Detection method and application of human mitochondrial gene ND1 mutation

Abstract

The invention discloses a detection method and application of human mitochondrial gene ND1 mutation. The mutation is m.3394 Tgt; and C, the mutation as the Parkinson's disease biomarker has high sensitivity and specificity, and has a good application prospect in early diagnosis of Parkinson's disease.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and medical clinical testing, and relates to the detection and application of human mitochondrial NADH-coenzyme Q reductase subunit 1 (ND1) gene mutation associated with Parkinson's disease. Background Art

[0002] Parkinson's disease (PD) is a complex, multifactorial degenerative neurological disorder characterized by bradykinesia, tremor, and rigidity. (In its early stages, PD presents with only mild tremor, bradykinesia, and unilateral limb stiffness, often mistaken for "natural aging." Non-motor symptoms such as constipation, hyposmia, and depression often precede motor symptoms by years, but lack specificity and are easily overlooked.) As the disease progresses, these symptoms gradually limit patients' ability to move freely, severely interfering with daily life and compromising their quality of life. However, the clinical symptoms of PD overlap significantly with those of many other neurodegenerative diseases, making accurate diagnosis based on clinical manifestations in the early stages of the disease difficult. The gold standard for confirming PD remains brain tissue pathology (detection of Lewy bodies), but this can only be performed after the patient's death. Furthermore, PD develops insidiously, often being diagnosed only after neuronal degeneration has reached a more advanced stage, missing the optimal time for intervention. Therefore, the development of early biomarkers that can specifically identify PD has become the key to preventing and effectively controlling the disease.

[0003] In recent years, extensive research has been conducted on biomarkers for Parkinson's disease (PD). While these biomarkers have provided auxiliary information for clinical diagnosis, their specificity is low, making them difficult to meet the needs of precision medicine. Currently, the search for PD biomarkers primarily focuses on the nuclear genome, but highly specific biomarkers derived from the nuclear genome are still lacking. The mitochondrial genome, another potential source for biomarkers, remains relatively underrepresented in current research regarding its genetic information and potential links to the pathogenesis of diseases such as PD.

[0004] Mutations in mitochondrial DNA (mtDNA), the genetic material found in mitochondria, are closely associated with mitochondrial dysfunction and dopaminergic neuron death. Several known mtDNA mutations are closely associated with human mitochondrial diseases, including the m.11778 G>A mutation causing Leber hereditary optic neuropathy (LHON), the m.3243 A>G mutation causing mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), the m.8344 A>G mutation causing myoclonic epilepsy with red tearing fibers (MERRF), and the m.8993 T>G / C mutations causing neuromyasthenia-ataxia-retinopathy (NARP) and Leigh syndrome. Furthermore, the m.1555 A>G mutation can cause nonsyndromic hearing loss associated with aminoglycoside antibiotics, while large mtDNA deletions are closely associated with various clinical phenotypes, including Kearns-Sayre syndrome, Pearson syndrome, and chronic progressive external ophthalmoplegia (CPEO). mtDNA mutations can damage the function of the mitochondrial electron transport chain, leading to energy metabolism disorders, increased oxidative stress and cell apoptosis, and thus may play an important role in the pathogenesis of neurodegenerative diseases, especially Parkinson's disease. For example, "Study on the Effect of LRRK2 G2385R Mutation on Mitochondrial Function and Its Mechanism" pointed out that the LRRK2 G2385R mutation, which is closely related to PD, can significantly downregulate the mitochondrial genome (including MT-ND1) encoding oxidative phosphorylation (OXPHOS) proteins.

[0005] Human mitochondrial genes ND1 ( hND1 ) mutations can cause neuronal degeneration. However, the currently available hND1 Gene mutations are not associated with the production of PD-related phenotypes, such as the various known hND1 Mutations are closely associated with Leber hereditary optic neuropathy (LHON), including m.3460 G>A (A52T); hND1 The mutation m.3394 T>C (Y30H) is associated with diabetes (reference: Mitochondrial haplotypes and mitochondrial-related human diseases. Progress in Biochemistry and Biophysics, 2016, 43 (11): 1070-1075.). Summary of the Invention

[0006] The purpose of the present invention is to provide a human mitochondrial gene ND1 The detection methods and applications of mutations can provide new ideas and basis for the development of early diagnostic reagents for Parkinson's disease, thereby accurately identifying patients through specific biomarkers in the early stages of the disease to achieve timely intervention.

[0007] To achieve the above object, the present invention adopts the following technical solutions: In a first aspect, a method for detecting human mitochondrial gene mutations is provided, comprising the following steps: Whole genome DNA was extracted from the specimens, and PCR amplification was performed using the DNA as a template and primers. The mutation at position 3394 of the mitochondrial DNA of the specimens was determined based on the amplification results.

[0008] Preferably, the primers are based on mitochondrial genes ND1 The mutation m.3394 T>C was designed at the site where it is located.

[0009] Preferably, the primers are specifically: Forward primer: 5'-AGGGTTTGTTAAGATGGCAGAG-3' (i.e. SEQ.ID.NO.1) Reverse primer: 5'-ATAGTAGAAGAGCGATGGTGAGA-3' (i.e., SEQ.ID.NO.2).

[0010] Preferably, when a mutation occurs at position 3394 of the mitochondrial DNA, the genotype of the site is a mutant type (ie, C / C), otherwise the genotype is a non-mutated type (ie, T / T). Preferably, the genotype is obtained by a typing method of sequencing the amplification result (ie, PCR product).

[0011] Preferably, the samples are selected from patients with neurodegenerative diseases such as Parkinson's disease (for example, patients with Parkinson's disease may show motor symptoms or may not show motor symptoms) and high-risk groups of related diseases (especially Parkinson's disease) (for example, patients who have already had mitochondrial gene deletion ND1 Any one of the following: susceptible persons with other pathogenic factors other than mutation m.3394 T>C.

[0012] Preferably, the sample is blood.

[0013] Preferably, the biological function verification of the mutation m.3394 T>C comprises the following steps: hND1 Overexpression vector of mutant and overexpression of corresponding mitochondrial mutant gene in Parkinson's disease model cells using the overexpression vector hND1 (T3394C).

[0014] Preferably, the construction hND1 The mutant overexpression vector specifically includes the following steps: first, transforming the pcDNA3.1(+) vector, specifically inserting the gene fragment of the mitochondrial localization signal (MTS) after the CMV promoter on the vector; then, hND1 (T3394C) was integrated into the modified pcDNA3.1(+) vector containing the MTS gene fragment (i.e., pcDNA3.1(+)-MTS).

[0015] Preferably, to verify the overexpression efficiency of the target gene, it is constructed into an expression vector containing a FLAG tag, and the expression of the FLAG-tagged protein is detected by Western blot.

[0016] Preferably, the overexpression specifically comprises the following steps: hND1 The mutant overexpression vector was transfected into SH-SY5Y cells.

[0017] Preferably, the biological function verification of the mutation m.3394 T>C further comprises the following steps: hND1 Transcriptome sequencing was performed on Parkinson's disease model cells (such as the above-mentioned SH-SY5Y cells) of T3394C, and overexpression was analyzed by mitochondrial function test and immunofluorescence staining. hND1 The difference between cells transfected with T3394C and control cells transfected with an empty vector was observed, thereby verifying the impact of this mutation on Parkinson's disease.

[0018] Preferably, the effects include a decrease in the expression level of tyrosine hydroxylase (TH) and an increase in the expression level of phosphorylated α-synuclein (α-Syn).

[0019] In a second aspect, a method for providing a detection reagent for a human mitochondrial gene mutation in the preparation of an early diagnosis reagent for Parkinson's disease is provided. ND1 Mutation m.3394 T>C.

[0020] Preferably, the detection reagent includes primers for amplifying the mitochondrial genomic site where the mutation is located (i.e., position 3394 of mitochondrial DNA) and its surrounding areas (e.g., primers for PCR-sequencing method, see SEQ.ID.NO.1 and SEQ.ID.NO.2).

[0021] The beneficial effects of the present invention are embodied in: The present invention identified a mtDNA mutation associated with Parkinson's disease by extracting genomic DNA, building a whole mitochondrial library and sequencing. ND1 mutation m.3394 T>C; and by transfecting Parkinson's disease model cells (such as SH-SY5Y cells) with the mutation ND1 Gene (i.e. hND1(T3394C)) was verified by combining the signaling pathways of differentially enriched genes in the transcriptome, mitochondrial function, and cell immunofluorescence. It is expected to provide new biomarkers for the early diagnosis of Parkinson's disease, accurately identify patients through specific biomarkers, thereby achieving timely intervention, changing the current situation of lagging diagnosis and treatment of Parkinson's disease, and promoting major progress in the prevention and treatment of Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the frequency distribution of Parkinson's disease-specific high-frequency SNPs.

[0023] Figure 2 For overexpression hND1 (T3394C) Transcriptome differential expression analysis in the experiment.

[0024] Figure 3 For overexpression hND1 (T3394C) Mitochondrial function test in the experiment.

[0025] Figure 4 For overexpression hND1 (T3394C) Immunofluorescence staining in the experiments.

[0026] Figure 5 for hND1 ROC curve of mutation m.3394 T>C in the diagnosis of Parkinson's disease.

[0027] Figure 6 The confusion matrix under the optimal threshold (5.25%) is as follows: True Negative (TN) - the actual negative is predicted to be negative; False Positive (FP) - the actual negative is predicted to be positive; False Negative (FN) - the actual positive is predicted to be negative; True Positive (TP) - the actual positive is predicted to be positive.

[0028] Figure 7 The sensitivity and specificity vary with the mutation load threshold. DETAILED DESCRIPTION

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and examples, which are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0030] (I) Construction of mitochondrial gene libraries based on PD patients and non-PD controls 1.1 Blood sample collection This experiment involved a total of 53 peripheral blood samples collected from PD patients and non-PD control subjects. All subjects signed informed consent forms and the results were approved by the ethics committee.

[0031] Inclusion criteria for subjects in the PD group included: (i) meeting the UK Parkinson's Disease Society Brain Bank Criteria; (ii) being diagnosed with idiopathic Parkinson's disease (PD) by a neurologist; and (iii) having no severe cognitive impairment or other major systemic diseases. Exclusion criteria included: (i) having secondary Parkinson's syndrome or other neurodegenerative diseases (such as MSA, PSP, etc.); (ii) having a family history of hereditary movement disorders or cerebrovascular diseases; and (iii) having recently taken medications that affect the function of the nervous system.

[0032] Inclusion criteria for the non-PD control group included: (i) healthy volunteers without neurological diseases or patients seeking medical treatment for non-neurological diseases whose age and gender matched those of the PD group; (ii) no history of Parkinson's disease or other neurodegenerative diseases. Exclusion criteria included: (i) a history of psychiatric or neurological diseases; (ii) a history of obvious movement disorders or anti-Parkinson's disease treatment.

[0033] Based on the study design and statistical requirements, the sample size for the two groups was set at 33 in the PD group and 20 in the non-PD control group to ensure the scientific and reproducible nature of subsequent group sequencing and data analysis. All samples were obtained from the Department of Neurosurgery, Second Affiliated Hospital, Air Force Medical University, and blood was collected between January 1, 2024, and December 31, 2024.

[0034] 1.2 Extraction of human peripheral blood genomic DNA The TIANGEN blood / cell / tissue genomic DNA extraction kit (DP304) was used for extraction. The specific steps are as follows: To a 1.5 mL centrifuge tube, add 200 µL of whole blood and 200 µL of Buffer GA containing a lysing agent and mix thoroughly. Next, add 20 µL of Proteinase K and mix thoroughly. Add 200 µL of Buffer GB and mix thoroughly by inversion. Incubate at 70°C for 10 min until the solution becomes clear and centrifuge briefly. Add 200 µL of anhydrous ethanol, vortex for 15 s, and centrifuge briefly. Transfer the entire mixture to an adsorption column (in a collection tube) and centrifuge with 500 µL of Buffer GD at 12,000 rpm for 30 s. Discard the waste solution. Add 600 µL of Buffer PW at 12,000 rpm for 30 s and discard the waste solution (repeat this step twice). Centrifuge the empty tube for 2 min. After incubating at room temperature for 2-5 min, place the adsorption column in a new 1.5 mL centrifuge tube and add 30 µL of Buffer TE. Incubate at room temperature for 2 min, then centrifuge at 12,000 rpm for 2 min to collect the eluted DNA.

[0035] 1.3 Construction of a complete mitochondrial DNA library 1.3.1 Sample processing For details on blood sample collection and genomic DNA extraction, see 1.1 and 1.2.

[0036] 1.3.2 First round of PCR When using high-fidelity enzymes for PCR, you need to use a pipette tip with a filter when adding primers and samples to the prepared PCR reaction system (see Table 1) to prevent aerosol contamination.

[0037] Table 1. Reaction system

[0038] Note: The high-fidelity enzyme in Table 1 is Phanta Max Super-Fidelity DNA Polymerase (Cat. No.: P505-d1) from Novozymes.

[0039] Table 2. Primers

[0040] Note: In Table 2, T200 / T201 (Forward Primer / Reverse Primer) amplifies the first segment, and T202 / T203 amplifies the second segment.

[0041] The first round of PCR was performed for 25 cycles using primers that amplify the full length of mitochondrial DNA (in two segments) (see Table 2). Specific PCR reaction conditions were: 95°C for 30 seconds, 25 cycles of (95°C for 15 seconds, 60°C for 15 seconds, 72°C for 8 minutes), 72°C for 5 minutes, and a hold temperature of 16°C.

[0042] 1.3.3 Transposase Tn5 interruption PCR products with bands were excised and recovered from the gel. After recovery, concentration was measured using a Qbit. A Tn5 reaction system (see Table 3) was prepared using the Novozymes kit (Cat. No. TD501 / 502 / 503). Reaction conditions were: 55°C for 10 min (with heated lid at 105°C), and a hold at 10°C.

[0043] Table 3. Reaction system

[0044] The product is recovered immediately, and the specific steps are as follows: (1) Vortex and mix the VAHTS DNA Clean Beads and pipette 10 μL into an RNase Free PCR tube pre-added with 10 μL of fragmented product. Vortex or pipette 10 times to mix thoroughly and incubate at room temperature for 5 minutes.

[0045] (2) Briefly centrifuge the RNase Free PCR tube and place it on a magnetic rack to separate the magnetic beads from the liquid. After the solution is clear (about 5 minutes), carefully remove the supernatant.

[0046] (3) Keep the RNase Free PCR tube on the magnetic stand at all times, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

[0047] (4) Repeat step 3 for a total of two rinses.

[0048] (5) Keep the RNase Free PCR tube on the magnetic rack at all times and air dry it with the lid open for about 5 minutes.

[0049] (6) Remove the RNase Free PCR tube from the magnetic stand, add 10.5 μL of sterile ultrapure water for elution, vortex or pipette 10 times to mix thoroughly, and incubate at room temperature for 5 minutes.

[0050] (7) Briefly centrifuge the RNase Free PCR tube and place it on a magnetic rack to separate the magnetic beads from the liquid. After the solution is clear (about 5 minutes), carefully pipette 8 μL of the supernatant into a new sterilized PCR tube.

[0051] 1.3.4 Enrichment and indexing The PCR reaction system is shown in Table 4. The reaction conditions are: 72°C, 3 min; 98°C, 30 s; (98°C, 15 s, 63°C, 30 s, 72°C, 1 min) × 6 cycles; 72°C, 5 min; hold at 4°C.

[0052] Table 4. Reaction system

[0053] The reference sequence of index is as follows: Each sample uses a specific index combination (N101 is fixed, and N7 is an index selected from N901 to N996 shown in Table 5). For example, sample 1 uses N101 and N901, sample 2 uses N101 and N925, and sample 3 uses N101 and N949. The purpose of amplifying and adding different indices is to achieve mixed sequencing of multiple samples, thereby improving sequencing efficiency and reducing costs.

[0054] Table 5. Index combinations and sequences (5'-3') of N7 and N101

[0055] 1.3.5 Double-round screening of amplified products (1) Transfer the PCR product to an RNase-Free PCR tube and add water to make up to 50 μL.

[0056] (2) Add 0.65×Beads (50×0.65=32.5 μL), vortex to mix, centrifuge briefly, and let stand for 5 minutes.

[0057] (3) Place the RNase Free PCR tube on a magnetic stand to separate the magnetic beads from the liquid (about 5 minutes), and aspirate the supernatant into a new RNase Free PCR tube (this step removes large fragments adsorbed by the beads).

[0058] (4) Add 0.15× beads (50×0.15=7.5 μL) to a new RNase-Free PCR tube, vortex to mix, centrifuge briefly, and let stand for 5 minutes.

[0059] (5) Briefly centrifuge the RNase Free PCR tube and place it on a magnetic rack to separate the magnetic beads from the liquid. After the solution is clear (about 5 minutes), carefully remove the supernatant (this step removes small fragments that are not adsorbed).

[0060] (6) Keep the RNase Free PCR tube on the magnetic stand at all times, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

[0061] (7) Repeat step 6 for a total of two rinses.

[0062] (8) Keep the RNase Free PCR tube on the magnetic rack at all times and air dry it with the lid open for about 5 minutes.

[0063] (9) Remove the RNase Free PCR tube from the magnetic stand, add 12.5 μL of sterile ultrapure water for elution, vortex or pipette 10 times to mix thoroughly, and incubate at room temperature for 5 minutes.

[0064] (10) Briefly centrifuge the RNase Free PCR tube and place it on a magnetic rack to separate the magnetic beads from the liquid. After the solution is clear (about 5 minutes), carefully pipette 10 μL of the supernatant into a new sterilized PCR tube.

[0065] (11) Use Qbit to measure concentration.

[0066] 1.3.6 Final library mixing The library output requirement is 6G, and 1G is sufficient for each sample. Therefore, multiple samples need to be mixed and mixed according to different N7 indices. Specific requirements for mixing: each mixed sample uses the same ng amount, the total volume is greater than 10 μL, and the total concentration is greater than 2ng / μL.

[0067] (2) Related to Parkinson's disease (PD) hND1 Identification of mutations The two sets of libraries were sequenced using the Illumina platform, with a sequencing strategy of PE150 and a data volume of 0.2 Gb.

[0068] By analyzing the sequencing data, the results are as follows Figure 1 As shown, compared with the non-PD control group, mutations unique to the PD group included synonymous mutations: m.6641 T>C, m.7142 T>C, m.9242 A>G, m.9545 A>G, and m.9950 T>C; mt-RNR1 mutation: m.1041 A>G; D-loop region mutations: m.16316 A>G, m.16179 C>A, m.16140 T>C, and m.153 A>G; and missense mutations: m.4491 G>A and m. 3394 T>C. Among these missense mutations, m. 3394 T>C was the most frequently detected site.

[0069] (three) hND1 Biological function of the mutation m. 3394T>C 3.1 Construction of pcDNA3.1(+)-MTS vector pcDNA3.1(+) plasmid (Addgene) Nhe Ⅰ / Hind Ⅲ Double enzyme digestion; the enzyme digestion system is shown in Table 6, and the reaction conditions are: 37°C, 2h.

[0070] Table 6. Enzyme digestion system

[0071] The MTS fragment was amplified using the following primers MTS-F and MTS-R: MTS-F: 5'-GCAGCTAGCattggggtttgtgggtcgG-3' MTS-R: 5'-GCTAAGCTTgccggggctcccgcctc-3' The PCR reaction system is shown in Table 7. The reaction conditions were: 98°C for 2 min, 30 cycles of (98°C for 10 s, 58°C for 5 s, and 72°C for 10 s), and 72°C for 5 min. After the reaction, the gel was recovered (TIANGEN DP209).

[0072] Table 7. Reaction system

[0073] The above-mentioned MTS plasmid (from the literature: A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. David R Liu et al. Nature. 2020. doi:10.1038 / s41586-020-2477-4) provides a template for cloning the gene fragment of the mitochondrial localization signal (MTS) (i.e., the MTS fragment).

[0074] Use T4 DNA ligase to ligate the digested pcDNA3.1(+) backbone and the amplified MTS fragment to create the pcDNA3.1(+)-MTS vector. The ligation system is shown in Table 8. The reaction conditions are: 16°C for at least 4 hours or overnight. After the reaction, place the vector in a refrigerator or on ice until ready for use.

[0075] Table 8. Connection system

[0076] Note: The molar ratio of pcDNA3.1(+) backbone to MTS fragment in Table 8 is 1:3.

[0077] The ligation product was transformed into Escherichia coli DH5a competent cells, and after the bacterial solution was revived, it was spread onto an ampicillin-resistant plate, cultured overnight, and screened; a single colony was picked for detection and subsequent experiments.

[0078] 3.2 Construction of pcDNA3.1(+)-MTS-hND1(T3394C)-FLAG vector pcDNA3.1(+)-MTS vector Kpn Ⅰ / Apa Ⅰ Double enzyme digestion; the enzyme digestion system is shown in Table 9, and the reaction conditions are: 37°C, 2h.

[0079] Table 9. Enzyme digestion system

[0080] Amplify the fragment hND1(T3394C)-FLAG. Specifically, use the following primers hND1(T3394C)-F1 and hND1(T3394C)-R1 to amplify fragment F1, and use the following primers hND1(T3394C)-F2 and hND1(T3394C)-R2 to amplify fragment F2: hND1(T3394C)-F1: 5'-AGCCCGGGCAAGCTTGGTAACCCCGATGGCCAATCTGCTGC-3' hND1(T3394C)-R1:5'-CTGGGC C ACATGCAACTGAG-3' hND1(T3394C)-F2:5'-CTCAGTTGCATGT G GCCCAG-3' hND1(T3394C)-R2: 5'-TCAGCGGGTTTTAAACGGGCCCTCACACCTTATCATCATCATCCTTG-3' The PCR reaction system is shown in Table 10. The reaction conditions were: 98°C for 2 min, 30 cycles of (98°C for 10 s, 55°C for 15 s, and 72°C for 15 s), and 72°C for 5 min. After completion of the reaction, the gel was recovered.

[0081] Table 10. Reaction system

[0082] The source of the hND1 plasmid: ND1 The gene sequence (NCBI NC_011137.1) was codon-optimized (because the coding sequence of mitochondrial genes is different from that of nuclear genes and the translation efficiency in the cell nucleus is very low, resulting in poor protein expression; reference: Amutha Boominathan et al. Redox Biol, 2020. doi: 10.1016 / j.redox.2020.101429.), and the sequence was synthesized by Qingke Biotechnology after adding a Flag tag at the end and constructed into the pll3.7 plasmid to obtain the hND1 plasmid.

[0083] The homologous recombination kit ClonExpress Ultra One Step Cloning Kit V2 (Vazyme, Product No. C116) was used to connect the pcDNA3.1(+)-MTS backbone and fragments F1 and F2 obtained by enzyme digestion to obtain the mutant gene for overexpression. hND1 (T3394C)-pcDNA3.1(+)-MTS-hND1(T3394C)-FLAG vector. The homologous recombination system is shown in Table 11. Reaction conditions are: 50°C, 5 min. After completion, place in a refrigerator or on ice until ready for use.

[0084] Table 11. Homologous recombination system

[0085] The ligation product obtained by homologous recombination was transformed into Escherichia coli DH5a competent cells. After the bacterial solution was revived, it was spread on ampicillin-resistant plates for overnight culture and screening. Single colonies were picked for detection and subsequent experiments.

[0086] 3.3 Cell culture and transfection (1) Culture of SH-SY5Y cells Cells were cultured in DMEM supplemented with 10% BI serum and 1% double-antibody. Culture conditions were 5% CO2 and 37°C. For passaging, cells were digested with 0.25% trypsin for 1 minute and centrifuged at 1000 rpm for 4 minutes.

[0087] (2) Transfection of SH-SY5Y cells Using Lipofect5000 transfection reagent (Bio-Gene), cells were transfected with the constructed pcDNA3.1(+)-MTS vector as a control group and with the pcDNA3.1(+)-MTS-hND1(T3394C)-FLAG vector as a treatment group. Experiments were performed in 6-well and 48-well plates, with triplicate transfections per group. Subsequent assays were performed 48 hours after transfection (two groups of cells on the 6-well plate were used for transcriptome sequencing, and two groups of cells on the 48-well plate were used for mitochondrial function testing and immunofluorescence staining).

[0088] 3.4 Transcriptome Sequencing After 48 hours of transfection, the culture medium was discarded, the cells were washed three times with PBS, and TRIzol was added. TM The cells were collected into 1.5 mL centrifuge tubes after thorough pipetting and frozen at -80°C and then sent to a company (Novogene) for transcriptome sequencing.

[0089] 3.5 Mitochondrial function test The Agilent Seahorse XFp experimental operation process was followed, including cell plating and culture, pretreatment with unbuffered assay solution, calibration and hydration of the probe card, loading of drugs into the injection port, connection to the analyzer and running the assay program, real-time monitoring of the cell oxygen consumption rate (OCR), and finally exporting and analyzing the data.

[0090] 3.6 Cell immunofluorescence After 48 hours of transfection, the culture medium was discarded from the cells, and the cells were washed twice with pre-cooled PBS, fixed with 4% paraformaldehyde for 20 minutes, and washed three times with PBS (5 minutes each time); then permeabilized with 0.5% Triton X-100 for 10 minutes, washed three times with PBS (5 minutes each time); then blocked with 5% BSA for 30 minutes, and washed three times with PBS (5 minutes each time); incubated with primary antibody at room temperature for 2 hours or in a refrigerator at 4°C overnight, and washed three times with PBS; incubated with secondary antibody at 37°C in the dark for 1.5 hours, and washed four times with PBS (5 minutes each time); stained with DAPI for 10 minutes, then washed three times with PBS (5 minutes each time); mounted with anti-fade mounting medium, stored at 4°C in the dark, and observed and photographed under a fluorescence microscope.

[0091] 3.7 Results (1) Transcriptome sequencing and differential expression analysis like Figure 2 As shown in the figure, using the KEGG functional annotation of the DAVID database, it was found that the differentially expressed genes were enriched in signal pathways related to neurodegenerative diseases (including Parkinson's disease, Huntington's disease and Alzheimer's disease), which verified hND1 The mutation m.3394 T>C is associated with the occurrence of neurodegenerative diseases such as Parkinson's disease (PD).

[0092] (2) Mitochondrial function test like Figure 3 As shown, overexpression of the gene carrying the mutation m.3394 T>C ND1 Gene hND1 (T3394C), the cell oxygen consumption rate decreased significantly, indicating hND1 The mutation m.3394 T>C affects mitochondrial function, and mitochondrial dysfunction is closely related to neurodegenerative diseases, especially Parkinson's disease (PD).

[0093] (3) Cell immunofluorescence like Figure 4 Compared with the control group, overexpression hND1 After (T3394C), the expression level of α-Syn increased significantly, and the expression level of TH decreased significantly. These results indicate that hND1 The mutation m.3394 T>C is closely related to the occurrence and development of Parkinson's disease (PD).

[0094] (Four) hND1 Specificity and sensitivity of the mutation m.3394 T>C in the diagnosis of Parkinson's disease 4.1 Case-control studies A total of 66 Parkinson's disease (PD) patients and 66 age- and sex-matched healthy controls were enrolled. Genomic DNA was extracted from the peripheral blood of 132 samples collected (samples were collected from the Department of Neurosurgery, Second Affiliated Hospital of Air Force Medical University, and blood was collected between January 1, 2024, and December 31, 2024). PCR amplification and sequencing were used to detect the presence of hND1 The mutation m.3394 T>C was analyzed and the percentage of mutation burden was calculated. The results showed that the average mutation burden in Parkinson's disease patients was 15.00±7.03%, while the average mutation burden in healthy controls was 3.00±1.96%, with a statistically significant difference between the two groups (t=13.35, p<0.00000001). The primers used for PCR amplification are as follows: ND1Seq-F: 5'-AGGGTTTGTTAAGATGGCAGAG-3' ND1Seq-R: 5'-ATAGTAGAAGAGCGATGGTGAGA-3' 4.2 Obtaining the ROC Curve (Receiver Operating Characteristic Curve) 4.2.1 Determination of diagnostic variables and reference standards Will target hND1 The mutation burden of mutation m.3394 T>C was used as the diagnostic variable, and clinically confirmed Parkinson's disease was used as the reference standard.

[0095] 4.2.2 Setting multiple threshold points One hundred different threshold points ranging from 0% to 40% were set to cover all possible clinical decision points.

[0096] 4.2.3 Calculation of sensitivity and specificity at each threshold For each threshold point, a 2×2 contingency table (confusion matrix) is constructed to calculate sensitivity and specificity: Sensitivity (true positive rate) = true positives / (true positives + false negatives) Specificity (true negative rate) = true negative / (true negative + false positive) 1-Specificity (false positive rate) = False Positives / (True Negatives + False Positives) 4.2.4 Drawing ROC Curve With "1-specificity" (false positive rate) as the horizontal axis and "sensitivity" (true positive rate) as the vertical axis, all threshold points are connected to form the ROC curve.

[0097] 4.2.5 Calculating the Area Under the Curve (AUC) The area under the ROC curve was calculated using the trapezoidal rule, and the AUC value was 0.987, which is close to 1. hND1 The mutation m.3394 T>C has extremely high diagnostic accuracy.

[0098] 4.2.6 Calculating Confidence Intervals To assess the stability of the AUC estimate, the 95% confidence interval was calculated using the bootstrap resampling method (1000 repetitions): samples were drawn from the original data with replacement each time; the AUC was calculated for each bootstrap sample; based on the AUC distribution of the 1000 bootstrap samples, the 2.5% and 97.5% quantiles were determined as the 95% confidence interval; the final 95% confidence interval of the AUC was 0.972–0.998.

[0099] 4.2.7 Determining the Optimal Threshold Point By calculating the Youden's J (sensitivity + specificity - 1), the optimal diagnostic threshold was determined to be 5.25%, at which point the sensitivity was 100% and the specificity was 89.4%.

[0100] When drawing the ROC curve ( Figure 5 ), the solid line represents the ROC curve itself; the gray shaded area represents the 95% confidence interval calculated by the Bootstrap method; the red dot marks the optimal threshold point (5.25%), at which the sensitivity is 100% and the specificity is 89.4%; the dotted line represents the reference line of random guessing (AUC=0.5). This ROC curve is clearly above the reference line and close to the upper left corner, indicating that hND1 The mutation m.3394 T>C has excellent discrimination ability. The AUC is 0.987, and the 95% confidence interval is 0.972-0.998. The interval is narrow and the lower limit is still high, indicating that even considering the sampling error, hND1 The diagnostic performance of the mutation m.3394 T>C is still very reliable. The optimal threshold point is selected based on the principle of maximizing the Youden index, and the optimal balance is achieved at 5.25%. The confusion matrix at this time shows ( Figure 6 ): 66 true positives, 7 false positives, 59 true negatives, and 0 false negatives. This means that using this threshold can detect all Parkinson's disease patients (i.e., no missed diagnosis), and 89.4% of healthy people can be correctly excluded. In order to more intuitively show the trend of diagnostic performance as the threshold changes, the curves of sensitivity and specificity as the mutation load threshold changes are plotted ( Figure 7 ), the results showed that as the threshold increased, the sensitivity gradually decreased, while the specificity gradually increased. When the threshold was 5.25% ( Figure 7 The combined performance of sensitivity and specificity is optimal.

[0101] 4.3 Conclusion Through systematic ROC curve analysis, it was confirmed that human mitochondrial genes ND1 The mutation m.3394 T>C has a very high diagnostic value in the diagnosis of Parkinson's disease: the AUC is 0.987 (95% confidence interval: 0.972-0.998), and at the optimal threshold of 5.25%, the diagnostic sensitivity is 100% and the specificity is 89.4%. hND1 The mutation m.3394 T>C can be used as an effective biomarker for the diagnosis of Parkinson's disease and provide a new diagnostic tool for clinical practice.

[0102] (V) Cases of early diagnosis of Parkinson’s disease 5.1 Acquisition of diagnostic samples A case-control study was conducted, involving 100 subjects, including 50 clinically diagnosed Parkinson's disease (PD) patients (experimental group) and 50 age-matched healthy controls (control group). The experimental group included two types of PD patients: 40 patients with idiopathic PD and 10 patients with familial PD carrying the LRRK2 kinase G2019S mutation.

[0103] In addition, 15 high-risk individuals who carried LRRK2 mutations but had not yet shown clinical symptoms were included to evaluate the diagnostic efficacy of the diagnostic method (and the above-mentioned biomarkers) in the early stages of PD.

[0104] All participants (115 samples) underwent clinical evaluation by neurologists, and the diagnosis of Parkinson's disease met the diagnostic criteria of the International Parkinson's and Movement Disorder Society (MDS). The control group had no history of neurological disease and was matched with the experimental group on demographic characteristics such as age, gender, and race. Blood samples for all samples were collected and stored under standardized conditions (samples were obtained from the Department of Neurosurgery, Second Affiliated Hospital, Air Force Medical University, and blood was collected between January 1, 2024, and March 30, 2025).

[0105] 5.2 Diagnostic Reagents 5.2.1 Primers Specifically, the primers used to amplify the region where the 3394th site of mtDNA is located are the above-mentioned primers ND1Seq-F and ND1Seq-R; the length of the amplified product is 340 bp.

[0106] 5.2.2 Other reagents TIANGEN Blood / Cell / Tissue Genomic DNA Extraction Kit (DP304); TIANGEN General Agarose Gel DNA Recovery Kit (DP209); qPCR SYBR Green Master Mix (Thermo Fisher Scientific, catalog number: A25742); Agarose (Sigma-Aldrich, catalog number: A9539); TAE Running Buffer (50×); DNA Molecular Weight Standard (100 bp DNA Ladder; Thermo Fisher Scientific, catalog number: SM0241); Gel Red Nucleic Acid Gel Stain (Biotium, catalog number: 41003); BigDye X Terminator Purification Kit (Applied Biosystems, catalog number: 4376486); BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, catalog number: 4337455).

[0107] 5.3 Diagnostic techniques 5.3.1 Pre-sequencing treatment (1) DNA extraction and purification Extract genomic DNA from the stored blood sample according to step 1.2; then perform gel electrophoresis and gel recovery and purification.

[0108] (2) PCR amplification The region where the mtDNA site 3394 is located was amplified using the above primers ND1Seq-F and ND1Seq-R. Table 12. Reaction system

[0109] The PCR reaction system is shown in Table 12. The reaction conditions were: 95°C for 5 min; (95°C for 30 s; 58°C for 30 s; 72°C for 30 s) × 35; 72°C for 7 min; hold at 4°C. PCR products were purified using TIANGEN DP209.

[0110] (3) Sequencing Sequencing platform: Applied Biosystems 3500xL Genetic Analyzer.

[0111] Sequencing reagents: BigDye™ Terminator v3.1 Cycle Sequencing Kit.

[0112] Data analysis software: Sequencing Analysis Software v6.0 (Applied Biosystems).

[0113] Sequence alignment software: SnapGene; detection position Chromosome MT, reference sequence NC_012920.1 (3307..4262).

[0114] The PCR product of each sample was sequenced. The reaction system is shown in Table 13. The sequencing reaction conditions were: 96°C, 1 min (initial denaturation); (96°C, 10 s; 50°C, 5 s; 60°C, 4 min) × 25; hold at 4°C. Product purification prior to sequencing was performed using the BigDye XTerminator Purification Kit.

[0115] Table 13. Reaction system

[0116] Typing: The sequencing results were organized into a table using R programming language. The data displayed the mutation frequency at each site. The mutation frequency was used to determine the mutation status of mtDNA site 3394 (see Table 14).

[0117] Table 14. Sample genotyping examples

[0118] 5.4 Accuracy of diagnostic results 5.4.1 Sample mutation detection results 100 research subjects (50 Parkinson's disease patients and 50 healthy controls) were tested for mutations at mtDNA site 3394. The results are shown in Tables 15 and 16: Table 15. Experimental group (Parkinson's disease patients, n = 50)

[0119] Table 16. Control group (healthy controls, n=50)

[0120] The mutation frequencies were compared based on the detected mutations. The results showed that the total frequency of the m.3394 T>C mutation in Parkinson's disease patients was 34% (17 / 50), while the total frequency in healthy controls was only 2% (1 / 50). The chi-square test results showed that the difference in mutation frequencies between the two groups was statistically significant (χ 2 =17.36, p <0.001).

[0121] 5.4.2 Diagnostic Accuracy Assessment Based on the mutation detection results, the accuracy of the m.3394 T>C mutation as an early diagnostic marker for Parkinson's disease was calculated: (1) Four-cell table analysis (see Table 17) Table 17. Analysis results

[0122] Note: In Table 17, TP = true positive; FP = false positive; FN = false negative; TN = true negative.

[0123] (2) Diagnostic accuracy indicators i. Positive Predictive Value (PPV) calculation formula: TP / (TP+FP); Result: 17 / 18 = 94.4%; Interpretation: Of all samples that tested positive for the mutation, 94.4% actually had Parkinson's disease.

[0124] ii. Negative Predictive Value (NPV) calculation formula: TN / (TN+FN); Result: 49 / 82 = 59.8%; Interpretation: Of all samples that tested negative for the mutation, 59.8% were indeed healthy controls.

[0125] iii. False Positive Rate (FPR) calculation formula: FP / (FP+TN); Result: 1 / 50 = 2%; Interpretation: Among healthy controls, 2% were falsely detected as mutation-positive.

[0126] iv. False Negative Rate (False Negative Rate) calculation formula: FN / (TP+FN); Result: 33 / 50=66%; Interpretation: Among Parkinson's disease patients, 66% were falsely tested as mutation-negative.

[0127] v. Overall Accuracy: (TP + TN) / (TP + TN + FP + FN); Result: (17 + 49) / 100 = 66%; Interpretation: Of all subjects, 66% were correctly classified (true positive or true negative).

[0128] 5.4.3 Subgroup Analysis (1) Stratification by age The study subjects (50 Parkinson's disease patients and 50 healthy controls) were divided into two groups according to age: ≤65 years old and >65 years old. The detection efficiency of the m.3394 T>C mutation was analyzed. The results are shown in Table 18: Table 18. Analysis results

[0129] The results ( Table 18 ) showed that the diagnostic efficacy of m.3394 T>C mutation detection was similar in different age groups.

[0130] (2) Stratification by gender The study subjects (50 Parkinson's disease patients and 50 healthy controls) were divided into two groups according to gender: male and female. The detection efficiency of the m.3394 T>C mutation was analyzed. The results are shown in Table 19: Table 19. Analysis results

[0131] The results (Table 19) showed that the diagnostic efficacy of m.3394 T>C mutation detection was similar in different gender groups.

[0132] In addition, 15 high-risk individuals who carried LRRK2 mutations but had not yet exhibited clinical symptoms were tested for the m.3394 T>C mutation. The results showed that four individuals (26.7%) carried the m.3394 T>C mutation, while 11 individuals (73.3%) did not. Compared with the control group, the positive predictive value for this high-risk group was 80.0%, the negative predictive value was 81.7%, and the overall accuracy was 81.5%.

[0133] These results suggest that m.3394 T>C provides a new molecular target for early warning and intervention of Parkinson's disease, especially for individuals known to carry genetic risk factors.

[0134] 5.5.4 Conclusion Based on the results of this study, the m.3394 T>C mutation has high sensitivity and specificity as an early diagnostic marker for Parkinson's disease and holds great promise for its application in the early diagnosis of Parkinson's disease. Considering the complex etiology and diverse clinical manifestations of Parkinson's disease, detection of the m.3394 T>C mutation could serve as an effective adjunct to clinical diagnosis, particularly for early screening and risk assessment in patients suspected of Parkinson's disease.

Claims

1. Use of a human mitochondrial gene mutation detection reagent in the preparation of an early diagnostic reagent for Parkinson's disease, characterized by: The mutation is m.3394 T>C.

2. The use according to claim 1, characterized in that: The detection reagent includes primers for amplifying the mitochondrial genome site where the mutation is located.

3. The use according to claim 2, characterized in that: The primers are shown as SEQ.ID.NO.1 and SEQ.ID.NO.

2.

4. The use according to claim 1, characterized in that: The genotype corresponding to the mutation is mutant type C / C.

5. The use according to claim 4, characterized in that: The genotype is obtained by sequencing the amplification results.

6. The use according to claim 1, characterized in that: The sample is selected from any one of patients with neurodegenerative diseases and high-risk populations of related diseases.

7. The use according to claim 1, characterized in that: The sample is selected from any one of Parkinson's disease patients and Parkinson's disease high-risk population.

8. The use according to claim 1, characterized in that: The sample is blood.

9. A method for detecting human mitochondrial gene mutations, characterized by: The following steps are involved: Genomic DNA was extracted from the specimens, and PCR amplification was performed using the DNA as a template and primers. The mutation at position 3394 of the mitochondrial DNA of the specimens was determined based on the amplification results.

10. The method for detecting human mitochondrial gene mutation according to claim 9, characterized in that: The primers were designed based on the site where the mutation m.3394 T>C was located.

Images