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Abstract：
BACKGROUND: The Rh blood group system is characterized by its complexity and polymorphism, encompassing 56 different antigens. Accurately predicting the presence of the C antigen using genotyping methods has been challenging. The objective of this study was to evaluate the accuracy of various genotyping methods for predicting the Rh C and to identify a suitable method for the Chinese Han population.
METHODS: In total, 317 donors, consisting 223 D+ (including 20 with the Del phenotype) and 94 D- were randomly selected. For RHC genotyping, 48C and 109bp insertion were detected on the Real-time PCR platform and -292 substitution was analyzed via restriction fragment length polymorphism (RFLP). Moreover, the promoter region of the RHCE gene was sequenced to search for other nucleotide substitutions between RHC and RHc. Agreement between prediction methods was evaluated using the Kappa statistic, and comparisons between methods were conducted via the χ2 test.
RESULTS: The analysis revealed that the 48C allele, 109bp insertion, a specific pattern observed in RFLP results, and wild-type alleles of seven single nucleotide polymorphisms (SNPs) were in strong agreement with the Rh C, with Kappa coefficients exceeding 0.8. However, there were instances of false positives or false negatives (0.6% false negative rate for 109bp insertion and 5.4-8.2% false positive rates for other methods). The 109bp insertion method exhibited the highest accuracy in predicting the Rh C, at 99.4%, compared to other methods (P values≤0.001). Although no statistical differences were found among other methods for predicting Rh C (P values>0.05), the accuracies in descending order were 48C (94.6%) > rs586178 (92.7%) > rs4649082, rs2375313, rs2281179, rs2072933, rs2072932, and RFLP (92.4%) > rs2072931 (91.8%).
CONCLUSIONS: None of the methods examined can independently and accurately predict the Rh C. However, the 109bp insertion test demonstrated the highest accuracy for predicting the Rh C in the Chinese Han population. Utilizing the 109bp insertion test in combination with other methods may enhance the accuracy of Rh C prediction.
METHODS: In total, 317 donors, consisting 223 D+ (including 20 with the Del phenotype) and 94 D- were randomly selected. For RHC genotyping, 48C and 109bp insertion were detected on the Real-time PCR platform and -292 substitution was analyzed via restriction fragment length polymorphism (RFLP). Moreover, the promoter region of the RHCE gene was sequenced to search for other nucleotide substitutions between RHC and RHc. Agreement between prediction methods was evaluated using the Kappa statistic, and comparisons between methods were conducted via the χ2 test.
RESULTS: The analysis revealed that the 48C allele, 109bp insertion, a specific pattern observed in RFLP results, and wild-type alleles of seven single nucleotide polymorphisms (SNPs) were in strong agreement with the Rh C, with Kappa coefficients exceeding 0.8. However, there were instances of false positives or false negatives (0.6% false negative rate for 109bp insertion and 5.4-8.2% false positive rates for other methods). The 109bp insertion method exhibited the highest accuracy in predicting the Rh C, at 99.4%, compared to other methods (P values≤0.001). Although no statistical differences were found among other methods for predicting Rh C (P values>0.05), the accuracies in descending order were 48C (94.6%) > rs586178 (92.7%) > rs4649082, rs2375313, rs2281179, rs2072933, rs2072932, and RFLP (92.4%) > rs2072931 (91.8%).
CONCLUSIONS: None of the methods examined can independently and accurately predict the Rh C. However, the 109bp insertion test demonstrated the highest accuracy for predicting the Rh C in the Chinese Han population. Utilizing the 109bp insertion test in combination with other methods may enhance the accuracy of Rh C prediction.
摘要：
背景：Rh血型系统的特征在于其复杂性和多态性，包含56种不同的抗原。使用基因分型方法准确预测C抗原的存在一直是具有挑战性的。这项研究的目的是评估各种基因分型方法预测RhC的准确性，并确定适合中国汉族人群的方法。
方法：总共，317个捐助者，随机选择由223个D+(包括20个具有Del表型)和94个D-组成。对于RHC基因分型，在实时PCR平台上检测到48C和109bp的插入，并通过限制性片段长度多态性（RFLP）分析了-292个取代。此外，对RHCE基因的启动子区进行测序以寻找RHC和RHc之间的其他核苷酸取代。使用Kappa统计量评估了预测方法之间的一致性，方法间比较采用χ2检验。
结果：分析显示48C等位基因，109bp插入，在RFLP结果中观察到的特定模式，7个单核苷酸多态性(SNPs)的野生型等位基因与RhC,Kappa系数超过0.8。然而,存在假阳性或假阴性的情况(109bp插入的假阴性率为0.6%,其他方法的假阳性率为5.4-8.2%).109bp插入法在预测RhC时表现出最高的准确性，99.4%，与其他方法相比（P值≤0.001）。尽管在其他预测RhC的方法中没有发现统计学差异（P值>0.05），准确度降序为48C(94.6%)>rs586178(92.7%)>rs4649082、rs2375313、rs2281179、rs2072933、rs2072932和RFLP(92.4%)>rs2072931(91.8%)。
结论：所检查的方法均不能独立且准确地预测RhC。109bp插入试验显示了在中国汉族人群中预测RhC的最高准确性。结合其他方法利用109bp插入测试可以提高RhC预测的准确性。
方法：总共，317个捐助者，随机选择由223个D+(包括20个具有Del表型)和94个D-组成。对于RHC基因分型，在实时PCR平台上检测到48C和109bp的插入，并通过限制性片段长度多态性（RFLP）分析了-292个取代。此外，对RHCE基因的启动子区进行测序以寻找RHC和RHc之间的其他核苷酸取代。使用Kappa统计量评估了预测方法之间的一致性，方法间比较采用χ2检验。
结果：分析显示48C等位基因，109bp插入，在RFLP结果中观察到的特定模式，7个单核苷酸多态性(SNPs)的野生型等位基因与RhC,Kappa系数超过0.8。然而,存在假阳性或假阴性的情况(109bp插入的假阴性率为0.6%,其他方法的假阳性率为5.4-8.2%).109bp插入法在预测RhC时表现出最高的准确性，99.4%，与其他方法相比（P值≤0.001）。尽管在其他预测RhC的方法中没有发现统计学差异（P值>0.05），准确度降序为48C(94.6%)>rs586178(92.7%)>rs4649082、rs2375313、rs2281179、rs2072933、rs2072932和RFLP(92.4%)>rs2072931(91.8%)。
结论：所检查的方法均不能独立且准确地预测RhC。109bp插入试验显示了在中国汉族人群中预测RhC的最高准确性。结合其他方法利用109bp插入测试可以提高RhC预测的准确性。
