WO2018228028A1 - Gene marker for use in detecting liver cancer and use thereof - Google Patents

Abstract

Disclosed in the present invention is a genetic marker for use in detecting liver cancer, comprising one or more genes selected from among: FAT atypical cadherin 1 (FAT1), estrogen-related receptor gamma (ESRRG), gamma aminobutyric acid class A receptor beta-3 subunit (GABRB3), TNF receptor superfamily member 11b (TNFRSF11B), receptor interacting serine/threonine kinase 4 (RIPK4), rearranged L-myc fusion protein (RLF), solute carrier family 13 member 5 (SLC13A5), cytochrome P450 oxidoreductase (POR) and Deltex E3 ubiquitin ligase (DTX1).

Description

Gene marker for detecting liver cancer and use thereof Technical field

The present invention relates to the field of clinical molecular diagnosis of liver cancer. In particular, the present invention relates to a method and kit for detecting the presence of liver cancer by detecting the 5-hydroxymethylcytosine content of a liver cancer gene marker by high-throughput sequencing.

Background technique

Liver cancer is one of the most common global malignancies. According to the statistics of the World Health Organization in 2008, there are 748,300 new cases each year and 695,900 deaths, of which more than 50% occur in China. In primary liver cancer, 70%-85% is hepatocellular carcinoma (HCC). At present, the 5-year survival rate of liver cancer is only 3%-5%, because most patients are in the middle and advanced stage of treatment, and they have lost the best treatment time. Therefore, early detection and early diagnosis and early treatment are the key to improve the quality of life and prolong survival.

At present, the detection of liver cancer mainly through imaging, tissue biopsy, serological testing and the like. However, imaging is susceptible to operator experience and relies on equipment, which is expensive, especially when medical resources are limited, and its accuracy is difficult to guarantee, and it is difficult to widely and routinely apply. Tissue biopsy is the gold standard for clinically diagnosed liver cancer, but there are many limitations in tissue biopsy, such as the difficulty of surgical sampling, or the inconvenience of puncture in some cancer sites, and the puncture itself will bring certain clinical risks, repeated puncture Screening can cause great pain to patients. The most widely used serological test is the detection of alpha fetoprotein (AFP), but the sensitivity and specificity of AFP for early liver cancer are not high, for example, in some patients with chronic liver disease other than liver cancer, such as many chronic hepatitis and cirrhosis In patients, serum AFP also increased.

In the early screening of liver cancer, the most difficult to screen for small liver cancer. Small liver cancer is also called subclinical liver cancer or early liver cancer. There are no obvious symptoms and signs of liver cancer in clinical practice. Generally speaking, the maximum diameter of a single cancer nodule in hepatocellular carcinoma is no more than 3 cm or the sum of the diameters of two cancer nodules is less than 3 cm. Liver cancer. The standard of small liver cancer in China is that the maximum diameter of a single cancer nodule is no more than 3 cm; the number of multiple cancer nodules is not more than two, and the sum of the maximum diameters should be less than 3 cm. The surgical resection rate of small liver cancer is as high as 93.6%, the prognosis is better, and the survival rate is higher. Therefore, early screening of small liver cancer has important clinical significance. At present, screening for small liver cancer is mainly performed by ultrasound examination, imaging diagnosis and serum alpha-fetoprotein detection. However, as mentioned above, the accuracy and specificity of these traditional methods for the diagnosis of small liver cancer are not high.

Therefore, the search for new markers of liver cancer, especially for early warning and early diagnosis, is of great significance for improving the diagnosis rate of early liver cancer, achieving early intervention and reducing the mortality of liver cancer.

Summary of the invention

The inventors unexpectedly discovered a number of highly informative samples by performing high-throughput sequencing of normal samples and liver cancer samples and analyzing the 5-hydroxymethylcytosine (5-hmC) content of each gene. It can be used to detect genetic markers of liver cancer.

Accordingly, a first aspect of the invention relates to a genetic marker for detecting liver cancer comprising one or more genes selected from the group consisting of FAT atypical cadherin 1 (FAT1) and estrogen-related receptor gamma (ESRRG) Γ-aminobutyric acid receptor A β3 subunit (GABRB3), TNF receptor superfamily member 11b (TNFRSF11B), receptor interaction serine/threonine kinase 4 (RIPK4), rearranged L-myc fusion protein (RLF), solute carrier family 13 member 5 (SLC13A5), cytochrome P450 oxidoreductase (POR) and Deltex E3 ubiquitin ligase (DTX1). Preferably, the genetic marker comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or at least nine genes selected from the group consisting of FAT1, ESRRG, GABRB3, TNFRSF11B, RIPK4, RLF, SLC13A5, POR and DTX1. More preferably, the genetic markers include FAT1, ESRRG, GABRB3, TNFRSF11B, RIPK4, RLF, SLC13A5, POR and DTX1.

The invention also relates to the use of the above genetic markers for detecting liver cancer.

A second aspect of the invention relates to a method for detecting liver cancer comprising the steps of:

(a) determining the content of 5-hmC of the genetic marker of the present invention in a normal sample and a subject sample;

(b) normalizing the 5-hmC content of the corresponding gene marker in the subject sample using the 5-hmC content of the gene marker in the normal sample as a reference;

(c) mathematically correlating the 5-hmC content of the standardized genetic marker and obtaining a score;

(d) Obtaining test results based on the score.

In one embodiment, the sample is a free DNA fragment in a subject or normal human body fluid, or is derived from intact genomic DNA in organelles, cells, and tissues. Among them, body fluids are blood, urine, sweat, sputum, feces, cerebrospinal fluid, ascites, pleural effusion, bile, pancreatic juice, and the like.

In one embodiment, the 5-hmC content of the genetic markers of the invention can be determined by any method known to those skilled in the art, including, for example, but not limited to, glucosylation, restriction endonucleases Method, chemical labeling method, precipitation method combined with high-throughput sequencing method, single molecule real-time sequencing method (SMRT), oxidized bisulfite sequencing method (OxBS-Seq), and the like. The principle of the glucosylation method is to transfer glucose to the hydroxyl group in the presence of glucose donor substrate uridine nucleoside diphosphate glucose (UDP-Glu) using T4 phage β-glucose transferase (β-GT). β-Glucosyl-5-hydroxymethylcytosine (5-ghmC) was produced. Isotopically labeled substrates can also be used for quantification. The restriction endonuclease method and the chemical labeling method were further developed on the basis of the glucosylation method. The principle of the restriction endonuclease method is that the glucosylation reaction changes the enzymatic cleavage properties of some restriction enzymes. The methylation-dependent restriction enzymes MspI and HpaII recognize the same sequence (CCGG), but their sensitivity to methylation status is different: MspI recognizes and cleaves 5-methylcytosine (5-mC) And 5-hmC, but not 5-ghmC; HpaII only cleaves completely unmodified sites, and any modification on cytosine (5-mC, 5-hmC, 5-ghmC) blocks cleavage. If the CpG site contains 5-hmC, the band can be detected after glycosylation and enzymatic hydrolysis, and there is no band in the unglycosylated control reaction; qPCR can also be used for quantitative analysis. In addition, other restriction enzymes also have a hindrance to 5-ghmC digestion, and can be applied to 5-hmC detection (e.g., GmrSD, MspJI, PvuRts1I, TaqI, etc.). The principle of the chemical labeling method is to chemically modify the glucose on the substrate of the enzyme reaction into UDP-6-N3-glucose, and transfer 6-N3-glucose to the position of the hydroxymethyl group to form N3-5ghmC. Subsequently, a single molecule of biotin was added to each 5-hmC by click chemistry, combined with next-generation high-throughput DNA sequencing technology or single-molecule sequencing technology to analyze the distribution of 5-hmC in genomic DNA. The precipitation method is to modify 5-hmC in a special way and then specifically capture it from genomic DNA and perform sequencing analysis. Oxidized bisulfite sequencing is the first method to quantify 5-hmC with single base resolution. Firstly, 5-hmC is subjected to KRuO4 oxidation treatment to produce 5-formylcytosine (5fC), and then heavy Sulfite sequencing. In this process, 5-hmC is first oxidized to 5fC, and then deaminated to form U. Usually, quantitative detection of 5-hmC is performed simultaneously using a variety of detection methods.

In one embodiment of the invention, the 5-hmC content of the genetic markers of the invention is determined using chemical labeling in conjunction with high throughput sequencing. In this particular embodiment, the method of determining the 5-hmC content of a genetic marker of the present invention comprises the steps of: fragmenting DNA from a sample of a liver cancer patient and a normal human; repairing the fragmented DNA end and The ends are filled; the end-filled DNA is ligated to the sequencing linker to obtain a ligation product; the 5-hydroxymethylcytosine in the ligation product is labeled by a labeling reaction; the DNA containing the 5-hydroxymethylcytosine tag is enriched The fragment is obtained, and the enriched product is obtained; the enriched product is subjected to PCR amplification to obtain a sequencing library; the sequencing library is subjected to high-throughput sequencing to obtain a sequencing result; and the content of 5-hydroxymethylcytosine is determined according to the sequencing result. Wherein the labeling reaction comprises: i) covalent attachment of a sugar having a modifying group to a methylol group of 5-hydroxymethylcytosine using a glycosyltransferase, and ii) direct or indirect attachment of biotin Click on the chemical substrate to react with 5-hydroxymethylcytosine with a modifying group. Wherein step i) and step ii) may be carried out sequentially or simultaneously in one reaction. This labeling method reduces the amount of sample required for sequencing, and the biotin tag on 5-hydroxymethylcytosine allows it to display higher kinetic signals in sequencing, improving the accuracy of nucleotide recognition. In this embodiment, the glycosyltransferase includes, but is not limited to, T4 phage β-glucosyltransferase (β-GT), T4 bacteriophage α-glucosyltransferase (α-GT), and the same Or a similarly active derivative, analog, or recombinase; the saccharide with a modifying group includes, but is not limited to, a saccharide with an azide modification (eg, 6-N3-glucose) or with other chemical modifications a saccharide (e.g., a carbonyl group, a thiol group, a hydroxyl group, a carboxyl group, a carbon-carbon double bond, a carbon-carbon triple bond, a disulfide bond, an amine group, an amide group, a diene, etc.), among which a saccharide modified with azide is preferred. The chemical group for indirectly linking the biotin and the click chemical substrate includes, but is not limited to, a carbonyl group, a thiol group, a hydroxyl group, a carboxyl group, a carbon-carbon double bond, a carbon-carbon triple bond, a disulfide bond, an amine group, Amido group, diene. In this embodiment, the DNA fragment containing the 5-hmC label is preferably enriched by a solid phase material. Specifically, a DNA fragment containing a 5-hydroxymethylcytosine label can be bound to a solid phase material by a solid phase affinity reaction or other specific binding reaction, and then the unbound DNA fragment can be removed by multiple washings. Solid phase materials include, but are not limited to, silicon wafers or other chips with surface modification, such as artificial polymer beads (preferably 1 nm to 100 um in diameter), magnetic beads (preferably 1 nm to 100 um in diameter), agarose beads, etc. (Preferably from 1 nm to 100 um in diameter). The washing liquid used for solid phase enrichment is a buffer well known to those skilled in the art, including but not limited to: containing Tris-HCl, MOPS, HEPES (pH=6.0-10.0, concentration between 1 mM and 1 M), NaCl (0) - 2M) or a buffer such as Tween 20 (0.01% - 5%). In this embodiment, PCR amplification is preferably performed directly on the solid phase to prepare a sequencing library. If necessary, after performing PCR amplification on a solid phase, the amplified product can be recovered and subjected to a second round of PCR amplification to prepare a sequencing library. The second round of PCR amplification can be performed using conventional methods known to those skilled in the art. Optionally, one or more purification steps may be further included in the process of preparing the sequencing library. Any purification kit known or commercially available to those skilled in the art can be used in the present invention. Purification methods include, but are not limited to, gel electrophoresis gel recovery, silica gel membrane spin column method, magnetic bead method, ethanol or isopropanol precipitation method, or a combination thereof. Optionally, the sequencing library is quality checked prior to high throughput sequencing. For example, the library is subjected to fragment size analysis and the concentration of the library is absolutely quantified using the qPCR method. Sequencing libraries that pass quality checks can be used for high throughput sequencing. Then, a certain number (1-96) of libraries containing different barcodes were mixed at the same concentration and sequenced according to the standard on-line method of the second generation sequencer to obtain sequencing results. Various second generation sequencing platforms and related reagents known in the art can be used in the present invention.

In one embodiment of the invention, the sequencing results are preferably aligned with a standard human genome reference sequence, and the sequences in which the gene markers of the invention are aligned are selected, ie, the alignment sites and gene features (eg, groups) are selected. The number of reads of the coincident region of the protein modification site, transcription factor binding site, gene exon intron region, and gene promoter, etc., to represent the level of modification of 5-hmC on the gene, thereby determining 5-hmC The amount on the genetic marker. Preferably, the sequencing results are first cleared of low-quality sequencing sites prior to the alignment, wherein factors that measure the quality of the sequencing sites include, but are not limited to, base quality, reads mass, GC content, repeat sequences, and number of Overrepresented sequences. The various alignment software and analytical methods involved in this step are known in the art.

In one embodiment of the present invention, determining the 5-hmC content of the gene marker means determining the 5-hmC content of the full length of the gene marker or determining the 5-hmC content of a fragment of the gene marker or combination.

According to the present invention, after determining the 5-hmC content of each gene marker, the 5-hmC content of the corresponding gene marker in the sample of the subject is used as a reference with the 5-hmC content of the gene marker in the normal sample. standardization. For example, the 5-hmC content of the same gene marker in the normal sample and the subject sample is X and Y, respectively, and the normalized 5-hmC content of the gene marker in the subject sample is Y/X.

According to the present invention, after the data is normalized, the standardized 5-hmC content of each gene marker is mathematically correlated to obtain a score, thereby obtaining a detection result based on the score. As used herein, "mathematical association" refers to any computational or machine learning method that correlates the 5-hmC content of a genetic marker from a biological sample with the diagnosis of liver cancer. One of ordinary skill in the art understands that different computing methods or tools can be selected to provide the mathematical associations of the present invention, such as elastic network regularization, decision trees, generalized linear models, logistic regression, highest score pairs, neural networks, linear and Quadratic Discriminant Analysis (LQA and QDA), Naive Bayes, Random Forest, and Support Vector Machines.

In one embodiment of the present invention, the specific steps for mathematically correlating the standardized 5-hmC content of each gene marker and obtaining a score are as follows: multiplying the normalized 5-hmC content of each gene marker by a weighting coefficient to obtain the gene The predictor of the marker t; the predictor t of each gene marker is added to obtain a total predictor T; the total predictor T is subjected to Logistic conversion to obtain a score P; if P>0.5, the subject sample suffers Liver cancer; if P ≤ 0.5, the subject sample is normal. The weighting factor described herein refers to the art by the art in consideration of factors that may affect the 5-hmC content (eg, subject area, age, sex, below, smoking history, drinking history, family history, etc.) The coefficients obtained by various advanced statistical analysis methods known to the person.

A third aspect of the present invention relates to a kit for detecting liver cancer using the above gene marker, which comprises a reagent and a specification for measuring a 5-hmC content of the above gene marker. Agents for determining the 5-hmC content of a genetic marker are known to those skilled in the art, such as T4 bacteriophage beta-glucose transferase and isotopic labeling (for glucosylation), restriction enzymes (for restriction) Endonuclease method), glycosyltransferase and biotin (for chemical labeling), reagents for PCR and sequencing, and the like.

The method for detecting liver cancer in the present invention is based on the 5-hmC content on the genetic marker as compared with the prior art, and thus a wider range of DNA sample sources can be used. Therefore, the method for detecting liver cancer in the present invention has the following advantages: (1) safe and non-invasive, and the acceptance of the test is high even in asymptomatic people; (2) the source of DNA is extensive, and there is no detection blind spot in imaging. (3) high accuracy, high sensitivity and specificity for early liver cancer, suitable for early screening of liver cancer; (4) easy to operate, user experience is good, easy to carry out dynamic monitoring of liver cancer recurrence and metastasis. The gene markers of the present invention can be combined with other clinical indicators to provide more accurate judgments for liver cancer screening, diagnosis, treatment and prognosis.

DRAWINGS

Figure 1: Results of differentiation of liver cancer samples and healthy controls using the liver cancer gene markers of the present invention.

Figure 2: Results of differentiation of small liver cancer samples and healthy controls using the liver cancer gene markers of the present invention.

detailed description

The invention will be described in detail below with reference to the drawings, in order to provide a better understanding of the invention. It should be understood that the drawings and the embodiments of the present invention are intended to be illustrative only and not restrictive. The embodiments of the present application and the features of the embodiments may be combined with each other without contradiction.

Example 1. Screening of liver cancer gene markers

(1) Extraction of plasma DNA:

10 ng of plasma DNA was extracted from samples from 20 liver cancer patients and 20 normal subjects, respectively. This step can be carried out using any method and reagent suitable for extracting plasma DNA well known to those skilled in the art.

(2) The plasma DNA was end-filled, suspended in A and ligated to the sequencing linker: prepared containing 50 uL of plasma DNA, 7 uL End Repair & A-Tailing Buffer and 3 uL End Repair & A-Tailing Enzyme mix according to the Kapa Hyper Perp Kit instructions. The reaction mixture (total volume 60 uL) was incubated at 20 ° C for 30 minutes and then at 65 ° C for 30 minutes. The following ligation reaction mixture was placed in a 1.5 mL low adsorption EP tube: 5 uL Nuclease free water, 30 uL Ligation Buffer and 10 uL DNA Ligase. 5 uL of the sequencing adaptor was added to the 45 uL ligation reaction mixture, mixed, heated at 20 °C for 20 minutes, and then kept at 4 °C. The reaction product was purified using Ampure XP beads, and eluted with 20 uL of a buffer containing Tris-HCl (10 mM, pH = 8.0) and EDTA (0.1 mM) to obtain a final DNA-ligated sample.

(3) Labeling 5-hydroxymethylcytosine:

Prepare a total of 26 uL of labeled reaction mixture: azide-modified uridine diphosphate glucose (ie UDP-N3-Glu, final concentration 50 uM), β-GT (final concentration 1 uM), Mg ²⁺ (final concentration) It was 25 mM), HEPES (pH = 8.0, final concentration of 50 mM) and 20 uL of DNA from the above procedure. The mixture was incubated at 37 ° C for 1 hour. The mixture was taken out and purified with Ampure XP beads to obtain purified 20 uL of DNA.

Then, 1 uL of biotin-containing diphenylcyclooctyne (DBCO-Biotin) was added to the above purified 20 uL DNA, and reacted at 37 ° C for 2 hours, followed by purification with Ampure XP beads to obtain a purified labeled product.

(4) Solid phase enrichment of DNA fragments containing labeled 5-hydroxymethylcytosine:

First, the magnetic beads were prepared as follows: 0.5 uL of C1 streptadvin beads (life technology) was taken out and 100 uL of buffer (5 mM Tris, pH = 7.5, 1 M NaCl, 0.02% Tween 20) was added, vortexed for 30 seconds, and then washed with 100 uL. The magnetic beads (5 mM Tris, pH = 7.5, 1 M NaCl, 0.02% Tween 20) were washed 3 times, and finally 25 uL of binding buffer (10 mM Tris, pH = 7.5, 2 M NaCl, 0.04% Tween 20 or other surfactant) was added. well mixed. Then, the purified labeled product obtained in the above procedure was added to the magnetic bead mixture, and mixed for 15 minutes in a rotary mixer to sufficiently bind.

Finally, the magnetic beads were washed 3 times with 100 uL of washing solution (5 mM Tris, pH = 7.5, 1 M NaCl, 0.02% Tween 20), the supernatant was removed by centrifugation, and 23.75 uL of nuclease-free water was added.

(5) PCR amplification:

To the final system of the above procedure, 25 uL of 2 X PCR master mix and 1.25 uL of PCR primers (total volume 50 uL) were added and amplified according to the temperature and conditions of the following PCR reaction cycle:

The amplified product was purified using Ampure XP beads to give a final sequencing library.

(6) High-throughput sequencing after sequencing the sequencing library:

The obtained sequencing library was subjected to concentration determination by qPCR, and the DNA fragment size content in the library was determined using Agilent 2100. The sequencing libraries passed the QC were mixed at the same concentration and sequenced using an Illumina Hiseq 4000.

(7) Determine the 5-hmC content and weighting coefficient of each gene marker

The obtained sequencing results were subjected to preliminary quality control evaluation, and after the low-quality sequencing sites were cleared, the readings that met the sequencing quality standards were compared with the human standard genomic reference sequences using the Bowtie 2 tool. The feature counts and HtSeq-Count tools were then used to count the number of reads to determine the 5-hmC content of each gene marker. At the same time, using high-throughput sequencing results, the factors that may affect the 5-hmC content were used as covariates, and the weighting coefficients of each gene marker were obtained by logistic regression and elastic network regularization. The results are shown in Table 1.

Table 1: Average normalized 5-hmC content and weighting coefficient of liver cancer gene markers of the present invention

As described above, the average normalized 5-hmC content refers to the ratio of the average 5-hmC content of the gene marker in the liver cancer sample to the average 5-hmC content of the same gene marker in the normal sample. As can be seen from Table 1, the 5-hmC content of the liver cancer gene marker of the present invention is significantly different between the normal sample and the liver cancer sample, and the 5-hmC content of the other genetic markers except the RLF is relative to the normal human population. A significant increase.

Example 2. Effectiveness of liver cancer gene markers

This example demonstrates the effectiveness of the liver cancer gene marker of the present invention for detecting liver cancer.

The 5-hmC content of the nine liver cancer gene markers of the present invention in the first batch of 96 samples (50 liver cancers and 46 healthy controls) was determined according to the method of Example 1, and the weighting coefficients of each gene marker were determined. .

Multiplying the normalized 5-hmC content of each gene marker by its corresponding weighting coefficient to obtain the predictive factor t of the gene marker, adding the predictor t of each gene marker to obtain the total predictor T, and then The total predictor T is scored by Logistic conversion according to the following formula:

If P > 0.5, the subject sample has liver cancer; if P < 0.5, the subject sample is normal.

Figure 1 shows the results of distinguishing the batch of samples in accordance with the method of the present invention. As shown in Figure 1, the method of the invention is capable of achieving 90% sensitivity and 91% specificity.

In addition, small liver cancers were also screened using the nine liver cancer gene markers of the present invention. As shown in Fig. 2, in the samples of 42 small liver cancer patients and 42 healthy controls, the screening of small liver cancer using the liver cancer gene marker of the present invention still has a sensitivity of about 83% and a specificity of about 83%.

Claims (10)

1. A genetic marker for detecting liver cancer, comprising one or more genes selected from the group consisting of FAT atypical cadherin 1 (FAT1), estrogen-related receptor gamma (ESRRG), gamma aminobutyric acid class A receptor β3 Subunit (GABRB3), TNF receptor superfamily member 11b (TNFRSF11B), receptor interaction serine/threonine kinase 4 (RIPK4), rearranged L-myc fusion protein (RLF), solute carrier family 13 member 5 (SLC13A5), cytochrome P450 oxidoreductase (POR) and Deltex E3 ubiquitin ligase (DTX1).
2. The genetic marker of claim 1 comprising FAT1, ESRRG, GABRB3, TNFRSF11B, RIPK4, RLF, SLC13A5, POR and DTX1.
3. Use of the gene marker of claim 1 or 2 in a method for detecting liver cancer.
4. A method for detecting liver cancer, comprising the steps of:

   (a) determining the content of 5-hydroxymethylcytosine (5-hmC) of the gene marker according to claim 1 or 2 in the normal sample and the subject sample;

   (b) normalizing the 5-hmC content of the corresponding gene marker in the subject sample using the 5-hmC content of the gene marker in the normal sample as a reference;

   (c) mathematically correlating the 5-hmC content of the genetic marker normalized in step (b) and obtaining a score P;

   (d) A test result is obtained based on the score P, and a score P greater than 0.5 indicates that the subject sample has liver cancer.
5. The method of claim 4, wherein step (a) is to determine the content of 5-hmC over the entire length of the gene marker or a fragment thereof.
6. The method of claim 4, wherein the sample is a free DNA fragment from a normal human or a subject's body fluid, or is derived from intact genomic DNA in organelles, cells, and tissues.
7. The method of claim 6 wherein said body fluid is blood, urine, sweat, sputum, feces, cerebrospinal fluid, ascites, pleural effusion, bile or pancreatic juice.
8. Use of an agent for determining the 5-hmC content of the gene marker of claim 1 or 2 in the preparation of a kit for detecting liver cancer.
9. A kit for detecting liver cancer, comprising:

   (a) an agent for determining a 5-hmC content of the gene marker of claim 1 or 2;

   (b) Instructions.
10. The kit according to claim 9, wherein the 5-hmC content means a content of 5-hmC on the entire length of the gene marker or a fragment thereof.

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