title: Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS) Gene.
abstract: Dihydropyrimidinase (DHP) deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R) in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients.
authors.affiliation: Department of Pediatrics, Fujita Health University School of Medicine, Toyoake 470-1192, Japan. ncu.metabolism@gmail.com.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Nakajima
authors.affiliation: Laboratory Genetic Metabolic Diseases, Academic Medical Center, Amsterdam 1105 AZ, The Netherlands. j.meijer@amc.uva.nl.
authors.forename: Judith
authors.initials: J
authors.lastname: Meijer
authors.affiliation: Department of Research and Development, MILS International, Kanazawa 921-8105, Japan. mils@yacht.ocn.ne.jp.
authors.forename: Chunhua
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Neurology, Beijing Children's Hospital Affiliated to Capital University of Medical Sciences, Beijing 100045, China. zfwx05@126.com.
authors.forename: Xu
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Pediatrics, Fujita Health University School of Medicine, Toyoake 470-1192, Japan. tomomi-h@fujita-hu.ac.jp.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Kondo
authors.affiliation: Department of Pediatrics, Fujita Health University School of Medicine, Toyoake 470-1192, Japan. itotetsu@fujita-hu.ac.jp.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Ito
authors.affiliation: Department of Chemistry-BMC, Uppsala University, Uppsala 75123, Sweden. doreen.dobritzsch@kemi.uu.se.
authors.forename: Doreen
authors.initials: D
authors.lastname: Dobritzsch
authors.affiliation: Laboratory Genetic Metabolic Diseases, Academic Medical Center, Amsterdam 1105 AZ, The Netherlands. a.b.vankuilenburg@amc.uva.nl.
authors.forename: André B P
authors.initials: AB
authors.lastname: Van Kuilenburg
mesh_terms: D017398:Alternative Splicing; D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D020134:Catalytic Domain; D002648:Child; D005091:Exons; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006579:Heterozygote; D006801:Humans; D007223:Infant; D007438:Introns; D008661:Metabolism, Inborn Errors / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D012322:RNA Precursors; D011994:Recombinant Proteins; D000581:Amidohydrolases; C020047:dihydropyrimidinase
keywords: DPYS; dihydropyrimidinase; minigene; splicing