荧光光谱分析鉴别SNP等位位点比例法产前诊断唐氏综合征方法的建立和应用
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摘要
目的:建立稳定的单碱基延伸结合荧光光谱分析鉴别SNP中2种碱基比例的方法,并应用于羊水标本的检测,探讨该技术产前诊断唐氏综合征的可行性,为最终建立一种简便、可靠的非创伤性诊断唐氏综合征技术打下基础。
方法:选取一个胎盘源性表达的基因PLAC4,利用分子“开/关”技术对其中一个SNP位点rs8130833(A和G)进行特异性扩增,最后采用荧光分光光度计对扩增产物进行光谱分析。在方法稳定的基础上,用于63例唐氏筛查高危孕妇的羊水标本检测,获得诊断唐氏胎儿的置信区间。
结果:在方法建立时,模板中A:G比例分别按1:1,1:2,2:1混匀,扩增后检测产物,A:G荧光光谱强度比例与原反应模板一致,表明我们建立的鉴别SNP等位位点比例的方法是可行的。63例羊水标本,经分子“开/关”鉴定,33例为杂合,进一步检测A:G比例,获得置信区间结果为正常胎儿为0.75-1.29,唐氏患儿(AAG)和(AGG)分别为1.85-2.29和0.39-0.67,与染色体核型分析的对比,诊断特异性达94.1%。
结论:本研究中建立的SNP等位位点比例法产前诊断唐氏综合征简便,直观,特异性强。可进一步优化、完善,用于疾病的非创伤性产前诊断。
Objective: To set up a simple, reliable non-invasive prenatal diagnosis technical for Down’s syndrome eventually, a stable method, which combined single base extension with fluorescence spectrometry, was established to identify SNP allelic ratios, and amniotic fluid samples were used to explore the feasibility of this method for prenatal diagnosing Down’s syndrome.
Methods: A placenta derived expression gene PLAC4 was selected, a SNP loci rs8130833 (A and G) in PLAC4 was amplified by on/off switch to eliminate non-specific amplification. Finally amplified products were analyzed by fluorescence spectrophotometer. On the basis of the method, 63 amniotic fluid samples obtained from Down’s high risk pregnant women were detected to get confidence intervals for diagnosing Down’s syndrome.
Results: When established the method, template’s A:G ratio were mixed as 1:1,1:2,2:1 respectively. After amplification, the products were detected. The A:G ratio of fluorescence intensity was consistent with the ratio of original template, which indicated that the method we established to identify SNP allelic ratio was feasible. Identified by on/off switch, 33 cases were heterozygous in 66 cases of amniotic fluid samples. Through detecting A:G ratio of heterozygous samples, the confidence intervals were obtained, the euploid was 0.75-1.29, Down’s samples(AAG or AGG) were 1.85-2.29 or 0.39-0.67. Compared with karyotype analysis, the specificity was 94.1%.
Conclusion: The method, which we established to identify SNP allelic ratios, was simple, direct and specific, and could be used to diagnose Down’s syndrome. Further optimized, it could be applied for non-invasive prenatal diagnosis.
方法:选取一个胎盘源性表达的基因PLAC4,利用分子“开/关”技术对其中一个SNP位点rs8130833(A和G)进行特异性扩增,最后采用荧光分光光度计对扩增产物进行光谱分析。在方法稳定的基础上,用于63例唐氏筛查高危孕妇的羊水标本检测,获得诊断唐氏胎儿的置信区间。
结果:在方法建立时,模板中A:G比例分别按1:1,1:2,2:1混匀,扩增后检测产物,A:G荧光光谱强度比例与原反应模板一致,表明我们建立的鉴别SNP等位位点比例的方法是可行的。63例羊水标本,经分子“开/关”鉴定,33例为杂合,进一步检测A:G比例,获得置信区间结果为正常胎儿为0.75-1.29,唐氏患儿(AAG)和(AGG)分别为1.85-2.29和0.39-0.67,与染色体核型分析的对比,诊断特异性达94.1%。
结论:本研究中建立的SNP等位位点比例法产前诊断唐氏综合征简便,直观,特异性强。可进一步优化、完善,用于疾病的非创伤性产前诊断。
Objective: To set up a simple, reliable non-invasive prenatal diagnosis technical for Down’s syndrome eventually, a stable method, which combined single base extension with fluorescence spectrometry, was established to identify SNP allelic ratios, and amniotic fluid samples were used to explore the feasibility of this method for prenatal diagnosing Down’s syndrome.
Methods: A placenta derived expression gene PLAC4 was selected, a SNP loci rs8130833 (A and G) in PLAC4 was amplified by on/off switch to eliminate non-specific amplification. Finally amplified products were analyzed by fluorescence spectrophotometer. On the basis of the method, 63 amniotic fluid samples obtained from Down’s high risk pregnant women were detected to get confidence intervals for diagnosing Down’s syndrome.
Results: When established the method, template’s A:G ratio were mixed as 1:1,1:2,2:1 respectively. After amplification, the products were detected. The A:G ratio of fluorescence intensity was consistent with the ratio of original template, which indicated that the method we established to identify SNP allelic ratio was feasible. Identified by on/off switch, 33 cases were heterozygous in 66 cases of amniotic fluid samples. Through detecting A:G ratio of heterozygous samples, the confidence intervals were obtained, the euploid was 0.75-1.29, Down’s samples(AAG or AGG) were 1.85-2.29 or 0.39-0.67. Compared with karyotype analysis, the specificity was 94.1%.
Conclusion: The method, which we established to identify SNP allelic ratios, was simple, direct and specific, and could be used to diagnose Down’s syndrome. Further optimized, it could be applied for non-invasive prenatal diagnosis.
引文
[1]吴刚,伦玉兰.中国优生科学[M].北京:科学技术文献出版社,2000.306.
[2] Tsukahara F,Hattori M,Muraki T,et al.Identification and cloning of a novel cDNA belonging to tetratricopeptide repeat gene family from Down syndrome-critical region 21q22.2[J].J Biochem,1996,120(4):820-7.
[3] Poon LL,Leung TN,Lau TK,et al.Prenatal detection of fetal Down’s syndrome from maternal plasma[J].Lancet,2000,356(9244):1819-20.
[4] Farina A,LeShane ES,Lambert-Messerlian GM,et al.Evaluation of cell-free fetal DNA as a second-trimester maternal serum marker of Down syndrome pregnancy[J].Clin Chem,2003,49(2):239-42.
[5] Poon LL, Leung TN,Lau TK,et al.Presence of fetal RNA in maternal Plasma[J].Clin Chem,2000,46(11):1832-4.
[6] Oudejans CB,Go AT,Visser A,et al.Detection of chromosome 21-encoded mRNA of placental origin in maternal plasma[J].Clin Chem,2003,49(9):1445-9.
[7] Lo YM,Tsui NB,Chiu RM,et al.Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection. NatMed. 2007,13(2):218 -23.
[8]陈琳玲,张佳,彭翠英等.DNA聚合酶高保真机理的新发现及其在SNP分析中的应用[J].遗传,2005,27(2):279-83.
[9] Dutton C,Sommer SS.Simultaneous detection of multiple single-base alleles at a polymorphic site[J].Biotechniques,1991,11(6):700-702.
[10] Ruano G,Kidd KK.Direct haplotyping of chromosomal segments from multiple heterozygotes via allele-specific PCR amplification[J]. Nucleic Acids Res, 1989,17(20)8392.
[11] Chan Yoke F,Tan Kim L,Wong Yean C et al.The use of the amplification refractory mutation system(arms) in the detection of rare beta-thalassemia mutations in the Malays and Chinese in Malaysia[J].Southeast Asian J Trop Med Public Health,2001,32(4):872-9.
[12] Bravo M,Salazar R,Arends A et al.[Detection of beta thalassemia by the technique of refractory amplification of mutation systems (ARMS-PCR)][J]. Invest Clin,1999,40(3):203-13.
[13] Zhang J,Li K,Deng Z,et al. Efficient mutagenesis method for producing the template of single nucleoctide polymoprhisms[J]. Mol Biotechnol,2003,24(2): 105-10.
[14] Zhang J,Chen LL,Guo ZF, et al.On/off switch mediated by exo+ polymerases: experimental analysis for its physiological and technological implications[J].J Biochem Mol Biol,2003,36(6):529-32.
[15] Zhang J,Li K,Pardinas JR,et al. SNP discrimination through proofreading and OFF-switch of exo+ polymerase[J]. Mol Biotechnol.2004:27(1):75-80.
[16] Zhang J and Li K.Single-Base Discrimination Mediated by Proofreading 3' Phos- phorothioate-Modified Primers[J]. Mol Biotechnol.2003,25(3):223-228.
[17] Chehab FF,Kan YM. Detection of specific DNA by fluoresecence amplification :a color complementation assay[J].Proc Natl Acad Sci USA.1989,86(23) : 9178-82.
[18] Go AT,Visser A,Betsale OT,et al. Measurement of allelic-expression ratios in tr- isomy21 placentas by quencher extension of heterozygous samples identified by partially denauturing HPLC[J].Clin Chem,2008,54(2):437-40.
[19] Chehab FF,Kan YM. Detection of specific DNA by fluoresecence amplification: a color complementation assay[J].Proc Natl Acad Sci USA.1989,86(23) : 9178-82.
[20] McClay JL,Sugden K,Koch HG,et al. High-throughput single-nucleotide polymorphism genotyping by fluorescent competitive allele-specific polymerase chain reaction(SniPTag)[J].Anal Biochem.2002,301(2):200-6.
[21] Pont-Kingdon G, Lyon E.Rapid detection of aneuploidy(trisomy 21) by allele quantification combined with melting curve analysis of single- nucleotide polymorphism loci[J].Clin Chem,2003,49(7):1087-94.
[22] Tsui NB,Chim SS,Chiu RW,et al.Systematic micro-array based identification of placental mRNA in maternal plasma:towards non-invasive prenatal gene expres- sion profiling[J].J Med Genet.2004,41(6):461-7.
[23] Go AT,vissei A,Mulder MA,Blankenstein MA,et al. 44 single-nucleotidepolymorphisms expressed by placental RNA: assessment for use in noninvasive prenatal diagnosis of trisomy21[J]. Clin Chem. 2007,53(12):2223-4.
[24] Tong YK,Ding C,Chiu RW,et al.Noninvasive prenatal detection of fetal trisomy 18 by epigenetic allelic ratio analysis in maternal plasma:Theoretical and empirical considerations[J].Clin Chem,2006,52(12):2194-202.
[1] Zijno A ,Andreoli C ,Leopardi P ,et al. Folate status,metabolic genotype and biomarkers of genotoxicity in healthy subjects[J].Carcinogenesis, 2003,24(6): 1097- 103.
[2] Noble J . Natural history of Down's Syndrome:a brief review for those involved in antenatal screening[J].J Med Screen, 1998,5(4) :172-7.
[3] Eiben B,Glaubitz R.First-trimester screening:an overview[J].J Histochem Cytochem,2005,53(3):281-3.
[4] Yraon Y,Wolman l,Kupferminc MJ,et al. Effect of fetal gender on first trimester markers and on Down syndrome screening[J].Prenat Diagn,2001,21(12):1027-30.
[5] Karidas CN,Michailidis GD,Spencer K,et al. Biochemical screening for Down Syndrome in pregnancy following renal transplantation[J].Prenat Diagn,2002,22 (3),226-30.
[6] Spencer K,Liao AW,Ong CY,et a1.Maternal serum activin A and inhibin A in trisomy 18 pregnancies at 10-14 weeks[J].Prenat Diagn,2001,21(7):571-4.
[7] Spencer K,Liao AW,Ong CY,et a1. Maternal serum levels of dimeric inhibin A in pregrance affected by trisomy21 in the first trimester[J].Prenat Diagn,2001,21 (6):441-44.
[8] Knight GJ, Palomaki GE,Neveux LM,et al. Clinical validation of new dimeric inhibin-A assay suitable for second trimester Donw’s syndrome screening[J].J Med screen,2001,8(1):2-7.
[9] Agostino O,Ciuli R,Tozzi P,et al.Maternal serum superoxide disumtase(SOD) : Apossible marker for screening Down Syndrome affected pregnancy[J].Prenat Diagn.1999, 19(11):1058-106.
[10] Spencer K,Liao AW,Ong CY,et al. Firster trimester maternal serum placenta growth factor(PLGF) concentration in pregnancy with fetal trimosy21 or trimosy18[J].Prenat Diagn.2001,21 (9):718-722.
[11] Su YN,Hsu JJ,Lee CN,et al. Raised matenral serum placent growth fator concent- ration during the second trimester durign the second trimester isassociated with Down syndrome[J].Prenat Diagn,2002,22(1):8-12.
[12] Zhou QC,Zhang J,Zhang M,et al.Utilising ducrus venosus Dorrpler wave form and four-chamber view to screen for fetal cardiac malformation in early second trimester of pregrancy[J].Chin Med,2005,118(21):1971-6.
[13] Locatelli A,Piccoli MG,Vergani P,et al. Critical apppraisal of the use of nuchal f- old thickness measurement for the prediction of Down’s syndrome[J].Am J Obstet Gynecol,2001,182(1 Pt 1):192-7.
[14] Bromley B,Shipp T,Benaerraf BR. Genetic sonogram scoring index: accuracy a- nd clinical utility[J].J Ultrasound Med,1999,18(8):523-8;quiz 529-30.
[15] Filly RA. Obstetrieal sonography: the best way to terrify a pregnant woman[J].J Ultrasound Med,2000,19(1):1-5.
[16] Vergani P, Locatelli A, Piccoli GM,et al. Best second trimester sonographic mar- kers for the detection of trisomy21[J].J Ultrasound Med,1999,18(7):469-73.
[17] Cicero S, Curcio P, Papageorghiou A,et al. Absence of nasal bone in fetuses with trisomy 21 at 11-14 weeks of gestation: an observational study[J].Lancet,2001, 358(9296):1665-7.
[18] Niemimaa M, Suonp?? M, Perheentupa A,et al. Evaluation of first trimester maternal serum and ultrasound screening for Down's syndrome in Eastern and Northern Finland[J].Eur J Hum Genet,2001,9(6):404-8.
[19] Meier C, Huang T, Wyatt PR,et al. Accuracy of expected risk of Down syndrome using the second-trimester triple test[J].Clin Chem.2002,48(4):653-5.
[20]李文典,吴玉萍,叶兹礼等.染色体13/21α卫星探针用于产前诊断21三体综合征[J].中华妇产科杂志,2001,36(2):76-8.
[21] Bryndorf T,Christensen B,Vad M,et al.Prenatal detection of chromosome aneuploidies aneuploidies in uncultured chorionic villus samples by FISH[J].Am J Hum Genet,1996,59(4):918-26.
[22] Witters I ,Devriendt K,Legius E,et al. Rapid prenatal diagnosis of trisomy21 in 5049 cosecutive unclultured amniotic fluid samples by fluorescence in situ hybridizatio (FISH)[J].Prenat Diagn ,2002 ,22 (1):29-33.
[23] Pellestor F ,Girardet A ,Lefort G,et al . Rapid in situ detection of chromosome 21 by PRINS technique[J].Am J Hum Genet,1995,56(4):393-7.
[24] Lee MH, Ryu HM, Kim DJ, et al. Rapid prenatal diagnosis of Down Syndrome using quantitative fluorescent PCR in uncultured amniocytes[J].J Korean Med Sci, 2004,19 (3):341-4.
[25] Pont-Kingdon G, Lyon E. Rapid detection of aneuploidy (trisomy 21) by allele quantification combined with melting curves analysis of single-nucleotide polymorphism loci[J].Clin Chem,2003,49(7):1087-94.
[26] Lo YM,Tsui NB,Chiu RM,et al. Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection[J].Nat Med, 2007,13(2) :218-23.
[27] Lee H,Chang JG,Lin SP,et al. Rapid detection of trisomy 21 by homologous gene quantitative PCR(HGQ-PCR)[J].Hum Gene,1997,99(3):364-7.
[28] Schouten JP, McElgunn CJ, Waaijer R,et al. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification[J].Nucleic Acids Res,2002,30(12):e57.
[29] Hochstenbach R, Meijer J, van de Brug J, et al. Rapid detection of chromosomal aneuploidies in uncultured amniocytes by multiplex ligation-dependent probe amplification(MLPA)[J].Prenat Diagn,2005,25(11):1032-9.
[30] Lapierre JM, Cacheux V, Luton D,et al. Analysis of uncultured amniocytes by comparative genomic hybridization: a prospective prenatal study[J].Prenat Diagn,2000,20(2):123-31.
[31]季修庆,邵振堂.染色体综合征的产前诊断分子技术研究进展[M].国外医学妇幼保健分册. 2003,14 (5) :292-4.
[32] Belaud-Rotureau MA,Elghezal H,Bernardin C,et al.Spectral karyotyping(SKY) principle,avantages and limitation[J].Ann Biol Clin,2003,61(2):139-46.
[2] Tsukahara F,Hattori M,Muraki T,et al.Identification and cloning of a novel cDNA belonging to tetratricopeptide repeat gene family from Down syndrome-critical region 21q22.2[J].J Biochem,1996,120(4):820-7.
[3] Poon LL,Leung TN,Lau TK,et al.Prenatal detection of fetal Down’s syndrome from maternal plasma[J].Lancet,2000,356(9244):1819-20.
[4] Farina A,LeShane ES,Lambert-Messerlian GM,et al.Evaluation of cell-free fetal DNA as a second-trimester maternal serum marker of Down syndrome pregnancy[J].Clin Chem,2003,49(2):239-42.
[5] Poon LL, Leung TN,Lau TK,et al.Presence of fetal RNA in maternal Plasma[J].Clin Chem,2000,46(11):1832-4.
[6] Oudejans CB,Go AT,Visser A,et al.Detection of chromosome 21-encoded mRNA of placental origin in maternal plasma[J].Clin Chem,2003,49(9):1445-9.
[7] Lo YM,Tsui NB,Chiu RM,et al.Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection. NatMed. 2007,13(2):218 -23.
[8]陈琳玲,张佳,彭翠英等.DNA聚合酶高保真机理的新发现及其在SNP分析中的应用[J].遗传,2005,27(2):279-83.
[9] Dutton C,Sommer SS.Simultaneous detection of multiple single-base alleles at a polymorphic site[J].Biotechniques,1991,11(6):700-702.
[10] Ruano G,Kidd KK.Direct haplotyping of chromosomal segments from multiple heterozygotes via allele-specific PCR amplification[J]. Nucleic Acids Res, 1989,17(20)8392.
[11] Chan Yoke F,Tan Kim L,Wong Yean C et al.The use of the amplification refractory mutation system(arms) in the detection of rare beta-thalassemia mutations in the Malays and Chinese in Malaysia[J].Southeast Asian J Trop Med Public Health,2001,32(4):872-9.
[12] Bravo M,Salazar R,Arends A et al.[Detection of beta thalassemia by the technique of refractory amplification of mutation systems (ARMS-PCR)][J]. Invest Clin,1999,40(3):203-13.
[13] Zhang J,Li K,Deng Z,et al. Efficient mutagenesis method for producing the template of single nucleoctide polymoprhisms[J]. Mol Biotechnol,2003,24(2): 105-10.
[14] Zhang J,Chen LL,Guo ZF, et al.On/off switch mediated by exo+ polymerases: experimental analysis for its physiological and technological implications[J].J Biochem Mol Biol,2003,36(6):529-32.
[15] Zhang J,Li K,Pardinas JR,et al. SNP discrimination through proofreading and OFF-switch of exo+ polymerase[J]. Mol Biotechnol.2004:27(1):75-80.
[16] Zhang J and Li K.Single-Base Discrimination Mediated by Proofreading 3' Phos- phorothioate-Modified Primers[J]. Mol Biotechnol.2003,25(3):223-228.
[17] Chehab FF,Kan YM. Detection of specific DNA by fluoresecence amplification :a color complementation assay[J].Proc Natl Acad Sci USA.1989,86(23) : 9178-82.
[18] Go AT,Visser A,Betsale OT,et al. Measurement of allelic-expression ratios in tr- isomy21 placentas by quencher extension of heterozygous samples identified by partially denauturing HPLC[J].Clin Chem,2008,54(2):437-40.
[19] Chehab FF,Kan YM. Detection of specific DNA by fluoresecence amplification: a color complementation assay[J].Proc Natl Acad Sci USA.1989,86(23) : 9178-82.
[20] McClay JL,Sugden K,Koch HG,et al. High-throughput single-nucleotide polymorphism genotyping by fluorescent competitive allele-specific polymerase chain reaction(SniPTag)[J].Anal Biochem.2002,301(2):200-6.
[21] Pont-Kingdon G, Lyon E.Rapid detection of aneuploidy(trisomy 21) by allele quantification combined with melting curve analysis of single- nucleotide polymorphism loci[J].Clin Chem,2003,49(7):1087-94.
[22] Tsui NB,Chim SS,Chiu RW,et al.Systematic micro-array based identification of placental mRNA in maternal plasma:towards non-invasive prenatal gene expres- sion profiling[J].J Med Genet.2004,41(6):461-7.
[23] Go AT,vissei A,Mulder MA,Blankenstein MA,et al. 44 single-nucleotidepolymorphisms expressed by placental RNA: assessment for use in noninvasive prenatal diagnosis of trisomy21[J]. Clin Chem. 2007,53(12):2223-4.
[24] Tong YK,Ding C,Chiu RW,et al.Noninvasive prenatal detection of fetal trisomy 18 by epigenetic allelic ratio analysis in maternal plasma:Theoretical and empirical considerations[J].Clin Chem,2006,52(12):2194-202.
[1] Zijno A ,Andreoli C ,Leopardi P ,et al. Folate status,metabolic genotype and biomarkers of genotoxicity in healthy subjects[J].Carcinogenesis, 2003,24(6): 1097- 103.
[2] Noble J . Natural history of Down's Syndrome:a brief review for those involved in antenatal screening[J].J Med Screen, 1998,5(4) :172-7.
[3] Eiben B,Glaubitz R.First-trimester screening:an overview[J].J Histochem Cytochem,2005,53(3):281-3.
[4] Yraon Y,Wolman l,Kupferminc MJ,et al. Effect of fetal gender on first trimester markers and on Down syndrome screening[J].Prenat Diagn,2001,21(12):1027-30.
[5] Karidas CN,Michailidis GD,Spencer K,et al. Biochemical screening for Down Syndrome in pregnancy following renal transplantation[J].Prenat Diagn,2002,22 (3),226-30.
[6] Spencer K,Liao AW,Ong CY,et a1.Maternal serum activin A and inhibin A in trisomy 18 pregnancies at 10-14 weeks[J].Prenat Diagn,2001,21(7):571-4.
[7] Spencer K,Liao AW,Ong CY,et a1. Maternal serum levels of dimeric inhibin A in pregrance affected by trisomy21 in the first trimester[J].Prenat Diagn,2001,21 (6):441-44.
[8] Knight GJ, Palomaki GE,Neveux LM,et al. Clinical validation of new dimeric inhibin-A assay suitable for second trimester Donw’s syndrome screening[J].J Med screen,2001,8(1):2-7.
[9] Agostino O,Ciuli R,Tozzi P,et al.Maternal serum superoxide disumtase(SOD) : Apossible marker for screening Down Syndrome affected pregnancy[J].Prenat Diagn.1999, 19(11):1058-106.
[10] Spencer K,Liao AW,Ong CY,et al. Firster trimester maternal serum placenta growth factor(PLGF) concentration in pregnancy with fetal trimosy21 or trimosy18[J].Prenat Diagn.2001,21 (9):718-722.
[11] Su YN,Hsu JJ,Lee CN,et al. Raised matenral serum placent growth fator concent- ration during the second trimester durign the second trimester isassociated with Down syndrome[J].Prenat Diagn,2002,22(1):8-12.
[12] Zhou QC,Zhang J,Zhang M,et al.Utilising ducrus venosus Dorrpler wave form and four-chamber view to screen for fetal cardiac malformation in early second trimester of pregrancy[J].Chin Med,2005,118(21):1971-6.
[13] Locatelli A,Piccoli MG,Vergani P,et al. Critical apppraisal of the use of nuchal f- old thickness measurement for the prediction of Down’s syndrome[J].Am J Obstet Gynecol,2001,182(1 Pt 1):192-7.
[14] Bromley B,Shipp T,Benaerraf BR. Genetic sonogram scoring index: accuracy a- nd clinical utility[J].J Ultrasound Med,1999,18(8):523-8;quiz 529-30.
[15] Filly RA. Obstetrieal sonography: the best way to terrify a pregnant woman[J].J Ultrasound Med,2000,19(1):1-5.
[16] Vergani P, Locatelli A, Piccoli GM,et al. Best second trimester sonographic mar- kers for the detection of trisomy21[J].J Ultrasound Med,1999,18(7):469-73.
[17] Cicero S, Curcio P, Papageorghiou A,et al. Absence of nasal bone in fetuses with trisomy 21 at 11-14 weeks of gestation: an observational study[J].Lancet,2001, 358(9296):1665-7.
[18] Niemimaa M, Suonp?? M, Perheentupa A,et al. Evaluation of first trimester maternal serum and ultrasound screening for Down's syndrome in Eastern and Northern Finland[J].Eur J Hum Genet,2001,9(6):404-8.
[19] Meier C, Huang T, Wyatt PR,et al. Accuracy of expected risk of Down syndrome using the second-trimester triple test[J].Clin Chem.2002,48(4):653-5.
[20]李文典,吴玉萍,叶兹礼等.染色体13/21α卫星探针用于产前诊断21三体综合征[J].中华妇产科杂志,2001,36(2):76-8.
[21] Bryndorf T,Christensen B,Vad M,et al.Prenatal detection of chromosome aneuploidies aneuploidies in uncultured chorionic villus samples by FISH[J].Am J Hum Genet,1996,59(4):918-26.
[22] Witters I ,Devriendt K,Legius E,et al. Rapid prenatal diagnosis of trisomy21 in 5049 cosecutive unclultured amniotic fluid samples by fluorescence in situ hybridizatio (FISH)[J].Prenat Diagn ,2002 ,22 (1):29-33.
[23] Pellestor F ,Girardet A ,Lefort G,et al . Rapid in situ detection of chromosome 21 by PRINS technique[J].Am J Hum Genet,1995,56(4):393-7.
[24] Lee MH, Ryu HM, Kim DJ, et al. Rapid prenatal diagnosis of Down Syndrome using quantitative fluorescent PCR in uncultured amniocytes[J].J Korean Med Sci, 2004,19 (3):341-4.
[25] Pont-Kingdon G, Lyon E. Rapid detection of aneuploidy (trisomy 21) by allele quantification combined with melting curves analysis of single-nucleotide polymorphism loci[J].Clin Chem,2003,49(7):1087-94.
[26] Lo YM,Tsui NB,Chiu RM,et al. Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection[J].Nat Med, 2007,13(2) :218-23.
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