EP3966347A1

EP3966347A1 - Method for the direct determination of fetal aneuploidies by non-invasive analysis of the fetal dna from maternal blood by means of dpcr

Info

Publication number: EP3966347A1
Application number: EP20725933.4A
Authority: EP
Inventors: Claudio DELLO RUSSO; Alvaro MESORACA; Claudio GIORLANDINO
Original assignee: Artemisia SpA
Current assignee: Artemisia SpA
Priority date: 2019-05-09
Filing date: 2020-04-20
Publication date: 2022-03-16
Also published as: WO2020225632A1; IT201900006679A1

Abstract

Method for determining the presence of fetal chromosomal aneuploidies by means of non-invasive analysis of the fetal DNA present in a sample of maternal blood previously taken from the pregnant woman, said method being characterized in that the DNA analysis is based on direct and quantitative cfDNA analysis using the ddPCR technique, said method providing for the use of a mathematical and bioinformatic analytical system which correlates the diagnostic result with the reciprocal ratios of the counts obtained for each chromosome of the fetal DNA.

Description

“Method for the direct determination of fetal aneuploidies bw non-invasive analysis of the fetal DNA from maternal blood by means of DPCR”

Description

Field of the art

The present description refers to the field of techniques for non-invasive prenatal analysis (NIPT) and in particular to a new non-invasive diagnostic methodology that, being based on the general principles of NIPT, allows executing the analysis of the fetal DNA in a more direct maimer due to an innovative system for the direct and quantitative analysis of the fragments of circulating free fetal DNA (cffDNA) in the maternal blood on dropped digital PCR (ddPCR or dPCR) platform. More in detail, the present description refers to an innovative fetal aneuploidy search method based on an original analytical work flow that exploits the conventional dPCR as analytical platform, overcoming all the biases of the preceding tests that have attempted to execute the same investigations on analogous dPCR platforms. Specifically, the present description refers to a non-invasive diagnostic test for searching for fetal chromosome aberrations (aneuploidies) in the circulating free fetal DNA in the maternal blood (cffDNA) through an innovative workflow and a new technique for the computer analysis of the data.

State of the art

Non-invasive prenatal tests (NIPT) are predictive and non-diagnostic tests carried out by means of taking maternal blood, based on the identification - in the drawn maternal blood - of fragments of fetal DNA and on the reading and analysis of their sequences. In some cases (4-5%), the genetic material of fetal origin is too scarce and the examination cannot therefore be executed. The reading of the fetal DNA, in addition to detecting the sex of the unborn child, today - for most of the tests currently on the market - allows recognizing the presence of chromosome anomalies (errors in the number of chromosomes), in particular for chromosome 21 (Down’s syndrome), and for the other most common trisomies (chromosomes 13, Patau, and 18, Edwards). The NIPT is an examination that can be executed starting from the tenth week of pregnancy, it has a high reliability but variable based on the many commercial tests presently offered. The percentage of the false positives is low, attested between 0.1 and 0.5%. The test is not diagnostic (i.e. its result cannot be considered definitive, mainly because several possible differences between the fetal DNA and the fetal part of the placenta can introduce errors) but it is very useful for identifying the women at high risk of having a baby with chromosome diseases. A possible positive result of the test on free DNA makes it necessary to carry out invasive prenatal diagnostic examinations, i.e. amniocentesis or chorionic villus sampling.

For the execution of such tests, the most diffused/commonly-used methods are those of Next Generation Sequencing (NGS), with high management costs and high diagnostic times.

Methods alternative to the NGS are represented by the use of array, economically even more disadvantageous than the NGS, and by the digital PCR(dPCR). The work flows applied up to now to the digital PCR have not met the criteria of reliability, speed and low cost.

The main technical problems that can be encountered in the execution of such tests lies in the fact that the tests of the fetal DNA on maternal blood, by means of analysis of the cffDNA, have several limits that regard the sensitivity (capacity of recognizing the pathology, if present) and the specificity (capacity of not giving false positives) of the test. Such sensitivity/specificity is not high for all chromosomes. Most of the NIPT tests present on the market, throughout the world, are characterized by the following main limits:

- Sensitivity that is not high for all the chromosomes: presently, the NIPTs identify about 50% of the irregularities routinely identified with the invasive prenatal diagnosis.

- They do not allow distinguishing all the possible aneuploidies (presence of specific chromosomes in an abnormal number).

- Presence of false positives and negatives. The first to a greater extent than the second.

- The result of the test is conditioned by the quantity of fetal DNA present in the maternal plasma, which must be greater than 5%. In the cases of twin pregnancies, it is not possible to distinguish the condition of the single fetus.

- The diagnosis of the fetal aneuploidies with certainty can therefore only be obtained with amniocentesis or chorionic villus sampling.

Up to present, the two main obstacles preventing a wider application of the dPCR in the diagnostic field are given by the fact that a simple, strong methodology has never been found that could examine the cfifDNA without having to recur to protocols that provide for several thousand PCRs, amplifications on magnetic balls, or artifices based, for example, on the delicate and imprecise methylation ratios. The first problem has been resolved by the recent technical discoveries in the field of digital PCR (dPCR), such as the Droplet digital PCR (ddPCR), in which thousands of individual PCRs are executed in the same reaction well ⁽¹⁾; the other methodologies remain up to now unresolved.

The limitations of the published methods, sometimes also patented, which are substantially different from the present invention, have never allowed the industrial and diagnostic use of such method.

The present applications of Digital PCR methods at the NIPT are highly difficult, imprecise and have very high attainment costs.

Preceding attempts to apply the digital PCR to the NIPT have, already in the past, provided that there have been numerous attempts to attain a test for the prenatal diagnosis of the fetal trisomies on the search for free fetal DNA fragments in the maternal blood by means of digital PCR. Such studies have not allowed identifying a diagnostic method that can be used clinically in Prenatal diagnosis since they are overly imprecise or totally ineffective.

- Hindson BJ ⁽²⁾ and coll were only able to demonstrate the existence of ffDNA with a dPCR.

- Ge Q,Bai Y and coll. ⁽³⁾ only obtained some results in Y chromosome search without obtaining any diagnostic value on the trisomies. - Fan HC and coll. ⁽⁴⁾ only showed that it was possible to use the dPCR to search for trisomy 21 in the maternal blood; their methodology was in any case too imprecise and complicated and was never used in diagnosis.

- Whale AS and coll. ⁽⁵⁾ test the possibility to evaluate by means of dPCR the Copy Number Variation which, normally, is executed by means of NGS, assuming the use thereof in Prenatal diagnosis.

- El Khatthabi L.A. ⁽⁶⁾ with his method only proposed a“concept study” on the trisomy 21 without appreciable results. The technique resulted extremely complex, on a very“weak” platform, and the results too imprecise to be able to be used in a clinic, since the differences between normal fetuses and aneuploidies were too small to be able to be diagnostic and up to now it has not been possible to transform it into an industrial product.

- Recently Lee Seung Yong and coll. ⁽⁷⁾ implemented a technique for searching, by analyzing the cffDNA in the maternal blood, for fetuses with trisomy 21. The method only allowed partial results. The technique was very complex and costly.

- Chianru Tan, and coll. ⁽⁸⁾ also recently attempted to implement a new method based on the study of the cffDNA in the maternal blood by means of dPCR but the results were disappointing and the method too complex for being applied in prenatal diagnosis; the authors were unable to resolve the bias represented by the variability present in the sample of cfDNA obtained maternal blood.

- Also other researchers, Kadoch first and Seung Yong more recently, have tried to execute diagnostic methods by patenting products which, however, given their technical limitations and diagnostic results limitations, have never found industrial application.

Two documents which describe methods that are totally different from that which is the object of the present invention, which in common with said known methods only have the common step of extraction of the cfDNA (moreover present in all non- invasive diagnosis methods) and of final reading based on digital PCR, also common to many applications, are the following: the document WO 2016/059601 and the document WO2017/074094 A1. The first, i.e. the document WO 2016/059601, discusses the research of the trisomies 21, 18 and 13 by means of dPCR. The method, object of said document, totally differs from that according to the present invention since it is based on a method that is completely different, through the research and comparison of the methylation of DNA fragments. The dPCR represents only the common method for analyzing the process. In this research, a quantitative analysis of the fetal DNA in the maternal plasma was calculated for each patient by using, for the search for fetuses affected by aneuploidy of chromosome 21, the difference in the methylation of the fetal DNA with respect to the material DNA. Such ratio was imprecise and biologically not constant. Therefore, this method was never used in diagnostics, and never reached the market since it was overly imprecise, costly and difficult.

Another limit of the methodology, object of the abovementioned prior art document, and which marks the profound difference with respect to the method according to the present invention, is the lack of any mathematical analysis that objectively and repeatably interprets the data, hence being based on precise algorithms.

The second document, i.e. said document WO2017/074094 A1 HWANG, Seung Yong) discusses a methodology which is not at all seen in the industrial application of the product. Indeed, for operation thereof, such workflow required use of the thirty- year-old method of enriching the cffDNA by means of Magnetic balls ⁽⁹⁾. This invention was therefore poorly usable for diagnostic purposes since the methodology introduced an alteration of the sample that determined artifacts. In addition, the test uses for the analysis of the data a control gene on a chromosome not involved in the aneuploidy, towards a gene present in the chromosome affected by aneuploidy; such method involves the risk that the reference chromosome also be characterized by a numerical irregularity, as often happens in placenta mosaicisms, introducing a new analysis element that increases the risk of error. A fundamental methodological limit that profoundly distinguishes said known methodology from that according to the present invention is the lack, in the known case, of any mathematical analysis that objectively and repeatably interprets the data, being based on precise algorithms. Up to know, the tests that have used the dPCR provided for extremely complex workflows, procedures that included methodological biases which then in reality proved unsuitable for industrial distribution. Therefore, up to now, no NIPT dPCR test has been introduced in the diagnostic routine since none has proven to be reliable and repeatable, but rather difficult and even costly with respect to the simple ddPCR. Specifically, the preceding tests, also patented, presented the following limits:

- Poor reliability due to complex sample enrichment methodologies, manipulation of the same cfDNA, and absence of a rational mathematical and bioinformatic analysis;

- Long execution times;

- High costs.

The invention, described in detail hereinbelow, lies in having created an innovative workflow, extremely simple and direct, which does not require amplification (e.g. on magnetic balls) and does not correlate non-constant and delicate biological differences such as methylations. The bioinformatic methodology, based on an agile algorithm, has allowed arranging a data analysis instrument that is absolutely precise. Description of the invention

The object of the present description is a new methodology for determining the presence of fetal chromosomal aneuploidies according to the principles of the NIPT technique. More in detail the invention according to the present description consists of a new NIPT methodology which, by overcoming the current critical features relative to the lack of a totally consistent reliability of the data and relatively long times for obtaining the test results, allows obtaining said results in relatively brief time periods and with increased precision and reliability.

More in detail, the core and the central structure of the entire work flow of the present invention is decidedly advantageous in terms of efficiency, savings and speed of execution of the test ⁽¹⁰⁾.

Still more in detail, the unique and absolutely original present invention provides for the following:

- The DNA analysis is based on direct and quantitative cfDNA analysis, without providing for any preliminary method which can alter the results thereof, such as the use of magnetic balls, nor associating the level of methylation between fetal and mother DNA, but ratiier it directly examines, through the work flow discussed hereinbelow, the quantity of circulating free DNA of the chromosomes that are being investigated and which an original bioinformatic analysis will then detect so as to evaluate the possible existence of an aneuploidy.

- The data obtained, using a pool of probes and primers which allow the amplification of highly unique chromosome regions, ensuring high sensitivity and specificity, is analyzed by means of two different statistical comparison systems: the first system uses the threshold value identified by the ratio of the number of counts obtained for positive and negative samples in a cohort of 600 samples; the second, essential, is based on the intrasample comparison, and such analysis is indispensable for overcoming the biases due to the variability in the quality and quantity of cfDNA in the different samples, bias which has rendered non-usable the previously proposed methods. Such second analysis system is based on the control of the three ratios of counts obtained per chromosome (chromosome 21 count/ chromosome 18 count, chromosome 21 count/ chromosome 13 count, chromosome 18 count/ chromosome 13 count); in the case of normal samples, the three ratios are in the same range, while in the case of presence of aneuploidy on one of the chromosomes (the simultaneously aneuploidy of two or three chromosomes is not compatible with life and does not allow reaching 10 weeks of pregnancy), one of the ratios will be positive while the others will keep the value of the normality range, representing the internal control necessary for obtaining a reliable result notwithstanding the inter sample variability.

- The optimization of the design of the probes and primers used in the assay, capable of covering unique regions of the chromosomes and whose redundancy allows obtaining a high level of specificity; for the mix of PCR primers, original probes were used in a primer assay designed by the system being used. In particular 13 probes were used for chromosome 21 (ABCG1, APP, BACE2, DOPEY2, DYRK1A, ERG, ETS2, LS5, NCAM2, OLIG2, PDE9A, RUNX1, WDR4) with a FAM fluorophore, 7 probes for chromosome 18 (ANKRD12, DCC, DSC1, GATA6, SMAD4, TWSG1, ZNF521) with FAM fluorophore, 14 probes for chromosome 13 (ATP7B, BRCA2, FLY1, FLT3, FOXOl, GPC5, HTR2A, IRS2, LCP1, NBEA, PCDH9, RBI, SPRY2, TFDP1) with HEX fluoroscope.

- the presented method allows the detection of chromosomes X and Y and consequently the exclusion of the associated aneuploidies. For the X chromosome, 3 probes (ED 12, AR, ATRX), and for the Y chromosome one probe (SRY).

- The bioinformatic component for the analysis of the data in the present method is capable of discriminating pathological events even with a percentage of fetal DNA lower than 1%, proving itself suitable for diagnostic used and overcoming the variability present in nature and the quality of the analyzed sample.

The non-invasive prenatal analysis test according to the present invention is therefore a non-invasive diagnostic test for the search for fetal chromosome aberrations (aneuploidies) in circulating free fetal DNA in maternal blood (cffDNA) through an innovative work flow of sampling and quantitative direct evaluation of the cflDNA on Digital PCR (dPCR). The analyses predicted by such tests, unlike that contemplated by preceding studies, are based on a new methodology for analyzing the cffDNA on maternal blood by means of dPCR. The absolute novelty lies in the fact that such method uses an analytical system for the data integrated with the technical method. The experiment is constituted by probes identifying the chromosomes involved in the main aneuploidies. The diagnostic result is obtained by means of reciprocal ratios of the counts obtained for each chromosome. The rapidity of the data processing reduces the biases associated with biostatistical calculations that are affected by the high intersample variability, hence for the first time they are usable in clinical diagnostics with the advantages of high sensitivity and specificity, speed of execution and low cost. Such original procedure is considerably more precise and less costly than the conventional NIPT of the 21, by means of Massive Parallel Sequencing (MPS) in Next Generation Sequencing (NGS). Advantageously the invention allows the application of Digital PCR methods in the NIPT, facilitating the speed of application, the reliability and the reduced management cost, which will allow offering the patient the same advantages.

Advantageously it is able to allow a quick increase of the tests carried out annually. The reduction of the cost for the patient, deriving from the reduction of the management costs of the test by the laboratory, may in fact allow the industrial extension of the test to the population, with great advantages for public health.

Description of the figures

FIG. 1A shows the representation of the amplification conditions used for the execution of the assay. The normal value between the ratios R1 / R2 / R3 (cfiDNA) is automatically calculated by a mathematical algorithm normalized for each subject. FIG. IB shows the representation of the method used for obtaining the ratios between the counts obtained for each chromosome; A normal sample, B pathological sample. From such scheme, it is inferred that the intrasample analysis allows overcoming the biases associated with the variability in the quantity and quality of the cfDNA extracted from different subjects, constituting a normalizing value essential for data reliability. The abnormal value between the ratios R1 and R3 with respect to R2 is automatically calculated by a mathematical algorithm normalized for each subject. Detailed description of the invention

As is multiple times repeated in the course of the present description, the non- invasive prenatal analysis test according to the present invention has application in the analysis of the aneuploidies of the chromosomes 21, 18, 13 and sex chromosomes on cfDNA.

Hereinbelow, the present invention will be described in the analytical details; the same technical approach can however be applied for any similar diagnostic objective. Still more specifically, the direct non-invasive prenatal analysis test according to the present invention can be correlated with a method for executing the digital PCR which is based on the technology of the water droplets and oil-emulsion. A sample is fractioned into about 20,000 droplets and the PCR amplification of the model molecules is verified in each single droplet. The ddPCR technology uses reagents and work flows similar to those used for most of the standard qPCR tests.

The massive sampling of the sample is a key aspect of the ddPCR technique. Each droplet of a sample is plotted on a graph of the fluorescence intensity with respect to the number of droplets. All the positive droplets (those above the threshold intensity) are evaluated as positive and each is assigned a value of 1. All the negative droplets (those under the threshold) are classified as negative and each is assigned a value equal to 0 (zero). This counting technique provides a digital signal from which the concentration of the initial target DNA is calculated by means of a statistical analysis of the numbers of positive and negative droplets in a given sample.

The direct and non-invasive prenatal analysis test according to the present invention is based on a new technology of amplification of the nucleic acid. The technology provides for revolutionary rules for designing the primer, which in the end resolve the formation of the dimer of the primer and non-specific problems of amplification in the conventional PCR technology. The method allows highly multiplexed nucleic acid amplification. In the direct prenatal non-invasive analysis system according to the present invention, 34 sets of primer and probes are multiplexed with detection targets on each of the chromosomes 21, 18 and 13. Facilitated by ddPCR, the present test allows an accurate quantification of three chromosomes in a single ddPCR reaction and provides a powerful method for screening for chromosomal aneuploidy. Said method substantially consists of two steps: a first step, Step 1, termed DNA extraction step, and a second analytical step, Step 2, for identifying the possible aneuploidy. The latter step, i.e. that comprising that particular characteristics of the method according to the present invention, in turn comprises a plurality of sequential sub-steps as described in detail hereinbelow according to an embodiment of the present invention.

Step 1 - Extraction of the cfDNA

The circulating fetal DNA is extracted from a drawing of a maternal plasma sample by using the Stretch test tubes. The invention provides for the use of an automated protocol by using the QiaSymphony (QIAGEN, Valencia, CA, USA) system. The QIAsymphony kits are used with completely automated and simultaneous procedures for removing the fetal blood from the total DNA, such fetal blood isolated from samples of maternal blood. The technology employed is that with magnetic particles, which allows purifying the high-quality nucleic acids which lack proteins, nucleases and other impurities. The purified nucleic acids are directly used in subsequent analyses.

Step 2 - Analytical method

Each sample was executed in eight replicates by following the following methodology: a) 90 mΐ of ddPCR™ Supermix for probes No dUTP (Biorad), 18 mΐ of primer mixture for PCR are added to 67.5 mΐ of sample and 117 mΐ of water.

b) After the mixing, proceed with the generation of droplets by following described methods (method described on the droplets generator QX200™ and droplets reader QX200™ and described method of the software QuantaSoft

™).

c) Then, proceed by inserting the DG8 cartridge in the support with the notch in the cartridge at the top left of the support. Each sample must be situated in a cartridge and provided with a seal.

d) Then, delicately, the 20 ml reaction mix is transferred into each of the 8 reaction wells (intermediate wells) on the cartridge.

e) Then, 70mL of droplet oil for the probes are added into each of the 8 oil wells (lower wells) on the cartridge.

f) After, one couples the seal on the support of the cartridge by using the holes on both sides.

g) Then, one positions the cartridge support in the droplets generator and one starts the generation of droplets.

h) At the end of the droplets generation, one must remove the cartridge support from the droplet generator. Once the support has been removed and the seal discarded, the droplets are transferred from the first rows to the PCR reaction plate.

i) Then, the PCR plate is sealed with a perforable film plate and it is situated in the PCR machine. j) At this point, the following program is followed for completing the PCR, with ramp speed for each passage at 2 °C, by following this scheme:

Table 1.

k) The plates were incubated at ambient temperature for one night before reading on the droplets reader.

Reading of the sample on the drop reader 0X200 (Biorad).

The instrument was set by selecting ABS in the experiment type and ddPCR Supermix For Probes (without dUTP) on the supermix. The target 1 was selected for chr21 + chr18 and target 2 for chrl3; FAM / HEX for the option Set with colors.

Analysis of the data with OuantaSoft Analysis Pro

Manual setting of the threshold: on the main interface, one must set the ID card “ Width ", selecting the reaction wells and the multiple threshold wells. On“ Graphic options ", both Log boxes must be controlled.

Two thresholds are necessary for Channel 1 and only one threshold is necessary for channel 2.

The following are exported in an Excel file:

Column A = Reaction Well.

Column C = Channel (FAM or HEX).

Column K = Copy Number in the Reaction Well.

In die data of threshold 1. the total of the Copy Numbers for the chromosomes 18 and 13 (C18 + C13) must be calculated by adding together the Copy Number (Column K) of Channel 1 (FAM) of the 8 wells. The total copy number of chromosome 21 (C21) is calculated by adding together the Copy Number (Column K) of Channel 2 (HEX) of the 8 wells.

In the data of threshold 2 the total of the Copy Numbers of chromosome 18 (C18) is calculated by adding together the Copy Number (Column K) of Channel 1 (FAM) of 8 wells. The total of the Copy Numbers of chromosome 13 (C13) is calculated on the ratio (C18 + C13) - C18. The ratio between the chromosomes, C21/C18, C21/C13, and C 18/C 13, is calculated by dividing the corresponding numbers.

For each sample, the absolute counts for the chromosomes 21, 18 and 13 are quantified and calculated by ddPCR and Quantasoft. Three ratios 21/18, 21/13 and 18/13 are then derived. The average of the ratios is plotted with respect to the fetal fractions.

Both the ratios 21/18 and 21/13 of all the fetal fractions show a statistically significant difference with respect to the negative samples (0%) as indicated by the asterisks (p <0.05 from Student t-test). The ratio 18/13 of all the fetal fractions is not significantly different from the negative samples. This is based on both ratios 21/18 and 21/13 in order to determine the state of aneuploidy of the chromosome 21, 21/18 and 18/13 in order to determine the state of aneuploidy of the chromosome 18 and the ratio 21/13 and 18/13 in order to determine the state of aneuploidy of the chromosome 13.

BIOINFORMATICS:

Mathematical algorithm based on the bioinformatics model:

On the basis of the chromosomes 21, 18 and 13, there is the possibility to show the presence of irregular concentrations of one of the aforesaid three chromosomes, being based on the following mathematical algorithm:

- One creates an archive of counts that have measurements greater than 10,000 counts per chromosome type, detected on different samples whose normality is certain for all three types of chromosomes; the names TotalChr21_obs(i), TotalChr18_obs(i) and TotalChr13_obs(i) indicate the totals of the counts that refer to the three types of chromosomes for the sample (i);

- For each sample (i), the relative ratios of the total counts are calculated, which are indicated with the names 21/18(i), 21/13(i) and 18/13(i):

Table 2.

- For each group of ratios, one calculates the average value and the relative standard deviation, which we indicate with the names 21/18m, 21/18s, 21/13m, 21/13s, 18/13m, 18/13s.

The statistical result shows that all samples, whose normality is certain, fall within the interval of two standard deviations from the average value, hence simultaneously complying with the following comparisons:

21/18m - 2 * 21/18s < 21/18(i) < 21/18m + 2 * 21/18s

21/13m - 2 * 21/13s < 21/13(i) < 21/13m + 2 * 21/13s

18/13m - 2 * 18/13s < 18/13 (i) < 18/13m + 2 * 18/13s

a) Excess Chr21 :

21/18(i) > 21/18m + 2 * 21/18s

21/13(i) > 21/13m + 2 * 21/13s

b) Excess Chrl8:

21/18(i) < 21/18m - 2 * 21/18s

18/13(i) > 18/13m + 2 * 18/13s

c) Excess Chrl3:

21/13(i) < 21/13m - 2 * 21/13s

18/13(i) < 18/13m - 2 * 18/13s

In support of the evaluation, a correlation was also introduced between the three ratios of the same sampling, which considers the rectification that can be operated on the evaluation of concentration of the single chromosome as a function of the ratio measured for the other two chromosomes.

Indicating with % the deviation from the average value of the single ratio, and ascribing 50% of this deviation to the numerator and 50% to the denominator, the correction factor is calculated and then applied as indicated in the following tables:

Table 3.

Table 4.

Using the values of the table, the possible irregular concentration is confirmed, upon verification of the pair of the following comparisons:

a) Excess Chr21:

21/18rl3(i) > 21/18m + 2 * 21/18s

21/13rl8(i) > 21/13m + 2 * 21/13s

b) Excess Chrl8:

18/21rl3(i) < 21/18m - 2 * 21/18s

18/13r21(i) > 18/13m + 2 * 18/13s

c) Excess Chrl3:

13/21rl8(i) < 21/13m - 2 * 21/13s

13/18r21(i) < 18/13m - 2 * 18/13s

The present invention is a prenatal diagnosis method. Such invention can remedy the risk for the fetus associated with invasive prenatal diagnosis. The International Scientific Societies deem that non-invasive prenatal tests must be executed in selected laboratories, accredited for Medical Genetics activities and qualified to carry out such investigations. The test is to be considered an“advanced screening” method for evaluating the risk of trisomies.

Bibliography

1. Multiplexed real-time polymerase chain reaction on a digital microfluidic platform. Hua Z, Rouse JL, Eckhardt AE, Srinivasan V, Pamula VK, Schell WA, et al. Analytical chemistry. 2010; 82(6):2310-6.

2. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, et al. Analytical chemistry. 2011; 83(22):8604-

10.

3. Detection of fetal DNA in maternal plasma by microarray coupled with emulsions PCR Ge Q.Bai Y, liu Z, Liu Q, Yan L, lu Z. 2006. Clin ChimActa 2006; 369: 82-88.

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

5. Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation Whale AS, Huggett JF, Cowen S, et al.. Nucleic Acids Res 2012:1-9.

6. Be an Alternative as a Non-Invasive Prenatal Test for Trisomy 21: A Proof of Concept Study. El Khattabi LA, Rouillac-Le Sciellour C, Le Tessier D, Luscan A, Coustier A, Porcher R, Bhouri R, Nectoux J, Sirazin V, Quibel T, Mandelbrot L, Tsatsaris V, Vialard F, Dupont JM, Could Digital PCR PLoS One. 2016 May 11; 11(5).

7. A new approach of digital PCR system for non-invasive prenatal screening of trisomy 21 Lee Seung Yong et al: " ", CLINIC A CHIMICA ACTA, ELSEVIER BV, AMSTERDAM, NL, voi. 476, 21 November 2017 (2017-11- 21), pages 75-80, XP085302848, ISSN: 0009-8981, DOl:

10.1016/J. CCA.2017.11.015.

8. A multiplex droplet digital PCR assay for noninvasive prenatal testing of fetal aneuploidies Chianru Tan, Xihua Fang Wang, a Dong

Wan,c Zongfii Cao, b Xiurui Zhu,a Chao Lu,b Wenjun Yang,c Na Gao,c Huafang Gao,b Yong Guo *a and Lingxiang Zhu DOI: 10.1039/c8an02018c. 9. Hultman T and coll Nucleic Acids Res. 1989 Jul 11; 17(13) .4937-46.)

10. Innovative model of non-invasive diagnosis for fetal aneuploidy through direct and quantitative analysis of cfDNA in maternal blood: a new digital PCR and bioinformatic work flow. Dello Russo C, Mesoraca A., Pasquali C., Longo S., Cima A., Viola A., Barone MA., Cesta A., Sparacino D., Giorlandino C. Vol.l3(No 1) Journal of prenatal medicine 2019 January.

Claims

1. Method for determining the presence of fetal chromosomal aneuploidies by non- invasive analysis of fetal DNA present in a sample of maternal blood previously taken from the pregnant woman, said method being characterized in that DNA analysis is based on a direct and quantitative analysis of cfDNA using the ddPCR technique, said method providing for the use of a mathematical and bioinformatic analytical system which correlates the diagnostic result with the reciprocal ratios of the counts obtained for each chromosome of the fetal DNA.

2. Method for determining the presence of fetal chromosomal aneuploidies by non- invasive analysis of fetal DNA present in a sample of maternal blood previously taken from the pregnant woman according to the preceding claim, wherein the data obtained, using a pool of probes and primers that allow the amplification of the chromosomal regions of interest, is analyzed by means of two statistical comparison systems, said method providing that of said two statistical comparison systems, the first system uses the threshold value identified by the ratio of the number of counts obtained for positive and negative samples in a cohort of 600 samples, while the second one is based on the intra-sample comparison which provides for the control of the three ratios of counts obtained per chromosome, said ratios being at least the ratio of chromosome 21 count/chromosome 18 count, chromosome 21 count/chromosome 13 count, chromosome 18 count/chromosome 13 count, said method providing that in the case of normal samples, the three ratios are in the same range, while in the case of the presence of aneuploidy on one of the chromosomes, one of the ratios will be positive while the others will keep the value of the normal range.

3. Method according to the preceding claim, wherein: thirteen probes are used, with a FAM fluorophore, for chromosome 21, said probes being ABCG1, APP, BACE2, DOPEY2, DYRK1A, ERG ETS2, LS5, NCAM2, OLIG2, PDE9A, RUNXl, WDR4; seven probes, with a FAM fluorophore, for chromosome 18, said probes being ANKRD12, DCC, DSC1, GATA6, SMAD4, TWSG1, ZNF521; fourteen probes, with HEX fluoroscope, for chromosome 13, said probes being ATP7B, BRCA2, FLY1, FLT3, FOXOl, GPC5, HTR2A, IRS2, LCP1, NBEA, PCDH9, RBI, SPRY2, TFDP1.

4. Method according to the preceding claims wherein the detection of the X and Y chromosomes and consequently the exclusion of the associated aneuploidies is further provided, said method providing that three probes are used for the X chromosome, said probes being MED 12, AR, ATRX, and one probe for the Y chromosome, said probe being SRY.

5. Method according to the preceding claims, wherein the direct and quantitative analysis of the cfDNA provides that the maternal plasma sample, previously taken by means of Stretch test tubes, is treated according to common automated DNA purification techniques which use magnetic particles for the purification of nucleic acids.

6. Method according to the preceding claims, wherein the accurate quantification of the investigated chromosomes follows a single ddPCR reaction, digital droplet PCR, whereby each sample droplet is plotted on a graph of the fluorescence intensity with respect to the number of droplets, of said droplets those above the threshold intensity are evaluated as positive and each is assigned a value of 1, those below the threshold are evaluated as negative and each is assigned a value of 0, said counting technique allowing a digital signal to be obtained, from which the concentration of the initial target DNA is calculated by means of statistical analysis of the numbers of positive and negative droplets in a given sample.