EP3149202A1

EP3149202A1 - Method of prenatal diagnosis

Info

Publication number: EP3149202A1
Application number: EP15736606.3A
Authority: EP
Inventors: Matteo BENELLI; Francesca GERUNDINO; Claudia GIACHINI; Chiara PESCUCCI
Original assignee: Ebios Futura Srl
Current assignee: Ebios Futura Srl
Priority date: 2014-05-26
Filing date: 2015-05-26
Publication date: 2017-04-05
Also published as: WO2015181718A1

Abstract

Method of prenatal diagnosis comprising the steps of: acquiring (1) reads of fragments of genomic libraries obtained from circulating free total DNA in a sample of maternal plasma, the DNA representing a plurality of chromosomes; aligning (2) the reads with respect to a predetermined reference genome obtaining a first dataset (SAM); converting (4) the first dataset (SAM) into a second dataset (BAM) so as to allow sorting thereof according to chromosomal coordinates and successive duplicate removal, obtaining a third dataset containing all the reads aligned and devoid of duplicates; subdividing (6) at least one chromosome into windows of predetermined dimensions, so as to count the reads which have a "mapping validity" greater than or equal to a threshold value in each reading window, obtaining a total number of above-threshold reads (N_W); normalizing, for each reading window, the total number of above-threshold reads (N_W) with respect to the total number (N_tot) of reads of the fragment of genomic library obtaining a first normalized datum (N_W,lib); normalizing (8), for each reading window, the first normalized datum (N_W,lib) with respect to a systematic error due to the content of G and C bases, obtaining a second normalized datum (N_W,lib,GC); calculating (10), for each chromosome, a median of all the second normalized data (N_W,lib,GC) obtaining a final value (N_ji); calculating (12) an aneuploidy parameter (Z) for at least one chromosome; determining the aneuploidy parameter (Z) of at least one chromosome so as to evaluate whether the sample of maternal plasma is aneuploid.

Description

Method of prenatal diagnosis

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. It is present starting from the fourth/fifth week of gestation and is quickly eliminated after birth, and so is pregnancy- specific. In the first and second trimesters of pregnancy, 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.

The development of massive parallel sequencing (MPS) techniques has moreover made screening of fetal chromosomal aneuploidy possible. The accuracy of the non-invasive prenatal test is influenced by the fetal fraction, which is given by the amount of fetal DNA relative to the total amount of circulating free DNA (maternal DNA plus fetal DNA).

In recent years, non-invasive prenatal tests (NIPT, non-invasive prenatal testing), based on massive parallel sequencing of circulating free DNA in the plasma of pregnant women, were quickly introduced in prenatal diagnostics throughout the world. Between 2008 and 2011, 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 counselling, as an option for women at risk of chromosomal aneuploidy. Pre-test counselling 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 counselling must emphasize that the positive results must be confirmed by invasive prenatal diagnosis.

Recent data from application of these tests in clinical practice suggest that the negative predictive value is approx. 99.6%. Data obtained from a first screening in women in all classes of risk for aneuploidy indicate that, with these tests, the rate of false positives is significantly lower compared to biochemical screening, with a consequent marked reduction in recourse to invasive procedures.

At present, the data show a diagnostic accuracy for trisomy 21 above 99% when the test is used in women at high risk, however, exclusive and specific preliminary data of this methodology indicate that the performance of the test might be the same even when applied in populations not selected on the basis of risk.

When the test is applied to the same cohort of patients, 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.

To summarize, a non- invasive test envisages the following steps:

1) taking a blood sample; 2) isolating the plasma;

3) extracting the circulating free DNA;

4) analytical procedures for quantification of the circulating total DNA and of the fetal fraction;

5) specific molecular procedures for sequencing the cffDNA;

6) bioinformatic processing of the data obtained.

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.

This and other aims are achieved with a method of prenatal diagnosis with the features defined in Claim 1.

Particular embodiments form the subject matter of the dependent claims, the contents of which are to be understood as an integral part of the present description.

Further features and advantages of the invention will become clear from the detailed description given hereunder, which is purely illustrative and non-limiting, referring to the appended figure that shows a block diagram of the operations of the method of prenatal diagnosis according to the invention.

To summarize, the non-invasive test according to the invention envisages the following steps:

1) taking a blood sample;

2) isolating the plasma;

3) extracting the circulating free DNA;

5) specific molecular procedures for sequencing the cffDNA;

6) bioinformatic processing of the data obtained. After taking a sample of peripheral blood from the pregnant woman, a process is applied for isolating the plasma through a centrifugation procedure, preferably for 10 minutes at 3200 rpm. The portion of plasma is then transferred to a test tube of the Falcon type and is submitted to refrigerated centrifugation preferably at 4°C, preferably for 10 minutes at 9700 rpm. Extraction of the cffDNA is performed using a known protocol, whereas the subsequent procedure of sample elution is specific to the present invention.

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/μΐ 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 90μ1, the sample is subdivided into two aliquots each of 40μ1. 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.

Early non-invasive determination of fetal sex is indicated in the case of pregnancies at risk for X-linked diseases, in cases of congenital adrenal hyperplasia or in cases with ambiguous genitalia. The investigation consists of detecting the presence/absence of specific sequences of the Y chromosome by the Real-Time PCR technique. The fragments present on the short arm of the Y chromosome that are amplified in duplex are SRY and DYS14. The presence of an amplification signal indicates that the fetus is male; in contrast, lack of amplification is an indicator of a female fetus. The concentrations of primers and probes used for the analysis were optimized experimentally to obtain optimum performance of the tests.

The 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.

In particular, in the "end repair" step, a sample volume of 40μ1 is used, and is increased to 50μ1 by adding H₂0 of molecular grade. After the "end repair" step, purification is carried out with the MinElute Qiagen kit (final elution 17μ1). 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.

There then follows a step of "A-tailing" and "Ligate adapters" performed according to the protocol with adapters diluted 1 :200 and purification with the MinElute Qiagen kit (final elution 20μ1). A step of "enrichment" of the ligated fragments is performed by carrying out 14 amplification cycles.

PCR is followed by purification with Ampure XP Beckman beads according to the Illumina protocol.

Validation of the genomic libraries is performed by a run with the instrument BioAnalyzer 2100 Agilent and quantification with the Qubit fluorometer (Invitrogen) using 5μ1 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. Referring to the figure, the method of prenatal diagnosis according to the invention 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.

Addition of short nucleotide sequences or adapters to the DNA fragments allows them to be anchored to the support on which the sequencing takes place. The purpose of hybridization on the flow cell is to keep each fragment physically separate from the others, in this way the PCR reaction makes it possible to obtain groups of DNA molecules spatially separate from one another. The purpose of obtaining these groups of identical molecules is to obtain a fluorescence signal that is detectable in the subsequent sequencing passes. Then the genomic libraries, composed of the DNA fragments bound to the adapters, are deposited and hybridized on a suitable glass support known per se (flow cell) and each fragment is amplified by a bridge PCR process. The amplified fragments allow numerous complementary copies of each of them to be obtained.

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.

Next, in 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.

In 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.

At this point, in step 6, for each i-th sample, 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'^hb ~ N_tot ^ where N_w is the number of above-threshold reads identified in each reading window W. Advantageously, this normalizing is strictly dependent on the reading window selected and analysed.

Next, in step 8, 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). For this purpose, for all the whole-number percentages of GC present in the reading windows W selected (0, 1 , 2,..., 100%), 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. In 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 Ν is obtained, which is used for estimating the chromosomal aneuploidy, as described below. In step 12, an aneuploidy parameter Z is calculated for each chromosome j of each i-th sample according to the following formula:

_ (Njj -Nj)

(3) where 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.

For identifying aneuploidy, 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.

Naturally, without prejudice to the principle of the invention, the embodiments and the details of execution can be varied widely from what has been described and illustrated purely as a non-limiting example, without going beyond the scope of protection of the present invention, defined by the appended claims.

Claims

1. Method of prenatal diagnosis comprising the steps of:

- acquiring (1) reads of fragments of genomic libraries obtained from circulating free total DNA in a sample of maternal plasma, said DNA representing a plurality of chromosomes;

- aligning (2) the reads with respect to a predetermined reference genome obtaining a first dataset (SAM);

- converting (4) the first dataset (SAM) into a second dataset (BAM) so as to allow sorting thereof according to chromosomal coordinates and successive duplicate removal, obtaining a third dataset containing all the reads aligned and devoid of duplicates;

- subdividing (6) at least one chromosome into windows of predetermined dimensions, so as to count the reads which have a "mapping validity" greater than or equal to a threshold value in each reading window, obtaining a total number of above-threshold reads (N );

- normalizing, for each reading window, the total number of above-threshold reads (Nw) with respect to the total number (N_tot) of reads of the fragment of genomic library obtaining a first normalized datum (Nw.iib);

- normalizing (8), for each reading window, the first normalized datum (Nw.iib) with respect to a systematic error due to the content of G and C bases, obtaining a second normalized datum (N_w,iib,Gc);

- calculating (10), for each chromosome, a median of all the second normalized data (Nw,iib,Gc) obtaining a final value (Ν );

- calculating (12) an aneuploidy parameter (Z) for at least one chromosome;

- determining the aneuploidy parameter (Z) of at least one chromosome so as to evaluate whether the sample of maternal plasma is aneuploid.

2. Method according to Claim 1, wherein the normalizing operation, for each reading window, of the total number of above-threshold reads (N_w) with respect to the total number (N_tot) of reads of the fragment of genomic library is performed according to the following formula:

3. Method according to Claim 2, in which the normalizing operation (8), for each reading window, of the first normalized datum (Nw,Hb) with respect to a systematic error due to the content of G and C bases is performed with the following formula:

m

Nw.lib.GC ⁼ N_WiUb *—

mGC where m and moc are respectively the median of all the first normalized data (Nw,iib) and the median of all the first normalized data (Nwji_b) having the same GC percentage.

4. Method according to any one of the preceding claims, wherein a fragment of genomic library is considered aneuploid if each aneuploidy parameter (Z) relating to chromosomes 21, 13 or 18 is, in absolute value, greater than or equal to 3.