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Franca et al. Cancer Drug Resist 2019;2:256-70 I http://dx.doi.org/10.20517/cdr.2019.004 Page 261

encoded by the TPMT*2 and TPMT*3A alleles [30-32] . These TPMT activity alterations are related to a high risk
of severe side effects during thiopurine treatment. Many studies have demonstrated that standard thiopurine
doses administration is correlated to a higher risk of developing severe and even fatal hematologic toxicity
in individuals with low TPMT activity compared to individuals without genetic variations in this gene. In
[33]
particular, this side effect is due to the accumulation of TGN . Murine models knock-out for Tpmt do not
show any apparent phenotype in the absence of drug challenge, whereas they present differences in TGN
concentrations, MMPN concentrations and toxicity among Tpmt(-/-), Tpmt(+/-), and Tpmt(+/+) genotypes
when thiopurines are administered, resembling the same phenotype of human individuals carrying TPMT
[34]
defective polymorphisms .

Clinical guidelines from the Clinical Pharmacogenetics Implementation Consortium (CPIC) are currently
available and recommend to assess TPMT status in term of either genetic variations or activity in ALL
and IBD patients before starting thiopurine therapy, in order to optimize dosages and thus drug efficacy
and tolerance [35,36] . Nonetheless, there are many cases of patients without TPMT defective variants who
are intolerant to a full dose of thiopurines and present leukopenia or other adverse effects, suggesting that
also other genes or environmental factors can contribute to the risk of these side effects during thiopurine
[37]
therapy .
NUDT15
Recent independent agnostic pharmacogenomic studies have identified a novel pharmacogene important for
thiopurines: the nudix hydrolase 15 gene (NUDT15, also known as MTH2), is ~10 kb long, and is located on
q region of chromosome 13 . NUDT15 (EC 3.6.1.9) is an ubiquitously expressed nucleotide triphosphate
[38]
diphosphatase that converts oxidized GTP to its monophosphate form, preventing the integration of the
damaged purine nucleotides into DNA and subsequent mismatch repair . The triphosphate thionucleotides
[39]
are potential substrate for NUDT15 that can hydrolyse and inactivate them. In patients with defective
NUDT15, treatment with a standard dose of MP leads to an excessive accumulation of tGTP/tdGTP and
[40]
consequent extensive DNA damage and cytotoxicity .

[38]
Yang et al. performed an Immunochip-based association study in 978 Korean CD patients treated
with thiopurines, investigating the association of 196524 genetic variants previously shown to be genetic
susceptibility loci for drug-induced leukopenia in autoimmune or inflammatory diseases. The missense
SNP 415C>T of NUDT15 gene (rs116855232), that induces p.Arg139Cys change, was associated with early
leukopenia in the combined analysis of the discovery and replication samples (OR = 35.6; P combined =
-94
4.88 × 10 ). Similarly, a GWAS study was conducted, using the HumanExomeBeadChip array (244770
SNPs), on a discovery cohort including 657 ALL children and a replication cohort of 371 ALL patients.
The association with MP dose intensity (defined as the ratio between clinician-prescribed MP dose and
protocol dose) was investigated, finding rs1142345 in TPMT and rs116855232 in NUDT15 as significantly
associated loci (P ~9 × 10 ) with independent replication. Patients that presented a homozygous TT
-9
genotype for NUDT15 rs116855232 were very sensitive to MP and they could tolerate only 8.3% of the
usual MP dose; this polymorphism explains 22% of variance in thiopurine-tolerance . Moriyama et al.
[42]
[41]
reported that NUDT15 variant p.Arg139Cys did not affect enzymatic activity but rather protein stability,
likely due to a loss of supportive intramolecular bonds that caused rapid proteasomal degradation in cells.
[47]
Moriyama et al. sequenced all exons of NUDT15 in 3 ALL cohorts including 270 children from Guatemala,
Singapore and Japan and identified 4 coding SNPs located in exons 1 and 3 associated with a loss of
enzymatic activity (from 74.4% to 100%): the missense SNP rs116855232, the 416G>A SNP (rs147390019) that
induces p.Arg139His and two other variants that affect the Val18 residue, the 52G>A resulting in a conversion
of valine into isoleucine (p.Val18Ile) and the 36_37insGGAGTC insertion that leads to an in frame addition
of a glycine and a valine residue (p.Val18_Val19insGlyVal). They also identified 5 NUDT15 haplotypes (*1-*5)
with distinct combinations of these 4 variants and they classified patients into 3 diplotypic groups: normal