葡萄胎的分子遗传学分析及基因NLRP7和KHDL3C的致病性研究

发布时间：2018-04-04 03:45

本文选题：复发性葡萄胎　切入点：双亲来源完全性葡萄胎　出处：《北京协和医学院》2014年博士论文

【摘要】：背景葡萄胎是一种良性滋养细胞疾病,其特征为绒毛水肿变性和滋养细胞增生。根据其组织病理学表现,葡萄胎包括完全性葡萄胎(complete hydatidiform mole, CHM)和部分性葡萄胎(partial hydatidiform mole, PHM)两类。CHM常为孤雄二倍体(androgenetic CHM, AnCHM),但近来发现了双亲来源的完全性葡萄胎(biparental CHM, BiCHM),这种特殊类型多见于家族性复发性葡萄胎(familial recurrent hydatidiform mole, FRHM),偶可见于散发的复发性葡萄胎(recurrent hydatidiform mole, RHM)。FRHM是一种罕见的疾病,可能为常染色体隐性遗传病,患者多有印记基因异常,并有NLRP7和KHDC3L不同位点的纯合突变。目的本研究拟通过短串联重复序列(short tandem repeat, STR)多态性检测对FRHM、 RHM和散发性葡萄胎及其双亲进行遗传学分析,确定葡萄胎的遗传学起源,研究FRHM是否与BiCHM存在相关性。并对BiCHM患者的NLRP7和KHDC3L基因编码序列进行测序,以AnCHM患者为对照,寻找上述基因编码序列的变异位点,并结合数据库检索的一般人群资料,探讨所发现位点的致病性。材料收集1个FRHM家系、5个RHM家系和6个散发性葡萄胎病例的临床资料,并收集葡萄胎组织新鲜清宫标本或蜡块标本及其切片,采集患者及其丈夫和母亲、姐妹的外周血。分别提取葡萄胎组织和外周血DNA。方法一、复核葡萄胎病理标本HE染色和p57KIP2免疫组化染色切片,再次明确葡萄胎诊断,并区分CHM和PHM。二、选取人不同染色体上用于多态性检测的STR位点,使用带有荧光标记的标准引物分别对上述DNA进行PCR扩增。使用聚丙烯氨酰胺凝胶电泳或DNA测序仪对扩增产物进行长度片段检测,并按葡萄胎及其双亲为单位进行分组分析,从而确定葡萄胎的遗传学起源。三、根据基因NLRP7和KHDC3L的编码区参考序列,分别设计引物扩增葡萄胎患者及其母亲或姐妹这两个基因的所有外显子,并使用DNA测序仪对扩增产物进行测序。将测序结果与参考序列进行比较,找出可改变表达产物氨基酸序列的突变或非同义异形体(non-synonymous variant, NSV)。在dbSNP和emsembl数据库中检索找到的位点,判断是否为单核苷酸多态性(single nucleotide polymorphism,SNP)位点。使用Poly-Phen2评估找到的位点损害蛋白功能的可能性。结果共收集15例葡萄胎妊娠的病理标本,其中1例为FRHM,8例为RHM,6例为散发性葡萄胎。1例(1／1,100%)FRHM和1例(1／8,12.5%)无正常妊娠患者的RHM为BiCHM;5例(5／8,62.5%)RHM和6例(6／6,100%)散发性葡萄胎为AnCHM;2例(2／8,25%%)RHM符合PHM。11例AnCHM中,8例(8／11,72.7%)为纯合子,3例(3／11,27.3%)为杂合子。在15例复发性及散发性葡萄胎患者中共筛查到NLRP7基因编码区内3个NSV：c.955GA、c.1280TC和c.1441GA;以及KHDC3L基因编码区内1个NSV：c.602CG。其中NLRP7c.1441GA只出现于BiCHM患者；NLRP7c.955GA和c.1280TC杂合状态在HM患者和健康女性中都存在；KHDC3L c.602CG纯合状态在HM患者和健康女性中都存在。结论 FRHM为BiCHM,无正常妊娠患者的RHM可能为BiCHM;有正常妊娠患者的RHM和散发性HM可为AnCHM或PHM。BiCHM与AnCHM的临床和组织病理学表现难以区分,需要分子遗传学分析以鉴别。 NLRP7的NSV c.1441GA可能对BiCHM的发生有一定的促进作用。但本研究中葡萄胎患者的NLRP7和KHDC3L基因编码序列没有明确的致病突变或NSV,说明BiCHM存在遗传异质性,除NLRP7和KHDC3L外,还可能存在其他致病基因。本研究的分子遗传学方法和基因测序方法可用于临床遇到反复发生葡萄胎(≥2次)的患者时,判断葡萄胎的遗传学起源及患者有无NLRP7和KHDC3L基因突变,从而指导临床处理。
[Abstract]:background
Hydatidiform mole is a benign trophoblastic disease, characterized by swelling of chorionic villi and trophoblastic hyperplasia. According to the findings of the pathology, including complete hydatidiform mole (complete hydatidiform, mole, CHM) and partial hydatidiform mole (partial hydatidiform, mole, PHM) two.CHM (androgenetic for androgenetic diploid CHM, AnCHM), but the recent discovery of complete hydatidiform mole (biparental CHM, the parental origin of BiCHM), this special type found in familial recurrent hydatidiform mole (familial recurrent hydatidiform mole, FRHM), occasionally seen in sporadic recurrent hydatidiform mole (recurrent hydatidiform mole, RHM.FRHM) is a rare the disease may be an autosomal recessive disease, many patients have imprinted gene abnormalities, and mutation of NLRP7 and KHDC3L was homozygous.
objective
This study by short tandem repeat (short tandem, repeat, STR) to detect the polymorphism of FRHM, RHM and sporadic hydatidiform mole and its parents for genetic analysis, to determine the genetic origin of hydatidiform mole, whether the presence of FRHM associated with BiCHM. And NLRP7 and KHDC3L gene encoding sequences of BiCHM were sequenced by AnCHM patients were mutation sites for the gene encoding sequence, and combining with the database retrieval data of the general population, found pathogenic sites.
Material Science
The clinical data of 1 FRHM families, 5 RHM families and 6 cases of sporadic hydatidiform mole were collected, and the fresh curettage specimens or wax samples and their slices were collected. The peripheral blood of the patients and their husbands and their mothers and sisters were collected. The DNA. of hydatidiform mole and peripheral blood was extracted respectively.
Method
First, the HE and p57KIP2 immunohistochemical staining sections of the histopathological specimens of hydatidiform mole were reviewed, and the diagnosis of hydatidiform mole was redefined, and CHM and PHM. were distinguished.
Two, selected for STR polymorphism detection of different chromosomes, the DNA was amplified by PCR with primers respectively using standard fluorescence labeling. The PCR products were detected using polypropylene amide fragment gel electrophoresis or DNA sequencing, and according to the mole and their parents as the unit was analyzed, so as to determine the genetics the origin of hydatidiform mole.
Three, according to the reference sequence of NLRP7 gene encoding region and KHDC3L respectively, all primers were designed to amplify hydatidiform mole patients and their mother or sister of the two gene exon, and sequenced the PCR products using DNA sequencing. The sequencing results were compared with the reference sequence, find out the change of amino acid sequence mutation or expression product nonsynonymous isoform (non-synonymous variant, NSV). Retrieved found sites in dbSNP and emsembl database, to determine whether the single nucleotide polymorphism (single nucleotide polymorphism, SNP) sites. Sites use Poly-Phen2 to assess the possibility of damage to find the protein function.
Result
Collected pathology specimens of 15 cases of hydatidiform mole, 1 cases of FRHM, 8 cases RHM, 6 cases of sporadic.1 cases of hydatidiform mole (1 / 1100%) and 1 cases of FRHM (1 / 8,12.5%) in patients without normal pregnancy RHM BiCHM; 5 cases (5 / 8,62.5%) and 6 RHM (6 / 6100%) cases of sporadic hydatidiform mole of AnCHM; 2 cases (2 / 8,25%%) with RHM PHM.11 AnCHM cases, 8 cases (8 / 11,72.7%) were homozygous and 3 cases (3 / 11,27.3%) was heterozygous.
In 15 cases of recurrent and sporadic patients with hydatidiform mole to the screening of NLRP7 gene encoding region of 3 NSV:c.955GA, c.1280TC and c.1441GA; and 1 NSV:c.602CG. encoding region of KHDC3L gene in the NLRP7c.1441GA only in BiCHM patients; NLRP7c.955GA and c.1280TC heterozygous state exists in HM patients and healthy women; KHDC3L homozygous c.602CG the state exists in HM patients and healthy women.
conclusion
FRHM is BiCHM. RHM may be BiCHM in patients without normal pregnancy. RHM and sporadic HM in normal pregnant women can be difficult to distinguish AnCHM and PHM.BiCHM from AnCHM's clinical and histopathological features. Molecular genetic analysis is needed to identify them.
NLRP7 NSV c.1441GA may play a role in the pathogenesis of BiCHM. But the NLRP7 and KHDC3L gene encoding sequence in hydatidiform mole, in this study no clear pathogenic mutations or NSV, BiCHM shows the existence of genetic heterogeneity, except NLRP7 and KHDC3L, there may be other genes.
Molecular genetics and gene sequencing methods in this study can be used in the clinical encounter recurrent hydatidiform mole (more than 2) of the patients, and determine the genetic origin of hydatidiform mole patients without NLRP7 and KHDC3L gene mutation, so as to guide the clinical treatment.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.33

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