Methods for Non-Invasive Prenatal Ploidy Calling

• US 20110288780A1
• Filed: 05/18/2011
• Published: 11/24/2011
• Est. Priority Date: 05/18/2010
• Status: Active Grant

• Current Assignee: Natera Incorporated (Natera)
• Original Assignee: Gene Security Network Inc
• Inventors: Baner, Johan, Hill, Matthew, Rabinowitz, Matthew, Zimmermann, Bernhard, Demko, Zachary, Ryan, Allison, Gemelos, George, Banjevic, Milena
• Granted Patent: US 8,825,412 B2
• Time in Patent Office: Days
• US Class Current: 702/19
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2537/16 Assays for determining copy number or wherein the copy number is of special importance
• CPC Class Codes: C12Q 2537/161 A competitive reaction step
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2600/112 Disease subtyping, staging or classification
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: C12Q 2600/172 Haplotypes
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/10 Ploidy or copy number detection
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 20/40 Population genetics; Linkage disequilibrium
• CPC Class Codes: G16B 30/00 : ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 : Sequence alignment; Homology search
• CPC Class Codes: G16B 5/00 : ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks
• CPC Class Codes: G16B 5/20 : Probabilistic models
• CPC Class Codes: G16H 10/40 : for data related to laboratory analysis, e.g. patient specimen analysis
• CPC Class Codes: G16H 50/30 : for calculating health indices; for individual health risk assessment

Abstract

Methods for non-invasive prenatal ploidy calling are disclosed herein. Methods for determining the ploidy status of a chromosome in a gestating fetus from genotypic data measured from a sample of DNA from the mother of the fetus and from the fetus, and from genotypic data from the mother and optionally also from the father are disclosed herein. The ploidy state is determined by using a joint distribution model to create a set of expected allele distributions for different possible fetal ploidy states given the parental genotypic data, and comparing the expected allelic distributions to the pattern of measured allelic distributions measured in the mixed sample, and choosing the ploidy state whose expected allelic distribution pattern most closely matches the observed allelic distribution pattern. In an embodiment, the mixed sample of DNA may be preferentially enriched at a plurality of polymorphic loci in a way that minimizes the allelic bias.

Claims

1. A method for determining a ploidy status of a chromosome in a gestating fetus, the method comprising:

obtaining a first sample that contains DNA from the mother of the fetus and DNA from the fetus;

obtaining genotypic data from one or both parents of the fetus;

processing the first sample by purifying the DNA to obtain a second sample;

measuring the DNA in the second sample at a set of polymorphic alleles;

calculating, on a computer, allele ratios at the set of polymorphic alleles from the DNA measurements made on the second sample;

creating, on a computer, a plurality of ploidy hypotheses concerning expected allele ratios at the set of polymorphic alleles on the chromosome for different possible ploidy states of the chromosome;

building, on a computer, a joint distribution model for heterozygosity rates of each polymorphic allele on the chromosome for each ploidy hypothesis using genotypic data from the one or both parents of the fetus;

determining, on a computer, a relative probability of each of the ploidy hypotheses using the joint distribution model and the allele ratios calculated for the second sample; and

calling the ploidy state of the fetus by selecting the ploidy state corresponding to the hypothesis with the greatest probability.

2. The method of claim 1, wherein the first sample has been isolated from maternal blood.

3. The method of claim 1, wherein processing the first sample further comprises amplifying the DNA.

4. The method of claim 1, wherein processing the first sample further comprises preferentially enriching the DNA at the plurality of polymorphic loci.

5. The method of claim 4, wherein the preferentially enriching the DNA at a plurality of polymorphic loci comprises:

obtaining a pre-circularized probe such that the 3′ and

5′

ends are designed to hybridize to a region of DNA that is separated from the polymorphic region of the allele by a small number of bases, where the small number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or a combination thereof;

hybridizing the pre-circularized probe to purified DNA from the first sample;

circularizing the pre-circularized probe; and

amplifying some or all of the circularized probe.

6. The method of claim 4, wherein the preferentially enriching the DNA at a plurality of polymorphic loci comprises:

obtaining a forward probe such that the 3′

end of the forward probe is designed to hybridize to the region of DNA immediately upstream from the polymorphic region, and separated from the polymorphic region by a small number of bases, where the small number is selected from the group consisting of 1, 2, 3, 4, 5, 6 to 10, and 11 to 20;

obtaining a reverse probe such that the 3′

end of the reverse probe is designed to hybridize to the region of DNA immediately downstream from the polymorphic region, and separated from the polymorphic region by a small number of bases, where the small number is selected from the group consisting of 1, 2, 3, 4, 5, 6 to 10, and 11 to 20;

hybridizing the two probes to DNA in the first sample of DNA; and

amplifying the DNA using the polymerase chain reaction.

7. The method of claim 4, wherein the preferentially enriching the DNA at a plurality of polymorphic loci comprises:

obtaining a set of hybrid capture probes;

hybridizing the hybrid capture probes to the DNA in the first sample; and

physically separating the hybridized DNA from the first sample of DNA from the unhybridized DNA from the first sample.

10. The method of claim 4, wherein the preferential enrichment results in average degree of allelic bias between the second sample and the first sample of a factor selected from the group consisting of no more than a factor of 2, no more than a factor of 1.5, no more than a factor of 1.2, no more than a factor of 1.1, no more than a factor of 1.05, no more than a factor of 1.02, no more than a factor of 1.01, no more than a factor of 1.005, no more than a factor of 1.002, no more than a factor of 1.001 and no more than a factor of 1.0001.

11. The method of claim 1, wherein the set of polymorphic alleles are SNPs.

12. The method of claim 1, wherein measuring the DNA in the second sample is done by sequencing.

13. The method of claim 12, wherein the method is executed for a plurality of gestating fetuses, the method further comprising:

determining the percent of DNA that is fetal in each of the second fractions; and

wherein measuring the DNA in the second sample is done by sequencing a number of DNA molecules in each of the second samples, where more molecules of DNA are sequenced from those second samples that have a smaller fraction of fetal DNA than those second samples that have a larger fraction of fetal DNA.

14. The method of claim 1, wherein the method is executed for a plurality of gestating fetuses, and where the measuring the DNA in the second sample is done, for each of the fetuses, by sequencing a fraction of the second sample of DNA to give a first set of measurements, the method further comprising:

making a first relative probability determination for each of the ploidy hypotheses for each of the fetuses, given the first set of DNA measurements;

resequencing a second fraction of the second sample from those fetuses where the first relative probability determination for each of the ploidy hypotheses indicates that a ploidy hypothesis corresponding to an aneuploid fetus has a significant probability, to give a second set of measurements;

making a second relative probability determination for ploidy hypotheses for the fetuses using the second set of measurements and optionally also the first set of measurements; and

calling the ploidy states of the fetuses whose second sample was resequenced by selecting the ploidy state corresponding to the hypothesis with the greatest probability as determined by the second relative probability determination.

15. The method of claim 1, wherein building a joint distribution model is done by using data about the probability of chromosomes crossing over at different crossover locations in a chromosome to model dependence between polymorphic alleles on the chromosome.

16. The method of claim 1, wherein building a joint distribution model and determining the relative probability of each hypothesis are done using a method that does not require the use of a reference chromosome.

17. The method of claim 1, wherein determining the relative probability of each hypothesis makes use of an estimated fraction of fetal DNA in the measured sample.

18. The method of claim 1, wherein the DNA measurements from the second sample used in calculating allele ratios and determining the relative probability of each hypothesis comprise primary genetic data.

19. The method of claim 1, wherein selecting the ploidy state corresponding to the hypothesis with the greatest probability is carried out using maximum likelihood estimates.

20. The method of claim 1, wherein calling the ploidy state of the fetus further comprises:

combining the relative probabilities of each of the ploidy hypotheses determined using the joint distribution model and the allele ratios with relative probabilities of each of the ploidy hypotheses that are calculated using statistical techniques taken from a group consisting of a read count analysis, comparing heterozygosity rates, a statistic that is only available when parental genetic information is used, the probability of normalized genotype signals for certain parent contexts, a statistic that is calculated using an estimated fetal fraction of the first or second mixture, and combinations thereof.

21. The method of claim 1, wherein a confidence estimate is calculated for the called ploidy state.

22. The method of claim 1 further comprising:

producing a report stating the called ploidy state of the fetus.

23. The method of claim 1 further comprising:

taking a clinical action based on the called ploidy state of the fetus, wherein the clinical action is selected from one of terminating the pregnancy or maintaining the pregnancy.

24. The method of claim 1, wherein the method can be performed at between 4 and 5 weeks gestation;

between 5 and 6 weeks gestation;

between 6 and 7 weeks gestation;

between 7 and 8 weeks gestation;

between 8 and 9 weeks gestation;

between 9 and 10 weeks gestation;

between 10 and 12 weeks gestation;

between 12 and 14 weeks gestation;

between 14 and 20 weeks gestation;

between 20 and 40 weeks gestation;

in the first trimester;

in the second trimester;

or in the third trimester.

8. The method of claim 8, wherein the set of hybrid capture probes are designed to hybridize to a region that is flanking but not crossing the polymorphic allele.

9. The method of claim 8, wherein the set of hybrid capture probes are designed to hybridize to a region that is flanking but not crossing the polymorphic allele, and wherein the length of the flanking capture probe may be selected from the group consisting of as low as about 120 bases, as low as about 110 bases, as low as about 100 bases, as low as about 90 bases, as low as about 80 bases, as low as about 70 bases, as low as about 60 bases, as low as about 50 bases, as low as about 40 bases, as low as about 30 bases, and as low as about 25 bases.

25. A composition comprising:

a sample of preferentially enriched DNA, wherein the sample of preferentially enriched DNA has been preferentially enriched at a plurality of polymorphic loci from a first sample of DNA, wherein the degree of enrichment is selected from the group consisting of at least 10, at least 100, at least 1,000, at least 10,000, at least 100,000, and at least 1,000,000, and wherein the allelic bias between the first sample and the preferentially enriched sample is, on average, selected from the group consisting of less than 1000%, less than 500%, less than 200%, less than 100%, less than 50%, less than 20%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.02%, and less than 0.01%.

26. A method to create a sample of preferentially enriched DNA as claimed in claim 25.

27. A method of determining a fetal aneuploidy by determining the number of copies of maternal and fetal target chromosomes, having target sequences in a mixture of maternal and fetal genetic material, comprising the steps of:

a) obtaining maternal tissue comprising both maternal and fetal genetic material;

b) obtaining a mixture of maternal and fetal genetic material from the maternal tissue;

c) distributing the mixture of maternal and fetal genetic material into a plurality of reaction samples, to randomly provide individual reaction samples that contain a target sequence from a target chromosome and individual reaction samples that do not contain a target sequence from a target chromosome;

d) analyzing the target sequences of genetic material present or absent in the individual reaction samples to provide a first number of binary results representing presence or absence of a presumably euploid fetal chromosome in the reaction samples and a second number of binary results representing presence or absence of a possibly aneuploid fetal chromosome in the reaction samples;

e) calculating an expected distribution of a number of binary results for a presumably euploid fetal chromosome in the reaction samples using the first number;

f) calculating an expected distribution of a number of binary results for a presumably aneuploid fetal chromosome in the reaction samples using the first number and an estimated fraction of fetal DNA found in the mixture of maternal and fetal genetic material; and

g) using a maximum likelihood approach to determine whether the second number indicates the presence of a fetal aneuploidy.