The Study Of Copy Number Variation, KCNJ10 Gene Screening And Expression In Patients With Non-syndromic Enlarged Vestibular Aqueduct

Non-syndromic Enlarged Vestibular Aqueduct (EVA) is one of the most common inner ear malformations, which account about 10%of patients with sensorineural hearing loss. Clinical manifestations of pre-onset are sensorineural hearing loss, and some patients have a poor low-frequency air-bone gap, or mixed hearing loss; the level of hearing loss ranged from mild to extremely severe, and often fluctuating or progressive, up to severe or very severe, affecting language learning or normal communication. The cold, fever, other predisposing factors such as mild head trauma and so on that can make intracranial pressure increased often occurred before hearing loss in patients with Enlarged vestibular aqueduct, so the effective measures including early detection of disease, early diagnosis and early intervention should be taken to prevent or delay its onset. Enlarged vestibular aqueduct is an autosomal recessive disease, which is closely related to the SLC26A4 gene mutation on the chromosome 7q31. About 65～92%of the patients with enlarged vestibular aqueduct can be examined the gene mutation. SLC26A4 gene belongs to the family of anion transport, with 12 transmembrane domains, N terminal and C terminal in the cell, and expresses in the thyroid, kidney and inner ear. It mediated the transformation of sulfate, bicarbonate, formate, oxalate, hydroxide, chlorine, iodine, and fructose and other unit or divalent ions,and it plays an important role in the balance of ion composition in the body. The protein expression and function of the mutant are changed once the SLC26A4 gene mutation, so as to lead to diseases. With the gradual in-depth study of SLC26A4 gene, the great heterogeneous of the mutation gene was found: including the form of gene mutation, gene mutation loci and gene pattern of the different ethnic groups. Recent studies showed that the SLC26A4 gene mutations was closely related to non-syndromic EVA, but the gene mutations was not found or only found single-allele mutation in a considerable part of patients with enlarged vestibular aqueduct in some studies at home and abroad.In order to clarify of the pathogenic mechanism of the patients, and the role of other genetic factors in the patients with hearing loss. The role of the copy number variation and KCNJ10 gene (participation in endolymphatic K+regulation) mutation in the pathogenesis of non-syndrome EVA was studied in the issue, and the study provided the basis for improving the procedures and methods of the genes diagnosis of deafness. At the same time for the Chinese population-specific SLC26A4 mutation pattern, several common hot spot mutations were selected and studied the expression of SLC26A4 gene mutation in order to further explore molecular mechanisms of pathogenesis of the SLC26A4 gene mutations.PartⅠThe screening of non-double-allele mutation of SLC26A4 gene copy number variation based on the MLPA methodCopy number variations (CNVs) mean that widespread exist in human genome, and deletions, insertions, duplications and complex multi-site variability from the range of 1 000bp to several million bp, which were found in recent years in the human genes in addition to SNPs, the other rich source of polymorphism. CNVs not only widespread exist in normal individuals'genes, but also closely related to the chromosomal rearrangement and a number of hereditary diseases. The presence of CNVs can not be detected by some conventional detection technology. In order to understand whether the copy number variation play a role in the pathogenesis of non-syndromic EVA, the study had a screening of SLC26A4 gene copy number variation in patients with non-syndromic EVA in which any of SLC26A4 gene mutations were not detected, or only a single allele mutation was detected in the PLA General Hospital deafness Molecular Diagnostics Centre application of. multiplex ligation-dependent probe amplification MLPA) technology, in order to find possible copy number variation resulting in pathogenicity. The significant copy number variation was not found in 39 cases of patients without SLC26A4 gene mutations and 68 cases of patients with single-allele SLC26A4 gene mutations. Based on the results, we conclude several possibilities:①there was not a copy number variation in the exon of SLC26A4 gene, therefore the copy number variation was not a pathogenic mechanism in non-syndromic EVA.②copy number variations present in SLC26A4 gene, the copy number variations do not exist in the open reading frame area, but in the intron or the control region of the upstream and downstream,which can not be detected with the MLPA test kit.③opy number variations exit in the surrounding of SLC26A4 gene or other regulation genes, these copy number variations indirectly affect SLC26A4 gene by changing the expression or function of these genes, leading to non-syndromic EVA. The CNVs were apply to screening of patients non-syndromic EVA for the first time in this study, and provide a new perspective of genetic diagnosis of non-syndromic EVA and improve the genetic diagnosis of the disease.Part II The screening of KCNJIO gene mutation in patients with large vestibular aqueduct syndrome carrying non-double-allele mutation of SLC26A4 gene mutationsKCNJ10 gene is located on human chromosome 1q23, encoding an inward rectifier K+channel, mainly expression in the brain, inner ear and kidney. In the inner ear, the potassium ion channel KCNJ10 gene encoded mainly expressed in the vascular pattern, which play an important role in the formation of the cochlear potential (EP). Vascular pattern produced cochlear potential (EP) and secreted potassium into the cochlear endolymph. Cochlear potential is the main driving force of cochlear transduction current; and K+current is the main current of cochlear transduction. Hearing generated from cochlear transduction current. KCNJ10 mutations can lead to the disappearance of cochlear potentials, resulting in hearing loss. Recent studies showed that KCNJ10 gene mutation was in the presence of in Pendred syndrome and non-syndromic EVA, which indicated that KCNJ10 gene mutation maybe play a role in the pathogenesis of non-syndromic EVA. In order to understand whether the KCNJ10 gene mutation play a role in the pathogenesis of non-syndromic EVA, the screening of KCNJ10 gene mutation in patients with non-syndromic EVA was conducted in Molecular Diagnostic Centre deafness of the People's Liberation Army General Hospital.4 cases of KCNJ10 gene mutation were found In 107 cases of patients carrying single heterozygous SLC26A4 mutation and without mutation, including 812 G> A heterozygous mutation in 3 patients,1042 C>T heterozygous mutation in 1 case.12 cases of KCNJ10 gene mutation were found in 311 patients with two-allele SLC26A4 gene mutation (homozygous mutant and compound heterozygous mutations); including 812G> A heterozygous mutation in 11 cases; 1042C> T heterozygous mutation 1 case.12 cases of KCNJ10 gene mutation were found in 229 control patients; including 812G> A heterozygous mutation in 11 cases,1042C> T heterozygous mutation in two cases,811C> T heterozygous mutation in 1 case. The above two genes were conducted to screening in the parents of patients with SLC26A4 biallelic mutations and KCNJ10 gene mutation. The results showed that one of their parents must be carrying SLC26A4 and KCNJ10 single heterozygous for the same time, but with normal clinical symptoms, neither the hearing decline nor vestibular aqueduct enlargement, which is different from foreign. Based on the above results we draw the following inclusions:①KCNJ10 single heterozygous mutation is a common phenomenon in the Chinese population.②812G> A KCNJ10 mutation is the most common mutations in the Chinese population.③there is no necessary link between the single heterozygous KCNJ10 mutation and Pendred syndrome and non-syndromic EVA, although KCNJ10 gene play an important role in the formation of cochlear potentials and auditory.④KCNJ10 and SLC26A4 compound heterozygous mutations may not result in Pendred syndrome or non-syndromic EVA; for patients with non-syndromic EVA carrying both SLC26A4 and KCNJ10 gene heterozygous mutations,the disease maybe not caused by the KCNI10 mutation, which may be due to other reasons, such as another unknown gene mutation or the interaction between the environment and the gene. The preliminary analysis of the relation of KCNJ10 and non-syndromic EVA in the study showed that there was no necessary relation between the KCNJ10 gene mutation and non-syndromic EVA, which provide a basis for in improve the genetic diagnosis of non-syndromic EVA.Part III The study of the expression of common SLC26A4 gene mutations in patients with large vestibular aqueduct syndrome in Chinese peopleSLC26A4 gene mutations can cause Pendred syndrome or non-syndromic EVA. SLC26A4 gene mutation has great heterogeneity. The form of gene mutation, gene mutation pattern in different ethnic exist great heterogeneity. This heterogeneity showed that the molecular mechanism of disease maybe also exist certain heterogeneity after SLC26A4 mutation. SLC26A4 mutations pattern in Chinese population has a significantly difference in the rest of the world population patterns. In the Chinese population, several common types of hot spot mutations are IVS7-2A> G; 2168A> G (H723R); 1174A> T (N392Y); 1229C> T (T410M); 2027T> A, while the specific mechanism that the molecular functions changes caused by these mutations has not been reported so far. Therefore, several common types of SLC26A4 mutations plasmid were constructed, and transfected into HEK293 cells, in order to observe the expression of cell after mutation. The results showed that the selected four SLC26A4 gene mutations, its encoded protein Pendrin were stranded in the cytoplasm, while the wild-type Pendrin protein expressed in the cell membrane, which indicated that after SLC26A4 mutations, its coding the Pendrin protein can not be located in the membrane, while stranded in the cytoplasm, and can not complete the normal anion exchange function, leading to diseases. This study identified protein expression of common type of SLC26A4 mutations in Chinese population, providing a basis for further study changes in ion transport function and the possibility of taken intervention measures and saving hearing.