[go: up one dir, main page]

WO2024263045A1 - Method of non-invasive fetal dna and fetal fraction analysis using multiplex digital pcr reaction - Google Patents

Method of non-invasive fetal dna and fetal fraction analysis using multiplex digital pcr reaction Download PDF

Info

Publication number
WO2024263045A1
WO2024263045A1 PCT/PL2024/050042 PL2024050042W WO2024263045A1 WO 2024263045 A1 WO2024263045 A1 WO 2024263045A1 PL 2024050042 W PL2024050042 W PL 2024050042W WO 2024263045 A1 WO2024263045 A1 WO 2024263045A1
Authority
WO
WIPO (PCT)
Prior art keywords
fetal
reaction
chromosome
digital pcr
copy number
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/PL2024/050042
Other languages
French (fr)
Inventor
Anna NYKEL
Agnieszka GACH
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Instytut Centrum Zdrowia Matki Polki
Original Assignee
Instytut Centrum Zdrowia Matki Polki
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Instytut Centrum Zdrowia Matki Polki filed Critical Instytut Centrum Zdrowia Matki Polki
Publication of WO2024263045A1 publication Critical patent/WO2024263045A1/en
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/143—Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/16—Assays for determining copy number or wherein the copy number is of special importance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/165—Mathematical modelling, e.g. logarithm, ratio

Definitions

  • Subject of the invention is a non-invasive method of fetal DNA analysis that allows for sex determination and fetal fraction evaluation in maternal peripheral blood.
  • the solution falls within the field of genetic non-invasive fetal screening tests.
  • Invasive methods constitute the golden standard in prenatal diagnosis; however, they are associated with a one percent risk of miscarriage.
  • the discovery of free circulating fetal cells in maternal peripheral blood unlocked new possibilities within the field of prenatal diagnosis. It is estimated that cell-free fetal DNA (cffDNA) accounts for approximately 10% of total DNA in maternal peripheral blood in the 9 th week of gestation and its amount increases throughout pregnancy. Calculation of fetal fraction content percentage and fetal sex determination using a non-invasive method bears great significance in clinical practice for monogenic disorders and X-linked disorders. Furthermore, since efficiency of non-invasive prenatal tests is dependent on the fraction level, calculation of fetal fraction is particularly important in the case of such testing.
  • Each method based on non-invasive testing has a predetermined detection threshold for fetal fraction, hence any results obtained with such method may be affected by calculation of the fraction in relation to the corresponding threshold.
  • Calculation of fetal fraction is essential for quality control and ensuring statistical significance of non-invasive prenatal tests’ results.
  • it confirms that DNA of fetal origin is present in the sample in an amount that guarantees a reliable result, thus minimizing the risk of obtaining false negatives.
  • fetal fraction analysis Several non-invasive methods of fetal fraction analysis are known from the state of the art; these are methods based on: methylation markers analysis (WO 2017/081047A1 ), fragment length difference analysis (US20130237431 A1 ), SNP markers analysis (PL/EP 2376661 B1 , CN114645079A), and methods based on Y chromosome copy number (AU2019283856B2).
  • Non-invasive analyses of cffDNA for aneuploidy using methods based on DNA sequencing and digital PCR are known from the state of the art.
  • Such solutions, as disclosed in the state of the art, that use digital PCR technology are based on chromosome 13, 18, 21 analysis for aneuploidy identification in a fetus by non-invasive prenatal testing (NIPT).
  • NIPT non-invasive prenatal testing
  • CN105695567A discloses a method for non-invasive analysis of chromosome 13, 18, 21 aneuploidy in a fetus by means of digital PCR on QuantStudio 3D Digital PCR platform.
  • PL/EP 1981995 discloses non-invasive genetic fetal screening tests carried out by digital analysis that include the steps of obtaining biological material, separating DNA particles, detecting target sequences across a large number of reaction samples (digital PCR), and performing quantitative analysis.
  • WO 2017/074094A1 discloses a method for prenatal diagnosis of aneuploidy by means of droplet digital PCR.
  • the invention comprises the steps of: DNA extraction, DNA classification according to fragment length, digital PCR, calculation of target gene to control gene ratio.
  • WO 2020225632A discloses a method for direct fetal DNA quantitation and aneuploidy determination by means of droplet digital PCR technology.
  • the invention comprises both an analytical method and a statistical tool for analysis.
  • CN1 14645079A discloses a method for fetal fraction analysis based on SNP by means of digital PCR technology.
  • PL/EP 2376661 B1 provides simultaneous determination of aneuploidy and fetal fraction by means of DNA sequencing technology based on SNP analysis.
  • US20130237431 A1 PL/EP 3301193 disclose methods for analysis of fetal DNA fraction in maternal plasma based on fragment size by DNA sequencing.
  • US2018/0105864A1 provides a method for evaluation of cffDNA quality in a biological sample by means of two-step amplification with probe/primer sets. The method also provides generation of a library with the cffDNA for DNA sequencing.
  • WO2017/081047A1 discloses a technique of aneuploidy analysis in a fetus based on methylation markers (fetal cells and maternal DNA).
  • AU2019283856B2 discloses a method for fetal sex and chromosomal abnormalities determination in view of the percentage contribution of fetal DNA from Y chromosome in a maternal peripheral blood sample based on polymorphic (SNP) sequences.
  • SNP polymorphic
  • the state of the art calls for a high-quality, non-invasive prenatal diagnostic test for pregnant women that allows for rapid, precise and specific analysis and evaluation of fetal fraction and determination of sex, while being insusceptible to false positive/negative results (100% sensitivity and specificity).
  • the reaction is performed on a reaction microchamber-based digital PCR chip platform.
  • the reference genes are RPPH1 , TERT, EIF2C1.
  • the PCR reaction is performed under the following conditions: initial denaturation in 96 °C for 10 min, annealing in 60 °C for 2 min, denaturation in 98 °C for 30 s, final extension in 60 °C for 2 min, wherein the reaction comprises 40 cycles.
  • the PCR reaction is performed under the following conditions: initial denaturation in 96 °C for 10 min, annealing in 60 °C for 15 s, denaturation in 96 °C for 5 s, wherein the reaction comprises 40 cycles.
  • the reaction is performed on QuantStudio 3D Digital PCR or QuantStudio Absolute Q Digital PCR platform.
  • the fetal sex determination is based on whether Y chromosome signal is present, wherein lack of Y chromosome signal indicates female sex.
  • the fetal fraction (FF) concentration (%) in peripheral blood of a pregnant female is calculated based on copy number of amplified fragments in fetal genome, wherein the FF concentration is calculated with the following formula: wherein
  • the present invention relates to a method of non-invasive fetal DNA analysis, including fetal fraction determination, from peripheral blood of a pregnant female by means of digital PCR technology, preferably using reaction microchambers on QuantStudio 3D Digital PCR and QuantStudio Absolute Q Digital PCR platforms.
  • Digital PCR is a modern technology characterized by sensitivity, ease of performance and accessibility that may be utilized in prenatal testing.
  • Digital PCR is a method of absolute quantification of nucleic acids that - unlike the routinely used real-time PCR - does not require referring the results to a standard curve or a reference. It enables absolute quantification of DNA, ensuring high sensitivity and specificity.
  • the method’s concept is based on sample partitioning, followed by amplification reaction being performed simultaneously across several dozens of thousands of microreactions. Due to sample partitioning, each microreaction comprises a single nucleic acid target molecule, a small amount of such molecules, or does not contain any of them.
  • QuantStudio 3D Digital PCR and QuantStudio Absolute Q Digital PCR employ a system of reaction microchambers on a chip or a microfluidic array plate (MAP), while the others generate small reaction droplets.
  • MAP microfluidic array plate
  • the solution according to the invention based on copy number analysis performed simultaneously for several targets, enables much more sensitive and precise copy number evaluation than in the case of a test based on a single target analysis. Simultaneous amplification of 6 targets in 1 reaction reduces testing cost. Fast protocol, testing sensitivity and low cost enhance the solution’s accessibility as a screening test for use in clinical practice, ensuring easy implementation even in low-throughput laboratories.
  • the instrument’s sensitivity enables cffDNA analysis evaluation at an early stage of pregnancy (around the 10 th week of gestation).
  • the solution according to the invention is characterized by early detection, testing non-invasiveness, ease of implementation, low analysis cost and - due to method’s sensitivity - reliability of results.
  • the present tool eliminates the need for invasive material collection methods, enabling easy evaluation and providing diagnostic value during routine collection of maternal peripheral blood for other tests.
  • the present tool is fast, precise, specific and insusceptible to false positive/negative results (100% sensitivity and specificity), ensuring enhanced reliability of the diagnostic test.
  • the presented data is indicative of an excellent screening test.
  • Fig. 1 Diagram of digital PCR technology’s concept based on sample partitioning, followed by reaction being performed simultaneously across several dozens of thousands of reaction microchambers.
  • Fig. 2A Non-invasive cffDNA analysis protocol on QuantStudio 3D Digital PCR platform.
  • Fig. 3 Example of sample partitioning after cffDNA extraction, analysis of fetal DNA fragment size.
  • Fig. 5 Calculation of fetal fraction (FF) on clinical samples.
  • Fig. 7 Example of female fetal clinical sample analysis on QuantStudio Absolute Q Digital PCR platform.
  • Testing is performed on maternal peripheral blood, comprising free circulating fetal cells, as biological material.
  • the material is collected into two EDTA tubes (2x5 ml), which are then processed within 2-4 h (the tubes are refrigerated until processing).
  • the tubes are centrifuged in a two-step protocol: 1 ,900 g x 10 min in 4 °C, followed by transferring 2 ml of each tube’s content with a pipette into new Eppendorf tubes for another centrifugation at 16,000 g x 10 min in 4 °C. Then, the plasma is stored in a freezer at -80 °C until further processing.
  • the frozen material is equilibrated to room temperature. Isolation is performed from approximately 4 ml of plasma according to the manufacturer’s instructions by a vacuum method, using a QIAmp Circulating Nucleic Acid kit and a QIAVac 24 Plus Vacuum System (QIAGEN). cffDNA was resuspended in a final elution volume of 50 pl and frozen at -20 °C until the next step. DNA concentration was measured by fluorometry using a QuantiFluor ONE dsDNA System kit on a Quantus Fluorometer (Promega).
  • Fetal fraction evaluation based on DNA fragments shorter than 200 bp constitutes the first step of analysis; it is performed using D1000 High Sensitivity ScreenTape Assay on TapeStation 4150 (Agilent). The step enables cffDNA fragment size evaluation and, if necessary, exclusion of the sample at this point based on the fragment length analysis (no band of approximately 160 bp).
  • - amplicons must have similar length and be shorter than 100 bp (cffDNA fragments length is approximately 166 bp, hence amplicons must be shorter);
  • - targets on the chromosome of interest should have different genomic locations; - the selected genes should have constant expression (e.g. the so-called “housekeeping genes”).
  • RPS4Y2 NCBI#NM_001039567.3, M IM: 400030
  • SRY NCBI#NM_003140.3, MIM:480000
  • USP9Y NCBI#NM_004654.4, MIM:400005
  • RPPH1 NCBI#NR_002312.1 , MIM:608513
  • TERT NCBI#NM_198253.3, MIM:187270
  • EIF2C1 NCBI#NM_012199.5, MIM:606228.
  • the method is based on QuantStudio 3D Digital PCR system (Life Technologies) using 20 thousand reaction microchambers on a chip.
  • the reaction mix consists of: 7.5 pl 20x QuantStudio 3D dPCR master mix, 1 .2 pl 20x FAMA/IC TaqMan assay (Life Technologies) (2.4 pl assay mix in total), 6 pl cffDNA, topped up with nuclease-free water (15 pl final volume).
  • the table below presents selected assays for the analyzed targets. Selected targets on Y chromosome were labelled with FAM dye, while references were labelled with VIC dye. All amplicons have similar size of approximately 80 bp (76-89 bp).
  • Probe CGCCCTGCCATGTGGAAGAT B
  • the chips are loaded into a QuantStudio 3D Digital PCR Instrument, data from each chip is read, and an .eds file is generated. Further analysis, including data quality and copy number analysis for selected targets on Y chromosome against references, is performed in cloud environment using QuantStudio 3D AnalysisSuite Software.
  • reaction quality analysis performed by means of chip coverage evaluation (more than 16 thousand reaction microchambers) and analysis of signals form FAM and VIC fluorochromes, constitutes the first step.
  • Analysis of copy number/pl for selected targets on Y chromosome and references according to the Poisson statistics and reaction quality evaluation parameters constitutes the next step.
  • the fetal sex determination is based on detection of amplification signals for sequences on Y chromosome. Lack of Y chromosome signal suggests female sex.
  • Test results have been confirmed using clinical samples.
  • the arithmetic mean value for fetal fraction was 4.9% (SD 0.027, range 0.017-0.121 ).
  • 19/33 cases of male sex (57.6% of all cases) and 14/33 cases of female sex (42.4% of all cases) were identified correctly.
  • the analysis results have been confirmed by a diagnostic method during invasive prenatal diagnostic testing, demonstrating 100% consistency. No false positives or negatives were identified. According to the presented data, the method’s sensitivity is estimated to be 100%.
  • Testing is performed on maternal peripheral blood, comprising free circulating fetal cells, as biological material.
  • the material is collected into two EDTA tubes (2x5 ml), which are then processed within 2-4 h (the tubes are refrigerated until processing).
  • the tubes are centrifuged in a two-step protocol: 1 ,900 g x 10 min in 4 °C, followed by transferring 2 ml of each tube’s content with a pipette into new Eppendorf tubes for another centrifugation at 16,000 g x 10 min in 4 °C. Then, the plasma is stored in a freezer at -80 °C until further processing.
  • the frozen material is equilibrated to room temperature. Isolation is performed from approximately 4 ml of plasma according to the manufacturer’s instructions by a vacuum method, using a QIAmp Circulating Nucleic Acid kit and a QIAVac 24 Plus Vacuum System (QIAGEN). cffDNA was resuspended in a final elution volume of 50 pl and frozen at -20 °C until the next step. DNA concentration was measured by fluorometry using a QuantiFluor ONE dsDNA System kit on a Quantus Fluorometer (Promega).
  • Fetal fraction evaluation based on DNA fragments shorter than 200 bp constitutes the first step of analysis; it is performed using D1000 High Sensitivity ScreenTape Assay on TapeStation 4150 (Agilent). The step enables cffDNA fragment size evaluation and, if necessary, exclusion of the sample at this point based on the fragment length analysis (no band of approximately 160 bp).
  • the method is based on QuantStudio Absolute Q Digital PCR system (Life Technologies) using more than 20 thousand microchambers on a MAP plate.
  • the reaction mix consists of: 2 pl 1X Combinati MasterMix, 1.2 pl 20x FAMA/IC TaqMan assay mix (3 targets on Y chromosome and 3 reference targets), 4 pl cffDNA, topped up with nuclease-free water (10 pl final volume).
  • Taqman assays used in the experiment were the same as the ones in example 1 (Table).
  • reaction mix is loaded into a reaction microchamber (9 pl) and topped up with isolation buffer 15 pl. The microchambers are then sealed with a rubber gasket cap. The plate is loaded into a QuantStudio Absolute Q Digital PCR instrument. Amplification is performed under the following conditions: Further analysis, including copy number calculations, is performed using QuantStudio Absolute Q Digital PCR Software (v.6.2.1 ).
  • the samples are processed using QuantStudio Absolute Q Digital PCR Software.
  • the first step consists of analyzing the reaction’s accuracy and efficiency by means of coefficient of variation (CV) evaluation (in order for a reaction to be accepted, CV must be less than 10%), and analyzing images taken before amplification that show coverage across microchambers. Copy number analysis for Y chromosome targets of interest, as compared to copy number of reference chromosomes, constitutes the next step.
  • CV coefficient of variation

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

A method of cell-free fetal DNA (cffDNA) analysis for sex determination and fetal fraction content percentage evaluation, characterized in that biological material is collected from a pregnant female patient, cffDNA is isolated, the fetal DNA is evaluated, a multiplex digital PCR reaction is performed in order to amplify genes on Y chromosome, including RPS4Y2, SRY, USP9Y, and reference genes RPPH1, TERT, EIF2C1, and the data is analyzed.

Description

Method of non-invasive fetal DNA and fetal fraction analysis using multiplex digital PCR reaction
Subject of the invention is a non-invasive method of fetal DNA analysis that allows for sex determination and fetal fraction evaluation in maternal peripheral blood. The solution falls within the field of genetic non-invasive fetal screening tests.
Background of the invention
Invasive methods constitute the golden standard in prenatal diagnosis; however, they are associated with a one percent risk of miscarriage. The discovery of free circulating fetal cells in maternal peripheral blood unlocked new possibilities within the field of prenatal diagnosis. It is estimated that cell-free fetal DNA (cffDNA) accounts for approximately 10% of total DNA in maternal peripheral blood in the 9th week of gestation and its amount increases throughout pregnancy. Calculation of fetal fraction content percentage and fetal sex determination using a non-invasive method bears great significance in clinical practice for monogenic disorders and X-linked disorders. Furthermore, since efficiency of non-invasive prenatal tests is dependent on the fraction level, calculation of fetal fraction is particularly important in the case of such testing. Each method based on non-invasive testing has a predetermined detection threshold for fetal fraction, hence any results obtained with such method may be affected by calculation of the fraction in relation to the corresponding threshold. Calculation of fetal fraction is essential for quality control and ensuring statistical significance of non-invasive prenatal tests’ results. In addition, it confirms that DNA of fetal origin is present in the sample in an amount that guarantees a reliable result, thus minimizing the risk of obtaining false negatives.
State of the art
Several non-invasive methods of fetal fraction analysis are known from the state of the art; these are methods based on: methylation markers analysis (WO 2017/081047A1 ), fragment length difference analysis (US20130237431 A1 ), SNP markers analysis (PL/EP 2376661 B1 , CN114645079A), and methods based on Y chromosome copy number (AU2019283856B2).
Non-invasive analyses of cffDNA for aneuploidy using methods based on DNA sequencing and digital PCR are known from the state of the art. Such solutions, as disclosed in the state of the art, that use digital PCR technology are based on chromosome 13, 18, 21 analysis for aneuploidy identification in a fetus by non-invasive prenatal testing (NIPT).
On the other hand, research efforts have been focused on different methods for determining fetal fraction and fetal sex (based on various diagnostic approaches and different molecular analysis methods).
Publications regarding non-invasive fetal DNA analysis for aneuploidy using digital PCR technology are presented below. CN105695567A discloses a method for non-invasive analysis of chromosome 13, 18, 21 aneuploidy in a fetus by means of digital PCR on QuantStudio 3D Digital PCR platform. PL/EP 1981995 discloses non-invasive genetic fetal screening tests carried out by digital analysis that include the steps of obtaining biological material, separating DNA particles, detecting target sequences across a large number of reaction samples (digital PCR), and performing quantitative analysis.
WO 2017/074094A1 discloses a method for prenatal diagnosis of aneuploidy by means of droplet digital PCR. The invention comprises the steps of: DNA extraction, DNA classification according to fragment length, digital PCR, calculation of target gene to control gene ratio. WO 2020225632A discloses a method for direct fetal DNA quantitation and aneuploidy determination by means of droplet digital PCR technology. The invention comprises both an analytical method and a statistical tool for analysis.
Publications regarding methods for fetal fraction analysis are presented below. CN1 14645079A discloses a method for fetal fraction analysis based on SNP by means of digital PCR technology. PL/EP 2376661 B1 provides simultaneous determination of aneuploidy and fetal fraction by means of DNA sequencing technology based on SNP analysis. US20130237431 A1 , PL/EP 3301193 disclose methods for analysis of fetal DNA fraction in maternal plasma based on fragment size by DNA sequencing. US2018/0105864A1 provides a method for evaluation of cffDNA quality in a biological sample by means of two-step amplification with probe/primer sets. The method also provides generation of a library with the cffDNA for DNA sequencing. The invention is based on multiplex droplet digital PCR method. WO2017/081047A1 discloses a technique of aneuploidy analysis in a fetus based on methylation markers (fetal cells and maternal DNA). AU2019283856B2 discloses a method for fetal sex and chromosomal abnormalities determination in view of the percentage contribution of fetal DNA from Y chromosome in a maternal peripheral blood sample based on polymorphic (SNP) sequences.
Therefore, the state of the art calls for a high-quality, non-invasive prenatal diagnostic test for pregnant women that allows for rapid, precise and specific analysis and evaluation of fetal fraction and determination of sex, while being insusceptible to false positive/negative results (100% sensitivity and specificity).
Subject of the invention is a method of non-invasive fetal fraction analysis for its content percentage calculation and fetal sex determination in a biological sample, comprising cell-free fetal DNA (cffDNA), from peripheral blood of a pregnant woman, wherein: a) cffDNA is isolated; b) length of cffDNA fragments is analyzed and samples that comprise cffDNA fragments not larger than 200 bp are selected for further analysis; c) a multiplex digital PCR reaction using reaction microchambers is performed in order to simultaneously amplify genes on Y chromosome, including RPS4Y2, SRY, USP9Y, and reference genes; d) absolute copy number/pl of fetal genome fragments (cffDNA), obtained in step c), is established in order to calculate the fetal fraction content percentage and determine the fetal sex; wherein Y chromosome copy number > 0 for male sex, and Y chromosome copy number = 0 for female sex.
Preferably, the reaction is performed on a reaction microchamber-based digital PCR chip platform.
Preferably, the reference genes are RPPH1 , TERT, EIF2C1. Preferably, the PCR reaction is performed under the following conditions: initial denaturation in 96 °C for 10 min, annealing in 60 °C for 2 min, denaturation in 98 °C for 30 s, final extension in 60 °C for 2 min, wherein the reaction comprises 40 cycles.
Preferably, the PCR reaction is performed under the following conditions: initial denaturation in 96 °C for 10 min, annealing in 60 °C for 15 s, denaturation in 96 °C for 5 s, wherein the reaction comprises 40 cycles.
Preferably, the reaction is performed on QuantStudio 3D Digital PCR or QuantStudio Absolute Q Digital PCR platform.
Preferably, the fetal sex determination is based on whether Y chromosome signal is present, wherein lack of Y chromosome signal indicates female sex.
Preferably, the fetal fraction (FF) concentration (%) in peripheral blood of a pregnant female is calculated based on copy number of amplified fragments in fetal genome, wherein the FF concentration is calculated with the following formula: wherein
Figure imgf000005_0001
P1 - fetal Y chromosome copy number
M - maternal reference chromosomes copy number
P2 - fetal reference chromosomes copy number, wherein it is assumed that P2=2*P1 (in genome, there is 1 copy of Y chromosome, and there are 2 copies of reference genes on autosomal chromosomes)
The present invention relates to a method of non-invasive fetal DNA analysis, including fetal fraction determination, from peripheral blood of a pregnant female by means of digital PCR technology, preferably using reaction microchambers on QuantStudio 3D Digital PCR and QuantStudio Absolute Q Digital PCR platforms.
Digital PCR is a modern technology characterized by sensitivity, ease of performance and accessibility that may be utilized in prenatal testing. Digital PCR is a method of absolute quantification of nucleic acids that - unlike the routinely used real-time PCR - does not require referring the results to a standard curve or a reference. It enables absolute quantification of DNA, ensuring high sensitivity and specificity. The method’s concept is based on sample partitioning, followed by amplification reaction being performed simultaneously across several dozens of thousands of microreactions. Due to sample partitioning, each microreaction comprises a single nucleic acid target molecule, a small amount of such molecules, or does not contain any of them. Then, based on amplification and signal detection performed in each microreaction, a result of absolute copy number/pl is generated. Several digital PCR platforms are currently available, each being characterized by partitioning of a sample into several dozens of thousands of microreactions: QuantStudio 3D Digital PCR and QuantStudio Absolute Q Digital PCR by ThermoFisher Scientific, Raindrop Digital PCR by RainDance, and Droplet Digital PCR by Bio-RAD. QuantStudio 3D Digital PCR and QuantStudio Absolute Q Digital PCR employ a system of reaction microchambers on a chip or a microfluidic array plate (MAP), while the others generate small reaction droplets. Such solution allows for a streamlined protocol and reduces risk of contamination in the case of microchamber reactions; also, reaction efficiency is not dependent on the droplet generation process. The method provides high sensitivity, whilst remaining fast and easy to perform. Contrary to digital PCR that employs the reaction droplet technology (droplet digital PCR platform), the present method does not require the droplet generation step, which results in an optimized protocol and reduced risk of contamination at the PCR reaction mix preparation step.
The solution according to the invention, based on copy number analysis performed simultaneously for several targets, enables much more sensitive and precise copy number evaluation than in the case of a test based on a single target analysis. Simultaneous amplification of 6 targets in 1 reaction reduces testing cost. Fast protocol, testing sensitivity and low cost enhance the solution’s accessibility as a screening test for use in clinical practice, ensuring easy implementation even in low-throughput laboratories. The instrument’s sensitivity enables cffDNA analysis evaluation at an early stage of pregnancy (around the 10th week of gestation). Thus, the solution according to the invention is characterized by early detection, testing non-invasiveness, ease of implementation, low analysis cost and - due to method’s sensitivity - reliability of results. It eliminates the need for invasive material collection methods, enabling easy evaluation and providing diagnostic value during routine collection of maternal peripheral blood for other tests. The present tool is fast, precise, specific and insusceptible to false positive/negative results (100% sensitivity and specificity), ensuring enhanced reliability of the diagnostic test. The presented data is indicative of an excellent screening test.
Description of figures
Fig. 1 Diagram of digital PCR technology’s concept based on sample partitioning, followed by reaction being performed simultaneously across several dozens of thousands of reaction microchambers.
Fig. 2A Non-invasive cffDNA analysis protocol on QuantStudio 3D Digital PCR platform.
Fig. 2B Non-invasive cffDNA analysis protocol on QuantStudio Absolute Q Digital PCR platform.
Fig. 3 Example of sample partitioning after cffDNA extraction, analysis of fetal DNA fragment size.
Samples:
A1 : 163 bp 67.6 pg/pl
B1 : 158 bp 47.9 pg/pl
C1 : 160 bp 238 pg/pl
D1 : 163 bp 236 pg/pl
Fig. 4 Example of reaction analysis using QuantStudio 3D AnalysisSuite Software.
Fig. 5 Calculation of fetal fraction (FF) on clinical samples.
Fig. 6 Example of reaction analysis using QuantStudio Absolute Q Digital PCR Software based on reaction quality parameters.
Fig. 7 Example of female fetal clinical sample analysis on QuantStudio Absolute Q Digital PCR platform.
Fig. 8 Example of male fetal clinical sample analysis on QuantStudio Absolute Q Digital PCR platform. Example 1
Testing is performed on maternal peripheral blood, comprising free circulating fetal cells, as biological material. The material is collected into two EDTA tubes (2x5 ml), which are then processed within 2-4 h (the tubes are refrigerated until processing). The tubes are centrifuged in a two-step protocol: 1 ,900 g x 10 min in 4 °C, followed by transferring 2 ml of each tube’s content with a pipette into new Eppendorf tubes for another centrifugation at 16,000 g x 10 min in 4 °C. Then, the plasma is stored in a freezer at -80 °C until further processing.
The frozen material is equilibrated to room temperature. Isolation is performed from approximately 4 ml of plasma according to the manufacturer’s instructions by a vacuum method, using a QIAmp Circulating Nucleic Acid kit and a QIAVac 24 Plus Vacuum System (QIAGEN). cffDNA was resuspended in a final elution volume of 50 pl and frozen at -20 °C until the next step. DNA concentration was measured by fluorometry using a QuantiFluor ONE dsDNA System kit on a Quantus Fluorometer (Promega).
Fetal DNA evaluation.
Fetal fraction evaluation based on DNA fragments shorter than 200 bp (150-170 bp) constitutes the first step of analysis; it is performed using D1000 High Sensitivity ScreenTape Assay on TapeStation 4150 (Agilent). The step enables cffDNA fragment size evaluation and, if necessary, exclusion of the sample at this point based on the fragment length analysis (no band of approximately 160 bp).
Multiplexing of targets on Y chromosome and reference sequences constitutes the second step of analysis.
Targets on Y chromosome and reference genes were selected. Features required for selection of assays:
- amplicons must have similar length and be shorter than 100 bp (cffDNA fragments length is approximately 166 bp, hence amplicons must be shorter);
- targets on the chromosome of interest should have different genomic locations; - the selected genes should have constant expression (e.g. the so-called “housekeeping genes”).
The following genes were selected for Y chromosome copy number analysis: RPS4Y2 (NCBI#NM_001039567.3, M IM: 400030), SRY (NCBI#NM_003140.3, MIM:480000), USP9Y (NCBI#NM_004654.4, MIM:400005). The following reference genes were selected: RPPH1 (NCBI#NR_002312.1 , MIM:608513), TERT (NCBI#NM_198253.3, MIM:187270), EIF2C1 (NCBI#NM_012199.5, MIM:606228).
A multiplex (six-plex) reaction - simultaneous analysis of 6 targets in 1 reaction - was developed and validated. The method is based on QuantStudio 3D Digital PCR system (Life Technologies) using 20 thousand reaction microchambers on a chip.
A) Preparation of reaction mix
The reaction mix consists of: 7.5 pl 20x QuantStudio 3D dPCR master mix, 1 .2 pl 20x FAMA/IC TaqMan assay (Life Technologies) (2.4 pl assay mix in total), 6 pl cffDNA, topped up with nuclease-free water (15 pl final volume).
The table below presents selected assays for the analyzed targets. Selected targets on Y chromosome were labelled with FAM dye, while references were labelled with VIC dye. All amplicons have similar size of approximately 80 bp (76-89 bp).
Figure imgf000009_0001
*the assay’s sequences were published in US2011/0151442A1
Seq 5’-3’ F:GTTCGGCTTTCACCAGTCT R:CTCCATAGCTCTCCCCACTC
Probe: CGCCCTGCCATGTGGAAGAT B) Loading the reaction mix onto a chip using a QuantStudio 3D Digital Chip Loader. The mix is loaded into the instrument, and the instrument presses the reaction mix into reaction microchambers on the chip.
C) Amplification reaction using ProFlex Thermal Cycler
Figure imgf000010_0001
Following the amplification reaction, the chips are loaded into a QuantStudio 3D Digital PCR Instrument, data from each chip is read, and an .eds file is generated. Further analysis, including data quality and copy number analysis for selected targets on Y chromosome against references, is performed in cloud environment using QuantStudio 3D AnalysisSuite Software.
Data analysis.
The samples are processed using QuantStudio 3D AnalysisSuite Software. Reaction quality analysis, performed by means of chip coverage evaluation (more than 16 thousand reaction microchambers) and analysis of signals form FAM and VIC fluorochromes, constitutes the first step. Analysis of copy number/pl for selected targets on Y chromosome and references according to the Poisson statistics and reaction quality evaluation parameters constitutes the next step.
The fetal sex determination is based on detection of amplification signals for sequences on Y chromosome. Lack of Y chromosome signal suggests female sex.
Y chromosome copy number > 0 for male sex
Y chromosome copy number = 0 for female sex
Calculation of fetal fraction based on the following formula:
Figure imgf000011_0001
P1 - fetal Y chromosome copy number
M - maternal reference chromosomes copy number
P2 - fetal reference chromosomes copy number, wherein it is assumed that P2=2*P1 (in genome, there is 1 copy of Y chromosome, and there are 2 copies of reference genes on autosomal chromosomes)
Test results have been confirmed using clinical samples. The arithmetic mean value for fetal fraction was 4.9% (SD 0.027, range 0.017-0.121 ). Among 33 clinical samples, 19/33 cases of male sex (57.6% of all cases) and 14/33 cases of female sex (42.4% of all cases) were identified correctly. The analysis results have been confirmed by a diagnostic method during invasive prenatal diagnostic testing, demonstrating 100% consistency. No false positives or negatives were identified. According to the presented data, the method’s sensitivity is estimated to be 100%.
Example 2
Testing is performed on maternal peripheral blood, comprising free circulating fetal cells, as biological material. The material is collected into two EDTA tubes (2x5 ml), which are then processed within 2-4 h (the tubes are refrigerated until processing). The tubes are centrifuged in a two-step protocol: 1 ,900 g x 10 min in 4 °C, followed by transferring 2 ml of each tube’s content with a pipette into new Eppendorf tubes for another centrifugation at 16,000 g x 10 min in 4 °C. Then, the plasma is stored in a freezer at -80 °C until further processing.
The frozen material is equilibrated to room temperature. Isolation is performed from approximately 4 ml of plasma according to the manufacturer’s instructions by a vacuum method, using a QIAmp Circulating Nucleic Acid kit and a QIAVac 24 Plus Vacuum System (QIAGEN). cffDNA was resuspended in a final elution volume of 50 pl and frozen at -20 °C until the next step. DNA concentration was measured by fluorometry using a QuantiFluor ONE dsDNA System kit on a Quantus Fluorometer (Promega).
Fetal DNA evaluation.
Fetal fraction evaluation based on DNA fragments shorter than 200 bp (150-170 bp) constitutes the first step of analysis; it is performed using D1000 High Sensitivity ScreenTape Assay on TapeStation 4150 (Agilent). The step enables cffDNA fragment size evaluation and, if necessary, exclusion of the sample at this point based on the fragment length analysis (no band of approximately 160 bp).
Multiplexing of targets on Y chromosome and reference sequences constitutes the second step of analysis.
A multiplex (six-plex) reaction - simultaneous analysis of 6 targets in 1 reaction - was developed and validated. The method is based on QuantStudio Absolute Q Digital PCR system (Life Technologies) using more than 20 thousand microchambers on a MAP plate.
A. Preparation of reaction mix
The reaction mix consists of: 2 pl 1X Combinati MasterMix, 1.2 pl 20x FAMA/IC TaqMan assay mix (3 targets on Y chromosome and 3 reference targets), 4 pl cffDNA, topped up with nuclease-free water (10 pl final volume).
Taqman assays used in the experiment were the same as the ones in example 1 (Table).
B. The reaction mix is loaded into a reaction microchamber (9 pl) and topped up with isolation buffer 15 pl. The microchambers are then sealed with a rubber gasket cap. The plate is loaded into a QuantStudio Absolute Q Digital PCR instrument. Amplification is performed under the following conditions:
Figure imgf000012_0001
Further analysis, including copy number calculations, is performed using QuantStudio Absolute Q Digital PCR Software (v.6.2.1 ).
Data analysis.
The samples are processed using QuantStudio Absolute Q Digital PCR Software. The first step consists of analyzing the reaction’s accuracy and efficiency by means of coefficient of variation (CV) evaluation (in order for a reaction to be accepted, CV must be less than 10%), and analyzing images taken before amplification that show coverage across microchambers. Copy number analysis for Y chromosome targets of interest, as compared to copy number of reference chromosomes, constitutes the next step.
Analogous results based on copy number of Y chromosome targets of interest in relation to copy number of reference chromosomes are obtained regardless of platform. For that reason, fetal sex determination and fetal fraction calculation are performed in a manner analogous to example 1. Results obtained from both platforms are consistent and have been confirmed using classical cytogenetic approach, i.e. karyotyping from biological material collected by an invasive method, as a reference method.
Sequence list
<110> Institute of the Polish Mother's Health Center
<120> Method of non-invasive fetal DNA and fetal fraction analysis using multiplex digital PCR reaction
<130> PK/9745/AR
<160> 3
<170> Patentin version 3.5
<210> 1
<211 > 19
<212> DNA
<213> artificial
<220>
<223> Primer F
<400> 1 gttcggcttt caccagtct 19
<210> 2
<211 > 20
<212> DNA
<213> artificial
<220>
<223> Primer R
<400> 2 ctccatagct ctccccactc 20
<210> 3
<211 > 20
<212> DNA
<213> artificial
<220>
<223> Probe
<400> 3 cgccctgcca tgtggaagat 20

Claims

Claims
1 . A method of non-invasive fetal fraction analysis for its content percentage calculation and fetal sex determination in a biological sample, comprising cell-free fetal DNA (cffDNA), from peripheral blood of a pregnant woman, characterized in that e) cffDNA is isolated; f) length of cffDNA fragments is analyzed and samples that comprise cffDNA fragments not larger than 200 bp are selected for further analysis; g) a multiplex digital PCR reaction using reaction microchambers is performed in order to simultaneously amplify genes on Y chromosome, including RPS4Y2, SRY, USP9Y, and reference genes; h) absolute copy number/pl of fetal genome fragments (cffDNA), obtained in step c), is established in order to calculate the fetal fraction content percentage and determine the fetal sex; wherein Y chromosome copy number > 0 for male sex, and Y chromosome copy number = 0 for female sex.
2. The method of claim 1 , characterized in that the reaction is performed on a reaction microchamber-based digital PCR chip platform.
3. The method of claim 1 , characterized in that the reference genes are RPPH1 , TERT, EIF2C1.
4. The method of claim 1 , characterized in that the PCR reaction is performed under the following conditions: initial denaturation in 96 °C for 10 min, annealing in 60 °C for 2 min, denaturation in 98 °C for 30 s, final extension in 60 °C for 2 min, wherein the reaction comprises 40 cycles.
5. The method of claim 1 , characterized in that the PCR reaction is performed under the following conditions: initial denaturation in 96 °C for 10 min, annealing in 60 °C for 15 s, denaturation in 96 °C for 5 s, wherein the reaction comprises 40 cycles.
6. The method of claim 1 , characterized in that the reaction is performed on QuantStudio 3D Digital PCR or QuantStudio Absolute Q Digital PCR platform.
7. The method of claim 1 , characterized in that the fetal sex determination is based on whether Y chromosome signal is present, wherein lack of Y chromosome signal indicates female sex.
8. The method of claim 1 , characterized in that the fetal fraction (FF) concentration (%) in peripheral blood of a pregnant female is calculated based on copy number of amplified fragments in fetal genome, wherein the FF concentration is calculated with the following formula: wherein
Figure imgf000016_0001
P1 - fetal Y chromosome copy number
M - maternal reference chromosomes copy number
P2 - fetal reference chromosomes copy number, wherein it is assumed that P2=2*P1 (in genome, there is 1 copy of Y chromosome, and there are 2 copies of reference genes on autosomal chromosomes)
PCT/PL2024/050042 2023-06-20 2024-06-20 Method of non-invasive fetal dna and fetal fraction analysis using multiplex digital pcr reaction Pending WO2024263045A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PL445283A PL445283A1 (en) 2023-06-20 2023-06-20 Method for non-invasive analysis of fetal DNA and fetal fractions using multiplex digital PCR
PLP.445283 2023-06-20

Publications (1)

Publication Number Publication Date
WO2024263045A1 true WO2024263045A1 (en) 2024-12-26

Family

ID=93936122

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/PL2024/050042 Pending WO2024263045A1 (en) 2023-06-20 2024-06-20 Method of non-invasive fetal dna and fetal fraction analysis using multiplex digital pcr reaction

Country Status (2)

Country Link
PL (1) PL445283A1 (en)
WO (1) WO2024263045A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013055817A1 (en) * 2011-10-11 2013-04-18 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
WO2014180910A1 (en) * 2013-05-09 2014-11-13 Roche Diagnostics Gmbh Method of determining the fraction of fetal dna in maternal blood using hla markers

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020512000A (en) * 2017-03-31 2020-04-23 プレマイサ リミテッド How to detect fetal chromosomal abnormalities

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013055817A1 (en) * 2011-10-11 2013-04-18 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
WO2014180910A1 (en) * 2013-05-09 2014-11-13 Roche Diagnostics Gmbh Method of determining the fraction of fetal dna in maternal blood using hla markers

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DEBRAND, E ET AL.: "A Non-Invasive Droplet Digital PCR (ddPCR) Assay to Detect Paternal CFTR Mutations in the Cell -Free Fetal DNA (cffDNA) of Three Pregnancies at Risk of Cystic Fibrosis via Compound Heterozygosity", PLOS ONE, vol. 10, no. 11, pages 0142729, XP055445510, DOI: 10.1371/journal.pone.0142729 *
IOANNIDES MARIOS, ACHILLEOS ACHILLEAS, KYRIAKOU SKEVI, KYPRI ELENA, LOIZIDES CHARALAMBOS, TSANGARAS KYRIAKOS, CONSTANTINOU LOUIZA,: "Development of a new methylation‐based fetal fraction estimation assay using multiplex ddPCR", MOLECULAR GENETICS & GENOMIC MEDICINE, vol. 8, no. 2, 1 February 2020 (2020-02-01), XP093256787, ISSN: 2324-9269, DOI: 10.1002/mgg3.1094 *

Also Published As

Publication number Publication date
PL445283A1 (en) 2024-12-23

Similar Documents

Publication Publication Date Title
US20250215495A1 (en) Noninvasive diagnosis of fetal aneuploidy by sequencing
CA2456140C (en) Methods for detecting dna originating from different individuals in one sample
US9784742B2 (en) Means and methods for non-invasive diagnosis of chromosomal aneuploidy
JP6092891B2 (en) Method and system for determining the presence or absence of genomic abnormalities
JP2019162102A (en) System and method of detecting rnas altered by cancer in peripheral blood
US20080108071A1 (en) Methods and Systems to Determine Fetal Sex and Detect Fetal Abnormalities
CZ20001934A3 (en) Process for preparing complex DNA of methylation peptide maps
Picchiassi et al. The best approach for early prediction of fetal gender by using free fetal DNA from maternal plasma
US20160012201A1 (en) Determination device, computer readable medium, and marker for obtaining information on lung cancer
WO2016059601A1 (en) Non-invasive methods for detection of genetic abnormalities in an unborn fetus, and primers, probes and kits for uses thereof
US20240384334A1 (en) Efficient digital measurement of long nucleic acid fragments
WO2024263045A1 (en) Method of non-invasive fetal dna and fetal fraction analysis using multiplex digital pcr reaction
KR102036609B1 (en) A method for prenatal diagnosis using digital PCR
KR101848438B1 (en) A method for prenatal diagnosis using digital PCR
CN112899358A (en) Noninvasive prenatal fetal chromosome aneuploidy detection method and kit thereof
CN111500696A (en) Kit for screening 21-trisomy syndrome by detecting free RNA in peripheral blood of pregnant woman based on flight time mass spectrum
Mousavi et al. Development of a PCR-based method to identify fetal sex during IVF cycles
Wu et al. Application of droplet digital PCR for prenatal screening of Down syndrome
AU2018201920B2 (en) Noninvasive diagnosis of fetal aneuploidy by sequencing
JP2016192908A (en) Gynecological cancer information acquisition method, gynecological cancer information marker and gynecological cancer detection kit
JP6551656B2 (en) Method for obtaining information on ovarian cancer, and marker for obtaining information on ovarian cancer and kit for detecting ovarian cancer
CN116606926A (en) Digital PCR (polymerase chain reaction) kit for noninvasively detecting fetal chromosomal aneuploidy
WO2011082386A1 (en) Specific amplification of fetal dna sequences from a mixed, fetal-maternal source
JP2012095554A (en) Method of synthesizing single-stranded cdna, method of preparing microarray sample, and method of detecting nucleic acid

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24826342

Country of ref document: EP

Kind code of ref document: A1