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Definitions

• the present invention relates to a method of prenatal diagnosis.
• Prenatal genetic diagnosis is performed in pregnancies that have an increased risk of genetic changes.
• the conventional methods for prenatal genetic diagnosis envisage an invasive sampling of fetal material (chorionic villus sampling or amniocentesis) with consequent risk of miscarriage associated with the invasive procedure (0.5-1%).
• the presence of circulating free fetal DNA (cffDNA) in the maternal plasma has aroused the interest of the scientific community for the possibility of developing non-invasive approaches for prenatal genetic diagnosis, from a sample of venous blood.
• the cffDNA consists of small DNA fragments, mostly with length less than 300 bp and derives from apoptosis of the Trophoblastic cells of the placenta.
• the cffDNA represents about 5- 10% of the total circulating DN A, and its quantity increases as the pregnancy progresses.
• One of the first applications of non-invasive prenatal diagnosis was determination of the sex of the fetus in women who are carriers of diseases associated with the X chromosome and of the fetal Rh factor in Rh-negative women.
• MPS massive parallel sequencing
• NIPT non-invasive prenatal tests
• NIPT non-invasive prenatal testing
• massive parallel sequencing of circulating free DNA in the plasma of pregnant women were quickly introduced in prenatal diagnostics throughout the world.
• numerous clinical trials were conducted, which demonstrated high levels of sensitivity and specificity of these tests, initially for analysis of trisomy 21 and then for trisomies 13 and 18.
• This type of test must be offered with the support of suitable genetic counseling, as an option for women at risk of chromosomal aneuploidy.
• Pre-test counseling must emphasize the high negative predictive power of the test, the low rate of false positives and the fact that the test (after the 10th week) does not depend on the gestational age.
• Post-test genetic counseling must emphasize that the positive results must be confirmed by invasive prenatal diagnosis.
• the non-invasive test makes it possible to obtain a clear separation of the patients into two distinct groups of "high risk” and "low risk” for trisomy 21.
• the most important limitations of this non-invasive test (failure of the test, results that cannot be interpreted owing to mosaicism, false positive results of about 0.1-0.2%, the need for more detailed investigation with amniocentesis, etc.) are also found in invasive investigation of the chorionic villus. Compared to amniocentesis, the non-invasive test shows slightly lower accuracy, but it is carried out at an early stage and does not present any risks for the fetus.
• a non- invasive test envisages the following steps:
• the aim of the present invention is to propose a method of non-invasive prenatal diagnosis that is able to identify the fetal Rh and, more quickly and efficiently, aneuploidy and fetal sex.
• the non-invasive test according to the invention envisages the following steps:
• Quantification of the total DNA and of the fetal fraction is performed on the samples as a preliminary step to the preparation of the genomic libraries. Quantification of the total DNA and of the circulating free fetal DNA is performed by methylation-sensitive enzymatic digestion followed by quantification by Digital PCR, quantification of the cffDNA and of the total DNA representing an important step for avoiding false negative results due to a scant amount or absence of cffDNA. Digital PCR is a technique for absolute quantification of specific target sequences and is also suitable for identifying and quantifying rare events with high levels of precision and sensitivity.
• the marker used for determining the fetal fraction is the RASSFIA gene, an oncosuppressor whose promoter is hypermethylated in the placenta and hypomethylated in the mother. This epigenetic modification makes it possible, using methylation-sensitive digestion, to eliminate the maternal contribution and identify and quantify only the sequence of fetal RASSFIA present in the maternal plasma.
• the technique used for determining the fetal fraction is Droplet Digital PCR (ddPCR), whose mix has been optimized for obtaining optimum performance of the tests used, setting up three duplexes for each sample.
• the fetal fraction is calculated from the ratio between copies/ ⁇ of RASSFIA (feta ⁇ )/TERT (maternal plus fetal). Samples with a fetal fraction below 4% are not processed for preparing the genomic libraries.
• the non-invasive test for chromosomal aneuploidy is performed by genomic sequencing at low coverage (0.8- IX) of the circulating free DNA (maternal plus fetal). After extraction of the circulating free DNA from the maternal plasma and elution in a final volume of 90 ⁇ 1, the sample is subdivided into two aliquots each of 40 ⁇ 1. One aliquot is used for the genomic sequencing, and the other aliquot is used for quantification of circulating DNA and determination of fetal sex and fetal Rh.
• Rh system consists of about 50 antigens expressed on the surface of red blood cells, encoded by two highly homologous genes: RHD and RHCE.
• the RHD gene codes for antigen D while the RHCE gene codes for the antigens CcEe. From the clinical standpoint, antigen D is the most important as it is the most immunogenic. 15% of the Caucasian population has the deletion in homozygosity of the RHD gene and is therefore RHD negative. As well as deletion of the gene, some variants of the RHD gene may also be observed.
• Rh-negative pregnant women already immunized are at risk of developing haemolytic disease of the fetus and of the neonate when the fetus is positive. The administration of immunoprophylaxis to all Rh-negative women in pregnancy reduces the risk of immunization.
• the clinical usefulness is that it reduces recourse to immunoprophylaxis and selects pregnancies that require close monitoring.
• the investigation consists of identifying the presence/absence of the RHD gene, by the Real-Time PCR technique in samples from Rh-negative women.
• the exons that are amplified in duplex are exon 5 and exon 7 and the presence of an amplification signal of both exons indicates that the fetus is Rh-positive.
• the genomic libraries are prepared using the TruSeq DNA sample preparation kit (Illumina), according to a modified protocol as indicated here.
• a sample volume of 40 ⁇ 1 is used, and is increased to 50 ⁇ 1 by adding H 2 0 of molecular grade.
• purification is carried out with the MinElute Qiagen kit (final elution 17 ⁇ 1). The aliquots thus obtained are processed, giving a greater probability of a result, since the entire process of amplification and sequencing is not controllable and does not have return points, avoiding sampling repetitions.
• Validation of the genomic libraries is performed by a run with the instrument BioAnalyzer 2100 Agilent and quantification with the Qubit fluorometer (Invitrogen) using 5 ⁇ 1 of the library. Before the sequence run, a step of library QC is performed on the MiSeq instrument (Illumina) to evaluate the quality of the libraries produced. The libraries produced are thus validated, supplying reliable results.
• the sequencing run is performed with protocol 100 cycles single read. The reads of each sample (small DNA sequences obtained from the sequencing) are processed by means of algorithms known per se for the purpose of identifying aneuploid samples.
• the method of prenatal diagnosis comprises a first step 1 of acquisition in a manner known per se of the reads of samples of cffDNA, where the samples are fragments of genomic libraries obtained from circulating free total DNA.
• the genomic libraries consist of modified DNA fragments, with the addition of molecules, "adapters", which serve to the next step of hybridization on the flow cell and to the amplification of the hybridized molecule.
• the resultant PCR colonies are read by a sequencer known per se (Illumina), with reads of about a hundred bases. These readings of each PCR colony are defined as reads.
• the sequencing takes place with a genome coverage of about 0.8 - IX.
• step 2 the reads are aligned with respect to a predetermined reference genome, such as for example the genome hgl9, using the BWA aligner known per se.
• step 4 a file of the SAM type (generic format used for organizing the alignment of sequences of reads) resulting from the alignment in step 2 is converted to a BAM file (conversion of SAM to binary format) and ordered according to chromosomal coordinates using a device known per se such as samtools, and then the duplicates are removed in a manner known per se, for example with the command "Markduplicate” of Picard tools.
• the resultant file is a new BAM file that contains all the reads aligned, except those identified as duplicates by the "Markduplicate" program.
• each chromosome j where for example j is j (21 ,13, 18), is subdivided into windows of predetermined dimensions, for example 50kb, and the readings are counted, these correspond to the reads that have a "mapping validity" greater than or equal to a threshold value, for example 20, in each reading window W.
• the "mapping validity" makes it possible to evaluate the reliability that the particular read actually comes from the position in which it is aligned by the mapping algorithm.
• the count (total number of above-threshold reads) for each reading window is then normalized with respect to the total number N tot of reads of the i-th sample, according to the following formula:
• N w is the number of above-threshold reads identified in each reading window W.
• this normalizing is strictly dependent on the reading window selected and analysed.
• each count is normalized with respect to the systematic error due to the content of GC, the density of which is increased in some genomic regions called CpG islands (percentage of G and C bases in the reading windows).
• CpG islands percentage of G and C bases in the reading windows.
• the deviation of Nw.ii b relative to the median is determined for all the windows and each N w, ii b is corrected with the following formula: where m and moc are respectively the median of all the Nw,iib and the median of all the Nwji b having the same GC percentage.
• step 10 for each chromosome j, the median of all the Nw,iib,GC is calculated, correlated with each individual reading window W selected, and a final value ⁇ is obtained, which is used for estimating the chromosomal aneuploidy, as described below.
• step 12 an aneuploidy parameter Z is calculated for each chromosome j of each i-th sample according to the following formula:
• N j and Oj are respectively the median and the standard deviation of the N y - of all the euploid samples used as reference dataset, these euploid samples having been created suitably by means of targeted sequencings.
• the value of the aneuploidy parameter (Z-score) of chromosomes 21 , 13, 18 is evaluated.
• a sample is considered to be aneuploid if each aneuploidy parameter Z relative to the chromosomes analysed, in the example chromosomes 21 , 13 or 18, is, in absolute value, greater than or equal to a threshold value, in the example 3, as these chromosomes are subject to numerical abnormalities defined as trisomy, i.e. the chromosomes are in three copies. Chromosomes 21 , 13 and 18 are analysed as they are responsible for the most frequent syndromes, thus allowing a more reliable datum to be obtained. This procedure may be repeated on the sex chromosomes, but in this case the aneuploidy parameter Z is found to be equal to 1 or is greater than 3.

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