title: Deep-intronic ATM mutation detected by genomic resequencing and corrected in vitro by antisense morpholino oligonucleotide (AMO).
abstract: 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.
authors.affiliation: Università di Torino, Department of Medical Sciences & Azienda Ospedaliera Città della Salute e della Scienza, Turin, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Cavalieri
authors.forename: Elisa
authors.initials: E
authors.lastname: Pozzi
authors.forename: Richard A
authors.initials: RA
authors.lastname: Gatti
authors.forename: Alfredo
authors.initials: A
authors.lastname: Brusco
mesh_terms: D001260:Ataxia Telangiectasia / Q000235:genetics* / Q000473:pathology / Q000628:therapy*; D064007:Ataxia Telangiectasia Mutated Proteins; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics*; D002460:Cell Line; D016373:DNA, Antisense / Q000008:administration & dosage; D004268:DNA-Binding Proteins / Q000235:genetics*; D015894:Genome, Human; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007438:Introns / Q000235:genetics; D060172:Morpholinos / Q000008:administration & dosage*; D009154:Mutation / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D022821:RNA Splice Sites / Q000235:genetics*; D025521:Tumor Suppressor Proteins / Q000235:genetics*
chemical_list: D018797:Cell Cycle Proteins; D016373:DNA, Antisense; D004268:DNA-Binding Proteins; D060172:Morpholinos; D022821:RNA Splice Sites; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases