The Phenotype And Genotype Of Spontaneous Hereditary Retinitis Pigmentosa/deafness Mouse Models

Retinitis pigmentosa（RP） is a common form of inherited retinal degeneration（rd） characterized by a gradual degeneration of rod and cone photoreceptors resulting in night blindness, loss of mid-peripheral visual field and eventually central vision loss. The prevalence of RP is 1/4000 worldwide. RP is a group of highly heterogeneous diseases with variable phenotypes involving a multiplicity of genes and mutations, with at least 100 genes and more than 4000 mutations related to it. RP is usually confined to eyes; some patients may also develop lesions in other organs, which is defined as syndromic RP, among which, the most prevalent form is Usher syndrome（USH）. USH is an autosomal recessive disorder characterized by combined RP and deafness, and is the most common cause of hereditary deafness-blindness. Similar to RP, USH is also genetically and clinically heterogeneous. USH can be divided into three clinical types according to the onsite time and the degree of deafness, and the occurrence of vestibular dysfunction. Type II USH（USH2） is the most prevalent form of USH. Patients with USH2 suffer from moderate and non-progressive hearing loss and normal vestibular dysfunction.For RP and USH, the clinical symptoms are complex, genes and proteins involved are not fully known, and few therapies can rescue or reverse photoreceptor loss. To understand the etiology of inherited retinal disease, animal models are excellent for mimicking diseases, among which, mouse models are most widely used. To date, there exits many RP mouse models, numerous studies have been conducted using these mouse models and have made some progress. However, most USH mice do not exhibit detectable ocular phenotype, which brings obstacles to the study of disease mechanisms.Through visual electrophysiology screening, our laboratory discovered several strains of mice with spontaneous RP derived from Kunming mice. In a preliminary study, one strain of mice showed lower levels of Pde6 b and Ush2 a expression, which we designated as KM^ush/ush and has been inbred to 27 generations with a stable phenotype. Another strain of mice only showed lower levels of Pde6 b expression, which we designated as rd/KM and has been inbred to 33 generations with a stable phenotype. In the meantime, we crossed rd/KM mice with C57BL/6J mice and generated a congenic-inbred strain, designated as rd/B6, which has inbred to 25 generations. After studied the phenotype and genotype of KM^ush/ush mice in the present study, we crossed the KM^ush/ush mice with CBA/Ca J, a strain with ‘‘gold standard’’ normal hearing, in order to study the genetic feature of pathogenic genes and further gene tests. In this study, we examined the development of phenotypes as well as gene sequencing of KM^ush/ush, rd/KM and rd/B6 mice, and analyze the pedigree of KM^ush/ush and CBA/Ca J mice.Objective: To examine the visual and auditory functions,morphology changes in development, and gene mutations of KM^ush/ush, rd/KM and rd/B6 mice, as well as the pedigree analysis of hybrids of KM^ush/ush and CBA/Ca J mice.Methods: Part I: The characterisation of phenotype and gene sequencing in KM^ush/ush mice.Randomly selected KM^ush/ush mice from P7 to P56, age-matched wild type Kunming mice as controls. Electroretinogram（ERG） and auditory brainstem response（ABR） were conducted at P14, P21, and P56. The amplitudes of scotopic 0.01 cd.s.m-2, 3.0 cd.s.m-2, 3.0 cd.s.m-2 OPs ERG, and photopic 3.0 cd.s.m-2, 3.0 cd.s.m-2 Flicker ERG and the thresholds of ABR were recorded. Fundus photography was taken at P21, paraffin section and H E staining was performed at P7, P14, P21 and P56. The expressions of Pde6 b and Ush2 a were quantitatively determined at P7, P14, P21, P28 and P56. Exon sequencing of Pde6 b and Ush2 a was carried out at P56.Part II: The pedigree analysis of hybrids of KM^ush/ush and CBA/Ca J mice.The wild type CBA/Ca J mice were detected with ERG and ABR before hybridized with KM^ush/ush mice. Randomly chose two female CBA/Ca J mice to cross with a male KM^ush/ush mouse. The remaining CBA/Ca J mice were kept to reproduce. The F1 hybrids were tested by ERG and ABR at P30, then, they continued to breed by sib mating. The F2 hybrids were also tested by ERG and ABR at P30. The amplitudes of scotopic 3.0 cd.s.m-2 ERG and the thresholds of ABR were recorded.Part III: The phenotype and gene expression in F3 hybrids of KM^ush/ush and CBA/Ca J mice.The F2 hybrids with the same positive auditory/visual changes screened out in Part II were to breed through sib mating separately. The F3 hybrids were also tested by ERG, ABR and optical coherence tomography（OCT） at P30. The amplitudes of scotopic 3.0 cd.s.m-2 ERG, ABR thresholds and the thickness of outer nuclear layer（ONL） were recorded. In addition, the expressions of Pde6 b and Ush2 a in F3 hybrids were quantitatively determined at P30.Part IV: The function and morphology changes and gene sequencing in KM/rd and B6/rd mice.Randomly chose rd/KM and rd/B6 mice from P14 to P56, age-matched wild type Kunming and C57/BL6 J mice as controls separately. ERG and paraffin section and H E staining was performed at P14 and P21. The amplitudes of scotopic 3.0 cd.s.m-2 ERG were recorded. Besides, the fundus photography was taken at P21, the exon sequencing of Pde6 b was performed at P56.Results: 1. The characterisation of phenotype and gene sequencing in KM^ush/ush mice: The ERG amplitudes of KM^ush/ush mice were declined significantly since P14, and no obvious waveforms could be recognized. The ABR thresholds elevated significantly compared with controls since P14. Fundus photography at P21 mainly showed the attenuation of retinas and retinal vessels in KM^ush/ush mice. The structure of retinas and the thickness of ONL in KM^ush/ush mice were comparable to those of age-matched controls at P7. While the number of ONL cells reduced sharply between P14 and P21. The levels of m RNA expression of Pde6 b and Ush2 a in KM^ush/ush mice were lower compared with controls since P7. The exon sequencing showed that Pde6 b had five point mutations, of which the nonsense mutation at exon 7 generated a termination codon that lead to premature transcription termination; Ush2 a had twenty-five point mutations, of which eight were synonymous mutations, seventeen were missense mutations.2. The pedigree analysis of hybrids of KM^ush/ush and CBA/Ca J mice: The F1 hybrids, denoted as CBAKMush/+ mice had normal ERGs and ABR thresholds. 113 F2 hybrids were tested with ERG and ABR at P30. The phenotypes of F2 hybrids segregated into four types, namely, declined ERG amplitudes（ERG（+））, elevated ABR thresholds（ABR（+））, both declined ERG amplitudes and elevated ABR thresholds（ABR/ERG（+）） and normal ERGs and ABR thresholds（ABR/ERG（-））. These four types of phenotypes were homogeneously distributed in gender and coat color, which also indicted that the mutations of Pde6 b and Ush2 a were in autosomal recessive inheritance.3. The phenotype and gene expression in F3 hybrids of KM^ush/ush and CBA/Ca J mice: ERG and ABR tests showed that the phenotypes of F3 hybrids were in accordance with their parental generation. The OCT showed that the ONL of F3 ERG（+） and F3 ABR/ERG（+） was thickened, and the thickness ONL of F3 ABR（+） was comparable to those of controls. The F3 hybrids with declined ERG amplitudes had low expression of Pde6 b and F3 hybrids with elevated ABR thresholds had low expression of Ush2 a.4. The function and morphology changes and gene sequencing in KM/rd and B6/rd mice: The ERG amplitudes of rd/KM and rd/B6 mice declined significantly since P14. Fundus photography showed attenuated retinas and retinal vessels in rd/KM and rd/B6 mice, and patchy depigmentation in rd/B6 mice. The thickness of retinas especially ONL declined remarkably in KM/rd and B6/rd mice at P14, and ONL cells were barely seen at P21. Exon sequencing of Pde6 b indicated that rd/KM and rd/B6 mice had the same mutations, five point mutations in total, of which the nonsense mutation at exon 7 generated a termination codon that lead to premature transcription termination.Conclusions: 1. The decline of visual and auditory functions and retinal morphology changes in KM^ush/ush mice were due to the mutations of Pde6 b and Ush2a; thus, KM^ush/ush mice might be a strain of spontaneous hereditary RP/USH mouse with digenic mutation（Pde6b and Ush2a）.2. KM^ush/ush mice were in autosomal recessive inheritance. Pde6 b and Ush2 a segregated and independently assorted in the process of hybridization. The F2 hybrids had four types of phenotypes and the phenotypes of F3 hybrids were in accordance with their parental generation. The F3 ERG（+） mice might have monogenic mutation for Pde6 b. The F3 ABR（+） mice might have monogenic mutation for Ush2 a, and whether it occurs retinal degeneration need further observations.3. The rd/KM and rd/B6 mice were in different genetic background, but shared the similar phenotypes and the same mutations. They were both homozygous for a ninsense mutation located at the exon 7-49 of Pde6 b that lead to premature transcription termination.