CN105586389A - Kit and application thereof in detection on hereditary bone disease genes - Google Patents
Kit and application thereof in detection on hereditary bone disease genes Download PDFInfo
- Publication number
- CN105586389A CN105586389A CN201410563503.XA CN201410563503A CN105586389A CN 105586389 A CN105586389 A CN 105586389A CN 201410563503 A CN201410563503 A CN 201410563503A CN 105586389 A CN105586389 A CN 105586389A
- Authority
- CN
- China
- Prior art keywords
- genes
- bone disease
- relevant
- probe
- specifically recognize
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kit, an application thereof in detection on hereditary bone disease genes, and also a method and an apparatus for detection on the hereditary bone disease genes. The kit includes a probe which is fixed on a solid-phase substrate or is free in a solution. The probe can specially recognize exon regions of 722 or 363 genes, for example, the exon regions of the following nine genes: PHEX, ENPP1, FGF23, CLCN5, SLC34A3, DMP1, VDR, CYP2R1 and CYP27B1. The kit, and as well as the method and the apparatus for detection on the hereditary bone disease genes are used for one-time acquiring and/or detecting related genes of the hereditary bone disease and mutation status thereof.
Description
Technical field
The present invention relates to genetic test field, particularly, the present invention relates to kit and the use in detection constitutional bone disease gene thereofMore specifically, the present invention relates to kit, kit at the purposes, the detection constitutional bone disease that detect in constitutional bone disease gene on the way,The device of the method for gene and detection constitutional bone disease gene.
Background technology
Constitutional bone disease is the congenital disorders (the overall incidence of disease > 1/400) of a large group height Clinical heterogeneity and genetic heterogeneity,Normally monogenic disease. That the common clinical sign of constitutional bone disease has is of short and small stature, skeleton deformity, disproportionate growth and single orOne group of skeleton development is bad. Constitutional bone disease cause of disease complexity, comes from the metabolism way that skeleton development, growth and homeostasis process relate toRegulation and control [MatthewL.Warman, Valerie mutually between footpath, signal path numerous and metabolic pathway or signal pathCormier-Daire.NosologyandClassificationofGeneticSkeletalDisorders:2010Revision.AmJMedGenetA.2011May；155A(5):943-68.；DustinBaldridge,OlegShchelochkov；SignalingPathwaysinHumanSkeletalDysplasias.Annu.Rev.GenomicsHum.Genet.2010.11:189 – 217.]. Because constitutional bone disease has clinical and genetic heterogeneity highly, multiple comprehensiveThe clinical sign of levying is overlapping, and traditional constitutional bone disease gene tester need to consume a large amount of time and cost [MariaLBran.Lessonsfromnext-generationsequencingingeneticskeletaldisorders.BonekeyRep.2014May14; 3:528.]. Therefore, exploitation efficient, quick, economical, can be simultaneously to multiple known constitutional bone diseases base that causes a diseaseThe method detecting because suddenling change is extremely important to its clinical diagnosis.
Summary of the invention
According to an aspect of of the present present invention, the invention provides a kind of kit, it comprises probe, described probe is fixed on solid-phase matrixOr be free in solution, described probe can specific recognition the exon region of following 9 genes: PHEX, ENPP1,FGF23, CLCN5, SLC34A3, DMP1, VDR, CYP2R1 and CYP27B1. Abnormal and the rickets phase of these 9 genesClose, wherein, PHEX, ENPP1, FGF23, CLCN5, the abnormal and low serum p type rickets of SLC34A3 and/or DMP1Relevant, VDR, the abnormal rickets relevant to vitamin D metabolism of CYP2R1 and/or CYP27B1 is relevant.
Optionally, above-mentioned probe can also specific recognition the exon region of following 20 genes: COMP, FGFR3, CHST3,EBP,ARSE,PEX7,IMPAD1,ABCC9,PTH1R,GDF5,AGPS,BMPR1B,EVC,EVC2,TRIP11,SLC26A2, COL2A1, GNPAT, LBR and NSDHL. These 20 genes are relevant to cartilage development, wherein, and COMP'sExtremely can cause pseudoachondroplasia, FGFR3, CHST3, EBP and/or ARSE's is abnormal relevant to achondroplasia;FGFR3, PEX7, EBP, ARSE, GNPAT, IMPAD1, ABCC9, PTH1R, GDF5, AGPS, BMPR1B, EVC and/Or EVC2 extremely can cause osteochondrodysplasia; TRIP11, SLC26A2 and/or COL2A1 can cause Chondrogenesis not extremelyEntirely, EBP, ARSE, GNPAT, LBR and/or NSDHL are relevant to chondrodysplasia punctata.
Optionally, described probe can specific recognition the exon region of following 65 genes: SERPINH1, CRTAP,BMP1,FKBP10,COL1A2,LEPRE1,IFITM5,SERPINF1,PPIB,COL1A1,SP7,TMEM38B,WNT1,SERPINH1,TCIRG1,CLCN7,OSTM1,TNFSF11,TNFRSF11A,SNX10,PLEKHM1,CA2,LRP5,IKBKG,CTSK,LEMD3,FAM123B,ANKH,TGFB1,TBXAS1,HPGD,SLCO2A1,GJA1,TNFRSF11B,SOST,LRP4,DLX3,COL3A1,COL5A2,COL5A1,CHST14,ADAMTS2,TNXB,B4GALT7,LMNA,ZMPSTE24,MMP2,NOTCH2,MAFB,TREM2,TYROBP,ITGB3,FERMT3,SLC34A1,SLC9A3R1,MCM6,PLOD2,ATP6V0A2,PYCR1,ALDH18A1,GORAB,AP2S1, CASR, VCP and SQSTM1. These 65 genes are relevant to the abnormal relevant disease of bone mineralising, wherein, and SERPINH1,CRTAP,BMP1,FKBP10,COL1A2,LEPRE1,IFITM5,SERPINF1,PPIB,COL1A1,SP7,TMEM38B, WNT1 and/or SERPINH1 can cause osteogenesis imperfecta extremely; TCIRG1, CLCN7, OSTM1,TNFSF11,TNFRSF11A,SNX10,PLEKHM1,CA2,LRP5,IKBKG,CTSK,LEMD3,FAM123B,ANKH, TGFB1, TBXAS1, HPGD, SLCO2A1, GJA1, TNFRSF11B, SOST, LRP4 and/or DLX3's is differentOften can cause osteosclerosis; COL3A1, COL5A2, COL1A1, COL5A1, CHST14, COL1A2, ADAMTS2, TNXBAnd/or B4GALT7 extremely can cause Ehlers-Danlos syndrome; TNFRSF11A, LMNA, ZMPSTE24, MMP2,NOTCH2, MAFB, TREM2 and/or TYROBP can cause osteolysis extremely; ITGB3, FERMT3, SLC34A1,SLC9A3R1, LRP5 and/or MCM6 can cause osteoporosis extremely; FKBP10 and/or PLOD2 can cause Bruck extremelySyndrome; ATP6V0A2, PYCR1 and/or ALDH18A1 can cause cutis laxa extremely; Extremely can causing of GORAB is oldYear shape skin osteodysplasty; AP2S1 and/or CASR's is abnormal relevant to familial hypocal ciuria hypercalcinemia; VCP, SQSTM1,TNFRSF11A and/or TNFRSF11B's is extremely sick relevant to Paget.
Optionally, described probe can specific recognition the exon region of following 22 genes: IDUA, IDS, SGSH,NAGLU,HGSNAT,GNS,GALNS,HYAL1,GLB1,ARSB,GUSB,FUCA1,MAN2B1,MANBA,AGA, GLB1, NEU1, SLC17A5, CTSA, SUFM1, GNPTAB and GNPTG. Various lysosomal diseases and this 22Individual gene-correlation, mucopolysaccharidosis wherein and IDUA, IDS, SGSH, NAGLU, HGSNAT, GNS, GALNS,HYAL1, GLB1, ARSB and/or GUSB are relevant; The generation of fucosidosis is extremely relevant to FUCA1; MannoseThe generation that glycosides is stored up disease is extremely relevant to MAN2B1 and/or MANBA's; The generation of asparatylglucosaminuria disease and AGARelevant; Gangliosidosis is relevant to GLB1; Sialidosis is relevant to NEU1 and/or SLC17A5; GalaThe generation of sugar sialidosis is relevant to CTSA; The generation of sulfatase defect is relevant to SUFM1; Sticky fat is stored up sending out of diseaseLife is extremely relevant to GNPTAB and/or GNPTG's.
Optionally, described probe can specific recognition the exon region of following 54 genes: DYNC2H1, IFT80, NEK1,WDR35,TCTN3,DDX59,DYNC2H1,WDR19,TTC21B,COL9A1,COL2A1,COL11A1,COL11A2,COL9A2,COMP,COL9A3,SLC26A2,MATN3,COL9A1,RMRP,MMP13,MMP9,COL10A1,RUNX2,PTH1R,KIF22,ACP5,ACAN,PAPSS2,DDR2,TRAPPC2,CHST3,FLNA, NKX3-2,SLC35D1,INPPL1,SBDS,CDKN1C,ANKH,TRPV4,EIF2AK3,TRAPPC,SMARCAL1,SLC39A13,WISP3,DYM,IFT122,IFT43,IHH,TRPS1,PRKAR1A,ROR2,GPC6And SHOX. Backbone, backbone, epiphysis, metaphysis dysplasia relevant disease and this 54 gene-correlations, such as short rib-refer to more/ toe syndrome and DYNC2H1, IFT80, NEK1 and/or WDR35's is extremely relevant; The syndromic generation of ear-face-refer toTCTN3 and/or DDX59's is extremely relevant; Asphyxiating thoracic dysplasia and IFT80, DYNC2H1, WDR19, IFT80And/or TTC21B's is extremely relevant; Stickler syndrome and COL9A1, COL2A1, COL11A1, COL11A2 and/orCOL9A2 is relevant; The generation of multiple epiphyseal dysplasia and COL2A1, COMP, COL9A2, COL9A3, SLC26A2,MATN3 and/or COL9A1's is extremely relevant; The generation of metaphyseal dysplasia and RMRP, MMP13, MMP9,COL10A1,RUNX2,PTH1R,KIF22,COL2A1,MATN3,ACP5,ACAN,PAPSS2,DDR2,TRAPPC2, CHST3, FLNA, NKX3-2, SLC35D1, INPPL1, SBDS, CDKN1C, ANKH and/or TRPV4Extremely relevant; The undergrown generation of vertebra epiphysis and COL2A1, ACAN, EIF2AK3, MATN3, DDR2, TRAPPC,SMARCAL1, SLC39A13, NKX3-2, KIF22, WISP3 and/or DYM's is extremely relevant; Skull ectoderm is grown notGood and IFT122, WDR35, IFT43 and/or WDR19's is extremely relevant; Acromion-head-femoral hypoplasia is relevant to IHH;Nasopharynx hair follicle development is bad relevant to TRPS1; Acrodysostosis is extremely relevant to PRKAR1A's; Rainbow syndromeExtremely relevant to ROR2; Shoulder hypoplasia is extremely relevant with GPC6's; The generation of dyschondrosteosis and SHOX'sExtremely relevant.
Optionally, described probe can specific recognition the exon region of following 12 genes: SOX9, LIFR, CUL7,OBSL1, CCDC8, TBCE, FAM111A, PCNT, RNU4ATAC, CDKN1C, GJA1 and CANT1. These 12 basesCause abnormal relevant to the generation of cyrtosis, abarticulation diseases related, wherein, brachydactylia depauperation is extremely relevant with SOX9's;Stuve-Wiedmann syndrome is extremely relevant with LIFR's; 3M syndrome and CUL7, OBSL1 and/or CCDC8's is differentChang Youguan; Kenny-Caffey depauperation is extremely relevant with TBCE and/or FAM111A's; Former dwarf's cerebellum development is notEntirely extremely relevant to PCNT and/or RNU4ATAC; IMAGE syndrome is extremely relevant with CDKN1C's;Hallermann-Streiff syndrome is extremely relevant with GJA1's; Desbuquois depauperation is extremely relevant with CANT1's.
Optionally, described probe can specific recognition the exon region of following 9 genes: EXT1, EXT2, SH3BP2,PTPN11, NF1, ANO5, GNAS, FGFR1 and ACVR1. This 9 genes abnormal relevant with bone element disorder of development,Wherein, many cartilages epostoma and EXT1 and/or EXT2's is extremely relevant; Cherubism is relevant to SH3BP2; MixProperty chondroma is extremely relevant to PTPN11's; Neurofibroma is relevant with NF1; Jaw growth bad with extremely the having of ANO5Close; Carrying out property osteodysplasty is extremely relevant with GNAS's; Osteoglophonic depauperation is extremely relevant with FGFR1's,And the generation of FOP and ACVR1's is extremely relevant.
Optionally, described probe can specific recognition the exon region of following 8 genes: EZH2, NSD1, NFIX, AKT1,FBN1, FBN2, TGFBR1 and TGFBR2. Mainly extremely relevant with these 8 genes of the relevant outgrowth syndrome of skeletal system,Wherein, Wei Fu (Weaver) syndrome and EZH2's is extremely relevant; Suo Tuosi (Sotos) Megalencephaly syndrome is relevant to NSD1;Ma-Shi Er Shi (Marshall-Smith) syndrome is relevant to NFIX; Proteus syndrome is relevant with AKT1; Marfan syndromeExtremely relevant with FBN1; Congenital contracture marfan's syndrome is extremely relevant with FBN2's; Loeys-Dietz syndrome withTGFBR1 and/or TGFBR2's is extremely relevant.
Optionally, described probe can specific recognition the exon region of following 6 genes: WISP3, CIAS1, IL1RN,LPIN2, GALNT3 and ANTXR2. Main and these 6 gene-correlations of the generation of struvite/rheumatic osteoarthropathy, such as,The false rheumatic depauperation of carrying out property is extremely relevant with WISP3's; The neural skin syndromes of chronic baby and CIAS1's is differentChang Youguan; Aseptic many kitchen ranges osteomyelitis, periostitis and impetigo are extremely relevant with IL1RN's; Majeed syndrome and LPIN2Extremely relevant; Osteoproliferation/hyperphosphatemia syndrome is extremely relevant with GALNT3's; Children's's systematicness is saturating hyaline degeneration/childhoodBright property fibroma is extremely relevant with ANTXR2's.
Optionally, described probe can specific recognition the exon region of following 87 genes: RUNX2, FIG4, MSX2,ALX4,FGFR1,FGFR2,FGFR3,POR,TWIST1,RECQL4,RAB23,TCOF1,POLR1D,POLR1C,OFD1,EVC,ICK,EFNB1,ALX1,ALX3,DHODH,SF3B4,HLXB9,DLL3,MESP2,LFNG,HES7,GDF6,GDF3,COG1,BMPER,TBX4,PITX1,LMX1B,KAT6B,ORC1,IHH,BMPR1B,BMP2,GDF5,ROR2,NOG,HOXD13,HDAC4,PIGV,MYCN,HOXA13,PITX1,MGP,GNAS1,CREBBP,EP300,CHSY1,TBX3,NIPBL,FANCA,RBM8A,THPO,TBX5,SALT4,TBX15,ESCO2,LMBR1,WNT3,TP63,FBXW4,WNT10B,DLX5,CDH3,WNT7A,ARHGAP31,GLI3,MKS1,TMEM216,TMEM67,CEP290,RPGRIP1L,CC2D2A,GJA1,SHH,FAM58A,KIF7,FGF10, SALL1, FBLN1, FGF9 and HOXA11. Abnormal and the cranium of these 87 genes, face, axial skeleton, limb end,Finger/toe and the joint development not generation of total correlation disease are relevant, such as, craneocleidol dysplasia is extremely relevant with RUNX2; Especially-Wa (Yunis-Varon) syndrome is extremely relevant with FIG4; Parietal bone hypoplasia is extremely relevant with MSX2 and/or ALX4; StrikingNot (Pfeiffer) syndrome is relevant with FGFR1/FGFR2; A Peiershi (Apert) syndrome is relevant to FGFR2; Bill-Shi DiVincent (Beare-Stevenson) syndrome is extremely relevant to FGFR2; Acker (Crouzon) syndrome is extremely relevant with FGFR2;Acker (Crouzon) syndrome with black sour jujube skin is extremely relevant to FGFR3; Abnormal and the synthetic imbalance of steroids with genitals(Antley-Bixler) syndrome is extremely relevant to POR; Craniosynostosis is extremely relevant to FGFR3 and/or MSX2; Sai-Qiao(Saethre-Chotzen) syndrome and TWIST1/FGFR2's is extremely relevant; Ba-Ge Er Shi (Baller-Gerold) syndrome withRECQL4 is extremely relevant; Ka Pengte (Carpenter) syndrome is relevant to RAB23; Special Ke Er Shi (Treacher-Collins) is comprehensiveLevy extremely relevant with TCOF1POLR1D and/or POLR1C; Mouth, face, refer to that syndrome type 1 is extremely relevant with OFD1;Wei Ye (Weyer) women's head-ornaments dysosteogenesis is relevant with EVC; Endocrine-brain-osteodysplasty is relevant with ICK; Cranium volume nose is comprehensiveThe generation of levying is extremely relevant to EFNB1; Volume nose hypoplasia and ALX1, ALX3 and/or ALX4 are extremely relevant; Miller(Miller) syndrome is extremely relevant with DHODH; Hull (Nager) the women's head-ornaments dysosteogenesis of receiving is extremely relevant to SF3B4;Currarino tri-levies relevant with HLXB9; Spondylocostal dysostosis and DLL3, MESP2, LFNG and/or HES7 are abnormalRelevant; Ke-Fu Er Shi (Klippel-Fell) syndrome GDF6 and/or GDF3 are extremely relevant; With the class brain-rib of vertebra defectBone-mandibular syndrome is extremely relevant to COG1's; Transparent vertebrae hypoplasia is relevant with BMPER; Ischium kneecap is grownIncomplete extremely relevant to TBX4; Congenital reel foot is relevant with PITX1; Nailpatella syndrome is relevant with LMX1B; ReproductionKneecap syndrome is relevant with KAT6B; Ear-kneecap-runting syndrome is relevant with ORC1; Brachycephalia and IHH, BMPR1B,BMP2, GDF5, ROR2, NOG, HOXD13 and/or PTHLH are extremely relevant; Brachydactylia/toe-feeblemindedness syndrome withHDAC4 is extremely relevant; High phosphatase syndrome with feeblemindedness is extremely relevant with PIGV; Feingold syndrome type 1Extremely relevant with MYCN; Hand-foot-reproduction syndrome is relevant to HOXA13; Companion's elbow hypoplasia brachydactyly is relevant to PITX1;Keutel syndrome is relevant with MGP; Ovshinsky albright syndrome disease and GNAS1 are extremely relevant; Lu-Tai(Rubinstein-Taybi) syndrome and CREBBP and/or EP300 are extremely relevant; Before TemtamyShi axle, brachycephalia is comprehensiveLevy relevant with CHSY1; Ulna-mammary gland syndrome and TBX3 are extremely relevant; Derain Ji Shi (delange) syndrome and NIPBLExtremely relevant; Fan Kenishi anaemia is relevant to FANCA; Lack-absence of radius of blood platelet disease is relevant to RBM8A; Blood platelet increasesHow relevant with THPO with limb defect far away; Huo-Ao Er Shi (Holt-Oram) syndrome is relevant with TBX5; Okihiro syndrome andSALT4 is relevant; Coushin syndrome is relevant with TBX15; Roberts syndrome and ESCO2 are extremely relevant; AcheiropodiaRelevant to LMBR1; Congenital four limbs cut-out disease is relevant with WNT3, Split hand foot malformation and TP63, FBXW4, WNT10BAnd/or DLX5's is extremely relevant; Brachycephalia-ectoderm spot shape undernutrition syndrome is relevant with CDH3, accompanies serious limb to lackSunken ulna shin bone defect is extremely relevant to WNT7A's; Adams-Oliver syndrome is relevant with ARHGAP31;RAPADILINO syndrome is relevant with RECQL4, and Fuhrmann syndrome is relevant with WNT7A; Preaxial polydactyly and LMBR1And/or GLI3 is relevant; Dysencephalia splanchnocystica and MKS1, TMEM216, TMEM67, CEP290, RPGRIP1L and/orCC2D2A's is extremely relevant; Acrosyndactylism and GJA1, SHH and/or HOXD13's is extremely relevant; STAR syndrome withFAM58A is relevant; Acra chest syndrome is relevant to KIF7; Lacrimo-auriculo-dento-digital syndrome and FGFR2, FGFR3 and/orFGF10 is extremely relevant; Tang-Bu (Townes-Brocks) syndrome is relevant to SALL1; Zygodactyly polydactyly and FBLN1 and/Or HOXD13 is extremely relevant; Multiple synostosis syndrome and NOG, GDF5 and/or FGF9 are extremely relevant; Near-end refers toSynarthrophysis syndrome is relevant with NOG and/or GDF5; Companion without megakaryocytic decrease of platelet radius chi synostosis andHOXA11 is extremely relevant.
Optionally, described probe can specific recognition the exon region of following 77 genes: TP63, JAG1, SLC12A1,KCNJ1,CLCNKB,BSND,CLCNKA,TBX1,SLC12A3,CTSC,ABCC9,IGF-1,COL18A1,SMAD4,KCNJ10,POC1A,TRPM6,FXYD2,CLDN16,EGF,CLDN19,CNNM2,MEN1,RET,CDKN1B,CHRNA1,CHRND,CHRNG,GBA,SEPN1,POMT1,TMEM5,B3GALNT2,SKG196,B3GNT1,POMT2,POMGNT1,FKTN,FKRP,LARGE,ISPD,GTDC2,DAG1,CDC73,GNAS,GNAS-AS1,STX16,VPS13B,SEC23A,FAH,GHR,NLPR3,TFAP2A,AGTR1,ACE,AGT,REN,SLC4A1,ATP6V0A4,ATP6V1B1,G6PC,WRN,PTPN11,TPM2,MYH3,TNNI2,TNNT3,SLC29A3, COL6A1, COL6A2, COL6A3, HSPG2, SH3PXD2B, FAM20C, DHCR24, SULF1 andSLCO5A1. These 77 genes are relevant to other constitutional bone diseases, such as, Hay-Wells syndrome is extremely relevant with TP63;Alagille syndrome is extremely relevant with JAG1; Batter syndrome and SLC12A1, KCNJ1, CLCNKB, BSND and/orCLCNKA is extremely relevant; Dent is sick relevant with CLCN5; Respectively, DiGeoge syndrome and TBX1, Gitelman combinesSimulator sickness and SLC12A3, Haim-Munk syndrome and CTSC, osteochondrodysplasia companion's hair densification and ABCC9, IGF-1Lack and IGF-1, Knobloch syndrome and COL18A1, Myhre syndrome and SMAD4, SESAME syndrome andKCNJ10 is short and small, finger/toenail depauperation is relevant with hypotrichosis and POC1A with facial deformity; Hypomagnesemia and TRPM6,FXYD2, CLDN16, EGF, CLDN19 and/or CNNM2 are extremely relevant; MEA and MEN1, RET,And/or CDKN1B is extremely relevant; Multiple pterygium syndrome and CHRNA1, CHRND and/or CHRNG have extremelyClose; Gaucher disease is extremely relevant with GBA; Muscular dystrophy and SEPN1, POMT1, TMEM5, B3GALNT2, SKG196,B3GNT1, POMT2, POMGNT1, FKTN, FKRP, LARGE, ISPD, GTDC2 and/or DAG1 are extremely relevant; FirstThe other gland cancer of shape is extremely relevant with CDC73; Pseudohypoparathyroidism and GNAS, GNAS-AS1 and/or STX16Extremely relevant; Koln (Cohen) syndrome is relevant to VPS13B; Sutura cranii depauperation is extremely relevant to SEC23A; TyrosineMass formed by blood stasis and FAH are extremely relevant; Lay Lun Shi (Laron) syndrome is relevant to GHR; The neural skin syndromes of chronic baby andNLPR3 is relevant; Gill slit-eye-face syndrome and TFAP2A are extremely relevant; Renal tubule hypoplasia and AGTR1, ACE, AGTAnd/or REN is extremely relevant; RTA and SLC4A1, ATP6V0A4 and/or ATP6V1B1 are extremely relevant; GlycogenThesaurismosis and G6PC are extremely relevant; Werner (Werner) syndrome is relevant to WRN; Inborn dementia-dwarf (Noonan) combinesSimulator sickness and PTPN11 are extremely relevant; Far end arthrosis bend and TPM2, MYH3, TNNI2 and/or TNNT3 are extremely relevant; DifferentNormal osteosclerosis is relevant with SLC29A3; Congenital muscular dystrophy and COL6A1, COL6A2 and/or COL6A3 are extremely relevant;Schwartz – Jampel syndrome is extremely relevant with HSPG2; Franck – terHaar is comprehensive relevant to SH3PXD2B; RAINESyndrome and FAM20C are extremely relevant; Fiber laydown is sick extremely relevant with DHCR24; In Verloes – David – Pfeiffer typeGerminal layer depauperation is extremely relevant to SULF1 and/or SLCO5A1.
Kit of the present invention, can based on can the specific recognition above partly or entirely constitutional bone disease Disease-causing gene of described probeThe exon region of whole 363 genes of disposable acquisition, these 363 genes relate to 497 kinds of monogenic inheritance osteopathy, profitWith the kit of this one side of the present invention, can be used in disposable accurate acquisition constitutional bone disease related gene, and then can be simultaneouslyDetect multiple constitutional bone disease.
In one embodiment of the invention, described probe can also specific recognition the exon region of following 713 genes:AGT、CLCNKB、FUCA1、GBA、PLOD1、CTSK、ALPL、REN、IL10、CSF1、CR2、DDR2、RABGAP1L、THEMIS2、CLCNKA、TNFRSF1B、TBCE、NLRP3、ECE1、CLDN19、SHC1、FDPS、VASH2、GORAB、LEPRE1、ARHGEF2、COL11A1、ORC1、TGFBR3、MCL1、SERINC2、CTSS、NOTCH2、PIGV、ARID4B、ASPM、AHCYL1、POMGNT1、CDC20、CREB3L4、TMCO1、RXRG、LMNA、PTPRC、CNR2、COL9A2、IFI6、JUN、KCNJ10、LBR、PIK3C2B、SKI、SLAMF1、TNFSF4、FCER1G、GALNT2、MAPKAPK2、AIM2、MTOR、RBM8A、HSPG2、SF3B4、ZMPSTE24、IFI44、ALX3、SLC2A1、IFI44L、DUSP10、GNPAT、DHCR24、SLC35D1、ADIPOR1、FAM46C、SEPN1、CDC73、TRAF3IP3、WNT3A、BSND、TBX15、B3GALNT2、CRTC2、BGLAP、NOTCH2NL、NBPF8、CHRNA1、COL3A1、COL5A2、HOXD13、IL1B、IL1RN、CHRND、IL1R1、POMC、SP140、WDR35、CXCR4、ITM2C、MAP3K19、SP100、ACVR1、CFLAR、SMARCAL1、CAB39、KDM3A、CXCR2、OBSL1、SP110、ATP6V1B1、IHH、LY75、MATN3、PDK1、AGPS、COL6A3、ACSL3、GALNT3、NMI、EIF2AK3、CHRNG、MSTN、MYCN、IRS1、SOS1、MCM6、HDAC4、QPCT、INSIG2、IFIH1、NPPC、TTC21B、RSAD2、GCC2、RNU4ATAC、MITF、PTH1R、SLC25A20、CASR、GLB1、THPO、VHL、 AGTR1、PLOD2、CD200、TGFBR2、XRN1、BTLA、CTNNB1、TP63、IQSEC1、POC1A、FLNB、SUMF1、DAG1、IFT80、UBE2E1、CD86、PIK3CB、IRAK2、AHSG、CD47、KAT2B、CAMP、ETV5、WNT7A、CD80、SLCO2A1、PIK3CA、PPP1R2、CRTAP、CLDN16、APPL1、LAMP3、ZNF589、IFT122、IL17RB、NKIRAS1、ARHGAP31、GTDC2、HYAL1、AGA、FGFR3、IDUA、SPP1、GC、HPGD、DDX60L、FBXW7、MSMO1、TLR10、SPOCK3、CC2D2A、SH3BP2、ELOVL6、ANTXR2、NFKB1、EVC2、EGF、NKX3-2、NEK1、BMPR1B、SCARB2、CD38、ACSL1、FGF2、BST1、FGF5、IBSP、MANBA、PPARGC1A、DSPP、DKK2、TMEM156、WDR19、TIFA、EVC、ADRB2、ARSB、SLC26A2、GHR、NR3C1、TCOF1、SH3PXD2B、CAST、ARHGEF28、HMGCS1、PDE4D、CDKL3、LIFR、PDLIM4、SQSTM1、SLC34A1、PIK3R1、CHD1、FBN2、FLT4、MSX2、PITX1、SPARC、FGF18、FGF10、SPOCK1、CSF1R、PRKAA1、B4GALT7、ADAMTS2、MAML1、NIPBL、NSD1、ANKH、LTC4S、ESR1、GJA1、PEX7、NEU1、COL10A1、TNF、LTA、RUNX2、VEGFA、TFAP2A、COL11A2、EDN1、CUL7、RXRB、BMP6、COL9A1、PTP4A1、WISP3、GCM2、MAN1A1、TRIM38、HIVEP2、CD2AP、SLC17A5、OSTM1、FIG4、ICK、ZNF292、RAB23、TREM2、TNXB、SERPINB1、MB21D1、SFT2D1、KDM1B、POLR1C、CD36、COL1A2、GLI3、GUSB、SHH、LEP、PON2、TWIST1、HOXA13、IL6、NOS3、CYP3A7、NPY、POR、LFNG、TBXAS1、ISPD、CDCA7L、CDK6、CALCR、MNX1、SNX10、CUX1、CYP3A4、EZH2、IKZF1、SRI、FZD1、DLX5、HOXA11、INSIG1、SBDS、FAM20C、ATP6V0A4、LMBR1、PRKRIP1、BMPER、NCF1B、NCF1C、HIPK2、TMEM243、CA2、EXT1、WRN、GDF6、ESCO2、UBXN2B、ASAH1、SULF1、TMEM67、SLCO5A1、MCPH1、FGFR1、IKBKB、BMP1、WISP1、PDE7A、TNFRSF11B、SQLE、RIPK2、RECQL4、BNIP3L、FDFT1、TRPS1、ZHX2、VPS13B、CHD7、IMPAD1、TRIB1、SGK196、ADHFE1、LONRF1、HGSNAT、COL5A1、SNX30、VPS13A、POMT1、FKTN、TGFBR1、SYK、ADAMTSL2、LMX1B、TRPM6、CD274、RXRA、TLR4、TPM2、PIP5K1B、NPR2、ROR2、JAK2、CREB3、VCP、PAX5、TMEM38B、TRAF2、GLIPR2、C9orf85、RMRP、CYP17A1、ERCC6、FGFR2、PLEKHA1、GATA3、PAPSS2、ALDH18A1、PSAP、NFKB2、SPOCK2、TCTN3、PLAU、SLC29A3、PDCD4、FGF8、KAT6B、IFIT1、CHUK、ACADSB、SRGN、MAPK8、BNIP3、CHST3、DKK1、KIN、CNNM2、DDIT4、ENTPD7、RET、AS3MT、FBXW4、IPMK、CDKN1C、KCNJ1、MEN1、PTH、EXT2、IGF2、DRD4、DKK3、IFITM5、CPT1A、CALCA、TNNT3、DYNC2H1、RASGRP2、CTSC、SLC39A13、LTBP3、HRAS、FAM111A、ANO5、SLC37A2、TNNI2、DHCR7、TMEM216、SERPINH1、RAB6A、INPPL1、FXYD2、LRP4、LRP5、MMP13、IFITM1、FADS2、TRAF6、TCIRG1、B3GNT1、B3GAT3、FADS1、 LGR4、ALX4、FERMT3、TBX5、IGF1、ALDH2、MGP、ERBB3、FRS2、TNFRSF1A、ACACB、TESPA1、RAD51AP1、FAM60A、LEMD3、SP7、TRPV4、CD69、COL2A1、GNS、LRP6、P2RX7、PTHLH、PTPN11、WNT10B、EEA1、CDKN1B、ABCD2、WNT1、ABCC9、TBX3、PRKAB1、ALX1、WIF1、ATP6V0A2、TMEM5、SCYL2、FAR2、GDF3、GNPTAB、ADIPOR2、VPS37B、CEP290、EPSTI1、FLT1、SPATA13、POLR1D、CDX2、FGF9、TNFSF11、KL、GPC6、MBNL2、LGMN、AKT1、C14orf28、PCK2、ESR2、IFT43、HIF1A、DAAM1、TRIP11、FOS、RNASE6、SEC23A、SOS2、POMT2、ATP6V1D、NFKBIA、PPM1A、CLMN、CYP19A1、FAH、FBN1、SLC12A1、CYP1A1、PPIB、MESP2、EFTUD1、CRTC3、FMN1、ACAN、DTWD1、NPTN、GREM1、IDH2、TCF12、EID1、CHSY1、KLF13、LACTB、CHST14、KIF7、WHAMMP3、FANCA、SLC12A3、GALNS、CREBBP、CLCN7、MMP2、SALL1、RPGRIP1L、AGRP、TNFRSF17、FAM65A、KIF22、PRRT2、ZNF267、DHODH、CDH3、IRF8、CCL22、TRADD、ZC3H7A、IFT140、TXNDC11、CRISPLD2、NLRC5、GNPTG、COL1A1、G6PC、SGSH、ITGB3、NAGLU、NF1、SLC4A1、SOX9、TP53、ACE、CCL4L1、SHBG、SLC2A4、RGS9、EFTUD2、CANT1、MKS1、HES7、SLC16A6、GRB2、MYH3、SERPINF1、PRKAR1A、CCL4、STAT3、SOCS3、SLC9A3R1、MED13、DLX3、NOG、CDRT1、GNA13、PYCR1、PLEKHM1、TBX4、COG1、FKBP10、SOST、WNT3、CCL4L2、SERPINB8、PTPN2、TNFRSF11A、SMAD4、ROCK1、NFATC1、LPIN2、DYM、APOE、COMP、ERCC2、LDLR、MAN2B1、IL11、TGFB1、CD97、FKRP、CRTC1、ACP5、SLC44A2、ZNF583、ERCC1、TYROBP、NFIX、CD79A、JUNB、KCNN4、PRKACA、AP2S1、NAPSA、DLL3、MKNK2、MCOLN1、CCDC8、CREB3L3、OSCAR、ZNF700、ZNF564、JAG1、CTSA、PRNP、HNF4A、GNAS、GDF5、CYP24A1、PLCB4、STX16、SULF2、NCOA3、BMP2、RIN2、CTSZ、COL9A3、PLCG1、MMP9、MAFB、PLCB1、SALL4、NAS-AS1、AIRE、COL6A1、COL6A2、PCNT、SLC5A3、COL18A1、MRPS6、CLIC6、COMT、DENND6B、PPARA、EP300、ATF4、FBLN1、PDGFB、RAC2、GALR3、LARGE、TBX1、PLXNB2、ARSE、F9、IDS、STS、SHOX、C1GALT1C1、TRAPPC2、GAGE13、IKBKG、FLNA、NSDHL、FAM58A、SAT1、OFD1、EFNB1、ACSL4、EBP、FAM123B、DDX26B and AMER1. These 713 genes, and aforementioned PHEX, ENPP1, FGF23, CLCN5, SLC34A3, DMP1,VDR, 9 genes relevant to rickets of CYP2R1 and CYP27B1,722 genes altogether, these genes abnormal withThe early stage generation of constitutional bone disease, development, later stage etc. are relevant. Utilize this kit of the present invention, obtain these constitutional bone diseasesRelevant gene and information thereof, can be in order to the correlation circumstance of comprehensive detection person under inspection's constitutional bone disease gene, to abnormal variationThe analysis of detoxifying, and then comprehensively judge that person under inspection suffers from the probability of relevant osteopathy, suffers from the situation of relevant osteopathy, and in order to estimate itThe situation of family member's osteopathy gene and ill probability, realize constitutional bone disease accurate judged, provide base for immunotherapy targeted autoantibodyPlinth and be beneficial to and prevent that disease from recurring in family.
In one embodiment of the invention, described probe can also specific recognition its can specific recognition each gene outer aobviousThe introne region of the each 30bp of upstream and downstream of subregion. The specificity or the probe that are conducive to like this to improve probe identification object region are caughtSpecificity and efficiency while obtaining. And along with cause the discovery of the new gene of constitutional bone disease later, kit of the present invention can be notUpgrade disconnectedly, add the probe that can catch new gene, thereby constantly promote the recall rate of constitutional bone disease.
According to another aspect of the present invention, the present invention also provides the kit in one aspect of the present invention or arbitrary embodiment examiningSurvey the purposes in constitutional bone disease gene. In the time detecting constitutional bone disease gene, kit can be caught heredity in order to specific recognitionProperty osteopathy gene region, with obtain target area related data information.
The invention provides a kind of method that detects constitutional bone disease gene more on the one hand according to of the present invention, the method comprises: (1)Obtain person under inspection's nucleic acid, described nucleic acid is genomic nucleic acids and/or free nucleic acid fragment; (2) catch described nucleic acid and obtain heredityProperty osteopathy gene region; (3) described constitutional bone disease gene region is carried out to sequencing, obtain sequence information; (4) based on instituteState sequence information and detect described constitutional bone disease gene; Wherein, step (2) is utilized aforementioned one aspect of the present invention or each enforcementArbitrary kit in example carries out. The advantage of the aforementioned kit about one aspect of the present invention and the description of technical characterictic, applicable equallyIn the method for this one side of the present invention. In one embodiment of the invention, (4) comprise based on described sequence information and detect institute simultaneouslyState SNP and the INDEL variation of osteopathy gene, detect SNP and the INDEL variation of described osteopathy gene simultaneously, comprising: willDescribed sequence information and reference sequences carry out first to be compared, and obtains the first comparison result; By described the first comparison result and described referenceA part for sequence is carried out the second comparison, obtains the second comparison result; And, based on described the first comparison result and the second comparison knotReally, detect SNP and the INDEL variation of described osteopathy gene simultaneously. Said the second comparison is Local Alignment, said firstComparison is conventional overall comparison, and the first comparison can utilize but be not limited to the software such as SOAP or BWA to be carried out according to its default setting,Obtain the first comparison result, the first comparison result comprises matched position and the match condition information of sequence information on reference sequences,In one embodiment of the present of invention, said reference sequences is hg19, and in the second comparison, a part for said reference sequences comprisesEach known INDEL site in the reference sequences corresponding with caught deaf-related gene region and the each 1000bp of upstream and downstream thereofReference sequences, carry out the second comparison based on the first comparison result, to the reference corresponding with caught deaf-related gene regionNear all sequences information (reads) all known INDEL in sequence is carried out part and is again compared, and can eliminate firstMistake in comparison, improves the accuracy rate that follow-up variation detects, and the second comparison can utilize GATK anharmonic ratio to software(https://www.broadinstitute.org/gatk/) carry out. In one embodiment of the invention, based on described the first comparison knotFruit and described the second comparison result detect described SNP and INDEL variation simultaneously, are soft by GATKUnifiedGenotyperPart carries out. This method on the one hand of the present invention be based target areas captured in conjunction with ultra-high throughput sequencing technologies platform development,Be a kind of efficient, quick, economic constitutional bone disease gene tester, the method can be disposable clear and definite to 363The code area of constitutional bone disease Disease-causing gene and flank ± 30bp region, or 722 genes that constitutional bone disease genesis and development is relevantOrder-checking is caught in flank ± 30bp region, Ji Ge code area, code area, ground, then based on catching sequencing data, these genetic mutations is enteredRow determination and analysis is understood, and the disposable clear and definite person under inspection's of energy all relevant sudden change, at least relates to 497 kinds of monogenic inheritance bonesSick detection diagnosis, utilizes the method for this one side of the present invention or the method for arbitrary above-mentioned detailed description of the invention, can realize heredityThe accurate detection of property osteopathy related gene, the Accurate Diagnosis to constitutional bone disease, be conducive to immunotherapy targeted autoantibody, the personnel of family are ill severalThe recurrence of such disease in family estimated and prevented to rate. Method of the present invention provides a kind of high flux, has covered comprehensive, high precisionThe constitutional bone disease gene tester of degree, can provide scientific basis and preparental instruction to the treatment of constitutional bone disease patient clinical.
According to another aspect of the present invention, the invention provides a kind of device that detects osteopathy gene, utilize this device to complete aforementionedThe all or part of step of method of the present invention, described device comprises: A. nucleic acid acquiring unit, for obtaining person under inspection's coreAcid, described nucleic acid is genomic nucleic acids and/or free nucleic acid fragment; B. capturing unit, is connected with A unit, for catchingNucleic acid in A unit, to obtain described osteopathy gene region; C. sequencing unit, is connected with B unit, for to comeOsteopathy gene region from B unit carries out sequencing, to obtain sequence information; D. detecting unit, is connected with C unit, usesIn based on detect described osteopathy gene from the sequence information of C unit; Wherein, catching in B unit utilized aforementioned the present invention one sideKit in face or above-mentioned arbitrary detailed description of the invention carries out. Fig. 1 is the apparatus structure signal in one embodiment of the present of inventionFigure. Advantage to method of the present invention and the description of technical characterictic, be suitable for equally device of the present invention, and those skilled in the art canTo understand, all or part of unit in device of the present invention, selectable, dismountablely comprises one or more subelements to holdRow or realize each detailed description of the invention of aforementioned the inventive method.
Brief description of the drawings
Above-mentioned and/or additional aspect of the present invention and advantage from conjunction with below accompanying drawing to the description of embodiment, will become obviously withEasily understand, wherein:
Fig. 1 is the apparatus structure schematic diagram in a specific embodiment of the present invention;
Fig. 2 is the flow chart that the target area in a specific embodiment of the present invention is caught order-checking and analyzed.
Detailed description of the invention
Gene title during the application describes all adopts the official's name (OfficialSymbol) in NCBI-Gene. In addition, forAssistant separates, and some scientific and technical terminologies are explained.
Same sense mutation: refer to because the change of certain base makes to represent that certain amino acid whose codon mutation is other codons, but stillSo same amino acid of coding.
Missense mutation: encode certain amino acid whose codon after Substitution, become the another kind of amino acid whose codon of coding,Thereby amino acid kind and the sequence of polypeptide chain are changed. Some missense mutation can make polypeptide chain lose original function, many eggsThe abnormal of white matter caused by missense mutation.
Terminator codon obtains sudden change: be also referred to as nonsense mutation, refer to because the change of certain base makes to represent that certain is amino acid whose closeNumeral sports terminator codon, thereby makes peptide chain synthesize premature termination.
Terminator codon loss mutation: refer to because the change of certain base makes not other codons of terminator codon sudden change, thereby make peptideChain is synthetic cannot fair termination.
According to conventional method, realize method of the present invention and mainly comprise establishing of constitutional bone disease genetic test solid phase chip or liquid-phase chipMeter, the exploitation of sequencing technologies and analysis process is caught in target area.
(1) constitutional bone disease genetic test liquid-phase chip design
Chromosomal variation and single-gene can cause constitutional bone disease, and chromosomal variation comprises again the change of chromosome number, structure,Uniparental disomy and chimerism also can cause constitutional bone disease. This example is for single-gene and micro-heredity of causing of disappearance that repeatsOsteopathy. By to the searching of omim database and pertinent literature, obtain have that 363 individual genes can cause altogether 497Plant the single-gene disorder relevant to constitutional bone disease. Table 1 shows said 363 genes, and table 2 is bases corresponding to each large class osteopathyBecause of number, the specifying information of refinement again of table 3 display list 2, all kinds of osteopathy in each large class osteopathy and each large class osteopathy are with concreteThe relevant information of gene.
According to human genome HG19, choose exon sequence and flank ± 30bp region of above-mentioned 363 genes, can thisSample design obtains each probe sequence: obtain the exon sequence of above-mentioned 363 genes and flank ± 30bp region from hg19Each section of reference sequences, to each section of reference sequences, all, since one end of one section of reference sequences, copies the reference of predetermined length successivelySequence obtains probe sequence, makes last total probe can cover this section of reference sequences at least one times, can between adjacent probe sequenceWith overlapping or not overlapping, here, the length that predetermined length is probe, can be 50-250nt. Then, synthetic or through committing others synthesizesPrepare designed probe or chip. Liquid-phase chip size in this example is about 3.6M. On this chip, cover abundant catchingProbe, probe overlay area reaches 99.0%, can be from complicated genome enrichment target dna fragment, on same chipCatch the genome area that is about 3.6M with high specific and high coverage rate.
Table 1
Table 2
Table 3
(2) order-checking and analysis process are caught in target area
As shown in Figure 2, from person under inspection's whole blood, extract genomic DNA, and carry out library preparation by detecting qualified DNA. Literary compositionStorehouse preparation is that 1 μ g genomic DNA is broken into master tape is 200-300bp small pieces segment DNAs, then will interrupt rear DNA fragmentationCarry out end-filling, add base " A " at 3' end, DNA fragmentation can be connected with the special joint of " T " base with 3' endConnect, the library having built through Non-CapturedPCR, by constitutional bone disease genetic test probe or chip by the spy who choosesDetermine gene Exon and and flank ± 30bp region carry out enrichment, then by pcr amplification enrichment afterproduct, finally by hybridizationFront and back PCR product QPCR detects and obtains sequence capturing hybridization efficiency. After QPCR detection is qualified, the library of some is enteredThe upper machine order-checking of row Hiseq, carries out Quality Control to lower data, then to data analysis and deciphering. Wherein, a sample is prepared in libraryThe product cycle is 5-7 days. Data analysis is understood and is adopted the information analysis flow process of independent development to carry out data processing, and that does not explain especially dividesAnalysing software, the script etc. that flow process comprises can obtain by the disclosed webpage of Shenzhen Hua Da gene or database, or customization Shenzhen ChinaThis data processing is carried out in the service of large gene, and data processing mainly comprises that filtration, comparison, deduplication, anharmonic ratio are to, Quality Control, SNV(SNP) steps such as+INDEL detection, CNV detection, annotation. The deciphering of annotation result, mainly based on HGMD, BGIGapAnd each large osteopathy pathogenic mutation database and literature search consult and carry out, simultaneously in conjunction with multiple function prediction software results and person under inspectionClinical sign is comprehensively understood, and primitive rule is with reference to United States Medicine science of heredity and the (AmericanCollegeof of genomics instituteMedicalGeneticsandGenomics, ACMG) associated guideline carries out. Known, traditional constitutional bone disease genetic testMethod relies on clinician's Precise Diagnosis, by the Sanger of candidate gene being verified to seek patient's pathogenesis. If waitSelect the Sanger of gene to be verified as feminine gender, need to again assess to find new candidate gene to clinical sign, waste is a large amount ofTime and cost. And this method adopts probe/chip to catch in conjunction with ultra-high throughput sequencing technologies, disposable to 363 heredityThe detection that suddenlys change of osteopathy Disease-causing gene code area, the expense taking Sanger method as basic first generation sequencing technologies of having made up is high,Flux is low, detection range is little, be difficult to realize many-sided deficiencies such as automation, and the inspection of genetic chip mutational site detection methodThe deficiency that location point is single, coverage is low. Utilize kit of the present invention and/or method, detect the sexual valence of constitutional bone disease geneThan high, with strong points, be applicable to large-scale clinical genetic test service.
Below in conjunction with concrete individual specimen, the operation result according to concrete detection method of the present invention is described in detail. Show belowExample, only for explaining the present invention, and can not be interpreted as limitation of the present invention. In description of the invention, except as otherwise noted,The implication of " multiple " is two or more.
Except as otherwise explaining, the reagent of not explaining especially, sequence (joint, label and primer), the software that in following examples, relate toAnd instrument, be all conventional commercially available prod or disclosed, such as the hiseq2000 order-checking platform purchased from Illumina company is built storehouse phaseClose kit and carry out library construction etc.
Embodiment
To 1 routine skeletal abnormality, doubt and examine nanism and micrognathia patient (Tianjin Association for the Handicapped) detects. This patient existsCannot be diagnosed as clinically which kind of disease, after constitutional bone disease NGSPanel (above-mentioned liquid-phase chip) detects, at bone developmentOn insufficiency disorder I type Disease-causing gene FLNB, find that not bright sudden change of clinical meaning is c.542G > A (p.Gly181Asp), finalPatient is diagnosed as Atelosteogenesis I type.
Extract specimen dna with salting out method, large fragment DNA carries out ultrasonic interrupting, and uses at present sample to interrupt method and beats for CovarisDisconnected method, smashes sample DNA to the fragment of 100-700bp scope. (note: interrupt effect generally with required preparation library InsertFragment master tape position is comparatively desirable in 200-250bp position, again interrupts if interrupt undesirable needs of effect. )
1. library preparation
1.1 end reparation and purifying
After the mix concussion configuring is mixed, each reaction adds 25 μ L enzyme reaction mixed liquors.
Reaction condition: 20 DEG C, 30min
Use 180 μ LAmpureBeads to carry out product purification, the DNA of recovery is dissolved in 30 μ L (wherein 1.9 μ L are loss)Water in.
1.2 ends add " A " (A-Tailing)
After the mix concussion configuring is mixed, every pipe adds 6.9 μ L enzyme reaction mixed liquors.
Reaction condition: 20 DEG C, 30min
Note: end adds after " A " not purifying
The connection of 1.3Adapter and purifying
The mix concussion configuring is mixed, and each reaction adds 15 μ L enzyme reaction mixed liquors.
Reaction condition: 16 DEG C, 12-16h (spending the night)
Use 75 μ LAmpureBeads to carry out product purification, the DNA of recovery is dissolved in 35 μ L's (wherein 2 μ L are loss)In water.
1.4Non-Captured sample P re-LM-PCR
PCR program:
94℃2min；
94℃15s,62℃30s，72℃30s，4cycles；
72℃5min；
4℃forever
2. Probe Hybridization, enrichment is caught in target area
In this experiment, carry out hybridization elution with reference to NimbleGen operation instructions, obtain genes of interest PCR enrichment.
3. go up machine order-checking
This experiment adopts hiseq2000 or hiseq2500PE101+8+101 program to carry out upper machine order-checking.
4. information analysis
4.1 obtain initial data (FASTQ data) from sequenator.
4.2 filter: original FASTQ data are carried out to quality control, remove conventional said low quality Value Data.
4.3 comparisons: utilize SOAP software and default parameters setting thereof, use Hg19 reference sequences to compare, parallelization placeReason task.
4.4 deduplications: the read deduplication algorithm based on Picard, from comparison result, find out repetition read also to parallelizationTag mode with SAM/BAM file is carried out mark.
4.5 anharmonic ratioes pair: use the heavy comparison model based on GATK, carry out anharmonic ratio pair. That is, on the basis of a upper comparison result,Near all reads INDEL are carried out to part and again compare, to eliminate the mistake of comparison, improve the accuracy rate that variation detects.
4.6 detect SNVINDEL: use on GATKUnifiedGenotyper basis, develop based on Hadoop platformParallelization variation detection module carries out the detection of SNP and Indel simultaneously. Compare the more complete error model meter of rear utilizationCalculate genotype likelihood value, considered PCR, base quality (BaseQuality) and comparison quality (MappingQuality)Deng many factors. UnifiedGenotyper is the one variation identification software (Variants that the multiple mutation detection method of set formsCaller), UnifiedGenotyper uses Bayes Maximum Likelihood Model, estimates genotype and gene frequency simultaneously, finally to oftenEach variant sites of a sample and genotype can provide an accurate posterior probability. This software can be by input bam literary compositionThe detection that makes a variation of sample in part, finally generates a vcf file, in vcf file, can comprise all samples variant sites andGenotype information. The result obtaining from vcf file is Variants set the most original, that do not pass through any filtration and correction.The variant sites that this step produces has very high false positive, especially indel, and therefore, we carry out anharmonic ratio to the greatest extent aboveAmount reduces the false positive results in this sudden change detection.
4.7 annotations: use human genome database NCBI104, frequency database dbSNP135,1000human, ESP6500,And BGI internal frequency database annotates; Use HGVS to carry out standard name to variation, use OMIM, HGMD simultaneouslyDisease database, the clinical genome database of CGD suddenlys change and disease annotation.
5. interpretation of result
Sequencing data statistics is in table 4, table 5 display-object area coverage information, and table 6 is the pathogenic mutations that detect, table7 show other variation information of part detecting.
Table 4
Table 5
Table 6
As shown in table 6, this genetic test with Atelosteogenesis I type (AtelosteogenesistypeI, AO1; OMIM:108720) c.542G relevant FLNB gene coding region detects not bright sudden change of heterozygosis clinical meaning > A (p.Gly181Asp).The missense mutation of FLNB gene coding region is c.542G > A (p.Gly181Asp) causes FLNB gene code mRNA close at 181Numeral becomes Asp from Gly. Mother's person under inspection sample is carried out to constitutional bone disease NGSpanel detection, missense mutationC.542G > A (p.Gly181Asp) is undetected. Farrington-Rock etc. 2006 are at a routine Atelosteogenesis I type patient sampleIn this, detect heterozygous mutant c.542G > A (p.Gly181Asp) [ClaireFarrington-Rock, etal.MutationsinTwoRegionsofFLNBResultinAtelosteogenesisIandIII.HumMutat.2006Jul；27(7):705-10.]。HogueIn two routine Atelosteogenesis I type clinical samples, detected respectively heterozygosis missense mutation c.542G Deng 2013 > A (p.Gly181Asp)With heterozygosis missense mutation c.542G > C (p.Gly181Arg) [Hogue, etal.Clinicalreport:TwopatientswithatelosteogenesistypeIcausedbymissensemutationsaffectingthesameFLNBresidue.AmJMedGenetA.2013Mar; 161A (3): 619-25.]. Therefore, heterozygosis missense mutation is c.542G > A (p.Gly181Asp) is likelyCause the reason of person under inspection's phenotype.
Table 7
* as space is limited, in this list, only list database medium frequency < 5%, and the variation type that may affect protein function and mRNA montage (comprises nonsynonymous mutation, albumenInsertion, deletion mutation and the montage ± 10bp of code area are with interior variation).
* hom/het:hom represents that this mutational site is homozygous mutation, and het represents that this mutational site is heterozygous mutant.
The frequency information about this SNP of * including in Fr.1:dbSNP database.
* Fr.2: the thousand people frequency information in asian ancestry's ethnic group about this SNP that all checks order in the works in sample.
The frequency information about this SNP of * including in Fr.3:ESP6500 database.
* Fr.4: in local data base about the frequency information of this SNP.
* Condel:Condel database predicts the outcome.
Claims (11)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410563503.XA CN105586389A (en) | 2014-10-21 | 2014-10-21 | Kit and application thereof in detection on hereditary bone disease genes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410563503.XA CN105586389A (en) | 2014-10-21 | 2014-10-21 | Kit and application thereof in detection on hereditary bone disease genes |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105586389A true CN105586389A (en) | 2016-05-18 |
Family
ID=55926290
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410563503.XA Withdrawn CN105586389A (en) | 2014-10-21 | 2014-10-21 | Kit and application thereof in detection on hereditary bone disease genes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105586389A (en) |
Cited By (32)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861514A (en) * | 2016-05-27 | 2016-08-17 | 山东省立医院 | New mutant pathogenic gene SLC12A3 of Gitelman syndrome as well as encoded protein and application thereof |
| CN105907760A (en) * | 2016-05-31 | 2016-08-31 | 东北师范大学 | shRNA for targeted silencing of TKS4 |
| CN105925584A (en) * | 2016-06-23 | 2016-09-07 | 山东省立医院 | Novel mutant pathogenic gene PLEKHM1 of osteopetrosis, and encoded protein and application thereof |
| CN105936942A (en) * | 2016-07-06 | 2016-09-14 | 上海市内分泌代谢病研究所 | LGR4 gene detection kit |
| CN106755395A (en) * | 2016-12-16 | 2017-05-31 | 山东省医药生物技术研究中心 | The mutational site of XI type osteogenesis imperfecta Disease-causing genes FKBP10 and its application |
| CN107090501A (en) * | 2017-04-26 | 2017-08-25 | 青海大学 | Applications of the C1GALT1C1 in osteoarthritis diagnosis and treatment |
| CN107312864A (en) * | 2017-08-14 | 2017-11-03 | 中国医学科学院北京协和医院 | A WISP3 gene mutation for auxiliary diagnosis of PPD and its application |
| CN108280325A (en) * | 2017-12-08 | 2018-07-13 | 北京雅康博生物科技有限公司 | Processing method, processing unit, storage medium and the processor of high-flux sequence data |
| CN108300779A (en) * | 2018-02-05 | 2018-07-20 | 广州和康医疗技术有限公司 | A kind of kit and method for predicting the SNP site of leflunomide curative effect of medication |
| CN108517365A (en) * | 2018-03-27 | 2018-09-11 | 东北农业大学 | A kind of molecular marker breeding method and its application improving pig natural immunity |
| CN108570496A (en) * | 2017-03-07 | 2018-09-25 | 上海交通大学医学院附属上海儿童医学中心 | A kind of molecular diagnosis method and kit of constitutional bone disease |
| CN108710782A (en) * | 2018-05-16 | 2018-10-26 | 为朔医学数据科技（北京）有限公司 | Genotype conversion method, device and electronic equipment |
| CN108841971A (en) * | 2018-07-13 | 2018-11-20 | 西北农林科技大学 | A method of detection ox SH3PXD2B gene insertion/deletion label |
| CN108893531A (en) * | 2018-06-12 | 2018-11-27 | 广州中安基因科技有限公司 | A kind of children's vitamin requirement gene detecting kit |
| CN108982868A (en) * | 2018-06-25 | 2018-12-11 | 哈尔滨医科大学 | The application of nucleome Protein S P110 and kit containing the albumen in preparation alcoholic myocardiopathy early diagnosis reagent |
| CN109055528A (en) * | 2018-08-21 | 2018-12-21 | 中国医学科学院北京协和医院 | It is a kind of predict congenital scoliosis risk mutated gene and application |
| CN109182273A (en) * | 2018-08-21 | 2019-01-11 | 中国医学科学院北京协和医院 | Express cell model, construction method and the application of the FLNB gene of missense mutation |
| CN109825508A (en) * | 2017-11-23 | 2019-05-31 | 深圳市第三人民医院 | A biomarker for assessing HIV-TB infection |
| CN110272994A (en) * | 2019-07-15 | 2019-09-24 | 中国医学科学院北京协和医院 | Diagnose the gene mutation and its application of CVM |
| CN110938627A (en) * | 2019-11-25 | 2020-03-31 | 西安交通大学第二附属医院 | Use of an ESCO2 inhibitor for the manufacture of a medicament for the treatment of hypopharyngeal cancer |
| CN111394451A (en) * | 2020-04-22 | 2020-07-10 | 石家庄诺道中科医学检验实验室有限公司 | Insulin resistance gene polymorphism detection primer, application and kit thereof |
| CN111690734A (en) * | 2020-06-22 | 2020-09-22 | 山东第一医科大学(山东省医学科学院) | Primer group for detecting human IFITM5 gene mutation and kit thereof |
| CN112626010A (en) * | 2021-01-12 | 2021-04-09 | 北京大学口腔医学院 | Application of CDC20 in osteogenic differentiation of mesenchymal stem cells |
| CN112813156A (en) * | 2021-02-03 | 2021-05-18 | 山东第一医科大学附属省立医院(山东省立医院) | DNA library for detecting and diagnosing bone development disorder pathogenic gene and application thereof |
| CN113308527A (en) * | 2021-03-09 | 2021-08-27 | 上海市第六人民医院 | Gene composition, chip and kit for screening refractory hereditary bone diseases |
| CN113416776A (en) * | 2021-06-21 | 2021-09-21 | 深圳市儿童医院 | Biomarker for detecting ventricular septal defect and application thereof |
| CN114176043A (en) * | 2016-06-14 | 2022-03-15 | 明尼苏达大学董事会 | Genetically modified cells, tissues and organs for the treatment of disease |
| CN114599370A (en) * | 2019-10-24 | 2022-06-07 | 国立大学法人京都大学 | Pharmaceutical composition for protecting cartilage |
| CN115354078A (en) * | 2022-09-02 | 2022-11-18 | 中山大学附属第一医院 | Osteosarcoma detection markers and their applications |
| CN115807077A (en) * | 2022-12-24 | 2023-03-17 | 湛江中心人民医院 | Application of ETS1 in preparation of products for diagnosing or treating cilium diseases |
| CN117248004A (en) * | 2021-11-23 | 2023-12-19 | 山东大学齐鲁医院 | Application of RUNX2 polypeptide mutants in the preparation of kits for the evaluation of patients with cranioclavicular dysplasia |
| CN117467761A (en) * | 2023-12-28 | 2024-01-30 | 湖南家辉生物技术有限公司 | FKTN gene mutant, mutant protein, reagent, kit and application |
-
2014
- 2014-10-21 CN CN201410563503.XA patent/CN105586389A/en not_active Withdrawn
Cited By (49)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861514A (en) * | 2016-05-27 | 2016-08-17 | 山东省立医院 | New mutant pathogenic gene SLC12A3 of Gitelman syndrome as well as encoded protein and application thereof |
| CN105861514B (en) * | 2016-05-27 | 2019-05-21 | 山东省立医院 | The new mutation Disease-causing gene SLC12A3 of Gitelman syndrome and its coding albumen and application |
| CN105907760A (en) * | 2016-05-31 | 2016-08-31 | 东北师范大学 | shRNA for targeted silencing of TKS4 |
| CN114176043B (en) * | 2016-06-14 | 2024-04-23 | 明尼苏达大学董事会 | Genetically modified cells, tissues and organs for the treatment of diseases |
| CN114176043A (en) * | 2016-06-14 | 2022-03-15 | 明尼苏达大学董事会 | Genetically modified cells, tissues and organs for the treatment of disease |
| CN105925584B (en) * | 2016-06-23 | 2019-06-21 | 山东省立医院 | A novel mutation causative gene PLEKHM1, its encoded protein and its application in osteosclerosis |
| CN105925584A (en) * | 2016-06-23 | 2016-09-07 | 山东省立医院 | Novel mutant pathogenic gene PLEKHM1 of osteopetrosis, and encoded protein and application thereof |
| CN105936942A (en) * | 2016-07-06 | 2016-09-14 | 上海市内分泌代谢病研究所 | LGR4 gene detection kit |
| CN106755395B (en) * | 2016-12-16 | 2020-05-12 | 山东第一医科大学(山东省医学科学院) | Mutation site of the causative gene FKBP10 in type XI osteogenesis imperfecta and its application |
| CN106755395A (en) * | 2016-12-16 | 2017-05-31 | 山东省医药生物技术研究中心 | The mutational site of XI type osteogenesis imperfecta Disease-causing genes FKBP10 and its application |
| CN108570496A (en) * | 2017-03-07 | 2018-09-25 | 上海交通大学医学院附属上海儿童医学中心 | A kind of molecular diagnosis method and kit of constitutional bone disease |
| CN107090501B (en) * | 2017-04-26 | 2020-08-04 | 青海大学 | Application of C1GA L T1C1 in osteoarthritis diagnosis and treatment |
| CN107090501A (en) * | 2017-04-26 | 2017-08-25 | 青海大学 | Applications of the C1GALT1C1 in osteoarthritis diagnosis and treatment |
| CN107312864A (en) * | 2017-08-14 | 2017-11-03 | 中国医学科学院北京协和医院 | A WISP3 gene mutation for auxiliary diagnosis of PPD and its application |
| CN107312864B (en) * | 2017-08-14 | 2021-03-05 | 中国医学科学院北京协和医院 | WISP3 gene mutation for PPD auxiliary diagnosis and application thereof |
| CN109825508A (en) * | 2017-11-23 | 2019-05-31 | 深圳市第三人民医院 | A biomarker for assessing HIV-TB infection |
| CN108280325A (en) * | 2017-12-08 | 2018-07-13 | 北京雅康博生物科技有限公司 | Processing method, processing unit, storage medium and the processor of high-flux sequence data |
| CN108280325B (en) * | 2017-12-08 | 2020-11-27 | 北京雅康博生物科技有限公司 | High-throughput sequencing data processing method, processing device, storage medium and processor |
| CN108300779A (en) * | 2018-02-05 | 2018-07-20 | 广州和康医疗技术有限公司 | A kind of kit and method for predicting the SNP site of leflunomide curative effect of medication |
| CN108517365A (en) * | 2018-03-27 | 2018-09-11 | 东北农业大学 | A kind of molecular marker breeding method and its application improving pig natural immunity |
| CN108517365B (en) * | 2018-03-27 | 2021-03-19 | 东北农业大学 | A kind of Sp100 molecular marker breeding method for improving natural immunity of pigs and its application |
| CN108710782A (en) * | 2018-05-16 | 2018-10-26 | 为朔医学数据科技（北京）有限公司 | Genotype conversion method, device and electronic equipment |
| CN108893531A (en) * | 2018-06-12 | 2018-11-27 | 广州中安基因科技有限公司 | A kind of children's vitamin requirement gene detecting kit |
| CN108982868A (en) * | 2018-06-25 | 2018-12-11 | 哈尔滨医科大学 | The application of nucleome Protein S P110 and kit containing the albumen in preparation alcoholic myocardiopathy early diagnosis reagent |
| CN108841971A (en) * | 2018-07-13 | 2018-11-20 | 西北农林科技大学 | A method of detection ox SH3PXD2B gene insertion/deletion label |
| CN108841971B (en) * | 2018-07-13 | 2021-11-23 | 西北农林科技大学 | Method for detecting cattle SH3PXD2B gene insertion/deletion marker |
| CN109055528A (en) * | 2018-08-21 | 2018-12-21 | 中国医学科学院北京协和医院 | It is a kind of predict congenital scoliosis risk mutated gene and application |
| CN109182273A (en) * | 2018-08-21 | 2019-01-11 | 中国医学科学院北京协和医院 | Express cell model, construction method and the application of the FLNB gene of missense mutation |
| CN110272994B (en) * | 2019-07-15 | 2021-01-26 | 中国医学科学院北京协和医院 | Gene mutation diagnosis of CVM and application thereof |
| CN110272994A (en) * | 2019-07-15 | 2019-09-24 | 中国医学科学院北京协和医院 | Diagnose the gene mutation and its application of CVM |
| CN114599370A (en) * | 2019-10-24 | 2022-06-07 | 国立大学法人京都大学 | Pharmaceutical composition for protecting cartilage |
| CN110938627A (en) * | 2019-11-25 | 2020-03-31 | 西安交通大学第二附属医院 | Use of an ESCO2 inhibitor for the manufacture of a medicament for the treatment of hypopharyngeal cancer |
| CN110938627B (en) * | 2019-11-25 | 2021-07-06 | 西安交通大学第二附属医院 | Use of ESCO2 inhibitor in the preparation of a medicament for the treatment of hypopharyngeal carcinoma |
| CN111394451A (en) * | 2020-04-22 | 2020-07-10 | 石家庄诺道中科医学检验实验室有限公司 | Insulin resistance gene polymorphism detection primer, application and kit thereof |
| CN111394451B (en) * | 2020-04-22 | 2021-04-02 | 石家庄诺道中科医学检验实验室有限公司 | Insulin resistance gene polymorphism detection primer, application and kit thereof |
| CN111690734A (en) * | 2020-06-22 | 2020-09-22 | 山东第一医科大学(山东省医学科学院) | Primer group for detecting human IFITM5 gene mutation and kit thereof |
| CN111690734B (en) * | 2020-06-22 | 2021-07-02 | 山东第一医科大学(山东省医学科学院) | Primer set and kit for detecting human IFITM5 gene mutation |
| CN112626010A (en) * | 2021-01-12 | 2021-04-09 | 北京大学口腔医学院 | Application of CDC20 in osteogenic differentiation of mesenchymal stem cells |
| CN112626010B (en) * | 2021-01-12 | 2022-05-17 | 北京大学口腔医学院 | Application of CDC20 in osteogenic differentiation of mesenchymal stem cells |
| CN112813156A (en) * | 2021-02-03 | 2021-05-18 | 山东第一医科大学附属省立医院(山东省立医院) | DNA library for detecting and diagnosing bone development disorder pathogenic gene and application thereof |
| CN113308527A (en) * | 2021-03-09 | 2021-08-27 | 上海市第六人民医院 | Gene composition, chip and kit for screening refractory hereditary bone diseases |
| CN113416776A (en) * | 2021-06-21 | 2021-09-21 | 深圳市儿童医院 | Biomarker for detecting ventricular septal defect and application thereof |
| CN113416776B (en) * | 2021-06-21 | 2022-04-01 | 深圳市儿童医院 | Biomarker for detecting ventricular septal defect and application thereof |
| CN117248004A (en) * | 2021-11-23 | 2023-12-19 | 山东大学齐鲁医院 | Application of RUNX2 polypeptide mutants in the preparation of kits for the evaluation of patients with cranioclavicular dysplasia |
| CN115354078A (en) * | 2022-09-02 | 2022-11-18 | 中山大学附属第一医院 | Osteosarcoma detection markers and their applications |
| CN115807077A (en) * | 2022-12-24 | 2023-03-17 | 湛江中心人民医院 | Application of ETS1 in preparation of products for diagnosing or treating cilium diseases |
| CN115807077B (en) * | 2022-12-24 | 2023-06-27 | 湛江中心人民医院 | Use of ETS1 in the preparation of a product for diagnosing or treating ciliated disorders |
| CN117467761A (en) * | 2023-12-28 | 2024-01-30 | 湖南家辉生物技术有限公司 | FKTN gene mutant, mutant protein, reagent, kit and application |
| CN117467761B (en) * | 2023-12-28 | 2024-05-31 | 湖南家辉生物技术有限公司 | FKTN gene mutant, mutant protein, reagent, kit and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105586389A (en) | Kit and application thereof in detection on hereditary bone disease genes | |
| Sindher et al. | Food allergy, mechanisms, diagnosis and treatment: innovation through a multi‐targeted approach | |
| AU2024219712A1 (en) | Interpretation of genetic and genomic variants via an integrated computational and experimental deep mutational learning framework | |
| Costello et al. | Dissection of the inflammatory bowel disease transcriptome using genome-wide cDNA microarrays | |
| Granada et al. | A genome-wide association study of plasma total IgE concentrations in the Framingham Heart Study | |
| US20110098193A1 (en) | Methods and Systems for Medical Sequencing Analysis | |
| Newman et al. | High-altitude pulmonary hypertension in cattle (brisket disease): Candidate genes and gene expression profiling of peripheral blood mononuclear cells | |
| US20210074378A1 (en) | Methods for Analyzing Genetic Data to Classify Multifactorial Traits Including Complex Medical Disorders | |
| CN109652533A (en) | A method of for detecting the Disease-causing gene for causing the other systems disease of cardiovascular symptom | |
| Zhang et al. | Identification of monocytes associated with severe COVID-19 in the PBMCs of severely infected patients through single-cell transcriptome sequencing | |
| Jin et al. | Integrative analysis of transcriptomic and proteomic profiling in inflammatory bowel disease colon biopsies | |
| Wang et al. | Genome-wide transcriptome analysis of hippocampus in rats indicated that TLR/NLR signaling pathway was involved in the pathogenisis of depressive disorder induced by chronic restraint stress | |
| CN112813156A (en) | DNA library for detecting and diagnosing bone development disorder pathogenic gene and application thereof | |
| US20240282449A1 (en) | Methods and systems for machine learning analysis of inflammatory skin diseases | |
| Eckenrode et al. | Gene expression profiles define a key checkpoint for type 1 diabetes in NOD mice | |
| Gavriilidis et al. | Neutrophil-fibroblast crosstalk drives immunofibrosis in Crohn’s disease through IFNα pathway | |
| Yang et al. | Bioinformatics analysis of ceRNA regulatory network of baicalin in alleviating pathological joint alterations in CIA rats | |
| Zhao et al. | How does orthodontic tooth movement influence the dental pulp? RNA‐sequencing on human premolars | |
| Wu et al. | Identification of molecular signatures in acute myocardial infarction based on integrative analysis of proteomics and transcriptomics | |
| Li et al. | Combined transcriptomics and TMT-proteomics reveal abnormal complement and coagulation cascades in cow's milk protein allergy | |
| Nematiasgarabad et al. | In silico elucidation of protein-protein interaction network in fish pathogen Flavobacterium Columnare | |
| Chang et al. | Uncovering the genetic link between acute myocardial infarction and ulcerative colitis co-morbidity through a systems biology approach | |
| Alradi et al. | A long-term high-fat diet induces differential gene expression changes in spatially distinct adipose tissue of male mice | |
| Pavlidis et al. | Cytokine responsive networks in human colonic epithelial organoids unveil a novel molecular stratification of inflammatory bowel disease | |
| CN113308527A (en) | Gene composition, chip and kit for screening refractory hereditary bone diseases |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C04 | Withdrawal of patent application after publication (patent law 2001) | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20160518 |