Marker for prenatal diagnosis and monitoring

Abstract

The present invention relates to new methods for diagnosing a pregnancy-associated disorder by analyzing fetal DNA present in the mother's blood. More specifically, this invention relies on the discovery that the maspin gene is differentially methylated in fetal DNA and in maternal DNA and provides these new diagnostic methods, which distinguish fetal DNA from maternal DNA and detect prenatal disorders based on abnormalities in fetal DNA level and methylation status.

1 . A method for detecting or monitoring trisomy 21 in a woman pregnant with a fetus, comprising the steps of:
 (a) obtaining a blood sample from the woman;   (b) determining the methylation status of at least a portion of the maspin gene in the blood sample, wherein the portion of the maspin gene from the fetus and the portion from the woman are differentially methylated, thereby distinguishing the maspin gene from the woman and the maspin gene from the fetus in the blood sample;   (c) determining the level of the fetal maspin gene; and   (d) comparing the level of the fetal maspin gene with a standard control, wherein an increase or decrease from the standard control indicates the presence or progression of a trisomy 21.   
     
     
         2 . The method of  claim 1 , wherein the blood sample is whole blood. 
     
     
         3 . The method of  claim 1 , wherein the blood sample is plasma or serum. 
     
     
         4 . The method of  claim 1 , wherein the portion of the maspin gene from the woman is methylated and the portion from the maspin gene from the fetus is less methylated. 
     
     
         5 . The method of  claim 4 , wherein step (b) is performed by treating DNA present in the blood sample with a reagent that differentially modifies methylated and non-methylated DNA. 
     
     
         6 . The method of  claim 5 , wherein the reagent comprises bisulfite. 
     
     
         7 . The method of  claim 5 , wherein the reagent comprises one or more enzymes that preferentially cleave methylated DNA. 
     
     
         8 . The method of  claim 5 , wherein the reagent comprises one or more enzymes that preferentially cleave unmethylated DNA. 
     
     
         9 . A method for detecting or monitoring trisomy 21 in a woman pregnant with a fetus, comprising the steps of:
 (a) obtaining DNA in a blood sample from the woman;   (b) treating the DNA from step (a) with bisulfite; and   (c) performing an amplification reaction using the DNA from step (b) and two primers to amplify at least a portion of the maspin gene, wherein the portion of the maspin gene from the fetal DNA and the portion of the maspin gene from the maternal DNA in the blood sample are differentially methylated, and wherein at least one of the two primers binds differentially to the portion of the maspin gene from the fetus; and   (d) comparing the level of the amplified portion of the maspin gene from step (c) with a standard control, wherein an increase or decrease from the standard control indicates the presence or progression of trisomy 21.   
     
     
         10 . The method of  claim 9 , wherein the blood sample is whole blood. 
     
     
         11 . The method of  claim 9 , wherein the blood sample is plasma or serum. 
     
     
         12 . The method of  claim 9 , wherein the amplification reaction is a polymerase chain reaction (PCR). 
     
     
         13 . The method of  claim 9 , wherein the amplification reaction is a nucleic acid sequence based amplification. 
     
     
         14 . The method of  claim 9 , wherein the amplification reaction is a strand displacement reaction. 
     
     
         15 . The method of  claim 9 , wherein the amplification reaction is a branched DNA amplification reaction. 
     
     
         16 - 27 . (canceled) 
     
     
         28 . A method for detecting and monitoring trisomy 21, comprising the steps of:
 (a) obtaining DNA in a blood sample from the woman;   (b) treating the DNA from step (a) with a reagent that differentially modifies methylated and non-methylated DNA;   (c) determining the nucleotide sequence of at least a portion of the maspin gene from step (b); and   (d) comparing the profile of the nucleotide sequences from step (c) with a standard control, wherein a change in the profile from the standard control indicates the presence or progression of trisomy 21.   
     
     
         29 . The method of  claim 28 , wherein the reagent comprises bisulfite. 
     
     
         30 . The method of  claim 28 , wherein the reagent comprises one or more enzymes that preferentially cleave methylated DNA. 
     
     
         31 . The method of  claim 28 , wherein the reagent comprises one or more enzymes that preferentially cleave unmethylated DNA. 
     
     
         32 . The method of  claim 28 , wherein the blood sample is plasma or serum. 
     
     
         33 . The method of  claim 28 , further comprising an amplification step of using the DNA from step (b) and two primers to amplify a portion of the maspin gene, wherein the portion of the maspin gene from the fetal DNA and the portion from the maternal DNA in the blood sample are differentially methylated, and wherein at least one of the two primers binds differentially to the portion of the maspin gene from the fetus. 
     
     
         34 . The method of  claim 33 , wherein the amplification step is performed by PCR. 
     
     
         35 . The method of  claim 33 , wherein the amplification step is performed by methylation-specific PCR. 
     
     
         36 . The method of  claim 28 , wherein step (c) is performed by mass spectrometry. 
     
     
         37 . The method of  claim 28 , wherein step (c) is performed by primer extension. 
     
     
         38 . The method of  claim 28 , wherein step (c) is performed by polynucleotide hybridization. 
     
     
         39 . The method of  claim 28 , wherein step (c) is performed by real-time PCR. 
     
     
         40 . The method of  claim 28 , wherein step (c) is performed by electrophoresis. 
     
     
         41 - 56 . (canceled)