WO2010045617A2 - Détection d'anomalies génétiques - Google Patents

Détection d'anomalies génétiques Download PDF

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WO2010045617A2
WO2010045617A2 PCT/US2009/061096 US2009061096W WO2010045617A2 WO 2010045617 A2 WO2010045617 A2 WO 2010045617A2 US 2009061096 W US2009061096 W US 2009061096W WO 2010045617 A2 WO2010045617 A2 WO 2010045617A2
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alleles
allele
maternal
molecules
paternally
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WO2010045617A9 (fr
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Aoy Tomita Mitchell
Michael Mitchell
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University Of Louisville Research Foundation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention provides methods and compositions for detecting genetic abnormalities
  • the present invention provides method for determining whether a fetus has at least one chromosomal abnormality This method includes the step of comparing at least three alleles, and this comparing identifies the at least one chromosomal abnormality
  • the present invention provides a method for determining whether a fetus has at least one chromosomal abnormality
  • This method includes the steps of ( ⁇ ) detecting a paternally- inherited fetal allele in a sample that includes both maternal and fetal nucleic acids, wherein that paternally-inherited fetal allele is not present in the maternal genome, ( ⁇ ) detecting a first maternal allele and a second maternal allele in the sample, and (in) comparing the paternally-inherited fetal allele to the first and second maternal alleles, where the comparing identifies the at least one chromosomal abnormality
  • FIG. 1 is a schematic illustration of an embodiment of an assay of the invention.
  • FIG. 2 is a schematic illustration of possible haplotypes in an exemplary embodiment of a tandem SNP.
  • FIG. 3 is a schematic illustration of an embodiment of an assay of the invention.
  • FIG. 4 is a schematic illustration of an embodiment of an assay of the invention.
  • FIG. 5A-B is a table of an exemplary list of tandem SNPs of the invention.
  • FIG. 6A-AO is a table of an exemplary list of tandem SNPs of the invention and primers directed to those tandem SNPs.
  • FIG. 7 provides a DNA melting map of a constant denaturant capillary electrophoresis target sequence covering a tandem SNP.
  • FIG. 8A and B provides data of a haplotype ratio analysis.
  • FIG. 9A-C is a table of an exemplary list of tandem SNPs of the invention.
  • the practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art.
  • Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the example herein below. However, other equivalent conventional procedures can, of course, also be used.
  • Such conventional techniques and descriptions can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (VoIs.
  • the present invention is directed to methods and compositions for detecting genetic abnormalities
  • genetic abnormalities can include without limitation chromosomal abnormalities, single point mutations, and any other variations that can result in changes in the levels of cDNA, RNA, mRNA, microRNA, and coding and non-coding RNA
  • genetic abnormalities can include without limitation chromosomal abnormalities, single point mutations, and any other variations that can result in changes in the levels of cDNA, RNA, mRNA, microRNA, and coding and non-coding RNA
  • nucleic acid abnormality “Chromosomal abnormalities” can include without limitation aneuploidy (including without limitation trisomy 13 (Patau Syndrome), trisomy 18 (Edward Syndrome) and sex chromosome aneuploidies such as XXY (Klinefelter's Syndrome), subchromosomal abnormalities, gross deletions, gross insertions, large deletions, large insertions, copy number variants, copy number variation, repeat variations, structural alterations, small deletion
  • methods and compositions of the invention are used to analyze a sample containing fetal and maternal genetic material
  • methods and compositions of the invention are used to compare alleles contained in such a sample to determine if the fetal genetic material comprises a genetic abnormality
  • allele is a variant form of a sequence at a particular region on a chromosome
  • the variants in the sequence can come as a result of single nucleotide polymorphisms ("SNPs"), combinations of SNPs 1 haplotype methylation patterns, insertions, deletions, and the like
  • An allele may comprise the variant form of a single nucleotide, a variant form of a contiguous sequence of nucleotides from a region of interest on a chromosome, or a variant form of multiple single nucleotides (not necessarily all contiguous) from a region of interest on a chromosome [0026]
  • the present invention provides methods and compositions for comparing alleles in a sample
  • sample in accordance with the present invention may comprise any number of substances, including, but not limited to, bodily fluids (including, but not limited to, blood, urine, serum, lymph, saliva, anal and vaginal secretions, perspiration and semen, of virtually any organism, with mammalian samples being preferred and human samples being particularly preferred), environmental samples (including, but not limited to, air, agricultural, water and soil samples), biological warfare agent samples, research samples ( ⁇ e in the case of nucleic acids, the sample may be the products of an amplification reaction, including both target and signal amplification as is generally described in PCT/US99/01705, such as PCR amplification reaction), purified samples, such as purified genomic DNA, RNA, proteins, etc , raw samples (bacteria, virus, genomic DNA, etc ), as will be appreciated by those in the art, virtually any experimental manipulation may have been conducted on the sample [0030]
  • samples of use in the present invention are obtained from a pregnant female Such samples can include without limitation
  • the present invention provides methods and compositions for comparing alleles in a sample Comparing alleles in accordance with the invention includes detecting the identity of the different alleles present in the sample Comparing alleles also includes detecting and/or quantifying the number of molecules of each allele of interest that is present in a sample
  • a maternal sample containing both fetal and maternal DNA may contain three different alleles for a particular chromosomal region of interest As discussed above, these three alleles would be variant sequences of the chromosomal region of interest Methods known in the art and described further herein can be used to determine how many molecules of each of the three different alleles are present in the sample
  • comparing the three alleles would include comparing the number of molecules of each of the three alleles Comparing alleles in further embodiments of the invention can include without limitation evaluating the concentration of alleles in a sample and evaluating relative concentrations and/or relative numbers of molecules of alleles in a sample
  • evaluating relative concentrations or numbers of molecules in a sample includes calculating an "allelic ratio" among the alleles in a sample
  • This allelic ratio is the ratio of the amount of molecules of each of the alleles of interest present in the sample, and may be calculated in any number of ways known in the art and described further herein
  • a sample comprises two alleles A and B
  • a standard method in the art such as sequencing
  • the allelic ratio may be the characterization of A B as 1 2
  • the allelic ratio may be a standard ratio - for example, for three alleles A, B, and C
  • the allelic ratio may be expressed as the number of molecules of A B C
  • a more complex relationship may be described as an allelic ratio, such as (A-B)/C
  • an allelic ratio such as (A-B)/C
  • Methods for detecting and quantifying alleles or any other sequence of interest include without limitation any methods that detect DNA (including without limitation genomic and cDNA) and RNA (including without limitation mRNA, microRNA, and silent RNA)
  • methods for detecting nucleic acids can include without limitation sequencing methods, gel electrophoresis, mass spectrometry, detection of methylation patterns, PCR methods, high performance liquid chromatography (HPLC) and the like
  • Methods for detecting and quantifying alleles in accordance with the present invention provide the sequence of the alleles present in the sample (or identify the presence of an allele of interest) and may also provide the number of molecules of each of the alleles in the sample
  • Methods for detecting and/or quantifying alleles that are of use in the present invention also include methods that quantify the relative amounts of two or more alleles of interest in a sample
  • Sequencing methods of use for detecting and quantifying alleles of interest include without limitation single molecule sequencing, sequencing by synthesis, sequencing using arrays (hybridization and/or ligation), capillary sequencers, Sanger sequencing, constant denaturant capillary electrophoresis (CDCE), cycling temperature capillary electrophoresis (CTCE), polony sequencing, pyrosequencing, shot-gun sequencing, and the like
  • Commercial high throughput sequencing platforms for detecting and quantifying alleles of interest and such platforms are known in the art and can include without limitation lllumina's GA, Life Technologies' SOLD, Roche's 454, Pacific Biosciences single molecule sequencing platform, Oxford Nanopore, Ion Torrent, Complete Genomics, Nimblegen, Helicos Biosciences, Lingvitae, Nabsys, and Visigen Biotechnologies
  • PCR methods of use for detecting and quantifying alleles of interest include without limitation single molecule sequencing, sequencing by synthesis, sequencing using arrays (hybridization and/or
  • methods of the invention include detecting alleles of a specific chromosomal region
  • chromosomal region refers to all or part of a chromosome Detection of such alleles can be conducted using any method known in the art, including the sequencing and PCR methods described above
  • the sample being analyzed is from a pregnant female and contains both maternal and fetal DNA Detecting and/or quantifying the number of different alleles in the sample and the number of molecules of those alleles that are present in the sample can identify the presence of a chromosomal abnormality in a fetus
  • a sample contains three alleles of a chromosomal region that is being analyzed Two of the alleles are from a known source, while the third allele is expected to come from a separate source Quantification of the number of molecules present in the sample of each allele allows comparison between the alleles from the known source and the third allele [0040]
  • the sample is a maternal sample containing both fetal and maternal nucleic acids
  • the sample again contains three alleles of a particular chromosomal region If the maternal genome is heterozygous for this allele, then two of the three alleles detected in the sample are from the maternal germline DNA, while the third allele is expected from the paternal DNA contribution (if the paternal DNA contains an allele not present in the maternal genome) Quantification of the three alleles therefore allows comparisons between the two maternal alleles from the mother, allowing the determination of which allele and how many molecules were contributed by the mother to the
  • FIG 1 is a schematic representation of the comparisons that can be made between different alleles detected in a sample
  • a maternal sample in which three alleles are detected is shown
  • the peaks illustrated in FIG 1 represent the number of molecules of each allele present in the sample
  • these "peaks" can be determined using methods well known in the art and described herein
  • the area under the peaks from an electropherogram from a CDCE analysis of a sample provides information on the number of molecules of each detected allele
  • the output of a sequencing platform will provide a count of the number of molecules for each allele, which can also be depicted by the peaks in FIG 1
  • genetic abnormalities are detected by comparing the number of molecules of alleles in a sample
  • a sample comprising both fetal and maternal nucleic acids will be expected to show specific relationships between the number of molecules for alleles of a particular chromosome
  • the number of molecules detected for those two alleles will include the numbers of molecules from the maternal DNA and the numbers of molecules for the maternally-inherited allele in the fetal DNA (also referred to herein as the "maternally-inherited fetal allele”).
  • the fetal genome will contain maternally and paternally-inherited alleles If the paternally-inherited allele in the fetal DNA (also referred to herein as the "paternally-inherited fetal allele”)
  • the present invention provides methods for detecting genetic abnormalities, including chromosomal abnormalities, in a fetus by detecting alleles for a location of interest on a chromosome (also referred to herein as a "genetic location"), where the maternal genome is heterozygous at that location of interest and the fetus inherits a different allele from the father at that same genetic location
  • methods of the invention detect a paternally- inherited fetal allele in a sample comprising both maternal and fetal nucleic acids, where that paternally- inherited fetal allele is not present in the maternal genome Detection of such a paternally-inherited fetal allele will in such embodiments indicate that the fetus does have a genetic abnormality
  • the term "maternal allele" may refer to the allele inherited by the fetus from the mother and/or the alleles in the maternal
  • the fetus inherits two copies of the chromosome from the mother
  • three alleles of that chromosome or a particular region of that chromosome are detected in the sample (represented by the three peaks in FIG 1 )
  • two of the alleles detected are the maternal alleles
  • the number of molecules detected for those two alleles will reflect the molecules from the maternal DNA in the sample plus the molecules of those same two alleles from the fetal DNA ( ⁇ e , the maternal alleles plus the maternally-inherited fetal alleles)
  • the third allele detected will be the fetal allele that is paternally inherited and is not present in the maternal genome
  • trisomy is detected if the number of molecules for the two maternal alleles is equal (first trace of FIG 1 B) or if all three alleles are present in different numbers in the relationship of peak, x and peak + 2x, where
  • analysis of the number of molecules of alleles in a sample provides a measure of "allelic dosage”
  • allelic dosage as used herein is meant the number or concentration (or relative number or concentration) of molecules of each allele that is present in a sample
  • calculation of an "allelic ratio" as discussed above provides a mechanism to measure the dosage of each allele
  • determining the number of molecules of paternally-inherited fetal alleles also provides a measure of the paternally-inherited fetal allelic dosage
  • This paternally-inherited fetal allelic dosage can serve as an internal standard that can be used to determine the maternally-inherited fetal allelic dosage
  • a calculation of allelic dosage provides a measure of the allelic dosage from each parent to the fetus Calculations of allelic dosage can be used to detect a genetic abnormality using any of the methods described herein, because the relationships expected for maternal and paternal (and maternally-
  • the deletion is a de novo event in 75% of the cases. If the de novo event is a maternally-derived deletion in the fetal genome, three alleles would again be detectable in a sample comprising both maternal and fetal DNA. However, the number of molecules of the three alleles will show a relationship in which two of the alleles are present in equal numbers and the third allele will be present in smaller numbers. In other words, two fetal alleles will not be detected in the sample, because one of those alleles would be deleted.
  • multiple assays directed to the detection of different alleles can be conducted to confirm that the lack of a third fetal allele is the result of a deletion rather than the result of a non-informative assay (i.e., an assay in which the maternal genome is not heterozygous for an allele or the paternal genome has an allele in common with the maternal genome).
  • methods of the present invention can be used to detect alleles that are inherited.
  • the maternal and paternal genomes are normal at the deletion/insertion site, but during meiosis, deletions or insertions occur and are passed to the fetus.
  • deletions and insertions and point mutations which would include basepair substitutions
  • small (less than 20 base pairs) deletions or insertions also referred to herein as "small-deletions” and "small-insertions” respectively
  • small-deletions also referred to herein as "small-deletions” and "small-insertions” respectively
  • the same comparisons described above for allelic comparisons can be used to determine allelic dosage inheritance if a neighboring SNP can be encompassed by the target sequence.
  • the delta F508 deletion in the CFTR gene is a small deletion that can be passed on to the fetus by either the mother or the father. In the case where the mother is a carrier of the delta F508 deletion, it will appear as if she has two peaks. If the father contributes a normal allele and the fetus inherits the delta F508 deletion, the maternal plasma would show two peaks of equal area and height. It would not be clear if there was no fetal DNA present in the sample, or was at levels too low to be detected.
  • peaks refers to the number of molecules of an allele and to a signal associated with the number of molecules of an allele.
  • the present invention provides methods and compositions for comparing alleles, where those alleles may be comprised of haplotypes.
  • haplotypes refers to groups or sets of markers (including without limitation SNPs, deletions, small deletions, insertions, small insertions, methylation, and short tandem repeats) that are inherited together as a unit.
  • markers including without limitation SNPs, deletions, small deletions, insertions, small insertions, methylation, and short tandem repeats
  • alleles detected and quantified as described herein encompass multiple markers, and thus different alleles comprise different combinations of these multiple markers.
  • alleles of use in the present invention may comprise multiple SNPs. These SNPs may be contiguous or non-contiguous.
  • alleles for comparison may include any combination of sites, including deletion sites, insertion sites, and sites which comprise variant sequences of two or more nucleotides in length.
  • alleles detected and compared in accordance with the present invention may include a deletion site and/or an insertion site and/or one or more SNPs and/or a variant sequence comprising two or more nucleotides.
  • multiple alleles are detected in a single reaction or assay.
  • detecting multiple alleles e.g., detecting both maternal alleles and a paternally-inherited fetal allele
  • the nucleic acids in a sample are in many embodiments amplified using methods known in the art. Such amplification methods include polymerase chain reaction (PCR), strand displacement amplification (SDA), multiple displacement amplification (MDA), rolling circle amplification (RCA), rolling circle amplification (RCR) and other amplification (including whole genome amplification) methodologies.
  • PCR polymerase chain reaction
  • SDA strand displacement amplification
  • MDA multiple displacement amplification
  • RCA rolling circle amplification
  • RCR rolling circle amplification
  • the chromosomes are amplified using primers directed to desired regions.
  • This amplification enriches the sample for the allelic sequences for those specific chromosomal regions, and then methods known in the art for detecting those sequences can be used to detect and quantify the different alleles present in the sample.
  • all nucleic acids in the sample are amplified, and then alleles of interest are detected using methods known in the art and described herein.
  • alleles of interest are amplified using primers with sequences such as those listed in FIGs 5 and 6 and then detected using methods well known in the art and described herein.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, made of monomers (nucleotides) containing a sugar, phosphate and a base that is either a purine or pyrimidme. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid sequence also encompasses conservatively modified variants thereof ⁇ e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues.
  • nucleotide sequence refers to a polymer of DNA or RNA which can be single- stranded or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.
  • nucleic acid refers to a polymer of DNA or RNA which can be single- stranded or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.
  • nucleic acid nucleic acid molecule
  • polynucleotide are used interchangeably herein.
  • High-Fidelity (Hi-Fi) PCR is used to amplify alleles of interest in a sample.
  • High-Fidelity PCR is an amplification method resulting in an error rate (in per basepair doubling) equal to or better than standard PCR.
  • Taq polymerase which is not a high fidelity polymerase, has an error rate of - 10 "4 per basepair doubling.
  • Pyrococcus furiosus (Pfu) is a high-fidelity polymerase, with a published error rate for Pfu is 1.3 x 10 "6 per basepair doubling (Cline et al, Nucleic Acids Res. 1996 September 15; 24(18): 3546-3551 ).
  • high-fidelity enzymes include Pfu and its derivations, or other enzymes with similar proofreading 3'->5' exonucleases. Mixed blends and fusions with enzymes with proof-reading capabilities can increase the fidelity of a polymerase. Use of such a high fidelity polymerase ensures that the alleles of interest are amplified efficiently and with minimal to no introduction of errors.
  • Methods for improving PCR fidelity and efficiency include, among others: A) using a high-fidelity polymerase enzyme; and B) the addition of chemical reagents (e.g., betaine) that can lower temperatures required during the PCR process.
  • Lowering temperatures required during the PCR products can increase efficiency and prevent damage to the amplification products, because prolonged heating of DNA and nucleotides during PCR can lead to damaged products, such as deaminated cytosines (uracils) and thus lead to misincorporation errors and miscopying errors during PCR (Andre, Kim, Khrapko, ThMIy. Genome Res. 1997 7- 843-852. Zheng, Khrapko, Coller, Thilly, Copeland. Mutat Res. 2006 JuI 25;599(1 - 2):11 -20).
  • chemical reagents e.g., betaine
  • amplification using HiFi-PCR is performed with primers present in molar excess (e.g., 10 12 copies/ ⁇ l of primer vs 10 6 or less of the template) so that it is more likely that primers will anneal with template DNA than with each other (see, e.g., Li-Sucholeiki XC, Thilly WG. Nucleic Acids Res. 2000 May 1 ;28(9):E44; Thompson JR, Marcelino L, PoIz M. Nucleic Acids Res. 2002 May 1 ; 30(9): 2083-2088.). This can significantly reduce the creation of heteroduplexes.
  • molar excess e.g. 10 12 copies/ ⁇ l of primer vs 10 6 or less of the template
  • comparisons of alleles as described above utilize alleles of tandem single nucleotide polymorphisms (referred to herein as “tandem SNPs”).
  • a “single nucleotide polymorphism (SNP)” is a single basepair variation in a nucleic acid sequence.
  • a “tandem SNP” is a variation in more than one nucleotide in a nucleic acid sequence, e.g. on a chromosome.
  • a tandem SNP is a pair of SNPs that are located in the nucleic acid sequence.
  • a tandem SNP comprises more than two SNPs.
  • tandem SNP any number of SNPs can be contained in a tandem SNP, limited only by the total number of nucleotides present in the nucleic acid.
  • a tandem SNP comprises from 2 to about 20 SNPs.
  • a tandem SNP comprises from about 3 to about 19, about 4 to about 18, about 5 to about 17, about 6 to about 16, about 7 to about 15, about 8 to about 14, about 9 to about 13, and about 10 to about 12 SNPs.
  • a tandem SNP comprises between two SNPs and the maximum number of SNPS that can be assayed in a single reaction, which may depend on the detection platform (e.g., the read length of the sequencing technology).
  • a tandem SNP comprises between two SNPs and the maximum number of SNPs contained within the segment of genetic material to be analyzed.
  • fetal DNA circulating in maternal serum is often fragmented and has a length of 200 - 400 bases - in such an exemplary embodiment, the number of SNPs contained in a tandem SNP would be limited by the maximum number of SNPs contained in that fragmented DNA.
  • the two or more single SNPs in a tandem SNP may be of any distance apart.
  • the multiple SNPs may be of equal distance apart, or there may be varying distances between them.
  • the distance between SNPs of a tandem SNP is generally about 350 basepairs or fewer.
  • the SNPs of tandem SNPs are about 5 to about 300, about 10 to about 250, about 15 to about 200, about 20 to about 150, about 25 to about 140, about 30 to about 130, about 35 to about 120, about 40 to about 110, about 45 to about 100, about 50 to about 90, about 55 to about 80, and about 60 to about 70 base pairs apart.
  • Increasing the number of SNPs that make up tandem SNPs of the invention increases the potential number of haplotypes, and thereby increases the likelihood that an assay detecting alleles will be informative. For example, if a tandem SNP contains 3 SNP sites, the potential number of haplotypes is 8 and if it contains 4 SNP sites, the potential number of haplotypes is 16. This increases the likelihood that an assay will be informative, because the mother is more likely to be heterozygous at such a tandem SNP, and the paternal contribution to the fetus (also referred to herein as the "paternally-inherited fetal allele") is likely to be different from that present in the mother's genome.
  • Tandem SNPs provide a particularly powerful tool for comparing alleles in a sample, because tandem SNPs allow the detection and quantification of haplotypes.
  • haplotypes in methods of the present invention provide an internal standard that eliminates the need for using reference markers across different chromosomes, thereby increasing specificity and/or minimizing or eliminating false positives.
  • haplotypes are groups or sets of markers (including without limitation SNPs, deletions, small deletions, insertions, small insertions, methylation, and short tandem repeats) that are inherited together as a unit. Different alleles of a tandem SNP are thus haplotypes, and haplotypes can be assigned to a chromosome ⁇ i.e., the maternally inherited or paternally inherited chromosome). Comparing alleles of a tandem SNP comprises far more information than is possible from comparing individual SNPs 1 because comparing alleles of a tandem SNP provides a comparison of haplotypes.
  • An exemplary embodiment of a tandem SNP is schematically illustrated in FIG.
  • the tandem SNP comprises a pair of SNPs.
  • SNP 1 two alleles are possible: G or T.
  • SNP 2 two alleles are possible: A or G
  • these alleles are defined by the four possible haplotypes: G — A, T — A, G — G, or T — G.
  • an individual may be heterozygous or homozygous for a tandem SNP, as is the case for a single SNP, but being heterozygous for a tandem SNP means that multiple different haplotypes are possible alleles for that tandem SNP.
  • tandem SNPs containing three or more SNPs the resultant number of possible haplotypes will also increase.
  • genetic abnormalities can be detected in a fetus by comparing the number of molecules of alleles between the fetal and maternal nucleic acids in a sample.
  • tandem SNPs the comparison can be used to both compare and quantify the differences between maternal and fetal DNA.
  • detecting the alleles of tandem SNPs in a sample containing both maternal and fetal DNA can be used to calculate a haplotype ratio.
  • these allele comparison assays are conducted for chromosomal regions of interest at which the maternal genome is heterozygous.
  • the maternal genome being heterozygous means that it has two different haplotypes for that particular tandem SNP. Detecting the number of molecules of alleles of the tandem SNP present in a sample is thus a detection of the number of molecules of the different haplotypes are present in the sample.
  • haplotype ratio (HR)
  • P1 and P2 represent the number of molecules of each of the maternal haplotypes
  • P3 represents the number of molecules of a third haplotype that is not present in the maternal genome.
  • P3 thus represents the number of molecules of the haplotype inherited by the fetus from the father.
  • the calculation of the haplotype ratio provides a way to determine if the fetus has a chromosomal abnormality, including without limitation trisomy, monosomy, partial duplication, partial deletion, microduplication, microdeletion, and the like.
  • P1 , P2 and P3 of Formula 1 represent the maternal alleles - i.e., “peak + x” (or “peak + 2x” in cases of trisomy) and "peak”, whereas P3 of Formula 1 is equivalent to the paternally- inherited fetal allele, i.e., "x".
  • the haplotype ratio calculation will produce the discrete results of 1 , 2, 0 or 0.5. These numbers are interpreted as follows:
  • paternal non-disjunction in which the fetus inherits two alleles from the father, may result in the haplotype ratios described in Table 1.
  • the haplotype ratio can be calculated according to the following formula:
  • P1 and P2 represent the number of molecules of each of the maternal haplotypes and P3 and P4 represent the number of molecules of a third and fourth haplotype that is not present in the maternal genome.
  • Formula Il is applicable to embodiments of paternal nondisjunction trisomy, in which two paternally-inherited fetal alleles are present in the sample.
  • Formula Il can be applied to calculate the haplotype ratio for any sample from a pregnant female carrying a single fetus.
  • calculation of the haplotype ratio provides discrete detection and quantification of fetal chromosomal/genetic abnormality/allelic dosage within a single measurement from a sample comprising both maternal and fetal DNA.
  • the present invention does not require measurements of alleles across different chromosomes in order to determine the amount of fetal DNA present in the sample in relation to the maternal DNA.
  • the present invention has an internal standard for each measurement.
  • detection of three alleles in a sample containing both maternal and fetal DNA means that one of the detected alleles is the paternally-inherited fetal allele. Since the molecules of the paternally- inherited fetal allele can only be from the fetal DNA, detection of the number of molecules of that paternally-inherited fetal allele provides a measurement of how much fetal DNA is present in the sample. This third allele serves as an internal standard of the amount of fetal DNA, and there is no need to compare the measurement of the number of molecules of fetal alleles against the number of molecules of fetal alleles present for another chromosome that is not expected to have an abnormality.
  • a single measurement of the number of molecules of an allele of a particular chromosome i.e., a chromosome expected to be subject to an abnormality
  • a chromosome expected to be subject to an abnormality
  • a further advantage of the methods of the present invention is that the ratios of the alleles and the calculation of the haplotype ratio will not vary based on the concentration in the sample of fetal genetic material.
  • Traditional methods for detecting chromosomal abnormalities must normalize calculations of ratios with respect to the amount of fetal genetic material present in the sample being analyzed
  • the methods of the present invention do not require such normalization, because comparing the number of molecules of the paternally-inherited fetal allele to the number of molecules of the maternal alleles in accordance with the present invention allows a calculation of allelic ratios that is independent of the amount of the total amount of fetal genetic material present in the sample.
  • the above methods for calculating haplotype ratios can be conducted using a single tandem SNP or using multiple tandem SNPs (also referred to herein as "panels" of tandem SNPs).
  • multiple tandem SNPs are used in any methods in accordance with the present invention, they can be utilized one at a time, in specific groupings, or all possible tandem SNPs can be analyzed at the same time in a multiplex assay.
  • panels of tandem SNPs comprise between 2 and 200 tandem SNPs.
  • each of these tandem SNPs define a haplotype and therefore may in turn comprise two or more SNPs.
  • tandem SNPs comprising between about 2-150, 10-140, 15-130, 20-120, 25-110, 30-100, 35-90, 40-80, 45-70, and 50-60 tandem SNPs are used in accordance with the present invention.
  • Multiple tandem SNPs may be assayed individually for a sample or multiplexed into a single assay.
  • the number of SNPs applied in a single assay may depend on the amount of nucleic acids that are present in and/or can be obtained from a sample.
  • all or a selected portion of the nucleic acids in a sample are amplified, which will allow multiple tandem SNPs directed to one or more chromosomes to be applied in a single multiplexed assay.
  • multiple tandem SNPs either sequentially or simultaneously, can be used in some embodiments to assay for abnormalities in multiple chromosomes.
  • the number of chromosomes that can be assayed using methods of the invention is limited only by the number of chromosomes present in the organism from which the sample is obtained.
  • a panel of tandem SNPs is assayed in a sample, but only a subset of the panel (anywhere from between 1 and up to the total number of tandem SNPs in the panel) is informative, meaning that not every tandem SNP in a panel will produce a "positive" result.
  • a positive result in assays utilizing tandem SNPs is generally the detection of at least three alleles for a particular tandem SNP For any particular sample, even a single positive result from a tandem SNP can allow detection of a genetic abnormality. Results from more than one tandem SNP can be used as further internal confirmation of an assay.
  • the analysis of the paternally inherited fetal allele is of use in detecting non-chromosomal mutations.
  • a point mutation that is present in the mother or father's genome A * in FIG. 4
  • this can be combined with a nearby SNP site to create at least three potential haplotypes.
  • the inheritance pattern of the point mutation(s) to the fetus can therefore be traced in accordance with the methods described herein.
  • tandem SNPs allows detection of a recessive mutation that would not be detectable in methods utilizing single SNPs.
  • detecting alleles for a single SNP in a sample comprising both maternal and fetal DNA does not allow one to determine whether the fetus has inherited the point mutation.
  • using the haplotype analysis possible by detecting alleles of tandem SNPs FIG. 4, right panel, it is possible to detect the third allele in the sample, which is the paternally-inherited fetal allele. If the paternally-inherited fetal haplotype comprises the recessive point mutation, detection of that third allele is informative.
  • tandem SNPs may comprise multiple single SNPs.
  • the single site would be tri-allelic and therefore it may not be necessary to combine the point mutation site with a nearby SNP. Then a simple comparison of detected alleles as described in further detail above would allow detection of the mutant allele.
  • STRs short tandem repeats
  • tandem SNPs do not comprise STRs.
  • the information gathered about the alleles of interest in a sample comprising both maternal and fetal DNA is compared to similar information gathered about the alleles of interest in a sample comprising maternal DNA but no fetal DNA.
  • the sample containing only the maternal DNA can be used as a reference for the sample containing both maternal and fetal DNA. Although such a reference sample is not generally necessary, it can provide additional substantiation of the results from the assays discussed herein.
  • the sample comprising maternal DNA but no fetal DNA is obtained using methods known in the art. Such samples can be obtained from a number of sources, including without limitation: maternal buccal swabs, maternal cells, including maternal white blood cells, and the like.
  • tandem SNPs for detection of genetic abnormalities. This is because one requirement of the methods described herein, particularly methods involving tandem SNPs, is that the maternal genome must be heterozygous for the allele of interest in order to allow the detection of a third, paternally-inherited allele. In addition, tandem SNPs for which the paternal genome comprises the same haplotypes as the maternal genome will not be informative, because it is the detection of three alleles for a particular tandem SNP that provides the internal standard of the paternally-inherited fetal allele.
  • the present invention provides methods and compositions for identifying tandem SNPs.
  • a chromosomal region of interest is studied to identify potential markers (i.e., alleles) for use as tandem SNPs.
  • Studying a chromosomal region of interest for identifying tandem SNPs in some embodiments involves analyzing a database comprising information on the occurrence of SNPs in one or more populations (such as the database from the International HapMap Project).
  • studying a chromosomal region of interest to identify tandem SNPs comprises collecting samples of nucleic acids for a number of individuals and amplifying the region of interest. The amplification product can then be sequenced or otherwise analyzed to identify potential markers for use as tandem SNPs.
  • a tandem SNP is chosen by identifying two or more neighboring SNPs. Such SNPs may be separated by any range of distances, and as will be appreciated, the distances between neighboring SNPs is limited only by the size of the chromosomal region of interest. In some embodiments, the two or more SNPs in a tandem SNP may be separated by a distance of about 5 to about 400 base pairs.
  • the two or more SNPs in a tandem SNP may be separated by a distance of about 10 to about 350, about 20 to about 300, about 30 to about 250, about 40 to about 200, about 50 to about 150, about 50 to about 140, about 60 to about 130, about 70 to about 120, about 80 to about 110, and about 80 to about 100 base pairs.
  • each individual SNP may be of equal distance from the other SNPs, or the SNPs may be at varying distances from each other.
  • tandem SNPs may also include any combination of SNPs, deletions, insertions, and sequence variants of two nucleotides or greater, and descriptions of tandem SNPs herein applies to all such combinations.
  • neighbored SNPs as described herein may have one or more SNPs (or deletions, insertions, sequence variants) that occur in the region between the SNPs that are part of a particular tandem SNP.
  • tandem SNP may be chosen such that A and C are the "neighboring" SNPs, and B is a SNP located in the intervening region between A and C.
  • tandem SNPs of the invention are selected such that the combination of SNPs (and/or deletions, insertions and sequence variants) results in more than two haplotypes being present in a population.
  • tandem SNPs are chosen that result in more than two haplotypes present in populations across different ethnic groups - as will be appreciated, such information can be generated from publicly available databases (such as the HapMap database) or from direct investigation of samples collected from multiple individuals.
  • tandem SNPs of the invention are chosen such that their component SNPs, deletions, insertions and sequence variants do not lie within a common, non-disease related CNV (copy number variation) region, such as those present in the Database of Genomic Variants from The Centre for Applied Genomics at http://projects.tcag.ca/variation/.)
  • CNV copy number variation
  • methods for identifying tandem SNPs utilize maternal buccal samples (or any other samples that contain maternal DNA but no fetal DNA) in order to identify regions at which the maternal genome is heterozygous.
  • tandem SNPs are identified from population data files (such as the HapMap database).
  • highly heterozygous (i.e., greater than 10% or more or "common") SNPs are identified.
  • likely candidates are chosen from SNPs that are highly heterozygous in a subset of the populations contained in the database. Tandem SNPs can then be provisionally selected by choosing highly heterozygous SNPs that are at a desired distance from each other, as is discussed above.
  • likely tandem SNPs may comprise a wide range of individual SNPs.
  • any likely tandem SNPs that occur in the list of SNPs from common CNV regions are removed from further consideration.
  • tandem SNPs that lie in regions with long stretches of homopolymeric sequences are also removed from further consideration.
  • primers are designed to amplify regions containing likely tandem SNPs. In some embodiments, these primers are optimized for efficiency and specificity using methods known in the art. In further embodiments, primers are tested to determine if they preferentially amplify one or more alleles of the likely tandem SNPs over others.
  • tandem SNPs of use in accordance with the present invention can be chosen using any combination of the above methods and characteristics.
  • tandem SNPs are chosen based solely on distance between each component.
  • a "component" of a tandem SNP as used herein refers to individual SNPs, deletions, insertions, or sequence variations that make up a tandem SNP.
  • a chromosomal region of interest can be directly sequenced from one or more samples to identify potential tandem SNPs.
  • genetic material from a number of individuals e.g., 8 individuals, 10 individuals, 96 individuals, even 20,000 individuals
  • the alleles present in this amplified sample can then be detected and quantified to identify sequences that can serve as tandem SNPs.
  • the genomic DNA from these individuals are pooled together and a target sequence of interest is amplified in a single PCR reaction.
  • tandem SNPs can be identified for any chromosome.
  • Exemplary tandem SNPs identified in accordance with the present invention for chromosome 21 are provided in FIG. 5.
  • FIG. 6 shows tandem SNPs for chromosome 21 as well as primers that can be used for amplification and detection of these tandem SNPs.
  • Exemplary tandem SNPs identified in accordance with the present invention for chromosomes 13, 18 and 22 are provided in FIG. 9.
  • Tandem SNPs identified in accordance with the present invention for any other chromosome are also encompassed by the present invention, as are tandem SNPs other than those in FIGs. 5, 6 and 9.
  • sequences that have at least 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to the sequences provided in FIGs. 5-6 and 9 are also encompassed by the present invention.
  • the present methods and compositions of the present invention can be used to evaluate tissue rejection in a patient who has received a tissue transplant.
  • detection of a third (and possibly fourth) allele frequency may come from the graft at the time of transplant.
  • the levels of allelic dosage at the time of transplant can then be compared to levels at later time points to determine if the graft is being rejected Such comparisons can be conducted by calculating allelic and/or haplotype ratios as discussed herein
  • An advantage of using tandem SNPs in this embodiment, particularly with tandem SNPs comprising more than two SNPs or other components, is that the likelihood of having an informative assay (/ e , one in which at least three alleles can be detected) is increased, and it would be possible to quantitatively measure both the maternally inherited and the paternally inherited alleles from both the recipient and the graft by calculating a Haplotype Ratio Such information can not be obtained from studies in which only individual SNPs are used to compare between the genetic material of the recipient and the graft [0100]
  • the methods and compositions can be used in transfusions, to determine if a donor is a match for the recipient
  • the allelic dosage can be calculated to determine if the transfusion
  • Target sequences covering tandem SNPs were designed using Vector NTI and WmMeIt software
  • the melting map of a CDCE or CTCE target covering two tandem SNPs (dbSNP rs2839416 and rs2839417) on chromosome 21 was calculated using WmMeIt according to the algorithm of Lerman and Silverstein (Lerman et al , Methods Enzymol, 1987 155 p 482-501) and is depicted in FIG 7
  • FIG 7 depicts a DNA melting map of a CDCE or CTCE target sequence covering tandem SNPs All four haplotypes can be theoretically separated according to DNA melting temperature The curves for the four different haplotypes (haplotype 1 (G 1 A), haplotype 2 (T,A), haplotype 3 (G, G), and haplotype 4 (T 1 G)) are identified on the figure
  • HIFI PCR optimization for each target sequence was performed using Pfu polymerase
  • One of primers flanking the target sequence was -20 bases in length and labeled 5' with a fluorescein molecule
  • the other primer was about 74 bases including a ⁇ 20-base target specific sequence and the 54-base clamp sequence
  • a standard HiFi PCR condition was applied to all target sequences, varying only annealing temperatures
  • These PCR amplicons were subjected to CDCE or CTCE electrophoretic separation
  • the resulting electropherograms were analyzed for yield and purity of the PCR products The purity was evaluated by comparing the peak area of the desired products to that of the byproducts and nonspecific amplification Target sequences that could be amplified with a high PCR efficiency ( ⁇ 45% per cycle) and low levels of byproducts and nonspecific amplification ( ⁇ 0 1 % of the desired products) were subjected to CDCE or CTCE optimization For those target sequences that did not have acceptable PCR products in the first stage, increasing amounts
  • the relevant haplotypes were created for the targets using pools of 96 individuals.
  • the optimal separation condition for each haplotype should provide the greatest resolution among the observed peaks
  • Initial optimization is done around the theoretical melting temperature (T m ) in a 2°C temperature range in increments of 0 2°C which covers (T m - 1 0 C ⁇ a predetermined offset) to (T n , + 1 °C ⁇ a predetermined offset)
  • Electropherogram and peak measurements were transferred to a spreadsheet for analysis To ensure the quality of the data, minimum and maximum peak heights were used Individual markers were failed if electrophoretic spikes occur Peak areas were used to calculate allele ratios A check for allelic preferential amplification was performed on all 96 tandem SNPs
  • PCR, and obtaining the greatest resolution among peaks during CDCE or CTCE Haplotypes may be separated as homoduplex peaks. However, if certain targets cannot be separated out as homoduplexes, maternal DNA can be separated from fetal DNA as heteroduplexes.
  • Genomic DNA samples from 300 anonymous subjects were obtained from healthy young adults who were less than 35 years old. The samples were anonymous as the only data obtained were the geographic location of the Red Cross blood donor center, donor gender, and whether or not the donor was 35 and under. These samples were reviewed to ensure that at least three haplotypes were present for a given target sequence of interest. These results were compared to haplotypes identified through analysis of the database from the HapMap project as described in Example 1 , and it was found that the same or similar haplotypes were identified using both methods.
  • Biological samples including a buccal (cheek) swab and a blood sample were collected from a cohort of pregnant women. Maternal buccal swab samples were compared to maternal serum to demonstrate that a third (paternal) peak was observed in several of the tandem SNP assays. Approximately 20 maternal buccal swab to maternal serum comparisons were made. To control for experimental artifacts, genomic DNA samples from maternal buccal swabs were utilized for each target sequence. The buccal samples were subjected to the process in parallel with the maternal blood sample. Any artifacts generated by the CDCE/CTCE/HiFi-PCR procedure (including nonspecific PCR amplification and polymerase-induced mutations) were revealed as background peaks in the buccal swab samples.
  • a blinded study is performed where the goal is to detect 20 known trisomy 21 fetuses by assaying maternal serum from 40 patients (previously determined by amniocentesis or CVS) (see Figure 3).
  • FIG. 1A depicts a schematic illustration of the output of detecting alleles in a sample from maternal buccal swab. Markers exhibiting two alleles were pursued. A baby with trisomy is expected to show either three alleles, evident by three peaks in a 1 :1 :1 ratio or two alleles in a 2:1 ratio.
  • FIG. 1 B depicts a sample from maternal serum. Markers exhibiting three alleles are informative.
  • Maternal serum from a woman carrying a baby with trisomy is expected to exhibit three alleles, evident by two equal peaks with a third smaller peak if the trisomy occurred during meiosis I (75% of T21 cases) or three alleles with different areas if the trisomy occurred during meiosis Il (20% of T21 cases) where areas are: peak, x, and peak + 2x.
  • FIG 1C is a schematic illustration of the analysis of a sample from maternal serum Markers exhibiting three alleles are informative
  • Maternal serum from a woman with a normal baby with three alleles has three different areas where areas are peak, x, and peak + x
  • FIG 8 shows data from a CDCE experiment for two different samples In the top sample, the mother is carrying a non-t ⁇ somy 21 fetus
  • the haplotype ratio calculated from comparing the peaks in the output (which each represent a different allele, and the area under each of which provides the number of molecules for each allele) is within a margin of error to 1 , the haplotype ratio expected for a normal fetus (see FIG 8A)
  • FIG 8B shows the results for a mother carrying a fetus with trisomy 21
  • the haplotype ratio calculated from these data was within a margin of error to 2, which is one of the haplotype ratios expected for a fetus with a chromosomal abnormality
  • the allele frequencies at each SNP loci are expected to be 85% and 15% for the majority and minority alleles, respectively, assuming Hardy-Weinberg equilibrium
  • the desired third haplotype is expected to be present at an average of 6 4 markers (15%) of per maternal-fetal sample tested Because most loci have a heterozygosity value greater than 25%, for every maternal-fetal sample tested using the panel of 96 tandem SNP assays, greater than about 6 4 markers are most informative
  • 6 or 7 of those tandem SNPs may be informative for any one specific maternal-fetal sample tested, and a 'positive' result from any one of those tandem SNPs is informative
  • tandem SNPs should be identified on both the p and the q arm of chromosome 21 Because of the comparative nature of the basic approach, the tandem SNP assay is predicted to have a detection rate of 95% (those that occur during maternal meiosis) for trisomy 21 If paternal samples are available, non-disjunctions that occur during paternal meiosis can also be detected Thus, detection rates would be higher (about ⁇ 99%) with a 0% false positive rate
  • Subject 8 Trisomy 0.05 0.05 5.5% Subject 9 Disomy 0.95 0.16

Abstract

La présente invention concerne des compositions et des méthodes de détection d’anomalies génétiques qui permettent de comparer des allèles dans un échantillon contenant des acides nucléiques tant maternels que fœtaux, dans le but d’identifier des anomalies génétiques.
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