title: Functional significance of a deep intronic mutation in the ATM gene and evidence for an alternative exon 28a.
abstract: Screening for ATM mutations is usually performed using genomic DNA as a template for PCR amplification across exonic regions, with the consequence that deep intronic sequences are not analyzed. Here we report a novel pseudoexon-retaining deep intronic mutation (IVS28-159A>G; g.75117A>G based on GenBank U82828.1) in a patient with ataxia-telangiectasia (A-T), as well as the identification of a previously unrecognized alternative exon in the ATM gene (exon 28a) expressed in lymphoblastoid cell lines (LCL) derived from normal individuals. cDNA analysis using the A-T patient's LCL showed the retention of two aberrant intronic segments of 112 and 190 nt between exons 28 and 29. Minigenes were constructed to determine the functional significance of two genomic changes in the region of aberrant splicing: IVS28-193C>T (g.75083C>T) and IVS28-159A>G, revealing that: 1) the first is a polymorphism; 2) IVS28-159A>G weakens the 5' splice site of the alternative exon 28a and activates a cryptic 5' splice site (ss) 83 nt downstream; and 3) wild-type constructs also retain a 29-nt segment (exon 28a) as part of both the 112- and 190-nt segments. Maximum entropy estimates of ss strengths corroborate the cDNA and minigene findings. Such mutations may prove relevant in planning therapy that targets specific splicing aberrations.
authors.affiliation: Department of Pathology and Laboratory Medicine, The David Geffen School of Medicine, University of California, Los Angeles 90095-1732, USA.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Coutinho
authors.forename: Jiuyong
authors.initials: J
authors.lastname: Xie
authors.forename: Liutao
authors.initials: L
authors.lastname: Du
authors.forename: Alfredo
authors.initials: A
authors.lastname: Brusco
authors.forename: Adrian R
authors.initials: AR
authors.lastname: Krainer
authors.forename: Richard A
authors.initials: RA
authors.lastname: Gatti
mesh_terms: D017398:Alternative Splicing; D001260:Ataxia Telangiectasia / Q000235:genetics*; D064007:Ataxia Telangiectasia Mutated Proteins; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D009154:Mutation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D022821:RNA Splice Sites; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D022821:RNA Splice Sites; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases