小鼠先天性白內障新型自發(fā)突變基因的定位克隆
發(fā)布時間：2019-05-27 01:22
【摘要】：先天性白內障(congenital cataract)為出生時或出生后第一年內發(fā)生的晶狀體混濁,臨床上0.4%的新生兒患有先天性白內障,是常見的兒童致盲疾病。大約26%～51%的先天性白內障是遺傳因素導致的,因此,從分子機制上揭示先天性白內障的發(fā)病原因是防治該疾病的根本途徑。 2006年在ICR×BALB/cJ的F1代群體中,發(fā)現(xiàn)2只自發(fā)突變導致白內障的小鼠。經與BALB/cJ進一步回交繁育,保持了穩(wěn)定的顯性常染色體遺傳特征。 本研究對雜合子與純合子小鼠進行了性狀鑒定,雜合子突變小鼠產生了核型和放射狀白內障,而純合子突變小鼠在剛出生就形成完全白內障�；疾⌒∈笤�13天左右出現(xiàn)晶狀體混濁、角膜無異常、無行為學異常、無生殖障礙、生長發(fā)育也無異常。 為定位突變基因,將攜帶白內障基因BALB/cJ品系與正常C3H/HeJ回交兩代,建立了F2代家系,共挑選了83只白內障表型小鼠用于基因鑒定。第一步,通過全基因組掃描,對平均覆蓋19條常染色體的共44個遺傳位標在上述群體中分型,將基因定位到1號染色體上D1Mit410(17cM)和D1Mit102(73cM)之間;第二步,在該區(qū)段內(56cM長)進一步挑選遺傳位標,單倍型分析結果將白內障基因定位到D1Mit236(25.7cM)和D1Mit46(43.1cM)之間;第三步,選擇D1Mit236和D1Mit46之間單倍型不同的樣本13個,再次挑選遺傳位標D1Mit19(36.9cM)和D1Mit7(41cM)進行分型,將白內障相關基因進一步定位到D1Mit236和D1Mit19之間約11cM內,此區(qū)間內存在晶狀體蛋白基因Cryg(32cM),故選擇Cryg為候選基因。第四步,反轉錄出Cryg六個亞基的cDNA,cDNA測序分析發(fā)現(xiàn)在白內障純合子樣本的Crygc亞基第3外顯子的209bp處缺失一個堿基,該突變在Crygc亞基第3外顯子的76位引入終止密碼子,形成截短蛋白。為進一步驗證缺失突變是引起白內障的原因,對大樣本進行測序和單核苷酸鑒定,結果表明缺失基因型和白內障表型是完全連鎖的,因此該突變應該是引起白內障的原因。 由于突變位于高度保守的第四個關鍵基序中,當其變短時,雖然RT-PCR半定量表明Crygc亞基mRNA能正常轉錄,但翻譯成蛋白時,就會影響γC-晶狀體蛋白的正常功能。生物信息分析突變的γC-晶狀體蛋白時,突變型蛋白第二結構域的HMM是從1～66,丟失了15個氨基酸,第二結構域中的保守區(qū)被破壞、變短,而保守序列對維持蛋白質的結構和功能是相當重要的,變短則影響了晶狀體蛋白的功能,可能是形成白內障的原因。在蛋白質三維結構分析中,γC-突變蛋白的C-末端缺失一個β-折疊,并且在檢測三級結構模型的健康度時,因突變型蛋白從第3外顯子的76位氨基酸后缺失,導致原子經驗平均力勢增加,蛋白結構不穩(wěn)定。 綜上所述,無論從基因型與白內障表型的遺傳連鎖、突變基因本身的性質變化均表明,單堿基缺失是形成該自發(fā)突變白內障小鼠的成因。
[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，
本文編號：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，
本文編號：2485789
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