CN1618814A - Method of diagnosing and treating heritage spasm paraplegia using NIPAl gene mutation hot point - Google Patents
Method of diagnosing and treating heritage spasm paraplegia using NIPAl gene mutation hot point Download PDFInfo
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Abstract
A mutational hotspot in gene NIPA1 and two novel mutational procedures in the hotspot are disclosed. Said mutation can lead to hereditary spastic paraplegia. The transcripton and translated product of said gene can be used for detecting and diagnosing said hereditary apastic paraplegia, developing its therapeutic technique, separating and preparing the protein coded by said gene, and configuring the transgenic animal model expressing said gene.
Description
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 (outsideregion) 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.
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 | The ankle 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 Lod Score value (Zmax): 4.55, and recombination fraction (θ) is 0.The Lod Score value of adjacent several polymorphism marks also greater than 3.0 (table 2, Fig. 3).
LOD?SCORE
Zmax?at?0＝
Marker?θ＝0.00????0.01????0.05????0.10???0.20????0.30????0.40
The polymorphism mark
D15S1021???4.55????4.51????4.27????3.89???2.96????1.89????0.74????????4.55???0.00
D15S128????2.92????2.87????2.67????2.41???1.83????1.20????0.55????????2.92???0.00
D15S646????2.48????2.45????2.32????2.13???1.69????1.19????0.63????????2.48???0.00
D15S210????4.55????4.51????4.27????3.89???2.96????1.89????0.74????????4.55???0.00
D15S122????4.34????4.32????4.15????3.82???2.97????1.94????0.81????????4.34???0.00
D15S986????4.04????3.98????3.71????3.36???2.60????1.75????0.82????????4.04???0.00
D15S97?????3.42????3.43????3.36????3.18???2.63????1.91????1.02????????3.43???0.01
D15S822???-1.02????1.08????1.66????1.74???1.48????1.0?????0.45????????1.74???0.10
D15S975???-2.59???-0.31????0.29????0.47???0.51????0.40????0.22????????0.51???0.20
D15S156????0.04????0.17????0.41????0.52???0.53????0.39????0.19????????0.53???0.20
D15S1002??-6.72???-2.20???-0.65???-0.05???0.36????0.37????0.22????????0.37???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 InstitutePhysical 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).
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 CAA CTC 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 l, 75% 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.
1. the website is passed through in the homology of the nucleotide sequence of human NIPA1 and mouse, rat comparison
Comparative Sequence among the http://genome.ucsu.edu/ carries out.
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.
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 Label1
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.
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
125?????????????????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
100?????????????????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?Ile?Ala?Pro?Ala?His?Gly?Pro?Thr?Asn?Ile?Met?Val?Tyr?Ile
180?????????????????185?????????????????190
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
210?????????????????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
<220>
<221>misc_feature
<222>(1)..(21)
＜223〉primer P1
<400>3
gtgctgcgcc?catttcagtc?a????????????????????????????????????????????21
<210>4
<211>21
<212>DNA
＜213〉synthetic
<220>
<221>misc_feature
<222>(1)..(21)
＜223〉primer P2
<400>4
gtgccatctc?aactcactgc?a????????????????????????????????????????????21
Claims (13)
1. a NIPA1 transgenation albumen is characterized in that, it has the aminoacid sequence shown in the SEQ ID NO:2, and wherein the 106th amino acids is an arginine.
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, it 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。
4. method that the hereditary spastic paraplegia of individuality is diagnosed, it is characterized in that, it 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.
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 compound is characterized in that, described compound suppresses the claim 1 proteic activation of described NIPA1 transgenation or suppresses the expression of the described NIPA1 mutator gene of claim 2.
8. the described compound of claim 7 is characterized in that, described compound is the antisense nucleotide of NIPA1 mutator gene.
9. the proteic purposes of NIPA1 transgenation is characterized in that, is used for screening or preparing the application of the composition of treatment, diagnosing hereditary Spastic Paraplegia.
10. the purposes of a NIPA1 mutator gene is characterized in that, is used for screening or preparing the application of the composition of treatment, diagnosing hereditary Spastic Paraplegia.
11. a test kit that detects hereditary spastic paraplegia is characterized in that it comprises the primer of specific amplification NIPA1 gene or transcript.
12. test kit as claimed in claim 11 is characterized in that, it also contains the reagent that is selected from down group:
(a) with mutable site bonded probe;
(b) restriction enzyme in identification mutational site.
13. test kit as claimed in claim 12 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.
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| CN 200310108710 CN1281621C (en) | 2003-11-19 | 2003-11-19 | Method of diagnosing and treating heritage spasm paraplegia using NIPAl gene mutation hot point |
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| CN 200310108710 CN1281621C (en) | 2003-11-19 | 2003-11-19 | Method of diagnosing and treating heritage spasm paraplegia using NIPAl gene mutation hot point |
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| CN1281621C CN1281621C (en) | 2006-10-25 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7332282B2 (en) * | 2003-08-19 | 2008-02-19 | The Regents Of The University Of Michigan | Compositions and methods for detecting and treating neurological conditions |
| CN102286629A (en) * | 2011-09-08 | 2011-12-21 | 中南大学湘雅医院 | Hereditary spastic paraplegia gene diagnosis chip |
-
2003
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7332282B2 (en) * | 2003-08-19 | 2008-02-19 | The Regents Of The University Of Michigan | Compositions and methods for detecting and treating neurological conditions |
| US8518638B2 (en) | 2003-08-19 | 2013-08-27 | The Regents Of The University Of Michigan | Compositions and methods for detecting and treating neurological conditions |
| US9285375B2 (en) | 2003-08-19 | 2016-03-15 | The Regents Of The University Of Michigan | Compositions and methods for detecting and treating neurological conditions |
| US9868990B2 (en) | 2003-08-19 | 2018-01-16 | The Regents Of The University Of Michigan | Compositions and methods for detecting and treating neurological conditions |
| CN102286629A (en) * | 2011-09-08 | 2011-12-21 | 中南大学湘雅医院 | Hereditary spastic paraplegia gene diagnosis chip |
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| Publication number | Publication date |
|---|---|
| CN1281621C (en) | 2006-10-25 |
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