Identification of epistatic SNP combinations in rheumatoid arthritis using LAMPLINK and Japanese cohorts.

Genome-wide association studies have enabled the identification of important genetic factors in many trait studies. However, only a fraction of the heritability can be explained by known genetic factors, even in the most common diseases. Genetic loci combinations, or epistatic contributions expressed by combinations of single nucleotide polymorphisms (SNPs), have been argued to be one of the critical factors explaining some of the missing heritability, especially in oligogenic/polygenic diseases. Rheumatoid arthritis (RA) is a complex disease with more than 100 reported SNP associations, as well as various HLA haplotypes and amino acids; however, many associations between RA and inter-chromosomal SNP combinations are unknown. To discover novel associations of epistatic interactions with high odds ratios in RA, we applied the LAMPLINK method, a systematic enumerative procedure for identifying high-order SNP combinations, to a Japanese RA cohort (discovery cohort; 4024 patients with RA and 7731 controls). We validated the identified associations in a different Japanese cohort (validation cohort; 810 RA patients and 6303 controls). In this study, we identified 90 significant genetic associations in the discovery cohort. Among these, 74 (82.2%) associations were replicated in the validation cohort, and eight combinations were inter-chromosomal, all of which comprised rs7765379 or rs35265698 located in the HLA region. These two SNPs exhibited strong correlations with valine at amino acid position 11 in HLA-DRB1 (HLA-DRB1-11-Val). Finally, we discovered that rs9624 showed an association with RA through an epistatic interaction with HLA-DRB1-11-Val. Overall, LAMPLINK showed high reliability for identifying epistatic genetic contributions hidden in complex traits.

Identification of the VLDLR locus associated with giant cell arteritis and the possible causal role of low-density lipoprotein cholesterol in its pathogenesis.

OBJECTIVES: To elucidate the association between genetic variants and the risk of giant cell arteritis (GCA) via large-scale genome-wide association studies (GWAS). In addition, to assess the causal effect of a specific molecule by employing the obtained GWAS results as genetic epidemiological tools. METHODS: We applied additional variant quality control to the publicly available GWAS results from the biobank of the United Kingdom (UKBB) and Finnish (FinnGen), which comprised 532 cases vs 408 565 controls and 884 cases vs 332 115 controls, respectively. We further meta-analyzed these two sets of results. We performed two-sample Mendelian randomization (MR) to test the causal effect of low-density lipoprotein (LDL) cholesterol on the risk of GCA. RESULTS: The MHC class II region showed significant associations in UKBB, FinnGen, and the meta-analysis. The VLDLR region was associated with GCA risk in the meta-analysis. The T allele of rs7044155 increased the expression of VLDLR, decreased the LDL cholesterol level, and decreased the disease risk. The subsequent MR results indicated that a 1-standard deviation increase in LDL cholesterol was associated with an increased risk of GCA (odds ratio [OR] 1.21, 95% confidence interval [CI] 1.01-1.45; p = 0.04). CONCLUSIONS: Our study identified associations between GCA risk and the MHC class II and VLDLR regions. Moreover, LDL cholesterol was suggested to have a causal effect on the risk of developing GCA.

Genome-Wide Association Study with Three Control Cohorts of Japanese Patients with Esotropia and Exotropia of Comitant Strabismus and Idiopathic Superior Oblique Muscle Palsy.

Esotropia and exotropia in the entity of comitant strabismus are multifactorial diseases with both genetic and environmental backgrounds. Idiopathic superior oblique muscle palsy, as the predominant entity of non-comitant (paralytic) strabismus, also has a genetic background, as evidenced by varying degrees of muscle hypoplasia. A genome-wide association study (GWAS) was conducted of 711 Japanese patients with esotropia (n= 253), exotropia (n = 356), and idiopathic superior oblique muscle palsy (n = 102). The genotypes of single nucleotide polymorphisms (SNPs) were determined by Infinium Asian Screening Array. Three control cohorts from the Japanese population were used: two cohorts from BioBank Japan (BBJ) and the Nagahama Cohort. BBJ (180K) was genotyped by a different array, Illumina Infinium OmniExpressExome or HumanOmniExpress, while BBJ (ASA) and the Nagahama Cohort were genotyped by the same Asian array. After quality control of SNPs and individuals, common SNPs between the case cohort and the control cohort were chosen in the condition of genotyping by different arrays, while all SNPs genotyped by the same array were used for SNP imputation. The SNPs imputed with R-square values ≥ 0.3 were used to compare the case cohort of each entity or the combined entity with the control cohort. In comparison with BBJ (180K), the esotropia group and the exotropia group showed CDCA7 and HLA-F, respectively, as candidate genes at a significant level of p < 5 × 10-8, while the idiopathic superior oblique muscle palsy group showed DAB1 as a candidate gene which is involved in neuronal migration. DAB1 was also detected as a candidate in comparison with BBJ (ASA) and the Nagahama Cohort at a weak level of significance of p < 1 × 10-6. In comparison with BBJ (180K), RARB (retinoic acid receptor-ß) was detected as a candidate at a significant level of p < 5 × 10-8 in the combined group of esotropia, exotropia, and idiopathic superior oblique muscle palsy. In conclusion, a series of GWASs with three different control cohorts would be an effective method with which to search for candidate genes for multifactorial diseases such as strabismus.

Common and distinct risk profiles of asymptomatic extra- and intracranial atherosclerosis in the Nagahama cohort.

BACKGROUND AND PURPOSE: Atherosclerotic burden increases the risk of both extracranial internal carotid artery stenosis (ICS) and intracranial large artery disease (ICAD). However, the differences in risk profiles have not been thoroughly investigated. METHODS: Participants were recruited from the Nagahama study cohort in Japan. Individuals over 60 years old who underwent 1.5-T head and neck magnetic resonance angiography (MRA) between July 2013 and February 2017 were included. ICAD was defined as WASID ≥ 50 %, and ICS was defined as NSCET ≥ 30 %. The prevalence and association of risk factors, including proatherogenic and proinflammatory factors, and the p.R4810K variant in the RNF213 gene, were investigated. Multivariable logistic regression analyses were performed. RESULTS: A total of 3089 individuals participated in the study, with a mean age of 68.1 ± 5.3 years, and 36.0 % were males. Among them, 52 (1.7 %) had ICS, 119 (3.8 %) had ICAD, and 15 (0.49 %) had both conditions. Alopecia areata was an independent predictor for both ICS (Odds ratio [OR] 3.5; 95 % CI 1.3-8.3) and ICAD (OR 2.1; 95 % CI 1.0-3.9). Diabetes (OR 3.7; 95 % CI 2.0-7.0) and older age (OR 2.4; 95 % CI 1.2-4.5) were associated only with ICS, while the RNF213 variant was associated with only ICAD (OR 5.7; 95 % CI 1.6-16.0). ICS and ICAD were also independently associated with each other. CONCLUSIONS: In this MRA-based large scale study, alopecia areata, known as a systemic inflammatory disease, was shown to be a common risk factor for ICS and ICAD. While conventional atherosclerotic factors were associated with ICS, non-atherosclerotic factors appear to contribute to ICAD in Japan.

Comprehensive HLA Typing from a Current Allele Database Using Next-Generation Sequencing Data.

HLA allele information is essential for a variety of medical applications, such as genomic studies of multifactorial diseases, including immune system and inflammation-related disorders, and donor selection in organ transplantation and regenerative medicine. To obtain this information, an accurate HLA typing method that is applicable for any allele registered in HLA allele databases is needed. Here we describe a method-called HLA-HD-for determining alleles from a current HLA database using next-generation sequencing (NGS) results.

Protocol for genome-wide association study of human blood metabolites.

Genetic variations influence the levels of blood metabolites. We present analytical pipelines for assessing genetic influences on human blood metabolites. We describe steps for the normalization of metabolome data, genome-wide association studies, and the identification of metabolite quantitative trait loci (mQTLs). We then detail procedures for functional enrichment analysis of mQTLs. This protocol could be applicable to other quantitative traits, such as clinical measurements or proteome data. For complete details on the use and execution of this protocol, please refer to Iwasaki et al.1.

Relationship of total sagittal spinal alignment index of thoracic kyphosis and lumbar lordosis with physical function in community-dwelling older adults.

BACKGROUND: Overall spinal curvature is evaluated by calculating the difference between the angles of lumbar lordosis (LL) and thoracic kyphosis (TK) and is expressed as LL minus TK (LL-TK). It is unclear whether LL-TK is associated with physical function in community-dwelling older adults and whether it is more relevant than TK or LL alone. OBJECTIVE: This study aimed to identify whether LL-TK is associated with physical function in community-dwelling older adults, and whether it is strongly associated than TK or LL alone. METHODS: The participants comprised 1,674 community-dwelling older adults who underwent physical assessments (women, n = 1,099; mean age, 67.4 ± 5.3 years). As spinal alignment indices, TK and LL were measured using skin surface methods, and LL-TK was calculated as the difference between them. Decreased LL-TK indicated increased overall spinal curvature. Physical function was determined by measuring single-leg standing, five-times chair-stand, and usual gait speed. Stepwise multiple regression analyses were performed with each physical function as the dependent variable and spinal alignment indices as the independent variables, with adjustments. RESULTS: Multiple regression analyses showed that single-leg standing (ß = 0.092, 95% confidence interval [CI] = 0.071 to 0.214, p < .001) and five-times chair-stand (ß=-0.142, 95% CI = -0.037 to -0.019, p < .001) were significantly associated with LL-TK, but not LL. Both LL-TK (ß = 0.121, 95% CI = 0.001 to 0.004, p < .001) and LL (ß = 0.087, 95% CI = 0.001 to 0.003, p = .003) were significant determinants of usual gait speed. CONCLUSIONS: This study showed that decreased LL-TK may be associated with poor physical function. This association may be stronger than that observed for TK or LL alone.

Association of muscle quantity and quality with knee extension strength in knee osteoarthritis: the Nagahama study.

OBJECTIVE: We aimed to examine whether lower-limb muscle quantity and quality assessed by bioelectrical impedance analysis (BIA) were associated with knee extension strength and if the association differed with knee osteoarthritis (OA) severity. METHODS: We included 1,525 participants (63.6% women; mean age, 68.0±5.3 years) from the Nagahama Prospective Cohort. Knee extension strength was measured during maximum voluntary isometric contraction. Lower limb muscle mass and extracellular-to-intracellular water (ECW/ICW) ratio were used as indicators of muscle quantity and quality, respectively, and assessed via a BIA device. We executed multiple linear regression analyses to investigate the association of muscle quantity and quality with knee extension strength. Additionally, participants were classified into three groups with respect to OA severity and symptoms: control, early, and advanced OA groups; subgroup analyses were also executed. RESULTS: The muscle mass (p<0.001) and ECW/ICW ratio (p=0.009) were significantly associated with knee extension strength. In the subgroup analysis, the muscle mass was significantly associated with knee extension strength (p<0.001), but there was no association between ECW/ICW ratio and knee extension strength (p=0.731) in the control group. In the early and advanced OA groups, the muscle mass (both p<0.001) and ECW/ICW ratio (early OA: p=0.034, advanced OA: p=0.015) were significantly associated with knee extension strength. CONCLUSIONS: Lower limb muscle quality was associated with knee extension strength, and the association was stronger in patients with knee OA. These findings suggest that both muscle quantity as well as quality should be assessed to better understand muscle function in patients with knee OA.

Transcriptomic Analysis Reveals Sixteen Potential Genes Associated with the Successful Differentiation of Antibody-Secreting Cells through the Utilization of Unfolded Protein Response Mechanisms in Robust Responders to the Influenza Vaccine.

The seasonal influenza vaccine remains one of the vital recommended infection control measures for the elderly with chronic illnesses. We investigated the immunogenicity of a single dose of influenza vaccine in 123 seronegative participants and classified them into four distinct groups, determined by the promptness of vaccine response, the longevity of humoral immunity, and the likelihood of exhibiting cross-reactivity. Subsequently, we used transcriptional profiling and differential gene expression analysis to identify potential genes directly associated with the robust response to the vaccine. The group of exemplary vaccine responders differentially expressed 16 genes, namely: MZB1, MYDGF, TXNDC5, TXNDC11, HSP90B1, FKBP11, PDIA5, PRDX4, CD38, SDC1, TNFRSF17, TNFRSF13B, PAX5, POU2AF1, IRF4, and XBP1. Our findings point out a list of expressed proteins that are related to B cell proliferation, unfolded protein response, and cellular haemostasis, as well as a linkage of these expressions to the survival of long-lived plasma cells.

Sleep blood pressure measured using a home blood pressure monitor was independently associated with cardiovascular disease incidence: the Nagahama study.

BACKGROUND: Nocturnal blood pressure (BP) is associated with cardiovascular disease independently of awake BP. However, nocturnal BP measured using an ambulatory monitoring device has limited reproducibility because it is a single-day measurement. We investigated the association between sleep BP measured on multiple days using a timer-equipped home BP monitor and cardiovascular diseases in a general population. METHODS: The study population comprised 5814 community residents. Participants were required to sleep with wrapping cuffs on their upper arm and BP was measured automatically at 0 : 00, 2 : 00, and 4 : 00. Actigraph was used to determine BP measured during sleep. Participants were also measured home morning and evening BP manually using the same device. RESULTS: During the 7.3-year mean follow-up period, we observed 117 cases of cardiovascular diseases. The association between sleep BP (per 10 mmHg hazard ratio = 1.31, P < 0.001) and cardiovascular events remained significant (hazard ratio = 1.22, P = 0.036) even after adjusting for office BP and confounding factors, such as sleep-disordered breathing. Individuals with sleep-only hypertension (n = 1047; hazard ratio = 2.23, P = 0.005) had a significant cardiovascular risk. Daytime-only hypertension (n = 264; hazard ratio = 3.57, P = 0.001) and combined sleep and daytime hypertension (n = 1216; hazard ratio = 3.69, P < 0.001) was associated with cardiovascular events to the same extent. Sleep BP dipping was not identified as a significant determinant of cardiovascular events. CONCLUSION: Sleep BP measured using a home BP monitor was independently associated with the incidence of cardiovascular disease in a general population.

Large-Scale Whole-Genome Analysis of HTLV-1-Associated Myelopathy Identified Hereditary Spastic Paraplegias.

Objectives: Distinguishing human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy from hereditary spastic paraplegia in patients infected with HTLV-1 is challenging due to overlapping clinical symptoms. The aim of this study was to explore the possibility that hereditary spastic paraplegia is inherently present in patients diagnosed with HTLV-1-associated myelopathy. Methods: We performed whole-genome sequencing on 315 unrelated patients registered in the HTLV-1-Associated Myelopathy patient registry "HAM-net," from 2013 to 2022 in Japan. CSF inflammatory biomarkers, including CXCL10, were measured. Results: We identified 5 patients with pathogenic variants in the genes RTN2, SPAST, VCP, and UBAP1, which are the known causes of hereditary spastic paraplegia. These patients had no family history of hereditary spastic paraplegia. The levels of CSF inflammatory biomarkers were lower than expected in these patients, compared with disease severity. Discussion: Genetic analysis is useful for the differentiation of hereditary spastic paraplegia patients from HTLV-1-associated myelopathy patients, especially for the patients with low levels of CSF inflammatory markers. Here we report the presence of hereditary spinal cord diseases in patients diagnosed with HTLV-1-associated myelopathy and provides evidence that genetic analysis would be helpful in the diagnostic workflow.

Monocyte-derived transcriptomes explain the ineffectiveness of abatacept in rheumatoid arthritis.

BACKGROUND: The biological mechanisms underlying the differential response to abatacept in patients with rheumatoid arthritis (RA) are unknown. Here, we aimed to identify cellular, transcriptomic, and proteomic features that predict resistance to abatacept in patients with RA. METHODS: Blood samples were collected from 22 RA patients treated with abatacept at baseline and after 3 months of treatment. Response to treatment was defined by the European League Against Rheumatism (EULAR) response criteria at 3 months, and seven patients were classified as responders and the others as non-responders. We quantified gene expression levels by RNA sequencing, 67 plasma protein levels, and the expression of surface molecules (CD3, 19, and 56) by flow cytometry. In addition, three gene expression data sets, comprising a total of 27 responders and 50 non-responders, were used to replicate the results. RESULTS: Among the clinical characteristics, the number of monocytes was significantly higher in the non-responders before treatment. Cell type enrichment analysis showed that differentially expressed genes (DEGs) between responders and non-responders were enriched in monocytes. Gene set enrichment analysis, together with single-cell analysis and deconvolution analysis, identified that Toll-like receptor 5 (TLR5) and interleukin-17 receptor A (IL17RA) pathway in monocytes was upregulated in non-responders. Hepatocyte growth factor (HGF) correlated with this signature showed higher concentrations in non-responders before treatment. The DEGs in the replication set were also enriched for the genes expressed in monocytes, not for the TLR5 and IL17RA pathway but for the oxidative phosphorylation (OXPHOS) pathway. CONCLUSIONS: Monocyte-derived transcriptomic features before treatment underlie the differences in abatacept efficacy in patients with RA. The pathway activated in monocytes was the TLR5 and IL17RA-HGF signature in the current study, while it was the OXPHOS pathway in the replication set. Elevated levels of HGF before treatment may serve as a potential biomarker for predicting poor responses to abatacept. These findings provide insights into the biological mechanisms of abatacept resistance, contributing valuable evidence for stratifying patients with RA.

Associations between Nocturnal Hypoxemia and Retinal Nerve Fiber Layer Thinning: The Nagahama Study.

Rationale: There have been meta-analyses that showed reduced retinal nerve fiber layer (RNFL) thickness, which is a surrogate marker of glaucoma, in patients with obstructive sleep apnea (OSA). However, the sample sizes in these reports were small (<300), and the mechanism of RNFL thinning in patients with OSA was not revealed.Objectives: To investigate the relationship of RNFL thickness with nocturnal hypoxemia or hypoxemic burden in a large-scale study.Methods: In this epidemiological study, 8,309 community residents were enrolled. The actigraphy-modified 3% oxygen desaturation index (acti-ODI3%) and cumulative percentage of sleep time with oxygen saturation <90% (acti-CT90) modified by objective sleep duration using actigraphy were measured. The hypoxemic burden is shown as acti-CT90. Circumpapillary RNFL thickness was determined using optical coherence tomography.Results: Multivariable logistic analysis models revealed that an increase in acti-CT90 was significantly associated with mean RNFL thinning after adjusting for several factors in participants without glaucoma diagnosed or treated previously (ß = -0.037; P = 0.009). There were significant differences in mean RNFL thickness among participants stratified according to acti-CT90 (>1.5 vs. ⩽1.5; P = 0.04). Although acti-ODI3% was significantly associated with acti-CT90 (ß = 0.72; P < 0.0001), acti-ODI3% was not significantly associated with mean RNFL thickness in the multivariable logistic analysis (ß = -0.011; P = 0.48). In addition, acti-CT90 was significantly associated with mean RNFL thickness both in the elderly (⩾60 yr; ß = -0.058; P = 0.002) and nonelderly (<60 yr; ß = -0.054; P = 0.007).Conclusions: Acti-CT90, but not acti-ODI3%, was associated with mean RNFL thinning in participants irrespective of age in the elderly or nonelderly. Further prospective studies are required to investigate whether the prevention of hypoxic burden, which was shown as acti-CT90 in this study, is favorable for RNFL thinning.

Genetic architecture of alcohol consumption identified by a genotype-stratified GWAS and impact on esophageal cancer risk in Japanese people.

An East Asian-specific variant on aldehyde dehydrogenase 2 (ALDH2 rs671, G>A) is the major genetic determinant of alcohol consumption. We performed an rs671 genotype-stratified genome-wide association study meta-analysis of alcohol consumption in 175,672 Japanese individuals to explore gene-gene interactions with rs671 behind drinking behavior. The analysis identified three genome-wide significant loci (GCKR, KLB, and ADH1B) in wild-type homozygotes and six (GCKR, ADH1B, ALDH1B1, ALDH1A1, ALDH2, and GOT2) in heterozygotes, with five showing genome-wide significant interaction with rs671. Genetic correlation analyses revealed ancestry-specific genetic architecture in heterozygotes. Of the discovered loci, four (GCKR, ADH1B, ALDH1A1, and ALDH2) were suggested to interact with rs671 in the risk of esophageal cancer, a representative alcohol-related disease. Our results identify the genotype-specific genetic architecture of alcohol consumption and reveal its potential impact on alcohol-related disease risk.

IgG4-related disease in the Japanese population: a genome-wide association study.

BACKGROUND: IgG4-related disease is a newly recognised immunopathological entity that includes autoimmune pancreatitis, IgG4-related sialadenitis, and IgG4-related kidney disease. To understand the genetic landscape of IgG4-related disease, we did a genome-wide association study. METHODS: We did a genome-wide association study of Japanese individuals, initially screening 857 patients with IgG4-related disease at 50 Japanese research institutions and DNA samples from 2082 healthy control participants from the Nagahama Prospective Genome Cohort for the Comprehensive Human Bioscience. From Oct 27, 2008, to July 22, 2014, we enrolled 835 patients and used data from 1789 healthy participants. Only patients with confirmed diagnosis of IgG4-related disease according to the international diagnostic criteria were included. Genotyping was done with the Infinium HumanOmni5Exome, HumanOmni2.5Exome, or HumanOmni2.5 Illumina arrays, and genomic distributions were compared between case and control samples for 958 440 single nucleotide polymorphisms. The HLA region was extensively analysed using imputation of HLA alleles and aminoacid residues. Fine mapping of the FCGR2B region was also done. Associations between clinical manifestations of disease and the genetic variations identified in these two genes were examined. FINDINGS: We identified the HLA-DRB1 (p=1·1×10-11) and FCGR2B (p=2·0×10-8) regions as susceptibility loci for IgG4-related disease. We also identified crucial aminoacid residues in the ß domain of the peptide-binding groove of HLA-DRB1, in which the seventh aminoacid residue showed the strongest association signal with IgG4-related disease (p=1·7×10-14), as has been reported with other autoimmune diseases. rs1340976 in FCGR2B showed an association with increased FCGR2B expression (p=2·7×10-10) and was in weak linkage disequilibrium with rs1050501, a missense variant of FCGR2B previously associated with systemic lupus erythematosus. Furthermore, rs1340976 was associated with the number of swollen organs at diagnosis (p=0·011) and IgG4 concentration at diagnosis (p=0·035). INTERPRETATION: Two susceptibility loci for IgG4-related disease were identified. Both FCGR2B and HLA loci might have important roles in IgG4-related disease development. Common molecular mechanisms might underlie IgG4-related disease and other immune-related disorders FUNDING: The Japanese Ministry of Health, Labour, and Welfare, the Japanese Agency of Medical Research and Development, and Kyoto University Grant for Top Global University Japan Project.