Application of RP1 gene as molecular marker for diagnosing glaucoma

Abstract

The invention discloses application of an RP1 gene as a molecular marker for diagnosing glaucoma, and relates to the technical field of gene diagnosis. The method comprises the following steps: S1, selecting a research object; s2, a sample of a research object is collected and processed; s3, observing a research object sample, and performing gene sequencing and analysis; by detecting specific gene variation, potential patients can be found in the early stage of diseases and even before clinical symptoms are not obviously shown, and a key basis is provided for taking treatment measures in time, so that disease progression is effectively delayed or prevented, the blindness risk of the patients is greatly reduced, and the life quality and prognosis of the patients are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of gene diagnosis, and particularly relates to the application of RP1 gene as a molecular marker for diagnosing glaucoma. Background Art

[0002] RP1 gene, also known as retinitis pigmentosa type 1-associated gene, is located on human chromosome 8q12.1. It is a photoreceptor microtubule-associated protein that plays an important role in the retina. The RP1 protein contains two doublecortin domains that bind to microtubules and regulate microtubule polymerization. The RP1 protein is essential for the correct stacking of outer segment discs and works in concert with another retina-specific protein, RP1L1, to affect the photosensitivity and outer segment morphogenesis of rod photoreceptors. Primary congenital glaucoma (PCG) is a hereditary eye disease that seriously endangers the vision of infants and young children. Its pathogenesis is complex and involves abnormalities of multiple genes. In the early research history of PCG, diagnosis mainly relied on clinical symptoms and signs, such as increased intraocular pressure, corneal enlargement, photophobia and tearing, decreased vision, etc., but these manifestations lack specificity and are often not obvious in the early stages of the disease, which can easily lead to misdiagnosis and missed diagnosis, delaying the best treatment time. With the development of molecular biology, genetic testing has gradually become a research hotspot, but there is still a lack of efficient, accurate and comprehensive detection methods. Although traditional Sanger sequencing has high accuracy, it has low throughput and high cost, making it difficult to comprehensively detect a large number of genes. Moreover, the verification and functional analysis methods of candidate genes are not perfect, and it is impossible to accurately judge the causal relationship between gene variation and disease phenotype. In the research of PCG molecular markers, markers with high specificity and sensitivity have not yet been found, which makes early diagnosis and disease risk assessment lack reliable basis. Therefore, we propose the application of RP1 gene as a molecular marker for diagnosing glaucoma. Summary of the invention

[0003] The purpose of the present invention is to provide an application of RP1 gene as a molecular marker for diagnosing glaucoma, which can be effectively used to diagnose glaucoma.

[0004] In order to solve the above technical problems, the present invention is achieved through the following technical solutions: The present invention is the application of RP1 gene as a molecular marker for diagnosing glaucoma, comprising the following steps: Step S1: Selection of research subjects; Step S2: Collect samples from research subjects and process them; Step S3: observe the samples of the research subjects for gene sequencing and analysis; The step S2 includes the following sub-steps: Step S21: draw 5 mL of venous blood from the research subject and put it into an EDTA-Na2 anticoagulant tube, and store it in a -80°C refrigerator for later use; Step S22: Use a DNA extraction kit to extract genomic DNA, and use a Nanodrop spectrophotometer to measure the concentration of the extracted DNA. The 260 / 280nm OD value is between 1.8 and 2.0, and the DNA concentration is above 100ng / µL. The integrity of the DNA is identified by 1% agarose gel electrophoresis.

[0005] Furthermore, in step S1, the selection of research subjects includes the following steps: Step S11: Fifty patients with primary congenital glaucoma were collected from the ophthalmology department of the hospital. The diagnostic criteria were based on internationally recognized standards, including intraocular pressure higher than 22 mmHg, abnormal corneal diameter, typical changes in the retinal optic nerve muscle fiber layer, optic disc changes, visual field defects, and abnormal chamber angle development. Patients with secondary glaucoma, open-angle glaucoma, and glaucoma complications caused by other diseases were excluded. Step S12: PCG families were selected. The inclusion criteria were no family history of hypertension, diabetes, coronary heart disease, or hyperlipidemia. Sporadic PCG patients and normal people were collected as controls. All subjects participating in the study were given eye examinations.

[0006] Furthermore, in step S3, gene sequencing and analysis includes the following steps: Step S31: performing exon sequencing on PCG family samples; Step S32: screening of candidate mutations; Step S33: verifying the pathogenicity of the candidate variant in the population; Step S34: bioinformatics analysis; The step S31: selecting PCG patients and a normal control group from the PCG family, performing whole exome sequencing on their DNA samples, filtering the raw sequence data obtained by sequencing, filtering out reads with adapters, reads with a base information ratio greater than 1% that cannot be determined, and reads with a base number ratio of ≥50% with a quality value Q≤1, and performing quality assessment after filtering, including counting the amount of sequencing data, assessing the composition distribution of bases at each position, checking the distribution of GC content, and assessing the distribution of the accuracy and error rate of base recognition by the Illumina software; The data that meet the quality standards are compared with the reference genome, sorted, deduplicated and quality corrected, and the sequencing alignment rate, average coverage depth and 1× to 100× coverage ratio of the exon region are counted. When the alignment rate is ≥95% and the base coverage depth reaches 10× or more, the variant detected by sequencing is reliable; The HaplotypeCaller algorithm of GATK software was used to identify single nucleotide variants and insertions and deletions, ExomeDepth software and CNVKit software were used to analyze copy number variations, Manta software was used to analyze structural variations, and ANNOVAR gene variation detection and annotation software was used to annotate the detected variants. The annotation content included the name of the gene, the specific location of the chromosome where the gene was located, the region where the variation was located, the frequency of mutations in the normal population in the database, and the score for harmfulness prediction.

[0007] Further, the step S32: based on the rare variation theory, filtering out mutations with a frequency higher than 0.01 in the 1000 Genomes database, ESP6500 database and ExAC database, preliminarily filtering out synonymous mutations, and filtering out mutations predicted by the dbscSNV database as having no effect on the splicing site; Priority was given to variants located in the exon region or in the region 10 bp upstream and downstream of the splice site. Five mainstream online analysis software, SIFT, PROVEAN, PolyPhen-2, MutationTaster, and CADD, were used to predict the pathogenicity of the mutation sites. Only sites predicted to be pathogenic or harmful by three or more of the software were retained. First, homozygous mutations were selected, and further screening was carried out from the perspective of gene function, including reading published literature, querying the NCBI Gene database and GeneCards database, and performing functional enrichment analysis through the GO database, protein interaction analysis through the STRING database, and pathway enrichment analysis through the KEGG database. The mRNA expression profiles of candidate genes were queried through the BioGPS website, and the MGI database was used to query whether the candidate genes had phenotypes related to PCG in gene knockout mouse models, and genes and mutations that may be related to the PCG phenotype were comprehensively screened.

[0008] Furthermore, the step S33: using Sanger sequencing to sequence the selected candidate genes in all members of the PCG family, to verify the accuracy of the whole exome sequencing results, and to perform family co-segregation verification of the candidate genes to screen the PCG family pathogenic mutations, specifically including obtaining the genomic sequence information of the candidate gene mutation site and its flanking region by querying the UCSC website; using Primer3.0 software to design primer sequences online; using the Insilico PCR module of the UCSC website to detect the specificity of the primers online; configuring primer working solution; PCR amplifying the target fragment; purifying the PCR amplification product; performing sequencing reaction, using Chromas software to read the sequencing sequence results, and determining the mutation site through the BLAT online comparison module of the UCSC database; Multiplex PCR amplicon sequencing of the exonic regions and 2000 bp region upstream of the transcription start site of the candidate genes was performed in sporadic PCG patients and normal controls to preliminarily verify the pathogenicity of the candidate variants in the population.

[0009] Furthermore, the step S34: performing pathogenicity prediction, conservation analysis, mutation site distribution analysis and mutant protein simulation modeling on the candidate gene mutation sites found by sequencing, using SIFT, PROVEAN, PolyPhen-2, MutationTaster and CADD, five mainstream online analysis software, to predict the harmfulness and pathogenicity of the mutation sites. When three or more of the five software predict that the results are harmful, the mutation is considered to be pathogenic; T-coffee software was used to analyze amino acid conservation, and BioEdit software was used to draw a protein sequence evolutionary tree. The conservation of mutation sites between humans and species closely related to humans was compared. It was believed that sites located in more conservative regions had greater pathogenicity caused by mutations. The protein secondary structure information was queried through the UniProt website, and the distribution of the amino acid residues at the mutation site in the entire protein was plotted using the IBS software to analyze whether the mutation site was located in an important protein functional domain and to speculate whether the mutation would cause consequences. Swiss-Model software was used to simulate and construct the three-dimensional structure of the mutated protein based on the principle of homology modeling, and PyMOL software was used to visualize the modeling results. Alphafold software was used based on an artificial intelligence algorithm to model the mutant protein in order to infer the impact of gene mutation on the structure and function of the protein it encodes.

[0010] The present invention has the following beneficial effects: 1. The present invention can detect potential patients in the early stages of the disease, even before clinical symptoms become apparent, by detecting specific gene mutations, providing a key basis for timely treatment measures, thereby effectively delaying or preventing disease progression, greatly reducing the risk of blindness in patients, and improving their quality of life and prognosis.

[0011] 2. The present invention helps to determine the genetic pathogenic factors of PCG patients and provide accurate genetic counseling services for family members. Through genetic testing and analysis of family members, the inheritance pattern and carrier status of the disease can be clarified, helping families understand the genetic risks of the disease, guiding fertility decisions, achieving familial prevention and control of primary congenital glaucoma, reducing the spread of the disease in the family, and reducing the overall disease burden of the family.

[0012] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0014] Figure 1 The figure is a schematic diagram of the process of using the RP1 gene of the present invention as a molecular marker for diagnosing glaucoma. DETAILED DESCRIPTION

[0015] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] See also Figure 1 As shown, the present invention is the application of RP1 gene as a molecular marker for diagnosing glaucoma, comprising the following steps: Step S1: Selection of research subjects; Step S2: Collect samples from research subjects and process them; Step S3: observe the samples of the research subjects for gene sequencing and analysis; Step S2 includes the following sub-steps: Step S21: draw 5 mL of venous blood from the research subject and put it into an EDTA-Na2 anticoagulant tube, and store it in a -80°C refrigerator for later use; Step S22: Use a DNA extraction kit to extract genomic DNA, and use a Nanodrop spectrophotometer to measure the concentration of the extracted DNA. The 260 / 280nm OD value is between 1.8 and 2.0, and the DNA concentration is above 100ng / µL. The integrity of the DNA is identified by 1% agarose gel electrophoresis.

[0017] In step S1, the selection of research objects includes the following steps: Step S11: Fifty patients with primary congenital glaucoma were collected from the ophthalmology department of the hospital. The diagnostic criteria were based on internationally recognized standards, including intraocular pressure higher than 22 mmHg, abnormal corneal diameter, typical changes in the retinal optic nerve muscle fiber layer, optic disc changes, visual field defects, and abnormal chamber angle development. Patients with secondary glaucoma, open-angle glaucoma, and glaucoma complications caused by other diseases were excluded. Step S12: PCG families were selected. The inclusion criteria were no family history of hypertension, diabetes, coronary heart disease, or hyperlipidemia. Sporadic PCG patients and normal people were collected as controls. All subjects participating in the study were given eye examinations.

[0018] In step S3, gene sequencing and analysis include the following steps: Step S31: performing exon sequencing on PCG family samples; Step S32: screening of candidate mutations; Step S33: verifying the pathogenicity of the candidate variant in the population; Step S34: bioinformatics analysis; Step S31: PCG patients and normal controls were selected from the PCG family, and their DNA samples were sequenced for whole exome sequencing. The original sequence data obtained by sequencing were filtered to filter out reads with adapters, reads with a base information ratio greater than 1% that could not be determined, and reads with a base number ratio of ≥50% with a quality value Q≤1. After filtering, quality assessment was performed, including counting the amount of sequencing data, evaluating the composition distribution of bases at each position, checking the distribution of GC content, and evaluating the distribution of the accuracy and error rate of base recognition by Illumina software; The data that meet the quality standards are compared with the reference genome, sorted, deduplicated and quality corrected, and the sequencing alignment rate, average coverage depth and 1× to 100× coverage ratio of the exon region are counted. When the alignment rate is ≥95% and the base coverage depth reaches 10× or more, the variant detected by sequencing is reliable; The HaplotypeCaller algorithm of GATK software was used to identify single nucleotide variants and insertions and deletions, ExomeDepth software and CNVKit software were used to analyze copy number variations, Manta software was used to analyze structural variations, and ANNOVAR gene variation detection and annotation software was used to annotate the detected variants. The annotation content included the name of the gene, the specific location of the chromosome where the gene was located, the region where the variation was located, the frequency of mutations in the normal population in the database, and the score for harmfulness prediction.

[0019] Step S32: Based on the rare variation theory, the mutations with a frequency higher than 0.01 in the 1000 Genomes database, ESP6500 database, and ExAC database were filtered out, synonymous mutations were initially filtered out, and mutations predicted by the dbscSNV database to have no effect on the splicing site were filtered out; Priority was given to variants located in the exon region or in the region 10 bp upstream and downstream of the splice site. Five mainstream online analysis software, SIFT, PROVEAN, PolyPhen-2, MutationTaster, and CADD, were used to predict the pathogenicity of the mutation sites. Only sites predicted to be pathogenic or harmful by three or more of the software were retained. First, homozygous mutations were selected, and further screening was carried out from the perspective of gene function, including reading published literature, querying the NCBI Gene database and GeneCards database, and performing functional enrichment analysis through the GO database, protein interaction analysis through the STRING database, and pathway enrichment analysis through the KEGG database. The mRNA expression profiles of candidate genes were queried through the BioGPS website, and the MGI database was used to query whether the candidate genes had phenotypes related to PCG in gene knockout mouse models, and genes and mutations that may be related to the PCG phenotype were comprehensively screened.

[0020] Step S33: Sanger sequencing is used to sequence the selected candidate genes in all members of the PCG family to verify the accuracy of the whole exome sequencing results, and the family co-segregation verification of the candidate genes is performed to screen the pathogenic mutations in the PCG family, specifically including obtaining the genomic sequence information of the candidate gene mutation site and its flanking region by querying the UCSC website; using Primer3.0 software to design primer sequences online; using the In silico PCR module of the UCSC website to detect the specificity of the primers online; preparing primer working solution; PCR amplifying the target fragment; purifying the PCR amplification product; performing sequencing reaction, using Chromas software to read the sequencing sequence results, and determining the mutation site through the BLAT online comparison module of the UCSC database; Multiplex PCR amplicon sequencing of the exonic regions and 2000 bp region upstream of the transcription start site of the candidate genes was performed in sporadic PCG patients and normal controls to preliminarily verify the pathogenicity of the candidate variants in the population.

[0021] Step S34: Pathogenicity prediction, conservation analysis, mutation site distribution analysis and mutant protein simulation modeling are performed on the candidate gene mutation sites found by sequencing, and harmfulness and pathogenicity prediction of the mutation sites are performed using five mainstream online analysis software, namely SIFT, PROVEAN, PolyPhen-2, MutationTaster and CADD. When three or more of the five software predict harmfulness, the mutation is considered to be pathogenic. T-coffee software was used to analyze amino acid conservation, and BioEdit software was used to draw a protein sequence evolutionary tree. The conservation of mutation sites between humans and species closely related to humans was compared. It was believed that sites located in more conservative regions had greater pathogenicity caused by mutations. The protein secondary structure information was queried through the UniProt website, and the distribution of the amino acid residues at the mutation site in the entire protein was plotted using the IBS software to analyze whether the mutation site was located in an important protein functional domain and to speculate whether the mutation would cause consequences. Swiss-Model software was used to simulate and construct the three-dimensional structure of the mutated protein based on the principle of homology modeling, and PyMOL software was used to visualize the modeling results. Alphafold software was used based on an artificial intelligence algorithm to model the mutant protein in order to infer the impact of gene mutation on the structure and function of the protein it encodes.

[0022] Through statistics, 87 patients with primary congenital glaucoma admitted to the hospital from September 2012 to September 2022 have been followed up. Among the 9 patients in the 4 PCG families, 4 have undergone whole exome sequencing of DNA samples. Tables 1 and 2 show the basic information of the 9 patients tested.

[0023] ; ; In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0024] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. The use of RP1 gene as a molecular marker for diagnosing glaucoma is characterized by: The steps include: Step S1: Selection of research subjects; Step S2: Collect samples from research subjects and process them; Step S3: observe the samples of the research subjects for gene sequencing and analysis; The step S2 includes the following sub-steps: Step S21: draw 5 mL of venous blood from the research subject and put it into an EDTA-Na2 anticoagulant tube, and store it in a -80°C refrigerator for later use; Step S22: Use a DNA extraction kit to extract genomic DNA, and use a Nanodrop spectrophotometer to measure the concentration of the extracted DNA. The 260 / 280nm OD value is between 1.8 and 2.0, and the DNA concentration is above 100ng / µL. The integrity of the DNA is identified by 1% agarose gel electrophoresis.

2. The use of the RP1 gene as a molecular marker for diagnosing glaucoma according to claim 1, characterized in that: In step S1, the selection of research subjects includes the following steps: Step S11: Fifty patients with primary congenital glaucoma were collected from the ophthalmology department of the hospital. The diagnostic criteria were based on internationally recognized standards, including intraocular pressure higher than 22 mmHg, abnormal corneal diameter, typical changes in the retinal optic nerve muscle fiber layer, optic disc changes, visual field defects, and abnormal chamber angle development. Patients with secondary glaucoma, open-angle glaucoma, and glaucoma complications caused by other diseases were excluded. Step S12: PCG families were selected. The inclusion criteria were no family history of hypertension, diabetes, coronary heart disease, or hyperlipidemia. Sporadic PCG patients and normal people were collected as controls. All subjects participating in the study were given eye examinations.

3. The use of the RP1 gene according to claim 1 as a molecular marker for diagnosing glaucoma, characterized in that: In step S3, gene sequencing and analysis include the following steps: Step S31: performing exon sequencing on PCG family samples; Step S32: screening of candidate mutations; Step S33: verifying the pathogenicity of the candidate variant in the population; Step S34: bioinformatics analysis; The step S31: selecting PCG patients and a normal control group from the PCG family, performing whole exome sequencing on their DNA samples, filtering the raw sequence data obtained by sequencing, filtering out reads with adapters, reads with a base information ratio greater than 1% that cannot be determined, and reads with a base number ratio of ≥50% with a quality value Q≤1, and performing quality assessment after filtering, including counting the amount of sequencing data, assessing the composition distribution of bases at each position, checking the distribution of GC content, and assessing the distribution of the accuracy and error rate of base recognition by the Illumina software; The data that meet the quality standards are compared with the reference genome, sorted, deduplicated and quality corrected, and the sequencing alignment rate, average coverage depth and 1× to 100× coverage ratio of the exon region are counted. When the alignment rate is ≥95% and the base coverage depth reaches 10× or more, the variant detected by sequencing is reliable; The HaplotypeCaller algorithm of GATK software was used to identify single nucleotide variants and insertions and deletions, ExomeDepth software and CNVKit software were used to analyze copy number variations, Manta software was used to analyze structural variations, and ANNOVAR gene variation detection and annotation software was used to annotate the detected variants. The annotation content included the name of the gene, the specific location of the chromosome where the gene was located, the region where the variant was located, the frequency of mutations in the normal population in the database, and the score for harmfulness prediction.

4. The use of the RP1 gene as a molecular marker for diagnosing glaucoma according to claim 3, characterized in that: The step S32: based on the rare variation theory, filtering out mutations with a frequency higher than 0.01 in the 1000 Genomes database, the ESP6500 database, and the ExAC database, preliminarily filtering out synonymous mutations, and filtering out mutations predicted by the dbscSNV database as having no effect on the splicing site; Priority was given to variants located in the exon region or in the region 10 bp upstream and downstream of the splice site. Five mainstream online analysis software, SIFT, PROVEAN, PolyPhen-2, MutationTaster, and CADD, were used to predict the pathogenicity of the mutation sites. Only sites predicted to be pathogenic or harmful by three or more of the software were retained. First, homozygous mutations were selected, and further screening was carried out from the perspective of gene function, including reading published literature, querying the NCBIGene database and GeneCards database, and performing functional enrichment analysis through the GO database, protein interaction analysis through the STRING database, and pathway enrichment analysis through the KEGG database. The mRNA expression profiles of candidate genes were queried through the BioGPS website, and the MGI database was used to query whether the candidate genes had phenotypes related to PCG in gene knockout mouse models, and genes and mutations that may be related to the PCG phenotype were comprehensively screened.

5. The use of the RP1 gene as a molecular marker for diagnosing glaucoma according to claim 3, characterized in that: The step S33: using Sanger sequencing to sequence the selected candidate genes in all members of the PCG family, verifying the accuracy of the whole exome sequencing results, and performing family co-segregation verification of the candidate genes to screen the PCG family pathogenic mutations, specifically including obtaining the genome sequence information of the candidate gene mutation site and its flanking region by querying the UCSC website; using Primer3.0 software to design primer sequences online; using the In silico PCR module of the UCSC website to detect the specificity of the primers online; and preparing the primer working solution; PCR amplification of target fragment; Purification of PCR amplification products; The sequencing reaction was performed, and the sequencing sequence results were read using Chromas software, and the mutation sites were determined using the BLAT online alignment module of the UCSC database; Multiplex PCR amplicon sequencing of the exonic regions and 2000 bp region upstream of the transcription start site of the candidate genes was performed in sporadic PCG patients and normal controls to preliminarily verify the pathogenicity of the candidate variants in the population.

6. The use of the RP1 gene as a molecular marker for diagnosing glaucoma according to claim 3, characterized in that: Step S34: performing pathogenicity prediction, conservation analysis, mutation site distribution analysis and mutant protein simulation modeling on the candidate gene mutation sites found by sequencing, and using five mainstream online analysis software, namely SIFT, PROVEAN, PolyPhen-2, MutationTaster and CADD, to predict the harmfulness and pathogenicity of the mutation sites. When three or more of the five software predict that the results are harmful, the mutation is considered to be pathogenic; T-coffee software was used to analyze amino acid conservation, and BioEdit software was used to draw a protein sequence evolutionary tree. The conservation of mutation sites between humans and species closely related to humans was compared. It was believed that sites located in more conservative regions had greater pathogenicity caused by mutations. The protein secondary structure information was queried through the UniProt website, and the distribution of the amino acid residues at the mutation site in the entire protein was plotted using the IBS software to analyze whether the mutation site was located in an important protein functional domain and to speculate whether the mutation would cause consequences. Swiss-Model software was used to simulate and construct the three-dimensional structure of the mutated protein based on the principle of homology modeling, and PyMOL software was used to visualize the modeling results. Alphafold software was used based on an artificial intelligence algorithm to model the mutant protein in order to infer the impact of gene mutation on the structure and function of the protein it encodes.

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