Naturally occurring UBIAD1 mutations differentially affect menaquinone biosynthesis and vitamin K-dependent carboxylation.

UbiA prenyltransferase domain-containing protein-1 (UBIAD1) is responsible for the biosynthesis of menaquinone-4 (MK-4), a cofactor for extrahepatic carboxylation of vitamin K-dependent (VKD) proteins. Genetic variations of UBIAD1 are mainly associated with Schnyder corneal dystrophy (SCD), a disease characterized by abnormal accumulation of cholesterol in the cornea. Results from in vitro studies demonstrate that SCD-associated UBIAD1 mutations are defective in MK-4 biosynthesis. However, SCD patients do not exhibit typical phenotypes associated with defects of MK-4 or VKD carboxylation. Here, we coupled UBIAD1's biosynthetic activity of MK-4 with VKD carboxylation in HEK293 cells that stably express a chimeric VKD reporter protein. The endogenous Ubiad1 gene in these cells was knocked out by CRISPR-Cas9-mediated genome editing. The effect of UBIAD1 mutations on MK-4 biosynthesis and VKD carboxylation was evaluated in Ubiad1-deficient reporter cells by determining the production of MK-4 or by measuring the efficiency of reporter-protein carboxylation. Our results show that the hot-spot mutation N102S has a moderate impact on MK-4 biosynthesis (retained ˜ 82% activity) but does not affect VKD carboxylation. However, the G186R mutation significantly affected both MK-4 biosynthesis and VKD carboxylation. Other mutations exhibit varying degrees of effects on MK-4 biosynthesis and VKD carboxylation. These results are consistent with in vivo results obtained from gene knock-in mice and SCD patients. Our findings suggest that UBIAD1's MK-4 biosynthetic activity does not directly correlate with the phenotypes of SCD patients. The established cell-based assays in this study provide a powerful tool for the functional studies of UBIAD1 in a cellular milieu.

A novel vitamin K derived anticoagulant tolerant to genetic variations of vitamin K epoxide reductase.

BACKGROUND: Vitamin K antagonists (VKAs), such as warfarin, have remained the cornerstone of oral anticoagulation therapy in the prevention and treatment of thromboembolism for more than half a century. They function by impairing the biosynthesis of vitamin K-dependent (VKD) clotting factors through the inhibition of vitamin K epoxide reductase (VKOR). The challenge of VKAs therapy is their narrow therapeutic index and highly variable dosing requirements, which are partially the result of genetic variations of VKOR. OBJECTIVES: The goal of this study was to search for an improved VKA that is tolerant to the genetic variations of its target enzyme. METHODS: A series of vitamin K derivatives with benzyl and related side-chain substitutions at the 3-position of 1,4-naphthoquinone were synthesized. The role of these compounds in VKD carboxylation was evaluated by mammalian cell-based assays and conventional in vitro activity assays. RESULTS: Our results showed that replacing the phytyl side-chain with a methylene cyclooctatetraene (COT) moiety at the 3-position of vitamin K1 converted it from a substrate to an inhibitor for VKD carboxylation. Strikingly, this COT-vitamin K derivative displayed a similar inhibition potency in warfarin-resistant VKOR mutations whose warfarin resistance varied more than 400-fold. Further characterization of COT-vitamin K for the inhibition of VKD carboxylation suggested that this compound targets multiple enzymes in the vitamin K redox cycle. Importantly, the anticoagulation effect of COT-vitamin K can be rescued with high doses of vitamin K1 . CONCLUSION: We discovered a vitamin K analogue that functions as a VKA and is tolerant to genetic variations in the target enzyme.

The Relationship Between Polypharmacy and Hospital-stay Duration: A Retrospective Study.

Background There have been very few studies on the association of polypharmacy with clinical course. In this paper, we seek to evaluate the relationship between polypharmacy and hospitalization period. Methods We retrospectively analyzed 322 patients hospitalized from February to September 2017, after excluding short-term and orthopedic cases. Patients with polypharmacy were defined as those who were prescribed more than five drugs at the time of admission. The primary endpoint for all subjects regardless of polypharmacy was the hospitalization period. Using Mann-Whitney U test results, we compared the average number of hospital days between patients with and without polypharmacy. Secondary endpoints were hospitalization period with and without polypharmacy for each disease type. Results The hospitalization period was significantly extended for patients with polypharmacy as compared to those without (31.6 vs. 23.2 days, p: 0.002). Those with an infection had significantly longer hospitalization than those without polypharmacy (27.6 vs. 18.1 days, p: 0.007). Malignancy, heart disease, and cerebrovascular disease did not have a significant effect on hospitalization regardless of polypharmacy. Conclusion Polypharmacy is related to an extended hospitalization period and is found to occur more frequently in patients hospitalized for an infection.