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Background: Knowledge of the molecular determinants driving antigen expression is critical to design, optimize, and implement a genotyping approach on a population‐specific basis. Although RHD gene variability has been extensively reported in Caucasians, Africans, and East‐Asians, it remains to be explored in Southeast Asia. Thus the molecular basis of non‐D+ blood donors was investigated in Thailand. Study Design and Methods: First, 1176 blood samples exhibiting an inconclusive or negative result by automated serological testing were collected in the 12 Regional Blood Centres of the Thai Red Cross located throughout Thailand. Second, the RHD gene was analyzed in all samples by 1) quantitative multiplex PCR of short fluorescent fragments, and 2) direct sequencing, when necessary, for identifying structural variants and single nucleotide variants, respectively. Results: Additional serological typing yielded 51 and 1125 samples with weak/partial D and D‐negative (D‐) phenotype, respectively. In the first subset, partial RHD*06.03 was the most common variant allele (allele frequency: 18.6%). In the second subset, the whole deletion of the gene is largely the most frequent (allele frequency: 84.9%), followed by the Asian DEL allele found in 15.6% of the samples. Eight novel alleles with various mutational mechanisms were identified. Conclusion: We report, for the first time at the national level, the molecular basis of weak/partial D and serologically D‐ phenotypes in Thai blood donors. The design and implementation of a dedicated diagnostic strategy in blood donors and patients are the very next steps for optimizing the management and supply of RBC units in Thailand. [ABSTRACT FROM AUTHOR]

Background: Molecular genetics of the Rh system has been extensively studied in Caucasians, Black Africans, East Asians, and Indians more recently. In this work, we sought to investigate the molecular basis of variant D expression in the Thai population, which remains unknown. Materials and Methods: Blood samples from 450 Thai donors showing the variant D phenotype were collected. The RHD gene was analyzed by quantitative multiplex polymerase chain reaction of short fluorescent fragments and/or Sanger sequencing. Results: The most frequent alleles in 200 D-negative and 121 DEL samples were the whole RHD gene deletion and the Asian DEL alleles, respectively. In 129 weak/partial D samples, 36 variant alleles were identified, including eight novel alleles. RHD*06.03, which is common in variant D samples from South China, is the most prevalent variant allele, followed by the recently reported Indian RHD*01W.150 allele. Discussion: For the first time, a comprehensive overview of the nature and distribution of variant RHD alleles in Thailand is reported. It is a milestone to pave the way towards improvement of the current screening strategy to identify DEL donors accurately. The next step will be the design and implementation of a simple molecular test for screening the most frequent alleles, specifically in this population. [ABSTRACT FROM AUTHOR]

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., (© 2019 AABB.)