**5. Single nucleotide polymorphism and genotyping in HLA class II allele and PLA2R1 antigen**

The immune system recognizes peptide sequences processed from a target antigen when presented by the antigen-presenting cells. In PLA2R associated membranous nephropathy, the immune system will interpret the PLA2R1 presented by the HLA Class II molecule as the target antigen. The GWAS (genome-wide

association studies) identified a strong association of single nucleotide polymorphisms (SNPs) on both HLA-DQA1, PLA2R1 antigen, and Primary Membranous nephropathy [4]. Due to the proximity of HLA-DQ and HLA-DR on chromosome 6 and the probability of linkage disequilibrium that can result in the coinheritance of common haplotype, both alleles were found to be associated with membranous nephropathy. When imputation was performed it has confirmed the same signals on the same loci identified in other studies [33]. That can also be interpreted from one study where they have identified a significant association between PLA2R1 antigen and both HLA DQA1/DRB1 [34]. In the former study, it was noted that using a combined genetic risk score and serum anti PLA2R antibody using the ELISA method can potentially mitigate the need for a diagnostic renal biopsy in high-risk patients.

Not all cases of primary membranous nephropathy are associated with PLA2R1 antigen. After the discovery of the PLA2R antigen, THSD7A was the 2nd antigen implicated in primary membranous nephropathy and is found in 2–5% of patients [35]. Investigators have identified similarities and differences between THSD7A and PLA2R antigens [36]. This can be extrapolated to suggest that there may be an HLA link with THSD7A either similar or different to PLA2R antigen. In a large case–control study, the link between the HLA-DQA1 and PLA2R1 antigen was found to be more prevalent in patients with confirmed PLA2R1 associated membranous nephropathy compared to PLA2R1 negative patients [37]. For now, the specific association is yet to be noted in anti-PLA2R1 negative cases. Also, association with these SNPs is seen in patients with Caucasian backgrounds compared to patients from Afro-American origins.

Another Asian study found a strong association between HLADQA1 and PLA2R1 antigen [38]. In this study investigators not only identified SNP variation on HLA-DQA1 but also found that AA and AG genotype carriers are at increased risk of developing primary membranous nephropathy compared to those carrying GG genotype. On the other hand, GG genotype on PLA2R1 antigen SNP and AA genotype on another were encountered more frequently in subjects with primary membranous nephropathy. Two other Asian studies have identified other HLA risk alleles other than HLA-DQA1 in their cohorts of patients with primary membranous nephropathy [39, 40]. Again this raises the possibility of linkage disequilibrium.

Correlating the treatment response to the underlying genetic polymorphism is another area that is being investigated. A Chinese-Taiwanese study observed that haplotype H1 might carry a higher risk for disease progression when compared to H3 haplotype. The group found no relation between disease progression and underlying genetic polymorphism in PLA2rR1 antigen without the incidence of ESRD or death after therapy [41].

In another Spanish study, they found no association between survival and single nucleotide polymorphism in PLA2R1 antigen. In the same study, AA and AG genotypes in HLA-DQA1 and AA genotype on PLA2R1 antigen were shown to be associated with a trend towards immunosuppression treatment response compared to other genotypes [42]. Moreover, AA and AG genotypes on HLA-DQA1 SNP have shown significant protection for doubling of serum creatinine and progression to end-stage renal disease, without identifying any protective genotypes on PLA2R1 antigen.

A Chinese paper has highlighted the ethnic distribution difference in membranous nephropathy based on their HLA types [34]. They have found that DRB1\*1501 is the major risk allele in the East Asian population, DQA1\*0501 in Europeans, and DRB1\*0301 in both ethnicities. This new finding can allow us to categorize high-risk patients from different background ethnicities based on their HLA type.
