CN104293813B - The new Disease-causing gene of Skin peeling syndrome and its encoding proteins matter and application - Google Patents

Abstract

The present invention identifies the new recessive Disease-causing gene related to Skin peeling syndrome.Specifically, inventor is using the ill family of Skin peeling syndrome as research object, sequencing of extron group has been carried out to the diseased individuals in the family and non-diseased individual and compared, a frameshift mutation (c.607_608insAfs) is found in CAST genes, the mutation causes CAST protein translations to interrupt in advance.On this basis, the invention provides the CAST genes of mutation and its encoding proteins matter and application, carrier, host cell and the kit of the CAST genes comprising mutation.Using the CAST genes of the mutation, molecule diagnosis and risk evaluation can be carried out to Skin peeling syndrome.The mutator and its encoding proteins matter are alternatively arranged as treating the drug target of Skin peeling syndrome.

Description

The new Disease-causing gene of Skin peeling syndrome and its encoding proteins matter and application

Technical field

The present invention relates to a kind of gene of human variation, more particularly to a kind of new Disease-causing gene of Skin peeling syndrome- CAST genes.The invention further relates to the CAST genes of mutation and its encoding proteins matter and application, the CAST genes comprising mutation Carrier, host cell and kit.

Background technology

Skin peeling syndrome (peeling skin syndrome, PSS) is one group of rare hereditary dermatosis, is faced Bed performance stripped off using continuation keratoderma or epidermis shallow-layer, the scales of skin that peel off, erythema as principal character, similar " casting off a skin " phenomenon. It is two kinds of different types of acra type and system type that PSS, which is divided to, and wherein acra type only involves hand, sufficient end, shows as hand, foot and takes off repeatedly Bits and erythema；System type can involve whole skin, according to whether merge inflammation sexually revise and allergic conditions be divided into inflammatory type and Noninflammatory type.Acra type generally merges other diseases, and system type can merge the other diseases such as allergic rhinitis, asthma.Suffer from " casting off a skin " symptom of person can change with season, and Xia Chongdong is light.Histopathologic appearance is hyperkeratinization, and cuticula There is crack with stratum granulosum, or particle shallow-layer.This disease cause can patient skin there is obvious itch, outward appearance and be badly damaged, with And merge various anaphylactias.Previously research is thought, PSS is autosomal recessive inheritance, principal causative gene be TGM5, CDSN, CSTA or CHST8, these genes are played an important role during epidermis terminal differentiation.

Recently, sequencing of extron group (exome sequencing) is successfully applied to find rare single-gene disorder The MYH3 genes of Disease-causing gene, such as Freeman-Sheldon syndromes, the SETBP1 bases of Schinzel-Giedion syndromes Cause, and seriously WDR62 mutation of big deformity of brain etc..Full extron sequencing technologies are proven the rare single-gene disorder of reduction Candidate gene even finds strong, the effective means of its Disease-causing gene, only by several seldom individuals (including patient and just Often control) full extron be sequenced to screen the variation related to disease, its success rate is significantly increased.

The content of the invention

It is a primary object of the present invention to differentiate the new Disease-causing gene of Skin peeling syndrome (PSS), the disease is oriented The gene mutation site of disease.On this basis there is provided PSS Disease-causing genes and its encoding proteins matter and application, the pathogenic bases of PSS are included Carrier, host cell and the kit of cause, to carry out molecule diagnosis and risk evaluation to Skin peeling syndrome, to enter One step treats the target spot that the disease provides design medicine, while being provided fundamental basis to illustrate the sick mechanism of causing a disease.

Therefore, on the one hand, the invention provides the CAST genes of mutation, CAST gene orders and the SEQ ID of the mutation NO:1 compares with the non-silent mutation of at least one, and the CAST genes of the mutation cause the generation of Skin peeling syndrome；Institute State one or more of the non-silent mutation in insertion, missing and displacement.

In a preferred embodiment, the non-silent mutation is SEQ ID NO:The 607th of 1 and 608 it Between insert A, its sequence such as SEQ ID NO:Shown in 2.The mutation belongs to frameshit insertion mutation so that the amino of CAST gene codes The isoleucine that acid sequence is the 203rd becomes asparagine, and the 203rd later amino acid sequence also changes, until 210th glutamic acid codon mutation is terminator codon so that polypeptide chain synthesis is interrupted.

Second aspect, present invention also offers SEQ ID NO:The protein of 2 sequential codings, the sequence occurs from 203 Change, is terminated, the protein only has 209 amino acid, its amino acid sequence such as SEQ ID NO to the 210th:3 institutes Show.And the CAST albumen of wild type has 750 amino acid, therefore the structure of the CAST protein of the saltant type is different from function In the CAST albumen of wild type, it is impossible to play normal effect in body

In the third aspect of the present invention, there is provided the carrier containing above-mentioned mutation CAST genes.

In the fourth aspect of the present invention there is provided the host cell for being converted or being transduceed by above-mentioned carrier or by above-mentioned mutation The CAST genes host cell that directly converts or transduce.

In the fifth aspect of the present invention treatment Skin peeling syndrome is being used as there is provided the CAST genes of above-mentioned mutation Drug target or prepare Skin peeling syndrome kit for diagnosing diseases in application.

In the sixth aspect of the present invention, there is provided a kind of kit for being used to diagnose Skin peeling syndrome, the reagent Box includes the primer for being capable of specific amplification CAST genes, or is capable of the probe of specific detection CAST gene mutations.

In a preferred embodiment, the primer or probe sequence are SEQ ID NO:In sequence shown in 1 or 2 The sequence of 15~30bp length before and after 607th or the 608th.

In the seventh aspect of the present invention there is provided a kind of antibody, the antibody and the CAST protein of above-mentioned saltant type are special The opposite sex is combined, and does not act on the protein of normal CAST coded by said gene.

In the eighth aspect of the present invention there is provided a kind of Skin peeling syndrome therapeutic agent, the therapeutic agent is comprising above-mentioned Described antibody.

The present invention is found that CAST gene mutations can cause Skin peeling to integrate first by sequencing of extron group technology The morbidity levied.On the one hand, can be with early screening Skin peeling by detecting whether subject carries CAST gene pathogenic mutations There is provided prenatal and postnatal care guidance by syndrome mutator carrier；Or Skin peeling syndrome patient provide molecule diagnosis according to According to.Especially, diagnostic kit provided by the present invention can be used for quickly and efficiently predicting or diagnosing Skin peeling syndrome. On the other hand, CAST genes are realized in human diseases first, provide brand-new logical for complicated skin keratin physiology course Road；The third aspect, the present invention can provide possible drug target for treatment Skin peeling syndrome.

Brief description of the drawings

Fig. 1 is certain six generation autosome Skin peeling syndrome pedigree chart, wherein square represents male, circle represents female Property；Band oblique line is death, and solid is diseased individuals, and hollow is normal individual.

Fig. 2 is PSS patient symptom figures.

Fig. 3 is the mutational site sequence chart of PSS patient's CAST genes.

Fig. 4 is the mRNA expression qRT-PCR results of the CAST genes of PSS patient and normal control.

Fig. 5 is perspective electron microscopy detection calpastatin result.

Fig. 6 is the expression in vivo of CAST genes and functional study analysis result.

Embodiment

The present invention is described in more detail with reference to embodiments, but following description is only used for entering the present invention Row indicative explaination, does not carry out any limitation, protection scope of the present invention is with claim to protection scope of the present invention The scope that book is limited is defined.

In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology The implication that personnel are generally understood that.Also, molecular genetics used herein, nucleic acid chemistry and molecular biology relational language It is widely used term and conventional steps in corresponding field with laboratory operation step.Meanwhile, in order to more fully understand this Invention, is provided below the definition and explanation of relational language.

In the present invention, " CAST (calpastatin) gene " is the target gene of protease inhibitors, and it is calcium albumen One of enzyme hydrolysis DBMS member.Its protein encoded, which is that endogenous is single-minded for calpastatin (CAST), suppresses calcium egg The protein of white enzyme (CAPN) activity, can single-minded identification CAPN caused conformation change is combined with calcium, and energy is in combination.Activity CAST contains 5 domains, and first domain is L domains, and its function is also not very clear.2nd~5 domain is 4 The similar recurring unit of individual structure.

In the present invention, term " extron " refers to the part being retained in ripe mRNA, i.e. maturation mRNA correspondences Part in gene.Introne is the part being cut away in mRNA process, is not present in ripe mRNA.It is outer aobvious Son and introne are all that for gene, the part of coding is extron, do not encode for introne, introne is not lost Pass effect.

In the present invention, term " exon trapping " is used interchangeably with " chip hybridization ", refers to probe in library The DNA fragmentation of exon region carries out specific selection and the process of combination.DNA molecular is double-strand under normal circumstances, therefore is caught Before obtaining, DNA molecular must be changed into single-stranded, be denatured it generally by heating and reach purpose of unwinding, the DNA molecular unwind It is rapidly cooled, that is, keeps single-chain state.After the denaturation of library capture hybridization is carried out in hybridization platform with chip.Contain exon 1 The DNA fragmentation in domain and it is fixed between the probe on chip and carries out molecule hybridization under strict conditions.It is preferred that being visited on chip The concentration of pin molecule will be significantly larger than concentration of target molecules.After hybridization is finished, the sequence of capture is collected simultaneously by methods such as denaturation Purifying, obtains the sequential mixture after capture.

In the present invention, " high-flux sequence " refers to carry out high-flux sequence using three kinds of second generation microarray datasets： 454FLX (Roche companies), Solexa Genome Analyzer (Illumina companies) and Applied Biosystems are public SOLID of department etc..The characteristics of these platforms are common is high sequencing throughput, and the 96 road capillaries relative to tradition sequencing are surveyed Sequence, high-flux sequence, which is once tested, can read 40 ten thousand to 400 ten thousand sequences, according to the difference of platform, read length from 25bp To 450bp, therefore different microarray dataset is in once testing, and can read the base number of 1G to 14G not grades.

In the present invention, term " DNA library " refers to enter the purpose fragment of genome Break Row, and obtaining one group has one Determine the DNA fragmentation mixture of size.The preparation method of DNA library is well known to those skilled in the art.In a preference, Stopping pregnancy thing, end reparation product, joint product and the enriched product of can also fighting each other are purified.Condition to reaction is carried out necessarily Change or optimize also within those skilled in the art's limit of power.

In the present invention, term " mutation " refers to that the CAST polynucleotide sequences of wild type change, as variant, Variant can naturally occur or non-natural occurs.Variant includes substitution variants, Deletion variants and insertion and become Allosome.In the present invention, a certain variant can be accomplished in several ways, for example, to obtain wild type gene coding not Complete variant, can make some codon be changed into terminator codon by inserting base in wildtype gene sequence, or The mode of the direct lack part sequence of person is realized.In the present invention, term " non-silent mutation " refers in addition to silent mutation Gene mutation, including but not limited to missense mutation, nonsense mutation and frameshift mutation etc..

The invention further relates to have at least 80% between described CAST nucleotide hybridizations and two sequences, preferably extremely Few 90%, more preferably at least 95%, at least polynucleotides of 99% homology.The present invention is more particularly directed under strict conditions with this Invent the interfertile polynucleotides of the polynucleotides.

Wild type or saltant type CAST nucleotides full length sequence of the present invention or its fragment can generally use PCR TRAPs, again Group method or artificial synthesized method are obtained., can be according to relevant nucleotide sequence disclosed in this invention, especially for PCR TRAPs It is open reading frame sequence to design primer, and with commercially available cDNA storehouses or by conventional method well known by persons skilled in the art Prepared cDNA storehouses obtain relevant sequence as template, amplification.Once obtain relevant sequence, it is possible to recombination method come Relevant sequence is obtained in large quantity.This is typically to be cloned into carrier, then is transferred to cell, then by conventional method from propagation Isolated relevant sequence in host cell afterwards.

In the present invention, " carrier " includes but is not limited to cloning vector and expression vector.In a preferred embodiment, The carrier is such as plasmid, clay, bacteriophage, coemid etc..In a preferred embodiment, the carrier is It is obtained commercially.In a preferred embodiment, the carrier operationally connects comprising the CAST genes with above-mentioned mutation The expression control sequence connect, such as, but not limited to promoter, enhancer and terminator.In a preferred embodiment, institute State carrier and optionally also include selected marker.

The carrier of above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence is included, can be used for conversion suitable When host cell, allow it to marking protein.Such host cell includes but is not limited to, prokaryotic such as large intestine bar Bacterium cell, and eukaryotic such as yeast cells, insect cell, plant cell and zooblast (such as mammalian cell, example Such as mouse cell, people's cell).The host cell of the present invention can also be cell line.

The invention further relates to CAST muteins, due in SEQ ID NO:The 607th in nucleotide sequence shown in l A (Fig. 3) is inserted between the nucleotides of the 608th so that the different bright ammonia of the amino acid sequence the 203rd of CAST gene codes The amino acid sequence that acid becomes after asparagine, the 203rd also changes, and causes the glutamic acid codon of the 210th Terminator codon is sported, CAST muteins of the invention have SEQ ID NO:Sequence shown in 3, with wild type CAST The difference of amino acid sequence is:Only 209 amino acid, have lacked 541 amino acid of rear part, therefore do not have The normal function of CAST protein.The CAST muteins of the present invention can be recombinant protein, native protein, synthesis egg White matter, preferably recombinant protein, i.e., using recombinant technique from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, elder brother Worm and mammalian cell) middle generation.

In the present invention, CAS T muteins further relate to have and CAST mutein identical functions, SEQ ID The variant form of NO.3 sequences.Variant form includes:Homologous sequence, conservative variant, induced mutants etc..Invention is further related to CAST mutein analogs.These analog skins and the difference that CAST mutains qualitative difference can be on amino acid sequence Difference on modified forms that are different or not influenceing sequence, or have both at the same time.

As well known to those skilled in the art, Disease-causing gene tool is of use in many ways.Therefore, the mutation CAST that the present invention is provided The purposes of gene includes but is not limited to：Drug target as treatment Skin peeling syndrome；Prepare Skin peeling syndrome In diagnostic kit；For producing disease animal model；New pathways of drug action is provided for treatment Skin peeling syndrome.

" kit " of the present invention refers to those skilled in the art using CAST wild types or mutant nucleotide as base Because of probe, according to the principle of gene recombination, it can detect and whether there is the nucleotides complementary with the probe sequence in biological specimen Sequence, therefore using this kit can detect to whether there is in sample the gene mutation site of the present invention.Kit Generally comprise：Primer, probe, nucleic acid chip, specification etc., it is also possible to including the buffer solution compatible with active component, carrier or Medium etc..

In the present invention, " primer " refers to the polynucleotide passage for expanding target nucleic acids in being reacted in PCR, it typically is Oligonucleotides, such as containing at least five base, such as 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20, 21st, 22,23,24,25,30,35,40, the 45, polynucleotide passage of 50 or more bases.Primer need not with it is to be amplified Target gene or its complementary strand complete complementary, as long as it being capable of specific amplification target gene.As used herein, art Language " specific amplification " refers to that primer can react amplifying target genes by PCR, without expanding other genes.For example, special Property amplification CAST genes refer to, PCR reaction in primer only expand CAST genes, without expanding other genes, such primer Design be well known to a person skilled in the art.

In the present invention, term " probe of specific detection CAST gene mutations " refers to that probe can be distinguished containing prominent The CAST genes of change and the CAST genes for not containing mutation.In general, can by controlling the stringencies of hybridization conditions, Enable the probe to distinguish the gene containing mutation and do not contain the gene of mutation.For example, under high stringency conditions, with The probe of CAST gene exact complementarities can hybridize with not containing the CAST genes of mutation, without with even only including one The CAST gene recombinations of the mutation of point mutation, so that the two be distinguished.Same way, it is also possible to design the CAST gene bases with mutation Because of the probe of exact complementarity, so that its CAST gene recombination under high stringency conditions with mutation, without with not containing mutation CAST gene recombinations.In biology field, the design and hybridization technique of probe are well known.

There is specific polyclonal antibody and monoclonal antibody the present invention relates to the CAST protein to mutation, especially Monoclonal antibody.Here, " specificity " refers to that antibody can be incorporated into the CAST protein of mutation.Can be with dashing forward it is preferred that referring to those The CAST proteins or fragment of change are combined but nonrecognition and the CAST protein associated antigen molecules for being incorporated into wild type Antibody.The present invention not only includes complete monoclonal or polyclonal antibody, but also including having immunocompetent antibody fragment, Such as Fab, or (Fab) 2 fragment；Heavy chain of antibody；Antibody light chain；Genetically engineered Single Chain Fv Molecule A or chimeric antibody.This hair Bright antibody can be prepared by various technologies known to a person skilled in the art.

The CAST protein that the CAST protein antibodies of the mutation of the present invention can be mutated for identification.For example, can use A kind of detectable molecule such as fluorescein isothiocyanate (FITC) marks the CAST protein specific antibodies of mutation, Ran Hourang The CAST protein specific antibodies of mutation are contacted with sample, then are gone out and mutation with fluorescence microscope or flow cytomery The sample that CAST protein specific antibodies are combined, so as to whether there is idiopathic Skin peeling syndrome to predict or diagnosing patient Foundation and guidance are provided.

The CAST protein antibodies of the mutation of the present invention can also be used to the CAST protein of neutral mutation.If enough The individual PSS of as shown by data it is related to the presence of present invention mutation CAST protein, it may be considered that with the CAST eggs being mutated White matter specific antibody neutralizes these pathogenic mutations CAST protein molecules, so that the illness of reduction of patient.

Embodiment 1：Sample acquisition

Inventor has found and identifies the Skin peeling syndrome family of an autosomal recessive inheritance (such as Fig. 1 institutes Show).In the family of patient (individual shown in solid black in Fig. 1) from close relative's marriage and childbirth, father and mother's phenotype is normal, and patient is except typical case Beyond PSS symptoms, patient, which also merges, there is shallow blister, Bai Jia, palm plantar angling, drying property cheilitis and knuckle pad infringement, Histopathologic appearance is epidermal hyperkeratosis, and stratum granulosum and stratum spinosum epidermidis top keratinocyte are loosened (as shown in Figure 2). Patient does not merge other systems exception, and nervous system and intelligence development are normal.

Inventor gathers the peripheral blood of every research object as grinding using this patient and its father and mother as research sample Study carefully sample, add EDTA anti-freezings, -80 DEG C of preservations.All blood samples sign category informed consent form, and obtain Ethics Committee's approval.

Embodiment 2：It is prepared by sample DNA

The peripheral blood of Skin peeling syndrome patient and its father and mother are gathered, it is complete using OMEGA Blood DNA Midi Kit Blood DNA extraction kit extracts DNA from peripheral blood sample, and extraction step is as follows：

(1) 2ml whole blood samples are taken, 150ul OB Protease, 2.1ml Buffer BL and 20ul RNase A are added, Maximal rate whirlpool 1 minute, is thoroughly mixed.

(2) 65 C water baths 15-20 minutes, and whirlpool 5 times during water-bath.

(3) 2.2ml absolute ethyl alcohols are added, maximal rate whirlpool 30 seconds is thoroughly mixed.

(4) 3.5ml lysates are moved into the 15ml centrifuge tubes with Filter column, 4000 leave the heart 5 minutes, take out Filter column, Filtered fluid is outwelled, Filter column is put back to.

(5) the remaining lysate of the 3rd step is added into the 15ml centrifuge tubes with Filter column, 4000 leave the heart 5 minutes, take out filtering Post, outwells filtered fluid, puts back to Filter column.

(6) addition 3ml HB Buffer, washing and filtering post, 4000 leave the heart 5 minutes, take out Filter column, outwell filtered fluid Body, puts back to Filter column.

(7) 3ml DNA Wash Buffer are added, 4000 leave the heart 5 minutes, takes out Filter column, outwell filtered fluid, put Return Filter column.

(8) 3ml DNA Wash Buffer are added again, 4000 leave the heart 5 minutes, take out Filter column, outwell filtered fluid Body, puts back to Filter column.

(9) 4000 leave the heart 15 minutes, dry Filter column.

(10) Filter column is moved to new 15ml centrifuge tubes, 70 degrees Celsius of 500ul Elution Buffer, room is added Temperature stands 5 minutes, and 4000 leave the heart 5 minutes, collects the filtered fluid containing DNA.

(11) Filter column is moved to new 15ml centrifuge tubes again, 70 degrees Celsius of 500ul Elution is added Buffer, is stored at room temperature 5 minutes, 4000 leave the heart 5 minutes, collects the filtered fluid containing DNA.

(12) spectrophotometer measurement DNA concentration and purity, the OD260/ of each sample genomic DNA of gained are utilized OD280 is respectively positioned between 1.7~2.0, and concentration is no less than 200ng/ul, and total amount is no less than 30 μ g.

Embodiment 3：Exon trapping and sequencing

Inventor combines Solexa high throughput sequencing technologies to choosing with NimbleGen SeqCap EZ Exome (64M) The extron group sequence of patient of embodiment 1 be sequenced, it is specific as follows：

1) genomic DNA is broken into 200-300bp or so fragment at random, then connects and connects respectively at fragment two ends Head prepares Hybrid Library (referring to http:The Illumina/Solexa standards that //www.illumina.com/ is provided build storehouse explanation Book).

2) by ligation-mediated PCR (ligation-mediated PCR (LM-PCR)) linear expansion after library is purified Increase and carry out hybridization enrichment with Biotinylated DNA Library, then upper machine is carried out after LM-PCR linear amplification and survey Sequence.Microarray dataset is Illumina Hiseq 2000, and reading length is 90bp, and the average sequencing depth of each sample is at least 50×。

3) initial data obtained after being sequenced is handled by Illumina basecalling Software 1.7, is passed through Filtering depollutes, compared using SOAPaligner 2.20# reference gene group (refer to Li R, Li Y, KristiansenK,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 reference to mode be incorporated by herein), compared to obtain to unique comparison sequence on genome Row.Then using SOAP snp (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 reference to mode be incorporated by herein) determine the genotype of target region.(insertion-deletion is inserted Indel Enter/lack mark) using BWA (by Burrows-Wheeler conversion ratios to) (version 0.5.9-r16) compare to reference Genome Hg19 (snp132), then using GATK (Genome Analysis Toolkit) (version v1.0.4705) really Determine indel type.(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；McKenna,A,Hanna M,Banks E,et al.The genome analysis toolkit:a MapReduce framework for analyzing next-generation DNA sequencing data.Genome Research 2010；20(9): 1297-1303)。

As a result, find that patient has inserting at 130437 SNPs (SNPs) and 10738 respectively in the sample Enter/lack (indel)；Then pass through dbSNP databases (http://www.ncbi.nlm.nih.gov/projects/SNP/ ), snp_summary.cgi thousand human genome databases (www.1000genomes.org/), HapMap databases (http:// The filtering of public database such as hapmap.ncbi.nlm.nih.gov/), removes all known and equipotential bases in database Because frequency is more than 0.005 variation.By comparing normal sample, remove all known variations, same sense mutation and noncoding region Variation, influence protein function less site, including intron, intergenic, UTR, same sense mutation, and soft using SIFT Part carries out SNP function predictions

Then it is autosomal recessive according to hereditary pattern and is assumed to be the situation of full genepenetrance, with reference to consanguineous marriage homozygosis Area positioning method, the Disease-causing gene that inventor finally determines this patient is CAST genes, and c.607_ its mutational site is 608insAfs, p.I203Nfs*8 (as shown in Figure 3), i.e., insert an alkali between the 607th of the gene and the 608th Base A, the site meets in family to be isolated (father and mother are heterozygote) with disease.In localization method, extron data are utilized Homozygosis segment area is detected, the SNPs or reference (reference point) that are recorded in dbsnp135 is selected as makers Site of the reads supporting rates more than or equal to 95% of (mark), reference allele or change heteroallele is considered pure And markers, site of the non-reference allele reads supporting rates between 30~70% is thought of as heterozygosis markers, non-ginseng Examining allele reads supports number to be dropped in the site of 5%~30% or 70%~95%., for homozygosis markers The coverage in site is asked to be more than or equal to 10X, the coverage for requiring site for heterozygosis markers is more than or equal to 5X.More than.We Using comprising 500 markers, as a sliding window, each sliding window allows the heterozygosis makers within 2, it is allowed to Maximum spacing between makers is 500Kb.Merge all qualified sliding windows, if the section is more than or equal to 1M length, It is homozygosis region then to think the region.By real-time PCR methods, inventor determines the mRNA of the CAST genes of patient Expression is significantly reduced (as shown in Figure 4) compared with normal patient.By having an X-rayed electron microscopy, inventor determines patient and lacks calpain Suppress albumen (as shown in Figure 5).Therefore, inventor's prediction CAST is PSS Disease-causing gene.

Embodiment 4：Pathogenic mutation gene Sanger methods are verified

Because sequencing of extron group has a certain degree of false positive, therefore we further verify, in embodiment 1 The CAST genes of PSS patient and its normal father and mother, the PSS patient of another family and 200 unrelated normal individuals are detected have Body step is as follows：

1.DNA is extracted

Method according to embodiment 2 gathers patient and its father and mother, the PSS of another family in embodiment 1 respectively Patient and the peripheral blood of 200 unrelated normal individuals, extract genomic DNA.

2. design of primers and PCR reactions

Design of primers refers to human gene data unit sequence storehouse hg19/build36.3, is specifically shown in down.

C.607_608insAfs, (sample genomic dna extracted to step 1 carries out CAST gene mutation sites P.I203Nfs*8) detect, design primer for the site, design of primers scheme meets the rule of this area design of primers , can be using conventional primer-design softwares such as Primer Premier or Oligo.

PCR reaction systems：

Reaction condition：

3.DNA is sequenced

The pcr amplification product of acquisition is purified with QIAquick PCR amplification kits (Qiagen companies), then Carry out DNA sequencing.

Mutation investigation is carried out to the frameshit insertion mutation site of CAST genes in patients' family member, patient is carrying phase The frameshit insertion mutation answered, patient father and mother are to carry corresponding heterozygous mutant, meet it is compound isolate principle, in another class The Disease-causing gene mutational site is have also discovered in patient like performance, does not find that this causes a disease in 200 agnate normal populations Mutation.Therefore, inventor thinks that CAST genes are PSS Disease-causing genes.

Inventor is also to researching and analysing that the expression in vivo of protease inhibitors target gene and function are carried out, as a result such as Under：As shown in A, B in Fig. 6, PSS patient is relative to normal control, it may appear that filaggrin is broken and expression weakens Phenomenon；As shown in C, D in Fig. 6, PSS normal control skins show the phenomenon of loricrin normal expression；In Fig. 6 Shown in E, the space between cells of PSS patient becomes big；As shown in the F in Fig. 6, the nuclear chromatin condensation of PSS patient and showing for fracture As apparent；As shown in G, H in Fig. 6, there is substantial amounts of apoptotic cell (band fluorescent core) in PSS patient thigh's skins, and just The superiors of the apoptotic cell often compareed in stratum granulosum.

Embodiment 5：The kit of the CAST genes of vitro detection PSS patient

In order to detect the pathogenic mutation of other PSS familys gene, can design all extrons in the gene coding region and The primer sequence of extron and introne intersection.In this embodiment, kit primer and amplification sequencing condition such as embodiment 4。

1st, kit forms：

Primer：As described in Example 4；

Taq enzyme

Buffer solution

dNTP

Extracted and purified genes material (DNA sample) from organism sample to be detected

2nd, application method：

(1) extracting genome DNA：Carried using the Wizard Genomic DNA extraction kits of Promega companies of the U.S. Take peripheral blood sample genomic dna.

(2) performing PCR amplification is first entered instead using above-mentioned PCR primer, Taq enzyme, sample genomic dna, buffer solution, dNTP etc. Should；

(3) pcr amplification product is purified；

(4) PCR primer to purifying carries out BigDye reactions；

(5) BiyDye reaction products are purified；

(6) BiyDye reaction products are sequenced, sequencing sequence is compared with normal sequence.

Claims (7)

1. a kind of CAST genes of mutation, it is characterised in that：The CAST gene orders of the mutation and SEQ ID NO:1 compared to tool There is the non-silent mutation of at least one, and the CAST genes of the mutation cause the generation of Skin peeling syndrome；The non-silence is dashed forward The one or more become in insertion, missing and displacement

Wherein, the non-silent mutation is SEQ ID NO:A base A, its sequence are inserted between the 607th of 1 and 608 Such as SEQ ID NO:Shown in 2.

2. a kind of protein for the CAST gene codes being mutated as claimed in claim 1, it is characterised in that：The protein Amino acid sequence such as SEQ ID NO:Shown in 3.

3. a kind of carrier, it is characterised in that：The carrier contains the CAST genes being mutated as claimed in claim 1.

4. a kind of genetically engineered host cell, it is characterised in that：The host cell is that the carrier described in claim 3 turns The host cell changed or transduceed.

5. the CAST genes being mutated as claimed in claim 1 answering in Skin peeling syndrome kit for diagnosing diseases is prepared With.

6. a kind of kit for being used to diagnose Skin peeling syndrome, it is characterised in that：The kit is comprising can be special The probe for the CAST gene mutations that opposite sex detection is mutated as claimed in claim 1.

7. kit as claimed in claim 6, it is characterised in that：The probe sequence is SEQ ID NO:Sequence shown in 1 or 2 In row before and after the 607th or the 608th 15~30bp length sequence.