Red Cross Transfusion Service for Upper Austria, Krankenhausstrasse 7, Linz, Austria.
Vox Sang. 2011 May;100(4):381-8. doi: 10.1111/j.1423-0410.2010.01444.x. Epub 2010 Dec 7.
Molecular variations of the RHD gene may result in the reduced expression of the D antigen and altered Rh phenotypes. In many occasions, they cannot be typed reliably by standard serological methods. Sequence-based typing is the gold standard to determine rare and unknown RHD genotypes. For this pilot study, sequence-based typing by standard Sanger sequencing was compared to a newly established next-generation sequencing approach based on pyrosequencing.
Twenty-six DNA samples were selected after primary serological testing exhibiting a weak reaction in Rh phenotype. Parallel sequence analysis of the complete coding sequence including adjacent intronic sequences allowed a comparison of the methodical potency in mutation detection of Sanger with next-generation sequencing.
Sanger sequencing revealed 39 RHD polymorphisms in 21 of 26 samples in the RHD coding region, while pyrosequencing detected all but two alterations resulting in a concordance rate of 94·9% and clearly revealed a heterozygous compound mutation in one sample with RHDψ and Weak D type 4 alleles. The resolution of cis/trans linkage of polymorphisms and exact characterization of a 37 bp duplication was achieved by next-generation sequencing.
Our data suggest that next-generation sequencing offers a new development for high-throughput and clonal sequencing for molecular RHD genotyping. However, further attempts in the methodical set-up have to be undertaken prior to validation and introduction as a routine service.
RHD 基因的分子变异可能导致 D 抗原表达减少和 Rh 表型改变。在许多情况下，它们无法通过标准血清学方法可靠地分型。基于序列的分型是确定罕见和未知 RHD 基因型的金标准。本研究采用标准 Sanger 测序的基于序列的分型与新建立的基于焦磷酸测序的新一代测序方法进行了比较。
在原发性血清学检测呈 Rh 表型弱阳性反应的 26 个 DNA 样本中进行选择。对包括相邻内含子序列在内的完整编码序列进行平行序列分析，以比较 Sanger 测序在突变检测中的方法学效能与新一代测序。
Sanger 测序在 26 个样本中的 21 个样本的 RHD 编码区中发现了 39 个 RHD 多态性，而焦磷酸测序检测到了所有除两个以外的改变，一致性率为 94.9%，并在一个具有 RHDψ和 Weak D 型 4 等位基因的样本中清晰地显示了杂合性复合突变。新一代测序实现了多态性的顺/反式连锁的分辨率和 37 bp 重复的精确特征描述。
我们的数据表明，新一代测序为分子 RHD 基因分型的高通量和克隆测序提供了新的发展。然而，在作为常规服务进行验证和引入之前，必须在方法设置方面进行进一步尝试。
