中国组织工程研究

中国组织工程研究 ›› 2020, Vol. 24 ›› Issue (1): 77-82.doi: 10.3969/j.issn.2095-4344.1859

• 造血干细胞 hematopoietic stem cells • 上一篇    下一篇

一例白血病患者及家系HLA-B基因全长序列及18个点突变分析

钟艳平，邹红岩，全湛柔，邓志辉，洪文旭   

  1. 深圳市血液中心输血医学研究所，广东省深圳市  518020
  • 收稿日期:2019-05-08 修回日期:2019-05-16 接受日期:2019-06-12 出版日期:2020-01-08 发布日期:2019-12-12
  • 通讯作者: 邹红岩，主任技师，深圳市血液中心输血医学研究所，广东省深圳市 518020
  • 作者简介:钟艳平，女，1992年生，广东省梅州市人，汉族，2015年南方医科大学毕业，初级检验技师，主要从事人白细胞抗原组织配型高分辨确认工作。
  • 基金资助:
    深圳市医疗卫生三名工程项目(SZSM201811092)；深圳市卫生计生系统科研项目(201506076)

Analysis of full-length sequence and 18 point mutations of HLA-B in a leukemia patient and her family

Zhong Yanping, Zou Hongyan, Quan Zhanrou, Deng Zhihui, Hong Wenxu   

  1. Shenzhen Institute of Transfusion Medicine, Shenzhen Blood Center, Shenzhen 518020, Guangdong Province, China
  • Received:2019-05-08 Revised:2019-05-16 Accepted:2019-06-12 Online:2020-01-08 Published:2019-12-12
  • Contact: Zou Hongyan, Chief technician, Shenzhen Institute of Transfusion Medicine, Shenzhen Blood Center, Shenzhen 518020, Guangdong Province, China
  • About author:Zhong Yanping, Technician, Shenzhen Institute of Transfusion Medicine, Shenzhen Blood Center, Shenzhen 518020, Guangdong Province, China
  • Supported by:
    Sanming Project of Medicine in Shenzhen, No. SZSM201811092; Research Project of Shenzhen Health and Family Planning System, No. 201506076

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摘要：

文题释义：

人类白细胞抗原B(human leucocyte antigen，HLA-B)：HLA-B属于HLA经典一类分子，位于人类6号染色体短臂，具有高度的基因多态性，其表达的产物分布在几乎所有有核细胞表面，参与抗原提呈和特异性免疫应答反应。

下一代测序技术：一种以“边合成边测序”为核心思想的高通量测序技术，能够在短时间内同时对上百亿碱基进行测序。在人类基因组、转录组、肿瘤等研究领域具有巨大的应用价值。

背景：人类白细胞抗原HLA经过长期进化形成丰富的多态性，近几年由于受检人数增加及HLA分型技术快速发展，HLA新基因不断被发现。

目的：采用下一代测序技术对1例白血病患者及家系HLA-B基因的全长序列及18个点突变进行分析。

方法：应用序列特异性寡核苷酸探针杂交(PCR-SSOP)及聚合酶链反应-直接测序法(PCR-SBT)发现患者的HLA-B结果异常。为了鉴定该基因，采用下一代测序技术对该基因全长进行测序，同时采集患者父亲、母亲及2位同胞姐妹的血样进行HLA基因的遗传学分析。

结果与结论：应用序列特异性寡核苷酸探针杂交及聚合酶链反应-直接测序法均提示该样本HLA-B无完全匹配的基因型。应用下一代测序技术分析发现，与同源性最高的等位基因B*15:09:01相比，该基因在外显子、内含子和3′UTR共存在18个碱基突变。5个外显子碱基突变位于第3，4外显子，分别为：第486位G→C、第583位T→C、第636位T→C、第652位A→G和第756位C→T，导致5个相应密码子发生改变，其中2个碱基替换为错义突变，第171位酪氨酸(Tyr)→组氨酸(His)、第194位异亮氨酸(Ile)→缬氨酸(Val)。家系调查显示患者HLA-B新基因来源于父亲。新基因序列已递交给Genbank数据库(MG595995)。应用下一代测序技术鉴定了1个HLA-B新等位基因，该基因于2017年12月被WHO HLA因子命名委员会正式命名为HLA-B*15:435。

ORCID: 0000-0002-2210-9889(钟艳平)

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 白血病, 人类白细胞抗原, 新等位基因, 下一代测序技术, 家系调查, 碱基突变, 序列特异性寡核苷酸探针杂交, 聚合酶链反应-直接测序法

Abstract:

BACKGROUND: The human leukocyte antigen (HLA) has undergone long-term evolution to form diverse polymorphisms. In recent years, due to the increase in the number of examinees and the rapid development of HLA typing technology, novel HLA alleles have been discovered constantly.

OBJECTIVE: To analyze the full-length sequence and 18 point mutations of HLA-B gene in a leukemia patient and her family using the next-generation sequencing technology.

METHODS: Polymerase chain reaction and sequence-specific oligonucleotide probes (PCR-SSOP) and polymerase chain reaction-sequence based typing (PCR-SBT) revealed abnormalities in the patient’s HLA-B. To identify the genotype, we sequenced the full length of the gene by next-generation sequencing technology and collected blood samples from the patient’s father, mother and two sisters for genetic analysis of HLA genes.

RESULTS AND CONCLUSION: Both PCR-SSOP and PCR-SBT indicated that the HLA-B sample had no perfectly matched genotype. Further detection using the next-generation sequencing technology revealed that the novel allele had 18 base mutations in the exon, intron and 3'UTR compared to the most homologous allele B*15:09:01. Five exon base mutations were located in the exons 3 and 4, which were: 486G→C, 583T→C, 636T→C, 652A→G, 756C→T, resulting in changes in the five corresponding codons, including 171 tyrosine (Tyr) → histidine (His) and 194 isoleucine (Ile) → valine (Val). A pedigree survey found that the patient’s novel HLA B allele was inherited from her father. The novel allele sequence was submitted to the Genbank database (MG595995). A novel HLA-B allele was confirmed by the next-generation sequencing, which was officially named HLA-B*15:435 by the World Health Organization HLA Factor Nomenclature Committee in December 2017.

Key words: leukemia, human leukocyte antigen, novel allele, next-generation sequencing, pedigree survey, base mutation, polymerase chain reaction and sequence-specific oligonucleotide probes, polymerase chain reaction-sequence based typing

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