CN103571854B - SUCLA2 gene mutation body and application thereof - Google Patents
SUCLA2 gene mutation body and application thereof Download PDFInfo
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- CN103571854B CN103571854B CN201210249131.4A CN201210249131A CN103571854B CN 103571854 B CN103571854 B CN 103571854B CN 201210249131 A CN201210249131 A CN 201210249131A CN 103571854 B CN103571854 B CN 103571854B
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Abstract
The present invention relates to the nucleic acid of the coding succinate coenzyme A ligase enzyme β subunit mutant of separation, method that isolated polypeptide, screening susceptible Mitochondrial DNA exhaust syndromic biological sample, screening susceptible Mitochondrial DNA exhaust the system of syndromic biological sample and exhaust the test kit of syndromic biological sample for screening susceptible Mitochondrial DNA.Wherein, the nucleic acid of the coding succinate coenzyme A ligase enzyme β subunit mutant of separation, with SEQ? ID? NO:1 compares, and has c.C308A sudden change.Whether exist in biological sample by detecting this mutant, whether susceptible Mitochondrial DNA exhausts syndrome effectively can to detect biological sample.
Description
Technical field
The present invention relates to SUCLA2 gene mutation body and application thereof.Particularly, the present invention relates to the nucleic acid of the coding succinate coenzyme A ligase enzyme β subunit mutant of separation, method that isolated polypeptide, screening susceptible Mitochondrial DNA exhaust syndromic biological sample, screening susceptible Mitochondrial DNA exhaust the system of syndromic biological sample and exhaust the test kit of syndromic biological sample for screening susceptible Mitochondrial DNA.
Background technology
Mitochondrial membrane fluidity (Multiplemitochondrialrespiratorychain, MRC) defect is the major reason causing mitochondrial disease, affect by different kinds of molecules genetic mechanism, as Mitochondrial DNA (mtDNA) disappearance, mitochondrial tRNA point mutation, mitochondrial RNA(mt RNA) translation defect, Mitochondrial DNA (mtDNA) is exhausted, the assembling of respiratory chain subunit or regulatory function defect, mitochondrial protein input is impaired.The mitochondrial disease of childhood onset is exhausted primarily of mtDNA and is caused (mitochondrialDNAdepletionsyndromes, MDS, MIM251880, Mitochondrial DNA exhausts syndrome), MDS is different from other mitochondrial diseases, it one quantitatively occurs the disease of defect but not qualitative problem, show as mtDNA copy number to reduce, and the number of plastosome own may be normal, lack or increase, cause the synthesis of electron transport chain complex in cell and ATP to synthesize to decline, reduce mitochondrial energy metabolism function, for autosomal recessive inheritance, there is heredity and heterogeneity clinically.Liver encephalopathic modification, myonosus modification, its alloytype can be divided into clinically as three types such as Alpers syndromes.Research shows, the transgenation of many nuclear gene encodings and above-mentioned disease-related.
But to Mitochondrial DNA, present stage exhausts that syndromic research still needs deeply.
Summary of the invention
The present invention is intended at least to solve one of technical problem existed in prior art.For this reason, one object of the present invention is that proposition Mitochondrial DNA exhausts the new mutant on the Disease-causing gene of syndrome (sometimes also referred to as " MDS ").
The present invention completes based on the following work of contriver: contriver determines Mitochondrial DNA by the method that the sudden change of high-throughput exon group order-checking associating candidate gene is verified and exhausts new mutant (SUCLA2, c.C308A) on syndromic Disease-causing gene.
According to a first aspect of the invention, the present invention proposes a kind of nucleic acid (i.e. SUCLA2 gene mutation body) of coding succinate coenzyme A ligase enzyme β subunit mutant of separation.According to embodiments of the invention, this nucleic acid, compared with SEQIDNO:1, has c.C308A sudden change.According to embodiments of the invention, contriver determines SUCLA2 gene mutation body, whether the morbidity of this mutant and MDS is closely related, thus exist in biological sample by detecting this mutant, and whether susceptible Mitochondrial DNA exhausts syndrome effectively can to detect biological sample.
According to a second aspect of the invention, the present invention proposes a kind of isolated polypeptide.According to embodiments of the invention, this polypeptide, compared with SEQIDNO:2, has p.Ala103Asp sudden change.By detecting in biological sample whether express this polypeptide, whether susceptible Mitochondrial DNA exhausts syndrome effectively can to detect biological sample.
According to a third aspect of the invention we, the present invention proposes and a kind ofly screen the method that susceptible Mitochondrial DNA exhausts syndromic biological sample.According to embodiments of the invention, the method comprises the following steps: from extraction from biological material sample of nucleic acid; Determine the nucleotide sequence of described sample of nucleic acid; The nucleotide sequence of described sample of nucleic acid is compared with SEQIDNO:1, and having c.C308A sudden change is that described biological sample susceptible Mitochondrial DNA exhausts syndromic instruction.By exhausting the method for the biological sample of syndrome (i.e. MDS) according to the screening susceptible Mitochondrial DNA of the embodiment of the present invention, the biological sample of susceptible MDS effectively can be screened.
According to a forth aspect of the invention, the present invention proposes and a kind ofly screen the system that susceptible Mitochondrial DNA exhausts syndromic biological sample.According to embodiments of the invention, this system comprises: nucleic acid-extracting apparatus, and described nucleic acid-extracting apparatus is used for from described extraction from biological material sample of nucleic acid; Nucleotide sequence determining device, described nucleotide sequence determining device is connected with described nucleic acid-extracting apparatus, for analyzing described sample of nucleic acid, to determine the nucleotide sequence of described sample of nucleic acid; Judgment means, described judgment means is connected with described nucleotide sequence determining device, so that based on the nucleotide sequence of described sample of nucleic acid compared with SEQIDNO:1, whether there is c.C308A sudden change, judge whether susceptible Mitochondrial DNA exhausts syndrome to described biological sample.Utilize this system, effectively can implement the method that aforementioned screening susceptible Mitochondrial DNA exhausts syndromic biological sample, thus effectively can screen susceptible Mitochondrial DNA and exhaust syndromic biological sample.
According to a fifth aspect of the invention, the present invention proposes a kind of test kit exhausting syndromic biological sample for screening susceptible Mitochondrial DNA.According to embodiments of the invention, this test kit contains: be suitable for the reagent detecting SUCLA2 gene mutation body, and wherein compared with SEQIDNO:1, described SUCLA2 gene mutation body has c.C308A sudden change.Utilize test kit according to an embodiment of the invention, effectively can screen susceptible Mitochondrial DNA and exhaust syndromic biological sample.
Additional aspect of the present invention and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage will become obvious and easy understand from accompanying drawing below combining to the description of embodiment, wherein:
Fig. 1 shows the system of biological sample according to the screening susceptible MDS of the embodiment of the present invention and the schematic diagram of integral part thereof, wherein, A is the schematic diagram of the system of the biological sample of screening susceptible MDS according to the embodiment of the present invention, B is the schematic diagram of the nucleic acid-extracting apparatus according to the embodiment of the present invention, and C is the schematic diagram of the nucleotide sequence determining device according to the embodiment of the present invention;
Fig. 2 shows the Sanger sequence verification peak figure in the SUCLA2 gene c.C308A mutational site to patient PI according to the embodiment of the present invention;
Fig. 3 shows the Sanger sequence verification peak figure in the SUCLA2 gene c.C308A mutational site to patient PII according to the embodiment of the present invention; And
Fig. 4 and Fig. 5 respectively illustrates the Sanger sequence verification peak figure in the SUCLA2 gene c.C308A mutational site of the father and mother to patient PI and PII according to the embodiment of the present invention.
Embodiment
Be described below in detail embodiments of the invention, the example of described embodiment is shown in the drawings, and wherein same or similar label represents same or similar element or has element that is identical or similar functions from start to finish.Being exemplary below by the embodiment be described with reference to the drawings, only for explaining the present invention, and can not limitation of the present invention being interpreted as.
SUCLA2 gene mutation body
According to a first aspect of the invention, the present invention proposes a kind of nucleic acid of coding succinate coenzyme A ligase enzyme β subunit mutant of separation.According to embodiments of the invention, compared with SEQIDNO:1, this nucleic acid has c.C308A sudden change.Phraseology " nucleic acid of coding succinate coenzyme A ligase enzyme β subunit mutant " used in this article, refer to the nucleic acid substances corresponding with the gene of succinate coenzyme A ligase enzyme β subunit mutant of encoding, namely the type of nucleic acid is not particularly limited, can be anyly comprise the deoxyribonucleotide corresponding with the encoding gene (i.e. SUCLA2 gene mutation body) of succinate coenzyme A ligase enzyme β subunit mutant and/or the polymkeric substance of ribonucleotide, include but not limited to DNA, RNA or cDNA.According to a concrete example of the present invention, the nucleic acid of foregoing coding succinate coenzyme A ligase enzyme β subunit mutant is DNA.According to embodiments of the invention, contriver determines the mutant of SUCLA2 gene, this mutant and Mitochondrial DNA exhaust that syndromic morbidity is closely related, thus whether exist in biological sample by detecting this mutant, whether susceptible Mitochondrial DNA exhausts syndrome effectively can to detect biological sample, whether also can exist in organism by detecting this mutant, whether susceptible Mitochondrial DNA exhausts syndrome effectively can to predict organism.
The nucleic acid of this coding succinate coenzyme A ligase enzyme β subunit mutant is present inventor exhausts on syndromic Disease-causing gene new mutant by the Mitochondrial DNA that the method for high-throughput exon group order-checking associating candidate gene sudden change checking is determined.Further, in the prior art and the c.C308A sudden change report relevant to MDS had no on SUCLA2 gene.
The cDNA of wild-type SUCLA2 gene has nucleotide sequence as follows:
ATGGCGGCCTCCATGTTCTACGGCAGGCTAGTGGCCGTGGCCACCCTTCGGAACCACCGGCCTCGGACGGCCCAGCGGGCTGCTGCTCAGGTTCTGGGAAGTTCTGGATTGTTTAATAACCATGGACTCCAAGTACAGCAGCAACAGCAAAGGAATCTCTCACTACATGAATACATGAGTATGGAATTATTGCAAGAAGCTGGTGTCTCCGTTCCCAAAGGATATGTGGCAAAGTCACCAGATGAAGCTTATGCAATTGCCAAAAAATTAGGTTCAAAAGATGTCGTGATAAAGGCACAGGTTTTAGCTGGTGGTAGAGGAAAAGGAACATTTGAAAGTGGCCTCAAAGGAGGAGTGAAGATAGTTTTCTCTCCAGAAGAAGCAAAAGCTGTTTCTTCACAAATGATTGGGAAAAAATTGTTTACCAAGCAAACGGGAGAAAAGGGCAGAATATGCAATCAAGTATTGGTCTGTGAGCGAAAATATCCCAGGAGAGAATACTACTTTGCAATAACAATGGAAAGGTCATTTCAAGGTCCTGTATTAATAGGAAGTTCACATGGTGGTGTCAACATTGAAGATGTTGCTGCTGAGTCTCCTGAAGCAATAATTAAAGAACCTATTGATATTGAAGAAGGCATCAAAAAGGAACAAGCTCTCCAGCTTGCACAGAAGATGGGATTTCCACCTAATATTGTGGAATCAGCAGCAGAAAACATGGTCAAGCTTTACAGCCTTTTTCTGAAATACGATGCAACCATGATAGAAATAAATCCAATGGTGGAAGATTCAGATGGAGCTGTATTGTGTATGGATGCAAAGATCAATTTTGACTCTAATTCAGCCTATCGCCAAAAGAAAATCTTTGATCTACAGGACTGGACCCAGGAAGATGAAAGGGACAAAGATGCTGCTAAGGCAAATCTCAACTACATTGGCCTCGATGGAAATATAGGCTGCCTAGTAAATGGTGCTGGTTTGGCTATGGCCACAATGGATATAATAAAACTTCATGGAGGGACTCCAGCCAACTTCCTTGATGTTGGTGGTGGTGCTACAGTCCATCAAGTAACAGAAGCATTTAAGCTTATCACTTCAGATAAAAAGGTACTGGCTATTCTGGTCAACATTTTTGGAGGAATCATGCGCTGTGATGTTATTGCACAGGGTATAGTCATGGCAGTAAAAGACTTGGAAATTAAAATACCTGTTGTGGTACGGTTACAAGGLACACGAGTCGATGATGCLAAGGCACTGATAGCGGACAGTGGACTTAAAATACTTGCTTGTGATGACTTGGATGAAGCTGCTAGAATGGTTGTAAAGCTCTCTGAAATAGTGACCTTAGCGAAGCAAGCACATGTGGATGTGAAATTTCAGTTGCCAATATGA（SEQIDNO：1), the coding of succinic acid coa ligase beta subunits with amino acid sequence as shown below:
MAASMFYGRLVAVATLRNHRPRTAQRAAAQVLGSSGLFNNHGLQVQQQQQRNLSLHEYMSMELLQEAGVSVPKGYVAKSPDEAYAIAKKLGSKDVVIKAQVLAGGRGKGTFESGLKGGVKIVFSPEEAKAVSSQMIGKKLFTKQTGEKGRICNQVLVCERKYPRREYYFAITMERSFQGPVLIGSSHGGVNIEDVAAESPEAIIKEPIDIEEGIKKEQALQLAQKMGFPPNIVESAAENMVKLYSLFLKYDATMIEINPMVEDSDGAVLCMDAKINFDSNSAYRQKKIFDLQDWTQEDERDKDAAKANLNYIGLDGNIGCLVNGAGLAMATMDIIKLHGGTPANFLDVGGGATVHQVTEAFKLITSDKKVLAILVNIFGGIMRCDVIAQGIVMAVKDLEIKIPVVVRLQGTRVDDAKALIADSGLKILACDDLDEAARMVVKLSEIVTLAKQAHVDVKFQLPI（SEQIDNO：2）
The SUCLA2 gene mutation body that contriver finds is compared with SEQIDNO:1, there is c.C308A sudden change, namely relative to wild-type SUCLA2 gene, in the cDNA of SUCLA2 gene mutation body of the present invention, the C of the 308th sports A, thus, product coded by it is compared with the β subunit (SEQIDNO:2) of wild-type succinate coenzyme A ligase enzyme, and have p.Ala103Asp sudden change, namely its 103 amino acids sports Asp from Ala.
SUCLA2(MIM:*603921) No. 13 karyomit(e)s are positioned at, comprise 11 exons, coding, containing the β subunit of 463 amino acid succinate coenzyme A ligase enzymes, forms succinate coenzyme A ligase enzyme with α subunit by different dimerization effect, participates in tricarboxylic acid cycle process.This transgenation can cause Mitochondrial DNA to exhaust syndrome (MIM:612073), the mitochondrial encephalomyopathy of companion's methylmalonic aciduria.By the end of at present, the patient only having 25 examples to carry SUCLA2 transgenation is in the news, and wherein major part is from the Faeroe Islands, is montage micro-some G>A homozygous mutation of No. 4 introns, causes 4 exons to be fallen by montage.The c.C308A of the SUCLA2 gene proposed about the present invention sudden change is not yet had to cause the report of MDS at present.
According to a second aspect of the invention, the present invention proposes a kind of isolated polypeptide.According to embodiments of the invention, compared with SEQIDNO:2, this isolated polypeptide has p.Ala103Asp sudden change.According to concrete examples more of the present invention, this polypeptide is encoded by the nucleic acid (i.e. SUCLA2 gene mutation body) of the coding succinate coenzyme A ligase enzyme β subunit mutant of aforementioned separation.By detecting in biological sample whether express this polypeptide, whether susceptible Mitochondrial DNA exhausts syndrome effectively can to detect biological sample, whether also can exist in organism by detecting these polypeptide, whether susceptible Mitochondrial DNA exhausts syndrome effectively can to predict organism.
Screening susceptible Mitochondrial DNA exhausts the method for syndromic biological sample
According to a third aspect of the invention we, the present invention proposes and a kind ofly screen the method that susceptible Mitochondrial DNA exhausts syndromic biological sample.According to embodiments of the invention, this screening susceptible Mitochondrial DNA exhausts that the method for syndromic biological sample can comprise the following steps:
First, from extraction from biological material sample of nucleic acid.According to embodiments of the invention, the type of biological sample is also not particularly limited, as long as can extract the sample of nucleic acid whether reflection biological sample SUCLA2 exists sudden change from this biological sample.According to embodiments of the invention, biological sample can for being selected from blood of human body, skin, hypodermic at least one.Thus, can carry out easily sampling and detecting, thus the efficiency that screening susceptible Mitochondrial DNA exhausts syndromic biological sample can be improved further.According to embodiments of the invention, here used term " sample of nucleic acid " should be interpreted broadly, it can be anyly can reflect in biological sample, whether SUCLA2 exists the sample of sudden change, it can be such as the complete genome DNA of extracting directly from biological sample, also can be the part comprising SUCLA2 encoding sequence in this full-length genome, can be the total serum IgE extracted from biological sample, also can be the mRNA extracted from biological sample.According to one embodiment of present invention, described sample of nucleic acid is complete genome DNA.Thus, what can expand biological sample carrys out source range, and can determine the much information of biological sample simultaneously, thus can improve the efficiency that screening susceptible Mitochondrial DNA exhausts syndromic biological sample.In addition, according to embodiments of the invention, for employing RNA as sample of nucleic acid, may further include from extraction from biological material sample of nucleic acid: from extraction from biological material RNA sample, preferred RNA sample is mRNA; And based on obtained RNA sample, by reverse transcription reaction, obtain cDNA sample, the cDNA composition of sample sample of nucleic acid obtained.Thus, can improve further and utilize RNA to exhaust the efficiency of syndromic biological sample as sample of nucleic acid screening susceptible Mitochondrial DNA.
Next, after obtaining sample of nucleic acid, can analyze sample of nucleic acid, thus the nucleotide sequence of obtained sample of nucleic acid can be determined.According to embodiments of the invention, determine the method and apparatus of the nucleotide sequence of obtained sample of nucleic acid and be not particularly limited.According to a particular embodiment of the invention, sequence measurement can be passed through, the nucleotide sequence of definite kernel acid sample.According to embodiments of the invention, may be used for the method and apparatus that carries out checking order and be not particularly limited.According to embodiments of the invention, s-generation sequencing technologies can be adopted, also can adopt the third generation and forth generation or more advanced sequencing technologies.According to concrete example of the present invention, can utilize be selected from Hiseq2000, SOLiD, 454 and at least one of single-molecule sequencing device nucleotide sequence is checked order.Thus, in conjunction with up-to-date sequencing technologies, can reach the higher order-checking degree of depth for Single locus, detection sensitivity and accuracy improve greatly, thus can utilize the feature that the high-throughput of these sequencing devices, the degree of depth check order, improve further and carry out detecting the efficiency analyzed to sample of nucleic acid.Thus, follow-up accuracy when sequencing data is analyzed and accuracy can be improved.Thus, according to embodiments of the invention, the nucleotide sequence of definite kernel acid sample may further include: first, for obtained sample of nucleic acid, builds nucleic acid sequencing library; And checked order in obtained nucleic acid sequencing library, to obtain the sequencing result be made up of multiple sequencing data.According to some embodiments of the present invention, can adopt be selected from Hiseq2000, SOLiD, 454 and at least one of single-molecule sequencing device checked order in obtained nucleic acid sequencing library.In addition, according to embodiments of the invention, can screen sample of nucleic acid, enrichment SUCLA2 exon, this screening enrichment before structure sequencing library, can build in sequencing library process, or carries out after building sequencing library.According to one embodiment of present invention, for sample of nucleic acid, build nucleic acid sequencing library and comprise further: utilize SUCLA2 gene extron Auele Specific Primer, pcr amplification is carried out to sample of nucleic acid; And for obtained amplified production, build nucleic acid sequencing library.Thus, can pcr amplification be passed through, enrichment SUCLA2 exon, thus the efficiency that screening susceptible Mitochondrial DNA exhausts syndromic biological sample can be improved further.According to embodiments of the invention, the sequence of SUCLA2 gene extron Auele Specific Primer is not particularly limited, and according to a preferred embodiment of the invention, these SUCLA2 gene extron Auele Specific Primers have the nucleotide sequence shown in SEQIDNO:3-4.
Contriver is surprised to find, and by adopting these primers, significantly effectively can complete the amplification to the sequence between 3 exons of SUCLA2 exon especially c.C308A place and 4 exons in PCR reaction system.It should be noted that, the nucleotide sequence shown in these SEQIDNO:3-4 be the present inventor after having paid arduous labor, unexpected to obtain.
About for sample of nucleic acid; build method and the flow process of sequencing library; those skilled in the art suitably can select according to different sequencing technologies; about the details of flow process; the code that can provide see the such as Illumina company of manufacturer of order-checking instrument, for example, see Illumina company MultiplexingSamplePreparationGuide(Part#1005361; Or Paired-EndSamplePrepGuide(Part#1005063 Feb2010); Feb2010), by referring to being incorporated to herein.According to embodiments of the invention, from the method and apparatus of extraction from biological material sample of nucleic acid, be also not particularly limited, commercial nucleic acid extraction kit can be adopted to carry out.
It should be noted that, term " nucleotide sequence " used here should make broad understanding, it can be after the sequencing data that obtains of checking order to sample of nucleic acid is assembled, the complete nucleic acid sequence information obtained, also can be directly adopt sequencing data (reads) by checking order obtained to sample of nucleic acid as nucleotide sequence, as long as the encoding sequence containing corresponding SUCLA2 in these nucleotide sequences.
Finally, after the nucleotide sequence of definite kernel acid sample, the nucleotide sequence of obtained sample of nucleic acid and the sequence of SEQIDNO:1 are compared.If have c.C308A sudden change in obtained nucleotide sequence, then indicator organism sample susceptible Mitochondrial DNA exhausts syndrome.Thus, by exhausting the method for syndromic biological sample according to the screening susceptible Mitochondrial DNA of the embodiment of the present invention, effectively can screen susceptible Mitochondrial DNA and exhaust syndromic biological sample.According to embodiments of the invention, the method and apparatus of compare to nucleotide sequence and SEQIDNO:1 being also not particularly limited, and the software of any conventional can be adopted to operate, and according to specific examples of the present invention, SOAP software can be adopted to compare.
It should be noted that, the purposes according to " screening susceptible Mitochondrial DNA exhausts the method for syndromic biological sample " of the embodiment of the present invention is not particularly limited, such as, can be used as the screening method of non-diagnostic object.
Screening susceptible Mitochondrial DNA exhausts system and the test kit of syndromic biological sample
According to a forth aspect of the invention, the present invention proposes and a kind ofly effectively can implement the system that above-mentioned screening susceptible Mitochondrial DNA exhausts the method for syndromic biological sample.
With reference to figure 1, according to embodiments of the invention, this screening susceptible Mitochondrial DNA exhausts that the system 1000 of syndromic biological sample comprises: nucleic acid-extracting apparatus 100, nucleotide sequence determining device 200 and judgment means 300.
According to embodiments of the invention, nucleic acid-extracting apparatus 100 is for from extraction from biological material sample of nucleic acid.As previously mentioned, according to embodiments of the invention, the type of sample of nucleic acid is also not particularly limited, and for employing RNA as sample of nucleic acid, then nucleic acid-extracting apparatus comprises RNA extraction unit 101 and reverse transcription unit 102 further, wherein, extraction unit 101 is for from extraction from biological material RNA sample, and reverse transcription unit 102 is connected with RNA extraction unit 101, for carrying out reverse transcription reaction to RNA sample, to obtain cDNA sample, the cDNA composition of sample sample of nucleic acid obtained.
According to embodiments of the invention, nucleotide sequence determining device 200 is connected with nucleic acid-extracting apparatus 100, for analyzing sample of nucleic acid, so that the nucleotide sequence of definite kernel acid sample.As previously shown, the nucleotide sequence of the method definite kernel acid sample of order-checking can be adopted.Thus, according to one embodiment of present invention, described nucleotide sequence determining device 200 may further include: library construction unit 201 and order-checking unit 202.Library construction unit 201, for for sample of nucleic acid, builds nucleic acid sequencing library; Order-checking unit 202 is connected with library construction unit 201, for checking order to nucleic acid sequencing library, to obtain the sequencing result be made up of multiple sequencing data.As previously mentioned, can pcr amplification be passed through, enrichment SUCLA2 exon, improve the efficiency that screening susceptible Mitochondrial DNA exhausts syndromic biological sample further.Thus, library construction unit 201 may further include pcr amplification module (not shown), SUCLA2 gene extron Auele Specific Primer is provided with in this pcr amplification module, to utilize SUCLA2 gene extron Auele Specific Primer, pcr amplification is carried out to described sample of nucleic acid, according to a particular embodiment of the invention, SUCLA2 gene extron Auele Specific Primer has the nucleotide sequence as shown in SEQIDNO:3-4.According to embodiments of the invention, order-checking unit 202 can comprise and is selected from HISEQ2000, SOLiD, 454 and at least one of single-molecule sequencing device.Thus, in conjunction with up-to-date sequencing technologies, can reach the higher order-checking degree of depth for Single locus, detection sensitivity and accuracy improve greatly, thus can utilize the feature that the high-throughput of these sequencing devices, the degree of depth check order, improve further and carry out detecting the efficiency analyzed to sample of nucleic acid.Thus, improve follow-up accuracy when sequencing data is analyzed and accuracy.
According to embodiments of the invention, judgment means 300 is connected with nucleotide sequence determining device 200, be suitable for the nucleotide sequence of sample of nucleic acid to compare, to judge whether susceptible Mitochondrial DNA exhausts syndrome to biological sample based on the nucleotide sequence of sample of nucleic acid and the difference of SEQIDNO:1.Particularly, based on the nucleotide sequence of sample of nucleic acid compared with SEQIDNO:1, whether there is c.C308A sudden change, judge biological sample whether susceptible Mitochondrial DNA exhaustion syndrome.As previously mentioned, according to one embodiment of present invention, the nucleotide sequence of sample of nucleic acid is compared with SEQIDNO:1, and having c.C308A sudden change, is that biological sample susceptible Mitochondrial DNA exhausts syndromic instruction.As previously mentioned, according to embodiments of the invention, the equipment of compare to nucleotide sequence and SEQIDNO:1 being also not particularly limited, and the software of any conventional can be adopted to operate, and according to specific examples of the present invention, SOAP software can be adopted to compare.
Thus, utilize this system, effectively can implement the method that aforementioned screening susceptible Mitochondrial DNA exhausts syndromic biological sample, thus effectively can screen susceptible Mitochondrial DNA and exhaust syndromic biological sample.
According to a fifth aspect of the invention, the present invention proposes a kind of test kit exhausting syndromic biological sample for screening susceptible Mitochondrial DNA.According to embodiments of the invention, this is used for screening susceptible Mitochondrial DNA and exhausts that the test kit of syndromic biological sample comprises: be suitable for the reagent detecting SUCLA2 gene mutation body, wherein compared with SEQIDNO:1, this SUCLA2 gene mutation body has c.C308A sudden change.Utilize test kit according to an embodiment of the invention, effectively can screen susceptible Mitochondrial DNA and exhaust syndromic biological sample.In this article, the term used " is suitable for detecting the reagent of SUCLA2 gene mutation body " and should be interpreted broadly, namely can be the reagent detecting SUCLA2 encoding gene, also can be the reagent detecting succinate coenzyme A ligase enzyme β subunit mutant, such as, can adopt the antibody in identification specificity site.According to one embodiment of present invention, described reagent is nucleic acid probe, thus, can screen susceptible Mitochondrial DNA efficiently and exhaust syndromic biological sample.
It should be noted that, the feature and advantage described in method part that susceptible Mitochondrial DNA exhausts syndromic biological sample are screened before this paper, be equally applicable to screen system or the test kit that susceptible Mitochondrial DNA exhausts syndromic biological sample, do not repeat them here.
In addition, it will be understood by those skilled in the art that term " nucleotide sequence " used in this article, actual any of comprising complementary double-strand, or two.For convenience, in the present specification and claims, in most cases only give a chain, but in fact also disclose another complementary with it chain.Particularly, such as, phraseology " nucleotide sequence of sample of nucleic acid is compared with SEQIDNO:1; having c.C308A sudden change is that described biological sample susceptible Mitochondrial DNA exhausts syndromic instruction " used in this article, wherein said " nucleotide sequence of sample of nucleic acid " comprises its complementary sequence.Those skilled in the art are further appreciated that and utilize a chain can detect another chain with its complementation, and vice versa.Further, in this article, " nucleotide sequence " comprises DNA form or rna form, and the sequence of open wherein a kind of form, then mean that the sequence of another kind of form is also disclosed.Such as, disclose the cDNA sequence of SUCLA2 gene, reality also discloses its corresponding RNA sequence.
Below with reference to specific embodiment, the present invention will be described, it should be noted that, these embodiments are only illustrative, and can not be interpreted as limitation of the present invention.
If do not specialize, the conventional means that the technique means adopted in embodiment is well known to those skilled in the art, can carry out with reference to " Molecular Cloning: A Laboratory guide " third edition or related products, the reagent adopted and product be also can business obtain.The various process do not described in detail and method are ordinary methods as known in the art, source, the trade(brand)name of agents useful for same and be necessary to list its moiety person, all indicate when occurring first, identical reagent used if no special instructions thereafter, all identical with the content indicated first.
Disease-causing gene and mutational site are determined in the full exon group order-checking of embodiment 1
1, sample collection:
Contriver collects a gondola consanguineous marriage family, and father and mother are cousin marriage, and father and mother are all normal, and two daughters are ill:
First disease person (PI) main clinical manifestation of first case is: pregnancy period and puerperium all no abnormality seens.Be born and within 1 month, occur myasthenia, growth retardation, body weight increase is comparatively slow, frequently vomits.Be born 8 months time, neurologic check point out: cranial nerve inspection is normal, visual inspection is good; But near-end trunk muscles is unable, is slow in action, tendon hyperreflexia; Dysphagia, starts nasal feeding.Biochemical analysis shows: in blood plasma and celiolymph, lactic acid salt (is 3057 μm of ol/l respectively, 193 μm of ol/l) and pyruvate salt (be 2262 μm of ol/l respectively, 144 μm of ol/l) content rising, short chain acyl carnitine is slightly elevated to 20nmol/ml (normal reference value: 3.4-10nmol/l), does not carry out the detection of methyl-prop two uric acid in urine.Listening property Evoked ptential test display maincenter conduction abnormalities.Brain MRI checks display: bilateral caudatum and dentate nucleus image abnormal (this point was done NMR inspection and made a definite diagnosis further two years old time).Patient be born 18 months time, the state of an illness is continuous worsening: Tendon defection, bilateral ptosis, and ballet's disease is obvious, with serious cognitive disorder and development of speech obstacle.Patient's nutrition intake for many years all relies on nasal feeding, has done through skin stomach ostomy when 7 years old.Although conditions of patients is serious, its electroencephalogram is always normal, and never occurs metabolism failure.10 years old start, and patient engenders multiple tendon retraction and scoliosis, and the state of an illness runs down afterwards.Clinical assessment display when 14 years old: patient's growth indexes is normal, and ptosis is serious, and extraocular muscle is paralysed completely, vision and tactile response are not good, diffusivity amyotrophy, trunk and four limbs hypotonia, tendon reflex disappears, and be slow in action and show myodystonia attitude, scoliosis is serious.Die of pneumonia during patient 15 years old initiation respiratory complication.
There is identical clinical symptom in the first disease person younger sister of 7 years old (PII): the pregnancy period and puerperium no abnormality seen, just show obvious diffusivity hypotonia after birth.Within 2 months, occur trunk and four limbs hypotonia, tendon hyperreflexia also shows myodystonia attitude.Blood plasma lactic acid salts contg reaches 3500 μm of ol/l, but the methyl-prop two uric acid MMA in urine is in normal range.During by 1 years old, its body weight does not almost increase, and engenders mental deterioration.Due to Recurrent Vomiting, needs of patients is taken food by nasal feeding.When 14 months, large Typical AVM inspection prompting bilateral caudatum, lenticular nucleus, dentate nucleus are abnormal.When 5 years old, its clinical symptom and its elder sister symptom at that time similar: bilateral ptosis is serious, incomplete ophthalmoplegia, tetraplegia, myodystonia, and head can not automatic control, and scoliosis, without metabolism failure.
Be mitochondrial disease from the clinical symptom preliminary judgement of patient, the multiple enzyme disappearance of muscle biopsy witness line mitochondrial respiratory chain, main manifestations is: prozyme I, III, IV disappearance of PI, and prozyme cI, III, IV, V disappearance of PII.Then get tissue when patient carries out muscle biopsy, extract its STb gene, be separated mtDNA.Whole mtDNA is checked order, does not find mtDNA transgenation, eliminate mtDNA transgenation and cause a disease.Following employing southern blotting technique and Real-TimePCR carry out quantitative analysis to mtDNA, and both results all show the mtDNA exhaustion of patient PI and PII.Can infer that patient's suffer from the disease is into MDS thus.
2, Disease-causing gene and mutational site are determined in full exon group order-checking
Contriver utilizes NimblegenEZSeqCap44MKit to carry out exon order-checking in conjunction with Solexa high throughput sequencing technologies to case PII, and concrete steps are as follows:
2.1 sample preparation
Get patient PII skin flbroblast, utilize conventional phenol-chloroform method extracting extracting genomic dna, for high-flux sequence.Utilize concentration and the purity of spectrophotometer measurement DNA, the OD260/OD280 of each sample genomic dna of gained is all between 1.7-2.0, and concentration is no less than 200ng/ μ l, and total amount is no less than 30 μ g.
2.2 library constructions and order-checking
Utilize ultrasonoscope (CovarisS2, Massachusetts, USA) each genomic dna sample is broken at random the fragment of about 200-300bp, subsequently according to the process specifications that manufacturers provides, connect respectively at fragment two ends top connection prepare library (can be see:
http:// www.illumina.com/the Illumina/Solexa standard provided builds storehouse specification sheets, is incorporated in full herein by referring to by it).Hybridization enrichment is carried out through the linear amplification of Ligation-mediatedPCR (LM-PCR) and SureSelectBiotinylatedRNALibrary (BAITS) after library is purified, again through the linear amplification of LM-PCR, namely be available on the machine after library detection is qualified order-checking, to obtain raw sequencing data.Wherein, check order with reference to the cluster of Illumina standard and the protocol of order-checking, order-checking platform is IlluminaHiseq2000, and reading length is 90bp, the average order-checking degree of depth of sample is 71.94 ×.
2.3 variations detect, annotate and database compares
The raw sequencing data of IlluminabasecallingSoftware1.7 to above-mentioned acquisition is utilized to process, after filtering and depolluting, use SOAPaligner/SOAP2(can be see: LiR, LiY, KristiansenK, etal, SOAP:shortoligonucleotidealignmentprogram.Bioinformatics 2008,24 (5): 713-714; LiR, YuC, LiY, eaal, SOAP2:animprovedultrafasttoolforshortreadalignment.Bioin formatics2009,25 (15): 1966-1967., to be incorporated in full herein by referring to by it) comparison to reference genome Hg19 (snp132), to obtain comparison to the unique aligned sequences on genome.Then utilize SOAPsnp (can be see: LiR, LiY, FangX, YangH, etal, SNPdetectionformassivelyparallelwhole-genomeresequencing .GenomeRes2009,19 (6): 1124-1132., is incorporated in full herein by referring to by it) determine the genotype of target region.
As a result, contriver finds that there is the insertion/deletion at 94363 single nucleotide polymorphism (SNPs) and 6733 places in case PII.Subsequently by dbSNP database (
http:// www.ncbi.nlm.nih.gov/proiects/SNP/snp_summary.cgi), thousand human genome databases (
www.1000genomes.org/), HapMap8 database (
http:// hapmap.ncbi.nlm.nih.gov/) etc. the filtration of public database, remove all known and gene frequency variations of being greater than 0.005 in a database.Remove same sense mutation, remaining non-synonym/splice site sudden change and small insertion and deletion are carried out prioritizing selection according to following characteristics: (a), according to recessive inheritance mode and father and mother's consanguineous marriage relation, pays the utmost attention to homozygous mutation; B () and mitochondrial protein database MitoCarta compare, obtain may or determining to be positioned mitochondrial albumen.
2.4 homozygote location
Contriver is by the bioinformatics method of close relative's family homozygote positioning analysis, reduce candidate gene scope further, method is as follows: the site selected the mononucleotide polymorphism site (SNP) in single sample and be arranged in public database dbSNP is as genetic marker.Hybrid marker selection standard: the order-checking degree of depth is more than or equal to 10X, the reads supporting rate of 30%-70%; Isozygoty mark: the order-checking degree of depth is more than or equal to 5X, the reads supporting rate of 95%.The impact of order-checking mistake effectively can be reduced by this standard.After genetic marker is chosen, allow at most the distance of 2 hybrid marker and adjacent marker to be no more than the standard of 500Kb according to the window of every 500 mark compositions, the region being greater than 1M is defined as homozygote region (region being greater than 5M thinks more credible).Consider the length of exon and distribute on the impact analyzed, after only having neighboring exons to connect, total length is just analyzed more than 1Mb, and the distance of neighboring exons is no more than 500Kb, and all the other exon regions are still left candidate region.
As a result, contriver finds that in case PII the zone location of about 399M is homozygote region, accounts for whole genomic 14.23%.The length of homozygote region averages and median is 1.71MB and 1.33MB respectively, finally has 25 variant sites of isozygotying to be positioned at homozygote region.2 mutational sites are wherein had to be positioned at the gene of coding mitochondrial protein: TOP1MT and SUCLA2.Wherein, TOP1MT genes encoding plastosome topoisomerase, TtoC missense mutation, causes the amino acid change of Ile448Met; The β subunit of SUCLA2 genes encoding succinate coenzyme A ligase enzyme, 308C>A missense mutation, causes the amino acid change of Ala103Asp.UCSC predicts that this site is arranged in the amino acid residue sequence of high conservative, suddenlys change and will the function of SUCLA2 albumen be caused to be affected.
Then, contriver by software predictions such as Polyphen, SIFT, Mutpred, Pmut and Panther, confirms above-mentioned 2 mutator genes again further.
Because the order-checking of exon group exists false positive to a certain degree, next, contriver utilizes again Sanger sequence measurement, verifies in family the mutational site of these two genes, TOPlMTATA=>ATG sudden change is heterozygous mutant in PI, is therefore excluded.SUCLA2GCT=>GAT is homozygous mutation in PI and PII, and in patient father and mother, be heterozygous mutant, and EVS exon database (
http:// evs.gs.washington.edu/EVS, by June 20th, 2012 this database comprise 2203 non-descendants Americans and 4300 American individual specimen of Europe descendants) in do not find this mutational site.In Sanger order-checking, 100% is to the order-checking coverage of 11 exons of SUCLA2 gene.
In addition, known, SUCLA2(MIM:*603921) be positioned at No. 13 karyomit(e)s, comprise 11 exons, coding, containing the β subunit of 463 amino acid succinate coenzyme A ligase enzymes, forms succinate coenzyme A ligase enzyme with α subunit by different dimerization effect, participates in tricarboxylic acid cycle process.This transgenation can cause Mitochondrial DNA to exhaust syndrome (MIM:612073), the mitochondrial encephalomyopathy of companion's methylmalonic aciduria.By the end of at present, the patient only having 25 examples to carry SUCLA2 transgenation is in the news, and wherein major part is from the Faeroe Islands, and montage micro-some G>A homozygous mutation of No. 4 introns, causes 4 exons to be fallen by montage.The reported first such as Elpeleg in 2005 find that g.32720del43ins5 the indel that isozygotys of SUCLA2 gene suddenlys change in from the MDS patient of Moslem.The symptom that 10 patients of the Faeroe Islands, Ostergaard etc. 2007 annual report road increase with mitochondrial encephalomyopathy and MMA content, wherein only has 1 routine patient NMR to check the abnormal image occurring basal nuclei.Another is from the large family of the Faeroe Islands, and 10 patients all have the symptom (18) of basal nuclei abnormal image.Afterwards in 3 Italian familys, found the sudden change of SUCLA2 gene.In the family that contriver studies, the symptom of patient is comparatively similar to the symptom of patient in Italian family.The suddenly change typical symptom that causes of SUCLA2 is that in urine, MMA occurs abnormal.And in the family studied contriver, patient PI does not carry out MMA detection, PII was once MMA and detected 2 months time, and result display is normal.The suddenly change MMA that causes of this and the SUCLA2 to have reported raises and is not inconsistent, and therefore contriver comes back to clinical analysis of getting on.Because patient PI is just in hospitalize, therefore MMA detection has been carried out to PI, has found that MMA is 11(reference value <5 in PI), this just with document in report basically identical.
Therefore, in sum, the c.C308A sudden change that contriver thinks on the SULCA2 gene that the present invention finds is another pathogenic mutation site of MDS.
Embodiment 2Sanger method sequence verification (in family, outside family, distributing equal samples checking situation)
The patient PI(2011 gathered in family obtains and its peripheral blood preserved) and the peripheral blood of PII and father and mother thereof, utilize the genomic dna in conventional phenol-chloroform method extracting peripheral blood leucocyte, and utilize concentration and the purity of spectrophotometer measurement DNA, the OD260/OD280 of each sample genomic dna of gained is all between 1.7-2.0, concentration is no less than 200ng/ μ l, and total amount is no less than 30 μ g.
Then, respectively to the normal people (father and mother of patient in 2 patients (PI and PII) in family and 2 familys, they all do not fall ill) detect, for the primers between No. 3, SUCLA2 gene and 4 exons, SUCLA2 gene relevant sequence is obtained by the method for pcr amplification, product purification, order-checking, and belong to saltant type or wild-type according to sequencing results, the dependency between checking SUCLA2 and MDS.Concrete grammar step is as follows:
1, DNA extraction:
Take the peripheric venous blood of normal people in 2 patients and 2 familys respectively, extract genomic dna, spectrophotometric determination DNA content according to the method for embodiment 1.
2, design of primers and PCR reaction
Reference men and women's genoid data unit sequence storehouse GRCh37/hg19 designs SUCLA2 gene extron Auele Specific Primer, specifically sees the following form.
A) primer sequence:
| Upstream primer (5 ' → 3 ', SEQ ID NO :) | Downstream primer (5 ' → 3 ', SEQ ID NO :) |
| CATGCTGCTCCATGCTTCAT(3) | CATGCTCATGACATACAAACA(4) |
B) then, the PCR reaction system of each genomic dna sample is configured according to the following ratio respectively:
C) then, each PCR reaction system is carried out PCR reaction respectively according to following reaction conditions: reaction conditions:
Thus, the pcr amplification product of normal people in patient and family is obtained.
3, check order
DNA sequencing (ABIPrism3130XL) is directly carried out available from the pcr amplification product of normal people in 2 routine patients, 2 familys by what obtain in step 2.
The Sanger sequence verification peak figure in the SUCLA2 gene c.C308A mutational site of patient PI and PII and father and mother thereof is shown in Fig. 2-Fig. 5 respectively.From sequencing result, patient PI(Fig. 2) and PII(Fig. 3) all carry c.C308A homozygous mutation, and the father and mother of patient (Fig. 4 and Fig. 5) are the heterozygosis carrier of corresponding sudden change.
To sum up, in patient family member, carry out sudden change investigation to the sequence between No. 3, SULCA2 gene and 4 exons to find: patient PI and PII is c.C308A homozygous mutation, and the father and mother of patient are the heterozygosis carrier of corresponding sudden change.And do not find this mutational site in EVS exon database.Thus, proving that the c.C308A of SULCA2 gene suddenlys change further is that Mitochondrial DNA exhausts syndromic pathogenic mutation.
In the description of this specification sheets, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referred to the schematic representation of above-mentioned term.And the specific features of description, structure, material or feature can combine in an appropriate manner in any one or more embodiment or example.
Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.
Claims (4)
1. the nucleic acid of coding succinate coenzyme A ligase enzyme β subunit mutant be separated, is characterized in that, the nucleic acid of described mutant exists the sudden change of C → A the 308th of SEQIDNO:1.
2. an isolated polypeptide, is characterized in that, described isolated polypeptide sports Asp at the 103rd Ala of SEQIDNO:2, and described polypeptide is by nucleic acid encoding according to claim 1.
3. exhausting a test kit for syndromic biological sample for screening susceptible Mitochondrial DNA, it is characterized in that, contain:
Being suitable for the reagent detecting SUCLA2 gene mutation body, there is the sudden change of C → A the 308th of SEQIDNO:1 in wherein said SUCLA2 gene mutation body.
4. test kit according to claim 3, is characterized in that, described reagent is nucleic acid probe.
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| WO2009145945A2 (en) * | 2008-02-28 | 2009-12-03 | Battelle Energy Alliance, Llc | Thermophilic and thermoacidophilic metabolism genes and enzymes from alicyclobacillus acidocaldarius and related organisms |
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