Functional Study of the P32T ITPA Variant Associated with Drug Sensitivity in Humans

Abstract

Sanitization of the cellular nucleotide pools from mutagenic base analogs is necessary for the accuracy of transcription and replication of genetic material and plays a substantial role in cancer prevention. The undesirable mutagenic, recombinogenic and toxic incorporation of purine base analogs (i.e. ITP, dITP, XTP, dXTP or 6-hydroxyaminopurine (HAP) deoxynucleoside triphosphate) into nucleic acids is prevented by inosine triphosphate pyrophosphatase (ITPA). The ITPA gene is a highly conserved, moderately expressed gene. Defects in ITPA orthologs in model organisms cause severe sensitivity to HAP and chromosome fragmentation. A human polymorphic allele 94C->A encodes for the enzyme with a P32T amino acid change and leads to accumulation of non-hydrolyzed ITP. ITPase activity is not detected in erythrocytes of these patients. The P32T polymorphism has also been associated with adverse sensitivity to purine base analog drugs. We have found that the ITPA-P32T mutant is a dimer in solution, as is wild-type ITPA, and has normal ITPA activity in vitro, but the melting point of ITPA-P32T is 5 degrees C lower than that of wild-type. ITPA-P32T is also fully functional in vivo in model organisms as determined by a HAP mutagenesis assay and its complementation of a bacterial ITPA defect. The amount of ITPA protein detected by western blot is severely diminished in a human fibroblast cell line with the 94C->A change. We propose that the P32T mutation exerts its effect in certain human tissues by cumulative effects of destabilization of transcripts, protein stability and availability.

Genetic polymorphism of inosine-triphosphate-pyrophosphatase influences mercaptopurine metabolism and toxicity during treatment of acute lymphoblastic leukemia individualized for thiopurine-S-methyl-transferase status

“…The SNP rs1127354 is a C/A transversion located in the exon 2 of the gene [55] that causes an amino-acid change (P32T), abolishing ITPA enzymatic activity in individuals homozygous and reducing it to 25% in heterozygous subjects [55,57,58]; this pattern is consistent with impaired assembly of the dimeric structure of the enzyme caused by the P32T amino-acid change [59]. A recent study has shown that ITPA P32T maintains enzymatic activity comparable to the wildtype enzyme and proposed that the rs1127354 variant exerts its effects in human tissues by the cumulative effects of destabilization of transcript and protein stability [60]. This genetic polymorphism, leading to lower ITPA enzyme activity, causes physiologically an abnormal accumulation of ITP in cells, which is by itself a clinically benign condition [55,61].…”

The human ITPA polymorphic variant P32T is destabilized by the unpacking of the hydrophobic core

“…One explanation for the apparent contradiction between these observations and the results seen in knockout mice is that the P32T mutation does not completely abolish the function of the enzyme. In fact, the P32T mutant has been shown to have in vitro activity comparable to wild-type (Herting et al, 2010; Stepchenkova et al, 2009). In other cells besides erythrocytes, partial loss of activity could be compensated by higher levels of ITPA, thus preventing the phenotype seen in mice.…”

Defects in purine nucleotide metabolism lead to substantial incorporation of xanthine and hypoxanthine into DNA and RNA

“…GMP is also derived from IMP but via xanthosine monophosphate (XMP) through the action of IMP dehydrogenase (guaB) and GMP synthetase (guaA). Xanthine and hypoxanthine thus play significant roles in purine metabolism as components of the cellular nucleotide pool.The concentrations of purine nucleotide pool components are highly regulated (11,12), with many human diseases caused by imbalances arising from genetic polymorphisms and mutations in purine metabolism (13)(14)(15)(16)(17). For example, loss of adenosine deaminase results in severe combined immunodeficiency (18), while loss of hypoxanthine-guanine phosphoribosyltransferase leads to the hyperuricemia and neurological symptoms of LeschNyhan syndrome (14), and increases in the activity of purine nucleotide metabolic enzymes, such as IMP dehydrogenase, are associated with several types of cancer (19).…”