GB2564846A - Prenatal screening and diagnostic system and method - Google Patents

Abstract

Described is a prenatal screening and diagnostic system. The prenatal screening and diagnostic system includes a wet-laboratory arrangement and a data processing arrangement. The data processing arrangement is operable to exchange instructions and data with the wet-laboratory arrangement. The data processing arrangement includes a database arrangement storing genetic information accessible to algorithms executable on the data processing arrangement. The wet-laboratory arrangement is operable to collect maternal blood samples from a pregnant mother and to identify free fetal DNA (ffDNA) fragments present in cell-free DNA (cfDNA) derived from plasma of the maternal blood sample, wherein the isolation utilizes baits or primers based upon coordinates of cell-free fetal DNA fragment preferred end-points; and the data processing arrangement is operable to analyse the identified free fetal DNA (ffDNA) and compare with one or more DNA templates stored in the data processing arrangement for determining occurrence of one or more biological characteristics of fetal DNA present in the maternal blood samples.

Description

The present disclosure relates to prenatal screening systems and methods; for example, the present disclosure relates to non-invasive prenatal screening and diagnosis systems and methods of prenatal screening and diagnosis. Furthermore, the present disclosure is concerned with computer program products comprising a non-transitory computer-readable storage medium having computer-readable instructions stored thereon, the computer-readable instructions being executable by a computerized device comprising processing hardware to execute aforesaid methods of prenatal screening and diagnosis using the prenatal screening systems.

BACKGROUND

Zygote formation and associated subsequent fetal (alternative spelling: foetal) development is a complex biological process that does not always occur without defects arising. It is of great societal benefit that such defects are detected reliably, for example as early as possible, during fetal growth.

Conventionally, antenatal or prenatal screening is provided to pregnant women to prevent or treat potential health problems that may occur during pregnancy. Such problems may affect both a given mother and/or the given mother's fetus (alternative spelling: foetus) and may be determined by factors such as lifestyle, environment or genetics; for example, environmental degradation from nuclear accidents (for example, resulting from Caesium-134, Caesium-137 and Strontium-90 ingestion, as well as ambient ionizing radiation), nuclear contamination and inherited genetic defects can give rise to the aforesaid problems. However, of particular importance are fetal abnormalities that are genetic in origin. These abnormalities may be caused by mutations inherited from one or both parents or may arise spontaneously in a stochastic manner (namely arising de novo). The nature of such mutations can range extensively from changes in single nucleotides (i.e. minor mutations) to the presence of additional whole chromosomes (i.e. major mutations); a nucleotide is an organic molecule consisting of a nitrogenous heterocyclic nucleobase (namely a purine or a pyrimidine), a pentose sugar (deoxyribose in DNA or ribose in R.NA), and a phosphate or polyphosphate group, wherein the nucleotides form rungs in a DNA double-helix. Of particular clinical significance are the chromosomal disorders known as aneuploidies that occur when there is an abnormal number of chromosomes (e.g. Down's Syndrome); aneuploidy is a presence of an abnormal number of chromosomes in a cell, for example a human cell having 45 or 47 chromosomes, instead of the usual 46 chromosomes. Many chromosomal disorders are incompatible with life or result in multiple congenital anomalies for a given new born child.

Conventionally known prenatal screening systems and methods for detecting fetal abnormalities use fetal samples derived by invasive techniques such as amniocentesis and chorionic villus sampling. These techniques require careful handling and present a degree of risk to the mother and to the mother's pregnancy.

Prenatal screening for specific fetal chromosomal abnormalities during pregnancy is widely available through public and private healthcare providers. This prenatal screening is normally carried out during a first trimester of a given pregnancy (namely, during first 10 to 14 weeks of the given pregnancy). A risk score is computed for prenatal screening purposes from the following:

(i) by measuring levels (namely concentrations) of human chorionic gonadotrophin (hCG, namely a hormone produced by the placenta after implantation of a zygote);

(ii) by measuring levels (namely concentrations) of pregnancy-associated plasma protein (PAPP-A, namely Pappaylsin-1 that is a protein encoded by the PAPPA gene in humans, wherein PAPP-A is a secreted protease, namely one or more growth factor binding proteins, used in screening tests for Down syndrome);

(iii) performing a nuchal translucency (NT) scan (namely a sonographic prenatal screening scan (using ultrasound) to detect cardiovascular abnormalities in a given fetus, as well as extracellular matrix composition and limited lymphatic drainage pertaining to the given fetus); and (iv) determining other medical factors to be taken into account (e.g. maternal age).

If a pregnancy is categorised as being 'high-risk', an invasive diagnostic procedure (e.g. chorionic villus sampling, amniocentesis, cordocentesis) is offered to the mother to confirm or rule out:

(a) Down's syndrome (trisomy chromosome 21 - T21);

(b) Edwards's syndrome (trisomy chromosome 18 - T18); and (c) Patau syndrome (trisomy chromosome 13 - T13).

Invasive tests such as chorionic villus sampling and amniocentesis involve sampling from chorionic villus (placental tissue) and amniotic sac containing fetal tissues for prenatal diagnosis of chromosomal abnormalities and fetal infections. If placental tissue is subject to confined placental mosaicism (CPM; represents a discrepancy between a chromosomal makeup of cells in a given placenta and cells of a corresponding fetus), results from such sampling of chorionic villus can be very difficult to assess accurately.

Pregnant women are also offered a second ultrasound scan at 18 to 21 weeks gestation to check for structural fetal anomalies such as cardiac malformations, brain malformations and skeletal abnormalities. This second scan can be used to direct antenatal treatments, for identifying anomalies that require early intervention following delivery or enable follow-on diagnostic testing and pregnancy management. Invasive tests such as chorionic villus sampling, amniocentesis and cordocentesis carry a 1% chance of miscarriage and are therefore only executed when there is an enhanced risk of abnormalities occurring.

During recent years, non-invasive techniques (without an associated risk of miscarriage) have been developed for the diagnoses of fetal chromosomal anomalies that rely on the presence of circulating cell free fetal DNA in the mother's blood. Such testing of cell-free fetal DNA (cffDNA) has now entered routine clinical practice for non-invasive prenatal testing (NIPT) for aneuploidy (T21, T18, T13) and has a broad application as a replacement for the aforementioned 'Combined Test'. The number of anomalies that can be tested by NIPT are increasing as methods are developed for the identification of sub-chromosomal rearrangements such as 22qll.2/DiGeorge syndrome and other microdeletion syndromes. However, the false positive rate (namely false positive assessment risk of there being a defect) for these sub-chromosomal anomalies is considered to be too high to offer on a screening basis and is only offered if there is an accompanying clinical indication such as a congenital heart defect. NIPT is classified as 'testing' rather than 'diagnosis' as the cffDNA which is measured is derived from the placenta rather than the fetus, meaning that false positives can occur due to CPM. For this reason, it is recommended that positive NIPT results are confirmed by an invasive amniocentesis.

Non-invasive prenatal diagnosis (NIPD) is generally classified as a diagnostic assay, wherein a subsequent invasive assay is not required to confirm results from the NIPD. The use of NIPD is more limited than aforementioned non-invasive prenatal testing (NIPT), and is used for fetuses at risk of single gene disorders (namely, inherited and 'de novo’ mutations) or who present with a suspicion of a monogenic disorder on fetal ultrasound.

It is known that cell-free fetal DNA (cffDNA) circulates in maternal blood at a concentration of approximately 10% of a maternal cell-free component. Such cell-free fetal DNA potentially results from fetal cell apoptosis and similar cellular metabolic processes. Coupled with low concentrations of total cell free DNA, using next generation sequencing library preparation methods for analysing such cell-free fetal DNA (cffDNA) is challenging due to the need for next generation sequencing library preparation methods to measure small quantities of fetal DNA. Furthermore, using next generation sequencing library preparation methods is challenging for two reasons:

(1) it is difficult to identify genuine 'de novo’ variants in fetal DNA (namely, there may arise problems of differentiating variants of DNA due to errors introduced in library preparation and sequencing); and (2) it is difficult to determine an overrepresentation of fetal alleles which are shared with the mother (wherein, an allele is a variant form of a given gene).

Such difficulties give rise to stochastic noise in measurements that are susceptible to being contributory factors that increase a risk of a false positive or false negative when computing a risk score during prenatal screening.

With respect to the aforementioned challenges, firstly due to errors that are introduced by Polymerase Chain Reaction (PCR) and bridge amplification in sequencing, the 'de novo' variant frequency may be higher or at the same level as the fetal fraction. This error can cause false-positive and false negative results in the aforesaid risk score. Secondly, the lower the fetal fraction, the greater sequencing depth is required to determine whether or not there is over-, under- or equal-representation of a mutation/allele, to establish the zygosity of a fetus at that point. The amount of sequencing performed can be increased, but this has a cost and time implication when seeking to deliver a prenatal screening service.

In known testing systems, the problem of there being only relatively small amounts of fetal DNA in the presence of excess maternal DNA has been addressed by employing several approaches:

(i) by using formaldehyde in blood collection tubes (Dhallan et al., 2004), wherein the use of formaldehydes reduces cell lysis (namely, the breaking down of a membrane of a given cell) and which relatively increases the percentage of free fetal DNA (cffDNA) in samples of maternal blood;

(ii) by using gel size selection to enrich for short fragments of cffDNA, it has been shown to improve the sensitivity of paternal allele detection for β-thalassemia mutations (Li et al., 2005), wherein DNA is size sorted by employing gel electrophoresis and subsequently performing gel excision and associated DNA extraction. However, such an approach is not an amenable procedure for high throughput diagnostics, and hence commercially unsuitable when performing prenatal screening;

(Hi) by counting short DNA molecules only, using PCR which amplifies short and long amplicons (Lun etat., 2008); and (iv) by employing enrichment via use of aforementioned PCR (Yang et al., 2017)

However, enrichment based on size has not yet found a place in contemporary routine clinical practice. Whilst there are distinct populations of maternal and fetal DNA fragment sizes, there is also a considerable region of overlap in fragment sizes, so a complete separation of the two populations is not possible. Such a lack of complete separation effectively increases stochastic noise in measurement that adversely influences a final risk score computed when performing prenatal screen, namely increases a risk of false-positives or false-negatives.

Therefore, in light of foregoing discussion, there exist problems associated with conventional prenatal screening methods.

SUMMARY

The present disclosure seeks to provide an improved prenatal screening system that is capable of providing a lower occurrence of false-positive and false-negatives when the system prenatal screening system is employed for providing a prenatal screening service.

Moreover, the present disclosure seeks to provide an improved method of using a prenatal screening system that is capable of providing a lower occurrence of false-positive and false-negatives when the system prenatal screening system is employed for providing a prenatal screening service.

In a first aspect, embodiments of the present disclosure provide a prenatal screening system including a wet-laboratory arrangement and a data processing arrangement that is operable to exchange instructions and data with the wet-laboratory arrangement, wherein the data processing arrangement includes a database arrangement in which there is stored genetic information accessible to one or more algorithms executable on the data processing arrangement, wherein the wet-laboratory arrangement is operable to collect one or more maternal blood samples from a pregnant mother, characterized in that:

(i) the wet-laboratory arrangement is operable to identify free fetal DNA (ffDNA) fragments present in cell-free DNA (cfDNA) derived from plasma of the one or more maternal blood samples, wherein the isolation utilizes an enrichment based upon coordinates of cell-free fetal DNA preferrred endpoints; and (ii) the data processing arrangement is operable to analyse the identified free fetal DNA (ffDNA) and compare them with one or more DNA templates stored in the data processing arrangement for determining an occurrence of one or more biological characteristics of fetal DNA present in the one or more maternal blood samples.

The present disclosure is of advantage in that it provides an improved personalized non-invasive system and method of identifying genetic abnormalities in a fetus. Moreover, the system disclosed herein is advantageous as it provides no increased risk of miscarriage and has a higher accuracy with false negative and false positive results prevention.

Embodiments of the disclosure are advantageous in terms of providing a rapid, simple, low cost, patient specific and highly efficient method and system for performing prenatal screening. Moreover, the method and system is helpful in making possible prenatal screening at an earlier time in pregnancy, and also reducing diagnosis time.

Optionally, the wet-laboratory arrangement is operable to enrich the free fetal DNA fragments for providing a plurality of copies of the free fetal DNA fragments for analysis. More optionally, the wet-laboratory arrangement is operable to enrich the free fetal DNA fragments by using nucleosome profile to determine the most likely start position and the fetal preferred end positions.

Optionally, the enrichment method is designed to avoid maternal DNA present in the plasma. More optionally, the enrichment design is in combination with targeting of genes, wherein the genes are relevant to genetic disorders.

Optionally, the wet-laboratory arrangement is operable to identify and analyse cfDNA fragments in the plasma that start within a nucleosome, wherein the cfDNA fragments correspond to a fetal fraction of the plasma of the one or more maternal blood samples, wherein the cfDNA fragments that start within a nucleosome are relatively shorter in nucleic acid base count than an average length in nucleic acid base count of cfDNA fragments present in the one or more maternal blood samples.

Optionally, the wet-laboratory arrangement is operable to perform a combined test for prenatal screening of fetal chromosomal abnormalities, wherein the combined test includes:

(i) at least one maternal blood test; and (ii) an ultrasound scan of a fetus.

Optionally, the wet-laboratory arrangement is operable to measure levels of human chorionic gonadotrophin (hCG) and pregnancy-associated plasma protein (PAPP-A).

Optionally, the data processing arrangement is operable to store genetic information extracted from the one or more maternal blood samples in a secondary database.

In a second aspect, embodiments of the present disclosure provide a method of using a prenatal screening system including a wet-laboratory arrangement and a data processing arrangement that is operable to exchange instructions and data with the wet-laboratory arrangement, wherein the data processing arrangement includes a database arrangement in which there is stored genetic information accessible to one or more algorithms executable on the data processing arrangement, characterized in that the method includes:

(i) using the wet-laboratory arrangement to collect one or more maternal blood samples from a pregnant mother;

(ii) using the wet-laboratory arrangement to identify free fetal DNA (ffDNA) fragments present in cell-free DNA (cfDNA) derived from plasma of the one or more maternal blood sample, wherein the isolation utilizes an enrichment method based upon coordinates of cell-free fetal DNA fragment specific end-points; and (iii) using the data processing arrangement to analyse the identified free fetal DNA (ffDNA) and compare them with one or more DNA templates stored in the data processing arrangement for determining an occurrence of one or more biological characteristics of fetal DNA present in the one or more maternal blood samples.

Optionally, the method includes using the wet-laboratory arrangement to enrich the free fetal DNA fragments for providing a plurality of copies of the free fetal DNA fragments for analysis. More optionally, the method includes enriching the free fetal DNA fragments by using nucleosome profile to determine the most likely start position and the fetal preferred end positions.

Optionally, the method includes designing the enrichment method to avoid maternal DNA present in the plasma. More optionally, the designed is in combination with targeting of genes, wherein the genes are relevant to genetic disorders.

Optionally, the method includes using the wet-laboratory arrangement to identify and analyse cfDNA fragments in the plasma that start within a nucleosome, wherein the cfDNA fragments correspond to a fetal fraction of the plasma of the maternal blood sample, wherein the cfDNA fragments that start within a nucleosome are relatively shorter in nucleic acid base count than an average length in nucleic acid base count of cfDNA fragments present in the one or more maternal blood sample.

io

Optionally, the method includes using the wet-laboratory arrangement to perform a combined test for prenatal screening of fetal chromosomal abnormalities, wherein the combined test includes:

(i) at least one maternal blood test; and (ii) an ultrasound scan of a fetus.

Optionally, the method includes using the wet-laboratory arrangement to measure levels of human chorionic gonadotrophin (hCG) and pregnancyassociated plasma protein (PAPP-A).

Optionally, the method includes using data processing arrangement to store genetic information extracted from the maternal blood samples in a secondary database.

In a third aspect, embodiments of the present disclosure provide a computer program product comprising a non-transitory computer-readable storage medium having computer-readable instructions stored thereon, the computer-readable instructions being executable by a computerized device comprising processing hardware to execute the aforementioned method.

Additional aspects, advantages, features and objects of the present disclosure would be made apparent from the drawings and the detailed description of the illustrative embodiments construed in conjunction with the appended claims that follow.

It will be appreciated that features of the present disclosure are susceptible to being combined in various combinations without departing from the scope of the present disclosure as defined by the appended claims.

A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as limiting the present invention.

BRIEF DESCRIPTION OF DRAWINGS

Embodiments of the present disclosure will be more fully understood from examples described herein below and the accompanying drawings, which is given by way of illustration only, and thus are not limitative of the present invention, and wherein:

FIG. 1 is a schematic illustration of a prenatal screening system, in accordance with an embodiment of the present disclosure;

FIG. 2 is a Kalman filter equivalent representation of the system of FIG. 1, in accordance with an embodiment of the present disclosure; and

FIG. 3 is an illustration of steps of a method of operating the system of FIG. 2 for providing prenatal screening, in accordance with an embodiment of the present disclosure.

In the accompanying diagrams, an underlined number is employed to represent an item over which the underlined number is positioned or an item to which the underlined number is adjacent. A non-underlined number relates to an item identified by a line linking the non-underlined number to the item. When a number is non-underlined and accompanied by an associated arrow, the non-underlined number is used to identify a general item at which the arrow is pointing.

LIST OF ABBREVIATIONS

Abbreviation Meaning

RMD

Relative Mutation Dosage

RHDO

PCR

Relative Haplotype Dosage

Polymerase Chain Reaction

NT

Nuchal Translucency hCG

Human Chorionic Gonadotrophin

PAPP-A

Pregnancy-Associated Plasma Protein cffDNA

Cell-Free Fetal DNA

NIPT

Non-Invasive Prenatal Testing cfDNA

Cell-Free DNA

DEFINITIONS

As used herein, the following terms shall have the following meanings:

As used herein, the term 'data processing arrangement' refers to a process and/or system that can be embodied in software that determines the biological significance of acquired data (i.e., the ultimate results of an assay). For example, a data processing arrangement can determine the amount of each nucleotide sequence species based upon the data collected. A data processing arrangement also may control an instrument and/or a data collection system based upon results determined. A data processing and a data collection arrangement often are integrated and provide feedback to operate data acquisition by the instrument, and hence provide assaybased judging methods provided herein.

As used herein, 'polymerase chain reaction (PCR.)' is a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA by several orders of magnitude, thereby generating potentially thousands of millions of copies of a particular given DNA sequence.

As used herein, 'bridge amplification' or 'amplification' is employed in massively parallel sequencing for DNA sequencing purposes using a concept of massively parallel processing, wherein use is made of miniaturized and parallelized platforms for sequencing of 1 million to 43 billion short reads (50 to 400 nucleic acid bases each) per instrument run.

As used herein, 'zygosity' refers to a degree of similarity of alleles for a trait in a given organism, for example a given fetus.

As used herein, the term 'database arrangement’ refers to a nucleic acid databases known in the art including, for example, GenBank, dbEST, dbSTS, EMBL (European Molecular Biology Laboratory) and DDBJ (DNA Databank of Japan). BLAST or similar tools can be used to search the identified sequences against a sequence database.

As used herein, the term 'genetic information' refers to information related to nucleic acids, altered nucleotide sequence, chromosomes, segments of chromosomes, polymorphic regions, translocated regions, the like or combinations of the foregoing. Furthermore, the nucleic acids may include, are but not limited to, DNA, cDNA, RNA, mRNA, t RNA and rRNA. Moreover the genetic information may include information related to mutations, copy number variations, transversions, translocations, inversion, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structure alterations, gene fusions, chromosome fusions, gene truncations, gene amplification, gene duplications, chromosomal lesions, DNA lesions, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns, abnormal changes in nucleic acid methylation infection or cancer.

As used herein, the term 'cell-free DNA' refers to DNA that is not within a cell. In one embodiment, cell free DNA includes DNA circulating in blood. In another embodiment, cell free DNA includes DNA existing outside of a cell. In yet another embodiment, cell free DNA includes DNA existing outside of a cell as well as DNA present in a blood sample after such blood sample has undergone partial or gentle cell lysing.

As used herein, the term 'free fetal DNA' refers to DNA that originated from a given fetus and not a mother of the given fetus, wherein the DNA is not within a cell. In one embodiment, cell free fetal DNA includes fetal DNA circulating in maternal blood. In another embodiment, cell free fetal DNA includes fetal DNA existing outside a cell, for example a fetal cell. In yet another embodiment, cell free fetal DNA includes fetal DNA existing outside a cell as well as fetal DNA present in maternal blood sample after such blood sample has undergone partial or gentle cell lysing. Herein, the term 'free fetal DNA' also refers to small DNA fragments (i.e. about <300 base pairs) circulating in maternal plasma, in other terms it is the excluding DNA contained in fetal cells that may circulate in the maternal plasma.

As used herein, the terms 'maternal sample' or 'maternal blood sample' refers to the sample obtained from a female who is pregnant, the sample may include, but is not limited to, plasma, serum, peripheral blood and urine. Typically, the sample is a maternal plasma sample, although other tissue sources that contain both maternal and fetal DNA can be used. Maternal plasma can be obtained from a peripheral whole blood sample from a pregnant woman and the plasma can be obtained by standard methods. A volume of 3 ml to 5 ml of plasma is sufficient to provide suitable DNA material for analysis. The cell free DNA can be extracted from the sample using standard techniques, non-limiting examples of which include a Qiasymphony protocol (Qiagen) suitable for free fetal DNA isolation or any other automated or manual extraction method suitable for cell free DNA isolation.

As used herein, the term 'biological characteristics' refers to the genetic variations, abnormalities, irregularities or mutations which range extensively from changes in single nucleotides to the presence of additional whole chromosomes or abnormal number of chromosomes. The chromosomal abnormality is a structural abnormality, including, but not limited to, copy number changes including microdeletions and microduplications, insertions, translocations, inversions and small-size mutations including point mutations and mutational signatures.

As used herein, the term 'wet-laboratory arrangement' refers to a facility, clinic and/or a setup of: instruments, equipment and/or devices used for extraction, collection, processing and/or analysis of body fluid samples; instruments, equipment and/or devices used for extraction, collection, processing and/or analysis of genetic material; instruments, equipment and/or devices used for amplification, enrichment and/or processing of genetic material received from the body fluid samples; instruments, equipment and/or devices used for extraction and/or analysis of the genetic information received from the amplified genetic material. Herein the instruments, equipment and/or devices may include but not limited to centrifuge, ELISA, spectrophotometer, PCR, RT- PCR, High-ThroughputScreening (HTS) system, Microarray system, Ultrasound, genetic analyser, deoxyribonucleic acid (DNA) sequencer and SNP analyser. The wetlaboratory arrangement may be operable to monitor and/or scan a fetus, for example using ultrasonic scanning apparatus providing animated images of the fetus (ultrasound scanner). Herein, the wet-laboratory arrangement may include equipment, instruments and/or devices for scanning the fetus. Such equipment, instruments and/or devices include ultrasound scanners (as aforementioned), presymptomatic genetic testing and/or combined tests.

The enrichment design will be in combination with the targeting of genes which are relevant to genetic disorders and for the enrichment of fetal DNA from a maternal plasma sample. The baits or primers (for example) for enrichment are, for example, prepared beforehand, and are optionally selected from a library of prepared baits or primers.

DETAILED DESCRIPTION

Practical implementation of the embodiments of the present disclosure are described in further detail below; these embodiments are operable to employ, unless otherwise indicated, conventional methods of diagnostics, molecular biology, cell biology, biochemistry and immunology within the skill of the art. Such techniques are explained fully in the literature, for example contemporary academic research literature pertaining to pregnancy and genetic material processing.

It will be appreciated that certain features of the present invention, which are for clarity described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely various features of the invention, which are for brevity, described in the context of a single embodiment, may also be provided separately and/or in any suitable sub-combination.

The following detailed description illustrates embodiments of the present disclosure and ways in which they can be implemented. Although some modes of carrying out the present disclosure have been described, those skilled in the art would recognize that other embodiments for carrying out or practicing the present disclosure are also possible.

In FIG. 1, there is shown an illustration of a prenatal screening system 100, in accordance with an embodiment of the present disclosure. The prenatal screening system 100 includes a wet-laboratory arrangement 102 and a data processing arrangement 104. The data processing arrangement 104 is operable to exchange instructions and data with the wet-laboratory arrangement 102. The data processing arrangement 104 is operable to access a database arrangement 106 and a secondary database 108. Furthermore, information stored in the database arrangement 106 is accessible to one or more algorithms executable on the data processing arrangement 104. Herein, the wet-laboratory arrangement 102 is operable to collect one or more maternal blood samples from a pregnant mother, for example a single blood sample or a plurality of blood samples. Moreover, the wet-laboratory arrangement 102 is operable to identify free fetal DNA (ffDNA) fragments present in cell-free DNA (cfDNA) derived from plasma of the maternal blood sample. The isolation of free fetal DNA (ffDNA) utilizes, for example, baits or primers based upon coordinates of cell-free fetal DNA fragment preferred end-points. Furthermore, the data processing arrangement 106 is operable to analyse the identified free fetal DNA (ffDNA) and compare them with one or more DNA templates stored in the data processing arrangement 106 for determining an occurrence of one or more biological characteristics of fetal DNA present in the maternal blood samples.

In an embodiment, the wet-laboratory arrangement 102 of the prenatal screening system 100 may be operable to amplify (i.e. enrich) free fetal DNA fragments for providing a plurality of copies of the free fetal DNA fragments for analysis by the data processing arrangement 104. In this exemplary embodiment, the wet-laboratory arrangement 102 may include a PCR or RT-PCR for amplifying the free fetal DNA fragments for providing a plurality of copies of the free fetal DNA to the data processing arrangement 104 for accessing genetic information in the database arrangement 106. In this embodiment, the wet-laboratory arrangement 102 may enrich the free fetal DNA by using nucleosome profile for determining the fetal specific start and end positions of the free fetal DNA fragments.

In another embodiment, the prenatal screening system 100 may be operable to design, for example, baits or primers for avoiding the contamination of maternal DNA present in the plasma extracted from the maternal blood sample; in other words, the baits or primers are employed as a form of biological filter for distinguishing between DNA fragments of fetal origin from those of maternal origin. In this embodiment, the designed baits or primers may be taken in combination with the targeted genes. For example, the targeted genes may include but not limited to the genes relevant to genetic disorders.

In an embodiment, the wet-laboratory arrangement 102 may be operable to identify and analyse cfDNA fragments in the plasma that start within a nucleosome. Moreover, the cfDNA fragments correspond to a fetal fraction of the plasma of the maternal blood sample. In this embodiment, the cfDNA fragments that start within a nucleosome may be relatively shorter in nucleic acid base count than an average length in nucleic acid base count representing the fetal component of cfDNA fragments present in the maternal blood sample (for example approx. 143 bases long for the fetus relative to approx. 166 bases long for a corresponding mother).

In another embodiment, the wet-laboratory arrangement 102 may be operable to perform a combined test for prenatal screening of fetal chromosomal abnormalities. In this embodiment, the combined test may include, but is not limited to, a maternal blood test and an ultrasound scan of a fetus.

In yet another embodiment, the wet-laboratory arrangement 102 may be operable to measure levels of human chorionic gonadotrophin (hCG) and pregnancy-associated plasma protein (PAPP-A).

In an example operation of the prenatal screening system 100, a mother with fetus is presented to the prenatal screening system 100. The prenatal screening system 100 is used to perform an ultrasonic scanning test on the fetus to generate an ultrasonic image or video of the fetus, and a cardiac abnormality in the fetus is identified from the ultrasonic test. It is deduced, for example using an expert system artificial intelligence (Al) or similar analysis tool, that there is a risk of the mother suffering a miscarriage of the fetus if an aforementioned invasive amniocentesis or chorionic villus were to be performed. Many mothers, alternatively parents, would in such a situation choose not to pursue such invasive sampling for purposes of performing genetic testing. However, beneficially, the prenatal screening system 100 is capable of providing non-invasive assay from which it is feasible to make a genetic diagnosis. The non-invasive assay includes enriching a proportion of free fetal DNA(cffDNA) fragments in cell free DNA that is derived from a maternal blood sample, wherein the assay employs coordinates of cell-free DNA fragment specific end-points and genes that are relevant to a given disorder under investigation. Information indicative of favoured fetal fragment end positions and nucleosome profiles may be accessed by the prenatal screening system 100 from its database arrangement 106. There are thereby provided, namely 'designed', for the enrichment of fetal DNA from a maternal plasma sample derived from the aforementioned maternal blood sample.

In yet another embodiment, the data processing arrangement 104 may be operable to store genetic information extracted from the maternal blood samples in a secondary database 108.

In FIG. 2, there is shown an illustration of a Kalman filter equivalent representation 200 of the system (such as prenatal screening system 100 of FIG. 1), in accordance with an embodiment of the present disclosure. The

Kalman filter equivalent representation 200 of the system 100 includes a combined feed of genetic information received from free fetal DNA fragments 202 and information received for combined test of a fetus 204 to a data processing arrangement 206 (such as data processing arrangement 104 of FIG. 1). The data processing arrangement 206 may be operable to implement a Kalman filter on the genetic information received from free fetal DNA fragments 202 and information received for combined test of a fetus 204. The data processing arrangement 206 further includes a fuzzy logic module 208 for, a processing module 210, a genetic algorithm 212 for matching the ffDNA fragment in a database arrangement 214 (such as database arrangement 106 of FIG. 1), a secondary database 216 (such as secondary database 108 of FIG. 1) for storing the risk score 218 received from the processing module 210. In this embodiment, the data processing system 206 may be operable to implement the Kalman filter on the genetic material received from the maternal blood sample and removing contamination. Furthermore, the genetic algorithm 212 may be operable to match the ffDNA fragments in the database arrangement 214 and predicting the risk score 218.

In an exemplary embodiment, the prenatal screening system 100 may be operable to execute the genetic algorithm 212 in the data processing arrangement 104 for using the information indicative of the favoured fetal fragment end positions and nucleosome profiles. In this embodiment, the maternal plasma sample derived from the aforementioned maternal blood sample includes DNA sequences that are enriched using an assay targeting favoured fetal fragment end-points. Furthermore, use of favoured positions of fetal reads derived from nucleosome positioning the prenatal screening system 100 is operable to enrich fragments of cfDNA. In this embodiment, the processing module 210 is operable to validate positions of the cffDNA fragments.

In an embodiment, the prenatal screening system 100 may be operable to differentiate maternal and fetal components of cfDNA. In this embodiment, such differentiation may be achieved by employing an assay design which enriches the fetal component and which aids in mapping of maternal and fetal reads.

In an exemplary embodiment, the prenatal screening system 100 may be operable to design, for example, baits or primers and to employ these at the feta I-preferred positions and fetal-maternally shared positions. Furthermore, the prenatal screening system 100 may be operable to avoid the maternal-preferred regions in the maternal blood sample. Moreover, the bait or primer (for example) designs may be made in combination with the targeting of genes which are relevant to genetic disorders.

In yet another exemplary embodiment, the positioning of the fragments at specific locations is due to non-random fragmentation of DNA and it has been postulated that plasma DNA fragments are cleaved in accessible parts of the genome. Furthermore, shorter cfDNA fragments start within the nucleosome and it has been shown that these fragments positively correlate with fetal fraction. Moreover, by using the nucleosome profile to determine the most likely start position and the fetal specific end positions, the prenatal screening system 100 may be operable to improve the enrichment of cffDNA.

In FIG. 3, there is shown a flow chart of a method 300 of using a prenatal screening system (such as prenatal screening system 100 of FIG. 1), in accordance with an embodiment of the present disclosure. At a step 302, the flow chart initiates. At the step 302, a maternal blood sample is collected from a pregnant mother using a wet-laboratory arrangement (such as the wet-laboratory arrangement 102 of FIG. 1). At a step 304, plasma is derived from the maternal blood sample, using the wet-laboratory arrangement. At a step 306, free fetal DNA (ffDNA) fragments present in cell-free DNA (cfDNA) are identified using the wet laboratory arrangement, wherein the isolation utilizes baits or primers based upon coordinates of cellfree fetal DNA fragment specific end-points. At a step 308, the identified free fetal DNA (ffDNA) are analysed and compared with one or more DNA templates stored in the data processing arrangement for determining an occurrence of one or more biological characteristics of fetal DNA present in the maternal blood samples

In an embodiment, the method 300 of using the prenatal screening system may include using the wet-laboratory arrangement for enriching the ffDNA fragments for providing a plurality of copies of the free fetal DNA fragments for analysis to the data processing arrangement. Furthermore, the method 300 may include enriching the free fetal DNA fragments by using nucleosome profile to determine the most likely start position and the fetal preferred end positions.

In another embodiment, the method 300 of using the prenatal screening system may include using the wet-laboratory arrangement for designing for example, baits or primers to avoid maternal DNA present in the plasma of the maternal blood sample. Furthermore, the designed baits or primers may be in combination with targeting of genes, wherein the genes are relevant to genetic disorders.

In yet another embodiment, the method 300 of using the prenatal screening system may include using the wet-laboratory arrangement for isolating and analysing cfDNA fragments in the plasma that start within a nucleosome. Furthermore, the cfDNA fragments correspond to a fetal fraction of the plasma of the maternal blood sample, wherein the cfDNA fragments that start within a nucleosome are relatively shorter in nucleic acid base count than an average length in nucleic acid base count of cfDNA fragments present in the maternal blood sample.

In yet another embodiment, the method 300 of using the prenatal screening system may include using the wet-laboratory arrangement for performing a combined test for prenatal screening of fetal chromosomal abnormalities. In this embodiment, the combined test may include, but is not limited to, a maternal blood test and an ultrasound scan of a fetus.

In another embodiment, the method 300 of using the prenatal screening system may include using the wet-laboratory arrangement for measuring levels of human chorionic gonadotrophin (hCG) and pregnancy-associated plasma protein (PAPP-A).

In yet another embodiment, the method 300 of using the prenatal screening system may include using the data processing arrangement for storing genetic information extracted from the maternal blood samples in a secondary database.

In another embodiment, the method 300 of using the prenatal screening system may include using the data processing arrangement for matching the identified ffDNA fragment in a data base arrangement (such as data base arrangement 106 of FIG. 1) by applying a genetic algorithm (such as genetic algorithm 212 of FIG. 2).

Although use of the prenatal screening system 100 described in the foregoing, it may be appreciated that the prenatal screening system may be used for investigating other types of biological problems, and not merely restricted to prenatal screening and diagnostic tasks, for example: cancer risk determination; autistic risk determination; verification of organism performance after performing gene therapy; ionizing radiation damage identification to cell DNA; and/or diabetes risk determination.

Modifications to embodiments described in the foregoing are possible without departing from the scope of the invention as defined by the accompanying claims. Expressions such as including, comprising, incorporating, consisting of, have, is used to describe and claim the present invention 5 are intended to be construed in a non-exclusive manner, namely allowing for items, components or elements not explicitly described also to be present. Reference to the singular is also to be construed to relate to the plural. Numerals included within parentheses in the accompanying claims are intended to assist understanding of the claims and should not be construed 10 in any way to limit subject matter claimed by these claims.

REFERENCES [1] Chan, K. C. A., Jiang, P., Sun, K., Cheng, Y. K. Y., Tong, Y. K., Cheng, S. H., ... Lo, Y. M. D. (2016). Second generation noninvasive fetal genome analysis reveals de novo mutations, single-base parental inheritance, and preferred DNA ends. Proceedings of the National Academy of Sciences, 113(50), E8159-E8168.

https://doi.org/10.1073/pnas.1615800113 [2] Chandrananda, D., Thorne, N. P., Bahlo, M., Tam, L.-S., Liao, G., & Li, E. (2015). High-resolution characterization of sequence signatures due to non-random cleavage of cell-free DNA. BMC Medical Genomics, 8(1), 29. https://doi.org/10.1186/sl2920-015-0107-z [3] Dhallan, R., Au, W.-C., Mattagajasingh, S., Emche, S., Bayliss, P., Damewood, M., ... Mohr, M. (2004). Methods to Increase the Percentage of Free Fetal DNA Recovered From the Maternal Circulation. JAMA, 291(9), 1114.

https://doi.Org/10.1001/jama.291.9.1114 [4] Li, Y., Di Naro, E., Vitucci, A., Zimmermann, B., Holzgreve, W., & Hahn,

S. (2005). Detection of Paternally Inherited Fetal Point Mutations for βThalassemia Using Size-Fractionated Cell-Free DNA in Maternal Plasma. JAMA, 293(7), 843.

https://doi.Org/10.1001/jama.293.7.843 [5] Lun, F. M. F., Tsui, N. B. Y., Chan, K. C. A., Leung, T. Y., Lau, T. K., Charoenkwan, P., ... Lo, Y. M. D. (2008). Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma. Proceedings of the National

Academy of Sciences of the United States of America, 105(50), 19920-

5.

https://doi.org/10.1073/pnas.0810373105 [6] Snyder, M. W., Kircher, M., Hill, A. J., Daza, R. M., & Shendure, J.

(2016). Cell-free DNA Comprises an In Vivo Nucleosome Footprint that Informs Its Tissues-Of-Origin. Cell, 164(1-2), 57-68.

https://doi.Org/10.1016/j.cell.2015.ll.050 [7] Straver, R., Oudejans, C. B. M., Sistermans, E. A., & Reinders, M. J. T. (2016). Calculating the fetal fraction for noninvasive prenatal testing based on genome-wide nucleosome profiles. Prenatal Diagnosis, 36(7), 614-621.

https://doi.org/10.1002/pd.4816 [8] Vainshtein, Y., Rippe, K., & Teif, V. B. (2017). NucTools: analysis of chromatin feature occupancy profiles from high-throughput sequencing data. BMC Genomics, 18(1), 158. https://doi.org/10.1186/sl2864-017-3580-2 [9] Yang, Q., Du, Z., Song, Y., Gao, S., Yu, S., Zhu, H., ... Zhang, G. (2017). Size-selective separation and overall-amplification of cell-free fetal DNA fragments using PCR-based enrichment. Scientific Reports, 7, 40936. httDs://doi.org/10.1038/sreD40936

Claims (19)

CLAIMS We claim:

1. A prenatal screening and diagnostic system (100) including a wetlaboratory arrangement (102) and a data processing arrangement (104) that is operable to exchange instructions and data with the wet-laboratory arrangement (102), wherein the data processing arrangement (104) includes a database arrangement (106) in which there is stored genetic information accessible to one or more algorithms executable on the data processing arrangement (104), wherein the wet-laboratory arrangement (102) is operable to collect one or more maternal blood samples from a pregnant mother, characterized in that:

(i) the wet-laboratory arrangement (102) is operable to identify free fetal DNA (ffDNA) fragments present in cell-free DNA (cfDNA) derived from plasma of the one or more maternal blood samples, wherein the isolation utilizes baits or primers based upon coordinates of cell-free fetal DNA fragment preferred end-points; and (ii) the data processing arrangement (106) is operable to analyse the identified free fetal DNA (ffDNA) and compare them with one or more DNA templates stored in the data processing arrangement (106) for determining an occurrence of one or more biological characteristics of fetal DNA present in the one or more maternal blood samples.

2. A prenatal screening and diagnostic system (100) according to claim 1, characterized in that the wet-laboratory arrangement (102) is operable to enrich the free fetal DNA fragments for providing a plurality of copies of the free fetal DNA fragments for analysis.

3. A prenatal screening and diagnostic system (100) according to claim 2, characterized in that the wet-laboratory arrangement (102) is operable to enrich the free fetal DNA fragments by using nucleosome profile to determine the most likely start position and the fetal preferred end positions.

4. A prenatal screening and diagnostic system (100) according to claim 1, 2 or 3, characterized in that the baits or primers are designed to avoid maternal DNA present in the plasma.

5. A prenatal screening and diagnostic system (100) according to the claim 4, characterized in that the designed are in combination with targeting of genes, wherein the genes are relevant to genetic disorders.

6. A prenatal screening system (100) according to claim 1, 2, 3 or 4, characterized in that the wet-laboratory arrangement (102) is operable to identify and analyse cfDNA fragments in the plasma that start within a nucleosome, wherein the cfDNA fragments correspond to a fetal fraction of the plasma of the one or more maternal blood samples, wherein the cfDNA fragments that start within a nucleosome are relatively shorter in nucleic acid base count than an average length in nucleic acid base count of cfDNA fragments present in the one or more maternal blood samples.

7. A prenatal screening and diagnostic system (100) according to any of the preceding claims, characterized in that the wet-laboratory arrangement (102) is operable to perform a combined test for prenatal screening of fetal chromosomal abnormalities, wherein the combined test includes:

(i) at least one maternal blood test; and (ii) an ultrasound scan of a fetus.

8. A prenatal screening and diagnostic system (100) according to any of the preceding claims, characterized in that the wet-laboratory arrangement (102) is operable to measure levels of human chorionic gonadotrophin (hCG) and pregnancy-associated plasma protein (PAPP-A).

9. A prenatal screening and diagnostic system (100) according to any of the preceding claims, characterized in that the data processing arrangement (104) is operable to store genetic information extracted from the one or more maternal blood samples in a secondary database (108).

10. A method of using a prenatal screening and diagnostic system (100) including a wet-laboratory arrangement (102) and a data processing arrangement (104) that is operable to exchange instructions and data with the wet-laboratory arrangement (102), wherein the data processing arrangement (104) includes a database arrangement (106) in which there is stored genetic information accessible to one or more algorithms executable on the data processing arrangement (104), characterized in that the method includes:

(i) using the wet-laboratory arrangement (106) to collect one or more maternal blood samples from a pregnant mother;

(ii) using the wet-laboratory arrangement (102) to identify free fetal DNA (ffDNA) fragments present in cell-free DNA (cfDNA) derived from plasma of the one or more maternal blood samples, wherein the isolation utilizes baits or primers based upon coordinates of cell-free fetal DNA fragment preffered end-points; and (Hi) using the data processing arrangement (104) to analyse the identified free fetal DNA (ffDNA) and compare them with one or more DNA templates stored in the data processing arrangement (106) for determining an occurrence of one or more biological characteristics of fetal DNA present in the one or more maternal blood samples.

11. A method of using a prenatal screening and diagnostic system (100) according to claim 10, characterized in that the method includes using the wet-laboratory arrangement (102) to enrich the free fetal DNA fragments for providing a plurality of copies of the free fetal DNA fragments for analysis.

12. A method of using a prenatal and diagnostic screening system (100) according to claim 11, characterized in that the method includes enriching the free fetal DNA fragments by using nucleosome profile to determine the most likely start position and the fetal specific end positions.

13. A method of using a prenatal screening and diagnostic system (100) according to claim 10, 11 or 12, characterized in that the method includes designing the baits or primers to avoid maternal DNA present in the plasma.

14. A method of using a prenatal screening and diagnostic system (100) according to claim 13, characterized in that the designed baits or primers are in combination with targeting of genes, wherein the genes are relevant to genetic disorders.

15. A method of using a prenatal screening and diagnostic system (100) according to any one of claims 10 to 14, characterized in that the method includes using the wet-laboratory arrangement (102) to identify and analyse cfDNA fragments in the plasma that start within a nucleosome, wherein the cfDNA fragments correspond to a fetal fraction of the plasma of the one or more maternal blood samples, wherein the cfDNA fragments that start within a nucleosome are relatively shorter in nucleic acid base count than an average length in nucleic acid base count of cfDNA fragments present in the one or more maternal blood samples.

16. A method of using a prenatal and diagnostic screening and diagnostic system (100) according to any one of claims 10 to 15, characterized in that the method includes using the wet-laboratory arrangement (102) to perform a combined test for prenatal screening of fetal chromosomal abnormalities, wherein the combined test includes:

(i) at least one maternal blood test; and (ii) an ultrasound scan of a fetus.

17. A method of using a prenatal screening and diagnostic system (100) according to any one of claims 10 to 16, characterized in that the method includes using the wet-laboratory arrangement (102) to measure levels of human chorionic gonadotrophin (hCG) and pregnancy-associated plasma

5 protein (PAPP-A).

18. A method of using a prenatal screening and diagnostic system (100) according to any one of claims 10 to 17, characterized in that the method includes using the data processing arrangement (104) to store genetic information extracted from the maternal blood samples in a secondary io database.

19. A computer program product comprising a non-transitory computerreadable storage medium having computer-readable instructions stored thereon, the computer-readable instructions being executable by a computerized device comprising processing hardware (104) to execute a is method as claimed in claim 10.