摘要
耳聋是困扰人类的最常见的感觉性疾病,每一千个新生儿就有一个存在先天性听力障碍,遗传原因在耳聋致病因素中达到60%,绝大部分病例为遗传和环境等多因素共同作用的结果。根据耳聋是否伴有其它系统疾病,将遗传性耳聋分为综合征型耳聋(约占30%)和非综合征型耳聋(约占70%)。在非综合征型耳聋中,按照耳聋的遗传方式,可以分为常染色体显性遗传(约占75-85%),常染色体隐性遗传(12-13%),X连锁遗传、Y连锁遗传和线粒体遗传(共占约2-3%)。X连锁遗传还可以细分为X连锁显性遗传和X连锁隐性遗传。我们依托已经建立起来的遗传性耳聋家系收集系统,根据两个非综合征型X连锁遗传性耳聋家系资料分两部分开展了耳聋致病基因的定位和鉴定研究。
第一部分GZ-Z001家系致病基因定位和鉴定研究
GZ-Z001家系采集自我国贵州省,共涉及五代106人(含己故和配偶),男性患者分布于第二代和第四代,女性患者分布于第二代和第三代。根据家系中患者分布和临床听力学检查结果,判定为非综合征型X连锁显性遗传耳聋家系。男性患者表现为10岁前发病并呈进行性加重的神经性耳聋,成年后呈现双侧对称重度至极重度神经性耳聋;女性患者表现为40岁后发病,呈现双侧对称或双侧不对称的轻度至极重度度神经性耳聋。采用ABI公司提供X染色体上的微卫星标记,依据连锁分析原理,进行X染色体基因组扫描,发现在微卫星标记DXS1106处最大LOD=2.45(theta=0),初步定位。随后在DXS1106上下游选取遗传距离更紧密的微卫星标记,并补充部分家系成员血样行精细定位,最终在DXS8096处得到最大LOD=4.25(theta=0.0),根据连锁分析结果和观察到的重组事件,构建致病单倍体,确定致病基因位于DXS8020和DXS1059之间5.41cM区域。选取两名家系患者,首先筛查非综合征型X连锁遗传性耳聋基因座DFN3致病基因POU3F4和MOHR-TRANEBJAERG综合征致病基因TIMM8A,没有发现致病突变。根据定位区域内基因信息,结合耳蜗内表达ESTs信息库,先后选定12个候选基因行突变筛查,在PRPS1基因第二外显子区域内发现一处单碱基突变(193G＞A),并导致编码氨基酸改变(65asp＞asn)。随后对采集到的家系中全体成员行此突变筛查,发现6名男性患者在此位点表现出同样突变,所有确定女性患者和携带者表现为193G/A杂合突变,家系中确定正常人无突变,临床表型和基因型共分离,选取80名家系外散发听力正常人行此突变位点直接测序,皆未发现突变。因即往文献中关于PRPS1基因的突变所导致的临床改变皆为综合征型表现,为确定家系患者临床表型,再赴当地对家系成员行进一步检查,未发现文献中曾报道的临床表现或除耳聋外的共同临床症状,最终确定此突变导致临床表型为非综合征型耳聋。
第二部分JX-L005家系致病基因定位研究
JX-L005家系采集自我国江西省,家系成员共4代31人(含已故和配偶),男性患者5名,分布于第三代,第二代女性患者一名。男性患者表现为先天性双侧对称极重度神经性耳聋,女性患者表现为双侧对称中度神经性耳聋。经分析将遗传方式定为X连锁显性遗传。通过X染色体基因组扫描和连锁分析,在微卫星标记DXS1227处得到最大LOD=2.04(tbeta=0)。选取此标记上下游精细微卫星标记行精细定位,未发现＞2的LOD值,根据观察到的重组事件,暂时将致病基因定位于DXS1205至DXS8106之间4.08cM区域。对家系患者行POU3F4基因和TIMM8A基因测序,未查见突变。
结论
本研究第一部分对非综合征型X连锁显性遗传耳聋GZ-Z001家系致病基因进行定位,并在定位区域内选取候选基因测序筛查,鉴定出PRPS1基因第2外显子193G＞A(asp65asn)突变,并确定此突变和GZ-Z001家系耳聋相关。第二部分对非综合征型X连锁显性遗传耳聋JX-L005家系致病基因进行定位,确定致病基因位于X染色体上4.08cM区域。
Hearing loss is the most common sensory disorder,one in every 1,000 newborn suffers from congenital hearing impairment.More than 60%of the congenital cases are caused by genetic factors.Non-syndromic forms are responsible for 70%of the cases of hereditary etiology and syndromic cases represent 30%of them.Among the forms of heritage,autonomic recessive is the most frequent one(75%-85%),followed by dominant heritage(12-13%),X-linked and Y-linked or mitochondrial,with 2-3%of the cases of non-syndromic hearing loss.Molecular genetics of deafness has experienced remarkable progress in the last decade.Genes responsible for hereditary hearing impairment are being mapped and cloned progressively.Our reach focuses on non-syndromic X-linked hereditary hearing impairment.
PartⅠ:Linkage analysis of an nonsyndromic X-linked hereditary deafness pedigree(GZ-2001)and identified the causative mutation in PRPS1 gene.
We paid our attention to a large X-linked deafness pedigree collected from guizhou province of southern china(GZ-Z001family).There are totally five generations 106 members in the family with character of non-syndromic X-linked dominant hearing loss.Preliminary X-chromosome mapping in GZ-Z001family was performed using the ABI Prism set version 2,panel 28,obtained from Applied Biosystems.The family was genotyped for a set of microsatellite markers evenly spaced at intervals of about 10 cM.we found the maximum lod score of 2.45 at theta=0.0 for DXS1106.markers for the fine mapping of the candidate regions were spaced at about 0.4-1cM from Meshfield website,the forward primers for these microsatellites were labeled with different fluorescents,and got the max lod score of 4.25 at theta=0.0 for DXS8096.The position of the deafness locus was refined by haplotype analysis,flanking recombinations were observed at DXS8020 and DXS1059,and to define the interval 5.41cM of the locus.We have direct sequenced 14 candidate genes in the family including 12 genes in the region,and identified a 193G＞A transversion in exon 2 of the PRPS1 gene in all male affected patients,resulting in a asp65-to-asn(D65N)substitution,the female carriers showed heterozygosis.Again,we confirmed that D65N was not observed in 80 unrelated control chromosomes outside the pedigree.The mutated amino acids showed perfect conservation from canis to human.
Part 2:mapping a disease locus for the second nonsyndromic X-linked genetic hearing impairment family(JX-L005).
we investigated a family from jiangxi province of china(JX-L005family). There are 5 male patients with profound deafness and 1 female member with moderate hearing loss in the pedigree.Materials and method paralleled with the first part.At last we found the max lod score at marker DXS1227=2.04 (theta=0.0).recombinations were found frequency around the marker and to define the interval 4.08 c M between DXS1205 and DXS8106.Directed sequencing analyses of the TIMM8A gene and POU3F4 gene had not identified mutations.
Conclusion
In part one,we mapping a locus for the nonsyndromic X-linked hereditary deafness pedigree(GZ-Z001)and found that the deafness was associated with a 193G＞C transversion in exon 2 of the PRPS1 gene that resulted in a asp65asn substitution.In part two,we found a locus on X chromosome of the nonsyndromic X-linked genetic hearing impairment family(JX-L005).
引文
1 Piatto,V.B.,E.C.Nascimento,F.Alexandrino,et al.Molecular genetics of non-syndromic deafness.Rev Bras Otorrinolaringol(Engl Ed),2005,71:216-223
2 Bitner-Glindzicz,M.Hereditary deafness and phenotyping in humans.Br Med Bull,2002,63:73-94
3 Van Laer,L.,K.Cryns,R.J.Smith,et al.Nonsyndromic hearing loss.Ear Hear,2003,24:275-288
4 Wrightson,A.S.Universal newborn hearing screening.Am Fam Physician,2007,75:1349-1352
5 Resendes,B.L.,R.E.Williamson,C.C.Morton.At the speed of sound:gene discovery in the auditory system.Am J Hum Genet,2001,69:923-935
6 Reardon,W.Genetics of deafness:clinical aspects.Br J Hosp Med,1992,47:507-511
7 Morton,N.E.Genetic epidemiology of hearing impairment.Ann N Y Acad Sci,1991,630:16-31
8 Morton,C.C.Genetics,genomics and gene discovery in the auditory system.Hum Mol Genet,2002,11:1229-1240
9 Kim,H.J.,K.M.Sohn,M.E.Shy,et al.Mutations in PRPS1,which encodes the phosphoribosyl pyrophosphate synthetase enzyme critical for nucleotide biosynthesis,cause hereditary peripheral neuropathy with hearing loss and optic neuropathy(cmtx5).Am J Hum Genet,2007,81:552-558
10 Tyson,J.,S.Bellman,V.Newton,et al.Mapping of DFN2 to Xq22.Hum Mol Genet,1996,5:2055-2060
11 Manolis,E.N.,R.D.Eavey,S.Sangwatanaroj,et al.Hereditary postlingual sensorineural hearing loss mapping to chromosome Xq21.Am J Otol,1999,20:621-626
12 Wallis,C.,R.Ballo,G.Wallis,et al.X-linked mixed deafness with stapes fixation in a Mauritian kindred:linkage to Xq probe pDP34.Genomics,1988,3:299-301
13 Brunner,H.G,A.van Bennekom,E.M.Lambermon,et al.The gene for X-linked progressive mixed deafness with perilymphatic gusher during stapes surgery(DFN3)is linked to PGK.Hum Genet,1988,80:337-340
14 de Kok,Y.J.,S.M.van der Maarel,M.Bitner-Glindzicz,et al.Association between X-linked mixed deafness and mutations in the POU domain gene POU3F4.Science,1995,267:685-688
15 Lalwani,A.K.,J.R.Brister,J.Fex,et al.A new nonsyndromic X-linked sensorineural heating impairment linked to Xp21.2.Am J Hum Genet,1994,55:685-694
16 Pfister,M.H.,F.Apaydin,O.Turan,et al.A second family with nonsyndromic sensorineural hearing loss linked to Xp21.2:refinement of the DFN4 locus within DMD.Genomics,1998,53:377-382
17 del Castillo,I.,M.Villamar,M.Sarduy,et al.A novel locus for nonsyndromic sensorineural deafness(DFN6)maps to chromosome Xp22.Hum Mol Genet,1996,5:1383-1387
18 Hereditary heating loss homepage,http://webhost.ua.ac.be/hhh/.last updated:July 16.
19 谢鼎华,杨伟炎.耳聋的基础与临床.长沙.湖南科学技术出版社.2004,
20 Morrell,C.H.,S.Gordon-Salant,J.D.Pearson,et al.Age- and genderspecific reference ranges for hearing level and longitudinal changes in heating level.J Acoust Soc Am,1996,100:1949-1967
21 Cui,B.,H.Zhang,Y.Lu,et al.Refinement of the locus for non-syndromic sensorineural deafness (DFN2). JGenet,2004,83:35-38
22 de Brouwer, A. P., K. L. Williams, J. A. Duley, et al.Arts syndrome is caused by loss-of-function mutations in PRPS1. Am J Hum Genet, 2007,81:507-518
23 Kim, H. J., S. H. Hong, C. S. Ki, et al.A novel locus for X-linked recessive CMT with deafness and optic neuropathy maps to Xq21.32-q24 .Neurology,2005,64:1964-1967
24 Pauli, R. M.Sensorineural deafness and peripheral neuropathy.Clin Genet, 1984,26:383 -384
25 Rosenberg, R. N.,A. Chutorian. Familial opticoacoustic nerve degeneration and polyneuropathy. Neurology, 1967,17:827-832
26 Kremer, H., B. C. Hamel, B. van den Helm, et al. Localization of the gene (or genes) for a syndrome with X-linked mental retardation, ataxia, weakness, hearing impairment, loss of vision and a fatal course in early childhood. Hum Genet,1996,98:513-517
27 Arts, W. F., M. C. Loonen, R. C. Sengers, et al.X-linked ataxia, weakness, deafness, and loss of vision in early childhood with a fatal course.Ann Neurol,1993,33:535-539
28 Becker, M. A., M. J. Losman, J. Wilson, et al.Superactivity of human phosphoribosyl pyrophosphate synthetase due to altered regulation by nucleotide inhibitors and inorganic phosphate. Biochim Biophys Acta, 1986, 882:168-176
29 Becker, M. A., K. O. Raivio, B. Bakay, et al.Variant human phosphori-bosylpyrophosphate synthetase altered in regulatory and catalytic functions. J Clin Invest, 1980,65:109-120
30 Elston, R. C.,M. Anne Spence.Advances in statistical human genetics over the last 25 years. Stat Med,2006,25:3049-3080
31 Becker, M. A., P. R. Smith, W. Taylor, et al.The genetic and functional basis of purine nucleotide feedback-resistant phosphoribosylpyrophosphate synthetase superactivity. J Clin Invest,1995,96:2133-2141
32 Roessler, B. J., J. M. Nosal, P. R. Smith, et al.Human X-linked phosphoribosylpyrophosphate synthetase superactivity is associated with distinct point mutations in the PRPS1 gene. J Biol Chem,1993,268:26476-26481
33 Zoref, E., A. De Vries,O. Sperling.Mutant feedback-resistant phosphoribosylpyrophosphate synthetase associated with purine overproduction and gout. Phosphoribosylpyrophosphate and purine metabolism in cultured fibroblasts.J Clin Invest, 1975,56:1093-1099
34 Roessler, B. J., N. Golovoy, T. D. Palella, et al. Identification of distinct PRS1 mutations in two patients with X-linked phosphoribosyl- pyrophos-phate synthetase superactivity.Adv Exp Med Biol,1991,3O9B: 125-128
1 Piatto,V.B.,E.C.Nascimento,F.Alexandrino,et al.Molecular genetics of non-syndromic deafness.Rev Bras Otorrinolaringol(Engl Ed),2005,71:216-223
2 Bitner-Glindzicz,M.Hereditary deafness and phenotyping in humans.Br Med Bull,2002,63:73-94
3 Van Laer,L.,K.Cryns,R.J.Smith,et al.Nonsyndromic hearing loss.Ear Hear,2003,24:275-288
4 Wrightson,A.S.Universal newborn heating screening.Am Faro Physician,2007,75:1349-1352
5 Resendes,B.L.,R.E.Williamson,C.C.Morton.At the speed of sound:gene discovery in the auditory system.Am J Hum Genet,2001,69:923-935
6 Reardon,W.Genetics of deafness:clinical aspects.Br J Hosp Med,1992,47:507-511
7 Morton,N.E.Genetic epidemiology of heating impairment.Ann N Y Acad Sci,1991,630:16-31
8 Morton,C.C.Genetics,genomics and gene discovery in the auditory system.Hum Mol Genet,2002,11:1229-1240
9 Petersen,M.B.,Q.Wang,p.J.Willems.Sex-linked deafness.Clin Genet,2008,73:14-23
10 Tyson,J.,S.Bellman,V.Newton,et al.Mapping of DFN2 to Xq22.Hum Mol Genet,1996,5:2055-2060
11 Manolis,E.N.,R.D.Eavey,S.Sangwatanaroj,et al.Hereditary postlingual sensorineural hearing loss mapping to chromosome Xq21.Am J Otol,1999,20:621-626
12 Wallis,C.,R.Ballo,G.Wallis,et al.X-linked mixed deafness with stapes fixation in a Mauritian kindred:linkage to Xq probe pDP34.Genomics,1988,3:299-301
13 Gekle,D.,H.Essler,Ho Brunner.[Familial nephropathy and impaired sound perception(Alport syndrome)].Arch Kinderheilkd,1969,178:290-302
14 de Kok,Y.J.,S.M.van der Maarel,M.Bitner-Glindzicz,et al.Association between X-linked mixed deafness and mutations in the POU domain gene POU3F4.Science,1995,267:685-688
15 Lalwani,A.K.,J.R.Brister,J.Fex,et al.A new nonsyndromic X-linked sensorineural hearing impairment linked to Xp21.2.Am J Hum Genet,1994,55:685-694
16 Pfister,M H.,F.Apaydin,O.Turan,et al.A second family with nonsyndromic sensorineural hearing loss linked to Xp21.2:refinement of the DFN4 locus within DMD.Genomics,1998,53:377-382
17 del Castillo,I.,M.Villamar,M.Sarduy,et al.A novel locus for nonsyndromic sensorineural deafness(DFN6)maps to chromosome Xp22.Hum Mol Genet,1996,5:1383-1387
18 Hereditary hearing loss homepage.http://webhost.ua.ac.be/hhh/.last updated:July 16.
19 谢鼎华,杨伟炎.耳聋的基础与临床.长沙.湖南科学技术出版社.2004,
20 Morrell,C.H.,S.Gordon-Salant,J.D.Pearson,et al.Age- and genderspecific reference ranges for hearing level and longitudinal changes in hearing level.J Acoust Soc Am,1996,100:1949-1967
21 Cui,B.,H.Zhang,Y.Lu,et al.Refinement of the locus for non-syndromic sensorineural deafness(DFN2).J Genet,2004,83:35-38
22 Kim,H.J.,S.H.Hong,C.S.Ki,et al.A novel locus for X-linked recessive CMT with deafness and optic neuropathy maps to Xq21.32-q24.Neurology,2005,64:1964-1967
23 Pauli,R.M.Sensorineural deafness and peripheral neuropathy.Clin Genet,1984,26:383-384
24 Rosenberg,R.N.,A.Chutorian.Familial opticoacoustic nerve degeneration and polyneuropathy.Neurology,1967,17:827-832
25 Kremer,H.,B.C.Hamel,B.van den Helm,et al.Localization of the gene(or genes)for a syndrome with X-linked mental retardation,ataxia,weakness,hearing impairment,loss of vision and a fatal course in early childhood.Hum Genet,1996,98:513-517
26 Arts, W. F., M. C. Loonen, R. C. Sengers, et al.X-linked ataxia, weakness, deafness, and loss of vision in early childhood with a fatal course. Ann Neurol, 1993,33.535-539
27 Becker, M. A., M. J. Losman, J. Wilson, et al. Superactivity of human phosphoribosyl pyrophosphate synthetase due to altered regulation by nucleotide inhibitors and inorganic phosphate. Biochim Biophys Acta, 1986, 882:168-176
28 Becker, M. A., K. O. Raivio, B. Bakay, et al.Variant human phosphoribosy-lpyrophosphate synthetase altered in regulatory and catalytic functions. J Clin Invest, 1980, 65: 109-120
29 Elston, R. C.,M. Anne Spence.Advances in statistical human genetics over the last 25 years.Stat Med,2006,25:3049-3080
30 Kim, H. J., K. M. Sohn, M. E. Shy, et al.Mutations in PRPS1, which encodes the phosphoribosyl pyrophosphate synthetase enzyme critical for nucleotide biosynthesis, cause hereditary peripheral neuropathy with hearing loss and optic neuropathy (cmtx5). Am J Hum Genet,2007,81:552-558
31 de Brouwer, A. P., K. L. Williams, J. A. Duley, et al.Arts syndrome is caused by loss-of-function mutations in PRPS1. Am J Hum Genet,2007,81:507-518
32 Becker, M. A., P. R. Smith, W. Taylor, et al.The genetic and functional basis of purine nucleotide feedback-resistant phosphoribosylpyrophosphate synthetase superactivity J Clin Invest, 1995,96:2133-2141
33 Roessler, B. J., J. M. Nosal, P. R. Smith, et al.Human X-linked phosphoribosylpyrophosphate synthetase superactivity is associated with distinct point mutations in the PRPS1 gene. J Biol Chem, 1993,268:26476-26481
34 Zoref, E., A. De Vries,O. Sperling.Mutant feedback-resistant phosphoribosylpyrophosphate synthetase associated with purine overproduction and gout. Phosphoribosylpyrophosphate and purine metabolism in cultured fibroblasts.J Clin Invest,1975,56: 1093-1099
35 Roessler, B. J., N. Golovoy, T. D. Palella, et al.Identification of distinct PRS1 mutations in two patients with X-linked phosphoribosylpyrophosphate synthetase superactivity. Adv Exp Med Biol, 1991, 309B: 125-128
36 Becker, M. A.Hyperuricemia and Gout.The Metabolic and Molecular Bases of Inherited Disease.McGraw-Hill 2001,n,(8).2625
37 Becker, M. A., S. A. Heidler, G. I. Bell, et al.Cloning of cDNAs for human phosphoribosylpyrophosphate synthetases 1 and 2 and X chromosome localization of PRPS1 and PRPS2 genes.Genomics,1990,8:555-561
38 Taira, M., T. Iizasa, K. Yamada, et al.Tissue-differential expression of two distinct genes for phosphoribosyl pyrophosphate synthetase and existence of the testis-specifictranscript.Biochim Biophys Acta, 1989, 1007:203-208
39 Becker, M. A., J. G. Puig, F. A. Mateos, et al.Inherited superactivity of phosphoribosylpyrophosphate synthetase: association of uric acid overproduction and sensorineural deafness Am J Med,1988,85:383-390
40 Yen, R. C, W. B. Adams, C. Lazar, et al.Evidence for X-linkage of human phosphoribosylpyrophosphate synthetase.Proc Natl Acad Sci U S A ,1978,75:482-485
41 Becker, M. A., R. C. Yen, P. Itkin, et al.Regional localization of the gene for human phosphoribosylpyrophosphate synthetase on the X chromosome. Science, 1979,203:1016-1019
42 Inuma, K., Y. Wada, A. Onuma, et al.Electroencephalographic study of an infant with phosphoribosylpyrophosphate synthetase deficiency. Tohoku J Exp Med, 1975, 116:53-55
43 Wada, Y., Y. Nishimura, M. Tanabu, et al.Hypouricemic, mentally retarded infant with a defect of 5-phosphoribosyl-1-pyrophosphate synthetase of erythrocytes. Tohoku J Exp Med, 1974, 113:149-157
44 Zoref, E., A. De Vries, O. Sperling.Evidence for X-linkage of phosphoribosylpyrophosphate synthetase in man. Studies with cultured fibroblasts from a gouty family with mutant feedback-resistant enzyme. Hum Hered, 1977,27:73-80
45 Sperling, O., S. Persky-Brosh, P. Boer, et al.Human erythrocyte phosphoribosylpyrophosphate synthetase mutationally altered in regulatory properties. Biochem Med, 1973,7:389-395
46 Sperling, O., G. Eilam, B. Sara Persky, et al.Accelerated erythrocyte 5-phosphoribosyl-1-pyrophosphate synthesis. A familial abnormality associated with excessive uric acid production and gout. Biochem Med, 1972,6:310-316
47 Nyhan, W. L., J. A. James, A. J. Teberg, et al.A new disorder of purine metabolism with behavioral manifestations.J Pediatr, 1969, 74:20-27
48 Simmonds, H. A., D. R. Webster, J. Wilson, et al.An X-linked syndrome characterised by hyperuricaemia, deafness, and neurodevelopmental abnormalities. Lancet, 1982,2:68-70
49 Rosenberg, A. L., L. Bergstrom, B. T. Troost, et al.Hyperuricemia and neurologic deficits. A family study .N Engl J Med, 1970, 282:992-997
50 Christen, H. J., F. Hanefeld, J. A. Duley, et al.Distinct neurological syndrome in two brothers with hyperuricaemia. Lancet, 1992,340:1167-1168
51 Becker, M. A., L. J. Meyer, A. W. Wood, et al.Purine overproduction in man associated with increased phosphoribosylpyrophosphate synthetase activity. Science, 1973,179:1123-1126
52 Atkin, C. L., S. J. Hasstedt, L. Menlove, et al.Mapping of Alport syndrome to the long arm of the X chromosome. Am J Hum Genet, 1988,42:249-255
53 Becker, M. A., W. Taylor, P. R. Smith, et al.Overexpression of the normal phosphoribosylpyrophosphate synthetase 1 isoform underlies catalytic superactivity of human phosphoribosylpyrophosphate synthetase.J Biol Chem, 1996,271:19894-19899
54 Becker, M. A., P. J. Kostel, L. J. Meyer, et al.Human phosphoribosylpyrophosphate synthetase: increased enzyme specific activity in a family with gout and excessive purine synthesis. Proc Natl Acad Sci U S A, 1973, 70:2749-2752
55 Becker, M. A., M. J. Losman, A. L. Rosenberg, et al .Phosphoribosylpyrophosphate synthetase superactivity. A study of five patients with catalytic defects in the enzyme. Arthritis Rheum, 1986,29:880-888