CN105779464B - The nucleic acid of the coding FHL1 mutant of separation and its application - Google Patents
The nucleic acid of the coding FHL1 mutant of separation and its application Download PDFInfo
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- CN105779464B CN105779464B CN201410836230.1A CN201410836230A CN105779464B CN 105779464 B CN105779464 B CN 105779464B CN 201410836230 A CN201410836230 A CN 201410836230A CN 105779464 B CN105779464 B CN 105779464B
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Abstract
The invention discloses FHL1 gene mutation body and its applications, more particularly to the nucleic acid of the coding FHL1 mutant of separation, isolated polypeptide, the system that screening is susceptible to suffer from the biological sample of ecuador's syndrome is malnourished disease, for screening the kit for being susceptible to suffer from the biological sample of ecuador's syndrome is malnourished disease, the kit for carrying out quantitative detection to FHL1 mutant, and the method for building medicaments sifting model.Wherein, the nucleic acid of the coding FHL1 mutant of the separation has c.502-2A > T mutation compared with SEQ ID NO:1.It whether there is by detecting the new mutant in the biological sample, can be effectively detected whether biological sample is susceptible to suffer from ecuador's syndrome is malnourished disease.
Description
Technical field
The present invention relates to field of biotechnology, and in particular, to the nucleic acid of the coding FHL1 mutant of separation and its application,
Ai-moral Er Shi myotrophy is susceptible to suffer from not more particularly, to the nucleic acid of the coding FHL1 mutant of separation, the polypeptide of separation, screening
The system of the biological sample of good disease is susceptible to suffer from the kit of the biological sample of ecuador's syndrome is malnourished disease, is used for for screening
Kit, construct, recombinant cell and the method for constructing medicaments sifting model of quantitative detection are carried out to FHL1 mutant.
Background technique
Ecuador's syndrome is malnourished disease (EDMD) is a kind of genetic muscle atrophy disease, it is main influence skeletal muscle and
Cardiac muscle, it is unrelated with nervous system.The heterogeneity of EDMD is high, and morbidity's time is early, and the initial stage state of an illness is relatively light.In general,
There is the muscle contracture at the positions such as neck, ancon and heel string first in patient, gradually appears neck buckling, finally influences whole ridge
Column.Then, there are the symptoms such as limb muscle atrophy and muscle inability in patient.It is passed finally, patient will appear different types of heart
Lead defect.The intellectual development of patient is unaffected.According to the difference of hereditary pattern, EDMD can be divided into three classes: stealthy with X chromosome
Hereditary (X-linked), autosomal dominant inheritance (AD) and autosomal recessive inheritance (AR).Only find 6 EDMD's at present
Disease-causing gene constrains the early diagnosis and therapy of EDMD.
Therefore, still need to be further discovered that new FHL1 mutant.
Summary of the invention
The present invention is directed at least solve one of the technical problems existing in the prior art.For this purpose, one object of the present invention
A kind of method for being to propose Effective selection ecuador's syndrome is malnourished disease biological sample.
According to the first aspect of the invention, the present invention provides a kind of nucleic acid of the coding FHL1 mutant of separation.According to
The embodiment of the present invention, compared with SEQ ID NO:1, the nucleic acid has c.502-2A > T mutation.Inventor has surprisingly found that,
The mutant and the morbidity of ecuador's syndrome is malnourished disease are closely related, thus by detecting the mutant in biological sample
In whether there is, can be effectively detected whether biological sample is susceptible to suffer from ecuador's syndrome is malnourished disease, reality according to the present invention
Example is applied, the nucleic acid of the mutant further enriches the pathogenic mutation map of FHL1 gene, deeper into illustrating Ai-moral Er Shi flesh
The Molecular pathogenesis of muscular dystrophy is early stage Disease-causing gene screening and the therapeutic intervention of ecuador's syndrome is malnourished disease
Scientific basis is provided.
According to the second aspect of the invention, the present invention also provides a kind of isolated polypeptides.According to an embodiment of the invention,
The polypeptide is by nucleic acid encode above-mentioned.By whether expressing the polypeptide in detection biological sample, can be effectively detected
Whether biological sample is susceptible to suffer from ecuador's syndrome is malnourished disease.
According to the third aspect of the invention we, the present invention also provides a kind of screenings to be susceptible to suffer from ecuador's syndrome is malnourished disease
Biological sample system.According to an embodiment of the invention, the system includes: nucleic acid-extracting apparatus, the nucleic acid-extracting apparatus
For from the extraction from biological material sample of nucleic acid；Nucleic acid sequence determining device, the nucleic acid sequence determining device and the core
Sour extraction element is connected, for analyzing the sample of nucleic acid, to determine the nucleic acid sequence of the sample of nucleic acid；Judgement
Device, the judgment means are connected with the nucleic acid sequence determining device, so as to based on the sample of nucleic acid nucleic acid sequence with
SEQ ID NO:1 is compared, if is had c.502-2A > T mutation, is judged whether the biological sample is susceptible to suffer from Ai-moral Er Shi flesh battalion
Support bad disease.It is surprisingly found by the inventors that using the system gene level can be carried out to ecuador's syndrome is malnourished disease
Detection helps more accurately to screen the biological sample for being susceptible to suffer from ecuador's syndrome is malnourished disease.
According to the fourth aspect of the invention, the present invention also provides one kind is susceptible to suffer from Ai-moral Er Shi myotrophy not for screening
The kit of the biological sample of good disease.According to an embodiment of the invention, the kit contains: being adapted to detect for FHL1 gene mutation body
Reagent, wherein compared with SEQ ID NO:1, the FHL1 gene mutation body has c.502-2A > T mutation.Inventor is surprised
Ground discovery can carry out high-precision detection to FHL1 gene mutation body, to seek to Ai-moral Er Shi flesh using the kit
The detection that bad disease carries out gene level is supported, helps more accurately to screen the biology for being susceptible to suffer from ecuador's syndrome is malnourished disease
Sample.
According to the fifth aspect of the invention, the present invention also provides a kind of for carrying out quantitative detection to FHL1 mutant
Kit.According to an embodiment of the invention, the kit may include: FHL1 mutant amplimer, with SEQ ID NO:1 phase
Than the FHL1 mutant has c.502-2A > T mutation, and the FHL1 mutant amplimer includes selected from following
It is at least one set of: (i) SEQ ID NO:4 and 5, (ii) SEQ ID NO:6 and 7, and (iii) SEQ ID NO:8 and 9；And ginseng
According to gene magnification primer, it is preferable that the reference gene is hprt gene, and the reference gene amplimer includes comprising SEQ
Nucleotide sequence shown in ID NO:10 and 11.FHL1 mutant can quickly and accurately be carried out using the kit as a result,
Quantitative detection.
According to the sixth aspect of the invention, the present invention also provides a kind of constructs.According to an embodiment of the invention, the structure
Build the nucleic acid that body includes the coding FHL1 mutant of separation above-mentioned.Construct transformed acceptor cell of the invention is utilized as a result,
The recombinant cell of acquisition can be efficiently used for the drug of screening treatment ecuador's syndrome is malnourished disease.
According to the seventh aspect of the invention, the present invention also provides a kind of recombinant cells.According to an embodiment of the invention, institute
Stating recombinant cell is obtained by construct transformed acceptor cell above-mentioned.According to some embodiments of the present invention, it utilizes
Recombinant cell of the invention can effectively screen the drug for the treatment of ecuador's syndrome is malnourished disease.
According to the eighth aspect of the invention, the present invention also provides a kind of methods for constructing medicaments sifting model.According to this
The embodiment of invention, this method comprises: at least part cell of animal is made to express polypeptide above-mentioned.It is more according to the present invention
Embodiment can effectively screen the drug for the treatment of ecuador's syndrome is malnourished disease using animal model of the invention.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Detailed description of the invention
Above-mentioned and/or additional aspect of the invention and advantage will become from the description of the embodiment in conjunction with the following figures
Obviously and it is readily appreciated that, in which:
Fig. 1 shows the biological sample according to an embodiment of the invention for screening and being susceptible to suffer from ecuador's syndrome is malnourished disease
The system of product and its schematic diagram of component part, wherein
Figure 1A is the system that the biological sample of ecuador's syndrome is malnourished disease is susceptible to suffer from according to the screening of the embodiment of the present invention
Schematic diagram,
Figure 1B is the schematic diagram according to the nucleic acid-extracting apparatus of the embodiment of the present invention,
Fig. 1 C is the schematic diagram according to the nucleic acid sequence determining device of the embodiment of the present invention；
Fig. 2 shows the pedigree chart of ecuador's syndrome is malnourished disease patient's family according to an embodiment of the invention；
Fig. 3 shows FHL1 gene and its variable sheer body schematic diagram according to an embodiment of the invention；
Fig. 4 shows the schematic diagram of influence RAS-MAPK access after FHL1C overexpression according to an embodiment of the invention；
Fig. 5 shows that different patients 2, patient 3 and the cDNA compareed according to an embodiment of the invention transduce at fibre
Tie up the schematic diagram of ERK protein phosphorylation level in cell；
Fig. 6 shows the plasmid transduction of FHL1A, FHL1C according to an embodiment of the invention and control at fiber finer
The schematic diagram of ERK protein phosphorylation level in born of the same parents.
Specific embodiment
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings, wherein from beginning to end
Same or similar label indicates same or similar element or element with the same or similar functions.Below with reference to attached
The embodiment of figure description is exemplary, and for explaining only the invention, and is not considered as limiting the invention.
FHL1 mutant
According to the first aspect of the invention, the present invention provides a kind of nucleic acid of the coding FHL1 mutant of separation.According to
The embodiment of the present invention, the nucleic acid have c.502-2A > T mutation compared with SEQ ID NO:1.Wherein c.502-2A > T table
To show, point mutation occurs for the 2nd bit base of inverse of the previous introne of saltant type EDMD correlation FHL1 the 6th exon of gene,
T is mutated by A.Inventor has surprisingly found that the mutant and the morbidity of ecuador's syndrome is malnourished disease are closely related, from
And whether there is in the biological sample by detecting the mutant, it can be effectively detected whether biological sample is susceptible to suffer from Ai-moral two
Family name's muscular dystrophy is dashed forward according to an embodiment of the invention, the nucleic acid of the mutant further enriches causing a disease for FHL1 gene
Become map, is ecuador's syndrome is malnourished deeper into the Molecular pathogenesis of ecuador's syndrome is malnourished disease is illustrated
The early stage Disease-causing gene screening of disease and therapeutic intervention provide scientific basis.
Expression way used in herein " nucleic acid of coding FHL1 mutant ", refers to and coding FHL1 mutant
The corresponding nucleic acid substances of gene, the i.e. type of nucleic acid are not particularly limited, and can be any volume comprising with FHL1 mutant
The polymer of the corresponding deoxyribonucleotide of code gene and/or ribonucleotide, including but not limited to DNA, RNA or
cDNA.Some specific examples according to the present invention, the nucleic acid are DNA.
For the nucleic acid mentioned in description of the invention and claims, it will be appreciated by those skilled in the art that real
Border includes any one or two of complementary double-strand.For convenience, in the present specification and claims, although it is most
In the case of only give a chain, but actually also disclose another chain complementary to it.For example, refer to SEQ ID NO:1,
Practical includes its complementary series.Those skilled in the art, which are further appreciated that, can detecte another chain using a chain, otherwise also
So.
The nucleic acid of coding FHL1 mutant is that present inventor passes through the candidate base of target area capture sequencing joint
Because of the new pathogenic mutation of the Disease-causing gene FHL1 of the determining ecuador's syndrome is malnourished disease of the method for mutation verifying.The cause
Sick mutational site is not mentioned in the prior art.It should be noted that expression way " target area used in herein
After domain capture sequencing " refers to and carries out capture enrichment using special probe certain section of particular sequence interested to client, recycle
The method that second generation sequencing technologies carry out high-flux sequence and genome analysis.Desired target area can be obtained using this method
Hereditary information, greatly improve the Efficiency of target area in genome, significantly reduce research cost.In the present invention
In, by the structure variation in this method for identification coding region relevant to disease with research, in turn, in conjunction with a large amount of public
The data that database provides are conducive to preferably explain the association between gained variant structure and pathogenesis.
Wherein, the cDNA of the FHL1 of wild type sequence (information of FHL1 gene is referring to transcript id:
ENST00000394155, http://asia.ensembl.org/Homo_sapiens/Transcript/
ProteinSummary? db=core；G=ENSG00000022267；R=X:136147400-136211359；T=
ENST00000394155) as follows, wherein c.502 bit base adds frame to show:
ATGGCGGAGAAGTTTGACTGCCACTACTGCAGGGATCCCTTGCAGGGGAAGAAGTATGTGCAAAAGGA
TGGCCACCACTGCTGCCTGAAATGCTTTGACAAGTTCTGTGCCAACACCTGTGTGGAATGCCGCAAGCCCATCGGT
GCGGACTCCAAGGAGGTGCACTATAAGAACCGCTTCTGGCATGACACCTGCTTCCGCTGTGCCAAGTGCCTTCACC
CCTTGGCCAATGAGACCTTTGTGGCCAAGGACAACAAGATCCTGTGCAACAAGTGCACCACTCGGGAGGACTCCCC
CAAGTGCAAGGGGTGCTTCAAGGCCATTGTGGCAGGAGATCAAAACGTGGAGTACAAGGGGACCGTCTGGCACAAA
GACTGCTTCACCTGTAGTAACTGCAAGCAAGTCATCGGGACTGGAAGCTTCTTCCCTAAAGGGGAGGACTTCTACT
GCGTGACTTGC
GAATCACTTACCAGGATCAGCCCTGGCATGCCGATTGCTTTGTGTGTGTTACCTGCTCTAAGAAGCTGGCTGGGCA
GCGTTTCACCGCTGTGGAGGACCAGTATTACTGCGTGGATTGCTACAAGAACTTTGTGGCCAAGAAGTGTGCTGGA
TGCAAGAACCCCATCACTGGGAAAAGGACTGTGTCAAGAGTGAGCCACCCAGTCTCTAAAGCTAGGAAGCCCCCAG
TGTGCCACGGGAAACGCTTGCCTCTCACCCTGTTTCCCAGCGCCAACCTCCGGGGCAGGCATCCGGGTGGAGAGAG
GACTTGTCCCTCGTGGGTGGTGGTTCTTTATAGAAAAAATCGAAGCTTAGCAGCTCCTCGAGGCCCGGGTTTGGTA
AAGGCTCCAGTGTGGTGGCCTATGAAGGACAATCCTGGCACGACTACTGCTTCCACTGCAAAAAATGCTCCGTGA
(SEQ ID NO:1)
The gDNA sequence of wild type FHL1 of the invention is as follows, wherein capitalization is exon, and lowercase is
Introne:
ATGGCGGAGAAGTTTGACTGCCACTACTGCAGGGATCCCTTGCAGGGGAAGAAGTATGTGCAAAAGGA
TGGCCACCACTGCTGCCTGAAATGCTTTGACAAGTTCTGTGCCAACACCTGTGTGGAATGCCGCAAGCCCATCGGT
GCGGACTCCAAGgtaacgggcatccccatgtgccaatgggaagggctgggttttggagtgtcctttgcccacaacc
atggcagcagcagctggctgttaggatttcccagcatcactgcagccaccttgaggcctcaaggaagcctcctcca
ctccccaggccacagtggcccgagctgtttaatgtggggcttgactggatgggcgccagcgcccttgccagctctt
ttgattgcattctaaatatttcaagaattgtgagatttttatcctcacctcagcgtccctcctataagaatagtca
ctgtggggcagtcccaggtgtaagggactgtgtcatctcagtggtcagtcccagggaaatcagccttatgggaggg
ctcctgccaccacccccagcacccctcatggtggcccaccctgtctgcttggtttccagGAGGTGCACTATAAGAA
CCGCTTCTGGCATGACACCTGCTTCCGCTGTGCCAAGTGCCTTCACCCCTTGGCCAATGAGACCTTTGTGGCCAAG
GACAACAAGATCCTGTGCAACAAGTGCACCACTCGGGAGGACTCCCCCAAGTGCAAGGGGTGCTTCAAGGCCATTG
TGGCAGgtactgcctccttcccaccccgggttcccagggaggaggccctgagggcagacgtgatgtgggcttgctt
atcatggtggggctaacgttgctctgagctgctttgagatcttagcatatatatgtacacatatacatacacgtat
atatgcgtacacatatatgctcgcacacacgcacacactcggagactaaagaacactggcgagaacagcctgtggc
aacagaatgaagtgaacagtatgtagcgctttctcatttgggcgtagtaagtgatgaaagcatgcttcttcctcag
ggtgtcattctgggccaggcagtccctgatttaatgtctaagtgcacgcagggtatagaggtgggggagtggggga
ttcaggcactggatcctaaaataataatgctggggtccccacccatgacagaaatcctgggttggcacaagcacaa
gtagaacacaggtaggttagttggaggtgtgaggccagtaactgcagggcctgcatcccctcacctctggagggcc
tggggaggggagctgagtggatgcagccccctgcagagcctgtcagtggggctatccaattgcttccctctgcagG
AGATCAAAACGTGGAGTACAAGGGGACCGTCTGGCACAAAGACTGCTTCACCTGTAGTAACTGCAAGCAAGTCATC
GGGACTGGAAGCTTCTTCCCTAAAGGGGAGGACTTCTACTGCGTGACTTGCCATGAGACCAAGTTTGCCAAGCATT
GCGTGAAGTGCAACAAGgtatgctttcaagggagttctgcattgaccgttgtttctagaagtgtttgacagttTGC
AGAGCACTTCCACACACACTATCCCATTCCATCCTCACGACAGCCCTGTGACGTAGGAATTATTATTCCCGTTTTA
CAGATGAGGAGATCGTGGATTAGGAAGCAGTGCCACAGCCAAGTCAGGCTGTCAGTACGCATCCTCAGCCTGGTGG
TGCGGGTGGCCCGTATGGCATGCTGAGCTGGGCAGCCCTGGCTCTAGAAGCCTGCCTCCACCACTTAGGGGCTGTG
TAACCTCACATAGGGGCTCACCTTCTCCGAGCCTTGGTGTCTTCATCTGTAAAGTGGGGATAATAATAGCCCCTGC
CTCCCATGGCTGTTGTGGAGATTAAATGAGATATTGTATACCAAAGCGCCCAGCACAGTGCCTGGCACGCAGTAGG
CATTCAACAAAATGGTTGTTGaatctgaatccggtgctacactccctggtctagGCCATCACATCTGGAGGAATCA
CTTACCAGGATCAGCCCTGGCATGCCGATTGCTTTGTGTGTGTTACCTGCTCTAAGAAGCTGGCTGGGCAGCGTTT
CACCGCTGTGGAGGACCAGTATTACTGCGTGGATTGCTACAAGAACTTTGTGGCCAAGAAGTGTGCTGGATGCAAG
AACCCCATCACTGgtaggctaaagagtccttgctaagtctgccaggctaggttttgcgcatggtaaccatctctca
ttttcctgtcgtcggtttcatccacaaaggcccccagagaatgcccttctccccctgctattgtggtcccaaaggc
cccccaagagtttggattcgtccccaggccaggttagcctttgcaatacagaacacttcctgactgttgactaaca
atgctgagagttcacaagcctgagacctgccagccaacaccggcaggcactgccactttgcagggggattctgggg
ggaggggtgggaggagggtagaaagaagggggtgggggagggtggggaaggggtaagggaggccgaagagtgataa
cccgggattgtaataccccatagctgaaggctagttcagagatgcctgttggcagggtttcttttttttttggttt
gctgtttatttgtttttgcgatcagcaaagctaatcacttcattcctcatctcgcggccgcgatccacgtgccctg
ggccctttcccttgcctccaattttcccatcttcccggggagccttgaaatgtacatttgagaagacttttgccat
cctcagGGAAAAGGACTGTGTCAAGAGTGAGCCACCCAGTCTCTAAAGCTAGGAAGCCCCCAGTGTGCCACGGGAA
ACGCTTGCCTCTCACCCTGTTTCCCAGCGCCAACCTCCGGGGCAGGCATCCGGGTGGAGAGAGGACTTGTCCCTCG
TGGGTGGTGGTTCTTTATAGAAAAAATCGAAGCTTAGCAGCTCCTCGAGGCCCGgtaagtgcacaccccacgaaca
gcccaagtttgcctcctggtggccactttgatgtcatggccctgacctaaatcaaagaaactggttatgctgggag
gtcggtgcgcgtcacagggcaatagcgtggtggcatgggaacgtggggtctttacatcagggaagcccttggccgg
gcttcagcatcttctcaggtccttgagagccacagcagggttgcggcggcatgggggacaatggcggcgtggggga
ctgtgcacgatggagtggaggagggggtctgggagccaggcagacctggctcttgcgtgcttgtcggtctgtgagt
ggggcaggtcgtcattttatctctctgaatcgtggtttcctcacctgtattcattcagctgtttctcttgttttct
tttcttttctttttttttccccccagGGTTTGGTAAAGGCTCCAGTGTGGTGGCCTATGAAGGACAATCCTGGCAC
GACTACTGCTTCCACTGCAAAAAATGCTCCGTGA (SEQ ID NO:12)
FHL1 normal amino acid sequences are as follows:
MAEKFDCHYCRDPLQGKKYVQKDGHHCCLKCFDKFCANTCVECRKPIGADSKEVHYKNRFWHDTCFRC
AKCLHPLANETFVAKDNKILCNKCTTREDSPKCKGCFKAIVAGDQNVEYKGTVWHKDCFTCSNCKQVIGTGSFFPK
GEDFYCVTCHETKFAKHCVKCNKAITSGGITYQDQPWHADCFVCVTCSKKLAGQRFTAVEDQYYCVDCYKNFVAKK
CAGCKNPITGKRTVSRVSHPVSKARKPPVCHGKRLPLTLFPSANLRGRHPGGERTCPSWVVVLYRKNRSLAAPRGP
GLVKAPVWWPMKDNPGTTTASTAKNAP (SEQ ID NO:13)
The gDNA sequence of FHL1 mutant is as follows after shearing site mutation, and wherein mutational site adds frame to show, wherein capitalization
Letter is exon, and lowercase is introne:
ATGGCGGAGAAGTTTGACTGCCACTACTGCAGGGATCCCTTGCAGGGGAAGAAGTATGTGCAAAAGGA
TGGCCACCACTGCTGCCTGAAATGCTTTGACAAGTTCTGTGCCAACACCTGTGTGGAATGCCGCAAGCCCATCGGT
GCGGACTCCAAGgtaacgggcatccccatgtgccaatgggaagggctgggttttggagtgtcctttgcccacaacc
atggcagcagcagctggctgttaggatttcccagcatcactgcagccaccttgaggcctcaaggaagcctcctcca
ctccccaggccacagtggcccgagctgtttaatgtggggcttgactggatgggcgccagcgcccttgccagctctt
ttgattgcattctaaatatttcaagaattgtgagatttttatcctcacctcagcgtccctcctataagaatagtca
ctgtggggcagtcccaggtgtaagggactgtgtcatctcagtggtcagtcccagggaaatcagccttatgggaggg
ctcctgccaccacccccagcacccctcatggtggcccaccctgtctgcttggtttccagGAGGTGCACTATAAGAA
CCGCTTCTGGCATGACACCTGCTTCCGCTGTGCCAAGTGCCTTCACCCCTTGGCCAATGAGACCTTTGTGGCCAAG
GACAACAAGATCCTGTGCAACAAGTGCACCACTCGGGAGGACTCCCCCAAGTGCAAGGGGTGCTTCAAGGCCATTG
TGGCAGgtactgcctccttcccaccccgggttcccagggaggaggccctgagggcagacgtgatgtgggcttgctt
atcatggtggggctaacgttgctctgagctgctttgagatcttagcatatatatgtacacatatacatacacgtat
atatgcgtacacatatatgctcgcacacacgcacacactcggagactaaagaacactggcgagaacagcctgtggc
aacagaatgaagtgaacagtatgtagcgctttctcatttgggcgtagtaagtgatgaaagcatgcttcttcctcag
ggtgtcattctgggccaggcagtccctgatttaatgtctaagtgcacgcagggtatagaggtgggggagtggggga
ttcaggcactggatcctaaaataataatgctggggtccccacccatgacagaaatcctgggttggcacaagcacaa
gtagaacacaggtaggttagttggaggtgtgaggccagtaactgcagggcctgcatcccctcacctctggagggcc
tggggaggggagctgagtggatgcagccccctgcagagcctgtcagtggggctatccaattgcttccctctgcagG
AGATCAAAACGTGGAGTACAAGGGGACCGTCTGGCACAAAGACTGCTTCACCTGTAGTAACTGCAAGCAAGTCATC
GGGACTGGAAGCTTCTTCCCTAAAGGGGAGGACTTCTACTGCGTGACTTGCCATGAGACCAAGTTTGCCAAGCATT
GCGTGAAGTGCAACAAGgtatgctttcaagggagttctgcattgaccgttgtttctagaagtgtttgacagttTGC
AGAGCACTTCCACACACACTATCCCATTCCATCCTCACGACAGCCCTGTGACGTAGGAATTATTATTCCCGTTTTA
CAGATGAGGAGATCGTGGATTAGGAAGCAGTGCCACAGCCAAGTCAGGCTGTCAGTACGCATCCTCAGCCTGGTGG
TGCGGGTGGCCCGTATGGCATGCTGAGCTGGGCAGCCCTGGCTCTAGAAGCCTGCCTCCACCACTTAGGGGCTGTG
TAACCTCACATAGGGGCTCACCTTCTCCGAGCCTTGGTGTCTTCATCTGTAAAGTGGGGATAATAATAGCCCCTGC
CTCCCATGGCTGTTGTGGAGATTAAATGAGATATTGTATACCAAAGCGCCCAGCACAGTGCCTGGCACGCAGTAGG
CATTCAACAAAATGGTT
GCCCTGGCATGCCGATTGCTTTGTGTGTGTTACCTGCTCTAAGAAGCTGGCTGGGCAGCGTTTCACCGCTGTGGAG
GACCAGTATTACTGCGTGGATTGCTACAAGAACTTTGTGGCCAAGAAGTGTGCTGGATGCAAGAACCCCATCACTG
gtaggctaaagagtccttgctaagtctgccaggctaggttttgcgcatggtaaccatctctcattttcctgtcgtc
ggtttcatccacaaaggcccccagagaatgcccttctccccctgctattgtggtcccaaaggccccccaagagttt
ggattcgtccccaggccaggttagcctttgcaatacagaacacttcctgactgttgactaacaatgctgagagttc
acaagcctgagacctgccagccaacaccggcaggcactgccactttgcagggggattctggggggaggggtgggag
gagggtagaaagaagggggtgggggagggtggggaaggggtaagggaggccgaagagtgataacccgggattgtaa
taccccatagctgaaggctagttcagagatgcctgttggcagggtttcttttttttttggtttgctgtttatttgt
ttttgcgatcagcaaagctaatcacttcattcctcatctcgcggccgcgatccacgtgccctgggccctttccctt
gcctccaattttcccatcttcccggggagccttgaaatgtacatttgagaagacttttgccatcctcagGGAAAAG
GACTGTGTCAAGAGTGAGCCACCCAGTCTCTAAAGCTAGGAAGCCCCCAGTGTGCCACGGGAAACGCTTGCCTCTC
ACCCTGTTTCCCAGCGCCAACCTCCGGGGCAGGCATCCGGGTGGAGAGAGGACTTGTCCCTCGTGGGTGGTGGTTC
TTTATAGAAAAAATCGAAGCTTAGCAGCTCCTCGAGGCCCGgtaagtgcacaccccacgaacagcccaagtttgcc
tcctggtggccactttgatgtcatggccctgacctaaatcaaagaaactggttatgctgggaggtcggtgcgcgtc
acagggcaatagcgtggtggcatgggaacgtggggtctttacatcagggaagcccttggccgggcttcagcatctt
ctcaggtccttgagagccacagcagggttgcggcggcatgggggacaatggcggcgtgggggactgtgcacgatgg
agtggaggagggggtctgggagccaggcagacctggctcttgcgtgcttgtcggtctgtgagtggggcaggtcgtc
attttatctctctgaatcgtggtttcctcacctgtattcattcagctgtttctcttgttttcttttcttttctttt
tttttccccccagGGTTTGGTAAAGGCTCCAGTGTGGTGGCCTATGAAGGACAATCCTGGCACGACTACTGCTTCC
ACTGCAAAAAATGCTCCGTGA (SEQ ID NO:14)
The amino acid sequence of FHL1 is as follows after shearing site mutation, wherein the amino acid being mutated adds frame to show: MAEKFDCH
YCRDPLQGKKYVQKDGHHCCLKCFDKFCANTCVECRKPIGADSKEVHYKNRFWHDTCFRCAKCLHPLANETFVAKD
NKILCNKCTTREDSPKCKGCFKAIVAGDQNVEY
(SEQ ID NO:15)
Inventor has surprisingly found that the mutant and the morbidity of ecuador's syndrome is malnourished disease are closely related, thus logical
It crosses and detects the mutant and whether there is in the biological sample, can be effectively detected whether biological sample is susceptible to suffer from Ai-moral Er Shi flesh
Muscular dystrophy, according to an embodiment of the invention, the nucleic acid of the mutant further enriches the pathogenic mutation figure of FHL1 gene
Spectrum is ecuador's syndrome is malnourished disease deeper into the Molecular pathogenesis of ecuador's syndrome is malnourished disease is illustrated
Early stage Disease-causing gene screening and therapeutic intervention provide scientific basis.
According to the second aspect of the invention, the present invention also provides a kind of isolated polypeptides.According to an embodiment of the invention,
The polypeptide is by nucleic acid encode above-mentioned.Compared with SEQ ID NO:1, the cDNA of FHL1 mutant has c.502-2A > T
Mutation, in turn, the product of coding can cause variable sheer body FHL1C to express compared with the FHL1 polypeptide sequence of wild type
Amount increases, and the patient with this mutation not only will appear the related symptoms of EDMD, there is also RASopathy related symptoms.
But the overexpression of FHL1C can't be such that the phosphorylation level of albumen ERK increases, and then activate RAS-MAPK access.Saltant type
Difference of the EDMD correlation FHL1 gene FHL1 gene related to wild type EDMD in sequence be c.502-2A > T (IVS6-2A >
T).By whether expressing the polypeptide in detection biological sample, it can be effectively detected whether biological sample is susceptible to suffer from Ai-moral Er Shi flesh
Muscular dystrophy.
Screen the system for being susceptible to suffer from the biological sample of ecuador's syndrome is malnourished disease and its corresponding kit
According to the third aspect of the invention we, the present invention provides a kind of screenings to be susceptible to suffer from ecuador's syndrome is malnourished disease
The system of biological sample.A referring to Fig.1, the system include:
Nucleic acid-extracting apparatus 100, the nucleic acid-extracting apparatus 100 are used for from the extraction from biological material sample of nucleic acid.Root
According to the embodiment of the present invention, B referring to Fig.1, the nucleic acid-extracting apparatus 100 further comprises: RNA extraction unit 101, described
RNA extraction unit 101 is used for from the extraction from biological material RNA sample；And reverse transcription unit 102, the reverse transcription unit
102 are connected with the RNA extraction unit 101, for carrying out reverse transcription reaction to the RNA sample, to obtain cDNA sample,
The cDNA sample constitutes the sample of nucleic acid.
Nucleic acid sequence determining device 200, the nucleic acid sequence determining device 200 are connected with the nucleic acid-extracting apparatus 100,
For analyzing the sample of nucleic acid, to determine the nucleic acid sequence of the sample of nucleic acid.According to an embodiment of the invention,
C referring to Fig.1, the nucleic acid sequence determining device 200 further comprises: library construction unit 201, the library construction unit
201, for being directed to the sample of nucleic acid, construct nucleic acid sequencing library；And sequencing unit 202, the sequencing unit 202 and institute
It states library construction unit 201 to be connected, for the nucleic acid sequencing library to be sequenced, to obtain by multiple sequencing data structures
At sequencing result.Some specific examples according to the present invention, the library construction unit further comprises: PCR amplification module,
FHL1 gene extron specific primer is provided in the PCR amplification module, to utilize the specific primer, to described
Sample of nucleic acid carries out PCR amplification.Wherein, the specific primer has the nucleotide sequence as shown in SEQ IDNO:2 and 3.It is excellent
Selection of land, the sequencing unit include at least one selected from HISEQ2000, SOLiD, 454 and single-molecule sequencing device.
Judgment means 300, the judgment means 300 are connected with the nucleic acid sequence determining device 200, so as to based on described
The nucleic acid sequence of sample of nucleic acid is compared with SEQ ID NO:1, if has c.502-2A > T mutation, judges that the biological sample is
It is no to be susceptible to suffer from ecuador's syndrome is malnourished disease.
It is surprisingly found by the inventors that using the system gene level can be carried out to ecuador's syndrome is malnourished disease
Detection helps more accurately to screen the biological sample for being susceptible to suffer from ecuador's syndrome is malnourished disease.
According to the fourth aspect of the invention, the present invention also provides one kind is susceptible to suffer from Ai-moral Er Shi myotrophy not for screening
The kit of the biological sample of good disease.According to an embodiment of the invention, the kit contains: being adapted to detect for FHL1 gene mutation body
Reagent, wherein compared with SEQ ID NO:1, the FHL1 gene mutation body has c.502-2A > T mutation.
In the present invention, used term " reagent for being adapted to detect for FHL1 gene mutation body " shall be understood in a broad sense, i.e.,
The reagent that can be detection FHL1 encoding gene is also possible to detect the reagent of FHL1 mutant polypeptide, such as can be using knowledge
The antibody of other specific position.Some specific examples according to the present invention, the reagent are nucleic acid probe or primer.As a result, can
The detection FHL1 gene of enough specificity.Preferably, the nucleic acid probe or primer have the core as shown in SEQ ID NO:2 and 3
Nucleotide sequence.Good to the specificity of FHL1 genetic test as a result, accuracy is high.
It is surprisingly found by the inventors that high-precision detection can be carried out to FHL1 gene mutation body using the kit, from
And the detection of gene level is carried out to ecuador's syndrome is malnourished disease, facilitate more accurately screening and is susceptible to suffer from Ai-moral Er Shi flesh
The biological sample of muscular dystrophy.
According to the fifth aspect of the invention, the present invention also provides a kind of for carrying out quantitative detection to FHL1 mutant
Kit.According to an embodiment of the invention, the kit may include: FHL1 mutant amplimer, with SEQ ID NO:1 phase
Than the FHL1 mutant has c.502-2A > T mutation, and the FHL1 mutant amplimer includes selected from following
It is at least one set of: (i) SEQ ID NO:4 and 5, (ii) SEQ ID NO:6 and 7, and (iii) SEQ ID NO:8 and 9；And ginseng
According to gene magnification primer, it is preferable that the reference gene is hprt gene, and the reference gene amplimer includes comprising SEQ
Nucleotide sequence shown in ID NO:10 and 11.
Wherein, it should be noted that FHL1 gene has 3 kinds of variable sheer bodies, and FHL1A, FHL1B and FHL1C, structure is as schemed
Shown in 3, wherein the c-terminus of FHL1B and FHL1C has RBP-J structural domain, which can be in conjunction with transcription factor RBP-J κ.
Expression quantity highest of the FHL1A in skeletal muscle is taken second place in the expression quantity of cardiac muscle, and amplimer has the institute of SEQ ID NO:4 and 5
Show nucleotide sequence.Expression quantity highest of the FHL1B in skeletal muscle, expression quantity in the brain take second place, the expression in cardiac muscle
Measure it is lower, amplimer have SEQ ID NO:6 and 7 shown in nucleotide sequence.FHL1C is in skeletal muscle, spermary and cardiac muscle
There is expression, wherein the expression quantity in cardiac muscle is relatively low, amplimer has nucleotides sequence shown in SEQ ID NO:8 and 9
Column.Present invention discover that FHL1 gene new mutation (c.502-2A > T (IVS6-2A > T)), 3 kinds for will lead to FHL1 gene can
Become spliced body relative expression quantity to change, the variable sheer body FHL1C expression quantity which will lead to FHL1 gene rises.Benefit
With kit of the invention, RT-PCR is carried out using the variable sheer body of above-mentioned primer pair FHL1, using HPRT as quantitative control,
By the variation for detecting FHL1 spliced body expression quantity, it can be determined that the catastrophe of FHL1 gene.It as a result, can using the kit
Quickly and accurately to carry out quantitative detection to FHL1 mutant.
Construct and recombinant cell
According to the sixth aspect of the invention, the present invention also provides a kind of constructs.According to an embodiment of the invention, the structure
Build the nucleic acid that body includes the coding FHL1 mutant of separation above-mentioned.Construct transformed acceptor cell of the invention is utilized as a result,
The recombinant cell of acquisition can be efficiently used for the drug of screening treatment ecuador's syndrome is malnourished disease.
Term " construct " as used in the present invention refers to a kind of such genetic carrier, and it includes specific nucleic acid sequences
Column, and purpose nucleic acid sequence can be transferred in host cell, to obtain recombinant cell.According to an embodiment of the invention, structure
The form for building body is not particularly limited.According to an embodiment of the invention, it can be plasmid, bacteriophage, artificial chromosome, clay
(Cosmid), viral at least one, preferred plasmid.Plasmid has easy to operate as genetic carrier, can carry larger piece
The property of section, convenient for operating and handling.The form of plasmid is also not particularly limited, either circular plasmids, are also possible to line
Property grain, it can be it is single-stranded, be also possible to double-strand.Those skilled in the art, which can according to need, to be selected.At this
Term used in invention " nucleic acid " can be any polymer comprising deoxyribonucleotide or ribonucleotide, packet
It includes but is not limited to by modification or unmodified DNA, RNA, length is not any particular limitation.For for constructing
The construct of recombinant cell, the preferably described nucleic acid is DNA, more stable because DNA is for RNA, and is easy to grasp
Make.
According to the seventh aspect of the invention, the present invention also provides a kind of recombinant cells.According to an embodiment of the invention, institute
Stating recombinant cell is obtained by construct transformed acceptor cell above-mentioned.According to some embodiments of the present invention, it utilizes
Recombinant cell of the invention can effectively screen the drug for the treatment of ecuador's syndrome is malnourished disease.
The method for constructing medicaments sifting model
According to the eighth aspect of the invention, the present invention also provides a kind of methods for constructing medicaments sifting model.According to this
The embodiment of invention, this method comprises: at least part cell of animal is made to express polypeptide above-mentioned.Implementation according to the present invention
Example, the animal are mouse, pig, dog, primate.According to some embodiments of the present invention, animal mould of the invention is utilized
Type can effectively screen the drug for the treatment of ecuador's syndrome is malnourished disease.
Wherein, it should be noted that the method that the present invention constructs medicaments sifting model is not particularly limited, as long as making
At least part cell of object expresses polypeptide above-mentioned.For example, generating essence to recipient cell using Protocols in Molecular Biology
True target gene rite-directed mutagenesis, obtains the animal for suffering from ecuador's syndrome is malnourished disease, which can be used as model use
The drug of the disease is treated in screening.For example, marker-free transgenic technology, site-directed integration technology, genome editor can be passed through
The methods of technology realizes the rite-directed mutagenesis of target gene, obtains the gene of coding FHL1 mutant of the invention, and then constructs
The recombinant vector is imported homologous embryo by certain mode (common electroporation) by the recombinant vector of FHL1 mutant gene
In tire stem cell, make corresponding portion in the exogenous DNA and embryonic stem cell genome of mutation that homologous recombination occur, by recombinant vector
In DNA sequence dna be integrated into endogenous gene group, so that FHL1 mutant gene is expressed in cell.Again by recombinant cell
Pseudo-pregnant mothers are implanted into, it is made to develop into chimeric animal.Using the mating between chimera, homozygous animals, chimera are generated
Animal and purifying body animal are used equally for medicaments sifting model, thus effectively screening treatment ecuador's syndrome is malnourished disease
Drug.
The present invention at least has the advantages that
Present invention discover that a new missense mutation of ecuador's syndrome is malnourished disease Disease-causing gene.Based on this, may be used
Early stage is carried out for early screening ecuador's syndrome is malnourished disease pathogenic mutation carrier, and then before carrier's morbidity
Therapeutic intervention；It can also be used for the molecular diagnosis of ecuador's syndrome is malnourished disease patient and the antidiastole with related disease.
The technology has many advantages, such as that quick, accurate, efficient, easy, early diagnostic rate is high, testing result Ke Yi Wei Ai-moral Er Shi flesh battalion
The early diagnosis, antidiastole and exploitation ecuador's syndrome is malnourished disease therapeutic agent for supporting bad disease provide scientific basis.
Below with reference to specific embodiment, the present invention will be described, it should be noted that these embodiments are only explanation
Property, and be not considered as limiting the invention.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment
Part, described technology or conditions (such as with reference to the work such as J. Pehanorm Brooker, translate by Huang Peitang etc. according to the literature in the art
" Molecular Cloning:A Laboratory guide ", the third edition, Science Press) or carry out according to product description.Agents useful for same or instrument
Production firm person is not specified, being can be with conventional products that are commercially available, such as can purchase from Illumina company.
Embodiment 1
1, sample collection
The sample of the present embodiment is the family of a Denmark, and pedigree chart is as shown in Figure 2, wherein square indicates male,
Circle indicates women, and it is that black indicates patient in figure, arrow indicates that the patient is that color is not filled by figure, which indicates normal person,
Propositus.Oblique line indicates that the patient is dead.The family has 4 patients, and respective parent's phenotype is normal, and patient shows as flesh
Meat atrophy class disease, but 4 patient clinical symptom severity are different.
Patient 1, male.The patient once had back complication (suffered from back complication), and 16 years old prominent
It is so dead, thus it is speculated that the cause of the death is cardiac arrest.It is described according to its relatives, the facial characteristics phase of other male patients of patient He the family
Seemingly.
Patient 2, male, and 33 years old.The patient be born when, that is, patent ductus arteriosus and pulmonary arteries it is bad, childhood lung continue
There is inflammation.In addition, the patient face mild malformation, chin is small, (tooth) malocclusion, slight thoracic kyphosis.Patient's intelligence is just
Often.
Patient 3, male, and 34 years old.Patient's face is long, periorbital edema (periorbital fullness), and frontal eminence is prominent, volume
Head height, nostril are slightly leaned forward, antimongoloid slant, thoracic kyphosis, narrow shoulder.There is contracture in patient's childhood heel string, and heart is asked within 30 years old
Topic, and because cardiac arrest is diagnosed as with hypertrophic cardiomyopathy.In addition, patient childhood Zeng Yin has cognitive disorder to be diagnosed as suffering from
There is William's syndrome.
Patient 4, male, and 6 years old.The patient epiglottis is in Ω type, scoliokyphosis occurs within 16 months.Patient's face is wide, and forehead is grand
It rises, antimongoloid slant, pigeon breast, high arches.In addition, individual with pulmonary's valve is narrow, and there is kinesitherapy nerve and aphasis.
There is the symptom of EDMD, such as humpback and muscle contracture in 4 patients.Furthermore patient also occurs and RAS-
Symptom when MAPK access is lacked of proper care.The patient of RAS-MAPK access imbalance generally has skin disease, but 4 patients of the family are equal
Do not occur symptom relevant to skin.
Take a normal member of phenotype (control i.e. in Fig. 2 family tree) in patient 2, patient 3, patient 4 and the family
Peripheral blood, utilize Magnetic Separation Module I extract leucocyte DNA.According to World Medical Association, " Hull is pungent
Base declaration " requirement, patient parent or sufferers themselves and Italy, BGI cooperative institution molecule neurogenetics research institute's ethics are entrusted
Member can endorsed informed consent form.
2, linkage analysis
Genotyping is carried out using DNA of the Affymetrix SNP6.0array to patient 2,3,4 and normal control,
And gained genotype is uploaded to BCSNP.Using Merlin (reference can be made to Abecasis et al.Merlin-rapid
analysis of dense genetic maps using sparse gene flow trees.Nat Genet(2002)
Linkage analysis 30:97-101) is carried out to patient and control.Due to the symptom phase of the certain symptoms and Noonan syndrome of patient
Seemingly, using linkage analysis and exon sequencing detection with Noonan syndrome related gene (BRAF, CBL, KRAS, MAP2K1,
MAP2K2, NRAS, PTPN11, RAF1, SHOC2, SOS1, HRAS, SPRED1, KAT6B) variation, but discovery is corresponding
It causes a disease and makes a variation.Further, confirm that Disease-causing gene is located at X chromosome 123.6 by the family map and linkage analysis of patient
To between 148.4Mb.
3, library construction and high-flux sequence
High-throughput exon is carried out to the genomic DNA of patient 2,3 and normal control's (control i.e. in Fig. 2 family tree)
Sequencing.
1. each genomic DNA sample to be broken into the segment of 100-200bp or so at random using ultrasonoscope, then according to
Manufacturer provide operational manual, segment both ends be separately connected top connection preparation library (reference can be made to: http: //
The Illumina/Solexa standard that www.illumina.com/ is provided builds library specification, by referring to being incorporated by this
Text).Be available on the machine sequencing after library detection is qualified, to obtain raw sequencing data.Wherein, referring to Illumina standard at
The step of cluster and sequencing, is sequenced, and microarray dataset is Illumina Hiseq 2000.Utilize Illumina base
Calling Software 1.7 handles the raw sequencing data of above-mentioned acquisition, after filtering depollutes, uses
SOAPaligner/SOAP2 (reference can be made to: Li R, Li Y, Kristiansen K, et al, SOAP:short
oligonucleotide alignment program.Bioinformatics 2008,24(5):713-714；Li R,Yu
C,Li Y,et al.,SOAP2:an improved ultrafast tool for short read
Alignment.Bioinformatics 2009,25 (15): 1966-1967., by referring to be incorporated by herein) compare
To reference genome Hg19 (snp132), compare to obtain to unique aligned sequences on genome.Then SOAPsnp is utilized
(reference can be made to: Li R, Li Y, Fang X, Yang H, et al, SNP detection for massively parallel
Whole-genome resequencing.Genome Res 2009,19 (6): 1124-1132., by referring to by its full text simultaneously
Enter herein) determine the genotype of target region.
2.Indel is using BWA (version 0.7.5) (reference can be made to Li H, Durbin R.Fast and accurate
long-read alignment with Burrows-Wheeler transform.Bioinformatics 2010；26(5):
589-595) and GATK (version2.8-1) is (reference can be made to DePristo MA, Banks E, Poplin R, et al.A
framework for variation discovery and genotyping using next-generation DNA
sequencing data.Nat Genet 2011；43:491-8., by referring to be incorporated by herein) determine indel's
Type.
3. being analyzed using CLC Bio the variation found.Filter out dbSNP database (http: //
Www.ncbi.nlm.nih.gov/projects/SNP/snp_summary.cgi), thousand human genome databases (http: //
) and the common datas such as ESP6500 database (http://evs.gs.washington.edu/) www.1000genomes.org/
Existing variation in library.Remove same sense mutation, the non-synonymous/splice site mutation and small insertion and deletion to desmic region is located at
Preferentially selected.
Found by above-mentioned analysis, X chromosome 123.6 to found between 148.4Mb 3 genes (BCORL1, FHL1 and
MAGEC1 5 variations that may cause a disease) are had.Mutation since FHL1 gene has been reported will lead to similar symptom, FHL1
Gene c.502-2A > T mutation most likely result in ecuador's syndrome is malnourished disease.
Embodiment 2
Utilize the genome of patient 2,3,4 and normal control's (control i.e. in Fig. 2 family tree) in family in embodiment 1
DNA verifies ecuador's syndrome is malnourished disease pathogenic mutation site using Sanger method, including design of primers, PCR
Amplification, product purification, sequencing obtain sequence, belong to saltant type or wild type according to sequencing results, verifying mutation with angstrom-
Correlation between moral Er Shi muscular dystrophy.Specific step is as follows:
1.DNA is extracted:
The peripheric venous blood of patient and control in family is taken respectively, utilizes Magnetic Separation Module
I extracts leucocyte DNA.
2. design of primers and PCR reaction
FHL1 gene-specific primer is designed with reference to human gene data unit sequence library GRCh37/hg19, specifically see the table below.
A) primer sequence:
B) then, enterprising in BigDye Terminator to each genomic DNA sample using Advantage-GC polymerase
The quasi- PCR reaction of rower.
Reaction system:
Reaction condition (5 μM):
The pcr amplification product of normal person in patient and family is obtained as a result,.
3. sequencing
The pcr amplification product of the patient obtained in step 2 and normal person are purified through ethanol precipitation and utilize Genetic
Analyzer 3500 carries out DNA sequencing.
Sanger sequencing result, which is shown in 2,3,4 body of patient, has c.502-2A > T mutation, and in family health member
It is to detect the mutation in control1 body.
Embodiment 3
Utilize the fibroblast and epithelial cells of patient 2,3 and control in family described in qRT-PCR detection embodiment 1
The relative expression quantity of middle three kinds of alternative splicing bodies of FHL1 gene.Steps are as follows for specific method:
1. taking patient 2, patient 3 and the fibroblast and the epithelial cells that compare 1, and cultivated with being placed in culture solution,
In, Fibroblast culture solution formula is RPMI1640 culture medium, 10% fetal calf serum, 2mM glutamine, 100U/ml mould
Element-streptomysin；Keratinocyte culture formula of liquid CNT57 culture medium and gentamicin.
2. taking reverse transcription after the RNA of fibroblast and epithelial cells specific as follows at cDNA:
(1) Maxwell 16simplyRNA Cells Kit (Promega Biotech AB, Nacka, Sweden) is used
RNA is extracted, concrete operation step illustrates to carry out according to kit.
(2) design of primers and PCR reaction
FHL1A, FHL1B and FHL1C specific primer are designed with reference to human gene data unit sequence library GRCh37/hg19,
Specifically it see the table below.
A) primer sequence:
B) RT-PCR:
RT-PCR is carried out using SYBRGreenmastermix, HPRT is as quantitative control.RNA extraction and application
Maxwell16simplyRNA Cells kit (Promega Biotech AB, Nacka, Sweden) kit is specific according to it
Step illustrates to carry out, and the RNA extracted is placed in -80 DEG C of preservations.CDNA synthesis uses 1st Strand cDNA Synthesis
Kit for RT-PCR (AMV) (Roche Diagnostics, Basel, Switzerland) kit uses oligo-p (dT)
15 primers carry out, and concrete operation step is referring to kit specification.
The present invention calculates the relative expression quantity of every kind of variable sheer body using delta-delta method.
Research finds that FHL1A, FHL1B and FHL1C are as shown in table 1 in the fibroblast of patient 2 and patient 3, FHL1C's
Expression is significantly higher than the expression (value < 0.0001 P) of FHL1A and FHL1B.
The expression of 1 patient 2 of table and FHL1 gene difference variable sheer body in 3 fibroblast of patient.
| Patient 2 | Patient 3 | |
| FHL1A | 0 | 0 |
| FHL1B | 0 | 0 |
| FHL1C | 188 | 176 |
The expression trend having the same of 3 kinds of variable sheer bodies of FHL1 gene in 3 epithelial cells of patient 2 and patient.
3, the phosphorylation level of ERK1/2 in patient fibroblasts and epithelial cells is detected.
Patient fibroblasts and epithelial cells are dyed with ERK1/2 antibody first, then utilize flow cytometer pair
The phosphorylation level of ERK1/2 is detected.It utilizes Phospho---p44/42MAPK (Erk1/2) (Thr202/Tyr204)
(D13.14.4E) XP Rabbit mAb (Cell Signaling Technology, Inc., Danvers, MA, USA) as at
The primary antibody of fibrocyte dyeing；By Alexa---goat--- α --- rabbit (Molecular Probes, Eugene,
OR, USA) it is diluted 1 hour with dyeing/prevention buffer (saponin of the bovine serum albumin(BSA)+2% of PBS water+0.1%)；Carefully
Born of the same parents are washed 3 times with dyeing/prevention buffer；Finally with FACSAria III (BD Biosciences, Franklin Lakes, NJ,
USA) Fluorescence Activated Cell classification (FACS) is carried out；With FlowJo software ver.10 (Tree Star Inc.,
Ashland, OR, USA) carry out data analysis.
As a result as shown in figure 5, patient 2 (curve 1), patient 3 (curve 2) and the fibroblast for compareing (curve 3) three
In, the phosphorylation level difference very little of ERK albumen shows that patient and the phosphorylation level for compareing ERK in fibroblast do not have
Difference
4, it clones and transduces
To further appreciate that whether the high expression of FHL1C can cause ERK phosphorylation level to increase, inventor by FHL1C and
The cDNA clone of FHL1A is to expression vector and is transferred to healthy fibroblast, the specific steps are as follows:
1. FHL1C the and FHL1A cDNA sequence using patient carries out PCR amplification as template, and purifies amplicon.
PCR reaction condition: with DreamTaq DNA Polymerase (Thermo Scientific, Waltham, MA,
USA) kit carries out.
Reaction condition:
Amplicon is purified with the graded alcohols precipitation method.
2. amplicon is inserted into carrier pcDNA3.1.With pcDNA 3.1Directional TOPO Expression
Kit (Invitrogen, Carlsbad, CA, USA) kit is carried out according to its specification.
3. cellular control unit is transfected with pcDNA3.1---FHL1A pcDNA3.1---FHL1C plasmid and is used in combination
X---tremeGENE9DNA transfection reagent (Roche Diagnostics, Basel, Switzerland) examination
The cotransfection of agent box progress eGFP---N1 plasmid.Concrete operations are carried out according to kit specification.It goes forward side by side within cell culture 48 hours
Row dyeing.Insert Fragment and its direction are judged whether there is using Bst restriction enzyme.
4. with pcDNA3.1-FHL1A, pcDNA3.1-FHL1C plasmid fibroblast and as control at fibre
Dimension cell is placed in flow cytometer the phosphorylation level for detecting ERK albumen.
Testing result is as shown in fig. 6, ERK protein phosphorylation level is close in different transformed cells, wherein curve 2 indicates
The cell of FHL1A is imported, curve 1 indicates to import the cell of FHL1C, and curve 3 indicates control, and FHL1A and FHL1C do not have found ERK
Phosphorylation occurs.
5, interpretation of result
The mutation of human body FHL1 gene mostly can cause the false folding of FHL1 albumen, and then protein masses occur.Small
In the model of mouse, the complete missing of FHL1 albumen will lead to slight, age-dependent muscular dystrophy.Symptom slightly changes possibility
Being is not in protein misfolding in Mice Body after lacking completely because of FHL1 albumen, so without protein masses.This hair
The FHL1 genetic mutation of bright middle discovery is not in the false folding of albumen, but variable sheer body FHL1C expression quantity can be made to increase.
FHL1C overexpression other negative effects may be generated to cell, as shown in figure 4, the overexpression of FHL1C can interfere RBP-J with
The combination of its target gene DUSP1 so that the expression quantity of MKP-1 declines, and then makes ERK albumen that phosphorylation occur.Phosphorylation
ERK albumen can activate RAS-MAPK access, and RAS-MAPK access is caused to be lacked of proper care, and RAS-MARK signal path is in the hair of embryo
It educates, all there is important adjustment effect in the biological processes such as differentiation, proliferation, death of cell.Experiment discovery, patient's
The overexpression of FHL1C, but the phosphorylation level of ERK does not increase, and FHL1C and FHL1A do not have found that phosphorylation occurs for ERK.
Therefore, in conclusion inventor thinks that the variation for the FHL1 gene that the present invention is found will lead to its variable sheer body
The expression quantity of FHL1C rises.Patient with this mutation not only will appear the related symptoms of EDMD, there is also
RASopathy related symptoms.But the overexpression of FHL1C can't be such that the phosphorylation level of albumen ERK increases, thus, it can not
RAS-MAPK access is activated, patient condition is in turn resulted in.Saltant type EDMD correlation FHL1 gene FHL1 related to wild type EDMD
Difference of the gene in sequence is c.502-2A > T, which is the pathogenic mutation of ecuador's syndrome is malnourished disease.
Embodiment 4
Prepare a detection kit, it includes be able to detect FHL1 gene c.502-2A > T mutation primer, for sieving
The biological sample of ecuador's syndrome is malnourished disease is selected, wherein these primers are FHL1 gene extron specific primer, sequence
It arranges shown in SEQ ID NO:2 and 3 as described in example 2 above.
Utilize the specific steps of the biological sample of mentioned reagent box screening ecuador's syndrome is malnourished disease are as follows: according to reality
It applies method described in the step 1 of example 2 and extracts person under test DNA, using extracted DNA as the exon of template and above-mentioned FHL1 gene
Specific primer carries out PCR reaction, and reaction system and reaction condition is as described in Example 2, and according to conventional method in that art pair
PCR product purifying, the product of purifying is sequenced, and is then based on whether sequencing sequence has c.502-2A > T mutation, effectively
Ground detects FHL1 gene mutation body of the invention whether there is in person under test DNA, be so as to which person under test is effectively detected
It is no to be susceptible to suffer from Keppen-Lubinsky syndrome, further, ecuador's syndrome is malnourished disease can be filtered out from person under test
Biological sample.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
Centainly refer to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be any
One or more embodiment or examples in can be combined in any suitable manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that: not
A variety of change, modification, replacement and modification can be carried out to these embodiments in the case where being detached from the principle of the present invention and objective, this
The range of invention is defined by the claims and their equivalents.
Claims (13)
1. a kind of nucleic acid of the coding FHL1 gene mutation body of separation, which is characterized in that compared with SEQ ID NO:1, the core
Acid has c.502-2A > T mutation.
2. nucleic acid according to claim 1, which is characterized in that the nucleic acid is DNA.
3. a kind of isolated polypeptide, which is characterized in that the polypeptide is by the described in any item nucleic acid encodes of claim 1~2
's.
4. a kind of for screening the kit for being susceptible to suffer from the biological sample of ecuador's syndrome is malnourished disease, which is characterized in that contain
Have:
It is adapted to detect for the reagent of FHL1 mutant, compared with SEQ ID NO:1, the FHL1 mutant is prominent with c.502-2A > T
Become.
5. kit according to claim 4, which is characterized in that the reagent is nucleic acid probe or primer.
6. kit according to claim 5, which is characterized in that the nucleic acid probe has shown in SEQ ID NO:2 or 3
Nucleotide sequence.
7. kit according to claim 5, which is characterized in that the primer has core shown in SEQ ID NO:2 and 3
Nucleotide sequence.
8. it is a kind of for FHL1 mutant carry out quantitative detection kit, characterized by comprising:
FHL1 mutant amplimer, compared with SEQ ID NO:1, the FHL1 mutant has c.502-2A > T mutation, and
And the FHL1 mutant amplimer includes selected from following at least one set:
(i) SEQ ID NO:4 and 5, and/or
(ii) SEQ ID NO:6 and 7, and/or
(iii) SEQ ID NO:8 and 9；And
Reference gene amplimer.
9. kit according to claim 8, which is characterized in that the reference gene is hprt gene, described referring to base
Gene-amplification primer is nucleotide sequence shown in SEQ ID NO:10 and 11.
10. a kind of construct, which is characterized in that include the described in any item nucleic acid of claim 1~2.
11. a kind of recombinant cell, which is characterized in that the recombinant cell is by construct transformation receptor described in claim 10
What cell obtained.
12. a kind of method for constructing medicaments sifting model, comprising:
At least part cell of animal is set to express the described in any item nucleic acid of claim 1~2.
13. according to the method for claim 12, which is characterized in that the animal includes mouse, and pig, dog and primate are dynamic
Object.
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