Fichou Yann, Gehannin Pierre, Corre Manon, Le Guern Alice, Le Maréchal Cédric, Le Gac Gérald, Férec Claude
Institut National de la Santé et de la Recherche Médicale (Inserm), UMR1078.
Etablissement Français du Sang (EFS)-Région Bretagne.
Transfusion. 2015 Jun;55(6 Pt 2):1432-43. doi: 10.1111/trf.13083. Epub 2015 Mar 21.
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.
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.
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.
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.
在目前已鉴定出的RHD基因内300多个突变等位基因中，近40个被认为会改变细胞剪接，因此可能在数量和质量水平上对Rh血型表型产生直接影响。然而，大多缺乏功能数据来评估剪接缺陷在潜在生理学中的直接作用。
我们构建了质粒载体，通过小基因剪接试验对位于外显子-内含子连接处或其附近的38个RHD变体进行了详尽研究，通过测序进一步表征转录本结构，并鉴定了由基因缺陷激活的隐蔽位点。同时进行了生物信息学预测并与功能数据进行比较。
我们首次证明，在具有DEL表型的亚洲人常见的c.1227G>A替换存在的情况下，会转录出包含全长外显子9的产物，并证实RHD*弱D 2型等位基因（一种在白种人中最常见的罕见变体）的剪接发生了改变。
总体而言，我们1）在有可用数据时，展示了功能分析、生物信息学预测和表型之间的显著相关性，特别是对于靠近共有剪接位点的变体；2）将这些变异分类为剪接或非剪接变异；3）提供功能数据以进一步改进生物信息学剪接工具。相反，对7个沉默外显子变异的评估主要没有定论。
