Novel variants in NUDT15 and thiopurine intolerance in children with acute lymphoblastic leukemia from diverse ancestry

Abstract

Prolonged exposure to thiopurines (eg, mercaptopurine [MP]) is essential for curative therapy in acute lymphoblastic leukemia (ALL), but is also associated with frequent dose-limiting hematopoietic toxicities, which is partly explained by inherited genetic polymorphisms in drug metabolizing enzymes (eg, TPMT). Recently, our group and others identified germ line genetic variants in NUDT15 as another major cause of thiopurine-related myelosuppression, particularly in Asian and Hispanic people. In this article, we describe 3 novel NUDT15 coding variants (p.R34T, p.K35E, and p.G17_V18del) in 5 children with ALL enrolled in frontline protocols in Singapore, Taiwan, and at St. Jude Children's Research Hospital. Patients carrying these variants experienced significant toxicity and reduced tolerance to MP across treatment protocols. Functionally, all 3 variants led to partial to complete loss of NUDT15 nucleotide diphosphatase activity and negatively influenced protein stability. In particular, the p.G17_V18del variant protein showed extremely low thermostability and was completely void of catalytic activity, thus likely to confer a high risk of thiopurine intolerance. This in-frame deletion was only seen in African and European patients, and is the first NUDT15 risk variant identified in non-Asian, non-Hispanic populations. In conclusion, we discovered 3 novel loss-of-function variants in NUDT15 associated with MP toxicity, enabling more comprehensive pharmacogenetics-based thiopurine dose adjustments across diverse populations.

Figure 1.

Functional characterization of 3 novel NUDT15 risk variants. (A) Shown are the positions of novel (red text) and known risk variants (black text) in the coding region of the NUDT15 gene. (B) Shown is the spatial distribution of amino acid residues affected by risk variants in the human NUDT15 protein. Presented as a homodimer, chains A and B are discriminated by color (green and cyan, respectively). Each risk variant is identified by letters as well as colors, and TGMP is shown in stick-ball presentation. The 3-dimensional structure was drawn by using PyMOL software (accession code 5LPG of the Protein Data Bank, http://www.rcsb.org/pdb). (C) NUDT15 nucleotide diphosphatase activity was determined by using the PiPer Pyrophosphate Assay (Life Technologies) for each of the 3 novel variants, with wild-type (WT) and p.R139C proteins included as controls. (D) NUDT15 protein thermostability was measured by using SYPRO Orange (Molecular Probes) for each of the 3 variants, with wild-type and the p.R139C variant included as controls. The inflection point of each curve indicates the temperature for protein unfolding and is thus a measurement of stability. RFU, relative fluorescence unit.