本文選題：復(fù)發(fā)性葡萄胎　切入點(diǎn)：雙親來源完全性葡萄胎　出處：《北京協(xié)和醫(yī)學(xué)院》2014年博士論文

【摘要】：背景 葡萄胎是一種良性滋養(yǎng)細(xì)胞疾病,其特征為絨毛水腫變性和滋養(yǎng)細(xì)胞增生。根據(jù)其組織病理學(xué)表現(xiàn),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)兩類。CHM常為孤雄二倍體(androgenetic CHM, AnCHM),但近來發(fā)現(xiàn)了雙親來源的完全性葡萄胎(biparental CHM, BiCHM),這種特殊類型多見于家族性復(fù)發(fā)性葡萄胎(familial recurrent hydatidiform mole, FRHM),偶可見于散發(fā)的復(fù)發(fā)性葡萄胎(recurrent hydatidiform mole, RHM)。FRHM是一種罕見的疾病,可能為常染色體隱性遺傳病,患者多有印記基因異常,并有NLRP7和KHDC3L不同位點(diǎn)的純合突變。 目的 本研究擬通過短串聯(lián)重復(fù)序列(short tandem repeat, STR)多態(tài)性檢測(cè)對(duì)FRHM、 RHM和散發(fā)性葡萄胎及其雙親進(jìn)行遺傳學(xué)分析,確定葡萄胎的遺傳學(xué)起源,研究FRHM是否與BiCHM存在相關(guān)性。并對(duì)BiCHM患者的NLRP7和KHDC3L基因編碼序列進(jìn)行測(cè)序,以AnCHM患者為對(duì)照,尋找上述基因編碼序列的變異位點(diǎn),并結(jié)合數(shù)據(jù)庫(kù)檢索的一般人群資料,探討所發(fā)現(xiàn)位點(diǎn)的致病性。 材料 收集1個(gè)FRHM家系、5個(gè)RHM家系和6個(gè)散發(fā)性葡萄胎病例的臨床資料,并收集葡萄胎組織新鮮清宮標(biāo)本或蠟塊標(biāo)本及其切片,采集患者及其丈夫和母親、姐妹的外周血。分別提取葡萄胎組織和外周血DNA。 方法 一、復(fù)核葡萄胎病理標(biāo)本HE染色和p57KIP2免疫組化染色切片,再次明確葡萄胎診斷,并區(qū)分CHM和PHM。 二、選取人不同染色體上用于多態(tài)性檢測(cè)的STR位點(diǎn),使用帶有熒光標(biāo)記的標(biāo)準(zhǔn)引物分別對(duì)上述DNA進(jìn)行PCR擴(kuò)增。使用聚丙烯氨酰胺凝膠電泳或DNA測(cè)序儀對(duì)擴(kuò)增產(chǎn)物進(jìn)行長(zhǎng)度片段檢測(cè),并按葡萄胎及其雙親為單位進(jìn)行分組分析,從而確定葡萄胎的遺傳學(xué)起源。 三、根據(jù)基因NLRP7和KHDC3L的編碼區(qū)參考序列,分別設(shè)計(jì)引物擴(kuò)增葡萄胎患者及其母親或姐妹這兩個(gè)基因的所有外顯子,并使用DNA測(cè)序儀對(duì)擴(kuò)增產(chǎn)物進(jìn)行測(cè)序。將測(cè)序結(jié)果與參考序列進(jìn)行比較,找出可改變表達(dá)產(chǎn)物氨基酸序列的突變或非同義異形體(non-synonymous variant, NSV)。在dbSNP和emsembl數(shù)據(jù)庫(kù)中檢索找到的位點(diǎn),判斷是否為單核苷酸多態(tài)性(single nucleotide polymorphism,SNP)位點(diǎn)。使用Poly-Phen2評(píng)估找到的位點(diǎn)損害蛋白功能的可能性。 結(jié)果 共收集15例葡萄胎妊娠的病理標(biāo)本,其中1例為FRHM,8例為RHM,6例為散發(fā)性葡萄胎。1例(1／1,100%)FRHM和1例(1／8,12.5%)無正常妊娠患者的RHM為BiCHM;5例(5／8,62.5%)RHM和6例(6／6,100%)散發(fā)性葡萄胎為AnCHM;2例(2／8,25%%)RHM符合PHM。11例AnCHM中,8例(8／11,72.7%)為純合子,3例(3／11,27.3%)為雜合子。 在15例復(fù)發(fā)性及散發(fā)性葡萄胎患者中共篩查到NLRP7基因編碼區(qū)內(nèi)3個(gè)NSV：c.955GA、c.1280TC和c.1441GA;以及KHDC3L基因編碼區(qū)內(nèi)1個(gè)NSV：c.602CG。其中NLRP7c.1441GA只出現(xiàn)于BiCHM患者；NLRP7c.955GA和c.1280TC雜合狀態(tài)在HM患者和健康女性中都存在；KHDC3L c.602CG純合狀態(tài)在HM患者和健康女性中都存在。 結(jié)論 FRHM為BiCHM,無正常妊娠患者的RHM可能為BiCHM;有正常妊娠患者的RHM和散發(fā)性HM可為AnCHM或PHM。BiCHM與AnCHM的臨床和組織病理學(xué)表現(xiàn)難以區(qū)分,需要分子遺傳學(xué)分析以鑒別。 NLRP7的NSV c.1441GA可能對(duì)BiCHM的發(fā)生有一定的促進(jìn)作用。但本研究中葡萄胎患者的NLRP7和KHDC3L基因編碼序列沒有明確的致病突變或NSV,說明BiCHM存在遺傳異質(zhì)性,除NLRP7和KHDC3L外,還可能存在其他致病基因。 本研究的分子遺傳學(xué)方法和基因測(cè)序方法可用于臨床遇到反復(fù)發(fā)生葡萄胎(≥2次)的患者時(shí),判斷葡萄胎的遺傳學(xué)起源及患者有無NLRP7和KHDC3L基因突變,從而指導(dǎo)臨床處理。
[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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.33

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 向陽,楊秀玉,宋鴻釗,李志英,崔竹梅,萬希潤(rùn);惡性滋養(yǎng)細(xì)胞腫瘤腦轉(zhuǎn)移的療效分析[J];中華婦產(chǎn)科雜志;2001年07期

2 趙峻;向陽;;妊娠滋養(yǎng)細(xì)胞腫瘤保留生育功能的治療[J];中國(guó)癌癥雜志;2012年06期

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本文編號(hào)：1708221