Single Nucleotide Polymorphisms In Linkage Disequilibrium Research Articles

The HLA-B*57:01 allele corresponds to a very large MHC haploblock likely explaining its massive effect for HIV-1 elite control.

We have reanalyzed the genomic data of the International Collaboration for the Genomics of HIV (ICGH), centering on HIV-1 Elite Controllers. We performed a genome-wide Association Study comparing 543 HIV Elite Controllers with 3,272 uninfected controls of European descent. Using the latest database for imputation, we analyzed 35,552 Single Nucleotide Polymorphisms (SNPs) within the Major Histocompatibility Complex (MHC) region. Our analysis identified 2,626 SNPs significantly associated (p<5. 10-8) with elite control of HIV-1 infection, including well-established MHC signals such as the rs2395029-G allele which tags HLA-B*57:01. A thorough investigation of SNPs in linkage disequilibrium with rs2395029 revealed an extensive haploblock spanning 1.9 megabases in the MHC region tagging HLA-B*57:01, comprising 379 SNP alleles impacting 72 genes. This haploblock contains damaging variations in proteins like NOTCH4 and DXO and is also associated with a strong differential pattern of expression of multiple MHC genes such as HLA-B, MICB, and ZBTB12. The study was expanded to include two cohorts of seropositive African-American individuals, where a haploblock tagging the HLA-B*57:03 allele was similarly associated with control of viral load. The mRNA expression profile of this haploblock in African Americans closely mirrored that in the European cohort. These findings suggest that additional molecular mechanisms beyond the conventional antigen-presenting role of class I HLA molecules may contribute to the observed influence of HLA-B*57:01/B*57:03 alleles on HIV-1 elite control. Overall, this study has uncovered a large haploblock associated with HLA-B*57 alleles, providing novel insights into their massive effect on HIV-1 elite control.

Abstract 16731: Post-GWAS Functional Analysis of CBLN2 Locus Reveals the Role of CUX1 in the Inflammatory Pathogenesis of Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is a complex and deadly pulmonary vascular disease highly relevant to genetic susceptibility and acquired inflammatory triggers. Chr18q22.3 near cerebellin 2 precursor (CBLN2) gene is the first genetic locus identified by Genome-wide Association Study (GWAS) that is significantly associated with PAH risk in the European population. This locus includes closely linked non-coding intergenic single nucleotide polymorphisms (SNPs) downstream of CBLN2. Although CBLN2 is upregulated in PAH and causes pulmonary vascular dysfunction, it remains unknown how these non-coding SNPs remotely affect CBLN2 and in turn, alter the genetic susceptibility of PAH. Methods and Results: We combined Hi-C chromatin interaction analysis, electrophoretic mobility shift assay, and SNP genotype-PAH correlation analysis with the All of Us database (N=20,343). In doing so, we identified rs11151770(A/G) as a PAH-associated functional SNP (fSNP), among 19 haplotype SNPs in linkage disequilibrium with the associated CBLN2 locus. The genomic region containing rs11151770(A/G) exhibited long-range chromatin interaction with the CBLN2 promotor. Proteomic analysis revealed that nuclear transcription factor Cut-like Homeobox 1 (CUX1) binds specifically to the risk allele A of rs11151770 but not to the non-risk allele G, regulating the expression of CBLN2 as an activator in pulmonary arterial endothelial cells. CUX1-CBLN2 was upregulated by acquired inflammatory PAH triggers IL-1β and IL-6. SNP genotyping in PAH patients (N=159) and non-PAH controls (N=87) showed a 2.2-fold enrichment of the risk allele A only in patients with PAH associated with connective tissue disorders (N=77, p=0.036). These findings supported a pathogenic mechanism of an inflammation-sensitive pathway (CUX1-CBLN2) controlled by an fSNP (risk allele A) underlying the reported genetic association of the CBLN2 locus with PAH risk. Conclusion: Our post-GWAS functional genomics analysis defined a pathogenic CUX1-mediated, fSNP rs11151770(A/G)-controlled, and inflammation-responsive pathway that provides a mechanistic explanation for the GWAS-identified association between genetic variants in CBLN2 locus and PAH susceptibility.

Single Nucleotide Polymorphism rs9277336 Controls the Nuclear Alpha Actinin 4-Human Leukocyte Antigen-DPA1 Axis and Pulmonary Endothelial Pathophenotypes in Pulmonary Arterial Hypertension.

Background Pulmonary arterial hypertension (PAH) is a complex, fatal disease where disease severity has been associated with the single nucleotide polymorphism (SNP) rs2856830, located near the human leukocyte antigen DPA1 (HLA-DPA1) gene. We aimed to define the genetic architecture of functional variants associated with PAH disease severity by identifying allele-specific binding transcription factors and downstream targets that control endothelial pathophenotypes and PAH. Methods and Results Electrophoretic mobility shift assays of oligonucleotides containing SNP rs2856830 and 8 SNPs in linkage disequilibrium revealed functional SNPs via allele-imbalanced binding to human pulmonary arterial endothelial cell nuclear proteins. DNA pulldown proteomics identified SNP-binding proteins. SNP genotyping and clinical correlation analysis were performed in 84 patients with PAH at University of Pittsburgh Medical Center and in 679 patients with PAH in the All of Us database. SNP rs9277336 was identified as a functional SNP in linkage disequilibrium (r2>0.8) defined by rs2856830, and the minor allele was associated with decreased hospitalizations and improved cardiac output in patients with PAH, an index of disease severity. SNP pulldown proteomics showed allele-specific binding of nuclear ACTN4 (alpha actinin 4) protein to rs9277336 minor allele. Both ACTN4 and HLA-DPA1 were downregulated in pulmonary endothelium in human patients and rodent models of PAH. Via transcriptomic and phenotypic analyses, knockdown of HLA-DPA1 phenocopied knockdown of ACTN4, both similarly controlling cell structure pathways, immune pathways, and endothelial dysfunction. Conclusions We defined the pathogenic activity of functional SNP rs9277336, entailing the allele-specific binding of ACTN4 and controlling expression of the neighboring HLA-DPA1 gene. Through inflammatory or genetic means, downregulation of this ACTN4-HLA-DPA1 regulatory axis promotes endothelial pathophenotypes, providing a mechanistic explanation for the association between this SNP and PAH outcomes.

Genetically predicted adipose tissue distribution influences the risk of atherosclerosis.

Truncal obesity is a key modifiable risk factor in cardiovascular disease (CVD). It is a highly complex phenotype, and numerous factors such as the distribution of adipose tissue may influence risk of CVD.1 However, while some studies have suggested that gluteofemoral fat deposits may serve a protective role, others have offered conflicting results.2,3 As observational studies may be vulnerable to confounding from environmental factors, we conducted a Mendelian randomization study investigating the effect of regional adipose tissue distribution on the risk of different types of atherosclerosis and heart failure (HF). The Mendelian randomization approach leverages genetic variation to infer causal associations as genetic variants in the human genome are inherited randomly, independently of environmental factors.4 All data used in this study were publicly available. First, we obtained summary statistics from a recent genome-wide association study on visceral adipose tissue (VAT), abdominal subcutaneous adipose tissue (AAT), and gluteofemoral adipose tissue (GFAT).5 Adipose tissue traits were determined by magnetic resonance imaging (MRI) of up to 39 076 individuals from the UK Biobank and were adjusted for age, sex, BMI, height, and 10 principal components. Instrument variables were selected from single-nucleotide polymorphisms (SNPs) that were robustly associated with the traits on a genome-wide significant level (P < 5 × 10−8). Instrument variables were pruned for linkage disequilibrium (LD), using a 10 megabase (Mb) window. For genetically correlated variants (R2 > 0.001), the variant with the smallest P-value was selected. We considered myocardial infarction (MI), large artery atherosclerotic stroke (LAS), and peripheral artery disease (PAD) as proxies for atherosclerosis. We acquired MI and LAS summary statistics from the CARDIoGRAMplusC4D consortium6 and the European subset of the GIGASTROKE consortium.7 Summary statistics for PAD and HF were obtained from the FinnGen study.8 There was no sample overlap between exposure and outcome datasets. For instrument variables not present in the outcome datasets, we identified suitable proxy SNPs in LD (r2 > 0.8).

Allelic expression imbalance in articular cartilage and subchondral bone refined genome-wide association signals in osteoarthritis.

To present an unbiased approach to identify positional transcript single nucleotide polymorphisms (SNPs) of osteoarthritis (OA) risk loci by allelic expression imbalance (AEI) analyses using RNA sequencing of articular cartilage and subchondral bone from OA patients. RNA sequencing from 65 articular cartilage and 24 subchondral bone from OA patients was used for AEI analysis. AEI was determined for all genes present in the 100 regions reported by the genome-wide association studies (GWAS) catalog that were also expressed in cartilage or bone. The count fraction of the alternative allele (φ) was calculated for each heterozygous individual with the risk SNP or with the SNP in linkage disequilibrium (LD) with it (r2 > 0.6). Furthermore, a meta-analysis was performed to generate a meta-φ (null hypothesis median φ = 0.49) and P-value for each SNP. We identified 30 transcript SNPs (28 in cartilage and two in subchondral bone) subject to AEI in 29 genes. Notably, 10 transcript SNPs were located in genes not previously reported in the GWAS catalog, including two long intergenic non-coding RNAs (lincRNAs), MALAT1 (meta-φ = 0.54, FDR = 1.7×10-4) and ILF3-DT (meta-φ = 0.6, FDR = 1.75×10-5). Moreover, 12 drugs were interacting with seven genes displaying AEI, of which seven drugs have been already approved. By prioritizing proxy transcript SNPs that mark AEI in cartilage and/or subchondral bone at loci harbouring GWAS signals, we present an unbiased approach to identify the most likely functional OA risk-SNP and gene. We identified 10 new potential OA risk genes ready for further translation towards underlying biological mechanisms.

Synonymous mutation rs1129293 is associated with PIK3CG expression and PI3Kγ activation in patients with chronic Chagas cardiomyopathy

Single nucleotide polymorphisms (SNPs) that do not change the composition of amino acids and cause synonymous mutations (sSNPs) were previously considered to lack any functional roles. However, sSNPs have recently been shown to interfere with protein expression owing to a myriad of factors related to the regulation of transcription, mRNA stability, and protein translation processes. In patients with Chagas disease, the presence of the synonymous mutation rs1129293 in phosphatidylinositol-4,5-bisphosphate 3-kinase gamma (PIK3CG) gene contributes to the development of the chronic Chagas cardiomyopathy (CCC), instead of the digestive or asymptomatic forms. In this study, we aimed to investigate whether rs1129293 is associated with the transcription of PIK3CG mRNA and its activity by quantifying AKT phosphorylation in the heart samples of 26 chagasic patients with CCC. Our results showed an association between rs1129293 and decreased PIK3CG mRNA expression levels in the cardiac tissues of patients with CCC. The phosphorylation levels of AKT, the protein target of PI3K, were also reduced in patients with this mutation, but were not correlated with PI3KCG mRNA expression levels. Moreover, bioinformatics analysis showed that rs1129293 and other SNPs in linkage disequilibrium (LD) were associated with the transcriptional regulatory elements, post-transcriptional modifications, and cell-specific splicing expression of PIK3CG mRNA. Therefore, our data demonstrates that the synonymous SNP rs1129293 is capable of affecting the PIK3CG mRNA expression and PI3Kγ activation.

Genetic risk score for risk stratification of patients positive for SARS-CoV-2 virus. The CARGENCORS study

Funding AcknowledgementsType of funding sources: Other. Main funding source(s): Carlos III Health Institute and the European Regional Development Fund Government of Catalonia through the Agency for Management of University and Research Grants Crue-CSIC-Santander FONDO SUPERA COVID-19BackgroundThe disease presentation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ranges from asymptomatic to fatal. COVID-19 patients with pre-existing coronary artery disease (CAD) risk factors or overt cardiovascular disease are at particular risk of severe disease. We hypothesised that a specific genetic risk score (GRS) based on single nucleotide polymorphisms (SNPs) allele count to score COVID-19 severity might include SNPs counts related to CAD incidence and to thrombosis, inflammation, and viral infectivity determinants involved in the severity of SARS-CoV-2. Such GRS could improve the early risk stratification of COVID-19 patients and optimize treatment strategies.PurposeTo evaluate the capacity of a genetic risk score (GRS) with candidate genes to predict COVID-19 severity.MethodsWe conducted an age- and sex-matched case-control study with 1:2 ratio recruitment involving 2454 patients from Catalan hospitals and primary care. Cases were hospitalized severe (requiring at least oxygen treatment) or fatal COVID-19 patients; and controls were moderate-symptom and asymptomatic patients treated at home. Standard parametric and non-parametric methods, as required, were used to compare patient characteristics by severity. Individual genotypes for 33 CAD, 14 thrombosis, 22 inflammation, 15 viral infectivity SNPs and 2 COVID-19 SNPs already published were tested for association with severity with Cochran-Armitage statistics and p-values corrected for multiple comparisons. GRS was computed as the unweighted count of adverse alleles (0, 1 or 2). The odds ratio of severe COVID-19 was analysed for GRS (and its component SNPs) with logistic regression models adjusted for potential confounding factors. Area under the curve (AUC) improvement and net reclassification index (NRI) for GRS was estimated from a basic model including CAD and COVID-19 severity risk factors. Models’ performance was measured with the Akaike information criterion.ResultsSNPs identifications are not shown to prevent patent conflict. Cases and control characteristics are compared in Table 1. Cases had a more adverse cardiovascular and anthropometric risk profile. After correcting for multiple testing by Benjamini-Hochberg criteria, we observed 13 SNPs to be significantly associated with severity. After excluding the close SNPs in linkage disequilibrium, 7 were retained in the GRS model, which yielded the discrimination and reclassification characteristics described in Table 2.ConclusionA GRS with 7 SNPs related to CAD, thrombosis and inflammation significantly improves the severe COVID-19 risk assessment done with age, sex, comorbidity, and anthropometry alone.CV and anthropometric risk profileOR of COVID-19 severity for a 7-SNP GRS

Polygenic mechanisms underpinning the response to exercise-induced muscle damage in humans: In vivo and in vitro evidence.

We investigated whether 20 candidate single nucleotide polymorphisms (SNPs) were associated with in vivo exercise-induced muscle damage (EIMD), and with an in vitro skeletal muscle stem cell wound healing assay. Sixty-five young, untrained Caucasian adults performed 120 maximal eccentric knee-extensions on an isokinetic dynamometer to induce EIMD. Maximal voluntary isometric/isokinetic knee-extensor torque, knee joint range of motion (ROM), muscle soreness, serum creatine kinase activity and interleukin-6 concentration were assessed before, directly after and 48 h after EIMD. Muscle stem cells were cultured from vastus lateralis biopsies from a separate cohort (n = 12), and markers of repair were measured in vitro. Participants were genotyped for all 20 SNPs using real-time PCR. Seven SNPs were associated with the response to EIMD, and these were used to calculate a total genotype score, which enabled participants to be segregated into three polygenic groups: 'preferential' (more 'protective' alleles), 'moderate', and 'non-preferential'. The non-preferential group was consistently weaker than the preferential group (1.93 ± 0.81 vs. 2.73 ± 0.59 N ∙ m/kg; P = 9.51 × 10-4 ) and demonstrated more muscle soreness (p = 0.011) and a larger decrease in knee joint ROM (p = 0.006) following EIMD. Two TTN-AS1 SNPs in linkage disequilibrium were associated with in vivo EIMD (rs3731749, p ≤ 0.005) and accelerated muscle stem cell migration into the artificial wound in vitro (rs1001238, p ≤ 0.006). Thus, we have identified a polygenic profile, linked with both muscle weakness and poorer recovery following EIMD. Moreover, we provide evidence for a novel TTN gene-cell-skeletal muscle mechanism that may help explain some of the interindividual variability in the response to EIMD.

Assessment of linkage disequilibrium patterns between structural variants and single nucleotide polymorphisms in three commercial chicken populations

BackgroundStructural variants (SV) are causative for some prominent phenotypic traits of livestock as different comb types in chickens or color patterns in pigs. Their effects on production traits are also increasingly studied. Nevertheless, accurately calling SV remains challenging. It is therefore of interest, whether close-by single nucleotide polymorphisms (SNPs) are in strong linkage disequilibrium (LD) with SVs and can serve as markers. Literature comes to different conclusions on whether SVs are in LD to SNPs on the same level as SNPs to other SNPs. The present study aimed to generate a precise SV callset from whole-genome short-read sequencing (WGS) data for three commercial chicken populations and to evaluate LD patterns between the called SVs and surrounding SNPs. It is thereby the first study that assessed LD between SVs and SNPs in chickens.ResultsThe final callset consisted of 12,294,329 bivariate SNPs, 4,301 deletions (DEL), 224 duplications (DUP), 218 inversions (INV) and 117 translocation breakpoints (BND). While average LD between DELs and SNPs was at the same level as between SNPs and SNPs, LD between other SVs and SNPs was strongly reduced (DUP: 40%, INV: 27%, BND: 19% of between-SNP LD). A main factor for the reduced LD was the presence of local minor allele frequency differences, which accounted for 50% of the difference between SNP – SNP and DUP – SNP LD. This was potentially accompanied by lower genotyping accuracies for DUP, INV and BND compared with SNPs and DELs. An evaluation of the presence of tag SNPs (SNP in highest LD to the variant of interest) further revealed DELs to be slightly less tagged by WGS SNPs than WGS SNPs by other SNPs. This difference, however, was no longer present when reducing the pool of potential tag SNPs to SNPs located on four different chicken genotyping arrays.ConclusionsThe results implied that genomic variance due to DELs in the chicken populations studied can be captured by different SNP marker sets as good as variance from WGS SNPs, whereas separate SV calling might be advisable for DUP, INV, and BND effects.

Genome-Wide Association Study on Two Immune-Related Traits in Jinghai Yellow Chicken

This study was designed to discover molecular marker associated with the interferon INF-γ and avian influenza (AI) antibody titer traits in Jinghai Yellow chicken (Gallus gallus). Serum samples were taken from 400 female chickens and the INF-γ concentrations and AI antibody titer levels were measured. A genome-wide association study was carried out using specific-locus amplified fragment (SLAF) sequencing. Bioinformatics analysis was applied to detect single-nucleotide polymorphisms (SNPs) associated with the two traits. After sequencing and quality control, 103,680 SLAFs and 90,961 SNPs were obtained. The 400 samples were divided into 10 subgroups to reduce the effects of group stratification. The Bonferroni adjusted P-value of genome-wide significance was set at 1.87E−06 according to the number of independent SNP markers and linkage disequilibrium blocks. A SNP that was significantly associated with INF-γ concentration was detected in the myomesin 1 (MYOM1) gene on chromosome 2, and another SNPthat was significantly associated with the AI antibody titer level was detected in an RNA methyltransferase gene (Nsun7), which was found to have an important biological function. We propose that MYOM1 and Nsun7 are valuable candidate genes that influence the disease resistance characters of chicken. However, in-depth investigations are needed to determine the essential roles of these genes in poultry disease resistance and their possible application in breeding disease resistant poultry.

Genetic overlap analysis of endometriosis and asthma identifies shared loci implicating sex hormones and thyroid signalling pathways.

Is there a shared genetic or causal association of endometriosis with asthma or what biological mechanisms may underlie their potential relationships? Our results confirm a significant but non-causal association of endometriosis with asthma implicating shared genetic susceptibility and biological pathways in the mechanisms of the disorders, and potentially, their co-occurrence. Some observational studies have reported a pattern of co-occurring relationship between endometriosis and asthma; however, there is conflicting evidence and the aetiology, as well as the underlying mechanisms of the relationship, remain unclear. We applied multiple statistical genetic approaches in the analysis of well-powered, genome-wide association study (GWAS) summary data to comprehensively assess the relationship of endometriosis with asthma. Endometriosis GWAS from the International Endogene Consortium (IEC, 17 054 cases and 191 858 controls) and asthma GWAS from the United Kingdom Biobank (UKB, 26 332 cases and 375 505 controls) were analysed. Additional asthma data from the Trans-National Asthma Genetic Consortium (TAGC, 19 954 cases and 107 715 controls) were utilized for replication testing. We assessed single-nucleotide polymorphism (SNP)-level genetic overlap and correlation between endometriosis and asthma using SNP effect concordance analysis (SECA) and linkage disequilibrium score regression analysis (LDSC) methods, respectively. GWAS meta-analysis, colocalization (GWAS-PW), gene-based and pathway-based functional enrichment analysis methods were applied, respectively, to identify SNP loci, genomic regions, genes and biological pathways shared by endometriosis and asthma. Potential causal associations between endometriosis and asthma were assessed using Mendelian randomization (MR) methods. SECA revealed significant concordance of SNP risk effects across the IEC endometriosis and the UKB asthma GWAS. Also, LDSC analysis found a positive and significant genetic correlation (rG = 0.16, P = 2.01 × 10-6) between the two traits. GWAS meta-analysis of the IEC endometriosis and UKB asthma GWAS identified 14 genome-wide significant (Pmeta-analysis < 5.0 × 10-8) independent loci, five of which are putatively novel. Three of these loci were consistently replicated using TAGC asthma GWAS and reinforced in colocalization and gene-based analyses. Additional shared genomic regions were identified in the colocalization analysis. MR found no evidence of a significant causal association between endometriosis and asthma. However, combining gene-based association results across the GWAS for endometriosis and asthma, we identified 17 shared genes with a genome-wide significant Fisher's combined P-value (FCPgene) <2.73 × 10-6. Additional analyses (independent gene-based analysis) replicated evidence of gene-level genetic overlap between endometriosis and asthma. Biological mechanisms including 'thyroid hormone signalling', 'abnormality of immune system physiology', 'androgen biosynthetic process' and 'brain-derived neurotrophic factor signalling pathway', among others, were significantly enriched for endometriosis and asthma in a pathway-based analysis. The GWAS for endometriosis data were sourced from the International Endogen Consortium (IEC) and can be accessed by contacting the consortium. The GWAS data for asthma are freely available online at Lee Lab (https://www.leelabsg.org/resources) and from the Trans-National Asthma Genetic Consortium (TAGC). Given we analysed GWAS datasets from mainly European populations, our results may not be generalizable to other ancestries. This study provides novel insights into mechanisms underpinning endometriosis and asthma, and potentially their observed relationship. Findings support a co-occurring relationship of endometriosis with asthma largely due to shared genetic components. Agents targeting 'selective androgen receptor modulators' may be therapeutically relevant in both disorders. Moreover, SNPs, loci, genes and biological pathways identified in our study provide potential targets for further investigation in endometriosis and asthma. National Health and Medical Research Council (NHMRC) of Australia (241,944, 339,462, 389,927, 389,875, 389,891, 389,892, 389,938, 443,036, 442,915, 442,981, 496,610, 496,739, 552,485, 552,498, 1,026,033 and 1,050,208), Wellcome Trust (awards 076113 and 085475) and the Lundbeck Foundation (R102-A9118 and R155-2014-1724). All researchers had full independence from the funders. Authors do not have any conflict of interest.

Abstract 11643: ACTN4 Regulates HLA-DPA1-Related Pulmonary Endothelial Pathophenotypes in Pulmonary Arterial Hypertension Through Allele-Specific Binding to Functional SNP rs9277336

Introduction: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by increased pulmonary arterial pressure and vascular resistance. This vascular injury promotes immune dysregulation, further worsening the vasculopathy. A genome-wide association study identified a single nucleotide polymorphism (SNP) associated with PAH risk (rs2856830) at Human Leukocyte Antigen DPA1 (HLA-DPA1) locus, a gene known to be involved in adaptive immunity. However, little is known about the mechanism underlying this clinical association. Hypothesis: Allele-specific binding of transcription factors to a disease-associated functional SNP (fSNP) controls HLA-DPA1 expression, driving pulmonary vascular cell dysfunction and PAH. Methods: Oligonucleotides containing alleles of rs2856830 and eight SNPs in linkage disequilibrium (LD) were subjected to electrophoretic mobility shift assays (EMSA) to identify fSNPs characterized by allele-imbalanced binding to human pulmonary arterial endothelial cells (PAEC) nuclear proteins. SNP genotyping was done using blood from PAH (n=134) and control patients (n=131) at UPMC. Transcriptomics, angiogenesis, and migration were assessed in siRNA mediated HLA-DPA1 and Alpha Actinin 4 (ACTN4) knockdown (KD) in PAECs. Results: Through EMSA, rs9277336 (LD r 2 =0.87) was identified as a fSNP in HLA-DPA1 locus. SNP pulldown and immunoblotting confirmed allele-specific binding of nuclear ACTN4 protein to rs9277336 risk allele A (3.26 ± 0.49, p=0.001). SNP genotyping revealed a 2.04 ± 0.67 fold enrichment of risk allele A in PAH patients (χ 2 test, p=0.025). ACTN4 KD in cultured human PAECs decreased HLA-DPA1 transcript (0.30 ± 0.10, p&lt;0.001) . Transcriptomics revealed overlap of several cell structure and immune system-related pathways by HLA-DPA1 and ACTN4 KD. HLA-DPA1 and ACTN4 KD affected PAH associated pathophenotypes, including angiogenesis and migration. Conclusions: Our functional analysis identified ACTN4 as a novel regulator to disease-associated gene HLA-DPA1, affecting the endothelial pathophenotypes in PAH. The allele-specific binding to fSNP rs9277336 provides a mechanistic explanation for the clinical association between HLA-DPA1 locus and PAH outcomes.

Polymorphisms in HIF-1a gene are not associated with diabetic retinopathy in China.

BACKGROUNDIt has been reported that vascular endothelial growth factor (VEGF) is a susceptibility gene for both type 2 diabetes mellitus (T2DM) and diabetic retinopathy (DR). In response to hypoxia, VEGF mRNA levels are increased, which is mainly mediated by the binding of hypoxia-inducible factor-1 (HIF-1) and hypoxia response element upstream of the transcriptional start site of VEGF. Therefore, HIF-1a is supposed to be involved in pathology of DR.AIMTo investigate whether the polymorphisms in HIF-1a gene are associated with DR.METHODSTwo hundred and ninety-nine type 2 diabetic patients (128 males and 171 females) and 144 healthy volunteers were recruited. Mean age was 56.04 ± 21.05 years. According to the results of fundus fluorescein angiography and examination of ophthalmoscopy, patients were divided into two groups, DNR group (diabetes without retinopathy) and DR group (diabetes with retinopathy). There are 150 cases in DNR group and 149 cases in DR group. Two single nucleotide polymorphisms (SNP) of the HIF-1a gene were tested using matrix-assisted laser desorption/Ionization time of flight mass spectrometry. The frequency of genotypes and alleles, and odds ratio were measured.RESULTSThe mean age of the cases with diabetes was 55.84 ± 3.66 years, the mean age of the cases with DR was 55.97 ± 4.66 years and that of controls was 56.32 ± 4.70 years. Two variations were found in 76 patients. Rs11549465 is the change of C-T base, rs11549467 is the change of G-A base. The rs11549467 G/A genotype was 5.33% in diabetes and 6.04% in DR patients, respectively. The rs11549465 C/T genotype was 10% and 12.75% in patients with diabetes and DR. The rs11549467 A allele frequencies and rs11549465 T frequencies was similar to that of controls. Paired SNP linkage disequilibrium analysis indicated that rs11549467 was in linkage disequilibrium with rs11549465. Haplotype association analysis denoted that the haplotype association exhibited similar distribution in the patients compared to the normal controls.CONCLUSIONThis study suggests that there is no relationship between the genetic variations of HIF1a and diabetes or DR.

SNPs associated with colorectal cancer at 15q13.3 affect risk enhancers that modulate GREM1 gene expression.

Several genome wide association studies of colorectal cancer (CRC) have identified single nucleotide polymorphisms (SNPs) on chromosome 15q13.3 associated with CRC risk. To identify functional variant(s) underlying this association, we investigated SNPs in linkage disequilibrium with the risk‐associated SNP rs4779584 that overlapped regulatory regions/enhancer elements characterized in colon‐related tissues and cells. We identified several SNP‐containing regulatory regions that exhibited enhancer activity in vitro, including one SNP (rs1406389) that correlated with allele‐specific effects on enhancer activity. Deletion of either this enhancer or another enhancer that had previously been reported in this region correlated with decreased expression of GREM1 following CRISPR/Cas9 genome editing. That GREM1 is one target of these enhancers was further supported by an expression quantitative trait loci correlation between rs1406389 and GREM1 expression in the transverse but not sigmoid colon in the Genotype‐Tissue Expression dataset. Taken together, we conclude that the 15q13.3 region contains at least two functional variants that map to distinct enhancers and impact CRC risk through modulation of GREM1 expression.

Two-stage genome-wide association study of chronic rhinosinusitis and disease subphenotypes highlights mucosal immunity contributing to risk.

Two-stage genome-wide association study of chronic rhinosinusitis and disease subphenotypes highlights mucosal immunity contributing to risk.

Identification of Single Nucleotide Polymorphisms and Analysis of Linkage Disequilibrium in Different Bamboo Species Using the Candidate Gene Approach

Bamboos are one of the most beautiful and useful plants on Earth. The genetic background and population structure of bamboos are well known, which helps accelerate the process of artificial domestication of bamboo. Partial sequences of six genes involved in nitrogen use efficiency in 32 different bamboo species were analyzed for occurrence of single nucleotide polymorphisms (SNPs). The nucleotide diversity θw and total nucleotide polymorphisms πT of the sequenced DNA regions was 0.05137 and 0.03332, respectively. Both πnonsyn /πsyn and Ka/Ks values were 0.1. Estimation and analysis of linkage disequilibrium of five genes revealed that with the increase in nucleotide sequence length, the degree of SNP linkage disequilibrium decreased rapidly. We inferred the population genetic structure of 32 bamboo species based on the SNP loci of six genes with frequencies >18%. 32 bamboo species were divided into five categories, which indicated that the combined population of all bamboo species had obvious multivariate characteristics and was heterogeneous; red (Group 1) and green (Group 2) were the main groups.

A functional variant on 20q13.33 related to glioma risk alters enhancer activity and modulates expression of multiple genes.

Genome‐wide association studies (GWAS) have identified single‐nucleotide polymorphisms (SNPs) associated with glioma risk on 20q13.33, but the biological mechanisms underlying this association are unknown. We tested the hypothesis that a functional SNP on 20q13.33 impacted the activity of an enhancer, leading to an altered expression of nearby genes. To identify candidate functional SNPs, we identified all SNPs in linkage disequilibrium with the risk‐associated SNP rs2297440 that mapped to putative enhancers. Putative enhancers containing candidate functional SNPs were tested for allele‐specific effects in luciferase enhancer activity assays against glioblastoma multiforme (GBM) cell lines. An enhancer containing SNP rs3761124 exhibited allele‐specific effects on activity. Deletion of this enhancer by CRISPR‐Cas9 editing in GBM cell lines correlated with an altered expression of multiple genes, including STMN3, RTEL1, RTEL1‐TNFRSF6B, GMEB2, and SRMS. Expression quantitative trait loci (eQTL) analyses using nondiseased brain samples, isocitrate dehydrogenase 1 (IDH1) wild‐type glioma, and neurodevelopmental tissues showed STMN3 to be a consistent significant eQTL with rs3761124. RTEL1 and GMEB2 were also significant eQTLs in the context of early CNS development and/or in IDH1 wild‐type glioma. We provide evidence that rs3761124 is a functional variant on 20q13.33 related to glioma/GBM risk that modulates the expression of STMN3 and potentially other genes across diverse cellular contexts.

Decryption of favourable haplotypes and potential candidate genes for five fibre quality properties using a relatively novel genome-wide association study procedure in upland cotton

To understand the genetic basis of crop economic characteristics, identifying whole-genome quantitative trait loci (QTLs) in breeding resources via genome-wide association studies (GWASs) is important, but conventional GWAS procedures have mainly concentrated on identifying regions of major QTLs. In this study, to reveal whole-genome QTLs controlling cotton fibre quality traits, a total of 1,236,418 single-nucleotide polymorphism (SNP) markers were explored in a natural population of 315 upland cotton accessions using the specific-locus amplified fragment sequencing (SLAF-seq) method, and 13,391 high-quality SNPs were organized into 9244 SNP linkage disequilibrium blocks (SNPLDBs) to develop molecular markers with multiple haplotypes/alleles. A relatively novel procedure for restricted two-stage multi-locus multi-allele GWAS (RTM-GWAS) involving two methods was applied, including multiple environments and four single environments. In total, 75, 77, 66, 66, and 60 significant SNPLDBs associated with the upper half mean length (UHML), fibre strength (FS), fibre micronaire (FM), fibre uniformity index (FUI), and fibre elongation (FE), respectively, were identified via the multiple-environment RTM-GWAS method. Based on these results, the haplotype/allele effects of the significant SNPLDB loci were estimated, and QTL-allele matrices were established to provide the abbreviated genetic composition of the population. Most importantly, nine stable SNPLDB loci were simultaneously detected in multiple environments and two or more single test environments with large –lg(P) values and percentages of explained phenotypic variation. Furthermore, favourable haplotypes/alleles of the nine stable SNPLDB loci were identified, and 79 potential candidate genes related to UHML and FS traits were predicted from RNA-seq data. Favourable haplotypes/alleles, potential candidate genes and QTL-allele matrices can offer important insights into the genetic basis of fibre quality traits in upland cotton and may facilitate the breeding of cotton varieties with excellent fibre quality properties.

Molecular characterization of QTL-allele system for drought tolerance at seedling stage and optimal genotype design using multi-locus multi-allele genome-wide association analysis in a half-sib population of soybean (Glycine max (L.) Merr.)

AbstractCharacterizing the whole genetic architecture of drought tolerance (DT) is a persistent challenge for the breeders. Here we developed a half-sib population comprising of 404 lines of two recombinant inbred line (RIL) populations with M8206 as the joint parent (M8206 × TongShan and ZhengYang × M8206) and tested for its DT under sand culture. The population was sequenced using restriction-site-associated DNA sequencing filtered with minor allele frequency ≥0.01; 55,936 single nucleotide polymorphisms (SNPs) were obtained and assembled into 6137 SNPLDBs (SNP linkage disequilibrium blocks). The restricted two-stage multi-locus genome-wide association analysis characterized with error and false-positive control identified 40 QTLs with 93 alleles on an average of 34.75% of the phenotypic variance (PV) collectively for relative root length (RRL) and relative shoot length (RSL) that served as potential DT indicators. Among these, eight loci corresponded to previously reported QTLs, whereas 32 loci were therefore novel. The identified QTLs with their corresponding alleles for RRL and RSL were organized into QTL-allele matrices, depicting the comprehensive DT genetic architecture of the three parents/half-sib population. From the matrices, we predicted the possible best/optimal genotype with weighted average value (WAV) 1.553 over two indicators, while for the top 10 single crosses among RILs with 95th percentile WAV was 1.218–1.257, transgressive over the parents (0.693–0.794) yet much less than 1.553. From the detected QTL-allele system, 65 potential candidate genes collectively for both indicators explaining on an average of 24.41% PV were annotated and χ2-tested as a DT candidate gene system involving nine biological processes.

Association analysis between constructed SNPLDBs and GCA effects of 9 quality-related traits in parents of hybrid rice (Oryza sativa L.)

BackgroundThe general combining ability (GCA) of parents in hybrid rice affects not only heterotic level of grain yield and other important agronomic traits, but also performance of grain quality traits of F2 bulk population which is the commodity consumed by humans. In order to make GCA improvement for quality traits in parents of hybrid rice by molecular marker assisted selection feasible, genome-wide GCA loci for quality traits in parents were detected through association analysis between the effects of GCA and constructed single nucleotide polymorphism linkage disequilibrium blocks (SNPLDBs), by using unhusked rice grains harvested from F1 plants of 48 crosses of Indica rice and 78 crosses of Japonica rice. GCA-SNPLDBs association analysis.ResultsAmong the 8 CMS and 6 restorer lines of indica rice subspecies, CMS lines Zhenpin A, Zhenshan97 A, and 257A, and restorers Kanghui98, Minghui63 and Yanhui559 were recognized as good general combiners based on their GCA effect values for the 9 quality traits (brown rice rate, milled rice rate, head rice rate, percentage of chalky grains, chalky area size, chalkiness degree, gelatinization temperature, gel consistency and amylose content). Among the 13 CMS and 6 restorer lines of japonica rice subspecies, CMS 863A, 6427A and Xu 2A, and restorers C418, Ninghui8hao and Yunhui4hao showed elite GCA effect values for the 9 traits. GCA-SNPLDB association analysis revealed 39 significant SNPLDB loci associated with the GCA of the 9 quality-related traits, and the numbers of SNPLDB loci located on chromosome 1, 2, 3, 4, 5, 8, 9, 11 and 12 were 1, 4, 3, 9, 6, 5, 5, 4 and 2, respectively. Number of superior GCA alleles for the 9 traits among the 33 parents ranged from 1 to 26.ConclusionsThirty-nine significant SNPLDBs loci were identified associated with the GCA of 9 quality-related traits, and the superior SNPLDB alleles could be used to improve the GCA of parents for the traits in the future by molecular marker assisted selection. The genetic basis of trait GCA in parents is different from that of trait itself.