Genotype Information and Courses from MediaLab, Inc.
These are the MediaLab courses that cover Genotype and links to relevant pages within the course.
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|Autoimmune Disease (continued)|
Why our immune system malfunctions is not completely understood. One current hypothesis is that the following series of events occurs resulting in the initiation of an autoimmune reaction. Gender and Genetic PredispositionA predisposition is usually the first step toward the development of an autoimmune reaction. Women are more likely to develop a systemic autoimmune disease than men. For example in SLE the female to male ratio is 9:1. The genotype of some individuals predetermines that their immune system will be more prone to a break in tolerance. This genetic susceptibility appears to be linked to multiple genes rather than a single gene. This is supported by evidence that some autoimmune diseases are more frequently encountered in certain ethnic groups compared to others. For example in American women between the ages of 15 and 64, the prevalence of SLE is 1 in 700 for Caucasians while it is 1 in 245 for African-American women.(Ref1) Evidence in one recent study suggests that the genes that impart an increased resistance to malaria unfortunately produce an increased susceptibility to the systemic autoimmune rheumatic diseases.(Ref2)Triggering eventThe second step is the occurrence of a triggering event that leads to a break in tolerance. For some very susceptible individuals this event might be exposure to an environmental trigger. These environmental triggers could be ubiquitous such as exposure to the Epstein Barr virus (EBV), or very limited, such as the exposure to leaking silicon from a breast implant. In others, the triggering event might be a change in hormonal balance. Whatever the case, the triggering event initiates the break in tolerance and the cascade of immunological events that eventually lead to the formation of an autoimmune disease begins.Development of autoantibodiesThe third step is the development of autoantibodies and subsequent development of clinical symptoms. Studies have shown that this process can take 3 years or longer and unfortunately, by the time the diagnosis is made, substantial damage to the body may have already occurred.
|The most frequent genotype among Rho (D) -negative persons is:||View Page|
|The hh genotype gives rise to:||View Page|
|What are the possible ABO genotypes of offspring of parents whose genotype is OO and AB:||View Page|
|What are the possible ABO genotypes of offspring of parents whose genotype is OA and OB:||View Page|
|What are the possible ABO genotypes of offspring of parents whose genotype is AA and BB:||View Page|
|Match appropriate genotype to its corresponding phenotype:||View Page|
Adverse drug reactions are a leading cause of morbidity and mortality in the United States. Drug metabolism is a process whereby drugs are delivered to the body, distributed, metabolized and then ultimately excreted. As there can be potentially significant differences between patient drug absorption, metabolism and excretion, molecular testing allows a physician to work with a patient's individual phenotype and/or genotype to deliver an optimum pharmaceutical selection and/or dosage.
|Follow-up Investigative Tests (Mother)|
If a pregnant woman is found to have an unexpected clinically significant antibody, routine antenatal serologic tests on the mother include Antibody identification to detect clinically significant antibodies. Antigen typing: Once the antibody is identified, the mother is tested for the corresponding antigen, which she should lack. Antibody titration: Laboratories have different protocols. Depending on the antibody titer, titration may be performed at 2 or 4 week intervals after 18 weeks gestation.Notes (titration): Maternal antibody titer is an unreliable indicator of fetal disease and is mainly done to determine if clinical fetal monitoring is warranted, e.g., Doppler ultrasonography of fetal cerebral blood flow or, more rarely, invasive monitoring such as amniocentesis. Careful quality control is needed for titrations. QC includes using red cells from donors with the same phenotype or likely genotype (e.g., R2r or R2R2) and titrating the new sample in parallel with the prior sample. A two-tube rise or more in a doubling dilution is considered a significant rise in titer. In the case of anti-D, a predetermined critical titer (often 16 or 32 for anti-D depending on the method) indicates the need for clinical fetal monitoring.
|Follow-up Investigative Tests (Father)|
Investigative tests on the father depend on which maternal antibodies are present.1. Mother has anti-D ABO and Rh typing with anti-D, -C, -E, -c,-e to determine probable Rh genotype* to predict the chance the fetus has of being Rh positive and affected by HDFN; Test for weak D if initial Rh typing appears to be D-negative. * For D+ fathers, the probable Rh genotype can be determined using serologic tests, i.e., DCEce typing to determine if the father is probably homozygous or heterozygous for D.2. Other maternal clinically significant antibodies Phenotype father for the corresponding antigen and its antithetical antigen (e.g., K and k)
|Molecular Genotyping - Introduction|
The application of DNA analysis to typing blood group antigens started in the early 1990s but is not yet widely available. Molecular methods exist for typing Rh (RHD and RHCE), Kell (K & k), Duffy (Fya & Fyb), and Kidd (Jka &Jkb) loci.In perinatal testing programs, molecular typing can determine the Rh type of the mother, father, and fetus and may be done if the mother has anti-D or another antibody known to cause HDFN. More specifically, if available, DNA methods are typically used in these circumstances: For women who type as weak D in serologic tests, to determine the Rh genotype of the mother to identify if she is partial D or weak D; For women who have made anti-D, to determine the Rh genotype of the father to see if fetal monitoring is needed; For women who have made anti-D, to determine the Rh type of the fetus if the father is heterozygous for RhD or unavailable for testing. Fetal blood typing can be done using fetal DNA from cells obtained by amniocentesis or by testing cell-free, fetal-derived DNA present in maternal plasma at 5 weeks gestation and later. Like all diagnostic methods, DNA typing has limitations and is not 100% sensitive and specific. For example: The blood group's molecular basis may be unknown; Not all alleles in ethnic populations are known; Rare mutations in the RHD and other genes may not be detected; Silencing changes (switching off of a gene) may affect antigen expression; Fetal typing using amniotic fluid may give false-negative results because of maternal cell contamination.
|Routine Serologic Tests - Father|
Policies for typing fathers vary widely and usually testing is not done unless the mother develops anti-D or another clinically significant antibody. However, for Rh negative women, some labs consider Rh typing the father if paternity is certain. For example: Tests on Father ABO and Rh type; Test for weak D if initial Rh typing appears to be D-negative. If father is Rh negative, the fetus will be Rh negative and antenatal RhIg is not needed. The purpose of DCEce typing Rh positive fathers is to determine if the father is homozygous or heterozygous for D in order to predict whether the fetus is Rh positive. The father's actual Rh genotype can be determined by molecular methods, if available.
|Molecular Genotyping - Father and Fetus|
Rh Genotype (Father and Fetus)As noted, usually molecular typing of the father is done only if the mother has anti-D or an antibody to another antigen for which molecular methods exist. In the case of a mother with anti-D and a father who is D+ using serologic methods, molecular typing can determine the father's RHD heterozygosity or homozygosity*. If the father is homozygous for the RHD allele, all of his offspring will be Rh positive, negating the need for fetal D testing, but indicating that the fetus should be monitored for HDFN. If the father is heterozygous for RHD, the Rh type of the fetus should be determined to see if HDFN is possible. * For D+ fathers, the probable Rh genotype can be determined using serologic tests, i.e., DCEce typing to determine if the father is probably homozygous or heterozygous for D (see later).
|Epidemiology of HFE Mutations|
The prevalence of common HFE mutations among persons with hereditary hemochromatosis (HH) has been reported in numerous studies conducted in the US, France, Australia, and other countries. Homozygous C282Y mutation (C282Y/C282Y) is present in 82% to 90% of Caucasian patients diagnosed with iron overload due to HH.(7) This suggests a strong link between the genotype and the phenotypic presentation of clinical iron overload. Much lower percentages of persons diagnosed with HH do not have two C282Y mutations. A small percentage of persons diagnosed with HH are compound heterozygotes for C282Y and H63D (C282Y/H63D), are homozygous for H63D (H63D/H63D), heterozygous for C282Y (C282Y/wild type) or for H63D (H63D/wild type), or carry S65C or other HFE mutations.It may be that symptomatic heterozygotes are actually HFE-compound heterozygotes with additional unidentified mutations modifying the expression of the more severe known mutation. It is quite possible that more mutations of HFE and elucidation of other gene mutations modifying HFE will be discovered in the future enabling scientists to better explain the phenotypic heterogeneity of this disorder.In the US, the C282Y mutation is most prevalent in the non-Hispanic white population. It is much less common among Hispanics and African Americans.
|Which genotype accounts for the greatest percentage of cases of hereditary hemochromatosis (HH)?||View Page|
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.
Those who type as group O must have two O genes present (since both the A and B genes would have produced recognizable antigens, neither of which is present on group O cells). Therefore, in the case of an AB individual or an O individual, we can tell exactly which genes are present, or a genotype. Group A or group B typing reveals only one gene product and thus only a phenotype can be determined. Persons of phenotype A can be genotype AA or AO , while those of phenotype B can be genotypically BB or BO. Family studies may be done to determine the genotype of an A or B individual. For example, if the mating of one A and one O parent produced a group O child, the second gene present in the A parent must have been O since the child has inherited one O gene from each parent.
|Determining Possible Offspring|
The mating of an A individual with another A individual can produce AA, AO, or OO offspring, depending on the genotype of the parents. This is illustrated by the Punnett squares on the next page. You can determine all possible offspring from ABO mating using these straightforward genetic principals.
|If an individual is type O, what is his/her ABO genotype?||View Page|
|CYP450 Induction and Inhibition|
Variables other than mutations also affect CYP450 enzymes. Many drugs are able to induce CYP450 enzymes, and CYP450s can be inhibited by a variety of substances. For example, CYP2D6 can be inhibited by the common medications cimetidine (Tagamet) and fluoxetine (Prozac). Since many patients are on multiple medications and since dietary and environmental factors can change, CYP450 expression levels cannot be solely predicted based on their genotype. Some CYP450 inducers and inhibitors are listed in the table on the following page.
|Genotype versus Phenotype|
Genotyping can give us a definitive profile of a given CYP450 enzyme's mutations. But since there are dozens of mutations usually associated with each enzyme, a complete characterization of a CYP450 is not always realistic. Without complete sequencing of the entire allele, it may not be possible to entirely rule out a mutation in a patient who shows none of the more common polymorphisms. If we consider the number of possible mutations and the possible presence of inducing/inhibiting substances, phenotyping for drug metabolism may sound more reasonable than genotyping.
|Warfarin Metabolism, continued|
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.
|Genotype versus Phenotype|
Phenotyping involves measuring the metabolism of a probe drug. For example, with CYP2D6, dextromethorphan or debrisoquine can be given to a patient to see how well the drug is metabolized. Both these drugs are safe and extensively metabolized by CYP2D6. By measuring the parent drug and the metabolite in urine, the metabolic capacity of a CYP450 enzyme can be estimated. Such testing is complex and tedious, however, and has not become routine in clinical laboratories. Therefore, genotyping is likely to be the main tool that is used for assessing the PGx of a patient.
Single nucleotide polymorphisms (SNP) genotype analysis looks for small changes in sequences instead of simply identifying the amplicon. One of the best ways to detect SNPs is to compare melting curves. The use of real-time PCR to create melting curves can detect differences as small as one base pair. Melting curve analysis is an assessment of the disassociation characteristics of double stranded DNA when exposed to heat. The strength of the hydrogen bond of each piece of DNA is dependent upon several different factors: the length of the DNA segment, the amount of guanine and cytosine pairs, and the degree of compliment. In real-time PCR the fluorescence that is given off by the probes will decrease once the strand of DNA disassociates. After the DNA or RNA is amplified, the temperature of the sample is slowly increased while the fluorescence is recorded. The melting points should show up as peaks when plotting temperature against fluorescence. These peaks allow one to differentiate between homozygous wild type, heterozygous, and homozygous mutant alleles. One clinical use of this method is to detect both HIV-1 and HIV-2 in samples.
|RhIg prophylaxis is typically given antenatally to Rh negative pregnant females without knowing the Rh of the fetus.||View Page|
|Routine Serologic Tests - Father|
FatherPolicies for Rh typing fathers vary widely and often Rh typing not done unless the mother develops anti-D.Some labs consider typing the father if paternity is certain. For example: ABO and Rh type father with anti-D, -C, -E, -c,-e to determine probable Rh genotype Test for weak D if initial Rh typing appears to be D-negative) If father is Rh negative, RhIg is not needed. The purpose of DCEce typing Rh positive fathers is to determine if the father is probably homozygous or heterozygous for D to predict the chance the fetus has of being Rh positive. For example: Rh Phenotype Results D C E c e + + –̶ + + For these results, the father could have one of three Rh genotypes: CDe/cde CDe/cDe cDe/Cde Because the most common is CDe/cde (R1r), the father would be assessed as probably heterozygous for D. The father's actual Rh genotype can be determined by molecular methods, if available.