Detection of two Alu insertions in the CFTR gene

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Detection of two Alu insertions in the CFTR gene Jian-Min Chen, Emmanuelle Masson, Milan Macek, Odile Raguénès, Tereza Piskackova, Brigitte Fercot, Libor Fila, David N. Cooper, Marie-Pierre Audrézet, Claude Férec Journal of Cystic Fibrosis Volume 7, Issue 1, Pages 37-43 (January 2008) DOI: 10.1016/j.jcf.2007.04.001 Copyright © 2007 European Cystic Fibrosis Society Terms and Conditions

Fig. 1 Identification of the #1 simple Alu insertion in the CFTR gene. (a) Employing QMPSF [5], the peak height of the exon 16 amplicon was found to be significantly reduced in a 24-year-old French girl with typical cystic fibrosis (in red) as compared with a normal control (in blue). [See d for primers used to amplify exon 16]. (b) QHPLC analysis of exon 16 of the CFTR gene in the patient and her relatives. Note the reduced height of the main peak and the appearance of an additional small peak (downward pointing arrow) in the patient and her father. (c) Electrophoretic profiles of PCR (performed under the same conditions as for the QMPSF analysis) products from the patient and her relatives run on a 2% agarose gel. Note the appearance of a longer (albeit weaker) band in the patient and her father. (d, e) Wild-type and mutant genomic sequences of exon 16 (in upper case) and its flanking sequence in the CFTR gene. Sequences are given in the 5′ to 3′ orientation with respect to the sense strand of the CFTR gene. Shaded sequences correspond to the primer pair used for the QMPSF analysis [5]. Underlined sequences denote the primer pair used for the QHPLC analysis (this study). The target site duplication (TSD), a characteristic hallmark of simple LINE-1 endonuclease-dependent retrotranspositional events, is highlighted in bold. Sequence in italics (e) indicates the 46 bp Alu element plus the 57 bp poly(A) tail that has been inserted in the antisense orientation with respect to the transcriptional direction of the target gene. The second t (barred) of the downstream TSD was deleted (e), probably by replication slippage. (f) Deduced top and bottom strand cleavage sites involved in the #1 simple Alu insertion in the CFTR gene. Note that although this figure was derived from d and e, the top strand represents the antisense strand of the CFTR gene. The TSD is shown in bold and the cleavage sites are indicated by arrows. Shaded sequences on the bottom strand indicate nucleotides matching the degenerate L1 endonuclease consensus cleavage site, 3′-A/TTTT-5′. Journal of Cystic Fibrosis 2008 7, 37-43DOI: (10.1016/j.jcf.2007.04.001) Copyright © 2007 European Cystic Fibrosis Society Terms and Conditions

Fig. 2 Schematic illustration of the locations of the primer pairs employed (indicated by pairs of arrows) with respect to their target exons (indicated by boxes) in QMPSF [5] and QHPLC (this study), respectively. Journal of Cystic Fibrosis 2008 7, 37-43DOI: (10.1016/j.jcf.2007.04.001) Copyright © 2007 European Cystic Fibrosis Society Terms and Conditions

Fig. 3 Identification of the #2 simple Alu insertion in the CFTR gene from a Czech man with cystic fibrosis. (a) QHPLC profiles of exon 17b of the CFTR gene. Note the reduced height of the main peak and the appearance of an additional small peak (downward pointing arrow) in the patient. [See c for the primers used to amplify exon 17b]. (b) Electrophoretic profiles of PCR (performed with a prolonged extension time as compared with that used for the QHPLC analysis; see text) products from the patient and his parents on a 2% agarose gel. Note the appearance of a reasonably strong band of ∼750 bp in the patient and his mother. See text regarding the additional band of <700 bp. (c, d) Wild-type and mutant genomic sequences of exon 17b (in upper case) and its flanking sequence in the CFTR gene. Shaded sequences correspond to the primer pair used for QMPSF analysis [5]. Underlined sequences indicate the primer pair used for the QHPLC analysis. Sequence in italics (d) identifies the 281 bp Alu insert plus the 56 bp poly(A) tail that has been inserted in a sense orientation with respect to the transcriptional orientation of the target gene. (e) Deduced top and bottom strand cleavage sites involved in the #2 simple Alu insertion. The target site duplication (TSD) is shown in bold. Cleavage sites are denoted by arrows. Shaded nucleotides in the bottom strand indicate a highly degenerate L1 endonuclease cleavage site. Journal of Cystic Fibrosis 2008 7, 37-43DOI: (10.1016/j.jcf.2007.04.001) Copyright © 2007 European Cystic Fibrosis Society Terms and Conditions