title: Extensive functional analyses of RHD splice site variants: Insights into the potential role of splicing in the physiology of Rh.
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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors: Institut National de la Santé et de la Recherche Médicale (Inserm), UMR1078.|Etablissement Français du Sang (EFS)-Région Bretagne.
authors: Yann
authors: Y
authors: Fichou
authors: Institut National de la Santé et de la Recherche Médicale (Inserm), UMR1078.|Etablissement Français du Sang (EFS)-Région Bretagne.
authors: Pierre
authors: P
authors: Gehannin
authors: Institut National de la Santé et de la Recherche Médicale (Inserm), UMR1078.|Etablissement Français du Sang (EFS)-Région Bretagne.
authors: Manon
authors: M
authors: Corre
authors: Institut National de la Santé et de la Recherche Médicale (Inserm), UMR1078.|Etablissement Français du Sang (EFS)-Région Bretagne.
authors: Alice
authors: A
authors: Le Guern
authors: 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.
authors: Cédric
authors: C
authors: Le Maréchal
authors: 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.
authors: Gérald
authors: G
authors: Le Gac
authors: 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.
authors: Claude
authors: C
authors: Férec
mesh_terms: D044466:Asian People / Q000235:genetics / Q000706:statistics & numerical data; D001483:Base Sequence; D003001:Cloning, Molecular; D019295:Computational Biology; D005787:Gene Frequency; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020641:Polymorphism, Single Nucleotide / Q000502:physiology; D022821:RNA Splice Sites / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics / Q000502:physiology*; D012204:Rh-Hr Blood-Group System / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D014162:Transfection; D044465:White People / Q000235:genetics / Q000706:statistics & numerical data
chemical_list: D022821:RNA Splice Sites; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen