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Leukaemia is a clonal malignant disorder of the blood and blood forming organs causing an accumulation of dysfunctional cells and a loss of cell division regulation. The excessive overcrowding in leukemic cells results in a congestion of bone marrow which causes a decreased production and function of normal hematopoietic cells. (McCance & Huether) It is an acute or chronic disease of the bone marrow which causes proliferation of white blood cells occurs and is usually accompanied by anaemia, impaired blood clotting and enlargement of the lymph nodes, liver and spleen (McCance & Huether).
To understand the mechanisms of polymorphs and thiopurine methyltransferase (TPMT); a perception of genes must be acquired. Each gene occupies a position along a chromosome called a locus. The genes at a particular locus can take different forms known as alleles. A locus that has two or more alleles that occur with an appreciable frequency in a population is called polymorphisms. (McCance & Huether). Polymorphism can be defined as a change in the sequence of DNA associated with a large portion of the population; and can be linked to specific diseases (Krynetski & Evans, 2003)
The TPMT enzyme appends to a methyl group onto the sulfhydryl group (Chowning, 2007).
This prevents thioguanine drugs into replicating DNA. TPMT enzyme in the body helps to metabolise the thiopurine drugs by limiting their toxicity; meaning it is deactivated. Several different genetic polymorphisms of the gene result in an enzyme that has low activity, due to the fact that the enzyme is more susceptible to degradation (Chowning, 2007). Those individuals homozygous for low activity alleles are not able to process the thioguanine nucleotides well, and when the thiopurine drug is administered at the usual level it can be toxic and consequentially result in death.
The thiopurine methyltransferase gene is location on chromosome six, at position 22.3
The TPMT gene is located on the short (p) arm of chromosome 6 at position 22.3.
Thiopurine drugs such as 6-mercaptopurine and azathioprine are used to treat Acute Lymphocytic Leukaemia. Thiopurines are prodrugs which mean they are inactive until they enter the body, where they become active. Thiopurines are converted in the body the nucleotides called thioguanine. Thioguanine can also be incorporated into DNA during replication (Chowning, 2007). When incorporated into replicating DNA, the altered nucleotides stop the process of replication from continuing. Thiopurines can also impact the purine biosynthesis pathway. Frequently replicating cells such as cancer cells are affected harshly by thiopurines. TPMT activity exhibits autosomal co dominant genetic polymorphism (Weinshilboum and Sladek (1980).
The TPMT enzyme appends a methyl group onto the sulfhydryl group (Chowning, 2007). This prevents thioguanine drugs into replicating DNA. TPMT enzyme in the body helps to metabolise the thiopurine drugs by limiting their toxicity; meaning it is deactivated. Several different genetic polymorphisms of the gene result in an enzyme that has low activity, probably because the enzyme is more susceptible to degradation (Chowning, 2007).
In the study of leukaemia, it is found that cancer drugs such as 6-mercaptopurine is changed in the body to the active form as it is a prodrug. The active form is thioguanine nucleotide (TGN). The TGN is important as it disrupts cell division of harmful cells while keeping healthy cells intact. The body excretes cancer drugs via the enzyme TPMT.
6-mercaptopurine (Marx, 2005)
An exon is a region of a gene that is present in the final functional transcript (mRNA) from that gene. The gene structure of TPMT is complex. At first, the gene structure of TPMT was resolute by Szumlanski. Szumlanski et al. (1996) determined that the TPMT gene is 34 kb long and contains 10 exons. Not long after this discovery, Krynetski showed that through isolating clones corresponding to human TPMT, it was determined that TPMT spanned 25kb and contained 9 exons. Krynetski (1997). This advancement was discovered by screening a phage artificial chromosome library. The difference between these two theories includes 17 additional nucleotides upstream from the transcription start site and a shorter intron 8. (Tai, Krynetski, & Scheutz, 1997)
Weinshilboum, 2001 The second gene is TPMT 3*A which is a TPMT gene associated with low enzyme activity. TPMT *3A has two single nucleotide polymorphisms. As noted before, a change in single DNA nucleotide bases Gïƒ A and Aïƒ G. This change results in dissimilar amino acids placed in the enzyme. As such, this affects the enzymatic function
The molecular biology of TPMT is intricate. It is apparent that a significantly lower accumulation of thioguanine nucleotides occurred in leukemic cells that had obtained an additional chromosome. The extra chromosome contained a wild type TMPT allele. This means that TPMT has a certain effect on individuals. According to numerous studies by Evans, McLeoud and Schutz, it is evident that people who are TPMT heterozygotes have an intermediate risk of haematological toxicity. On the other hand, people who are TPMT deficient are at a high risk for severe and sometimes fatal haematological toxicity. This means patients who inherit two mutant alleles should be treated with six-ten percent of the standard dose of thiopurines, while heterozygous patients can start on full doses. In addition, heterozygous patients may require a dose reduction of thiopurines. This is to prevent toxicity. (Krynetski & Evans, 2003)
PCR is an enzyme-mediated reaction, the reaction must occur at the enzyme's ideal operating temperature. The enzymes that are used for the PCR are DNA-dependent DNA polymerases (DDDP) (Virology Down Under, 2009). As well as DNA polymerase, a DNA template is needed for the PCR process to begin. The template serves as a basis to copy and pair short DNA sequences which are known as primers.
Polymerase chain reaction method is used for TPMT genotype to determine if antileukemic therapy is practical. Yates(1997) reported that PCR based method for detection of TPMT variants in patients genomic DNA show an excellent correlation between genotype and phenotype, based on assay of TPMT activity in erythrocytes. More specifically, patients with low TPMT activity may accumulate a higher level of 6- thioguanine nucleotides metabolite (Henderson & O'Brien, 2011). As such these patients also have a high risk of hematologic toxicity than those with higher TPMT activity. It is evident that TPMT is located on chromosome six, there are two mutant TMPT alleles; at position 460, Guanineïƒ Adenine and at position 719, Adenineïƒ Guanine. In a stance to avoid and prevent toxicity, it is suggested to have complete blood count monitoring on a regular basis. (Troy, 2006)
Guanine nucleotide Thioguanine nucleotide
Oxygen circled Sulphur circled
There are many issues that arise from gene testing, research and being a frontline healthcare professional in the pharmaceutical industry. Such concerns must be addressed to fully comprehend the consequences of research on aspects of the pharmaceutical industry. Such issues include ethical issues, healthcare insurance, dosage/ treatment issues and the TPMT screening tests in the pharmacy that may happen in the future.
In regards to ethical issues, privacy and control in pharmaceutical research produce many implication for patients of DNA samples. In focusing on TPMT screening test, confidentiality and privacy play a crucial role in positive research (Henderson & O'Brien, 2011). Some people may choose not to participate in tests due to the privacy factors which are determined specifically for each test. An example of this may be how easily their samples may be traced back, and would the information give rise o personal clinical significance. An approach to avoid this may be to make samples anonymous by linking codes with samples. As such, individuals with pre existing conditions could be prohibited from forms of private health care cover.
The next issue arises which is healthcare insurance. Insurance companies cannot discriminate against individuals through reduced coverage or pricing factors. This is a fear from the companies' point of view that predisposing genetic factors may cause a loss in productivity which causes a loss in profitability. Employers may want to discriminate in an attempt to minimise their companies cost relating to health. This may also increase insurance premiums, insurers can't demand genetic tests for individuals and employers cannot discriminate against individuals with predisposing diseases. As such, people can have genetic testing for diseases such as cancer, and TPMT activity with reduced fear of insurance companies. In turn, this will increase the chances of early treatment with possible prolonged quality of life.
As a primary healthcare professional, we must be aware of correct dosage amounts of the thiopurine drugs and the level that the person is high or low TPMT activity. A problem that may arise is dosage abuse which can lead to haematological toxicity. A patient with high TPMT activity can start on a regular dosage. As a pharmacist must take control to progressively reduce the dosage amount over the period of time, because a pharmacist, we hold a duty of care over this.
For example, patients on coexisting therapy with drugs which may inhibit TPMT (olsalazine) may be susceptible to myelosuppressive effects. Patients with low or absent TPMT activity may require further dose reductions or discontinuation. Furthermore, thiopurines have a 'narrow therapeutic index', which means that they can be very toxic if not administered correctly (Guggenheim, 1999). The ideal dosage is high enough to damage frequently dividing cancer cells but low enough that excess drug can be cleared with the aid of the TPMT enzyme. This is a decrease in the production of white blood cells, red blood cells, and plasma cells in the bone marrow (Guggenheim, 1999).
When looking toward to future, we must understand the ever growing demand of pharmacists in both the community and in research. The TPMT test may be able to be done in the pharmacy. We must understand the later to provide an acceptable test which can be standardised by the above. We must be able to regulate the tests; and know eligibility of having such a test.
In conclusion, TPMT plays a vital role in the treatment of leukaemia. Polymorphisms of TPMT can have effects on patients with both high and low TPMT activity. Drug doses need to be regulated to ensure that patients to do not experience any haematological toxic effects. This brings about many pharmaceutical issues including ethics, treatment issues, insurance and tests in the future. It is important for primary healthcare professionals to understand the implications of TPMT screening and how it affects individuals.
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