Processes and compositions for methylation-based acid enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses

• US 8,476,013 B2
• Filed: 09/16/2009
• Issued: 07/02/2013
• Est. Priority Date: 09/16/2008
• Status: Active Grant

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Ehrich, Mathias, Nygren, Anders Olof Herman
• Primary Examiner(s): SITTON, JEHANNE SOUAYA
• Application Number:
  US12/561,241
• Publication Number:
  US 20100105049A1
• Time in Patent Office: 1,385 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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 determining the presence or absence of a fetal aneuploidy comprising:

a) digesting nucleic acid from a pregnant female, which nucleic acid comprises fetal nucleic acid and maternal nucleic acid, with a methylation sensitive restriction enzyme that specifically digests non-methylated maternal nucleic acid at a plurality of loci, wherein the plurality of loci comprises the locus of SEQ ID NO;

38, thereby enriching the fetal nucleic acid;

b) determining the amount of fetal nucleic acid from a target chromosome;

c) determining the amount of fetal nucleic acid from a reference chromosome and;

d) comparing the amount of fetal nucleic acid determined in step (b) to the amount of fetal nucleic acid determined in step (c), whereby a statistically significant difference between the amount of fetal nucleic acid determined in step (b) and the amount of fetal nucleic acid determined in step (c) determines the presence of a fetal aneuploidy.

2. 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 reference chromosome is determined.

3. The method of claim 1, wherein the digestion efficiency of the methylation sensitive restriction enzyme is determined.

4. The method of claim 1, wherein determining the amount of fetal nucleic acid in (b) and/or (c) comprises use of a competitor-based amplification method.

5. The method of claim 1, wherein the method further comprises determining the presence or absence of Y-chromosome nucleic acid present in the nucleic acid.

6. The method of claim 5, wherein the amount of Y-chromosome nucleic acid present in the nucleic acid is determined for a male fetus.

7. The method of claim 6, wherein the amount of fetal nucleic acid is compared to the amount of Y-chromosome nucleic acid.

8. The method of claim 1, wherein the total amount of nucleic acid is determined.

9. The method of claim 1, wherein the total amount of nucleic acid and the amount of Y-chromosome nucleic acid for a male fetus are determined.

10. The method of claim 1, 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 determined.

11. The method of claim 10, 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.

12. The method of claim 11, wherein the amount of fetal nucleic acid at 3 or more loci is determined.

13. 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.

14. The method of claim 1, wherein determining the amount of fetal nucleic acid in (b) and/or (c) comprises use of a mass spectrometry method.

15. The method of claim 1, wherein determining the amount of fetal nucleic acid in (b) and/or (c) 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 plurality of loci further comprises one or more loci selected from loci of SEQ ID NOs:

1 to 37 and SEQ ID NOs;

39 to 59.

18. The method of claim 1, wherein the plurality of loci further comprises one or more loci selected from loci of SEQ ID NOs:

1 to 37 and SEQ ID NO;

39.

19. The method of claim 1, wherein the plurality of loci further comprises one or more loci selected from loci of SEQ ID NOs:

33, 34, 35, 36, 37, and 39.

20. 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 reference chromosome is determined.