Processes and kits for identifying aneuploidy

• US 9,926,593 B2
• Filed: 12/20/2010
• Issued: 03/27/2018
• Est. Priority Date: 12/22/2009
• Status: Active Grant

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Ehrich, Mathias, Mistro, Guy Del, Deciu, Cosmin, Chen, Yong Qing, McCullough, Ron Michael, Tim, Roger Chan
• Primary Examiner(s): Weiler, Karen S.
• Application Number:
  US13/518,368
• Publication Number:
  US 20130130923A1
• Time in Patent Office: 2,654 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation

Abstract

Provided are methods for identifying the presence or absence of a chromosome abnormality by which a cell-free sample nucleic acid from a subject is analyzed. In certain embodiments, provided are methods for identifying the presence or absence of a fetal chromosome abnormality in a nucleic acid from cell-free maternal blood.

Claims

1. A multiplex method for identifying the presence or absence of an aneuploidy of a target chromosome in a sample from a pregnant female subject, which comprises:

a. providing about 20 to about 56 amplification primer pairs, wherein each amplification primer pair specifically hybridizes with polynucleotides of a nucleotide sequence species set, wherein;

i) the polynucleotides of a nucleotide sequence species set are present on two or more different chromosomes at different loci, comprising a target chromosome and one or more reference chromosomes not associated with the aneuploidy;

(ii) the polynucleotides of a nucleotide sequence species set differ by one or more mismatch nucleotides;

(iii) the polynucleotides of a nucleotide sequence species set are reproducibly amplified by a single pair of amplification primers relative to each other;

(iv) each amplified polynucleotide in a nucleotide sequence species set comprises a nucleotide sequence having the one or more mismatch nucleotides and (v) the polynucleotides of the nucleotide sequence species sets have nucleotide sequences corresponding to SEQ ID NOs;

4736, 4808, 4258, 4413, 4537, 4692, 4203, 4077, 4697, 4436, 3869, 4475, 4078, 4396, 4782, 4683, 4279, 3940, 4236, 3865, 4733, 4168, 3818, 4563, 4906, 3807, 3849, 4594, 4022, 4538, 4219, 4548, 4382, 4374, 4444, 4305, 4403, 4307, 4059, 4767, 3994, 4561, 4209, 3850, 4686, 3985, 4608, 3963, 4038, 4948, 4468, 4879, 4196, 3967, 4150 and 4102;

b. contacting in one or more reaction vessels under amplification conditions, extracellular nucleic acid of the sample comprising fetally derived and maternally derived nucleic acid with amplification primer pairs consisting of the about 20 to about 56 amplification primer pairs of (a), wherein about 10 to about 25 of the amplification primer pairs are in each of the one or more reaction vessels, and each primer pair amplifies only a single nucleotide species set in the reaction vessel, thereby producing about 20 to about 56 species sets of amplified polynucleotides;

c. determining the amount of each amplified polynucleotide in each set by detecting the one or more mismatch nucleotides in each amplified polynucleotide by primer extension, sequencing, Q-PCR or mass spectrometry;

d. determining a ratio between the relative amount of (i) an amplified target polynucleotide and (ii) an amplified reference polynucleotide, for each set; and

e. identifying the presence or absence of an aneuploidy of a target chromosome based on the ratios from the about 20 to about 56 species sets of amplified polynucleotides.

2. The method of claim 1, wherein the extracellular nucleic acid is from blood, blood plasma or blood serum of the pregnant female subject.

3. The method of claim 2, wherein the extracellular nucleic acid is from a female subject in the first trimester of pregnancy, second trimester of pregnancy or third trimester of pregnancy.

4. The method of claim 1, wherein the fetal nucleic acid is about 5% to about 40% of the extracellular nucleic acid;

or the number of fetal nucleic acid copies is about 10 copies to about 2000 copies of the total extracellular nucleic acid.

5. The method of claim 1, which comprises enriching the extracellular nucleic acid for fetal nucleic acid.

6. The method of claim 1, which comprises determining the fetal nucleic acid concentration in the extracellular nucleic acid.

7. The method of claim 1, wherein the amounts of the amplified polynucleotides in each set vary by up to a value that permits detection of the aneuploidy of a target chromosome with a confidence level of about 95% or more.

8. The method of claim 1, wherein the amounts of the amplified polynucleotides in each set vary by up to a value that permits detection of the aneuploidy of a target chromosome with a sensitivity of about 90% or more, and a specificity of about 95% or more.

9. The method of claim 1, wherein the number of sets of amplified polynucleotides is based on (i) the number of sets that provides a 85% or greater sensitivity for determining the absence of the aneuploidy of a target chromosome, (ii) the number of sets that provides a 85% or greater specificity for determining the presence of the aneuploidy of a target chromosome or (i) the number of sets that provides a 85% or greater sensitivity for determining the absence of the aneuploidy of a target chromosome and (ii) the number of sets that provides a 85% or greater specificity for determining the presence of the aneuploidy of a target chromosome.

10. The method of claim 1, wherein identifying the presence or absence of an aneuploidy of a target chromosome is based on the ratios from about 56 species sets of amplified polynucleotides.

11. The method of claim 1, wherein detecting the one or more mismatch nucleotides in each amplified polynucleotide in a set is by primer extension.

12. The method of claim 1, wherein detecting the one or more mismatch nucleotides in each amplified polynucleotide in a set is by sequencing.

13. The method of claim 1, wherein detecting the one or more mismatch nucleotides in each amplified polynucleotide in a set is by Q-PCR.

14. The method of claim 1, wherein detecting the one or more mismatch nucleotides in each amplified polynucleotide in a set is by mass spectrometry.

15. The method of claim 1, wherein 56 amplification primer pairs are provided.

16. The method of claim 1, wherein the amplification primer pairs are selected from the primer pairs of the group consisting of: