Table_4_Analysis of the Genomic Sequence of ABO Allele Using Next-Generation Sequencing Method.xls

BackgroundAlthough many molecular diagnostic methods have been used for ABO genotyping, there are few reports on the full-length genomic sequence analysis of the ABO gene. Recently, next-generation sequencing (NGS) has been shown to provide fast and high-throughput results and is widely used in the clinical laboratory. Here, we established an NGS method for analyzing the sequence of the start codon to the stop codon in the ABO gene.
Study Design and MethodsTwo pairs of primers covering the partial 5’-untranslated region (UTR) to 3’-UTR of the ABO gene were designed. The sequences covering from the start codon to the stop codon of the ABO gene were amplified using these primers, and an NGS method based on the overlap amplicon was developed. A total of 110 individuals, including 88 blood donors with normal phenotypes and 22 ABO subtypes, were recruited and analyzed. All these specimens were first detected by serological tests and then determined by polymerase chain reaction sequence-based typing (PCR-SBT) and NGS. The sequences, including all the intron regions for the specimens, were analyzed by bioinformatics software.
ResultsAmong the 88 blood donors with a normal phenotype, 48 homozygous individuals, 39 heterozygous individuals, and one individual with a novel O allele were found according to the results of the PCR-SBT method. Some single-nucleotide variants (SNV) in intronic regions were found to be specific for different ABO alleles from 48 homozygous individuals using the NGS method. Sequences in the coding region of all specimens using the NGS method were the same as those of the PCR-SBT method. Three intronic SNVs were found to be associated with the ABO subtypes, including one novel intronic SNV (c.28+5956T>A). Moreover, six specimens were found to exhibit DNA recombination.
ConclusionAn NGS method was established to analyze the sequence from the start codon to the stop codon of the ABO gene. Two novel ABO alleles were identified, and DNA recombination was found to exist in the ABO alleles.

研究背景：尽管诸多分子诊断方法已应用于ABO基因分型，但目前针对ABO基因全长基因组序列分析的相关报道仍较为匮乏。近年来，新一代测序（next-generation sequencing, NGS）技术凭借其快速、高通量的检测优势，已在临床实验室中得到广泛应用。本研究建立了一种可分析ABO基因从起始密码子至终止密码子区域序列的NGS检测方法。
研究设计与方法：设计了两对引物，可覆盖ABO基因部分5'非翻译区（UTR）至3'非翻译区（UTR）区域。利用该引物扩增ABO基因从起始密码子至终止密码子的目标序列，进而开发出基于重叠扩增子的NGS检测方法。本研究共招募110名受试者，其中包括88名表型正常的献血者及22名ABO亚型携带者，所有标本均先通过血清学检测进行初筛，随后分别采用基于测序的聚合酶链反应分型（polymerase chain reaction sequence-based typing, PCR-SBT）及NGS技术进行检测。利用生物信息学软件对所有标本的内含子区域序列进行分析。
研究结果：基于PCR-SBT的检测结果显示，在88名表型正常的献血者中，共检出48名纯合子个体、39名杂合子个体及1名携带新型O等位基因的个体。通过NGS技术对48名纯合子个体的序列分析发现，部分内含子区域的单核苷酸变异（single-nucleotide variants, SNV）可作为不同ABO等位基因的特异性标志物。所有标本的编码区序列经NGS检测所得结果与PCR-SBT检测结果完全一致。本研究共发现3个与ABO亚型相关的内含子单核苷酸变异，其中包括1个新型内含子单核苷酸变异（c.28+5956T>A）。此外，共6份标本被检出存在DNA重组现象。
研究结论：本研究成功建立了一种可分析ABO基因从起始密码子至终止密码子区域序列的NGS检测方法，鉴定出2个新型ABO等位基因，并证实ABO等位基因中存在DNA重组现象。