CN116287214A

CN116287214A - Pathogenic gene for Dysferlin myopathy, detection and application

Info

Publication number: CN116287214A
Application number: CN202310313351.7A
Authority: CN
Inventors: 曾桥; 范园; 罗娇娇; 刘亚宁
Original assignee: Hunan Jiahui Biotechnology Co Ltd
Current assignee: Hunan Jiahui Biotechnology Co Ltd
Priority date: 2023-03-27
Filing date: 2023-03-27
Publication date: 2023-06-23

Abstract

The invention provides a pathogenic gene for causing Dysferlin myopathy, detection and application thereof; the pathogenic gene is deleted at bases 1535 to 1538 of exon17 of the wild-type DYSF gene and/or base C is inserted at bases 5527 to 5528 of exon49 of the wild-type DYSF gene, compared with the wild-type DYSF gene. The pathogenic gene mutation can be used as a biomarker for diagnosing Dysferlin myopathy. The invention can be used for screening or diagnosing the genetic diagnosis of Dysferlin myopathy by detecting whether a subject carries the mutation or not so as to guide treatment. The detection kit provided by the invention can be used for rapidly and effectively predicting or diagnosing Dysferlin myopathy.

Description

Pathogenic gene for Dysferlin myopathy, detection and application

Technical Field

The invention relates to the field of detection reagents, in particular to a pathogenic gene for causing Dysferlin myopathy, detection and application.

Background

Dysferlin myopathy (Dysferlingphy) is a group of autosomal recessive inherited skeletal muscle diseases characterized by progressive muscle weakness, atrophy, caused by a complete or partial loss of the Dysferlin protein by mutation of the DYSSF gene (MIM 603009). The clinical phenotypes are complex and diverse, and are classified into limb-girdle muscular dystrophy type 2B (limb girdle muscular dystrophy 2B,LGMD2B,MIM 253601) which is mainly caused by weakness of the proximal end of the lower limb, miyoshi distal end muscular dystrophy (Miyoshi myopathy dystrophy 1,MMD1,MIM 254130), distal anterior group myopathy (distal anterior compartment myopathy, DACM, MIM 606768) which is caused by weakness of the anterior portion of the lower leg, proximal and distal end simultaneously involved proximal and distal end myopathies, congenital myopathies, lumbar muscle weakness and asymptomatic hypercreatine kinase. LGMD2B, MMD and DACM are common, patients have more diseases before and after puberty, no obvious sex difference, and diseases are caused by limb proximal muscle, gastrocnemius muscle or tibialis anterior muscle weakness, progressive exacerbation is slow, and the patients can reach the whole body and lose walking ability in late stage of disease.

The DYSferlin gene (MIM 603009) is located on chromosome 2p13.2, comprises 56 exons and 55 introns, is 233.2kb in length, encodes Dysferlin (DYSF) protein of 2119 amino acid sequences, belongs to the ferlin family member, has a relative molecular mass of 230 000 and consists of 6 or 7 Ca2+ dependent lipid binding domains C2, 1 transmembrane domains T and ferl, ferA, ferB, dysf _N and Dysf_C domains. Dysferlin protein is widely expressed in various tissues, is mainly accumulated in the inner face of skeletal muscle and myocardial cell membrane or in cytoplasmic vesicles, and participates in repair of muscle cell membrane injury by regulating fusion process of vesicles and membranes. When normal skeletal muscle fiber membranes are broken by mechanical stress, calcium ion inner flow aggregated outside the envelope forms a short-term high-calcium area near the damaged part, and vesicles rich in Dysferlin protein are induced to migrate to the damaged part under the synergistic effect of annexin A1/A2 (annexin A1), so that fusion between vesicles and plasma membranes is mediated, and a patch membrane is formed to repair damaged cell membranes. DYSSF gene mutation causes deletion or dysfunction of Dysferlin proteins with different degrees, causes dysfunction of membrane repair process, continuous damage of muscle cell membranes, causes degeneration or necrosis of muscle cells, and thus clinical manifestations of progressive muscle weakness and muscle atrophy.

Thus, gene mutation is an important genetic basis for the development of diseases, and gene diagnosis is an important genetic criterion for the diagnosis of Dysferlin myopathy. The invention discovers a novel compound heterozygous mutation of DYSferlin for the first time, can cause Dysferin myopathy, develops a corresponding diagnosis kit according to the novel compound heterozygous mutation, and can assist screening and diagnosing Dysferin myopathy gene mutation, and provides a novel technical support for drug screening, drug effect evaluation and targeted therapy.

Disclosure of Invention

The invention mainly aims to provide pathogenic genes, detection and application for Dysferlin myopathy, so as to solve the technical problems of screening and diagnosis of Dysferlin myopathy.

To achieve the above object, the present invention provides a pathogenic gene causing Dysferlin myopathy, which is mutated at a site corresponding to bases 1535 to 1538 of exon17 of a wild-type DYSF gene as compared with a wild-type DYSF gene;

and/or, the pathogenic gene is mutated at a site corresponding to bases 5527 to 5528 of exon49 of the wild-type DYSF gene as compared to the wild-type DYSF gene.

The invention provides a detection reagent for Dysferlin myopathy caused by pathogenic genes, which comprises a specific amplification primer designed for a site where the pathogenic genes are mutated.

Further, the specific amplification primer comprises DYSF-1F, DYSF-1R, DYSF-2F, DYSF-2R, the nucleotide sequence of DYSF-1F is shown as SEQ ID NO.1, the nucleotide sequence of DYSF-1R is shown as SEQ ID NO.2, the nucleotide sequence of DYSF-2F is shown as SEQ ID NO.3, and the nucleotide sequence of DYSF-2R is shown as SEQ ID NO. 4.

The invention provides a detection kit for Dysferlin myopathy, which comprises the detection reagent.

Further, reagents for PCR amplification reactions, and/or reagents and sequencing primers required for DNA sequencing are included.

Further, the sequencing primer comprises DYSF-Seq1F, DYSF-Seq1R, DYSF-Seq2F and DYSF-Seq2R, wherein the nucleotide sequence of DYSF-Seq1F is shown as SEQ ID NO.5, the nucleotide sequence of DYSF-Seq1R is shown as SEQ ID NO.6, the nucleotide sequence of DYSF-Seq2F is shown as SEQ ID NO.7, and the nucleotide sequence of DYSF-Seq2R is shown as SEQ ID NO. 8.

The invention also provides an application of the detection reagent or the detection kit in a detection method of Dysferlin myopathy.

Further, the test sample of the test reagent includes blood.

The application proposes a pathogenic gene causing Dysferlin myopathy, which can effectively distinguish Dysferlin myopathy patients from normal human groups, so that the pathogenic gene mutation can be used as a biomarker for diagnosing Dysferlin myopathy. The invention can be used for genetic diagnosis, screening and prenatal and postnatal guidance of Dysferlin myopathy by detecting whether a subject carries the mutation. The detection kit provided by the invention can be used for rapidly and effectively predicting or diagnosing Dysferlin myopathy. The invention lays a new foundation and a new path for researching pathogenesis of Dysferlin myopathy and provides a brand new theoretical basis for treating Dysferlin myopathy patients. The invention can provide a possible drug target for treating Dysferlin myopathy.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to the structures shown in these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows a family genetic map of Dysferlin myopathy No. 1; wherein,,

representing a male carrier, is->

Representing female carriers, +. representing female patients, ↗ representing first-evidence.

FIG. 2 shows a graph of the results of detection of DYSF: NM-001130987.2: exo17: c.1535-1538 delTGAG: p.S 513Cfs.11 locus genotype using Sanger sequencing, wherein ancestor No.1, ancestor father is "c.1535-1538 delTGAG heterozygous mutation", ancestor mother is wild type (the position of the mutation indicated by the arrow in the sequencing diagram).

FIG. 3 shows a graph of the results of detection of DYSF: NM-001130987.2:exon49:c.5527_5528insC:p.R1845Tf s*22 locus genotype by Sanger sequencing, wherein the ancestor No.1 family, the mother of the ancestor, is "c.5527-5528 insC heterozygous mutation", and the father of the ancestor is wild type (the position of the mutation is indicated by the arrow in the sequencing).

FIG. 4 shows a genetic map of Dysferlin myopathy No.2 family; wherein,,

representing a male carrier, is->

Representing female carriers, ■ male patients, +.female patients, ↗ forerunner.

FIG. 5 shows a graph of the results of the detection of genotype at position 11 of line 2 DYSF: NM-001130987.2: exo17: c.1535-1538 delTGAG: p.S513Cfs using a kit, wherein line 2 ancestor, brother, mother is "c.1535-1538 delTGAG heterozygous mutation", the ancestor father is wild type (the position of the mutation occurrence indicated by the arrow in the sequencing diagram).

FIG. 6 shows a graph of the results of the detection of genotype at position 22 of line 2 DYSF: NM-001130987.2: exo49: c.5527-5528 insC: p.R185Tfs using a kit, wherein line 2 ancestor, brother, father is "c.5527-5528 insC heterozygous mutation", the mother of the ancestor is wild type (the position of the mutation indicated by the arrow in the sequencing diagram).

The achievement of the object, functional features and advantages of the present invention will be further described with reference to the drawings in connection with the embodiments.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are intended to fall within the scope of the present invention.

Moreover, the technical solutions of the embodiments of the present invention may be combined with each other, but it is necessary to be based on the fact that those skilled in the art can implement the embodiments, and when the technical solutions are contradictory or cannot be implemented, it should be considered that the combination of the technical solutions does not exist, and is not within the scope of protection claimed by the present invention.

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, the terms related to molecular genetics, nucleic acid chemistry and molecular biology and laboratory procedures used herein are all widely used terms and conventional procedures in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

The term "diagnosis" herein includes prediction of disease risk, diagnosis of the onset or absence of a disease, and also the assessment of disease prognosis.

The term "mutation" as used herein refers to the alteration of a wild-type polynucleotide sequence into a variant, which may be naturally occurring or non-naturally occurring.

In the present invention, the term "heterozygous mutation" means that the mutation exists in only one gene of a pair of alleles.

In the present invention, the term "complex heterozygous mutation" means a heterozygous mutation in which 1 or more parts of alleles occur, that is, a double allelic mutation, each chromosome being mutated.

The term "prenatal diagnosis" herein refers to definitive diagnosis of a high-risk fetus based on genetic counseling, mainly through genetic detection and imaging examination, and achieves the purpose of fetal selection through selective abortion of a diseased fetus, thereby reducing birth defect rate and improving prenatal quality and population quality.

In the present invention, a "primer" refers to a polynucleotide fragment, typically an oligonucleotide, containing at least 5 bases, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more bases, for amplifying a target nucleic acid in a PCR reaction. The primer need not be completely complementary to the target gene to be amplified or its complementary strand, as long as it can specifically amplify the target gene. The term "specifically amplify" as used herein refers to the ability of a primer to amplify a gene of interest by a PCR reaction without amplifying other genes. For example, specifically amplifying the DYSF gene means that the primer amplifies only the DYSF gene and not the other genes in the PCR reaction.

The research thought of the invention is as follows: the pathogenic gene mutation highly related to Dysferlin myopathy is first screened by using exon sequencing, and then verified by Sanger sequencing to finally obtain the pathogenic gene mutation of Dysferlin myopathy, DYSF: NM_001130987.2: exo17: c.1535_1538delTGAG: p.S513Cfs 11 and e xon49: c.5527_5528insC: p.R1845Tfs 22.

The selected pathogenic gene composite heterozygous mutation can distinguish Dysferlin myopathy patients, carriers and normal human groups, so that the pathogenic gene composite heterozygous mutation can be used as a biomarker for diagnosing Dysferlin myopathy.

Specifically, the invention provides a Dysferlin myopathy related gene composite heterozygous mutation, DYSSF: NM_001130987.2: exo17: c.1535_1538delTGAG: p.S 513Cfs.11 and exon49: c.5527_5528insC: p.R1845 Tfs.22.

The c.1535_1538delTGAG mutation refers to the deletion of the 1535 th to 1538 th bases of the 17 th exon of the wild DYSF gene to form DYSF gene mutant, the DYSF gene mutantThe nucleotide sequence of (a) is preferably as shown in SEQ ID NO.11 (tacc ≡ ≡tatgt) (the box is a 4-base deletion mutation position). Compared with the protein encoded by the wild-type DYSF gene, the DYSF mutant protein provided by the invention has the advantages that the 513 th amino acid is mutated from serine (S) to cysteine (C), and the frame shift mutation occurs, namely the DYSF mutant protein contains the mutation of p.S513Cfs 11, and the mutation is caused by missense mutation of c.1535_1538 delTGAG; the amino acid sequence of the DYSF mutant protein is shown as SEQ ID NO.12

The (bolded underlined letters are amino acid sequences after frame shift mutation, x is stop code).

The c.5527-5528 insC mutation according to the present invention means that the 5527 th to 5528 th nucleotides of the 49 th exon of the wild-type DYSF gene are inserted into base C to form a DYSF gene mutant, and the nucleotide sequence of the DYSF gene mutant is preferably as shown in SE Q ID No.13 (atcaccccca) (the underlined letters are the inserted mutant bases). Compared with the protein encoded by the wild-type DYSF gene, the DYSF mutant protein provided by the invention has the advantages that the 1845 th site is mutated from arginine (R) to threonine (T) and the frame shift mutation occurs, namely, the DYSF mutant protein contains the mutation of p.R185Tfs 22, and the mutation is caused by the frame shift mutation of c.5527_5528insC; the amino acid sequence of the DYSF mutant protein is shown as SEQ ID NO.14

Based on the above discussion, the present invention screens out and provides a pathogenic gene causing Dysferlin myopathy, which causes heterozygous mutation at the site corresponding to bases 1535 to 1538 of exon17 of the wild-type DYSSF gene compared with the wild-type DYSSF gene;

and/or, the pathogenic gene is heterozygous mutated at the site corresponding to bases 5527 to 5528 of exon49 of the wild-type DYSF gene as compared to the wild-type DYSF gene.

The invention also provides a detection reagent for Dysferlin myopathy induced by any of the pathogenic genes, which comprises a specific amplification primer designed for a site where the pathogenic genes are mutated. The detection reagent may be a gene mutation site for detecting the therapeutic gene.

The specific amplification primer comprises DYSF-1F, DYSF-1R, DYSF-2F, DYSF-2R, the nucleotide sequence of DYSF-1F is shown as SEQ ID NO.1, the nucleotide sequence of DYSF-1R is shown as SEQ ID NO.2, the nucleotide sequence of DYSF-2F is shown as SEQ ID NO.3, and the nucleotide sequence of DYSF-2R is shown as SEQ ID NO. 4.

Preferably, the reagent comprises a specific amplification primer of the mutation site of the gene, and the base sequence of a PCR primer of the reagent against the c.1535_1538d elTGAG site is as follows:

DYSF-1F:5’-ATGGGATGCTGGTGGTTC-3’(SEQ ID NO.1)

DYSF-1R:5’-GAAAGGTCTCGGAGTGCTA-3’(SEQ ID NO.2)

the base sequence of PCR primer aiming at the c.5527_5528insC site is as follows:

DYSF-2F:5’-CTTTGACACGCCACGACT-3’(SEQ ID NO.3)

DYSF-2R:5’-GCTCCCTACCCTTTCACATA-3’(SEQ ID NO.4)

the invention also provides a detection kit for Dysferlin myopathy, which comprises any detection reagent. In addition to specific amplification primers, the detection kit may also include conventional reagents for PCR amplification reactions, and/or reagents and sequencing primers required for DNA sequencing. The detection kit diagnoses whether an individual suffers from Dysferlin myopathy by detecting the genotype of a mutation site in a sample.

The sequencing primer comprises DYSF-Seq1F, DYSF-Seq1R, DYSF-Seq2F and DYSF-Seq2R, wherein the nucleotide sequence of the DYSF-Seq1F is shown as SEQ ID NO.5, the nucleotide sequence of the DYSF-Seq1R is shown as SEQ ID NO.6, the nucleotide sequence of the DYSF-Seq2F is shown as SEQ ID NO.7, and the nucleotide sequence of the DYSF-Seq2R is shown as SEQ ID NO. 8.

Specifically, the sequencing primer base sequence for the c.1535_1538delTGAG site is:

DYSF-Seq1F:5’-CCAAAGTGAAGACCAGAAGC-3’(SEQ ID NO.5)

DYSF-Seq1R:5’-TCCTCTTTGTGCCCTTC-3’(SEQ ID NO.6)

the sequencing primer base sequence for the c.5527_5528insC site is as follows:

DYSF-Seq2F:5’-ACCCTGTAGTCCCCTGTT-3’(SEQ ID NO.7)

DYSF-Seq2R:5’-GCCCAAACGAGAACTGTAT-3’(SEQ ID NO.8)

other conventional reagents in the PCR amplification reaction include, but are not limited to dNTPs, PCR buffers, magnesium ions, tap polymerase, and the like. The PCR buffer is 10 XPCR buffer: 500mmol/L KCl,100mmol/L Tris-Cl (pH 8.3), 15mmol/L MgCl ₂ 。

The detection reagent and the detection kit belong to the application of the disease curing gene and the gene mutation site thereof, and the invention can design a specific amplification primer or a specific detection probe according to the upstream and downstream sequences of the gene mutation site.

The invention also provides the application of the detection reagent or the detection kit in the detection method of Dysferlin myopathy. The detection reagent or the detection kit is used for diagnosing whether the male individual suffers from Dysferlin myopathy by detecting the genotype of the gene mutation site in the sample.

In a specific application, the test sample of the test reagent may be blood.

In a specific application process, the invention also provides a Dysferlin myopathy diagnostic kit, which comprises a reagent for detecting the mutation site of the gene.

In a specific application process, the method for detecting whether the DYSF gene has the gene mutation comprises the following steps:

1) Extracting sample genome DNA;

2) Amplifying DYSF gene sequence;

3) Sequencing DNA;

4) Comparing the DNA sequencing result of the sample to be detected with the genome DNA sequence of a normal person:

when the genotype of the c.1535_1538delTGAG site is wild type (i.e., no c.1535_1538delTGAG mutation has occurred), the genotype of the c.5527_5528insC site is wild type (i.e., no c.5527_5528insC mutation has occurred), the individual providing the test sample is a normal individual;

when the genotype of the c.1535_1538delTGAG site is a c.1535_1538delTGAG heterozygous mutation (one gene has a c.1535_1538delTGAG mutation, its allele has no c.1535_1538delTGAG mutation), the genotype of the c.5527_5528insC site is a c.5527_5528insC heterozygous mutation (one gene has a c.5527_5528insC mutation, its allele has no c.5527_5528insC mutation), and the two mutated sites are on both chromosomes,

or the genotype of the c.1535_1538delTGAG site is a c.1535_1538delTGAG homozygous mutation, the genotype of the c.5527_5528i nsC site is a c.5527_5528insC heterozygous mutation,

or the genotype of the c.1535_1538delTGAG site is a homozygous mutation of the c.1535_1538delTGAG, the genotype of the c.5527_5528i nsC site is a wild type,

or the genotype of the c.1535_1538delTGAG site is wild type, the genotype of the c.5527_5528insC site is a homozygous mutation of c.5527_5528insC,

or the genotype of the c.1535_1538delTGAG site is a c.1535_1538delTGAG heterozygous mutation, the genotype of the c.5527_5528i nsC site is a c.5527_5528insC homozygous mutation,

or when the genotype of the c.1535-1538 delTGAG site is a c.1535-1538 delTGAG homozygous mutation and the genotype of the c.5527-5528 i nsC site is a c.5527-5528 insC homozygous mutation, the individual providing the sample to be tested is a Dysferlin myopathy patient;

when the genotype of the c.1535_1538delTGAG site is the c.1535_1538delTGAG heterozygous mutation, the genotype of the c.5527_5528i nsC site is the c.5527_5528insC heterozygous mutation, and the two mutated sites are on the same chromosome,

or when the genotype of the c.1535_1538delTGAG site is wild type and the genotype of the c.5527_5528insC site is a c.5527_5528insC heterozygous mutation,

or the genotype of the c.1535_1538delTGAG locus is a c.1535_1538delTGAG heterozygous mutation, and the genotype of the c.5527_5528i nsC locus is a wild type, the individual providing the sample to be tested is a Dysferlin myopathy carrier.

The invention has the advantages that: the invention discovers for the first time that DYSferlin myopathy can be caused by the complex heterozygous mutation of DYSSF: NM_001130987.2: exo17: c.1535_1538delTGAG: p.S 513Cfs.11 and exon49: c.5527_5528insC: p.R1845 Tfs.22 sites through an exome sequencing technology. In one aspect, the genetic diagnosis for screening or diagnosing Dysferlin myopathy is used to guide the treatment by detecting whether the subject carries the mutation described above. In particular, the diagnostic kit provided by the invention can be used for rapidly and effectively predicting or diagnosing Dysferlin myopathy. On the other hand, the invention lays an important foundation for researching pathogenesis of Dysferlin myopathy and provides a brand new theoretical basis for treating Dysferlin myopathy patients. In a third aspect, the invention may provide a potential drug target for the treatment of Dysferlin myopathy.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally followed by conventional conditions such as those described in Sambrook et al, molecular cloning, A laboratory Manual (Molecular Cloning A LABORATORY MANUAL 1SECOND EDITION;New York:Cold Spring Harbor LaboratoryPress,2014), or by the manufacturer's recommendations.

Example 1 sample acquisition

The inventors found 1 Dysferlin myopathy family (abbreviated as family 1), and the clinical information of part members of the family is shown in Table 1. FIG. 1 is a family chart, wherein,

representing a male carrier, is->

1. Diagnostic criteria:

reference may be made to the 2010 version of the human monogenic genetic disease.

The limb belted muscular dystrophy in Dysferlin myopathy is mainly characterized by muscle weakness and atrophy, and is mainly represented by proximal muscle weakness, the first symptoms are pelvic girdle muscular atrophy and lower limb proximal weakness, shoulder be improperly familiar with belted muscular atrophy gradually occurs, serum CK is obviously increased, and myogenic damage is visible by electromyography. The incidence rate is about 1/10 ten thousand when the patients are seen. Muscular dystrophy of limb area type is ill in children, teenagers or adults, the ill ages are greatly different, men and women can ill after 10 to 50 years old, the first symptoms are usually muscular atrophy of pelvic girdle and shoulder swelling, lumbar lordosis, difficult upstairs, gait is duck steps, weakness of the proximal end of lower limb and frequent falling. The patient has difficulty in going upstairs and standing from the sitting position; knee reflection disappears earlier than stepping reflection; difficulty in lifting arm, wing-shaped shoulder enlargement: the head, face and neck muscles are not affected generally, and sometimes can be accompanied by the pseudohypertrophy of the intestinal muscles, the serum creatine kinase is obviously increased, but the serum creatine kinase is usually lower than the level of DMD, the electrogram indicates irregular, the electromyogram indicates myogenic damage, and the pathological result of the muscle biopsy accords with the change of the grab malnutrition.

TABLE 1 clinical information of Dysferlin myopathy No.1 family members

As shown in FIG. 1, the numbers I (first generation) and II (second generation) are adopted.

The peripheral blood DNA of family 1 personnel I1 (male parent of the first-wittedly), I2 (mother of the first-wittedly) and II 1 (mother of the first-wittedly) were used for sequencing analysis.

Example 2 exon sequencing

1. The instrument is shown in table 2.

Table 2 list of instruments and devices

2. Reagent consumable

Human whole exon sequencing kit (Agilent), DNA 1000 kit (Agilent), 96 well plate (Axygen), different model tips (Axygen), 200 μl centrifuge tube (Eppendorf), 1.5mL centrifuge tube (Eppendorf), capillary electrophoresis buffer (Thermo), sequencing standard (Thermo), absolute ethanol (Thermo), bigDye Terminator V3.1.3.1 (Thermo), peripheral blood gDNA extraction kit (TIANGEN), agarose (TIANGE N), EB dye solution (amerco).

3. Reagent formulation

A5 XTBE stock solution of electrophoresis liquid was prepared in accordance with Table 3.

Table 35 XTBE electrophoresis liquid formula

Reagent(s)	Tris	Boric acid	EDTA(pH 8.0,0.5mol/L)	ddH ₂ O
					Volume/weight	5.4g	750mg	2mL	90mL

With ddH ₂ O adjusts the final volume to 100mL.

0.5 XTBE working solution was run on ddH ₂ O is diluted by 10 times.

10 Xerythrocyte lysate was prepared according to Table 4.

TABLE 4 10 Xerythrocyte lysate formula

Reagent(s)	NH ₄ Cl	KHCO ₃	EDTA	Adding ddH ₂ O
					Volume/weight	82.9g	10g	0.37g	To 1000mL

Autoclaving and storing at 4deg.C.

1 Xnuclear lysate was prepared according to Table 5.

Table 51 XNuclear lysate formula

Reagent(s)	2M Tris-HCl，pH8.2	4M NaCl	2mM EDTA
				Volume/weight	0.5mL	10mL	0.4mL

4. Experimental procedure

After signing the informed consent, 3-5mL of peripheral blood of I1 (forerunner father), I2 (forerunner mother) and II 1 (forerunner) in family 1 were collected.

4.1 sample DNA extraction

1) 3-5mL of the sample is put into a 15mL centrifuge tube, and 2-3 times of 1 Xerythrocyte lysate is added, and the mixture is uniformly mixed and kept stand on ice for 30 minutes until the solution becomes transparent.

2) Centrifuge at 4℃for 10 min at 3000 rpm, carefully remove the supernatant. 1mL of 1 Xcell nucleus lysate was added to the pellet, mixed well, and 2mL of 1 Xcell nucleus lysate and 150. Mu.L of 20% SDS were added thereto, and shaken well until a viscous transparent state appeared. Add 10. Mu.L of 20mg/mL proteinase K and shake well. Digestion is performed at 37℃for more than 6 hours or overnight.

3) Adding saturated phenol with equal volume, mixing by shaking, and centrifuging at room temperature of 3000 rpm for 10 min.

4) The supernatant was carefully transferred to another centrifuge tube, mixed with an equal volume of phenol/chloroform (1:1 v/v) and centrifuged at 3000 rpm for 10 minutes at room temperature.

5) The supernatant was carefully removed and if not clear, extracted once more with an equal volume of chloroform.

6) Transferring the supernatant into another centrifuge tube, adding diploid absolute ethanol, shaking, and obtaining white flocculent DNA. The DNA was hooked with a flame sterilized glass crochet, washed twice with 70% ethanol, dried at room temperature for 5 minutes, and then dissolved in 200. Mu.L of 1 XTE and drum-dissolved overnight. OD was measured by uv.

7) The TE-dissolved DNA can be preserved for one year at 4deg.C, and if long-term preservation is required, 2 times volume of absolute ethanol is added for preservation at-70deg.C.

4.2 exon sequencing

Reference is made to the manual of the human whole exon sequencing kit (Agilent) and the manual of the molecular cloning laboratory (third edition; mol ecular Cloning A LABORATORY MANUAL 1SECOND EDITION;New York:Cold Spri ng Harbor LaboratoryPress,2014) for instructions.

1) Taking 2 mug DNA, mechanically breaking to ensure that the fragment size is about 200bp, cutting gel, and recovering 150-250bp fragments;

2) DNA fragment is used for terminal repair and A is added to the 3' -terminal;

3) Connecting sequencing joints, purifying the connection products, performing PCR amplification, and purifying the amplified products;

4) Adding the purified amplification product into an Agilent kit probe for hybridization capture, eluting and recovering the hybridization product, performing PCR amplification, recovering the final product, and performing quality control analysis by agarose gel electrophoresis on a small sample;

5) NextSeq500 sequencer sequencing and data analysis.

4.3 results

Finally, 1 gene composite heterozygous mutation DYSF with pathogenic significance is obtained, wherein the gene composite heterozygous mutation DYSF is NM_001130987.2, exo17, c.1535_1538delTGAG, p.S513Cfs 11 and exon49, c.5527_5528insC, p.R1845Tfs 22; 1535_1538delTG AG is mutated to a deletion of bases 1535 to 1538 TGAG, resulting in a mutation of amino acid 513 from serine (S) to cysteine (C) and a frame shift mutation, ending after amino acid 11 later; the 5527-5528 insC mutation is an insertion of a base C at positions 5527 to 5528, resulting in a mutation from arginine (R) to threonine (T) at position 1845 and a frameshift mutation, ending after the last 22 amino acids. The genotype of the DYSF: NM-001130987.2: exo17: c.1535-1538 delTGAG: p.S513Cfs x 11 and exon49: c.5527-5528insC: p.R 185Tfs x 22 sites in family 1 patient (pre-evidence) is the "c.1535-1538 delTGAG+c.5527-5528insC" composite heterozygous mutation; the genotype of this site in line 1 carrier was either the "c.1535_1538delTGAG" heterozygous mutation or the "c.5527_5528insC" heterozygous mutation.

Example 3Sanger sequencing validation

For the exome sequencing results, the DYSF: nm_001130987.2: exo17: c.1535_1538deltgag: p.s513cfs x 11 and exon49: c.5527_5528insc: p.r1845tfs x 22 sites were further verified using Sanger sequencing. DYSF: NM-001130987.2: exo17: c.1535-1538 delTGAG: p.S 513Cfs.11 and exon49: c.5527-5528insC: p.R1845 Tfs.22 site genotypes were respectively performed on 3 persons such as I1 (forerunner father), I2 (forerunner mother), II 1 (forerunner) and 100 normal persons outside the line in example 1.

The specific method comprises the following steps:

1. DNA extraction

Genomic DNA was extracted according to the method of example 1.

2. Candidate primer design, verification and preference

2.1 candidate primer design references the human genome sequence database hg19/build36.3 (https:// www.ncbi.nlm.nih.gov/genome, or http:// genome. Ucsc. Edu/cgi-bin/hgGateway.

2.2 design 17 and 18 pairs of candidate primers for the c.1535_1538delTGAG and c.5527_5528insC sites, respectively (see Table 6), and use PCR experiments to verify and evaluate the merits of each pair of candidate primers

TABLE 6 list of candidate primer base conditions and validation experiment results for each pair

Note that: after electrophoresis, the normal PCR amplification result has only one specific band, and if the primer dimer band and the non-specific product band are all the results of abnormal reaction of the primer; the target primer avoids such a situation as much as possible. The optimal primer pairs were also comprehensively evaluated and selected with reference to the following principles:

(1) the length of the primer is 15-30nt, and is usually about 20 nt;

(2) the content of G+C is preferably 40-60%, too little G+C has poor amplification effect, and excessive G+C is easy to generate nonspecific bands. TGC is preferably randomly distributed;

(3) avoiding a serial alignment of more than 5 purine or pyrimidine nucleotides;

(4) complementary sequences should not occur inside the primer;

(5) no complementary sequences should exist between the two primers, in particular to avoid complementary overlapping of the 3' ends;

(6) the homology of the primer and the sequence of the non-specific amplification region is not more than 70 percent, the continuous 8 bases at the 3' -end of the primer cannot have a complete complementary sequence outside the region to be amplified, otherwise, the non-specific amplification is easy to cause;

2.3 candidate primer PCR verification reaction

PCR was performed according to the reaction system in Table 7 and the reaction system was kept on ice; each pair of primers was provided with 8 reaction test tubes (SEQ ID NOS 1 to 8in Table 7).

TABLE 7 primer detection PCR reaction System

Reaction conditions: the test reaction tube was placed in a PCR instrument and the following reaction procedure was performed:

the first step: 95 ℃ for 5 minutes;

and a second step of: 30 cycles (95 ℃,30 seconds→tm,30 seconds→72 ℃,60 seconds); (the PCR amplification parameters were set according to the T m values of the primers shown in Table 6, and the Tm was averaged for the double primers).

And a third step of: 72 ℃,7 minutes;

fourth step: 4℃until sampling.

2.4 candidate primer PCR results agarose gel electrophoresis detection was performed to evaluate the effectiveness, specificity of the primer reactions:

1) Sealing the two ends of the gel sampler with adhesive tape, placing on a horizontal table, and placing a comb at about 1cm position at one end of the sampler.

2) Weighing 2g of agar powder in a conical flask, adding 100mL of 0.5 XTBE electrophoresis buffer, shaking uniformly, heating on a microwave oven or an electric furnace (adding asbestos gauze), taking out after boiling, shaking uniformly, reheating until the gel is completely melted, taking out and cooling at room temperature.

3) After the gel is cooled to about 50 ℃, pouring the gel into a sealed gel sampler to enable the thickness to be about 5 mm.

4) Gel is solidified and the adhesive tape is removed, and the gel and the sampler are put into an electrophoresis tank together.

5) Adding electrophoresis buffer solution to make the liquid level 1-2 mm higher than the rubber surface, and pulling out the comb upwards; and (3) uniformly mixing the sample and the DNA size standard substance with the sample loading liquid by using a micropipette, and adding the mixture into each sample loading hole, wherein the DNA is sunk into the hole bottom due to the fact that the sucrose in the sample loading liquid has a larger specific gravity.

6) And (5) covering an electrophoresis tank, switching on a power supply, adjusting to a proper voltage, and starting electrophoresis. And judging the approximate position of the sample according to the indication of bromophenol blue in the sample carrying liquid, and determining whether to terminate electrophoresis.

7) The power supply is cut off, the gel is taken out, and the gel is put into an EB water solution with the concentration of 0.5g/mL for dyeing for 10 to 15 minutes.

8) The gel was observed under a transmissive ultraviolet irradiator at 254nm and the electrophoresis results were recorded either with a camera with a red filter or with a gel scanning system.

2.5 evaluation of results:

1) If only one bright and clear target strip appears in the tube No.7 and no other strip exists, judging that the pair of primers and a reaction system are good in effectiveness and strong in specificity;

2) If no target band appears in the tube 7, judging that the pair of primers and the reaction system are invalid;

3) If the No.7 tube has a primer dimer band outside the target band and also has a primer dimer band in the No.2, 3, 4, 5 and 6 partial tubes, judging that the effectiveness of the pair of primers and the reaction system is poor;

4) If the No.7 tube has a nonspecific band outside the target band and also has a nonspecific band in the No.5 and 6 partial tubes, judging that the specificity of the pair of primers and the reaction system is poor;

5) If primer dimer and non-specific band outside the target band appear in the tube No.7, and primer dimer and non-specific band also appear in the tube No.2, 3, 4, 5, 6, the effectiveness and specificity of the pair of primers and the reaction system are judged to be poor.

2.6 based on the results of statistics after the verification test in Table 6, the optimal pair (No. 1 in Table 6) was selected as the primers for mutation family detection.

The PCR primer sequences for DYSF: NM-001130987.2: exo17: c.1535-1538 delTGAG: p.S 513Cfs. Times.11 sites are as follows:

DYSF-1F：5’-ATGGGATGCTGGTGGTT-3’(SEQ ID NO.1)

DYSF-1R：5’-GAAAGGTCTCGGAGTGCTA-3’(SEQ ID NO.2)

the primer sequences for DYSF: NM-001130987.2: exo49: c.5527_5528insC: p.R1845 Tfs. Times.22 sites are as follows:

DYSF-2F：5’-CTTTGACACGCCACGACT-3’(SEQ ID NO.3)

DYSF-2R：5’-GCTCCCTACCCTTTCACATA-3’(SEQ ID NO.4)

3. PCR amplification of mutation sites in family 1 personnel and 100 off-family personnel

PCR was performed according to the reaction system in Table 8 and the reaction system was kept on ice.

TABLE 8 mutation site PCR reaction system

Reaction conditions: the reaction system was put into a PCR instrument, and the following reaction procedure was performed:

for DYSF: NM-000400.4: exo18: c.1666-1G > A site reaction procedure is as follows:

the first step: 95 ℃ for 5 minutes;

and a second step of: 30 cycles (95 ℃,30 seconds- > 52 ℃,30 seconds- > 72 ℃,60 seconds);

and a third step of: 72 ℃,7 minutes;

fourth step: 4℃until sampling.

For DYSF: NM-000400.4: exo21: c.1922G > C: p.R641P site reaction procedure was as follows:

the first step: 95 ℃ for 5 minutes;

and a second step of: 30 cycles (95 ℃,30 seconds- > 54 ℃,30 seconds- > 72 ℃,60 seconds);

and a third step of: 72 ℃,7 minutes;

fourth step: 4℃until sampling.

4. Agarose gel electrophoresis detection

Refer to step 2.4 above.

5. Purifying a PCR product by an enzymolysis method: to 5. Mu.L of the PCR product, 0.5. Mu.L of exonuclease I (Exo I), 1. Mu.L of alkaline phosphatase (AIP) was added, and the mixture was digested at 37℃for 15 minutes and inactivated at 85℃for 15 minutes.

6. BigDye reaction

The BigDye reaction system is shown in Table 9.

TABLE 9BigDye reaction System

Sequencing PCR cycling conditions:

the first step: 96℃for 1 minute;

and a second step of: 33 cycles (96 ℃,30 seconds- > 55 ℃,15 seconds- > 60 ℃,4 minutes);

and a third step of: 4℃until sampling.

7. And (3) purifying a BigDye reaction product:

1) mu.L of 125mM EDTA (pH 8.0) was added to each tube, and 1. Mu.L of 3mol/L NaAc (pH 5.2) was added to the bottom of the tube;

2) Adding 70 mu L of 70% alcohol, shaking and mixing for 4 times, and standing at room temperature for 15 minutes;

3) 3000g, centrifugation at 4℃for 30 minutes; immediately inverting the 96-well plate and centrifuging 185g for 1 minute;

4) After 5 minutes at room temperature, the residual alcohol was allowed to evaporate at room temperature, 10. Mu.L Hi-Di formamide was added to dissolve DNA, denatured at 96℃for 4 minutes, quickly placed on ice for 4 minutes, and sequenced on the machine.

8. Sequencing

DNA sequencing the purified BigDye reaction product, wherein sequencing primers are designed based on the PCR preferred primers (the second set of primers are designed within the range of the product sequence obtained by amplifying the first set of primers) as sequencing primers, and the sequencing primer sequences of the sites of NM_001130987.2:exo17:c.1535_1538 delTGAG: p.S513Cfs are as follows:

DYSF-Seq1F：5’-CCAAAGTGAAGACCAGAAGC-3’(SEQ ID NO.5)

DYSF-Seq1R：5’-TCCTCTTTGTGCCCTTC-3’(SEQ ID NO.6)

the sequencing primer sequences for DYSF: NM-001130987.2: exo49: c.5527_5528insC: p.R185Tfs. Times.22 sites are as follows:

DYSF-Seq2F:5’-ACCCTGTAGTCCCCTGTT-3’(SEQ ID NO.7)

DYSF-Seq2R:5’-GCCCAAACGAGAACTGTAT-3’(SEQ ID NO.8)

9. analysis of results

The Sanger sequencing results of FIG. 2 show that the DYSF: NM-000400.4: exo18: c.1666-1G > A locus genotype of 2 persons of family 1 is "c.1666-1G > A heterozygote". The position indicated by the arrow in the sequencing diagram of FIG. 2 shows that the B, C layer DYSF: N M _000400.4: exo18: c.1666-1G > A locus genotype is a "c.1666-1G > A heterozygote" mutation; the position indicated by the arrow in the sequencing diagram of FIG. 2 shows that the A-layer individual genotype is wild type.

The Sanger sequencing results of FIG. 3 show that the genotype of the 2 members DYSF: NM-000400.4: exo21: c.1922G > C: p.R641P locus is "c.1666-1G > A heterozygote". The position indicated by the arrow in the sequencing diagram of FIG. 3 shows that the A, C-layer individual DYSF: NM-000400.4: exo21: c.1922G > C: p.R641P locus genotype is a "c.1922G > C heterozygote" mutation; the position indicated by the arrow in the sequencing diagram of FIG. 3 shows that the B-layer individual genotype is wild type.

Based on the detection results, the DYSF genotype of the precursor is c.1666-1G > A and c.1922G > C composite heterozygous mutation. And prompting the patient with the first syndrome to be a Dysferlin myopathy patient according to the detection result.

EXAMPLE 4DYSF genes c.1535_1538delTGAG, c.5527_5528insC mutation diagnostic kit and application

1. The kit comprises the following components:

1) Amplification primers (1.2. Mu.g per primer): as shown in example 3

2) Buffer (500 μl of 10 XPCR buffer: 500mmol/L KCl,100mmol/L Tris-Cl (pH 8.3), 15mmol/L MgCl 2)

3) Taq enzyme (20U)

4) dNTPs (four kinds of dNTPs 4mM each)

5) DYSF: c.1535_1538delTGAG, c.5527_5528insC positive mutant reference DNA the reference is a double-stranded DNA, the specific sequence of c.1535_1538delTGAG positive mutant reference DNA is as follows:

specific sequences of the 5527_5528insC positive mutant reference DNA are as follows:

wherein, single underlined bases are positions of the upstream and downstream primers of PCR amplification, boxes are mutation occurrence sites, and double underlined bases are positions of the upstream and downstream sequencing primers.

6) Sequencing primer: as shown in example 3

2. The using method comprises the following steps:

a total of 402 individuals of the myodystrophy family, 1329 individuals, with 1 more family-matched mutation site to be protected, were screened and tested, and the kit was applied to the patient test of family 2 (see table 10).

Table 10 clinical information of Dysferlin myopathy No.2 family members

As shown in FIG. 4, the numbers I (first generation) and II (second generation) are used.

Family members No. 2I 1 (father), I2 (mother), II 1 (brother) and II 2 (forerunner) peripheral blood DNA were used for the kit detection.

1) Genomic DNA extraction: and extracting the genomic DNA of the sample.

2) Firstly, carrying out PCR amplification reaction by using the PCR amplification primer, taq enzyme, buffer solution, dNTPs, sample genome DNA and the like, as in the example 3;

3) Purifying the PCR amplification product;

4) Performing BigDye reaction on the purified PCR product by using the sequencing primer;

5) Purifying the BiyDye reaction product;

6) The biydiye reaction products were sequenced and the sequenced sequences were compared to the normal sequences.

The results of the kit test of FIG. 5 show that the genotype of the DYSF: NM-001130987.2:exo n17:c.1535_1538delTGAG:p.S513Cfs*11 locus of family 2 mother, brother and forerunner is "c.1535-1538 delTGAG heterozygote". The position indicated by the arrow in the sequencing diagram of FIG. 5 shows B, C and D-layer DYSF. NM-001130987.2:exon17:c.1535_1538delTGA G:p.S513Cfs*11 locus genotype is the "c.1535-1538 delTGAG heterozygote" mutation; the position indicated by the arrow in the sequencing diagram of FIG. 5 shows that the A-layer individual genotype is wild type. The detection results of the kit of FIG. 6 show that the genotype of the line 2 father, brother and forerunner DYSF: NM-001130987.2: exo49: c.5527-5528insC: p.R185Tfs x 22 locus is "c.5527-5528 insC heterozygote". The position indicated by the arrow in the sequencing diagram of FIG. 6 shows A, C and D-layer DYSF: NM-001130987.2: exo49: c.5527-5528insC: p.R185Tfs. 22 locus genotype is the "c.5527-5528 insC heterozygote" mutation; the position indicated by the arrow in the sequencing diagram of FIG. 6 shows that the B-layer individual genotype is wild-type. The detection result confirms that the first evidence and the brother are Dysferlin myopathy patients, and the mother and father are mutation gene carriers; genetic counseling opinion is that a forerunner, forerunner go, and when coming wedding and giving birth, a spouse is recommended to carry out DYSF gene mutation detection in hospitals.

From the results of the above examples, it was found that novel DYSSF gene mutants were discovered and confirmed that the novel mutants are closely related to the onset of Dysferlin myopathy, which can be used for molecular diagnosis of Dysferlin myopathy and differential diagnosis of related diseases.

In the above technical solution of the present invention, the above is only a preferred embodiment of the present invention, and therefore, the patent scope of the present invention is not limited thereto, and all the equivalent structural changes made by the description of the present invention and the content of the accompanying drawings or the direct/indirect application in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A pathogenic gene causing Dysferlin myopathy, characterized in that the pathogenic gene is mutated at a site corresponding to bases 1535 to 1538 of exon17 of the wild-type dyssf gene compared to the wild-type dyssf gene;

2. A detection reagent for Dysferlin myopathy induced by the pathogenic gene according to claim 1, wherein the detection reagent comprises a specific amplification primer designed for a site where mutation of the pathogenic gene occurs.

3. The detection reagent according to claim 2, wherein the specific amplification primer comprises DYSF-1F, DYSF-1R, DYSF-2F, DYSF-2R, the DYSF-1F has a nucleotide sequence shown in SEQ ID NO.1, the DYSF-1R has a nucleotide sequence shown in SEQ ID NO.2, the DYSF-2F has a nucleotide sequence shown in SEQ ID NO.3, and the DYSF-2R has a nucleotide sequence shown in SEQ ID NO. 4.

4. A kit for detecting Dysferlin myopathy, comprising the detection reagent of claim 2 or 3.

5. The kit of claim 4, further comprising reagents for PCR amplification reaction, and/or reagents and sequencing primers required for DNA sequencing.

6. The detection kit of claim 5, wherein the sequencing primer comprises DYSF-Seq1F, DYSF-Seq1R, DYSF-Seq2F and DYSF-Seq2R, wherein the DYSF-Seq1F has a nucleotide sequence shown in SEQ ID NO.5, the DYSF-Seq1R has a nucleotide sequence shown in SEQ ID NO.6, the DYSF-Seq2F has a nucleotide sequence shown in SEQ ID NO.7, and the DYSF-Seq2R has a nucleotide sequence shown in SEQ ID NO. 8.

7. Use of the detection reagent according to claim 2 or 3 or the detection kit according to any one of claims 4 to 6 in a method for detecting Dysferlin myopathy.

8. The use of claim 7, wherein the test sample of the test agent comprises blood.