Clinical Research In Chinese Patients With Spinocerebellar Ataxia Type 3 And Generation Of A New BAC-SCA3 Mice Model

Spinocerebellar ataxias(SCAs)is a group of neurodegenerative diseases,which are autosomal dominant,with highly genetic and clinical heterogeneity.Spinocerebellar ataxia type 3(SCA3)is the most prevalent SCA in China,contributing to 48-73% of SCAs.SCA3 is a highly disabling disease which is caused by ATXN3 gene.There are no effective treatments,and the mechanism is still elusive.Patients progressively worsen after the onset and eventually die,which seriously affects the quality of patients’ life and survival time.Thus,clinical research is beneficial to understand deeply about Chinese SCA3 disease and provide guides to symptomatic treatments and prognosis.Generation a new transgenic mice model is helpful to uncover the mechanism of SCA3 and find the therapeutic targets in the future.Part One Clinical research in Chinese patients with spinocerebellar ataxia type 3Background and purpose: Due to high prevalence of SCA3 in China,a more in-depth clinical study of Chinese SCA3 patients is well-merited.Method: 722 SCA3 patients and 141 premanifest individuals from 667 families were enrolled.The patients were clinically diagnosed with SCA3 and genetically confirmed.The premanifest individuals were genetically diagnosed with SCA3.We analyzed genotype-phenotype correlation,intergenerational instability and phenotype-associated factors of the patients and premanifest individuals with SCA3.Results: Most(92.1%)patients initially had gait ataxia.Sleep disorder was the most common non-ataxia symptom in SCA3 patients(55.6%).Nystagmus happened most frequently(14.2%)in premanifest individuals,followed by sleep disorder(7.8%).SCA3 individuals had shorter normal CAG repeats(nor CAGs)than healthy controls(Mann-Whitney,p<0.0001).Parturition triggered the onset of gait ataxia in 10 female patients with longer expanded CAG repeats(exp CAGs)(77.4 ± 1.4 repeats)and earlier age at onset(AAO)(26.7 ± 4.3 years).A quadratic equation best explained the relationship between the logarithmically transformed AAO(LOA)and exp CAG(r2=0.664,p<0.001).During intergenerational transmissions of SCA3,the exp CAG was larger and anticipation was earlier in offspring than those in parents.Additionally,paternal transmission to son showed more unstable than the others.However,the anticipation was not related to the intergenerational instability.Conclusion: Parturition and long nor CAG are new phenotype-associated factors.The transmissions are more unstable during paternal transmissions to sons.Part Two Generation of a new BAC-SCA3 mice modelBackground and purpose: SCA3 is a highly disabling disease which is caused by ATXN3 gene.There are no effective treatments,and the mechanism is still elusive.The existed SCA3 mice model have some limitations and cannot simulate SCA3 disease very well.Therefore,generation a new BAC-SCA3 transgenic mice model is necessary,which will be helpful to uncover the mechanism of SCA3 and find the therapeutic targets in the future.Method: Bacteria artificial chromosome(BAC)was used to generate the new SCA3 transgenic mice model.The chosen SCA3 BAC(RP11-779H17)covered the full-length wildtype(WT)ATXN3 gene as well as the other two non-target genes which were NDUFB1 and CPSF2.The shuttle vector p LD 53 was used to modify the SCA3 BAC through homologous recombination.We aimed to delete the two non-target genes and insert long pure CAG repeats into WT ATXN3.Results: To modify the SCA3 BAC,the exon 1 in NDUFB1 and the exon 6 in CPSF2 were deleted meanwhile 120 pure CAG repeats were inserted into exon 10 at ATXN3,successively.After microinjection to Fv B/NJ mice fertilized egg,there were four positive founders(BAC-SCA3 A-D)and two of four were SCA3 germline transmitted.Therefore,we got BAC-SCA3 line C and D.Line C harbored three different length of CAG repeats,which were 46,75 and 110 repeats.Line D had two CAG repeats of 46 and 110 repeats.We found that the founder A suffered severe cerebellum atrophy,weight loss and movement disorder at 7 months.Robust ATXN3 aggregates were observed in the cerebellum,brain stem,hippocampus of BAC-SCA3 founder A by immunofluorescence.Additionally,the 2-month and 10-month BAC-SCA3 mice manifested significant weight loss comparing to WT mice with the same age.The rotarod showed that the latency on rotarod was shorter in 2-month,6-month and10-month BAC-SCA3 F1 mice than in WT mice with the same age.Conclusion: A new BAC-SCA3 transgenic mouse model was successfully established.