Prenatal Genetic Testing and Screening

Multiplex fluorescent PCR for noninvasive prenatal detection of fetal-derived paternally inherited diseases using circulatory fetal DNA in maternal plasma

New technologies pave way for fetal personalized medicine

To clarify the origin of cell-free fetal DNA in maternal plasma, we analyzed bidirectional transfer of plasma DNA between fetus and mother. We analyzed maternal and fetal plasma DNA obtained from 15 pregnant women at the time of Cesarean section. The subjects were five patients with preeclampsia and 10 gestational-age-matched normal controls. DNA was extracted from 1.5-ml plasma samples and the cellular fraction of maternal and umbilical blood. Seven polymorphic marker genes were analyzed. The relative concentration of fetal DNA in maternal plasma and maternal DNA in cord blood were evaluated. The relative concentration of maternal DNA in fetal circulation (median, 0.9%; range, 0.2-8.4%) was significantly lower than that of fetal DNA in maternal blood (14.3%, 2.3-64%), with P=0.007. The relative concentration of maternal DNA in fetal blood was not affected by preeclampsia. These findings indicate that cell-free DNA is unequally transferred through the placenta. The structural characteristics of the placenta suggest that the majority of cell-free fetal DNA in maternal plasma is derived from villous trophoblasts.

We assessed the effect of freezing on fragmentation of fetal DNA in maternal plasma and differences in DNA fragmentation between plasma and urine from pregnant women. 1. We prepared seven kinds of real-time PCR assays to amplify different-sized amplicons targeting the SRY gene. Fragmentation of fetal DNA in maternal plasma was compared between new (n=10) and 4-year-old samples (n=10). 2. To investigate differences in fragmentation of fetal DNA between plasma and urine from pregnant women, we amplified three different-sized amplicons and compared DNA fragmentation between plasma and urine (n=7). 1. Relative concentrations of fetal DNA compared to a 63-bp amplicon in new samples were 53.1, 42.0, 9.2 and 2.0% (median) for PCR amplicons of 107, 137, 193 and 313 bp, respectively. Concentrations in 4-year-old samples were 70.4, 40.9, 11.9 and 2.3%, respectively. 2. Although fetal DNA in urine was not detected for 107- and 137-bp amplicons of the SRY sequence, fetal DNA using a 63-bp amplicon was detectable in five of seven cases (71.4%). Cell-free fetal DNA in maternal plasma is stable under cryopreservation at -20 degrees C for at least 4 years. Approximately, 60% of fetal DNA in maternal plasma was fragmented to <100-bp long, and fetal DNA in urine was further fragmented. Maternal urine may be usable for detection of fetal DNA, although smaller target size is more important for PCR amplification of fetal DNA in urine than in the analysis of plasma from pregnant women.

Objective To assess the clinical value of fetal chromosomal aneuploidy diagnosed by free fetal DNA in maternal plasma in 11-13+6 gestational weeks.Methods A total of 2 650 pregnant women who had prenatal care in Tianjin Center Hospital of Obstetrics and Gynecology from January 1,2010 to December 31,2010 were included.Each of them had an ultrasound scan to measure fetal nuchal translucency thickness.Maternal serum free β-human chorionic gonadotropin and pregnancy-associated plasma protein A test was performed as part of screening for chromosomal abnormalities.Results of ultrasound and maternal plasma biochemical analysis were entered into the database,and converted into multiple of median (MoM) by factors such as maternal age,weight,ethnicity,smoking history and mode of conception.The cutoff value was 1 ∶ 270.Meanwhile,20 cases had cell free fetal DNA (cffDNA) test and the ratio of the single nucleotide polymorphism on two alleles of plancenta-specific 4 (PLAC4) were measured in 16 cases.T-test,rank sum test,MannWhitney U test and Chi-square test were used as statistical methods.Results (1) A total of 74 cases were judged as high-risk,among which 35 cases underwent transabdominal chorionic villus sampling (18 cases had cffDNA test),37 cases underwent amniocentesis at the week of 20,and two cases of Rh negative did not receive the invasive examination.Totally 20 cases,including two Rh negative cases,had the cell-free fetal DNA test.(2) By cffDNA test of maternal plasma,two cases of 21 trisomy,one case of 18 trisomy,two cases of 45,XO and one case of balanced translocation were diagnosed.(3) In the two cases of 21 trisomy,maternal plasma G/A ratio ofPL4C4 RNA-single nucleotide polymorphism alleles was 1.00 (0.98,1.02) ; in 14 pregnancies with normal chromosome,the ratio was 1.055 (1.02,1.13,Z=3.5).There was no significant difference (P=0.066).Conclusion Diagnosing of fetal chromosomal aneuploidy by cffDNA in maternal plasma is feasible and noninvasive with high negative predictive value,and can be used in Rh-negative pregnant women for prenatal screening and diagnosis. Key words: Aneuploidy; Prenatal diagnosis; DNA; Pregnancy trimester, first

Detection of fetal DNA in maternal plasma by microarray coupled with emulsions PCR

The discovery of fetal DNA in maternal plasma has opened up an approach for noninvasive prenatal diagnosis. Despite the rapid expansion in clinical applications, the molecular characteristics of plasma DNA in pregnant women remain unclear. We investigated the size distribution of plasma DNA in 34 nonpregnant women and 31 pregnant women, using a panel of quantitative PCR assays with different amplicon sizes targeting the leptin gene. We also determined the size distribution of fetal DNA in maternal plasma by targeting the SRY gene. The median percentages of plasma DNA with size >201 bp were 57% and 14% for pregnant and nonpregnant women, respectively (P <0.001, Mann-Whitney test). The median percentages of fetal-derived DNA with sizes >193 bp and >313 bp were 20% and 0%, respectively, in maternal plasma. Plasma DNA molecules are mainly short DNA fragments. The DNA fragments in the plasma of pregnant women are significantly longer than those in the plasma of nonpregnant women, and the maternal-derived DNA molecules are longer than the fetal-derived ones.

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Early detection of cell-free fetal DNA in maternal plasma

To develop a standardized blood collection device that preserves fetal cell-free DNA and minimizes the cell-free DNA background in maternal plasma. Blood samples were drawn from healthy pregnant donors into K(3)EDTA (BD vacutainer) and Cell-free DNA BCT tubes (Streck, Inc.) and kept at ambient temperature. Plasma was separated by centrifugation and cell-free DNA was extracted. Cell-free DNA from plasma was quantified by quantitative real-time polymerase chain reaction. Blood drawn into Cell-free DNA BCT tubes showed no change in the original proportion of fetal cell-free DNA during a 14-day storage period at ambient temperature. Conversely, maternal blood drawn into K(3)EDTA tubes showed a steady reduction in the original proportion of fetal cell-free DNA over the same time period. Using maternal plasma stored in Cell-free DNA BCT tubes for 14 days, fetal cell-free DNA was amplified 80-fold using whole genome amplification (WGA). Using Streck's Cell-free DNA BCT tubes, it is possible to preserve the original proportion of fetal cell-free DNA for extended times as well as minimize the post-sampling maternal cell-free DNA background. Preserved in this way, fetal cell-free DNA can be amplified by WGA technology to be used in prenatal diagnostic tests.

The aim of this study was to explore the feasibility of non-invasive prenatal genetic diagnosis of β-thalassemia with small fragments of cell-free fetal DNA (cffDNA) in peripheral blood of pregnant women. It was an observational study carried out at Department of Obstetrics, The People's Hospital of Guangxi Zhuang Autonomous Region, Nanning, Guangxi, China, from January 2016 to March 2018. A total of 40 pregnant women, who were likely to give birth to babies with severe β-thalassemia, were selected, and β-globin genotype of the fetus was non-invasively detected by cffDNA in peripheral blood of their mothers. Small fragments of cffDNA from all specimens were successfully amplified. Compared with the results of traumatic prenatal diagnosis, 37 cases (92.50%) were diagnosed and 3 cases (7.50%) were misdiagnosed. The cffDNA in maternal plasma can be used for non-invasive prenatal genetic diagnosis of β-thalassemia, and is worthy of promotion.

The launch of the genomics and postgenomics era has greatly expanded our understanding of the genetic basis of many diseases. In conjunction with the sociocultural trend to delay childbirth and to maintain smaller family units, extra demand may be placed on the existing prenatal diagnostic services. The inherent risk of fetal loss associated with current prenatal diagnostic procedures, such as amniocentesis and chorionic villus sampling, has spurred research into non-invasive prenatal diagnosis. Much research has been conducted on the exploitation of fetal genetic material present in the maternal circulation. The initial focus was on the isolation of intact fetal cells and subsequently, the existence of extracellular fetal DNA in maternal plasma was realized. Exciting developments have been achieved in recent years. A large-scale trial to evaluate the clinical utility of fetal cell isolation from maternal blood for fetal aneuploidy diagnosis was launched and data were recently published. Much has taken place in the research of fetal DNA analysis in maternal plasma and in one example, namely prenatal RhD determination, this type of analysis has been used in the clinical setting. This paper reviews the technological developments in non-invasive prenatal diagnosis.

Prenatal genetic screening is designed to assess whether a patient is at increased risk of having a fetus affected by a genetic disorder. In contrast, prenatal genetic diagnostic testing is intended to determine, with as much certainty as possible, whether a specific genetic disorder or condition is present in the fetus. The purpose of prenatal screening for aneuploidy is to provide an assessment of the woman's risk of carrying a fetus with one of the more common fetal aneuploidies. This is in contrast to prenatal diagnostic testing for genetic disorders, in which the fetal chromosomes are evaluated for the presence or absence of abnormalities in chromosome number, deletions, and duplications, or the fetal DNA is evaluated for specific genetic disorders. The wide variety of screening test options, each offering varying levels of information and accuracy, has resulted in the need for complex counseling by the health care provider and complex decision making by the patient. No one screening test is superior to other screening tests in all test characteristics. Each test has relative advantages and disadvantages. It is important that obstetrician-gynecologists and other obstetric care providers be prepared to discuss not only the risk of aneuploidy but also the benefits, risks, and limitations of available screening tests. Screening for aneuploidy should be an informed patient choice, with an underlying foundation of shared decision making that fits the patient's clinical circumstances, values, interests, and goals.The purpose of this Practice Bulletin is to provide current information regarding the available screening test options for fetal aneuploidy and to review their benefits, accuracy, and limitations. For information regarding prenatal diagnostic testing for genetic disorders, refer to Practice Bulletin No. 162, Prenatal Diagnostic Testing for Genetic Disorders.

The presence of cell-free fetal DNA in maternal plasma could allow performing a non-invasive prenatal diagnosis of Huntington disease (HD). The great advantage of this diagnosis is the absence of risk of fetal loss that it entails. Maternal plasma from four pregnant women in their first trimester of gestation with a fetus at-risk was studied. In all the four cases, the father was affected. The diagnosis was performed both by a direct study of the mutation and an indirect haplotype study. By the direct analysis, three out of the four fetuses could be correctly diagnosed whilst the indirect analysis was only conclusive in one case. Non-invasive prenatal diagnosis of HD is possible by the analysis of fetal DNA in maternal plasma. Direct analysis of the mutation has shown higher accuracy than the haplotype analysis except for long expansions. Haplotype analysis would need to be improved for the study of Juvenile-onset HD. This diagnostic method would be limited to those couples with an affected male however this situation represents 80-90% of the pregnancies at-risk of HD. Moreover, it could be used as a confirmation test of healthy embryos transferred on pre-implantation genetic studies of HD.

Detection of circulating fetal nucleic acids in maternal plasma is a promising approach to noninvasive prenatal assessment and fetal monitoring. Cell-free fetal/placental (cff) DNA concentrations in maternal plasma undergo transient increases after amniocentesis, suggesting that cff DNA in maternal plasma is a sensitive marker for fetal-maternal hemorrhage (1). However, cff DNA measurement is a sex-dependent marker used only in pregnancies with male fetuses, whereas cell-free placental (cff) mRNA in maternal plasma, recently quantified with real-time reverse-transcription PCR, is considered a sex-independent marker for monitoring placental conditions (2). Evidence suggests that cff mRNA release into the maternal circulation is comparable to cff DNA release, but it is not known which molecular marker is more sensitive for detection of fetal-maternal hemorrhage. To investigate this question, we performed a quantitative analysis of cff DNA and cff mRNA in maternal plasma collected before and 10-min after amniocentesis and evaluated which molecular marker was more sensitive for detection of fetal-maternal hemorrhage. Study participants were 32 pregnant women, at 15–17 weeks of gestation, who underwent amniocentesis at …