Genetic cerebral calcification disease pathogenic gene cmpk2 and application thereof
By identifying pathogenic mutations in the CMPK2 gene and developing detection methods, the challenges of early diagnosis and treatment of hereditary cerebral calcification have been solved. This provides a target for the detection and treatment of CMPK2 mutant genes, enabling effective diagnosis and treatment of hereditary cerebral calcification.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- THE FIRST AFFILIATED HOSPITAL OF FUJIAN MEDICAL UNIV
- Filing Date
- 2022-08-25
- Publication Date
- 2026-06-19
Smart Images
Figure CN115896119B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedicine, specifically to the pathogenic mutation form of the novel pathogenic gene CMPK2 for hereditary cerebral calcification and its application, and also to a detection kit and detection method for the CMPK2 mutant gene. Background Technology
[0002] Cerebral calcification is a relatively common imaging finding associated with aging, with an incidence of approximately 6.6 / 1000. In middle-aged and elderly individuals, over 10% of cranial CT scans show punctate calcifications, and over 1% show patchy calcifications. However, this finding is often overlooked by clinicians and patients. Furthermore, over 20% of patients with some neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD), also have cerebral calcification, and the lesions tend to enlarge with age. Some patients with cerebral calcification show dopaminergic neurotransmitter system dysfunction on SPECT (single-photon emission computed tomography) imaging. Therefore, research on cerebral calcification helps to elucidate the pathogenesis of some neurodegenerative diseases.
[0003] Hereditary cerebral calcification is a neurodegenerative disease caused by gene mutations. Its pathological features include bilateral calcification of the basal ganglia, thalamus, and cerebellum, occasionally with subcortical calcifications. As the disease progresses, these calcifications can merge into large patches, pathologically manifesting as the deposition of calcium salt particles in terminal arterioles, veins, and capillaries. Clinical symptoms of hereditary cerebral calcification are diverse, mainly including motor disorders, mental disorders, cognitive impairment, seizures, dizziness, and headaches. In severe cases, personality and behavioral changes may occur, eventually leading to psychosis or dementia. Some patients remain asymptomatic throughout their lives. Hereditary cerebral calcification occurs sporadically or familially, exhibiting high genetic heterogeneity. Treatment is primarily symptomatic; currently, there are no effective drugs to remove or reduce abnormal calcium deposits. Therefore, only by systematically identifying the pathogenic genes of hereditary cerebral calcification can we achieve early and accurate diagnosis, thereby effectively controlling the patient's clinical symptoms and slowing disease progression; and guide prenatal diagnosis or assisted reproductive technology to prevent the birth of affected children.
[0004] Hereditary brain calcification includes several types, such as primary familial brain calcification (PFBC), Aicardi-Goutières syndrome (AGS), and mitochondrial encephalomyopathy with hyperlactatemia and stroke-like episodes (MELAS). Over the past decade, human genetic studies have identified genes responsible for PFBC including SLC20A2, XPR1, PDGFB, PDGFRB, MYORG, and JAM2. Currently, there are two main hypotheses regarding the pathogenesis of brain calcification: First, impaired BBB (blood-brain barrier) structure or reduced pericyte coverage leads to increased BBB permeability. Patients associated with this pathway may have mutations in genes such as PDGFRB, PDGFB, JAM2, or MYORG, all of which are expressed in cells that constitute neurovascular units. The second pathway involves disruption of phosphate transport in the brain. Patients belonging to this pathway may have mutations in the SLC20A2 or XPR1 genes and typically show significantly elevated inorganic phosphorus levels in CSF (cerebrospinal fluid). In addition, AGS also presents with very obvious signs of brain calcification, along with leukodystrophy, microcephaly, and elevated interferon-α levels. Its pathogenic genes include ADAR, SAMHD1, IFIH1, TREX1, RNASEH2A, RNASEH2B, RNASEH2C, LSM11, and RNU7-1. Currently, it is believed that the pathogenesis of AGS is due to mutations in genes encoding exonucleases, leading to the accumulation of nucleic acid products such as DNA or RNA in the body, which activates the type I interferon pathway. MELAS, on the other hand, is a systemic syndrome characterized by mitochondrial encephalomyopathy, lactic acidosis, stroke-like episodes, and often accompanied by brain calcification; it is primarily caused by mitochondrial gene mutations.
[0005] This invention, through genetic analysis of two specific families of hereditary cerebral calcification, identified a CMPK2 mutant gene co-segregated with cerebral calcification. Related experiments showed that the CMPK2 mutant gene led to a decrease in mitochondrial DNA copy number, mitochondrial morphology and functional impairment, and disrupted the mitochondrial targeting of the CMPK2 protein. Furthermore, age-promoting cerebral calcification deposition occurred in homozygous CMPK2 knockout mice and CMPK2 knock-in mice carrying mutations from this cerebral calcification family. Therefore, CMPK2 has been confirmed as a genetic pathogenic factor for cerebral calcification, further extending the pathogenic mechanism of hereditary cerebral calcification to mitochondrial dysfunction caused by CMPK2 gene defects. This will provide a foundation and basis for the functional analysis and application of the CMPK2 gene, particularly providing new molecular targets and theoretical foundations for exploring the pathogenesis, diagnosis, and screening of cerebral calcification. Summary of the Invention
[0006] This invention discloses a novel pathogenic mutant form of the human CMPK2 gene. The presence of this mutant gene leads to impairment of mitochondrial copy number, mitochondrial morphology, and function, which in turn triggers mitochondrial disorder-related inflammation and diseases, including brain calcification. Furthermore, this invention provides related applications of the CMPK2 mutant gene, as well as a CMPK2 mutant gene detection kit and detection method.
[0007] The technical solution adopted by this invention to solve its technical problem is as follows:
[0008] One objective of this invention is to provide novel human CMPK2 mutant genes, corresponding CMPK2 mutant proteins, and their applications. This invention provides three pathogenic CMPK2 mutant gene forms, namely c.1A>C, c.2T>C, and c.1241A>G. Referring to the CMPK2 wild-type gene coding sequence shown in SED ID NO: 1, when the CMPK2 gene is mutated to c.1A>C, that is, the A base at position 1 of the wild-type CMPK2 gene coding sequence is mutated to a C base, the corresponding mutant gene coding sequence is shown in SEQ ID NO: 2; when the CMPK2 gene is mutated to c.2T>C, that is, the T base at position 2 of the wild-type CMPK2 gene coding sequence is mutated to a C base, the corresponding mutant gene coding sequence is shown in SEQ ID NO: 3; when the CMPK2 gene is mutated to c.1241A>G, that is, the A base at position 1241 of the wild-type CMPK2 gene coding sequence is mutated to a G base, the corresponding mutant gene coding sequence is shown in SEQ ID NO: 4. Mutations in the CMPK2 gene, such as c.1A>C, c.2T>C, or c.1241A>G, lead to a decrease in mitochondrial copy number, mitochondrial morphology and dysfunction, and consequently trigger related inflammation or diseases. Any diploid homozygous or compound heterozygous genotype of the CMPK2 mutant gene corresponding to the coding sequence shown in SEQ ID NO: 2-4 can cause human cerebral calcification. Therefore, the CMPK2 mutant gene or its encoded protein can serve as a target for the diagnosis and treatment of related inflammation or diseases, as well as for the development of diagnostic kits and therapeutic drugs.
[0009] Changes in the gene coding sequence will lead to changes in the encoded protein sequence. Corresponding to the above-mentioned CMPK2 gene mutation forms, this invention also provides its encoded protein sequence. The wild-type CMPK2 gene coding sequence shown in SED ID NO: 1 encodes a protein sequence shown in SED ID NO: 5. Using the protein sequence shown in SED ID NO: 5 as a reference, the encoded protein sequences of the CMPK2 mutant genes shown in SED ID NO: 2 and SED ID NO: 3 are both shown in SED ID NO: 6. Specifically, the amino acid fragment at positions 1-26 of the CMPK2 wild-type gene encoded protein is deleted, i.e., the p.1_26delMAFARRLLRGPLSGPLLGRRGVCAGA mutation occurs. Using the protein sequence shown in SED ID NO: 5 as a reference, the encoded protein sequence of the CMPK2 mutant gene coding sequence shown in SED ID NO: 4 is shown in SED ID NO: 7. Specifically, the amino acid at position 414 of the CMPK2 wild-type gene encoded protein changes from tyrosine to cysteine, i.e., the p.Y414C mutation occurs. The absence of the wild-type CMPK2 gene-encoded protein shown in SEQ ID NO:5 in nerve cells, and the presence of the mutant CMPK2 gene-encoded protein shown in SEQ ID NO:6 or SEQ ID NO:7, can lead to cerebral calcification.
[0010] Given the identification of pathogenic CMPK2 mutant genes, molecular biological methods and procedures can be followed to amplify or synthesize CMPK2 gene fragments containing any of the mutations c.1A>C, c.2T>C, and c.1241A>G. The mutant gene fragments can then be introduced into vectors to obtain recombinant vectors containing the mutant gene fragments. Furthermore, the recombinant vectors can be introduced into cells to obtain cells containing the recombinant vectors. The vectors and cells can be used in areas including but not limited to the investigation of the mechanisms of cerebral calcification, the development of diagnostic kits, and the development of drugs for diagnosis and treatment.
[0011] A second objective of this invention is to provide a CMPK2 mutation gene detection kit and detection method. For CMPK2 mutation genes with c.1A>C, c.2T>C, and c.1241A>G mutations, PCR primers as shown in SEQ ID NO:8-9 can be used to amplify the fragment containing the c.1A>C mutation, PCR primers as shown in SEQ ID NO:8-9 can be used to amplify the fragment containing the c.2T>C mutation, and PCR primers as shown in SEQ ID NO:10-11 can be used to amplify the fragment containing the c.1241A>G mutation. The amplified products can then be sequenced using Sanger sequencing to determine whether the individual contains the corresponding CMPK2 gene mutation. In addition, the kit also contains other necessary components for PCR, mainly DNA polymerase, PCR buffer, dNTP mix, and other related reagents.
[0012] Finally, this invention also provides a method for detecting CMPK2 mutant gene fragments based on the above-mentioned kit, the main steps of which are as follows:
[0013] (1) Extracting genomic DNA from biological samples;
[0014] (2) PCR amplification of the CMPK2 gene mutation fragment was performed using the kit of the present invention;
[0015] (3) Sequencing analysis was performed on the PCR products in (2) to determine the mutation information of the CMPK2 gene in the sample.
[0016] The biological sample mentioned in step (1) is a body fluid or tissue cell, including but not limited to blood, serum, amniotic fluid, lymph, and oral mucosal cells. Preferably, the body fluid is blood.
[0017] In step (2), the primer combination used for PCR amplification can be as follows: PCR primers as shown in SEQ ID NO:8-9 can be used to amplify the fragment containing the c.1A>C mutation of the CMPK2 gene; PCR primers as shown in SEQ ID NO:8-9 can be used to amplify the fragment containing the c.2T>C mutation of the CMPK2 gene; and PCR primers as shown in SEQ ID NO:10-11 can be used to amplify the fragment containing the c.1241A>G mutation of the CMPK2 gene. The amplified products can be sequenced by Sanger sequencing to determine whether the person being tested has the corresponding CMPK2 gene mutation.
[0018] The mutation information in step (3) is determined by referring to the CMPK2 wild-type gene coding sequence shown in SEQ ID NO: 1.
[0019] Based on the pathogenic mutations of the novel pathogenic gene CMPK2 in hereditary cerebral calcification, the corresponding encoded protein sequence, and its impact on mitochondrial function revealed in this invention, it can be used as a target for the diagnosis and treatment of related inflammation or diseases. In particular, it can serve as a detection target for diagnostic kits for cerebral calcification and a target for the development of preventive and therapeutic drugs. Furthermore, it can provide an experimental basis and theoretical foundation for research on the pathogenesis of cerebral calcification, as well as clinical diagnosis and treatment methods and drug development. For the detection of CMPK2 gene mutations, this invention has developed corresponding detection kits and methods based on PCR amplification and Sanger sequencing, which can easily and effectively detect CMPK2 gene mutations. Attached Figure Description
[0020] Figure 1 These are experimental results related to the identification of CMPK2 gene mutations in patients with cerebral calcification. (a) Pedigree mutations in families with hereditary cerebral calcification carrying CMPK2 gene mutations (c.2T>C, p.M1; c.1A>C, p.M1 and c.1241A>G, p.Y414C). Fill or empty symbols represent individuals with or without cerebral calcification, respectively. "+ / -" indicates a heterozygous mutation carrier, and "- / -" indicates an individual without the mutation. Arrows represent probands. (b) Sanger sequencing peak diagrams of members in pedigrees 1 and 2, including homozygous c.2T>C mutations, compound heterozygous c.1A>C and c.1241A>G mutations, with reference alleles below. Mutations are marked in red. het represents heterozygotes, and hom represents homozygotes. (c) Homology alignment of the CMPK2 protein sequence, showing the α7b helix of the core domain including p.Y414, and the conformational change of Y414C. (d) Schematic diagram of CMPK2 gene start codon loss in a family. CMPK2 (c.2T>C, p.M1 and c.1A>C, p.M1) mRNA transcription may be restarted from a subsequent ATG codon (e.g., p.M27), resulting in mTP loss (ΔN26) in the CMPK2 protein. mTP refers to the mitochondrial targeting peptide sequence; CMPK refers to the CMP-UMP kinase domain. (e) Immunofluorescence analysis of subcellular localization of the CMPK2 protein. CMPK2 wild-type (WT) or c.1A>C, c.2T>C, c.1241A>G mutant plasmids labeled with flag were immunostained with antibodies against flag (green) and the mitochondrial marker COX IV (red). DAPI (blue) was used for nuclear staining. Scale bar, 5 μm. (f) Western blot analysis of CMPK2 mitochondrial and cytoplasmic expression. Primary cultured rat neurons were transfected with flag-labeled CMPK2-WT or c.1A>C, c.2T>C, and c.1241A>G mutant plasmids. CMPK2 protein quantification statistics are shown in (g). **** indicates p<0.0001.
[0021] Figure 2 These are representative axial brain CT images of family members. Plain CT scans of the head of I-1, I-2, II-1, II-2, II-3, and II-5 in family 1, and I1, I2, and II1 in family 2. Typical axial images of the cerebellum, basal ganglia, and subcortical areas. Calcified deposits are detected as high-density lesions within the brain parenchyma, distributed in the globus pallidus, thalamus, cerebellum, caudate nucleus, periventricular white matter, and cerebral cortex.
[0022] Figure 3 This is a summary of the genetic and clinical characteristics of patients with CMPK2 gene mutations. PTH (parathyroid hormone); CSF (cerebrospinal fluid); WBC (white blood cell count); ECG (electrocardiogram); NA indicates absence. Normal laboratory test results are as follows: serum calcium 2.15-2.55 mmol / L, serum phosphorus 0.87-1.45 mmol / L, parathyroid hormone 15-65 ng / L, cerebrospinal fluid white blood cell count 0-8 × 10⁶ / L, cerebrospinal fluid lactate 0.7-2.1 mmol / L. aPosition on Genome Reference Consortium human genome build38 (GRCh38); bTCS (Total calcification score); cMDC Mitochondrial Disease Criteria 29: Score 1: Non-mitochondrial disorder; Score 2-4: Possible mitochondrial disorder; Score 5-7: Highly probable mitochondrial disorder; Score 8-12: Definite mitochondrial disorder.
[0023] Figure 4 This is a transcriptomic analysis of PBMCs. RNA sequencing analysis was performed on peripheral blood mononuclear cells from patients with brain calcification (P1 corresponds to II-3, P2 corresponds to II-5) compared to three unaffected controls (C1-C3). (ab) shows the upregulated (a) and downregulated (b) GO pathways. (ce) shows the gene expression patterns of representative downregulated pathways in PBMCs from patients and the three unaffected controls, including energy homeostasis (c), mitochondrial tissue regulation (d), and ATP-dependent helicase activity (e). (fg) shows the gene expression patterns of representative upregulated pathways in PBMCs from patients and the three unaffected controls, including nucleoside diphosphate metabolism (f) and carbohydrate phosphorylation (g).
[0024] Figure 5This section presents the results of Cmpk2 expression in mouse neurons. (a) In situ hybridization of Cmpk2 expression in sagittal sections of adult mouse brain. (b) Magnified view of Cmpk2 expression in the hippocampus, thalamus, and cerebellum; nuclear DAPI (blue) staining. (c and d) Double in situ hybridization of Cmpk2 (red) with the glutamatergic neuron marker Vglut2 (green) in the mouse thalamus (c) and dentate nucleus (d). (e and f) Double in situ hybridization of Cmpk2 (red) with the GABAergic neuron marker Gad1 (green) in the PCL of the mouse thalamus (e) and cerebellum (f). Dual fluorescent labeling of Cmpk2 (red) and the astrocyte marker S100β (green) in the DG of the thalamus and hippocampus. Ctx, cortex; Hip, hippocampus; Thal, thalamus; Crb, cerebellum; DG, dentate gyrus; PCL, Pukinje cell layer; GCL, granular cell layer. Scale bar, 500 μm (a); 50 μm (bf).
[0025] Figure 6 These are single-cell transcriptome sequencing data of Cmpk2 in the mouse brain. (A) Expression of Cmpk2 scRNA in blood vessels and perivascular cells of adult mouse brain and lung (http: / / betsholtzlab.org / VascularSingleCells / database.html) (B) Expression of Cmpk2-scRNA in different cell types of the brain (http: / / celltypes.org / brain).
[0026] Figure 7These are the results of experiments on mitochondrial function in neurons from Cmpk2-deficient mice. (a) Mitochondrial DNA copy number analysis between cortical tissues of WT and Cmpk2-KO mice based on ddPCR. n=7-8, triple replicates. (b) Representative images of mitochondria in primary cultured cortical neurons of WT and Cmpk2-KO mice, with antibodies against mitochondrial markers ATP synthase β chain (green) and cytoskeletal protein MAP2 (red); DAPI (blue) was used for nuclear staining. (c) Western blot analysis of COX IV and cytochrome c in primary cultured cortical neurons of WT and Cmpk2-KO mice. (d) Quantification of changes in COX IV and cytochrome c protein levels. n=3, triple replicates. (e) Measurement of ATP levels in neurons of WT and Cmpk2-KO mice. (fg) Pi levels in neurons (f), cerebrospinal fluid, and serum (g) of WT and Cmpk2-KO mice. (h) Representative transmission electron micrographs of mitochondrial morphology in primary cultured cortical neurons of WT and Cmpk2-KO mice. (ij) Statistical graph of crest coverage of primary cultured cortical neurons in WT and Cmpk2 KO mice. CC represents crest coverage. Error bars represent mean ± SEM. Ns indicates no significance, *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001; t-test. Scale bars are 2μm (h, left side of each image) and 0.2μm (h, right side of each image).
[0027] Figure 8 This is an experimental result showing extensive calcification deposition in the brains of Cmpk2 KO and KI mice. (a) Brain sections from WT and Cmpk2-KO mice stained with H&E, Von Kossa, Alizarin Red, Periodic Schiff (PAS), and Alsin Blue. (b) Coronal and sagittal micro-CT scans of the brains of 13-month-old Cmpk2 KO mice. Arrows indicate high-density lesions (these are calcification deposits). Ctx, cortex; Crb, cerebellum; Thal, thalamus. (c) Progressive development of calcification deposition in the brains of Cmpk2-KO mice at 10, 12, and 14 months of age. Alizarin Red and Von Kossa staining show calcification. Scale bar, 100 μm (a and c). Detailed Implementation
[0028] The present invention will be further illustrated below with reference to specific embodiments.
[0029] The applicant collected a large number of patient and family samples with cerebral calcification. The total calcification score (TCS) of the patients was assessed according to accepted clinical standards, excluding secondary pathogenic factors. Furthermore, in 117 families and 365 sporadic cerebral calcification patients, six reported PFBC pathogenic genes (SLC20A2, PDGFRB, PDGFB, XPR1, MYORG, and JAM2) were screened. The results showed that 44 families and 334 sporadic patients tested negative for all six known pathogenic genes (i.e., all were wild-type genes). In the process of discovering new genetic pathogenic genes for hereditary cerebral calcification, a potential pathogenic mutation in CMPK2 was identified in two FBC families. This was then extended to all 43 families and 334 sporadic patients who tested negative for all six genes via Sanger sequencing, as well as 500 ethnically matched healthy controls. The relevant research was approved by the ethics committees of the First Affiliated Hospital of Fujian Medical University (Approval No.:
[2019] 198) and the Second Affiliated Hospital of Zhejiang University School of Medicine (Approval No.: I2019001149), and written informed consent was obtained from all participants before the research was conducted. Data in the examples are presented as mean ± standard error of mean (SEM). Statistical significance was assessed using a t-test, with differences considered statistically significant at <0.05, specifically including: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Experimental methods not specified in the examples were generally performed under standard conditions or according to the manufacturer's recommendations.
[0030] Example 1: Identification of homozygous CMPK2 gene mutations in families with hereditary cerebral calcification
[0031] By performing whole-exome sequencing on samples from selected family members with cerebral calcification and conducting functional analysis on specific candidate pathogenic mutant genes, the inventors have for the first time identified CMPK2, a new potential autosomal recessive pathogenic gene for hereditary cerebral calcification, and its pathogenic mutant forms.
[0032] (1) Patient inclusion and genetic analysis
[0033] The pedigrees of the two specific families with cerebral calcification are as follows: Figure 1 As shown in figure a, the brother and sister (II-3 and II-5) in family 1 on the left side of the figure show obvious calcifications in the bilateral globus pallidus, thalamus, and cerebellum. Figure 2For II-5, extensive calcification was also observed in the caudate nucleus, periventricular white matter, and cerebral cortex, with a total calcification (TCS) value of 52. At her initial presentation (age 40), II-5 presented with moderate motor dysfunction, speech impairment, and cognitive deficits, as well as involuntary laughter and crying. Two years later, her symptoms gradually worsened, with impaired verbal communication, difficulty standing, and loss of self-care abilities. At that time, she was unable to participate in standard scoring measurements, indicating a further significant decline in cognitive function. Her electrocardiogram showed mild pulmonary hypertension, and cerebrospinal fluid examination showed a slightly elevated white blood cell count (value 10) and a low lactate level (2.2 mmol / L), but her echocardiography, abdominal ultrasound, antinuclear antibody, blood, and urine tests were all within normal ranges. Figure 3 Although II-3, the older brother of II-5, had a TCS value of 20, he was asymptomatic and no abnormalities were found on neurological examination. Serum calcium and phosphate levels were normal in both II-3 and II-5. There was no consanguineous marriage between I-1 and I-2. No other family members examined showed any significantly elevated brain calcifications above the physiological threshold (I-1, I-2, II-1, II-2, and III-1). Figure 2 In the second family of brain calcifications ( Figure 1 a. Right side family lineage 2, Figure 2 In this study, the brain calcification pattern (TCS 57) of proband II-1 was similar to that of II-5 in family 1. This 39-year-old woman presented with progressive dysphagia and cognitive impairment (MMSE score 21 and Moca score 24) for 7 years; in the past two years, she developed severe motor impairment and urinary and fecal incontinence. Her EEG, EMG, serum calcium, phosphate, PTH, and other biochemical markers were normal, but her anti-SSA levels were elevated. Figure 3 Neither of her parents reported any symptoms that could be related to brain calcification.
[0034] First, known pathogenic genes associated with brain calcification were excluded using Sanger sequencing. Based on this, to determine the potential genetic factors contributing to the familial brain calcification observed in II-3 and II-5, whole-exome sequencing (WES) was performed on I-1, I-2, II-3, and II-5 in family 1. Assuming an autosomal recessive inheritance pattern, an algorithm was used to search for biallelic mutations present in the two brain calcification patients but absent in their other siblings. In this way, two candidate gene mutations containing homozygous mutations (LPIN1 (c.1100C>G, p.P367R) and CMPK2 (c.2T>c, p.M1)) were identified, showing complete cosegregation with the brain calcification phenotype. Furthermore, non-standard mutants of known genes were screened using whole-genome sequencing (WGS) to identify pathogenic gene mutations causing FBC in II-5 of family 1; however, no suspicious mutations were detected. Previously, biallelic loss-of-function mutations in LPIN1 have been found to be associated with myoglobinuria19, which is not associated with brain calcification. Furthermore, the amino acid composition of the LPIN1 missense mutant (c.1100C>G, p.P367R) in this family is not conserved across different species, and it is not located in the core domain of the LPIN1 protein, leading to a lower likelihood of pathogenicity.
[0035] WES analysis of autosomal recessive family 2 also revealed compound heterozygous mutants in the CMPK2 gene (c.1A>C, p.M1; c.1241A>G, p.Y414C) in II-1 patients with severe brain calcification. Sanger sequencing was performed. Figure 1 (b) The co-segregation of biallelic CMPK2 variants with brain calcification phenotypes was successfully confirmed in both families. Note that the three CMPK2 mutants were either absent from the ESP, gnomAD, ExAC, and 1000 Genomes databases (c.1A>C and c.2T>C) or had extremely low frequencies (c.1241A>G), and were not detected in 500 healthy controls at the Department of Neurology, First Affiliated Hospital of Fujian Medical University. Relevant primers are detailed in Table 1. Furthermore, the gnomAD database reported 43 heterozygous mutants of the CMPK2 gene with termination, frameshift, and loss-of-initiation, and their allele frequencies were very low. Therefore, it is preliminarily inferred that the detected CMPK2 mutant gene is a hereditary pathogenic gene in this family.
[0036] Table 1. Primer and oligonucleotide sequences involved in this invention.
[0037]
[0038]
[0039] In this invention, three pathogenic CMPK2 gene mutations were detected in patients with hereditary cerebral calcification: c.1A>C, c.2T>C, and c.1241A>G. Referring to the wild-type CMPK2 gene coding sequence shown in SED ID NO: 1, when the CMPK2 gene mutation is c.1A>C, that is, the A base at position 1 of the wild-type CMPK2 gene coding sequence is mutated to a C base, the corresponding coding sequence is shown in SEQ ID NO: 2; when the CMPK2 gene mutation is c.2T>C, that is, the T base at position 2 of the wild-type CMPK2 gene coding sequence is mutated to a C base, the corresponding coding sequence is shown in SEQ ID NO: 3. Corresponding to the coding sequences of the CMPK2 mutant genes shown in SEQ ID NO: 2 and SEQ ID NO: 3, one protein sequence of these two mutant genes is shown in SEQ ID NO: 6. Referring to the coding sequence of the wild-type CMPK2 gene shown in SEQ ID NO: 5, specifically, the amino acid segment at positions 1-26 of the wild-type CMPK2 gene coding protein is deleted, i.e., the p.1_26delMAFARRLLRGPLSGPLLGRRGVCAGA mutation occurs. When the CMPK2 gene mutates to c.1241A>G, i.e., the A base at position 1241 of the wild-type CMPK2 gene coding sequence is mutated to a G base, the corresponding coding sequence is shown in SEQ ID NO: 4. Corresponding to the coding sequence of the CMPK2 mutant gene shown in SEQ ID NO: 4, one protein sequence of this mutant gene is shown in SEQ ID NO: 7. Referring to the coding sequence of the wild-type CMPK2 gene shown in SEQ ID NO: 5, specifically, the amino acid at position 414 of the wild-type CMPK2 gene coding protein changes from tyrosine to cysteine, i.e., the p.Y414C mutation occurs. The PCR primers shown in SEQ ID NO:8-9 can be used to amplify the fragment containing the c.1A>C mutation in the CMPK2 gene, the PCR primers shown in SEQ ID NO:8-9 can be used to amplify the fragment containing the c.2T>C mutation in the CMPK2 gene, and the PCR primers shown in SEQ ID NO:10-11 can be used to amplify the fragment containing the c.1241A>G mutation in the CMPK2 gene. Sanger sequencing of the amplified products can then determine whether the individual contains the corresponding CMPK2 gene mutation. The selected biological sample can be body fluid or tissue cells, including but not limited to blood, serum, amniotic fluid, lymph, and oral mucosal cells. Preferably, the body fluid is blood.
[0040] (2) The CMPK2 mutant gene affects the mitochondrial targeting of its encoded protein.
[0041] The human Cmpk2 gene is located on chromosome 2 (6840570-6866635, with GRCh genome 38 as a reference), encoding a 449-amino acid protein named UMP-CMP kinase 2. It functions as a monophosphate kinase and participates in the salvage pathway of dUTP and dCTP required for mitochondrial genomic DNA replication. Mutations in the c.1A>C (p.M1) and c.2T>C (p.M1) genes of the Cmpk2 gene clearly disrupt the start codon of the open reading frame of the Cmpk2 gene. Figure 1 c) suggests that any translation of the mRNA transcript of this CMPK2 mutant gene may be disrupted, or translation may be restarted from subsequent ATG codons (e.g., c.79-81, p.M27). Figure 1 d). This would result in the truncation of the N-terminal domain (1-26aa) of CMPK2, which is known to be crucial for its targeting of mitochondria (see Xu, Y., Johansson, M. & Karlsson, A. Human UMP-CMP kinase 2, a novel nucleoside monophosphate kinase localized in mitochondria. J. Biol. Chem. 2008, 283, 1563-1571). The missense mutation c.1241A>G would result in a highly conserved tyrosine residue within the α7b helix of the CMPK2 core domain being mutated to cysteine (p. Y414C). This tyrosine residue may provide crucial structural support for nearby R407 and R416, which serve as positively charged docking sites for negatively charged phosphate groups in ATP; their mutation to cysteine could disrupt the ATP-binding capacity of CMPK2. Figure 1 c). To verify this, the N-terminal truncated variant CMPK2 (abbreviated as CMPK2.ΔN26) and the missense mutation CMPK2.Y414C, labeled with the Flag polypeptide tag (the gene coding sequence corresponding to the Flag polypeptide tag is: 5'-GATTACAAGGATGACGACGATAAG-3'), were overexpressed in Cos-7 cells. The relevant operations and detection analysis are as follows.
[0042] A human CMPK2 cDNA sequence tagged with a flag was cloned into the pCMV-Entry vector (Origene). Following the manufacturer's instructions, Mut... The CMPK2 point mutation was introduced into the template using the II Fast Mutagenesis Kit V2 (Vazyme). Table 1 lists the primers used for plasmid construction. Cos-7 cells were cultured in 12-well plates (Corning) with DMEM supplemented with 10% (v / v) fetal bovine serum (FBS), including both with and without coverslips. The CMPK2 plasmid was transfected using Lipofectamine 3000 (Invitrogen). Primary culture and electroporation of cortical neurons were performed as described in the literature (Yang, Z. et al. ADAM10-initiated release of notch intracellular domain regulates microtubule stability and radial migration of cortical neurons. Cereb Cortex. 2017, 27(2): 919–932). Rat neurons isolated using an AMAXA nuclear transfection instrument (Lonza) were co-transfected with 6 μg of CMPK2-Flag (or CMPK2-c.1A>C-Flag, CMPK2-c.2T>C-Flag, or CMPK2-c.1241A>G-Flag plasmid) and GFP. During electroporation, 1×10⁻⁶ PCR PCR was used. 7 The cells were then subjected to subsequent biochemical analyses, including immunofluorescence and Western blotting.
[0043] For cellular immunofluorescence analysis, coverslips were first fixed with 4% PFA for 15 minutes, then infiltrated and blocked with 0.1% Triton X-100 in PBS containing 5% BSA for 15 minutes. Next, the coverslips were incubated overnight at 4°C with primary antibody against Flag (1:2000, F7425, Sigma) and COX IV (1:2000, ab33985, Abcam). The next day, the coverslips were washed three times with PBS and then incubated for 2 hours at room temperature with the corresponding secondary antibody (Alexa Fluor, Invitrogen). Finally, the coverslips were stained with Hoechst (Beyotime), washed three more times with PBS, and then mounted on glass slides. Fluorescence images were captured using an Olympus FV3000 laser confocal microscope with a 60× objective lens in 488 / 468 nm laser and 0.4 μm Z-axis superposition mode. Each sample underwent triple-parallel imaging analysis.
[0044] For Western blotting analysis, cultured cells were collected in lysis buffer containing protease inhibitors and centrifuged at 14000g for 15 minutes. Protein extracts were separated using a 13% SDS-PAGE gel and blotted onto a PVDF membrane. The cell membrane was blocked with 5% nonfat milk and then detected with Flag (1:5000, F1804, Sigma-Aldrich), COXIV (1:2000, #4850, Cell Signaling), cytochrome c (1:250, sc13156, Santa Cruz) or GFP (1:3000, B-2, Santa Cruz) and the corresponding HRP-binding secondary antibody (1:2000, Cell Signaling). Banding was performed using ECL (Beijing Tiangen Biotech Co., Ltd.) and measured using ImageJ software.
[0045] Immunofluorescence analysis using Flag antibody and the mitochondrial marker COX IV showed that wild-type (WT) CMPK2 was localized to mitochondria, while CMPK2.ΔN26 could not be localized to mitochondria, and CMPK2.Y414C also showed partial mitochondrial localization abnormalities. Figure 1 e). Western blot results of mitochondrial components in nerve cells transfected with CMPK2.ΔN26 and CMPK2.Y414C also confirmed the disruption of mitochondrial localization. Figure 1 f, 1g). Furthermore, compared to the wild type, protein expression was significantly reduced in all three mutants, with CMPK2-ΔN26 resulting in smaller protein products (f, 1g). Figure 1 f). These results indicate that CMPK2 deficiency impairs its location and function in mitochondria. Therefore, it is hypothesized that CMPK2 mutations are a genetic pathogenic factor leading to brain calcification in members of both families.
[0046] Example 2: Transcriptome analysis showed abnormal energy metabolism in the patient.
[0047] To further investigate whether the c.2T>C(p.M1) homozygous mutation in the CMPK2 gene is... Figure 1Genetic factors leading to brain calcification within families were investigated. Transcriptome analysis of peripheral blood mononuclear cells (PBMCs) was performed to explore differential gene expression between patients (patients II-3 and II-5 in family 1) and healthy controls. Total RNA was extracted from PBMCs of patients and healthy controls using TRIzol (Invitrogen, USA), and treated with DNase I to remove DNA contamination. For RNA library preparation and sequencing, mRNA was first obtained by processing with the TruSeq RNA Sample Preparation Kit, followed by mRNA library construction and quality testing. The RNA library was sequenced on the Illumina Hiseq 4000 platform (150 bp paired-end sequencing).
[0048] The data processing quality of raw reads was checked using FastQC v0.10.1 software (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ), and low-quality target and adapter data were adjusted using TrimGalore software. Qualified reads were mapped to a reference genome using HISAT2 v2.0.4 software, and the mapped reads for each sample were assembled using StringTie v1.3.1 software. Differentially expressed genes were analyzed using the DESeq2 software package; differentially expressed genes were defined as genes with an expression level change >2-fold and p < 0.05. Differential gene expression (DEG) enrichment analysis was performed using the DAVID (Database for Annotation, Visualization and Integrated Discovery) and GO (Gene Ontology) databases.
[0049] Transcriptome sequencing results showed that, compared with healthy controls, patients (II-3, II-5) had 4579 differentially expressed genes identified by DEGs analysis (differential expression criteria: log2 fold change >1, p-value <0.05), including 2572 upregulated genes and 2007 downregulated genes. Further GO analysis was used to categorize the differentially expressed genes. Among the 241 downregulated biological processes (BP) categories in GO, the top two subcategories both belonged to energy homeostasis regulation (…). Figure 4(a, b, c). Four biological processes are known to be involved with ribosomes (e.g., ribosome biogenesis, rRNA metabolism, ribonucleoprotein complex biogenesis, and regulation of ribosome biogenesis). It is generally believed that changes in ribosome-related pathway homeostasis are the main response to mitochondrial dysfunction. Impaired regulation of steroid metabolism was also found in the patient group, which is related to the crucial role of mitochondria in steroid synthesis. Furthermore, several other pathways related to mitochondrial function were downregulated in the patient group (e.g., ATP-dependent helicase activity, purine NTP-dependent helicase activity, and positive regulation of mitochondrial tissue). Figure 4 (b, d, e). In response to these downregulated pathways, several energy metabolism-related pathways, such as superoxide metabolism and reactive oxygen species reactions, carbohydrate derivative catabolism, regulation of fatty acid biosynthesis, and nucleoside diphosphate metabolism, were correspondingly upregulated. Figure 4 a, f, g).
[0050] The high energy demands of the mammalian central nervous system rely on the efficient energy production of mitochondria. Therefore, impaired mitochondrial function is closely associated with neurodegenerative diseases such as Alzheimer's and Parkinson's. While the causal relationship between mitochondrial stress and neurodegenerative diseases is largely established, the impact of upstream molecular processes in mitochondria (especially those factors that disrupt normal mitochondrial DNA synthesis) on neurodegenerative diseases remains unclear. This embodiment, through transcriptomic analysis of peripheral blood mononuclear cells from patients, reveals significant energy metabolism dysregulation, providing strong evidence for the association between mitochondrial dysfunction and brain calcification, and further confirms that functional loss-of-function mutations in the CMPK2 gene are a key genetic factor leading to the aforementioned symptoms.
[0051] Example 3: Expression of the Cmpk2 gene in the mouse brain
[0052] To further elucidate the function of the Cmpk2 gene, in situ hybridization (ISH) was used to analyze the expression pattern of Cmpk2 mRNA in the brains of WT mice. Prior to ISH, all instruments and reagents were washed or pretreated with diethyl pyrocarbonate (DEPC). Two-month-old mice were perfused with 4% paraformaldehyde (PFA) and dehydrated sequentially with 15% and 30% sucrose to prepare 20 μm thick brain slices, which were then mounted on Superfrost slides (Fisher). For double ISH, the RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics, Cat. 323100) was used in conjunction with the Cmpk2 probe C1, with or without the Gad1 C2, Slc17a6 C2 (Vglut2), or Aldh1i1 C2 probes. For dual labeling of in situ hybridization and immunofluorescence, brain slices were first in situ hybridized with a Cmpk2 gene probe, followed by incubation with anti-S100β antibody (1:300, ab868, Abcam). Whole-brain in situ hybridization images were acquired using a VS120 microscope with a 40× objective (Olympus). For colocalization imaging, in situ hybridization slices were imaged on a 40× objective Olympus FV3000 laser confocal microscope using a 488 / 468 nm laser and a 0.4 μm Z-axis overlay mode. In situ hybridization analysis was repeated three times for each sample. Single probe hybridization showed that the Cmpk2 gene was widely expressed in all brain regions. Figure 5 a). In the cell body layer of CA1 (Cornu Ammonis 1) pyramidal neurons, the granule cell layer (GCL) of the hippocampal dentate gyrus (DG), and the Purkinje cell layer (PCL) and GCL of the cerebellum. Figure 5 Strong enrichment of Cmpk2 signal was observed in both (b), indicating that it is abundant in neurons.
[0053] To determine the cell type expressing the Cmpk2 gene, a two-hybrid assay was performed by combining the Cmpk2 gene probe with marker gene probes targeting other cell types. The Cmpk2 gene marker also overlapped with a glutamatergic neuron-specific Slc17a6 (Vglut2) positive cell marker in the dentate nucleus of the thalamus and cerebellum. Figure 5 (c, d). Furthermore, Cmpk2 signals co-localize with GABAergic neuron-specific Gad1-positive cells on the thalamus and cerebellum PCL ( Figure 5(e, f). Conversely, no significant Cmpk2 signal was detected in cells that were positive for the astrocyte marker protein S100β immunostaining. Furthermore, based on published single-cell transcriptome data, the Cmpk2 gene was not significantly expressed in pericytes or microglia (http: / / betsholtzlab.org / VascularSingleCells / database.html). Figure 6 A). In contrast, this single-cell RNA-seq study found strong expression of the Cmpk2 gene in vascular endothelial cells (vECs), a finding further confirmed by another independent study (http: / / celltypes.org). Figure 6 B). In summary, the high expression of the Cmpk2 gene in neurons and cerebral vascular endothelial cells supports the possibility that Cmpk2 gene defects may lead to brain diseases, including brain calcification.
[0054] Example 4: Construction of Cmpk2 KO mouse and Cmpk2 c.2T>C(p.M1)KI mouse models
[0055] Furthermore, Cmpk2 KO mice and Cmpk2 c.2T>C(p.M1)KI mouse models were constructed to further investigate the function of the Cmpk2 mutant gene. Cmpk2 KO mice and Cmpk2c.2T>C(p.M1)KI mice were constructed using the CRISPR / Cas9 method (see Yao, XP et al. Biallelic Mutations in MYORG Cause Autosomal Recessive Primary Familial Brain Calcification. Neuron. 2018, 98:1116-1123 and Desai, N. et al. Elongational stalling activates mitoribosome-associated quality control. Science. 2020, 370:1105–1110), and genotyping was performed on the F0 generation mice. For KO mice, MEGAshortscript was used to... TM andmESSAGEmMACHINE TMsgRNAs and Cas9 mRNA (100 ng / μL) were synthesized using the T7 ultratranscription kit (Invitrogen) and co-injected into C57BL / 6J fertilized eggs, which were then implanted into surrogate female mice during the blastocyst stage. For KI mice, donor oligonucleotides were co-injected with sgRNA and Cas9 mRNA. Genomic DNA was extracted from the tails of F0 generation mice, and the gene-edited Cmpk2 genome sequence was amplified by PCR and cloned into a T vector (Promega, A137A). At least 20 clones from each F0 mouse were sequenced to assess Cmpk2 gene knockout. Cmpk2 KO F0 mice and homozygous Cmpk2 c.2T>C(p.M1)KI homozygous mice were selected for subsequent experiments. Table 1 lists the sgRNA sequence, donor oligonucleotide sequence, and genotyping primers. All P0Sprague-Dawley (SD) rats were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. All mice were housed at the Animal Center of the Shanghai Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences. Mice were kept in a standard 12-hour light / dark cycle environment with free access to standard rodent food and water. All studies included both male and female mice, and no sex bias was observed. All experimental procedures were approved by the Animal Husbandry and Use Committee of the Institute of Neuroscience, Chinese Academy of Sciences.
[0056] Example 5: Cmpk2 gene deficiency reduces mitochondrial DNA copy number in mouse neurons and impairs mitochondrial inorganic phosphorus (Pi) homeostasis.
[0057] Based on the Cmpk2 KO mouse and Cmpk2 KI mouse models, we further explored the mitochondrial functional impairment caused by the Cmpk2 mutant gene from the perspectives of mitochondrial DNA copy number, phosphorus and energy balance, and maintenance of normal cristae morphology.
[0058] (1) Mitochondrial DNA (mtDNA) copy number analysis
[0059] Mitochondrial DNA copy number was quantified by droplet digital PCR (ddPCR). Total DNA (TIANGEN and Gene Tech) was extracted from brain tissue of Cmpk2-KO mice and control mice. ddPCR was then performed on calibrated DNA samples using a QX200 AutoDG droplet digital PCR system (Bio-Rad) with a TaqMan primer / probe set. The probes and primers for ddPCR (ThermoFisher, 4458367) are listed in Table 1. mtDNA copy number was determined by the normalized level of total mtDNA relative to genomic DNA (Nd1-ms / β-actin or Nd1-ms / Tfrc-ms). Triples of each sample were analyzed. Compared with wild-type littermates, the ratio of mitochondrial DNA (Nd1) to nuclear DNA (β-actin or Tfrc) in the brains of Cmpk2-KO mice was reduced by 25-30%. Figure 7 a).
[0060] Furthermore, at the protein expression level, primary cultured rat neurons (cultured in vitro for 4 days, DIV 4) after electroporation were collected using the Cell Mitochondria Isolation Kit (Beyotime). Primary mouse neurons from WT and Cmpk2-KO mice (DIV 5) were collected in lysis buffer containing protease inhibitors. The remaining Western blotting steps were performed as described in Example 1. The levels of respiratory chain enzyme complex IV (COX IV) and cytochrome C encoded by mitochondrial DNA were also decreased in Cmpk2-KO mouse neurons. Figure 7 c, d).
[0061] At the immunofluorescence level, coverslips were fixed with 4% PFA for 15 minutes, then infiltrated and blocked with 0.1% Triton X-100 in PBS containing 5% BSA for 15 minutes. Next, the coverslips were incubated overnight at 4°C with antibodies against ATP synthase (1:500, MAB3494, Sigma) and MAP2 (1:1000, SC20172, Santa Cruz). The next day, the coverslips were washed three times with PBS and then incubated for 2 hours at room temperature with the corresponding secondary antibody (Alexa Fluor, Invitrogen). Finally, the coverslips were stained with Hoechst (Beyotime), washed three more times with PBS, and then mounted on glass slides. Fluorescence images were captured using an Olympus FV3000 laser confocal microscope with a 60× objective lens in 488 / 468 nm laser and 0.4 μm Z-axis superposition mode. Each sample was analyzed in triplicate. Immunofluorescence staining of the mitochondrial marker ATP synthase β chain showed a decrease in its signal intensity. Figure 7 b) Morphologically supports the reduction of mitochondria in neurons of Cmpk2-KO mice.
[0062] (2) Measurement of intracellular phosphorus and ATP
[0063] To measure intracellular phosphorus, primary cultured WT and Cmpk2 KO neurons were washed three times with phosphorus-free DMEM (11971025, Gibco). Cells were then lysed with phosphorus-free lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100). The lysates were then used to detect the release of inorganic phosphate (Pi) (303-0030, Innova Biosciences). To measure intracellular ATP, primary cultured WT and Cmpk2 KO neurons were placed in 96-well cell culture plates. ATP levels were detected at DIV 6 by luminescence assay (ab113849, Abcam).
[0064] Cmpk2-related dysfunction in mitochondrial genomic DNA synthesis involves aberrant phosphorylation of mitochondrial nucleotides. Indeed, significantly reduced ATP levels were observed in neurons of Cmpk2-KO mice. Figure 7 e), indicating that Cmpk2-KO does indeed lead to mitochondrial dysfunction. Furthermore, intracellular Pi levels in neurons of Cmpk2-KO mice were found to be significantly increased compared to WT mice (e). Figure 7 f). However, no significant changes in Pi concentration were found in the cerebrospinal fluid and serum of Cmpk2 mice (f). Figure 7Therefore, these results indicate that the absence of CMPK2 disrupts mitochondrial function and intracellular Pi homeostasis.
[0065] (3) Transmission electron microscopy (TEM) analysis
[0066] To further elucidate the role of CMPK2 in mitochondrial function, the mitochondrial morphology of primary neurons from cultured WT and CMPK2-KO mice was further evaluated using transmission electron microscopy (TEM).
[0067] Primary cultured pyramidal neurons derived from P0-P1 WT and Cmpk2 KO mice were imaged using transmission electron microscopy (TEM). Neurons were encased in Thermanox plastic coverslips (Thermanox, ThermoFisher) and fixed at DIV7 with 2.5% glutaraldehyde, followed by post-fixation with 1% OsO4 (TED PELLA, 18451), and then stained with 4% uranium acetate. They were then dehydrated by increasing the concentrations of ethanol and propylene oxide, and then embedded in resin. Ultrathin sections (70 nm) were cut using a superdiamond scalpel (Diatome, Switzerland) on an ultratissue microtome (EM UC7, Leica, Germany) and mounted on a copper grid. These sections were then observed and photographed using a TEM (JEM-1230, JEOL, Japan) and a CCD camera (Gatan Orius SC, 200w, 2048×2048). Mitochondrial cristae were observed in a double-blind study, and their coverage area was assessed by trained observers. The length and width of the cristae-covered regions were measured, and their areas, along with the mitochondrial area, were calculated. The cristae coverage area was represented as the ratio of cristae coverage area to the total mitochondrial area. Mitochondria were then categorized into different types based on cristae coverage, such as <40%, 40–60%, and >60%. Quantitative mitochondrial morphology analysis of primary cultured cortical neurons was performed from 40 WT neurons and 48 Cmpk2 KO neurons (derived from TEM images, independently repeated three times).
[0068] Crimson lines are specialized structures in which the respiratory chain supercomplex assembles, ensuring efficient energy production. Normal neurons have mitochondria with neatly arranged cristae extending throughout the mitochondrial cavity. Compared to normal controls, neurons cultured from Cmpk2-KO mice showed a higher proportion of mitochondria with cristae coverage below 40% (proportions: 68.8% in KO neurons and 37.8% in WT neurons). Figure 7 This indicates that the Cmpk2 gene maintains the cristae morphology of normal neuronal mitochondria in some way.
[0069] In summary, Cmpk2 gene mutations leading to reduced mitochondrial DNA synthesis result in insufficient synthesis of proteins encoded by mitochondrial DNA, thus causing inadequate energy production. Since mitochondrial cristae are specifically designed for efficient energy production, cristae disruption or reduced septal coverage implies potential impairment of mitochondrial energy production. Within mitochondria, various phosphates are used in a variety of metabolic pathways. For example, phosphates are essential substrates for the synthesis of CDP / dCDP and UDP / dUDP; therefore, a deficiency in CMPK2 may trigger a homeostatic upregulation of mitochondrial phosphate levels. Furthermore, insufficient ATP production may also stimulate homeostatic processes to upregulate intracellular phosphate levels. Considering that neuronal firing and adaptive activation in vECs involve active calcium ion activity in their mitochondria, the long-term involvement of these phosphate-related homeostatic responses may somehow lead to the accumulation and eventual precipitation of calcium phosphate in both cell types.
[0070] In summary, Cmpk2 gene dysfunction leads to a reduction in mitochondrial DNA copy number and impairs phosphorus and energy balance as well as the normal morphology of cristae. These findings are consistent with PBMC transcriptome analysis of patients with cerebral calcification, which showed energy metabolism dysfunction. This suggests that mitochondrial dysfunction promotes the occurrence and progressive development of cerebral calcification, and CMPK2 gene deficiency-related mitochondrial dysfunction expands the pathogenic mechanism of cerebral calcification.
[0071] Example 6: Calcification Deposition in the Brains of Cmpk2-KO and KI Mice
[0072] To test whether a deficiency in the Cmpk2 gene in mice leads to brain calcification similar to that observed in patients, Cmpk2-KO and Cmpk2-KI mice, prepared using the CRISPR-Cas9 strategy in Example 4, were used as research models. Cmpk2-KO mice were periodically analyzed for calcification deposition using mico-CT and various histological staining methods, including alizarin red, von Kossa, Alcian blue, periodic acid-Schiff (PAS), and H&E. Mice were anesthetized with chloral hydrate and perfused with 4% PFA in PBS. Prior to paraffin embedding, the brains of homozygous Cmpk2-KO and WT mice underwent a series of ethanol dehydration processes to prepare 4 μm thick sections. Brain calcification levels were assessed using histological staining (using alizarin red, von Kossa, Alcian blue, and periodic acid-Schiff (PAS) stains). Three-dimensional images of mouse brains were obtained using X-ray-based micro-CT scanning (Quantum GX, PerkinLemer). Paraffin-embedded mouse brains were evaluated in high-resolution mode with an X-ray tube voltage of 90 kV, a current of 88 μA, and a voxel size of 72 μm. Each sample was analyzed three times.
[0073] The results showed that dense calcification deposits were observed in the thalamus of Cmpk2-KO mice at 14 months of age, but not in WT mice. Figure 8 a). Furthermore, calcified nodules can be detected by micro-CT in 13-month-old Cmpk2 KO mice. Figure 8 b).
[0074] To further investigate the potential causal relationship between Cmpk2 gene mutations and brain calcification, a Cmpk2 KI mouse model was constructed by knocking in the coding sequence of the patient's Cmpk2 gene mutation (c.2T>C, p.M1). This mutation leads to the loss of mTP, and it is speculated that protein translation will be restarted at the subsequent ATG codon (p.M31). From 12 months onwards, significant calcification deposits were also found in the thalamus of homozygous Cmpk2 KI mice. Figure 8 c). These results confirm that Cmpk2 gene defects are the cause of calcification deposits in the mouse brain and further support that Cmpk2 gene mutations can explain the brain calcification phenotype in patients.
[0075] Example 7: Analysis of the CMPK2 gene-related interferon pathway and its applications
[0076] In the context of brain calcification, the CMPK2 gene-related interferon pathway warrants attention. In the human genome, the CMPK2 gene is located between the RSAD2 and NRIR genes. RSAD2 and NRIR are interferon-related factors, and CMPK2 gene expression has been found to be regulated upon stimulation by type I interferon, suggesting a potential interaction between CMPK2 and the interferon response. Since interferon stimulation has previously been shown to promote the progression of brain calcification (see Chakrabarty, P. et al. Interferon-γ induces progressive nigrostriatal degeneration and basalganglia calcification. Nat. Neurosci. 2011, 14, 694–696), investigating the effects of the CMPK2 gene on the interferon pathway in the human or mouse brain contributes to a comprehensive understanding of how CMPK2 gene defects lead to brain calcification. A recent study reported that CMPK2-dependent mitochondrial DNA synthesis can activate the NLRP3 inflammasome in macrophages, and NLRP3 has also been found to trigger neuroinflammation in Parkinson's disease and Alzheimer's disease. Based on evidence from families with latent cerebral calcification and KO and KI mouse models, this study supports the view that disruption of CMPK2 gene function leads to brain calcification. This can be explained by the fact that long-term inhibition of CMPK2-related NLRP3 inflammasome signaling may also interfere with the mitochondrial signaling network for nucleotide synthesis, thereby leading to intracranial calcification deposition. Furthermore, pharmacological manipulation of the NLRP3 inflammasome has attracted attention as a potential therapeutic intervention for cancer and various inflammatory diseases; however, this study indicates that this strategy may carry risks: inhibition of upstream regulatory signaling of NLPR3 in mitochondria may lead to brain calcification and / or mitochondrial dysfunction. Therefore, monitoring for mitochondrial dysfunction and brain calcification should be considered when investigating these innovative anti-inflammatory therapies.
[0077] The above findings reveal a novel pathogenic mechanism of brain calcification caused by mitochondrial dysfunction resulting from CMPK2 gene mutations. In Cmpk2-KO and KI mice, age-progressive brain calcification deposition was observed, mimicking the pathological development of human patients carrying Cmpk2 gene mutations. More specifically, reduced mitochondrial genomic DNA copy number, decreased ATP production, intracellular Pi abnormalities, and cristae disruption were observed in Cmpk2 gene-deficient neurons, potentially contributing to the development of new treatments for brain calcification. This invention broadens the potential cellular and molecular mechanisms leading to brain calcification. Previously, only two main pathogenic pathways were identified as contributing to brain calcification. First, impaired BBB structure integrity or reduced pericyte coverage leads to increased BBB permeability; patients associated with this pathway may have mutations in genes such as PDGFRB, PDGFB, JAM2, or MYORG, all of which are expressed in the units of cells that constitute neurovascular bundles. The second pathway involves disruption of phosphate transport in the brain. Patients exhibiting this pathway may have mutations in the SLC20A2 or XPR1 genes and typically show significantly elevated CSF Pi levels. In contrast, mitochondria, targeted by the CMPK2 gene product, are unique organelles involved in the pathogenesis of cerebral calcification and are not significantly associated with previously identified pathways related to cerebral calcification. Furthermore, the expression of the Cmpk2 gene in neurons and vascular endothelial cells suggests that mitochondrial defects induced by the Cmpk2 gene may affect neuronal and endothelial cell function. In conclusion, the CMPK2 gene mutations found in patients with hereditary cerebral calcification reveal that its mitochondrial damage could serve as a novel cellular pathological mechanism for cerebral calcification.
[0078] Although the present invention has been described through preferred embodiments, various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A mutant gene of CMPK2, characterized in that: Referring to the CMPK2 wild-type gene coding sequence shown in SEQ ID NO: 1, the CMPK2 mutant gene coding sequence is shown in SEQ ID NO: 2, that is, the CMPK2 mutant gene is formed by a c.1A>C mutation in the wild-type CMPK2 gene; the CMPK2 mutant gene can be used as a target in the development of diagnostic kits and preventive drugs for cerebral calcification, as well as in the research on the pathogenesis of cerebral calcification and clinical diagnosis and treatment.
2. The protein encoded by the CMPK2 mutant gene as described in claim 1, characterized in that: The coding sequence of the CMPK2 mutant gene corresponding to SEQ ID NO: 2 is shown in SEQ ID NO:
6. Referring to the coding sequence of the wild-type CMPK2 gene shown in SEQ ID NO: 5, specifically, the amino acid fragment at positions 1-26 of the wild-type CMPK2 gene coding protein is deleted, i.e., the p.1_26delMAFARRLLRGPLSGPLLGRRGVCAGA mutation occurs. The deletion of the wild-type CMPK2 gene coding protein shown in SEQ ID NO: 5 and the presence of the CMPK2 mutant gene coding protein shown in SEQ ID NO: 6 in nerve cells lead to the occurrence of cerebral calcification.
3. A detection kit for the CMPK2 mutant gene as described in claim 1, characterized in that: The kit contains PCR primer pairs as shown in SEQ ID NO:8-9, which can amplify the c.1A>C mutation contained in the coding sequence of the CMPK2 mutant gene as shown in SEQ ID NO:
2.
4. A recombinant vector, characterized in that: The recombinant vector contains the CMPK2 mutant gene fragment as shown in SEQ ID NO: 2.