PROCESSES AND COMPOSITIONS FOR METHYLATION-BASED ENRICHMENT OF FETAL NUCLEIC ACID FROM A MATERNAL SAMPLE USEFUL FOR NON INVASIVE PRENATAL DIAGNOSES

• US 20100105049A1
• Filed: 09/16/2009
• Published: 04/29/2010
• Est. Priority Date: 09/16/2008
• Status: Active Grant

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Original Assignee: Sequenom Center for Molecular Medicine LLC (Laboratory Corporation Of America Holdings), Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Ehrich, Mathias, Nygren, Anders Olof Herman
• Granted Patent: US 8,476,013 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6879 for sex determination
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2600/154 Methylation markers
• CPC Class Codes: G01N 2800/385 Congenital anomalies
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

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 aneuplodies.

Claims

1. A method for preparing fetal nucleic acid, which comprises:

a) providing a sample from a pregnant female;

b) separating fetal nucleic acid from maternal nucleic acid from the sample of the pregnant female according to a different methylation state between the fetal nucleic acid and the maternal nucleic acid counterpart, wherein the fetal nucleic acid comprises one or more CpG sites from one or more of the polynucleotide sequences of SEQ ID NOs;

1-89; and

c) preparing nucleic acid comprising fetal nucleic acid by a process in which fetal nucleic acid separated in part (b) is utilized as a template.

2. The method of claim 1, wherein the fetal nucleic acid is separated from the maternal nucleic acid by an agent that specifically binds to methylated nucleotides.

3. The method of claim 2, wherein the agent that binds to methylated nucleotides is a methyl-CpG binding protein (MBD) or fragment thereof.

4. The method of claim 2, wherein the agent that binds to methylated nucleotides binds to methylated fetal nucleic acid.

5. The method of claim 2, wherein the agent that binds to methylated nucleotides binds to methylated maternal nucleic acid.

6. The method of claim 2, wherein the fetal nucleic acid is separated from the maternal nucleic acid by an agent that specifically binds to non-methylated nucleotides.

7. The method of claim 1, wherein the fetal nucleic acid is separated from the maternal nucleic acid by an agent that specifically digests non-methylated maternal nucleic acid.

8. The method of claim 7, wherein the agent that specifically digests non-methylated maternal nucleic acid is a methyaltion-sensitive restriction enzyme.

9. The method of claim 8, wherein two or more methyaltion-sensitive restriction enzymes are used in the same reaction.

10. The method of claim 1, wherein the process of step c) is an amplification reaction.

11. The method of claim 1 wherein the process of step c) is a method for determining the absolute amount of fetal nucleic acid.

12. The method of claim 1, wherein three or more of the polynucleotide sequences of SEQ ID NOs:

1-89 are prepared.

13. A method for determining the absolute amount of fetal nucleic acid in a maternal sample, wherein the maternal sample comprises differentially methylated maternal and fetal nucleic acid, comprising:

a) digesting the maternal nucleic acid in a maternal sample using one or more methylation sensitive restriction enzymes, thereby enriching the fetal nucleic acid; and

b) determining the absolute amount of fetal nucleic acid from step a) using a non-polymorphic-based and non-bisulfite-based quantitative method.

14. The method of claim 13, wherein the absolute amount or concentration of fetal nucleic acid is used in conjunction with a diagnostic method to determine a fetal trait, wherein the diagnostic method requires a given absolute amount or concentration of fetal nucleic acid to meet certain clinical sensitivity or specificity requirements.

19. The method of claim 13, 15 or 17, wherein the digestion efficiency of the methylation sensitive restriction enzyme is determined.

20. The method of claim 13, 15 or 17, wherein a non-polymorphic-based and non-bisulfite-based method for performing said quantification uses a competitor-based method to determine the amount of fetal nucleic acid.

21. The method of claim 13, 15 or 17, wherein the method further comprises determining the presence or absence of Y-chromosome nucleic acid present in a maternal sample.

22. The method of claim 21, wherein the amount of Y-chromosome nucleic acid present in a maternal sample is determined for a male fetus.

23. The method of claim 22, wherein the amount of fetal nucleic acid is compared to the amount of Y-chromosome nucleic acid.

24. The method of claim 13 or 15, wherein the amount of fetal nucleic acid at 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more loci is determined.

25. The method of claim 13 or 17, wherein the total amount of nucleic acid present in a maternal sample is determined.

26. The method of claim 13, 15 or 17, wherein the total amount of nucleic acid and the amount of Y-chromosome nucleic acid for a male fetus are determined.

27. The method of claim 13, 15 or 17, wherein the total amount of nucleic acid, the amount of Y-chromosome nucleic acid for a male fetus, and the digestion efficiency of the methylation sensitive restriction enzyme are all determined.

28. The method of claim 27, 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.

29. The method of claim 28, wherein the amount of fetal nucleic acid at 3 or more loci is determined.

30. The method of claim 13, 15 or 17, 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.

15. A method for determining the concentration of fetal nucleic acid in a maternal sample, wherein the maternal sample comprises differentially methylated maternal and fetal nucleic acid, comprising:

a) determining the total amount of nucleic acid present in the maternal sample;

b) digesting the maternal nucleic acid in a maternal sample using a methylation sensitive restriction enzyme thereby enriching the fetal nucleic acid;

c) determining the amount of fetal nucleic acid from step b) using a non-polymorphic-based and non-bisulfite-based quantitative method; and

d) comparing the amount of fetal nucleic acid from step c) to the total amount of nucleic acid from step a), thereby determining the concentration of fetal nucleic acid in the maternal sample.

16. The method of claim 15, wherein the absolute amount or concentration of fetal nucleic acid is used in conjunction with a diagnostic method to determine a fetal trait, wherein the diagnostic method requires a given absolute amount or concentration of fetal nucleic acid to meet certain clinical sensitivity or specificity requirements.

17. A method for determining the presence or absence of a fetal aneuploidy using fetal nucleic acid from a maternal sample, wherein the maternal sample comprises differentially methylated maternal and fetal nucleic acid, comprising:

a) digesting the maternal nucleic acid in a maternal sample using a methylation sensitive restriction enzyme thereby enriching the fetal nucleic acid;

b) determining the amount of fetal nucleic acid from a target chromosome using a non-polymorphic-based and non-bisulfite-based quantitative method;

c) determining the amount of fetal nucleic acid from a reference chromosome using a non-polymorphic-based and non-bisulfite-based quantitative method;

d) comparing the amount of fetal nucleic acid from step b) to step c), wherein a statistically significant difference between the amount of target and reference fetal nucleic acid is indicative of the presence of a fetal aneuploidy.

18. The method of claim 17, wherein the amount of fetal nucleic acid at between 3 and 15 loci on each of the target chromosome and reference chromosome is determined.