Extensive functional analyses of RHD splice site variants: Insights into the potential role of splicing in the physiology of Rh
Corresponding Author
Yann Fichou
Institut National de la Santé et de la Recherche Médicale (Inserm), UMR1078
Etablissement Français du Sang (EFS)–Région Bretagne
Address reprint requests to: Yann Fichou, PhD, Etablissement Français du Sang (EFS)–Région Bretagne, Inserm UMR1078, 46 rue Félix Le Dantec, CS 51819, 29218 Brest Cedex, France; e-mail: [email protected]Search for more papers by this authorPierre Gehannin
Institut National de la Santé et de la Recherche Médicale (Inserm), UMR1078
Etablissement Français du Sang (EFS)–Région Bretagne
Search for more papers by this authorManon Corre
Institut National de la Santé et de la Recherche Médicale (Inserm), UMR1078
Etablissement Français du Sang (EFS)–Région Bretagne
Search for more papers by this authorAlice Le Guern
Institut National de la Santé et de la Recherche Médicale (Inserm), UMR1078
Etablissement Français du Sang (EFS)–Région Bretagne
Search for more papers by this authorCédric Le Maréchal
Institut National de la Santé et de la Recherche Médicale (Inserm), UMR1078
Etablissement Français du Sang (EFS)–Région Bretagne
Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Centre Hospitalier Régional Universitaire (CHRU), Hôpital Morvan
Faculté de Médecine et des Sciences de la Santé, Université de Bretagne Occidentale, Brest, France.
Search for more papers by this authorGérald Le Gac
Institut National de la Santé et de la Recherche Médicale (Inserm), UMR1078
Etablissement Français du Sang (EFS)–Région Bretagne
Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Centre Hospitalier Régional Universitaire (CHRU), Hôpital Morvan
Faculté de Médecine et des Sciences de la Santé, Université de Bretagne Occidentale, Brest, France.
Search for more papers by this authorClaude Férec
Institut National de la Santé et de la Recherche Médicale (Inserm), UMR1078
Etablissement Français du Sang (EFS)–Région Bretagne
Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Centre Hospitalier Régional Universitaire (CHRU), Hôpital Morvan
Faculté de Médecine et des Sciences de la Santé, Université de Bretagne Occidentale, Brest, France.
Search for more papers by this authorCorresponding Author
Yann Fichou
Institut National de la Santé et de la Recherche Médicale (Inserm), UMR1078
Etablissement Français du Sang (EFS)–Région Bretagne
Address reprint requests to: Yann Fichou, PhD, Etablissement Français du Sang (EFS)–Région Bretagne, Inserm UMR1078, 46 rue Félix Le Dantec, CS 51819, 29218 Brest Cedex, France; e-mail: [email protected]Search for more papers by this authorPierre Gehannin
Institut National de la Santé et de la Recherche Médicale (Inserm), UMR1078
Etablissement Français du Sang (EFS)–Région Bretagne
Search for more papers by this authorManon Corre
Institut National de la Santé et de la Recherche Médicale (Inserm), UMR1078
Etablissement Français du Sang (EFS)–Région Bretagne
Search for more papers by this authorAlice Le Guern
Institut National de la Santé et de la Recherche Médicale (Inserm), UMR1078
Etablissement Français du Sang (EFS)–Région Bretagne
Search for more papers by this authorCédric Le Maréchal
Institut National de la Santé et de la Recherche Médicale (Inserm), UMR1078
Etablissement Français du Sang (EFS)–Région Bretagne
Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Centre Hospitalier Régional Universitaire (CHRU), Hôpital Morvan
Faculté de Médecine et des Sciences de la Santé, Université de Bretagne Occidentale, Brest, France.
Search for more papers by this authorGérald Le Gac
Institut National de la Santé et de la Recherche Médicale (Inserm), UMR1078
Etablissement Français du Sang (EFS)–Région Bretagne
Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Centre Hospitalier Régional Universitaire (CHRU), Hôpital Morvan
Faculté de Médecine et des Sciences de la Santé, Université de Bretagne Occidentale, Brest, France.
Search for more papers by this authorClaude Férec
Institut National de la Santé et de la Recherche Médicale (Inserm), UMR1078
Etablissement Français du Sang (EFS)–Région Bretagne
Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Centre Hospitalier Régional Universitaire (CHRU), Hôpital Morvan
Faculté de Médecine et des Sciences de la Santé, Université de Bretagne Occidentale, Brest, France.
Search for more papers by this authorThis work was supported by the Association Recherche et Transfusion (ART; Contract 63-2012); the Etablissement Français du Sang (EFS)–Région Bretagne; and the Institut National de la Santé et de la Recherche Médicale (Inserm), France.
Abstract
BACKGROUND
Among more than 300 mutated alleles identified so far within the RHD gene, almost 40 are assumed to alter cellular splicing and therefore may have a direct effect on Rh phenotype both at the quantitative and at the qualitative levels. Functional data are, however, mostly unavailable to assess the direct involvement of splicing defect in the underlying physiology.
STUDY DESIGN AND METHODS
We generated plasmid constructs to carry out an exhaustive investigation of 38 RHD variants located within or in the vicinity of exon–intron junctions by a minigene splicing assay, further characterized the transcript structures by sequencing, and identified cryptic sites activated by the genetic defect. Bioinformatics predictions were carried out in parallel and compared with the functional data.
RESULTS
For the first time we demonstrate that a product including the full-length Exon 9 is transcribed in the presence of the c.1227G>A substitution frequently carried by Asians with DEL phenotype and confirmed that splicing is altered in the RHD*weak D Type 2 allele, a rare variant most commonly found in Caucasians.
CONCLUSION
Overall we 1) show significant correlation between functional analyses, bioinformatics predictions, and phenotypes, when available, especially for variants in close proximity of the consensus splice sites; 2) classify the variations as splicing or nonsplicing variants; and 3) provide functional data to further improve bioinformatics splicing tools. Conversely assessment of seven silent exonic variants was mainly inconclusive.
Supporting Information
Additional Supporting Information may be found in the online version of this article at the publisher's Web site:
| Filename | Description |
|---|---|
| trf13083-sup-0001-suppinfo.docx13.2 KB | Supporting Information |
| trf13083-sup-0002-suppfigs.doc26.7 MB |
Fig. S1. Schematic representation of the strategy used to generate the c.1154G>C construct. (A) Two products were first amplified by PCRs 1 and 2 by using wild-type and RHD*weak D type 2 genomic DNAs as templates, respectively, resulting in the generation of partially overlapping wild-type and variant sequences. PCR products 1 and 2 were then diluted at 1/100th, and mixed together for PCR 3. Arrows with extensions: PCR primers for exon 9 amplification (Table SI, available as supporting information in the online version of this paper); arrows: complementary primers 5′-CTGTTTAAATGCATAATTTAATGTTAAAAG-3′ in PCR 1, and 5′-CTTTTAACATTAAATTATGCATTTAAACAG-3′ in PCR 2; nucleotides to incorporate are bold red underlined; nucleotides to remove are bold black underlined. PCR conditions are those defined in the manuscript. (B) Control of PCR amplifications by agarose gel electrophoresis; bp: base pairs. Fig. S2. Sequencing profiles of the RT-PCR products including full-length and alternative products of the respective exons (A-H). Arrowheads in the sequencing profiles indicate the position of the variant of interest; bp: base pairs. Fig. S3. Functional analysis of silent exonic RHD variants by minigene splicing assay in K-562 cells. NT = not transfected; pSP = pSplicePOLR2G vector; WT = wild-type construct; bp = base pairs. |
| trf13083-sup-0003-supptables.doc66 KB |
Table S1. Primer sequences for PCR amplification of RHD exons and flanking intronic regions. Table S2. Primer sequences for site-directed mutagenesis. |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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