Application of ABCA6 gene or expression product thereof as marker in preparation of product for evaluating patella dysplasia, molecular marker and product

Abstract

The invention belongs to the technical field of biology, and discloses application of an ABCA6 gene or an expression product of the ABCA6 gene as a marker in preparation of a product for evaluating patella dysplasia, a molecular marker and a product. The ABCA6 gene or an expression product thereof provided by the invention is remarkably related to patella dysplasia, and the problems of abnormal cartilage generation, abnormal joint tissue development and the like are caused by influence on the expression levels of cartilage matrix proteins and cartilage degeneration markers in cartilage tissues due to down-regulation or deletion of the expression of the ABCA6. The molecular marker has obvious correlation with patella dysplasia and the disease process thereof, can be used as a marker for early diagnosis of patella dysplasia, disease process prediction, treatment effect expectation and disease molecular typing, and has important significance for promoting precise medical development of patella dysplasia.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to the use of ABCA6 gene or its expression product as a marker in the preparation of a product for evaluating patellar dysplasia, as well as molecular markers and products. Background Art

[0002] Patellar dysplasia is a common pathological manifestation of abnormal development of the patella and knee joint, often accompanied by symptoms such as patellar dislocation and knee pain. Long-term untreated may lead to complications such as osteoarthritis, seriously affecting the patient's knee stability and daily activities, thereby significantly reducing the quality of life and joint function. As a disease with high genetic heterogeneity, its etiology is complex and diverse, involving a variety of potential genetic and molecular mechanisms. At present, the diagnosis of patellar dysplasia in clinical practice mainly relies on imaging analysis and family history tracking. However, these traditional methods have problems with insufficient sensitivity and specificity, especially in early or mild cases, and it is difficult to fully reveal the underlying cause of the disease.

[0003] Therefore, developing a molecular biological diagnostic tool for early detection and etiology tracking of patellar dysplasia is of great significance for the treatment of patellar dysplasia. Summary of the invention

[0004] After extensive and in-depth research, the inventors of the present invention creatively discovered that the downregulation or loss of the expression of ABCA6 protein (NP_525023.2) in the ATP-binding cassette transporter A family will affect the expression levels of cartilage matrix proteins and cartilage degeneration markers in cartilage tissue, thereby leading to problems such as abnormal cartilage formation and abnormal joint tissue development. It is significantly correlated with patellar dysplasia and its disease progression, and can be used as a marker for early diagnosis of patellar dysplasia, prediction of disease progression, anticipation of treatment effects and molecular typing of the disease.

[0005] In addition, the inventors of the present invention also found that there is a missense mutation p.Asp797Asn in the ABCA6 gene in patients of a certain family with multiple patellar dysplasia in China. The occurrence of the missense mutation p.Asp797Asn causes the amino acid residues at the splicing site of the ABCA6 protein to change, affecting the formation of mature ABCA6 protein, resulting in the loss of ABCA6 protein expression, and showing a significant correlation with patellar dysplasia. That is, the ABCA6 gene including the missense mutation p.Asp797Asn can be used as a biomarker for patellar dysplasia, providing a new target for molecular biological detection of early detection and etiology tracking of patellar dysplasia, which is of great significance. Based on this, the technical solution of the present invention is obtained.

[0006] The first object of the present invention is to provide the use of ABCA6 gene or its expression product as a marker in the preparation of a product for evaluating patellar dysplasia.

[0007] The second object of the present invention is to provide a molecular marker for evaluating patellar dysplasia.

[0008] A third object of the present invention is to provide a product for evaluating patellar dysplasia.

[0009] Specifically, the present invention provides the use of the ABCA6 gene or its expression product as a marker in the preparation of a product for evaluating patellar dysplasia.

[0010] Furthermore, the ABCA6 gene is associated with cartilage formation and / or normal development of joint tissue.

[0011] Furthermore, the expression of the ABCA6 gene is positively correlated with the expression of cartilage matrix protein.

[0012] Furthermore, the cartilage matrix protein includes ACAN and / or Col2a1.

[0013] Furthermore, the expression of the ABCA6 gene is negatively correlated with the expression of cartilage degeneration markers.

[0014] Furthermore, the cartilage degeneration markers include MMP13 and / or Col10a1.

[0015] The molecular markers for evaluating patellar dysplasia provided by the present invention include ABCA6 gene and / or the expression product of the ABCA6 gene.

[0016] Furthermore, the ABCA6 gene includes a missense mutation p.Asp797Asn.

[0017] The product for evaluating patellar dysplasia provided by the present invention comprises a reagent for detecting the molecular marker described in claim 7 or 8.

[0018] Furthermore, the reagent is selected from one or more of primers, probes and antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A pedigree diagram of a family with multiple patellar dysplasia provided in Example 1 of the present invention;

[0020] Figure 2 The result diagram of the genetic linkage analysis provided in Example 1 of the present invention;

[0021] Figure 3 This is one of the result graphs of clinical patient disease research provided in Example 2 of the present invention (immunofluorescence staining);

[0022] Figure 4 The second result graph of the clinical patient disease study provided in Example 2 of the present invention (relative expression level of ACAN);

[0023] Figure 5 Figure 3 (relative expression level of Col10a1) of the clinical patient disease research results provided in Example 2 of the present invention;

[0024] Figure 6 Figure 4 (relative expression level of ABCA6) of the clinical patient disease research results provided in Example 2 of the present invention;

[0025] Figure 7 Figure 5 (MPP13 relative expression level) of the clinical patient disease study results provided in Example 2 of the present invention;

[0026] Figure 8 This is one of the result graphs of the mouse model validation provided in Example 2 of the present invention (immunofluorescence staining);

[0027] Fig. 9 This is the second result diagram of the mouse model validation provided in Example 2 of the present invention (OARSI score). DETAILED DESCRIPTION

[0028] Based on the significant correlation between the ABCA6 gene and patellar dysplasia, the present invention first provides the use of the ABCA6 gene or its expression product as a marker in the preparation of a product for evaluating patellar dysplasia.

[0029] In the present invention, the ABCA6 gene itself can be used as a marker for molecular biological detection of patellar dysplasia. More specifically, ABCA6 genes with mutations that can lead to loss of ABCA6 protein expression and / or loss of structural function all have the potential to be used as targets for patellar dysplasia detection, and can provide analytical tools for early diagnosis of patellar dysplasia and molecular mechanism research, which is conducive to promoting the development of precision medicine for patellar dysplasia.

[0030] In the present invention, specific examples of the mutations that can lead to loss of ABCA6 protein expression and / or loss of structural function include but are not limited to: one or more of missense mutations, nonsense mutations, frameshift mutations and non-frameshift mutations.

[0031] In the present invention, the expression product of the ABCA6 gene can be used as a marker for molecular biological detection of patellar dysplasia. More specifically, the expression of the expression product and its expression level can be used as an evaluation index for the occurrence, progression of the disease, and expected treatment effect of patellar dysplasia, and can provide an analytical tool for early diagnosis of patellar dysplasia and molecular mechanism research, which is conducive to promoting the development of precision medicine for patellar dysplasia.

[0032] In the present invention, specific examples of the expression product of the ABCA6 gene include but are not limited to: mRNA and / or ABCA6 protein.

[0033] In the present invention, the significant correlation between the ABCA6 gene and patellar dysplasia specifically includes: the ABCA6 gene is related to cartilage formation and / or joint tissue development.

[0034] In the present invention, the expression of the ABCA6 gene is positively correlated with the expression of cartilage matrix proteins. More specifically, the reduction or loss of the ABCA6 gene expression is accompanied by a decrease in the expression of cartilage matrix proteins such as ACAN and Col2a1, affecting processes such as cartilage formation and joint tissue development, resulting in damage to the structure and function of cartilage tissue.

[0035] In the present invention, the expression of the ABCA6 gene is negatively correlated with the expression of cartilage degeneration markers. More specifically, the reduction or loss of the ABCA6 gene expression is accompanied by an increase in the expression of cartilage degeneration markers such as MMP13 and Col10a1, which induces or aggravates cartilage degeneration and causes damage to the structure and function of cartilage tissue.

[0036] Based on the significant correlation between the ABCA6 gene and patellar dysplasia, the present invention also provides a molecular marker for evaluating patellar dysplasia.

[0037] In the present invention, the molecular marker specifically includes the ABCA6 gene and / or the expression product of the ABCA6 gene.

[0038] In the present invention, the ABCA6 gene includes a mutation that can lead to loss of ABCA6 protein expression and / or loss of structural function. More specifically, the mutation may lead to abnormal transcription and / or abnormal translation of the ABCA6 gene, resulting in misfolded or incomplete ABCA6 protein during the translation process, so that the ABCA6 protein cannot be expressed normally.

[0039] In the present invention, when the ABCA6 gene includes a missense mutation p.Asp797Asn, the base pair substitution occurring in the ABCA6 gene causes the codon encoding the 797th amino acid residue in the mRNA to change - from a codon encoding aspartic acid to a codon encoding asparagine, and the 797th amino acid residue in the ABCA6 protein is predicted to be a protein splicing site. The change in the amino acid residue leads to abnormal protein self-splicing, and the mature ABCA6 protein cannot be formed, resulting in the inability of the ABCA6 protein to be expressed normally, thereby affecting the expression of cartilage matrix proteins and cartilage degeneration markers, resulting in damage to the structure and function of the cartilage tissue, and showing a significant correlation with patellar dysplasia.

[0040] In the present invention, the occurrence, disease progression and expected treatment effect of patellar dysplasia are evaluated by detecting the molecular markers, which is of great significance for the clinical diagnosis and treatment of patellar dysplasia.

[0041] Based on the significant correlation between the ABCA6 gene and patellar dysplasia, the present invention also provides a product for assessing patellar dysplasia.

[0042] In the present invention, the product specifically includes a reagent for detecting the above-mentioned molecular markers. More specifically, the specific examples of the detection method for the above-mentioned molecular markers to be detected by the reagent include but are not limited to: one or more of immunohistochemistry, fluorescence in situ hybridization, Sanger sequencing, fluorescence quantitative PCR and NGS sequencing.

[0043] In some specific embodiments, the reagents vary depending on the method used, and specific examples thereof include but are not limited to: one or more of primers, probes, and antibodies.

[0044] Embodiments of the present invention are described in detail below, and the examples of the embodiments are intended to be used to explain the present invention, but should not be construed as limiting the present invention. In the embodiments, if specific techniques or conditions are not specified, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be obtained commercially.

[0045] Example 1

[0046] This example is used to illustrate the correlation between the ABCA6 gene and patellar dysplasia. A family with multiple patellar dysplasia was used as the research object. The family pedigree of the family is as follows: Figure 1 As shown. Figure 1 It is known that there are 6 patients with patellar dysplasia in this family. The tests include:

[0047] (1) Whole exome sequencing: Whole exome sequencing (WES) was performed on two patients with patellar dysplasia and one healthy member of the family. A total of 111 new mutation sites were obtained by WES sequencing, including 6 homozygous mutations and 105 heterozygous mutations. Among them, there were 85 missense mutations, 12 non-frameshift deletion mutations, 6 frameshift deletion mutations, 4 non-frameshift insertion mutations, 2 non-frameshift conversion mutations, 1 premature stop mutation, and 1 frameshift insertion mutation.

[0048] It is worth noting that the abnormal expression or mutation of TBX4, LMX1B and RECQL4 genes known to be associated with patellar dysplasia was not detected in WES sequencing, suggesting that the cause of patellar dysplasia may involve unknown pathogenic genes.

[0049] (2) Linkage analysis: Genetic linkage analysis is performed on all members of the family. The results are as follows: Figure 2 As shown. Figure 2 It can be seen that there are chromosomal regions with linkage disequilibrium (LOD)>1 on chromosomes 11 and 17, and combined with the WES sequencing results shown in Table 1, six mutation sites were confirmed, as shown in Table 1.

[0050] Table 1.

[0051] chromosome chr11 chr11 chr17 chr17 chr17 chr21 Start site 66114017 65687908 67103837 67145196 67190540 34003929 Termination site 66114017 65687908 67103837 67145197 67190543 34003929 Original base T A C AG GACA - Mutated base G G T - - GATTTA Mutation type Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Gene region Exon Exon Exon Exon Exon Exon, splicing Gene B3GNT1 DRAP1 ABCA6 ABCA10 ABCA10 SYNJ1 Mutation type NS-SNV NS-SNV NS-SNV FD FD NF-I Protein changes K334Q M102V D797N 1504del 443_444del V1405delinesV

[0052] (3) In situ Sanger sequencing: In situ Sanger sequencing was performed on all members of the family, and 5 mutations were detected in all patients with patellar dysplasia in the family, including ABCA10: p.1504del, ABCA10: p.443_444del, B3GNT1: p.Lys334Gln, DRAP1: P.Met102Val and ABCA6: p.Asp797Asn. Among them, ABCA10: p.1504del and ABCA10: p.443_444del were excluded as polymorphic sites; while B3GNT1: p.Lys334Gln, DRAP1: P.Met102Val and ABCA6: p.Asp797Asn may be the pathogenic genes causing patellar dysplasia in this family.

[0053] (4) Verification of independent healthy samples: 200 independent Chinese healthy samples were used as research subjects and subjected to in situ Sanger sequencing. The results showed that ABCA6:p.Asp797Asn was not detected in these 200 independent Chinese healthy volunteers, which means that ABCA6:p.Asp797Asn is not a common polymorphism in the general population and may cause disease.

[0054] (5) Prediction of protein function: Poly-Phen and Mutation-Taster software were used to predict the effects of B3GNT1:p.Lys334Gln, DRAP1:P.Met102Val and ABCA6:p.Asp797Asn on the functions of the corresponding proteins. The prediction results showed that DRAP1:P.Met102Val and B3GNT1:p.Lys334Gln had no negative impact on the final protein function; however, in the ABCA6 protein, the 797th amino acid is a protein splicing site, and the missense mutation ABCA6:p.Asp797Asn will lead to the inability to form mature ABCA6 protein, which may cause disease. In other words, the ABCA6 gene is associated with patellar dysplasia and is a potential marker for molecular biological detection of patellar dysplasia.

[0055] Example 2

[0056] This example is used to illustrate the correlation between ABCA6 gene expression level and cartilage formation and joint tissue development, specifically including:

[0057] 1. Clinical patient disease research

[0058] A sample population independent of the multiple patellar dysplasia family in Example 1 was used as the research object, and the sample population included: TPD group (traumatic patellar dislocation, 4 patients), SPD group (sporadic patellar dislocation, 4 patients), FPD group (familial patellar dysplasia, 4 patients) and Con group (healthy control group, 6 volunteers), and the following tests were performed:

[0059] (1) In situ Sanger sequencing: In situ Sanger sequencing was performed on 16 patients in the sample. The results showed that none of the TPD, SPD, and Con groups carried the missense mutation ABCA6:p.Asp797Asn, while all four patients in the FPD group carried the missense mutation ABCA6:p.Asp797Asn.

[0060] (2) Cartilage tissue sample detection: Cartilage tissue samples obtained from 18 patients in the group were stained and immunofluorescence tested to obtain the expression levels of mature ABCA6 protein, MMP13, Col10a1, ACAN and Col2a1 in the cartilage tissue of the patients. Figures 3 to 7 shown.

[0061] Depend on Figures 3 to 7The results shown show that compared with the Con group, the expression level of mature ABCA6 protein in the joint tissues of patients in the TPD group, SPD group and FPD group was decreased, accompanied by a decrease in cartilage matrix proteins ACAN and Col2a1 and an increase in cartilage degeneration markers MMP13 and Col10a1; and the decrease in the expression level of mature ABCA6 protein in the FPD group was significantly higher than that in the TPD group and SPD group.

[0062] 2. Mouse model validation

[0063] (1) Construction of mouse model: ABCA6 knockout mice (ABCA6-KO, N=12) and wild-type mice (WT, N=12) of the same strain constructed by an external biological company were used as research subjects. ABCA6-KO mice (N=12) and WT mice (N=12) were fed with standard food and water for 22 months.

[0064] (2) Cartilage tissue sample detection: At 6, 14, 18, and 22 months of feeding, the knee joint cartilage tissues of each group of mice were subjected to Saf-O staining and OARSI scoring. The results are as follows: Figure 8 and 9 shown.

[0065] Depend on Figure 8 and 9 The test results shown show that compared with WT mice, ABCA6-KO mice had significantly increased OARSI scores under the same feeding conditions and showed more obvious joint degenerative changes, that is, the knockout of the ABCA6 gene caused spontaneous patellar dysplasia in mice, that is, the ABCA6 gene was highly correlated with patellar dysplasia and its disease progression.

[0066] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. Use of the ABCA6 gene or its expression product as a marker in the preparation of products for evaluating patellar dysplasia.

2. The use according to claim 1, characterized in that: The ABCA6 gene is involved in cartilage formation and / or joint tissue development.

3. The use according to claim 1, characterized in that: The expression of the ABCA6 gene is positively correlated with the expression of cartilage matrix protein.

4. The use according to claim 3, characterized in that: The cartilage matrix proteins include ACAN and / or Col2a1.

5. The use according to claim 1, characterized in that: The expression of the ABCA6 gene is negatively correlated with the expression of cartilage degeneration markers.

6. The use according to claim 5, characterized in that: The cartilage degeneration markers include MMP13 and / or Col10a1.

7. A molecular marker for evaluating patellar dysplasia, characterized in that: The molecular markers include the ABCA6 gene and / or the expression product of the ABCA6 gene.

8. The molecular marker for evaluating patellar dysplasia according to claim 7, characterized in that: The ABCA6 gene includes a missense mutation p.Asp797Asn.

9. A product for evaluating patellar dysplasia, characterized in that: The product comprises a reagent for detecting the molecular marker according to claim 7 or 8.

10. The product for evaluating patellar dysplasia according to claim 9, characterized in that: The reagent is selected from one or more of a primer, a probe and an antibody.

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