CN115141837B

CN115141837B - Novel SLC9A6 mutant gene and diagnostic reagent thereof

Info

Publication number: CN115141837B
Application number: CN202210769201.2A
Authority: CN
Inventors: 曾桥; 薛斌; 李婵艺
Original assignee: Hunan Jiahui Biotechnology Co Ltd
Current assignee: Hunan Jiahui Biotechnology Co Ltd
Priority date: 2022-06-30
Filing date: 2022-06-30
Publication date: 2023-06-23
Anticipated expiration: 2042-06-30
Also published as: CN115141837A

Abstract

The invention provides a novel SLC9A6 mutant gene and a diagnostic reagent thereof, belonging to the technical field of medical diagnosis. The invention discovers that the mutation of SLC9A6: NM_001177651.2: exon2: c.169+2C > T site can cause Christianson syndrome for the first time through an exome sequencing technology. The research result of the invention can be used for genetic diagnosis of Christianson syndrome. Provides a new basis and a new path for researching pathogenesis of the Christianson syndrome, provides a new theoretical basis for treating the Christianson syndrome, and can provide a possible drug target for treating the Christianson syndrome.

Description

Novel SLC9A6 mutant gene and diagnostic reagent thereof

Technical Field

The invention belongs to the technical field of medical diagnosis, and particularly relates to a novel SLC9A6 mutant gene and a diagnostic reagent thereof.

Background

X chromosome-linked dysnoesia (X-1inked intellectual disability,XLID) is a congenital dysnoesia caused by mutation of genes located on the X chromosome, and the related congenital dysnoesia accounts for about 15% of all congenital dysnoesia. XLID is divided into two categories depending on whether there are other physiological defects besides mental retardation: S-XLID (syndromic forms) and NS-XLID (non-synthromic forms), which are manifested by abnormal or defective bone, craniofacial, neurological features or other signs in addition to mental disorders

According to the definition of the united states mental disorder association (American Association on Mental Retardation) for mental disorders (mental retardation, MR), congenital mental disorders are complex diseases mainly caused by central nervous system dysplasia and possibly accompanied by symptoms such as metabolic disorders, and patients often show significant defects in terms of mental and behavioral before 18 years of age. According to statistics, the mental disorder patients account for 1% -3% of the general population, and the proportion of men and women is 1.4-1.6:1. To date, studies have found that a loss of function of 102 genes can lead to 81S-XLID syndromes and 50 more families of NS-XLID, and another 30S-XLID syndromes and 48 families carrying NS-XLID are associated with a specific region of the X chromosome. Factors causing congenital mental disorders include changes in gene copy number, deletion or insertion of small nucleotide fragments, abnormal functions of regulatory elements, epigenetic changes, etc., 10% -15% of mental disorders being linked to the X chromosome.

Mutation of the SLC9A6 (MIM 300231) gene results in Christianson syndrome type X-linked dysnoesia characterized by small head deformity, impaired eye movement, severe global developmental retardation, developmental reversal, low muscle tone, motor abnormalities and various types of early onset seizures. The seizures are all manifested as Lennox-Gastaut syndrome (LGS), which is a severe age-dependent epileptic encephalopathy (epileptic encephalopathy, EE) with a peak of 3-5 years of onset accounting for 1% -10% of children's seizures.

Christianson syndrome is a dominant genetic disorder of the X chromosome. Typically, the gene on the male X chromosome is hemizygous, and the SLC9A6 mutation can lead to male morbidity; whether a carrier of a SLC9A6 mutant female heterozygote has clinical manifestations depends not only on the expression status of the causative gene, but also on whether the X chromosome is inactivated. Thus, some female heterozygotes have clinical manifestations, others do not, and some female carriers may be mildly affected.

The SLC9A6 gene is located on chromosome Xq26.3, comprising 16 exons and 15 introns, is 73.4kb in length, encodes intracellular sodium hydrogen ion exchange pump 6 (NHE 6) of 649 amino acids, and NHE6 is critical for neuronal dendrite and synapse formation. NHE6 is an endosomal transmembrane protein that regulates endosomal pH and endosomal transport and signaling. It was found that the deletion of NHE6 resulted in lysosomal dysfunction and impairment of endosomal maturation and transport, and that targeted modulation of NHE6 also altered amyloid β (aβ) levels. In addition, NHE6 expression levels appear to be inversely related to tau deposition. SLC9A6 gene mutation can affect NHE6 function, thereby leading to neurodevelopment and neurodegenerative disease, ultimately leading to mental disorder and epileptic occurrence.

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 Christianson syndrome. There is a clinical need to establish corresponding detection techniques for different mutations and for clear etiology and disease diagnosis.

Disclosure of Invention

Therefore, the invention aims to provide a novel SLC9A6 mutant gene and a diagnostic reagent thereof, and the novel SLC9A6 mutant gene can be discovered for the first time to cause Christianson syndrome, and a corresponding diagnostic kit is developed according to the novel SLC9A6 mutant gene, so that screening and diagnosis of the gene mutation of the assisted Christianson syndrome can be realized, and a novel technical support is provided for drug screening, drug effect evaluation and targeted treatment of the novel SLC9A6 mutant gene.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a novel mutant gene of Christianson syndrome, which comprises mutation at SLC9A6: NM_001177651.2: exon2: c.169+2C > T site.

The c.169+2C > T mutation refers to that the 2 nd position of the 2 nd intron of the wild SLC9A6 gene, which is next to the 2 nd exon, is mutated from C to T, so that a gene mutant is formed, and the nucleotide sequence of the gene mutant is preferably shown as SEQ ID NO.6 (GATTTATGGTAAGTTCCT).

The invention also provides a detection reagent for the Christianson syndrome caused by the novel mutant gene, which comprises a specific amplification primer designed for a mutation site of the novel mutant gene.

Preferably, the specific amplification primers comprise SLC9A6-F and SLC9A6-R, the nucleotide sequence of SLC9A6-F is shown as SEQ ID NO.1, and the nucleotide sequence of SLC9A6-R is shown as SEQ ID NO. 2.

The invention also provides a detection kit for Christianson syndrome, which comprises the detection reagent.

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

Preferably, the sequencing primer comprises SLC9A6-SeqF and SLC9A6-SeqR, the nucleotide sequence of the SLC9A6-SeqF is shown as SEQ ID NO.3, and the nucleotide sequence of the SLC9A6-SeqR is shown as SEQ ID NO. 4.

The invention also provides an application of the detection reagent or the detection kit in preparing a diagnostic reagent for Christianson syndrome.

Preferably, the test sample of the diagnostic reagent comprises blood or amniotic fluid.

The beneficial effects are that: the invention utilizes exon sequencing to screen pathogenic gene mutation highly related to Christianson syndrome, in order to avoid false positive results, and then verifies through Sanger sequencing to finally obtain the pathogenic gene mutation of Christianson syndrome, which is SLC9A6:NM_001177651.2:exon2:c.169+2C > T. The pathogenic gene mutation screened by the invention can distinguish the Christianson syndrome patients from normal people, so that the pathogenic gene mutation can be used as a biomarker for diagnosing the Christianson syndrome. The invention can be used to screen or diagnose genetic diagnosis of Christianson syndrome by detecting whether a subject carries the mutation described above to guide treatment. The diagnosis kit provided by the invention can be used for rapidly and effectively predicting or diagnosing the Christianson syndrome. The invention lays an important foundation for researching pathogenesis of the Christianson syndrome and provides a brand new theoretical basis for treating patients with the Christianson syndrome. The invention can provide a possible drug target for treating Christianson syndrome.

Drawings

FIG. 1 shows a family genetic map of Christianson syndrome No. 1; wherein, it represents normal male, by which it represents female carrier, ■ male patient, ↗ forerunner, and fetus;

FIG. 2 shows a graph of the results of detection of genotype at the SLC9A 6:NM-001177651.2:exo2:c.169+2C > T locus of line 1 by Sanger sequencing, with the first evidence in line 1 being the patient (the position of mutation by arrow in the sequencing);

FIG. 3 shows a Christianson syndrome No.2 family genetic map; wherein, it represents normal male individuals, it represents female carriers, ■ represents male patients, ↗ represents forerunner;

FIG. 4 shows a graph of the results of the detection of genotype at SLC9A 6:NM-001177651.2:exon2:c.169+2C > T locus in line 2, with the patient being the first patient in line 2 (the position of mutation indicated by the arrow in the sequencing diagram).

Detailed Description

The novel mutant gene provided by the invention is used for screening pathogenic gene mutation highly related to Christianson syndrome by utilizing exon sequencing, and is finally obtained by verifying through Sanger sequencing in order to avoid false positive results. In the invention, the DNA sequencing result of a sample to be detected is compared with the genome DNA sequence of a normal person, if the genotype of SLC9A6: NM_001177651.2: exo2: c.169+2C > T site is 'c.169+2C > T hemizygous', the mutation of SLC9A6 gene is judged, and a male individual is a patient; if the locus is not mutated, the SLC9A6 gene is judged to be wild type, and the individual is normal.

The specific amplification primer of the present invention preferably comprises:

SLC9A6-F(SEQ ID NO.1):CAACCTGCTCATCTTCATCC；

SLC9A6-R(SEQ ID NO.2):GCCCACTCGTTTTCCATC。

The detection kit of the present invention preferably further comprises reagents for PCR amplification reaction, and/or reagents and sequencing primers required for DNA sequencing. Wherein the sequencing primer preferably comprises:

SLC9A6-SeqF (SEQ ID NO. 3): ATCCTGCTGCTCACCCTC and SLC9A6-SeqR (SEQ ID NO. 4): CCACTCGTTTTCCATCCTCA. The detection kit of the invention preferably also comprises other conventional reagents in PCR amplification reaction, such as dNTP, PCR buffer solution, magnesium ion, tap polymerase and the like.

The test sample of the diagnostic reagent of the present invention preferably includes blood or amniotic fluid. When the diagnostic reagent of the present invention is used for diagnosing Christianson syndrome, the diagnostic reagent preferably comprises: 1) Extracting sample genome DNA;

2) Amplifying SLC9A6 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, and judging that mutation exists in SLC9A6 gene and male individuals are patients if the genotype of SLC9A6: NM_001177651.2: exon2: c.169+2C > T locus is 'c.169+2C > T hemizygous'; if the locus is free of mutation, the SLC9A6 gene is judged to be wild type, and the individual is normal.

The method for amplifying SLC9A6 gene sequence is preferably a PCR amplification method, and the system comprises the following steps of: 10 XPCR buffer 2.0. Mu.L, 10mmol/L dNTPs

0.4. Mu.L, 100 ng/. Mu.L SLC9A 6-F0.5. Mu.L, 100 ng/. Mu.L SLC9A 6-R0.5. Mu.L, 100 ng/. Mu.L peripheral blood extracted DNA 1.0. Mu.L, 5 u/. Mu.L Taq enzyme 0.2. Mu.L and the balance ddH ₂ O. The system is placed in a PCR instrument, and the program is set as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95℃for 30 seconds, annealing at 55℃for 30 seconds, elongation at 72℃for 60 seconds, 30 cycles; the extension was carried out at 72℃for 7 minutes. The method of the present invention for sequencing DNA is not particularly limited, and it is preferable to use a conventional sequencing means in the art for sequencing, such as sanger sequencing.

The novel SLC9A6 mutant gene and its diagnostic reagents provided in the present invention will be described in detail with reference to the examples, but they should not be construed as limiting the scope of the invention. In the present invention, the term "diagnosis" includes prediction of disease risk, diagnosis of disease onset or non-disease onset, and also includes assessment of disease prognosis; the term "mutation" refers to the alteration of a wild-type polynucleotide sequence into a variant, which may be naturally occurring or non-naturally occurring; "primer" refers to a polynucleotide fragment, typically an oligonucleotide, such as a polynucleotide fragment containing at least 5 bases, e.g., 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. In the present invention, the term "specifically amplifying" means that a primer is capable of amplifying a gene of interest by a PCR reaction without amplifying other genes. For example, specifically amplifying the SLC9A6 gene refers to the primers amplifying only the SLC9A6 gene and not the other genes in the PCR reaction.

The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor LaboratoryPress, 1989) or as recommended by the manufacturer.

Example 1 sample acquisition

The inventor has found a family of Christianson syndromes (abbreviated as SLC9A6 family) and the clinical information of the SLC9A6 family members is shown in table 1. FIG. 1 shows a SLC9A6 family map in which ∈two represents a normal male subject, "∈two represents a female carrier, ■ represents a male patient, ↗ represents a forerunner, and indicates a fetus.

1. Diagnostic criteria:

reference may be made to the "human monogenic genetic disease" 2010 edition and the "diagnosis of childhood dysnoesia or general developmental delay etiology diagnosis policy expert consensus" 2018 edition:

clinical characteristics: small head deformity, impaired eye movement, severe overall developmental retardation, reversal of development, hypotonia, dyskinesia, and various types of early onset seizures.

TABLE 1 clinical information of Christianson syndrome family members

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

Family members I1 (father), I2 (mother), II 1 (forerunner) peripheral blood and II 2 (fetus) amniotic fluid DNA were used for sequencing analysis.

Example 2 exon sequencing

1. The instrument is shown in table 2.

Table 2 list of instruments and devices

Instrument name	Manufacturing factories
		High throughput sequencer NextSeq500	Illumina
QubitFluometer nucleic acid metering meter	Invitrogen
		PCR instrument	Bio-RAD
Centrifuge 5810R	Eppendorf
		Centrifuge 5424	Eppendorf
5418 small-sized high-speed centrifugal machine	Eppendorf
		Biological safety cabinet	Sujing (Sujing)
Super clean bench	Sujing (Sujing)
		Ice machine	Grant
UPS power supply	Santa
		MilliQ ultrapure water instrument	Millipore
High performance computers (including servers, cabinets, switches, storage, etc.)	DELL
		-25 degree refrigerator	Meilishi (Meilishi)
Ultralow temperature refrigerator	Eppendorf
		Microwave oven	Beautiful appearance

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 (Thermo), peripheral blood gDNA extraction kit (TIANGEN), agarose (TIANGEN), EB dye solution (amerco).

3. Reagent formulation

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

Table 3 5 XTBE electrophoresis liquid formula

Reagent(s)	Volume/weight
		Tris	5.4g
Boric acid	750mg
		EDTA(pH8.0,0.5mol/L)	2mL
ddH ₂ O	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)	Volume/weight
		NH4Cl	82.9g
KHCO ₃	10g
		EDTA	0.37g
Adding ddH ₂ O	To 1000mL

Autoclaving and storing at 4deg.C.

1 Xnuclear lysate was prepared according to Table 5.

Table 51 XNuclear lysate formula

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

4. Experimental procedure

After signing informed consent, 3-5mL of peripheral blood of members such as I1 (father), I2 (mother), II 1 (forerunner) and 5-10mL of amniotic fluid of II 2 (fetus) in the family are collected.

4.1 sample DNA extraction

1) If the sample is heparin anticoagulated peripheral blood sample, 3-5mL of peripheral blood 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. The amniotic fluid specimen directly enters the next step.

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

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 pathogenic gene mutation SLC9A6:NM_001177651.2:exon2:c.169+2C > T is obtained; c.169+2C is located at base 2 of intron No.2, and forms a cleavage signal together with base 1 of intron (c.169+1) and the last 2 bases of the preceding exon No.2 (c.168, c.169), and the cleavage site sequence is destroyed by the c.169+2C > T mutation, so that the cleavage signal is abnormal, and mRNA is abnormal in cleavage. The genotype of SLC9A6: NM-001177651.2: exon2: c.169+2C > T site in individuals from family 1 male patient is a "c.169+2C > T hemizygous" mutation; the genotype of the SLC9A 6:NM-001177651.2:exo2:c.169+2C > T locus in 1 female carrier individual of family is a "c.169+2C > T heterozygote" mutation; the genotype is a wild type with no mutation in normal individuals of the SLC9A6 family.

Example 3Sanger sequencing validation

The SLC9A6: NM-001177651.2: exo2: c.169+2C > T sites were further verified using Sanger sequencing for exome sequencing results. SLC9A 6:NM-001177651.2:exo2:c.169+2C > T locus genotype was examined for family members such as I1, I2, II 1, II 2 and 100 extrapedigree normal persons in example 1, respectively.

The specific method comprises the following steps:

1. DNA extraction

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

2. Candidate primer design, verification and preference

2.1 primer design reference human genome sequence database hg19/build36.3, primer sequences were synthesized by Shanghai Biotechnology Inc.

2.2 20 pairs of candidate primers were designed for the c.169+2C > T site (see Table 6), and the merits of each pair of candidate primers were verified and evaluated using PCR experiments.

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. ATGC 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 8 in 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 Tm value is calculated for each primer in Table 6 by setting PCR amplification parameters based on the Tm value of each primer).

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 1-2mm 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 the statistics after the verification test of Table 6, the optimal pair (No. 1in Table 6) was selected as the primers for mutation family detection, and the primer sequences for SLC9A6: NM-001177651.2: exo2: c.169+2C > T sites were as follows:

5’-CAACCTGCTCATCTTCATCC-3’(SEQ ID NO.1)

5’-GCCCACTCGTTTTCCATC-3’(SEQ ID NO.2)

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

Reagent(s)	Volume of
		10 XPCR buffer	2.0μL
10mmol/L dNTPs	0.4μL
		100ng/μL SLC9A6-F	0.5μL
100ng/μL SLC9A6-R	0.5μL
		100 ng/. Mu.L of peripheral blood extract DNA	1.0μL
5 u/. Mu.L Taq enzyme	0.2μL
		ddH ₂ O	15.4μL

Reaction conditions: the reaction system was put into 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- > 55 ℃,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 7.

TABLE 9 BigDye reaction System

Reagent(s)	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: 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 is carried out on the purified BigDye reaction product, and a nest primer (a second set of primers are designed within the range of the product sequence obtained by amplifying the first set of primers) is designed on the basis of the PCR preferred primers as a sequencing primer, wherein the sequence of the sequencing primer is as follows: .

5’-ATCCTGCTGCTCACCCTC-3’(SEQ ID NO.3)

5’-CCACTCGTTTTCCATCCTCA-3’(SEQ ID NO.4)

9. Analysis of results

The Sanger sequencing results of FIG. 2 show that the genotype of SLC9A6: NM-001177651.2: exon2: c.169+2C > T site in 3 patients of family is "c.169+2C > T heterozygote". The position indicated by the arrow in the sequencing diagram of FIG. 2 shows that the SLC9A6:NM_001177651.2:exon2:c.169+2C > T locus genotype of the patient with C-layer Christianson syndrome is a "c.169+2C > T hemizygous" mutation. The position indicated by the arrow in the sequencing diagram of FIG. 2 shows that the B-layer carrier SLC9A6: NM-001177651.2: exon2: c.169+2C > T-site genotype is the "c.169+2C > T-heterozygote" mutation.

EXAMPLE 4SLC9A6 Gene c.169+2C > T mutation diagnostic kit and application

1. The kit comprises the following components:

1) Amplification primers: as shown in example 3

2) Buffer solution

3) Taq enzyme

4)dNTPs

5) SLC9A6, c.169+2C > T positive mutation reference DNA the reference is a double-stranded DNA, and the specific sequence is shown in SEQ ID NO. 5.

6) Sequencing primer: as shown in example 3

2. The using method comprises the following steps:

the method is applied to the detection of the gene mutation of the family 2.

TABLE 10 clinical information of Christianson syndrome 2 good family members

Family members I1 (father), I2 (mother) and II 1 (forerunner) peripheral blood were used for detection and analysis of the kit.

FIG. 4 shows the result of detecting genotype of SLC9A6:NM_001177651.2:exon2:c.169+2C > T locus of family 2 by using the kit, wherein the genotype of the locus of the ancestor in family 2 is c.169+2C > T hemizygous, the ancestor mother is c.169+2C > T heterozygous, and the male parent of the ancestor is wild type; the detection result confirms that the first person is a Christianson syndrome patient; the detection result shows that the mother of the forerunner is a carrier, the probability of the child of the same kind of birth is 1/4, the probability of the carrier of the birth female is 1/4, and the probability of the normal birth individual is 1/2; genetic counseling suggests that parents may conduct pre-embryo implantation genetic diagnosis or prenatal diagnosis.

From the results of the above examples, it can be seen that the present invention has found a novel SLC9A6 gene mutation, and confirmed that the novel mutation is closely related to the onset of Christianson syndrome, which can be used for molecular diagnosis of Christianson syndrome and differential diagnosis of related diseases.

1) Genomic DNA extraction: 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 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 foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

<110> Hunan Jiahui biotechnology Co., ltd

<120> a novel SLC9A6 mutant gene and diagnostic reagent therefor

<160> 43

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

caacctgctc atcttcatcc 20

<210> 2

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

gcccactcgt tttccatc 18

<210> 3

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

atcctgctgc tcaccctc 18

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

ccactcgttt tccatcctca 20

<210> 5

<211> 430

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

caacctgctc atcttcatcc tgctgctcac cctcaccatt ctcacaatct ggctcttcaa 60

gcaccgccgg gcccgcttcc tgcacgaaac cggcctggct atgatttatg gtaagttcct 120

caacccttgt cagccccttg gcgctgcccc tttctctgcc cgccggctgc ttcgcctcct 180

ctgctggccc tgctcggcct acgttcggct ccccttctaa ttccttccat tttctgcctc 240

gccttccccc taccccgcgt ttctctgcct cacccccttt cctcttcagc ctcgcgcccc 300

attttatctg cctctccaca cctttttcgc ttccgacccc accccctttt tcctccgcac 360

ccccagcccc ccaccctttc cctgcctacc aagctcggga cccggggctg aggatggaaa 420

acgagtgggc 430

<210> 6

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

gatttatggt aagttcct 18

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

acctgctcat cttcatcctg 20

<210> 8

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

gcccactcgt tttccatc 18

<210> 9

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

atcctgctgc tcaccctc 18

<210> 10

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

gcccactcgt tttccatc 18

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

acctgctcat cttcatcctg 20

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

accttaaaca aactgacacc c 21

<210> 13

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

atcctgctgc tcaccctc 18

<210> 14

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

accttaaaca aactgacacc c 21

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

caacctgctc atcttcatcc 20

<210> 16

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

gaaaggacag ggcgtagc 18

<210> 17

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

ctgggacaga ggggcaaag 19

<210> 18

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

tggggtcgga agcgaaaa 18

<210> 19

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

atcgtgtccg agaagcaa 18

<210> 20

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

ctccaaaaga acgccaga 18

<210> 21

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

atcgtgtccg agaagcaa 18

<210> 22

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

aaggacaggg cgtagcag 18

<210> 23

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

atcgtgtccg agaagcaa 18

<210> 24

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

atggatcgca atggtgtc 18

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

caacctgctc atcttcatcc 20

<210> 26

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

accttaaaca aactgacacc c 21

<210> 27

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

acctgggaca gaggggcaaa g 21

<210> 28

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

tggggtcgga agcgaaaa 18

<210> 29

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

caacctgctc atcttcatcc 20

<210> 30

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

agcccaattc cagacacc 18

<210> 31

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 31

acctgctcat cttcatcctg 20

<210> 32

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 32

atcgcaatgg tgtctccc 18

<210> 33

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 33

acctgctcat cttcatcctg 20

<210> 34

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 34

ccagcccaat tccagaca 18

<210> 35

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 35

cgtgtccgag aagcaagc 18

<210> 36

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 36

tcgaaaggac agggcgta 18

<210> 37

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 37

cgtgtccgag aagcaagc 18

<210> 38

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 38

cagggcgtag caggagct 18

<210> 39

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 39

acctgctcat cttcatcctg 20

<210> 40

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 40

ctccaaaaga acgccaga 18

<210> 41

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 41

caacctgctc atcttcatcc 20

<210> 42

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 42

aacaaactga caccccagag 20

<210> 43

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 43

acctgctcat cttcatcctg 20

Claims

1. A mutant gene for inducing Christianson syndrome is characterized in that the nucleotide sequence of the mutant gene is shown as SEQ ID NO. 5.

2. A detection reagent for Christianson syndrome triggered by the mutant gene according to claim 1, wherein the detection reagent comprises a specific amplification primer designed for a mutation site of the mutant gene;

the specific amplification primer comprises SLC9A6-F and SLC9A6-R, the nucleotide sequence of the SLC9A6-F is shown as SEQ ID NO.1, and the nucleotide sequence of the SLC9A6-R is shown as SEQ ID NO. 2.

3. A kit for the detection of Christianson syndrome comprising the detection reagent of claim 2.

4. The kit of claim 3, further comprising reagents for PCR amplification reaction, and/or reagents and sequencing primers required for DNA sequencing.

5. The detection kit according to claim 4, wherein the sequencing primer comprises SLC9A6-SeqF and SLC9A6-SeqR, the nucleotide sequence of SLC9A6-SeqF is shown as SEQ ID NO.3, and the nucleotide sequence of SLC9A6-SeqR is shown as SEQ ID NO. 4.

6. Use of the detection reagent of claim 2 or the detection kit of any one of claims 3 to 5 for the preparation of a diagnostic reagent for Christianson syndrome.

7. The use according to claim 6, wherein the test sample of the diagnostic reagent is blood or amniotic fluid.