Prenatal paternity testing using maternal blood, free floating fetal DNA and SNP genotyping

• US 10,113,196 B2
• Filed: 12/22/2011
• Issued: 10/30/2018
• Est. Priority Date: 05/18/2010
• Status: Active Grant

• Current Assignee: Natera Incorporated (Natera)
• Original Assignee: Natera Incorporated (Natera)
• Inventors: Ryan, Allison, Sigurjonsson, Styrmir, Banjevic, Milena, Gemelos, George, Hill, Matthew, Baner, Johan, Rabinowitz, Matthew, Demko, Zachary
• Primary Examiner(s): Vanni, G. Steven
• Application Number:
  US13/335,043
• Publication Number:
  US 20120122701A1
• Time in Patent Office: 2,504 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G16B 10/00 ICT specially adapted for evolutionary bioinformatics, e.g. phylogenetic tree construction or analysis

Abstract

Methods for non-invasive prenatal paternity testing are disclosed herein. The method uses genetic measurements made on plasma taken from a pregnant mother, along with genetic measurements of the alleged father, and genetic measurements of the mother, to determine whether or not the alleged father is the biological father of the fetus. This is accomplished by way of an informatics based method that can compare the genetic fingerprint of the fetal DNA found in maternal plasma to the genetic fingerprint of the alleged father.

Claims

1. A method for establishing whether an alleged father is the biological father of a fetus that is gestating in a pregnant mother, the method comprising:

obtaining genotypic measurements of single nucleotide polymorphism (SNP) alleles at a plurality of polymorphic loci on genetic material from the alleged father;

obtaining fetal and maternal genotypic measurements of SNP alleles at the plurality of polymorphic loci from genetic material isolated from a blood sample from the pregnant mother, wherein the blood sample comprises a mixture of free floating DNA of fetal origin and free floating DNA of maternal origin, wherein the obtaining of fetal and maternal genotypic measurements comprises amplifying the plurality of polymorphic loci from the free floating DNA of fetal origin and the free floating DNA of maternal origin together in a single reaction and measuring the genotypes of the SNP alleles in the amplified DNA by a technique selected from the group consisting of quantitative PCR, digital PCR, SNP microarrays, DNA microarrays, and sequencing;

determining a probability that the alleged father is the biological father of the fetus by calculating a test statistic for the alleged father and the fetus, wherein the test statistic for the alleged father and the fetus indicates a degree of genetic similarity between the alleged father and the fetus, and wherein the test statistic for the alleged father and the fetus is based on the genotypic measurements of SNP alleles made from genetic material from the alleged father and the genotypic measurements of SNP alleles made from genetic material isolated from the blood sample from the pregnant mother;

determining a distribution of test statistics for a plurality of unrelated individuals and the fetus, where each of the test statistics for the plurality of unrelated individuals and the fetus indicates a degree of genetic similarity between an individual unrelated to the fetus and the fetus, wherein the test statistic is based on genotypic measurements of SNP alleles made from genetic material from the unrelated individual and the genotypic measurements of SNP alleles made from genetic material isolated from the blood sample from the pregnant mother;

determining if the test statistic for the alleged father and the fetus belongs to the distribution of the test statistics for the plurality of unrelated individuals and the fetus; and

outputting that the alleged father is the biological father of the fetus if the test statistic for the alleged father and the fetus does not belong to the distribution of the test statistics for the plurality of unrelated individuals and the fetus.

2. The method of claim 1, wherein determining the probability that the alleged father is the biological father is performed using a maximum likelihood estimation, or a maximum a posteriori technique.

3. The method of claim 1, wherein the polymorphic loci correspond to chromosomes that are disomic.

4. The method of claim 1, wherein the method further comprises isolating the genetic material from the alleged father from a tissue selected from the group consisting of blood, somatic tissue, sperm, hair, buccal sample, skin, forensic samples, and combinations thereof.

5. The method of claim 1, further comprising determining a confidence for the establishment of whether the alleged father is the biological father of the fetus.

6. The method of claim 1, wherein the genotypes of the SNP alleles in the amplified DNA are measured using a technique or technology selected from the group consisting of Sanger DNA sequencing, pyrosequencing, high throughput sequencing, targeted sequencing using circularizing probes, targeted sequencing using capture by hybridization probes, reversible dye terminator sequencing, sequencing by ligation, and sequencing by hybridization.

7. The method of claim 1, wherein the free floating DNA of fetal origin and the free floating DNA of maternal origin are amplified using a technique or technology that is selected from the group consisting of Polymerase Chain Reaction (PCR), ligation mediated PCR, degenerative oligonucleotide primer PCR, targeted amplification, PCR, mini-PCR, universal PCR amplification, Multiple Displacement Amplification (MDA), linear amplification methods, ligation of substrate DNA followed by another method of amplification, bridge amplification, padlock probes, circularizing probes, and capture by hybridization probes.

8. The method of claim 1, further comprising generating a report comprising the probability of paternity of the fetus.

9. The method of claim 1, wherein the polymorphic loci comprise short tandem repeats (STRs).

10. The method of claim 1, further comprising outputting the determined probability that the alleged father is the biological father of the fetus.

11. The method of claim 1, wherein the test statistic calculated for the alleged father and the fetus does not belong to the distribution of the test statistics calculated for the plurality of unrelated individuals and the fetus if the P value of the alleged father'"'"'s test statistic according to the distribution of unrelated individuals is less than 10⁻

4.

12. The method of claim 1, wherein the test statistic calculated for the alleged father and the fetus does belong to the distribution of the test statistics calculated for the plurality of unrelated individuals and the fetus if the P value of the alleged father'"'"'s test statistic according to the distribution of unrelated individuals is greater than 0.02.

13. A method for establishing whether an alleged father is the biological father of a fetus that is gestating in a pregnant mother, the method comprising:

obtaining genotypic measurements of SNP alleles at a plurality of polymorphic loci on genetic material from the alleged father;

obtaining genotypic measurements of SNP alleles at the plurality of polymorphic loci on genetic material from a plurality of individuals who are genetically unrelated to the fetus;

obtaining fetal and maternal genotypic measurements of SNP alleles at the plurality of polymorphic loci from genetic material isolated from a blood sample from the pregnant mother, wherein the blood sample comprises a mixture of free floating DNA of fetal origin and free floating DNA of maternal origin, wherein the obtaining of fetal and maternal genotypic measurements comprises amplifying the plurality of polymorphic loci from the free floating DNA of fetal origin and the free floating DNA of maternal origin together in a single reaction and measuring the genotypes of the SNP alleles in the amplified DNA by a technique selected from the group consisting of quantitative PCR, digital PCR, SNP microarrays, DNA microarrays, and sequencing;

calculating a test statistic for the alleged father, wherein the test statistic for the alleged father indicates a degree of genetic similarity between the alleged father and the fetus, and wherein the test statistic for the alleged father is a function of the genotypic measurements of SNP alleles made on the genetic material from the alleged father and the genetic material isolated from the blood sample from the pregnant mother;

calculating a test statistic for each individual in the plurality of unrelated individuals, wherein the test statistic for each unrelated individual indicates a degree of genetic similarity between the unrelated individual and the fetus, and wherein the test statistic for each unrelated individual is a function of genotypic measurements of SNP alleles made on the genetic material from the unrelated individual and the genetic material isolated from the blood sample from the pregnant mother;

determining whether the test statistic for the alleged father belongs to a distribution of the test statistic for the plurality of unrelated individuals; and

outputting that the alleged father is the biological father if the test statistic for the alleged father does not belong to the distribution of the test statistic for the plurality of unrelated individuals.

14. The method of claim 13, wherein establishing whether the alleged father is the biological father of the fetus further comprises:

establishing that the alleged father is the biological father of the fetus by rejecting a hypothesis that the alleged father is unrelated to the fetus if the probability that the test statistic for the alleged father belongs to the distribution of the test statistic for the plurality of unrelated individuals is below a lower threshold;

or

establishing that the alleged father is not the biological father of the fetus by not rejecting a hypothesis that the alleged father is unrelated to the fetus if the probability is above an upper threshold;

or

not establishing whether an alleged father is the biological father of the fetus if the probability is between the lower threshold and the upper threshold, or if the probability is not determined with sufficiently high confidence.

15. The method of claim 13, further comprising obtaining genotypic measurements at the plurality of polymorphic loci from a sample that consists essentially of genetic material from the mother;

wherein the test statistic for the alleged father is a function of the genotypic measurements made on the genetic material from the alleged father, the sample that consists essentially of genetic material from the mother, and the genetic material isolated from the blood sample from the pregnant mother and

wherein the test statistic for the unrelated individual is a function of genotypic measurements made on the genetic material from the unrelated individual, the sample that consists essentially of genetic material from the mother, and the genetic material isolated from the blood sample from the pregnant mother;

calculating a probability that the test statistic for the alleged father is part of the distribution of the test statistic for the plurality of unrelated individuals; and

determining a probability that the alleged father is the biological father of the fetus using the probability that the test statistic for the alleged father is part of the distribution of the test statistic for the plurality of unrelated individuals.

16. The method of claim 13, wherein the test statistic calculated for the alleged father and the fetus does not belong to the distribution of the test statistics calculated for the plurality of unrelated individuals and the fetus if the P value of the alleged father'"'"'s test statistic according to the distribution of unrelated individuals is less than 10⁻

4.

17. The method of claim 13, wherein the test statistic calculated for the alleged father and the fetus does belong to the distribution of the test statistics calculated for the plurality of unrelated individuals and the fetus if the P value of the alleged father'"'"'s test statistic according to the distribution of unrelated individuals is greater than 0.02.