CN104099349A - Maple syrup urine disease related gene mutation, and detection method and purpose thereof - Google Patents

Abstract

The invention relates to the field of disease related gene mutation, especially gene mutation of autosomal recessive hereditary amino acid metabolism disease, maple syrup urine disease related gene mutation, and more particularly maple syrup urine disease related gene mutation, and a detection method and purpose thereof.

Description

Maple syrup urine disease associated gene mutation, its detection method and uses thereof

Technical field

The present invention relates to disease-related mutator gene field, the particularly transgenation of recessive hereditary aminoacidopathy, maple syrup urine disease associated gene mutation.

Background technology

Maple syrup urine disease (Maple Syrup Urine Disease, MSUD) be a kind of recessive hereditary aminoacidopathy, it is mainly due to due to following reason: the genetic flaw of branched-chain amino acid alpha-ketoacid dehydrogenase complex, make the corresponding branched-chain alpha-ketoacid that forms after branched-chain amino acid (mainly comprising leucine, Isoleucine and α-amino-isovaleric acid) transamination can not oxidative decarboxylation, in tissue, branched-chain amino acid and branched-chain alpha-ketoacid increase extremely, body fluid is discharged alpha-ketoacid, thereby occur the smell of similar maple sugar, and gain the name therefrom.Meanwhile, owing to accumulating branched-chain amino acid in body and alpha-ketoacid derivative thereof to the toxic effect of cerebral tissue, can suppress myelin and generate, disturb in brain albumen synthetic, suppress neurotransmitter function, make developmental cerebral tissue be subject to grievous injury.Special odor and serious encephalopathic are very obvious two features of this disease.At present, this disease is divided into into following 5 types: classic, osculant, clearance type, VITMAIN B1 are treated effective type and dihydrolipoamide Acyl dehydrogenase (E3) shortage type, and wherein last type is very rare.

The morbidity of maple syrup urine disease in newborn infant is 1,/18 ten thousand.Hereditary defect is E1, E2 in the BCKD multienzyme complex existing in plastosome and the transgenation of E33 subunit.BCKD multienzyme complex is a kind of multienzyme complex being present in plastosome, and its function is the oxidative decarboxylation of 3 kinds of side chain α ketone acids producing from branched-amino acid degradation of catalysis.Combined enzyme agent contains 24 identical two hydrogen acyl swivelases (dihydrolipoyltranscylase, E2) subunits around a cubes core, and these subunits link together by ionic interaction.In addition, also have side chain α keto acid decarboxylase (E1), specificity kinases (E3) and specificity Phosphoric acid esterase.Specificity kinases and Phosphoric acid esterase regulate BCKD complex activity by reversible phosphorylation.E1 is the assorted tetramer, 2 E1 α and 2 E1 β subunits, consists of.E3 is homodimer.The encoding gene of E1 α, E1 β, E2 and E3 all can be undergone mutation and be caused BCKD multienzyme complex activity decreased.E1 is diphosphothiamine (TPP) dependent enzyme, by 2 E1 α and 2 E1 β, forms α 2 β 2 tetramers, and molecular weight is 170kD.E1 α and E1 β locus are positioned at respectively 19q and 6p, all can undergo mutation.E1 α transgenation can hinder it and aggregate into the tetramer with normal E1 β, E1 α is degraded rapidly, thereby makes side chain α keto acid decarboxylase activity decreased or completely lose; Or only form α β dimer, also can form the low-molecular-weight tetramer.Wynn etc. point out after having studied the polymerization obstacle of E1 α and E1 β in IA type maple syrup urine disease patient: if in studied E1 α sudden change the diphosphothiamine binding pocket in supposition, on the polymerization of E1 subunit without impact; If relate to the sudden change of C-terminal aromaticity residue, hinder the formation of polymeric subunit kinetics and natural α 2 β 2 structures.The sudden change of E1 α subunit makes branched-chain α-keto acid decarboxylase activity decreased or completely loses.

Determine the pathogenic mutation of new MSUD gene, significant to carrying out the molecular diagnosis of MUSD.

Summary of the invention

The inventor, by extensive and deep research, has found the compound heterozygous mutations of the gene BCKDHA gene of encode side chain alpha-ketoacid dehydrogenase complex E1-α subunit: c.392A>G(missense mutation is the sudden change p.Tyr131Cys of E1-α protein subunit in a MUSD patient) and EX2-4DUP.This compound heterozygous mutations causes BCKDHA gene to lose normal function, thereby causes MUSD to occur.Contriver has completed this invention on this basis.

The present invention relates to the compound heterozygous mutations of MUSD ospc gene, be specially: c.392A>G(the missense mutation of BCKDHA gene is the missense mutation p.Tyr131Cys of branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit) and EX2-4DUP(refer on item chromosome,, there is the structure of exon 2-3-4-2-3-4 in 1 times of the gene copy number increase of the 2-4 exon of BCKDHA gene).Wherein, BCKDHA genes encoding branched-chain alpha-ketoacid dehydrogenase complex E1-α subunit.

In first aspect, the present invention relates to the biomarker of MUSD disease, be the heterozygous mutant of BCKDHA gene or branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit, described biomarker is following sudden change BCKDHA gene or E1-α protein subunit: c.392A>G(the missense mutation of BCKDHA gene is the missense mutation p.Tyr131Cys of branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit) and EX2-4DUP.

C.392A>G and EX2-4DUP in one embodiment, sudden change BCKDHA gene of the present invention is the SEQ ID NO:1 with following sudden change: missense mutation.

In one embodiment, the branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit of sudden change of the present invention is the SEQ ID NO:2 with following sudden change: missense mutation p.Tyr131Cys and EX2-4DUP.

In second aspect, the present invention relates to a kind of MUSD of detection sick or check the method for its susceptibility, described method comprises detecting in experimenter's BCKDHA gene or branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit whether have mutational site, if there is mutational site, described experimenter is accredited as and suffers from MUSD disease or easily suffer from MUSD disease, or its offspring can suffer from MUSD disease or easily suffer from MUSD disease, c.392A>G(, the missense mutation of the described BCKDHA of sporting gene is the missense mutation p.Tyr131Cys of branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit) and/or EX2-4DUP.

In one embodiment, the cDNA sequence of BCKDHA gene is that the sequence of SEQ ID NO:1 represents.

In one embodiment, E1-α protein subunit is that the sequence of SEQ ID NO:2 represents.

In one embodiment, the method for detection MUSD disease of the present invention comprises the step of following two groups of primer amplifications:

SEQ ID NO:3 and SEQ ID NO:4;

SEQ ID NO:5 to SEQ ID NO:10.

In one embodiment, in the method for detection MUSD disease of the present invention, detecting mutational site is undertaken by being selected from following technology: order-checking, electrophoresis, nucleic acid hybridization, PCR, reversed transcriptive enzyme chain reaction and sex change high performance liquid chromatography, the DNA sequencing based on fluorescent mark technology.

In the method for second aspect present invention, c.392A>G(the missense mutation of preferred detection BCKDHA gene is the missense mutation p.Tyr131Cys of branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit) and/or EX2-4DUP.

In the third aspect, the present invention relates to a kind of method that detects sudden change BCKDHA gene or branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit, described method comprises that in the BCKDHA gene (being branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit) that detects experimenter, whether having the missense mutation of BCKDHA gene is c.392A>G(the missense mutation p.Tyr131Cys of branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit) and/or EX2-4DUP.

In one embodiment, BCKDHA gene is that the sequence of SEQ ID NO:1 represents.

In one embodiment, E1-α protein subunit is that the sequence of SEQ ID NO:2 represents.

In one embodiment, the method for detection sudden change BCKDHA gene of the present invention or E1-α protein subunit comprises the step of following two groups of primer amplifications:

SEQ ID NO:3 and SEQ ID NO:4;

SEQ ID NO:5 to SEQ ID NO:10.

In one embodiment, in the method for detection sudden change BCKDHA gene of the present invention or E1-α protein subunit, detecting mutational site is undertaken by being selected from following technology: order-checking, electrophoresis, nucleic acid hybridization, PCR, reversed transcriptive enzyme chain reaction and sex change high performance liquid chromatography, the DNA sequencing based on fluorescent mark technology.

In fourth aspect, the present invention relates to detect by PCR the primer pair using in sudden change BCKDHA gene or branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit, described sudden change is that c.392A>G(the missense mutation of BCKDHA gene is the missense mutation p.Tyr131Cys of branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit) and/or EX2-4DUP.

Wherein said primer pair is respectively based on being selected from following position Ge front and back design in genome sequence or cDNA sequence, and this Ge makes to increase: the 392nd of BCKDHA gene cDNA sequence and/or exon 2-4.

Aspect the 5th, the present invention relates to the nucleic acid probe with the BCKDHA gene complementation that suddenlys change, described sudden change be BCKDHA gene missense mutation c.392A>G and/or EX2-4DUP.

The complementary district of described probe and sudden change BCKDHA gene comprises and is selected from Wei Ge in following genome sequence or cDNA sequence: the 392nd of BCKDHA gene cDNA sequence and/or exon 2-4.

Aspect the 6th, the present invention relates to detect the test kit of sudden change BCKDHA gene or branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit, comprise one or more groups primer pair, wherein said sudden change is that c.392A>G(the missense mutation of BCKDHA gene is the missense mutation p.Tyr131Cys of branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit) and/or EX2-4DUP.

Wherein said primer pair designs in genome sequence or cDNA sequence based on being selected from following Wei Ge respectively, makes its amplified production contain Gai Wei Ge: the 392nd of BCKDHA gene cDNA sequence and/or exon 2-4.

In one embodiment, the test kit of described detection sudden change BCKDHA gene or branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit comprises following two groups of primers:

SEQ ID NO:3 and SEQ ID NO:4;

SEQ ID NO:5 to SEQ ID NO:10.

Aspect the 7th, the present invention relates to detect the test kit of sudden change BCKDHA gene or branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit, comprise one or more nucleic acid probes, described sudden change be BCKDHA gene missense mutation c.392A>G and/or EX2-4DUP.

On described probe and sudden change BCKDHA gene, comprise the regional complementarity being selected from as in the genome sequence of the next Ge or cDNA sequence: the 392nd of BCKDHA gene cDNA sequence and/or exon 2-4.

The inventor finds, c.392A>G the causing a disease with the compound heterozygous mutations of EX2-4DUP of BCKDHA gene of the present invention; And BCKDHA gene of the present invention c.392A>G with any heterozygous mutant not pathogenic of EX2-4DUP, as heterozygosis missense mutation c.392A>G(p.Tyr131Cys) be present in mother of patient, heterozygous mutant EX2-4DUP is present in patient's father, and they are not ill.

Compound heterozygous mutations in BCKDHA gene of the present invention, can be for the diagnosis and detection of maple syrup urine disease pathology.The present invention has found the compound heterozygous mutations of the sick genes involved of MUSD, may be for MUSD patient's auxiliary diagnosis to the detection of these transgenations, and may be conducive to clear and definite MUSD patient's molecular diagnosis.Therefore, the method of detection compound heterozygous mutations BCKDHA gene of the present invention (or E1-α protein subunit) and/or EX2-4DUP can for example, for the object of diagnosis MUSD disease or its susceptibility, antenatal diagnosis, PGD (PGD), patient's examination.Yet method of the present invention is not limited in the object for diagnosing the illness.

In addition, the method for detection sudden change BCKDHA gene of the present invention and/or E1-α protein subunit also can be for the non-object diagnosing the illness.The object of described non-detection disease includes but not limited to study SNP and distributes and polymorphism, for family, develops and studies.The present invention can provide important clue to the pathogeny of MUSD disease, and the diagnoses and treatment tool of MUSD disease is of great significance.Such application also it will be appreciated by those skilled in the art that.

For example, according to description herein, can find out, some individuality carries sudden change BCKDHA gene of the present invention (or branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit) or carries EX2-4DUP of the present invention but do not suffer from MUSD disease, and for example only item chromosome carries the heterozygous genes type of sudden change.This part crowd's detection can anyly not related to the object diagnosing the illness, because these individualities are not ill.But the result detecting for them can be used as for example useful information and uses, for example, as the important indicator of educating front inspection, instruct fertility, or for carriers of mutation examination, or distribute and instrument or tracking transgenation or family's evolution of polymorphism research as SNP.

Therefore, the pathogenic mutation spectrum of MSUD gene has been enriched in this research, significant to carrying out the molecular diagnosis of MUSD.

Accompanying drawing explanation

Fig. 1: the sanger order-checking of patient's (being propositus P610) and its father and mother's (P609 is father, and P611 is mother) BCKDHA gene

Fig. 2: the QPCR sequencing result of the BCKDHA gene of patient, its father and mother and normal controls.Wherein P610 is patient, and P611 is mother patient, and P609 is father patient; N108 is normal people.

The cDNA sequence of BCKDHA gene (SEQ ID NO:1; Underscore is partly EX2-4; What add frame is the 392nd):

ATGGCGGTAGCGATCGCTGCAGCGAGGGTCTGGCGGCTAAACCGTGGTTTGAGCCAGGCTGCCCTCCTGCTGCTGCGGCAGCCTGGGGCTCGGGGACTGGCTAGATCTCACCCCCCCAGGCAGCAGCAGCAGTTTTCATCTCTGGAT GACAAGCCCCAGTTC CCAGGGGCCTCGGCGGAGTTTATAGATAAGTTGGAATTCATCCAGCCCAACGTCATC TCTGGAATCCCCATCTACCGCGTCATGGACCGGCAAGGCCAGATCATCAACCCCAG CGAGGACCCCCACCTGCCGAAGGAGAAGGTGCTGAAGCTCTACAAGAGCATGACAC TGCTTAACACCATGGACCGCATCCTCTATGAGTCTCAGCGGCAGGGCCGGATCTCCT TCT CATGACCAACTATGGTGAGGAGGGCACGCACGTGGGGAGTGCCGCCGCCCTGGACAACACGGACCTGGTGTTTGGCCAGTACCGGGAGGCAGGTGTGCTGATGTATCGGGACTACCCCCTGGAACTATTCATGGCCCAGTGCTATGGCAACATCAGTGACTTGGGCAAGGGGCGCCAGATGCCTGTCCACTACGGCTGCAAGGAACGCCACTTCGTCACTATCTCCTCTCCACTGGCCACGCAGATCCCTCAGGCGGTGGGGGCGGCGTACGCAGCCAAGCGGGCCAATGCCAACAGGGTCGTCATCTGTTACTTCGGCGAGGGGGCAGCCAGTGAGGGGGACGCCCATGCCGGCTTCAACTTCGCTGCCACACTTGAGTGCCCCATCATCTTCTTCTGCCGGAACAATGGCTACGCCATCTCCACGCCCACCTCTGAGCAGTATCGCGGCGATGGCATTGCAGCACGAGGCCCCGGGTATGGCATCATGTCAATCCGCGTGGATGGTAATGATGTGTTTGCCGTATACAACGCCACAAAGGAGGCCCGACGGCGGGCTGTGGCAGAGAACCAGCCCTTCCTCATCGAGGCCATGACCTACAGGATCGGGCACCACAGCACCAGTGACGACAGTTCAGCGTACCGCTCGGTGGATGAGGTCAATTACTGGGATAAACAGGACCACCCCATCTCCCGGCTGCGGCACTATCTGCTGAGCCAAGGCTGGTGGGATGAGGAGCAGGAGAAGGCCTGGAGGAAGCAGTCCCGCAGGAAGGTGATGGAGGCCTTTGAGCAGGCCGAGCGGAAGCCCAAACCCAACCCCAACCTACTCTTCTCAGACGTGTATCAGGAGATGCCCGCCCAGCTCCGCAAGCAGCAGGAGTCTCTGGCCCGCCACCTGCAGACCTACGGGGAGCACTACCCACTGGATCACTTCGATAAGTGA

The aminoacid sequence of E1-α protein subunit (SEQ ID NO:2; Underscore is the amino acid of EX2-4 coding; What add frame is the 131st):

MAVAIAAARVWRLNRGLSQAALLLLRQPGARGLARSHPPRQQQQFSSLD DKPQFP GASAEFIDKLEFIQPNVISGIPIYRVMDRQGQIINPSEDPHLPKEKVLKLYKSMTLLNTMD RIL ESQRQGRISFYMTNYGEEGTHVGSAAALDNTDLVFGQYREAGVLMYRDYPLELFMAQCYGNISDLGKGRQMPVHYGCKERHFVTISSPLATQIPQAVGAAYAAKRANANRVVICYFGEGAASEGDAHAGFNFAATLECPIIFFCRNNGYAISTPTSEQYRGDGIAARGPGYGIMSIRVDGNDVFAVYNATKEARRRAVAENQPFLIEAMTYRIGHHSTSDDSSAYRSVDEVNYWDKQDHPISRLRHYLLSQGWWDEEQEKAWRKQSRRKVMEAFEQAERKPKPNPNLLFSDVYQEMPAQLRKQQESLARHLQTYGEHYPLDHFDK

Embodiment

The order-checking of catching of BCKDHA, BCKDHB, DBT, DLD gene is carried out in this research to a patient who is diagnosed as MSUD.The laggard row variation that checks order detects.Then, the variation of detection and dbSNP database, thousand human genome databases, HapMap database, HGMD database, LSMD database and normal people's data are compared, finally find the compound heterozygous mutations of not reporting of BCKDHA gene: the missense mutation of BCKDHA gene c.392A>G(or the missense mutation p.Tyr131Cys of branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit) and EX2-4DUP.Wherein, gene BCKDHA encode side chain alpha-ketoacid dehydrogenase complex E1-α subunit.

Sample collection

We accepted one go to see a doctor from the MUSD patient of Pekinese, 3 years old, its production process situation of normally not suffocating, 6 months was be diagnosed as amyotonia, through a series of inspections, its MRI and electroencephalogram are all undesired.The quantitative amino acid analysis result demonstration of its blood plasma, leucine+Isoleucine total amount is 715.4808 μ M(reference value 120-190 μ M), tyrosine total amount is 452.3789 μ M(reference value 200-425 μ M).We have gathered this patient and its parental peripheral blood and have extracted genomic dna.In addition, this research has also gathered 455 normal peoples' peripheral blood and has extracted its genomic dna.

Sign written informed consent with all experimenters or its guardian.

Chip design, library construction and high-flux sequence

Contriver has carried out catch order-checking in conjunction with Solexa Hiseq2000 high throughput sequencing technologies to this patient's BCKDHA, BCKDHB, DBT, DLD gene with the chip (NimblGen, Roche) of catching of customization, specific as follows:

1) we have designed one and catch chip (NimblGen, Roche) exon of 4 genes, splice site and contiguous intron sequences are caught.

2) genomic dna is broken at random to the fragment of 150-200bp left and right, at fragment two ends, connects respectively top connection subsequently, preparation library (the Illumina/Solexa standard providing referring to http://www.illumina.com/ is built storehouse specification sheets).

3) enrichment is hybridized through the linear amplification of ligation-mediated PCR (LM-PCR) and the capture array of customization after purified in library, then after the linear amplification of LM-PCR, carries out upper machine order-checking.Order-checking platform is Illumina Hiseq2000, and reading length is 90bp.

Variation detects, annotation and with database comparison

1) raw data obtaining after order-checking is processed by Illumina basecalling Software1.7.Then filter the inferior quality section of reading and comprise joint and pollute the section of reading.Use BWA(version0.5.9-r16) (can be referring to Li H, Durbin R.Fast and accurate long-read alignment with Burrows-Wheeler transform.Bioinformatics2010; 26 (5): 589-595) comparison of the high quality section of reading is arrived with reference to genome, obtain unique comparison section of reading of comparing on genome.Then utilize the SOAPsnp(can be referring to: Li R, Li Y, Fang X, Yang H, et al, SNP detection for massively parallel whole-genome resequencing.Genome Res2009, 19 (6): 1124-1132., by reference, it is incorporated to herein in full) and GATK(version v1.0.4705) (can be referring to McKenna, A, Hanna M, Banks E, et al.The genome analysis toolkit:a MapReduce framework for analyzing next-generation DNA sequencing data.Genome Research2010, 20 (9): 1297-1303) carry out respectively the detection of SNP and indel.

2) for the SNP detecting and indel variation, we utilize inner flow process to annotate, i.e. definitive variation genome environment of living in, and the type of variation.We pay close attention to nonsynonymous mutation, and insert the sudden change of acceptor splicing site/donor site and coding region and this three class of deletion mutantion is most possible and the sudden change of disease-related.We compare the variation detecting and dbSNP database, thousand human genome databases, HapMap database, HGMD database and LSMD database public database and normal people's data.We have selected the variation comprising in HGMD database and LSMD database and have been positioned at coding region and the variation of previously not reporting in splice site district, then compare with our internal contrast database.For the candidate's pathogenic mutation being positioned on disease known, we have carried out sanger checking.

By above-mentioned analysis, we find the compound heterozygous mutations of not reporting in this patient's BCKDHA gene: missense mutation is c.392A>G(p.Tyr131Cys) and EX2-4DUP.Wherein, heterozygosis missense mutation is c.392A>G(p.Tyr131Cys) be present in mother of patient, heterozygous mutant EX2-4DUP is present in patient's father, therefore can be judged as compound heterozygous mutations.Our result shows that the afunction sudden change of BCKDHA gene is the reason that causes maple syrup urine disease (MUSD).

The target using is in this article caught order-checking and is called again target area and catches order-checking, exactly the interested genome area of investigator customized to probe and carries out chip hybridization (NimbleGen Sequence Capture array) or solution hybridization (Agilent Sure-Select System) with genomic dna, will after the enrichment of target gene regional DNA, recycle the research strategy that new-generation sequencing technology checks order.Target area can be continuous DNA sequence dna, can be also the fragment being distributed on same karyomit(e) different zones or coloured differently body.At present, due to its high-throughput, low cost and the experimental period that greatly shortens, target area order-checking is used widely gradually in clinical molecular diagnosis.

In the present invention, adopt this area generic representation method to represent sudden change.C.392A>G represent that the 392nd Nucleotide of cDNA becomes G by A; P.Tyr131Cys represents that protein level the 131st bit codon becomes Cys by Tyr; EX2-4DUP represents cDNA and/or the repetition of protein level exon 2-4.

In the present invention, unless otherwise indicated, otherwise A and/or B are equivalent to following three kinds of situation: A; B; A and B.

In specification sheets of the present invention and claims, mention gene order, it will be appreciated by those skilled in the art that actual any of comprising complementary two strands, or two.For convenient, in the present specification and claims, although in most cases only provided a chain, another chain of complementation with it is in fact also disclosed.For example mention the cDNA sequence of BCKDHA gene, in fact can comprise/or do not comprise this sequence with and complementary sequence.For example, mention SEQ ID NO:1, actually can comprise its complementary sequence.Those skilled in the art are further appreciated that and utilize a chain can detect another chain, and vice versa.

Gene order in the application comprises DNA form or rna form, open wherein a kind of, means that another kind is also disclosed.For example mention the cDNA sequence of BCKDHA gene, reality also comprises corresponding RNA sequence.

Below in conjunction with embodiment, embodiment of the present invention are described in detail.It will be understood to those of skill in the art that the following examples are only for the present invention is described, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, according to the described technology of the document in this area or condition (such as with reference to works such as J. Pehanorm Brookers, the < < molecular cloning experiment guide > > that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.

Embodiment 1

Genome extracts

Gathering all experimenters' peripheral blood, utilize QIAamp DNA BloodMiNi Kit(Qiagen, Hilden, Germany) scheme that provides according to manufacturers extracts genomic dna.

Embodiment 2

Chip design, library construction and high-flux sequence

The inventor has designed one and has caught chip (NimblGen, Roche) exon of these 4 genes of BCKDHA, BCKDHB, DBT and DLD, splice site and contiguous intron sequences are caught.

The scheme that the method for library construction can provide with reference to Illumina company (http://www.illumina.com/).Now be briefly described as follows: utilize ultrasonoscope (Covaris S2, Massachusetts, USA) genomic dna to be broken at random to the fragment of 200-300bp.Get 1 μ g(quantitative with Nanodrop) for purifying DNA fragment, the Klenow fragment of T4DNA polysaccharase, T4 phosphoric acid nucleoside acid kinase and intestinal bacteria (Escherichia coli) archaeal dna polymerase is processed, and fill 5 ' protruding terminus and remove 3 ' protruding terminus.At 3 ' end, add A base and reconnect joint sequence later.After adding joint, the PCR that utilizes the PCR primer that comprises 8bp sequence label (barcode sequence) to carry out 4 circulations obtains catching front library.10 libraries are mixed rear and caught chip and carry out hybridization in 72 hours.After having hybridized, utilize 300ml elution buffer (SeqCap EZ Hybridization and Wash Kit; Qiagen, Valencia, CA, USA) DNA fragmentation being attached on chip is carried out to wash-out.On homemade wash-out equipment, 95 ℃ of wash-outs are 20 minutes, then add 200ml elution buffer and once repeat wash-out.With reference to the cluster of Illumina standard and the scheme of order-checking, we utilize Illumina Hiseq2000 to check order, and reading length is 90bp.

Embodiment 3

Variation detects, annotation and with database comparison

1) after having checked order, utilize Illumina1.3.4 to carry out image analysis, error rate estimation and base and read, obtain raw data.Then filter the inferior quality section of reading (read) and comprise joint and pollute the section of reading.We are less than 5 base by the section of reading that comprises the N base that surpasses 10%, 50% mass value and account for the section of reading and use BWA that the comparison of the high quality section of reading is arrived with reference to genome, and the unique comparison section of reading on genome is compared in acquisition.Then utilize SOAPsnp and GATK to carry out respectively the detection of SNP and indel.

2) for the SNP detecting and indel variation, we utilize inner flow process to annotate, i.e. definitive variation genome environment of living in, and the type of variation.We pay close attention to nonsynonymous mutation, and insert the sudden change of acceptor splicing site/donor site and coding region and this three class of deletion mutantion is most possible and the sudden change of disease-related.We compare the variation detecting and dbSNP database, thousand human genome databases, HapMap database, HGMD database and LSMD database public database.We have selected the variation comprising in HGMD database and LSMD database and have been positioned at coding region and the variation of previously not reporting in splice site district, then compare with our internal contrast database.

Find the compound heterozygous mutations of not reporting of BCKDHA gene: missense mutation is c.392A>G(p.Tyr131Cys) and EX2-4DUP.

Embodiment 4

Respectively 1 clinical samples and 2 non-clinical samples in family are carried out to Sanger order-checking, determine that missense mutation c.392A>G(p.Tyr131Cys).Concrete grammar step is as follows:

1) DNA extraction:

The peripheric venous blood that gathers patient and its father and mother extracts genomic dna by QIAamp DNA BloodMiNi Kit (Qiagen, Hilden, Germany) method, and DNA is for following steps 2) DNA profiling of reaction system.

2) design of primers and pcr amplification reaction

A) primer sequence

For increasing this c.392A>G primer in site that suddenlys change:

Upstream primer F:AGCAGTCTGGCAGCGTCTTCTTA(SEQ ID NO:3)

Downstream primer R:GCTCCTTAGACCTTCCTGGCACTA(SEQ ID NO:4)

B) reaction system:

Reaction adopts 25 μ l systems, wherein: enzyme and buffered soln are all purchased from KATARA company.

C) PCR response procedures:

95 ℃ 5 minutes;

30 circulations:

94 ℃ 30 seconds,

Tm30 second,

72 ℃ are extended 30 seconds;

72 ℃ 10 minutes;

15 ℃ of maintenances.

D) after pcr amplification, carry out detected through gel electrophoresis, confirm that the fragment amplifying is the object fragment needing.The Marker using in electrophoresis is DL2000DNA Marker and the 50bp DNA Marker of 5 μ l.The sepharose of electrophoresis employing 2% carries out, and under 135V, electrophoresis is 35 minutes.

3) by step 2) in the pcr amplification product that obtains with QIAquick pcr amplification test kit (Qiagen), carry out purifying, then utilize the 3730xl of ABI to carry out DNA sequencing (Fig. 1).

Confirm in patient and mother patient with the missense mutation of not reporting of BCKDHA gene c.392A>G(p.Tyr131Cys), patient's dad is this missense mutation not.

Embodiment 5

Respectively 1 clinical samples and 2 non-clinical samples in family are carried out to QPCR, determine heterozygous mutant EX2-4DUP.Concrete grammar step is as follows:

1) DNA extraction:

The peripheric venous blood that gathers patient and its father and mother extracts genomic dna by QIAamp DNA BloodMiNi Kit (Qiagen, Hilden, Germany) method.

2) design of primers and pcr amplification reaction

A) QPCR primer:

BCKDHA-qex2-F	CCAGGCAGCAGCAGCAGTTTTC（SEQ?ID?NO:5）
		BCKDHA-qex2-R	GATGGGGATTCCAGAGATGACG（SEQ?ID?NO:6）
BCKDHA-qin3-F	AGGACAGATTCTTTTTTGGGGG（SEQ?ID?NO:7）
		BCKDHA-qin3-R	CAGGACTGCTCTCACACTTCTC（SEQ?ID?NO:8）
BCKDHA-qin4-F	CCTGTGGTCCCCATTGAAGTGT（SEQ?ID?NO:9）
		BCKDHA-qin4-R	TCCTGGCACTATGCTGCTCCTG（SEQ?ID?NO:10）

Wherein:

BCKDHA-qex2-F and BCKDHA-qex2-R No. 2, BCKDHA gene extron that are used for increasing.

BCKDHA-qin3-F and BCKDHA-qin3-R No. 3, BCKDHA gene extron that are used for increasing.

BCKDHA-qin4-F and BCKDHA-qin4-R No. 4, BCKDHA gene extron that are used for increasing.

B) QPCR laboratory apparatus and condition: ABI7500 quantitative real time PCR Instrument

95 ℃ 30 seconds;

40 circulations:

95 ℃ 30 seconds,

60 ℃ 15 seconds,

72 ℃ of 30s second (collection fluorescence);

The step that obtains solubility curve as follows:

95 ℃ 15 seconds

60 ℃ 1 minute

95 ℃ 30 seconds (collection fluorescence),

60 ℃ 15 seconds.

C) experiment reagent and amount thereof, wherein template is 1 μ l, and experiment is done to three Duplicate Samples.

3) minimum cycling numerical value (Ct) relative method is calculated the relative copy number application 2-Δ Δ Ct method of 2,3 exons of BCKDHA gene, can draw the relative copy number (Fig. 2) of 2,3 exons of BCKDHA gene with internal reference ALB genetic comparison.

Confirm the heterozygous mutant of not reporting of BCKDHA gene: EX2-4DUP, in patient and father patient with heterozygous mutant EX2-4DUP, and mother of patient does not carry heterozygous mutant EX2-4DUP.

Embodiment 6

For 455 normal specimens, according to the step of embodiment 4 and 5, carry out identical experiment, find that c.392A>G(p.Tyr131Cys they do not carry missense mutation), do not carry heterozygous mutant EX2-4DUP yet.

In crowd, carry heterozygosis missense mutation c.392A>G or the people of heterozygous mutant EX2-4DUP very rare, carry the rare especially of the two simultaneously.The inventor finds that a patient simultaneously carrying the two suffers from maple syrup urine disease, has further proved that these two kinds of sudden changes that this patient carries are respectively from its father and mother simultaneously.Two kinds of sudden changes occur in and it is believed that on the branched-chain alpha-ketoacid dehydrogenase complex E1-α subunit relevant to maple syrup urine disease, the father and mother that carry respectively these two kinds of sudden changes all do not fall ill, and the patient who carries the two falls ill simultaneously, this has proved the relation of simultaneously carrying two kinds of sudden changes and maple syrup urine disease.

Claims (9)

1. a biomarker for maple syrup urine disease, described biomarker comprises BCKDHA gene and/or the branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit of sudden change,

C.392A>G the sudden change of BCKDHA gene is, the sudden change of branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit is p.Tyr131Cys.

2. the biomarker of claim 1, described biomarker also comprises EX2-4DUP.

3. claim 1 or 2 biomarker, have following sudden change in the sequence that described sudden change BCKDHA gene is SEQ ID NO:1:

c.392A>G。

4. detect a method that whether has mutational site and copy number variation in experimenter's BCKDHA gene and/or branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit, described mutational site and copy number variation are selected from following any or its combination:

BCKDHA transgenation c.392A>G, branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit sudden change p.Tyr131Cys; With

EX2-4DUP。

5. the method for claim 4, comprises the step of following at least one group of primer amplification:

SEQ ID NO:3 and SEQ ID NO:4;

SEQ ID NO:5 to SEQ ID NO:10.

6. detect the primer pair using in sudden change BCKDHA gene and/or branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit, described sudden change is to be selected from following any or its combination:

BCKDHA transgenation c.392A>G, branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit sudden change p.Tyr131Cys; With

EX2-4DUP，

Wherein said primer pair is respectively based on being selected from following position Ge front and back design in genome sequence or cDNA sequence, and this Ge makes to increase: the 392nd of BCKDHA gene cDNA sequence.

7. with the nucleic acid probe of sudden change BCKDHA gene complementation, described sudden change is to be selected from following any or its combination:

BCKDHA transgenation c.392A>G,

The complementary district of described probe and sudden change BCKDHA gene comprises and is selected from Wei Ge in following genome sequence or cDNA sequence: BCKDHA gene cDNA sequence the 392nd.

8. detect the test kit of sudden change BCKDHA gene and/or branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit, comprise one or more groups primer pair, and/or comprise one or more nucleic acid probes, wherein said sudden change is to be selected from following any or its combination:

BCKDHA transgenation c.392A>G, branched-chain alpha-ketoacid dehydrogenase complex E1-α protein subunit sudden change p.Tyr131Cys; With

EX2-4DUP，

Wherein said primer pair designs in genome sequence or cDNA sequence based on being selected from following Wei Ge respectively, makes its amplified production contain Gai Wei Ge: the 392nd of BCKDHA gene cDNA sequence.

9. the test kit of claim 8, described primer pair is selected from following one group or two groups:

SEQ ID NO:3 and SEQ ID NO:4;

SEQ ID NO:5 to SEQ ID NO:10.