Multiplex fluorescent quantitative PCR (polymerase chain reaction) primer, probe and kit for detecting neurofibroma I-type NF1 gene variation sites

Abstract

The invention relates to a multiplex fluorescent quantitative PCR (polymerase chain reaction) primer, a probe and a kit for detecting neurofibroma I type NF1 gene variation sites, and provides four NF1 gene mutation sites found for the first time, the four variation sites are NF1 gene c.21002105delinsAG, c.4561dup, c.6421del and c.6797dup variation sites respectively, and the mutation sites of the NF1 gene c.21002105delinsAG, the c.4561dup, the c.6421del and the c.6797dup can be used for detecting neurofibroma I type NF1 gene mutation sites. Meanwhile, the invention also provides a specific primer, a probe composition and a kit for detecting the NF1 gene variation site of the neurofibromatosis I type for screening or diagnosing the neurofibromatosis I type. The NF1 gene pathogenic variation spectrum is expanded, and a basis is provided for diagnosis and genetic counseling of the neurofibroma type I. The kit disclosed by the invention can comprehensively cover four new pathogenic variation sites of the NF1 gene related to the neurofibroma type I, is high in accuracy, and can specifically detect the pathogenic variation sites.

Description

Technical Field

[0001] This invention relates to the field of gene diagnostic technology, specifically to a primer and probe composition and kit for detecting NF1 gene mutation sites in neurofibromatosis type I. Background Technology

[0002] Neurofibromatosis type 1 (NF1; MIM# 162200) is a common autosomal dominant genetic syndrome with an incidence of approximately 1 in 3000 (Evans et al., 2010; Kallionpaa et al., 2018; Miller et al., 2019; Uusitalo et al., 2015). NF1 presents with diverse clinical manifestations, including café-au-lait macules (CALMs), cutaneous and plexiform neurofibromas, freckle-like pigmentation in the axilla and groin, optic pathway gliomas, Lisch nodules in the iris, and an increased risk of both benign and malignant tumors. Although the diagnostic criteria of the National Institutes of Health (NIH) remain the basis for clinical diagnosis (1987 NIH Consensus Statement on Neurofibromatosis), NF1 presents diagnostic challenges, particularly in pediatric patients, due to its high clinical variability and age-dependent expression (DeBella et al., 2000; Ferner & Gutmann, 2013). In many cases, early manifestations may be limited to café-au-lait spots, while other clinical signs gradually appear later in life. Therefore, for pediatric patients who do not yet fully meet the NIH consensus criteria, molecular genetic testing is crucial in diagnosing NF1 and differentiating it from other diseases with similar phenotypes, such as RASopathy syndrome (Tidyman & Rauen, 2009).

[0003] Neurofibromatosis type I is caused by mutations in the NF1 gene located on chromosome 17q11.2. This gene encodes neurofibromin, an important regulator of the Ras signaling pathway. Due to the large size of the NF1 gene, the presence of homologous sequences, and the lack of mutation hotspots, mutation screening for the NF1 gene faces significant challenges (Zhang et al., 2015).

[0004] In-depth research on neurofibromatosis type I (NF1) helps to understand its genetic basis and pathogenesis. Early intervention can reduce the risk of complications, especially malignant tumors, in patients with NF1. Currently, according to the NIH diagnostic criteria for NF1, a positive NF1 gene mutation test is the most important diagnostic criterion for NF1 type I. Early diagnosis is crucial not only for genetic counseling for patients and their families but also for developing targeted management and treatment plans, thus improving prognosis. With the development of genome sequencing technology, scientists are constantly discovering new mutants in the NF1 gene. Many of these mutants are believed to be associated with different phenotypes and symptom severity of NF1. Accurate detection and analysis of these new mutants can not only improve the accuracy of early diagnosis of NF1 but also lay the foundation for the development of personalized treatment plans.

[0005] Therefore, studying the variant profile of the NF1 gene is crucial for understanding the etiology of neurofibromatosis type I. These studies have revealed the correlation and related mechanisms between gene mutations and phenotypes such as neurofibromatosis, growth and development, and skeletal deformities, providing clues for developing potential therapeutic strategies. Although several known mutations have been identified in the NF1 gene, many undiscovered mutation sites remain. Expanding our comprehensive understanding of NF1 gene mutants will provide solid support for future clinical diagnosis, treatment, and research into the molecular mechanisms of neurofibromatosis type I. Summary of the Invention

[0006] The purpose of this invention is to provide four newly discovered NF1 gene mutation sites to enrich the NF1 gene variant spectrum. These four mutation sites are the NF1 gene c.2100_2105delinsAG, c.4561dup, c.6421del, and c.6797dup mutation sites. This invention also provides a specific primer and probe composition and kit for detecting NF1 gene mutation sites in neurofibromatosis type I for screening or diagnosis of neurofibromatosis type I; including specific primers and TaqMan probe compositions targeting the NF1 gene c.2100_2105delinsAG, c.4561dup, c.6421del, and c.6797dup mutation sites respectively.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] The specific primer and probe composition described in this invention was designed using Primer 5.0 software. Specifically:

[0009] 1) Targeting the c.2100_2105delinsAG site of the NF1 gene:

[0010] Forward primer A: 5'-TGGTAGCATTATGTTGGTCCAGAT-3' (SEQ ID NO.1);

[0011] Reverse primer A: 5'-AGCTCCACCCAAAAGGAGTAG-3' (SEQ ID NO.2);

[0012] NF1 gene wild-type probe Aw: 5'-CCCTGACACTGAAGCTGTTCTGGT-3' (SEQ ID NO.3);

[0013] NF1 gene mutant probe Am: 5'-CCCTGACACAGTGTTCTGGT-3' (SEQ ID NO.4).

[0014] 2) Targeting the c.4561dup site of the NF1 gene:

[0015] Forward primer B: 5'-GCTGTTTACAAATCAGCTGACAG-3' (SEQ ID NO.5);

[0016] Reverse primer B: 5'-ATCATATCTAACAAGTGGCCTGGT-3' (SEQ ID NO.6);

[0017] NF1 gene wild-type probe Bw: 5'-GATGGCAACACTTCTTGCAT-3' (SEQ ID NO.7);

[0018] NF1 gene mutant probe Bm: 5'-GATGGCAACACCTTCTTGC-3' (SEQ ID NO.8);

[0019] 3) Targeting the c.6421del site of the NF1 gene:

[0020] Forward primer C: 5'-GACCATGTTCAGTTACCAGCAC-3' (SEQ ID NO.9);

[0021] Reverse primer C: 5'-CCACTTGCACGTTGGAATATCT-3' (SEQ ID NO.10);

[0022] NF1 gene wild-type probe Cw: 5'-TACCCAAATTTTACTTGCTG-3' (SEQ ID NO.11);

[0023] NF1 gene mutant probe Cm: 5'-TACCCAAATTTACTTGCTGT-3' (SEQ ID NO.12);

[0024] 4) Targeting the c.6797dup site of the NF1 gene:

[0025] Forward primer D: 5'-GCTAGCTACCAAGATCACCATAGC-3' (SEQ ID NO.13);

[0026] Reverse primer D: 5'-CACCCCAGAAAGTAAGCTCCATG-3' (SEQ ID NO.14);

[0027] NF1 gene wild-type probe Dw: 5'-ACTTACAACAGTCAAGTTCT-3' (SEQ ID NO.15);

[0028] NF1 gene mutant probe Dm: 5'-TTACAACAGGTCAAGTTCTG-3' (SEQ ID NO.16).

[0029] The probe has different reporter fluorescent groups at its 5' end and an MGB quencher fluorescent group at its 3' end.

[0030] In the same reaction system, the 5' ends of the probes are each equipped with different reporter fluorescent groups. For example, in reaction system A, the 5' ends of six probes detecting three sites are modified with different identifiable fluorescent dyes, including but not limited to one of FAM, SYBR, Fluorescein, SYPRO Orange, VIC, JOE, TET, HEX, TAMRA, Texas Red, Alexa Fluor633, ResoLight, EvaGreen, LC Green, Cy3, Cy5, Yellow555, LC Red610, ROX, SYPRO Ruby, LCRed640, Snarf 1, Acid Fuchsin, Cy5.5, LC Red670, and LC Red705.

[0031] A detection kit for detecting NF1 gene mutation sites in neurofibromatosis type I based on TaqMan probe dual-tube multiplex real-time quantitative PCR technology, comprising reaction system A and reaction system B;

[0032] The reaction system A includes the detection primers and detection probes shown below:

[0033] Detection primers:

[0034] Targeting the c.2100_2105delinsAG site of the NF1 gene:

[0035] Forward primer A: 5'-TGGTAGCATTATGTTGGTCCAGAT-3';

[0036] Reverse primer A: 5'-AGCTCCACCCAAAAGGAGTAG-3';

[0037] Targeting the c.4561dup site of the NF1 gene:

[0038] Forward primer B: 5'-GCTGTTTACAAATCAGCTGACAG-3';

[0039] Reverse primer B: 5'-ATCATATCTAACAAGTGGCCTGGT-3';

[0040] Targeting the c.6797dup site of the NF1 gene:

[0041] Forward primer D: 5'-GCTAGCTACCAAGATCACCATAGC-3';

[0042] Reverse primer D: 5'-CACCCCAGAAAGTAAGCTCCATG-3';

[0043] Detection probe:

[0044] Targeting the c.2100_2105delinsAG site of the NF1 gene:

[0045] NF1 gene wild-type probe Aw: 5'-CCCTGACACTGAAGCTGTTCTGGT-3';

[0046] NF1 gene mutant probe Am: 5'-CCCTGACACAGTGTTCTGGT-3';

[0047] Targeting the c.4561dup site of the NF1 gene:

[0048] NF1 gene wild-type probe Bw: 5'-GATGGCAACACTTCTTGCAT-3';

[0049] NF1 gene mutant probe Bm: 5'-GATGGCAACACCTTCTTGC-3';

[0050] Targeting the c.6797dup site of the NF1 gene:

[0051] NF1 gene wild-type probe Dw: 5'-ACTTACAACAGTCAAGTTCT-3';

[0052] NF1 gene mutant probe Dm: 5'-TTACAACAGGTCAAGTTCTG-3';

[0053] The probe has a reporter fluorescent group at its 5' end and a quencher fluorescent group at its 3' end. In reaction system A, the reporter fluorescent groups at the 5' end of each probe are different and can be distinguished from each other by a real-time PCR instrument based on their different spectral wavelengths.

[0054] The reaction system A is 20 μL, and each 20 μL of reaction system A contains:

[0055] 10 μL of 2×premix Taq™ buffer, 0.5 μL of 10 μmol / L Forward primer A, 0.5 μL of 10 μmol / L Reverse primer A, 0.5 μL of 10 μmol / L Forward primer B, 0.5 μL of 10 μmol / L Reverse primer B, 0.5 μL of 10 μmol / L Forward primer D, 0.5 μL of 10 μmol / L Reverse primer D, 0.5 μL of 10 μmol / L probe Aw, 0.5 μL of 10 μmol / L probe Am, 0.5 μL of 10 μmol / L probe Bw, 0.5 μL of 10 μmol / L probe Bm, 0.5 μL of 10 μmol / L probe Dw, 0.5 μL of 10 μmol / L probe Dm, template DNA 2 μL (≥10 ng), remove RNase and DNase from water 2 μL.

[0056] The reaction system B includes the detection primers and detection probes shown below:

[0057] Detection primers:

[0058] Targeting the c.6421del site of the NF1 gene:

[0059] Forward primer C: 5'-GACCATGTTCAGTTACCAGCAC-3';

[0060] Reverse primer C: 5'-CCACTTGCACGTTGGAATATCT-3';

[0061] Detection probe:

[0062] Targeting the c.6421del site of the NF1 gene:

[0063] NF1 gene wild-type probe Cw: 5'-TACCCAAATTTTACTTGCTG-3';

[0064] NF1 gene mutant probe Cm: 5'-TACCCAAATTTACTTGCTGT-3';

[0065] The probe has a reporter fluorescent group at its 5' end and a quencher fluorescent group at its 3' end.

[0066] The reaction system B is 20 μL, and each 20 μL of reaction system B contains:

[0067] 10 μL of 2×premix Taq™ buffer, 0.5 μL of 10 μmol / L Forward primer C, 0.5 μL of 10 μmol / L Reverse primer C, 0.5 μL of 10 μmol / L probe Cw, 0.5 μL of 10 μmol / L probe Cm, 2 μL of template DNA (≥10 ng), and 6 μL of RNase- and DNase-free water.

[0068] The reporter fluorescent group is one of FAM, SYBR, Fluorescein, SYPRO Orange, VIC, JOE, TET, HEX, TAMRA, Texas Red, Alexa Fluor 633, ResoLight, EvaGreen, LC Green, Cy3, Cy5, Yellow555, LC Red610, ROX, SYPRO Ruby, LC Red640, Snarf 1, Acid Fuchsin, Cy5.5, LCRed670, LC Red705, etc., and the quencher fluorescent group is MGB. In the same reaction system (reaction system A, reaction system B), the 5' end of the probe has different reporter fluorescent groups, and they can be distinguished by a real-time PCR instrument based on their different spectral wavelengths.

[0069] The detection kit, when using TaqMan probes for dual-tube multiplex real-time quantitative PCR amplification, has the following amplification reaction procedure:

[0070] .

[0071] The present invention also provides the application of reagents for detecting NF1 gene variant sites in the preparation of a diagnostic reagent for neurofibromatosis type I, wherein the NF1 gene variant sites include at least one of the following sites: NF1 gene c.2100_2105delinsAG site, NF1 gene c.4561dup site, NF1 gene c.6421del site, and NF1 gene c.6797dup site;

[0072] The wild-type NF1 gene has the gene number NM_000267.3 in the NCBI database;

[0073] Among them, the c.2100_2105delinsAG site of the NF1 gene is a heterozygous mutation. The NF1 gene has a deletion of bases from position 2100 to 2105 and an insertion of AG. The mutation causes a frameshift mutation of the amino acid encoding the protein from position 701 (glutamic acid is mutated into valine). Translation terminates after 44 amino acids of frameshift mutation.

[0074] The c.4561dup site of the NF1 gene is a heterozygous variant. The NF1 gene has a base duplication at position 4561. The variant causes a frameshift mutation (leucine to proline) in the amino acid encoding the protein from position 1521. Translation terminates after the frameshift mutation of 21 amino acids.

[0075] The c.6421del site of the NF1 gene is a heterozygous variant, which is a deletion of the 6421st base in the NF1 gene. The variant causes a frameshift mutation (tyrosine to threonine) of the amino acid encoding the protein from the 2141st position. Translation terminates after the frameshift mutation of 37 amino acids.

[0076] The c.6797dup site of the NF1 gene is a heterozygous variant. The NF1 gene has a base duplication at position 6797. The variant causes a frameshift mutation (serine to arginine) in the amino acid encoding the protein from position 2266. Translation terminates after the frameshift mutation of 19 amino acids.

[0077] The NF1 gene c.2100_2105delinsAG site, NF1 gene c.4561dup site, NF1 gene c.6421del site, and NF1 gene c.6797dup site are NF1 gene variant sites discovered in this invention using high-throughput sequencing technology after excluding other neurofibromatosis pathogenic genes. These variants are not yet included in the gnomAD database and are the first pathogenic variant sites discovered.

[0078] The reagents for detecting NF1 gene variant sites are one or more of the probes and primers used to detect NF1 gene variant sites.

[0079] The primers include primers targeting at least one variant site among the following: the NF1 gene c.2100_2105delinsAG site, the NF1 gene c.4561dup site, the NF1 gene c.6421del site, and the NF1 gene c.6797dup site. The primers targeting these sites are as follows:

[0080] Targeting the c.2100_2105delinsAG site of the NF1 gene:

[0081] Forward primer A: 5'-TGGTAGCATTATGTTGGTCCAGAT-3';

[0082] Reverse primer A: 5'-AGCTCCACCCAAAAGGAGTAG-3'

[0083] Targeting the c.4561dup site of the NF1 gene:

[0084] Forward primer B: 5'-GCTGTTTACAAATCAGCTGACAG-3';

[0085] Reverse primer B: 5'-ATCATATCTAACAAGTGGCCTGGT-3'

[0086] Targeting the c.6421del site of the NF1 gene:

[0087] Forward primer C: 5'-GACCATGTTCAGTTACCAGCAC-3';

[0088] Reverse primer C: 5'-CCACTTGCACGTTGGAATATCT-3'

[0089] Targeting the c.6797dup site of the NF1 gene:

[0090] Forward primer D: 5'-GCTAGCTACCAAGATCACCATAGC-3';

[0091] Reverse primer D: 5'-CACCCCAGAAAGTAAGCTCCATG-3'.

[0092] The probes include probes targeting at least one variant site among the following: the NF1 gene c.2100_2105delinsAG site, the NF1 gene c.4561dup site, the NF1 gene c.6421del site, and the NF1 gene c.6797dup site. The probes targeting these sites are as follows:

[0093] Targeting the c.2100_2105delinsAG site of the NF1 gene:

[0094] NF1 gene wild-type probe Aw: 5'-CCCTGACACTGAAGCTGTTCTGGT-3';

[0095] NF1 gene mutant probe Am: 5'-CCCTGACACAGTGTTCTGGT-3',

[0096] Targeting the c.4561dup site of the NF1 gene:

[0097] NF1 gene wild-type probe Bw: 5'-GATGGCAACACTTCTTGCAT-3';

[0098] NF1 gene mutant probe Bm: 5'-GATGGCAACACCTTCTTGC-3'

[0099] Targeting the c.6421del site of the NF1 gene:

[0100] NF1 gene wild-type probe Cw: 5'-TACCCAAATTTTACTTGCTG-3';

[0101] NF1 gene mutant probe Cm: 5'-TACCCAAATTTACTTGCTGT-3';

[0102] Targeting the c.6797dup site of the NF1 gene:

[0103] NF1 gene wild-type probe Dw: 5'-ACTTACAACAGTCAAGTTCT-3';

[0104] NF1 gene mutant probe Dm: 5'-TTACAACAGGTCAAGTTCTG-3'.

[0105] Compared to existing technologies, the advantages of this invention are as follows: This invention utilizes high-throughput sequencing technology to discover, for the first time, four NF1 gene mutation sites after excluding other neurofibromatosis-causing genes: the NF1 gene c.2100_2105delinsAG site, c.4561dup site, c.6421del site, and c.6797dup site. These mutation sites are novel pathogenic variants associated with neurofibromatosis type I. The results are accurate, based on population frequency, family information, computer bioinformatics analysis, genetic patterns, and clinical phenotypes, expanding the spectrum of NF1 gene pathogenic mutations and providing a basis for the diagnosis and genetic counseling of neurofibromatosis type I. Furthermore, this invention provides primer and probe compositions and kits for detecting NF1 gene mutation sites in neurofibromatosis type I for screening or diagnosis of neurofibromatosis type I; including specific primers and TaqMan probe compositions targeting the NF1 gene c.2100_2105delinsAG, c.4561dup, c.6421del, and c.6797dup mutation sites respectively. The present invention provides a detection kit and method for detecting NF1 gene variants in neurofibromatosis type I using a dual-tube multiplex real-time quantitative PCR technique based on TaqMan probes. This kit comprehensively covers four novel pathogenic variants of the NF1 gene associated with neurofibromatosis type I. The multiplex TaqMan real-time quantitative PCR technique is time-efficient and simple to operate. The subject's DNA sample is added to one of the two reaction systems (reaction system A and reaction system B) of the present invention for detection. No post-processing such as gel electrophoresis, purification, or sequencing is required. The results are accurate and reliable, meeting the current clinical need for a simple, rapid, and accurate diagnostic method for patient and prenatal samples. Furthermore, the primers and probes designed in this invention have high specificity, specifically detecting the four newly discovered pathogenic variants, enabling the diagnosis of neurofibromatosis type I and providing a basis for guiding clinical diagnosis and genetic counseling. Attached Figure Description

[0106] Figure 1 It is a candidate sequence variant detected by proband 1 through high-throughput sequencing in Example 2.

[0107] Figure 2 This is a BAM file image of the high-throughput sequencing results of the c.2100_2105delinsAG variant of the NF1 gene in proband 1 in Example 2.

[0108] Figure 3 It is a candidate sequence variant detected by high-throughput sequencing in the proband 2 in Example 3.

[0109] Figure 4 This is a BAM file image of the high-throughput sequencing results of the c.4561dup variant of the NF1 gene in proband 2 in Example 3.

[0110] Figure 5 It is a candidate sequence variant detected by high-throughput sequencing in the proband 3 in Example 4.

[0111] Figure 6 This is a BAM file image of the high-throughput sequencing results of the c.6421del variant of the NF1 gene in proband 3 in Example 4.

[0112] Figure 7 It is a candidate sequence variant detected by high-throughput sequencing in the proband 4 in Example 5.

[0113] Figure 8 This is a BAM file image of the high-throughput sequencing results of the c.6797dup variant of the NF1 gene in proband 4 in Example 5.

[0114] Figure 9 The results show the functional study of the effects of the four variants on NF1 gene mRNA expression in Example 6. A is the result of qRT-PCR experiment, and B is the gel electrophoresis analysis of qRT-PCR products.

[0115] Figure 10 This is the analysis result of the interaction between probe C and probes A, B, and D using Primerselect software in Example 7.

[0116] Figure 11 This is a real-time quantitative PCR result of the TaqMan probe at the c.2100_2105delinsAG site of the NF1 gene in Example 7; the right figure is the amplification curve result of the heterozygote at the c.2100_2105delinsAG site of the NF1 gene; the left figure is the amplification curve result of the wild type c.2100_2105.

[0117] Figure 12 The images show the real-time quantitative PCR results of the TaqMan probe at the c.4561dup site of the NF1 gene in Example 7. The right image shows the amplification curve of the heterozygote at the c.4561dup site of the NF1 gene, while the left image shows the amplification curve of the wild-type c.4561.

[0118] Figure 13 The image shows the real-time quantitative PCR results of the TaqMan probe at the c.6421del site of the NF1 gene in Example 7. The right image shows the amplification curve of the heterozygote at the c.6421del site of the NF1 gene, while the left image shows the amplification curve of the wild-type c.6421.

[0119] Figure 14The image shows the real-time quantitative PCR results of the TaqMan probe at the c.6797dup site of the NF1 gene in Example 7. The right image shows the amplification curve of the heterozygote at the c.6797dup site of the NF1 gene, while the left image shows the amplification curve of the wild-type at the c.6797 site.

[0120] Figure 15 This is a Sanger sequencing result of NF1: NM_000267.3:c.2100_2105delinsAG in Example 8; the red boxes represent the detection sites.

[0121] Figure 16 This is a Sanger sequencing result of NF1: NM_000267.3:c.4561dup in Example 8; the red boxes represent the detection sites.

[0122] Figure 17 This is a Sanger sequencing result image of NF1: NM_000267.3:c.6421del in Example 8, where the red box represents the detection site.

[0123] Figure 18 This is a Sanger sequencing result image of NF1: NM_000267.3:c.6797dup in Example 8, where the red box represents the detection site. Detailed Implementation

[0124] The present invention will now be described in detail with reference to the accompanying drawings and embodiments:

[0125] Example 1: Detection of pathogenic variants in patients with neurofibromatosis type I using the Customized Clinical Exome Capture Kit (TWIST).

[0126] This study included 536 peripheral blood samples from patients clinically diagnosed with or suspected of having neurofibromatosis type I and submitted to Furui Medical Laboratory. The samples collected included clinical symptoms and signs (skin, nervous system, growth and development history, intelligence, skeletal system, hearing, vision), laboratory tests, and imaging examinations. High-throughput sequencing using a customized clinical exome capture kit (TWIST) was performed on the probands and some family members of candidate neurofibromatosis type I patients. The sequencing process included the following steps:

[0127] (1) Sample collection and extraction of genomic DNA.

[0128] Collect blood samples (EDTA anticoagulated) from the proband and / or family members. Extract genomic DNA from each family member's blood according to the instructions of the blood DNA extraction kit (Magen, HiPure Blood & Tissue DNA Kit). Measure DNA purity using Nanodrop One; the OD260nm / OD280nm of the obtained genomic DNA should be between 1.7 and 2.0. Measure DNA concentration using Nanodrop One; the concentration of the obtained genomic DNA should be 50-100 ng / μL, with a total volume of 5-10 μg. Store at -20°C.

[0129] (2) High-throughput sequencing and bioinformatics analysis of neurofibromatosis type I gene mutations.

[0130] First, the extracted genomic DNA was fragmented using the KAPA HyperPlus Library Preparation Kit. The fragmented DNA underwent enzyme digestion, end repair, 3' end A addition, adapter linking, and PCR amplification. A custom-designed Clinical Exome Capture Kit (TWIST) was used to capture the exon regions and flanking ±20 bp intron regions of Mendelian disease-causing genes indexed in the OMIM database, as well as other regions containing pathogenic variants of HMGD and ClinVar. The library was sequenced on a Novaseq sequencer (Illumina, San Diego, CA, USA) (sequencing depth ≥150X, 20X coverage ≥96%). Before data interpretation, all sequenced samples underwent quality control analysis to ensure 100% 20X coverage of the major neurofibromatosis-causing genes NF1, NF2, and SPRED1. If 100% coverage was not achieved, Sanger sequencing was used to supplement the missing 20X regions to ensure the detection rate of pathogenic variants. According to statistics from the internationally authoritative database Genereviews, the positive rate of NF1, NF2, and SPRED1 is 93.7% in all patients with the solitary neurofibromatosis phenotype, and the positive rate is greater than 97% in neurofibromatosis patients who meet the NIH diagnostic criteria.

[0131] Secondary and tertiary analyses of the sequencing data were performed using the ISoGenetic v1.2.6 system (Shanghai Fujun Gene Biotechnology Co., Ltd.). Specifically, Sentieon BWA v0.7.15 was used to align the NGS sequencing results with the human reference genome UCSCNCBI37 / hg19 to obtain unique aligned sequences on the genome; SAMtools v1.9 and Sentieon GATK software v4.1.4.0 were used to detect and identify variants in the target regions; Remove Run Common Variants and Remove Global Common Variants software were used to remove common variants (>5%) from the dbSNP and gnomAD databases; and Alamut-Batch standalone version v1.9 was used. The software annotates variants (annotation databases include: dbSNP, gnomAD, 1000g, ClinVar, OMIM, HGMD Professional Edition, etc.); using filterAlamut.py, the annotated variants are sorted according to high, medium, and low priorities. Within the high and medium groups, variants are assigned a priority value and a reason for classification. All variants initially fall into the low group; when a variant meets the preset pathogenicity criteria, it can be classified into a higher-level variant. The software provided by the VarSome database, including FATHMM, FATHMMMKL, METALR, METASVM, MUTATIONASSESSOR, MUTATIONTASTERAGVGD, AGVGD, LRT, PROVEAN, SIFT, REVEL, and SpliceAI, is used for bioinformatics prediction of SNP function and splicing activity; CNV... TM DNA copy number variation (CNV) analysis was performed using the exon tool (resolution ≥1 exon).

[0132] (3) Interpretation of candidate gene variations

[0133] First, candidate variants in the NF1, NF2, and SPRED1 genes are analyzed. If a positive pathogenic / probable pathogenic variant (P / LP) is found, a positive test result will be reported. If no pathogenic variant is found among the candidate variants, candidate reportable variants will be searched among those in the clinical exome scope. Sequence variant interpretation follows the internationally authoritative 2015 ACMG Guidelines for Clinical Interpretation of Genetic Variations (ACMG Guidelines) and various guidelines from the ClinGen Sequence Variation Interpretation (SVI) Working Group (SVI). CNV variant interpretation follows the 2019 ACMG Standards for Interpretation and Reporting of Chromosomal Copy Number Variations. The various variants detected by the sequencing are classified as pathogenic variants (P), probable pathogenic variants (LP), variants of unknown significance (VUS), possibly benign variants (LB), and benign variants (B). Clinical reports include pathogenic variants (P), probable pathogenic variants (LP), and variants of unknown significance (VUS) that can explain the subject's phenotype and family history.

[0134] Example 2: A novel pathogenic variant of the NF1 gene, NM_000267.3:c.2100_2105delinsAG (p.Glu701Valfs*46), was discovered in a case of neurofibromatosis type I.

[0135] The proband, a male aged 4 years and 5 months, was clinically diagnosed with neurofibromatosis. He had been exhibiting growth retardation for approximately 2 years and weighed 15.5 kg. His general condition was typically around 100 cm tall. He had Tanner stage I external genitalia, 1-2 ml of testes, and numerous café-au-lait spots on skin examination. He was born full-term via cesarean section, weighing approximately 3.55 kg and measuring about 50 cm in length. He showed no delays in walking or speaking, but was prone to colds. He had a history of ventricular septal defect surgery. His birth length was 74.8 cm at 1 year, 82 cm at 2 years, and 91 cm at 3 years. His father is 168 cm tall, and his mother is 158 cm tall. Both his mother and maternal grandfather also have multiple café-au-lait spots.

[0136] In this embodiment, high-throughput sequencing analysis of genomic variant information of samples was performed using a customized clinical exome capture kit (TWIST). Firstly, among the neurofibromatosis candidate genes NF1, NF2, and SPRED1, it was found that the proband carried two heterozygous sequence variants (population frequency ≤5%) and 0 candidate CNV variants. Figure 1The sequence variations were: the heterozygous NF1 gene variant NM_000267.3:c.2100_2105delinsAG (p.Glu701Valfs*46) and NM_000267.3:c.*871del. First, according to the ACMG guidelines, the NF1 gene variant NM_000267.3:c.*871del was classified as a benign variant and was initially excluded. The relevant classification criteria were: the NF1 gene variant NM_000267.3:c.*871del is located in the UTR region, and the relevant evidence for this variant is BS1 (frequency 4.1% in African populations, higher than the incidence rate) and BS2 (6 homozygous variants found in African populations). According to the ACMG guidelines, this variant was classified as a benign variant.

[0137] The NF1 gene heterozygous variant NM_000267.3:c.2100_2105delinsAG (p.Glu701Valfs*46), derived from adjacent cis-arranged NM_000267.3:c.2105del and NM_000267.3:c.2100_2102del, was obtained by HGVS merging and naming. Its original NGS BAM file is as follows: Figure 2 As shown, the mutation involves a deletion of bases 2100 to 2105 in the coding region, followed by the insertion of AG. This mutation results in a frameshift mutation at position 701 (glutamate to valine), and translation terminates after 44 amino acids of frameshift mutation. According to the ACMG guidelines, this mutation is classified as a pathogenic variant. The relevant evidence for this variant is PVS1+PM2+PP1+PP4, specifically:

[0138] PVS1: This frameshift variant is located in exon 18 (out of 57 exons, encoding 2818 amino acids) of the NF1 gene transcript NM_000267.3, resulting in an out-of-frame transcript and an early stop codon. This variant may cause protein truncation or activate nonsense-mediated mRNA degradation, leading to loss of function of the gene's protein products. Downstream truncation variants are known to be pathogenic (PubMed: 25525159, 30308447, 25403449, 9475595).

[0139] PP1: This variant was inherited from the mother and maternal grandfather who were both affected by the disease, confirming that the variant is a co-segregation of the disease and the family lineage.

[0140] PM2: The variant is not currently included in the gnomAD database.

[0141] PP4: The subject’s phenotype and family history meet the NIH diagnostic criteria for NF1.

[0142] Therefore, the NF1 gene mutant c.2100_2105delinsAG (p.Glu701Valfs*46) was discovered using high-throughput sequencing technology after excluding other neurofibromatosis-causing genes. It is a novel pathogenic variant associated with neurofibromatosis type I, and this variant can explain the inherited neurofibromatosis type I phenotype in the progenitor's family, providing a diagnostic result for the progenitor. Based on population frequency, family information, computational bioinformatics analysis, and clinical phenotype, the result is accurate, expanding the spectrum of pathogenic variants of the NF1 gene and providing a basis for the diagnosis and genetic counseling of neurofibromatosis type I.

[0143] Example 3: A novel pathogenic variant of the NF1 gene, NM_000267.3:c.4561dup (p.Leu1521Profs*22), was discovered in a case of neurofibromatosis type I.

[0144] The proband 2 was a 1-year-old male clinically diagnosed with neurofibromatosis. He had been diagnosed with café-au-lait spots all over his body for more than a year. After birth, he was found to have scattered café-au-lait spots of varying sizes all over his body, which gradually increased in number (N>6). He denied any family history of neurofibromatosis.

[0145] In this embodiment, high-throughput sequencing analysis of genomic variant information of samples was performed using a customized clinical exome capture kit (TWIST). Firstly, among the neurofibromatosis candidate genes NF1, NF2, and SPRED1, it was found that the proband carried two heterozygous sequence variants (population frequency ≤5%) and 0 candidate CNV variants. Figure 3 The sequence variations were: a heterozygous variant NM_000267.3:c.4561dup (p.Leu1521Profs*22) in the NF1 gene and a heterozygous variant NM_000268.3:c.*2780dup in the NF2 gene. First, according to the ACMG guidelines, the NF2 gene variant NM_000268.3:c.*2780dup was classified as a benign variant and was excluded. The relevant classification criteria were: the NF2 gene variant NM_000268.3:c.*2780dup is located in the UTR region, and the relevant evidence for this variant is BS1 (frequency 1.7% in African populations, higher than the incidence rate) and BS2 (two homozygous variants found in African populations). According to the ACMG guidelines, this variant was classified as a benign variant.

[0146] The heterozygous variant of the NF1 gene, NM_000267.3:c.4561dup (p.Leu1521Profs*22), has its original NGS BAM file as follows: Figure 4As shown, the mutation involves a base repetition at position 4561 of the coding region. This mutation results in a frameshift mutation (leucine to proline) at position 1521 of the encoded protein. Translation terminates after this 21-amino-acid frameshift. According to the ACMG guidelines, this mutation is classified as a pathogenic variant. The relevant evidence for this variant is PVS1+PM2+PS2, specifically:

[0147] PVS1: This frameshift variant is located in exon 34 (out of 57) of transcript NM_000267.3, resulting in a premature stop codon that may activate nonsense-mediated mRNA degradation, thereby affecting the function of the protein product encoded by this gene. Several truncation variants of this gene have been reported as pathogenic variants (PubMed: 24789688, 28961165, 23656349, 22106164).

[0148] PS2: Not detected in the peripheral blood of the subject's parents, suggesting a possible new variant.

[0149] PM2: The variant is not currently included in the gnomAD database.

[0150] Therefore, the NF1 gene mutant NM_000267.3:c.4561dup (p.Leu1521Profs*22) was discovered using high-throughput sequencing technology after excluding other neurofibromatosis-causing genes. This novel pathogenic variant associated with neurofibromatosis type I was found, and it explains the sporadic neurofibromatosis type I phenotype in the progenitor. This was a diagnostic result for the progenitor, and according to the neurofibromatosis type I treatment guidelines, the patient was ultimately diagnosed with neurofibromatosis type I from a suspected case. Based on population frequency, family information, computational bioinformatics analysis, and clinical phenotype, the results were accurate, expanding the spectrum of pathogenic variants in the NF1 gene and providing a basis for the diagnosis and genetic counseling of neurofibromatosis type I.

[0151] Example 4: A novel pathogenic variant of the NF1 gene, NM_000267.3:c.6421del (p.Tyr2141Thrfs*38), was discovered in a case of neurofibromatosis type I.

[0152] The proband 3 was a 2-month-old female with a suspected clinical diagnosis of neurofibromatosis. She was found to have brown spots all over her body 1.5 months after birth, which gradually increased in number (N>6). She denied any family history of neurofibromatosis.

[0153] In this embodiment, high-throughput sequencing analysis of the sample genome variation information was performed using a customized clinical exome capture kit (TWIST). Firstly, it was found that the proband carried three heterozygous sequence variations (population frequency ≤5%) and zero candidate CNV variations in the neurofibromatosis candidate genes NF1, NF2, and SPRED1. Figure 5 The sequence variations were: NF1 gene heterozygous variant NM_000267.3:c.6421del (p.Tyr2141Thrfs*38), SPRED1 gene heterozygous variant NM_152594.2:c.*3976C>T, and NF2 gene heterozygous variant NM_000268.3:c.*2780dup. First, according to the ACMG guidelines, the NF2 gene variant NM_000268.3:c.*2780dup and the SPRED1 gene heterozygous variant NM_152594.2:c.*3976C>T were excluded. The relevant classification criteria are as follows: The NF2 gene variant NM_000268.3:c.*2780dup is located in the UTR region. The relevant evidence for this variant is BS1 (frequency of 1.7% in African populations, higher than the incidence rate) and BS2 (two homozygous variants found in African populations). According to the ACMG guidelines, this variant is classified as a benign variant. The SPRED1 gene heterozygous variant NM_152594.2:c.*3976C>T is located in the UTR region with a priority of 9 (the lowest level). The relevant evidence for this variant is BP4 (predicted as benign by bioinformatics tools such as CADD) + BP7 (synonymous or non-coding region variants are predicted by bioinformatics tools not to affect splicing) and BS4 (inherited from a non-affected father). According to the ACMG guidelines, this variant is classified as a possibly benign variant.

[0154] The heterozygous variant of the NF1 gene, NM_000267.3:c.6421del (p.Tyr2141Thrfs*38), has the following original BAM file from NGS: Figure 6 As shown, the deletion occurs at position 6421 of the coding region. This mutation results in a frameshift mutation (tyrosine to threonine) at position 2141 of the encoded protein. Translation terminates after 37 amino acids of frameshift mutation. According to the ACMG guidelines, this mutation is classified as a pathogenic mutation. The relevant evidence for this mutation is: PVS1+PS2+PM2.

[0155] PVS1: This frameshift variant is located in exon 42 (out of 57) of transcript NM_000267.3, resulting in a premature stop codon that may activate nonsense-mediated mRNA degradation, thereby affecting the function of the protein product encoded by this gene. Several truncation variants of this gene have been reported as pathogenic variants (PubMed: 18643859, 29914388, 30014477, 30308447).

[0156] PS2: Not detected in the peripheral blood of the subject's parents, suggesting a possible new variant.

[0157] PM2: The variant is not currently included in the gnomAD database.

[0158] Therefore, the NF1 gene mutant NM_000267.3:c.6421del (p.Tyr2141Thrfs*38) was discovered using high-throughput sequencing technology after excluding other neurofibromatosis-causing genes. This novel pathogenic variant associated with neurofibromatosis type I was found, and it explains the sporadic neurofibromatosis type I phenotype in the progenitor. This serves as a diagnostic result for the progenitor, and according to the neurofibromatosis type I treatment guidelines, the patient was ultimately diagnosed with neurofibromatosis type I from a suspected case. Based on population frequency, family information, computational bioinformatics analysis, and clinical phenotype, the results are accurate, expanding the spectrum of pathogenic variants in the NF1 gene and providing a basis for the diagnosis and genetic counseling of neurofibromatosis type I.

[0159] Example 5: A novel pathogenic variant of the NF1 gene, NM_000267.3:c.6797dup (p.Ser2266Argfs*20), was discovered in a case of neurofibromatosis type I.

[0160] The proband 4 was a 7-year-old female clinically diagnosed with neurofibromatosis. She had been diagnosed with café-au-lait spots on her skin for 7 years, which had been present since birth and gradually increased in number (N>6). A cranial MRI showed high signal intensity in the cerebellum and basal ganglia (neurofibroma). She denied any family history of neurofibromatosis.

[0161] In this embodiment, high-throughput sequencing analysis of the sample genome variation information was performed using a customized clinical exome capture kit (TWIST). Firstly, among the neurofibromatosis candidate genes NF1, NF2, and SPRED1, it was found that the proband carried one heterozygous sequence variant (population frequency ≤5%) and zero candidate CNV variants. Figure 7 The sequence variation is a heterozygous variant of the NF1 gene: NM_000267.3:c.6797dup (p.Ser2266Argfs*20), and its original BAM file from NGS is as follows. Figure 8 As shown, the base repeat at position 6797 in the coding region causes a frameshift mutation (serine to arginine) in the encoded protein, starting from position 2266. Translation terminates after 19 amino acids of frameshift mutation. According to the ACMG guidelines, this mutation is classified as a pathogenic mutation. The relevant evidence for this mutation is: PVS1+PS2+PM2+PP4.

[0162] PVS1: The frameshift variant is located at exon 45 (out of 57) of the NF1 gene transcript NM_000267.3, resulting in a premature stop codon that may activate nonsense-mediated mRNA degradation, thereby affecting the function of the protein product encoded by this gene. Downstream truncation variants of this variant have been reported as pathogenic (PubMed: 25525159, 30308447, 25403449, 9475595).

[0163] PS2: Not detected in the peripheral blood of the subject's parents, suggesting a possible new variant.

[0164] PM2: The variant is not currently included in the gnomAD database.

[0165] PP4: The subject’s phenotype and family history meet the NIH diagnostic criteria for NF1.

[0166] Therefore, the NF1 gene mutant NM_000267.3:c.6797dup (p.Ser2266Argfs*20) was discovered using high-throughput sequencing technology after excluding other neurofibromatosis-causing genes. It is a novel pathogenic variant associated with neurofibromatosis type I, and this variant can explain the sporadic neurofibromatosis type I phenotype in progenitors, providing a diagnostic result for progenitors. Based on population frequency, family information, computational bioinformatics analysis, and clinical phenotype, the result is accurate, expanding the spectrum of pathogenic variants in the NF1 gene and providing a basis for the diagnosis and genetic counseling of neurofibromatosis type I.

[0167] Example 6: qRT-PCR experiment to analyze the effect of variations on NF1 gene function / expression

[0168] To verify the effects of the four NF1 gene variants described in this invention on the function / expression of the gene product, this embodiment used peripheral blood cells from probands as clinical analysis samples. The relative expression levels of NF1 gene mRNA in peripheral blood samples from individuals carrying different variants were detected using qRT-PCR to determine the impact of the variants on NF1 gene function / expression. Peripheral blood samples (collected in PAX gene tubes) from four probands carrying the c.2100_2105delinsAG, c.4561dup, c.6421del, and c.6797dup variants of the NF1 gene, respectively, were selected as the experimental group. Peripheral blood samples (collected in PAX gene tubes) from six healthy individuals collected concurrently served as the control group (n=6).

[0169] Peripheral blood total RNA was extracted using the PAXgene Blood RNA Extraction Kit (Thermo Fisher Scientific). RNA concentration and purity were measured (A260 / 280 ratio 1.8–2.1). Residual genomic DNA was removed by DNase I treatment. cDNA was synthesized using 1 µg of total RNA as a template with the Superscript® IV Reverse Transcriptase Kit (Thermo Fisher Scientific). qPCR was performed using the SYBR Green Master Mix system (Thermo Fisher Scientific), specifically comprising a 20 µL reaction volume: 10 µL 2×Master Mix, 1 µL each of 0.4 µL 10 µM forward and reverse primers, 2 µL cDNA template, and deionized water to make up the volume. The internal control gene was GAPDH (NM_002046). The qPCR reaction conditions were as follows: incubation at 50°C for 2 minutes, followed by pre-denaturation at 95°C for 10 minutes; then cyclic amplification, including denaturation at 95°C for 15 seconds and annealing at 60°C for 1 minute; finally, melting curve analysis was performed to confirm amplification specificity, with incubation at 95°C for 15 seconds, 60°C for 15 seconds, and 95°C for 15 seconds respectively. Each sample was analyzed in triplicate. The yield and purity of the amplified products were analyzed by gel electrophoresis.

[0170] NF1 gene primers are designed to target exon regions far from mutation sites to ensure that the detection results reflect the overall expression of the full-length transcript. Primers span exon linkers to avoid genomic DNA contamination. Primer sequences are as follows:

[0171] NF1_qF: 5′-GCCTTGAGGAAAACCAGCGGAA-3′;

[0172] NF1_qR: 5′-TCAAGCCCCTTTCGATTCTAGG-3′;

[0173] GAPDH_qF: 5′-AGGGCATCCTGGGCTACACTGAG-3′;

[0174] GAPDH_qR: 5′-ACCACCCTGTTGCTGTAGCCAA-3′.

[0175] The relative expression levels were calculated using the ΔΔCt method. First, the NF1 Ct values ​​were normalized to the internal reference (ΔCt = Ct_NF1 - Ct_GAPDH). Then, ΔΔCt was calculated using the mean ΔCt of the healthy control group as a reference (ΔΔCt = ΔCt_sample - mean ΔCt_control). The relative expression level was calculated as 2^(-ΔΔCt). Statistical analysis was performed using the two-tailed Student's t-test (P < 0.05 was considered statistically significant).

[0176] The results are as follows Figure 9 A showed that qRT-PCR experiments revealed significantly lower NF1 gene mRNA levels in individuals with the c.2100_2105delinsAG (proband 1), c.4561dup (proband 2), c.6421del (proband 3), and c.6797dup (proband 4) variants compared to the healthy control group, with mean relative expression levels downregulated by 47.6%, 45.3%, 48.6%, and 50.9%, respectively (P<0.05). The NF1 gene mRNA expression levels of the mother of proband 1, who also had neurofibromatosis, were comparable to those of proband 1; the NF1 gene mRNA expression levels of the parents of probands 2-4 (who were asymptomatic) were comparable to those of other healthy controls collected during the same period. Figure 9 B shows that gel electrophoresis analysis of qRT-PCR products revealed that the NF1 gene band was the same size (264 bp) in the patient group (P) and the control group (C), but its brightness was significantly reduced. The internal reference GAPDH gene band was the same size (163 bp) in the patient group (P) and the control group (C), with similar brightness. These results indicate that all four variants can lead to a decrease of approximately 50% in NF1 transcript expression, suggesting that the variants can trigger nonsense-mediated mRNA degradation or affect transcriptional stability, thereby causing insufficient NF1 gene product dosage.

[0177] According to the ClinGen database and literature reports, the pathogenic mechanism of NF1 in neurofibromatosis is haplo-insufficiency (HI), meaning that the loss of function in one of the two alleles of the NF1 gene is pathogenic. Therefore, the qRT-PCR results obtained in this embodiment directly prove that the NF1 gene variants c.2100_2105delinsAG, c.4561dup, c.6421del, and c.6797dup can lead to loss of NF1 gene function by significantly reducing its mRNA level. This is consistent with the genetic laws and pathogenic mechanisms of NF1 gene pathogenesis and has a direct causal relationship with the occurrence of neurofibromatosis.

[0178] Example 7: Primer and probe compositions and kits for detecting NF1 gene variants in neurofibromatosis type I.

[0179] (1) The primer and probe composition and kit for detecting NF1 gene mutation sites in neurofibromatosis type I are used for screening or diagnosis of neurofibromatosis type I; including specific primers and TaqMan probe compositions targeting the c.2100_2105delinsAG, c.4561dup, c.6421del, and c.6797dup mutation sites of the NF1 gene respectively.

[0180] The specific primer and probe composition was designed using Primer 5.0 software, specifically:

[0181] 1) Targeting the c.2100_2105delinsAG site of the NF1 gene:

[0182] Forward primer A: 5'-TGGTAGCATTATGTTGGTCCAGAT-3';

[0183] Reverse primer A: 5'-AGCTCCACCCAAAAGGAGTAG-3'

[0184] NF1 gene wild-type probe Aw: 5'-CCCTGACACTGAAGCTGTTCTGGT-3';

[0185] NF1 gene mutant probe Am: 5'-CCCTGACACAGTGTTCTGGT-3',

[0186] 2) Targeting the c.4561dup site of the NF1 gene:

[0187] Forward primer B: 5'-GCTGTTTACAAATCAGCTGACAG-3';

[0188] Reverse primer B: 5'-ATCATATCTAACAAGTGGCCTGGT-3'

[0189] NF1 gene wild-type probe Bw: 5'-GATGGCAACACTTCTTGCAT-3';

[0190] NF1 gene mutant probe Bm: 5'-GATGGCAACACCTTCTTGC-3'

[0191] 3) Targeting the c.6421del site of the NF1 gene:

[0192] Forward primer C: 5'-GACCATGTTCAGTTACCAGCAC-3';

[0193] Reverse primer C: 5'-CCACTTGCACGTTGGAATATCT-3'

[0194] NF1 gene wild-type probe Cw: 5'-TACCCAAATTTTACTTGCTG-3';

[0195] NF1 gene mutant probe Cm: 5'-TACCCAAATTTACTTGCTGT-3';

[0196] 4) Targeting the c.6797dup site of the NF1 gene:

[0197] Forward primer D: 5'-GCTAGCTACCAAGATCACCATAGC-3';

[0198] Reverse primer D: 5'-CACCCCAGAAAGTAAGCTCCATG-3'

[0199] NF1 gene wild-type probe Dw: 5'-ACTTACAACAGTCAAGTTCT-3';

[0200] NF1 gene mutant probe Dm: 5'-TTACAACAGGTCAAGTTCTG-3'

[0201] The probe has different reporter fluorescent groups at its 5' end and an MGB quencher fluorescent group at its 3' end.

[0202] In the same reaction system, the 5' ends of the probes are each equipped with different reporter fluorescent groups. For example, in reaction system A, the 5' ends of the six probes detecting three sites are modified with different identifiable fluorescent dyes, including but not limited to one of FAM, SYBR, Fluorescein, SYPRO Orange, VIC, JOE, TET, HEX, TAMRA, Texas Red, Alexa Fluor633, ResoLight, EvaGreen, LC Green, Cy3, Cy5, Yellow555, LC Red610, ROX, SYPRO Ruby, LCRed640, Snarf 1, Acid Fuchsin, Cy5.5, LC Red670, and LC Red705.

[0203] (2) Analysis of probe interactions and optimization of combinations using bioinformatics software: Using Primerselect software in DNAstar 7.1, open the multiplex probe file saved in the same folder, then select each of the designed multiplex probes in pairs, and under the "report" menu, select "primer pair dimers" to analyze the dimer structures between the probes. The pop-up window shows the formation of dimers between the probe pairs and evaluates the probe pair combination using dG values ​​(usually giving the worst dG value; theoretically, the higher the dG value, the better). Combinations that form dimers are indicated as "bad," meaning the result is "poor," otherwise it is "no effect," and dimer structures may form. The analysis results are as follows: Figure 10 As shown, there is potential mutual interference between the probes.

[0204] (3) Construction of the multiplex reaction system: Since bioinformatics analysis indicated the possibility of dimer formation between the probes of this invention, the multiplex reaction system was determined through mutual interference experiments. Quantitative reaction systems for single sites and two or more sites were constructed according to the following reaction system: 10 μL of 2×premix Taq™ buffer, 0.5 μL of forward primer (10 μmol), 0.5 μL of reverse primer (10 μmol), 2 μL of DNA, 0.5 μL of wild-type (w) TaqMan probe (10 μmol), 0.5 μL of wild-type (m) TaqMan probe (10 μmol), and ddH2O was added to bring the total to 20 μL. The Ct values ​​of each reaction system were recorded. If there was no statistically significant difference between the Ct values ​​of the two or more site reaction systems and the single site reaction systems (p > 0.05), then the probes in the multiplex system did not interfere with each other; conversely, if there was a statistically significant difference (p ≤ 0.05), then the probes in the multiplex system interfered with each other. The specific results are shown in the table below: Probe C interacts with probes A, B, and D, while probes A, B, and D do not interfere with each other. Therefore, reaction system A includes probes A, B, and D; reaction system B includes probe C.

[0205]

[0206] (4) The reagent components of the kit may be general PCR amplification reaction reagents, including buffer, ions, dNTPs, PCR polymerase, water or other PCR additives, including but not limited to 2×premix Taq™ buffer (LA Taq™ Version 2.0) and RNase- and DNase-free water used in this embodiment.

[0207] (5) The kit uses a dual-tube multiplex fluorescent PCR reaction system, as detailed below:

[0208] Reaction system A: NF1 gene c.2100_2105delinsAG site, c.4561dup site, and c.6797dup site

[0209]

[0210] Reaction system B: NF1 gene c.6421del site

[0211]

[0212] (4) The amplification reaction procedure is as follows:

[0213]

[0214] (5) Procedure for detecting the c.2100_2105delinsAG, c.4561dup, c.6421del, and c.6797dup variant sites of the neurofibromatosis type I NF1 gene using multiplex probe reagents:

[0215] As described in Example 1, clinical tissue samples were collected from the proband of neurofibromatosis or their family members, including but not limited to peripheral blood anticoagulated with EDTA / sodium citrate.

[0216] As described in Example 1, genomic DNA was extracted from clinical tissue samples and quality control was performed.

[0217] Using genomic DNA samples, a multiplex fluorescent PCR reaction system was configured, and the amplification reaction was carried out on a real-time PCR instrument according to the above reaction procedure;

[0218] Result Interpretation: After the quantitative real-time PCR is completed, data analysis and processing are performed using software to obtain the results for each subject. This is done by determining whether there are amplification peaks of wild-type and mutant specific probes at each detection site for each subject. Figure 11-14 According to the standards in the table below, determine whether the subject carries the pathogenic locus and genotype of the NF1 gene.

[0219]

[0220] Example 8: Performance study results of the kit for detecting NF1 gene variant sites in neurofibromatosis type I.

[0221] (1) Performance analysis scheme

[0222] To investigate the analytical performance of the dual-tube multiplex fluorescent TaqMan probe PCR detection kit for neurofibromatosis type I NF1 gene variant sites in this invention, 544 clinical samples were used as the research subjects in this example, including: 536 clinical samples that underwent NGS testing in Example 1, and 8 clinical samples (EDTA-anticoagulated peripheral blood) from family members of probands 1-4. All positive samples were compared with NGS analysis results and Sanger sequencing verification results. Samples that did not undergo NGS analysis were all verified by Sanger sequencing. The sensitivity and specificity of the kit for detecting the c.2100_2105delinsAG, c.4561dup, c.6421del, and c.6797dup variant sites of the neurofibromatosis type I NF1 gene were calculated using the following formulas:

[0223] Sensitivity = Number of true positive results / (Number of true positive results + Number of false negative results)

[0224] Specificity = Number of true negative results / (Number of true negative results + Number of false positive results)

[0225] (2) The system and steps for the Sanger sequencing validation:

[0226] PCR amplification system (20 μl) for c.2100_2105delinsAG, c.4561dup, c.6421del, and c.6797dup variant sites. Each PCR reaction was a single-site single-tube amplification system, for a total of 4 amplification systems: 10 μL of 2×premix Taq™ buffer, 1 μL of forward primer (10 μmol), 1 μL of reverse primer (10 μmol), 6 μL of ddH2O, and 2 μL of DNA. The primers were the amplification primers corresponding to each variant site provided in Example 6 above. PCR reaction program: 95℃ for 5 min, 35 cycles (95℃ for 5 min, 95℃ for 30 s, 65℃ for 1 min), 72℃ for 10 min, and storage at 4℃. After PCR amplification, the results were detected by 1% agarose gel electrophoresis. After gel excision, the DNA was recovered using the "Common Agarose Gel DNA Recovery Kit (DP209)" and diluted to 10 ng / μL. The recovered product was then purified using Taq polymerase. All PCR products were analyzed by Sanger sequencing on the ABI 3730XL (Applied Biosystems) platform using amplification primers.

[0227] (3) Analysis of performance study results

[0228] The kit for detecting NF1 gene variants in neurofibromatosis type I of this invention has 100% sensitivity and specificity for detecting the c.2100_2105delinsAG variant, as detailed in the table below and the Sanger sequencing verification results. Figure 15 ):

[0229]

[0230] The kit for detecting NF1 gene variants in neurofibromatosis type I of this invention has 100% sensitivity and specificity for detecting the c.4561dup variant, as detailed in the table below and the Sanger sequencing verification results. Figure 16 ):

[0231]

[0232] The kit for detecting NF1 gene variants in neurofibromatosis type I of this invention has 100% sensitivity and specificity for detecting the c.6421del variant, as detailed in the table below and the Sanger sequencing verification results. Figure 17 ):

[0233]

[0234] The kit for detecting NF1 gene variants in neurofibromatosis type I of this invention has 100% sensitivity and specificity for detecting the c.6797dup variant, as detailed in the table below and the Sanger sequencing verification results. Figure 18 ):

[0235]

[0236] The embodiments of this example have been described above. However, this example is not limited to the specific implementation methods described above. The specific implementation methods described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms based on the guidance of this example, and all of them are within the protection scope of this example.

Claims

1. A detection kit for detecting NF1 gene variants in neurofibromatosis type I using a dual-tube multiplex real-time quantitative PCR technique based on TaqMan probes, characterized in that: It comprises reaction system A and reaction system B; The reaction system A comprises detection primers and detection probes as shown below: Detection primers: For the site of c.2100_2105delinsAG of the NF1 gene: Forward primer A: 5'-TGGTAGCATTATGTTGGTCCAGAT-3'; Reverse primer A: 5'-AGCTCCACCCAAAAGGAGTAG-3'; For the site of c.4561dup of the NF1 gene: Forward primer B: 5'-GCTGTTTACAAATCAGCTGACAG-3'; Reverse primer B: 5'-ATCATATCTAACAAGTGGCCTGGT-3'; For the site of c.6797dup of the NF1 gene: Forward primer D: 5'-GCTAGCTACCAAGATCACCATAGC-3'; Reverse primer D: 5'-CACCCCAGAAAGTAAGCTCCATG-3'; Detection probes: For the site of c.2100_2105delinsAG of the NF1 gene: Wild type probe Aw of the NF1 gene: 5'-CCCTGACACTGAAGCTGTTCTGGT-3'; Mutant type probe Am of the NF1 gene: 5'-CCCTGACACAGTGTTCTGGT-3'; For the site of c.4561dup of the NF1 gene: Wild type probe Bw of the NF1 gene: 5'-GATGGCAACACTTCTTGCAT-3'; Mutant type probe Bm of the NF1 gene: 5'-GATGGCAACACCTTCTTGC-3'; For the site of c.6797dup of the NF1 gene: Wild type probe Dw of the NF1 gene: 5'-ACTTACAACAGTCAAGTTCT-3'; Mutant type probe Dm of the NF1 gene: 5'-TTACAACAGGTCAAGTTCTG-3'; The 5' end of the probe is provided with a reporter fluorescent group, and the 3' end is provided with a quencher fluorescent group; in the reaction system A, the reporter fluorescent groups at the 5' end of each probe are different; The reaction system B comprises detection primers and detection probes as shown below: Detection primers: For the site of c.6421del of the NF1 gene: Forward primer C: 5'-GACCATGTTCAGTTACCAGCAC-3'; Reverse primer C: 5'-CCACTTGCACGTTGGAATATCT-3'; Detection probes: For the site of c.6421del of the NF1 gene: Wild type probe Cw of the NF1 gene: 5'-TACCCAAATTTTACTTGCTG-3'; Mutant type probe Cm of the NF1 gene: 5'-TACCCAAATTTACTTGCTGT-3'; The 5' end of the probe is provided with a reporter fluorescent group, and the 3' end is provided with a quencher fluorescent group.

2. The test kit according to claim 1, characterized in that: The reaction system A is 20 μL, and each 20 μL reaction system A contains: 2×premix Taq™ buffer 10 μL, 10 μmoL / L of Forward primer A 0.5 μL, 10 μmoL / L of Reverse primer A 0.5 μL, 10 μmoL / L of Forward primer B 0.5 μL, 10 μmoL / L of Reverse primer B 0.5 μL, 10 μmoL / L of Forward primer D 0.5 μL, 10 μmoL / L of Reverse primer D 0.5 μL, 10 μmoL / L of probe Aw 0.5 μL, 10 μmoL / L of probe Am 0.5 μL, 10 μmoL / L of probe Bw 0.5 μL, 10 μmoL / L of probe Bm 0.5 μL, 10 μmoL / L of probe Dw 0.5 μL, 10 μmoL / L of probe Dm 0.5 μL, template DNA 2 μL (≥10 ng), RNase-free and DNase-free water 2 μL.

3. The test kit according to claim 1, characterized in that: The reaction system B is 20 μL, and each 20 μL reaction system B contains: 2×premix Taq™ buffer 10 μL, 10 μmoL / L of Forward primer C 0.5 μL, 10 μmoL / L of Reverse primer C 0.5 μL, 10 μmoL / L of probe Cw 0.5 μL, 10 μmoL / L of probe Cm 0.5 μL, template DNA 2 μL (≥10 ng), RNase-free and DNase-free water 6 μL.

4. The test kit of claim 1, wherein: The reporter fluorescent group is one of FAM, SYBR, Fluorescein, SYPRO Orange, VIC, JOE, TET, HEX, TAMRA, Texas Red, Alexa Fluor 633, ResoLight, EvaGreen, LC Green, Cy3, Cy5, Yellow555, LC Red610, ROX, SYPRO Ruby, LCRed640, Snarf 1, Acid Fuchsin, Cy5.5, LC Red670, and LC Red705, and the quencher fluorescent group is MGB.

5. The test kit according to claim 1, characterized in that: When the TaqMan probe is subjected to double-tube multiplex real-time fluorescent quantitative PCR amplification, the amplification reaction program is as follows:

6. Use of a reagent for detecting a mutation site of the NF1 gene in the preparation of a test reagent for neurofibromatosis type I, characterized in that: The NF1 gene mutation sites include the following sites: The NF1 gene c.2100_2105delinsAG site, the NF1 gene c.4561dup site, the NF1 gene c.6421del site, and the NF1 gene c.6797dup site; The wild type NF1 gene has a gene number of NM_000267.3 in the NCBI database, The NF1 gene c.2100_2105delinsAG site is a heterozygous variation, the 2100th to 2105th base of the NF1 gene is deleted and AG is inserted, the variation causes a frameshift mutation of the amino acid of the encoded protein from the 701st position (glutamic acid is changed to valine), and the translation is terminated after 44 amino acids of the frameshift mutation; The NF1 gene c.4561dup site is a heterozygous variation, the 4561st base of the NF1 gene is repeated, the variation causes a frameshift mutation of the amino acid of the encoded protein from the 1521st position (leucine is changed to proline), and the translation is terminated after 21 amino acids of the frameshift mutation; The NF1 gene c.6421del site is a heterozygous variation, the 6421st base of the NF1 gene is deleted, the variation causes a frameshift mutation of the amino acid of the encoded protein from the 2141st position (tyrosine is changed to threonine), and the translation is terminated after 37 amino acids of the frameshift mutation; The NF1 gene c.6797dup site is a heterozygous variation, the 6797th base of the NF1 gene is repeated, the variation causes a frameshift mutation of the amino acid of the encoded protein from the 2266th position (serine is changed to arginine), and the translation is terminated after 19 amino acids of the frameshift mutation.

7. Use according to claim 6, characterized in that: The reagent for detecting the NF1 gene variation site is a probe and a primer for detecting the NF1 gene variation site.

8. Use according to claim 7, characterized in that: The primer includes a primer for the NF1 gene c.2100_2105delinsAG site, the NF1 gene c.4561dup site, the NF1 gene c.6421del site, and the NF1 gene c.6797dup site, and the primers for the sites are as follows: For the NF1 gene c.2100_2105delinsAG site: Forward primer A: 5 '-TGGTAGCATTATGTTGGTCCAGAT-3 '; Reverse primer A: 5 '-AGCTCCACCCAAAAGGAGTAG-3 ' For the NF1 gene c.4561dup site: Forward primer B: 5 '-GCTGTTTACAAATCAGCTGACAG-3 '; Reverse primer B: 5 '-ATCATATCTAACAAGTGGCCTGGT-3 ' For the NF1 gene c.6421del site: Forward primer C: 5 '-GACCATGTTCAGTTACCAGCAC-3 '; Reverse primer C: 5 '-CCACTTGCACGTTGGAATATCT-3 ' For the NF1 gene c.6797dup site: Forward primer D: 5 '-GCTAGCTACCAAGATCACCATAGC-3 '; Reverse primer D: 5 '-CACCCCAGAAAGTAAGCTCCATG-3 ' 9. Use according to claim 7, characterized in that: The probes include probes for NF1 gene c.2100_2105delinsAG site, NF1 gene c.4561dup site, NF1 gene c.6421del site, NF1 gene c.6797dup site, the probes for the sites are as follows respectively: For NF1 gene c.2100_2105delinsAG site: NF1 gene wild type probe Aw: 5 '-CCCTGACACTGAAGCTGTTCTGGT-3 '; NF1 gene mutant probe Am: 5 '-CCCTGACACAGTGTTCTGGT-3 ', For NF1 gene c.4561dup site: NF1 gene wild type probe Bw: 5 '-GATGGCAACACTTCTTGCAT-3 '; NF1 gene mutant probe Bm: 5 '-GATGGCAACACCTTCTTGC-3 ' For NF1 gene c.6421del site: NF1 gene wild type probe Cw: 5 '-TACCCAAATTTTACTTGCTG-3 '; NF1 gene mutant probe Cm: 5 '-TACCCAAATTTACTTGCTGT-3 '; For NF1 gene c.6797dup site: NF1 gene wild type probe Dw: 5 '-ACTTACAACAGTCAAGTTCT-3 '; NF1 gene mutant probe Dm: 5 '-TTACAACAGGTCAAGTTCTG-3 '.

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