Patent Application
20180340223
The present invention pertains to a method for identifying a greater risk for developing bronchopulmonary dysplasia (BPD) of preterm birth. The present invention also relates to a primer pair for genotyping rs1800566 SNP in the NQO1 gene, and a method thereof.
Preterm birth (PTB), or birth before 37 weeks of gestation period, is the major cause of neonatal mortality and morbidity worldwide. Approximately 70% of the neonatal deaths are due to preterm delivery.
Bronchopulmonary dysplasia (BPD), a common chronic inflammatory lung disease of very-low-birth-weight (VLBW) preterm infants, is associated with arrested lung development and treatment of supplemental oxygen [1]. Due to the influences of long-term oxygen therapy and mechanical ventilation, many of these preterm infants consequently acquire different types of problems, such as highly reactive airway diseases, recurrent lower respiratory tract infections, abrupt alveolar development, growth retardation, and feeding difficulties [2, 3]. While early detection of BPD is crucial to prevent chronic symptoms and complications later in life, diagnosis and prevention of this disease remains challenging due to the lack of good biomarkers for identification of infants at risk [1]. It has been reported that interleukin-8 (IL-8) and C-reactive protein (CRP), two recently identified preterm biomarkers, can also be biomarkers for BPD [4, 5].
There is still an urgent need for novel and efficient biomarkers for BPD.
In one aspect, the present invention provides a method for identifying a greater risk for developing bronchopulmonary dysplasia (BPD) of a preterm infant, comprising obtaining a genomic DNA sample from the preterm infant's mother, genotyping rs1800566 SNP in the NQO1 gene, and determining the preterm infant as being at risk of developing BPD if the genotype of the rs1800566 SNP is TT.
According to certain preferred embodiments of the present invention, the method comprises genotyping the rs1800566 SNP using a primer pair comprising a forward primer of SEQ ID NO: 1, and a reverse primer of SEQ ID NO: 2.
In one embodiment of the present invention, the genotype of the rs1800566 SNP is determined by a process comprising performing qPCR using the primer pair to obtain a first melting curve of a first reference sample for the genotype CC, a second melting curve of a second reference sample for the genotype TT, and a third melting curve of the genomic DNA sample; subtracting the first melting curve from each of the first, second and third melting curves to obtain a first, second, and third difference curves, respectively; and comparing the third difference curve with the first and second difference curves, respectively, so as to determine the genotype of the rs1800566 SNP.
In another embodiment, the genotype of the rs1800566 SNP is determined by a process comprising performing qPCR using the primer pair to obtain a plurality of first melting curves of a first reference sample for the genotype CC, a plurality of second melting curves of a second reference sample for the genotype TT, and a plurality of third melting curves of the genomic DNA sample; subtracting the average of the plurality of first melting curves from each of the plurality of first, second and third melting curves to obtain a plurality of first, second, and third difference curves, respectively; and comparing the plurality of third difference curves with the plurality of first and second difference curves, respectively, so as to determine the genotype of the rs1800566 SNP.
In another aspect, the present invention provides a primer pair for genotyping rs1800566 SNP in the NQO1 gene, comprising a forward primer of SEQ ID NO: 1, and a reverse primer of SEQ ID NO: 2.
In a further aspect, provided is a method for genotyping rs1800566 SNP in the NQO1 gene. The method comprises performing qPCR using a primer pair according to the present invention to obtain a first melting curve of a first reference sample for the genotype CC, a second melting curve of a second reference sample for the genotype TT, and a third melting curve of the genomic DNA sample; subtracting the first melting curve from each of the first, second and third melting curves to obtain a first, second, and third difference curves, respectively; and comparing the third difference curve with the first and second difference curves, respectively, so as to determine the genotype of the rs1800566 SNP.
According to certain preferred embodiments of the present invention, the genotype of the rs1800566 SNP is determined as CC if the third difference curve fits better with the first difference curve than the second difference curve; the genotype of the rs1800566 SNP is determined as TT if the third difference curve fits better with the second difference curve than the first difference curve; and if otherwise, the third difference curve is in between the first difference curve and the second difference curve, the genotype of the rs1800566 SNP is determined as TC.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention.
The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred.
In the drawings:
In one aspect, the present invention provides a method for identifying a greater risk for developing bronchopulmonary dysplasia (BPD) of a preterm infant. The method comprises the following steps: obtaining a genomic DNA sample from the preterm infant's mother; genotyping rs1800566 SNP in the NQO1 (NAD(P)H quinone dehydrogenase 1) gene; and determining the preterm infant as being at risk of developing BPD if the genotype of the rs1800566 SNP is TT.
The term “SNP” as used herein means a single nucleotide polymorphism which is a single nucleotide position in a nucleotide sequence for which two or more alternative alleles are present in a given population.
According to the present invention, the genomic DNA sample may be derived from a tissue selected from the group consisting of blood, placenta, amniotic membrane, chorionic disk, chorionic membrane, and umbilical cord, but is not limited thereto. In one embodiment of the present invention, the blood is umbilical cord blood. In another embodiment, the blood is peripheral blood.
According to certain preferred embodiments of the present invention, the method comprises genotyping the rs1800566 SNP using a primer pair comprising a forward primer of SEQ ID NO: 1 (TGAGAAGCCCAGACCAACTT), and a reverse primer of SEQ ID NO: 2 (CCATCCTTCCAGGATTTGAA).
In certain embodiments of the present invention, the genotype of the rs1800566 SNP in the NQO1 gene of the genomic DNA sample is determined by a process comprising the following steps: (a) performing qPCR using the primer pair to obtain a melting curve of a first reference sample for the genotype CC (the “first melting curve”), a melting curve of a second reference sample for the genotype TT (the “second melting curve”), and a melting curve of the genomic DNA sample (the “third melting curve”); (b) subtracting the first melting curve from each of the first, second and third melting curves to obtain a first, second, and third difference curves, respectively; and (c) comparing the third difference curve with the first and second difference curves, respectively, so as to determine the genotype of the rs1800566 SNP.
According to one embodiment of the present invention, the genotype of the rs1800566 SNP is determined as CC if the third difference curve fits better with the first difference curve than the second difference curve; the genotype of the rs1800566 SNP is determined as TT if the third difference curve fits better with the second difference curve than the first difference curve; and if otherwise, the third difference curve is in between the first difference curve and the second difference curve, the genotype of the rs1800566 SNP is determined as TC.
In some other embodiments, the genotype of the rs1800566 SNP is determined based on an arithmetic mean of SP1/RC1 and SP2/RC2, where SP1 and SP2 are the smallest and largest extreme values, respectively, of the third difference curve, and RC1 and RC2 are the smallest and largest extreme values, respectively, of the second difference curve. A lower arithmetic mean indicates that the genotype is CC, a moderate arithmetic mean indicates that the genotype is TC, and a higher arithmetic mean indicates that the genotype is TT. According to one specific example, the genotype is determined as CC if the arithmetic mean is smaller than 0.32, the genotype is determined as TC if the arithmetic mean is in the range of 0.32 to 1.1, and the genotype is determined as TT if the arithmetic mean is larger than 1.1.
In another embodiment, the genotype of the rs1800566 SNP is determined by a process comprising the following steps: (a) performing qPCR using the primer pair to obtain a plurality of melting curves of a first reference sample for the genotype CC (the “first melting curves”), a plurality of melting curves of a second reference sample for the genotype TT (the “second melting curves”), and a plurality of melting curves of the genomic DNA sample (the “third melting curves”); (b) subtracting the average of the plurality of first melting curves from each of the plurality of first, second and third melting curves to obtain a plurality of first, second, and third difference curves, respectively; and (c) comparing the plurality of third difference curves with the plurality of first and second difference curves, respectively, so as to determine the genotype of the rs1800566 SNP.
According to one embodiment of the present invention, the genotype of the rs1800566 SNP is determined as CC if the plurality of third difference curves fits better with the plurality of first difference curves than the plurality of second difference curves; the genotype of the rs1800566 SNP is determined as TT if the plurality of third difference curves fits better with the plurality of second difference curves than the plurality of first difference curves; and if otherwise, the plurality of third difference curves is in between the plurality of first difference curves and the plurality of second difference curves, genotype of the rs1800566 SNP is determined as TC.
In some other embodiments, the genotype of the rs1800566 SNP is determined based on an arithmetic mean of SP1/RC1 and SP2/RC2, where SP1 and SP2 are the smallest and largest extreme values, respectively, of the third difference curve, and RC1 and RC2 are the smallest and largest extreme values, respectively, of the second difference curve. A lower arithmetic mean indicates that the genotype is CC, a moderate arithmetic mean indicates that the genotype is TC, and a higher arithmetic mean indicates that the genotype is TT. According to one specific example, the genotype is determined as CC if the arithmetic mean is smaller than 0.32, the genotype is determined as TC if the arithmetic mean is in the range of 0.32 to 1.1, and the genotype is determined as TT if the arithmetic mean is larger than 1.1.
In another aspect, the present invention provides a primer pair for genotyping rs1800566 SNP in the NQO1 gene, comprising a forward primer of SEQ ID NO: 1, and a reverse primer of SEQ ID NO: 2.
In a further aspect, provided is a method for genotyping rs1800566 SNP in the NQO1 gene. The method comprises (a) performing qPCR using a primer pair according to the present invention to obtain a first melting curve of a first reference sample for the genotype CC, a second melting curve of a second reference sample for the genotype TT, and a third melting curve of the genomic DNA sample; (b) subtracting the first melting curve from each of the first, second and third melting curves to obtain a first, second, and third difference curves, respectively; and (c) comparing the third difference curve with the first and second difference curves, respectively, so as to determine the genotype of the rs1800566 SNP.
According to one embodiment of the present invention, the genotype of the rs1800566 SNP is determined as CC if the third difference curve fits better with the first difference curve than the second difference curve; the genotype of the rs1800566 SNP is determined as TT if the third difference curve fits better with the second difference curve than the first difference curve; and if otherwise, the third difference curve is in between the first difference curve and the second difference curve, the genotype of the rs1800566 SNP is determined as TC.
In some other embodiments, the genotype of the rs1800566 SNP is determined based on an arithmetic mean of SP1/RC1 and SP2/RC2, where SP1 and SP2 are the smallest and largest extreme values, respectively, of the third difference curve, and RC1 and RC2 are the smallest and largest extreme values, respectively, of the second difference curve. A lower arithmetic mean indicates that the genotype is CC, a moderate arithmetic mean indicates that the genotype is TC, and a higher arithmetic mean indicates that the genotype is TT. According to one specific example, the genotype is determined as CC if the arithmetic mean is smaller than 0.32, the genotype is determined as TC if the arithmetic mean is in the range of 0.32 to 1.1, and the genotype is determined as TT if the arithmetic mean is larger than 1.1.
In a still further aspect, the present invention provides a method for genotyping rs1800566 SNP in the NQO1 gene, comprising (a) performing qPCR using the primer pair to obtain a plurality of melting curves of a first reference sample for the genotype CC (the “first melting curves”), a plurality of melting curves of a second reference sample for the genotype TT (the “second melting curves”), and a plurality of melting curves of the genomic DNA sample (the “third melting curves”); (b) subtracting the average of the plurality of first melting curves from each of the plurality of first, second and third melting curves to obtain a plurality of first, second, and third difference curves, respectively; and (c) comparing the plurality of third difference curves with the plurality of first and second difference curves, respectively, so as to determine the genotype of the rs1800566 SNP.
According to one embodiment of the present invention, the genotype of the rs1800566 SNP is determined as CC if the plurality of third difference curves fits better with the plurality of first difference curves than the plurality of second difference curves; the genotype of the rs1800566 SNP is determined as TT if the plurality of third difference curves fits better with the plurality of second difference curves than the plurality of first difference curves; and if otherwise, the plurality of third difference curves is in between the plurality of first difference curves and the plurality of second difference curves, genotype of the rs1800566 SNP is determined as TC.
In some other embodiments, the genotype of the rs1800566 SNP is determined based on an arithmetic mean of SP1/RC1 and SP2/RC2, where SP1 and SP2 are the smallest and largest extreme values, respectively, of the average of the plurality of third difference curves, and RC1 and RC2 are the smallest and largest extreme values, respectively, of the average of the plurality of second difference curves. A lower arithmetic mean indicates that the genotype is CC, a moderate arithmetic mean indicates that the genotype is TC, and a higher arithmetic mean indicates that the genotype is TT. According to one specific example, the genotype is determined as CC if the arithmetic mean is smaller than 0.32, the genotype is determined as TC if the arithmetic mean is in the range of 0.32 to 1.1, and the genotype is determined as TT if the arithmetic mean is larger than 1.1.
According to the present invention, the first reference sample for the genotype CC and the second reference sample for the genotype TT each may be a synthesized polynucleotide comprising a fragment of the NQO1 gene which includes the rs1800566 SNP site (with the nucleotide being a C or T), or a plasmid inserted with such synthesized polynucleotide. In one specific example, the first reference sample is a plasmid inserted with a polynucleotide of SEQ ID NO: 3, and the second reference sample is a plasmid inserted with a polynucleotide of SEQ ID NO: 4.
The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation.
Perform qPCR using the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 on a first reference sample for the genotype CC and a second reference sample for the genotype TT. The first reference sample contains a plasmid inserted with a polynucleotide of SEQ ID NO: 3, the second reference sample contains a plasmid inserted with a polynucleotide of SEQ ID NO: 4. The nucleotide sequence of SEQ ID NO: 3 is that of base 20166 to base 20715 of the NQO1 gene (NCBI Reference Sequence: NG_011504.1) where base 20390 is a C, and the nucleotide sequence of SEQ ID NO: 4 is that of base 20166 to base 20715 of the NQO1 gene (NCBI Reference Sequence: NG_011504.1) where base 20390 is a T.
The nucleotide sequence SEQ ID NO: 3 is as follows:
The nucleotide sequence of SEQ ID NO: 4 is as follows:
A specific melting curve was observed for each of the first and second reference samples (data not shown), demonstrating the specificity of the primer pair. The PCR products are expected to be 195 bp. The results of gel electrophoresis analysis showed single bands (see
Perform qPCR using the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 on a first reference sample and a second reference sample as described in Example 1 and on a genomic DNA sample isolated from mesenchymal stem cells derived from the placenta of a female subject. The experiments were done in triplicate for each sample, and two melting curves were shown for each sample. Calculate the average of the three melting curves of the first reference sample, and subtract it from the three melting curves of the first reference sample, the three melting curves of the second reference sample, and the three melting curves of the genomic DNA sample to obtain three first difference curves, three second difference curves, and three third difference curves, respectively. The results are shown in
Briefly, the genomic DNAs were isolated from mesenchymal stem cells derived from the placenta of 15 mothers of respective preterm infants. In a parallel test, genomic DNAs were isolated from umbilical cord blood samples (data not shown). The genotype of the rs1800566 SNP in the NQO1 gene of each genomic DNA sample was determined through the process as described in Example 2.
The results of statistical analysis are shown in Table 2 below.
7(38.9)
The p value of 0.04 in the Chi-square test show that there is a statistically significant relationship between the genotype TT and BPD.
It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.
