A genome-wide association study identifies new loci for factor VII and implicates factor VII in ischemic stroke etiology.

Factor VII (FVII) is an important component of the coagulation cascade. Few genetic loci regulating FVII activity and/or levels have been discovered to date. We conducted a meta-analysis of 9 genome-wide association studies of plasma FVII levels (7 FVII activity and 2 FVII antigen) among 27 495 participants of European and African ancestry. Each study performed ancestry-specific association analyses. Inverse variance weighted meta-analysis was performed within each ancestry group and then combined for a trans-ancestry meta-analysis. Our primary analysis included the 7 studies that measured FVII activity, and a secondary analysis included all 9 studies. We provided functional genomic validation for newly identified significant loci by silencing candidate genes in a human liver cell line (HuH7) using small-interfering RNA and then measuring F7 messenger RNA and FVII protein expression. Lastly, we used meta-analysis results to perform Mendelian randomization analysis to estimate the causal effect of FVII activity on coronary artery disease, ischemic stroke (IS), and venous thromboembolism. We identified 2 novel (REEP3 and JAZF1-AS1) and 6 known loci associated with FVII activity, explaining 19.0% of the phenotypic variance. Adding FVII antigen data to the meta-analysis did not result in the discovery of further loci. Silencing REEP3 in HuH7 cells upregulated FVII, whereas silencing JAZF1 downregulated FVII. Mendelian randomization analyses suggest that FVII activity has a positive causal effect on the risk of IS. Variants at REEP3 and JAZF1 contribute to FVII activity by regulating F7 expression levels. FVII activity appears to contribute to the etiology of IS in the general population.

Genetic susceptibility to radiation-induced breast cancer after Hodgkin lymphoma.

Female Hodgkin lymphoma (HL) patients treated with chest radiotherapy (RT) have a very high risk of breast cancer. The contribution of genetic factors to this risk is unclear. We therefore examined 211 155 germline single-nucleotide polymorphisms (SNPs) for gene-radiation interaction on breast cancer risk in a case-only analysis including 327 breast cancer patients after chest RT for HL and 4671 first primary breast cancer patients. Nine SNPs showed statistically significant interaction with RT on breast cancer risk (false discovery rate, <20%), of which 1 SNP in the PVT1 oncogene attained the Bonferroni threshold for statistical significance. A polygenic risk score (PRS) composed of these SNPs (RT-interaction-PRS) and a previously published breast cancer PRS (BC-PRS) derived in the general population were evaluated in a case-control analysis comprising the 327 chest-irradiated HL patients with breast cancer and 491 chest-irradiated HL patients without breast cancer. Patients in the highest tertile of the RT-interaction-PRS had a 1.6-fold higher breast cancer risk than those in the lowest tertile. Remarkably, we observed a fourfold increased RT-induced breast cancer risk in the highest compared with the lowest decile of the BC-PRS. On a continuous scale, breast cancer risk increased 1.4-fold per standard deviation of the BC-PRS, similar to the effect size found in the general population. This study demonstrates that genetic factors influence breast cancer risk after chest RT for HL. Given the high absolute breast cancer risk in radiation-exposed women, these results can have important implications for the management of current HL survivors and future patients.

Genome-Wide Association Transethnic Meta-Analyses Identifies Novel Associations Regulating Coagulation Factor VIII and von Willebrand Factor Plasma Levels.

BACKGROUND: Factor VIII (FVIII) and its carrier protein von Willebrand factor (VWF) are associated with risk of arterial and venous thrombosis and with hemorrhagic disorders. We aimed to identify and functionally test novel genetic associations regulating plasma FVIII and VWF. METHODS: We meta-analyzed genome-wide association results from 46 354 individuals of European, African, East Asian, and Hispanic ancestry. All studies performed linear regression analysis using an additive genetic model and associated ≈35 million imputed variants with natural log-transformed phenotype levels. In vitro gene silencing in cultured endothelial cells was performed for candidate genes to provide additional evidence on association and function. Two-sample Mendelian randomization analyses were applied to test the causal role of FVIII and VWF plasma levels on the risk of arterial and venous thrombotic events. RESULTS: We identified 13 novel genome-wide significant ( P≤2.5×10-8) associations, 7 with FVIII levels ( FCHO2/TMEM171/TNPO1, HLA, SOX17/RP1, LINC00583/NFIB, RAB5C-KAT2A, RPL3/TAB1/SYNGR1, and ARSA) and 11 with VWF levels ( PDHB/PXK/KCTD6, SLC39A8, FCHO2/TMEM171/TNPO1, HLA, GIMAP7/GIMAP4, OR13C5/NIPSNAP, DAB2IP, C2CD4B, RAB5C-KAT2A, TAB1/SYNGR1, and ARSA), beyond 10 previously reported associations with these phenotypes. Functional validation provided further evidence of association for all loci on VWF except ARSA and DAB2IP. Mendelian randomization suggested causal effects of plasma FVIII activity levels on venous thrombosis and coronary artery disease risk and plasma VWF levels on ischemic stroke risk. CONCLUSIONS: The meta-analysis identified 13 novel genetic loci regulating FVIII and VWF plasma levels, 10 of which we validated functionally. We provide some evidence for a causal role of these proteins in thrombotic events.

Genome-wide association study with additional genetic and post-transcriptional analyses reveals novel regulators of plasma factor XI levels.

Coagulation factor XI (FXI) has become increasingly interesting for its role in pathogenesis of thrombosis. While elevated plasma levels of FXI have been associated with venous thromboembolism and ischemic stroke, its deficiency is associated with mild bleeding. We aimed to determine novel genetic and post-transcriptional plasma FXI regulators.We performed a genome-wide association study (GWAS) for plasma FXI levels, using novel data imputed to the 1000 Genomes reference panel. Individual GWAS analyses, including a total of 16,169 European individuals from the ARIC, GHS, MARTHA and PROCARDIS studies, were meta-analysed and further replicated in 2,045 individuals from the F5L family, GAIT2 and MEGA studies. Additional association with activated partial thromboplastin time (aPTT) was tested for the top SNPs. In addition, a study on the effect of miRNA on FXI regulation was performed using in silico prediction tools and in vitro luciferase assays.Three loci showed robust, replicating association with circulating FXI levels: KNG1 (rs710446, P-value = 2.07 × 10-302), F11 (rs4253417, P-value = 2.86 × 10-193), and a novel association in GCKR (rs780094, P-value = 3.56 ×10-09), here for the first time implicated in FXI regulation. The two first SNPs (rs710446 and rs4253417) also associated with aPTT. Conditional and haplotype analyses demonstrated a complex association signal, with additional novel SNPs modulating plasma FXI levels in both the F11 and KNG1 loci. Finally, eight miRNAs were predicted to bind F11 mRNA. Over-expression of either miR-145 or miR-181 significantly reduced the luciferase activity in cells transfected with a plasmid containing FXI-3'UTR.These results should open the door to new therapeutic targets for thrombosis prevention.

Discovery of novel heart rate-associated loci using the Exome Chip.

Resting heart rate is a heritable trait, and an increase in heart rate is associated with increased mortality risk. Genome-wide association study analyses have found loci associated with resting heart rate, at the time of our study these loci explained 0.9% of the variation. This study aims to discover new genetic loci associated with heart rate from Exome Chip meta-analyses.Heart rate was measured from either elecrtrocardiograms or pulse recordings. We meta-analysed heart rate association results from 104 452 European-ancestry individuals from 30 cohorts, genotyped using the Exome Chip. Twenty-four variants were selected for follow-up in an independent dataset (UK Biobank, N = 134 251). Conditional and gene-based testing was undertaken, and variants were investigated with bioinformatics methods.We discovered five novel heart rate loci, and one new independent low-frequency non-synonymous variant in an established heart rate locus (KIAA1755). Lead variants in four of the novel loci are non-synonymous variants in the genes C10orf71, DALDR3, TESK2 and SEC31B. The variant at SEC31B is significantly associated with SEC31B expression in heart and tibial nerve tissue. Further candidate genes were detected from long-range regulatory chromatin interactions in heart tissue (SCD, SLF2 and MAPK8). We observed significant enrichment in DNase I hypersensitive sites in fetal heart and lung. Moreover, enrichment was seen for the first time in human neuronal progenitor cells (derived from embryonic stem cells) and fetal muscle samples by including our novel variants.Our findings advance the knowledge of the genetic architecture of heart rate, and indicate new candidate genes for follow-up functional studies.

Correction to: Assessing the causal relationship between obesity and venous thromboembolism through a Mendelian Randomization study.

The co-author name Immaculata DeVivo was incorrectly published. The correct name is given below.

Meta-analysis of 65,734 individuals identifies TSPAN15 and SLC44A2 as two susceptibility loci for venous thromboembolism.

Venous thromboembolism (VTE), the third leading cause of cardiovascular mortality, is a complex thrombotic disorder with environmental and genetic determinants. Although several genetic variants have been found associated with VTE, they explain a minor proportion of VTE risk in cases. We undertook a meta-analysis of genome-wide association studies (GWASs) to identify additional VTE susceptibility genes. Twelve GWASs totaling 7,507 VTE case subjects and 52,632 control subjects formed our discovery stage where 6,751,884 SNPs were tested for association with VTE. Nine loci reached the genome-wide significance level of 5 × 10(-8) including six already known to associate with VTE (ABO, F2, F5, F11, FGG, and PROCR) and three unsuspected loci. SNPs mapping to these latter were selected for replication in three independent case-control studies totaling 3,009 VTE-affected individuals and 2,586 control subjects. This strategy led to the identification and replication of two VTE-associated loci, TSPAN15 and SLC44A2, with lead risk alleles associated with odds ratio for disease of 1.31 (p = 1.67 × 10(-16)) and 1.21 (p = 2.75 × 10(-15)), respectively. The lead SNP at the TSPAN15 locus is the intronic rs78707713 and the lead SLC44A2 SNP is the non-synonymous rs2288904 previously shown to associate with transfusion-related acute lung injury. We further showed that these two variants did not associate with known hemostatic plasma markers. TSPAN15 and SLC44A2 do not belong to conventional pathways for thrombosis and have not been associated to other cardiovascular diseases nor related quantitative biomarkers. Our findings uncovered unexpected actors of VTE etiology and pave the way for novel mechanistic concepts of VTE pathophysiology.

Assessing the causal relationship between obesity and venous thromboembolism through a Mendelian Randomization study.

Observational studies have shown an association between obesity and venous thromboembolism (VTE) but it is not known if observed associations are causal, due to reverse causation or confounding bias. We conducted a Mendelian Randomization study of body mass index (BMI) and VTE. We identified 95 single nucleotide polymorphisms (SNPs) that have been previously associated with BMI and assessed the association between genetically predicted high BMI and VTE leveraging data from a previously conducted GWAS within the INVENT consortium comprising a total of 7507 VTE cases and 52,632 controls of European ancestry. Five BMI SNPs were associated with VTE at P < 0.05, with the strongest association seen for the FTO SNP rs1558902 (OR 1.07, 95% CI 1.02-1.12, P = 0.005). In addition, we observed a significant association between genetically predicted BMI and VTE (OR = 1.59, 95% CI 1.30-1.93 per standard deviation increase in BMI, P = 5.8 × 10-6). Our study provides evidence for a causal relationship between high BMI and risk of VTE. Reducing obesity levels will likely result in lower incidence in VTE.

Identification of coagulation gene 3'UTR variants that are potentially regulated by microRNAs.

MicroRNAs have been recognized as critical regulators of gene expression and might affect the risk of venous thrombosis. We aimed to identify 3' untranslated region (UTR) variants in coagulation genes that influence coagulation factor levels and venous thrombosis risk. The 3'UTR of coagulation genes were sequenced in subjects with extremely high or low plasma levels of these factors in two case-control studies. In total, 28 variants were identified. Five single nucleotide polymorphisms (SNPs) were predominantly present in one extreme level group (F2 rs1799963, F8 rs1050705 and F11 rs4253429, rs4253430 and rs1062547). Additional to F2 rs1799963, F8 rs1050705 (in men) and F11 rs4253430 were associated with an increased risk of venous thrombosis albeit confidence intervals were wide. The three F11 SNPs were in high linkage disequilibrium with functional variants rs2289252 and rs2036914. Rs1062547 and rs4253430 were associated with a significant increase of plasma FXI activity in heterozygotes and homozygotes in wild-type controls. In silico prediction revealed that these SNPs might disturb the binding sites of miR-544 and miR-513a-3p. Only miR-544 provoked a significant decrease of the luciferase activity that was not observed with a rs4253430 mutated vector. In conclusion, these results reinforce that microRNAs are candidates to play a role in haemostasis and complex disorders, such as thrombosis.

Multiple SNP testing improves risk prediction of first venous thrombosis.

There are no risk models available yet that accurately predict a person's risk for developing venous thrombosis. Our aim was therefore to explore whether inclusion of established thrombosis-associated single nucleotide polymorphisms (SNPs) in a venous thrombosis risk model improves the risk prediction. We calculated genetic risk scores by counting risk-increasing alleles from 31 venous thrombosis-associated SNPs for subjects of a large case-control study, including 2712 patients and 4634 controls (Multiple Environmental and Genetic Assessment). Genetic risk scores based on all 31 SNPs or on the 5 most strongly associated SNPs performed similarly (areas under receiver-operating characteristic curves [AUCs] of 0.70 and 0.69, respectively). For the 5-SNP risk score, the odds ratios for venous thrombosis ranged from 0.37 (95% confidence interval [CI], 0.25-0.53) for persons with 0 risk alleles to 7.48 (95% CI, 4.49-12.46) for persons with more than or equal to 6 risk alleles. The AUC of a risk model based on known nongenetic risk factors was 0.77 (95% CI, 0.76-0.78). Combining the nongenetic and genetic risk models improved the AUC to 0.82 (95% CI, 0.81-0.83), indicating good diagnostic accuracy. To become clinically useful, subgroups of high-risk persons must be identified in whom genetic profiling will also be cost-effective.

Mutations in NSUN3, a Mitochondrial Methyl Transferase Gene, Cause Inherited Optic Neuropathy.

Inherited optic neuropathies (IONs) are rare genetic diseases characterized by progressive visual loss due the atrophy of optic nerves. The standard diagnostic workup involving next-generation sequencing panels has a diagnostic yield of about forty percent. In the other 60% of the patients with a clinical diagnosis of ION, the underlying genetic variants remain unknown. In this case study, we describe a potentially new disease-associated gene, NSUN3, for IONs. The proband was a young woman with consanguineous parents. She presented with bilateral optic atrophy and nystagmus at the age of seven years. Genetic testing revealed the homozygous variant c.349_352dup p.(Ala118Glufs*45) in NSUN3, with a segregation in the family compatible with autosomal recessive inheritance. Additional functional analysis showed decreased NSUN3 mRNA levels, slightly diminished mitochondrial complex IV levels, and decreased cell respiration rates in patient fibroblasts compared to healthy controls. In conclusion, pathogenic variants in NSUN3 can cause optic neuropathy. Trio whole-exome sequencing should be considered as a diagnostic strategy in ION cases where standard diagnostic analysis does not reveal disease-causing variants.

Associations of autozygosity with a broad range of human phenotypes.

In many species, the offspring of related parents suffer reduced reproductive success, a phenomenon known as inbreeding depression. In humans, the importance of this effect has remained unclear, partly because reproduction between close relatives is both rare and frequently associated with confounding social factors. Here, using genomic inbreeding coefficients (FROH) for >1.4 million individuals, we show that FROH is significantly associated (p < 0.0005) with apparently deleterious changes in 32 out of 100 traits analysed. These changes are associated with runs of homozygosity (ROH), but not with common variant homozygosity, suggesting that genetic variants associated with inbreeding depression are predominantly rare. The effect on fertility is striking: FROH equivalent to the offspring of first cousins is associated with a 55% decrease [95% CI 44-66%] in the odds of having children. Finally, the effects of FROH are confirmed within full-sibling pairs, where the variation in FROH is independent of all environmental confounding.

Next-generation DNA sequencing to identify novel genetic risk factors for cerebral vein thrombosis.

BACKGROUND: Cerebral vein thrombosis (CVT) is a rare, life-threatening disease affecting one adult per 100,000 per year. Genetic risk factors are deficiencies of the natural anticoagulant proteins antithrombin, protein C, protein S or single nucleotide polymorphisms such as factor V Leiden and prothrombin 20210A. In 20% of patients, the cause of CVT remains unknown. AIM: To identify novel genetic risk factors for CVT using targeted next-generation DNA sequencing (NGS). METHODS: We investigated 171 CVT patients and 298 healthy controls. Patients were selected using the following criteria: objective diagnosis of CVT, no active cancer. We performed targeted NGS analysis of the protein-coding regions of 734 candidate genes related to hemostasis and inflammation, 150 ancestry informative markers and 28 thrombosis-associated variants. RESULTS: We identified 3723 common and low frequency variants with minor allele frequency (MAF) >1% in 590 genes. Single variant association testing using logistic regression analysis identified rs8176719 insertion/deletion (indel) variant in the ABO gene associated with CVT (age and sex adjusted OR 2.03; 95% CI 1.52-2.73; P = 2.07 × 10-6; Bonferroni P = 0.008). In addition, we identified 8839 rare variants (MAF ≤ 1%) in 723 genes. Gene-based association analysis of these rare variants using a burden test revealed only a tentative association of non-coding variants located in the F8 locus with CVT. CONCLUSION: Targeted NGS identified a common indel variant rs8176719 in the ABO gene. Gene-based tests of association failed to reveal genomic loci with a cumulative burden of rare variants associated with CVT.

Next-Generation Sequencing and In Vitro Expression Study of ADAMTS13 Single Nucleotide Variants in Deep Vein Thrombosis.

BACKGROUND: Deep vein thrombosis (DVT) genetic predisposition is partially known. OBJECTIVES: This study aimed at assessing the functional impact of nine ADAMTS13 single nucleotide variants (SNVs) previously reported to be associated as a group with DVT in a burden test and the individual association of selected variants with DVT risk in two replication studies. METHODS: Wild-type and mutant recombinant ADAMTS13 were transiently expressed in HEK293 cells. Antigen and activity of recombinant ADAMTS13 were measured by ELISA and FRETS-VWF73 assays, respectively. The replication studies were performed in an Italian case-control study (Milan study; 298/298 patients/controls) using a next-generation sequencing approach and in a Dutch case-control study (MEGA study; 4306/4887 patients/controls) by TaqMan assays. RESULTS: In vitro results showed reduced ADAMTS13 activity for three SNVs (p.Val154Ile [15%; 95% confidence interval [CI] 14-16], p.Asp187His [19%; 95%[CI] 17-21], p.Arg421Cys [24%; 95%[CI] 22-26]) similar to reduced plasma ADAMTS13 levels of patients carriers for these SNVs. Therefore these three SNVs were interrogated for risk association. The first replication study identified 3 heterozygous carriers (2 cases, 1 control) of p.Arg421Cys (odds ratio [OR] 2, 95%[CI] 0.18-22.25). The second replication study identified 2 heterozygous carriers (1 case, 1 control) of p.Asp187His ([OR] 1.14, 95%[CI] 0.07-18.15) and 10 heterozygous carriers (4 cases, 6 controls) of p.Arg421Cys ([OR] 0.76, 95%[CI] 0.21-2.68). CONCLUSIONS: Three SNVs (p.Val154Ile, p.Asp187His and p.Arg421Cys) showed reduced ex vivo and in vitro ADAMTS13 levels. However, the low frequency of these variants makes it difficult to confirm their association with DVT.

Single Nucleotide Variant rs2232710 in the Protein Z-Dependent Protease Inhibitor (ZPI, SERPINA10) Gene Is Not Associated with Deep Vein Thrombosis.

Rare mutations in PROC, PROS1 or SERPINC1 as well as common variants in F5, F2, F11 and SERPINC1 have been identified as risk factors for deep vein thrombosis (DVT). To identify novel genetic risk factors for DVT, we have developed and applied next-generation DNA sequencing (NGS) of the coding area of hemostatic and proinflammatory genes. Using this strategy, we previously identified a single nucleotide variant (SNV) rs6050 in the FGA gene and novel, rare SNVs in the ADAMTS13 gene associated with DVT. To identify novel coding variants in the genetic predisposition to DVT, we applied NGS analysis of the coding area of 186 hemostatic and proinflammatory genes in 94 DVT cases and 98 controls and we identified 18 variants with putative role in DVT. A group of 585 Italian idiopathic DVT patients and 550 healthy controls was used to genotype all the 18 risk-associated variants identified by NGS. Replication study in the Italian population identified the rs2232710 variant in the protein Z-dependent protease inhibitor (ZPI) gene to be associated with an increased risk of DVT (OR 2.74; 95% CI 1.33-5.65; P = 0.0045; Bonferroni P = 0.081). However, the rs2232710 SNV showed no association with DVT in two Dutch replication cohorts the LETS study (454 patients and 451 controls) and the MEGA study (3799 patients and 4399 controls), indicating that the rs2232710 variant is not a risk factor for DVT.

[Mendelian randomisation]. / Mendeliaanse randomisatie.

Classical observational studies into the causal relationship between a risk factor and a disease sometimes result in contradictory and spurious findings. This is due to confounding factors. It is not possible to conclude from the results of classical observational studies whether a specific risk factor may be a suitable target for future treatments. A solution is to conduct a Mendelian randomization analysis, which uses genetic variation as a surrogate marker for the risk factor. Mendelian randomisation is based on the idea that characteristics and environmental factors are proportionately divided into carriers and non-carriers of various genetic variants. Mendelian randomisation can be used only if there is a robust relationship between the genetic variant and the risk factor, if the genetic variant is not associated with other factors that confound the relationship between the risk factor and the disease, and if the genetic variant has an effect on the disease only via the risk factor, i.e. not via other biological mechanisms.

Genetic variants in Cell Adhesion Molecule 1 (CADM1): a validation study of a novel endothelial cell venous thrombosis risk factor.

INTRODUCTION: In a protein C deficient family, we recently identified a candidate gene, CADM1, which interacted with protein C deficiency in increasing the risk of venous thrombosis (VT). This study aimed to determine whether CADM1 variants also interact with protein C pathway abnormalities in increasing VT risk outside this family. MATERIALS AND METHODS: We genotyped over 300 CADM1 variants in the population-based MEGA case-control study. We compared VT risks between cases with low protein C activity (n=194), low protein S levels (n=23), high factor VIII activity (n=165) or factor V Leiden carriers (n=580), and all 4004 controls. Positive associations were repeated in all 3496 cases and 4004 controls. RESULTS: We found 22 variants which were associated with VT in one of the protein C pathway risk groups. After mutual adjustment, six variants remained associated with VT. The strongest evidence was found for rs220842 and rs11608105. For rs220842, the odds ratio (OR) for VT was 3.2 (95% CI 1.2-9.0) for cases with high factor VIII activity compared with controls. In addition, this variant was associated with an increased risk of VT in the overall study population (OR: 1.5, 95% CI 1.0-2.2). The other variant, rs11608105, was not associated with VT in the overall study population (OR: 1.0, 95% CI 0.8-1.1), but showed a strong effect on VT risk (OR: 21, 95% CI 5.1-88) when combined with low protein C or S levels. CONCLUSIONS: In a population-based association study, we confirm a role for CADM1 variants in increasing the risk of VT by interaction with protein C pathway abnormalities.