OPN1LW mutation as a marker for the diagnosis of high myopia and related products

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to the diagnostic application of OPN1LW mutation as a marker in high myopia and related products. The present invention provides a reagent for detecting the OPN1LW c.843G>A mutation (NM_020061.6:exon5:c.843G>A mutation) of the OPN1LW gene, including a specific primer pair with a nucleotide sequence such as SEQ ID NO.1-SEQ ID NO.4. The kit of the present invention has high sensitivity and strong specificity. Only a small amount of DNA sample is needed to determine whether the mutation site is mutated, thereby determining whether high myopia or potential risks exist. It is easy to use and simple to operate, and the purpose of early screening is achieved, and it has good market application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the diagnostic application of OPN1LW mutation as a marker in high myopia and related products. Background Art

[0002] Normal color vision is three primary colors, which require the participation of S (short wavelength), M (medium wavelength), and L (long wavelength) cones. The vast majority (~94%) of cones in the retina are L or M cones. In addition, cones are essential for regulating refractive development. The OPN1LW gene encodes the long-wavelength photopigment gene for cone cells. Mutations in the OPN1LW gene may lead to cone dysfunction. Complete loss of OPN1LW gene function usually leads to red blindness, high myopia, cone dysfunction, etc.

[0003] OPN1LW and OPN1MW are located on the X chromosome, usually arranged in tandem end to end. Since the L gene and the M gene are similar in structure and at the nucleotide level (more than 96%), non-homologous recombination is prone to occur between these genes. Since high myopia and color blindness are more common in the general population, such patients are not easy to identify in the early stage, and there are limited reports on related cases in the Chinese population.

[0004] According to the age of onset, high myopia can be divided into late-onset high myopia (loHM) occurring after school age and early-onset high myopia (eoHM) occurring before school age. A large number of genetic studies have shown that eoHM is different from loHM. The onset age of eoHM is before school age (<7 years old), and it is least affected by environmental factors (such as close work) and is mainly determined by genetic factors. Therefore, eoHM is an ideal model for studying the pathogenesis of high myopia and a unique resource for finding genes related to high myopia. eoHM is closely related to some hereditary eye diseases, and is often the earliest feature of some hereditary eye diseases. It is the main reason for children to seek medical treatment as early as possible and a clue for clinicians to discover potential eye diseases. In order to improve the clinical diagnostic efficiency of eoHM, high-throughput second-generation sequencing technology is increasingly widely used. Second-generation sequencing usually uses designed DNA sequence probes to capture the exon region of the target gene or the whole genome, and then performs deep sequencing on each exon. Using this technical process to find the pathogenic gene of eoHM has become a research hotspot. Li et al. (Li J, Gao B, Guan L, Xiao X, Zhang J, Li S, Jiang H, Jia X, Yang J, Guo X, Yin Y, Wang J, Zhang Q. Unique Variants in OPN1LW Cause Both Syndromic and Nonsyndromic X-Linked High Myopia Mapped to MYP1. Invest Ophthalmol Vis Sci. 2015 Jun; 56(6): 4150-5.) used second-generation sequencing technology to detect two haplotypes and one frameshift variant in three families, which were co-segregated with high myopia.

[0005] The pathogenesis of myopia is complex and has not yet been elucidated. A large number of research results have confirmed the role of heredity, and environmental factors will play a regulatory and modifying role. In this context, choosing to study the cause of the disease from the perspective of genetics is of great significance for the prevention and treatment of the disease. At present, some institutions have used genetic testing methods to detect susceptibility to high myopia. For example, one or more sites in the ZNF644 gene are used to predict the susceptibility of the sample to be tested to high myopia, but studies have confirmed that only some of the gene mutations are related to the onset of hereditary high myopia. In short, although there is currently a prediction of the risk of high myopia through the detection of a certain gene, its effectiveness needs to be further verified, the prediction accuracy is low, and it is difficult to effectively assess the risk of high myopia caused by the combined effects of genetic factors and other factors. Therefore, other genetic testing methods need to be developed to facilitate early diagnosis and early intervention, delay the progression of the disease, and preserve the patient's vision. Summary of the invention

[0006] In order to solve the above problems, the present invention provides an isolated nucleic acid encoding an OPN1LW mutant, which has a c.843G>A mutation, namely NM_020061.6:exon5:c.843G>A mutation. The mutant is closely related to the onset of early-onset high myopia, so by detecting whether the mutant is mutated in a biological sample, it is possible to effectively detect whether the organism is susceptible to high myopia. Furthermore, a method for screening biological samples for high myopia is proposed, which can be used to detect whether the biological sample has an OPN1LW gene mutation, thereby effectively screening out biological individuals susceptible to high myopia.

[0007] The NM_020061.6 in the NM_020061.6:exon5:c.843G>A mutation of the present invention refers to the number in the RefSeq database of NCBI.

[0008] On the one hand, the present invention provides the use of a reagent for detecting the OPN1LW gene NM_020061.6:exon5:c.843G>A mutation in the preparation of a kit for myopia screening or preventive screening.

[0009] Specifically, the NM_020061.6:exon5:c.843G>A mutation is a nonsense mutation, and the associated disease is LOF.

[0010] Specifically, the myopia is high myopia, and the high myopia is early-onset myopia of more than 600 degrees.

[0011] Specifically, the reagents include an amplification reagent for the OPN1LW gene NM_020061.6:exon5 gene fragment;

[0012] The OPN1LW gene NM_020061.6:exon5 gene fragment is a fragment containing the c.843G>A mutation.

[0013] More specifically, the amplification reagent includes primers for amplifying the NM_020061.6:exon5 fragment of the OPN1LW gene.

[0014] More specifically, the primers include first-round amplification primers and second-round amplification primers; the substrate of the second-round amplification primers is the product of the first-round amplification.

[0015] Preferably, the sequence of the first round amplification primers is shown as SEQ ID NO.1-SEQ ID NO.2; the sequence of the second round amplification primers is shown as SEQ ID NO.3-SEQ ID NO.4.

[0016] Specifically, the amplification reagents also include but are not limited to: enzymes, substrates and metal ions.

[0017] Specifically, the first round of amplification reagents adopt the following reaction system:

[0018] Reagents concentration Dosage (μL) KOD enzyme 5U / μL 1-2 μL KOD Buffer 10× 20-30μL SEQ ID NO.1 10μM 0.5-2μL SEQ ID NO.2 10μM 0.5-2μL Nuclease-free water / 15-20μL

[0019] The second round of amplification reagents uses the following reaction system:

[0020] Reagents concentration Dosage <![CDATA[TaKaRa Ex Premier TM DNA]]> 2× 20-30μL SEQ ID NO.3 10μM 0.5-2μL SEQ ID NO.4 10μM 0.5-2μL Template DNA 200ng 3-8μL Nuclease-free water / 15-20μL

[0021] Specifically, the first round amplification system adopts the following reaction procedure:

[0022]

[0023] The second round of amplification system uses the following reaction procedure:

[0024]

[0025] Specifically, the kit also includes a sequencing reagent for determining the sequence of the amplified product.

[0026] More specifically, the sequencing reagents include but are not limited to: sequencing primers, nucleic acid bases, enzymes and buffers.

[0027] Specifically, the sample detected by the kit is selected from any one or more of oral swabs, tissues, blood, urine, saliva, semen, milk, cerebrospinal fluid, tears, sputum, mucus, ascites, pleural effusion, amniotic fluid, bladder washing fluid and bronchoalveolar lavage fluid.

[0028] Specifically, the kit also includes a DNA extraction reagent and / or an RNA reverse transcription reagent.

[0029] More specifically, the DNA extraction reagent includes but is not limited to: lysis solution and detergent.

[0030] More specifically, the RNA reverse transcription reagent includes but is not limited to: reverse transcription primers, buffer, and enzyme.

[0031] Specifically, the kit also includes a sample preservation reagent.

[0032] Preferably, the sample preservation reagent includes but is not limited to physiological saline and DNA cell preservation solution.

[0033] Specifically, the myopia screening is to screen individuals who have already developed myopia; the preventive screening is to screen individuals who are likely to develop myopia in the later stage and issue risk warnings.

[0034] On the other hand, the present invention provides a primer for amplifying and detecting the OPN1LW gene NM_020061.6:exon5:c.843G>A mutation, characterized in that the primer sequence is shown in SEQ ID NO.1-SEQ ID NO.4.

[0035] Specifically, the amplification primers include a first amplification primer and a second amplification primer; the substrate of the second amplification primer is the product of the first amplification.

[0036] More specifically, the primer sequences SEQ ID NO.1-SEQ ID NO.2 are primers for the first amplification; and the primer sequences SEQ ID NO.3-SEQ ID NO.4 are primers for the second amplification.

[0037] In yet another aspect, the present invention provides a myopia screening or preventive screening kit, comprising amplification primers for detecting the OPN1LW gene NM_020061.6:exon5:c.843G>A mutation.

[0038] Specifically, the amplification primers include a first amplification primer and a second amplification primer; the substrate of the second amplification primer is the product of the first amplification.

[0039] Preferably, the sequence of the primers for the first amplification is shown as SEQ ID NO.1-SEQ ID NO.2; the sequence of the primers for the second amplification is shown as SEQ ID NO.3-SEQ ID NO.4.

[0040] The technical effects achieved by the present invention are:

[0041] (1) By detecting whether the OPN1LW gene NM_020061.6:exon5:c.843G>A mutation occurs, patients with high myopia or potential risk of high myopia can be effectively identified;

[0042] (2) The use of PCR amplification technology has the characteristics of high detection efficiency and low cost, which is more conducive to promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the gel electrophoresis diagram of the first round of amplification products, where 1-8 are samples; M is marker HindⅢ.

[0044] Figure 2 This is a sample base sequence map of high myopia.

[0045] Figure 3 This is the sample base sequence map of control 1.

[0046] Figure 4 This is the sample base sequence map of control 2. DETAILED DESCRIPTION

[0047] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0048] The OPN1LW c.843G>A mutation described in the present invention is the OPN1LW gene NM_020061.6:exon5:c.843G>A mutation.

[0049] Example 1 Screening of gene mutations

[0050] Oral swab genomic DNA extraction kit, manufacturer: Shanghai Lianmai Bioengineering Co., Ltd., catalog number: LM-0179.

[0051] An 8-year-old male patient with severe myopia (proband) from Beijing, China, OD: -15.25; OS: -14 (OD: naked eye visual acuity of the right eye; OS: naked eye visual acuity of the left eye), with no myopia in his family. Informed consent was obtained from all participants, and oral swabs were collected.

[0052] (1) Gene extraction of the sample to be tested. Genetic material DNA is extracted from the patient's oral swab sample according to the instructions of the oral swab genomic DNA extraction kit.

[0053] (2) Whole exome sequencing of DNA samples. Whole exome sequencing was performed using the Twist Human Core Exon Kit and an Illumina NovaSeq 6000 sequencer. The sequence fragments obtained by sequencing were aligned to the human genome sequence (GRCh37) using the Burrows-Wheeler Aligner (BWA 0.7.12). Subsequently, Sambamba0.6.6 was used to sort the chromosome coordinates of the sequenced fragments and mark the repetitive sequences. Finally, a series of genome analysis tool combinations (GATK 4.0.11.0) were used to distinguish single nucleotide variations (SNVs) and insertion or deletion variations (indels).

[0054] (3) Annotation of variant sites. The experiment used Ensembl's variant effect prediction software (VEP v.99) to annotate variants in the human genome GRCh37. Protein-coding variants were divided into the following four categories: a) synonymous mutations; b) benign missense mutations; c) destructive missense mutations; d) protein truncating variants (PTV).

[0055] (4) Variant interpretation. The analysis focused on 807 genes related to high myopia, including ADIPOR1, ARL3, BEST1, CA4, CRX, FSCN2, GUCA1B, HK1, IMPDH1, IMPG1, KIF3B, KLHL7, etc.

[0056] The NM_020061.6:exon5:c.843G>A:p.W281X mutant was found, that is, it has the OPN1LW c.843G>A mutation compared to the OPN1LW gene. This variant is a nonsense variant, and the associated disease is LOF pathogenic. The transcript in which it is located is a biologically significant transcript, and it is not in the last 50bp of the last coding exon or the second-to-last coding exon. It is predicted that it may activate nonsense-mediated mRNA degradation, thereby affecting the function of the gene encoding protein; the frequency of occurrence of this variant in ExAC, gnomAD, and the 1000 Genomes Asian population database is extremely low or not included. This mutant is closely related to the onset of early-onset high myopia, so by detecting whether this mutant is mutated in a biological sample, it is possible to effectively detect whether an organism is susceptible to high myopia.

[0057] Example 2 Mutation gene detection method and verification

[0058] The main reagents are shown in Table 1:

[0059] Table 1

[0060]

[0061]

[0062] The samples of this embodiment are derived from samples that were clearly diagnosed as high myopia and two control samples (control 1 and control 2) with healthy vision.

[0063] Since the OPN1LW and OPN1MW genes are highly homologous (96%), conventional first-generation sequencing will amplify the products of both genes simultaneously. In order to ensure that the amplified fragment is the target fragment, two rounds of amplification are performed. The first round is long-fragment specific amplification to ensure that the amplified gene is the first gene downstream of the LCR at the OPN1LW / OPN1MW locus; the second round is amplification using the amplified product of the first round as a substrate.

[0064] According to the mutant genes screened, the present invention designed two pairs of primers, long fragment specific amplification of the first gene (15022bp) located downstream of LCR on the OPN1LW / OPN1MW locus, the primer sequence is SEQ ID NO.1-SEQ ID NO.2; PCR amplification of exon 5 (398bp), the primer sequence is SEQ ID NO.3-SEQ ID NO.4. Primer sequence information is shown in Table 2:

[0065] Table 2

[0066]

[0067] The primer pairs in Table 1 were used to perform PCR amplification on the DNA extracted from the high myopia sample and the DNA of the two control samples. The amplification reaction system and reaction procedure of the first round are shown in Tables 3 and 4.

[0068] Table 3 Reaction system

[0069] Reagents concentration Dosage (μL) KOD enzyme 5U / μL 1.5μL KOD Buffer 10× 25μL SEQ ID NO.1 10μM 1μL SEQ ID NO.2 10μM 1μL Nuclease-free water / 16.5μL

[0070] Table 4 Reaction procedure

[0071]

[0072] Experimental steps:

[0073] (1) Prepare the system of the required detection sites according to Table 3. Prepare N reactions for each system, where N = the number of negative quality control products (nuclease-free water) + the number of samples to be tested + 1.

[0074] (2) The prepared system was vortexed for 30 seconds, centrifuged for 10 seconds, and dispensed into a PCR reaction plate at a rate of 45 μL / well.

[0075] (3) Add 5 μL of the sample to be tested and the negative control sample to the PCR reaction plate containing the detection system. After adding the samples, affix the sealing film and place the PCR plate in a plate centrifuge for 10 seconds to remove bubbles in the liquid.

[0076] (4) Place the PCR reaction plate in a fluorescent PCR instrument and set the total volume to 50 μL. Set the parameters of the PCR instrument according to Table 4 and start the instrument after setting the parameters.

[0077] (5) The product after PCR (about 15K) was recovered by gel cutting. Figure 1 .

[0078] The amplification reaction system and reaction procedures for the first and second rounds are shown in Tables 5 and 6.

[0079] Table 5 Reaction system

[0080] Reagents concentration Dosage <![CDATA[TaKaRa Ex Premier TM DNA]]> 2× 25μL SEQ ID NO.3 10μM 1μL SEQ ID NO.4 10μM 1μL Template DNA 200ng 5μL Nuclease-free water / 18μL

[0081] Table 6 Reaction procedure

[0082]

[0083]

[0084] Experimental steps:

[0085] (1) Gel excision was performed to recover samples from the first round of amplification. The system was prepared according to Table 5 (Exon 5 reaction system, excluding template DNA). N reactions were prepared for each system, where N = the number of negative quality control products + the number of samples to be tested + 1.

[0086] (2) Vortex the prepared system to mix evenly for 30 seconds, centrifuge for 10 seconds, and dispense into the PCR reaction plate at a volume of 45 μL / well.

[0087] (3) Add 5 μL of template DNA (i.e., the recovered DNA sample from the first round of amplification) to each PCR reaction plate containing the detection system.

[0088] (4) Perform PCR amplification according to Table 6.

[0089] (5) The PCR product (about 398 bp) was directly sent for first-generation sequencing after being identified by 2% agarose gel.

[0090] Sequencing of the above amplified bands showed that the OPN1LW gene mutant c.843G>A mutation did exist in the high myopia sample, while the control 1 and control 2 did not have mutations at this site. Figure 2-Figure 4 The above results illustrate that the high myopia screening method provided by the present invention has high sensitivity and specificity, and can be used for screening or preventive screening of myopia.

[0091] In summary, the specific primer pairs designed by the present invention, whose nucleotide sequences are respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the specific primer pairs whose nucleotide sequences are respectively shown in SEQ ID NO: 3 and SEQ ID NO: 4, as well as the reaction system and amplification conditions, are used for PCR detection, and can detect whether the OPN1LW gene has a c.843G>A mutation to predict high myopia, and can effectively identify high-risk patients who develop high myopia or hereditary high myopia at an early stage, and has the characteristics of high detection efficiency and low cost.

Claims

1. Detection OPN1LW The use of a reagent for the gene NM_020061.6:exon5:c.843G>A mutation in the preparation of a kit for high myopia screening or preventive screening; the high myopia screening is to screen individuals who have already developed high myopia; the preventive screening is to screen individuals who have a probability of developing high myopia in the later stage and issue a risk warning.

2. The use according to claim 1, characterized in that: The reagents include OPN1LW Amplification reagent for gene NM_020061.6:exon5 fragment.

3. The use according to claim 2, characterized in that: The amplification reagent includes amplification OPN1LW Primers for gene NM_020061.6:exon5 fragment.

4. The use according to claim 3, characterized in that: The primers include first-round amplification primers and second-round amplification primers; the substrate of the second-round amplification primers is the product of the first amplification.

5. The use according to claim 4, characterized in that: The sequences of the primers for the first round of amplification are shown in SEQ ID NO.1-SEQ ID NO.2; the sequences of the primers for the second round of amplification are shown in SEQ ID NO.3-SEQ ID NO.

4.

6. The use according to claim 1, characterized in that: The kit also includes a sequencing reagent for determining the sequence of the amplified product.

7. The use according to claim 6, characterized in that: The kit also includes a DNA extraction reagent and / or an RNA reverse transcription reagent.

Images