Processes and Compositions for Methylation-Based Enrichment of Fetal Nucleic Acid From a Maternal Sample Useful for Non-Invasive Prenatal Diagnoses

Abstract

Provided are compositions and processes that utilize genomic regions differentially methylated between a mother and her fetus to separate, isolate or enrich fetal nucleic acid from a maternal sample. The compositions and processes described herein are useful for non-invasive prenatal diagnostics, including the detection of chromosomal aneuploidies.

What is claimed is: 
     
         1 . A method for determining the amount of fetal nucleic acid of a target chromosome comprising:
 a) contacting nucleic acid from a pregnant female, which nucleic acid comprises differentially methylated fetal nucleic acid and maternal nucleic acid, with an agent that specifically binds methylated nucleotides; thereby producing bound nucleic acid;   b) isolating the bound nucleic acid of (a), thereby enriching the fetal nucleic acid; and   c) determining from the products of (b) the amount of fetal nucleic acid for a plurality of loci of a target chromosome comprising at least one locus selected from loci of SEQ ID NOs: 1-59 or portions thereof.   
     
     
         2 . The method of  claim 1 , wherein the agent that binds methylated nucleotides is a methyl-CpG binding protein (MBD) or fragment thereof. 
     
     
         3 . The method of  claim 2 , wherein the MBD or fragment thereof is fused to an Fc fragment of an antibody. 
     
     
         4 . The method of  claim 1 , wherein the agent that binds methylated nucleotides is a methylation specific antibody. 
     
     
         5 . The method of  claim 1 , wherein determining the amount of fetal nucleic acid comprises use of a sequencing method. 
     
     
         6 . The method of  claim 1 , wherein the plurality of loci of a target chromosome comprises the locus of SEQ ID No. 33 or a portion thereof. 
     
     
         7 . The method of  claim 1 , wherein the amount of fetal nucleic acid of a target chromosome is indicative of a fetal aneuploidy, the amount of fetal nucleic acid from a reference chromosome is determined and the presence or absence of a fetal aneuploidy is determined by comparing the amount of fetal nucleic acid from the target chromosome and the reference chromosome. 
     
     
         8 . The method of  claim 1 , wherein after (b), the isolated nucleic acid is amplified and the amplified products are used in (c). 
     
     
         9 . A method for determining the presence or absence of a fetal aneuploidy, comprising:
 a) contacting nucleic acid from a pregnant female, which nucleic acid comprises fetal nucleic acid and maternal nucleic acid with an agent that specifically binds methylated nucleotides; thereby enriching the fetal nucleic acid;   b) amplifying the bound methylated nucleic acid or a fraction thereof, thereby generating amplification products;   c) determining from the products of (b) the amount of fetal nucleic acid for a plurality of loci of a target chromosome comprising a locus comprising SEQ ID NO: 33;   d) determining the amount of fetal nucleic acid from a control nucleic acid; and   e) comparing the amount of fetal nucleic acid for the plurality of loci the target chromosome to the amount of fetal nucleic acid for the control nucleic acid to determine the presence or absence of a fetal aneuploidy, wherein a statistically significant difference between the amount of fetal nucleic acid for the plurality of loci of the target chromosome and the amount of fetal nucleic acid for the control nucleic acid indicates the presence of a fetal aneuploidy.   
     
     
         10 . The method of  claim 9 , wherein the agent that binds methylated nucleotides is a methylation specific antibody. 
     
     
         11 . The method of  claim 9 , wherein determining the amount of fetal nucleic acid comprises use of RT-PCR. 
     
     
         12 . The method of  claim 9 , wherein the control nucleic acid is obtained from a female with a normal euploid pregnancy. 
     
     
         13 . The method of  claim 12 , wherein the statistically significant difference is a deviation from the euploid absolute copy number. 
     
     
         14 . A method for determining the presence or absence of a fetal aneuploidy, comprising:
 a) contacting nucleic acid from a pregnant female, which nucleic acid comprises fetal nucleic acid and maternal nucleic acid with an agent that specifically binds methylated nucleotides; thereby enriching the fetal nucleic acid;   b) amplifying the bound methylated nucleic acid or a fraction thereof, thereby generating amplification products;   c) determining from the products of (b) the amount of fetal nucleic acid for a plurality of loci of a target chromosome comprising at least one locus selected from loci of SEQ ID NOs: 1-59 or portions thereof;   d) determining the amount of fetal nucleic acid from a control nucleic acid; and   e) comparing the amount of fetal nucleic acid for the plurality of loci the target chromosome to the amount of fetal nucleic acid for the control nucleic acid to determine the presence or absence of a fetal aneuploidy, wherein a statistically significant difference between the amount of fetal nucleic acid for the plurality of loci of the target chromosome and the amount of fetal nucleic acid for the control nucleic acid indicates the presence of a fetal aneuploidy.   
     
     
         15 . The method of  claim 14 , wherein the agent that binds methylated nucleotides is a methyl-CpG binding protein (MBD) or fragment thereof. 
     
     
         16 . The method of  claim 15 , wherein the MBD or fragment thereof is fused to an Fc fragment of an antibody. 
     
     
         17 . The method of  claim 14 , wherein determining the amount of fetal nucleic acid comprises use of a sequencing method. 
     
     
         18 . The method of  claim 14 , wherein the control nucleic acid is obtained from a female with a normal euploid pregnancy. 
     
     
         19 . The method of  claim 14 , wherein the amount of fetal nucleic acid at 3 or more loci is determined. 
     
     
         20 . The method of  claim 14 , wherein the statistically significant difference is a deviation from the euploid absolute copy number.