Document Detail

Deep-intronic ATM mutation detected by genomic resequencing and corrected in vitro by antisense morpholino oligonucleotide (AMO).
MedLine Citation:
PMID:  23211698     Owner:  NLM     Status:  MEDLINE    
Recent development of next-generation DNA sequencing (NGS) techniques is changing the approach to search for mutations in human genetic diseases. We applied NGS to study an A-T patient in which one of the two expected mutations was not found after DHPLC, cDNA sequencing and MLPA screening. The 160-kb ATM genomic region was divided into 31 partially overlapping fragments of 4-6 kb and amplified by long-range PCR in the patient and mother, who carried the same mutation by segregation. We identified six intronic variants that were shared by the two genomes and not reported in the dbSNP(132) database. Among these, c.1236-405C>T located in IVS11 was predicted to be pathogenic because it affected splicing. This mutation creates a cryptic novel donor (5') splice site (score 1.00) 405 bp upstream of the exon 12 acceptor (3') splice site. cDNA analysis showed the inclusion of a 212-bp non-coding 'pseudoexon' with a premature stop codon. We validated the functional effect of the splicing mutation using a minigene assay. Using antisense morpholino oligonucleotides, designed to mask the cryptic donor splice-site created by the c.1236-405C>T mutation, we abrogated the aberrant splicing product to a wild-type ATM transcript, and in vitro reverted the functional ATM kinase impairment of the patients' lymphoblasts. Resequencing is an effective strategy for identifying rare splicing mutations in patients for whom other mutation analyses have failed (DHPLC, MLPA, or cDNA sequencing). This is especially important because many of these patients will carry rare splicing variants that are amenable to antisense-based correction.
Simona Cavalieri; Elisa Pozzi; Richard A Gatti; Alfredo Brusco
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2012-12-05
Journal Detail:
Title:  European journal of human genetics : EJHG     Volume:  21     ISSN:  1476-5438     ISO Abbreviation:  Eur. J. Hum. Genet.     Publication Date:  2013 Jul 
Date Detail:
Created Date:  2013-06-13     Completed Date:  2013-11-04     Revised Date:  2014-07-01    
Medline Journal Info:
Nlm Unique ID:  9302235     Medline TA:  Eur J Hum Genet     Country:  England    
Other Details:
Languages:  eng     Pagination:  774-8     Citation Subset:  IM    
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MeSH Terms
Ataxia Telangiectasia / genetics*,  pathology,  therapy*
Ataxia Telangiectasia Mutated Proteins
Base Sequence
Cell Cycle Proteins / genetics*
Cell Line
DNA, Antisense / administration & dosage
DNA-Binding Proteins / genetics*
Genome, Human
High-Throughput Nucleotide Sequencing
Introns / genetics
Morpholinos / administration & dosage*
Mutation / genetics
Protein-Serine-Threonine Kinases / genetics*
RNA Splice Sites / genetics*
Tumor Suppressor Proteins / genetics*
Reg. No./Substance:
0/Cell Cycle Proteins; 0/DNA, Antisense; 0/DNA-Binding Proteins; 0/Morpholinos; 0/RNA Splice Sites; 0/Tumor Suppressor Proteins; EC protein, human; EC Telangiectasia Mutated Proteins; EC Kinases

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