Multiplex fluorescent quantitative PCR (polymerase chain reaction) primer, probe and kit for detecting variation site of KMT2D gene of Pansheng syndrome

Abstract

The invention relates to a primer, a probe composition and a kit for detecting variation sites of KMT2D genes of the Pantoea syndrome, and provides three types of KMT2D gene mutation sites found for the first time so as to enrich the variation spectrum of the KMT2D genes, the three types of variation sites are respectively the variation sites of the KMT2D genes c.3247dup, c.1101611019del and c.15545del, and the variation spectrum of the KMT2D genes is enriched by using the primer, the probe composition and the kit for detecting the variation sites of the KMT2D genes of the Pantoea syndrome, namely the mutation sites of the KMT2D genes of the Pantoea syndrome, of the KMT2D genes of the Pantoea syndrome, of the Pantoea syndrome. Meanwhile, the invention also provides a primer, a probe composition and a kit for detecting the variation site of the KMT2D gene of the Pansheng syndrome. The primer, the probe composition and the kit are used for screening or diagnosing the Pansheng syndrome. The mutation sites are proved to be new pathogenic mutations related to the Pansher syndrome, the KMT2D gene pathogenic variation spectrum is expanded, the kit can comprehensively cover the three new pathogenic variation sites of the KMT2D gene related to the Pansher syndrome, and the three pathogenic variation sites found for the first time can be specifically and accurately detected.

Description

Technical Field

[0002] This invention relates to the field of gene diagnostic technology, specifically to a primer and probe composition and kit for detecting KMT2D gene variant sites in Kabuki syndrome. Background Technology

[0003] Kabuki syndrome (KS) is a rare congenital developmental disorder characterized by distinctive facial features, intellectual disability, skeletal deformities, abnormal fingerprints, and multi-systemic symptoms including immunodeficiency. Its facial features primarily include arched eyebrows, thick eyelashes, and downward-sloping eyes, resembling the makeup of traditional Japanese Kabuki actors, hence the name. The immune dysfunction present in Kabuki syndrome requires particular attention. Frequent and recurrent infections caused by immune dysfunction such as hypogammaglobulinemia and IgA deficiency can be a major early mortality factor in children with Kabuki syndrome. Intravenous immunoglobulin administration and peripheral blood immune cell count monitoring are considered important measures to improve the quality of life for affected children. The incidence of Kabuki syndrome is approximately 1 in 32,000, occurring in various ethnic and regional populations. Due to the complex and variable nature of the symptoms, and significant differences in severity among patients, genetic testing is often necessary for accurate diagnosis in clinical practice.

[0004] The international consensus released in 2019 clarified the diagnostic criteria for Kabuki syndrome (PMID: 30514738), including hypotonia, developmental delay or intellectual disability in infancy, and meeting one or more of the following characteristics: (1) pathogenic or probable pathogenic mutations in the KMT2D or KDM6A gene; (2) typical facial features, such as long palpebral fissures, ectropion of the outer third of the lower eyelid, arched eyebrows, cleft or sparse eyebrows, short nasal bridge, flat nasal tip, large ears, etc. Although typical facial features are a significant marker of Kabuki syndrome, some patients may exhibit different clinical symptoms and varying degrees of developmental delay, therefore genetic testing is particularly important.

[0005] The main pathogenic genes for Kabuki syndrome are mutations in the KMT2D and KDM6A genes, with KMT2D gene mutations accounting for approximately 75% of cases. The KMT2D gene, located on chromosome 12, encodes a histone lysine methyltransferase whose primary function is to methylate the 4th lysine residue (H3K4) of histone H3 in chromatin. H3K4 methylation is a crucial step in gene expression activation. Defects or mutations in the KMT2D gene affect chromatin structure, leading to abnormalities in the regulation of various gene expression, impacting embryonic development, nervous system development, and immune system function. Therefore, mutations in the KMT2D gene are usually loss-of-function mutations, resulting in partial or complete loss of function of the encoded methyltransferase, leading to abnormalities in neurodevelopment, skeletal development, the immune system, and other systems. Patients exhibit typical facial features, developmental delays, skeletal deformities, and immunodeficiency.

[0006] In-depth research into Kabuki syndrome helps to understand its genetic basis and pathogenesis, providing support for improving patients' quality of life. Currently, the detection of KMT2D gene mutations is the main method for diagnosing this syndrome. 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 KMT2D gene, many of which are believed to be associated with different Kabuki syndrome phenotypes and symptom severity. Accurate detection and analysis of these new mutants can not only improve the accuracy of early diagnosis of Kabuki syndrome but also lay the foundation for the development of personalized treatment plans.

[0007] Therefore, studying the variant profile of the KMT2D gene is crucial for understanding the etiology of Kabuki syndrome. These studies have revealed how gene mutations affect chromatin remodeling and gene expression, providing clues for developing potential therapeutic strategies. Although several known mutations have been identified in the KMT2D gene, many undiscovered mutation sites remain. Expanding our comprehensive understanding of KMT2D gene mutants will provide solid support for future clinical diagnosis, treatment, and research into the molecular mechanisms of Kabuki syndrome. Summary of the Invention

[0008] The purpose of this invention is to provide three newly discovered KMT2D gene mutation sites to enrich the variation spectrum of the KMT2D gene. These three mutation sites are the KMT2D gene c.3247dup, c.11016_11019del, and c.15545del mutation sites. This invention also provides a primer and probe composition and kit for detecting KMT2D gene mutation sites in Kabuki syndrome for screening or diagnosis of Kabuki syndrome; including specific primers and TaqMan probe compositions targeting the KMT2D gene c.3247dup, c.11016_11019del, and c.15545del mutation sites respectively.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] The specific primer and probe composition described in this invention was designed using Primer Premier 6.0 software. Specifically:

[0011] 1) Targeting the c.3247dup site of the KMT2D gene:

[0012] Forward primer A: 5'-TCGCCACTCCTTCAGCATTC-3' (SEQ ID NO.1);

[0013] Reverse primer A: 5'-CGTGGCTCTTCCTGTTCTTCA-3' (SEQ ID NO.2);

[0014] KMT2D gene wild-type probe A:

[0015] 5'-TCCAAGGCTGGGCATTCAGGTTCTGAAA-3' (SEQ ID NO. 3);

[0016] KMT2D gene mutant probe A:

[0017] 5'-TCCAAGGCTGGGCAATTCAGGTTCTGAAA-3' (SEQ ID NO.4),

[0018] 2) Targeting the c.11016_11019del site of the KMT2D gene:

[0019] Forward primer B: 5'-ACAGAGCAGCAGAGCAAGATCCAG-3' (SEQ ID NO.5);

[0020] Reverse primer B: 5'-GGTCATAAGCACCTGTCTGTGAGG-3' (SEQ ID NO.6);

[0021] KMT2D gene wild-type probe B:

[0022] 5'-AGAGCTGTATTAAGGAAGGGGCCACC-3' (SEQ ID NO. 7);

[0023] KMT2D gene mutant probe B:

[0024] 5 '-AGAGCTGTATT----AAGGGGCCACC-3 ' (SEQ ID NO.8);

[0025] 3) Targeting the c.15545del site of the KMT2D gene:

[0026] Forward primer C: 5'-TGAACCTGGACCTGGACCTGTG-3' (SEQ ID NO.9);

[0027] Reverse primer C: 5'-AGCGATAGCAGCAGCGACGAT-3' (SEQ ID NO.10);

[0028] KMT2D gene wild-type probe C:

[0029] 5'-AACACAAGGCCCCCCACACGGAACA-3' (SEQ ID NO. 11);

[0030] KMT2D gene mutant probe C:

[0031] 5'-AACACAAGGCC-CCCACACGGAACA-3' (SEQ ID NO. 12);

[0032] The probe has different reporter fluorescent groups at its 5' end and an MGB quencher fluorescent group at its 3' end.

[0033] In the same multiplex reaction system, the 5' end of the probe is equipped with different reporter fluorescent groups. For example, the reporter fluorescent group can be one of FAM, SYBR, Fluorescein, SYPRO Orange, VIC, JOE, TET, HEX, TAMRA, Texas Red, Alexa Fluor 633, ResoLight, EvaGreen, LC Green, Cy3, Cy5, Yellow555, LCRed610, ROX, SYPRO Ruby, LC Red640, Snarf 1, Acid Fuchsin, Cy5.5, LC Red670, and LCRed705.

[0034] A detection kit for detecting the KMT2D gene variant site in Kabuki syndrome using a single-tube multiplex real-time quantitative PCR technique based on TaqMan probes, comprising reaction system A;

[0035] The reaction system A includes the detection primers and detection probes shown below:

[0036] Detection primers:

[0037] Targeting the c.3247dup site of the KMT2D gene:

[0038] Forward primer A: 5'-TCGCCACTCCTTCAGCATTC-3';

[0039] Reverse primer A: 5'-CGTGGCTCTTCCTGTTCTTCA-3';

[0040] Targeting the c.11016_11019del site of the KMT2D gene:

[0041] Forward primer B: 5'-ACAGAGCAGCAGAGCAAGATCCAG-3';

[0042] Reverse primer B: 5'-GGTCATAAGCACCTGTCTGTGAGG-3';

[0043] Targeting the c.15545del site of the KMT2D gene:

[0044] Forward primer C: 5'-TGAACCTGGACCTGGACCTGTG-3';

[0045] Reverse primer C: 5'-AGCGATAGCAGCAGCGACGAT-3';

[0046] Detection probe:

[0047] Targeting the c.3247dup site of the KMT2D gene:

[0048] KMT2D gene wild-type probe A:

[0049] 5'-TCCAAGGCTGGGCATTCAGGTTCTGAAA-3';

[0050] KMT2D gene mutant probe A:

[0051] 5'-TCCAAGGCTGGGCAATTCAGGTTCTGAAA-3';

[0052] Targeting the c.11016_11019del site of the KMT2D gene:

[0053] KMT2D gene wild-type probe B:

[0054] 5'-AGAGCTGTATTAAGGAAGGGGCCACC-3';

[0055] KMT2D gene mutant probe B:

[0056] 5 '-AGAGCTGTATT----AAGGGGCCACC-3 ';

[0057] Targeting the c.15545del site of the KMT2D gene:

[0058] KMT2D gene wild-type probe C:

[0059] 5'-AACACAAGGCCCCCCACACGGAACA-3';

[0060] KMT2D gene mutant probe C:

[0061] 5'-AACACAAGGCC-CCCACACGGAACA-3';

[0062] The probe has a reporter fluorescent group at its 5' end and a quencher fluorescent group at its 3' end. In the reaction system, the reporter fluorescent group at the 5' end of each probe is different. The reporter fluorescent group is one of FAM, SYBR, Fluorescein, SYPROOrange, VIC, JOE, TET, HEX, TAMRA, Texas Red, Alexa Fluor 633, ResoLight, EvaGreen, LCGreen, Cy3, Cy5, Yellow555, LC Red610, ROX, SYPRO Ruby, LC Red640, Snarf 1, AcidFuchsin, Cy5.5, LC Red670, and LC Red705. The quencher fluorescent group is MGB.

[0063] The reaction system A is 30 μL, and each 30 μL of reaction system A contains:

[0064] 15 μL of 2×premix Taq™ buffer; 1 μL of 10 μmol / L Forward primer A, 1 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; 1 μL of 10 μmol / L Forward primer c, 1 μL of 10 μmol / L Reverse primer c; 1 μL of 10 μmol / L Wild-type probe A, 1 μL of 10 μmol / L Wild-type probe B, 1 μL of 10 μmol / L Wild-type probe C; 1 μL of 10 μmol / L Mutant probe a, 1 μL of 10 μmol / L Mutant probe b, 1 μL of 10 μmol / L Mutant probe c; 2 μL of template DNA; 2 μL of RNase- and DNase-free water.

[0065] The detection kit, when using TaqMan probes for single-tube multiplex real-time quantitative PCR amplification, has the following amplification reaction procedure:

[0066] .

[0067] The present invention also provides the application of reagents for detecting KMT2D gene variant sites in the preparation of Kabuki syndrome detection reagents, wherein the KMT2D gene variant sites include at least one of the following sites: KMT2D gene c.3247dup site, KMT2D gene c.11016_11019del site, and KMT2D gene c.15545del site;

[0068] The wild-type KMT2D gene has the gene ID NM_003482.4 in the NCBI database.

[0069] Among them, the c.3247dup site of the KMT2D gene is a heterozygous variant. The base duplication at position 3247 of the KMT2D gene leads to a frameshift mutation of the amino acid encoding the protein from position 1083 (cysteine ​​is replaced by leucine). Translation terminates after 31 amino acids of frameshift mutation, i.e. p.Cys1083Leufs*32.

[0070] The KMT2D gene c.11016_11019del site is a heterozygous variant, which is a deletion of bases from position 11016 to 11019 in the KMT2D gene. This mutation causes a frameshift mutation (phenylalanine to leucine) in the encoded protein from position 3672. Translation terminates after 75 amino acids of frameshift mutation, i.e. p.Phe3672Leufs*76;

[0071] The KMT2D gene c.15545del site is a heterozygous variant, which is a deletion of the base at position 15545 of the KMT2D gene. This variant causes a frameshift mutation (glycine to alanine) of the amino acid encoded by the protein from position 5182. Translation terminates after 60 amino acids of frameshift mutation, i.e. p.Gly5182Alafs*61.

[0072] The three KMT2D gene variant sites, c.3247dup, c.11016_11019del, and c.15545del, were discovered using high-throughput sequencing technology in this invention after excluding other pathogenic genes for Kabuki syndrome. These variants are not yet included in the gnomAD database and are the first pathogenic variant sites discovered.

[0073] The reagents for detecting KMT2D gene variant sites are one or more of the probes and primers used to detect KMT2D gene variant sites.

[0074] The primers include primers targeting at least one variant site among the KMT2D gene c.3247dup site, KMT2D gene c.11016_11019del site, and KMT2D gene c.15545del site. The primers targeting the aforementioned sites are as follows:

[0075] Targeting the c.3247dup site of the KMT2D gene:

[0076] Forward primer A: 5'-TCGCCACTCCTTCAGCATTC-3';

[0077] Reverse primer A: 5'-CGTGGCTCTTCCTGTTCTTCA-3'

[0078] Targeting the c.11016_11019del site of the KMT2D gene:

[0079] Forward primer B: 5'-ACAGAGCAGCAGAGCAAGATCCAG-3';

[0080] Reverse primer B: 5'-GGTCATAAGCACCTGTCTGTGAGG-3'

[0081] Targeting the c.15545del site of the KMT2D gene:

[0082] Forward primer C: 5'-TGAACCTGGACCTGGACCTGTG-3';

[0083] Reverse primer C: 5'-AGCGATAGCAGCAGCGACGAT-3'.

[0084] The probes include probes targeting at least one variant site among the KMT2D gene c.3247dup site, the KMT2D gene c.11016_11019del site, and the KMT2D gene c.15545del site. The probes targeting the aforementioned sites are as follows:

[0085] Targeting the c.3247dup site of the KMT2D gene:

[0086] KMT2D gene wild-type probe A:

[0087] 5'-TCCAAGGCTGGGCATTCAGGTTCTGAAA-3';

[0088] KMT2D gene mutant probe A:

[0089] 5'-TCCAAGGCTGGGCAATTCAGGTTCTGAAA-3',

[0090] Targeting the c.11016_11019del site of the KMT2D gene:

[0091] KMT2D gene wild-type probe B:

[0092] 5'-AGAGCTGTATTAAGGAAGGGGCCACC-3';

[0093] KMT2D gene mutant probe B:

[0094] 5'-AGAGCTGTATT----AAGGGGCCACC-3',

[0095] Targeting the c.15545del site of the KMT2D gene:

[0096] KMT2D gene wild-type probe C:

[0097] 5'-AACACAAGGCCCCCCACACGGAACA-3';

[0098] KMT2D gene mutant probe C:

[0099] 5'-AACACAAGGCC-CCCACACGGAACA-3'.

[0100] 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, three KMT2D gene variant sites after excluding other pathogenic genes for Kabuki syndrome: the KMT2D gene c.3247dup site, the KMT2D gene c.11016_11019del site, and the KMT2D gene c.15545del site. These variant sites are novel pathogenic variants associated with Kabuki syndrome. Based on population frequency, family information, computer bioinformatics analysis, genetic patterns, and clinical phenotypes, the results are accurate, expanding the spectrum of pathogenic variants of the KMT2D gene and providing a basis for the diagnosis and genetic counseling of Kabuki syndrome. Furthermore, this invention provides primer and probe compositions and kits for detecting KMT2D gene variant sites in Kabuki syndrome for screening or diagnosis of Kabuki syndrome; including specific primers and TaqMan probe compositions targeting the KMT2D gene c.3247dup, c.11016_11019del, and c.15545del variant sites respectively. The present invention provides a detection kit and method for detecting KMT2D gene variants in Kabuki syndrome using a single-tube multiplex real-time quantitative PCR technique based on TaqMan probes. This kit comprehensively covers the three novel pathogenic variants of the KMT2D gene associated with Kabuki syndrome. The multiplex TaqMan real-time quantitative PCR technique is time-efficient and simple to operate. The DNA sample from the subject is added to one reaction system 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 three newly discovered pathogenic variants, enabling the diagnosis of Kabuki syndrome and providing a basis for guiding clinical diagnosis and genetic counseling. Attached Figure Description

[0101] Figure 1 This is the data quality control result of 72 suspected Kabuki syndrome patients tested using the "Kabuki Syndrome Related Gene Mutation Detection Kit (Liquid Hybridization Capture Sequencing Method)".

[0102] Figure 2 This is a photograph of the probationer 1's eye features (palpebral fissure, brow bone) in Example 2. The red arrow indicates the characteristic lateral extension of the palpebral fissure accompanied by ectropion of the lower eyelid in the outer 1 / 3.

[0103] Figure 3 This is the pedigree of the Kabuki syndrome family to which the proband 1 in Example 2 belongs.

[0104] Figure 4 This is a BAM file diagram of the c.3247dup variant of the KMT2D gene in proband 1 of the Kabuki syndrome family in Example 2.

[0105] Figure 5 This is the pedigree of the suspected Kabuki syndrome proband 2 in Example 3.

[0106] Figure 6 This is the list of candidate sequence variations detected by high-throughput sequencing in the suspected Kabuki syndrome proband 2 in Example 3.

[0107] Figure 7 This is a BAM file image of the c.11016_11019del KMT2D gene of the suspected Kabuki syndrome proband 2 in Example 3.

[0108] Figure 8 This is the pedigree of the suspected Kabuki syndrome proband 3 in Example 4.

[0109] Figure 9 This is the list of candidate sequence variations detected by high-throughput sequencing in the suspected Kabuki syndrome proband 3 in Example 4.

[0110] Figure 10 This is a BAM file diagram of the c.15545del variant of the KMT2D gene in the suspected Kabuki syndrome proband 3 in Example 4.

[0111] Figure 11 This is a qRT-PCR and gel electrophoresis analysis diagram of the effect of three variants on the expression level of KMT2D gene mRNA in Example 5.

[0112] Figure 12The images show the real-time quantitative PCR results of the TaqMan probe at the c.3247dup site of the KMT2D gene in Example 6; the upper image shows the amplification curve of the heterozygote at the c.3247dup site of the KMT2D gene; the lower image shows the amplification curve of the wild-type at the c.3247T site.

[0113] Figure 13 The images show the real-time quantitative PCR results of the TaqMan probe at the c.11016_11019del site of the KMT2D gene in Example 6. The upper image shows the amplification curve of the heterozygote at the c.11016_11019del site of the KMT2D gene; the lower image shows the amplification curve of the wild-type at the c.11016_11019CCTT site.

[0114] Figure 14 The images show the real-time quantitative PCR results of the TaqMan probe at the c.15545del site of the KMT2D gene in Example 6. The top image shows the amplification curve of the heterozygote at the 937del site of the KMT2D gene, and the bottom image shows the amplification curve of the wild-type at the c.15545G site.

[0115] Figure 15 This is a Sanger sequencing result of the NM_003482.4:c.3247dup variant of the KMT2D gene in Example 7; the red box indicates the detection site.

[0116] Figure 16 This is a Sanger sequencing result of the NM_003482.4:c.11016_11019del variant of the KMT2D gene in Example 7; the red box indicates the detection site.

[0117] Figure 17 This is a Sanger sequencing result of the NM_003482.4:c.15545del variant of the KMT2D gene in Example 7, where the red box represents the detection site. Detailed Implementation

[0118] The present invention will now be described in detail with reference to the accompanying drawings and embodiments:

[0119] Example 1: Detection of pathogenic variants in Kabuki syndrome patients using the "Kabuki Syndrome Gene Mutation Detection Kit (Liquid Hybridization Capture Sequencing)".

[0120] This study used peripheral blood samples from 72 patients with Kabuki syndrome or suspected Kabuki syndrome submitted to Furui Medical Laboratory as research subjects. Data collected included patients' growth and development history, intelligence, skeletal system, nervous system, digestive and endocrine system, reproductive and urinary system, circulatory system, teeth, nail development, sweating, hearing, vision, and other clinical symptoms and signs, laboratory tests, and imaging examinations. High-throughput sequencing using a "Kabuki Syndrome Gene Mutation Detection Kit (Liquid Hybridization Capture Sequencing)" was performed on the probands of candidate Kabuki syndrome and some family members. The sequencing included the following steps in sequence:

[0121] (1) Sample collection and extraction of genomic DNA.

[0122] 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.

[0123] (2) High-throughput sequencing and bioinformatics analysis of gene mutations in Kabuki syndrome.

[0124] 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. The Kabuki Syndrome Related Gene Mutation Detection Kit (Liquid Hybridization Capture Sequencing) developed by Furui Medical Laboratory was used to capture the exon regions of the KMT2D, KDM6A, CHD7, IRF6, EYA1, FLNB, SIX1, and SIX5 genes, as well as other regions containing pathogenic variants of HMGD and ClinVar. These eight genes include all candidate genes for the diagnosis and differential diagnosis of Kabuki Syndrome in the internationally authoritative database Genereviews. The library was sequenced using a Novaseq sequencer (Illumina, San Diego, CA, USA) (sequencing depth ≥150X, 20X coverage ≥98%). Based on historical cumulative data, the 20X coverage of the KMT2D, KDM6A, CHD7, IRF6, EYA1, FLNB, SIX1, and SIX5 genes reached 100% in all sequencing samples. Performance study results indicate that the "Kabuki Syndrome Related Gene Mutation Detection Kit (Liquid Hybridization Capture Sequencing)" can detect all candidate variants of the above eight genes in clinical samples. Figure 1 ).

[0125] 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).

[0126] (3) Interpretation of candidate gene variations

[0127] Sequence variations were interpreted according to 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 variations were interpreted according to the "2019 ACMG Chromosomal Copy Number Variation Interpretation and Reporting Technical Standards Interpretation". The various variations detected by the sequencing were classified as pathogenic variations (P), possibly pathogenic variations (LP), variations of unknown significance (VUS), possibly benign variations (LB), and benign variations (B).

[0128] Example 2: Pathogenic variant of KMT2D gene NM_003482.4:c.3247dup (p.Cys1083Leufs*32) in Kabuki syndrome cases

[0129] Proband 1 is a 4-year-old male with developmental delay and distinctive facial features (wide-set eyes, narrow palpebral fissures, eversion of the outer third of the lower eyelid, and high brow ridges). Figure 2 The patient has experienced afebrile seizures for 2 years and 9 months. Developmental delay began at 1 year and 3 months; the seizures occur while the patient is awake, with both eyes veering to one side, resolving within 20 seconds, 3 times / day. Preliminary diagnosis: Kabuki syndrome, epilepsy. Both parents are healthy; see family pedigree for details. Figure 3 ).

[0130] In this embodiment, high-throughput sequencing of the hereditary bone disease gene using the "Kabuki Syndrome Related Gene Mutation Detection Kit (Liquid Hybridization Capture Sequencing Method)" revealed that the proband carried one sequence variant (population frequency ≤5%) and 0 candidate CNV variants. The sequence variant was a heterozygous variant at the c.3247dup site of the KMT2D gene. Figure 4 The mutation, with a priority of 1 (highest priority), causes a frameshift mutation (cysteine ​​to leucine) at position 1083 of the encoded protein. Translation terminates after 31 amino acids of frameshift. According to the ACMG guidelines, this variant is classified as pathogenic. The relevant evidence for this variant is PVS1+PS2+PM2, specifically:

[0131] PS2: Not detected in the peripheral blood of the subject's parents, suggesting a possible new variant.

[0132] PVS1: This variant occurs in exon 11 of transcript NM_003482.3 (54 exons in total), which may cause protein truncation or activate nonsense-mediated mRNA degradation, thereby affecting the function of the gene-encoded protein product. Furthermore, there are multiple loss-of-function variants (DM) recorded in HGMD downstream of this variant.

[0133] PM2: The variant is not currently included in the gnomAD database.

[0134] Therefore, the KMT2D gene mutant c.3247dup (p.Cys1083Leufs*32) was discovered using high-throughput sequencing technology after excluding other pathogenic genes for Kabuki syndrome. This novel pathogenic variant associated with Kabuki syndrome explains the distinctive facial features and epileptic phenotype of the progenitor, 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 KMT2D gene and providing a basis for the diagnosis and genetic counseling of Kabuki syndrome.

[0135] Example 3: Pathogenic variant of KMT2D gene NM_003482.4: c.11016_11019del (p.Phe3672Leufs*76) in suspected Kabuki syndrome

[0136] Proband 2 is a 6-year-old male, clinically suspected of having Kabuki syndrome, exhibiting developmental delays, multiple congenital malformations, transient neonatal hypoglycemia, acute respiratory failure, and cleft palate. The child was found to have cleft palate, occult spina bifida, low nasal bridge, elongated palpebral fissures, arched eyebrows, webbed neck, low-set ears, aortic arch coarctation, patent ductus arteriosus, atrial septal defect, pulmonary hypertension, and smaller than average size of both kidneys. The father, mother, and sister are healthy; see family pedigree for details. Figure 5 ).

[0137] In this embodiment, high-throughput sequencing of the hereditary bone disease gene was performed using the "Kabuki Syndrome Related Gene Mutation Detection Kit (Liquid Hybridization Capture Sequencing Method)" to identify that the proband carried three sequence variations (population frequency ≤5%). Figure 6 ), 0 candidate CNV variants. Of the three candidate sequence variants, two FLNB gene sequence variants, NM_001457.3:c.2576-64C>T (p.?) and NM_001457.3:c.2482C>G (p.Gln828Glu), were first excluded. The NM_001457.3:c.2576-64C>T variant is located in a non-coding region and, according to the ACMG guidelines, is classified as a possibly benign variant. The relevant evidence for this variant is BS1 (population frequency of 0.94%, allele frequency greater than disease incidence) + BP4 (predicted benign by bioinformatics tools such as CADD) + BP7 (synonymous or non-coding region variants are predicted by bioinformatics tools not to affect splicing). The NM_001457.3:c.2482C>G variant, according to the ACMG guidelines, is also classified as a possibly benign variant. The relevant evidence for this variant is BS1 (local population frequency of 0.33%). (62 / 18968, allele frequency greater than disease incidence) + BP4 (REVEL=0.193 predicts benign).

[0138] Heterozygous variant at the c.11016_11019del site of the KMT2D gene ( Figure 7 This mutation, with a priority of 1 (highest priority), causes a frameshift mutation (phenylalanine to leucine) at position 3672 of the encoded protein. Translation terminates after 75 amino acids of frameshift. This variant is not yet included in the HGMD database. According to the ACMG guidelines, this variant is classified as a pathogenic variant. The relevant evidence for this variant is PM2_Supporting+PS2+PVS1, specifically:

[0139] PVS1: This variant occurs in exon 39 of transcript NM_003482.3 (54 exons in total), which may cause protein truncation or activate nonsense-mediated mRNA degradation, thereby affecting the function of the gene-encoded protein product. Furthermore, there are multiple loss-of-function variants (DM) recorded in HGMD downstream of this variant.

[0140] PM2: This variant has not yet been included in gnomAD v2.1.1.

[0141] PS2: Not detected in the peripheral blood of the subject's parents, suggesting a possible de novo variant (PS2).

[0142] Therefore, the KMT2D gene mutant c.11016_11019del (p.Phe3672Leufs*76) was discovered using high-throughput sequencing technology after excluding other pathogenic genes for Kabuki syndrome. This novel pathogenic variant associated with Kabuki syndrome explains the sporadic multiple congenital malformations in the proband, providing a diagnostic result for the proband. According to the Kabuki syndrome diagnosis and treatment guidelines, after the positive detection of this mutation, proband 2 was ultimately diagnosed with Kabuki syndrome 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 KMT2D gene and providing a basis for the diagnosis and genetic counseling of Kabuki syndrome.

[0143] Example 4: Pathogenic variant of KMT2D gene NM_003482.4:c.15545del (p.Gly5182Alafs*61) in suspected Kabuki syndrome

[0144] The proband, a 7-year-old male, was initially diagnosed with suspected Kabuki syndrome. His clinical presentation included obesity, intellectual disability, and distinctive facial features. He could lift his head at 3 months, could not sit at 9 months, and could speak at around 1 year old. He currently cannot write and cannot perform addition and subtraction within 10. He has an obese physique, a long face, long eye slits, large protruding ears, visible freckles on his face, a small penis, and small testes. Both parents were previously healthy. See the family pedigree for details. Figure 8 ).

[0145] In this embodiment, high-throughput sequencing of the hereditary bone disease gene was performed using the "Kabuki Syndrome Related Gene Mutation Detection Kit (Liquid Hybridization Capture Sequencing Method)" to identify that the proband carried two sequence variations (population frequency ≤5%). Figure 9), 0 candidate CNV variants. Among the 2 candidate sequence variants, the KMT2D gene sequence variant NM_003482.4:c.12764G>A (p.Gly4255Asp) was first excluded because the variant was predicted by the REVEL software to not affect protein function, was inherited from a healthy father, suggesting that the disease was not segregated from the family lineage. According to the ACMG guidelines, this variant was classified as a possibly benign variant. The relevant evidence for this variant was BS4 (lack of cosegregation in a family member) + BP5 (a variant found in cases where another molecular pathogenic factor, c.15545del, already exists). Heterozygous variant at the c.15545del site of the KMT2D gene ( Figure 10 This mutation, with a priority of 1 (highest), results in the deletion of base 15545 in the coding region. The mutation causes a frameshift mutation (glycine to alanine) at position 5182 of the encoded protein, terminating translation after 60 amino acids. According to the ACMG guidelines, this mutation is recommended to be classified as pathogenic. The relevant evidence for this mutation is PVS1+PS2+PM2, specifically:

[0146] PVS1: This frameshift variant is a 1-base pair deletion, resulting in out-of-frame transcripts and premature stop codons. This variant may activate nonsense-mediated mRNA degradation, leading to loss of function of the protein product of the KMT2D gene. This variant has not yet been reported in the literature, and downstream truncation variants are known to be pathogenic (PubMed: 22126750, 21671394, 21280141).

[0147] PS2: This variant was verified to be undetectable in the samples of the father and mother of the proband 3, indicating that it is a novel variant.

[0148] PM2: The variant has not yet been included in the gnomAD database.

[0149] Therefore, the KMT2D gene mutant c.15545del was discovered using high-throughput sequencing technology after excluding other pathogenic genes for Kabuki syndrome. It is a novel pathogenic variant associated with Kabuki syndrome, and this variant can explain the sporadic Kabuki syndrome phenotype in the progenitor, serving as a diagnostic result for the progenitor. Based on the Kabuki syndrome diagnosis and treatment guidelines, this patient had suspected Kabuki syndrome and was ultimately diagnosed with Kabuki syndrome. The results were accurate, based on population frequency, family information, and clinical phenotype, expanding the spectrum of pathogenic variants in the KMT2D gene and providing a basis for the diagnosis and genetic counseling of Kabuki syndrome. In terms of genetic counseling, Kabuki syndrome caused by the KMT2D gene mutation is a clinically variable and heterogeneous genetic disorder. Comprehensive monitoring of Kabuki syndrome patients is recommended: measuring height and weight at each visit to monitor growth and development; conducting an ophthalmological or optometric examination at least annually to assess vision; performing regular hearing tests; conducting a clinical assessment of scoliosis at each visit before skeletal maturity; testing thyroid function every 2 to 3 years to assess endocrine health; and performing regular complete blood counts to monitor the immune system. Furthermore, each visit during childhood and adolescence should focus on developmental progress and educational needs to provide timely support.

[0150] Example 5: qRT-PCR experiment to analyze the effect of variation on KMT2D gene function / expression

[0151] To verify the effects of the three KMT2D 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 KMT2D gene mRNA in peripheral blood samples from individuals carrying different variants were detected using qRT-PCR to determine the impact of the variants on KMT2D gene function / expression. Peripheral blood samples (collected in PAX gene tubes) from three probands carrying the c.3247dup, c.11016_11019del, and c.15545del variants of the KMT2D gene, respectively, were selected as the experimental group. Peripheral blood samples (collected in PAX gene tubes) from healthy individuals collected concurrently served as the control group. Three independent biological replicates (n=3) were collected from each proband sample, and n=6 healthy control samples (including the proband's non-affected parents) were collected.

[0152] Peripheral blood total RNA was extracted using the PAXgene Blood RNA Extraction Kit (Thermo Fisher Scientific). RNA concentration and purity (A260 / 280 ratio 1.8–2.1) were measured. 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 a 20 µL reaction mixture: 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 a final volume. GAPDH was used as the internal control gene. qPCR conditions were: 95 °C for 3 min; 95 °C for 10 s, 60 °C for 30 s, for 40 cycles. Melting curve analysis was performed at the ends to confirm amplification specificity. Each sample was tested in triplicate. The yield and purity of the amplified products were analyzed by gel electrophoresis.

[0153] KMT2D 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:

[0154] KMT2D_qF: 5′-GGAATGGGTAGCTCTTTGGCGA-3′;

[0155] KMT2D_qR: 5′-TGCCGAATCAGCAGCTCTCGTA-3′;

[0156] GAPDH_qF: 5′-GCACCGTCAAGGCTGAGAAC-3′;

[0157] GAPDH_qR: 5′-TGGTGAAGACCGCCAGTGGA-3′.

[0158] The relative expression levels were calculated using the ΔΔCt method. First, the KMT2D Ct values ​​were normalized to the internal reference (ΔCt = Ct_KMT2D - 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 a two-tailed Student's t-test (P < 0.05 was considered statistically significant).

[0159] The results are as follows Figure 11The qRT-PCR experiment showed that the KMT2D mRNA levels in individuals with the c.3247dup (proband 1), c.11016_11019del (proband 2), and c.15545del (proband 3) variants were significantly lower than those in the healthy control group, with mean relative expression levels downregulated by 59.82%, 57.23%, and 50.35%, respectively (P<0.001). The KMT2D mRNA expression levels in the parents of asymptomatic probands were comparable to those in other healthy controls collected at the same time. Gel electrophoresis analysis of the qRT-PCR products showed that the bands in the patient group were in the same position as those in the control group, but with significantly reduced brightness. These results indicate that all three variants can lead to a decrease in KMT2D transcript expression by approximately 50%, suggesting that the variants can trigger nonsense-mediated mRNA degradation or affect transcriptional stability, thereby resulting in insufficient KMT2D gene product dosage.

[0160] According to the ClinGen database and literature reports, the pathogenic mechanism of KMT2D leading to Kabuki syndrome is haplo-insufficiency (HI), meaning that the loss of function in one of the two alleles of the KMT2D gene is pathogenic. Therefore, the qRT-PCR results obtained in this embodiment directly prove that the KMT2D gene variants c.3247dup, c.11016_11019del, and c.15545del can significantly reduce its mRNA level, which is consistent with the genetic rules and pathogenic mechanism of KMT2D gene pathogenesis and has a direct causal relationship with the occurrence of Kabuki syndrome.

[0161] Example 6: Primer and probe composition and kit for detecting KMT2D gene variant sites in Kabuki syndrome

[0162] (1) The primer and probe composition and kit for detecting the KMT2D gene variant sites of Kabuki syndrome are used for screening or diagnosis of Kabuki syndrome; including specific primers and TaqMan probe compositions for the KMT2D gene variant sites c.3247dup, c.11016_11019del and c.15545del respectively.

[0163] The specific primer and probe composition was designed using Primer Premier 6.0 software, specifically:

[0164] 1) Targeting the c.3247dup site of the KMT2D gene:

[0165] Forward primer A: 5'-TCGCCACTCCTTCAGCATTC-3';

[0166] Reverse primer A: 5'-CGTGGCTCTTCCTGTTCTTCA-3'

[0167] KMT2D gene wild-type probe A:

[0168] 5'-TCCAAGGCTGGGCATTCAGGTTCTGAAA-3';

[0169] KMT2D gene mutant probe A:

[0170] 5'-TCCAAGGCTGGGCAATTCAGGTTCTGAAA-3',

[0171] 2) Targeting the c.11016_11019del site of the KMT2D gene:

[0172] Forward primer B: 5'-ACAGAGCAGCAGAGCAAGATCCAG-3';

[0173] Reverse primer B: 5'-GGTCATAAGCACCTGTCTGTGAGG-3'

[0174] KMT2D gene wild-type probe B:

[0175] 5'-AGAGCTGTATTAAGGAAGGGGCCACC-3';

[0176] KMT2D gene mutant probe B:

[0177] 5 '-AGAGCTGTATT----AAGGGGCCACC-3 '

[0178] 3) Targeting the c.15545del site of the KMT2D gene:

[0179] Forward primer C: 5'-TGAACCTGGACCTGGACCTGTG-3';

[0180] Reverse primer C: 5'-AGCGATAGCAGCAGCGACGAT-3'

[0181] KMT2D gene wild-type probe C:

[0182] 5'-AACACAAGGCCCCCCACACGGAACA-3';

[0183] KMT2D gene mutant probe C:

[0184] 5'-AACACAAGGCC-CCCACACGGAACA-3';

[0185] The probe has different reporter fluorescent groups at its 5' end and an MGB quencher fluorescent group at its 3' end.

[0186] The 5' end of the probe is equipped with different reporter fluorescent groups, such as 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, Snarf1, Acid Fuchsin, Cy5.5, LC Red670, and LC Red705.

[0187] (2) The reagent components of the kit may be general PCR amplification reaction reagents, including buffer, ions, dNTP, 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.

[0188] (3) The kit uses a single-tube multiplex fluorescent PCR reaction system, as detailed below:

[0189] Reaction system: KMT2D gene c.3247dup site, c.11016_11019del site, and c.15545del site

[0190]

[0191] (4) The amplification reaction procedure is as follows:

[0192]

[0193] (5) Procedure for detecting the c.3247dup, c.11016_11019del, and c.15545del variant sites of the KMT2D gene in Kabuki syndrome using multiplex probe reagents:

[0194] As described in Example 1, clinical tissue samples were collected from the proband of Kabuki syndrome or their family members, including but not limited to peripheral blood anticoagulated with EDTA / sodium citrate.

[0195] As described in Example 1, genomic DNA was extracted from clinical tissue samples and quality control was performed.

[0196] 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;

[0197] 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 12-14 The following criteria are used to determine whether the subject carries the pathogenic locus of the KMT2D gene.

[0198]

[0199] Example 7: Performance Study Results of the Kit for Detecting KMT2D Gene Variants in Kabuki Syndrome

[0200] (1) Performance analysis scheme

[0201] To investigate the analytical performance of the single-tube multiplex fluorescent TaqMan probe PCR detection kit for Kabuki syndrome KMT2D gene variant sites in this invention, 120 peripheral blood samples were used as the research subjects in this example. These included: clinical samples from 72 candidate Kabuki syndrome patients who underwent NGS testing in Example 1, 42 normal control samples, and 6 clinical samples (EDTA-anticoagulated peripheral blood) from family members of the proband 1-3. All positive samples were compared with NGS analysis results and Sanger sequencing validation results. The sensitivity and specificity of the kit for detecting the c.3247dup, c.11016_11019del, and c.15545del variant sites of the Kabuki syndrome KMT2D gene were calculated using the following formulas:

[0202] Sensitivity = Number of true positive results / (Number of true positive results + Number of false negative results)

[0203] Specificity = Number of true negative results / (Number of true negative results + Number of false positive results)

[0204] (2) The system and steps for the Sanger sequencing validation:

[0205] PCR amplification systems (20 μL) for the c.3247dup, c.11016_11019del, and c.15545del variant sites were performed. Each PCR reaction was a single-site, single-tube amplification system, for a total of three 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. The PCR reaction program was: 95℃ for 5 min, 35 cycles (95℃ for 5 min, 95℃ for 30 s, 62℃ 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.

[0206] (3) Analysis of performance study results

[0207] The kit for detecting the KMT2D gene variant site in Kabuki syndrome of this invention has 100% sensitivity and specificity for detecting the c.3247dup variant site, as detailed in the table below and the Sanger sequencing verification results. Figure 15 ):

[0208]

[0209] The kit for detecting the KMT2D gene variant site in Kabuki syndrome of this invention has 100% sensitivity and specificity for detecting the c.11016_11019del variant site, as detailed in the table below and the Sanger sequencing verification results. Figure 16 ):

[0210]

[0211] The kit for detecting the KMT2D gene variant site in Kabuki syndrome of this invention has 100% sensitivity and specificity for detecting the c.15545del variant site, as detailed in the table below and the Sanger sequencing verification results. Figure 17 ):

[0212]

[0213] 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 TaqMan probe-based single-tube multiplex real-time fluorescent quantitative PCR technique for detecting Kabuki syndrome KMT2D gene variation site, characterized in that: It includes reaction system A; The reaction system A includes the detection primers and detection probes shown below: Detection primers: Targeting the c.3247dup site of the KMT2D gene: Forward primer A: 5'-TCGCCACTCCTTCAGCATTC-3'; Reverse primer A: 5'-CGTGGCTCTTCCTGTTCTTCA-3'; Targeting the c.11016_11019del site of the KMT2D gene: Forward primer B: 5'-ACAGAGCAGCAGAGCAAGATCCAG-3'; Reverse primer B: 5'-GGTCATAAGCACCTGTCTGTGAGG-3'; Targeting the c.15545del site of the KMT2D gene: Forward primer C: 5'-TGAACCTGGACCTGGACCTGTG-3'; Reverse primer C: 5'-AGCGATAGCAGCAGCGACGAT-3'; Detection probe: Targeting the c.3247dup site of the KMT2D gene: KMT2D gene wild-type probe A: 5'-TCCAAGGCTGGGCATTCAGGTTCTGAAA-3'; KMT2D gene mutant probe A: 5'-TCCAAGGCTGGGCAATTCAGGTTCTGAAA-3'; Targeting the c.11016_11019del site of the KMT2D gene: KMT2D gene wild-type probe B: 5'-AGAGCTGTATTAAGGAAGGGGCCACC-3'; KMT2D gene mutant probe B: 5'-AGAGCTGTATT----AAGGGGCCACC-3'; Targeting the c.15545del site of the KMT2D gene: KMT2D gene wild-type probe C: 5'-AACACAAGGCCCCCCACACGGAACA-3'; KMT2D gene mutant probe C: 5'-AACACAAGGCC-CCCACACGGAACA-3'; 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.

2. The test kit according to claim 1, characterized in that: The reaction system A is 30 μL, and each 30 μL of reaction system A contains: 2 x premix Taq TM Buffer 15 μL; 10 μmoL / L of Forward primer A 1 μL, 10 μmoL / L of Reverse primer A 1 μL; 10 μmoL / L of Forward primer B 0.5 μL, 10 μmoL / L of Reverse primer B 0.5 μL; 1 μL of 10 μmol / L Forward primer C, 1 μL of 10 μmol / L Reverse primer C; 1 μL of 10 μmol / L Wild-type probe A, 1 μL of 10 μmol / L Wild-type probe B, 1 μL of 10 μmol / L Wild-type probe C; 1 μL of 10 μmol / L Mutant probe A, 1 μL of 10 μmol / L Mutant probe B, 1 μL of 10 μmol / L Mutant probe C; 2 μL of template DNA; 2 μL of RNase- and DNase-free water.

3. The test kit according to claim 1, characterized in that: 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.

4. The detection kit according to claim 1, characterized in that: When using TaqMan probes for single-tube multiplex real-time quantitative PCR amplification, the amplification reaction procedure is as follows:

5. The application of reagents for detecting KMT2D gene variant sites in the preparation of reagents for the detection of Kabuki syndrome, characterized in that: The KMT2D gene variant sites include at least one of the following sites: KMT2D gene c.3247dup site, KMT2D gene c.11016_11019del site, and KMT2D gene c.15545del site; The wild-type KMT2D gene has the gene ID NM_003482.4 in the NCBI database. Among them, the c.3247dup site of the KMT2D gene is a heterozygous variant. The base duplication at position 3247 of the KMT2D gene leads to a frameshift mutation (cysteine ​​to leucine) of the amino acid encoding the protein from position 1083. After the frameshift mutation of 31 amino acids, translation terminates, i.e. p.Cys1083Leufs*32. The KMT2D gene c.11016_11019del site is a heterozygous variant, which is a deletion of bases from position 11016 to 11019 in the KMT2D gene. This mutation causes a frameshift mutation (phenylalanine to leucine) in the encoded protein from position 3672. Translation terminates after 75 amino acids of frameshift mutation, i.e. p.Phe3672Leufs*76; The KMT2D gene c.15545del site is a heterozygous variant, which is a deletion of the base at position 15545 of the KMT2D gene. This variant causes a frameshift mutation (glycine to alanine) of the amino acid encoded by the protein from position 5182. Translation terminates after 60 amino acids of frameshift mutation, i.e. p.Gly5182Alafs*61.

6. The application according to claim 5, characterized in that: The reagents for detecting KMT2D gene variant sites are probes and primers for detecting KMT2D gene variant sites.

7. The application according to claim 6, characterized in that: The primers include primers targeting the KMT2D gene c.3247dup site, the KMT2D gene c.11016_11019del site, and the KMT2D gene c.15545del site, and the primers targeting these sites are as follows: Targeting the c.3247dup site of the KMT2D gene: Forward primer A: 5'-TCGCCACTCCTTCAGCATTC-3'; Reverse primer A: 5'-CGTGGCTCTTCCTGTTCTTCA-3' Targeting the c.11016_11019del site of the KMT2D gene: Forward primer B: 5'-ACAGAGCAGCAGAGCAAGATCCAG-3'; Reverse primer B: 5'-GGTCATAAGCACCTGTCTGTGAGG-3' Targeting the c.15545del site of the KMT2D gene: Forward primer C: 5'-TGAACCTGGACCTGGACCTGTG-3'; Reverse primer C: 5'-AGCGATAGCAGCAGCGACGAT-3'.

8. The application according to claim 6, characterized in that: The probes include probes targeting the KMT2D gene c.3247dup site, the KMT2D gene c.11016_11019del site, and the KMT2D gene c.15545del site, and the probes targeting these sites are as follows: Targeting the c.3247dup site of the KMT2D gene: KMT2D gene wild-type probe A: 5'-TCCAAGGCTGGGCATTCAGGTTCTGAAA-3'; KMT2D gene mutant probe A: 5'-TCCAAGGCTGGGCAATTCAGGTTCTGAAA-3', Targeting the c.11016_11019del site of the KMT2D gene: KMT2D gene wild-type probe B: 5'-AGAGCTGTATTAAGGAAGGGGCCACC-3'; KMT2D gene mutant probe B: 5'-AGAGCTGTATT----AAGGGGCCACC-3', Targeting the c.15545del site of the KMT2D gene: KMT2D gene wild-type probe C: 5'-AACACAAGGCCCCCCACACGGAACA-3'; KMT2D gene mutant probe C: 5'-AACACAAGGCC-CCCACACGGAACA-3'.

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