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

Patent metadata

Field	Value
Publication number	US-10113196-B2
Application number	US-201113335043-A
Country	US
Kind code	B2
Filing date	Dec 22, 2011
Priority date	May 18, 2010
Publication date	Oct 30, 2018
Grant date	Oct 30, 2018

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.

First claim

What is claimed is:

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

Assignees

• Ryan Allison
• Sigurjonsson Styrmir
• Banjevic Milena
• Gemelos George
• Hill Matthew
• Baner Johan
• Rabinowitz Matthew
• Demko Zachary
• Natera Inc

Inventors

• Ryan Allison
• Sigurjonsson Styrmir
• Banjevic Milena
• Gemelos George
• Hill Matthew
• Baner Johan
• Rabinowitz Matthew
• Demko Zachary

Classifications

• C12Q1/6876 Primary

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• G16B10/00

  ICT specially adapted for evolutionary bioinformatics, e.g. phylogenetic tree construction or analysis · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• G16B30/00

  ICT specially adapted for sequence analysis involving nucleotides or amino acids · CPC title