Search

BackgroundLigation of CD28 with ligands such as CD80 or CD86 provides a critical second signal alongside antigen presentation by class II major histocompatibility complex expressed on antigen-presenting cells through the T cell antigen receptor for naïve T cell activation. A number of studies suggested that CD28 plays an important role in the pathogenesis of various human diseases. Recent genome-wide association studies (GWASs) identifiedCD28as a susceptibility locus for lymphocyte and eosinophil counts, multiple sclerosis, ulcerative colitis, celiac disease, rheumatoid arthritis, asthma, and primary biliary cholangitis. However, the primary functional variant and molecular mechanisms of disease susceptibility in this locus remain to be elucidated. This study aimed to identify the primary functional variant from thousands of genetic variants in theCD28locus and elucidate its functional effect on the CD28 molecule.ResultsAmong the genetic variants exhibiting stronger linkage disequilibrium (LD) with all GWAS-lead variants in theCD28locus, rs2013278, located in the Rbfox binding motif related to splicing regulation, was identified as a primary functional variant related to multiple immunological traits. Relative endogenous expression levels ofCD28splicing isoforms (CD28i and CD28Δex2) compared with full-length CD28 in allele knock-in cell lines generated using CRISPR/Cas9 were directly regulated by rs2013278 (P ConclusionThe present study demonstrated for the first time thatCD28has a shared disease-related primary functional variant (i.e., rs2013278) that regulates the CD28 alternative splicing that generates loss-of-function isoforms. They reduce disease risk by inducing anergy of effector T cells that over-react to autoantigens and allergens.

Genome‐wide association studies (GWASs) in European and East Asian populations have identified more than 40 disease‐susceptibility genes in primary biliary cholangitis (PBC). The aim of this study is to computationally identify disease pathways, upstream regulators, and therapeutic targets in PBC through integrated GWAS and messenger RNA (mRNA) microarray analysis. Disease pathways and upstream regulators were analyzed with ingenuity pathway analysis in data set 1 for GWASs (1,920 patients with PBC and 1,770 controls), which included 261 annotated genes derived from 6,760 single‐nucleotide polymorphisms (P 2 versus controls (P

To identify novel host genetic variants that predispose to hepatitis B virus (HBV) persistence, we performed the first genome-wide association study in the Thai population involving 318 cases of chronic hepatitis B and 309 healthy controls after quality control measures. We detected the genome-wide significant association of the HLA class II region (HLA-DPA1/DPB1, rs7770370, p-value = 7.71 × 10−10, OR = 0.49) with HBV chronicity. Subsequent HLA allele imputation revealed HLA-DPA1*01:03 (Pc = 1.21 × 10−6, OR = 0.53), HLA-DPB1*02:01 (Pc = 2.17 × 10−3, OR = 0.50), and HLA-DQB1*06:09 (Pc = 2.17 × 10−2, OR = 0.07) as protective alleles, and HLA-DPA1*02:02 (Pc = 6.32 × 10−5, OR = 1.63), HLA-DPB1*05:01 (Pc = 1.13 × 10−4, OR = 1.72), HLA-DPB1*13:01 (Pc = 4.68 × 10−2, OR = 1.60), and HLA-DQB1*03:03 (Pc = 1.11 × 10−3, OR = 1.84) as risk alleles for HBV persistence. We also detected suggestive associations in the PLSCR1 (rs35766154), PDLIM5 (rs62321986), SGPL1 (rs144998273), and MGST1 (rs1828682) loci. Among single-nucleotide polymorphisms in the PLSCR1 locus, rs1061307 was identified as the primary functional variant by in silico/in vitro functional analysis. In addition to replicating the association of the HLA class II region, we detected novel candidate loci that provide new insights into the pathophysiology of chronic hepatitis B.

Primary biliary cholangitis (PBC) is a chronic, progressive cholestatic liver disease in which the small intrahepatic bile ducts are destroyed by autoimmune reactions. Among autoimmune diseases, which are polygenic complex traits caused by the combined contribution of genetic and environmental factors, PBC exhibits the strongest involvement of genetic heritability in disease development. As at December 2022, genome-wide association studies (GWASs) and associated meta-analyses identified approximately 70 PBC susceptibility gene loci in various populations, including those of European and East Asian descent. However, the molecular mechanisms through which these susceptibility loci affect the pathogenesis of PBC are not fully understood. This study provides an overview of current data regarding the genetic factors of PBC as well as post-GWAS approaches to identifying primary functional variants and effector genes in disease-susceptibility loci. Possible mechanisms of these genetic factors in the development of PBC are also discussed, focusing on four major disease pathways identified by in silico gene set analyses, namely, (1) antigen presentation by human leukocyte antigens, (2) interleukin-12-related pathways, (3) cellular responses to tumor necrosis factor, and (4) B cell activation, maturation, and differentiation pathways.

Primary biliary cholangitis (PBC) is a chronic, progressive cholestatic liver disease in which intrahepatic bile ducts are destroyed by an autoimmune reaction. Our previous genome-wide association study (GWAS) identified chromosome 11q23.1 as a susceptibility gene locus for PBC in the Japanese population. Here, high-density association mapping based on single nucleotide polymorphism (SNP) imputation and in silico/in vitro functional analyses identified rs1944919 as the primary functional variant. Expression-quantitative trait loci analyses showed that the PBC susceptibility allele of rs1944919 was significantly associated with increased COLCA1/COLCA2 expression levels. Additionally, the effects of rs1944919 on COLCA1/COLCA2 expression levels were confirmed using genotype knock-in versions of cell lines constructed using the CRISPR/Cas9 system and differed between rs1944919-G/G clones and -T/T clones. To our knowledge, this is the first study to demonstrate the contribution of COLCA1/COLCA2 to PBC susceptibility.

Guillain-Barre syndrome (GBS), including its variant Miller Fisher syndrome (MFS), is an acute peripheral neuropathy that involves autoimmune mechanisms leading to the production of autoantibodies to gangliosides; sialic acid-containing glycosphingolipids. Although association with various genetic polymorphisms in the major histocompatibility complex (MHC) is shown in other autoimmune diseases, GBS is an exception, showing no such link. No significant association was found by genome wide association studies, suggesting that GBS is not associated with common variants. To address the involvement of rare variants in GBS, we analyzed Siglec-10, a sialic acid-recognizing inhibitory receptor expressed on B cells. Here we demonstrate that two rare variants encoding R47Q and A108V substitutions in the ligand-binding domain are significantly accumulated in patients with GBS. Because of strong linkage disequilibrium, there was no patient carrying only one of them. Recombinant Siglec-10 protein containing R47Q but not A108V shows impaired binding to gangliosides. Homology modeling revealed that the R47Q substitution causes marked alteration in the ligand-binding site. Thus, GBS is associated with a rare variant of the SIGLEC10 gene that impairs ligand binding of Siglec-10. Because Siglec-10 regulates antibody production to sialylated antigens, our finding suggests that Siglec-10 regulates development of GBS by suppressing antibody production to gangliosides, with defects in its function predisposing to disease.

Stevens–Johnson syndrome (SJS) and its severe condition with extensive skin detachment and a poor prognosis, toxic epidermal necrolysis (TEN), are immunologically mediated severe cutaneous reactions of the skin and mucous membranes such as the ocular surface. Genetic variations on the HLA-A and other autosomal genes have been identified as risk factors for cold medicine-related SJS/TEN with severe ocular complications (CM-SJS/TEN with SOC). Using a whole-genome sequencing (WGS) approach, we explored other susceptible variants of CM-SJS/TEN with SOC, especially among rare variants and structural variants (SVs). WGS was performed on samples from 133 patients with CM-SJS/TEN with SOC and 418 healthy controls to obtain single nucleotide polymorphisms (SNPs) and SVs. Genome-wide association tests were performed with these variants. Our genome-wide association test reproduced the associations of the common variants of HLA-A and loci on chromosome 16q12.1. We also identified novel associations of SVs on these loci and an aggregation of rare coding variants on the TPRM8 gene. In silico gene expression analysis on the HLA-A locus revealed that the SNP (rs12202296), which was significantly associated with susceptibility to CM-SJS/TEN with SOC, was correlated to an increase in HLA-A expression levels in the whole blood (P = 2.9 × 10−17), from the GTEx database. The majority of variants that were significantly associated with CM-SJS/TEN with SOC were found in non-coding regions, indicating the regulatory role of genetic variations in the pathogenesis of CM-SJS/TEN with SOC.

Croissamide, a proline-rich cyclic peptide that contains an N-prenylated tryptophan, was isolated from a marine cyanobacterium Symploca sp. Its gross structure was determined by spectroscopic analyses, and the absolute configuration was established based on chiral HPLC analyses of acid hydrolysates.

Background Nephrotic syndrome is the most common cause of chronic glomerular disease in children. Most of these patients develop steroid-sensitive nephrotic syndrome (SSNS), but the loci conferring susceptibility to childhood SSNS are mainly unknown.Methods We conducted a genome-wide association study (GWAS) in the Japanese population; 224 patients with childhood SSNS and 419 adult healthy controls were genotyped using the Affymetrix Japonica Array in the discovery stage. Imputation for six HLA genes (HLA-A, -C, -B, -DRB1, -DQB1, and -DPB1) was conducted on the basis of Japanese-specific references. We performed genotyping for HLA-DRB1/-DQB1 using a sequence-specific oligonucleotide-probing method on a Luminex platform. Whole-genome imputation was conducted using a phased reference panel of 2049 healthy Japanese individuals. Replication was performed in an independent Japanese sample set including 216 patients and 719 healthy controls. We genotyped candidate single-nucleotide polymorphisms using the DigiTag2 assay.Results The most significant association was detected in the HLA-DR/DQ region and replicated (rs4642516 [minor allele G], combined Pallelic=7.84×10-23; odds ratio [OR], 0.33; 95% confidence interval [95% CI], 0.26 to 0.41; rs3134996 [minor allele A], combined Pallelic=1.72×10-25; OR, 0.29; 95% CI, 0.23 to 0.37). HLA-DRB1*08:02 (Pc=1.82×10-9; OR, 2.62; 95% CI, 1.94 to 3.54) and HLA-DQB1*06:04 (Pc=2.09×10-12; OR, 0.10; 95% CI, 0.05 to 0.21) were considered primary HLA alleles associated with childhood SSNS. HLA-DRB1*08:02-DQB1*03:02 (Pc=7.01×10-11; OR, 3.60; 95% CI, 2.46 to 5.29) was identified as the most significant genetic susceptibility factor.Conclusions The most significant association with childhood SSNS was detected in the HLA-DR/DQ region. Further HLA allele/haplotype analyses should enhance our understanding of molecular mechanisms underlying SSNS.

Primary biliary cholangitis (PBC) is a chronic cholestatic autoimmune liver disease that appears to be strongly influenced by genetic factors. Recently, an international meta-analysis of genome-wide association studies (GWAS) identified CC-Motif Chemokine Receptor-6 (CCR6) and FGFR1 Oncogene-Partner (FGFR1OP) as PBC-susceptibility genes. However, the lead single nucleotide polymorphisms (SNPs) of CCR6/FGFR1OP showed low linkage disequilibrium with each other in East Asian and European populations. Additionally, the primary functional variants and the molecular mechanisms responsible for PBC-susceptibility remain unclear. Here, among the PBC-susceptibility SNPs identified by high-density association mapping in our previous meta-GWAS (Patients: n = 10,516; healthy controls: n = 20,772) within the CCR6/FGFR1OP locus, rs9459874 and rs1012656 were identified as primary functional variants. These functional variants accounted for the effects of GWAS-identified lead SNPs in CCR6/FGFR1OP. Additionally, the roles of rs9459874 and rs1012656 in regulating FGFR1OP transcription and CCR6 translation, respectively, were supported by expression quantitative trait loci (eQTL) analysis and gene editing technology using the CRISPR/Cas9 system. Immunohistochemistry showed higher expression of CCR6 protein in the livers of patients with PBC than in those of a non-diseased control. In conclusion, we identified primary functional variants in CCR6/FGFR1OP and revealed the molecular mechanisms by which these variants confer PBC-susceptibility in an eQTL-dependent or -independent manner. The approach in this study is applicable for the elucidation of the pathogenesis of other autoimmune disorders in which CCR6/FGFR1OP is known as a susceptibility locus, as well as PBC.

Coronin-1, a hematopoietic cell-specific actin-binding protein, is thought to be involved in the phagocytic process through its interaction with actin filaments. The dissociation of coronin-1 from phagosomes after its transient accumulation on the phagosome surface is associated with lysosomal fusion. We previously reported that 1) coronin-1 is phosphorylated by protein kinase C (PKC), 2) coronin-1 has two phosphorylation sites, Ser-2 and Thr-412, and 3) Thr-412 of coronin-1 is phosphorylated during phagocytosis. In this study, we examined which PKC isoform is responsible for the phosphorylation of coronin-1 at Thr-412 by using isotype-specific PKC inhibitors and small interfering RNAs (siRNAs). Thr-412 phosphorylation of coronin-1 was suppressed by Gö6976, an inhibitor of PKCα and PKCβI. This phosphorylation was attenuated by siRNA for PKCα, but not by siRNA for PKCβ. Furthermore, Thr-412 of coronin-1 was phosphorylated by recombinant PKCα in vitro, but not by recombinant PKCβ. We next examined the effects of Gö6976 on the intracellular distribution of coronin-1 in HL60 cells during phagocytosis. The confocal fluorescence microscopic observation showed that coronin-1 was not dissociated from phagosomes in Gö6976-treated cells. These results indicate that phosphorylation of coronin-1 at Thr-412 by PKCα regulates intracellular distribution during phagocytosis., Highlights • Phosphorylation of coronin-1 at Thr-412 is suppressed by PKCα/β inhibitor. • PKCα not PKCβ phosphorylates coronin-1 at Thr-412 in vitro. • Dissociation of coronin-1 from phagosome is regulated by PKCα. • Phosphorylation of coronin-1 at Thr-412 may trigger phagosome maturation.

Background Our genome-wide association study documented an association between cold medicine related Stevens-Johnson syndrome / toxic epidermal necrolysis (CM-SJS/TEN) and Ikaros Family Zinc Finger 1 (IKZF1). Few studies examined biological and pathological functions of IKZF1 in mucosal immunity. We hypothesized that IKZF1 contributes to the mucocutaneous inflammation. Methods Human skin and conjunctival tissues were obtained for immunohistological studies. Primary human conjunctival epithelial cells (PHCjECs) and adult human epidermal keratinocytes (HEKa) also used for gene expression analysis. We also generated K5-Ikzf1-EGFP transgenic mice (Ikzf1 Tg) by introducing the Ik1 isoform into cells expressing keratin 5, which is expressed in epithelial tissues such as the epidermis and conjunctiva and then examined them histologically and investigated gene expression of the epidermis. Moreover, Ikzf1 Tg were induced allergic contact dermatitis. Results We found that human epidermis and conjunctival epithelium expressed IKZF1, and in PHCjECs and HEKa, the expression of IKZF1 mRNA was up-regulated by stimulation with polyI:C, a TLR3 ligand. In Ikzf1 Tg, we observed dermatitis and mucosal inflammation including the ocular surface. In contact dermatitis model, inflammatory infiltrates in the skin of Ikzf1 Tg were significantly increased compared with wild type. Microarray analysis showed that Lcn2, Adh7, Epgn, Ifi202b, Cdo1, Gpr37, Duoxa1, Tnfrsf4, and Enpp5 genes were significantly up-regulated in the epidermis of Ikzf1 Tg compared with wild-type. Conclusion Our findings support the hypothesis that Ikaros might participate in mucocutaneous inflammation. This article is protected by copyright. All rights reserved.

Lipopolysaccharides (LPS) are associated with various inflammatory diseases; therefore, the inhibition of LPS-induced nitric oxide (NO) production may have extensive therapeutic applications. We searched for inhibitors of NO production in the LPS-stimulated murine macrophage-like cell line RAW264.7 from MeOH extracts of marine organisms. The MeOH extract of the marine cyanobacterium Okeania sp., collected in Okinawa, Japan, showed inhibitory activity. Biseokeaniamide A was isolated from the MeOH extract by chromatographic separation. Biseokeaniamide A inhibited NO production without cytotoxicity. It reduced inducible nitric oxide synthase levels and suppressed the expression of IL-1β in LPS-stimulated RAW264.7 cells. Biseokeaniamide A did not inhibit IκBα degradation but inhibited IκBα expression. Thus, biseokeaniamide A, a naturally occurring lipopeptide, was identified as a selective inhibitor of LPS signal transduction.

HLA-DQB1*05:03:01:04 differs from HLA-DQB1*05:03:01:01 by one nucleotide difference at position 5075(A > G) with no amino acid change.

HLA-B*51:01:01:36 differs from HLA-B*51:01:01:01 by one nucleotide difference at position -230(G>T) with no amino acid change.

HLA-A*02:01:01:58 differs from HLA-A*02:01:01:01 by one nucleotide difference at position 2126(G>T) with no amino acid change.

Human genome variation may cause differences in traits and disease risks. Disease-causal/susceptible genes and variants for both common and rare diseases can be detected by comprehensive whole-genome analyses, such as whole-genome sequencing (WGS), using next-generation sequencing (NGS) technology and genome-wide association studies (GWAS). Here, in addition to the application of an NGS as a whole-genome analysis method, we summarize approaches for the identification of functional disease-causal/susceptible variants from abundant genetic variants in the human genome and methods for evaluating their functional effects in human diseases, using an NGS and in silico and in vitro functional analyses. We also discuss the clinical applications of the functional disease causal/susceptible variants to personalized medicine.

The present study investigated the association between single nucleotide polymorphisms (SNPs) in immune- or DNA repair-related genes and the integration pattern of human papillomavirus (HPV), a promising prognostic marker in cervical cancer. The HPV integration patterns of cervical cancer patients were determined by polymerase chain reaction and in situ hybridization, and categorized as episomal (group A), single-copy or multi-copy tandem repetition integrated (group B), and undetectable HPV types (group C). After sample and SNP quality control, 166,505 SNPs in 161 samples (38, 111, and 12 patients in groups A, B, and C, respectively) were examined. None of the SNPs reached genome-wide significance, and several candidate SNPs for future study were selected, including rs10999435 on chromosome 10q22, rs1322054 on chromosome 9q32-33, and rs10902171 on chromosome 11p15. Luciferase assay identified rs1322054 as the primary functional variant to regulate gene expression in immune cell. Further studies are needed to determine the genetic background of different integration patterns of HPV in cervical cancer patients.

Our recent genome-wide association study found that the NELFCD/CTSZ locus was significantly associated with progression of primary biliary cholangitis (PBC) to jaundice stage in the Japanese population. In this study, we investigated the role of cathepsin Z in the etiology and pathology of PBC. Serum cathepsin Z levels were measured using enzyme-linked immunosorbent assay. The expression and localization of cathepsin Z in liver specimens were analyzed by western blotting and immunohistochemistry. In PBC patients, serum cathepsin Z levels were significantly increased with disease progression. In addition, its levels were positively correlated with alanine transaminase, aspartate transaminase and total bilirubin, and were negatively correlated with platelet count and albumin. Cathepsin Z expression was markedly increased in hepatocytes at later stages of PBC, and its localization was altered from the peri-bile canaliculus to the cytoplasm, where a fraction was no longer colocalized with endosomal/lysosomal vesicles. Similar altered expression of cathepsin Z was observed in end-stage of other cholestatic liver diseases including sepsis, obstructive jaundice, and Alagille syndrome. Our results indicate that altered expression and localization of cathepsin Z in hepatocytes are characteristic features of PBC and other cholestatic liver diseases, and are implicated in the progression of PBC.

The associations of single nucleotide polymorphisms (SNPs) in PLA2R1 and HLA-DQA1, as well as HLA-DRB1*15:01-DQB1*06:02 haplotype with idiopathic membranous nephropathy (IMN) is well known. However, the primary associations of these loci still need to be determined. We used Japanese-specific SNP genotyping array and imputation using 2,048 sequenced Japanese samples to fine-map PLA2R1 region in 98 patients and 413 controls. The most significant SNPs were replicated in a separate sample set of 130 patients and 288 controls. A two-SNP haplotype of intronic and missense SNPs showed the strongest association. The intronic SNP is strongly associated with PLA2R1 expression in the Genotype-Tissue Expression (GTEx) database, and the missense SNP is predicted to alter peptide binding with HLA-DRB1*15:01 by the Immune Epitope Database (IEDB). In HLA region, we performed relative predispositional effect (RPE) tests and identified additional risk alleles in both HLA-DRB1 and HLA-DQB1. We collapsed the risk alleles in each of HLA-DRB1 and HLA-DQB1 into single risk alleles. Reciprocal conditioning of these collapsed risk alleles showed more residual significance for HLA-DRB1 collapsed risk than HLA-DQB1 collapsed risk. These results indicate that changes in the expression levels of structurally different PLA2R protein confer risk for IMN in the presence of risk HLA-DRB1 alleles.

We previously identified TNFSF15 as the most significant susceptibility gene at non-HLA loci for both primary biliary cirrhosis (PBC) and Crohn's diseases (CD) in the Japanese population. The aim of this study is to identify further disease susceptibility genes shared by PBC and CD. We selected 15 and 33 genetic variants that were significantly associated with PBC and CD, respectively, based on previously reported genome-wide association studies of the Japanese population. Next, an association study was independently performed for these genetic variants in CD (1312 CD patients and 3331 healthy controls) and PBC (1279 PBC patients and 1015 healthy controls) cohorts. Two CD susceptibility genes, ICOSLG rs2838519 and IL12B rs6556412, were also nominally associated with susceptibility to PBC (P=3.85 × 10(-2) and P=8.40 × 10(-3), respectively). Three PBC susceptibility genes, CXCR5 rs6421571, STAT4 rs7574865 and NFKB1 rs230534, were nominally associated with susceptibility to CD (P=2.82 × 10(-2), P=3.88 × 10(-2) and P=2.04 × 10(-2), respectively). The effect of ICOSLG and CXCR5 variants were concordant but the effect of STAT4, NFKB1 and IL12B variants were discordant for PBC and CD. TNFSF15 and ICOSLG-CXCR5 might constitute a shared pathogenic pathway in the development of PBC and CD in the Japanese population, whereas IL12B-STAT4-NFKB1 might constitute an opposite pathogenic pathway, reflecting the different balance between Th1 and Th17 in the two diseases.

A genome-wide association study (GWAS) identified tumor necrosis factor superfamily member 15 (TNFSF15) as the strongest associated gene with susceptibility to primary biliary cirrhosis (PBC) outside the HLA loci in the Japanese population. However, causal functional variants of the TNFSF15 locus and the molecular mechanism underlying disease susceptibility have not been clarified. Here, to identify the functional causal variants of the TNFSF15 locus, integrated analysis comprising in silico analysis, a case–control association study and in vitro functional analysis was performed. Initially, 32 functional candidate single-nucleotide polymorphisms (SNPs) in the expression regulatory motifs, the coding region, or the untranslated regions (UTRs) of the TNFSF15 locus were selected by in silico analysis. By the case–control association studies using PBC patients (n = 1279) and healthy controls (n = 1091) in the Japanese population, rs4979462 [P = 1.85 × 10−14 (our previous study)], rs56211063 (P = 2.21 × 10−14), and rs55768522 (r 2 = 1 with rs4979462) were likely candidates for causal variants. Among these SNPs, rs4979462 was identified as the causal variant by in vitro functional analysis using luciferase assay and electrophoretic mobility shift assay (EMSA). Super-shift assay clarified that PBC-susceptible allele of rs4979462 generated a novel NF-1 binding site. Moreover, higher endogenous TNFSF15 protein and mRNA expression levels were observed in individuals with the PBC-susceptible allele of rs4979462. This study identified the causal variant for PBC susceptibility in the TNFSF15 locus and clarified its underlying molecular mechanism. TNFSF15 and NF-1 are considered to be potential targets for the treatment of PBC.

Background Nephrotic syndrome is the most common cause of chronic glomerular disease in children. Most of these patients develop steroid-sensitive nephrotic syndrome (SSNS), but the loci conferring susceptibility to childhood SSNS are mainly unknown. Methods We conducted a genome-wide association study (GWAS) in the Japanese population; 224 patients with childhood SSNS and 419 adult healthy controls were genotyped using the Affymetrix Japonica Array in the discovery stage. Imputation for six HLA genes (HLA-A, -C, -B, -DRB1, -DQB1, and -DPB1) was conducted on the basis of Japanese-specific references. We performed genotyping for HLA-DRB1/-DQB1 using a sequence-specific oligonucleotide-probing method on a Luminex platform. Whole-genome imputation was conducted using a phased reference panel of 2049 healthy Japanese individuals. Replication was performed in an independent Japanese sample set including 216 patients and 719 healthy controls. We genotyped candidate single-nucleotide polymorphisms using the DigiTag2 assay. Results The most significant association was detected in the HLA-DR/DQ region and replicated (rs4642516 [minor allele G], combined P(allelic)=7.84×10(−23); odds ratio [OR], 0.33; 95% confidence interval [95% CI], 0.26 to 0.41; rs3134996 [minor allele A], combined P(allelic)=1.72×10(−25); OR, 0.29; 95% CI, 0.23 to 0.37). HLA-DRB1*08:02 (Pc=1.82×10(−9); OR, 2.62; 95% CI, 1.94 to 3.54) and HLA-DQB1*06:04 (Pc=2.09×10(−12); OR, 0.10; 95% CI, 0.05 to 0.21) were considered primary HLA alleles associated with childhood SSNS. HLA-DRB1*08:02-DQB1*03:02 (Pc=7.01×10(−11); OR, 3.60; 95% CI, 2.46 to 5.29) was identified as the most significant genetic susceptibility factor. Conclusions The most significant association with childhood SSNS was detected in the HLA-DR/DQ region. Further HLA allele/haplotype analyses should enhance our understanding of molecular mechanisms underlying SSNS.

We identified a small family with autosomal recessive, infantile onset epilepsy and intellectual disability. Exome sequencing identified a homozygous missense variant in the gene TNK2, encoding a brain-expressed tyrosine kinase. Sequencing of the coding region of TNK2 in 110 patients with a similar phenotype failed to detect further homozygote or compound heterozygote mutations. Pathogenicity of the variant is supported by the results of our functional studies, which demonstrated that the variant abolishes NEDD4 binding to TNK2, preventing its degradation after epidermal growth factor stimulation. Definitive proof of pathogenicity will require confirmation in unrelated patients.

Genetic polymorphism within the 9q32 locus is linked with increased risk of several diseases, including Crohn’s disease (CD), primary biliary cholangitis (PBC) and leprosy. The most likely disease-causing gene within 9q32 is TNFSF15, which encodes the pro-inflammatory cytokine TNF super-family member 15, but it was unknown whether these disparate diseases were associated with the same genetic variance in 9q32, and how variance within this locus might contribute to pathology. Using genetic data from published studies on CD, PBC and leprosy we revealed that bearing a T allele at rs6478108/rs6478109 (r2 = 1) or rs4979462 was significantly associated with increased risk of CD and decreased risk of leprosy, while the T allele at rs4979462 was associated with significantly increased risk of PBC. In vitro analyses showed that the rs6478109 genotype significantly affected TNFSF15 expression in cells from whole blood of controls, while functional annotation using publicly-available data revealed the broad cell type/tissue-specific regulatory potential of variance at rs6478109 or rs4979462. In summary, we provide evidence that variance within TNFSF15 has the potential to affect cytokine expression across a range of tissues and thereby contribute to protection from infectious diseases such as leprosy, while increasing the risk of immune-mediated diseases including CD and PBC.

A previous genome-wide association study (GWAS) performed in 963 Japanese individuals (487 primary biliary cholangitis [PBC] cases and 476 healthy controls) identified TNFSF15 (rs4979462) and POU2AF1 (rs4938534) as strong susceptibility loci for PBC. In this study, we performed GWAS in additional 1,923 Japanese individuals (894 PBC cases and 1,029 healthy controls), and combined the results with the previous data. This GWAS, together with a subsequent replication study in an independent set of 7,024 Japanese individuals (512 PBC cases and 6,512 healthy controls), identified PRKCB (rs7404928) as a novel susceptibility locus for PBC (odds ratio [OR] = 1.26, P = 4.13 × 10-9). Furthermore, a primary functional variant of PRKCB (rs35015313) was identified by genotype imputation using a phased panel of 1,070 Japanese individuals from a prospective, general population cohort study and subsequent in vitro functional analyses. These results may lead to improved understanding of the disease pathways involved in PBC, forming a basis for prevention of PBC and development of novel therapeutics.

Activating and inhibitory signal transducers, CD19 and CD22, have been substantially implicated both in human systemic sclerosis (SSc) and tight-skin mouse, a model for SSc. We previously showed that a single nucleotide polymorphism (SNP) in CD19 promoter region was significantly associated with increased CD19 expression level and with susceptibility to SSc. In the present study, we examined whether CD22 polymorphisms were associated with susceptibility to SSc. CD22 variations were genotyped in 126 Japanese patients with SSc [47 diffuse cutaneous SSc and 79 limited cutaneous SSc (lcSSc)] and 93 unrelated healthy controls. At the c.2304C > A SNP coding for a synonymous substitution in exon 13, A/A genotype was observed in six patients with SSc (4.8 %) but none in the controls (P=0.040). All six patients with A/A genotype belonged to the lcSSc subgroup (7.6%, P=0.008 vs controls). Surface expression level of CD22 tended to be lower in B cells from the patients with A/A genotype (n=5) as compared with C/A (n=7) or C/C (n=14) genotype (17% decrease, P=0.0032). Taken together with our previous observation on CD19 polymorphism, intrinsic difference in the expression level of CD19 and CD22 was suggested to play a causative role in a proportion of patients with lcSSc.

We previously reported association of FCGR2B-Ile232Thr with systemic lupus erythematosus (SLE) in three Asian populations. Because polymorphism of CD72, another inhibitory receptor of B cells, was associated with murine SLE, we identified human CD72 polymorphisms, tested their association with SLE and examined genetic interaction with FCGR2B in the Japanese (160 SLE, 277 controls), Thais (87 SLE, 187 controls) and Caucasians (94 families containing SLE members). Four polymorphisms and six rare variations were detected. The former constituted two major haplotypes that contained one or two repeats of 13 nucleotides in intron 8 (designated as *1 and *2, respectively). Although association with susceptibility to SLE was not detected, the *1 allele was significantly associated with nephritis among the Japanese patients (P=0.024). RT-PCR identified a novel alternatively spliced (AS) transcript that was expressed at the protein level in COS-7 transfectants. The ratio of AS/common isoforms was strikingly increased in individuals with *2/*2 genotype when compared with *1/*1 (P=0.000038) or *1/*2 (P=0.0085) genotypes. Using the two Asian cohorts, significant association of FCGR2B-232Thr/Thr with SLE was observed only in the presence of CD72-*1/*1 genotype (OR 4.63, 95% CI 1.47-14.6, P=0.009 versus FCGR2B-232Ile/Ile plus CD72-*2/*2). Minigene assays demonstrated that the 13-nucleotide repeat and 4 bp deletion within the same haplotype of intron 8 could regulate alternative splicing. The AS isoform lacks exon 8, and is deduced to contain 49 amino acid changes in the membrane-distal portion of the extracellular domain, where considerable amino acid changes are known in CD72(c) allele associated with murine SLE. These results indicated that the presence of CD72-*2 allele decreases risk for human SLE conferred by FCGR2B-232Thr, possibly by increasing the AS isoform of CD72.

Aim The human MHC region has been shown to be associated with a wide range of diseases.The recent advancement in the next generation sequencing (NGS) technology has definitely helped in increasing the resolution (up to 4-field) of HLA genotyping. However, it remains a challenge in interpreting the results from NGS platform due to biases including systematic sequencing error by sequencing platform, difference in the sensitivity and specificity of HLA calling algorithm, ability to resolve HLA alleles ambiguity by incorporating phasing information, and more importantly incompleteness of the current IMGT database. Methods In order to access the sensitivity and specificity of the NXTypeTM NGS HLA typing kit (One Lambda), a total of 105 Japanese samples were sequenced for HLA class I genes using the Ion Torrent Personal Genome Machine (PGM) following the NXType protocol. HLA calling was performed using the HLATypeStream v1.0.0.86 with IMGT/HLA databses of 3.21.0. Concordance rates were evaluated by comparing HLATypeStream results with Luminex-based HLA typing result for up to 2-field result. Results With the default setting of HLATypeStream and without manual interpretation of the results, the allelic concordance is more than 98% for HLA-A, HLA-B and HLA-C. Other commercially available HLA calling software and open-access software were also evaluated. Conclusions Careful inspection of the final results and occasionally manual interpretations of results are necessary especially for rare or novel HLA alleles.

Background There is considerable interest in the use of next-generation sequencing to help diagnose unidentified genetic conditions, but it is difficult to predict the success rate in a clinical setting that includes patients with a broad range of phenotypic presentations. Methods The authors present a pilot programme of whole-exome sequencing on 12 patients with unexplained and apparent genetic conditions, along with their unaffected parents. Unlike many previous studies, the authors did not seek patients with similar phenotypes, but rather enrolled any undiagnosed proband with an apparent genetic condition when predetermined criteria were met. Results This undertaking resulted in a likely genetic diagnosis in 6 of the 12 probands, including the identification of apparently causal mutations in four genes known to cause Mendelian disease ( TCF4, EFTUD2, SCN2A and SMAD4 ) and one gene related to known Mendelian disease genes ( NGLY1 ). Of particular interest is that at the time of this study, EFTUD2 was not yet known as a Mendelian disease gene but was nominated as a likely cause based on the observation of de novo mutations in two unrelated probands. In a seventh case with multiple disparate clinical features, the authors were able to identify homozygous mutations in EFEMP1 as a likely cause for macular degeneration (though likely not for other features). Conclusions This study provides evidence that next-generation sequencing can have high success rates in a clinical setting, but also highlights key challenges. It further suggests that the presentation of known Mendelian conditions may be considerably broader than currently recognised.

IL-18 is a unique cytokine that enhances innate immunity and both Th1- and Th2-driven immune responses. Recent murine and human genetic studies have shown its role in the pathogenesis of asthma.We conducted an association study in a Japanese population to discover variants of IL-18 that might have an effect on asthma susceptibility and/or progression and conducted functional analyses of the related variants.The IL-18 gene locus was resequenced in 48 human chromosomes. Asthma severity was determined according to the 2002 Global Initiative for Asthma Guidelines. Association and haplotype analyses were performed using 1,172 subjects.Although no polymorphisms differed significantly in frequency between the control and adult asthma groups, rs5744247 CG was significantly associated with the severity of adult asthma (steps 1, 2 vs. steps 3, 4; P = 0.0034). We also found a positive association with a haplotype (P = 0.0026). By in vitro functional analyses, the rs5744247 variant was found to increase enhancer-reporter activity of the IL-18 gene in bronchial epithelial cells. Expression levels of IL-18 in response to LPS stimulation in monocytes were significantly greater in subjects homozygous for the susceptibility G allele at rs5744247 CG. Furthermore, we found a significant correlation between the serum IL-18 level and the genotype of rs5744247 (P = 0.031).Although the association results need to be replicated by other studies, IL-18 variants are significantly associated with asthma severity, and the rs5744247 variant reflects higher transcriptional activity and higher expression of IL-18 in LPS-stimulated monocytes and a higher serum IL-18 level.

Background Stevens-Johnson syndrome (SJS) and its severe form, toxic epidermal necrolysis (TEN), are acute inflammatory vesiculobullous reactions of the skin and mucous membranes, including the ocular surface, oral cavity, and genitals. These reactions are very rare but are often associated with inciting drugs, infectious agents, or both. Objective We sought to identify susceptibility loci for cold medicine–related SJS/TEN (CM-SJS/TEN) with severe mucosal involvement (SMI). Methods A genome-wide association study was performed in 808 Japanese subjects (117 patients with CM-SJS/TEN with SMI and 691 healthy control subjects), and subsequent replication studies were performed in 204 other Japanese subjects (16 cases and 188 control subjects), 117 Korean subjects (27 cases and 90 control subjects), 76 Indian subjects (20 cases and 56 control subjects), and 174 Brazilian subjects (39 cases and 135 control subjects). Results In addition to the most significant susceptibility region, HLA-A , we identified IKZF1 , which encodes Ikaros, as a novel susceptibility gene (meta-analysis, rs4917014 [G vs T]; odds ratio, 0.5; P = 8.5 × 10 −11 ). Furthermore, quantitative ratios of the IKZF1 alternative splicing isoforms Ik1 and Ik2 were significantly associated with rs4917014 genotypes. Conclusion We identified IKZF1 as a susceptibility gene for CM-SJS/TEN with SMI not only in Japanese subjects but also in Korean and Indian subjects and showed that the Ik2/Ik1 ratio might be influenced by IKZF1 single nucleotide polymorphisms, which were significantly associated with susceptibility to CM-SJS/TEN with SMI.

Background NLR family, pyrin domain containing 3 (NLRP3), controls the activity of inflammatory caspase-1 by forming inflammasomes, which leads to cleavage of the procytokines IL-1β and IL-18. Recent studies have shown associations of human NLRP3 polymorphisms with susceptibility to various inflammatory diseases; however, the association with allergic diseases remains unclear. Objective We sought to examine whether NLRP3 polymorphisms are associated with susceptibility to food allergy, food-induced anaphylaxis, and aspirin-induced asthma (AIA). Methods We selected 15 tag single nucleotide polymorphisms (SNPs) of NLRP3 and conducted association analyses of NLRP3 using 574 and 1279 samples for food allergy and AIA, respectively. We further performed functional analyses of the susceptible SNPs. Results Two NLRP3 SNPs (rs4612666 and rs10754558) were significantly associated with susceptibility to food-induced anaphylaxis ( P = .00086 and P = .00068, respectively). The NLRP3 haplotype of the 2 SNPs also showed a significant association ( P = .000098). We could confirm the association with susceptibility to another hypersensitivity phenotype, AIA (rs4612666, P = .0096). Functional analysis revealed that the risk alleles of rs4612666 and rs10754558 increased the enhancer activity of NLRP3 expression and NLRP3 mRNA stability, respectively. Conclusion Our results indicate that the NLRP3 SNPs might play an important role in the development of food-induced anaphylaxis and AIA in a gain-of-function manner. Further research on the NLRP3 inflammasome will contribute to the development of novel diagnostic and therapeutic methods for food-induced anaphylaxis and AIA.

Background & Aims Genetic variation of inosine triphosphatase ( ITPA ) causing an accumulation of inosine triphosphate (ITP) has been shown to protect patients against ribavirin (RBV)-induced anemia during treatment for chronic hepatitis C infection by genome-wide association study (GWAS). However, the biologic mechanism by which this occurs is unknown. Methods We examined whether ITP can be used by adenosine triphosphatase (ATPase) in human erythrocytes or recombinant human adenylosuccinate synthase (ADSS). RBV-induced adenosine triphosphate (ATP) reduction in erythrocytes was compared with the genetically determined low or normal activity of ITPA , leading respectively to high or normal ITP levels. Results Although ITP is not used directly by human erythrocyte ATPase, it can be used for ATP biosynthesis via ADSS in place of guanosine triphosphate (GTP). With RBV challenge, erythrocyte ATP reduction was more severe in the wild-type ITPA genotype than in the hemolysis protective ITPA genotype. This difference also remains after inhibiting adenosine uptake using nitrobenzylmercaptopurine riboside (NBMPR). Interestingly, the alleviation of ATP reduction by the hemolysis protective ITPA genotype was canceled by the ADSS inhibitor 6-mercaptoethanol (6-MP). Conclusions ITP confers protection against RBV-induced ATP reduction by substituting for erythrocyte GTP, which is depleted by RBV, in the biosynthesis of ATP. Because patients with excess ITP appear largely protected against anemia, these results confirm that RBV-induced anemia is due primarily to the effect of the drug on GTP and consequently ATP levels in erythrocytes.