A spontaneous Kunming mouse for Usher syndrome caused by homozygous mutations of Ush2a and Pde6b

Abstract

Purpose : Our lab discovered a spontaneously hereditary deafness and retinitis pigmentosa mouse origened from Kunming mouse and designated it KM^ush/ush temporarily, which has been inbred to 26 generations. To test if the ocular and auditory defeats were due to homozygous for separate mutations and generate congenic inbred strain of CBA/CaJ background, we crossed it with CBA/CaJ strain. This study was to identify the phenotype and genotype of KM^ush/ush mouse and screen the F1 and F2 hybrids of KM^ush/ush and CBA/CaJ strain.

Methods : We examined the visual and auditory function of KM^ush/ush and age-matched WT controls with Electroretinogram (ERG) and auditory brainstem response (ABR) at postnatal day (P) 14,21 and 56. Retinal sections and hematoxylin and eosin staining were performed at P7,14,21 and 56. The Pde6b and Ush2a mRNA was evaluated by quantitative real time-PCR at P7, 14, 21, 28 and 56. The coding region of Pde6b and Ush2a was sequenced at P56. F1 and F2 hybrids were screened with Electroretinogram (ERG) and auditory brainstem response (ABR) at P30.

Results : ERG tests showed low amplitudes and no obvious waveforms since P14 (n=6, p=0.0019) compared with controls. ABR thresholds were higher than those of controls since P14 (n=10, p<0.0001). The outer nuclear layer (ONL) had no differences between KM^ush/ush and controls at P7 (n=6, p=0.33). While the ONL of KM^ush/ush mouse attenuated significantly at P14 (n=6, p<0.0001) and was barely seen P21 (n=6, p<0.0001). The expression quantity of Pde6b (n=3, p=0.001) and Ush2a (n=3, p=0.008) mRNA declined significantly compared with controls since P7. DNA sequencing showed that KM^ush/ush mouse carried the rd1 allele, and Ush2a had 25 mutations in 13 exons. The F1 hybrids were normal in ERG and ABR tests. The phenotypes of F2 hybrids segregated into four types, of which 32 had elevated ABR thresholds, 9 showed declined ERG amplitudes, 9 presented both declined ERG amplitudes and elevated ABR thresholds, and 63 were normal in ERG and ABR tests.

Conclusions : Our study showed that changes of ocular and auditory phenotypes of KM^ush/ush mouse exhibited a mixture of rd1 mouse and Usher II patients. The genotype analysis demonstrated that KM^ush/ush mouse was homozygous for mutations at Pde6b and Ush2a loci. These results lay the foundation for our future jobs to generate Ush2a mouse of CBA/CaJ background and further study the Ush2a phenotype and genotype.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.