CN117625632A - P.L3832 CfsTer13 mutant pathogenic gene of SQSTM1 for amyotrophic lateral sclerosis and application thereof - Google Patents
P.L3832 CfsTer13 mutant pathogenic gene of SQSTM1 for amyotrophic lateral sclerosis and application thereof Download PDFInfo
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Abstract
The invention provides a new mutant pathogenic gene of amyotrophic lateral sclerosis and a kit thereof, belonging to the field of molecular biology, wherein a nucleic acid sample of the new mutant pathogenic gene and an SQSTM1 gene are shown as SEQ ID NO:1, nucleotide changes compared to the nucleotide sequence shown in seq id no: a deletion mutation of base C at 179260760 of human chromosome 5. The invention discloses that the p.L3832 CfsTer13 locus mutation of the SQSTM1 gene is related to amyotrophic lateral sclerosis for the first time, and further provides a kit for screening amyotrophic lateral sclerosis, which can be used for screening amyotrophic lateral sclerosis caused by the mutation pathogenic gene and provides guidance for timely discovery and timely treatment of patients. Meanwhile, provides new evidence for the SQSTM1 being an ALS pathogenic gene and a pathogenic mechanism thereof, and provides a new target for the accurate treatment of ALS.
Description
Technical Field
The invention relates to the technical field of biomedical detection, in particular to a novel mutant pathogenic gene for amyotrophic lateral sclerosis and application thereof.
Background
Amyotrophic Lateral Sclerosis (ALS) is a progressive, limb-disabled, fatal disabling, neurodegenerative disease, and currently no effective treatment. Pathogenic genes include those commonly foundSOD1，C9ORF72Etc., identification of ALS causative genes and mutations is beneficial to help clarify the pathogenesis of the disease. Based on the complexity of Amyotrophic Lateral Sclerosis (ALS) pathogenesis and the malignancy of clinical prognosis, identification of ALS pathogenic genes is very important for early diagnosis, and if the possibility of pathological changes can be detected by applying medical detection means early, therapeutic intervention can be performed timely, so that the decline of the motor functions of patients is delayed as far as possible, which is an unprecedented wish of current scientific researchers.
Therefore, it is necessary to perfect new mutant pathogenic genes of amyotrophic lateral sclerosis, and more timely find ALS mutation and pathogenic mechanism by biological gene screening means in early stage of clinical discovery, and timely perform therapeutic intervention.
Disclosure of Invention
The invention aims to provide a mutation pathogenic gene of amyotrophic lateral sclerosis and a kit thereof, and provides a mutation pathogenic site p.L382CfsTer13 of SQSTM1 gene of amyotrophic lateral sclerosis, so that more and more powerful detection tools are provided for more timely finding amyotrophic lateral sclerosis by a biological gene screening means in the early stage of clinical discovery, and therapeutic intervention can be timely carried out.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, there is provided a p.L3832 CfsTer13 mutant pathogenic gene of SQSTM1 for amyotrophic lateral sclerosis, a nucleic acid sample of the mutant pathogenic gene is identical to the SQSTM1 gene as set forth in SEQ ID NO:1, the nucleotide changes are: a deletion mutation of the 179260760 th base C of the human chromosome 5, wherein the site mutation is chr5:179260760: 1143del.
The mutant pathogenic gene and the SQSTM1 gene are shown as SEQ ID NO:1, resulting in a stop codon for leucine at position 382, resulting in a frame shift mutation resulting in a change of the last 13 amino acids. Such mutations may cause muscle disorders or neurodegenerative diseases. The nucleotide sequence of the SQSTM1 gene before mutation is shown as SEQ ID NO:1, the amino acid sequence is shown as SEQ ID NO:2 is shown in the figure; the nucleotide sequence of the mutation pathogenic site p.L3832 CfsTer13 of the SQSTM1 gene after mutation is shown as SEQ ID NO:3, the amino acid sequence is shown as SEQ ID NO: 4.
In a second aspect of the invention, there is provided the use of an agent for detecting the p.L3832 CfsTer13 mutant pathogenic gene of SQSTM1 for amyotrophic lateral sclerosis in the manufacture of a diagnostic product for amyotrophic lateral sclerosis.
In a third aspect of the invention, there is provided a kit for screening amyotrophic lateral sclerosis, the kit comprising reagents for detecting the p.L3832 CfsTer13 mutant pathogenic gene of SQSTM1 for said amyotrophic lateral sclerosis.
Further, the kit comprises: primer pairs for amplifying p.L3832 CfsTer13 mutant pathogenic genes of SQSTM1 for amyotrophic lateral sclerosis.
Further, the nucleotide sequence of the primer pair is shown as SEQ ID NO:7-SEQ ID NO: shown at 8.
Further, the kit for use further comprises: dNTPs, taq enzyme, mg 2+ And a PCR reaction buffer.
One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:
1. the p.L3832 CfsTer13 mutant pathogenic gene of SQSTM1 and application thereof provided by the invention detect SQSTM1 mutant patients in ALS queue gene screening, the patients show typical upper and lower motor neuron damage, the p.L3832 CfsTer13 site mutation is found for the first time through gene detection for definite ALS cases, the mutation site is located in the 7 th exon, the species high conservation is achieved, and the close correlation between the site mutation and amyotrophic lateral sclerosis diseases is verified.
2. The invention designs a kit for directly detecting the mutant gene. The kit can directly pass through the first-generation sequencing, reduce complicated procedures, skip high-throughput screening and directly target gene sequencing. The primer design provided by the invention detects the gene mutation by the previous generation of sequencing through PCR amplification, has simple operation, mature technology and extremely low cost, is not influenced by the mutation size (20 bp), and can detect single base variation and large fragment deletion insertion. The kit has wide application range, and the detected DNA can be extracted from fresh tissues, peripheral blood and paraffin section samples.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the sequencing results of the SQSTM1 mutant Sanger.
FIG. 2 shows the quantitative results of SQSTM1 protein induced by SQSTM1 mutant patients. Mutations resulted in reduced expression of the SQSTM1 protein compared to the control.
FIG. 3 shows the results of RT-qPCR of SQSTM1 gene induced pluripotent stem cells from SQSTM1 mutant patients. Mutations resulted in a half reduction in the mRNA expression of SQSTM1 compared to the control.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.
Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, etc., used in the present invention are commercially available or may be obtained by existing methods.
The technical scheme of the embodiment of the application aims to solve the technical problems, and the overall thought is as follows:
we have found for the first time through experiments that 1 patient with SQSTM1 mutation (p.L3832 CfsTer13) was detected in ALS queue gene screening, and the patient exhibited typical damage to the upper and lower motor neurons, the case of ALS being diagnosed. The mutation is located in exon 7, which is highly conserved among different species, and is chr5:179260760: 1143del, and the SQSTM1 gene as shown in SEQ ID NO:1, resulting in a stop codon for leucine at position 382, resulting in a frame shift mutation resulting in a change of the last 13 amino acids. The mutation has not been reported before. There are no frequency reports in databases such as gnomeAD, exAC, etc. that bioinformatics predicts harm, ACMG suggests possible pathogenicity, and predicts that a premature termination of the SQSTM1 protein will be produced.
Thus, a kit for screening amyotrophic lateral sclerosis can be prepared, which comprises reagents for detecting the novel mutated SQSTM1 pathogenic gene of amyotrophic lateral sclerosis.
The present application will be described in detail with reference to examples and experimental data.
EXAMPLE 1 discovery of the p.L382CfsTer13 mutant pathogenic Gene of SQSTM1 for amyotrophic lateral sclerosis
1. Obtaining biological samples
Male patients developed disease at 56 years of age with no family history of degenerative disease. Muscle weakness begins with the two upper limbs and then progresses slowly to the extremities. Neurologic examination: shenqing Liu, MMSE 30/30 min, limb myodynamia, tendon reflex active, bisbalinski+. Electromyography suggests extensive neurogenic lesions. No evidence of other diseases such as inflammation, tumor and the like is found in blood, lumbar puncture cerebrospinal fluid and whole body screening, and ALS is diagnosed. Symptoms progress slowly and life can be self-care after 53 months of onset.
2. DNA extraction
Peripheral Blood mononuclear cells were extracted, and DNA was extracted using DNA isolation Kit (Blood DNA Kit V2, # CW2553).
3. Constructing a library and sequencing
Genomic DNA was randomly fragmented and sheared into fragments of 180-280bp in length using a Covaris disrupter. Libraries were prepared using the Biorupter UCD-200 (Diagenode), KAPA library preparation kit (Kapa Biosystems, # KR0453) and SureSelect XT2 target enrichment System (Agilent). DNA library sequencing was performed using Illumina NovaSeq platform and downstream analysis was performed using Illumina Sequence Control Software (SCS). Variations are retained only when the read depth is greater than or equal to 10. High quality reads were aligned with the USCS human genome (build 37.1 version hg19) using the Burrows-Wheeler alignment tool.
Genomic Analysis Toolkit (GATK) and ANNOVAR (version: 2016-05-1110:54:48-0700) were used for variant calls and comments. All subjects were screened for C9ORF72 repeat amplification using standard repeat primer PCR. Mutation frequencies were initially determined in gnomeAD and Exome Aggregation Consotium (ExAC) to remove common Single Nucleotide Polymorphisms (SNPs). Only Minor Allele Frequencies (MAF) <0.5% or non-synonymous, splice and frame shift variations that are not present in the population database were selected for further evaluation.
As shown in FIG. 1, the deletion of base C at c.1143 was observed in the patient, and the position was heterozygous.
Example 2 SQSTM1 Gene p.L382CfsTer13 site mutation was associated with amyotrophic lateral sclerosis
1. To confirm the frequency of these mutations in the chinese population, we also examined the frequency of these rare mutations in the chinese healthy population. The deleterious effects of variation were evaluated by three online software of SIFT (https:// SIFT. Bii. A-star. Edu. Sg/www/SIFT4G_vcf_subset. Html), polyPhen-2 (http:// genetics. Bwh. Harvard. Edu/pph2 /) and Mutation Taster (http:// www.mutationtaster.org).
Referring to ACMG genetic variation classification standards and guidelines, wherein PM2 and BS1 are rating indexes of variation frequency and use of control population, and PP3 and BP4 are indexes of bioinformatic analysis data. PM2 is an index of variation not found in normal control populations in ESP database, thousand people database, EXAC database. BS1 is an indicator that allele frequency is greater than the incidence of disease. PP3 is a statistical approach to predict the deleterious effects of this variation on genes or gene products, including conservative predictions, evolutionary predictions, splice site effects, etc. BP4 is a statistical measure of the fact that the variation is predicted to have no effect on the gene or gene product, including conservation prediction, evolution prediction, splice site effect, etc.
The evaluation results were:
(1) Frequency of people
PM2: allele frequencies are extremely rare in the control database compared to the prevalence of gene-related diseases. Variations below this threshold may be pathogenic.
BS1: allele frequencies in the control database were higher than expected for the gene-related disease. Variations above this threshold are more likely to be benign.
(2) Detrimental effects PP3: allele frequencies are extremely rare in the control database compared to the prevalence of gene-related diseases, above which
Threshold variation may be pathogenic.
BP4: allele frequencies in the control database were higher than expected for the gene-related disease. Variants below this threshold are more likely to be benign.
2. Potential ALS pathogenic variations were confirmed by Sanger sequencing. The variants are classified (pathogenic, potentially pathogenic, ambiguous, potentially benign and benign) according to the variant interpretation guidelines of the american college of medical genetics and genomics (ACMG). Application of SWISS-MODEL predictions resulted in changes in protein structure.
SQSTM1; chr5:179260735:c.1118dup (p.L3832 CfsTer13) variation was detected and no correlation was reported for this variation. According to ACMG guidelines (appendix), this variation was judged as a suspected pathogenic variation, with pvs1+pm2 evidence as follows:
PVS1 when the pathogenic mechanism of a disease is loss of function (LOF), the detected mutation is a nonsense mutation, a frameshift mutation, a classical.+ -.1 or 2 splice mutation, an initial codon mutation, a single or multiple exon deletion).
PM2 variation not found in normal control population (or very low frequency locus in recessive genetic disease) in ESP database, thousand person database, EXAC database
The site can confirm that the new mutation pathogenic site p.L382CfsTer13 is amyotrophic lateral sclerosis through bioinformatics analysis
Markers for symptoms of metaplasia.
3. RT-qPCR results of SQSTM1 mutant patient induced pluripotent stem cell SQSTM1 gene
The operation method comprises the following steps: RNA of induced pluripotent stem cells of SQSTM1 normal control and mutant patients is extracted, then reverse transcribed into cDNA, and finally RT-qPCR detection is performed by the following primers:
forward direction: 5'-CACTACCGCGATGAGGAC-3' (SEQ ID NO: 5);
reversing: 5'-CATCCTTCACGTAGGACATGG-3' (SEQ ID NO: 6);
the results are shown in FIG. 3, and it is found that the p.L3832 CfsTer13 mutation results in a decrease in the mRNA level of SQSTM 1. May be caused by nonsense-mediated mRNA degradation.
4. Quantitative results of protein
The operation method comprises the following steps: extracting whole cell protein lysate of induced pluripotent stem cells of SQSTM1 normal control and mutant patients, carrying out denaturation on the protein, and then carrying out Western blot detection.
The results are shown in FIG. 2, and it is seen that the p.L3832 CfsTer13 mutation results in partial degradation of the SQSTM1 protein. The size of the wild-type SQSTM1 protein was 62kd, and no truncated protein was produced after mutation, but the normal size protein was degraded.
Example 3 primer design and PCR kit
1. Referring to the human genome sequence database, a PCR specific primer pair corresponding to the p.L382CfsTer13 locus is designed, and is specifically shown as 1.
TABLE 1 primer pairs
| Name of the name | Sequence (5 '-3') |
| Upstream primer | TGACTGGACCCATCTGTCTTC（SEQ ID NO：7） |
| Downstream primer | TTCAGAACTGCATTAGCAGAGC（SEQ ID NO：8） |
2. Next, PCR reaction systems of the respective genomic DNA samples were prepared and PCR reactions were carried out in the following proportions, and the reaction systems are shown in Table 2 below.
TABLE 2 reaction System
Note that: recommended amount of template DNA used in 50ul PCR reaction system: the human genome DNA is 0.1 ug-1 ug.
3. Sanger method sequencing verification
The obtained PCR amplified product was directly subjected to Sanger sequencing verification.
After PCR amplification is finished, taking 5 mu L of amplified products, carrying out 1% agarose gel electrophoresis, carrying out electrophoresis for 30min, dyeing for 20min, then placing gel blocks into a gel imager for observation, and preliminarily judging amplified fragments according to the fragment size condition of a comparison Marker so as to purify the amplified products meeting the requirements: adopts a Mag-BindOligonucleotidePurific a t i o n K i t reagent box and operates according to the requirements of the reagent box.
And (5) loading and sequencing: adopting an ABI BigDye3.1Sequencingkit kit, and operating according to the kit requirement; sequencing was performed using an ABI model 3730 sequencer.
4. Analysis of results
The sequencing result is compared with a standard sequence by means of Chromas sequence analysis software, and mutation sites are searched for to see whether the mutation is p.L3832 CfsTer13 site mutation. Thereby further confirming that the mutation site can be used for auxiliary diagnosis such as detection, diagnosis, prognosis evaluation and the like of amyotrophic lateral sclerosis.
Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (8)
1. A p.l3832 cfsster13 mutant pathogenic gene of SQSTM1 for amyotrophic lateral sclerosis, wherein the nucleotide sequence of the mutant pathogenic gene is as shown in SEQ ID NO:3, and the SQSTM1 gene is shown as SEQ ID NO:1, the nucleotide changes are: a deletion mutation of the 179260760 th base C of the human chromosome 5, wherein the site mutation is chr5:179260760: 1143del.
2. Use of an agent for detecting the p.l3832 cfsster13 mutant pathogenic gene of SQSTM1 for amyotrophic lateral sclerosis according to claim 1 for the preparation of a diagnostic product for amyotrophic lateral sclerosis.
3. A kit for screening amyotrophic lateral sclerosis, comprising a reagent for detecting the p.l3832 cfsster13 mutant pathogenic gene of SQSTM1 for amyotrophic lateral sclerosis according to claim 1.
4. A kit according to claim 3, characterized in that it comprises: primer pairs for amplifying p.L3832 CfsTer13 mutant pathogenic genes of SQSTM1 for amyotrophic lateral sclerosis.
5. The kit of claim 4, wherein the primer pair has a nucleotide sequence set forth in SEQ ID NO:7-SEQ ID NO: shown at 8.
6. A kit according to claim 3, wherein the kit further comprises: dNTPs, taq enzyme, mg 2+ And a PCR reaction buffer.
7. A kit according to claim 3, wherein the kit further comprises: premix Taq.
8. A polypeptide mutated in p.l3832 cfseter 13 of SQSTM1 for amyotrophic lateral sclerosis, wherein the amino acid sequence of said polypeptide is as set forth in SEQ ID NO: 4.
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