| Auditory neuropathy(AN)is a kind of auditory dysfunction disorder characterized by impaired speech comprehension.The relevant understanding of its clinical manifestations and diagnosis has been deepened,but the lesions and specific pathological mechanisms are still the hot spots and difficulties in domestic and foreign research,and the intervention is extremely difficult.With the continuous development of molecular diagnostic technology,it has been recognized that AN is genetically related,and a variety of AN-related genes have been found which AIFM1(apoptosis-inducing factor,mitochondria-associated,1)is one of them.Our team firstly found that AIFM1 gene is related to AN.More and more patients with late-onset AN have detected variations in the AIFM1 gene,and it is of great significance to carry out genotypic correlation analysis and follow-up analysis on these patients.In addition,there is no animal model of Aimf1 AN related missense mutation at present,and the preliminary study on the construction of specific animal models will provide a basis for further studies on pathological changes and pathogenesis,as well as a reference for the construction of animal models of other mutation types.This study is divided into two parts:the first part is the genotype and phenotype analysis of patients with AN related to the variations of AIFM1 gene;The second part is a preliminary study on the construction of specific mutation of Aifm1 gene in mice.Part1:Genotype and phenotype analysis of patients with auditory neuropathy related to the variations of AIFM1 geneObjective To analyze the genotype and phenotype relationship of AIFM1 gene variations,and the follow-up results,in order to gain a deeper understanding of the characteristics of AIFM1 gene-positive AN patients from clinical characteristics,genetic characteristics and natural course changes,and to provide a reference for clinical diagnosis and thinking of location-based type.Methods 1.Analyzed the genotypes and phenotypes of 50 AN patients from 36 families with AIFM1 variations,including 30 patients from 16 reported families.The basic characteristics and the results of audiological examination were analyzed,such as pure tone audiometry(PTA),speech recognition score(SRS)and various auditory evoked potential test;2.Summarized the mutation spectrum of AIFM1 gene,analyzed the location of the variations,and summarized the proportion of each mutation;3.Divided the follow-up patients into two groups by less than 10 years and greater than 10 years,and analyzed the changes of PTA;4.Compared the hearing differences between male and female with the same mutation and the genetic characteristics of a special family were analyzed.Results 1.This study analyzed 50 AN patients with AIFM1 mutations were analyzed in this study,including 45 male and 5 female.They all presented delayed-onset AN.The degree of hearing loss varies,mainly moderate to moderately severe.The age of onset was 13.4±3.9 years old and the age of first visit was 23.2±8.4.All of the patients had no neonatal risk factors.Accompanying symptoms included tinnitus,numbness of limbs,dizziness and unsteady gait.Tinnitus was the most common symptom(74%).VEMP test was performed in 15 patients(30 ears),and 19 ears were abnormality(63.3%).ECochG was performed in 27 patients(51 ears)at the first diagnosis,and 48(94.1%)ears were elicited waves while 3 ears were not.The-SP waves were found in all of the 48 ears,and AP was extracted in 40 ears(78.4%).All of the ears showed-SP/AP>0.4.Cerebral magnetic resonance imaging(MRI)were carried out in 14 patients,7 of whom presented with bilateral cochlear nerve hypoplasia.2.In the 20 new patient,7 novel and 3 reported variations were found;the novel variations are all missense variations,including c.547A>T(p.Thr183Ser),c.881G>A(p.Arg294Gln),c.890A>T(p.Lys297Ile),c.912C>G(p.Ile304Met),c.997C>T(p.Leu333Phe),c.1394C>T(p.Ala465Val)and c.1678T>C(p.Tyr560His).In all of the 50 AN patients,there are 18 variation in total,with 6 in NADH,9 in FAD and 3 in C-terminal region.Among the 36 families,the most common variant was p.Leu344Phe.3.For the patients with less than 10-year follow-up period(14 ears),there was no significant difference in all frequencies.But for the more than 10 years patients(18 ears),the hearing thresholds of low-frequency and high-frequency significantly increased(p<0.01).4.All of the 5 female showed p.Leu344Phe.One family was considered for X-linked dominant inheritance pattern.Conclusion AIFM1 gene is the mainly related gene in Chinese patients with late-onset AN,and the most common variation is p.Leu344Phe;the AIFM1 gene mutation-positive AN patients' hearing gradually becomes worse;in addition,the AIFM1 gene variation may lead to an X-linked dominant inheritance pattern in AN.Part 2:Preliminary study on the construction of specific mutation of Aifml gene in miceObjective To build Aifm1-related AN-specific mutation mouse model and evaluate the model,to determine whether the animals can be used for further exploration of pathological mechanisms.Methods 1.According to the clinical case and protein structure prediction,we select a specific site p.Thr260Ala(corresponding to p.Thr259Ala in mice)to construct animal models,design gRNAs,construct mutation targeting vectors,and use microinjection technology to inject modified gene fragments into small Mouse fertilized eggs,transplanted surrogate mother mice,and finally got p.T259A mutant mice,this step was completed with National Institute of Biological Sciences of Beijing;2.Primer design,genotyping and breeding of the mice obtained;3.Select mice around 14 days of gestation,take mouse embryonic fibroblasts(MEF)for genotyping,culture MEF cells of different genotypes,and analyze AIF expression levels of different genotype cells by qPCR and Western Blot;4.The expression level of AIF in cochlea of wild-type mice and Aifm1 positive mice was analyzed;Click ABR and TB-ABR tests were initially performed on 15 2-month-old positive mice and 17 wild-type mice,to observe the waveform and threshold of the two groups.Results 1.We obtained Aifm1 p.Thr259Ala mouse model,and have different genotype mice.2.Compared with the wild type,there was no significant difference in the transcription level of Aifm1 in the positive MEF cells,and the protein expression level was lower than that of the wild type.The cochlea observation was consistent with the results.3.There was no significant difference in hearing frequency between the positive mice and the wild mice(p>0.05).Conclusion Different genotype mice with Aifm1 specific point mutation(p.Thr259Ala)have been identified,but can not be ensured to conform to the delayed-onset AN phenotype of clinical patients,so further evaluation is needed. |
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