5.2 N&V 493 MH

relevant DNA segment. To winnow the list, they turned to patients and rats with inherited defects in VKOR activity. They found that mutations in one particular gene occurred in two families with defective vitamin-K-dependent clotting factors, in four families with hereditary warfarin resistance and in numerous rat strains with resistance to warfarin-like poisons.They also identified similar genes in fish, frogs and even mosquitoes (although not in fruitflies). Rost et al. show that this candidate VKOR gene encodes a small transmembrane protein that is found in the endoplasmic reticulum, a subcellular compartment. Leaving open the possibility that this protein might be only one component of a larger complex, they name it vitamin K epoxide reductase complex subunit 1 (VKORC1). The protein produced from the normal gene shows VKOR activity that is inhibited by warfarin. As expected, the mutant protein encoded by the gene from patients with a clotting-factor deficiency is inactive. Surprisingly, though, the mutant proteins from warfarin-resistant people are fairly sensitive to warfarin when tested in cultured cells; it remains to be seen how VKORC1 mutations cause warfarin resistance in vivo. Li et al. did not have the benefit of access to patients with VKOR mutations, but overcame this obstacle in an ingenious way. They first ruled out genes from the relevant section of chromosome 16 that code for proteins with known or predicted functions,and then selected the 13 remaining candidates that encode proteins containing potential transmembrane portions (given that VKOR seemed to be a membrane protein). Their subsequent identification of the VKOR gene relied on the use of RNA interference. In this technique, double-stranded small interfering RNA molecules (siRNAs) are inserted into cells, where they target matching messenger RNAs for degradation. The result is that the gene encoding those messenger RNAs is effectively silenced. Li et al. designed siRNAs to silence each of the 13 candidate genes in human cells with a high level of VKOR activity. They found that targeting only one gene — identical to Rost and colleagues’ VKORC1 gene — caused a marked reduction in VKOR activity. When expressed in insect cells, this gene produced active VKOR, which was inhibited by warfarin. As the authors point out, this siRNA strategy should be useful in other positional cloning projects for which a functional assay can be devised, even if the activity of interest depends on many gene products. VKOR was previously thought to be a large multiprotein complex. But Li et al. found that expressing just one protein could confer VKOR activity on insect cells that lacked it. Although it seems a heavy burden for a small protein without obvious relationship to other reductases, this molecule alone may be responsible for recycling vitamin K. Now that we have tracked down the VKOR gene, it should be possible to identify the chemical source of the electrons that actually reduce vitamin K epoxide in living animals. Although various sulphydryl compounds work in the laboratory, a role for other common reductase cofactors, containing nicotinamide or flavin,can now be tested. It will also be important to characterize the structure of this apparent warfarin target, perhaps leading to the identification of better vitamin K antagonists for treating blood clots or, possibly, for poisoning rats. Finally, we now know that deficiency in multiple vitamin-K-dependent clotting factors can be caused by mutations in either of two genes — that for vitamin K carboxylase or, as shown by Rost et al., for VKORC1. Yet this may not be an exhaustive list. If it is not, then linkage studies in affected families might quickly lead to the discovery of yet more components of the vitamin K cycle. ■ J. Evan Sadler is at the Howard Hughes Medical Institute, Washington University School of Medicine, St Louis, Missouri 63110, USA. e-mail: esadler@im.wustl. edu