Determination of NUDT15 variants by targeted sequencing can identify compound heterozygosity in pediatric acute lymphoblastic leukemia patients

Mercaptopurine intolerance is an adverse effect of mercaptopurine administration in childhood acute lymphoblastic leukemia. NUDT15 variants were recently identified as a major determinant of mercaptopurine intolerance. Two NUDT15 variants, c.36_37insGGAGTC and c.415C>T, are located on exons 1 and 3, respectively. Sanger sequencing cannot distinguish heterozygous patients with both variants on the same allele from compound heterozygous patients with two variants on different alleles. Because patients with biallelic NUDT15 variants are extremely sensitive to mercaptopurine, clinical identification of the diplotype of NUDT15 would be advantageous. A cohort of 37 patients with c.36_37insGGAGTC and c.415C>T NUDT15 variants were selected for haplotyping using targeted sequencing. NUDT15 complementary DNA was amplified and sequenced by 300-bp paired-end sequencing on an Illumina MiSeq. Of the 37 patients carrying NUDT15 variants, 35 were heterozygous NUDT15*1/*2 variants and two were compound heterozygous NUDT15*3/*6 and NUDT15*2/*7 variants. These two patients with compound heterozygous variants could only tolerate low doses of mercaptopurine, similar to patients with homozygous NUDT15 variants. Targeted sequencing of NUDT15 cDNA can be used to determine the diplotype of NUDT15 and identify patients with compound heterozygous NUDT15 variants.

巯嘌呤（Mercaptopurine）不耐受是儿童急性淋巴细胞白血病患者接受巯嘌呤治疗时产生的不良反应。近期研究鉴定出，NUDT15变异是决定巯嘌呤不耐受的主要因素。两种NUDT15变异分别为c.36_37insGGAGTC与c.415C>T，分别定位在外显子1和外显子3上。桑格测序无法区分两类患者：一类是同一等位基因携带两种变异的杂合子患者，另一类是不同等位基因各携带一种变异的复合杂合子患者。由于双等位基因NUDT15变异患者对巯嘌呤的敏感性极高，精准鉴定NUDT15双倍型具有重要临床价值。本研究纳入37例携带c.36_37insGGAGTC与c.415C>T NUDT15变异的患者队列，采用靶向测序开展单倍型分析。实验中，我们对NUDT15互补DNA（cDNA）进行扩增，并使用伊卢米纳（Illumina）MiSeq测序仪完成300碱基对双端测序。在37例携带NUDT15变异的患者中，35例为NUDT15*1/*2杂合变异携带者，剩余2例分别为NUDT15*3/*6与NUDT15*2/*7复合杂合变异携带者。这2例复合杂合变异患者仅能耐受低剂量巯嘌呤，其临床表型与纯合子NUDT15变异患者高度相似。对NUDT15 cDNA进行靶向测序可精准鉴定NUDT15双倍型，从而识别出携带复合杂合NUDT15变异的患者。