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 in a sample comprising:
 a) contacting nucleic acid present in a sample from a pregnant female, which sample comprises differentially methylated fetal nucleic acid and maternal nucleic acid, the combination of the fetal nucleic acid and the maternal nucleic acid comprising total nucleic acid in the sample, with an agent that specifically digests non-methylated maternal nucleic acid at a plurality of loci, thereby enriching the fetal nucleic acid; and   b) determining the amount of fetal nucleic acid in (a).   
     
     
         2 . The method of  claim 1 , wherein the agent that specifically digests non-methylated maternal nucleic acid is a methylation-sensitive restriction enzyme. 
     
     
         3 . The method of  claim 1 , further comprising determining the amount of total nucleic acid present in the sample. 
     
     
         4 . The method of  claim 3 , comprising determining the concentration of fetal nucleic acid in the sample by comparing the amount of fetal nucleic acid in (b) to the total amount of nucleic acid. 
     
     
         5 . The method of  claim 1 , comprising after (a) introducing one or more competitors at known concentrations. 
     
     
         6 . The method of  claim 5 , comprising determining the absolute amount of fetal nucleic acid in the sample. 
     
     
         7 . The method of  claim 1 , wherein the plurality of loci are selected from loci of SEQ ID NO:1-89. 
     
     
         8 . The method of  claim 1 , wherein the plurality of loci are selected from loci of SEQ ID NO:1-59 and SEQ ID NO:86-89. 
     
     
         9 . The method of  claim 1 , wherein the plurality of loci are selected from loci of SEQ ID NO:1-59. 
     
     
         10 . The method of  claim 1 , wherein the plurality of loci comprises a locus of SEQ ID NO:42. 
     
     
         11 . The method of  claim 1 , wherein the plurality of loci comprises a locus of SEQ ID NO:52. 
     
     
         12 . The method of  claim 1 , wherein the concentration of fetal nucleic acid is used in conjunction with a diagnostic method to determine a fetal trait, wherein the diagnostic method requires a concentration of fetal nucleic acid to meet certain clinical sensitivity or specificity requirements. 
     
     
         13 . The method of  claim 1 , wherein determining the amount of fetal nucleic acid comprises use of one or more processes selected from mass spectrometry methods, RT-PCR, digital PCR, array-based methods, sequencing methods, nanopore-based methods, nucleic acid-bound bead-based counting methods, competitor-based methods and methods for separating nucleic acid using agents that bind nucleic acid based on methylation status. 
     
     
         14 . The method of  claim 1 , wherein determining the amount of fetal nucleic acid comprises use of a mass spectrometry method. 
     
     
         15 . The method of  claim 1 , wherein determining the amount of fetal nucleic acid comprises use of a sequencing method. 
     
     
         16 . The method of  claim 15 , wherein the sequencing method comprises sequencing by synthesis. 
     
     
         17 . The method of  claim 1 , wherein the digestion efficiency of the methylation sensitive restriction enzyme is determined. 
     
     
         18 . The method of  claim 1 , wherein the method further comprises determining the presence or absence of Y-chromosome nucleic acid present in a sample from a pregnant female. 
     
     
         19 . The method of  claim 18 , wherein the amount of Y-chromosome nucleic acid present in a sample from a pregnant female is determined for a male fetus. 
     
     
         20 . The method of  claim 19 , wherein the amount of fetal nucleic acid is compared to the amount of Y-chromosome nucleic acid. 
     
     
         21 . The method of  claim 1 , wherein the amount of fetal nucleic acid at 10 or more loci is determined. 
     
     
         22 . The method of  claim 1 , wherein the amount of Y-chromosome nucleic acid for a male fetus and the digestion efficiency of the methylation sensitive restriction enzyme are determined. 
     
     
         23 . The method of  claim 22 , wherein two or more assays are used to determine the total amount of nucleic acid, one or more assays are used to determine the amount of Y-chromosome nucleic acid for a male fetus, and one or more assays are used to determine the digestion efficiency of the methylation sensitive restriction enzyme. 
     
     
         24 . The method of  claim 23 , wherein the amount of fetal nucleic acid at 3 or more loci is determined. 
     
     
         25 . The method of  claim 1 , wherein the amount of fetal nucleic acid is determined by an amplification reaction that generates amplicons larger than the average length of the digested maternal nucleic acid, thereby further enriching the fetal nucleic acid.