WO1992012987A1 - Locus genetique ugt1 et presence d'une mutation - Google Patents

Locus genetique ugt1 et presence d'une mutation Download PDF

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WO1992012987A1
WO1992012987A1 PCT/US1992/000282 US9200282W WO9212987A1 WO 1992012987 A1 WO1992012987 A1 WO 1992012987A1 US 9200282 W US9200282 W US 9200282W WO 9212987 A1 WO9212987 A1 WO 9212987A1
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hug
dna
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bilirubin
crigler
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Ida S. Owens
Joseph K. Ritter
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The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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    • C—CHEMISTRY; METALLURGY
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    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the subject invention relates to the isolated gene locus UGT1, a family of UDP- glucuronosyl transferase isozymes encoded by this locus, and to the uses of both the locus as well as the isozymes themselves.
  • the locus encodes at least six transferase isoforms two of which metabolize bilirubin.
  • the six isoforms share four common exons.
  • the present inventors have discovered that in a patient having Crigler-Najjar Syndrome (CN) Type I, a mutation is present in the second common exon.
  • CN Crigler-Najjar Syndrome
  • Bilirubin an extremely lipophilic-like and toxic metabolite formed by the ongoing turnover of hemoproteins (most significantly hemoglobin) is normally metabolized to excretable derivatives by conjugation of one or both of the carboxyl groups of its propionyl side chains (C-8 or C-12) with glucuronic acid (Ostrow et al., Biochem. J. 120:311- 327 (1970)). Both the formation of the C-8 or C-12 monoglucuronide, or the C-8, C-12 diglucuronide, is catalyzed by hepatic bilirubin UDP-glucurono- syltransferase (Ritter, et al., J. Biol. Chem. 266:1043-1047 (1991)). Inadequate bilirubin glucuronidation is commonly observed in neonates (Roy Chowdhury, et al. in The Metabolic Basis of
  • Unconjugated bilirubin in cholestatic liver diseases is associated with the accumulation of serum bilirubin and the appearance of the clinical features of jaundice.
  • Coincident with the continuous and marked elevations (>20 mg%) in serum bilirubin concentrations seen in serious diseased states is the risk of kernicterus, the degenerative changes associated with bilirubin deposition in the brain (Roy Chowdhury, et al. 1367-1408 (1989)) in The Metabolic Basis of Inherited Disease, supra (1989) .
  • Bilirubin glucurono- syltransferase Deficient activity of bilirubin glucurono- syltransferase has been described in each of the three unconjugated hyperbilirubinemias.
  • Bilirubin glucuronosyltransferase is absent in Crigler-Najjar syndrome, Type I and is partially deficient in Crigler-Najjar syndrome. Type II, and Gilbert's syndrome.
  • Crigler-Najjar syndrome In contrast to patients suffering from Gilbert's syndrome, most patients suffering from Crigler-Najjar syndrome, Type I, die between the ages of 3 and 20. This syndrome is characterized by chronic nonhemolytic unconjugated hyperbilirubinemia in which hepatic bilirubin UDP-glucurono- syltransferase activity is absent (Crigler et al., Pediatrics 10:169-180 (1952)). Crigler Najjar syndrome (CN) Type I, is inherited in an autosomal recessive pattern (Childs et al.. Pediatrics. 23:903 (1959); Szabo et al., Acta Paediatr. Hung.. 4:153 (1963); and Arias et al..
  • the cause of death is the deposit of unconjugated, lipid-soluble bilirubin in the gray matter of the Central Nervous System, leading to seizures, kernicteric damage, and ultimately death.
  • the only effective long-term treatment that has been used successfully in patients with Crigler-Najjar syndrome, Type I, is liver transplantation, although this procedure is not without risk in these individuals.
  • the fatalities associated with this condition confirm the need to detoxify bilirubin. Characterization of the bilirubin transferase gene is, therefore, critical to making progress in the development of therapies for the Type I disease.
  • Crigler-Najjar syndrome Type II
  • Crigler-Najjar syndrome Type II
  • Administration of phenobarbital or other microsomal enzyme inducers is known to reduce the hyperbilirubinemia in Crigler- Najjar syndrome, Type II.
  • the present invention relates to an isolated gene locus, referred to as UGTl, comprising the nucleotide sequence shown in Figure 1. Within this locus are 6 transcriptional units or DNA segments referred to as UGT1A, UGT1BP, UGT1C, UGT1D, UGT1E, and UGT1F. Each segment encodes a unique transferase isoform.
  • the present invention also encompasses recombinant DNA molecules comprising a vector and exon 1 of each of the 6 DNA segments.
  • Another aspect of the invention relates to a DNA segment that codes for a polypeptide having an amino acid sequence corresponding to human bilirubin UDP-glucuronosyltransferase type I or to human bilirubin UDP-glucuronosyltransferase type II.
  • the present invention also includes the human liver cDNA segment HUG-Brl, ATCC accession under 68510, and the human liver cDNA segment HUG- Br2, ATCC accession under 68509.
  • the present invention encompasses recombinant DNA molecules comprising all of the DNA segments referred to above, as well as procaryotic or eukaryotic cells transformed or transfected with these recombinant DNA molecules.
  • the present invention also includes uses of the above-described clones or DNA segments.
  • the invention relates to a diagnostic probe of at least 18 bases for the diagnosis of a syndrome selected from the group consisting of Gilbert's syndrome, Crigler-Najjar syndrome Type I, and Crigler-Najjar syndrome Type II comprising a specific sequence of a cDNA encoding a mammalian bilirubin UDP-glucuronosyltransferase enzyme, mutant form, or variant thereof, said diagnostic probe identifying the corresponding mammalian genomic DNA.
  • the invention also encompasses, in particular, a diagnostic probe for the detection of Crigler-Najjar. Type I syndrome, wherein said probe comprises a DNA sequence corresponding to the 13-bp nucleotide deletion present in exon 2 of the UGTl complex.
  • the invention also relates to a diagnostic probe of at least 18 bases for the diagnosis of a syndrome selected from the group consisting of Gilbert's syndrome, Crigler-Najjar syndrome Type I and Crigler-Najjar syndrome Type II comprising a DNA sequence having sufficient homology to a cDNA encoding a mammalian bilirubin UDP-glucurono ⁇ syltransferase form, mutant form, or variant thereof to identify the mammalian genomic DNA corresponding to said cDNA.
  • a syndrome selected from the group consisting of Gilbert's syndrome, Crigler-Najjar syndrome Type I and Crigler-Najjar syndrome Type II comprising a DNA sequence having sufficient homology to a cDNA encoding a mammalian bilirubin UDP-glucurono ⁇ syltransferase form, mutant form, or variant thereof to identify the mammalian genomic DNA corresponding to said cDNA.
  • the invention also includes a PCR primer pair designed to amplify a specific portion of mammalian genomic DNA, wherein each member of said primer pair is from 17 to 20 bases, and further wherein each member of said primer pair comprises a specific sequence of a cDNA encoding a mammalian bilirubin UDP-glucuronosyltransferase form, mutant form, or variant thereof.
  • the invention includes a diagnostic assay for a syndrome selected from the group consisting of Gilbert's disease, Crigler— Najjar syndrome Type I and Crigler-Najjar syndrome Type II comprising the steps of:
  • PCR product would be expected from template genomic DNA from a normal individual and from both heterozygous parents, but not from genomic DNA from the homozygous defective individual. The failure to generate product with the homozygous defective genome is diagnostic.
  • the invention includes a diagnostic assay for the detection of Crigler-Najjar, Type I syndrome in a patient comprising the steps of: a) altering one of a pair of normal PCR primers such that said altered primer contains the deletion mutation present in exon 2 of UGT1F of the genome of Crigler-Najjar, Type I patients; b) adding said primers to a DNA sample of a patient suspecting of having said syndrome; and c) determining whether hybridization occurs such that a PCR product results, lack of said product indicating presence of Crigler-Najjar. Type I syndrome.
  • the present invention includes the a ino acid sequences as shown in Figure 9.
  • One sequence corresponds to human bilirubin UDP- glucuronosyltransferase, Type I (HUG-Brl) .
  • the other sequence corresponds to human bilirubin UDP- glucuronosyltransferase, Type II (HUG-Br2) .
  • Figure 1 Nucleotide sequence of the exons, intron-exon injunctions, and 5'- and 3'- flanking regions of the human UGTl gene complex.
  • the exonic sequences (in capitals) were determined by using a nested sequencing strategy with double- stranded plasmid DNA as template as described in Ritter et al., supra (1990) and by comparison to the HUG-Brl, HUG-Br2, and HLUG Pl cDNA sequences.
  • the TATA boxes are underlined, and the polyadenylation consensus signal sequence is in bold.
  • FIG. 1 represents a schematic diagram of the UGTl gene locus showing the exon/intron arrangement.
  • UGTl is a complex of nested transcription units featuring multiple first exons of UGTIA through UGTIF and their associated promoters (right-angled arrows) , and four common exons (2-5) .
  • the solid black lines indicate intervening DNA.
  • the complex is not drawn to scale and covers at least 85-kb region of chromosome 2 (Harding et al., Ann. Hum. Genet. 54:17-21 (1990)).
  • First exons are spaced an average of 7-kb from each other and the four common exons are clustered in a 6-kb region at the 3' end of the complex.
  • RNA B represents the predicted primary transcripts produced from the UGTl gene locus. Wavy black lines indicate segments of RNA excised from the transcript during splicing and polyadenylation.
  • the RNAs encode a family of UDP-glucurono- syltransferases with unique amino termini (286-289 residues) and identical carboxyl termini (246 amino acids) .
  • the products of UGTIA and UGT1D are the bilirubin UDP-glucuronosyltransferases encoded by HUG-Brl and HUG Br2, respectively (Ritter et al., J. Biol. Chem. 266:1043 (1991)).
  • Figure 3 Detailed illustration of the
  • UGTl gene locus Three overlapping clones [44(39.8 kb) , 37-1 (43.1 kb) , and 42-1 (36 kb) ] were isolated from a leukocyte DNA cosmid library constructed in Sp cos2 (Hilor et al.. Nucleic Acids Res. , 15:9129 (1987)) and transformed into E. coli 490A cells. Approximately 2 x 10 5 colonies were analyzed by the Southern blot technique via hybridization to the unique region of either HUGBrl (1-470-bp fragment) or HUG-Br2 (1-450-bp fragment) (Ritter et al. , J. Biol. Chem.. 266:1043 (1991)).
  • the probes were labelled by random primed synthesis in the presence of [ ⁇ 32 P]dCTP. Hybridization conditions used were previously described (Ritter et al., J. Biol. Chem.. 266:1043 (1991)). Clones 44 and 37-1 hybridized to the HUGBr2-specific probe and not to the HUG-Brl- or to the common end-one. Clone 42-1 hybridized to the HUGBrl- and the 3' common end-[1836 to 2167 bp of the cDNAs] probes (Ritter et al., J. Biol. Chem. 266:1043 (1991)). The sites for 6 different restriction endonucleases and their relative positions among the three clones were determined as shown in Figure 3B.
  • B Positive subclones which hybridized to HUG-Brl-, HUG-Br2-, or to the common end-probes were mapped in greater detail and sequenced as previously described (Ritter et al. supra, (1991)).
  • B,C Four common exons (solid boxes) encoding the common region of the cDNAs and six exon Is of UGTIA to UGTIF (patterned boxes specifying uniqueness) were identified and are shown according to their distribution in the cosmid clones. Introns are shown as lines.
  • Endonucleases used were: Pstl (P) , EcoRI (E) , Xhol (Xh) , Xbal (X) , Clal (C) , BamHI (B) , Hindlll (H) , and Sail (S) .
  • Exon 1 of UGT1BP or UGT1C contains a Kpnl or a Sphl site, respectively, which was used to establish orientation.
  • E, Cap sites for the UGTIA, UGT1D, and UGTIF mRNAs were determined by primer extension.
  • the cap sites were predicted for the UGT1BP, UGT1C, hand UGTl mRNAs based on 90% sequence identity to exon 1 of UGT1D in the coding and in at least 200 bp of the flanking regions.
  • Each of the six unique exon Is contained a 5' TATA box ( ⁇ ) .
  • C According to the maps for endonucleases and sequence data, the UGTl gene locus kb.
  • FIG. 4 Autoradiogra s of Sanger nucleotide sequencing reactions of normal and CN, Type I patient DNA. Plasmid DNA containing the entire UGTl exon 2 from a normal (A) and the CN, Type I patient FB (B) was sequenced using the
  • Bluescript primer KS.
  • the sequence ladders were generated by electrophoresis through a denaturing 6% polyacrylamide gel, after which the gels were transferred to blotting paper, dried, and exposed overnight to X-ray film to generate the autoradiogram shown.
  • A, C, T and G are lanes corresponding to separate reactions with added dideoxy derivatives of ATP, CTP, TTP and GTP, respectively.
  • the boundary between the first intron and the second exon is shown.
  • the shaded vertical line designates the 13-bp segment in normal DNA which is deleted from the DNA of the CN, Type I patient.
  • N-termini (288 or 289 amino acids, respectively) and identical C-termini (246 amino acids) .
  • these proteins are predicted to be severely truncated; N and C, the amino and carboxyl termini of bilirubin transferase; CS (unknown function) and MAD (membrane anchoring domain) are conserved domains present in all known transferases.
  • FIG. 5 PCR analysis of genomic DNA from family members.
  • Samples of PCR reactions generated from genomic DNA (0.1 ⁇ g) of the CN, Type I individual, FB (Lane 3) , his parents (Lanes 2 and 4) , and an unrelated normal individual (Lane 1) with oligonucleotides PXG3 and J127 as primers were analyzed by electrophoresis through nondenaturing polyacrylmide (6%) for 5 hr. at 50V in 0.5X TBE.
  • the gel was stained with ethidium bromide, irradiated with UV, and photographed.
  • the primers amplify a fragment representing the last 45-bp of the first intron and the first 154-bp of exon 2.
  • Lane 5 is a sample of a control reaction in which
  • FIG. 6 A, Northern blot analysis of human liver mRNA and kidney and skin total RNA samples using UGTIA-, UGT1D-, and UGTIF-specific probes. Triplicate sets of two different liver mRNA (2 ⁇ g each) samples, and one kidney and one skin total RNA (25 ⁇ g each) samples were prepared for analysis according to published procedures. (Ritter et al., J. Biol. Chem.. 265:7900 (1990)). Corrections for differences in probe lengths were made in calculations of relative amounts of mRNA. A human B-actin probe was hybridized (Church et al., Proc. Natl Acad.
  • Figure 7 The conjugation of glucuronic acid (donated by UDP-glucuronic acid) to bilirubin- iX ⁇ to produce water-soluble IX ⁇ C12 Bilirubin- ⁇ - glucuronide.
  • Figure 8 Three different Bilirubin- ⁇ - glucuronide conjugates: bilirubin-IX ⁇ C-8 monoconjugate, bilirubin-IX ⁇ .C-12 monoconjugate, and bilirubin-IX ⁇ dico jugate.
  • Figure 9 The nucleotide and deduced amino acid sequences of HUG-Brl and HUG-Br2. Nucleotide and deduced amino acid residues in HUG- Brl which differ from HUG-Br2 are shown in reverse font. The start and stop codons are designated by open boxes. Putative membrane-insertion signal and membrane-anchoring peptides are indicated by dashed and solid lines, respectively. Predicted asparagine-linked glycosylation sites are denoted by solid triangles and consensus polyadenylation signals by dashed boxes.
  • Figure 10 (Panel A) Hybridization of HUG-Brl and HUG-Br2 to liver mRNA isolated from normal human and from a Crigler-Najjar syndrome patient. Duplicate sets of mRNA samples (2 ug each) from two normal (N) and a type I Crigler-Najjar patient (CN) were electrophoresed as described (Ritter, et al. J. Biol. Chem. 265: 7900-7906
  • the TLC plates were exposed to X-ray film (10 days) .
  • Panels A and B represent two different experiments. Bilirubin glucuronides formed by mouse microsomes (2.9 mg) in the presence (A, Lane 3) and absence (A, Lane 4) of UDP-glucuronic acid are shown. Sensitivity of the reaction product generated in 6.5 hr. to glucuronidase treatment for 1 hr. at 37°C is shown in A, Lane 2.
  • the methyl esters of bilirubin corresponding to the IX ⁇ C8 and the IX ⁇ C12 isomeric monoglucuronides (BMEs) , the diglucuronide (BDE) , and unconjugated bilirubin (Br) are designated.
  • the rank in mobility is: Br > IX ⁇ C8 > IX C12 > IX ⁇ C8 ,IX ⁇ C12.
  • a (Lane 1) and B (Lane 2) Cells transfected with pHUG- Br2 or pHUG-Brl, which contained the insert in the correct orientation with respect to the promoter element in pSVL, are shown in A (Lane 1) and B (Lane 2) , respectively. Cell homogenates were assayed as described for microsomes. Control homogenate from cells transfected with pHUG-Brl in the reversed orientation is shown in B (Lane 3) . The sensitivity of the product generated by HUG-Brl-encoded activity to B-glucuronidase is shown in B, Lane 1. Unconjugated bilirubin is more sensitive to oxygen than its glucuronide or methyl ester derivatives. and the oxidized bilirubin will remain at the origin in this system. A nonspecific band (NS) is present in each reaction.
  • NS
  • FIG. 12 A comparison of the amino acid sequences of various UDP-glucuronosyltransferase forms: rat phenol UDP-glucuronosyltransferase (Rat PHENOL) , human phenol UDP-glucuronosyltransferase (HLUGP1) , human bilirubin UDP-glucurono ⁇ syltransferase, type I (HUB-BR1) , human bilirubin, type II (HUG-BR2) , and rat bilirubin UDP- glucuronosyltransferase (Rat Bil) .
  • the carboxyl terminus of the various UDP- glucuronosyltransferase forms is conserved as between the human and rat species and the phenol and bilirubin acceptor substrate forms.
  • the human forms, HLUGP1, HUG-Brl and HUG-Br2 show exact homology between amino acids 283-531, 285-533, and 286-534, respectively.
  • Rat PHENOL and Rat Bil show exact homology between amino acids 281-529 and 283-531, respectively. Further, there is a 90.6% conserved amino acid sequence between the two species.
  • the subject invention relates to the UGTl locus.
  • the nucleotide sequence of UGTl is shown in Figure 1.
  • UGTl is a unique gene complex featuring six nested transferase transcriptional units, which are depicted in Figure 2 (panel A) . Each of these units, UGT1A-UGT1F, encodes a unique transferase isoform.
  • the complex contains a tandem array of six promoters (Fig. 2A, arrows) with each positioned adjacent to a first exon (Fig. 2A, designated boxes, «880-960-bp each) , thereby defining the transcriptional start site for the unit.
  • the six unique amino acid termini of 286- 289 amino acids are encoded by the six different first exons and identical carboxyl termini of 246 amino acids are encoded by the common exons 2-5. It is thought that critical mutations introduced into any of the common exons 2-5 will result in the inactivation of all encoded isoforms. Alternatively, a critical mutation in exon 1 or a specific regulatory region can generate a specific defect in that encoded isoform or responsiveness to drug treatment, respectively. Mutations in sequences necessary for RNA splicing (e.g., upstream of a common exon) can generate a defect in all the transferase isoforms.
  • liver microsomal bilirubin UDP glucuronosyltransferase is necessarily composed of at least two distinct isozymes encoded by the cDNAs HUG-Brl and HUG-Br2 (Ritter et al., J. Biol. Chem. 266:1043-47 (1991)) which are encoded by the UGTl locus.
  • the present invention relates to the isolation and characterization of these two human liver bilirubin UDP-glucurono ⁇ syltransferase cDNAs, referred to as HUGBr1 and HUG-Br2 (Ritter, et al., J. Biol. Chem. 266:1043-1047 (1991)) which, upon expression individually in COS-1 cells, encode isoforms that catalyze the formation of the two bilirubin monoglucuronides and the diglucuronide.
  • HLUGP1 The phenol isozyme specifically conjugates a series of hydroxyl- substituted aromatic compounds.
  • HLUGP1, HUG-Brl and HUG-Br2 have unique 5' ends (879, 882- and 912-bp) which encode the first 286 of 534, 288 of 533, and 289- amino acids of 534, respectively.
  • the identity in the 3' region of the 3 clones suggested that a gene arrangement exists such that primary transcripts containing unique exons are alternatively spliced to one or more common exons to account for the structural similarities. Sato et al. (Biochem. Biophvs. Res. Comm.
  • rat bilirubin transferase cDNA contains a 3* end identical to that of the rat phenol transferase cDNA (Iyanagi et al., J. Biol. Chem. 261:15607 (1986)).
  • the carboxyl-termini between rat and human isoforms are 90% identical.
  • Phenobarbital has been the agent for treating and/or diagnosing patients with certain defects with bilirubin glucuronidating activity.
  • the cDNAs provided the necessary probes for isolating, sequencing and describing the genes encoding the isoforms.
  • the purification and nucleotide sequencing of the genes encoding the bilirubin and phenol transferase isoforms and all flanking regions provide all the necessary information to describe and verify all critical and non-critical mutations which may lead to an impairment or total loss of bilirubin glucuronidating activity in affected individuals.
  • the gene complex arrangement with an independent promoter for each encoded isoform and the sharing of 4 common exons provide a basis for the human phenotypes seen with respect to impaired bilirubin and phenol transferase activities.
  • the sequence data of the normal gene allows one to amplify and sequence all relevant regions of a defective gene to determine alterations and to confirm the effect of the mutation on activity by altering the cDNA before expression in an appropriate host cell.
  • Prior to the present invention there was no available gene-based method for approaching or developing an analysis of abnormal detoxification of bilirubin in humans.
  • Primary messenger RNA transcripts are predicted by each unit as shown in Figure 2 (panel B) and are further predicted to contain different 5' termini but identical 3' termini.
  • each transcript undergoes differential splicing of the first exon to exon 2 (common exon) and then is further extended with exons 3, 4, and 5 (common exons) to produce mature RNAs approximately 2.6-kb in length.
  • UGTIA and UGT1D represent the transcriptional units which encode mRNAs corresponding to HUG-Brl and HUG-Br2 bilirubin transferase cDNAs prepared from mRNA isolated from normal human liver.
  • a third unit, UGTIF encodes a phenol-specific isoform (Dutton, Glucuronidation of Drugs and Other Compounds, CRC Press, Boca Raton, Fl. , pp. 3-78 (1980)) .
  • Exon 1 of UGTIA, UGT1D, and UGTIF is located 5.6-, 49-, and 73-kb, respectively, upstream of the 4 common exons (See Fig.
  • UGT1BP Three additional unique exon Is of UGT1BP, UGT1C, and UGT1E which are -90% identical to and of the same length as that of the HUG-Br2 exon (UGT1D) , but never before uncovered, were identified 19-, 38-, and 54-kb upstream of the 4 common exons (Fig. 3B and 3C) .
  • the first exons of UGT1C and UGT1E are predicted to encode the unique regions of proteins which are similar to the HUG-Br2 protein.
  • the first exon of UGT1BP has a frame-shift mutation which creates a premature stop codon and, presumably, a truncated protein. It is, therefore, designated a pseudogene (P) . Because of the 90% identity of the exons Is with that of UGT1D, it is possible that UGT1C and UGT1E encode bilirubin transferase isoforms with properties similar to that encoded by HUG-Br2.
  • Each of the 6 unique exon Is of UGTIA to UGTIF has an RNA polymerase II transcriptional promoter element, TATATATATATATAA, TAATTAA, or TATCAAA, upstream of the predicted cap site of the mRNA as detailed in Fig. 3C and 3E.
  • TATATATATATATAA TAATTAA
  • TATCAAA RNA polymerase II transcriptional promoter element
  • the presence of these elements at position - 37 to -23 bp (UGTIA) or -31 to -25 bp (all other exon Is) from the cap site is consistent with the typical location of TATA boxes.
  • the presence of individual promoters create the condition whereby each encoded isoform can have independent regulation and tissue-specific expression. Hence, an independent mutation in a promoter region can specifically affect the expression of that isoform.
  • Each exon 1 has a 3* exon/intron junction consistent with a donor splice site (data not shown).
  • the common exons 2, 3, and 4 contain an acceptor and donor splice site, common exon 5 contains only an acceptor site.
  • the model predicts the synthesis of at least 6 nested primary transcripts ranging from 15- to 95-kb (Fig. 3D) where each exon 1 is differentially spliced to the 4 common exons to generate 6 different mRNAs.
  • exons Is uncovered in this invention allow one to explain substrate specificity of the isoforms and to explain defects in humans where only bilirubin transferase activity is impaired and not phenol transferase activity.
  • UGT1C and UGT1E bilirubin transferase-like encoded isoforms
  • UGT1C and UGT1E are now known isoforms which detoxify bilirubin.
  • UGT1C and UGT1E are possibly (based on 90% identity to UGT1D) bilirubin transferase isoforms.
  • nucleotide oligos can be designed and applied to the diagnostic screening of this population of individuals.
  • Figure 6 shows that UGTIF mRNA is expressed in kidney and skin at levels equal to or higher than that in liver, whereas the mRNAs coding for UGTIA and UGTID are not detected in kidney and skin.
  • Human liver is the only known site for bilirubin transferase activity (Chowdhury et al., The Metabolic Basis of Inherited Diseases (Scriver et al., eds.) McGraw Hill, N.Y. pp. 1367-1408 (1989)).
  • the multiple initiation sites and splicing to produce alternative protein products have major consequences for cellular phenotype.
  • the liver selectively processes bilirubin for clearance through the biliary system, while phenol clearance is not restricted and takes place in many different tissues.
  • the presumed similarities in the chemical nature of bilirubin and phenol and/or the biological circumstances leading to the establishment and evolution of a single gene complex to encode detoxifying enzymes are not obvious.
  • the implication of this gene arrangement is that multiple bilirubin transferase isozymes have evolved to cope with and prevent the toxic effects of the ever-present waste product derived from he oprotein turnover. It is known that bilirubin represents the only biological toxin in mammals undergoing constant replenishment: a) from dying red blood cells and, b) from episodic increases due to red blood cell hemolysis.
  • the toxin most likely represents a powerful selective pressure to evolve an efficient detoxifying system.
  • Duplication events within the UGTID-like alleles have likely been in response to the need to expand the only mechanism for detoxifying this heme derivative.
  • the syndromes in humans attributed to mild elevations in serum bilirubin are in response to a modest decrease in bilirubin transferase activity.
  • This reciprocal relationship likely signals that the constitutive level of the enzyme is not in excess for metabolizing this endogenous toxin.
  • the inducibility of the mRNA encoiding the UGTID gene by phenobarbitol expands the glucoronidating capacity and provides greater flexibility to the organism in responding to environmental factors.
  • FIG. 4A shows the site leading to a change in its predicted translation.
  • the FB sequence would, therefore, be very different from that of a normal individual after codon 294.
  • Continuation of translation of the open-reading- frame shows a TGA stop signal after the 365th codon (illustrated schematically in Fig. 4B) ; thus, a 72-residue unrelated peptide is present after codon 294.
  • the bilirubin transferases represented by UGTIA and UGTID are predicted to be missing the final 240 amino acids of the wild type proteins, a region in the phenol/bilirubin transferase gene which is highly conserved across species (90% identity between rat and human) (Ritter et al. J. Biol. Chem. 266:1043-1047 (1991)). This region contains two smaller domains/motifs that have been recognized as highly conserved among transferase cDNAs encoding members of both major subgroups so far identified, the steroid and bilirubin/phenol subfamilies (Tephly et al., TIPS 11:276-279 (1990)).
  • the truncated proteins are predicted to contain a foreign 72- residue carboxyl terminus, resulting from the -1 shift in reading frame.
  • a 1-bp deletion with a similar consequence i.e., the introduction of a premature stop codon was described at the cDNA levels for phenol and bilirubin transferase mRNAs of the hyperbilirubinemic Gunn rat, considered a model of CN, Type I disease (Schmid et al., J. Clin.
  • Type I is a rare disorder which was first described in the literature in 1952 (Roy Chowdhury et al., In The Metabolic Basis of Inherited Disease, pp. 1367-1408 (1989)) and is often a consequence of consanguineous parents.
  • the heterozygosity and consanguinity of the parents are the basis of the homozygosity of FB at the UGT1*FB locus.
  • PCR assay used herein may be utilized as one in a series of diagnostic markers—among others to emerge from future characterization of the different defects associated with heritable hyperbilirubinemic disorders (e.g. CN, Type II or Gilbert's syndrome) .
  • Type I Although it is known that a 13-basepair deleteion accounts for the UGT1*FB CN, Type I case, the existence of multiple phenotypes associated with CN, Type I (Arias et al., Am. J. Med. 47:395-409 (1969) ; Robertson et al. J. Inher. Metab. Pis. 14:563-579 (1991)) supports the notion that multiple types of mutations in UGTl are involved.
  • Type I patients a) a single nucleotide change in common exon 2 which creates an amino acid change at residue 309, a conserved region of the molecule, and b) a codon deletion in exon 1 of UGTIA leading to the deletion of residue 170.
  • Van Es et al. (Van Es, et al., J. Clin. Invest. 85:1199-1205 (1990)) performed a biochemical and immunochemical analysis of four different (unrelated) CN patients with no detectable transferase activity toward bilirubin.
  • FB has been described in detail in example IV with confirmation that each parent's DNA is heterozygous for the defect.
  • two other genetic alterations have been determined, it has not been confirmed that either the single nucleotide change leading to an amino acid change or a codon deletion leading to an amino acid deletion generates a loss of enzyme activity, with the use of pairs of primers (at least 17 nucleotides in length) in the UGTl gene complex, one can amplify any target region of the genome from normal and defective DNA by the PCR methodology using both a Perkin-Elmer kit and a thermocycler, for example, to generate sufficient
  • Subclones will be sequenced by the method described above to establish sequence data for comparison to normal by using, for example, the IBI Pustell sequence analysis analysis software package. Each defect will provide a site for a diagnostic probe.
  • Each blot may be made in duplicate.
  • An approximately 18-base oligo reflecting the altered sequence in the Crigler-Najjar. Type I, as well as an approximately 18-base oligonucleotide reflecting this sequence in normal DNA can be made and each can be radiolabelled to at least 2 x 10 8 dpm/ug by T4 polynucleotide kinase and/or terminal transferase to be used as a probe.
  • Preliminary studies can be carried out to establish wash conditions for each normal and altered sequence (see Sambrook et al. , In Molecular Cloning-A Laboratory Manual) . Following hybridization, high stringency wash conditions (as established for the filters) can be carried out in combination with the test sample of DNA present on each filter.
  • the validity of the diagnostic probe can be established by the selective hybridization (under high stringency wash conditions) of each probe to target DNA containing a perfect match.
  • the PCR methodology could also be utilized diagnostically in a slightly different manner.
  • One of a pair of normal primers (not altered to match the defect) could be selected to include the defect in the genome. It would be predicted that PCR product could be generated from the genome of the normal individual and that of both heterozygous parents for the defect, but not that homozygous for the defect.
  • the failure to generate product would be diagnostic for a defect at the site of the specified primer.
  • the present invention can also be used with respect to gene therapy.
  • Crigler- Najjar Type I disease, a disease which is fatal for most patients between the ages of 3-20, could potentially be treated using the claimed invention.
  • Liver transplantation has been the only effective long-term treatment.
  • the present invention provides two cDNAs which encode two different bilirubin UDP-glucuronosyltransferases which can be used as agents for gene therapy in order to correct the defect in the expression of this activity.
  • gene therapy research is in its infancy, progress is being made with animal models.
  • the protocols relevant for the bilirubin transferase gene therapy model necessarily deal with liver.
  • amphotropic retroviral vectors which have been engineered to be non-disease producing are utilized as agents for the transmission of foreign DNA into cultured primary liver cells prepared from a portion of the liver from an affected animal (individual) . After the ex vivo gene replacement, the cells are injected into the spleen of the same animal. From this site, most of the cells migrate to and remain in the liver.
  • CN Type I Human Samples
  • Blood samples were collected from FB, a 4 year old male CN, Type I patient, his parents, and an unrelated normal female, AR. FB, the offspring of consanguineous parents, had average
  • transaminases alkaline phosphatases, 5'-nucleotidase, total bile acids, thyroid function tests, PT, PTT, G6PD screen, pyruvate kinase screen, ⁇ -antitrypsin levels, and various bacterial cultures.
  • Unconjugated hyperbilirubinemia persisted despite repeated exchange transfusions and phototherapy.
  • a liver biopsy at 6 weeks of age confirmed normal histology and the absence of bilirubin UDP-glucurono ⁇ syltransferase activity. Bilirubin glucuronides were absent in duodenal bile, serum, and urine.
  • the blood samples were transferred to 50-ml Falcon tubes and spun at 4000 rpm for 30 min in a HS-4 rotor (Dupont-Sorvall) .
  • the pellet was resuspended in 40 ml of prechilled RSB (10 mM Tris-Cl, pH 7.5, 10 mM NaCl, 3 mM MgCl, and 0.2%
  • Triton X-100 Triton X-100
  • SDS solution 10 mM Tris-Cl, pH 7.5, 20 mM NaCl, 5 mM MgCl, 2% SDS
  • Proteinase K 900 ⁇ g/10 ml volume blood was added, and the tubes were incubated at 37°C for 2 hr and then at room temperature overnight. The samples were then extracted five times with 1:1 volume of phenol:chloroform and dialyzed at 4°C overnight against 10 mM Tris, pH 7.5, 1 mM EDTA with several changes.
  • Oligonucleotide primer synthesis Oligonucleotides for PCR amplification of the four UGTl common exons [PXG3-PXG4 for exon 2, PXG5 and PXG6 for exon 3, PXG7 and PXG8 for exon 4, PXG9 and PXG10 for the protein coding region of exon 5] were synthesized using a Milligen/Biosearch Cyclone DNA synthesizer, Model 8400 (Milligen/Biosearch, Bedford, MA) and a 200-coupling ⁇ -phosphoamidite kit (Milligen/Biosearch) .
  • oligonucleotides and their sequences are as follows: PXG3 5'-CTATCTCAAACACGCATGCC-3' , PXG4 5'- GGATTAGCGCTCCTGTGAA-3' , PXG5 5'- GTCTTTCTTTACGTTCTGCTC-3' , PXG6 5'- GACCCTGGTTTGACCTATAC-3' , PXG7 5'- CTCAGAGATGTAACTGCTGAC-3' , PXG8 5'- CATGAATGCCATGACCAAAG-3 ' , PXG9 5 1 - GTTCATACCACAGGTGTTCCA-3' , PXG10 5'-GGAAATGACTAGGGAATGGTTC-3' , J127 5'- TCTGAGACCATTGATCC-3' .
  • the oligos were cleaved and deprotected with concentrated aqueous ammonia (29%) as suggested by the manufacturer, dried and resuspended in sterile water, re-precipitated with ethanol, and resuspended in 10 mM Tris, pH 8.0, 1 mM EDTA. Oligos were analyzed by electrophoresis through a 12% polyacrylamide gel.
  • Primer combinations PXG3 and PXG4, PXG5 and PXG6, PXG7 and PXG8, or PXG9 and PXGIO were used to amplify exons 2, 3, 4, or the first 312-bp of exon 5, respectively (along with 30- 50 bases, on average, of flanking intron.
  • Each PCR reaction contained genomic DNA (0.1 ⁇ g) , each of the designated primer combinations (lO ⁇ M each) , 10 mM Tris-HCl, pH 8.3 (at 25°C) , 50 mM KC1, 1.5 mM MgCl, 0.001% (w/v) gelatin (Sigma, Cat. No. G2500, St. Louis, MO), 0.2 mM each dNTP and 2.5 units of
  • Amplitaq polymerase Reaction mixes were overlaid with mineral oil (50 ⁇ l) and subjected to thirty cycles of 94°C for 1 min, 56°C for 1.5 min, and 72°C for 3 min, using a Perkin-Elmer Cetus thermocycler (Emeryville, CA) . The final extension step at 72°C was for 30 min. Using normal DNA, these primer combinations result in the amplification of fragments which are 311, 164, 309, and 398-bp in length for exons 2 through 5, respectively. PCR products were polished with Klenow, phosphorylated with T4 polynucleotide kinase, and subjected to electrophoresis through 1% low melting point agarose.
  • Amplitaq polymerase was from Cetus (Emeryville, CA) , T4 polynucleotide kinase from Bethesda Research Laboratories (BRL, Gaithersburg, MD) , and the Large Klenow fragment from U.S. Biochemical (Cleveland, OH). All restriction endonucleases were from either New England Biolabs (Beverly, MA) , Pharmacia-LKB (Piscataway, NJ) , or BRL. Calf intestinal alkaline phosphatase was from Boehringer-Mannheim
  • the vector pBluescript was purchased from Stratagene.
  • Example II Identification of a 13-Base Pair Deletion in Exon 2
  • the exons 2-5 (of Ex. I) were amplified individually using the polymerase chain reaction and subcloned as described in Example I. Analysis of exon 3, 4, and 5 subclones showed that each had the normal sequence (data not shown) . However, a 13-bp deletion (Fig. 4) was observed in each of four independent exon-2 subclones. To confirm the mutation, four additional exon 2 subclones were tested from a second PcR reaction, indicating that the mutation was not due to errors by Taq polymerase. The deletion is located 12-bp (downstream) from the intron 1/exon 2 junction (Fig.
  • UGT1A-, UGT1P-, UGTIF-, and common exon-probes were used to analyze a set of Southern blots containing genomic PNA digested with three different enzymes predicted not to cleave exon 1 of UGTIA, UGTIF, or exons 2, 3, 4, or 5 (the common ones) .
  • RNA was prepared from human adult liver biopsy and converted to double-stranded cDNA using the Boehringer Mannheim cDNA synthesis kit and oligo (dT) as primer.
  • the cDNA was ligated with EcoRI-Sall adaptors and cloned into the EcoRI site of lambda-ZAP bacteriophage (Stratagene, San Diego,
  • the probe was labeled to a specific activity of 0.22 X 10' dpm/ug by fill-in of overlapping sense and antisense oligonucleotides.
  • the probe was hybridized as described (Jaye et al., Nucleic Acids Research 11, 2325-2335 (1983)) except that 45°C and 30°c were the hybridization and wash temperatures, respectively. All manipulations of lambda-ZAP and excision of the Bluescript plasmid containing positive inserts were performed as recommended by the manufacturer. Four clones representing independent copies of the HUG-Brl cDNA and one clone of HUG-Br2 were isolated and characterized.
  • Complementary DNAs containing the remaining portion of the Z6 and Z17 coding and 5' untranslated regions were generated using the anchored PCR (RACE) method of Frohman et al. (PRC Protocols: A Guide to Methods and Applications pp. 28-38, Academic Press, New York (1990)).
  • the filters were hybridized with 32 p-labeled probes corresponding to the most 5' 725-bp EcoRI-frag ent of HUG-Brl and the most 5' 400-bp-fragment of HUG- Br2 (Church, et al., Proc. Nat. Acad. Sci. U.S.A. 81:1991-1995).
  • the amounts of RNA applied to the gel were normalized (McKinnon, et al., Mol. Cell. Biol. 7: 2148-2154 (1987)) by hybridization to 3 p- human cyclophilin cDNA. All probes were radiolabeled by random primed synthesis using a kit (Pharmacia) .
  • SUBSTITUTESHEET exon or a highly mutable region of a multigene model underlies the defect in Type I Crigler-Najjar.
  • the present inventors compared the levels from the liver of an Old World monkey maintained on phenobarbital in the drinking water. The results show that HUG-Br2, the lower abundance mRNA, was induced 2-fold but not that for HUG-Brl (Fig. 10, Panel B) based on normalization to cyclophilin mRNA. The existence of two bilirubin transferase isozymes and the responsiveness of one to phenobarbital administration suggest that elevation in activity after this treatment is due to the increased expression of the low constitutive isoenzyme encoded by HUG-Br2.
  • the expression units pHUG-Brl and pHUG-Br2, contained HUG-Brl and HUG-Br2, respectively, in the correct orientation with respect to the promoter element of the pSVL plasmid, and a control unit contained HUG- Brl in the reversed orientation.
  • Plasmid preparations and transfection studies were performed as previously described (Ritter, et al., J. Biol. Chem. 265: 7900-7906). Cells were incubated 72 hour at 37°C, harvested, and stored at -70°C or assayed fresh.
  • Microsomes (2.9 mg) from 3-methylcholan- threne-treated CDl mice and cell homogenates (0.8 or 1.9 mg) from transfected cells were assayed in a 200-uL reaction mixture containing 50mM Tris pH 7.7, 5mM MgCl, 20 mM UDP-glucuronic acid, 8.3 mM saccharic 1,4-lactone, and 25 uM C-bilirubin (74,000 CPM, solubilized in 10 uL of di ethylsulfoxide) and incubated for 6.5 hour at 37°C.
  • the sensitivity of the reaction product to ⁇ - glucuronidase was then determined by adjusting the reaction to pH 7.0 with 1 M Na 2 HP0, and adding 5 units of enzyme plus 3 mM ascorbic acid and continuing the incubation for 1 hour at 37°C.
  • Microsomes and cell homogenates were preactivated with 0.5 mg of [3-(3-cholamidopropyl)- dimethylammonio]-l-propane (CHAPS) detergent to 1 mg protein before adding to the reaction mixture. All reagents used in the assay were made fresh in argonized double-distilled water, and all incubations were flushed with argon, sealed and incubated in the dark.
  • the relative mobility of bilirubin and its methyl ester derivatives in this stem is Br > IX ⁇ C ⁇ > IX ⁇ C12 > IX ⁇ C8, C12 according to the published method (Blanckaert, N. (1980) Biochem. J. 185: 115-128).
  • Lane 2 shows that the product was sensitive to ⁇ -glucuronidase after one hour incubation.
  • the band intensities also show that bilirubin diglucuronide was the primary conjugate in mouse microsomes.
  • Lane 1 in Figure 11 shows that HUG-Br2- encoded activity in 1.9 mg of cell homogenate yielded the same three converted bilirubin methyl ester isomeric products in about equal amounts.
  • Example VIII Seguence Homology A comparison of the amino acid sequences of various UDP-glucuronosyltransferase forms: rat phenol UDP- glucuronosyltransferase (Rat PHENOL) , human phenol UDP-glucuronosyltransferase (HLUGP1) , human bilirubin UDP-glucuronosyltransferase, type I (HUG-Brl) , human bilirubin UDP-glucuronosyl- transferase, type II (HUG-Br2) , and rat bilirubin
  • UDP-glucuronosyltransferase (Rat Bil) , as set forth in Figure 12, shows that the carboxyl terminus of the various UDP-glucuronosyltransferase forms is conserved as between the human and the rat species and the phenol and bilirubin acceptor substrate forms.
  • the human forms, HLUGP1, HUG-Brl and HUG-Br2 show exact homology between amino acids 283-531, 285-533, and 296-534, respectively.
  • Rat PHENOL and Rat Bil show exact homology between amino acids 281-529 and 283-531, respectively. Further, there is a 90.6% conserved amino acid sequence between the two species.
  • Rat PHENOL, HLUGP1, HUG-Brl, HUG-Br2 and Rat Bil corresponding to amino acids 1-280, 1-282, 1-284, 1- 285, and 1-282, respectively.
  • there is 66% homology between HUG-Brl and HUG-Br2 70% homology between HUG-Brl and Rat Bil, 76% homology between HUG-BR2 and Rat Bilo, 84% homology between Rat PHENOL and HLUGP 1, and 60% homology between Rat Bil and HLUGP 1.
  • Comparisons of the rat bilirubin and phenol transferase amino acid sequences supports an association between the amino terminal sequences and substrate specificity.
  • the 470-bp 5' end of HUG-Brl and the 450- bp 5• end of HUG Br2 were used as probes to screen a human genomic cosmid library.
  • One clone was selected (approximately 40kb) which hybridized avidly to HUG-Brl and very weakly to HUG-Br2 and 2 other clones (45 and 44kb) which hybridized to the unique region of HUG-Br2 and not to the unique region of HUG-Brl or to the common fragment of HUG- Brl and HUG-Br2 coding for amino acids 292-533.
  • the three clones were determined by restriction enzyme analysis to overlap to generate a linear consensus fragment (97kb) (see Figure 13A and 13B) .
  • Each one of the eight subclones was probed with the three fragments of HUG-Br2 (respectively, 1-450 bp; 454- 666 bp; and 666-863 bp fragments representing the unique end of HUG-Br2) .
  • Each of the HUG-Br2 fragments (defined above) was separately hybridized to one of the Southern blots and the fourth blot was hybridized to full-length HUG-Brl to confirm that only the HUG-Br2 (1-863-bp) region hybridized to these subclones.
  • Five separate clones were identified (1, 4, 6, 7, and 8), which hybridized to the 3 separate fragments of HUG-Br2. Other subclones were all introns.
  • Each positive subclones was further mapped in detail by restriction enzymes.
  • Each subclone was digested with two to three sets of two enzymes each and again analyzed by Southern blot analysis
  • the nucleotide sequence of the clones derived from the Crigler-Najjar Type I patient were compared to the sequences of the normal HUG-Brl, HUG-Br2 and HLUGP1 to determine if there are altered bases, deletions or insertions. The comparisons were made using the IBI Pustell Sequence software computer package. The location of a modified nucleotide sequence was assessed at the level of the gene in the same patient's genomic DNA. Opposite strand primers (5—3') were synthesized with an l ⁇ based complementary sequence to HUG-Brl, HUG-Br2, and
  • HLGUPl at positions 100 bases upstream and 100 bases downstream of the altered nucleotides.
  • sufficient quantities of the target DNA from normal genome and from the defective genome were generated by PCR using a Perkin-Elmer Kit (Norwalk, CT) .
  • the two types of amplified DNA as well as samples of the HUG-Brl, HUG-Br2 and HLUGP1 cDNA previously isolated above from the Crigler-Najjar Type I cDNA library and the normal HUG-Brl and HUG-Br2 cDNA clones (as heretofore described) were analyzed by Southern blot analysis. Each blot was made in duplicate.
  • Genomic DNA from patients with Gilbert's disease and Crigler-Najjar Type II syndrome can be analyzed for altered DNA sequence(s) compared to normal.
  • the DNA will be analyzed for alterations by first making PCR products to exonic regions of the HUG-Brl and HUG-Br2 and other bilirubin genes as yet to be determined (e.g., HUG-Br3) . Plasmid subclones of the PCR products will be made and sequenced. Upon making comparisons to normal DNA any altered sequences will be subjected to the same analyses already described for Crigler-Najjar Type I as above.
  • the validity of the diagnostic probe is established by the selective hybridization under high stringency conditions) of each probe to target DNA containing a perfect match.
  • clones according to the invention have utilized as diagnostic probes or for use in a diagnostic assay as described above, said clones would also have utility in gene therapy techniques for Crigler-Najjar. Type I disease.

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Abstract

L'invention se rapporte au locus du gène isolé UGT1, à une famille d'isozymes UDP-glucuronosyltransférase codées par ledit locus, ainsi qu'aux utilisations à la fois du locus du gène et des isozymes elles-mêmes. En particulier, le locus code au moins six isoformes de transférase, parmi lesquelles deux métabolisent la bilirubine. Les six isoformes ont quatre exons communs. Les inventeurs ont découvert, chez un patient atteint du syndrom de Crigler-Najjar (CN) type I, la présence d'une mutation dans le deuxième exon commun.
PCT/US1992/000282 1991-01-10 1992-01-10 Locus genetique ugt1 et presence d'une mutation WO1992012987A1 (fr)

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WO1997031111A2 (fr) * 1996-02-22 1997-08-28 Introgene B.V. Famille de transporteurs d'anions organiques, acides nucleiques codant ceux-ci, cellules les comprenant et procedes d'utilisation associes
WO1997032042A2 (fr) * 1996-03-01 1997-09-04 The University Court Of The University Of Dundee Systeme de dosage pour des essais de medicaments
WO1999057322A2 (fr) * 1998-05-07 1999-11-11 Axys Pharmaceuticals, Inc. Genotypage du gene de l'udp-glucuronosyltransferase 1 humain (ugt1)
WO2000006776A1 (fr) * 1998-07-28 2000-02-10 Axys Pharmaceuticals, Inc. Etablissement du genotype de genes humains de l'udp-glucoronosyl-transferase 2b4 (ugt2b4), 2b7 (ugt2b7) et 2b15 (ugt2b15)
WO2001079230A2 (fr) * 2000-04-18 2001-10-25 Genaissance Pharmaceuticals, Inc. Haplotypes du gene ugt1a1
WO2002006523A2 (fr) 2000-07-14 2002-01-24 F. Hoffmann-La Roche Ag Detection d'une predisposition a l'hepatotoxicite

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FEDERATION OF EUROPEAN BIOLOGICAL SOCIETIES LETTERS, Vol. 243, No. 2, issued 1989, FOURNELL-GIGLEUX et al., "Expression of a Human Liver cDNA Encoding a UDP-Glucuronosyltransferase Catalyzing the Glucuronidation of Hyodeoxycholic Acid in Cell Culture", pages 119-122. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 265, No. 14, issued 1990, RITTER et al., "Cloning and Expression of Human Liver UDP-Glucuronosyltransferase in COS-1 Cells, 3', 4'-Catechol Estrogens and Estrol as Primary Substrates". *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, Vol. 85, No. 22, issued 1988, HARDING et al., "Cloning and Substrate Specificity of a Human Phenol UDP-Glucuronosyltransferase Expressed in COS-7 Cells - DNA Sequence Determination", pages 8381-8385. *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997031111A3 (fr) * 1996-02-22 1997-11-27 Introgene Bv Famille de transporteurs d'anions organiques, acides nucleiques codant ceux-ci, cellules les comprenant et procedes d'utilisation associes
WO1997031111A2 (fr) * 1996-02-22 1997-08-28 Introgene B.V. Famille de transporteurs d'anions organiques, acides nucleiques codant ceux-ci, cellules les comprenant et procedes d'utilisation associes
WO1997032042A2 (fr) * 1996-03-01 1997-09-04 The University Court Of The University Of Dundee Systeme de dosage pour des essais de medicaments
WO1997032042A3 (fr) * 1996-03-01 1997-11-20 Univ Dundee Systeme de dosage pour des essais de medicaments
US6479236B2 (en) 1998-05-07 2002-11-12 Dna Sciences Laboratories, Inc. Genotyping the human UDP-glucuronosyltransferase 1 (UGT1) gene
WO1999057322A2 (fr) * 1998-05-07 1999-11-11 Axys Pharmaceuticals, Inc. Genotypage du gene de l'udp-glucuronosyltransferase 1 humain (ugt1)
WO1999057322A3 (fr) * 1998-05-07 2000-01-13 Axys Pharm Inc Genotypage du gene de l'udp-glucuronosyltransferase 1 humain (ugt1)
WO2000006776A1 (fr) * 1998-07-28 2000-02-10 Axys Pharmaceuticals, Inc. Etablissement du genotype de genes humains de l'udp-glucoronosyl-transferase 2b4 (ugt2b4), 2b7 (ugt2b7) et 2b15 (ugt2b15)
US6586175B1 (en) 1998-07-28 2003-07-01 Dna Sciences Laboratories, Inc. Genotyping the human UDP-glucuronosyltransferase 2B7 (UGT2B7) gene
WO2001079230A3 (fr) * 2000-04-18 2002-03-21 Genaissance Pharmaceuticals Haplotypes du gene ugt1a1
WO2001079230A2 (fr) * 2000-04-18 2001-10-25 Genaissance Pharmaceuticals, Inc. Haplotypes du gene ugt1a1
WO2002006523A2 (fr) 2000-07-14 2002-01-24 F. Hoffmann-La Roche Ag Detection d'une predisposition a l'hepatotoxicite
WO2002006523A3 (fr) * 2000-07-14 2003-04-17 Hoffmann La Roche Detection d'une predisposition a l'hepatotoxicite

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