Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non invasive prenatal diagnoses
Provided are compositions and processes that utilize genomic regions that are 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 particularly useful for non-invasive prenatal diagnostics, including the detection of chromosomal aneuplodies.
1. 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 a methylation sensitive restriction enzyme that digests the maternal nucleic acid at a plurality of loci, wherein the plurality of loci comprises loci that are hypermethylated in fetal nucleic acid, thereby enriching the fetal nucleic acid;
b) amplifying the loci, or portion thereof, not digested in (a) in an amplification reaction, thereby generating amplification products;
c) sequencing the amplification products of (b), thereby generating sequencing products;
d)determining from the sequencing products of (c) the amount of fetal nucleic acid for a plurality of loci of a target chromosome comprising a locus comprising SEQ ID NO: 211;
e) determining the amount of fetal nucleic acid from a reference chromosome; and
f) comparing the amount of fetal nucleic acid for the plurality of loci of the target chromosome to the amount of fetal nucleic acid for the reference chromosome, whereby 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 reference chromosome determines the presence of a fetal aneuploidy.
2. The method of claim 1 , wherein the plurality of loci comprises one or more loci selected from loci of SEQ ID NOs: 90-163, 176, 179, 180, 184, 188, 189, 190, 191, 193, 195, 198, 199, 200, 201, 202, 203, 205, 206, 207, 208, 209, 210, 212, 213, 214, 221, 223, 225, 226, 231, 232, 233, 235, 239, 241, 257, 258, 259, and 261.
3. The method of claim 1 , wherein the plurality of loci comprises one or more loci selected from loci of SEQ ID NOs: 176, 179, 180, 184, 188, 189, 190, 191, 193, 195, 198, 199, 200, 201, 202, 203, 205, 206, 207, 208, 209, 210, 212, 213, 214, 221, 223, 225, 226, 231, 232, 233, 235, 239, 241, 257, 258, 259, and 261.
4. The method of claim 1 , wherein the plurality of loci comprises one or more loci selected from loci of SEQ ID NOs: 193, 200, 208, 209, 213, 214, 231, 232, 235, and 241.
5. The method of claim 1 , wherein the plurality of loci comprises the locus of SEQ ID NO:213.
6. The method of claim 1 , wherein the plurality of loci comprises one or more loci selected from loci of SEQ ID NOs: 200, 208, 231, 232, and 241.
7. The method of claim 1 , wherein the plurality of loci comprises the locus of SEQ ID NO:209.
8. The method of claim 1 , wherein the plurality of loci comprises the locus of SEQ ID NO:214.
9. The method of claim 1 wherein the amount of fetal nucleic acid at between 3 and 15 loci on each of the target chromosome and the reference chromosome is determined.
10. The method of claim 1 , wherein the amount of fetal nucleic acid at about 16 or more loci on each of the target chromosome and the reference chromosome is determined.
11. The method of claim 1 , wherein determining the amount of fetal nucleic acid for the target chromosome and the reference chromosome comprises use of a competitor-based amplification method.
12. The method of claim 1 , further comprising:
i) determining the amount of total nucleic acid;
ii) determining the digestion efficiency of the methylation sensitive restriction enzyme; and
iii) determining the presence or absence of Y-chromosome nucleic acid in the nucleic acid.
13. The method of claim 12 , wherein the amount of Y-chromosome nucleic acid present in the nucleic acid is determined for a male fetus.
14. The method of claim 13 , wherein the amount of fetal nucleic acid is compared to the amount of Y-chromosome nucleic acid.
15. The method of claim 13 , 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.
16. The method of claim 1 , wherein the sequencing method comprises sequencing by synthesis.