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  本文關鍵詞： BPES FOXL2基因 突變分析　出處：《鄭州大學》2007年碩士論文　論文類型：學位論文

【摘要】： 瞼裂狹小、倒轉型內眥贅皮和上瞼下垂綜合征(Blepharophimosis-ptosis-epicanthus inversus syndrome，BPES)是一種少見的常染色體顯性遺傳疾病，在普通人群中的發(fā)病率大約為1／100，000。臨床上以瞼裂狹小、倒轉型內眥贅皮及上瞼下垂三聯(lián)征為主要特征。少數病例還存在其他異常，如智力低下、發(fā)育遲緩、心臟缺損、小頭及低位耳等。Vonammon于1841年最先描述此病。BPES可分為兩種類型：Ⅰ型患者除了具有典型的BPES臉部特征外，女性患者不孕，原發(fā)性閉經或提前絕經，小子宮及卵巢早衰，而男性患者可生育，但可將這一性狀傳遞給下一代。Ⅱ型患者男女均可生育。研究表明位于3q23的FOXL2基因是該病的致病基因，BPESⅠ型和Ⅱ型患者均可檢測到FOXL2基因突變。 FOXL2基因由長2.7kb的單個外顯子組成。其編碼蛋白質屬于forkhead轉錄因子大家族，由376個氨基酸組成，其中包含一個由101個氨基酸構成的forkhead DNA結合區(qū)，位置在第54～152殘基區(qū)域。在此下游還存在一個與此分離且功能尚不明確的多聚丙氨酸肽段。多聚丙氨酸肽段可能與轉錄活性抑制有關。FOXL2基因是第一個被證實的在卵巢功能維持和卵巢分化中發(fā)揮重要作用的人類常染色體基因。 迄今為止臨床尚無有效的方法可以治療BPESⅠ型女性患者的不孕癥。雖然應用外科整形手術可以矯正BPES患者的眼部及顏面部畸形，從而改善其外觀，但是BPES患者一般視力都受影響，有些還合并有全身其他系統(tǒng)不同程度的疾病，使患者及其家庭承受著軀體、精神及經濟上的多重痛苦。另外，該病患者的臨床表型變異性較大，給臨床診斷也帶來一定困難，根據孟德爾遺傳規(guī)律，本病患者下一代再發(fā)風險率為50％，故對BPES患者進行遺傳咨詢尤為重要。目前產前診斷的方法有細胞遺傳學檢查及基因分析。患者染色體核型不正常者，妊娠后可做細胞遺傳學檢查進行排查。但對核型正常者，只能通過基因分析做產前診斷。因此，應用遺傳學方法對BPES致病基因FOXL2突變類型的檢測是對該病進行產前基因診斷的基礎。 目的 對一個來自中國連續(xù)傳遞5代的Ⅰ型BPES大家系中的患者進行FOXL2基因突變研究，尋找其突變位點。為進一步的遺傳咨詢提供指導和理論依據。 方法 1．抽取該家系中患病個體Ⅳ8及Ⅲ3的外周靜脈血進行染色體核型分析。檢測該家系中患者是否存在染色體缺失。 2．采用經典鹽析法提取該家系中患者及來我院健康體檢的正常人外周血基因組DNA，并進行DNA純化和定量。 3．根據參考文獻設計4對引物，來對FOXL2基因的編碼區(qū)進行PCR擴增。 4．對擴增產物進行直接測序，檢測是否存在突變及突變位點。 5．聚丙烯酰胺電泳進一步驗證直接測序的結果。 結果 1．家系分析結果表明該BPES家系的遺傳方式為常染色體顯性遺傳，并且此家系為BPESⅠ型。 2．染色體檢測顯示該家系中患者Ⅳ8及Ⅲ3分別為正常男性染色體核型：46，XY和正常女性染色體核型：46，XX。 3．直接測序顯示，，該家系中患病個體的FOXL2基因存在1080-1096dup17突變。 4．聚丙烯酰胺電泳驗證結果顯示該家系中患者的在FOXL2基因的CD和EF區(qū)域為雜合子。 結論 1．正常個體的FOXL2全基因序列與GenBank數據庫相符，而BPES家系患者的FOXL2基因引物CD PCR擴增產物的測序結果顯示該患者存在1080-1096dup17這一重復突變。從而導致該家系中患病個體呈現出BPESⅠ型的表型。 2．該BPESⅠ型家系中發(fā)現的1080-1096dup17重復突變，引起讀碼框第287位密碼子之后發(fā)生移碼，從而導致編碼蛋白質在第361位密碼子處提前終止。這個突變雖然未影響FOXL2基因的forkhead區(qū)域和多聚丙氨酸區(qū)域的完整性，但將影響蛋白質的三級結構，從而影響蛋白質的功能。 3．本研究首次報道了中國人群中的1080-1096dup17重復突變，這種突變可以發(fā)生在不同種族的BPES家族性或散發(fā)性病例中，因此它可能是一種獨立起源的突變，并且是BPES患病個體的一個突變熱點，也是進行BPES患者突變分析的重要部位。
[Abstract]:Blepharophimosis, epicanthus inversus and ptosis syndrome (Blepharophimosis-ptosis-epicanthus inversus, syndrome, BPES) is a rare autosomal dominant disease incidence in the general population rate of about 1 / 100000. in clinical blepharophimosis, epicanthus inversus and ptosis triple sign as the main feature. In some cases there are other abnormalities, such as mental retardation, developmental delay, heart defects, head and low ear.Vonammon in 1841 was the first to describe the.BPES can be divided into two types: type I patients except with BPES typical facial features, female patients with infertility, primary amenorrhea or early menopause, uterine and ovarian failure, and men with fertility, but this trait can be passed on to the next generation. Type II patients of both men and women. Family study showed that the FOXL2 gene is located in 3q23 is the base of the disease pathogenesis The FOXL2 gene mutation can be detected in patients with type BPES I and type II.
The FOXL2 gene consists of a single long 2.7kb exons. Its encoding protein belongs to forkhead transcription factor family, consisting of 376 amino acids, which contains a 101 amino acid forkhead DNA binding region, position in fifty-fourth ~ 152 residues in this region. There are downstream polyalanine peptides with a this separation and function is not clear. Polyalanine peptide may inhibit the.FOXL2 gene and transcriptional activity is to play an important role in maintaining the first confirmed ovarian function and ovarian differentiation in human autosomal genes.
So far there is no effective method to clinical treatment of BPES type of female patients with infertility. Although the application of plastic surgery can correct BPES patients with eye and facial deformity, so as to improve their appearance, but most of the BPES patients eyesight are affected, some patients also have different degrees of systemic disease, the patients and their families a body, multiple psychic pain and economic terms. In addition, the disease phenotype variability for clinical diagnosis is difficult, according to the law of Mendel inheritance, the patients of next generation recurrence rate was 50%, the genetic counseling of BPES patients is particularly important. The current method of prenatal diagnosis analysis of cytogenetics and gene. The karyotype in patients with abnormal pregnancy, after do cytogenetic examination investigation. But on normal karyotypes, only through the Genetic analysis has been used for prenatal diagnosis. Therefore, the detection of the FOXL2 mutation type of the BPES pathogenic gene by genetic method is the basis for the prenatal gene diagnosis of the disease.
objective
In order to provide guidance and theoretical basis for further genetic counseling, we conducted a FOXL2 gene mutation study in a Chinese BPES family from 5 consecutive generations in China.
Method
1. the chromosome karyotype of the peripheral venous blood of the sick individuals in the family was analyzed. The chromosome deletion was detected in the patients in the family.
2. the genomic DNA of the peripheral blood of the patients in the family and the normal people from our hospital were extracted by the classical salting out method, and the DNA was purified and quantified.
3. according to the reference literature, 4 pairs of primers were designed to amplify the coding region of the FOXL2 gene by PCR.
4. the amplified products were directly sequenced, and the presence of mutation and mutation sites was detected.
5. polyacrylamide gel electrophoresis was used to further verify the results of direct sequencing.
Result
The results of the 1. family analysis showed that the hereditary mode of the BPES family was autosomal dominant, and the family was type BPES I.
2. chromosome tests showed that the patients in the family were normal male chromosome karyotypes: 46, XY and normal female chromosome karyotype: 46, XX..
3. direct sequencing showed that there was a 1080-1096dup17 mutation in the FOXL2 gene of the individuals in the family.
The results of 4. polyacrylamide gel electrophoresis showed that the CD and EF regions of the FOXL2 gene in the family were heterozygotes.
conclusion
1. normal individuals of FOXL2 gene sequences and GenBank database with BPES family and sequencing of PCR products with FOXL2 gene primers CD PCR results showed that the patients with 1080-1096dup17. The repeat mutations resulting in the pedigree of affected individuals showed the phenotype of BPES type.
2. the BPES type 1080-1096dup17 family repeat mutation, caused by ORF after codon 287th frameshift occurred, resulting in 361st codon encoding proteins at early termination. Although this mutation did not affect the forkhead region of the FOXL2 gene and polyalanine region integrity, but will be three level structure the effect of protein, thus affecting the protein function.
3. this is the first report of the China crowd 1080-1096dup17 duplication, this mutation can occur in the BPES family of different races or sporadic cases, so it may be an independent origin of the mutation, and is a hot spot mutation BPES prevalence of individuals, but also an important part of patients with BPES mutation analysis.

【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R394

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