WO2021114422A1 - 淋巴管平滑肌瘤病联合检测方法及其应用 - Google Patents

淋巴管平滑肌瘤病联合检测方法及其应用 Download PDF

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WO2021114422A1
WO2021114422A1 PCT/CN2019/129173 CN2019129173W WO2021114422A1 WO 2021114422 A1 WO2021114422 A1 WO 2021114422A1 CN 2019129173 W CN2019129173 W CN 2019129173W WO 2021114422 A1 WO2021114422 A1 WO 2021114422A1
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gene
genes
detection
lam
tsc1
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French (fr)
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欧小华
刘菲菲
孙明明
胡昌明
邓俊豪
于世辉
赵薇薇
黄晓强
王春晖
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广州金域医学检验集团股份有限公司
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Definitions

  • the invention relates to the technical field of gene detection, in particular to a combined detection method for lymphangiomyomatosis and its application.
  • Lymphangioleio-myomatosis (Lymphangioleio-myomatosis, LAM) is a rare multi-system neoplastic disease. Almost all cases occur in women and are characterized by chronically progressive diffuse parenchymal cystic lesions in both lungs. Patients with LAM have mild early symptoms, usually with increasing dyspnea and recurring pneumothorax and chylothorax. About 60-70% of patients will develop pneumothorax at a certain stage, and about 30% of patients will develop chylothorax. Common extrapulmonary manifestations include renal angiomyolipoma, retroperitoneal lymphatic involvement, and pelvic lymph node involvement.
  • LAM is divided into sporadic LAM (S-LAM) and tuberous sclerosis (TSC-LAM) related LAM.
  • S-LAM sporadic LAM
  • TSC-LAM tuberous sclerosis
  • the pathogenesis of LAM is not yet fully understood.
  • mTOR mammalian target of rapamycin
  • Sirolimus can inhibit the mTOR pathway, so it has become an effective drug for the treatment of LAM, and it is also the first drug approved by the FDA for the treatment of this disease.
  • LAM lymphangiomyomatosis
  • the method first uses probe capture to obtain the exon coding region including the TSC1/2 gene and its relationship with the tumor.
  • the sequence of key gene hotspots related to development occurred, and the Illumina sequencing platform was used for paired-end sequencing to detect the sequence variation in the target region of the specimen.
  • CMA, MLPA and Sanger were used for supplementary detection and result verification, which improved LAM patients.
  • the detection rate of positive mutations can be used for auxiliary diagnosis and pre-pregnancy genetic counseling based on the test results.
  • a combined detection method for lymphangiomyomatosis including the following steps:
  • Liquid phase capture and targeted sequencing Design Panel for the whole exon coding regions of TSC1 and TSC2 genes that are highly related to LAM and mutant genes closely related to solid tumors, construct a gDNA library, and perform hybrid capture and sequence on the computer;
  • Sorting Analyze the above-mentioned sequencing data through bioinformatics processing. If the TSC1 and TSC2 gene test results are negative or there are only single-hit mutation sites, then the chromosome chip analysis method and the multiple connection probe amplification method are used for supplementary detection; Sanger sequencing method is used to verify the site that is not clear whether it is a somatic mutation or a germline mutation source is detected;
  • Chromosome microarray analysis Obtain the results of loss of heterozygosity and copy number variation through chromosome microarray analysis
  • Multiple ligation probe amplification use multiple ligation probe amplification method to obtain large indel results
  • Sanger sequencing Take the white blood cell samples corresponding to the sample to be tested for Sanger detection, and determine whether it belongs to the S-LAM type or TSC-LAM type.
  • the inventors discovered in the research that the TSC1 and TSC2 genes are relatively large, with 23 and 42 exons, respectively, and the total length of the coding region is about 9kb, and there are regions with complex sequences.
  • Traditional detection schemes such as the Sanger method need to be performed multiple times. Sequencing after segmented amplification requires a lot of effort, cost, and sample demand. For FFPE samples of relatively poor quality, traditional amplification methods cannot achieve full area coverage.
  • LAM loss of heterozygosity
  • LAM is hereditary and sporadic, and hereditary patients with unobvious symptoms are often clinically misdiagnosed as sporadic LAM.
  • the inventors proposed the above-mentioned combined detection method.
  • another mechanism may be involved in the occurrence and development of LAM. Therefore, the detection of other tumor-related genes is included in the above-mentioned LAM.
  • CMA, MLPA and Sanger methods are used for supplementary testing and result verification, which improves the detection rate of positive mutations in LAM patients, and can perform auxiliary diagnosis and pre-pregnancy genetic counseling based on the test results.
  • the panel design covers the following genes: ALDH1 gene, EGFR gene, FLT3 gene, MYC gene, PTEN gene, SDHD gene, AQP9 gene, ERBB2 gene, HRAS gene, MYCN gene, RET gene, TP53 gene, AR gene, ESR1 gene, KIT gene, NF1 gene, RICTOR gene, TSC1 gene, ATRX gene, FGFR1 gene, KRAS gene, NRAS gene, RUNX1 gene, TSC2 gene, BCL2 gene , FGFR2 gene, MDM2 gene, PDGFRA gene, SDHA gene, VHL gene, BRAF gene, FGFR3 gene, MAP2K1 gene, PGR gene, SDHB base, CCND1 gene, FGFR4 gene, MET gene, POLE gene, SDHC gene; ABL1 gene, CDKN2A Genes, FBXW7 genes, IDH2 genes, NOTCH1 genes, SMAD
  • the above-mentioned panels refer to COSMIC, TCGA and other databases, combined with the latest NCCN guidelines/consensus, to screen protocarcinoma and tumor suppressor genes that are closely related to the occurrence and development of solid tumors, and combine them into gene panels.
  • the probe sequences for the TSC1 gene and the TSC2 gene are as shown in SEQ ID No. 1-SEQ ID No. 276.
  • the sequencing depth is greater than 1000x. Output for mutation sites with mutation frequency above 1%, avoiding the missed detection of low-frequency mutations
  • the present invention also discloses the application of the above-mentioned combined detection method of lymphangiomyomatosis in studying the pathogenesis of LAM and/or the diagnosis and treatment of LAM.
  • the specific detection reagent in the detection method is used in the preparation of a diagnostic reagent or diagnostic equipment for the combined detection of lymphangioleiomyomatosis.
  • the present invention also discloses a lymphangioleiomyomatosis joint detection kit, including a detection panel covering the following genes: ALDH1 gene, EGFR gene, FLT3 gene, MYC gene, PTEN gene, SDHD gene, AQP9 gene, ERBB2 gene, HRAS gene, MYCN gene, RET gene, TP53 gene, AR gene, ESR1 gene, KIT gene, NF1 gene, RICTOR gene, TSC1 gene, ATRX gene, FGFR1 gene, KRAS gene, NRAS gene, RUNX1 gene, TSC2 gene, BCL2 gene , FGFR2 gene, MDM2 gene, PDGFRA gene, SDHA gene, VHL gene, BRAF gene, FGFR3 gene, MAP2K1 gene, PGR gene, SDHB base, CCND1 gene, FGFR4 gene, MET gene, POLE gene, SDHC gene; ABL1 gene, CDKN2A Genes, FBXW7 genes, IDH2 genes, NOT
  • the probe sequence for detecting the Panel is as shown in SEQ ID No. 1-SEQ ID No. 276.
  • the kit further includes chromosome chip analysis reagents. Understandably, the chromosome chip analysis reagent can be selected according to specific experimental needs, such as CNV FFPE Assay Kit.
  • the kit further includes multiple ligation probes. Understandably, the probe can also be selected according to specific experimental requirements, such as MRC-Holland's TSC1&TSC2 Probe.
  • the invention also discloses a combined detection system for lymphangiomyomatosis, which includes the following modules:
  • Detection modules including liquid phase capture targeted sequencing module, chromosome chip analysis module, multiple connection probe amplification module, Sanger sequencing module, the liquid phase capture targeted sequencing module includes: TSC1 and TSC2 that are highly related to LAM Panels designed with all exon coding regions of genes and mutant genes closely related to solid tumors;
  • the analysis module first obtains the liquid phase capture targeted sequencing results. If the TSC1 and TSC2 gene detection results are negative or there are only single-strike mutation sites, it is prompted to use the chromosome chip analysis module and the multiple connection probe amplification module to supplement Detection; if it is not clear whether it is a somatic mutation or a germline mutation source site is detected, it is prompted to use the Sanger sequencing module for verification; then the results detected by the detection module are comprehensively analyzed and judged, and a combination of lymphangioleiomyomatosis is obtained Test results.
  • the Panel covers the following genes: ALDH1 gene, EGFR gene, FLT3 gene, MYC gene, PTEN gene, SDHD gene, AQP9 gene, ERBB2 gene, HRAS gene, MYCN gene, RET gene, TP53 gene, AR gene, ESR1 gene, KIT gene, NF1 gene, RICTOR gene, TSC1 gene, ATRX gene, FGFR1 gene, KRAS gene, NRAS gene, RUNX1 gene, TSC2 gene, BCL2 gene, FGFR2 gene, MDM2 gene, PDGFRA gene, SDHA gene , VHL gene, BRAF gene, FGFR3 gene, MAP2K1 gene, PGR gene, SDHB base, CCND1 gene, FGFR4 gene, MET gene, POLE gene, SDHC gene; ABL1 gene, CDKN2A gene, FBXW7 gene, IDH2 gene, NOTCH1 gene, SMAD4 Genes, AKT1 gene, CSF1
  • the probe sequences for the TSC1 gene and the TSC2 gene are as shown in SEQ ID No. 1-SEQ ID No. 276.
  • the above-mentioned joint detection system can be used in conjunction with equipment or instrument reagents that can achieve liquid phase capture targeted sequencing, chromosome chip analysis, multiple connection probe amplification, and Sanger sequencing, and only need to be able to achieve what the system requires.
  • the achieved function is sufficient.
  • conventional equipment and instruments can be used with the specific Panel of the present invention to obtain exon coding regions including TSC1/2 genes and key gene hotspot sequences related to tumor occurrence and development, and can be used at the same time CMA, MLPA, Sanger and other methods are used for the purpose of supplementary testing and result verification.
  • the present invention has the following beneficial effects:
  • the method for combined detection of lymphangioleiomyomatosis of the present invention first uses probe capture to obtain exon coding regions including TSC1/2 genes and key gene hotspot sequences related to tumor occurrence and development, using Illumina
  • the sequencing platform performs paired-end sequencing to detect the sequence variation in the target area of the specimen.
  • CMA, MLPA and Sanger are used for supplementary testing and result verification, which improves the detection rate of positive mutations in LAM patients and can be based on the test results. Carry out auxiliary diagnosis and genetic counseling before pregnancy.
  • the lymphangioleiomyomatosis joint detection kit of the present invention includes the detection of the exon coding region including the TSC1/2 gene and the sequence of the key gene hotspots related to the occurrence and development of tumors, and the chromosome chip analysis is selected according to needs Reagents and/or multiple ligation probes, etc., can simultaneously use CMA, MLPA, and Sanger methods for supplementary testing and result verification, which improves the detection rate of positive mutations in LAM patients, and assists diagnosis and pre-pregnancy genetic counseling based on the test results .
  • a combined detection system for lymphangiomyomatosis of the present invention is provided with a detection module and an analysis module, wherein the detection module includes a liquid phase capture targeted sequencing module, a chromosome chip analysis module, a multiple connection probe amplification module, and a Sanger sequencing module , Can detect the exon coding region of TSC1/2 gene and the sequence of key gene hot spots related to tumor occurrence and development, and select chromosome chip analysis reagents and/or multiple ligation probes according to needs, and can use CMA, Methods such as MLPA and Sanger perform supplementary testing and result verification, which increase the detection rate of positive mutations in LAM patients, and can perform auxiliary diagnosis and pre-pregnancy genetic counseling based on the test results.
  • the detection module includes a liquid phase capture targeted sequencing module, a chromosome chip analysis module, a multiple connection probe amplification module, and a Sanger sequencing module , Can detect the exon coding region of TSC1/2 gene and the sequence of key gene hot spots related to tumor
  • Figure 1 is the flow chart of the multi-method combined detection scheme for lymphangiomyomatosis
  • FIG. 2 is a diagram of the design scheme of TSC1&2 gene targeting probe
  • Figure 3 is a graph showing the proportion of variants detected by different detection methods
  • Figure 4 is a diagram showing the advantages of combined detection schemes over single method detection
  • Figure 5 shows the LOH phenomenon of cases carrying the TSC2 gene detected by the CMA method.
  • the shingled design is carried out for the whole exon coding regions of TSC1 and TSC2 genes which are basically highly related to LAM.
  • the probe design of TSC1 and TSC2 genes is shown in Figure 1 to ensure that the target regions of these two genes are covered by at least 2 times. , Each probe is 100bp long.
  • the fragmented PCR tube is prepared as follows:
  • DNA library pooling The concentration is measured according to Qubit2.0, and the total amount of 100ng of each sample is mixed in a PCR tube, and 5 samples are subjected to a hybridization reaction;
  • Reagent name Volume of stock solution ( ⁇ l) ddH 2 O volume ( ⁇ l) 1X Buffer total ( ⁇ l) xGen 10X Wash Buffer I 30 270 300 xGen 10X Wash Buffer II 20 180 200 xGen 10X Wash Buffer III 20 180 200 xGen 10X Stringent Wash Buffer 40 360 400 xGen 2X Bead Wash Buffer 250 250 500
  • TSC1 and TSC2 gene test results are negative or there are only single-strike mutation sites (that is, only one gene in TSC1&TSC2 has a mutation, or fragment deletion/insertion, or copy number variation, etc.)
  • chromosome chip analysis method and multiple connection are used
  • the probe amplification method is used for supplemental detection, and the kits used are as follows.
  • a site that is not clear whether it is a somatic mutation or a germline mutation refers to a mutation with a mutation frequency of about 50%.
  • Sanger sequencing is used to verify the patient's white blood cell specimen (ABI 3730XL), and the verification result is used to distinguish whether the patient belongs to S-LAM or TSC-LAM.
  • the patient’s leukocytes also have the above-mentioned mutations, it can be determined that the patient belongs to TSC-LAM, otherwise, it can be determined that the patient belongs to S-LAM.
  • Example 1 The method of Example 1 was used for the LAM joint detection study.
  • the single detection method is a method that only uses NGS targeted sequencing.
  • the overall positive detection rate for TSC1 and TSC2 genes was 72.13%, and the number of patients with 1-hit and 2-hits were 15 and 29, respectively.
  • the combined detection scheme resulted in the overall positive detection rate. It was 75.41%, and the number of patients with 1-hit and 2-hits were 8 and 38, respectively. The results are shown in Figure 4.
  • the combined detection program not only improves the detection rate of positive mutations in LAM patients, but more importantly, the test results are more in line with Knudson's "second hit” theory.
  • the sample came from a fixed tissue specimen from the Department of Respiratory Medicine of a Class-A hospital in Guangzhou, and was clinically diagnosed as S-LAM.
  • Example 1 supplementary detection was performed using the chromosome chip analysis method.
  • the results showed that the patient found a loss of heterozygosity (LOH) mutation in the TSC2 gene: arr ⁇ GRCh37>16p13.3p11.2(83886_30809063)x2moshmz, mutation
  • LOH heterozygosity
  • the test results of this patient showed that the premature stop coding signal was generated at codon 1138 of the TSC2 gene, and the mutation frequency was 50.9%.
  • the Sanger method determined that the mutation did not exist in white blood cells and was a somatic mutation, which would lead to normal protein function. Loss, more than 50 cases have been reported in individuals with LAM in the past. It is a pathogenic mutation.
  • Supplementary test results show that this patient also has the phenomenon of LOH in the TSC2 gene. Studies have shown that LOH can cause the inactivation of suppressor genes. Affect the occurrence and progress of cancer. Through this joint detection program, the pathogenesis of the patient can be completely restored, and based on the results, it can be judged as sporadic LAM, which is not hereditary.

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Abstract

一种淋巴管平滑肌瘤病联合检测方法及其应用，属于基因检测技术领域。该方法包括以下步骤：液相捕获靶向测序：针对与LAM高度相关的TSC1和TSC2基因全外显子编码区和与实体肿瘤密切相关的突变基因设计Panel，构建gDNA文库，进行杂交捕获后，上机测序；分选：通过生物信息学处理分析上述测序数据，如检测到TSC1和TSC2基因阴性，则采用染色体芯片分析方法进行补充检测；如检测到单打击位点，则采用多重连接探针扩增进行补充检测；如检测到不明确体细胞突变和胚系突变来源的位点，则采用Sanger测进行补充检测。该方法提高了LAM患者的阳性变异检出率。

Description

淋巴管平滑肌瘤病联合检测方法及其应用
优先权
本申请要求2019年12月13号提交到中国专利局的专利的优先权，具体如下：申请号为CN 201911281998.6、申请名称为淋巴管平滑肌瘤病联合检测方法及其应用。其全部内容通过引用结合在本申请中。
技术领域
本发明涉及基因检测技术领域，特别是涉及一种淋巴管平滑肌瘤病联合检测方法及其应用。
背景技术
淋巴管平滑肌瘤病(Lymphangioleio-myomatosis,LAM)是一种罕见的多系统肿瘤性疾病，几乎所有病例均发生于女性，以慢性进展的双肺弥漫性薄壁囊性病变为主要特征。LAM患者早期症状轻微，通常有逐渐加重的呼吸困难和反复发生的气胸及乳糜胸。约有60-70％的患者会在某一个阶段出现气胸，约30％的患者会出现乳糜胸。常见的肺外表现包括肾脏血管肌脂瘤，腹膜后淋巴管受累，盆腔淋巴结受累等。
LAM分为散发的LAM(S-LAM)和结节性硬化症(TSC-LAM)相关的LAM两大类。LAM发病机制尚不十分清楚，目前认为，LAM患者存在TSC1、TSC2基因突变，可以导致哺乳动物雷帕霉素靶蛋白(mTOR)所介导的细胞信号通路持续活化。而西罗莫司可抑制mTOR通路，因此其成为治疗LAM的有效药物，也是FDA首个批准的用于治疗此疾病的药物。据研究报道，采用西罗莫司治疗淋巴管肌瘤病(LAM)使得其中一部分患者的肺功能得以改善，减少血清中VEGF-D的含量，减轻患者症状和改善患者的生命质量；而一些接受西罗莫司治疗的LAM患者病情持续恶化，而导致西罗莫司反应差异的根本原因尚不清楚。同时研究显示，部分患者在TSC1和TSC2基因均未检测到突变，这意味着，另一种机制可能参与了LAM的发生与发展。
因此，开发一套完善的针对LAM的基因检测方案，绘制大标本量LAM疾病患者的基因变异图谱，充分了解LAM的发病机制及对LAM的诊断和治疗具有重要意义。
发明内容
基于此，有必要针对上述问题，提供一种淋巴管平滑肌瘤病联合检测方法及其应用，该方法先利用探针捕获方式获取包括TSC1/2基因在内的外显子编码区及与肿瘤发生发展相关的关键基因热点区序列，采用Illumina测序平台进行双端测序，以检测标本目标区域发生的序列变异，同时，采用CMA、MLPA及Sanger等方法进行补充检测和结果验证，提高了LAM患者的阳性变异检出率，并可依据检测结果进行辅助诊断及孕前遗传咨询。
一种淋巴管平滑肌瘤病联合检测方法，包括以下步骤：
液相捕获靶向测序：针对与LAM高度相关的TSC1和TSC2基因全外显子编码区和与实体肿瘤密切相关的突变基因设计Panel，构建gDNA文库，进行杂交捕获后上机测序；
分选：通过生物信息学处理分析上述测序数据，如检测到TSC1和TSC2基因检测结果阴性或只有单打击突变位点，则采用染色体芯片分析方法及多重连接探针扩增法进行补充检测；如检测到不明确属于体细胞突变还是胚系突变来源的位点，则采用Sanger测序法进行验证；
染色体芯片分析：通过染色体芯片分析获得杂合性缺失及拷贝数变异结果；
多重连接探针扩增：以多重连接探针扩增法获得大片段插入缺失结果；
Sanger测序：取待测样本对应的白细胞标本进行Sanger检测，判断属于S-LAM型或TSC-LAM型。
本发明人在研究中发现，TSC1和TSC2基因较大，分别有23和42个外显子，编码区总长约9kb，且存在序列复杂区域，传统的检测方案如Sanger方法，则需要进行多次分段扩增后进行测序，在工作量、成本和标本需求量上需要较多的耗费，且针对质量相对较差的FFPE标本，传统扩增方法无法实现全区域覆盖。
同时研究显示，约50％LAM患者TSC1和TSC2基因体细胞突变频率分布在<10％，传统的Sanger方法检测 灵敏度在10％以上，将导致漏检现象的发生。而LAM的发生发展基于双打击的模型，其突变形式多样性如杂合性缺失(LOH)和大片段插入缺失，单一的检测手段无法满足高效的检出。而LAM具有遗传性和散发性，当表症不明显的遗传性患者常常被临床误诊为散发性LAM。
基于上述研究基础，本发明人提出上述联合检测方法，除了TSC1和TSC2基因突变，还考虑到LAM的发生与发展可能存在另一种机制可能参与，因此将其它肿瘤相关基因检测包含在上述LAM的综合检测中，同时CMA、MLPA及Sanger等方法进行补充检测和结果验证，提高了LAM患者的阳性变异检出率，并可依据检测结果进行辅助诊断及孕前遗传咨询。
在其中一个实施例中，所述液相捕获靶向测序步骤中，所述Panel设计覆盖如下基因：ALDH1基因，EGFR基因，FLT3基因，MYC基因，PTEN基因，SDHD基因，AQP9基因，ERBB2基因，HRAS基因，MYCN基因，RET基因，TP53基因，AR基因，ESR1基因，KIT基因，NF1基因，RICTOR基因，TSC1基因，ATRX基因，FGFR1基因，KRAS基因，NRAS基因，RUNX1基因，TSC2基因，BCL2基因，FGFR2基因，MDM2基因，PDGFRA基因，SDHA基因，VHL基因，BRAF基因，FGFR3基因，MAP2K1基因，PGR基因，SDHB基，CCND1基因，FGFR4基因，MET基因，POLE基因，SDHC基因；ABL1基因，CDKN2A基因，FBXW7基因，IDH2基因，NOTCH1基因，SMAD4基因，AKT1基因，CSF1R基因，GNA11基因，JAK2基因，NPM1基因，SMARCB1基因，ALK基因，CTNNB1基因，GNAQ基因，JAK3基因，PIK3CA基因，SMO基因，APC基因，DDR2基因，GNAS基因，KDR基因，PTPN11基因，SRC基因，ATM基因，ERBB4基因，HNF1A基因，MLH1基因，RB1基因，STK11基因，CDH1基因，EZH2基因，IDH1基因，MPL基因，ROS1基因，TET2基因。
上述panel是参考COSMIC、TCGA等数据库，结合最新NCCN指南/共识，从中筛选与实体肿瘤发生、发展密切相关的原癌、抑癌基因，组合成基因panel。
在其中一个实施例中，所述液相捕获靶向测序步骤中，针对TSC1基因和TSC2基因的探针序列如SEQ ID No.1-SEQ ID No.276所示。
对于上述Panel的设计，针对与LAM基本高度相关的TSC1和TSC2基因全外显子编码区进行叠瓦式设计，以保证这两个基因目标区域至少2倍覆盖，每条探针100bp长，可进一步提高检出率。
在其中一个实施例中，所述液相捕获靶向测序步骤中，测序深度大于1000x。针对突变频率1％以上的变异位点进行输出，避免了低频突变漏检的现象
本发明还公开了上述的淋巴管平滑肌瘤病联合检测方法在研究LAM的发病机制和/或LAM的诊断和治疗中的应用。
在其中一个实施例中，所述检测方法中的特异性检测试剂在制备淋巴管平滑肌瘤病联合检测诊断试剂或诊断设备中的应用。
本发明还公开了一种淋巴管平滑肌瘤病联合检测试剂盒，包括覆盖如下基因的检测Panel：ALDH1基因，EGFR基因，FLT3基因，MYC基因，PTEN基因，SDHD基因，AQP9基因，ERBB2基因，HRAS基因，MYCN基因，RET基因，TP53基因，AR基因，ESR1基因，KIT基因，NF1基因，RICTOR基因，TSC1基因，ATRX基因，FGFR1基因，KRAS基因，NRAS基因，RUNX1基因，TSC2基因，BCL2基因，FGFR2基因，MDM2基因，PDGFRA基因，SDHA基因，VHL基因，BRAF基因，FGFR3基因，MAP2K1基因，PGR基因，SDHB基，CCND1基因，FGFR4基因，MET基因，POLE基因，SDHC基因；ABL1基因，CDKN2A基因，FBXW7基因，IDH2基因，NOTCH1基因，SMAD4基因，AKT1基因，CSF1R基因，GNA11基因，JAK2基因，NPM1基因，SMARCB1基因，ALK基因，CTNNB1基因，GNAQ基因，JAK3基因，PIK3CA基因，SMO基因，APC基因，DDR2基因，GNAS基因，KDR基因，PTPN11基因，SRC基因，ATM基因，ERBB4基因，HNF1A基因，MLH1基因，RB1基因，STK11基因，CDH1基因，EZH2基因，IDH1基因，MPL基因，ROS1基因，TET2基因。
在其中一个实施例中，所述检测Panel的探针序列如SEQ ID No.1-SEQ ID No.276所示。
在其中一个实施例中，该试剂盒还包括染色体芯片分析试剂。可以理解的，该染色体芯片分析试剂可根据具体实验需求选择，如
Figure PCTCN2019129173-appb-000001
CNV FFPE Assay Kit。
在其中一个实施例中，该试剂盒还包括多重连接探针。可以理解的，该探针也可根据具体实验需求选择，如 MRC-Holland的TSC1&TSC2 Probe。
本发明还公开了一种淋巴管平滑肌瘤病联合检测系统，包括以下模块：
检测模块，包括液相捕获靶向测序模块、染色体芯片分析模块、多重连接探针扩增模块、Sanger测序模块，所述液相捕获靶向测序模块中包括：针对与LAM高度相关的TSC1和TSC2基因全外显子编码区和与实体肿瘤密切相关的突变基因设计的Panel；
分析模块，首先获取所述液相捕获靶向测序结果，如检测到TSC1和TSC2基因检测结果阴性或只有单打击突变位点，则提示采用染色体芯片分析模块及多重连接探针扩增模块进行补充检测；如检测到不明确属于体细胞突变还是胚系突变来源的位点，则提示采用Sanger测序模块进行验证；再以检测模块检测到的结果综合分析判断，得出淋巴管平滑肌瘤病联合检测结果。
在其中一个实施例中，所述Panel覆盖如下基因：ALDH1基因，EGFR基因，FLT3基因，MYC基因，PTEN基因，SDHD基因，AQP9基因，ERBB2基因，HRAS基因，MYCN基因，RET基因，TP53基因，AR基因，ESR1基因，KIT基因，NF1基因，RICTOR基因，TSC1基因，ATRX基因，FGFR1基因，KRAS基因，NRAS基因，RUNX1基因，TSC2基因，BCL2基因，FGFR2基因，MDM2基因，PDGFRA基因，SDHA基因，VHL基因，BRAF基因，FGFR3基因，MAP2K1基因，PGR基因，SDHB基，CCND1基因，FGFR4基因，MET基因，POLE基因，SDHC基因；ABL1基因，CDKN2A基因，FBXW7基因，IDH2基因，NOTCH1基因，SMAD4基因，AKT1基因，CSF1R基因，GNA11基因，JAK2基因，NPM1基因，SMARCB1基因，ALK基因，CTNNB1基因，GNAQ基因，JAK3基因，PIK3CA基因，SMO基因，APC基因，DDR2基因，GNAS基因，KDR基因，PTPN11基因，SRC基因，ATM基因，ERBB4基因，HNF1A基因，MLH1基因，RB1基因，STK11基因，CDH1基因，EZH2基因，IDH1基因，MPL基因，ROS1基因，TET2基因。
在其中一个实施例中，所述液相捕获靶向测序模块中，针对TSC1基因和TSC2基因的探针序列如SEQ ID No.1-SEQ ID No.276所示。
可以理解的，上述联合检测系统，可通过将能够实现液相捕获靶向测序、染色体芯片分析、多重连接探针扩增、Sanger测序的设备或仪器试剂进行配合使用，仅需能够实现该系统所要达到的功能即可。如可借助常规设备仪器，配合本发明特定的Panel等，以实现即可获取包括TSC1/2基因在内的外显子编码区及与肿瘤发生发展相关的关键基因热点区序列，又可同时采用CMA、MLPA及Sanger等方法进行补充检测和结果验证的目的。
与现有技术相比，本发明具有以下有益效果：
本发明的一种淋巴管平滑肌瘤病联合检测方法，先利用探针捕获方式获取包括TSC1/2基因在内的外显子编码区及与肿瘤发生发展相关的关键基因热点区序列，采用Illumina测序平台进行双端测序，以检测标本目标区域发生的序列变异，同时，采用CMA、MLPA及Sanger等方法进行补充检测和结果验证，提高了LAM患者的阳性变异检出率，并可依据检测结果进行辅助诊断及孕前遗传咨询。
并且，通过采用2x 100bp的探针设计方案，结合液相杂交捕获的方法，可实现在质量较差的FFPE标本中单次实验中将TSC1和TSC2基因以及实体肿瘤相关基因关键区域完好地获得并进行测序。
本发明的一种淋巴管平滑肌瘤病联合检测试剂盒，包括检测TSC1/2基因在内的外显子编码区及与肿瘤发生发展相关的关键基因热点区序列，以及根据需要选取染色体芯片分析试剂和/或多重连接探针等，能够同时采用CMA、MLPA及Sanger等方法进行补充检测和结果验证，提高了LAM患者的阳性变异检出率，并可依据检测结果进行辅助诊断及孕前遗传咨询。
本发明的一种淋巴管平滑肌瘤病联合检测系统，具备检测模块和分析模块，其中检测模块包括液相捕获靶向测序模块、染色体芯片分析模块、多重连接探针扩增模块、Sanger测序模块，可检测TSC1/2基因在内的外显子编码区及与肿瘤发生发展相关的关键基因热点区序列，以及根据需要选取染色体芯片分析试剂和/或多重连接探针等，能够同时采用CMA、MLPA及Sanger等方法进行补充检测和结果验证，提高了LAM患者的阳性变异检出率，并可依据检测结果进行辅助诊断及孕前遗传咨询。
附图说明
图1为淋巴管平滑肌瘤病多方法联合检测方案流程图；
图2为TSC1&2基因靶向探针设计方案图；
图3为采用不同检测方法所检测变异占比图；
图4为联合检测方案比单一方法检测优势图；
图5为采用CMA方法检测到病例携带TSC2基因LOH现象图。
具体实施方式
为了便于理解本发明，下面将参照相关附图对本发明进行更全面的描述。附图中给出了本发明的较佳实施例。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
实施例1
按照如图1所示的淋巴管平滑肌瘤病多方法联合检测方案，对淋巴管平滑肌瘤病标本进行下述检测。
一、采用探针进行液相捕获靶向测序(Target Capture Sequencing,NGS)；
1、设计Panel
针对与LAM基本高度相关的TSC1和TSC2基因全外显子编码区进行叠瓦式设计，其中TSC1和TSC2基因的探针设计如图1所示，以保证这两个基因目标区域至少2倍覆盖，每条探针100bp长。
并同时参考COSMIC、TCGA等数据库，结合最新NCCN指南/共识，从中筛选与实体肿瘤发生、发展密切相关的原癌、抑癌基因，按照常规方法设计探针，即探针以末端相接的方式对目标区域进行1倍覆盖，组合成基因panel列表如下：
表1.Panel基因列表
Figure PCTCN2019129173-appb-000002
表2.TSC1和TSC2探针设计表
Figure PCTCN2019129173-appb-000003
Figure PCTCN2019129173-appb-000004
Figure PCTCN2019129173-appb-000005
Figure PCTCN2019129173-appb-000006
Figure PCTCN2019129173-appb-000007
Figure PCTCN2019129173-appb-000008
Figure PCTCN2019129173-appb-000009
Figure PCTCN2019129173-appb-000010
Figure PCTCN2019129173-appb-000011
Figure PCTCN2019129173-appb-000012
Figure PCTCN2019129173-appb-000013
Figure PCTCN2019129173-appb-000014
Figure PCTCN2019129173-appb-000015
2、gDNA文库构建
按照常规方法进行文库构建：
1)取样。
a)用Qubit2.0检测各样本DNA浓度并记录；
b)取样品50ng放置0.2ml PCR管中，
2)DNA片段化
a)往已取好样品的PCR管按下表配制片段化体系：
组分 体积(μL)
In put Double-stranded DNA 35
KAPA Frag Buffer(10X) 5
KAPA Frag Enzyme 10
Total 50
b)按以下反应程序进行打断：
Figure PCTCN2019129173-appb-000016
3)末端修复及A-Tailing
a)已片段化的PCR管按下表配制片段化体系：
组分 体积(μL)
Fragmented DNA 50
End Repair & A-Tailing Buffer 7
End Repair & A-Tailing Enzyme Mix 3
Total 60
b)按以下程序进行反应：
Figure PCTCN2019129173-appb-000017
4)接头连接
a)往已修复加A尾的PCR管按下表配制片段化体系：
组分 体积(μL)
End repair and A-tailing product 60
Index Adapter 2.5
PCR-grade water 7.5
Ligation Buffer 30
DNA Ligase 10
Total 110
b)按以下程序进行反应：
Figure PCTCN2019129173-appb-000018
5)连接产物纯化及片段筛选
a)将连接产物取出，转移到含88μL Ampure xp Beads的1.5mL EP管中，充分混匀，微离心，室温放置5min，磁力架上放置至澄清后去除上清液体；
b)加入200μL新鲜配制80％乙醇，转动EP管几次，澄清后弃上清，室温稍晾干；
c)加入50.0μL ddH 2O充分混匀，微离心，室温放置5min，磁力架上放置至澄清后转移上清到含50μL Ampure xp Beads的1.5mL EP管中，充分混匀，微离心，室温放置5min，磁力架上放置至澄清后去除上清液体；
d)加入200μL新鲜配制80％乙醇，转动EP管几次，澄清后弃上清；
e)加入200μL新鲜配制80％乙醇，转动EP管几次，澄清后弃上清，室温稍晾干；
f)用21μL ddH 2O洗脱，待下步PCR。
6)PCR反应
a)取一新的PCR管按下表配制PCR体系：
组分 体积(μL)
Adapter-ligated library 20
KAPA HiFi HotStart ReadyMix(2X) 25
Library Amplification Primer Mix(10X) 5
Total 50
b)按以下反应程序扩增：
98℃,45s→(98℃,15s,60℃,30s,72℃,30s)8个循环→72℃,1min→4℃,∞。
7)PCR产物纯化
a)将PCR产物取出后，转移到含50μL Ampure xp Beads的1.5mL EP管中，充分混匀，微离心，室温放置10-15min，磁力架上放置至澄清后去除上清液体；
b)加入200μL新鲜配制80％乙醇，转动EP管几次，澄清后弃上清；
c)加入200μL新鲜配制80％乙醇，转动EP管几次，澄清后弃上清，室温静置晾干；
d)加入21μL ddH 2O洗脱文库；
e)用Qubit2.0检测各样本文库浓度并记录；
f)用2100芯片分析仪检测各样本文库片段大小(Optional)。
3、杂交捕获
1)探针杂交
a)DNA文库Pooling：根据Qubit2.0测得浓度，按每个标本100ng总量混合于PCR管中，5个样品进行一次杂交反应；
b)往Pooling好文库的PCR管中加入封闭Oligo，
Figure PCTCN2019129173-appb-000019
c)充分吹打混匀后，用真空抽滤系统将混合溶液抽干(温度设置60℃)；
d)往抽干的PCR管中添加以下杂交缓冲液，充分吹打混匀，室温静置5-10min：
组分 体积(μL)
xGen 2X Hybridization Buffer 8.5
xGen Hybridization Buffer Enhancer 2.7
Nuclease-Free Water 1.8
e)将PCR管置PCR仪上95℃温浴10min变性；
f)变性完毕立即将PCR管取出，放置预冷金属板上，立即添加4μL xGen Lockdown Probe pool(探针)；
g)按下表程序进行杂交：
温度 时间 热盖温度
65℃ >14h 75℃
2)捕获洗脱
a)按下表比例将各Wash Buffer进行稀释(每份满足一次捕获洗脱的量)：
试剂名称 原液体积(μl) ddH 2O体积(μl) 1X Buffer总(μl)
xGen 10X Wash Buffer I 30 270 300
xGen 10X Wash Buffer II 20 180 200
xGen 10X Wash Buffer III 20 180 200
xGen 10X Stringent Wash Buffer 40 360 400
xGen 2X Bead Wash Buffer 250 250 500
b)将400μl 1X Stringent Wash Buffer放置65℃金属温浴器预热；
c)分装100μl 1X Wash Buffer I放置65℃温浴器预热，剩余200μl 1X Wash Buffer I放置室温待用；
d)将
Figure PCTCN2019129173-appb-000020
M-270 Streptavidin beads从4℃取出平衡至室温，充分震荡后吸取100μl至1.5ml离心管中，放置磁力架上，澄清后去除上清；
e)将离心管从磁力架上取出，加入200μl 1X Bead Wash Buffer，充分震荡10s，微离后放回磁力架上，澄清后去除上清；；
f)重复上述步骤一次；
g)加入100μl 1X Bead Wash Buffer重悬
Figure PCTCN2019129173-appb-000021
M-270 Streptavidin beads，并转至新的200μl PCR管中，放置磁力架上，澄清后去除上清待用；
h)将过夜杂交的样本取出(PCR仪维持程序65℃，热盖75℃)，把全部液体转至上一步清洗好的
Figure PCTCN2019129173-appb-000022
M-270 Streptavidin beads中，充分吹打混匀，微离心后重新放回PCR仪中；
i)反应12min后取出吹打混匀，共3次；
j)反应完毕，将PCR管取出，添加100μl 1X Wash Buffer I(65℃预热)，充分震荡10s后将液体转至1.5ml离心管中，放置磁力架上，澄清后去除上清；
k)将离心管从磁力架上取出，加入200μl 1X Stringent Buffer(65℃预热)，充分吹打混匀后迅速放回65℃水浴锅中，温浴5min；
l)重复操作步骤k一次
m)温浴后放置磁力架上，澄清后去除上清；
n)将离心管从磁力架上取出，加入200μl 1X Wash Buffer I(常温)，震荡2min，微离心后放置磁力架上，澄清后去除上清；
o)将离心管从磁力架上取出，加入200μl 1X Wash Buffer II，震荡1min，微离心后放置磁力架上，澄清后去除上清；
p)将离心管从磁力架上取出，加入200μl 1X Wash Buffer III，震荡30s，微离心后放置磁力架上，澄清后去除上清；
q)加入20μl ddH 2O重悬洗脱
Figure PCTCN2019129173-appb-000023
M-270 Streptavidin beads待PCR。
3)第二次PCR(Post-PCR)
a)取一新的PCR管按下表分别配制PCR体系：
体系组成 体积(μl)
上一步捕获洗脱产物 20
KAPA HiFi HotStart ReadyMix 25
10μM Illumina P5 primer 2.5
10μM Illumina P7 primer 2.5
总体积 50
b)按以下反应程序扩增文库：
98℃,45s→(98℃,15s,60℃,30s,72℃,30s)11个循环→72℃,1min→4℃,∞。
4)PCR产物纯化
a)将PCR产物取出后，转移到含75μL Ampure xp Beads的1.5mL EP管中，充分混匀，微离心，室温放置10-15min，磁力架上放置至澄清后去除上清液体；
b)加入200μL新鲜配制80％乙醇，转动EP管几次，澄清后弃上清；
c)加入200μL新鲜配制80％乙醇，转动EP管几次，澄清后弃上清，室温静置晾干；
d)加入21.0μL ddH 2O洗脱文库。
5)文库质控
a)用Qubit2.0将终文库进行浓度测定；
b)采用Agilent 2100生物分析仪检测文库片段大小(Optional)；
6)上机测序
Illumina Nextseq500
二、分选。
通过生物信息学处理分析上述测序数据，如检测到TSC1和TSC2基因检测结果阴性或只有单打击突变位点，则采用染色体芯片分析方法及多重连接探针扩增法进行补充检测；针对所检测到的不明确属于体细胞突变还是胚系突变来源的位点，则采用Sanger测序法进行验证。
三、染色体芯片分析及多重连接探针扩增法。
如检测到TSC1和TSC2基因检测结果阴性或只有单打击突变位点(即TSC1&TSC2基因中只有一个基因产生突变、或片段缺失/插入、或拷贝数变异等)，则采用染色体芯片分析方法及多重连接探针扩增法进行补充检测，所用试剂盒如下。
表3.补充检测分析试剂盒
Figure PCTCN2019129173-appb-000024
四、Sanger测序。
如检测到不明确属于体细胞突变还是胚系突变来源的位点，具体的，不明确属于体细胞突变还是胚系突变来源的位点指突变频率在50％左右的变异。则采用Sanger测序进行对该患者白细胞标本进行验证(ABI 3730XL)，通过验证结果以辨别该患者属于S-LAM还是TSC-LAM。
如经检测，该患者白细胞也具有上述突变，可判定该患者属于TSC-LAM，否则，可判定该患者属于S-LAM。
实施例2
采用实施例1的方法进行LAM联合检测研究。
1、单一检测方法和联合检测法对比。
本研究中，共有61名LAM入组患者，同时采用单一检测方法和实施例1中的方法进行检测。
单一检测方法为只采用NGS靶向测序的方法。
结果如图3-4所示，其中图3为采用不同检测方法变异位点的检出比例，其中NGS表示液相捕获靶向测序，表示染色体芯片分析，MLPA表示多重连接探针扩增检测。上述结果说明，通过多方法联合检测，针对LAM患者标本全面输出SNV、INDEL、CNV、LOH等变异类型，如使用单一NGS检测方法，将有12.8％的变异丢失
采用单一检测方法，针对TSC1和TSC2基因整体阳性检出率为72.13％，分别检测到1-hit和2-hits的患者分别为15人、29人，而采用联合检测方案，整体阳性检出率为75.41％，分别检测到1-hit和2-hits的患者分别为8人、38人，结果如图4所示。
联合检测方案不但提高了LAM患者的阳性变异检出率，更重要的是检测结果更符合Knudson的“二次打击”学说。
2、研究发现。
采用实施例1的联合检测方法，在61名入组的LAM患者中找到了30个新发的突变，具体如下：
Figure PCTCN2019129173-appb-000025
这些新发突变将大大丰富LAM这种罕见疾病的基因数据库，对推动该疾病的研究进展及治疗指导有重要临床价值。
实施例3
采用实施例1的方法进行LAM联合检测示例。
一、样本来源
样本来自广州市某三甲医院呼吸内科的固定组织标本，临床诊断为S-LAM。
二、检测方法及结果
1、采用探针进行液相捕获靶向测序(Target Capture Sequencing,NGS)
测序结果显示，该样本具有单一位点突变，TSC2：NM_000548.4:c.3412C＞T(p.Arg1138*)，变异频率为50.9％。
2、染色体芯片分析及多重连接探针扩增
按照实施例1的方法，采用染色体芯片分析方法进行补充检测，结果显示，该患者在TSC2基因发现杂合性缺失(LOH)变异：arr<GRCh37>16p13.3p11.2(83886_30809063)x2mos hmz，变异大小为30.73Mb，其LOH片段如图5所示；而采用多重连接探针扩增法的检测结果为阴性。
3、Sanger测序
由于NGS检测到的位点变异频率为50.9％，无法判定该位点来源为体细胞突变还是胚系突变，针对该位点设计引物并对该患者白细胞标本进行测序，验证结果表示该患者白细胞不存在上述突变，从而验证为体细胞突变，即为散发的LAM(S-LAM)。
三、结论
该名患者检测结果显示在TSC2基因密码子1138处产生过早停止编码信号，变异频率为50.9％，通过Sanger法确定该突变不存在于白细胞中，属于体细胞突变，该变异会导致正常蛋白质功能丧失，在往前患有LAM的个体中报道了50多次，为致病性突变，补充检测结果显示该患者还存在TSC2基因LOH现象，已有研究表明LOH会导致抑制基因的失活，从而影响癌症的发生与进行。通过本联合检测方案，可以完整地对该患者的发病机制进行还原，并依据结果判定为散发性LAM，不具有遗传性。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (13)

  1. 一种淋巴管平滑肌瘤病联合检测方法，其特征在于，包括以下步骤：
    液相捕获靶向测序：针对与LAM高度相关的TSC1和TSC2基因全外显子编码区和与实体肿瘤密切相关的突变基因设计Panel，构建gDNA文库，进行杂交捕获后上机测序；
    分选：通过生物信息学处理分析上述测序数据，如检测到TSC1和TSC2基因检测结果阴性或只有单打击突变位点，则采用染色体芯片分析方法及多重连接探针扩增法进行补充检测；如检测到不明确属于体细胞突变还是胚系突变来源的位点，则采用Sanger测序法进行验证；
    染色体芯片分析：通过染色体芯片分析获得杂合性缺失及拷贝数变异结果；
    多重连接探针扩增：以多重连接探针扩增法获得大片段插入缺失结果；
    Sanger测序：取待测样本对应的白细胞标本进行Sanger检测，判断属于S-LAM型或TSC-LAM型。
  2. 根据权利要求1所述的淋巴管平滑肌瘤病联合检测方法，其特征在于，所述液相捕获靶向测序步骤中，所述Panel设计覆盖如下基因：ALDH1基因，EGFR基因，FLT3基因，MYC基因，PTEN基因，SDHD基因，AQP9基因，ERBB2基因，HRAS基因，MYCN基因，RET基因，TP53基因，AR基因，ESR1基因，KIT基因，NF1基因，RICTOR基因，TSC1基因，ATRX基因，FGFR1基因，KRAS基因，NRAS基因，RUNX1基因，TSC2基因，BCL2基因，FGFR2基因，MDM2基因，PDGFRA基因，SDHA基因，VHL基因，BRAF基因，FGFR3基因，MAP2K1基因，PGR基因，SDHB基，CCND1基因，FGFR4基因，MET基因，POLE基因，SDHC基因；ABL1基因，CDKN2A基因，FBXW7基因，IDH2基因，NOTCH1基因，SMAD4基因，AKT1基因，CSF1R基因，GNA11基因，JAK2基因，NPM1基因，SMARCB1基因，ALK基因，CTNNB1基因，GNAQ基因，JAK3基因，PIK3CA基因，SMO基因，APC基因，DDR2基因，GNAS基因，KDR基因，PTPN11基因，SRC基因，ATM基因，ERBB4基因，HNF1A基因，MLH1基因，RB1基因，STK11基因，CDH1基因，EZH2基因，IDH1基因，MPL基因，ROS1基因，TET2基因。
  3. 根据权利要求2所述的淋巴管平滑肌瘤病联合检测方法，其特征在于，所述液相捕获靶向测序步骤中，针对TSC1基因和TSC2基因的探针序列如SEQ ID No.1-SEQ ID No.276所示。
  4. 根据权利要求2所述的淋巴管平滑肌瘤病联合检测方法，其特征在于，所述液相捕获靶向测序步骤中，测序深度大于1000x。
  5. 权利要求1-4任一项所述的淋巴管平滑肌瘤病联合检测方法在研究LAM的发病机制和/或LAM的诊断和治疗中的应用。
  6. 根据权利要求5所述的应用，其特征在于，所述检测方法中的特异性检测试剂在制备淋巴管平滑肌瘤病联合检测诊断试剂或诊断设备中的应用。
  7. 一种淋巴管平滑肌瘤病联合检测试剂盒，其特征在于，包括覆盖如下基因的检测Panel：ALDH1基因，EGFR基因，FLT3基因，MYC基因，PTEN基因，SDHD基因，AQP9基因，ERBB2基因，HRAS基因，MYCN基因，RET基因，TP53基因，AR基因，ESR1基因，KIT基因，NF1基因，RICTOR基因，TSC1基因，ATRX基因，FGFR1基因，KRAS基因，NRAS基因，RUNX1基因，TSC2基因，BCL2基因，FGFR2基因，MDM2基因，PDGFRA基因，SDHA基因，VHL基因，BRAF基因，FGFR3基因，MAP2K1基因，PGR基因，SDHB基，CCND1基因，FGFR4基因，MET基因，POLE基因，SDHC基因；ABL1基因，CDKN2A基因，FBXW7基因，IDH2基因，NOTCH1基因，SMAD4基因，AKT1基因，CSF1R基因，GNA11基因，JAK2基因，NPM1基因，SMARCB1基因，ALK基因，CTNNB1基因，GNAQ基因，JAK3基因，PIK3CA基因，SMO基因，APC基因，DDR2基因，GNAS基因，KDR基因，PTPN11基因，SRC基因，ATM基因，ERBB4基因，HNF1A基因，MLH1基因，RB1 基因，STK11基因，CDH1基因，EZH2基因，IDH1基因，MPL基因，ROS1基因，TET2基因。
  8. [根据细则26改正13.01.2020]　
    根据权利要求1所述的淋巴管平滑肌瘤病联合检测试剂盒，其特征在于，所述检测Panel的探针序列如SEQ ID No.1-SEQ ID No.276所示。
  9. 根据权利要求7所述的淋巴管平滑肌瘤病联合检测试剂盒，其特征在于，还包括染色体芯片分析试剂。
  10. 根据权利要求7所述的淋巴管平滑肌瘤病联合检测试剂盒，其特征在于，还包括多重连接探针。
  11. 一种淋巴管平滑肌瘤病联合检测系统，其特征在于，包括以下模块：
    检测模块，包括液相捕获靶向测序模块、染色体芯片分析模块、多重连接探针扩增模块、Sanger测序模块，所述液相捕获靶向测序模块中包括：针对与LAM高度相关的TSC1和TSC2基因全外显子编码区和与实体肿瘤密切相关的突变基因设计的Panel；
    分析模块，首先获取所述液相捕获靶向测序结果，如检测到TSC1和TSC2基因检测结果阴性或只有单打击突变位点，则提示采用染色体芯片分析模块及多重连接探针扩增模块进行补充检测；如检测到不明确属于体细胞突变还是胚系突变来源的位点，则提示采用Sanger测序模块进行验证；再以检测模块检测到的结果综合分析判断，得出淋巴管平滑肌瘤病联合检测结果。
  12. 根据权利要求11所述的淋巴管平滑肌瘤病联合检测系统，其特征在于，所述Panel覆盖如下基因：ALDH1基因，EGFR基因，FLT3基因，MYC基因，PTEN基因，SDHD基因，AQP9基因，ERBB2基因，HRAS基因，MYCN基因，RET基因，TP53基因，AR基因，ESR1基因，KIT基因，NF1基因，RICTOR基因，TSC1基因，ATRX基因，FGFR1基因，KRAS基因，NRAS基因，RUNX1基因，TSC2基因，BCL2基因，FGFR2基因，MDM2基因，PDGFRA基因，SDHA基因，VHL基因，BRAF基因，FGFR3基因，MAP2K1基因，PGR基因，SDHB基，CCND1基因，FGFR4基因，MET基因，POLE基因，SDHC基因；ABL1基因，CDKN2A基因，FBXW7基因，IDH2基因，NOTCH1基因，SMAD4基因，AKT1基因，CSF1R基因，GNA11基因，JAK2基因，NPM1基因，SMARCB1基因，ALK基因，CTNNB1基因，GNAQ基因，JAK3基因，PIK3CA基因，SMO基因，APC基因，DDR2基因，GNAS基因，KDR基因，PTPN11基因，SRC基因，ATM基因，ERBB4基因，HNF1A基因，MLH1基因，RB1基因，STK11基因，CDH1基因，EZH2基因，IDH1基因，MPL基因，ROS1基因，TET2基因。
  13. 根据权利要求11所述的淋巴管平滑肌瘤病联合检测系统，其特征在于，所述液相捕获靶向测序模块中，针对TSC1基因和TSC2基因的探针序列如SEQ ID No.1-SEQ ID No.276所示。
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