WO2013129727A1

WO2013129727A1 - Analysis method for determining fetal gender and apparatus therefor

Info

Publication number: WO2013129727A1
Application number: PCT/KR2012/002301
Authority: WO
Inventors: Hyun Mee Ryu; So Yeon Park; Ji Hyae Lim; Shin Young Kim
Original assignee: Cheil General Hospital & Women's Healthcare Center
Priority date: 2012-03-02
Filing date: 2012-03-29
Publication date: 2013-09-06
Also published as: KR101256206B1

Abstract

An analysis method for determining fetal gender, an apparatus therefor, and more particularly, a method of determining fetal gender by using a concentration ratio of DYS14/GAPDH in maternal plasma. According to the method, fetal gender may be accurately determined as early as 5 to 6 weeks when a fetal DNA concentration in maternal plasma is very low.

Description

ANALYSIS METHOD FOR DETERMINING FETAL GENDER AND APPARATUS THEREFOR

The present invention relates to an analysis method for determining fetal gender and an apparatus therefor.

Early prenatal determination of fetal gender is important for pregnant women at risk of X-linked diseases, such as hemophilia, X-linked mental retardation, and retinitis pigmentosa. There is a higher possibility that a male fetus may have these diseases. Fetal gender determination is also required for some endocrine diseases, such as congenital adrenal hyperplasia, where there is masculinization of the female fetus, which is preventable with antenatal treatment. Reliable determination of fetal gender using ultrasonography cannot be performed in the first trimester, because the development of external genitalia is not complete.

Generally, early fetal gender determination has been performed by invasive procedures such as chorionic villus sampling or amniocentesis. However, these invasive procedures still carry a 1% to 2% risk of miscarriage and cannot be performed until 11 weeks of gestation.

Since it has been reported that circulating fetal DNA arises from trophoblasts in the placenta, is released directly into the circulation system of the mother, and represents around 3 to 6% of the total cell-free DNA that is present in maternal circulation, research into non-invasive fetal gender detection using the circulating fetal DNA has been conducted. Prior reports have used a polymerase chain reaction (PCR)-based detection using Y chromosome-specific sequences, including DYS14, a multicopy marker located within a TSPY(testis specific protein, Y-linked 1) gene, and a sex-determining region Y (SRY). However, the circulating fetal DNA has an undetectably low concentration, particularly, in the first trimester and may be non-specifically amplified under PCR conditions as shown in a DYS14 assay, and thus accuracy decreases when using the circulating fetal DNA.

Meanwhile, research has been conducted to find a fetal DNA identifier using epigenetic differences between maternal DNA and fetal DNA. For example, PDE9A, SERPINB5, RASSF1A, APC, CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1, TMEFF2, and PYCARD genes are present regardless of fetal gender and are methylated differently in maternal blood and placentas. Thus, they are used to identify the presence of the circulating fetal DNA in maternal plasma. PDE9A and SERPINB5 genes are found methylated in maternal blood cells and unmethylated in placentas (Biol Reprod. 82(4):745-50, 2010). On the other hand, RASSF1A, APC, CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1, TMEFF2, and PYCARD genes are found unmethylated in maternal blood cells and methylated in placentas (US Patent No. 7754428).

The present invention provides an accurate, simple, and effective method for non-invasively detecting fetal gender and an apparatus therefor.

According to an aspect of the present invention, there is provided a method of analyzing a sample for providing information required to determine fetal gender, the method including: extracting fetal DNA from collected maternal plasma; measuring concentrations of DYS14 and GAPDH(glyceraldehye-s-phosphate dehydrogenase) in the extracted fetal DNA; and calculating a concentration ratio of DYS14/GAPDH from the measured concentrations of DYS14 and GAPDH.

The method may further include measuring a concentration of a fetal DNA marker regardless of fetal gender.

The method may further include determining an optimal cutoff value by measuring concentrations of DYS14 and GAPDH in maternal blood of a group of pregnant women, the cutoff value of which is calculated, and by assessing a concentration ratio of DYS14/GAPDH at 100% specificity where the detection exactly matches gender of female fetus.

According to another aspect of the present invention, there is provided a method of determining fetal gender using the above analysis method.

According to another aspect of the present invention, there is provided an apparatus for determining fetal gender including: a device for amplifying DYS14 and GAPDH genes or portions thereof using fetal DNA as a template, a device for measuring concentrations of DYS14 and GAPDH; a device for calculating a concentration ratio of DYS14/GAPDH; and a device for printing the calculated concentration ratio of DYS14/GAPDH.

The apparatus may further include a device for amplifying a U-PDE9A gene of bisulfite-treated fetal DNA and measuring a concentration of a PDE9A(phosphodiesterase 9A) gene. The apparatus may further include a device for determining fetal gender by comparing the concentration ratio of DYS14/GAPDH and the concentration of the U-PDE9A gene with cutoff values of the concentration ratio of DYS14/GAPDH and the concentration of the U-PDE9A gene which are input in advance.

According to another aspect of the present invention, there is provided a reagent package for determining fetal gender including: a PCR reagent set including primer pairs capable of amplifying DYS14 of fetal DNA or portions thereof; a reagent set including primer pairs capable of amplifying GAPDH of fetal DNA or portions thereof. The reagent package may further includes a PCR reagent set including primer pairs capable of discriminating methylated CpG sites and unmethylated CpG sites of U-PDE9A of bisulfite-treated fetal DNA.

The method of non-invasively detecting fetal gender by using the concentration ratio of DYS14/GAPDH according to the present invention is effective, simple, technically easy, cost-effective, and accessible in all basic diagnostic laboratories. Therefore, this approach can be applied routinely in clinical work. In addition, this method can reduce the need for invasive procedures in pregnant women carrying an X-linked chromosomal abnormality and clarify inconclusive ultrasound readings.

The above and other features and advantages of the present invention will become more apparent by describing in detail exemplary embodiments thereof with reference to the attached drawings in which:

Figures 1a and 1b show bisulfite genomic sequencing of PDE9A. The arrows indicate methylation sites of a PDE9A gene. Figure 1a refers to methylated CpG sites of PDE9A which is detected in maternal blood cells and placental tissues. Figure 1b refers to unmethylated CpG sites of PDE9A which is detected only in placental tissues.

Figures 2a, 2b and 2c show graphs illustrating correlation of DYS14, U-PDE9A, and GAPDH in the male-bearing participants. DYS14, U-PDE9A, and GAPDH concentrations showed significantly positive associations with each other (P<0.001 in all). Correlations among the concentrations were estimated using Spearman’s rank correlation. Figure 2a refers to DYS14 and U-PDE9A. Figure 2b refers to DYS14 and GAPDH. Figure 2c refers to U-PDE9A and GAPDH.

Figures 3a, 3b and 3c show graphs illustrating comparisons of U-PDE9A, DYS14, and GAPDH concentrations between the false-negative results and the correct results. The upper and lower limits of the boxes and the lines across the boxes indicate the 75th/25th percentiles and the medians, respectively. The upper and lower error bars indicate the 90th and 10th percentiles, respectively. The circles indicate outliers. Data was compared by Mann Whitney U-test. Figure 3a refers to U-PDE9A. Figure 3b refers to DYS14. Figure 3c refers to GAPDH.

Figures 4a and 4b show graphs illustrating comparison of DYS14/GAPDH and U-PDE9A/GAPDH ratios in the false-negative results and the correct results. The upper and lower limits of the boxes and the lines across the boxes indicate the 75th/25th percentiles and the medians, respectively. The upper and lower error bars indicate the 90th and 10th percentiles, respectively. The circles indicate outliers. Data was compared by Mann Whitney U-test. Figure 4a refers to a concentration ratio of DYS14/GAPDH. Figure 4b refers to a concentration ratio of U-PDE9A/GAPDH.

Hereinafter, the present invention will now be described more fully with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown.

According to an embodiment of the present invention, there is provided an analysis method for determining fetal gender, an apparatus therefor, and a method of determining fetal gender. In non-invasive prenatal diagnosis, fetal gender determination is still inaccurate because circulating fetal DNA in maternal plasma is only present in small amounts. To solve this problem, the present inventors have conducted research and found that fetal gender may accurately be detected by using a concentration ratio of DYS14/GAPDH in maternal plasma. Particularly, fetal gender was accurately determined as early as 5 to 6 weeks when a fetal DNA concentration in maternal plasma is very low.

At the same time, the presence of circulating fetal DNA was confirmed by using a U-PDE9A gene. Thus, the possibility of false-negative results, where a fetus is falsely diagnosed as a female fetus, due to undetectable low concentrations of circulating fetal DNA, can be precluded.

More particularly, circulating fetal DNA was extracted from maternal plasma obtained from 203 participants with singleton pregnancies at or before 12 weeks of gestation, multiplex quantitative real-time PCR was performed using Y chromosome-specific DYS14 and reference GAPDH genes as molecular markers, and then concentrations thereof were respectively measured and expressed as copies/mL to obtain the concentration ratio of DYS14/GAPDH (Table 3). The fetal gender was confirmed at birth, and a cutoff value for each of a DYS14 Quantification cycle (Cq), a DYS14 concentration, and a concentration ratio of DYS14/GAPDH was set at 100% specificity where the detection exactly matched gender of female fetus (Table 4). Diagnostic results of fetal gender determined by considering the cutoff value were compared with actual gender at birth. As a result, it was confirmed that false-negative results of the DYS14 Cq or DYS14 concentration were observed only at and before 8 weeks of gestational age, whereas the concentration ratio of DYS14/GAPDH showed 100% detective accuracy regardless of gestational weeks (Table 5).

In addition, the present inventors have identified the presence of unmethylated PDE9A (U-PDE9A) that is not present in maternal blood cells but present in placentas. Generally, in the majority of studies using circulating fetal DNA for gender determination, a female fetus cannot be detected directly but only inferred by a negative result for Y chromosome-specific sequences. Therefore, confirming the presence of fetal DNA is of utmost importance when a negative result for Y chromosome-specific sequences such as DYS14 and SRY is found. The U-PDE9A gene used herein was detected in all fetal DNA regardless of fetal gender, and there was no concentration difference according to the fetal gender (Table 3). Furthermore, the concentration of U-PDE9A was significantly positively correlated with the concentration of DYS14 (FIG. 2). These results showed that the possibility of false-negative results due to undetectable low concentrations of circulating fetal DNA may be precluded, and U-PDE9A is effective for quantifying the circulating fetal DNA regardless of fetal gender according to the present invention.

According to an embodiment of the present invention, there is provided a method of analyzing a sample for providing information required to determine fetal gender, the method including: extracting fetal DNA from collected maternal plasma; measuring concentrations of DYS14 and GAPDH in the extracted fetal DNA; and calculating a concentration ratio of DYS14/GAPDH from the measured concentrations of DYS14 and GAPDH.

According to another embodiment of the present invention, there is provided a method of determining fetal gender, the method including: extracting fetal DNA from collected maternal plasma; measuring concentrations of DYS14 and GAPDH in the extracted fetal DNA; and calculating a concentration ratio of DYS14/GAPDH from the measured concentrations of DYS14 and GAPDH.

Plasma may be obtained by collecting blood using a syringe treated with an anti-coagulant and centrifuging the blood. The extracting of fetal DNA from the maternal plasma may be performed by using, for example, a QIAamp Kit including QIAGEN protease, or any method commonly known in the art. In certain embodiments, the fetal DNA be extracted from maternal whole blood or plasma using e.g. DNA extraction methods such as, but not limited to, gelatin extraction method; silica, glass bead, or diatom extraction method; guanidinium thiocyanate acid based extraction methods; guanidine-hydrochloride based extraction methods; methods using centrifugation through cesium chloride or similar gradients; phenol-chloroform based extraction methods; and/or other available DNA extraction methods, as are known in the art.

In the measuring of concentrations of DYS14 and GAPDH in the extracted fetal DNA, polymerase chain reaction (PCR), preferably multiplex real-time PCR, may be performed in advance using a solution including primers and probes in order to amplify DYS14, GAPDH, or portions thereof. Examples of primer pairs used in the PCR are shown in Table 2, but are not limited thereto. Any primers capable of amplifying the DYS14, GAPDH, or portions thereof may also be used.

The concentration of DYS14 or GAPDH may be obtained by performing serial dilutions of standard DNA including target DNA, the concentration of which is known, performing PCR, and calculating the concentration by using calibration curves.

The method of analyzing a sample for providing information required for determining fetal gender or the method of determining fetal gender may further include measuring a concentration of a fetal DNA marker regardless of fetal gender in addition to the extracting fetal DNA from collected maternal plasma; measuring concentrations of DYS14 and GAPDH in the extracted fetal DNA; and calculating a concentration ratio of DYS14/GAPDH from the measured concentrations of DYS14 and GAPDH. The fetal DNA marker regardless of fetal gender may include one selected from the group consisting of unmethylated PDE9A (U-PDE9A), unmethylated SERPINB 5, methylated RASSF1A, methylated APC, methylated CASP8, methylated RARB, methylated SCGB3A1, methylated DAB2IP, methylated PTPN6, methylated THY1, methylated TMEFF2, and methylated PYCARD, but is not limited thereto.

In order to determine whether the fetus is male or female based on the concentration ratio of DYS14/GAPDH obtained from a sample used to predict fetal gender, a reference value, or cutoff value, is required to be set. For this, a cutoff value is calculated in advance by using a plurality of samples. For example, an optimal cutoff value may be obtained by measuring concentrations of DYS14 and GAPDH in maternal plasma of a group of pregnant women, the cutoff value of which is calculated, and assessing a concentration ratio of DYS14/GAPDH at 100% specificity where the detection exactly matches gender of female fetus.

The method of determining fetal gender by using the concentration ratio of DYS14/GAPDH in maternal plasma is accurate not only during the second and third trimesters, but also during the first trimester.

According to another embodiment of the present invention, there is provided an apparatus for determining fetal gender including a device for amplifying DYS14 and GAPDH genes or portions thereof using fetal DNA as a template, a device for measuring concentrations of DYS14 and GAPDH, a device for calculating a concentration ratio of DYS14/GAPDH, and a device for printing the calculated concentration ratio of DYS14/GAPDH. The apparatus may further include a device for amplifying a U-PDE9A gene of bisulfite-treated fetal DNA using primers capable of discriminating methylated CpG sites and unmethylated CpG sites of the U-PDE9A gene of the bisulfite-treated fetal DNA, and a device for measuring a concentration of the U-PDE9A gene. The apparatus may further include a device for determining fetal gender by comparing the concentration ratio of DYS14/GAPDH and the concentration of the U-PDE9A gene with cutoff values of the concentration ratio of DYS14/GAPDH and the concentration of the U-PDE9A gene, which are input in advance.

The device for amplifying the DYS14 and GAPDH genes or portions thereof using fetal DNA as a template, or the device for amplifying the U-PDE9A gene of bisulfite-treated fetal DNA may include a PCR device including a reaction chamber and a heat block, but is not limited thereto. The device for measuring the concentration of DYS14, GAPDH, or U-PDE9A genes may include a means for measuring absorbency, fluorescence, radial rays, or the like, but is not limited thereto.

The inventions of the present application is not limited by the detection method; therefore, the amplification products may be detected by any detection method, which includes but is not limited to, the use of hybridization probes and quantitative real time polymerase chain reaction, digital PCR, electrophoresis, pyrosequencing, primer extension,microarrays, chips and sequencing.

The device for determining fetal gender by comparing the concentration ratio of DYS14/GAPDH and the concentration of the U-PDE9A gene with cutoff values of the concentration ratio of DYS14/GAPDH and the concentration of the U-PDE9A gene which are input in advance may include a computing device such as a computer, but is not limited thereto.

The device for printing the calculated concentration ratio of DYS14/GAPDH may include a memory device, a screen, a monitor, a facsimile, a printer, a terminal unit, a cellular phone, or the like, but is not limited thereto.

According to another embodiment of the present invention, there is provided a reagent package for an apparatus for determining fetal gender including: a PCR reagent set including primer pairs capable of amplifying DYS14 of fetal DNA or portions thereof; a PCR reagent set including primer pairs capable of amplifying GAPDH of fetal DNA or portions thereof; and a PCR reagent set including primer pairs capable of discriminating methylated CpG sites and unmethylated CpG sites of PDE9A of bisulfite-treated fetal DNA. Examples of primer pairs used herein are shown in Table 2, but are not limited thereto. The PCR reagent set includes primer pairs with respect to a target gene, a hydrolysis probe, a buffer solution, and other reagents required for PCR.

The term "primer" as used herein refers to a nucleic acid that comprises a nucleotide sequence capable of hybridizing or annealing to a target nucleic acid, at or near(e.g. adjacent to) a specific region of interest. Primers used in the inventions of this application may have nucleotide sequences substantially idential to a nucleotide sequence of primers provided herein, for example, about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical and further where the primers still speicifically hybridize to a target region (e.g., gene).

The present invention will now be described in greater detail with reference to the following examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

Statistical analyses were performed using the Statistical Package for Social Sciences 10.0 (SPSS Inc., Chicago, IL, USA). In all tests, a threshold of P<0.05 was set for statistical significance.

Example 1: Clinical characteristics of study population

A nested cohort study was performed with women who enrolled in the Cheil General Hospital Non-invasive Prenatal Diagnosis Study (CNIPD). The participants in this study were healthy pregnant woman who delivered a healthy singleton at term (37 weeks of gestation or more) without medical or obstetric complications. No participants had a history of preexisting hypertension, diabetes mellitus, liver disease, or chronic kidney disease.

Clinical characteristics of the study population were compared between fetal genders using the Mann-Whitney U test and χ2 test (Table 1). From blood sampling, maternal age and gestational age were found not to be significantly different between male and female-bearing participants. In 18% of male-bearing participants and 21% of female bearing participants, testing was performed before 7 weeks of gestation (range 5 to 6 weeks). 22% of male-bearing participants and 23% of female-bearing participants were pregnant women older than 35 years.

At delivery, birth weight and gestational age also did not differ between the two groups.

Table 1

	Characteristic	Fetal gender		P-value
	Characteristic	Male (n=99)	Female (n=104)	P-value
At blood sampling	Maternal age (yr)	33 (29-35)	32 (29-34)	0.379^a
	≥ 35 years [n.(%)]	22 (22.3)	24 (23.1)	0.599^b
	Gestational age (wk)	8.1 (7.2-12.0)	8.1 (7.1-11.3)	0.890^a
	≤ 6 weeks [n.(%)]	18 (18.2)	21 (20.2)	0.716^b
At delivery	Gestational age (wk)	39.0 (37.6-40.3)	39.0 (38.3-40.2)	0.428^a
At delivery	Birth weight (g)	3,365 (3,080-3,555)	3,362 (3,097-3,553)	0.920^a

Values are median with interquartile range in parentheses or as indicated. a: Mann-Whitney U test, b:χ2 test

Example 2: Sample collection and processing

Before blood sampling, ultrasonography was performed to establish viability of the singleton pregnancy and to confirm gestational age calculated from last menstruation.

Maternal blood samples were obtained from all participants at or before 12 weeks of gestation. 10 mL of peripheral blood was obtained using ethylenediaminetetraacetic acid (EDTA) as an anti-coagulant. Immediately after blood sampling, plasma was separated from whole blood by centrifugation at 2,500 g for 10 minutes. Recovered plasma was then centrifuged for an additional 10 minutes at 16,000 g to minimize any additional release of maternal DNA. Circulating fetal DNA from 1 mL of maternal plasma was extracted using a QIAamp DSP Virus Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The DNA was eluted into 30 μL sterile, DNase-free water. All samples were coded for a subsequent blinded analysis with respect to fetal gender.

Example 3: Tissue-specific epigenetic characteristics of PDE9A gene by bisulfite sequencing

The PDE9A gene was found to be completely methylated in maternal blood cells and unmethylated in placentas obtained from both the first and third trimesters. This epigenetic characteristic of the PDE9A gene was confirmed by bisulfite sequencing.

DNA samples extracted from maternal blood cells and placental tissues were subjected to bisulfite conversion using a CpGenome universal DNA modification kit (Chemicon, USA) according to the manufacturer’s instructions. The bisulfite-treated DNA was then amplified by PCR with primers capable of discriminating the methylated and unmethylated CpG sites of the PDE9A gene.

The primers used for sequencing the methylated CpG sites of PDE9A gene were as follows:

5’-CGGTGAGTGCGCGTCGC-3’ (forward primer)

5’-CCAACCATCCCGAAAAAGCG-3’ (reverse primer)

The primers used for sequencing the unmethylated CpG sites of the PDE9A gene were as follows:

5'-GGTTTGTTTTGGTGAGTGTGTGTCGT-3’ (forward primer)

5’-CCCAACCATCCCAAAAAAGCA-3’ (reverse primer)

PCR reaction solutions included 10 ng genomic DNA, 10 pM primers, 0.25 mM dNTPs, 1.5 mM MgCl2, 1 X buffer, and 0.25 U Taq polymerase per 50 μL of total reaction volume. PCR conditions included pre-denaturation at 94°C for 5 minutes, 35 cycles of 94°C for 45 seconds, 60°C for 45 seconds, 72°C for 45 seconds, and final extension at 60°C for 30 minutes.

After PCR amplification, PCR products were purified using a PCR purification kit (BIONEER, Korea) and sequenced using a PRISM Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems Inc., Forster City, CA, USA). Sequencing products were analyzed using a PRISM 3100 Genetic Analyzer (Applied Biosystems Inc., USA) and electropherogram traces were interpreted with Genescan software version 3.7 (Applied Biosystems Inc., USA). Genotypes thereof were assigned using Genotyper software version 3.7 (Applied Biosystems Inc., USA).

The tissue-specific epigenetic characteristics of PDE9A gene are shown in FIGs. 1a and 1b. The methylated pattern of the PDE9A gene was detected in both maternal blood cells and placental tissues. However, an unmethylated pattern of the PDE9A gene was detected only in placental tissues. Therefore, according to an embodiment of the present invention, the U-PDE9A gene was used as a marker to confirm the presence of fetal DNA in maternal plasma.

Example 4: Detection of unmethylated PDE9A (U-PDE9A) in maternal plasma by qMSP

In order to identify the presence of fetal DNA in maternal plasma, quantitative methylation-specific PCR (qMSP) was used to measure U-PDE9A.

The qMSP assay was performed according to the method disclosed in Clin. Chem, 54, 500-511 (2008). Sequences of circulating fetal DNA were converted with an EZ DNA methylation kit (Zymo Research, Irvine, CA, USA). The EZ DNA methylation kit, including sodium bisulfite, converts unmethylated cytosine into uracil residues, whereas methylated cytosine residues keeps cytosine residues unchanged. Therefore, circulating fetal DNA in maternal plasma exists in a form which is converted by sodium bisulfite. For validation of bisulfite conversion, the synthetic DNA oligonucleotide of a U-PDE9A region was used as the positive control and DNA extracted from maternal blood cells was used as the negative control.

Total DNA (30 μL) extracted from 1 mL of maternal plasma was bisulfite-converted using the EZ DNA methylation kit and eluted with 25 μL of DNase-free water. Eluted DNA was used as a template for each PCR reaction. The qMSP assay for the U-PDE9A was preformed with a primer set and a dual-labeled fluorescent hydrolysis probe. Sequences of primers and probes are shown in Table 2.

Table 2

Target		Sequences
U-PDE9A	Forward primer	5'- GGT TTG TTT TGG TGA GTG TGT GTC GT-3'
	Reverse primer	5'- CCC AAC CAT CCC AAA AAA GCA-3'
	Hydrolysis probe	5'-(FAM)-TTT GTT TGG TGA TGT TAT GTG GTT T-(MGBNFQ) -3'
DYS14	Forward primer	5'-GGG CCA ATG TTG TAT CCT TCT-3'
	Reverse primer	5'-GCC CAT CGG TCA CTT ACA CTT C-3 '
	Hydrolysis probe	5'-(FAM)-TCT AGT GGA GAG GTG CTC-(TAMRA)-3'
GAPDH	Forward primer	5'-CCC CAC ACA CAT GCA CTT ACC-3'
	Reverse primer	5'-CCT AGT CCC AGG GCT TTG ATT-3 '
	Hydrolysis probe	5'-(HEX)-AAA GAG CTA GGA AGG ACA GGC AAC TTG GC-(TAMRA)-3'

FAM: 6-Carboxyfluorescein ,

MGBNFQ: minor groove-binding nonfluorescent quencher

HEX: 2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein

TAMRA: 6-carboxytetramethyl-rhodamine

< Sequences of primers and probes according to target genes>

A PCR reaction solution included 12.5 μL iQ Supermix (Bio-Rad Laboratories, Hercules, CA, USA), a 200 nM hyprolysis probe (Applied Biosystems Inc., USA), 400 nM primers, and 5 μL converted DNA per 25 μL total reaction volume. The thermal profile for the qMSP assay consisted of an initial renaturation step of 95°C for 10 minutes followed by 50 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds.

Calibration curves for the assay were prepared by serial dilutions of single-stranded synthetic DNA oligonucleotide calibrators specific to U-PDE9A. Used calibrator sequence is as follows:

5’-GGT TTG TTT TGG TGA GTG TGT GTT GTG GGT TTT GTT TGG TGA TGT TAT GTG GTT TTT TTG TTT TTT TGG GAT GGT TGG G-3’

The U-PDE9A gene was detected in all maternal blood samples used herein, and concentrations thereof in male and female fetuses are shown in Table 3 below. Since the presence of the circulating fetal DNA was confirmed by using the U-PDE9A gene, the possibility of false-negative results due to undetectable low concentrations of circulating fetal DNA may be precluded.

Example 5: Multiplex quantitative real-time polymerase chain reaction (PCR) of DYS14 and GAPDH

Multiplex quantitative real-time PCR was performed for fetal gender determination using Y chromosome-specific DYS14 and the reference GAPDH genes as the molecular marker. The multiplex quantitative real-time PCR was performed using DNA Engine Opticon 2 system (MJ Research, Waltham, MA, USA).

The multiplex quantitative real-time PCR was performed in a volume of 25 μL, using 12.5 μL of iQ Supermix (Bio-Rad Laboratories, USA) and 5 μL of the extracted plasma DNA. Sequences of primers and probes are shown in Table 2. Primers and probes were used at final concentrations of 400 nM and 200 nM for DYS14 and 300 nM and 100 nM for GAPDH. Initial denaturation cycling conditions were 95°C for 10 minutes, followed by 50 cycles of 15 seconds at 95°C and 1 minute at 60°C.

The level of male fetal DNA that was present in the plasma sample was determined by comparison with a standard dilution curve using a known concentration of a commercial male genomic DNA (Promega, Madison, WI, USA). A standard curve was made by amplification of reference male genomic DNA at serial 10-fold dilutions such as 1, 0.1, 0.01, 0.001, and 0.0001 ng/μL. Each standard was amplified in triplicate and included on every PCR plate.

In standard curves, slope values and R²-values were -3.371 and 0.996 for DYS14 and -3.337 and 0.997 for GAPDH, respectively. The PCR efficiency was calculated from the slope of the curve by the following formula: Efficiency =10^-(1/slope) - 1. Both assays amplified with close to optimal efficiencies of 98% in DYS14 and 99% in GAPDH.

Strict precautions were taken against contamination, and multiple negative-control water blanks were included in every analysis. To reduce inter-experimental variation, PCR was performed triplicate. The final data reflected the mean of the results.

Example 6: Concentrations of DYS14, U-PDE9A, and GAPDH according to fetal gender

Concentrations of factors, DYS14, U-PDE9A, and GAPDH measured after the PCR of Examples 4 and 5 were expressed as copies/mL and a standard factor of 6.6 pg was used to convert the data to copy numbers (Am. J. Hum. Genet., 62, 768-775).

Concentrations and ratios of DYS14, U-PDE9A, and GAPDH according to the fetal gender were compared with each other using the Mann-Whitney U test. Ratios of DYS14/GAPDH and U-PDE9A/GAPDH were calculated as (DYS14 concentration/GAPDH concentration) X 100 and (U-PDE9A concentration/GAPDH concentration) X 100, respectively.

Concentrations and ratio of DYS14, U-PDE9A, and GAPDH according to the fetal gender are shown in Table 3 below.

Table 3

		Male (n=99)	Female (n=104)	P-value
Concentration(copies/ml)	DYS14	177.2 (108.1-325.9)	36.5 (16.0-58.3)	<0.001
	U-PDE9A	110.2 (74.8-172.6)	118.8 (82.4-179.7)	0.427
	GAPDH	3285.3 (1988.5-5516.0)	3491.4 (2530.5-5393.2)	0.168
Ratio	U-PDE9A/GAPDH	3.7 (2.9-4.8)	3.4 (2.7-4.1)	0.108
Ratio	DYS14/GAPDH	5.7 (4.8-7.5)	0.8 (0.3-2.1)	<0.001

Values are median with interquartile range in parentheses, and data was compared by Mann-Whitney U-test.

Concentrations of U-PDE9A and GAPDH were not significantly different between male and female-bearing participants (P>0.05 in both). However, the concentration of DYS14 was significantly higher in male-bearing participants than in female-bearing participants (P<0.001).

Correlations among the concentrations were estimated using Spearman’s rank correlation. In male-bearing participants, the concentration of DYS14 was positively correlated with the concentrations of U-PDE9A and GAPDH (r=0.831 for U-PDE9A and r=0.889 for GAPDH; P<0.001 in both, Figures 2A and 2B). The concentration of U-PDE9A was also positively correlated with the concentration of GAPDH (r=0.823; P<0.001, Figure 2C).

A strong positive correlation between circulating fetal DNA concentrations was found based on the results obtained using DYS14 and U-PDE9A. A strong positive correlation was also observed when comparing DYS14 with GAPDH or comparing U-PDE9A with GAPDH, used as fetal DNA and total DNA identifiers. These findings suggest that the amount of fetal DNA extracted from maternal plasma is proportional to the amount of total DNA extracted from maternal plasma. Therefore, a concentration ratio of DYS14/GAPDH was calculated by adjusting the concentration of DYS14 with the GAPDH concentration amplified simultaneously in the same PCR conditions, and thus the false-negative results may be removed.

These findings indicate that a total DNA identifier extracted from maternal plasma, such as GAPDH, is an important factor in fetal gender detection, and the concentration ratio of DYS14/GAPDH may be the most effective biomarker for early detection of fetal gender. In analysis of the concentration ratio, the ratio of U-PDE9A/GAPDH did not differ between fetal genders (P=0.108). However, the ratio of DYS14/GAPDH was significantly higher in male-bearing participants than in female-bearing participants (P<0.001).

Example 7: Limit of detection for quantitative PCR

The mean fetal DNA concentration in maternal plasma was previously estimated to be 25.4 copies/mL (range 3.3 to 69.4) in early pregnancy (Am. J. Hum. Genet., 62, 768-775).

In qMSP of the PDE9A gene, 1 mL of maternal plasma is extracted, eluted in 25 μL, then 5 μL is used for each PCR. Thus, each PCR has fetal DNA of more than 4 copies. Therefore, a PCR is sensitive enough because at least 3 copies of the target are required at a real-time PCR according to the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines. A minimum value to consider the presence of circulating fetal and total DNA was 20 copies/mL for DYS14 and U-PDE9A and 1,000 copies/mL for GAPDH.

Example 8: Accuracy of factors for detecting fetal gender

The accuracy of detecting fetal gender was analyzed according to factors such as a DYS14 quantification cycle (Cq), a DYS14 concentration, and a ratio of DYS14/GAPDH. Accuracy of fetal gender detection was determined with the final delivery record.

Receiver operating characteristics (ROC) curve analysis was performed to assess the optimal cutoff value (Obstet. Gynecol. 111, 1403-1409, 2008). The optimal cutoff was set at 100% specificity where the detection exactly matched gender of female fetus for each factor. The false-negative rate, positive predictive value (PPV), and negative predictive value (NPV) were calculated to consider the diagnostic efficiency using the EpiMax Table Calculator (http://www.healthstrategy.com/epiperl/epiperl.htm). Overall accuracy was estimated by measuring the area under the ROC curve (AUC) with a 95% confidence interval (CI).

The accuracy of each factor for the detection of fetal gender is shown in Table 4. PPVs of the DYS14 concentration, the DYS14 Cq, and the DYS14/GAPDH ratio were 100.0%. The DYS14/GAPDH ratio showed the highest value in NPV and AUC and the lowest value in the false-negative rate. For the DYS14 Cq, the DYS14 concentration, and the DYS14/GAPDH ratio, concordances of 95.6% (194/203), 96.6% (196/203), and 100.0% (203/203) were observed with the results confirmed phenotype at birth, respectively.

Table 4

Parameter	Cutoff	Specificity (%)	False-Negative Rate (%)	PPV(%)	NPV(%)	AUC and95% CI
DYS14 Cq	37.0	100	9.1	100	92.0	0.994(0.988-1.000)
DYS14 concentration	85.0	100	7.1	100	93.7	0.997(0.993-1.001)
DYS14/GAPDH ratio	4.0	100	0.0	100	100	1.000(1.000-1.000)

In male-bearing participants, concentrations of U-PDE9A, DYS14, and GAPDH were significantly lower in the false-negative results than in the correct results (113.0 versus 49.1 copies/mL in U-PDE9A, 184.9 versus 76.6 copies/mL in DYS14, and 3449.9 versus 1414.2 copies/mL in GAPDH, P≤0.001 in all, Figures 3a, 3b, 3c). However, the DYS14/GAPDH ratio and the U-PDE9A/GAPDH ratio did not significantly differ between the participants showing false-negative results and the participants showing correct results (5.2 versus 5.8 in the DYS14/GAPDH ratio and 4.2 versus 3.7 in the U-PDE9A/GAPDH ratio, P>0.05 in both, Figures 4a, 4b).

Detective accuracies of fetal gender were calculated based on gestational weeks when blood sampling was performed (Table 5). The false-negative results of the DYS14 assay were observed only at and before 8 weeks of gestational age. The concentration ratio of DYS14/GAPDH showed 100.0% detective accuracy regardless of gestational weeks.

Table 5

Gestational age (wk)	Total number	Parameters
Gestational age (wk)	Total number	DYS14 Cq	DYS14 concentration	DYS14/GAPDH ratio
5-6	39	87.2 (34/39)	89.7 (35/39)	100.0 (39/39)
7	35	91.4 (32/35)	94.3 (33/35)	100.0 (35/35)
8	33	97.0 (32/33)	97.0 (32/33)	100.0 (33/33)
9	31	100.0 (31/31)	100.0 (31/31)	100.0 (31/31)
10-11	24	100.0 (24/24)	100.0 (24/24)	100.0 (24/24)
12	41	100.0 (41/41)	100.0 (41/41)	100.0 (41/41)

The numbers in parentheses indicate case numbers showing the correct results/total number.

In prior studies, it was reported that male fetal DNA in maternal plasma may be identified by amplifying a sequence with multiple copies per male genome (Prenat. Diagn. 28, 525-530, 2008). The DYS14 assay targets a multicopy sequence and, therefore, has a higher sensitivity than a single-copy gene, such as SRY. However, Scheffer et al. have emphasized that the SRY assay should be used to increases the specificity of the test as a whole, because of amplification signals in the DYS14 assay obtained in female-bearing pregnancies, albeit at a high Cq value (Obstet Gynecol, 115, 117-126, 2010). The present inventors confirmed amplification signals of DYS14 in female-bearing participants at a high Cq value such as at more than 40 Cq. This result may be induced by non-specific amplification of artifacts such as primer dimerization and instability of the fluorescence probe at high Cq. However, this condition did not affect the detection of female-fetal gender, because a higher Cq and lower concentration than the cutoff value was used in the DYS14 assay. In contrast, the false-negative results were found at a high Cq and low concentration of DYS14 in male-bearing participants before 8 weeks of gestation.

The concentrations of U-PDE9A and DYS14 used as fetal DNA identifiers were significantly lower in the false-negative results than in the correct results. The concentration of GAPDH used as a total DNA identifier also showed the same pattern. However, the values of all factors in false-negative results were above a minimum value used to consider the presence of circulating fetal and total DNA. These results indicate that the false-negative results may have been due to a lower quantity of circulating fetal and total DNA than the cutoff value used for fetal gender detection, rather than the undetectable concentration of circulating fetal DNA. Moreover, the false-negative results were observed in the early first trimester such as before 8 weeks of gestation. Therefore, fetal gender detection may be performed by using the concentration ratio of DYS14/GAPDH rather than by only the DYS14 assay during the 5 to 7 weeks of gestation.

The inventions of the present application may applicable to fetus of animals including human being.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.

The present invention is applicable for determining fetal gender non-ivasively.

Claims

A method of analyzing a sample for providing information required to determine fetal gender, the method comprising:

extracting fetal DNA from collected maternal plasma;

measuring concentrations of DYS14 and GAPDH in the extracted fetal DNA; and

calculating a concentration ratio of DYS14/GAPDH from the measured concentrations of DYS14 and GAPDH.
The method of claim 1, further comprising measuring a concentration of a fetal DNA marker regardless of fetal gender.
The method of claim 2, wherein the fetal DNA marker regardless of fetal gender comprises one selected from the group consisting of unmethylated PDE9A (U-PDE9A), unmethylated SERPINB 5, methylated RASSF1A, methylated APC, methylated CASP8, methylated RARB, methylated SCGB3A1, methylated DAB2IP, methylated PTPN6, methylated THY1, methylated TMEFF2, and methylated PYCARD.
The method of any one of claims 1 to 3, further comprising determining an optimal cutoff value by measuring concentrations of DYS14 and GAPDH in maternal blood of a group, the cutoff value of which is calculated, and assessing a concentration ratio of DYS14/GAPDH at 100% specificity where the determination exactly matches gender of female fetus.
The method of any one of claims 1 to 3, wherein the fetal gender determination is performed during a first trimester pregnancy.
The method of any one of claims 1 to 3, further comprising amplifying DYS14, GAPDH, a fetal DNA marker regardless of fetal gender, or portions thereof.
The method of claim 6, wherein the amplifying is performed by a polymerase chain reaction (PCR).
An apparatus for determining fetal gender, the apparatus comprising: a device for amplifying DYS14 and GAPDH genes or portions thereof using fetal DNA as a template, a device for measuring concentrations of DYS14 and GAPDH; a device for calculating a concentration ratio of DYS14/GAPDH; and a device for printing the calculated concentration ratio of DYS14/GAPDH.
The apparatus of claim 8, further comprising a device for amplifying a PDE9A gene of bisulfite-treated fetal DNA using a primer capable of discriminating between methylated CpG sites and unmethylated CpG sites of the PDE9A gene of the bisulfite-treated fetal DNA, and a device for measuring a concentration of the U-PDE9A gene.
The apparatus of claim 9, further comprising a device for determining fetal gender by comparing the concentration ratio of DYS14/GAPDH and the concentration of the U-PDE9A gene with cutoff values of the concentration ratio of DYS14/GAPDH and the concentration of the U-PDE9A gene which are input in advance.
A reagent package for determining fetal gender for the apparatus of any one of claims 8 to 10, the reagent package comprising:

a PCR reagent set comprising primer pairs capable of amplifying DYS14 of fetal DNA or portions thereof;

a PCR reagent set comprising primer pairs capable of amplifying GAPDH of fetal DNA or portions thereof;

a PCR reagent set comprising primer pairs capable of discriminating between methylated CpG sites and unmethylated CpG sites of PDE9A of bisulfite-treated fetal DNA.
A method of determining fetal gender, the method comprising:

extracting fetal DNA from collected maternal plasma;

measuring concentrations of DYS14 and GAPDH in the extracted fetal DNA; and

calculating a concentration ratio of DYS14/GAPDH from the measured concentrations of DYS14 and GAPDH.
The method of claim 12, further comprising measuring a concentration of a fetal DNA marker regardless of fetal gender.
The method of any one of claims 12 to 13, further comprising determining an optimal cutoff value by measuring concentrations of DYS14 and GAPDH in maternal blood of a group, the cutoff value of which is calculated, and assessing a concentration ratio of DYS14/GAPDH at 100% specificity where the determination exactly matches gender of female fetus.
The method of any one of claims 12 to 13, wherein the fetal gender determination is performed during a first trimester pregnancy.