WO2000075294A1 - Glycogenine, pseudogenes de glycogenine et leurs utilisations pour la determination des risques de developper le diabete de type ii - Google Patents
Glycogenine, pseudogenes de glycogenine et leurs utilisations pour la determination des risques de developper le diabete de type ii Download PDFInfo
- Publication number
- WO2000075294A1 WO2000075294A1 PCT/US2000/015685 US0015685W WO0075294A1 WO 2000075294 A1 WO2000075294 A1 WO 2000075294A1 US 0015685 W US0015685 W US 0015685W WO 0075294 A1 WO0075294 A1 WO 0075294A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- glycogenin
- pseudogene
- human
- polynucleotide
- mutation
- Prior art date
Links
- 108010062764 glycogenin Proteins 0.000 title claims abstract description 160
- 102000011054 glycogenin Human genes 0.000 title claims abstract description 136
- 108091008109 Pseudogenes Proteins 0.000 title claims abstract description 91
- 102000057361 Pseudogenes Human genes 0.000 title claims abstract description 91
- 208000001072 type 2 diabetes mellitus Diseases 0.000 title abstract description 20
- 230000035772 mutation Effects 0.000 claims abstract description 74
- 230000014509 gene expression Effects 0.000 claims abstract description 36
- 238000000034 method Methods 0.000 claims abstract description 29
- 102000040430 polynucleotide Human genes 0.000 claims description 91
- 108091033319 polynucleotide Proteins 0.000 claims description 91
- 239000002157 polynucleotide Substances 0.000 claims description 91
- 108090000623 proteins and genes Proteins 0.000 claims description 56
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 49
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 47
- 229920001184 polypeptide Polymers 0.000 claims description 44
- 239000000523 sample Substances 0.000 claims description 43
- 239000002773 nucleotide Substances 0.000 claims description 38
- 125000003729 nucleotide group Chemical group 0.000 claims description 38
- 108020004705 Codon Proteins 0.000 claims description 33
- 239000002299 complementary DNA Substances 0.000 claims description 27
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 25
- 238000013518 transcription Methods 0.000 claims description 25
- 230000035897 transcription Effects 0.000 claims description 25
- 108020004999 messenger RNA Proteins 0.000 claims description 22
- 238000009396 hybridization Methods 0.000 claims description 19
- 230000009870 specific binding Effects 0.000 claims description 18
- 108020004485 Nonsense Codon Proteins 0.000 claims description 14
- 238000003780 insertion Methods 0.000 claims description 12
- 230000037431 insertion Effects 0.000 claims description 12
- 239000000758 substrate Substances 0.000 claims description 12
- 241001465754 Metazoa Species 0.000 claims description 9
- 239000013598 vector Substances 0.000 claims description 6
- 230000037432 silent mutation Effects 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 230000009261 transgenic effect Effects 0.000 claims description 5
- 230000000295 complement effect Effects 0.000 claims description 4
- 238000006467 substitution reaction Methods 0.000 claims description 4
- 108020005038 Terminator Codon Proteins 0.000 claims 1
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 9
- 102000039446 nucleic acids Human genes 0.000 abstract description 3
- 108020004707 nucleic acids Proteins 0.000 abstract description 3
- 150000007523 nucleic acids Chemical class 0.000 abstract description 3
- 229920002527 Glycogen Polymers 0.000 description 28
- 229940096919 glycogen Drugs 0.000 description 28
- 230000027455 binding Effects 0.000 description 26
- 206010012601 diabetes mellitus Diseases 0.000 description 26
- 210000004027 cell Anatomy 0.000 description 22
- 239000013615 primer Substances 0.000 description 19
- 102000004169 proteins and genes Human genes 0.000 description 19
- 239000000047 product Substances 0.000 description 18
- 235000018102 proteins Nutrition 0.000 description 18
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 17
- 239000008103 glucose Substances 0.000 description 17
- 108020004414 DNA Proteins 0.000 description 15
- 239000012634 fragment Substances 0.000 description 14
- 230000002068 genetic effect Effects 0.000 description 14
- 238000003757 reverse transcription PCR Methods 0.000 description 14
- 239000000126 substance Substances 0.000 description 14
- 102000004190 Enzymes Human genes 0.000 description 13
- 108090000790 Enzymes Proteins 0.000 description 13
- 150000001413 amino acids Chemical class 0.000 description 13
- 230000008569 process Effects 0.000 description 13
- 230000014616 translation Effects 0.000 description 13
- 238000001514 detection method Methods 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- 238000013519 translation Methods 0.000 description 11
- 230000015572 biosynthetic process Effects 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 238000003786 synthesis reaction Methods 0.000 description 10
- 239000000427 antigen Substances 0.000 description 9
- 108091007433 antigens Proteins 0.000 description 9
- 102000036639 antigens Human genes 0.000 description 9
- 210000004369 blood Anatomy 0.000 description 9
- 239000008280 blood Substances 0.000 description 9
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 8
- 230000000875 corresponding effect Effects 0.000 description 8
- 210000000265 leukocyte Anatomy 0.000 description 8
- 229940024606 amino acid Drugs 0.000 description 7
- 235000001014 amino acid Nutrition 0.000 description 7
- 230000003321 amplification Effects 0.000 description 7
- 238000003556 assay Methods 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 201000001421 hyperglycemia Diseases 0.000 description 7
- 230000001771 impaired effect Effects 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 238000001964 muscle biopsy Methods 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- 230000001575 pathological effect Effects 0.000 description 7
- 230000002829 reductive effect Effects 0.000 description 7
- 210000002027 skeletal muscle Anatomy 0.000 description 7
- 229930024421 Adenine Natural products 0.000 description 6
- 108700026244 Open Reading Frames Proteins 0.000 description 6
- 229960000643 adenine Drugs 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 230000015556 catabolic process Effects 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000012217 deletion Methods 0.000 description 6
- 230000037430 deletion Effects 0.000 description 6
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 6
- 238000012544 monitoring process Methods 0.000 description 6
- 210000003205 muscle Anatomy 0.000 description 6
- 230000001105 regulatory effect Effects 0.000 description 6
- 108020004635 Complementary DNA Proteins 0.000 description 5
- 108091092195 Intron Proteins 0.000 description 5
- 102100041030 Pancreas/duodenum homeobox protein 1 Human genes 0.000 description 5
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 5
- 230000002159 abnormal effect Effects 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000037433 frameshift Effects 0.000 description 5
- -1 glyco- sylase Proteins 0.000 description 5
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 5
- 238000002955 isolation Methods 0.000 description 5
- 230000011987 methylation Effects 0.000 description 5
- 238000007069 methylation reaction Methods 0.000 description 5
- 238000003752 polymerase chain reaction Methods 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 101710163270 Nuclease Proteins 0.000 description 4
- 108091034117 Oligonucleotide Proteins 0.000 description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- 101710144033 Pancreas/duodenum homeobox protein 1 Proteins 0.000 description 4
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 4
- 230000001594 aberrant effect Effects 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000006957 competitive inhibition Effects 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 231100000221 frame shift mutation induction Toxicity 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 230000000977 initiatory effect Effects 0.000 description 4
- 210000004698 lymphocyte Anatomy 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 230000002503 metabolic effect Effects 0.000 description 4
- 230000004060 metabolic process Effects 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 239000002777 nucleoside Substances 0.000 description 4
- 230000006337 proteolytic cleavage Effects 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000005215 recombination Methods 0.000 description 4
- 230000006798 recombination Effects 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 238000011282 treatment Methods 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 230000004543 DNA replication Effects 0.000 description 3
- 108010017544 Glucosylceramidase Proteins 0.000 description 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 3
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 3
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 3
- 102100029237 Hexokinase-4 Human genes 0.000 description 3
- 102000004877 Insulin Human genes 0.000 description 3
- 108090001061 Insulin Proteins 0.000 description 3
- 102100034343 Integrase Human genes 0.000 description 3
- 108060001084 Luciferase Proteins 0.000 description 3
- 239000005089 Luciferase Substances 0.000 description 3
- 208000035180 MODY Diseases 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 108091092878 Microsatellite Proteins 0.000 description 3
- OKIZCWYLBDKLSU-UHFFFAOYSA-M N,N,N-Trimethylmethanaminium chloride Chemical compound [Cl-].C[N+](C)(C)C OKIZCWYLBDKLSU-UHFFFAOYSA-M 0.000 description 3
- 239000004677 Nylon Substances 0.000 description 3
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 3
- 238000002105 Southern blotting Methods 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 3
- 230000010933 acylation Effects 0.000 description 3
- 238000005917 acylation reaction Methods 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 230000008021 deposition Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 239000005090 green fluorescent protein Substances 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 229940125396 insulin Drugs 0.000 description 3
- 238000013507 mapping Methods 0.000 description 3
- 201000006950 maturity-onset diabetes of the young Diseases 0.000 description 3
- 230000037230 mobility Effects 0.000 description 3
- 230000037434 nonsense mutation Effects 0.000 description 3
- 229920001778 nylon Polymers 0.000 description 3
- 230000026731 phosphorylation Effects 0.000 description 3
- 238000006366 phosphorylation reaction Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 230000019635 sulfation Effects 0.000 description 3
- 238000005670 sulfation reaction Methods 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- 102000003925 1,4-alpha-Glucan Branching Enzyme Human genes 0.000 description 2
- 108090000344 1,4-alpha-Glucan Branching Enzyme Proteins 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 2
- 102100026189 Beta-galactosidase Human genes 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 102000053642 Catalytic RNA Human genes 0.000 description 2
- 108090000994 Catalytic RNA Proteins 0.000 description 2
- 244000138502 Chenopodium bonus henricus Species 0.000 description 2
- 235000008645 Chenopodium bonus henricus Nutrition 0.000 description 2
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 239000004971 Cross linker Substances 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 108700024394 Exon Proteins 0.000 description 2
- 208000015872 Gaucher disease Diseases 0.000 description 2
- 102000004547 Glucosylceramidase Human genes 0.000 description 2
- 108010001483 Glycogen Synthase Proteins 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 2
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 2
- 108700005084 Multigene Family Proteins 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 2
- 108010038807 Oligopeptides Proteins 0.000 description 2
- 102000015636 Oligopeptides Human genes 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 2
- 108700008625 Reporter Genes Proteins 0.000 description 2
- 108091035242 Sequence-tagged site Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- HSCJRCZFDFQWRP-JZMIEXBBSA-N UDP-alpha-D-glucose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-JZMIEXBBSA-N 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- HSCJRCZFDFQWRP-UHFFFAOYSA-N Uridindiphosphoglukose Natural products OC1C(O)C(O)C(CO)OC1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-UHFFFAOYSA-N 0.000 description 2
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 238000001261 affinity purification Methods 0.000 description 2
- 210000004436 artificial bacterial chromosome Anatomy 0.000 description 2
- 210000001106 artificial yeast chromosome Anatomy 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 108010005774 beta-Galactosidase Proteins 0.000 description 2
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000002759 chromosomal effect Effects 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000006735 deficit Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 210000004602 germ cell Anatomy 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 238000001114 immunoprecipitation Methods 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- FOMCONPAMXXLBX-MQHGYYCBSA-N isopanose Chemical group O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H](O)[C@H]([C@H](O)[C@@H](O)C=O)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 FOMCONPAMXXLBX-MQHGYYCBSA-N 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 201000002273 mucopolysaccharidosis II Diseases 0.000 description 2
- 208000022018 mucopolysaccharidosis type 2 Diseases 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 150000003833 nucleoside derivatives Chemical class 0.000 description 2
- 125000003835 nucleoside group Chemical group 0.000 description 2
- 239000002751 oligonucleotide probe Substances 0.000 description 2
- 210000003463 organelle Anatomy 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 230000002974 pharmacogenomic effect Effects 0.000 description 2
- 150000004713 phosphodiesters Chemical class 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 230000004481 post-translational protein modification Effects 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 238000006722 reduction reaction Methods 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 108091092562 ribozyme Proteins 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 2
- 239000012064 sodium phosphate buffer Substances 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- 239000011593 sulfur Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000000123 temperature gradient gel electrophoresis Methods 0.000 description 2
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- 230000005945 translocation Effects 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 230000018412 transposition, RNA-mediated Effects 0.000 description 2
- 230000007306 turnover Effects 0.000 description 2
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 description 1
- AOFUBOWZWQFQJU-SNOJBQEQSA-N (2r,3s,4s,5r)-2,5-bis(hydroxymethyl)oxolane-2,3,4-triol;(2s,3r,4s,5s,6r)-6-(hydroxymethyl)oxane-2,3,4,5-tetrol Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O.OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1O AOFUBOWZWQFQJU-SNOJBQEQSA-N 0.000 description 1
- ZDSRFXVZVHSYMA-CMOCDZPBSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-carboxybutanoyl]amino]pentanedioic acid Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O)C1=CC=C(O)C=C1 ZDSRFXVZVHSYMA-CMOCDZPBSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 description 1
- UFBJCMHMOXMLKC-UHFFFAOYSA-N 2,4-dinitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O UFBJCMHMOXMLKC-UHFFFAOYSA-N 0.000 description 1
- HKJKONMZMPUGHJ-UHFFFAOYSA-N 4-amino-5-hydroxy-3-[(4-nitrophenyl)diazenyl]-6-phenyldiazenylnaphthalene-2,7-disulfonic acid Chemical compound OS(=O)(=O)C1=CC2=CC(S(O)(=O)=O)=C(N=NC=3C=CC=CC=3)C(O)=C2C(N)=C1N=NC1=CC=C([N+]([O-])=O)C=C1 HKJKONMZMPUGHJ-UHFFFAOYSA-N 0.000 description 1
- 108091032151 5-hydroxytryptamine receptor family Proteins 0.000 description 1
- 206010069754 Acquired gene mutation Diseases 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- 102000014654 Aromatase Human genes 0.000 description 1
- 108010078554 Aromatase Proteins 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 102000001189 Cyclic Peptides Human genes 0.000 description 1
- 108010069514 Cyclic Peptides Proteins 0.000 description 1
- 108010015742 Cytochrome P-450 Enzyme System Proteins 0.000 description 1
- 102100025621 Cytochrome b-245 heavy chain Human genes 0.000 description 1
- 102000018832 Cytochromes Human genes 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 238000000018 DNA microarray Methods 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 108090000204 Dipeptidase 1 Proteins 0.000 description 1
- 238000004435 EPR spectroscopy Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 108091008794 FGF receptors Proteins 0.000 description 1
- 102000044168 Fibroblast Growth Factor Receptor Human genes 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 102000014630 G protein-coupled serotonin receptor activity proteins Human genes 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 101000867891 Gallus gallus Calmodulin Proteins 0.000 description 1
- 206010071602 Genetic polymorphism Diseases 0.000 description 1
- 108090000079 Glucocorticoid Receptors Proteins 0.000 description 1
- 102100033417 Glucocorticoid receptor Human genes 0.000 description 1
- 108010021582 Glucokinase Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 102000005720 Glutathione transferase Human genes 0.000 description 1
- 108010070675 Glutathione transferase Proteins 0.000 description 1
- 102000006752 Hepatocyte Nuclear Factor 4 Human genes 0.000 description 1
- 108010086524 Hepatocyte Nuclear Factor 4 Proteins 0.000 description 1
- 102100022054 Hepatocyte nuclear factor 4-alpha Human genes 0.000 description 1
- 108091027305 Heteroduplex Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 101000617823 Homo sapiens Solute carrier organic anion transporter family member 6A1 Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 108010001127 Insulin Receptor Proteins 0.000 description 1
- 102100036721 Insulin receptor Human genes 0.000 description 1
- 101710203526 Integrase Proteins 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 108010047357 Luminescent Proteins Proteins 0.000 description 1
- 102000006830 Luminescent Proteins Human genes 0.000 description 1
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 1
- 102000003792 Metallothionein Human genes 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 241000713333 Mouse mammary tumor virus Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 102000008934 Muscle Proteins Human genes 0.000 description 1
- 108010074084 Muscle Proteins Proteins 0.000 description 1
- WGZDBVOTUVNQFP-UHFFFAOYSA-N N-(1-phthalazinylamino)carbamic acid ethyl ester Chemical compound C1=CC=C2C(NNC(=O)OCC)=NN=CC2=C1 WGZDBVOTUVNQFP-UHFFFAOYSA-N 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 206010067482 No adverse event Diseases 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 238000002944 PCR assay Methods 0.000 description 1
- 239000007990 PIPES buffer Substances 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108091093037 Peptide nucleic acid Proteins 0.000 description 1
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 description 1
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 108091036407 Polyadenylation Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 101710086015 RNA ligase Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 241000714474 Rous sarcoma virus Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 102100021991 Solute carrier organic anion transporter family member 6A1 Human genes 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- PQISXOFEOCLOCT-UUOKFMHZSA-N [[(2r,3s,4r,5r)-5-(6-amino-8-azidopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound [N-]=[N+]=NC1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O PQISXOFEOCLOCT-UUOKFMHZSA-N 0.000 description 1
- DPKHZNPWBDQZCN-UHFFFAOYSA-N acridine orange free base Chemical compound C1=CC(N(C)C)=CC2=NC3=CC(N(C)C)=CC=C3C=C21 DPKHZNPWBDQZCN-UHFFFAOYSA-N 0.000 description 1
- 230000002730 additional effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000002820 assay format Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000005844 autocatalytic reaction Methods 0.000 description 1
- 230000005784 autoimmunity Effects 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 1
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 1
- 102000006635 beta-lactamase Human genes 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000008512 biological response Effects 0.000 description 1
- 238000009534 blood test Methods 0.000 description 1
- 230000000981 bystander Effects 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 238000007707 calorimetry Methods 0.000 description 1
- 238000001818 capillary gel electrophoresis Methods 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 150000001721 carbon Chemical group 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 108091092328 cellular RNA Proteins 0.000 description 1
- 230000030570 cellular localization Effects 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000007073 chemical hydrolysis Effects 0.000 description 1
- 208000016532 chronic granulomatous disease Diseases 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000012411 cloning technique Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000002559 cytogenic effect Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000003935 denaturing gradient gel electrophoresis Methods 0.000 description 1
- 239000005549 deoxyribonucleoside Substances 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000005014 ectopic expression Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000009088 enzymatic function Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 230000010502 episomal replication Effects 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 238000003804 extraction from natural source Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 238000005189 flocculation Methods 0.000 description 1
- 230000016615 flocculation Effects 0.000 description 1
- 238000002875 fluorescence polarization Methods 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000004545 gene duplication Effects 0.000 description 1
- 238000010359 gene isolation Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 238000012248 genetic selection Methods 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 210000003917 human chromosome Anatomy 0.000 description 1
- 210000004754 hybrid cell Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000001571 immunoadjuvant effect Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 239000000568 immunological adjuvant Substances 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 238000005462 in vivo assay Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000003914 insulin secretion Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 210000004153 islets of langerhan Anatomy 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 150000003951 lactams Chemical class 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 210000000633 nuclear envelope Anatomy 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000000816 peptidomimetic Substances 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- UEZVMMHDMIWARA-UHFFFAOYSA-M phosphonate Chemical compound [O-]P(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-M 0.000 description 1
- 150000008300 phosphoramidites Chemical class 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 238000000206 photolithography Methods 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000003169 placental effect Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 108091008077 processed pseudogenes Proteins 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000007363 regulatory process Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000002342 ribonucleoside Substances 0.000 description 1
- 150000003290 ribose derivatives Chemical class 0.000 description 1
- 235000002020 sage Nutrition 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000003196 serial analysis of gene expression Methods 0.000 description 1
- 229940076279 serotonin Drugs 0.000 description 1
- 230000003584 silencer Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 230000037439 somatic mutation Effects 0.000 description 1
- 210000001988 somatic stem cell Anatomy 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000005382 thermal cycling Methods 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 230000005758 transcription activity Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 108010068794 tyrosyl-tyrosyl-glutamyl-glutamic acid Proteins 0.000 description 1
- 230000009452 underexpressoin Effects 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
Definitions
- This invention relates to human health, particularly in the area of endocrinology and metabolism. Glycogen synthesis may be impaired in some diabetics or people at risk for developing diabetes due to reduced expression of glycogenin.
- This protein serves as the scaffolding upon which glucose is polymerized, and thereby serves as a substrate for the production of glycogen.
- the invention relates to a diagnostic procedure for determining whether a human subject has a predisposition to develop Type 2 diabetes (e.g., he or she has an identified risk for becoming diabetic that is increased as compared to a control group of the general population) or is a candidate for preventative treatment.
- Maintaining a stable blood glucose level in mammals is an important, multifaceted regulatory process, as chronic hyperglycemia promotes well-recognized adverse consequences.
- the level of blood glucose in mammals is ordinarily under strict hormonal control.
- the principal ways in which excess glucose can be removed from the circulation are the storage of glucose as glycogen and the breakdown of glucose to lactate, or to water and carbon dioxide, in peripheral tissues.
- glucose levels rise insulin is released from the pancreas and most of the excess glucose is deposited in skeletal muscle in the form of glycogen.
- an inability to form glycogen in instances of chronic hyperglycemia may lead to pathologic conditions.
- Persistent hyperglycemia is a symptom of diabetes mellitus, which afflicts more than 100 million people worldwide.
- Type 1 diabetes Patients with diabetes mellitus are divided into two groups: those with deficient insulin secretion due to an autoimmune disorder (Type 1 diabetes) and those who secrete insulin but still develop hyperglycemia (Type 2 diabetes).
- Type 2 diabetes The latter form accounts for almost 90% of human diabetic patients.
- a generally accepted principle is that both genetic and environmental factors contribute to the etiology of Type 2 diabetes. The genetics of the disease are complex and despite much effort and achievement during recent years, the basic etiology of Type 2 diabetes has been poorly understood. See Permutt et al. (1998).
- MODY maturity-onset diabetes of the young
- MODY can be caused by an impairment or change in a protein encoded by one of at least five different genes (i.e., at least five different genetic mutations): the enzyme glucokinase enzyme (GCK/MODY2) or the transcription factors hepatocyte nuclear factor 4 alpha (HNF-4 ⁇ / MODY1), HNF-lo/MODY3, insulin promoter factor 1 (IPF-1/MODY4), and HNF-l ⁇ / MODY5.
- GCK/MODY2 glucokinase enzyme
- HNF-4 ⁇ / MODY1 the transcription factors hepatocyte nuclear factor 4 alpha
- HNF-lo/MODY3 insulin promoter factor 1
- HNF-l ⁇ / MODY5 HNF-l ⁇ / MODY5.
- Glycogenin is a muscle protein which plays a crucial role in nonoxidative glucose disposal in mammals, such as humans.
- Glycogenin is an enzyme which autocatalytically creates on itself a maltosacchari.de primer for glycogen synthase which, together with branching enzyme, forms the mature glycogen molecule.
- Glycogenin covalently binds glycogen at a 1 :1 molar ration and glycogenin remains an integral part of the growing polysaccharide in all steps of glycogen synthesis.
- the first glucose unit in glycogen is joined to glycogenin via an alpha-glucosidic bond to tyrosine at position 194 (Tyr-194, ⁇ .B. the IUPAC-IUBMB one- and three-letter abbreviations for amino acids with the associated position as numbered in the mature polypeptide are used herein) of glycogenin in a unique carbohydrate-protein linkage.
- tyrosine ⁇ .B.
- glycogenin builds autocatalytically on itself a maltosaccharide chain long enough to serve as a primer for glycogen synthase which, together with branching enzyme, creates the mature glycogen molecule.
- the glycogenin so released may again form the maltosaccharide primer for synthesis of a new glycogen molecule.
- pseudogenes may be involved in the etiology of a disease
- some variants of Hunter syndrome deficiency of iduronate-2-sulphatase
- a rare inherited syndrome chronic granulomatous disease, wherein patients' phagocytes fail to generate superoxide, making those patients very susceptible to microbial infection
- the formation of abnormal mRNA is thought to be due to recombination between the wild type gene and the pseudogene(s).
- these mutations occur and accu- mulate in glycogenin pseudogenes which prematurely terminate translation of the open reading frame (e.g., missense or frameshift mutations that create a premature stop codon).
- the invention relates to the isolation of genomic and cDNA clones corresponding to the human gene encoding the glyocogenin enzyme, which has permitted the identification of human glycogenin pseudogenes.
- these pseudogenes can be transcribed in diabetic patients but, in general, they would code for truncated forms of glycogenin that appear to be enzymatically inactive.
- Increased transcription of glycogenin pseudogenes may be correlated in diabetic patients or other subjects at risk for the development of diabetes with decreased glycogen biosynthesis and hyperglycemia (i.e., a method of genetic forecasting).
- Such correlations may also be used in pharmacogenomics to correlate transcription with the bioavailability and/or biologic response of a drug that is or will be administered to subjects who have had sequences determined at the gene or transcript level, or both (i.e., a method of genetic profiling or monitoring at the DNA and/or RNA level).
- the invention may relate to using glycogenin pseudogenes in the diagnosis and possible treatment of Type 1 and/or Type 2 diabetes.
- a further embodiment of the invention is products and processes useful in the aforementioned embodiments, especially with respect to isolation, detection, and identification of glycogenin mutations in the human genome and transcripts from the glycogenin pseudogenes.
- the mutations disclosed herein may be used in genetic fingerprinting, mapping, DNA (genomic) profiling, RNA transcript monitoring, and forecasting as single nucleotide polymorphisms.
- Kits comprised of the aforementioned product are also provided to practice the described processes. Such kits are preferably further comprised of instructions for performing processes, standards to calibrate processes and quantitate their results, reagents to perform the processes, or combinations thereof.
- Figure 1 shows the normal nucleotide sequence and the predicted amino acid sequence of the open reading frame (ORF) for a cDNA clone of human glycogenin (SEQ ID NOS: 1-2, respectively).
- T thymidine
- U uracil
- T base a DNA polynucleotide
- U uracil
- FIG. 2 shows a physical map of the human glycogenin gene.
- Figure 3A-K shows the nucleotide sequences for the normal human glycogenin gene and its pseudogenes (SEQ ID NOS:3-19).
- the numbers enclosed in parentheses at the beginning and end of the listed sequences shows the positions of the 5'- and 3 '-termini, respectively, relative to positions 1 to 999 of the normal sequence.
- Nucleotides appearing in bold type (A) represent a sequence difference between the pseudogene and the normal sequence; inserted nucleotides appear in italicized, bold type (A); underlined triplet bases represent a stop codon; deleted nucleotides appear as dashes.
- glycogenin an enzyme involved in glycogen synthesis, allowed us to determine whether under-expression of this protein occurs in patients with Type 2 diabetes. Because glycogen cannot be synthesized without glycogenin and no free glycogenin exists in muscle, a decrease in the supply of glycogenin would be expected to lower the amount of glycogen synthesized by muscle in response to elevation of blood glucose. In particular, factors which could decrease the amount or activity of glycogenin may be expected to lead to proportional decreases in glucose deposition in the form of glycogen.
- One molecule of skeletal glycogen contains up to 60,000 glucose residues. Accordingly, one molecule of glycogenin can initiate the deposition of this amount of glucose. Because there is no free glycogenin in skeletal muscle under normal circumstances, the available quantity of this protein may limit the amount of glucose that can be stored as glycogen. Decreases in glucose deposition can result in elevated blood glucose levels, the characteristic symptom of diabetes.
- Factors reducing glycogenin activity may include, for example, mutations in the glycogenin gene in one or both alleles (i.e., phenotypes of heterozygotes or homozygotes, respectively, may be an quantitative impairment and/or qualitative change), impaired or changed transcription of that glycogenin gene into RNAs, impaired or changed processing of those glycogenin RNAs, impaired or changed translation of a glycogenin mRNA into polypeptides, impaired or changed post-translational modification of a polypeptide of the glycogenin gene, or combinations thereof.
- mutations in the glycogenin gene in one or both alleles i.e., phenotypes of heterozygotes or homozygotes, respectively, may be an quantitative impairment and/or qualitative change
- impaired or changed transcription of that glycogenin gene into RNAs i.e., impaired or changed processing of those glycogenin RNAs, impaired or changed translation of a glycogenin mRNA into polypeptides, impaired or changed post-translational modification of
- Genetic mutation may contribute to the initiation, establishment, progression, or maintenance of the diabetic state, or a symptom or pathologic condition associated with diabetes.
- glycogenin pseudogenes may be the pathognomonic event for this disease or a related pathologic condition, it might not be the only event or other events might combine in its natural history or pathogenesis: DNA replication, methylation, unwinding, topology, conformation, packing, or other higher-order structures above the linear sequence; competitive inhibition in or interference with RNA transcription, turnover, transport, editing, splicing, or other processing; competitive inhibition in or interference with protein translation, degradation, proteolytic cleavage, reduction, glycosylation, phosphorylation, methylation, sulfation, acylation, other covalent modifications, translo- cation across a cell membrane, targeting to an organelle or other subcellular location, splicing, folding or achieving higher-order structures above the linear sequence, conformation, binding to another protein, substrate binding, autocatalytic or catalytic activity; or combinations thereof.
- Glycogenin pseudogenes were discovered during chromosomal mapping of the human gene for glycogenin. Some pseudogenes may represent duplication of a functional gene and subsequent mutation, but other pseudogenes appear to be "processed" (i.e., they lack the introns present in most eukaryotic genes) and then integrated into the germ line (e.g., by retrotransposition). The former class of pseudogenes would still contain introns, although mutation may reduce the ability of a splice donor or acceptor to function, and the non-transcribed regulatory sequences might also still function. Therefore transcription of pseudogenes initially created by gene duplication would be expected.
- pseudogenes that have integrated into the genome would lack regulatory regions outside the transcribed region or those that were located in an intron removed by splicing. Therefore, in general, transcription of intron-less pseudogenes would not be expected. In either case, missense or nonsense mutations that reduced the activity of the translated protein might make the pseudogene a neutral player in the process of genetic selection.
- Such multigene families are also of interest to the study of evolution and genome dynamics, where pseudogenes may act as neutral accumulators of deleterious mutations or bystanders to the selective process.
- the human glycogenin gene is located on chromosome 3q24 (Lomako et al., 1996). The gene spans about 7 Kb and contains five exons ranging in length from 129 (exon 2) to 315 (exon 1) base pairs.
- Four introns are located within the human glycogenin genome which have been substantially sequenced. A high degree of homology with rabbit glycogenin has been observed, and both human and rabbit glycogenins are comprised of 332 amino acids. The two proteins differ by about 10% in amino acid sequence, although the sequences between Leu-63 and Lys-201 and between Glu-295 and the terminal Glu-332 display essentially 91% identity. Intron 1 has been completely sequenced, while most of introns 2-4 has been sequenced.
- the glycogenin genome exhibits an unusual intron boundary between exons 3 and 4, wherein the 5 '-end of the intron begins with AA and terminates with GC.
- This unusual intron may be connected with a sequence encoding human glycogenin in the EMBL databank (publicly accessible as X79537), which is missing exon 4.
- Intron 3 of the glycogenin gene has an unusual splicing site (i.e., GTGT/aag . . . ggc/TTGG), but introns 1, 2 and 4 of the glycogemn gene comply with the gt/ag acceptor and donor rule.
- the presence of this unusual splicing site promotes alternative splicing in certain circumstances when there are particular mutations of the glycogenin gene. This may generate pseudogenes which are inactive but stable components of the genome derived by mutation of a previously active ancestral gene. Transcribing these pseudogenes might be involved in the etiology of the diabetic state, especially Type 2 diabetes.
- FIG. 2 A detailed physical map has been constructed (Figure 2) which identifies features of interest within the human genome in the vicinity of the glycogenin gene.
- Figure 2 A detailed physical map has been constructed (Figure 2) which identifies features of interest within the human genome in the vicinity of the glycogenin gene.
- a muscle biopsy from a Type 2 diabetic patient a cDNA corresponding to a four amino acid deletion from the wild type sequence was identified and further analyzed. Expressed recombinant protein having this deletion is enzymatically inactive. This inactivation was determined to result from absence of the Asp- 159 residue because mutants in which this residue was changed to Ala, Glu, or Lys by genetic manipulation were also inactive. Therefore, Asp-159 may be part of glycogenin' s active site.
- Recombinantly produced muscle glycogenin from rabbit was labeled with a photo- affinity probe (8-azidoadenosine triphosphate) and subjected to tryptic digestion. Peptides obtained therefrom were fractionated and the sequence Ser-Nal-Arg was detected. This oligopeptide corresponds to positions 229-231 of the glycogenin polypeptide sequence. Substitution of Ser-229 by Arg (Ser229Arg) or Arg-231 by Met (Arg23 lMet) allows expression of an active enzyme which is inhibited by ATP.
- Manipulation of such binding sites may prove useful in producing better reagents to detect the presence of glycogenin and modulate its enzymatic activity.
- Therapeutic treatments may be devised for controlling the activity of glycogenin, thereby alleviating hyperglycemia associated with Type 1 and/or Type 2 diabetes.
- gene-based therapies could inhibit the expression of pseudogenes of glycogenin; preventing their interference in the normal pathway of glycogen synthesis and/or the expression of the normal glycogenin gene may be increased to overcome expression of pseudogenes.
- assays for the amount of glycogenin present in a sample from a patient or the glycogenin activity therein could be applied in a clinical setting.
- Enzymatic activity of glycogenin could be measured using in vitro or in vivo assays (e.g., glycogenin autocatalysis).
- the present invention contemplates testing individuals of any age group for predisposition to Type 1 and/or Type 2 diabetes.
- the present invention may also involve detecting a predisposition to diabetes in humans and animals by their aberrant transcription of one or more glycogenin pseudogenes. It is believed on the basis of the present disclosure that there are at least ten different pseudogenes corresponding to the glycogenin gene in the human genome. Given the large size of this multigene family, it is apparent that expansion and contraction of the family might occur by genomic recombination or retrotransposition of transcripts into the genome.
- a polynucleotide, polypeptide, or specific binding molecule according to the present invention may be used to identify and detect a genetic marker in family pedigrees (e.g., CEPH/NIH or Utah projects), radiation hybrids, or human-rodent somatic cell hybrids. Fingerprinting would allow identification of an individual within a genetically similar population or construction of a genealogy among genetically related individuals. Detection of a germline or somatic mutation will determine that a disease is inherited or acquired, respectively.
- Identification of mutations by molecular genetic or cytogenetic techniques may also determine how glycogenin expression, especially at the level of transcriptional activity, is regulated during development.
- Genetic polymorphism may be used in linkage mapping, genetic fingerprinting, and molecular taxonomy.
- a polymorphism as restriction fragment length polymo ⁇ hism (RFLP), by heteroduplex analysis, random amplified polymorphic DNA (RAPD), amplified fragment length polymo ⁇ hism (AFLP), denaturing gradient gel electrophoresis (DGGE), single- strand conformation poly-mo ⁇ hism (SSCP), temperature gradient gel electrophoresis (TGGE), single nucleotide polymo ⁇ hism (SNP), short tandem repeat (STR), variable nucleotide tandem repeat (NNTR), or micro-satellite length heterogeneity may be used.
- RAPD random amplified polymorphic DNA
- AFLP amplified fragment length polymo ⁇ hism
- DGGE denaturing gradient gel electrophoresis
- SSCP single- strand conformation poly-mo ⁇ hism
- TGGE temperature gradient gel electrophoresis
- SNP single nucleotide polymo ⁇ hism
- STR short tandem repeat
- NTR variable nucleotide
- Such polymo ⁇ hisms may be linked to a genetic trait or phenotype, or correlated with gene expression or development.
- a complementary DNA sequence (either single- or double-stranded cDNA) representing a messenger RNA (mRNA) transcript of glycogenin genes and related pseudogenes in human cells may be monitored by polynucleotide detection techniques.
- Nucleotide sequence specific for glycogenin can be used as a probe. Such probes could be full length covering the entire transcribed message or gene, at least one coding region, or a shorter length fragment which is unique to the glycogenin transcript or gene but contains only a portion of same.
- the polynucleotide may be at least about 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 750, 1000, 2500, 5000, 10K, or 20K bases long; intermediate size ranges consisting of these independently selected lower and upper limits (e.g., between about 10 to about 100 bases) are also preferred.
- the Sanger and Maxam-Gilbert sequencing reactions produce a collection of polynucleotide fragments by enzymatic and chemical methods, respectively. These fragments may be separated by slab or capillary gel electrophoresis as a ladder of bands with different mobilities, detected by labeling, and isolated by collecting the moving zone containing the desired fragment with a relative mobility predicted from comparison to a standard.
- polynucleotide fragments may also be produced by limited or complete nuclease digestion.
- Organic synthesis with phosphonate or Caruthers' phosphoramidite may also be used to produce short oligonucleotides.
- High-performance liquid chromatography and mass spectroscopy are alternative methods of separation and detection, respectively.
- a recombinant clone or expression construct containing a nucleotide sequence is one preferred form of the polynucleotide of the present invention.
- the recombinant clone or expression construct may be an episome, phagemid, plasmid, bacteriophage, cosmid, yeast artificial chromosome (YAC), or bacterial artificial chromosome (BAC).
- YAC yeast artificial chromosome
- BAC bacterial artificial chromosome
- nucleotides may be deoxyribonucle- osides, ribonucleosides, nucleoside analogs and variants, nucleosides with a modified base, nucleotides with a modified ribose sugar, or combinations thereof; linkages between nucleosides may be comprised of phosphorus, nitrogen, sulfur, oxygen, carbon, or combinations thereof (e.g., phosphorothioate, peptide nucleic acids or PNA).
- the expression construct may be further comprised of a regulatory region for gene expression (e.g., promoter, enhancer, silencer, splice donor and acceptor sites, polyadeny- lation signal, cellular localization sequence) and, optionally, an origin of replication that allows chromosomal or episomal replication in a selected host cell.
- a regulatory region for gene expression e.g., promoter, enhancer, silencer, splice donor and acceptor sites, polyadeny- lation signal, cellular localization sequence
- an origin of replication that allows chromosomal or episomal replication in a selected host cell.
- the expression construct may be based on a general-pu ⁇ ose vector with at least one regulatory region from a mammalian gene (e.g., actin, hormone responsive element of glucocorticoid receptor, histone, metallothionein) or a virus (e.g., adenovirus, baculovirus, cytomegalo- virus, he ⁇ es virus, Moloney leukemia virus, mouse mammary tumor virus, Rous sarcoma virus, SV40 virus), as well as regions that facilitate engineering a polynucleotide or poly- peptide (e.g., selectable marker, linker with multiple recognition sites for restriction endo- nucleases, promoter for in vitro transcription, primer annealing sites for in vitro replication, consensus site for initiation of translation, recognition site mediating site-specific recombination, site for proteolytic cleavage or post-translational modification).
- a mammalian gene e.g., actin
- Advantages of such clones or constructs include ease of genetic or proteomic mani- pulation, abundance of additional copies of polynucleotide or polypeptide, convenience of producing fragments and fusions thereof, and ability to shuttle among different host cells or organisms.
- telomeres and constructs like any other recombinant molecule, are known in the art and typically involves enzymes, such as Taq polymerase, DNA and RNA polymerases, DNA and RNA ligases, restriction endonucleases, SI nuclease, glyco- sylase, reverse transcriptase, and ribonuclease H. See Kornberg and Baker, DNA Replication, Freeman, 1991.
- the recombinant molecule may be transfected into a host, selected positively or negatively, and further manipulated. Examples of drugs for which selectable marker genes exist are ampicillin, hygromycin, kanamycin/neomycin, puromycin, and tetracycline.
- a metabolic enzyme e.g., dihydrofolate reductase, thymidine kinase may be used as a selectable marker in sensitive host cells or auxotrophs.
- Vectors, reagents, and other supplies are commercially available. See, for example, the catalogs and product information contained therein of Amersham Pharmacia Biotech, Biol 01, Bio-Rad, CLONTECH, Invitrogen, Molecular Probes, New England Biolabs, Novagen, PharMingen, Pierce Chemical, Promega, Roche Molecular Biochem- icals, Sigma-Aldrich, Stratagene, and United States Biological.
- a host cell may be transfected with an expression construct comprised of the polynucleotide of the invention.
- the host cell may be a human primary culture or cell line, bacterium, yeast, other fungus, insect cell, plant cell, rodent cell, cell in a primary culture, established cell line, somatic cell, or stem cell.
- a heterologous promoter may be used to regulate expression in a host cell or transgenic animal. See No et al. (1996); Rivera et al. (1996); Allgood and Eastman (1997); U.S. Pat. Nos.
- Transgenic and gene knockout non-human animals may be used to produce an animal model of diabetes or other pathologic conditions according to mechanisms disclosed herein for the effects of actively transcribing human glycogenin pseudogenes (e.g., ectopic expression of mutated host glycogemn sequences, normal human glycogenin and modulated pseudogene expression in a transgenic or knockout animal).
- human glycogenin pseudogenes e.g., ectopic expression of mutated host glycogemn sequences, normal human glycogenin and modulated pseudogene expression in a transgenic or knockout animal.
- the invention also provides primer pairs and other polynucleotides for use in amplifying polynucleotides (e.g., polymerase chain reaction or PCR, ligation chain reaction or LCR, transcription-mediated amplification or TMA, other thermal cycling or isothermal reactions) and hybridization probes.
- a set of such primers may be selected and used for PCR assays to quantitate transcript abundance within cells.
- Oligonucleotide sequences may be selected using procedures such as those described in U.S. Pat. Nos. 5,556,749, 5,639,612, or others known to the skilled artisan.
- This set of primers will be specific for amplification of the glycogenin gene or pseudogenes, and can be used as a pair for PCR and RT-PCR amplification of DNA and RNA, respectively.
- a hybridization probe with a sequence complementary to at least one of the mutations disclosed herein preferably, the mutated position is located near the middle of an oligonucleotide probe
- One or more primer probes with a sequence complementary to at least one of the mutation disclosed herein preferably, the mutated position is located near the 3 'end of an oligonucleotide probe
- the present invention will be useful for development and utilization of primers and other polynucleotide probes to quantitate cognate RNA and DNA within cells. This information may then be used to correlate glycogen catabolism and metabolism, or diabetes and other related pathologic conditions, with glycogenin transcription activity or other modes of gene expression. Primers that specifically amplify sequences in the vicinity of the glycogenin genetic locus also serve as a sequence tagged site (STS) for human chromosome 3q24-25.
- STS sequence tagged site
- glycogenin nucleotide sequences Based on the glycogenin nucleotide sequences, other specific-binding molecule (e.g., antisense, ribozyme) can be used to detect gene expression. Alternatively, specific- binding molecules developed to detect glycogenin protein activity may be produced in or administered to an organism.
- the amino acid sequence of glycogenin antigen can be used for preparation of specific-binding molecules (e.g., polyclonal or monoclonal antibody, antibody fragment, humanized antibody, single chain antibody, phage hybrid protein or other members of a combinatorial library) for monitoring protein expression, affinity puri- fication, and functional studies.
- Antibody may be produced by immunizing an animal (e.g., chicken, goat, hamster, horse, mouse, rabbit, rat, sheep) with antigen.
- the immune response may be potentiated by immunoadjuvant, conjugation of antigen to a multivalent carrier, booster immunization, or combinations thereof.
- Antibody fragments may be prepared by proteolytic cleavage or genetic engineering; humanized antibody and single chain antibody may be prepared by transplanting sequences from the antigen binding regions of antibodies to framework molecules.
- Antigen may be full-length polypeptide or a fragment thereof. Algorithms to guide the selection of hybridization probes, oligonucleotide primers, polypeptide binding molecules, and antigenic peptides have also been implemented in computer software packages.
- Specific binding molecules may also be generally produced by screening a combinatorial library for a clone which specifically binds glycogenin antigen or mutants thereof (e.g., phage display library). See U.S. Pat. Nos. 5,403,484, 5,723,286, 5,733,743, 5,747,334, and 5,871,974. Preferred are molecules that specifically bind to mutant polynucleotide or polypeptide in preference to the native glycogemn gene, transcript, or enzyme to distinguish mutant from normal. For immunological screening methods, antibody preparations, either monoclonal or polyclonal, may be utilized. Polyclonal antibodies, although generally less specific, typically are more useful in gene isolation.
- Immunizing an animal may produce polyclonal antibody which recognizes multiple epitopes of the glycogenin antigen or at least one immunodominant epitope.
- Monoclonal antibody may be produced by fusing lymphocytes of an immunized animal with a myeloma or other immortalized cell, and selecting clones producing antibody that recognizes a desired immunogenic epitope or possesses a desired properties (e.g., specifically binding denatured, hydrolyzed, and/or truncated polypeptide instead of native enzyme, recognizing structural differences therebetween, precipitating glycogenin polypeptides or fragments thereof).
- the epitope bound may be present in the native polypeptide or only a denatured, hydrolyzed, and/or truncated form.
- a molecule able to specifically hybridize to a polynucleotide of the invention is also considered a specific binding molecule.
- Hybridization conditions are preferably chosen with a stringency that uniquely identifies the human glycogenin gene or pseudogenes in a population of genomic DNA or any of the human transcript in a population of cellular RNA. Conditions may also have to be chosen to distinguish human nucleotide sequences from those of other species by a physical criterion like sedimentation velocity or electrophoretic mobility. Alternatively, hybridization conditions may be relaxed to identify orthologs or paralogs in human or other mammalian species. Specific hybridization by such a molecule is also useful for monitoring gene expression, genetic profiling and finge ⁇ rinting, and functional studies.
- the specific binding molecule of the invention may be a chemical mimetic; for example, an aptamer or peptidomimetic. It is preferably a short oligomer selected for binding affinity and bioavailability (e.g., passage across the plasma and nuclear membranes, resistance to hydrolysis of oligomeric linkages, adsorbance into cellular tissue, and resistance to metabolic breakdown).
- the chemical mimetic may be chemically synthesized with at least one non-natural analog of a nucleoside or amino acid (e.g., modified base or ribose, designer or non-classical amino acid, D or L optical isomer).
- Modification may also take the form of acylation, glycosylation, methylation, phosphory- lation, sulfation, or combinations thereof.
- Oligomeric linkages may be phosphodiester or peptide bonds; linkages comprised of a phosphorus, nitrogen, sulfur, oxygen, or carbon atom (e.g., phosphorothioate, disulfide, lactam, lactone bond); or combinations thereof.
- the chemical mimetic may have significant secondary structure (e.g., ribozymes) or be constrained (e.g., cyclic peptides). Solid-phase synthesis is preferred to avoid representa- tional bias and to generate chemical diversity in making a library of non-natural mimetics. See, for example, U.S. Pat. Nos. 5,650,489 and 5,877,030. Cleavage from the solid support would produce a solution library or selectively release/retain the mimetic.
- the antibody is labeled using radioactivity or any one of a variety of second antibody/enzyme conjugate systems that are commercially available (e.g., alkaline phosphatase, ⁇ -galactosidase, horseradish peroxidase).
- Chemical staining may be used to detect polynucleotide (e.g., intercalators like acridine orange and ethidium bromide) or polypeptide (e.g., stains like amido black, dyes like coomassie brilliant blue).
- polynucleotide, polypeptides, and specific binding molecule are labeled for use as probes in assays of the invention.
- the probe is preferably labeled with a small molecule (e.g., biotin, chromochrome, colloidal gold, digoxygenin, dinitrophenol, fluorochrome, radio- isotope, spin label), fusion protein (e.g., avidin or biotin-binding analogs, immunogenic epitope), or enzyme (e.g., fluorescent proteins like GFP, hydrolases and transferases, kinases and phosphatases, luminescent proteins like LUC) to detect its presence.
- a small molecule e.g., biotin, chromochrome, colloidal gold, digoxygenin, dinitrophenol, fluorochrome, radio- isotope, spin label
- fusion protein e.g., avidin or biotin-binding analogs, immunogenic epitope
- enzyme e.g., fluorescent proteins like GFP, hydrolases and transferases, kinases and phosphatases, luminescent proteins like LUC
- Detection may involve, for example, chemical or physical phenomena like aggluti- nation or flocculation, autoradiography, chemiluminescence or electrochemiluminescence (ECL), calorimetry, colorimetry, electron spin resonance, charge or energy transfer; fluorescence polarization or quenching, liquid scintillation, nuclear magnetic resonance, surface plasmon resonance, and other spectroscopic measures of radiation adso ⁇ tion, emission, reflection, or refraction.
- ECL electrochemiluminescence
- nucleotide sequences may be defined by structural and/ or functional criteria.
- related nucleotide sequences derived from the human glycogenin nucleotide sequence may hybridize under stringent conditions known in the art.
- Suitable conditions for oligonucleotides of about 20 or about 50 bases could be 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C (see Beltz et al., 1983); suitable conditions for polynucleotides longer than 50 bases could be 500 mM NaHPO 4 pH 7.2, 7% (w/v) sodium dodecyl sulfate (SDS), 1% bovine serum albumin (BSA), 1 mM EDTA, 65°C (Church and Gilbert, 1984). Short, conserved peptide domains may be used to design amplification primers which probe for related nucleotide sequences (Gould et al., 1989).
- hybridization and washing conditions are described herein. Thus, relatedness may be found when there is similarity or identity of sequence and this may be determined by comparison of sequence information or through hybridization between a probe and a source (e.g., Southern or Northern blots, genomic or cDNA libraries).
- a source e.g., Southern or Northern blots, genomic or cDNA libraries.
- Nonsense mutations are easily produced by changing the nucleotide at each position between the native initiation of translation Met codon and the native termination of translation stop codon to the other three nucleotides (i.e., all bases except the base normally found at that position) and determining whether a premature stop codon was created in the normal open reading frame.
- frameshift mutations are easily produced by inserting any one of the four possible nucleotides (i.e., single insertion) or any one of the 16 possible dinucleotides (i.e., double insertion) at each position between the native initiation of translation Met codon and the native termination of translation stop codon and determining whether a premature stop codon was created in the normal open reading frame.
- Functional criteria for mutated sequences include effects of the mutant such as alterations in DNA replication, methylation, unwinding, topology, conformation, packing, or other higher-order structures above the linear sequence; competitive inhibition in or interference with RNA transcription, folding, turnover, transport, editing, splicing, or other processing; competitive inhibition in or interference with protein translation, degradation, proteolytic cleavage, reduction, glycosylation, phosphorylation, methylation, sulfation, acylation, other covalent modifications, translocation across a cell membrane, targeting to an organelle or other subcellular location, splicing, folding or achieving higher-order structures above the linear sequence, conformation, binding to another protein, substrate binding, autocatalytic or catalytic activity; or combinations thereof.
- a reference sequence according to the invention may be defined as a polynucleotide sequence, preferably at least 20 nucleotides in length, used as a basis for a comparison between sequences.
- the reference sequence may comprise a full-length cDNA or gene sequence in a sequence listing, or the reference sequence may comprise a portion of a full-length DNA or gene sequence.
- a comparison of the sequences of two polynucleotides may be restricted to a "comparison window" in order to determine sequence similarity in a local region of the two polynucleotides.
- a “comparison window” is defined to be a continuous series of at least 10 nucleotides in a first polynucleotide sequence, which when aligned with a reference sequence of at least 10 contiguous nucleotides, enables a comparison of the first polynucleotide and reference sequences.
- the segment of contiguous nucleotides in the "comparison window" of the first polynucleotide may comprise additions and/or deletions, such as, for example, gaps, of up to 20% of the reference sequence.
- Local homology or sequence identity within the "comparison window'Of the first polynucleotide and reference polynucleotide may be achieved via alignment of the sequences according to the algorithm of Smith and Waterman, or by another suitable alignment method or algorithm (Needleman and Wunsch 1970; Pearson and Lipman, 1988). Sequence alignment also may be objectively achieved and optimized by other known algorithms. These algorithms may be inco ⁇ orated into commercial computer packages and implemented using default parameters.
- Sequence identity may be established when the first polynucleotide and reference polynucleotide exhibit complete sequence similarity in a nucleotide by nucleotide basis over the span of the "comparison window".
- the invention contemplates polynucleotides having substantial identity with a reference polynucleotide, wherein a polynucleotide having substantial identity with the reference polynucleotide exhibits the same function as the reference polynucleotide.
- substantially identity is meant that a polynucleotide had nearly identical nucleic acid bases in a "comparison window" located in positions corresponding to those of the reference polynucleotide.
- the invention contemplates a reference sequence having a "comparison window" of at least 10 nucleotides (preferably 20-50 nucleotides), for example a gene comprising the sequence identity of SEQ ID NO:3, and a first, second or other sequence having at least 80% sequence identity to the gene comprising SEQ ID NO: 3, preferably at least 95% sequence identity, more preferably 97%, and most preferably 98%-99% sequence identity with SEQ ID NO.3.
- nucleotide sequence may show as little as 80% sequence identity, and more preferably at least 90% sequence identity, between the target sequence and the human polynucleotide excluding any deletions or additions which may be present, and still be considered related.
- Nucleotide sequence identity may be at least 95% and, most preferably, nucleotide sequence identity is at least 98% or 99%.
- compositions or extracts of the invention may be made substantially pure by controlled expression of the polynucleotide or polypeptide, and isolating same. Expression may be accomplished by extraction from natural sources, recombinant technology, or total or partial chemical synthesis.
- compositions containing a molecule are described as being at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 99%o pure by weight as compared to other substances (i.e., contaminants) of the same chemical character as the recited molecule (e.g., nucleotide, amino acid).
- purified polynucleotide or polypeptide could be assessed relative to the starting source (e.g., cytoplasm, nucleoplasm, cellular or nuclear lysate, cellular or tissue extract) from which purification was initiated.
- such compositions are reduced by at least 95% of the initial number of intact cells (i.e., 95%) free).
- a substantially cell-free composition is reduced by at least 99% of the initial number of intact cells, and a reduction of at least 99.99% may also be achieved.
- Compositions may also be cleared so that they are substantially free of membranes or other coated structures (reduced by at least 95% of the initial content by weight).
- heterologous polynucleotide regions or polypeptide domains may mean that some regions/domains are not found in the same species in nature (e.g., a human polynucleotide encoding glycogenin and a prokaryotic promoter).
- heterologous polynucleotide regions or polypeptide domains may mean that the regions/domains are not found joined together in nature (e.g., human glycogenin structural gene and tetracycline- or FK506-responsive regulatory region; human glycogenin polypeptide and epitope tag, signal for localization in the cell, or specific-binding domain).
- Ligation of polynucleotide regions or fusion of polypeptide domains occurs by inventive manipulation, such as by de novo synthesis or recombination.
- such joining may be preceded or followed by fragmentation (e.g., hydrolysis of a phosphodiester or peptide bond) through enzymatic (e.g., nuclease or protease) or chemical hydrolysis.
- a human polynucleotide or polypeptide it may mean that the polynucleotide/polypeptide was purified from a human source, has a sequence identical to a non-mutant human glycogenin gene or protein (in contrast to a pseudogene), shares a conformation with properly folded polynucleotide/ polypeptide, or is not denatured in its chemical structure.
- Binding is described as "specific" for binding which is able to discriminate human glycogenin polynucleotide or polypeptide from a mixture of other chemical substances which are not related to glycogenin. Processes of isolation, detection, and identification may depend on specific binding of human glycogenin polynucleotide or polypeptide in the mixture. The skilled artisan would be able to determine appropriate process conditions to achieve specific binding by choice of length of time, temperature, ionic strength, pH, addition of surfactant, pre-treatment (e.g., adso ⁇ tion, affinity purification, subtraction), and post-treatment (e.g., additional rounds of binding, signal amplification, washing).
- pre-treatment e.g., adso ⁇ tion, affinity purification, subtraction
- post-treatment e.g., additional rounds of binding, signal amplification, washing.
- binding specific for a glycogenin mutation in a polynucleotide- containing sample or a glycogenin mutant in a polypeptide-containing sample would detect the mutation or mutant preferentially in comparison to the normal polynucleotide or polypeptide. Binding could be quantitated by appropriate correction for the manner in which a sample was prepared or conditions of the reaction, comparison to a standard curve of known amounts or types of glycogenin, competitive or other assay formats, or combinations thereof. Washing and separation is facilitated by immobilization to an insoluble polymeric support (e.g., acrylamide, agarose. cellulose, nylon, plastics) or other supports such as glass.
- an insoluble polymeric support e.g., acrylamide, agarose. cellulose, nylon, plastics
- binding in solution has favorable kinetics
- prior or subsequent binding to a support allows increasing detection sensitivity and/or decreasing non-specific binding or background.
- the reaction components may be separated, non-binding components may be removed, and the support may be washed. Washing may also be facilitated by forming the support into a bilious strip with one or more zones; a well of a 96-, 192- or 384-well plate; a magnetic or non-magnetic bead; a chromatographic plate, resin, or column; or a porous or non-porous membrane.
- immobilization of a probe on a glass or silanized slide, nylon or nitrocellulose membrane, magnetic bead, or microtiter plate is preferred. Binding will result in capture of the solution species. Homogeneous assays will generally not require immobilization to a support.
- Isolation, detection, purification, or quantitation of mutated polynucleotide or mutant polypeptide may involve conventional techniques such as in vitro transcription, in vitro translation, Northern hybridization, reverse transcription-polymerase chain reaction (RT-PCR), run-on transcription, solution hybridization, nuclease protection, Southern hybridization, metabolic protein labeling, antibody binding, enzyme linked immunosorbent assay (ELISA), immunofluorescence, immunoprecipitation (IP), fluorescence activated cell analysis (FACS), radioimmunoassay (RIA), and Western blotting.
- ELISA enzyme linked immunosorbent assay
- IP immunofluorescence
- FACS fluorescence activated cell analysis
- RIA radioimmunoassay
- Western blotting The presence of mutated polynucleotide or mutant polypeptide may be assayed by use of a reporter whose product is easily assayed.
- reporter genes include alkaline phosphatase, ⁇ -galacto- sidase (LacZ), chloramphenicol acetyltransferase (CAT), ⁇ -glucoronidase (GUS), green fluorescent protein (GFP), ⁇ -lactamase, luciferase (LUC), or derivatives thereof.
- Such reporter genes would use cognate substrates that are preferably assayed by a chromogen, fluorescent, or luminescent signal.
- the assayed product may be tagged with a heterologous polypeptide epitope (e.g., FLAG, MYC, SN40 T antigen, glutathione-S- transferase or GST, oligohistidine, maltose binding protein or MBP) for which cognate antibodies or affinity resins are commercially available.
- a heterologous polypeptide epitope e.g., FLAG, MYC, SN40 T antigen, glutathione-S- transferase or GST, oligohistidine, maltose binding protein or MBP
- Changes in transcription may be detected qualitatively and/or quantitated with, for example, differential message display (U.S. Pat. ⁇ os. 5,459,037; 5,599,672; 5,665,544; 5,707,807; 5,807,680; 5,814,445; 5,851,805; and 5,876,932); subtractive hybridization (U.S. Pat. ⁇ os. 5,316,925; 5,643,761; 5,804,382; 5,830,662; 5,837,468; 5,846,721; and 5,853,991); computer-assisted comparison with an electronic database (e.g., U.S. Pat. No.
- differential message display U.S. Pat. ⁇ os. 5,459,037; 5,599,672; 5,665,544; 5,707,807; 5,807,680; 5,814,445; 5,851,805; and 5,876,932
- subtractive hybridization U.S. Pat. ⁇
- Biochips or microarrays oligo- nucleotides and/or oligopeptides arranged at high density on a solid substrate.
- biochips or microarrays oligo- nucleotides and/or oligopeptides arranged at high density on a solid substrate.
- Such reagents allow capture of a molecule in solution by a specific interaction between the cognate molecules and immobilization of the solution molecule on the solid substrate. See, for example, U.S. Pat. Nos. 5,143,854, 5,639,603, 5789,162, and 5,789,172.
- multiplex analyses to monitor gene expression employ simultaneous solution methods such as multi-probe ribonuclease protection assay or multi- primer pair polynucleotide amplification.
- glycogenin other genes or proteins which may be monitored are those involved in autoimmunity (e.g., pancreatic islet antigen, glutamic acid dehydrogenase); pharmacogenomics (e.g., cytochrome P450 genes); sugar catabolism and metabolism (e.g., insulin, insulin receptor, glucokinase); transcription of the aforementioned genes (e.g., HNF-l , HNF-l ⁇ , HNF-4 ⁇ , IPF-1, and IPF-1); and combinations thereof.
- autoimmunity e.g., pancreatic islet antigen, glutamic acid dehydrogenase
- pharmacogenomics e.g., cytochrome P450 genes
- sugar catabolism and metabolism e.g., insulin, insulin receptor, glucokina
- Nucleotide and amino acid sequences may be synthesized in situ on the substrate by solid-phase chemistry or photolithography. In situ synthesis attaches the nucleotides or amino acids directly to the substrate.
- the polynucleotide, polypeptide, or specific binding molecule may be attached by interaction of a specific binding pair (e.g., antibody-digoxygenin/hapten/peptide, biotin-avidin/streptavidin, GST-glutathione, MBP- maltose, polyhistidine-chelated nickel, protein A/G-Fc domain); crosslinking may be used if covalent attachment to the substrate is desired.
- a specific binding pair e.g., antibody-digoxygenin/hapten/peptide, biotin-avidin/streptavidin, GST-glutathione, MBP- maltose, polyhistidine-chelated nickel, protein A/G-Fc domain
- crosslinking may be used if covalent attachment to the substrate
- Glutaraldehyde is a covalent bifunctional crosslinker suitable for immobilization on a substrate, but a photoactivatable, reversible crosslinker is preferred to identify and isolate molecules interacting in a complex (e.g., a thiol linkage that may be reduced).
- Polynucleotides or polypeptides may also be delivered by replicating from a reference plate, dotting with a pen, or spraying from a reservoir and then attached to a nitrocellulose or nylon membrane.
- An overlapping set of polypeptides which define all possible linear epitopes of glycogenin may be arranged on a solid substrate to map the epitope specifically bound by a binding molecule (e.g., polyclonal or monoclonal antibody). See U.S. Pat. No. 5,194,392. Once a reactive epitope is defined, it may be used to isolate the specific binding molecule or to inhibit binding between glycogenin and the specific binding molecule. A polypeptide or specific binding molecule thereof may be used to establish a profiling reference panel. See U.S. Pat. Nos. 5,384,263, 5,541,070, and 5,798,275.
- the density of polynucleotides and/or polypeptides may be arrayed at a density of at least about 10 2 molecules per cm 2 , about 10 3 molecules per cm 2 , about 10 4 molecules per cm 2 , or about 10 5 molecules per cm 2 .
- the array may contain at least 10 different sequences, 10 2 different sequences, 10 3 different sequences, 10 4 different sequences, 10 5 different sequences, or 10 6 different sequences. Some or all of the arrayed molecules have sequences which correspond to a mutation of a glycogenin pseudogene or its product (e.g., 5, 10).
- SNPs single nucleotide polymo ⁇ hisms
- Total RNA was isolated from white blood cells of healthy control and Type 2 diabetic individuals, and was transcribed by reverse transcriptase using nucleic acid primers derived from the 5' and 3' ends of the glycogenin gene and amplified by RT-PCR.
- the forward 5'-end primer (ACAGATCAGGCCTTTGTGAC, SEQ ID NO:20) and the reverse 3'-end primer (CTGGAGGTAAGTGTCGTCAAGTTTCC, SEQ ID NO:21) were synthesized using the TITANTM single tube RT-PCR system of Roche Molecular Bio- chemicals according to the manufacturer's protocol.
- DNA was isolated from a single bacterial colony transfected by the recombinant clone with a WIZARD DNA purification kit from Promega following the manufacturer's methodology, and then sequenced using the AMPLI CYCLE sequencing kit from Perkin Elmer according to the manufacturer's instructions.
- the RT-PCR products of Example I were cloned to the TA CLONING vector, manually sequenced by the dideoxy chain termination, and separated by polyacrylamide thin gel electrophoresis.
- the entire cDNAs isolated from three Type 2 patients and from two healthy subjects were sequenced.
- One clone from each of the three Type 2 patients presented numerous mutations in glycogenin cDNA, while preserving almost 90% of sequence identity compared to normal glycogenin cDNA.
- no such mutations were found in one colony isolated from each of two healthy human subjects.
- the presence of cDNA mutations generally implies that mutations are present in corresponding mRNA. The most frequent mutations were silent, but also present were several premature stop codons.
- An addi- tional stop codon is due to a single adenine insertion in the triplet AGC coding for Ser- 171, causing a shift in the reading frame, followed by a second adenine insertion in the triplet ATC coding for isoleucine at position 178 (Ile-178), creating the TAA stop codon.
- At least two of the mutations described lead to expression of a protein lacking Tyr- 19, to which the first glucose unit of glycogen normally attaches. See Figure 3 which shows the sequences in the context of the rest of the human glycogenin gene or related pseudogenes as SEQ ID OS:3-19.
- RT reverse transcription
- PCR polymerase chain reaction
- RNA transcripts Their detection as RNA transcripts indicates that the glycogenin pseudogenes in the human genome are actively transcribed.
- the numbers in parentheses correspond to total numbers of analyzed subjects (top horizontal row) and the subject carrying a particular set of mutations. The mutations are described in more detail below and in Figure 3 where they are displayed in the context of the glycogenin sequence.
- glycogenin pseudogenes i.e., sequences with premature stop codons
- Type 2 diabetes is not unqualified.
- RNA transcripts were further investigated using the standard procedures of colony lifting, hybridization, and dot blotting which are known in the art (Table 3). Probes labeled with 32 P were used to detect cDNA obtained from RT-PCR of human lymphocyte mRNA from Type 2 patients. Three probes were constructed, one corresponding to the normal glycogenin sequence (AGTAT- GCCTTGGTCCCCACCA, SEQ ID NO:22) and two corresponding to the same region but carrying pseudogene mutations (AGTAAGACTTGGTCTCCACTG for probe A and CGT- ATGCCTTGTTCTCCACCA for probe B, SEQ ID NO:23-24, respectively).
- Type 2 405 20.5 58.5 30.0
- Type 2 392 11.0 13.5 75.5
- the silent mutation gives rise to three genotypes, one of which is found in 11 ) of Type 2 diabetic patients studied, but not in any control subjects.
- the normal triplet can be either CCG (termed G) or CCA (termed A) and the mutation creates three genotypes: G/A, G/G and A/A. Differences between the two subject types in terms of the relative frequency of occurrence of the genotypes are shown in Table 4.
- a skeletal muscle biopsy from a Type 2 diabetic patient was analyzed for the presence of nonsense mutations (i.e., the creation of a stop codon). Skeletal muscle was chosen for sampling due to the reduced muscle glycogen levels associated with hyperglycemia in Type 2 patients.
- cDNA sequences isolated from the muscle biopsy generated 20 clones, three of which had a mutated stop codon in the triplet TGG coding for T ⁇ -89, followed by two adenine insertions.
- Another clone displayed a deletion of 12 base pairs coding for Ser-Phe-Asp-Gly located at positions 157-160 of the human glycogenin amino acid sequence, a mutation not observed when leukocyte cDNA sequences were analyzed. Sixteen clones from the muscle biopsy were normal.
- a simple blood test would be desirable for detection of abnormal glycogenin expression related to the observed pseudogene expression.
- leukocyte proteins were analyzed for the expression of truncated glycogenin proteins. No truncated glycogenin proteins were identified by Western blotting, using polyclonal antibodies against recombinant skeletal muscle glycogenin. These results suggest that truncated glycogenin proteins were not present in the blood samples tested.
- RNA transcripts Both normal subjects and diabetic patients have genomic DNA with pseudogenes that are indistinguishable from our analyses. Extrapolating from the central dogma of molecular biology (i.e., DNA ⁇ RNA ⁇ protein), this implies that any difference would be attributable to the RNA or protein produced if the effect is not due to DNA.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
L'invention concerne des acides nucléiques comportant des séquences nucléotidiques liées à des pseudogènes de glycogénine, des méthodes permettant de détecter l'expression de ces pseudogènes par leurs mutations spécifiques, ainsi que des méthodes permettant d'utiliser cette expression pour identifier des sujets présentant une prédisposition au diabète sucré de type 2.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13809699P | 1999-06-08 | 1999-06-08 | |
US60/138,096 | 1999-06-08 | ||
US15060399P | 1999-08-26 | 1999-08-26 | |
US60/150,603 | 1999-08-26 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2000075294A1 true WO2000075294A1 (fr) | 2000-12-14 |
Family
ID=26835861
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2000/015685 WO2000075294A1 (fr) | 1999-06-08 | 2000-06-08 | Glycogenine, pseudogenes de glycogenine et leurs utilisations pour la determination des risques de developper le diabete de type ii |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2000075294A1 (fr) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998047527A1 (fr) * | 1997-04-21 | 1998-10-29 | Advanced Research & Technology Institute | Compositions a base de glycogenine-2 et procedes correspondants |
WO1999014374A2 (fr) * | 1997-09-18 | 1999-03-25 | Joslin Diabetes Center | Procede de determination de susceptibilite genetique a une nephropathie diabetique |
-
2000
- 2000-06-08 WO PCT/US2000/015685 patent/WO2000075294A1/fr active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998047527A1 (fr) * | 1997-04-21 | 1998-10-29 | Advanced Research & Technology Institute | Compositions a base de glycogenine-2 et procedes correspondants |
WO1999014374A2 (fr) * | 1997-09-18 | 1999-03-25 | Joslin Diabetes Center | Procede de determination de susceptibilite genetique a une nephropathie diabetique |
Non-Patent Citations (7)
Title |
---|
BARBETTI ET AL: "The human skeletal muscle glycogenin gene: cDNA, tissue expression, and chromosomal location", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 220, 1996, pages 72 - 77, XP002078436 * |
DATABASE EMHUM1 EMBL; 1 September 1999 (1999-09-01), ROACH P J ET AL.: "Isoforms of glycogenin-1, the self-glycosylating initiator of glycogen synthesis", XP002149902 * |
DATABASE GENBANK NCBI; 3 June 1994 (1994-06-03), LEFFERS H: "H. sapiens mRNA for glycogenin", XP002149901 * |
HANSEN L ET AL.: "Mutational analysis of the coding regions of the genes encoding protein kinase B-alpha and -beta, phosphoinositide-dependent protein kinase-1, phosphatase targeting to glycogen, protein phosphatase inhibitor-1, and glycogenin: lessons from a search for genetic variability of the insulin-stim... etc.", DIABETES, vol. 48, no. 2, February 1999 (1999-02-01), pages 403 - 407, XP000946889 * |
LOMAKO J ET AL.: "The human intron-containing gene for glycogenin maps to chromosome 3, band q24.", GENOMICS, vol. 33, no. 3, 1 May 1996 (1996-05-01), pages 519 - 522, XP000946882 * |
LOMAKO, J. ET AL: "The possible role of glycogenin in the etiology of type 2 diabetes", NUCLEIC ACIDS SYMP. SER. 38 (ADVANCES IN GENE TECHNOLOGY: MOLECULAR BIOLOGY IN THE CONQUEST OF DISEASE), vol. 38, 1998, pages 77 - 78, XP000956289 * |
ROACH P J ET AL: "Self-glycosylating initiator proteins and their role in glycogen biosynthesis", PROGRESS IN NUCLEIC ACID RESEARCH AND MOLECULAR BIOLOGY, vol. 57, 1997, pages 289 - 316, XP002910362 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6448149B2 (ja) | 肝線維症に関連する遺伝的多型、その検出方法および使用 | |
JP3372044B2 (ja) | 薬物の貧メタボライザーの検出 | |
JP2018057395A (ja) | 心筋梗塞に関連する遺伝的多型、その検出方法および使用 | |
US20040096885A1 (en) | Loci for idiopathic generalized epilepsy, mutations thereof and method using same to assess, diagnose, prognose or treat epilepsy | |
US5879884A (en) | Diagnosis of depression by linkage of a polymorphic marker to a segment of chromosome 19P13 bordered by D19S247 and D19S394 | |
Tolan | Molecular basis of hereditary fructose intolerance: mutations and polymorphisms in the human aldolase B gene | |
JP2003529315A (ja) | 病気の診断および治療のための成分および方法 | |
JP2009520460A (ja) | 心筋梗塞に関連する遺伝的多型、その検出方法および使用 | |
US6551812B1 (en) | Compositions and methods relating to the peroxisomal proliferator activated receptor-α mediated pathway | |
US6297014B1 (en) | Genetic test to determine non-responsiveness to statin drug treatment | |
US6586175B1 (en) | Genotyping the human UDP-glucuronosyltransferase 2B7 (UGT2B7) gene | |
CA2243191A1 (fr) | Procede et appareil de diagnostic | |
US20070277251A1 (en) | Diagnosis and Prediction of Parkinson's Disease | |
KR20200024772A (ko) | B4galt1 변이체 및 이의 용도 | |
EP1203827A2 (fr) | Polymorphismes dans le gène humain de KDR | |
EP1194595A1 (fr) | Polymorphismes du gene de hmg-coa reductase humaine | |
SK5972003A3 (en) | Genetic test for the identification of carriers of complex vertebral malformations in cattle | |
US6242181B1 (en) | Methods for diagnosing hypertension by detecting a mutation in the human G protein β3 subunit gene | |
US6942967B1 (en) | Target for treating athersclerosis, obesity and type II diabetes | |
KR20060116825A (ko) | 바이러스 감염에 대한 저항성과 관련된 유전자인oas1에서의 돌연변이의 검출 | |
US20040086886A1 (en) | Polymorphisms associated with cardiac arrythmia | |
WO2000075294A1 (fr) | Glycogenine, pseudogenes de glycogenine et leurs utilisations pour la determination des risques de developper le diabete de type ii | |
Seyrantepe et al. | Identification of mutations in the galactose‐1‐phosphate uridyltransferase (GALT) gene in 16 Turkish patients with galactosemia, including a novel mutation of F294Y | |
EP1206578A2 (fr) | Polymorphisme de bdnf et association avec la maladie affective bipolaire | |
US8518644B2 (en) | Method of judging inflammatory disease by using single nucleotide polymorphism |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): CA JP |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
122 | Ep: pct application non-entry in european phase | ||
NENP | Non-entry into the national phase |
Ref country code: JP |