Methylation marker for diagnosis of Down's syndrome

Abstract

The invention relates to the technical field of molecular biology, in particular to a methylation marker for Down's syndrome diagnosis. The invention particularly relates to application of a detection reagent for detecting the methylation level of a CpG site of a nucleic acid marker in preparation of a Down's syndrome diagnostic kit. The nucleic acid marker comprises a) an SIM2 gene segment as shown in SEQ ID NO: 1 or a polynucleotide segment complementary to the SIM2 gene segment, and / or b) a CRYBG1 gene segment as shown in SEQ ID NO: 2 or a polynucleotide segment complementary to the CRYBG1 gene segment.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to methylation markers for the diagnosis of Down syndrome. Background Technology

[0002] Down syndrome, also known as trisomy 21, is a disorder caused by the presence of an extra copy of chromosome 21. It is the most common chromosomal abnormality syndrome. The incidence of Down syndrome in newborns is approximately 1 in 600, while the incidence during fetal development can be as high as 1 in 225. In recent years, with advancements in technology and increased emphasis on prenatal screening for Down syndrome, the incidence of Down syndrome in live births in my country has decreased to 0.05%-0.06%.

[0003] The main clinical features of children with Down syndrome include distinctive facial features, intellectual disability, growth retardation, and may be accompanied by various malformations such as congenital heart disease. Down syndrome is incurable. Down syndrome patients who survive to adulthood are unable to care for themselves and require lifelong care, placing a heavy burden on their families and society.

[0004] Down syndrome occurs randomly and unpredictably, and even healthy parents can have a child with Down syndrome. Therefore, routine prenatal screening and necessary prenatal diagnosis during pregnancy, along with timely intervention for any Down syndrome cases detected during pregnancy, are effective means of preventing Down syndrome. Currently, widely accepted prenatal screening techniques for Down syndrome include serological screening and NIPT based on next-generation sequencing technology.

[0005] Serological Down syndrome screening utilizes multiple biochemical markers in maternal serum, combined with information such as the pregnant woman's age, weight, and gestational age, to calculate the risk rate of Down syndrome. However, the accuracy rate of serological screening methods is relatively low, only about 60%-80%, which can lead to missed detections of some positive fetuses and also produce many false-positive results for normal fetuses.

[0006] NIPT based on next-generation sequencing (NGS) technology leverages the fact that cell-free DNA in maternal peripheral blood contains fetal cell-free DNA. It uses NGS to perform large-scale sequencing of this DNA and calculates the contribution of each chromosome to the overall genome. Despite interference from a large number of maternal background genes, even minor changes in the overall proportions due to fetal chromosomal variations can be detected with the high precision of NGS, thus identifying aneuploidy. While NGS-based NIPT has high accuracy, it is costly, requiring expensive sequencers; the testing cycle is long, generally at least one week; and it requires a very complex sample processing procedure before analysis.

[0007] Given the limitations of the aforementioned technologies, some researchers have begun to seek alternative methods that are more cost-effective and faster to implement. Among these, the use of placental tissue-specific methylation sites for trisomy 21 detection has proven feasible. Methylation markers exist on chromosome 21, exhibiting a hypermethylated state in placental tissue but a demethylated state in maternal leukocytes. By utilizing the difference in methylation status between the fetus and mother, methods such as sulfite conversion or restriction endonuclease digestion can be used to completely remove the unmethylated DNA (maternal background) from the maternal plasma cfDNA, retaining only the methylated DNA containing fetal information. These methylated DNAs can then be quantitatively analyzed using methods such as real-time quantitative PCR or digital PCR.

[0008] Although fetal-specific methylation sites were discovered as early as 2002, and research on them predates the currently popular next-generation sequencing-based NIPT, this method has not yet been widely adopted. The main reason is the lack of a sufficient number of reliable and stable methylation biomarkers; existing biomarkers cannot fully meet clinical requirements. Summary of the Invention

[0009] This invention covers the following technical solutions: One aspect of the present invention relates to a polynucleotide selected from: a) The SIM2 gene fragment shown in SEQ ID NO: 1 or its complementary polynucleotide fragment, and / or b) The CRYBG1 gene fragment shown in SEQ ID NO: 2 or its complementary polynucleotide fragment.

[0010] Another aspect of the present invention relates to the use of a detection reagent for detecting the methylation level of CpG sites of nucleic acid biomarkers in the preparation of a diagnostic kit for Down syndrome; The nucleic acid markers include polynucleotides as described above.

[0011] Another aspect of the present invention relates to a kit for detecting Down syndrome, comprising the detection reagents as defined above.

[0012] Another aspect of the invention relates to a diagnostic device comprising one or more separate modules, at least one of which contains a detection reagent as defined above, or a component of a kit as described above.

[0013] This invention provides a Down syndrome detection platform based on a combination of fetal-specific methylation markers. By optimizing the selection of target / reference methylation sites, a T21 screening method with high sensitivity, high specificity, low cost, and easy promotion can be achieved. Attached Figure Description

[0014] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0015] Figure 1 Scatter plot of correlation analysis between copy number of reference methylation markers (CRYBG1, TBX3, PCDHGA1) and copy number of fetal DNA-specific marker SRY shows the correlation between each reference gene and fetal DNA, and verifies its stability as a reference methylation marker.

[0016] Figure 2 Box plots (with AUC values) comparing the ratios of the two biomarkers SIM2 / CRYBG1 and SIM2 / TBX3 demonstrate the detection performance of different combinations in distinguishing T21 positive and negative samples, reflecting the superiority of CRYBG1 over TBX3.

[0017] Figure 3 The scatter plot based on the joint discrimination of the two ratios SIM2 / CRYBG1 and DSCAM / TBX3 shows the distribution of each sample in a two-dimensional plane, visualizing the discrimination effect of T21 positive and negative samples. The dashed line represents the discrimination threshold line of linear fitting.

[0018] Figure 4 The box plot of R values ​​(SIM2 / CRYBG1 + 1.46 × DSCAM / TBX3) in 40 samples shows the ability of all samples to distinguish between positive and negative results, intuitively reflecting the discrimination performance. Detailed Implementation

[0019] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0020] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0021] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, immunology-related terms and laboratory procedures used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0022] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this invention, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0023] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0024] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0025] As used in this invention, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5%, of a given value or range. It also includes specific numbers, such as about 20 including 20.

[0026] Furthermore, in describing representative embodiments of the invention, this specification may present the methods and / or processes of the invention as a specific sequence of steps. However, the method or process should not be limited to the specific order of the steps described herein, to the extent that the method or process does not depend on the specific order of the steps presented herein. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps presented in the specification should not be construed as a limitation of the claims. Additionally, the claims relating to the methods and / or processes of the invention should not be limited to the execution of their steps in the order they are written, and those skilled in the art will readily recognize that the sequence can be changed while still remaining within the spirit and scope of the invention.

[0027] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% can fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 100mM can fluctuate within ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations of ±10% are allowed.

[0028] As used in this invention, unless otherwise stated, the singular forms of the articles “a,” “an,” and “the” include plural referents.

[0029] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0030] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0031] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" merely describe implementation methods or embodiments with better effects and should be understood not to limit the scope of protection of this invention. In this invention, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" statements appear in a technical solution, unless otherwise specified and without contradiction or mutual constraint, each "optional" statement is independent.

[0032] In this invention, the term "diagnosis" refers to the process of determining or predicting whether a subject individual (including but not limited to human or other mammalian patients) has a certain disease (especially Down syndrome), including its status, risk, subtype, or development trend, based on the detection results of one or more biological indicators in their sample, including but not limited to methylation sites. Diagnosis can be qualitative (e.g., presence / absence of disease), quantitative (e.g., risk score), or subtyping (e.g., different subtypes), including medical judgment behaviors such as initial screening, retesting, risk assessment, and disease monitoring. In this invention, "diagnosis" particularly refers to prenatal screening.

[0033] In this invention, the terms "Down syndrome," "trisomy 21," and "fetal trisomy 21 abnormality" are considered equivalent and used interchangeably. Down syndrome refers to a common congenital chromosomal abnormality syndrome caused by aneuploidy of chromosome 21. The pathogenesis of Down syndrome is the presence of an extra complete or partial copy of chromosome 21 in the patient's somatic cells, leading to disordered expression of specific genes. Its clinical manifestations may include intellectual disability, facial abnormalities, growth retardation, and various congenital structural malformations (such as heart and digestive tract defects). In the prenatal screening application exemplified in this invention, Down syndrome specifically refers to the situation where the fetus carries the trisomy 21 chromosomal abnormality. The diagnostic goal is to infer the risk of trisomy 21 by detecting fetal DNA information in the sample.

[0034] In this invention, the term "nucleic acid biomarker" refers to a nucleic acid molecule used to diagnose Down syndrome, which includes a target methylation biomarker and a reference methylation biomarker.

[0035] In this invention, the terms "target methylation marker", "methylation target gene", "target gene" or similar expressions refer to methylation sites used to determine the presence of trisomy 21 abnormalities, which are methylated in T21-positive fetuses but have lower methylation levels in T21-negative fetuses or mothers.

[0036] In this invention, the terms "reference methylation marker", "methylation reference gene", "reference gene" or similar expressions refer to sites located on non-chromosome 21 that are detectable in both the mother and fetus and have a stable methylation state, used as internal reference genes in the detection process for signal normalization or standardization.

[0037] In this invention, the term "methylation-sensitive restriction enzyme (MSRE)" refers to a class of restriction endonucleases capable of recognizing specific DNA sequences and cleaving them when those sequences are in an unmethylated state, but failing to cleave or exhibiting significantly reduced cleavage efficiency when methylation modifications (typically 5-methylcytosine) are present in the recognized sequences. This enzyme selectively cleaves DNA based on the methylation status of CpG sites in the sample DNA, thereby preserving the difference between methylated and unmethylated DNA fragments, and is suitable for the fetal-specific methylation biomarker detection platform described in this invention.

[0038] All references to this invention are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this invention, the referenced documents are incorporated herein by reference in their entirety and for all purposes. When references are made in this invention, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the referenced technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this invention. It should be understood that when the cited content conflicts with the description in this invention, this invention shall prevail or modifications shall be made adaptively according to the description in this invention.

[0039] To achieve sensitive and highly specific screening for fetal trisomy 21, this invention develops a multi-marker detection platform based on target / reference methylation ratio analysis, which relies on the detection of methylation status of specific nucleotide segments. Therefore, the first aspect of this invention relates to a polynucleotide selected from: a) The SIM2 gene fragment shown in SEQ ID NO: 1 or its complementary polynucleotide fragment, and / or b) The CRYBG1 gene fragment shown in SEQ ID NO: 2 or its complementary polynucleotide fragment.

[0040] This invention has discovered novel target / reference methylation biomarkers for Down syndrome screening. These biomarkers provide target and reference methylation biomarker fragments that can be used to determine fetal T21 status, laying the foundation for subsequent diagnostic methods and kit development.

[0041] The complementary polynucleotide fragments mentioned in this invention include DNA sequences capable of hybridizing with DNA having a corresponding sequence under stringent conditions. The “stringent conditions” used in this invention are well-known and include, for example, hybridization at 60°C for 12–16 hours in a hybridization solution containing 400 mM NaCl, 40 mM PIPES (pH 6.4), and 1 mM EDTA, followed by washing at 65°C for 15–60 minutes with a washing buffer containing 0.1% SDS and 0.1% SSC. The “complementarity” can be integral to the sequence or local complementarity within a subsequence range.

[0042] The polynucleotides described in this invention are isolated polynucleotides. The term "isolated polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules isolated or purified from their naturally occurring environment. These can be genomic DNA, cDNA, synthetic oligonucleotides, PCR products, transcripts, or in vitro synthesized fragments, etc. The isolation can be achieved through physical, chemical, or enzymatic methods, such as centrifugation, gel electrophoresis, column chromatography, synthesis, or amplification techniques. "Isolation" does not require the nucleic acid to be chemically pure, but it must be structurally or functionally independent of most of the biomolecules that coexist with it naturally (such as other genomic DNA fragments, tissue proteins, cell membrane components, etc.). This term also includes nucleic acid fragments that have been modified, labeled, or linked to other functional groups (such as probes, fluorescent labels, primers, etc.).

[0043] A second aspect of the present invention relates to the use of a detection reagent for detecting the methylation level of CpG sites of nucleic acid biomarkers in the preparation of a diagnostic kit for Down syndrome; The nucleic acid markers include polynucleotides as described above.

[0044] In this invention, the methylation level of the nucleic acid biomarker can be analyzed by any known methylation detection method, and this invention is not limited thereto. Methylation detection methods suitable for this invention include, but are not limited to: bisulfite conversion combined with sequencing (e.g., BS-seq, RRBS), methylation-specific PCR (MSP), high-resolution melting analysis (HRM), methylation microarray hybridization, methylation-specific probe hybridization, methylation-sensitive restriction endonuclease (MSRE) methods, enrichment methods (e.g., MeDIP), and mass spectrometry (e.g., MALDI-TOF MS). Any of the above methods can be used to determine the status of the target methylation biomarker and the reference methylation biomarker described in this invention.

[0045] In some embodiments, the nucleic acid biomarker includes at least one target methylation biomarker other than SIM2. Exemplarily, it includes one or more of the following gene fragments or complementary polynucleotide fragments: DSCAM, EP1, EP6, EP7, EP10, CGI149, CGI045, the HLCS gene, or its sub-segment HLCS-1 / 2.

[0046] DSCAM is referenced from Chinese patent application 201680078311.7, published on April 26, 2019. Regions EP1, EP6, EP7, and EP10 are fetal-specific differentially methylated regions (FSERs) screened by Lim et al. in *BMC Medical Genomics* (2014) based on placental / maternal blood methylation differences, exhibiting good stability and significant differences. Sites CGI149, CGI045, and HLCS-1 / 2 are T21 placental hypermethylated regions validated by Yin et al. and Zhang et al. using the MSRE-qPCR system; related studies have been published in *Genet Test MolBiomarkers* (2019) and other literature. These regions are significantly hypermethylated in T21 fetal placental tissue and hypomethylated in maternal blood, making them particularly suitable for constructing target / reference ratio models using enzyme digestion + digital PCR methods.

[0047] In some specific embodiments, the nucleotide sequence of the DSCAM gene fragment is shown in SEQ ID NO: 3.

[0048] In some embodiments, the nucleic acid biomarker further includes at least one reference methylation biomarker other than CRYBG1. Exemplary examples include one or more of the following gene fragments or their complementary polynucleotide fragments: TBX3, PCDHGA1, RASSF1A, TFAP2C.

[0049] TBX3 and PCDHGA1 are referenced from Chinese Patent Application 201680078311.7, which was published on April 26, 2019.

[0050] In some specific embodiments, the nucleotide sequence of the gene fragment of TBX3 is shown in SEQ ID NO: 4; In some specific embodiments, the nucleotide sequence of the PCDHGA1 gene fragment is shown in SEQ ID NO: 5.

[0051] It is readily understood that, in a further preferred embodiment of the present invention, the target methylation marker and the reference methylation marker are not limited to a single combination of SIM2 and CRYBG1. Based on the detection results in the examples, combining other highly methylated fragments derived from chromosome 21, such as DSCAM, EP6, CGI149, and HLCS, as additional target markers, with SIM2 as the target marker, can further enhance the signal differentiation between Down syndrome samples and normal samples. Similarly, using CRYBG1 as the reference marker, supplementing it with stable, low-variance methylation fragments located on non-chromosome 21, such as TBX3, PCDHGA1, RASSF1A, or TFAP2C, as controls, can also improve the anti-interference capability and detection stability of the ratio model. For those skilled in the art, based on the proven effectiveness of markers such as SIM2 and CRYBG1, selecting other methylation fragments that are complementary or synergistic with them, according to their chromosomal location, placental-specific methylation characteristics, and known literature data, is an obvious way to extend the technology.

[0052] The combination of target and reference biomarkers described in this invention can be flexibly configured according to the detection platform, sample type, and algorithm model to adapt to the needs of different screening accuracies and procedures. By detecting the copy number relationship between the target methylation site and the reference site, a highly sensitive and specific trisomy 21 risk discrimination model can be constructed, which is particularly suitable for the deployment of automated prenatal screening systems on digital PCR platforms.

[0053] Preferably, the present invention employs a method combining methylation-sensitive restriction endonuclease with amplification detection technology to identify whether the target fragment has been methylated.

[0054] After enzyme digestion, the amplification of the nucleic acid fragments can be performed in various ways, including but not limited to conventional quantitative PCR (qPCR), real-time quantitative PCR (RT-qPCR), multiplex PCR, and high-fidelity amplification (such as the Q5 system). More preferably, a digital PCR (dPCR) platform is used for absolute quantification of fragment copy number. By establishing the signal ratio between the target biomarker and the reference biomarker (such as SIM2 / CRYBG1), the risk of fetal chromosomal abnormalities (such as Down syndrome) can be accurately assessed. The digital PCR method has extremely high sensitivity and quantitative accuracy, and is particularly suitable for non-invasive prenatal screening scenarios with complex cfDNA backgrounds and scarce templates. Therefore, it is the preferred detection strategy recommended by this invention.

[0055] In some specific embodiments, the detection reagent includes primers and probes. The primers and probes, as detection reagents, are targeted amplifications of the nucleic acid biomarker. As used in this disclosure, the term "targeted amplification" refers to the process of amplifying a target nucleic acid. The target nucleic acid acts as a template in a DNA amplification reaction. A portion of the nucleic acid biomarker or an entire region of the target nucleic acid can be amplified by DNA polymerase in a DNA amplification reaction to generate an amplification product or amplicon. The amplicon may include multiple copies of the target nucleic acid or multiple copies of a sequence complementary to the target nucleic acid. The target nucleic acid can be obtained from a biological sample. The isolated nucleic acid biomarker can be dispersed in solution or immobilized on a solid support such as a speckle, array, slide, microtiter plate, beads, or ELISA plate.

[0056] Primers and probes may be modified, and modifications may be performed using known methods. Modified versions of these primer and / or probe sequences may include, by non-limiting examples, adding one or more nucleotides to the 5' end, adding one or more nucleotides to the 3' end, adding one or more nucleotides to both the 5' and 3' ends, adding a tail, shortening the sequence, lengthening the sequence, shifting the sequence upstream or downstream by several bases, or any combination thereof.

[0057] Base modifications, such as 3'P, 5'P, 5-nitroindole, 2-aminopurine, 8-amino-2'-deoxyadenosine, C-5-propynyl-deoxycytidine, C-5-propynyl-deoxyuridine, 2-amino-2'-deoxyadenosine-5'-triphosphate, 2,6-diaminopurine (2-amino-dA), reversed-dT, reversed-dideoxy-T, hydroxymethyl-dC, iso-dC, 5-methyl-dC, aminoethyl-phenoxazine-deoxycytidine, and locked nucleic acids (LNAs), including at least one mismatched base at one of the bases, or replacing at least one of the bases with an RNA base, can achieve, for example, increased nucleic acid interaction at the 3' end of mutant-specific primers to increase Tm. The addition of stable double-stranded base modifications has a positive effect on PCR, enabling it to be performed at higher temperatures, within which Taq polymerase is known to exhibit maximum activity. Modified probes should retain the ability to distinguish between the mutant and wild-type sites to be detected.

[0058] In some embodiments, the probe is labeled with a detectable signal substance. In some embodiments, the signal substance is a fluorophore, colorimetric label, colloidal gold, quantum dots, biotin, and other tagged molecules that can be used for detection (such as alkyne groups for Raman diffraction imaging, cycloalkenes for click reactions, and initiating groups for polymer labeling). It may also be selected from peptide / protein molecules, LNA / PNA, non-natural amino acids and their analogs (e.g., peptide-like substances), non-natural nucleic acids and their analogs (nucleotide-like substances), and nanostructures (including inorganic nanoparticles, NV-centers, aggregation / assembly-induced luminescent molecules, rare earth ion ligand molecules, polyoxometalates, etc.).

[0059] In some specific embodiments, the detection reagent includes: a′) The upstream primer shown in SEQ ID NO: 6, the downstream primer shown in SEQ ID NO: 7, and the probe shown in SEQ ID NO: 8 for SIM2; and / or b′) The upstream primer shown in SEQ ID NO: 9, the downstream primer shown in SEQ ID NO: 10, and the probe shown in SEQ ID NO: 11 for CRYBG1.

[0060] In some specific embodiments, the detection reagent further includes one or more of the following primer-probe combinations: c′) The upstream primer shown in SEQ ID NO: 12, the downstream primer shown in SEQ ID NO: 13, and the probe shown in SEQ ID NO: 14 for DSCAM; d′) For TBX3, the upstream primer shown in SEQ ID NO: 15, the downstream primer shown in SEQ ID NO: 16, and the probe shown in SEQ ID NO: 17; e′) The upstream primer shown in SEQ ID NO: 18, the downstream primer shown in SEQ ID NO: 19, and the probe shown in SEQ ID NO: 20 for PCDHGA1.

[0061] Useful sequences also include functional equivalents of any one of the sequences SEQ ID NO: 1-SEQ ID NO: 20 above. A functional equivalent is a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the corresponding sequence and retains the relevant function (such as function as a primer, probe, or nucleic acid marker). Functional equivalents also include modified nucleic acids. The percentage of sequence identity can be obtained using well-known bioinformatics algorithms, including the Myers and Miller algorithm (Bioinformatics, 4(1): 11-17, 1988), the Needleman-Wunsch global alignment method (J.Mol.Biol., 48(3): 443-53, 1970), the Smith-Waterman local alignment method (J.Mol.Biol., 147: 195-197, 1981), the Pearson and Lipman similarity search method (PNAS, 85(8): 2444-2448, 1988), and the Karlin and Altschul algorithm (Altschul et al., J.Mol.Biol., 215(3): 403-410, 1990; PNAS, 90: 5873-5877, 1993). This is familiar to those skilled in the art.

[0062] Preferred methods for removing unmethylated background DNA include using restriction endonucleases (MSREs) sensitive to the methylation status of CpG sites. These enzymes recognize and cleave specific sites in unmethylated DNA, while recognition is inhibited in methylated DNA, thus preserving fetal-derived hypermethylated fragments during cleavage. Therefore, in some embodiments, the detection reagent further includes methylation-sensitive restriction endonucleases. Preferred MSREs include, but are not limited to: HpaII (recognition sequence: CCGG), HhaI (recognition sequence: GCGC), and BstUI (recognition sequence: CGCG). These enzymes are commercially available, such as from New England Biolabs (NEB). In other embodiments, enzymes such as AciI, HpyCH4IV, NotI, and BssHII may also be used. The specific enzyme selection depends on the distribution of cleavage sites contained in the target or reference methylation marker sequence.

[0063] In this disclosure, the terms "biological sample," "sample," etc., refer to animal samples; which may be derived from bodily fluids, tissues, or organs of animals (preferably including at least mammals, such as primates, including humans), tissue lysates, cells (in the subject, directly taken from the subject, or held in culture, or derived from a cultured cell line), cell lysates (or portions of lysates), or cell extracts. In some embodiments, the sample contains nucleic acids. In some embodiments, the sample detected by the kit is peripheral blood, peripheral plasma, peripheral serum, amniotic fluid, cervical mucus, urine, umbilical cord blood, chorionic villus tissue, placental tissue, free nucleic acid extracts, or combinations thereof.

[0064] This invention can be used for raw body fluid samples as well as their extracts. As an extract, "extracted cell-free nucleic acid" refers to a mixture of nucleic acids obtained from biological body fluid samples by extraction methods, free of cellular components, primarily including cell-free DNA (cfDNA) or cell-free RNA (cfRNA). The body fluid samples from which the extract is derived include, but are not limited to, plasma, serum, peripheral blood, urine, amniotic fluid, or other available biological body fluids. In addition to cfDNA, "extracted cell-free nucleic acid" in this invention may also encompass cfRNA extracted in certain applications, particularly when it is necessary to analyze differences in RNA methylation or stability. Unless otherwise stated, this invention primarily addresses the analysis of cfDNA for fetal methylation status.

[0065] The preferred application of the present invention is non-invasive diagnosis, and the corresponding sample is preferably peripheral blood, peripheral plasma, peripheral serum, free nucleic acid extract or a combination thereof.

[0066] A third aspect of the invention relates to a kit for detecting Down syndrome, comprising the detection reagents as defined in the second aspect above.

[0067] In this disclosure, the term "kit" may refer to any article (e.g., packaging or container) that includes at least one device and comprises the detection reagents as described in this disclosure. The kit may further include instructions for use, supplementary reagents, and / or components or parts used in the methods or steps described in this disclosure.

[0068] In some embodiments, the kit further comprises one or more of the following components: • Reaction buffer suitable for enzyme digestion and / or amplification reactions; • Hot-start DNA polymerase; • Fluorescent probes or fluorescent signal labeling systems; • Oil-phase reagents used to form droplets; • Negative control DNA; • Positive control template; • cfDNA extraction reagent.

[0069] The reaction buffer is used to maintain the optimal enzyme reaction system and usually contains Tris-HCl, KCl, MgCl2 and stabilizers to ensure that the pH, ionization intensity and metal ion concentration of the reaction system are maintained at the optimal level during enzyme digestion, amplification or fluorescence signal reading, thereby ensuring the consistency and repeatability of the amplification reaction.

[0070] To meet the amplification requirements of high sensitivity and low template conditions in digital PCR systems, the kit preferably includes a hot-start DNA polymerase. Suitable polymerases include, but are not limited to: Hot Start Taq DNA Polymerase, Q5 Hot Start DNA Polymerase, AmpliTaq Gold, Platinum Taq, and Phusion Hot Start High-Fidelity DNA Polymerase. These polymerases are capable of activation at high temperatures, providing stable amplification efficiency while avoiding low-temperature nonspecific amplification, making them particularly suitable for single-molecule level detection in the droplet environment of digital PCR.

[0071] Fluorescent probes or fluorescent signal labeling systems are used for identification in the signal acquisition module of PCR (especially digital PCR) platforms. TaqMan probe labeling is preferred, where the probes may carry fluorescent groups such as FAM, HEX, VIC, and ROX, and combine with polymerases possessing 5'-3' exonuclease activity to release fluorescent signals during target amplification. Some platforms also support indirect dye labeling methods such as SYBR Green, but this invention prefers the TaqMan system to improve the specificity of ratio discrimination.

[0072] For use with droplet digital PCR platforms (such as the Bio-Rad QX200), the kit may further include an oil phase system or emulsifier component to form the droplet reaction environment. For example, it may include a fluid oil for stabilizing the oil-in-water structure, or a surfactant (such as a PEG derivative or fluorocarbon oil), allowing each reaction unit to be amplified independently in a closed system for high-throughput signal reading.

[0073] In addition, to improve clinical operability, the kit may also include a positive control template and / or a negative control DNA. The former may contain an artificial standard fragment derived from fetal DNA or a synthetic sequence carrying a Y chromosome marker (such as SRY) to verify the performance of the kit; the latter may be a cfDNA extract from peripheral blood of healthy women to determine background interference. The control template may be packaged together with the amplification reagent or separately lyophilized to support standardized quality control of the platform.

[0074] In some embodiments, the kit is a digital PCR detection kit.

[0075] Components within reagents or kits can be packaged in the form of solutions, solids, or test strips. In some preferred embodiments, at least one component of the reagent or kit is a solid, including at least one of lyophilized microspheres, lyophilized cakes, lyophilized powders, and spots dependent on a solid medium. Therefore, components required for nucleic acid amplification (and preferably nucleic acid detection), particularly various enzymes, nucleic acid components, and reaction buffer components, can be provided in lyophilized form. In this way, the nucleic acid amplification (and preferably nucleic acid detection) process can be directly initiated in a very user-friendly manner by adding the sample to be quantified and optionally other required components.

[0076] According to a fourth aspect of the invention, a diagnostic device is provided, comprising one or more separate modules, at least one of which contains a detection reagent as defined in the second aspect, or a component of a kit as described in the third aspect.

[0077] The diagnostic device is preferably a digital PCR diagnostic device.

[0078] The device can be pre-loaded or loaded with the detection reagents described in this invention, realizing an integrated operation of fetal cfDNA enzyme digestion, amplification, fluorescence signal reading, and ratio model determination. The diagnostic device is typically beneficial for nucleic acid detection, for example, preventing contamination during the detection process, accelerating the detection speed (e.g., simultaneously detecting multiple samples), facilitating the preservation of components in the reagent kit during the detection process, or otherwise improving the convenience and / or accuracy of the detection. The use of these diagnostic devices is generally common practice among those skilled in the art and can be modified from commercial diagnostic platforms, such as, but not limited to, devices like the Bio-Rad QX200, Thermo Fisher QuantStudio Absolute Q, and QIAGEN QIAcuity.

[0079] According to a fifth aspect of the present invention, there is a method for detecting the presence of trisomy 21 in a fetus, the method comprising the following steps: i) Extract total cfDNA from the maternal sample; ii) Detect the CpG site methylation level of nucleic acid biomarkers therein, wherein the nucleic acid biomarkers are as defined in the second aspect of the invention; iii) Determine the risk of Down syndrome in a sample based on the ratio of expression levels of methylation markers and reference methylation markers.

[0080] In some embodiments, step ii) includes: treating the cfDNA with at least one methylation-sensitive restriction endonuclease; amplifying specific nucleic acid fragments of the target methylation marker and a reference methylation marker; and determining the amplification signal of the marker fragment.

[0081] In some implementations, the amplification method is digital PCR.

[0082] In some implementations, the reagents used for amplification include primers and probes.

[0083] The methylation-sensitive restriction endonuclease, the sample, the primers and probes (primer-probe combination) are defined in the second aspect of the present invention.

[0084] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0085] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0086] Example 1: Screening of methylation markers for prenatal fetal T21 screening The inventors collected chorionic villus sampling tissues from 4 T21-positive and 4 T21-negative samples, as well as maternal blood samples from 4 samples. Genomic DNA was extracted from the tissue samples using a tissue extraction kit, and from the blood samples using a blood extraction kit. All genomic DNA samples were treated with bisulfite and then used to construct single-stranded libraries. Methylation regions were captured using a whole-genome methylation panel. The captured methylated libraries underwent quality control and were then sequenced.

[0087] Based on the high-throughput sequencing data above, differentially methylated regions were screened: Target differentially methylated regions: fetal-specific methylation markers located on target chromosomes (chromosome 21 in this invention), which are unmethylated (methylation level close to 0) in maternal blood cells, but methylated in the fetus. Optimally, the methylation level of T21-positive markers is more than 20% higher than that of T21-negative markers.

[0088] Reference differentially methylated regions: These markers are located on non-target chromosomes, are unmethylated in maternal blood cells (methylation level close to 0), and are methylated in fetuses, and remain relatively stable across samples.

[0089] This invention screened an ideal reference differentially methylated region located on the CRYBG1 gene on chromosome 6. The methylation level was 0 in the maternal background, while the methylation level in the fetal sample was between 0.6 and 0.65, and it was relatively stable among different samples.

[0090] Furthermore, this invention identifies a target differentially methylated region for trisomy 21 screening, located on the SIM2 gene on chromosome 21. The methylation level in the maternal background is close to 0, the methylation level in T21-negative samples is between 0.3 and 0.4, and the methylation level in T21-positive fetal samples is between 0.57 and 0.66.

[0091] Table: Methylation levels of the differentially methylated regions (DMRs) identified during screening in the samples.

[0092] In summary, this embodiment constructs a screening system for target and reference methylation markers based on fetal-maternal differential methylation regions.

[0093] Example 2: Comparison of the reference methylation marker of the present invention with existing reference methylation markers When using methylation markers for fetal chromosomal aneuploidy screening, the proportion of fetal-derived nucleic acids in the pregnant woman is uncertain. Therefore, a reference gene is needed to standardize the amount of the target gene. This reference gene is also located in a region of differential methylation between the fetus and the mother, and this region is not on the chromosome being tested. Theoretically, for normal fetuses, the ratio of the target gene to the reference gene is 1:1 or 2:2, while for fetuses with trisomy syndrome, the ratio is 3:2. Of course, due to differences in methylation frequency and amplification efficiency of the target fragment between the target gene and the reference gene, the actual ratio will deviate from the theoretical ratio. The stability of the reference methylation marker and the consistency between samples play a crucial role in the accuracy of the detection in this method.

[0094] For plasma samples from pregnant women with male fetuses, genes on the Y chromosome (such as SRY, DYS14, etc.) can be used as reference markers. In this embodiment, a target fragment is designed and amplified on the SRY gene. This amplified fragment does not contain any methylation-sensitive restriction enzyme sites, so it will not be cleaved in subsequent enzyme digestion reactions and will be retained. The SRY fragment exists only in male chromosomes, and the amount of this fragment does not contain any maternal background, which can truly reflect the amount of fetal DNA fragments in the plasma. In this embodiment, the correlation between the number of SRY fragments and the number of reference methylation markers will be used as the evaluation criterion to compare the performance of the reference methylation marker CRYBG1 of this invention with two existing reference markers (TBX3 and PCDHGA1).

[0095] Materials and methods: (1) Sample collection and processing Two ml of plasma from a pregnant woman with a male fetus was collected, and free nucleic acids were extracted using the Tiangen Enhanced Magnetic Bead Large Volume Free Nucleic Acid Extraction Kit (DP720-01) according to the manufacturer's instructions. The extracted nucleic acid solution was digested with a methylation-sensitive restriction endonuclease (NEB). Digestion was performed for 4 hours at the temperature recommended in the manufacturer's instructions using 10 U HpaII, 10 U HhaI, and 10 U BstUI. After digestion, the nucleic acid was inactivated at 80°C for 30 min.

[0096] (2) Digital PCR The enzyme digestion products from the previous step were amplified and analyzed using a Sniper DQ24 digital PCR instrument. The digital PCR reaction conditions were as follows: steady-state phase: 60℃ for 5 min, 95℃ for 5 min; 50 cycles: 95℃ for 20 s, 60℃ for 30 s. All samples were run in duplicate, and the copy numbers were summed.

[0097] (3) Primer-probe combination The amplification reaction was carried out in a quadruple digital PCR system, and the primers and probes are as follows:

[0098] (4) Results Analysis: Digital PCR was used to detect the copy number of each gene in the sample. Correlation analysis was performed between the copy numbers of methylation reference genes (CRYBG1, TBX3, and PCDHGA1) and the copy number of the Y chromosome gene SRY. The correlation coefficients between CRYBG1, TBX3, and PCDHGA1 and SRY were 0.92, 0.85, and 0.82, respectively. The correlation between CRYBG1 and SRY was higher than that between TBX3 and PCDHGA1. This indicates that CRYBG1 has better consistency with SRY and is more suitable as a reference methylation marker.

[0099] Example 3: Combination of methylation biomarkers for prenatal screening of trisomy 21 (1) Sample collection and processing From NGS-based non-invasive prenatal testing (NIPT) samples, plasma samples were collected from 79 patients at 12-18 weeks of gestation, of whom 59 were T21 negative and 20 were T21 positive. All positive samples were confirmed by amniocentesis, and all negative samples were confirmed by telephone follow-up.

[0100] Two ml of plasma was extracted from each sample, and free nucleic acids were extracted using the Tiangen Enhanced Magnetic Bead Large Volume Free Nucleic Acid Extraction Kit (DP720-01) according to the manufacturer's instructions. The extracted nucleic acid solution was digested with a methylation-sensitive restriction endonuclease (NEB). Digestion was performed for 4 hours at the temperature recommended in the manufacturer's instructions using 10 U HpaII, 10 U HhaI, and 10 U BstUI. After digestion, the nucleic acid was inactivated at 80°C for 30 min.

[0101] (2) Digital PCR The enzyme digestion products from the previous step were amplified and analyzed using a Sniper DQ24 digital PCR instrument. The digital PCR reaction conditions were as follows: steady-state phase: 60℃ for 5 min, 95℃ for 5 min; 50 cycles: 95℃ for 20 s, 60℃ for 30 s. All samples were run in duplicate, and the copy numbers were summed.

[0102] (3) Primer-probe combination The amplification reaction was performed in a triple digital PCR system, with the following primers and probes:

[0103] (4) Results Analysis This embodiment uses the same target differential methylation marker SIM2, paired with two different reference methylation markers TBX3 and CRYBG1. The T21 detection performance of the two methylation marker pairs in plasma samples from 79 pregnant women was evaluated. The AUC value of SIM2 / CRYBG1 was 0.9992, while the AUC value of SIM2 / TBX3 was 0.9771, indicating that the CRYBG1 group was significantly superior to the TBX3 group.

[0104] Example 4: Prenatal Trisomy 21 Detection Using a Differential Methylation Marker Kit Using only a single methylation marker combination to detect trisomy 21 often falls short of performance requirements. Combining it with other methylation marker combinations can effectively improve detection performance. This embodiment adds a methylation marker combination DSCAM / TBX3 to the S2 / CRYBG1 methylation marker combination; the combination of these two combinations enables the detection of trisomy 21.

[0105] (1) Sample collection and processing Plasma was collected from 40 pregnant women at 12-20 weeks of gestation, including 28 normal samples and 12 T21 positive samples. All positive fetuses were subsequently verified for T21 and the diagnosis results were true positive.

[0106] Two ml of plasma was extracted from each sample, and free nucleic acids were extracted using the Tiangen Enhanced Magnetic Bead Large Volume Free Nucleic Acid Extraction Kit (DP720-01) according to the manufacturer's instructions. The extracted nucleic acid solution was digested with a methylation-sensitive restriction endonuclease (NEB). Digestion was performed for 4 hours at the temperature recommended in the manufacturer's instructions using 10 U HpaII, 10 U HhaI, and 10 U BstUI. After digestion, the nucleic acid was inactivated at 80°C for 30 min.

[0107] (2) Methylation marker kit In this embodiment, the copy number of two pairs of methylation markers was detected by digital PCR. Combination 1: the target gene is the SIM2 gene located on chromosome 21, and the reference gene is the CRYBG1 gene located on chromosome 6; Combination 2: the target gene is the DSCAM gene located on chromosome 21, and the reference gene is the TBX3 gene located on chromosome 3.

[0108] (3) Primer-probe combination The amplification reaction was carried out in a quadruple digital PCR system, and the primers and probes are as follows:

[0109] (4) Digital PCR The copy numbers of four genes (SIM2, CRYBG1, DSCAM, and TBX3) in a methylation biomarker kit were detected using digital PCR. The amplification reactions were performed in a quadruple digital PCR system, with fluorescent labels ROX, VIC, cy5, and FAM, respectively. The digital PCR reaction conditions were as follows: steady-state phase: 60℃ for 5 min, 95℃ for 5 min; 50 cycles: 95℃ for 20 s, 60℃ for 30 s. All samples were run in duplicate, and the copy numbers were summed.

[0110] (5) Results Analysis The copy number ratios of the two pairs of methylation markers (SIM2 / CRYBG1 and DSCAM / TBX3) were calculated, and the two ratios for each plasma sample were plotted as a scatter plot. The scatter plots show that the combined use of the two sets of methylation markers can easily distinguish between T21 positive and negative samples. A linear regression fit yielded an ideal threshold line (dashed line in the figure): x + 1.46y = 2.14. Here, an index R can be defined. Calculation formula: R = ratio1 + 1.46 × ratio2 Where ratio1 = SIM2 / CRYBG1, ratio2 = DSCAM / TBX3.

[0111] In this embodiment, when R is greater than 2.14, the sample can be identified as T21 positive, while when R is less than 2.14, the sample is identified as T21 negative.

[0112] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. Polynucleotides, selected from: a) The SIM2 gene fragment shown in SEQ ID NO: 1 or its complementary polynucleotide fragment, and / or b) The CRYBG1 gene fragment shown in SEQ ID NO: 2 or its complementary polynucleotide fragment.

2. The application of reagents for detecting the methylation level of CpG sites of nucleic acid biomarkers in the preparation of diagnostic kits for Down syndrome; in, The nucleic acid biomarker includes the polynucleotide of claim 1.

3. The use according to claim 2, wherein the nucleic acid marker comprises at least one target methylation marker other than SIM2, preferably comprising one or more of the following gene fragments or complementary polynucleotide fragments: DSCAM, EP1, EP6, EP7, EP10, CGI149, CGI045, HLCS gene or its sub-segment HLCS-1 / 2; more preferably, the nucleotide sequence of the DSCAM gene fragment is shown in SEQ ID NO:

3.

4. The use according to claim 2, wherein the nucleic acid marker further comprises at least one reference methylation marker other than CRYBG1, preferably including one or more of the following gene fragments or complementary polynucleotide fragments: TBX3, PCDHGA1, RASSF1A, TFAP2C; More preferably, the nucleotide sequence of the TBX3 gene fragment is shown in SEQ ID NO: 4; More preferably, the nucleotide sequence of the PCDHGA1 gene fragment is shown in SEQ ID NO:

5.

5. The use according to any one of claims 2-4, wherein the detection reagent comprises primers and probes.

6. The use according to claim 5, wherein the detection reagent comprises: a′) The upstream primer shown in SEQ ID NO: 6, the downstream primer shown in SEQ ID NO: 7, and the probe shown in SEQ ID NO: 8 for SIM2; and / or b′) The upstream primer shown in SEQ ID NO: 9, the downstream primer shown in SEQ ID NO: 10, and the probe shown in SEQ ID NO: 11 for CRYBG1.

7. The detection reagent according to claim 6 further comprises one or more of the following primer-probe combinations: c′) The upstream primer shown in SEQ ID NO: 12, the downstream primer shown in SEQ ID NO: 13, and the probe shown in SEQ ID NO: 14 for DSCAM; d′) The upstream primer shown in SEQ ID NO: 15, the downstream primer shown in SEQ ID NO: 16, and the probe shown in SEQ ID NO: 17 for TBX3; e′) The upstream primer shown in SEQ ID NO: 18, the downstream primer shown in SEQ ID NO: 19, and the probe shown in SEQ ID NO: 20 for PCDHGA1.

8. In the use according to claim 6 or 7, the detection reagent further comprises a methylation-sensitive restriction endonuclease; preferably at least one of HpaII, HhaI, and BstUI.

9. The use according to any one of claims 2-4, 6, and 7, wherein the sample detected by the kit is peripheral blood, peripheral plasma, peripheral serum, amniotic fluid, cervical mucus, urine, umbilical cord blood, chorionic villus tissue, placental tissue, free nucleic acid extract, or a combination thereof.

10. A kit for detecting Down syndrome, comprising the detection reagents as defined in any one of claims 2-9.

11. The kit according to claim 10, further comprising one or more of the following components: • Reaction buffer suitable for enzyme digestion and / or amplification reactions; • Hot-start DNA polymerase; • Fluorescent probes or fluorescent signal labeling systems; • Oil-phase reagents used to form droplets; • Negative control DNA; • Positive control template; and • cfDNA extraction reagent.

12. The kit according to claim 10 or 11 is a digital PCR detection kit.

13. A diagnostic device comprising one or more independent modules, at least one independent module comprising a detection reagent as defined in any one of claims 2-9, or a component of a kit as described in any one of claims 10-12.

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