Establishment of a medium‐throughput approach for the genotyping of <i><scp>RHD</scp></i> variants and report of nine novel rare alleles

Fichou, Yann; Maréchal, Cédric Le; Jamet, Déborah; Bryckaert, Laurence; Ka, Chandran; Audrézet, Marie-Pierre; Gac, Gérald Le; Dupont, Isabelle; Chen, Jian‐Min; Férec, Claude

doi:10.1111/trf.12009

Cited by 34 publications

(51 citation statements)

References 19 publications

(28 reference statements)

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“…When hybrid genes were suspected, RHCE QMPSF was carried out [25]. Sanger sequencing was performed as previously described when QMPSF analysis was inconclusive [26]. …”

Section: Methodsmentioning

confidence: 99%

RHD-Positive Alleles among D- C/E+ Individuals from India

Kulkarni

Gogri

Parchure

et al. 2018

Transfus Med Hemother

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Background: Molecular bases of blood group systems, including Rh blood group, have been poorly studied in the Indian population so far, while specificities of Europeans, East Asians and Africans have been well known for years. In order to gain insights into the molecular bases of this population, we sought to characterize the RHD allele in D- Indian donors expressing C and/or E antigen(s). Methods:RHD gene was analyzed in 171 serologically D-, C/E+ samples by standard molecular methods such as quantitative, multiplex PCR of short fluorescent fragments (QMPSF) and direct sequencing when necessary. Results:RHD whole gene deletion at the homozygous state was found to be the most common genotype associated with D- phenotype (118/171, 69.0%). Nonfunctional, negative hybrid genes with reported molecular backgrounds were observed in approximately one-third of the samples, while only four samples carry single-nucleotide variations, including one novel nonsense (RHD(Y243X)), one novel frameshift (RHD(c.701delG)), and two missense (RHD(T148R) and RHD(T148R, T195M)) alleles. Conclusion: Overall we report for the first time the molecular bases of D antigen negativity in the D-, C/E+ Indian population, which appears to be qualitatively similar to other populations, but with a population-specific, quantitative distribution of D-- alleles.

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“…When hybrid genes were suspected, RHCE QMPSF was carried out [25]. Sanger sequencing was performed as previously described when QMPSF analysis was inconclusive [26]. …”

Section: Methodsmentioning

confidence: 99%

RHD-Positive Alleles among D- C/E+ Individuals from India

Kulkarni

Gogri

Parchure

et al. 2018

Transfus Med Hemother

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“…In an attempt to characterize the effect of selected variations at the molecular level, we demonstrated previously, by using a functional test, namely minigene splicing assay (MSA), that several genetic defects in the vicinity of consensus splice sites in the RHD gene alter splicing . Indeed splicing of pre‐messenger RNAs (mRNA) is a fundamental key‐step in the eukaryotic gene expression pathway.…”

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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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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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“…DNA was extracted by using a centrifugation technique (FlexiGene DNA kit, Qiagen). RHD and RHCE genes were analyzed by quantitative multiplex polymerase chain reaction (PCR) of short fluorescent fragments (QMPSF) for exon copy number quantitation and Sanger sequencing, as previously described . All 10 exons of the RHAG gene were amplified by PCR in the same conditions as above and PCR products were loaded onto a 2% agarose gel (primer sequences are provided in Table S2, available as supporting information in the online version of this paper).…”

Section: Methodsmentioning

confidence: 99%

“…Effect of the potential splice site variant was assessed by minigene splicing assay as described previously . Briefly the pSplice POLR2G vector was digested with Eco RI restriction enzyme (New England Biolabs) and column purified (Amicon Ultra‐0.5 centrifugal filter unit with Ultracel‐100 membrane, Millipore).…”

Section: Methodsmentioning

confidence: 99%

First report of Rh_null individuals in the Indian population and characterization of the underlying molecular mechanisms

et al. 2017

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Establishment of a medium‐throughput approach for the genotyping of RHD variants and report of nine novel rare alleles

Abstract: The new two-step analysis proved to be much easier and cheaper than the DHPLC method and therefore is convenient to be used as a routine, medium-throughput approach for RHD genotyping.

Cited by 34 publications

References 19 publications

RHD-Positive Alleles among D- C/E+ Individuals from India

RHD-Positive Alleles among D- C/E+ Individuals from India

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

First report of Rh_null individuals in the Indian population and characterization of the underlying molecular mechanisms

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Establishment of a medium‐throughput approach for the genotyping of RHD variants and report of nine novel rare alleles

Abstract: The new two-step analysis proved to be much easier and cheaper than the DHPLC method and therefore is convenient to be used as a routine, medium-throughput approach for RHD genotyping.

Cited by 34 publications

References 19 publications

RHD-Positive Alleles among D- C/E+ Individuals from India

RHD-Positive Alleles among D- C/E+ Individuals from India

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

First report of Rhnull individuals in the Indian population and characterization of the underlying molecular mechanisms

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First report of Rh_null individuals in the Indian population and characterization of the underlying molecular mechanisms