小鼠先天性白内障新型自发突变基因的定位克隆
发布时间：2019-05-27 01:22
【摘要】：先天性白内障(congenital cataract)为出生时或出生后第一年内发生的晶状体混浊,临床上0.4%的新生儿患有先天性白内障,是常见的儿童致盲疾病。大约26%～51%的先天性白内障是遗传因素导致的,因此,从分子机制上揭示先天性白内障的发病原因是防治该疾病的根本途径。 2006年在ICR×BALB/cJ的F1代群体中,发现2只自发突变导致白内障的小鼠。经与BALB/cJ进一步回交繁育,保持了稳定的显性常染色体遗传特征。 本研究对杂合子与纯合子小鼠进行了性状鉴定,杂合子突变小鼠产生了核型和放射状白内障,而纯合子突变小鼠在刚出生就形成完全白内障。患病小鼠在13天左右出现晶状体混浊、角膜无异常、无行为学异常、无生殖障碍、生长发育也无异常。 为定位突变基因,将携带白内障基因BALB/cJ品系与正常C3H/HeJ回交两代,建立了F2代家系,共挑选了83只白内障表型小鼠用于基因鉴定。第一步,通过全基因组扫描,对平均覆盖19条常染色体的共44个遗传位标在上述群体中分型,将基因定位到1号染色体上D1Mit410(17cM)和D1Mit102(73cM)之间;第二步,在该区段内(56cM长)进一步挑选遗传位标,单倍型分析结果将白内障基因定位到D1Mit236(25.7cM)和D1Mit46(43.1cM)之间;第三步,选择D1Mit236和D1Mit46之间单倍型不同的样本13个,再次挑选遗传位标D1Mit19(36.9cM)和D1Mit7(41cM)进行分型,将白内障相关基因进一步定位到D1Mit236和D1Mit19之间约11cM内,此区间内存在晶状体蛋白基因Cryg(32cM),故选择Cryg为候选基因。第四步,反转录出Cryg六个亚基的cDNA,cDNA测序分析发现在白内障纯合子样本的Crygc亚基第3外显子的209bp处缺失一个碱基,该突变在Crygc亚基第3外显子的76位引入终止密码子,形成截短蛋白。为进一步验证缺失突变是引起白内障的原因,对大样本进行测序和单核苷酸鉴定,结果表明缺失基因型和白内障表型是完全连锁的,因此该突变应该是引起白内障的原因。 由于突变位于高度保守的第四个关键基序中,当其变短时,虽然RT-PCR半定量表明Crygc亚基mRNA能正常转录,但翻译成蛋白时,就会影响γC-晶状体蛋白的正常功能。生物信息分析突变的γC-晶状体蛋白时,突变型蛋白第二结构域的HMM是从1～66,丢失了15个氨基酸,第二结构域中的保守区被破坏、变短,而保守序列对维持蛋白质的结构和功能是相当重要的,变短则影响了晶状体蛋白的功能,可能是形成白内障的原因。在蛋白质三维结构分析中,γC-突变蛋白的C-末端缺失一个β-折叠,并且在检测三级结构模型的健康度时,因突变型蛋白从第3外显子的76位氨基酸后缺失,导致原子经验平均力势增加,蛋白结构不稳定。 综上所述,无论从基因型与白内障表型的遗传连锁、突变基因本身的性质变化均表明,单碱基缺失是形成该自发突变白内障小鼠的成因。
[Abstract]:Congenital cataract (congenital cataract) is a kind of lens opacification occurred at birth or in the first year after birth. 0.4% of newborns suffer from congenital cataract, which is a common blinding disease in children. About 26% of congenital cataracts are caused by genetic factors, so it is the fundamental way to prevent and cure congenital cataracts to reveal the cause of congenital cataracts from molecular mechanism. In 2006, two mice with spontaneous mutation were found in F1 generation of ICR 脳 BALB/cJ. After further backcross with BALB/cJ, the stable dominant autosomal genetic characteristics were maintained. In this study, the characters of heterozygous and homozygous mice were identified. The karyotype and radial cataract were produced in the mutant mice, while the complete cataracts were formed in the homozygous mutant mice at birth. Lens opacification, no corneal abnormality, no behavioral abnormality, no reproductive disorder and no abnormal growth and development occurred in the diseased mice at about 13 days. In order to locate the mutant gene, the BALB/cJ strain carrying cataract gene was backcrossed with normal C3H/HeJ for two generations, and a F2 generation family was established. A total of 83 cataract phenotypic mice were selected for gene identification. In the first step, a total of 44 genetic loci covering an average of 19 autosomes were typed in the above populations by whole genome scanning, and the genes were mapped between D1Mit410 (17cM) and D1Mit102 (73cM) on chromosome 1. In the second step, the genetic markers were further selected in this section (56cM length). The results of haplotype analysis mapped the cataract gene between D1Mit236 (25.7cM) and D1Mit46 (43.1cM). In the third step, 13 samples with different haplotypes between D1Mit236 and D1Mit46 were selected, and the genetic markers D1Mit19 (36.9cM) and D1Mit7 (41cM) were selected for typing, and the cataract related genes were further mapped to about 11cM between D1Mit236 and D1Mit19. There is lens protein gene Cryg (32cM) in this region, so Cryg is selected as the candidate gene. In the fourth step, cDNA,cDNA sequencing analysis of Cryg six subunits showed that a base was missing in the 209bp of exon 3 of Crygc subunit in cataract homozygote samples, and the mutation introduced termination codon at position 76 in exon 3 of Crygc subunit. A truncated protein is formed. In order to further verify that deletion mutation is the cause of cataract, the large samples were sequenced and identified by single nucleotides. The results showed that the deletion genotype and cataract phenotype were completely linked, so the mutation should be the cause of cataract. Because the mutation is located in the highly conserved fourth key motif, when it becomes shorter, although RT-PCR semi-quantitatively indicates that Crygc subunit mRNA can be transcribed normally, it will affect the normal function of gamma C-lens protein when it is translated into protein. When the mutant gamma-lens protein was analyzed by biological information, the HMM of the second domain of the mutant protein was 1 鈮，
本文编号：2485789
[Abstract]:Congenital cataract (congenital cataract) is a kind of lens opacification occurred at birth or in the first year after birth. 0.4% of newborns suffer from congenital cataract, which is a common blinding disease in children. About 26% of congenital cataracts are caused by genetic factors, so it is the fundamental way to prevent and cure congenital cataracts to reveal the cause of congenital cataracts from molecular mechanism. In 2006, two mice with spontaneous mutation were found in F1 generation of ICR 脳 BALB/cJ. After further backcross with BALB/cJ, the stable dominant autosomal genetic characteristics were maintained. In this study, the characters of heterozygous and homozygous mice were identified. The karyotype and radial cataract were produced in the mutant mice, while the complete cataracts were formed in the homozygous mutant mice at birth. Lens opacification, no corneal abnormality, no behavioral abnormality, no reproductive disorder and no abnormal growth and development occurred in the diseased mice at about 13 days. In order to locate the mutant gene, the BALB/cJ strain carrying cataract gene was backcrossed with normal C3H/HeJ for two generations, and a F2 generation family was established. A total of 83 cataract phenotypic mice were selected for gene identification. In the first step, a total of 44 genetic loci covering an average of 19 autosomes were typed in the above populations by whole genome scanning, and the genes were mapped between D1Mit410 (17cM) and D1Mit102 (73cM) on chromosome 1. In the second step, the genetic markers were further selected in this section (56cM length). The results of haplotype analysis mapped the cataract gene between D1Mit236 (25.7cM) and D1Mit46 (43.1cM). In the third step, 13 samples with different haplotypes between D1Mit236 and D1Mit46 were selected, and the genetic markers D1Mit19 (36.9cM) and D1Mit7 (41cM) were selected for typing, and the cataract related genes were further mapped to about 11cM between D1Mit236 and D1Mit19. There is lens protein gene Cryg (32cM) in this region, so Cryg is selected as the candidate gene. In the fourth step, cDNA,cDNA sequencing analysis of Cryg six subunits showed that a base was missing in the 209bp of exon 3 of Crygc subunit in cataract homozygote samples, and the mutation introduced termination codon at position 76 in exon 3 of Crygc subunit. A truncated protein is formed. In order to further verify that deletion mutation is the cause of cataract, the large samples were sequenced and identified by single nucleotides. The results showed that the deletion genotype and cataract phenotype were completely linked, so the mutation should be the cause of cataract. Because the mutation is located in the highly conserved fourth key motif, when it becomes shorter, although RT-PCR semi-quantitatively indicates that Crygc subunit mRNA can be transcribed normally, it will affect the normal function of gamma C-lens protein when it is translated into protein. When the mutant gamma-lens protein was analyzed by biological information, the HMM of the second domain of the mutant protein was 1 鈮，
本文编号：2485789
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