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:  Publisher    
Abstract/OtherAbstract:
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.European Journal of Human Genetics advance online publication, 5 December 2012; doi:10.1038/ejhg.2012.266.
Authors:
Simona Cavalieri; Elisa Pozzi; Richard A Gatti; Alfredo Brusco
Related Documents :
24706558 - Novel tubb4a mutations and expansion of the neuroimaging phenotype of hypomyelination w...
24426208 - Usage of conventional pcr technology for the detection of hla-b27 allele: a significant...
23246278 - High prevalence of neonatal presentation in korean patients with citrullinemia type 1, ...
23624138 - Prop-1 gene mutations in a 63-year-old woman presenting with osteoporosis and hyperlipi...
14970678 - Sequence analysis of myostatin promoter in cattle.
15548288 - The rare silver gum, eucalyptus cordata, is leaving its trace in the organellar gene po...
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-12-05
Journal Detail:
Title:  European journal of human genetics : EJHG     Volume:  -     ISSN:  1476-5438     ISO Abbreviation:  Eur. J. Hum. Genet.     Publication Date:  2012 Dec 
Date Detail:
Created Date:  2012-12-5     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9302235     Medline TA:  Eur J Hum Genet     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
Università di Torino, Department of Medical Sciences & Azienda Ospedaliera "Città della Salute e della Scienza", Torino, Italy.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


Previous Document:  Refining genome-wide linkage intervals using a meta-analysis of genome-wide association studies iden...
Next Document:  Chromosomal microarray analysis as a first-line test in pregnancies with a priori low risk for the d...