2例罕见RhD变异型RHD*DEL37的分子生物学分析

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doi:10.19723/j.issn.1671-167X.2024.02.024

摘要：

Rh血型系统是输血医学中重要的常规检测血型系统, 因RhD血型不合引起的溶血性输血反应及新生儿溶血病一直以来倍受临床重视。本研究报道了2例罕见的RHD基因变异型RHD*DEL37个体的血清学和基因特征, 这2例个体的血液样本经血清学方法(试管法和微柱凝胶法)鉴定为RhD阴性。采用聚合酶链反应-序列特异性引物(polymerase chain reaction-sequence specific prime, PCR-SSP)对样本进行RHD基因分型和合子型分析, 基因分型结果为RHD基因阳性, 并排除了常见的几种RHD基因变异体的可能。RHD基因合子型检测阳性, 证明其中一条RHD等位基因1~10外显子全部缺失。进一步对样本RHD基因1~10外显子基因进行Sanger法测序, 测序结果显示在另一条等位基因第8内含子上的第1154-31号碱基发生了T>C突变, 国际输血协会(International Society of Blood Transfusion, ISBT)将该突变等位基因命名为RHD*DEL37, 表型为RhD放散型(D-elute, D^el)。本研究中, 2例血清学初筛RhD阴性的样本通过分子生物学检测, 其基因型为RHD*DEL37/RHD-(RHD*01N.01)。由于这2例个体无血缘关系, 提示我国可能存在携带该基因突变的人群。本研究提示分子生物学方法可辅助鉴别血清学初筛为阴性的RhD变异体样本, 联用分子生物学方法和血清学方法在准确鉴定血型、保障患者输血安全方面有重要意义。

关键词: Rh-Hr血型系统, 血清学, 基因分型技术, 等位基因

Abstract:

The Rh blood grouping system is a critical standardized test in transfusion medicine, especially for the cases related to haemolytic transfusion reactions and neonatal haemolytic disease caused by clinical RhD blood group incompatibility. In the present case report, we presented two cases with the uncommon RHD gene variation RHD*DEL37. The blood samples of the two subjects were mistakenly identified as RhD-negative through conventional serological testing. Firstly, both blood samples were tested negative for the RhD antigen using traditional tube test and gel microcolumn methods. The phenotyping of RhCE were identified as ccEe and ccee for each sample, respectively. Secondly, genetic analysis was performed using polymerase chain reaction-sequence specific prime (PCR-SSP) which revealed that neither sample belonging to the several common RHD gene variants which was found in Asia. Moreover, they turned out to be positive for the RHD haplotype, which indicated that exons 1-10 on one of the RHD alleles were entirely absent. In addition, a T>C mutation was observed at bases 1154-31 in intron 8 of the other allele, which was located at the intron 8 breakpoint. This result was obtained after further Sanger sequencing of exons 1-10 of the RHD gene. The mutant allele was designated as RHD*DEL37 by the International Society of Blood Transfusion (ISBT) and was identified as D-elute(D^el) by phenotype ana-lysis. Both samples were genotyped as RHD*DEL37 and showed positive results. In summary, the true genotype of the two blood samples, of which the screening results only using serological testing method was negative, were RHD*DEL37 /RHD-(RHD*01N.01). Notably, this kind of genotype was reported for the first time in Chinese population. Moreover, the two individuals did not have ties of consanguinity, indicating that some of the Chinese individuals could be carriers of the genetic mutation. Therefore, it might be necessary to further confirm the frequency of this mutation in the Chinese population and the possibility of homozygosity for this mutation. This report identifies infrequent RHD gene mutation samples by coupling molecular biology and serological methods to prevent misclassification of blood groups. Combining serological and molecular biology test results to determine blood group is critical in protecting patients during clinical transfusion procedures.

Key words: Rh-Hr blood-group system, Serology, Genotyping techniques, Alleles

中图分类号:

R446.6

王鹏,杨子瑶,王萌,王巍,李爱芝. 2例罕见RhD变异型RHD*DEL37的分子生物学分析[J]. 北京大学学报（医学版）, 2024, 56(2): 352-356.

Peng WANG,Ziyao YANG,Meng WANG,Wei WANG,Aizhi LI. Molecular biology analysis of 2 rare RhD variant individuals with RHD*DEL37[J]. Journal of Peking University (Health Sciences), 2024, 56(2): 352-356.

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1	Flegel WA , Chen Q , Castilho L , et al. Molecular immunohaematology round table discussions at the AABB Annual Meeting, Orlando 2016[J]. Blood Transfus, 2018, 16 (5): 447- 456.
2	杨光远, 朱健, 郑朝晖, 等. 浙江台州地区血清型RhD初筛阴性的血型基因分析[J]. 中国卫生检验杂志, 2022, 32 (5): 555- 558.
3	邹彬彬, 谢毓滨, 阳智芬, 等. 湖南省RhD阴性献血人群11种红细胞血型系统基因频率及多态性研究[J]. 中国输血杂志, 2022, 35 (2): 144- 148.
4	Zhang J , Zeng Y , Wang Y , et al. RHD genotypes in a Chinese cohort of pregnant women[J]. Front Genet, 2021, 12, 752485. doi: 10.3389/fgene.2021.752485
5	Ding M , Li Q , Zhang H , et al. A novel RHD (c.509T>G, p.Met170Arg) allele shows weakened D expression[J]. Transfusion, 2022, 62 (3): E17- E18.
6	Wagner FF , Mardt I , Bittner R , et al. RhD PCR of blood donors in northern Germany: Use of adsorption/elution to determine D antigen status[J]. Vox Sanguinis, 2012, 103 (Suppl 1): 15.
7	Fukumori Y , Hori Y , Ohnoki S , et al. Further analysis of D^el (D-elute) using polymerase chain reaction (PCR) with RHD gene-specific primers[J]. Transfus Med, 1997, 7 (3): 227- 231.
8	王敏, 王保龙, 蒋光明, 等. D^el红细胞膜表面D抗原表位及抗原强度分析[J]. 中国输血杂志, 2011, 24 (1): 23- 26.
9	邵超鹏, 徐华, 徐群, 等. 汉族DEL型孕妇可免去抗-D产前检查[J]. 中国输血杂志, 2010, 23 (9): 671- 674.

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Case	RhD blood type		Antibody screening test^*			Self control^*	ABO blood type	RhCE phenotype
Case	-D screen^*	-D screen^**	Ⅰ	Ⅱ	Ⅲ	Self control^*	ABO blood type	RhCE phenotype
Case 1	-	-	-	-	-	-	O	ccEe
Case 2	-	-	-	-	-	-	A	ccee

Detection of mutation sites	Tm value/℃	Positive standard^*/℃	Negative standard^*/℃	Detection result
RhD exon 1	85.73	86.0	76.5	+
RhD exon 5	82.04	82.5	76.5	+
RhD exon 6	84.33	84.5	76.5	+
RhD exon 7	83.63	84.5	76.5	+
RhD exon 10	79.34	79.5	76.5	+
RhD 845A	71.57	84.0	71.0-73.0	-
RhD 1227A	72.26	82.0	71.0-73.0	-
RhD zygoslty	83.13	84.0-86.0	76.5	+

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