TPMT Human

What is TPMT and what is its primary function in human metabolism?

TPMT (S-adenosyl L-methionine:thiopurine S-methyltransferase; EC 2.1.1.67) is a cytosolic enzyme that catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds. In human metabolism, TPMT plays a crucial role in the biotransformation of thiopurine drugs, including azathioprine (AZA), mercaptopurine, and thioguanine, which are widely used as immunosuppressants and chemotherapeutic agents. The enzyme mediates the rate-limiting step in converting these thiopurine medications to inactive metabolites, thereby regulating their therapeutic and toxic effects. TPMT activity is primarily found in the liver, but is also present in erythrocytes, making blood samples a convenient source for measuring enzyme activity in clinical settings .

How does TPMT polymorphism manifest in human populations?

TPMT activity exhibits autosomal codominant genetic polymorphism in human populations, resulting in a trimodal distribution of enzyme activity. Approximately 89-94% of individuals display normal or high enzyme activity (homozygous wild-type), 6-11% have intermediate activity (heterozygous), and about 0.3-0.5% exhibit very low or undetectable activity (homozygous variant). This polymorphic distribution is clinically significant as it correlates directly with the amount of TPMT protein present in tissues. Studies using Western blot analysis of erythrocytes from patients with varying TPMT activities (0.4-23 units/ml pRBC) have demonstrated a significant correlation (rs=0.99; P<0.001) between enzyme activity and protein quantity. Population studies in different ethnic groups, including Eastern Europeans, have established the frequency of these polymorphisms, with notable variations across different populations .

What are the major TPMT alleles associated with altered enzyme activity?

The TPMT gene has numerous allelic variants that affect enzyme activity. The wild-type allele, designated as TPMT*1, encodes fully functional enzyme, while multiple variant alleles have been identified that result in reduced or absent enzymatic activity. The most prevalent alleles associated with TPMT deficiency include:

Additional alleles have been identified (TPMT4 through TPMT18), each characterized by specific point mutations or combinations of mutations. The biochip technology developed for TPMT genotyping can detect six critical point mutations (G238C, G292T, G460A, G644A, T681G, and A719G) that correspond to seven inactive alleles: TPMT*2, *3A, *3B, *3C, *3D, *7, and *8 .

What molecular mechanisms underlie the reduced catalytic activity in TPMT variants?

The primary molecular mechanism responsible for reduced TPMT activity in variant alleles is enhanced protein degradation rather than impaired protein synthesis. Research utilizing heterologous expression of wild-type (TPMT1) and mutant (TPMT2 and TPMT3A) human cDNAs in yeast and COS-1 cells demonstrated comparable levels of TPMT mRNA but significantly lower TPMT protein with the mutant cDNAs. Pulse-chase experiments revealed dramatically shorter degradation half-lives for TPMT2 and TPMT3A proteins (approximately 0.25 hours) compared to wild-type TPMT1 (18 hours). This accelerated degradation appears to be mediated by the proteasome pathway, as the process is impaired by ATP depletion and in yeast with mutant proteasomes (pre-1 strain), but unaffected by the lysosomal inhibitor chloroquine. These findings establish that the predominant mechanism for TPMT deficiency is enhanced proteasomal degradation of variant proteins, resulting in lower TPMT protein levels and consequently reduced catalytic activity .

Why is TPMT testing relevant in thiopurine drug administration?

TPMT testing has significant clinical utility in thiopurine drug administration because patients with TPMT deficiency face a substantially higher risk of severe, potentially fatal hematopoietic toxicity when treated with standard doses of thiopurines. Azathioprine (AZA), a commonly prescribed thiopurine, is widely used for immunosuppression following organ transplantation and for treating autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, and various dermatological conditions. In patients with low or absent TPMT activity, thiopurine drugs accumulate as active metabolites, leading to myelosuppression, leukopenia, and potentially neutropenic sepsis. TPMT testing before initiating therapy allows for personalized dosing strategies: patients with intermediate activity may require dose reductions (30-70% of standard dose), while those with deficient activity might need either extreme dose reductions (≤10% of standard) or alternative therapies. Data from clinical studies support that routine TPMT testing before AZA prescribing can reduce AZA-related adverse events, avert deaths from neutropenic sepsis, and improve health-related quality of life .

How does TPMT deficiency impact patient outcomes in thiopurine therapy?

TPMT deficiency significantly impacts patient outcomes in thiopurine therapy through multiple mechanisms. Patients with complete TPMT deficiency (homozygous variant) who receive standard thiopurine doses experience rapid and severe myelosuppression, typically within 1-2 weeks of starting therapy, with potentially fatal consequences. Those with intermediate activity (heterozygous) face a moderate but still significant risk of toxicity, often requiring dose adjustments. The impact on patient outcomes includes:

• Increased risk of severe neutropenia and related infections

• Extended hospitalization periods for toxicity management

• Therapy interruptions or discontinuations affecting disease control

• Potential mortality from neutropenic sepsis

• Compromised quality of life during toxicity episodes

What techniques are available for TPMT genotyping in research settings?

Multiple methodological approaches have been developed for TPMT genotyping in research settings, each with specific advantages and limitations:

• PCR-RFLP (Restriction Fragment Length Polymorphism): A conventional method involving PCR amplification followed by restriction enzyme digestion and gel electrophoresis. While reliable, this technique is labor-intensive and time-consuming.

• Biochip-based Analysis: A newer approach utilizing oligonucleotide DNA probes immobilized in gel pads on a biochip. The specially designed TPMT biochip can simultaneously detect six point mutations (G238C, G292T, G460A, G644A, T681G, and A719G) and seven corresponding alleles. The procedure involves two-round nested multiplex PCR followed by hybridization of fluorescently labeled amplified DNA to the biochip. This method offers advantages in throughput, standardization, and efficiency of sample analysis.

• Real-time PCR Assays: Methods using fluorescent probes that can detect TPMT*2, *3, *4, *5, and *6 alleles. While offering rapid results, the reagent and equipment costs may limit widespread implementation in standard clinical laboratories.

• Pyrosequencing: A technique capable of genotyping multiple single-nucleotide polymorphisms including TPMT*1S, *2, *3A, *3B, *3C, *3D, *4, *5, *6, *7, and *8. This approach offers comprehensive analysis but requires specialized equipment typically found only in large research centers.

• Sanger Sequencing: The gold standard for mutation detection, providing comprehensive analysis of the entire coding region but at higher cost and with longer turnaround time.

For large-scale population screening, the biochip approach offers significant advantages, including multiplex PCR for all six mutations in one tube, minimal DNA requirements (1 μl), and rapid analysis (approximately 1 minute per biochip) with automatic image analysis and genotype assignment. In one study implementing the biochip method for population screening in Russia, operator-to-operator reproducibility was approximately 99% .

How do phenotyping and genotyping approaches compare in TPMT assessment?

Phenotyping and genotyping represent complementary approaches to TPMT assessment, each with distinct advantages and limitations:

TPMT Phenotyping:

• Measures actual enzyme activity, typically in erythrocytes

• Directly reflects the functional capacity of TPMT in the patient

• Can detect deficiency regardless of the underlying genetic cause

• May be affected by recent blood transfusions, certain medications, or disease states

• Requires specialized laboratory capabilities for enzymatic analysis

• Results may be influenced by sample handling and storage conditions

TPMT Genotyping:

• Detects specific mutations in the TPMT gene

• Results are not affected by external factors like medications or transfusions

• Can be performed on any nucleated cell sample

• Provides stable, lifetime prediction of TPMT status

• May miss rare or novel mutations not included in the testing panel

• Does not account for potential post-translational modifications or other factors affecting protein function

Research has established a strong correlation between TPMT genotype and phenotype, particularly for the most common variant alleles. Studies have demonstrated that erythrocyte TPMT activity is significantly related to the amount of TPMT protein detected by Western blotting, with correlation coefficients as high as 0.99. This correlation provides the theoretical basis for using either approach in clinical practice.

In the UK and Spain, both methods are utilized, with an increased uptake of testing in recent years. The choice between phenotyping and genotyping often depends on local expertise, laboratory capabilities, and specific clinical scenarios. For comprehensive patient assessment, some centers employ both methods in select cases, particularly when results from one approach are ambiguous or when clinical presentation doesn't align with initial test results .

What are the current challenges in correlating TPMT genotype with clinical phenotype?

Despite significant advances in understanding TPMT polymorphisms, several challenges persist in correlating genotype with clinical phenotype:

Research approaches to address these challenges include comprehensive sequencing of the TPMT gene, analysis of gene-gene interactions, metabolomic profiling, and prospective clinical studies correlating multiple genetic markers with clinical outcomes. Some investigators advocate for a combined approach using both genotyping and phenotyping in select clinical scenarios to enhance predictive accuracy .

How might other genetic factors interact with TPMT polymorphisms to influence thiopurine metabolism?

Thiopurine metabolism represents a complex pathway influenced by multiple enzymes beyond TPMT, creating potential for gene-gene interactions that modify clinical outcomes. Current research indicates several important interactions:

Methodological approaches to study these interactions include genome-wide association studies (GWAS), targeted gene panels examining multiple enzymes in thiopurine pathways, physiologically-based pharmacokinetic modeling, and ex vivo cellular systems to assess combined effects of multiple genetic variants. Advanced machine learning approaches are also being employed to develop predictive algorithms incorporating multiple genetic markers for optimized thiopurine dosing regimens.

The clinical implementation of these findings remains challenging but represents an important frontier in pharmacogenomics. Future therapeutic algorithms may incorporate multiple genetic markers beyond TPMT to further personalize thiopurine therapy and minimize adverse events while maintaining efficacy .