Methylenetetrahydrofolate reductase gene polymorphisms and their association with trisomy 21

OBJECTIVES: To verify whether the frequencies of 5,10-methylenotetrahydrofolate reductase (MTHFR) polymorphisms at positions 677 and 1298 are higher in women with children affected by trisomy 21 than in those with chromosomally normal offspring. METHODS: A case-control study was carried out with 70 women whose children had trisomy 21 and 88 controls whose children were chromosomally normal. The frequencies of polymorphisms of points C677T and A1298C of MTHFR gene coding were studied in these two groups. Odds ratios (OR) for having a child affected by trisomy 21 were estimated for homozygosis, heterozygosis or the absence of the above-mentioned MTHFR polymorphisms. Logistic regression models were used to control for the effect of confounding variables on these odds ratios. RESULTS: The frequency of joint heterozygotic polymorphism (677 and 1298) was significantly higher in women with children affected by trisomy 21 than in those with chromosomally normal offspring (OR: 5.7). CONCLUSIONS: The presence of joint heterozygotic polymorphism in the codifying gene for MTHFR was a risk factor for having a child with trisomy 21.     

Ultrasound findings and multiple marker screening in trisomy 18

OBJECTIVE: To compare detection of trisomy 18 in the second trimester by ultrasound and multiple-marker testing. METHODS: A computerized genetics database was used to identify fetuses of 14-22 weeks' gestation who had comprehensive ultrasound examinations, multiple-marker screening tests (alpha-fetoprotein [AFP]), hCG, unconjugated estriol [E3], and trisomy 18 karyotype. A positive trisomy 18 screen was defined as AFP up to 0.75 multiples of the median (MoM), hCG up to 0.55 MoM, and unconjugated E3 up to 0.60 MoM. A risk of at least 1:190 defined a positive Down syndrome screen. Ultrasound abnormalities were diagnosed prospectively and were confirmed later by retrospective review of sonographic images. RESULTS: From 1988-1997, 30 trisomy 18 fetuses who had comprehensive ultrasounds and multiple-marker testing were identified. Twenty-one (70%) had abnormalities detected by ultrasound, of which the most common isolated finding was choroid plexus cyst. Eleven fetuses (37%) had positive trisomy 18 screens, and two had positive Down syndrome screens, for a total of 13 of 30 (43%) fetuses with positive multiple-marker screening tests. CONCLUSION: We found that ultrasound was more likely to be abnormal than multiple-marker screening tests in fetuses with trisomy 18 (70%) (95% confidence interval [CI] 54, 86 versus 43% CI 25, 61). However, combining the two testing methods yielded the highest detection rate (80% [CI 66%, 94%]).     

Diagnosis of trisomy 21 in preimplantation embryos by single-cell DNA fingerprinting

Many couples presenting for preimplantation genetic diagnosis (PGD) for a single gene disorder are of advanced reproductive age (>35 years) and have a greater chance of producing embryos with chromosomal aneuploidies. The most common chromosomal aneuploidy observed in newborns is trisomy 21, or Down's syndrome. Consequently, the availability of a highly reliable system that simultaneously detects the heritable gene disorder and trisomy 21 would be beneficial to couples at specific risk. A pentaplex chromosome 21 (Ch 21) single-cell DNA fingerprinting system was developed in a multiplex fluorescence polymerase chain reaction (FL-PCR) on single cells. High reliability and accuracy rates were observed, together with low allele dropout (ADO) and preferential amplification rates on diploid buccal cells, trisomy 21 buccal cells and blastomeres derived from Ch 21 aneuploid embryos. A combined multiplex FL-PCR format was optimized with the common cystic fibrosis delta F508 mutation and validated on single buccal cells from a carrier of the cystic fibrosis delta F508 mutation. This new test is a very powerful technique, which also allows confirmation of the embryo parentage and the identification of extraneous DNA contamination that could cause a misdiagnosis in PGD cases.     

Accuracy of trisomy 18 screening using the second-trimester triple test

OBJECTIVE: To assess the accuracy of the calculated risk for trisomy 18 assigned to individual women screened with the second-trimester triple test. METHODS: The study was based on 382598 women screened in the Ontario Maternal Serum Screening Programme between October 1993 and September 2000. Of the women screened, 111 cases of trisomy 18 were identified. Originally, 92874 women were screened using a risk cut-off level method. Estimated risks of trisomy 18 were calculated by applying published population parameters for the remaining women screened using a fixed analyte cut-off method.Women were ranked according to their individual risk for trisomy 18 syndrome in decreasing order and divided into 12 groups. The mean calculated risks of having an affected pregnancy at term for each group were compared with the birth prevalence of the corresponding group after allowing for spontaneous fetal losses. RESULTS: Agreement between the mean calculated risks and the observed prevalence was seen across the entire risk range, although women identified as having high-risk pregnancies had an actual prevalence that was somewhat lower than that estimated by the screen. CONCLUSION: The calculated risk for trisomy 18 syndrome assigned to the individual woman on the basis of the risk cut-off method accurately reflects their risk of having a term trisomy 18 syndrome pregnancy.     

Comprehensive investigation of DNA methylation and gene expression in trisomy 21 placenta

IntroductionTrisomy 21 (T21) is the most common aneuploidy affecting humans and is caused by an extra copy of all or part of chromosome 21 (chr21). DNA methylation is an epigenetic event that plays an important role in human diseases via regulation of gene expression. However, the integrative association between DNA methylation and gene expression in T21 fetal placenta has yet to be determined.MethodsWe profiled expression of 207 genes on chr21 and their DNA methylation patterns in placenta samples from normal and DS fetuses using microarray analysis and predicted the functions of differentially expressed genes using bioinformatics tools.ResultsWe found 47 genes with significantly increased expression in the T21 placenta compared to the normal placenta. Hypomethylation of the 47 genes was observed in the T21 placenta. Most of hypomethylated DNA positions were intragenic regions, i.e. regions inside a gene. Moreover, gene expression and hypomethylated DNA position showed significantly positive associations. By analyzing the properties of the gene-disease network, we found that increased genes in the T21 placenta were significantly associated with T21 and T21 complications such as mental retardation, neurobehavioral manifestations, and congenital abnormalities.DiscussionTo our knowledge, this is the first study to comprehensively survey the association between gene expression and DNA methylation in chr21 of the T21 fetal placenta. Our findings provide a broad overview of the relationships between gene expression and DNA methylation in the placentas of fetuses with T21 and could contribute to future research efforts concerning genes involvement in disease pathogenesis.     

STR-PCR检测孕妇外周血浆中胎儿DNA基因型的初步研究

目的采用短串联重复序列(STR)多态位点的复合扩增方法,研究孕妇血浆中胎儿DNA基因型。方法采用STR多态位点即CSF1PO、TPOX、TH01(简称CTT)三个位点的复合扩增方法,对30例孕妇孕中期的血浆DNA进行扩增,并对扩增产物进行变性聚丙烯酰胺凝胶电泳,银染后观察结果。结果30例孕中期的孕妇中,16例从母体血浆中检出了父源性等位基因,5例可能检出了胎儿DNA,3例未检出胎儿DNA,6例扩增失败。结论STR多态位点的复合扩增,判断基因型简便、快速,结果容易判断,费用低,可用于父源性遗传病的产前诊断。     

Apparent confined placental mosaicism of trisomy 16 and multiple fetal anomalies: case report

Trisomy 16 is frequently found confined to the placenta (confined placental mosaicism (CPM)), with a structurally normal fetus. In some cases of trisomy 16, the fetus has uniparental disomy for chromosome 16 (UPD16) which is associated with intrauterine growth restriction (IUGR) and fetal anomalies. We report a case of apparent confined placental mosaicism for trisomy 16, using standard cytogenetic techniques, but with multiple fetal abnormalities including congenital diaphragmatic hernia in which there was no evidence of UPD in the disomic tissues examined. Subsequent examination of fetal tissues using fluorescent in situ hybridization (FISH) demonstrated low levels of mosaicism for trisomy 16 in all the tissues examined. The use of FISH permits identification of mosaicism which conventional techniques may not identify.     

Second trimester two-step trisomy 18 screening using maternal serum markers

Trisomy 21 maternal serum marker screening has led to screening for other anomalies, including trisomy 18. Trisomy 18 is generally prenatally diagnosed because of major morphological defects. However, in up to 30% of cases ultrasound signs are unclear, and in most cases diagnosis is performed late in pregnancy. Of the different maternal serum markers, PAPP-A is now considered as the best for trisomy 18 screening. However, pregnancy-associated plasma protein A (PAPP-A) is of value in first trimester screening for trisomy 21, but not in the second trimester. We therefore propose a two-step screening strategy. Based on 45 trisomy 18 cases, we confirm the values of alpha-fetoprotein (AFP) (median 0.61 MoM), free beta-human chorionic gonadotrophin (beta-hCG) (median 0.24 MoM) and of PAPP-A (median 0.08 MoM). In the first step, a 0.5 MoM cut-off for AFP or for free beta-hCG resulted in detection of 37/45 trisomy 18 cases (82%) with a 10% false-positive rate. The second step consisted of the measurement of PAPP-A for all these false-positive cases. Using a PAPP-A cut-off of 0.5 MoM, all the 37 trisomy 18 cases were detected, but now with a 0.1-0.2% false-positive rate. Amniocentesis was only offered to these few patients. This two-step second trimester screening will be of value for patients who have not been included in first trimester screening based on nuchal translucency (NT) measurement combined with the first trimester markers, PAPP-A and free beta-hCG.     

Overlapping DNA methylation profile between placentas with trisomy 16 and early-onset preeclampsia

Introduction

Maternal preeclampsia is associated with altered placental development in the first trimester of pregnancy. Confined placental trisomy 16 mosaicism (CPM16) is a genetic abnormality of the placenta that is highly predisposing to preeclampsia. We previously demonstrated widespread alterations in DNA methylation in 3rd trimester placentae associated with chromosomally normal early-onset preeclampsia (EOPET) and questioned whether similar changes would be associated with CPM16, making this condition a potential model for studying EOPET-associated changes early in pregnancy.

Methods

Using the Illumina Infinium HumanMethylation450 BeadChip, 3rd trimester CPM16 placental samples (N = 10) were compared to gestational age matched controls, and to 1st trimester trisomy 16 placentae (N = 5).

Results

DNA methylation differences associated with CPM16 were identified at 2254 CpGs using stringent criteria (FDR < 0.01, Δβ > 0.15). A subset of these differences (11%; p < 0.0001) overlapped those observed in chromosomally normal EOPET using similarly stringent criteria (FDR < 0.01; Δβ > 0.125). Importantly, the majority of EOPET-associated CpGs were significantly altered (p < 0.05) in CPM16 with a similar Δβ distribution. This was true for CPM16 with (N = 5) and without (N = 5) EOPET, although EOPET cases showed a tendency towards larger changes. Of the shared CPM16/EOPET associated changes, three CpGs near two genes (ARGHEF37 and JUNB) were also altered in 1st trimester trisomy 16 placentae.

Discussion

Despite the limited sample size, widespread DNA methylation changes are observed in Trisomy 16 that overlap those seen previously in chromosomally normal EOPET. Hence, Trisomy 16 may provide a model to study the progression of placental changes that occurs in EOPET across different gestational ages.     

利用短串联重复序列无创性产前诊断21-三体综合征

目的:探索短串联重复序列用于无创性产前诊断21号染色体非整倍体异常的可行性。方法:选择进行遗传咨询的孕妇及其配偶共50对,以血红蛋白γ链染色结合显微操作分离获取母血中胎儿有核红细胞,经PEP扩增细胞基因组后,对3个21染色体位点D21S11、D21S1411、D21S1412进行PCR扩增。同时对各对夫妻外周血DNA进行上述基因位点扩增,聚丙烯酰胺凝胶电泳后胎儿及其双亲的基因型对比分析。结果:50例均获取胎儿细胞样本,平均4.55±2.60个/ml。其中3例扩增失败,其余47例D21S11、D21S1411及D21S1412位点的有效信息率分别为85.11%(40/47)、78.72%(37/47)、76.60%(36/47)。3个STR位点联合应用,对45例样本成功鉴定了细胞的胎儿源性并准确判断胎儿21号染色体的数量,符合率为95.74%(45/47)。检出2例21-三体综合征胎儿,与其实际核型相符。结论:PEP结合多个染色体特异性STR位点分型可用于非整倍体异常的无创性产前诊断。     

Rapid determination of trisomy 21 from amniotic fluid cells using single-nucleotide polymorphic loci

OBJECTIVES: Rapid detection of trisomy 21 is an important goal for prenatal genetic centers. Fluorescent-PCR and DNA fragment analysis was developed a decade ago and thousands of samples were analyzed in routine practice using this method. Quantitative real-time PCR with melting curve analysis using SNP markers for trisomy 21 detection was described recently. We studied the reliability of this method on a cohort of samples of Hungarian patients. METHODS: DNA was isolated with silica adsorption method from amniotic fluid cells. We investigated 67 trisomy 21 and 62 diploid samples in the study. Quantitative real-time PCR was performed using hybridization probes combined with melting curve analysis. Peak areas under the derivative curves were determined and analyzed. RESULTS: The SNP marker WIAF 899 was informative in 41.86% of cases and WIAF 2643 in 48.83%. The melting curve area ratios were significantly different between trisomic and normal cases for WIAF 899 (trisomic 0.5246 +/- 0.2498 vs 0.8347 +/- 0.5234; p < 0.001), while in the case of WIAF 2643, they were not different. CONCLUSION: Combined and selected SNP markers could be valuable tools for rapid trisomy 21 detection in prenatal genetic screening.     

Detection of fetal trisomy 9 mosaicism by noninvasive prenatal testing through maternal plasma DNA sequencing

Objective

Noninvasive prenatal testing (NIPT) is widely used as a powerful screening tool to detect common aneuploidies. However, its application for detection of rare chromosomal abnormalities remains inconclusive.

Maternal serum-integrated screening for trisomy 18 using both first- and second-trimester markers

OBJECTIVES: To estimate the prenatal screening performance of an integrated serum test for detecting trisomy 18, which combines measurements of first- and second-trimester markers with maternal age to assign patient-specific risks. METHODS: Published and new observations of maternal serum marker levels in trisomy 18 and unaffected pregnancies are used to derive population parameters. These parameters are then combined in a multivariate Gaussian model to assign patient-specific risks for trisomy 18. RESULTS: The best combination of serum markers includes pregnancy-associated plasma protein-A in the first trimester and alpha-fetoprotein, unconjugated estriol and human chorionic gonadotropin in the second trimester. At a second-trimester risk cutoff of 1 : 100, these 4 markers, in combination with maternal age, detect 90% of trisomy 18 pregnancies at a false-positive rate of 0.1%. The odds of a trisomy 18 pregnancy among screen-positive women are 1 : 4. Without the first-trimester marker, detection is reduced to 67% at about the same false-positive rate. CONCLUSION: The algorithm described here is highly efficient for detecting trisomy 18 and should be considered by programs that offer serum-integrated screening for Down syndrome.