## 3 Results
### Enrichment Analysis of HLA Variants in the Pustular Skin Diseases  
We further performed a variant enrichment analysis to determine whether a particular variant is consistently abundant in the patient group in comparison to the general population. Here, the frequency of variants, including single nucleotide variants (SNVs), insertion/deletion (INDEL), and frameshift variants found in the HLA candidate genes (_HLA-B_, \(C\), and _DQB1_), was compared to that of the East Asian population (EAS) consisting of Han Chinese, Japanese, Southern Han Chinese, Chinese Dai, and Kinh populations from a catalog of common human genetic variation (1000 genome project database) [23] and the Genome Aggregation Database (gnomAD) [24]. From a total of 216 missense and 23 frameshift variants identified in these three HLA genes, 52 missense and 8 frameshift variants were observed to be significantly enriched in the subjects presented with pustular skin diseases (Supplementary Table S5). To further investigate the potential effect of these variants on HLA protein function, we reconstructed HLA protein structures, mapped the variants, accordingly, and studied them in the context of the annotated HLA functional domains (Supplementary Table S5). Interestingly, many of those variants were identified in the antigen-binding groove (11 out of 23 variants in _HLA-B_, 9 out of 17 variants in _HLA-C_, 12 out of 20 variants in _HLA-DQB1_) (Figure 3 and Supplementary Table S5). Of note, a primary activity of HLA is to load endogenous and exogenous peptides into the binding groove and present them to immune cells. Therefore, variants leading to amino acid change in the binding groove are likely to impact the HLA function and, thus, the regulation of immune response. \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline
**Allele** & **2n = 82** & \
\begin{tabular}{c} **AF (2n = \\ **82** \\ \end{tabular} &
\begin{tabular}{c} **AF Thai Healthy** \\ **(2n = 940)** \\ \end{tabular} & **2n = 940** & **Odds Ratio** & **95\% CI** & \(p\)**-Value** \\ \hline _A*_33:03 & 4 & 0.049 & 0.1117 & 105 & 0.408 & 0.146 to 1.137 & 0.086 \\ \hline _B*_13:01 & 16 & 0.195 & 0.0596 & 56 & 3.825 & 2.08 to 7.035 & **<0.001** \\ \hline _B*_18:02 & 4 & 0.049 & 0.016 & 15 & 3.154 & 1.022 to 9.73 & **0.046** \\ \hline _C*_03:04 & 20 & 0.244 & 0.081 & 76 & 3.665 & 2.102 to 6.39 & **<0.001** \\ \hline _DPB1*05:01_* & 25 & 0.305 & NA & NA & NA & NA \\ \hline _DQB1*03:01_ & 7 & 0.085 & 0.1723 & 162 & 0.448 & 0.203 to 0.991 & **0.047** \\ \hline _DQB1*05:01_ & 13 & 0.159 & 0.1404 & 132 & 1.154 & 0.62 to 2.145 & 0.652 \\ \hline _DQB1*05:02_ & 30 & 0.366 & 0.2128 & 200 & 2.134 & 1.326 to 3.434 & **0.002** \\ \hline _DRB1*15:01_ & 17 & 0.207 & 0.081 & 76 & 2.971 & 1.658 to 5.324 & **<0.001** \\ \hline _DRB1*15:02_ & 19 & 0.232 & 0.145 & 136 & 1.783 & 1.034 to 3.072 & **0.037** \\ \hline _DRB1*16:02_ & 9 & 0.11 & 0.0596 & 56 & 1.945 & 0.925 to 4.09 & 0.079 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Odd ratio of HLA alleles in 41 patients with pustular skin diseases in comparison with Thai healthy controls ([PERSON] et al., 2020) [21]. We further identified the variants that were enriched in _DPB1_ and _DRB1_ genes as they can potentially lead to separate entities of AOID and PP. It was found that 13 missense (5 in _DPB1_ and 8 in _DRB1_) and 4 frameshift variants (1 in _DPB1_ and 3 in _DRB1_) out of 90 missense and 21 frameshift variants were observed to be significantly enriched in _DPB1_ and _DRB1_ genes in the subjects presented with pustular skin diseases (Supplementary Table S5). Among these, four missense variants of _DPB1_ (Figure 4A) and four missense and three frameshift variants of _DRB1_ were localized in the binding groove (Figure 4B). Figure 4: Detected missense SNVs in the peptide-binding groove of _HLA-DRB1_ (**A**) and _HLA-DPB1_ (**B**) molecules. HLA molecules are shown in a black ribbon, while peptide antigens are shown in the yellow line.