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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/6851—Quantitative amplification
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• 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.
• NIPT non-invasive prenatal analysis
• 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.
• Non-invasive prenatal tests 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.
• NGS Next Generation Sequencing
• 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:
• the NIPTs identify about 50% of the irregularities routinely identified with the invasive prenatal diagnosis.
• 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.
• dPCR digital PCR
• ddPCR Droplet digital PCR
• the dPCR represents only the common method for analyzing the process.
• 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.
• 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.
• the invention 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.
• 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) .
• 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.
• chromosome 21 ABCG1, APP, BACE2, DOPEY2, DYRK1A, ERG, ETS2, LS5, NCAM2, OLIG2, PDE9A, RUNX1, WDR4
• 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.
• 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).
• dPCR Digital PCR
• 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).
• MPS Massive Parallel Sequencing
• NGS Next Generation Sequencing
• 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.
• 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
• 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.
• 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.
• 34 sets of primer and probes are multiplexed with detection targets on each of the chromosomes 21, 18 and 13.
• 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.
• 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.
• 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.
• 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.
• the total of the Copy Numbers of chromosome 18 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.
• 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.
• 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.

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