A kit for detecting a pathogenic gene of phenylketonuria and use thereof
By designing a kit for PAH gene mutations in the Chinese population, and employing multiplex PCR and high-throughput sequencing technologies, the problems of low detection throughput and insufficient coverage in existing technologies have been solved, achieving efficient and accurate gene mutation detection, which is suitable for newborn screening and prenatal diagnosis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- THE THIRD AFFILIATED HOSPITAL OF ZHENGZHOU UNIVERSITY
- Filing Date
- 2026-04-30
- Publication Date
- 2026-06-05
AI Technical Summary
Existing commercial PAH gene detection kits do not fully cover the mutation spectrum of the Chinese population, have low throughput, cannot simultaneously detect point mutations and copy number variations, and the traditional Sanger sequencing method is costly and time-consuming, making it unsuitable for large-scale screening.
A kit containing specific primer compositions and detection probe compositions was designed to target the PAH gene mutation characteristics of the Chinese population. Multiplex PCR technology is used to amplify multiple exons and adjacent regions at once. Combined with high-throughput sequencing or microarray hybridization, point mutations and small fragment insertions/deletions are detected simultaneously. Large fragment deletions/duplications are quantitatively analyzed. Specific probes are used to ensure the accuracy of the detection results.
It improves the coverage and detection efficiency of PAH gene mutations in the Chinese population, achieving high-throughput and accurate gene mutation detection. It is applicable to newborn screening, clinical diagnosis, and prenatal diagnosis, meeting the needs of the entire chain from screening to diagnosis, and reducing costs and time.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of gene detection and molecular diagnostic technology, specifically relating to a kit for detecting the pathogenic gene of phenylketonuria and its uses. Background Technology
[0002] Phenylketonuria (PKU) is a common autosomal recessive inherited amino acid metabolism disorder, primarily caused by mutations in the phenylalanine hydroxylase (PAH) gene, leading to decreased or absent enzyme activity. This results in the accumulation of phenylalanine and its metabolites in the body, causing irreversible damage to the nervous system. Early diagnosis and lifelong dietary therapy are crucial for improving prognosis. Currently, PKU is included in newborn screening programs in my country. Traditional screening methods involve measuring serum phenylalanine concentrations, but these methods have a certain false-positive rate and cannot differentiate between classic PKU, mild hyperphenylalaninemia, and tetrahydrobiopterin deficiency. Furthermore, they cannot identify the pathogenic mutation for genetic counseling and prenatal diagnosis.
[0003] PAH gene mutations exhibit high heterogeneity, with over 1000 reported mutations, and the mutation spectrum shows significant ethnic and regional differences. More than 100 PAH gene mutations have been identified in the Chinese population, among which c.1222C>T, c.1068-11G>A, c.728G>A, and c.1162G>A are relatively common hotspot mutations. However, existing commercial kits are mostly designed based on the mutation spectrum of Western populations, lacking sufficient coverage and detection efficiency for specific hotspot mutations in the Chinese population. Furthermore, traditional Sanger sequencing methods suffer from low throughput, high cost, and long processing times, making them unsuitable for large-scale screening. Therefore, there is an urgent need to develop an integrated detection kit that specifically targets the PAH gene mutation characteristics of the Chinese population and can efficiently detect multiple hotspot mutations and copy number variations in a single test. Summary of the Invention
[0004] To address the problems of incomplete coverage of the mutation spectrum in the Chinese population, low detection throughput, and inability to simultaneously detect point mutations and copy number variations in existing PAH gene detection kits, this invention provides a kit for detecting the pathogenic gene of phenylketonuria and its application.
[0005] To achieve the above objectives, the present invention adopts the following technical solution: The present invention first provides a kit for detecting the pathogenic gene of phenylketonuria, comprising a primer composition for multiplex amplification of the coding region of the phenylalanine hydroxylation (PAH) gene and key regions of adjacent introns, and a detection probe composition for identifying specific mutation sites.
[0006] Preferably, in some embodiments of the present invention, the primer composition comprises multiple pairs of specific primers (specifically 8 pairs) for amplifying genomic DNA fragments containing exons 3, 5, 6, 7, 11, and 12 of the PAH gene and their adjacent splice sites.
[0007] Preferably, in some embodiments of the present invention, the primer composition further comprises primer pairs for amplifying conserved sequences in the 5' and 3' untranslated regions of the PAH gene, so as to assist in the identification of large fragment deletions or duplications through quantitative analysis.
[0008] Preferably, in some embodiments of the present invention, the detection probe composition comprises wild-type and mutant-specific probes complementary to hotspot mutation sites of the PAH gene, wherein the hotspot mutation sites preferably include c.1222C>T (p.Arg408Trp), c.1068-11G>A (IVS10-11G>A), c.728G>A (p.Arg243Gln), or c.1162G>A (p.Glu388Lys).
[0009] Preferably, in some embodiments of the present invention, the probe may be in the form of a TaqMan probe or a molecular beacon, labeled with different fluorescent reporter groups; more preferably, the fluorescent reporter group is at least one of FAM, TET, VIC, HEX, ROX, JOE, CY3 and CY5; the quenching group is BHQ1, DABCYL or TAMRA.
[0010] Specifically, the detection probe composition is as follows: 1) For c.1222C>T (p.Arg408Trp): The wild-type probe sequence is shown in SEQ ID NO.1, with a fluorescent reporter group FAM at the 5' end and a quencher group BHQ1 at the 3' end; the mutant probe sequence is shown in SEQ ID NO.2, with a fluorescent reporter group HEX at the 5' end and a quencher group BHQ1 at the 3' end. 2) For c.1068-11G>A (IVS10-11G>A): The wild-type probe sequence is shown in SEQ ID NO.3, with a fluorescent reporter group FAM at the 5' end and a quencher group BHQ1 at the 3' end. The mutant probe sequence is shown in SEQ ID NO.4, with a fluorescent reporter group HEX at the 5' end and a quencher group BHQ1 at the 3' end. 3) For c.728G>A (p.Arg243Gln) and c.1162G>A (p.Glu388Lys): Design similar allele-specific probes.
[0011] Preferably, in some embodiments of the present invention, the kit further comprises: an optimized PCR reaction buffer, a mixture of dNTPs, a thermostable DNA polymerase, a positive control, a negative control, a nucleic acid extraction reagent, and a DNA molecular weight standard.
[0012] Preferably, in some embodiments of the present invention, the positive control is a plasmid DNA or a synthetic DNA fragment containing at least one of the four mutations c.1222C>T, c.1068-11G>A, c.728G>A, and c.1162G>A.
[0013] Furthermore, the present invention also provides the use of the kit in the preparation of products for the diagnosis or screening of phenylketonuria.
[0014] Specifically, the diagnosis or screening includes, but is not limited to: genetic confirmation of positive samples for neonatal phenylketonuria screening, molecular diagnosis of clinically suspected phenylketonuria patients, carrier screening of families of phenylketonuria patients, and prenatal diagnosis of phenylketonuria.
[0015] Furthermore, the present invention also provides a method for detecting the pathogenic gene of phenylketonuria (PAH), using the above-mentioned kit, comprising the following steps: (1) extracting genomic DNA from the subject's biological sample; (2) using the primer composition in the kit to perform multiplex PCR amplification on the target region of the PAH gene; (3) performing high-throughput sequencing on the amplification product or hybridizing it with the detection probe composition; (4) analyzing the sequencing results or hybridization signal to determine whether there is a pathogenic mutation in the PAH gene.
[0016] Specifically, in the analysis step (4), if two pathogenic or potentially pathogenic mutations verified by the database are detected in the alleles of the PAH gene, the subject is determined to be a patient with phenylketonuria; if only one pathogenic mutation is detected, the subject is determined to be a carrier.
[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Highly targeted: The primers and probes of the kit described in this invention are designed based on big data analysis of the PAH gene mutation spectrum in the Chinese population, focusing on common hotspot mutation regions in the Chinese population, thus improving the targeting and effectiveness of the detection.
[0018] 2. Comprehensive and efficient: Multiplex PCR technology amplifies multiple key exons at once. Combined with high-throughput sequencing or microarray hybridization, it can simultaneously detect point mutations, small fragment insertions / deletions, and indicate large fragment deletions / duplications through quantitative analysis, achieving "one tube reaction, comprehensive detection".
[0019] 3. Accurate and sensitive: The kit contains rigorously validated positive controls and specific probes, ensuring the accuracy and reproducibility of the test results and effectively distinguishing pathogenic mutations, polymorphic sites, and false positive signals.
[0020] 4. Wide range of applications: The kit described in this invention has standardized operation and is suitable for clinical molecular diagnostic laboratories, newborn screening centers and prenatal diagnostic centers. It meets the needs of the entire chain from screening and diagnosis to clinical diagnosis, genetic counseling and prenatal diagnosis, and strongly supports the precise prevention and control of phenylketonuria. Attached Figure Description
[0021] Figure 1 This is a schematic diagram of the overall process of the phenylketonuria pathogenic gene detection method provided in an embodiment of the present invention. Detailed Implementation
[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
[0024] In the following examples, room temperature or normal temperature refers to 25±5℃. Example 1
[0025] Components of the reagent kit This embodiment provides a kit for detecting the pathogenic gene of phenylketonuria, the core components of which are as follows: 1. Multiplex PCR Primer Composition: Contains 8 pairs of specific primers for amplifying exons 3, 5, 6, 7, 11, and 12 of the PAH gene and their flanking intron sequences (approximately 50 bp), as well as conserved regions of the 5' and 3' UTRs. The primer sequences have been carefully designed and optimized to ensure uniform amplification efficiency in a single reaction system, free from primer dimer interference. Specific primer sequences were obtained using professional primer design software after alignment with human PAH gene sequences in public databases. 2. Detection Probe Composition: Designed to target four of the most frequent hotspot mutations in the Chinese population: 2.1 For c.1222C>T (p.Arg408Trp): Wild-type probe (5'-FAM-CTACCGCCACC-BHQ1-3'), Mutant probe (5'-HEX-CTACCGTCACC-BHQ1-3').
[0026] 2.2 For c.1068-11G>A (IVS10-11G>A): wild-type probe (5'-FAM-AGGTAAGGGT-BHQ1-3'), mutant probe (5'-HEX-AGGTAAGAGT-BHQ1-3').
[0027] 2.3 For c.728G>A (p.Arg243Gln) and c.1162G>A (p.Glu388Lys): similar allele-specific probes were designed.
[0028] The probes can be in the form of TaqMan probes or molecular beacons, labeled with different fluorescent reporter groups.
[0029] 3. Reaction system and core components: Includes 2× multiplex PCR premix (DNA polymerase, dNTPs Mixture, MgCl2, reaction buffer) and sterile nuclease-free water. See Table 1 below for details.
[0030] Table 1.
[0031]
[0032] 4. Reference standard: 4.1 Positive control: Artificial plasmid DNA containing a double-site compound heterozygous mutation of c.1222C>T and c.1068-11G>A.
[0033] 4.2 Negative control: Genomic DNA of healthy individuals without PAH gene mutations, verified by sequencing.
[0034] 4.3 Blank control: sterile water without nuclease.
[0035] 5. Auxiliary reagents: Genomic DNA extraction reagents for blood / dried blood spots, reagents for agarose gel electrophoresis, and DNA molecular weight markers. Example 2
[0036] Detection methods See Figure 1 This is the overall process of the phenylketonuria pathogenic gene detection method described in this invention.
[0037] like Figure 1 As shown, this embodiment provides a detection method, which specifically includes the following steps: S100: Sample Collection and DNA Extraction. Collect 2-3 mL of peripheral venous blood from the subject (EDTA anticoagulated). Extract genomic DNA using the kit provided or a universal genomic DNA extraction kit (DP348 Blood / Cell / Tissue Genomic Extraction Kit, Tiangen Biotech Co., Ltd.), following the instructions. Determine the DNA concentration and purity using a UV spectrophotometer and adjust to the working concentration (e.g., 10-20 ng / μL).
[0038] S200: Multiplex PCR amplification. Prepare a 25 μL reaction mixture in a PCR tube containing 12.5 μL of 2× premix (DNA polymerase, dNTPs Mixture, MgCl2, reaction buffer), 1 μL of primer mixture, 1 μL of probe mixture, 2 μL of template DNA, and 8.5 μL of sterile water. Perform amplification on a standard PCR instrument. The amplification program is as follows: 98℃ pre-denaturation for 1 minute; followed by 40 cycles: 98℃ denaturation for 20 seconds, 60℃ annealing / extension for 30 seconds; and a final extension at 72℃ for 30 seconds. If sequencing is subsequently used, no probe needs to be added to the PCR mixture; only primers are used for amplification.
[0039] S300: Product Analysis and Mutation Detection. This step offers two optional pathways: Path A (based on real-time fluorescence PCR and melting curve analysis): After amplification, melting curve analysis is performed. Based on the fluorescence signal (FAM vs HEX) and melting temperature peak of each mutation site probe, the genotype of the sample at each site is automatically determined (wild-type homozygous, mutant homozygous, heterozygous).
[0040] Pathway B (based on high-throughput sequencing): After purifying the multiplex PCR products, a sequencing library is constructed and sequenced using a next-generation sequencing platform (see references 1. Podnar, J, Deiderick, H, Hunicke-Smith, S. Next-generation sequencing fragment library construction. Curr Protoc Mol Biol. 2014; 107 7.17.1-7.17.16; 2. Kidder, BL, Zhao, K. Efficient library preparation for next-generation sequencing analysis of genome-wide epigenetic and transcriptional landscapes in embryonic stem cells. Methods Mol Biol. 2014; 1150 3-20.). The sequencing data is compared with the PAH gene reference sequence, and bioinformatics software is used to identify single nucleotide variants and small fragment insertions / deletions. Simultaneously, by comparing the sequencing depth of each amplicon, the presence of large fragment deletions or duplications can be preliminarily determined (further verification using techniques such as MLPA is required).
[0041] S400: Result Interpretation and Reporting. Detected variants are compared with professional databases such as HGMD, ClinVar, and PAHvdb, and pathogenicity is classified according to ACMG guidelines.
[0042] - If pathogenic or potentially pathogenic variants are found in both PAH alleles, a report stating "molecular diagnosis consistent with classic phenylketonuria" will be issued.
[0043] - If only one pathogenic variant is found, a report of "PAH gene variant carrier" will be issued, and it will be recommended that the patient's parents be verified to clarify the variant phase.
[0044] - If no pathogenic variants are found, but there is a high clinical suspicion, the report should state "Not detected within the scope of this test".
[0045] "Identify pathogenic mutations within the blood," and suggest combining results such as blood Phe concentration and BH4 load test for a comprehensive judgment, or consider whole exome sequencing to find potential new mutations. Example 3
[0046] Performance validation of the reagent kit To verify the performance of the kit described in this invention, the inventors collected 100 clinically and biochemically diagnosed PKU patient samples and 100 healthy control samples for blinded testing (experimental method as described in Example 2).
[0047] 1. Specificity and Sensitivity: Four hotspot mutations were detected using pathway A (fluorescent PCR). Results showed that the kit described in this invention detected 78 of the mutated alleles from 80 patients with known genotypes, achieving a sensitivity of 97.5%. No false-positive mutations were detected in the alleles of 200 healthy controls, demonstrating a specificity of 100%.
[0048] 2. Consistency: Thirty samples were randomly selected and subjected to whole-exome sequencing using both the kit described in this invention (path B, sequencing method) and the gold standard Sanger sequencing method. The two methods showed completely consistent results in detecting hotspot mutations. Furthermore, this kit also detected a low proportion of chimeric mutations in two additional samples, which might have been missed by the Sanger method due to its sensitivity limitations.
[0049] 3. Detection Scope: In 100 patients, the kit described in this invention successfully identified biallelic pathogenic mutations in 95 patients (95%). Among them, 65 cases (65%) were diagnosed using only four hotspot mutation probes. Through sequencing, more than 20 other mutations, including c.721C>T and c.1197A>T, were additionally discovered, as well as 3 samples suspected of having exon deletions (later confirmed by MLPA). Example 4
[0050] Application in the recall and confirmation of positive newborn screening results At a newborn screening center, 50 newborns with serum Phe concentrations consistently above 120 μmol / L for six consecutive months were recalled and their genetic diagnosis was performed using the kit described in Example 1. The results showed that 32 cases (64%) were carriers of the biallelic pathogenic mutation in the PAH gene, diagnosed as classic PKU; 10 cases (20%) were single heterozygous carriers, classified as mild HPA or requiring exclusion of BH4D; and 8 cases (16%) showed no pathogenic mutation and require further follow-up. The average time for genetic diagnosis was 3 working days, significantly shorter than traditional external sequencing (which typically takes 2-3 weeks), allowing for faster implementation of early dietary interventions. Example 5
[0051] Application in prenatal diagnosis A couple who had previously given birth to a child with classic PKU (the proband's genotype was c.1222C>T / c.1068-11G>A complex heterozygous) requested prenatal diagnosis during their second pregnancy. At 18 weeks of gestation, amniocentesis was performed to obtain amniotic fluid cells. The extracted fetal DNA was analyzed using the kit described in Example 1. Since the parental mutations were clearly identified, fluorescent PCR was performed directly using probes targeting these two sites. The results showed that the fetus carried only the c.1222C>T mutation from the father and not the c.1068-11G>A mutation from the mother, with a genotype of c.1222C>T / wild-type, classifying the fetus as a carrier. No special dietary treatment was required after birth. This diagnostic result was consistent with the fetus's postnatal blood Phe concentration monitoring results, avoiding unnecessary family anxiety and intervention.
[0052] In summary, the reagent kit and detection method provided by this invention have advantages such as high specificity, high sensitivity, high throughput, speed, and relatively low cost. They can effectively meet the current urgent clinical needs for precise molecular diagnosis of PKU and are particularly suitable for promotion and application in medical and screening institutions at all levels in China. They have important social and economic value for reducing birth defects and improving population quality.
[0053] It should be noted that the order of the above embodiments is for descriptive purposes only and does not represent superiority or inferiority. The processes depicted in the accompanying drawings do not necessarily require the specific order shown to achieve the desired results. In some embodiments, multitasking and parallel processing are also feasible or advantageous.
[0054] The various embodiments in this specification are described in a progressive manner. Similar or identical parts between embodiments can be referred to mutually. Each embodiment focuses on describing the differences from other embodiments. In particular, the system embodiments are basically similar to the method embodiments, so the description is relatively simple; relevant parts can be referred to the descriptions in the method embodiments.
[0055] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.
Claims
1. A kit for detecting the pathogenic gene of phenylketonuria, characterized in that, The invention comprises a primer composition for multiplex amplification of the coding region of the phenylalanine hydroxylase gene and key regions of adjacent introns, and a detection probe composition for identifying specific mutation sites. The primer composition comprises multiple pairs of specific primers for amplifying genomic DNA fragments containing exons 3, 5, 6, 7, 11, and 12 of the PAH gene and their adjacent splicing sites. The detection probe composition comprises wild-type and mutant-specific probes complementary to the following PAH gene hotspot mutation sites: c.1222C>T (p.Arg408Trp), c.1068-11G>A (IVS10-11G>A), c.728G>A (p.Arg243Gln), and c.1162G>A (p.Glu388Lys).
2. The reagent kit according to claim 1, characterized in that, The primer composition also includes primer pairs for amplifying conserved sequences in the 5' and 3' untranslated regions of the PAH gene to aid in the detection of large deletion / duplication variants.
3. The reagent kit according to claim 1, characterized in that, The kit also includes: PCR reaction buffer, dNTPs mixture, thermostable DNA polymerase, positive control, negative control, nucleic acid extraction reagent, and DNA molecular weight standard.
4. The reagent kit according to claim 1, characterized in that, The positive control is a plasmid DNA or a synthetic DNA fragment containing at least one of the four mutations described in claim 1: c.1222C>T, c.1068-11G>A, c.728G>A, and c.1162G>A.
5. Use of the kit as described in any one of claims 1-4 in the preparation of a product for the diagnosis or screening of phenylketonuria.
6. The use according to claim 5, characterized in that, The diagnosis or screening includes: genetic confirmation of positive samples for neonatal phenylketonuria screening, molecular diagnosis of clinically suspected phenylketonuria patients, carrier screening of families with phenylketonuria patients, and prenatal diagnosis of phenylketonuria.
7. A method for detecting the pathogenic gene of phenylketonuria, characterized in that, Using the kit as described in any one of claims 1-4, the steps include: (1) Extract genomic DNA from the subject's biological samples; (2) Using the primer composition in the kit, perform multiplex PCR amplification of the target region of the PAH gene; (3) Perform high-throughput sequencing on the amplification products or hybridize them with the detection probe composition for detection; (4) Analyze the sequencing results or hybridization signals to determine whether there are pathogenic mutations in the PAH gene.
8. The detection method according to claim 7, characterized in that, In step (4), if two pathogenic or potentially pathogenic mutations verified by the database are detected in the alleles of the PAH gene, the subject is determined to be a patient with phenylketonuria; if only one pathogenic mutation is detected, the subject is determined to be a carrier.