The landscape of the immunoglobulin repertoire in endemic pemphigus foliaceus.

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.

Persistence of foetal testicular features in patients with defective androgen signalling.

OBJECTIVE: Congenital defects of androgen synthesis or action in 46,XY individuals can result in impaired virilisation, despite the apparent testicular development. In a recent case, report of a young adult with complete androgen insensitivity syndrome (CAIS), tumourous gonadal tissue was shown to express HSD17B3 in Sertoli cells (SCs) and not in Leydig cells (LCs). This expression pattern differs from the typical adult human testis and resembles a foetal mouse testis, suggesting an underlying testicular development and function defect. Here, we investigate the effect of altered androgen signalling in gonads from five 46,XY individuals with defects in androgen synthesis or action. METHODS: Gonadal tissue sections from four patients with CAIS, one with CYP17A1 deficiency, and one control were immunostained for LC developmental and steroidogenic markers. The expression of some of these markers during development was investigated by reanalysing previously published single-cell RNA sequencing (scRNA-seq) data from normal human testicular tissues. RESULTS: All gonadal tissues from the patients show an exclusive expression of HSD17B3 in SCs and an expression of the foetal/immature LC marker DLK1 in a subset of LCs, suggesting an androgen-dependent differentiation defect of adult SCs and LCs. Furthermore, reanalysis of scRNA-seq data reveals an expression of HSD17B3 in foetal and neonatal SCs that is downregulated in adult SCs. CONCLUSIONS: Androgen signalling may affect the differentiation of adults, but possibly not foetal SCs or LCs, and may induce a shift of testosterone production from the tubular compartment in the foetal phase to the interstitial compartment in the adult phase.

MYRF: A New Regulator of Cardiac and Early Gonadal Development-Insights from Single Cell RNA Sequencing Analysis.

De novo variants in the myelin regulatory factor (MYRF), a transcription factor involved in the differentiation of oligodendrocytes, have been linked recently to the cardiac and urogenital syndrome, while familiar variants are associated with nanophthalmos. Here, we report for the first time on a patient with a de novo stop-gain variant in MYRF (p.Q838*) associated with Scimitar syndrome, 46,XY partial gonadal dysgenesis (GD) and severe hyperopia. Since variants in MYRF have been described in both 46,XX and 46,XY GD, we assumed a role of MYRF in the early development of the bipotential gonad. We used publicly available single cell sequencing data of human testis and ovary from different developmental stages and analysed them for MYRF expression. We identified MYRF expression in the subset of coelomic epithelial cells at stages of gonadal ridge development in 46,XX and 46,XY individuals. Differential gene expression analysis revealed significantly upregulated genes. Within these, we identified CITED2 as a gene containing a MYRF binding site. It has been shown that Cited2-/- mice have gonadal defects in both testis and ovary differentiation, as well as defects in heart development and establishment of the left-right axis. This makes MYRF a potential candidate as an early regulator of gonadal and heart development via upregulation of the transcriptional cofactor CITED2.

Population structure and forensic genetic analyses in Guarani and Kaingang Amerindian populations from Brazil.

When DNA profile comparisons between a crime scene trace and a reference sample generate correspondence, the match probability has to be estimated, so that evaluation of the strength of the forensic DNA evidence can be made. The random match probability estimations require information on allele frequencies and an adjustment factor, referred to as theta (θ) or Fst, a co-ancestry correction factor for subpopulation effects. The θ value has been standardized for urban and isolated populations, but inconsistencies have been reported when it is specifically calculated for smaller and isolated populations, including Amerindian populations. Notably, attempts to characterize forensic markers of these minor populations have been extensively limited and more conservative estimates of the correction factor may be generated for each of them. Therefore, we estimate allele frequencies of 21 autosomal STR markers used for forensic testing and calculated relevant forensic parameters for the set. In addition, we featured the possible structure of five Brazilian Amerindian populations that have been genetically isolated for centuries so we could obtain the appropriate θ value for them. The sample consisted of 319 individuals: (1) 121 Kaingang, from two communities: Ivaí (KIV=61) and Rio das Cobras (KRC=60); and (2) 198 Guaranis from three communities: Mbya from Rio das Cobras (GRC=51), Guarani Ñandeva (GND=71) and Guarani Kaiowá (GKW=76). Between Guarani populations low (Rst=0.0402, p < 10-4) to high (Rst=0.1557, p < 10-5) differentiation was found. Regarding Guarani and Kaingang populations, intermediate (Rst=0.0590, p < 10-5) to high (Rst=0.1604, p < 10-5) differentiation was found. The two Kaingang populations showed very low differentiation between them (Rst=0.0017, p = 0.27), which justifies the union of both genetic data for forensic databases and calculations. The combined power of discrimination (PD) and the combined power of exclusion (PE) were calculated for each population, demonstrating the usefulness of this set of markers in forensic and kinship analysis regarding these populations. Considering the demographic heterogeneity of Amerindian populations in general, the Fst mean value (0.03) was evaluated regarding 43 different indigenous populations from the Americas, including Guaranis and Kaingangs. This result confirms the adequacy of the standardized θ value for the forensic random match probability estimations involving Amerindian populations.

Uniparental markers reveal new insights on subcontinental ancestry and sex-biased admixture in Brazil.

The Brazilian population is a product of asymmetric admixture among European men and Amerindian and African women. However, Brazilian subcontinental ancestry is scarcely documented, especially regarding its African roots. Here, we aimed to unveil the uniparental continental and subcontinental contributions from distinct Brazilian regions, including South (n = 43), Southeast (n = 71), the poorly genetically characterized Central-Western region (n = 323), and a subset of unique Brazilian Amerindians (n = 24), in the context of their genome-wide ancestral contributions. The overwhelming majority of European Y haplogroups (85%) contrast sharply with the predominant African and Amerindian mtDNA haplogroups (73.2%) in admixed populations, whereas in Amerindians, non-Native haplogroups could only be detected through the paternal line. Our in-depth investigation of uniparental markers showed signals of an Andean and Central-Brazilian Amerindian maternal contribution to Southeastern and Central-Western Brazil (83.1 ± 2.1% and 56.9 ± 0.2%, respectively), the last having the highest paternal Amerindian ancestry yet described for an admixed Brazilian region (9.7%) and contrasting with higher Southern-Brazilian Amerindian contribution to Southern Brazil (59.6 ± 1%). Unlike the higher African Bantu contribution previously reported for the South and Southeast, a relevant Western African non-Bantu contribution was detected in those regions (85.7 ± 5% and 71.8 ± 10.8% respectively). In contrast, a higher Bantu contribution was described for the first time in the Central-West (64.8 ± 1.3% maternal and 86.9 ± 9.6% paternal). We observed sex-biased signatures consistent with the historically recorded Brazilian colonization and added new insights in the subcontinental maternal ancestry of Brazilians from regions never studied at this level.

Genetic association and differential expression of HLAComplexGroup lncRNAs in pemphigus.

BACKGROUND: Pemphigus is a group of bullous diseases characterized by acantholysis and skin blisters. As for other autoimmune diseases, the strongest genetic associations found so far for pemphigus foliaceus (PF) and vulgaris (PV) are with alleles of HLA genes. However, apart from protein-coding genes, the MHC region includes a set of poorly explored long non-coding RNA (lncRNA) genes, the HLA complex group (HCG). OBJECTIVES: To investigate if HCG lncRNA alleles are associated with pemphigus susceptibility. METHODS AND RESULTS: We analyzed SNPs in 13 HCG lncRNA genes, both in PV (Germany: 241 patients; 1,188 controls) and endemic PF (Brazil: 227 patients; 194 controls), applying multivariate logistic regression. We found 55 associations with PV (pcorr < 0.01) and nine with endemic PF (pcorr < 0.05), the majority located in TSBP1-AS1 (which includes HCG23) and HCG27 lncRNA genes, independently of HLA alleles previously associated with pemphigus. The association of TSBP1-AS1 rs3129949*A allele was further replicated in sporadic PF (p = 0.027, OR = 0.054; 75 patients and 150 controls, all from Germany). Next, we evaluated the expression levels of TSBP1-AS1, TSBP1, HCG23, and HCG27 in blood mononuclear cells of Brazilian patients and controls. HCG27 was upregulated in endemic PF (p = 0.035, log2 FC = 1.3), while TSBP1-AS1 was downregulated in PV (p = 0.029, log2 FC = -1.29). The same expression patterns were also seen in cultured keratinocytes stimulated with IgG antibodies from patients and controls from Germany. TSBP1 mRNA levels were also decreased in endemic PF blood cells (p = 0.042, log2 FC = -2.14). TSBP1-AS1 and HCG27 were also observed downregulated in CD19+ cells of endemic PF (p < 0.01, log2 FC = -0.226 and -0.46 respectively). CONCLUSIONS: HCG lncRNAs are associated with susceptibility to pemphigus, being TSBP1-AS1 and HCG27 also differentially expressed in distinct cell populations. These results suggest a role for HCG lncRNAs in pemphigus autoimmunity.

Variation in genes implicated in B-cell development and antibody production affects susceptibility to pemphigus.

Pemphigus foliaceus (PF) is an autoimmune blistering skin disease characterized by the presence of pathogenic autoantibodies against desmoglein 1, a component of intercellular desmosome junctions. PF occurs sporadically across the globe and is endemic in some Brazilian regions. Because PF is a B-cell-mediated disease, we aimed to study the impact of variants within genes encoding molecules involved in the different steps of B-cell development and antibody production on the susceptibility of endemic PF. We analysed 3,336 single nucleotide polymorphisms (SNPs) from 167 candidate genes genotyped with Illumina microarray in a cohort of 227 PF patients and 193 controls. After quality control and exclusion of non-informative and redundant SNPs, 607 variants in 149 genes remained in the logistic regression analysis, in which sex and ancestry were included as covariates. Our results revealed 10 SNPs within or nearby 11 genes that were associated with susceptibility to endemic PF (OR >1.56; p < 0.005): rs6657275*G (TGFB2); rs1818545*A (RAG1/RAG2/IFTAP);rs10781530*A (PAXX), rs10870140*G and rs10781522*A (TRAF2); rs535068*A (TNFRSF1B); rs324011*A (STAT6);rs6432018*C (YWHAQ); rs17149161*C (YWHAG); and rs2070729*C (IRF1). Interestingly, these SNPs have been previously associated with differential gene expression, mostly in peripheral blood, in publicly available databases. For the first time, we show that polymorphisms in genes involved in B-cell development and antibody production confer differential susceptibility to endemic PF, and therefore are candidates for possible functional studies to understand immunoglobulin gene rearrangement and its impact on diseases.

An integrated personal and population-based Egyptian genome reference.

A small number of de novo assembled human genomes have been reported to date, and few have been complemented with population-based genetic variation, which is particularly important for North Africa, a region underrepresented in current genome-wide references. Here, we combine long- and short-read whole-genome sequencing data with recent assembly approaches into a de novo assembly of an Egyptian genome. The assembly demonstrates well-balanced quality metrics and is complemented with variant phasing via linked reads into haploblocks, which we associate with gene expression changes in blood. To construct an Egyptian genome reference, we identify genome-wide genetic variation within a cohort of 110 Egyptian individuals. We show that differences in allele frequencies and linkage disequilibrium between Egyptians and Europeans may compromise the transferability of European ancestry-based genetic disease risk and polygenic scores, substantiating the need for multi-ethnic genome references. Thus, the Egyptian genome reference will be a valuable resource for precision medicine.

Single Nucleotide Polymorphism in KIR2DL1 Is Associated With HLA-C Expression in Global Populations.

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.

Unveiling the Diversity of Immunoglobulin Heavy Constant Gamma (IGHG) Gene Segments in Brazilian Populations Reveals 28 Novel Alleles and Evidence of Gene Conversion and Natural Selection.

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.

High-resolution characterization of 12 classical and non-classical HLA loci in Southern Brazilians.

The human leukocyte antigen (HLA) are the most polymorphic genes in the human genome. Because of their importance for antigen recognition, HLA molecules play a central role in host defense and graft rejection upon transplantation. The aim of this study was to characterize allelic diversity of the classical HLA genes HLA-A, -B, -C, -DRA, -DRB1, -DQA1, -DQB1, -DPA1, -DPB1, and the non-classical class I genes HLA-E, -F and -G at high-resolution for a population of predominantly European ancestry from Curitiba, Brazil. Genotyping of 108 individuals was performed by next-generation sequencing on the MiSeq platform and also by Sanger sequencing. The genotype distributions of all loci were in accordance with Hardy-Weinberg equilibrium (P > 0.05) and a total of 202 HLA variants at second field resolution were observed for the 12 loci. The strongest linkage disequilibrium (r2 = 1.0, P < 10-5 ) was observed for the following pairs of alleles: HLA-B*42:01:01 ~ HLA-DRB1*03:02:01; HLA-B*14:02:01 ~ HLA-C*08:02:01; B*42:01:01 ~ HLA-C*17:01:01; HLA-DRB1*03:01:01 ~ HLA-DQB1*02:01:01 ~ DRB1*03:01:01 ~ HLA-DQB1*02:01:01; DRB1*13:01:01~ HLA-DQB1*06:03:01 and HLA-DRB1*09:01:02 ~ HLA-DQA1*03:02. This is the first study to characterize all 12 HLA genes at high resolution in a single population. On the basis of the allelic frequencies of worldwide populations and principal component analysis, we confirmed the similarity of the study population to European and other Euro-descendant populations.