Screen And Identification Of The Causative Gene In The Retinal Cone Dysfunction Rat

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. Methods1. 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.Results1. 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.ConclusionA 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.