A novel double deletion underscores the importance of characterizing end points of the CFTR large rearrangements

Abstract

Large genomic rearrangements in patients with cystic fibrosis (CF) account for up to 16 -24% of CF alleles negative for point mutations in European populations. Herein, we identified a new large rearrangement removing exon 19 in a young CF patient, who hitherto harbored only the F508del mutation. By using LightCycler technology, we successfully and rapidly delineated the deletion end points by determining the relative copy number of a set CFTR sequence from introns 18 to 19. Fine mapping of the sequences bordering its break points was achieved using direct sequencing. We reported the first complex CFTR rearrangement containing two successive deletion events putatively linked. We evidenced the presence of short direct repeats in the vicinity of the deletions suggesting a possible replication slippage model. In this report, we also discussed the putative molecular mechanism and consequences of this complex gene rearrangement, unprecedented in CF. This complex deletion illustrates the importance of delineating the genomic rearrangement to improve our knowledge of the CFTR mutational spectrum and to better understand the molecular mechanism controlling the CFTR expression.

Identification of a novel duplication CFTRdup2 and functional impact of large rearrangements identified in the CFTR gene

“…We next used a previously developing and practical technique to determine the endpoints using quantitative PCR technology (Taulan et al, 2009). With reductions in gene copy number, from two to one, more readily identifiable, we successfully revealed this duplication using our previously developed and practiced quantitative PCR technology (Taulan et al, 2009), followed by direct DNA sequencing to precisely map the duplication breakpoint junctions Such precise mapping is widely accepted as indispensable for both diagnostic and global understanding of the large rearrangements identified at the CFTR locus. Majority of deletions/duplications can be classified as severe mutations as associated with severe phenotype.…”

“…Informed consent to CFTR studies has been previously obtained from parents at time of referral. A SQF-PCR assay was used to detect large rearrangements in the CFTR gene as previously reported (primers on demand) (Taulan et al, 2009). The method relies on the comparison of the fluorescent profiles of multiplex PCR fragments obtained from different samples, the amplification being stopped at the exponential phase.…”

“…To identify the break-end junctions, a set of amplification using Light Cycler technology (Roche Diagnosis) was realized (Taulan et al, 2009). We performed the relative quantification of several sequences from intron 1 to intron 2 and their copy number was assessed.…”

“…Then, in addition to delineating the exact breakpoint junctions of the duplication, we used functional approaches to study the impact of naturally occurring large rearrangements, CFTR-dup2 and two previously reported deletions, CFTR-del2 and CFTR-del19 (Ferec et al, 2006;Taulan et al, 2007Taulan et al, , 2009). …”

Hereditary Hemorrhagic Telangiectasia: Breakpoint Characterization of a Novel Large Deletion in ACVRL1 Suggests the Causing Mechanism

“…This sequence includes the 'immunoglobulin heavy chain class switch repeat', a short direct repeat of 5 bp [CCCCA] [Chen et al, 2005a, b], which has been reported to mediate slippage mispairing and to cause deletions and translocations [Demura et al, 2002;Abeysinghe et al 2003]. It is well documented that the slippage mechanism during replication process is mediated by the presence of direct repeats (2-8 bp) at the end points of deletions/duplications and that only one short repeat is retained in the rearrangement [Ketterling et al, 1994;Chen et al, 2005a, b;Taulan et al, 2009]. Several characterized deletions showed locus variability both in the sequence and length of the direct repeats.…”