Abstract:
The present invention provides a method for non-invasively detecting, expecting, or diagnosing fetal single nucleotide polymorphisms and the resultant monogenic disorders, through maternal cell-free DNA sequencing. The diagnosis method according to the present invention does not harm mothers or fetuses and is convenient, in that analysis is possible using maternal blood samples; and can be favorably used for a prenatal diagnosis method capable of determining at an early stage whether single nucleotide polymorphisms causing monogenic disorders occur or not

Description:
TECHNICAL FIELD 
       [0001]    The present disclosure relates to a method for predicting fetal monogenic genetic variation through the sequencing of maternal serum DNA. More particularly, the present disclosure relates to a method for the non-invasive prenatal prediction of fetal monogenic genetic variation and a resultant monogenic disorder from the sequencing of maternal serum DNA. 
       BACKGROUND ART 
       [0002]    A recent increase in the incidence of congenital anomalies has focused attention on prenatal diagnosis, resulting in the development of various kinds of diagnostic equipment. Particularly, a pregnant women who 35 years old or older, who has delivered fetusren with chromosomal abnormalities, who was born with a structural chromosomal anomaly or whose fetus was conceived with a man with a structural chromosomal anomaly, who has a family history of genetic disorders, who is at risk of neural tube defect, or who is suspected of fetal deformity is recommended to receive prenatal diagnosis. 
         [0003]    Prenatal diagnosis can be performed by invasive or non-invasive methods. Imposing an impact on the fetus, invasive diagnosis methods are in danger of causing miscarriage, disorders or deformity. Hence, non-invasive diagnosis methods have been developed as alternatives to overcome such problems. 
         [0004]    The discovery of cell-free fetal DNA (cffDNA) in the cell-free DNA (cfDNA) of maternal plasma has provided a powerful tool for developing non-invasive prenatal genetic diagnosis methods. The application of cffDNA to prenatal diagnosis is further accelerated by the introduction of massively parallel sequencing, also called next-generation sequencing. 
         [0005]    In addition, some studies showed that fetal and maternal DNAs are uniformly distributed throughout the entire genome, as analyzed by whole genome sequencing (WGS) and sequencing following the target enrichment of cffDNA (Lo Y M et al., Science Translational Medicine 2010; 2:61ra91; Liao G J et al., Clinical Chemistry 2011; 57:92-101; Kitzman J O et al., Science Translational Medicine 2012; 4:137ra76). These results provide a basis on which detection can be extended to monogenic disorders, which are more common than chromosomal aneuploidy. 
         [0006]    However, there are still many intricate obstacles to be overcome for application to monogenic disorders, whereas aneuploidy detection can be readily applied to clinical practice. Since the content of cffDNA in maternal plasma is low and very differs from one maternal plasma to another versatile, fetal variants are difficult to detect with reliability at the mononucleotide level. 
       DISCLOSURE 
     Technical Problem 
       [0007]    The present disclosure aims to provide a method for sensing, detecting, or diagnosing fetal monogenic variation through the sequencing of maternal serum DNA, or a method for predicting or diagnosing a monogenic disorder related to the monogenic variation or for providing information about the prediction/diagnosis of a monogenic disorder related to the monogenic variation. 
       Technical Solution 
       [0008]    The present disclosure addresses a method for predicting fetal monogenic genetic variation through the sequencing of maternal serum DNA. More particularly, the present disclosure addresses a method for the non-invasive prediction, detection or diagnosis of fetal monogenic genetic variation and a resultant monogenic disorder through the sequencing of maternal serum DNA. 
         [0009]    An embodiment of the present disclosure provides a method for detecting a fetal genetic variation, using maternal serum RNA, the method comprising defining a genetic variation in a maternal single gene, with regard to a deletion- or duplication-type genetic mutation and a region where the genetic variation occurs, by use of sequencing analysis data of maternal serum DNA, and measuring an allele frequency of heterozygous SNP in the variation region from a maternal serum DNA having the genetic variation to determine whether or not the fetus has a deletion- or duplication-type genetic variation in the single gene linked to the monogenic disorder. 
         [0010]    Another embodiment of the present disclosure addresses a method for detecting a fetal genetic variation, using maternal serum RNA, the method comprising defining a genetic variation in a maternal single gene, with regard to a deletion- or duplication-type genetic mutation and a region where the genetic variation occurs, by use of sequencing analysis data of maternal serum DNA, analyzing serum DNA and corpuscle DNA of the pregnant woman having the genetic variation through next-generation sequencing to acquire read depth ratios (serum read depth/corpuscle read depth) and applying the read depth ratios to determining whether or not the fetus has a deletion- or duplication-type genetic variation in the single gene linked to the monogenic disorder. 
         [0011]    With the advance development of DNA sequencing techniques, massive genomic information can be deciphered. Particularly, next-generation sequencing (NGS)-based genomic analysis techniques are applied to the prenatal analysis. Based on the fact that maternal blood contains a fetal genome at a certain level, a maternal blood sample can be used to analyze fetal genomic information. This method is simple without impacts on the pregnant woman or the fetus, and can be useful as a prenatal diagnosis method by which a fetal genetic variation can be predicted in an early stage. 
       Advantageous Effects 
       [0012]    Capable of predicting a fetal monogenic genetic variation before birth (from 6 weeks after conception) in a non-invasive manner through the sequencing of a serum DNA taken from a carrier pregnant woman, the method of the present disclosure is safe, effective and simple, and thus can be applied to prenatal diagnosis instead of conventional non-invasive methods, such as chorionic villus sampling and amniocentesis. 
     
    
     
       DESCRIPTION OF DRAWINGS 
         [0013]      FIG. 1  shows plots of allele frequencies versus fetal DNA fractions, wherein the upper line accounts for a fetus having a mutation in a single gene and the lower line accounts for a fetus having no mutations in a single gene; and 
           [0014]      FIG. 2  shows ratios of serum read depth to corpuscle read depth (serum read depth/corpuscle read depth, γ) in variation and non-variation regions in a single gene, wherein the X-axis and Y-axis respectively represent nucleotide positions on an X chromosome and relative read depth ratios (γ) in variation regions (closed circles) and non-variation regions (open circles), together with a representative value in each region (dotted lines) 
       
    
    
     DETAILED DESCRIPTION 
       [0015]    The present invention will be described in more details. 
         [0016]    The present disclosure addresses a method for sensing, detecting, or diagnosing fetal monogenic variation through the sequencing of maternal serum DNA. The method is simple because it does not need patrilineal or male-line DNA. 
         [0017]    Particularly, the method for detecting fetal monogenic variation using maternal serum DNA comprises 
         [0018]    defining a genetic variation in a maternal single gene linked to a monogenic disorder, with regard to a deletion- or duplication-type genetic mutation and a region where the genetic variation occurs, by use of sequencing analysis data of maternal serum DNA (step A), 
         [0019]    using a maternal serum DNA having a genetic variation to analyze a discriminate genetic trait depending on fetal genetic variation in the variation region or non-variation region (normal region)(step B), and 
         [0020]    determining whether or not the fetus has a deletion- or duplication-type genetic variation in the single gene linked to the monogenic disorder (step C). 
         [0021]    In accordance with some embodiments of the present disclosure, the step B of using a maternal serum DNA having a genetic variation to analyze a discriminate genetic trait can be carried out in two manners. First, the maternal serum DNA having a genetic variation is measured for the allele frequency of heterozygous SNP in the variation region to give a measurement distribution of the allele frequency. A fetal DNA fraction (f) of the maternal serum DNA is obtained, and used, together with Mendel&#39;s Law, to calculate expectation values of the allele frequency of heterozygous SNP in the variation region of the single gene for the presence and absence of fetal genetic variation. Then, the measurement distribution and the expectation values are used to determine whether or not the fetus has a genetic mutation. 
         [0022]    Second, maternal serum DNA and corpuscle DNA (genomic DNA) is analyzed through next-generation sequencing to acquire read depths in a variation region and a non-variation region of each of the serum DNA and the corpuscle DNA, and ratios of serum read depth to corpuscle read depth (serum read depth/corpuscle read depth) are compared to determine the presence or absence of a fetal genetic mutation. 
         [0023]    In accordance with some embodiments of the present disclosure, the method of using an allele frequency of heterozygous SNP in a genetic variation region may be carried out as follows: 
         [0024]    analyzing the maternal serum DNA to give maternal sequencing data; 
         [0025]    defining a deletion- or duplication-type genetic mutation and a variation region in a single gene linked to a maternal monogenic disorder from the sequencing data; 
         [0026]    measuring an allele frequency of heterozygous SNP in the variation region from a maternal serum DNA having the genetic variation to give a measurement distribution of the allele frequency; 
         [0027]    obtaining a fetal DNA fraction (f) of the maternal serum DNA, and applying Mendel&#39;s Law to calculate an expectation value of the allele frequency of heterozygous SNP in the variation region of the single gene as a first expectation value (θ exp|aff ) when the fetus has the genetic variation of the single gene and as a second expectation value (θ exp|unaff ) when the fetus does not have the genetic variation of the single gene; and determining whether the fetus has a monogenic deletion or duplication mutation for a monogenic disorder, based on whether the first expectation value (θ exp|aff ) and the second expectation value (θ exp|unaff ) fall within a statistically significant interval of the measurement distribution of the allele frequency. 
         [0028]    The detecting method of the present disclosure will be stepwise explained in more detail. 
         [0029]    (Step A) 
         [0030]    This step is adapted to define a genetic variation in a maternal single gene linked to a monogenic disorder, with regard to a deletion- or duplication-type genetic mutation and a region where the genetic variation occurs, by use of sequencing analysis data of maternal serum DNA 
         [0031]    Since the method of the present disclosure is based on the analysis of an allele frequency for an X-linked disorder, that is, a monogenic disorder, from a maternal serum, it will be readily understood to those skilled in the art that the method of the present disclosure can be applied to an X-linked recessive disorder or an autosomal recessive disorder. 
         [0032]    Examples of the disorders caused by monogenic genetic variations may be monogenic disorders including, but not limited to, Duchenne muscular dystrophy, Pelizaeus-Merzbacher disease, myotubular myopathy, Lowe syndrome, Menkes syndrome, X-linked adrenoleukodystrophy, Hoyeraal-Hreidarsson syndrome, spinal muscular atrophy, metachromatic leukodystrophy, and Krabbe disease. 
         [0033]    As used herein, the term “carrier pregnant woman” or “target pregnant woman” refers to a pregnant woman having a single-gene mutation in an X chromosome. Because the carrier pregnant woman has a recessive single-gene mutation (X′) in only one of her two X (XX) chromosomes, she does not express the monogenic disorder. In the present disclosure, the carrier pregnant woman is particularly one at 6 weeks or longer after conception. The cell-free DNA of the carrier pregnant woman includes cell-free fetal DNA. 
         [0034]    Whether or not a pregnant woman is a carrier can be determined by investigating the family story of a corresponding single-gene disease or sequencing genomic DNA and comparing the sequence with a reference DNA sequence, which is a normal DNA sequence or has a single-gene mutation. 
         [0035]    A pregnant woman who is a carrier and from whom cell-free DNA can be obtained during a pregnancy period is an experimental target in the present disclosure 
         [0036]    The sequencing of serum DNA may be performed by a next-generation sequencing method, examples of which include, but are not limited to, target enrichment and massively parallel sequencing. 
         [0037]    Types (insertion or deletion) and regions of genetic mutations can be revealed using many well-known structural variation-detecting programs. Examples of such structural variation-detecting programs include Delly, Pindell, BreakDancer, GASV, Hydra, and CNVnator, but are not limited thereto. In a particular embodiment, the type and region of a genetic mutation may be defined by calculating a moving average from a read depth for an overlapping sliding window having a window size of 10 kb read points after massively parallel sequencing; and applying a CBS (circular binary segmentation) algorithm. For example, the definition of a deletion- or duplication-type genetic mutation and a variation region in a single gene can be performed by calculating a moving average from a read depth obtained from the analysis of maternal serum DNA through next-generation sequencing, and applying CBS (circular binary segmentation). Optionally, the method may further comprise a step of eliminating variation attributed to GC content differences according to gene regions from the read depth data, using a known method, in advance. 
         [0038]    (Steps B and C) 
         [0039]    Step B utilizes the allele frequency of heterozygous SNP in the genetic variation region of maternal serum DNA, and is largely divided into the utilization of SNP allele frequency (B-1) and the utilization of a ratio of serum read depth to corpuscle read depth in variation and non-variation regions of a single gene (serum read depth/corpuscle read depth) (B-2). 
         [0040]    According to a particular embodiment, the alleles used to obtain a measurement distribution and an expectation value of the allele frequency are the same. For instance, a minor allele with an allele frequency less than 0.5 or a major allele with an allele frequency greater than 0.5 may be used in both steps of obtaining a measurement distribution of the allele frequency and an expectation value of the allele frequency. When the minor allele and the major allele are used together to obtain a measurement distribution and an expectation value of the allele frequency, both the measurement value and the expectation value converged at 0.5, regardless of fetal genetic variation, thus making not only the difference based on the assumption small but also complicating the calculation of the expectation value. 
         [0041]    A first substep (B-1) of the step B, which is adapted to utilize an SNP allele frequency, may be carried out as follows. 
         [0042]    (B-11) measuring maternal serum DNA having a genetic variation for an allele frequency of heterozygous single nucleotide polymorphism (SNP) in the variation region to give a measurement distribution of the allele frequency, 
         [0043]    (B-12) obtaining a fetal DNA fraction (t) of the maternal serum DNA, and applying Mendel&#39;s Law to calculating an expectation value of the allele frequency of heterozygous SNP in the variation region of the single gene as a first expectation value (θ exp|aff ) when the fetus has a genetic variation in the single gene and as a second expectation value (θ exp|unaff ) when the fetus does not have a genetic variation in the single gene, and 
         [0044]    (B-13) determining whether the fetus has a monogenic deletion or duplication mutation for a monogenic disorder on the basis of the fact that the first expectation value (θ exp|aff ) and the second expectation value (θ exp|unaff ) fall within a statistically significant interval of the measurement distribution of the allele frequency. 
         [0045]    Optionally, substep B-11 may further comprise eliminating outliers from the allele frequency values using a known process. 
         [0046]    Substep B-11 is designed to measure an average allele frequency (θ obs ) of heterozygous SNP in a DNA region having a monogenic genetic variation (duplication/deletion) from maternal serum DNA sequence data. The allele frequency can be calculated by counting the number of each allele in next-generation sequencing data by use of a conventional program such as Samtools. 
         [0047]    In substep B-12, a fetal DNA fraction (f) in the serum DNA of a carrier pregnant woman is obtained. The fetal DNA fraction may be measured using one of various conventional methods. 
         [0048]    By way of example, a male fetal DNA fraction (f) of maternal serum DNA may be obtained by capturing ZFX and ZFY genes with capture probes that can respectively capture an X-linked zinc finger (Zfx) gene region and a Y-linked zinc finger (Zfy) gene region. For a male fetus, in greater detail, custom capture probes that target ZFX (X-linked zinc finger protein) and ZFY (Y-linked zinc finger protein) gene regions may be used to capture ZFX and ZFY genes, followed by calculation according to the following equation: 
         [0049]    Fetal DNA fraction: 
         [0000]    
       
         
           
             f 
             = 
             
               
                 2 
                  
                 
                   ZFY 
                   
                     
                         
                     
                      
                     ′ 
                   
                 
               
               
                 
                   ZFX 
                   
                     
                         
                     
                      
                     ′ 
                   
                 
                 + 
                 
                   ZFY 
                   
                     
                         
                     
                      
                     ′ 
                   
                 
               
             
           
         
       
     
         [0050]    wherein, ZFX′ and ZFY′ are the quotients of the number of total mapped reads divided by the number of probes. 
         [0051]    A female fetal DNA fraction (f) of the maternal serum DNA can be obtained using a distribution of a SNP allele frequency, formed over an allele frequency range from 0.02 to 0.3, with 0.1 as a median in a non-variation region. 
         [0052]    In substep B-12, a fetal DNA fraction (f) of the maternal serum DNA is obtained, and an expectation value of the allele frequency of heterozygous SNP in the variation region of the single gene is calculated as a first expectation value (θ exp|aff ) when the fetus has a genetic variation in the single gene and as a second expectation value (θ exp|unaff ) when the fetus does not have a genetic variation in the single gene, using Mendel&#39;s Law. The equations according to which the first expectation value and the second expectation values are calculated can be established depending on the kind of chromosome (e.g., autosomes and allosomes), the kind of Mendelian inheritance, and the sex of the fetus in the case of allosomes, under the assumption that the pregnant woman is the carrier. 
         [0053]    For example, when the fetus is male with an X-linked recessive disorder, the first expectation value (θ exp|aff ) and the second expectation value (θ exp|unaff ) can be calculated according to the following Equations 1 and 2: 
         [0000]      θ exp|aff =2/(3− f )  &lt;Equation 1&gt;
 
         [0000]      θ exp|unaff =2(1− f )/3−2 f   &lt;Equation 2&gt;
 
         [0054]    Substep B-13 is designed to determine whether the fetus has a monogenic deletion or duplication mutation for a monogenic disorder based on the fact that the first expectation value (θ exp|aff ) and the second expectation value (θ exp|unaff ) fall within a statistically significant interval of the measurement distribution of the allele frequency. 
         [0055]    For instance, when the measured average allele frequency (θ obs ) is nearer to the expectation value (θ exp|aff ) of the allele frequency for a fetus that has a monogenic genetic variation, the fetus in the carrier pregnant woman is predicted to have a monogenic genetic variation. On the other hand, when the measured average allele frequency (θ obs ) is nearer to the expectation value (θ exp|unaff ) of the allele frequency for a fetus that does not have a monogenic genetic variation, the fetus in the carrier pregnant woman is predicted not to have the monogenic genetic variation. For statistical significance, the region of significance of the measured allele frequency (θ obs ) is calculated under the assumption that it is binomially distributed. If both the expectation values are within the region of significance, the prediction is determined to be without statistical significance. 
         [0056]    In accordance with another embodiments of the present disclosure, the second substep B-2 of the step B, which is designed to use the ratio of serum read depth to corpuscle read depth in variation and non-variation regions of a single gene (serum read depth/corpuscle read depth), may be carried out by: 
         [0057]    defining a genetic variation in a single gene for a maternal monogenic disorder, with regard to the kind of genetic mutation (deletion or duplication) and the region where the genetic variation occurs, by use of the sequencing analysis data of maternal serum DNA; 
         [0058]    acquiring, from the serum DNA sequencing data of the pregnant woman having the genetic variation, respective serum read depths in a variation region and a non-variation region in a single gene; 
         [0059]    acquiring, from the corpuscle DNA sequencing data of the pregnant woman having the genetic variation, respective corpuscle read depths in a variation region and a non-variation region in the single gene; 
         [0060]    calculating a first ratio of the serum read depth to the corpuscle read depth (serum read depth/corpuscle read depth) in the variation region in the single gene and a second ratio of the serum read depth to the corpuscle read depth (serum read depth/corpuscle read depth) in the non-variation region in the single gene; and 
         [0061]    comparing the first ratio with the second ratio to determine whether or not the fetus has a deletion- or duplication-type genetic mutation, which may cause a monogenic disorder, in the single gene. 
         [0062]    Optionally, the substeps of acquiring the first and second ratios may further comprise eliminating variation attributed to GC content differences according to gene regions from the read depth data, using a known method, and eliminating an outlier of the allele frequency values from the read depth data, using a known method. 
         [0063]    In the step of defining genetic variation with regard to kind and region by use of the sequencing analysis data of maternal serum DNA, the sequence information of maternal serum DNA is analyzed and used to define the kind of genetic variation (deletion or duplication) and the region of the genetic variation in the single gene, which may account for the monogenic disorder of the pregnant woman. The sequence analysis of maternal serum DNA may be carried out by obtaining maternal serum DNA and sequencing the serum DNA. The sequence analysis of serum DNA and the determination of the kind and region of genetic variation may be carried out in the same manner as in step A. 
         [0064]    The serum DNA and genome DNA of a carrier pregnant woman having a monogenic genetic variation may be analyzed using next-generation sequencing (NGS) to determine the kind and region of monogenic genetic variation, as described above. This method may be applied to both duplication and deletion mutations. All of the disorder, the pregnant woman, and the single gene that are used in step B-2 can be treated in the same manner as in step A. 
         [0065]    In detail, the utilization of a ratio of serum read depth to corpuscle read depth in variation and non-variation regions of a single gene (serum read depth/corpuscle read depth) may be implemented by: 
         [0066]    (B-21) acquiring, from the serum DNA sequencing data of the pregnant woman having the genetic variation, respective serum read depths in a variation region and a non-variation region in the single gene; 
         [0067]    (B-22) acquiring, from the corpuscle DNA sequencing data of the pregnant woman having the genetic variation, respective corpuscle read depths in a variation region and a non-variation region in the single gene; 
         [0068]    (B-23) calculating a first ratio of the serum read depth to the corpuscle read depth (serum read depth/corpuscle read depth) in the variation region in the single gene and a second ratio of the serum read depth to the corpuscle read depth (serum read depth/corpuscle read depth) in the non-variation region in the single gene; and 
         [0069]    (B-24) comparing the first ratio with the second ratio to determine whether or not the fetus has a deletion- or duplication-type genetic mutation, which may cause a monogenic disorder, in the single gene. 
         [0070]    For use in substep B-21 of acquiring, from the serum DNA sequencing data of the pregnant woman having the genetic variation, respective serum read depths in a variation region and a non-variation region in the single gene, the sequence information and the read depths may be obtained using next-generation sequencing, as described in step A. For example, a next-generation sequencing method including target enrichment and massively parallel sequencing may be used. 
         [0071]    Substep B-22 of acquiring, from the corpuscle DNA sequencing data of the pregnant woman having the genetic variation, respective corpuscle read depths in a variation region and a non-variation region in the single gene may be carried out in the same manner as in substep B-21, with the exception of using corpuscle DNA instead of serum DNA. For example, maternal corpuscle DNA is obtained and then analyzed for sequence and read depth using next-generation sequencing. 
         [0072]    Maternal serum and corpuscle DNA may be obtained by separating a maternal blood sample into a serum and corpuscles and isolating respective DNA from the serum and the corpuscles. The corpuscles can be obtained by, without limitation thereto, centrifuging a blood sample from the pregnant women, and the corpuscles may be lysed to extract genome DNA. DNA isolation may be performed using a conventional method well known in the art. Separation of maternal blood samples into serum and corpuscles makes it possible to obtain both serum and corpuscles from a blood sample taken only once from a pregnant woman, so that the method is simple. In addition, the method of the present disclosure enjoys the advantage of requiring no patrilineal DNA, and detecting fetal genetic variation with high sensitivity and accuracy. 
         [0073]    According to a particular embodiment, the maternal serum and corpuscle DNA may be analyzed using next-generation sequencing with the same custom capture probes under the same conditions in order to minimize measurement errors. When target single genes are determined, the custom capture probes can be easily selected, and thus are not particularly limited. 
         [0074]    In substeps B-23 and B-24, the first ratio of serum read depth to corpuscle read depth in a variation region of the single gene (serum read depth/corpuscle read depth) and the second ratio of serum read depth to corpuscle read depth in a non-variation region of the single gene (serum read depth/corpuscle read depth) are calculated and compared in order to determine whether or not the fetus has a deletion- or duplication-type genetic mutation, which is likely to cause a monogenic disorder, in the single gene. 
         [0075]    In detail, a relative serum read depth is calculated by dividing a read depth for maternal serum DNA by a read depth for maternal genome DNA in a variation region. Likewise, a relative serum read depth is calculated by dividing a read depth for maternal serum DNA by a read depth for maternal genome DNA in a non-variation region. Read depths may be calculated using conventional programs, such as Samtools, and relative read depths are calculated according to base sequence positions. 
         [0076]    According to some embodiments of the present disclosure, the first ratio may be a mean or median value of two or more measurements obtained from individual nucleotides in the variation region in the single gene, while the second ratio may be a mean or median value of two or more measurements obtained from individual nucleotides in the non-variation region in the single gene. 
         [0077]    Alternatively, the first ratio may be a mean or median value of two or more measurements of the moving average of read depth obtained from individual nucleotide bins of 5 to 100,000 bases in the variation region in the single gene while the second ratio may be a mean or median value of two or more measurements of the moving average of read depth obtained from individual nucleotide bins of 5 to 100,000 bases in the non-variation region in the single gene. For computation of the moving average of read depths, the nucleotide bins may or may not be set to overlap. 
         [0078]    The first ratio is compared with the second ratio to determine whether or not the fetus has a deletion- or duplication-type genetic mutation, which may cause a monogenic disorder, in the single gene. In this regard, when the maternal genetic variation is of a deletion type and the first ratio for the variation region is smaller than the second ratio for the non-variation region, the fetus can be determined to have a deletion variation in the single gene. On the other hand, when the maternal genetic variation is of a duplication type and the first ratio for the variation region is larger than the second ratio for the non-variation region, the fetus can be determined to have a duplication variation in the single gene. 
         [0079]    For example, when a woman with a monogenic genetic variation conceives a male fetus, the ratio of serum DNA read depth to genome DNA read depth (relative read depth: γ) varies depending on whether or not the fetus has a monogenic genetic variation and on the kind of fetal monogenic genetic variation, if present (see Table 5). 
         [0080]    As can be seen in Table 5, when the fetus has a deletion mutation, γ in the variation region is smaller than that in a normal region. When the fetus has no deletion mutations, γ in the variation region is larger than that in a normal region. For a fetal duplication mutation, γ in the variation region is larger than that in a normal region. On the other hand, when the fetus has no duplication mutations, γ in the variation region is smaller than that in a normal region. With respect to a pregnant woman having a deletion mutation, the fetus is determined to have a deletion genetic mutation when the first ratio for the variation region is smaller than the second ratio for the non-variation region. The fetus in a pregnant woman having a duplication mutation can be determined to have a duplication mutation in a single gene when the first ratio for the variation region is larger than the second ratio for the non-variation region. 
         [0081]    In greater detail, when the first ratio is compared to the second ratio for a pregnant woman having a deletion mutation, a comparison result in which a relative read depth in a variation region in the single gene is smaller than that in a non-variation region in the single gene makes it possible to predict that the fetus might have a monogenic genetic variation. On the other hand, a comparison result in which a relative read depth in a variation region in the single gene is larger than that in a non-variation region in the single gene leads to the prediction that the fetus might have no monogenic genetic variations. 
         [0082]    For a pregnant woman having a duplication mutation, when a relative read depth in a variation region in the single gene is found to be larger than that in a non-variation region in the single gene as the result of comparison between the first ratio and the second ratio, the fetus is predicted to have a monogenic genetic variation. In contrast, the fetus is predicted to have no monogenic genetic variations when a relative read depth in a variation region in the single gene is smaller than that in a non-variation region in the single gene. 
         [0083]    As described above, the method of the present disclosure is carried out by determining whether or not a pregnant woman has a deletion or duplication mutation in a single gene, followed by comparing γ in a normal region with γ in a variation region, whereby the inheritance by the fetus of the single-gene mutation, that is, whether the fetus has the single-gene mutation, can be easily determined. 
         [0084]    As such, the method according to some embodiments of the present disclosure is designed to precisely predict the single-gene mutation of a fetus by analyzing maternal serum and genome DNA using next-generation sequencing. 
         [0085]    Hence, the present disclosure can provide diagnostic information about fetal monogenic disorders using maternal serum DNA in determining a deletion or duplication mutation in a single gene. 
         [0086]    A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as limiting the present invention. 
       Example 1: Prediction of Fetal Monogenic Variation 
       [0087]    &lt;1-1&gt; Sequencing of Maternal Serum DNA 
         [0088]    As the monogenic disorder that was handled in this experiment, an X-linked recessive disorder was selected. 
         [0089]    Maternal serum DNA was assumed to have a duplication mutation, causative of a genetic disorder, in some region. 
         [0090]    It was assumed that a pregnant woman showed a target sequencing coverage of about 95%, an average serum DNA read depth of about 100, and an average genome DNA read depth of about 100. 
         [0091]    &lt;1-2&gt; Identification of Kind and Region of Genetic Variation 
         [0092]    To identify the kind and region of a single-gene mutation, the moving average of read depths was computed and processed using a CBS algorithm. As a result, the region was determined and the pregnant woman was found to have a duplication mutation. 
         [0093]    &lt;1-3&gt; Measurement of Fetal DNA Fraction 
         [0094]    The fetal DNA content of maternal DNA was measured as in Example &lt;1-1&gt;. To this end, ZFX and ZFY genes were captured using an Agilent SureSelect Custom Kit containing capture probes that target ZFX (X-linked zinc finger protein) and ZFY (Y-linked zinc finger protein) regions. 
         [0095]    Fetal DNA fraction: 
         [0000]    
       
         
           
             f 
             = 
             
               
                 2 
                  
                 
                   ZFY 
                   
                     
                         
                     
                      
                     ′ 
                   
                 
               
               
                 
                   ZFX 
                   
                     
                         
                     
                      
                     ′ 
                   
                 
                 + 
                 
                   ZFY 
                   
                     
                         
                     
                      
                     ′ 
                   
                 
               
             
           
         
       
     
         [0096]    wherein, ZFX′ and ZFY′ are the quotients of the number of total mapped reads divided by the number of probes. 
         [0097]    Results of prediction of fetal DNA fraction (f) using ZFX and ZFY genes are given in Table 1, below. 
         [0000]    
       
         
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
             
             
               
                   
                   
               
               
                   
                 number of total mapped reads 
                 Predicted fetal 
               
             
          
           
               
                   
                 Sample 
                 ZFX 
                 ZFY 
                 DNA fraction 
               
               
                   
                   
               
             
          
           
               
                   
                 Mother I-2 
                 24242485 
                 878522 
                 0.08604 
               
               
                   
                 Mother I-1 
                 44040616 
                 1175572 
                 0.06484 
               
               
                   
                   
               
             
          
         
       
     
         [0098]    As can be seen in Table 1, the fetal DNA fraction was 8.6% in the serum of pregnant woman I-1 and 6.4% in the serum of pregnant woman II-2. 
         [0099]    &lt;1-4&gt; Prediction of Allele Frequency of Heterozygous Single-Nucleotide Polymorphism 
         [0100]    Assuming that each of the pregnant women had a duplication mutation carrier and conceived a male fetus, the fetal DNA fraction f may be used to derive an allele frequency (θ exp/aff ) of heterozygous SNP in a variation region in a single gene according to the following Equation 1 and an allele frequency (θ exp/unaff ) of heterozygous SNP in a non-variation region in the single gene according to the following Equation 2: 
         [0000]      θ exp/aff =2/(3− f )  &lt;Equation 1&gt;
 
         [0000]      θ exp/unaff =2(1− f )/(3−2 f )  &lt;Equation 2&gt;
 
         [0101]    In  FIG. 1 , allele frequencies are plotted versus fetal DNA fractions. The upper line indicates a fetus having a single-gene mutation while the lower line accounts for a fetus having no single-gene mutations. The gray regions along the upper line show a confidence interval calculated from an allele frequency of 12 SNPs in the variation region and standard error resulting from 100 rounds of calculation of random samples of consecutive 6 and 3 SNPs. 
         [0102]    When the fetal DNA fractions calculated in Example 1-3 were input into the equations, allele frequencies were predicted as given in Table 2, below. 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Item 
                 Mother I-1 
                 Mother I-2 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 fetal DNA fraction (f, %) 
                 8.604% 
                 6.484% 
               
               
                 θ exp/aff  (fetus with genetic mutation) 
                 0.6863512 
                 0.6813939 
               
               
                 θ exp/unaff  (fetus without genetic mutation) 
                 0.6463832 
                 0.6516068 
               
               
                   
               
             
          
         
       
     
         [0103]    &lt;1-5&gt; Measurement of Average Allele Frequency (θ obs ) of Heterozygous SNP in DNA Region Having Single-Gene Mutation 
         [0104]    From next-generation sequencing data of maternal serum DNA, the average allele frequency (θ obs ) of heterozygous SNP in a DNA region having a single-gene mutation can be calculated by counting the number of each allele gene. The calculation results from the data of Examples are given in Table 3, below. 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 Mother I-1 
                 Mother I-2 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 Number of heterozygous SNP 
                 12 
                 10 
               
               
                 θ obs  (observed average allele 
                 0.6782778 
                 0.6826352 
               
               
                 frequency) (95% confidence interval) 
                 (0.6606647- 
                 (0.69998752- 
               
               
                   
                 0.6958908) 
                 0.6653952 
               
               
                   
               
             
          
         
       
     
         [0105]    Because the calculated average allele frequency (θ obs ) of SNP in Table 3 was nearer to the calculated θ exp/aff  values for mutation regions in Table 2, the fetus conceived by the pregnant woman can be predicted to have a single-gene mutation. 
       Example 2: Prediction of Fetal Monogenic Variation 
       [0106]    &lt;2-1&gt; Sequencing of Maternal Serum DNA 
         [0107]    It was assumed that a pregnant women had a deletion mutation causative of a recessive genetic disorder and conceived a male fetus. 
         [0108]    Serum and genome DNAs from the pregnant woman were analyzed using massively parallel sequencing, a kind of next-generation sequencing, in the same manner as in Example 1-1. 
         [0109]    The pregnant woman showed a target sequencing coverage of about 97.7%, an average serum DNA read depth of from about 465 to about 530, and an average genome DNA read depth of about 1210. 
         [0110]    &lt;2-2&gt; Identification of Kind and Region of Genetic Variation 
         [0111]    To identify the kind and region of a single-gene mutation, the moving average of read depths was computed and subjected to CBS algorithm. 
         [0112]    &lt;2-3&gt; Measurement of Fetal DNA Fraction 
         [0113]    The fetal DNA content of maternal DNA was measured as in Example &lt;1-3&gt;. The measurement results are shown in Table 4, below. 
         [0000]    
       
         
               
               
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 4 
               
             
             
               
                   
                   
               
               
                   
                 number of total mapped reads 
                 Predicted fetal 
               
             
          
           
               
                 Sample 
                 ZFX 
                 ZFY 
                 DNA fraction 
               
               
                   
               
             
          
           
               
                 Mother II-1 
                 26051623 
                 567051 
                 0.05352 
               
               
                 Mother II-2 
                 29480725 
                 888942 
                 0.07264 
               
               
                   
               
             
          
         
       
     
         [0114]    As can be seen in Table 4, the fetal DNA fraction was 5.4% in the serum of pregnant woman II-1 and 7.3% in the serum of pregnant woman II-2. 
         [0115]    &lt;2-4&gt; Ratio of Read Depth Between Serum DNA and Corpuscle DNA (Genome DNA) in Normal (Non-Variation) and Variation Regions 
         [0116]    Assuming that each of the pregnant women had a single-gene mutation carrier and was carrying a male fetus, the ratio of serum DNA read depth to genome DNA read depth may be predicted, as shown in Table 5, depending on whether the fetus has a single-gene mutation. 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                 Mutation status 
                 normal 
                 deletion 
                 duplication 
               
               
                   
               
             
             
               
                 Read depth of mother gDNA 
                 1 
                 0.5 
                 1.5 
               
               
                 cfDNA 
                   
                 Fetus status 
                 Fetus status            
               
             
          
           
               
                   
                   
                 With 
                 Without 
                 With 
                 Without 
               
               
                   
                   
                 mutation 
                 mutation 
                 mutation 
                 mutation 
               
               
                 Read depth of mother DNA 
                 1 − f 
                 (1 − f)/2 
                 (1 − f)/2 
                 3(1 − f)/2 
                 3(1 − f)/2 
               
               
                 Read depth of fetus DNA 
                 f/2 
                 0 
                     f/2 
                 f 
                 f 
               
               
                 Read depth of cfDNA 
                 (2 − f)/2 
                 (1 − f)/2 
                 1/2 
                 (3 − f)/2 
                 (3 − 2f)/2 
               
               
                 γ(cfDNA/gDNA) 
                 (2 − f)/2 = 
                 1 − f 
                 1 
                 (3 − f)/3 = 
                 (3 − 2f)/3 = 
               
               
                   
                 1 − (f/2) 
                   
                   
                 1 − (f/3) 
                 1 − (2f/3) 
               
               
                 Comparison of read ratio 
                   
                 Lower than 
                 Higher than 
                 Higher than 
                 Lower than 
               
               
                   
                   
                 normal 
                 normal 
                 normal 
                 normal 
               
               
                   
               
               
                 f: fetal DNA fraction 
               
             
          
         
       
     
         [0117]    As can be seen in Table 5, when the fetus has a deletion mutation, γ in a variation region is smaller than that in a normal region. On the other hand, when the fetus has no deletion mutations, γ in a variation region is larger than that in a normal region. Hence, when the maternal single-gene mutation is of a deletion type, comparison of γ between a normal region and a variation region allows for determining the inheritance of the single-gene mutation from the pregnant woman to the fetus, that is, whether or not the fetus has the single-gene mutation. 
         [0118]    In addition, when the fetus has a duplication mutation, γ in a variation region is larger than that in a normal region. On the other hand, in the absence of fetal deletion mutations, γ in a variation region is smaller than that in a normal region. Hence, when the maternal single-gene mutation is of a duplication type, comparison of γ between a normal region and a variation region allows for determining the inheritance of the single-gene mutation from the pregnant woman to the fetus, that is, whether or not the fetus has the single-gene mutation. 
         [0119]    Based on the prediction, ratios of serum DNA read depth to corpuscle DNA read depth were measured in normal and variation regions. 
         [0120]    As an experimental result, the relative read depths (γ) were 0.99632 and 1.06031 in a normal region and a variation region of pregnant woman II-1, respectively. For pregnant woman II-2, the relative read depths (γ) were 0.99647 in a normal region and 1.07075 in a variation region. Relative read depths (γ) for pregnant woman II-2 are depicted in  FIG. 2 . In  FIG. 2 , the X-axis represents nucleotide positions on an X-chromosome while the Y-axis accounts for relative read depths (γ). Read depth ratios in variation regions (closed circles) and non-variation regions (open circles) are given, together with a representative value (dotted lines) in each region. 
         [0121]    For pregnant woman II-1 having a deletion mutation, the fetus was predicted to be normal as the relative read depths in variation regions were larger than those in normal regions.