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. According to the present invention, the mutation sites are the new pathogenic mutation related to the Pansher syndrome, the KMT2D gene pathogenic mutation spectrum is expanded, and the kit can completely cover the three new pathogenic mutation sites of the KMT2D gene related to the Pansher syndrome, and can specifically and accurately detect the three pathogenic mutation sites found for the first time.

Description

Technical Field

[0001] The present invention relates to the technical field of gene diagnosis, and in particular to a primer and probe combination and a kit for detecting a Kabuki syndrome KMT2D gene mutation site. Background Art

[0002] Kabuki syndrome (KS) is a rare congenital developmental disorder with multi-system symptoms such as characteristic facial features, intellectual retardation, skeletal deformities, abnormal fingerprints, and immune deficiency. Its facial features mainly include arched eyebrows, thick eyelashes, and downward slanting palpebral fissures, which are similar to the makeup of traditional Japanese Kabuki actors, hence the name. The immune dysfunction in Kabuki syndrome requires special attention. Frequent and recurrent infections caused by immune dysfunction such as hypogammaglobulinemia and IgA deficiency may become the main early death factor in children with Kabuki syndrome. Intravenous immunoglobulin injection and examination of the number of peripheral blood immune cells are considered to be important guarantees for improving the quality of life of children. The incidence of Kabuki syndrome is about 1 case per 32,000 people, and it is seen in people of different races and regions. Because the symptoms of this syndrome are complex and variable, and the severity of the symptoms varies significantly among different patients, genetic testing is needed to assist in clinical diagnosis.

[0003] The international consensus published in 2019 clarified the diagnostic criteria for Kabuki syndrome (PMID: 30514738), including hypotonia, developmental delay or intellectual disability in infancy, and one or more of the following characteristics: (1) pathogenic or likely pathogenic mutations in the KMT2D or KDM6A genes; (2) typical facial features, such as long palpebral fissures, eversion of the outer third of the lower eyelid, and arched eyebrows, notched eyebrows or sparse eyebrows, short nose bridge, flat nose tip, large ears, etc. Although typical facial features are the hallmarks of Kabuki syndrome, some patients may show different clinical symptoms and varying degrees of developmental delay, so genetic testing is particularly important.

[0004] The main pathogenic genes of Kabuki syndrome are mutations of KMT2D and KDM6A genes, of which KMT2D gene mutations account for about 75% of cases. The KMT2D gene is located on chromosome 12 and encodes a histone lysine methyltransferase, whose main function is to methylate the 4th lysine residue (H3K4) of chromatin histone H3. H3K4 methylation is one of the key steps in gene expression activation. Functional defects or mutations of the KMT2D gene can affect chromatin structure, leading to abnormal regulation of multiple gene expression, affecting embryonic development, nervous system development and immune system function. Therefore, mutations in the KMT2D gene are usually loss-of-function mutations, which cause the methyltransferase it encodes to partially or completely lose its function, thereby causing abnormalities in neural development, bone development, immune system and other systems. Patients show symptoms such as typical facial features, developmental delay, bone deformities and immunodeficiency.

[0005] In-depth research on Kabuki syndrome helps to understand its genetic basis and pathogenesis, and provides support for improving the quality of life of patients. Currently, the detection of KMT2D gene mutations is the main means of diagnosing the syndrome. Early diagnosis is not only crucial for genetic counseling for patients and their families, but also helps to develop targeted management and treatment plans and improve prognosis. With the development of genome sequencing technology, scientists continue to discover new mutants in the KMT2D gene, many of which are considered 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.

[0006] Therefore, studies on the variation spectrum of the KMT2D gene are crucial to understanding the etiology of Kabuki syndrome. These studies reveal how gene mutations affect chromatin remodeling and gene expression, providing clues for the development of potential therapeutic strategies. Although several known mutations have been identified in the KMT2D gene, there are still many undiscovered mutation sites. Expanding the comprehensive understanding of KMT2D gene mutants will provide solid support for future clinical diagnosis, treatment, and research on the molecular mechanisms of Kabuki syndrome. Summary of the invention

[0007] The purpose of the present invention is to provide three KMT2D gene mutation sites discovered for the first time to enrich the variation spectrum of the KMT2D gene, and the three variation sites are KMT2D gene c.3247dup, c.11016_11019del, and c.15545del variation sites, respectively. The present invention also provides a primer and probe combination and a kit for detecting Kabuki syndrome KMT2D gene variation sites for screening or diagnosing Kabuki syndrome; comprising specific primers and TaqMan probe combinations for KMT2D gene c.3247dup, c.11016_11019del, and c.15545del variation sites, respectively.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] The specific primer and probe combination of the present invention is designed using PrimerPremier 6.0 software, specifically:

[0010] 1) Targeting the c.3247dup site of the KMT2D gene:

[0011] Forward primer A: 5'-TCGCCACTCCTTCAGCATTC-3' (SEQ ID NO. 1);

[0012] Reverse primer A: 5'-CGTGGCTCTTCCTGTTCTTCA-3' (SEQ ID NO. 2);

[0013] KMT2D gene wild-type probe A:

[0014] 5'-TCCAAGGCTGGGCATTCAGGTTCTGAAA-3' (SEQ ID NO. 3);

[0015] KMT2D gene mutation probe A:

[0016] 5'-TCCAAGGCTGGGCAATTCAGGTTCTGAAA-3' (SEQ ID NO.4),

[0017] 2) Targeting the KMT2D gene c.11016_11019del site:

[0018] Forward primer B: 5'-ACAGAGCAGCAGAGCAAGATCCAG-3' (SEQ ID NO. 5);

[0019] Reverse primer B: 5'-GGTCATAAGCACCTGTCTGTGAGG-3' (SEQ ID NO. 6);

[0020] KMT2D gene wild-type probe B:

[0021] 5'-AGAGCTGTATTAAGGAAGGGGCCACC-3' (SEQ ID NO. 7);

[0022] KMT2D gene mutation probe B:

[0023] 5'-AGAGCTGTATT----AAGGGGCCACC-3'(SEQ ID NO.8);

[0024] 3) Targeting the c.15545del site of the KMT2D gene:

[0025] Forward primer C: 5'-TGAACCTGGACCTGGACCTGTG-3' (SEQ ID NO. 9);

[0026] Reverse primer C: 5'-AGCGATAGCAGCAGCGACGAT-3' (SEQ ID NO. 10);

[0027] KMT2D gene wild-type probe C:

[0028] 5'-AACACAAGGCCCCCCACACGGAACA-3' (SEQ ID NO. 11);

[0029] KMT2D gene mutation probe C:

[0030] 5'-AACACAAGGCC-CCCACACGGAACA-3' (SEQ ID NO. 12);

[0031] The 5' end of the probe carries a different reporter fluorescent group, and the 3' end carries an MGB quencher fluorescent group.

[0032] In the same multiplex reaction system, the 5' ends of the probes respectively carry 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, AlexaFluor 633, ResoLight, EvaGreen, LC Green, Cy3, Cy5, Yellow555, LCRed610, ROX, SYPRO Ruby, LC Red640, Snarf 1, Acid Fuchsin, Cy5.5, LC Red670, and LCRed705.

[0033] A detection kit for detecting Kabuki syndrome KMT2D gene mutation sites by single-tube multiplex real-time fluorescence quantitative PCR technology based on TaqMan probes, comprising a reaction system A;

[0034] The reaction system A includes the detection primers and detection probes as shown below:

[0035] Detection primers:

[0036] Targeting the KMT2D gene c.3247dup site:

[0037] Forward primer A: 5′-TCGCCACTCCTTCAGCATTC-3′;

[0038] Reverse primer A: 5′-CGTGGCTCTTCCTGTTCTTCA-3′;

[0039] Targeting the KMT2D gene c.11016_11019del site:

[0040] Forward primer B: 5′-ACAGAGCAGCAGAGCAAGATCCAG-3′;

[0041] Reverse primer B: 5′-GGTCATAAGCACCTGTCTGTGAGG-3′;

[0042] Targeting the KMT2D gene c.15545del site:

[0043] Forward primer C: 5'-TGAACCTGGACCTGGACCTGTG-3';

[0044] Reverse primer C: 5′-AGCGATAGCAGCAGCGACGAT-3′;

[0045] Detection probe:

[0046] Targeting the KMT2D gene c.3247dup site:

[0047] KMT2D gene wild-type probe A:

[0048] 5'-TCCAAGGCTGGGCATTCAGGTTCTGAAA-3';

[0049] KMT2D gene mutation probe A:

[0050] 5'-TCCAAGGCTGGGCAATTCAGGTTCTGAAA-3';

[0051] Targeting the KMT2D gene c.11016_11019del site:

[0052] KMT2D gene wild-type probe B:

[0053] 5'-AGAGCTGTATTAAGGAAGGGGCCACC-3';

[0054] KMT2D gene mutation probe B:

[0055] 5'-AGAGCTGTATT----AAGGGGCCACC-3';

[0056] Targeting the KMT2D gene c.15545del site:

[0057] KMT2D gene wild-type probe C:

[0058] 5'-AACACAAGGCCCCCCACACGGAACA-3';

[0059] KMT2D gene mutation probe C:

[0060] 5'-AACACAAGGCC-CCCACACGGAACA-3';

[0061] The 5' end of the probe carries a reporter fluorescent group, and the 3' end carries a quencher fluorescent group; in the reaction system, the 5' end of each probe carries a different reporter fluorescent group, and 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, and the quencher fluorescent group is MGB;

[0062] The reaction system A is 30 μL, and each 30 μL of reaction system A contains:

[0063] 2×premix Taq TM15μL of buffer; 1μL of 10μmoL / L Forward primer A, 1μL of 10μmoL / L Reverse primer A; 0.5μL of 10μmoL / L Forward primer B, 0.5μL of 10μmoL / L Reverse primer B; 1μL of 10μmoL / L Forward primer C, 1μL of 10μmoL / L Reverse primer C; 1μL of 10μmoL / L wild-type probe A, 1μL of 10μmoL / L wild-type probe B, 1μL of 10μmoL / L wild-type probe C; 1μL of 10μmoL / L mutant probe A, 1μL of 10μmoL / L mutant probe B, 1μL of 10μmoL / L mutant probe C; 2μL of template DNA, 2μL of RNase and DNase-free water.

[0064] In the detection kit, when the TaqMan probe is amplified in a single tube multiplex real-time fluorescence quantitative PCR, the amplification reaction procedure is as follows:

[0065]

[0066] The present invention also provides the use of a reagent for detecting a KMT2D gene mutation site in preparing a Kabuki syndrome detection reagent, wherein the KMT2D gene mutation site comprises at least one of the following sites: a KMT2D gene c.3247dup site, a KMT2D gene c.11016_11019del site, and a KMT2D gene c.15545del site;

[0067] The gene number of the wild-type KMT2D gene in the NCBI database is: NM_003482.4,

[0068] Among them, the c.3247dup site of the KMT2D gene is a heterozygous mutation, and the base at position 3247 of the KMT2D gene is repeated. This mutation causes a frameshift mutation in the amino acid of the encoded protein from position 1083 (cysteine ​​mutates to leucine). After the frameshift mutation of 31 amino acids, the translation is terminated, i.e., p.Cys1083Leufs*32;

[0069] The c.11016_11019del site of the KMT2D gene is a heterozygous mutation, with bases from positions 11016 to 11019 deleted. This mutation causes a frameshift mutation (phenylalanine to leucine) in the amino acid encoding the protein from position 3672. After the frameshift mutation of 75 amino acids, translation is terminated, i.e., p.Phe3672Leufs*76;

[0070] The c.15545del site of the KMT2D gene is a heterozygous mutation, and the base at position 15545 of the KMT2D gene is deleted. This mutation causes a frameshift mutation in the amino acid encoded by the protein from position 5182 (glycine mutates to alanine). After 60 amino acids of the frameshift mutation, translation is terminated, i.e., p.Gly5182Alafs*61.

[0071] The three KMT2D gene variation sites, namely, the KMT2D gene c.3247dup site, the KMT2D gene c.11016_11019del site, and the KMT2D gene c.15545del site, were discovered by the present invention using high-throughput sequencing technology after excluding other Kabuki syndrome pathogenic genes. These variations have not yet been included in the gnomAD database and are the first pathogenic variation sites discovered.

[0072] The reagent for detecting the KMT2D gene variation site is one or more of a probe and a primer for detecting the KMT2D gene variation site.

[0073] The primers include primers for 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, and the primers for the sites are as follows:

[0074] Targeting the KMT2D gene c.3247dup site:

[0075] Forward primer A: 5′-TCGCCACTCCTTCAGCATTC-3′;

[0076] Reverse Primer A: 5'-CGTGGCTCTTCCTGTTCTTCA-3'

[0077] Targeting the KMT2D gene c.11016_11019del site:

[0078] Forward primer B: 5′-ACAGAGCAGCAGAGCAAGATCCAG-3′;

[0079] Reverse primer B: 5'-GGTCATAAGCACCTGTCTGTGAGG-3'

[0080] Targeting the KMT2D gene c.15545del site:

[0081] Forward primer C: 5'-TGAACCTGGACCTGGACCTGTG-3';

[0082] Reverse primer C: 5'-AGCGATAGCAGCAGCGACGAT-3'.

[0083] The probes include probes for 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, and the probes for the sites are as follows:

[0084] Targeting the KMT2D gene c.3247dup site:

[0085] KMT2D gene wild-type probe A:

[0086] 5'-TCCAAGGCTGGGCATTCAGGTTCTGAAA-3';

[0087] KMT2D gene mutation probe A:

[0088] 5'-TCCAAGGCTGGGCAATTCAGGTTCTGAAA-3',

[0089] Targeting the KMT2D gene c.11016_11019del site:

[0090] KMT2D gene wild-type probe B:

[0091] 5'-AGAGCTGTATTAAGGAAGGGGCCACC-3';

[0092] KMT2D gene mutation probe B:

[0093] 5'-AGAGCTGTATT----AAGGGGCCACC-3',

[0094] Targeting the KMT2D gene c.15545del site:

[0095] KMT2D gene wild-type probe C:

[0096] 5'-AACACAAGGCCCCCCACACGGAACA-3';

[0097] KMT2D gene mutation probe C:

[0098] 5'-AACACAAGGCC-CCCACACGGAACA-3'.

[0099] Compared with the prior art, the advantages of the present invention are: the present invention uses high-throughput sequencing technology to discover for the first time three KMT2D gene mutation sites on the basis of excluding other Kabuki syndrome pathogenic genes: KMT2D gene c.3247dup site, KMT2D gene c.11016_11019del site, KMT2D gene c.15545del site, these mutation sites are new pathogenic mutations associated with Kabuki syndrome, according to population frequency, family information, computer bioinformatics analysis, inheritance pattern, clinical phenotype, etc., the results are accurate, the KMT2D gene pathogenic mutation spectrum is expanded, and the diagnosis and genetic counseling of Kabuki syndrome are provided. In addition, the present invention provides primers and probe compositions and kits for detecting Kabuki syndrome KMT2D gene mutation sites for screening or diagnosing Kabuki syndrome; including specific primers and TaqMan probe compositions for KMT2D gene c.3247dup, c.11016_11019del, c.15545del mutation sites, respectively. Among them, the detection kit and detection method of the single-tube multiplex real-time fluorescence quantitative PCR technology based on TaqMan probe for detecting the mutation site of the KMT2D gene of Kabuki syndrome can comprehensively cover the three new pathogenic mutation sites of the KMT2D gene related to Kabuki syndrome. The multiplex TaqMan real-time fluorescence quantitative PCR technology is short in time and simple to operate. It only needs to add the DNA specimens of the subject to one reaction system of the present invention respectively, and then detect it on the machine without gel electrophoresis, purification recovery and sequencing and other post-processing. The results are accurate and reliable, which can meet the current clinical urgent need for a simple, fast and accurate patient and prenatal sample diagnosis and detection method. In addition, the primers and probes designed by the present invention have strong specificity, can specifically detect the three pathogenic mutation sites discovered for the first time, realize the diagnosis of Kabuki syndrome, and provide a basis for guiding clinical diagnosis and treatment and genetic counseling. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 These are the data quality control results of 72 suspected Kabuki syndrome patients tested with the Kabuki Syndrome-related Gene Mutation Detection Kit (Liquid Phase Hybridization Capture Sequencing Method).

[0101] Figure 2 This is a photograph of the eye features (palpebral fissure, superciliary arch) of Proband 1 in Example 2. The red arrow indicates the characteristic palpebral fissure extending outward with eversion of the outer 1 / 3 of the lower eyelid.

[0102] Figure 3 This is the pedigree of the Kabuki syndrome family to which Proband 1 in Example 2 belongs.

[0103] Figure 4This is a BAM file diagram of the c.3247dup mutation in the KMT2D gene of proband 1 in the Kabuki syndrome family in Example 2.

[0104] Figure 5 This is the pedigree of the proband 2 suspected of Kabuki syndrome in Example 3.

[0105] Figure 6 This is a list of candidate sequence variations detected by high-throughput sequencing of the suspected Kabuki syndrome proband 2 in Example 3.

[0106] Figure 7 This is a BAM file map of c.11016_11019del of the KMT2D gene of the suspected Kabuki syndrome proband 2 in Example 3.

[0107] Figure 8 This is the pedigree of the proband 3 suspected of Kabuki syndrome in Example 4.

[0108] Fig. 9 This is a list of candidate sequence variations detected by high-throughput sequencing of proband 3 suspected of Kabuki syndrome in Example 4.

[0109] Fig.10 This is a BAM file diagram of the c.15545del mutation of the KMT2D gene in the suspected Kabuki syndrome proband 3 in Example 4.

[0110] Fig.11 It is the result diagram of real-time fluorescence quantitative PCR of TaqMan probe at c.3247dup site of KMT2D gene in Example 5; wherein, the upper diagram is the result diagram of amplification curve of heterozygote at c.3247dup site of KMT2D gene; and the lower diagram is the result diagram of amplification curve of wild type at c.3247T site.

[0111] Fig.12 It is the real-time fluorescence quantitative PCR result diagram of the TaqMan probe at the c.11016_11019del site of the KMT2D gene in Example 5, wherein the upper figure is the amplification curve result diagram of the heterozygote at the c.11016_11019del site of the KMT2D gene; the lower figure is the amplification curve result diagram of the wild type at the c.11016_11019CCTT site.

[0112] Fig.13 It is the real-time fluorescence quantitative PCR result diagram of the TaqMan probe at the c.15545del site of the KMT2D gene in Example 5, wherein the upper figure is the amplification curve result diagram of the heterozygote at the 937del site of the KMT2D gene; the lower figure is the amplification curve result diagram of the wild type at the c.15545G site.

[0113] Fig.14This is the Sanger sequencing result diagram of the KMT2D gene NM_003482.4:c.3247dup variant in Example 6; wherein the red box is the detection site.

[0114] Fig.15 This is the Sanger sequencing result diagram of the KMT2D gene NM_003482.4:c.11016_11019del variation in Example 6; wherein the red box is the detection site.

[0115] Fig.16 This is a diagram of the Sanger sequencing results of the KMT2D gene NM_003482.4:c.15545del mutation in Example 6, wherein the red box is the detection site. DETAILED DESCRIPTION

[0116] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0117] Example 1 Detection of pathogenic mutations in patients with Kabuki syndrome using the Kabuki syndrome gene mutation detection kit (liquid phase hybridization capture sequencing)

[0118] This example uses 72 clinical peripheral blood samples sent to Furui Medical Laboratory for Kabuki syndrome or suspected Kabuki syndrome as research objects, and collects clinical symptoms and signs, laboratory tests, and imaging examinations such as patient growth and development history, intelligence, skeletal system, nervous system, digestive endocrine system, reproductive and urinary system, circulatory system, teeth, nail development, sweating, hearing, and vision. The proband of candidate Kabuki syndrome and some family members were subjected to high-throughput sequencing detection of "Kabuki syndrome gene mutation detection kit (liquid hybridization capture sequencing)", which includes the following steps performed in sequence:

[0119] (1) Sample collection and genomic DNA extraction.

[0120] Blood samples (EDTA anticoagulation) were collected from the proband and / or family members, and genomic DNA from the blood of each family member was extracted according to the instructions of the blood DNA extraction kit (Magen, HiPure Blood & Tissue DNA Kit). The purity of the DNA was measured using Nanodrop one, and the OD260nm / OD280nm of the obtained genomic DNA was between 1.7 and 2.0. The concentration of the DNA was measured using Nanodrop one, and the concentration of the obtained genomic DNA was 50-100 ng / μL, and the total amount was 5-10μg. Store at -20°C.

[0121] (2) High-throughput sequencing and bioinformatics analysis of Kabuki syndrome gene mutations.

[0122] First, the extracted genomic DNA was fragmented using KAPA's kit (KAPA HyperPlus Library Preparation Kit), and the genomic DNA was subjected to enzyme fragmentation, end repair, 3' end A addition, linker connection and PCR amplification; the "Kabuki syndrome-related gene mutation detection kit (liquid phase hybridization capture sequencing)" developed by Furui Medical Laboratory was used to capture the exon regions of KMT2D, KDM6A, CHD7, IRF6, EYA1, FLNB, SIX1, SIX5 genes, and other regions with HMGD and ClinVar pathogenic variants. These 8 genes include all candidate genes for the diagnosis and differential diagnosis of Kabuki syndrome in the international authoritative database Genereviews. The library was sequenced on a Novasek sequencer (Illumina, San Diego, CA, USA) (sequencing depth ≥150X, 20X coverage ≥98%). According to historical data, the 20X coverage of KMT2D, KDM6A, CHD7, IRF6, EYA1, FLNB, SIX1, and SIX5 genes in all sequenced samples reached 100%. The performance study results showed that the Kabuki Syndrome-Related Gene Mutation Detection Kit (Liquid Hybridization Capture Sequencing) can detect all candidate variants of the above 8 genes in clinical samples ( Figure 1 ) 。

[0123] The secondary and tertiary analysis of sequencing data was completed using the ISoGenetic v1.2.6 system (Shanghai Fujun Gene Biotechnology Co., Ltd.). Specifically, the NGS sequencing results were aligned with the human reference genome UCSCNCBI37 / hg19 using SentieonBWAv0.7.15 to obtain the unique alignment sequence aligned to the genome; SAMtools v1.9 and Sentieon GATK software v4.1.4.0 were used to detect and determine the variation in the target region; Remove RunCommonVariants and Remove Global CommonVariants software were used to remove common variations (>5%) in the dbSNP and gnomAD databases; Alamut-Batch standalone version v1.9 was used to analyze the sequence of the target region; the sequence of the target region was analyzed using the Alamut-Batch standalone version v1.9. software to annotate variants (annotation databases include: dbSNP, gnomAD, 1000g, ClinVar, OMIM, HGMD Professional Edition, etc.); use filterAlamut.py to sort the annotated variants according to high, medium, and low priority. In the high and medium groups, give the variants a priority value and a grading reason. All variants are initially in the low group. When a variant meets the preset pathogenicity criteria, it can be classified as a higher-level variant; use FATHMM, FATHMMMKL, METALR, METASVM, MUTATIONASSESSOR, MUTATIONTASTERAGVGD, AGVGD, LRT, PROVEAN, SIFT, REVEL, and SpliceAI software provided by the VarSome database to perform bioinformatics prediction of SNP function and splicing activity; CNV TM The exon tool performs DNA copy number variation (CNV) analysis (resolution of ≥1 exon).

[0124] (3) Interpretation of candidate gene variants

[0125] The interpretation of sequence variation is based on the internationally authoritative "2015 ACMG Guidelines for Clinical Interpretation of Genetic Variation" (ACMG Guidelines) and various guidelines of the ClinGen Sequence Variant Interpretation (SVI) Working Group (SVI); the interpretation of CNV variation is based on the "2019 ACMG Interpretation of Chromosome Copy Number Variation Interpretation and Reporting Technical Standards". The various variations detected by the above sequencing are classified as pathogenic variation (P), possible pathogenic variation (LP), variation of unknown significance (VUS), possible benign variation (LB), and benign variation (B).

[0126] Example 2 KMT2D gene NM_003482.4:c.3247dup (p.Cys1083Leufs*32) pathogenic variant in Kabuki syndrome cases

[0127] Proband 1 is a 4-year-old male with developmental delay and a unique facial appearance (wide-set eyes, narrow palpebral fissures, ectropion of the lateral third of the lower eyelid, and high brow ridges) ( Figure 2 ), afebrile seizures for 2 years and 9 months. Developmental delay, onset at 1 year and 3 months, conscious, eyes slanting to one side, relieved in 20 seconds, 3 times / day. Preliminary diagnosis: Kabuki syndrome, epilepsy. Father and mother are healthy, see pedigree for details ( Figure 3 ).

[0128] In this example, the high-throughput sequencing of the genetic bone disease gene was performed using the "Kabuki syndrome-related gene mutation detection kit (liquid hybridization capture sequencing method)" and found that the proband carried 1 sequence variation (population frequency ≤ 5%) and 0 candidate CNV variation. The sequence variation was a heterozygous variation at the c.3247dup site of the KMT2D gene ( Figure 4 ), with a priority of 1 (highest level). This mutation causes a frameshift mutation in the amino acid encoding the protein from position 1083 (cysteine ​​mutates to leucine). Translation terminates 31 amino acids after the frameshift mutation. According to the ACMG guidelines, this variant is classified as a pathogenic variant. The relevant evidence for this variant is PVS1+PS2+PM2, specifically:

[0129] PS2: The gene was not detected in the peripheral blood of the parents of the subject, suggesting that it may be a new mutation.

[0130] PVS1: This variation occurs in exon 11 of the 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. There are multiple loss-of-function variations (DM) included in HGMD downstream of this variation.

[0131] PM2: This variant is not currently included in the gnomAD database.

[0132] Therefore, the KMT2D gene mutant c.3247dup (p.Cys1083Leufs*32) was discovered by high-throughput sequencing technology after excluding other Kabuki syndrome pathogenic genes. It is a new pathogenic variant associated with Kabuki syndrome, and this variant can explain the special facial features and epilepsy phenotype of the proband, which is a diagnostic result for the proband. According to the population frequency, family information, computer bioinformatics analysis, clinical phenotype, etc., the results are accurate, expanding the spectrum of KMT2D gene pathogenic variants, and providing a basis for the diagnosis and genetic counseling of Kabuki syndrome.

[0133] Example 3 Pathogenic variant of KMT2D gene NM_003482.4:c.11016_11019del (p.Phe3672Leufs*76) in suspected Kabuki syndrome

[0134] Proband 2 is a 6-year-old male with a suspected diagnosis of Kabuki syndrome, growth retardation, multiple congenital malformations, transient neonatal hypoglycemia, acute respiratory failure, and cleft palate. After birth, the child was found to have cleft palate, spina bifida occulta, low nose bridge, extended palpebral fissures, arched eyebrows, webbed neck, low ears, aortic arch stenosis, patent ductus arteriosus, atrial septal defect, pulmonary hypertension, and small kidneys. The father, mother, and sister are healthy. For details, see the pedigree chart ( Figure 5 ).

[0135] In this example, the high-throughput sequencing of the genetic bone disease gene was performed using the "Kabuki syndrome-related gene mutation detection kit (liquid hybridization capture sequencing method)" and found that the proband carried 3 sequence variations (population frequency ≤ 5%) ( Figure 6 ), 0 candidate CNV variants. Among the 3 candidate sequence variants, 2 FLNB gene sequence variants NM_001457.3:c.2576-64C>T (p.?) and NM_001457.3:c.2482C>G (p.Gln828Glu) were excluded first, among which NM_001457.3:c.2576-64C>T variant is located in the non-coding region. According to the ACMG guidelines, this variant is classified as a possible benign variant. The relevant evidence of this variant is BS1 (population frequency is 0.94%, allele Frequency is greater than the disease incidence) + BP4 (CADD and other bioinformatics tools predict it to be benign) + BP7 (synonymous or non-coding region variants are predicted by bioinformatics tools not to affect splicing); NM_001457.3:c.2482C>G variant, according to the ACMG guidelines, this variant is classified as a possible benign variant, and the relevant evidence for this variant is BS1 (the frequency in the local population is 0.33% (62 / 18968), and the allele frequency is greater than the disease incidence) + BP4 (REVEL=0.193 predicted to be benign).

[0136] Heterozygous mutation of KMT2D gene c.11016_11019del site ( Figure 7 ), with a priority of 1 (highest level). This mutation causes a frameshift mutation in the amino acid encoding the protein from position 3672 (phenylalanine mutates to leucine). Translation terminates 75 amino acids after the frameshift mutation. This variant has not yet been 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:

[0137] PVS1: This variation occurs in exon 39 of the 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. There are multiple loss-of-function variations (DM) included in HGMD downstream of this variation.

[0138] PM2: This variant has not yet been included in gnomAD v2.1.1.

[0139] PS2: It was not detected in the peripheral blood of the parents of the subject, suggesting that it may be a new mutation (PS2).

[0140] Therefore, the KMT2D gene mutant c.11016_11019del (p.Phe3672Leufs*76) was discovered by high-throughput sequencing technology after excluding other Kabuki syndrome pathogenic genes. It is a new pathogenic variant associated with Kabuki syndrome, and this variant can explain the multiple congenital malformations sporadically in the proband. It is 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 finally diagnosed with Kabuki syndrome from suspected Kabuki syndrome. According to the population frequency, family information, computer bioinformatics analysis, clinical phenotype, etc., 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.

[0141] Example 4 KMT2D gene NM_003482.4:c.15545del (p.Gly5182Alafs*61) pathogenic variant in suspected Kabuki syndrome

[0142] Proband 3 is a 7-year-old male with suspected Kabuki syndrome as the main diagnosis. The clinical manifestations are obesity, mental retardation, and special facial features. He raised his head at 3 months old, could not sit at 9 months old, could speak at 1 year old, but now cannot write and cannot do addition and subtraction within 10. He has an obese body, a long face, long eye slits, large protruding ears, freckles on the face, a small penis, and small testicles. The father and mother were healthy in the past. See the pedigree for details ( Figure 8 ).

[0143] In this example, the high-throughput sequencing of the genetic bone disease gene was performed using the "Kabuki syndrome-related gene mutation detection kit (liquid hybridization capture sequencing method)" and found that the proband carried 2 sequence variations (population frequency ≤ 5%) ( Fig. 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 REVEL software to have no effect on protein function and was inherited from a healthy father, indicating that the disease was not isolated from the family. According to the ACMG guidelines, the variant was classified as a possible benign variant. The relevant evidence for the variant was BS4 (lack of co-segregation in a family member) + BP5 (a variant found in a case with another molecular pathogenic cause c.15545del). Heterozygous variant of the KMT2D gene c.15545del site ( Fig.10 ), with a priority of 1 (highest level). This variant causes a deletion of the 15545th base in the coding region, which causes a frameshift mutation (glycine to alanine) in the amino acid encoding the protein from the 5182th position. After 60 amino acids of the frameshift mutation, translation is terminated. According to the ACMG guidelines, it is recommended that this variant be rated as a pathogenic variant. The relevant evidence for this variant is PVS1+PS2+PM2, specifically:

[0144] PVS1: This frameshift variant is a 1-base pair deletion, resulting in an out-of-frame transcript and a premature stop codon. This variant may activate nonsense-mediated mRNA degradation, resulting in loss of function of the protein product of the KMT2D gene. Currently, this variant has not been reported in the literature, and downstream truncating variants of this variant are known to be pathogenic variants (PubMed: 22126750, 21671394, 21280141).

[0145] PS2: This variant was verified to be a novel variant, as it was not detected in the samples of the father and mother of proband 3;

[0146] PM2: The variant has not yet been included in the gnomAD database.

[0147] Therefore, the KMT2D gene mutant c.15545del was discovered using high-throughput sequencing technology after excluding other Kabuki syndrome pathogenic genes. It is a new pathogenic variant associated with Kabuki syndrome, and this variant can explain the sporadic Kabuki syndrome phenotype of the proband. It is a diagnostic result for the proband. According to the Kabuki syndrome diagnosis and treatment guidelines, the patient was suspected of Kabuki syndrome and was finally diagnosed with Kabuki syndrome. According to the population frequency, family information, clinical phenotype, etc., the results are accurate, expanding the spectrum of KMT2D gene pathogenic variants and providing a basis for the diagnosis and genetic counseling of Kabuki syndrome. In terms of genetic counseling, Kabuki syndrome caused by KMT2D gene mutations is a clinically variable genetic heterogeneous disease. Comprehensive monitoring of patients with Kabuki syndrome is recommended: measuring height and weight at each visit to monitor growth and development; performing an ophthalmological or optometric examination at least once a year to assess vision; performing hearing tests regularly; performing clinical assessments of scoliosis at each visit before skeletal maturity; testing thyroid function every 2 to 3 years to assess endocrine health; and regularly checking complete blood counts to monitor the status of the immune system. In addition, attention should be paid to developmental progress and educational needs at each visit during childhood and adolescence to provide timely support.

[0148] Example 5 Primer and probe composition and kit for detecting Kabuki syndrome KMT2D gene mutation site

[0149] (1) The primer and probe combination and kit for detecting the mutation site of the KMT2D gene of Kabuki syndrome are used for screening or diagnosing Kabuki syndrome; they include specific primers and TaqMan probe combinations for the mutation sites of the KMT2D gene c.3247dup, c.11016_11019del, and c.15545del, respectively.

[0150] The specific primer and probe combination was designed using Primer Premier 6.0 software, specifically:

[0151] 1) Targeting the c.3247dup site of the KMT2D gene:

[0152] Forward primer A: 5′-TCGCCACTCCTTCAGCATTC-3′;

[0153] Reverse Primer A: 5'-CGTGGCTCTTCCTGTTCTTCA-3'

[0154] KMT2D gene wild-type probe A:

[0155] 5'-TCCAAGGCTGGGCATTCAGGTTCTGAAA-3';

[0156] KMT2D gene mutation probe A:

[0157] 5'-TCCAAGGCTGGGCAATTCAGGTTCTGAAA-3',

[0158] 2) Targeting the KMT2D gene c.11016_11019del site:

[0159] Forward primer B: 5′-ACAGAGCAGCAGAGCAAGATCCAG-3′;

[0160] Reverse primer B: 5'-GGTCATAAGCACCTGTCTGTGAGG-3'

[0161] KMT2D gene wild-type probe B:

[0162] 5'-AGAGCTGTATTAAGGAAGGGGCCACC-3';

[0163] KMT2D gene mutation probe B:

[0164] 5'-AGAGCTGTATT----AAGGGGCCACC-3'

[0165] 3) Targeting the c.15545del site of the KMT2D gene:

[0166] Forward primer C: 5'-TGAACCTGGACCTGGACCTGTG-3';

[0167] Reverse primer C: 5'-AGCGATAGCAGCAGCGACGAT-3'

[0168] KMT2D gene wild-type probe C:

[0169] 5'-AACACAAGGCCCCCCACACGGAACA-3';

[0170] KMT2D gene mutation probe C:

[0171] 5'-AACACAAGGCC-CCCACACGGAACA-3';

[0172] The 5' end of the probe carries a different reporter fluorescent group, and the 3' end carries an MGB quencher fluorescent group.

[0173] The 5' end of the probe carries a different reporter fluorescent group, for example, 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.

[0174] (2) 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 the 2× premix Taq TM Buffer (LATaq TM Version 2.0) and RNase- and DNase-free water.

[0175] (3) The kit adopts a single-tube multiplex fluorescent PCR reaction system, which is as follows:

[0176] Reaction system: KMT2D gene c.3247dup site, c.11016_11019del site and c.15545del site

[0177]

[0178] (4) The amplification reaction procedure is as follows:

[0179]

[0180] (5) Operational procedures for multiplex probe reagent detection of Kabuki syndrome KMT2D gene c.3247dup, c.11016_11019del, c.15545del mutation sites:

[0181] As described in Example 1, clinical tissue samples are collected from the proband or family members of Kabuki syndrome, including but not limited to EDTA / sodium citrate anticoagulated peripheral blood;

[0182] As described in Example 1, genomic DNA from clinical tissue samples was obtained and quality controlled;

[0183] Using genomic DNA samples, a multiplex fluorescent PCR reaction system is configured, and an amplification reaction is performed on a fluorescent quantitative PCR instrument according to the above reaction procedure;

[0184] Result interpretation: After the fluorescence quantitative PCR is completed, the software is used to analyze the data and obtain the results of each subject. By judging whether there are amplification peaks of wild-type and mutant-type specific probes at each detection site of the subject ( Figure 11-13 ), and determine whether the subject carries the KMT2D gene pathogenic site according to the standards in the table below.

[0185]

[0186] Example 6 Performance study results of a kit for detecting Kabuki syndrome KMT2D gene mutation sites

[0187] (1) Analytical performance research plan

[0188] In order to study the analytical performance of the single-tube multiplex fluorescent TaqMan probe PCR detection kit for the Kabuki syndrome KMT2D gene variant site in the present invention, 120 peripheral blood samples were used as research objects for performance research in this example, including: clinical samples of 72 candidate Kabuki syndrome patients for NGS detection in Example 1, 42 normal control samples, and 6 clinical samples (EDTA anticoagulated peripheral blood) from 1-3 family members of the proband. All positive samples were compared with the NGS analysis results and Sanger sequencing verification results. The sensitivity (Sensitivity) and specificity (Specificity) of the kit of the present invention for detecting the Kabuki syndrome KMT2D gene c.3247dup, c.11016_11019del, and c.15545del variant sites were calculated respectively, and the formula is:

[0189] Sensitivity = number of true positive results / (number of true positive results + number of false negative results)

[0190] Specificity = number of true negative results / (number of true negative results + number of false positive results)

[0191] (2) The system and steps of Sanger sequencing verification:

[0192] PCR amplification system (20 μL) for c.3247dup, c.11016_11019del, and c.15545del variant sites. Each PCR reaction was a single-site single-tube amplification system, with a total of 3 amplification systems: 2× premix Taq TMBuffer 10μL, Forward primer (10μmoL) 1μL, Reverse primer (10μmoL) 1μL, ddH2O6μL, DNA2μL; the primers are the amplification primers corresponding to each variant site provided in Example 5 above; PCR reaction program: 95℃5min, 35 cycles (95℃5min, 95℃30s, 62℃1min), 72℃10min, 4℃ storage. After PCR amplification, 1% agarose gel electrophoresis was used for detection. After gel cutting, the product was recovered with the "Ordinary Agarose Gel DNA Recovery Kit (DP209)", and the product was diluted to 10ng / μL, and the product was purified and recovered with Taq enzyme. All PCR products were subjected to Sanger sequencing analysis on the ABI3730XL (Applied Biosystems) platform using amplification primers.

[0193] (3) Analyze performance research results

[0194] The sensitivity and specificity of the Kabuki syndrome KMT2D gene mutation site detection kit of the present invention for detecting the c.3247dup mutation site are both 100%, as shown in the following table and the Sanger sequencing verification results ( Fig.14 ):

[0195]

[0196] The sensitivity and specificity of the Kabuki syndrome KMT2D gene mutation site detection kit of the present invention for detecting the c.11016_11019del mutation site are both 100%, as shown in the following table and the Sanger sequencing verification results ( Fig.15 ):

[0197]

[0198] The sensitivity and specificity of the Kabuki syndrome KMT2D gene mutation site detection kit of the present invention for detecting the c.15545del mutation site are both 100%, as shown in the following table and the Sanger sequencing verification results ( Fig.16 ):

[0199]

[0200] The above describes an embodiment of the present embodiment, but the present embodiment is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present embodiment, ordinary technicians in this field can also make many forms, all of which are within the protection of the present embodiment.

Claims

1. A single-tube multiplex real-time fluorescence quantitative PCR detection kit for detecting Kabuki syndrome KMT2D gene mutation sites based on TaqMan probes, characterized in that: It includes a reaction system A; The reaction system A includes the detection primers and detection probes as shown below: Detection primers: Targeting the KMT2D gene c.3247dup site: Forward primer A: 5′-TCGCCACTCCTTCAGCATTC-3′; Reverse primer A: 5′-CGTGGCTCTTCCTGTTCTTCA-3′; Targeting the KMT2D gene c.11016_11019del site: Forward primer B: 5′-ACAGAGCAGCAGAGCAAGATCCAG-3′; Reverse primer B: 5′-GGTCATAAGCACCTGTCTGTGAGG-3′; Targeting the KMT2D gene c.15545del site: Forward primer C: 5'-TGAACCTGGACCTGGACCTGTG-3'; Reverse primer C: 5′-AGCGATAGCAGCAGCGACGAT-3′; Detection probe: Targeting the KMT2D gene c.3247dup site: KMT2D gene wild-type probe A: 5'-TCCAAGGCTGGGCATTCAGGTTCTGAAA-3'; KMT2D gene mutation probe A: 5'-TCCAAGGCTGGGCAATTCAGGTTCTGAAA-3'; Targeting the KMT2D gene c.11016_11019del site: KMT2D gene wild-type probe B: 5'-AGAGCTGTATTAAGGAAGGGGCCACC-3'; KMT2D gene mutation probe B: 5'-AGAGCTGTATT----AAGGGGCCACC-3'; Targeting the KMT2D gene c.15545del site: KMT2D gene wild-type probe C: 5'-AACACAAGGCCCCCCACACGGAACA-3'; KMT2D gene mutation probe C: 5'-AACACAAGGCC-CCCACACGGAACA-3'; The 5' end of the probe carries a reporter fluorescent group, and the 3' end carries a quencher fluorescent group; in reaction system A, the 5' end of each probe carries a different reporter fluorescent group.

2. The detection 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×premix Taq TM Buffer 15μL; 10μmoL / L Forward Primer A 1μL, 10μmoL / L Reverse Primer A 1μL; 10μmoL / L Forward Primer B 0.5μL, 10μmoL / L Reverse Primer B 0.5μL; 10μmoL / L Forward primer C1μL, 10μmoL / L Reverse primer C1μL; 10μmoL / L wild-type probe A1μL, 10μmoL / L wild-type probe B1μL, 10μmoL / L wild-type probe C1μL; 10μmoL / L mutant probe A1μL, 10μmoL / L mutant probe B1μL, 10μmoL / L mutant probe C1μL; 2μL template DNA, 2μL RNase and DNase-free water.

3. The detection 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 quenching fluorescent group is MGB.

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

5. Use of a reagent for detecting KMT2D gene mutation sites in the preparation of a Kabuki syndrome detection reagent, characterized in that: The KMT2D gene mutation site includes at least one of the following sites: KMT2D gene c.3247dup site, KMT2D gene c.11016_11019del site, KMT2D gene c.15545del site; The gene number of the wild-type KMT2D gene in the NCBI database is: NM_003482.4, Among them, the c.3247dup site of the KMT2D gene is a heterozygous mutation, and the base at position 3247 of the KMT2D gene is repeated. This mutation causes a frameshift mutation in the amino acid of the encoded protein from position 1083 (cysteine ​​mutates to leucine). After the frameshift mutation of 31 amino acids, the translation is terminated, i.e., p.Cys1083Leufs*32; The c.11016_11019del site of the KMT2D gene is a heterozygous mutation, with bases from positions 11016 to 11019 deleted. This mutation causes a frameshift mutation (phenylalanine to leucine) in the amino acid encoding the protein from position 3672. After the frameshift mutation of 75 amino acids, translation is terminated, i.e., p.Phe3672Leufs*76; The c.15545del site of the KMT2D gene is a heterozygous mutation, and the base at position 15545 of the KMT2D gene is deleted. This mutation causes a frameshift mutation in the amino acid encoded by the protein from position 5182 (glycine mutates to alanine). After 60 amino acids of the frameshift mutation, translation is terminated, i.e., p.Gly5182Alafs*61.

6. The use according to claim 5, characterized in that: The reagents for detecting the KMT2D gene mutation site are probes and primers for detecting the KMT2D gene mutation site.

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

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

Images