Characterization of a recurrent novel large duplication in the cystic fibrosis transmembrane conductance regulator gene

Abstract

Recently, DNA rearrangements in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been described with increasing frequency. These large DNA rearrangements are not detected using conventional methods of DNA sequencing, single-strand conformational polymorphism, or denaturing high-performance liquid chromatography. We and others have described methods to detect such rearrangements in the CFTR gene. With one exception, all rearrangements reported thus far are single or multiple exon deletions, whereas only one report has described a large duplication. We describe here the detection and characterization of a novel large duplication in the CFTR gene. This duplication, referred to as gIVS6a + 415_IVS10 + 2987Dup26817bp, was detected in a classic CF female patient whose other mutation was DeltaF508. The duplication was inherited paternally. The duplication encompassed exons 6b to 10 and occurred on the IVS8-11TG/IVS8-7T/G1540 haplotype. This large duplication is predicted to result in the production of a truncated CFTR protein lacking the terminal part of NBD1 domain and beyond and thus can be considered a null allele. The combination of the DeltaF508 and gIVS6a + 415_IVS10 + 2987Dup26817bp mutation probably causes the severe CF phenotype in this patient. We designed a simple polymerase chain reaction test to detect the duplication, and we further detected the same duplication from another independent laboratory. The duplication breakpoint is identical in all three patients, suggesting a likely founder mutation.

Figure 1

Detection of duplication of exons 6b to 10 in the proband. A: DNA sample with no exon deletions or duplications; B: probands’ DNA. A and B: SQF PCR analysis of proband DNA for exon deletions or duplications. Arrows in B indicated duplicated exons. The duplicated fragments included 6b, 7, 8, UpEx 9 (a fragment in IVS8 upstream of exon 9), 9, and 10. The asterisk in the proband’s DNA indicate the detection of ΔF508 mutation, a faster migrating fragment related to exon 10.

Figure 2

Detection of duplication junction fragment by PCR amplification and DNA sequencing. A: Schematic representation of the duplicated region, where duplicated exons are referred to with 6b′, 7′, etc. The figure also shows the primer walking strategy (arrows) used to amplify the junction fragment in B. B: Under PCR conditions using primers 10F and 6bR, a fragment of ∼3.8 kb was detected from the proband (lane 1) but not from two random DNA samples (lanes 2 and 3). When primers from 10F, 6bF, and 6bR were mixed together in a separate PCR, conditions of the PCR allowed the amplification only of the fragment representing exon 6b. C: A DNA sequencing trace showing sequences of IVS10 and IVS6a at the breakpoint of the duplication.

Figure 3

Detection of downstream (A) and upstream (B) junction fragments in three probands harboring duplication of exons 6b to 10. Sample 1 is from the proband from Quest Diagnostics. Samples 2 to 4 are from Ambry Genetics. Samples 1, 2, and 4 harbor the duplications, whereas sample 3 is a normal sample. All samples showed amplification of sequences in IVS10 (A), whereas only the probands showed the amplification of upstream junction fragment (B) between IVS10 and IVS6a. DNA sequencing on junction fragment products (data not shown) showed all probands harboring the same breakpoint, suggesting a founder duplication.