CN115873938A

CN115873938A - LRPRC gene recombination mutant leading to French-Canadian type Leigh syndrome

Info

Publication number: CN115873938A
Application number: CN202211140345.8A
Authority: CN
Inventors: 曾桥; 伍亮; 邓亚兰; 吴伟娟
Original assignee: Hunan Jiahui Biotechnology Co Ltd
Current assignee: Hunan Jiahui Biotechnology Co Ltd
Priority date: 2022-09-20
Filing date: 2022-09-20
Publication date: 2023-03-31
Anticipated expiration: 2042-09-20

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a LRPRC gene complex mutant causing French-Canadian type Leigh syndrome. The invention provides an LRPRC gene mutant causing French-Canadian type Leigh syndrome, which comprises c.3259A > G mutation compared with a wild type LRPRC gene; the c.3259A > G mutation is compounded with the existing c.4078G > A mutation to form a compound heterozygous mutation, and then an LRPRC gene compound mutant is formed. By detecting whether the LRPRC complex mutant protein exists in a biological sample, whether a subject carries the mutation can be effectively detected, and the LRPRC complex mutant protein is used for screening or diagnosing a French-Canadian type Leigh syndrome pathogenic gene mutation carrier or patient.

Description

LRPRC gene recombination mutant leading to French-Canadian type Leigh syndrome

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a LRPRC gene complex mutant causing French-Canadian type Leigh syndrome.

Background

French-Canadian type Leigh syndrome (Leigh syndrome, french-Canadian type, LSFC, MIM 220111), namely French-Canadian type Leigh syndrome, is a rare autosomal recessive hereditary severe nervous system disease and develops in infancy. It is characterized in that bilateral symmetry necrosis focus exists in subcortical brain area. Patients often exhibit developmental delay, hypotonia, mild facial deformity, chronic benign compensatory metabolic acidosis, with severe acidotic episodes and coma being the leading cause of high mortality.

LSFC is one of Leigh syndromes, the clinical symptoms of patients depend on damaged central nervous system areas, the main clinical characteristics are poor sucking ability of infants and children, head control loss and motor skill loss, the infants and children can have the symptoms of inappetence, vomiting, dysphoria, continuous crying, mental motor retardation, dysplasia, ataxia, epileptic seizure and the like, the electroencephalogram is mostly provided with acanthosis and slowness, and the patients can have the symptoms of general weakness, low muscle tension and lactic acidosis along with the development of diseases until the respiratory and renal functions are damaged. Most LSFC patients clinically manifest lactic acidosis and encephalopathy, and the amount of metabolic and/or neurological crisis that manifests determines the death of the patient. Metabolic crises are characterized by high serum lactate, hyperglycemia, hypotonia, coma, liver dysfunction, shock, respiratory distress and multiple organ failure. Crises in the nervous system are mainly manifested as hypotonia, ataxia, coma, abnormal breathing pattern, seizures and stroke-like seizures.

The LRPRC gene (MIM 607544) is positioned on chromosome 2p21, has a full length of 109.8kb, comprises 38 exons and 37 introns, and codes 130kD LRP130 consisting of 1394 amino acids. The LRPPRC gene encodes a leucine-rich protein with multiple pentapeptide repeats (PPR), which is mainly localized to the mitochondria, regulates mitochondrial gene protein expression at post-transcriptional levels, and plays an important role in regulating mitochondrial autophagy. Ten mutations of LRPPRC genes for LSFC included in the ClinVar database, wherein the pathogenic and possibly pathogenic mutations mainly cover the variant types of fragment deletion, minimal duplication, minimal deletion, missense mutation, splice site mutation, etc.

The gene mutation is an important genetic basis for generating and developing the French-Canadian type Leigh syndrome, and the gene diagnosis is an important standard for accurately diagnosing the French-Canadian type Leigh syndrome. Corresponding detection technologies are clinically needed to be established aiming at different mutations and used for determining causes and disease diagnosis, the detection of the genotype of a gene mutation site in the prior art can adopt other methods such as restriction fragment length polymorphism, single-strand conformation polymorphism, allele-specific oligonucleotide hybridization and the like, but the detection methods can not simultaneously meet the aims of determining the sequence of a mutant gene qualitatively, quantitatively and definitely.

Disclosure of Invention

The invention aims to provide an LRPRC gene complex mutant causing French-Canadian type Leigh syndrome, complex mutant protein and application.

The invention provides a mutant of LRPRC gene, which comprises c.3259A > G mutation compared with wild type LRPRC gene.

Preferably, the LRPPRC mutant protein comprises a p.m1087v mutation compared to the wild type LRPPRC protein.

The invention also provides an LRPRC gene compound mutant causing French-Canadian type Leigh syndrome, which comprises compound heterozygous mutations comprising the c.3259A > G mutation and the c.4078G > A mutation in the technical scheme.

The invention also provides the LRPRC complex mutant protein coded by the LRPRC gene complex mutant in the technical scheme, which comprises the p.M1087V mutation and p.A1360T in the technical scheme.

The invention also provides application of the LRPRC gene complex mutant or the LRPRC complex mutant protein in the technical scheme as a detection target in preparation of a kit for one or more of the following I to VII:

i: french-Canadian type Leigh syndrome diagnostic kit;

II: french-Canadian type Leigh syndrome screening kit;

III: a French-Canadian type Leigh syndrome antenatal diagnostic kit;

IV: a French-Canadian type Leigh syndrome prenatal screening kit;

v: a French-Canadian type Leigh syndrome pre-pregnancy diagnosis kit;

VI: a French-Canadian type Leigh syndrome pre-pregnancy screening kit;

VII: a kit for assisting in preventing and treating French-Canadian Leigh syndrome.

The invention also provides a reagent for detecting the LRPRC gene complex mutant or the LRPRC complex mutant protein in the technical scheme, which comprises at least one of a primer, a probe, an antibody and a mass spectrometry detection reagent.

The invention also provides a primer group for detecting the LRPRC gene complex mutant, which comprises a primer pair LRPRC-1 for detecting the c.3259A > G mutation and a primer pair LRPRC-2 for detecting the c.4078G > A mutation;

the nucleotide sequence of an upstream primer LRPRC-1F of the primer pair LRPRC-1 is shown as SEQ ID No.1, and the nucleotide sequence of a downstream primer LRPRC-1R of the primer pair LRPRC-1 is shown as SEQ ID No. 2;

the nucleotide sequence of an upstream primer LRPRC-2F of the primer pair LRPRC-2 is shown as SEQ ID No.3, and the nucleotide sequence of a downstream primer LRPRC-2R of the primer pair LRPRC-2 is shown as SEQ ID No. 4.

The invention also provides a reagent for diagnosing and/or screening French-Canadian type Leigh syndrome, which comprises the primer group in the technical scheme.

Preferably, the reagents further comprise sequencing primers and reagents required for amplification;

the sequencing primers comprise the c.3259A > G mutant sequencing primer LRPRC-Seq 1 and the c.4078G > A mutant sequencing primer LRPRC-Seq 2;

the nucleotide sequence of an upstream primer LRPRC-Seq 1F of the sequencing primer LRPRC-Seq 1 is shown as SEQ ID NO.5, and the nucleotide sequence of a downstream primer LRPRC-Seq 1R of the sequencing primer LRPRC-Seq 1 is shown as SEQ ID NO. 6;

the nucleotide sequence of an upstream primer LRPRC-Seq 2F of the sequencing primer LRPRC-Seq 2 is shown as SEQ ID No.7, and the nucleotide sequence of a downstream primer LRPRC-Seq 2R of the sequencing primer LRPRC-Seq 2 is shown as SEQ ID No. 8.

The invention also provides application of the reagent in the technical scheme in preparation of any one or more kits for diagnosing, screening and assisting in preventing and treating French-Canadian Leigh syndrome.

Has the advantages that:

the invention provides a mutant of LRPRC gene, which comprises c.3259A > G mutation compared with wild type LRPRC gene. The LRPRC gene mutant is the LRPRC gene mutant discovered for the first time, the c.3259A > G mutation means that the 3259 th base A of the No. 30 exon of the wild type LRPRC gene is mutated into the base G to form the LRPRC gene mutant, and the amino acid 1087 th of the encoded LRPRC protein is changed into valine (V) from methionine (M) to form the LRPRC mutant protein.

The invention also provides an LRPRC gene composite mutant causing French-Canadian Leigh syndrome, which comprises the c.3259A > G mutation and the c.4078G > A mutation in the technical scheme. The c.4078G > A mutation in the LRPRC gene complex mutant is the mutation site of the existing LRPRC gene, namely the 4078 th G mutation on the 37 th chromosome of the wild LRPRC gene is A, and the mutation can cause the 1360 th amino acid residue of the encoded protein to be changed from alanine (A) to threonine (T). The compound heterozygous mutation is formed by compounding the c.3259A > G mutation discovered for the first time and the existing c.4078G > A mutation, and then the LRPRC gene compound mutant is formed. The LRPPRC gene complex mutants of the present invention may form LRPPRC complex mutant proteins, including the p.m1087v and p.a1360t mutations. The invention discovers for the first time that the LRPRC gene complex mutant or the LRPRC complex mutant protein can cause French-Canadian Leigh syndrome and is closely related to the pathogenesis of French-Canadian Leigh syndrome, and whether the LRPRC mutant protein exists in a biological sample or not can be effectively detected, so that whether a subject carries the mutation or not can be effectively detected, the LRPRC mutant protein is used for screening or diagnosing a carrier or a patient of the French-Canadian Leigh syndrome pathogenic gene mutation, and the LRPRC gene complex mutant protein or the LRPRC complex mutant protein can provide prenatal and postnatal care and therapeutic intervention guidance to provide possible drug targets for treating French-Canadian Leigh syndrome.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments will be briefly described below.

FIG. 1 shows a French-Canadian type Leigh syndrome No.1 family genetic map; wherein, the first and the second end of the pipe are connected with each other,

indicating male carrier, \\ indicating female normal individual, \9632 \ indicating male patient, \8599andindicating proband;

figure 2 shows a plot of results from Sanger sequencing for the genotype of family 1 LRPPRC: NM — 133259.4: those mutated in family 1; layer a and layer B: genotype in family 1 is wild type (arrow in the sequencing panel indicates site of mutation);

figure 3 shows a graph of the results of the detection of family No.1 LRPPRC: NM — 133259.4: those mutated in family 1; layer B: genotype in pedigree No.1 is wild type (the arrow in the sequencing map indicates the site where the mutation occurred);

FIG. 4 shows a French-Canadian type Leigh syndrome No.2 family genetic map; wherein, the male carrier is shown in the figure,

indicating a female carrier, \ 9632while indicating a male patient,o denotes a fetus, \\99indicatesa proband patient;

FIG. 5 is a graph showing the results of the kit for detecting the genotype of the p.A1360T locus of family No.2 LRPRC: NM-133259.4; wherein, A and C: mutant in family 2; layer B: genotype in pedigree No.2 is wild type (the arrow in the sequencing plot indicates the site where the mutation occurred);

FIG. 6 shows graphs indicating the results of the detection of the LRPRC family No.2, NM-133259.4; wherein, layers B and C: mutant in family 2; layer A: the genotype in family 2 is wild type (the arrow in the sequencing panel indicates the site where the mutation occurs).

Detailed Description

The cDNA sequence of the wild type LRPRC gene is shown in Genbank with the accession number of NM-133259.4. The c.3259A > G mutation refers to the fact that the 4078 th G on the 37 th chromosome of a wild type LRPRC gene is mutated into A to form an LRPRC gene mutant.

The invention also provides a LRPRC mutant protein coded by the LRPRC gene mutant in the technical scheme, and the LRPRC mutant protein comprises p.M1087V compared with wild LRPRC protein. P.m1087v in the LRPPRC mutant protein described in the invention was caused by the c.3259a > G mutation.

The invention provides LRPRC gene complex mutants causing French-Canadian type Leigh syndrome, which comprise complex heterozygous mutations including the c.3259A > G mutation and the c.4078G > A mutation described in the technical scheme.

The invention aims at one French-Canadian Leigh syndrome family (wherein the family 1 comprises proband, proband father and mother) collected by self to carry out pathogenic mutation detection and verification on the family by a method of sequencing all exons, family analysis and Sanger sequencing verification. Novel compound heterozygous mutations are determined in the LRPRC gene, including the newly discovered c.3259A > G mutation and the existing c.4078G > A mutation, which constitute novel compound heterozygous mutations that can lead to the development of French-Canadian type Leigh syndrome. The invention discovers C.3259A > G mutation on LRPRC gene for the first time, confirms the close relation between the compound heterozygous mutation consisting of the C.3259A > G mutation and the C.4078G > A mutation and French-Canadian type Leigh syndrome, and can be used for molecular genetic research of the French-Canadian type Leigh syndrome and diagnosis of related diseases of the French-Canadian type Leigh syndrome.

The c.4078g > a mutation of the present invention refers to the mutation of 4078G to a on chromosome 37 of the wild-type LRPPRC gene, which may result in the 1360 th amino acid residue of the encoded protein being changed from alanine (a) to threonine (T). The c.4078G > A mutation and the c.3259A > G mutation found for the first time form a compound heterozygous mutation, and then form the LRPRC gene compound mutant. By detecting whether the subject carries the compound heterozygous mutation, the carrier or the patient of the mutation of the pathogenic gene of the French-Canadian Leigh syndrome can be screened or diagnosed to provide the direction of bearing and rearing and therapeutic intervention. In particular, the diagnostic kit provided by the invention can be used for quickly and effectively predicting or diagnosing French-Canadian type Leigh syndrome. On the other hand, the invention lays an important foundation for the research of pathogenesis of French-Canadian type Leigh syndrome and provides a brand new theoretical basis for the treatment of patients with French-Canadian type Leigh syndrome. In a third aspect, the invention can provide a possible drug target for treating French-Canadian type Leigh syndrome.

The invention also provides LRPPRC complex mutant proteins encoded by the LRPPRC gene complex mutant according to the above technical scheme, which comprise the p.m1087v and p.a1360t mutations described in the above technical scheme. The p.a1360t mutation in the lrppprc complex mutant protein of the invention is caused by the c.4078g > a mutation; and said p.m1087v caused by the c.3259a > G mutation constitute the LRPPRC complex mutant protein of the invention.

The invention also provides the application of the LRPRC gene complex mutant or the LRPRC complex mutant protein in the technical scheme as a detection target point in preparing the following kit: i: french-Canadian type Leigh syndrome diagnostic kit; II: french-Canadian type Leigh syndrome screening kit; III: a French-Canadian type Leigh syndrome antenatal diagnostic kit; IV: a French-Canadian type Leigh syndrome antenatal screening kit; v: a French-Canadian type Leigh syndrome pre-pregnancy diagnosis kit; VI: a French-Canadian type Leigh syndrome pre-pregnancy screening kit; VII: a kit for assisting in preventing and treating French-Canadian type Leigh syndrome. According to the invention, whether the biological sample is suffered from French-Canadian type Leigh syndrome or is susceptible to French-Canadian type Leigh syndrome or not can be effectively detected by detecting whether the LRPRC gene complex mutant is contained in the biological sample or not. According to the invention, whether the LRPRC complex mutant protein is expressed in a biological sample or not is detected, so that whether the biological sample is suffered from French-Canadian Leigh syndrome or is susceptible to French-Canadian Leigh syndrome or not can be effectively confirmed.

The invention also provides a reagent for detecting the LRPRC gene complex mutant or the LRPRC complex mutant protein in the technical scheme, wherein the reagent comprises at least one of a primer, a probe, an antibody and a mass spectrometry detection reagent, further preferably comprises the primer and/or the probe, and more preferably comprises the primer.

The invention also provides a primer group for detecting the LRPRC gene complex mutant, which comprises a primer pair LRPRC-1 for detecting the c.3259A > G mutation and a primer pair LRPRC-2 for detecting the c.4078G > A mutation; the nucleotide sequence of an upstream primer LRPRC-1F of the primer pair LRPRC-1 is shown as SEQ ID NO.1, and the nucleotide sequence of a downstream primer LRPRC-1R of the primer pair LRPRC-1 is shown as SEQ ID NO. 2; the nucleotide sequence of an upstream primer LRPRC-2F of the primer pair LRPRC-2 is shown as SEQ ID No.3, and the nucleotide sequence of a downstream primer LRPRC-2R of the primer pair LRPRC-2 is shown as SEQ ID No. 4.

The nucleotide sequences of SEQ ID NO. 1-4 of the invention are as follows: 1, SEQ ID NO.1:5 'CTTCAGGCACCATCCATTC-3'; SEQ ID No.2:5 'TTCTCAGGCTGCTCCCAC-3'; SEQ ID NO.3:5 'GTTCCTGGATTATCTTGA-3'; SEQ ID No.4:5 'GAGCTATGTTCTCCGACT-3'.

The invention also provides a reagent for diagnosing and/or screening French-Canadian type Leigh syndrome, which comprises the reagent in the technical scheme. The reagent of the invention preferably also comprises a sequencing primer and a reagent required for amplification; the sequencing primer preferably comprises the c.3259A > G mutant sequencing primer LRPRC-Seq 1 and the c.4078G > A mutant sequencing primer LRPRC-Seq 2; the nucleotide sequence of an upstream primer LRPRC-Seq 1F of the sequencing primer LRPRC-Seq 1 is preferably shown as SEQ ID No.5, and the nucleotide sequence of a downstream primer LRPRC-Seq 1R of the sequencing primer LRPRC-Seq 1 is preferably shown as SEQ ID No. 6; the nucleotide sequence of an upstream primer LRPRC-Seq 2F of the sequencing primer LRPRC-Seq 2 is preferably shown as SEQ ID No.7, and the nucleotide sequence of a downstream primer LRPRC-Seq 2R of the sequencing primer LRPRC-Seq 2 is preferably shown as SEQ ID No. 8. The nucleotide sequences of SEQ ID NO. 5-8 of the invention are as follows: SEQ ID No.5:5 'TTTTCCATCCTTTGGCATT-doped 3'; SEQ ID NO.6:5'-TTTAGATGTTGAACAAGGAA-3'; SEQ ID NO.7:5 'TCAAAGTGAGTGGATGCCGAATG-3'; SEQ ID NO.8:5' TCAGTCCCTCAAGCCATC-.

The reagents required for PCR amplification according to the present invention preferably include, but are not limited to dNTPs, PCR buffer, magnesium ions and Tap polymerase. The PCR buffer solution of the present invention is preferably a 10 XPCR buffer solution, and specifically includes 500mmol/LKCl,100mmol/L Tris-Cl (pH 8.3) and 15mmol/LMgCl ₂ . The reagent of the present invention preferably further comprises a DNA sequencing reagent. The type of the DNA sequencing reagent is not particularly limited in the present invention, and a conventional DNA sequencing reagent in the art may be used. The sequencing primer in the reagent can sequence an amplification product of a primer group for amplifying the LRPRC gene complex mutant so as to judge whether c.3259A exists on the LRPRC gene>G mutation and c.4078G>Mutation A, and fast and accurately diagnosing French-Canadian Leigh syndrome.

The invention also provides application of the reagent in the technical scheme in preparation of any one or more kits for diagnosing, screening and assisting in preventing and treating French-Canadian Leigh syndrome. The kit of the invention particularly preferably comprises I: french-Canadian type Leigh syndrome diagnostic kit; II: french-Canadian Leigh syndrome screening kit; III: a French-Canadian type Leigh syndrome prenatal diagnosis kit; IV: a French-Canadian type Leigh syndrome antenatal screening kit; v: a French-Canadian type Leigh syndrome pre-pregnancy diagnostic kit; VI: a French-Canadian type Leigh syndrome pre-pregnancy screening kit; VII: a kit for assisting in preventing and treating French-Canadian Leigh syndrome.

The invention also provides a method for detecting French-Canadian Leigh syndrome, which comprises the following steps: and amplifying the DNA of a sample to be detected by adopting the primer pair of the LRPRC gene mutant, sequencing and comparing products obtained by amplification, and judging the result.

The method for obtaining the DNA of the sample to be detected is not particularly limited, and a conventional DNA extraction method in the field can be adopted. The primer pair of the pathogenic gene mutant is preferably the same as the technical scheme, and is not described again. The steps and specific processes of amplification, sequencing and alignment are not particularly limited in the present invention, and may be performed by conventional methods in the art. The source of the DNA of the sample to be tested is preferably blood. The result judgment of the present invention preferably includes: judging the presence of a compound hybrid mutation in the LRPPRC gene if the genotype at the LRPPRC: NM — 133259.4; if the genotype at the two sites is "c.4078G > A heterozygous mutation" or "c.3259A > G heterozygous mutation", judging that the LRPRC gene has a single heterozygous mutation and the individual is a carrier; if the locus has c.4078G > A homozygous mutation + c.3259A > G wild type or c.4078G > A wild type + c.3259A > G homozygous mutation or c.4078G > A homozygous mutation + c.3259A > G heterozygous mutation or c.4078G > A heterozygous mutation + c.3259A > G homozygous mutation, judging that the LRPRC gene has compound heterozygous mutation and the individual is a patient; if the genotype of the locus is "wild type", the LRPRC gene is judged to be wild type, and the individual is a normal person.

In order to further illustrate the present invention, the following detailed description of the technical solutions provided by the present invention is made with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.

Experimental procedures for specific conditions not specified in the following examples are generally performed according to conventional conditions such as those described in Sambrook et al, molecular Cloning A LABORATORY Manual 1 SECOND edition, new York.

Example 1 sample acquisition

The inventor finds a French-Canadian Leigh syndrome family (called 1 family for short), and the clinical information of partial members of the 1 family is shown in Table 1. FIG. 1 shows a family map of LRPRC gene mutation No.1, in which,

indicates male carriers, \\ 9632indicating female normal individuals, indicates male patients, \8599indicatingprobands.

1. Diagnostic criteria:

reference may be made to "human monogenic genetic diseases" 2010 edition.

Leigh syndrome patients are clinically diagnosed mostly according to postmortem neuropathological examination results, but with the accumulation of clinical experience and the improvement of diagnosis technology, the diagnosis standard of the syndrome is continuously clear. The clinical diagnosis criteria mainly comprise: (1) Clinically, progressive nervous system diseases are shown to be accompanied by motor and/or intelligence development retardation; (2) biochemical examination: in most cases, the lactic acid and the pyruvic acid in blood, urine and cerebrospinal fluid are obviously increased, and the increase of the lactic acid and the pyruvic acid in the cerebrospinal fluid is more obvious; (3) imaging examination: double basal ganglia and/or brainstem symmetric long T1 and long T2 lesions in most cases; (4) special inspection: the gene detection finds the pathogenic mutation site, and is the gold standard for disease diagnosis.

LSFC is one of Leigh syndromes, the clinical symptoms of patients of the LSFC depend on damaged central nervous system areas, the LSFC is mainly clinically characterized by poor sucking ability of infants and children, head loss control and motor skill loss, and can be accompanied by inappetence, vomiting, dysphoria, continuous crying and screaming, mental motor retardation, dysplasia, ataxia, epileptic attack and the like, an electroencephalogram is mostly provided with acantha and slow waves, and the symptoms of general weakness, low muscle tension and lactic acidosis can appear along with the development of diseases until the respiratory and renal functions are damaged.

TABLE 1 clinical information of family Member of French-Canadian type Leigh syndrome No.1

As shown in FIG. 1, I (first generation) and II (second generation) are used as the numbering.

The family member No. 1I: 1, I: 2, II: 1 peripheral blood DNA was used for sequencing.

Example 2 exon sequencing

1. The instrumentation is shown in table 2.

Table 2 Instrument and Equipment List

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2. Reagent consumable

Human whole exon sequencing kit (Agilent), DNA 1000 kit (Agilent), 96-well plate (Axygen), different model tips (Axygen), 200 μ L centrifuge tube (Eppendorf), 1.5mL centrifuge tube (Eppendorf), capillary electrophoresis buffer (Thermo), sequencing standard (Thermo), absolute ethanol (Thermo), bigDye Terminator V3.1 (Thermo), peripheral blood gDNA extraction kit (TIANGEN), agarose (TIANGEN), EB stain (amereco).

3. Reagent formulation

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

TABLE 3 formulation of 5 XTBE electrophoresis solution

Reagent	Volume/weight
		Tris	5.4g
Boric acid	750.0mg
		EDTA(pH8.0,0.5mol/L)	2.0mL
ddH ₂ O	90.0mL

By ddH ₂ O the final volume was adjusted to 100mL.

Working solution of 0.5 XTBE electrophoresis solution by ddH ₂ Diluting with O by 10 times.

10 × erythrocyte lysates were prepared according to table 4.

TABLE 4 erythrocyte lysate recipe

Reagent	Volume/weight
		NH4Cl	82.9g
KHCO ₃	10.0g
		EDTA	0.37g
Adding dH ₂ O	To 1000mL

Autoclaving, and storing at 4 deg.C.

1 × cell nucleus lysate was prepared according to Table 5.

TABLE 5 cell nucleus lysate recipe

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

4. Experimental procedure

After signing an informed consent, 3-5mL of peripheral blood of members I: 1, I: 2 and II: 1 in the family is collected as a research sample.

4.1 sample DNA extraction

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

2) Centrifuge at 3000 rpm for 10 minutes at 4 ℃ and carefully remove the supernatant. The pellet was mixed with 1mL of 1 Xcell nucleus lysate, followed by addition of 2mL of 1 Xcell nucleus lysate and 150. Mu.L of 20% SDS, and the mixture was shaken until it became viscous and transparent. Add 10. Mu.L of 20mg/mL proteinase K and shake well. Digestion was carried out at 37 ℃ for more than 6 hours or overnight.

3) Adding equal volume of saturated phenol, shaking gently, mixing, and centrifuging at 3000 r/min for 10 min.

4) Carefully move the supernatant to another centrifuge tube, add the mixture of phenol and chloroform and mix well, phenol: the chloroform volume ratio was 1.

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

6) The supernatant was transferred to another centrifuge tube, and two times the volume of absolute ethanol was added thereto, followed by shaking to obtain white flocculent DNA. The DNA was hooked out using a flame-sterilized glass hook needle, 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. Measuring OD value by ultraviolet.

7) TE-dissolved DNA can be stored at 4 ℃ for one year, and if long-term storage is required, 2 times volume of absolute ethyl alcohol is added for storage at-70 ℃.

4.2 exon sequencing

Reference is made to the human whole exon sequencing kit (Agilent) instructions and Molecular Cloning LABORATORY Manual (third edition; molecular Cloning A LABORATORY MANUAL 1 SECOND EDITION.

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

2) Carrying out end repair on the DNA fragment and adding A at the 3' end;

3) Connecting a sequencing joint, purifying a connecting product, performing PCR amplification, and purifying an amplification product;

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 agarose gel electrophoresis on a small sample for quality control analysis;

5) NextSeq500 sequencer and data analysis.

4.3 results

Finally, a pathogenic gene complex hybrid mutation LRPRC, NM-133259.4; wherein the mutation of c.3259A > G is found for the first time, and the mutation can cause that the amino acid 1087 of the encoded protein is changed from methionine to valine; another c.4078g > a mutation, which has been found to result in the amino acid residue 1306 of the encoded protein changing from alanine to threonine; the genotype of LRPPRC NM _ 133259.4.

Example 3 Sanger sequencing validation

The LRPPRC: NM — 133259.4. LRPPRC: NM _133259.4exon 37 c.4078g > a and exon30: c.3259a > G: p.m1087v site genotype were performed on3 persons in family 1 (proband, proband father, proband mother) and 100 normal persons out of family 1 in example 1, respectively. A (c)

The method comprises the following specific steps:

1. DNA extraction

Genomic DNA was extracted according to the method of example 2.

2. Candidate primer design, validation and optimization

2.1 candidate primer design reference human genome sequence database hg 19/built 36.3.

2.2 for the old mutation site c.4078G > A and the newly found mutation site c.3259A > G, 15 pairs of candidate primers are designed respectively (see Table 6 and Table 7), and PCR experiments are used to verify and evaluate the advantages and disadvantages of each pair of candidate primers.

TABLE 6 c.3259A >

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TABLE 7 c.4078G >

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Note: only one specific band exists after electrophoresis of a normal PCR amplification result, and if a primer dimer band and a non-specific product band appear, the primer dimer band and the non-specific product band are both the results of primer abnormal reaction; the target primer avoids this as much as possible.

2.3 candidate primer PCR validation reactions

PCR was performed according to the reaction system in table 8 and kept on ice; 8 reaction test tubes (Nos. 1 to 8 in Table 8) were provided for each pair of primers.

TABLE 8 primer detection PCR reaction System

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Reaction conditions are as follows: placing the test reaction tube into a PCR instrument, and executing the following reaction procedures:

the first step is as follows: 95 ℃ for 5min; the second step: 30 cycles (95 ℃,30sec → Tm,30sec → 72 ℃,60 sec); (PCR amplification parameters are set according to Tm values of the primers in Table 8, and if the primers are double primers, the average Tm value is taken); the third step: 72 ℃,7min; the fourth step: 4 ℃ until sampling.

2.4 agarose gel electrophoresis detection of the candidate primer PCR results to evaluate the effectiveness and specificity of the primer reaction:

1) The two ends of the cleaned and dried gel sample applicator are sealed by an adhesive tape, the gel sample applicator is placed on a horizontal table, and a comb is placed at a position of about 1cm of one end of the sample applicator.

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

3) And when the gel is cooled to about 50 ℃, pouring the gel into a sealed gel sample injector to ensure that the thickness is about 5 mm.

4) The gel is solidified, the adhesive tape is removed, and the gel and the sample injector are placed into an electrophoresis tank.

5) Adding electrophoresis buffer solution to make the liquid surface 1-2 mm higher than the glue surface, and pulling out the comb upwards; and (3) respectively mixing the sample and the DNA size standard substance with the sample carrying liquid by using a micropipettor, and adding the mixture into each sample adding hole, wherein the DNA sinks into the bottom of the hole due to the large specific gravity of the sucrose in the sample carrying liquid.

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

7) The power was turned off, the gel was taken out and stained in 0.5g/ml EB aqueous solution for 10 to 15 minutes.

8) The gel was placed under a transmission ultraviolet irradiator to observe the result at a wavelength of 254nm, and photographed with a camera with a red color filter or the electrophoresis result was recorded with a gel scanning system.

2.5 evaluation of results:

1) If only one bright and clear target band appears in the No.7 tube and no other band exists, the pair of primers and the reaction system are judged to have good effectiveness and strong specificity;

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

3) If the primer-primer dimer band outside the target entry appears in the No.7 tube and the primer-dimer band also appears in the No.2, 3, 4, 5 and 6 tubes, the effectiveness of the pair of primers and the reaction system is judged to be poor;

4) If the non-specific band outside the target band appears in the No.7 tube and the non-specific band also appears in the No.5 and No.6 tubes, the specificity of the pair of primers and the reaction system is judged to be poor;

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

2.6 according to the results of statistics after the verification test in tables 6 to 7, the most preferable pair (SEQ ID NO.1 and SEQ ID NO.2 in Table 6, and SEQ ID NO.3 and SEQ ID NO.4 in Table 7) among them was selected as a primer for detecting a mutant family.

Primer sequences for LRPPRC: NM — 133259.4: 5 'GTTCCTGGATTATCTTGA-3' (SEQ ID NO. 3) 5 'GAGCTATGTTCCGACT-3' (SEQ ID NO. 4)

The primer sequences for the newly found mutant LRPPRC: NM — 133259.4exon30 c.3259a > -g: 5 'CTTCAGGCACCATCCATTC-3' (SEQ ID NO. 1) 5 'TTCTCAGGCTGCTCCCAC-3' (SEQ ID NO. 2)

3. PCR amplification of mutation sites of family No.1 and 100 family members

PCR was performed according to the reaction system in Table 9 while keeping the reaction system on ice.

TABLE 9 mutant site PCR reaction System

Reagent	Volume of
		10 XPCR buffer	2.0μL
10mmol/L dNTPs	0.4μL
		100 ng/. Mu.L LRPRC-1F (or LRPRC-2F)	0.5μL
100 ng/. Mu.L LRPRC-1R (or LRPRC-2R)	0.5μL
		DNA extraction from 100 ng/. Mu.L peripheral blood	1.0μL
5 u/. Mu.L Taq enzyme	0.2μL
		ddH2O	15.4μL

The reaction conditions are as follows: the reaction system was placed in a PCR instrument and the following reaction procedure was performed: for the c.4078g > a site, the reaction procedure was: the first step is as follows: 5 minutes at 95 ℃; the second step: 30 cycles (95 ℃,30sec → 46 ℃,30sec → 72 ℃,60 sec); the third step: 72 ℃ for 7 minutes; the fourth step: 4 ℃ until sampling.

For c.3259a > G sites, the reaction program was: the first step is as follows: 5 minutes at 95 ℃; the second step: 30 cycles (95 ℃,30 seconds → 56 ℃,30 seconds → 72 ℃,60 seconds); the third step: 7 minutes at 72 ℃; the fourth step: 4 ℃ until sampling.

4. Agarose gel electrophoresis detection

Refer to step 2.4 above.

5. And (3) carrying out enzymolysis purification on the PCR product: mu.L of each of exonuclease I (Exo I) and alkaline phosphatase (AIP) was added to 5. Mu.L of the PCR product, 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 10.

TABLE 10 BigDye reaction System

Reagent	Dosage of
		DNA after purification of PCR product	2.0μL
3.2 pmol/. Mu.L sequencing primer	1.0μL
		BigDye	0.5μL
5 xBigDye sequencing buffer	2.0μL
		ddH ₂ O	4.5μL

Sequencing PCR cycling conditions: the first step is as follows: at 96 ℃ for 1 minute; the second step: 33 cycles (96 ℃,30sec → 55 ℃,15 sec → 60 ℃,4 min); the third step: 4 ℃ until sampling.

7. Purification of BigDye reaction product:

1) Add 1. Mu.L 125mM EDTA (pH8.0) to each tube, add to the bottom of the tube, add 1. Mu.L 3mol/L NaAc (pH5.2);

2) Adding 70 μ L70% ethanol, shaking and mixing for 4 times, standing at room temperature for 15 min;

3) 3000g, centrifuging at 4 ℃ for 30 minutes; immediately invert the 96-well plate and centrifuge at 185g for 1 min;

4) Standing at room temperature for 5min, allowing residual ethanol to evaporate at room temperature, adding 10 μ L Hi-Di formamide to dissolve DNA, denaturing at 96 deg.C for 4 min, rapidly standing on ice for 4 min, and sequencing on computer.

8. Sequencing

And (3) carrying out DNA sequencing on the purified BigDye reaction product, and designing a nested primer (the second group of primers are designed in the sequence range of the product obtained by amplifying the first group of primers) as a sequencing primer on the basis of the PCR optimal primer by using a sequencing primer.

Sequencing primer sequences for LRPPRC: NM — 133259.4: 5 'TCAAAGTGAGTGGATGCCGAATG-3' (SEQ ID NO. 7); 5 'TCAGTCCCTCAAGCCATC-3' (SEQ ID NO. 8)

Sequencing primer sequences for the newly found mutation sites LRPPRC: NM — 133259.4exon30: 5-; 5' TTTAGATGTTGAACAAGGAA-

9. Analysis of results

Sanger sequencing results showed that the LRPPRC: NM — 133259.4; the genotype of the carrier at this site in family 1 is a single heterozygous mutation "c.3259A > G heterozygous mutation", respectively; proband parental affinity LRPPRC for 100 normal controls without kindred NM — 133259.4. The C-layer at the position indicated by the arrow in the profile in fig. 2 shows that the genotype at the LRPPRC: NM _ 133259.4. The positions indicated by the arrows in the sequencing diagram of figure 3, layers a and C, show that the individual LRPPRC: NM _ 133259.4. Example 4 French-Canadian type Leigh syndrome diagnostic kit and application

1. The kit comprises the following components:

1) An amplification primer: the sequences shown in Table 7 SEQ ID NO.1 and SEQ ID NO.2 and Table 8 SEQ ID NO.3 and SEQ ID NO.4 in example 3, 2) buffer: the 10 × PCR buffer specific components were not: 500mmol/LKCl,100mmol/LTris-Cl (pH 8.3), 15mmol/L MgCl ₂ 3) Taq enzyme, 4) dNTPs, 5) LRPRC c.4078G>A and c.3259A>G-Positive mutant reference DNA, c.4078G>The A positive reference substance is a section of double-stranded DNA, and the specific sequence is shown as follows: GTTCCTGGATTATCTTGATTCACAGTGGTTTTAAGCCAAAGGGGATTGAATGGTTGATTATTTTATAACTGAGGACTTCAGTTTGTGGTCACTTGAACTTGAACTATATATATATATGGTATCATATTATTCTAAAGTCCTTAAATGTGTGTAGCCATTTGTCATTCCTTCAAAGTGAGGTGATGAATGAGTGAGTGAGGTTGAGGTATCTTCTGAAATTGAATGATCTGTGAATCTTGAAGTGAATCTTGAAGTTGAGCTTGAAGTTGAAGTGTTGATCTTGAACTGTTGAACTGTTGAGCTTGAACTGTTGAACTGTCATGTGAAGGAATTGAAGTCATCTGAATTGAAGTTGAAGTTGAGTGAATGAGCTGTCATCTGTCATTGAATGAATGAAGTTGAGATTGAAGTGTGTGTGTTGAAGTTGAAGTTGAAGTTGAATGAATGAGCTTGAATGAATGAGCTGTTGAATGAATGAATGAAGTGTGTGTTGAATGAATGAATGAAGTCATTGAAGTTGAAGTCATTGAAGTCATTGAAGTCATTGAAGTCATTGAAGTCATTGAAGTCATTGAAGTCATGTCATTGAGGAAGGAAGGATGAAGTCATGTCATTGAGGAAGGAAGGAAGATGAAGATGAAGATGATGAAGTCATTGAAGTCATGTCATTGAAGTCATGTCATTGAAGTCATGTCATTGAAGTCATTGAAGTCATTGAAGTGTGTCATGTCATGTCATGTCATTGAAGTCATTGAAGTGTGTGTGTCATGTGTGTGTGTCATGTCATGTCATGTCATGTCATGTCATGTCATTGAAGTCATTGAAGTGTGTGTCATTGAAGTCATGTCATGTCATGTCATGTCATGTCATTGAAGTGTGTGTGTCATGTCATGTCATGTCATGTCATTGAAGTCATGTTGAAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTGAAGTGTGTGTGTGTGTGTTGAAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTGAAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTGAAGTTGAAGTGTTGAAGTTGAAGTTGAAGTTGAAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTGAAGTGTTGAAGTTGAAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTGAAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTGAAGTTGAAGTTGAAGTTGAAGTGTTTCATTGAAGTGTTGAAGTTGAAGTTGAAGTGTGTGTGTGTGTTGAAGTGTGTGTTGAAGTTGAAGTTGAAGTTGAAGTGTTGAAGTTGAAGTGTGTGTTGAAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTTTTTTTGAAGTTGAAGTGTGTTGAAGTGTGTGTGTTGAAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTGAAGTTGAAGTTGAAGTGTGTTGAAGTTGAAGTGTTGAAGTTGAAGTTGAAGTTGAAGTTGAAGTTGAAGTGTGTGTGTGTGTGTGTTGAAGTTGAAGTTGAAGTTGAAGTTGAAGTTGAAGTGTTGAAGTTGAAGTTGAAGTTGAAGTGTGTTGAAGTTGAAGTTGAAGTTGAAGTTGAAGTTGAAGTTGAAGTTGAAGCGGCAGTCGGAGAACATAGCTC(SEQ ID NO.65)

The 3259A > G positive reference substance is a section of double-stranded DNA, and the specific sequence is shown as follows:

CTTCAGGCACCATCCATTCAGATTAAGGTGTAGTTTCCAAACCTGTAGCTTTGTCTTTAAAATATTTGAAGAATTATGTCAGTGTGTCTCTCTATAGAGTGGTAACAGTGGGTTGGTATTAAAAAGCAAAAATATTTGGGTTTAGGTCTTTGTTTCAGGATTTTTTCCATCCTTTTGGCATTTCAGAATAATGTCATCTCATTTTTTTTGTCTGTTTTCTCTCATAGGGGCATATGATATTTTCCTGAATGCAAAAGAGCAAAACATTGTGTTTAATGCTGAAACCTACAGCAATCTCATTAAATTACTGATGTCAGAAGATTATTTTACACAAGCAGTGGAAGTGAAAGCATTGTAAGTGTTAGTCCATTTATTAAACCTCTTGTTTAAAAAAGAATATTGGCTGCCATTAGCCAATAATTATAATAGTAATGTGCTGTGTAGTAAAATTGCTTGATTGGCCTCAGCTACCCATAAAAATCATATGAAATGGTTAACTAACTTTATCAGGTTTTTTCACCTTCAAAAAAAAACACCTTTACTGGCCATATACAGTGCAGTACATTTTGCTTACTAAGTTATATTCCATCACAATTCATTTAAGATATCCATGCACAGAAAAGTAATTTTATTATATTCTGTTCCTTGTTCAACATCTAAATCCATGTACCTTTTGTGGTGGGAGCAGCCTGAGAA(SEQ ID NO.66)

6) Sequencing primers: as shown in example 3

2. The using method comprises the following steps:

applied to the test of family 2 patients (see table 11).

TABLE 11 clinical information of family Member of French-Canadian type Leigh syndrome No.2

/>

As shown in FIG. 4, I (first generation) and II (second generation) are used as the numbers. Wherein, the first and the second end of the pipe are connected with each other,

indicates a male carrier>

Indicating female carrier, \ 9679, female patient, \ 8599and probation patient.

The peripheral blood DNA of family personnel No. 2I 1, I2, II 1 and II 2 is used for the detection of the kit.

1) Extracting genome DNA: and extracting the genomic DNA of the sample.

2) Firstly, carrying out PCR amplification reaction by adopting the PCR amplification primer, taq enzyme, buffer solution, dNTPs, sample genome DNA and the like;

3) Purifying PCR amplification products;

4) Carrying out BigDye reaction on the purified PCR product by adopting the sequencing primer;

5) Purifying the BiyDye reaction product;

6) The BiyDye reaction products were sequenced and the sequence compared to the normal sequence.

The test results of the kit show that the genotype of the proband LRPRC: NM-133259.4. The positions indicated by the arrows in the sequencing map of fig. 5 show that the pro, proband father lrppprc of family is "c.4078g > a heterozygous mutation" at the site genotype of NM — 133259.4; the positions indicated by the arrows in the sequencing graph of fig. 6 show that the proband, proband mother LRPPRC: NM — 133259.4; the results in fig. 5 and fig. 6 suggest that the parents of the proband are French-Canadian type Leigh syndrome carriers, and the parents of the proband need to come to the hospital for prenatal diagnosis after genetic consultation when in later pregnancy.

From the above embodiments, it follows that: the invention confirms that the novel LRPRC gene complex mutant comprises c.3259A > G mutation and c.4078G > A mutation, confirms that the novel mutant is closely related to the pathogenesis of French-Canadian Leigh syndrome, and can be used for molecular diagnosis of French-Canadian Leigh syndrome and differential diagnosis of related diseases.

Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and all of the embodiments are included in the scope of the present invention.

Claims

1. A mutant of the LRPRC gene comprising a c.3259A > G mutation compared to the wild type LRPRC gene.

2. The LRPRC mutant protein encoded by the LRPRC gene mutant of claim 1, wherein the LRPRC mutant protein comprises a p.M1087V mutation compared to the wild type LRPRC protein.

3. A complex mutant of the LRPPRC gene resulting in French-Canadian type Leigh syndrome, wherein said complex mutant of the LRPPRC gene comprises a compound heterozygous mutation comprising the c.3259a > G mutation and the c.4078g > a mutation of claim 1.

4. The LRPRC complex mutant protein encoded by the LRPRC gene complex mutant of claim 3, wherein the LRPRC complex mutant protein comprises the p.M1087V mutation and p.A1360T of claim 2.

5. Use of the LRPPRC gene complex mutant according to claim 3 or of the LRPPRC complex mutant protein according to claim 4 as a detection target in a kit for the preparation of one or more of the following i to vii:

i: french-Canadian Leigh syndrome diagnostic kit;

II: french-Canadian Leigh syndrome screening kit;

III: a French-Canadian type Leigh syndrome antenatal diagnostic kit;

IV: a French-Canadian type Leigh syndrome antenatal screening kit;

v: a French-Canadian type Leigh syndrome pre-pregnancy diagnosis kit;

VI: a French-Canadian type Leigh syndrome pre-pregnancy screening kit;

6. A reagent for the detection of the LRPRC gene complex mutant of claim 3 or the LRPRC complex mutant protein of claim 4, wherein the reagent comprises at least one of a primer, a probe, an antibody, and a mass spectrometry detection reagent.

7. A primer set for detecting the LRPPRC gene complex mutant of claim 3, wherein the primer set comprises the primer pair LRPPRC-1 that detects the c.3259a > G mutation and the primer pair LRPPRC-2 that detects the c.4078g > a mutation;

8. A reagent for diagnosing and/or screening French-Canadian type Leigh syndrome, comprising the primer set of claim 7.

9. The reagent of claim 8, wherein the reagent further comprises a sequencing primer and reagents required for amplification;

10. Use of a reagent according to claim 8 or 9 for the manufacture of any one or more of a kit for the diagnosis, screening and adjunct treatment of French-Canadian-type Leigh syndrome.