Seik-Soon Khor

High-accuracy imputation for HLA class I and II genes based on high-resolution SNP data of population-specific references.

Statistical imputation of classical human leukocyte antigen (HLA) alleles is becoming an indispensable tool for fine-mappings of disease association signals from case–control genome-wide association studies. However, most currently available HLA imputation tools are based on European reference populations and are not suitable for direct application to non-European populations. Among the HLA imputation tools, The HIBAG R package is a flexible HLA imputation tool that is equipped with a wide range of population-based classifiers; moreover, HIBAG R enables individual researchers to build custom classifiers. Here, two data sets, each comprising data from healthy Japanese individuals of difference sample sizes, were used to build custom classifiers. HLA imputation accuracy in five HLA classes (HLA-A, HLA-B, HLA-DRB1, HLA-DQB1 and HLA-DPB1) increased from the 82.5–98.8% obtained with the original HIBAG references to 95.2–99.5% with our custom classifiers. A call threshold (CT) of 0.4 is recommended for our Japanese classifiers; in contrast, HIBAG references recommend a CT of 0.5. Finally, our classifiers could be used to identify the risk haplotypes for Japanese narcolepsy with cataplexy, HLA-DRB1*15:01 and HLA-DQB1*06:02, with 100% and 99.7% accuracy, respectively; therefore, these classifiers can be used to supplement the current lack of HLA genotyping data in widely available genome-wide association study data sets.

New susceptibility variants to narcolepsy identified in HLA class II region

Narcolepsy, a sleep disorder characterized by excessive daytime sleepiness, cataplexy and rapid eye movement sleep abnormalities, is tightly associated with human leukocyte antigen HLA-DQB1*06:02. DQB1*06:02 is common in the general population (10-30%); therefore, additional genetic factors are needed for the development of narcolepsy. In the present study, HLA-DQB1 in 664 Japanese narcoleptic subjects and 3131 Japanese control subjects was examined to determine whether HLA-DQB1 alleles located in trans of DQB1*06:02 are associated with narcolepsy. The strongest association was with DQB1*06:01 (P = 1.4 × 10(-10), odds ratio, OR = 0.39), as reported in previous studies. Additional predisposing effects of DQB1*03:02 were also found (P = 2.5 × 10(-9), OR = 1.97). A comparison between DQB1*06:02 heterozygous cases and controls revealed dominant protective effects of DQB1*06:01 and DQB1*05:01. In addition, a single-nucleotide polymorphism-based conditional analysis controlling for the effect of HLA-DQB1 was performed to determine whether there were other independent HLA associations outside of HLA-DQB1. This analysis revealed associations at HLA-DPB1 in the HLA class II region (rs3117242, P = 4.1 × 10(-5), OR = 2.45; DPB1*05:01, P = 8.1 × 10(-3), OR = 1.39). These results indicate that complex HLA class II associations contribute to the genetic predisposition to narcolepsy.

Phenome-wide association study maps new diseases to the human major histocompatibility complex region

Background Over 160 disease phenotypes have been mapped to the major histocompatibility complex (MHC) region on chromosome 6 by genome-wide association study (GWAS), suggesting that the MHC region as a whole may be involved in the aetiology of many phenotypes, including unstudied diseases. The phenome-wide association study (PheWAS), a powerful and complementary approach to GWAS, has demonstrated its ability to discover and rediscover genetic associations. The objective of this study is to comprehensively investigate the MHC region by PheWAS to identify new phenotypes mapped to this genetically important region. Methods In the current study, we systematically explored the MHC region using PheWAS to associate 2692 MHC-linked variants (minor allele frequency ≥0.01) with 6221 phenotypes in a cohort of 7481 subjects from the Marshfield Clinic Personalized Medicine Research Project. Results Findings showed that expected associations previously identified by GWAS could be identified by PheWAS (eg, psoriasis, ankylosing spondylitis, type I diabetes and coeliac disease) with some having strong cross-phenotype associations potentially driven by pleiotropic effects. Importantly, novel associations with eight diseases not previously assessed by GWAS (eg, lichen planus) were also identified and replicated in an independent population. Many of these associated diseases appear to be immune-related disorders. Further assessment of these diseases in 16 484 Marshfield Clinic twins suggests that some of these diseases, including lichen planus, may have genetic aetiologies. Conclusions These results demonstrate that the PheWAS approach is a powerful and novel method to discover SNP–disease associations, and is ideal when characterising cross-phenotype associations, and further emphasise the importance of the MHC region in human health and disease.

A polymorphism in CCR1/CCR3 is associated with narcolepsy.

Etiology of narcolepsy-cataplexy involves multiple genetic and environmental factors. While the human leukocyte antigen (HLA)-DRB1*15:01-DQB1*06:02 haplotype is strongly associated with narcolepsy, it is not sufficient for disease development. To identify additional, non-HLA susceptibility genes, we conducted a genome-wide association study (GWAS) using Japanese samples. An initial sample set comprising 409 cases and 1562 controls was used for the GWAS of 525,196 single nucleotide polymorphisms (SNPs) located outside the HLA region. An independent sample set comprising 240 cases and 869 controls was then genotyped at 37 SNPs identified in the GWAS. We found that narcolepsy was associated with a SNP in the promoter region of chemokine (C-C motif) receptor 1 (CCR1) (rs3181077, P=1.6×10(-5), odds ratio [OR]=1.86). This rs3181077 association was replicated with the independent sample set (P=0.032, OR=1.36). We measured mRNA levels of candidate genes in peripheral blood samples of 38 cases and 37 controls. CCR1 and CCR3 mRNA levels were significantly lower in patients than in healthy controls, and CCR1 mRNA levels were associated with rs3181077 genotypes. In vitro chemotaxis assays were also performed to measure monocyte migration. We observed that monocytes from carriers of the rs3181077 risk allele had lower migration indices with a CCR1 ligand. CCR1 and CCR3 are newly discovered susceptibility genes for narcolepsy. These results highlight the potential role of CCR genes in narcolepsy and support the hypothesis that patients with narcolepsy have impaired immune function.

Understanding of HLA-conferred susceptibility to chronic hepatitis B infection requires HLA genotyping-based association analysis

Associations of variants located in the HLA class II region with chronic hepatitis B (CHB) infection have been identified in Asian populations. Here, HLA imputation method was applied to determine HLA alleles using genome-wide SNP typing data of 1,975 Japanese individuals (1,033 HBV patients and 942 healthy controls). Together with data of an additional 1,481 Japanese healthy controls, association tests of six HLA loci including HLA-A, C, B, DRB1, DQB1, and DPB1, were performed. Although the strongest association was detected at a SNP located in the HLA-DP locus in a SNP-based GWAS using data from the 1,975 Japanese individuals, HLA genotyping-based analysis identified DQB1*06:01 as having the strongest association, showing a greater association with CHB susceptibility (OR = 1.76, P = 6.57 × 10−18) than any one of five HLA-DPB1 alleles that were previously reported as CHB susceptibility alleles. Moreover, HLA haplotype analysis showed that, among the five previously reported HLA-DPB1 susceptibility and protective alleles, the association of two DPB1 alleles (DPB1*09:01, and *04:01) had come from linkage disequilibrium with HLA-DR-DQ haplotypes, DRB1*15:02-DQB1*06:01 and DRB1*13:02-DQB1*06:04, respectively. The present study showed an example that SNP-based GWAS does not necessarily detect the primary susceptibility locus in the HLA region.

Genome-wide association study of HLA-DQB1*06:02 negative essential hypersomnia

Essential hypersomnia (EHS), a sleep disorder characterized by excessive daytime sleepiness, can be divided into two broad classes based on the presence or absence of the HLA-DQB1*06:02 allele. HLA-DQB1*06:02-positive EHS and narcolepsy with cataplexy are associated with the same susceptibility genes. In contrast, there are fewer studies of HLA-DQB1*06:02 negative EHS which, we hypothesized, involves a different pathophysiological pathway than does narcolepsy with cataplexy. In order to identify susceptibility genes associated with HLA-DQB1*06:02 negative EHS, we conducted a genome-wide association study (GWAS) of 125 unrelated Japanese EHS patients lacking the HLA-DQB1*06:02 allele and 562 Japanese healthy controls. A comparative study was also performed on 268 HLA-DQB1*06:02 negative Caucasian hypersomnia patients and 1761 HLA-DQB1*06:02 negative Caucasian healthy controls. We identified three SNPs that each represented a unique locus— rs16826005 (P = 1.02E-07; NCKAP5), rs11854769 (P = 6.69E-07; SPRED1), and rs10988217 (P = 3.43E-06; CRAT) that were associated with an increased risk of EHS in this Japanese population. Interestingly, rs10988217 showed a similar tendency in its association with both HLA-DQB1*06:02 negative EHS and narcolepsy with cataplexy in both Japanese and Caucasian populations. This is the first GWAS of HLA-DQB1*06:02 negative EHS, and the identification of these three new susceptibility loci should provide additional insights to the pathophysiological pathway of this condition.

Immune-related pathways including HLA-DRB1(∗)13:02 are associated with panic disorder.

Panic disorder (PD) is an anxiety disorder characterized by panic attacks and anticipatory anxiety. Both genetic and environmental factors are thought to trigger PD onset. Previously, we performed a genome-wide association study (GWAS) for PD and focused on candidate SNPs with the lowest P values. However, there seemed to be a number of polymorphisms which did not reach genome-wide significance threshold due to their low allele frequencies and odds ratios, even though they were truly involved in pathogenesis. Therefore we performed pathway analyses in order to overcome the limitations of conventional single-marker analysis and identify associated SNPs with modest effects. Each pathway analysis indicated that pathways related to immunity showed the strongest association with PD (DAVID, P=2.08×10(-6); i-GSEA4GWAS, P<10(-3); ICSNPathway, P<10(-3)). Based on the results of pathway analyses and the previously performed GWAS for PD, we focused on and investigated HLA-B and HLA-DRB1 as candidate susceptibility genes for PD. We typed HLA-B and HLA-DRB1 in 744 subjects with PD and 1418 control subjects. Patients with PD were significantly more likely to carry HLA-DRB1(∗)13:02 (P=2.50×10(-4), odds ratio=1.54). Our study provided initial evidence that HLA-DRB1(∗)13:02 and genes involved in immune-related pathways are associated with PD. Future studies are necessary to confirm these results and clarify the underlying mechanisms causing PD.

Evaluation of polygenic risks for narcolepsy and essential hypersomnia

In humans, narcolepsy is a sleep disorder that is characterized by sleepiness, cataplexy and rapid eye movement (REM) sleep abnormalities. Essential hypersomnia (EHS) is another type of sleep disorder that is characterized by excessive daytime sleepiness without cataplexy. A human leukocyte antigen (HLA) class II allele, HLA-DQB1*06:02, is a major genetic factor for narcolepsy. Almost all narcoleptic patients are carriers of this HLA allele, while 30–50% of EHS patients and 12% of all healthy individuals in Japan carry this allele. The pathogenesis of narcolepsy and EHS is thought to be partially shared. To evaluate the contribution of common single-nucleotide polymorphisms (SNPs) to narcolepsy onset and to assess the common genetic background of narcolepsy and EHS, we conducted a polygenic analysis that included 393 narcoleptic patients, 38 EHS patients with HLA-DQB1*06:02, 119 EHS patients without HLA-DQB1*06:02 and 1582 healthy individuals. We also included 376 individuals with panic disorder and 213 individuals with autism to confirm whether the results were biased. Polygenic risks in narcolepsy were estimated to explain 58.1% (PHLA-DQB1*06:02=2.30 × 10−48, Pwhole genome without HLA-DQB1*06:02=6.73 × 10−2) including HLA-DQB1*06:02 effects and 1.3% (Pwhole genome without HLA-DQB1*06:02=2.43 × 10−2) excluding HLA-DQB1*06:02 effects. The results also indicated that small-effect SNPs contributed to the development of narcolepsy. Reported susceptibility SNPs for narcolepsy in the Japanese population, CPT1B (carnitine palmitoyltransferase 1B), TRA@ (T-cell receptor alpha) and P2RY11 (purinergic receptor P2Y, G-protein coupled, 11), were found to explain 0.8% of narcolepsy onset (Pwhole genome without HLA-DQB1*06:02=9.74 × 10−2). EHS patients with HLA-DQB1*06:02 were estimated to have higher shared genetic background to narcoleptic patients than EHS patients without HLA-DQB1*06:02 even when the effects of HLA-DQB1*06:02 were excluded (EHS with HLA-DQB1*06:02: 40.4%, PHLA-DQB1*06:02=7.02 × 10−14, Pwhole genome without HLA-DQB1*06:02=1.34 × 10−1, EHS without HLA-DQB1*06:02: 0.4%, Pwhole genome without HLA-DQB1*06:02=3.06 × 10−1). Meanwhile, the polygenic risks for narcolepsy could not explain the onset of panic disorder and autism, suggesting that our results were reasonable.

An association analysis of HLA-DQB1 with narcolepsy without cataplexy and idiopathic hypersomnia with/without long sleep time in a Japanese population

Narcolepsy without cataplexy (NA w/o CA) (narcolepsy type 2) is a lifelong disorder characterized by excessive daytime sleepiness and rapid eye movement (REM) sleep abnormalities, but no cataplexy. In the present study, we examined the human leukocyte antigen HLA-DQB1 in 160 Japanese patients with NA w/o CA and 1,418 control subjects. Frequencies of DQB1*06:02 were significantly higher in patients with NA w/o CA compared with controls (allele frequency: 16.6 vs. 7.8%, P=1.1×10−7, odds ratio (OR)=2.36; carrier frequency: 31.3 vs. 14.7%, P=7.6×10−8, OR=2.64). Distributions of HLA-DQB1 alleles other than DQB1*06:02 were compared between NA w/o CA and narcolepsy with cataplexy (NA-CA) to assess whether the genetic backgrounds of the two diseases have similarities. The distribution of the HLA-DQB1 alleles in DQB1*06:02-negative NA w/o CA was significantly different from that in NA-CA (P=5.8×10−7). On the other hand, the patterns of the HLA-DQB1 alleles were similar between DQB1*06:02-positive NA w/o CA and NA-CA. HLA-DQB1 analysis was also performed in 186 Japanese patients with idiopathic hypersomnia (IHS) with/without long sleep time, but no significant associations were observed.

Genome-wide association study of self-reported food reactions in Japanese identifies shrimp and peach specific loci in the HLA-DR / DQ gene region

Food allergy is an increasingly important health problem in the world. Several genome-wide association studies (GWAS) focused on European ancestry samples have identified food allergy-specific loci in the HLA class II region. We conducted GWAS of self-reported reactivity with common foods using the data from 11011 Japanese women and identified shrimp and peach allergy-specific loci in the HLA-DR/DQ gene region tagged by rs74995702 (P = 6.30 × 10−17, OR = 1.91) and rs28359884 (P = 2.3 × 10−12, OR = 1.80), respectively. After HLA imputation using a Japanese population-specific reference, the most strongly associated haplotype was HLA-DRB1*04:05-HLA-DQB1*04:01 for shrimp allergy (P = 3.92 × 10−19, OR = 1.99) and HLA-DRB1*09:01-HLA-DQB1*03:03 for peach allergy (P = 1.15 × 10−7, OR = 1.68). Additionally, both allergies’ associated variants were eQTLs for several HLA genes, with HLA-DQA2 the single eQTL gene shared between the two traits. Our study suggests that allergy to certain foods may be related to genetic differences that tag both HLA alleles having particular epitope binding specificities as well as variants modulating expression of particular HLA genes. Investigating this further could increase our understanding of food allergy aetiology and potentially lead to better therapeutic strategies for allergen immunotherapies.