential publication bias was assessed by funnel plots (figure 6, figure S106-112).
There was substantial heterogeneity for most of the alleles with the exception of HLA-DRB1*14 which showed a low level of heterogeneity only pemphigus allele frequency, but was highly heterogenic otherwise. HLA-DRB1*13 showed low heterogeneity in TTP and IgG4-AID excluding pemphigus, but moderate heterogeneity in all IgG4 AID collectively.
Due to the high level of heterogeneity between the studies, the publication bias was assessed only by funnel plots. We found a low to moderate and mostly symmetrical publication bias in HLA-DQB1*05 and HLA-DRB1*14, with very few outliers in both directions, while 1-2 outliers towards lower ORs were found for HLA-DRB1*13.

Discussion
In recent years it was appreciated for the first time that IgG4 autoimmune diseases may be considered as a collective group of autoimmune diseases that share distinct pathophysiological and therapeutic commonalities (1,3,4), setting them apart from classical antibody mediated autoimmune diseases. Based on preliminary observations (3), we hypothesized that IgG4 AID may also share genetic associations with polymorphisms in the HLA class II genes. To address this possibility, we conducted a systematic review and metaanalysis on the genotype, haplotype and allele frequency of reported HLA class II alleles across IgG4-AID and found that HLA-DQB1*05, an allele that is not typically associated with autoimmunity, is significantly more frequent in patients with IgG4-AID. This suggests it may be a genetic susceptibility factor for IgG4-AID. In addition, HLA-DRB1*14, a known genetic susceptibility factor for autoimmunity, is also associated with IgG4 autoimmunity, as is the HLA-DQB1*05-DRB1*14 haplotype. HLA-DRB1*13, which is considered as protective for autoimmunity in general, is also negatively associated with IgG4-AID. HLA-DRB1*03 and *04, which are often associated with autoimmunity, did not correlate with IgG4-AID with the notable exception of pemphigus, which showed a strong association with HLA-DRB1*04.

Genetic associations with individual IgG4 autoimmune diseases
This is to the best of our knowledge the first systematic review and meta-analysis investigating a potential association of HLA class II alleles with IgG4-AID. Systematic reviews on individual IgG4-AID (Pemphigus, MuSK MG) are in agreement with our findings (23,24). A significant positive association of MuSK MG with HLA-DRB1*14, HLA-DRB1*16 and HLA-DQB1*05 could be confirmed in our study (24). In contrast, a significant negative association for HLA-DQB1*03 reported in the MuSK MG study could not be reproduced in our analysis, and the reported negative association with HLA-DQB1*06 did not reach significance in our study. Possible reasons for this might be 1) the exclusion of one Italian study (30) from our analysis that was included in the Hong study as it did not match our inclusion/exclusion criteria and 2) the use of different statistical methodology (randomvs fixed-effects model).
Our analysis of pemphigus data is in line with previous meta-analyses. Increased There were only few studies with haplotype data in IgG4-AID available, but the HLA-DRB1*14 also is strongly associated with IgG4-AID in our study, and was also reported as increased in patients with rheumatoid arthritis, Guillain-Barré syndrome and MuSK MG (40), suggesting it may be a genetic risk factor to develop autoimmune diseases.
HLA-DRB1*13 was found to be less frequent in IgG4-AID in our study, and this was also observed in classical AID, including T1D and autoimmune hepatitis (41-43).

Study limitations
The main limitation of the study was owed to the low prevalence of IgG4-AID, including 1) small numbers of patient per individual study (mostly between 30-100 patients), and 2) a low number of available studies, leading to 3) substantial heterogeneity, which was especially pronounced in studies on TTP. Pooling of data was not always possible due to different types of analysis and the differential use of nomenclature (e.g. genotype, haplotype, allele and phenotype frequency). Lack of information on homozygosity or heterozygosity in studies with genotype frequencies prevented a combined analysis for allele and genotype frequency, and since the HLA genes are in linkage disequilibrium (51), homo-and heterozygosity cannot be "re-calculated" by using the Hardy-Weinberg equilibrium. Therefore, we only included studies where the frequency was given in absolute and relative numbers and data for allele and genotype frequency were analyzed individually. Several studies used a single control group for two different datasets, and to avoid overestimating the number of controls, data of these studies were pooled where possible (52-55) (exception: two studies from Serbia (56,   57)). All studies included in the meta-analysis reported that the controls and patients derived from the same geographic location or that the controls were ethnically matched to the controls, but most studies did not provide further details on the ethnical matching. which was more frequent in pemphigus patients, was not associated with the other diseases.
Antibody tests were not described in a substantial number of studies on pemphigus, but histopathologic diagnosis implicates the presence of the relevant IgG4 autoantibodies (mostly desmoglein 1 and desmoglein 3, < 0.5% of patients desmocollin), the inclusion criteria were changed during the second round of screening to include the pemphigus studies 13 in the quantitative analysis. The PRISMA statement acknowledges this iterative process and accepts that modifications in the review protocol during the synthesis may sometimes be inevitable (29).

Conclusions
With the limitations of this study in mind, we observed an increased frequency of HLA-  polyneuropathy; TTP, thrombotic thrombocytopenic purpura; MuSK MG, muscle-specific kinase myasthenia gravis; PCR, polymerase chain reaction; SSP, sequence specific primer; SSO(P), sequence specific oligonucleotides (probes); RFLP, restriction fragment length polymorphism