摘要
研究目的:收集遗传性耳聋家系,对一个非综合症遗传性耳聋家系进行了临床表型及致病基因的研究,以确定这个遗传性耳聋家系的致病基因在染色体上的位置,为家系的基因诊断及遗传咨询提供目的基因。
研究方法:研究对象为一个临床表现为很少见的遗传性传导性耳聋家系(GY 家系)。对这个家系绘制系谱图;进行一般情况调查;临床听力检查;颞骨CT 检查;免疫学检查和抗核抗体系列检查;染色体核型分析;人工镫骨手术;PCR-SSCP 检测耳聋基因POU3F4;全基因组扫描微卫星多态标记的连锁分析,采用优化的基因定位试剂盒ABI PRISM?Linkage Mapping Sets2 MD10,应用这个试剂盒进行多重PCR 反应,反应产物进行聚丙烯酰胺凝胶电泳;应用ABI PRISM?377XL Collection 软件收集数据;GeneScanAnalysis3.0 软件进行泳道校正;Genotyper?2.1 软件进行各样本的基因分型;应用Linkage version5.1 软件的MLINK 程序进行两点连锁分析,计算LOD 值(LOD Scores);应用Linkage version5.1软件的ILINK 程序计算最大LOD 值(Zmax)。
研究结果:临床研究表明GY 家系为一个镫骨发育畸形的X-连锁遗传性传导性耳聋,人工镫骨手术可以使听力提高,POU3F4基因的二个外显子131bp 和297 片断经PCR-SSCP 检测正常。全基因组扫描微卫星多态标记的连锁分析显示基因组382 对标记中在常染色体上有6 个标记在θ=0.00 时其LOD 值大于1,分别为
Introduce and Objectives: Hearing impairment is a commonsensory disorder in the human population. The incidence of congenitalhearing impairment is estimated at 1 in 1000 births, of whichapproximately equal numbers of cases are attributed to environmentaland genetic factors. Of the hearing-impairment disorders attributableto genetic causes, 70% are classified as nonsyndromic and theremaining 30% as syndromic. Among the many disorders classified assyndromic hearing impairment, the pathology varies widely, but, innonsyndromic deafness, the defect is generally sensorineural.Nonsyndromic hearing impairment can be further subdivided by modeof inheritance: 70% of cases are autosomal recessive, 22% areautosomal dominant, 1% are X-linked, and<1% are duo tomitochondrial inheritance. Dominant loci are denoted with the prefix“DFNA’’, recessive loci with “DFNB’’, X-linked loci with “DFN’’,and modifying loci with “DFNM’’. Generally, patients with autosomalrecessive hearing impairment have prelingual and profound deafness,and patients with autosomal dominant hearing impairment haveprogresswive and postlingual hearing impairment. This observationmay be explained by the complete absence of functional protein inpatients with recessive disorders, dominant mutations may beconsistent with initial function and subsequent hearing impairment due
to accumulation of pathology. over the past 5 years, remarkableprogress has been made in the identification of new loci fornonsyndromic hearing impairment (NSHL) and in the cloning ofdeafness genes. To date, 77 loci have been reported for nonsyndromicdeafness: 40 autosomal dominant, 30 autosomal recessive, and 7X-linked. In addition, 51 auditory genes have been identified: 15 forautosomal dominant NSHI loci, 9 for autosomal recessive NSHL loci,2 for X-linked NSHL loci, 5 mitochondrial, and>=32 genes forsyndromic hearing impairment (some genes cause multiple forms ofdeafness). Although significant advances have been made, there is nodoubt that many more genes await discovery. Identifying these genesand characterizing the proteins they encode will increase ourknowledge of the molecular processes involved in the auditory systemand will improve our understanding of how such processes canbecome altered and lead to hearing impairment. Now we collect adeafness pedigree that have less characters of hereditary conductivehearing impairment, so we apply clinic and biologic methods to searchrelated gene for gene diagnosis and therapy. Study Design and methods: All subjects in the pedigree wasinterviewed personally and their clinical data and genetic backgrounddata are collected; Audiological Examination (pure-tone audiometry,Acoustic Immittance measurement), Supplementary Examination(High-resolution CT, Immunologic tests, Chromatosome Caryotype)
and Surgery are applied. Peripheral blood samples were obtained fromavailable family members for isolating genomic DNA. we selected 1candidate gene (POU3F4) playing important roles in X-linkedhereditary conductive hearing impairment from genomic databases inhttp://www.hereditary hearing loss.org/. we use PCR-SSCP methods todetect POU3F4 gene in order to certify that it is not related to deafnessof this pedigree. Finally two-point linkage analysis was conductedusing the MLINK and ILINK programs from the Linkage version5.1software. Results: Pedigree Analysis: Pedigree analysis indicated thatpatients distributed four continuous generations and each one nomatter they were male or female had 50% chance of recieving theautosome bearing the mutant gene and being affected. And theprevalence rate of female patients was larger than it of male patients.Furthermore, only female patients can transmit the mutant gene to herlater generations, males’were not observed this phenomenon.Although it maybe shows an autosome dominant inheritance, wesuspect that it is mostly an X-linked dominant inheritance.Audiological Examination Data: 1.pure-tone audiometry: Audiogramin the 9 hearing-impaired patients identified in the present study,pure-tone audiograms showed conductive deafness. 2.AcousticImmittance measurement: Acoustic Immittance measurement in the 7patients showed all of them are both ears’A form curve, and indicated
that the pressure of tympanic cavity was normal. This examinationexcludes hearing loss caused by secretory otitis media or suppurativeotitis media. Supplementary Examination 1. High-resolution CT:High-resolution CT scan of temporal bonein two patients showed normal inner structures including the vestibularaqueduct 、semicircular canals and internal auditory canal. 2.Immunologic tests: All of the immunologic tests to the members ofthis pedigree including RHF, CRP, ASO, HLA-27 and antinuclearantibody examinations were negtive. 3. Chromatosome Caryotype: Ⅲ2 was accepted chromatosome caryotype analysis. The chromatosomeshowed 46, XX. Surgery: In the patients described here, tympanotomyshowed her stapes was flat cylinder shape, stape footplate fix onfossulae fenestrae vestabuli and parallel to the stapedial tendon,respectively, which excludes otosclerosis. After this operation, theprobands’left ear hearing achieved normal level one month later.Tinnitus and vertigo appearing after operation were disappeared twoweeks later. candidate gene detection: It does not detect mutation ofPOU3F4 gene in this Pedigree. None of the patients was affected byPOU3F4 gene. genome scan: Two-point LOD scores of the dxs1060microsatellite marker and disease gene is 2.044489 at recombinationfraction 0.00. The loci dxs1060 microsatellite marker is on x-chromosome (Xp22). CONCLUSION:In this study, we have confirmed this family as
引文
1. Steel K.P.& Kros C.L. A genetic approach to understanding auditory function Nat Genet 2001,27:143-149
2. Barbara L, Resendes, Robin E, et al. At the speed of sound: Gene discovery in the auditory system[J].Am J Hum Genet, 2001,69:923.
3. 杜传书. 医学遗传学基础, 人民卫生出版社, 北京,1989
4. runner HG, van Bennekom A,The gene for X-linked progressive mixed deafness with perilymphatic gusher during stapes surgery DFN3 is linked to PGK Hum Genet 1988;30:337-40.
5. eckman JS, Weber JL. Survey of human and rat microsatellites. Genomics 1992,Apr;12(4):627-31.
6. PE, Raventos H. The gene for an inherited form of deafness maps to chromosome 5q31. Proc natl Acad Sci U.S.A. 1992;89:5181-4
7. an L, Yates PA.A POU do main transcription factor-dependent program regulates axon pathfinding in the vertebrate visual system. Neuron 2000,Dec;28(3):779-792.
8. Kok, Y.J.M. Association between X-linked mixed deafness and mutations in the POU domain gene POU3F4. Science 1995;267:685-688
9. llis C.X-linked mixed deafness with stapes fixation in a Mauritian kindred: linkage to Xq probe pDP34.Genomic1988,3,299-301
10.CL and Young WS. a POU domain-containing gene, and its