Anditory Neuropathy Patients' Fellow-up Study And Related Pathogenetic Genes Sequencing

Auditory neuropathy (AN) is a specific hearing disorder. The main clinicalcharacters include auditory brainstem responses (ABR) absent or abnormalseverely and evoked otoacoustic emissions (EOAE) normal, which reflects thefunction of auditory nerve with inner hair cells and outer hair cells, respectively.AN was first named by Starr in 1996, and interested a lot of researchers studyingthis special disease. Auditory tests results in AN patients distinguished fromother sensorineural hearing loss also include acoustic reflex (AR) absence orthreshold elevation, pure tone auditory threshold elevation mainly at lowerfrequencies and vestibular affection as vestibular evoked myogenic potentials(VEMP) and/or electronystagram (ENG) abnormal. The core complain of ANpatients is speech discrimination disability and difficulty of verbalcommunication especially in noises. However, there is no effective treatment forAN patients, and we still have no idea of the causes and the changes in thecourse of this disease. Therefore we follow up a bunch of adult AN patients andanalyse the changes in auditory tests, hoping to understand the natural processand the prognosis of the disease. Neonatal hyperbilirubinemia, anoxianeonatorum, premature birth and ototoxic drug history can explain the causes in some congenital AN, but most posteriority AN patients has no clear history ofsuch risk factors at all. Some AN patients had been detected genes mutations ascause of their disease, but we do not know if genes variations are the cause ofsporadic adult patients. Thus, we designed this study to explore whether ourpatients carry variations in related genes.MethodMethods and results1. Follow-up study of auditory neuropathy patientsThe main body of this study is 74 out patients diagnosed as AN during 2001January till 2009 July. All the patients test pure tone audiometry, acousticimpedance, ABR, DPOAE, ENG, VEMP and speech discrimination during thefollow-up period. The average ages of AN onset are 16.69±4.42 years and thecourse of follow up are 3.74±1.96 years. There is no difference in the pure toneaudiometry between left and right ears of the patients, and both ears affect lowfrequencies mostly (70.1%). During the course of follow up, the pure tonethreshold has no significant changes and has no trend affecting higherfrequencies. Word discrimination score has no changes during follow up, but thenumber of ears with 0% word discrimination rate increased during the followingcourse. AR, DPOAE, ENG and VEMP have no statistic differences between thefirst and last visits, but the recordable VEMP threshold and latency areapparently different from control. In conclusion, during following course, thereis no big difference in almost all the auditory tests. We need to visit more ANpatients and follow up a longer period to understand better about the specialdisease.2. OTOF gene and PJVK gene variations sequencing in late onset ANpatientsOTOF and PJVK are the pathogenetic genes detected in some AN patients.However, almost all the mutations are detected in early onset patients, who hadlittle ability to hear and speak. There is no variation map in adult AN patients,and we wonder if OTOF and/or PJVK are the pathogenetic genes of late onset AN patients. We tested 70 sporadic acquired AN patients, and sequenced all theexons as well as introns near exons of OTOF and PJVK. We have detected 10variations that altered amino acid coding, 4 out of which had not reported before,including 4023+1 G>A, G368R, N727S and A809D. We have detected 2 novelmissense mutations in PJVK gene. However, all of the new variations are thesingle heterozygous in single patients, so we can not say that these variations arethe only causes of these patients. We still need to sequence more patients anddetect more related genes.3. Hereditary AN families clinical and genetic features analyzingSome AN patients has clear family history. In clinical study, we collectedsome precious hereditary AN families as follow. We collected 7 AN familiesappearing autosomal dominant inheritance phenotype and 2 AN families withperipheral neuropathy showing X-linked recessive inheritance phenotype. Weevaluated the phenotype and genetic characters of these families in detail.4. Pathogenetic gene sequencing of an X-linked recessive AN familyWe focused on an X-linked recessive AN family searching for theirpathogenetic gene, which had not detected yet. We used the exome capture chip(Nimblgen) and sequencing chip (Solexa) in the propositus for variations in theentire chromosome. Then we narrow the scope of candidate genes step by step,and finally confirmed gene AIF as the pathogenetic gene of the family, whosepathogenetic mutation is S314N.5. AIF gene sequencing in late onset AN patientsWe sequenced the 70 AN patients for all the AIF exons and introns near exons.Interestingly, we deteceed 9 missense mutations in 9 male patients as follow:L191P, I287T, Q332H, G360R, R422W, A440V, P475L, E558V and D559G, aswell as a variation in the 5'UTR of the upper stream in a boy. All the abovevariations are novel and did not detected in control. We believe that AIF gene iscritical in AN pathogenesis, and the mutations of AIF gene could be the causesof the patients. 6. Fluorescence dye and mRNA express of AIF in normal rat inner earIn order to make sure the location of AIF staining in normal cochlea, we usedimmunofluorescent and RT-PCR techniques. We observed that AIF wasdistributed broadly in the inner ear cells, especially in hair cells. There is novisible difference among the expression of AIF mRNA among neonatal andadult cochlea basilar membranes and neonatal spiral ganglion cells. Thus, weknow that AIF is a important molecule in mitochondria and expressed stably inthe inner ear, and we should do more work to fully understand the function ofAIF in hearing loss. AIF could be the very key to the AN.ConclusionWe are still at the beginning of AN studying. In this study, we get the generalknowledge of the natural out come of AN, we realized the variations carryingstates of OTOF gene and PJVK gene in late onset AN patients, we found a newAN related pathogenetic gene in an X-linked AN family with peripheralneuropathy, we confirmed the importance of AIF in AN pathogenesis as detected10 novel pathogenetic mutations, and we make clear the expression of AIF inneonatal and adult rat inner ear. All our work urged to reveal the pathogenesis ofAN, so that to find some curable methods to make the patients recover.