Identification Of Pathogenic Genes In Four Patients With Usher Syndrome And A Preliminary Phenotype Observation In Ush2a Knockout Mice

Usher syndrome（USH）is an autosomal recessive disorder with congenital sensorineural deafness and progressive retinitis pigmentosa（RP）.The prevalence of USH in different populations ranges from 3.5 to 6.2 per 100000,and it is the most common cause of deafness and blindness in adult.According to the severity of hearing impairment,the onset age of retinitis pigmentosa and vestibular dysfunction,Usher syndrome is classified into three types:type I（USH1）,type II（USH2）and type III（USH3）.USH1:congenital severe-to-profound sensorineural hearing loss,absent vestibular responses,and prepubertal onset of retinitis pigmentosa（usually before 18 years of age）;USH2:congenital moderate-to-severe non-progressive sensorineural hearing loss,normal vestibular responses（in most cases）,and later（postpuberal）onset of retinitis pigmentosa（usually about 20 years of age）;USH3:postverbal hearing loss,progressive exacerbation of hearing impairment,normal vestibular function（in most cases）,varying age and severity of retinitis pigmentosa.Currently,14causative genes of Usher syndrome have been identified,of which MYO7A,USH2A and CLRN1 are the most common causative genes for USH1,USH2 and USH3,respectively.The high heterogeneity of clinical characteristics and genetic inheritance of Usher syndrome makes it difficult to diagnose.The pathogenesis of Usher syndrome is still largely unknown and the treatment of retinitis pigmentosa in Usher syndrome is still unavailable.In this study,the pathogenic genes in four Chinese families with Usher Syndrome（family USH-001,002,003,004）were screened by targeted sequence capture array technique and confirmed by Sanger sequencing.We also made a preliminary phenotype observation in Ush2a knockout mice,laying a foundation for exploring the role of USH2A in the pathogenesis of Usher syndrome.The genetic pattern of the four families is autosomal recessive inheritance.All Usher syndrome patients presented with binocular night blindness and hearing loss.Visual field tests revealed concentric contraction of visual field（tunnel vision）in both eyes in all patients.The electroretinogram（ERG）showed extinguished rod and cone responses in both eyes in all patients.Fundal photographs from a proband（USH-001 family）showed tiny pigments in the periphery retina.The fundus showed none of proliferative pigmentation in a proband （USH-002 family）.Examination of the dilated binocular fundus from a proband（USH-003family）demonstrated typical pigmentary degeneration with multiple bone spicule pigmentations in over the entire retina.The fundoscopic finding of both eyes from a proband（USH-004 family）showed bone spicule pigmentation variation in the periphery retina.The proband of USH-001 family,USH-002 family and USH-003 family carry mutations in USH2A,respectly.They were diagnosed as Usher syndrome typeⅡ.We found that the proband of USH-001 family carries three point mutations in USH2A,including c.2187C>A（p.C729X）,c.997T>C（p.S333P）and c.538T>C（p.S180P）.The c.2187C>A（p.C729X）and c.997T>C（p.S333P）mutation inherited from her father,the c.538T>C（p.S180P）mutation inherited from her mother.The compound heterozygous mutation has not been reported.The proband of USH-002 family carries two point mutations in USH2A,including c.8559-2A>G（splicing）and a deletion between exon 28 to 33.The c.8559-2A>G（splicing）mutation inherited from his father.The location of both ends of the deletion exons in the patient is not known,which can not to determine the deletion between exon 28 to 33 was inherited from his mother.The mutation is being confirmed by whole-genome sequencing（WGS）.The proband of USH-003 family carries three point mutations in USH2A,including c.3996C>A（p.Y1332X）,c.2187C>A（p.C729X）and c.997T>C（p.S333P）.The c.3996C>A（p.Y1332X）mutation inherited from her mother.It is unclear whether the c.2187C>A（p.C729X）and c.997T>C（p.S333P）mutation inherited from her father or not because of lacking blood sample from her father.The compound heterozygous mutation has not been reported.The proband of USH-004 family carries two point mutations in MYO7A,including c.1003+1G>A（splicing）and c.5957＿5958del（p.G1987Lfs*50）.The c.5957＿5958del（p.G1987Lfs*50）mutation inherited from his father,the c.1003+1G>A（splicing）mutation inherited from his mother.The proband was diagnosed as Usher syndrome typeⅠ.The compound heterozygous mutation has not been reported.We found two new compound heterozygous mutations in USH2A and one new compound heterozygous mutations in MYO7A which were new pathogenic causes of Usher syndrome.In this study,the Ush2a knockout mice were bred and a preliminary phenotype analysis was performed.At the age of 6 months,visual behavioral pattern in WT,Ush2a^+/-and Ush2a^-/-mice was measured by the black and white box tests,respectively.However,no statistically significant differences in the time of dark room were found（P>0.05）.ERG was performed on these mice at 3 months and 6 months of age,respectively.No significant differences in the amplitudes of a-and b-waves during light or dark adaptation conditions were found in all groups（P>0.05）.The results indicated that no significant changes in retina function in Ush2a^-/-mice before 6 months of age.At the age of 6 months,auditory brainstem response（ABR）tests were performed on WT,Ush2a^+/-and Ush2a^-/-mice.The ABR thresholds of Ush2a^-/-mice on the stimulation of short sound（click）and tone burst（4 kHz,8kHz,12 kHz and 16kHz）are significantly higher than WT and Ush2a^+/-mice.The results suggested that the hearing function of Ush2a^-/-mice was damaged.These results indicated the retinal impairment in Ush2a^-/-mice may occur later than hearing impairmet and develop slowly.In this study,we found new compound heterozygous mutations in USH2A and MYO7A which expand mutation spectrums of Usher syndrome in Chinese people.Our findings provide evidence for genetic diagnosis,genetic counseling and gene therapy of Usher syndrome.Meanwhile,the Ush2a knockout mice were bred and a preliminary phenotype observation was performed in this study.Our results indicate the hearing function of Ush2a^-/-mice was damaged at the age of 6 months and the retinal impairment of Ush2a^-/-mice was not observed at the age of 3 months and 6 months.The Ush2a knockout mice provide a basis for the further exploration of pathogenesis of USH2A in Usher syndrome.