Method for evaluating risk of developing hereditary disease

Abstract

The present invention provides a method for evaluating the risk of developing a hereditary disease in an individual by a variant of uncertain clinical significance (VUS), the method comprising: introducing DNA for a genetic factor for a hereditary disease, including at least one VUS, into a fertilized egg of zebrafish under the control of a promoter of a gene involved in a phenotype; and evaluating the risk of developing the hereditary disease by the VUS, by observing the change in the phenotype in the zebrafish.

Description

Methods for assessing the risk of developing genetic diseases

[0001] The present invention relates to a method for assessing the risk of developing a genetic disease.

[0002] In routine clinical practice, when a genetic disease is suspected, genetic testing involves determining the base sequence of the target gene in the subject. Generally, variants are extracted by comparing the subject's base sequence with a reference sequence, and the pathogenic significance of the variants is interpreted using various criteria. A final report is then prepared. According to the ACMG-AMP guidelines, the pathogenicity of variants is classified into five types: pathogenic, likely pathogenic, variant of uncertain significance (VUS), likely benign, and benign (Non-Patent Document 1).

[0003] Taking cancer as an example, approximately 10% of cancer cases are thought to be hereditary tumors caused by genetic factors, and definitive diagnosis relies on genetic testing of the relevant genes. Traditionally, risk assessment based on medical history and tumor history in family members was the standard approach, followed by genetic testing of genes likely to be causative. However, recent advances in next-generation sequencing (NGS) have led to the practical application of multi-gene panel testing (MGPT), which allows for simultaneous genetic testing of multiple genes. MGPT offers a higher detection rate of pathogenic variants in causative genes compared to traditional single-gene sequential testing, enabling rapid and cost-effective detection of a wide range of disease risk factors. It is likely to become a leading tool for hereditary tumor diagnosis in the future. However, MGPT faces challenges, including the inclusion of gene variants for which cancer risk and medical management evidence are uncertain, as well as a high frequency of variants classified as variants of uncertain significance (VUS). For example, variants in the BRCA1 or BRCA2 genes (BRCA1 / 2), which are responsible for hereditary breast and ovarian cancer (HBOC), a highly prevalent disease with a pathogenic variant carrier frequency of 1 in 200-500 individuals in the general population, have been the most widely shared, but a few percent of variants are identified as VUS. Other hereditary tumor-related genes are known to still have VUS at a high level of around 10%, making their functional annotation problematic. Currently, cases identified as VUS are further interpreted through experimental studies and surveillance using additional cell lines and experimental animals, and subsequent testing, treatment, and observation strategies are determined, but these verification procedures typically require a significant amount of time and expense.

[0004] PMID: 25741868; 2015, Genet Med;17(5):405-24

[0005] One object of the present invention is to provide a method for easily determining the pathological significance of a VUS.

[0006] Another object of the present invention is to correctly evaluate an individual's risk of developing a genetic disease based on the results of evaluation of VUS whose pathogenic significance has been clarified and known variants.

[0007] The present invention provides the following methods for assessing the risk of developing a VUS or a genetic disease in an individual: [1] A transformed zebrafish fertilized egg, obtained by introducing into a zebrafish fertilized egg DNA of a genetic factor for a genetic disease, the genetic factor comprising a mutant NRAS gene under the control of the mitfa promoter and at least one variant of unknown pathogenicity (VUS) under the control of the mitfa promoter; and a transformed zebrafish obtained from the fertilized egg. [2] A transformed zebrafish fertilized egg and a transformed zebrafish obtained from the fertilized egg according to [1], wherein the mutant NRAS gene is a human mutant NRAS gene (NRAS Q61K). [3] A transformed zebrafish fertilized egg and a transformed zebrafish obtained from the fertilized egg according to [1] or [2], obtained by introducing into a zebrafish fertilized egg DNA of an expression vector comprising a mutant NRAS gene under the control of the mitfa promoter and at least one variant of unknown pathogenicity (VUS) under the control of the mitfa promoter. [4] The transformed zebrafish fertilized egg of [3], wherein the expression vector comprises medaka Tol2 element, and the expression vector and mRNA encoding a transposon transferase are introduced into a zebrafish fertilized egg, and the transformed zebrafish obtained from the fertilized egg. [5] A combined preparation for transforming zebrafish, comprising a mutant NRAS gene under the control of the mitfa promoter and DNA of a genetic factor for a genetic disease comprising at least one variant of unknown pathogenicity (VUS) under the control of the mitfa promoter. [6] The combined preparation of [5], comprising an expression vector comprising a mutant NRAS gene under the control of the mitfa promoter and DNA of a genetic factor for a genetic disease comprising at least one variant of unknown pathogenicity (VUS) under the control of the mitfa promoter. [7] The combined preparation of [5] or [6], wherein the mutant NRAS gene is a human mutant NRAS gene (NRAS Q61K).[8] A kit for transforming zebrafish, comprising a mutant NRAS gene under the control of the mitfa promoter and DNA of a genetic factor for a genetic disease, the genetic factor comprising at least one variant of unknown pathogenicity (VUS) under the control of the mitfa promoter. [9] The kit according to [8], comprising an expression vector comprising a mutant NRAS gene under the control of the mitfa promoter and DNA of a genetic factor for a genetic disease, the genetic factor comprising at least one variant of unknown pathogenicity (VUS) under the control of the mitfa promoter.

[10] The kit according to [8] or [9], wherein the mutant NRAS gene is a human mutant NRAS gene (NRAS Q61K).

[11] A method for assessing an individual's risk of developing a genetic disease caused by a VUS, comprising introducing DNA of a genetic factor for a genetic disease, the genetic factor comprising any one variant of unknown pathogenicity (VUS), under the control of a promoter of a gene involved in the phenotype, into a fertilized zebrafish egg, and observing changes in the phenotype in the zebrafish to assess the risk of developing the VUS-related genetic disease.

[12] The method for assessing the risk of an individual developing a hereditary disease caused by a VUS according to

[11] , wherein the hereditary disease is cancer.

[13] The method for assessing the risk of an individual developing a hereditary disease caused by a VUS according to

[11] or

[12] , comprising: introducing DNA of a genetic factor for a hereditary disease, which contains any one variant of unknown pathogenic significance (VUS) under the control of the mitfa promoter, together with a mutant NRAS gene, into a fertilized egg of a zebrafish, wherein the promoter of a gene involved in a phenotype is the mitfa promoter; and assessing the risk of developing the hereditary disease caused by the VUS by observing the development of pigmented macules and / or melanoma in the zebrafish.

[14] The method for assessing the risk of developing a VUS-induced genetic disease in an individual according to

[11] , wherein the promoter of the gene involved in the phenotype is the col2a1a promoter, and the method comprises introducing DNA of a genetic factor for a genetic disease comprising any one variant of unknown pathogenicity (VUS) under the control of the col2a1a promoter together with a gene involved in an osteochondropathy into a fertilized zebrafish egg, and assessing the risk of developing the VUS-induced genetic disease by observing the morphology of the cartilage in the zebrafish.

[15] The method for assessing the risk of developing a VUS-induced genetic disease in an individual according to

[11] , wherein the promoter of the gene involved in the phenotype is the sp7 promoter, and the method comprises introducing DNA of a genetic factor for a genetic disease comprising any one variant of unknown pathogenicity (VUS) under the control of the sp7 promoter together with a gene involved in an osteochondropathy into a fertilized zebrafish egg, and assessing the risk of developing the VUS-induced genetic disease by observing the morphology of the bone in the zebrafish.

[16] The method for assessing an individual's risk of developing a genetic disease caused by a VUS according to

[11] , comprising: introducing DNA of a genetic factor for a genetic disease, including one variant of unknown pathogenic significance (VUS), under the control of the myl7 promoter, together with a gene involved in a heart disease, into a fertilized zebrafish egg; and assessing the risk of developing the genetic disease caused by the VUS by observing the heart morphology of the zebrafish.

[17] A method for assessing an individual's risk of developing a genetic disease for at least one VUS in at least one gene involved in a genetic disease by the methods of

[11] to

[16] , and evaluating the individual's risk of developing a genetic disease based on the evaluation results of the variant with known pathogenic significance and the at least one VUS.

[18] The method for assessing an individual's risk of developing a genetic disease according to

[17] , wherein the genetic disease is cancer.

[0008] According to the present invention, it is now possible to evaluate the contribution of genetic mutations in multifunctional genes, which are genetic factors in hereditary diseases such as cancer, osteochondral disease, heart disease, and neurodegenerative disease, to susceptibility (whether or not having the mutation makes one more susceptible to diseases such as cancer), which was previously impossible to determine using existing in vitro systems that output specific functions.

[0009] The technology is also fast enough to be used as a clinical diagnostic aid.

[0010] The procedure for evaluating the human mutant NRAS gene (NRAS Q61K) is shown. The results of the pigmented spot hyperplasia test are shown. The results of the pigmented spot hyperplasia test are shown. Photographs of zebrafish evaluated for pigmented spot hyperplasia are shown. (a) to (g) show the method for calculating the pigmented spot area. (a) Original image. (b) Draw a straight line from the center of the eye closest to the ventral side to the apex upward. (c) Draw a straight line from the apex upward to the maximum point of the swim bladder. (d) Draw a straight line from the maximum point of the swim bladder to the anus of the zebrafish (directly above where the fins separate). (e) Enclose the entire area, including the eyes and belly, to set the area to be excluded from analysis (vertical line). (f) The remaining area is the total area (horizontal line), and the area in μm is calculated. ２ (g) The area of ​​melanocyte area (hatched) in μm of the total area (horizontal line) ２ Calculated.

[0011] As used herein, the singular forms (a, an, the, etc.) include both the singular and the plural unless otherwise specified herein or clearly contradictory in context. As used herein, "comprise" is a concept that encompasses "consist essentially of" and "consist of."

[0012] In this specification, a VUS (variant of uncertain significance) is a variant of unknown pathogenic significance. Variants are extracted by comparing the base sequence of a subject with a reference sequence, and the interpretation of the pathogenic significance is unclear even when various criteria are used.

[0013] A genetic disease refers to a disease that is likely to be contracted due to a genetic factor, and at least one genetic factor (gene) is known for each disease. Specific examples of genetic diseases include cancer, osteochondral disease, heart disease, and neurodegenerative disease, with cancer being preferred.

[0014] Representative genetic factors associated with each genetic disease are shown in Table 1 below, but the present invention is also applicable to VUS in genetic factors for diseases other than those shown in Table 1.

[0015]

[0016] Genes involved in the phenotype include mitfa, which is involved in melanoma, col2a1a, which is specifically expressed in chondrocytes, sp7, which is specifically expressed in bone cells, and myl7, which is specifically expressed in cardiac muscle cells.

[0017] In one preferred embodiment of the present invention, DNA of a genetic factor for a genetic disease, including any one variant of unknown pathogenic significance (VUS), is introduced under the control of a promoter of a gene involved in the phenotype, together with a gene that facilitates the evaluation or observation of the phenotype.

[0018] For example, by introducing DNA of a genetic factor for a genetic disease containing a variant of unknown pathogenic significance (VUS) into a mutant NRAS gene under the control of the mitfa promoter, the risk of developing a VUS-related genetic disease can be assessed by observing the development of pigmented spot hyperplasia and / or melanoma in zebrafish.

[0019] Alternatively, DNA of a genetic factor for a genetic disease containing any one variant of unknown pathogenic significance (VUS) can be introduced together with a gene involved in osteochondropathy (e.g., COL1A1, COL2, COMP, COL9, etc.) under the control of the col2a1a promoter. In this case, the risk of developing the VUS-related genetic disease can be assessed by observing the cartilage morphology in zebrafish.

[0020] Alternatively, DNA of a genetic factor for a genetic disease containing any one variant of unknown pathogenic significance (VUS) can be introduced together with a gene involved in osteochondropathy (e.g., COL1A1, COL2, COMP, COL9, etc.) under the control of the sp7 promoter. In this case, the risk of developing the VUS-related genetic disease can be assessed by observing bone morphology in zebrafish.

[0021] Alternatively, DNA of a genetic factor for a genetic disease containing any one variant of unknown pathogenic significance (VUS) can be introduced under the control of the myl7 promoter together with a gene involved in cardiac or cardiomyopathy (e.g., MYH7, MYBPC3, MYL2, MYL3, TTN, etc.). In this case, the risk of developing the VUS-related genetic disease can be assessed by observing cardiac morphology in zebrafish.

[0022] The subject's base sequence can be obtained using next-generation sequencing (NGS), for example, multi-gene panel testing (MGPT), which allows genetic testing of multiple genes simultaneously.

[0023] There are many variants that are determined to be VUS by comparing the nucleotide sequence of a subject with a reference sequence. The pathological significance of these variants is determined by the method of the present invention. The pathological significance of VUS may be determined in four stages: pathogenic, likely pathogenic, likely benign, and benign, or in two stages: pathogenic and benign. The pathological significance of VUS can be evaluated by visually observing the morphological characteristics of zebrafish or by optical observation using a microscope, etc., with reference to the ACMG-AMP guidelines.

[0024] The method of the present invention for assessing an individual's risk of developing a genetic disease due to a VUS is simple and easy, allowing the pathological significance of all VUS related to all currently known genetic factors for genetic diseases to be determined. By conducting pathological evaluations of as many VUS as possible in advance, and further combining the pathological evaluations with information on known variants and optionally creating a database, nucleotide sequence information for any subject can be obtained. By comparing the subject's nucleotide sequence with a reference sequence, the possibility of the subject having any genetic disease of which the subject is interested can be quickly determined, assessed, detected, or evaluated.

[0025] When the mitfa promoter is used as the promoter of a gene involved in a phenotype, the method of the present invention for assessing the risk of an individual developing a genetic disease caused by a VUS comprises introducing DNA of a genetic factor for a genetic disease containing either one variant of unknown pathogenic significance (VUS) together with a mutant NRAS gene under the control of the mitfa promoter into a fertilized zebrafish egg, and observing the development of pigmented macules and / or melanoma in the zebrafish introduced with DNA containing the VUS. Zebrafish introduced with DNA of a wild-type genetic factor not containing a VUS can be used as a comparison subject.

[0026] The DNA of a genetic factor for a genetic disease and a mutant NRAS gene can be introduced into zebrafish by, for example, microinjection.

[0027] The pathological significance of VUS can be evaluated by observing pigmented spot hyperplasia and melanoma development in zebrafish with the naked eye or using optical equipment such as a microscope.

[0028] A preferred embodiment of the method of the present invention for assessing an individual's risk of developing a genetic disease due to a VUS is shown below.

[0029] A preferred method of the present invention uses transposon transposition. For example, when a human mutant NRAS gene (NRASQ61K) carrying a VUS is microinjected into fertilized eggs under the control of the mitfa promoter, a promoter specific to zebrafish pigmentation, pigmentation hyperplasia and melanoma develop at 7 days after birth. Microinjection of the wild-type BRCA2 gene (BRCA2 WT) together with the mutant NRAS gene, also under the control of the mitfa promoter, suppresses pigmentation hyperplasia and melanoma development (Figure 2). This property can be exploited to establish a system for assessing the susceptibility of individual BRCA2_VUS by determining how pigmentation hyperplasia and melanoma development change when individual BRCA2_VUS are co-injected instead of the wild-type BRCA2 gene. In one embodiment of the present invention, the pathogenicity of the following two types of variants can be estimated using BRCA2_WT (wild-type) as a positive control. (i) Q1502X: Likely Pathogenic (suspected pathogenic) according to the ACMG / AMP classification. (ii) I1929V: Likely Benign (suspected non-pathogenic) according to the ACMG / AMP classification. As a result, Q1502X exhibited significantly increased pigmented macular hyperplasia and melanoma incidence compared to WT, and was therefore classified as pathogenic, consistent with the ACMG / AMP classification. I1929V exhibited no significant differences in pigmented macular hyperplasia or melanoma incidence compared to WT, and was therefore classified as non-pathogenic, consistent with the ACMG / AMP classification (Figure 3). These results demonstrate that our novel evaluation system can accurately mimic the results of existing systems and is effective as a novel VUS diagnostic aid, using susceptibility as an output.

[0030] The above results are from using the mitfa promoter as the promoter of a gene involved in the phenotype and BRCA2 as the genetic factor for the disease, but the present invention can also be implemented in the same way when a promoter other than the mitfa promoter is used as the promoter of a gene involved in the phenotype, and a VUS of another genetic factor or another VUS of BRCA2 is used.

[0031] The present invention will be described in more detail below with reference to the following examples. Example 1 (1) Methods and Results The transposon transposition method is a system for inserting foreign genes into the zebrafish genome by transposon transposition using the medaka Tol2 element (Kawakami et al., Proc Natl Acad Sci USA, 2000, JP4629162B2). First, the inventors constructed a vector (mitfa:NRAS Q61K) incorporating the human mutant NRAS gene (NRAS Q61K), known as a driver gene for malignant melanoma, under the control of the promoter region of the mitfa gene, which is specifically expressed in zebrafish pigment spots (for details, see *1: Construction of zebrafish expression vectors). Subsequently, we microinjected tp53 mutant zebrafish (tp53M214K) (Berghmans et al. Proc Natl Acad Sci USA, 2009) into 1- to 2-cell stage fertilized eggs with mRNA encoding a transposon transferase (transposase mRNA) using a picopump (SYS-PV820, WPI) to establish a system that induces pigmented spot hyperplasia and melanoma within 3 weeks of birth (Fig. 1, Hosono et al. Cell, 2017).

[0032] In this study, the inventors created a vector (mitfa:BRCA2_WT) incorporating the wild-type BRCA2 gene (BRCA2 WT), a tumor suppressor gene, under the control of the mitfa promoter. This vector was then microinjected into 1- to 2-cell-stage fertilized eggs together with the aforementioned mitfa:NRAS_Q61K and transposase mRNA in the following composition: First, microinjection of 10 ng / ul of mitfa:NRAS_Q61K together with 40 ng / ul of transposase mRNA resulted in the development of pigmented macules and melanomas at 5 days after birth (Figure 2, center; hereafter, all data refer to pigmented macules as hyperplasia). On the other hand, microinjection of 20 ng / ul of mitfa_BRCA2_WT together with 10 ng / ul of mitfa:NRAS_Q61K and 40 ng / ul of transposase mRNA suppressed pigmented macules at 5 days after birth (Figure 2, right lane). Pigmented macular hyperplasia was evaluated using a deep learning model with cellSens Dimension based on image data captured with a microscope (digital imaging system APX100: APX100-SU) (for details, see Evaluation of Pigmented Macular Hyperplasia Using a Deep Learning Model below).

[0033] The clinical application of next-generation sequencing (NGS), exemplified by the practical application of multi-gene panel testing (MGPT), has dramatically increased the detection rate of pathogenic variants in causative genes. However, this has also led to the accumulation of a huge number of variants of uncertain significance (VUS), and rapid assessment of the susceptibility of these variants has been impossible using existing systems. In this study, we took advantage of the fact that co-injection of the wild-type BRCA2 gene suppresses pigmented macules (melanoma hyperplasia), and established a system for rapid assessment of the susceptibility of individual BRCA2_VUS variants by measuring the change in pigmented macules (melanoma hyperplasia) when co-injected with individual BRCA2_VUS variants instead of the wild-type BRCA2 gene. In this assessment, BRCA2_WT (wild-type) was used as a positive control, and the pathogenicity of the following two BRCA2 variants was estimated. Vectors incorporating individual BRCA2 variants were constructed using the same method as described above. (i) Q1502X: Likely Pathogenic (suspected to be pathogenic) according to the ACMG / AMP classification (ii) I1929V: Likely Benign (suspected to be non-pathogenic) according to the ACMG / AMP classification The results are shown in Figure 3. First, as in previous results, when mitfa:NRAS_Q61K 10 ng / ul was microinjected together with transposase mRNA 40 ng / ul, hyperplasia of pigmented spots was observed at 5 days after birth (Figure 3, second lane from the left), and when mitfa_BRCA2_WT 20 ng / ul was microinjected together with mitfa:NRAS_Q61K 10 ng / ul and transposase mRNA 40 ng / ul, hyperplasia of pigmented spots at 5 days after birth was suppressed (Figure 3, third lane from the left).Next, when a vector (mitfa:BRCA2_ Q1502X) carrying the variant Q1502X, which is classified as pathogenic according to the ACMG / AMP classification, was microinjected together with mitfa:NRAS_Q61K at 10 ng / ul and transposase mRNA at 40 ng / ul, the hyperplasia of pigmented spots was significantly enhanced compared to BRCA2_WT. Therefore, Q1502 was classified as pathogenic in our system, as in the ACMG / AMP classification (Figure 3, fourth lane from the left). Furthermore, when a vector carrying the I1929V variant (determined non-pathogenic by the ACMG / AMP classification) (mitfa:BRCA2_I1929V) was co-injected with mitfa:NRAS_Q61K at 10 ng / ul and transposase mRNA at 40 ng / ul, there was no significant difference in pigmented macular hyperplasia compared to BRCA2_WT, and I1929V was also determined to be non-pathogenic in our system, consistent with the ACMG / AMP classification (Figure 3, fifth lane from the left). These results demonstrate that our novel evaluation system can accurately mimic the results of existing systems and is effective as a novel VUS diagnostic aid with susceptibility as an output.

[0034] (2) Construction of Zebrafish Expression Vectors. A vector for transposon transposition was created using the MultiSite Gateway system (Thermo). First, a human mutant NRAS gene (NRAS Q61K), known as a driver gene for malignant melanoma, was amplified by PCR and cloned into the multiple cloning site (MCS) of a middle entry (pME) vector. Next, the promoter region of the mitfa gene, which is specifically expressed in zebrafish pigment spots, was amplified by PCR and cloned into the MCS of a 5' entry vector (p5E). The pME vector, p5E vector, and a p3E vector with a polyA fragment inserted into the MSC of the 3' entry vector (p3E) were then cloned into a destination vector (Addgene #64023) containing a Tol2 recognition sequence to create the vector (mitfa:NRAS_Q61K).

[0035] (3) Evaluation of pigmentation hyperplasia using a deep learning model. First, 5-day-old zebrafish were photographed using the APX100 digital imaging system (APX100-SU). Images in which the zebrafish were clearly facing directly upwards, images with unclear images, or malformations could result in an apparent underestimation (or overestimation) of pigmentation hyperplasia. These images were visually inspected and excluded from analysis. Next, total body surface area was calculated, and the area of ​​pigmentation in the region excluding the eyes and swimbladder (Figure 4, vertical lines) was extracted (Figure 4, diagonal lines). A deep learning model based on cellSens Dimension was used to extract total body surface area and pigmentation. The number of images used for training was 205, and the number of iterations was set to 300,000. Using data that showed a similarity of 86% after 250,000 iterations, the total body surface area and pigmented area of ​​zebrafish were calculated, and the percentage of pigmented areas was calculated.

Claims

1. A transformed zebrafish fertilized egg, which is obtained by introducing into a zebrafish fertilized egg DNA of a genetic factor for a genetic disease, which includes a mutant NRAS gene under the control of the mitfa promoter and at least one variant of unknown pathogenic significance (VUS) under the control of the mitfa promoter, and a transformed zebrafish obtained from the fertilized egg.

2. A transformed fertilized egg of zebrafish according to claim 1 and a transformed zebrafish obtained from said fertilized egg, wherein the mutant NRAS gene is a human mutant NRAS gene (NRAS Q61K).

3. A transformed fertilized egg of zebrafish described in claim 1 and a transformed zebrafish obtained from said fertilized egg, which are obtained by introducing into a fertilized egg an expression vector containing a mutant NRAS gene under the control of the mitfa promoter and an expression vector containing DNA of a genetic factor for a genetic disease including at least one variant of unknown pathological significance (VUS) under the control of the mitfa promoter.

4. A transformed zebrafish fertilized egg and a transformed zebrafish obtained from the fertilized egg described in claim 3, wherein the expression vector contains medaka Tol2 element and the expression vector and mRNA encoding a transposon transferase are introduced into a fertilized zebrafish egg.

5. A combination preparation for transformation of zebrafish, comprising a mutant NRAS gene under the control of the mitfa promoter and DNA of a genetic factor for a genetic disease, comprising at least one variant of unknown pathogenic significance (VUS) under the control of the mitfa promoter.

6. The combination preparation described in claim 5, comprising an expression vector containing a mutant NRAS gene under the control of the mitfa promoter and an expression vector containing DNA of a genetic factor for a genetic disease containing at least one variant of unknown pathogenic significance (VUS) under the control of the mitfa promoter.

7. The combination preparation according to claim 5 or 6, wherein the mutant NRAS gene is a human mutant NRAS gene (NRAS Q61K).

8. A kit for transforming zebrafish containing a mutant NRAS gene under the control of the mitfa promoter and DNA of a genetic disease factor containing at least one variant of unknown pathogenic significance (VUS) under the control of the mitfa promoter.

9. The kit described in claim 8, comprising an expression vector containing a mutant NRAS gene under the control of the mitfa promoter, and an expression vector containing DNA of a genetic factor for a genetic disease containing at least one variant of unknown pathogenic significance (VUS) under the control of the mitfa promoter.

10. The kit according to claim 8 or 9, wherein the mutant NRAS gene is a human mutant NRAS gene (NRAS Q61K).

11. A method for assessing an individual's risk of developing a genetic disease caused by a VUS, comprising introducing DNA of a genetic factor for a genetic disease, including any one type of variant of unknown pathological significance (VUS) under the control of the mitfa promoter, together with a mutant NRAS gene, into a fertilized zebrafish egg, and assessing the risk of developing the genetic disease caused by the VUS by observing the development of pigmentation spot hyperplasia and / or melanoma in the zebrafish.

12. A method for assessing an individual's risk of developing a genetic disease due to a VUS as described in claim 11, wherein the genetic disease is cancer.

13. A method for assessing an individual's risk of developing a genetic disease caused by a VUS as described in claim 11, comprising: introducing DNA of a genetic factor for a genetic disease containing any one of a variant of unknown pathogenic significance (VUS) under the control of the col2a1a promoter together with a gene involved in an osteochondropathy into a fertilized zebrafish egg; and assessing the risk of developing the genetic disease caused by the VUS by observing the morphology of the cartilage in the zebrafish.

14. A method for assessing an individual's risk of developing a genetic disease caused by a VUS as described in claim 11, comprising: introducing DNA of a genetic factor for a genetic disease containing any one of a variant of unknown pathogenic significance (VUS) under the control of the sp7 promoter together with a gene involved in an osteochondropathy into a fertilized egg of a zebrafish; and assessing the risk of developing the genetic disease caused by the VUS by observing bone morphology in the zebrafish.

15. A method for assessing an individual's risk of developing a genetic disease caused by a VUS as described in claim 11, comprising: introducing DNA of a genetic factor for a genetic disease containing any one of a variant of unknown pathogenic significance (VUS) under the control of the myl7 promoter together with a gene involved in heart disease into a fertilized zebrafish egg, wherein the promoter of the gene involved in the phenotype is the myl7 promoter; and assessing the risk of developing the genetic disease caused by the VUS by observing the cardiac morphology in the zebrafish.

16. A method for assessing the risk of developing a genetic disease in an individual based on the evaluation results of variants with known pathological significance and at least one of the VUS, by using the method of claim 11 to evaluate the risk of developing a genetic disease for at least one VUS in at least one gene involved in the genetic disease.

17. The method for assessing an individual's risk of developing a genetic disease as described in claim 16, wherein the genetic disease is cancer.

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