Thiopurine methyltransferase pharmacogenetics: Cloning of human liver cDNA and a processed pseudogene on human chromosome 18q21.1

D. Lee, C. Szumlanski, J. Houtman, R. Honchel, K. Rojas, J. Overhauser, E. D. Wieben, R. M. Weinshilboum

Research output: Contribution to journalArticlepeer-review

66 Scopus citations

Abstract

Thiopurine methyltransferase (TPMT) is a genetically polymorphic enzyme that catalyzes the S-methylation of thiopurine drugs such as 6- mercaptopurine. This genetic polymorphism is an important factor responsible for individual variation in thiopurine drug response. A cDNA for TPMT has been cloned from T84 human colon carcinoma cells. Northern blot analysis of multiple human tissues was performed with the T84 human colon carcinoma TPMT cDNA open reading frame (ORF) as a probe as one step toward understanding the molecular basis for the TPMT genetic polymorphism. Three mRNA species (~1.4, 2.0, and 3.6 kb in length) were present in all tissues studied, including liver. However, none of these mRNAs matched the length of the 2.7 kb T84 TPMT cDNA. Therefore, it was important to clone a TPMT cDNA from a human drug- metabolizing organ such as the liver to determine whether its sequence matched that of the cDNA cloned from the T84 cell line. A human liver cDNA library was screened with the T84 TPMT CDNA ORF as a probe, and a 1.8 kb cDNA was isolated with a coding region sequence identical to that of the T84 TPMT cDNA. The TPMT cDNA ORF was then used to screen a human lymphocyte genomic DNA library in an attempt to clone the TPMT gone(s) in humans. Three intronless clones were isolated with identical ORF sequences that were 96% identical to that of the TPMT cDNA, but which contained multiple nucleotide substitutions and one deletion, The 3'- and 5'-flanking regions of one of the genomic DNA clones were sequenced. The clone had a 3'-poly(A) tract, with a 10 nucleotide direct repeat, CAATTTGCTT, flanking the gone at both the 5'- and 3'-ends. All of these properties are characteristics of a processed pseudogene. This apparent processed pseudogene for TPMT was mapped to human chromosome 18q21.1. The cloning of a human liver TPMT cDNA and the discovery of a TPMT processed pseudogene on chromosome 18 represent significant steps toward the goal of determining the molecular basis for the TPMT genetic polymorphism in humans.

Original languageEnglish (US)
Pages (from-to)398-405
Number of pages8
JournalDrug Metabolism and Disposition
Volume23
Issue number3
StatePublished - 1995

ASJC Scopus subject areas

  • Pharmacology
  • Pharmaceutical Science

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