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Introduction: Primarily driven by autoreactive B cells, pemphigus foliaceus (PF) is an uncommon autoimmune blistering skin disease of sporadic occurrence worldwide. However, PF reaches a prevalence of 3% in the endemic areas of Brazil, the highest ever registered for any autoimmune disease, which indicates environmental factors influencing the immune response in susceptible individuals. We aimed to provide insights into the immune repertoire of patients with PF living in the endemic region of the disease, compared to healthy individuals from the endemic region and a non-endemic area., Methods: We characterized the B-cell repertoire in i) nontreated patients (n=5); ii) patients under immunosuppressive treatment (n=5); iii) patients in remission without treatment (n=6); and two control groups iv) from the endemic (n=6) and v) non-endemic areas in Brazil (n=4). We used total RNA extracted from peripheral blood mononuclear cells and performed a comprehensive characterization of the variable region of immunoglobulin heavy chain (IGH) in IgG and IgM using next-generation sequencing., Results: Compared to individuals from a different area, we observed remarkably lower clonotype diversity in the B-cell immune repertoire of patients and controls from the endemic area ( p < 0.02), suggesting that the immune repertoire in the endemic area is under geographically specific and intense environmental pressure. Moreover, we observed longer CDR3 sequences in patients, and we identified differential disease-specific usage of IGHV segments, including increased IGHV3-30 and decreased IGHV3-23 in patients with active disease ( p < 0.04). Finally, our robust network analysis discovered clusters of CDR3 sequences uniquely observed in patients with PF., Discussion: Our results indicate that environmental factors, in addition to disease state, impact the characteristics of the repertoire. Our findings can be applied to further investigation of the environmental factors that trigger pemphigus and expand the knowledge for identifying new targeted and more effective therapies., Competing Interests: 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., (Copyright © 2023 Calonga-Solís, Olbrich, Ott, Adelman Cipolla, Malheiros, Künstner, Farias, Camargo, Petzl-Erler, Busch, Fähnrich and Augusto.)

The KIR (killer-cell immunoglobulin-like receptor ) region is characterized by structural variation and high sequence similarity among genes, imposing technical difficulties for analysis. We undertook the most comprehensive study to date of KIR genetic diversity in a large population sample, applying next-generation sequencing in 2,130 United States European-descendant individuals. Data were analyzed using our custom bioinformatics pipeline specifically designed to address technical obstacles in determining KIR genotypes. Precise gene copy number determination allowed us to identify a set of uncommon gene-content KIR haplotypes accounting for 5.2% of structural variation. In this cohort, KIR2DL4 is the framework gene that most varies in copy number (6.5% of all individuals). We identified phased high-resolution alleles in large multi-locus insertions and also likely founder haplotypes from which they were deleted. Additionally, we observed 250 alleles at 5-digit resolution, of which 90 have frequencies ≥1%. We found sequence patterns that were consistent with the presence of novel alleles in 398 (18.7%) individuals and contextualized multiple orphan dbSNPs within the KIR complex. We also identified a novel KIR2DL1 variant, Pro151Arg, and demonstrated by molecular dynamics that this substitution is predicted to affect interaction with HLA-C. No previous studies have fully explored the full range of structural and sequence variation of KIR as we present here. We demonstrate that pairing high-throughput sequencing with state-of-art computational tools in a large cohort permits exploration of all aspects of KIR variation including determination of population-level haplotype diversity, improving understanding of the KIR system, and providing an important reference for future studies., Competing Interests: 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., (Copyright © 2021 Amorim, Augusto, Nemat-Gorgani, Montero-Martin, Marin, Shams, Dandekar, Caillier, Parham, Fernández-Viña, Oksenberg, Norman and Hollenbach.)

Regulation of NK cell activity is mediated through killer-cell immunoglobulin-like receptors (KIR) ability to recognize human leukocyte antigen (HLA) class I molecules as ligands. Interaction of KIR and HLA is implicated in viral infections, autoimmunity, and reproduction and there is growing evidence of the coevolution of these two independently segregating gene families. By leveraging KIR and HLA-C data from 1000 Genomes consortium we observed that the KIR2DL1 variant rs2304224 * T is associated with lower expression of HLA-C in individuals carrying the ligand HLA-C2 ( p = 0.0059). Using flow cytometry, we demonstrated that this variant is also associated with higher expression of KIR2DL1 on the NK cell surface ( p = 0.0002). Next, we applied next generation sequencing to analyze KIR2DL1 sequence variation in 109 Euro and 75 Japanese descendants. Analyzing the extended haplotype homozygosity, we show signals of positive selection for rs4806553 * G and rs687000 * G , which are in linkage disequilibrium with rs2304224 * T . Our results suggest that lower expression of HLA-C2 ligands might be compensated for higher expression of the receptor KIR2DL1 and bring new insights into the coevolution of KIR and HLA ., (Copyright © 2020 Vargas, Dourado, Amorim, Ho, Calonga-Solís, Issler, Marin, Beltrame, Petzl-Erler, Hollenbach and Augusto.)

Pemphigus foliaceus (PF) is an autoimmune blistering skin disease that occurs sporadically across the globe and is endemic in Brazil. Keratinocyte adhesion loss (acantholysis) is associated with high levels of anti-desmoglein 1 IgG autoantibodies, but the role of cell death is poorly understood in PF. Current evidence disqualifies apoptosis as the major cell death mechanism and no other process has yet been investigated. To approach the role of variation in genes responsible for cell death pathways in pemphigus susceptibility, we systematically investigated the frequencies of 1,167 polymorphisms from genes encoding products of all 12 well-established cell death cascades (intrinsic and extrinsic apoptosis, necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic, NETotic, lysosome-dependent, autophagy-dependent, and immunogenic). By multivariate logistic regression, we compared allelic and genotypic frequencies of 227 PF patients and 194 controls obtained by microarray hybridization. We found 10 variants associated with PF ( p < 0.005), belonging to six cell death pathways: apoptosis ( TNF, TRAF2, CD36 , and PAK2 ), immunogenic cell death ( EIF2AK3, CD47 , and SIRPA ), necroptosis ( TNF and TRAF2 ), necrosis ( RAPGEF3 ), parthanatos ( HK1 ), and pyroptosis ( PRKN ). Five polymorphisms were associated with susceptibility: TNF rs1800630 * A ( OR = 1.9, p = 0.0003), CD36 rs4112274 * T ( OR = 2.14, p = 0.0015), CD47 rs12695175 * G ( OR = 1.77, p = 0.0043), SIRPA rs6075340 * A/A ( OR = 2.75, p = 0.0009), and HK1 rs7072268 * T ( OR = 1.48, p = 0.0045). Other five variants were associated with protection: TRAF2 rs10781522 * G ( OR = 0.64, p = 0.0014), PAK2 rs9325377 * A/A ( OR = 0.48, p = 0.0023), EIF2AK3 rs10167879 * T ( OR = 0.48, p = 0.0007), RAPGEF3 rs10747521 * A/A ( OR = 0.42, p = 0.0040), and PRKN rs9355950 * C ( OR = 0.57, p = 0.0004). Through functional annotation, we found that all associated alleles, with the exception of PRKN rs9355950 * C , were previously associated with differential gene expression levels in healthy individuals (mostly in skin and peripheral blood). Further functional validation of these genetic associations may contribute to the understanding of PF etiology and to the development of new drugs and therapeutic regimens for the disease., (Copyright © 2019 Bumiller-Bini, Cipolla, Spadoni, Augusto, Petzl-Erler, Beltrame and Boldt.)

Even though immunoglobulins are critical for immune responses and human survival, the diversity of the immunoglobulin heavy chain gene ( IGH ) is poorly known and mostly characterized only by serological methods. Moreover, this genomic region is not well-covered in genomic databases and genome-wide association studies due to particularities that impose technical difficulties for its analysis. Therefore, the IGH gene has never been systematically sequenced across populations. Here, we deliver an unprecedented and comprehensive characterization of the diversity of the IGHG1, IGHG2 , and IGHG3 gene segments, which encode the constant region of the most abundant circulating immunoglobulins: IgG1, IgG2, and IgG3, respectively. We used Sanger sequencing to analyze 357 individuals from seven different Brazilian populations, including five Amerindian, one Japanese-descendant and one Euro-descendant population samples. We discovered 28 novel IGHG alleles and provided evidence that some of them may have been originated by gene conversion between common alleles of different gene segments. The rate of synonymous substitutions was significantly higher than the rate of the non-synonymous substitutions for IGHG1 and IGHG2 ( p = 0.01 and 0.03, respectively), consistent with purifying selection. Fay and Wu's test showed significant negative values for most populations ( p < 0.001), which indicates that positive selection in an adjacent position may be shaping IGHG variation by hitchhiking of variants in the vicinity, possibly the regions that encode the Ig variable regions. This study shows that the variation in the IGH gene is largely underestimated. Therefore, exploring its nucleotide diversity in populations may provide valuable information for comprehension of its evolution, its impact on diseases and vaccine research.

The killer-cell immunoglobulin-like receptor ( KIR ) region comprises a fast-evolving family of genes that encode receptors for natural killer (NK) cells and have crucial role in host defense. Evolution of KIR was examined in the context of the human genome. Gene-content diversity and single nucleotide polymorphisms (SNP) in the KIR genes and flanking regions were compared to >660,000 genome-wide SNPs in over 800 individuals from 52 populations of the human genome diversity panel (HGDP). KIR allelic diversity was further examined using next generation sequencing in a subset of 56 individuals. We identified the SNP rs587560 located in KIR3DL3 as a marker of KIR2DL2 and KIR2DL3 and, consequently, Cen A and Cen B haplotypes. We also show that combinations of two KIR2DL4 SNPs ( rs35656676 and rs592645 ) distinguish KIR3DL1 from KIR3DS1 and also define the major KIR3DL1 high- and low-expressing alleles lineages. Comparing the diversity of the SNPs within the KIR region to remainder of the genome, we observed a high diversity for the centromeric KIR region consistent with balancing selection ( p < 0.01); in contrast, centromeric KIR diversity is significantly reduced in East Asian populations ( p < 0.01), indicating purifying selection. By analyzing SNP haplotypes in a region spanning ~500 kb that includes the KIR cluster, we observed evidence of strong positive selection in Africa for high-expressing KIR3DL1 alleles, favored over the low-expressing alleles ( p < 0.01). In sharp contrast, the strong positive selection ( p < 0.01) that we also observed in the telomeric KIR region in Oceanic populations tracked with a high frequency of KIR3DS1 . In addition, we demonstrated that worldwide frequency of high-expression KIR3DL1 alleles was correlated with virus with virus (r = 0.64, p < 10 -6 ) and protozoa (r = 0.69, p < 10 -6 ) loads, which points to selection globally on KIR3DL1 high-expressing alleles attributable to pathogen exposure.

Skin blisters of pemphigus foliaceus (PF) present concomitant deposition of autoantibodies and components of the complement system (CS), whose gene polymorphisms are associated with susceptibility to different autoimmune diseases. To investigate these in PF, we evaluated 992 single-nucleotide polymorphisms (SNPs) of 44 CS genes, genotyped through microarray hybridization in 229 PF patients and 194 controls. After excluding SNPs with minor allele frequency <1%, out of Hardy-Weinberg equilibrium in controls or in strong linkage disequilibrium ( r 2  ≥ 0.8), 201 SNPs remained for logistic regression. Polymorphisms of 11 genes were associated with PF. MASP1 encodes a crucial serine protease of the lectin pathway (rs13094773: OR = 0.5, p  = 0.0316; rs850309: OR = 0.23, p  = 0.03; rs3864098: OR = 1.53, p  = 0.0383; rs698104: OR = 1.52, p  = 0.0424; rs72549154: OR = 0.55, p  = 0.0453). C9 (rs187875: OR = 1.46, p  = 0.0189; rs700218: OR = 0.12, p  = 0.0471) and C8A (rs11206934: OR = 4.02, p  = 0.0323) encode proteins of the membrane attack complex (MAC) and C5AR1 (rs10404456: OR = 1.43, p  = 0.0155), a potent anaphylatoxin-receptor. Two encode complement regulators: MAC-blocking CD59 (rs1047581: OR = 0.62, p  = 0.0152) and alternative pathway-blocking CFH (rs34388368: OR = 2.57, p  = 0.0195). One encodes opsonin: C3 (rs4807895: OR = 2.52, p  = 0.0239), whereas four encode receptors for C3 fragments: CR1 (haplotype with rs6656401: OR = 1.37, p  = 0.0382), CR2 (rs2182911: OR = 0.23, p  = 0.0263), ITGAM (CR3, rs12928810: OR = 0.66, p  = 0.0435), and ITGAX (CR4, rs11574637: OR = 0.63, p  = 0.0056). Associations reinforced former findings, regarding differential gene expression, serum levels, C3, and MAC deposition on lesions. Deregulation of previously barely noticed processes, e.g., the lectin and alternative pathways and opsonization-mediated phagocytosis, also modulate PF susceptibility. The results open new crucial avenues for understanding disease etiology and may improve PF treatment through additional therapeutic targets.