Variant screening of the <i>RHD</i> gene in a large cohort of subjects with D phenotype ambiguity: report of 17 novel rare alleles

Fichou, Yann; Maréchal, Cédric Le; Bryckaert, Laurence; Guerry, Christine; Bénech, Caroline; Dupont, Isabelle; Jamet, Déborah; Férec, Claude; Chen, Jian‐Min

doi:10.1111/j.1537-2995.2011.03350.x

Cited by 23 publications

(34 citation statements)

References 22 publications

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“…An up‐to‐date review of the literature and the RhesusBase was performed . On the basis of their position relative to the constitutive SSs, 15 variations of the RHD gene, including eight exonic (i.e., first, antepenultimate and penultimate position of exons) and seven intronic variants were selected for functional analysis (Table ).…”

Section: Methodsmentioning

confidence: 99%

“…An up-to-date review of the literature and the RhesusBase was performed. 1,[13][14][15][16][17][18][19][20][21][22][23][24] On the basis of their position relative to the constitutive SSs, 15 variations of the RHD gene, including eight exonic (i.e., first, antepenultimate and penultimate position of exons) and seven intronic variants were selected for functional analysis (Table 1). In an effort to elucidate the potential effect of more internal synonymous variants on transcript expression, as reported for the c.960G>A variation by two independent studies, 4,13 the c.1056C>G and c.1065C>T variants, both located within exon 7 of the RHD gene, were also selected for functional study.…”

Section: Selection Of Rhd Variantsmentioning

confidence: 99%

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Functional analysis of novel RHD variants: splicing disruption is likely to be a common mechanism of variant D phenotype

Raud

Gourlaouen

et al. 2019

Transfusion

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BACKGROUND We previously showed that several variations in the RHD gene, including synonymous changes, can be classified as splice site variants and may play a direct role in D variant phenotype expression. We sought to extend our study to additional candidates, notably in the first and last exons of the gene, by engineering a novel universal splice reporting vector, i.e., minigene. STUDY DESIGN AND METHODS Our previous plasmid construct was modified to allow subcloning of any exon(s) of interest for assessing effect of variations on splicing. Seventeen novel and/or uncharacterized variations of the RHD gene were selected for the study and tested in our novel model. RESULTS We engineered and validated a novel universal minigene for assessing virtually any variations of interest for splicing defect. Of the 17 variants tested in the novel model, 11 were shown to alter splicing either totally or partially, including the silent c.1065C>T variation, which induces major skipping of exon 7, and may therefore be responsible for reducing D antigen expression. We also showed that while all three missense variations c.1154G>C, c.1154G>T, and c.1154G>A in exon 9 are splice site variants, splicing is differentially altered and D‐negative phenotype observed in the presence of the latter substitution is likely due to a defect in RhD protein folding. CONCLUSION Overall, we hypothesize that splicing alteration is likely to be a common mechanism of D phenotype variation that has been underestimated so far. Further large‐scale studies are necessary to demonstrate this statement definitely.

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Section: Methodsmentioning

confidence: 99%

Section: Selection Of Rhd Variantsmentioning

confidence: 99%

Functional analysis of novel RHD variants: splicing disruption is likely to be a common mechanism of variant D phenotype

Raud

Gourlaouen

et al. 2019

Transfusion

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“…The allele DWN was named after the two characteristic amino acid substitutions tryptophan (W) and asparagine (N) and has been observed before without attribution of a name . The designation DNT was derived from the amino acid substitution asparagine (N) to threonine (T).…”

Section: Methodsmentioning

confidence: 99%

D category IV: a group of clinically relevant and phylogenetically diverse partial D

et al. 2013

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Background The D typing strategies in several European countries protect carriers of D category VI (DVI) from anti-D immunization but not carriers of other partial D. Besides DVI, one of the clinically most important partial D is D category IV (DIV). A detailed description and direct comparison of the different DIV types was missing. Study design and methods RHD nucleotide sequences were determined from genomic DNA. D epitope patterns were established with commercial monoclonal anti-D panels. Results DIV comprises several variants of the D antigen with distinct serology, molecular structures, evolutionary origins and ethnic prevalences. The DIV phenotype is determined by 350H shared by all, but not limited to, DIV variants which are further divided into DIVa and DIVb. The DIVa phenotype is expressed by DIV type 1.0 harboring 350H and the dispersed amino acids 62F, 137V and 152T. The DIVb phenotype is expressed by DIV type 3 to type 5 representing RHD-CE-D hybrids. 4 of the 6 postulated DIV variants were encountered among 23 DIV samples analyzed. Of 12 DIV carriers with anti-D, 10 were female and 7 likely immunized by pregnancy. 2 DIV related alleles are newly described: DWN which differs from DIV type 4 by 350D and epitope pattern. DNT carries 152T, known to cause a large D antigen density. Conclusion DIV alleles arose from at least 2 independent evolutionary events. DIV type 1.0 with DIVa phenotype belongs to the oldest extant human RHD alleles. DIV type 2 to type 5 with DIVb phenotype arose from more recent gene conversions. Anti-D immunization, especially dreaded in pregnancies, will be avoided not only in carriers of DVI but also in carriers of other D variants like DIV, if our proposed D typing strategy is adopted.

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“…It is characterized by the single‐nucleotide polymorphism (SNP) c.1136C>T (p.Thr379Met) in Exon 8 of the RHD gene . Only nine DAU alleles have formally been published between 2002 and 2009, with documented anti‐D in carriers of DAU‐3 and DAU‐4 , although several more DAU allele candidates have since accrued in online repositories …”

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The DAU cluster: a comparative analysis of 18 RHD alleles, some forming partial D antigens

et al. 2016

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Background The Rh system is the most complex and polymorphic blood group system in humans with more than 460 alleles known for the RHD gene. The DAU cluster of RHD alleles is characterized by the single nucleotide change producing the p.Thr379Met amino acid substitution. It is called the DAU-0 allele and has been postulated to be the primordial allele, from which all other alleles of the DAU cluster have eventually evolved. Study design and methods For 2 novel DAU alleles, the nucleotide sequences of all 10 exons as well as adjacent intronic regions, including the 5’ and 3’ untranslated regions (UTR), were determined for the RHD and RHCE genes. A phylogenetic tree for all DAU alleles was established using the neighbor-joining method with Pan troglodytes as root. Standard hemagglutination and flow cytometry tests were performed. Results We characterized 2 DAU alleles, DAU-11 and DAU-5.1, closely related to DAU-3 and DAU-5 respectively. A phylogenetic analysis of the 18 known DAU alleles indicated point mutations and interallelic recombination contributing to diversification of the DAU cluster. Conclusions The DAU alleles encode a group of RhD protein variants, some forming partial D antigens known to permit anti-D in carriers; all are expected to cause anti-D alloimmunization in recipients of red cell transfusions. The DAU alleles evolved through genomic point mutations and recombination. These results suggest that the cluster of DAU alleles represent a clade, which is concordant with our previous postulate that they derived from the primordial DAU-0 allele.

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Variant screening of the RHD gene in a large cohort of subjects with D phenotype ambiguity: report of 17 novel rare alleles

Abstract: This study further increased the already large repertoire of RHD allelic variants. Whereas most of the newly found variants are putative weak or partial D alleles, most of the complex alleles are readily understandable in the present phylogenetic model of RHD.

Cited by 23 publications

References 22 publications

Functional analysis of novel RHD variants: splicing disruption is likely to be a common mechanism of variant D phenotype

Functional analysis of novel RHD variants: splicing disruption is likely to be a common mechanism of variant D phenotype

D category IV: a group of clinically relevant and phylogenetically diverse partial D

The DAU cluster: a comparative analysis of 18 RHD alleles, some forming partial D antigens

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