WO2022078099A1 - Mrkh综合征诊断标记物及其在制备mrkh综合征诊断试剂盒中的应用 - Google Patents

Mrkh综合征诊断标记物及其在制备mrkh综合征诊断试剂盒中的应用 Download PDF

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WO2022078099A1
WO2022078099A1 PCT/CN2021/116122 CN2021116122W WO2022078099A1 WO 2022078099 A1 WO2022078099 A1 WO 2022078099A1 CN 2021116122 W CN2021116122 W CN 2021116122W WO 2022078099 A1 WO2022078099 A1 WO 2022078099A1
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mrkh
mrkh syndrome
syndrome diagnostic
mutation site
diagnostic marker
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朱兰
马聪聪
陈娜
吴南
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中国医学科学院北京协和医院
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  • the invention belongs to the technical field of medical detection, and particularly relates to a MRKH syndrome diagnostic marker and its application in the preparation of a MRKH syndrome diagnostic kit.
  • the abnormal development of the Müllerian duct during the embryonic period of females leads to congenital absence of vagina with no uterus or only primordial uterus, namely MRKH syndrome (Mayer-Rokitansky-Küster-Hauser syndrome), with an incidence rate of 1/5000-1/4000.
  • the main clinical features include: no uterus or solid primordial uterine nodules, a small number of patients have functional endometrium but uterine dysplasia; complete absence of vagina, or absence of upper 2/3 and lower 1/3 of acupoints The top is a blind end; the fallopian tubes and ovaries are normally developed, the secondary sexual characteristics are normal female, and the chromosomal karyotype is female karyotype 46, XX.
  • MRKH syndrome is mainly divided into two types, among which type I (simple type) is characterized by simple abnormal development of the uterus and vagina, while the development of the urinary system and skeletal system is normal, which is the most common type; type II (complex type) is characterized by the removal of the uterus , In addition to vaginal dysplasia, it is also accompanied by urinary, skeletal or other system development abnormalities.
  • chromosomal aberrations may be the pathogenic factors of MRKH syndrome.
  • the researchers screened related genes by detecting chromosomal aberration sites and identified the pathogenicity of related genes in MRKH syndrome.
  • Previous literature reported that segmental deletion of chromosome 16p11.2 region was found in about 2% of patients with MRKH syndrome, which was one of the most common chromosomal aberrations in MRKH syndrome, and the TBX6 gene located in this region was considered to be more likely. Large suspected causative genes. Sandakan et al.
  • splice site mutation (622-2A>T) of the TBX6 gene in patients with MRKH syndrome, which is located at the splice acceptor site of exon 5, so this mutation is likely to reduce the splicing of exon 5.
  • Correct splicing; Tewes et al. found 5 missense mutations in the TBX6 gene among 167 MRKH patients; Tbx6 knockout female mice also showed genitourinary malformations. Therefore, researchers speculate that TBX6 gene mutation may be one of the causes of MRKH syndrome, and early detection of gene mutation sites can achieve early and early diagnosis.
  • the purpose of the present invention is to provide a diagnostic marker for MRKH syndrome and its application in the preparation of a diagnostic kit for MRKH syndrome.
  • a MRKH syndrome diagnostic marker is a TBX6 gene fragment with a mutation site c.423G>A.
  • the present invention provides the application of the above-mentioned MRKH syndrome diagnostic markers in the preparation of MRKH syndrome diagnostic kits.
  • MRKH can be assisted. Rapid diagnosis of the syndrome.
  • the present invention does not require the length of the TBX6 gene fragment for which the primers are designed (ie, the length of the MRKH syndrome diagnostic marker), as long as the TBX6 gene fragment carries the mutation site c.423G>A.
  • a TBX6 gene fragment with 200 bp bases before and after the mutation site c.423G>A can be selected as a template to design specific amplification primers.
  • the present invention also provides a MRKH syndrome diagnostic kit, which at least comprises specific amplification primers for amplifying the above MRKH syndrome diagnostic markers.
  • the specific amplification primers are:
  • Upstream primer 5'-AACCCTATCCTGGAGTCCCA-3';
  • Downstream primer 5'-TTTCTCTGACAGCTCTGGGG-3'.
  • the present invention also provides the application of the above-mentioned MRKH syndrome diagnostic marker in preparing a fetal MRKH syndrome prenatal screening kit.
  • prenatal screening of MRKH syndrome during pregnancy it is possible to know whether the fetus has MRKH syndrome as early as possible, and reduce the incidence of the disease from the source.
  • the present invention also provides a fetal MRKH syndrome prenatal screening kit, which at least comprises the specificity for amplifying the above-mentioned MRKH syndrome diagnostic markers Amplification primers.
  • the present invention also provides the application of the above-mentioned MRKH syndrome diagnostic marker in the preparation of a male pre-pregnancy genetic disease screening kit.
  • the present invention also provides a male pre-pregnancy genetic disease screening kit, which at least comprises specific amplification primers for amplifying the above-mentioned MRKH syndrome diagnostic markers.
  • the present invention provides the application of the above-mentioned MRKH syndrome diagnostic markers in the preparation of MRKH syndrome diagnostic kits, by collecting peripheral blood of a subject, obtaining genomic DNA, and detecting whether the mutation exists in the TBX6 gene by Sanger sequencing
  • the present invention also provides a fetal MRKH syndrome prenatal screening kit, which can be used to perform prenatal screening of MRKH syndrome during pregnancy, so as to understand the fetus as soon as possible. Whether there is the possibility of suffering from MRKH syndrome, reduce the birth rate of babies with birth defects, and reduce the incidence of the disease from the source.
  • the test kit can be used to screen whether the male TBX6 gene carries the mutation site c.423G>A before pregnancy or marriage, and it can also prevent the birth of children with MRKH syndrome as soon as possible.
  • Fig. 1 is a graph showing the results of Sanger sequencing on peripheral blood DNA of patients with MRKH;
  • the point of the arrow is the mutation site c.423G>A;
  • Fig. 2 is a graph showing the results of agarose gel electrophoresis analysis of RNA extraction from peripheral blood of tested MRKH patients and their parents and reverse transcription into cDNA for PCR amplification;
  • Figure 3 is a graph showing the results of sequencing analysis of the gel electrophoresis products of the tested MRKH patient and the father carrying the TBX6 gene mutation (c.423G>A);
  • WT indicates the normal TBX6 gene without mutation
  • c.423G>A indicates the TBX6 gene with the mutation site c.423G>A
  • Exon indicates the exon.
  • the patentee recruited a total of 442 MRKH patients from Peking Union Medical College Hospital (PUMCH) and Shenzhen Luohu Hospital, who were diagnosed with MRKH by gynecological ultrasound, pelvic MRI, karyotype analysis and collected medical records. Each subject signed an informed consent form, and the ethics committees of Peking Union Medical College Hospital and Shenzhen Luohu Hospital approved the study.
  • Peripheral blood DNA was extracted from each subject separately, and exome sequencing was performed on all subjects.
  • Illumina paired-end libraries were prepared from DNA samples and exome-captured, then sequenced on the Illumina HiSeq 4000 platform; variants were called and filtered using an in-house developed analysis strategy (Peking Union Medical College Pipeline, PUMP).
  • the patentee first confirmed that the mutation site was a true mutation through Sanger sequencing.
  • the sequencing results are shown in Figure 1, and the point indicated by the arrow is the mutation site; the primers used for Sanger sequencing are shown in Table 1.
  • upstream primer 5 -AACCCTATCCTGGAGTCCCA-3’ downstream primer 5'-TTTCTCTGACAGCTCTGGGG-3'
  • the verification method includes: collecting the peripheral blood of patients and their parents, extracting RNA using a conventional kit, and reverse transcribing it into cDNA, and using pre-designed primers to amplify the cDNA by nested PCR. Nested PCR amplification primers are shown in Table 2.
  • the amplified product of the second round of PCR reaction was used for agarose gel electrophoresis to analyze PCR product fragments, and at the same time for sequencing analysis.
  • the patentee has launched a MRKH syndrome diagnostic kit, a fetal MRKH syndrome prenatal screening kit and a male pre-pregnancy genetic disease screening kit, all of which are in the kits.
  • a MRKH syndrome diagnostic kit a fetal MRKH syndrome prenatal screening kit and a male pre-pregnancy genetic disease screening kit, all of which are in the kits.
  • the MRKH syndrome diagnostic kit can be used for MRKH screening in female patients with suspected MRKH syndrome, as an auxiliary means for the diagnosis of MRKH syndrome.
  • the fetal MRKH syndrome prenatal screening kit is used for prenatal screening of MRKH syndrome during pregnancy, which can identify whether the fetus has MRKH syndrome as soon as possible, and reduce the incidence of MRKH syndrome from the source.
  • the male pre-pregnancy genetic disease screening kit is used to screen men for genetic diseases before pregnancy, and can also prevent the birth of children with MRKH syndrome as early as possible.

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Abstract

一种MRKH综合征诊断标记物及其在制备MRKH综合征诊断试剂盒中的应用。该MRKH综合征诊断标记物为带有突变位点c.423G>A的TBX6基因片段。专利权人通过对442名MRKH受试患者进行外显子组测序，首次在TBX6基因上发现了该突变位点c.423G>A；经验证，该突变位点c.423G>A会导致TBX6基因转录过程中mRNA上四号外显子起始端71bp碱基缺失，引起剪接异常，使基因表达产物因丧失功能而引发MRKH。因此通过采集受检者外周血，获取DNA，通过Sanger测序检测TBX6基因上是否存在该突变位点c.423G>A，即可辅助MRKH综合征的快速诊断。

Description

MRKH综合征诊断标记物及其在制备MRKH综合征诊断试剂盒中的应用 技术领域
本发明属于医学检测技术领域，特别涉及一种MRKH综合征诊断标记物及其在制备MRKH综合征诊断试剂盒中的应用。
背景技术
女性胚胎期苗勒管发育异常，导致先天性无阴道合并无子宫或仅有始基子宫，即MRKH综合征(Mayer-Rokitansky-Küster-Hauser syndrome)，发病率为1/5000-1/4000。主要临床特征包括：无子宫或实性始基子宫结节，极少部分患者虽有功能性子宫内膜但子宫发育不良；阴道完全缺失，或阴道上2/3缺失、下1/3呈穴状，其顶端为盲端；输卵管、卵巢发育正常，第二性征为正常女性，染色体核型为女性核型46，XX。MRKH综合征主要分为两种类型，其中Ⅰ型(单纯型)表现为单纯子宫、阴道发育异常，而泌尿系统、骨骼系统发育正常，此型最为常见；Ⅱ型(复杂型)表现为除子宫、阴道发育异常外，还伴有泌尿、骨骼系统或其他系统发育异常。
MRKH综合征的患者女性第二性征发育正常，多因青春期原发性闭经就诊发现，少数患者以婚后性生活困难或周期性下腹痛为主诉症状。因患者青春期后无月经来潮、无法正常性生活、不具生育能力，仅可成年后通过人工阴道成型术解决性生活的困难，对患者身心影响巨大。
应用微阵列、比较基因组杂交(array-CGH)、DNA测序等技术已检测出某些染色体畸变(chromosomal aberration)、基因突变等可能是MRKH综合征的致病因素。研究者通过检测染色体畸变位点来筛选相关的基因，并判别相关基因 在MRKH综合征中的致病性。既往文献报道，染色体16p11.2区域的片段缺失在约2％的MRKH综合征患者中发现，是MRKH综合征最常见的染色体畸变之一，而定位于该区域的TBX6基因被认为是可能性较大的可疑致病基因。Sandakan等在MRKH综合征患者中发现TBX6基因的剪切位点突变(622-2A>T)，该突变位于五号外显子的剪接受体位点，因此该突变很可能降低了五号外显子的正确剪接；Tewes等人在167名MRKH患者中发现5例携带TBX6基因的错义突变；Tbx6基因敲除的雌性小鼠中也表现出泌尿生殖系统畸形。因此研究人员猜想TBX6基因突变可能是MRKH综合征的致病原因之一，在早期对基因突变位点进行检测能够实现尽早、尽快确诊。
发明内容
本发明的发明目的是提供一种MRKH综合征诊断标记物及其在制备MRKH综合征诊断试剂盒中的应用。
为实现上述发明目的，本发明的技术方案如下：
一种MRKH综合征诊断标记物，该MRKH综合征诊断标记物为带有突变位点c.423G>A的TBX6基因片段。
专利权人通过对442名MRKH受试患者进行外显子组测序，首次在TBX6基因上发现了突变位点c.423G>A(p.Leu141＝)，该突变位点经Sanger测序确认为真性突变；经验证，该突变位点c.423G>A会导致TBX6基因在转录过程中mRNA上四号外显子起始端71bp碱基缺失，引起剪接异常，使TBX6基因的表达产物因丧失功能而导致MRKH综合征。
基于此，本发明提供了上述的MRKH综合征诊断标记物在制备MRKH综合征诊断试剂盒中的应用。通过采集受检者外周血，提取基因组DNA，针对携 带该突变位点c.423G>A的TBX6基因片段设计引物，检测TBX6基因上是否存在该突变位点c.423G>A，即可辅助MRKH综合征的快速诊断。
本发明对设计引物所针对的TBX6基因片段长度(即MRKH综合征诊断标记物的长度)没有要求，只要该TBX6基因片段携带该突变位点c.423G>A即可。作为优选，可以选择该突变位点c.423G>A前后200bp碱基的TBX6基因片段作为模板设计特异性扩增引物。
基于此，本发明还提供了一种MRKH综合征诊断试剂盒，该MRKH综合征诊断试剂盒中至少包含用于扩增上述MRKH综合征诊断标记物的特异性扩增引物。
在上述的MRKH综合征诊断试剂盒中，所述的特异性扩增引物为：
上游引物：5’-AACCCTATCCTGGAGTCCCA-3’；
下游引物：5’-TTTCTCTGACAGCTCTGGGG-3’。
为了实现MRKH综合征的早期诊断，本发明还提供了上述的MRKH综合征诊断标记物在制备胎儿MRKH综合征产前筛查试剂盒中的应用。通过在妊娠期进行MRKH综合征产前筛查，能够尽早了解胎儿是否患有MRKH综合征，从源头上降低该病的发生率。
基于此，本发明还提供了一种胎儿MRKH综合征产前筛查试剂盒，该胎儿MRKH综合征产前筛查试剂盒中至少包含用于扩增上述的MRKH综合征诊断标记物的特异性扩增引物。
专利权人还发现，TBX6基因上的突变位点c.423G>A(p.Leu141＝)遗传自父亲，该基因突变在父亲中未发现致病性，但在其女性后代中却是潜在的致病因素，表明该突变位点c.423G>A的遗传可能是限性遗传。
基于此，本发明还提供了上述的MRKH综合征诊断标记物在制备男性孕前 遗传病筛查试剂盒中的应用。通过在孕前或婚前对男性TBX6基因上是否携带的突变位点c.423G>A进行筛查，可以及早地避免MRKH综合征患儿的降生。
本发明还提供了一种男性孕前遗传病筛查试剂盒，该男性孕前遗传病筛查试剂盒中至少包含用于扩增上述的MRKH综合征诊断标记物的特异性扩增引物。
与现有技术相比，本发明的有益效果体现在：
(1)专利权人通过对442名MRKH受试患者进行外显子组测序，首次在TBX6基因上发现了该突变位点c.423G>A(p.Leu141＝)；经验证，该突变位点c.423G>A(p.Leu141＝)会导致TBX6基因转录过程中mRNA上四号外显子起始端71bp碱基缺失，引起剪接异常，使TBX6基因的表达产物因丧失功能而导致MRKH综合征。基于此，本发明提供了上述的MRKH综合征诊断标记物在制备MRKH综合征诊断试剂盒中的应用，通过采集受检者外周血，获取基因组DNA，通过Sanger测序检测TBX6基因上是否存在该突变位点c.423G>A(p.Leu141＝)，即可辅助MRKH综合征的快速诊断。
(2)为了实现MRKH综合征的早期诊断，本发明还提供了一种胎儿MRKH综合征产前筛查试剂盒，利用该试剂盒在妊娠期进行MRKH综合征产前筛查，能够尽早了解胎儿是否有患MRKH综合征的可能，降低出生缺陷婴儿的降生率，从源头上降低该病的发生率。
(3)本发明还发现，TBX6基因上的突变位点c.423G>A(p.Leu141＝)遗传自父亲，且致病性较强，因此本发明还提供了一种男性孕前遗传病筛查试剂盒，利用该试剂盒在孕前或婚前对男性TBX6基因上是否携带突变位点c.423G>A进行筛查，也可以及早地避免MRKH综合征患儿的降生。
附图说明
图1为对受试MRKH患者外周血DNA进行Sanger测序结果图；
其中，箭头所指处即为突变位点c.423G>A；
图2为对受试MRKH患者及其双亲外周血提取RNA并反转录为cDNA进行PCR扩增的琼脂糖凝胶电泳分析结果图；
图3为受试MRKH患者及携带TBX6基因突变(c.423G>A)的父亲凝胶电泳产物的测序分析结果图；
其中，“WT”表示未发生突变的正常TBX6基因，“c.423G>A”表示存在突变位点c.423G>A的TBX6基因，“Exon”表示外显子。
具体实施方式
下面结合附图和具体实施方式对本发明的技术方案做进一步详细说明。
实施例
本专利权人共招募了来自北京协和医院(PUMCH)和深圳罗湖医院的442名MRKH患者，通过妇科超声、盆腔MRI、染色体核型分析和收集的医疗记录确诊为MRKH。每个受试者均签署了知情同意书，北京协和医院和深圳罗湖医院伦理委员会批准了这项研究。
分别提取每个受试者的外周血DNA，并对所有受试者进行了外显子组测序。从DNA样品制备Illumina配对末端文库并进行外显子组捕获，然后在Illumina HiSeq 4000平台上测序；使用内部开发的分析策略(Peking Union Medical College Pipeline，PUMP)调用和过滤变体。
从外显子组数据中提取了所有TBX6基因(正常TBX6基因的核苷酸序列如 SEQ No.1所示)存在的意义未明的基因突变，通过SpliceAI预测软件鉴定出一个可能影响剪切的突变位点c.423G>A(p.Leu141＝)。该突变位点在ExAC、gnomAD数据库中均未报道，为专利权人首次发现的突变位点(突变后TBX6基因的核苷酸序列如SEQ No.2所示)。出现该突变位点的患者均为MRKH I型，即除了无子宫无阴道生殖系统畸形外，其他系统未发现异常。
专利权人首先通过Sanger测序确认该突变位点为真性突变，测序结果见图1，箭头所指处即为突变位点；Sanger测序所采用的引物如表1所示。
表1
上游引物 5’-AACCCTATCCTGGAGTCCCA-3’
下游引物 5’-TTTCTCTGACAGCTCTGGGG-3’
然后对上述基因突变是否能致MRKH进行验证。验证方法包括：分别采集患者及其父母外周血，采用常规试剂盒提取RNA，并反转录为cDNA，采用预先设计的引物对cDNA进行巢式PCR扩增。巢式PCR扩增引物如表2所示。
表2
Figure PCTCN2021116122-appb-000001
巢式PCR的反应体系及反应条件如表3所示。
表3
Figure PCTCN2021116122-appb-000002
取第二轮PCR反应的扩增产物作琼脂糖凝胶电泳分析PCR产物片段，同时作测序分析，电泳分析结果见图2，测序分析结果见图3。
由图2可见，受试患者与其父亲的PCR扩增产物中出现了两个条带，而在其母亲的PCR扩增产物正常，为一条条带，表明受试患者TBX6基因上发生的c.423G>A突变是遗传自其父亲，且该突变位点影响了剪接。
而由图3可见，与未发生基因突变的TBX6基因相比，受试患者及其父亲的TBX6基因上存在的突变位点c.423G>A导致TBX6基因在转录过程中mRNA上四号外显子起始部71bp碱基缺失，引起剪接异常，使TBX6基因的表达产物因丧失功能而导致MRKH综合征。
在上述实验结果的基础上，专利权人推出一种MRKH综合征诊断试剂盒、一种胎儿MRKH综合征产前筛查试剂盒和一种男性孕前遗传病筛查试剂盒，上述试剂盒中均包含有表1所示的特异扩增引物，用以Sanger测序。
其中，MRKH综合征诊断试剂盒可用于对疑似MRKH综合征女性患者进行MRKH筛查，作为MRKH综合征确诊的辅助手段。
而胎儿MRKH综合征产前筛查试剂盒则用于在妊娠期进行MRKH综合征产前筛查，能够尽早了解胎儿是否患有MRKH综合征，从源头上降低MRKH综合征的发生率。
男性孕前遗传病筛查试剂盒则用于在孕前对男性进行遗传病筛查，也可以及早地避免MRKH综合征患儿的降生。

Claims (10)

  1. MRKH综合征诊断标记物，其特征在于，为带有突变位点c.423G>A的TBX6基因片段。
  2. 如权利要求1所述的MRKH综合征诊断标记物在制备MRKH综合征诊断试剂盒中的应用。
  3. 一种MRKH综合征诊断试剂盒，其特征在于，至少包含用于扩增如权利要求1所述的MRKH综合征诊断标记物的特异性扩增引物。
  4. 如权利要求3所述的MRKH综合征诊断试剂盒，其特征在于，所述的特异性扩增引物为：
    上游引物：5’-AACCCTATCCTGGAGTCCCA-3’；
    下游引物：5’-TTTCTCTGACAGCTCTGGGG-3’。
  5. 如权利要求1所述的MRKH综合征诊断标记物在制备胎儿MRKH综合征产前筛查试剂盒中的应用。
  6. 一种胎儿MRKH综合征产前筛查试剂盒，其特征在于，至少包含用于扩增如权利要求1所述的MRKH综合征诊断标记物的特异性扩增引物。
  7. 如权利要求6所述的胎儿MRKH综合征产前筛查试剂盒，其特征在于，所述的特异性扩增引物为：
    上游引物：5’-AACCCTATCCTGGAGTCCCA-3’；
    下游引物：5’-TTTCTCTGACAGCTCTGGGG-3’。
  8. 如权利要求1所述的MRKH综合征诊断标记物在制备男性孕前遗传病筛查试剂盒中的应用。
  9. 一种男性孕前遗传病筛查试剂盒，其特征在于，至少包含用于扩增如权利要求1所述的MRKH综合征诊断标记物的特异性扩增引物。
  10. 如权利要求9所述的男性孕前遗传病筛查试剂盒，其特征在于，所述 的特异性扩增引物为：
    上游引物：5’-AACCCTATCCTGGAGTCCCA-3’；
    下游引物：5’-TTTCTCTGACAGCTCTGGGG-3’。
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