CN116445597A - Human CMT pathogenic gene SARS1 with 1187 position mutation and its application - Google Patents

Abstract

The invention provides an application of a detection reagent of human CMT pathogenic gene SARS1 with mutation at 1187 site in preparing a CMT diagnostic kit, wherein the nucleotide sequence of the human CMT pathogenic gene SARS1 with mutation at 1187 site is shown as SEQ ID NO.1, and the 1187 site is located in Exon9, in particular the C mutation at 1187 site of a wild gene is T. The invention can provide basis and lay foundation for the gene screening of the undiagnosed CMT patient, the analysis of pathogenesis, the drug development and the formulation of treatment scheme.

Description

Human CMT pathogenic gene SARS1 with mutation at the 1187th position and its application

technical field

The disclosure relates to the technical field of genes, in particular to the human CMT pathogenic gene SARS1 with a mutation at the 1187th site and its application.

Background technique

Charcot-Marie-Tooth (CMT) is a group of the most common monogenic genetic diseases of peripheral nerves, with an incidence rate of about 1/2500. This type of disease has a high degree of clinical heterogeneity and genetic heterogeneity, and its typical clinical features are mainly manifested as onset in adolescence, slowly progressive progressive muscle weakness and atrophy of distal extremities with sensory disturbance.

CMT usually develops in childhood or adolescence, and its clinical features are progressive and symmetrical distal limb muscle weakness and muscle atrophy, as well as muscle weakness and atrophy in the foot, calf muscles, and muscles below the lower third of the thigh, forming "crane legs" Or inverted wine bottle deformity, often with varus and clubbed toes, difficulty walking and running, straddling gait, atrophy of interosseous muscles and large and hypothenar muscles of the hand, showing claw or ape hand Deformity, muscular atrophy generally does not exceed the elbow joint, and fine hand movements cannot be performed; the limbs are distributed in a glove-sock pattern, the pain, temperature, and vibration sensations in the area are decreased, the tendon reflexes are weakened or disappeared, and the ankle reflexes usually disappear. Signs of neurological dysfunction and nutritional disorders, often accompanied by high arched feet, scoliosis and other skeletal deformities. Although CMT primarily involves peripheral sensory and motor nerves, a lesser proportion of patients with predominantly axonal involvement also present with pyramidal tract signs, including mildly elevated muscle tone, preserved/active/hyperflexic tendon reflexes in the lower extremities, and pathological A positive sign indicates involvement of the central nervous system. In addition, many other symptoms may occur in CMT, such as cranial nerve involvement, vocal cord paralysis, pupil abnormalities, glaucoma, optic atrophy, hearing loss, mental decline and other non-motor symptoms. These specific accompanying symptoms often provide important clues for molecular diagnosis. The severity of clinical heterogeneity also varied widely, ranging from mildly symptomatic forms to those resulting in severe disability. Most patients have slow disease progression and mild to moderate functional impairment without affecting life expectancy.

According to the electrophysiological and pathological characteristics, it is mainly divided into demyelinating type, axonal type and intermediate type. Among them, the most common subtype of CMT is demyelinating type, which is mainly characterized by median nerve motor conduction velocity (MNCV) less than 25m/ s, The typical pathological change is an onion-like structure of myelinated nerve fibers. The axonal type is characterized by MNCV greater than 45m/s, and the typical pathological changes are a large number of myelinated fibers with reduced density. The intermediate type is characterized by MNCV of 25-45m/s, and the pathological changes have the characteristics of both types. With the increasing refinement of CMT causative genes, it is now tending to combine electrophysiological, genetic and genetic methods for accurate typing. This method can facilitate rapid individualized genetic screening for patients after medical history and electrophysiological typing.

In recent years, research on the molecular mechanism of CMT has become more and more in-depth. Its occurrence is closely related to specific gene mutations. More than 100 pathogenic genes have been discovered. In addition, there are various forms of inheritance, including autosomal dominant inheritance, autosomal recessive inheritance, X-linked dominant inheritance and X-linked recessive inheritance, among which autosomal dominant (autosomal dominant, AD) inheritance Most common, found in most CMT1 and CMT2 family patients. Molecular genetic studies have confirmed that the disease-causing gene plays an important role in maintaining the structural integrity and functional integrity of peripheral nerve axons and myelin, protein synthesis, cell signal transduction, and mitochondrial function. Among the pathways that have been identified to play a role in CMT are transcriptional regulation, protein turnover, Schwann cell-axonal interactions, axonal transport, and mitochondrial fusion and fission.

SARS1 is a kind of aminoacyl-tRNA synthases (Aminoacyl-tRNA synthases, ARSs), which consists of three functional domains: tRNA binding domain, aminoacylation domain and UNE-S, and its main function is to bind serine to its corresponding tRNA involved in body protein synthesis. In the study of the mechanism of CMT, it was found that the ARSs family played an important role in it. To date, at least 6 heterozygous mutations in ARSs lead to CMT, and most mutations are located in the aminoacylation domain of ARSs.

At present, the mutation of SARS1, the causative gene of CMT, has not been reported yet. This application, combined with functional experiments, can provide potentially important value for the analysis, screening, detection and treatment of the disease.

Contents of the invention

In view of this, the purpose of the present invention is to provide a detection reagent for a gene mutation form of a new CMT pathogenic gene in the preparation of a CMT diagnostic kit, and provide a detection method based on Sanger sequencing, and verify its pathogenicity through functional experiments. Sickness. These can provide evidence and lay the foundation for genetic screening of undiagnosed CMT patients, analysis of pathogenesis, drug development and formulation of treatment plans.

The purpose of the present invention is achieved by the following technical solutions:

The use of the detection reagent of the human CMT pathogenic gene SARS1 mutated at the 1187th position in the preparation of a CMT diagnostic kit, the nucleotide sequence of the human CMT pathogenic gene SARS1 mutated at the 1187th position is as SEQ ID NO. 1, the 1187th position is located in Exon 9, specifically the 1187th C of the wild-type gene is mutated to T.

As a preferred embodiment of the embodiment, the CMT diagnostic kit includes at least a pair of primer sequences, the base sequence of the upstream primer is shown in SEQ ID NO.2, and the base sequence of the downstream primer is shown in SEQ ID NO.3.

As a preferred embodiment of the embodiment, the CMT diagnostic kit includes at least one amplification system, the amplification system includes: KOD one Mix 12.5 μl, Nuclease-free water 10 μl, upstream primer 1 μl, downstream primer 1 μl, concentration > 20 μg/μl 0.5 μl of sample genomic DNA.

As a preferred embodiment of the embodiment, when the amplification system performs amplification, the amplification conditions are: pre-denaturation at 98°C for 1 min; denaturation at 98°C for 5s, annealing and extension at 68°C for 30s, 30 cycles.

As a preferred mode of the embodiment, the use includes the analysis of the pathogenic mechanism of CMT and its drug development.

SARS1 is used as a detection target in the preparation of a CMT diagnostic kit. The nucleotide sequence of the SARS1 is shown in SEQ ID NO.1, and its mutation is that the C mutation at position 1187 is T.

A CMT diagnostic kit, comprising a reagent capable of detecting the 1187th C mutation of SARS1 as shown in SEQ ID NO.1.

As a preferred embodiment of the embodiment, the reagents include reagents for detecting whether SARS1 is mutated by PCR.

As a preferred embodiment of the embodiment, the primers used in the PCR include at least a pair of primer sequences, the base sequence of the upstream primer is shown in SEQ ID NO.2, and the base sequence of the downstream primer is shown in SEQ ID NO.3.

As a preferred embodiment of the embodiment, the PCR amplification system includes: 12.5 μl of KOD one Mix, 10 μl of Nuclease-free water, 1 μl of upstream primer, 1 μl of downstream primer, and 0.5 μl of sample genomic DNA with a concentration > 20 μg/μl.

Description of drawings

The accompanying drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the disclosure, are included to provide a further understanding of the disclosure, and are incorporated in and constitute this specification. part of the manual.

Fig. 1 is a Sanger sequencing verification partial map of the c.1187C>T mutation form of the SARS1 gene according to some embodiments.

FIG. 2 is an image of peroneal muscle atrophy in a CMT patient according to some embodiments.

FIG. 3 is a genetic map of a pedigree of a CMT patient according to some embodiments. Among them, squares represent males, circles represent females; arrows represent probands, solids represent CMT affected individuals, hollows represent normal individuals; I, II, and III represent the 1st, 2nd, and 3rd generations, respectively, and the numbers are member numbers

Fig. 4 is the linkage analysis result of the family according to some embodiments. Among them, the shared interval carries the c.1187C>T mutation of the SARS1 gene.

Figure 5 is the results of an aminoacylation functional assay according to some embodiments.

Figure 6 is a graph of total protein synthesis rate results, according to some embodiments.

Figure 7 is a graph of ISR pathway activation results according to some embodiments.

Detailed ways

The present disclosure will be further described in detail below with reference to the drawings and embodiments. It can be understood that the specific implementation manners described here are only used to explain relevant content, rather than to limit the present disclosure. It should also be noted that, for ease of description, only parts related to the present disclosure are shown in the drawings.

It should be noted that, in the case of no conflict, the implementation modes and the features in the implementation modes in the present disclosure can be combined with each other. The present disclosure will be described in detail below with reference to the drawings and embodiments.

This disclosure provides the use of a detection reagent for the human CMT pathogenic gene SARS1 mutated at the 1187th position in the preparation of a CMT diagnostic kit, the nucleotide sequence of the human CMT pathogenic gene SARS1 mutated at the 1187th position As shown in SEQ ID NO.1, the 1187th position is located in Exon 9, specifically, the C at the 1187th position of the wild-type gene is mutated to T.

As a preferred embodiment of the embodiment, the CMT diagnostic kit includes at least a pair of primer sequences, the base sequence of the upstream primer is shown in SEQ ID NO.2, and the base sequence of the downstream primer is shown in SEQ ID NO.3.

As a preferred embodiment of the embodiment, the CMT diagnostic kit includes at least one amplification system, the amplification system includes: KOD one Mix 12.5 μl, Nuclease-free water 10 μl, upstream primer 1 μl, downstream primer 1 μl, concentration > 20 μg/μl 0.5 μl of sample genomic DNA.

As a preferred embodiment of the embodiment, when the amplification system performs amplification, the amplification conditions are: pre-denaturation at 98°C for 1 min; denaturation at 98°C for 5s, annealing and extension at 68°C for 30s, 30 cycles.

As a preferred mode of the embodiment, the use includes the analysis of the pathogenic mechanism of CMT and its drug development.

SARS1 is used as a detection target in the preparation of a CMT diagnostic kit. The nucleotide sequence of the SARS1 is shown in SEQ ID NO.1, and its mutation is that the C mutation at position 1187 is T.

A CMT diagnostic kit, comprising a reagent capable of detecting the 1187th C mutation of SARS1 as shown in SEQ ID NO.1.

As a preferred embodiment of the embodiment, the reagents include reagents for detecting whether SARS1 is mutated by PCR.

As a preferred embodiment of the embodiment, the primers used in the PCR include at least a pair of primer sequences, the base sequence of the upstream primer is shown in SEQ ID NO.2, and the base sequence of the downstream primer is shown in SEQ ID NO.3.

As a preferred embodiment of the embodiment, the PCR amplification system includes: 12.5 μl of KOD one Mix, 10 μl of Nuclease-free water, 1 μl of upstream primers, 1 μl of downstream primers, and 0.5 μl of sample genomic DNA with a concentration > 20 μg/μl.

The SARS1 gene detection method based on next-generation sequencing provided by the present invention includes the following main steps:

(1) Next-generation sequencing PCR capture amplification, analysis of gene mutation spectrum

Use a sequencing chip for capture sequencing, and find out the mutation site through bioinformatics analysis.

(2) Verification of mutation sites

After designing primers for the mutation sites obtained from the analysis, amplify the mutant fragments and perform Sanger sequencing directly, and then analyze the genotype-phenotype co-segregation of the samples of related members of the patient family through Sanger sequencing;

The amplification system of step (2):

Element Amount added (μl) KOD one Mix 12.5 Nuclease-free water 10 Primer F 1 Downstream primer Primer R 1 Sample genomic DNA with a concentration > 20 μg/μl 0.5

Amplification stage and conditions of step (2):

(3) Linkage analysis

Next-generation sequencing was used to extract the SNPs of the patient's family, and the bioinformatics software Plink and MERLIN were used for linkage analysis to obtain the linkage interval shared by the members of the family.

(4) Function verification experiment

A skin biopsy was taken from the patient, and skin fibroblasts were cultured, and the following experiments were performed:

I. Aminoacyl function test: by extracting the total protein of skin fibroblasts from patients and normal controls, and continuing in the reaction solution for 30 minutes at a temperature of 37 ° C, the pyrophosphate produced by the reaction is hydrolyzed by the coupling enzyme pyrophosphatase, and then used Colorimetric determination of free phosphate.

II. Detection of total protein synthesis rate: add 5 μg/ml puromycin to skin fibroblasts, culture in the cell incubator for 30 minutes, extract the total protein of the cells, perform SDS-PAGE electrophoresis, and use anti- puromycin for incubation, followed by detection of gray value.

III. Detection of ISR pathway activation: extract the total protein of skin fibroblasts from patients and normal controls, quantify the BCA protein, perform SDS-PAGE electrophoresis, detect protein expression with p-eIF2α antibody, and use Vinculin as an internal reference.

Among them, the next-generation sequencing and biological information analysis by PCR capture amplification described in (1) can be performed according to the standard operation analysis if there is a second-generation sequencer and supporting analysis software, or it can be completed by a commercial high-throughput sequencing company In the end, it is necessary to obtain the information about the SARS1 gene mutation of the test sample.

Among them, the description in (2) refers to the use of specific primers to amplify the mutant fragment and perform Sanger sequencing to verify whether it is consistent with the mutation result detected by the second-generation sequencing; the Sanger sequencer performs sequencing according to the standard operation or can be sent for commercial sequencing The company completes, and finally needs to obtain the corresponding amplicon sequence. The amplification of the mutant fragment of the original sample refers to c.1187C>T of the SARS1 gene, and the amplification primer pair is SEQ ID NO.2 and SEQ ID NO.3.

Among them, the related members of the patient family mentioned in (2) must include CMT and non-CMT patients, and the genotype-phenotype co-segregation means that the genotype determines the phenotype, so the two meet the common corresponding genetic transmission phenomenon and regularity.

Among them, the quality of the polynucleotide polymorphic sites extracted by next-generation sequencing in (3) needs to satisfy the square root of the mapping quality (MQ) should be greater than 40, the combined depth (DP) should be greater than 10, and the gene The genotype quality (GQ) should be greater than 80, and the maximum missing value (max missing) should be 0.8.

Wherein, the volume of the reaction solution in (4) I is 50 μ l, including 100 mM Tris-HCl (PH7.6), 10 mM magnesium chloride, 40 mM KCl ₂ , 1 mM dithiothreitol, 40 U/ml yeast tRNA (Invitgen, AM7119), 1 mM ATP, 0.2 mM tryptophan, and 5 U/ml yeast inorganic pyrophosphatase. Corresponding reagents are available from commercial companies.

Among them, the reagents for protein extraction in (4) II and III, the BCA reagent for protein quantification and the corresponding antibodies can be purchased by commercial companies.

A new mutation type of SARS1, a new pathogenic gene of CMT provided by the present invention, can provide a basis and lay a foundation for the analysis of the pathogenic mechanism of the disease, the development of drugs, the screening and detection of pathogenic genes, and the formulation of treatment plans; In addition, the detection method based on next-generation sequencing provided by the present invention covers all exons of the gene, and can obtain mutation information efficiently, comprehensively, quickly and accurately.

Example:

(1) Patient information collection

According to the detailed medical history and family history investigation, standardized nervous system physical examination and related auxiliary examination information, the CMT patients in the hospital were selected to undergo disease-causing gene detection under the condition that they signed the informed consent.

Among them, the image of peroneal muscle atrophy of patients with CMT in the hospital is shown in Figure 2. It can be seen that patients carrying the c.1187C>T mutation of the SARS1 gene have distal limb muscle atrophy, specifically manifested as bilateral peroneal muscle atrophy and large and small thenars in both hands Muscular atrophy; the genetic map of the CMT patient's family is shown in Figure 3. It can be seen that a total of 6 members of the family are CMT patients in three generations.

(2) Next-generation sequencing linkage analysis and verification

Take 10ml of cubital venous blood (anticoagulated with EDTA) from the patient, and use QIAamp DNA Mini Kit (Qiagen, Germany) to extract genomic DNA. Send the genomic DNA to Shanghai Jingneng Biotechnology Co., Ltd. for sequencing reaction, obtain raw data and conduct biological information analysis to obtain mutation information. Sanger verification of the same mutation fragments was performed on the rest of the family members. The sequencing results are shown in Figure 1. It can be seen that the patient has a C>T heterozygous mutation at c.1187 of the SARS1 gene. The original data were extracted and further assembled according to the standard process, and Plink and MERLIN were set and analyzed according to the relevant parameters. The linkage analysis results are shown in Figure 4. It can be seen that the family has a linkage sharing interval on chromosome 1, and the specific range is chr1: 102271774-112539061 ; and the LOD value of the linkage interval is 1.8.

(3) Function verification experiment

According to the aforementioned methods of aminoacyl function detection, total protein synthesis rate detection, and ISR pathway activation detection methods, functional verification experiments were carried out in this family of CMT patients. The aminoacyl function test results are shown in Figure 5. Compared with normal human skin fibroblasts, it can be seen that the aminoacyl function of skin fibroblasts derived from patients carrying the c.1187C>T mutation of the SARS1 gene is reduced. The detection results of the total protein synthesis rate are shown in Figure 6. Compared with normal human skin fibroblasts, it can be seen that the total protein synthesis rate of skin fibroblasts derived from patients carrying the SARS1 gene c.1187C>T mutation is reduced. The results of the ISR pathway activation test are shown in Figure 7. Compared with normal human skin fibroblasts, the ISR pathway of skin fibroblasts derived from patients carrying the SARS1 gene c.1187C>T mutation was significantly activated.

In the description of this specification, descriptions referring to the terms "one embodiment/mode", "some embodiments/modes", "examples", "specific examples", or "some examples" mean that the embodiments/modes are combined The specific features, structures, materials or characteristics described in or examples are included in at least one embodiment/mode or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment/mode or example. Moreover, the described specific features, structures, materials or characteristics may be combined in any one or more embodiments/modes or examples in an appropriate manner. In addition, those skilled in the art may combine and combine different embodiments/modes or examples and features of different embodiments/modes or examples described in this specification without conflicting with each other.

In addition, in the description of the present disclosure, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined. "And/or" only describes the association relationship of associated objects, indicating three kinds of relationships, for example, A and/or B, expressed as: A exists alone, A and B exist simultaneously, and B exists independently. Those of ordinary skill in the art can understand the specific meanings of the above terms in the present disclosure according to specific situations.

It should be understood by those skilled in the art that the above-mentioned embodiments are only for clearly illustrating the present disclosure, rather than limiting the scope of the present disclosure. For those skilled in the art, other changes or modifications can be made on the basis of the above disclosure, and these changes or modifications are still within the scope of the present disclosure.

Claims (10)

1. The use of the detection reagent of the human CMT pathogenic gene SARS1 with the 1187 locus mutation in the preparation of the CMT diagnostic kit is characterized in that the nucleotide sequence of the human CMT pathogenic gene SARS1 with the 1187 locus mutation is shown as SEQ ID NO.1, and the 1187 locus is positioned in Exon9, in particular the C mutation of the 1187 locus of the wild type gene is T.

2. The use according to claim 1, wherein the CMT diagnostic kit comprises at least one pair of primer sequences, the upstream primer base sequence being shown in SEQ ID No.2 and the downstream primer base sequence being shown in SEQ ID No.3.

3. The use of claim 1, wherein the CMT diagnostic kit comprises at least one amplification system comprising: KOD one Mix 12.5. Mu.l, nucleic-free water 10. Mu.l, upstream primer 1. Mu.l, downstream primer 1. Mu.l, sample genomic DNA at a concentration > 20. Mu.g/. Mu.l 0.5. Mu.l.

4. The use of claim 1, wherein the amplification system, when amplified, provides the following amplification conditions: pre-denaturation at 98℃for 1min; denaturation at 98℃for 5s, annealing extension at 68℃for 30s,30 cycles.

5. The use according to claim 1, wherein the use comprises the resolution of CMT pathogenesis and drug development thereof.

The use of sars1 as a detection target in the preparation of a CMT diagnostic kit characterized in that: the nucleotide sequence of SARS1 is shown in SEQ ID NO.1, and its mutation is 1187 th C mutation to T.

7. A CMT diagnostic kit, characterized in that: comprising an agent capable of detecting the mutation of C at 1187 of SARS1 as indicated by SEQ ID NO.1 to T.

8. The diagnostic kit of claim 7, wherein: the reagent comprises a reagent for detecting whether SARS1 is mutated or not by PCR.

9. The diagnostic kit of claim 8, wherein: the primer used for PCR at least comprises a pair of primer sequences, wherein the base sequence of an upstream primer is shown as SEQ ID NO.2, and the base sequence of a downstream primer is shown as SEQ ID NO.3.

10. The diagnostic kit of claim 9, wherein: the PCR amplification system comprises: KOD one Mix 12.5. Mu.l, nucleic-free water 10. Mu.l, upstream primer 1. Mu.l, downstream primer 1. Mu.l, sample genomic DNA at a concentration > 20. Mu.g/. Mu.l 0.5. Mu.l.