应用引物原位标记技术快速检测18三体综合征

目的　探索应用改良的引物原位标记技术快速检测未培养羊水细胞间期核的可能性。方法采用改良的引物原位标记 (primed in situ labeling,PRINS)技术 ,对 2 6 2份羊水细胞中 18号染色体进行分析检测。结果　在未培养羊水细胞间期核和培养细胞的中期分裂相中 ,PRINS技术均能特异性地检测 18号染色体 ,PRINS反应的成功率为 95 %。并发现两个样本为 18三体综合征。结论　引物原位标记技术是一种简便、快速、经济的染色体检测方法 ,具有较强的敏感性和特异性 ,有助于对传统细胞遗传学的分析。     

应用双色引物原位标记技术快速检测X和Y染色体

目的发展快速检测染色体的技术，探索应用改良的双色引物原位标记（primed in situlabeling，PRINS）技术快速检测未培养细胞间期核可能性。方法采用改良的双色技术，对205份羊水细胞中X和Y染色体进行分析检测。结果在未培养羊水细胞间期核和培养细胞的中期分裂相中，PRINS技术均能特异性地检测X和Y染色体，PRINS反应的成功率为98％，1个样本检出为47，XXY。结论双色引物原位标记技术是一种快速、简便、经济的染色体检测方法，具有较强的特异性和敏感性，有助于快速诊断染色体畸变。     

人类中期染色体三色引物原位标记技术研究

目的探索应用优化的三色引物原位标记技术同时标记多条人类外周血淋巴细胞培养中期染色体的可行性。方法采用双色引物原位标记预实验检测外周血中期淋巴细胞的X、Y染色体以摸索和优化引物应用条件和反应顺序。以优化的三色技术同时标记正常样本及克氏综合征样本(47,XXY)的18、X、Y染色体以验证其有效性。结果在3h之内特异性的同时标记了18、X、Y染色体,双色和三色反应的标记率均达到90%以上。克氏综合征样本准确显示了2条18号染色体,2条X染色体和1条Y染色体。结论非ddNTP阻断的多色引物原位标记技术能够快速而准确地判别染色体数目异常的外周血淋巴细胞样本,原位标记由于有内对照而更加具有说服力。     

应用荧光原位杂交技术诊断未培养羊水细胞染色体异常

目的建立运用荧光原位杂交技术(fluorescence in situ hybridization,FISH)检测未培养羊水细胞染色体异常的方法。方法对24例未培养羊水细胞进行FISH检测,其中用21号染色体荧光探针检测19例,X/Y染色体探针检测3例,13、18号染色体探针各检测1例。结果24例产前诊断者未培养羊水细胞检测出一例21三体。X/Y染色体探针检测时1例为2个绿色的X信号,2例均为1个红色Y信号和1个绿色X信号。检测结果与培养后羊水细胞检测结果一致,并与外周血染色体核型检测结果相符。结论本研究方法快速、简便、准确可靠,适于临床运用推广。     

89份羊水细胞的荧光原位杂交分析

目的 探讨荧光原位杂交(fluorescent in situ hybridization,FISH)技术在产前诊断中的应用价值.方法 应用FISH对82例未培养的羊水细胞、2例培养成功的羊水细胞上清液及5例培养未成功的羊水细胞上清液进行染色体非整倍体检测,以其中79例未培养羊水细胞和2例培养成功羊水细胞的常规G显带核型分析结果作为对照.结果 89例标本均成功杂交,共检出21-三体3例,超雄综合征1例,三倍体1例.羊水培养细胞核型分析结果与FISH检测结果一致.结论 将FISH应用于染色体非整倍体的产前检测具有快速、直观的优点.FISH也可作为羊水标本未培养成功的补救诊断措施.     

荧光原位杂交技术在未培养羊水快速产前诊断中的应用

目的建立运用荧光原位杂交技术（fluorescence in situ hybridization,FISH）检测未培养羊水细胞染色体数目的产前诊断方法。方法对100例妊娠15—26周抽取的羊水未经培养进行FISH检测,均采用13、21、18、X、Y号染色体荧光探针检测,同时行羊水培养染色体核型分析,比较两种检测方法结果的一致性。结果100例产前诊断者中未培养羊水细胞FISH检测均于48h内完成,发现6例21-三体和1例18-三体,与羊水培养染色体核型分析结果一致,后者显示6例21-三体中4例为完全型、2例为易位型21-三体。结论FISH技术与传统的羊水培养染色体核型分析相比较,具有方法快速、简便、准确可靠的特点,但无法完全取代传统的染色体核型分析,应两者结合应用于临床。     

3728例遗传咨询者羊水细胞产前诊断结果分析

目的探讨羊水细胞在染色体疾病产前诊断的应用价值。方法抽取孕中期孕妇羊水,采用羊水细胞原位培养、染色体制备、核型分析。结果3728例标本,培养成功率为99.5%,检出异常核型108例。结论采用羊水细胞原位培养,在产前诊断的应用中获得了满意的结果;有针对性地进行产前诊断,可有效地控制和减少出生缺陷的发生。     

淮安地区322例羊水细胞荧光原位杂交技术与核型分析结果分析

目的探讨荧光原位杂交(fluorescence in situ hybridization,FISH)技术结合羊水细胞培养在检测胎儿染色体异常中的临床应用价值。方法用FISH技术检测322例孕17-28周孕妇的未培养羊水细胞,同时进行常规羊水细胞培养核型分析,将两者结果进行对照分析。结果 322例未培养羊水细胞FISH检测成功321例,其中检出正常303例,数目异常18例,与常规细胞培养核型分析结果一致,另外4例正常变异、1例嵌合体以及1例罗伯逊易位型21三体未被FISH技术检测出。结论 FISH技术检测未培养羊水细胞染色体数目异常具有快速、简便、所用样本量少的优势,可以作为羊水细胞染色体核型分析的补充,但对于检测同源罗伯逊易位型21三体有一定局限性。     

应用引物原位标记技术快速检测X和18号染色体

目的为发展快速检测人类染色体的技术。方法应用原位引物标记（ＰＲＩＮＳ）在８例女性外周血淋巴细胞培养标本中检测了Ｘ、１８号染色体。结果在间期核及分裂相中均能特异地检测Ｘ、１８号染色体，Ｘ染色体的标记效率为８４％～９２％（平均８９％），１８号染色体为７３％～８７％（平均８４％）。标本经适当浓度的蛋白酶Ｋ处理后可以明显提高标记效率，增强标记信号。标记反应可利用原位ＰＣＲ仪进行，不到一小时即可完成。结论原位引物标记是一种快速、特异的染色体检测方法，利用这一技术将有可能在产前快速诊断染色体非整倍体。     

应用引物原位标记技术快速诊断先天愚型

目的 :为发展快速诊断先天愚型的方法 ,方法 :利用 2 1号染色体α -卫星序列 ,设计特异性引物直接在分裂间期和中期细胞上作引物原位标记。结果 :先天愚型三体型间期细胞核中和中期染色体上观察到 3个标记信号。正常对照组为2个标记信号 ,平均标记效率分别分 90 .8%、91.3%。先天愚型嵌合型显示 2个和 3个标记信号 ,并且各占一定的比例。结论 :引物原位标记技术可直接在淋巴细胞间期核中进行 ,4～ 5h内能对先天愚型患者作出快速、准确的诊断。为先天愚型的快速产前诊断开辟了新的途径     

X-连锁肾上腺脑白质营养不良产前诊断探讨

目的探讨X-连锁肾上腺脑白质营养不良(adrenoleukodystrophy,ALD)产前诊断的方法。方法应用气相色谱-质谱联用法对17例ALD高危孕妇进行了18次羊水细胞中极长链脂肪酸(very long chain fatty acids,VLCFAs)水平测定,其中8例胎儿出生后或引产后进行了血浆VLCFAs水平检测。应用PCR和测序方法对8例胎儿羊水细胞或生后外周血细胞DNA进行了基因突变分析(其中4例羊水细胞VLCFAs水平增高,4例VLCFAs正常)。应用Western杂交对同一家系的两例胎儿羊水细胞进行了ALD蛋白(ALD protein,ALDP)的检测(两例胎儿VLCFAs均增高,1例女性,1例男性)。结果18例胎儿中,11例羊水细胞VLCFAs水平正常,7例增高(3例男性,4例女性)。8例胎儿出生后或引产后血浆VLCFAs水平检测,3例增高,5例正常,与产前诊断结果相一致。其中4例羊水细胞VLCFAs水平增高的胎儿,均有ABCD1基因突变,4例羊水细胞VLCFAs水平正常者,均未发现突变。VLCFAs增高的男性胎儿,未检测到ALDP,VLCFAs增高的女性胎儿,可检测到ALDP。结论羊水细胞中VLCFAs水平检测可以准确地进行X-ALD产前诊断,结合基因突变分析及ALDP的测定,可进一步保证产前诊断的准确性。     

Telomere aggregates in trisomy 21 amniocytes

Trisomy 21 is the most common chromosomal abnormality among persons with intellectual disability, with a live birth rate of 1 in 800–1,000. As such, this abnormality may serve as a model for human disorders that result from supernumerary copies of a genomic region. Down syndrome carries an increased risk of developing acute leukemia and other malignancies. Telomeres of tumor cells nuclei tend to form aggregates (TA). This study evaluated TA formation in amniocytes from trisomy 21 pregnancies, compared with amniocytes from normal euploid pregnancies. A commercially available peptide nucleic acid telomere kit was used to evaluate TA formation, using two-dimensional fluorescence microscopy. Significantly higher frequencies of TA were found in trisomy 21 amniocytes than in amniocytes from normal pregnancies. The TAs found in trisomy 21 amniocytes apparently represent an additional parameter that reflects the high genetic instability of this syndrome and its recognized predisposition to develop leukemia and other malignancies.     

光原位杂交技术与羊水染色体核型分析在产前诊断中的作用和意义

目的探讨荧光原位杂交技术（fluorescence in situ hybridization,FISH）与羊水染色体核型分析在产前诊断中的应用价值。方法对未培养羊水间期细胞进行荧光原位杂交;同时进行羊水细胞培养和染色体核型分析,比较两种方法所得到的结果。结果在染色体核型正常的标本中,也可以出现少量带有异常杂交信号的核。这个研究中对1例21-三体的胎儿作出正确诊断。但FISH对1例染色体结构异常胎儿无法诊断。结论FISH技术对检出染色体非整倍体有较强的特异性。较适用于高龄妇女的产前诊断。对于胎儿有B超异常和有过流产史的孕妇应采取染色体核型分析进行产前诊断。     

Difference in constitutive heterochromatin behaviour between human amniocytes and lymphocytes detected by a sequential in situ exonuclease III digestion-random primer extension procedure.

Fixed chromosomes from human amniotic fluid cells and peripheral blood lymphocytes were digested in situ with exonuclease III and the single stranded DNA obtained was used as template for an in situ random primer extension. Under these conditions an R banding pattern, more evident in lymphocytes than in amniocytes, was obtained. Nevertheless, constitutive heterochromatin of chromosomes 1, 16, Yq, and mainly the pericentromeric region of chromosome 9 was far more intensely labelled in amniocytes than in lymphocytes. Fluorescence in situ hybridisation with a specific classical satellite DNA probe, showed that this differential labelling was dependent on a greater sensitivity of chromosome 9 constitutive heterochromatin to exonuclease III digestion in amniocytes than in lymphocytes, thus indicating qualitative differences in this region between both human cellular materials.     

An improved method for the detection of Down's syndrome aneuploidy in uncultured amniocytes

We report a modified method for the rapid detection of aneuploidies directly on human uncultured amniocytes that simplifies and shortens the entire experimental procedure, yielding signals which allow correct diagnosis of trisomy 21 in 97% of cases. The improvement is based on two points: 1) use of cosmid pockets specific for the Down's syndrome minimal region as FISH probes, and 2) a modified protocol for the fixation and preparation of amniocytes.     

Second trimester prenatal diagnosis of the fragile X

The fra(X) chromosome was detected in 5 samples of amniotic fluid cells in a series of 23 pregnancies at risk. The prenatal results were confirmed in 2 male abortuses, one with a relatively high and one with a very low frequency of expression in both amniocytes and fetal tissue. In a third male fetus with low expression in amniocytes, the fra(X) was not detected in the fetal tissues tested. In another male with low expression in amniocytes the fra(X) was not detected after birth. In one female with a low expression in amniocytes, a very high frequency (28%) was detected in cord blood after birth. Low expression of the fra(X) was found in a 4-year-old normally developed girl, where the prenatal results had been negative. In 4 males and 4 females the negative prenatal diagnoses were confirmed after birth. This study indicates that prenatal diagnosis of the fragile X after amniocentesis may be complicated, either due to technical problems related to the use of amniotic fluid cells, or due to genetic heterogeneity, or both. Part of this heterogeneity could be due to the existence of normal male transmitters. Also, it seems that the frequency of expression in amniocytes from female carriers can not be used for the prediction of the frequency in blood after birth.     

Prenatal diagnosis of congenital myotonic dystrophy and counseling of the pregnant mother: Case report and literature review

The molecular basis of the myotonic dystrophy (MD) kinase gene is expansion of the CTG repeat at the 3′-untranslated region of the MD gene. Variability of the CTG repeat size in different tissues of affected individuals has been demonstrated. The objective of this report was to examine and review the feasibility of prenatal diagnosis of congenital myotonic dystrophy (CMD) in pregnant women with MD using CTG repeat sizes in amniocytes or villi. We present a case of a pregnant woman with MD who underwent prenatal diagnosis of MD using amniocytes. The repeat size in the amniocytes was smaller than the repeat size in the maternal leukocytes and smaller than the repeat size in the infant blood. The infant had CMD. We also reviewed the literature for reports on MD cases that were prenatally tested for CTG repeat size using amniocytes or chorionic villi. Data were tabulated based on the number of maternal CTG repeats, prenatal procedure [amniocentesis or chorionic villus sampling (CVS)], CTG repeat size in fetal tissue, fetal/infant blood, and pregnancy outcome. Twenty-seven pregnancies at risk for MD that underwent prenatal diagnosis were reported. Eleven (40.7%) of the 27 pregnancies underwent amniocentesis, and 16 (59.3%) underwent CVS. Fourteen patients (61%) demonstrated an increase in CTG repeat size in the amniocytes or villi compared with the maternal repeat size. Nine (33%) of the 27 pregnancies were terminated because of CMD risk. The outcomes of 11 (40.7%) pregnancies were consistent with diagnosis of CMD. CMD was diagnosed in fetuses demonstrating expansion or contraction of the CTG mutation in the amniocytes. Prenatal diagnosis of MD is possible by using mutation analysis on maternal and fetal DNA and detection of the CTG repeat expansion. Prenatal diagnosis of CMD is more complex. The possible lack of correlation between CTG repeat size in amniocytes, villi, and other fetal tissues is a potential limitation in prenatal diagnosis and counseling of CMD using CTG repeat size. Thus, prenatal diagnosis of CMD should be based on a combination of factors, including maternal pregnancy history, clinical findings, and cautious interpretation of maternal and fetal DNA analysis. Am. J. Med. Genet. 78:250–253, 1998. © 1998 Wiley-Liss, Inc.     

Phosphorylation of 3'-azidothymidine in maternal and fetal peripheral blood mononuclear cells during gestation and at term

The present study compared the phosphorylation rate of 3'-azidothymidine (AZT) in isolated maternal and fetal peripheral blood mononuclear cells (PBMCs) with that in amniocytes obtained during gestation and at term. Maternal PBMCs were isolated from venous blood samples obtained from HIV-seronegative pregnant women during delivery. Immediately after delivery, cord blood specimens were collected, and fetal PBMCs were isolated. In a separate set of experiments, maternal and fetal PBMCs and amniocytes were obtained at 17-21 weeks of gestation. The fresh isolated PBMCs and amniocytes were maintained in RPMI 1640 medium until incubation with 10 microM tritiated AZT (10 microCi/mL). Thereafter, methanolic cell extracts were prepared for determination of AZT phosphates by high-performance liquid chromatography. Fetal PBMCs can efficiently convert AZT to its antivirally active metabolite. There were no significant differences after 6 or 12 hours of incubation with AZT between AZT phosphate levels in maternal and fetal PBMCs isolated at term or at 17-21 weeks of gestation: AZT monophosphate was found to be the major metabolite (about 95%). AZT phosphate levels in the amniocytes were up to sevenfold higher than those in the maternal or fetal PBMCs. These results show that during pregnancy and at term, fetal PBMCs-like maternal PBMCs-are able to take up AZT and to efficiently generate the active metabolite AZT triphosphate. These results are of major significance both in enabling efficient treatment of the fetuses of HIV-infected women and in the prediction and understanding of the efficacy and toxicity of AZT in pregnant women and their fetuses.     

Prenatal detection of Salla disease based upon increased free sialic acid in amniocytes

Salla disease is an autosomal recessive lysosomal storage disease relatively common in the Finnish population. The main manifestations of more than 70 patients detected to date are severe psychomotor retardation and ataxia of early onset. Intracellular free N-acetylneuraminic acid (sialic acid) is increased 10-20-fold and localized in the lysosomes. Four pregnancies at risk were monitored by quantitation of free and total sialic acid in amniocytes and supernatant amniotic fluid by high-performance liquid chromatography. In 3 children results were normal. Free sialic acid content of the amniocytes from one affected child was 2.6 nmol/mg protein, which was approximately 5 times higher than that of the 3 unaffected children (0.3 to 0.8) and 14 control samples (0.3 to 0.9). The ratio of free/total sialic acid of the amniocytes also clearly distinguished the affected pregnancy (13.8%) from the unaffected (2.3-4.8%) and control individuals (1.8-5.3%). This represents the first successful prenatal identification of a patient with Salla disease and indicates that both free sialic acid and free/total sialic acid ratio should be monitored in pregnancies at risk for the disease.     

Amniocytes can serve a dual function as a source of iPS cells and feeder layers

Clinical barriers to stem-cell therapy include the need for efficient derivation of histocompatible stem cells and the zoonotic risk inherent to human stem-cell xenoculture on mouse feeder cells. We describe a system for efficiently deriving induced pluripotent stem (iPS) cells from human and mouse amniocytes, and for maintaining the pluripotency of these iPS cells on mitotically inactivated feeder layers prepared from the same amniocytes. Both cellular components of this system are thus autologous to a single donor. Moreover, the use of human feeder cells reduces the risk of zoonosis. Generation of iPS cells using retroviral vectors from short- or long-term cultured human and mouse amniocytes using four factors, or two factors in mouse, occurs in 5-7 days with 0.5% efficiency. This efficiency is greater than that reported for mouse and human fibroblasts using similar viral infection approaches, and does not appear to result from selective reprogramming of Oct4(+) or c-Kit(+) amniocyte subpopulations. Derivation of amniocyte-derived iPS (AdiPS) cell colonies, which express pluripotency markers and exhibit appropriate microarray expression and DNA methylation properties, was facilitated by live immunostaining. AdiPS cells also generate embryoid bodies in vitro and teratomas in vivo. Furthermore, mouse and human amniocytes can serve as feeder layers for iPS cells and for mouse and human embryonic stem (ES) cells. Thus, human amniocytes provide an efficient source of autologous iPS cells and, as feeder cells, can also maintain iPS and ES cell pluripotency without the safety concerns associated with xenoculture.