CN1281621C - Method of diagnosing and treating heritage spasm paraplegia using NIPAl gene mutation hot point - Google Patents

Abstract

The present invention relates to a process for discovering a mutation hotspot in an NIPA1 gene and two new mutations in the mutation hotspot. The mutations in the gene can cause hereditary spastic paraplegia and can be simultaneously compared with NIPA1 homologous genes of mice, rats, globefishes and zebra fish by homoeology; transcripton and translation products of the gene are useful for detection and diagnosis of the hereditary spastic paraplegia, development of iatrotechnics of the hereditary spastic paraplegia, separation and production of protein of NIPA1 mutant gene codes, and construction of transgenic animal models for expressing the mutant gene.

Description

Utilize the method for the diagnosis of NIPA1 transgenation focus, treatment hereditary spastic paraplegia

Technical field

The present invention relates to biotechnology and medical field.More specifically, the present invention relates to utilize the method for people NIPA1 gene and coded product diagnosis and treatment hereditary spastic paraplegia, and contain NIPA1 gene and/or proteic pharmaceutical composition.

Background technology

Hereditary spastic paraplegia is the disease that is caused by the pathologic sudden change of individual gene, is one group of disease that genetic heterogeneity is very strong, and promptly this disease can be caused by heterogeneic different sudden changes.Therefore, looking for and screen this sick Disease-causing gene and mutation type thereof and be not only understanding, illustrate the pathogenetic necessary approach of this disease, also is simultaneously to set up the prerequisite of diagnosing before this sick gene diagnosis and the antenatal/plantation.After understanding this sick Disease-causing gene and mutation type thereof, just can set up gene diagnosis technology based on this gene and mutation type thereof, in the hope of suspicious clinically patient being carried out the diagnosis of conclusive gene level, can also be by the detection of amniotic fluid and bleeding of the umbilicus, judge whether fetus carries the sudden change of this gene, thereby whether can suffer from this disease after the birth of prediction fetus, and this information is offered pregnant woman and household thereof, thereby decision continues or termination of pregnancy.Owing to the sudden change of gene is this pathogenetic reason, in the long term, the gene therapy of correcting sudden change is used in the screening of sudden change, or other method at the biological effect of this sudden change (as the protein antagonist) is gone to treat this disease and is become possibility.Therefore, in the long term, this discovery has the application prospect of treatment aspect.Rainier S equals this month and has reported the T45R of NIPA1 gene (sudden change of 159C → G) can cause this disease (Rainier S, et al:NIPA1 gene mutations cause autosomal dominanthereditary spastic paraplegia (SPG6) .Am J Hum Genet, 73:967-971,2003).

Because this sick clinical diagnosis is not now had special methods, only depend on and get rid of other disease that can cause the same symptoms sign and carry out (referring to table 1), thus the diagnosis of gene level not only clear and definite the concrete position and the kind of sudden change, also have accurately, fast, low consumed characteristics.Therefore, this area presses for the effective ways of the new early diagnosis of exploitation, treatment hereditary spastic paraplegia.

Summary of the invention

The purpose of this invention is to provide morbidity or relevant polynucleotide and the albumen of onset risk with hereditary spastic paraplegia.

Another target of the present invention is to regulate the compound of these albumen or polynucleotide expression or function with these albumen and polynucleotide screening.

Further aim of the present invention is to regulate or albumen and the polynucleotide interaction relevant with described hereditary spastic paraplegia with these compounds, to treat and to diagnose this disease.

Further purpose of the present invention provides the method for controlling the diagnosing hereditary Spastic Paraplegia.

A first aspect of the present invention provides a kind of NIPA1 transgenation albumen, and described NIPA1 transgenation albumen has the aminoacid sequence shown in the SEQID NO:2, and wherein the 106th amino acids is an arginine.

A second aspect of the present invention provides a kind of isolating NIPA1 mutator gene, it is characterized in that its coding said mutation albumen.

Preferably, described NIPA1 mutator gene has the nucleotide sequence shown in the SEQ ID NO:1, wherein undergos mutation for the 341st, and its sudden change is selected from:

G→C：

G→A。

A third aspect of the present invention, a kind of method that the hereditary spastic paraplegia of individuality is diagnosed is provided, described method comprises step: detect this individual NIPA1 gene, transcript and/or albumen, and compare with normal N IPA1 gene, transcript and/or albumen, just there are differences and show that this individuality suffers from the possibility of hereditary spastic paraplegia and be higher than normal population.Preferably, detection be gene or the transcript of NIPA1, and with normal NIPA1 nucleotide sequence (SEQ ID NO:1) comparing difference.Preferable, described difference is selected from:

The 341st G → C among the SEQ ID NO:1;

The 341st G → A among the SEQ ID NO:1;

The 106th glycine becomes arginine among the SEQ ID NO:2.

A fourth aspect of the present invention provides a kind of compound, and described compound suppresses the proteic activation of above-mentioned NIPA1 transgenation or suppresses the expression of above-mentioned NIPA1 mutator gene.Preferable, described compound is the antisense nucleotide of NIPA1 mutator gene.

A fifth aspect of the present invention provides a kind of NIPA1 transgenation proteic purposes, and described NIPA1 transgenation albumen is used to prepare the composition of treatment or diagnosing hereditary Spastic Paraplegia.

A sixth aspect of the present invention provides a kind of purposes of NIPA1 mutator gene, described NIPA1 mutator gene to be used to prepare the composition of treatment or diagnosing hereditary Spastic Paraplegia.

A seventh aspect of the present invention provides a kind of test kit that detects hereditary spastic paraplegia, and described test kit comprises the primer of specific amplification NIPA1 gene or transcript.Preferable described test kit also contains the reagent that is selected from down group:

(a) with mutable site bonded probe;

(b) restriction enzyme in identification mutational site.

Be selected from the sudden change described in the preference:

The 341st G → C among the SEQ ID NO:1;

The 341st G → A among the SEQ ID NO:1;

The 106th glycine becomes arginine among the SEQ ID NO:2.

The present invention has also comprised the compound of using described albumen and polynucleotide screening treatment and/or diagnosing hereditary Spastic Paraplegia, and these compounds can be used as the activator or the antagonist of NIPA1 genetic expression or function.Both as the target of SCREENED COMPOUND, for synthetic derivative compound provides raw material, the latter can be as the activator or the antagonist of NIPA1 genetic expression or function simultaneously for these albumen and polynucleotide.

Especially, the present invention also comprises the unconventionality expression level at NIPA1 gene or gene product, the sudden change of gene even the detection method of aberrant gene or gene product.Above-mentioned coming across unusually has hereditary spastic paraplegia or has in people's the cell or tissue of hereditary spastic paraplegia onset risk, perhaps in the cell of suffering from this disease or animal model.

Thereby the present invention is used for by detecting the onset risk that NIPA1 gene or gene product instruct screening patients with hereditary spastic paraplegia or hereditary spastic paraplegia.The detection of unconventionality expression level, transgenation or other unusual phenomenon allowed the risk of patients with hereditary spastic paraplegia or hereditary spastic paraplegia morbidity is diagnosed.In one embodiment, the detection of passing through abnormal level, transgenation or other unusual phenomenon of NIPA1 gene has been indicated in this invention.

Therefore, the present invention also comprises suffering from hereditary spastic paraplegia, or the people's of hereditary spastic paraplegia onset risk methods of treatment arranged, present method is with unconventionality expression level, sudden change or other the unusual phenomenon of NIPA1 gene or gene product index or the target as treatment.

The present invention has also comprised with NIPA1 gene or gene product and regulates the factor of expression level as index or target screening or effectively reverse sudden change in NIPA1 gene or gene product or the method for other off-notes.Therefore, the present invention includes screening NIPA1 gene or the activator of gene product and the method for antagonist.These factors can be used for diagnosis or treatment hereditary spastic paraplegia to the expression level of NIPA1 gene or gene product or the influence of function according to them.The expression of NIPA1 gene or gene product or the factor of function be can regulate by differentiating, just expression level by regulating NIPA1 gene or gene product or hereditary spastic paraplegia pathogenic process that function influences a people or the method for occurring degree obtained.Further, by obtaining the factor that these genetic expressions are regulated, just formed the method for the result of treatment of assessment pair cell and animal model.

Can interact by screening, perhaps allow the factor that unconventionality expression or function to NIPA1 gene or gene product detect, so just form the morbidity of diagnosing hereditary Spastic Paraplegia or the method for onset risk.

The present invention has further comprised the composition that obtains on NIPA1 gene or gene product basis, they are all very useful for the expression or the function that detect or regulate NIPA1 gene or gene product.So these compositions are very useful for diagnosis and/or treatment hereditary spastic paraplegia.

The present invention has also comprised cell and the animal model according to NIPA1 gene in the model or gene product research hereditary spastic paraplegia.

Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.

Description of drawings

Fig. 1. family 1 D15S1021～D15S1002 mark monoploid analytical results.

Black pattern symbolic representation patient, the rectangular representative pathology of black monoploid.The box indicating male sex, circle is represented the women.Reorganization has appearred in IV-7, V-1 and V-7 among the kinsfolk.

Fig. 2. family 2 pedigrees.

Distance between 2 linkage analysis Lod of Fig. 3 .D15S1021～D15S1002 Scores value and polymorphism mark (family 1).

The cDNA sequence of Fig. 4 .NIPA1.

The sudden change that the contriver finds is the sudden change of the 341st alkali base in this sequence, and the alkali base of this position has added frame)

Fig. 5: patient's forward sequencing result in the family 1 (arrow is depicted as the mutational site, G → C).

Fig. 6: patient's backward sequencing result in the family 1 (arrow is depicted as the mutational site, C → G).

Fig. 7: normal people's forward sequencing result (arrow is depicted as the place, mutational site, but does not undergo mutation, and is G) among normal people and the crowd in the family 1.

Fig. 8: normal people's backward sequencing result (arrow is depicted as the place, mutational site, but does not undergo mutation, and is C) among normal people and the crowd in the family 1.

Fig. 9: patient's forward sequencing result in the family 2 (arrow is depicted as the mutational site, G → A).

Figure 10: patient's backward sequencing result in the family 2 (arrow is depicted as the mutational site, C → T).

Figure 11: the sequence alignment result of human NIPA1 gene and mouse nipal gene.

Grayish alkali basis representation sudden change place codon, what add a horizontal line down is the place, mutational site.The result shows that this codon is identical in mouse and people.

Figure 12: the sequence alignment result of human NIPA1 gene and rat nipal gene.

Grayish alkali basis representation sudden change place codon, what add a horizontal line down is the place, mutational site.The result shows that this codon is identical in rat and people.

Figure 13: the sequence alignment result of human NIPA1 gene and zebra fish (zebrafish) gene.

Grayish alkali basis representation sudden change place codon, what add a horizontal line down is the place, mutational site.The result shows that this codon is identical in zebra fish and people.

Figure 14: the comparison result of the amino acid of human NIPA1 genes encoding and mouse, filefish aminoacid sequence.

Place, box indicating mutational site amino acid.Comparison result shows is at mouse, filefish (Fugul), philtrum, and this site all is glycine (Glysine).

The biological structure prognostic chart of Figure 15: NIPA1.

329 in the total amino acid of the proteic aminoacid sequence of NIPA1, the contriver is divided into four sections arrangements to it in this figure:

1. the first line number word of each section is the amino acid counting of this Argine Monohydrochloride sequence.

2. second of each section row is this proteic aminoacid sequence.

3. the M of the third line of each section represents that this place may be a cross-cell membrane functional domain (transmembranemotif), and the whole piece sequence has 8 possible cross-cell membrane functional domains.

4. the O of the fourth line of each section, M, i represent the possible film outside part (outsiceregion) of this aminoacid sequence respectively, stride part (inside region) in membrane portions (membrane helix) and the film.

The new mutant that the contriver finds is positioned at the 106th of this aminoacid sequence, is the change from glycine (G) to arginine (R), underlines in the drawings.

Embodiment

NIPA1 mutain of the present invention is meant the albumen with the aminoacid sequence shown in the SEQ ID NO:2, and wherein the 106th amino acids is an arginine.NIPA1 mutator gene coding NIPA1 mutain of the present invention.It has the nucleotide sequence shown in the SEQ ID NO:1, wherein undergos mutation for the 341st.Preferable, said mutation is selected from:

G→C：

G→A。

People NIPA1 Nucleotide full length sequence of the present invention (NIPA1 mutator gene) or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be according to the relevant nucleotide sequence of NIPA1, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This is normally with its human cloning carrier; Change cell again over to; Separate from the host cell after the propagation by ordinary method then and obtain relevant sequence.

In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually by earlier synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

NIPA1 encoding sequence of the present invention is inserted suitable expression vector, change host cell again over to, just can isolate the NIPA1 mutain.

The present invention also comprises NIPA1 mutator gene of the present invention or the albumen of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people NIPA1 mutator gene product or fragment.Preferably, refer to that those can combine with people NIPA1 mutator gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.

This type of monoclonal antibody can utilize hybridoma technology to prepare.Antibody of the present invention comprises the antibody that can block people NIPA1 mutain function and the antibody that does not influence people NIPA1 protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people NIPA1 mutator gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize the albumen synthesizer synthetic.

The present invention includes NIPA1 mutain and antibody thereof, inhibitor, agonist, antagonist etc.When in treatment, using (administration), can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intravenously, subcutaneous or topical (comprising affected area).Be preferably topical.

The present invention also provides a kind of pharmaceutical composition, and it contains antagonist and the pharmaceutically acceptable carrier or the vehicle of the NIPA1 mutain of the present invention of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.When making pharmaceutical composition, be that antagonist antagonist with the NIPA1 mutain of safe and effective amount is applied to Mammals, wherein the safety significant quantity is usually at least about 0.1 microgram/kg body weight, and in most of the cases be no more than about 10 mg/kg body weight, preferably this dosage is about 0.1 microgram/kg body weight one about 100 micrograms/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization people NIPA1 mutain level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.

The method that whether has the NIPA1 mutain in a kind of test sample is to utilize the proteic specific antibody of NIPA1 to detect, and it comprises: sample is contacted with the NIPA1 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample NIPA1 mutain.

The proteic polynucleotide of NIPA1 can be used for the diagnosis and the treatment of NIPA1 protein related diseases.Aspect diagnosis, the proteic polynucleotide of NIPA1 can be used for detecting the proteic expression of NIPA1 NIPA1 abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of NIPA1 as the NIPA1 dna sequence dna.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA one polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of NIPA1 albumen and also can detect the proteic transcription product of NIPA1.

The invention provides the whether unusual method of a kind of people of detection NIPA1 genetic expression; It comprises step: (a) determine the 341st Nucleotide in sequence shown in the SEQ ID of the people NIPA1 gene NO:1; And b) detects the missense mutation that whether has alkali base G in described position.

The sudden change that detects the NIPA1 gene also can be used for the diagnosing hereditary Spastic Paraplegia.Detection can be at DNA, also can be at genomic dna.The form of NIPA1 protein mutation comprises that the point mutation compared with normal wild type NIPA1 dna sequence dna, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.

The contriver has collected two hereditary spastic paraplegia patients' big family, and has used the karyomit(e) sections that full genome scanning, linkage analysis technology are positioned Disease-causing gene 15q11.1-q12 first through extensive and deep research.The contriver has carried out the detection of NIPA1 transgenation in a big family (family 1) that contains 3 generations, 14 these patients (still survive and algebraically and patient's number of blood sample are arranged), and the method by order-checking, found the sudden change of the 106th codon G → C (G106R) that this is at first screened by the contriver in the world.Simultaneously, the contriver also contains (family 2) in 3 generations, 7 patients' (still survive and the numeral of blood sample is arranged) the family at another and has found to be positioned at G → A of same alkali base and change.These two kinds of changes are G106R at amino acid levels.This makes the neutral hydrophobicity glycine (Glycine) in the peptide chain become alkaline hydrophilic arginine (Arginine).Because mutating acid is arranged in the functional group that the coding peptide chain is striden the film district, it is unusual that this change makes that the function of NIPA1 coded by said gene peptide chain takes place, thereby cause this disease.The conservative property of sudden change alkali base G in vertebrates also proved the importance of this alkali base on function.Because the sudden change of NIPA1 gene betides same alkali base site in the family of these 2 consanguinity-less relations, and the alkali base difference after the sudden change, show that this Nucleotide is the mutantional hotspot of Ben Jiyin.On this basis, the contriver has finished the present invention.

The present invention has found an intragenic mutantional hotspot of NIPA1 and has been positioned at 2 new mutants of this focus.This discovery can be applicable to hereditary spastic paraplegia diagnosis, the treatment in; And confirmed that further the NIPA1 gene is the disease gene that causes hereditary spastic paraplegia.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only are used to illustrate the present invention, limit the scope of the invention and be not used in.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1 family is collected, full genome scanning and linkage analysis

1. family is collected

By patient (McDermott CJ, White k, the et al:Hereditaryspastic paraparesis:a review of new developments.J Neurol Neurosurg Psychiary2000 in the improvement Harding standard screening family; 69:150-160).Two family patients' phenotype all is simple form, symptoms (family 1 phenotype sees Table 1, and pedigree is seen Fig. 1, and family 2 pedigrees are seen Fig. 2) such as no dysnoesia, cerebellum symptom, deafness, eye.Two familys provide by the one attached Song Chun doctor of institute of Harbin Medical University.After all family member's informed consents, get peripheral blood in the EDTA pipe.

Case	Sex	Age	Age of onset	Paraplegia	Gait changes	Quadriceps reflex is hyperfunction	Hypermyotonia	Hot-tempered spasm	Babinski sign	chadolock
											III-2	The woman	72	25	+	+	+++	+++	+	+	+
III-5	The woman	63	16	+	+	+++	+++	+	+	+
											IV-2	The woman	56	30	-	+	++	++	+	+	+
IV-4	The woman	51	35	-	+	++	++	+	+	+
											IV-6	The woman	37	30	-	+	++	++	+	+	+
IV-9	The woman	52	24	+	+	+++	+++	+	+	+
											IV-14	The man	44	25	-	+	++	++	+	+	+
V-1	The man	33	15	-	+	+	++	+	+	+
											V-2	The woman	31	25	-	+	+	++	+	+	+
V-3	The woman	31	30	-	+	++	+	-	+	+
											V-4	The man	28	25	-	+	++	++	-	+	+
V-5	The man	26	20	-	+	+	+	+	+	+
											V-6	The man	26	13	-	-	+	+	-	+	+
V-9	The man	26	20	-	+	++	+	-	+	+

The individual disease phenotype of getting involved in table 1 family

2.DNA extract

The QIAamp DNA Blood Maxi Kits that produces with Qiagen company extracts DNA from anticoagulation cirumferential blood, TE dissolving ,-70 ℃ of preservations.

3. full genome scanning

1. polymorphism mark primer sequence: be 400 little satellite repeat sequence primers of the polymorphism dinucleotide of from Genethon human inheritance collection of illustrative plates, selecting, fluorescent mark.Every pair of marker spacing is about 10cM, and ordering and the distance of the used polymorphism mark of linkage analysis on karyomit(e) comes from Http:// www.marshfieldclinic.org/The website data.

2. PCR reaction conditions: 5-μ l reaction system contains 50ng of DNA, 2.5mM MgCl ₂, 10mM Tris-HCl pH8.3,50mM KCl, 250mM each dNTP, 0.625pmol primer and the 0.25 Hotstart Taq of unit archaeal dna polymerase (QIANGEN GmbH, Hilden, Germany).Perkin-Elmer 9700 thermal cyclers are adopted in reaction.Employing standard Touchdown program: 95 ℃ of pre-sex change 12 minutes: Touchdown circulation 14 times, 95 ℃ of sex change 30 seconds, annealing temperature 1 minute, by 63 ℃ initial, each circulation is later on successively decreased 0.5 ℃, up to 56 ℃, 72 ℃ were extended 1 minute and 30 seconds; Keeping annealing temperature afterwards is 56 ℃, 1 minute, carries out 30 circulations again; Final product extended 10 minutes for 72 ℃.

3. genotype screening: the PCR product carries out genotype screening on ABI377 or 3100 sequenators, with GeneScan, and Genotyper software analysis result.

4. linkage analysis

2 linkage analysises: adopt LINKAGE software (Version 5.10), suppose that the Disease-causing gene frequency is 10 ^-4, the gene frequency of polymorphism mark equates that men and women's recombination fraction equates, penetrance 0.9.2 linkage analysis results obtain maximum Led Score value (Zmax): 4.55, and recombination fraction (e) is 0.The Led Score value of adjacent several polymorphism marks also greater than 3.0 (table 2, Fig. 3).

Marker polymorphism mark	LOD SCORE	Zmax at	θ＝
				θ＝0.00 0.01 0.05 0.10 0.20 0.30 0.40
	D15S1021 D15S128 D15S646 D15S210 D15S122 D15S986 D15S97 D15S822 D15S975 D15S156 D15S1002				4.55 4.51 4.27 3.89 2.96 1.89 0.74 2.92 2.87 2.67 2.41 1.83 1.20 0.55 2.48 2.45 2.32 2.13 1.69 1.19 0.63 4.55 4.51 4.27 3.89 2.96 1.89 0.74 4.34 4.32 4.15 3.82 2.97 1.94 0.81 4.04 3.98 3.71 3.36 2.60 1.75 0.82 3.42 3.43 3.36 3.18 2.63 1.91 1.02 -1.02 1.08 1.66 1.74 1.48 1.0 0.45 -2.59 -0.31 0.29 0.47 0.51 0.40 0.22 0.04 0.17 0.41 0.52 0.53 0.39 0.19 -6.72 -2.20 -0.65 -0.05 0.36 0.37 0.22	4.55 2.92 2.48 4.55 4.34 4.04 3.43 1.74 0.51 0.53 0.37	0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.10 0.20 0.20 0.30

Table 2 D15S1021～2 linkage analysis results of D15S1002 polymorphism mark

5. Fine Mapping and monoploid make up

Behind the positive chain site of preliminary acquisition, according to Genome Datebase and Genethon, database datas such as Whitehead InstitutePhysicai Map are done the intensive scanning of trying one's best with all known polymorphism marks in this sections, and method is the same.From making up, monoploid seeks possible reorganization (cross-over) simultaneously, in the hope of shortening the zone at candidate gene place to greatest extent.Monoploid makes up ordering and the distance of polymorphism mark on karyomit(e) and also comes from Http:// www.marshfieldclinic.org/The website data.Family 1 is still survived and had the monoploid of 3 generations of blood sample, 34 individualities analyze to find, reorganization between homologous chromosomes appears respectively in IV-7, V-1 and V-7 individuality, but do not find reorganization in the kinetochore direction, therefore, the crucial sections of Disease-causing gene is between No. 15 karyomit(e) kinetochores and D15S97 in about 9.85cM zone (Fig. 1).

Embodiment 2 NIPA1 detection in Gene Mutation

Its mRNA complete cDNA sequence from GeneBank (accession number BK001020) (Fig. 4) for NIPA1 gene (Homo sapiens non-imprinted in Prader-Willi/Angleman syndrom 1).The genomic dna sequence dna from Http:// genome.ucsc.edu

Key step:

1.PCR amplification: the PCR reaction volume is 50 μ l, includes 50mmol/L Tris-HCl (pH8.8), 16.6mmol/L (NH ₄) ₂SO ₄, 0.10mg/mlBSA, 3.0mmol/L MgCl ₂, 200 μ mol/L dNTPs, each 0.2 μ mol/L of primer P1, P2, Taq enzyme 2U (MBI company), about 0.1～0.7 μ g of human gene group DNA.PCR reaction conditions: 94 ℃ of 3min:94 ℃ of 45s, 60 ℃ of 1min, 72 ℃ of 1min, 35 circulations; Last 72 ℃ are extended 10min.The sequence of primer P1 (SEQ ID NO:3) is: 5 '-GTG CTG CGC CCA TTT CAG TCA-3 ', the sequence of P2 (SEQ ID NO:4) is: 5 '-GTG CCA TCT CAACTC ACT GCA-3 '.

2. sequencing reaction scheme:

(1) pre-reaction: reaction volume is totally 3 μ l, includes PCR product 5-10ng (about 0.5-1.0 μ l), and shrimp alkaline phosphotase (SAP), each 0.25 μ l (USB company) of excision enzyme I (Exo I) mend ultrapure water to 3 μ l.Reaction mixture reacts on the PCR instrument, and 37 ℃ of temperature are bathed 15min, 85 ℃ of 15min inactivators then.Reacting the afterreaction mixture that finishes directly uses as the template of sequencing reaction.

(2) sequencing reaction: 96 orifice plates with ABI company, carry out on GeneAmp 9700 PCR instrument.Reaction volume is totally 10 μ l, includes the pre-reaction product of 3 μ l, 5 * buffer of 2 μ l, the Big-Dye of 1 μ l (ABI), 1 μ l primer (3.2pmol/ μ l), the ultrapure water of 3 μ l.Reaction conditions: 98 ℃ of 2min:96 ℃ of 10s, 50 ℃ of 5s, 60 ℃ of 4min, 30 circulations; Last 4 ℃ of preservations.

(3) order-checking purifying: directly on 96 orifice plates, carry out.

A) after sequencing reaction finished, each sample added 90 μ l75% ethanol, sticks and seals aluminium foil, the whirlpool mixing;

B) room temperature lucifuge 20min;

C) 20 ℃, the centrifugal 30min of 3500rpm (carrying out on the 5810R whizzer);

D) remove supernatant, tip upside down on four layers of thieving paper the centrifugal 30s of 350rpm;

E) room temperature lucifuge 20min is to dry ethanol;

F) every sample adds 10 μ l ultrapure waters, and last ABI3100 sequenator checks order.

3. sequencing result analysis: analyze with the ABI sequence analysis software

The contriver has found the sudden change of the 341st alkali base (cDNA sequence) G → C in family 1, found the sudden change of same site G → A in family 2.2 sudden changes all are arranged in NIPA1 gene the 3rd exon, and these 2 kinds of changes all cause the 106th amino acid to become alkaline hydrophilic arginine by the hydrophobic glycine of neutrality.So this sudden change is G341C (family 1) or G341A (family 2) on the cDNA level; In total length genomic sequence is that G25596C (family 1) or G25596A (family 2) change; Being G106R in amino acid levels two familys changes.

The contriver is to the patient of totally 14 blood samples of still surviving and adopted and 37 non-patients have all carried out forward and reverse order-checking in the family 1.All patients all contain G → C in this mutantional hotspot (G341C, G106R) (Fig. 5 Fig. 6) changes, and this sudden change and disease phenotype be divided into from.And all non-patients all do not contain this sudden change (Fig. 7,8) in the family.

The contriver is also to still surviving in the family 2 and having 7 patients of blood sample and the normal people of 13 blood samples of still surviving and have to carry out two-way order-checking.All patients all contain G in the mutantional hotspot → A change (G341A, G106R) (Fig. 9, Figure 10) and the normal people does not have this change (chart saves).

The contriver also checks order to 100 normal peoples with this family consanguinity-less relation, does not also contain this sudden change (Fig. 7,8) entirely.

Embodiment 3 strides the comparison of kind homology

1. the homology of the nucleotide sequence of human NIPA1 and mouse, rat comparison is undertaken by the Comparative Sequence among the http://genome.ucsu.edu/ of website.

2. the sequence analysis of the amino acid sequence of human NIPA1 and filefish, mouse is with reference to the people's such as Chai JH article " Identification of four highly conserved genes between breakpoint hotspots BP1 andBP2 of the Prader-Willi/Angleman syndromes deletion region that have undergoneevolutionary transposition mediated by flanking duplicons " Am J Hum Genet 2003; The comparison result of 73:898-925.

3. the homology of the aminoacid sequence of the sequence of human NIPA1, NIPA1 and zebra fish comparison utilizes software clustalx to carry out, and corresponding Nucleotide of zebra fish and aminoacid sequence derive from the website Http:// www.ensembl.org

The result is presented in the animal of all comparisons and is equivalent to the 341st alkali base of human NIPA1 gene cDNA, and promptly first alkali base of No. 106 codon is G, and the coded amino acid of codon is glycine (Figure 11-14) entirely.The high conservative of this alkali base in organic evolution illustrated that it has important biological function in the coding peptide chain.

Embodiment 4 NIPA1 protein secondary structure predictions

Utilize website http://www.embl-heidelberg.de/predictprotein/predictprotein.htm l to carry out, and examine with software TMHMM2.0.The aminoacid sequence of corresponding N IPA1 obtains from website http://www.ncbi.nlm.nih.gov.

The glycine that studies show that the 106th codon (GGG) coding of the biological structure of NIPA1 genes encoding peptide chain is arranged in the 2nd and strides film district functional group, is arranged in alpha-helix, so have important biological function (seeing Figure 15).And the glycine (GGG) that sudden change is caused is to arginine (CGG, family 1; AGG family 2)) change has obviously changed the normal physiological function of this functional group, thereby causes clinically symptom, sign.

The preparation of embodiment 5 hereditary spastic paraplegia detection kit

Prepare a test kit, it contains:

Title sequence (5 ' → 3 ') numbering concentration

Forward primer GTG CTG CGC CCA TTT CAG TCA SEQ ID NO:3 dry powder 2 OD

Reverse primer GTG CCA TCT CAA CTC ACT GCA SEQ ID NO:4 dry powder 2 OD

The PCR reaction solution contains Taq enzyme, dNTP, magnesium ion, PCR reaction buffer

PCR product purification box contains the solution of PCR product purification in a small amount, DNA adsorption column Labell

Sequencing reaction liquid contains Big Dye

Extract patients'blood 3ml to be detected, use ordinary method (or using specific test kit) from blood, to extract DNA.PCR primer in the hereditary spastic paraplegia detection kit is diluted to 0.2 μ mol/L, is that template is carried out the PCR reaction with the primer that is provided with the DNA that is extracted.Use the PRC product purification box that detection kit provided that the PCR product is carried out purifying.The product of purifying is carried out directly checking order behind the sequencing reaction.Whether the resulting sequence of observation order-checking has missense mutation.

Embodiment 6 preparation of drug combination

The antagonist of NIPA1 mutain and common ointment material are pressed specific mixed, make the ointment of the antagonist that contains the NIPA1 mutain.During use, ointment is imposed on the affected part, the affected part coded mutain of NIPA1 mutator gene is suppressed, and makes patient owing to the hereditary spastic paraplegia that the coded normal protein of shortage NIPA1 causes is alleviated, until last disappearance.Or the mutain that the NIPA1 mutator gene is coded makes injection, during use subcutaneous injection carried out in the affected part, directly suppresses the NIPA1 mutain of affected area, patient's hereditary spastic paraplegia symptom improved, thereby reach the purpose of treatment.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table

＜110〉Zhongshan University

＜120〉utilize NIPA1 transgenation focus to diagnose, treat the method for hereditary spastic paraplegia

<130>031112

<160>4

<170>PatentIn version 3.1

<210>1

<211>6565

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>CDS

<222>(26)..(1015)

<223>

<400>1

ggctcggagg gcgggcgcgg gcgga atg ggg act gca gct gcg gca gcg gcg 52

Met Gly Thr Ala Ala Ala Ala Ala Ala

1 5

gcg gcg gcg gcg gcg gcg gcc ggg gag ggg gcg cgt agc ccg agc ccc 100

Ala Ala Ala Ala Ala Ala Ala Gly Glu Gly Ala Arg Ser Pro Ser Pro

10 15 20 25

gcc gcc gtg tcg ctc ggc ctg ggc gtg gcc gtc gtg tcg agc ctg gtg 148

Ala Ala Val Ser Leu Gly Leu Gly Val Ala Val Val Ser Ser Leu Val

30 35 40

aac ggg tcc acg ttc gtg cta cag aag aag ggc atc gtg cgt gcc aag 196

Asn Gly Ser Thr Phe Val Leu Gln Lys Lys Gly Ile Val Arg Ala Lys

45 50 55

cgg cga ggt act tcc tat tta aca gac att gtg tgg tgg gct ggc aca 244

Arg Arg Gly Thr Ser Tyr Leu Thr Asp Ile Val Trp Trp Ala Gly Thr

60 65 70

atc gca atg gct gtt ggc cag att gga aac ttc ctg gct tac acg gcg 292

Ile Ala Met Ala Val Gly Gln Ile Gly Asn Phe Leu Ala Tyr Thr Ala

75 80 85

gtc ccc acg gtc ctg gta acc ccc ctg ggc gcc ctt gga gta ccg ttc 340

Val Pro Thr Val Leu Val Thr Pro Leu Gly Ala Leu Gly Val Pro Phe

90 95 100 105

ggg tcc att tta gct tcc tat ctc ctg aag gaa aag ctc aac atc ttg 388

Gly Ser Ile Leu Ala Ser Tyr Leu Leu Lys Glu Lys Leu Asn Ile Leu

110 115 120

ggc aag ttg ggg tgc ctg cta agc tgt gca ggc tcc gtc gtg ctg att 436

Gly Lys Leu Gly Cys Leu Leu Ser Cys Ala Gly Ser Val Val Leu Ile

l25 130 135

atc cac tcc cca aag tct gag agt gtg acg act cag gct gag ctg gag 484

Ile His Ser Pro Lys Ser Glu Ser Val Thr Thr Gln Ala Glu Leu Glu

140 145 150

gaa aag ctg acc aac cca gtg ttt gtg ggc tac ctg tgc atc gtg ctg 532

Glu Lys Leu Thr Asn Pro Val Phe Val Gly Tyr Leu Cys Ile Val Leu

155 160 165

ctc atg ctg ctg ctg ctc atc ttc tgg atc gcg ccg gcc cat ggg ccc 580

Leu Met Leu Leu Leu Leu Ile Phe Trp Ile Ala Pro Ala His Gly Pro

170 175 180 185

acc aac atc atg gtc tac atc agc atc tgc tcc ttg ctg ggc agt ttc 628

Thr Asn Ile Met Val Tyr Ile Ser Ile Cys Ser Leu Leu Gly Ser Phe

190 195 200

acc gtg cct tcc acc aag ggc atc ggg ctg gcg gcc caa gac atc ttg 676

Thr Val Pro Ser Thr Lys Gly Ile Gly Leu Ala Ala Gln Asp Ile Leu

205 210 215

cat aac aac ccg tcc agt cag aga gcc ctc tgc ctg tgc ctg gta ctc 724

His Asn Asn Pro Ser Ser Gln Arg Ala Leu Cys Leu Cys Leu Val Leu

220 225 230

ctg gcc gtg ctc ggc tgc agc atc atc gtc cag ttc agg tac atc aac 772

Leu Ala Val Leu Gly Cys Ser Ile Ile Val Gln Phe Arg Tyr Ile Asn

235 240 245

aag gcg ctg gag tgc ttc gac tcc tcg gtg ttc ggg gcc atc tac tac 820

Lys Ala Leu Glu Cys Phe Asp Ser Ser Val Phe Gly Ala Ile Tyr Tyr

250 255 260 265

gtc gtg ttt acc acg ctg gtc ctg ctg gcc tca gcc atc ctc ttc cgg 868

Val Val Phe Thr Thr Leu Val Leu Leu Ala Ser Ala Ile Leu Phe Arg

270 275 280

gag tgg agc aac gtg ggc ctg gtg gac ttc ttg ggg atg gcc tgt gga 916

Glu Trp Ser Asn Val Gly Leu Val Asp Phe Leu Gly Met Ala Cys Gly

285 290 295

ttc acg acc gtc tcc gtg ggg att gtc ctt ata cag gtg ttc aaa gag 964

Phe Thr Thr Val Ser Val Gly Ile Val Leu Ile Gln Val Phe Lys Glu

300 305 310

ttc aat ttc aac ctt ggg gag atg aac aaa tct aat atg aaa aca gac 1012

Phe Asn Phe Asn Leu Gly Glu Met Asn Lys Ser Asn Met Lys Thr Asp

315 320 325

tagattgcaa taggagcttg gatggttcga ggaataggca ttggaggtgg tttctggccg 1072

tgattggatg tgaagtagaa gaggtcctcg atcatggtgt tagaattgac tggatagtaa 1132

caggtggtct ggtggatagc ggggagcatg gctcagcacc agagcagagg cccagccagc 1192

cctctgcagc ccaaacgtcc ccaacggttg cctggcacca tctctctctg atgagacgaa 1252

tctcattttc atttccatta acctggaagc tttcatgaat attctcttct tttaaaacat 1312

tttaacatta tttaaacaga aaaagatggg ctctttctgg ttagttgtta catgatagca 1372

gagatatttt tacttagatt actttgggaa tgagagattg ttgtcttgaa ctctggcact 1432

gtacagtgaa tgtgtctgta gttgtgttag tttgcattaa gcatgtataa cattcaagta 1492

tgtcatccaa ataagaggca tatacattga attgttttta atcctctgac aagttgactc 1552

ttcgaccccc acccccaccc aagacatttt aatagtaaat agagagagag agaagagtta 1612

atgaacatga ggtagtgttc cactggcagg atgacttttc aatagctcaa atcaatttca 1672

gtgcctttat cacttgaatt attaacttaa tttgactctt aatgtgtata tgttcttaga 1732

ttagaataat gcaacttcga gtatgcttta atatttcaat attcaagtta caaatgtata 1792

aggcagttag aaataataca gtcacatgtc acttaatgat agggaaacat tctgagaaat 1852

gcattgtaag gtgactttat tgtgtgaaca tcatggagtg cacttataca aacctagatg 1912

ggacacctat gacccaccca ggccagatgg tacagcctgt tgctcctggg ccacacacct 1972

gtacagcatg tgaccgcact gaataccgca ggcaattgta acacagtggt gagtatttgt 2032

gtttacaaac ataggaaagg tacagtaaaa ctatggtatt acaatgttat gggaccaccg 2092

tcatgtaagt ggtatgtctt tgacagaaac atggttacgt ggttcatgac tgtatattca 2152

ctggaagata gtcaagacta aagacacatt agagcaaatt gaccccttta acatgtgatt 2212

attgtccaat taaagacagt tgatttaagt agcatgaggt attattttat ttgtattcga 2272

tctgtgttac ctgggatcca gtatcaaata tatccacatt ctttatcagc aagcattcat 2332

ggccattcag aagaaataaa ttaggtaact tgataataag gctaagtggg agagtacctg 2392

ttcaatagct catatatcga gtaccctgtc atacaggaac aagttaaagg acacaattga 2452

ggttaggcta gcttctacaa attgcaatat gcagtttttg aaagattttc taacaaaaag 2512

ccaataaatg tagccatctc cttgttgttt gcaatggcag agcatcctag agttcctcag 2572

ctaacctctc attatgtgtc ttaaatgcaa aagagccatt aattatgcca gtatttgaat 2632

caaagaggtc attctctgtc tatagtgttc ccatccatgt gttccaaatg ggagcatagc 2692

atgaagtgat gcacatattt caccacggta tcatgtactt catggccagt gttttatctc 2752

agcagggaac tacgccaagt tgaaagatgg ggttgggtaa agtagattag gtgaagtaga 2812

acataaaatt gaatagtacc caattaaagt tcctcagtaa gaaaaaaaat gtgtttttgt 2872

aggcaaaaag aacatttcta aagtctcaag gaatagcttc ctaaagtgtt gagtaaagag 2932

gctaaataaa atgagactag tttaatatag agagaaaaat acctttatgg agtaaacgtg 2992

tacatgatga tcatgggttg tcagtgattt gtgaactgag agcagcaaca acattatttt 3052

ttaaaaatct taaatcctct caatggatgg ttaacaaatg ctcaaagtcc attactcttt 3112

ttattggctc ttgcaggttt tgtgttttat catcagtgct tttagaaatg caggccttaa 3172

cttactgaac tgaactttct gaaaacgtaa tgtagcagta tcaatatact tttgggcata 3232

aaaatagttt cctaggtaag gggtgtgaga tattcaaaga atacatgtgg ctaacaagtg 3292

taatgagaaa gttcatgtgt cacatgaaaa tgatcatgtt tgtgttgcta cagcttttgt 3352

gggaaattta gtttaaaggc agctcttggt gtaccttagt atattttaat ccacaattat 3412

accattgata ctgagaggtg atacccgatg atcttctcta taatattctt agagtaaaac 3472

aaaatctcaa aagtattaat agctcttcta cccttgaagg tgactggtcc tgggacagtt 3532

agaatctttc aggtttacct ctgttcagca gatacttcag taggatacat agcttttctt 3592

ccagtgaaac aaagttcata tcatccattg tttttcaagc acgtgacacc agcctcaaag 3652

taaatgacat gaccagtggt tgaacagtct aattttcaaa tttaatatag agcatataac 3712

ttctgatttg atagtattta ttttaaaaaa ttatgttttc atcattcatt tgaaaatgaa 3772

aaagccccaa agtgagaact ttgggggagg gcctagaaca tggatagatc tcttagtggt 3832

ctttccaaaa gtacatgtac ttgaaatatt ttcattatca tactattctt tgaaaaaaaa 3892

gatgcttact gtatacttgt tttcaagcat cctctaaaat caaaggtttt gatcacaata 3952

tgcagatttc tcttgataga tacttaaata ggctatttct ctcctcttct tgggcaatgc 4012

cttgttttct cctctgaata tttgcatttg aaaggattgc ttcctgttct gctcattgat 4072

caaaggtagg gccaattaag gattctaacc ctaacccagc accacaaagc ccccctggag 4132

catcttcccg gctggcagga ccatgccatc tctgtggaga aggtgctggg gagggaagtc 4192

cttccagtgc cacatggagt gaggccctgc ccatgctggg gactttgggg aggaatttgg 4252

tattctggtg gccttgctca gctctcattg agatcttttc ctatcagaat gttagtgaat 4312

atacttcgca gctctttgtt cagcaataag gaatattctt tcaattcctg ctcttcaagc 4372

caatttacta cacccagttg tctttccaga agttcatccc agcggtaata tgttggtgtt 4432

tgttcttctt tggatttcac atctgttttc tggtagaagt gagcactgtt cacttgtgca 4492

gtcgtcttat tttccttctt cctagatgac tcagctcttt gtaaatgttg tgctcaactt 4552

ctaggggcca gttctagact ttggagatgc agtgtctccc aggtgtgcac ggacacctgg 4612

tccgtggaaa caggtgtgat gggcacaggc tgctgccctt ctgtctggtc gggggattcc 4672

tcttcttcaa gctgctcagc taacccagaa gaggggagag agtactccgg tggttcccag 4732

agcccctccc gttgtgccgc ttcgacctga cacctgctcg atgctgactt aggcttcctg 4792

ccaccaagca ggaaactaga aagagaacat ttcagtgtaa ggtctgttcc cgacagcatg 4852

gattagcttc cgtgttctga agttgttctt ttcatggtgt ctgacaccga gggcgttgtt 4912

cgtccatcag gcgggattgg atggagtctt ggtgttttgc cttctcaggg accaaaaatg 4972

tatcattgac tccttaacag tgaccttcct cccaaggaca tatccgtgtt catttttcat 5032

aggttttact catattcata ggtagattct gttaatgtga gttggaaaga aaagaccaat 5092

ttgtacacca gtcacaccac aagacagttt atcatataaa atacctcaat tttttgtatt 5152

cctcatttcc acctcacaat tgtactggtg atgaatttta agggtctgtc ctttagctta 5212

taggtgatgt ttcacatctg gccagattct tatacctcca ttgtatactt gaaaaggttc 5272

agaattacag gaacagcagt gagaatttgg cccactacca cgactcattt gtttcattca 5332

cattcctcac gtgcaacaac ataattatat tttaagaaaa tgtaactttg ttacatcaaa 5392

atatgttgtc tagtaaaaag ttgatattca gtagaacaag gatcatgtaa ataaacatct 5452

atttcacatg tacccaaaag catttaaaaa gcagaatcca gggcccagag catgagccag 5512

ggaggaggat gtttttcttc ttttctctat ttttccctaa attgtgcaaa cataggtgag 5572

tctcttaacc tttctgtgcc tcagtttttc tacctctaaa ggggtgggat ggttcttcaa 5632

attgtttcta aaacaccggc actttcagca gtgttctggt ggcctgagat gagagcaccg 5692

tgttcagaag tgcctgggag tggcacagtg gaaactccgc ttgcacggac catggagtct 5752

gctcaggacc atgctgtagg acacacagcc tcatgcgctg agaaagcaaa ggaagtgctg 5812

ggtgtaaagt ttgcatgatt ccatgaagct ttagttttcc tttttttgtt ttaaaagaaa 5872

gggttttata tgttctattg taaaatatgg aaattaaaca gggacttcag aaagccgcac 5932

agaaagatca ccttccgatg gtgtgatgtg ctcctgacat tcggccgagg tctgtattct 5992

gaaaaagatt taatggcctg tgaaacacgt ggattctgtt gcactggatt tgtaataaat 6052

gacgctgaac ttcctgcttc caagcagctc aaccctgatg ctgaactgac accaggcgaa 6112

tgtcagggct cccaaaccac tagtgccaaa gggtcatgtt gaaaagttca gaatatttat 6172

ttgtcagaat ataataattg ccccccacct tagtattttt gcactttaca gaaatttaga 6232

tactgttttt cagtggcttg agcgttttgc cttttcaaag gataactatt attttcttga 6292

aaatggaata taatcatgag aggaagaaga tgtaaaaaat gtcaaatgtt gattggttgt 6352

gtaaaagttt tgtcatagac atgtattggg gagcttccaa ttagcataca tagacacatg 6412

tgtcagtggc caagacctgc ttatattttg ctttatagat gtagtcatag catgttgtta 6472

ttgcctcatg taaataaaaa ggctattaag ttttccagta atatttatta atctgtatgt 6532

gttttaaaat aaaataactt atttctagct gaa 6565

<210>2

<211>329

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>2

Met Gly Thr Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

1 5 10 15

Gly Glu Gly Ala Arg Ser Pro Ser Pro Ala Ala Val Ser Leu Gly Leu

20 25 30

Gly Val Ala Val Val Ser Ser Leu Val Asn Gly Ser Thr Phe Val Leu

35 40 45

Gln Lys Lys Gly Ile Val Arg Ala Lys Arg Arg Gly Thr Ser Tyr Leu

50 55 60

Thr Asp Ile Val Trp Trp Ala Gly Thr Ile Ala Met Ala Val Gly Gln

65 70 75 80

Ile Gly Asn Phe Leu Ala Tyr Thr Ala Val Pro Thr Val Leu Val Thr

85 90 95

Pro Leu Gly Ala Leu Gly Val Pro Phe Gly Ser Ile Leu Ala Ser Tyr

l00 105 110

Leu Leu Lys Glu Lys Leu Asn Ile Leu Gly Lys Leu Gly Cys Leu Leu

115 120 125

Ser Cys Ala Gly Ser Val Val Leu Ile Ile His Ser Pro Lys Ser Glu

130 135 140

Ser Val Thr Thr Gln Ala Glu Leu Glu Glu Lys Leu Thr Asn Pro Val

145 150 155 160

Phe Val Gly Tyr Leu Cys Ile Val Leu Leu Met Leu Leu Leu Leu Ile

165 170 175

Phe Trp IIe Ala Pro Ala His Gly Pro Thr Asn Ile Met Val Tyr Ile

180 185 l90

Ser Ile Cys Ser Leu Leu Gly Ser Phe Thr Val Pro Ser Thr Lys Gly

195 200 205

Ile Gly Leu Ala Ala Gln Asp Ile Leu His Asn Asn Pro Ser Ser Gln

2l0 215 220

Arg Ala Leu Cys Leu Cys Leu Val Leu Leu Ala Val Leu Gly Cys Ser

225 230 235 240

Ile Ile Val Gln Phe Arg Tyr Ile Asn Lys Ala Leu Glu Cys Phe Asp

245 250 255

Ser Ser Val Phe Gly Ala Ile Tyr Tyr Val Val Phe Thr Thr Leu Val

260 265 270

Leu Leu Ala Ser Ala Ile Leu Phe Arg Glu Trp Ser Asn Val Gly Leu

275 280 285

Val Asp Phe Leu Gly Met Ala Cys Gly Phe Thr Thr Val Ser Val Gly

290 295 300

Ile Val Leu Ile Gln Val Phe Lys Glu Phe Asn Phe Asn Leu Gly Glu

305 310 315 320

Met Asn Lys Ser Asn Met Lys Thr Asp

325

<210>3

<211>21

<212>DNA

＜213〉synthetic

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<221>misc_feature

<222>(1)..(21)

＜223〉primer P1

<400>3

gtgctgcgcc catttcagtca 21

<210>4

<211>21

<212>DNA

＜213〉synthetic

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<221>misc_feature

<222>(1)..(21)

＜223〉primer P2

<400>4

gtgccatctc aactcactgc a 21

Claims (8)

1. a NIPA1 transgenation albumen is characterized in that, the 106th amino acids sports arginine in the aminoacid sequence shown in the SEQ ID NO:2.

2. an isolating NIPA1 mutator gene is characterized in that, the described mutain of its coding claim 1.

3. NIPA1 mutator gene as claimed in claim 2 is characterized in that, undergos mutation for the 341st in the nucleotide sequence shown in the SEQ ID NO:1, and its sudden change is selected from:

G→C；

G→A。

4. method that detects individual NIPA1 transgenation, it comprises step: detect this individual NIPA1 gene, transcript and/or albumen, and compare with normal N IPA1 gene, transcript and/or albumen, it is characterized in that, described difference be among the SEQ ID NO:1 the 341st undergo mutation or SEQ ID NO:2 in the 106th undergo mutation.

5. method as claimed in claim 4 is characterized in that, detection be gene or the transcript of NIPA1, and with normal NIPA1 nucleotide sequence (SEQ ID NO:1) comparing difference.

6. method as claimed in claim 5 is characterized in that, described difference is selected from:

The 341st G → C among the SEQ ID NO:1;

The 341st G → A among the SEQ ID NO:1;

The 106th glycine becomes arginine among the SEQ ID NO:2.

7. a test kit that detects the NIPA1 transgenation is characterized in that, it comprises the primer of specific amplification NIPA1 gene or transcript, and described primer is:

Forward primer: SEQ ID NO:3;

Reverse primer: SEQ ID NO:4.

8. test kit as claimed in claim 7 is characterized in that, described sudden change is selected from:

The 341st G → C among the SEQ ID NO:1;

The 341st G → A among the SEQ ID NO:1;

The 106th glycine becomes arginine among the SEQ ID NO:2.

Images