一中国BPES家系的基因突变分析

发布时间：2018-02-09 13:57

本文关键词： BPES FOXL2基因突变分析　出处：《郑州大学》2007年硕士论文　论文类型：学位论文

【摘要】： 睑裂狭小、倒转型内眦赘皮和上睑下垂综合征(Blepharophimosis-ptosis-epicanthus inversus syndrome，BPES)是一种少见的常染色体显性遗传疾病，在普通人群中的发病率大约为1／100，000。临床上以睑裂狭小、倒转型内眦赘皮及上睑下垂三联征为主要特征。少数病例还存在其他异常，如智力低下、发育迟缓、心脏缺损、小头及低位耳等。Vonammon于1841年最先描述此病。BPES可分为两种类型：Ⅰ型患者除了具有典型的BPES脸部特征外，女性患者不孕，原发性闭经或提前绝经，小子宫及卵巢早衰，而男性患者可生育，但可将这一性状传递给下一代。Ⅱ型患者男女均可生育。研究表明位于3q23的FOXL2基因是该病的致病基因，BPESⅠ型和Ⅱ型患者均可检测到FOXL2基因突变。 FOXL2基因由长2.7kb的单个外显子组成。其编码蛋白质属于forkhead转录因子大家族，由376个氨基酸组成，其中包含一个由101个氨基酸构成的forkhead DNA结合区，位置在第54～152残基区域。在此下游还存在一个与此分离且功能尚不明确的多聚丙氨酸肽段。多聚丙氨酸肽段可能与转录活性抑制有关。FOXL2基因是第一个被证实的在卵巢功能维持和卵巢分化中发挥重要作用的人类常染色体基因。迄今为止临床尚无有效的方法可以治疗BPESⅠ型女性患者的不孕症。虽然应用外科整形手术可以矫正BPES患者的眼部及颜面部畸形，从而改善其外观，但是BPES患者一般视力都受影响，有些还合并有全身其他系统不同程度的疾病，使患者及其家庭承受着躯体、精神及经济上的多重痛苦。另外，该病患者的临床表型变异性较大，给临床诊断也带来一定困难，根据孟德尔遗传规律，本病患者下一代再发风险率为50％，故对BPES患者进行遗传咨询尤为重要。目前产前诊断的方法有细胞遗传学检查及基因分析。患者染色体核型不正常者，妊娠后可做细胞遗传学检查进行排查。但对核型正常者，只能通过基因分析做产前诊断。因此，应用遗传学方法对BPES致病基因FOXL2突变类型的检测是对该病进行产前基因诊断的基础。目的对一个来自中国连续传递5代的Ⅰ型BPES大家系中的患者进行FOXL2基因突变研究，寻找其突变位点。为进一步的遗传咨询提供指导和理论依据。方法 1．抽取该家系中患病个体Ⅳ8及Ⅲ3的外周静脉血进行染色体核型分析。检测该家系中患者是否存在染色体缺失。 2．采用经典盐析法提取该家系中患者及来我院健康体检的正常人外周血基因组DNA，并进行DNA纯化和定量。 3．根据参考文献设计4对引物，来对FOXL2基因的编码区进行PCR扩增。 4．对扩增产物进行直接测序，检测是否存在突变及突变位点。 5．聚丙烯酰胺电泳进一步验证直接测序的结果。结果 1．家系分析结果表明该BPES家系的遗传方式为常染色体显性遗传，并且此家系为BPESⅠ型。 2．染色体检测显示该家系中患者Ⅳ8及Ⅲ3分别为正常男性染色体核型：46，XY和正常女性染色体核型：46，XX。 3．直接测序显示，，该家系中患病个体的FOXL2基因存在1080-1096dup17突变。 4．聚丙烯酰胺电泳验证结果显示该家系中患者的在FOXL2基因的CD和EF区域为杂合子。结论 1．正常个体的FOXL2全基因序列与GenBank数据库相符，而BPES家系患者的FOXL2基因引物CD PCR扩增产物的测序结果显示该患者存在1080-1096dup17这一重复突变。从而导致该家系中患病个体呈现出BPESⅠ型的表型。 2．该BPESⅠ型家系中发现的1080-1096dup17重复突变，引起读码框第287位密码子之后发生移码，从而导致编码蛋白质在第361位密码子处提前终止。这个突变虽然未影响FOXL2基因的forkhead区域和多聚丙氨酸区域的完整性，但将影响蛋白质的三级结构，从而影响蛋白质的功能。 3．本研究首次报道了中国人群中的1080-1096dup17重复突变，这种突变可以发生在不同种族的BPES家族性或散发性病例中，因此它可能是一种独立起源的突变，并且是BPES患病个体的一个突变热点，也是进行BPES患者突变分析的重要部位。
[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.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R394

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