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Functional significance of a deep intronic mutation in the ATM gene and evidence for an alternative exon 28a
Author(s) -
Coutinho Gabriela,
Xie Jiuyong,
Du Liutao,
Brusco Alfredo,
Krainer Adrian R.,
Gatti Richard A.
Publication year - 2005
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.20170
Subject(s) - minigene , exon , biology , genetics , rna splicing , splice site mutation , microbiology and biotechnology , exon trapping , gene , splice , intron , alternative splicing , rna
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. Hum Mutat 25:118–124, 2005. © 2005 Wiley‐Liss, Inc.