Methods for the Diagnosis of Fetal Abnormalities

• US 20080138809A1
• Filed: 06/14/2007
• Published: 06/12/2008
• Est. Priority Date: 06/14/2006
• Status: Active Grant

• Current Assignee: Gpb Scientific, LLC, Verinata Health Incorporated (Illumina Incorporated), Mehmet Toner
• Original Assignee: Mehmet Toner
• Inventors: Toner, Mehmet, Kapur, Ravi, Wang, Zihua, Fuchs, Martin
• Granted Patent: US 8,137,912 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 a condition in a patient or a fetus of a patient comprising:

enriching one or more cells from a sample from said patient using size-based separation,

obtaining one or more nucleic acid molecules from said enriched cells;

detecting said one or more nucleotides in said nucleic acid molecules by high throughput sequencing.

2. The method of claim 1, wherein said sample is a blood sample.

3. The method of claim 2, wherein said blood sample is a maternal blood sample.

4. The method of claim 1, wherein said one or more cells are selected from the group consisting of fetal cells, epithelial cells, endothelial cells, progenitor stem cells, or a combination thereof.

6. The method of claim 1, wherein said one or more cells are fetal cells and said condition is selected from the group consisting of trisomy 13, trisomy 18, trisomy 21, Klinefelter Syndrome, dup(17)(p11.2p11.2) syndrome, Down syndrome, Pre-eclampsia, Pre-term labor, Edometriosis, Pelizaeus-Merzbacher disease, dup(22)(q11.2q11.2) syndrome, Cat eye syndrome, Cri-du-chat syndrome, Wolf-Hirschhorn syndrome, Williams-Beuren syndrome, Charcot-Marie-Tooth disease, neuropathy with liability to pressure palsies, Smith-Magenis syndrome, neurofibromatosis, Alagille syndrome, Velocardiofacial syndrome, DiGeorge syndrome, steroid sulfatase deficiency, Kallmann syndrome, microphthalmia with linear skin defects, Adrenal hypoplasia, Glycerol kinase deficiency, Pelizaeus-Merzbacher disease, testis-determining factor on Y, Azospermia (factor a), Azospermia (factor b), Azospermia (factor c), 1p36 deletion, or a combination thereof.

9. The method of claim 1, wherein said size-based separation is performed by flowing said sample or a fraction thereof through an array of obstacles that selectively directs cells larger than a predetermined size to a first outlet and cells smaller than said predetermined size to a second outlet.

12. The method of claim 1 wherein said enriching step further comprises one or more of the following steps:

(i) flowing said sample or a fraction thereof through one or more magnetic fields that selectively retain paramagnetic components; and

(ii) applying hyperbaric or hypobaric pressure to said sample or a fraction thereof.

5. (canceled)

7-8. -8. (canceled)

10-11. -11. (canceled)

13. A method for determining a condition in a patient or a fetus of a patient comprising:

enriching one or more cells from a sample from said patient by flowing said sample or a fraction thereof through an array of obstacles, wherein at least a portion of said obstacles are coated with binding moieties that specifically bind to one or more cell populations in said sample,

obtaining one or more nucleic acid molecules from said enriched cells;

detecting said one or more nucleotides in said nucleic acid molecules by high throughput sequencing.

14-23. -23. (canceled)

24. A method for determining a fetal abnormality comprising:

obtaining a sample other than amniotic fluid sample from a pregnant female,

enriching one or more fetal cells from said sample using size-based separation,

obtaining one or more nucleic acid molecules from said enriched fetal cells;

amplifying said one or more nucleic acid molecules.

25. The method of claim 24, wherein said size-based separation is performed by flowing said sample or a fraction thereof through an array of obstacles that selectively directs cells larger than a predetermined size to a first outlet and cells smaller than said predetermined size to a second outlet.

26. The method of claim 25, wherein said array of obstacles for size-based separation is fluidly coupled with a second array of obstacles, wherein at least a portion of said obstacles of said second array are coated with binding moieties that specifically bind to one or more cell populations in said sample

28. The method of claim 24, wherein said abnormality is aneuploidy.

30. The method of claim 24, wherein said amplifying occurs on a bead.

31. The method of claim 24, wherein said one or more nucleic acid molecules comprises genomic DNA.

27. (canceled)

29. (canceled)

32-35. -35. (canceled)

36. A method for determining an abnormality of a fetus comprising:

enriching one or more fetal cells from a blood sample from a female pregnant with said fetus by flowing said sample through an array of obstacles that selectively directs cells smaller than a predetermined size to a first outlet and cells larger than a predetermined size to a second outlet,

flowing sample enriched according to step (i) through one or more magnetic fields; and

applying hyperbaric or hypobaric pressure to sample enriched according to step (ii).

37. The method of claim 36, further comprising performing one or more genetic analyses on sample enriched according to step (iii), wherein said genetic analyses are selected from the group consisting of:

SNP detection, RNA expression detection, sequence detection, or a combination thereof.

38. The method of claim 36, further comprising amplifying of one or more nucleic acid fragments from sample enriched according to step (iii).

39. (canceled)

40. A method for determining a fetal abnormality comprising:

enriching one or more fetal cells from a blood sample from a female pregnant with said fetus by flowing said sample through an array of obstacles that selectively directs cells smaller than a predetermined size to a first outlet and cells larger than a predetermined size to a second outlet; and

performing one or more genetic analyses on enriched sample, wherein said genetic analyses are selected from the group consisting of SNP detection, RNA expression detection, and sequence detection.

43. The method of claim 40, wherein said genetic analyses are performed on DNA or RNA from chromosome X, Y, 13, 18, or 21.

44. The method of claim 40, wherein said sequence detection involves sequencing by synthesis.

45. The method of claim 40, wherein said enriched sample comprises one or more fetal cells and one or more non-fetal cells.

46. The method of claim 40, further comprising performing one or more genetic analyses on a control sample.

47. The method of claim 46, wherein said control sample is a maternal only cell sample.

49. The method of claim 40, wherein said sequence detection involves adding a plurality of labeled nucleotides or nucleotides analogs to a nucleotide sequence complementary to enriched nucleic acid molecules.

51. The method of claim 40, further comprising amplifying target nucleic acids from enriched sample.

41-42. -42. (canceled)

48. (canceled)

50. (canceled)

52-57. -57. (canceled)