Variants of Unknown Significance in Genes Associated with Heritable Thoracic Aortic Disease Can Be Low Penetrant "Risk Variants".

Thoracic aortic aneurysms leading to acute aortic dissections are a preventable cause of premature deaths if individuals at risk can be identified. Individuals with early-onset aortic dissections without a family history or syndromic features have an increased burden of rare genetic variants of unknown significance (VUSs) in genes with pathogenic variants for heritable thoracic aortic disease (HTAD). We assessed the role of VUSs in the development of disease using both in vitro enzymatic assays and mouse models. VUSs in LOX and MYLK identified in individuals with acute aortic dissections were assayed to determine whether they disrupted enzymatic activity. A subset of VUSs reduced enzymatic activity compared to the wild-type proteins but less than pathogenic variants. Additionally, a Myh11 variant, p.Arg247Cys, which does not cause aortic disease in either humans or mice, was crossed with the Acta2-/- mouse, which has aortic enlargement with age while Acta2+/- mice do not. Acta2+/-Myh11R247C/R247C mice have aortic dilation by 3 months of age without medial degeneration, indicating that two variants not known to cause disease do lead to aortic enlargement in combination. Furthermore, the addition of Myh11R247C/R247C to the Acta2-/- mouse model accelerates aortic enlargement and increases medial degeneration. Therefore, our results emphasize the need for a classification system for variants in Mendelian genes that goes beyond the 5-tier system of pathogenic, likely pathogenic, VUS, likely benign, and benign, and includes a designation for low-penetrant "risk variants" that trigger disease either in combination with other risk factors or in a stochastic manner.

MYLK pathogenic variants aortic disease presentation, pregnancy risk, and characterization of pathogenic missense variants.

PURPOSE: Heritable thoracic aortic disease can result from null variants in MYLK, which encodes myosin light-chain kinase (MLCK). Data on which MYLK missense variants are pathogenic and information to guide aortic disease management are limited. METHODS: Clinical data from 60 cases with MYLK pathogenic variants were analyzed (five null and two missense variants), and the effect of missense variants on kinase activity was assessed. RESULTS: Twenty-three individuals (39%) experienced an aortic event (defined as aneurysm repair or dissection); the majority of these events (87%) were aortic dissections. Aortic diameters were minimally enlarged at the time of dissection in many cases. Time-to-aortic-event curves showed that missense pathogenic variant (PV) carriers have earlier-onset aortic events than null PV carriers. An MYLK missense variant segregated with aortic disease over five generations but decreases MYLK kinase acitivity marginally. Functional Assays fail to identify all pathogenic variants in MYLK. CONCLUSION: These data further define the aortic phenotype associated with MYLK pathogenic variants. Given minimal aortic enlargement before dissection, an alternative approach to guide the timing of aortic repair is proposed based on the probability of a dissection at a given age.

Development of Poly Unsaturated Fatty Acid Derivatives of Aspirin for Inhibition of Platelet Function.

The inhibition of platelet aggregation is key to preventing conditions such as myocardial infarction and ischemic stroke. Aspirin is the most widely used drug to inhibit platelet aggregation. Aspirin absorption can be improved further to increase its permeability across biologic membranes via esterification or converting the carboxylic acid to an anhydride. There are several reports indicating that ω-3 and ω-6 fatty acids such as linoleic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) separately inhibit platelet aggregation. Herein, we synthesize anhydride conjugates of aspirin with linoleic acid, EPA, and DHA to form aspirin anhydrides that are expected to have higher permeability across cellular membranes. These aspirin-fatty acid anhydrides inhibited platelet aggregation in washed human platelets and platelet-rich plasma in a dose-dependent manner. In particular, the aspirin-DHA anhydride displayed similar effectiveness to aspirin. Platelet aggregation studies conducted in the presence of various platelet agonists indicated that the aspirin-lipid conjugates act through inhibition of the cyclooxygenase (COX)-thromboxane synthase (TXAS) pathway. Hence, we performed detailed biochemical studies using purified COX-1 as well as TXAS stabilized in nanoscale lipid bilayers of nanodiscs to confirm results from the platelet aggregation studies. We show that although all of the aspirin conjugates act through the COX-TXAS pathway by inhibiting COX-1, the parent fatty acids do not act via this pathway. Finally, we studied the hydrolysis of these compounds in buffer and human plasma, and we demonstrate that all of the aspirin-fatty acid conjugates hydrolyze to the parent molecules aspirin and fatty acid in a controlled manner.

Genetic variation in UGT genes modify the associations of NSAIDs with risk of colorectal cancer: colon cancer family registry.

The use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with reduced risk of colorectal neoplasia. Previous studies have reported that polymorphisms in NSAID-metabolizing enzymes central to NSAID metabolism including UDP-glucuronosyltransferases (UGT) and cytochrome P450 (CYP) 2C9 may modify this protective effect. We investigated whether 35 functionally relevant polymorphisms within CYP2C9 and UGT genes were associated with colorectal cancer risk or modified the protective effect of NSAIDs on colorectal cancer susceptibility, using 1,584 colorectal cancer cases and 2,516 unaffected sibling controls from the Colon Cancer Family Registry. A three-SNP genotype in UGT1A6 (G-A-A; Ala7-Thr181-Arg184) and the Asp85 variant in UGT2B15 increased the risk of colorectal cancer (OR 3.87; 95% CI 1.04-14.45 and OR 1.34; 95% CI 1.10-1.63, respectively). We observed interactions between UGT1A3 Thr78Thr (A>G) and NSAID use (P-interaction = 0.02), a three-SNP genotype within UGT2B4 and ibuprofen use (P-interaction = 0.0018), as well as UGT2B15 Tyr85Asp (T>G) and aspirin use (P-interaction = 0.01). The interaction with the UGT2B4 and the UGT2B15 polymorphisms were noteworthy at the 25% FDR level. This study highlights the need for further pharmacogenetic studies to identify individuals who might benefit from NSAID use as part of developing effective strategies for prevention of colorectal neoplasia. © 2014 Wiley Periodicals, Inc.

IκBKß and NFκB1, NSAID use and risk of colorectal cancer in the Colon Cancer Family Registry.

The NFκB-signaling pathway regulates cell proliferation and inflammation. Activation of the pathway is implicated in the etiology of colorectal cancer (CRC). NSAIDs may reduce CRC risk partially through a nuclear factor-kappa B (NFκB)-dependent pathway. In this study, we investigated associations between 34 NFκB1 and 8 IκBKß tagSNPs and CRC risk and examined interactions with non-steroidal anti-inflammatory drug (NSAID) use. Using conditional logistic regression, we investigated these associations among 1584 incident CRC cases and 2516 sibling controls from the Colon Cancer Family Registry. Three IκBKß SNPs were associated with a statistically significant lower colorectal or colon cancer risk: rs9694958 (A>G intron 5) (colorectal: OR(hzv) = 0.26(0.07-0.99), P(trend) = 0.048, P(adj) = 0.25), rs10958713 (A>C intron 19) (colon: OR(hzv) = 0.62(0.42-0.92), P(trend) = 0.005, P(adj) = 0.03) and rs5029748 (C>A intron 2) (colon: OR(het) = 0.72(0.56-0.91), P(trend) = 0.01, P(adj) = 0.08). We replicated trends associated with NFκB1 and IκBKß variants identified in a previous study (rs4648110 (T>A intron 22), rs13117745 (G>A intron 5) and rs3747811 (T>A intron 1)). IκBKß's rs6474387 (C>T intron 20) and rs11986055 (A>C intron 2) showed substantially lower colon cancer risk among current NSAID users (P(interaction) = 0.01 and P(interaction) = 0.045, respectively), whereas NFκB1's rs230490 (G>A 5' (outside UTR)) and rs997476 (C>A 3' (outside UTR)) showed higher CRC risk among current NSAID users (P(interaction) = 0.01 and P(interaction) = 0.03, respectively). These findings suggest that variants in NFκB1 and IκBKß are associated with CRC risk and NSAIDs may function partially through an NFκB-dependent pathway. The SNPs identified here should be considered for future functional studies and may be useful in designing a pharmacogenetic approach to preventive NSAID use.

COX-1 (PTGS1) and COX-2 (PTGS2) polymorphisms, NSAID interactions, and risk of colon and rectal cancers in two independent populations.

PURPOSE: Nonsteroidal anti-inflammatory drugs (NSAIDs) target the prostaglandin H synthase enzymes, cyclooxygenase (COX)-1 and COX-2, and reduce colorectal cancer risk. Genetic variation in the genes encoding these enzymes may be associated with changes in colon and rectal cancer risk and in NSAID efficacy. METHODS: We genotyped candidate polymorphisms and tag SNPs in PTGS1 (COX-1) and PTGS2 (COX-2) in a population-based case­control study (Diet, Activity and Lifestyle Study, DALS) of colon cancer (n = 1,470 cases/1,837 controls) and rectal cancer (n = 583/775), and independently among cases and controls from the Colon Cancer Family Registry (CCFR; colon n = 959/1,535, rectal n = 505/839). RESULTS: In PTGS2, a functional polymorphism (-765G[C; rs20417) was associated with a twofold increased rectal cancer risk (p = 0.05) in the DALS. This association replicated with a significant nearly fivefold increased risk of rectal cancer in the CCFR study (ORCC vs. GG = 4.88; 95 % CI 1.54­15.45; ORGC vs. GG = 1.36; 95 %CI 0.95­1.94). Genotype­NSAID interactions were observed in the DALS for PTGS1 and rectal cancer risk and for PTGS2 and colon cancer risk, but were no longer significant after correcting for multiple comparisons and did not replicate in the CCFR. No significant associations between PTGS1 polymorphisms and colon or rectal cancer risk were observed.

Glutathione peroxidase tagSNPs: associations with rectal cancer but not with colon cancer.

Glutathione peroxidases (GPXs) are selenium-dependent enzymes that reduce and, thus, detoxify hydrogen peroxide and a wide variety of lipid hydroperoxides. We investigated tagSNPs in GPX1-4 in relation to colorectal neoplasia in three independent study populations capturing the range of colorectal carcinogenesis from adenoma to cancer. A linkage-disequilibrium (LD)-based tagSNP selection algorithm (r(2) ≥ 0.90, MAF ≥ 4%) identified 21 tagSNPs. We used an identical Illumina platform to genotype GPX SNPs in three population-based case-control studies of colon cancer (1,424 cases/1,780 controls), rectal cancer (583 cases/775 controls), and colorectal adenomas (485 cases/578 controls). For gene-level associations, we conducted principal component analysis (PCA); multiple logistic regression was used for single SNPs. Analyses were adjusted for age, sex, and study center and restricted to non-Hispanic white participants. Analyses of cancer endpoints were stratified by molecular subtypes. Without correction for multiple testing, one polymorphism in GPX2 and three polymorphisms in GPX3 were associated with a significant risk reduction for rectal cancer at α = 0.05, specifically for rectal cancers with TP53 mutations. The associations regarding the three polymorphisms in GPX3 remained statistically significant after adjustment for multiple comparisons. The PCA confirmed an overall association of GPX3 with rectal cancer (P = 0.03). No other statistically significant associations were observed. Our data provide preliminary evidence that genetic variability in GPX3 contributes to risk of rectal cancer but not of colon cancer and thus provide additional support for differences in underlying pathogenetic mechanisms for colon and rectal cancer.

Decreased cyclooxygenase inhibition by aspirin in polymorphic variants of human prostaglandin H synthase-1.

OBJECTIVES: Aspirin (ASA), a major antiplatelet and cancer-preventing drug, irreversibly blocks the cyclooxygenase (COX) activity of prostaglandin H synthase-1 (PGHS-1). Considerable differences in ASA effectiveness are observed between individuals, and some of this variability may be due to PGHS-1 protein variants. Our overall aim is to determine which, if any, of the known variants in the mature PGHS-1 protein lead to functional alterations in COX catalysis or inhibition by ASA. The present study targeted four PGHS-1 variants: R53H, R108Q, L237M, and V481I. METHODS: Wild-type human PGHS-1 and the four polymorphic variants were expressed as histidine-tagged, homodimeric proteins in insect cells using baculovirus vectors, solubilized with a detergent, and purified by affinity chromatography. The purified proteins were characterized in vitro to evaluate COX and peroxidase (POX) catalytic parameters and the kinetics of COX inhibition by ASA and NS-398. RESULTS: Compared with the wild type, several variants showed a higher COX/POX ratio (up to 1.5-fold, for R108Q), an elevated arachidonate Km (up to 1.9-fold, for R108Q), and/or a lower ASA reactivity (up to 60% less, for R108Q). The decreased ASA reactivity in R108Q reflected both a 70% increase in the Ki for ASA and a 30% decrease in the rate constant for acetyl group transfer to the protein. Computational modeling of the brief ASA pulses experienced by PGHS-1 in circulating platelets during daily ASA dosing predicted that the 60% lower ASA reactivity in R108Q yields a 15-fold increase in surviving COX activity; smaller, approximately two-fold increases in surviving COX activity were predicted for L237M and V481I. NS-398 competitively inhibited COX catalysis of the wild type (Ki=6 µmol/l) and inhibited COX inactivation by 1.0 mmol/l ASA in both the wild type (IC50=0.8 µmol/l) and R108Q (IC50=2.1 µmol/l). CONCLUSION: Of the four PGHS-1 variants examined, R108Q exerts the largest functional effects, with evidence for impaired interactions with a COX substrate and inhibitors. As Arg108 is located on the protein surface and not in the active site, the effects of R108Q suggest a novel, unsuspected mechanism for the modulation of the PGHS-1 active site structure. The lower intrinsic ASA reactivity of R108Q, V481I, and L237M, combined with the rapid hydrolysis of ASA in the blood, suggests that these variants decrease the antiplatelet effectiveness of the drug. These PGHS-1 variants are uncommon but ASA is used widely; hence, a considerable number of individuals could be affected. Further examination of these and other PGHS-1 variants will be needed to determine whether PGHS-1 genotyping can be used to personalize anti-COX therapy.

Functional analysis of human thromboxane synthase polymorphic variants.

BACKGROUND: Thromboxane A synthase (TXAS) metabolizes the cyclooxygenase product prostaglandin (PG) H2 into thromboxane H2 (TXA2), a potent inducer of blood vessel constriction and platelet aggregation. Nonsynonymous polymorphisms in the TXAS gene have the potential to alter TXAS activity and affect TXA2 generation. OBJECTIVES: The aim of this study was to assess the functional effects of genetic variants in the TXAS protein, including K258E, L357V, Q417E, E450K, and T451N. METHODS: Wild-type TXAS and the variant proteins were expressed in a bacterial system and purified by affinity and hydroxyapatite chromatography. The two characteristic catalytic activities of TXAS were assayed in each of the purified recombinant proteins: isomerization of PGH2 to TXA2 and fragmentation of PGH2 to 12-hydroxyheptadecatrienoic acid and malondialdehyde. RESULTS: All of the variants showed both isomerization and fragmentation activities. The Km values of the variants ranged from 27 to 52 µmol/l PGH2 (wild-type value: 32 µmol/l PGH2); the Vmax values of the variants ranged from 18 to 40 U/mg (wild-type value: 41 U/mg). The kinetic differences were largest for the L357V variant, whose Vmax/Km ratio was just 27% of the wild-type value. CONCLUSION: The increased Km and decreased Vmax values observed with L357V suggest that this variant may generate less TXA2 at the low levels of PGH2 expected in vivo, raising the possibility of attenuated signaling through the thromboxane pathway.

Functional and structural roles of residues in the third extramembrane segment of adrenal cytochrome b561.

Several residues in the third extramembrane segment (EM3) of adrenal cytochrome b(561) have been proposed to be involved in this cytochrome's interaction with ascorbate, but there has been no systematic evaluation of residues in the segment. We used alanine scanning mutagenesis to assess the functional and structural roles of the EM3 residues and several adjacent residues (residues 70-85) in the bovine cytochrome. Each alanine mutant was expressed in a bacterial system, solubilized with detergent, and affinity-purified. The recombinant proteins contained approximately two hemes per monomer and, except for R74A, retained basic functionality (≥ 94% reduced by 20 mM ascorbate). Equilibrium spectrophotometric titrations with ascorbate were used to analyze the α-band line shape and amplitude during reduction of the high- and low-potential heme centers (b(H) and b(L), respectively) and the midpoint ascorbate concentrations for the b(H) and b(L) transitions (C(H) and C(L), respectively). Y73A and K85A markedly narrowed the b(H) α-band peak; other mutants had weaker effects or no effect on b(H) or b(L) spectra. Relative changes in C(H) for the mutants were larger than changes in C(L), with 1.5-2.9-fold increases in C(H) for L70A, L71A, Y73A, R74A, N78A, and K85A. The amounts of functional b(H) and b(L) centers in additional Arg74 mutants, assessed by ascorbate titration and EPR spectroscopy, declined in concert in the following order: wild type > R74K > R74Q > R74T and R74Y > R74E. The results of this first comprehensive experimental test of the proposed roles of EM3 residues have identified residues with a direct or indirect impact on ascorbate interactions, on the environment of the b(H) heme center, and on formation of the native b(H)-b(L) unit. Surprisingly, no individual EM3 residue was by itself indispensable for the interaction with ascorbate, and the role of the segment appears to be more subtle than previously thought. These results also support our topological model of the adrenal cytochrome, which positions b(H) near the cytoplasmic side of the membrane.