Genetic association to autoimmune diseases (ADs) includes variants of genes coding for proteins involved in B lymphocyte antigen receptor signaling, of which BLK and BANK1 are components of the B-cell signalosome (Suthers and Sarantopoulos, 2017). The BLK protein is a src family non-receptor tyrosine kinase mainly expressed by B-cells, where besides B-cell receptor signaling, it plays a role in development (Akerblad and Sigvardsson, 1999; Tretter et al., 2003), meanwhile, the BANK1 protein is an adaptor/scaffold primarily expressed in B-lymphocytes, which plays an important role in activation and signaling (Kozyrev et al., 2008; Castillejo-Lopez et al., 2012). It has been shown that BLK, similar to other members of the src family interacts with BANK1 (Castillejo-Lopez et al., 2012). Thus, BLK and BANK1 proteins have an important role in both B-cell signaling and activation.

In 2009 and 2011, two genome wide association studies (GWAS) carried out in European and Asian derived-populations, respectively, identified some single nucleotide variants (SNVs) in the BLK gene to be associated with risk for rheumatoid arthritis (RA) (Gregersen et al., 2009; Freudenberg et al., 2011). However, other GWA or candidate gene studies have failed to replicate these findings (Génin et al., 2013; Jiang et al., 2014; Orozco et al., 2014; Bossini-Castillo et al., 2015; Zhu et al., 2016; Julià et al., 2016; Saxena et al., 2017), except in patients from United Kingdom, Colombia, and China (Deshmukh et al., 2011; Orozco et al., 2011; Viatte et al., 2012; Huang et al., 2017). On the other hand, the BANK1 gene, which was identified as an important risk factor for systemic lupus erythematosus (SLE) (Kozyrev et al., 2008), does not appear to be associated with RA through GWA (Gregersen et al., 2009; Freudenberg et al., 2011; Jiang et al., 2014; Orozco et al., 2014; Bossini-Castillo et al., 2015; Julià et al., 2016; Saxena et al., 2017) or candidate gene studies (Suarez-Gestal et al., 2009; Orozco et al., 2011; Génin et al., 2013; Huang et al., 2017), except for the BANK1 rs3733197G/A (Ala383Thr; non-synonymous polymorphism) SNV, which showed an association with RA in Spanish and Argentine patients (Orozco et al., 2009).

Although different BLK and BANK1 SNVs have been examined in patients with RA, the results are uncertain because some reports show an association with this AD (Gregersen et al., 2009; Orozco et al., 2009; Deshmukh et al., 2011; Freudenberg et al., 2011; Orozco et al., 2011; Viatte et al., 2012; Huang et al., 2017), while others have not replicated this finding (Suarez-Gestal et al., 2009; Génin et al., 2013; Jiang et al., 2014; Orozco et al., 2014; Bossini-Castillo et al., 2015; Julià et al., 2016; Zhu et al., 2016; Saxena et al., 2017). In addition, BLK (except in patients from Colombia) and BANK1 (except in one study which evaluated patients from Argentina and Mexico) variants have been scarcely explored in different Latin-American populations with RA (Orozco et al., 2009; Deshmukh et al., 2011). Therefore, our aim was to examine the possible associations of BLK rs13277113A/G and rs2736340T/C and BANK1 rs10516487C/T (R61H) and rs3733197G/A (A383T) SNVs with RA in Mexican patients, in addition to the interactions between BLK and BANK1 genotypes and this AD.

The study of gene polymorphism associations to diseases in different ethnic groups is an important means to close down on the likelihood that particular genes and specific variants to the disease. Here we studied two SNVs of the BLK gene, and two of BANK1 (both of them part of the B cell signalosome) for their association to RA in Mexican patients. Association of BLK SNVs to RA was initially documented in North American and Korean patients (Gregersen et al., 2009; Freudenberg et al., 2011), and later in British, Colombian, and Han Chinese patients (Deshmukh et al., 2011; Orozco et al., 2011; Viatte et al., 2012; Huang et al., 2017). Nevertheless, other GWA or candidate gene studies carried out in some Arab countries and Europeans from the Netherlands and Spain, and even another Chinese group failed to replicate this finding (Suarez-Gestal et al., 2009; Génin et al., 2013; Jiang et al., 2014; Bossini-Castillo et al., 2015; Julià et al., 2016; Zhu et al., 2016; Saxena et al., 2017). As far as we know, the only study conducted in Latin-Americans thus far, found an association of both BLK rs13277113A/G and rs2736340T/C SNVs and risk for RA in Colombians (Deshmukh et al., 2011). Our current findings confirm such association in Mexican RA patients. On the other hand, in European-derived populations, BLK association to RA has only been identified in patients from the UK (Orozco et al., 2011; Viatte et al., 2012). It is important to note that in Chinese population, one candidate gene study found an association between BLK rs13277113A/G and RA (Huang et al., 2017), which was not previously identified in a GWAS (Jiang et al., 2014). GWAS allow the identification of many genes associated with any disease, including RA or other ADs (Gregersen et al., 2009; Freudenberg et al., 2011; Kozyrev et al., 2008; Rodríguez-Elías et al., 2016). However, in these studies, several hundreds or thousands of SNVs are removed, particularly because of their high error rates, such as those with an excess missing genotype (Anderson et al., 2010). Possible explanations for this are: a) the probes for the genotyping of BLK rs13277113A/G and rs2736340T/C (or SNVs in high LD for these variants) SNVs might have been lost in that process, b) the controls had a deviation from the HWE, c) these variants had no associations at genome-wide significance level (p < 5×10⁻⁸), among others. All these caveats must be considered to explain the controversial results found among the aforementioned studies. To solve this, it would be useful to carry out replications with candidate gene studies in the different populations to confirm or rule out these findings. It is important to note that in controls from North America, Spain (or European-derived populations), Colombia, and Africa, BLK rs2736340T or the BLK rs13277113A are the minor alleles (Hom et al., 2008; Gregersen et al., 2009; Orozco et al., 2009; Deshmukh et al., 2011; Sánchez et al., 2011; Génin et al., 2013; Guthridge et al., 2014; Martínez-Bueno et al., 2018) whereas in our population these were the major alleles as reported in the 1000 Genomes Project for Mexicans (Mexican-Americans from Los Angeles, CA) (https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/) as well as in other Asian populations like Japanese and Chinese (Génin et al., 2013; Dang et al., 2016; Huang et al., 2017) ( Table 5 ). We conclude, hence, that in our population the BLK rs2736340T and/or rs13277113A major alleles are associated to risk for RA.

Functionally, the rs13277113A allele has been associated with lower levels of BLK mRNA in transformed B-cell lines (Hom et al., 2008). However, it is possible that this BLK SNV is in LD with another BLK variant truly associated with risk for RA. In this manner, BLK rs13277113A appears to belong to a haplotype associated with RA, such as the BLK rs922483 variant (which is in high LD; r² > 0.7 with both BLK rs13277113 and rs2736340T/C), which affects the BLK mRNA and protein expression early in B cell ontogeny (Simpfendorfer et al., 2012). Moreover, another study found that the rs922483 and rs1382568 variants located within the proximal BLK promoter and in the upstream alternative BLK promoter, respectively (both BLK variants are in high LD with rs13277113; r² > 0.74 in Europeans), affect the promoter activity in B progenitor cell lines (Guthridge et al., 2014).

On the other hand, the two BANK1 rs10516487C/T (R61H) and rs3733197G/A (A383T) variants have been scarcely studied in patients with RA (Orozco et al., 2009; Suarez-Gestal et al., 2009; Orozco et al., 2011; Génin et al., 2013; Huang et al., 2017), having found only one association with BANK1 rs3733197G/A in RA patients from Spain and Argentina (Orozco et al., 2009). In that same study, a sample of Mexican RA patients was examined for BANK1 rs10516487C/T, rs3733197G/A, and rs17266594T/C showing no association. There was, however, an association of RA with BANK1 rs10516487G/A and rs3733197G/A when the cases and controls of all the countries were pooled. Our current results identified an individual association between BANK1 rs10516487G/A and RA in Mexican patients, which is at a variance with what was published by Orozco et al. (2009). A possible explanation for this discrepancy could be the different sample sizes between both studies. Our study included 470 RA patients and 487 controls, whereas they studied 278 patients and 272 controls. In addition, our cases and controls are from Central Mexico, whereas Orozco et al. did not reveal the place of origin of their cases and controls (Orozco et al., 2009). The Mexican Republic is a large country with a great population admixture of several ethnic origins, which are not evenly distributed, e.g. Mexico City, located in central Mexico, is formed by approximately 50% Amerindian, 45% Caucasian, and 5% African ancestry (Martinez-Marignac et al., 2007). On the other hand, regions located in Western or Southeastern Mexico have a markedly different ethnic composition (Martínez-Cortés et al., 2012; Rincón et al., 2016). As far as we can tell, this is the first study showing an individual association between BANK1 rs1051648T/C and RA. Although some authors have not found association between different BANK1 or BLK alleles and risk for RA, in Spanish and Chinese patients an association of an interaction of certain BANK1-BLK genotype with RA has been reported (Génin et al., 2013; Huang et al., 2017). Moreover, is has been documented a physical and genetic interaction between BLK and BANK1 (Castillejo-Lopez et al., 2012), which would support our finding of a BLK-BANK1 genetic interaction associated to RA (Castillejo-Lopez et al., 2012; Génin et al., 2013; Huang et al., 2017). According to our results, the best model of interaction was BLK rs2736340T/C-BANK1 rs10516487G/A.

Among the four studied SNPs, it has been reported that rs13277113A/G and rs2736340T/C could have a potential eQTL effect on FAM167A and BLK genes in monocytes and lymphoblastic cell lines suggesting that these variants are involved in the physiopathology of RA and SLE (Deng et al., 2013a; Deng et al., 2013b). In addition, because both BLK variants studied here map near the promoter, they were examined in silico by means of expression quantitative trait locus (eQTL) studies (Schmiedel et al., 2018), which point out to a possible overrepresentation of T helper cells, predominantly Th17 and almost any other T helpers, and for the BLK-FAM167A locus for naïve B cells (for rs13277113A/G). Although for rs2736340T/C the eQTL associations are similar, there is a slight difference for the BLK only on that T cell overrepresentation is Th17 followed by T follicular helper cells. Thus, we can conclude that these associations clearly point to a role of an altered adaptive immunity conferring risk to develop RA. For the BANK1 variants we did not do an eQTL analysis, because both of them affect the ORF and not the regulatory regions.

A study about the functional consequence of the non-synonymous BANK1 rs10516487G/A variant (located in exon 2 with a change of arginine to histidine at amino acid position 61 [R61H] of BANK1) reported that the product of the BANK1 rs10516487A allele lacks a binding site for the SRp40 splicing enhancer protein, thereby affecting the splicing efficiency of BANK1 isoforms (Kozyrev et al., 2012). In addition, the BANK1 rs10516487GG genotype produces more BANK1 mRNA and protein than the rs10516487AA genotype. Moreover, the full-length isoform transcript BANK1-R61 generates a larger protein complex and self-association and multimerization than BANK1-H61 (Kozyrev et al., 2012). Another study showed the functional effect of the SLE-associated TGG risk haplotype which is formed by BANK1 rs17266594T/C, rs10516487G/A, and rs3733197G/A. The first variant is located in a putative branch point of intron 1, the second and third variants (R61H and A383T) are located in exon 2 and 7, respectively. Thus, the TGG risk haplotype is formed by T allele of the rs17266594T/C variant and by Arg61 and Ala383. That study showed that this risk haplotype leads to a decrease of B cell receptor signaling in Ramos B cells and in peripheral B cells. Finally, it was also described in BANK1 risk carriers that this haplotype appears to result on an increased number of memory B cells (Dam et al., 2016).

Of note, the lack of clinical and laboratory information (bone erosions, rheumatoid nodules, autoantibodies such as anti-CCPs, rheumatoid factor) as well as the absence of mRNA or protein expression studies to correlate with the four BLK and BANK1 variants studied, are some limitations of our study. Thus, at present, it is not possible to determine if these variants are associated with particular traits of RA patients.

In summary, our data indicates that both BLK as BANK1 SNVs studied herein are associated with risk to RA in Mexican patients. On the other hand, the individual association of BANK1 rs1054857G/A with RA found in the present study had not been identified in a particular population, although one study reported association between this variant and RA only in pooled patients from several countries. The main limitations in our study is the lack of clinical traits and serological markers in or RA patients, as well as the absence of ancestry informative-markers.

The data has been uploaded to EVA repository Project: PRJEB36187 Analyses: ERZ1284135 www.ebi.ac.uk/eva/?eva-study=PRJEB36187.

This study was conducted in compliance with the Declaration of Helsinki. All individuals included in our study signed a written informed consent. This protocol was approved by the Ethics and Research Committees of the HJM (registry number 0446/18-I). The patients/participants provided their written informed consent to participate in this study.

JR-B contributed to the study design, acquired the funding, data analyzed, and wrote the article. IA-Á performed the methodology and contributed with the experiments. JF and AC-P performed the data analysis. JM contributed to the data analysis and wrote the article. SJ-M contributed to the data analysis. RB-C contributed in the explanation of the project to the patients and in the collection of samples.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.