WO2013130857A1

WO2013130857A1 - Defining diagnostic and therapeutic targets of conserved fetal dna in maternal circulating blood

Info

Publication number: WO2013130857A1
Application number: PCT/US2013/028388
Authority: WO
Inventors: Andrew Brooks
Original assignee: Bio Dx, Inc.
Priority date: 2012-02-29
Filing date: 2013-02-28
Publication date: 2013-09-06

Abstract

The present invention provides methods and materials useful for detecting fetal DNA as well as markers for fetal conditions by using biological samples of a maternal host.

Description

DEFINING DIAGNOSTIC AND THERAPEUTIC TARGETS OF CONSERVED FETAL DNA IN MATERNAL CIRCULATING BLOOD

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S.

Provisional Application No. 61/604,984 filed February 28, 2012, which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION The present invention provides for detecting and characterizing fetal genetic material, e.g., fetal DNA in maternal samples, e.g., maternal blood as well as identification of fetal conditions based on non-invasive prenatal testing.

BACKGROUND OF THE INVENTION

The challenges associated with DNA diagnostics from fetal DNA present in maternal biological samples, in particular, blood samples, are many. Issues associated with the amount of DNA, enrichment of fetal specific DNA, nucleic acid purity and understanding the specific fetal DNA sequences that are conserved between samples, across pregnancies and subjects are among the largest hurdles. Currently there is no satisfactory methodology for determining the presence of fetal DNA prior to diagnostic testing which adversely affects the ability to report consistent and reliable data. There is also lack of sufficient

characterization of fetal DNA derived from maternal biological samples, in particular blood samples, that can be used to identify specific sequences (in addition to disease targets) that can be used to obtain a high rate of success in assay development across pregnancies.

Sequence and mutation specific assay development is currently difficult to carry out given the variability associated with prenatal nucleic acid analysis from maternal whole blood.

As such, there remains a need in the art for methods and approaches of detecting fetal DNA and related fetal conditions. The present invention describes a technological approach for detecting and characterizing fetal genetic material in maternal samples. In addition, the present invention provides methods and related materials for identifying fetal conditions based on fetal genetic materials in maternal samples.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery that certain fetal genetic materials are conserved in maternal biological samples, e.g., maternal blood. Accordingly the present invention provides methods and materials useful for detecting fetal genetic material as well as for identification of fetal conditions.

In one aspect, the present invention provides a method for detecting the presence of fetal DNA in a biological sample of a maternal host. In one embodiment, the method comprises identifying the genotype of at least one conserved genomic segment in a biological sample of a maternal host and comparing the genotype to the corresponding maternal genotype to determine the presence of fetal DNA based on one or more differences between the genotype of the sample and the genotype of the maternal host.

In one embodiment, the conserved genomic segment is a genomic segment provided in Table 1. In one embodiment, the conserved genomic segment includes any probe identified in Table 1. In another embodiment, the conserved genomic segment includes any gene identified in Table 1. In yet another embodiment, the conserved genomic segment is a fragment of a gene identified in Table 1, e.g., a fragment associated with any genotype marker of a gene identified in Table 1. In still another embodiment, the conserved genomic segment is any gene identifiable by the probe or associated with the probe identified in Table 1. In one embodiment, the method comprises detecting the genotypes of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 20, at least 50, at least 100, at least 150, at least 200, at least 250, at least 500, at least 600, at least 700, or at least 800 conserved genomic segments provided in Table 1 in a biological sample of a maternal host and comparing the genotypes to the corresponding maternal genotypes to determine the presence of fetal DNA based on one or more differences between the genotype of the sample and the genotype of the maternal host . In one embodiment, the genotype of a conserved genomic segment comprises the profile of any one or more genetic makeup suitable for distinguishing one genome from another genome. For example, the genotype of a conserved genomic segment can comprise the profile of single nucleotide polymorphism (SNP), restriction fragment length

polymoprhism (RFLP), short tandem repeats (STR), DNA sequence, or any combination thereof. In one embodiment, the genotype of a conserved genomic segment comprises the profile of SNP. In yet another embodiment, the genotype of one or more conserved genomic segments comprises the profile of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 SNPs in one or more conserved genomic segments. In one embodiment, the biological sample of a maternal host includes any processed or unprocessed, solid, semi-solid, or liquid biological sample, e.g., blood, urine, saliva, mucosal samples (such as samples from uterus or vagina, etc.). For example, the biological sample of a maternal host can be a sample of whole blood, partially lysed whole blood, plasma, partially processed whole blood. In one embodiment, the biological sample of a maternal host is a sample comprising cell free DNA or free floating DNA from the whole blood of the maternal host.

In one embodiment, the biological sample of a maternal host is enriched for fetal DNA. In one embodiment, the biological sample of a maternal host is enriched for fetal DNA by DNA size fractionation. In one embodiment the fraction of DNA containing fetal DNA is characterized by having a size of about less than 500 base pairs, or about 50 to about 500 base pairs or about 50 to about 400 base pairs, or about 50 to about 300 base pairs, or about 50 to about 200 base pairs, or about 50 to about 100 base pairs.

In one embodiment, the genotype of at least one conserved genomic segment in a biological sample of a maternal host that has been enriched for fetal DNA is determined and compared to a maternal genotype for the same conserved genomic segments in a maternal cell sample. In one embodiment, the maternal biological sample enriched for fetal DNA is a whole blood sample. In a further embodiment, the maternal cell sample is derived from a maternal whole blood sample, e.g., prior to pregnancy.

In another aspect, the invention provides for a method of detecting the presence or absence of a genetic condition in a fetus comprising detecting the presence or absence of a genetic marker in a biological sample obtained from the maternal host of a fetus. In one embodiment, the genetic marker is within a chromosomal location conserved in fetal DNA in the biological sample of the maternal host. In one embodiment, the chromosomal location is selected from the chromosomal locations listed in Table 2. In one embodiment, the presence or absence of the genetic marker indicates the presence or absence of the genetic condition in the fetus.

In one embodiment, the biological sample of a maternal host includes any processed or unprocessed, solid, semi-solid, or liquid biological sample, e.g., blood, urine, saliva, mucosal samples (such as samples from uterus or vagina, etc.). For example, the biological sample of a maternal host can be a sample of whole blood, partially lysed whole blood, plasma, partially processed whole blood. In one embodiment, the biological sample of a maternal host is a sample of cell free DNA or free floating DNA from the whole blood of the maternal host.

In one embodiment, prior to, concurrent with or subsequent to the detection of the presence or absence of a genetic marker, the presence of fetal DNA is confirmed in the biological sample. In one embodiment, the presence of fetal DNA is confirmed in the biological sample by identifying the genotype of at least one conserved genomic segment in the biological sample and comparing the genotype to the corresponding maternal genotype to determine the presence of fetal DNA based on one or more differences between the genotype of the sample and the genotype of the maternal host.

In one embodiment, the genetic marker is a combination of a first genetic marker from a first chromosomal location conserved in fetal DNA and a second genetic marker from a second chromosomal location conserved in fetal DNA. In another embodiment, the first and second chromosomal locations are different. In a further embodiment, the method further includes a third genetic marker from a third chromosomal location in fetal DNA. In still another embodiment, the method further includes a fourth genetic marker from a fourth chromosomal location in fetal DNA. In yet another embodiment, the method further includes a fifth genetic marker from a fifth chromosomal location in fetal DNA. In one embodiment, the third, fourth and/or fifth chromosomal locations are different from the first two and each other. In another embodiment, the first and second chromosomal locations, and optionally the third, fourth, and fifth chromosomal locations are on the same or different chromosomes.

In one embodiment, the genetic marker is associated with skeletal dysplasia. In a further embodiment, the genetic marker is associated with spinal muscular atrophy. In yet another embodiment, the genetic marker is located within the chromosomal location 5ql3- 5ql3.

In one embodiment, the genetic maker is associated with an aneuploidy. In one embodiment, the aneuploidy is a trisomy. In a further embodiment, the genetic marker associated with a trisomy is within one or more of the chromosomal locations selected from the group consisting of X21.2-Xp21.1, 17ql 1.2-17ql 1.2, 3p26-3p25, 5ql3-5ql3, 16q24.3- 16q24.3, Iq24.2-lq23 and/or 1 lq22-l lq23. In one embodiment, the genetic marker associated with a trisomy is within a chromosomal location of chromosome 13, 14, 15, 16, 18, 21, 22, X or Y. In another embodiment, the genetic marker includes a panel of genetic markers from a chromosomal location of chromosome 13, 14, 15, 16, 18, 21, 22, X, Y, or any combination thereof. In yet another embodiment, the generic marker includes a panel of genetic markers from one or more chromosomal locations of X21.2-Xp21.1, 17ql 1.2- 17ql l .2, 3p26-3p25, 5ql3-5ql3, 16q24.3-16q24.3, Iq24.2-lq23, I lq22-l lq23 or any combination thereof.

In one aspect, the current invention provides a method for selecting a genetic marker for determining a genetic condition of a fetus in a biological sample of a maternal host of the fetus by identifying a group of genetic markers associated with a genetic condition to be determined for the fetus in a biological sample of a the maternal host, identifying within the group of genetic markers, a subset of genetic makers that are within one or more chromosomal locations conserved in fetal DNA in the biological sample of the maternal host, selecting the subset of genetic markers for assay testing and determining the genetic condition of the fetus based on the results obtained from assay testing.

In another aspect, the current invention provides for a databases in a computer readable medium comprising conserved genomic segments. In one embodiment, the conserved genomic segments are those conserved genomic segments provided for in Table 1. In a further embodiment, the database is searchable based on an identifier for each chromosomal location or gene provided in Table 1. In one aspect, the current invention provides for a computer readable medium comprising chromosomal locations provided in Table 2. In one embodiment, the database is searchable based on an identifier for each chromosomal location provided in Table 2.

In one aspect, the current invention provides an array of probes useful for detecting a panel of genetic markers within one or more chromosomal locations provided in Table 2.

Another aspect of the invention provides a method of detecting a genetic disorder in a fetus comprising: separating methylated DNA from unmethylated DNA in a biological sample obtained from a maternal host of the fetus to provide a methylated DNA fraction and an unmethylated DNA fraction; detecting a first genetic marker in the methylated DNA fraction or a second genetic marker in the unmethylated DNA fraction; wherein the first and second genetic marker each are associated with a genetic disorder listed in Table 3 and are within one or more conserved genomic segments; and wherein the first genetic marker is predetermined to be methylated in fetal DNA while the second genetic marker is

predetermined to be unmethylated in fetal DNA; wherein the presence of the first or second genetic marker is indicative of the genetic disorder. In this regard, in certain embodiments, a genetic marker of interest is predetermined to be unmethylated in fetal DNA. In another embodiment, a genetic marker of interest is predetermined to be methylated in fetal DNA. In certain embodiments, the genetic marker of interest is a genetic marker that is methylated in fetal DNA and unmethylated in maternal DNA. In other embodiments, the genetic marker of interest is a genetic marker that is unmethylated in fetal DNA and methylated in maternal DNA. Thus, in certain embodiments of the methods, a genetic marker of interest is detected in the methylated fraction of DNA. In another embodiment, a genetic marker of interest is detected in the unmethylated fraction of DNA. As discussed further herein, methods for determining the methylation pattern in fetal DNA for particular genetic markers present within conserved segments as compared to the corresponding segment of DNA in maternal DNA to determine whether the genetic markers of interest are methylated or unmethylated in fetal DNA are known in the art. In one embodiment of the method, the biological sample is a biological sample of the maternal host enriched for fetal DNA. In another embodiment, the biological sample is confirmed for the presence of fetal DNA. In yet a further embodiment of the method, the genetic marker is associated with skeletal dysplasia and is present in the methylated DNA. In this regard, the genetic marker is a mutation in the FGFR3 gene. In certain embodiments, methylated DNA is separated from unmethylated DNA using an antibody. In other embodiments, the methylated DNA can be separated from unmethylated DNA by hybridization with nucleic acids that specifically hybridize to uracil DNA

conversion products. In one embodiment, the presence or absence of the genetic marker is detected using PCR or sequencing.

One aspect of the present invention provides a method for enriching fetal DNA from a biological sample from a maternal host of a fetus comprising: contacting DNA extracted from the biological sample obtained from the maternal host of the fetus, wherein the biological sample comprises fetal DNA, with a nucleic acid, wherein the nucleic acid specifically hybridizes to a conserved genomic segment and wherein the nucleic acid is attached to a support. In one embodiment of the method, the support is a plate, a bead, a microsphere, a nanoparticle, or a colloidal particle. In another embodiment of the method, the nucleic acid is single stranded DNA.

Another aspect of the present invention provides a method for determining aneuploidy in a fetus comprising: (a) contacting DNA extracted from a biological sample obtained from a maternal host of a fetus with a first nucleic acid, wherein the first nucleic acid specifically hybridizes to a conserved genomic segment comprising a genetic marker for aneuploidy; (b) contacting the DNA extracted from the biological sample with a second nucleic acid, wherein the second nucleic acid specifically hybridizes to a disomy reference DNA sequence; (c) isolating the DNA extracted from the biological sample that specifically hybridizes to the first nucleic acid and the second nucleic acid; wherein the isolated DNA comprises fetal DNA; (d) quantifying the amount of isolated fetal DNA that specifically hybridizes to the first nucleic acid; (e) quantifying an amount of the isolated fetal DNA that specifically hybridizes to the second nucleic acid; (f) comparing the amount of isolated fetal DNA from (d) to an amount of isolated fetal DNA from (e), wherein a 50% increase in fetal DNA in (d) as compared to the amount of fetal DNA from (e) indicates the presence of a trisomic chromosome and wherein a 50% decrease in fetal DNA in (d) as compared to the amount of fetal DNA from (e) indicates the presence of a monosomic chromosome; thereby determining aneuploidy in the fetus. In one embodiment of the method, the first nucleic acid and the second nucleic acid may be attached to a support. In this regard, a suitable support is a bead. In another embodiment, the quantifying is performed using digital PCR and/or nextgen sequencing. In yet another embodiment, the conserved genomic segment comprising a genetic marker for aneuploidy is selected from the group consisting of 13ql2-13ql3, 13q34, 18ql l, 18ql l .3, 18q21, 21q22.1-21q22.3, 15ql l-15ql2, 15q22.3-15q23, 15ql5.1-15ql5.3, 17ql 1.2, 17pl 1.2, and 17ql2. In an additional embodiment, the disomy reference DNA sequence is on chromosome 15 or 17. DETAILED DESCRIPTION

It is frequently of medical importance to know the presence of genetic mutations affecting a fetus carried by a maternal host. However, most reliable tests for fetal genetic diseases, amniocentesis and chronic villus sampling, involve invasive sampling techniques that are difficult for the mother and carry some risk of fetal loss. It has been known for many years that fetal DNA can be isolated from maternal blood during pregnancy. It is also known that there is a small number of intact fetal cells circulating within the maternal system during pregnancy. The methods described herein utilize DNA isolated from maternal blood and from circulating fetal cells for the analysis of fetal genetic mutations early in the pregnancy, especially during the first trimester when the fetus is too small for effective invasive sampling.

The maternal circulation has free fetal DNA circulating along with free maternal DNA and in addition has some circulating nucleated fetal cells, both nucleated red cells and trophoblasts. The fetal cells are to some degree compromised, less hardy and easier to lyse than the maternal hosts own cells. By treating the blood sample with a mild lysis buffer, the compromised fetal cells undergo lysis while leaving the bulk of maternal cells intact.

Treating a maternal blood sample with a gentle lysis buffer consisting of, e.g., 0.3 M sucrose, 5mM MgCl₂, 3% Triton X-100, 0.1% saponin and 10 mM Tris-HCLpH 7.3 for 5 minutes at 37° C is effective at lysing compromised fetal cells, releasing their DNA into the blood sample, while leaving the majority of maternal cells intact. The cellular debris and remaining intact maternal cells are centrifuged off and the resulting supernatant digested with proteinase K to digest the protein and the DNA extracted on to charged beads or other suitable support. The lysis methods described herein increase the total fetal DNA by approximately 15% over that which can be extracted from an unlysed blood sample. The resulting extracted DNA contains both fetal and maternal DNA components, with the majority being maternal DNA. Genetic sequences not present in the maternal DNA but which are unique to the fetus can be directly assayed from the maternal-fetal DNA mix. Fetal DNA circulating in the maternal system is badly degraded as the mother's body attempts to clear it from her system. As a result not all segments of the fetal genome are present in any given maternal blood sample and no segment is present in every maternal blood sample. Without additional knowledge, a reliable assay for a fetal genetic condition would have to assay a great many different sequences to assure that at least one sequence representing a condition of interest is present in the sample. This is only possible if the affected genetic region is large with a number of different regions that can be analyzed, such as the Y chromosome for a gender assay. However, if the genetic marker of a condition of interest is a small region such as a specific mutation at a single locus, whether fetal DNA having that specific site will be present in the maternal blood sample is uncertain from patient to patient.

The present invention is based in part on the discovery that certain fetal DNA segments are more reliably present circulating in the maternal system and are thus relatively "conserved", that is occur in a higher percentage of patient samples. Using these conserved DNA segments allows fewer individual target sequences to be used for a fetal genetic assay because there is a higher probability that the selected sequences will be in the sample. For example, because there are no conserved segments on the Y chromosome, at least 12 different sequences must be utilized at different sites on the Y chromosome to reliably determine the presence of a Y chromosome and thus if the fetus is male or female. However, for relatively conserved segments the number of required different sequences can be reduced to three.

Thus, in one aspect of the invention, "conserved genomic fragments" or "conserved genomic segments" means the entire length or a fragment thereof of a genetic DNA segment that is found in at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or at least 95% of maternal samples, or a higher percentage of maternal samples. Illustrative conserved genomic fragments include the entire length or a fragment thereof of the genomic segments listed in Table 1 , any gene identified in Table 1 , or any fragment of a gene identified in Table 1. In one step of the invention, the presence of fetal DNA is detected in a biological sample of a maternal host of a fetus. Specifically, fetal DNA is detected in a whole blood sample of a pregnant female. By "fetal DNA" is meant, DNA that is derived from the fetus and not the mother. In some embodiments, fetal DNA may be derived from cell free DNA circulating in the maternal blood and may also be derived from fetal cells (e.g., fetal trophoblasts or nucleated red blood cells) circulating in the maternal blood. Thus, in some embodiments, fetal DNA includes fetal DNA existing outside of a cell, for example circulating in the maternal blood, and DNA that is derived from a fetal cell that has been lysed for example, using the gentle lysis methods described herein. In yet another embodiment, fetal DNA includes fetal DNA existing outside of a cell as well as fetal DNA present in maternal blood sample after such blood sample has been subjected to partial or gentle cell lysing procedures.

In one aspect of the invention, a biological sample, such as a whole blood sample, is obtained from the maternal host of a fetus, and the genotype of at least one conserved genomic segment in the biological sample of the maternal host is determined. The one or more conserved genomic segment is one or more of the identified conserved genomic segments listed in Table 1. The genotype of the biological sample of the maternal host is then compared with the genotype of the same conserved genomic segment of the mother. A difference in maternal genotype and the genotype determined from the biological sample of the maternal host of the fetus indicates the presence of fetal DNA in the biological sample of the maternal host.

In another aspect of the invention, the biological sample from the maternal host can be enriched for fetal DNA by any means known in the art. In the first trimester fetal DNA is approximately 6% of the total DNA found in maternal blood. This percentage increases as gestation ages progresses. However, the entire fetal DNA genome is not present in any given sample, e.g., only certain fragments of fetal DNA genome are consistently present or conserved in maternal biological samples. In addition, the fetal DNA species that are found in circulating maternal blood are generally smaller in size than that of maternal DNA.

Therefore, fetal DNA may be enriched by DNA size fractionation. In this method, DNA is separated based on size. The fetal DNA fraction is characterized as the fraction of DNA having a size of less than about 500 base pairs, for example about 50 to about 500 base pairs or about 50 to about 400 base pairs, or about 50 to about 300 base pairs or about 50 to about 200 base pairs or about 50 to about 100 base pairs. Thus, isolating the fraction of DNA having a size of less than about 500 base pairs, particularly the fraction having a size of about 50 to about 300 enriches the fetal DNA in a biological sample of maternal host. The enriched fetal DNA fraction can then be used to determine the genotype of the fetus by determining the genotype of at least one conserved genomic segment listed in Table 1. This genotype is then compared to the genotype of the same one or more conserved genomic segments from the mother. The maternal genotype can be determined by determining the genotype of the one ore more conserved genomic segments in the biological sample prior to enriching for fetal DNA or by determining the genotype of the one or more conserved genomic segments in the fraction of DNA containing DNA larger than about 250 base pairs after size fractionation. Alternatively, the genotype can be compared to a maternal genotype of the conserved genomic segments determined prior to the pregnancy.

In certain embodiments, the biological sample from the maternal host can be enriched for fetal DNA using nucleic acid sequence based isolation of specific DNA segments. Single strand nucleic acids that hybridize to the desired target DNA sequences (e.g., a genetic marker for a particular disease, such as a genetic marker found within a conserved genomic segment as described herein) can be bound to beads or other suitable supports and exposed to the sample DNA. The desired target DNA sequences specifically hybridize to the single strand DNA attached to the beads or other suitable support. Because fetal DNA is degraded and broken into small segments, it preferentially hybridizes to the nucleic acid attached to the suitable support due to reaction kinetics, yielding a higher purity fetal DNA sample. Thus, such enriched fetal DNA can then be used in many settings, including diagnostic settings where quantitation (which may require more pure DNA) is required, such as for detection of aneuploidy and deletions. In such instances, a reference sequence (e.g., a disomy reference sequence) can be used to compare against the target sequence to determine quantities of target sequence. As one illustrative example, in certain embodiments, assays for trisomy 13, 18 and 21 are performed by binding sequences that hybridize to conserved fetal genomic regions 13ql2-13ql3, 13q34 on chromosome 13, 18ql l , 18ql 1.3 and 18q21 on chromosome 18, 21q22.1 to 21q22.3 on chromosome 21 , 15ql l-15ql2, 15q22.3-15q23, 15ql5.1-15ql5.3 on chromosome 15 and 17ql l .2, 17p l 1.2, 17ql2 to beads or other suitable support. A maternal blood sample is lysed using gentle lysis as described herein and the extracted DNA isolated by hybridizing to the prepared beads having attached thereto sequences that hybridize to the target conserved fetal DNA segments. The selected (hybridized) DNA is eluted and quantified using digital PCR (see e.g., Proc Natl Acad Sci USA 1999, 96:9236-9241 ; US patent 6143496; Proc Natl Acad Sci USA 100 (15): 8817- 22), other quantitative PCR methods, and/or any of a variety of nextgen sequencing technologies known to the skilled person (e.g. , commercially available from Illumina, San Diego, CA; ABI, Foster City, CA; and others). Using chromosomes 15 and 17 (or other suitable control reference sequence) as a disomy reference, the amount of DNA quantitated in the sample for chromosomes 13, 18 and 21 is compared to the amount of reference DNA of chromosome 15 and 17, thereby allowing for the detection of the 50% increase in DNA for a trisomic chromosome (3 chromosomes instead of two) or a 50% decrease in DNA for a monsomic chromosome (one chromosome instead of two).

Illustrative supports that can be used for the methods described herein include but are not limited to plastic, glass, silica, silicon, collagen, hydroxyapatite, hydrogels, PTFE, polypropylene, polystyrene, nylon, or polyacrylamide. Yet additional embodiments include wherein the suitable support comprises a lipid, a plate, a bag, a rod, a pellet, a fiber, or a mesh. Other embodiments include wherein the support is a particle and additionally wherein the particle comprises a bead, a microsphere, a nanoparticle, or a colloidal particle. In certain embodiments, the biological sample from the maternal host can be enriched for fetal DNA by separating methylated DNA from unmethylated DNA. In this regard, it has been discovered that there are DNA methylation differences between fetal DNA and maternal DNA. Methylation occurs on the cytosine bases that are adjacent to a guanine base (CpG). Where CpGs are formed in groups or clusters, all of the cytosine bases in the group are methylated if methylation is present. Treatment of unmethylated CG with bisulfide converts the unmethylated cytosine to Uracil which can be detected by Polymerase Chain Reaction (PCR) techniques or hybridized and isolated using sequence-specific hybridization techniques. Methylated DNA can be separated from non-methylated DNA by either binding with antibodies to methylated DNA or by hybridizing to single strand DNA sequences specific to the uracil DNA conversion product. Separating the methylated DNA from non-methylated DNA increases the concentration of fetal DNA in a sample and increases the detectability of fetal genetic mutations. As would be understood by the skilled person, some genetic markers of interest are unmethylated in fetal DNA and thus will be present in the unmethylated fraction of fetal DNA while others are methylated in fetal DNA and thus will be present in the methylated fraction of DNA. Determining the methylation pattern of particular genetic markers of interest can be carried out using techniques known in the art, such as via bisulfite modification of the template DNA and sequencing (whole genome bisulfite sequencing or BS-seq), or by methylation-specific PCR (MSP), which amplifies the DNA depending on the methylation status of the primer-binding regions (see, e.g., Proc Natl Acad Sci USA. 1996;93 :9821-9826), or differential cleavage by restriction enzymes. Illustrative restriction enzymes useful for determining methylation at bases in their recognition sequence include, but are not limited to, BstXJ I or Hpall. In certain

embodiments, methylated DNA immunoprecipitation may be used in combination with other techniques for determining methylation patterns of genetic markers of interest. See also, Nucleic Acids Res. 1994;22:2990-2997; Chimerism. 2010 Jul-Sep; 1(1): 30-35.

Table 3 herein shows a list of genetic disorders that are particularly amenable to detection using a combination of extraction of fetal DNA from a maternal host sample following gentle lysis of compromised fetal cells and enriching methylated and/or unmethylated DNA followed by detection of conserved fetal DNA regions in the methylated and/or unmethylated DNA fractions. Other genetic disorders, such as those listed in Table 2, may also be amenable to this process.

A "CpG-containing genomic sequence" as used herein refers to a segment of DNA sequence at a defined location in the genome of an individual such as a human fetus or a pregnant woman. Typically, a "CpG-containing genomic sequence" is at least 15 nucleotides in length and contains at least one cytosine. In certain embodiments, it can be at least 30, 50, 80, 100, 150, 200, 250, or 300 nucleotides in length and contains at least 2, 5, 10, 15, 20, 25, or 30 cytosines. For any one "CpG-containing genomic sequence" at a given location, e.g., within a region centering around a given genetic locus associated with a disease (such as those listed in Table 3), nucleotide sequence variations may exist from individual to individual and from allele to allele even for the same individual. Typically, such a region centering around a defined genetic locus (e.g., a CpG island) contains the locus as well as upstream and/or downstream sequences. Each of the upstream or downstream sequence (counting from the 5 ' or 3' boundary of the genetic locus, respectively) can be as long as 10 kb, in other cases may be as long as 5 kb, 2 kb, 1 kb, 500 bp, 200 bp, or 100 bp. Furthermore, a "CpG-containing genomic sequence" may encompass a nucleotide sequence transcribed or not transcribed for protein production, and the nucleotide sequence can be a protein-coding sequence, a non protein-coding sequence (such as a transcription promoter), or a combination thereof.

A "CpG island" describes a segment of DNA sequence found in a genome that has a minimal length, a minimal GC content, and a minimal ratio of observed CpG

frequency/expected CpG frequency (OCF/ECF). Yamada et al. (Genome Research 14:247- 266, 2004) have described a set of standards for determining a CpG island: at least 400 nucleotides in length, greater than 50% GC content, and an OCF/ECF ratio greater than 0.6. Others (Takai et al, Proc. Natl. Acad. Sci. U.S.A. 99:3740-3745, 2002) have defined a CpG island less stringently as a sequence at least 200 nucleotides in length, having a greater than 50% GC content, and an OCF/ECF ratio greater than 0.6. The concept of a "CpG island", such as on chromosome 21, is one that fits the CpG island profiles provided by any one of the currently available computational programs designed for scanning chromosomes based on the above stated criteria and/or those known to the skilled artisan.

The term "epigenetic state" or "epigenetic status" refers to any structural feature at a molecular level of a nucleic acid (e.g., DNA or RNA) other than the primary nucleotide sequence. For instance, the epigenetic state of a genomic DNA may include its secondary or tertiary structure determined or influenced by, e.g., its methylation pattern or its association with cellular proteins.

The term "methylation profile" or "methylation status," refers to the characteristics of a DNA segment at a particular genomic locus relevant to methylation. Such characteristics include, but are not limited to, whether any of the cytosine (C) residues within this DNA sequence are methylated, location of methylated C residue(s), percentage of methylated C at any particular stretch of residues, and allelic differences in methylation due to, e.g., difference in the origin of the alleles. The term "methylation" profile" or "methylation status" also refers to the relative or absolute concentration of methylated C or unmethylated C at any particular stretch of residues in a biological sample.

The term "bisulfite" encompasses all types of bisulfites, such as sodium bisulfite, that are capable of chemically converting a cytosine (C) to a uracil (U) without chemically modifying a methylated cytosine and therefore can be used to differentially modify a DNA sequence based on the methylation status of the DNA.

As used herein, a reagent that "differentially modifies" methylated or non- methylated DNA encompasses any reagent that modifies methylated and/or unmethylated DNA in a process through which distinguishable products result from methylated and non- methylated DNA, thereby allowing the identification of the DNA methylation status. Such processes may include, but are not limited to, chemical reactions (such as a C to U conversion by bisulfite) and enzymatic treatment (such as cleavage by a methylation-dependent endonuclease). Thus, an enzyme that preferentially cleaves or digests methylated DNA is one capable of cleaving or digesting a DNA molecule at a much higher efficiency when the DNA is methylated, whereas an enzyme that preferentially cleaves or digests unmethylated DNA exhibits a significantly higher efficiency when the DNA is not methylated.

Other methods useful for the detection of methylated DNA are known in the art, such as those described in US patent numbers 6,893,820 and 7,906,288. The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acids (DNA) or ribonucleic acids (R A) and polymers thereof in either single-or double-stranded form. 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. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences as well as the sequence explicitly indicated. Specifically, 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 (Batzer et al, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al, Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene. Two nucleic acid sequences are said to be capable of specifically hybridizing to one another if the two molecules are capable of forming an anti-parallel, double-stranded nucleic acid structure or hybrid under hybridizing conditions, whereas they are substantially unable to form a double-stranded structure or hybrid when incubated with a non-target nucleic acid sequence under the same conditions. A nucleic acid molecule is said to be the "complement" of another nucleic acid molecule if it exhibits complete Watson-Crick base pair

complementarity. Two molecules are said to be "substantially complementary" if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional low-stringency conditions. Similarly, the molecules are said to be "complementary" if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional high-stringency conditions. Stringency conditions in referring to homology or substantial similarity in the hybridization context, can be combined conditions of salt, temperature, organic solvents or other parameters that are typically known to influence hybridization. Typically, high stringency conditions include conditions selected to be 5 or more degrees higher than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched sequence. These techniques are well known in the art. For example, conventional stringency conditions are described in Sambrook, J., et al., Molecular Cloning, a Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and Haymes, B. D., et al, Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985).

By "genotype" is meant the genetic makeup of a cell or an individual (i.e. a fetus or the maternal host of a fetus). The genotype may be determined by any method known in the art. For example, the genotype of the fetus or the maternal host of a fetus may be determined by DNA sequencing, for example NextGen sequencing, SNP, RFLP or STR analysis. For SNP analysis any number of SNPs may be used to determine the genotype. For example, a panel of 96 SNPs allows for the SNP pattern to repeat in every 2 x 10²³ individuals, thereby giving a high probability of genetic identity. Methods of determining genotypes by DNA sequencing, SNP, RFLP, and STR are well known in the art.

In one aspect of the invention, the genotype of one or more of the conserved genomic fragments listed in Table 1 is determined. In one embodiment, conserved genomic fragments include a panel of fragments within one or more probes or genes identified in Table 1. In one aspect of the invention, the genotypes of about 5 to about 500 of the conserved genomic fragments given in Table 1 are determined. In another aspect of the invention, the genotypes of about 10 to about 400 of the conserved genomic fragments given in Table 1 are determined. In yet another example, the genotype of about 20 to about 300 of the conserved genomic fragments given in Table 1 is determined. In still another

embodiment, the genotypes of about 30 to about 200 of the conserved genomic fragments given in Table 1 are determined. In another embodiment, the genotypes of about 40 to about 100 of the conserved genomic fragments given in Table 1 are determined. In certain embodiments, as noted elsewhere herein, the genotyping of one or more of the conserved genomic fragments listed in Table 1 is determined in a sample in which methylated DNA has been separated from unmethylated DNA. In this regard, the genotyping of one or more of the conserved genomic fragments listed in Table 1 may be determined in the methylated fraction of DNA and/or in the unmethylated fraction of DNA. In another embodiment, the genotyping of one or more of the conserved genomic fragments listed in Table 1 is determined in a sample in which fetal DNA has been enriched by nucleic acid sequence based isolation using known methods. By "maternal host of a fetus" is meant the woman who is pregnant with the fetus whose DNA is sought to be detected and/or tested for a genetic condition. The term

"maternal host of a fetus," "maternal host" and "mother" are used interchangeably. By "fetus" is meant in uterus developing offspring of any gestational stage. Fetal DNA can be detected prior to the "fetal period" which begins at 11 weeks of gestation in human.

Therefore, "fetus" encompasses not only the developing offspring in the fetal period but also in the earlier embryonic stages of development prior to the 11^th week of human gestation.

By "biological sample" is meant any sample that is derived from the maternal host of the fetus. In one embodiment, the biological sample of a maternal host includes any processed or unprocessed, solid, semi-solid, or liquid biological sample, e.g., blood, urine, saliva, mucosal samples (such as samples from uterus or vagina, etc.). For example, the biological sample of a maternal host can be a sample of whole blood, partially lysed whole blood, plasma, serum, partially processed whole blood. In one embodiment, the biological sample of a maternal host is a sample of cell free DNA or free floating DNA from the whole blood of the maternal host.

In a further aspect, the current invention provides for a method of non-invasive genetic testing of a fetus by detecting the presence or absence of a genetic marker associated with a genetic condition in a fetus. For example, a method is provided for the detection of the presence or absence of a genetic marker in a fetus by detecting the presence or absence of the genetic marker in a biological sample obtained from a maternal host of a fetus. The presence or absence of the genetic marker indicates the presence or absence of the genetic condition.

In some aspects, the invention provides first detecting the presence of fetal DNA in a sample from a maternal host of fetus by the methods described herein, then testing the detected fetal DNA for the presence or absence of a genetic marker associated with a disease or condition. In certain embodiments, the methods include separating methylated DNA from unmethylated DNA, detecting the presence of the fetal DNA in a sample from a maternal host of fetus by the methods described herein, then testing the detected fetal DNA for the presence or absence of a genetic marker associated with a disease or condition, such as those listed in Tables 2 and/or 3.

By "genetic marker" is meant any genetic marker known to be associated with a disease or condition. In one embodiment, the genetic marker is located within a

chromosomal location conserved in fetal DNA in the biological sample of the maternal host. For example, the chromosomal location is one or more of the chromosomal locations/genes listed in Table 2 or Table 3. In some embodiments, a condition is detected in a fetus by detecting the presence or absence of a marker located in just one chromosomal location/genes listed in Table 2 or Table 3. In other embodiments, a condition is detected in a fetus by detecting the presence or absence of more than one genetic markers, for example more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or more than 20 markers in one or more chromosomal locations/genes listed in Table 2 or Table 3. In further embodiments, a condition is detected in a fetus by detecting the presence or absence of more than five, more than 10 or more than 15 markers in one or more chromosomal locations/genes listed in Table 2 or Table 3. In some embodiments, the genetic marker can be a mutation in the one or more chromosomal locations or genes listed in Table 2 or Table 3. The mutation can be an insertion, deletion, frame shift, substitution, or any other mutations known in the art.

The presence or absence of the genetic marker can be determined by any method known in the art, for example, DNA sequencing, hybridization assays (e.g. FISH), or PCR, or a combination of such methods. Other methods known to the skilled artisan are useful herein including digital PCR (see e.g. , Proc Natl Acad Sci USA 1999, 96:9236-9241 ; US patent 6143496; Proc Natl Acad Sci USA 100 (15): 8817-22), other quantitative PCR methods, and/or any of a variety of nextgen sequencing technologies known to the skilled person {e.g. , commercially available from Illumina, San Diego, CA; ABI, Foster City, CA; and others). In some embodiments, the presence or absence of the one or more genetic markers can be detected in enriched fetal DNA derived from a whole blood sample from the maternal host of the fetus. By way of example, a whole blood sample may be taken from the maternal host of the fetus and size fractionated as described above, to obtain a sample of enriched fetal DNA. The enriched fetal DNA is then tested by any method known in the art, for example, DNA hybridization, sequencing or PCR, to detect the presence or absence of a genetic marker within one or more chromosomal locations listed in Table 2 or Table 3. The results of the fetal DNA testing done by this method may be further compared against the same genetic marker testing of un-enriched whole blood derived from the mother, or fractionated DNA of larger size containing maternal DNA or a DNA sample obtained from the maternal host prior to pregnancy to confirm the presence or absence of the genetic marker is being detected in the fetal DNA and not the maternal DNA.

The genetic condition to be detected can be any condition listed in Table 2 or Table 3. For example, the condition can be spinal muscular atrophy and may be detected by detecting the presence of one or more genetic markers within the 5ql3-5ql3 chromosomal location.

The methods of the present invention are also useful in detecting the presence or absence of aneuploidies, including monosomies or trisomies. For example, the methods of the current invention are useful in detecting trisomy 13, 14, 15, 16, 18, 21 , 22, X, and/or Y. In a specific embodiment, trisomy 21 is detected by measuring the DCR gene located at chromosome 21q22.2-21q22.3, the CBS gene located at chromosome 21q22.2-21q22.3, the KNO gene at 21q22.3-21q22.3 and/or the SOD1 gene at chromosoome 21q22.1-21q22.1 or any combination thereof. The current invention further provides for a method for selecting a genetic marker for determining the genetic condition of a fetus in a biological sample of a maternal host of a fetus. In this aspect of the invention, a genetic marker is selected by first identifying a group of genetic markers associated with the genetic condition to be determined for the fetus followed by determining which of these markers among the group of genetic markers identified as being associated with the particular condition fall within one or more

chromosomal locations conserved in fetal DNA in the maternal host of the mother. Next, the subset of markers that fall within these one or more chromosomal locations is selected for assay testing, for example, PCR or DNA sequencing analysis to determine the presence or absence of the marker. Lastly, the biological sample is assayed for the presence or absence of the selected genetic marker and the genetic condition of the fetus is determined based on the results of the assay. In certain embodiments, fetal DNA is enriched from the biological sample by separating methylated DNA from unmethylated DNA, then the methylated and/or the unmethylated DNA fraction is assayed for the presence or absence of the selected genetic marker and the genetic condition of the fetus is determined based on the results of the assay. In certain embodiments, the selected genetic marker is one or more markers that indicates a genetic condition as listed in Table 2 and/or Table 3.

In addition to methods of detecting and characterizing fetal DNA and methods of selecting genetic markers, the invention also provides for a database in a computer readable medium comprising the conserved genomic segments in Table I. In a particular embodiment, the database is searchable based on an identifier for each conserved genomic segment provided in Table 1. Such identifiers include, but are not limited to, the chromosomal location, the alignment probe ID, the sequence of the segment, gene symbol, the accession number, the segment description, and any other useful identifier.

The invention also provides for a computer readable medium comprising the chromosomal locations provided for in Table 2 and/or Table 3. In a particular embodiment, the database is searchable based on identifiers for each of the chromosomal locations provided in Table 2 and/or Table 3. Such identifiers include, but are not limited to, gene name, genbank ID number, gene sequence, chromosomal location, associated genetic condition, and any other useful identifier.

The invention also provides arrays of probes useful for genetic testing of fetal DNA and/or fetal conditions. In one embodiment, the array of the present invention includes probes useful for detecting one or more genetic markers within one or more chromosomal locations listed in Table 2 and/or Table 3. In one embodiment, the array of the present invention includes probes useful for detecting one or more conserved segments, such as those provided in Table 1. In another embodiment, the array contains one or more, or 10 or more or 50 or more or 100 or more defined DNA probes selected from those listed in Table 1 which can be hybridized to the DNA derived from the maternal biological sample to detect and increase or decrease in copy number changes in the DNA. In this embodiment, the array can detect an increase or decrease in the copy number of any particular DNA region encompassed within a particular probe, thereby signifying an increased copy number and the presence of fetal DNA. In some embodiments, the array is customized to detect only certain chromosomal locations corresponding to particular genetic markers in Table 2 and/or Table 3 which are useful in detecting a particular condition, for example, trisomy. In this

embodiment, probes from Table 1 are selected which correspond to the chromosomal locations encompassing the genetic markers of the particular genes of interest listed in Table 2 and/or 3. In other embodiments, the array contains a random sampling of the probes listed in Table 1. In another embodiment, the array contains all of the probes listed in Table 1. In some embodiments, the probes are attached to the array ready for hybridization of DNA from the maternal biological sample. In other embodiments, the probes are contained in solution ready for attachment by the end user. In this embodiment, the array may be customized by the end user to allow attachment of only particular probes of interest. Table 1

( hromosoma Agilent (■cue

1 Location Probe I I) SMiibol Accession Description

hs|chrl : 1 1493 A 16 P15 entg|DENN ref]NM_19 ref] Homo sapiens DENN/M ADD

1604- 266059 D2C 8459 domain containing 2C

1 14931662 (DENND2C), mRNA.

hs|chrl : 1 1501 A 14 P10 entg AMPD ref|NM 00 ref|Homo sapiens adenosine

7266- 5570 1 0036 monophosphate deaminase 1

1 15017326 (isoform M) (AMPD 1), mRNA. hs|chrl : 1 1505 A 14 P10 entg NRAS ref|NM 00 ref|Homo sapiens neuroblastoma

3836- 3853 2524 RAS viral (v-ras) oncogene

1 15053896 homolog (NRAS), mRNA.

hs|chrl : 1 1507 A 16 P00 entg CSDE ref|NM_00 ref|Homo sapiens cold shock

0345- 151987 1 1007553 |ref domain containing E 1 , RNA-

1 15070405 NM 00715 binding (CSDEl), transcript variant

8 1 , mRNA.

hs|chrl : 1 1512 A 14 P10 entg SIKE ref|NM 02 ref|Homo sapiens suppressor of

0477- 3084 5073 IKK epsilon (SIKE), mRNA.

1 15120537

hs|chrl : 12214 A 16 P35 entg VPS 13 ref|NM 01 ref|Homo sapiens vacuolar protein 130-12214190 031747 D 5378|ref]N sorting 13 homolog D (S.

M O 18156 cerevisiae) (VPS 13D), transcript variant 1 , mRNA.

hs|chrl : 12550 A 16 POO entg|DHRS ref|NM_00 refjHomo sapiens

506-12550566 016149 3 4753 dehydrogenase/reductase (SDR family) member 3 (DHRS3), mRNA.

hs|chrl : 12632 A 14 P12 entg AADA ref|NM 00 ref|Homo sapiens arylacetamide 722-12632782 7931 CL4 1013630 deacetylase-like 4 (AADACL4), mRNA.

3627- 281639 DIP 4644 ref|N phosphodiesterase 4D interacting

143623687 M 0010028 protein (myomegalin) (PDE4DIP),

1 1 ref|NM transcript variant 1 , mRNA.

001002812

hs|chrl : 14381 A 14 P10 entg|SEC22 ref]NM_00 ref]Homo sapiens SEC22 vesicle

5232- 5608 B 4892 trafficking protein homolog B (S.

143815289 cerevisiae) (SEC22B), mRNA. hs|chrl : 14412 A 14 P13 entg HFE2 ref|NM 14 ref|Homo sapiens hemochromatosis

4744- 1300 5277 ref|N type 2 (juvenile) (HFE2), transcript

144124804 M 2020041 variant b, mRNA.

ref]NM 21

3652|ref|N

M 213653

hs|chrl : 14415 A 14 P13 entg TXNIP ref|NM 00 ref|Homo sapiens thioredoxin

1603- 7169 6472 interacting protein (TXNIP),

144151663 mRNA.

( hromosoma Agilent (■cue

1 Location Probe I I) SMiibol Accession Description

hs|chrl : 15092 A 14 P13 entg LCE2 ref|NM 01 ref|Homo sapiens late cornified

6290- 3620 B 4357 envelope 2B (LCE2B), mR A.

150926344

hs|chrl : 15093 A 14 P10 entg LCE2 ref|NM 17 ref|Homo sapiens late cornified

7952- 0722 A 8428 envelope 2A (LCE2A), mRNA.

150938009

hs|chrl : 15099 A 16 P00 entg KPPvP ref|NM 00 ref|Homo sapiens keratinocyte

9073- 165508 1025231 proline-rich protein (KPRP),

150999133 mRNA.

hs|chrl : 15102 A 14 P13 entg LCEl ref|NM 17 ref|Homo sapiens late cornified

6227- 1413 E 8353 envelope IE (LCE1E), mRNA.

151026285

hs|chrl : 15103 A 16 P35 entg LCEl ref|NM 17 ref|Homo sapiens late cornified

6434- 310000 D 8352 envelope ID (LCE ID), mRNA.

151036494

hs|chrl : 15104 A 14 P13 entg LCEl ref|NM 17 ref|Homo sapiens late cornified

4658- 8003 C 8351 envelope 1C (LCE 1C), mRNA.

151044718

hs|chrl : 15105 A 14 P13 entg LCEl ref|NM 17 ref|Homo sapiens late cornified

1979- 4645 B 8349 envelope IB (LCE IB), mRNA.

151052027

hs|chrl : 151 12 A 14 P13 entg SMCP ref|NM 03 ref|Homo sapiens sperm

1644- 1912 0663 mitochondria-associated cysteine -

151 121704 rich protein (SMCP), nuclear gene encoding mitochondrial protein, mRNA.

hs|chrl : 151 14 A 14 P12 entg IVL ref|NM 00 ref|Homo sapiens involucrin (IVL),

8957- 0925 5547 mRNA.

151 149005

hs|chrl : 15121 A 14 P12 entg SPRR4 ref|NM 17 ref|Homo sapiens small proline-rich

0846- 2655 3080 protein 4 (SPRR4), mRNA.

151210906

hs|chrl : 15122 A 14 P12 entg SPRR1 ref|NM 00 ref|Homo sapiens small proline-rich

4196- 4410 A 5987 protein 1A (SPRR1A), mRNA.

151224256

hs|chrl : 15124 A 14 Pl l entg SPRR3 ref|NM 00 ref|Homo sapiens small proline-rich

2009- 6391 5416 protein 3 (SPRR3), mRNA.

151242069

hs|chrl : 15127 A 14 P12 entg SPRR1 ref|NM 00 ref|Homo sapiens small proline-rich

1033- 0073 B 3125 protein 1 B (cornifm) (SPRR1B),

151271088 mRNA.

hs|chrl : 15128 A 16 P15 entg SPRR2 ref|NM 00 ref|Homo sapiens small proline-rich

0809- 297784 D 6945 protein 2D (SPRR2D), mRNA.

151280869

hs|chrl : 15144 A 16 P00 entg LELPl ref|NM 00 ref|Homo sapiens late cornified

3781- 166054 1010857 envelope-like proline-rich 1

151443841 (LELP1), mRNA. ( hromosoma Agilent (■cue

1 Location Probe II) SMiihol Accession Description

hs|chrl:15150 A 14 Pll entg LOR ref|NM 00 ref|Homo sapiens loricrin (LOR),

0023- 0433 0427 mRNA.

151500072

hs|chrl:15154 A 16 P15 entg PGLY ref|NM 05 ref|Homo sapiens peptidoglycan

1460- 298291 RP3 2891 recognition protein 3 (PGLYRP3),

151541519 mRNA.

hs|chrl:15157 A 14 P10 entg PGLY ref|NM 02 ref|Homo sapiens peptidoglycan

6489- 2942 RP4 0393 recognition protein 4 (PGLYRP4),

151576549 mRNA.

hs|chrl:15159 A 14 P10 entg|S100A ref|NM 00 ref|Homo sapiens SI 00 calcium

9178- 0749 9 2965 binding protein A9 (S 100A9),

151599238 mRNA.

hs|chrl:15161 A 14 P12 entg|S100A ref|NM 00 ref|Homo sapiens SI 00 calcium

2984- 4338 12 5621 binding protein A12 (S100A12),

151613042 mRNA.

hs|chrl:15162 A 14 P10 entg|S100A ref|NM 00 ref|Homo sapiens SI 00 calcium

9265- 5363 8 2964 binding protein A8 (S 100A8),

151629319 mRNA.

hs|chrl:15165 A 14 P10 entg SlOOA ref|NM 17 ref|Homo sapiens SI 00 calcium

7016- 6240 7A 6823 binding protein A7A (S 100A7A),

151657076 mRNA.

hs|chrl:15167 A 16 P15 entg|S100A ref|NM_00 ref|Homo sapiens SI 00 calcium

6848- 298617 7L2 1045479 binding protein A7-like 2

151676908 (S100A7L2),mRNA.

hs|chrl:15169 A 14 P12 entg|S100A ref|NM 00 ref|Homo sapiens SI 00 calcium

8217- 5828 7 2963 binding protein A7 (S 100A7),

151698277 mRNA.

hs|chrl:15178 A 16 POO entg|S100A ref|NM 00 ref|Homo sapiens SI 00 calcium

0744- 166502 5 2962 binding protein A5 (S 100A5),

151780804 mRNA.

hs|chrl:15180 A 16 P15 entg|S100A ref|NM 00 ref|Homo sapiens SI 00 calcium

0251- 298906 2 5978 binding protein A2 (S 100A2),

151800311 mRNA.

hs|chrl:15185 A 14 Pll entg|S100A ref|NM 08 ref|Homo sapiens SI 00 calcium

1895- 8231 16 0388 binding protein Al 6 (S100A16),

151851947 mRNA.

hs|chrl:15185 A 14 P10 entg|S100A ref|NM 02 ref|Homo sapiens SI 00 calcium

4423- 7098 14 0672 binding protein A14 (S100A14),

151854471 mRNA.

hs|chrl:15185 A 14 P10 entg|S100A ref|NM 00 ref|Homo sapiens SI 00 calcium

9028- 5005 13 1024213|ref binding protein Al 3 (SI 00A13),

151859088 |NM 00102 transcript variant 5, mRNA.

4212|ref]N

M 0010242

ll|ref]NM

005979|ref]

NM 00102

4210

( hromosoma Agilent (■cue

1 Location Probe I I) SMiibol Accession Description

hs|chr4: 16701 A 14 P10 entg TLLl ref|NM 01 ref|Homo sapiens tolloid-like 1

4608- 1657 2464 (TLL1), mR A.

167014666

hs|chr4:39875 A 16 P00 entg RHOH ref|NM 00 ref|Homo sapiens ras homolog gene 242-39875302 960225 4310 family, member H (RHOH),

mRNA.

hs|chr4:40032 A 14 P20 entg CHRN ref|NM 01 ref|Homo sapiens cholinergic 601-40032660 2451 A9 7581 receptor, nicotinic, alpha 9

(CHRNA9), mRNA.

hs|chr4:40130 A 16 P00 entg FLJ20 ref|NM 01 refj Homo sapiens RNA-binding 619-40130679 960548 273 9027 protein (FLJ20273), mRNA.

hs|chr4:98699 A 14 P13 entg MGC4 ref|NM 17 refj Homo sapiens hypothetical 087-98699147 8919 6496 4952 protein MGC46496 (MGC46496), mRNA.

hs|chr5 : 1 1998 A 16 P01 entg PRR16 ref|NM 01 ref|Homo sapiens proline rich 16

9315- 322931 6644 (PRR16), mRNA.

1 19989375

hs|chr5 : 12121 A 14 P12 entg FTMT ref|NM 17 ref|Homo sapiens ferritin

6336- 3018 7478 mitochondrial (FTMT), mRNA.

121216396

hs|chr5 : 12133 A 14 Pl l entg SRFB ref|NM 15 ref|Homo sapiens serum response

001 1- 4585 PI 2546 factor binding protein 1 (SRFBP1),

121330071 mRNA.

hs|chr5 : 14121 A 16 P01 entg PCDH ref|NM 03 ref|Homo sapiens protocadherin 1

7581- 352316 1 2420 (PCDH1), transcript variant 2,

141217641 mRNA.

hs|chr5 : 14128 A 16 P01 entg|KIAA ref]NM 01 refj Homo sapiens KIAAO 141

6271- 352410 0141 4773 (KIAAO 141), mRNA.

141286331

hs|chr5 : 14130 A 16 P01 entg PCDH ref|NM 01 ref|Homo sapiens protocadherin 12

4698- 352447 12 6580 (PCDH 12), mRNA.

141304756

hs|chr5 : 14133 A 16 P17 entg RNF14 ref|NM 18 refj Homo sapiens ring finger

0366- 326902 3399|ref]N protein 14 (RNF14), transcript

141330426 M 0042901 variant 3, mRNA.

ref]NM 18

3398|ref|N

M 1834001

ref]NM 18

3401

hs|chr5 : 14136 A 14 Pl l entg GNPD ref|NM 00 ref|Homo sapiens glucosamine-6-

0756- 0042 Al 5471 phosphate deaminase 1 (GNPDAl),

141360816 mRNA.

hs|chr5 : 14146 A 16 P37 entg NDFIP ref|NM_03 ref|Homo sapiens Nedd4 family

9990- 392339 1 0571 interacting protein 1 (NDFIP 1),

141470050 mRNA.

hs|chr5 :36542 A 16 P01 entg IRX1 ref|NM 02 ref|Homo sapiens iroquois

12-3654272 168082 4337 homeobox protein 1 (IRX1),

mRNA.

( hromosoma Agilent (■cue

1 Location Probe I I) SMiibol Accession Description

hs|chr9: 12261 A 14 P10 entg PSMD ref|NM 00 ref|Homo sapiens proteasome

8558- 2384 5 5047 (prosome, macropain) 26S subunit,

122618618 non-ATPase, 5 (PSMD5), mR A. hs|chr9: 12265 A 16 P38 entg|PHF19 ref|NM_01 refjHomo sapiens PHD finger

7730- 863797 5651 protein 19 (PHF 19), transcript

122657790 variant 1 , mRNA.

hs|chr9: 12271 A 14 P13 entg TRAF ref|NM 00 ref|Homo sapiens TNF receptor-

4143- 8468 1 5658 associated factor 1 (TRAF 1),

122714203 mRNA.

hs|chr9: 12275 A 14 Pl l entg C5 ref|NM 00 ref|Homo sapiens complement

5812- 6555 1735 component 5 (C5), mRNA.

122755872

hs|chr9: 12289 A 16 P02 entg CEP 1 1 ref|NM 00 ref|Homo sapiens centrosomal

2410- 172604 0 7018 protein 1 lOkDa (CEP 1 10), mRNA.

122892470

hs|chr9: 12298 A 16 P38 entg RAB 1 ref|NM 01 ref|Homo sapiens RAB 14, member

1254- 864720 4 6322 RAS oncogene family (RAB 14),

122981314 mRNA.

hs|chr9: 12307 A 16 P02 entg GSN ref|NM_19 ref|Homo sapiens gelsolin

9770- 172892 8252 (amyloidosis, Finnish type) (GSN),

123079830 transcript variant 2, mRNA.

hs|chr9: 12314 A 14 P10 entg STOM ref|NM 00 ref|Homo sapiens stomatin

6242- 3990 4099|ref]N (STOM), transcript variant 1 ,

123146302 M 198194 mRNA.

hs|chr9: 12688 A 14 P10 entg TYRP ref|NM 00 ref|Homo sapiens tyrosinase-related 541-12688598 1680 1 0550 protein 1 (TYRP1), mRNA.

6723- 9649 8A 3387 sequence similarity 78, member A

133126775 (FAM78A), mRNA.

hs|chr9: 13315 A 16 P02 entg PPAP ref|NM 03 ref|Homo sapiens phosphatidic acid

8121- 186691 DC3 2728 phosphatase type 2 domain

133158181 containing 3 (PPAPDC3), mRNA. hs|chr9: 13337 A 14 Pl l entg POMT ref|NM 00 ref|Homo sapiens protein-O-

1617- 4470 1 1077365 |ref mannosyltransferase 1 (POMT1),

133371677 NM 00107 transcript variant 2, mRNA.

7366|ref|N

M 007171

hs|chr9: 13339 A 14 P10 entg UCK1 ref|NM 03 ref|Homo sapiens uridine-cytidine

1218- 6183 1432 kinase 1 (UCK1), mRNA.

133391273

hs|chr9: 13344 A 14 Pl l entg RAPG ref|NM 19 ref|Homo sapiens Rap guanine

3193- 871 1 EF1 8679 ref|N nucleotide exchange factor (GEF) 1

133443253 M 005312 (RAPGEFl), transcript variant 2, mRNA.

hs|chr9: 19040 A 14 P10 entg|RRAG ref]NM 00 ref] Homo sapiens Ras-related GTP 854-19040914 2520 A 6570 binding A (RRAGA), mRNA.

( hromosoma Agilent (■cue

1 Location Probe I I) SMiihol Accession Description

hs|chrl0:8191 A 16 P39 entg ANXA ref|NM 00 ref|Homo sapiens annexin Al 1

5876- 103644 1 1 1 157|ref]N (ANXA1 1), transcript variant a,

81915936 M 1458681 mR A.

ref]NM 14

5869

hs|chrl0:8202 A 16 P39 entg MAT 1 ref|NM 00 ref|Homo sapiens methionine

1610- 103937 A 0429 adenosyltransferase I, alpha

82021670 (MAT1A), mRNA.

hs|chrl0:8210 A 14 Pl l entg|DYDC ref]NM 03 ref] Homo sapiens DP Y30 domain

9832- 6526 2 2372 containing 2 (DYDC2), mRNA.

82109885

hs|chrl0:8221 A 16 P18 entg TSPA ref|NM 03 ref|Homo sapiens tetraspanin 14

1903- 994582 N14 0927 (T SPAN 14), mRNA.

8221 1963

hs|chrl 1 :4009 A 16 P02 entg LRRC ref|NM 02 ref|Homo sapiens leucine rich

4067- 433770 4C 0929 repeat containing 4C (LRRC4C),

40094127 mRNA.

hs|chrl 1 :4060 A 16 P19 entg STIM1 ref|NM 00 ref|Homo sapiens stromal

785-4060845 134535 3156 interaction molecule 1 (STIM1), mRNA.

hs|chrl 1 :4083 A 14 Pl l entg RRMl ref|NM 00 ref] Homo sapiens ribonucleotide 821-4083881 7324 1033 reductase Ml polypeptide (RRMl), mRNA.

618-4564678 383787 2 1005170 receptor, family 52, subfamily I, member 2 (OR52I2), mRNA.

306-4631366 1 1 1 1 El 2430 receptor, family 51 , subfamily E, member 1 (OR51E1), mRNA. hs|chrl 1 :4800 A 16 P02 entg OR51F ref|NM 00 ref|Homo sapiens olfactory

123-4800183 384155 2 1004753 receptor, family 51 , subfamily F, member 2 (OR51F2), mRNA. hs|chrl 1 :4826 A 16 P39 entg OR51 S ref|NM 00 ref|Homo sapiens olfactory

070-4826130 251 123 1 1004758 receptor, family 51 , subfamily S, member 1 (OR51 S 1), mRNA. hs|chrl 1 :4892 A 16 P02 entg OR51 ref|NM 00 ref|Homo sapiens olfactory

946-4893006 384301 G2 1005238 receptor, family 51 , subfamily G, member 2 (OR51G2), mRNA. hs|chrl 1 :4967 A 14 P20 entg MMP2 ref|NM 02 ref|Homo sapiens matrix

429-4967485 1783 6 1801 metallopeptidase 26 (MMP26), mRNA.

hs|chrl 1 :4977 A 16 P19 entg OR51 ref|NM 00 ref|Homo sapiens olfactory

379-4977439 136601 LI 1004755 receptor, family 51 , subfamily L, member 1 (OR51L1), mRNA. ( hromosoma Agilent (■ene

1 Location Probe I I) SMiibol Accession Description

hs|chrl 1 :5024 A 16 P02 entg OR52J ref|NM 00 ref|Homo sapiens olfactory

325-5024385 384493 3 1001916 receptor, family 52, subfamily J, member 3 (OR52J3), mRNA. hs|chrl 1 :7278 A 14 P20 entg TNFR ref|NM 15 ref|Homo sapiens tumor necrosis

4964- 1861 SF19L 2222 ref|N factor receptor superfamily,

72785022 M 032871 member 19-like (TNFRSF 19L), transcript variant 2, mRNA.

hs|chrl 1 :7280 A 16 P02 entg|KIAA ref]NM 01 ref] Homo sapiens KIAA0280

3772- 468705 0280 5159 (KIAA0280), mRNA.

72803832

hs|chrl 1 :7303 A 14 Pl l entg PLEK ref|NM 02 ref|Homo sapiens pleckstrin

6040- 2326 HB1 1200 homology domain containing,

73036100 family B (evectins) member 1

(PLEKHB1), mRNA.

hs|chrl 1 :7306 A 14 Pl l entg RAB6 ref|NM 00 ref|Homo sapiens RAB6A, member

8322- 2013 A 2869|ref]N RAS oncogene family (RAB6A),

73068382 M 198896 transcript variant 1 , mRNA.

hs|chrl l :7317 A 14 P12 entg MRPL ref|NM 01 ref|Homo sapiens mitochondrial

7046- 4013 48 6055 ribosomal protein L48 (MRPL48),

73177106 nuclear gene encoding

mitochondrial protein, mRNA. hs|chrl 1 :8370 A 14 P10 entg STK33 ref|NM 03 ref|Homo sapiens serine/threonine 101-8370161 4176 0906 kinase 33 (STK33), mRNA.

hs|chrl 1 :8663 A 14 P10 entg RPL27 ref|NM 00 ref|Homo sapiens ribosomal protein 250-8663310 0322 A 0990 L27a (RPL27A), mRNA.

hs|chrl 1 :8671 A 14 Pl l entg ST5 ref|NM_21 ref|Homo sapiens suppression of 715-8671775 4318 3618 ref|N tumorigenicity 5 (ST5), transcript

M 1391571 variant 3, mRNA.

ref]NM 00

5418

hs|chrl2: 1 169 A 16 P19 entg WSB2 ref|NM 01 ref|Homo sapiens WD repeat and

61616- 698214 8639 SOCS box-containing 2 (WSB2),

1 16961676 mRNA.

hs|chrl2: 1 169 A 14 P12 entg FLJ20 ref|NM 01 ref] Homo sapiens hypothetical

87912- 1974 674 9086 protein FLJ20674 (FLJ20674),

1 16987972 mRNA.

hs|chrl2: 1 170 A 14 P13 entg|PEBPl ref|NM_00 refjHomo sapiens

662 lO- 5050 2567 phosphatidylethanolamme binding l l 7066270 protein 1 (PEBP1), mRNA.

hs|chrl2: 1 170 A 14 P10 entg|TAOK ref]NM 01 ref]Homo sapiens TAO kinase 3

74739- 2661 3 6281 (TAOK3), mRNA.

1 17074791

hs|chrl2: 1 173 A 16 P19 entg SUDS ref|NM_02 ref|Homo sapiens suppressor of

05180- 698995 3 2491 defective silencing 3 homo log (S.

1 17305240 cerevisiae) (SUDS3), mRNA. hs|chrl2: 1 179 A 14 P13 entg|KIAA ref]NM 19 reflHomo sapiens KIAA 1853

13638- 6339 1853 4286 (KIAA1853), mRNA.

1 17913698

( hromosoma Agilent (■cue

1 Location Probe I I) SMiihol Accession Description

hs|chr 13 :4543 A 16 P19 entg ZC3H ref|NM 01 ref| Homo sapiens zinc finger

4467- 800508 13 5070 CCCH-type containing 13

45434527 (ZC3H13), mRNA.

hs|chrl3 :6088 A 14 P10 entg PCDH ref|NM 02 ref|Homo sapiens protocadherin 20

1928- 0254 20 2843 (PCDH20), mRNA.

60881988

hs|chrl3 :7316 A 14 P12 entg KLF12 ref|NM 00 ref|Homo sapiens Kruppel-like

0850- 6532 7249 factor 12 (KLF 12), mRNA.

73160910

hs|chrl3 :9334 A 16 P19 entg GPC6 ref|NM 00 ref|Homo sapiens glypican 6

7774- 915260 5708 (GPC6), mRNA.

93347834

hs|chrl4: 1862 A 16 P02 entg|ACTB ref]NM_00 reflHomo sapiens ACTBLl protein

4382- 870240 LI 1005356 (ACTBL 1 ), transcript variant

18624442 POTE-14A, mRNA.

hs|chrl4:41 15 A 16 P20 entg LRFN ref|NM 15 ref|Homo sapiens leucine rich

1632- 021229 5 2447 repeat and fibronectin type III

41 151692 domain containing 5 (LRFN5), mRNA.

hs|chrl4:5103 A 16 P40 entg|FRMD ref|NM_00 refjHomo sapiens FERM domain

7524- 185330 6 1042481 containing 6 (FRMD6), transcript

51037584 variant 1 , mRNA.

hs|chrl4:5140 A 16 P20 entg GNG2 ref|NM 05 ref|Homo sapiens guanine

2028- 043525 3064 nucleotide binding protein (G

51402088 protein), gamma 2 (GNG2),

mRNA.

hs|chrl4:5180 A 14 P10 entg PTGD ref|NM 00 ref|Homo sapiens prostaglandin D2

9818- 21 13 R 0953 receptor (DP) (PTGDR), mRNA.

51809878

hs|chrl4:5185 A 16 P40 entg PTGE ref|NM 00 ref|Homo sapiens prostaglandin E

2545- 187454 R2 0956 receptor 2 (subtype EP2), 53kDa

51852605 (PTGER2), mRNA.

hs|chrl4:5196 A 14 Pl l entg|KIAA ref]NM 02 ref] Homo sapiens KIAA 1344

7479- 8876 1344 0784 (KIAA1344), mRNA.

51967539

hs|chrl4:5217 A 14 P13 entg|ERO 1 ref]NM_01 ref]Homo sapiens ERO 1 -like (S .

8964- 9028 L 4584 cerevisiae) (ERO 1L), mRNA.

52179024

hs|chrl4:5224 A 16 P40 entg PSMC ref|NM 00 ref|Homo sapiens proteasome

7459- 188360 6 2806 (prosome, macropain) 26S subunit,

52247519 ATPase, 6 (PSMC6), mRNA. hs|chrl4:5226 A 16 P20 entg|STYX ref|NM_14 refjHomo sapiens

9683- 045531 5251 serine/threonine/tyrosine

52269743 interacting protein (STYX),

mRNA.

hs|chrl4:5231 A 14 Pl l entg GNPN ref|NM 19 ref|Homo sapiens glucosamine -

5755- 2387 AT1 8066 phosphate N-acetyltransferase 1

52315815 (GNPNAT1), mRNA.

( hromosoma Agilent (■cue

1 Location Probe I I) SMiibol Accession Description

hs|chrl6:8062 A 14 Pl l entg HSDl ref|NM 00 ref| Homo sapiens hydroxy steroid

6347- 2039 7B2 2153 (17-beta) dehydrogenase 2

80626407 (HSD17B2), mR A.

hs|chrl6:8074 A 16 P03 entg|MPHO ref|NM 00 ref|Homo sapiens M-phase

2547- 187927 SPH6 5792 phosphoprotein 6 (MPHOSPH6),

80742607 mRNA.

hs|chrl7: 1613 A 14 P13 entg SERPI ref|NM_00 ref|Homo sapiens serpin peptidase 875-1613934 2429 NF1 2615 inhibitor, clade F (alpha-2

antiplasmin, pigment epithelium derived factor), member 1

(SERPINF1), mRNA.

hs|chrl7: 1629 A 16 P03 entg|SMYD ref]NM_05 reflHomo sapiens SET and MYND 727-1629787 201775 4 2928 domain containing 4 (SMYD4), mRNA.

hs|chrl7: 1684 A 16 P03 entg RPA1 ref|NM 00 ref|Homo sapiens replication 634-1684694 201824 2945 protein Al , 70kDa (RPA1),

mRNA.

hs|chrl7: 1784 A 14 Pl l entg RTN4 ref|NM 17 ref] Homo sapiens reticulon 4 695-1784755 7472 RL1 8568 receptor-like 1 (RTN4RL1),

mRNA.

hs|chrl7: 1912 A 16 P40 entg SMG6 ref|NM_01 ref|Homo sapiens Smg-6 homo log, 423-1912483 730036 7575 nonsense mediated mRNA decay factor (C. elegans) (SMG6), mRNA.

hs|chrl7: 1965 A 16 P20 entg ULK2 ref|NM 01 ref|Homo sapiens unc-51 -like

4761- 607805 4683 kinase 2 (C. elegans) (ULK2),

19654821 mRNA.

hs|chrl7: 1974 A 14 P12 entg|AKAP ref|NM 00 refjHomo sapiens A kinase (PRKA)

9897- 7035 10 7202 anchor protein 10 (AKAP 10),

19749957 nuclear gene encoding

mitochondrial protein, mRNA. hs|chrl7: 1993 A 14 P13 entg SPEC ref|NM 15 ref|Homo sapiens sperm antigen

9012- 4326 C I 2904 ref|N with calponin homology and

19939072 M 0010335 coiled-coil domains 1 (SPECC1),

53 transcript variant NSP5beta3 alpha, mRNA.

hs|chrl7:2097 A 14 Pl l entg DHRS ref|NM_01 ref|Homo sapiens

4662- 1 1 15 7B 5510 dehydrogenase/reductase (SDR

20974722 family) member 7B (DHRS7B), mRNA.

hs|chrl7:2104 A 16 P03 entg TME ref|NM 00 ref|Homo sapiens transmembrane

2200- 225976 Mi l 3876 protein 1 1 (TMEM 1 1 ), mRNA.

21042260 ( hromosoma Agilent (■cue

1 Location Probe I I) SMiihol Accession Description

hs|chrl7:2108 A 16 P20 entg MGC3 ref|NM 15 ref] Homo sapiens transcript

8749- 610978 3894 2914 expressed during hematopoiesis 2

21088809 (MGC33894), mRNA.

hs|chrl7:21 13 A 16 P03 entg MAP2 ref|NM 14 ref|Homo sapiens mitogen-

0610- 226092 K3 5109 activated protein kinase kinase 3

21 130664 (MAP2K3), transcript variant B, mRNA.

hs|chrl7:2122 A 16 P20 entg KCNJ ref|NM 02 ref|Homo sapiens potassium

1644- 61 1320 12 1012 inwardly-rectifying channel,

21221703 subfamily J, member 12 (KCNJ 12), mRNA.

hs|chrl7:231 1 A 14 Pl l entg NOS2 ref|NM 00 ref|Homo sapiens nitric oxide

5967- 9698 A 0625|ref|N synthase 2A (inducible,

231 16019 M_l 53292 hepatocytes) (NOS2A), transcript variant 1 , mRNA.

hs|chrl7:3077 A 16 P03 entg SLFNl ref|NM 01 ref] Homo sapiens schlafen family

2407- 237885 2 8042 member 12 (SLFN12), mRNA.

30772467

hs|chrl7:3078 A 16 P40 entg SLFN1 ref|NM 14 ref|Homo sapiens schlafen family

7395- 801937 3 4682 member 13 (SLFN 13), mRNA.

30787455

hs|chrl7:3092 A 14 P13 entg PEX12 ref|NM 00 ref|Homo sapiens peroxisomal

6420- 1358 0286 biogenesis factor 12 (PEX12),

30926480 mRNA.

hs|chrl7:3094 A 14 P12 entg AP2B l ref|NM 00 ref|Homo sapiens adaptor-related

5164- 1424 1030006 ref protein complex 2, beta 1 subunit

30945224 NM 00128 (AP2B 1 ), transcript variant 1 ,

2 mRNA.

hs|chrl7:3108 A 16 P03 entg RASL ref|NM 03 ref|Homo sapiens RAS-like, family

7715- 238336 10B 3315 10, member B (RASL 10B),

31087775 mRNA.

hs|chrl7:3109 A 14 P10 entg GAS2 ref|NM 13 ref] Homo sapiens growth arrest-

6790- 6138 L2 9285 specific 2 like 2 (GAS2L2),

31096839 mRNA.

hs|chrl7:31 1 1 A 14 P13 entg MMP2 ref|NM 02 ref|Homo sapiens matrix

8123- 6665 8 4302 metallopeptidase 28 (MMP28),

31 1 18181 transcript variant 1 , mRNA.

hs|chrl7:31 16 A 14 Pl l entg|TAF15 ref]NM_00 ref] Homo sapiens TAF 15 RN A

1335- 5859 3487 ref|N polymerase II, TATA box binding

31 161395 M 139215 protein (TBP)-associated factor,

68kDa (TAF 15), transcript variant

2, mRNA.

hs|chrl7:4148 A 14 Pl l entg|KIAA ref]NM 01 ref]Homo sapiens KIAA1267

3739- 0538 1267 5443 (KIAA1267), mRNA.

41483793

hs|chrl7:4198 A 16 P20 entg LRRC ref|NM 00 ref|Homo sapiens leucine rich

3406- 659819 37A2 1006607 repeat containing 37, member A2

41983466 (LRRC37A2), mRNA.

( hromosoma Agilent (■cue

1 Location Probe I I) SMiibol Accession Description

hs|chr 18:2792 A 14 P12 entg RNF13 ref|NM 01 ref] Homo sapiens ring finger

9522- 1996 8 6271 |ref]N protein 138 (RNF 138), transcript

27929582 M 198128 variant 1 , mRNA.

hs|chr 18:3269 A 16 P20 entg|KIAA ref]NM 02 reflHomo sapiens KIAA 1328

3151- 82391 1 1328 0776 (KIAA1328), mRNA.

3269321 1

hs|chrl 8:3308 A 16 P03 entg BPvUN ref|NM 02 ref|Homo sapiens bruno-like 4,

2621- 345423 OL4 0180 RNA binding protein (Drosophila)

33082681 (BRUNOL4), mRNA.

hs|chr 18:7007 A 16 P03 entg CYB5 ref|NM 00 ref|Homo sapiens cytochrome b5

3944- 402184 A 1914|ref]N type A (microsomal) (CYB5A),

70074004 M 148923 transcript variant 2, mRNA.

hs|chr 18:7031 A 14 P13 entg|CNDP ref]NM_01 ref]Homo sapiens CNDP

6045- 3122 2 8235 dipeptidase 2 (metallopeptidase

70316105 M20 family) (CNDP2), mRNA. hs|chrl 8:7035 A 16 P03 entg CNDP ref|NM 03 ref|Homo sapiens carnosine

3477- 402618 1 2649 dipeptidase 1 (metallopeptidase

70353537 M20 family) (CNDP1), mRNA. hs|chr 18:7041 A 16 P20 entg LOC4 ref|NM 00 ref|Homo sapiens hypothetical gene

1563- 919551 00657 1008234 supported by BC036588

7041 1623 (LOC400657), mRNA.

hs|chr 18:7090 A 14 P13 entg ZNF40 ref|NM 01 ref] Homo sapiens zinc finger

5729- 4173 7 7757 protein 407 (ZNF407), mRNA.

70905783

hs|chr 18:7523 A 14 Pl l entg|ATP9 ref]NM 19 ref]Homo sapiens ATPase, Class II,

5681- 1672 B 8531 type 9B (ATP9B), mRNA.

75235741

hs|chr 18:7525 A 16 P03 entg NF AT ref|NM 17 ref|Homo sapiens nuclear factor of

9663- 41061 1 C I 2390 ref|N activated T-cells, cytoplasmic,

75259723 M 006162 calcineurin-dependent 1

ref]NM 17 (NF ATC 1 ), transcript variant 1 , 2388 mRNA.

hs|chrl 8:7560 A 16 P03 entg PTPR ref|NM 00 ref|Homo sapiens protein tyrosine 828-7560888 312395 M 2845 phosphatase, receptor type, M

(PTPRM), mRNA.

hs|chrl9: 1915 A 14 P12 entg MEF2 ref|NM 00 ref|Homo sapiens myocyte

4706- 5162 B 5919 enhancer factor 2B (MEF2B),

19154766 mRNA.

hs|chrl9: 1916 A 16 P03 entg RFXA ref|NM 00 ref|Homo sapiens regulatory factor

5678- 431888 NK 3721 ref|N X-associated ankyrin-containing

19165738 M_l 34440 protein (RFXANK), transcript variant 1 , mRNA.

hs|chrl9: 1918 A 16 P20 entg NCAN ref|NM 00 ref|Homo sapiens neurocan

8967- 975002 4386 (NCAN), mRNA.

19189027

hs|chrl9: 1923 A 14 P12 entg HAPL ref|NM 02 ref|Homo sapiens hyaluronan and

2502- 9282 N4 3002 proteoglycan link protein 4

19232547 (HAPLN4), mRNA.

( hromosoma Agilent (■ene

1 Location Probe I I) SMiibol Accession Description

hs|chrl9:5882 A 16 P21 entgpPRX ref|NM 00 ref|Homo sapiens divergent-paired

9751- 036530 1012728 related homeobox (DPRX),

5882981 1 mRNA.

hs|chrl9:5898 A 16 P21 entg NLRP ref|NM_14 ref| Homo sapiens NLR family,

9173- 036862 12 4687 ref|N pyrin domain containing 12

58989233 M_033297 (NLRP12), transcript variant 2, mRNA.

hs|chrl9:5906 A 14 Pl l entg MY A ref|NM 00 ref|Homo sapiens myeloid-

9708- 2678 DM 1020820 ref associated differentiation marker

59069759 NM 00102 (MY ADM), transcript variant 4,

0819|ref]N mRNA.

M 1383731

ref]NM 00

102082 l |ref

NM 00102

0818

hs|chrl9:5908 A 16 P21 entg PRKC ref|NM 00 ref|Homo sapiens protein kinase C,

2056- 036989 G 2739 gamma (PRKCG), mRNA.

590821 16

hs|chrl9:5910 A 16 P41 entg CACN ref|NM 03 ref|Homo sapiens calcium channel,

8290- 227373 G7 1896 voltage-dependent, gamma subunit

59108349 7 (CACNG7), mRNA.

hs|chrl9:5916 A 16 P03 entg CACN ref|NM 03 ref|Homo sapiens calcium channel,

4790- 464185 G8 1895 voltage-dependent, gamma subunit

59164845 8 (CACNG8), mRNA.

hs|chrl9:5919 A 14 P10 entg CACN ref|NM 03 ref|Homo sapiens calcium channel,

4401- 7943 G6 1897 ref|N voltage-dependent, gamma subunit

59194461 M 1458141 6 (CACNG6), transcript variant 3, ref]NM 14 mRNA.

5815

hs|chrl9:5923 A 16 P21 entg|VSTM ref|NM_19 refjHomo sapiens V-set and

7251- 037291 1 8481 transmembrane domain containing

59237309 1 (VSTM1), mRNA.

hs|chrl9:5929 A 14 P10 entg OSCA ref|NM_13 ref|Homo sapiens osteoclast-

4757- 5349 R 0771 |ref]N associated receptor (OSCAR),

59294809 M 133168 transcript variant 3, mRNA.

ref]NM 13

3169|ref|N

M 206818

hs|chrl9:5930 A 14 P13 entg|NDUF ref]NM_00 ref]Homo sapiens NADH

0843- 3007 A3 4542 dehydrogenase (ubiquinone) 1

59300897 alpha subcomplex, 3, 9kDa

(NDUFA3), mRNA.

hs|chrl9:5930 A 16 P03 entg|TFPT ref]NM_01 ref]Homo sapiens TCF3 (E2A)

6164- 464303 3342 fusion partner (in childhood

59306215 Leukemia) (TFPT), mRNA.

( hromosoma Agilent (■cue

1 Location Probe I I) SMiibol Accession Description

hs|chr20:5670 A 16 P03 entg NPEP ref|NM 02 ref|Homo sapiens aminopeptidase-

4228- 541605 Ll 4663 like 1 (NPEPL1), mRNA.

56704287

hs|chr20:6055 A 16 P41 entg FLJ30 ref|NM 15 ref] Homo sapiens hypothetical

2910- 3801 17 313 2757 protein FLJ30313 (FLJ30313),

60552955 mRNA.

hs|chr20:6075 A 16 P03 entg SLCO ref|NM 01 ref|Homo sapiens solute carrier

3459- 547933 4A1 6354 organic anion transporter family,

60753516 member 4A1 (SLC04A1), mRNA. hs|chr20:6081 A 16 P03 entg NTSR ref|NM 00 ref|Homo sapiens neurotensin

4249- 548043 1 2531 receptor 1 (high affinity) (NTSRl),

60814309 mRNA.

hs|chr20:6091 A 16 P03 entg OGFR ref|NM 00 ref|Homo sapiens opioid growth

1384- 548202 7346 factor receptor (OGFR), mRNA.

6091 1434

hs|chr20:6092 A 16 P41 entg COL9 ref|NM 00 ref|Homo sapiens collagen, type

0275- 380946 A3 1853 IX, alpha 3 (COL9A3), mRNA.

60920320

hs|chr20:6700 A 14 Pl l entg BMP2 ref|NM 00 ref|Homo sapiens bone

675-6700735 8128 1200 morphogenetic protein 2 (BMP2), mRNA.

hs|chr21 : 1602 A 16 P03 entg USP25 ref|NM 01 ref|Homo sapiens ubiquitin specific

6984- 553415 3396 peptidase 25 (USP25), mRNA.

16027044

hs|chr21 :2130 A 14 P13 entg NCA ref|NM 00 ref|Homo sapiens neural cell

1277- 6854 M2 4540 adhesion molecule 2 (NCAM2),

21301337 mRNA.

hs|chr21 :2588 A 16 P03 entg MRPL ref|NM 01 ref|Homo sapiens mitochondrial

3439- 567193 39 7446 |ref]N ribosomal protein L39 (MRPL39),

25883499 M 080794 nuclear gene encoding

mitochondrial protein, transcript variant 1 , mRNA.

hs|chr21 :2593 A 16 P21 entg JAM2 ref|NM 02 ref|Homo sapiens junctional

7744- 219885 1219 adhesion molecule 2 (JAM2),

25937804 mRNA.

hs|chr21 :2602 A 14 P12 entg|ATP5J ref|NM_00 refjHomo sapiens ATP synthase,

0409- 9661 1003696 ref H+ transporting, mitochondrial F0

26020469 |NM_00100 complex, subunit F6 (ATP5J),

3697 ref|N nuclear gene encoding

M 0010037 mitochondrial protein, transcript 01 |ref]NM variant 3 , mRNA.

0010037031

ref]NM 00

1685

hs|chr21 :2603 A 16 P03 entg GABP ref|NM_00 ref|Homo sapiens GA binding

4503- 567438 A 2040 protein transcription factor, alpha

26034563 subunit 60kDa (GABPA), mRNA. ( hromosoma Agilent (■cue

1 Location Probe I I) SMiibol Accession Description

hs|chr21 :2617 A 16 P21 entg APP ref|NM 00 ref|Homo sapiens amyloid beta

5054- 220493 0484 ref|N (A4) precursor protein (peptidase

261751 12 M 201413 nexin-II, Alzheimer disease)

ref|NM 20 (APP), transcript variant 1 , mR A. 1414

hs|chr21 :3061 A 16 P03 entg KRTA ref|NM 20 ref|Homo sapiens keratin associated

4342- 574153 P26-1 3405 protein (K TAP26-1), mRNA.

30614402

hs|chr21 :3066 A 14 Pl l entg KRTA ref|NM 18 ref|Homo sapiens keratin associated

5869- 4884 P 13-2 1621 protein 13-2 (KRTAP 13-2), nuclear

30665920 gene encoding mitochondrial

protein, mRNA.

hs|chr21 :3069 A 14 Pl l entg KRTA ref|NM 18 ref|Homo sapiens keratin associated

0772- 1688 P13-1 1599 protein 13-1 (KRTAP 13-1),

30690832 mRNA.

hs|chr21 :3073 A 14 P12 entg KRTA ref|NM 18 ref|Homo sapiens keratin associated

4699- 3396 P15-1 1623 protein 15-1 (KRTAP 15-1),

30734746 mRNA.

hs|chr21 :3078 A 14 P20 entg KRTA ref|NM 18 ref|Homo sapiens keratin associated

5733- 0585 PI 9-3 1609 protein 19-3 (KRTAP 19-3),

30785793 mRNA.

hs|chr21 :3079 A 14 P10 entg KRTA ref|NM 18 ref|Homo sapiens keratin associated

1066- 4353 P 19-4 1610 protein 19-4 (KRTAP 19-4),

30791 126 mRNA.

hs|chr21 :3083 A 14 P13 entg KRTA ref|NM 18 ref|Homo sapiens keratin associated

5781- 9606 P 19-6 1612 protein 19-6 (KRTAP 19-6),

30835829 mRNA.

hs|chr21 :3420 A 16 P03 entg ATP5 ref|NM 00 ref|Homo sapiens ATP synthase,

6536- 579493 O 1697 H+ transporting, mitochondrial Fl

34206595 complex, O subunit (oligomycin sensitivity conferring protein) (ATP50), nuclear gene encoding mitochondrial protein, mRNA. hs|chr21 :3437 A 16 P21 entg MRPS ref|NM 03 ref|Homo sapiens mitochondrial

0842- 240974 6 2476 ribosomal protein S6 (MRPS 6),

34370902 nuclear gene encoding

mitochondrial protein, mRNA. hs|chr21 :3466 A 14 P10 entg KCNE ref|NM 17 ref|Homo sapiens potassium

2787- 6199 2 2201 voltage-gated channel, Isk-related

34662842 family, member 2 (KCNE2),

mRNA.

hs|chr21 :3481 A 14 P13 entg DSCR ref|NM 20 ref|Homo sapiens Down syndrome

0803- 9150 1 3418|ref]N critical region gene 1 (DSCR1),

34810863 M 2034171 transcript variant 3, mRNA.

ref]NM 00

4414

hs|chr21 :3496 A 16 P21 entg CLIC6 ref|NM 05 ref|Homo sapiens chloride

5868- 242477 3277 intracellular channel 6 (CLIC6),

34965928 mRNA.

mitochondrial protein, mRNA. ( hromosoma Agilent (■cue

1 Location Probe I I) SMiibol Accession Description

hs|chr22:3955 A 16 P21 entg ST 13 ref|NM_00 ref|Homo sapiens suppression of

2993- 331714 3932 tumorigenicity 13 (colon

39553053 carcinoma) (Hsp70 interacting protein) (ST 13), mRNA.

hs|chr22:3958 A 14 P12 entg|XPNP ref|NM_02 refjHomo sapiens X-prolyl

6378- 3506 EP3 2098 ref|N aminopeptidase (aminopeptidase P)

39586438 M 145174 3, putative (XPNPEP3), mRNA. hs|chr22:4242 A 16 P41 entg|FLJ23 ref]NM_02 reflHomo sapiens CAP-binding

8563- 540805 588 2785 ref|N protein complex interacting protein

42428623 M_198856 1 (FLJ23588), transcript variant 1 , mRNA.

hs|chr22:4255 A 14 P12 entg SULT ref|NM 01 ref|Homo sapiens sulfotransferase

4803- 7520 4A1 4351 family 4A, member 1 (SULT4A1),

42554850 mRNA.

hs|chr22:4857 A 16 P03 entg BRDl ref|NM 01 ref|Homo sapiens bromodomain

8748- 640757 4577 containing 1 (BRD1), mRNA.

48578802

hs|chr22:4863 A 16 P21 entg ZBED ref|NM 01 ref|Homo sapiens zinc finger, BED-

4916- 353790 4 4838 type containing 4 (ZBED4),

48634976 mRNA.

hs|chr22:4868 A 16 P41 entg ALG1 ref|NM 02 ref|Homo sapiens asparagine-linked

2858- 556699 2 4105 glycosylation 12 homo log (S.

48682918 cerevisiae, alpha-1 ,6- mannosyltransferase) (ALG12), mRNA.

hs|chr22:4869 A 14 Pl l entg CREL ref|NM 02 ref|Homo sapiens cysteine-rich

9807- 6178 D2 4324 with EGF-like domains 2

48699863 (CRELD2), mRNA.

hs|chr22:4874 A 16 P21 entg PIM3 ref|NM 00 ref|Homo sapiens pim-3 oncogene

2058- 354055 1001852 (PIM3), mRNA.

48742103

hs|chr22:4878 A 14 P10 entg|FLJ41 ref]NM 00 ref]Homo sapiens FLJ41993 protein

5453- 7287 993 1001694 (FLJ41993), mRNA.

48785513

hs|chr22:4879 A 16 P21 entg|LOCl refjNM 00 refj Homo sapiens similar to RIKEN

8354- 354142 64714 1080447 cDNA 1700019P01 (LOCI 64714),

48798413 mRNA.

hs|chr22:4883 A 16 P03 entg MLC l ref|NM 13 ref|Homo sapiens megalencephalic

9988- 641053 9202 ref|N leukoencephalopathy with

48840048 M 015166 subcortical cysts 1 (MLC1),

transcript variant 2, mRNA.

hs|chr22:4887 A 16 P41 entg|MOVl ref|NM_01 ref|Homo sapiens MovlOll ,

2720- 557230 0L1 8995 Moloney leukemia virus 10-like 1 ,

48872776 homolog (mouse) (MOV10L1), mRNA.

hs|chrX: 10006 A 14 Pl l entg|WWC ref|NM 01 refj Homo sapiens WWC family 847-10006907 2163 3 5691 member 3 (WWC3), mRNA.

hs|chrX: 10085 A 14 P10 entg CLCN ref|NM 00 ref|Homo sapiens chloride channel 077-10085133 81 16 4 1830 4 (CLCN4), mRNA.

( hromosoma Agilent (■ene

1 Location Probe I I) SMiibol Accession Description

Table 2

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein as well as all accession numbers, Agilent probe IDs and GenBank IDs, particularly those referenced in Tables 1-3, are hereby incorporated by reference in their entirety for all purposes.

EXAMPLES

In order to identify conserved regions of DNA of fetal origin in maternal whole blood the following experimental design was employed. The culmination of the process described below has yielded both regional and sequence specific targets that are used for the identification of fetal DNA in the context of maternal DNA. The experimental process has four major components including: (1) gentle lysis of maternal whole blood DNA and size specific bead-based DNA extraction, (2) fetal DNA enrichment and detection using size selection and digital PCR, (3) subtractive hybridization of maternal, fetal fractionated and fetal DNA using array comparative genomic hybridization (CGH) to identify conserved genomic regions in fetal DNA and (4) target specific next generation sequencing to identify condition/disease related loci for diagnostic assay development.

EXAMPLE I: Dx Lysis for fetal DNA Extraction

Isolation of fetal DNA from whole blood presents unique challenges. The two confounding variables in maximizing the yield of fetal DNA from whole blood is the selective lysis and disaggregation of target specific cells and DNA in order to efficiently extract them from the background of maternal genomic DNA. To accomplish this task a buffer and protocol that accomplishes two critical goals was formulated. First, the gentle lysis procedure selectively lyses cells that are not in their optimal growth environment (i.e. fetal trophoblasts) allowing for the release of nucleic acid from these cells that are otherwise not present in the non-cellular DNA fraction and secondly disaggregate small DNA molecules that are not available for efficient extraction in its normal state. This lysis buffer and procedure increases the yield of fetal DNA in any given maternal whole blood sample by approximately 15%. Following lysis an automated process for DNA extraction was employed on the Qiagen Symphony Dx instrument. This instrument utilizes bead based chemistry to extract high quality DNA from whole blood (or in this case gently lysed produced) samples. The chemistry being used for extraction was modified to work in concert with the Dx lysed product and is optimized to preferentially isolate "small" DNA products over high molecular weight genomic DNA species. This led to an enrichment of fetal DNA in each sample when compared to standard practice for DNA extraction which is critical to maximize detection of mutations that are fetal specific.

Briefly, samples consist of 8mL to lOmL of whole blood in an ACD tube. The samples were stored at 2°-8° C and were processed within 8 hours of receipt. The ACD tubes were gently inverted three times to mix the blood and 10 mL of whole blood is then removed and placed in a clean 15mL conical-bottom tube. The BioDx 20 buffer (0.32M sucrose, 5mM MgCl₂, 3% Triton X-100, Saponin 0.1%, lOmM Tris-HCl, pH 7.3) was then added at 10%) by volume, for example, for 10 mL of blood, 1 mL of buffer was added. The tubes were then inverted at least 4 times and centrifuged at 3000 rpm for 5 minutes to separate the liquid layer from the lysed cell debris at the bottom of the tube. The top liquid layer of cell lysate was then removed to a second clean 15 mL conical-bottom tube taking care to not disrupt the cell debris layer. The lysate was then aliquoted into 1.2 mL aliquots and frozen for future use. A 1.2 mL aliquot of cell lysate prepared above was pipetted into a clean 2 mL tube and an automated process for DNA extraction was employed on the Qiagen Symphony Dx instrument to separate the DNA.

EXAMPLE 2: Characterization of Conserved Fetal DNA Sequences

A subtractive hybridization approach was utilized to identify fetal specific sequences in Dx lysed, size fractionated free floating DNA. Briefly, the subtractive hybridization approach requires that two CGH arrays be run for each clinical case. The first array analyzes maternal DNA against fetal DNA (a product of conception) to identify differences in fetal genomic DNA. The second array analyzes maternal DNA against enriched free floating fetal DNA (a product of maternal whole blood) to identify regions present in free floating fetal DNA. A comparative analysis of unique fetal segments from both arrays identifies regions of conservation in free floating fetal DNA samples in each case analyzed. By following this hybridization scheme in we can confirm which sequences are present in the free floating fetal DNA fraction when compared to the entire fetal genome. This is the first step in the conserved sequence identification process.

Differences in the free floating fetal genome relative to intact maternal and fetal DNA were identified by array CGH analysis using microarray slides, which contain 244 000 (244 K) and one million (l x l M) oligonucleotide probes (Agilent Technologies, Santa Clara, CA, USA). For sample preparation and hybridization we have followed the protocol developed and described in detail by Agilent. Briefly, genomic DNA was extracted from as described above. The integrity of DNA was confirmed with nanodrop and agarose gel electrophoresis. For array CGH without WGA, we used 2.5 μg of fetal DNA and 2.5 μg of maternal DNA for each analysis. DNA was digested with Rsa I and Alu I and labeled by random priming using either Cy5-dUTP or Cy3-dUTP. Following purification with Microcon Centrifugation Filters, Ultracel YM-30 (Millipore, Billerica, Ma, USA), probes were denatured and pre-annealed with 50 μg of human Cot-1 DNA (Invitrogen, Burlington, Ontario, Canada). Hybridization was performed at 65 °C for 40 h with constant rotation. After hybridization, slides were washed according to the manufacturer's instructions and scanned immediately with a DNA Microarray Scanner (Agilent Technologies). Data were extracted from scanned images using Feature Extraction software, version 10.7.3.1 (Agilent). The text files were then imported for analysis into Genomic Workbench, standard edition 5.0.14 (Agilent). We used the reference maternal DNA to identify DNA copy number aberrations. The algorithm used identifies all aberrant intervals in a given sample with consistently high or low log ratios based on the statistical score. It then samples adjacent probes to arrive at an estimation of the true range of the aberrant segment (aberrant being under represented as is the case with fetal fractionated samples). The statistical score represents the deviation of the average of the log ratios from the expected value of zero, in units of standard deviation. The algorithm searches for intervals in which a statistical score based on the average quality weighted log ratio of the sample and reference channels exceeds a user specified threshold. We applied a filtering option of minimum of 5 probes in region and minimum absolute average log2 ratio > 0.3. USCS human genome assembly hgl8 was used as a reference and copy number variations (CNV) were identified with a database integrated in the Agilent Genomic Workbench analytic software.

During analysis with CGH analytics software, the sensitivity threshold was 6.0 and the moving average window was 1 Mb. In order to determine that there was a change in a particular locus, three criteria must have been met. These were positive call by the software, presence of 10 consecutive probes pointing out the same direction, and 1.5-fold average fold difference in the test DNA compared to the reference normal DNA. EXAMPLE 3: NextGen Sequencing

In order to fully understand the length and fidelity of sequence identified by array CGH this NextGeneration sequencing approach is employed to validate and finally map conserved loci in the free floating fetal genome. The loci sequenced are derived from the conserved probed sequences identified with array CGH described above. Briefly, the conserved probe sequences identified to be present in free floating fetal DNA were used as "bait" to create the capture libraries used for sequencing the entire segments of conserved free floating fetal DNA. The extent of natural genomic variation between individuals creates an additional problem when predicting conservation of fetal DNA between individuals. Hence, it is prudent to have available constitutional ("normal") DNA as well as fetal DNA from the same individual as a potential reference, in this instance it is maternal DNA. For DNA analysis, a targeted sequencing approach using paired end genomic libraries was used. Sequence capture of conserved array CGH was performed by solution hybridization and recovered using the Agilent SureSelectXTTM system. The bait for the 30 target genes selected for this application covers all conserved fetal regions and the flanking 10 bp for interrogating splice/donor/acceptor sites and branch site mutations, and was designed using Agilent's eArray https://earray.chem.agilent.com/earray.

In brief, isolated DNA was sheared to a target size of 150-200bp with a Covaris AFA instrument, purified with Agencourt AMPure™ XP beads, and quantified using cuvetteless spectroscopy and quality determined with the Agilent 2100 bioanalyzer. The DNA ends are blunt-ended with T4 polymerase, repurified and modified by 3' addition of an A nucleotide. Following one more round of bead purification, bar-coded paired-end adapters were ligated to the DNA fragments which are then PCR amplified for five cycles using the SureSelect™ Indexing Pre-Capture PCR (reverse) primer. After another purification round, the libraries were hybridized to biotinylated bait in solution and recovered on streptavidin-coated paramagnetic beads. Hybridization was carried out in the presence of oligonucleotide blockers complementary to minimize the formation of chains or circles which can potentially reduce enrichment levels. Genomic fragments were index tagged by post-hybridization amplification and pooled in equimolar concentrations for balanced sequencing. Sequencing was done with paired lOObp read at a density of about 700 clusters/mm². All sequence analysis and mutation detection was performed using commercially available software (e.g.

SeqNext, NextGene, ZOOM, MAQ). These approaches were used to verify the primary sequence data alignments and reports the genotype at all dbSNP130 on the depth of coverage and improved concordance rates with other genotyping platforms (e.g. illumina HumanOmni 1 million SNP chip) from 96% to >99%. The primary sequencer output is in *.bcl binary files (base calls per cycle) which are converted to complete reads with quality scores (*.qseq files or quality and sequence files) each read and a third for the indexing read per tile. This is a necessary but relatively quick process and was done using the BCL converter provided with the software package. The 32 qseq files/lane were then converted to .fastq (text-based format for storing nucleotide sequence) as they undergo demultiplexing into their individual sample data and combined into 2 files per sample, one for each read of the paired run. Files were given unique names according to the convention sampleID_flowcellID_lane#_read#. fastq so that sample data collected on different runs and/or different lanes can be placed at the same file structure level. Once all the runs/lanes scheduled to contain data for a given sample have been demultiplexed the reads were aligned to the reference genome, chosen through the web interface for each sample. We used the Burrows-Wheeler Transform method implemented in the BWA (Burrows-Wheeler Alignment) package which we find as having better

performance than other aligners we have tested (ELAND, Bowtie, Zoom, MAQ) in terms of quality of alignments, number of reads aligned and capacity to open gaps. Upon alignment request .fastq files are split into 10M reads chunks and a BWA process is spawned on the cluster. Each instance of BWA produces an alignment in .SAM format and all .SAM files for single samples are concatenated into a final alignment result file for that sample with a unique naming following the convention sampleID_flowcellID_lane#.sam. Collectively, these methods have identified 67,848 conserved regions across 30 different independent subjects and correlated the conserved regions to 157 unique disease mutations. Furthermore, the methods have identified 70% of prenatal markers currently used in standard genetic analysis and conserved regions across the entire genome providing for novel targets of investigation. The vast amount of data uncovered from the methods of the current invention are useful in targeted diagnostics by identifying targets for assay development, global screens to explore the fetal DNA genome as a screening tool for early risk assessment, as well as for "follow up" diagnostics employing fetal DNA as a tool for postnatal analysis. EXAMPLE 4: Identification of Fetal Genetic Disorders Using Combination of

Preserved DNA Regions, Gentle Lysis and Differential Methylation Analysis

It has been discovered that there are DNA methylation differences between fetal DNA and Maternal DNA. Methylation occurs on the cytosine bases that are adjacent to a guanine base (CpG). Where CpGs are formed in groups or clusters, all of the cytosine bases in the group are methylated if methylation is present. Treatment of unmethylated CG with bisulfide converts the unmethylated cytosine to Uracil which can be detected by Polymerase Chain Reaction (PCR) techniques. In addition methylated DNA can be separated from non- methylated DNA by either binding with antibodies to methylated DNA or by hybridizing to single strand DNA sequences specific to the uracil DNA conversion product. Separating the methylated DNA from non-methylated DNA increases the concentration of fetal DNA in a sample and increases the detectability of fetal genetic mutations. Table 3 shows a list of genetic disorders that are particularly amenable to detection using a combination of extraction of fetal DNA from a maternal host sample following gentle lysis of compromised fetal cells and enriching methylated and/or unmethylated DNA followed by PCR detection of conserved fetal DNA regions in the methylated and/or unmethylated DNA fractions. As would be understood by the skilled person, some genetic markers of interest will be present in the unmethylated fraction of fetal DNA while others will be present in the methylated fraction of DNA.

Table 3 - Diseases amenable to analysis by methylation isolation.

Disease gene(s)

Marfan Syndrome FBN1 gene

Hemophilia

Duchene Muscular Dystrophy Dystrophin gene

Familial Hypercholesterolemia CYP21B, C4 and LDLRAP-1 genes

Canavan IKBKAP gene chromosome 9

Fanconi Anemia FA genes chromosome 15

Gaucher Dosease beta-glucosidase

Mucolipidosis Type IV MCOLN1 gene

Niemann Pick SMPD1 gene

Neurofibromatosis Neurofibromin 17ql l .2, merlin 22ql 2

EXAMPLE 5: Identification of Fetal Trisomy or Monosomy by Quantitative DNA

Assay A biological sample from the maternal host is enriched for fetal DNA using nucleic acid sequence based isolation of specific DNA segments. Single strand nucleic acids that hybridize to a desired target DNA sequences (e.g., a genetic marker for a particular disease) is bound to beads or other suitable support and exposed to the sample DNA. The desired target DNA sequences specifically hybridizes to the single strand DNA attached to the beads or other suitable support. Because fetal DNA is degraded and broken into small segments, it preferentially hybridizes to the nucleic acid attached to the suitable support due to reaction kinetics, yielding a higher purity fetal DNA sample. Thus, such enriched fetal DNA can then be used in many settings, including diagnostic settings where quantitation (which may require more pure DNA) is required, such as for detection of aneuploidy and deletions. In such instances, a reference sequence (e.g., a disomy reference sequence) is used to compare against the target sequence to determine quantities of target sequence.

Assays for trisomy 13, 18 and 21 are performed by attaching sequences that hybridize to conserved fetal genomic regions 13ql2-13ql3, 13q34 on chromosome 13, 18ql l , 18ql 1.3 and 18q21 on chromosome 18, 21q22.1 to 21q22.3 on chromosome 21 , 15ql l-15ql2, 15q22.3-15q23, 15ql5.1-15ql5.3 on chromosome 15 and 17ql l .2, 17p l l .2, 17ql2 to beads or other suitable support. A maternal blood sample is lysed using gentle lysis as described herein and the extracted DNA is mixed with the prepared beads. The selected (hybridized) DNA is eluted and quantified using digital PCR (see e.g. , Proc Natl Acad Sci USA 1999, 96:9236-9241 ; US patent 6143496; Proc Natl Acad Sci USA 100 (15): 8817-22), other quantitative PCR methods, and/or any of a variety of nextgen sequencing technologies known to the skilled person (e.g. , commercially available from Illumina, San Diego, CA; ABI, Foster City, CA; and others). Single strand nucleic acids that hybridize to chromosome 15 and 17 disomy reference sequences (or other suitable control reference sequence) are used as control reference sequences. The amount of DNA quantified in the sample for chromosomes 13, 18 and 21 is compared to the amount of disomy reference DNA, thereby allowing for the detection of the 50% increase in DNA for a trisomic chromosome (3 chromosomes instead of two) or a 50% decrease in DNA for a monsomic chromosome (one chromosome instead of two).

Claims

CLAIMS: What is claimed is:

1. A method of detecting a genetic disorder in a fetus comprising:

separating methylated DNA from unmethylated DNA in a biological sample obtained from a maternal host of the fetus to provide a methylated DNA fraction and an unmethylated DNA fraction;

detecting a first genetic marker in the methylated DNA fraction or a second genetic marker in the unmethylated DNA fraction;

wherein the first and second genetic marker each are associated with a genetic disorder listed in Table 3 and are within one or more conserved genomic segments; and

wherein the first genetic marker is predetermined to be methylated in fetal DNA while the second genetic marker is predetermined to be unmethylated in fetal DNA;

wherein the presence of the first or second genetic marker is indicative of the genetic disorder.

2. The method of claim 1, wherein the biological sample is a biological sample of the maternal host enriched for fetal DNA.

3. The method of claim 1, wherein the biological sample is confirmed for the presence of fetal DNA.

4. The method of claim 1, wherein the genetic marker is associated with skeletal dysplasia and is present in the methylated DNA.

5. The method of claim 4, wherein the genetic marker is a mutation in the FGFR3 gene.

6. The method of claim 1, wherein the methylated DNA is separated using an antibody.

7. The method of claim 1, wherein the methylated DNA is separated using DNA hybridization.

8. The method of claim 1 wherein the presence or absence of the genetic marker is detected using PCR or sequencing.

9. The method of claim 1 wherein the genetic marker associated with the genetic disorder is predetermined to be methylated in fetal DNA.

10. The method of claim 1 wherein the genetic marker associated with the genetic disorder is predetermined to be unmethylated in fetal DNA.

11. A method for determining aneuploidy in a fetus comprising:

a. contacting DNA extracted from a biological sample obtained from a maternal host of a fetus, wherein the biological sample comprises fetal DNA, with a first nucleic acid, wherein the first nucleic acid specifically hybridizes to a conserved genomic segment comprising a genetic marker for aneuploidy;

b. contacting the DNA extracted from the biological sample with a second nucleic acid, wherein the second nucleic acid specifically hybridizes to a disomy reference DNA sequence;

c. isolating the DNA extracted from the biological sample that specifically hybridizes to the first nucleic acid and the second nucleic acid;

d. quantifying the amount of isolated DNA that specifically hybridizes to the first nucleic acid;

e. quantifying the amount of the isolated DNA that specifically hybridizes to the second nucleic acid;

f. comparing the amount of isolated DNA from (d) to an amount of isolated DNA from (e), wherein a 50% increase in DNA in (d) as compared to the amount of DNA from (e) indicates the presence of a trisomic chromosome and wherein a 50% decrease in DNA in (d) as compared to the amount of DNA from (e) indicates the presence of a monosomic chromosome; thereby determining aneuploidy in the fetus.

12. The method of claim 11 wherein the first nucleic acid is attached to a support.

13. The method of claim 12 wherein the support is a bead.

14. The method of claim 11 wherein the quantifying is performed using digital PCR or nextgen sequencing.

15. The method of claim 11, wherein the conserved genomic segment comprising a genetic marker for aneuploidy is selected from the group consisting of 13ql2-13ql3, 13q34, 18ql l, 18ql l .3, 18q21, 21q22.1-21q22.3, 15ql l-15ql2, 15q22.3-15q23, 15ql5.1-15ql5.3, 17ql l .2, 17pl l .2, and 17ql2.

16. The method of claim 11, wherein the disomy reference DNA sequence is on chromosome 15 or 17.

17. A method for enriching fetal DNA from a biological sample from a maternal host of a fetus comprising: contacting DNA extracted from the biological sample obtained from the maternal host of the fetus, wherein the biological sample comprises fetal DNA, with a nucleic acid, wherein the nucleic acid specifically hybridizes to a conserved genomic segment and wherein the nucleic acid is attached to a support.

18. The method of claim 17 wherein the support is a plate, a bead, a microsphere, a nanoparticle, or a colloidal particle.

19. The method of claim 17 wherein the nucleic acid is single stranded DNA.