WO2017166030A1 - 分离的核酸及其在筛查肾上腺皮质腺瘤中的应用 - Google Patents

分离的核酸及其在筛查肾上腺皮质腺瘤中的应用 Download PDF

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WO2017166030A1
WO2017166030A1 PCT/CN2016/077562 CN2016077562W WO2017166030A1 WO 2017166030 A1 WO2017166030 A1 WO 2017166030A1 CN 2016077562 W CN2016077562 W CN 2016077562W WO 2017166030 A1 WO2017166030 A1 WO 2017166030A1
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gene
nucleic acid
mutations
mutation
chr11
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PCT/CN2016/077562
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French (fr)
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吴松
黄培德
张蒙
黄毅
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深圳市众循精准医学研究院
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Priority to CN201680084151.7A priority Critical patent/CN109196101B/zh
Priority to PCT/CN2016/077562 priority patent/WO2017166030A1/zh
Publication of WO2017166030A1 publication Critical patent/WO2017166030A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

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  • the present invention relates to the field of biomedicine, in particular, the present invention relates to isolated nucleic acids and their use in screening adrenocortical adenomas, in particular, the present invention relates to an isolated nucleic acid, an isolated polypeptide, an isolated nucleic acid And/or use of a polypeptide, a kit capable of detecting the nucleic acid and/or polypeptide, and a method and apparatus for screening a biological sample of an adrenocortical adenoma.
  • Adrenal adenoma is the most common cause of primary aldosteronism and causes 10% of patients to have hypertension.
  • APA is one of the most common genetic determinants of secondary hypertension. Patients with APA tend to have a higher incidence of cardiovascular disease and mortality than those with a similar severity of essential hypertension.
  • the present invention is directed to solving one of the above technical problems at least to some extent or to provide a commercial choice.
  • an isolated nucleic acid comprising a gene sequence comprising at least one of the following mutations compared to a corresponding reference gene sequence: DNMT3A gene chr2: 25463248G>C, CIC gene chr19: 42790976G>T, CTNNB1 gene chr3: 41266136T>C, RAD50 gene chr5: 131926953T>C, RAD50 gene chr4:83782843G>A and KCNA1 gene chr12:5021496A>G.
  • the corresponding reference gene sequence refers to the reference sequence of the gene in which the mutation is located, for example, the sequence in which the DNMT3A gene chr2:25463248G>C mutation is present: the DNMT3A of the nucleic acid sequence compared to the DNMT3A gene in the human reference genome HG19 There is a mutation at the position of the gene chr2: 25463248 - the wild G base is mutated to the C base.
  • the six mutations corresponding to the above five genes are unsuccessful single-sense single nucleotide mutations (SNPs), or unreported APA-related missense mutations, and the gene with the missense mutation is an invention.
  • SNPs single-sense single nucleotide mutations
  • Humans have discovered and identified validated mutant genes associated with APA pathogenicity in East Asian populations. These six mutant genes and their encoded proteins are associated with the control of ionic balance between the cytoplasm and the extracellular matrix.
  • the inventors conducted a study on a population of APA patients from China based on whole exome sequencing, and found candidate somatic mutations and mutant genes that may be associated with APA. The analysis confirmed that these mutant genes are associated with the occurrence of APA.
  • the use of these mutant genes, mutation sites as the standard of APA
  • it can provide rich and reliable information for the mechanism of APA and clinical diagnosis and development of target drugs, and assist in the diagnosis and treatment of APA.
  • the identified markers are used for the detection of APA susceptibility and the results of individualized tests, and are capable of supporting a wide range of personalized APA tests, particularly APA testing in East Asian populations.
  • any one or more of the six mutated gene sequences can be used to assist in the diagnosis of APA, and the one or more mutated gene sequences are present in the sample to be tested, and the sample can be determined to be an APA susceptible sample. It should be noted that APA is a complex disease, and it is difficult to determine the occurrence or development of individual APA by using the presence of a single or combined mutant gene.
  • Detecting one, two, three, four, five or all six of the above six mutant genes in the test sample can indicate that the sample is an APA susceptible sample, and generally, the more than one exists
  • the above mutated gene sequence indicates that the sample has a higher probability of being an APA sample, and the detection of the above marker can assist in diagnosing the occurrence, development and treatment of the individual APA, especially in APA patients or susceptible populations in East Asia.
  • the above six SNP mutations are expressed by labeling the position of the site on the reference genomic DNA (gDNA, HG19 version) according to the HGVS nomenclature, and those skilled in the art can know that one SNP position
  • the point may also have other representations, and the above-mentioned mutations which are otherwise expressed in the same manner as the present invention are still within the scope of the present invention, for example, the naming method of the GenBank SNP database - rs plus 7 digits of Arabic numerals to represent a SNP, Further, for example, the same SNP site or SNP combination as described above is indicated by the position of the SNP on the reference cDNA.
  • an isolated nucleic acid comprising a DNMT3A gene sequence in the presence of chr2:25463248G>C.
  • the DNMT3A gene with the chr2:25463248G>C mutation is a mutated gene that has not been reported, and the gene carrying the mutation is a mutant gene discovered and identified by the inventors to be associated with the occurrence or development of APA.
  • the nucleic acid can be used as a marker for detecting APA, and can be used for assisting diagnosis of APA.
  • the marker is present in the sample to be tested, and the sample can be determined to be an APA susceptible sample.
  • the nucleic acid is a combination of mutated sequences, which further comprises a corresponding gene having one, two, three, four, five, six, seven or all eight mutations
  • KCNJ5 gene chr11: 128781671T>G Sequence: CIC gene chr19: 42790976G>T, CTNNB1 gene chr3: 41266136T>C, RAD50 gene chr5: 131926953T>C, RAD50 gene chr4:8378
  • the corresponding gene sequence refers to the reference sequence of the gene in which the mutation is located, for example, the sequence in which the DNMT3A gene chr2:25463248G>C mutation is present: the DNMT3A gene of the nucleic acid sequence is compared with the DNMT3A gene in the human reference genome HG19. There is a mutation at the position of chr2:25463248 - the wild G base is mutated to the C base. It should be noted that APA is a complex disease, and it is difficult to determine the occurrence or development of individual APA by using the presence of a single or combined mutant gene.
  • the sample is an APA susceptible sample, and generally, the more the above-mentioned mutant gene exists in the sample, indicating that the sample has a higher probability of being an APA sample, and the detection of the above marker can assist in diagnosing the occurrence, development and development of the individual APA. Treatment, especially for East Asian APA patients or susceptible populations. It should also be noted that the above 9 SNP mutations are expressed by reference to the genomic DNA (gDNA, HG19) according to the HGVS nomenclature.
  • SNP site may have other representations, and the above-mentioned mutations which are otherwise expressed in the same manner as the present invention are still within the scope of the present invention, for example, GenBank.
  • GenBank The naming method of the SNP database - a sNP is represented by rs plus 7 digits of Arabic numerals, and the SNP locus or SNP combination referring to the same is described, for example, by the position of the SNP on the reference cDNA.
  • an isolated nucleic acid comprising a CIC gene sequence in the presence of chr19: 42790976G>T.
  • the CIC gene carrying the chr19:42790976G>T mutation is a mutated gene that has not been reported, and the gene carrying the mutation is a mutated gene which the inventors discovered and identified to be related to the occurrence or development of APA.
  • the nucleic acid can be used as a marker for detecting APA, and can be used for assisting diagnosis of APA.
  • the marker is present in the sample to be tested, and the sample can be determined to be an APA susceptible sample.
  • the nucleic acid is a combination of mutated sequences, the nucleic acid further comprising a corresponding gene sequence in which at least one mutation is present: DNMT3A gene chr2: 25463248G>C, CTNNB1 gene chr3: 41266136T>C, RAD50 Gene chr5: 131926953T>C, RAD50 gene chr4:83782843G>A and KCNA1 gene chr12:5021496A>G, KCNJ5 gene chr11:128781619G>C, KCNJ5 gene chr11:128781619G>A and KCNJ5 gene chr11:128781671T>G.
  • the corresponding gene sequence refers to a reference sequence of the gene in which the mutation is located, for example, the sequence in which the CIC gene chr19:42790976G>T mutation is present: the CIC gene of the nucleic acid sequence compared with the CIC gene in the human reference genome HG19 There is a mutation in the position of chr19:42790976 - the wild G base is mutated to the T base.
  • APA is a complex disease, and it is difficult to determine the occurrence or development of individual APA by using the presence of a single or combined mutant gene.
  • the sample is an APA susceptible sample, and generally, the more the above-mentioned mutant gene exists in the sample, indicating that the sample has a higher probability of being an APA sample, and the detection of the above marker can assist in diagnosing the occurrence, development and development of the individual APA. Treatment, especially for East Asian APA patients or susceptible populations.
  • the above nine SNP mutations are expressed by labeling the position of the site on the reference genomic DNA (gDNA, HG19 version) according to the HGVS nomenclature, and those skilled in the art can know that one SNP position
  • the point may also have other representations, and the above-mentioned mutations which are otherwise expressed in the same manner as the present invention are still within the scope of the present invention, for example, the naming method of the GenBank SNP database - rs plus 7 digits of Arabic numerals to represent a SNP, Further, for example, the same SNP site or SNP combination as described above is indicated by the position of the SNP on the reference cDNA.
  • an isolated nucleic acid comprising a CTNNB1 gene sequence in the presence of chr3: 41266136T>C.
  • the CTNNB1 gene with the chr3:41266136T>C mutation is a mutated gene that has not been reported, and the gene carrying the mutation is a mutant gene discovered and identified by the inventors to be associated with the occurrence or development of APA.
  • the nucleic acid can be used as a marker for detecting APA, and can be used for assisting diagnosis of APA.
  • the marker is present in the sample to be tested, and the sample can be determined to be an APA susceptible sample.
  • the nucleic acid is a combination of mutated sequences, and the nucleic acid further comprises a gene sequence corresponding to at least one of the following mutations: DNMT3A gene chr2: 25463248G>C, CIC gene chr19: 42790976G>T, RAD50 gene chr5: 131926953T>C, RAD50 gene chr4:83782843G>A and KCNA1 gene chr12:5021496A>G, KCNJ5 gene chr11:128781619G>C, KCNJ5 gene chr11:128781619G>A and KCNJ5 gene chr11:128781671T>G.
  • the corresponding gene sequence refers to the reference sequence of the gene in which the mutation is located, for example, the sequence in which the CTNNB1 gene chr3: 41266136T>C mutation is present: the CTNNB1 gene of the nucleic acid sequence compared with the CTNNB1 gene in the human reference genome HG19 There is a mutation in the position of chr3: 41266136 - the wild T base is mutated to the C base.
  • APA is a complex disease, and it is difficult to determine the occurrence or development of individual APA by using the presence of a single or combined mutant gene.
  • the sample is an APA susceptible sample, and generally, the more the above-mentioned mutant gene exists in the sample, indicating that the sample has a higher probability of being an APA sample, and the detection of the above marker can assist in diagnosing the occurrence, development and development of the individual APA. Treatment, especially for East Asian APA patients or susceptible populations.
  • the above nine SNP mutations are expressed by labeling the position of the site on the reference genomic DNA (gDNA, HG19 version) according to the HGVS nomenclature, and those skilled in the art can know that one SNP position
  • the point may also have other representations, and the above-mentioned mutations which are otherwise expressed in the same manner as the present invention are still within the scope of the present invention, for example, the naming method of the GenBank SNP database - rs plus 7 digits of Arabic numerals to represent a SNP, Further, for example, the same SNP site or SNP combination as described above is indicated by the position of the SNP on the reference cDNA.
  • a nucleic acid according to any of the above embodiments for the detection of adrenocarcinoma and/or for the preparation of a medicament for the treatment of adrenal sebaceous adenoma.
  • the KCNJ5 gene chr11:128781619G>C the KCNJ5 gene chr11:128781619G>A and the KCNJ5 gene chr11:128781671T>G
  • the other 6 mutations are unreported single nucleotide mutations which are unreported single nucleotides.
  • SNP acid mutation
  • Any one or more of the 9 mutant genes can be used to assist in the diagnosis of APA, and the one or more mutant gene sequences are present in the sample to be tested, and the sample can be determined to be an APA susceptible sample. Information on any one or more of these 9 mutant genes can be used to prepare APA-targeted drugs. It should be noted that APA is a complex disease, and it is difficult to determine the occurrence or development of individual APA by using the presence of a single or combined mutant gene.
  • Detection of one, two, three, four, five, six, seven, eight or all nine of the above 9 mutant genes in the test sample can indicate that the sample is an APA susceptible sample And, in general, the more mutated genes are present, indicating that the sample has a relatively high probability of being an APA sample, and the detection marker can aid in diagnosing the occurrence, development, and treatment of individual APA.
  • a kit comprising an agent capable of detecting a nucleic acid in any of the above embodiments, the reagent comprising a primer sequence.
  • primers capable of binding and detecting the mutation site of the above nucleic acid by using primer design software according to the information of the mutation site existing on the isolated nucleic acid provided above, including the position of the mutation, the sequence near the mutation, and the like. sequence. It should be noted that detecting the above nucleic acid with a specific mutation is substantially detecting the SNP, and those skilled in the art can understand that one or more SNPs can be utilized.
  • the sequence of the position where the SNP is located is designed to be detected by PCR, and the amplification primer and the extension primer can be designed by using the sequence of the position where the mutation is located, and mass spectrometry is used to detect the presence or absence of the SNP.
  • the so-called reagents include, but are not limited to, primer sequences.
  • kits for detecting any of the isolated nucleic acids and/or detecting an adrenocortical adenoma in vitro are provided.
  • a kit comprising an agent capable of detecting a nucleic acid in any of the above embodiments can be conveniently and efficiently used for detecting an adrenocortical adenoma in vitro.
  • an isolated polypeptide encoded by the isolated nucleic acid of any of the above embodiments is provided.
  • the so-called polypeptide is encoded by the above-mentioned nucleic acid with a specific mutation. Due to the specific mutation on the nucleic acid, the so-called polypeptide also has a variation of the amino acid residue at the corresponding site as compared with the wild protein sequence.
  • the so-called isolated nucleic acids and the proteins encoded thereby are associated with the control of the ionic balance between the cytoplasm and the extracellular matrix.
  • any of the above polypeptides for the detection of adrenal sebaceous adenoma and/or for the preparation of a medicament for the treatment of adrenosecretory adenoma.
  • the so-called polypeptide is encoded by the above-mentioned nucleic acid with a specific mutation. Due to the specific mutation on the nucleic acid, the so-called polypeptide also has a variation of the amino acid residue at the corresponding site as compared with the wild protein sequence.
  • the presence of the so-called polypeptide in the test sample can be detected, and the sample can be described as an APA susceptible sample.
  • the polypeptide contains information that can be used to prepare APA-targeted drugs.
  • the polypeptide can be used in conjunction with the diagnosis of the occurrence, development and treatment of APA in a subject.
  • kits comprising an agent capable of detecting any of the above polypeptides.
  • the so-called reagents include, but are not limited to, western blot and protein sequencing related reagents.
  • kits for detecting any of the above polypeptides and/or detecting adrenal sebaceous adenomas in vitro comprising an agent capable of detecting the polypeptide of any of the above embodiments can be conveniently and efficiently used for the detection of adrenal adenomas in vitro.
  • a method for screening a biological sample of an adrenal sebaceous adenoma comprising: (1) obtaining exome sequencing data of a sample to be tested, the sequencing data comprising a plurality of reads (2) Based on the sequencing data of (1), the following (a)-(c) are performed: (a) determining the frequency of somatic mutations present on the exome, and (b) detecting the exons Whether the somatic mutation present on the group includes at least one of the following mutations: KCNJ5 gene chr11:128781619G>C, KCNJ5 gene chr11:128781619G>A and KCNJ5 gene chr11:128781671T>G, and (c) detection of the exon Whether the isolated nucleic acid in any of the above embodiments is present on the group; (3) determining that the frequency of (a) in (2) is greater than 1 individual cell mutation per 10 Mb and (b) and/or
  • the frequency of somatic mutations present on the so-called exome refers to the number of somatic mutations present on the exome per unit length.
  • the method is capable of efficiently and conveniently detecting and screening APA samples based on the use of the isolated nucleic acid in any of the above embodiments as an APA marker, and the frequency of binding to somatic mutations present on the exon.
  • sequencing data is obtained by sequencing the nucleic acid sequence in the sample, and sequencing is based on the selected sequencing platform.
  • semiconductor sequencing technology platforms such as PGM, Ion Proton, BGISEQ-100 platform, synthetic side sequencing technology platforms such as Illumina's Hiseq, Miseq sequence platform and single molecule real-time sequencing platform such as PacBio sequence platform.
  • the sequencing method can be either single-ended sequencing or double-end sequencing, and the obtained offline data is a segment read out, which is called a read.
  • somatic mutation is a mutation that occurs in normal body cells, such as a mutation that occurs in the skin or organs, and such mutations are not passed on to the offspring.
  • germline mutations also known as germ cell mutations, germline mutations, or germline mutations, are mutations that occur in cells that will become gametes (sperms and eggs). Mutations in germ cells can be passed on to future generations.
  • the mutation information utilized in this method is somatic mutation information, and somatic mutation is important for the development of cancer.
  • step (2) comprises: comparing the reads to a reference genome to obtain a comparison result; determining a mutation based on the comparison result, including, removing the Germ cell mutations in the mutation are obtained to obtain the somatic mutation; the frequency of the somatic mutation on the exome is determined.
  • germ cell mutations therein are removed by annotating the identified mutations against known germ cell mutations, for example, by annotating the mutations with dbSNP.
  • the reference genome-predetermined genomic sequence may be any reference template in the biological category to which the target individual belongs, such as a published genome assembly sequence of the same biological category, if the target individual is a human, its genome reference
  • the sequence also known as the reference genome
  • a resource library containing more reference sequences may be pre-configured, and before the sequence comparison, the target individual's gender, race, region, and other factors are selected or determined to assemble a closer sequence as a reference sequence. To help get more accurate screening results.
  • the so-called alignment can be performed by using known comparison software, such as SOAP, BWA, and TeraMap, etc., which is not limited in the present invention.
  • comparison software such as SOAP, BWA, and TeraMap, etc.
  • the comparison process generally compare the parameters, set one or a pair of reads (reads) to allow up to s base mismatch, for example, set s ⁇ 2, if there are more than s base in the reads If a mismatch occurs, the read or the pair of reads cannot be compared (on the alignment) to the reference sequence.
  • the somatic mutation of (a) in the step (2) is selected from the mutations in Table 1 below.
  • the somatic mutations in Table 1 were determined by the inventors based on the analysis of exome data of a large number of adrenocortical adenoma samples, and the somatic mutations often found in adrenocortical adenoma samples, which need to be explained. Most of the somatic mutations are mutations that have not been reported.
  • step (3) includes determining that when the frequency of (a) in (2) is greater than 2 individual cell mutations per 10 Mb and (b) and/or (c) is YES,
  • the biological sample is a biological sample of adrenal sebaceous adenoma. In this way, the accuracy of the determined result can be made higher.
  • the device includes: a data input unit, configured to acquire exome sequencing data of the sample to be tested, the sequencing data includes a plurality of reading segments; and the detecting unit is composed of an A module, a B module, and a C module, and is configured to: Based on the sequencing data from the data input unit, the module is used to perform the following: A module, determining the frequency of somatic mutations present on the exome, B module, detecting the body present on the exome Whether the cell mutation includes at least one of the following mutations: CNJ5 gene chr11:128781619G>C, KCNJ5 gene chr11:128781619G>A and KCNJ5 gene chr11
  • the detection unit A module is configured to: compare the reads to a reference genome to obtain a comparison result; based on the comparison result, determine a mutation, including removing the mutation Germ cell mutation in to obtain the somatic mutation; determine the frequency of said somatic mutation on said exome.
  • the somatic mutation in the module of detection unit A is selected from the mutations in Table 1.
  • the somatic mutations in Table 1 were determined by the inventors based on the analysis of exome data of a large number of adrenocortical adenoma samples, and the somatic mutations often found in adrenocortical adenoma samples, which need to be explained. Most of the somatic mutations are mutations that have not been reported.
  • the biological sample is determined to be an adrenal sebaceous adenoma biological sample.
  • the frequency in the detection unit A module is greater than 2 individual cell mutations per 10 Mb and the output of the B module and/or C module is YES
  • the biological sample is determined to be an adrenal sebaceous adenoma biological sample.
  • Figure 1 is a schematic representation of a mutated gene associated with the development or progression of APA in one embodiment of the invention.
  • Figure 2 is a graph showing the results of a Sanger verification mutation in one embodiment of the present invention.
  • Fig. 3 is a schematic diagram showing the effect of somatic mutation of the potassium ion channel gene KCNA1 in one embodiment of the present invention.
  • a missense mutation refers to a mutation in a DNA that causes a change in the codon in the mRNA, encoding another amino acid.
  • Glu glutamic acid
  • Change to lysine (Lys) silent mutation, also known as a sense-sense mutation or a synonymous mutation, refers to a mutation in DNA that causes a codon change in mRNA to another code, but The encoded amino acid does not change due to the codonation of the codon;
  • a nonsense mutation refers to a mutation in the DNA that causes the codon in the mRNA to change to a stop codon. Both missense mutations and nonsense mutations can be referred to as nonsynonymous mutations.
  • APA detection according to the method/device of the present invention will be described in detail below in conjunction with general steps and specific parameter settings.
  • “multiple” or “multiple sets” means two (groups) or two (groups) or more unless otherwise stated.
  • APA tissue samples and peripheral blood samples were obtained from 22 patients with progressive adrenal hypertension who had primary aldosteronism and adrenocortical tumors from the Chinese Genitourinary Cancer Genome Association (UCGC).
  • Primary aldosteronism is diagnosed by a significantly elevated ratio of aldosterone to renin (ARR). Renin is a plasma renin concentration (PRC; unit mIU/L).
  • PRC plasma renin concentration
  • Adrenal tumors were diagnosed by CT scan. When the most likely diagnosis is APA, A single-sided adrenalectomy is needed. All specimens should be immediately frozen in liquid nitrogen and stored immediately at -80 °C for further study. Tumor sections were stained with hematoxylin-eosin (HE) and then independently microscopically evaluated by two pathologists. All patients showed normal blood potassium and improved blood pressure and control after surgery.
  • HE hematoxylin-eosin
  • APA01-22 is the number of 22 patients, and APA01N and APA01C represent normal tissue samples and tumor tissue samples of APA01, respectively.
  • Tumor genomic DNA and matched peripheral blood samples from 22 APA patients were isolated using QIAGEN's DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). The full exome was obtained from the SureSelect Human Whole Exon 50Mb kit according to the agreement of the manufacturer (Agilent Technologies). 2 ⁇ 90 double-end sequencing was performed according to the manufacturer's instructions and was performed on the HiSeq2000 platform.
  • high-quality sequencing data is obtained by removing reads containing sequencing primers and reads containing more than five unknown bases N in the sequencing data.
  • the paired reads in the high quality sequencing data were aligned to the human reference genome hg19 of NCBI using BWA to obtain a first alignment result.
  • at least a portion of the first alignment results are re-aligned by a Genomic Analysis Toolkit (GATK) to obtain a second alignment result.
  • GATK Genomic Analysis Toolkit
  • VarScan was used to identify potential somatic mutations to obtain a raw list of somatic mutations.
  • the preliminary list of somatic insertion/deletion mutations (INDEL) is based on the second pair of results, identified by GATK.
  • single nucleotide variants (SNVs) and INDEL in tumor samples were identified using the SAMtools software package. The inventors removed somatic mutations that met the following criteria: (i) base mass value ⁇ 20; (ii) aligned mass fraction ⁇ 30; (iii) INDEL only on one DNA strand; (iv) located in suspected insertion Missing mutations in the range of around 30BP.
  • Non-synonymous substitution mutations were verified by Sanger sequencing based on PCR. PCR was performed on a dual 96-96 GeneAmp PCR system (Applied Biosystems) with 20 ng of DNA template per sample. The amplified product was sequenced by a 3730 ⁇ 1 sequencing DNA analyzer (Applied Biosystems). Sequencing sequence analysis was performed using sequencing analysis software (version 5.2). If the mutation in the tumor sample is successfully confirmed, the same primer will be used to amplify the DNA sample from the same patient's blood to determine the state of the observed mutant cells.
  • a gene with FDR q ⁇ 0.1 is considered to be a gene that is significantly mutated in the APA sample.
  • the average sequencing depth of the target region was 71.2 ⁇ (50.75-103.6 ⁇ ), of which 88.5% (81%-92%) was covered by at least 10 ⁇ , as shown in Table 2.
  • the inventors identified a total of 205 individual cell mutation candidates, as shown in Table 1, which included 62 synonymous, 129 missense, 11 nonsense and 2 splice site mutations.
  • C:G>T:A is dominant, which is consistent with most other cancer types. No somatic insertions and deletions were found in the exon region in this mutation cohort.
  • KCNJ5 somatic mutations were clustered in two hot regions, p.G151R and p.L168R, each showing 27.3% (6/22) and 45.5% (10/22) mutations, respectively. frequency.
  • KCNA1 encodes a voltage-gated potassium channel Kv1.1. This protein exhibits a moderate level of expression in the adrenal cortex compared to other potassium channel genes. This mutation results in the appearance of highly conserved threonine and alanine (T318A) at position 318, as shown in Figure 3.
  • T318A is located near the fourth and fifth transmembrane segments (S4 and S5) between the voltage sensors. The inventors hypothesized that this mutation may increase the voltage-activated Ca 2+ influx by disrupting the structure of the Kv1.1 channel, thereby promoting aldosterone synthesis and tumor cell proliferation.
  • the inventors also identified non-synonymous mutations that do not directly control the steady state of ions, but have been shown to frequently mutate in APA, as shown in Figure 1.
  • the CTNNB1 activating mutation is an early and common condition of adrenal tumorigenesis.
  • the inventors identified an activated CTNNB1 mutation (S45P) in a KCNJ5 wild-type APA.
  • S45P activated CTNNB1 mutation
  • the DNA methyltransferase encoded by DNMT3A is responsible for the establishment of a genome-wide nascent methyl group. Mutations in DNMT3A are associated with AML, previously reported and development of lung cancer.
  • CIC is a transcriptional repressor that plays a role in the development of the central nervous system. Mutations in CIC are associated with the origin of oligodendroglioma. These results suggest that multiple other genes may also contribute to the development or progression of APA tumors.
  • somatic mutations (G151R and L168R) in KCNJ5 were first identified by Choi et al. as approximately 40% of APA, which they considered to be the main cause of these tumors. These two mutations are reported to be localized on or near the selective filter of KCNJ5 and result in an increase in sodium conductance and membrane depolarization, resulting in increased production of aldosterone and proliferation of APA cells.
  • 34%-46% of KCNJ5 hotspot mutation patients were identified as European descendants of APA (including patients in France, Germany, Italy, the United Kingdom, and Australia). It is worth noting that in the inventors' study, ultra-high frequency mutations were found in the KCNJ5 gene, and about 72.7% of APA tumors were derived from G151R or L168R proteins to alter somatic mutations (16/22).
  • the KCNJ5 gene was further screened from 23 Japanese APA patients, and the mutation rate of the KCNJ5 gene was found to be 65%, which was also significantly higher than that in APA patients in Europe.
  • the inventors found that in East Asian patients (including at least Japan and China), the frequency of KCNJ5 somatic mutations was significantly higher than that of European descendants, while ATP1A1, ATP2A3 and CACNA1D mutations were more Seen in Europe APA patient.
  • the prevalence of KCNJ5 mutations in East Asian APA still needs to be confirmed in a larger population, the results of this example strongly suggest that KCNJ5 mutations are ethnically related to APA patients.

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Abstract

本发明公开一种分离的核酸，与相应的参考基因序列相比，所述核酸包括存在以下至少一种突变的基因序列：DNMT3A基因chr2：25463248G>C、CIC基因chr19：42790976G>T、CTNNB1基因chr3：41266136T>C、RAD50基因chr5：131926953T>C、RAD50基因chr4：83782843G>A和KCNA1基因chr12：5021496A>G。本发明还公开一种筛查肾上腺皮质腺瘤生物样本的方法及装置。

Description

分离的核酸及其在筛查肾上腺皮质腺瘤中的应用 技术领域
本发明涉及生物医学领域，具体的，本发明涉及分离的核酸及其在筛查肾上腺皮质腺瘤中的应用，具体的，本发明涉及一种分离的核酸、一种分离的多肽、分离的核酸和/或多肽的用途、能够检测该核酸和/或多肽的试剂盒、筛查肾上腺皮质腺瘤生物样本的方法和装置。
背景技术
肾上腺皮质腺瘤(APA)是原发性醛固酮增多症最常见的原因，并且导致10％的患者因此患有高血压，APA是继发性高血压最常见的遗传决定因素之一。比起那些患有类似严重程度的原发性高血压患者，APA的患者往往有更高的心血管疾病发病率和死亡率。
为了揭开APA的的发病机理，几个基因组的研究已经在欧洲患病人群中进行。这些研究证实，几个基因频繁的突变可能导致盐皮质激素醛固酮的生产过剩。对于来自于西方国家的APA患者中，获得性功能突变的钾通道基因KCNJ5的患病率和在P型ATP酶基因家族的两个成员(ATP1A1和ATP2A3)的功能丧失突变的患病率，分别为30-40％和6.8％。最近报道发现，在欧洲7.8％的APA患者是由于CACNA1D中的体细胞突变后，由该基因编码的电压门控钙通道改变所造成。
而对于亚洲人群，目前由于缺乏系统的基因组研究，APA在东亚人群，包括在中国，日本和韩国人群中的突变情况仍不清楚。
发明内容
本发明旨在至少在一定程度上解决上述技术问题之一或者提供一种商业选择。
依据本发明的第一方面，提供一种分离的核酸，与相应的参考基因序列相比，所述核酸包括存在以下至少一种突变的基因序列：DNMT3A基因chr2：25463248G>C、CIC基因chr19：42790976G>T、CTNNB1基因chr3：41266136T>C、RAD50基因chr5：131926953T>C、RAD50基因chr4：83782843G>A和KCNA1基因chr12：5021496A>G。其中，所称的相应的参考基因序列指突变所在的基因的参考序列，例如存在DNMT3A基因chr2：25463248G>C突变的序列为：与人参考基因组HG19中的DNMT3A基因相比，该核酸序列的DNMT3A基因chr2：25463248位置上存在突变——野生的G碱基突变为C碱基。
上述5个基因对应的6个突变是未见报道过的错义单核苷酸突变(SNP)、或者为未报道过的与APA相关的错义突变，带有该错义突变的基因是发明人发现且鉴定验证的与东亚人群APA致病相关的突变基因，这6个突变基因及其编码的蛋白均与细胞质和细胞外基质之间的离子体平衡的控制相关。发明人采取基于全外显子组测序对来自中国的APA患者群体进行研究，找到可能与APA相关的候选体细胞突变以及突变基因，通过分析鉴定验证了这些突变基因与APA的发生有关联。由此，利用这些突变基因、突变位点作为APA的标 志物来检测APA，能够为APA的发生机制和临床诊断及靶点药物的开发提供丰富可靠的信息，辅助诊断治疗APA。更具体地，所确定的标志物用于对APA易感性的检测和个体化检测的结果，能够支持广泛的、个性化的APA检测，特别是东亚人群的APA检测。
6个突变基因序列中的任意一个或者多个都能够用于辅助诊断APA，待测样本中存在上述一个或多个突变基因序列，能够判定该样本为APA易感样本。需要说明的是，APA为复杂疾病，难以利用单一或者组合突变基因的存在，来确切判断检测个体APA的发生或发展。检测待测样本中存在上述6个突变基因中的一个、两个、三个、四个、五个或者全部六个，都能够说明该样本为APA易感样本，而且一般地，存在越多个上述突变基因序列，说明该样本具有越高的几率是APA样本，上述标志物的检测能够辅助诊断个体APA的发生、发展和治疗，特别是东亚APA患者或易感人群。还需要说明的是，上述6个SNP突变的表示方式是根据HGVS命名法标注该位点在参考基因组DNA(gDNA，HG19版本)上的位置来显示的，本领域技术人员能够知道，一个SNP位点还可以有其它表示方式，以其它方式表示的与本发明相同的上述突变，仍旧属于本发明的范围，例如以GenBank SNP数据库的命名方法——以rs加7位阿拉伯数字来表示一个SNP，再例如以该SNP在参考cDNA上的位置来标注指代与上述一样的SNP位点或SNP组合。
依据本发明的第二方面，提供一种分离的核酸，所述核酸包括存在chr2：25463248G>C的DNMT3A基因序列。带有chr2：25463248G>C突变的DNMT3A基因是未见报道过的突变基因，带有该突变的基因是发明人发现且鉴定验证的与APA发生或发展相关的突变基因。该核酸能够作为检测APA的标志物，能够用于辅助诊断APA，待测样本中存在该标志物，能够判定该样本为APA易感样本。
根据本发明的实施例，所述核酸是突变序列的组合，其还包括存在以下一种、两种、三种、四种、五种、六种、七种或者全部八种突变的对应的基因序列：CIC基因chr19：42790976G>T、CTNNB1基因chr3：41266136T>C、RAD50基因chr5：131926953T>C、RAD50基因chr4：83782843G>A、KCNA1基因chr12：5021496A>G、KCNJ5基因chr11：128781619G>C、KCNJ5基因chr11：128781619G>A和KCNJ5基因chr11：128781671T>G。其中，所称的对应的基因序列指突变所在的基因的参考序列，例如存在DNMT3A基因chr2：25463248G>C突变的序列为：与人参考基因组HG19中的DNMT3A基因相比，该核酸序列的DNMT3A基因chr2：25463248位置上存在突变——野生的G碱基突变为C碱基。需要说明的是，APA为复杂疾病，难以利用单一或者组合突变基因的存在，来确切判断检测个体APA的发生或发展。检测待测样本中存在上述9个突变基因中除了DNMT3A基因chr2：25463248G>C突变的序列以外的一个、两个、三个、四个、五个、六个、七个或全部八个，能够进一步说明该样本为APA易感样本，而且一般地，样本存在越多个上述突变基因，说明该样本具有越高的几率是APA样本，上述标志物的检测能够辅助诊断个体APA的发生、发展和治疗，特别是对东亚APA患者或易感人群。还需要说明的是，上述9个SNP突变的表示方式是根据HGVS命名法标注该位点在参考基因组DNA(gDNA，HG19 版本)上的位置来显示的，本领域技术人员能够知道，一个SNP位点还可以有其它表示方式，以其它方式表示的与本发明相同的上述突变，仍旧属于本发明的范围，例如以GenBank SNP数据库的命名方法——以rs加7位阿拉伯数字来表示一个SNP，再例如以该SNP在参考cDNA上的位置来标注指代与上述一样的SNP位点或SNP组合。
依据本发明的第三方面，提供一种分离的核酸，所述核酸包括存在chr19：42790976G>T的CIC基因序列。带有chr19：42790976G>T突变的CIC基因是未见报道过的突变基因，带有该突变的基因是发明人发现且鉴定验证的与APA发生或发展相关的突变基因。该核酸能够作为检测APA的标志物，能够用于辅助诊断APA，待测样本中存在该标志物，能够判定该样本为APA易感样本。
根据本发明的实施例，所述核酸是突变序列的组合，所述核酸还包括存在以下至少一种突变的对应的基因序列：DNMT3A基因chr2：25463248G>C、CTNNB1基因chr3：41266136T>C、RAD50基因chr5：131926953T>C、RAD50基因chr4：83782843G>A和KCNA1基因chr12：5021496A>G、KCNJ5基因chr11：128781619G>C、KCNJ5基因chr11：128781619G>A和KCNJ5基因chr11：128781671T>G。其中，所称的对应的基因序列指突变所在的基因的参考序列，例如存在CIC基因chr19：42790976G>T突变的序列为：与人参考基因组HG19中的CIC基因相比，该核酸序列的CIC基因chr19：42790976位置上存在突变——野生的G碱基突变为T碱基。需要说明的是，APA为复杂疾病，难以利用单一或者组合突变基因的存在，来确切判断检测个体APA的发生或发展。检测待测样本中存在上述9个突变基因中除了CIC基因chr19：42790976G>T突变的序列以外的一个、两个、三个、四个、五个、六个、七个或全部八个，能够进一步说明该样本为APA易感样本，而且一般地，样本存在越多个上述突变基因，说明该样本具有越高的几率是APA样本，上述标志物的检测能够辅助诊断个体APA的发生、发展和治疗，特别是对东亚APA患者或易感人群。还需要说明的是，上述9个SNP突变的表示方式是根据HGVS命名法标注该位点在参考基因组DNA(gDNA，HG19版本)上的位置来显示的，本领域技术人员能够知道，一个SNP位点还可以有其它表示方式，以其它方式表示的与本发明相同的上述突变，仍旧属于本发明的范围，例如以GenBank SNP数据库的命名方法——以rs加7位阿拉伯数字来表示一个SNP，再例如以该SNP在参考cDNA上的位置来标注指代与上述一样的SNP位点或SNP组合。
依据本发明的第四方面，提供一种分离的核酸，所述核酸包括存在chr3：41266136T>C的CTNNB1基因序列。带有chr3：41266136T>C突变的CTNNB1基因是未见报道过的突变基因，带有该突变的基因是发明人发现且鉴定验证的与APA发生或发展相关的突变基因。该核酸能够作为检测APA的标志物，能够用于辅助诊断APA，待测样本中存在该标志物，能够判定该样本为APA易感样本。
根据本发明的实施例，所述核酸是突变序列的组合，所述核酸还包括存在以下至少一种突变对应的基因序列：DNMT3A基因chr2：25463248G>C、CIC基因chr19：42790976G>T、 RAD50基因chr5：131926953T>C、RAD50基因chr4：83782843G>A和KCNA1基因chr12：5021496A>G、KCNJ5基因chr11：128781619G>C、KCNJ5基因chr11：128781619G>A和KCNJ5基因chr11：128781671T>G。其中，所称的对应的基因序列指突变所在的基因的参考序列，例如存在CTNNB1基因chr3：41266136T>C突变的序列为：与人参考基因组HG19中的CTNNB1基因相比，该核酸序列的CTNNB1基因chr3：41266136位置上存在突变——野生的T碱基突变为C碱基。需要说明的是，APA为复杂疾病，难以利用单一或者组合突变基因的存在，来确切判断检测个体APA的发生或发展。检测待测样本中存在上述9个突变基因中除了CTNNB1基因chr3：41266136T>C突变的序列以外的一个、两个、三个、四个、五个、六个、七个或全部八个，能够进一步说明该样本为APA易感样本，而且一般地，样本存在越多个上述突变基因，说明该样本具有越高的几率是APA样本，上述标志物的检测能够辅助诊断个体APA的发生、发展和治疗，特别是对东亚APA患者或易感人群。还需要说明的是，上述9个SNP突变的表示方式是根据HGVS命名法标注该位点在参考基因组DNA(gDNA，HG19版本)上的位置来显示的，本领域技术人员能够知道，一个SNP位点还可以有其它表示方式，以其它方式表示的与本发明相同的上述突变，仍旧属于本发明的范围，例如以GenBank SNP数据库的命名方法——以rs加7位阿拉伯数字来表示一个SNP，再例如以该SNP在参考cDNA上的位置来标注指代与上述一样的SNP位点或SNP组合。
依据本发明的第五方面，提供上述任一实施例中的核酸在检测肾上腺皮脂腺瘤和/或制备治疗肾上腺皮脂腺瘤的药物中的用途。除了KCNJ5基因chr11：128781619G>C、KCNJ5基因chr11：128781619G>A和KCNJ5基因chr11：128781671T>G，其它的6个突变是未见报道过的单核苷酸突变是未见报道过的单核苷酸突变(SNP)、或者为未报道过的与APA相关的突变，带有该突变的基因是发明人发现且鉴定验证的与东亚人群APA致病相关的突变基因。9个突变基因中的任意一个或者多个都能够用于辅助诊断APA，待测样本中存在上述一个或多个突变基因序列，能够判定该样本为APA易感样本。利用这9个突变基因中的任意一个或者多个的信息，可以用于制备APA靶向药物。需要说明的是，APA为复杂疾病，难以利用单一或者组合突变基因的存在，来确切判断检测个体APA的发生或发展。检测待测样本中存在上述9个突变基因中的一个、两个、三个、四个、五个、六个、七个、八个或者全部九个，都能够说明该样本为APA易感样本，而且一般地，存在越多个突变基因，说明该样本具有相对越高的几率是APA样本，检测标志物能够辅助诊断个体APA的发生、发展和治疗。
依据本发明的第六方面，提供一种试剂盒，其包括能够用于检测上述任一实施例中的核酸的试剂，所述试剂包括引物序列。本领域技术人员根据上述提供的分离的核酸上存在的突变位点的信息，包括突变的位置、突变附近的序列等，利用引物设计软件能够设计出能够结合并且检测上述核酸的突变位点的引物序列。需要说明的是，检测上述带有特定突变的核酸，实质上是检测SNP，本领域技术人员可以理解，检测一个或多个SNP可以利用 SNP所在的位置的序列来设计引物，利用PCR来检测；也可以利用突变所在的位置的序列设计扩增引物和延伸引物，利用质谱来检测是否存在该SNP。所称的试剂包括但不限于引物序列。
依据本发明的第七方面，提供上述试剂盒在检测上述任一分离的核酸和/或体外检测肾上腺皮质腺瘤中的用途。包含能够检测上述任一实施例中的核酸的试剂的试剂盒，能够方便高效地用于体外检测肾上腺皮质腺瘤。
依据本发明的第八方面，提供一种分离的多肽，所述多肽由上述任一实施例中的分离的核酸所编码。所称多肽是上述带特定突变的核酸所编码的，由于核酸上带有特定突变，与野生蛋白序列相比，所称的多肽也在相应的位点存在氨基酸残基的变异。上述所称分离的核酸及其所编码的蛋白均与细胞质和细胞外基质之间的离子体平衡的控制相关。上述对任一实施例中的分离的核酸的优点和技术特征的描述，同样适用本发明这一方面的多肽，在此不再赘述。
依据本发明的第九方面，提供上述任一多肽在检测肾上腺皮脂腺瘤和/或制备治疗肾上腺皮脂腺瘤的药物中的用途。所称多肽是上述带特定突变的核酸所编码的，由于核酸上带有特定突变，与野生蛋白序列相比，所称的多肽也在相应的位点存在氨基酸残基的变异。检测待测样本中存在所称的多肽，能够说明该样本为APA易感样本。另外，该多肽包含的信息，可以用于制备APA靶向药物。该多肽能够同于辅助诊断个体APA的发生、发展和治疗。
依据本发明的第十方面，提供一种试剂盒，其包括能够用于检测上述任一多肽的试剂。所称试剂包括但不限于蛋白质免疫印迹(western blot)和蛋白质测序相关试剂。
依据本发明的第十一方面，提供一种上述试剂盒在检测上述任一多肽和/或体外检测肾上腺皮脂腺瘤中的用途。包含能够检测上述任一实施例中的多肽的试剂的试剂盒，能够方便高效地用于体外检测肾上腺皮质腺瘤。
依据本发明的第十二方面，提供一种筛查肾上腺皮脂腺瘤生物样本的方法，该方法包括：(1)获取待测样本的外显子组测序数据，所述测序数据包括多个读段；(2)基于(1)的测序数据，进行以下(a)-(c)：(a)确定所述外显子组上存在的体细胞突变的频率，(b)检测所述外显子组上存在的体细胞突变是否包括以下突变中的至少一个：KCNJ5基因chr11：128781619G>C，KCNJ5基因chr11：128781619G>A和KCNJ5基因chr11：128781671T>G，以及(c)检测所述外显子组上是否存在上述任一实施例中的分离的核酸；(3)判定(2)中的(a)的频率为大于1个体细胞突变每10Mb并且(b)和/或(c)的检测结果为是的生物样本为肾上腺皮脂腺瘤生物样本。所称的外显子组上存在的体细胞突变的频率是指单位长度上外显子组上的存在的体细胞突变的个数。该方法，基于利用上述任一实施例中的分离的核酸作为APA标志物、以及结合外显子上存在的体细胞突变的频率，能够有效方便的检测筛查出APA样本。
所称的测序数据通过对样本中的核酸序列进行测序得来，测序依据所选的测序平台的 不同，可选择但不限于半导体测序技术平台比如PGM、Ion Proton、BGISEQ‐100平台，合成边测序的技术平台比如Illumina公司的Hiseq、Miseq序列平台以及单分子实时测序平台比如PacBio序列平台。测序方式可以选择单端测序，也可以选择双末端测序，获得的下机数据是测读出来的片段，称为读段(reads)。
所称的体细胞突变(somatic mutation)是发生在正常机体细胞中的突变，比如发生在皮肤或器官中的突变，这样的突变不会传给后代。与体细胞突变不同的是种系突变(germline mutation)，也称为生殖细胞突变、生殖系突变或者胚系突变，是指发生在将成为配子(gametes)(精子和卵子)的细胞中的突变，生殖细胞的突变可传递给后代。该方法中利用的突变信息为体细胞突变信息，体细胞突变对癌症的发生发展很重要。
根据本发明的实施例，步骤(2)中的(a)包括：将所述读段比对至参考基因组，获得比对结果；基于所述比对结果，确定突变，其中包括，去除掉所述突变中的生殖细胞突变，以获得所述体细胞突变；确定在所述外显子组上所述体细胞突变的频率。根据本发明的一个实施例，通过将确定出的突变对比已知的生殖细胞突变，例如利用dbSNP对突变进行注释，将其中的生殖细胞突变去除掉。
所称的参考基因组预先确定的基因组序列，可以是预先获得的目标个体所属生物类别中的任意的参考模板，例如，同一生物类别的已公开的基因组组装序列，若目标个体是人类，其基因组参考序列(也称为参考基因组)可选择NCBI数据库提供的HG19。进一步地，也可以预先配置包含更多参考序列的资源库，在进行序列比对前，先依据目标个体的性别、人种、地域等因素选择或是测定组装出更接近的序列来作为参考序列，有助于获得更准确的筛查结果。
所称的比对可以利用已知比对软件进行，例如SOAP、BWA和TeraMap等，本发明对此不作限制。在比对过程中，一般对比对参数进行设置，设置一个或者一对读段(reads)最多允许有s个碱基错配(mismatch)，例如设置s≤2，若reads中有超过s个碱基发生错配，则视为该reads或者该对reads无法比对到(比对上)参考序列上。
根据本发明的实施例，步骤(2)中的(a)的体细胞突变选自以下表1中的突变。表1中的体细胞突变，是发明人基于分析大量肾上腺皮质腺瘤样本的外显子组数据而确定下来的，集合了肾上腺皮质腺瘤样本中常存在的体细胞突变，需要说明的是，其中的大部分体细胞突变都是未见报道过的突变。
表1
Figure PCTCN2016077562-appb-000001
Figure PCTCN2016077562-appb-000002
Figure PCTCN2016077562-appb-000003
Figure PCTCN2016077562-appb-000004
Figure PCTCN2016077562-appb-000005
Figure PCTCN2016077562-appb-000006
Figure PCTCN2016077562-appb-000007
Figure PCTCN2016077562-appb-000008
Figure PCTCN2016077562-appb-000009
Figure PCTCN2016077562-appb-000010
Figure PCTCN2016077562-appb-000011
Figure PCTCN2016077562-appb-000012
步骤(3)中的参数或条件，是经大量样本验证确定的，能够有效准确的用于APA生物样本的判断筛查。根据本发明的实施例，(3)包括：当(2)中的(a)的频率为大于2个体细胞突变每10Mb并且(b)和/或(c)的检测结果为是，判定所述生物样本为肾上腺皮脂腺瘤生物样本。如此，能够使判定出的结果准确性更高。
依据本发明的第十三方面，提供一种筛查肾上腺皮质腺瘤生物样本的装置，该装置能够用以实施上述本发明任一实施例中的筛查肾上腺皮质腺瘤的方法的全部或部分步骤，该装置包括：数据输入单元，用于获取待测样本的外显子组测序数据，所述测序数据包括多个读段；检测单元，由A模块、B模块和C模块组成，用于基于来自数据输入单元的测序数据，利用所述模块分别进行以下：A模块，确定所述外显子组上存在的体细胞突变的频率，B模块，检测所述外显子组上存在的体细胞突变是否包括以下突变中的至少一个：CNJ5基因chr11：128781619G>C，KCNJ5基因chr11：128781619G>A和KCNJ5基因chr11：128781671T>G，以及C模块，检测所述外显子组上是否存在上述任一实施例中的核酸；判定单元，用于判定来自检测单元A模块的频率为大于1个体细胞突变每10Mb并且B模块和/或C模块的输出结果为是的生物样本为肾上腺皮脂腺瘤生物样本。上述对本发明一方面的方法的有点和技术特征的描述，同样适用本发明这一方面的装置，在此不再赘述。
根据本发明的实施例，检测单元A模块用于进行以下：将所述读段比对至参考基因组，获得比对结果；基于所述比对结果，确定突变，其中包括，去除掉所述突变中的生殖细胞突变，以获得所述体细胞突变；确定在所述外显子组上所述体细胞突变的频率。
根据本发明的实施例，检测单元A模块中的体细胞突变选自表1中的突变。表1中的体细胞突变，是发明人基于分析大量肾上腺皮质腺瘤样本的外显子组数据而确定下来的，集合了肾上腺皮质腺瘤样本中常存在的体细胞突变，需要说明的是，其中的大部分体细胞突变都是未见报道过的突变。
根据本发明的实施例，当检测单元A模块中的频率为大于2个体细胞突变每10Mb并且B模块和/或C模块的输出结果为是，判定所述生物样本为肾上腺皮脂腺瘤生物样本。如此，更为严格的条件能够使判定出的结果准确性更高。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1是本发明的一个实施例中的与APA发生或发展相关的突变基因示意图。
图2是本发明的一个实施例中的Sanger验证突变的结果图。
图3是本发明的一个实施例中的钾离子通道基因KCNA1的体细胞突变的影响的示意图。
具体实施方式
本文中，错义突变(missense mutation)是指DNA的突变引起mRNA中密码子改变,编码另一种氨基酸.如DNA中某GAA发生转换突变成AAA后，使原编码的谷氨酸(Glu)改变为赖氨酸(Lys)；沉默突变(silent mutation)也称同义突变(same-sense mutation或synonymous mutation)，是指DNA的突变虽引起mRNA中密码子改变为另一种密码，但由于密码子的兼并作用，并未使编码的氨基酸改变；无义突变(nonsense mutation)指DNA的突变引起mRNA中的密码子改变为一种终止密码子。错义突变和无义突变均可称为非同义突变(nonsynonymous mutation)。
以下结合一般步骤及具体参数设置对依据本发明的方法/装置进行APA检测进行详细描述。本发明的描述中，除非另有说明，“多个”、“多组”的含义是两个(组)或两个(组)以上。
除另有交待，以下实施例中涉及的未特别交待的软件、统计检验方法及其参数设置，都是公开的且可利用默认设置进行。
实施例
1、试验
1.1样本的收集
对来自中国泌尿生殖系统癌症基因组协会(UCGC)的22名患有性肾上腺高血压且患有原发性醛固酮增多症和肾上腺皮质肿瘤的患者中获得APA组织样本和外周血样本。原发性醛固酮增多症是通过显著升高的醛固酮与肾素的比值(ARR)来诊断的。肾素为血浆肾素浓度(PRC；单位mIU/L)。肾上腺肿瘤是通过CT扫描确诊的。当最可能诊断为APA时， 就需要进行单侧肾上腺切除术了。所有的标本应立刻冷冻在液氮中，并立即存储于-80℃低温下用于进一步研究。肿瘤切片组织进行苏木精-伊红(HE)染色，然后由两个病理学家进行独立的显微镜下评估。所有患者术后显示血钾正常，血压得到改善和控制。
APA01-22为22名患者的编号，APA01N以及APA01C分别表示APA01的正常组织样品和肿瘤组织样品。
1.2基因组DNA的提取和Illumina基于全外显子组测序
来自于22个APA患者中的肿瘤基因组DNA和匹配外周血样本，用QIAGEN公司的DNeasy血液和组织试剂盒(Qiagen，Hilden，德国)进行分离。按照制造商(安捷伦科技)的协议，由SureSelect人类全外显子50Mb试剂盒获取全外显子组。根据制造商的说明进行2×90双末端测序，在HiSeq2000平台上进行。
1.3比对以及体细胞突变检测
首先，除去测序数据中包含测序接头的读段和包含五个以上未知碱基N的读段，获得高质量测序数据。接着，利用BWA将高质量测序数据中的成对读段比对到NCBI的人类参考基因组hg19上，获得第一比对结果。然后，通过基因组分析工具包(GATK)对至少一部分第一比对结果进行重新比对，获得第二比对结果。
基于第一和第二比对结果，利用VarScan识别潜在的体细胞突变，获得体细胞突变原始列表。体细胞插入/缺失突变(INDEL)初步列表是基于第二笔对结果、通过GATK识别出的。进一步地，为了减少假阳性的出现，利用SAMtools软件包对肿瘤样本中的单核苷酸变异(SNVs)和INDEL进行识别。发明人除去了符合下列标准的体细胞突变：(i)碱基质量值<20；(ii)比对质量分数<30；(iii)仅在一条DNA链上的INDEL；(iv)位于疑似插入缺失的30BP左右范围的置换突变。为了消除任何上述的种系突变，将体细胞突变原始列表中的突变与dbSNP数据库(132版)和千人基因组项目中的SNP数据集(http://www.1000genomes.org)进行对比，去除任何存在于上述已知数据集中的突变，利用原始列表中剩余的突变进行后续分析。
1.4Sanger测序验证突变
基于PCR利用Sanger测序对非同义置换突变(non-silent体细胞突变)进行验证。在双96-96GeneAmp PCR系统(应用生物系统公司)进行PCR，每个样本20ng DNA模板。扩增产物通过3730xl测序DNA分析仪(应用生物系统公司)进行测序。利用测序分析软件(版本5.2)进行测序序列分析。如果肿瘤样本中的突变被成功确认，同样的引物将被用来扩增来自同一病人血液的DNA样本，以确定所观察到的突变体细胞的状态。
1.5显著的突变基因分析
简单地说，基于前面步骤获得的体细胞突变(SNV和INDEL)的基因列表，利用MutSigCV11方法来确定显著突变基因。FDR q<0.1的基因被认为是在APA样本中显著突变的基因。
2、结果
2.1东亚APA突变的情况
目标区的平均测序深度为71.2×(50.75-103.6×)，其中88.5％(81％-92％)被覆盖至少10×，如表2所示。
发明人总共确定了205个体细胞突变候选者，如表1所示，其中包括62个同义，129个错义，11个无义和2个剪接位点突变。基因突变谱中，C：G>T：A占主导地位用，这一点与大多数其他癌症类型是一致的。在该突变队列中，外显子区域没有发现体细胞插入和缺失。
表2
Figure PCTCN2016077562-appb-000013
Figure PCTCN2016077562-appb-000014
在突变列表中，为了识别候选突变基因与APA的发展是否存在关系，发明人参照使用已报道的方法【Cao Y,He M,Gao Z,Peng Y,Li Y,Li L,et al.Activating hotspot L205R mutation in PRKACA and adrenal Cushing's syndrome.Science.2014；344:913-7.】，研究确定了 六个与APA存在潜在功能相关的基因，其中包括两个钾通道基因和其他四个基因，这些基因已被存放在肿瘤基因普查(Cancer Gene Census)或COSMIC数据库中，如图1所示。通过PCR Sanger测序证实，所有潜在的功能突变均为体细胞突变，如图2所示。通过MutSigCV的计算，在突变队列中，KCNJ5基因表现出显著升高的突变率(Q＝1.47×10-11)；而在其他三个已知的APA驱动基因中(ATP1A1，ATP2A3和CACNA1D)，没有鉴定出体细胞突变。在这次示例的APA患者中，KCNJ5体细胞突变聚集在两个热区，p.G151R和p.L168R，每个分别显示出27.3％(6/22)和45.5％(10/22)的突变频率。
2.2参与离子稳态或其他生物过程的体细胞突变基因
为了调查是否有任何以前没有发现的，参与跨膜离子平衡的APA相关基因，发明人系统地分析了体细胞突变基因的功能，并在一个KCNJ5野生型的APA患者和另一个钾通道基因KCNQ1中确定了有害突变(SIFT评分＝0)。KCNA1编码电压门控钾离子通道Kv1.1。该蛋白相比于其他钾离子通道的基因，在肾上腺皮质中呈现中等水平表达。这种突变导致在位置318处出现高度保守的苏氨酸和丙氨酸(T318A)，如图3所示。该T318A突变定位在接近电压传感器之间的第四和第五跨膜区段(S4和S5)。发明人推测该突变可能通过破坏Kv1.1通道的结构而增加电压激活的Ca2+内流，从而促进醛固酮的合成和肿瘤细胞增殖。
在其他几个基因中，发明人还鉴定了非同义突变，它们并不直接控制离子的稳态，但被证明为在APA中经常发生变异，如图1所示。CTNNB1激活突变是肾上腺肿瘤发生的早期且常见的情况。发明人在一个KCNJ5野生型APA中，确定了一个活化的CTNNB1突变(S45P)。DNMT3A编码的DNA甲基转移酶，负责建立全基因组新生甲基。DNMT3A的突变与AML，之前报道和肺癌的发展有关。RAD50编码涉及DNA双链断裂修复的蛋白质，已有报道其为乳腺癌易感基因，与基因组不稳定性相关。CIC是转录抑制因子，可在中枢神经系统的发展中发挥作用。CIC的突变与少突胶质细胞瘤的起源相关。这些结果表明，多个其他基因也可能促进APA肿瘤的发生或发展。
2.3相比欧洲的病人，东亚APA患者中KCNJ5基因表现出了超高的突变率
据发明人所知，在2011年，KCNJ5中的体细胞突变(G151R和L168R)首次由Choi等人鉴定为约40％的APA，他们认为这是导致这些肿瘤的主要原因。据报道，这两个突变定位在或接近KCNJ5的选择性过滤器上，并导致钠电导和膜去极化的增加，从而造成醛固酮的生产增加和APA细胞增殖。既这一初步研究之后，进一步研究发现，KCNJ5热点突变患者中34％-46％被认定为欧洲后裔APA患者(包括法国，德国，意大利，英国和澳大利亚的患者)。值得注意的是，在发明人的研究中，在KCNJ5基因中发现超高频突变，约72.7％APA肿瘤源于G151R或L168R蛋白改变体细胞突变(16/22)。
为了支持发明人的发现，进一步从23位日本APA患者筛选KCNJ5基因，发现KCNJ5基因的突变率为65％，这也比在欧洲APA患者明显更高。通过总结对比欧洲后裔APA组群的研究证据，发明人发现，东亚患者(至少包括日本和中国)，KCNJ5体细胞突变的频率都显著高于欧洲后裔患者，而ATP1A1，ATP2A3和CACNA1D突变则更多见于欧洲APA 患者。尽管，东亚APA的KCNJ5突变患病率仍然需要在更大的人群得到证实，该示例的结果强有力地表明了，对于APA患者，其KCNJ5突变与种族相关。
3、验证
混合其它APA样本和正常样本，按照该发明方法进行筛查：(1)获取待测样本的外显子组测序数据，所述测序数据包括多个读段；(2)基于(1)的测序数据，进行以下(a)-(c)：(a)确定所述外显子组上存在的体细胞突变的频率，(b)检测所述外显子组上存在的体细胞突变是否包括以下突变中的至少一个：KCNJ5基因chr11：128781619G>C，KCNJ5基因chr11：128781619G>A和KCNJ5基因chr11：128781671T>G，以及(c)检测所述外显子组上是否存在上述任一分离的核酸(图1中的任一突变基因序列)；(3)判定(2)中的(a)的频率为大于1个体细胞突变每10Mb并且(b)和/或(c)的检测结果为是的生物样本为肾上腺皮脂腺瘤生物样本。对患病样本的筛查检测结果70％以上与该样本的来源或临床信息上的记录一致。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
尽管已经示出和描述了本发明的实施例，本领域的普通技术人员可以理解：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。

Claims (22)

  1. 一种分离的核酸，其特征在于，与相应的参考基因序列相比，所述核酸包括存在以下至少一种突变的基因序列：
    DNMT3A基因chr2：25463248G>C、CIC基因chr19：42790976G>T、CTNNB1基因chr3：41266136T>C、RAD50基因chr5：131926953T>C、RAD50基因chr4：83782843G>A和KCNA1基因chr12：5021496A>G。
  2. 权利要求1的核酸，其特征在于，所述核酸包括存在chr2：25463248G>C的DNMT3A基因序列。
  3. 权利要求2的核酸，其特征在于，所述核酸还包括存在以下至少一种突变的基因序列：CIC基因chr19：42790976G>T、CTNNB1基因chr3：41266136T>C、RAD50基因chr5：131926953T>C、RAD50基因chr4：83782843G>A、KCNA1基因chr12：5021496A>G、KCNJ5基因chr11：128781619G>C、KCNJ5基因chr11：128781619G>A和KCNJ5基因chr11：128781671T>G。
  4. 权利要求1的核酸，其特征在于，所述核酸包括存在chr19：42790976G>T的CIC基因序列。
  5. 权利要求4的核酸，其特征在于，所述核酸还包括存在以下至少一种突变的基因序列：DNMT3A基因chr2：25463248G>C、CTNNB1基因chr3：41266136T>C、RAD50基因chr5：131926953T>C、RAD50基因chr4：83782843G>A和KCNA1基因chr12：5021496A>G、KCNJ5基因chr11：128781619G>C、KCNJ5基因chr11：128781619G>A和KCNJ5基因chr11：128781671T>G。
  6. 权利要求1的核酸，其特征在于，所述核酸包括存在chr3：41266136T>C的CTNNB1基因序列。
  7. 权利要求6的核酸，其特征在于，所述核酸还包括存在以下至少一种突变的基因序列：DNMT3A基因chr2：25463248G>C、CIC基因chr19：42790976G>T、RAD50基因chr5：131926953T>C、RAD50基因chr4：83782843G>A和KCNA1基因chr12：5021496A>G、KCNJ5基因chr11：128781619G>C、KCNJ5基因chr11：128781619G>A和KCNJ5基因chr11：128781671T>G。
  8. 权利要求1-7任一核酸在检测肾上腺皮质腺瘤和/或制备治疗肾上腺皮质腺瘤的药物中的用途。
  9. 一种试剂盒，其包括能够用于检测权利要求1-7任一核酸的试剂，所述试剂包括引物序列。
  10. 权利要求9的试剂盒在检测权利要求1-7任一核酸和/或体外检测肾上腺皮质腺瘤中的用途。
  11. 一种分离的多肽，其特征在于，所述多肽由权利要求1-7任一核酸所编码。
  12. 权利要求11的多肽在检测肾上腺皮脂腺瘤和/或制备治疗肾上腺皮质腺瘤的药物 中的用途。
  13. 一种试剂盒，其包括能够用于检测权利要求11的多肽的试剂。
  14. 权利要求13的试剂盒在检测权利要求11的多肽和/或体外检测肾上腺皮质腺瘤中的用途。
  15. 一种筛查肾上腺皮质腺瘤生物样本的方法，其特征在于，包括：
    (1)获取待测样本的外显子组测序数据，所述测序数据包括多个读段；
    (2)基于(1)的测序数据，进行以下(a)-(c)：
    (a)确定所述外显子组上存在的体细胞突变的频率，
    (b)检测所述外显子组上存在的体细胞突变是否包括以下突变中的至少一个：KCNJ5基因chr11：128781619G>C，KCNJ5基因chr11：128781619G>A和KCNJ5基因chr11：128781671T>G，以及
    (c)检测所述外显子组上是否存在权利要求1-7任一核酸；
    (3)判定(2)中的(a)的频率为大于1个体细胞突变每10Mb并且(b)和/或(c)的检测结果为是的生物样本为肾上腺皮质腺瘤生物样本。
  16. 权利要求15的方法，其特征在于，(2)中的(a)包括：
    将所述读段比对至参考基因组，获得比对结果；
    基于所述比对结果，确定突变，其中包括，
    去除掉所述突变中的生殖细胞突变，以获得所述体细胞突变；
    确定在所述外显子组上所述体细胞突变的频率。
  17. 权利要求15或16的方法，其特征在于，(2)中的(a)的体细胞突变选自表2中的突变。
  18. 权利要求15的方法，其特征在于，当(2)中的(a)的频率为大于2个体细胞突变每10Mb并且(b)和/或(c)的检测结果为是，判定所述生物样本为肾上腺皮质腺瘤生物样本。
  19. 一种筛查肾上腺皮脂腺瘤生物样本的装置，其特征在于，包括：
    数据输入单元，用于获取待测样本的外显子组测序数据，所述测序数据包括多个读段；
    检测单元，由A模块、B模块和C模块组成，用于基于来自数据输入单元的测序数据，利用所述模块分别进行以下：
    A模块，确定所述外显子组上存在的体细胞突变的频率，
    B模块，检测所述外显子组上存在的体细胞突变是否包括以下突变中的至少一个：KCNJ5基因chr11：128781619G>C，KCNJ5基因chr11：128781619G>A和KCNJ5基因chr11：128781671T>G，以及
    C模块，检测所述外显子组上是否存在权利要求1-7任一核酸；
    判定单元，用于判定来自检测单元A模块的频率为大于1个体细胞突变每10Mb并且B模块和/或C模块的输出结果为是的生物样本为肾上腺皮质腺瘤生物样本。
  20. 权利要求19的装置，其特征在于，检测单元A模块用于进行以下：
    将所述读段比对至参考基因组，获得比对结果；
    基于所述比对结果，确定突变，其中包括，
    去除掉所述突变中的生殖细胞突变，以获得所述体细胞突变；
    确定在所述外显子组上所述体细胞突变的频率。
  21. 权利要求19或20的装置，其特征在于，检测单元A模块中的体细胞突变选自表2中的突变。
  22. 权利要求19的装置，其特征在于，当检测单元A模块中的频率为大于2个体细胞突变每10Mb并且B模块和/或C模块的输出结果为是，判定所述生物样本为肾上腺皮质腺瘤生物样本。
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