Establishment of a medium-throughput approach for the genotyping of RHD variants and report of nine novel rare alleles.
Transfusion
; 53(8): 1821-8, 2013 Aug.
Article
em En
| MEDLINE
| ID: mdl-23228153
ABSTRACT
BACKGROUND:
The routinely used serologic methods are robust in accurately typing standard D- or D+ blood. However, they result in discrepancy in weak or partial D blood, which requires genetic analysis. We have previously used denaturing high-performance liquid chromatography (DHPLC) to screen the entire RHD-coding sequence. However, DHPLC is technically challenging, labor-intensive, and time-consuming. To overcome these inconveniences, we sought to develop a new two-step approach. STUDY DESIGN ANDMETHODS:
A total of 430 blood samples with D phenotype ambiguity were recruited for this study. The three most frequent weak D alleles (i.e., weak D, Type 1; weak D, Type 2; and weak D, Type 3), which altogether account for 60% to 90% of the atypical RHD alleles in the Caucasian population, were first identified by Tm-shift genotyping. The remaining unidentified samples were then subjected to a single-tube multiplex polymerase chain reaction (PCR) amplification of all 10 RHD exons followed by direct sequencing.RESULTS:
Optimal conditions for efficient and reliable identification of the three most common weak D variants by Tm-shift genotyping were established. All 10 RHD exons were successfully amplified in a single-multiplex PCR procedure. Employment of the two-step analysis identified RHD variants in 91.6% of the 430 studied samples. Two of the nine previously undescribed variants, c.335G>T and c.939G>A, were found to cause aberrant mRNA splicing by means of a splicing minigene assay.CONCLUSION:
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Sistema do Grupo Sanguíneo Rh-Hr
/
Alelos
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Técnicas de Genotipagem
Tipo de estudo:
Etiology_studies
/
Incidence_studies
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Observational_studies
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Prognostic_studies
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Risk_factors_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article