Kit for noninvasive prenatal detection of single-gene inherited metabolic disease and detection and analysis method of single-gene inherited metabolic disease

Abstract

The invention relates to a kit for noninvasive prenatal detection of a single gene inherited metabolic disease and a detection analysis method of the single gene inherited metabolic disease, related pathogenic genes detected by the kit comprise MMUT, MMACHC and PAH, the diseases caused by the related pathogenic genes comprise Mut type methylmalonic acidmia, cblC type methylmalonic acidmia and phenylketonuria, and the sequences of adopted primers are shown as SEQ ID No.1-SEQ ID No.106. The invention further discloses a kit for noninvasive prenatal detection of the single gene inherited metabolic disease and a detection analysis method of the single gene inherited metabolic disease. According to the kit and the detection and analysis method, the haplotype of family parents can be constructed by utilizing target region enrichment, a Linked-reads technology and a probe hybridization capture method under the condition that no precursor DNA exists, and the genotype and haplotype information of a fetus are analyzed by combining the genotype of the plasma cfDNA of the mother. Compared with a traditional invasive prenatal detection technology, the method constructed by the invention is noninvasive, low in risk and high in sensitivity, can be widely applied to prenatal screening of pregnant women, expands the range of disease species detection, does not depend on DNA information of prenatal patients, and has a better application prospect in noninvasive prenatal detection research of monogenic diseases.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, in particular to the field of non-invasive prenatal detection technology, and specifically to a kit for non-invasive prenatal detection of single gene genetic metabolic diseases and a detection and analysis method for single gene genetic metabolic diseases. Background Art

[0002] Monogenic inherited metabolic diseases are a type of disease that is caused by a single gene mutation that changes the structure and function of enzymes, receptors, carriers and other proteins, thereby causing metabolic disorders in the body. Traditional prenatal diagnosis methods such as amniocentesis, chorionic villus sampling and cord blood puncture are invasive examinations, which carry certain risks of miscarriage and infection. Non-invasive prenatal testing (NIPT) is a technology that uses a new generation of high-throughput sequencing technology to detect cell-free fetal DNA (cffDNA) in maternal peripheral plasma by drawing peripheral blood from pregnant women. It is used to assess the risk of the fetus developing certain diseases. It can avoid the risks of traditional prenatal diagnosis to a certain extent and provide a safer way for prenatal diagnosis of monogenic inherited metabolic diseases.

[0003] Currently, there are methods for non-invasive prenatal testing (NIPT) using second-generation sequencing technology, which mainly detects large-fragment variations such as chromosomal abnormalities. For example, the VerifiNIPT product developed by Illumina can detect common chromosomal abnormalities such as Down syndrome and Edwards syndrome by sequencing free fetal DNA in maternal plasma. However, the accuracy of this method in detecting small-fragment genetic defects such as single-gene inherited metabolic diseases is low, and there are certain false positives and false negatives. In addition, improvements have been made to related second-generation sequencing technologies, for example: (1) Using 10X genomics' linked-read technology combined with Illumina sequencing, the haplotypes of the parents were analyzed and the SNPs enriched in the pathogenic gene loci were determined. The relative haploid dosage test and K-Stest were used to detect the source of DNA (maternal DNA + fetal DNA) in the mother's plasma to infer the mutation status of the fetus; (2) The haplotypes of the parental genome were directly sequenced through linked-read sequencing, and combined with allele-specific PCR, the target genes in the maternal plasma DNA were sequenced, and the posterior risk calculation and Anderson-Darling test were performed to infer the maternal and paternal genetic information respectively. The use of linked-read sequencing combined with posterior risk calculation can expand the range of diseases detected; (3) Beagle 4.0 was used to deduce the parental haplotypes based on the screening data set; based on this, a hidden Markov model and Viterbi algorithm were established to infer the fetal haplotype.

[0004] However, the types of diseases involved in the above-mentioned second-generation sequencing methods are very limited. They are mainly used for prenatal screening and diagnosis of single-gene inherited metabolic diseases, including thalassemia, cystic fibrosis, Duchenne muscular dystrophy, etc., and do not involve prenatal screening for diseases such as methylmalonic acidemia, phenylketonuria, propionic acidemia and glutaric acidemia type I. Specifically, the above-mentioned second-generation sequencing methods have the following shortcomings: (1) The technology that mainly focuses on relative mutation dose (RMD) analysis and directly targets mutation site detection, such as cSMART technology quantification, cfBEST technology quantification, and digital PCR quantification, has a high risk and can only target a single disease; (2) The detection technology that mainly focuses on relative haplotype dose (RHDO) analysis and uses multiple single nucleotide polymorphism sites (SNPs) near the mutation site to construct linked haplotypes depends on the presence of the proband's DNA, which has certain limitations for families without probands; (3) For families without proband DNA, the NIPT based on third-generation sequencing technology has a high error rate, bioinformatics analysis is difficult, and the price is expensive. Summary of the invention

[0005] In view of at least one deficiency in the prior art, the present invention has developed a non-invasive prenatal genetic testing method for the more common single-gene inherited metabolic diseases in China. It can target a variety of diseases (including methylmalonic acidemia and phenylketonuria), and is independent of the proband's DNA. The detection and analysis method has high sensitivity and accuracy. It can construct the haplotypes of the family parents in the absence of proband DNA by using target region enrichment, linked-reads technology, and probe hybridization capture methods, and combine the genotype of the mother's plasma cfDNA to determine the genotype and haplotype information of the fetus.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention is to provide a kit for non-invasive prenatal detection of monogenic inherited metabolic diseases, which includes a primer combination, wherein the primer combination includes a primer combination for amplifying a target gene fragment of at least one of the pathogenic genes MMUT, MMACHC, PAH and a regional sequence of 180 to 210 kb upstream and downstream thereof (for example, preferably a 190 to 200 kb regional sequence).

[0008] Furthermore, based on the human reference genome hg38, the MMUT target gene fragment includes Chr6:49345400-49541567; the MMACHC target gene fragment includes Chr1:45410655-45602552; and the PAH target gene fragment includes Chr12:102796508-102995704.

[0009] Furthermore, the single gene genetic metabolic disease includes at least one of methylmalonic acidemia (Mut-type methylmalonic acidemia, cblC-type methylmalonic acidemia) and phenylketonuria.

[0010] Furthermore, the primer composition comprises:

[0011] MMUT primers: as shown in SEQ ID No.1 to SEQ ID No.36;

[0012] MMACHC primers: as shown in SEQ ID No.37 to SEQ ID No.70;

[0013] PAH primers: shown in SEQ ID No.71 to SEQ ID No.106.

[0014] Furthermore, the kit also includes a probe for hybridization capture of the whole genome library of pregnant women's plasma cfDNA, and the above-mentioned probe can be designed using conventional methods in the art.

[0015] The second aspect of the present invention is to provide a detection and analysis method for single-gene inherited metabolic diseases for non-diagnostic purposes, which includes the following steps: based on a second-generation sequencing platform, in the absence of proband DNA, using any of the kits described in the first aspect, utilizing target region enrichment, linked-reads method, and probe hybridization capture method to construct the haplotypes of the family parents, combined with the genotype of the mother's plasma cfDNA, thereby analyzing the genotype and haplotype information of the fetus.

[0016] Furthermore, the second-generation sequencing platforms include Illumina and BGI-SEQ second-generation sequencing platforms.

[0017] Furthermore, the detection and analysis method specifically comprises the steps of:

[0018] Step S1, using the target region PCR enrichment method, using the specific primers shown in SEQ ID No.1 to SEQ ID No.106 to amplify the genomic DNA of the father and the mother by multiple long-fragment PCR, and constructing a linked-read library for the long-fragment amplification products. The DNA fragments from the same source will be added with the same barcode fragments, and the constructed Tell-seq library will be used for second-generation sequencing;

[0019] Step S2, splicing the short sequences obtained by the second-generation sequencing into long sequences according to the barcode information on each DNA fragment, and obtaining the haplotype information of the target regions of the father and the mother;

[0020] Step S3, constructing a whole genome library of maternal peripheral plasma cfDNA, using probe hybridization capture and targeted next-generation sequencing to obtain genotype information of maternal plasma cfDNA;

[0021] Step S4: Based on the hidden Markov model and Viterbi algorithm, the genotype information of the mother's plasma cfDNA and the haplotype information of the father's and mother's target regions are combined to analyze the genotype and haplotype information of the fetus.

[0022] Furthermore, the preparation of the sequencing library includes:

[0023] Tell-seq library: The starting amount of gDNA was 100 ng, and PCR amplification was performed using KOD FX reagent. After amplification, the amplified fragments were checked by agarose gel electrophoresis and quantified by Qubit. 0.1 ng of mixed PCR products was taken and TELL-Seq TM WGS LibraryReagent Box 1 and TELL-Seq TM WGS Library Reagent Box 2 kit was used to build the library; each gene had 18 or 17 pairs of amplification primers, of which the odd-numbered primer pairs were mixed into one tube, and the even-numbered primer pairs were mixed into one tube. Each gene was amplified using the above two tubes of primers, and after the amplification, the two tubes of PCR products from the same sample were mixed in equal amounts;

[0024] cfDNA whole genome library: The starting amount for library construction is 5-15 ng, and the library is constructed using the Hieff NGS MaxUp IIDNA LibraryPrep Kit for Illumina universal DNA library construction kit (final library volume 25 uL).

[0025] Furthermore, the probe hybridization capture includes: using Twist Standard Hybridization and WashKit to perform hybridization capture on the established whole genome library, the starting amount of cfDNA library hybridization is 300 ng, and the hybridization time is 16 hours.

[0026] Furthermore, the next generation sequencing of the library includes:

[0027] Tell-seq library was sequenced by Illumina NextSeq500, with 500Mb of data per sample, and sequencing mode PE150;

[0028] The cfDNA library was sequenced using MGISEQ-T7, with 7000Mb of data per cfDNA library and sequencing mode PE100.

[0029] Furthermore, the libraries were all analyzed for fragments using QSEP100 and quantified using Qubit 4.0.

[0030] Furthermore, the hidden Markov model and Viterbi algorithm specifically include: taking each allele information site as an observation sequence, taking the corresponding parental genotype at the site, the sequencing depth information of the reference allele and the variant allele in the cfDNA as the observation state, and taking the haplotype inherited by the fetus as the hidden state to be analyzed; the initial state of the two alleles is defined as {1 / 2, 1 / 2}, the observation state probability matrix is ​​composed of the conditional probability of the fetus inheriting the corresponding allele when the alleles in the peripheral blood cfDNA at the site are in different observation states, and the hidden state transition probability matrix is ​​composed of the corresponding recombination rate information obtained according to the genetic distance information corresponding to the physical distance of the alleles on the chromosome; using the Viterbi algorithm to infer the optimal hidden state according to the model, combined with the haplotype information of the parents, to analyze the haplotype of the fetus.

[0031] Furthermore, the detection and analysis method also includes the collection and extraction of samples, specifically including:

[0032] Peripheral blood collection: 10 mL of peripheral blood was collected from the pregnant woman and 5 mL of peripheral blood was collected from the pregnant woman's husband;

[0033] Genomic DNA extraction: The peripheral venous blood of the pregnant woman is centrifuged in a low-speed refrigerated centrifuge, and the upper plasma is drawn into a centrifuge tube. The separated plasma is centrifuged in a low-speed refrigerated centrifuge, and the supernatant is drawn into a centrifuge tube for subsequent extraction of free fetal DNA; the remaining white blood cell-containing blood of the pregnant woman is mixed and divided into another centrifuge tube for subsequent extraction of maternal genomic DNA; the peripheral venous blood of the father is used for subsequent extraction of genomic DNA of the father. The above DNA extraction can use conventional kits in the field.

[0034] The third aspect of the present invention is to provide the use of any of the kits described in the first aspect in the detection of single gene inherited metabolic diseases for non-diagnostic purposes. In this application, the detection and analysis method used is any of the detection methods described in the second aspect.

[0035] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0036] (1) By enriching the target gene region, unnecessary whole genome sequencing can be avoided, so that more targeted testing plans can be developed according to different diseases or genes, reducing testing costs;

[0037] (2) Multiple long fragment amplification combined with linked-read technology is used to detect haplotypes in the target region, which reduces detection costs and improves detection efficiency compared to whole-genome haplotype sequencing;

[0038] (3) Without relying on the proband or other family members, the haplotypes of the parents can be directly obtained through targeted haplotype detection technology, providing a non-invasive prenatal diagnosis method for families without a proband.

[0039] The non-invasive prenatal test for single-gene inherited metabolic diseases established by the present invention provides pregnant women with more accurate and comprehensive non-invasive prenatal screening, helps to discover potential inherited metabolic diseases early and improve the timeliness of diagnosis; shortens the diagnosis cycle, reduces the economic burden on families, and improves the accessibility of prenatal testing; promotes early prevention and intervention of single-gene inherited metabolic diseases and improves the quality of life of patients; provides clinicians with more valuable diagnostic basis and helps to formulate more accurate treatment plans; promotes the further development of non-invasive prenatal testing technology and lays the foundation for the screening and diagnosis of other genetic diseases. DETAILED DESCRIPTION

[0040] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0041] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with specific embodiments, but is not intended to be a limitation of the present invention.

[0042] In certain preferred embodiments of the present invention, a kit for non-invasive prenatal detection of single gene inherited metabolic diseases is provided, which is used to detect methylmalonic acidemia and phenylketonuria, and the pathogenic genes involved include MMUT, MMACHC, and PAH. The above-mentioned kit includes a primer combination as shown in SEQ ID No.1 to SEQ ID No.106 for amplifying the target gene and its upstream and downstream 190 to 200 kb regional sequence; it may also include a panel (probe) covering the target gene and its upstream and downstream 190 to 200 kb; it may also include a peripheral blood collection device and a gene DNA extraction reagent, etc.

[0043] The present invention is exemplified below.

[0044] Example 1 - Establishment of an analytical method for single gene inherited metabolic diseases

[0045] This embodiment constructs a preferred non-invasive prenatal detection and analysis method for detecting single gene genetic metabolic diseases such as methylmalonic acidemia and phenylketonuria, which specifically includes the following steps:

[0046] (1) Peripheral blood collection;

[0047] After informed consent was obtained from the pregnant woman and her family, 10 mL of peripheral blood was collected from the pregnant woman (in a plasma collection tube) and 5 mL of peripheral blood was collected from the pregnant woman's husband (in an EDTA anticoagulant tube).

[0048] (2) Genomic DNA extraction;

[0049] The peripheral venous blood of pregnant women was centrifuged at 4℃ low speed, 1600g, for 10 minutes, and the upper plasma was aspirated into a centrifuge tube. The separated plasma was centrifuged at 4℃ low speed, 16000g, for 10 minutes, and the supernatant was aspirated into a centrifuge tube for subsequent extraction of fetal free DNA. The remaining white blood cell-containing blood of the pregnant woman was mixed and divided into another centrifuge tube for subsequent extraction of maternal genomic DNA; the peripheral venous blood of the father was used for subsequent extraction of paternal genomic DNA.

[0050] DNA extraction: Tiangen's blood genomic DNA extraction kit (catalog number: DP348-03) was used to extract the genomic DNA of the father and the mother, and Laifeng's trace DNA extraction kit (catalog number: DK803-02) was used to extract the genomic DNA in the mother's amniotic fluid. Agarose gel electrophoresis was used to detect the integrity of the DNA fragments, and Qubit4.0 (invitrogen) was used to detect the DNA concentration; Laifeng's free DNA extraction kit (catalog number: DK607-1) was used to extract the plasma free DNA of the pregnant woman, and Qubit4.0 (invitrogen) was used to detect the DNA concentration and determine whether there was genomic DNA contamination.

[0051] (3) Calculation of fetal free DNA concentration;

[0052] The concentration of free fetal DNA in maternal plasma (fetal fraction, FF) refers to the proportion of cffDNA derived from the fetus in the free DNA in maternal plasma, which can be calculated by selecting homologous SNPs of different alleles of both parents. The formula is as follows: f = 2p / (p+q), where p is the reading of a specific allele of the fetus, and q is the reading of other alleles common to the mother and the fetus. The concentration of free fetal DNA is the average of the calculation results of all the above gene readings.

[0053] (4) Design of detection-specific primers;

[0054] a) Select target genes, specifically: based on the human reference genome hg38, the MMUT target gene fragment includes Chr6:49345400-49541567; the MMACHC target gene fragment includes Chr1:45410655-45602552; the PAH target gene fragment includes Chr12:102796508-102995704,

[0055] b) Primer design: Based on the target gene fragment and its upstream and downstream 190-200kb base sequence, primer3 was used to design multiple pairs of specific primers. The primers involved are suitable for long fragment amplification, and the Tm value is higher than that of conventional primers. The position of the primers and the amplification length are suitable for the Linked-read mentioned below. The pathogenic genes detected above include: MMUT, MMACHC, and PAH. This method can design multiple pairs of primers at the same time, which is efficient and effective, and through computer-assisted optimization of the primer design process, its specificity and amplification efficiency are improved. Primer design needs to ensure that the length of a single amplified fragment is greater than 20kb, and at the same time, the two adjacent amplified fragments have an overlapping sequence of 10kb, which ultimately covers a region of about 200kb upstream and downstream of the target gene. The specific information of the above primers is shown in the following table:

[0056] Table 1 - List of MMUT primers

[0057]

[0058]

[0059] Table 2 - MMACHC primer list

[0060]

[0061]

[0062] Table 3-PAH primer list

[0063]

[0064]

[0065] (5) Establishment of non-invasive prenatal diagnosis methods for single-gene genetic diseases without a proband

[0066] a) Use the primer sequences designed in (4) to perform multiple long-fragment PCR amplification of the genomic DNA of the father and mother; construct a linked-read library of the long-fragment amplification products, and the DNA fragments from the same source will be added with the same barcode fragments. The constructed library will be used for next-generation sequencing.

[0067] b) In the data analysis phase, the short sequences obtained by the second-generation sequencing are spliced ​​into long sequences through the barcode information on each DNA fragment to obtain the haplotype information of the target regions of the father and mother.

[0068] c) Construct a whole-genome library of maternal peripheral plasma cfDNA, use a panel (probe) covering the target gene (designed by conventional methods) for hybridization capture and targeted second-generation sequencing to obtain the genetic information of cfDNA.

[0069] d) The method for fetal haplotype inference is to use an inference method based on the hidden Markov model. This method uses the corresponding parental genotype at the site, the sequencing depth of the reference allele and variant allele in cfDNA as the observed state, and the haplotype inherited by the fetus as the hidden state to be analyzed. The initial state of the two alleles is defined as {1 / 2,1 / 2}, the observed state probability matrix is ​​composed of the conditional probability of the fetus inheriting the corresponding allele when the different alleles in the peripheral blood cfDNA are observed at the site, and the hidden state transition probability matrix is ​​composed of the corresponding recombination rate information obtained according to the genetic distance information corresponding to the physical distance of the alleles on the chromosome. The Viterbi algorithm is used to infer the optimal hidden state according to the model, combined with the haplotype information of the parents, to determine the haplotype of the fetus.

[0070] Example 2 - Non-invasive prenatal testing analysis without proband DNA

[0071] This example provides a better non-invasive prenatal test for the more common single gene inherited metabolic diseases in China, specifically, provides gene mutation detection for three pathogenic genes related to diseases such as methylmalonic acidemia and phenylketonuria. This example tests samples without proband DNA. The number of samples in this example is 39 families with probands with inherited metabolic diseases. The sample collection, extraction and detection methods refer to Example 1. The specific detection steps are briefly described as follows:

[0072] (1) PCR enrichment of target regions and Tell-seq library preparation (father’s gDNA, mother’s gDNA);

[0073] As shown in Example 1, there are 18 or 17 pairs of amplification primers for each gene, among which odd-numbered primer pairs are mixed into one tube (e.g., MMUT-1F / 1R, MMUT-3F / 3R, MMUT-5F / 5R...), and even-numbered primer pairs are mixed into one tube (e.g., MMUT-2F / 2R, MMUT-4F / 4R, MMUT-6F / 6R...), and each gene is amplified using the above two tubes of primers, with a gDNA starting amount of 100ng, and PCR amplification is performed using TOYOBO's KOD FX reagent (Toyobo (Shanghai) Biotechnology Co., Ltd., KFX-201). After amplification, the amplified fragments are quality checked by agarose gel electrophoresis and quantified by Qubit, and the two tubes of PCR products from the same sample are mixed in equal amounts. Take 0.1ng of the mixed PCR product and use the TELL-Seq from Universal Sequencing Technology. TM WGSLibrary Reagent Box 1 and TELL-Seq TM The WGS Library Reagent Box 2 kit (Universal Sequencing Technology, 100011, 100012) was used to build the library, and all experimental operations were performed according to the kit instructions. The library was analyzed for fragments using QSEP100 (BIOptic, China), and quantified using Qubit 4.0 (Thermo, USA).

[0074] (2) Preparation of plasma cfDNA whole genome library;

[0075] The starting amount of cfDNA whole genome library construction was 5-15 ng, and the library was constructed using the Yisheng HieffNGS MaxUp IIDNALibrary Prep Kit for Illumina Universal DNA Library Construction Kit (Yisheng Biotechnology (Shanghai) Co., Ltd., 12200ES08), with a final library volume of 25 μL. All experimental operations were performed according to the kit instructions. The library was analyzed by QSEP100 (BIOptic, China) and quantified by Qubit 4.0 (Thermo, USA).

[0076] (3) Probe hybridization capture (plasma cfDNA whole genome library);

[0077] The Twist Standard Hybridization and Wash Kit (Twist Bioscience, USA) was used to perform hybridization capture on the established cfDNA library. The starting amount of cfDNA library hybridization was 300 ng, and the hybridization time was 16 hours. All experimental operations were performed according to the instructions of the kit. The library was analyzed by QSEP100 (BIOptic, China) and quantified by Qubit 4.0 (Thermo, USA).

[0078] (4) Next-generation sequencing;

[0079] The Tell-seq library was sequenced using Illumina NextSeq500, with 500Mb of data loaded per sample and sequencing mode PE150.

[0080] The cfDNA capture library was sequenced using MGISEQ-T7, with 1500Mb of data per cfDNA library and sequencing mode PE100.

[0081] (5) Data analysis;

[0082] 1) Calculation of sequencing error rate and fetal cffDNA concentration;

[0083] First, the sequencing error rate was analyzed and evaluated as a quality control. Based on the SNP sites where the father and mother had the same homozygous genotype, the percentage of genotype sequencing depth at the same site in the free DNA that did not conform to Mendel's law of inheritance was analyzed as an estimate of the sample sequencing error rate, with 0.5% as the upper threshold limit.

[0084] Secondly, calculate the concentration of fetal free DNA. The concentration of fetal free DNA in maternal plasma (fetal fraction, FF) refers to the proportion of cffDNA derived from the fetus in the free DNA in maternal plasma. It can be calculated by selecting homologous SNPs of different alleles of both parents. The formula is as follows: f = 2p / (p+q), where p is the reading of the fetal specific allele, and q is the reading of other alleles common to the mother and fetus. The concentration of fetal free DNA is the average of the calculation results of all the above gene readings.

[0085] 2) construct family haplotypes;

[0086] Based on Linked-read, the haplotypes of the target regions of the father and mother are constructed, and effective SNP sites are selected: for sites where one parent is heterozygous and the other is homozygous (the carrier of the mutation is heterozygous and the other is homozygous). Based on these effective SNP information, the haplotypes carrying the target mutation and the normal haplotype are determined. At the same time, by analyzing the relative sequencing depth of the alleles at these sites in the free DNA, combined with the fetal free concentration and sequencing error rate information, the fetal genotype in the free DNA is estimated according to the binomial distribution model and the hidden Markov model (HMM).

[0087] 3) Fetal haplotype prediction;

[0088] The inference results are judged based on the continuity of the inferred fetal haplotype and the log odds ratio of the fetal haplotype inherited from the two haplotypes of the mutation source to predict whether the fetus inherits the mutation haplotype.

[0089] 4) Results:

[0090] The specific test results are shown in Table 4. Among the 39 fetuses undergoing NIPD, 14 could not be haplotyped because their fathers or mothers did not have suitable effective points. Two alleles were detected in the remaining 25 fetuses, of which 4 fetuses were affected, 13 fetuses only carried one gene mutation site and were carriers, and 8 fetuses were normal. A total of 25 families underwent haplotype judgment, with an effectiveness of 64.1% (25 / 39), of which 23 had consistent results with amniocentesis gene testing, with a consistency of 92% (23 / 25).

[0091] Table 4 - Non-invasive prenatal results without proband DNA

[0092]

[0093]

[0094] Note: "F" means father, "M" means mother; F0 / M0 means the fetus inherits the pathogenic haplotype of the father / mother; F1 / M1 means the fetus inherits the normal haplotype of the father / mother. " / " means there is no suitable valid point and haplotype judgment cannot be performed. #: MLPA shows that there is an abnormality between the first 3238nt of exon 1 and the first 191nt of exon 2 of the PAH gene, and the heterozygous deletion is as long as 7.9Kb.

[0095] As can be seen from the above embodiments, the non-invasive prenatal detection method for monogenic inherited metabolic diseases developed by the present invention is used to determine whether the fetus suffers from related genetic diseases by detecting whether there are gene mutations in the fetal genes of pregnant women's peripheral blood. Detection of related pathogenic genes includes MMUT, MMACHC, and PAH. The method is based on the second-generation sequencing platform of Illumina and BGI-SEQ. Through targeted enrichment combined with linked-reads technology, a panel combination (gene package combination) containing three pathogenic genes related to diseases such as methylmalonic acidemia and phenylketonuria is designed, thereby directly obtaining the haplotypes of the couple located in the upstream and downstream regions of the relevant pathogenic genes without relying on the proband, and combining the genotype information of the fetal free DNA in the plasma of pregnant women to infer the possible genotype of the fetus and determine whether it carries pathogenic mutations. Compared with traditional invasive prenatal testing techniques, this method is non-invasive, low-risk, and highly sensitive, and can be widely used in prenatal screening of pregnant women; compared with previous non-invasive prenatal testing technologies, this method expands the scope of detected diseases, does not rely on the DNA information of the proband, and has good application prospects in non-invasive prenatal testing research for single-gene diseases.

[0096] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification of the present invention should be included in the protection scope of the present invention.

Claims

1. A kit for non-invasive prenatal detection of monogenic inherited metabolic diseases, characterized in that The kit comprises a primer composition, which comprises a primer composition for amplifying a target gene of at least one of the pathogenic genes MMUT, MMACHC and PAH and a 180-210 kb regional sequence upstream and downstream thereof; wherein, based on the human reference genome hg38, the MMUT target gene fragment comprises Chr6:49345400-49541567; the MMACHC target gene fragment comprises Chr1:45410655-45602552; and the PAH target gene fragment comprises Chr12:102796508-102995704.

2. The kit according to claim 1, characterized in that The primer composition comprises: MMUT primers: as shown in SEQ ID No.1 to SEQ ID No.36; MMACHC primers: as shown in SEQ ID No.37 to SEQ ID No.70; PAH primers: as shown in SEQ ID No.70 to SEQ ID No.

106.

3. The kit according to claim 1, characterized in that The kit also includes a probe for hybridization capture of a whole genome library of maternal plasma cfDNA.

4. A method for detecting and analyzing a single gene inherited metabolic disease for non-diagnostic purposes, characterized in that: The detection and analysis method comprises the steps of: based on a second generation sequencing platform, in the absence of a proband's DNA, using a kit as described in any one of claims 1 to 3, utilizing target region enrichment, linked-reads method, and probe hybridization capture method to construct the haplotypes of the family's parents, and combining the genotype of the mother's plasma cfDNA to analyze the genotype and haplotype information of the fetus.

5. The detection and analysis method according to claim 4, characterized in that: The detection and analysis method specifically comprises the steps of: Step S1, adopting the target region PCR enrichment method, using the specific primers described in claim 2 to amplify the genomic DNA of the father and the mother by multiple long-fragment PCR, and constructing a linked-read library for the long-fragment amplification products. The DNA fragments from the same source will be added with the same barcode fragments, and the constructed Tell-seq library will be used for second-generation sequencing; Step S2, splicing the short sequences obtained by the second-generation sequencing into long sequences according to the barcode information on each DNA fragment, and obtaining the haplotype information of the target regions of the father and the mother; Step S3, constructing a whole genome library of maternal peripheral plasma cfDNA, using probe hybridization capture and targeted next-generation sequencing to obtain genotype information of maternal plasma cfDNA; Step S4: Based on the hidden Markov model and Viterbi algorithm, the genotype information of the mother's plasma cfDNA and the haplotype information of the father's and mother's target regions are combined to analyze the genotype and haplotype information of the fetus.

6. The detection and analysis method according to claim 5, characterized in that: Preparation of sequencing libraries includes: Tell-seq library: The starting amount of gDNA was 100 ng, and PCR amplification was performed using KOD FX reagent. After amplification, the amplified fragments were checked by agarose gel electrophoresis and quantified by Qubit. 0.1 ng of mixed PCR products was taken and TELL-Seq TM WGS LibraryReagent Box 1 and TELL-Seq TM WGS Library Reagent Box 2 kit to build the library; cfDNA whole genome library: The starting amount for library construction is 5-15 ng, and the library is constructed using the Hieff NGS MaxUp IIDNA Library PrepKit for Illumina universal DNA library construction kit.

7. The detection and analysis method according to claim 5, characterized in that: Probe hybridization capture includes: using TwistStandard Hybridization and Wash Kit to perform hybridization capture on the established whole genome library, the starting amount of cfDNA library hybridization is 300 ng, and the hybridization time is 16 hours.

8. The detection and analysis method according to claim 5, characterized in that: Next-generation sequencing of the library includes: Tell-seq library was sequenced by Illumina NextSeq500, with 500Mb of data per sample, and sequencing mode PE150; The cfDNA library was sequenced using MGISEQ-T7, with 7000Mb of data per cfDNA library and sequencing mode PE100.

9. The detection and analysis method according to claim 5, characterized in that: The hidden Markov model and Viterbi algorithm specifically include: taking each allele information site as an observation sequence, taking the corresponding parental genotype at the site, the sequencing depth information of the reference allele and the variant allele in cfDNA as the observation state, and taking the haplotype inherited by the fetus as the hidden state to be analyzed; the initial state of the two alleles is defined as {1 / 2, 1 / 2}, the observation state probability matrix is ​​composed of the conditional probability of the fetus inheriting the corresponding allele when the alleles in the peripheral blood cfDNA at the site are in different observation states, and the hidden state transition probability matrix is ​​composed of the corresponding recombination rate information obtained according to the genetic distance information corresponding to the physical distance of the alleles on the chromosome; using the Viterbi algorithm to infer the optimal hidden state according to the model, combined with the haplotype information of the parents, to analyze the haplotype of the fetus.

10. Use of the kit according to any one of claims 1 to 3 in the detection of single gene inherited metabolic diseases for non-diagnostic purposes.

Images