Thiopurine therapy, commonly used in autoimmune conditions, can be complicated by life-threatening leukopenia. This leukopenia is associated with genetic variation in TPMT (encoding thiopurine ...S-methyltransferase). Despite a lower frequency of TPMT mutations in Asians, the incidence of thiopurine-induced leukopenia is higher in Asians than in individuals of European descent. Here we performed an Immunochip-based 2-stage association study in 978 Korean subjects with Crohn's disease treated with thiopurines. We identified a nonsynonymous SNP in NUDT15 (encoding p.Arg139Cys) that was strongly associated with thiopurine-induced early leukopenia (odds ratio (OR) = 35.6; P(combined) = 4.88 × 10(-94)). In Koreans, this variant demonstrated sensitivity and specificity of 89.4% and 93.2%, respectively, for thiopurine-induced early leukopenia (in comparison to 12.1% and 97.6% for TPMT variants). Although rare, this SNP was also strongly associated with thiopurine-induced leukopenia in subjects with inflammatory bowel disease of European descent (OR = 9.50; P = 4.64 × 10(-4)). Thus, NUDT15 is a pharmacogenetic determinant for thiopurine-induced leukopenia in diverse populations.
Objective Crohn's disease (CD) is an intractable inflammatory bowel disease (IBD) of unknown cause. Recent meta-analysis of the genome-wide association studies (GWAS) and Immunochip data identified ...163 susceptibility loci to IBD in Caucasians, however there are limited studies in other populations. Methods We performed a GWAS and two validation studies in the Korean population comprising a total of 2311 patients with CD and 2442 controls. Results We confirmed four previously reported loci: TNFSF15, IL23R, the major histocompatibility complex region, and the RNASET2-FGFR1OP-CCR6 region. We identified three new susceptibility loci at genome-wide significance: rs6856616 at 4p14 (OR=1.43, combined p=3.60×10−14), rs11195128 at 10q25 (OR=1.42, combined p=1.55×10−10) and rs11235667 at 11q13 (OR=1.46, combined p=7.15×10−9), implicating ATG16L2 and/or FCHSD2 as novel susceptibility genes for CD. Further analysis of the 11q13 locus revealed a non-synonymous single nucleotide polymorphism (SNP) (R220W/rs11235604) in the evolutionarily conserved region of ATG16L2 with stronger association (OR=1.61, combined p=2.44×10−12) than rs11235667, suggesting ATG16L2 as a novel susceptibility gene for CD and rs11235604 to be a potential causal variant of the association. Two of the three SNPs (rs6856616 (p=0.00024) and rs11195128 (p=5.32×10−5)) showed consistent patterns of association in the International IBD Genetics Consortium dataset. Together, the novel and replicated loci accounted for 5.31% of the total genetic variance for CD risk in Koreans. Conclusions Our study provides new biological insight to CD and supports the complementary value of genetic studies in different populations.
Abstract Neoantigens are ideal targets for cancer immunotherapy because they are expressed de novo in tumor tissue but not in healthy tissue and are therefore recognized as foreign by the immune ...system. Advances in next-generation sequencing and bioinformatics technologies have enabled the quick identification and prediction of tumor-specific neoantigens; however, only a small fraction of predicted neoantigens are immunogenic. To improve the predictability of immunogenic neoantigens, we developed the in silico neoantigen prediction workflows VACINUS pMHC and VACINUS TCR: VACINUS pMHC incorporates physical binding between peptides and MHCs (pMHCs), and VACINUS TCR integrates T cell reactivity to the pMHC complex through deep learning-based pairing with T cell receptors (TCRs) of putative tumor-reactive CD8 tumor-infiltrating lymphocytes (TILs). We then validated our neoantigen prediction workflows both in vitro and in vivo in patients with hepatocellular carcinoma (HCC) and in a B16F10 mouse melanoma model. The predictive abilities of VACINUS pMHC and VACINUS TCR were confirmed in a validation cohort of 8 patients with HCC. Of a total of 118 neoantigen candidates predicted by VACINUS pMHC , 48 peptides were ultimately selected using VACINUS TCR . In vitro validation revealed that among the 48 predicted neoantigen candidates, 13 peptides were immunogenic. Assessment of the antitumor efficacy of the candidate neoepitopes using a VACINUS TCR in vivo mouse model suggested that vaccination with the predicted neoepitopes induced neoantigen-specific T cell responses and enabled the trafficking of neoantigen-specific CD8 + T cell clones into the tumor tissue, leading to tumor suppression. This study showed that the prediction of immunogenic neoantigens can be improved by integrating a tumor-reactive TIL TCR-pMHC ternary complex.
BackgroundThe efficacy of therapeutic cancer vaccines mainly depends on the selection of neoantigen that can induce strong cytotoxic CD8 T-cell response required to reject tumors. Previously, we ...developed a neoantigen prediction platform called ’VACINUS,’ which predicts neoantigens based on the binding between tumor-reactive tumor-infiltrating lymphocytes (TILs) TCR and peptide-MHC(pMHC). In this study, we investigated anti-tumor immune responses mediated by neoantigens predicted by VACINUS using a mouse tumor model.MethodsSeven-week-old male mice were implanted subcutaneously on the flank with B16F10 cells (C57BL/6). After two rounds of vaccination with tier 1 neoantigens, we compared tumor growth and obtained splenocytes to examine the tier 1 neoantigen-specific T cell response. We performed single-cell analysis on tumor tissue to analyze TILs. Splenocytes were sorted for tier 1 neoantigen-specific T cells using pMHC-dextramer and subjected to single-cell sequencing for comparative analysis.ResultsTo assess the anti-tumor efficacy, tumor size and survival rate were measured. The vaccinated groups showed tumor control and longer survival than unvaccinated groups. To investigate mechanisms regarding response to our vaccine, we compared the extent of TCR repertoire overlap in CD8 T cells between TIL and splenic T cells using single-cell TCR sequencing. In the vaccinated group, around 20% of T cells in the spleen and tumor overlap. These clones were more prevalent in tumors than in spleens, and more abundant in the vaccinated group than in the unvaccinated group. An analysis of a total of 10,000 single cells for distributions of phenotypes showed 13 subtypes of T cells based on gene expresison markers. The overlapping clones were found in effector/exhausted CD8 types. Most of the enriched cell types in tumor cells were exhausted cells. Terminally exhausted T cells were dominant in the vaccinated group, whereas pre-exhausted T cells were dominant in the combination treatment group.ConclusionsNeoantigens derived from VACINUS showed better anti-tumor effects than anti-PD-1 alone. An increase in overlapping TCR clones of spleen and tumors in vaccinated group suggested that our vaccines induce infiltration of T cells into tumor. The overlapping T cell clonotypes were enriched in effector and/or exhausted types which recognize and respond to antigens. A neoantigen prediction platform ‘VACINUS’ with high hit rate shed light on this field.
Expression quantitative trait loci (eQTL) datasets have extensively been used to help interpret genome-wide association study signals. Most eQTL analyses have been conducted with populations of ...European ancestry.
To determine the most functionally relevant genes at the Crohn's disease (CD) loci identified in genome-wide association studies (GWAS) involving Asian populations and to find novel disease-associated genes, we conducted an eQTL analysis.
eQTL analysis was performed using whole-blood RNA-sequencing of 101 Korean patients with CD. FastQTL was used for a pair-wise genome analysis of ∼ 6.5 M SNPs and ∼ 22 K transcripts.
We identified 135,164
eQTL and 3,816 eGenes with a false discovery rate less than 0.05. A significant proportion of the genes identified in our study overlapped with those identified in previous studies. The significantly enriched pathways of these 3,816 eGenes included neutrophil degranulation and small molecule biosynthetic process. Integrated analysis of CD GWAS with Korean eQTL revealed two putative target genes,
and
, at two previously reported loci, whereas
only with the whole blood data from the Genotype-Tissue Expression (GTEx) project, highlighting the utility of building a population-specific data set, even of modest size. The risk alleles of these genes were found to be associated with lower expression levels of
and
, respectively. Our eQTL browser can be accessed at "http://asan.crohneqtl.com/".
This resource would be useful for studies that need to employ genome-wide association analyses involving Asian populations.
The majority of colon cancers develop from pre-existing adenomas. We analyzed the expression profiles in the sequence of normal colon crypts, adenomas and early-stage carcinomas using microdissected ...cells from tubular adenomas with foci of malignant transformation. Differentially expressed genes were detected between normal-adenoma and adenoma-carcinoma, and were grouped according to the patterns of expression changes in the sequence. Down-regulated genes in the sequence included PLA2G2A, TSPAN1, PDCD4, FCGBP, AATK, EPLIN, FABP1, AGR2, MTUS1, TSC1, galectin 4 and MT1F. PLA2G2A has been shown to suppress colon tumorigenesis in mice, but the pathobiological role in humans has been controversial. Our data showed continuous down-regulation of PLA2G2A in the sequence supporting an implication in human colon cancer. Tumor suppressor and/ or proapoptotic activities have also been reported in other genes. Up-regulated genes included ribosomal proteins, IER3 and TPR. TGF-beta2 and matrix metalloproteinase 23B were up-regulated in carcinoma but not in adenoma, supporting the pathobiological roles in malignant transformation. Differentially expressed genes partly coincided with those in the adenoma-carcinoma sequence of the stomach, which was published previously, suggesting a partial overlap between the adenoma-carcinoma sequences of the colon and stomach.
Abstract
Crohn's disease (CD) and ulcerative colitis (UC) are the major types of chronic inflammatory bowel disease (IBD) characterized by recurring episodes of inflammation of the gastrointestinal ...tract. Although it is well established that human leukocyte antigen (HLA) is a major risk factor for IBD, it is yet to be determined which HLA alleles or amino acids drive the risks of CD and UC in Asians. To define the roles of HLA for IBD in Asians, we fine-mapped HLA in 12 568 individuals from Korea and Japan (3294 patients with CD, 1522 patients with UC and 7752 controls). We identified that the amino acid position 37 of HLA-DRβ1 plays a key role in the susceptibility to CD (presence of serine being protective, P = 3.6 × 10−67, OR = 0.48 0.45-0.52). For UC, we confirmed the known association of the haplotype spanning HLA-C*12:02, HLA-B*52:01 and HLA-DRB1*1502 (P = 1.2 × 10−28, OR = 4.01 3.14-5.12).
Chronic enteropathy associated with
gene (CEAS), an inherited disease characterized by nonspecific intestinal ulcers, has emerged in the Japanese population via loss-of-function mutations in the
...gene. We aimed to investigate the clinical and genetic characteristics of Korean patients diagnosed with CEAS.
From July 2018 to July 2021, we performed Sanger sequencing of the
gene in 46 patients with chronic intestinal ulcers. CEAS was confirmed based on known
mutations. We summarized the clinical characteristics of patients with confirmed CEAS.
Fourteen out of 46 patients (30.4%) had genetically confirmed CEAS, and two
variants were detected (splicing site variant c.940+1G>A and nonsense mutation p.R603X in
). Twelve patients (85.7%) were females and the median age at diagnosis of CEAS was 44.5 years. All patients presented with abdominal pain, and 13 patients (92.9%) presented with anemia (median hemoglobin, 9.6 g/dL). Ten patients (71.4%) had hypoalbuminemia (median, 2.7 g/dL). The most commonly involved site was the ileum (13/14, 92.9%). Manifestations of primary hypertrophic osteoarthropathy (PHO), such as digital clubbing, pachydermia, and periostosis were observed in five patients (28.6%) and two male patients and one female patient satisfied all major PHO diagnostic criteria.
The clinical and genetic characteristics of Korean patients with confirmed CEAS were similar to those reported in the literature. CEAS should be considered in the differential diagnosis for patients with unexplained chronic nonspecific ulcers of the small intestine.
Crohn's disease (CD) is an intractable inflammatory bowel disease of unknown cause. Recent genome-wide association studies of CD in Korean and Japanese populations suggested marginal sharing of ...susceptibility loci between Caucasian and Asian populations. As the 7 identified loci altogether explain 5.31% of the risk for CD, the objective of this study was to identify additional CD susceptibility loci in the Korean population.
Using the ImmunoChip custom single-nucleotide polymorphism array designed for dense genotyping of 186 loci identified through GWAS, we analyzed 722 individuals with CD and 461 controls for 96,048 SNP markers in the discovery stage, followed by validation in an additional 948 affected individuals and 977 controls.
We confirmed 6 previously reported loci in Caucasian: GPR35 at 2q37 (rs3749172; P = 5.30 × 10, odds ratio OR = 1.45), ZNF365 at 10q21 (rs224143; P = 2.20 × 10, OR = 1.38), ZMIZ1 at 10q22 (rs1250569; P = 3.05 × 10, OR = 1.30), NKX2-3 at 10q24 (rs4409764; P = 7.93 × 10, OR = 1.32), PTPN2 at 18p11 (rs514000; P = 9.00 × 10, OR = 1.33), and USP25 at 21q11 (rs2823256; P = 2.49 × 10, OR = 1.35), bringing the number of known CD loci (including 3 in the HLA) in Koreans to 15. The 6 additional loci increased the total genetic variance for CD risk from 5.31% to 7.27% in Koreans.
Although the different genetic backgrounds of CD between Asian and Western countries has been well established for the major susceptibility genes, our findings of overlapping associations offer new insights into the genetic architecture of CD.
Caspase recruitment domain (CARD)-containing protein 8 (CARD8) is a potential candidate risk gene for inflammatory bowel disease (IBD) because of its role as a component of the NALP3 inflammasome and ...as an inhibitor of nuclear factor-kappa B. Previous studies examining the association of a CARD8 single-nucleotide polymorphism (SNP) (rs2043211, p.Cys10X) with IBD yielded mixed results in Caucasians that may result from interaction with NALP3 or NOD2 (nucleotide-binding oligomerization domain 2) variants. To understand the genetic association between CARD8/NALP3 and IBD in Koreans, we investigated seven CARD8, four NALP3 and four NOD2 SNPs in 650 Crohn's disease (CD), 660 ulcerative colitis (UC) patients and 688 controls from the Korean population. rs2043211 of CARD8 showed significant association with UC (P = 0.011; odds ratio = 1.50, 95% confidence intervals = 1.12-2.00, P = 0.006 under recessive model). In contrast, an SNP in intron 1, rs1972619, was associated with CD only (P = 0.033). None of the NALP3 or NOD2 SNPs was significantly associated with CD or UC in the Korean populations. The stop allele of rs2043211 was associated with higher serum interleukin-1β levels only in female patients with UC (P = 0.027). Our data suggest that CARD8 variants might have roles in the pathogenesis of CD and UC in Koreans.
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