NON-INVASIVE FETAL SEX DETERMINATION

• US 20140256572A1
• Filed: 03/08/2013
• Published: 09/11/2014
• Est. Priority Date: 03/08/2013
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Ariosa Diagnostics Incorporated (Roche Holding AG)
• Inventors: Struble, Craig, Oliphant, Arnold, Wang, Eric
• Granted Patent: US 9,994,897 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• 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 2600/156 Polymorphic or mutational markers

Abstract

The present invention provides methods for non-invasive determination of sex in a fetus or of Y chromosomal frequency abnormalities—indicative of aneuploidy or sex mosaicisms in a fetus—by detecting and determining the relative contribution genetic sequences from the Y chromosome in view of the percent fetal contribution in a maternal mixed sample.

Claims

1. A method for determining a risk for Y chromosomal frequency abnormalities in a fetus comprising the steps of:

obtaining maternal samples comprising maternal and fetal nucleic acids;

annealing sets of two fixed sequence oligonucleotides specific to selected nucleic acid regions on the Y chromosome and to polymorphic and non-polymorphic selected nucleic acid regions on at least one non-Y chromosome to the maternal and fetal nucleic acids;

selectively amplifying the selected nucleic acid regions from the Y chromosome and the at least one non-Y chromosome to generate amplified selected nucleic acid regions;

sequencing the amplified selected nucleic acid regions;

quantifying the sequenced nucleic acid regions;

determining a frequency of the quantified nucleic acid regions from the Y chromosome and the at least one non-Y chromosome;

determining a percent of fetal nucleic acids in the maternal samples by looking at the frequency of the quantified polymorphic selected nucleic acid regions from the at least one non-Y chromosome; and

determining a probability that the fetus is a normal male fetus and the risk for Y chromosomal frequency abnormalities in the fetus by assessing the frequency of the quantified nucleic acid regions from the Y chromosome in view of the frequency of the quantified nucleic acid regions from the non-Y chromosome and the percent fetal nucleic acids in the maternal samples.

2. The method of claim 1, wherein at least eight selected nucleic acid regions from the Y chromosome and the at least one non-Y chromosome are amplified.

3. The method of claim 2, wherein at least forty-eight selected nucleic acid regions from the Y chromosome and the at least one non-Y chromosome are amplified.

4. The method of claim 3, wherein at least ninety-six selected nucleic acid regions from the Y chromosome and the at least one non-Y chromosome are amplified.

5. The method of claim 1, wherein the Y chromosomal frequency abnormality arises from a Y chromosome aneuploidy, a Y chromosome mosaicism of the fetus or sample contamination.

6. The method of claim 1, wherein selected nucleic acid regions from at least two non-Y chromosomes are selectively amplified, sequenced and quantified.

7. The method of claim 6, wherein selected nucleic acid regions from at least four non-Y chromosomes are selectively amplified, sequenced and quantified.

8. The method of claim 7 wherein selected nucleic acid regions from at least six non-Y chromosomes are selectively amplified, sequenced and quantified.

9. The method of claim 1, wherein the at least one non-Y chromosome is selected from chromosome 13, 18 or 21.

10. The method of claim 1, wherein at least one of the fixed oligonucleotides comprises at least one identifying index.

11. The method of claim 10, wherein the at least one index comprises a sample index.

12. The method of claim 11, wherein before the selective amplification step the maternal samples are in different vessels for the reactions, and after the selective amplification step the maternal samples are pooled.

13. The method of claim 10, wherein the at least one index comprises a locus index.

14. The method of claim 13, further comprising a sample index, where the locus index and sample index are located on a same fixed sequence oligonucleotide in a set.

15. The method of claim 13, further comprising a sample index, where the locus index and sample index are located on different fixed sequence oligonucleotides in a set.

16. The method of claim 13, wherein the determining the frequency of the quantified nucleic acid regions from the Y chromosome and the at least one non-Y chromosome step is performed by hybridization to an array.

17. The method of claim 1, wherein at least one of the fixed sequence oligonucleotides interrogating the polymorphic selected nucleic acid regions in the at least one non-Y chromosome comprises an allele index.

18. The method of claim 1, where the DNA is selectively amplified in a single vessel.

19. The method of claim 1, where the selected nucleic acid regions are each counted an average of at least five times.

20. The method of claim 19, where the selected nucleic acid regions are each counted an average of at least 250 times.

21. The method of claim 1, wherein the quantifying step is performed using next generation sequencing.

22. The method of claim 1, wherein the sets of oligonucleotide primers further comprise a bridging oligonucleotide.

23. A method for determining the risk of Y chromosomal frequency abnormalities in a fetus comprising the steps of:

obtaining at least five maternal samples comprising maternal and fetal nucleic acids;

placing each of the maternal samples in a separate reaction vessel;

annealing sets of two fixed sequence oligonucleotides specific to selected nucleic acid regions on the Y chromosome and to polymorphic and non-polymorphic selected nucleic acid regions on at least two non-Y chromosomes to the maternal and fetal nucleic acids, wherein at least one of the fixed sequence oligonucleotides comprises a sample index;

selectively amplifying the selected nucleic acid regions from the Y chromosome and the at least two non-Y chromosomes to generate amplified selected nucleic acid regions;

pooling the at least five maternal samples into a single reaction vessel;

sequencing the amplified selected nucleic acid regions;

using software executed on a computer, quantifying the sequenced nucleic acid regions;

using software executed on a computer, determining a frequency of the quantified selected nucleic acid regions from the Y chromosome and the at least two non-Y chromosomes;

using software executed on a computer, determining a percent of fetal nucleic acids in the maternal sample by looking at the frequency of the quantified polymorphic selected nucleic acid regions from the at least two non-Y chromosomes; and

using software executed on a computer, determining the probability that the fetus is a normal male fetus and the risk for Y chromosomal frequency abnormalities in the fetus by assessing the frequency of the selected nucleic acid regions from the Y chromosome in view of the quantified selected nucleic acid regions from the at least two non-Y chromosomes and the percent fetal nucleic acids in the maternal sample.

24. The method of claim 23, wherein at least one of the fixed oligonucleotides comprises at least one additional index.

25. The method of claim 24, wherein the at least one index comprises a locus index.

26. The method of claim 25, wherein the determining the frequency of the quantified nucleic acid regions from the Y chromosome and the at least two non-Y chromosomes step is performed by hybridization to an array.

27. The method of claim 24, where the locus index and sample index are located on a same fixed sequence oligonucleotide in a set.

28. The method of claim 24, where the locus index and sample index are located on different fixed sequence oligonucleotides in a set.

29. The method of claim 23, wherein at least one of the fixed sequence oligonucleotides interrogating the polymorphic selected nucleic acid regions in the at least one non-Y chromosome comprises an allele index.

30. The method of claim 23, wherein the sets of oligonucleotide primers further comprise a bridging oligonucleotide.