Sunday 29 January 2017

Thiopurine S-methyltransferase (TPMT) Deficiency



Introduction:

Thiopurine S-methyltransferase (TPMT) is an enzyme that catalyses the methylation of aromatic or heterocyclic thiol (sulphydryl) compounds (Peng et al., 2008).This function is ubiquitous, therefore the enzyme could be active in methylating toxic compounds as part of immunity such as against bacterial toxins. Its ubiquitous function is mimicked by its tissue expression and expression levels (Figure 1A). RNA-sequencing reveals that not only is it present in all tissues but it is present in specialised areas within those tissues (Figure 1B) and there is little variability between the expression levels in all tissues (Figure 1A and B vertical black lines show range), with the average expression around 0.55 FPKM (Figure 1A and B vertical red lines). The narrow range and ubiquitous nature of this enzyme show that TPMT has been highly conserved throughout all cell types and plays an important role in cell protection. The TPMT protein is coded for by the TPMT gene and has numerous polymorphisms which cause varying levels of loss of function of the expressed protein (Otterness et al., 1997). Interestingly, even those that are homozygous seem to live normal lives regardless of complete TPMT deficiency, unless they are treated during their life with the immunosuppressive thiopurine drugs, which in the absence of TPMT lead to extensive cell apoptosis. This report will discuss the TPMT gene and its mutations and assess the pharmacogenetic interaction between thiopurine drugs and the mutated alleles of this gene. 


TPMT Genotype:

TPMT is located at position 22.3 on the P-arm of chromosome-6 (figure 2a), it is therefore autosomal so a person can be homozygous for one allele or heterozygous. In heterozygotes the two alleles will be expressed equally so the alleles are co-dominant but the majority of the global population are homozygotes (Weinshilboum and Sladek, 1980). Complete TPMT deficiency is autosomal recessive as it requires two copies of the mutant to produce low to absent function, however heterozygotes with one mutant allele will have a varying response to thiopurines. In the general population 89% are homozygous, 11% heterozygous and 0.3% homozygous mutant (Weinshilboum and Sladek, 1980). In 2011, Booth included evidence of 30 alleles (Booth et al., 2011) with relatively few mutations in total suggesting the allele differences are due to the combination of mutations rather than separate mutations. This observation is supported by genetic information from the Uniprot database which also shows a limited number of mutations (Uniprot, 2015). Despite a limited sample, Stanulla provides evidence that TPMT2 and TPMT3A-D account for 95% of TPMT deficiency (Stanulla et al., 2005), which is supported by equivalent studies (Collie-Duguid et al., 1999; McLeod et al., 1999). TPMT*3A is most common in Caucasians with Tai identifying 75% of the sample with TPMT*3A allele (Tai et al., 1996).In contrast, TPMT*3C is the most common allele in Asia (Collie-Duguid et al., 1999; Hiratsuka et al., 2000; Lu et al., 2006) and Africa (McLeod et al., 1999; Ameyaw et al., 1999). TPMT*3C is characterised by a single missense point mutation of tyrosine to cysteine at position 240 due to an adenine to guanine substitution in exon 10 (Figure 2B). TPMT*3A has this same mutation, in addition to an alanine to threonine mutation at position 154 (Figure 2B).



Protein structure and function in wildtype and mutant alleles:
TPMT is a 245 amino acid protein consisting of 10 exons that form 9 beta sheets and 8 alpha helices (figure 3A) that fold to form the tertiary structure in Figure 3B. This figure also shows the binding site for adenosyl methionine and an adjacent binding site for 6-mercaptopurine from Peng’s study, identifying that adenosyl methionine forms at least seven hydrogen bonds here but 6-mercaptopurine only forms one (Peng et al., 2008). This study of the structures and binding sites concurs with the predicted function that adenosyl methionine binds as a methyl donor to the thiopurine, producing methylated thiopurine and adenosyl homocysteine in the reaction (figure 3C) (Peng et al., 2008). The same mechanism is expected for endogenous substances, however, no endogenous substances have been identified. It is possible that TPMT is a cellular defence to exogenous substances that attack through DNA integration. Evidence for this is highlighted by the toxic effects of thiopurine drugs in those with TPMT deficiency. In wildtype, TPMT inactivates prodrug thiopurines before they can be converted to active compounds (figure 3D) (Mlakar et al., 2016). Active compounds such as methylthioinosinemonophosphate inhibit the purine biosynthesis pathway which prevents DNA synthesis and repair or other active metabolites that insert into DNA forming interstrand crosslinks and single strand breaks that lead to apoptosis due to extensive changes in gene expression and DNA damage (figure 3D).

The tyrosine to cysteine mutation in TPMT*3C changes the structure because tyrosine has a large side chain that forms hydrogen bonds and Van der Waals interactions with residues on beta7, beta9 and alpha8. Mutation to cysteine means alpha8 interactions are lost, causing it to pull away (Rutherford and Daggett, 2008). This makes the thiopurine binding site more flexible so the thiopurine is exposed to solution. Making the thiopurine less likely to accept methylation and instead become unbound. In the TPMT*3A allele this structural deformation is coupled with a loss of hydrogen bonding between alanine and tyrosine, the double mutation forms a flattened protein and destabilises the protein causing it to be degraded by proteasomes (Rutherford and Daggett, 2008). Therefore has a half-life of 20 minutes compared to 11 hours in TPMT*3C allele (Rutherford and Daggett, 2008). This seems to be the major contributing factor as to why TPMT activity is almost absent in TPMT*3A homozygotes rather than changes in enzyme action which is the explanation for TPMT*3C allele loss of function.



Clinical features, treatments and diagnosis of TPMT deficiency:

Clinical features of TPMT deficiency are the same as the toxicities of the drugs, only the incidence and severity is higher in those with the disorder so fatality is higher (Dewit et al., 2010). To control the toxicity in those with the deficiency the dose is reduced by 50% for heterozygous and 90% for homozygous mutants (Coenen et al., 2015).

The main adverse effect is myelosuppression, a reduction in erythrocytes (anaemia) and leukocytes (leukopenia) due to the destruction of newly differentiated stem cells in bone marrow (Colombel et al., 2000). The crossover between efficacy and toxicity is evident here because when thiopurines are used to treat lymphoblastic leukaemia (increased level leukocyte derived cells), they act to reduce the number of these cells but the white blood cell loss can be excessive, causing leukopenia. This means there is a narrow therapeutic window. In those with the deficiency, leukopenia is more severe due to the excess active metabolites.

Approaches to diagnosis include phenotype testing which surveys TPMT enzyme activity by adding thiopurines and measuring the metabolite level in a purified sample (Burnett et al., 2014). Or genotype tests which sequence the gene to identify mutations that could change TPMT activity. However, a novel mutation may not be picked up (Burnett et al., 2014).

Treatments tend to deal with the toxicities as there has been no co-agonist identified that can increase the endogenous activity of the non-mutant allele in heterozygotes. Future therapy may be to reduce the degradation rate of TPMT allowing it to have a longer action. However, the most effective way would be through inhibition of the protease pathway which is ubiquitous and heavily relied on in the body. This would likely lead to cell apoptosis due to a build-up of toxic substances, so is not a viable option until another way to reduce its degradation can be found. Myelosuppression can be managed through managing anaemia, by stimulating erythropoietin with Darbepoetin alpha and neutropenia can be rescued with granulocyte colony stimulating factors as well as prophylactic treatments of infection. There has been a suggestion that treating with thioguanine may provide a less toxic alternative to other thiopurines. This has shown to be the case for inflammatory bowel syndrome, unfortunately, the licencing has been rejected due to hepatotoxicity but a derivative with some structural changes could be a future therapy. 

Conclusion:

In summary, the limited treatments and genetic variation of the TPMT gene in the population highlight the need for individualised treatments especially in pharmacogenetics. The wildtype allele functions as a methylating enzyme expressed ubiquitously throughout the body and may have a role in foreign immunity as no endogenous substrates have been identified. Mutant alleles for TPMT lead to damaging effects due to an inability to reduce the cytotoxic metabolites of thiopurine prodrugs and therefore cause fatal infections through myelosuppression. TPMT*3A and C are the most common mutant alleles but TPMT*3A disrupts function primarily due to an increased degradation rate, whereas TPMT*3C has a lack of function due to a change in protein structure. The negative impacts of this drug to mutant interaction are severe but their benefit outweighs the negatives because the majority of the population do not have any mutation and they are used in the treatment of conditions that are fatal.

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