CN113604561B - Application of SF3B1 gene mutation in auxiliary diagnosis of hybrid PRL positive pituitary adenoma - Google Patents

Abstract

The invention discloses an application of SF3B1 gene mutation in auxiliary diagnosis of mixed PRL positive pituitary adenoma. The invention can make diagnosis of mixed PRL positive pituitary adenoma more convenient and easy to implement by detecting SF3B1 gene c.1874G > A mutation and developing and applying a diagnostic kit, so that a patient can know disease risk at early stage of disease, and a simple and effective auxiliary means is provided for drug screening, drug effect evaluation and targeted treatment of pituitary adenoma.

Description

Application of SF3B1 gene mutation in auxiliary diagnosis of hybrid PRL positive pituitary adenoma

Technical Field

The invention relates to the field of auxiliary diagnosis of pituitary adenoma, in particular to application in auxiliary diagnosis and parting products of mixed PRL positive pituitary adenoma.

Background

Pituitary tumors are one of the most common intracranial tumors, accounting for about 10-15% of intracranial tumors, and the incidence rate of people is about 8-9/ten thousand. With the development of image technology, the detection rate of the image is in a trend of increasing year by year. Pituitary tumors can be divided into several subtypes of prolactin type, growth hormone type, adrenocorticotropic hormone type, thyrotropin type, nonfunctional type, mixed type and the like according to different endocrine characteristics. Tumors secrete various hormones abnormally, so that patients have obvious endocrine disturbance symptoms such as infertility, acromegaly, cushing syndrome and hyperthyroidism.

Polyhydrogen pituitary adenomas (Plurihomonal Pituitary Adenomas (PPAs)) are combinations of different hormone immunohistochemical staining positive types, expressed as Prolactin (PRL), growth Hormone (GH), thyroid Stimulating Hormone (TSH), follicle Stimulating Hormone (FSH)/Luteinizing Hormone (LH) or adrenocorticotropic hormone (ACTH), which can be clinically manifested as pituitary dysfunction and tumor compression symptoms including headache, vision impairment, etc., wherein PRL and ACTH mixed cell adenomas are the most common polyhydrogen and di-hormone adenomas, and PRL, GH and TSH immunohistochemical staining positive is classified as pith-1 positive. For patients clinically diagnosed with nonfunctional adenomas, if compression symptoms occur, surgical treatment is the preferred treatment regimen.

PRL/GH mixed adenomas are mixed pituitary adenomas positive for PRL and GH staining, including PRL-GH cell adenomas and PRL-GH mixed cell adenomas, which are clinically manifested as signs and symptoms of high levels of serum GH and IGF-1 and acromegaly or giant, and some patients may have symptoms and signs of hyperprolactinemia such as menopause, lactation, hyposexuality, and the like. The immunohistochemical staining of the two pituitary adenomas shows positive PRL and positive GH, wherein the PRL-GH cell adenomas are positive in the same cell and the PRL and GH are visible; PRL-GH mixed cell adenomas are a mixture of cells that secrete different hormones, i.e., a mixture of PRL-secreting cells and GH-secreting cells. The preferred treatment regimen for acromegaly is surgical treatment, and somatostatin drugs including octreotide and lanreotide may be used.

Mixed PRL positive pituitary adenomas are a collective term for pituitary adenomas that have two or more hormone-staining positives that are positive for PRL staining, and that have, in addition to PRL staining positives, at least one hormone-staining positivity selected from the group consisting of: GH. TSH, FSH, LH and ACTH. Mixed PRL positive pituitary adenomas include PRL positive multi-hormonal pituitary adenomas, PRL-GH cell adenomas, and PRL-GH mixed cell adenomas, and the like. Part of the tumors in this class appear to be voluminous and grow invasively to the periphery, complete tumor resection is extremely difficult to obtain during surgery, and the prognosis of this part of the patients is poor, presumably they are likely to fall into two different subtypes of mixed PRL positive pituitary adenomas, for which different clinical treatment strategies should be adopted. There is currently no specific molecular diagnostic marker that can diagnose, or even differentially diagnose, them.

As a key step in RNA processing, RNA splicing accuracy is one of the prerequisites for normal expression of genes, and gene expression disorder caused by RNA splicing abnormality is an important cause of occurrence of various diseases. In recent years, high-throughput sequencing technology detects and discovers that various splicing factor mutations exist in various tumor cells, and the mutations affect RNA splicing to different degrees, so that the downstream gene expression is abnormal.

Splice factor SF3B is a multiprotein complex that includes subunits such as SF3B1, SF3B3, and PHF5A. The SF3b complex is part of a U2 snRNA-protein complex (snRNP) assembled from U2 snRNA, splicing factors SF3a and SF3b, and other related proteins. Together they form 17s U2 snRNP, which assembles in an ATP-dependent manner on the 3' side of the intron to form an a complex. The SF3B core complex contains several splice-related proteins (SAP), including SF3B1/SAP155, SF3B2/SAP145, SF3B3/SAP130, SF3B4/SAP49, SF3B5/SAP10, SF3B6/SAP14a, and PHF5A/SAP14B.

Studies have shown that splicing factors such as SF3B1, U2AF1 and SRSF2 are involved in hematological malignancies, including chronic lymphocytic leukemia and myelodysplastic syndrome, breast, pancreatic, gastric, prostate and uveal melanoma. Chinese patent application CN107405383a discloses that SF3B1 mutations identified in both myelodysplastic syndrome (MDS) and cancer include, for example, R625H.

However, no report has been made on the application of the splicing factor subunit SF3B1 mutation to the diagnosis of pituitary adenomas, particularly mixed PRL positive pituitary adenomas. Based on the limitations of the means for diagnosing the diseases in the prior art, if the susceptible SF3B1 mutation can be screened out as a molecular diagnostic marker, an effective and low-cost effective treatment method capable of assisting in diagnosis of pituitary adenoma, especially for mixed PRL positive pituitary adenoma is needed to be solved.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to screen out pituitary adenomas, in particular to the SF3B1 mutation which is susceptible to the mixed PRL positive pituitary adenomas as a molecular diagnosis marker, and develops a corresponding diagnosis kit to detect the pituitary adenomas, thereby leading the diagnosis of the pituitary adenomas to be more convenient and easy, leading a patient to know the disease risk at early stage of the disease, simultaneously being applicable to the tumor typing and malignancy evaluation of the pituitary adenomas, in particular to the mixed PRL positive pituitary adenomas, providing a simple and effective auxiliary means for drug screening, drug effect evaluation and targeted treatment thereof, and opening up a new way.

Specifically, the invention provides the following technical scheme:

in one aspect, the invention provides the use of a product for detecting mutations in the SF3B1 gene c.1874g > a in the preparation of a reagent or kit for aiding in the diagnosis of mixed PRL positive pituitary adenoma, distinguishing between mixed PRL positive pituitary adenoma SF3B1 mutant and wild type, or predicting whether mixed PRL positive pituitary adenoma is malignant.

In one aspect, the invention provides the use of a product for detecting an SF3B1 protein R625H mutation in the preparation of a reagent or kit for aiding in the diagnosis of mixed PRL positive pituitary adenoma, distinguishing between mixed PRL positive pituitary adenoma SF3B1 mutant and wild type, or predicting whether mixed PRL positive pituitary adenoma is malignant.

In one aspect, the invention provides a primer for detecting a mutation of the SF3B1 gene c.1874G > A.

In one aspect, the invention provides a kit for aiding in the diagnosis of mixed PRL positive pituitary adenomas, distinguishing between mixed PRL positive pituitary adenomas SF3B1 mutant and wild type, or predicting whether mixed PRL positive pituitary adenomas are malignant.

The invention and its advantageous technical effects are described in detail below with reference to the attached drawings and to the various embodiments, wherein:

drawings

FIG. 1 shows the specificity of probe detection.

Fig. 2 shows the sensitivity of probe detection.

FIG. 3 shows the proliferation capacity of CCK-8 activity in cells infected with SF3B1 mutant adenovirus.

FIG. 4 shows the proliferation capacity of cells infected with SF3B1 mutant adenovirus tested by the colony formation assay.

Figure 5 shows the apoptotic capacity of SF3B1 mutant adenovirus-infected cells.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For the purposes of the present invention, the following terms are defined as follows.

The term "mixed PRL positive pituitary adenoma" refers to a collective term for pituitary adenomas that are hormone-immunohistochemical staining positive for PRL, and that are two or more hormone-staining positive, and which have, in addition to PRL staining positive, at least one hormone-staining positive selected from the group consisting of: GH. TSH, FSH, LH and ACTH. Mixed PRL positive pituitary adenomas include PRL positive multi-hormonal pituitary adenomas, PRL-GH cell adenomas, and PRL-GH mixed cell adenomas, and the like.

The term "gene" refers to a DNA segment involved in the production of a polypeptide chain; it includes regions preceding and following the coding regions involved in transcription/translation of the gene product and regulation of said transcription/translation (leader and trailer), as well as intervening sequences (introns) between individual coding regions (exons).

The term "allele" is one of many alternatives to a gene or non-coding region of DNA that occupies the same position on the chromosome. The term allele may be used to describe DNA from any organism including, but not limited to, bacteria, viruses, fungi, protozoa, molds, yeasts, plants, humans, non-humans, animals, and archaea.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term is applicable to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the term encompasses amino acid chains of any length, including full-length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.

The term "SF3B1" refers to splicing factor 3B1. The SF3B1 gene is located on chromosome 2q33.1, encoding subunit 1 of splicing factor 3B, which is the core part of the U2 micronuclear ribonucleoprotein complex. SF3B1 is necessary for recognition and binding of the branching point sequence near the 3' splice site and plays an important role in the precise excision of introns from pre-mRNA to form mature mRNA. SF3B1 mutations may be driving events during tumorigenesis, not only leading to splicing abnormalities in transcriptional coupling, but also affecting genomic instability and stem cell differentiation.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, and subsequently modified amino acids such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. In this context, amino acids may be represented by the universal three-letter code or one-letter code recommended by the IUPAC-IUB biochemical nomenclature committee. Also, nucleotides may be referred to by their commonly accepted single letter codes.

The term "template" refers to any nucleic acid molecule that can be used for amplification according to the invention. RNA or DNA that is not naturally double-stranded can be made into double-stranded DNA to serve as double-stranded DNA. Any double-stranded DNA or preparation containing a plurality of different double-stranded DNA molecules may be used as template DNA to amplify one or more loci of interest contained within the template DNA.

The term "primer" refers to an oligonucleotide that can be used in an amplification method, such as the Polymerase Chain Reaction (PCR), for amplifying a nucleotide sequence based on a polynucleotide sequence, which corresponds to a particular genomic sequence. At least one PCR primer for amplifying a polynucleotide sequence is sequence specific for the sequence.

The term "probe" refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that binds to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Probes can bind target polynucleotides that lack complete sequence complementarity to the probe, depending on the stringency of the hybridization conditions. The probes may be labeled directly or indirectly.

The term "amplification reaction" refers to a process for copying nucleic acid one or more times. In embodiments, the amplification methods include, but are not limited to: polymerase chain reaction, self-sustaining sequence reaction, ligase chain reaction, rapid amplification of cDNA ends, polymerase chain reaction and ligase chain reaction, Q-beta phage amplification, strand displacement amplification, or overlap extension splice polymerase chain reaction. In some embodiments, single molecule nucleic acids are amplified, for example, by digital PCR.

The term "amplification product" refers to a nucleic acid product produced by a nucleic acid amplification technique.

The term "wild-type" refers to a gene or gene product that, when isolated from a naturally occurring source, has the characteristics of a gene or gene product. Wild-type genes are the most common genes in a population and are therefore arbitrarily referred to as the "normal" or "wild-type" form of the gene. In contrast, the term "modified," "mutated," or "polymorphic" refers to a gene or gene product that exhibits modification of sequence and/or functional properties (i.e., altered characteristics) as compared to the wild-type gene or gene product. It should be noted that naturally occurring mutants can be isolated; their identification is based on the following facts: the characteristics are altered compared to the wild-type gene or gene product.

The term "biomarker" refers to a biomolecule or fragment of a biomolecule, the change and/or detection of which may be associated with a particular physical condition or state. Throughout the disclosure, the terms "marker" and "biomarker" are used interchangeably. For example, the biomarkers of the invention are related to Prolactin (PRL) type adenomas. These biomarkers include, but are not limited to, biomolecules including nucleotides, nucleic acids, nucleosides, amino acids, sugars, fatty acids, steroids, metabolites, peptides, polypeptides, proteins, carbohydrates, fats, hormones, antibodies, regions of interest as substitutes for biological macromolecules, and combinations thereof (e.g., glycoproteins, ribonucleoproteins, lipoproteins). The term also includes portions or fragments of a biomolecule, e.g., peptide fragments of a protein or polypeptide comprising at least 5 consecutive amino acid residues, at least 6 consecutive amino acid residues, at least 7 consecutive amino acid residues, at least 8 consecutive amino acid residues, or more consecutive amino acid residues.

The terms "patient" and "subject" are used interchangeably to refer to patients and subjects of humans or other mammals, and include any individual that is examined or treated using the methods of the present invention. However, it should be understood that "patient" does not mean that symptoms are present. Suitable mammals within the scope of the invention include, but are not limited to, primates, farm animals (e.g., sheep, cattle, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs), and wild animals in containment (e.g., koala, bear, wild dogs, wolves, wild dogs, foxes, etc.).

The term "kit" refers to any delivery system for delivering a substance. In a reaction assay, such delivery systems include systems that store, transport or deliver reagents (e.g., oligonucleotides, enzymes, etc. in suitable containers) and/or support materials (e.g., buffers, instructions for performing the assay, etc.) from one location to another. For example, the kit comprises one or more housings (e.g., cassettes) containing the relevant reagents and/or support materials.

In one aspect, the invention provides the use of a product for detecting mutations in the SF3B1 gene c.1874g > a in the preparation of a reagent or kit for aiding in the diagnosis of mixed PRL positive pituitary adenoma, distinguishing between mixed PRL positive pituitary adenoma SF3B1 mutant and wild type, or predicting whether mixed PRL positive pituitary adenoma is malignant.

In a preferred embodiment, the mixed PRL positive pituitary adenoma is a hormone-stained including PRL positive and at least one hormone-stained positive pituitary adenoma selected from the group consisting of: GH. TSH, FSH, LH and ACTH; preferably, the mixed PRL positive pituitary adenoma is selected from PRL positive multi-hormonal pituitary adenoma or mixed PRL and GH staining positive pituitary adenoma; preferably, the PRL positive multi-hormonal pituitary adenomas are TSH, PRL and ACTH hormone-staining positive PRL positive multi-hormonal pituitary adenomas; preferably, the PRL and GH staining positive mixed pituitary adenomas are selected from PRL-GH cell adenomas or PRL-GH mixed cell adenomas.

In a preferred embodiment, the product comprises reagents for detecting the mutation of the SF3B1 gene c.1874G > A by PCR techniques.

In a preferred embodiment, the product comprises a mixture of PCR amplification reagents for SF3B1 gene c.1874G>A primer pair for mutation and a mutant allele probe; preferably, the mixed solution of the PCR amplification reaction reagent comprises Taq DNA polymerase, dNTP mixed solution and MgCl ₂ Solution, fluorescent quantitative PCR reaction buffer and deionized water.

In a preferred embodiment, the product further comprises a wild-type allele probe.

In a preferred embodiment, the PCR is digital PCR or fluorescent quantitative PCR (qPCR).

In a preferred embodiment, the probe is a TaqMan MGB probe, more preferably, the TaqMan MGB probe has a fluorescent reporter group labeled at the 5 'end and a non-fluorescent quenching group MGB labeled at the 3' end; preferably, the fluorescent reporter is a FAM or HEX fluorescent reporter.

In a preferred embodiment, the nucleotide sequence of the wild-type gene of the SF3B1 gene is shown in SEQ ID NO. 1.

In a preferred embodiment, the nucleotide sequence of the forward primer in the primer pair is shown in SEQ ID NO. 3, and the nucleotide sequence of the reverse primer in the primer pair is shown in SEQ ID NO. 4.

In a preferred embodiment, the nucleotide sequence of the mutant allele probe is shown in SEQ ID NO. 5; preferably, the nucleotide sequence of the wild-type allele probe is shown in SEQ ID NO. 6.

In a specific embodiment, the mutant allele probe has the structure:

HEX-5'-ATGTCCATAACACAAC-3'-MGB；

in a specific embodiment, the wild-type allele probe is of the structure:

FAM-5'-AGTATGTCCGTAACACA-3'-MGB。

in one aspect, the invention provides the use of a product for detecting an SF3B1 protein R625H mutation in the preparation of a reagent or kit for aiding in the diagnosis of mixed PRL positive pituitary adenoma, distinguishing between mixed PRL positive pituitary adenoma SF3B1 mutant and wild type, or predicting whether mixed PRL positive pituitary adenoma is malignant.

In a preferred embodiment, the product comprises an antibody and/or protein chip for detecting the R625H mutation of SF3B1 protein; preferably, the amino acid sequence of the wild type protein of the SF3B1 protein is shown as SEQ ID NO. 2.

In a specific embodiment, the SF3B1c.1874G > A mutation of the SF3B1 gene results in the mutation of amino acid 625 of the wild type SF3B1 protein encoded by the gene from arginine (R) to histidine (H), i.e., the R625H mutation.

In one aspect, the invention provides a primer for detecting a mutation of the SF3B1 gene c.1874g > a, comprising a forward primer and a reverse primer; the nucleotide sequence of the forward primer is shown as SEQ ID NO. 3, and the nucleotide sequence of the reverse primer in the primer pair is shown as SEQ ID NO. 4.

In one aspect, the invention provides a kit for aiding in the diagnosis of, distinguishing between, or predicting malignancy of a mixed PRL positive pituitary adenoma SF3B1 mutant and wild type, the kit comprising a primer pair for the SF3B1 gene c.1874g > a mutation and a mutant allele probe, and/or the kit comprising an antibody and/or protein chip for detecting the SF3B1 protein R625H mutation.

In a preferred embodiment, the kit further comprises a mixture of reagents for PCR amplification reaction; preferably, the mixed solution of the PCR amplification reaction reagent comprises Taq DNA polymerase, dNTP mixed solution and MgCl ₂ Solution, fluorescent quantitative PCR reaction buffer and deionized water.

In a preferred embodiment, the kit further comprises a wild-type allele probe.

In a preferred embodiment, the probe in the kit is a TaqMan MGB probe, more preferably the TaqMan MGB probe is labeled with a fluorescent reporter group at the 5 'end and a non-fluorescent quenching group MGB at the 3' end. It should be noted that the 5 'fluorescent reporter group and its corresponding 3' non-fluorescent quenching group can be any available fluorescent reporter group or non-fluorescent quenching group in the art, and that the selection of these fluorescent reporter groups and their corresponding non-fluorescent quenching groups is also a well-known routine technique to those skilled in the art. For example, the fluorescent dyes FAM and HEX may be replaced by other fluorophores or dyes, such as VIC, JOE, etc.

In a preferred embodiment, the nucleotide sequence of the wild-type gene of the SF3B1 gene is shown in SEQ ID NO. 1.

In a preferred embodiment, the nucleotide sequence of the forward primer in the primer pair in the kit is shown in SEQ ID NO. 3, and the nucleotide sequence of the reverse primer in the primer pair is shown in SEQ ID NO. 4.

In a preferred embodiment, the nucleotide sequence of the mutant allele probe in the kit is shown in SEQ ID NO. 5; in a preferred embodiment, the nucleotide sequence of the wild-type allele probe is as shown in SEQ ID NO. 6.

In a preferred embodiment, the mutant allele probe in the kit has the following structure:

HEX-5'-ATGTCCATAACACAAC-3'-MGB；

in a preferred embodiment, the structure of the wild-type allele probe in the kit is as follows:

FAM-5'-AGTATGTCCGTAACACA-3'-MGB。

the beneficial effects of the invention include:

(1) Mixed PRL positive pituitary adenomas are classified into two subtypes, mutant (where SF3B1 gene is mutated) and wild-type (where SF3B1 gene is not mutated) based on whether the SF3B1c.1874G > A gene is mutated or not. Different subtypes of mixed PRL positive pituitary adenoma can be judged by detecting whether SF3B1 gene is mutated or not, so that different clinical treatment strategies can be adopted in time.

(2) SF3B1c.1874G > A gene mutation can be used as a sign of malignancy prediction.

(3) The detection probe provided by the invention has high sensitivity, good selectivity and low detection limit, and can still detect when the concentration of the sample template is as low as 1.875 ng/mu l.

Examples

The invention will now be described in more detail with reference to specific examples, which are, however, given for illustrative purposes only and are not limiting of the invention. Reagents and biological materials described in the examples below, unless otherwise indicated, are all commercially available.

Example 1 primers and TaqMan-MGB Probe design for detection of SF3B1c.1874G > A Gene mutations

According to the disclosed SF3B1 gene sequence (GenBank ^TM Accession No. NM-012433.4, the nucleotide sequence of which is shown as SEQ ID NO: 1) and SF3B1c.1874G>A mutation primers for SF3B1 gene, probes for SF3B1 mutant allele (hereinafter referred to as mutant allele probes) and probes for SF3B1 wild type allele (hereinafter referred to as wild type allele probes) were designed to detect SF3B1c.1874G>A mutation. Wherein:

the primer for SF3B1 gene is a primer for complementing the probe region and TaqMan MGB probe (TsingKe BiologicalTechnology), and comprises a primer pair consisting of a forward primer SF3B1-PF and a reverse primer SF3B1-PR, wherein the specific sequences are as follows:

SF3B1-PF：5'-CTGGCTACTATGATCTCTACCATGAGA-3'(SEQ ID NO:3)

SF3B1-PR：5'-GAGGCTACAACAGCAAAAGCTCTA-3'(SEQ ID NO:4)

mutant allele probes were labeled with 5-Hexachlorofluorofluorescein (HEX) fluorescent label (available from Applied Biosystems company) and had the following specific structure:

mutant allele probe: HEX-5' -ATGTCCATAACACAAC-3'-MGB(SEQ ID NO:5)。

The wild type allele probe was labeled with a 6-carboxyfluorescein (FAM) fluorescent label (available from the biotechnology company, beginnings, department of the general name) and its specific structure was as follows:

wild type allele probe: FAM-5' -AGTATGTCCGTAACACA-3'-MGB(SEQ ID NO:6)。

Probe-specific detection:

a wild-type template for a wild-type allele probe was designed according to SF3B1c.1874G > A as a positive control (the nucleotide sequence of which is shown in SEQ ID NO:7,TsingKe Biological Technology), and water as a negative control, and the detection results are shown in FIG. 1A. FIG. 1A shows the specificity of the wild-type probe.

A mutant template for the mutant allele probe was designed according to SF3B1c.1874G > A as a positive control (the nucleotide sequence of which is shown in SEQ ID NO:8,TsingKe Biological Technology), and water as a negative control, and the detection results are shown in FIG. 1B. FIG. 1B shows the specificity of the mutant probe.

Probe sensitivity detection:

the mutant samples were used as templates, and their DNA concentrations were diluted in a double ratio of 30 ng/. Mu.l, 15 ng/. Mu.l, 7.5 ng/. Mu.l, 3.75 ng/. Mu.l, and 1.875 ng/. Mu.l, respectively. Digital PCR amplification and detection was performed and the amount of fluorescence was found to decrease with decreasing concentration of template sample (fig. 2). Fig. 2 shows the sensitivity of the probe.

EXAMPLE 2 detection of Mixed PRL-positive pituitary adenoma SF3B1c.1874G > A mutation

Surgical specimens of neurosurgery via pituitary adenoma radical surgery were collected and each patient signed an informed consent. This study was approved by the ethics committee.

The mixed PRL positive pituitary adenoma inclusion criteria were as follows:

1. PRL positive multi-hormonal pituitary adenomas (Plurihomonal pituitary adenomas (PPAs)) into a panel of standards:

(1) Abnormal pituitary function and tumor occupancy effect, including sexual hypofunction, amenorrhea, hypothyroidism, headache, vision disorder, etc.;

(2) Pathological diagnosis of pituitary adenoma, hormone immunohistochemical staining of PRL positive and other hormones GH, TSH, FSH/LH and ACTH, etc.

(3) Skull MRI/CT shows lesions in the saddle area.

2. PRL-GH cell adenoma/PRL-GH mixed cell adenoma

(1) Serum Human Growth Hormone (HGH) is greater than normal, positive in glucose inhibition test or serum insulin-like growth factor-1 (IGF-1) is greater than 2 standard deviations above the average of normal persons of the same age and sex;

(2) Manifestations associated with acromegaly and clinical symptoms associated in part with hyperlactinemia, and tumor occupancy effects, including menopause, lactation, infertility, sexual hypofunction, headache, vision impairment, and the like.

(3) Pathologically diagnosed pituitary adenomas, immunohistochemistry showed PRL positive and GH positive;

(4) Skull MRI/CT shows lesions in the saddle area.

In this example, 28 samples of pooled mixed PRL positive pituitary adenoma surgical specimens were examined for mutations in the SF3B1 gene. The specific method for detecting SF3B1c.1874G > A mutation in the case group detection sample by using the primers and TaqMan-MGB probes designed in the example 1 and taking the control group detection sample as a control is as follows:

step 1: extraction of genomic DNA from a sample to be tested

Genomic DNA from the samples was extracted using the AllPrep DNA/RNA Mini kit (80204, qiagen). Genomic DNA was extracted from formalin-fixed, paraffin-embedded tumor tissue using GeneRead DNA FFPE Kit (180134, qiagen).

Step 2: PCR amplification

The DNA extracted in step 1 was used as a template, and the amplification reaction was performed under the guidance of the primers designed in example 1 and TaqMan-MGB probe, and passed through EP1 ^TM The mutant of SF3B1 was detected by digital chip (Fluidigm) ddPCR system.

Two different chips were used in the experiment.

1) 12.765 chip (BMK-M10-12.765, fluidigm)

The reaction system was 8. Mu.L, and the mixture contained:

(1) the DNA sample was 0.8. Mu.L,

②20×GE Sample Loading Reagent(PN 85000746，Fluidigm)0.4μL，

(3) 10. Mu.M Gene-specific reagent (primers and TaqMan Probe specific for mutation and wild-type SF3B 1)

2.8μL

④Gene Expression Master Mix(PN 4369016，Life Technologies)4μL；

2) 48.770 chip (dPCR 37k IFC 100-6151 fluidigm)

The reaction system was 4. Mu.L, and the mixture included:

(1) the DNA sample was 0.88. Mu.L,

②20×GE Sample Loading Reagent(PN 85000746，Fluidigm)0.4μL，

(3) 20. Mu.M Gene-specific reagent (primers and TaqMan Probe specific for mutant and wild-type SF3B 1)

0.72μL

④Gene Expression Master Mix 2μL。

The loaded chip was placed in an EP1 Cycler.

The PCR process was as follows: denaturation at 95℃for 15s at 120s at 50℃for 10 min, denaturation at 95℃and annealing and extension at 60℃for 1 min (40 cycles of this step).

Step 3: allele discrimination analysis

And (3) carrying out allele discrimination analysis on the amplified product in the step (2). If the HEX fluorescent signal is detected by the fluorescent detection channel, namely the mutant allele is detected, and the mutation is judged according to the result; if the fluorescence detection channel detects FAM fluorescence signal, namely the wild type allele is detected, the mutation is judged according to the result.

Data were processed using EP1 data collection and analysis software, PCR amplification was analyzed and the number of HEX and FAM positives in each panel was calculated. 6-carboxy-X-rhodamine (6-carboxy-X-Rhodamine (ROX)) signal was used as an internal positive PCR control; double distilled water (dd water) was used as a negative control. The feasibility of the system, amplification protocol, established Cq range and quantification threshold were evaluated using positive and negative controls.

A450 bp DNA fragment from SF3B1c.1874G > A (the nucleotide sequence of which is shown in SEQ ID NO: 7) was used as a positive control (TsingKe Biological Technology). DNA samples from healthy individuals of the control group served as negative controls. To determine the specificity of the assay, ddPCR amplification was performed using water and healthy tissue DNA as negative controls. In control experiments without template addition, generally no HEX positive signal was detected. 1 to 2 HEX positive signals were detected in a small portion of healthy tissue control DNA samples. The minimum cut-off frequency was set to 0.5% (1 mutant per 200 total alleles) to detect SF3B1 mutation positive DNA samples by ddPCR. For all samples, c.1874g > a ddPCR was performed at least 3 times.

Of the 28 mixed PRL positive pituitary adenoma specimens tested, 10 were PRL positive multi-hormone pituitary adenomas, 18 were PRL/GH mixed adenomas (including PRL-GH mixed cell adenomas and PRL-GH cell adenomas), 2 were found to have the SF3B1c.1874G > A mutant shown in Table 1, with a mutation rate of about 7.14% (2/28) for SF3B1R 625H.

TABLE 1

Nucleotide(s)	Nucleotide substitutions	Amino acid substitutions	Exons
				1874	G→A	R265H	14

The hormonal staining of the 10 PRL positive multi-hormonal pituitary adenomas is shown in table 2.

TABLE 2

Positive hormone staining	Number of cases
		PRL+ACTH	3
PRL+FSH	2
		TSH+PRL+ACTH	1
TSH+GH+PRL	3
		TSH+PRL	1

Of these, 1 patient was clinically diagnosed as non-functional, i.e., free of symptoms of increased hormone secretion. Post-operative tumor tissue hormone immunohistochemical staining was TSH+PRL+ACTH positive, other staining was negative. As can be seen, this example of a multi-hormonal pituitary adenoma is a mixed PRL positive prolapseAnd (3) body adenoma. SF3B1 was detected in this case ^R625H Mutation.

The hormonal staining of the 18 PRL/GH mixed adenomas is shown in Table 3.

TABLE 3 Table 3

Positive hormone staining	Number of cases
		PRL-GH mixed cell adenoma	14
PRL-GH cell adenoma	4

Of these, 1 patient clinically showed increased growth hormone secretion, acromegaly and altered appearance. Serum prolactin>200ng/ml, HGH of 16.681ng/m, post-operative tumor tissue hormone immunohistochemical staining of GH and PRL positive, LH, FSH, TSH, ACTH negative, diagnosis of PRL and GH mix. It can be seen that this mixed pituitary adenoma belongs to mixed PRL positive pituitary adenomas. SF3B1 was detected in this case ^R625H Mutation.

As a result of combining the above 28 cases of pituitary adenoma gene mutation detection, SF3B1 was found ^R625H Mutations can occur in mixed PRL positive pituitary adenomas, SF3B1 ^R625H The mutation rate of (2/28) was about 7.14%.

Example 3SF3B1 ^R625H Effect of mutations on Mixed PRL-positive pituitary adenomas

The GH3 cell line ATCC CCL-82.1 (purchased from American type culture Collection ATCC (American TypeCulture Collection)) was selected as the experimental cell line, and the GH3 cell line was a rat pituitary tumor cell line which can express and secrete PRL and GH, and was used as a mixed cell line in the subsequent experiments.

Viral fluids of adenovirus expressing SF3B1 wild type and mutant (SF 3B1c.1874g > a) were purchased from beijing bai biotechnology, inc.

GH3 cell line cells are divided into the following three groups:

(1) SF3B1 wild type group: infection with SF3B1 wild-type adenovirus (adenovirus-SF 3B 1) ^WT ) Is a cell obtained by infecting GH3 cells with a virus solution expressing SF3B1 wild-type adenovirus;

(2) SF3B1 mutant group: infection with SF3B1 mutant adenovirus (adenovirus-SF 3B 1) ^R625H ) Is a cell obtained by infecting GH3 cells with a virus solution expressing SF3B1 mutant adenovirus;

(3) Control group: GH3 cells infected with adenovirus empty vector (adenovirus-empty vector).

The proliferation activity of cells was observed after infection with 30MOI virus and 100MOI virus, respectively; where MOI (multiplicity of infection ) indicates how many viable viruses each cell is infected with.

1. Measurement of cell proliferation Capacity by CCK-8 Activity

SF3B1 wild type cells, SF3B1 mutant cells and control cells were each used at 4X 10 per well ³ 100. Mu.L of medium (the medium composition: F12K medium (ATCC 30-2004), fetal bovine serum (Gibco Co.) at a final concentration of 2.5%, horse serum (Gibco Co.) at a final concentration of 15%) per well was inoculated into 96-well plates. After 48h of incubation of infected cells 10. Mu.L of CCK-8 reagent (Beyotime, C0039) was added to each well and the wells were incubated at 37℃with 5% CO ₂ Incubate for 4h. OD value was measured with an ELISA reader, and absorption wavelength was 450nm. The detection results are shown in FIG. 3. Experimental results show that cells infected with SF3B1 mutant adenovirus have stronger proliferation capacity than cells infected with wild-type SF3B1 adenovirus and cells of a control group.

2. Clonogenic assay to measure cell proliferation

SF3B1 wild type group cells, SF3B1 mutant group cells and control group cells were used in 1X 10, respectively ³ Single cell suspensions of cells were plated in 2mL of DMEM containing 10% Fetal Bovine Serum (FBS). During 3 weeks of infected cells, the medium was changed every 3 days. The colonies formed were then fixed with 4% paraformaldehyde and stained with 0.04% crystal violet in PBS for 15 min at room temperature. After extensive washing and air drying, colony count was measured with ImageJ. The detection results are shown in FIG. 4. Experimental results show that the cell infected by SF3B1 mutant adenovirus has more cell colonies, namely stronger cell proliferation capacity, compared with the cell infected by wild SF3B1 adenovirus and the cell colony of the control group.

3. Cell flow technology detects apoptosis.

Apoptosis was analyzed by the Annexin V-FITC/propidium ion double staining method provided by Beyotime corporation's kit (C1062M). SF3B1 wild-type, SF3B1 mutant and control cells 48h after adenovirus infection were collected and analyzed, respectively. Centrifugation to collect about 5X 10 of each group ⁵ Mu.l of binding buffer (1X Annexin V Binding Buffer) was resuspended in each cell. Mu.l of Annexin-V-FITC (Annexin V-FITC) and 5. Mu.l of propidium iodide (propidium iodide) were added to each tube, followed by incubation in the dark at room temperature for 15 minutes. Stained cells were analyzed by flow cytometry in FITC and PE channels. The detection results are shown in FIG. 5. Experimental results show that the total apoptosis number of the cells infected with SF3B1 mutant adenovirus is the lowest, and the cells infected with SF3B1 mutant adenovirus have weak apoptosis capacity compared with the cells infected with wild type SF3B1 adenovirus and the cells infected with the control group.

The experimental results show that mutant SF3B1 promotes cell proliferation and inhibits apoptosis in a mixed pituitary adenoma cell line GH3 cell line. The above cell experiments prove that the SF3B1 mutation has the effect of promoting proliferation and inhibiting apoptosis of cells, and the case of SF3B1 mutation is suggested to have malignant performance compared with the wild type. Therefore, the new treatment method and the medicine obtained by researching the mutation site have great significance for treating the mixed PRL positive pituitary adenoma of SF3B1 mutant.

SEQUENCE LISTING

<110> Beijing city neurosurgery institute

<120> application of SF3B1 gene mutation in auxiliary diagnosis of mixed PRL positive pituitary adenoma

<130> MTI20071

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 6463

<212> DNA

<213> Homo sapiens (Homo sapiens)

<400> 1

agttccgtct gtgtgttcga gtggacaaaa tggcgaagat cgccaagact cacgaagata 60

ttgaagcaca gattcgagaa attcaaggca agaaggcagc tcttgatgaa gctcaaggag 120

tgggcctcga ttctacaggt tattatgacc aggaaattta tggtggaagt gacagcagat 180

ttgctggata cgtgacatca attgctgcaa ctgaacttga agatgatgac gatgactatt 240

catcatctac gagtttgctt ggtcagaaga agccaggata tcatgcccct gtggcattgc 300

ttaatgatat accacagtca acagaacagt atgatccatt tgctgagcac agacctccaa 360

agattgcaga ccgggaagat gaatacaaaa agcataggcg gaccatgata atttccccag 420

agcgtcttga tccttttgca gatggaggga aaacccctga tcctaaaatg aatgctagga 480

cttacatgga tgtaatgcga gaacaacact tgactaaaga agaacgagaa attaggcaac 540

agctagcaga aaaagctaaa gctggagaac taaaagtcgt caatggagca gcagcgtccc 600

agcctccatc aaaacgaaaa cggcgttggg atcaaacagc tgatcagact cctggtgcca 660

ctcccaaaaa actatcaagt tgggatcagg cagagacccc tgggcatact ccttccttaa 720

gatgggatga gacaccaggt cgtgcaaagg gaagcgagac tcctggagca accccaggct 780

caaaaatatg ggatcctaca cctagccaca caccagcggg agctgctact cctggacgag 840

gtgatacacc aggccatgcg acaccaggcc atggaggcgc aacttccagt gctcgtaaaa 900

acagatggga tgaaaccccc aaaacagaga gagatactcc tgggcatgga agtggatggg 960

ctgagactcc tcgaacagat cgaggtggag attctattgg tgaaacaccg actcctggag 1020

ccagtaaaag aaaatcacgg tgggatgaaa caccagctag tcagatgggt ggaagcactc 1080

cagttctgac ccctggaaag acaccaattg gcacaccagc catgaacatg gctaccccta 1140

ctccaggtca cataatgagt atgactcctg aacagcttca ggcttggcgg tgggaaagag 1200

aaattgatga gagaaatcgc ccactttctg atgaggaatt agatgctatg ttcccagaag 1260

gatataaggt acttcctcct ccagctggtt atgttcctat tcgaactcca gctcgaaagc 1320

tgacagctac tccaacacct ttgggtggta tgactggttt ccacatgcaa actgaagatc 1380

gaactatgaa aagtgttaat gaccagccat ctggaaatct tccattttta aaacctgatg 1440

atattcaata ctttgataaa ctattggttg atgttgatga atcaacactt agtccagaag 1500

agcaaaaaga gagaaaaata atgaagttgc ttttaaaaat taagaatgga acaccaccaa 1560

tgagaaaggc tgcattgcgt cagattactg ataaagctcg tgaatttgga gctggtcctt 1620

tgtttaatca gattcttcct ctgctgatgt ctcctacact tgaggatcaa gagcgtcatt 1680

tacttgtgaa agttattgat aggatactgt acaaacttga tgacttagtt cgtccatatg 1740

tgcataagat cctcgtggtc attgaaccgc tattgattga tgaagattac tatgctagag 1800

tggaaggccg agagatcatt tctaatttgg caaaggctgc tggtctggct actatgatct 1860

ctaccatgag acctgatata gataacatgg atgagtatgt ccgtaacaca acagctagag 1920

cttttgctgt tgtagcctct gccctgggca ttccttcttt attgcccttc ttaaaagctg 1980

tgtgcaaaag caagaagtcc tggcaagcga gacacactgg tattaagatt gtacaacaga 2040

tagctattct tatgggctgt gccatcttgc cacatcttag aagtttagtt gaaatcattg 2100

aacatggtct tgtggatgag cagcagaaag ttcggaccat cagtgctttg gccattgctg 2160

ccttggctga agcagcaact ccttatggta tcgaatcttt tgattctgtg ttaaagcctt 2220

tatggaaggg tatccgccaa cacagaggaa agggtttggc tgctttcttg aaggctattg 2280

ggtatcttat tcctcttatg gatgcagaat atgccaacta ctatactaga gaagtgatgt 2340

taatccttat tcgagaattc cagtctcctg atgaggaaat gaaaaaaatt gtgctgaagg 2400

tggtaaaaca gtgttgtggg acagatggtg tagaagcaaa ctacattaaa acagagattc 2460

ttcctccctt ttttaaacac ttctggcagc acaggatggc tttggataga agaaattacc 2520

gacagttagt tgatactact gtggagttgg caaacaaagt aggtgcagca gaaattatat 2580

ccaggattgt ggatgatctg aaagatgaag ccgaacagta cagaaaaatg gtgatggaga 2640

caattgagaa aattatgggt aatttgggag cagcagatat tgatcataaa cttgaagaac 2700

aactgattga tggtattctt tatgctttcc aagaacagac tacagaggac tcagtaatgt 2760

tgaacggctt tggcacagtg gttaatgctc ttggcaaacg agtcaaacca tacttgcctc 2820

agatctgtgg tacagttttg tggcgtttaa ataacaaatc tgctaaagtt aggcaacagg 2880

cagctgactt gatttctcga actgctgttg tcatgaagac ttgtcaagag gaaaaattga 2940

tgggacactt gggtgttgta ttgtatgagt atttgggtga agagtaccct gaagtattgg 3000

gcagcattct tggagcactg aaggccattg taaatgtcat aggtatgcat aagatgactc 3060

caccaattaa agatctgctg cctagactca cccccatctt aaagaacaga catgaaaaag 3120

tacaagagaa ttgtattgat cttgttggtc gtattgctga caggggagct gaatatgtat 3180

ctgcaagaga gtggatgagg atttgctttg agcttttaga gctcttaaaa gcccacaaaa 3240

aggctattcg tagagccaca gtcaacacat ttggttatat tgcaaaggcc attggccctc 3300

atgatgtatt ggctacactt ctgaacaacc tcaaagttca agaaaggcag aacagagttt 3360

gtaccactgt agcaatagct attgttgcag aaacatgttc accctttaca gtactccctg 3420

ccttaatgaa tgaatacaga gttcctgaac tgaatgttca aaatggagtg ttaaaatcgc 3480

tttccttctt gtttgaatat attggtgaaa tgggaaaaga ctacatttat gccgtaacac 3540

cgttacttga agatgcttta atggatagag accttgtaca cagacagacg gctagtgcag 3600

tggtacagca catgtcactt ggggtttatg gatttggttg tgaagattcg ctgaatcact 3660

tgttgaacta tgtatggccc aatgtatttg agacatctcc tcatgtaatt caggcagtta 3720

tgggagccct agagggcctg agagttgcta ttggaccatg tagaatgttg caatattgtt 3780

tacagggtct gtttcaccca gcccggaaag tcagagatgt atattggaaa atttacaact 3840

ccatctacat tggttcccag gacgctctca tagcacatta cccaagaatc tacaacgatg 3900

ataagaacac ctatattcgt tatgaacttg actatatctt ataattttat tgtttatttt 3960

gtgtttaatg cacagctact tcacacctta aacttgcttt gatttggtga tgtaaacttt 4020

taaacattgc agatcagtgt agaactggtc atagaggaag agctagaaat ccagtagcat 4080

gatttttaaa taacctgtct ttgtttttga tgttaaacag taaatgccag tagtgaccaa 4140

gaacacagtg attatataca ctatactgga gggatttcat ttttaattca tctttatgaa 4200

gatttagaac tcattccttg tgtttaaagg gaatgtttaa ttgagaaata aacatttgtg 4260

tacaaaatgc taatttgtgt gtgttttttg aacatgactt gtaaaatgcg gaactttgat 4320

aaagtattgg tttatgtgga ataagtggct taatttcatt ttctgtcaca tggtttatag 4380

aaagtagtta gctgaataaa aactatataa agtgatggca tctttgtcaa aattccattg 4440

ttgtgttaat aatgacagca agaagagtag cctcagggga tgttcccctc aaactagcac 4500

aaccattcca tcatcgtaga aaagtagcac ttttgctaaa ctgtcttgaa tattttgtac 4560

ttacatagcg cctttcatct cttgatttct caaaatgctt tatgaacaca tttaaagaaa 4620

gtggtttaag tcttgtccaa cacttgacag gtctgctgtg tttagcaagt gaggaattta 4680

actttacttc aaaactgctt tctgcctatt aggagtgagg atacctaagt aatgctgata 4740

gaacaggaca atgttgggct tttctccatg ttataagcca ctactcagca atgcatcagt 4800

aaatacctat tcacccactg tatgccagcc actgtgctta tatgcagggg atgcaaaggt 4860

gcatgagacc tgccctctac ctttaagaac agtataatgg agaaggggag acaaaccttg 4920

tgacagattc cttatgtact gtgataatac acagtggcag cagctaaaat gtctaggttt 4980

gcttagcttt gtattcagta ataataagtt gatctaagag ttcagcataa actgaatgaa 5040

atgccattta atggtagagg aaccaagcat taaggcagta ctacacttaa ttttttaagc 5100

aaatatgtaa agtatatttt caaacttttc taatgttatg gccccaaatt tcttagtttt 5160

ggccttttta ctacctatac tatttttact gttttgtttt gtctccatgt ggtagtactt 5220

ctgtgaactc taaagggaaa aaaaattctg caagcagcat tagtatataa ctactactgt 5280

aagtaaaact gcctattgac actttaggag ttcctgcttc agaagcttag ttaagaaaca 5340

gcttgtggcc gggtgtggtg gctcatgcct gtaatcccag cactttggga ggctgaggcg 5400

ggcggatcac gaggtcagga gatcgagacc atcctggcta acgcggtgaa gtcccgtcta 5460

ctaaaaatac aaaaattagc caggcgtggt ggcgggcgcc tgtagtccca gctactaggg 5520

aggctgaggc aggagaatgg cttgaacccg ggaggcggag cttgcagtga gccgagatag 5580

cgctgctaca ctccagcctg ggcgacagag actccgtctc aaaaaaaaaa aaaaaattga 5640

ctttcaacaa attgatagtg agcattaagg gtttccaagt tggatttgta actcctcatc 5700

attccttgta tgacaacttt ctgaatatat gtcactatgt agtaaaatta aacactccaa 5760

actcatcttt ctgttgttag aagttttcag cggtacttcc atgcaacttt aaatctcact 5820

gctctctatg gttgatgtca aatgaccttc agtaatgact gagaattgaa tacaaataga 5880

ttacaaagcc aaaatttgat gttaaatgac tcaggaaatt ttagttgtat tttcaattca 5940

agtacttagt agcctacgtt tgcttggcct ctggttcttt atggaaaata ggctttgtag 6000

tggcattgtg gagcaaagga gactgttaca ccttaattaa ctttttttac tgatgcaaat 6060

aatttgagga tagagaggag ggaagtagtg aaagctatga cctaaaacat tgggaccaaa 6120

tagaggctca cagatatttg gattatttta tgtgcttatt attaaataag gaaagcattt 6180

tgtgatatgt ggaagacgct atgtgaagtt ttacctatct tctcaaagac cttttctttt 6240

gtattttctt ttggtgtttc ttaaagccaa acaaagaaat gttcttaagg agacagggtg 6300

ggtttttctg tgggcctttg ttggtttttc tgtgggccat cgccctctaa tggaattgat 6360

ctctggctgt ttgatttttt tcatattgta tttttaaaat ttgttgtaca gtgccctgtg 6420

agcaccaagt accactagat gaataaaacg tattatatct aaa 6463

<210> 2

<211> 1304

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 2

Met Ala Lys Ile Ala Lys Thr His Glu Asp Ile Glu Ala Gln Ile Arg

1 5 10 15

Glu Ile Gln Gly Lys Lys Ala Ala Leu Asp Glu Ala Gln Gly Val Gly

20 25 30

Leu Asp Ser Thr Gly Tyr Tyr Asp Gln Glu Ile Tyr Gly Gly Ser Asp

35 40 45

Ser Arg Phe Ala Gly Tyr Val Thr Ser Ile Ala Ala Thr Glu Leu Glu

50 55 60

Asp Asp Asp Asp Asp Tyr Ser Ser Ser Thr Ser Leu Leu Gly Gln Lys

65 70 75 80

Lys Pro Gly Tyr His Ala Pro Val Ala Leu Leu Asn Asp Ile Pro Gln

85 90 95

Ser Thr Glu Gln Tyr Asp Pro Phe Ala Glu His Arg Pro Pro Lys Ile

100 105 110

Ala Asp Arg Glu Asp Glu Tyr Lys Lys His Arg Arg Thr Met Ile Ile

115 120 125

Ser Pro Glu Arg Leu Asp Pro Phe Ala Asp Gly Gly Lys Thr Pro Asp

130 135 140

Pro Lys Met Asn Ala Arg Thr Tyr Met Asp Val Met Arg Glu Gln His

145 150 155 160

Leu Thr Lys Glu Glu Arg Glu Ile Arg Gln Gln Leu Ala Glu Lys Ala

165 170 175

Lys Ala Gly Glu Leu Lys Val Val Asn Gly Ala Ala Ala Ser Gln Pro

180 185 190

Pro Ser Lys Arg Lys Arg Arg Trp Asp Gln Thr Ala Asp Gln Thr Pro

195 200 205

Gly Ala Thr Pro Lys Lys Leu Ser Ser Trp Asp Gln Ala Glu Thr Pro

210 215 220

Gly His Thr Pro Ser Leu Arg Trp Asp Glu Thr Pro Gly Arg Ala Lys

225 230 235 240

Gly Ser Glu Thr Pro Gly Ala Thr Pro Gly Ser Lys Ile Trp Asp Pro

245 250 255

Thr Pro Ser His Thr Pro Ala Gly Ala Ala Thr Pro Gly Arg Gly Asp

260 265 270

Thr Pro Gly His Ala Thr Pro Gly His Gly Gly Ala Thr Ser Ser Ala

275 280 285

Arg Lys Asn Arg Trp Asp Glu Thr Pro Lys Thr Glu Arg Asp Thr Pro

290 295 300

Gly His Gly Ser Gly Trp Ala Glu Thr Pro Arg Thr Asp Arg Gly Gly

305 310 315 320

Asp Ser Ile Gly Glu Thr Pro Thr Pro Gly Ala Ser Lys Arg Lys Ser

325 330 335

Arg Trp Asp Glu Thr Pro Ala Ser Gln Met Gly Gly Ser Thr Pro Val

340 345 350

Leu Thr Pro Gly Lys Thr Pro Ile Gly Thr Pro Ala Met Asn Met Ala

355 360 365

Thr Pro Thr Pro Gly His Ile Met Ser Met Thr Pro Glu Gln Leu Gln

370 375 380

Ala Trp Arg Trp Glu Arg Glu Ile Asp Glu Arg Asn Arg Pro Leu Ser

385 390 395 400

Asp Glu Glu Leu Asp Ala Met Phe Pro Glu Gly Tyr Lys Val Leu Pro

405 410 415

Pro Pro Ala Gly Tyr Val Pro Ile Arg Thr Pro Ala Arg Lys Leu Thr

420 425 430

Ala Thr Pro Thr Pro Leu Gly Gly Met Thr Gly Phe His Met Gln Thr

435 440 445

Glu Asp Arg Thr Met Lys Ser Val Asn Asp Gln Pro Ser Gly Asn Leu

450 455 460

Pro Phe Leu Lys Pro Asp Asp Ile Gln Tyr Phe Asp Lys Leu Leu Val

465 470 475 480

Asp Val Asp Glu Ser Thr Leu Ser Pro Glu Glu Gln Lys Glu Arg Lys

485 490 495

Ile Met Lys Leu Leu Leu Lys Ile Lys Asn Gly Thr Pro Pro Met Arg

500 505 510

Lys Ala Ala Leu Arg Gln Ile Thr Asp Lys Ala Arg Glu Phe Gly Ala

515 520 525

Gly Pro Leu Phe Asn Gln Ile Leu Pro Leu Leu Met Ser Pro Thr Leu

530 535 540

Glu Asp Gln Glu Arg His Leu Leu Val Lys Val Ile Asp Arg Ile Leu

545 550 555 560

Tyr Lys Leu Asp Asp Leu Val Arg Pro Tyr Val His Lys Ile Leu Val

565 570 575

Val Ile Glu Pro Leu Leu Ile Asp Glu Asp Tyr Tyr Ala Arg Val Glu

580 585 590

Gly Arg Glu Ile Ile Ser Asn Leu Ala Lys Ala Ala Gly Leu Ala Thr

595 600 605

Met Ile Ser Thr Met Arg Pro Asp Ile Asp Asn Met Asp Glu Tyr Val

610 615 620

Arg Asn Thr Thr Ala Arg Ala Phe Ala Val Val Ala Ser Ala Leu Gly

625 630 635 640

Ile Pro Ser Leu Leu Pro Phe Leu Lys Ala Val Cys Lys Ser Lys Lys

645 650 655

Ser Trp Gln Ala Arg His Thr Gly Ile Lys Ile Val Gln Gln Ile Ala

660 665 670

Ile Leu Met Gly Cys Ala Ile Leu Pro His Leu Arg Ser Leu Val Glu

675 680 685

Ile Ile Glu His Gly Leu Val Asp Glu Gln Gln Lys Val Arg Thr Ile

690 695 700

Ser Ala Leu Ala Ile Ala Ala Leu Ala Glu Ala Ala Thr Pro Tyr Gly

705 710 715 720

Ile Glu Ser Phe Asp Ser Val Leu Lys Pro Leu Trp Lys Gly Ile Arg

725 730 735

Gln His Arg Gly Lys Gly Leu Ala Ala Phe Leu Lys Ala Ile Gly Tyr

740 745 750

Leu Ile Pro Leu Met Asp Ala Glu Tyr Ala Asn Tyr Tyr Thr Arg Glu

755 760 765

Val Met Leu Ile Leu Ile Arg Glu Phe Gln Ser Pro Asp Glu Glu Met

770 775 780

Lys Lys Ile Val Leu Lys Val Val Lys Gln Cys Cys Gly Thr Asp Gly

785 790 795 800

Val Glu Ala Asn Tyr Ile Lys Thr Glu Ile Leu Pro Pro Phe Phe Lys

805 810 815

His Phe Trp Gln His Arg Met Ala Leu Asp Arg Arg Asn Tyr Arg Gln

820 825 830

Leu Val Asp Thr Thr Val Glu Leu Ala Asn Lys Val Gly Ala Ala Glu

835 840 845

Ile Ile Ser Arg Ile Val Asp Asp Leu Lys Asp Glu Ala Glu Gln Tyr

850 855 860

Arg Lys Met Val Met Glu Thr Ile Glu Lys Ile Met Gly Asn Leu Gly

865 870 875 880

Ala Ala Asp Ile Asp His Lys Leu Glu Glu Gln Leu Ile Asp Gly Ile

885 890 895

Leu Tyr Ala Phe Gln Glu Gln Thr Thr Glu Asp Ser Val Met Leu Asn

900 905 910

Gly Phe Gly Thr Val Val Asn Ala Leu Gly Lys Arg Val Lys Pro Tyr

915 920 925

Leu Pro Gln Ile Cys Gly Thr Val Leu Trp Arg Leu Asn Asn Lys Ser

930 935 940

Ala Lys Val Arg Gln Gln Ala Ala Asp Leu Ile Ser Arg Thr Ala Val

945 950 955 960

Val Met Lys Thr Cys Gln Glu Glu Lys Leu Met Gly His Leu Gly Val

965 970 975

Val Leu Tyr Glu Tyr Leu Gly Glu Glu Tyr Pro Glu Val Leu Gly Ser

980 985 990

Ile Leu Gly Ala Leu Lys Ala Ile Val Asn Val Ile Gly Met His Lys

995 1000 1005

Met Thr Pro Pro Ile Lys Asp Leu Leu Pro Arg Leu Thr Pro Ile

1010 1015 1020

Leu Lys Asn Arg His Glu Lys Val Gln Glu Asn Cys Ile Asp Leu

1025 1030 1035

Val Gly Arg Ile Ala Asp Arg Gly Ala Glu Tyr Val Ser Ala Arg

1040 1045 1050

Glu Trp Met Arg Ile Cys Phe Glu Leu Leu Glu Leu Leu Lys Ala

1055 1060 1065

His Lys Lys Ala Ile Arg Arg Ala Thr Val Asn Thr Phe Gly Tyr

1070 1075 1080

Ile Ala Lys Ala Ile Gly Pro His Asp Val Leu Ala Thr Leu Leu

1085 1090 1095

Asn Asn Leu Lys Val Gln Glu Arg Gln Asn Arg Val Cys Thr Thr

1100 1105 1110

Val Ala Ile Ala Ile Val Ala Glu Thr Cys Ser Pro Phe Thr Val

1115 1120 1125

Leu Pro Ala Leu Met Asn Glu Tyr Arg Val Pro Glu Leu Asn Val

1130 1135 1140

Gln Asn Gly Val Leu Lys Ser Leu Ser Phe Leu Phe Glu Tyr Ile

1145 1150 1155

Gly Glu Met Gly Lys Asp Tyr Ile Tyr Ala Val Thr Pro Leu Leu

1160 1165 1170

Glu Asp Ala Leu Met Asp Arg Asp Leu Val His Arg Gln Thr Ala

1175 1180 1185

Ser Ala Val Val Gln His Met Ser Leu Gly Val Tyr Gly Phe Gly

1190 1195 1200

Cys Glu Asp Ser Leu Asn His Leu Leu Asn Tyr Val Trp Pro Asn

1205 1210 1215

Val Phe Glu Thr Ser Pro His Val Ile Gln Ala Val Met Gly Ala

1220 1225 1230

Leu Glu Gly Leu Arg Val Ala Ile Gly Pro Cys Arg Met Leu Gln

1235 1240 1245

Tyr Cys Leu Gln Gly Leu Phe His Pro Ala Arg Lys Val Arg Asp

1250 1255 1260

Val Tyr Trp Lys Ile Tyr Asn Ser Ile Tyr Ile Gly Ser Gln Asp

1265 1270 1275

Ala Leu Ile Ala His Tyr Pro Arg Ile Tyr Asn Asp Asp Lys Asn

1280 1285 1290

Thr Tyr Ile Arg Tyr Glu Leu Asp Tyr Ile Leu

1295 1300

<210> 3

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

ctggctacta tgatctctac catgaga 27

<210> 4

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

gaggctacaa cagcaaaagc tcta 24

<210> 5

<211> 16

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

atgtccataa cacaac 16

<210> 6

<211> 17

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

agtatgtccg taacaca 17

<210> 7

<211> 450

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

tctaagatgt ggcaagatgg cacagcccat aagaatagct atctgttgta caatcttaat 60

accagtgtgt ctcgcttgcc aggacttctt gcttttgcac acagctttta agaagggcaa 120

taaagaagga atgcccaggg cagaggctac aacagcaaaa gctctagctg ttgtgttacg 180

gacatactca tccatgttat ctatatcagg tctcatggta gagatcatag tagccagacc 240

agcagcctaa aatgtaaaca aagaaaggac agtcatgagt tggtaatatt aatcttcaac 300

catttctttc cataatcaat tccataaaca gatataaatt ttcttctcta gaaattaaat 360

gtaaatacct ttgccaaatt agaaatgatc tctcggcctt ccactctagc atagtaatct 420

tcatcaatca atagcggttc aatgaccacg 450

<210> 8

<211> 450

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

tctaagatgt ggcaagatgg cacagcccat aagaatagct atctgttgta caatcttaat 60

accagtgtgt ctcgcttgcc aggacttctt gcttttgcac acagctttta agaagggcaa 120

taaagaagga atgcccaggg cagaggctac aacagcaaaa gctctagctg ttgtgttatg 180

gacatactca tccatgttat ctatatcagg tctcatggta gagatcatag tagccagacc 240

agcagcctaa aatgtaaaca aagaaaggac agtcatgagt tggtaatatt aatcttcaac 300

catttctttc cataatcaat tccataaaca gatataaatt ttcttctcta gaaattaaat 360

gtaaatacct ttgccaaatt agaaatgatc tctcggcctt ccactctagc atagtaatct 420

tcatcaatca atagcggttc aatgaccacg 450

Claims (15)

1. Use of a product for detecting a mutation of the SF3B1 gene c.1874g > a in the preparation of a reagent or kit for aiding in the diagnosis of mixed PRL positive pituitary adenomas, distinguishing between mixed PRL positive pituitary adenomas SF3B1 mutant and wild type, or predicting whether mixed PRL positive pituitary adenomas are malignant, wherein the mixed PRL positive pituitary adenomas are PRL and GH staining positive mixed pituitary adenomas.

2. The use according to claim 1, wherein the PRL and GH staining positive mixed pituitary adenoma is selected from PRL-GH cell adenoma or PRL-GH mixed cell adenoma.

3. Use according to claim 1, wherein the product comprises reagents for detecting the SF3B1 gene c.1874g > a mutation by PCR techniques.

4. The use according to claim 3, wherein the product comprises a mixture of PCR amplification reagents, a primer pair for the SF3B1 gene c.1874G > A mutation, and a mutant allele probe; the PCR amplification reaction reagent mixed solution comprises TaqDNA polymerase, dNTP mixed solution, mgCl2 solution, fluorescent quantitative PCR reaction buffer solution and deionized water.

5. The use of claim 1, wherein the product further comprises a wild-type allele probe.

6. The use according to claim 3, wherein the PCR is digital PCR or fluorescent quantitative PCR.

7. The use according to claim 4, wherein the probe is a TaqMan MGB probe.

8. The use according to claim 7, wherein the TaqMan MGB probe is labeled with a fluorescent reporter group at the 5 'end and a non-fluorescent quenching group MGB at the 3' end.

9. The use of claim 8, wherein the fluorescent reporter is a FAM or HEX fluorescent reporter.

10. The use according to claim 4, wherein the nucleotide sequence of the forward primer in the primer pair is shown in SEQ ID NO. 3 and the nucleotide sequence of the reverse primer in the primer pair is shown in SEQ ID NO. 4.

11. The use according to claim 4, wherein the nucleotide sequence of the mutant allele probe is shown in SEQ ID NO. 5, and the structure of the mutant allele probe is as follows:

HEX-5'-ATGTCCATAACACAAC-3'-MGB。

12. the use according to claim 4, wherein the nucleotide sequence of the wild-type allele probe is shown in SEQ ID NO. 6, and the structure of the wild-type allele probe is as follows:

FAM-5'-AGTATGTCCGTAACACA-3'-MGB。

13. use of a product for detecting an SF3B1 protein R625H mutation in the preparation of a reagent or kit for aiding in the diagnosis of mixed PRL positive pituitary adenomas, distinguishing between mixed PRL positive pituitary adenomas SF3B1 mutant and wild type, or predicting whether mixed PRL positive pituitary adenomas are malignant, wherein the mixed PRL positive pituitary adenomas are PRL and GH staining positive mixed pituitary adenomas.

14. The use of claim 13, wherein the product comprises an antibody and/or protein chip to detect SF3B1 protein R625H mutation.

15. The use according to claim 13, wherein the amino acid sequence of the wild type protein of the SF3B1 protein is shown in SEQ ID No. 2.