| Objective:Large vestibular aqueduct syndrome(LVAS) or enlarged vestibular aqueduct syndrome(EVAS) is an autosomal recessive nonsyndromic hearing loss which is characterized as enlarged vestibular aqueduct and Sensorineural hearing loss(SNHL). To study its clinical features,genetic mutation and gene function systematically, We analyzed the serial characters of clinic,audiometry and imageology of 19 EVAS patients, carried gene mutation detections on SLC26A4 and FOXI1 to find specific and frequent mutation map, constructed mutant and transfected HEK293T cell to observe the influence of gene mutation on Pendrin' s expression to study the pathopoiesis mechanism of SLC26A4 gene mutation.Methods:The audiometry included pure tone audiometry(PTA) or behavior audiometry(BA), acoustic immitance (tympanometry, acoustic reflex thresholds and stapedius muscle reflex), otoacoustic emissions (OAE), auditory brain stem responses (ABR), 40Hz auditory event related potential(40Hz AERP), auditory steady-state response (ASSR). Different tests were chosen according to different ages and circumstances to get the audiological datas. Computed tomography scan(CT) of the temporal bone and magnetic resonance imaging(MRI) were used for understanding the structure of cochlea and endolymphatic sac. All above methods were used in clinic.Mutation analysis was carried out by the method of polymerase chain reaction(PCR) and direct sequencing on 21 exons of SLC26A4 gene and 2 exons of FOXI1 gene and their consecutive part of introns. Then we used the software of DNAStar (EditSeq and SeqMan) to analyze the sequence.Mutant and wild type of PDS were reconstructed with pEGFP-N1 expression vector and transfected HEK293T cell. By the method of western blot and immunofluorescence , we tested the expression protein and got the photo through Confocal microscopy.Results:1. The classic performance of EVAS is bilateral hearing loss, especially SNHL with character of progressive or fluctuate hearing loss in childhood. The majority is worse than moderate severe hearing loss with decreasing in high frequency. Air-bone gap and ASNR account for 44.1 % and 42.1% , which are the characters of EVAS. While the imageology is the golden standard of diagnosis of EVAS. Stapedial gusher always happened in cochlear implantation, while otologists should carefully deal with it to get a good result.2. There were 17 EVAS patients who had SLC26A4 gene mutations in 19 EVAS patients. The mutation rate reached up to 84.2%. 12 were complex heterozygosis mutations, 3 were homozygosis mutations, 2 were heterozygosis mutations and there were no mutations in the 2 patients left. 17 different pathopoiesis mutations of SLC26A4 gene were detected. Among these mutations, 13 were missense mutations, 2 were splicing site mutations, 1 was nonsense mutation and the other 1 was klenow fragment deletion. 16 mutations have been reported before, IVS7-2A>G was at most, which accounted 34.4%. While a novel mutation 1716A>T /F572L was found in this examination. There were 2 polymorphisms of FOXI1 in 19 EVAS patients: 279 G>A,1044T>C.3. We successfully constructed SLC26A4 gene wild-type and S448X mutant green fluorescent vectors. They were named as pEGFP N1 PDS WT and pEGFP N1 PDS S448X. HEK293T cells were successfully transfected with pEGFP N1 PDS WT and pEGFP N1 PDS S448X. The expression of PDS WT and PDS S448X protein were detected by the method of western blot. With the help of immunofluorescence staining and laser Confocal microscopy, we founded that pEGFP N1 PDS S448X was limited in cytoplasm, there wasn't limpid green fluorescence on cytomembrane. while the majority of pEGFP N1 PDS WT has clear green fluorescence on cytomembrane.Conclusions:1. EVAS is an autosomal recessive nonsyndromic hearing loss which is characterized as enlarged vestibular aqueduct and Sensorineural hearing loss(SNHL). Characteristics of audiology such as air-bone gap at low frequency and acoustically evoked short latency negative response(ASNR) can help us to discover this disease. The golden standard of EVAS is imageology. Stapedial gusher always happened in cochlear implantation, while otologists should carefully deal with it to get a good result.2. In this study, the mutation rate of SLC26A4 reached up to 84.2%. This data demonstrated that SLC26A4 gene mutation had close correlation with EVAS. Since gene mutation can prognosticate EVAS, then clinic disease can be found by gene detection. The novel mutation 1716A>T /F572L multiplied Chinese mutation map. We can use the hot spot mutation IVS7-2A>G to develop fast screening of SLC26A4. FOXI1 was not the unique transcriptional control factor of SLC26A4. Other etiological factors maybe exist in the development of EVAS.3. The mutation of S448X of SLC26A4 gene caused the result that mutein was limited in cytoplasm, it couldn't locate at cytomembrane correctly. |
