Noninvasive detection of fetal aneuploidy in egg donor pregnancies
The present invention provides assay systems and methods for determining the percent fetal contribution of cell-free DNA in a maternal sample from a pregnant female with an egg donor pregnancy. Further provided, are assay systems and methods for determining a statistical likelihood of the presence or absence of a fetal aneuploidy in a maternal sample using a determined percent fetal cell-free DNA in the sample.
1. A method for determining the percent fetal cell-free DNA in a maternal sample from a pregnant female with an egg donor pregnancy, comprising:
providing a maternal sample comprising maternal and fetal cell-free DNA;
interrogating two or more selected polymorphic nucleic acid regions;
detecting the interrogated nucleic acid regions;
determining a relative frequency of polymorphisms in the interrogated nucleic acid regions to identify egg donor informative loci;
calculating a percent fetal cell-free DNA using the identified egg donor informative loci.
2. The method of claim 1 , wherein the egg donor informative loci include loci in which the maternal DNA and the fetal DNA differ in at least one allele.
3. The method of claim 1 , wherein the egg donor informative loci include loci in which the maternal DNA and the fetal DNA differ in both alleles.
4. The method of claim 1 , wherein calculating the percent fetal cell-free DNA comprises comparing a relative frequency of low frequency alleles to a relative frequency of both the high and low frequency alleles.
5. The method of claim 1 , wherein the step of interrogating two or more selected polymorphic nucleic acid regions comprises:
introducing a first set of fixed sequence oligonucleotides to the maternal sample under conditions that allow the fixed oligonucleotides to specifically hybridize to complementary regions of a first polymorphic nucleic acid region;
introducing a second set of fixed sequence oligonucleotides to the maternal sample under conditions that allow the fixed oligonucleotides to specifically hybridize to complementary regions of a second polymorphic nucleic acid region; and
ligating the hybridized oligonucleotides to create contiguous ligation products complementary to the nucleic acids.
6. The method of claim 5 , wherein the fixed sequence oligonucleotides comprise a universal primer region.
7. The method of claim 6 , wherein the first set of fixed sequence oligonucleotides and the second set of fixed sequence oligonucleotides comprises the same universal primer region.
8. The method of claim 6 , wherein the first set of fixed sequence oligonucleotides comprises a first universal primer region and the second set of fixed sequence oligonucleotides comprises a second universal primer region.
9. The method of claim 6 , and further comprising amplifying the contiguous ligation products using the universal primer region to create amplification products.
10. The method of claim 9 , wherein the amplifying comprises performing polymerase chain reaction (PCR) amplification on the contiguous ligation product templates.
11. The method of claim 1 , wherein the maternal sample is maternal plasma or serum.
12. The method of claim 1 , wherein interrogating two or more selected polymorphic nucleic acid regions comprises interrogating at least ten nucleic acid regions.
13. The method of claim 12 , wherein interrogating two or more selected polymorphic nucleic acid regions comprises interrogating at least twenty nucleic acid regions.
14. The method of claim 13 , wherein interrogating two or more selected polymorphic nucleic acid regions comprises interrogating at least forty nucleic acid regions.
15. The method of claim 14 , wherein interrogating two or more selected polymorphic nucleic acid regions comprises interrogating at least ninety nucleic acid regions.
16. The method of claim 15 , wherein interrogating two or more selected polymorphic nucleic acid regions comprises interrogating at least 300 nucleic acid regions.
17. A method for identifying the presence or absence of a fetal aneuploidy in sample from a female with an egg donor pregnancy, comprising
providing a maternal sample comprising maternal and fetal cell-free DNA;
calculating a percent fetal cell-free DNA in the sample using identified egg donor informative loci;
demonstrating the sample comprises fetal cell-free DNA above a threshold level;
interrogating two or more nucleic acid regions from a first chromosome;
interrogating two or more nucleic acid regions from at least a second chromosome
determining a frequency of the interrogated nucleic acid regions from the first and at least second chromosome;
identifying the presence or absence of a fetal aneuploidy based on the determined relative frequencies of the nucleic acid regions from the first and at least second chromosome.
18. The method of claim 17 , wherein the threshold value is between 1.0 and 5.0 percent.
19. The method of claim 18 , wherein the threshold value is between 1.5 and 4.5 percent.
20. The method of claim 19 , wherein the threshold value is between 2.0 and 4.0 percent.