唐氏综合征的基因诊断及其无创性产前基因诊断的初步研究
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摘要
唐氏综合征(Down Syndrome，Ds，又称先天愚型或21三体综合征)是—种最常见的先天性染色体数目畸形，发病率高达0.12％。目前该病的诊断是通过羊水穿刺、绒毛膜活检或脐静脉穿刺术获取胎儿细胞，经培养后进行染色体核型分析。它存在以下缺点：1、羊水细胞培养难度大，周期长(2周多)，不易得到普及而影响产前诊断的开展。2、羊水穿刺等技术是有创性检查，可导致1％的胎儿流产率，故不易被孕妇所接受而漏诊。由于上述原因的影响，我国每年约有25000个DS患儿出生，给家庭和社会带来严重经济负担，并降低国民的人口素质。故迫切需要一种对孕妇无创伤，对胎儿无风险，且能准确快速简便地进行DS诊断的新方法。
短串联重复序列(STR)广泛分布人类基因组中，具有操作简便，分型准确和丰富的多态信息量等特点而成为第二代DNA分析技术的核心。STR位点基因型在正常二倍体胎儿中常表现为1：1的二条带图谱，而三体的胎儿常表现为1：1：1三体图谱或2：1双倍剂量图谱，3条带图谱是诊断三体的金标准，双倍剂量图谱为辅助诊断指标。故在21号染色体上DS关键区内选择10个STR位点(D21S11、D21S1432、D21S1413、D21S1414、D21S1446、D21S1437、D21S2039、D21S1270、D21S141 1、D21S1412)，根据STR基因型的表现，可进行DS的基因诊断。由于不同民族具有不同的基因库，在应用STR位点进行基因诊断时，首先必须阐明该民族的STR结构分布特征。故在北京地区汉族人群中随机抽取200多例外周血对其中未知结构特征的9个STR位点进行PCR-STR分型。根据基因序列，设计1对引物，进行PCR反应，其产物经聚丙烯凝胶电泳和银染后与等位基因标准混合物对比，或运用基因片段扫描技术，确定各个样本的基因型，最后进行统计学处理。结果首次阐明了中国汉族人群该9个STR位点的结构分布符合Hardy-weinberg定律，具有较高的杂合度和丰富的多态信息量，证实为21号染色体的良好的遗传标记，为基因诊断、亲子鉴定等DNA分析技术提供理论和概率计算依据。
联合运用~匕违10个STR位点对23例可疑患者和26例DS筛查出高危孕妇的羊水
细胞进行DS的基因诊断。将10个STR位点的陀R产物用聚丙烯铣胺凝胶电泳和银染
法或基因片段扫描法幽神卸叮，结果从23例可疑患者中发现7例璐患者，其中6例
均出现三条带图谱，1例虽未发现3条带图谱，但除纯合子外其余位点均表现为双倍
剂量(2:1)的2条带图谱。26例胎少公准水细胞盯R基因型均表现为正常2条带图谱
(1:1)，表明为非璐胎儿。将基因诊断结果与染色体核型分析结果对比，二者完全
一致‘但基因诊断仅需耗时2~3天，操作简便，适宜推广应用。以上结果表明联合应
用10个STR位点司稚确、快速简便地进行璐产前基因诊断。国内少数单位曾仅根据
l~2个STR位点的基因型进行基因诊断声聪汰大。国外近几年来采用荧光陀R对1~
4个STR位点基因型幽予分析，根据3条带或2:1的2条带图谱进往基因诊断。为此，
本文在国内首次建立了联合应用10个SIR位点的荧光陀R，几乎使所有的DS患者出
现3条带型而得到明确诊断。
近年来发现孕妇夕调血中存在微壕幼台少嘟胞，本文通过密度梯度离心获取有核血
细胸舌，应用磁激活细胞分选法富集纯化夕调血中的有核红细胞，Kl eihauer染色鉴
定出胎少断育核红细胞(刚砒犯)，应用显微切害g技才彬瞰单个胎儿有核红细胞，进行单
细胞靶基因的预扩增和巢式咒R，最后应用基因片段扫描技术进行检测。靶基因的筛
选是应用唐氏综合症基因诊断中的10个位点，通过卜眺绷台少以姆涂基因型完全不同或
部分不同的盯R位点确定的，例如有3个，这样对刚班犯进行分析时就只作这3个STR
位点的分析，同时也可进一步断定有核红细胞的来源。应用上述方法对20例DS筛查
阳性的孕妇夕调血的胎少嘟胞幽于DS基因诊断，结果均成功分离出脚七有核红细胞，
12例单细胞陀R成功扩增出靶基因片段，结果为非21三体胎儿。其结果与临床细胞
遗传学检查一致，初步表明运用此策略可成功训称陇d性产前DS的基因诊断。无创性
产前DS的单细胞基因诊断国内外未仄甘团道，偶见国夕游7一O个胎儿有核幻细胞混合
在洲起进行天沧d性产前DS基因诊断，但有母喇翻台少断育核乡闯胞污染的可能。本研究
策略在国内外尚属首见，有待于进一步深入研究。
孕妇外周血中除胎少图胞外，其血浆中还存在微量游离的胎儿侧A，是天沧!胜产
前基因诊断的另一重要材料来源。对73例孕妇夕调血浆通过柱分离提取血桨总侧A
后，应用巢式代R进布到治儿性别决定基因(SRY)的扩增，其灵敏度为97.36%，特异
性85.71%，总符合率91 .7少y0(6认勺3)。表明应用孕妇夕调血游翻台儿洲A可进布孙咙d
性产前性别鉴定，有助于性连锁遗传病高危胎儿的早期性拐lJ诊断。
总之，本辛首次阐明了21号染色体上9个弧位点在中国汉族人群中的杂合频
率和多态信息含量，并建立了准确、快速、简便的璐产前基因诊断方注拜口无创性产前
性别基因诊断方法，可及时进行DS的产前诊断和性别鉴定，从而有效预防DS和性连
锁遗传病的患儿出生，具有明显的经济效益和社会效益。本文采用新策略初步对DS
进行无创性产前基因诊
Down syndrome(DS) is the most common congenital chromosome aneuploidies and its incidence reaches to 0.12%. At present, the diagnosis of DS mainly depends on cytogenetic analysis through amniocentesis, chorionic villus sampling or puncture of umbilical vein to recovery fetal cell. There are a few drawbacks: Firstly, the amniocyte culture is quite difficult and it cost more than two weeks to complete. So the prenatal diagnosis of DS is not easily to perform in many hospitals, especially in country hospital. Secondly, those inspection techniques are belong to invasive manipulation which may lead to 1% fetus abortioa So many pregnant women with high risk of DS refuse to accept those inspection. Owing to the above deficiency, the incidence of DS remains in high level every year in China, which give heavy burden for family and country. Under this situation, it is urgent to create a rapid, reliable technique to diagnosis of DS and non-invasive prenatal diagnosis of DS.
Short tandem repeats (STR) exists in widespread genome and easily carry out to judge STR marker. STR marker shows normal diallelic pattern with a ratio of 1:1 or homozygous in normal human beings. Trisomic triallelic pattern is a "Gold standard" for diagnosis of DS and trisomic diallelic pattern with a ratio of 2:1 also is a powerful indicator for DS. Therefore, we can use of DNA polymorphism to diagnosis of DS if we chose some STR loci in or near to DS critical region on chromosome 21. Because different nations have different distribution structures of STR marker, it is premise to elucidate local people's structure of STR loci before genetic diagnosis. More than 200 people without relationship were carried out randomly to elucidate the structure of nine loci of D21S1432, D21S1413, D21S1414, D21S1446, D21S1437, D21S2039, D21S1270, D21S1411 and 21S1412 in Chinese Han population in Beijing. According to sequence of STR loci provided by genebank in MEDLINE, we design nine pair of primers to perform PCR and judg
e genetypes using polyacrylamide gel electrophoresis and silver staining or automated fluorescent laser activated detection sequencer.
The statistical result using SPSS software is in agreement with Haidy-Weinberg equatioa Those 9 STR loci were verified as good genetic markers with high heterozygosity and polymorphism information contents and also provide date for paternity identification.
23 suspects of DS and 26 cases of fetal amniocytes come from pregnant woman with high risk of DS were undertook to analysis 10 STR loci simultaneously by PAGE and silver staining or automated fluorescent laser activated detector sequencer(ABI310). 6 suspects of DS were determined as DS by trisomic triallelic with a ratio of 1:1:1 and the other STR loci show normal diallelic pattern with a ratio of 1:1 or homozygous with two alldes of the same size. The other one suspect determined as DS by 7 STR marker showing trisomic diallelic band or peak. The results of genetic diagnosis were identical to cytogenetic analysis which cost more than two weeks to complete. But it just cost within 2 days to complete the genetic diagnosis. Therefore, genetic diagnosis of DS is a rapid and reliable techniques and it promote development of prenatal diagnosis of DS. But it is risk to carry out gene diagnosis just depended on one or two STR loci. Quantitative fluorescent PCR was used to detect triallellic peaks or trisomic diallelic peaks to diagnosis DS depended on 1 to 4 STR loci. We also established a quantitative fluorescent PCR method to detect triallelic peaks in almost 100% DS depended on 10 STR loci.
In the past few year fetal cells were discovered in maternal blood and then non-invasive prenatal diagnosis was paid more attention. The total nucleated cells were collected by gradient centrifuge from peripheral blood, and then nucleated red blood cells were enriched from the total nucleated cell by magnetic activated cell sorting. The fetal erythrocytes were identified by Kleihauer test and microdissected on slide under micromanipulation. From the single fetal erythrocyt
短串联重复序列(STR)广泛分布人类基因组中，具有操作简便，分型准确和丰富的多态信息量等特点而成为第二代DNA分析技术的核心。STR位点基因型在正常二倍体胎儿中常表现为1：1的二条带图谱，而三体的胎儿常表现为1：1：1三体图谱或2：1双倍剂量图谱，3条带图谱是诊断三体的金标准，双倍剂量图谱为辅助诊断指标。故在21号染色体上DS关键区内选择10个STR位点(D21S11、D21S1432、D21S1413、D21S1414、D21S1446、D21S1437、D21S2039、D21S1270、D21S141 1、D21S1412)，根据STR基因型的表现，可进行DS的基因诊断。由于不同民族具有不同的基因库，在应用STR位点进行基因诊断时，首先必须阐明该民族的STR结构分布特征。故在北京地区汉族人群中随机抽取200多例外周血对其中未知结构特征的9个STR位点进行PCR-STR分型。根据基因序列，设计1对引物，进行PCR反应，其产物经聚丙烯凝胶电泳和银染后与等位基因标准混合物对比，或运用基因片段扫描技术，确定各个样本的基因型，最后进行统计学处理。结果首次阐明了中国汉族人群该9个STR位点的结构分布符合Hardy-weinberg定律，具有较高的杂合度和丰富的多态信息量，证实为21号染色体的良好的遗传标记，为基因诊断、亲子鉴定等DNA分析技术提供理论和概率计算依据。
联合运用~匕违10个STR位点对23例可疑患者和26例DS筛查出高危孕妇的羊水
细胞进行DS的基因诊断。将10个STR位点的陀R产物用聚丙烯铣胺凝胶电泳和银染
法或基因片段扫描法幽神卸叮，结果从23例可疑患者中发现7例璐患者，其中6例
均出现三条带图谱，1例虽未发现3条带图谱，但除纯合子外其余位点均表现为双倍
剂量(2:1)的2条带图谱。26例胎少公准水细胞盯R基因型均表现为正常2条带图谱
(1:1)，表明为非璐胎儿。将基因诊断结果与染色体核型分析结果对比，二者完全
一致‘但基因诊断仅需耗时2~3天，操作简便，适宜推广应用。以上结果表明联合应
用10个STR位点司稚确、快速简便地进行璐产前基因诊断。国内少数单位曾仅根据
l~2个STR位点的基因型进行基因诊断声聪汰大。国外近几年来采用荧光陀R对1~
4个STR位点基因型幽予分析，根据3条带或2:1的2条带图谱进往基因诊断。为此，
本文在国内首次建立了联合应用10个SIR位点的荧光陀R，几乎使所有的DS患者出
现3条带型而得到明确诊断。
近年来发现孕妇夕调血中存在微壕幼台少嘟胞，本文通过密度梯度离心获取有核血
细胸舌，应用磁激活细胞分选法富集纯化夕调血中的有核红细胞，Kl eihauer染色鉴
定出胎少断育核红细胞(刚砒犯)，应用显微切害g技才彬瞰单个胎儿有核红细胞，进行单
细胞靶基因的预扩增和巢式咒R，最后应用基因片段扫描技术进行检测。靶基因的筛
选是应用唐氏综合症基因诊断中的10个位点，通过卜眺绷台少以姆涂基因型完全不同或
部分不同的盯R位点确定的，例如有3个，这样对刚班犯进行分析时就只作这3个STR
位点的分析，同时也可进一步断定有核红细胞的来源。应用上述方法对20例DS筛查
阳性的孕妇夕调血的胎少嘟胞幽于DS基因诊断，结果均成功分离出脚七有核红细胞，
12例单细胞陀R成功扩增出靶基因片段，结果为非21三体胎儿。其结果与临床细胞
遗传学检查一致，初步表明运用此策略可成功训称陇d性产前DS的基因诊断。无创性
产前DS的单细胞基因诊断国内外未仄甘团道，偶见国夕游7一O个胎儿有核幻细胞混合
在洲起进行天沧d性产前DS基因诊断，但有母喇翻台少断育核乡闯胞污染的可能。本研究
策略在国内外尚属首见，有待于进一步深入研究。
孕妇外周血中除胎少图胞外，其血浆中还存在微量游离的胎儿侧A，是天沧!胜产
前基因诊断的另一重要材料来源。对73例孕妇夕调血浆通过柱分离提取血桨总侧A
后，应用巢式代R进布到治儿性别决定基因(SRY)的扩增，其灵敏度为97.36%，特异
性85.71%，总符合率91 .7少y0(6认勺3)。表明应用孕妇夕调血游翻台儿洲A可进布孙咙d
性产前性别鉴定，有助于性连锁遗传病高危胎儿的早期性拐lJ诊断。
总之，本辛首次阐明了21号染色体上9个弧位点在中国汉族人群中的杂合频
率和多态信息含量，并建立了准确、快速、简便的璐产前基因诊断方注拜口无创性产前
性别基因诊断方法，可及时进行DS的产前诊断和性别鉴定，从而有效预防DS和性连
锁遗传病的患儿出生，具有明显的经济效益和社会效益。本文采用新策略初步对DS
进行无创性产前基因诊
Down syndrome(DS) is the most common congenital chromosome aneuploidies and its incidence reaches to 0.12%. At present, the diagnosis of DS mainly depends on cytogenetic analysis through amniocentesis, chorionic villus sampling or puncture of umbilical vein to recovery fetal cell. There are a few drawbacks: Firstly, the amniocyte culture is quite difficult and it cost more than two weeks to complete. So the prenatal diagnosis of DS is not easily to perform in many hospitals, especially in country hospital. Secondly, those inspection techniques are belong to invasive manipulation which may lead to 1% fetus abortioa So many pregnant women with high risk of DS refuse to accept those inspection. Owing to the above deficiency, the incidence of DS remains in high level every year in China, which give heavy burden for family and country. Under this situation, it is urgent to create a rapid, reliable technique to diagnosis of DS and non-invasive prenatal diagnosis of DS.
Short tandem repeats (STR) exists in widespread genome and easily carry out to judge STR marker. STR marker shows normal diallelic pattern with a ratio of 1:1 or homozygous in normal human beings. Trisomic triallelic pattern is a "Gold standard" for diagnosis of DS and trisomic diallelic pattern with a ratio of 2:1 also is a powerful indicator for DS. Therefore, we can use of DNA polymorphism to diagnosis of DS if we chose some STR loci in or near to DS critical region on chromosome 21. Because different nations have different distribution structures of STR marker, it is premise to elucidate local people's structure of STR loci before genetic diagnosis. More than 200 people without relationship were carried out randomly to elucidate the structure of nine loci of D21S1432, D21S1413, D21S1414, D21S1446, D21S1437, D21S2039, D21S1270, D21S1411 and 21S1412 in Chinese Han population in Beijing. According to sequence of STR loci provided by genebank in MEDLINE, we design nine pair of primers to perform PCR and judg
e genetypes using polyacrylamide gel electrophoresis and silver staining or automated fluorescent laser activated detection sequencer.
The statistical result using SPSS software is in agreement with Haidy-Weinberg equatioa Those 9 STR loci were verified as good genetic markers with high heterozygosity and polymorphism information contents and also provide date for paternity identification.
23 suspects of DS and 26 cases of fetal amniocytes come from pregnant woman with high risk of DS were undertook to analysis 10 STR loci simultaneously by PAGE and silver staining or automated fluorescent laser activated detector sequencer(ABI310). 6 suspects of DS were determined as DS by trisomic triallelic with a ratio of 1:1:1 and the other STR loci show normal diallelic pattern with a ratio of 1:1 or homozygous with two alldes of the same size. The other one suspect determined as DS by 7 STR marker showing trisomic diallelic band or peak. The results of genetic diagnosis were identical to cytogenetic analysis which cost more than two weeks to complete. But it just cost within 2 days to complete the genetic diagnosis. Therefore, genetic diagnosis of DS is a rapid and reliable techniques and it promote development of prenatal diagnosis of DS. But it is risk to carry out gene diagnosis just depended on one or two STR loci. Quantitative fluorescent PCR was used to detect triallellic peaks or trisomic diallelic peaks to diagnosis DS depended on 1 to 4 STR loci. We also established a quantitative fluorescent PCR method to detect triallelic peaks in almost 100% DS depended on 10 STR loci.
In the past few year fetal cells were discovered in maternal blood and then non-invasive prenatal diagnosis was paid more attention. The total nucleated cells were collected by gradient centrifuge from peripheral blood, and then nucleated red blood cells were enriched from the total nucleated cell by magnetic activated cell sorting. The fetal erythrocytes were identified by Kleihauer test and microdissected on slide under micromanipulation. From the single fetal erythrocyt
引文
1、Walsh PS, Metzger DA, Higuchi R. Chelex-100 as a mediumfor simple extration of DNA for PCR-based typing from forensic material. Bio Techniques, 1991,10:506-513.
2、郑秀芬主编，概率统计学基础与DNA遗传标记系统参考计算，法医DNA分析，中国人民公安大学出版社，2002年，374-387。
3、候一平，李英碧，唐剑频等，成都汉族群体八个STR基因座遗传多态性研究。中华医学遗传学杂志，2000，17：236-240。
4、李生斌，郑海波，冯继东等，中华民族STR遗传结构及变化规律的研究(Ⅰ)。西安医科大学学报，2000，21：1-5。
5、俞建昆，诸嘉佑，钱亚屏等，应用30个常染色体STR位点研究中国6个民族群体的遗传关系。遗传学报，2001，28：699-706。
2、郑秀芬主编，概率统计学基础与DNA遗传标记系统参考计算，法医DNA分析，中国人民公安大学出版社，2002年，374-387。
3、候一平，李英碧，唐剑频等，成都汉族群体八个STR基因座遗传多态性研究。中华医学遗传学杂志，2000，17：236-240。
4、李生斌，郑海波，冯继东等，中华民族STR遗传结构及变化规律的研究(Ⅰ)。西安医科大学学报，2000，21：1-5。
5、俞建昆，诸嘉佑，钱亚屏等，应用30个常染色体STR位点研究中国6个民族群体的遗传关系。遗传学报，2001，28：699-706。