CN114196736A - 一种同步检测多种出生缺陷遗传病全染色体基因分型芯片及其方法和应用 - Google Patents
一种同步检测多种出生缺陷遗传病全染色体基因分型芯片及其方法和应用 Download PDFInfo
- Publication number
- CN114196736A CN114196736A CN202111307383.3A CN202111307383A CN114196736A CN 114196736 A CN114196736 A CN 114196736A CN 202111307383 A CN202111307383 A CN 202111307383A CN 114196736 A CN114196736 A CN 114196736A
- Authority
- CN
- China
- Prior art keywords
- gene
- probe
- copy number
- sack
- probes
- 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.)
- Granted
Links
- 208000032170 Congenital Abnormalities Diseases 0.000 title claims abstract description 47
- 230000007698 birth defect Effects 0.000 title claims abstract description 47
- 208000026350 Inborn Genetic disease Diseases 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 27
- 238000003205 genotyping method Methods 0.000 title claims abstract description 20
- 210000000349 chromosome Anatomy 0.000 title claims abstract description 19
- 208000024556 Mendelian disease Diseases 0.000 title abstract description 10
- 239000000523 sample Substances 0.000 claims abstract description 198
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 128
- 238000001514 detection method Methods 0.000 claims abstract description 54
- 208000016361 genetic disease Diseases 0.000 claims abstract description 33
- 230000001717 pathogenic effect Effects 0.000 claims abstract description 12
- 230000035945 sensitivity Effects 0.000 claims abstract description 9
- -1 CYP21a2 Proteins 0.000 claims description 55
- 238000009396 hybridization Methods 0.000 claims description 40
- 239000002773 nucleotide Substances 0.000 claims description 27
- 125000003729 nucleotide group Chemical group 0.000 claims description 27
- 238000013467 fragmentation Methods 0.000 claims description 21
- 238000006062 fragmentation reaction Methods 0.000 claims description 21
- 238000001556 precipitation Methods 0.000 claims description 16
- 230000003321 amplification Effects 0.000 claims description 14
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 14
- 230000036425 denaturation Effects 0.000 claims description 11
- 238000012217 deletion Methods 0.000 claims description 10
- 230000037430 deletion Effects 0.000 claims description 10
- 238000002493 microarray Methods 0.000 claims description 10
- 238000003908 quality control method Methods 0.000 claims description 10
- 238000004925 denaturation Methods 0.000 claims description 9
- 239000011259 mixed solution Substances 0.000 claims description 9
- 108020004414 DNA Proteins 0.000 claims description 7
- 102100040202 Apolipoprotein B-100 Human genes 0.000 claims description 6
- 102100022317 Dihydropteridine reductase Human genes 0.000 claims description 6
- 102100024108 Dystrophin Human genes 0.000 claims description 6
- 102100027685 Hemoglobin subunit alpha Human genes 0.000 claims description 6
- 101000889953 Homo sapiens Apolipoprotein B-100 Proteins 0.000 claims description 6
- 101000902365 Homo sapiens Dihydropteridine reductase Proteins 0.000 claims description 6
- 101001053946 Homo sapiens Dystrophin Proteins 0.000 claims description 6
- 101001009007 Homo sapiens Hemoglobin subunit alpha Proteins 0.000 claims description 6
- 102100038223 Phenylalanine-4-hydroxylase Human genes 0.000 claims description 6
- 101710125939 Phenylalanine-4-hydroxylase Proteins 0.000 claims description 6
- 101001053942 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) Diphosphomevalonate decarboxylase Proteins 0.000 claims description 6
- 102100034803 Small nuclear ribonucleoprotein-associated protein N Human genes 0.000 claims description 6
- 210000001766 X chromosome Anatomy 0.000 claims description 6
- 238000004364 calculation method Methods 0.000 claims description 6
- 101150055297 SET1 gene Proteins 0.000 claims description 5
- 230000002159 abnormal effect Effects 0.000 claims description 5
- 210000002230 centromere Anatomy 0.000 claims description 5
- 108091008794 FGF receptors Proteins 0.000 claims description 4
- 101000851054 Homo sapiens Elastin Proteins 0.000 claims description 4
- 101000687673 Homo sapiens Small integral membrane protein 6 Proteins 0.000 claims description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 4
- 102100024806 Small integral membrane protein 6 Human genes 0.000 claims description 4
- 102000052178 fibroblast growth factor receptor activity proteins Human genes 0.000 claims description 4
- 230000007918 pathogenicity Effects 0.000 claims description 4
- 210000003765 sex chromosome Anatomy 0.000 claims description 4
- PCTMTFRHKVHKIS-BMFZQQSSSA-N (1s,3r,4e,6e,8e,10e,12e,14e,16e,18s,19r,20r,21s,25r,27r,30r,31r,33s,35r,37s,38r)-3-[(2r,3s,4s,5s,6r)-4-amino-3,5-dihydroxy-6-methyloxan-2-yl]oxy-19,25,27,30,31,33,35,37-octahydroxy-18,20,21-trimethyl-23-oxo-22,39-dioxabicyclo[33.3.1]nonatriaconta-4,6,8,10 Chemical compound C1C=C2C[C@@H](OS(O)(=O)=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2.O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 PCTMTFRHKVHKIS-BMFZQQSSSA-N 0.000 claims description 3
- KUBWJGWIWGGEPZ-UHFFFAOYSA-N 1-[amino(ethoxy)phosphoryl]oxy-4-nitrobenzene Chemical compound CCOP(N)(=O)OC1=CC=C([N+]([O-])=O)C=C1 KUBWJGWIWGGEPZ-UHFFFAOYSA-N 0.000 claims description 3
- 102100035352 2-oxoisovalerate dehydrogenase subunit alpha, mitochondrial Human genes 0.000 claims description 3
- 102100035315 2-oxoisovalerate dehydrogenase subunit beta, mitochondrial Human genes 0.000 claims description 3
- MXCVHSXCXPHOLP-UHFFFAOYSA-N 4-oxo-6-propylchromene-2-carboxylic acid Chemical compound O1C(C(O)=O)=CC(=O)C2=CC(CCC)=CC=C21 MXCVHSXCXPHOLP-UHFFFAOYSA-N 0.000 claims description 3
- 101150091481 ATP7 gene Proteins 0.000 claims description 3
- 102100034561 Alpha-N-acetylglucosaminidase Human genes 0.000 claims description 3
- 102100025511 Anti-Muellerian hormone type-2 receptor Human genes 0.000 claims description 3
- 101100225890 Aplysia californica ENPP gene Proteins 0.000 claims description 3
- 102100031491 Arylsulfatase B Human genes 0.000 claims description 3
- 102100025142 Beta-microseminoprotein Human genes 0.000 claims description 3
- 101000796712 Canis lupus familiaris Mastin Proteins 0.000 claims description 3
- 102100029297 Cholinephosphotransferase 1 Human genes 0.000 claims description 3
- 102100027591 Copper-transporting ATPase 2 Human genes 0.000 claims description 3
- 108010016788 Cyclin-Dependent Kinase Inhibitor p21 Proteins 0.000 claims description 3
- 102100033270 Cyclin-dependent kinase inhibitor 1 Human genes 0.000 claims description 3
- 102000004328 Cytochrome P-450 CYP3A Human genes 0.000 claims description 3
- 108010081668 Cytochrome P-450 CYP3A Proteins 0.000 claims description 3
- 102100024329 Cytochrome P450 11B2, mitochondrial Human genes 0.000 claims description 3
- 102100029363 Cytochrome P450 2C19 Human genes 0.000 claims description 3
- 230000004544 DNA amplification Effects 0.000 claims description 3
- 101100055841 Danio rerio apoa1 gene Proteins 0.000 claims description 3
- 102100029792 Dentin sialophosphoprotein Human genes 0.000 claims description 3
- 102100033189 Diablo IAP-binding mitochondrial protein Human genes 0.000 claims description 3
- 102100023319 Dihydrolipoyl dehydrogenase, mitochondrial Human genes 0.000 claims description 3
- 102100037713 Down syndrome cell adhesion molecule Human genes 0.000 claims description 3
- 101001037071 Drosophila melanogaster GIGYF family protein Gyf Proteins 0.000 claims description 3
- 102100030695 Electron transfer flavoprotein subunit alpha, mitochondrial Human genes 0.000 claims description 3
- 102100023593 Fibroblast growth factor receptor 1 Human genes 0.000 claims description 3
- 102100027346 GTP cyclohydrolase 1 Human genes 0.000 claims description 3
- 102100025894 Glomulin Human genes 0.000 claims description 3
- 102100029481 Glycogen phosphorylase, liver form Human genes 0.000 claims description 3
- 102100021519 Hemoglobin subunit beta Human genes 0.000 claims description 3
- 102100026265 Histone-lysine N-methyltransferase ASH1L Human genes 0.000 claims description 3
- 101000597665 Homo sapiens 2-oxoisovalerate dehydrogenase subunit alpha, mitochondrial Proteins 0.000 claims description 3
- 101000597680 Homo sapiens 2-oxoisovalerate dehydrogenase subunit beta, mitochondrial Proteins 0.000 claims description 3
- 101100001532 Homo sapiens AMHR2 gene Proteins 0.000 claims description 3
- 101000784014 Homo sapiens Argininosuccinate synthase Proteins 0.000 claims description 3
- 101000901140 Homo sapiens Arylsulfatase A Proteins 0.000 claims description 3
- 101000923070 Homo sapiens Arylsulfatase B Proteins 0.000 claims description 3
- 101000576812 Homo sapiens Beta-microseminoprotein Proteins 0.000 claims description 3
- 101000933545 Homo sapiens Biotinidase Proteins 0.000 claims description 3
- 101000859570 Homo sapiens Carnitine O-palmitoyltransferase 1, liver isoform Proteins 0.000 claims description 3
- 101000909313 Homo sapiens Carnitine O-palmitoyltransferase 2, mitochondrial Proteins 0.000 claims description 3
- 101000989606 Homo sapiens Cholinephosphotransferase 1 Proteins 0.000 claims description 3
- 101000936280 Homo sapiens Copper-transporting ATPase 2 Proteins 0.000 claims description 3
- 101000761960 Homo sapiens Cytochrome P450 11B1, mitochondrial Proteins 0.000 claims description 3
- 101000761956 Homo sapiens Cytochrome P450 11B2, mitochondrial Proteins 0.000 claims description 3
- 101000896586 Homo sapiens Cytochrome P450 2D6 Proteins 0.000 claims description 3
- 101000804964 Homo sapiens DNA polymerase subunit gamma-1 Proteins 0.000 claims description 3
- 101000865404 Homo sapiens Dentin sialophosphoprotein Proteins 0.000 claims description 3
- 101000871228 Homo sapiens Diablo IAP-binding mitochondrial protein Proteins 0.000 claims description 3
- 101000880945 Homo sapiens Down syndrome cell adhesion molecule Proteins 0.000 claims description 3
- 101001010541 Homo sapiens Electron transfer flavoprotein subunit alpha, mitochondrial Proteins 0.000 claims description 3
- 101000862581 Homo sapiens GTP cyclohydrolase 1 Proteins 0.000 claims description 3
- 101000857303 Homo sapiens Glomulin Proteins 0.000 claims description 3
- 101000700616 Homo sapiens Glycogen phosphorylase, liver form Proteins 0.000 claims description 3
- 101000785963 Homo sapiens Histone-lysine N-methyltransferase ASH1L Proteins 0.000 claims description 3
- 101001122174 Homo sapiens Lipoamide acyltransferase component of branched-chain alpha-keto acid dehydrogenase complex, mitochondrial Proteins 0.000 claims description 3
- 101001098256 Homo sapiens Lysophospholipase Proteins 0.000 claims description 3
- 101000760730 Homo sapiens Medium-chain specific acyl-CoA dehydrogenase, mitochondrial Proteins 0.000 claims description 3
- 101001013648 Homo sapiens Methionine synthase Proteins 0.000 claims description 3
- 101000651201 Homo sapiens N-sulphoglucosamine sulphohydrolase Proteins 0.000 claims description 3
- 101000979216 Homo sapiens Necdin Proteins 0.000 claims description 3
- 101001111328 Homo sapiens Nuclear factor 1 A-type Proteins 0.000 claims description 3
- 101000595929 Homo sapiens POLG alternative reading frame Proteins 0.000 claims description 3
- 101000611202 Homo sapiens Peptidyl-prolyl cis-trans isomerase B Proteins 0.000 claims description 3
- 101001137939 Homo sapiens Phosphorylase b kinase regulatory subunit beta Proteins 0.000 claims description 3
- 101001066701 Homo sapiens Pogo transposable element with ZNF domain Proteins 0.000 claims description 3
- 101001003584 Homo sapiens Prelamin-A/C Proteins 0.000 claims description 3
- 101001098989 Homo sapiens Propionyl-CoA carboxylase alpha chain, mitochondrial Proteins 0.000 claims description 3
- 101001098982 Homo sapiens Propionyl-CoA carboxylase beta chain, mitochondrial Proteins 0.000 claims description 3
- 101001125901 Homo sapiens Pterin-4-alpha-carbinolamine dehydratase Proteins 0.000 claims description 3
- 101000896576 Homo sapiens Putative cytochrome P450 2D7 Proteins 0.000 claims description 3
- 101000703464 Homo sapiens SH3 and multiple ankyrin repeat domains protein 2 Proteins 0.000 claims description 3
- 101000631760 Homo sapiens Sodium channel protein type 1 subunit alpha Proteins 0.000 claims description 3
- 101000685323 Homo sapiens Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial Proteins 0.000 claims description 3
- 101000874160 Homo sapiens Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial Proteins 0.000 claims description 3
- 101000934888 Homo sapiens Succinate dehydrogenase cytochrome b560 subunit, mitochondrial Proteins 0.000 claims description 3
- 101000617738 Homo sapiens Survival motor neuron protein Proteins 0.000 claims description 3
- 101000585635 Homo sapiens Unconventional myosin-XV Proteins 0.000 claims description 3
- 101000844802 Lacticaseibacillus rhamnosus Teichoic acid D-alanyltransferase Proteins 0.000 claims description 3
- 102100027064 Lipoamide acyltransferase component of branched-chain alpha-keto acid dehydrogenase complex, mitochondrial Human genes 0.000 claims description 3
- 102100037611 Lysophospholipase Human genes 0.000 claims description 3
- 108700012912 MYCN Proteins 0.000 claims description 3
- 101150022024 MYCN gene Proteins 0.000 claims description 3
- 102100024590 Medium-chain specific acyl-CoA dehydrogenase, mitochondrial Human genes 0.000 claims description 3
- 101710122877 Muellerian-inhibiting factor Proteins 0.000 claims description 3
- 108700026495 N-Myc Proto-Oncogene Proteins 0.000 claims description 3
- 102100030124 N-myc proto-oncogene protein Human genes 0.000 claims description 3
- 102100027661 N-sulphoglucosamine sulphohydrolase Human genes 0.000 claims description 3
- 108091008652 NR0B Proteins 0.000 claims description 3
- 108091008637 NR5A Proteins 0.000 claims description 3
- 102000007530 Neurofibromin 1 Human genes 0.000 claims description 3
- 108010085793 Neurofibromin 1 Proteins 0.000 claims description 3
- 102100024006 Nuclear factor 1 A-type Human genes 0.000 claims description 3
- 108010011536 PTEN Phosphohydrolase Proteins 0.000 claims description 3
- 102100040283 Peptidyl-prolyl cis-trans isomerase B Human genes 0.000 claims description 3
- 102100020854 Phosphorylase b kinase regulatory subunit beta Human genes 0.000 claims description 3
- 102100034345 Pogo transposable element with ZNF domain Human genes 0.000 claims description 3
- 101710189720 Porphobilinogen deaminase Proteins 0.000 claims description 3
- 102100034391 Porphobilinogen deaminase Human genes 0.000 claims description 3
- 101710170827 Porphobilinogen deaminase, chloroplastic Proteins 0.000 claims description 3
- 102100026531 Prelamin-A/C Human genes 0.000 claims description 3
- 101710119292 Probable D-lactate dehydrogenase, mitochondrial Proteins 0.000 claims description 3
- 101710100896 Probable porphobilinogen deaminase Proteins 0.000 claims description 3
- 102100039022 Propionyl-CoA carboxylase alpha chain, mitochondrial Human genes 0.000 claims description 3
- 102100039025 Propionyl-CoA carboxylase beta chain, mitochondrial Human genes 0.000 claims description 3
- 102100029333 Pterin-4-alpha-carbinolamine dehydratase Human genes 0.000 claims description 3
- 102100021702 Putative cytochrome P450 2D7 Human genes 0.000 claims description 3
- 101001030849 Rhinella marina Mesotocin receptor Proteins 0.000 claims description 3
- 102100030680 SH3 and multiple ankyrin repeat domains protein 2 Human genes 0.000 claims description 3
- 102100022978 Sex-determining region Y protein Human genes 0.000 claims description 3
- 102100028910 Sodium channel protein type 1 subunit alpha Human genes 0.000 claims description 3
- 102100023155 Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial Human genes 0.000 claims description 3
- 102100035726 Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial Human genes 0.000 claims description 3
- 102100025393 Succinate dehydrogenase cytochrome b560 subunit, mitochondrial Human genes 0.000 claims description 3
- 102100021947 Survival motor neuron protein Human genes 0.000 claims description 3
- 102100029836 Unconventional myosin-XV Human genes 0.000 claims description 3
- 210000002593 Y chromosome Anatomy 0.000 claims description 3
- 108010016200 Zinc Finger Protein GLI1 Proteins 0.000 claims description 3
- 108010009380 alpha-N-acetyl-D-glucosaminidase Proteins 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 101150055214 cyp1a1 gene Proteins 0.000 claims description 3
- 108010012052 cytochrome P-450 CYP2C subfamily Proteins 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 238000001917 fluorescence detection Methods 0.000 claims description 3
- 239000007850 fluorescent dye Substances 0.000 claims description 3
- 238000001215 fluorescent labelling Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims description 3
- CXVGEDCSTKKODG-UHFFFAOYSA-N sulisobenzone Chemical compound C1=C(S(O)(=O)=O)C(OC)=CC(O)=C1C(=O)C1=CC=CC=C1 CXVGEDCSTKKODG-UHFFFAOYSA-N 0.000 claims description 3
- AMQVHASIFJZFOS-UHFFFAOYSA-N 3-[(4-chlorophenyl)-(4-hydroxy-2-oxochromen-3-yl)methyl]-4-hydroxychromen-2-one Chemical compound O=C1OC=2C=CC=CC=2C(O)=C1C(C=1C(OC2=CC=CC=C2C=1O)=O)C1=CC=C(Cl)C=C1 AMQVHASIFJZFOS-UHFFFAOYSA-N 0.000 claims description 2
- 102100022385 Activity-dependent neuroprotector homeobox protein Human genes 0.000 claims description 2
- 102100026277 Alpha-galactosidase A Human genes 0.000 claims description 2
- 101100002343 Arabidopsis thaliana ARID1 gene Proteins 0.000 claims description 2
- 102100025621 Cytochrome b-245 heavy chain Human genes 0.000 claims description 2
- 102100038002 Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit STT3A Human genes 0.000 claims description 2
- 102100028554 Dual specificity tyrosine-phosphorylation-regulated kinase 1A Human genes 0.000 claims description 2
- 102100031804 Electron transfer flavoprotein-ubiquinone oxidoreductase, mitochondrial Human genes 0.000 claims description 2
- 102100025403 Epoxide hydrolase 1 Human genes 0.000 claims description 2
- 102100026561 Filamin-A Human genes 0.000 claims description 2
- 102100029492 Glycogen phosphorylase, muscle form Human genes 0.000 claims description 2
- 102100032610 Guanine nucleotide-binding protein G(s) subunit alpha isoforms XLas Human genes 0.000 claims description 2
- 101000901099 Homo sapiens Achaete-scute homolog 1 Proteins 0.000 claims description 2
- 101000755474 Homo sapiens Activity-dependent neuroprotector homeobox protein Proteins 0.000 claims description 2
- 101000838016 Homo sapiens Dual specificity tyrosine-phosphorylation-regulated kinase 1A Proteins 0.000 claims description 2
- 101000920874 Homo sapiens Electron transfer flavoprotein-ubiquinone oxidoreductase, mitochondrial Proteins 0.000 claims description 2
- 101001077852 Homo sapiens Epoxide hydrolase 1 Proteins 0.000 claims description 2
- 101000913549 Homo sapiens Filamin-A Proteins 0.000 claims description 2
- 101000700475 Homo sapiens Glycogen phosphorylase, muscle form Proteins 0.000 claims description 2
- 101001014590 Homo sapiens Guanine nucleotide-binding protein G(s) subunit alpha isoforms XLas Proteins 0.000 claims description 2
- 101001014594 Homo sapiens Guanine nucleotide-binding protein G(s) subunit alpha isoforms short Proteins 0.000 claims description 2
- 101000687968 Homo sapiens Membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase Proteins 0.000 claims description 2
- 101000587058 Homo sapiens Methylenetetrahydrofolate reductase Proteins 0.000 claims description 2
- 101001014610 Homo sapiens Neuroendocrine secretory protein 55 Proteins 0.000 claims description 2
- 101001126783 Homo sapiens Phosphorylase b kinase gamma catalytic chain, skeletal muscle/heart isoform Proteins 0.000 claims description 2
- 101000945267 Homo sapiens Phosphorylase b kinase regulatory subunit alpha, skeletal muscle isoform Proteins 0.000 claims description 2
- 101000797903 Homo sapiens Protein ALEX Proteins 0.000 claims description 2
- 101000588019 Homo sapiens Shadow of prion protein Proteins 0.000 claims description 2
- 101000664527 Homo sapiens Spastin Proteins 0.000 claims description 2
- 108010007013 Melanocyte-Stimulating Hormones Proteins 0.000 claims description 2
- 102100024262 Membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase Human genes 0.000 claims description 2
- 102100031551 Methionine synthase Human genes 0.000 claims description 2
- 102100029684 Methylenetetrahydrofolate reductase Human genes 0.000 claims description 2
- 101710132427 Mevalonate 3,5-bisphosphate decarboxylase Proteins 0.000 claims description 2
- 101100079084 Mus musculus Myo7a gene Proteins 0.000 claims description 2
- 108010082739 NADPH Oxidase 2 Proteins 0.000 claims description 2
- 101000797638 Oryctolagus cuniculus Alveolar macrophage chemotactic factor Proteins 0.000 claims description 2
- 102100030278 Phosphorylase b kinase gamma catalytic chain, skeletal muscle/heart isoform Human genes 0.000 claims description 2
- 102100033547 Phosphorylase b kinase regulatory subunit alpha, skeletal muscle isoform Human genes 0.000 claims description 2
- 102100027467 Pro-opiomelanocortin Human genes 0.000 claims description 2
- 102100031579 Shadow of prion protein Human genes 0.000 claims description 2
- 102100038829 Spastin Human genes 0.000 claims description 2
- 102100029823 Tyrosine-protein kinase BTK Human genes 0.000 claims description 2
- 108010039827 snRNP Core Proteins Proteins 0.000 claims description 2
- 108700031620 S-acetylthiorphan Proteins 0.000 claims 6
- 150000003839 salts Chemical class 0.000 claims 3
- 108091006208 SLC3 Proteins 0.000 claims 2
- NPOAOTPXWNWTSH-UHFFFAOYSA-N 3-hydroxy-3-methylglutaric acid Chemical compound OC(=O)CC(O)(C)CC(O)=O NPOAOTPXWNWTSH-UHFFFAOYSA-N 0.000 claims 1
- SVYBEBLNQGDRHF-UHFFFAOYSA-N 4-amino-N-(5-ethyl-1,3,4-thiadiazol-2-yl)benzenesulfonamide Chemical compound S1C(CC)=NN=C1NS(=O)(=O)C1=CC=C(N)C=C1 SVYBEBLNQGDRHF-UHFFFAOYSA-N 0.000 claims 1
- 102100033714 40S ribosomal protein S6 Human genes 0.000 claims 1
- 102100033391 ATP-dependent RNA helicase DDX3X Human genes 0.000 claims 1
- 101001005951 Acidaminococcus fermentans (strain ATCC 25085 / DSM 20731 / CCUG 9996 / CIP 106432 / VR4) (R)-2-hydroxyglutarate dehydrogenase Proteins 0.000 claims 1
- 101100168911 Arabidopsis thaliana CUL4 gene Proteins 0.000 claims 1
- 101150013553 CD40 gene Proteins 0.000 claims 1
- 101710176668 Cartilage oligomeric matrix protein Proteins 0.000 claims 1
- 102100028914 Catenin beta-1 Human genes 0.000 claims 1
- 102100038018 Corticotropin-releasing factor receptor 1 Human genes 0.000 claims 1
- 101100168913 Dictyostelium discoideum culD gene Proteins 0.000 claims 1
- 102100039825 G protein-regulated inducer of neurite outgrowth 2 Human genes 0.000 claims 1
- 102000009012 HMGA Proteins Human genes 0.000 claims 1
- 108010049069 HMGA Proteins Proteins 0.000 claims 1
- 108090000353 Histone deacetylase Proteins 0.000 claims 1
- 102100038720 Histone deacetylase 9 Human genes 0.000 claims 1
- 102100035009 Holocytochrome c-type synthase Human genes 0.000 claims 1
- 101000656896 Homo sapiens 40S ribosomal protein S6 Proteins 0.000 claims 1
- 101000870662 Homo sapiens ATP-dependent RNA helicase DDX3X Proteins 0.000 claims 1
- 101000916173 Homo sapiens Catenin beta-1 Proteins 0.000 claims 1
- 101000878678 Homo sapiens Corticotropin-releasing factor receptor 1 Proteins 0.000 claims 1
- 101001034045 Homo sapiens G protein-regulated inducer of neurite outgrowth 2 Proteins 0.000 claims 1
- 101000946589 Homo sapiens Holocytochrome c-type synthase Proteins 0.000 claims 1
- 101000840540 Homo sapiens Iduronate 2-sulfatase Proteins 0.000 claims 1
- 101000982538 Homo sapiens Inositol polyphosphate 5-phosphatase OCRL Proteins 0.000 claims 1
- 101000994815 Homo sapiens Interleukin-1 receptor accessory protein-like 1 Proteins 0.000 claims 1
- 101001055427 Homo sapiens Mediator of RNA polymerase II transcription subunit 13 Proteins 0.000 claims 1
- 101000581402 Homo sapiens Melanin-concentrating hormone receptor 1 Proteins 0.000 claims 1
- 101000720704 Homo sapiens Neuronal migration protein doublecortin Proteins 0.000 claims 1
- 101001121709 Homo sapiens Nyctalopin Proteins 0.000 claims 1
- 101000595489 Homo sapiens Phosphatidylinositol N-acetylglucosaminyltransferase subunit A Proteins 0.000 claims 1
- 101001123986 Homo sapiens Protein-serine O-palmitoleoyltransferase porcupine Proteins 0.000 claims 1
- 101001120726 Homo sapiens Pyruvate dehydrogenase E1 component subunit alpha, somatic form, mitochondrial Proteins 0.000 claims 1
- 101000634060 Homo sapiens Sterol-4-alpha-carboxylate 3-dehydrogenase, decarboxylating Proteins 0.000 claims 1
- 101000951145 Homo sapiens Succinate dehydrogenase [ubiquinone] cytochrome b small subunit, mitochondrial Proteins 0.000 claims 1
- 101000611194 Homo sapiens Trinucleotide repeat-containing gene 6A protein Proteins 0.000 claims 1
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 claims 1
- 102100029098 Hypoxanthine-guanine phosphoribosyltransferase Human genes 0.000 claims 1
- 102100029199 Iduronate 2-sulfatase Human genes 0.000 claims 1
- 102100034413 Interleukin-1 receptor accessory protein-like 1 Human genes 0.000 claims 1
- 102100026161 Mediator of RNA polymerase II transcription subunit 13 Human genes 0.000 claims 1
- 108010050345 Microphthalmia-Associated Transcription Factor Proteins 0.000 claims 1
- 102100030157 Microphthalmia-associated transcription factor Human genes 0.000 claims 1
- 108010012255 Neural Cell Adhesion Molecule L1 Proteins 0.000 claims 1
- 102100024964 Neural cell adhesion molecule L1 Human genes 0.000 claims 1
- 102100025929 Neuronal migration protein doublecortin Human genes 0.000 claims 1
- 102100027096 Nucleotide exchange factor SIL1 Human genes 0.000 claims 1
- 102100035196 POLG alternative reading frame Human genes 0.000 claims 1
- 102000014160 PTEN Phosphohydrolase Human genes 0.000 claims 1
- 102100036050 Phosphatidylinositol N-acetylglucosaminyltransferase subunit A Human genes 0.000 claims 1
- 102100028119 Protein-serine O-palmitoleoyltransferase porcupine Human genes 0.000 claims 1
- 101000947436 Psychotria longipes Cyclopsychotride-A Proteins 0.000 claims 1
- 102100026067 Pyruvate dehydrogenase E1 component subunit alpha, somatic form, mitochondrial Human genes 0.000 claims 1
- 102000037055 SLC1 Human genes 0.000 claims 1
- 102000037062 SLC2 Human genes 0.000 claims 1
- 108091006209 SLC2 Proteins 0.000 claims 1
- 101100168914 Schizosaccharomyces pombe (strain 972 / ATCC 24843) pcu4 gene Proteins 0.000 claims 1
- 102100029238 Sterol-4-alpha-carboxylate 3-dehydrogenase, decarboxylating Human genes 0.000 claims 1
- 101000745670 Streptomyces venezuelae (strain ATCC 10712 / CBS 650.69 / DSM 40230 / JCM 4526 / NBRC 13096 / PD 04745) Chloramphenicol 3-O phosphotransferase Proteins 0.000 claims 1
- 102100038014 Succinate dehydrogenase [ubiquinone] cytochrome b small subunit, mitochondrial Human genes 0.000 claims 1
- 101710145783 TATA-box-binding protein Proteins 0.000 claims 1
- 101000699803 Talaromyces verruculosus Geranylgeranyl diphosphate synthase Proteins 0.000 claims 1
- 102100040241 Trinucleotide repeat-containing gene 6A protein Human genes 0.000 claims 1
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 claims 1
- 101710148271 UDP-glucose:glycoprotein glucosyltransferase 1 Proteins 0.000 claims 1
- 102100029151 UDP-glucuronosyltransferase 1A10 Human genes 0.000 claims 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims 1
- 208000027393 severe congenital neutropenia 5 Diseases 0.000 claims 1
- 239000011701 zinc Substances 0.000 claims 1
- 229910052725 zinc Inorganic materials 0.000 claims 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 12
- 201000010099 disease Diseases 0.000 abstract description 11
- 238000013461 design Methods 0.000 abstract description 8
- 238000011282 treatment Methods 0.000 abstract description 8
- 206010010356 Congenital anomaly Diseases 0.000 abstract description 3
- 238000003759 clinical diagnosis Methods 0.000 abstract description 3
- 230000002068 genetic effect Effects 0.000 abstract description 3
- 238000009223 counseling Methods 0.000 abstract description 2
- 238000004393 prognosis Methods 0.000 abstract description 2
- 208000026487 Triploidy Diseases 0.000 abstract 1
- 239000000203 mixture Substances 0.000 description 68
- 239000000243 solution Substances 0.000 description 51
- 239000000975 dye Substances 0.000 description 34
- 238000002156 mixing Methods 0.000 description 34
- 239000000872 buffer Substances 0.000 description 16
- 239000012192 staining solution Substances 0.000 description 16
- 230000035772 mutation Effects 0.000 description 14
- 238000004458 analytical method Methods 0.000 description 13
- 238000005516 engineering process Methods 0.000 description 12
- 239000007853 buffer solution Substances 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 239000000499 gel Substances 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 239000003085 diluting agent Substances 0.000 description 8
- 238000010186 staining Methods 0.000 description 8
- 238000005406 washing Methods 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 230000000087 stabilizing effect Effects 0.000 description 7
- 238000012795 verification Methods 0.000 description 7
- DIQHTDHADJUONG-UHFFFAOYSA-N C.C.C.C.C.C.C.C.C.C.C.C.C.C.C.C.C.C.C.C Chemical compound C.C.C.C.C.C.C.C.C.C.C.C.C.C.C.C.C.C.C.C DIQHTDHADJUONG-UHFFFAOYSA-N 0.000 description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 6
- 238000003745 diagnosis Methods 0.000 description 6
- 238000001502 gel electrophoresis Methods 0.000 description 6
- 230000010355 oscillation Effects 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 108090000364 Ligases Proteins 0.000 description 4
- 102000003960 Ligases Human genes 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000007812 deficiency Effects 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000012165 high-throughput sequencing Methods 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 230000002265 prevention Effects 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 239000012160 loading buffer Substances 0.000 description 3
- 238000003793 prenatal diagnosis Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 239000012089 stop solution Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000010200 validation analysis Methods 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- 102100028780 AP-1 complex subunit sigma-2 Human genes 0.000 description 2
- 102100024643 ATP-binding cassette sub-family D member 1 Human genes 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 2
- 101000690509 Aspergillus oryzae (strain ATCC 42149 / RIB 40) Alpha-glucosidase Proteins 0.000 description 2
- 108010004586 Ataxia Telangiectasia Mutated Proteins Proteins 0.000 description 2
- 102100036951 DNA polymerase subunit gamma-1 Human genes 0.000 description 2
- 102100021757 E3 ubiquitin-protein ligase RNF135 Human genes 0.000 description 2
- 102100027280 Fanconi anemia group A protein Human genes 0.000 description 2
- 208000028782 Hereditary disease Diseases 0.000 description 2
- 101000768016 Homo sapiens AP-1 complex subunit sigma-2 Proteins 0.000 description 2
- 101000893559 Homo sapiens Amylo-alpha-1,6-glucosidase Proteins 0.000 description 2
- 101001106984 Homo sapiens E3 ubiquitin-protein ligase RNF135 Proteins 0.000 description 2
- 101000938776 Homo sapiens ETS domain-containing transcription factor ERF Proteins 0.000 description 2
- 101000938790 Homo sapiens Eukaryotic peptide chain release factor subunit 1 Proteins 0.000 description 2
- 101000914673 Homo sapiens Fanconi anemia group A protein Proteins 0.000 description 2
- 101000827746 Homo sapiens Fibroblast growth factor receptor 1 Proteins 0.000 description 2
- 101000918487 Homo sapiens Fumarylacetoacetase Proteins 0.000 description 2
- 101000596925 Homo sapiens Homeobox protein TGIF1 Proteins 0.000 description 2
- 101001033233 Homo sapiens Interleukin-10 Proteins 0.000 description 2
- 101001051093 Homo sapiens Low-density lipoprotein receptor Proteins 0.000 description 2
- 101000883014 Homo sapiens Protein capicua homolog Proteins 0.000 description 2
- 101000599843 Homo sapiens RelA-associated inhibitor Proteins 0.000 description 2
- 101000807859 Homo sapiens Vasopressin V2 receptor Proteins 0.000 description 2
- 102100039068 Interleukin-10 Human genes 0.000 description 2
- 102100024640 Low-density lipoprotein receptor Human genes 0.000 description 2
- 108010049137 Member 1 Subfamily D ATP Binding Cassette Transporter Proteins 0.000 description 2
- 102100032543 Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN Human genes 0.000 description 2
- 102100028680 Protein patched homolog 1 Human genes 0.000 description 2
- 108010048349 Steroidogenic Factor 1 Proteins 0.000 description 2
- 102100029856 Steroidogenic factor 1 Human genes 0.000 description 2
- 102100037108 Vasopressin V2 receptor Human genes 0.000 description 2
- 230000005856 abnormality Effects 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 210000004381 amniotic fluid Anatomy 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000001186 cumulative effect Effects 0.000 description 2
- 230000006806 disease prevention Effects 0.000 description 2
- 230000001605 fetal effect Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000001360 synchronised effect Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- IOSROLCFSUFOFE-UHFFFAOYSA-L 2-nitro-1h-imidazole;platinum(2+);dichloride Chemical compound [Cl-].[Cl-].[Pt+2].[O-][N+](=O)C1=NC=CN1.[O-][N+](=O)C1=NC=CN1 IOSROLCFSUFOFE-UHFFFAOYSA-L 0.000 description 1
- 102100030310 5,6-dihydroxyindole-2-carboxylic acid oxidase Human genes 0.000 description 1
- 101150101112 7 gene Proteins 0.000 description 1
- 102100024378 AF4/FMR2 family member 2 Human genes 0.000 description 1
- 102100024408 AT-hook DNA-binding motif-containing protein 1 Human genes 0.000 description 1
- 102100034580 AT-rich interactive domain-containing protein 1A Human genes 0.000 description 1
- 102100024645 ATP-binding cassette sub-family C member 8 Human genes 0.000 description 1
- 102100033106 ATP-binding cassette sub-family G member 5 Human genes 0.000 description 1
- 102100033350 ATP-dependent translocase ABCB1 Human genes 0.000 description 1
- 101150020330 ATRX gene Proteins 0.000 description 1
- 102100040191 Alpha-tectorin Human genes 0.000 description 1
- 102100040894 Amylo-alpha-1,6-glucosidase Human genes 0.000 description 1
- 102100030988 Angiotensin-converting enzyme Human genes 0.000 description 1
- 101710185050 Angiotensin-converting enzyme Proteins 0.000 description 1
- 102100023086 Anosmin-1 Human genes 0.000 description 1
- 101000686547 Arabidopsis thaliana 30S ribosomal protein S1, chloroplastic Proteins 0.000 description 1
- 101100429155 Arabidopsis thaliana XTH4 gene Proteins 0.000 description 1
- 206010003805 Autism Diseases 0.000 description 1
- 102100035553 Autism susceptibility gene 2 protein Human genes 0.000 description 1
- 208000020706 Autistic disease Diseases 0.000 description 1
- 102100027954 BAG family molecular chaperone regulator 3 Human genes 0.000 description 1
- 102100021247 BCL-6 corepressor Human genes 0.000 description 1
- 102100025423 Bone morphogenetic protein receptor type-1A Human genes 0.000 description 1
- 108010014064 CCCTC-Binding Factor Proteins 0.000 description 1
- 102100032937 CD40 ligand Human genes 0.000 description 1
- 102100034279 Calcium-binding mitochondrial carrier protein Aralar2 Human genes 0.000 description 1
- 102100025232 Calcium/calmodulin-dependent protein kinase type II subunit beta Human genes 0.000 description 1
- 102100033561 Calmodulin-binding transcription activator 1 Human genes 0.000 description 1
- 102100032765 Chordin-like protein 1 Human genes 0.000 description 1
- 102100023470 Cobalamin trafficking protein CblD Human genes 0.000 description 1
- 102100040453 Connector enhancer of kinase suppressor of ras 2 Human genes 0.000 description 1
- 108010022637 Copper-Transporting ATPases Proteins 0.000 description 1
- 102100027587 Copper-transporting ATPase 1 Human genes 0.000 description 1
- 102100023376 Corrinoid adenosyltransferase Human genes 0.000 description 1
- 102100023381 Cyanocobalamin reductase / alkylcobalamin dealkylase Human genes 0.000 description 1
- 101710164985 Cyanocobalamin reductase / alkylcobalamin dealkylase Proteins 0.000 description 1
- 102100034746 Cyclin-dependent kinase-like 5 Human genes 0.000 description 1
- 102100030878 Cytochrome c oxidase subunit 1 Human genes 0.000 description 1
- 108010014790 DAX-1 Orphan Nuclear Receptor Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 230000009946 DNA mutation Effects 0.000 description 1
- 101000923091 Danio rerio Aristaless-related homeobox protein Proteins 0.000 description 1
- 206010058314 Dysplasia Diseases 0.000 description 1
- 102100037024 E3 ubiquitin-protein ligase XIAP Human genes 0.000 description 1
- 102100037241 Endoglin Human genes 0.000 description 1
- 102100031785 Endothelial transcription factor GATA-2 Human genes 0.000 description 1
- 102100021793 Epsilon-sarcoglycan Human genes 0.000 description 1
- 101100406415 Escherichia coli (strain K12) ompR gene Proteins 0.000 description 1
- 102100029074 Exostosin-2 Human genes 0.000 description 1
- 101150021185 FGF gene Proteins 0.000 description 1
- 102100027909 Folliculin Human genes 0.000 description 1
- 102100028496 Galactocerebrosidase Human genes 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 102100028603 Glutaryl-CoA dehydrogenase, mitochondrial Human genes 0.000 description 1
- 206010053759 Growth retardation Diseases 0.000 description 1
- 102100034472 H(+)/Cl(-) exchange transporter 4 Human genes 0.000 description 1
- 102100034471 H(+)/Cl(-) exchange transporter 5 Human genes 0.000 description 1
- 102100031561 Hamartin Human genes 0.000 description 1
- 102100022057 Hepatocyte nuclear factor 1-alpha Human genes 0.000 description 1
- 102100028909 Heterogeneous nuclear ribonucleoprotein K Human genes 0.000 description 1
- 102100033071 Histone acetyltransferase KAT6A Human genes 0.000 description 1
- 102100031470 Homeobox protein ARX Human genes 0.000 description 1
- 101000773083 Homo sapiens 5,6-dihydroxyindole-2-carboxylic acid oxidase Proteins 0.000 description 1
- 101000833172 Homo sapiens AF4/FMR2 family member 2 Proteins 0.000 description 1
- 101000833502 Homo sapiens AT-hook DNA-binding motif-containing protein 1 Proteins 0.000 description 1
- 101000924266 Homo sapiens AT-rich interactive domain-containing protein 1A Proteins 0.000 description 1
- 101000760570 Homo sapiens ATP-binding cassette sub-family C member 8 Proteins 0.000 description 1
- 101000598552 Homo sapiens Acetyl-CoA acetyltransferase, mitochondrial Proteins 0.000 description 1
- 101000718525 Homo sapiens Alpha-galactosidase A Proteins 0.000 description 1
- 101000889766 Homo sapiens Alpha-tectorin Proteins 0.000 description 1
- 101001050039 Homo sapiens Anosmin-1 Proteins 0.000 description 1
- 101000874361 Homo sapiens Autism susceptibility gene 2 protein Proteins 0.000 description 1
- 101000697871 Homo sapiens BAG family molecular chaperone regulator 3 Proteins 0.000 description 1
- 101100165236 Homo sapiens BCOR gene Proteins 0.000 description 1
- 101000934638 Homo sapiens Bone morphogenetic protein receptor type-1A Proteins 0.000 description 1
- 101000868215 Homo sapiens CD40 ligand Proteins 0.000 description 1
- 101001077352 Homo sapiens Calcium/calmodulin-dependent protein kinase type II subunit beta Proteins 0.000 description 1
- 101000945309 Homo sapiens Calmodulin-binding transcription activator 1 Proteins 0.000 description 1
- 101000941971 Homo sapiens Chordin-like protein 1 Proteins 0.000 description 1
- 101000777079 Homo sapiens Chromodomain-helicase-DNA-binding protein 2 Proteins 0.000 description 1
- 101000977167 Homo sapiens Cobalamin trafficking protein CblD Proteins 0.000 description 1
- 101000749824 Homo sapiens Connector enhancer of kinase suppressor of ras 2 Proteins 0.000 description 1
- 101001114650 Homo sapiens Corrinoid adenosyltransferase Proteins 0.000 description 1
- 101000945692 Homo sapiens Cyclin-dependent kinase-like 5 Proteins 0.000 description 1
- 101000919849 Homo sapiens Cytochrome c oxidase subunit 1 Proteins 0.000 description 1
- 101000661592 Homo sapiens Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit STT3A Proteins 0.000 description 1
- 101000881679 Homo sapiens Endoglin Proteins 0.000 description 1
- 101001066265 Homo sapiens Endothelial transcription factor GATA-2 Proteins 0.000 description 1
- 101000616437 Homo sapiens Epsilon-sarcoglycan Proteins 0.000 description 1
- 101000918275 Homo sapiens Exostosin-2 Proteins 0.000 description 1
- 101000914679 Homo sapiens Fanconi anemia group B protein Proteins 0.000 description 1
- 101000917159 Homo sapiens Filaggrin Proteins 0.000 description 1
- 101001060703 Homo sapiens Folliculin Proteins 0.000 description 1
- 101000860395 Homo sapiens Galactocerebrosidase Proteins 0.000 description 1
- 101001058943 Homo sapiens Glutaryl-CoA dehydrogenase, mitochondrial Proteins 0.000 description 1
- 101000710229 Homo sapiens H(+)/Cl(-) exchange transporter 4 Proteins 0.000 description 1
- 101000710225 Homo sapiens H(+)/Cl(-) exchange transporter 5 Proteins 0.000 description 1
- 101000795643 Homo sapiens Hamartin Proteins 0.000 description 1
- 101000899111 Homo sapiens Hemoglobin subunit beta Proteins 0.000 description 1
- 101001045751 Homo sapiens Hepatocyte nuclear factor 1-alpha Proteins 0.000 description 1
- 101000838964 Homo sapiens Heterogeneous nuclear ribonucleoprotein K Proteins 0.000 description 1
- 101000944179 Homo sapiens Histone acetyltransferase KAT6A Proteins 0.000 description 1
- 101000923090 Homo sapiens Homeobox protein ARX Proteins 0.000 description 1
- 101001011446 Homo sapiens Interferon regulatory factor 6 Proteins 0.000 description 1
- 101000984044 Homo sapiens LIM homeobox transcription factor 1-beta Proteins 0.000 description 1
- 101000620503 Homo sapiens LIM/homeobox protein Lhx4 Proteins 0.000 description 1
- 101000780208 Homo sapiens Long-chain-fatty-acid-CoA ligase 4 Proteins 0.000 description 1
- 101000997662 Homo sapiens Lysosomal acid glucosylceramidase Proteins 0.000 description 1
- 101000783776 Homo sapiens Lysosomal cobalamin transporter ABCD4 Proteins 0.000 description 1
- 101000834118 Homo sapiens Malonate-CoA ligase ACSF3, mitochondrial Proteins 0.000 description 1
- 101001126977 Homo sapiens Methylmalonyl-CoA mutase, mitochondrial Proteins 0.000 description 1
- 101000957756 Homo sapiens Microtubule-associated protein RP/EB family member 2 Proteins 0.000 description 1
- 101000939438 Homo sapiens Mitochondrial brown fat uncoupling protein 1 Proteins 0.000 description 1
- 101001028702 Homo sapiens Mitochondrial-derived peptide MOTS-c Proteins 0.000 description 1
- 101000590830 Homo sapiens Monocarboxylate transporter 1 Proteins 0.000 description 1
- 101000982010 Homo sapiens Myelin proteolipid protein Proteins 0.000 description 1
- 101001066305 Homo sapiens N-acetylgalactosamine-6-sulfatase Proteins 0.000 description 1
- 101001124388 Homo sapiens NPC intracellular cholesterol transporter 1 Proteins 0.000 description 1
- 101001109579 Homo sapiens NPC intracellular cholesterol transporter 2 Proteins 0.000 description 1
- 101000854060 Homo sapiens Oxygen-regulated protein 1 Proteins 0.000 description 1
- 101000922137 Homo sapiens Peripheral plasma membrane protein CASK Proteins 0.000 description 1
- 101000731078 Homo sapiens Phosphorylase b kinase gamma catalytic chain, liver/testis isoform Proteins 0.000 description 1
- 101000945272 Homo sapiens Phosphorylase b kinase regulatory subunit alpha, liver isoform Proteins 0.000 description 1
- 101001047090 Homo sapiens Potassium voltage-gated channel subfamily H member 2 Proteins 0.000 description 1
- 101000610107 Homo sapiens Pre-B-cell leukemia transcription factor 1 Proteins 0.000 description 1
- 101000695187 Homo sapiens Protein patched homolog 1 Proteins 0.000 description 1
- 101000620777 Homo sapiens Rab proteins geranylgeranyltransferase component A 1 Proteins 0.000 description 1
- 101000641879 Homo sapiens Ras/Rap GTPase-activating protein SynGAP Proteins 0.000 description 1
- 101000927773 Homo sapiens Rho guanine nucleotide exchange factor 9 Proteins 0.000 description 1
- 101001095807 Homo sapiens Ribonuclease inhibitor Proteins 0.000 description 1
- 101001125551 Homo sapiens Ribose-phosphate pyrophosphokinase 1 Proteins 0.000 description 1
- 101000707152 Homo sapiens SH2B adapter protein 1 Proteins 0.000 description 1
- 101100477520 Homo sapiens SHOX gene Proteins 0.000 description 1
- 101000628575 Homo sapiens Serine/threonine-protein kinase 19 Proteins 0.000 description 1
- 101000826130 Homo sapiens Sex-determining region Y protein Proteins 0.000 description 1
- 101000685990 Homo sapiens Specifically androgen-regulated gene protein Proteins 0.000 description 1
- 101000616167 Homo sapiens Splicing factor 3B subunit 4 Proteins 0.000 description 1
- 101000861263 Homo sapiens Steroid 21-hydroxylase Proteins 0.000 description 1
- 101000617830 Homo sapiens Sterol O-acyltransferase 1 Proteins 0.000 description 1
- 101000648077 Homo sapiens Syntaxin-binding protein 1 Proteins 0.000 description 1
- 101000799388 Homo sapiens Thiopurine S-methyltransferase Proteins 0.000 description 1
- 101001074042 Homo sapiens Transcriptional activator GLI3 Proteins 0.000 description 1
- 101001098093 Homo sapiens Transcriptional repressor p66-beta Proteins 0.000 description 1
- 101000864342 Homo sapiens Tyrosine-protein kinase BTK Proteins 0.000 description 1
- 101000819146 Homo sapiens UDP-glucose 4-epimerase Proteins 0.000 description 1
- 101000841498 Homo sapiens UDP-glucuronosyltransferase 1A1 Proteins 0.000 description 1
- 101000954820 Homo sapiens WD repeat domain phosphoinositide-interacting protein 4 Proteins 0.000 description 1
- 101000916523 Homo sapiens Zinc finger C4H2 domain-containing protein Proteins 0.000 description 1
- 101000964479 Homo sapiens Zinc finger and BTB domain-containing protein 18 Proteins 0.000 description 1
- 101000782483 Homo sapiens Zinc finger protein 462 Proteins 0.000 description 1
- 101000964741 Homo sapiens Zinc finger protein 711 Proteins 0.000 description 1
- 101000931371 Homo sapiens Zinc finger protein ZFPM2 Proteins 0.000 description 1
- 101000976645 Homo sapiens Zinc finger protein ZIC 3 Proteins 0.000 description 1
- 102000043138 IRF family Human genes 0.000 description 1
- 108091054729 IRF family Proteins 0.000 description 1
- 201000006347 Intellectual Disability Diseases 0.000 description 1
- 102100030130 Interferon regulatory factor 6 Human genes 0.000 description 1
- 102100029874 Kappa-casein Human genes 0.000 description 1
- 102100025457 LIM homeobox transcription factor 1-beta Human genes 0.000 description 1
- 102100022257 LIM/homeobox protein Lhx4 Human genes 0.000 description 1
- 102100034319 Long-chain-fatty-acid-CoA ligase 4 Human genes 0.000 description 1
- 102100033342 Lysosomal acid glucosylceramidase Human genes 0.000 description 1
- 102100020978 Lysosomal cobalamin transporter ABCD4 Human genes 0.000 description 1
- 101150083522 MECP2 gene Proteins 0.000 description 1
- 102100026665 Malonate-CoA ligase ACSF3, mitochondrial Human genes 0.000 description 1
- 101001129124 Mannheimia haemolytica Outer membrane lipoprotein 1 Proteins 0.000 description 1
- 108010047230 Member 1 Subfamily B ATP Binding Cassette Transporter Proteins 0.000 description 1
- 108010090837 Member 5 Subfamily G ATP Binding Cassette Transporter Proteins 0.000 description 1
- 102100039124 Methyl-CpG-binding protein 2 Human genes 0.000 description 1
- 102100030979 Methylmalonyl-CoA mutase, mitochondrial Human genes 0.000 description 1
- 102000008071 Mismatch Repair Endonuclease PMS2 Human genes 0.000 description 1
- 108010074346 Mismatch Repair Endonuclease PMS2 Proteins 0.000 description 1
- 102100029820 Mitochondrial brown fat uncoupling protein 1 Human genes 0.000 description 1
- 102100037173 Mitochondrial-derived peptide MOTS-c Human genes 0.000 description 1
- 102100034068 Monocarboxylate transporter 1 Human genes 0.000 description 1
- 102100030173 Muellerian-inhibiting factor Human genes 0.000 description 1
- 206010028182 Multiple congenital abnormalities Diseases 0.000 description 1
- 102100026784 Myelin proteolipid protein Human genes 0.000 description 1
- 108010009047 Myosin VIIa Proteins 0.000 description 1
- 102100031688 N-acetylgalactosamine-6-sulfatase Human genes 0.000 description 1
- 102100029565 NPC intracellular cholesterol transporter 1 Human genes 0.000 description 1
- 102100022737 NPC intracellular cholesterol transporter 2 Human genes 0.000 description 1
- 102100023210 Necdin Human genes 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102100039019 Nuclear receptor subfamily 0 group B member 1 Human genes 0.000 description 1
- 101000761187 Odontomachus monticola U-poneritoxin(01)-Om1a Proteins 0.000 description 1
- 102100030275 PH-interacting protein Human genes 0.000 description 1
- 101710119304 PH-interacting protein Proteins 0.000 description 1
- 102100023498 Palmitoyltransferase ZDHHC9 Human genes 0.000 description 1
- 108010065129 Patched-1 Receptor Proteins 0.000 description 1
- 101710203703 Peptidyl-lysine N-acetyltransferase YjaB Proteins 0.000 description 1
- UQVKZNNCIHJZLS-UHFFFAOYSA-N PhIP Chemical compound C1=C2N(C)C(N)=NC2=NC=C1C1=CC=CC=C1 UQVKZNNCIHJZLS-UHFFFAOYSA-N 0.000 description 1
- 101100541055 Phaseolus angularis XTHA gene Proteins 0.000 description 1
- 102100032391 Phosphorylase b kinase gamma catalytic chain, liver/testis isoform Human genes 0.000 description 1
- 102100033548 Phosphorylase b kinase regulatory subunit alpha, liver isoform Human genes 0.000 description 1
- 102100022807 Potassium voltage-gated channel subfamily H member 2 Human genes 0.000 description 1
- 102100040171 Pre-B-cell leukemia transcription factor 1 Human genes 0.000 description 1
- 102100022881 Rab proteins geranylgeranyltransferase component A 1 Human genes 0.000 description 1
- 102000002490 Rad51 Recombinase Human genes 0.000 description 1
- 108010068097 Rad51 Recombinase Proteins 0.000 description 1
- 102100033428 Ras/Rap GTPase-activating protein SynGAP Human genes 0.000 description 1
- 102100037875 RelA-associated inhibitor Human genes 0.000 description 1
- 102100033221 Rho guanine nucleotide exchange factor 9 Human genes 0.000 description 1
- 101001031757 Rhodococcus jostii (strain RHA1) F420-dependent glucose-6-phosphate dehydrogenase 2 Proteins 0.000 description 1
- 102100029508 Ribose-phosphate pyrophosphokinase 1 Human genes 0.000 description 1
- 102100031770 SH2B adapter protein 1 Human genes 0.000 description 1
- 108091006736 SLC22A5 Proteins 0.000 description 1
- 108091006418 SLC25A13 Proteins 0.000 description 1
- 108091006296 SLC2A1 Proteins 0.000 description 1
- 101001128051 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 60S ribosomal protein L3 Proteins 0.000 description 1
- 102100026757 Serine/threonine-protein kinase 19 Human genes 0.000 description 1
- 108700025071 Short Stature Homeobox Proteins 0.000 description 1
- 102100029992 Short stature homeobox protein Human genes 0.000 description 1
- 102100023536 Solute carrier family 2, facilitated glucose transporter member 1 Human genes 0.000 description 1
- 102100036924 Solute carrier family 22 member 5 Human genes 0.000 description 1
- 102100021796 Sonic hedgehog protein Human genes 0.000 description 1
- 101710113849 Sonic hedgehog protein Proteins 0.000 description 1
- 102100023355 Specifically androgen-regulated gene protein Human genes 0.000 description 1
- 102100021815 Splicing factor 3B subunit 4 Human genes 0.000 description 1
- 102100027545 Steroid 21-hydroxylase Human genes 0.000 description 1
- 102100021993 Sterol O-acyltransferase 1 Human genes 0.000 description 1
- 102100025293 Syntaxin-binding protein 1 Human genes 0.000 description 1
- 102100033456 TGF-beta receptor type-1 Human genes 0.000 description 1
- 102100033455 TGF-beta receptor type-2 Human genes 0.000 description 1
- 101150112897 TS gene Proteins 0.000 description 1
- 108010027179 Tacrolimus Binding Proteins Proteins 0.000 description 1
- 102000018679 Tacrolimus Binding Proteins Human genes 0.000 description 1
- 102100034162 Thiopurine S-methyltransferase Human genes 0.000 description 1
- 102100030140 Thiosulfate:glutathione sulfurtransferase Human genes 0.000 description 1
- 108090001039 Transcription factor AP-2 Proteins 0.000 description 1
- 102100033348 Transcription factor AP-2-beta Human genes 0.000 description 1
- 102100035559 Transcriptional activator GLI3 Human genes 0.000 description 1
- 102100027671 Transcriptional repressor CTCF Human genes 0.000 description 1
- 102100037556 Transcriptional repressor p66-beta Human genes 0.000 description 1
- 108010011702 Transforming Growth Factor-beta Type I Receptor Proteins 0.000 description 1
- 108010082684 Transforming Growth Factor-beta Type II Receptor Proteins 0.000 description 1
- 206010051956 Trichorhinophalangeal syndrome Diseases 0.000 description 1
- 101100541059 Triticum aestivum XTH gene Proteins 0.000 description 1
- 102100021436 UDP-glucose 4-epimerase Human genes 0.000 description 1
- 102100029152 UDP-glucuronosyltransferase 1A1 Human genes 0.000 description 1
- 102100031835 Unconventional myosin-VIIa Human genes 0.000 description 1
- 102100037048 WD repeat domain phosphoinositide-interacting protein 4 Human genes 0.000 description 1
- 108700031544 X-Linked Inhibitor of Apoptosis Proteins 0.000 description 1
- 108091007416 X-inactive specific transcript Proteins 0.000 description 1
- 102000056014 X-linked Nuclear Human genes 0.000 description 1
- 108700042462 X-linked Nuclear Proteins 0.000 description 1
- 108091035715 XIST (gene) Proteins 0.000 description 1
- 101150086051 XTH1 gene Proteins 0.000 description 1
- 108091009220 ZDHHC9 Proteins 0.000 description 1
- 102100028880 Zinc finger C4H2 domain-containing protein Human genes 0.000 description 1
- 102100040762 Zinc finger and BTB domain-containing protein 18 Human genes 0.000 description 1
- 102100035849 Zinc finger protein 462 Human genes 0.000 description 1
- 102100040724 Zinc finger protein 711 Human genes 0.000 description 1
- 102100020996 Zinc finger protein ZFPM2 Human genes 0.000 description 1
- 102100023495 Zinc finger protein ZIC 3 Human genes 0.000 description 1
- ABUGVBRDFWGJRD-CHOYNLESSA-N [9-[(2r,3r,4s,5r)-3,4-dihydroxy-5-methyloxolan-2-yl]-2-(2,4-dinitrophenyl)sulfanylpurin-6-yl] [hydroxy(phosphonooxy)phosphoryl] hydrogen phosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](C)O[C@H]1N1C2=NC(SC=3C(=CC(=CC=3)[N+]([O-])=O)[N+]([O-])=O)=NC(OP(O)(=O)OP(O)(=O)OP(O)(O)=O)=C2N=C1 ABUGVBRDFWGJRD-CHOYNLESSA-N 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 208000036878 aneuploidy Diseases 0.000 description 1
- 231100001075 aneuploidy Toxicity 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 238000003766 bioinformatics method Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 101150015424 dmd gene Proteins 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 231100000001 growth retardation Toxicity 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- ONCZDRURRATYFI-QTCHDTBASA-N methyl (2z)-2-methoxyimino-2-[2-[[(e)-1-[3-(trifluoromethyl)phenyl]ethylideneamino]oxymethyl]phenyl]acetate Chemical compound CO\N=C(/C(=O)OC)C1=CC=CC=C1CO\N=C(/C)C1=CC=CC(C(F)(F)F)=C1 ONCZDRURRATYFI-QTCHDTBASA-N 0.000 description 1
- 238000010208 microarray analysis Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000036438 mutation frequency Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 238000007481 next generation sequencing Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000009933 reproductive health Effects 0.000 description 1
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 238000012106 screening analysis Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- G16B20/50—Mutagenesis
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Medical Informatics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Theoretical Computer Science (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Evolutionary Biology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- Artificial Intelligence (AREA)
- Bioethics (AREA)
- Computer Vision & Pattern Recognition (AREA)
- Data Mining & Analysis (AREA)
- Databases & Information Systems (AREA)
- Epidemiology (AREA)
- Evolutionary Computation (AREA)
- Public Health (AREA)
- Software Systems (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明提供了一种同步检测多种出生缺陷遗传病全染色体基因分型芯片及其方法和应用。该芯片用于检测由基因拷贝数变异和单核苷酸变异引起的多种出生缺陷遗传病。本发明在全基因组水平覆盖的基因拷贝数变异探针基础上，针对性选择基因组不重要区域减少或不设计探针，并针对性对相关单倍体剂量不足及三倍体剂量敏感性致病基因进行加密，在相关常见出生缺陷遗传病基因相关单核苷酸变异位点设计探针，增加相应基因区域探针密度，从而在减少临床意义不明基因拷贝数变异检出的同时，增加致病性基因单核苷酸变异检出，延展传统芯片的高性能，提高检出有效性，提高临床对于出生缺陷相关基因拷贝数变异和单核苷酸变异疾病的诊断、治疗、预后及遗传咨询水平。
Description
技术领域
本发明属于出生缺陷综合防治领域，特别涉及一种同步检测多种出生缺陷遗传病全染色体基因分型芯片及其方法和应用。
背景技术
我国每年新增出生缺陷患儿约90万例，约80％是由基因组变异造成的，涉及变异类型多、表型症状复杂、临床诊断困难，是产后儿童致残、致死的主要原因之一。其中，约24.3％是由于基因拷贝数变异(CNV)而引起的微缺失微重复综合征，包括很多不明原因的智力障碍、生长发育迟缓、自闭症和多发性先天异常；约17.4％是严重的单基因遗传病，其发病机制是单核苷酸变异(SNV)，广义上包括点突变和小片段***/缺失(InDel)。基因拷贝数变异(CNV)和单核苷酸变异(SNV)导致的累计婴儿致死率约为19.1％，累计儿科住院率约为18％。可见，精准同步检测基因拷贝数变异(CNV)和单核苷酸变异(SNV)对于出生缺陷遗传病的早发现、早防控和早治疗具有重要的临床意义。
基因拷贝数变异(CNV)主要检测方法有G显带，荧光原位杂交(Fluorescence InSitu Hybridization，FISH)和染色体微阵列分析技术(Chromosome MicroarrayAnalysis,CMA)。G显带和FISH可检测部分染色体区域，但难以提供基因组水平更高分辨率的变异信息，且操作繁琐，通量低，整体阳性检出率仅3％。CMA技术包括比较基因组杂交(Array Comparative Genomic Hybridization，aCGH)和单核苷酸多态性微阵列芯片(Single Nucleotide Polymorphism-based Array，SNP array)，国内各大医院普遍开展了CMA技术作为基因拷贝数变异(CNV)一线临床诊断策略，用于产前诊断以及产后不明原因的发育异常检测，全基因组水平的阳性基因拷贝数变异(CNV)检出率可以达到31.6％。但是无论是G显带、FISH还是CMA均不能用于检测引起单基因遗传病的单核苷酸变异(SNV)，单核苷酸变异(SNV)目前通过Sanger测序、定量PCR、长PCR以及二代测序等方法来检测，这些技术不能检测基因拷贝数变异(CNV)导致的遗传病，目前临床策略是先检测CNV再检测SNV，所以开发同时检测CNV和SNV的方法对出生缺陷遗传病的早发现、早防控和早治疗及降低成本均有具有重要的意义。
有报道记载，对于113例患儿的对比检测中，高通量测序技术CNV检出率为81.4％，Array-CGH的检出率为67.3％[1]；采用荧光定量PCR联合全基因组拷贝数变异测序(CNV-Seq)技术对270例胎儿常见染色体非整倍体进行快速产前诊断并检测>100kb的全基因组拷贝数变异，荧光定量PCR共计检测19例，检出率7.03％；CNV-Seq检测异常总数43例，检出率15.9％[2]；对52例孕妇进行羊水穿刺，利用CMA技术进行产前诊断，共检出与NIPT结果较一致的拷贝数变异15例，检出率达28.85％[3]；1674例孕中期孕妇样本中G带核型分析异常206例(12.31％)，其中73例CMA未检出异常；CMA分析异常147例(8.81％)，其中26例G带核型分析结果正常，多为小片段的重复或缺失[4]。
此外，很多单基因遗传病的变异热点既包括基因拷贝数变异(CNV)也包含单核苷酸变异(SNV)，例如杜氏肌营养不良DMD基因的突变中约70％都是大片段外显子缺失/重复，约23％是由DMD内部外显子区域和侧翼区域单核苷酸变异(SNV)导致。此类疾病临床检测不到点突变的情况下，还需要额外用到多重连接探针扩增(MLPA)、聚合酶链式限制性片段长度多态性分析(PCR-RFLP)等方法来检测基因内部的基因拷贝数变异(CNV)，大大增加了检测的时间成本和经济成本。
综上可知，由于现有技术的局限性，临床检查时往往需要联合运用多种技术手段，如CMA结合MLPA，PCR结合Sanger等方式，才能检测出基因拷贝数变异(CNV)和单核苷酸变异(SNV)。CN110592213A公开的预测新抗原负荷和检测基因组突变的基因Panel，基于二代测序，主要用于一次性检测肿瘤细胞多个位点的DNA突变，也可用于肿瘤免疫领域测量SNV、InDel、CNV、HLA等多种类型的突变，但仅能测量肿瘤细胞各种变异类型的突变频率(VAF)，无法用于出生缺陷遗传疾病的诊断检测。
参考文献：
[1]秦谦,刘博,杨琳等.基于高通量测序技术的拷贝数变异筛选分析流程的建立及应用[J].中国循证儿科杂志,2018,08(13):275.
[2]李琴，魏兆莲，乔金平等.高通量测序技术在胎儿染色体拷贝数变异检测中的应用[J].国际生殖健康/计划生育杂志,2020,39(6):450-455.
[3]姜楠，张印帅，宋丽杰等.高通量测序技术在胎儿染色体拷贝数变异检测中的应用[J].中华医学遗传学杂志,2020,37(7):779-784.
[4]靳倩，令狐克燕，卓召振等.1674例孕妇羊水细胞G显带核型与染色体微阵列结果对比分析[J].中国计划生育学杂志,2021,29(05):1046-1049.
发明内容
鉴于上述现有遗传性出生缺陷疾病的诊断检测中存在的问题，本发明针对引起出生缺陷遗传病的基因拷贝数变异(CNV)和单核苷酸变异(SNV)，提供了一种同步检测多种出生缺陷遗传病全染色体基因分型芯片和方法。基于SNV-array技术，针对16个单基因和已经公开的350个致病性单倍剂量不足(HI)基因以及三倍剂量敏感(TS)基因设计了genome_framework探针组，同时在genome_framework探针组基础之上，进一步针对190个重要出生缺陷遗传病基因的致病性单核苷酸变异(SNV)设计了special_gene探针组，通过这样的设计，有效克服现有检测方法仅能单一性检测基因拷贝数变异(CNV)/单核苷酸变异(SNV)的不足，实现引起出生缺陷遗传病的基因拷贝数变异(CNV)和单核苷酸变异(SNV)的同步检测。
本发明的一个目的是提供一种同步检测多种出生缺陷遗传病全染色体基因分型芯片，用于同步检测基因拷贝数变异(CNV)和单核苷酸变异(SNV)，仅需一次实验，就可实现多重结果，有效减轻部分患者家庭的经济负担，提升临床经济适用性，该芯片包括genome_framework探针组和special_gene探针组；
所述genome_framework探针组覆盖染色体全基因组，探针总数为642338个，探针平均分布密度为1个/5Kb；所述染色体全基因组覆盖探针数目如下表1所示；
表1全基因组覆盖探针数目(基因组版本：Hg38)
所述genome_framework探针组包括针对常染色体以及针对性染色体按照一定间距设计的骨架探针；所述骨架探针减少/去除各条染色体首、末端区域，着丝粒区域和其他无关键基因区域探针；不重要基因组区域不设计或少设计探针，以减少临床意义不明的所述基因拷贝数变异(CNV)区域，
所述genome_framework探针组包括针对16个单基因水平的所述基因拷贝数变异区域设计的探针，探针平均分布密度≥15个/5Kb；该探针密度为高度加密，根据各疾病的遗传特征，由单基因内部CNV报道的相关疾病进行设置；
所述16个单基因包括如下表2所示的VHL、QDPR、SMN1、CYP21A2、HBB、PTS、PAH、ATP7B、GCH1、HBA1、HBA2、NF1、PHEX、DMD、OTC、MECP2；
表2 16个重要单基因水平CNV区域覆盖探针数目及探针平均分布密度(基因组版本：Hg38)
所述genome_framework探针组包括针对致病性单倍剂量不足基因以及三倍剂量敏感基因的所述基因拷贝数变异区域设计的探针，根据Clingen数据库和文献报道，设置探针平均分布密度≥1个/5Kb，为中度加密，超过全基因组覆盖探针平均分布密度；所述致病性单倍剂量不足(HI)基因以及三倍剂量敏感(TS)基因为350个，包括如下表3所示的AHDC1、ARID1A、ASH1L、CAMTA1、FH、FLG、GATAD2B、GLMN、IRF6、LHX4、LMNA、NFIA、PBX1、POGZ、SDHB、SDHC、SF3B4、SLC2A1、ZBTB18、BAG3、BMPR1A、EMX2、GATA3、KAT6B、KIF11、PAX2、PTEN、RPS24、WAC、ZMYND11、ALX4、ARCN1、ATM、CDKN1C、EXT2、FZD4、HMBS、KCNQ1、KCNQ1OT1、KMT2A、LRP5、MEN1、MYBPC3、PAX6、PHF21A、SDHAF2、SDHD、SHANK2、WT1、ACVRL1、ARID2、CDKN1B、GRIN2B、HMGA2、KMT2D、LEMD3、MED13L、PTHLH、PTPN11、RPS26、SLC17A8、SOX5、TBX3、BRCA2、CHAMP1、EDNRB、NBEA、POLR1D、RB1、ZIC2、BMP4、CHD8、DICER1、FOXG1、GPHN、PAX9、AAGAB、CHD2、FBN1、IGF1R、MEIS2、RPS17、SIN3A、SMAD3、SPRED1、TCF12、UBE3A、ANKRD11、CDH1、CREBBP、CTCF、FOXC2、FOXF1、PKD1、SALL1、SETD1A、SH2B1、TSC2、AXIN2、BRCA1、BRIP1、COL1A1、EFTUD2、FLCN、HNF1B、KANSL1、PAFAH1B1、PMP22、RAD51C、RAD51D、RAI1、RNF135、TBX4、TP53、ASXL3、DSC2、GATA6、SETBP1、SMAD4、TCF4、TGIF1、CACNA1A、CIC、ERF、KMT2B、LDLR、RPS19、SMARCA4、STK11、APOB、BARD1、BCL11A、CFC1、COL3A1、CUL3、GIGYF2、GLI2、MBD5、MSH2、MSH6、MYCN、MYT1L、NCKAP1、NRXN1、PAX3、PAX8、SATB2、SCN1A、SCN2A、SIX3、SOX11、SPAST、TBR1、TRIP12、ZEB2、ADNP、ASXL1、GDF5、GNAS、JAG1、KCNQ2、SALL4、APP、DSCAM、DYRK1A、RUNX1、SON、CHEK2、EP300、NF2、SHANK3、SMARCB1、SOX10、TBX1、TCF20、TNRC6B、BAP1、CTNNB1、FOXL2、FOXP1、GATA2、MITF、MLH1、SCN5A、SETD2、SETD5、SLC6A1、SOX2、TBL1XR1、TGFBR2、ZIC1、ZIC4、ANK2、NAA15、NR3C2、PITX2、PKD2、APC、CTNND2、FGF10、GABRA1、LMNB1、MEF2C、NKX2-5、PURA、RASA1、SPINK1、TRIO、ARID1B、EYA4、FOXC1、HIVEP2、PHIP、SYNGAP1、TFAP2B、AUTS2、CAMK2B、ELN、GLI3、KCNH2、KMT2C、MNX1、PMS2、SGCE、SHH、TWIST1、CHD7、EXT1、GATA4、KAT6A、TRPS1、ZFPM2、COL5A1、DMRT1、EHMT1、ENG、HNRNPK、LMX1B、NR5A1、PTCH1、STXBP1、TGFBR1、TSC1、ZNF462、ABCD1、ACSL4、AFF2、ANOS1、AP1S2、AR、ARHGEF9、ARX、ATP7A、ATRX、AVPR2、BCOR、BRWD3、BTK、CASK、CD40LG、CDKL5、CHM、CHRDL1、CLCN4、CLCN5、CNKSR2、COL4A5、CUL4B、CYBB、DCX、DDX3X、DLG3、EBP、EDA、EFNB1、F8、F9、FANCB、FGD1、FLNA、FMR1、FRMD7、FTSJ1、GDI1、GK、GLA、GPC3、GRIA3、HCCS、HDAC8、HPRT1、IDS、IKBKG、IL1RAPL1、IQSEC2、KDM5C、KDM6A、L1CAM、LAMP2、MAGT1、MID1、MTM1、NDP、NHS、NR0B1、NSDHL、NYX、OCRL、OFD1、OPHN1、PAK3、PCDH19、PDHA1、PGK1、PHF6、PIGA、PLP1、PORCN、PQBP1、PRPS1、PTCHD1、RAB39B、RP2、RPS6KA3、RS1、SH2D1A、SLC16A2、SLC35A2、SLC6A8、SLC9A6、SMC1A、SMS、STS、SYN1、TBX22、TIMM8A、TRAPPC2、TSPAN7、UBE2A、UPF3B、USP9X、WDR45、XIAP、XIST、ZC4H2、ZDHHC9、ZIC3、ZNF711、SHOX、SRY；
表3 350个致病性HI基因和TS基因区域覆盖探针数目及探针平均分布密度(基因组版本：Hg38)
所述special_gene探针组覆盖190个重要出生缺陷遗传病基因的单核苷酸变异位点37083个，对应探针总数为330272个，平均每个所述单核苷酸变异位点设计9个重复探针；
所述190个重要出生缺陷遗传病基因是结合临床实验室的文献报道，针对出生缺陷产后遗传病防治领域，依据等位基因携带率、外显率、发病率、患者发病时间、临床可防可治措施等原则挑选而出，通过ClinVar、HGMD、gnomAD和本地临床实验室的数据库，对所述190个重要出生缺陷遗传病基因的相关位点进行人群频率的统计和筛选，确认了每个基因的中国人群高发SNV位点，为了增加对应SNV位点的结果准确度和阳性检出率，在所述芯片不同位置随机放置这些探针，并保证每个SNV位点最低4个重复探针，所述190个重要出生缺陷遗传病基因为ABCB1、ABCC8、ABCD1、ABCD4、ABCG5、ABCG8、ACADM、ACADS、ACADVL、ACAT1、ACE、ACSF3、AGL、AMH、AMHR2、APOA5、APOB、APOC3、AR、ARG1、ARSA、ARSB、ASS1、ATP7A、ATP7B、BCKDHA、BCKDHB、BMP1、BTD、CBS、CD320、CLCN5、COCH、COL1A1、COL1A2、COL2A1、COMP、CPS1、CPT1A、CPT2、CRHR1、CYP11B1、CYP17A1、CYP1A1、CYP21A2、CYP2C19、CYP2D6、CYP3A5、DBT、DIABLO、DLD、DMD、DMP1、DNAJC12、DRD2、DSPP、ELN、ENPP1、EPHX1、ETFA、ETFDH、FAH、FGF23、FGFR1、FGFR2、FGFR3、FKBP5、G6PC、G6PD、GAA、GALC、GALE、GALK1、GALNS、GALT、GBA、GBE1、GCDH、GCH1、GCK、GJB2、GJB3、GLA、GLB1、GNPTAB、GNPTG、GNS、GUSB、GYS2、HADH、HBA1、HBA2、HBB、HEXA、HEXB、HGSNAT、HLA_B、HLCS、HNF4A、HPD、HSD3B2、HTR2C、HYAL1、IDS、IDUA、IFITM5、IFNL4、IVD、KCNJ11、LDLR、LHCGR、LIPI、LMBRD1、LPL、MC4R、MCCC1、MCCC2、MCEE、MCOLN1、MLYCD、MMAA、MMAB、MMACHC、MMADHC、MMUT、MT-CO1、MT-RNR1、MT-TH、MT-TL1、MT-TS1、MTHFR、MTR、MYO15A、MYO7A、NAGLU、NDN、NPC1、NPC2、NR0B1、NR5A1、OTC、PAH、PCBD1、PCCA、PCCB、PHEX、PHKA2、PHKB、PHKG2、PLA2G4A、PLP1、POLG、PPIB、PRPS1、PTS、PYGL、PYGM、QDPR、RP1、SDHA、SGSH、SLC16A1、SLC22A5、SLC25A13、SLC25A15、SLC25A20、SLC26A4、SLC34A3、SLC37A4、SLCO2B1、SMN1/SMN2、SMPD1、SNRPN、SPR、SPRN、SRD5A2、SRY、STAR、SUMF1、TAT、TBX1、TECTA、TMC1、TPMT、TYRP1、UCP2、UGT1A、UGT1A4、VKORC1、WFS1；所述芯片针对所述190个重要出生缺陷遗传病基因的37083个人致病性SNV统计信息如下表4所示。
表4出生缺陷遗传病190个基因的SNV数目
优选地，所述special_gene探针组兼顾提升所述genome_framework探针组对应基因区域的探针分布密度。
优选地，所述genome_framework探针组和special_gene探针组的核苷酸序列包括如SEQ ID No.1～SEQ ID No.720所示的探针序列，但该部分序列仅为小部分示例。
优选地，所述genome_framework探针组针对所述16个单基因、所述致病性单倍剂量不足基因以及所述三倍剂量敏感基因的变异，计算每个探针拷贝数CN；常染色体和女性X染色体正常所述探针拷贝数CN1为2，计算出现的所述探针拷贝数CN1变异情况为2种，其中所述探针拷贝数CN1为0或1属于缺失，为3或4属于重复；男性X和Y染色体正常所述探针拷贝数CN2为1，计算出现的所述探针拷贝数CN2变异情况为2种，其中所述探针拷贝数CN2为0属于缺失，为2属于重复；
当出现连续50个探针的所述探针拷贝数CN异常情况，判定为基因拷贝数变异；
计算拷贝数度量值是log2 Ratio，其中Ratio＝每个探针拷贝数的观测值÷每个探针拷贝数的参考值，每个探针拷贝数的参考值是来自于正常人群的一组数据：
当log2 Ratio≈-1.5±0.05时，CN＝0；
当log2 Ratio≈-1±0.05时，CN＝1；
当log2 Ratio≈0±0.05时，CN＝2；
当log2 Ratio≈0.58±0.05时，CN＝3；
当log2 Ratio≈1±0.05时，CN＝4。
优选地，所述special_gene探针组针对所述190个重要出生缺陷遗传病基因的相应SNV位点进行检测，结果的判读基于该部分探针的检测值来实现的，所用的方法为Genotype_SNV和ES聚类算法。
Genotype_SNV方法是通过对SNV array探针进行双色荧光标记实现的，所述探针会得到两种荧光检测值A和B，通过对A和B值进行计算得到基因分型结果，计算公式如下：
t(snv)＝(A-B)/(A+B)
当t(snv)≈1±0.05时，分型结果为AA；
当t(snv)≈-1±0.05时，分型结果为BB；
当t(snv)≈0±0.05时，分型结果为AB；
基因分型结果包括三种，AA、AB和BB，其中所述AA和BB代表纯和类型，一个代表野生型，一个代表纯合子，根据聚类结果判读野生型和纯合子，所述AB代表杂合子。
所述ES聚类算法用于对所述基因分型结果进行聚类，计算公式如下：
t(x)＝log2(A/B)；
t(y)＝[log2(A)+log2(B)]/2；
所述ES聚类算法以t(x)为横轴，t(y)为纵轴进行聚类，所述检测结果的阳性或阴性根据样本分型结果和聚类数目进行判断。
优选地，所述芯片还包括变性混合液、扩增混合液、片段化混合液、沉淀混合液和杂交混合液。
所述变性混合液由10×变性液用纯水稀释10倍而成。
所述扩增混合液由1体积的扩增酶加45体积的扩增液混合制备而成。
所述片段化混合液由1体积的片段化酶、10.3体积的片段化稀释液加45.7体积的10×片段化缓冲液混合制备所得。
所述沉淀混合液由1体积的沉淀溶液2加119体积的沉淀溶液1混合制备所得。
所述杂交混合液由1体积的杂交液1、18体积的杂交液2和140体积的杂交缓冲液混合制备所得。
优选地，所述芯片在
微阵列芯片扫描仪中进行检测使用。
所述
微阵列芯片扫描仪配套试剂包括染料混合物A、染料混合物B、稳定混合物、连接混合物A和连接混合物B。
所述染料混合物A由1体积的染色液1-A、1体积的染色液1-B、2体积的染色缓冲液和96体积的洗液A混合制备所得。
所述染料混合物B由1体积的染色液2-A、1体积的染色液2-B、2体积的染色缓冲液和96体积的洗液A混合制备所得。
所述稳定混合物由1体积的稳定液、8体积的固定稀释液用纯水稀释7.9倍而成。
所述连接混合物A由1体积的连接液1、0.4体积的连接液2和5体积的连接缓冲液混合制备所得。
所述连接混合物B由1体积的连接酶、6.6体积的探针混合液1、6.6体积的探针混合液2和52体积的连接混合物A混合制备所得。
本发明的另一个目的是提供一种同步检测多种出生缺陷遗传病基因的方法，
所述方法包括以下步骤，
S1、采集样品；
S2、样品制备与质控；
S3、检测反应：
S31、DNA扩增：取100ng DNA样本(5ng/μL)，加入2μl 10×变性混合液和18μl纯水，室温孵育10min；加入130μl中和液，混匀后加入225μl扩增混合液和5μl扩增酶，37℃孵育23±1h，然后转移到65℃孵育20min，最后在37℃继续孵育45min；
S32、DNA片段化和沉淀：对步骤S31孵育的样本加入45.7μl 10×片段化缓冲液、10.3μl片段化稀释液、1.0μl片段化酶混匀，简短离心后37℃孵育30min，而后室温下加入19μl终止液，震荡混匀离心；在室温环境下加入238μl沉淀溶液1、2μl沉淀溶液2和600μl异丙醇，用移液器上下充分混匀，-20℃冰冻16～24h；
S33、干燥、重悬及质控：对步骤S32处理后的样本进行4℃3200rpm离心40min，倒出废液后置于37℃晾干20min，加入35μl重悬缓冲液振荡混匀，震荡10min后，加入70.5μl杂交缓冲液、0.5μl杂交液1、9μl杂交液2，震荡混匀离心，即得115μl变性杂交液；质控使用10μl的所述变性杂交液，包括用Nanodrop测量吸光度和凝胶电泳，其中胶上样染料为InvitrogenTMTrackIt Cyan/Orange Loading Buffer；25bp Invitrogen Ladder；凝胶电泳***为Invitrogen E-GelTM48agarose gels 4％，G8008-04；
S34、变性和杂交：将步骤S33产生的所述变性杂交液放于杂交板，而后放置于PCR仪中，开始变性程序(Denature program)：95℃变性10min，48℃变性3min，冷却。用移液器小心将剩余105μl变性杂交液转移至杂交盘中，载入
微阵列芯片扫描仪，持续杂交23.5h～24h；
S35、芯片扫描仪检测：对步骤S34产生的混合物进行染料盘对应分装：其中染料混合物A的染料盘2个，其中提前准备好100.8μl洗液A、2.1μl染色缓冲液、1.05μl染色液1-A和1.05μl染色液1-B；染料混合物B的染料盘1个，其中提前准备好100.8μl洗液A、2.1μl染色缓冲液、1.05μl染色液2-A和1.05μl染色液2-B；稳定混合物的染料盘1个，其中提前准备好93.2μl纯水、10.5μl固定稀释液和1.3μl稳定液；连接混合物的染料盘1个，其中提前准备好66.2μl连接缓冲液、13.1μl连接液1、3.1μl连接液2、10.5μl探针混合液1、10.5μl探针混合液2和-20℃现取出的连接酶1.6μl；后直接载入芯片扫描仪，同时向上述各扫描盘每孔中加入150μl固定缓冲液，避免接触扫描盘底部。将盖子的切口角对准扫描盘的切口边缘，盖住扫描盘，而后放置于工作台顶部，最后开始扫描检测流程；
S4、数据质控和分析；
S5、结果分析与注释。
本发明还提供一种上述的芯片在同步检测多种出生缺陷遗传病CNV和SNV中的应用。
本发明提供的一种同步检测多种出生缺陷遗传病全染色体基因分型芯片，能够同步检测基因拷贝数变异和单核苷酸变异，操作简单，诊断高效，仅需一次实验实现对全基因组范围的CNV和190个常见出生缺陷遗传病基因相关SNV位点进行针对性检测。
本发明提供的一种同步检测多种出生缺陷遗传病基因的方法，采用全染色体基因分型芯片，能够显著提升医生对出生缺陷相关CNV和单基因疾病临床检测的标准化程度，是一种高时效性、高准确率和高操作性的分子诊断方法，能够显著提升此类疾病的诊断、治疗、预后及遗传咨询水平。
附图说明
图1为本发明的方法检测流程图；
图2为本发明的芯片的杂交信号图；
图3-1为本发明的基因拷贝数缺失结果图，纵坐标代表log2Ratio；
图3-2为本发明的基因拷贝数重复结果图，纵坐标代表log2Ratio；
图4-1为单核苷酸变异阳性结果图；
图4-2为单核苷酸变异阳性结果图；
图5-1为单核苷酸变异阴性结果图；
图5-2为单核苷酸变异阴性结果图。
具体实施方式
下面结合具体实施例，进一步阐述本发明设计、阳性样本验证和结果分析。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1
本实施例的一种同步检测多种出生缺陷遗传病全染色体基因分型芯片，该芯片包括genome_framework探针组和special_gene探针组。
genome_framework探针组包括针对常染色体以及针对性染色体设计的骨架探针；骨架探针减少/去除各条染色体首、末端区域，着丝粒区域和其他无基因区域探针；
genome_framework探针组包括针对16个单基因水平的基因拷贝数变异(CNV)区域设计的探针，16个单基因包括VHL、QDPR、SMN1、CYP21A2、HBB、PTS、PAH、ATP7B、GCH1、HBA1、HBA2、NF1、PHEX、DMD、OTC、MECP2；
genome_framework探针组包括针对350个致病性单倍剂量不足(HI)基因以及三倍剂量敏感(TS)基因的基因拷贝数变异(CNV)区域设计的探针，致病性单倍剂量不足(HI)基因以及三倍剂量敏感(TS)基因包括AHDC1、ARID1A、ASH1L、CAMTA1、FH、FLG、GATAD2B、GLMN、IRF6、LHX4、LMNA、NFIA、PBX1、POGZ、SDHB、SDHC、SF3B4、SLC2A1、ZBTB18、BAG3、BMPR1A、EMX2、GATA3、KAT6B、KIF11、PAX2、PTEN、RPS24、WAC、ZMYND11、ALX4、ARCN1、ATM、CDKN1C、EXT2、FZD4、HMBS、KCNQ1、KCNQ1OT1、KMT2A、LRP5、MEN1、MYBPC3、PAX6、PHF21A、SDHAF2、SDHD、SHANK2、WT1、ACVRL1、ARID2、CDKN1B、GRIN2B、HMGA2、KMT2D、LEMD3、MED13L、PTHLH、PTPN11、RPS26、SLC17A8、SOX5、TBX3、BRCA2、CHAMP1、EDNRB、NBEA、POLR1D、RB1、ZIC2、BMP4、CHD8、DICER1、FOXG1、GPHN、PAX9、AAGAB、CHD2、FBN1、IGF1R、MEIS2、RPS17、SIN3A、SMAD3、SPRED1、TCF12、UBE3A、ANKRD11、CDH1、CREBBP、CTCF、FOXC2、FOXF1、PKD1、SALL1、SETD1A、SH2B1、TSC2、AXIN2、BRCA1、BRIP1、COL1A1、EFTUD2、FLCN、HNF1B、KANSL1、PAFAH1B1、PMP22、RAD51C、RAD51D、RAI1、RNF135、TBX4、TP53、ASXL3、DSC2、GATA6、SETBP1、SMAD4、TCF4、TGIF1、CACNA1A、CIC、ERF、KMT2B、LDLR、RPS19、SMARCA4、STK11、APOB、BARD1、BCL11A、CFC1、COL3A1、CUL3、GIGYF2、GLI2、MBD5、MSH2、MSH6、MYCN、MYT1L、NCKAP1、NRXN1、PAX3、PAX8、SATB2、SCN1A、SCN2A、SIX3、SOX11、SPAST、TBR1、TRIP12、ZEB2、ADNP、ASXL1、GDF5、GNAS、JAG1、KCNQ2、SALL4、APP、DSCAM、DYRK1A、RUNX1、SON、CHEK2、EP300、NF2、SHANK3、SMARCB1、SOX10、TBX1、TCF20、TNRC6B、BAP1、CTNNB1、FOXL2、FOXP1、GATA2、MITF、MLH1、SCN5A、SETD2、SETD5、SLC6A1、SOX2、TBL1XR1、TGFBR2、ZIC1、ZIC4、ANK2、NAA15、NR3C2、PITX2、PKD2、APC、CTNND2、FGF10、GABRA1、LMNB1、MEF2C、NKX2-5、PURA、RASA1、SPINK1、TRIO、ARID1B、EYA4、FOXC1、HIVEP2、PHIP、SYNGAP1、TFAP2B、AUTS2、CAMK2B、ELN、GLI3、KCNH2、KMT2C、MNX1、PMS2、SGCE、SHH、TWIST1、CHD7、EXT1、GATA4、KAT6A、TRPS1、ZFPM2、COL5A1、DMRT1、EHMT1、ENG、HNRNPK、LMX1B、NR5A1、PTCH1、STXBP1、TGFBR1、TSC1、ZNF462、ABCD1、ACSL4、AFF2、ANOS1、AP1S2、AR、ARHGEF9、ARX、ATP7A、ATRX、AVPR2、BCOR、BRWD3、BTK、CASK、CD40LG、CDKL5、CHM、CHRDL1、CLCN4、CLCN5、CNKSR2、COL4A5、CUL4B、CYBB、DCX、DDX3X、DLG3、EBP、EDA、EFNB1、F8、F9、FANCB、FGD1、FLNA、FMR1、FRMD7、FTSJ1、GDI1、GK、GLA、GPC3、GRIA3、HCCS、HDAC8、HPRT1、IDS、IKBKG、IL1RAPL1、IQSEC2、KDM5C、KDM6A、L1CAM、LAMP2、MAGT1、MID1、MTM1、NDP、NHS、NR0B1、NSDHL、NYX、OCRL、OFD1、OPHN1、PAK3、PCDH19、PDHA1、PGK1、PHF6、PIGA、PLP1、PORCN、PQBP1、PRPS1、PTCHD1、RAB39B、RP2、RPS6KA3、RS1、SH2D1A、SLC16A2、SLC35A2、SLC6A8、SLC9A6、SMC1A、SMS、STS、SYN1、TBX22、TIMM8A、TRAPPC2、TSPAN7、UBE2A、UPF3B、USP9X、WDR45、XIAP、XIST、ZC4H2、ZDHHC9、ZIC3、ZNF711、SHOX、SRY；
special_gene探针组覆盖190个重要出生缺陷遗传病基因的单核苷酸变异位点37083个，对应探针总数为330272个，平均每个所述单核苷酸变异位点设计9个重复探针；
90个重要出生缺陷遗传病基因为ABCB1、ABCC8、ABCD1、ABCD4、ABCG5、ABCG8、ACADM、ACADS、ACADVL、ACAT1、ACE、ACSF3、AGL、AMH、AMHR2、APOA5、APOB、APOC3、AR、ARG1、ARSA、ARSB、ASS1、ATP7A、ATP7B、BCKDHA、BCKDHB、BMP1、BTD、CBS、CD320、CLCN5、COCH、COL1A1、COL1A2、COL2A1、COMP、CPS1、CPT1A、CPT2、CRHR1、CYP11B1、CYP17A1、CYP1A1、CYP21A2、CYP2C19、CYP2D6、CYP3A5、DBT、DIABLO、DLD、DMD、DMP1、DNAJC12、DRD2、DSPP、ELN、ENPP1、EPHX1、ETFA、ETFDH、FAH、FGF23、FGFR1、FGFR2、FGFR3、FKBP5、G6PC、G6PD、GAA、GALC、GALE、GALK1、GALNS、GALT、GBA、GBE1、GCDH、GCH1、GCK、GJB2、GJB3、GLA、GLB1、GNPTAB、GNPTG、GNS、GUSB、GYS2、HADH、HBA1、HBA2、HBB、HEXA、HEXB、HGSNAT、HLA_B、HLCS、HNF4A、HPD、HSD3B2、HTR2C、HYAL1、IDS、IDUA、IFITM5、IFNL4、IVD、KCNJ11、LDLR、LHCGR、LIPI、LMBRD1、LPL、MC4R、MCCC1、MCCC2、MCEE、MCOLN1、MLYCD、MMAA、MMAB、MMACHC、MMADHC、MMUT、MT-CO1、MT-RNR1、MT-TH、MT-TL1、MT-TS1、MTHFR、MTR、MYO15A、MYO7A、NAGLU、NDN、NPC1、NPC2、NR0B1、NR5A1、OTC、PAH、PCBD1、PCCA、PCCB、PHEX、PHKA2、PHKB、PHKG2、PLA2G4A、PLP1、POLG、PPIB、PRPS1、PTS、PYGL、PYGM、QDPR、RP1、SDHA、SGSH、SLC16A1、SLC22A5、SLC25A13、SLC25A15、SLC25A20、SLC26A4、SLC34A3、SLC37A4、SLCO2B1、SMN1/SMN2、SMPD1、SNRPN、SPR、SPRN、SRD5A2、SRY、STAR、SUMF1、TAT、TBX1、TECTA、TMC1、TPMT、TYRP1、UCP2、UGT1A、UGT1A4、VKORC1、WFS1。
special_gene探针组兼顾提升genome_framework探针组对应基因区域的探针分布密度。
其中，genome_framework探针组针对基因拷贝数变异(CNV)设计，基本设计原则而如下：
(1)必须保证全基因组覆盖，以实现基本的检测性能，因此对常染色体和性染色体分别按照一定的间距设计骨架探针，探针总数为642338个；
(2)不重要基因组区域不设计或少设计探针，以减少临床意义不明的基因拷贝数变异(CNV)区域，主要位于各条染色体首、末端区域、着丝粒区域和其他无关键基因区域；
(3)根据各疾病的遗传特征，有单基因内部的基因拷贝数变异(CNV)报道的相关疾病进行了高度加密，保证探针平均分布密度≥15个/5Kb；
(4)根据Clingen数据库和文献报道，对已知的350个致病性单倍剂量不足(HI)基因以及三倍剂量敏感(TS)基因进行了中度加密，保证这些区域探针平均分布密度≥1个/5Kb，即超过全基因组覆盖探针平均分布密度。
special_gene探针组针对单核苷酸变异(SNV)设计，基本设计原则而如下：
结合临床实验室的文献报道，针对出生缺陷产后遗传病防治领域，依据等位基因携带率、外显率、发病率、患者发病时间、临床可防可治措施等原则，挑选出190个基因，通过ClinVar、HGMD、gnomAD和本地临床实验室的数据库，对这些基因相关位点进行人群频率的统计和筛选，确认了每个基因的中国人群高发SNV位点，为了增加对应SNV位点的结果准确度和阳性检出率，在芯片不同位置随机放置这些探针，并保证每个SNV位点最低4个重复探针；
genome_framework探针组和special_gene探针组的核苷酸序列包括如下表5中SEQ ID No.1～SEQ ID No.720所示的探针序列，但该部分序列仅为小部分示例。
表5部分genome_framework探针组和special_gene探针组的核苷酸序列
genome_framework探针组针对16个单基因、350个致病性单倍剂量不足(HI)基因、三倍剂量敏感(TS)基因的变异，计算每个探针拷贝数CN；常染色体和女性X染色体正常探针拷贝数CN1为2，计算出现的探针拷贝数CN1变异情况为2种，其中探针拷贝数CN1为0或1属于缺失，为3或4属于重复；男性X和Y染色体正常探针拷贝数CN2为1，计算出现的探针拷贝数CN2变异情况为2种，其中探针拷贝数CN2为0属于缺失，为2属于重复；当出现连续50个探针的探针拷贝数CN异常情况，判定为基因拷贝数变异；
计算拷贝数度量值是log2 Ratio，其中Ratio＝每个探针拷贝数的观测值÷每个探针拷贝数的参考值，每个探针拷贝数的参考值是来自于正常人群的一组数据：
当log2 Ratio≈-1.5±0.05时，CN＝0；
当log2 Ratio≈-1±0.05时，CN＝1；
当log2 Ratio≈0±0.05时，CN＝2；
当log2 Ratio≈0.58±0.05时，CN＝3；
当log2 Ratio≈1±0.05时，CN＝4。
special_gene探针组针对190个重要出生缺陷遗传病基因的相应SNV位点进行检测，结果的判读基于该部分探针的检测值来实现的，所用的方法为Genotype_SNV和ES聚类算法。
Genotype_SNV方法是通过对SNV array探针进行双色荧光标记实现的，所述探针会得到两种荧光检测值A和B，通过对A和B值进行计算得到基因分型结果，计算公式如下：
t(snv)＝(A-B)/(A+B)
当t(snv)≈1±0.05时，分型结果为AA；
当t(snv)≈-1±0.05时，分型结果为BB；
当t(snv)≈0±0.05时，分型结果为AB；
基因分型结果包括三种，AA、AB和BB，其中AA和BB代表纯和类型，一个代表野生型，一个代表纯合子，根据聚类结果判读野生型和纯合子，AB代表杂合子。
ES聚类算法对基因分型结果进行聚类，计算公式如下：
t(x)＝log2(A/B)；
t(y)＝[log2(A)+log2(B)]/2；
ES聚类算法以t(x)为横轴，t(y)为纵轴进行聚类，检测结果的阳性或阴性根据样本分型结果和聚类数目进行判断，如图4-1和图4-2所示，检测结果属于单核苷酸变异阳性，如图5-1和图5-2所示，检测结果属于单核苷酸变异阴性。
芯片的组分还包括变性混合液、扩增混合液、片段化混合液、沉淀混合液和杂交混合液。
变性混合液由10×变性液用纯水稀释10倍而成。
扩增混合液由1体积的扩增酶加45体积的扩增液混合制备而成。
片段化混合液由1体积的片段化酶、10.3体积的片段化稀释液加45.7体积的10×片段化缓冲液混合制备所得。
沉淀混合液由1体积的沉淀溶液2加119体积的沉淀溶液1混合制备所得。
杂交混合液由1体积的杂交液1、18体积的杂交液2和140体积的杂交缓冲液混合制备所得。
芯片在
微阵列芯片扫描仪中进行检测使用，
微阵列芯片扫描仪配套试剂包括染料混合物A、染料混合物B、稳定混合物、连接混合物A和连接混合物B。
染料混合物A由1体积的染色液1-A、1体积的染色液1-B、2体积的染色缓冲液和96体积的洗液A混合制备所得。
染料混合物B由1体积的染色液2-A、1体积的染色液2-B、2体积的染色缓冲液和96体积的洗液A混合制备所得。
稳定混合物由1体积的稳定液、8体积的固定稀释液用纯水稀释7.9倍而成。
连接混合物A由1体积的连接液1、0.4体积的连接液2和5体积的连接缓冲液混合制备所得。
连接混合物B由1体积的连接酶、6.6体积的探针混合液1、6.6体积的探针混合液2和52体积的连接混合物A混合制备所得。
试剂盒中所用试剂可以选自下列产品(购自江苏源隆医疗科技有限公司)，如下表6所示：
表6试剂名称及货号
实施例2
本实施例中例中的一种同步检测多种出生缺陷遗传病CNV和SNV的方法包括以下步骤，如图1所示，
S1：采集样品：共计96例样本，男性54例，女性42例，其中48例为已经验证过的携带CNV的患者，另外48例为已经验证过的携带SNV的患者，作为发明的阳性验证样品。
S2：样品制备与质控，验证样品进行DNA抽提后通过凝胶电泳，Nanodrop等进行质控，确保每个DNA样品无降解，无杂质污染，高纯度。光密度(Optical Density,OD)260/280nm比值介于1.8～2.0，OD 260/230nm比值介于1.5～2.0，不满足任一条件的样品需进行纯化等处理；其中胶上样染料为InvitrogenTMTrackIt Cyan/Orange Loading Buffer(Invitrogen P/N 10482-028)；25bp Invitrogen Ladder(Invitrogen P/N10488-022)；凝胶电泳***为Invitrogen E-GelTM48agarose gels 4％，G8008-04。
S3：检测反应：
S31、DNA扩增：取100ng DNA样本(5ng/μL)，加入2μl 10×变性混合液和18μl纯水，室温孵育10min；加入130μl中和液，混匀后加入225μl扩增混合液和5μl扩增酶，37℃孵育23±1h，然后转移到65℃孵育20min，最后在37℃继续孵育45min100ng DNA样本(5ng/μL)中加入20μl变性混合液，室温孵育10min；加入130μl中和液，震荡混匀加入230μl扩增混合液，震荡混匀1000rpm离心1min；37℃孵育箱中孵育23±1h；65℃孵育箱中孵育20min；37℃孵育箱中继续孵育45min；
S32、DNA片段化和沉淀：对步骤S31孵育的样本加入45.7μl 10×片段化缓冲液、10.3μl片段化稀释液、1.0μl片段化酶混匀，简短离心后37℃孵育30min，而后室温下加入19μl终止液，震荡混匀离心；在室温环境下加入238μl沉淀溶液1、2μl沉淀溶液2和600μl异丙醇，用移液器上下充分混匀，-20℃冰冻16～24h加入57μl片段化混合液，混匀、简短离心；37℃孵育30min，室温环境下加入19μl终止液，进行震荡混匀离心；室温环境下加入240μl沉淀混合液和600μl异丙醇，充分混匀；-20℃环境放置16～24h；
S33、干燥、重悬及质控：对步骤S32处理后的样本进行4℃3200rpm离心40min，倒出废液后置于37℃晾干20min，加入35μl重悬缓冲液振荡混匀，震荡10min后，加入70.5μl杂交缓冲液、0.5μl杂交液1、9μl杂交液2，震荡混匀离心，即得115μl变性杂交液；4℃3200rpm离心40min，去除废液，置于37℃杂交炉中20min晾干，加入35μl重悬缓冲液进行振荡混匀，震荡仪中震荡10min，加入80μl杂交混合液，进行震荡混匀离心，产生115μl变性杂交液；
质控使用10μl的所述变性杂交液，包括用Nanodrop测量吸光度和凝胶电泳，其中胶上样染料为InvitrogenTMTrackIt Cyan/Orange Loading Buffer；25bp InvitrogenLadder；凝胶电泳***为Invitrogen E-GelTM48agarose gels 4％，G8008-04；
S34、变性和杂交：将步骤S33产生的所述变性杂交液放于杂交板，而后放置于PCR仪中，开始变性程序(Denature program)：95℃变性10min，48℃变性3min，冷却。用移液器小心将剩余105μl变性杂交液转移至杂交盘中，载入
微阵列芯片扫描仪，持续杂交23.5h～24h将所述杂交板放置于PCR仪中进行变性程序：95℃变性10min；48℃变性3min；冷却；使用移液器转移105μl变性杂交液至杂交盘，载入
微阵列芯片处理仪，持续杂交23.5h～24h；
S35、芯片扫描仪检测：对步骤S34产生的混合物进行染料盘对应分装：其中染料混合物A的染料盘2个，其中提前准备好100.8μl洗液A、2.1μl染色缓冲液、1.05μl染色液1-A和1.05μl染色液1-B；染料混合物B的染料盘1个，其中提前准备好100.8μl洗液A、2.1μl染色缓冲液、1.05μl染色液2-A和1.05μl染色液2-B；稳定混合物的染料盘1个，其中提前准备好93.2μl纯水、10.5μl固定稀释液和1.3μl稳定液；连接混合物的染料盘1个，其中提前准备好66.2μl连接缓冲液、13.1μl连接液1、3.1μl连接液2、10.5μl探针混合液1、10.5μl探针混合液2和-20℃现取出的连接酶1.6μl；后直接载入芯片扫描仪，同时向上述各扫描盘每孔中加入150μl固定缓冲液，避免接触扫描盘底部。将盖子的切口角对准扫描盘的切口边缘，盖住扫描盘，而后放置于工作台顶部，最后开始扫描检测流程其中染料混合物A的染料盘2个，每孔105μl；染料混合物B的染料盘1个，每孔105μl；稳定混合物的染料盘1个，每孔105μl；连接混合物B的染料盘1个，每孔105μl；将分装后的所述染料盘载入芯片扫描仪，同时向所述染料盘每孔加入150μl固定缓冲液后盖住，放置于工作台顶部进行检测；
S4：数据质控和分析：如图2所示，成像完成后会得到每块杂交板的原始荧光信号数据，利用自动分析软件对其进行质控和分析，包括整板质控、样品质控、探针序列识别、样品比对和生物信息学分析等流程，即可得到相应样品的变异位点信息结果文件。
S5：结果分析与注释：结果包含每个样品检测到的拷贝数变异信息和基因突变位点，这些信息再经过进一步的注释分析，包括突变评级和医学解读，最终得到的明确的真实的位点信息。
本次基因拷贝数变异(CNV)验证样品位点信息如下表7所示；基因拷贝数变异(CNV)分析结果如下表8所示。
表7基因拷贝数变异(CNV)验证样品位点信息
表8基因拷贝数变异(CNV)分析结果
注：NA表示无结果，Noise表示评判CNV真实性的密度图谱有杂乱信号，无法判断。实测大小是指软件分析出来的CNV的长度范围。
本发明对48个阳性CNV样本，共计62个CNV进行检测，共检测出60例CNV，CNV检出率为96.8％。进一步分析可发现，CNV-42样本CNV密度图有Noise，如附图3-1、附图3-2所示，CNV-47检测到CNV的探针数小于50(阈值为50)，CNV-48样本没有通过QC(可能是实验或者样品本身的问题)，其余45个阳性样本的CNV均被有效检测到，则CNV阳性验证率为93.75％。
本次单核苷酸变异(SNV)验证样品位点信息如下表9所示；单核苷酸变异(SNV)分析结果如下表10所示。
表9单核苷酸变异(SNV)验证样品位点信息
表10单核苷酸变异(SNV)分析结果
注：Normal表示该SNV位点未检测为突变；Affected表示检测到杂合突变或纯合突变，且与患者基因型一致；Carrier表示携带者；Not designed表示芯片上未设计探针。
本发明对48例阳性SNV位点进行验证试验，结果表明CNV-09位点未设计探针，其余样本位点均被检测到，SNV检出率达到97.9％，如附图4-1、代表1个样本被判读为AB(杂合子)，考虑为携带者，其余94个样本均为BB，考虑为野生型；如附图4-2所示代表1个样本被判读为BB(纯合子)，考虑为携带者，其余84个样本均为BB，考虑为野生型。进一步分析结果的准确性，发现CNV-19、CNV-30两个样本通过本发明试剂盒检测的结果均为纯合且解读结果为Normal，即未检测到发生对应的突变，而实际上这三个样本均为杂合突变，且为对应的疾病患者，因此可认为这两个样本验证失败。而CNV-32和CNV-38样本通过本发明试剂盒检测的结果均为纯合且解读结果为Affected，说明检测到发生了纯合突变，而不是原来结果提供的杂合突变患者。在此，均认为原来的结果是正确的，则CNV-32和CNV-38样本结果也认为是验证失败，因此SNV阳性验证率为91.5％。
综上，本发明产品对96例阳性样本进行验证，包括48例CNV样本和48例SNV样本，CNV检出率为96.8％、阳性率为93.75％。SNV检出率为97.9％、阳性率为91.5％。说明本发明产品所设计的探针可同步检测出出生缺陷遗传病基因相关CNV和SNV，充分说明了本发明芯片的优势，主要体现在：
(1)临床通用性：通过同步检测出生缺陷遗传病基因相关CNV和SNV，操作简单，诊断高效，仅需一次实验就可以对全基因组范围的CNV和190个单基因疾病进行检测，适合大部分临床遗传检测使用。
(2)有效性：采用了全新的探针设计方法，传统方案是设计骨架探针实现全基因组覆盖即可。本方案在骨架探针基础上，减少或去除了各条染色体首、末端区域、着丝粒区域和其他无基因区域等不重要区域的探针，并对已经公开的350个致病性单倍剂量不足(HI)基因以及三倍剂量敏感(TS)基因进行了加密，这样即减少了临床意义不明CNV的检出，同时显著增加致病性CNV的检出。特别的，还针对16个单基因内部CNV区域高度加密，以保证单基因内部较小片段CNV的检出率，这样可以最大限度保证产品的有效性。
(3)创造性：通过增加190个单基因疾病相关SNV，共计37083个，结合改良的基因分型算法和聚类方法，创造性的实现了利用全染色体基因分型芯片检测点突变和InDel，并衍生了SNV-array技术。
还需要注意的是，以上列举的实施例仅是本发明的一个具体实施例子，仅选用了部分已经鉴定的CNV患者和SNV患者，还可以检测更多的CNV和上述37083个SNV。本领域的其他技术人员能从本发明公开的内容中利用一次实验就可同步检测全基因组水平的CNV和上述37083个SNV，但应认为此种设计理念是本发明的保护范围。本发明并不限于上述实施方式和实施例，在本领域技术人员所具备的知识范围内，还可以在不脱离本发明构思的前提下作出各种变化。
Claims (9)
1.一种同步检测多种出生缺陷遗传病全染色体基因分型芯片，所述出生缺陷遗传病由基因拷贝数变异和单核苷酸变异引起，其特征在于：
所述芯片包括genome_framework探针组和special_gene探针组；
所述genome_framework探针组覆盖染色体全基因组，探针总数为643228个，探针平均分布密度为1个/5Kb；
所述genome_framework探针组包括针对常染色体以及针对性染色体设计的骨架探针；所述骨架探针减少/去除各条染色体首、末端区域，着丝粒区域和其他无基因区域探针；
所述genome_framework探针组包括针对16个单基因水平的所述基因拷贝数变异区域设计的探针，探针平均分布密度≥15个/5Kb；所述16个单基因包括：VHL、QDPR、SMN1、CYP21A2、HBB、PTS、PAH、ATP7B、GCH1、HBA1、HBA2、NF1、PHEX、DMD、OTC、MECP2；
所述genome_framework探针组包括针对致病性单倍剂量不足基因以及三倍剂量敏感基因的所述基因拷贝数变异区域设计的探针，探针平均分布密度≥1个/5Kb；所述致病性单倍剂量不足基因以及三倍剂量敏感基因为350个，包括：AHDC1、ARID1A、ASH1L、CAMTA1、FH、FLG、GATAD2B、GLMN、IRF6、LHX4、LMNA、NFIA、PBX1、POGZ、SDHB、SDHC、SF3B4、SLC2A1、ZBTB18、BAG3、BMPR1A、EMX2、GATA3、KAT6B、KIF11、PAX2、PTEN、RPS24、WAC、ZMYND11、ALX4、ARCN1、ATM、CDKN1C、EXT2、FZD4、HMBS、KCNQ1、KCNQ1OT1、KMT2A、LRP5、MEN1、MYBPC3、PAX6、PHF21A、SDHAF2、SDHD、SHANK2、WT1、ACVRL1、ARID2、CDKN1B、GRIN2B、HMGA2、KMT2D、LEMD3、MED13L、PTHLH、PTPN11、RPS26、SLC17A8、SOX5、TBX3、BRCA2、CHAMP1、EDNRB、NBEA、POLR1D、RB1、ZIC2、BMP4、CHD8、DICER1、FOXG1、GPHN、PAX9、AAGAB、CHD2、FBN1、IGF1R、MEIS2、RPS17、SIN3A、SMAD3、SPRED1、TCF12、UBE3A、ANKRD11、CDH1、CREBBP、CTCF、FOXC2、FOXF1、PKD1、SALL1、SETD1A、SH2B1、TSC2、AXIN2、BRCA1、BRIP1、COL1A1、EFTUD2、FLCN、HNF1B、KANSL1、PAFAH1B1、PMP22、RAD51C、RAD51D、RAI1、RNF135、TBX4、TP53、ASXL3、DSC2、GATA6、SETBP1、SMAD4、TCF4、TGIF1、CACNA1A、CIC、ERF、KMT2B、LDLR、RPS19、SMARCA4、STK11、APOB、BARD1、BCL11A、CFC1、COL3A1、CUL3、GIGYF2、GLI2、MBD5、MSH2、MSH6、MYCN、MYT1L、NCKAP1、NRXN1、PAX3、PAX8、SATB2、SCN1A、SCN2A、SIX3、SOX11、SPAST、TBR1、TRIP12、ZEB2、ADNP、ASXL1、GDF5、GNAS、JAG1、KCNQ2、SALL4、APP、DSCAM、DYRK1A、RUNX1、SON、CHEK2、EP300、NF2、SHANK3、SMARCB1、SOX10、TBX1、TCF20、TNRC6B、BAP1、CTNNB1、FOXL2、FOXP1、GATA2、MITF、MLH1、SCN5A、SETD2、SETD5、SLC6A1、SOX2、TBL1XR1、TGFBR2、ZIC1、ZIC4、ANK2、NAA15、NR3C2、PITX2、PKD2、APC、CTNND2、FGF10、GABRA1、LMNB1、MEF2C、NKX2-5、PURA、RASA1、SPINK1、TRIO、ARID1B、EYA4、FOXC1、HIVEP2、PHIP、SYNGAP1、TFAP2B、AUTS2、CAMK2B、ELN、GLI3、KCNH2、KMT2C、MNX1、PMS2、SGCE、SHH、TWIST1、CHD7、EXT1、GATA4、KAT6A、TRPS1、ZFPM2、COL5A1、DMRT1、EHMT1、ENG、HNRNPK、LMX1B、NR5A1、PTCH1、STXBP1、TGFBR1、TSC1、ZNF462、ABCD1、ACSL4、AFF2、ANOS1、AP1S2、AR、ARHGEF9、ARX、ATP7A、ATRX、AVPR2、BCOR、BRWD3、BTK、CASK、CD40LG、CDKL5、CHM、CHRDL1、CLCN4、CLCN5、CNKSR2、COL4A5、CUL4B、CYBB、DCX、DDX3X、DLG3、EBP、EDA、EFNB1、F8、F9、FANCB、FGD1、FLNA、FMR1、FRMD7、FTSJ1、GDI1、GK、GLA、GPC3、GRIA3、HCCS、HDAC8、HPRT1、IDS、IKBKG、IL1RAPL1、IQSEC2、KDM5C、KDM6A、L1CAM、LAMP2、MAGT1、MID1、MTM1、NDP、NHS、NR0B1、NSDHL、NYX、OCRL、OFD1、OPHN1、PAK3、PCDH19、PDHA1、PGK1、PHF6、PIGA、PLP1、PORCN、PQBP1、PRPS1、PTCHD1、RAB39B、RP2、RPS6KA3、RS1、SH2D1A、SLC16A2、SLC35A2、SLC6A8、SLC9A6、SMC1A、SMS、STS、SYN1、TBX22、TIMM8A、TRAPPC2、TSPAN7、UBE2A、UPF3B、USP9X、WDR45、XIAP、XIST、ZC4H2、ZDHHC9、ZIC3、ZNF711、SHOX、SRY；
所述special_gene探针组覆盖190个重要出生缺陷遗传病基因的单核苷酸变异位点37083个，对应探针总数为330272个，平均每个所述单核苷酸变异位点设计9个重复探针；
所述190个重要出生缺陷遗传病基因包括：ABCB1、ABCC8、ABCD1、ABCD4、ABCG5、ABCG8、ACADM、ACADS、ACADVL、ACAT1、ACE、ACSF3、AGL、AMH、AMHR2、APOA5、APOB、APOC3、AR、ARG1、ARSA、ARSB、ASS1、ATP7A、ATP7B、BCKDHA、BCKDHB、BMP1、BTD、CBS、CD320、CLCN5、COCH、COL1A1、COL1A2、COL2A1、COMP、CPS1、CPT1A、CPT2、CRHR1、CYP11B1、CYP17A1、CYP1A1、CYP21A2、CYP2C19、CYP2D6、CYP3A5、DBT、DIABLO、DLD、DMD、DMP1、DNAJC12、DRD2、DSPP、ELN、ENPP1、EPHX1、ETFA、ETFDH、FAH、FGF23、FGFR1、FGFR2、FGFR3、FKBP5、G6PC、G6PD、GAA、GALC、GALE、GALK1、GALNS、GALT、GBA、GBE1、GCDH、GCH1、GCK、GJB2、GJB3、GLA、GLB1、GNPTAB、GNPTG、GNS、GUSB、GYS2、HADH、HBA1、HBA2、HBB、HEXA、HEXB、HGSNAT、HLA_B、HLCS、HNF4A、HPD、HSD3B2、HTR2C、HYAL1、IDS、IDUA、IFITM5、IFNL4、IVD、KCNJ11、LDLR、LHCGR、LIPI、LMBRD1、LPL、MC4R、MCCC1、MCCC2、MCEE、MCOLN1、MLYCD、MMAA、MMAB、MMACHC、MMADHC、MMUT、MT-CO1、MT-RNR1、MT-TH、MT-TL1、MT-TS1、MTHFR、MTR、MYO15A、MYO7A、NAGLU、NDN、NPC1、NPC2、NR0B1、NR5A1、OTC、PAH、PCBD1、PCCA、PCCB、PHEX、PHKA2、PHKB、PHKG2、PLA2G4A、PLP1、POLG、PPIB、PRPS1、PTS、PYGL、PYGM、QDPR、RP1、SDHA、SGSH、SLC16A1、SLC22A5、SLC25A13、SLC25A15、SLC25A20、SLC26A4、SLC34A3、SLC37A4、SLCO2B1、SMN1/SMN2、SMPD1、SNRPN、SPR、SPRN、SRD5A2、SRY、STAR、SUMF1、TAT、TBX1、TECTA、TMC1、TPMT、TYRP1、UCP2、UGT1A、UGT1A4、VKORC1、WFS1。
2.根据权利要求1所述的芯片，其特征在于：
所述special_gene探针组兼顾提升所述genome_framework探针组对应基因区域的探针分布密度。
3.根据权利要求1所述的芯片，其特征在于：
所述genome_framework探针组和special_gene探针组的核苷酸序列包括如SEQ IDNo.1～SEQ ID No.720所示的探针序列。
4.根据权利要求1所述的芯片，其特征在于：
所述genome_framework探针组针对所述16个单基因、所述致病性单倍剂量不足基因以及所述三倍剂量敏感基因的变异，计算每个探针拷贝数CN；常染色体和女性X染色体正常所述探针拷贝数CN1为2，计算出现的所述探针拷贝数CN1变异情况为2种，其中所述探针拷贝数CN1为0或1属于缺失，为3或4属于重复；男性X和Y染色体正常所述探针拷贝数CN2为1，计算出现的所述探针拷贝数CN2变异情况为2种，其中所述探针拷贝数CN2为0属于缺失，为2属于重复；当出现连续50个探针的所述探针拷贝数CN异常情况，判定为基因拷贝数变异；
计算拷贝数度量值是log2 Ratio，其中Ratio＝每个探针拷贝数的观测值÷每个探针拷贝数的参考值，每个探针拷贝数的参考值是来自于正常人群的一组数据：
当log2 Ratio≈-1.5±0.05时，CN＝0；
当log2 Ratio≈-1±0.05时，CN＝1；
当log2 Ratio≈0±0.05时，CN＝2；
当log2 Ratio≈0.58±0.05时，CN＝3；
当log2 Ratio≈1±0.05时，CN＝4。
5.根据权利要求1所述的芯片，其特征在于：
所述special_gene探针组针对所述190个重要出生缺陷遗传病基因的相应SNV位点进行检测，结果的判读基于该部分探针的检测值来实现的，所用的方法为Genotype_SNV和ES聚类算法。
Genotype_SNV方法是通过对SNV array探针进行双色荧光标记实现的，所述探针会得到两种荧光检测值A和B，通过对A和B值进行计算得到基因分型结果，计算公式如下：
t(snv)＝(A-B)/(A+B)
当t(snv)≈1±0.05时，分型结果为AA；
当t(snv)≈-1±0.05时，分型结果为BB；
当t(snv)≈0±0.05时，分型结果为AB；
所述基因分型结果包括三种，AA、AB和BB，其中所述AA和BB代表纯和类型，一个代表野生型，一个代表纯合子，根据聚类结果判读野生型和纯合子，所述AB代表杂合子。
所述ES聚类算法用于对所述基因分型结果进行聚类，计算公式如下：
t(x)＝log2(A/B)；
t(y)＝[log2(A)+log2(B)]/2；
所述ES聚类算法以t(x)为横轴，t(y)为纵轴进行聚类，所述检测结果的阳性或阴性根据样本分型结果和聚类数目进行判断。
6.根据权利要求1所述的芯片，其特征在于：
所述芯片还包括变性混合液、扩增混合液、片段化混合液、沉淀混合液和杂交混合液。
7.根据权利要求1所述的芯片，其特征在于：
所述芯片在
微阵列芯片扫描仪中进行检测使用。
8.一种应用权利要求1所述芯片同步检测多种出生缺陷遗传病CNV和SNV的方法，其特征在于包括以下步骤：
S1、采集样品；
S2、样品制备与质控；
S3、应用权利要求1所述芯片的检测反应：
S31、DNA扩增；
S32、DNA片段化和沉淀；
S33、干燥、重悬及质控；
S34、变性和杂交；
S35、芯片扫描仪检测；
S4、数据质控和分析；
S5、结果分析与注释。
9.一种如权利要求1所述的芯片在同步检测多种出生缺陷遗传病CNV和SNV中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111307383.3A CN114196736B (zh) | 2021-11-05 | 2021-11-05 | 一种同步检测多种出生缺陷遗传病全染色体基因分型芯片及其方法和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111307383.3A CN114196736B (zh) | 2021-11-05 | 2021-11-05 | 一种同步检测多种出生缺陷遗传病全染色体基因分型芯片及其方法和应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114196736A true CN114196736A (zh) | 2022-03-18 |
| CN114196736B CN114196736B (zh) | 2024-04-02 |
Family
ID=80646941
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111307383.3A Active CN114196736B (zh) | 2021-11-05 | 2021-11-05 | 一种同步检测多种出生缺陷遗传病全染色体基因分型芯片及其方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114196736B (zh) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115216532A (zh) * | 2022-06-30 | 2022-10-21 | 湖南家辉生物技术有限公司 | 一种短链酰基辅酶a脱氢酶缺乏症的诊断试剂和试剂盒 |
| CN116042811A (zh) * | 2022-12-15 | 2023-05-02 | 湖南家辉生物技术有限公司 | 一种导致mrd26型神经发育迟滞的致病基因auts2突变的应用及检测试剂 |
| CN116083458A (zh) * | 2023-02-20 | 2023-05-09 | 中南大学湘雅医院 | 黏多糖贮积症iiic型的致病突变基因及其应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282349A (zh) * | 2016-08-18 | 2017-01-04 | 陈晓丽 | 一种x染色体高密度探针定制的全基因组拷贝数检测芯片 |
| US20170037475A1 (en) * | 2014-04-09 | 2017-02-09 | Lineagen, Inc. | Genetic markers associated with asd and other childhood developmental delay disorders |
| CN110592213A (zh) * | 2019-09-02 | 2019-12-20 | 深圳市新合生物医疗科技有限公司 | 预测新抗原负荷和检测基因组突变的基因panel |
-
2021
- 2021-11-05 CN CN202111307383.3A patent/CN114196736B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170037475A1 (en) * | 2014-04-09 | 2017-02-09 | Lineagen, Inc. | Genetic markers associated with asd and other childhood developmental delay disorders |
| CN106282349A (zh) * | 2016-08-18 | 2017-01-04 | 陈晓丽 | 一种x染色体高密度探针定制的全基因组拷贝数检测芯片 |
| CN110592213A (zh) * | 2019-09-02 | 2019-12-20 | 深圳市新合生物医疗科技有限公司 | 预测新抗原负荷和检测基因组突变的基因panel |
Non-Patent Citations (1)
| Title |
|---|
| 秦谦 等: "基于高通量测序技术的拷贝数变异筛选分析流程的建立及应用", 中国循证儿科杂志, vol. 13, no. 4, 31 August 2018 (2018-08-31), pages 275 - 279 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115216532A (zh) * | 2022-06-30 | 2022-10-21 | 湖南家辉生物技术有限公司 | 一种短链酰基辅酶a脱氢酶缺乏症的诊断试剂和试剂盒 |
| CN116042811A (zh) * | 2022-12-15 | 2023-05-02 | 湖南家辉生物技术有限公司 | 一种导致mrd26型神经发育迟滞的致病基因auts2突变的应用及检测试剂 |
| CN116083458A (zh) * | 2023-02-20 | 2023-05-09 | 中南大学湘雅医院 | 黏多糖贮积症iiic型的致病突变基因及其应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114196736B (zh) | 2024-04-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7343563B2 (ja) | 高度多重ｐｃｒ法および組成物 | |
| US11390916B2 (en) | Methods for simultaneous amplification of target loci | |
| Esteki et al. | Concurrent whole-genome haplotyping and copy-number profiling of single cells | |
| JP6386494B2 (ja) | 母親生物試料の胎児ゲノム分析 | |
| CN114196736A (zh) | 一种同步检测多种出生缺陷遗传病全染色体基因分型芯片及其方法和应用 | |
| CA2874195C (en) | Methods and processes for non-invasive assessment of genetic variations | |
| Oliver et al. | New insights into human nondisjunction of chromosome 21 in oocytes | |
| JP6153874B2 (ja) | 非侵襲的出生前倍数性呼び出しのための方法 | |
| CN106591441B (zh) | 基于全基因捕获测序的α和/或β-地中海贫血突变的检测探针、方法、芯片及应用 | |
| González et al. | A fast and accurate method to detect allelic genomic imbalances underlying mosaic rearrangements using SNP array data | |
| Lau et al. | Birth of a healthy infant following preimplantation PKHD1 haplotyping for autosomal recessive polycystic kidney disease using multiple displacement amplification | |
| Bahrambeigi et al. | Distinct patterns of complex rearrangements and a mutational signature of microhomeology are frequently observed in PLP1 copy number gain structural variants | |
| WO2024027569A1 (zh) | 一种不依赖于先证者的单体型构建方法 | |
| Coutton et al. | Fine characterisation of a recombination hotspot at the DPY19L2 locus and resolution of the paradoxical excess of duplications over deletions in the general population | |
| KR20230038263A (ko) | 무세포 핵산에 대한 뉴클레아제 관련 말단 특징 분석 | |
| WO2018090991A1 (en) | Universal haplotype-based noninvasive prenatal testing for single gene diseases | |
| Geng et al. | Sequence and structure characteristics of 22 deletion breakpoints in intron 44 of the DMD gene based on long-read sequencing | |
| CN114512183B (zh) | 一种预测met基因扩增或多倍体的方法及装置 | |
| CN114540479A (zh) | 用于检测与耳聋相关的基因snp的组合物、试剂盒及检测方法 | |
| Amr et al. | Targeted hybrid capture for inherited disease panels | |
| CN112735518B (zh) | 基于染色体微阵列的roh数据分析*** | |
| Oliva Pavia | Functional impact of polymorphic inversions in the human genome | |
| Lähdesmäki | CS-E5875 High-Throughput Bioinformatics Genotype calling and de novo assembly | |
| Chia | DNA Copy Number Variation in a Cohort of Healthy Australian Women | |
| Loovers et al. | Validation and implementation of a custom 21-gene panel next-generation sequencing assay for myeloid neoplasms |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |