rs9459874 and rs1012656 in CCR6/FGFR1OP confer susceptibility to primary biliary cholangitis.

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.

An international genome-wide meta-analysis of primary biliary cholangitis: Novel risk loci and candidate drugs.

BACKGROUNDS & AIMS: Primary biliary cholangitis (PBC) is a chronic liver disease in which autoimmune destruction of the small intrahepatic bile ducts eventually leads to cirrhosis. Many patients have inadequate response to licensed medications, motivating the search for novel therapies. Previous genome-wide association studies (GWAS) and meta-analyses (GWMA) of PBC have identified numerous risk loci for this condition, providing insight into its aetiology. We undertook the largest GWMA of PBC to date, aiming to identify additional risk loci and prioritise candidate genes for in silico drug efficacy screening. METHODS: We combined new and existing genotype data for 10,516 cases and 20,772 controls from 5 European and 2 East Asian cohorts. RESULTS: We identified 56 genome-wide significant loci (20 novel) including 46 in European, 13 in Asian, and 41 in combined cohorts; and a 57th genome-wide significant locus (also novel) in conditional analysis of the European cohorts. Candidate genes at newly identified loci include FCRL3, INAVA, PRDM1, IRF7, CCR6, CD226, and IL12RB1, which each play key roles in immunity. Pathway analysis reiterated the likely importance of pattern recognition receptor and TNF signalling, JAK-STAT signalling, and differentiation of T helper (TH)1 and TH17 cells in the pathogenesis of this disease. Drug efficacy screening identified several medications predicted to be therapeutic in PBC, some of which are well-established in the treatment of other autoimmune disorders. CONCLUSIONS: This study has identified additional risk loci for PBC, provided a hierarchy of agents that could be trialled in this condition, and emphasised the value of genetic and genomic approaches to drug discovery in complex disorders. LAY SUMMARY: Primary biliary cholangitis (PBC) is a chronic liver disease that eventually leads to cirrhosis. In this study, we analysed genetic information from 10,516 people with PBC and 20,772 healthy individuals recruited in Canada, China, Italy, Japan, the UK, or the USA. We identified several genetic regions associated with PBC. Each of these regions contains several genes. For each region, we used diverse sources of evidence to help us choose the gene most likely to be involved in causing PBC. We used these 'candidate genes' to help us identify medications that are currently used for treatment of other conditions, which might also be useful for treatment of PBC.

Comparing genomic studies in animal breeding and human genetics: Focus on disease-related traits in livestock.

Genomic studies of diseases can be divided into two types: (1) analyses that reveal causal genes by focusing on linkage disequilibrium between observed and causal variants and (2) those that simultaneously assess numerous genetic markers to estimate the polygenic effects of a particular genomic region or entire genome. The field of human genetics has emphasized the discovery of causal genes, but these represent only a fraction of the total genetic variance. Therefore, alternative approaches, such as the polygenic risk score, which estimates the genetic risk for a given trait or disease on the basis of all genetic markers (rather than on known causal variants only), have begun to garner attention. In many respects, these human genetic methods are similar to those originally developed for the estimation of breeding values (i.e., total additive genetic effects) in livestock. However, despite these similarities in methods, the fields of human and animal genetics still differ markedly in terms of research objectives, target populations, and other characteristics. For example, livestock populations have continually been selected and inbred throughout their history; consequently, their effective population size has shrunk and preferred genes (such as those influencing disease resistance and production traits) have accumulated in the modern breeding populations. By examining the characteristics of these two fields, particularly from the perspectives of disease and disease resistance, this review aims to improve understanding of the intrinsic differences between genomic studies using human compared with livestock populations.

Regional heritability mapping identifies several novel loci (STAT4, ULK4, and KCNH5) for primary biliary cholangitis in the Japanese population.

While the advent of GWAS more than a decade ago has ushered in remarkable advances in our understanding of complex traits, the limitations of single-SNP analysis have also led to the development of several other approaches. Simulation studies have shown that the regional heritability mapping (RHM) method, which makes use of multiple adjacent SNPs jointly to estimate the genetic effect of a given region of the genome, generally has higher detection power than single-SNP GWAS. However, thus far its use has been mostly limited to agricultural settings, and its potential for the discovery of new genes in human diseases is yet to be fully exploited. In this study, by applying the RHM method to primary biliary cholangitis (PBC) in the Japanese population, we identified three novel loci (STAT4, ULK4, and KCNH5) at the genome-wide significance level, two of which (ULK4 and KCNH5) have not been found associated with PBC in any population previously. Notably, these genes could not be detected by using conventional single-SNP GWAS, highlighting the potential of the RHM method for the detection of new susceptibility loci in human diseases. These findings thereby provide strong empirical evidence that RHM is an effective and practical complementary approach to GWAS in this context. Also, liver tissue mRNA microarray analysis revealed higher gene expression levels in ULK4 in PBC patients (P < 0.01). Lastly, we estimated the common SNP heritability of PBC in the Japanese population (0.210 ± 0.026).

rs1944919 on chromosome 11q23.1 and its effector genes COLCA1/COLCA2 confer susceptibility to primary biliary cholangitis.

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.

Genomic Heritabilities and Correlations of 17 Traits Related to Obesity and Associated Conditions in the Japanese Population.

Over the past few decades, obesity has become a public health issue of global concern. Even though disparities exist between human populations, e.g., the higher liver fat content of the Japanese despite a lower body mass index (BMI), studies on the genetics of obesity still largely focus on populations of European descent, leading to a dearth of genetic data on non-European populations. In this context, this study aimed to establish a broad picture of the genetic attributes of the Japanese population, by examining a representative sample of 18,889 individuals participating in the Tohoku Medical Megabank Project cohort. We applied linear mixed model methods to 17 traits related to obesity and associated diseases to estimate the heritabilities explained by common genetic variants and the genetic correlations between each pair of traits. These analyses allowed us to quantify the SNP heritability of health indicators such as BMI (0.248 ± 0.032) and HDL cholesterol (0.324 ± 0.031), and to provide one of the few estimates of the SNP heritability of cystatin C in unrelated individuals (0.260 ± 0.025). We discuss potential differences between the Japanese and people of European ancestry with respect to the genetic correlations between urinary biomarkers and adiposity traits, for which large estimates were obtained. For instance, the genetic correlations between urine potassium level and the values for weight, BMI, waist circumference, and waist-to-height ratio ranged from 0.290 to 0.559, much higher than the corresponding estimates in the UK Biobank.

Integrated GWAS and mRNA Microarray Analysis Identified IFNG and CD40L as the Central Upstream Regulators in Primary Biliary Cholangitis.

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 < 0.00001), and data set 2 for mRNA microarray analysis of liver biopsy specimens (36 patients with PBC and 5 normal controls), which included 1,574 genes with fold change >2 versus controls (P < 0.05). Hierarchical cluster analysis and categorization of cell type-specific genes were performed for data set 2. There were 27 genes, 10 pathways, and 149 upstream regulators that overlapped between data sets 1 and 2. All 10 pathways were immune-related. The most significant common upstream regulators associated with PBC disease susceptibility identified were interferon-gamma (IFNG) and CD40 ligand (CD40L). Hierarchical cluster analysis of data set 2 revealed two distinct groups of patients with PBC by disease activity. The most significant upstream regulators associated with disease activity were IFNG and CD40L. Several molecules expressed in B cells, T cells, Kupffer cells, and natural killer-like cells were identified as potential therapeutic targets in PBC with reference to a recently reported list of cell type-specific gene expression in the liver. Conclusion: Our integrated analysis using GWAS and mRNA microarray data sets predicted that IFNG and CD40L are the central upstream regulators in both disease susceptibility and activity of PBC and identified potential downstream therapeutic targets.

HLA-VBSeq v2: improved HLA calling accuracy with full-length Japanese class-I panel.

HLA-VBSeq is an HLA calling tool developed to infer the most likely HLA types from high-throughput sequencing data. However, there is still room for improvement in specific genetic groups because of the diversity of HLA alleles in human populations. Here, we present HLA-VBSeq v2, a software application that makes use of a new Japanese HLA reference panel to enhance calling accuracy for Japanese HLA class-I genes. Our analysis showed significant improvements in calling accuracy in all HLA regions, with prediction accuracies achieving over 99.0, 97.8, and 99.8% in HLA-A, B and C, respectively.

Population-scale whole genome sequencing identifies 271 highly polymorphic short tandem repeats from Japanese population.

Forensic DNA typing is widely used to identify missing persons and plays a central role in forensic profiling. DNA typing usually uses capillary electrophoresis fragment analysis of PCR amplification products to detect the length of short tandem repeat (STR) markers. Here, we analyzed whole genome data from 1,070 Japanese individuals generated using massively parallel short-read sequencing of 162 paired-end bases. We have analyzed 843,473 STR loci with two to six basepair repeat units and cataloged highly polymorphic STR loci in the Japanese population. To evaluate the performance of the cataloged STR loci, we compared 23 STR loci, widely used in forensic DNA typing, with capillary electrophoresis based STR genotyping results in the Japanese population. Seventeen loci had high correlations and high call rates. The other six loci had low call rates or low correlations due to either the limitations of short-read sequencing technology, the bioinformatics tool used, or the complexity of repeat patterns. With these analyses, we have also purified the suitable 218 STR loci with four basepair repeat units and 53 loci with five basepair repeat units both for short read sequencing and PCR based technologies, which would be candidates to the actual forensic DNA typing in Japanese population.

Long-term selection using a single trait criterion, non-destructive deformation, in White Leghorns: Effect over time on genetic parameters for traits related to egg production.

Although non-destructive deformation is relevant for assessing eggshell strength, few long-term selection experiments are documented which use non-destructive deformation as a selection criterion. This study used restricted maximum likelihood-based methods with a four-trait animal model to analyze the effect of non-destructive deformation on egg production, egg weight and sexual maturity in a two-way selection experiment involving 17 generations of White Leghorns. In the strong shell line, corresponding to the line selected for low non-destructive deformation values, the heritability estimates were 0.496 for non-destructive deformation, 0.253 for egg production, 0.660 for egg weight and 0.446 for sexual maturity. In the weak shell line, corresponding to the line selected for high non-destructive deformation values, the heritabilities were 0.372, 0.162, 0.703 and 0.404, respectively. An asymmetric response to selection was observed for non-destructive deformation, egg production and sexual maturity, whereas egg weight decreased for both lines. Using non-destructive deformation to select for stronger eggshell had a small negative effect on egg production and sexual maturity, suggesting the need for breeding programs to balance selection between eggshell traits and egg production traits. However, the analysis of the genetic correlation between non-destructive deformation and egg weight revealed that large eggs are not associated with poor eggshell quality.

Antagonistic genetic correlations for milking traits within the genome of dairy cattle.

Genome-wide association studies can be applied to identify useful SNPs associated with complex traits. Furthermore, regional genomic mapping can be used to estimate regional variance and clarify the genomic relationships within and outside regions but has not previously been applied to milk traits in cattle. We applied both single SNP analysis and regional genomic mapping to investigate SNPs or regions associated with milk yield traits in dairy cattle. The de-regressed breeding values of three traits, total yield (kg) of milk (MLK), fat (FAT), and protein (PRT) in 305 days, from 2,590 Holstein sires in Japan were analyzed. All sires were genotyped with 40,646 single-nucleotide polymorphism (SNP) markers. A genome-wide significant region (P < 0.01) common to all three traits was identified by regional genomic mapping on chromosome (BTA) 14. In contrast, single SNP analysis identified significant SNPs only for MLK and FAT (P < 0.01), but not PRT in the same region. Regional genomic mapping revealed an additional significant region (P < 0.01) for FAT on BTA5 that was not identified by single SNP analysis. The additive whole-genomic effects estimated in the regional genomic mapping analysis for the three traits were positively correlated with one another (0.830-0.924). However, the regional genomic effects obtained by using a window size of 20 SNPs for FAT on BTA14 were negatively correlated (P < 0.01) with the regional genomic effect for MLK (-0.940) and PRT (-0.878). The BTA14 regional effect for FAT also showed significant negative correlations (P < 0.01) with the whole genomic effects for MLK (-0.153), FAT (-0.172), and PRT (-0.181). These negative genomic correlations between loci are consistent with the negative linkage disequilibrium expected for traits under directional selection. Such antagonistic correlations may hamper the fixation of the FAT increasing alleles on BTA14. In summary, regional genomic mapping found more regions associated with milk production traits than did single SNP analysis. In addition, the existence of non-zero covariances between regional and whole genomic effects may influence the detection of regional effects, and antagonistic correlations could hamper the fixation of major genes under intensive selection.

Effect of Long-Term Selection for Non-Destructive Deformation on Egg Shape in White Leghorns.

Several conventional traits, including eggshell thickness, are commonly being improved genetically as a means to increase eggshell strength. At the same time, researchers have come to recognize that factors related to egg geometry, such as egg shape, are important determinants of the variability remaining in eggshell strength, after conventional traits have been considered. Therefore, given that the value of the egg shape index -the egg's width to length ratio-depends highly on the hen strain, it is necessary to examine the relationship between eggshell strength and shape index more closely in a variety of breeds. From this perspective, by using REML methodology under a five-trait animal model, we analyzed a two-way selection experiment for non-destructive eggshell deformation in 31 generations of White Leghorns, to evaluate the effect of selection for eggshell strength on egg shape. In the strong line, which refers to the line that was selected for decreased non-destructive deformation value, the genetic correlation between eggshell breaking strength and shape index was 0.285±0.055, whereas that between non-destructive deformation and shape index was -0.021±0.063. In the weak line, these values were 0.244±0.055 and -0.093±0.060, respectively. The heritability estimates were 0.381±0.033 for non-destructive deformation, 0.349±0.029 for eggshell breaking strength, and 0.544 ±0.027 for shape index in the strong line, and 0.408±0.031, 0.468±0.032, and 0.484±0.028, respectively, in the weak line. The genetic correlation between eggshell breaking strength and shape index suggests that rounder eggs are somewhat more resistant to breakage than more elongated eggs. The moderately high heritability estimates for shape index indicate the potential to improve egg shape through genetic gain.