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Missense RHD single nucleotide variants induce weakened D antigen expression by altering splicing and/or protein expression
Author(s) -
Raud Loann,
Le Tertre Marlène,
Vigneron Léonie,
Ka Chandran,
Richard Gaëlle,
Callebaut Isabelle,
Chen JianMin,
Férec Claude,
Le Gac Gérald,
Fichou Yann
Publication year - 2021
Publication title -
transfusion
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.045
H-Index - 132
eISSN - 1537-2995
pISSN - 0041-1132
DOI - 10.1111/trf.16538
Subject(s) - missense mutation , minigene , rna splicing , exon , biology , alternative splicing , genetics , in silico , gene , phenotype , microbiology and biotechnology , rna
Background Although D variant phenotype is known to be due to genetic defects, including rare missense single nucleotide variants (SNVs), within the RHD gene, few studies have addressed the molecular and cellular mechanisms driving this altered expression. We and others showed previously that splicing is commonly disrupted by SNVs in constitutive splice sites and their vicinity. We thus sought to investigate whether rare missense SNVs located in “deep” exonic regions could also impair this mechanism. Study design and methods Forty‐six missense SNVs reported within exons 6 and 7 were first selected from the Human RhesusBase. Their respective effect on splicing was assessed by using an in vitro assay. An RhD‐negative cell model was further generated by using the CRISPR‐Cas9 approach. RhD‐mutated proteins were overexpressed in the newly created model, and cell membrane expression of the D antigen was measured by flow cytometry. Results Minigene splicing assay showed that 14 of 46 (30.4%) missense SNVs alter splicing. Very interestingly, further investigation of two missense SNVs, which both affect codon 338 and confer a weak D phenotype, showed various mechanisms: c.1012C>G (p.Leu338Val) disrupts splicing only, while c.1013T>C (p.Leu338Pro) alters only the protein structure, in agreement with in silico prediction tools and 3D protein structure visualization. Conclusion Our functional data set suggests that missense SNVs damage quantitatively D antigen expression by, at least, two different mechanisms (splicing alteration and protein destabilization) that may act independently. These data thereby contribute to extend the current knowledge of the molecular mechanisms governing weakened D expression.