A PKHD1L1 mutant gene inducing familial cortical tremor with epilepsy and its application

Abstract

The present invention provides a PKHD1L1 mutant gene and application that induces familial cortical tremor with epilepsy, and belongs to the field of gene detection and application technology. The present invention provides a human PKHD1L1 mutant gene, including a point mutation at the 801bp site in the 3'-UTR region. The present invention discovered the PKHD1L1c.*801G>A mutation through gene sequencing in a family with familial cortical tremor with epilepsy. The mutation is located in the 3'-UTR region, and the difference between the patient and the normal person is found through in vivo expression and luciferase experiments; and combined with molecular dynamics research, it is found that at the position of the mutant base, the helical region is opened, and the change in structure may affect the expression of the gene. Therefore, it is proved that the mutant gene is the pathogenic gene of the family.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection and application, and specifically relates to a PKHD1L1 mutant gene inducing familial cortical tremor with epilepsy and an application thereof. Background Art

[0002] Epilepsy is a serious chronic disease of the central nervous system. It is a temporary brain dysfunction caused by repeated and highly synchronized abnormal discharges of brain neurons. The clinical manifestations are different disorders of movement, sensation, consciousness, autonomic nerves and spirit. It is one of the more common diseases of the nervous system, endangering more than 65 million people worldwide. The disability, death and complications related to epilepsy have placed a heavy burden on patients and society.

[0003] Familial cortical myoclonic tremor and epilepsy (FCMTE) is a rare chromosomal dominant hereditary disease with cortical tremor and distal limb clonus with or without epileptic seizures as the main symptoms, and has significant genetic heterogeneity. According to linkage mapping of pathogenic regions, it can be divided into four subtypes: FCMTE1 (8q24, OMIM: 601068), FCMTE2 (2p11.1-q12.2, OMIM: 607876), FCMTE3 (5p15.31-p15.1, OMIM: 613608) and FCMTE4 (3q26.32-3q28, OMIM: 615127). No confirmed pathogenic gene has been reported so far.

[0004] The PKHD1L1 gene (NM_001034931) is located on human chromosome 8q23 and occupies about 168kb. The RNA of the PKHD1L1 gene has multiple splicing modes, producing multiple transcripts. The longest transcript consists of 78 exons, totaling 13079bp, encoding a protein Fibrocystin-L composed of 4243 amino acids. Bioinformatics prediction results show that Fibrocystin-L is a single transmembrane receptor-like protein. Its carboxyl terminus is located intracellularly and consists of 8 amino acids. It contains a potential protein kinase C (PKC) phosphorylation site. The extracellular region of Fibrocystin-L consists of 4212 amino acids, and from the N-terminus, there are a signal peptide, 14 tandem TIG domains, and a TEMEE homology region composed of 9 PbH1 repeat sequences. Preliminary RT-PCR research results indicate that PKHD1L1 is widely expressed in various tissues and organs of humans and mice. Genetic studies have shown that the heterozygous mutation of PKHD1L1 gene exon23:c.2602A>T may be the causative gene of FAME (familial adult myoclonic epilepsy, FAME), but the functions of mutations at other sites of the PKHD1L1 gene have not been predicted. Summary of the invention

[0005] The purpose of the present invention is to provide a PKHD1L1 mutant gene and application that induces familial cortical tremor with epilepsy. The PKHD1L1 mutant gene is a pathogenic gene of familial cortical tremor with epilepsy and can be used to quickly and effectively predict or diagnose familial cortical tremor with epilepsy.

[0006] The present invention provides a human PKHD1L1 mutant gene, comprising a point mutation at the 801 bp site in the 3'-UTR region of the human PKHD1L1 gene.

[0007] Preferably, the point mutation comprises mutating the G at the 801 bp position in the 3'-UTR region of the human PKHD1L1 gene to A.

[0008] The present invention also provides the protein expressed by the human PKHD1L1 mutant gene.

[0009] The present invention also provides a use of a reagent for detecting the human PKHD1L1 mutant gene in preparing a tool for predicting and / or diagnosing familial cortical tremor with epilepsy.

[0010] Preferably, the reagent for detecting the human PKHD1L1 mutant gene includes a reagent for detecting the expression amount of the human PKHD1L1 mutant gene, and / or a reagent for detecting the luciferase activity of the human PKHD1L1 mutant gene.

[0011] The present invention also provides a reagent for detecting a human PKHD1L1 mutant gene, comprising at least one of the following: a specific primer pair and a luciferase detection reagent;

[0012] The specific primer pair includes a first specific primer pair consisting of PKHD1L1-qpcr-1F with a nucleotide sequence as shown in SEQ ID No.1 and PKHD1L1-qpcr-1R with a nucleotide sequence as shown in SEQ ID No.2, and / or a second specific primer pair consisting of PKHD1L1-qpcr-2F with a nucleotide sequence as shown in SEQ ID No.3 and PKHD1L1-qpcr-2R with a nucleotide sequence as shown in SEQ ID No.4.

[0013] Preferably, the luciferase detection reagent includes a reagent for constructing a human PKHD1L1 gene and the above-mentioned human PKHD1L1 mutant gene transformation vector, a reagent for transforming eukaryotic cells, and a reagent for detecting luciferase activity.

[0014] The present invention also provides a prediction and / or diagnosis kit for familial cortical tremor associated with epilepsy, comprising the above reagent.

[0015] Preferably, detection primers for internal reference genes are also included.

[0016] Preferably, the detection primers of the internal reference gene include Actin-F with a nucleotide sequence as shown in SEQ ID No.5 and Actin-R with a nucleotide sequence as shown in SEQ ID No.6.

[0017] Beneficial effects: The present invention provides a human PKHD1L1 mutant gene, including a point mutation at the 801bp site in the 3'-UTR region. The present invention discovered the PKHD1L1 c.*801G>A mutation in a family with familial cortical tremor and epilepsy through gene sequencing. The mutation is located in the 3'-UTR region. The difference between patients and normal people was found through in vivo expression and luciferase experiments; and combined with molecular dynamics research, it was found that at the position of the mutant base, the helical region was opened, and the change in structure may affect the expression of the gene. Therefore, it is proved that the mutant gene is the pathogenic gene of the family. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the amplification curve of the internal reference Actin;

[0019] Figure 2is the melting curve of the internal reference Actin;

[0020] Figure 3 is the amplification curve of PKHD1L1-qpcr-1F / PKHD1L1-qpcr-1R;

[0021] Figure 4 This is the melting curve of PKHD1L1-qpcr-1F / PKHD1L1-qpcr-1R;

[0022] Figure 5 is the amplification curve of PKHD1L1-qpcr-2F / PKHD1L1-qpcr-2R;

[0023] Figure 6 This is the melting curve of PKHD1L1-qpcr-2F / PKHD1L1-qpcr-2R;

[0024] Figure 7 It is the three-dimensional result diagram of wild-type 3'UTR;

[0025] Figure 8 It is the three-dimensional result diagram of mutant 3'UTR;

[0026] Fig. 9 It is an information diagram of molecular simulation process parameters;

[0027] Fig.10 This is the result diagram after wild-type kinetic simulation;

[0028] Fig.11 This is the result diagram after the mutant dynamics simulation;

[0029] Fig.12 A pedigree diagram of the patient involved in the present invention;

[0030] Fig.13 The results of PKHD1L1 luciferase activity detection are shown in Figure 1. (A) The sequencing verified that the vector was successfully constructed; (B) The PKHD1L1 luciferase activity was detected in 293T cells; (C) The PKHD1L1 luciferase activity was detected in HeLa cells. DETAILED DESCRIPTION

[0031] The present invention provides a human PKHD1L1 mutant gene, comprising a point mutation at the 801bp site in the 3'-UTR region.

[0032] The point mutation of the present invention includes mutating the G at the 801bp site to A, that is, PKHD1L1 c.*801G>A mutation, and the transcript of the PKHD1L1 gene of the present invention is preferably NM_177531.6.

[0033] The present invention also provides the protein expressed by the human PKHD1L1 mutant gene.

[0034] Since the mutation described in the present invention occurs in the 3'-UTR region, which is not a protein coding region, the mutation in the UTR region does not cause changes in the protein structure, but only regulates the expression of the protein; the expression level of the mutant gene in human blood is also detected in the embodiment of the present invention, confirming that the point mutation will lead to a decrease in the expression level of the gene. The three-dimensional structure of the mutated protein of the present invention is as follows Figure 8 As shown, its structure after dynamic simulation is as follows Fig.11 shown.

[0035] The present invention also provides a reagent for detecting a human PKHD1L1 mutant gene, comprising at least one of the following: a specific primer pair and a luciferase detection reagent;

[0036] The specific primer pair includes a first specific primer pair consisting of PKHD1L1-qpcr-1F with a nucleotide sequence as shown in SEQ ID No.1 and PKHD1L1-qpcr-1R with a nucleotide sequence as shown in SEQ ID No.2, and / or a second specific primer pair consisting of PKHD1L1-qpcr-2F with a nucleotide sequence as shown in SEQ ID No.3 and PKHD1L1-qpcr-2R with a nucleotide sequence as shown in SEQ ID No.4.

[0037] Since the mutation site of the mutant gene described in the present invention is located in the UTR region, qRT-PCR and luciferase detection are generally required to determine whether it is a pathogenic gene. Therefore, the reagent described in the present invention includes at least one type of reagent for qRT-PCR quantitative detection and luciferase activity detection.

[0038] The primer sequences of qRT-PCR used in the present invention are preferably as shown below:

[0039] PKHD1L1-qpcr-1F (SEQ ID No. 1): GGGGAAGGTTTTTCTCAAGC;

[0040] PKHD1L1-qpcr-1R (SEQ ID No. 2): TCTTCCGGCATTGCTCTAGT;

[0041] PKHD1L1-qpcr-2F (SEQ ID No. 3): CAGCAAAGCATCAACTGTGG;

[0042] PKHD1L1-qpcr-2R (SEQ ID No. 4): GCCTATTCTTCGGAACAGCA.

[0043] When the present invention uses qRT-PCR to detect the expression change of the mutant gene, Actin is preferably used as an internal reference, and the primer sequence of the internal reference is preferably as follows:

[0044] Actin-F (SEQ ID No. 5): TGACGTGGACATCCGCAAAG;

[0045] Actin-R (SEQ ID No. 6): CTGGAAGGTGGACAGCGAGG.

[0046] The present invention preferably uses the cDNA obtained by reverse transcription of RNA from a whole blood sample as a template for PCR amplification. The PCR amplification system preferably includes 2×PCRMix 10 μL, 10 mM Primer-F 1 μL, 10 mM Primer-R 1 μL, cDNA 1 μL and ddH 2 O 7 μL. The PCR amplification program of the present invention is preferably as follows: 95°C for 5 min; 95°C for 30 s, 57°C for 30 s, 72°C for 5 min, 30 cycles.

[0047] When the present invention predicts and / or diagnoses familial cortical tremor with epilepsy by detecting luciferase activity, it preferably includes reagents for constructing human PKHD1L1 gene and the above-mentioned human PKHD1L1 mutant gene transformation vector, reagents for transforming eukaryotic cells and reagents for detecting luciferase activity.

[0048]

[0049] 166918-PKHD1L1-F (SEQ ID No. 8): tgcattttgcattgcctcat;

[0050] 169714-PKHD1L1-R (SEQ ID No. 9): CATGGCAAATGTTCCTCATG;

[0051] 167162-PKHD1L1-F (SEQ ID No. 10):ggattaaaatgctgggttct;

[0052] 168854-PKHD1L1-R (SEQ ID No. 11): CCTGAGACAGTGATCATGTT;

[0053] pmirGLO-3'MCS-PKHD1L1-NheI-F (SEQ ID No. 12): GCTCGCTAGCGAAGCTACTAAAGTGCTGTT;

[0054] pmirGLO-3'MCS-PKHD1L1-SalI-R (SEQ ID No. 13):GCAGGTCGACAAGTAAGACTAACAATGATC.

[0055] The present invention preferably uses the vector pmirGLO-3'MCS (article number VT1439) purchased from Youbao Bio as the basic vector, connects the NheI-wt-SalI fragment shown in SEQ ID No.7 with the vector pmirGLO-3'MCS linearized with NheI and SalI, transforms the DH5α competent cells with the connection product, selects the positive clones for sequencing after colony PCR identification, and obtains the pmirGLO-3'MCS-PKHD1L1-wt vector with correct sequencing. The colony PCR identification of the present invention preferably includes the following steps: after monoclonal expansion culture, using the bacterial solution as a template, using pmirGLO-PKHD1L1-NheI-F and PKHD1L1-mut-R as primers for PCR amplification, and the obtained PCR product is subjected to agarose gel electrophoresis, and the positive clone is in accordance with the expected size of 843bp. The system and procedure of PCR amplification of the present invention are preferably the same as those of the above-mentioned PCR, and will not be repeated here.

[0056] When constructing a transformation vector of a human PKHD1L1 mutant gene, the present invention preferably uses the fragment PKHD1L1-wt shown in SEQ ID No. 7 as a template, uses primers pmirGLO-3'MCS-PKHD1L1-NheI-F and PKHD1L1-mut-R as primers to amplify the mut-1 fragment, and uses primers PKHD1L1-mut-F and pmirGLO-3'MCS-PKHD1L1-SalI-R as primers to amplify the mut-2 fragment; then, the mut-1 fragment and the mut-2 fragment are mixed in equal amounts and used as a template, and pmirGLO-3'MCS-PKHD1L1-NheI-F and pmirGLO-3'MCS-PKHD1L1-SalI-R are used as primers to amplify the NheI-mut-SalI fragment, and the nucleotide sequence is preferably as shown in SEQ IDAs shown in No.14: GCTCGCTAGCGAAGCTACTAAAGTGCTGTTCCGAAGAATAGGCTGAAACAAAAATATAAGAATTATTAGCTACTTTGTTGGGCAATAGGCAAAAGTCTATAGCATTTTCATGAAAATATACTAAAAATATTTTTATGATATATAAAATGTACTAATTAGCTTTAAACACTAAAATCAGATTTCTTCAAAATATAAATTTGTTTTGATTCTTTATATTTATATGTTTTTATTTCATTTCAATAAACTTCCAGAAATTTGTCATTTGGAAGTAGATATGACACCTCTAAGTTATTGTACCAACAAATCATAGAATCCTTTAAATAATGGAAAGAGCTTGTCTAGTTCCGACTTAAGCTCTTTACATCAGATTGGAAGCATTTTAAGTATTCTTTTTAATACTTGAGGGGGGGGGTTCTTATTTTCTCTATCCATTTACAAGGTTTTGATTTCCAATAATCTAAGCCATTCTAGTTTCTAAGGATTTTGGAGAGACGAGATTGTCTTCACACAATCTGAGATGACAATCTCATCTCAGATTTTTCACACTGAGTTCCTGTCTTTCAGATTCACAGTAATCCCAGGAATTCTAGACTGTCCTGTATTTCTGGATCTGCCCAACTCTGGCCAATATTGGGAAAAGTTTTCTTGTCCTTGGAATGAACTTTTGGAACAAGTCCAAACTCCTTCTCTGCCTACCCCTCCTTCCCACTCAAAACTGCCACTACTAAGAGCAGGACTCCCCTCAACCCCGGGCATGAGTTGAGGGTTGAGGGGTTCCCTTAACTGGTGGATGCTTATTCACTGTTTACTGGATGAATGAAATATAAAGTGTGGGAAGTCAACAACAGAACAGAACTTATTTTCAAGAAGATAAATTAGAGAATGCAAAAAAGCTACAGACTAAGGTAGCTAAGTTAACCGAACTCTCTAACAGTATTGAAAATAGCAATTCTTTACTCAGAAAATTCTAAAGGGAATACTTAATTTGACAGAACTCCTAATAAAGACATTGTAGCCAGATCCAGAGCCTTCCAGCAAGTGACACTCAGCAAACATTTGGGACAGCTGAGAGAATTCAACAAAGCCCATACCGTTGTCTCTTGTATAACATCTCCTAGTACATTCACCTCAGATCATTGTTAGTCTTACTTGTCGACCTGC, a total of 1161 bp. The primer sequences used in the construction of the transformation vector of the present invention are preferably as follows:

[0057] PKHD1L1-mut-R (SEQ ID No. 15): CTGTTTACTGGATGAATGAAATATAAAGTGTGGGAAGTCAAC;

[0058] PKHD1L1-mut-F (SEQ ID No. 16): GTTGACTTCCCACACTTTATATTTCATTCATCCAGTAAACAG.

[0059] The present invention preferably uses the vector pmirGLO-3'MCS as the basic vector, connects the NheI-mut-SalI fragment with the vector pmirGLO-3'MCS linearized by NheI and SalI, transforms the connection product into DH5α competent cells, and selects positive clones for sequencing after colony PCR identification, and obtains the pmirGLO-3'MCS-PKHD1L1-mut vector after correct sequencing.

[0060] After constructing the pmirGLO-3'MCS-PKHD1L1-wt vector and the pmirGLO-3'MCS-PKHD1L1-mut vector, the two vectors are preferably transformed into eukaryotic cells, such as 293T and HeLa cells, respectively, and luciferase activity is detected 48 hours after the transformation. The present invention does not specifically limit the method for detecting the luciferase activity. In the embodiment, the dual- ReporterAssay System (Promega), just follow the instructions.

[0061] The present invention also provides a use of a reagent for detecting the human PKHD1L1 mutant gene in preparing a tool for predicting and / or diagnosing familial cortical tremor with epilepsy.

[0062] In the embodiment of the present invention, the mRNA expression level of the human PKHD1L1 mutant gene was determined using the primers shown in SEQ ID No.1 to SEQ ID No.6. The results showed that in the detection of multiple samples, both pairs of primers and the internal reference had single bands without mixed bands, which met the requirements of Q-PCR. Compared with the normal group, the expression level of the human PKHD1L1 mutant gene in patients with familial cortical tremor and epilepsy was significantly decreased; and the luciferase activity detection showed that the luciferase activity of patients with familial cortical tremor and epilepsy was also significantly reduced. Therefore, the present invention uses the human PKHD1L1 mutant gene as a marker for predicting and / or diagnosing familial cortical tremor and epilepsy.

[0063] The present invention also provides a prediction and / or diagnosis kit for familial cortical tremor associated with epilepsy, comprising the above reagent.

[0064] The kit of the present invention preferably further comprises detection primers for internal reference genes, wherein the detection primers for internal reference genes comprise Actin-F having a nucleotide sequence as shown in SEQ ID No.5 and Actin-R having a nucleotide sequence as shown in SEQ ID No.6.

[0065] To further illustrate the present invention, a PKHD1L1 mutant gene for inducing familial cortical tremor with epilepsy and its application provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0066] The diseased families involved in the embodiments of the present invention are as follows: Fig.12 shown.

[0067] In the embodiment of the present invention, the RNA extraction method can be carried out according to the instructions of PAXgene BloodRNA kit purchased from QIAGEN (company), the product number is 761604;

[0068] Genome digestion and cDNA synthesis were performed according to the kit purchased from Takara (company): PrimeScript TM Just follow the instructions of RTreagent Kit with gDNA Eraser (Perfect Real Time), the catalog number is RR047A.

[0069] Example 1

[0070] by Fig.12The cDNA obtained by reverse transcription of RNA extracted from whole blood samples of II2: B2A (report number), II 3: B3A (report number), II5: B4A (report number) and II8: B1A (report number) of the family shown was used as a template, and PCR amplification was performed using primer pairs consisting of SEQ ID No.1 and SEQ ID No.2, and primer pairs consisting of SEQ ID No.3 and SEQ ID No.4, respectively, and SEQ ID No.5 and SEQ ID No.6 were used as internal reference genes to verify the mRNA expression levels of B1A, B2A, B3A and B4A. The results are shown in Figures 1 to 6 shown.

[0071] Two pairs of detection primers were designed to detect the mRNA expression of PKHD1L1 gene in four people, B1A, B2A, B3A and B4A in the family. Taking B3A as the control, the detection results of PKHD1L1-1 (SEQ ID No.1~SEQ ID No.2) showed that the expression level of B4A was 0.32, the expression level of B1A was 0.04, and the expression level of B2A was 0.17; the detection results of PKHD1L1-2 (SEQ ID No.3~SEQ ID No.4) showed that the expression level of B4A was 0.35, the expression level of B1A was 0.17, and the expression level of B2A was 0.23. The mRNA expression level in the case group was significantly lower than that in the control group.

[0072] Example 2

[0073] Luciferase Assay

[0074] 2.1 Vector construction

[0075] 2.1.1 Vector construction

[0076] (1) Construction of pmirGLO-3'MCS-PKHD1L1-wt vector

[0077] Primer design: Two pairs of nested primers 166918-PKHD1L1-F and 169714-PKHD1L1-R, 167162-PKHD1L1-F and 168854-PKHD1L1-R, pmirGLO-3'MCS-PKHD1L1-NheI-F and pmirGLO-3'MCS-PKHD1L1-SalI-R were designed and nested PCR was performed using DNA as template.

[0078] Fragment amplification: Using normal human DNA as a template, using nested primers 166918-PKHD1L1-F and 169714-PKHD1L1-R to amplify the first round of PCR products; using the nested first round of PCR products as a template, using nested primers 167162-PKHD1L1-F and 168854-PKHD1L1-R to amplify the second round of PCR products; using the nested second round of PCR products as a template, using pmirGLO-3'MCS-PKHD1L1-NheI-F and pmirGLO-3'MCS-PKHD1L1-SalI-R as primers to amplify the NheI-wt-SalI fragment SEQ ID No.7;

[0079] Enzyme digestion: The vector pmirGLO-3'MCS and the fragment NheI-wt-SalI were digested with NheI and SalI, respectively;

[0080] Ligation: After the digested vector and fragments are recovered, they are ligated in a ratio of 3:1 between fragment and vector;

[0081] Transformation: The ligation product was transformed into DH5α competent medium;

[0082] Select bacteria for colony PCR identification and sequence the positive clones.

[0083] The constructed pmirGLO-3'MCS-PKHD1L1-wt vector was transformed and sequenced after bacterial liquid PCR, and sequence alignment was performed to select wild-type positive clones.

[0084] (2) Construction of pmirGLO-3'MCS-PKHD1L1-mut vector

[0085] Fragment amplification: Using the fragment PKHD1L1-wt as a template and pmirGLO-3'MCS-PKHD1L1-NheI-F / PKHD1L1-mut-R as primers, the mut-1 fragment was amplified, and the mut-2 fragment was amplified by PKHD1L1-mut-F / pmirGLO-3'MCS-PKHD1L1-SalI-R as primers. Then, using a mixture of mut-1 and mut-2 1:1 as a template, pmirGLO-3'MCS-PKHD1L1-NheI-F and pmirGLO-3'MCS-PKHD1L1-SalI-R as primers, the NheI-mut-SalI fragment SEQ ID No.14 was amplified;

[0086] Enzyme digestion: The vector pmirGLO-3'MCS and the fragment NheI-mut-SalI were digested with NheI and SalI;

[0087] Ligation: After the digested vector and fragments are recovered, they are ligated in a ratio of 3:1 between fragment and vector;

[0088] Transformation: The ligation product was transformed into DH5α competent medium;

[0089] Select bacteria for colony PCR identification and sequence the positive clones.

[0090] The constructed pmirGLO-3'MCS-PKHD1L1-mut vector was transformed and sequenced after bacterial liquid PCR, and sequence alignment was performed to select mutant positive clones.

[0091] 2.2 Cell culture and transfection

[0092] 293T and HeLa cells were cultured in DMEM medium containing 10% fetal bovine serum, and the constructed pmirGLO-3'MCS-PKHD1L1-wt / mut recombinant vector was transiently co-transfected into 293T and HeLa cells according to the instructions of Lipo2000 liposomes, and luciferase activity was detected 48 hours after transfection.

[0093] 2.3 Luciferase activity detection

[0094] 48 hours after transfection, remove the culture medium in the culture plate, wash the cells with 1×PBS, and remove the culture medium. According to the instructions of the Reporter Assay System (E1910, Promega), 250 μL of freshly prepared 1×PLB lysis buffer was added to each well of a 12-well plate, and the culture plate was gently shaken for 15 minutes at room temperature to collect the lysate. The detection was performed using a full-function microplate reader (PerkinElmer). 20 ul of cell lysate was added to each well, 100 μL of LARII was added to detect firefly luciferase, and then 100 μL of freshly prepared 1×Stop& The luciferase activity was detected by using a reagent. The luciferase activity was calculated according to Formula I using the luciferase activity as a control.

[0095] Formula I: Luciferase expression activity = average firefly luciferase activity / average Renilla luciferase activity

[0096] The results are as follows Fig.13 As shown: 48 hours after transfection, in 293T cells, the activity of mutant mut luciferase decreased by 40% compared with wild-type wt; in HeLa cells, the activity of mutant mut luciferase decreased by 35% compared with wild-type wt; quantitative cell detection showed that the activity of mutant luciferase was lower than that of wild-type, suggesting that mutation may affect gene expression.

[0097] Example 3

[0098] Construction of PKHD1L1 gene 3'UTR region structure using RNAcomposer

[0099] 1. Construct 3'UTR wild-type and mutant structures based on the PKHD1L1 c.*801G>A site mutation information;

[0100] 2. Use the RNAFold algorithm in RNAcomposer software to predict the secondary structure and three-dimensional structure of the 3'UTR region;

[0101] 3. The constructed three-dimensional structure was preliminarily optimized using Gromacs, and the optimized wild-type structure and mutant structure were used as the initial structure for subsequent molecular dynamics;

[0102] The wild-type sequence is shown in SEQ ID No. 17: UGAGGGGAGUUGGGGCCCGUACUCAACUCCCAACUCCCCAAGGGAAUUGACCACCUACGAAUAAGUGACAAAUGACCUACUUACUUCAUAUUUCACACCCUUCAGUUGUUGUCUUGUCUUGAAUAAAAGUUCUUCUAUUUAAUCUCUUA, and the structure is shown in Figure 7 As shown in the figure, the position marked in red is the location where the base mutation occurs. The location of the mutation indicates the structural importance in the 3'UTR region. This position is exposed in the spatial structure and may be easily recognized by regulatory molecules. From the structural point of view, this mutation may affect the gene function.

[0103] The mutant (Mut) sequence is shown in SEQ ID No. 18: UGAGGGGAGUUGGGGCCCGUACUCAACUCCCAACUCCCCAAGGGAAUUGACCACCUACGAAUAAGUGACAAAUGACCUACUUACUUUAUUUCACACCCUUCAGUUGUUGUCUUGUCUUGAAUAAAAGUUCUUCUAUUUAAUCUCUUA, and the structure is shown in Figure 8 shown.

[0104] 3.2 Molecular dynamics simulation

[0105] GROMACS is a high-performance molecular dynamics software used to simulate the motion of particles under Newton's equations. When simulating the 3'UTR region of RNA, several steps need to be followed. The following is the general process of simulating the 3'UTR partial structure using GROMACS version 5.1, which includes selecting an appropriate force field, preparing an initial structure, minimizing energy, performing equilibrium, and producing simulations.

[0106] 1. Prepare the initial structure: First, use the structural model obtained by RNAcomposer prediction software as the initial structure for molecular simulation.

[0107] 2. Select force field:

[0108] GROMACS provides a variety of force field options for simulating different types of molecules. The force field used in the present invention is the AMBER ff14SB force field.

[0109] 3. Create topology file:

[0110] The topology files were generated using the GROMACS pdb2gmx tool and integrated into one topology file according to the selected force field.

[0111] 4. Define simulation box and solvation:

[0112] Define the simulation box using the editconf tool and solvate the RNA structure using gmx solvate. This typically involves adding water molecules and possibly ions to neutralize the charge of the system.

[0113] 5. Energy minimization: Before the dynamics simulation, energy minimization must be performed to eliminate any unreasonable contacts and steric conflicts. This can be done using grompp (preprocessor) and mdrun tools.

[0114] 6. Temperature and pressure balance: After energy minimization, the system needs to be balanced to ensure that the temperature and pressure are stable. The simulation is performed using the NVT (constant volume and temperature) and NPT (constant pressure and temperature) ensembles.

[0115] 7. Production simulation: Finally, actual production simulation is performed to collect data for analysis. Molecular simulation process parameter information such as Fig. 9 shown.

[0116] The results of the wild type (Wild) kinetic simulation are as follows Fig.10 As shown, the green three-dimensional structure at the end of the dynamics simulation is Figure 7The comparison of the initial structures shown in the figure shows that the wild type has little structural change before and after the dynamics. The red sticks show the bases corresponding to the mutation positions. The detailed diagram also shows that the structural changes are small. The results after the dynamics simulation of the mutant (Mut) are as follows Fig.11 As shown, the green three-dimensional structure is the structure of the mutant structure at the end of molecular dynamics simulation. Figure 8 By comparison, it was found that during the dynamic process, the mutant structure underwent significant changes. At the position of the mutant base, the helical region was opened, and the structural changes may affect gene expression.

[0117] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a reagent for detecting a human PKHD1L1 mutant gene in the preparation of a kit for predicting and / or diagnosing familial cortical tremor with epilepsy, characterized in that: The human PKHD1L1 mutant gene is a human PKHD1L1 mutated gene in which the G at the 801 bp site in the 3'-UTR region is mutated to A. The accession number of the PKHD1L1 transcript in NCBI is NM_177531.

6.

2. A prediction and / or diagnosis kit for familial cortical tremor with epilepsy, characterized in that: The invention comprises a reagent for detecting a human PKHD1L1 mutant gene, wherein the G at the 801bp site of the 3'-UTR region of the human PKHD1L1 is mutated to A, and the accession number of the PKHD1L1 transcript in NCBI is NM_177531.6.

Images