Methods for the diagnosis of fetal abnormalities

• US 10,041,119 B2
• Filed: 05/06/2015
• Issued: 08/07/2018
• Est. Priority Date: 06/14/2006
• Status: Active Grant

• Current Assignee: Gpb Scientific, LLC, Mehmet Toner
• Original Assignee: Gpb Scientific, LLC, Verinata Health Incorporated (Illumina Incorporated), The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Kapur, Ravi, Toner, Mehmet, Wang, Zihua, Fuchs, Martin
• Primary Examiner(s): Thomas, David C
• Application Number:
  US14/705,239
• Publication Number:
  US 20150344956A1
• Time in Patent Office: 1,189 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 2800/385 Congenital anomalies
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere

Abstract

The present invention relates to methods for detecting, enriching, and analyzing rare cells that are present in the blood, e.g. fetal cells. The invention further features methods of analyzing rare cell(s) to determine the presence of an abnormality, disease or condition in a subject, e.g. a fetus by analyzing a cellular sample from the subject.

Claims

1. A method for determining the presence or absence of a fetal trisomy or monosomy using a maternal blood sample comprising a mixture of fetal and maternal genomic nucleic acids, the method comprising:

obtaining a test sample comprising a mixture of fetal and maternal genomic nucleic acids from a maternal blood sample;

selectively amplifying a plurality of target nucleic acids from the test sample and a plurality of target nucleic acids from a reference sample, wherein the target nucleic acids are selected from one or more chromosomes to be tested for trisomy or monosomy, and wherein the reference sample is diploid for the one or more chromosomes to be tested for trisomy or monosomy;

detecting the amplified target nucleic acids from the test sample and from the reference sample;

quantifying the target nucleic acids from the test sample and from the reference sample by highly parallel sequencing;

analyzing the test sample for the presence or absence of a fetal trisomy or monosomy by comparing the quantity of the detected amplified target nucleic acids obtained from the test sample for the one or more chromosomes tested for trisomy or monosomy to the quantity of the detected amplified target nucleic acids obtained from the reference sample for the one or more chromosomes tested for trisomy or monosomy; and

generating a report on the presence or absence of trisomy or monosomy in the maternal blood sample.

2. The method of claim 1, wherein the maternal blood sample is from a pregnant woman.

3. The method of claim 1, wherein the fetal and maternal genomic nucleic acids are enriched from the maternal blood sample.

4. The method of claim 1, wherein the target nucleic acids are selected from one or more of chromosomes X, Y, 13, 18, and 21.

5. The method of claim 1, wherein the highly parallel sequencing is selected from high-throughput sequencing, ultra-deep sequencing, and sequencing-by-synthesis.

6. The method of claim 1, wherein the fetal trisomy is trisomy 13, trisomy 18, or trisomy 21.

7. The method of claim 1, wherein the target nucleic acids are SNPs, STRs, or RNA transcripts.

8. The method of claim 1, wherein the amplification comprises:

hybridizing two or more oligonucleotide probes to each of the target nucleic acids;

ligating the two or more hybridized oligonucleotide probes at each of the target nucleic acids to create amplification templates for each of the target nucleic acids; and

amplifying the amplification templates.

9. The method of claim 8, further comprising attaching biotin to the target nucleic acids to generate biotin-labeled target nucleic acids.

10. The method of claim 9, further comprising attaching the biotin-labeled target nucleic acids to streptavidin.

11. The method of claim 1, wherein quantifying the target nucleic acids further comprises determining gene or allele copy number or both gene and allele copy number.

12. The method of claim 11, further comprising determining fetal trisomy or monosomy based on the gene or allele copy number or gene and allele copy number determination.