KR102952443B1 - Methylation marker for diagnosing colorectal cancer and uses thereof - Google Patents

Abstract

The invention relates to a methylation marker for diagnosing colorectal cancer and its use, and more specifically, to a methylation marker that specifically shows reduced methylation in the genes of colorectal cancer patients, a combination thereof, and a method for diagnosing lung cancer using the same.
Each methylation marker or combination thereof according to the present invention can be very usefully utilized to accurately diagnose whether colorectal cancer has developed.

Description

Methylation marker for diagnosing colorectal cancer and uses thereof

The invention relates to a methylation marker for diagnosing colorectal cancer and its use, and more specifically, to a methylation marker that specifically shows reduced methylation in the genes of colorectal cancer patients, a combination thereof, and a method for diagnosing lung cancer using the same.

Colorectal cancer (CRC) is a common cancer worldwide. In Korea, it is the second most common cancer in men after stomach, lung, and liver cancer, and in women after breast and stomach cancer. Recently, as dietary habits have become Westernized, its incidence has increased even more sharply. Over the past 10 years, the mortality rate from colorectal cancer has increased by approximately 80% and continues to rise.

The International Agency for Research on Cancer (IARC), under the World Health Organization, investigated 36 types of cancer in 185 countries worldwide. The results showed that the incidence of colorectal cancer was 10.2% of the total, and that 9.2% of cancer deaths were attributed to colorectal cancer. Additionally, according to the 2017 report on the incidence of major cancers in Korea released by the National Cancer Information Center (NCIC, Korea), the incidence of colorectal cancer in Korea was reported to be 12.1%, which is higher than the global average.

Meanwhile, although the specific pathways and mechanisms by which cancer cells develop have not yet been definitively elucidated, the general consensus is that cancer arises when mutations in genes responsible for regulating normal cell proliferation lead to the emergence of cells with uncontrolled proliferation. Consequently, many cancer researchers are increasingly utilizing DNA chips designed for genes suspected of being associated with cancer. By comparing expression levels between cancerous and normal tissues, they are employing genes specifically overexpressed in cancerous tissues as tumor markers for cancer screening. Of course, while such diagnostic methods have limitations in accuracy and can sometimes result in positive findings in the absence of cancer, causing confusion, it remains necessary to approach the issue at the genetic level to fundamentally diagnose and treat cancer.

Numerous diseases are caused by genetic abnormalities, and the most frequent form of genetic abnormality is a change in the genetic code sequence. Such genetic changes are called mutations. If a mutation occurs in a specific gene, the structure and function of the protein encoded by that gene change, abnormalities and cleavages occur, and these mutated proteins cause disease. However, even without a mutation in a specific gene, abnormalities in gene expression can cause disease. A typical example is methylation, in which a methyl group is attached to a cytosine base region of a gene transcription regulatory site, such as a CpG island. This is called an epigenetic change, which is transmitted to progeny cells in a manner similar to mutations and produces the same effects, such as the loss of expression of the corresponding protein. In cancer cells, the expression of tumor suppressor genes is inhibited by the methylation of promoter CpG islands, and this inhibited expression is recognized as one of the important mechanisms for inducing cancer (Robertson, K. and Jones, P., Carcinogen, 21, 461, 2000). In fact, abnormal methylation/demethylation in CpG islands has been reported in various cancer cells, including prostate, colon, uterine, and breast cancers, and it has been revealed that they play an important role in the early stages of cancer formation, so DNA methylation trends are receiving attention as a promising marker for early cancer diagnosis. However, there is a shortage of markers sufficient to diagnose cancer, so there is a need for the continuous development of markers that exhibit cancer-specific DNA methylation trends.

Accordingly, the inventors conducted research to develop DNA methylation markers specific to colorectal cancer and confirmed that four types of genes exhibit a colorectal cancer-specific hypomethylation tendency, and that colorectal cancer can be diagnosed by measuring these methylation levels, thereby completing the present invention.

Accordingly, the objective of the present invention is to provide a composition for diagnosing colorectal cancer comprising a preparation for measuring the methylation level of one or more genes selected from the group consisting of NEK3 (NIMA related kinase 3), KSR1 (Kinase suppressor of ras 1), GALNT17 (Polypeptide N-acetylgalactosaminyltransferase 17) and BOC (BOC cell adhesion associated, oncogene regulated).

Another objective of the present invention is to provide a colorectal cancer diagnostic kit comprising the above composition.

Another objective of the present invention is to provide a method for providing information for the diagnosis of colorectal cancer, comprising the steps of: (a) extracting DNA from a biological sample of a subject; and measuring the methylation level of one or more genes selected from the group consisting of NEK3 (NIMA related kinase 3), KSR1 (Kinase suppressor of ras 1), GALNT17 (Polypeptide N-acetylgalactosaminyltransferase 17) and (b) BOC (BOC cell adhesion associated, oncogene regulated).

To achieve the aforementioned objective of the present invention, the present invention provides a composition for diagnosing colorectal cancer comprising a preparation for measuring the methylation level of one or more genes selected from the group consisting of NEK3 (NIMA related kinase 3), KSR1 (Kinase suppressor of ras 1), GALNT17 (Polypeptide N-acetylgalactosaminyltransferase 17) and BOC (BOC cell adhesion associated, oncogene regulated).

To achieve another objective of the present invention, the present invention provides a colorectal cancer diagnostic kit comprising the above composition.

To achieve another objective of the present invention, the present invention provides a method for providing information for the diagnosis of colorectal cancer, comprising: (a) a step of extracting DNA from a biological sample of a subject; and (b) a step of measuring the methylation level of one or more genes selected from the group consisting of NEK3 (NIMA related kinase 3), KSR1 (Kinase suppressor of ras 1), GALNT17 (Polypeptide N-acetylgalactosaminyltransferase 17) and BOC (BOC cell adhesion associated, oncogene regulated).

The present invention will be described in detail below.

The present invention provides a composition for diagnosing colorectal cancer comprising a preparation for measuring the methylation level of one or more genes selected from the group consisting of NEK3 (NIMA related kinase 3), KSR1 (Kinase suppressor of ras 1), GALNT17 (Polypeptide N-acetylgalactosaminyltransferase 17) and BOC (BOC cell adhesion associated, oncogene regulated).

In the present invention, diagnosis of colorectal cancer refers to confirming the existence or characteristics of a pathological state in a subject, and for the purposes of one aspect of the present invention, diagnosis may mean confirming whether colorectal cancer has developed. A composition, kit, or method according to one aspect of the present invention may be used to delay the onset of or prevent the onset of colorectal cancer in any specific patient with a high risk of developing colorectal cancer through special and appropriate management. Additionally, a composition, kit, or method according to one aspect of the present invention may be used clinically to determine treatment by diagnosing lung cancer early and selecting the most appropriate treatment method.

In the present invention, the nucleotide sequence of the human genome chromosome region is expressed according to the February 2009 Human reference sequence (GRCh37/hg19); however, the specific sequence of the human genome chromosome region may be slightly modified as genome sequence research results are updated, and the representation of the human genome chromosome region in the present invention may differ as a result of such modifications. Therefore, it is evident that even if the representation of the human genome chromosome region expressed according to the February 2009 Human reference sequence (GRCh37/hg19) in the present invention changes differently from the present due to updates in the human reference sequence after the filing date of the present invention, the scope of the present invention extends to the modified human genome chromosome region. Such modifications are matters that anyone with ordinary knowledge in the technical field to which the present invention belongs can easily recognize.

In the present invention, "methylation" refers to the addition of a methyl group to the fifth carbon of a cytosine residue of DNA. In the present invention, the term "methylation" refers to the attachment of a methyl group to a base constituting DNA. Preferably, in the present invention, the presence of methylation refers to whether methylation occurs at the fifth carbon of a cytosine residue of a specific CpG site of a specific gene. When methylation occurs, the binding of transcription factors is hindered as a result, thereby suppressing the expression of the specific gene; conversely, when non-methylation or hypomethylation occurs, the expression of the specific gene increases.

In addition to A, C, G, and T, the genomic DNA of mammalian cells contains a fifth base called 5-methylcytosine (5-mC), in which a methyl group is attached to the fifth carbon of the cytosine ring. Methylation of 5-methylcytosine occurs only at the C of a CG dinucleotide (5'-mCG-3') called CpG, and methylation of CpG inhibits the expression of alu or transposons and repetitive sequences of the genome. Furthermore, because the 5-mC of the CpG is prone to naturally deamination to become thymine (T), CpG is the site where most epigenetic changes frequently occur in mammalian cells.

In the present invention, the "CpG region" or "CpG island" refers to a genomic region in which CpGs are clustered at an exceptionally high frequency, and refers to a region of average length of 0.2 to 3 kb in which the C+G content is 50% or more and the CpG ratio is 3.75% or more.

In CpG, C represents cytosine and G represents guanine, and p signifies the phosphodiester bond between cytosine and guanine.

There are about 45,000 CpG islands in the human genome, most of which are found in promoter regions that regulate gene expression, and in fact, CpG islands are found in the promoters of housekeeping genes, which make up about 50% of human genes.

In the somatic cells of normal humans, the CpG islands of the housekeeping gene promoter regions are unmethylated, while genes that are not expressed during development, such as imprinted genes and genes on the X chromosome in an inactive state, are methylated.

In the present invention, if the methylation level within the CpG region of at least one gene selected from the group consisting of NEK3, KSR1, GALNT17, and BOC is relatively hypomethylated compared to a normal control group, it can be diagnosed that colorectal cancer has developed or is highly likely to develop. Specifically, by measuring the methylation levels of one, two, three, or four of the four genes and combining the results, it is possible to predict whether colorectal cancer has developed or the likelihood of developing it.

In the present invention, the CpG region of the gene refers to a CpG region existing on the DNA of the gene. The DNA of the gene is a concept that includes all of a series of constituent units that are necessary for the expression of the gene and are operably connected to one another, such as, for example, a promoter region, a protein-coding region (open reading frame, ORF), an intron, an exon, a 5'-UTR, and a terminator region. Accordingly, the CpG region of each gene may exist in the promoter region, protein-coding region (open reading frame, ORF), intron, exon, 5'-UTR, and terminator region of the corresponding gene.

Preferably, in the present invention, the methylation of the NEK3 gene may be characterized as being at the 52733728th base of chromosome 13, the methylation of the KSR1 gene as being at the 25783763rd base of chromosome 17, the methylation of the GALNT17 gene as being at the 70061252nd base of chromosome 7, and the methylation of the BOC gene as being at the 112930505th base of chromosome 3.

In the present invention, a preparation for measuring the methylation level of the gene may include a compound that modifies a non-methylated cytosine base, a methylation-sensitive restriction enzyme, a primer capable of amplifying a fragment containing a methylated base, a probe capable of hybridizing to a fragment containing a methylated base, a methylation-specific binding protein capable of binding to a methylated base, a methylation-specific binding antibody or aptamer, a methylation-sensitive restriction endonuclase, a sequencing primer, a sequencing biosynthesis primer, and a sequencing bioligation primer.

The compound that modifies the above-mentioned unmethylated cytosine base may be bisulfite or a salt thereof, but is not limited thereto, and preferably may be sodium bisulfite. A method of detecting whether a CpG site is methylated by modifying an unmethylated cytosine residue using such bisulfite is widely known in the art.

In addition, the above-mentioned methylation-sensitive restriction enzyme is a restriction enzyme capable of specifically detecting methylation of CpG sites, and may be a restriction enzyme containing CG as its recognition site. Examples include, but are not limited to, SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI, NotI, etc. Depending on the methylation or non-methylation at the C of the restriction enzyme recognition site, the cleavage by the restriction enzyme varies, and this can be detected through PCR or Southern Blot analysis. Other methylation-sensitive restriction enzymes other than the above-mentioned restriction enzyme are well known in the industry.

An exemplary method for measuring the methylation level in each of the above genes according to the present invention can be measured by obtaining DNA from a biological sample of a subject, treating the obtained DNA with a compound that modifies unmethylated cytosine bases or a methylation-sensitive restriction enzyme, amplifying the treated DNA by PCR using primers, and confirming the presence or absence of the amplified product.

Accordingly, the formulation of the present invention may include a primer specific to the methylated allele sequence of each gene and a primer specific to the unmethylated allele sequence. In the present invention, the term "primer" refers to a short nucleic acid sequence having a short free 3-terminal hydroxyl group, capable of forming base pairs with a complementary template, and functioning as a starting point for template strand replication. The primer may initiate DNA synthesis in the presence of a reagent for a polymerization reaction (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer solution and temperature. Additionally, the primer may incorporate additional features that do not alter the basic properties of the primer acting as a starting point for DNA synthesis, such as sense and antisense nucleic acids having a sequence of 7 to 50 nucleotides.

The primers of the present invention can preferably be designed according to the sequence of a specific CpG site to be analyzed for methylation status, and may be a primer pair capable of specifically amplifying cytosine that is methylated and not modified by bisulfite, and a primer pair capable of specifically amplifying cytosine that is not methylated and is modified by bisulfite.

In addition to the above preparation, the above composition and kit may additionally include polymerase, agarose, a buffer solution required for electrophoresis, etc.

According to one embodiment of the present invention, the composition may be provided in the form of a “kit.”

In the present invention, the “kit” refers to a set of reagents for performing nucleic acid amplification or methylation level analysis, and the kit may be characterized as being any one selected from the group consisting of RT-PCR kits, microarray chip kits, DNA kits, and protein chip kits, but is not limited thereto.

The present invention also comprises (a) a step of extracting DNA from a biological sample of a subject; and

(b) A method for providing information for the diagnosis of colorectal cancer, comprising the step of measuring the methylation level of one or more genes selected from the group consisting of NEK3 (NIMA related kinase 3), KSR1 (Kinase suppressor of ras 1), GALNT17 (Polypeptide N-acetylgalactosaminyltransferase 17) and BOC (BOC cell adhesion associated, oncogene regulated).

In the present invention, the term "biological sample" includes, but is not limited to, samples such as tissues, cells, blood (including whole blood, serum, and plasma), and body fluids (saliva, sputum, or urine). Preferably, it may be a blood or body fluid sample.

First, to obtain DNA from a subject and measure the methylation level, DNA can be obtained using the phenol/chloroform extraction method, SDS extraction method, CTAB separation method, or commercially available DNA extraction kit commonly used in the industry, but is not limited thereto.

The step of measuring the methylation level of the gene in step (b) above can be performed by a method selected from the group consisting of PCR, methylation specific PCR, real-time methylation specific PCR, MethyLight PCR, MethyLight digital PCR, EpiTYPER, PCR using methylated DNA-specific binding protein, quantitative PCR, DNA chip, pyrosequencing, bisulfite sequencing, Southern blot, RLGS, SNuPE, CpG island microarray, single-nucleotide primer extension, COBRA (a combined bisulfite-restriction analysis), MIRA (methylated-CpG island recovery assay), mass spectroscopy, and next-generation sequencing.

In one embodiment of the present invention, the step of measuring the methylation level of the gene in step (b) may be performed using a compound that modifies non-methylated cytosine bases or a methylation-sensitive restriction enzyme, a primer specific to the methylated sequence of the gene CpG site, and a primer specific to the non-methylated sequence.

More specifically, the step of treating DNA in a obtained sample with a compound that modifies non-methylated cytosine bases or a methylation-sensitive restriction enzyme; and

The above-mentioned processed DNA can be measured by selecting one or more methods from the group consisting of methylation-specific polymerase chain reaction, real-time methylation-specific polymerase chain reaction, PCR using methylation DNA-specific binding proteins, quantitative PCR, pyrosequencing, and bisulfite sequencing using primers capable of amplifying methylation sites of gene CpG regions.

In the above, the compound that modifies the unmethylated cytosine base may be a bisulfite, preferably a sodium bisulfite. A method of detecting gene methylation by modifying unmethylated cytosine residues using such a bisulfite is widely known in the industry.

In addition, the above-mentioned methylation-sensitive restriction enzyme is a restriction enzyme capable of specifically detecting methylation of a specific CpG site as described above, and may be a restriction enzyme containing CG as a recognition site of the restriction enzyme, such as SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI, NotI, etc., but is not limited thereto.

As described above, the primers can be preferably designed according to the sequence of a specific CpG site to be analyzed for methylation, and may be a primer pair capable of specifically amplifying cytosine that is methylated and not modified by bisulfite, and a primer pair capable of specifically amplifying cytosine that is not methylated and is modified by bisulfite.

The measurement of the methylation level can be performed by methods known in the art, for example, by performing electrophoresis and determining whether a band at a desired location is detected. For example, when a compound that modifies non-methylated cytosine residues is used, the degree of methylation can be determined based on the presence or absence of PCR products amplified by two types of primer pairs: a primer pair capable of specifically amplifying cytosine that is methylated and not modified by bisulfite, and a primer pair capable of specifically amplifying cytosine that is not methylated and is modified by bisulfite. Preferably, the presence of methylation can be determined by using a bisulfite genome sequencing method (e.g., next-generation sequencing), which involves treating sample genomic DNA with bisulfite, amplifying the CpG region of the corresponding gene by PCR, and analyzing the nucleotide sequence of the amplified region.

In addition, even when using restriction enzymes, the presence of methylation can be determined by methods known in the art, for example, when a PCR result appears in mock DNA, if a PCR result appears in DNA treated with restriction enzymes, it is determined that the gene is methylated, and if no PCR result appears in DNA treated with restriction enzymes, it is determined that the gene is non-methylated; this is obvious to those skilled in the art. In the above, mock DNA refers to sample DNA that has been isolated from a sample and has not undergone any treatment.

The information providing method of the present invention may additionally include a step of comparing the methylation level of the gene of the subject with the methylation level of a control group, and may be characterized by determining that the subject has colorectal cancer if, as a result of the comparison, the methylation level of the gene of the subject is reduced compared to the control group.

Meanwhile, the method of the present invention may additionally include a step of associating the methylation level of each gene or a combination thereof with the determination of whether colorectal cancer develops.

That is, since the methylation level of each of the above genes varies depending on the patient's condition, the quantitative analysis level is not easily used to determine whether depression has developed based on only the fragmentary quantitative analysis level of the above proteins. Therefore, by analyzing the quantitative analysis levels of each of the above proteins in combination, it can be used to determine whether depression has developed.

As an example of a method for analyzing by combining the results of quantitative analysis of each of the above proteins, a method for determining whether depression has developed can be used by using the quantitative analysis levels of each protein measured in a serum sample, either individually or in combination.

As an example of a method for determining colorectal cancer by combining the results of the analysis of the methylation levels of the above genes, conventional statistical analysis methods may be used. At this time, the statistical analysis methods that can be used are not particularly limited thereto, but as examples, linear or non-linear regression analysis methods; forward or non-linear classification analysis methods; ANOVA; neural network analysis methods; genetic analysis methods; support vector machine analysis methods; hierarchical analysis or clustering analysis methods; hierarchical algorithms using decision trees, or kernel principal components analysis methods; Markov Blanket analysis methods; recursive feature elimination or entropy-based recursive feature elimination analysis methods; forward floating search or backward floating search analysis methods, etc., may be used alone or in combination.

In addition, the combination of the analysis results of the methylation levels of each of the above genes may also be performed using a computer algorithm capable of automatically performing the above statistical method.

Each methylation marker or combination thereof according to the present invention can be very usefully utilized to accurately diagnose whether colorectal cancer has developed.

Figure 1 is the result of analyzing one or more combinations of two types of four types of colorectal cancer-specific methylation markers selected in an embodiment of the present invention using a deep neural network (1a) and logistic regression analysis (1b) to show the colorectal cancer diagnostic ability as an AUC value.
Figure 2 is a heatmap showing the degree of hypomethylation of four types of colorectal cancer-specific methylation markers selected in an embodiment of the present invention compared to a normal sample group.

The present invention will be explained in detail below by the following examples. However, the following examples are merely illustrative of the present invention and do not limit the present invention.

To select cancer-specific markers, an analysis was conducted by comparing the colorectal cancer sample group with the normal sample group.

The values represented by each marker were normalized using the mean of the InterQuartile Range (IQR) and standardized using Z-scores to reduce potential differences between sequencing. Subsequently, a t-test was performed between the cancer group and the normal group for each marker to calculate the p-value. Among these, only hypomethylation-related markers were selected, and the top four markers were chosen and are listed in order in Table 1 below (Table 1).

Subsequently, the performance of marker combinations was evaluated using a Deep Neural Network and a Logistic Regression model among machine learning models.

As can be seen in Figure 1, the combination of the selected colorectal cancer markers was evaluated using machine learning models (deep neural network (DNN) and regression analysis models), and it was confirmed that the highest diagnostic ability was achieved when four markers were combined compared to when only one marker was used.

Figure 2 shows how hypomethylated the selected markers are compared to the normal sample group. It is represented as a heatmap with high values in red and low values in blue.

Each methylation marker or combination thereof according to the present invention can be very usefully utilized to accurately diagnose whether colorectal cancer has developed, and thus has very high industrial applicability.

Claims (14)

A composition for diagnosing colorectal cancer comprising a preparation for measuring the methylation levels of NEK3 (NIMA related kinase 3), KSR1 (Kinase suppressor of ras 1), GALNT17 (Polypeptide N-acetylgalactosaminyltransferase 17) and BOC (BOC cell adhesion associated, oncogene regulated) genes.
A composition for diagnosing colorectal cancer according to claim 1, wherein the agent capable of measuring the methylation level is selected from the group consisting of a compound that modifies a non-methylated cytosine base, a methylation-sensitive restriction enzyme, a primer capable of amplifying a fragment containing a methylated base, a probe capable of hybridizing to a fragment containing a methylated base, a methylation-specific binding protein capable of binding to a methylated base, a methylation-specific binding antibody or aptamer, a methylation-sensitive restriction endonuclase, a sequencing primer, a sequencing biosynthesis primer, and a sequencing bioligation primer.
A composition for diagnosing colorectal cancer according to claim 1, characterized in that the methylation of the NEK3 gene is at the 52733728th base of chromosome 13, the methylation of the KSR1 gene is at the 25783763rd base of chromosome 17, the methylation of the GALNT17 gene is at the 70061252nd base of chromosome 7, and the methylation of the BOC gene is at the 112930505th base of chromosome 3.
A colorectal cancer diagnostic kit comprising the composition of any one of claims 1 to 3.
A kit according to claim 4, characterized in that the kit is selected from the group consisting of an RT-PCR kit, a microarray chip kit, a DNA kit, and a protein chip kit.
(a) a step of extracting DNA from a biological sample of a subject; and
(b) a method for providing information for the diagnosis of colorectal cancer, comprising the step of measuring the methylation levels of NEK3 (NIMA related kinase 3), KSR1 (Kinase suppressor of ras 1), GALNT17 (Polypeptide N-acetylgalactosaminyltransferase 17) and BOC (BOC cell adhesion associated, oncogene regulated) genes.
In claim 6, the information providing method is characterized by additionally including the step of comparing the methylation level of the genes of the subject with the methylation level of the control group.
A method of providing information according to claim 7, characterized by determining colorectal cancer when the methylation level of the genes of the subject is reduced compared to the control group.
A method for providing information according to claim 6, characterized in that the biological sample is one or more selected from the group consisting of a subject's tissue, cells, blood, plasma, urine, and body fluids.
In claim 6, the step of measuring the methylation level of the above genes is performed using a method selected from the group consisting of PCR, methylation specific PCR, real-time methylation specific PCR, MethyLight PCR, MethyLight digital PCR, EpiTYPER, PCR using a methylated DNA-specific binding protein, quantitative PCR, DNA chip, pyrosequencing, bisulfite sequencing, Southern blot, RLGS, SNuPE, CpG island microarray, single-nucleotide primer extension, COBRA (a combined bisulfite-restriction analysis), MIRA (methylated-CpG island recovery assay), mass spectroscopy, and next-generation sequencing.
A method for providing information according to claim 6, characterized in that the methylation of the NEK3 gene is at the 52733728th base of chromosome 13, the methylation of the KSR1 gene is at the 25783763rd base of chromosome 17, the methylation of the GALNT17 gene is at the 70061252nd base of chromosome 7, and the methylation of the BOC gene is at the 112930505th base of chromosome 3.
A method for providing information according to claim 6, characterized by additionally including a step of associating the methylation levels of the measured genes with the determination of whether colorectal cancer has developed.
In claim 12, the method of providing information is characterized in that the associated step is performed by combining the results of the methylation level analysis of the genes.
In claim 13, the combination of the above results is characterized by being performed using an analysis method selected from a group consisting of: a linear or non-linear regression analysis method; a leading or non-linear classification analysis method; ANOVA; a neural network analysis method (Deep neural network); a genetic analysis method; a support vector machine analysis method; a hierarchical analysis or clustering analysis method; a hierarchical algorithm using a decision tree, or a kernel principal components analysis method; a Markov Blanket analysis method; a recursive feature elimination or entropy-based recursive feature elimination analysis method; a forward floating search or backward floating search analysis method; and a combination thereof.