视网膜锥细胞失功能大鼠致病基因的鉴定
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摘要
视网膜包括视杆与视锥两种神经回路,分别负责暗(弱光)视觉、无色视觉和明(强光)视觉、色觉信息的接受、加工和传导,这两种感光系统之间存在着复杂的联系。色觉是视觉的高级功能,色觉异常疾病在男性中的发病率可高达8%,是一种常见的遗传疾病,被认为是视觉传导通路中一些基因的重组或点突变导致的。但由于缺乏先天性色觉异常疾病的模式动物,对其发病机制的研究难以深入,因此,目前色觉异常疾病的发病机制尚不完全清楚。本实验室发现并培育了一种先天性视网膜锥细胞失功能(retinal cone dysfunction, RCD)大鼠,其遗传表型为暗适应视网膜电图基本正常,明适应视网膜电图消失;组织结构学结果显示视网膜结构层次完整,光感受器数量无明显变化。本研究将对此模式动物的致病基因、突变方式及功能等进行研究,旨在了解该模式动物的发病机制。
方法
1.建立突变型大鼠与野生型BN大鼠的杂交品系,得到的F1代大鼠雌雄配对,获得F2代大鼠。通过建立遗传家系,明确遗传方式。后续研究主要以F2代大鼠为研究对象。
2.采用国际标准临床视网膜电图记录方法,进行表型鉴定。
3.应用序列标签位点(sequence tagged site,STS)和单倍体型(haplotype)分析方法,定位突变基因。
4.针对候选基因,采用基因克隆、测序等方法确定基因的突变方式,并观察突变基因是否与表型一致。
5.应用荧光实时定量PCR方法和免疫荧光方法,观察突变基因在模式大鼠中的mRNA表达水平以及蛋白表达水平。
6.采用苏木精-伊红染色及全视网膜铺片方法比较突变是否会影响不同年龄段的大鼠视网膜结构和锥体细胞的数量。
7.记录不同光强、频率及颜色光刺激下的视觉电生理反应,分析光感受器细胞的功能。
结果
1.将雌性突变型大鼠与雄性野生型BN大鼠杂交,子一代互交,收集到杂交二代大鼠共441只,所有大鼠均经过临床视觉电生理学检测确定其表型。根据NCBI网站中提供的序列标签位点作为分子标记物设计引物,对441只F2代大鼠分别进行聚合酶链式反应扩增。通过比较分子标记物在不同个体中的扩增产物,分析突变基因与分子标记物间的交换重组率,结果显示突变基因与DXRat21和DXRat96有连锁关系,并位于X染色体的末端端粒区27.8 Mb的区域内。此区域内共有169个候选基因,只有中波长视蛋白基因与视觉有关。
2.针对中波长视蛋白基因的的6个外显子分别设计引物、扩增并测序,结果显示第四内含子3’端剪接位点处保守的AG双碱基突变为AT双碱基。此突变在441只F2代大鼠中,除雄性正常表型大鼠外,其余个体中均存在;且在50只野生型SD大鼠和156种近交系大鼠中均未被发现。
3.反转录聚合酶链式反应和荧光实时定量聚合酶链式反应结果显示,野生型SD大鼠中可以检测到中波长视蛋白基因mRNA的表达,但是突变型大鼠中无法检测到该基因mRNA的表达,表明其转录过程受到抑制;免疫荧光结果显示,花生凝集素特异性标记的锥体细胞在野生型和突变型大鼠中均可以检测到,且正常表型的野生型SD大鼠中还可以检测到中波长视蛋白的表达,但突变型大鼠中无法检测到,中波长视蛋白在突变型大鼠中未被表达。
4.苏木精-伊红染色方法观察1个月、3个月、6个月、9个月和12个月野生型SD大鼠和突变型大鼠的视网膜结构,结果显示突变型大鼠的视网膜结构、光感受器细胞层的厚度与同龄的野生型SD大鼠相比均未发生明显变化;全视网膜铺片结果表明,同龄的突变型大鼠和野生型大鼠的锥体细胞的数量无明显差异。中波长视蛋白在野生型大鼠中表达,而突变型大鼠中无法检测到该蛋白的表达。
5.视觉电生理学记录结果显示,在暗适应条件下,随着刺激光强的增强,记录到的视网膜电图(electroretinogram,ERG)反应的波幅值在突变型和正常表型大鼠中均逐渐增大;明适应条件下,不同频率、光强的光刺激均显示出随着光强的增强,正常表型大鼠的波幅值逐渐增大,直至达到饱和状态,而突变型大鼠即使在最大光强刺激下也无法记录到任何波形。颜色光刺激后,在明适应环境中,突变型大鼠仍然无法记录到任何波形。
结论
中波长视蛋白基因中内含子和外显子间保守的剪接位点发生了点突变,导致该基因无法被正常的剪接,不能转录成成熟的mRNA,蛋白质的翻译过程也被终止。感光视蛋白与11-顺式视黄醛结合形成视锥细胞中的感光色素,由于基因突变致使蛋白无法合成,锥体细胞感光后的感光换能机制受到了抑制,所以在视觉电生理学检测中显示为无法检测到锥体细胞的功能,表现为明适应下无任何波形。中波长视蛋白基因的突变会导致绿色盲或者绿色弱疾病的产生,所以模式大鼠为绿色色觉异常大鼠。
迄今为止发现的中波长视蛋白基因的突变或是由于基因重组,或是由于外显子中的点突变引起,而该基因内含子的剪接位点突变属于首例,这为研究中波长视蛋白基因的功能、结构等方面提供了更多的机遇。蓝色锥体全色盲(blue cone monochromatism,BCM)疾病表现为感红(长波长)和感绿(中波长)锥体细胞功能丧失,而感蓝(短波长)锥体细胞和视杆细胞的功能正常。由于大鼠中只存在感绿(中波长)和感蓝(短波长)两种锥体细胞,所以,本模式大鼠可能为国际首例发现的自发突变BCM模式动物,因此具有重要的研究意义。同时,光学显微镜和全视网膜铺片实验结果显示模式大鼠视网膜结构无明显改变,光感受器细胞层的厚度及锥体细胞的数量并未发生明显变化,这为研究色觉异常机制、视网膜信号回路及基因治疗色觉异常疾病提供了理想的动物模型。
There are two signal circuits in retina: rod and cone neural circuits. Rod signal system is in charge of receiving, procession and transmission of visual information under scotopic condition, while cone signal system plays an important role under photopic condition. There is a complicated linkage in the rod and cone neural circuits. Color vision is one of the highest levels of vision. The color anomalopia is a very common inherited disease, which its prevalence is about 8% in western European men. It is presumably caused by mutations of genes in visual pathway. However, so far, the mechanisms of color anomalopia are not totally understood due to the lack of congenital color anomalopia animal model. The retinal cone dysfunction rat is a naturally occurring mutant model of X-linked cone dysfunction. In this model, the photopic electroretinogram (ERG) is abnormal, while there are no obvious histopathological changes. Here, we will screen and identify the causative gene and its function, aim to unserstand the mechanism of this disease.
Methods
1. Constructed a hybrid rat family by crossing the affected female rat with wild-type male BN rat. The F1 rats were intercrossed to get the F2 rats. The genetic mode was testified after the family was constructed.
2. Identified the phenotypes of the family members by ERG recordings.
3. Analyzed and located the mutant gene by using the sequence tagged site (STS) and haplotype method.
4. Cloned and sequenced the candidate gene. The mutant site was determined. Checked the relationship of the mutant gene and the phenotype.
5. Applied real-time quantitative PCR (RQ-PCR) and immunohistochemistry (IHC) methods to profile the expression levels of mRNA and protein in the retina of the affected rat.
6. Utilized hematoxylin and eosin (HE) staining and wholemount immunocytochemistry methods to compare the retinal structures and quantities of cones in affected rats with wild rats at the same ages.
7. Recorded electroretinograms with different intensities, frequencies and colors. With these results, functions of photoreceptors were analyzed.
Results
1. Cross family members were collected and 441 rats were obtained in F2 generation. Phenotypes were determined by using the ERG recordings. According to the sequence tagged sites provided by the NCBI website, 441 rats were amplified by using PCR method. After the amplified products were compared, the recombinant rates were analyzed. The affected gene showed a linkage relationship with the markers DXRat21 and DXRat96. The gene was mapped to the telomeric region of chromosome X and spanned the 27.8-Mb region. 169 candidate genes were located in this region, only the opsin 1, medium-wavelength sensitive (Opn1mw) gene had a relationship with vision.
2. Six primers were designed to amplify the six exons of Opn1mw gene. After sequencing, the presence of a G-to-T substitution in the invariant AG dinucleotide at the 3’splcing acceptor site of intron 4 was found. This substitution showed a specific relationship to the affected rat and could not be found in 50 wild-type SD rats and 156 inbred rats.
3. The results of reverse transcription-PCR (RT-PCR) and real-time quantitative PCR showed that the transcriptional expression of Opn1mw could be detected in wild rat but in mutant rat. The transcription was refrained. In immunohistochemistry, the marked cones with specific peanut agglutinin (PNA) could be found both in wild and mutant rats, but the protein of Opn1mw gene only were detected in wild rats, but not in mutant rats.
4. The results of hematoxylin and eosin (HE) staining demonstrated that there were no significant change in the structure and thickness of photoreceptors between the wild and mutant rats. The wholemount immunocytochemistry showed no obvious changes of numbers of cones in these two kinds of rats. Middle-wavelength opsin (M-opsin) could be expressed in wild rat, but not in mutant rat.
5. Under scotopic conditions, the amplitudes in wild and affected rats increased with the intensities increasing gradually and got the saturation state at last. Under photopic conditions, the same phenomenon could be seen in wild-type rats. No wave could be recorded with any intensity flashes and colorful lights in mutant rats.
Conclusion
A point mutation was proved to happen in the splicing acceptor site in intron 4 of Opn1mw gene. After mutation, the gene could not be spliced according to the usual way and no mRNA or protein could be synthesized. The visual pigment is consisted of opsin and 11-cis retinal. Due to the lack of M-opsin, the function of visual pathway was restrained and the cone response could not be recorded. The previous studies showed that mutations of Opn1mw gene caused deutan, so we inferred that the mutant rat would be a deuteranopia animal.
As far as we know, the mutations of Opn1mw gene that have been reported are all rearrangements or point mutations in exons. It is the first time that the mutation was found in splcing site. This would provide an important model for researching on the structure and function of Opn1mw gene. BCM is known as a rare X-linked disorder of color vision characterized by the absence of both red and green cone sensitivities, and have only rods and blue cones. Rat has only two kinds of cones, green cones and blue cones. So, this rat model would be also a blue cone monochromatism (BCM) animal model. If that, it would be the first animal model of BCM disease reported in the world. Optical microscope and wholemount immunocytochemistry showed no change could be found in structure, thickness of photoreceptors and the quantity of cones, which hinted that this rat could be an idealized animal model for gene therarpy for curing color anomalopia diseases in human.
方法
1.建立突变型大鼠与野生型BN大鼠的杂交品系,得到的F1代大鼠雌雄配对,获得F2代大鼠。通过建立遗传家系,明确遗传方式。后续研究主要以F2代大鼠为研究对象。
2.采用国际标准临床视网膜电图记录方法,进行表型鉴定。
3.应用序列标签位点(sequence tagged site,STS)和单倍体型(haplotype)分析方法,定位突变基因。
4.针对候选基因,采用基因克隆、测序等方法确定基因的突变方式,并观察突变基因是否与表型一致。
5.应用荧光实时定量PCR方法和免疫荧光方法,观察突变基因在模式大鼠中的mRNA表达水平以及蛋白表达水平。
6.采用苏木精-伊红染色及全视网膜铺片方法比较突变是否会影响不同年龄段的大鼠视网膜结构和锥体细胞的数量。
7.记录不同光强、频率及颜色光刺激下的视觉电生理反应,分析光感受器细胞的功能。
结果
1.将雌性突变型大鼠与雄性野生型BN大鼠杂交,子一代互交,收集到杂交二代大鼠共441只,所有大鼠均经过临床视觉电生理学检测确定其表型。根据NCBI网站中提供的序列标签位点作为分子标记物设计引物,对441只F2代大鼠分别进行聚合酶链式反应扩增。通过比较分子标记物在不同个体中的扩增产物,分析突变基因与分子标记物间的交换重组率,结果显示突变基因与DXRat21和DXRat96有连锁关系,并位于X染色体的末端端粒区27.8 Mb的区域内。此区域内共有169个候选基因,只有中波长视蛋白基因与视觉有关。
2.针对中波长视蛋白基因的的6个外显子分别设计引物、扩增并测序,结果显示第四内含子3’端剪接位点处保守的AG双碱基突变为AT双碱基。此突变在441只F2代大鼠中,除雄性正常表型大鼠外,其余个体中均存在;且在50只野生型SD大鼠和156种近交系大鼠中均未被发现。
3.反转录聚合酶链式反应和荧光实时定量聚合酶链式反应结果显示,野生型SD大鼠中可以检测到中波长视蛋白基因mRNA的表达,但是突变型大鼠中无法检测到该基因mRNA的表达,表明其转录过程受到抑制;免疫荧光结果显示,花生凝集素特异性标记的锥体细胞在野生型和突变型大鼠中均可以检测到,且正常表型的野生型SD大鼠中还可以检测到中波长视蛋白的表达,但突变型大鼠中无法检测到,中波长视蛋白在突变型大鼠中未被表达。
4.苏木精-伊红染色方法观察1个月、3个月、6个月、9个月和12个月野生型SD大鼠和突变型大鼠的视网膜结构,结果显示突变型大鼠的视网膜结构、光感受器细胞层的厚度与同龄的野生型SD大鼠相比均未发生明显变化;全视网膜铺片结果表明,同龄的突变型大鼠和野生型大鼠的锥体细胞的数量无明显差异。中波长视蛋白在野生型大鼠中表达,而突变型大鼠中无法检测到该蛋白的表达。
5.视觉电生理学记录结果显示,在暗适应条件下,随着刺激光强的增强,记录到的视网膜电图(electroretinogram,ERG)反应的波幅值在突变型和正常表型大鼠中均逐渐增大;明适应条件下,不同频率、光强的光刺激均显示出随着光强的增强,正常表型大鼠的波幅值逐渐增大,直至达到饱和状态,而突变型大鼠即使在最大光强刺激下也无法记录到任何波形。颜色光刺激后,在明适应环境中,突变型大鼠仍然无法记录到任何波形。
结论
中波长视蛋白基因中内含子和外显子间保守的剪接位点发生了点突变,导致该基因无法被正常的剪接,不能转录成成熟的mRNA,蛋白质的翻译过程也被终止。感光视蛋白与11-顺式视黄醛结合形成视锥细胞中的感光色素,由于基因突变致使蛋白无法合成,锥体细胞感光后的感光换能机制受到了抑制,所以在视觉电生理学检测中显示为无法检测到锥体细胞的功能,表现为明适应下无任何波形。中波长视蛋白基因的突变会导致绿色盲或者绿色弱疾病的产生,所以模式大鼠为绿色色觉异常大鼠。
迄今为止发现的中波长视蛋白基因的突变或是由于基因重组,或是由于外显子中的点突变引起,而该基因内含子的剪接位点突变属于首例,这为研究中波长视蛋白基因的功能、结构等方面提供了更多的机遇。蓝色锥体全色盲(blue cone monochromatism,BCM)疾病表现为感红(长波长)和感绿(中波长)锥体细胞功能丧失,而感蓝(短波长)锥体细胞和视杆细胞的功能正常。由于大鼠中只存在感绿(中波长)和感蓝(短波长)两种锥体细胞,所以,本模式大鼠可能为国际首例发现的自发突变BCM模式动物,因此具有重要的研究意义。同时,光学显微镜和全视网膜铺片实验结果显示模式大鼠视网膜结构无明显改变,光感受器细胞层的厚度及锥体细胞的数量并未发生明显变化,这为研究色觉异常机制、视网膜信号回路及基因治疗色觉异常疾病提供了理想的动物模型。
There are two signal circuits in retina: rod and cone neural circuits. Rod signal system is in charge of receiving, procession and transmission of visual information under scotopic condition, while cone signal system plays an important role under photopic condition. There is a complicated linkage in the rod and cone neural circuits. Color vision is one of the highest levels of vision. The color anomalopia is a very common inherited disease, which its prevalence is about 8% in western European men. It is presumably caused by mutations of genes in visual pathway. However, so far, the mechanisms of color anomalopia are not totally understood due to the lack of congenital color anomalopia animal model. The retinal cone dysfunction rat is a naturally occurring mutant model of X-linked cone dysfunction. In this model, the photopic electroretinogram (ERG) is abnormal, while there are no obvious histopathological changes. Here, we will screen and identify the causative gene and its function, aim to unserstand the mechanism of this disease.
Methods
1. Constructed a hybrid rat family by crossing the affected female rat with wild-type male BN rat. The F1 rats were intercrossed to get the F2 rats. The genetic mode was testified after the family was constructed.
2. Identified the phenotypes of the family members by ERG recordings.
3. Analyzed and located the mutant gene by using the sequence tagged site (STS) and haplotype method.
4. Cloned and sequenced the candidate gene. The mutant site was determined. Checked the relationship of the mutant gene and the phenotype.
5. Applied real-time quantitative PCR (RQ-PCR) and immunohistochemistry (IHC) methods to profile the expression levels of mRNA and protein in the retina of the affected rat.
6. Utilized hematoxylin and eosin (HE) staining and wholemount immunocytochemistry methods to compare the retinal structures and quantities of cones in affected rats with wild rats at the same ages.
7. Recorded electroretinograms with different intensities, frequencies and colors. With these results, functions of photoreceptors were analyzed.
Results
1. Cross family members were collected and 441 rats were obtained in F2 generation. Phenotypes were determined by using the ERG recordings. According to the sequence tagged sites provided by the NCBI website, 441 rats were amplified by using PCR method. After the amplified products were compared, the recombinant rates were analyzed. The affected gene showed a linkage relationship with the markers DXRat21 and DXRat96. The gene was mapped to the telomeric region of chromosome X and spanned the 27.8-Mb region. 169 candidate genes were located in this region, only the opsin 1, medium-wavelength sensitive (Opn1mw) gene had a relationship with vision.
2. Six primers were designed to amplify the six exons of Opn1mw gene. After sequencing, the presence of a G-to-T substitution in the invariant AG dinucleotide at the 3’splcing acceptor site of intron 4 was found. This substitution showed a specific relationship to the affected rat and could not be found in 50 wild-type SD rats and 156 inbred rats.
3. The results of reverse transcription-PCR (RT-PCR) and real-time quantitative PCR showed that the transcriptional expression of Opn1mw could be detected in wild rat but in mutant rat. The transcription was refrained. In immunohistochemistry, the marked cones with specific peanut agglutinin (PNA) could be found both in wild and mutant rats, but the protein of Opn1mw gene only were detected in wild rats, but not in mutant rats.
4. The results of hematoxylin and eosin (HE) staining demonstrated that there were no significant change in the structure and thickness of photoreceptors between the wild and mutant rats. The wholemount immunocytochemistry showed no obvious changes of numbers of cones in these two kinds of rats. Middle-wavelength opsin (M-opsin) could be expressed in wild rat, but not in mutant rat.
5. Under scotopic conditions, the amplitudes in wild and affected rats increased with the intensities increasing gradually and got the saturation state at last. Under photopic conditions, the same phenomenon could be seen in wild-type rats. No wave could be recorded with any intensity flashes and colorful lights in mutant rats.
Conclusion
A point mutation was proved to happen in the splicing acceptor site in intron 4 of Opn1mw gene. After mutation, the gene could not be spliced according to the usual way and no mRNA or protein could be synthesized. The visual pigment is consisted of opsin and 11-cis retinal. Due to the lack of M-opsin, the function of visual pathway was restrained and the cone response could not be recorded. The previous studies showed that mutations of Opn1mw gene caused deutan, so we inferred that the mutant rat would be a deuteranopia animal.
As far as we know, the mutations of Opn1mw gene that have been reported are all rearrangements or point mutations in exons. It is the first time that the mutation was found in splcing site. This would provide an important model for researching on the structure and function of Opn1mw gene. BCM is known as a rare X-linked disorder of color vision characterized by the absence of both red and green cone sensitivities, and have only rods and blue cones. Rat has only two kinds of cones, green cones and blue cones. So, this rat model would be also a blue cone monochromatism (BCM) animal model. If that, it would be the first animal model of BCM disease reported in the world. Optical microscope and wholemount immunocytochemistry showed no change could be found in structure, thickness of photoreceptors and the quantity of cones, which hinted that this rat could be an idealized animal model for gene therarpy for curing color anomalopia diseases in human.
引文
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