Pharmacogenetics
Pharmacogenetics studies how genes influence the efficacy and side-effects of drugs. It explains why different people react differently to medicines. Other related terms are pharmacogenomics (the interactions between drug and the genome), pharmacokinetics (metabolism of drugs) and pharmacodynamics (the interactions between drugs and their molecular targets). |
| Stages of drug metabolism (c) Elsevier |
Drug metabolism occurs mainly in the liver, where glucuronide conjugation and acetylation are the most common mechanisms of metabolising drugs. Metabolism is usually two-phased: First a polar group is added to the drug molecule to make it water-soluble. This is usually due to P450 enzyme, and includes hydroxylation or oxidation.Then the drug is metabolised into an excretable product. This phase often uses N-acetyltransferase (NAT) or glutathione S-transferase (GST).
Isoniazid is a drug used to treat tuberculosis. It absorbs quickly from the gut, and the level of isoniazid in the blood rises fast. The speed of metabolization depends on the enzyme NAT2, which has two alleles: fast acetylation and slow acetylation. Users homozygous for the slow acetylation metabolize isoniazid very slowly. For them the drug may cause liver damage and neural inflammation, because the toxic metabolites accumulate to the body. Certain genotypes concerning CYP2E1 and GSTM1 enzymes are also connected to slow metabolization of isoniazid and therefore hepatotoxicity. 50 % of West Europeans are homozygous slow acetylators.
Some hemolytic anemias such as favism are caused by deficiency of the enzyme G6PD, glucose-6-phosphate dehydrogenase. It also causes sensitivity against primaquine, a malaria drug. G6PD deficiency is inherited as X-linked recessive trait. If G6PD deficient people use primaquine, they develop black urine, jaundice and low hemoglobine due to destruction of red cells. The condition is not lethal. The deficiency is very common in Africa where malaria is common, which may support the claim that the deficiency protects against malaria.
Coumarin and other anti-blood-clotting drugs mut be very carefully dosed to each patient. There is extiensive variation between individual reactions to the drug, and a relatively high risk of severe bleeding complications if the dose is incorrect. Genes, ethnicity, the intake of vitamin K, diet, health and other drugs all have an impact on coumarin. Genetically the most important factors are polymorphism is CYP2C9 and VKORC1. The enzyme CYP2C9 has over 29 variants, and accounts for 15 % of all drug metabolism in the liver. Its most common variants are 20-70 % less effective enzymatically, and therefore need smaller dosages than fast metabolizers. VKORC1 is a vitamin K reductase, which reduces a by-product of blood-clotting factors into vitamin K. Vitamin K is essential in blood clotting. Warfarin, a drug similar to coumarin, inhibits VKORC1.
More on dosing blood clotting drugs can be found from http://www.warfarindosing.org.
Ethical dilemmas associated with gene tests and prenatal screening
Prenatal screening means the screening for inherited diseases in fetuses. It is a manner of genetic testing. Gene tests can also be done to humans of all ages to- detect carriers of inherited disorders
- predict late-onset diseases, which cause symptoms only as the patient grows older
- detect of increased risk of multifactorial diseases
- detect of risk to adverse drug effects
- diagnose cancer and determine most effective medication.
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| (c) McGraw-Hill |
Ethical dilemmas arise from several factors. One is the clinical validity: how trustworthy the test is? Ultrasonography images and test results may show unclear results, subtle anomalies of unknown significance or offer mild indications of a disease. In which case should these findings be concidered strong enough to decide whether to keep or abort the fetus? Laws may dictate that abortion is either illegal or legal only under certain conditions. Invasive tests also increase the risk of miscarriage. Lastly, how to estimate the risk of anxiety if the test results are inconclusive, or a sick child is born despite negative test results?
There is also a possibility that the result is erroneous, but it can be easily checked by taking another test.
Fundamental ethical principles are
- autonomy: the respect for privacy and absolute confidentiality. The patient is informed of the risks and validity of the offered test, and other options available.
- beneficence: the test is done for the best interest of the patient.
- non-maleficence: the principle of not doing harm.
- justice: fairness, equity of access and opportunity. This is especially important when concerning countries without government-supported healthcare, where all mothers should have equal opportunities to participate to gene tests.
Gene therapy
Gene therapy is the deliberate introduction of genetic material into human somatic cells for therapeutic, prophylactic or diagnostic purposes. Currently gene therapy is still only experimental, and tried mostly on cancers. No major breakthroughs have been achieved.Gene therapy can be either done on germline cells or autosomal cells. Germline mutations are permanent and inherited, due to which they are banned in many countries. No current trials use germline therapy. Autosomal cell therapies aim to modifying specific cells or tissues of the patient.
There are a variety of techniques used for gene therapy:
– Delivery of synthetic or recombinant nucleic acids into humans
– Genetically modified vectors (viruses or plasmids). This is the most used technique, covering over 60 % of all gene therapy trials. The usability of a given virus depends on the amount of foreign DNA it can carry, how it interacts with the host genome and how the transgene is expressed.
– Genetically modified stem cells
– Oncolytic viruses, which target only cancel cells, proliferate inside it and kill the cell after releasing new virusparticles to another cancer cells.
– Nucleic acids associated with delivery vehicles
– Naked nucleic acids
– Antisense techniques
– Genetic vaccines
– RNA interference
– Xenotransplantation of animal cells (but not solid organs)
For somatic cell therapy, the techniques can be divided into four methods. In gene supplementation a working copy of a gene is inserted to its target cells to treat loss-of-function conditions.Gene replacement replaces a mutant gene, and targeted inhibition silences certain genes. Lastly, gene therapy can be used for targeted killing of specific cells. In all cases the target gene can be inserted either directly to the patient cells (in vivo), or by growing and infection the patient's own cells with the target gene in a cell culture before injecting them to the patient (ex vivo).
All the previous techniques can be used to treat cancer. Supplementation can restore tumor suppressor gene function. Inactivation can silence an oncogene, and manipulation of tumor cells can lead to apoptosis. Cells can also be made more antigenic, and thus promote the immune system to target the cancer cells. Cell killing can be done by using oncolytic viruses, which targets certain cells. Another method is the suicide gene therapy. In suicide gene therapy the cells are modified to turn a safe substance into a toxic one. For example, a retrovirus is modified to infect cells with herpes virus kinase. The virus is injected directly to the tumor, where it infects only dividing cells. When the cells have been infected, they are treated with a drug, which the kinase metabolizes into a toxic substance. The infected and drug-treated cells die.
Problems in gene therapy arise from several factors. Most serious problems are
- temporary effects: to be effective, some therapies need constant injections or "rounds" of gene therapy.
- immune response: patient's body may recognize the engineered cells and destroy them. Since the immune response is stronger in later infections, the patient may become immune to gene therapy.
- usage of viral vectors carries a risk of viral infections and mutated viruses in addition to possible toxicity and immune responses.
- multigene disorders cannot at the moment be treated with gene therapy at all. It is suitable only for monogenic diseases.
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| Ups and downs of gene therapy (c) Elsevier |
RNA-therapy is a form of genetic therapy which uses RNA to affect genes and genetic expression. One example is the antisense nucleotide therapy used to treat Duchenne muscular dystrophy.
The principle of antisense oligonucleotide therapy for Duchenne muscular dystrophy
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| Dystrophin in a DMD patient and patients treated with AON. (c) Garland science |
A milder version of DMD is the Becker syndrome, where the patients have a normal life expectancy. Becker syndrome patients have a partially functioning dystrophin, which can be smaller in size than normal, and/or have reduced abundance. The difference of DMD and Becker is caused by a genetic mutation. In DMD, the dystrophin protein has an out-of-frame deletion, which leads to an altered reading frame and eventually to a truncate and unstable protein. Becker patients have an in-frame deletion, which leads to a shorter but partly functional protein.
The antisense oligoribonucleotide (AON) therapy targets at fixing the broken reading frame. The deletion between exons 48 and 52 is severe, because the exons are not compatible. Therefore the spliceosome stops at the exon 48, unable to continue to 52. The gene therapy aims at bridging this cap. The antisense oligoribonucleotides are tailored to hybridize to exon 51 and hide it from the spliceosome. The exons 48 and 52 are compatible, so the spliceosome continues and runs to the end of the protein. The final product is a shortened protein, which is however party functional. Gene therapy does not cure DMD - it merely makes the disease milder and increases the lifespan of the patients.
Currently gene therapy has been tested on 150 antisense oligonucleotides. The deletion of exons 49-50 is just one possible mutation causing DMD, and other mutations need their own oligonucleotides. First clinical studies have been performed on three AON therapy methods to hide the exon 51. One of these requires weekly abdominal injections.
Examples of genetic diseases
| Disease name | Cause | Inheritance | Symptoms |
| Hutchinson-Gilford Progreria Syndrome | de novo point mutations in LMNA gene in 1q22 | - | Growth failure, loss of body fat and hair, aged-looking skin, stiffness of joints, hip dislocation, generalized atherosclerosis, cardiovascular (heart) disease and stroke |
| Huntington disease | CAG-repeat expansion in 4p16.3 | AD | Movement abnormality, involuntary movements, memory impairment, dementia |
| Spinal and bulbar muscular atrophy | Expansion of a CAG-repeat in androgen receptor | X-linked | Muscle wasting, weakness, contractions, swallowing difficulties |
| Myotonic dystrophy | CTG-repeat expansion in 3'UTR or CCTG-repeat expansion in ZNF9 (leads to gain-of-function RNA) | AD | Progressive muscle weakness and myotonia. DM1 allele causes a more severe disease than DM2 |
| Fragile-X | CGG-repeat in promoter region of FMR1 (suppresses transcription due to hypermethylation -> slow development of cerebral neurons) | X-linked | Learning difficulties, typical facial features (large ears, high forehead, long face) |
| Facioscapulohumeral muscular dystrophy (FSHD) | Shortened megasatellite repeat in 4q35 leading to hypomethylation and cascading effects (deregulation of several muscle genes) | AD | Striking asymmetry of muscle involment from side to side, unbalance between right and left side muscles |
| Achondroplasia | De novo mutations in FGFR3 gene in 4p16.3, usually a missense mutation Gly380Arg | AD | Short stature (120-130cm), characteristic facial features |
| Cartilage-hair hypoplasia (CHH) | Mutation in RMRP (insertion which prevents transcription or SNP), leads to lack of RNAse required for cell growth | - | Short-limbed dwarfism (100-140 cm), sparse hair, immunodeficencies |
| Diastrophic dysplasia (DTD) | SNP mutation in SLC26A2, leads to undersulphation of cartilage matrix | - | Short stature (100-160 cm), deformities of joints, hip dysplasia, hand deformities |
| Tibial muscular dystrophy | Heterozygous deletion-insertion in titin gene | AD | Mild weakness of tibial muscles (calves) |
| Down syndrome | Trisomy 21 (47, +21). Can be due to translocation (t14;21) or t(21;21) or mosaicism. | Extra chromosome often from mother | Hypotonia, mental retardation, characteristic facial features, adult height of ~150 cm. Lifetime expectancy of 50-60 years. |
| Patau syndrome | Trisomy 13 (47,+13). Can be due to translocation (t13;14) | - | Central nervous system malformations, heart defects, growth retardation, cleft lip and palate. Most die as newborns. |
| Edwards syndrome | Trisomy 18 | - | Malformations in many organs, elfin features, mental deficiency. Most die within a week from birth. |
| Klinefelter syndrome (47, XXY) | Extra X chromosome(s) in males. Can also be (48, XXXY) or (49, XXXXY). | - | Taller than average, long lower limbs. All patients are infertile. |
| Turner syndrome (45, X | Lack of chromosome X in women | - | Short stature, infertility, no puberty. |
| XXX females | More severe symptoms if more than one extra X chromosome | - | Possible mild reduction in intellectual skills |
| Hermafroditism | Paternal X-chromosome has Y-chromosome sequences; Chimerism of XX and XY cell lines; androgen insensitivity in males; adrenal hypoplasia in females | - | Possibly taller stature |
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