Diagnosis Of Deafness Dystonia Optic Neuropathy(DDON) Syndrome And Establishment Of A Mouse Disease Model

BackgroundDeafness-Dystonia-Optic Neuropathy?DDON?Syndrome,also known as Mohr-Tranebjaerg Syndrome?MTS;MIM 304700?,is a rare progressive X-linked recessive genetic disorder.There were only 91 cases of DDON patients in 37 families reported worldwide.The main clinical features are acquired post-natal aggravation of sensorineural hearing loss,progressively increased dystonia,and ataxia,most in male.The causitive gene of DDON syndrome is DDP1?Deafness/Dystonia Peptide 1?,which is also named as TIMM8A?Translocase of Inner Mitochondrial Membrane 8A?.It is located on the 2 arms,2 bands and 1 sub-band of the long arm of the X chromosome,Xq22.1.The DDP1 protein encoded by the DDP1/T1MM8A gene contains 97 amino acid residues and is predicted to have a molecular weight of approximately 11 kDa.It is a highly conserved mitochondrial protein which abundantly expressed in the human brain.Due to the lack of corresponding animal models,the molecular mechanism study mainly performed with cell and yeast system.Although Tim8 and Timl3 are not essential in yeast,the mutation or deletion of Tim8 will affect Tim23 transport ability,impaired cell membrane potential and loss of temperature-sensitive growth characteristics.Similarly,human DDP1 locates in the intermitochondrial membrane space?IMS?and forms a 70 kDa complex with Tim 13,which regulating thetranslocation of Tim23 precursor protein and the biosynthesis of mitochondrial inner membrane.However,the pathological mechanism of DDP1 is still unclear.In this study,we indentified a clinically suspected DDON patient with a novel hemizygous likely pathogoical variant of the DDP1 gene?NM₀04085.3?:c.82C>T?p.Q28*?.To further investigate the role of DDP1 on the mechanism of the disease,we used CRISPR/Cas9 technology to construct a DDP1?p.123fs49X?gene knock-in mouse model that mimics DDP1?p.Q28X?,and conducted behavioral studies on this mouse.We observed the phenotype differences between this mouse model and DDON syndrome.Further study of the pathogical mechanism of DDON syndrome and possible targeted therapy will be continued.Purposes1.Clinical diagnosis and genetic analysis of DDON patient.2.Construction of a mouse line bearing DDP1?p.123fs49X?variant which mimics DDP1?p.Q28X?variant.3.To observe the expression and distribution of DDP1 in different tissues of DDP1123fs49X/y mice.4.To observe the phenotype differences between DDP1?p.I23fs49X?gene knock-in mice and DDON syndrome through behavioral tests.5.Assesment of the morphological changes of DDP1123fs49X/y mice.Methods1.To clinically diagnose and take genetic analysis of a patient with suspected DDON syndrome.Summarize the characteristics of clinical manifestiation of the patient.To identify the causitive gene of the disease by targeted capture and high-throughput next generation sequencing,which underwent family segregation and functional prediction of variant.2.DDP1?p.123fs49X?gene knock-in mice were constructed using CRISPR/Cas9 gene editing technology and was verified by PCR sequencing.3.Detection of DDP1 expression in 24 weeks old DDP1+/y mice and DDP1123fs49X/y mice by real-time quantitative PCR.4.Behavioral testingExperimental animals were divided into four groups:male wild-type DDP1+/y?WT-M?,female wild-type DDP1+/+?WT-F?,DDP1123fs49X/y?MUT-M?and DDP123fs49X/I23fs49X?MUT-F?.The behavioral experiments are as follows:4.1 Weighing four groups of mice and compared.4.2 Auditory brainstem response test to detect the auditory function of 4-5 weeks-old mice.4.3 Grip test to detect muscle strength of 9-10 weeks-old mice.4.4 Rotarod test to detect the motor function of 9-10 weeks old and 24-week-old mice.4.5 Balance beam test to detect movement coordination function of 9-10 weeks-old and 24-week-old mice.4.6 Hot plate test to detect the pain perception ability in 9-10 weeks-old and 24-week-old mice.5.The morphological changes of DDP1123ts49X/y mice were observed by MRI of mouse brain and Nissl staining of brain tissues.6.Statistical analysisAll data in this study were analyzed using SPSS20.0 and GraphPad Prism 7 statistical analysis software.Independent sample t-test and Wilcoxon-Mann-Whitney rank sum test were used for comparison between the two groups.All the experimental data were expressed by the mean±standard error of mean?X ± SEM?.P<0.05 was considered significant.Results:1.According to the patient's manefistation and genetic analysis,the patient with DDON was diagnosed.2.The genetic test showed that the patient had a novel hemizygous mutation of DDP1 gene?NM₀04085.3,c.82C>T,p.Q28*?.3.Genomic sequencing of the F0 mice confirmed the gene variant mice carrying the DDP1?p.I23fs49X?gene,indicating successful generation of a DDP1 variant mouse model with CRISPR/Cas9 technology.4.QPCR assessment indicated that the expression of DDP1 increased in the DDP 123fs49X/y mice compared to DDP1+/y mice.5.Up to now,after 1 year of observation,no significant changes in the survival rate was obsereved in the mutant mice,but the body weight decreased significantly since 3 weeks.6.Behavioral testing:6.1 Auditory brainstem response tests revealed that the DDP1?p.123fs49X?mice had impaired hearing.6.2 In grip test,the muscle strength of the DDP1?p.123fs49X?mice was significantly reduced.6.3 Rotarod test showed there was no change in the motion of DDP1?p.123fs49X?mice.6.4 Balance beam test showed that 24-week-old DDPl?p.123fs49X?mice had reduced coordination function.6.5 Hot plate test showed that the 9-10 weeks old DDP1123fs49X/y mice had impaired pain perception.7.Compared with the DDPl+/y mice,the organs size of DDP1123fs49x/y mice at 24 weeks of age was reduced.There was no brain parenchymal changes,such as atrophy,and the brain neuronal structure was unchanged.Conclusion1.Clinical diagnosis of a patient with DDON2.The new mutation p.Q28X of the DDP1 gene is the cause of the disease in this DDON patient3.DDP1?p.I23fs49X?mice partial repeated the phenotype of DDON syndrome4.DDP1?p.I23fs49X?variant resulted in reduced tissue weight in mice but do not affect the development of neurons in the brain of 24-week old DDP1123fs49X/y mice.