Polymorphism Information and Courses from MediaLab, Inc.

Molecular diagnostic techniques utilize CE extensively. Automation, microvolume sample, increased sensitivity, immediate detection, and the computerization provided by CE enhance the analysis of nucleic acids. A multiple fluorescence detection system available with CE is also valuable.CE analysis of short tandem repeat polymorphisms is used in forensics, parentage testing, bone marrow engraftment analysis and other identification assays. Other testing for diagnosis of genetic diseases, oncology studies and DNA sequencing frequently utilize CE. DNA sequencing uses CE for separation of nucleotides labeled with multiple colored fluorescence dyes; CE and these markers enable computerized determination of the nucleotide sequence of DNA segments.

The reason to chose a particular molecular method can be influenced by disease detection, monitoring or therapy in certain patient populations. Molecular methodologies can be used to identify alterations or variations or changes in DNA sequencing that can cause disease. Sequence alterations that are known to cause disease are termed mutations. These changes or mutations can be applied to areas of the clinical lab such as infectious disease, paternity, genetic testing, and pharmacogenetics. Some of the more common alterations are:Deletion: a missing nucleotide or other portion of DNA sequence Insertion: an extra DNA nucleotide or other portion of DNA sequence Missense: a nucleotide or sequence substitution that codes for a different amino acidNonsense: a nucleotide substitution that ends in early termination of the protein manufacturing process; usually due to a stop codon.The most common alteration is a single base change or single nucleotide polymorphism (SNP)

For reasons as yet unknown, not all individuals who are homozygous for the C282Y mutation display phenotypic features of HH, and persons with H63D polymorphisms rarely develop iron overload. The penetrance (percentage of individuals with a specific genotype who express the associated phenotype) of HFE mutations is generally considered to be low. Results of a recent meta analysis by the US Preventive Services Task Force conclude that 38% to 50% of all C282Y homozygotes develop some evidence of iron overload, but that only 10% to 33% develop clinical disease due to HH. (8) In other words, some individuals may have elevated iron test results such as transferrin saturation, but do not demonstrate significant organ damage. Estimates of penetrance in some studies have found it to be even lower. Penetrance of HFE mutations is currently a controversial subject among experts, and the significance of finding HFE mutations in a given individual is often unclear. The probability that a given individual with HFE mutations will develop clinical disease from iron overload cannot be determined at this time.

To discuss PGx, we must first define two terms - polymorphism and cytochrome P450 (CYP450).A polymorphism is a variation in a gene (allele) that affects at least 1% of the population. CYP450 refers to a family of enzymes found predominantly in the liver. CYP450 enzymes work on a variety of substrates (drugs), altering their chemical structures to facilitate excretion in the urine and feces. There are many known polymorphisms in CYP450 enzymes.

The ultimate goal in measuring CYP450 function or identifying polymorphisms is to predict effective therapeutic doses and responses in patients.Polymorphisms are identified using molecular techniques (allele-specific PCR, restriction digests, sequencing, hybridization assays, bead-based systems, microarrays, pyrosequencing, et al).Although most clinical labs do not offer PGx testing, reference labs are beginning to market these tests. For example, one reference laboratory in the Midwest that offers CYP2D6 profiling measures about one dozen of the most common and significant mutation sites on this enzyme. This allows for detection of approximately 98% of the known CYP2D6 polymorphisms. The laboratory then generates a report which will advise the physician on the patient's drug-metabolizing status.Estimates show that 6-10% of the general population have a complete deficiency of CYP2D6, with the prevalence of mutations varying from <1% to as much as 21% within a given population.

By knowing a patient's disposition to specific drugs, the physician should be able to start the patient on an appropriate regimen rather than perfecting treatment based on trial and error. Drugs whose metabolism may prove to be problematic can be avoided, and second-line therapies that are metabolized by different, unaffected enzymes can be chosen. Clinical chemists, pharmacologists, and physicians need to translate knowledge of CYP450 polymorphisms into clinically-validated treatment algorithms. Dosing recommendations for PM, EM, IM and UM patients are beginning to appear in the literature for various classes of drugs, and the FDA is encouraging the incorporation of pharmacogenomic testing in the development process for new drugs.

The genes involved in warfarin metabolism are CYP2C9 and vitamin K epoxide reductase complex subunit 1 (VKOR). Warfarin owes its anticoagulant action to its inhibition of VKOR. This enzyme recycles vitamin K, a critical element for the clotting factors II, VII, IX, and X, as well as for proteins C, S, and Z. There are six CYP2C9 alleles that are known to cause prolonged metabolism of warfarin: CYP2C9 *2, *3, *4, *5, *6, and *11. (Polymorphisms in CYP450 genes are denoted with asterisks.)One-third of the patients that receive warfarin metabolize it differently than expected and experience a higher risk of bleeding.Genetic testing for the two most common polymorphisms (CYP2C9*2 and *3) as well as for VKOR may be able to reduce the variability associated with warfarin dosing response. Labs performing PGx testing can provide general warfarin dosing recommendations based on the patient's genotype analysis. The lab report will indicate whether a patient has a normal, mild, moderate, high, or very high sensitivity to warfarin. For example, a patient who has one CYP2C9 normal wild-type allele (CYP2C9 *1), one polymorphism (CYP2C9*3), and also a VKOR polymorphism is predicted to have a moderate sensitivity to warfarin. This patient should have frequent INR monitoring and possible warfarin dose reduction. It is important to recognize that knowing a genotype does not necessarily guarantee accurate dose prediction; other drugs and/or environmental or disease factors can also alter CYP2C9 activity. Therefore, monitoring the INR is still very important.