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

Abstract

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. (J Mol

Complete ascertainment of intragenic copy number mutations (CNMs) in the CFTR gene and its implications for CNM formation at other autosomal loci

“…CNMs in the CFTR gene have been extensively studied over the past 5 years by means of quantitative PCR techniques [Audrézet et al, 2004; Bombieri et al, 2005; Chevalier-Porst et al, 2005; Férec et al, 2006; Hantash et al, 2006; Loumi et al, 2008; Niel et al, 2004, 2006; Paracchini et al, 2008; Schneider et al, 2007; Schrijver et al, 2008; Taulan et al, 2009]. An array CGH method has recently been developed that has been used to screen CNMs in eight human disease genes including CFTR [Saillour et al, 2008].…”

Cystic fibrosis in Chilean patients: Analysis of 36 common CFTR gene mutations

“…Direct gene sequencing [17,18] and search for CFTR genomic rearrangements [19,20] are needed to detect other possible regionally prevalent mutations and rare (private) mutations not found with mutation-specific tests. This would allow us to improve diagnostic confirmation, carrier analysis and genetic counselling, and assist in the future development of a newborn screening program and cost-effective tests.…”

CFTR Rearrangements in Spanish Cystic Fibrosis Patients: First New Duplication (35kb) Characterised in the Mediterranean Countries

“…CFTR rearrangements account for around 2% of CF alleles (Lerer et al, 1999;Niel et al, 2004;Bombieri et al, 2005;Schrijver et al, 2008). Nevertheless, an estimate of their frequency in the Spanish CF population had not yet been made.…”