CN116445597A

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

Info

Publication number: CN116445597A
Application number: CN202211107182.3A
Authority: CN
Inventors: 何瑾; 陈万金; 王柠
Original assignee: First Affiliated Hospital of Fujian Medical University
Current assignee: First Affiliated Hospital of Fujian Medical University
Priority date: 2022-09-10
Filing date: 2022-09-10
Publication date: 2023-07-18

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 1187 position mutation and its application

Technical Field

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

Background

Fibular muscular dystrophy (CMT) is the most common group of peripheral monogenic genetic diseases with a morbidity of about 1/2500. The disease has high clinical heterogeneity and genetic heterogeneity, and typical clinical characteristics of the disease are mainly represented by onset of teenagers, progressive muscle weakness and atrophy with sensory disturbance at the distal ends of limbs which progress slowly.

CMT usually develops in childhood or adolescence and is characterized clinically by progressive, symmetric limb distal muscle weakness and muscle atrophy, foot, calf muscle and less than 1/3 of the lower thigh muscle weakness and atrophy, forming "crane" or inverted wine bottle-like deformities, frequently presenting varus foot deformity and pestle-like toe, difficulty walking and running, cross-threshold gait; interosseous muscle and large and small thenar muscle atrophy of the hand, which is in claw shape or ape hand deformity, and the muscle atrophy generally does not exceed the elbow joint, and the fine motion of the hand cannot be realized; the limbs are distributed in glove-sock shape, pain sense, temperature sense and vibration sense are reduced in the region, tendon reflex is weakened or disappeared, ankle reflex is generally disappeared, and skeletal deformity such as high arcus foot and scoliosis can be accompanied with autonomic nerve dysfunction and nutritional disorder sign. Although CMT primarily involves peripheral sensory and motor nerves, a small proportion of patients with predominantly axonal involvement are simultaneously presented with symptoms of pyramidal signs, including mild elevated muscle tone, retention/activation/hyperactivity of lower limb tendon reflex, and pathological positive, suggesting the presence of central nervous system involvement. In addition, many other symptoms may occur in CMT, such as non-motor symptoms like cranial nerve involvement, vocal cord paralysis, pupil abnormalities, glaucoma, optic atrophy, hearing loss, impaired intelligence, etc. These specific accompanying symptoms can often provide important clues for molecular diagnostics. The severity of clinical heterogeneity also varies greatly, ranging from a form of mild symptoms to a form that results in severe disability. Most patients develop slow disease progression with mild to moderate functional impairment, but do not affect life expectancy.

The most common CMT subtypes are demyelinating, axonal and intermediate, based on electrophysiological and pathological characteristics, and are mainly characterized by median nerve motor conduction velocity (MNCV) less than 25m/s, and the pathology is typically altered by the presence of onion bulb-like structures in the medullary nerve fibers. The axonal type is characterized by MNCV greater than 45m/s, and the pathology typically changes into a large number of marrow-like fiber density decreases. The intermediate type is characterized by MNCV of 25-45 m/s, and the pathological change has the characteristics of both types. With the increasing refinement of CMT pathogenic genes, there is a current trend to combine electrophysiological, genetic and genetic means for precise typing. The method can facilitate the rapid individual gene screening of patients after medical history and electrophysiological typing.

In recent years, the molecular mechanism of CMT has been studied more and more, its occurrence has a close relationship with specific genetic variation, and more than 100 pathogenic genes have been found. In addition, the genetic forms are also diverse, with autosomal dominant inheritance, autosomal recessive inheritance, X-chromosome linked dominant inheritance and X-chromosome linked recessive inheritance, most commonly seen in most CMT1 and CMT2 family patients with autosomal dominant (autosomal dominant, AD) inheritance. Through research in molecular genetics, it is proved that the pathogenic genes play an important role in maintaining the structural integrity and functional integrity of peripheral nerve axons and myelin, protein synthesis, cell signal transduction, mitochondrial function and the like. Among the pathways that have been identified to play a role in CMT are transcriptional regulation, protein turnover, schwann cell axon interactions, axon transport, and mitochondrial fusion and fission.

SARS1 is one of Aminoacyl tRNA synthetases (ARSs) consisting of three domains, tRNA binding domain, aminoacylation domain and UNE-S, which are responsible for binding serine to its corresponding tRNA and participating in protein synthesis in the body. In the study of the mechanism of CMT, the ARSs family has been found to play an important role therein. To date, at least 6 ARSs heterozygous mutations result in CMT, and most mutations are located in the aminoacylation domain of the ARSs.

At present, mutation of CMT pathogenic gene SARS1 has not been reported, and the application can provide potential important value for analysis, screening, detection and treatment of the disease in combination with functional experiments.

Disclosure of Invention

In view of this, the object of the present invention is to provide the use of a reagent for detecting 1 gene mutant form of a novel pathogenic gene of CMT in the preparation of a CMT diagnostic kit, and to provide a detection method based on Sanger sequencing, and to verify its pathogenicity by functional experiments. The method can provide basis and lay foundation for gene screening of undiagnosed CMT patients, analysis of pathogenesis, drug development and formulation of treatment schemes.

The invention aims at realizing the following technical scheme:

the use of a detection reagent of human CMT pathogenic gene SARS1 with mutation at 1187 position in preparing CMT diagnosis kit, wherein the nucleotide sequence of human CMT pathogenic gene SARS1 with mutation at 1187 position is shown as SEQ ID NO.1, and the 1187 position is located in Exon9, specifically, the C mutation at 11 th 87 position of wild gene is T.

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

As a preferred mode of embodiment, 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.

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

As a preferred mode of embodiment, the uses include the resolution of CMT pathogenesis and drug development thereof.

The SARS1 as detection target is used in preparing CMT diagnosis kit, and the nucleotide sequence of SARS1 is shown in SEQ ID NO.1, and the mutation is 1187 th C mutation into T.

A CMT diagnostic kit comprising reagents capable of detecting the mutation of C to T at position 1187 of SARS1 as shown in SEQ ID No. 1.

As a preferred mode of the embodiment, the reagent comprises a reagent for PCR detection of SARS1 mutation.

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

As a preferred mode of embodiment, the amplification system of PCR 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.

Drawings

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

FIG. 1 is a Sanger sequencing validation local map of a mutant form of the SARS1 gene, c.1187C > T, according to some embodiments.

Fig. 2 is a fibular muscle atrophy image of a CMT patient according to some embodiments.

Fig. 3 is a genetic map of the family of CMT patients according to some embodiments. Wherein, square represents male, and circle represents female; arrows indicate the first witness, filled indicates the CMT afflicted individual, and open indicates the normal individual; I. II and III represent the 1 st, 2 nd and 3 rd generation respectively, and the numbers are member numbers

FIG. 4 is a linkage analysis result for the family, according to some embodiments. Wherein the shared interval carries mutation of SARS1 gene c.1187C > T.

FIG. 5 is an aminoacylation function test result according to some embodiments.

FIG. 6 is a total protein synthesis rate result according to some embodiments.

FIG. 7 is an ISR pathway activation result according to some embodiments.

Detailed Description

The present disclosure is described in further detail below with reference to the drawings and the embodiments. It is to be understood that the specific embodiments described herein are merely illustrative of the relevant content and not limiting of the present disclosure. It should be further noted that, for convenience of description, only a portion relevant to the present disclosure is shown in the drawings.

In addition, embodiments of the present disclosure and features of the embodiments may be combined with each other without conflict. The present disclosure will be described in detail below with reference to the accompanying drawings in conjunction with embodiments.

The present disclosure 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, specifically, C mutation at 1187 site of a wild gene is T.

The SARS1 gene detection method based on the second generation sequencing comprises the following main steps:

(1) Second generation sequencing PCR capture amplification and analysis of gene mutation spectrum

The sequencing chip is used for capturing sequencing, and mutation sites are found out through bioinformatic analysis.

(2) Verification of mutation sites

After the mutation sites obtained by analysis are subjected to primer design, the mutation fragments are amplified and then directly subjected to Sanger sequencing, and then genotype-phenotype co-segregation conditions of the patient family related member samples are analyzed through Sanger sequencing;

amplification system of step (2):

composition of the components	Additive amount (μl)
		KOD one Mix	12.5
Nuclease-free water	10
		Primer F as an upstream Primer	1
Downstream Primer R	1
		Sample genomic DNA at a concentration > 20. Mu.g/. Mu.l	0.5

Amplification stage and conditions of step (2):

(3) Linkage analysis

Single nucleotide polymorphism sites SNPs of a patient family are extracted by utilizing second generation sequencing, and linkage analysis is performed by utilizing bioinformatics software Plink and MERLIN, so that a linkage interval shared by family members is obtained.

(4) Functional verification experiment

Skin biopsies were taken from the patient and skin fibroblasts were cultured and the following experiments were performed:

I. detection of aminoacyl function: the free phosphate was determined colorimetrically by extracting total skin fibroblast proteins from patients and normal controls, and allowing the reaction to proceed at 37 ℃ for 30 minutes in the reaction solution, wherein pyrophosphate produced by the reaction was hydrolyzed by the enzyme pyrophosphatase coupled thereto.

And II, detecting the total protein synthesis rate: the skin fibroblasts were added with 5. Mu.g/ml puromycin, and after culturing in a cell incubator for 30 minutes, the total cell proteins were extracted, SDS-PAGE electrophoresis was performed, and after transfer, incubation was performed with anti-puromycin, followed by detection of gray scale values.

Isr pathway activation detection: the total protein of the skin fibroblasts of the patients and normal controls is extracted, BCA protein is quantified, SDS-PAGE electrophoresis is performed, p-eIF2 alpha antibody is used for detecting protein expression, and Vinculin is used as an internal reference.

The second generation sequencing and biological information analysis by PCR capture amplification described in (1) is performed by a second generation sequencer and matched analysis software according to standard operation analysis or can be completed by commercial high-throughput sequencing companies, and finally the mutation information of SARS1 gene in the detection sample is required to be obtained.

Wherein, (2) amplifying the mutant fragment by using a specific primer and performing Sanger sequencing to verify whether the mutation result detected by the second generation sequencing is consistent; sequencing by Sanger sequencer according to standard operation or can be completed by commercial sequencing company, and the correspondent amplicon sequence can be obtained. The original mutant fragment amplification refers to c.1187C > T of SARS1 gene, and the amplification primer pair is SEQ ID NO.2 and SEQ ID NO.3.

Wherein the patient family-related members described in (2) must include CMT and non-CMT patients, and said genotype-phenotype co-segregation refers to genotype-determining phenotypes whereby both conform to the common corresponding genetic transmission phenomena and laws.

Wherein the quality of the polymorphic site of the polynucleotide extracted by second generation sequencing in (3) should satisfy the square root of the localization quality (MQ) of more than 40, the combined Depth (DP) of more than 10, the Genotype Quality (GQ) of more than 80, and the maximum deletion value (max deletion) of 0.8.

Wherein the reaction solution in (4) I has a volume of 50. Mu.l and comprises 100mM Tris-HCl (pH 7.6), 10mM magnesium chloride, 40mM KCl ₂ 1mM dithiothreitol, 40U/ml yeast tRNA (Invitrogen, AM 7119), 1mM ATP,0.2mM tryptophan, and 5U/ml yeast inorganic pyrophosphatase. The corresponding reagents are available from commercial companies.

Among them, (4) reagents for protein extraction, BCA reagents for protein quantification and corresponding antibodies in II and III are commercially available.

The invention provides a new mutation type of SARS1, which can provide basis and lay foundation for analyzing the pathogenesis of the disease, developing medicine, screening and detecting the pathogenesis gene, preparing therapeutic scheme, etc; in addition, the detection method based on the second generation sequencing provided by the invention covers all exons of the gene, and can be used for efficiently, comprehensively, quickly and accurately acquiring mutation information.

Examples:

(1) Patient information acquisition

And selecting the CMT patient in the hospital to detect the pathogenic genes under the condition that the CMT patient signs an informed consent form according to detailed medical history and family history investigation conditions and standardized physical examination of the nervous system and related auxiliary examination information.

The fibular muscle atrophy image of the patient with CMT in the hospital is shown in fig. 2, and it can be seen that the patient carrying mutation of SARS1 gene c.1187C > T has limb distal muscle atrophy, which is specifically represented by bilateral fibular muscle atrophy and large and small thenar muscle atrophy of both hands; the genetic map of the family of CMT patients is shown in figure 3, and 6 human CMT patients in the third generation family can be seen.

(2) Second generation sequencing linkage analysis and verification

10ml of elbow vein blood (EDTA anticoagulation) of the patient was taken and genomic DNA was extracted with QIAamp DNA Mini Kit (Qiagen, germany). And sending the genome DNA to Shanghai crystal energy biotechnology limited company for sequencing reaction, obtaining the original data, and performing biological information analysis to obtain mutation information. And Sanger verification of the same mutant fragments was performed on the remaining family members, and the sequencing results are shown in FIG. 1, which shows that the patient had a C > T heterozygous mutation at SARS1 gene c.1187. Extracting original data according to a standard flow, further assembling, setting Plink and MERLIN according to related parameters, and analyzing, wherein a linkage analysis result is shown in fig. 4, and the fact that the family has a linkage sharing interval in chromosome 1 is shown, and the specific range is chr1:102271774-112539061; and the LOD value of the linkage interval is 1.8.

(3) Functional verification experiment

According to the aminoacyl function detection, total protein synthesis rate detection and ISR pathway activation detection methods described above, a function verification experiment was performed on the family of CMT patients. As shown in FIG. 5, the results of aminoacyl function test revealed that the aminoacyl function of the skin fibroblasts derived from the patient carrying the mutation of SARS1 gene c.1187C > T was decreased as compared with that of the skin fibroblasts of the normal person. As shown in FIG. 6, the results of the total protein synthesis rate test showed that the total protein synthesis rate of skin fibroblasts derived from patients carrying the SARS1 gene c.1187C > T mutation was decreased as compared with that of skin fibroblasts of normal persons. As shown in FIG. 7, it was found that the ISR pathway was significantly activated in the skin fibroblasts derived from patients carrying the mutation of SARS1 gene c.1187C > T, as compared with the skin fibroblasts of normal persons.

In the description of the present specification, reference to the terms "one embodiment/manner," "some embodiments/manner," "example," "specific example," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment/manner or example is included in at least one embodiment/manner or example of the present application. In this specification, the schematic representations of the above terms are not necessarily for the same embodiment/manner or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments/modes or examples. Furthermore, the various embodiments/modes or examples described in this specification and the features of the various embodiments/modes or examples can be combined and combined by persons skilled in the art without contradiction.

Furthermore, in the description of the present disclosure, the meaning of "a plurality" is at least two, such as two, three, etc., unless specifically defined otherwise. "and/or" is merely an association relationship describing an association object, and represents three relationships, for example, a and/or B, expressed as: a exists alone, A and B exist together, and B exists alone. The specific meaning of the terms in this disclosure will be understood by those of ordinary skill in the art as the case may be.

It will be appreciated by those skilled in the art that the above-described embodiments are merely for clarity of illustration of the disclosure, and are not intended to limit the scope of the disclosure. Other variations or modifications will be apparent to persons skilled in the art from the foregoing disclosure, and such variations or modifications are intended to be within the scope of the present disclosure.

Claims

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.