粘脂累积病和先天性眼球震颤相关致病基因突变分析及功能研究

发布时间：2018-06-13 06:17

  本文选题：粘脂累积病 + 先天性眼球震颤　； 参考：《华中科技大学》2012年博士论文

【摘要】：本文收集并鉴定了一个粘脂累积病及两个先天性眼球震颤家系,通过连锁分析(Linkage Analysis)与定位克隆(Positional Cloning)分子遗传学技术对这三个家系进行了遗传学分析,确定了它们的致病原因。同时对一个功能不清楚的眼球震颤致病基因FRMD7进行了初步的功能分析。其结果如下： 1、粘脂累积病家系的分子遗传学分析 对收集和鉴定的一对夫妇(非近亲婚配)生育了三个粘脂累积病孩子(两女一男)进行微卫星标记的连锁分析,发现GNPTG基因附近的微卫星标记D16S3024和选择对先证者GNPTG基因所有外显子和外显子／内含子交界处进行直接测序,发现了先征者GNPTG基因存在两个未报导的杂合突变(即一个复合型杂合突变)IVS4-1GC(剪切突变)和c.471de1C(单碱基缺失突变)。对其双亲测序发现,先征者携带的一个杂合IVS4-1GC突变是来自其母亲,而另一个杂合的c.471de1C缺失突变是来自父亲。同时对另外的两位患者进行上述两个突变位置的测序检测,发现这两位患者携带与先征者相同的突变。；通过体外和体内反转录-聚合酶链式反应(RT-PCR)及测序实验,发现IVS4-1GC突变导致增加第4内含子74个碱基的InRNA的产生,该mRNA翻译的GNPTG蛋白在第4个外显子编码开始处产生一个终止密码子,因此也产生一个可能无功能的蛋白。说明GNPTG基因的IVS4-1GC和c.471de1C这个复合型杂合突变是导致该家系三个患者的发病原因。 2、X连锁先天性眼球震颤家系的分子遗传学分析 收集并鉴定了两个X-连锁的先天性眼球震颤家系。家系1的遗传方式为X-连锁隐性遗传,通过微卫星标记的连锁分析,发现不能排除GPR143基因突变是导致该家系眼球震颤的原因,对先证者GPR143基因所有外显子和外显子／内含子交界处直接测序,在该基因编码框819到822位的四个碱基(AACA)缺失(c.819822de1),这是一个新的突变,该突变造成GPR143蛋白在274位移码,移码7个氨基酸后产生一个终止密码子(p.T274Ifs*8),产生一个截短蛋白；运用单链构象多态性(SSCP)分析,家系中所有男性患者和女性携带者都存在该突变,而家系外100例(50例男性和50女性)正常人不存在该突变。家系2的遗传方式为X-连锁显性遗传,先证者及其母亲眼球震颤并伴随头震颤和斜视等症状,根据这些临床症状不能排除FRMD7基因突变所致；因此选择先征者基因组DNA,对FRMD7基因所有外显子和外显子交界处直接测序,在该基因的580位发现单个碱基改变即c.580GA(p.A194T)；并且该突变存在男性患者和女性患者,且女性患者为杂合突变,不存在于100例(50例男性和50女性)正常人群；通过生物信息学氨基酸保守性分析,发现194位的丙氨酸从线虫到人类都是高度保守的。以上研究推测GPR143基因p.T274IfsX7突变和FRMD7基因c.580GA(p.A194T)突变分别是导致眼球震颤家系1和家系2的致病原因。3、眼球震颤致病基因FRMD7初步的功能分析 为了初步探讨FRMD7如何参与细胞分化功能,本研究构建了FRMD7野生型、突变体p.A194T、p.H333fs(本实验室之前所发现突变)和p.G24R(文献中报道的突变)分别与EGFP形成融合蛋白载体,观察各类突变体和野生型在真核细胞中定位情况,发现突变体p.A194T和野生型类似,主要定位于细胞质,而突变体p.G24R和p.H333fs发生核转位现象,绝大部分融合蛋白集中在细胞核中。通过生物信息学预测NLS位于FRMD7氨基酸序列的234位KRKH,NES位于93位的LTRYLFTLQI。并进行了功能验证。为了进一步分析FRMD7蛋白入核后的功能,本实验分析了其与p53蛋白的共定位分析,结果没有发现明显的共定位情况；另外通过免疫荧光技术证明FRMD7在核内过表达形成的点状结构也不是细胞核常见结构Cajal body。说明FRMD7对于神经元细胞分化的促进作用可能不是通过与p53蛋白及Cajal body实现的。 总之,本研究主要是以粘脂累积病及先天性眼球震颤家系为对象,通过分子遗传学及分子生物学等手段,分析一个粘脂累积病家系和两个先天性眼球震颤的致病原因,同时对一个眼球震颤基因FRMD7蛋白亚细胞定位的可能机制进行了初步探讨。在粘脂累积病家系发现的致病基因GNPTG的杂合突变是首次在中国人群中检测到的基因变异,扩充了该基因的突变谱；这一研究结果可以将该疾病与其症状相近(粘多糖累积病Ⅱ、粘脂累积病ⅢA)的其他疾病准确区分开来,有利于实现对该家系病人的个体化治疗方案的制定。对于X连锁的先天性眼球震颤家系1的研究结果再一次支持了在中国人群中,GPR143基因的突变所导致的先天性眼球震颤疾病与在白种人群中表现的I型白化病不同。对于X连锁的先天性眼球震颤家系2的研究结果扩展了眼球震颤致病基因FRMD7的突变谱。这些遗传学研究结果对于疾病的准确分型至关重要,是实施个体化针对性治疗的基础,而且可以作为遗传病家系产前诊断的遗传学基础。对于FRMD7蛋白功能的初步分析结果则提示了部分FRMD7的突变所导致的亚细胞定位的改变可能是导致蛋白在细胞质中的功能丧失的主要原因。这一结果有助于加深对FRMD7蛋白突变的致病机制的认识。
[Abstract]:In this paper , we collected and identified a myxosis and two congeners of ocular tremor , and analyzed these three families by linkage analysis and molecular genetic technique . The pathogenic factors were determined . At the same time , a preliminary functional analysis was carried out on a functional unknown pathogenic gene FRMD7 . The results were as follows :
1 . Molecular genetic analysis of myxolipid accumulation disease
Two unrelated heterozygous mutations ( i.e . , a compound heterozygous mutation ) IVS4 - 1GC ( shear mutation ) and c . 471de1C ( single base deletion mutation ) were detected by the microsatellite markers D16S3024 in the vicinity of GNPTG gene .
In vitro and in vivo reverse transcription - polymerase chain reaction ( RT - PCR ) and sequencing experiments , it was found that the IVS4 - 1GC mutation resulted in an increase in the production of InRNA of 74 bases in the 4th intron , which translated GNPTG proteins at the beginning of the 4th exon coding to produce a stop codon , thus also producing a protein that may not function . This compound heterozygous mutation of the GNPTG gene IVS4 - 1GC and c.471de1C is the cause of the three patients in the family .
Molecular genetic analysis of 2 , X - linked congenital exophthalmos
The genetic pattern of family 1 was X - linked recessive inheritance , and the linkage analysis of microsatellite markers found that it was not possible to exclude the gene mutation of the gene . The deletion of the four bases ( AACA ) in the gene coding block 819 to 822 was a new mutation which resulted in a stop codon ( p.T274Ifs * 8 ) at the position 274 of the gene encoding the amino acids of the gene coding block 819 to 822 , resulting in a truncated protein .
Single - strand conformation polymorphism ( SSCP ) analysis showed that the mutation was found in all male and female carriers in the family , but there were 100 cases ( 50 males and 50 females ) in the family without the mutation .
Therefore , the genomic DNA of the precursor was selected and sequenced directly at the junction of all exon and exon of the FRMD7 gene , and a single base change , i.e . , c.580 GA ( p . A194T ) was found at 580 bits of the gene ;
and the mutation existed in male and female patients , and female patients were heterozygous mutations , and did not exist in 100 cases ( 50 males and 50 females ) in the normal population ;
Through the analysis of amino acid conservation in bioinformatics , it was found that the 194 - position alanine was highly conserved from nematodes to humans . The above studies have speculated that the mutation of the p . T274Ifs7 mutation and the c . 580 GA ( p . A194T ) mutation of the gene of the pH.580 GA ( p . A194T ) , respectively , are responsible for the pathogenesis of the ocular tremor family 1 and the family 2 . 3 . The preliminary functional analysis of the pathogenic gene FRMD7 of the eyeball tremor
In order to investigate the role of FRMD7 in cell differentiation , we constructed FRMD7 wild - type , mutant p . A194T , p . H333fs ( mutation identified earlier in the laboratory ) and p . G24R ( the mutation reported in the literature ) . We observed the localization of various mutants and wild type in eukaryotic cells .
In addition , the dot - like structure formed by overexpression of FRMD7 in the nucleus by immunofluorescence technique is not a common structure of the nucleus of the nucleus . It is suggested that the promotion of FRMD7 to neuronal cell differentiation may not be achieved by interaction with p53 protein and CaSO body .
In conclusion , this study was mainly focused on the pathogenesis of myxolipid accumulation disease and congenital eyeball tremor , and the possible mechanism for the localization of a myxomil gene FRMD7 protein was investigated by molecular genetics and molecular biology . The mutation of GNPTG gene was the first to be detected in Chinese population , which extended the mutation spectrum of the gene .
The results of this study can distinguish the disease from other diseases closely related to its symptoms ( mucopolysaccharidosis 鈪，

本文编号：2013028