一个中国Alport综合征家系中剪接突变及致病性分析
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摘要
目的对一个中国Alport综合征家系进行基因变异检测,并对发现的基因变异进行致病性分析。方法采用目标序列捕获芯片联合高通量测序技术对先证者进行基因变异检测,应用Sanger测序技术对可疑位点进行家系验证,通过体外Mini基因实验分析基因变异对pre-mRNA剪接过程的影响。结果先证者COL4A5基因32号外显子发现一杂合变异c.2767G>T(p.Gly923Cys),为新发现的变异。Sanger测序验证此变异在家系中与疾病呈现共分离。体外Minigene实验表明,c.2767G>T突变可造成COL4A5基因32号外显子缺失。结论通过目标序列捕获芯片联合高通量测序技术发现一个新的COL4A5基因突变,丰富了Alport综合征突变谱;通过体外Minigene实验证实c.2767G>T突变为一剪接突变,促进了对Alport综合征分子发病机制的理解。
Objective The purpose of this study was to identify a pathogenic variant in a Chinese family with Alport syndrome and analyze the pathogenicity of the variant.Methods Using targeted region capture and high-throughput sequencing technology,we identified the genetic variant of the proband with Alport syndrome,verified the variant in the family members by Sanger sequencing,and analyzed its influence on the pre-mRNA splicing process by in vitro minigene assay.Results A heterozygous variant c.2767 G>T(p.Gly923 Cys)was identified as a novel variant in exon 32 of the COL4 A5 gene in the proband,which was confirmed by Sanger sequencing to be cosegregated with disease in the family. The minigene assay demonstrated that the c.2767 G>T variant induced deletion of exon 32 of the COL4 A5 gene.Conclusion A novel COL4 A5 mutation was identified by targeted region capture and high-throughput sequencing,which has enriched the gene mutation spectrum of Alport syndrome. The exonic mutation c.2767 G>T confirmed to be a splicing mutation by in vitro minigene assay,which may lead to a deeper insight into the molecular pathogenesis of Alport syndrome.
Objective The purpose of this study was to identify a pathogenic variant in a Chinese family with Alport syndrome and analyze the pathogenicity of the variant.Methods Using targeted region capture and high-throughput sequencing technology,we identified the genetic variant of the proband with Alport syndrome,verified the variant in the family members by Sanger sequencing,and analyzed its influence on the pre-mRNA splicing process by in vitro minigene assay.Results A heterozygous variant c.2767 G>T(p.Gly923 Cys)was identified as a novel variant in exon 32 of the COL4 A5 gene in the proband,which was confirmed by Sanger sequencing to be cosegregated with disease in the family. The minigene assay demonstrated that the c.2767 G>T variant induced deletion of exon 32 of the COL4 A5 gene.Conclusion A novel COL4 A5 mutation was identified by targeted region capture and high-throughput sequencing,which has enriched the gene mutation spectrum of Alport syndrome. The exonic mutation c.2767 G>T confirmed to be a splicing mutation by in vitro minigene assay,which may lead to a deeper insight into the molecular pathogenesis of Alport syndrome.
引文
[1]Flinter F.Alport's syndrome[J].J Med Genet,1997,34(4):326-330.
[2]高春林,夏正坤,樊忠民,等.新X连锁缺失突变致Alport综合征的研究[J].医学研究生学报,2015,28(9):929-933.
[3]Hudson BG.The molecular basis of Goodpasture and Alport syndromes:beacons for the discovery of the collagen IV family[J].J Am Soc Nephrol,2004,15(10):2514-2527.
[4]Hudson BG,Tryggvason K,Sundaramoorthy M,et al.Alport‘s syndrome,Goodpasture’s syndrome,and type IV collagen[J].N Engl J Med,2003,348(25):2543-2556.
[5]Barker DF,Hostikka SL,Zhou J,et al.Identification of mutations in the COL4A5 collagen gene in Alport syndrome[J].Science,1990,248(4960):1224-1227.
[6]Pirson Y.Making the diagnosis of Alport's syndrome[J].Kidney Int,1999,56(2):760-775.
[7]Hertz JM,Thomassen M,Storey H,et al.Clinical utility gene card for:Alport syndrome-update 2014[J].Eur J Hum Genet,2015,23(9):e1-e4.
[8]McAlinden A,Majava M,Bishop PN,et al.Missense and nonsense mutations in the alternatively-spliced exon 2 of COL2A1cause the ocular variant of Stickler syndrome[J].Hum Mutat,2008,29(1):83-90.
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[11]Liu JH,Wei XX,Li A,et al.Novel mutations in COL4A3,COL4A4,and COL4A5 in Chinese patients with Alport Syndrome[J].PLoS One,2017,12(5):e0177685.
[12]Richards S,Aziz N,Bale S,et al.Standards and Guidelines for the Interpretation of Sequence Variants:A Joint Consensus Recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology[J].Genet Med,2015,17(5):405-424.
[13]Gregory MC,Terreros DA,Barker DF,et al.Alport syndrome--clinical phenotypes,incidence,and pathology[J].Contrib Nephrol,1996,117(117):1-28.
[14]何威,夏正坤,高春林.常见遗传性肾小球疾病的研究进展[J].医学研究生学报,2015,28(3):308-312.
[15]何威,高春林,夏正坤.Alport综合征家系的基因突变及临床表型分析[J].医学研究生学报,2016,29(5):508-513.
[16]Li A,Cui YX,Lv X,et al.The COL4A3 and COL4A4 Digenic Mutations in cis Result in Benign Familial Hematuria in a Large Chinese Family[J].Cytogenet Genome Res,2018,154(3):132-136.
[17]King K,Flinter FA,Green PM,et al.A two-tier approach to mutation detection in the COL4A5 gene for Alport syndrome[J].Hum Mutat,2006,27(10):1061.
[18]Wang F,Zhao D,Ding J,et al.Skin biopsy is a practical approach for the clinical diagnosis and molecular genetic analysis of X-linked Alport's syndrome[J].J Mol Diagn,2012,14(6):586-93.
[19]Goyal S,J?ger M,Robinson PN,et al.Confirmation of TTC8 as a disease gene for nonsyndromic autosomal recessive retinitis pigmentosa(RP51)[J].Clin Genet,2016,89(4):454-460.
[20]Lopez-Bigas N,Audit B,Ouzounis C,et al.Are splicing mutations the most frequent cause of hereditary disease?[J]FEBSLett,2005,579(9):1900-1903.
[21]Anna A,Monika G.Splicing mutations in human genetic disorders:examples,detection,and confirmation[J].J Appl Genet,2018,59(3):253-268.
[22]Warf MB,Berglund JA.Role of RNA structure in regulating premRNA splicing[J].Trends Biochem Sci,2010,35(3):169-178.
[23]Tazónvega B,Ars E,Burset M,et al.Genetic testing for X-linked Alport syndrome by direct sequencing of COL4A5 cDNAfrom hair root RNA samples[J].Am J Kidney Dis,2007,50(2):257.e1-e14.
[24]Malone AF,Funk SD,Alhamad T,et al.Functional assessment of a novel COL4A5 splice region variant and immunostaining of plucked hair follicles as an alternative method of diagnosis in X-linked Alport syndrome[J].Pediatr Nephrol,2017,32(6):997-1003.
[25]Chiereghin C,Robusto M,Mastrangelo A,et al.Alport syndrome cold cases:Missing mutations identified by exome sequencing and functional analysis[J].PLoS One,2017,12(6):e0178630.
[26]Nozu K,Iijima K,Kawai K,et al.In vivo and in vitro splicing assay of SLC12A1 in an antenatal salt-losing tubulopathy patient with an intronic mutation[J].Hum Genet,2009,126(4):533-538.
[27]Nakanishi K,Nozu K,Hiramoto R,et al.A comparison of splicing assays to detect an intronic variant of the OCRL gene in Lowe syndrome[J].Eur J Med Genet,2017,60(12):631-634.
[2]高春林,夏正坤,樊忠民,等.新X连锁缺失突变致Alport综合征的研究[J].医学研究生学报,2015,28(9):929-933.
[3]Hudson BG.The molecular basis of Goodpasture and Alport syndromes:beacons for the discovery of the collagen IV family[J].J Am Soc Nephrol,2004,15(10):2514-2527.
[4]Hudson BG,Tryggvason K,Sundaramoorthy M,et al.Alport‘s syndrome,Goodpasture’s syndrome,and type IV collagen[J].N Engl J Med,2003,348(25):2543-2556.
[5]Barker DF,Hostikka SL,Zhou J,et al.Identification of mutations in the COL4A5 collagen gene in Alport syndrome[J].Science,1990,248(4960):1224-1227.
[6]Pirson Y.Making the diagnosis of Alport's syndrome[J].Kidney Int,1999,56(2):760-775.
[7]Hertz JM,Thomassen M,Storey H,et al.Clinical utility gene card for:Alport syndrome-update 2014[J].Eur J Hum Genet,2015,23(9):e1-e4.
[8]McAlinden A,Majava M,Bishop PN,et al.Missense and nonsense mutations in the alternatively-spliced exon 2 of COL2A1cause the ocular variant of Stickler syndrome[J].Hum Mutat,2008,29(1):83-90.
[9]Wimmer K,Roca X,Beiglbock H,et al.Extensive in silico analysis of NF1 splicing defects uncovers determinants for splicing outcome upon 5'splice-site disruption[J].Hum Mutat,2007,28(6):599-612.
[10]Nozu K,Vorechovsky I,Kaito H,et al.X-linked Alport syndrome caused by splicing mutations in COL4A5[J].Clin J Am Soc Nephrol,2014,9(11):1958-1964.
[11]Liu JH,Wei XX,Li A,et al.Novel mutations in COL4A3,COL4A4,and COL4A5 in Chinese patients with Alport Syndrome[J].PLoS One,2017,12(5):e0177685.
[12]Richards S,Aziz N,Bale S,et al.Standards and Guidelines for the Interpretation of Sequence Variants:A Joint Consensus Recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology[J].Genet Med,2015,17(5):405-424.
[13]Gregory MC,Terreros DA,Barker DF,et al.Alport syndrome--clinical phenotypes,incidence,and pathology[J].Contrib Nephrol,1996,117(117):1-28.
[14]何威,夏正坤,高春林.常见遗传性肾小球疾病的研究进展[J].医学研究生学报,2015,28(3):308-312.
[15]何威,高春林,夏正坤.Alport综合征家系的基因突变及临床表型分析[J].医学研究生学报,2016,29(5):508-513.
[16]Li A,Cui YX,Lv X,et al.The COL4A3 and COL4A4 Digenic Mutations in cis Result in Benign Familial Hematuria in a Large Chinese Family[J].Cytogenet Genome Res,2018,154(3):132-136.
[17]King K,Flinter FA,Green PM,et al.A two-tier approach to mutation detection in the COL4A5 gene for Alport syndrome[J].Hum Mutat,2006,27(10):1061.
[18]Wang F,Zhao D,Ding J,et al.Skin biopsy is a practical approach for the clinical diagnosis and molecular genetic analysis of X-linked Alport's syndrome[J].J Mol Diagn,2012,14(6):586-93.
[19]Goyal S,J?ger M,Robinson PN,et al.Confirmation of TTC8 as a disease gene for nonsyndromic autosomal recessive retinitis pigmentosa(RP51)[J].Clin Genet,2016,89(4):454-460.
[20]Lopez-Bigas N,Audit B,Ouzounis C,et al.Are splicing mutations the most frequent cause of hereditary disease?[J]FEBSLett,2005,579(9):1900-1903.
[21]Anna A,Monika G.Splicing mutations in human genetic disorders:examples,detection,and confirmation[J].J Appl Genet,2018,59(3):253-268.
[22]Warf MB,Berglund JA.Role of RNA structure in regulating premRNA splicing[J].Trends Biochem Sci,2010,35(3):169-178.
[23]Tazónvega B,Ars E,Burset M,et al.Genetic testing for X-linked Alport syndrome by direct sequencing of COL4A5 cDNAfrom hair root RNA samples[J].Am J Kidney Dis,2007,50(2):257.e1-e14.
[24]Malone AF,Funk SD,Alhamad T,et al.Functional assessment of a novel COL4A5 splice region variant and immunostaining of plucked hair follicles as an alternative method of diagnosis in X-linked Alport syndrome[J].Pediatr Nephrol,2017,32(6):997-1003.
[25]Chiereghin C,Robusto M,Mastrangelo A,et al.Alport syndrome cold cases:Missing mutations identified by exome sequencing and functional analysis[J].PLoS One,2017,12(6):e0178630.
[26]Nozu K,Iijima K,Kawai K,et al.In vivo and in vitro splicing assay of SLC12A1 in an antenatal salt-losing tubulopathy patient with an intronic mutation[J].Hum Genet,2009,126(4):533-538.
[27]Nakanishi K,Nozu K,Hiramoto R,et al.A comparison of splicing assays to detect an intronic variant of the OCRL gene in Lowe syndrome[J].Eur J Med Genet,2017,60(12):631-634.