WO2016059601A1

WO2016059601A1 - Non-invasive methods for detection of genetic abnormalities in an unborn fetus, and primers, probes and kits for uses thereof

Info

Publication number: WO2016059601A1
Application number: PCT/IB2015/057945
Authority: WO
Inventors: Isaac Jacques KADOCH; Dominique BÉRUBÉ; Maryse GAGNÉ; Cécile Le Saint
Original assignee: Group Ovo Inc
Current assignee: Group Ovo Inc
Priority date: 2014-10-16
Filing date: 2015-10-15
Publication date: 2016-04-21
Anticipated expiration: 2017-04-16

Abstract

Described herein are methods, primers, probes and kits useful in the prenatal detection of undesirable genetic abnormalities in an unborn fetus, such as aneuploidies. The methods render possible the detection of the genetic abnormalities in the unborn fetus without having to sequence fetal DNA but instead by measuring accurately in maternal plasma levels of free fetal DNA corresponding to the genetic abnormality. The measuring comprises amplifying free fetal DNA from two different regions (DNA associated to the abnormality and DNA associated to a reference gene) and, though calculations and statistics, obtaining a relative ratio of abundance for free fetal DNA corresponding to the genetic abnormality. This relative ratio of abundance is indicative of presence or absence of the predefined genetic abnormality to be detected. In one embodiment, the amplifying comprises employing a droplet digital PCR (ddPCR).

Description

NON-INVASIVE METHODS FOR DETECTION OF GENETIC ABNORMALITIES IN AN UNBORN FETUS, AND PRIMERS, PROBES AND KITS FOR USES THEREOF

FIELD OF THE INVENTION

The invention relates to the field of medicine, and more particularly to non-invasive prenatal detection of undesirable genetic abnormalities in an unborn fetus, such as aneuploidies.

BACKGROUND OF THE INVENTION

Modern prenatal screening and diagnostic began in the 60's and 70's with the deployment of the techniques of amniocentesis and echography. In 1997, it was reported for the first time that free fetal DNA could be detected in the plasma of the mother. That discovery opened up a new field of non-invasive prenatal testing (NIPT) directed in identifying undesired genes or mutations in the genome of the fetus. It is now known that free fetal DNA can be detected from the 4^th week of pregnancy and that free fetal DNA can constitute more than 4% of the DNA circulating in the plasma of the mother from the 10^th week of pregnancy.

Different approaches have been used for NIPT, including shotgun massively parallel sequencing (s-MPS), targeted massively parallel sequencing (t-MPS), single nucleotide polymorphism (SNP) based approaches, methylated DNA-based approaches, digital PCR and RNA-based testing.

However, these approaches have different limitations such as costs, delays, lack of sensitivity, false positives or false negatives, impossibility to use for twin or multiple pregnancies, impossibility to use when an egg is donated, or they have been validated only in high risks pregnant women.

There is thus a need for more reliable, effective and cheaper NIPT tools and methods and allowing prenatal detection of undesirable genetic abnormalities, including tools and methods which can be used in the clinic.

One of the problems limiting the analysis of fetal DNA is related to the fact that the fraction of fetal DNA circulating in the mother's plasma is really poor compared to maternal DNA fraction (14, 15). Accordingly, many researchers worked on increasing free fetal DNA in maternal plasma by standardization of blood-processing (16-19), by genome amplification (20-22), by improving sampling maternal plasma (23-25) and DNA extraction (26).

Digital PCR was first introduced in 1992 (1 ). Since the purpose of Digital PCR was to quantify PCR targets rather than the PCR amplified products in a sample, the technique was limited in terms of dilution, PCR and Poisson statistics. Further evolutions of the technology allowed for more practical use of this method with a wider audience with small partitions created by emulsion droplets and/or microfluidics (2-5). Microfluidic chamber digital PCR (cdPCR) has represented an improvement for quantifying small fraction of cell free DNA (6-10). However, there have been practical limitations, primarily cost to the number of technical replicates that can be analyzed by cdPCR. Using these two technologies, detection of trisomy 21 in maternal plasma has been demonstrated feasible but it was labor-consuming and needed enrichment step for increasing fetal DNA quantity (27). Recently, a new dPCR format called droplet digital PCR (ddPCR) has been commercialized by Bio-Rad (QX100™ Droplet Digital PCR System) (1 1 ,12). A single ddPCR well may contain approximately 20,000 partitioned droplets corresponding to about 25 times the 765 chambers of a single sample panel on a microfluidic cdPCR array. However, no one has ever envisioned employing ddPCR for the analysis of free fetal DNA, let alone for NIPT or prenatal screening of aneuploidies.

The present invention addresses these needs, as it relates to methods, primers, probes and kits useful in the prenatal detection of undesirable genetic abnormalities in an unborn fetus.

Additional features of the invention will be apparent from review of the disclosure, figures, and description of the invention below.

BRIEF SUMMARY OF THE INVENTION

According to a first aspect, the invention is concerned with a non-invasive method for detecting a predefined genetic abnormality in an unborn fetus, comprising the steps of:

(a) obtaining a biological sample from a pregnant subject carrying said unborn fetus, wherein said biological sample comprises free fetal DNA and DNA from said pregnant subject;

(b) isolating the free fetal DNA and the DNA from the pregnant subject and quantifying said DNA for calculating a percentage of free fetal DNA in said biological sample;

(c) amplifying isolated fetal DNA comprising genomic sequences for a first genomic region and a second genomic region, the first region comprising a genomic sequence associated with a predefined genetic abnormality to be detected, the second region comprising a reference genomic sequence associated with a genomic region for which no genetic abnormality is expected;

(d) calculating a relative ratio of abundance of amplified DNA corresponding to the first and second regions;

(e) adjusting said relative ratio of abundance in function of said percentage of free fetal DNA for obtaining an adjusted ratio; wherein said adjusted ratio is indicative of presence or absence of the predefined genetic abnormality to be detected.

According to a second aspect, the invention is concerned with a non-invasive method for detecting aneuploidy in an unborn fetus, comprising the steps of:

(a) obtaining a biological sample from a pregnant subject carrying said unborn fetus, wherein said biological sample comprises free fetal DNA and DNA from said pregnant subject ;

(b) detecting presence or absence of an abnormally elevated ratio of abundance for free fetal DNA;

wherein said abnormally elevated ratio is indicative of aneuploidy.

According to a preferred embodiment, the detecting does not comprise sequencing free fetal DNA. Instead, the detecting comprises employing a droplet digital PCR (ddPCR).

According to a third aspect, the invention is concerned with a method for detecting aneuploidy in an unborn human fetus, comprising the step of measuring abundance of free fetal DNA in plasma from a pregnant woman carrying said unborn fetus, wherein said measuring comprises employing a droplet digital PCR (ddPCR), and wherein abnormally elevated abundance of free fetal DNA is indicative of aneuploidy.

According to a further aspect, the invention is concerned with a primer for DNA amplification, said primer comprising a sequence selected from the group consisting of SEQ ID NOs 1 to 72.

According to a further aspect, the invention is concerned with a probe for DNA detection, said probe comprising a sequence selected from the group consisting of SEQ ID NOs 83 to 1 12.

According to a further aspect, the invention is concerned with the general use of a droplet digital PCR (ddPCR) apparatus, for the detection of a predefined genetic abnormality in an unborn fetus.

The invention further relates to a kit for the detection of a predefined genetic abnormality in an unborn fetus. In embodiments, the kit is a diagnostic kit which comprises a primer and/or a probe as defined herein.

An advantage of the invention is that it provides means and nucleic acid molecules suitable for non-invasive, quick and reliable detection of undesirable genetic abnormalities in an unborn fetus. According to preferred embodiments, the present invention allows discrimination between normal and trisomic fetuses. Additional aspects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments which are exemplary and should not be interpreted as limiting the scope of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In the following description of the embodiments, references to the accompanying drawings are by way of illustration of an example by which the invention may be practiced. It will be understood that other embodiments may be made without departing from the scope of the invention disclosed.

A) General overview of the invention

To date, non-invasive prenatal testing (NIPT) is relatively costly, unreliable and mainly based on sequencing of free fetal DNA circulating in the plasma of the mother.

The inventors have demonstrated that it is possible to detect undesirable genetic abnormalities in an unborn fetus without sequencing the free fetal DNA techniques. Sequencing can be avoided by measuring accurately levels of the free fetal DNA corresponding to the genetic abnormality (e.g. extra DNA from chromosome 21 for trisomy 21). The measurements comprise amplifying free fetal DNA from two different regions (abnormality and reference) and, through calculations and statistics, obtaining a relative ratio of abundance for free fetal DNA corresponding to the genetic abnormality, that ratio being indicative of presence or absence of the predefined genetic abnormality to be detected.

The inventors have also designed primers and probes for amplifying specific free fetal DNA fragments, e.g. DNA fragments for chromosomes 13, 18 and 21 which are associated with trisomy 13, trisomy 18, and trisomy 21 , respectively.

The inventors have also demonstrated that droplet digital PCR (ddPCR) may be successfully used for the analysis of free fetal DNA, including NIPT and prenatal screening of aneuploidies.

B) Diagnostic methods

As indicated hereinbefore and exemplified hereinafter, one aspect of the invention concerns methods for prenatal detection of undesirable genetic abnormalities in an unborn fetus.

According to a first aspect, the invention relates to a method for detecting a predefined genetic abnormality in an unborn fetus, comprising the steps of:

(a) obtaining a biological sample from a pregnant subject carrying said unborn fetus, wherein said biological sample comprises free fetal DNA and DNA from said pregnant subject; (b) isolating the free fetal DNA and the DNA from the pregnant subject and quantifying said DNA for calculating a percentage of free fetal DNA in said biological sample;

(e) adjusting said relative ratio of abundance in function of said percentage of free fetal DNA for obtaining an adjusted ratio;

wherein said adjusted ratio is indicative of presence or absence of the predefined genetic abnormality to be detected.

In one embodiment, the adjusted ratio is greater than 1 :1 (first region : second region). In other embodiments, the adjusted ratio is greater than 1 .2:1 ; or greater than 1 .3:1 ; or greater than 1 .4:1 ; or greater than 1 .5:1 ; or greater than 1 .6:1 ; or greater than 1 .7:1 .

In one embodiment, the amplifying comprises employing a droplet digital PCR (ddPCR).

In one embodiment, the predefined genetic abnormality is aneuploidy (e.g. trisomy 13, trisomy 18, or trisomy 21).

In embodiments, targeted genes (i.e. first genomic region) for aneuploidies of chromosome 21 are selected from SOD1 , DSCR8 and PLAC4. In embodiments, the targeted genes for aneuploidies of the chromosome 18 are selected from LPIN2, TGIF1 , PHLPP1 and TAF4B. In embodiments, the targeted genes for aneuploidies of the chromosome 13 are selected from SLITRK1 , RB1 and ITM2B.

A single or a plurality of genes can be used for reference purposes according to the invention (i.e. second genomic region). The reference gene(s) can be found on any chromosome other than the targeted chromosome (e.g. 13, 18, 21). Preferably, the reference chromosome is a "conserved" chromosome for which aneuploidy is rare or unlikely. In one embodiment, the reference chromosome is chromosome 1 , and the reference gene is selected from FM03, EIF2C4, PTBP2 and ZBTB18. In another embodiment, the reference chromosome is chromosome 12 and the reference gene is GAPDH. In one embodiment, step (c) of amplifying comprises amplifying DNA corresponding to the genes listed in Table 1.

In embodiments, the first genomic region is selected from SLITRK, LPIN2, SOD1 , DSCR8, RB1 , PHLPP1 , PLAC4, TGIF1 , TAF4B and ITM2B. In embodiments, the second genomic region is selected from GAPDH, FM03, ZBTB18, EIF2C4, andPTBP2.

In one embodiment, step (b) of quantifying free fetal DNA in the biological sample in the mother's blood comprises using methylation-sensitive restriction enzymes for specifically cutting maternally-derived DNA while leaving intact placental or foetus-derived DNA. In embodiments, the DNA is DNA from RASSF1 (chromosome 3) and/or SERPINB5 (chromosome 18). In embodiments, the ZFX and ZFY genes are used to determine the sexual chromosomes.

According to a further aspect, the invention relates to a non-invasive method for detecting aneuploidy in an unborn fetus, comprising the steps of:

wherein said abnormally elevated ratio is indicative of aneuploidy.

In one embodiment, the detecting does not comprise sequencing free fetal DNA, but comprises employing a droplet digital PCR (ddPCR).

In one embodiment, the biological sample is plasma. However, it is conceivable that other biological samples such as whole blood, serum, urine and saliva may be used.

In one embodiment, the detecting comprises measuring a relative ratio of abundance of fetal DNA comprising genomic sequences from a first and a second genomic region, the first region comprising a DNA sequence associated with a predefined genetic abnormality to be detected, the second region comprising a reference DNA sequence for which no genetic abnormality is expected;

wherein a ratio abundance of DNA for the first region : DNA for the second region greater than 1 :1 is indicative of aneuploidy. In other embodiments, a ratio abundance that is indicative of aneuploidy is a ratio abundance of DNA for the first region : DNA for the second region greater than 1 .2:1 ; or greater than 1 .3:1 ; or greater than 1 .4:1 ; or greater than 1 .5:1 ; or greater than 1 .6:1 ; or greater than 1 .7:1 .

According to a further aspect, the invention relates to a method for detecting aneuploidy in an unborn human fetus, comprising the step of measuring in plasma from a pregnant woman carrying said unborn fetus abundance of free fetal DNA, wherein said measuring comprises employing a droplet digital PCR (ddPCR), and wherein abnormally elevated abundance of free fetal DNA is indicative of aneuploidy.

In embodiments, the aneuploidy is trisomy 13, trisomy 18, or trisomy 21 .

As used herein, the term "free fetal DNA" refers to small DNA fragments (i.e. about <300 base pairs) circulating in maternal plasma (i.e. excluding DNA contained in fetal cells that may circulate in the maternal plasma).

As used herein, the term "subject" includes female viviparous animals carrying an embryo or fetus inside. The term "subject" includes animals such as mammals. Preferably, the subject is a mammal, including, but not limited to, species such as a human, a dog, a cat, a horse, a bovine, a rabbit, a rat, a mouse, and wild animals living in zoos (e.g. lion, tiger, elephant, panda, bear, etc.). More preferably, the subject is a woman. The subject may share genetic information with the fetus (i.e. the subject is the parent), or the subject may have no direct genetic connection with the fetus (e.g. the subject is a carrying mother or the fetus originates from a donated egg).

In some embodiments, the subject may be at risk of carrying an abnormal fetus. For instance, risk factors for congenital anomalies (also referred as birth defects) include, mother's age (e.g. > 35 years), genetic factors (e.g. consanguinity), infections (e.g. syphilis and rubella), maternal nutritional status (e.g. iodine deficiency, folate insufficiency, obesity, or diabetes mellitus), environmental factors (e.g. maternal exposure to pesticides, medications, alcohol, tobacco, and other psychoactive substances, certain chemicals, high doses of radiation) and socioeconomic factors.

In some embodiments, the subject has a low risk of carrying an abnormal fetus (e.g. women < 25 years old).

As used herein, the term "predefined genetic abnormality" refers to a particular genetic disease, condition or birth defects to be detected in an unborn fetus. In one embodiment, the predefined genetic abnormality is aneuploidy, i.e. a condition in which the number of chromosomes in the nucleus of a fetus is not an exact multiple of the monoploid number of a particular species. In one embodiment, the predefined genetic abnormality is an extra copy of a chromosome, including but not limited to chromosome 13 (trisomy 13), chromosome 18 (trisomy 18), and chromosome 21 (trisomy 21). In other embodiments, the predefined genetic abnormality is an extra copy of chromosome X (Klinefelter syndrome), or any other chromosome (triploidy). In other embodiments, the predefined genetic abnormality is one copy of chromosome X (Turner syndrome. The present invention may also be useful for the detection of tetrasomy and pentasomy (i.e. presence of four or five copies of a chromosome).

In some embodiments, two or more predefined genetic abnormalities are detected or assessed in combination. As used herein, the term "combination" refers to a test or method where two or more genetic abnormalities are assessed together. As used herein, "combination" also encompasses tests or methods where two or more genetic abnormalities are assessed separately. For instance, "combination" encompasses means in which the first (or second or third) abnormality is assessed, wherein second (or first or third) abnormality may have been previously been assessed. The assessment of the two or more abnormalities may also be executed step-wise by the same or by different actors and by similar or by different techniques. For example, one actor may assess the first abnormality with one given technique, and a second actor may assess the second and/or third abnormality by using the same or different technique(s). The assessment steps may be executed at the same time, or nearly the same time, or at distant times, in the same or in different sample(s) so long as it is possible to obtain a combined assessment on the fetal condition. In a preferred embodiment, the two or more abnormalities are assessed simultaneously in the same biological sample, or in different samples within a relatively short period of time (i.e. less than 1 week). In particular embodiments, the two or more abnormalities are assessed starting from a single biological sample. In particular embodiments, the two or more abnormalities are assessed simultaneously or in parallel during the same amplification procedure. In specific embodiments, the methods comprise detecting within the same clinical test or clinical analysis presence or absence of trisomy 13, trisomy 15, and trisomy 21 .

In a preferred embodiment, the biological sample is plasma. Typically blood samples are drawn to preserve free fetal DNA (e.g. cell-free DNA BCT tubes) according to known techniques (Fernando et al, 2010, Das et al, 2012). Small DNA fragments (i.e. about <300 base pairs) are extracted (free fetal DNA is mainly composed of small DNA fragments) using suitable DNA extraction protocols (e.g. Qiagen™). Quantitative analysis of fetal DNA in maternal plasma is calculated for each patient and for the selected gene(s), using the principles there exist a difference in DNA methylation of fetal DNA vs. maternal DNA. For instance, for RASSF1 fetal DNA is hypermethylated in the placenta while RASSF1 DNA is hypomethylated in maternal bood cells (see for instance Chan et al, 2006). Accordingly, RASSF1 DNA fragments hypermethylated from the fetus and RASSF1 DNA fragments hypomethylated from the mother are circulating in the maternal blood. For other genes like SERPIN it is the opposite: maternal DNA is methylated or hypermethylated whereas fetal DNA is not methylated (Tong Y.K et al, Clinical Chemistry, 2007 nov, 53(1 1 ), 1906-1914)).

In a preferred embodiment, the present invention is based on the detection of hypermethylated placental (fetal) DNA sequences circulating in maternal blood like RASSF1 . The methylation pattern of the RASSF1A promoter in the placenta and maternal blood cells allows the use of methylation-sensitive restriction enzymes for specifically cutting maternally-derived background RASSF1A sequences while leaving intact the RASSF1A sequences originating from the placenta (i.e. foetus).

Preferably, the biological sample contains at least about 4% free fetal DNA In some embodiments, the sample may comprises 5%, 6%, 7%, 8% 9%, 10%, 15%, 20% or more free fetal DNA. In some embodiments, the sample may comprise less than 4% free fetal DNA (e.g. 3.5%, 3%, 2.5%, 2%, 1 .5%. 1 % or lower).

It is known that the amount free fetal DNA is increasing with gestation. Is some embodiments, the gestational age of the subject is between 12 to 26 weeks of gestation. In other embodiments, the gestational age of the subject may be as low as 5 weeks of gestation or as high as 37 weeks. In embodiments, the gestational age is within the first trimester (about <13- 15 weeks).

Any suitable method or technique may be used for detecting, measuring or calculating abnormally elevated ratio of abundance for free fetal DNA associated with the predefined genetic abnormality to be detected.

In one embodiment, the detecting comprises employing a droplet digital PCR (ddPCR).

An advantage of using ddPCR according to the present invention is that no sequencing is requires. It is also possible to generate a great number of reactions (i.e. each individual droplet) which provides the possibility of measuring many thousands of copies (number of events) with a high level of precision, even when starting with a small amount or low concentration of DNA. In the examples, the measurements comprised amplifying free fetal DNA from two different regions (interest gene and reference gene) and, through calculations and statistics, it was possible to obtain a relative ratio of abundance for free fetal DNA corresponding to the genetic abnormality. The relative ratio is a value (e.g. number of events, concentration) adjusted according to the percentage of the fetal DNA in the original plasma sample (e.g. 90% maternal DNA; 10% fetal DNA). Accordingly, the relative ratio is indicative of presence or absence of the predefined genetic abnormality to be detected. In embodiments, a ratio abundance that is indicative of aneuploidy is a ratio abundance of DNA for the interest gene : DNA for the reference gene that is greater than 1 .1 :1 ; or greater than 1 .2:1 ; or greater than 1 .3:1 ; or greater than 1 .4:1 ; or greater than 1 .5:1 ; or greater than 1 .6:1 ; or greater than 1 .7:1 . For instance, in Example 2, the fetus of two patients were considered to be negative for trisomy 21 because the adjusted ratio was 1 .007 and 0.98 respectively, whereas the fetus of two other patients were considered to be positive for trisomy 21 because the adjusted ratio was >1 .5 and >1 .7, respectively. It is conceivable that the methods, primers, probes and kits according to the present invention may also be useful for detecting an extra or a missing copy of a sexual chromosome, including but not limited to chromosome X (Turner's syndrome), and chromosome Y. In one embodiment, the following primers are used for common amplification of the X and Y chromosomes: Forward = GGACTCAGATGTAACTGAA (SEQ ID NO: 124); Reverse = AG AAG CTAAAAC ATC ATCTG (SEQ ID NO: 125).

The methods, primers, probes and kits, according to the present invention, may also be useful for detecting the presence of the chromosomes X and Y, and accordingly, determine the sex of the unborn fetus.

It is conceivable that the methods, primers, probes and kits, according to the present invention, may also be useful in the case of twins or multiple gestations. Accordingly, in some embodiment the subject may be carrying two or more fetuses.

It is further envisioned that the methods, according to the invention, may be amenable to detection of a variety of genetic diseases, including but not limited to various chromosomal abnormality, genetic mutations (e.g. nonsense mutation or a frameshift mutation (e.g. addition, deletion, or substitution of nucleotide(s)).

D) Primers and probes

Also contemplated in the scope of the present invention are oligonucleotide primers and probes which specifically hybridize with genes located on particular chromosomes (e.g. a chromosome for which aneuploidy is common). The exact size of the primer or probe will depend on various factors and on the particular application and use of the oligonucleotide.

The term "primer" as used herein refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis. When presented with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as appropriate temperature and pH, the primer may be extended at its 3' terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product. The primer may vary in length depending on the particular conditions and requirement of the application. For example, in diagnostic applications, the oligonucleotide primer is typically 15-25 or more nucleotides in length. The primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able to anneal with the desired template strand in a manner sufficient to provide the 3' hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template. For example, a non- complementary nucleotide sequence may be attached to the 5' end of an otherwise complementary primer. Alternatively, non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.

The term "probe", as used herein, refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. The probes herein are selected to be complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to "specifically hybridize" or anneal with their respective target strands under a set of predetermined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5' or 3' end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically. Furthermore, it might be possible to use clustered regularly interspaced short palindromic repeats (CRISPR) as probe with the methods according to present invention, including ddPCR related techniques.

Nucleic acid molecules of the invention (e.g. primers and probes) may be prepared using general methods well known in the art such as synthesis from appropriate nucleotide triphosphates and isolation from biological sources. Synthetic oligonucleotides may be DNA synthesizers or similar devices. The resulting construct may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC). Long, double- stranded polynucleotides may be synthesized in stages, due to any size limitations inherent in the oligonucleotide synthetic methods.

The probes and primers, according to the invention, may be useful in various methods of molecular biology, including but not limited to, PCR amplification, including multiplex PCR amplification, Mutational Analysis/Conformation Sensitive Gel Electrophoresis (CSGE), Isolation and amplification of DNA, Allele Specific PCR, Oligonucleotide Screening Methods, Ligase Mediated Allele Detection Method, Denaturing Gradient Gel Electrophoresis, Gradient Gel Electrophoresis, Single-Strand Conformation Polymorphism Analysis.

The probes, according to the present invention, are preferably labeled, directly or indirectly, such that by assaying for the presence or absence of the probe, one can detect the presence or absence of the target sequence. Direct labeling methods include radioisotope labeling, such as with ³²P or ³⁵S. Indirect labeling methods include fluorescent tags, biotin complexes which may be bound to avidin or streptavidin, or peptide or protein tags. Examples of fluorochromes that may be coupled to probes include, but are not limited to, 56-FAM (excited at 495 nm, emitting at 520 nm), HEX (excited at 538 nm, emitting at 520 nm), VIC (excited at 538 nm, emitting at 554 nm). The probes may also be coupled to known quenchers such as 3'-Black Hole Quencher™-1 (3BHQ_1 ), 3'-lowa Black FQ™ (3IABkFQ), 3MGB, 3TAMRA, QSY and the like. Visual detection methods include, without limitation, photoluminescence, chemoluminescence, horse radish peroxidase, alkaline phosphatase, and the like. Table 1 hereinafter provides information on selected examples of probes and primers according to the invention. In particular embodiments, the methods, primers, probes and kits according to the invention comprise the use of these particular examples.

Table 1 : Selected sequences for primers and probes

* 56-FAM and 5HEX are fluorochomes coupled to the probes; 3BHQ_1 and 3IABkFQ are quenchers coupled to the probes; ZEN is minor bonding groove.

Kits irther aspect of the invention relates to kits, e.g. diagnostic kits. The kits of the invention may be eful for the practice of the methods of the invention, particularly for diagnostic applications in mans according to the prenatal detection of undesirable genetic abnormalities in an unborn us, such as aneuploidies, as described herein. kit of the invention may comprise one or more of the following elements: blood samples llecting tubes (e.g. ten ml cell-free DNA BCT tubes), a buffer for the homogenization of the blood mple(s), purified DNA to be used as controls, incubation buffer(s), substrate and assay buffer(s), jdulator buffer(s) and modulators (e.g. enhancers, inhibitors), standards, detection materials g. antibodies, fluorochromes, fluorescein-labelled derivatives, luminogenic substrates, detection lutions, scintillation counting fluid, etc.), laboratory supplies (e.g. desalting columns, reaction )es or microplates (e.g. 96- or 384-well plates), a user manual or instructions, etc. Preferably, the and methods of the invention are configured such as to permit a quantitative detection or ;asurement of amplicons, DNA or desired gene(s). In one embodiment, the kit is optimized for jplet digital PCR (ddPCR) and it further comprises components for droplet generation, lplification and visualization and digital droplet classification. r instance, a kit of the invention may comprise at least one primer or probe which specifically bridizes with nucleic acid molecules for the genetic abnormality of interest (e.g. primers or )bes as defined herein in Table 1 , reaction buffers, and instructional material. Optionally, the mer or probe may contain a detectable tag. Certain kits may contain two or more of such primers probes. In particular embodiments, a kit of the invention comprises components of the lplification system, including PCR reaction materials such as buffers and a thermostable lymerase. In other embodiments, the kit of the present invention can be used in conjunction with mmercially available amplification kits. The kit may optionally include instructional material, sitive or negative control reactions, templates, or markers, molecular weight size markers for gel ictrophoresis, and the like. ose skilled in the art will recognize, or be able to ascertain, using no more than routine perimentation, numerous equivalents to the specific procedures, embodiments, claims, and amples described herein. Such equivalents are considered to be within the scope of this 'ention, and covered by the claims appended hereto. The invention is further illustrated by the lowing example, which should not be construed as further or specifically limiting. [AMPLES e Examples set forth herein below provide exemplary methods and results showing feasibility, )idity and accuracy for detection of aneuploidies. ample 1 : Non-invasive detection of aneuploidies in maternal plasma using droplet digital :R system (ddPCR) iterials and methods:

5 patients at high risk for trisomy, after prenatal screening test, were recruited by a genetic unsellor. 25 women we excluded from the study. Women were between 21 to 50 years old, with riean age of 36 years old. The gestational age was between 12 to 26 weeks of gestation, with an erage of 17 weeks.

:er being informed by the genetic counsellor and after having obtained consent 'signature, blood mples were drawn, using one ten ml cell-free DNA BCT tubes. For confirmation of the high risk, 3H analysis was done and NIPT results were compared with FISH results.

JA extraction was carried out using the QIASymphony™ apparatus using a DNA extraction jtocol designed by QIAgen™ (Germany). Quantitative analysis of fetal DNA in maternal plasma is calculated for each patient. PCR reactions included, probes, forward and reverse primers quences that are specific for chromosomes 13, 18 and 21 , as well as reference genes, as fined in Tables 1 and 2. The probes and primers used in this validation were designed by the ;sent inventors and synthesized by Integrated DNA technologies (IDT, USA). The probes were lelled with FAM and HEX fluorochromes. oplet digital PCR Instrumentation, workflow and Data analysis laction mixture of 20 μΙ volume comprising ddPCR Master Mix™ (Bio-Rad™), relevant forward, /erse primers and probes for the reference genes or the genes of interest (see Tables 1 and 2) ire prepared and added to DNA sampling. Each 20 μΙ was dispensed into a separate well of a ;posable eight channel generator cartridge according to a ddPCR workflow schematized and scribed previously (Pinheiro et al, 2012). r the PCR amplification, two different apparatus were used: C1000 Touch (Bio-Rad™) and the istercycler^® (Eppendorf™). Thermal cycling conditions were adjusted according to the genes to amplified and particular primers and probes used in the procedures (see Table 2).

:er amplification, determination of the resulting amount of amplified DNA in each well was done ing an automated droplet reader singulating droplets before reading (Bio-Rad QX100™ Droplet gital PCR System). Discrimination between droplets that did not contain target (negatives) terest gene or reference gene] and those which did contain target (positives) [interest gene or erence gene] was achieved manually in QuantaSoft™ (Bio-Rad™), the software provided with ; ddPCR system for acquisition and analysis. oplets appeared stable through the entire process including pipet manipulations, thermal cycling d reading. For better accuracy, the analysis included a rejection criteria for excluding wells ving a low number of droplets (<10,000) per 20 μΙ. Typically, for each experiment, the droplet ader counted over 13,000 gated droplets per well (ranging from 12,000 to 16,000 droplets per ill). For each one of the experiments, at least four wells per probe were analysed for each patient d for each sequence, and the results of the four individual wells were then merged together. :er the merger, the ratio of interest gene on reference gene was calculated automatically by the ftware (QuantaSoft™) and thereafter, adjusted for each probe in function of the percentage of e fetal DNA (i.e. see above, free fetal DNA (methylated) vs. total circulating DNA). isults: this cohort of 275 patients with high risk for fetal trisomy were recruited. 25 patients were eluded for different reasons such that 250 patients remained in the study.

:er ddPCR with probes specific for chromosomes 13, 18 and 21 , we found 24 trisomies 21 , 5 iomies 18 and 2 trisomies 13. With ddPCR technology, the final result was available after 3 work ys. The cut-off value discriminating, high risk was set to >1/100 and from low risk at < 1/1000. e same cut-off is commonly used in prenatal screening with biochemical markers. r patients at risk for trisomy 21 , the sensibility of the method was 100% and the specificity was .1 %. When the NIPT results were compared with FISH results, only 2 false positive were found there was 22 trisomies 21 , not 24). For women at risk for trisomy 18 (5 positives) and trisomy (2 positives), the results are concordant at 100% with the FISH results. The sensitivity and ecificity obtained in our method is similar to those of other laboratories using other, different PT technologies. scussion: oplet digital PCR is a very sensitive technique because it requires a very low quantity of DNA lile providing a very high number of reactions in a single well (each droplet generated by the jplet generator cartridge). s well known that it is not possible to differentiate with accuracy between aneuploid and euploid us when using a small quantity of cfDNA or a reduced numbers of reactions. With ddPCR, it has en demonstrated (1 1) that with a number of reactions under 1 ,000, the estimated copy number ve a larger contribution to uncertainty than the partition volume. When the number of reactions is er 10,000, copy number measurement gives a very high level of precision eliminating other urces of variability. Accordingly, the results obtained according to the present experiment are pected to be very accurate and with a high level of precision because it analysed four wells for ch probe and for each patients, generating a total of about 50,000 to 64,000 reactions. A very |h constancy was obtained in the number of droplets produced between assays translating the provement in the concentration estimate compared to digital PCR.

/vas also possible to obtain a high percentage in sensibility for each trisomy An explanation for ding 2 false positives for trisomy 21 could be explained by the fact that confined placental jsaicism (CPM) with trisomic rescue has been proven with chorionic villi sampling (CVS). This enomenon has also been suggest as an explanation for false positives and false negative cases NIPT (30,31 ). e present study has demonstrated that non-invasive prenatal testing with ddPCR is feasible and curate for screening aneuploidies in maternal plasma. Two of the most interesting advantages ire the short time-response and the non-restriction for pregnant woman with a natural singleton ;gnancy or pregnancy by assisted procreation. Indeed, the final results could be obtained within ee work days, without decreasing the precision of results, a time period faster than other known PT technology. Providing a quick result is beneficial for the patients since it reduces anxiety of le waiting. The time-saving is due to the non-sequencing DNA and the rapidity of analysing by PCR. ample 2: Calculations for obtaining an adjusted ratio and for determining presence or sence of a fetal abnormalityTable 2 hereinafter provides an example of real life calculations d statistical analysis wherein it was possible to determine the presence or absence of Trisomy in fetuses of four pregnant patients, according to the present invention. Because the assessed romosome is chromosome 21 , the targeted gene was SOD1 and the reference gene was FM03.

Table 2: Examples of calculations

iferring to the columns in Table 2 hereinabove:

(A) : refers to the patient number;

(B) : refers to the number of droplet positives for RASSF1 amplified products from maternal DNA (non-methylated);

(C) : refers to the number of droplet positives for RASSF1 amplified products from fetal (methylated);

(D) : proportion of fetal DNA in the sample = C/B

(E) : CNV (Copy Number Variation): CNV analysis by ddPCR involves quantification of target and reference loci through the use of duplex target and reference assay. In Quantasoft™ software, copy number is determined by calculating the ratio of the target molecule concentration to the reference molecule concentration, times the number of copies of reference chromosome in the genome (i.e. usually 2). Accordingly, CNV = ([T]/[R] x Nb, where [T] = concentration of target chromosome; [R] = concentration of reference chromosome; and Nb = number of copies of reference chromosome in the genome (usually 2). (F): Genomic Ratio Adjusted (GRA): GRA = 1.5 x D + (1-D)

(G) : Adjusted ratio: E/F

(H) : Result: Fetus is Negative for trisomy 21 (G is closed to or below 1 ) or positive for trisomy 21 (G is clearly above 1 ). course, it is also possible to make additional calculations and statistical analysis (e.g. Poisson itistics) to determine a P value, intervals of confidence, standard deviations and the like. It is also ssible to include additional risk factors in the calculations (e.g. age).

:FERENCES

1 . Sykes PJ, Neoh SH, Brisco MJ, Hughes E, Condon J, Morley AA. , Quantitation of targets for PCR by use of limiting dilution Biotechniques 1992; 13,444-449.

2. Kalinina, O; Brown J; Silver J "Nanoliter scale PCR with TaqMan detection" Nucleic Acids Research 1999; 25 (10): 1999-2004.

3. .Vogelstein, B; Kinzler KW. Digital PCR. Proc Natl Acad Sci USA 1999; 96 : 9236-41 .

4. Pohl, G ; Shih, I. Principle and applications of digital PCR. Expert Rev Mol Diagn 2004;

4 (1 ): 41-7,

5. Dressman D, Yan H, Traverso G , Kinzler KW; Vogelstein B. Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proc Natl Acad Sci USA 2003; 100 (15): 8817-22.

6. Liu J, Hansen C, Quake Sr. Solving the" world-to-chip" interface problem with a microfluid c matrix. Anal Chem 2003; 75: 4718-4723.

7. Ge Q,Bai Y, iiu Z, Liu Q, Yan L, iu Z. 2006 Dtection of fetal DNA in maternal plasma by microarray coupled with emulsions PGR. Clin Chim Acta 2006; 369: 82-88,

8. Fan HC, Quake SR. Detection of aneupioidy with digital polymerase chain reaction. Anal Chem 2007;79:7576-7579.

9. Lun F , Chiu RW , Allen Chan KC, et al. icrofluidics Digital PCR reveals a higher than expected fraction of fetal DNA in maternal plasma. Clin Chem 2008; 54:10 1664-1672.

10. Whale AS, Huggett JF, Cowen S, et al. Comparaison of microfluidic digital PCR and conventional quantitative PGR for measuring copy number variation. Nucleic Acids Res2012; 1 -9.

1 1 . Leonardo B Pinbeiro, Victoria A.olemen, Christopher . Hindson, Jan Herrmann, Benjamin J Hindson, Somanath Bhat, and Kerry R Emsiie. Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification. Anal. Chem 2012;84, 1003- 101 1. Hindson B J, Ness K. D, Masquelier D. A, Belgrader P, et al. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal. Chem. 201 1 ; 83, 8604- 8610.

Lo YMD, Patel P, Wainscoat JS, et al. Prenatal sex determination by DNA amplification from maternal peripheral blood. Lancet 1989;2:1363-1365.

Lo YMD, Corbetta N, chamberlain PF, et al. Presence of fetal DNA in maternal plasma and serum. Lancet 1997;350: 485-487

Lo YMD, Tein MS, Lau TK, et al. Quantitative analysis of fetal DNA in maternal plasma and serum. Implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998; 62:768-775. Chiu RWK, PoonLLM, Lau TK, Leung TN, Wong EMC, Lo YMD. Effects of blood-processing protocols on fetal and total DNA quantification in maternal plasma. Clin Chem 2001 ;47;9 1607-1613.

Chan AKC, Yeung SW, Lui WB, et al. Effects of preanalatycal factors on the molecular size of cell-free DNA in blood. Clin Chem 2005;51 :4 781 -784

Lo YMD and Chiu RWK. Prenatal diagnosis: progress through plasma nucleic acids. Nat Rev Genet 2007; 8 :71 -77.

Lun FM, Chiu RW , Allen Chan KC, et al. Microfluidics Digital PCR reveals a higher than expected fraction of fetal DNA in maternal plasma. Clin Chem 2008; 54:10 1664-1672 Fernando, .R., Chen, K, Norton, S., Ryan, W.L., and Bassett, C.(2009) Preservation and amplification of fetal cell-free DNA in maternal plasma for noninvasive prenatal diagnosis.2009 AACC/ASCLS Clinical Lab Expo July 23.

Lu Y, Gioa-Patricola L.Vivas Gomez J, Piummer M, et al. of whole genome amplification to rescue DNA from plasma samples. Bio Techniques 2005;39: 4 51 1 -515.

Jorgez CJ, Bischoff FZ. Improving enrichment of circulating Fetal DNA for genetic testing:size fractionation followed by whole genome amplification. Fetal Diagnosis Therapy 2009;25:314-319.

McDonnell T, Barrett A, Baker T, Chitty LS: The use of formaldehyde to stabilise the percentage of fetal DNA from cell-free DNA. North East Thames Regional Genetics Service Annual Report 201 1 -2012 ,

Das, K, Fernando, R., Basiaga, S., Dumais, J., Krzyzanowski, G, and Ryan, W.L. (2012) A cell stabilizing reagent in cell-free DNA™ BCT is formaldehyde free and has no adverse effects on DNA integrity. AACC annual meeting. 25. Norton SE, Lechner JM,, Williams T, Fernando MR. A stabilizing reagent prevents cell-free DNA contamination by cellular DNA in plasma during blood sample storage and shipping as determined by digital PCR. Clin Chem 2013;46:1551 -1565.

26. Clausen FB, Krog GR, Klaus R, Dziegiel MH. Improvement in fetal DNA extraction from maternal plasma. Evaluation of the nuclisens magnetic extraction system and the QIAamp DSP virus kit in comparaison with the QIAamp blood mini kit. Prenat Diagn 2007;27:6-10.

27. Go , A.T.J. I, Van Vugt, JMG and Oudejans C.B.M.201 1 . Non-invasive aneuploidy detection using free fetal DNA and RNA in maternal plasma: recent progress and future possibilities. Human Reproduction Update, Vol.17, No.3 pp.372-382.

28. Choi.H, Lau TK, Jiang FM et aL Fetal aneuploidy screening by maternal plasma DNA sequencing « false positive » due to confined placental mosaicism. Prenat Diagn 2013;33: 198-200.

29. asuzaki H, Miura K, Yoshiura ei al.(2004) Detection of cell free placental DNA in maternal plasma : direct evidence from three cases of confined placental mosaicism. J Med Genet; 41 :289-92

30. Zimmerman BG, Grill S, Holzgreve.W, et al. Digital PCR: a powerful new tool for noninvasive prenatal diagnosis. Prenat Diagn 2008;28:1087-1093.

31 . Benn P., Cuckle, H and Pergament, E (2013) Non-invasive prenatal testing for aneuploidy: current status and future prospect. Ultrasound Obstet Gynecol; 42: 15-33.

32. Chan KCA, Ding C, Gerovassiii A, Yeung SW, Chiu, RWK, Leung, TN, Lau, T.K., Chim, S.S.C, Chung, GTY, Nicolaides, KH and Lo, Y D (2006) Hypermethyiated RASSF1 A in maternal plasma: a universal fetal DNA marker that improves the reliability of non-invasive prenatal diagnosis Clinical Chemistry 52: 12, 221 1 -2218 (2006).

ladings are included herein for reference and to aid in locating certain sections. These headings ; not intended to limit the scope of the concepts described therein under, and these concepts ly be applicable in other sections throughout the entire specification. Thus, the present invention not intended to be limited to the embodiments shown herein but is to be accorded the widest Dpe consistent with the principles and novel features disclosed herein. used herein and in the appended claims, the singular forms "a", "an", and "the" include plural erents unless the context clearly indicates otherwise. Thus, for example, reference to "a gene" :ludes one or more of such genes, and reference to "the method" includes reference to uivalent steps and methods known to those of ordinary skill in the art that could be modified or bstituted for the methods described herein. iless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, ncentrations, properties, and so forth used in the specification and claims are to be understood being modified in all instances by the term "about". At the very least, each numerical parameter ould at least be construed in light of the number of reported significant digits and by applying linary rounding techniques. Accordingly, unless indicated to the contrary, the numerical rameters set forth in the present specification and attached claims are approximations that may ry depending upon the properties sought to be obtained. Notwithstanding that the numerical lges and parameters setting forth the broad scope of the embodiments are approximations, the merical values set forth in the specific examples are reported as precisely as possible. Any merical value, however, inherently contains certain errors resulting from variations in periments, testing measurements, statistical analyses and so forth. s understood that the examples and embodiments described herein are for illustrative purposes ly and that various modifications or changes in light thereof will be suggested to persons skilled the art and are to be included within the present invention and scope of the appended claims.

Claims

CLAIMS:

1 . A non-invasive method for detecting a predefined genetic abnormality in an unborn fetus, comprising the steps of:

2. The method of claim 1 , wherein the adjusted ratio is greater than 1 :1 (first region : second region), or greater than 1 .2:1 ; or greater than 1 .3:1 ; or greater than 1 .4:1 ; or greater than 1 .5:1 ; or greater than 1 .6:1 ; or greater than 1 .7:1 .

3. The method of claim 1 or 2, wherein said amplifying comprises employing a droplet digital PCR (ddPCR).

4. The method of any one of claims 1 -3, wherein the predefined genetic abnormality is aneuploidy.

5. The method of claim 4, wherein the aneuploidy is trisomy 13, trisomy 18, or trisomy 21 .

6. The method of claim 5, wherein step (c) of amplifying comprises amplifying DNA corresponding to the genes listed in Table 1 and/or Table 2.

7. The method of claim 6, wherein step (c) of amplifying comprises employing forward and/or reverse primers comprising a sequence selected from the group consisting of SEQ ID NOS 1 to 82.

8. The method of claim 6 or 7, wherein step (d) of calculating a relative ratio of abundance comprises employing a probe comprising a sequence selected from the group consisting of SEQ ID NOS 83 to 123.

9. The method of any one of claims 1 -8, wherein said subject is a woman.

10. A non-invasive method for detecting aneuploidy in an unborn fetus, comprising the steps of:

wherein said abnormally elevated ratio is indicative of aneuploidy.

1 1 . The method of claim 10, wherein said detecting does not comprises sequencing free fetal DNA.

12. The method of claim 10 or 1 1 , wherein said detecting comprises employing a droplet digital PCR (ddPCR).

13. The method of any one of claims 10 to 12, wherein said biological sample is plasma.

14. The method of any one of claims 10 to 13, wherein said detecting comprises measuring a relative ratio of abundance of fetal DNA comprising genomic sequences from a first and a second genomic region, the first region comprising a DNA sequence associated with a predefined genetic abnormality to be detected, the second region comprising a reference DNA sequence for which no genetic abnormality is expected;

wherein a ratio abundance of DNA for the first region : DNA for the second region greater than 1 :1 is indicative of aneuploidy.

15. The method of any one of claims 10 to 12, wherein said first genomic region is selected from SLITRK, LPIN2, SOD1 , DSCR8, RB1 , PHLPP1 , PLAC4, TGIF1 , TAF4B and ITM2B; and wherein said second genomic region is selected from GAPDH, FM03, ZBTB18, EIF2C4 and PTBP2.

16. The method of any one of claims 1 to 15, wherein step (b) of quantifying free fetal DNA comprises using methylation-sensitive restriction enzymes for specifically cutting maternally- derived DNA while leaving intact placental or foetus-derived DNA.

17. A method for detecting aneuploidy in an unborn human fetus, comprising the step of measuring abundance of free fetal DNA in plasma from a pregnant woman carrying said unborn fetus, wherein said measuring comprises employing a droplet digital PCR (ddPCR), and wherein abnormally elevated abundance of free fetal DNA is indicative of aneuploidy.

18. The method of claim 17, wherein the aneuploidy is trisomy 13, trisomy 18, or trisomy 21 .

19. A primer for DNA amplification, said primer comprising a sequence selected from the group consisting of SEQ ID NOS 1 to 72.

20. A probe for DNA detection, said probe comprising a sequence selected from the group consisting of SEQ ID NOS 83 to 1 12.

21 . Use of a primer according to claim 19 or a probe according to claim 20 for the detection of aneuploidy in an unborn fetus.

22. Use of a droplet digital PCR (ddPCR) apparatus, for the detection of a predefined genetic abnormality in an unborn fetus.

23. The use of claim 22, comprising detecting abundance of predefined free fetal DNA obtained from a biological sample from a pregnant subject carrying said unborn fetus.

24. The use of claim 23, wherein the predefined genetic abnormality is aneuploidy.

25. The use of claim 24, wherein the aneuploidy is trisomy 13, trisomy 18, or trisomy 21 .

26. The use of any one of claims 22 to 25, comprising employing a primer according to claim 19 or a probe according to claim 20.

27. A diagnostic kit comprising a primer according to claim 19 or a probe according to claim 20.

28. The kit of claims 27, wherein said kit further comprises instructions for the detection of aneuploidy in an unborn fetus.