Primer group and kit for detecting various mutations of Gaucher disease GBA1 gene and pseudogene GBAP1 thereof and application of primer group and kit

Abstract

The invention provides a primer group and a kit for detecting various mutations of a Gaucher disease GBA1 gene and a pseudogene GBAP1 of the Gaucher disease GBA1 gene, and application of the primer group and the kit. Specifically, the invention provides a primer group which is used for simultaneously amplifying or detecting various mutations of a GBA1 gene and a pseudogene GBAP1 thereof, and the primer group comprises a GBA-F primer, a GBA-R primer and a GBAP-R primer and further comprises a Gap-F primer. The invention also provides a kit which comprises the primer group. Based on a specific combination of homologous region multiple long fragment GAP PCR amplification and single molecule length high-throughput sequencing, the kit can be used for simultaneously detecting multiple mutations of GD-related GBA1 pathogenic genes and pseudogenes GBAP1 thereof in multiple samples in a high-specificity, accurate and rapid manner.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a method for detecting Gaucher disease-related GBA1 Genes and their pseudogenes GBAP1 Primer sets, kits and applications for multiple mutations. Background Art

[0002] Gaucher disease (GD) is a rare autosomal recessive genetic disease caused by GBA1 Gaucher disease is caused by a gene mutation. It is characterized by hepatosplenomegaly, decreased cellularity, sometimes severe bone involvement, and, in some forms, nerve damage. 1 . GD can be divided into 3 types based on clinical manifestations. Type 1 GD (GD1, chronic non-neuropathy, accounting for: 90%-95%) is the most common phenotypic form of the disease. Its clinical manifestations are variable, ranging from lifelong asymptomatic to early-onset forms that appear in childhood. The overall average age of onset of patients is 20.4 years 2 GD1 usually affects quality of life but is rarely life-threatening. Common phenotypes include fatigue, growth retardation and delayed puberty, hepatosplenomegaly, gallstones, bone involvement, etc. 2,3,4 Type 2 GD (GD2, acute neuropathy, <5%) is characterized by early and severe phenotypic symptoms beginning in infants aged 3-6 months, brainstem involvement (i.e., eye movement abnormalities, spasticity, hypotonia) and seizures, as well as life-threatening systemic symptoms such as respiratory distress and aspiration pneumonia, with a life expectancy of less than 2 years. 5 Type 3 GD (GD3, chronic neuropathy, accounting for 5%), the phenotype of GD3 is also very heterogeneous, mainly manifested in liver and spleen involvement, heart involvement (valve calcification), aortic calcification, etc., in the nervous system, including progressive myoclonic epilepsy, cerebellar ataxia, spasticity or dementia 6,7,8 . It has a significant negative impact on the child's health-related quality of life. In addition to the above three, fetal GD is the rarest (<1%) and most serious form of the disease. It usually manifests as fetal edema, hepatosplenomegaly, ichthyosis, joint contractures, facial deformities, and death usually occurs in utero or shortly after birth. 9 The incidence of GD is approximately 1 / 40,000 to 1 / 60,000 in the general population, but can reach 1 / 800 in Ashkenazi Jews. 10 Due to clinical heterogeneity, patients are often misdiagnosed and there are delays in obtaining an accurate diagnosis, with the diagnosis of GD patients often delayed by up to 10 years. Early diagnosis is important to initiate appropriate treatment and prevent complications and disease progression. 11 .

[0003] GBA1The gene is located on the long arm of chromosome 1 (1q21) and contains 11 exons. A highly homologous pseudogene ( GBAP1 ), which leads to GBA1 and GBAP1 The sites of these recombination events are variable, ranging from intron 2 to exon 11. 12 So far, GBA1 Approximately 400 pathogenic mutations have been identified in the gene. These include substitutions, insertions, deletions, and complex alleles. The most common mutations are missense mutations, of which point mutations c.1226A>G (N370S) and c.1448T>C (L444P) are the most common. However, the N370S mutation is rarely found in Chinese and Japanese patients, and in Asian ethnic groups, c.1448T>C (L444P / L483P) and c.754T>A (F252I) are more common. 13 .

[0004] Initially, the laboratory used PCR-based screening techniques to identify a limited number of known mutations. This was a fairly efficient approach in patients with GD1 of Ashkenazi Jewish descent. 14 However, especially in patients with neuropathic GD, rarer pathological variants may be missed when based on limited mutations, leading to false negatives. DNA sequencing using the Sanger method has been the gold standard for mutation identification in GD patients for the past 30 years, but due to the limitations of Sanger sequencing, it is not suitable for GBA1 Conduct system analysis 15 .

[0005] The advent of NGS, increased availability, and reduced costs have been important factors in its wider use to establish a challenging diagnosis. Wider inclusion of NGS in genotyping GD patients has led to the discovery of many new GBA1 mutation. However, when new GBA1 In addition, when highly homologous GBA1 Recombinant alleles generated by genomic recombination of pseudogenes may be difficult to detect using this method 16 Therefore, in addition to NGS analysis, at least 30 regions of the gene should be analyzed by Sanger sequencing. Without the support of Sanger sequencing, many studies using NGS technology do not report the presence of recombination mutations, which increases the possibility that the presence of these alleles is underestimated. 15 .

[0006] In recent years, with the emergence and development of SMRT sequencing and Nanopore sequencing, it is easy to achieve long-read sequencing of several kb and complete GBA1 Sequencing of genomic regions 17 But currently GBA1 Gene long fragment enrichment systems focus only on GBA1 Gene region, this enrichment method has certain defects and cannot detect all GBA1 and GBAP1 The types of structural variations formed between 18 The existing enrichment methods also exclude GBAP1 Genetic testing, studies have shown that GBAP1 May act as ceRNA regulator GBA1 Gene expression 19 None of the existing systems have simultaneously enriched GBA1 and GBAP1 Although it reduces the difficulty of amplification and avoids non-specific amplification of homologous regions, it can more accurately enrich functional genes; however, due to the lack of enrichment of pseudogenes, the detection system is more complicated and cannot comprehensively screen and display mutations in the region.

[0007] Existing technology can achieve GBA1 Some structural variations and point mutations are detected, but the detection has the following main limitations: It is impossible to detect them simultaneously in the same system GBA1 All point mutations and structural variation types of genes; GBA1 and GBAP1 The high homology between them may affect the design of Sanger sequencing primers and the sequencing results of NGS, resulting in a certain degree of false negative or false positive; when two or more mutations exist at the same gene locus at the same time, the existing methods cannot distinguish between cis or trans mutations, and whether the mutations have a chain effect; the existing GBA1 Long fragment amplification technology is only suitable for GBA1 Gene regions are enriched if GBA1 and GBAP1 The interaction between non-allelic genes caused by homologous recombination cannot be detected; the existing technology ignores the non-functional genes. GBAP1 The detection of GBA1 "Comprehensive" testing of the region; because traditional methods require the combination of multiple technologies, they are labor-intensive, have higher requirements on sample quality, take a long time for genetic diagnosis, and are expensive. Summary of the invention

[0008] As mentioned above, existing long fragment amplification technologies are usually only GBA1 Gene regions were enriched and pairs were excluded GBAP1Although the amplification of genes reduces the difficulty of amplification and avoids non-specific amplification of homologous regions, it will make it impossible to detect the interaction between non-allelic genes caused by homologous recombination. In view of this, the present invention provides a method based on homologous region multiple long fragment GAP PCR amplification and single molecule sequencing to detect GD-related GBA1 Genes and their pseudogenes GBAP1 multiple mutations.

[0009] Based on the special design and setting of the primer set, the present invention can not only restore the entire GBA1 and GBAP1 Variations in gene regions, and overcome GBA1 and GBAP1 The amplification preference problem of simultaneous enrichment of homologous regions of genes reduces the problem of background generation in homologous regions, and realizes multiple long-fragment GAP PCR amplification of homologous regions in one reaction tube, while amplifying the exon SNVs and InDels. GBA1 Gene fragments, for detection GBA1 The absence and fusion of GBAP1 Pseudogene fragments and Gap fragments for detecting large fragment deletions. Combined with the long read length and other characteristics of the single-molecule sequencing platform, the present invention can achieve accurate, rapid and high-throughput detection GBA1 Genes and their pseudogenes GBAP1 The method of the present invention is simple to operate, and the homologous region multiple long-fragment GAP PCR and single-molecule sequencing library have reliable quality and strong repeatability, which is conducive to the application of single-molecule sequencing technology in clinical detection.

[0010] The purpose of the present invention is to solve the current GBA1 Incomplete coverage of pathogenic gene detection, cumbersome genetic diagnosis and detection methods, and single detection of variant types by different methods lead to clinical problems of missed detection and misdiagnosis. GBA1 Pathogenic genes and their pseudogenes GBAP1 Multiple fragments and preparation of single-molecule sequencing libraries to achieve comprehensive, accurate and rapid detection of GD-related genes in multiple samples GBA1 and its pseudogenes GBAP1 multiple mutation targets.

[0011] In one aspect, the present invention provides a primer set for simultaneously amplifying or detecting GBA1 Genes and their pseudogenes GBAP1The primer set comprises a GBA-F primer, a GBA-R primer and a GBAP-R primer; wherein the nucleotide sequence of the GBA-F primer is shown in SEQ ID NO: 1-2, the nucleotide sequence of the GBA-R primer is shown in SEQ ID NO: 5-7, and the nucleotide sequence of the GBAP-R primer is shown in SEQ ID NO: 3-4. In some embodiments, the primer set further comprises a Gap-F primer, wherein the nucleotide sequence of the Gap-F primer is shown in SEQ ID NO: 8-11. The positions of all primers are as shown in Figure 1 shown.

[0012] In some embodiments, the GBA1 Genes and their pseudogenes GBAP1 Multiple mutations include GBA1 SNV and InDel on gene exons, GBA1 and GBAP1 Deletions and fusions caused by gene recombination and their corresponding GBA1 Gene copy number variation, GBA1 Large fragment deletion variation within 20kb upstream. In some embodiments, the GBA1 The gene mutations include at least 640 mutations as shown in Table 1.

[0013]

[0014]

[0015]

[0016]

[0017]

[0018] In some preferred embodiments, the primer set is used for multiple long fragment GAP PCR amplification of homologous regions, and simultaneously amplifies in one reaction system GBA1、GBAP1 Therefore, the primer set can simultaneously detect the gene fragment and the gap fragment in the same reaction system. GBA1 SNV and InDel on gene exons, GBA1 and GBAP1 Deletions and fusions caused by gene recombination and their corresponding GBA1 Gene copy number variation, GBA1 Large deletion mutation within 20 kb upstream. The point mutations and structural variations at the gene loci described in this article can be queried in LOVD, ClinVar and references. Some mutation information is shown in Table 1.

[0019] In some embodiments, the amplification product of the primer set is 10 kb-18 kb. In some preferred embodiments, if there is a SNP at the primer position, a degenerate base primer is used.

[0020] In some embodiments, the primer set is used to detect whether multiple mutations within the amplified product fragment are linked.

[0021] In some embodiments, the 5' end of the primer of the primer set also contains a DNA barcode for distinguishing different samples. In some preferred embodiments, the 5' end barcodes of the F and R primers can be the same or different, and those skilled in the art can choose as needed. In some preferred embodiments, the length of the DNA barcode is 5-50nt.

[0022] In another aspect, the present invention provides a method for simultaneously amplifying or detecting GBA1 Genes and their pseudogenes GBAP1 A kit for multiple mutations, comprising the following reagents: (1) reagents for multiple long-fragment GAP PCR amplification of homologous regions; (2) reagents for constructing a single-molecule sequencing library; wherein the reagents for multiple long-fragment GAP PCR amplification of homologous regions comprise the primer set described in the present invention.

[0023] In some embodiments, the GBA1 Genes and their pseudogenes GBAP1 Multiple mutations include GBA1 SNV and InDel on gene exons, GBA1 and GBAP1 Deletions and fusions caused by gene recombination and their corresponding GBA1 Gene copy number variation, GBA1 In some preferred embodiments, the GBA1 The gene mutations include at least 640 mutations as shown in Table 1.

[0024] In some embodiments, the reagents for multiple long-fragment GAP PCR amplification of homologous regions are used to simultaneously amplify GBA1、GBAP1 Gene fragments and Gap fragments. In some embodiments, the reagents for homologous region multiple long fragment GAP PCR amplification further include DNA polymerase and reaction buffer. For the kit, the PCR amplification product can be purified or not before the next step of reaction, and those skilled in the art can choose according to needs.

[0025] In some embodiments, the reagents for constructing a single-molecule sequencing library include an adapter, a ligase, DNA purification magnetic beads, a reaction buffer, and an exonuclease.

[0026] In another aspect, the present invention provides a method for simultaneously amplifying or detecting GBA1 Genes and their pseudogenes GBAP1 A system for multiple mutations, comprising the following modules: (1) an acquisition module: used to obtain and prepare subject samples; (2) an amplification module: used to perform homologous region multiple long-fragment GAP PCR amplification on the sample; (3) a library construction module: used to construct a single-molecule sequencing library; (4) a sequencing module: used to sequence and analyze mutation types; wherein the homologous region multiple long-fragment GAP PCR amplification in the acquisition module is performed using the primer set described in the present invention or the kit described in the present invention.

[0027] In some embodiments, the GBA1 Genes and their pseudogenes GBAP1 Multiple mutations include GBA1 SNV and InDel on gene exons, GBA1 and GBAP1 Deletions and fusions caused by gene recombination and their corresponding GBA1 Gene copy number variation, GBA1 Large fragment deletion variation within 20kb upstream. In some embodiments, the GBA1 The gene mutations include at least 640 mutations as shown in Table 1.

[0028] In some embodiments, the sample is selected from a biological sample or gDNA extracted from the biological sample. Preferably, the biological sample comprises cultured cell lines, blood, amniotic fluid, chorionic villi, gametes, blastocyst cells, joint fluid, urine, sweat, saliva, feces, cerebrospinal fluid, ascites, pleural effusion, bile and pancreatic fluid.

[0029] In some embodiments, the sequencing module is selected from PacBio sequencing of Pacific Biosciences or Nanopore sequencing of ONT.

[0030] In a preferred embodiment, the single-molecule gene sequencing is selected from PacBio sequencing of Pacific Biosciences or Nanopore sequencing of ONT.

[0031] In a specific embodiment, PacBio library adapter ligation can be performed using blunt-end ligation or TA ligation.

[0032] In a specific embodiment, the PacBio universal blunt-end adapter sequence is 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGAT-3' (SEQ ID NO: 12), and a blunt-end stem-loop structure adapter adapter is formed by annealing. DNA (barcode) of different sequences of 5-50nt can be added to the stem to form adapter adapters with different barcodes. PacBio libraries with different barcodes can be mixed together for sequencing.

[0033] In a specific embodiment, the PacBio universal TA adapter sequence is 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGATT-3' (SEQ ID NO: 13), and a blunt-ended stem-loop structure adapter adapter is formed by annealing. DNA (barcode) of different sequences of 5-50nt can be added to the stem to form adapter adapters with different barcodes. PacBio libraries with different barcodes can be mixed together for sequencing.

[0034] In one embodiment, the PacBio connector may or may not have a barcode. Preferably, the PacBio connector has a barcode designed by PacBio or a barcode designed by the company itself, and those skilled in the art can choose according to their needs.

[0035] In a preferred embodiment, the PacBio library is compatible with the Pacific Biosciences sequencing platform.

[0036] In a preferred embodiment, the reagents used to construct the Nanopore library include end repair enzymes, linkers, ligases, DNA purification magnetic beads, 80% ethanol and reaction buffer.

[0037] In one embodiment, Nanopore library adapter ligation can be performed using blunt-end ligation or TA ligation.

[0038] In one embodiment, the Nanopore connector may or may not have a barcode. Preferably, the Nanopore connector has a barcode designed by ONT or a self-designed barcode, and those skilled in the art can select one according to their needs.

[0039] In a preferred embodiment, the Nanopore library matches the sequencing platform of ONT.

[0040] The excellent technical effects of the primer set, kit, system and method provided by the present invention mainly lie in the following aspects: (1) Wide detection range. The present invention can simultaneously detect the GD-related genes that have been discovered GBA1 All point mutations and InDels on exons include more than 640 types in total, and the cis-trans relationship of all mutations on the same gene can be distinguished. GBA1 and GBAP1 Deletions and fusions caused by gene recombination, GBA1 Large deletion variants within 20 kb upstream, and the corresponding deletion and fusion genes GBA1 Increase and decrease of gene copy number, as well as accurate determination of GBA1 The location of the fusion and deletion. GBAP1 Genes can further restore the entire GBA1 and GBAP1 The variation in gene regions provides convenience for subsequent scientific research and screening work.

[0041] (2) Single-tube detection of multiple mutation types. Traditional methods require setting up a detection system for each mutation type, but the present invention overcomes this problem. GBA1 and GBAP1 The amplification preference problem of enriching homologous regions of genes together reduces the problem of background easily generated by homologous regions and realizes the simultaneous detection of multiple mutations in one reaction primer system, including SNV, Indel, gene recombination and copy number variation.

[0042] (3) Low false positive and missed detection rates. Currently, the most common pathogenic gene for GD is detected GBA1 The method of point mutation is PCR+Sanger. GBA1 The gene is huge and there are pseudogenes GBAP1 Interference, as well as the limited detection range of Sanger and the cumbersome detection experiments, can easily lead to missed diagnosis of pathogenic mutations and result in false negative judgments. GBA1 and GBAP1 All exon regions of the gene, all GBA1 Related missing fragments, GBA1 Related fusion fragments greatly reduce the risk of misdiagnosis and missed diagnosis of patients, greatly improve the simplicity of GD gene diagnosis, and greatly reduce the time and labor costs of GD gene diagnosis.

[0043] (4) Sample diversification. The template used for PCR can be peripheral blood, dried blood spots or extracted genomic DNA, or human cell lines or other specific tissues.

[0044] (5) High adaptability to sequencing platforms. The single-molecule sequencing library prepared by the amplification and library construction method provided by the present invention is compatible with all SMRT sequencing and Nanopore sequencing platforms on the market.

[0045] (6) High-throughput detection. Single-molecule sequencing can achieve 384 types of barcode adapters, and more types of barcode adapters can be designed according to needs. Or a dual barcode system with primers and adapters can be used to achieve more barcode combinations. The high-throughput characteristics of the single-molecule sequencing platform determine that high-throughput sample detection can be achieved.

[0046] (7) High accuracy. The dumbbell-shaped library of the SMRT library can be read multiple times during sequencing, and the base accuracy of the sequencing results after correction is greater than 99%. In addition, SMRT sequencing errors are random, and the base accuracy is greater than 99.9% after correction through sequencing depth. Therefore, gene mutations within the primer detection range can be accurately read.

[0047] (8) Flexible detection time. The Nanopore platform can generate data within minutes, and data analysis can be started within minutes or hours depending on the actual data volume. When the detection timeliness requirements are relatively high, the Nanopore platform has a time advantage. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Schematic diagram of multiplex PCR primer design.

[0049] Figure 2 It is a DNA gel electrophoresis diagram of the sample amplified by the multiplex PCR method in Example 1.

[0050] Figure 3 yes GBA1 Figure 2. SMRT sequencing results of healthy human samples.

[0051] Figure 4 yes GBA1 Figure 1. SMRT sequencing results of E8-E11 deletion samples.

[0052] Figure 5 yes GBA1 SMRT sequencing results of point mutation samples. DETAILED DESCRIPTION

[0053] While describing in detail exemplary embodiments of the present invention, it should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0054] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope. Although any methods and materials similar or equivalent to those described herein can be used in the practice or test of the present disclosure, what is described below is preferred materials and methods.

[0055] The sequences used in the present invention are shown in Table 2.

[0056]

[0057] Example 1: Using the long fragment multiplex PCR method of the present invention to amplify GD-related gene mutations using different primer combinations The collection module is used to obtain and prepare blood, dried blood spots and genomic DNA samples, and the amplification module is used to perform long-fragment multiplex PCR amplification on the samples. The reaction system is prepared according to Table 3 below to amplify peripheral blood, dried blood spots and genomic DNA samples:

[0058] On the PCR instrument, pre-amplification was performed according to the conditions shown in Table 4 below:

[0059] After amplification, 5 mL of each sample was taken and tested on a 1% DNA gel. The results were as follows: Figure 2 As shown, using different samples as templates, GD-related GBA1 Genes and their pseudogenes GBAP1 can be effectively amplified.

[0060] Example 2: Construction of SMRT sequencing library using the homologous region multiple long fragment GAP PCR method of the present invention

[0061] Prepare the reaction system according to Table 5 below to amplify the related GBA1 Peripheral blood samples for gene mutations:

[0062] On the PCR instrument, pre-amplification was performed according to the conditions shown in Table 6 below:

[0063] After amplification, the amplified product was placed in a centrifuge at 10,000 rpm for 20 minutes. After centrifugation, the product was placed horizontally and 4 mL of supernatant was added to a new tube.

[0064] Step 2: Construction of SMRT sequencing library The single-molecule sequencing library was constructed using the library construction module, and the sequencing module was used to sequence and analyze the mutation type. The reaction system was prepared according to Table 7 below:

[0065] The reaction was carried out in a PCR machine under the following conditions: 37ºC 20 min; 25ºC 15 min; 65ºC 10 min. After the reaction was completed, 0.5 mL Exonuclease III (NEB, Cat#M0206L) and 0.5 mL Exonuclease VII (NEB, Cat#M0379L) were added and the reaction was continued at 37ºC for 1 hour. The DNA was purified twice using 0.6x AMpure PB magnetic beads (PacBio, Cat#100-265-900) according to the manufacturer's instructions, and finally eluted with 10 mL Elution Buffer. The obtained DNA eluate is the target SMRT sequencing library. The DNA concentration was measured on a Qubit 3 Fluoromter (ThermoFisher, Cat#Q33216) using Qubit dsDNA HS reagent (ThermoFisher, Cat# Q32851). When there are multiple sample SMRT libraries, equal amounts of the libraries can be mixed together to prepare a mixed library.

[0066] Step 3: SMRT sequencing and analysis According to the total concentration and molar concentration of the library, an appropriate volume of the library was reacted with the binding reagent (PacBio, Cat#101-820-200) and primer (PacBio, Cat#100-970-100) to prepare the final library that can be used in the machine. Representative sequencing results are shown in Figure 3-5 As shown, Figure 3 For healthy people GBA1 Schematic diagram of gene IGV, Figure 4 for GBA1 Schematic diagram of homologous recombination variant IGV, Figure 5 for GBA1 Schematic diagram of gene point mutation IGV.

[0067] Embodiment 3: GBA1 Testing and validation of gene mutation detection Peripheral blood genomic DNA of 15 subjects was collected as 15 verification samples. Referring to Example 2, the method (and kit) of the present invention was used to simultaneously detect GD-related GBA1 Multiple mutations at gene loci. Detected using PCR+Sanger sequencing GBA1 Gene point mutation. The results obtained by the present invention were compared with the control results, and the results are shown in Table 8. The results of the 15 samples are completely consistent.

[0068]

[0069] Therefore, the results of the detection by the method of the present invention, after comparison with the PCR+Sanger sequencing method, both the specificity and sensitivity reached 100%. Moreover, the allele position relationship of each SNV was clearly defined in all four samples.

[0070] It should be noted that although the above implementation cases have demonstrated a series of features of the present invention, researchers and technicians in this field can use the ideas of the present invention to adjust and change the reaction reagents, reaction conditions, etc. involved in the multiple PCR reaction and the construction of the single molecule sequencing library according to specific needs. Therefore, for those skilled in the art, several simple substitutions can be made without departing from the concept and principle of the present invention, which should be included in the protection scope of the present invention.

[0071] References [1] Hruska KS, LaMarca ME, Scott CR et al. Gaucher disease: Mutationand polymorphism spectrum in the glucocerebrosidase gene (GBA). Hum Mutat.2008, 29, 567–583. [2] Kaplan P, Andersson HC, Kacena KA, et al. The clinical anddemographic characteristics of nonneuronopathic Gaucher disease in 887children at diagnosis. Arch. Pediatrics Adolesc. Med. 2006, 160, 603–608. [3] Taddei TH, Dziura J, Chen S et al. High incidence of cholesterolgallstone disease in type 1 Gaucher disease: Characterizing the biliaryphenotype of type 1 Gaucher disease. J Inherit Metab Dis. 2010, 33, 291–300. [4] Rosenbaum H. Hemorrhagic aspects of Gaucher disease. RambamMaimonides Med J. 2014, 5, e0039. [5] Mignot C, Doummar D, Maire I et al. French Type 2 Gaucher DiseaseStudy Group. Type 2 Gaucher disease: 15 new cases and review of theliterature. Brain Dev. 2006, 28, 39–48. [6] Bennett LL, Mohan D. Gaucher disease and its treatment options.Ann Pharm. 2013, 47, 1182–1193. [7] Cindik N, Ozcay F, Suren D et al. Gaucher disease withcommunicating hydrocephalus and cardiac involvement. Clin Cardiol. 2010, 33,e26–e30. [8] Abdelwahab M, Blankenship D, Schiffmann R. Long-term follow-upand sudden unexpected death in Gaucher disease type 3 in Egypt. Neurol Genet.2016, 2, e55. [9] Mignot C, Gelot A, Bessieres B et al. Perinatal-lethal Gaucherdisease. Am J Med Genet A. 2003, 120A, 338-344.

[10] Stirnemann J, Vigan M, Hamroun D et al. The French Gaucher’sdisease registry: Clinical characteristics, complications and treatment of562 patients. Orphanet J Rare Dis. 2012, 7, 77.

[11] Mistry PK, Sadan S, Yang R et al. Consequences of diagnosticdelays in type 1 Gaucher disease: the need for greater awareness amonghematologists-oncologists and an opportunity for early diagnosis andintervention. Am J Hematol. 2007, 82, 697-701.

[12] Tayebi N, Stubblefield BK, Park JK et al. Reciprocal andnonreciprocal recombination at the glucocerebrosidase gene region:implications for complexity in Gaucher disease. Am J Hum Genet. 2003, 72,519-534.

[13] Laura S, Anthony HVS. GBA Variants and Parkinson Disease:Mechanisms and Treatments. Cell. 2022, 11, 1261.

[14] Koprivica V, Stone DL, Park JK et al. Analysis andclassification of 304 mutant alleles in patients with type 1 and type 3Gaucher disease. Am J Hum Genet. 2000, 66, 1777-1786.

[15] Stefania Z, Silvia C, Bruno B et al. GBA Analysis in Next-Generation Era: Pitfalls, Challenges, and Possible Solutions. J Mol Diagn.2017, 19, 733-741.

[16] Weiss K, Gonzalez A, Lopez G et al. The clinical management ofType 2 Gaucher disease. Mol Genet Metab. 2015, 114, 110-122.

[17] Guillermo ID, Nicolas FE, Barbara CS et al. Long-read singlemolecule real-time (SMRT) sequencing of GBA1 locus in Gaucher diseasenational cohort from Argentina reveals high frequency of complex alleleunderlying severe skeletal phenotypes: Collaborative study from the ArgentineGroup for Diagnosis and Treatment of Gaucher Disease. Mol Genet Metab Rep.2021, 29, 100820.

[18] Marco T, Xiao C, Fritz JS et al. Comprehensive short and longread sequencing analysis for the Gaucher and Parkinson's disease-associatedGBA gene. Commun Biol. 2022, 5, 670.

[19] Letizia S, Valeria R, Maura S et al. The GBAP1 pseudogene actsas a ceRNA for the glucocerebrosidase gene GBA by sponging miR-22-3p. SciRep. 2017, 7, 12702.

Claims

1. A primer set for simultaneous amplification or detection GBA1 Genes and their pseudogenes GBAP1 multiple mutations, the primer set comprising a GBA-F primer, a GBA-R primer and a GBAP-R primer; The nucleotide sequence of the GBA-F primer is shown in SEQ ID NO: 1-2, the nucleotide sequence of the GBA-R primer is shown in SEQ ID NO: 5-7, and the nucleotide sequence of the GBAP-R primer is shown in SEQ ID NO: 3-4.

2. The primer set according to claim 1, further comprising a Gap-F primer, wherein the nucleotide sequence of the Gap-F primer is shown in SEQ ID NO: 8-11.

3. The primer set according to claim 1 or 2, wherein GBA1 Genes and their pseudogenes GBAP1 Multiple mutations include GBA1 SNV and InDel on gene exons, GBA1 and GBAP1 Deletions and fusions caused by gene recombination and their corresponding GBA1 Gene copy number variation, GBA1 Large deletion variant within 20kb upstream.

4. The primer set according to claim 3, wherein the GBA1 The gene mutations include at least 640 mutations as shown in Table 1.

5. The primer set according to claim 1 or 2, wherein the primer set is used for multiple long fragment GAPPCR amplification of homologous regions, and amplification is performed simultaneously in one reaction system. GBA1, GBAP1 Gene fragments and Gap fragments. The primer set according to claim 1 or 2, wherein the amplification product of the primer set is 10 kb-18 kb.

7. The primer set according to claim 1 or 2, wherein the primer set is used to detect whether multiple mutations within the amplified product fragment are linked. The primer set according to claim 1 or 2, wherein the 5' end of the primer in the primer set further comprises a DNA barcode. 9 . The primer set according to claim 8 , wherein the DNA barcodes are the same or different, and the length of the DNA barcodes is 5-50 nt.

10. A simultaneous amplification or detection GBA1 Genes and their pseudogenes GBAP1 A kit for multiple mutations, comprising the following reagents: (1) Reagents for multiple long-fragment GAP PCR amplification of homologous regions; (2) Reagents for constructing single-molecule sequencing libraries; The reagents for multiple long-fragment GAP PCR amplification of homologous regions comprise the primer set according to any one of claims 1 to 9.

11. The kit according to claim 10, wherein the GBA1 Genes and their pseudogenes GBAP1 Multiple mutations include GBA1 SNV and InDel on gene exons, GBA1 and GBAP1 Deletions and fusions caused by gene recombination and their corresponding GBA1 Gene copy number variation, GBA1 Large deletion variant within 20kb upstream.

12. The kit according to claim 11, wherein the GBA1 The gene mutations include at least 640 mutations as shown in Table 1.

13. The kit according to claim 10, wherein the reagents for multiple long fragment GAP PCR amplification of homologous regions are simultaneously amplified in one reaction system GBA1, GBAP1 Gene fragments and Gap fragments. 14 . The kit according to claim 10 , wherein the reagents for multiple long-fragment GAP PCR amplification of homologous regions further comprise DNA polymerase and a reaction buffer.

15. The kit according to claim 10, wherein the reagents for constructing a single-molecule sequencing library comprise adapters, ligase, DNA purification magnetic beads, reaction buffer and exonuclease.

16. A simultaneous amplification or detection GBA1 Genes and their pseudogenes GBAP1 A system of multiple mutations, which includes the following modules: (1) Collection module: used to obtain and prepare subject samples; (2) Amplification module: used to perform multiple long-fragment GAP PCR amplification of homologous regions on the sample; (3) Library construction module: used to construct single-molecule sequencing libraries; (4) Sequencing module: used for sequencing and analyzing mutation types; The homologous region multiple long fragment GAP PCR amplification in the acquisition module is performed using the primer set described in any one of claims 1 to 9 or the kit described in any one of claims 10 to 15.

17. The system according to claim 16, wherein the GBA1 Genes and their pseudogenes GBAP1 Multiple mutations include GBA1 SNV and InDel on gene exons, GBA1 and GBAP1 Deletions and fusions caused by gene recombination and their corresponding GBA1 Gene copy number variation, GBA1 Large deletion variant within 20kb upstream.

18. The system according to claim 17, wherein the GBA1 The gene mutations include at least 640 mutations as shown in Table 1.

19. The system according to claim 16, wherein the sample is selected from a biological sample or gDNA extracted from the biological sample.

20. The system of claim 19, wherein the biological sample comprises cultured cell lines, blood, amniotic fluid, chorionic villi, gametes, blastocyst cells, joint fluid, urine, sweat, saliva, feces, cerebrospinal fluid, ascites, pleural effusion, bile, and pancreatic fluid.

21. The system according to claim 16, wherein the sequencing module is selected from PacBio sequencing of Pacific Biosciences or Nanopore sequencing of ONT.

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