24-hour urine oxalate and risk of chronic kidney disease.

BACKGROUND: To assess whether 24-hr urine oxalate (UOx) excretion is a risk factor for incident chronic kidney disease (CKD). METHODS: This longitudinal observational US-based study included 426,896 individuals age ≥ 18 years with no CKD at baseline and with at least one UOx and at least 6 months of baseline and 6 months of follow-up data. Of these, 11,239 (2.6%) had an underlying malabsorptive condition. Incident CKD, defined by relevant ICD codes, was identified from a multi-source data cloud containing individual-level healthcare claims and electronic medical records data. The association between categories of UOx and incident CKD was modeled using logistic regression adjusting for age, sex, race, BMI, baseline urine calcium, urine citrate, urine volume, tobacco use, hypertension, diabetes, malabsorption, and cardiovascular disease. RESULTS: Mean follow-up time was 38.9 months (SD 21.7). Compared with individuals with UOx <20 mg/24-hr, the odds of developing incident CKD increased for UOx 20-29 mg/24-hr (multivariate-adjusted (MV) OR: 1.14, 95% CI: 1.07, 1.21) through 80+ mg/24-hr (MVOR: 1.35 [1.21, 1.50] and was statistically significant for each UOx category. A similar pattern was seen in the subgroup with a malabsorptive condition though the magnitudes of association were larger, with the odds of developing incident CKD increased for UOx 20-29 mg/24-hr (MVOR: 1.50 [1.03, 2.20] through 80+ mg/24-hr (MVOR: 2.34 [1.50, 3.63] as compared with UOx <20 mg/24-hr. CONCLUSIONS: The risk of incident CKD increases with increasing 24-hr urine oxalate excretion. Future studies should examine whether reducing urine oxalate diminishes the risk of developing CKD.

Efficacy and safety of a synthetic biotic for treatment of phenylketonuria: a phase 2 clinical trial.

Despite available treatment options, many patients with phenylketonuria (PKU) cannot achieve target plasma phenylalanine (Phe) levels1. We previously modified Escherichia coli Nissle 1917 to metabolize Phe in the gut after oral administration (SYNB1618) and designed a second strain (SYNB1934) with enhanced activity of phenylalanine ammonia lyase2,3. In a 14-day open-label dose-escalation study (Synpheny-1, NCT04534842 ), we test a primary endpoint of change from baseline in labeled Phe (D5-Phe AUC0-24; D5-Phe area under the curve (AUC) over 24 hours after D5-Phe administration) in plasma after D5-Phe challenge in adult participants with screening Phe of greater than 600 µM. Secondary endpoints were the change from baseline in fasting plasma Phe and the incidence of treatment-emergent adverse events. A total of 20 participants (ten male and ten female) were enrolled and 15 completed the study treatment. Here, we show that both strains lower Phe levels in participants with PKU: D5-Phe AUC0-24 was reduced by 43% from baseline with SYNB1934 and by 34% from baseline with SYNB1618. SYNB1934 led to a decrease in fasting plasma Phe of 40% (95% CI, -52, -24). There were no serious adverse events or infections. Four participants discontinued because of adverse events, and one withdrew during the baseline period. We show that synthetic biotics can metabolize Phe in the gut, lower post-prandial plasma Phe levels and lower fasting plasma Phe in patients with PKU.

Influence of polymorphisms in CYP2C9, VKORC1, MDR1 and APOE genes on the warfarin maintenance dose in Brazilian patients.

Background: Polymorphisms in the CYP2C9, VKORC1, MDR1 and APOE genes may impact warfarin dose. Aim: To investigate the influence of sociodemographic, clinical factors and polymorphisms *1, *2 and *3 for CYP2C9, -1639G>A for VKORC1, 3435C>T for MDR1, and ϵ2, ϵ3 and ϵ4 for APOE genes on the mean weekly warfarin maintenance dose in adults. Methods: This cross-sectional study recruited a calculated sample of 315 patients in three anticoagulation clinics in Brazil. A model containing the variables significantly associated with warfarin dose was estimated. Results: The mean age of patients was 64.1 ± 13.1 years, with 173 (54.9%) women. Age, use of amiodarone, genotype VKORC1 GA, genotype VKORC1 AA, genotypes CYP2C9*1/*2 or *1/*3 and genotypes CYP2C9*2/*2 or *2/*3 or *3/*3 were associated with a reduced warfarin dose. Conclusion: This study pointed out factors that could impact the management of oral anticoagulation.

Complications of the Low Phenylalanine Diet for Patients with Phenylketonuria and the Benefits of Increased Natural Protein.

Phenylketonuria (PKU) is an inherited disorder in which phenylalanine (Phe) is not correctly metabolized leading to an abnormally high plasma Phe concentration that causes profound neurologic damage if left untreated. The mainstay of treatment for PKU has centered around limiting natural protein in the diet while supplementing with medical foods in order to prevent neurologic injury while promoting growth. This review discusses several deleterious effects of the low Phe diet along with benefits that have been reported for patients with increased natural protein intake while maintaining plasma Phe levels within treatment guidelines.

Long-Read Sequencing Identifies the First Retrotransposon Insertion and Resolves Structural Variants Causing Antithrombin Deficiency.

The identification of inherited antithrombin deficiency (ATD) is critical to prevent potentially life-threatening thrombotic events. Causal variants in SERPINC1 are identified for up to 70% of cases, the majority being single-nucleotide variants and indels. The detection and characterization of structural variants (SVs) in ATD remain challenging due to the high number of repetitive elements in SERPINC1. Here, we performed long-read whole-genome sequencing on 10 familial and 9 singleton cases with type I ATD proven by functional and antigen assays, who were selected from a cohort of 340 patients with this rare disorder because genetic analyses were either negative, ambiguous, or not fully characterized. We developed an analysis workflow to identify disease-associated SVs. This approach resolved, independently of its size or type, all eight SVs detected by multiple ligation-dependent probe amplification, and identified for the first time a complex rearrangement previously misclassified as a deletion. Remarkably, we identified the mechanism explaining ATD in 2 out of 11 cases with previous unknown defect: the insertion of a novel 2.4 kb SINE-VNTR-Alu retroelement, which was characterized by de novo assembly and verified by specific polymerase chain reaction amplification and sequencing in the probands and affected relatives. The nucleotide-level resolution achieved for all SVs allowed breakpoint analysis, which revealed repetitive elements and microhomologies supporting a common replication-based mechanism for all the SVs. Our study underscores the utility of long-read sequencing technology as a complementary method to identify, characterize, and unveil the molecular mechanism of disease-causing SVs involved in ATD, and enlarges the catalogue of genetic disorders caused by retrotransposon insertions.

Development of a mechanistic model to predict synthetic biotic activity in healthy volunteers and patients with phenylketonuria.

The development of therapeutics depends on predictions of clinical activity from pre-clinical data. We have previously described SYNB1618, an engineered bacterial therapeutic (synthetic biotic) for the treatment of Phenylketonuria (PKU), a rare genetic disease that leads to accumulation of plasma phenylalanine (Phe) and severe neurological complications. SYNB1618 consumes Phe in preclinical models, healthy human volunteers, and PKU patients. However, it remains unclear to what extent Phe consumption by SYNB1618 in the gastrointestinal tract lowers plasma Phe levels in PKU patients. Here, we construct a mechanistic model that predicts SYNB1618 function in non-human primates and healthy subjects by combining in vitro simulations and prior knowledge of human physiology. In addition, we extend a model of plasma Phe kinetics in PKU patients, in order to estimate plasma Phe lowering by SYNB1618. This approach provides a framework that can be used more broadly to define the therapeutic potential of synthetic biotics.

Safety and pharmacodynamics of an engineered E. coli Nissle for the treatment of phenylketonuria: a first-in-human phase 1/2a study.

Phenylketonuria (PKU) is a rare disease caused by biallelic mutations in the PAH gene that result in an inability to convert phenylalanine (Phe) to tyrosine, elevated blood Phe levels and severe neurological complications if untreated. Most patients are unable to adhere to the protein-restricted diet, and thus do not achieve target blood Phe levels. We engineered a strain of E. coli Nissle 1917, designated SYNB1618, through insertion of the genes encoding phenylalanine ammonia lyase and L-amino acid deaminase into the genome, which allow for bacterial consumption of Phe within the gastrointestinal tract. SYNB1618 was studied in a phase 1/2a randomized, placebo-controlled, double-blind, multi-centre, in-patient study ( NCT03516487 ) in adult healthy volunteers (n = 56) and patients with PKU and blood Phe level ≥600 mmol l-1 (n = 14). Participants were randomized to receive a single dose of SYNB1618 or placebo (part 1) or up to three times per day for up to 7 days (part 2). The primary outcome of this study was safety and tolerability, and the secondary outcome was microbial kinetics. A D5-Phe tracer (15 mg kg-1) was used to study exploratory pharmacodynamic effects. SYNB1618 was safe and well tolerated with a maximum tolerated dose of 2 × 1011 colony-forming units. Adverse events were mostly gastrointestinal and of mild to moderate severity. All participants cleared the bacteria within 4 days of the last dose. Dose-responsive increases in strain-specific Phe metabolites in plasma (trans-cinnamic acid) and urine (hippuric acid) were observed, providing a proof of mechanism for the potential to use engineered bacteria in the treatment of rare metabolic disorders.

Variability and Lability of Ammonia Levels in Healthy Volunteers and Patients With Cirrhosis: Implications for Trial Design and Clinical Practice.

INTRODUCTION: Ammonia levels are used to assess hepatic encephalopathy, but their levels are highly variable in clinical practice. METHODS: We studied factors associated with variation in ammonia values in cirrhotic patients without previous hepatic encephalopathy and healthy volunteers (HVs). RESULTS: Ammonia increased by 12% and 18% at 1 and 2 hour, respectively, after a protein meal in 64 cirrhotic patients (P < 0.001). In 237 HVs, ammonia levels varied significantly between sites (P < 0.0001). New site-specific ammonia upper limits based on HV levels using a strict analysis protocol differed from routinely used values. Correlation between paired fresh samples was high (r = 0.83) but modest between fresh and frozen samples (r = 0.62). DISCUSSION: Sample handling, processing, and protein intake impact ammonia levels across sites.

Genomic and transcriptomic association studies identify 16 novel susceptibility loci for venous thromboembolism.

Venous thromboembolism (VTE) is a significant contributor to morbidity and mortality. To advance our understanding of the biology contributing to VTE, we conducted a genome-wide association study (GWAS) of VTE and a transcriptome-wide association study (TWAS) based on imputed gene expression from whole blood and liver. We meta-analyzed GWAS data from 18 studies for 30 234 VTE cases and 172 122 controls and assessed the association between 12 923 718 genetic variants and VTE. We generated variant prediction scores of gene expression from whole blood and liver tissue and assessed them for association with VTE. Mendelian randomization analyses were conducted for traits genetically associated with novel VTE loci. We identified 34 independent genetic signals for VTE risk from GWAS meta-analysis, of which 14 are newly reported associations. This included 11 newly associated genetic loci (C1orf198, PLEK, OSMR-AS1, NUGGC/SCARA5, GRK5, MPHOSPH9, ARID4A, PLCG2, SMG6, EIF5A, and STX10) of which 6 replicated, and 3 new independent signals in 3 known genes. Further, TWAS identified 5 additional genetic loci with imputed gene expression levels differing between cases and controls in whole blood (SH2B3, SPSB1, RP11-747H7.3, RP4-737E23.2) and in liver (ERAP1). At some GWAS loci, we found suggestive evidence that the VTE association signal for novel and previously known regions colocalized with expression quantitative trait locus signals. Mendelian randomization analyses suggested that blood traits may contribute to the underlying risk of VTE. To conclude, we identified 16 novel susceptibility loci for VTE; for some loci, the association signals are likely mediated through gene expression of nearby genes.

A large-scale exome array analysis of venous thromboembolism.

Although recent Genome-Wide Association Studies have identified novel associations for common variants, there has been no comprehensive exome-wide search for low-frequency variants that affect the risk of venous thromboembolism (VTE). We conducted a meta-analysis of 11 studies comprising 8,332 cases and 16,087 controls of European ancestry and 382 cases and 1,476 controls of African American ancestry genotyped with the Illumina HumanExome BeadChip. We used the seqMeta package in R to conduct single variant and gene-based rare variant tests. In the single variant analysis, we limited our analysis to the 64,794 variants with at least 40 minor alleles across studies (minor allele frequency [MAF] ~0.08%). We confirmed associations with previously identified VTE loci, including ABO, F5, F11, and FGA. After adjusting for multiple testing, we observed no novel significant findings in single variant or gene-based analysis. Given our sample size, we had greater than 80% power to detect minimum odds ratios greater than 1.5 and 1.8 for a single variant with MAF of 0.01 and 0.005, respectively. Larger studies and sequence data may be needed to identify novel low-frequency and rare variants associated with VTE risk.

An engineered E. coli Nissle improves hyperammonemia and survival in mice and shows dose-dependent exposure in healthy humans.

The intestine is a major source of systemic ammonia (NH3); thus, capturing part of gut NH3 may mitigate disease symptoms in conditions of hyperammonemia such as urea cycle disorders and hepatic encephalopathy. As an approach to the lowering of blood ammonia arising from the intestine, we engineered the orally delivered probiotic Escherichia coli Nissle 1917 to create strain SYNB1020 that converts NH3 to l-arginine (l-arg). We up-regulated arginine biosynthesis in SYNB1020 by deleting a negative regulator of l-arg biosynthesis and inserting a feedback-resistant l-arg biosynthetic enzyme. SYNB1020 produced l-arg and consumed NH3 in an in vitro system. SYNB1020 reduced systemic hyperammonemia, improved survival in ornithine transcarbamylase-deficient spfash mice, and decreased hyperammonemia in the thioacetamide-induced liver injury mouse model. A phase 1 clinical study was conducted including 52 male and female healthy adult volunteers. SYNB1020 was well tolerated at daily doses of up to 1.5 × 1012 colony-forming units administered for up to 14 days. A statistically significant dose-dependent increase in urinary nitrate, plasma 15N-nitrate (highest dose versus placebo, P = 0.0015), and urinary 15N-nitrate was demonstrated, indicating in vivo SYNB1020 activity. SYNB1020 concentrations reached steady state by the second day of dosing, and excreted cells were alive and metabolically active as evidenced by fecal arginine production in response to added ammonium chloride. SYNB1020 was no longer detectable in feces 2 weeks after the last dose. These results support further clinical development of SYNB1020 for hyperammonemia disorders including urea cycle disorders and hepatic encephalopathy.

ADP Platelet Hyperreactivity Predicts Cardiovascular Disease in the FHS (Framingham Heart Study).

BACKGROUND: Platelet function is associated with adverse events in patients with cardiovascular disease (CVD). METHODS AND RESULTS: We examined associations of baseline platelet function with incident CVD events in the community-based FHS (Framingham Heart Study). Participants free of prevalent CVD and without recent aspirin treatment with available data in the Framingham Offspring cohort (1991-1995) and Omni cohort (1994-1998) were included. Platelet function was measured with light transmission aggregometry using collagen (1.9 µg/mL), ADP (0.05-15 µmol/L), and epinephrine (0.01-15 µmol/L). We used proportional hazards models to analyze incident outcomes (myocardial infarction/stroke, CVD, and CVD mortality) with respect to platelet measures. The study sample included 2831 participants (average age, 54.3 years; 57% women). During follow-up (median, 20.4 years), we observed 191 composite incident myocardial infarction or stroke events, 432 incident CVD cases, and 117 CVD deaths. Hyperreactivity to ADP and platelet aggregation at ADP concentration of 1.0 µmol/L were significantly associated with incident myocardial infarction/stroke in a multivariable model (hazard ratio, 1.68 [95% confidence interval, 1.13-2.50] [P=0.011] for hyperreactivity across ADP doses; and hazard ratio, 1.16 [95% confidence interval, 1.02-1.33] [P=0.029] for highest quartile of ADP response at 1.0 µmol/L versus others). No association was observed for collagen lag time or any epinephrine measures with incident myocardial infarction or stroke. CONCLUSIONS: Intrinsic hyperreactivity to low-dose ADP in our community-based sample, who were free of CVD and any antiplatelet therapy, is associated with future arterial thrombosis during a 20-year follow-up. These findings reinforce ADP activation inhibition as a critical treatment paradigm and encourage further study of ADP inhibitor-refractive populations.

Familial Hypercholesterolemia and Type 2 Diabetes in the Old Order Amish.

Alleles associated with lower levels of LDL cholesterol (LDL-C) have recently been associated with an increased risk of type 2 diabetes (T2D), highlighting the complex relationship between LDL-C and diabetes. This observation begs the question of whether LDL-C-raising alleles are associated with a decreased risk of T2D. This issue was recently addressed in a large familial hypercholesterolemia (FH) screening study, which reported a lower prevalence of self-reported diabetes in FH subjects than in age-matched relatives without FH. To extend this observation, we tested the association of FH with diabetes status and glycemia in a large Amish population enriched for the FH-associated APOB R3527Q variant that included 640 APOB R3527Q carriers and 4,683 noncarriers. Each copy of the R3527Q T allele was associated with a 74.9 mg/dL increase in LDL-C. There was little difference in T2D prevalence between subjects with (5.2%) and without (4.5%) the R3527Q allele (P = 0.23), and there was no association between R3527Q variant and impaired fasting glucose, fasting glucose or insulin, or oral glucose tolerance test-derived measures. Our data provide no evidence supporting an association between the APOB R3527Q variant and T2D or glycemia and highlight the asymmetry of the LDL-C-T2D relationship and/or the gene/variant-dependent specificity of the LDL-C-T2D association.

Platelet function as a risk factor for venous thromboembolism in the Framingham Heart Study.

INTRODUCTION: The relationship of venous thromboembolism (VTE) with platelet reactivity is unclear. Platelet function plays a key role in arterial thrombosis. Evidence suggests antiplatelet agents also effects in reducing VTE. Our aim is to describe the role of baseline platelet function in development of VTE in the community-based Framingham Heart Study (FHS) cohort. MATERIALS AND METHODS: Participants in the Framingham Offspring cohort fifth examination and Omni cohort first examination were eligible. We used light transmission aggregometry to measure platelet aggregation in response to collagen and a range of ADP and epinephrine doses. The study population consisted of 2831 participants [average age 54.3years; 57% female]. RESULTS AND CONCLUSIONS: During a median follow-up of 20.4years, we observed 138 incident VTE events. In age-, sex- and cohort-adjusted analysis an increase in collagen lag time was associated with increased risk for incident VTE (HR 1.01 [1.00-1.02]; p=0.049). Increased maximal aggregation to low dose epinephrine (1.0µM) was associated with lower VTE risk (HR 0.84 [0.71-0.99]; p=0.042]). However, additional multivariable analyses attenuated the collagen-VTE and epinephrine-VTE associations to trends, primarily due to adjustment for baseline body mass index (BMI), a VTE risk factor and potential modifier of platelet function. Secondary analyses considering varying follow-up periods, cancer incidence and interim aspirin use did not dramatically affect the collagen and epinephrine trends observed. Baseline platelet aggregability was only weakly associated with incident VTE, and in a paradoxical direction, in a community-based population. Other markers of platelet function and hemostasis could prove to be more useful predictors.

Association of Traditional Cardiovascular Risk Factors With Venous Thromboembolism: An Individual Participant Data Meta-Analysis of Prospective Studies.

BACKGROUND: Much controversy surrounds the association of traditional cardiovascular disease risk factors with venous thromboembolism (VTE). METHODS: We performed an individual level random-effect meta-analysis including 9 prospective studies with measured baseline cardiovascular disease risk factors and validated VTE events. Definitions were harmonized across studies. Traditional cardiovascular disease risk factors were modeled categorically and continuously using restricted cubic splines. Estimates were obtained for overall VTE, provoked VTE (ie, VTE occurring in the presence of 1 or more established VTE risk factors), and unprovoked VTE, pulmonary embolism, and deep-vein thrombosis. RESULTS: The studies included 244 865 participants with 4910 VTE events occurring during a mean follow-up of 4.7 to 19.7 years per study. Age, sex, and body mass index-adjusted hazard ratios for overall VTE were 0.98 (95% confidence interval [CI]: 0.89-1.07) for hypertension, 0.97 (95% CI: 0.88-1.08) for hyperlipidemia, 1.01 (95% CI: 0.89-1.15) for diabetes mellitus, and 1.19 (95% CI: 1.08-1.32) for current smoking. After full adjustment, these estimates were numerically similar. When modeled continuously, an inverse association was observed for systolic blood pressure (hazard ratio=0.79 [95% CI: 0.68-0.92] at systolic blood pressure 160 vs 110 mm Hg) but not for diastolic blood pressure or lipid measures with VTE. An important finding from VTE subtype analyses was that cigarette smoking was associated with provoked but not unprovoked VTE. Fully adjusted hazard ratios for the associations of current smoking with provoked and unprovoked VTE were 1.36 (95% CI: 1.22-1.52) and 1.08 (95% CI: 0.90-1.29), respectively. CONCLUSIONS: Except for the association between cigarette smoking and provoked VTE, which is potentially mediated through comorbid conditions such as cancer, the modifiable traditional cardiovascular disease risk factors are not associated with increased VTE risk. Higher systolic blood pressure showed an inverse association with VTE.

Biomarkers for the prediction of venous thromboembolism in the community.

BACKGROUND &OBJECTIVES: Venous and arterial thrombosis share common pathophysiology. Multiple biomarkers reflecting various biological pathways can predict arterial thrombosis. We studied whether this approach could identify persons at risk of first venous thromboembolism (VTE).

Epidemiology of venous thromboembolism in the Framingham Heart Study.

BACKGROUND: Reports of the crude incidence of venous thromboembolism (VTE) in Western countries vary widely. Data regarding risk factors, incidence and recurrence of VTE from deeply-phenotyped community-based cohort studies are needed. OBJECTIVES: To study the incidence, associated mortality, and predisposing factors of VTE in the prospective, longitudinal community-based Framingham Heart Study. PATIENTS/METHODS: The study sample consisted of the Framingham Heart Study Original, Offspring, Third Generation, and Omni cohorts (N=9754). Incidence rates (IR) were standardized to the 2000 US population. Cox proportional hazards regression models were used to study risk factor associations. RESULTS: During 1995-2014 (total follow-up time 104,091 person-years [median 9.8 (range 0-20) years]), 297 incident VTE events were observed. Age-adjusted IR of VTE was 20.3/10,000 (95% CI 17.9-22.6). Of the events 120 (40%) were pulmonary embolism (PE) and 177 (60%) were deep venous thrombosis (DVT); 29% were unprovoked, 40% provoked, and 31% cancer-related. Cancer-related VTE was associated with high mortality at 30days (24.2%), 1year (66.3%), and 5years (75.6%). In multivariable models, age and obesity, but no other traditional cardiovascular risk factors, were significantly associated with VTE (hazard ratio [HR] per 10-year increase in age 1.69, 95% CI 1.48-1.92; HR for obesity (BMI≥30kg/m(2)) 1.88, 95% CI 1.44-2.45). CONCLUSIONS: We provide data on the epidemiology of VTE. VTE is associated with significant mortality, and prognosis after cancer-related VTE is particularly poor. Traditional cardiovascular risk factors beyond age and obesity are not associated with VTE.

Renal Impairment and Prognosis of Patients with Atrial Fibrillation Undergoing Coronary Intervention - The AFCAS Trial.

BACKGROUND: Renal impairment is a well-known risk factor for cardiovascular complications, but the effect of different stages of renal impairment on thrombotic/thromboembolic and bleeding complications in patients with atrial fibrillation (AF) undergoing percutaneous coronary intervention (PCI) remains largely unknown. We sought to evaluate the incidence and clinical impact of four stages of renal impairment in patients with AF undergoing PCI. METHODS: We assessed renal function by estimated glomerular filtration rate (eGFR) and outcomes in 781 AF patients undergoing PCI by using the data from a prospective European multicenter registry. End-points included all-cause mortality, major adverse cardiac and cerebrovascular events (MACCE) and bleeding events at 12 months. RESULTS: A total of 195 (25%) patients had normal renal function (eGFR ≥90 mL/min), 290 (37%) mild renal impairment (eGFR 60-89), 263 (34%) moderate renal impairment (eGFR 30-59) and 33 (4%) severe renal impairment (eGFR <30). Degree of renal impairment remained an independent predictor of mortality and MACCE in an adjusted a Cox regression model. Even patients with mild renal impairment had a higher risk of all-cause mortality (HR 2.25, 95%CI 1.02-4.98, p=0.04) and borderline risk for MACCE (HR 1.56, 95%CI 0.98- 2.50, p=0.06) compared to those with normal renal function. CONCLUSIONS: Renal impairment is common in patients with AF undergoing PCI and even mild renal impairment has an adverse prognostic effect in these patients requiring multiple antithrombotic medications.