Published online before print June 13, 2008

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Journal of Molecular Diagnostics 2008, Vol. 10, No. 4
Copyright © 2008 American Society for Investigative Pathology & Association for Molecular Pathology
DOI: 10.2353/jmoldx.2008.080004

Multiplex Ligation-Dependent Probe Amplification Identification of Whole Exon and Single Nucleotide Deletions in the CFTR Gene of Hispanic Individuals with Cystic Fibrosis

Iris Schrijver^*, Krista Rappahahn^*, Lynn Pique^*, Martin Kharrazi and Lee-Jun Wong

From the Departments of Pathology * and Pediatrics, Stanford University Medical Center, Stanford, California; the Genetic Disease Screening Program, California Department of Public Health, Richmond, California; and the Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas

A disparity between Caucasian and Hispanic mutation detection for cystic fibrosis continues to exist, although the carrier frequency is only moderately lower in Hispanics. We aimed to identify exonic rearrangements that remained undetected by conventional methods. In seven of 32 cystic fibrosis-affected self-identified Hispanics for whom only one or no mutations were identified by extensive molecular testing, exon deletions appeared to be present with a multiplex ligation-dependent probe amplification (MLPA) assay. Two recurrent deletions (of exons 2–3 and exons 22–23) were identified in one and three patients, respectively (12.5%, 11.1% of unidentified alleles). Two apparently novel deletions (exons 6b and 20) were identified in three additional patients. Subsequent sequencing to characterize deletion breakpoints, however, identified single nucleotide deletions at the probe binding sites close to the ligation point. All resulted in false positive MLPA deletion signals. Interestingly, these mutations were not common in Caucasians, and one (935delA) was common in U.S. Hispanics. On examination of all probe binding sites, we identified a total of 76 reported mutations and five silent variants that immediately surrounded the MLPA ligation sites, with 22 occurring in non-Caucasians. These mutations are not all rare. Thus, apparent exon deletions by MLPA may indicate the presence of both large deletions and point mutations, with important implications for pan-ethnic MLPA testing in cystic fibrosis and other genetic conditions.