A keratoconus detection panel and its application

Abstract

The present invention provides a detection panel for keratoconus and its application, belonging to the field of biotechnology. The detection panel includes the following genes: VSX1, COL4A3, COL4A4, HGF, DOCK9, COL4A1, TUBA3D, KRT12, COL4A2, MPDZ, POLG, PARP1, ERCC8, MAP3K19, TGFBI, TNF‑α, TIMP‑1, MMP‑9, WNT10A, FNDC3B, IPO5, CENPF, CNBD1 and ZNF469. The detection panel of the present invention can detect mutant genes in a targeted manner, can realize accurate detection of keratoconus, and can effectively reduce the cost of detection.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a keratoconus detection panel and application thereof. Background Art

[0002] Keratoconus (KC) is a corneal disease characterized by corneal ectasia, thinning of the central corneal stroma, conical protrusions, and highly irregular myopic astigmatism. It is a primary degenerative disease and one of the important blinding eye diseases in clinical practice. It often occurs in young people and can also be seen in minors. The main manifestations of patients are progressive corneal morphological changes, highly irregular astigmatism, and decreased vision. It is characterized by corneal ectasia, which can cause the central part of the cornea to bulge forward, become thin and cone-shaped, and produce highly irregular astigmatism. At present, the treatment methods for early keratoconus are mainly corneal contact lenses and corneal collagen cross-linking. The most effective treatment for late keratoconus is corneal transplantation. However, on the one hand, there may be immune rejection reactions and corneal graft turbidity after surgery, requiring reoperation. On the other hand, corneal donors in my country are scarce, which undoubtedly prolongs the waiting time for patients and misses the best treatment opportunity.

[0003] The pathological mechanism of keratoconus is still unclear, and there is currently no therapeutic target closely related to the pathogenesis of the disease. The pathogenesis of keratoconus is quite complex, and there is no recognized, systematic theory to explain the occurrence and development of the disease. Related research has mostly focused on genetic factors, corneal biomechanics, and oxidative stress. Zhang Dongyan et al. (Correlation between aqueous humor sFas and sFasL levels and corneal morphological indicators in patients with keratoconus [J], Chinese Laboratory Diagnostics, November 2022, Vol. 26, No. 11) disclosed the correlation between aqueous humor sFas and sFasL levels and corneal morphological indicators in patients with keratoconus, and analyzed the correlation between aqueous humor sFas and sFasL levels and corneal morphological indicators in patients with keratoconus. With the decrease of aqueous humor sFas and sFasL levels, the corneal morphological changes of patients gradually intensified. There was downregulated expression of sFas and sFasL in the aqueous humor of patients with keratoconus, and their levels were correlated with the degree of corneal morphological changes in patients, suggesting that hyperfunction of the Fas and FasL system in corneal tissue caused by decreased aqueous humor sFas and sFasL levels may be the mechanism that promotes the occurrence and development of keratoconus. This conclusion was supported in subgroup analysis of patients of different ages and genders.

[0004] Chinese patent CN105256059B discloses an application of the TUBA3D gene in the preparation of a diagnostic product for detecting keratoconus (KC), providing a new use of the TUBA3D gene, thereby providing an effective way to perform genetic diagnosis of KC disease, prenatal genetic screening and genetic counseling. The application results show that the SNP sites and detection primers of the gene provided by the invention can be effectively used for rapid detection of TUBA3D gene mutation sites in clinical patients and fetal chorionic villi or amniotic fluid.

[0005] Studies have shown that high-throughput sequencing technology provides a convenient and effective detection method, which is of great significance for the improvement of genetic diagnosis. However, there is currently no dedicated panel for keratoconus detection. Summary of the invention

[0006] Terminology explanation:

[0007] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise. Numerical ranges recited by endpoints include all numbers and fractions within the corresponding ranges, as well as the recited endpoints.

[0008] Genetic testing panel refers to the collection of probes corresponding to several genes on the same capture chip to capture target DNA for subsequent gene sequencing. Genetic testing panel does not just detect one site or one gene, but detects multiple sites and multiple genes at the same time. These sites and genes need to be selected and combined according to specific standards.

[0009] In view of the problems existing in the prior art, the present invention provides a keratoconus detection panel and application thereof.

[0010] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0011] On the one hand, the present invention provides a detection panel for keratoconus, which includes the following genes: VSX1, COL4A3, COL4A4, HGF, DOCK9, COL4A1, TUBA3D, KRT12, COL4A2, MPDZ, POLG, PARP1, ERCC8, MAP3K19, TGFBI, TNF-α, TIMP-1, MMP-9, WNT10A, FNDC3B, IPO5, CENPF, CNBD1 and ZNF469.

[0012] Preferably, the detection reagents of the panel include: specific antibodies for the detection panel, specific binding molecules, primers or primer pairs for specific amplification of the detection panel, specific probes or chips.

[0013] In yet another aspect, the present invention provides use of the reagents of the above detection panel in the preparation of a keratoconus detection product.

[0014] Preferably, the product is a detection device or a detection kit.

[0015] In yet another aspect, the present invention provides a kit for detecting keratoconus, wherein the kit comprises reagents for detecting a panel.

[0016] Preferably, the kit comprises detection probes, and the detection probes are directed to the exon region and part of the intron region of the gene in the detection panel.

[0017] In some embodiments, the detection probe comprises a 5' detection probe and a 3' detection probe.

[0018] In some embodiments, the detection probes include one or more internal detection probes that are complementary to a region of the target nucleic acid between the detection targeting sequences of the 5' detection probe and the 3' detection probe.

[0019] In some embodiments, the 5' detection probe and any internal detection probe are phosphorylated at the 5' end. In some embodiments, the 5' detection probe and any internal detection probe are not phosphorylated at the 5' end.

[0020] Preferably, the detection probe is a probe targeting the gene mutation or copy number variation of the above detection panel.

[0021] Further preferably, the gene mutation includes point mutation, deletion, insertion and duplication.

[0022] Preferably, the kit further comprises a nucleic acid extraction reagent;

[0023] Further preferably, the nucleic acid extraction reagent includes but is not limited to tissue DNA extraction and purification reagent.

[0024] Preferably, the kit further comprises one or more of a fragmentation enzyme, T4 DNA ligase, DNA polymerase, PCR buffer, dNTP, and a quality control product.

[0025] Preferably, the detection method of the kit comprises the following steps:

[0026] (1) Extraction of sample DNA;

[0027] (2) Construction of sample DNA library: DNA fragmentation into small fragments, and library preparation using a library construction kit.

[0028] (3) constructing detection probes for the detection panel, hybridizing and capturing with the DNA library, and sequencing;

[0029] (4) Perform biological information analysis on the sequencing results to obtain sample sequence information;

[0030] (5) Compare the sample mutation result with the detection panel to obtain the gene mutation result of the sample, and combine it with clinical information to obtain the final diagnosis.

[0031] Specifically, the preparation of the Chinese library in step (2) mainly includes the following steps: DNA shearing, end filling, 3' end "A" addition, adapter ligation, purification, library amplification, purification. After the library is prepared, it is stored at -20°C.

[0032] Specifically, the sequencing result in step (4) is a fastq file, and the sequencing result is analyzed using a gene mutation algorithm to obtain the gene mutation result and annotate it.

[0033] More specifically, the step (4) includes quality control of fastq files, genome alignment, analysis of somatic mutations and germ cell mutations, and annotation.

[0034] Further preferably, the evaluation includes comparing sequencing data of tissues of keratoconus patients and healthy controls to determine gene mutations.

[0035] In another aspect, the present invention provides a use of the detection reagent for the detection panel as described above, wherein the use is one or more of the following:

[0036] (1) Used to assess the risk of keratoconus;

[0037] (2) Used for prognostic assessment of patients with keratoconus;

[0038] (3) Used for screening patients with keratoconus.

[0039] Preferably, the sample to be tested is selected from one or more of a tissue sample, a blood sample, an aqueous humor sample, and a cell sample.

[0040] In another aspect, the present invention provides a device for detecting keratoconus, the device comprising:

[0041] (1) A sequencing module is used to extract DNA from the sample to be tested and perform high-throughput sequencing to obtain sequencing results;

[0042] (2) a comparison module, which is used to process the results of high-throughput sequencing and compare the data with the panel to obtain mutation information;

[0043] (3) Analysis module, used to analyze the acquired mutation information and derive clinical guidance plans.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The present invention discloses a keratoconus detection panel, which can detect keratoconus-related mutant genes in a targeted manner, thereby achieving accurate detection of keratoconus, effectively reducing detection costs, and having wide universality. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Frequency distribution of gene mutations detected for 90 subjects. DETAILED DESCRIPTION

[0047] The present invention is described below by specific examples to make the technical solution of the present invention easier to understand and grasp, but the present invention is not limited thereto, and the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work should belong to the scope of protection of this application. The experimental methods described in the following examples, unless otherwise specified, are conventional methods; the reagents and materials, unless otherwise specified, can be obtained from commercial channels.

[0048] Example 1

[0049] In Example 1 of the present invention, a keratoconus detection panel is provided, comprising the following genes (Table 1):

[0050] Table 1.

[0051]

[0052]

[0053] Example 2

[0054] The next generation sequencing method for detecting keratoconus comprises the following steps:

[0055] 1. Extraction of sample DNA

[0056] DNA extraction from samples: DNA was extracted from the whole blood samples of the subjects using a DNA extraction kit.

[0057] Quality testing of sample DNA: Nanodrop was used to test the purity of DNA, and Qubit was used to test the concentration of DNA.

[0058] DNA samples with no degradation, no RNA contamination, OD260 / OD280 ratio between 1.8-2.0, and a total amount of more than 1.5 μg were used to construct the library.

[0059] 2. DNA library preparation

[0060] (1) Genomic DNA fragmentation

[0061] In this case, the extracted DNA samples were fragmented using a Covaris M220 ultrasonic disruptor. The sample was diluted to 6 ng / μL with TE in a 130 μL fragmentation tube, with a total volume of 55 μL; the fragmentation time was 50 s. The fragmentation product can be stored at -20°C.

[0062] (2) End repair and adding "A"

[0063] a. Take out the broken sample to be tested, dissolve it, mix it evenly, centrifuge it, and place it on ice.

[0064] b. Turn on the PCR instrument and set the end repair and A addition program: 20℃ for 15 min, 72℃ for 15 min, and finally standby at 4℃.

[0065] c. Prepare the end repair and 3' end A tailing buffer, including 16μL End Repair & A Tailing Buffer and 4μL End Repair & A Tailing Enzyme Mix. Vortex the end repair and 3' end A tailing buffer at high speed for 5-10 seconds to mix thoroughly, centrifuge briefly, and place on ice.

[0066] d. Add 20 μL of end repair solution and 3' end addition A mixture to the sample tubes respectively, vortex for 5-10 seconds to mix thoroughly, and centrifuge instantly.

[0067] e. After checking that the PCR program is correct, place the sample tube on the PCR instrument and start the program with the lid of the PCR instrument open.

[0068] f. When the PCR program temperature drops to 4°C, remove the sample and place it on ice for later use.

[0069] (3) Connector

[0070] a. After the above procedures are completed, set the connection joint program: 20℃30min, 4℃ standby.

[0071] b. Prepare the adapter ligation mixture, which includes: 23 μL of Ligation Buffer and 2 μL of T4 DNA Ligase, with a total volume of 25 μL.

[0072] c. Vortex the adapter ligation mixture at high speed for 15 seconds to mix thoroughly and centrifuge briefly.

[0073] d. Add 5 μL of Adaptor Oligo Mix to the 75 μL PCR tube containing the product after addition of A and vortex for 5-10 seconds.

[0074] e. Add 25 μL of adapter ligation mix to each sample tube, vortex for 5-10 seconds, and centrifuge briefly.

[0075] f. After checking that the PCR program is correct, place the sample tube into the PCR instrument and run the program.

[0076] g. When the temperature of the PCR instrument drops to 4°C, take out the reaction tube and centrifuge briefly.

[0077] (4) Purification of ligation product

[0078] a. Take the ligation product and centrifuge briefly, then add 50 μL XP magnetic beads (equilibrated at room temperature for 30 min in advance);

[0079] b. Mix by shaking, incubate at room temperature for 10 minutes to allow DNA to fully bind to the magnetic beads, centrifuge briefly, place the beads on a magnetic rack for 5 minutes, and discard the supernatant;

[0080] c. Add 200 μL 80% ethanol and incubate for 30 seconds, then discard and repeat the 200 μL 80% ethanol washing step;

[0081] d. After briefly centrifuging the centrifuge tube, place it on a magnetic rack and use a gun to suck out all the ethanol at the bottom of the centrifuge tube. Dry it at room temperature for 3-5 minutes until the ethanol is completely evaporated.

[0082] e. Remove the centrifuge tube, add 22 μL ultrapure water, shake to mix, and incubate at room temperature for 5 minutes;

[0083] f. Centrifuge briefly, place on a magnetic rack for 3 minutes, and transfer 20 μL of the supernatant to a new centrifuge tube.

[0084] (5) Prelibrary preparation

[0085] a. Set the PCR reaction program: 98℃ for 2 min, then enter 6 cycles: 98℃ for 30 s, 60℃ for 30 s, 72℃ for 1 min. After the cycle, 72℃ for 5 min, and finally standby at 4℃.

[0086] b. Take out the purified ligation product, flick it to mix, centrifuge it instantly, and place it on ice for later use.

[0087] c. Assign a different Index Primer to each sample.

[0088] d. Prepare the pre-library amplification mixture including: 5×Herculase II Reaction Buffer 10μL, Forward Primer 2μL, 25mM dNTP Mix 0.5μL, Herculase II Fusion DNA Polymerase 1μL and nuclease-free water 14.5μL, with a total volume of 28μL.

[0089] e. Add 28 μL of pre-library amplification mixture to the sample tube, then add 2 μL of IndexPrimer to the sample tube, vortex to mix, and centrifuge to collect the liquid.

[0090] f. After checking that the PCR program is correct, place the sample tube into the PCR instrument, cover the PCR instrument's hot cover and run the program.

[0091] g. After the reaction is completed, take out the reaction tube, centrifuge it instantly, and place it on ice for later use.

[0092] (6) Pre-library purification

[0093] a. Place the XP magnetic beads at room temperature for 30 minutes, prepare fresh 80% ethanol, and mix thoroughly.

[0094] b. Fully oscillate the XP magnetic beads for at least 1 minute to mix them completely.

[0095] c. Add 50 μL XP magnetic beads to the pre-library amplification product, vortex for 5-10 seconds to mix, and incubate at room temperature for 10 minutes.

[0096] d. Centrifuge the sample tube instantaneously and place it on a magnetic rack for 5 minutes until the solution is completely clear. Use a pipette to carefully discard the supernatant, making sure the pipette tip does not touch the magnetic beads.

[0097] e. Add 200 μL of 80% ethanol, incubate for 30 seconds, then discard, and repeat the 200 μL 80% ethanol washing step;

[0098] f. After instant centrifugation, place the tube on a magnetic rack for adsorption, use a pipette to absorb all the ethanol at the bottom of the tube, and dry it at room temperature for 3-5 minutes until the ethanol is completely evaporated.

[0099] g. Remove the sample tube from the magnetic rack, add 22 μL of ultrapure water, vortex until the magnetic beads are completely mixed, centrifuge briefly, and incubate at room temperature for 5 minutes.

[0100] h. Briefly centrifuge and place the centrifuge tube on a magnetic rack for 3 minutes. When the liquid is clear, transfer 21 μL of the supernatant to a new centrifuge tube and store it at -20°C.

[0101] (7) Library quality control

[0102] Take 1 μL of DNA library to detect its concentration.

[0103] 3. Library hybridization capture

[0104] The detection panel and hybridization capture reagent of the present invention are used to perform library hybridization capture, and the operation process is carried out according to the product instructions.

[0105] (1) The total amount of library sample loaded is 1 μg. Calculate the volume of each library added. Volume of library added (μL)

[0106] = 1000ng / capture library number / library concentration (ng / μL). Add 2.5μL blocking oligonucleotide to the library mixture, add 5μL COT Human DNA, shake and mix, and centrifuge briefly.

[0107] (2) Library and probe hybridization: Add the reaction system (the reaction system includes 7.5 μL of hybridization buffer, 3 μL of hybridization enhancer, and a total volume of 10.5 μL) to the above system, vortex to mix, centrifuge briefly, and place in a 95°C heating block for denaturation for 10 min. Take out 4.5 μL of probe and add it to the system, vortex to mix, centrifuge briefly, place in a PCR instrument, and hybridize at 47°C.

[0108] (3) Library capture

[0109] a. Prepare the elution working solution according to the instructions;

[0110] b. Preparation of capture magnetic beads: Each capture aliquot is 100 μL capture magnetic beads, and 100 μL capture magnetic beads are placed on a magnetic rack until the liquid is clear, and the supernatant is discarded. Add 200 μL 1× magnetic bead washing solution, shake and mix, place on a magnetic rack until the liquid is clear, and then discard the supernatant. Add 100 μL 1× magnetic bead washing solution, shake and mix, place on a magnetic rack until the liquid is clear, and discard the supernatant for later use.

[0111] c. Transfer the hybridization liquid captured overnight into the washed magnetic beads, place in a PCR instrument and incubate at 47°C for 45 minutes, shaking every 15 minutes.

[0112] d. After incubation, add 100 μL of 1× elution working solution 1 preheated at 47°C to each tube and shake to mix. Place on a magnetic rack until the liquid is clear and discard the supernatant.

[0113] e. Add 200 μL of 1× elution working solution 4 preheated at 47°C, pipette and mix ten times. Incubate at 47°C for 5 min, place on a magnetic rack until the liquid is clear, and discard the supernatant. Repeat the above steps once.

[0114] f. Add 200 μL of 1× elution working solution 4 preheated at 47°C and mix well by pipetting ten times. Incubate at 47°C for 5 min, place on a magnetic rack until the liquid becomes clear, and discard the supernatant.

[0115] g. Add 1× elution working solution 1 according to the above steps, oscillate for 2 minutes, and follow the remaining steps as above.

[0116] h. Add 1× elution working solution 2 according to the above steps, shake for 1 min, and follow the rest of the steps as above.

[0117] i. Add 1× elution working solution 3 according to the above steps, shake for 30 seconds, and follow the rest of the steps as above.

[0118] j. Add 22 μL of ultrapure water to the centrifuge tube for elution, shake and mix, let stand for 5 minutes, then adsorb on a magnetic rack for 3 minutes, and aspirate 20 μL of supernatant for the next amplification test.

[0119] (4) Post-capture PCR:

[0120] Add the reaction system (50 μL of amplification premix, 10 μL of primer (5 μM), 20 μL of nuclease-free water) to the captured product, vortex to mix, centrifuge, and place in a PCR instrument. The reaction program is as follows: 98°C, 45 s; (98°C, 15 s; 60°C, 30 s; 72°C, 30 s; 13 cycles); 72°C, 1 min, and store at 4°C.

[0121] Post-amplification purification:

[0122] Take 180μL of purified magnetic beads in a 1.5mL centrifuge tube, add 100μL of amplified captured DNA library, shake to mix, and incubate at room temperature for 15min. Place on a magnetic stand until the liquid is clear and discard the supernatant. Add 200μL 80% ethanol and incubate for 30sec and discard. Repeat the 200μL 80% ethanol washing step. Use a 10μL pipette tip to discard the residual ethanol at the bottom of the centrifuge tube and dry at room temperature until the ethanol is completely evaporated. Remove the centrifuge tube from the magnetic stand, add 50μL ultrapure water, and shake to mix. Incubate at room temperature for 2min. Centrifuge briefly, place on a magnetic stand until the liquid is clear, and transfer the captured sample to a new centrifuge tube. Take 1μL of the captured sample for Qubit concentration detection.

[0123] Example 3

[0124] According to the method of Example 2, 90 subjects were subjected to DNA extraction, shearing, end repair, ligation, first PCR, specific capture of gene panel, and second PCR amplification to obtain DNA samples to be sequenced. The DNA samples were sequenced after meeting the requirements of the machine. The sequencing results were evaluated. The gene mutation frequencies detected in the 90 subjects were as follows: Figure 1As shown in Table 2. The test results are consistent with the actual situation, proving that the gene panel for detecting keratoconus prepared in this example can detect whether a subject suffers from keratoconus or the risk of suffering from keratoconus.

[0125] Table 2.

[0126]

[0127]

[0128] The details of the genetic test results are shown in Table 3 below:

[0129] Table 3.

[0130]

[0131]

[0132] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A kit for detecting keratoconus, characterized in that: The kit includes a reagent for detecting a panel, and the reagent detects the gene mutation site of the panel; the mutation site of the VSX1 gene is c.5427G>C, c.452A>G, c.7672G>T, c.496C>T, c.650G>A, c.740C>G, c.715G>C, the mutation site of the COL4A3 gene is c.976G>T, c.422T>C, the mutation site of the COL4A4 gene is c.1444C>T, c.3979G>A, c.4548A>G, c.4932C>T, c.3636_3637del, the mutation site of the HGF gene is c.1757+143G>A, c.1041-116A>T, the mutation site of the DOCK9 gene is c.262A>C, c.1940C>T, the mutation sites of COL4A1 gene are c.19G>C, c.1663A>C, c.4002A>C, c.3693G>A, the mutation sites of TUBA3D gene are c.31C>T, c.201_202insT, c.1355G>A, the mutation sites of KRT12 gene are c.1456_1457insGAT, the mutation sites of COL4A2 gene are c.574G>T, c.1550G>A, c.2048G>C, c.2102A>G, c.2152C>T, the mutation sites of MPDZ gene are c.5015G>T, c.3798dupA, c.28G>A, c.394G>A, the mutation sites of POLG gene are c.127_128insGGC, c.3139C>T, c.2890C>T, the mutation sites of PARP1 gene are c.2440A>G, c.114G>A, c.14C>G; The detection reagents of the panel include: primers or primer pairs for specific amplification of the detection panel, specific probes or chips.

2. The kit according to claim 1, characterized in that The kit comprises a detection probe, and the specific probe is a probe for the gene mutation or copy number variation of the detection panel according to claim 1.

3. The kit according to claim 1, characterized in that The detection method of the kit comprises the following steps: (1) Extraction of sample DNA; (2) Construction of sample DNA library: fragment the DNA into small fragments and prepare the library using a library construction kit; (3) Construct detection probes for the detection panel, hybridize with the DNA library for capture, and sequence; (4) Perform biological information analysis on the sequencing results to obtain sample sequence information; (5) Compare the sample mutation results with the detection panel to obtain the gene mutation results of the sample, and combine them with clinical information to obtain the final diagnosis.

4. The kit according to claim 1, characterized in that The sample to be tested is selected from one or more of tissue samples, blood samples, aqueous humor samples, and cell samples.

5. A keratoconus detection device, characterized in that: The detection device comprises: (1) Sequencing module, used to extract DNA from the sample to be tested and perform high-throughput sequencing to obtain sequencing results; (2) Comparison module, which is used to process the results of high-throughput sequencing and compare the data with the panel to obtain mutation information; (3) Analysis module, used to analyze the acquired mutation information and derive clinical guidance plans; The panel described in step (2) includes the following gene mutation sites: The mutation sites of VSX1 gene are c.5427G>C, c.452A>G, c.7672G>T, c.496C>T, c.650G>A,c.740C>G, c.715G>C, the mutation sites of COL4A3 gene are c.976G>T, c.422T>C, the mutation sites of COL4A4 gene are c.1444C>T, c.3979G>A, c.4548A>G, c.4932C>T, c.3636_3637del, the mutation sites of HGF gene are c.1757+143G>A, c.1041-116A>T, the mutation sites of DOCK9 gene are c.262A>C, c.1940C>T, the mutation sites of COL4A1 gene are c.19G>C, The mutation sites of TUBA3D gene are c.31C>T, c.201_202insT, c.1355G>A, the mutation sites of KRT12 gene are c.1456_1457insGAT, the mutation sites of COL4A2 gene are c.574G>T, c.1550G>A, c.2048G>C, c.2102A>G,c.2152C>T, the mutation sites of MPDZ gene are c.5015G>T, c.3798dupA, c.28G>A, c.394G>A, the mutation sites of POLG gene are c.127_128insGGC, c.3139C> T, c.2890C>T, the mutation sites of PARP1 gene are c.2440A> G, c.114G>A, c.14C>G.

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