Hum. Reprod. Advance Access originally published online on June 21, 2007
Human Reproduction 2007 22(8):2267-2272; doi:10.1093/humrep/dem154
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Elevated levels of total (maternal and fetal) 
-globin DNA in maternal blood from first trimester pregnancies with trisomy 21
1 Department of Obstetrics and Gynecology, Baylor College of Medicine, 2450 Holcombe Blvd, Houston, TX 77054, USA 2 Department of Obstetrics and Gynecology, Columbia University, New York City, NY, USA 3 Department of Obstetrics and Gynecology, University of Bologna, Italy 4 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, USA
5 Correspondence address. Tel: +1-713-798-8885; Fax: +1-713-798-5575; E-mail: bischoff{at}bcm.tmc.edu
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Abstract |
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BACKGROUND: Elevated levels of circulating fetal DNA have been observed in maternal plasma when a trisomy 21 fetus is confirmed. However, these studies have been limited to pregnancies carrying a male fetus. We sought to quantify total (fetal and maternal) DNA from dried blood spots (DBS) for use as an additional factor in multi-parameter prenatal screening for aneuploidy.
METHODS: Maternal DBS were obtained from the NICHD-sponsored multi-center cohort (BUN) study. Seventeen confirmed trisomy 21 (mean gestational age 12.23 ± 0.77 weeks) cases were each matched by gestational age to euploid controls (n = 30). DNA was extracted and quantitative PCR was performed to measure four non-chromosome 21 loci, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (12p13), 
-globin (11p15.5), -actin (7p15–12) and p53 (17p13.1).
RESULTS: -Globin DNA levels were significantly elevated (P = 0.003) in 13 of 17 trisomy 21 cases (4.08 ± 1.78 Geq/ml x 105) compared with matched controls (2.35 ± 1.84 Geq/ml x 105). Following conversion of -globin concentrations into multiples of the median (MoM), MoM for trisomy 21 cases was 2.8 compared with 1.0 in euploid cases. No significant differences in levels of circulating GAPDH, -actin and p53 sequences were detected.
CONCLUSIONS: This work demonstrates differential levels of circulating -globin DNA in maternal blood of euploid and trisomy 21 cases. Sequence-specific quantification could provide an additional measure to improve non-invasive methods of prenatal screening to detect trisomy 21 using dried blood. -Globin in particular is an attractive biomarker that could contribute to enhance multiple serum parameter testing in the first trimester.
Key words:
-globin/cell-free fetal DNA/maternal dried blood spot/non-invasive prenatal diagnosis/trisomy 21
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Introduction |
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Definitive prenatal genetic diagnosis requires invasive procedures such as amniocentesis or chorionic villus sampling. These procedures carry a small but finite risk of fetal loss and/or injury, thus limiting the number of women who undergo direct testing. Non-invasive screening is thus often performed initially to identify pregnant women whose risk for a Down syndrome (DS) fetus is sufficiently high to justify an invasive procedure. Multiple non-invasive options exist in both first and second trimesters (Snijders et al., 1998
; Malone et al., 2005; Simpson, 2005). These non-invasive screening methods can identify 80–93% of trisomy 21 fetuses, given a 5% false-positive rate. To increase sensitivity, or lower procedure rate, additional parameters are continually sought.
One additional option involves the analysis of circulating intact fetal cells and/or cell-free fetal DNA. Fetal cells in maternal circulation have long been recovered and used to correctly diagnose fetal aneuploidy (Price et al., 1991; Elias et al., 1992; de la Cruz et al., 1995); however, enrichment methods remain complex and inefficient in the absence of a fetal-specific marker. The number of fetal chromosomal aneuploidy (trisomy 21) cases diagnosed in first and second trimester pregnancies is at best 75% with a false-positive rate estimated between 0.6 and 4.1% (de la Cruz et al., 1998; Bianchi et al., 2002).
Cell-free fetal DNA also exists in plasma of pregnant women as early as 6 weeks of gestation. Concentrations rise during pregnancy and peak prior to parturition (Lo et al., 1998). Many laboratories, including ours, have shown the utility of fetal circulating DNA as a unique source of genetic material for non-invasive prenatal evaluation of fetal gender, genetic diseases and aneuploidy using quantitative PCR (Bischoff et al., 1999, 2002, 2003, 2005; Johnson et al., 2004). Moreover, cell-free DNA has been used to distinguish trisomy 21 from euploid pregnancies. Studies indicate increased levels of cell-free fetal DNA with trisomy 21. These studies have been limited to pregnancies carrying a male fetus, given that Y-sequence detection is marker for fetal DNA (Lo et al., 1999; Zhong et al., 2000; Ohashi et al., 2001; Hromadnikova et al., 2002; Lee et al., 2002; Farina et al., 2003; Spencer et al., 2003; Bauer et al., 2006). Of course, a marker applicable for pregnancies carrying either a male or female fetus is necessary for clinical applications. Thus, we propose the use of maternal blood DNA quantification as an additional factor in multi-parameter prenatal screening for aneuploidy. This approach involves comparing total blood DNA levels between euploid and trisomy 21 cases. Moreover, this approach is perhaps more practical given that analysis of maternal dried blood spots (DBS) has been developed (Bischoff et al., 2003).
In this study, we employ real-time PCR to quantify circulating levels of four non-chromosome 21 loci [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), -globin, -actin and p53] using DBS from pregnant women carrying euploid and trisomy 21 affected fetuses. We found the levels of -globin DNA to be significantly increased in trisomy 21 pregnancies.
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Materials and Methods |
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Sample collection
We obtained Institutional Review Board approval from Baylor College of Medicine for this study. DBS from 47 pregnant women were obtained from the NICHD-sponsored multi-center cohort (BUN study) (Table 1). Their ethnicities were Caucasian (38), Hispanic (6), Asian (1) and African American (2). Drops of blood from a finger stick were spotted onto a sterile S&S 903 specimen collection card (Schleicher and Schuell, Keene, NH, USA, lot #W0131). Filter cards containing blood samples were all coded and allowed to air-dry overnight and then transferred to sterile specimen plastic bags for storage at room temperature. Blood spots were delivered as blinded matched sets (based on gestational age) and coded such that the technologist was unaware of the karyotype. Following real-time PCR quantification, the karyotype of each paired set was revealed.
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DNA extraction
Our laboratory received a single DBS from each case. From each blood spot sample, six 3 mm punched-out circles were placed together in a 1.5 ml microcentrifuge tube. On the basis of product information, 100 µl of blood is estimated to be absorbed per 16 mm diameter of filter paper. Therefore, for the six 3 mm punched-out sections, 112 µl of blood was estimated to be used for DNA extraction. DNA was extracted using the QIAamp DNA blood kit for DBS (Qiagen, Valencia, CA, USA) according to manufacturer's instructions. All DNA samples were stored at 4°C prior to analysis.
PCR analysis
Quantitative real-time PCR using the TaqMan Assay to measure total DNA levels using four non-chromosome 21 loci [GAPDH (12p13); beta-globin (11q21); beta-actin (7ptel) and p53 (17p13)] were performed using the Applied Biosystems 7700 sequence detection system (Foster City, CA, USA). Quantification of DNA as genome equivalents per milliliter of blood was based on the copies of the gene sequences detected per microliter of blood. Each reaction plate was run simultaneously with a duplicate calibration curve of titrated DNA (standard curve). Each sample (5 µl) was run in triplicate for each locus, and the mean of the values was determined using the 7700 software and the standard curve of known DNA concentrations. All samples were analyzed blindly with respect to genotype (trisomy 21) and gestational age. Control specimens included filter paper alone (processed without blood) and PCR blanks (no DNA). The standard factor of 6.6 pg was used to convert the data to genome equivalents (Geq).
Statistics
Two sample t-tests analysis were used to compare the total levels of DNA between the matched trisomy 21 and the control euploid cases at each locus. Fisher's exact test was used to calculate an exact P-value. Data were converted in log scale and plotted versus gestational age expressed in days. Finally a multiples of the mean (MoM) of the controls conversion was performed. P-plot analysis was used to evaluate the normal distribution of the MoM in both cases and controls. A receiver operating characteristic (ROC) curve was also used to test the discriminate power of the available variables. Total DNA levels between the two groups were stratified based on maternal age, gestational age and other non-invasive first trimester screen results (triple screen).
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Results |
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DBS from a total of 47 pregnant women were evaluated. Of these women, 17 carried a trisomic fetus (47, XX, +21 or 47, XY, +21) and were all reported to have an uneventful pregnancy. In the remaining 30 cases, the fetus was 46, XY. Analysis of DNA was performed by laboratory staff unaware of fetal karyotype, gestational age or result of first trimester non-invasive screenings. Table 1 summarizes the age, weight, gestational age and calculated first trimester risk based on serum screening. There were no significant differences in age, weight or gestational age among the trisomy 21 and control groups (P > 0.05). As expected, a significant difference in the levels of first trimester risk based on serum screening and NT measurement was observed between the two groups (P = 0.00003).
Circulating levels of four different specific gene sequences (p53, GAPDH, -globin and -actin) were determined by real-time PCR. Circulating levels of DNA for each of the four different loci are shown in Fig. 1. A significant difference (P = 0.003) in the mean levels of -globin between the two groups was observed. No significant differences in circulating levels of the remaining three loci (GAPDH, -actin or p53) were observed (P > 0.05) (Table 1, Fig. 1).
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All 17 confirmed trisomy 21 cases were matched blindly, by gestational age to one or two euploid control cases (Fig. 2). In 13 of the trisomics cases matched to euploid control(s), a significant difference was observed between the levels of
-globin, P-values ranging from 0.9401 to 0.0003. Though overlap exists between the values observed in trisomic and euploid pregnancies (Figs 1 and 2), 50% of the euploid samples had -globin values lower than any of the trisomic samples. After log conversion, circulating DNA levels for each locus were converted to MoM value by using a weighted log-linear regression. When plotting MoM DNA levels for -globin versus first trimester serum screen and NT risk, a significant correlation was also found (P = 0.026). Figure 3 demonstrates the overlap between the values measured with -globin levels distinctly greater among the trisomic cases compared with control euploid cases. The median MoM was 2.8 for trisomy 21 cases and 1.0 for the euploid controls, which is representative of the MoM of the general population. The distribution of circulating DNA levels in both trisomic and euploid pregnancies followed a log-Gaussian pattern at least between the 10th and 90th centiles of the unaffected cases, as judged by a probability plot, even given relatively small numbers (Fig. 3).
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Using > 400 000 Geq/ml
-globin level as a cut-off for positive detection, the optimal sensitivity of the test was 59% with a specificity of 83% (Table 2). The cut-off for a 5% false-positive rate and sensitivity of 35% was 3.00 MoM. The ROC curve yielded a P-value of 0.002 with the area under the curve calculated as 0.775 (0.639–0.910 95%CI) (Fig. 4).
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Discussion |
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DS is caused by over-expression of genes located within the long arm of chromosome 21. DS affects all major organ systems including the skeletal, immune, central nervous and cardiovascular systems (Korenberg et al., 1994
). DS abnormalities of the central nervous system that result in mental retardation and precocious Alzheimer-type neurodegeneration are associated with enhanced apoptosis (Sawa, 2001). Some pathophysiological features of DS are indeed associated with increased expression of genes located on chromosome 21. Among those genes are amyloid precursor protein (Oyama et al., 1994), superoxide dismutase I (Groner et al., 1994) and S100-beta (Marks et al., 1996). However, neurodegeneration in DS is also associated with aberrant expression of genes not present on chromosome 21, including nitric oxide synthase 3 (de la Monte and Bloch, 1997), GAPDH (Cumming and Schubert, 2005) and p53 and Bax (de la Monte et al., 1998; Seidl et al., 1999). The additional copy of human chromosome 21 presumably induces changes in several genes located on other chromosomes in DS. Therefore, we were interested in determining whether differences in the levels of circulating DNA involving non-chromosome 21 loci also exist. We selected the tumor-suppressor p53 gene and the ‘housekeeping’ genes, GAPDH, -globin and -actin, all of which are routinely used in our laboratory. -Globin is a housekeeping gene located on chromosome 11, and there are no previous reports proposing an association of -globin with DS pathology (Farina et al., 2004; Sekizawa et al., 2004).We found the levels of total cell-free -globin to be significantly increased by 2–3-fold in maternal blood of trisomy 21 cases compared with gestational age-matched controls (P = 0.003). The optimal sensitivity of the test was 59% with a specificity of 83%. The MoM was 2.8 in trisomy 21 cases. Even though these results are insufficient for -globin to stand alone as a unique test, these findings indicate great potential for the utility of -globin as a novel serum marker to increase the sensitivity of non-invasive diagnosis of fetal trisomy 21 by maternal blood analysis, combined with other first or second trimester parameters. Before the true practical value of this new test can be judged with more certainty, further prospective studies in a consecutive set of patients attending for antenatal DS screening is required.
Few studies have addressed the utility of circulating fetal DNA concentrations combined with serum markers. Farina et al. (2003) reported a moderate improvement when fetal DNA was added to the quadruple marker screen test, increasing detection rate from 81 to 86% with a 5% false-positive rate. However, this study was limited to pregnancies carrying a male fetus (Farina et al., 2003). In our study, total DNA quantification is independent of fetal sex. In plotting MoM DNA levels for -globin versus first trimester risks (first trimester serum screen and NT), a significant correlation was found (P = 0.026).
Our study differs from others that have correlated cell-free DNA levels with trisomy 21. These studies show discrepancies with some reporting significant differences (Lo et al., 1999; Zhong et al., 2000; Lee et al., 2002; Farina et al., 2003; Bauer et al., 2006), whereas others do not (Ohashi et al., 2001; Spencer et al., 2003; Hromadnikova et al., 2002). In two studies from Hromadnikova et al. and Spencer et al., results involving quantification of total and fetal DNA levels were included. Using albumin to quantify total DNA, the concentration of total DNA was significantly increased in maternal serum of pregnancies with trisomy 21 (Spencer et al., 2003). In contrast, using -globin as a measure for total DNA, no significant difference was observed; however, the total median DNA levels in patients bearing a fetus affected with DS were higher (10 165 Geq) than that in controls (7330 Geq) carrying fetuses with a normal karyotype (Hromadnikova et al., 2002). This latter study differs from our study in which a significant difference in the levels of -globin between the two populations was found. One possible difference is sample size; in our study, the control group included 30 gestational age-matched cases in contrast to the other study with only 21 euploid cases. In addition, plasma, not whole blood, was used to extract DNA in the Hromadnikova study. The higher levels of -globin that we observed is likely explained by the fact that we processed whole dried blood that consists of circulating free DNA as well as cellular DNA. Moreover, free nucleic acids are more stable when absorbed onto filter paper, thus preserving free DNA more efficiently. That only we observed a difference between the -globin levels suggests that the target sequences are more stable and/or more susceptible to PCR-based amplification. A third study also reports an increase in the levels of -globin among a mixed population of chromosomal aberrations and other disorders, including two cases of trisomy 21 (Bauer et al., 2006).
Two recent studies report on the use of single-nucleotide polymorphisms (SNPs) and allelic ratios for detection of trisomy 21(Dhallan et al., 2007; Lo et al., 2007). Lo et al. describe an RNA allelic ratio strategy using an SNP in PLAC4, a placental expressed mRNA transcribed from chromosome 21, to detect differential chromosome dosage (Lo et al., 2007). In this study, 9 of 10 (90%) trisomy 21 cases were correctly detected with a 3.5% false-positive rate. Dhallan et al. describe the use of multiple SNPs to calculate the ratio of a unique fetal DNA allele versus the combined maternal and fetal DNA alleles based on pixel density measurement of the amplified PCR products. DNA ratio of SNPs on chromosomes 13 and 21 were evaluated and correctly detected two of three (67%) trisomy 21 cases with one false positive in 57 euploid cases (Dhallan et al., 2007). Several concerns perhaps arise from these high-tech and costly studies. SNPs are population-specific (Halushka et al., 1999), therefore variability among ethnicities are expected. Also, SNPs generally have a low average rate of heterozygosity, thus a high number of SNPs are required for an informative result. In addition, it remains unclear what portion of the fetal genome is universally represented in circulating DNA among pregnant women or whether certain sequences are more stable and therefore more suitable for PCR-based detection. Therefore, quantification based on ratio calculations may be unreliable or difficult to replicate between cases.
The DBS samples were obtained as part of the NICHD-sponsored multi-center cohort (BUN study) (Wapner et al., 2003), which were also used to measure PAPP-A and hCG to determine first trimester risk. The DBS approach clearly facilitates transport and generalized application. Thus, the same blood sample obtained for first or second trimester test can be used for quantitative DNA measurement. Samples can be archived and shipped to a central test site. DBS collection requires only a small amount of blood that can be stored at room temperature for a surprising length of time with little loss in sensitivity. It has been shown that the use of filter paper and corresponding matrix stabilizes many molecules, including DNA and RNA (Mei et al., 2001). Our group was the first to prove reliable detection of DNA for fetal gender determination from DBS (Bischoff et al., 2003). Furthermore, we recently reported isolation and detection of placental RNA accurately from DBS (Jorgez et al., 2006). These results validate the stable nature of fetal and placental nucleic acids from DBS, making this an attractive alternative approach for prenatal testing. By adding -globin to the existing screening test, increased sensitivity or decreased false-positive rates could be expected. The possible increase in the detection rate provided by the addition of -globin to a screening program would be affected by the correlation found with NT and biochemical markers.
Our study represents an innovative, economical and practical approach for prenatal genetic diagnosis. The current preliminary report is the largest study using dried maternal blood to compare circulating DNA levels among trisomy 21 and euploid cases. However, further prospective studies in a consecutive set of patients will be required before considering adding -globin to first trimester screenings.
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Acknowledgement |
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This study was supported in part by grant NIH/NICHD HDO46623.
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References |
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Submitted on February 23, 2007; resubmitted on April 20, 2007; accepted on May 10, 2007.
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