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THE PRIMER :: MOLECULAR DIAGNOSTICS


Gene differences lead to variations in drug response


Lessons from warfarin pharamacogenomics By John Brunstein, PhD


T


he term “pharmacogenomics” came into vogue roughly 15 years ago or so, when the dream of the Human Genome Project had just com-


pleted its first release. It was becoming apparent that novel sequencing technologies were going to make it feasible to sequence, if not entire genomes of patients, at least selected highly informative loci at a low enough cost and fast enough turn around time to have potential for front-line clini- cal utility. Many drugs have well understood and narrowly defined targets (such as a particular cel- lular receptor or intracellular enzyme) and may also have known specific interactions with enzymes responsible for activation (conversion of prodrug forms to active forms) and/or enzymes respon- sible for breakdown and clearance of the drug. This creates three obvious steps where individual variations in genetic sequence can alter response


to the drug: t Target molecule – changes in binding efficiency


and degree of activity modulation; t activation pathway – changes in kinetics of active


drug availability; and t degradation pathway – changes in clearance rates, impacting steady state levels and duration of impact.


There are additional possible chances for interplay between individual genetic variation and pharmacodynamics/pharmacokinetics, such as specific transport molecules, but the general gist remains the same. Genetic differences can lead to individual variations in dose responsiveness to a given drug, and for drugs with a narrow therapeutic window (the dosing level which maximizes benefits and minimizes side effects), knowledge of this should be applicable in determining aappropriate dosing regimens.


Warfarin—variations in target and clearance The simpler a given drug’s metabolism, the better understood the impact of various genetic varia- tion on this metabolism, and the narrower the therapeutic window the more this pharmacoge- nomic approach seems attractive. Warfarin (the name, as many readers may know, comes from Wisconsin Alumni Research Foundation WARF; also known under trademark as “Coumadin”) provided a convenient intersection of these attributes. Warfarin, and in particular its “S” stereoiso- mer form, acts indirectly as an anticoagulant by inhibiting the activity of the enzyme VKORC1, the vitamin K epoxide reductase. Reduced vita- min K is needed in the clotting cascade to convert


60 JULY 2019 MLO-ONLINE.COM


inactive Factors II, VII, IX, and X to their active forms during clot formation, so a lack of reduced vitamin K slows the clotting process. The “S” enantiomer of warfarin is in turn degraded by a cytochrome CYP2C9, a member of the Cytochrome P450 family which is active in degrading a num- ber of drugs (such as NSAIDs and angiotensin II receptor blockers).


Not all VKCOR1 genes are identical. In fact, there are quite a few known allelic variations in this gene but two in particular—1173 C>T and 1639 G>A— are associated with less translation of the VKORC1 mRNA into protein, leading to lower levels of enzyme available. (A quick aside here, those codes aren’t as mysterious as they look. They’re the number of the nucleotide in the gene changed, the wild type or “normal” base found there, and the mutated form.) None of us need special training in enzyme kinetics or pharmacology to grasp the key point here: If you have less enzyme around, and your goal is to partially block or slow down clotting (not just stop it altogether, which would be very dangerous), then you want less enzyme inhibitor in the system than you would want in the case of someone starting off with more enzyme. There’s additional nuances related to whether people are homozygous or heterozygous for these mutations to factor in as well, but to a first approximation we have a pretty good idea of how much VKORC1 is present in each of these genetic scenarios, and thus, some idea of how much we’d like to suppress it in order for all of these cases to end up with the same optimal therapeutic window of VKCOR1 activity. We also need to think about genetic variations in CYP2C9, though, because these will influence what the duration and effective level of warfarin is in the system. A faster version of the enzyme will require more frequent or larger doses to maintain the same drug level then a sluggish version of the enzyme, where a dose will linger around longer. It turns out there’s a whole bunch of known CYP2C9 single nucleotide genetic variants which impact speed of warfarin breakdown, but two are particularly signifi- cant. CYP2C9*2 (these mutations get their own spe- cial names; in the coding system referenced above, this would be 430 C>T) has only about 70 percent of normal activity for warfarin clearance, and CYP2C9*3 (aka 1075 A>C) is a dismal 20 percent of normal rate. The caveats above regarding homo- zygous vs. heterozygous apply here as well, but the clear bottom line is if you blindly give the same (optimal) dose you’d give on a CYP2C9 wild type individual to someone homozygous for CYP2C9*3,


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