Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) and depression frequently occur together. Identifying the genes that influence both MASLD and depression may facilitate the discovery of biological pathways associated with disease risk. Methods: We recruited 525 participants from Mexican American families living in the Rio Grande Valley of south Texas. We collected clinical data, biometric measurements, hepatic health assessments using Vibration-Controlled Transient Elastography (VCTE), and depression evaluations determined with the Beck Depression Inventory-II. We estimated the heritability (h2) of MASLD-related measures, depression status, aspartate aminotransferase (AST), alanine aminotransferase (ALT), the AST/ALT ratio, and Vibration-Controlled Transient Elastography measurements. For each gene, we derived a genetic endophenotype representing its expression level. We then performed functional network and gene ontology enrichment analyses to characterize the underlying protein pathways. Results: We observed significant associations between the expression of two genes, Thyroid Hormone Receptor-Associated Protein 3 (THRAP3) (h2 = 0.56 [0.45, 0.67]) and ADAM Metallopeptidase with Thrombospondin Type 1 Motif 7 (ADAMTS7) (h2 = 0.66 [0.55, 0.77]), with depression and multiple MASLD-related phenotypes. We identified 351 genes with expression levels significantly correlated with one or more MASLD phenotypes and depression. Among these, five genes-ADAMTS7, THRAP3, CHPM4A, RAB9A, and PDIA3-were jointly associated with three phenotypes: AST/ALT, ALT, and Controlled Attenuation Parameter (CAP kPa). Based on the Fisher Combined Test, only THRAP3 (p = 3.0 × 10-2) and ADAMTS7 (p = 2 × 10-2) were jointly significant for depression (BDI-II) and AST, ALT, AST/ALT ratio, FAST, and CAP (kPa). We present a protein-protein interaction network comprising nodes (proteins) and edges (interactions), and a gene ontology enrichment analysis of cellular components. Discussion: Our findings highlight pleiotropic genes underlying MASLD and depression. Two genes, ADAMTS7 and THRAP3, warrant further investigation as potential targets for therapeutic interventions to manage MASLD and depression among Mexican Americans. These results may improve our understanding of the pathways involved in these two diseases, advance current research, and contribute to improvements in personalized medicine. Conclusion: We identified possible shared gene expression phenotypes linking MASLD and depression, which may provide insight into a common molecular underpinning. Pathway enrichment and gene analysis were used to help refine networks and enhance our understanding of complex gene-environmental interactions and their implications for precision medicine.

BACKGROUND:von Willebrand disease (VWD) is a common inherited bleeding disorder caused by low levels or activity of circulating von Willebrand factor (VWF). Genetic susceptibility to VWF antigen (VWF:Ag) below normal (≤ 50 IU/dL) in the general population is underexplored. OBJECTIVES:To identify genetic variants influencing VWF:Ag levels ≤ 50 IU/dL. METHODS:We performed a genome-wide association study in 926 cases with VWF:Ag levels ≤ 50 IU/dL and 12 846 controls from 7 studies from the Trans-Omics for Precision Medicine program. We then examined whether significant genome-wide findings were also associated with clinical diagnosis of VWD in 5 biobanks with 708 VWD cases and 1 286 069 controls, and with 6 bleeding and thrombotic disorders in FinnGen. RESULTS:Variants at 2 loci were associated (P < 5 × 10-9) with VWF:Ag levels ≤ 50 IU/dL: ABO and VWF. The VWF index variant, p.Tyr1584Cys, is a rare (0.22%) missense variant with odds ratio (OR) of 78.58, while the ABO index variant is a common intronic variant with a smaller effect (OR = 2.52). Notably, both VWF (OR = 7.16) and ABO (OR = 1.57) variants were also associated (P < .025) with diagnosed VWD. Among p.Tyr1584Cys heterozygotes, the penetrance of VWF:Ag levels ≤ 50 IU/dL was 24.2% and the penetrance of diagnosed VWD was 0.3%. p.Tyr1584Cys was associated (P < .0042) with increased odds of heavy menstrual bleeding (OR = 1.27), iron deficiency anemia (OR = 1.55), and intrapartum hemorrhage (OR = 2.20), but decreased odds of deep vein thrombosis (OR = 0.54). CONCLUSIONS:Although there are currently conflicting interpretations of pathogenicity p.Tyr1584Cys, our results suggest that it is a low penetrance pathogenic variant that contributes to VWF:Ag levels ≤ 50 IU/dL, bleeding, and VWD.

Introduction:Hemophilia A (HA) patients (HAPs) with the human leukocyte antigen (HLA)-class-II (HLAII) haplotype DRB1*15:01/DQB1*06:02, and thus antigen presenting cells which express HLAII β-polypeptide chains that form heterodimers of DR15- and DQ6-serotypes, respectively, have an increased risk of developing factor (F)VIII inhibitors (FEIs)-neutralizing antibodies against the therapeutic-FVIII-proteins (tFVIIIs) infused to prevent/arrest bleeding. As DRB1*15:01 and DQB1*06:02 exist in strong linkage disequilibrium, association analysis cannot determine which is the actual risk allele. Methods:To establish the true risk allele of this haplotype, we analyzed the tFVIII-derived peptides (tFVIII-dPs) bound to either the DR or DQ molecules that comprise the individual HLAII repertoires expressed by monocyte-derived dendritic cells obtained from 25 normal blood donors and six HAPs, four without and two with FEIs. We performed log-linear mixed model analyses, where the dependent variable is the log of the measured peptide count. Under Model 1, we analyzed an HLAII allele predictor consisting of ten levels (four DRB1 and six DQB1 alleles) in the fixed effects and variables in the random effects to account for non-independence. Model 2-where the HLAII allele variable consisted of only DRB1*15:01 and DQB1*06:02-compares the HLAII alleles. Results:Relative to the Model 1 reference, DRB1*15:01 and DQB1*06:02 significantly increased tFVIII-derived peptide counts, and DRB1*15:01 contributed significantly more than DQB1*06:02. Reported as risk ratios (RRs) and their 95% confidence interval (CI) lower- (LB) and upper-bound (UB), we found a RR (95% CI-LB, -UB) of 14.16 (10.38, 19.33) and 1.76 (1.24, 2.50) for DRB1*15:01 and DQB1*06:02, respectively. Under Model 2, we found an RR for DRB1*15:01 against DQB1*06:02 of 7.00 (5.80, 8.44). Discussion/conclusion:Our results suggest that DRB1*15:01 is the offending HLAII allele and that DR15 allotypes underlie the increased FEI risk in HAPs.

Abstract Hemophilia-A (HA) is caused by heterogeneous loss-of-function factor (F)VIII gene (F8)-mutations and deficiencies in plasma-FVIII-activity that impair intrinsic-pathway-mediated coagulation-amplification. The standard-of-care for severe-HA-patients is regular infusions of therapeutic-FVIII-proteins (tFVIIIs) but ~30% develop neutralizing-tFVIII-antibodies called “FVIII-inhibitors (FEIs)” and become refractory. We used the PATH study and ImmunoChip to scan immune-mediated-disease (IMD)-genes for novel and/or replicated genomic-sequence-variations associated with baseline-FEI-status while accounting for non-independence of data due to genetic-relatedness and F8-mutational-heterogeneity. The baseline-FEI-status of 450 North American PATH subjects—206 with black-African-ancestry and 244 with white-European-ancestry—was the dependent variable. The F8-mutation-data and a genetic-relatedness matrix were incorporated into a binary linear-mixed model of genetic association with baseline-FEI-status. We adopted a gene-centric-association-strategy to scan, as candidates, pleiotropic-IMD-genes implicated in the development of either ³2 autoimmune-/autoinflammatory-disorders (AADs) or ³1 AAD and FEIs. Baseline-FEI-status was significantly associated with SNPs assigned to NOS2A (rs117382854; p=3.2E-6) and B3GNT2 (rs10176009; p=5.1E-6), which have functions in anti-microbial-/-tumoral-immunity. Among IMD-genes implicated in FEI-risk previously, we identified strong associations with CTLA4 assigned SNPs (p=2.2E-5). The F8-mutation-effect underlies ~15% of the total heritability for baseline-FEI-status. Additive genetic heritability and SNPs in IMD-genes account for >50% of the patient-specific variability in baseline-FEI-status. Race is a significant determinant independent of F8‑mutation-effects and non-F8-genetics.

ABSTRACT:Coagulation factor VIII (FVIII) and its carrier protein von Willebrand factor (VWF) are critical to coagulation and platelet aggregation. We leveraged whole-genome sequence data from the Trans-Omics for Precision Medicine (TOPMed) program along with TOPMed-based imputation of genotypes in additional samples to identify genetic associations with circulating FVIII and VWF levels in a single-variant meta-analysis, including up to 45 289 participants. Gene-based aggregate tests were implemented in TOPMed. We identified 3 candidate causal genes and tested their functional effect on FVIII release from human liver endothelial cells (HLECs) and VWF release from human umbilical vein endothelial cells. Mendelian randomization was also performed to provide evidence for causal associations of FVIII and VWF with thrombotic outcomes. We identified associations (P < 5 × 10-9) at 7 new loci for FVIII (ST3GAL4, CLEC4M, B3GNT2, ASGR1, F12, KNG1, and TREM1/NCR2) and 1 for VWF (B3GNT2). VWF, ABO, and STAB2 were associated with FVIII and VWF in gene-based analyses. Multiphenotype analysis of FVIII and VWF identified another 3 new loci, including PDIA3. Silencing of B3GNT2 and the previously reported CD36 gene decreased release of FVIII by HLECs, whereas silencing of B3GNT2, CD36, and PDIA3 decreased release of VWF by HVECs. Mendelian randomization supports causal association of higher FVIII and VWF with increased risk of thrombotic outcomes. Seven new loci were identified for FVIII and 1 for VWF, with evidence supporting causal associations of FVIII and VWF with thrombotic outcomes. B3GNT2, CD36, and PDIA3 modulate the release of FVIII and/or VWF in vitro.

Genetic studies have identified numerous regions associated with plasma fibrinogen levels in Europeans, yet missing heritability and limited inclusion of non-Europeans necessitates further studies with improved power and sensitivity. Compared with array-based genotyping, whole genome sequencing (WGS) data provides better coverage of the genome and better representation of non-European variants. To better understand the genetic landscape regulating plasma fibrinogen levels, we meta-analyzed WGS data from the NHLBI’s Trans-Omics for Precision Medicine (TOPMed) program (n=32,572), with array-based genotype data from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium (n=131,340) imputed to the TOPMed or Haplotype Reference Consortium panel. We identified 18 loci that have not been identified in prior genetic studies of fibrinogen. Of these, four are driven by common variants of small effect with reported MAF at least 10 percentage points higher in African populations. Three signals (SERPINA1, ZFP36L2, and TLR10) contain predicted deleterious missense variants. Two loci, SOCS3 and HPN, each harbor two conditionally distinct, non-coding variants. The gene region encoding the fibrinogen protein chain subunits (FGG;FGB;FGA), contains 7 distinct signals, including one novel signal driven by rs28577061, a variant common in African ancestry populations but extremely rare in Europeans (MAFAFR=0.180; MAFEUR=0.008). Through phenome-wide association studies in the VA Million Veteran Program, we found associations between fibrinogen polygenic risk scores and thrombotic and inflammatory disease phenotypes, including an association with gout. Our findings demonstrate the utility of WGS to augment genetic discovery in diverse populations and offer new insights for putative mechanisms of fibrinogen regulation.

Hemophilia-A (HA) is caused by heterogeneous loss-of-function factor (F) VIII gene ( F8) mutations and deficiencies in plasma FVIII coagulant activity (FVIII:C) that impair intrinsic pathway coagulation amplification. The standard-of-care for severe HA patients is regular infusions of therapeutic FVIII proteins (tFVIIIs) but ~25-30% of all such subjects develop neutralizing anti-tFVIII-antibodies called “FVIII inhibitors (FEIs)” that leave them refractory to treatment and necessitate use of alternative therapies that are less safe and effective, and extremely expensive. We used the ImmunoChip genotyping platform and subjects in the Personalized Alternative Therapies for Hemophilia (PATH) study to scan immune-mediated disease (IMD) genes for novel and/or replicated genomic sequence variations associated with FEI risk while accounting for non-independence of data due to genetic relatedness and F8 mutational heterogeneity. The “enrollment-FEI-status” of 450 North American PATH study subjects-which include 446 that self-identified as being only either black-African (n=204) or white-European (n=242), and four that self-identified as being both white-European and either black-African (n=3) or Asian (n=1)-was the dependent variable. The F8 mutation data and a genetic relatedness matrix were incorporated into a binary linear mixed model of genetic association with the novel FEI outcome of enrollment-FEI-status based on whether or not FEIs of any titer (AT) developed-Yes (AT:FEIs+) versus No (AT:FEI-)-regardless of their “historical-FEI-status', i.e., whether or not they had ever developed FEIs of AT prior to study entry. We adopted a gene-centric association strategy to scan as ”candidates“ a subset of 101 genes-of the ~2,000 total immune system genes that are interrogated by the naturally occurring sequence variations able to be genotyped simultaneously on the ImmunoChip-which comprise what we refer to herein as ”pleiotropic-IMD-genes“ because they have been implicated previously in the development of either (i) two or more autoimmune diseases (n=80) or (ii) at least one autoimmune disease if they were also implicated in FEI risk previously (n=25). (Note that four of the pleiotropic-IMD-genes previously implicated in FEI risk were also in the 80 genes implicated previously in the development of multiple autoimmune diseases such that we scanned 101 distinct pleiotropic-IMD-genes for association with FEI development in the PATH study.) Enrollment-FEI-status was significantly associated with SNPs in NOS2A ( Figure 1) and B3GNT2 ( Figure 2), which are involved directly and indirectly, respectively, in anti-microbial-/-tumoral-immunity. Among the pleiotropic-IMD-genes previously implicated in FEI risk, we identified strong associations with SNPs in CTLA4 (p=2.2E-5). We also quantified the influence of the different F8 mutation types on FEI risk for the first time and demonstrated that the F8-mutation-effect underlies ~15% of the total heritability of FEI development. The additive genetic heritability together with the SNPs in the pleiotropic-IMD-genes were found to account for >50% of the patient-specific variability in FEI risk. Finally, race was shown to be a significant determinant of FEI risk independent of the effects of F8 mutation types and non- F8-genetics.

Hemophilia-A (HA) is caused by heterogeneous factor (F) VIII gene ( F8) mutations, variably deficient plasma FVIII activity and reduced to absent intrinsic-pathway amplification of coagulation. Infused therapeutic-FVIII-proteins (tFVIIIs) prevent bleeding in all HA patients but ~30% with severe HA and ~7.5% with non-severe HA become refractory with the development of neutralizing anti-tFVIII-antibodies called “FVIII inhibitors (FEIs)”. HLA-class-II (HLAII) molecules are critical for the development of FEIs and several association studies on different HA patient populations clearly implicate an important role for the highly polymorphic encoding HLA-D loci in the variable frequency of immunogenicity observed for tFVIIIs infused in different subjects with HA. However, the results from these studies were often conflicting, and none have undergone true independent confirmation. We use the ImmunoChip to genotype each subject in the PATH study simultaneously for the >900 single-nucleotide-variations (SNVs) distributed across the extended MHC-class-II (MHCII) region-which includes the classical- and non-classical-HLAII-genes and -pseudogenes-and then evaluated each independently for associations with FEI risk while accounting for the non-independence of data due to genetic relatedness and F8 mutational heterogeneity using novel statistical methods. The “lifetime-FEI-status” of the 438 North American HA patients in PATH-whose racial-identity/ethnic-ancestry was self-reported as either black-African (n=200) or white-European (n=238)-was the dependent variable of interest. The F8-mutation data and a genetic-relatedness matrix were incorporated into a binary linear mixed model of genetic association with lifetime-FEI-status (Yes vs. No), with ‘Yes’ designating those patients having FEIs of either any titer (AT), i.e., >0.4 Bethesda Units (BUs) mL -1, or only high-titer (HT), i.e., ≥5.0 BUs mL -1. Following the analytical procedure used in prior studies designed to identify determinants of FEI risk, we conducted the extended-MHCII-region-wide association screen-against the 926 distinct SNVs with high-quality genotypes that passed QC-of lifetime-FEI-status on two groups of FEI-positive patients, i.e., those with AT-FEIs, or those with only HT-FEIs. HA patients in the HT-FEI group are suspected to be more homogeneous with respect to the underlying immunobiology as they appear clinically to have induced full adaptive immunity. In contrast, HA patients in the AT-FEI group are heterogenous as some will have transient FEIs, which spontaneously disappear, and others will have FEIs that may remain low-titer (LT) despite continued infusions of tFVIIIs. We found several SNPs that were not only significantly associated in both FEI groups-or significant and suggestive respectively in the HT- and AT-groups-but also increased in significance (i.e., their p-values decreased) under the HT analysis ( Figure 1). The latter observation indicates that these results correspond to true associations that became more apparent as sources of “noise” were removed upon going from the AT to HT analysis. These included a SNP in the 3'-UTR of DQB1 (rs1049225, p-value=5.7E-7) and two intergenic HLAII region SNPs (rs2647012, p-value=1.1E-5; and rs2858324, p-value=1.1E-5). The DQB1 SNP reached an ImmunoChip-wide significance threshold in the HT analysis (i.e., a p-value <5.9E-7) and extended-MHCII-region-wide significance in the AT analysis (i.e., p-value <5.4E-5). Although the two HLAII-intergenic SNPs were not ImmunoChip-wide significant, they were significant across the MHCII region (i.e., p-value <5.4E-5). We found two SNPs that were significant and suggestive, respectively, in the HT- and AT-FEI analyses (rs9276189, p-value=1.3E-5; and rs2856717, p-value=3.3E-5), as well as one SNP that was significant in the HT-FEI analyses but not significant or suggestive in the AT analysis (rs9271366, p-value=1.9E-6). Our results establish that a novel DQB1 genetic variant is associated with FEI risk and confirm that HLA-DQ-allotypes and race independently influence the rate of FEI development in HA ( Figure 2).

The ns-SNP, rs7586970 (T>C), which results in an asparagine (Asn [N]) to serine (Ser [S]) substitution at amino acid 221 (221 N>S) in TFPI, has been shown to be associated with coronary heart disease and T2D. We performed an exome scan of plasma TFPI levels in 706 participants in our study of genetic determinants of cardiovascular disease in Mexican Americans of South Texas. Using a linear mixed model approach, while accounting for age, sex, and their interactions, we found a heritability of 34% for plasma TFPI levels (p=4.4E-08). The only variant showing exome-wide significance was the rs7586970 (T>C) variant on chromosome 2 at the TFPI gene (p=5.1E-07; Figure 1A). The quantile-quantile distribution of the observed p-values from all the exome-wide tests against the p-values expected under the null hypothesis of no association shows an overall agreement, indicating that the p-value for the association test for the 221 N>S variant is likely not an artifact (data not shown). The regression coefficient for the polymorphism as a predictor of TFPI levels showed them to be increasing from the individuals homozygous for the major T-allele (T/T: N/N), to the heterozygous individuals (T/C: N/S), to the individuals homozygous for the minor C-allele (C/C: S/S) (Figure 1B). Using a statistical genetic model for the liability to disease conditional on a threshold, which is equivalent to a probit mixed model, we found that the rs7586970 (T>C) polymorphism is significantly associated with a reduction in the risk of T2D (p=0.009) (data not shown). While not shown, the effect of the TFPI variant is to upwardly displace the liability threshold to the right, which results in a reduced prevalence of T2D from 6% to just below 3% in the rare allele homozygotes. While the reduction is a small change, it is statistically significant, nonetheless. Moreover, our observed effect size in terms of T2D risk is consistent with the widely held view that T2D has a multifactorial etiology involving a complex combination of environmental risk factors and genes with small effects sizes. A plausible mechanism for the reduced risk of T2D involves tissue factor (TF), which is the physiologic initiator of coagulation that is normally found outside the vasculature. As such, coagulation is initiated when the blood vessel is disturbed or when vascular and blood cells are induced to secrete and express surface membrane TF, as occurs in inflammation. TF then binds its primary cofactor, FVIIa, which circulates in the blood, TF-FVIIa activates FIX and FX, and the small amount FXa generated converts prothrombin to thrombin. TFPI is the primary inhibitor of TF-induced coagulation by binding and inhibiting TF-FVIIa in a FXa-dependent manner. Coagulation only proceeds when enough FXa is generated to overwhelm the inhibitory potential of TFPI. The rs7586970 ns-SNP conferred substitution of Ser for Asn at amino acid 221, the putative glycosyl-phosphatidylinositol anchor site of TFPI-β, its phospholipid bound form, potentially disrupts the binding of TFPI-β to the surface of endothelial cells resulting in higher levels of free form which has more potent anticoagulant activity. In conclusion, we found that TFPI 221 N>S ns-SNP is pleiotropically associated with plasma TFPI levels and risk of T2D. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Studies have reported that the G505A nonsynonymous (ns)-SNP encoding the Ala147Thr amino acid substitution in TAFI, is associated with a reduction in risk of venous thrombosis and myocardial infarction. Interestingly, homozygosity for the A-allele (AA) of G505A is associated with increased TAFI antigen (TAFI:Ag) levels in plasma. In our study of the genetic determinants of cardiovascular disease (CVD) in Mexican Americans of South Texas, we performed an exome-wide scan in relation to plasma TAFI antigen levels in 784 individuals. While accounting for age, sex, and their interactions as confounders in linear mixed model, we found a heritability of 59% for TAFI:Ag levels (p=1.4E-19), and that ns-SNP G505A was the only variant showing exome-wide significance (p=3.5E-14; Figure A). Figure B shows the quantile-quantile distribution of the p-values from all the exome-wide tests. Clearly, the p-value distribution reveals that there is no systematic bias that may act to skew the p-values, and that the lone exception¾in agreement between observed and expected quantiles¾is due to this TAFI SNP, which strongly suggests a truly significant effect. Consistent with previous reports, the regression coefficient for G505A as a predictor of TAFI:Ag levels showed them to be increasing from the homozygous for the major G-allele (GG), to the heterozygous individuals (GA), to the individuals homozygous for the minor A-allele (AA) (Figure B). We also investigated if G505A is associated with our CHD variable. Using a statistical genetic model for the liability to disease conditional on a threshold, which is equivalent to a probit mixed model, we found that the G505A ns-SNP in TAFI, encoding TAFI Ala147Thr, is significantly associated with a reduction in the risk of CHD (p=0.002). As observed in existing literature, potential limitations of this study include the ELISA assay used for the quantification of TAFI levels. Some ELISA assays measure proTAFI, TAFIa, and TAFIi. However, recent evidence suggests that there may be cross reactivity between TAFIa and TAFIi. This can result in measuring ongoing TAFI activation peptides and elevated levels of TAFIa which ultimately goes on to make resistant fibrin. This is likely the marker that results in the increased risk of venous thromboembolism. To mitigate this confounding factor, an ELISA assay specific to measuring TAFI antigen is needed. In conclusion, we found that TAFI G505A is pleiotropically associated with TAFI:Ag levels and risk of CHD.

Myelodysplastic syndrome (MDS) is an onco-hematologic disease with distinct levels of peripheral blood cytopenias, dysplasias in cell differentiation and various forms of chromosomal and cytogenomic alterations. In this study, the Chromosomal Microarray Analysis (CMA) was performed in patients with primary MDS without numerical and/or structural chromosomal alterations in karyotypes. A total of 17 patients was evaluated by GTG banding and eight patients showed no numerical and/or structural alterations. Then, the CMA was carried out and identified gains and losses CNVs and long continuous stretches of homozygosity (LCSHs). They were mapped on chromosomes 1, 2, 3, 4, 5, 6, 7, 9, 10, 12, 14, 16, 17, 18, 19, 20, 21, X, and Y. Ninety-one genes that have already been implicated in molecular pathways important for cell viability were selected and in-silico expression analyses demonstrated 28 genes differentially expressed in mesenchymal stromal cells of patients. Alterations in these genes may be related to the inactivation of suppressor genes or the activation of oncogenes contributing to the evolution and malignization of MDS. CMA provided additional information in patients without visible changes in the karyotype and our findings could contribute with additional information to improve the prognostic and personalized stratification for patients.

The literature on risk factors for venous thromboembolism (VTE) is replete with complex combinations of genetic and environmental determinants. Here we apply statistical genetic models to data from Mexican Americans of South Texas participating in the San Antonio Family Study (SAFS) to help disentangle some of this complexity. The SAFS has data for nearly 50 large extended pedigrees (Figure A) that are extensively phenotyped, especially for traits related to the pathophysiology of cardiovascular disease. Using a linear mixed model approach, while accounting for age, sex, and their interactions (including age-squared, age-by-sex, and age-squared-by-sex) as confounders, we found significant heritability for plasma FII (46%; N=640; p=6.9E-12) and fibrinogen (28%; N=759; p=1.6E-06) coagulant activity levels and that the Prothrombin G20210A mutation was significantly associated with both traits (FII: p=0.002; fibrinogen: p=0.037; Figure B). We also examined a dichotomous obesity variable based on the Adult Treatment Panel III criterion of sex-specific waist circumferences (>102 cm in males; >88 cm females) denoted as OBWC. Under a threshold and liability model, we found a significant heritability of the liability of OBWC (71%; N=654; p=4.4E-08) and that Prothrombin G20210A was a significant predictor (p=0.031), while still adjusting for age, sex, and their interactions. As can be seen in Figure C, the G20210A mutation profoundly impacts the liability of OBWC such that obesity prevalence, where the prevalence parameter is denoted by Kp in the figure, increases by 27% from individuals homozygous for the major G-allele (G/G) with a prevalence of 34% to heterozygous (G/A) individuals with a prevalence of 61%. To the best of our knowledge, this appears to be the largest single-allele-dose effect for obesity reported in the literature. We next performed bivariate trait analysis (each time accounting for age, sex, and their interactions as confounders) to discover potentially meaningful correlations between the three traits of interest and to see if these would influence their association with G20210A. Under a bivariate model for any two traits, denoted as trait A and B say, the parameters of the following equation are estimated:

Background: Hemostasis variables represent well-known pathogenic determinants of venous-thromboembolism and are hypothesized to influence risk for cardiometabolic outcomes (CMOs) by effecting susceptibility to vascular inflammation and/or endothelial dysfunction.

A heterogeneous collection of >1500 distinct causative factor (F) VIII gene (F8) mutations have been identified thus far in unrelated severe Hemophilia A (HA) patients, who have less than 1% of normal FVIII activity in their plasmas and experience recurrent bleeding that often results in crippling arthropathies and can be life threatening. Curative gene therapies are being pursued intensely as the current standard of care, which involves 2-3 infusions/week of therapeutic FVIII (tFVIII) proteins throughout a patient's life, is extremely expensive and very demanding. Moreover, ~25% of patients with severe HA (PSHA) develop anti-tFVIII antibodies that neutralize the efficacy of their tFVIII proteins. Despite remarkable progress in clinical trials of various adeno-associated viruses (AAVs) as vectors for in vivo delivery of therapeutic F8 genes, it is not clear how widespread viral-mediated gene replacement therapy (GRT) will become due to current limitations that include the: (1) presence of existing immunity to the AAV capsid protein (CP) in ~30-70% of PSHA for whom GRT is contraindicated; (2) immunity to AAV-CP induced in all PSHA during the initial GRT that precludes subsequent dosing; (3) use of heterologous promoters which drive F8 expression in non-physiologic cells that may increase the encoded tFVIII protein's immunogenicity; and 4) episomal location of AAV-genome replication, which, together with "(2)" and "(3)", precludes GRT in children. These important unmet needs require new gene-based therapeutic strategies for HA. Our goal was to develop a virus-free, ex vivo personalized gene repair therapy that minimally manipulates the mutant F8 in autologous patient-derived blood outgrowth endothelial cells (BOECs)-the physiologically relevant cell type for FVIII production in vivo-followed by their expansion and reinfusion into the same individual patient. We chose to focus initially on the intron (I) 22 inversion (I22I) mutation initially as it is causative in >40% of all PSHA. CRISPR/Cas9 guide RNAs were designed to target the 3' end of F8 exon (E) 22. For initial experiments in K562 cells, a donor plasmid containing a restriction enzyme site was nucleofected with the CRISPR system encoding plasmid, which triggered successful homology-directed repair (HDR) at the target site (efficiency of 18.2%, n=2). Subsequently, a cDNA-based therapeutic donor plasmid was constructed containing all F8 coding sequences in E23-E26 followed by a bGH polyA signal. After appropriate informed consent was obtained, BOECs were cultured from the blood of 3 severe pediatric HA patients with the I22I (ages 6 years, 7 years, and 13 years). After nucleofecting the BOECs with the F8-specific CRISPR and HDR constructs, site-specific knock-in of the cDNA at the 3' end of E22 was confirmed via PCR (n=3). Direct Sanger sequencing of the resultant amplicons from the repaired I22-inverted F8 locus in treated BOECs from one representative patient confirmed complete and seamless knock-in of the therapeutic cDNA at the endogenous site of the mutant F8. Current efforts are to isolate clonal populations of the repaired BOECs and characterize their ability to secrete active FVIII in vitro. Similar experiments are underway using canine BOECs in the canine model of HA. The use of clonal populations of gene corrected autologous BOECs as the infused therapy allows whole genome sequencing analysis to be performed to confirm that no off-target cutting or integration occurred in the therapeutic cell preparation prior to infusion into the recipient patient, further strengthening the safety profile of this proposed autologous cell therapy. Overall, these current results lay promising proof-of-concept data for a potential new curative therapeutic alternative approach for HA which should overcome drawbacks of the current generations of AAV-based treatments. Disclosures Dinh: Haplogenics Corporation: Current Employment. Luu:Haplogenics Corporation: Current Employment. Mead:CSL Behring: Current Employment. Escobar:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; National Hemophilia Foundation: Consultancy, Membership on an entity's Board of Directors or advisory committees. Powell:Haplogenics Corporation: Membership on an entity's Board of Directors or advisory committees. Howard:Haplogenics Corporation: Membership on an entity's Board of Directors or advisory committees.

miRNA regulates the expression of protein coding genes and plays a regulatory role in human development and disease. The human iPSCs and their differentiated progenies provide a unique opportunity to identify these miRNA-mediated regulatory mechanisms. To identify miRNA–mRNA regulatory interactions in human nervous system development, well characterized NSCs were differentiated from six validated iPSC lines and analyzed for differentially expressed (DE) miRNome and transcriptome by RNA sequencing. Following the criteria, moderated t statistics, FDR-corrected p-value ≤ 0.05 and fold change—absolute (FC-abs) ≥2.0, 51 miRNAs and 4033 mRNAs were found to be significantly DE between iPSCs and NSCs. The miRNA target prediction analysis identified 513 interactions between 30 miRNA families (mapped to 51 DE miRNAs) and 456 DE mRNAs that were paradoxically oppositely expressed. These 513 interactions were highly enriched in nervous system development functions (154 mRNAs; FDR-adjusted p-value range: 8.06 × 10−15–1.44 × 10−4). Furthermore, we have shown that the upregulated miR-10a-5p, miR-30c-5p, miR23-3p, miR130a-3p and miR-17-5p miRNA families were predicted to down-regulate several genes associated with the differentiation of neurons, neurite outgrowth and synapse formation, suggesting their role in promoting the self-renewal of undifferentiated NSCs. This study also provides a comprehensive characterization of iPSC-generated NSCs as dorsal neuroepithelium, important for their potential use in in vitro modeling of human brain development and disease.