[Pharmacogenetic testing for prevention of severe cutaneous adverse drug reactions].

Pharmacogenetic testing benefits patients by predicting drug efficacy and risk of adverse drug reactions (ADRs). Pharmacogenetic biomarkers useful in clinical practice include drug-metabolizing enzyme and drug transporter genes and human leukocyte antigen (HLA) genes. HLA genes, which are important molecules involved in human immunity, have long been analyzed for associations with ADRs, such as skin rash, drug-induced liver injury, and agranulocytosis. HLA is composed of many genes, each of which has dozens of different types (alleles), and many HLA alleles associated with ADRs have been reported. The odds ratios in the association of HLA alleles range from approximately 5 to several thousand, indicating a very large impact on the risk of ADRs. Thus, HLA genetic testing prior to initiation of drug therapy is expected to make a significant contribution to avoiding ADRs, but to demonstrate the clinical utility, it is necessary to prospectively show the effects of medical interventions based on the test results. We conducted the GENCAT study, a prospective, multicenter, single-arm clinical trial to investigate the impact of a therapeutic intervention based on the HLA-A*31:01 test on the incidence of carbamazepine-induced skin rash. HLA-A*31:01-positive patients were treated with an alternative drug such as valproic acid, and the study showed an approximately 60% reduction in the incidence of carbamazepine-induced skin rash. It is expected that the genetic test, which has demonstrated clinical utility, will lead to the establishment of safer and more appropriate stratified medicine by reflecting the information in clinical practice guidelines.

The HLA-DRB1*09:01-DQB1*03:03 haplotype is associated with the risk for late-onset Alzheimer's disease in APOE [Formula: see text]4-negative Japanese adults.

Late-onset Alzheimer's disease (LOAD) is the most common cause of dementia among those older than 65 years. The onset of LOAD is influenced by neuroinflammation. The human leukocyte antigen (HLA) system is involved in regulating inflammatory responses. Numerous HLA alleles and their haplotypes have shown varying associations with LOAD in diverse populations, yet their impact on the Japanese population remains to be elucidated. Here, we conducted a comprehensive investigation into the associations between LOAD and HLA alleles within the Japanese population. Using whole-genome sequencing (WGS) data from 303 LOAD patients and 1717 cognitively normal (CN) controls, we identified four-digit HLA class I alleles (A, B, and C) and class II alleles (DRB1, DQB1, and DPB1). We found a significant association between the HLA-DRB1*09:01-DQB1*03:03 haplotype and LOAD risk in APOE [Formula: see text]4-negative samples (odds ratio = 1.81, 95% confidence interval = 1.38-2.38, P = 2.03[Formula: see text]). These alleles not only showed distinctive frequencies specific to East Asians but demonstrated a high degree of linkage disequilibrium in APOE [Formula: see text]4-negative samples (r2 = 0.88). Because HLA class II molecules interact with T-cell receptors (TCRs), we explored potential disparities in the diversities of TCR α chain (TRA) and ß chain (TRB) repertoires between APOE [Formula: see text]4-negative LOAD and CN samples. Lower diversity of TRA repertoires was associated with LOAD in APOE [Formula: see text]4-negative samples, irrespective of the HLA DRB1*09:01-DQB1*03:03 haplotype. Our study enhances the understanding of the etiology of LOAD in the Japanese population and provides new insights into the underlying mechanisms of its pathogenesis.

Association of Genetic Variants of HLA-DQA1 with Bullous Pemphigoid Induced by Dipeptidyl Peptidase-4 Inhibitors.

Bullous pemphigoid (BP) is the most common autoimmune blistering disorder. Several factors, including an antidiabetic (dipeptidyl peptidase-4 inhibitor [DPP-4i]), have been reported to trigger BP. To identify the genetic variants associated with BP, GWAS and HLA fine-mapping analyses were conducted. The 21 cases of noninflammatory BP induced by DPP-4i (i.e., DPP-4i-induced noninflammatory BP) and 737 controls (first cohort) and the 8 cases and 164 controls (second cohort) were included in the GWAS. Combining GWAS satisfied the genome-wide significant association of HLA-DQA1 (chromosome 6, rs3129763 [T/C]) with the risk of DPP-4i-induced noninflammatory BP (allele T carrier of 72.4% [21 of 29] in cases vs. 15.3% [138 of 901] in controls; dominant model, OR = 14, P = 1.8 × 10-9). HLA fine mapping revealed that HLA-DQA1∗05 with serine at position 75 of HLA-DQα1 (Ser75) had the most significant association with the combined cohort of DPP-4i-induced noninflammatory BP (79.3% [23 of 29] cases vs. 16.1% [145 of 901] controls; dominant model, OR = 21, P = 2.0 × 10-10). HLA-DQα1 Ser75 polymorphism was located inside the functional pocket of HLA-DQ molecules, suggesting the impact of HLA-DQα1 Ser75 on DPP-4i-induced noninflammatory BP.

Genotyping, characterization, and imputation of known and novel CYP2A6 structural variants using SNP array data.

CYP2A6 metabolically inactivates nicotine. Faster CYP2A6 activity is associated with heavier smoking and higher lung cancer risk. The CYP2A6 gene is polymorphic, including functional structural variants (SV) such as gene deletions (CYP2A6*4), duplications (CYP2A6*1 × 2), and hybrids with the CYP2A7 pseudogene (CYP2A6*12, CYP2A6*34). SVs are challenging to genotype due to their complex genetic architecture. Our aims were to develop a reliable protocol for SV genotyping, functionally phenotype known and novel SVs, and investigate the feasibility of CYP2A6 SV imputation from SNP array data in two ancestry populations. European- (EUR; n = 935) and African- (AFR; n = 964) ancestry individuals from smoking cessation trials were genotyped for SNPs using an Illumina array and for CYP2A6 SVs using Taqman copy number (CN) assays. SV-specific PCR amplification and Sanger sequencing was used to characterize a novel SV. Individuals with SVs were phenotyped using the nicotine metabolite ratio, a biomarker of CYP2A6 activity. SV diplotype and SNP array data were integrated and phased to generate ancestry-specific SV reference panels. Leave-one-out cross-validation was used to investigate the feasibility of CYP2A6 SV imputation. A minimal protocol requiring three Taqman CN assays for CYP2A6 SV genotyping was developed and known SV associations with activity were replicated. The first domain swap CYP2A6-CYP2A7 hybrid SV, CYP2A6*53, was identified, sequenced, and associated with lower CYP2A6 activity. In both EURs and AFRs, most SV alleles were identified using imputation (>70% and >60%, respectively); importantly, false positive rates were <1%. These results confirm that CYP2A6 SV imputation can identify most SV alleles, including a novel SV.

Genome-wide host-pathogen analyses reveal genetic interaction points in tuberculosis disease.

The genetics underlying tuberculosis (TB) pathophysiology are poorly understood. Human genome-wide association studies have failed so far to reveal reproducible susceptibility loci, attributed in part to the influence of the underlying Mycobacterium tuberculosis (Mtb) bacterial genotype on the outcome of the infection. Several studies have found associations of human genetic polymorphisms with Mtb phylo-lineages, but studies analysing genome-genome interactions are needed. By implementing a phylogenetic tree-based Mtb-to-human analysis for 714 TB patients from Thailand, we identify eight putative genetic interaction points (P < 5 × 10-8) including human loci DAP and RIMS3, both linked to the IFNγ cytokine and host immune system, as well as FSTL5, previously associated with susceptibility to TB. Many of the corresponding Mtb markers are lineage specific. The genome-to-genome analysis reveals a complex interactome picture, supports host-pathogen adaptation and co-evolution in TB, and has potential applications to large-scale studies across many TB endemic populations matched for host-pathogen genomic diversity.

Augmenting Granzyme B-Expressing NK Cells by Invariant NKT Ligand-Loaded APCs in Patients with Postoperative Early Stage Non-Small Cell Lung Cancer: Results of a Randomized Phase II Study.

NK cells are major effector cells involved in the elimination of early tumors and prevent metastasis. They often have an impaired function in patients with cancer. Preclinical studies have demonstrated NK cell activation as the adjunctive effect of invariant NKT (iNKT) cells. Activation of iNKT cells after administration of the glycolipid ligand α-galactosylceramide, loaded with CD1d-expressing human PBMC-derived APCs (APC/Gal), is an attractive cancer therapy to optimize the use of NK cells. However, the subsets of NK cells that are activated following iNKT cell activation as well as the period of NK cell activation remain unclear. In this study, we report that the granzyme B-expressing NK cell response in postoperative lung cancer patients was enhanced 49 d after administration of APC/Gal in a phase II study. We found maximum IFN-γ production on day 49 in 13 out of 27 APC/Gal-treated patients. On day 49, 14 out of 27 patients (51.9%) had higher IFN-γ production by iNKT cells (>6-fold higher than the baseline level). This increment significantly correlated with granzyme B-expressing NK cells. Although IFN-γ production was lower in patients in the nontreated group, we detected maximum IFN-γ production 12 mo after the resection of lung cancer (9 out of 29 patients [31%]). These findings suggest that elimination of cancer cells leads to increased NK cell function, which can be further enhanced by APC/Gal therapy.

Avoidance of cutaneous adverse drug reactions induced by antiepileptic drugs based on pharmacogenomics.

Pharmacogenomics (PGx) is a research area aimed at identifying genetic factors that are associated with drug responses, including drug efficacy, adverse drug reactions, and the appropriate drug dosage on a case-to-case basis. To promote the clinical implementation of PGx testing, which is currently of limited use in clinical practice, recent research has focused on providing reliable evidence for its clinical utility. In neurology, psychiatry, and neurosurgery, several human leukocyte antigen (HLA) alleles have been reportedly associated with cutaneous adverse drug reactions (cADRs) induced by antiepileptic drugs, which significantly carry the risk of developing cADRs. Prior to using antiepileptic drugs such as carbamazepine and lamotrigine, which are prone to cause severe cADRs, preemptive HLA genetic testing and therapeutic interventions such as drug selection and dosage adjustment based on the results of the tests can reduce the incidence of cADRs in the population before the initiation of treatment.

Association between genetic variants and the risk of nivolumab-induced immune-related adverse events.

Aim: We sought to identify the variants that could predict the risk of nivolumab-induced immune-related adverse events (irAEs) in patients with cancer. Patients & methods: We enrolled 622 Japanese patients and carried out a genome-wide association study. The associations for 507 single nucleotide polymorphisms (SNPs) showing p < 0.001 were further investigated using an independent cohort. Results: In the combined analysis, possible associations were found for a total of 90 SNPs. Although no SNPs were identified to be significantly associated with nivolumab-induced irAEs, the SNP most strongly associated with nivolumab-induced irAEs was rs469490. Conclusion: This study is an important hypothesis-generating study to guide future studies in larger and/or other ethnic cohorts.

Replication Study for the Association of Five SNPs Identified by GWAS and Trastuzumab-Induced Cardiotoxicity in Japanese and Singaporean Cohorts.

Trastuzumab (herceptin) is an effective drug for human epidermal growth factor receptor type 2 (HER2)-positive cancer. However, cardiotoxicity remains a serious complication. In our previous genome-wide association study (GWAS), we identified potential associations for five single nucleotide polymorphisms (SNPs) with trastuzumab-induced cardiotoxicity in a Japanese population. To validate this association, here we performed replication studies using Japanese and Singaporean case-control cohorts (Japan: 6 cases and 206 controls; Singapore: 22 cases and 178 controls). Although none of the SNPs showed a statistically significant association with trastuzumab-induced cardiotoxicity, we show that three (rs8032978, rs7406710 and rs9316695) and four (rs8032978, rs7406710, rs28415722 and rs11932853) SNPs had an effect in the same direction in the Japanese and the Singaporean cohort, respectively, as that in our previous study. Combining the previous study with the current replication studies, we find a strong association for two SNPs, rs8032978 and rs7406710, with trastuzumab-induced cardiotoxicity (Pcombined = 4.92 × 10-5 and 5.50 × 10-5, respectively). These data suggest that rs8032978 and rs7406710 could be predictive markers of trastuzumab-induced cardiotoxicity in Japanese and Singaporean populations, and support their potential use in clinical risk assessment. These findings offer a first step toward the development of clinically available markers for the potential risk of trastuzumab-induced cardiotoxicity as well as an improved understanding of the pathogenesis of this complication.

Genome-Wide Meta-Analysis Identifies Variants in DSCAM and PDLIM3 That Correlate with Efficacy Outcomes in Metastatic Renal Cell Carcinoma Patients Treated with Sunitinib.

Individual response to sunitinib in metastatic renal cell carcinoma (mRCC) patients is highly variable. Earlier, sunitinib outcome was related to single nucleotide polymorphisms (SNPs) in CYP3A5 and ABCB1. Our aim is to provide novel insights into biological mechanisms underlying sunitinib action. We included mRCC patients from the European EuroTARGET consortium (n = 550) and the RIKEN cohort in Japan (n = 204) which were analysed separately and in a meta-analysis of genome-wide association studies (GWAS). SNPs were tested for association with progression-free survival (PFS) and overall survival (OS) using Cox regression. Summary statistics were combined using a fixed effect meta-analysis. SNP rs28520013 in PDLIM3 and the correlated SNPs rs2205096 and rs111356738 both in DSCAM, showed genome-wide significance (p < 5 × 10−8) with PFS and OS in the meta-analysis. The variant T-allele of rs28520013 associated with an inferior PFS of 5.1 months compared to 12.5 months in non-carriers (p = 4.02 × 10−10, HR = 7.26). T-allele carriers of rs28520013 showed an inferior OS of 6.9 months versus 30.2 months in non-carriers (p = 1.62 × 10−8, HR = 5.96). In this GWAS we identified novel genetic variants in PDLIM3 and DSCAM that impact PFS and OS in mRCC patients receiving sunitinib. The underlying link between the identified genes and the molecular mechanisms of sunitinib action needs to be elucidated.

The Association of HLA-B*35 and GSTT1 Genotypes and Hepatotoxicity in Thai People Living with HIV.

Glutathione s-transferase (GST) is a family of drug-metabolizing enzymes responsible for metabolizing and detoxifying drugs and xenobiotic substances. Therefore, deletion polymorphisms of GSTs can be implicated in developing several pathological conditions, including antiretroviral drug-induced liver injury (ARVDILI). Notably, GST polymorphisms have been shown to be associated with ARVDILI risk. However, data on GST polymorphisms in the Thai population are limited. Therefore, this study investigated possible associations between GST genetic polymorphisms and ARVDILI development. A total of 362 people living with HIV (PLHIV) and 85 healthy controls from multiple centers were enrolled. GSTM1 and GSTT1 genetic polymorphisms were determined using polymerase chain reactions. In addition, HLA genotypes were determined using a sequence-based HLA typing method. After comparing GST genotypic frequencies, there was no significant difference between PLHIV and healthy volunteers. However, while observing the PLHIV group, GSTT1 wild type was significantly associated with a 2.04-fold increased risk of ARVDILI (95%CI: 1.01, 4.14; p = 0.045). Interestingly, a combination of GSTT1 wild type and HLA-B*35:05 was associated with a 2.28-fold higher risk of ARVDILI (95%CI: 1.15, 4.50; p = 0.02). Collectively, GSTT1 wild type and a combination of GSTT1 wild type plus HLA-B*35:05 were associated with susceptibility to ARVDILI in the Thai population.

Genome-wide association study of platelet factor 4/heparin antibodies in heparin-induced thrombocytopenia.

Heparin, a widely used anticoagulant, carries the risk of an antibody-mediated adverse drug reaction, heparin-induced thrombocytopenia (HIT). A subset of heparin-treated patients produces detectable levels of antibodies against complexes of heparin bound to circulating platelet factor 4 (PF4). Using a genome-wide association study (GWAS) approach, we aimed to identify genetic variants associated with anti-PF4/heparin antibodies that account for the variable antibody response seen in HIT. We performed a GWAS on anti-PF4/heparin antibody levels determined via polyclonal enzyme-linked immunosorbent assays. Our discovery cohort (n = 4237) and replication cohort (n = 807) constituted patients with European ancestry and clinical suspicion of HIT, with cases confirmed via functional assay. Genome-wide significance was considered at α = 5 × 10-8. No variants were significantly associated with anti-PF4/heparin antibody levels in the discovery cohort at a genome-wide significant level. Secondary GWAS analyses included the identification of variants with suggestive associations in the discovery cohort (α = 1 × 10-4). The top variant in both cohorts was rs1555175145 (discovery ß = -0.112 [0.018], P = 2.50 × 10-5; replication ß = -0.104 [0.051], P = .041). In gene set enrichment analysis, 3 gene sets reached false discovery rate-adjusted significance (q < 0.05) in both discovery and replication cohorts: "Leukocyte Transendothelial Migration," "Innate Immune Response," and "Lyase Activity." Our results indicate that genomic variation is not significantly associated with anti-PF4/heparin antibody levels. Given our power to identify variants with moderate frequencies and effect sizes, this evidence suggests genetic variation is not a primary driver of variable antibody response in heparin-treated patients with European ancestry.

ABO O blood group as a risk factor for platelet reactivity in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is an unpredictable, potentially catastrophic adverse effect resulting from an immune response to platelet factor 4 (PF4)/heparin complexes. We performed a genome-wide association study (GWAS) with positive functional assay as the outcome in a large discovery cohort of patients divided into 3 groups: (1) functional assay-positive cases (n = 1269), (2) antibody-positive (functional assay-negative) controls (n = 1131), and (3) antibody-negative controls (n = 1766). Significant associations (α = 5 × 10-8) were investigated in a replication cohort (α = 0.05) of functional assay-confirmed HIT cases (n = 177), antibody-positive (function assay-negative) controls (n = 258), and antibody-negative controls (n = 351). We observed a strong association for positive functional assay with increasing PF4/heparin immunoglobulin-G (IgG) level (odds ratio [OR], 16.53; 95% confidence interval [CI], 13.83-19.74; P = 1.51 × 10-209) and female sex (OR, 1.15; 95% CI, 1.01-1.32; P = .034). The rs8176719 C insertion variant in ABO was significantly associated with positive functional assay status in the discovery cohort (frequency = 0.41; OR, 0.751; 95% CI, 0.682-0.828; P = 7.80 × 10-9) and in the replication cohort (OR, 0.467; 95% CI, 0.228-0.954; P = .0367). The rs8176719 C insertion, which encodes all non-O blood group alleles, had a protective effect, indicating that the rs8176719 C deletion and the O blood group were risk factors for HIT (O blood group OR, 1.42; 95% CI, 1.26-1.61; P = 3.09 × 10-8). Meta-analyses indicated that the ABO association was independent of PF4/heparin IgG levels and was stronger when functional assay-positive cases were compared with antibody-positive (functional assay-negative) controls than with antibody-negative controls. Sequencing and fine-mapping of ABO demonstrated that rs8176719 was the causal single nucleotide polymorphism (SNP). Our results clarify the biology underlying HIT pathogenesis with ramifications for prediction and may have important implications for related conditions, such as vaccine-induced thrombotic thrombocytopenia.

Accuracy and applications of sequencing and genotyping approaches for CYP2A6 and homologous genes.

OBJECTIVES: We evaluated multiple genotyping/sequencing approaches in a homologous region of chromosome 19, and investigated associations of two common 3'-UTR CYP2A6 variants with activity in vivo. METHODS: Individuals (n = 1704) of European and African ancestry were phenotyped for the nicotine metabolite ratio (NMR), an index of CYP2A6 activity, and genotyped/sequenced using deep amplicon exon sequencing, SNP array, genotype imputation and targeted capture sequencing. Amplicon exon sequencing was the gold standard to which other methods were compared within-individual for CYP2A6, CYP2A7, CYP2A13, and CYP2B6 exons to identify highly discordant positions. Linear regression models evaluated the association of CYP2A6*1B and rs8192733 genotypes (coded additively) with logNMR. RESULTS: All approaches were ≤2.6% discordant with the gold standard; discordant calls were concentrated at few positions. Fifteen positions were discordant in >10% of individuals, with 12 appearing in regions of high identity between homologous genes (e.g. CYP2A6 and CYP2A7). For six, allele frequencies in our study and online databases were discrepant, suggesting errors in online sources. In the European-ancestry group (n = 935), CYP2A6*1B and rs8192733 were associated with logNMR (P < 0.001). A combined model found main effects of both variants on increasing logNMR. Similar trends were found in those of African ancestry (n = 506). CONCLUSION: Multiple genotyping/sequencing approaches used in this chromosome 19 region contain genotyping/sequencing errors, as do online databases. Gene-specific primers and SNP array probes must consider gene homology; short-read sequencing of related genes in a single reaction should be avoided. Using improved sequencing approaches, we characterized two gain-of-function 3'-UTR variants, including the relatively understudied rs8192733.

Identification and functional validation of novel pharmacogenomic variants using a next-generation sequencing-based approach for clinical pharmacogenomics.

Inter-individual variability in pharmacokinetics and drug response is heavily influenced by single-nucleotide variants (SNVs) and copy-number variations (CNVs) in genes with importance for drug disposition. Nowadays, a plethora of studies implement next generation sequencing to capture rare and novel pharmacogenomic (PGx) variants that influence drug response. To address these issues, we present a comprehensive end-to-end analysis workflow, beginning from targeted PGx panel re-sequencing to in silico analysis pipelines and in vitro validation assays. Specifically, we show that novel pharmacogenetic missense variants that are predicted or putatively predicted to be functionally deleterious, significantly alter protein activity levels of CYP2D6 and CYP2C19 proteins. We further demonstrate that variant priorization pipelines tailored with functional in vitro validation assays provide supporting evidence for the deleterious effect of novel PGx variants. The proposed workflow could provide the basis for integrating next-generation sequencing for PGx testing into routine clinical practice.

Genome-wide association study of colorectal polyps identified highly overlapping polygenic architecture with colorectal cancer.

No genome-wide association studies (GWAS) were reported for colorectal polyps and the overlap in polygenic backgrounds conferring risk of colorectal cancer and polyps remains unclear. We performed GWAS on subjects with colorectal polyps using the BioBank Japan data with 4447 cases and 157,226 controls. We evaluated genetic correlations between colorectal polyps and cancer, and effects on colorectal polyps of single nucleotide polymorphisms (SNPs) known to be associated with colorectal cancer. We identified CUX2, a known genetic locus to colorectal cancer, as a susceptibility locus to colorectal polyps (p value = 1.1 × 10-15). Subsequent fine-mapping analysis indicated that rs11065828 in CUX2 is the causal variant for colorectal polyps. We found that known colorectal cancer-susceptible SNPs were also associated with colorectal polyps. The genetic correlation between colorectal cancer and polyps is very high (r = 0.98 and p value = 0.0006). We additionally identified 14 significant loci of colorectal polyps and three significant loci of colorectal cancer by applying the multi-trait analysis of GWAS of colorectal cancer and colorectal polyps. We showed very similar germline polygenic features, which gives us the additional insight into potential cancers at polygenic levels for patients with polyps who are followed up at outpatients' clinic; thus, close observation and polypectomy is critical to prevent colorectal cancers.

Functional characterization of novel rare CYP2A6 variants and potential implications for clinical outcomes.

CYP2A6 activity, phenotyped by the nicotine metabolite ratio (NMR), is a predictor of several smoking behaviors, including cessation and smoking-related disease risk. The heritability of the NMR is 60-80%, yet weighted genetic risk scores (wGRSs) based on common variants explain only 30-35%. Rare variants (minor allele frequency <1%) are hypothesized to explain some of this missing heritability. We present two targeted sequencing studies where rare protein-coding variants are functionally characterized in vivo, in silico, and in vitro to examine this hypothesis. In a smoking cessation trial, 1687 individuals were sequenced; characterization measures included the in vivo NMR, in vitro protein expression, and metabolic activity measured from recombinant proteins. In a human liver bank, 312 human liver samples were sequenced; measures included RNA expression, protein expression, and metabolic activity from extracted liver tissue. In total, 38 of 47 rare coding variants identified were novel; characterizations ranged from gain-of-function to loss-of-function. On a population level, the portion of NMR variation explained by the rare coding variants was small (~1%). However, upon incorporation, the accuracy of the wGRS was improved for individuals with rare protein-coding variants (i.e., the residuals were reduced), and approximately one-third of these individuals (12/39) were re-assigned from normal to slow metabolizer status. Rare coding variants can alter an individual's CYP2A6 activity; their integration into wGRSs through precise functional characterization is necessary to accurately assess clinical outcomes and achieve precision medicine for all. Investigation into noncoding variants is warranted to further explain the missing heritability in the NMR.