Intergenic Variants Research Articles

PsychArray-Based Genome Wide Association Study of Suicidal Deaths in India.

Suicide is a preventable but escalating global health crisis. Genome-wide association studies (GWAS) studies to date have been limited, and some are underpowered. In this study, we aimed to perform the PsychArray-based GWAS study to identify single nucleotide variations associated with suicide in the Indian population. We recruited unrelated subjects who died by suicide as cases (N = 313) and the non-suicidal deaths as controls (N = 294). The 607 samples were genotyped, including cases and controls using the Illumina Infinium PsychArray-24 BeadChip v1.3 Results: In our study, four single nucleotide polymorphisms (SNPs) crossed the threshold of significance level <1 × 10-5. One of them is intronic at Chromosome2:rs1901851 and three are intergenic at Chromosome12:rs3847911, Chromosome8:rs2941489, Chromosome8:rs1464092. At a significance level of 5 × 10-5, we found a few more SNPs, with the majority of them being intergenic variants. The associated genes were associated with various important functions ranging from cell signaling, GTP binding, GPCR binding, and transcription factor binding. The SNPs identified in our study were not reported earlier. To our best knowledge, this study is one of the first GWAS for suicide in the Indian population. The results indicate few novel SNPs that may be associated with suicide and require further investigation. Their clinical significance is to be studied in the future.

206. Using Genome-Wide Association Analysis of Pneumococcal Isolates to Identify Novel Gene Variants in Central Nervous System Infection

Abstract Background Streptococcus pneumoniae is the leading cause of meningitis in children and is associated with significant morbidity and mortality. Despite conjugate pneumococcal vaccines that have otherwise been successful in reducing invasive disease caused by vaccine serotypes, the incidence of pneumococcal meningitis remains relatively unchanged. Using genome-wide association, we identified sequence elements associated with pneumococcal central nervous system (CNS) infection. Methods From 1996 to 2018, 366 clinical pneumococcal isolates were collected from pediatric patients and paired with clinical metadata. All isolates underwent whole-genome sequencing and in silico serotyping. Fifty-four clinical isolates were from children with CNS infection. Sequence element enrichment analysis was performed using elastic net and linear mixed modeling statistical methods to find genetic variants associated with CNS infection. We identified associated genes by mapping statistically associated variants to a serotype 19F reference S. pneumoniae genome. Results CNS isolates spanned 22 unique serotypes; the most common were 22F (n = 6), 7F (n = 5), 18C (n = 4), and 35B (n = 4). We identified two intergenic sequence variants and one intragenic sequence variant significantly associated with CNS infection (p < 1.48 × 10-7). The top intragenic candidate in our analysis was located within cpsC, a gene involved in regulation of capsule synthesis. The CNS infection-associated cpsC variant was found in serotypes 22F (n = 1), 18C (n = 4), and 14 (n = 2). Conclusion The diversity of pneumococcal isolates associated with CNS infection suggests the importance of genetic determinants beyond capsular serotype. We identified a number of novel microbial genetic variants significantly associated with pneumococcal CNS infection across serotypes, including a coding mutation in cpsC. Functional characterization of cpsC variants is needed to inform mechanisms contributing to pneumococcal CNS virulence. Disclosures Anne J. Blaschke, MD, PhD, BioFire Diagnostics/Biomerieux: Advisor/Consultant|BioFire Diagnostics/Biomerieux: Grant/Research Support|BioFire Diagnostics/Biomerieux: IP licensed to BioFire Diagnostics through the University of Utah and royalties received through the University of Utah related to the FilmArray|Merck: Advisor/Consultant.

GWAS of Genetic Resilience to Age‐Related Risk for Alzheimer’s Disease in Admixed African Ancestry Individuals

AbstractBackgroundWe sought to identify genetic variants that confer resilience to age‐related Alzheimer’s disease (AD) risk in subjects of admixed African ancestry.MethodsParticipants were ages 60+, of African ancestry (> = 25%), and diagnosed as cases or controls. Genetic data were available from SNP arrays imputed to TOPMed, whole genome sequencing (WGS), or whole exome sequencing (WES). Genome‐wide association studies (GWAS) were performed per data type, split by Hispanic/non‐Hispanic participants (Table‐1), followed by meta‐analysis (Plink v2.0; GWAMA v2.2.2). GWAS performed multiple linear regression on an AD‐age score that models resilience to age‐related risk for AD (Le Guen & Belloy et al. 2021; Figure‐1). Models adjusted for sex, APOE*4/APOE*2 dosage, the first five genetic principal components (PC‐AiR; GENESIS; R v3.6), and array/sequencing center. Brain amyloid Positron Emission Tomography (PET) qualitative reads together with SNP array data imputed to TOPMed were also available for African American individuals in A4. These data were independent from the primary GWAS and were used to evaluate significant GWAS variants for their association with binarized amyloid outcome (negative/positive), using a consistent model that additionally adjusted for age‐at‐scan.ResultsWe found a novel protective genome‐wide significant intergenic variant (rs77450754) ∼200kb downstream of ATXN8OS/KLHL1 (Figure‐2‐3). It has a MAF of 13% in African ancestry samples, but is rare (0.1%) in Europeans and (<2%) Latino/Admixed Americans in gnomAD v.3. The latter may explain why effects appeared less pronounced in the considered Hispanic samples (since linkage to potential causal nearby variants may differ) (Figure‐3). The variant also displayed a protective, non‐significant, association with brain amyloid in African ancestry samples (Table‐2). Interestingly, KLHL1, also known as MRP2, is a member of the superfamily of ATP‐binding cassette (ABC) transporters, and has potential functions related to neurite outgrowth and efflux transport at the blood‐brain barrier, supporting its potential relevance to AD pathogenesis.ConclusionsOur results provide new insights into genetic resilience to AD across aging and emphasize the importance of including ancestrally‐diverse populations in genetic studies. Our results also highlight the need for the field to compose ancestrally‐diverse multi‐omics and endophenotype data to support further validation analyses of genetic risk loci prioritized from GWAS.

Genome-wide discovery for diabetes-dependent triglycerides-associated loci.

We aimed to discover loci associated with triglyceride (TG) levels in the context of type 2 diabetes (T2D). We conducted a genome-wide association study (GWAS) in 424,120 genotyped participants of the UK Biobank (UKB) with T2D status and TG levels. We stratified the cohort based on T2D status and conducted association analyses of TG levels for genetic variants with minor allele count (MAC) at least 20 in each stratum. Effect differences of genetic variants by T2D status were determined by Cochran's Q-test and we validated the significantly associated variants in the Mass General Brigham Biobank (MGBB). Among 21,176 T2D and 402,944 non-T2D samples from UKB, stratified GWAS identified 19 and 315 genomic risk loci significantly associated with TG levels, respectively. Only chr6p21.32 exhibited genome-wide significant heterogeneity (I2 = 98.4%; pheterogeneity = 2.1x10-15), with log(TG) effect estimates of -0.066 (95%CI: -0.082, -0.050) and 0.002 (95%CI: -0.002, 0.006) for T2D and non-T2D, respectively. The lead variant rs9274619:A (allele frequency 0.095) is located 2Kb upstream of the HLA-DQB1 gene, between HLA-DQB1 and HLA-DQA2 genes. We replicated this finding among 25,137 participants (6,951 T2D cases) of MGBB (pheterogeneity = 9.5x10-3). Phenome-wide interaction association analyses showed that the lead variant was strongly associated with a concomitant diagnosis of type 1 diabetes (T1D) as well as diabetes-associated complications. In conclusion, we identified an intergenic variant near HLA-DQB1/DQA2 significantly associates with decreased triglycerides only among those with T2D and highlights an immune overlap with T1D.

Insights From a Large-Scale Whole-Genome Sequencing Study of Systolic Blood Pressure, Diastolic Blood Pressure, and Hypertension.

The availability of whole-genome sequencing data in large studies has enabled the assessment of coding and noncoding variants across the allele frequency spectrum for their associations with blood pressure. We conducted a multiancestry whole-genome sequencing analysis of blood pressure among 51 456 Trans-Omics for Precision Medicine and Centers for Common Disease Genomics program participants (stage-1). Stage-2 analyses leveraged array data from UK Biobank (N=383 145), Million Veteran Program (N=318 891), and Reasons for Geographic and Racial Differences in Stroke (N=10 643) participants, along with whole-exome sequencing data from UK Biobank (N=199 631) participants. Two blood pressure signals achieved genome-wide significance in meta-analyses of stage-1 and stage-2 single variant findings (P<5×10-8). Among them, a rare intergenic variant at novel locus, LOC100506274, was associated with lower systolic blood pressure in stage-1 (beta [SE]=-32.6 [6.0]; P=4.99×10-8) but not stage-2 analysis (P=0.11). Furthermore, a novel common variant at the known INSR locus was suggestively associated with diastolic blood pressure in stage-1 (beta [SE]=-0.36 [0.07]; P=4.18×10-7) and attained genome-wide significance in stage-2 (beta [SE]=-0.29 [0.03]; P=7.28×10-23). Nineteen additional signals suggestively associated with blood pressure in meta-analysis of single and aggregate rare variant findings (P<1×10-6 and P<1×10-4, respectively). We report one promising but unconfirmed rare variant for blood pressure and, more importantly, contribute insights for future blood pressure sequencing studies. Our findings suggest promise of aggregate analyses to complement single variant analysis strategies and the need for larger, diverse samples, and family studies to enable robust rare variant identification.

Genetic Contributors of Efficacy and Adverse Metabolic Effects of Chlorthalidone in African Americans from the Genetics of Hypertension Associated Treatments (GenHAT) Study.

Hypertension is a leading risk factor for cardiovascular disease mortality. African Americans (AAs) have the highest prevalence of hypertension in the United States, and to alleviate the burden of hypertension in this population, better control of blood pressure (BP) is needed. Previous studies have shown considerable interpersonal differences in BP response to antihypertensive treatment, suggesting a genetic component. Utilizing data from 4297 AA participants randomized to chlorthalidone from the Genetics of Hypertension Associated Treatments (GenHAT) study, we aimed to identify variants associated with the efficacy of chlorthalidone. An additional aim was to find variants that contributed to changes in fasting glucose (FG) in these individuals. We performed genome-wide association analyses on the change of systolic and diastolic BP (SBP and DBP) over six months and FG levels over 24 months of treatment. We sought replication in the International Consortia of Pharmacogenomics Studies. We identified eight variants statistically associated with BP response and nine variants associated with FG response. One suggestive LINC02211-CDH9 intergenic variant was marginally replicated with the same direction of effect. Given the impact of hypertension in AAs, this study implies that understanding the genetic background for BP control and glucose changes during chlorthalidone treatment may help prevent adverse cardiovascular events in this population.

Genome-wide association study of aromatase inhibitor discontinuation due to musculoskeletal symptoms.

Aromatase inhibitors (AIs) are commonly used to treat hormone receptor positive (HR +) breast cancer. AI-induced musculoskeletal syndrome (AIMSS) is a common toxicity that causes AI treatment discontinuation. The objective of this genome-wide association study (GWAS) was to identify genetic variants associated with discontinuation of AI therapy due to AIMSS and attempt to replicate previously reported associations. In the Exemestane and Letrozole Pharmacogenetics (ELPh) study, postmenopausal patients with HR + non-metastatic breast cancer were randomized to letrozole or exemestane. Genome-wide genotyping of germline DNA was conducted followed by imputation. Each imputed variant was tested for association with time-to-treatment discontinuation due to AIMSS using a Cox proportional hazards model assuming additive genetic effects and adjusting for age, baseline pain score, prior taxane treatment, and AI arm. Secondary analyses were conducted within each AI arm and analyses of candidate variants previously reported to be associated with AIMSS risk. Four hundred ELPh participants were included in the combined analysis. Two variants surpassed the genome-wide significance level in the primary analysis (p value < 5 × 10-8), an intronic variant (rs79048288) within CCDC148 (HR = 4.42, 95% CI: 2.67-7.33) and an intergenic variant (rs912571) upstream of PPP1R14C (HR = 0.30, 95% CI: 0.20-0.47). In the secondary analysis, rs74418677, which is known to be associated with expression of SUPT20H, was significantly associated with discontinuation of letrozole therapy due to AIMSS (HR = 5.91, 95% CI: 3.16-11.06). We were able to replicate associations for candidate variants previously reported to be associated with AIMSS in this cohort, but were not able to replicate associations for any other variants previously reported in other patient cohorts. Our GWAS findings identify several candidate variants that may be associated with AIMSS risk from AI generally or letrozole specifically. Validation of these associations in independent cohorts is needed before translating these findings into clinical practice to improve treatment outcomes in patients with HR + breast cancer.

Associated regions for multiple birth in Brown Swiss and Original Braunvieh cattle on chromosomes 15 and 11.

Twin and multiple births have negative effects on the performance and health of cows and calves. To decipher the genetic architecture of this trait in the two Swiss Brown Swiss cattle populations, we performed various association analyses based on de‐regressed breeding values. Genome‐wide association analyses were executed using ~600 K imputed SNPs for the maternal multiple birth trait in ~3500 Original Braunvieh and ~7800 Brown Swiss animals. Significantly associated QTL were observed on different chromosomes for both breeds. We have identified on chromosome 11 a QTL that explains ~6% of the total genetic variance of the maternal multiple birth trait in Original Braunvieh. For the Brown Swiss breed, we have discovered a QTL on chromosome 15 that accounts for ~4% of the total genetic variance. For Original Braunvieh, subsequent haplotype analysis revealed a 90‐kb window on chromosome 11 at 88 Mb, where a likely regulatory region is located close to the ID2 gene. In Brown Swiss, a 130‐kb window at 75 Mb on chromosome 15 was identified. Analysis of whole‐genome sequence data using linkage‐disequilibrium estimation revealed possible causal variants for the identified QTL. A presumably regulatory variant in the non‐coding 5′ region of the ID2 gene was strongly associated with the haplotype for Original Braunvieh. In Brown Swiss, an intron variant in PRDM11, one 3′ UTR variant in SYT13 and three intergenic variants 5′ upstream of SYT13 were identified as candidate variants for the trait multiple birth maternal. In this study, we report for the first time QTL for the trait of multiple births in Original Braunvieh and Brown Swiss cattle. Moreover, our findings are another step towards a better understanding of the complex genetic architecture of this polygenic trait.

Commentary: The genetics of pseudoexfoliation syndrome/glaucoma.

Genetics forms the basis of all life and evolution. The study of genetics primarily deals with trait inheritance and gene expression. Medical genetics has revolutionized the understanding of health and disease and opened several new prospects in prevention, diagnostics, and therapy. Genetic determinants of several ocular diseases are now well elucidated, with work in progress in many others. Ocular pseudoexfoliation (PXF) results from the accumulation of abnormal fibrillar aggregates in the anterior segment of the eye, blocking the trabecular meshwork and leading to glaucoma. These fibrils consist of elastic fiber components of elastin, fibrillin-1, fibulins, latent TGF-ß binding proteins, and the crosslinking enzyme lysyl oxidase-like 1 (LOXL1), produced by the epithelial cells in the anterior segment (lens epithelial cells, ciliary epithelial cells, and trabecular meshwork cells).[1] The LOXL1 locus on chromosome 15q24.1 has shown the most significant genetic association in PXF glaucoma, similar to the current study’s findings.[23] LOXL1 encodes a member of the lysyl oxidase (LOX) family of enzymes (LOX and LOXL1-4) required for lysine-derived covalent crosslinking in collagen and elastin, contributing to the tensile strength and elasticity of connective tissues. The LOXL1 isoform catalyzes the crosslinking of soluble tropoelastin into insoluble elastin in elastic fibers. Two common nonsynonymous protein-coding variants in exon 1 of the LOXL1 gene, rs1048661 (p.R141L) and rs3825942 (p.G153D), have consistently been associated with PXF in numerous population-based and well-powered genome-wide association studies (GWASs).[3] However, the phenomenon of allele reversal of these two variants in different ethnic populations reflects our ignorance of the exact biological mechanism underlying PXF and the need for further work in elucidating it.[4] Studies in transgenic mice models showed an inverse relationship between LOXL1 expression and intraocular pressure (IOP), with increased IOP in Loxl1-/- mice from elevated episcleral venous pressure.[5] LOXL1 animal models have been unsuccessful in duplicating the PXF phenomenon seen in humans, indicating additional unknown mechanisms. A deep resequencing approach has identified a single common variant (minor allele G of the intergenic sequence variant rs7173049: A>G) downstream of LOXL1 that downregulates two genes affecting vitamin A metabolism in ocular tissues in PXF patients. This allele did not show any reversal in different populations and suggested potential therapeutic targets.[6] The GWAS method identified another polymorphism outside of LOXL1, at 19p13, rs4926244, in the intronic region of CACNA1A, affecting calcium metabolism.[7] Another locus at 13q12 (proteasome maturation protein, POMP) showed increased strength of genetic association with increasing distance from the equator, consistent with the observed prevalence of ocular PXF, indicative of gene-environment interaction.[4] It is interesting to note that several other single nucleotide polymorphisms (SNPs) associated with PXF glaucoma have been identified in different ethnic groups. Multiple genetic loci identified in PXF open the prospect of developing polygenic risk scores (PRS) for PXF in the future. The prospect of gene–environment interactions has spawned several epigenetic investigations with exciting results. These include DNA hypermethylation in the promoter of the LOXL1 gene, downregulation of HSP70 (heat shock protein 70), long non-coding RNAs such as LOXL1 antisense RNA 1 (evidence for regulatory function in immortalized lens epithelial cells), and microRNAs, particularly miR-122-5p (shown to regulate the TGF-b/Smad signaling pathway and lipoprotein metabolism).[3] MicroRNAs are involved in the epigenetic regulation of gene expression and are potential biomarkers of disease identifiable in blood and aqueous humor. Mass spectrometric proteomic analyses of various biological samples have also revealed cellular stress/inflammation, lipid metabolism, coagulation, and dietary factors (altered vitamin D and retinol-binding proteins) contributing to PEX development. Specific metabolomic profiles in plasma and aqueous humor have been identified and validated using machine-learning prediction models.[3] These analyses implicate oxidative stress in the pathophysiology of PXF. However, there is a significant knowledge gap in the exact pathomechanism of the PXF syndrome that requires well-designed and extensive functional investigations. The vast proportion of work done in genetics in PXF comes from the developed world. Studies from India are extremely few, underlining the need to cover a lot of ground, particularly as genetics and epigenetics vary with ethnicity and region. Studies similar to this one and more extensive/multicentric investigations from different parts of India will go a long way in unraveling the specifics of PXF pathobiology among South Asians.[2] The primary challenge in conducting genetic research is the relative scarcity of geneticists/trained researchers in the developing world and funding requirements. Stalwarts in the field have called for equity in genomics.[8] The onus of responsibility lies on clinician-scientists to convince policy-makers and funding agencies for more significant funding in core research in this field.

Insights into Lichen Planus Pigmentosus Inversus using Minimally Invasive Dermal Patch and Whole Transcriptome Analysis.

Lichen Planus Pigmentosus inversus (LPPi) is a rare interface and lichenoid dermatitis (ILD) and supposed variant of lichen planus (LP) that presents as well-demarcated brown to grey macules in flexural and intertriginous areas. LPPi is deemed 'inversus' because its anatomical distribution in skin folds is opposite that seen in lichen planus pigmentosus (LPP) whose pigmented lesions arise on sun-exposed skin. Biopsy is required for the clinical diagnosis of all ILDs. Though multiple clinically-oriented studies have reported differences between LPP, LPPi, and LP, few molecular studies have been performed. In this case study, 3 patients, 2 with LPPi and one with LP, provided samples using minimally invasive whole transcriptome analysis using a dermal biomarker patch. This study confirms the involvement of interferon signaling and T-cell activation in LPPi and suggests an expression profile distinct from LP. Specific genes significantly upregulated in LPPi vs LP include an intergenic splice variant of the primary pigmentation determining receptor in humans and dysregulation of genes essential for ceramide synthesis and construction of the cornified envelope. This work expands upon our knowledge of the pathogenesis of LPPi vs LP, and supports the potential use of this technology in the diagnostic clinical setting to mitigate the need for invasive procedures.

Discovering metabolite quantitative trait loci in asthma using an isolated population

Integration of metabolomics with genetics may advance understanding of disease pathogenesis but has been underused in asthma genetic studies. We sought to discover new genetic effects in asthma and to characterize the molecular consequences of asthma genetic risk through integration with the metabolome in a homogeneous population. From fasting serum samples collected on 348 Tangier Island residents, we quantified 2612 compounds using untargeted metabolomics. Genotyping was performed using Illumina's MEGA array imputed to the TOPMed reference panel. To prioritize metabolites for genome-wide association analysis, we performed a metabolome-wide association study with asthma, selecting asthma-associated metabolites with heritability q value less than 0.01 for genome-wide association analysis. We also tested the association between all metabolites and 8451 candidate asthma single nucleotide polymorphisms previously associated with asthma in the UK Biobank. We followed up significant associations by characterizing shared genetic signal for metabolites and asthma using colocalization analysis. For detailed Methods, please see this article's Online Repository at www.jacionline.org. A total of 60 metabolites were associated with asthma (P< .01), including 40 heritable metabolites tested in genome-wide association analysis. We observed a strong association peak for the endocannabinoid linoleoyl ethanolamide on chromosome 6 in VNN1 (P< 2.7×10-9). We found strong evidence (colocalization posterior probability >75%) for a shared causal variant between 3 metabolites and asthma, including the polyamine acisoga and variants in LPP, and derivative leukotriene B4 and intergenic variants in chr10p14. We identified novel metabolite quantitative trait loci with asthma associations. Identification and characterization of these genetically driven metabolites may provide insight into the functional consequences of genetic risk factors for asthma.

An Intergenic Noncoding Chromosome 18 Variant Suppresses Lethal Thrombosis in Mice By Normalizing Blood Coagulation and Reducing Platelet Reactivity

Background: Factor V Leiden (FVL) is a common thrombosis susceptibility variant in humans. It is incompletely penetrant; this indicates that there are modifiers of FVL that alter thrombosis susceptibility. We used mouse models of FVL (F5L) and heterozygous tissue factor pathway inhibitor deficiency (Tfpi+/-), to identify a perinatal lethal genetic interaction when mice inherited F5L/L Tfpi+/-. This phenotype was used as the basis for a sensitized genome wide ENU mutagenesis screen to identify mutations suppressing lethal thrombosis in F5L/L Tfpi+/- mice. From this screen, we generated multiple independent lines of thrombosuppressed mice, called MF5L, for Modifier of F5L. MF5L16 was a large, highly penetrant (77.2%), multigenerational pedigree containing 136 viable F5L/L Tfpi+/- mice.Aims: In the present study, we aimed to identify and functionally characterize the thrombosuppressor mutation present in MF5L16.Methods: Genomic analyses: We performed whole genome sequencing (WGS) on four MF5L16 F5L/L Tfpi+/- mice. We used comparative bioinformatic analyses to identify variants inherited by all four mice and compiled these variants into candidate variant list. PCR and Sanger sequencing were used to analyze the 136 F5L/L Tfpi+/- mice for inheritance of each of the candidate variants. Functional analyses: We performed biochemical blood coagulation and platelet assays of blood from the Chr18 A mice . Complete blood counts were measured using the Advia 2120 with settings optimized for C57BL/6 mouse blood. Platelet aggregation studies were performed using the Roche Multiplate Aggregometer with ADP and type 1 collagen as the aggregating agents.Results: We analyzed four MF5L16 mice by WGS and identified seven spontaneous mutations that arose in our F5L/L breeding colony that were introduced into MF5L16. Importantly, no coding variants were linked to these variants. Analysis of these seven mutations in all 136 MF5L16 F5L/L Tfpi+/- mice revealed a significant association between a Chromosome 18 intergenic variant (Chr18 G to A, Chr18 A) and F5L/L Tfpi+/- mouse survival (p=0.003). To re-create the suppression of the lethal F5L/L Tfpi+/- phenotype, we bred F5+/L Tfpi+/- Chr18 +/A triple heterozygous mice to F5L/L Chr18 A/A mice to observe the effects of Chr18 A on F5L/L Tfpi+/- mouse survival. Out of 109 mice from this cross, two F5L/L Tfpi+/- Chr18 +/A mice were produced (expected ratio ~1:8). This suggests that the Chr18 A variant suppresses F5L/L Tfpi+/- lethal thrombosis at ~15% penetrance. Complete blood count analysis on Chr18 +/+,Chr18 +/A, and Chr18 A/A mice determined that Chr18 A/A mice had reduced platelet count and distribution width and increased variability in red blood cell (RBC) mean corpuscular volume (n≥4; p<0.05). The Chr18 A/A mice did not display differences in PT or aPTT assays, but had significantly reduced platelet aggregation velocity when stimulated by both ADP and collagen agonists (n≥4; p=0.0002). Additionally, blood smears revealed the presence of poikilocytic RBCs in the Chr18 A/A mice.Conclusions and future directions: Our results establish that a noncoding intergenic Chr18 variant at nucleotide position 62,970,011 (G>A, Chr18 A) contributes to thrombosuppression by reducing platelet reactivity. The observed platelet and RBC phenotypes suggest that a major mechanism of Chr18 A thrombosuppression could be through regulation of gene expression in cells of the myeloid lineage. We are performing additional platelet and blood coagulation analyses to refine the phenotypic differences due to the Chr18 A variant. Comparative transcriptomic analyses are also being performed to identify the genetic pathways involved. Understanding the mechanism in which this intergenic mutation suppresses thrombosis could provide insights into human thrombosis regulation. DisclosuresNo relevant conflicts of interest to declare.

Genome-wide association study of problematic opioid prescription use in 132,113 23andMe research participants of European ancestry

The growing prevalence of opioid use disorder (OUD) constitutes an urgent health crisis. Ample evidence indicates that risk for OUD is heritable. As a surrogate (or proxy) for OUD, we explored the genetic basis of using prescription opioids ‘not as prescribed’. We hypothesized that misuse of opiates might be a heritable risk factor for OUD. To test this hypothesis, we performed a genome-wide association study (GWAS) of problematic opioid use (POU) in 23andMe research participants of European ancestry (N = 132,113; 21% cases). We identified two genome-wide significant loci (rs3791033, an intronic variant of KDM4A; rs640561, an intergenic variant near LRRIQ3). POU showed positive genetic correlations with the two largest available GWAS of OUD and opioid dependence (rg = 0.64, 0.80, respectively). We also identified numerous additional genetic correlations with POU, including alcohol dependence (rg = 0.74), smoking initiation (rg = 0.63), pain relief medication intake (rg = 0.49), major depressive disorder (rg = 0.44), chronic pain (rg = 0.42), insomnia (rg = 0.39), and loneliness (rg = 0.28). Although POU was positively genetically correlated with risk-taking (rg = 0.38), conditioning POU on risk-taking did not substantially alter the magnitude or direction of these genetic correlations, suggesting that POU does not simply reflect a genetic tendency towards risky behavior. Lastly, we performed phenome- and lab-wide association analyses, which uncovered additional phenotypes that were associated with POU, including respiratory failure, insomnia, ischemic heart disease, and metabolic and blood-related biomarkers. We conclude that opioid misuse can be measured in population-based cohorts and provides a cost-effective complementary strategy for understanding the genetic basis of OUD.

Genetic Analyses of Amphotericin B Susceptibility in Aspergillus fumigatus.

Aspergillus fumigatus is a ubiquitous saprophytic mold that can cause a range of clinical syndromes, from allergic reactions to invasive infections. Amphotericin B (AMB) is a polyene antifungal drug that has been used to treat a broad range of systemic mycoses since 1958, including as a primary treatment option against invasive aspergillosis in regions with high rates (≥10%) of environmental triazole resistance. However, cases of AMB-resistant A. fumigatus strains have been increasingly documented over the years, and high resistance rates were recently reported in Brazil and Canada. The objective of this study is to identify candidate mutations associated with AMB susceptibility using a genome-wide association analysis of natural strains, and to further investigate a subset of the mutations in their putative associations with differences in AMB minimum inhibitory concentration (MIC) and in growths at different AMB concentrations through the analysis of progeny from a laboratory genetic cross. Together, our results identified a total of 34 candidate single-nucleotide polymorphisms (SNPs) associated with AMB MIC differences—comprising 18 intergenic variants, 14 missense variants, one synonymous variant, and one non-coding transcript variant. Importantly, progeny from the genetic cross allowed us to identify putative SNP–SNP interactions impacting progeny growth at different AMB concentrations.

A pair of long intergenic non-coding RNA LINC00887 variants act antagonistically to control Carbonic Anhydrase IX transcription upon hypoxia in tongue squamous carcinoma progression

BackgroundLong noncoding RNAs (lncRNAs) are important regulators in tumor progression. However, their biological functions and underlying mechanisms in hypoxia adaptation remain largely unclear.ResultsHere, we established a correlation between a Chr3q29-derived lncRNA gene and tongue squamous carcinoma (TSCC) by genome-wide analyses. Using RACE, we determined that two novel variants of this lncRNA gene are generated in TSCC, namely LINC00887_TSCC_short (887S) and LINC00887_TSCC_long (887L). RNA-sequencing in 887S or 887L loss-of-function cells identified their common downstream target as Carbonic Anhydrase IX (CA9), a gene known to be upregulated by hypoxia during tumor progression. Mechanistically, our results showed that the hypoxia-augmented 887S and constitutively expressed 887L functioned in opposite directions on tumor progression through the common target CA9. Upon normoxia, 887S and 887L interacted. Upon hypoxia, the two variants were separated. Each RNA recognized and bound to their responsive DNA cis-acting elements on CA9 promoter: 887L activated CA9’s transcription through recruiting HIF1α, while 887S suppressed CA9 through DNMT1-mediated DNA methylation.ConclusionsWe provided hypoxia-permitted functions of two antagonistic lncRNA variants to fine control the hypoxia adaptation through CA9.

Cis-regulatory variants affect gene expression dynamics in yeast.

Evolution of cis-regulatory sequences depends on how they affect gene expression and motivates both the identification and prediction of cis-regulatory variants responsible for expression differences within and between species. While much progress has been made in relating cis-regulatory variants to expression levels, the timing of gene activation and repression may also be important to the evolution of cis-regulatory sequences. We investigated allele-specific expression (ASE) dynamics within and between Saccharomyces species during the diauxic shift and found appreciable cis-acting variation in gene expression dynamics. Within-species ASE is associated with intergenic variants, and ASE dynamics are more strongly associated with insertions and deletions than ASE levels. To refine these associations, we used a high-throughput reporter assay to test promoter regions and individual variants. Within the subset of regions that recapitulated endogenous expression, we identified and characterized cis-regulatory variants that affect expression dynamics. Between species, chimeric promoter regions generate novel patterns and indicate constraints on the evolution of gene expression dynamics. We conclude that changes in cis-regulatory sequences can tune gene expression dynamics and that the interplay between expression dynamics and other aspects of expression is relevant to the evolution of cis-regulatory sequences.

Association of Variants Near the Bradykinin Receptor B2 Gene With Angioedema in Patients Taking ACE Inhibitors

BackgroundAngioedema is a rare but potentially life-threatening adverse reaction associated with angiotensin-converting enzyme (ACE) inhibitors. Identification of potential genetic factors related to this adverse event may help identify at-risk patients. ObjectivesThe aim of this study was to identify genetic factors associated with ACE inhibitor–associated angioedema. MethodsA genomewide association study involving patients of European descent, all taking ACE inhibitors, was conducted in a discovery cohort (Copenhagen Hospital Biobank), and associations were confirmed in a replication cohort (Swedegene). Cases were defined as subjects with angioedema events and filled prescriptions for ACE inhibitors ≤180 days before the events. Control subjects were defined as those with continuous treatment with ACE inhibitors without any history of angioedema. Odds ratios (ORs) and 95% confidence intervals (CIs) were computed for angioedema risk using logistic mixed model regression analysis. Summary statistics from the discovery and replication cohorts were analyzed using a fixed-effects meta-analysis model. ResultsThe discovery cohort consisted of 462 cases and 53,391 ACE inhibitor–treated control subjects. The replication cohort consisted of 142 cases and 1,345 ACE inhibitor–treated control subjects. In the discovery cohort, 1 locus, residing at chromosome 14q32.2, was identified that associated with angioedema at the genomewide significance level of P <5 × 10–8. The lead variant at this locus, rs34485356, is an intergenic variant located 60 kb upstream of BDKRB2 (OR: 1.62; 95% CI: 1.38 to 1.90; P = 4.3 × 10−9). This variant was validated in our replication cohort with a similar direction and effect size (OR: 1.60; 95% CI: 1.13 to 2.25; P = 7.2 × 10−3). We found that carriers of the risk allele had significantly lower systolic (−0.46 mm Hg per T allele; 95% CI: −0.83 to −0.10; P = 0.013) and diastolic (−0.26 mm Hg per T allele; 95% CI: −0.46 to −0.05; P = 0.013) blood pressure. ConclusionsIn this genomewide association study involving individuals treated with ACE inhibitors, we found that common variants located in close proximity to the bradykinin receptor B2 gene were associated with increased risk for ACE inhibitor–related angioedema.

Novel genetic variants associated with brain functional networks in 18,445 adults from the UK Biobank

Here, we investigated the genetics of weighted functional brain network graph theory measures from 18,445 participants of the UK Biobank (44–80 years). The eighteen measures studied showed low heritability (mean h2SNP = 0.12) and were highly genetically correlated. One genome-wide significant locus was associated with strength of somatomotor and limbic networks. These intergenic variants were located near the PAX8 gene on chromosome 2. Gene-based analyses identified five significantly associated genes for five of the network measures, which have been implicated in sleep duration, neuronal differentiation/development, cancer, and susceptibility to neurodegenerative diseases. Further analysis found that somatomotor network strength was phenotypically associated with sleep duration and insomnia. Single nucleotide polymorphism (SNP) and gene level associations with functional network measures were identified, which may help uncover novel biological pathways relevant to human brain functional network integrity and related disorders that affect it.

Assessing the Subclonal Genetic Landscape of KMT2A -Rearranged Infant Acute Lymphoblastic Leukemia By Single-Cell Genomics

Acute lymphoblastic leukemia (ALL) arising in infants less than 1 year of age is characterized by genetic rearrangements of the KMT2A gene (previously MLL) and an exceedingly poor prognosis. We have previously shown that infant KMT2A -rearranged (KMT2A -R) ALL has one of the lowest numbers of somatic mutations of any sequenced cancer with a mean of only 1.3 non-silent mutations being present in all leukemia cells per patient (Andersson et al., Nat Genet, 2017). Despite the paucity of mutations, activating mutations within the PI3K/RAS signaling pathway were present in about half of cases, most of which were subclonal with a mutant allele frequency (MAF) <0.30. In addition, some patients harbored multiple activating mutations at varying MAFs, suggesting the presence of multiple low-frequency leukemia clones. Based on these findings, we hypothesized that the genetic landscape of infant KMT2A -R ALL is more clonally heterogeneous than other pediatric leukemias, which might contribute to its poor prognosis as current treatment regimens may not eliminate all clones.To test our hypothesis, high coverage bulk and single-cell genomics were used to study the mutation profiles of subclones in diagnostic samples from four infants younger than 6 months of age with KMT2A -R ALL. This included patients with t(4;11)(q21;q23) KMT2A-AFF1 (n=3) of which two relapsed and a patient with t(11;19)(q23;p13.3) KMT2A - MLLT1 (n=1) that remains in remission. DNA was isolated from bulk leukemia cells and high-depth paired-end whole-genome sequencing was performed on paired diagnostic and remission samples at an average haploid coverage of 160x and 35x, respectively. In addition, whole-exome sequencing was performed on the diagnostic samples at an average exon coverage of 375x. Somatic alterations, including single-nucleotide variations (SNVs), insertions-deletions (indels), structural variations (SVs), and copy number alterations, were detected using multiple analytical pipelines. In parallel, approximately 75 viable single cells from each diagnostic sample were isolated and the DNA from the cells underwent whole genome amplification using the Fluidigm C1 system.Analyses of the whole-genome sequencing data from the four infant cases revealed an average of 715 putative sequence variants (range of 54-1199) per case across the genome. Using the combined data from whole-genome- and exome sequencing, an average of 55 (range 51-65) non-silent mutations affecting coding genes or non-coding RNAs were identified, with 83% having support by both sequencing methods, validating their presence. An average of 63% and 92% of the non-silent mutations detected by whole-genome and exome sequencing, respectively, were subclonal, demonstrating the enhanced ability to detect low frequency variants with higher sequencing depth.Subclonal mutations in genes within the PI3K/RAS signaling pathway were detected in 2/4 cases with each patient harboring both a KRASG12D and a FLT3V491L as well as an additional NRASG13D for one of the cases. The recurrent FLT3V491L mutation is present in COSMIC and has been previously detected in leukemia, but to the best of our knowledge, it has not been experimentally verified to be activating.We are now performing high-throughput amplicon-based sequencing at the single cells from those patients using 150 targets/cell to determine the number of distinct clones into which the bulk mutations segregate. The targets include a mixture of coding, intergenic variants and SVs. In addition, we are determining if each cell contains a mutation at a selected number of loci that are leukemia-associated mutational hotspots. After acquiring the data, we will reconstruct the clonal architecture of the samples, enabling us to assess the clonal heterogeneity and mutational histories of the leukemias.Taken together, these data will provide a unique look into the underlying biology of infant KMT2A -R ALL by providing new details about the population genetic diversity and temporal genetic changes that occur during KMT2A -R infant leukemogenesis. These data may also provide insight into the universally poor prognosis of patients with KMT2A -R infant ALL. DisclosuresNo relevant conflicts of interest to declare.

Novel prostate cancer susceptibility gene SP6 predisposes patients to aggressive disease

Prostate cancer (PrCa) is one of the most common cancers in men, but little is known about factors affecting its clinical outcomes. Genome-wide association studies have identified more than 170 germline susceptibility loci, but most of them are not associated with aggressive disease. We performed a genome-wide analysis of 185,478 SNPs in Finnish samples (2738 cases, 2400 controls) from the international Collaborative Oncological Gene-Environment Study (iCOGS) to find underlying PrCa risk variants. We identified a total of 21 common, low-penetrance susceptibility loci, including 10 novel variants independently associated with PrCa risk. Novel risk loci were located in the 8q24 (CASC8 rs16902147, OR 1.86, padj = 3.53 × 10−8 and rs58809953, OR 1.71, padj = 4.00 × 10−6; intergenic rs79012498, OR 1.81, padj = 4.26 × 10−8), 17q21 (SP6 rs2074187, OR 1.66, padj = 3.75 × 10−5), 11q13 (rs12795301, OR 1.42, padj = 2.89 × 10−5) and 8p21 (rs995432, OR 1.38, padj = 3.00 × 10−11) regions. Here, we describe SP6, a transcription factor gene, as a new, potentially high-risk gene for PrCa. The intronic variant rs2074187 in SP6 was associated not only with overall susceptibility to PrCa (OR 1.66) but also with a higher odds ratio for aggressive PrCa (OR 1.89) and lower odds for non-aggressive PrCa (OR 1.43). Furthermore, the new intergenic variant rs79012498 at 8q24 conferred risk for aggressive PrCa. Our findings highlighted the power of a population-stratified approach to identify novel, clinically actionable germline PrCa risk loci and strongly suggested SP6 as a new PrCa candidate gene that may be involved in the pathogenesis of PrCa.