CN111690729A

CN111690729A - Method for diagnosing wet age-related macular degeneration by measuring NPVF protein in peripheral blood

Info

Publication number: CN111690729A
Application number: CN201910186248.4A
Authority: CN
Inventors: 许迅; 于复东; 刘堃; 成璐; 郑颖; 沈胤忱; 陈翀; 熊淑毓
Original assignee: Shanghai First Peoples Hospital
Current assignee: Shanghai First Peoples Hospital
Priority date: 2019-03-12
Filing date: 2019-03-12
Publication date: 2020-09-22

Abstract

The invention provides a method for determining the NPVF gene of peripheral blood or the NPVF protein coded by the NPVF gene to diagnose age-related macular degeneration. Specifically, the invention provides an NPVF gene or an NPVF protein coded by the NPVF gene, which is used for preparing a reagent or a kit, wherein the reagent or the kit is used for evaluating the risk of a subject to be detected to suffer from age-related macular degeneration or diagnosing the age-related macular degeneration. The invention diagnoses age-related macular degeneration by detecting the expression of NPVF gene in blood or serum, and has the advantages of high operation feasibility, convenient sampling and high accuracy.

Description

Method for diagnosing wet age-related macular degeneration by measuring NPVF protein in peripheral blood

Technical Field

The invention relates to the field of biomedicine, in particular to a serological diagnosis method of age-related macular degeneration of a human. The invention also relates to the identification and characterization of the serological diagnostic method.

Background

Age-related macular degeneration (AMD) is one of the leading causes of blindness in patients over the age of 55, which affects approximately 30 million people by the year 2040. Risk factors for the onset of AMD include age, smoking, obesity, etc., and its onset is related to various factors such as genetic inheritance and environment. Wet AMD (wet-AMD, wAMD) can cause sub-macular fluid and lipid leakage, fibrous scarring due to Choroidal Neovascularization (CNV), and ultimately severe visual impairment. Early diagnosis of wAMD is particularly important for effective treatment in time, mainly by intravitreal injection of anti-Vascular Endothelial Growth Factor (VEGF) drugs, to avoid irreversible vision impairment.

Currently, diagnosis of AMD relies heavily on imaging examinations, such as: optical Coherence Tomography (OCT), blood flow OCT (octa), Fundus Fluorography (FFA), indocyanine green angiography (ICGA), and the like. OCT and OCTA are non-invasive tests, and can quickly and effectively provide high-definition images of fundus retina, choroid and even sclera; in FA and ICGA examinations, fluorescein sodium or indocyanine green contrast agent is injected into the vein of a patient and is commonly used for detecting the blood circulation of the retina and choroid of the fundus oculi of the patient. However, the typical pathological changes of the ocular fundus occur because the AMD detected by the imaging is often damaged in visual function, and the treatment effect of the patient is limited. Therefore, there is an urgent need to find other diagnostic means for AMD, so as to prevent and intervene in time for the high risk group who finds AMD at an early stage.

Therefore, there is a strong need in the art to develop an accurate and early diagnosis of AMD.

Disclosure of Invention

The invention aims to provide a method for accurately and early diagnosing AMD.

Another object of the present invention is to provide a method for diagnosing age-related macular degeneration by measuring the NPVF gene in the peripheral blood or the NPVF protein encoded by the gene.

It is another object of the invention to provide identification and characterization methods comprising such serological assays.

In a first aspect of the invention, the use of an NPVF gene or its encoded NPVF protein is provided for preparing a detection reagent or a kit for assessing the risk of a subject for developing age-related macular degeneration or diagnosing age-related macular degeneration.

In another preferred embodiment, the detection reagent comprises a primer, a probe, and/or a chip, preferably a primer or primer pair, a probe, and/or a chip (e.g., a nucleic acid chip) for specifically amplifying NPVF mRNA or NPVF cDNA.

In another preferred example, the detection reagent comprises a detection reagent for detecting the expression level (expression amount) of the NPVF gene or the NPVF protein encoded by the same.

In another preferred example, the detection reagent is a detection reagent for an ex vivo sample.

In another preferred embodiment, the ex vivo sample comprises: a blood sample, a serum sample, a tissue sample, a bodily fluid sample, or a combination thereof.

In another preferred example, the ex vivo sample is a peripheral blood serum sample.

In another preferred embodiment, the detection reagent comprises a reagent for detecting the expression level of NPVF gene or its encoded NPVF protein in a peripheral blood serum sample.

In another preferred embodiment, the kit is a peripheral blood serum detection kit.

In another preferred embodiment, the kit is used for detecting a peripheral blood serum sample.

In another preferred example, the NPVF gene or the encoded NPVF protein thereof is derived from a peripheral blood serum sample.

In another preferred embodiment, the kit contains one or more reagents selected from the group consisting of:

(A) a specific primer pair for amplifying the NPVF gene;

(B) enzyme for amplifying NPVF gene, buffer solution;

(C) a chip for PCR amplification;

(D) primers for gene sequencing.

In another preferred embodiment, the sequence of the specific primer pair for amplifying the NPVF gene is shown as SEQ ID NO. 1 and SEQ ID NO. 2, or as shown as SEQ ID NO. 3 and SEQ ID NO. 4, or as shown as SEQ ID NO. 5 and SEQ ID NO. 6, or as shown as SEQ ID NO. 7 and SEQ ID NO. 8.

In another preferred embodiment, the sequence of the primer for gene sequencing is shown as SEQ ID NO. 7 and SEQ ID NO. 8, or as shown as SEQ ID NO. 9 and SEQ ID NO. 10.

In another preferred embodiment, the kit comprises a reagent selected from the group consisting of: antibodies, protein chips or probes for NPVF proteins.

In another preferred embodiment, the kit comprises: an Elisa-related reagent for detecting NPVF protein;

in another preferred embodiment, the kit further comprises a label or instructions for use in assessing the risk of or diagnosing age-related macular degeneration in a subject.

In another preferred embodiment, the kit further comprises a label or a specification, wherein the label or the specification describes a detection method, and the detection method comprises the following steps:

(1) providing a test sample from a subject, said test sample selected from the group consisting of: a blood sample, a serum sample, or a combination thereof;

(2) detecting the expression level of the NPVF gene or the encoded NPVF protein thereof in the test sample; and optionally

(3) The test results in the test sample are compared to a control reference value.

In another preferred embodiment, the following are also noted in the label or the specification:

if the expression level C1 of the NPVF gene or the NPVF protein coded by the NPVF gene in the test sample is obviously higher than the control reference value C0, the risk of the age-related macular degeneration of the test object is higher than that of the general population.

In another preferred example, the control reference value C0 is the expression level of NPVF gene or its encoded NPVF protein in the same sample of normal population (healthy population).

In another preferred embodiment, the "significantly higher" means that the ratio (beta value) of C1/C0 is ≧ 0.5, preferably ≧ 0.2, more preferably ≧ 0.05.

In another preferred embodiment, the evaluating the risk of the subject for age-related macular degeneration includes:

(a) whether the subject suffers from age-related macular degeneration; and/or

(b) Probability of developing age-related macular degeneration.

In another preferred embodiment, said assessing the risk of developing age-related macular degeneration in a subject comprises early screening for age-related macular degeneration.

In another preferred embodiment, the age-related macular degeneration (AMD) comprises wet age-related macular degeneration (wetAMD).

In another preferred embodiment, the diagnosis comprises an early diagnosis.

In another preferred example, the diagnosis is an auxiliary diagnosis.

In another preferred embodiment, the diagnosis is a serological diagnosis.

In another preferred embodiment, the subject of said detection comprises a human or non-human mammal.

In another preferred embodiment, the non-human mammal comprises: rodents (e.g., rats, mice), primates (e.g., monkeys).

In a second aspect of the invention, there is provided a kit comprising detection reagents for detecting the expression level of an NPVF gene or an NPVF protein encoded thereby.

In another preferred embodiment, the kit is a serum detection kit.

In another preferred embodiment, the detection reagent comprises a reagent for detecting the NPVF gene or its encoded NPVF protein (e.g. expression level) in a peripheral blood serum sample.

In another preferred embodiment, the kit is used for evaluating the risk of the subject to be diagnosed for age-related macular degeneration or diagnosing age-related macular degeneration.

(a) whether the subject suffers from age-related macular degeneration; and/or

(b) Probability of developing age-related macular degeneration.

In a third aspect of the present invention, there is provided a method for assessing the risk of developing age-related macular degeneration or diagnosing age-related macular degeneration in a subject, the method comprising the steps of:

In another preferred example, if the expression level C1 of the NPVF gene or the NPVF protein encoded by the NPVF gene in the test sample is significantly higher than the control reference value C0, it indicates that the risk of the age-related macular degeneration in the test subject is higher than that in the general population.

(a) whether the subject suffers from age-related macular degeneration; and/or

(b) Probability of developing age-related macular degeneration.

In another preferred embodiment, the diagnosis comprises an early diagnosis.

In another preferred example, the diagnosis is an auxiliary diagnosis.

In another preferred embodiment, the diagnosis is a serological diagnosis.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In another preferred embodiment, the test subject includes human and non-human mammals.

In another preferred embodiment, the assay is an in vitro assay.

In a fourth aspect of the present invention, there is provided a method of screening a candidate compound for the treatment of age-related macular degeneration, comprising the steps of:

(1) in a test group, administering a test compound to a sample derived from a subject to be tested, and detecting the expression level of the NPVF gene or its encoded NPVF protein in the sample derived from the subject in the test group, V1; in the control group, a blank control (comprising a solvent) is applied to a sample derived from the object to be tested, and the expression level V2 of the NPVF gene or the NPVF protein coded by the NPVF gene in the sample derived from the object to be tested in the control group is detected;

(2) comparing the level V1 and the level V2 detected in the previous step to determine whether the test compound is a candidate compound for reducing the risk of developing age-related macular degeneration.

In another preferred embodiment, the candidate compound is a candidate compound that reduces the risk of developing age-related macular degeneration.

In another preferred example, the sample is an ex vivo sample.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

The following drawings are included to illustrate specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.

Figure 1 shows the results of NPVF gene expression in the study tissue samples control group versus AMD group (, p < 0.01).

FIG. 2 shows the results of NPVF gene expression in the study tissue sample control group and pre-AMD/dry-AMD/wet-AMD group (. about. about.p. < 0.01).

FIG. 3 shows statistics among the NPVF gene expression groups in the tissue samples of this study.

Figure 4 shows the expression levels of NPVF in serum samples from this study (×, p < 0.001).

Detailed Description

The present inventors have made extensive and intensive studies and, as a result of extensive screening, have unexpectedly found a relationship between the expression of the NPVF gene in peripheral blood and wet age-related macular degeneration for the first time. Specifically, the inventors obtained paired expression data of 83 samples from the gene expression data set of Iowa AMD patient tissues, and performed differential expression gene analysis to find that the NPVF gene differentially upregulated in AMD and wAMD and normal groups. Further by Elisa measurements of peripheral blood serum of wAMD patients and control patients (30vs30), NPVF expression was found to be significantly upregulated in wAMD patients compared to normal. The present invention has been completed based on this finding.

The invention unexpectedly discovers a key gene NPVF which can easily detect AMD in peripheral blood or a coding protein thereof, provides a new diagnosis and typing means for AMD, and lays a foundation for further clarifying the occurrence and development mechanism of the diseases.

Term(s) for

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.

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

As used herein, the term "reference value", "control reference value" or "reference value" refers to a value that is statistically correlated with a particular result when compared to the results of an analysis. In a preferred embodiment, the reference value is determined from a statistical analysis of studies comparing the expression level (expression level) of the NPVF gene or its encoded NPVF protein in a normal population (healthy population) or a normal sample (sample from a healthy population) with known clinical outcomes. Some of these studies are shown in the examples section herein. However, studies from the literature and user experience with the methods disclosed herein can also be used to produce or adjust the reference values. The reference value may also be determined by considering conditions and outcomes particularly relevant to the patient's medical history, genetics, age, and other factors.

In one embodiment of the invention, the reference value refers to a reference value or normal value of a healthy control. It is clear to those skilled in the art that the range of normal values (absolute values) of the expression level (expression amount) of the NPVF gene in a sufficient number of samples can be obtained by a test and calculation method. Therefore, when the expression level (expression amount) of the NPVF gene is detected by a method other than PCR, the absolute values of these expression levels can be directly compared with normal values to evaluate the risk of AMD and diagnose or early diagnose AMD. Statistical methods may also be used in the present invention.

The term "sample" or "specimen" as used herein refers to a material that is specifically associated with a subject from which specific information about the subject can be determined, calculated, or inferred. The sample may be composed in whole or in part of biological material from the subject. The sample may also be a material that has been contacted with the subject in a manner such that the test performed on the sample provides information about the subject. The sample may also be a material that has been contacted with other materials that are not the subject, but that enable the first material to be subsequently tested to determine information about the subject, e.g., the sample may be a probe or scalpel wash. The sample can be a source of biological material other than that contacted with the subject, so long as one of skill in the art is still able to determine information about the subject from the sample.

As used herein, the term "individual" refers to an animal, particularly a mammal, such as a primate, preferably a human.

As used herein, terms such as "a," "an," and "the" refer not only to the singular, but also include the general class that can be used to describe a particular embodiment.

As used herein, the term "expression" includes the production of mRNA from a gene or portion of a gene, and includes the production of protein encoded by an RNA or gene or portion of a gene, as well as the presence of a test substance associated with expression. For example, cDNA, binding of a binding partner (e.g., an antibody) to a gene or other oligonucleotide, protein or protein fragment, and chromogenic moieties of the binding partner are included within the scope of the term "expression". Thus, an increase in the density of half-spots on immunoblots such as western blots is also within the scope of the term "expression" based on biological molecules.

It should be noted that the explanation of the terms provided herein is only for the purpose of better understanding the present invention by those skilled in the art, and is not intended to limit the present invention.

NPVF gene

In the invention, through public database and AMD database modeling, a plurality of wAMD related genes are determined, wherein one of the wAMD related genes is a neuropeptide VF (neuropeptide VF) gene.

This gene is located at the 7p15.3 site and is also referred to as RFRP/C7orf9 gene base. The protein encoded by the gene is precursor protein of a plurality of RF amino-peptides (RFRPs), including RFRP-1 (neuropeptide FF, NPFF), RFRP-2 (neuropeptide SF, NPSF) and RFRP-3 (neuropeptide VF, NPVF). RFRPs are major members of the neuropeptide family, and play a major role in neurotransmitter and neuromodulation. RFRP gene mutation is known to be associated with the onset of dominant cystic macular dystrophy (CYMD), but no report has been found in AMD, and its function in the eye, particularly in the retina, is still unknown at present.

In one embodiment of the invention, NPVF expression in peripheral blood of wAMD patients and control patients (30vs30) was measured by Elisa method, and its phenotype was verified in ARPE19, HUVECs two cell lines by high content imaging/live cell imaging by means of plasmid transfection of over-expression/knock-out of knock-down NPVF gene, etc. to elucidate its role in AMD and its biological function.

In one embodiment of the present invention, the kit for detecting NPVF gene comprises one or more reagents selected from the group consisting of:

(A) a specific primer pair for amplifying the NPVF gene;

(B) enzyme for amplifying NPVF gene, buffer solution;

(C) a chip for PCR amplification;

(D) primers for gene sequencing.

In a preferred embodiment, the sequence of the specific primer pair for amplifying the NPVF gene is shown as SEQ ID NO. 1 and SEQ ID NO. 2, or as shown as SEQ ID NO. 3 and SEQ ID NO. 4, or as shown as SEQ ID NO. 5 and SEQ ID NO. 6, or as shown as SEQ ID NO. 7 and SEQ ID NO. 8.

Table 1 amplification primer pair sequences:

in another preferred embodiment, the sequence of the primer for gene sequencing is shown as SEQ ID NO. 9 and SEQ ID NO. 10, or as shown as SEQ ID NO. 11 and SEQ ID NO. 12.

Table 2 gene sequencing primer pair sequences:

primer name	Sequence of	SEQ ID NO:
			A129F	5'-TCT GAG CCT AGA GGA TAC C-3'	9
A129R	5'-GAT CTC AGA GGC AGG TTG-3'	10
			A129E1F	5'-ACA TTG GGC TGC ACA TAG-3'	11
A129E3R	5'-TTT TCT TTT CTC CCT AAA GTC-3'	12

Age-related macular degeneration (AMD)

Age-related macular degeneration (AMD) is the leading cause of blindness in patients over the age of 55; by 2040 years, it will affect about 17 billion population, 11 billion of which are asian population; abnormal neovascularization from age-related macular degeneration can cause fluid and lipid leakage under the macula, fibrous scarring, and ultimately severe visual impairment. The onset of AMD is related to genetic inheritance, environment and other factors.

Wet AMD (wet-AMD, wAMD) can cause sub-macular fluid and lipid leakage, fibrous scarring due to Choroidal Neovascularization (CNV), and ultimately severe visual impairment. Early diagnosis of wAMD is particularly important for effective treatment in time, mainly by intravitreal injection of anti-Vascular Endothelial Growth Factor (VEGF) drugs, to avoid irreversible vision impairment.

In the present invention, by performing the analysis of the differentially expressed genes, the NPVF gene was found to be differentially up-regulated in AMD patients group versus normal group, and differentially up-regulated in wAMD group versus normal group. Based on this, the present invention provides a method for diagnosing age-related macular degeneration by measuring the peripheral blood NPVF protein, and provides a method for screening a candidate compound for treating age-related macular degeneration.

The main advantages of the invention include:

(a) the invention discovers a method for diagnosing age-related macular degeneration by measuring the NPVF gene expression of peripheral blood for the first time. The NPVF gene is used as a marker for AMD screening (including early screening) and diagnosis, has the advantages of high sensitivity, high specificity and high accuracy, and has important application value.

(b) The invention diagnoses age-related macular degeneration by detecting the expression of NPVF gene in blood or serum, and has high operation feasibility and convenient sampling.

(c) The methods and kits of the invention are effective in diagnosing (or aiding in the diagnosis of) early stage AMD, dry AMD and wet AMD, as well as in preventing or intervening in therapy.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

The materials and reagents used in the examples were all commercially available products unless otherwise specified.

Example 1: analysis of Gene expression

From the gene expression data set of the tissues of Iowa AMD patients, 837 samples (of which the normal group 42, the pre-AMD group 16, the dry-AMD group 16 and the wet-AMD group 9) were paired (Cy5/Cy3) by redundancy sample screening, and differential gene expression analysis was performed (t-test).

The results are shown in FIGS. 1-3, and FIGS. 1-2 show the gene expression of NPVF in the tissue samples of the study; fig. 3 shows statistical results between the NPVF gene expression groups in the study tissue samples, where the NPVF gene was differentially up-regulated in AMD patients (p.value 0.0095) and wet-AMD (p.value 0.0012) compared to normal groups, whereas there was no statistical difference between pre-AMD, dry-AMD and normal groups.

Example 2: results of Gene expression analysis

The expression level of the protein product NPVF encoded by the NPVF gene in the serum of 30 wAMD patients and 30 age-and sex-matched normal control groups was detected by the Elisa technology, and differential expression analysis was performed (t-test).

The results are shown in FIG. 4. Fig. 4 shows that the peripheral blood NPVF levels in wAMD patients were significantly higher than those in normal controls (p.value ═ 0.0005).

Example 3 preparation and validation of the kit

This example provides a kit for detecting NPVF gene expression. The kit can detect the NPVF gene expression level:

the kit comprises the following main reagents:

(1) an amplification primer: an upstream primer (F) and a downstream primer (R);

(2) the main PCR reagents include Pfu high fidelity enzyme, 10 × PCR Buffer, dNTPMixtur, ddH₂O；

(3) The gene expression level detection main reagent.

The invention has also been tested and validated in a population of samples. The results indicate that the expression level of the NPVF gene can provide useful auxiliary diagnostic information for age-related macular degeneration, particularly for early and/or auxiliary diagnosis.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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Claims

1. Use of the NPVF gene or the NPVF protein coded by the NPVF gene for preparing a detection reagent or a kit, wherein the reagent or the kit is used for evaluating the risk of the subject to be detected to suffer from the age-related macular degeneration or diagnosing the age-related macular degeneration.

2. The use of claim 1, wherein the detection reagent comprises a reagent that detects the expression level of the NPVF gene or its encoded NPVF protein in a peripheral blood serum sample.

3. The use of claim 1, wherein the kit comprises one or more reagents selected from the group consisting of:

(A) a specific primer pair for amplifying the NPVF gene;

(B) enzyme for amplifying NPVF gene, buffer solution;

(C) a chip for PCR amplification;

(D) primers for gene sequencing.

4. The use of claim 1, wherein the kit comprises an agent selected from the group consisting of: antibodies, protein chips or probes for NPVF proteins.

5. The use of claim 1, wherein the age-related macular degeneration (AMD) comprises wet age-related macular degeneration (wetAMD).

6. A kit comprising detection reagents for detecting the expression level of an NPVF gene or an NPVF protein encoded thereby.

7. A method of assessing the risk of developing or diagnosing age-related macular degeneration in a subject, comprising the steps of:

8. The method of claim 7, wherein the method is non-diagnostic and non-therapeutic.

9. The method of claim 7, wherein the assay is an in vitro assay.

10. A method of screening a candidate compound for the treatment of age-related macular degeneration comprising the steps of: