title: The ITPA c.94C>A and g.IVS2+21A>C sequence variants contribute to missplicing of the ITPA gene.
abstract: Inosine triphosphate pyrophosphatase (ITPase) catalyzes the conversion of inosine triphosphate (ITP) to the correspondent monophosphate. The ITPA c.94C>A and g.IVS2+21A>C allelic variants are associated with decreased red cell enzyme activity. The ITPA c.94C>A [P32T] sequence variant is associated with an increased risk of adverse drug reactions in patients treated with the thiopurine drug azathioprine. The aim of this study was to explore the molecular mechanisms of ITPase deficiency. ITPA mRNA was extracted from peripheral blood leukocytes (PBL), Epstein-Barr virus transformed lymphoblast cell cultures, reticulocytes, and cultured fibroblast from patients with known ITPA genotypes. ITPA mRNA was reversed transcribed, sequenced and the relative amounts of misspliced transcripts quantitated from three independent experiments. The ITPA g.IVS2+21A>C sequence variant resulted in missplicing of exon 3. The ITPA c.94C>A allelic variant resulted in missplicing of exons 2 and 3 representing, in PBL samples, 61% of the total mRNA expressed in ITPA c.94C>A homozygotes. We proposed that the ITPA c.94C>A allelic variant destroys an exonic splicing silencing (ESS) element in exon 2, resulting in the activation of two nearby upstream 5' splice sites and missplicing of the exons 2 and 3 cassette causing structural changes to the enzyme and contributing to ITPase deficiency.
authors.affiliation: Purine Research Laboratory, Department of Chemical Pathology, Guy's Hospital, London, SE1 9RT, UK, and Department of Paediatrics, Neonatology and Congenital Disorders, Nagoya City University, Japan.
authors.forename: Monica
authors.initials: M
authors.lastname: Arenas
authors.forename: John
authors.initials: J
authors.lastname: Duley
authors.forename: Satoshi
authors.initials: S
authors.lastname: Sumi
authors.forename: Jeremy
authors.initials: J
authors.lastname: Sanderson
authors.forename: Anthony
authors.initials: A
authors.lastname: Marinaki
mesh_terms: D000483:Alleles; D001483:Base Sequence; D002478:Cells, Cultured; D004342:Drug Hypersensitivity / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D011755:Pyrophosphatases / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D000097630:Inosine Triphosphatase
chemical_list: D011755:Pyrophosphatases; D012333:RNA, Messenger; C511829:ITPA protein, human; D000097630:Inosine Triphosphatase