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WO2006084133A2 - Methodes et compositions destinees au dosage de la l-thyroxine - Google Patents

Methodes et compositions destinees au dosage de la l-thyroxine Download PDF

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WO2006084133A2
WO2006084133A2 PCT/US2006/003846 US2006003846W WO2006084133A2 WO 2006084133 A2 WO2006084133 A2 WO 2006084133A2 US 2006003846 W US2006003846 W US 2006003846W WO 2006084133 A2 WO2006084133 A2 WO 2006084133A2
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repeats
patient
alleles
thyroxine
ugtlal
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WO2006084133A3 (fr
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Mark J. Ratain
Andrea Graber
Federico Innocenti
Jacqueline Ramirez
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University of Chicago
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University of Chicago
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    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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

Definitions

  • the present invention relates generally to the fields of molecular genetics, pharmacogenetics, and hormone therapy.
  • the present invention is directed to methods and compositions for evaluating how much L-thyroxine, a particular drug which undergoes glucuronidation, is to be prescribed to a patient in need of the drug.
  • the present invention is directed to methods and compositions for achieving a euthyroid state in a patient who suffers from hyperthyroidism.
  • Thyroxine (T 4 ) is prescribed in patients with hypothyroidism, and is known to undergo glucuronidation. Previous studies propose IAl and 1A9 as the main UGT isoforms responsible for T 4 G formation, but a complete IA screening has not been performed.
  • Hyperthyroidism occurs in approximately 5% of the US population (Hollowell et ah, 2002). L-thyroxine is the most widely used treatment for hypothyroidism and the third prescribed drug in USA in 2003. Titration of L-thyroxine dosage in hypothyroid patients is required to prevent continued hypothyroidism or iatrogenic hyperthyroidism which can lead to cardiac dysfunction (Klein et ah, 2001). There is a high variability in the L-thyroxine dosage required to achieve a euthyroid state. L-thyroxine is inactivated by metabolism via glucuronidation in the liver.
  • the present invention is based on identification and characterization of correlations between the genotype of the UGTlAl gene and phenotype relating to the inactivation of L-thyroxine.
  • the inventors have identified the UGT enzymes involved in the hepatic and extrahepatic metabolism of L-thyroxine. Using liver specimens from donors, the inventors have demonstrated a significant correlation between a common polymorphism in the UGTlAl gene (a TA indel in the gene promoter, also referred to as (TA) n ) and L-thyroxine glucuronidation rates.
  • the present invention provides methods and compositions that exploit correlations between genotype and phenotype concerning L-thyroxine. It is contemplated that such methods and compositions can be used to optimize dosing in patients. Moreover, such methods and conipostions can prevent or reduce the risk of iatrogenic hyperthyroidism and/or its side effects.
  • the present invention involves methods for determining or evaluating the level of L-thyroxine glucuronidation activity in a patient. This is accomplished by directly or indirectly determining the number of TA repeats in the promoter of the UGTlAl promoter. Additionally, the claims provide a summary of the invention. It will be understood that the term “determine” is used according to its ordinary and plain meaning to indicate “to ascertain definitely by observation, examination, calculation, etc.,” according to the Oxford English Dictionary (2 nd ed.). It will also be understood that the phrase "determining the sequence at position X" means that the nucleotide(s) at that position is directly or indirectly identified.
  • the sequence at a particular position is determined, while in other embodiments, what is determined at a particular position is that a particular nucleotide is not at that position. It is specifically contemplated that embodiments of the invention include methods in which a medical practitioner determines information regarding a patient's genotype by performing a test or assay or ordering that the patient have the assay or test performed, such as by a laboratory. Therefore, it is contemplated that a person may "determine" information by receiving the results of an assay or test revealing the results of a determination. Based on the level of activity determined, it is contemplated that a patient is given a different dosage than he or she would have otherwise received had the genotyping not been performed. Thus, in some embodiments of the invention, a typical dosage is adjusted for a particular person (individualized therapy).
  • some methods of the invention are practiced by a laboratory and include, but are not limited to, the following steps: receiving or obtaining a biological sample from a patient; assaying the sample for one or more polymorphisms (such as are described herein); providing the results of the assay to a medical practitioner of the patient's. It is contemplated that the results may be provided in the form of a report or lab result.
  • Certain methods of the invention concern prescribing an amount of L- thyroxine for a patient comprising: a) determining the number of TA repeats in one or both alleles of the uridine diphosphate glucuronosyltransf erase IAl (UGTlAl) promoter, wherein the presence of seven TA repeats correlates with decreased inactivation of L-thyroxine; and, b) prescribing an average daily dose of L-thyroxine for the patient.
  • the term "average daily dose” means an amount of L-thyroxine that is the average amount of L-thyroxine on a daily basis.
  • a patient may be prescribed X amount to be taken over a three day period, which amounts to X/3 as the average daily dose over that period even though the patient may have taken 0.5X on day 1 and 0.5X on day 3.
  • an average daily dose can be expressed as a total amount to be taken in a day or as one or more amounts that are to be taken a certain number of times a day.
  • a patient is prescribed L-thyroxine having a particular dose, such as 50 meg, to be taken twice a day (b.i.d.).
  • L-thyroxine is taken once a day, so any dose prescribed is the total amount for the day.
  • there are methods for reducing the risk of hypothyroidism or hyperthyroidism in a patient comprising: a) predicting the level of L-thyroxine glucuronidation in the patient; and, b) prescribing an average daily dose of L-thyroxine to the patient based on the level of L-thyroxine glucuronidation predicted in the patient.
  • the risk of either hyperthyroidism or hypothyroidism is reduced by prescribing a therapeutically appropriate amount of L-thyroxine to address the patient's thyroid problem based on the predicted level of L-thyroxine glucuronidation in that patient.
  • the present invention also concerns methods for evaluating a patient's L- thyroxine glucuronidation capacity comprising: a) obtaining DNA from the patient; b) determining from the DNA the number of TA repeats in one or both alleles of the UGTlAl promoter; and, c) informing the patient or the patient's physician about the patient's L-thyroxine glucuronidation capacity. It is contemplated that either instead of c) or as a way of achieving c), a report or other document providing the results from steps a) and b) is generated. The report or document can be provided to the patient and/or the patient's caregiver (physician or nurse).
  • there are methods for optimizing the dose of L-thyroxine for a patient comprising: a) determining from the patient's DNA the number of TA repeats in one or both alleles of the UGTlAl promoter; and b) prescribing a dose of L-thyroxine to the patient based on the number of TA repeats.
  • the level of L-thyroxine glucuronidation is predicted by determining the number of TA repeats in one or both alleles of the UGTlAl promoter in some embodiments of the invention.
  • the presence of seven TA repeats in a single allele indicates the same or a lower level of L-thyroxine glucuronidation than the presence of six TA repeats in a single allele.
  • the presence of six TA repeats in one allele and seven TA repeats in the other allele indicates a lower level than the presence of six TA repeats in both alleles, but a higher level than the presence of seven TA repeats in both alleles.
  • only one allele is or can be evaluated, while in other embodiments, at least one allele or both alleles is or can be evaluated.
  • the number of TA repeats correlates with the level of inactivation of L- thyroxine inactivation as a result of glucuronidation by the gene product of UGTlAl. Consequently, patients having at least one UGTlAl allele with seven TA repeats in the promoter region generally have a lower level of L-thyroxine inactivation than patients who do not have a (TA) 7 . Consequently, the highest doses will generally be prescribed for those with six TA repeats in both alleles, the lowest doses will generally be prescribed for those with seven TA repeats in both alleles, and doses between the highest and lowest will generally be prescribed to those with six TA repeats in one allele and seven TA repeats in the other allele.
  • the dose prescribed for the patient will be lower if the patient is found to have seven TA repeats in at least one allele than the dose prescribed for a patient who has six TA repeats in both alleles. Conversely, in further embodiments, the dose prescribed for the patient will be higher if the patient has six TA repeats in one allele than the dose prescribed for a patient who has a seven TA repeats in both alleles. Moreover, the dose prescribed for the patient will be lower if the patient has seven TA repeats in both alleles than the dose prescribed for a patient who has six TA repeats in one allele and seven TA repeats in the other allele.
  • the dose prescribed for the patient will be higher if the patient has six TA repeats in both alleles than the dose prescribed for a patient who has six TA repeats in one allele and seven TA repeats in the other allele.
  • a person of ordinary skill in the art would be able to adjust the amount typically prescribed in the absence of evaluating the number of TA repeats.
  • the dose may be adjusted by about, at least about or at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300% or more, or any range derivable therein.
  • methods include determining the presence of seven TA repeats in at least one allele. In certain embodiments, methods include determining the presence of six TA repeats in at least one allele, hi other embodiments, methods including determining the presence of six or seven TA repeats in both alleles or six repeats in one and seven repeats in the other, hi some cases, methods include prescribing a dose of L-thyroxine for a patient having six TA repeats in both alleles, wherein the dose is higher than the dose for a patient having seven TA repeats in one or both alleles.
  • Other methods involve prescribing a dose of L-thyroxine for a patient having seven TA repeats in both alleles, wherein the dose is lower than the dose for a patient having six TA repeats in one or both alleles. Additional methods include prescribing a dose of L-thyroxine for a patient having six TA repeats in one allele and seven TA repeats in the other allele, wherein the dose is lower than the dose for a patient having seven TA repeats in both alleles but higher than the dose for a patient having having six TA repeats in both alleles. It is contemplated that a dose may be adjusted with respect to the amount of L- thyroxine that a patient takes at any one time and/or the frequency of the intake.
  • the dose will be adjusted with respect to the total amount a patient takes a day (24-hour period) (referred to as average daily dose), while in others, the adjustment is with respect to the total amount a patient takes in a week (seven 24- hours periods) (average weekly dose). It is contemplated that any embodiment discussed with respect to "dose” can be implemented with respect to "average daily dose” and "average weekly dose.” In other words, if embodiments concern a first patient who is prescribed a lower "dose” than a second patient with a different genotype, then the embodiment will be understood to describe also a first patient who is prescribed a lower "average daily dose” or an "average weekly dose” than the second patient with a different genotype.
  • any embodiment discussed with respect to "dose” can be implemented with respect to frequency of intake. For example, if embodiments concern a first patient who is prescribed a lower "dose" than a second patient with a different genotype, then the embodiment will be understood to describe also a first patient who is prescribed to intake L-thyroxine less frequently than the second patient with a different genotype.
  • Frequency can be discussed in terms of any measurement of time, but in specific embodiments, the measurements are expressed in terms of number of times a day (e.g., one, two, three, four, five, or more times) or number of times a week (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more times). It is contemplated that adjustments can be such that a patient takes L-thyroxine less frequently or more frequently than someone else with a different genotype.
  • the invention is not limited by the way in which the number of TA repeats in one or both alleles is determined.
  • the number of TA repeats is determined by evaluating directly the TA repeats in one or both alleles of the UGTlAl promoter. This can be accomplished, for example, by sequencing one or both alleles at that location in the gene or by amplifying the number of TA repeats.
  • the number of TA repeats can be amplified by polymerase chain reaction.
  • the number of TA repeats can be determined using an invasive cleavage assay.
  • the number of TA repeats is determined by using gel electrophoresis as part of the assay. In certain embodiments, it is contemplated that only one allele is evaluated, while in other embodiments, both alleles are evaluated.
  • embodiments further include obtaining DNA from the patient. This may be accomplished by obtaining any biological sample from the patient so long as the sample contains DNA from the patient.
  • the polymorphism may be in the gene of any other UGT enzyme.
  • the polymorphism is in the UGTlA gene.
  • the polymorphism is in the UGTl A3 gene.
  • the UGTl A3 polymorphism may be one or more of the following polymorphisms: 17(A>G), 31(T>C), 81(G>A), 133(OT), 140(T>C), or 477(A>G), or a combination thereof.
  • the polymorphism is in the UGT1A8 gene.
  • the UGT1A8 polymorphism may be one or more of the following polymorphisms: 518(C>G), 765(A>G), 830(G>A), or a combination thereof. It is contemplated that the polymorphism is selected from the group consisting of: UGTl A9 -118(T) 9>10 , UGTl A9 -275T>A, and UGTl A9 -2152OT. In other embodiments, the polymorphism is in the UGTlAlO gene.
  • the UGTlAlO polymorphism may be one or more of the following polymorphisms: 126(G>A), 177(G>A), 415(G>A), 597(T>C), 605(OT), 622 (T>C), 693(OT), 719(OT), 730(OA), or a combination thereof. It is contemplated that a combination of polymorphisms or a specific haplotype may be evaluated in certain embodiments of the invention.
  • Methods of the present invention involve determining the number of TA repeats in the promoter region of the UGTlAl gene by evaluating a polymorphism in linkage disequilibrium (LD) with the number' of repeats. In certain embodiments, this involves evaluating at least one of the following polymorphisms: UGTlAl - 3156G>A, UGTlAl -3279T>G, UGTlAl 211G>A, UGTlAl 686OA. Other polymorphisms in UGTlAl may also be included in embodiments of the invention.
  • the term "linkage disequilibrium" is used according to its ordinary meaning in the art.
  • the polymorphism is in complete LD with the number of TA repeats.
  • Other embodiments also involve identifying a patient in need of L-thyroxine. Methods and compositions of the present invention are of particular interest to such patients.
  • the patient does not have or is not suspected of having Gilbert's Syndrome.
  • Gilbert's Syndrome is correlated with the number of repeats, however, it is not clear that the presence or absence of clinical Gilbert's Syndrome indicates a particular phenotype with respect to a particular UGTlAl drug substrate.
  • Methods also include identifying a starting daily dose or starting average daily dose for a patient in need of L-thyroxine.
  • a “starting daily dose” refers to the amount of L-thyroxine a patient is initially prescribed on a daily basis after the prescriber takes into consideration the number of TA repeats in one or both alleles of the patient's UGTlAl genes.
  • a “starting average daily dose” is the same as a daily dose except that it is expressed as the average amount that is take as an initial dose. It is contemplated that the dose prescribed for a patient may subsequently be altered depending on the patient's health, the prognosis of the patient's thyroid conditions, any side effects, etc.
  • dosages were altered as a result of titrating the amount of L-thyroxine in the patient.
  • further refinements may involve titrating the amount of L-thyroxine in the patient. Such refinements may be implemented to address issues concerning specific patients.
  • DNA from multiple patients is obtained and the number of TA repeats determined for each patient.
  • Assays may be carried out concurrently for multiple samples ⁇ i.e., patients).
  • assays may be fully or partially automated.
  • Methods of the invention also include monitoring for toxicity or adverse events once L-thyroxine is administered, and possibly, adjusting or modifying dosage based on those results.
  • nucleotides or residues may be according to their well known abbreviations.
  • a “C” refers to a cytosine;
  • T refers to "thymine”;
  • A refers to adenine; and
  • G refers to guanine.
  • n ⁇ RNA is used to determine a nucleotide sequence,
  • U refers to uracil. Positions are indicated by conventional numbering where a negative sign (-) refers to nucleotides upstream (5') from the transcriptional start site (+1) (these sequences are in the promoter), unless otherwise designated.
  • Sequences in the 5' untranslated region may also be referred to using a negative sign, and in these cases, the positioning is with respect to the translated portion, where the first nucleotide of a codon is understood as +1.
  • Positions downstream of the translational start site may or may not have a plus sign (+).
  • identification of a position downstream of the transcriptional start site refers to a position with respect to only the coding region of the gene, that is, its exons and not the introns. hi some instances, positions within introns are referred to and the numbering for these positions is typically with respect to that intron alone, and not the gene as a whole.
  • methods of the invention one or more sequences in one or both alleles of the XJGTlAl gene is determined. In some embodiments, both alleles of the patient are evaluated, while in others, only one allele is evaluated. hi further embodiments of the invention, methods also include obtaining a sample from a patient and using the sample to determine the sequence at a particular position or the number of repeats at a particular position.
  • the sample may contain whole blood, serum, plasma, or a tissue biopsy. Sequences may be determined by performing or conducting a hybridization assay, an amplification assay, particularly one that is allele-specific, a sequencing or microsequencing assay.
  • the number of TA repeats in the UGTlAl gene may be determined directly or indirectly.
  • a direct determination involves performing an assay with respect to that position.
  • An indirect determination means that the sequence or number of repeats at that position is determined based on data regarding a different position, particularly by evaluating the sequence of a position in linkage disequilibrium (LD) with that polymorphism, hi some embodiments, the sequence in LD with the number of TA repeats is in complete linkage disequilibrium with another polymorphism, hi additional embodiments, the position in linkage disequilibrium with the number of TA repeats is selected from the group consisting of positions -3156G>A, -3279T>G, - 3156OA, 211G>A, and/or 686OA of the UGTlAl gene.
  • a haplotype that includes the number of TA repeats is evaluated, hi these embodiments, a determination of one or more sequences in one or both alleles of a gene in the haplotype is included in methods of the invention. hi methods of the invention, in some embodiments, an additional step of administering L-thyroxine to the patient is included, hi some cases, the amount, formulation, or timing of the administration is based on the analysis of the number of TA repeats or the level of L-thyroxine glucuronidation capacity. In some embodiments of the invention, a patient is also provided additional thyroid therapy.
  • Kits can include reagents for detecting the number of TA repeats and one or more of the polymorphisms discussed above and elsewhere in the application.
  • reagents may be nucleic acids, for example, sequencing primers, amplification primers, or probes.
  • the reagents may be compatible with specific cleavage assays.
  • FIG. 1 UGTlAl (TA) n Genotype versus L-thyroxine glucuronidation.
  • FIG. 2 Screen of UGTs for T 4 G formation. All of the UGTlA activities were normalized by relative protein expression as determined by western blot.
  • FIG. 3A-B Correlation of T 4 with SN-38 (A) and Flavopiridol glucuronidation (B). Data represent means of duplicate determinations from a single experiment.
  • FIG. 5 Comparison of T 4 G formation between UGT1A9 -118T9>10 genotypes. Data represent means of duplicate determinations from a single experiment. Kruskal-Wallis test p > 0.05.
  • FIG. 6 Michaelis-Menten kinetics of T 4 glucuronidation in HLM. Data represent the mean ⁇ SE of a single experiment performed in triplicate.
  • FIG. 7A-B Screen of recombinant microsomes expressing functional human UGTs for T 4 G formation (A) non-normalized (B) normalized by relative protein expression. Data represent means of duplicate determinations from a single experiment. UGTlA activities were normalized for relative protein expression by western blot. The UGTlA standard was designated as the basal UGT expression level.
  • FIG. 8A-B Correlation of T 4 glucuronidation with (A) SN-38 and (B) flavopiridol glucuronidation. Data represent means of duplicate determinations from a single experiment.
  • FIG. 9 Comparison of T 4 G formation between UGTlAl -53(TA) 6>7 genotypes. Data represent means of duplicate determinations from a single experiment. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
  • the present invention provides methods and compositions for evaluating the level of L-thyroxine glucuronidation in a subject who will be administered L- thyroxine (or has already been administered L-thyroxine).
  • the development of these methods and compositions allows for the use of such an evaluation to optimize treatment of a patient and to lower the risk of toxicity or adverse events.
  • L-thyroxine is the generic name for a commonly prescribed drug to treat hypothyroidism. Hypothyroidism is a condition caused by the effects of too little thyroid hormone on tissues of the body. L-thyroxine may also be used to treat Pituitary TSH Suppression. L-thyroxine is the most widely used treatment for hypothyroidism, a condition which occurs in approximately 5% of the U.S. population (Hollowell et al., 2002). Due to a low therapeutic index, T 4 dosage requires titration in small increments (Roberts and Ladenson, 2004). Interindividual differences in T 4 metabolism could result in variation in the patient exposure to T 4 . This variation could potentially affect the steady state dose required to reach a euthyroid state.
  • T 4 is metabolized by deiodination, sulfation, and glucuronidation. Deiodination acts to regulate the bioactivity of the molecule. Outer ring deiodination converts T 4 to the biologically active form, 3,3',5-triiodothyronine (T 3 ). Deiodination of the inner ring produces the biologically inactive 3,3',5'-triiodothyronine (rT 3 ). Conjugation of T 4 with a glucuronide or sulfate group increases urinary and biliary clearance by rendering the molecule more water-soluble.
  • T 4 glucuronide (T 4 G) is stable and may undergo enterohepatic recirculation through conversion to T 4 by bacterial ⁇ - glucuronidase in the intestine (Visser, 1996).
  • T 4 sulfate is very unstable because sulfation accelerates inner ring deiodination by approximately 200-fold. Thus, little T 4 sulfate is found in bile, urine, or serum.
  • T 4 glucuronidation is widely considered to be the major route of T 4 metabolism.
  • mass balance studies including conjugates are not currently available.
  • animal studies suggest that T 4 glucuronidation may significantly affect T 4 clearance.
  • Gunn rats, which are UGTlA deficient have circulating T 4 concentrations that are 1.8 fold higher than those in normal rats, suggesting that decreased UGT activity leads to higher T 4 levels (Kato et al., 2005).
  • several other studies have demonstrated that the administration of hepatic inducers of UGTs leads to a dose-dependent decrease in serum T 4 concentrations in rats (Beetstra et al, 1991; Liu et al., 1995; Hood et al., 1999).
  • T 3 undergoes glucuronidation by UGT2B2 in rats (Visser et al, 1993b). However, no significant T 3 glucuronidation has been observed in human liver or kidney microsomes, suggesting that glucuronidation is a minor pathway of T 3 metabolism in humans (Findlay et al., 2000).
  • L-thyroxine is sold in the United States under a variety of names including, but not limited to, Synthroid (levothyrozine sodium tablets), Levothroid, Levoxyl, Unithroid, and Levo-T. It is typically taken once a day. It is contemplated that the daily average dose of L-thyroxine may be adjusted after the level of a patient is evaluated using methods and compositions of the invention.
  • UGT enzymes are broadly classified into two distinct gene families.
  • the UGTl locus codes for multiple isoforms of UGT, all of which share a C-terminus encoded by a unique set of exons 2-5, but which have a variable N-terminus encoded by different first exons, each with its own independent promoter (Bosma et al., 1992; Ritter et al, 1992).
  • the variable first exons confer the substrate specificity on the enzyme.
  • Isoforms of the UGT2 family are unique gene products of which at least eight isozymes have been identified (Clarke et al, 1994).
  • the UGTlAl isoform is the major bilirubin glucuronidation enzyme. Genetic defects in the UGTlAl gene can result in decreased glucuronidation activity which leads to abnormally high levels of unconjugated serum bilirubin that may enter the brain and cause encephalopathy and kernicterus (Owens & Ritter, 1995). As described above, this condition is commonly known as Gilbert's syndrome (which is frequently diagnosed based on elevated total bilirubin levels — a biochemical diagnosis).
  • the molecular defect in Gilbert's Syndrome is a change in the TATA box within the UGTlAl promoter (Bosma et al, 1995; Monaghan et al, 1996).
  • This promoter usually contains a (TA) 6 TAA element, but another allele, termed UGTlAl *28 or allele 7, is also present in human populations at high frequencies, and contains the sequence (TA) 7 TAA.
  • UGTlAl *28 or allele 7 is also present in human populations at high frequencies, and contains the sequence (TA) 7 TAA.
  • This polymorphism in the promoter of the UGTlAl gene results in reduced expression of the gene and accounts for most cases of Gilbert's Syndrome (Bosma et al, 1995).
  • gene expression levels for the UGTlAl promoter alleles are inversely related to the length of the TA repeat in the TATA box.
  • UGTs have been shown to contribute to the detoxification and elimination of both exogenous and endogenous compounds. Examples of how UGT polymorphisms affect drugs other than L-thyroxine are provided in, for example, U.S. Patent Nos. 6,472,157 and 6,395,481, which are both incorporated by reference with respect to their teaching about UGTlAl sequences. II. NUCLEIC ACIDS
  • the TA repeats in the UGTlAl gene of humans have been reported as GenBank Accession numbers AY533181 (eight TA repeats) (SEQ ID NO:4); AY533180 (seven TA repeats) (SEQ ID NO:3); AY533179 (six TA repeats) (SEQ ID NO:2); and AY533178 (five TA repeats) (SEQ ID NO:1), which are all incorporated by reference.
  • GenBank Accession numbers AY533181 (eight TA repeats) (SEQ ID NO:4)
  • AY533180 single TA repeats
  • AY533179 single TA repeats
  • SEQ ID NO:2 six TA repeats
  • AY533178 five TA repeats
  • nucleic acids including amplification primers, oligonucleotide probes, and other nucleic acid elements involved in the analysis of genomic DNA.
  • a nucleic acid comprises a wild-type, a mutant, or a polymorphic nucleic acid.
  • nucleic acid is well known in the art.
  • a “nucleic acid” as used herein will generally refer to a molecule ⁇ i.e., a strand) of DNA, RJSfA or a derivative or analog thereof, comprising a nucleobase.
  • a nucleobase includes, for example, a naturally occurring purine or pyrimidine base found in DNA ⁇ e.g., an adenine "A,” a guanine “G,” a thymine “T” or a cytosine "C”) or RNA ⁇ e.g., an A, a G, an uracil "U” or a C).
  • nucleic acid encompasses the terms “oligonucleotide” and “polynucleotide,” each as a subgenus of the term “nucleic acid.”
  • oligonucleotide refers to a molecule of between about 3 and about 100 nucleobases in length.
  • polynucleotide refers to at least one molecule of greater than about 100 nucleobases in length.
  • a “gene” refers to coding sequence of a gene product, as well as introns and the promoter of the gene product. In addition to the UGTlAl gene, other regulatory regions such as enhancers for UGTlAl are contemplated as nucleic acids for use with compositions and methods of the claimed invention.
  • nucleic acids of the invention comprise or are complementary to all or 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,
  • a nucleic acid may encompass a double-stranded molecule or a triple-stranded molecule that comprises one or more complementary strand(s) or "complement(s)" of a particular sequence comprising a molecule.
  • a single stranded nucleic acid may be denoted by the prefix "ss”, a double stranded nucleic acid by the prefix "ds”, and a triple stranded nucleic acid by the prefix "ts.”
  • a nucleic acid encodes a protein, polypeptide, or peptide.
  • the present invention concerns novel compositions comprising at least one proteinaceous molecule.
  • a "proteinaceous molecule,” “proteinaceous composition,” “proteinaceous compound,” “proteinaceous chain,” or “proteinaceous material” generally refers, but is not limited to, a protein of greater than about 200 amino acids or the full length endogenous sequence translated from a gene; a polypeptide of greater than about 100 amino acids; and/or a peptide of from about 3 to about 100 amino acids. All the "proteinaceous” terms described above may be used interchangeably herein.
  • embodiments of the invention concern evaluating a patient's UGTlAl genes and determining how many TA repeats the patient has in each allelle.
  • other polymorphisms in UGTlAl may be evaluated and/or polymorphisms in other UGT genes may be evaluated. These include the following polymorphisms discussed herein.
  • UGTlAl polymorphisms are discussed in published PCT application number WO 04/108954, which is hereby incorporated by reference in its entirety.
  • the polymorphism is the following: -3440C>A, -3401T>C, - 3279G>T, -3177OG, -3175A>G, -3156G>A, or any combination thereof.
  • the notation -3440C>A indicates that cytosine nucleotide (C) at the -3440 position is replaced by an Adenosine (A), where the negative sign indicates what residue upstream from the mRNA start site (+1) the residue is.
  • a UGTlAl polymorphism is one or more of the following: -3401T>C, -3345C/-, -3279T>G (originally reported as T>G but G is the ancestral allele), - 3177OG, -3175G>A, -3156G>A, -2951A>G, -2828G>C, -2473G>T, -2353C>T, - 1516T>G, -1352A>C, -484G>A, -364OT, -129C>A, -106T>C, -53(TA)5-8, 141OT, 211G>A, 674T>G, 686OA, 924OT, 953OT, 1206OT, 1395A>G, 3705G/T, 3726A>G, 3729G/T, 3788T>G, 4254OT, 4305G>A, 4375OT, 4528T>C, 4578A/AAAAGGGAGGGA
  • the present invention in some embodiments also concerns other UGT genes, such as UGT1A3, UGT1A8, UGT1A9, and UGTlAlO.
  • UGT1A3, UGT1A8, UGT1A9, and UGTlAlO UGT genes
  • UGT1A3, UGT1A8, UGT1A9, and UGTlAlO UGT genes
  • the following tables describe polymorphisms, genotypes, and/or haplotypes that may also be evaluated in the context of the invention. It is contemplated that one or more of the polymorphisms, genotypes, or haplotypes identified below may be evaluated as part of methods of the invention or that kits of the invention will include a nucleic acid(s) capable of detecting one or more of such polymorphisms, genotypes, or haplotypes.
  • GenBank or other accession numbers provided in the tables below are specifically incorporated by reference. TABLE l
  • any of the following polymorphisms, or a combination thereof, may be implemented in the context of the present invention: -2208(C>T), - 2152(OT), -2141(OT), -1887(T>G), -1818(T>C), -665(OT), -440 (T>C), - 331(OT), -275(T>A), -109 to -98 T ⁇ ( 9 >io), -87 (G>A), and codon 33 (T>C).
  • Any embodiment discussed in the context of UGTlAl may be applied with respect to any other UGT polymorphism discussed herein using the relevant sequence or sequences.
  • a nucleic acid may be made by any technique known to one of ordinary skill in the art, such as for example, chemical synthesis, enzymatic production or biological production.
  • a synthetic nucleic acid e.g., a synthetic oligonucleotide
  • Non-limiting examples of a synthetic nucleic acid include a nucleic acid made by in vitro chemical synthesis using phosphotriester, phosphite or phosphoramidite chemistry and solid phase techniques such as described in European Patent 266,032, incorporated herein by reference, or via deoxynucleoside H-phosphonate intermediates as described by Froehler e? ⁇ l., 1986 and U.S. Patent 5,705,629, each incorporated herein by reference.
  • one or more oligonucleotide may be used.
  • Various different mechanisms of oligonucleotide synthesis have been disclosed in for example, U.S. Patents 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which is incorporated herein by reference.
  • a non-limiting example of an enzymatically produced nucleic acid include one produced by enzymes in amplification reactions such as PCRTM (see for example, U.S. Patent 4,683,202 and U.S. Patent 4,682,195, each incorporated herein by reference), or the synthesis of an oligonucleotide described in U.S. Patent 5,645,897, incorporated herein by reference.
  • a non-limiting example of a biologically produced nucleic acid includes a recombinant nucleic acid produced (i.e., replicated) in a living cell, such as a recombinant DNA vector replicated in bacteria (see for example, Sambrook et ⁇ l. 2001, incorporated herein by reference).
  • a nucleic acid may be purified on polyacrylamide gels, cesium chloride centrifugation gradients, chromatography columns or by any other means known to one of ordinary skill in the art (see for example, Sambrook et ⁇ l., 2001, incorporated herein by reference).
  • a nucleic acid is a pharmacologically acceptable nucleic acid.
  • Pharmacologically acceptable compositions are known to those of skill in the art, and are described herein.
  • the present invention concerns a nucleic acid that is an isolated nucleic acid.
  • isolated nucleic acid refers to a nucleic acid molecule (e.g., an RNA or DNA molecule) that has been isolated free of, or is otherwise free of, the bulk of the total genomic and transcribed nucleic acids of one or more cells, hi certain embodiments, "isolated nucleic acid” refers to a nucleic acid that has been isolated free of, or is otherwise free of, bulk of cellular components or in vitro reaction components such as for example, macromolecules such as lipids or proteins, small biological molecules, and the like. D. Nucleic Acid Segments
  • the nucleic acid is a nucleic acid segment.
  • nucleic acid segment are fragments of a nucleic acid, such as, for a non-limiting example, those that encode only part of a UGTlAl gene locus or a UGTlAl gene sequence.
  • a “nucleic acid segment” may comprise any part of a gene sequence, including from about 2 nucleotides to the full length gene including promoter regions to the polyadenylation signal and any length that includes all the coding region.
  • nucleic acid segments may be designed based on a particular nucleic acid sequence, and may be of any length.
  • an algorithm defining all nucleic acid segments can be created: n to n + y where n is an integer from 1 to the last number of the sequence and y is the length of the nucleic acid segment minus one, where n + y does not exceed the last number of the sequence.
  • the nucleic acid segments correspond to bases 1 to 10, 2 to 11, 3 to 12 ... and so on.
  • nucleic acid segments correspond to bases 1 to 15, 2 to 16, 3 to 17 ... and so on.
  • the nucleic segments correspond to bases 1 to 20, 2 to 21, 3 to 22 ... and so on.
  • the nucleic acid segment may be a probe or primer.
  • a probe generally refers to a nucleic acid used in a detection method or composition.
  • a primer generally refers to a nucleic acid used in an extension or amplification method or composition.
  • the present invention also encompasses a nucleic acid that is complementary to a nucleic acid.
  • a nucleic acid is "complement(s)" or is “complementary” to another nucleic acid when it is capable of base-pairing with another nucleic acid according to the standard Watson-Crick, Hoogsteen or reverse Hoogsteen binding complementarity rules.
  • another nucleic acid may refer to a separate molecule or a spatial separated sequence of the same molecule.
  • a complement is a hybridization probe or amplification primer for the detection of a nucleic acid polymorphism.
  • complementary also refers to a nucleic acid comprising a sequence of consecutive nucleobases or semiconsecutive nucleobases (e.g., one or more nucleobase moieties are not present in the molecule) capable of hybridizing to another nucleic acid strand or duplex even if less than all the nucleobases do not base pair with a counterpart nucleobase.
  • semiconsecutive nucleobases e.g., one or more nucleobase moieties are not present in the molecule
  • completely complementary nucleic acids are preferred.
  • Some embodiments of the invention concern identifying polymorphisms in UGTlAl such as the number of TA repeats, correlating genotype or haplotype to phenotype, wherein the phenotype is altered UGTlAl activity or expression, and then identifying such polymorphisms in patients who have or will be given L-thyroxine or other drugs or compounds that are UGTlAl substrates.
  • the present invention involves assays for identifying polymorphisms and other nucleic acid detection methods. Such assays involve identifying the number of TA repeats in the UGTlAl gene, as shown in SEQ ID NOs: 1-4.
  • Other embodiments also involve identifying other polymorphisms in UGTlAl or in other UGT genes, including, but not limited to UGT1A3, UGTl A8, UGT1A9, and UGTAlO.
  • probes and primers can be prepared using the sequences disclosed in SEQ ID NOs: 1-4.
  • Nucleic acids therefore, have utility as probes or primers for embodiments involving nucleic acid hybridization. They may be used in diagnostic or screening methods of the present invention. Detection of nucleic acids encoding UGTlAl, as well as nucleic acids involved in the expression or stability of UGTlAl polypeptides or transcripts, are encompassed by the invention. General methods of nucleic acid detection methods are provided below, followed by specific examples employed for the identification of polymorphisms, including single nucleotide polymorphisms (SNPs) or the number of TA repeats.
  • SNPs single nucleotide polymorphisms
  • nucleotides preferably between 17 and 100 nucleotides in length, or in some aspects of the invention up to 1- 2 kilobases or more in length, allows the formation of a duplex molecule that is both stable and selective.
  • Molecules having complementary sequences over contiguous stretches greater than 20 bases in length are generally preferred, to increase stability and/or selectivity of the hybrid molecules obtained.
  • Such fragments may be readily prepared, for example, by directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.
  • amplification is employed to determine the number of TA repeats. See, e.g., U.S. Patent Nos. 6,472,157 and 6,395,481; Te et al, 2000; and, Innocenti et ah, 2004, all of which are hereby incorporated by reference for their teachings regarding determining the number of TA repeats in the UGTlAl gene.
  • nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNAs and/or RNAs or to provide primers for amplification of DNA or RNA from samples.
  • relatively high stringency conditions For applications requiring high selectivity, one will typically desire to employ relatively high stringency conditions to form the hybrids.
  • relatively low salt and/or high temperature conditions such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50 0 C to about 70 0 C.
  • Such high stringency conditions tolerate little, if any, mismatch between the probe or primers and the template or target strand and would be particularly suitable for isolating specific genes or for detecting a specific polymorphism. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.
  • hybridization to filter-bound DNA may be carried out in 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 0 C 5 and washing in 0.1 x SSC/0.1% SDS at 68°C (Ausubel et al, 1989).
  • Conditions may be rendered less stringent by increasing salt concentration and/or decreasing temperature.
  • a medium stringency condition could be provided by about 0.1 to 0.25M NaCl at temperatures of about 37°C to about 55°C
  • a low stringency condition could be provided by about 0.15M to about 0.9M salt, at temperatures ranging from about 20°C to about 55°C.
  • the washing may be carried out for example in 0.2 x SSC/0.1% SDS at 42 0 C (Ausubel et al, 1989).
  • Hybridization conditions can be readily manipulated depending on the desired results.
  • hybridization may be achieved under conditions of, for example, 5OmM Tris-HCl (pH 8.3), 75mM KCl, 3mM MgCl 2 , 1.OmM dithiothreitol, at temperatures between approximately 2O 0 C to about 37°C.
  • Other hybridization conditions utilized could include approximately 1OmM Tris-HCl (pH 8.3), 5OmM
  • nucleic acids of defined sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization.
  • appropriate indicator means include fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of being detected.
  • colorimetric indicator substrates are known that can be employed to provide a detection means that is visibly or spectrophotometrically detectable, to identify specific hybridization with complementary nucleic acid containing samples, hi other aspects, a particular nuclease cleavage site may be present and detection of a particular nucleotide sequence can be determined by the presence or absence of nucleic acid cleavage.
  • the probes or primers described herein will be useful as reagents in solution hybridization, as in PCR, for detection of expression or genotype of corresponding genes, as well as in embodiments employing a solid phase.
  • the test DNA or RNA
  • the test DNA is adsorbed or otherwise affixed to a selected matrix or surface.
  • This fixed, single-stranded nucleic acid is then subjected to hybridization with selected probes under desired conditions.
  • the conditions selected will depend on the particular circumstances (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.).
  • hybridization After washing of the hybridized molecules to remove non-specifically bound probe molecules, hybridization is detected, and/or quantified, by determining the amount of bound label.
  • Representative solid phase hybridization methods are disclosed in U.S. Patents 5,843,663, 5,900,481 and 5,919,626.
  • Other methods of hybridization that may be used in the practice of the present invention are disclosed in U.S. Patents 5,849,481, 5,849,486 and 5,851,772. The relevant portions of these and other references identified in this section of the Specification are incorporated herein by reference.
  • Nucleic acids used as a template for amplification may be isolated from cells, tissues or other samples according to standard methodologies (Sambrook et ah, 2001). hi certain embodiments, analysis is performed on whole cell or tissue homogenates or biological fluid samples with or without substantial purification of the template nucleic acid.
  • the nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to first convert the RNA to a complementary DNA.
  • primer is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process.
  • primers are oligonucleotides from ten to twenty and/or thirty base pairs in length, but longer sequences can be employed.
  • Primers may be provided in double-stranded and/or single-stranded form, although the single-stranded form is preferred. Pairs of primers designed to selectively hybridize to nucleic acids corresponding to the UGTlAl gene locus, or variants thereof, and fragments thereof are contacted with the template nucleic acid under conditions that permit selective hybridization.
  • high stringency hybridization conditions may be selected that will only allow hybridization to sequences that are completely complementary to the primers. In other embodiments, hybridization may occur under reduced stringency to allow for amplification of nucleic acids that contain one or more mismatches with the primer sequences.
  • the template- primer complex is contacted with one or more enzymes that facilitate template- dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles," are conducted until a sufficient amount of amplification product is produced.
  • the amplification product may be detected, analyzed or quantified. In certain applications, the detection may be performed by visual means. hi certain applications, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical and/or thermal impulse signals (Affymax technology; Bellus, 1994).
  • PCRTM polymerase chain reaction
  • LCR ligase chain reaction
  • European Application No. 320 308 incorporated herein by reference in its entirety.
  • U.S. Patent 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.
  • a method based on PCRTM and oligonucleotide ligase assay (OLA) (described in further detail below), disclosed in U.S. Patent 5,912,148, may also be used.
  • An isothermal amplification method in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[alpha-thio]-triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention (Walker et al., 1992).
  • Strand Displacement Amplification (SDA) disclosed in U.S. Patent 5,916,779, is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation.
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al., 1989; PCT Application WO 88/10315, incorporated herein by reference in their entirety).
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR 3SR
  • European Application 329 822 disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA”), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention.
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter region/primer sequence to a target single-stranded DNA ("ssDNA”) followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts.
  • Other amplification methods include "RACE” and “one-sided PCR” (Frohman, 1990; Ohara et ⁇ /., 1989).
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et al, 2001). Separated amplification products may be cut out and eluted from the gel for further manipulation. Using low melting point agarose gels, the separated band may be removed by heating the gel, followed by extraction of the nucleic acid.
  • Separation of nucleic acids may also be effected by spin columns and/or chromatographic techniques known in art.
  • chromatographic techniques There are many kinds of chromatography which may be used in the practice of the present invention, including adsorption, partition, ion-exchange, hydroxylapatite, molecular sieve, reverse-phase, column, paper, thin-layer, and gas chromatography as well as HPLC.
  • the amplification products are visualized, with or without separation.
  • a typical visualization method involves staining of a gel with ethidium bromide and visualization of bands under UV light.
  • the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the separated amplification products can be exposed to x-ray film or visualized under the appropriate excitatory spectra.
  • a labeled nucleic acid probe is brought into contact with the amplified marker sequence.
  • the probe preferably is conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antibody or biotin, or another binding partner carrying a detectable moiety.
  • detection is by Southern blotting and hybridization with a labeled probe.
  • the techniques involved in Southern blotting are well known to those of skill in the art (see Sambrook et al. , 2001).
  • U.S. Patent 5,279,721, incorporated by reference herein discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids. The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.
  • DGGE denaturing gradient gel electrophoresis
  • RFLP restriction fragment length polymorphism analysis
  • SSCP single-strand conformation polymorphism analysis
  • mismatch is defined as a region of one or more unpaired or mispaired nucleotides in a double-stranded RNA/RNA, RNA/DNA or DNA/DNA molecule. This definition thus includes mismatches due to insertion/deletion mutations, as well as single or multiple base point mutations.
  • U.S. Patent 4,946,773 describes an RNase A mismatch cleavage assay that involves annealing single-stranded DNA or RNA test samples to an RNA probe, and subsequent treatment of the nucleic acid duplexes with RNase A.
  • the single-stranded products of the RNase A treatment electrophoretically separated according to size, are compared to similarly treated control duplexes. Samples containing smaller fragments (cleavage products) not seen in the control duplex are scored as positive.
  • RNase I in mismatch assays.
  • the use of RNase I for mismatch detection is described in literature from Promega Biotech. Promega markets a kit containing RNase I that is reported to cleave three out of four known mismatches. Others have described using the MutS protein or other DNA-repair enzymes for detection of single-base mismatches.
  • VNTRs variable nucleotide type polymorphisms
  • RFLPs restriction fragment length polymorphisms
  • SNPs single nucleotide polymorphisms
  • SNPs single nucleotide polymorphisms
  • SNPs are the most common genetic variations and occur once every 100 to 300 bases and several SNP mutations have been found that affect a single nucleotide in a protein-encoding gene in a manner sufficient to actually cause a genetic disease.
  • SNP diseases are exemplified by hemophilia, sickle-cell anemia, hereditary hemochromatosis, late-onset alzheimer disease etc.
  • SNPs can be the result of deletions, point mutations and insertions and in general any single base alteration, whatever the cause, can result in a SNP.
  • the greater frequency of SNPs means that they can be more readily identified than the other classes of polymorphisms.
  • the greater uniformity of their distribution permits the identification of SNPs "nearer" to a particular trait of interest.
  • the combined effect of these two attributes makes SNPs extremely valuable. For example, if a particular trait (e.g., inability to efficiently metabolize irinotecan) reflects a mutation at a particular locus, then any polymorphism that is linked to the particular locus can be used to predict the probability that an individual will exhibit that trait.
  • SNPs or other polymorphisms relating to UGTlAl, or any other UGTl enzyme can be characterized by the use of any of these methods or suitable modification thereof.
  • Such methods include the direct or indirect sequencing of the site, the use of restriction enzymes where the respective alleles of the site create or destroy a restriction site, the use of allele-specific hybridization probes, the use of antibodies that are specific for the proteins encoded by the different alleles of the polymorphism, or any other biochemical interpretation.
  • the most commonly used method of characterizing a polymorphism is direct DNA sequencing of the genetic locus that flanks and includes the polymorphism. Such analysis can be accomplished using either the "dideoxy-mediated chain termination method,” also known as the “Sanger Method” (Sanger et ah, 1975) or the “chemical degradation method,” also known as the “Maxam-Gilbert method” (Maxam et ah, 1977). Sequencing in combination with genomic sequence-specific amplification technologies, such as the polymerase chain reaction may be utilized to facilitate the recovery of the desired genes (Mullis et ah, 1986; European Patent Application 50,424; European Patent Application. 84,796, European Patent Application 258,017, European Patent Application. 237,362; European Patent Application. 201,184; U.S. Patents 4,683,202; 4,582,788; and 4,683,194), all of the above incorporated herein by reference. ii) Exonuclease Resistance
  • French Patent 2,650,840 and PCT Application WO91/02087 discuss a solution-based method for determining the identity of the nucleotide of a polymorphic site.
  • a primer complementary to allelic sequences immediately 3'-to a polymorphic site is used.
  • the identity of the nucleotide of that site is determined using labeled dideoxynucleotide derivatives which are incorporated at the end of the primer if complementary to the nucleotide of the polymorphic site.
  • v) Genetic Bit Analysis or Solid-Phase Extension PCT Application WO92/15712 describes a method that uses mixtures of labeled terminators and a primer that is complementary to the sequence 3' to a polymorphic site.
  • the labeled terminator that is incorporated is complementary to the nucleotide present in the polymorphic site of the target molecule being evaluated and is thus identified.
  • the primer or the target molecule is immobilized to a solid phase.
  • oligonucleotides capable of hybridizing to abutting sequences of a single strand of a target DNA are used.
  • One of these oligonucleotides is biotinylated while the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate. Ligation permits the recovery of the labeled oligonucleotide by using avidin.
  • Other nucleic acid detection assays, based on this method, combined with PCR have also been described (Nickerson et al., 1990).
  • Invasive cleavage reactions can be used to evaluate cellular DNA for a particular polymorphism.
  • a technology called INVADER® employs such reactions ⁇ e.g., de Arruda et al, 2002; Stevens et al, 2003, which are incorporated by reference).
  • upstream oligo an oligonucleotide upstream of the target site
  • probe a probe oligonucleotide covering the target site
  • target a single-stranded DNA with the the target site
  • the upstream oligo and probe do not overlap but they contain contiguous sequences.
  • the probe contains a donor fluorophore, such as fluoroscein, and an acceptor dye, such as Dabcyl.
  • a donor fluorophore such as fluoroscein
  • an acceptor dye such as Dabcyl
  • the nucleotide at the 3' terminal end of the upstream oligo overlaps ("invades") the first base pair of a probe-target duplex. Then the probe is cleaved by a structure-specific 5' nuclease causing separation of the fluorophore/quencher pair, which increases the amount of fluorescence that can be detected. See Lu et al., 2004. In some cases, the assay is conducted on a solid-surface or in an array format. ix) Other Methods To Detect SNPs
  • extended haplotypes may be determined at any given locus in a population, which allows one to identify exactly which SNPs will be redundant and which will be essential in association studies.
  • the latter is referred to as 'haplotype tag SNPs (htSNPs)', markers that capture the haplotypes of a gene or a region of linkage disequilibrium.
  • htSNPs 'haplotype tag SNPs
  • the VDA-assay utilizes PCR amplification of genomic segments by long PCR methods using TaKaRa LA Taq reagents and other standard reaction conditions.
  • the long amplification can amplify DNA sizes of about 2,000-12,000 bp.
  • Hybridization of products to variant detector array (VDA) can be performed by a Affymetrix High Throughput Screening Center and analyzed with computerized software.
  • Chip Assay uses PCR amplification of genomic segments by standard or long PCR protocols. Hybridization products are analyzed by VDA, Halushka et al. (1999), incorporated herein by reference. SNPs are generally classified as “Certain” or “Likely” based on computer analysis of hybridization patterns. By comparison to alternative detection methods such as nucleotide sequencing, “Certain” SNPs have been confirmed 100% of the time; and “Likely” SNPs have been confirmed 73% of the time by this method. Other methods simply involve PCR amplification following digestion with the relevant restriction enzyme. Yet others involve sequencing of purified PCR products from known genomic regions.
  • individual exons or overlapping fragments of large exons are PCR-amplified.
  • Primers are designed from published or database sequences and PCR-amplification of genomic DNA is performed using the following conditions: 200 ng DNA template, 0.5 ⁇ M each primer, 80 ⁇ M each of dCTP, dATP, dTTP and dGTP, 5% formamide, 1.5mM MgCl 2 , 0.5U of Taq polymerase and 0.1 volume of the Taq buffer.
  • PCR-SSCP PCR-single strand conformation polymorphism
  • filters that: (i) exclude sequences in any given slice from SNP consideration where neighboring sequence quality scores drop 40% or more; (ii) exclude calls in which peak amplitude is below the fifteenth percentile of all base calls for that nucleotide type; (iii) disqualify regions of a sequence having a high number of disagreements with the consensus from participating in SNP calculations; (iv) removed from consideration any base call with an alternative call in which the peak takes up 25% or more of the area of the called peak; (v) exclude variations that occur in only one read direction.
  • PHRED quality scores were converted into probability-of-error values for each nucleotide in the slice.
  • Standard Baysian methods are used to calculate the posterior probability that there is evidence of nucleotide heterogeneity at a given location.
  • CU-RDF RESEQ
  • PCR amplification is performed from DNA isolated from blood using specific primers for each SNP, and after typical cleanup protocols to remove unused primers and free nucleotides, direct sequencing using the same or nested primers.
  • DEBNICK DEBNICK
  • DEBNICK (METHOD-C)
  • no mismatches in 5 bases 5' and 3' to the SNP at least two occurrences of each allele is performed and confirmed by examining traces.
  • ERO ERO
  • new primers sets are designed for electronically published STSs and used to amplify DNA from 10 different mouse strains.
  • the amplification product from each strain is then gel purified and sequenced using a standard dideoxy, cycle sequencing technique with 33 P-labeled terminators.
  • AU the ddATP terminated reactions are then loaded in adjacent lanes of a sequencing gel followed by all of the ddGTP reactions and so on. SNPs are identified by visually scanning the radiographs.
  • ERO RESEQ-HT
  • new primers sets are designed for electronically published murine DNA sequences and used to amplify DNA from 10 different mouse strains.
  • the amplification product from each strain is prepared for sequencing by treating with Exonuclease I and Shrimp Alkaline Phosphatase. Sequencing is performed using ABI Prism Big Dye Terminator Ready Reaction Kit (Perkin-Elmer) and sequence samples are run on the 3700 DNA Analyzer (96 Capillary Sequencer).
  • FGU-CBT (SCA2-SNP) identifies a method where the region containing the SNP were PCR amplified using the primers SCA2-FP3 and SCA2-RP3. Approximately 100 ng of genomic DNA is amplified in a 50 ml reaction volume containing a final concentration of 5mM Tris, 25mM KCl, 0.75mM MgCl 2 , 0.05% gelatin, 20pmol of each primer and 0.5U of Taq DNA polymerase.
  • Samples are denatured, annealed and extended and the PCR product is purified from a band cut out of the agarose gel using, for example, the QlAquick gel extraction kit (Qiagen) and is sequenced using dye terminator chemistry on an ABI Prism 377 automated DNA sequencer with the PCR primers.
  • Qiagen QlAquick gel extraction kit
  • two independent PCR reactions are performed with genomic DNA. Products from the first reaction are analyzed by sequencing, indicating a unique Fspl restriction site. The mutation is confirmed in the product of the second PCR reaction by digesting with Fsp I.
  • SNPs are identified by comparing high quality genomic sequence data from four randomly chosen individuals by direct DNA sequencing of PCR products with dye-terminator chemistry (see Kwok et al., 1996).
  • SNPs are identified by comparing high quality genomic sequence data from overlapping large-insert clones such as bacterial artificial chromosomes (BACs) or Pl-based artificial chromosomes (PACs). An STS containing this SNP is then developed and the existence of the SNP in various populations is confirmed by pooled DNA sequencing (see Taillon-Miller et al., 1998).
  • SNPs are identified by comparing high quality genomic sequence data from overlapping large-insert clones BACs or PACs.
  • the SNPs found by this approach represent DNA sequence variations between the two donor chromosomes but the allele frequencies in the general population have not yet been determined.
  • SNPs are identified by comparing high quality genomic sequence data from a homozygous DNA sample and one or more pooled DNA samples by direct DNA sequencing of PCR products with dye- terminator chemistry.
  • the STSs used are developed from sequence data found in publicly available databases.
  • these STSs are amplified by PCR against a complete hydatidiform mole (CHM) that has been shown to be homozygous at all loci and a pool of DNA samples from 80 CEPH parents (see Kwok et al., 1994).
  • CHM complete hydatidiform mole
  • KWOK OverlapSnpDetectionWithPolyBayes
  • SNPs are discovered by automated computer analysis of overlapping regions of large-insert human genomic clone sequences.
  • clone sequences are obtained directly from large-scale sequencing centers. This is necessary because base quality sequences are not present/available through GenBank.
  • Raw data processing involves analyzed of clone sequences and accompanying base quality information for consistency. Finished ('base perfect', error rate lower than 1 in 10,000 bp) sequences with no associated base quality sequences are assigned a uniform base quality value of 40 (1 in 10,000 bp error rate). Draft sequences without base quality values are rejected. Processed sequences are entered into a local database.
  • a version of each sequence with known human repeats masked is also stored. Repeat masking is performed with the program "MASKERAID.” Overlap detection: Putative overlaps are detected with the program "WUBLAST.” Several filtering steps followed in order to eliminate false overlap detection results, i.e. similarities between a pair of clone sequences that arise due to sequence duplication as opposed to true overlap. Total length of overlap, overall percent similarity, number of sequence differences between nucleotides with high base quality value "high-quality mismatches.” Results are also compared to results of restriction fragment mapping of genomic clones at Washington University Genome Sequencing Center, finisher's reports on overlaps, and results of the sequence contig building effort at the NCBI.
  • SNP detection Overlapping pairs of clone sequence are analyzed for candidate SNP sites with the 'POLYBAYES' SNP detection software. Sequence differences between the pair of sequences are scored for the probability of representing true sequence variation as opposed to sequencing error. This process requires the presence of base quality values for both sequences. High-scoring candidates are extracted. The search is restricted to substitution-type single base pair variations. Confidence score of candidate SNP is computed by the POLYBAYES software. hi method identified by KWOK (TaqMan assay), the TaqMan assay is used to determine genotypes for 90 random individuals. hi method identified by KYUGEN(Ql) 5 DNA samples of indicated populations are pooled and analyzed by PLACE-SSCP.
  • KYUGEN Method identified as KYUGEN (Methodl)
  • PCR products are post-labeled with fluorescent dyes and analyzed by an automated capillary electrophoresis system under SSCP conditions (PLACE-SSCP).
  • Samples of DNA (10 ng/ul) are amplified in reaction mixtures containing the buffer (1OmM Tris-HCl, pH 8.3 or 9.3, 5OmM KCl, 2.OmM MgCl 2 ), 0.25 ⁇ M of each primer, 200 ⁇ M of each dNTP, and 0.025 units/ ⁇ l of Taq DNA polymerase premixed with anti-Taq antibody.
  • the two strands of PCR products are differentially labeled with nucleotides modified with RIlO and R6G by an exchange reaction of Klenow fragment of DNA polymerase I.
  • the reaction is stopped by adding EDTA 5 and unincorporated nucleotides are dephosphorylated by adding calf intestinal alkaline phosphatase.
  • SSCP For the SSCP: an aliquot of fluorescently labeled PCR products and TAMRA-labeled internal markers are added to deionized formamide, and denatured. Electrophoresis is performed in a capillary using an ABI Prism 310 Genetic Analyzer. Genescan softwares (P-E Biosystems) are used for data collection and data processing. DNA of individuals (two to eleven) including those who showed different genotypes on SSCP are subjected for direct sequencing using big-dye terminator chemistry, on ABI Prism 310 sequencers. Multiple sequence trace files obtained from ABI Prism 310 are processed and aligned by Phred/Phrap and viewed using Consed viewer. SNPs are identified by PolyPhred software and visual inspection.
  • KYUGEN Method 2
  • individuals with different genotypes are searched by denaturing HPLC (DHPLC) or PLACE-SSCP (hiazuka et al., 1997) and their sequences are determined to identify SNPs.
  • PCR is performed with primers tagged with 5'-ATT or 5'-GTT at their ends for post-labeling of both strands.
  • DHPLC analysis is carried out using the WAVE DNA fragment analysis system (Transgenomic). PCR products are injected into DNASep column, and separated under the conditions determined using WAVEMaker program (Transgenomic).
  • the reaction is stopped by adding EDTA, and unincorporated nucleotides are dephosphorylated by adding calf intestinal alkaline phosphatase.
  • SSCP followed by electrophoresis is performed in a capillary using an ABI Prism 310 Genetic Analyzer. Genescan softwares (P-E Biosystems). DNA of individuals including those who showed different genotypes on DHPLC or SSCP are subjected for direct sequencing using big-dye terminator chemistry, on ABI Prism 310 sequencer.
  • SNPs are identified by PolyPhred software and visual inspection. Trace chromatogram data of EST sequences in Unigene are processed with PHRED. To identify likely SNPs, single base mismatches are reported from multiple sequence alignments produced by the programs PHRAP, BRO and POA for each Unigene cluster. BRO corrected possible misreported EST orientations, while POA identified and analyzed non-linear alignment structures indicative of gene mixing/chimeras that might produce spurious SNPs.
  • Bayesian inference is used to weigh evidence for true polymorphism versus sequencing error, misalignment or ambiguity, misclustering or chimeric EST sequences, assessing data such as raw chromatogram height, sharpness, overlap and spacing; sequencing error rates; context-sensitivity; cDNA library origin, etc.
  • MARSHFIELD Method identified as MARSHFIELD(Method-B)
  • overlapping human DNA sequences which contained putative insertion/deletion polymorphisms are identified through searches of public databases.
  • PCR primers which flanked each polymorphic site are selected from the consensus sequences.
  • Primers are used to amplify individual or pooled human genomic DNA. Resulting PCR products are resolved on a denaturing polyacrylamide gel and a Phosphorhnager is used to estimate allele frequencies from DNA pools.
  • Linkage disequilibrium (“LD” as used herein, though also referred to as “LED” in the art) refers to a situation where a particular combination of alleles (i.e., a variant form of a given gene) or polymorphisms at two loci appears more frequently than would be expected by chance.
  • Haplotype is used according to its plain and ordinary meaning to one skilled in the art. It refers to a collective genotype of two or more alleles or polymorphisms along one of the homologous chromosomes. 03846
  • L-thyroxine is commercially available as SYNTHROID®.
  • SYNTHROID® is supplied as a tablet containing synthetic crystalline L-3,3',5,5'-tetraiodothyronine sodium salt [levoxythyroxine (T 4 ) sodium].
  • Synthetic T 4 is chemically identical to the hormone produced in the thyroid gland of a human.
  • SYNTHROID® typically, a patient takes a single daily dose of SYNTHROID®. Dosages of SYNTHROID® include 100-125 meg ( ⁇ g)/day for a 70 kg adult. Occasionally, patients take doses above that, such as between 200 and 300 ⁇ g/day, or lower.
  • Average daily dosages that are taken include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,
  • a typical amount for an adult patient is adjusted based on the predicted L-thyroxine glucuronidation activity in the patient. If the activity is relatively high, the dosage will be higher than the dosage for a patient whose activity is predicted to be relatively low or at least lower. It is contemplated that dosages may be adjusted by timing as well so that the patient effectively receives relatively more or less on an average daily basis.
  • doses can be adjusted to be less than or more than the concentrations discussed above or less frequently or more frequently than the timing discussed above. It is contemplated treatment cycles may be repeated and that there may be a respite between cycles.
  • dosages regimens One of ordinary skill in the art is familiar with dosages regimens.
  • the overall amount of the drug administered to the patient in a single regimen or for the treatment overall may be increased or decreased by about, by at least about, or by at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000 meg/day, or any ranges derivable therein.
  • dose and "dosage" are used interchangeably as synonyms herein.
  • L-thyroxine that are administered to patients is well known to those of skill in the art. These dosages may be reduced or increased relative to a dosage that would have been adminstered in the absence of genotyping. It is specifically contemplated that the dosages of any of those drugs may be similarly altered or modified based on genotypic analysis described herein.
  • compositions described herein may be comprised in a kit.
  • reagents for determining the sequence or number one or more polymorphisms are included in a kit.
  • the kit includes reagents for determining the number of TA repeats in the upstream region of the UGTlAl gene.
  • kits may include nucleic acids for amplifying, priming, or hybridizing to any of the polymorphisms discussed herein, such as the following: UGTlAl -3156G>A, UGTlAl -3279T>G, UGTlAl 211G>A, UGTlAl 686OA, UGT1A3 17(A>G), UGT1A3 31(T>C), UGT1A3 81(G>A), UGT1A3 133(OT), UGTl A3 140(T>C), or UGTl A3 477(A>G), UGTl A8 518(OG), UGTl A8 765(A>G), UGTl A8 83O(G>A), UGTl A9 -118(T) 9 >IQ, UGTl A9 -275T>A, and UGT1A9 -2152OT.
  • nucleic acids for amplifying, priming, or hybridizing to any of the polymorphisms discussed herein, such as
  • kits for implementing methods of the invention described herein are specifically contemplated.
  • the kits will thus comprise in some embodiments, in suitable container means, one or more of the following: an enzyme for amplifying or detecting a nucleic acid sequence with the polymorphism or capable of detecting whether a nucleic acid sequence has or does not have the polymorphism, one or more buffers, such as reaction buffer, or a hybridization buffer, compounds for preparing amplified nucleic acid sequences or detecting nucleic acid sequences, and components for isolating sequences.
  • an enzyme for amplifying or detecting a nucleic acid sequence with the polymorphism or capable of detecting whether a nucleic acid sequence has or does not have the polymorphism one or more buffers, such as reaction buffer, or a hybridization buffer, compounds for preparing amplified nucleic acid sequences or detecting nucleic acid sequences, and components for isolating sequences.
  • kits of the invention may include components for making a nucleic acid array comprising polymorphic sequences or sequences that will allow the detection of polymorphisms and/or the absence of such polymorphisms, and thus, may include, for example, a solid support.
  • kits may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial.
  • the kits of the present invention also will typically include a means for containing the nucleic acids, and any other reagent containers in close confinement for commercial sale. 6 003846
  • Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
  • the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the components of the kit may be provided as dried powder(s).
  • the powder can be reconstituted by the addition of a suitable solvent.
  • the solvent may also be provided in another container means.
  • labeling dyes are provided as a dried power. It is contemplated that 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 ⁇ g or at least or at most those amounts of dried dye are provided in kits of the invention.
  • the dye may then be resuspended in any suitable solvent, such as DMSO.
  • the container means will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the nucleic acid formulations are placed, preferably, suitably allocated.
  • the kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.
  • the kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection and/or blow-molded plastic containers into which the desired vials are retained.
  • kits may also include components that facilitate isolation of nucleic acids. It may also include components that preserve or maintain the nucleic acids or that protect against their degradation. Such components may be nuclease-free or protect against nucleases, such as DNAses or RNAses.
  • Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or solution.
  • kits will also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented.
  • Kits of the invention may also include one or more of the following: Control nucleic acids; nuclease-free water; PEG or dextran; ethanol; acetic acid; sodium acetate; ammonium acetate; guanidinium; detergent; nucleic acid size marker; tube tips; and RNase or DNase inhibitors.
  • kits of the invention are embodiments of kits of the invention.
  • Such kits are not limited to the particular items identified above and may include any reagent used for the manipulation or characterization of nucleic acids that determine or detect polymorphisms discussed herein.
  • the highest T 4 G formation was observed with 1A3 followed by 1A8, IAl, IAlO, 1A9, and 1A7 and was undetectable with 1A4 and 1A6.
  • HLMs human liver microsomes
  • Microsomes from cDNA-transfected baculovirus-insect cells expressing human UGTs (supersomes) and control insect cell microsomes from wild-type baculo virus-infected cells were obtained from BD Gentest (Woburn, MA).
  • HLMs and supersomes were screened for T4 glucuronidation levels by incubation with T4 under the following conditions: 0.2 mg of HLMs, 5 mM UDPGA, 1 mM MgCl 2 , 5 mM sacharolactone, 10 ⁇ g alamethicin, and 100 mM Tris-HCl pH 7.1 in a final volume of 200 ⁇ L.
  • AU screening and correlation incubations were performed with 91 ⁇ M T4.
  • Kinetics experiments in HLMs were performed with 0- 300 ⁇ M T4. Incubations were carried out for 90 minutes at 37°C.
  • T 4 G was measured by HPLC. HPLC was implemented as follows:
  • the K m for T4 in HLMs was determined to be 91 ⁇ M.
  • UGT screening showed UGTl A8 to have the highest T4G formation followed by UGTlAl > 1A3 > IAlO > 1A7 > 1A9 (FIG. 2).
  • UGTlAlO, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 as well as control insect cell microsomes from wild-type baculovirus-infected cells were obtained from BD Gentest (Woburn, MA).
  • Rabbit anti-human UGTlA primary and secondary antibodies were also purchased from BD Gentest.
  • Pre-cast 10% acrylamide gels were obtained from BioRad (Hercules, CA). Blotto was purchased from Pierce (Rockford,
  • Chemiluminescence reagents were obtained from Amersham Biosciences
  • T 4 incubations with HLM described above were dried down under nitrogen gas and reconstituted in 0.2 mL of 0.1 M sodium phosphate buffer, pH 6.4 containing 2000 units of /3-glucuronidase (Type VII, from Escherichia coli, Sigma, St Louis, MO) and incubated for 24 hours at 37°C. The reaction was stopped with 0.4 mL of ice-cold methanol. Samples were processed and analyzed by HPLC as described above. Control incubations containing no /3-glucuror ⁇ dase were treated identically. Incubation with /3-glucuronidase resulted in the disappearance of T 4 G peaks. T 4 G peaks were observed in control incubations with the buffer when /S- glucuronidase was absent (data not shown).
  • T 4 G formation was measured with a modification of a method previously published (Jemnitz and Vereczkey, 1996). Separation was achieved using a reverse- phase ⁇ Bondapak C 18 column (10 ⁇ M, 3.9 X 300 mm, Waters Corp., Milford, MA). The mobile phase consisted of acetonitrile (A) and 20 mM potassium phosphate monobasic, pH 2 (B). The following gradient was applied at a flow rate of 1 mL/min: 0-25 min 25% A and 75% B, 25.1-30 min from 25% A and 75% B to 70% A and 30% B, 30.1-45 min 70% A and 30% B, and 45.1-55 min 25% A and 75% B. The eluate was monitored using UV detection at 234 nm. Retention times for CPT-Il, T 4 G, and T 4 were 11.4, 16.0 and 33.4 min, respectively.
  • T 4 G is not commercially available, so T 4 G formation was calculated using a T 4 standard curve made by combining T 4 stock in DMSO (0.1% final concentration) with incubation buffer, incubated at 37°C for 90 min and processed as described above. The quantitation limit was 32.2 nM. hitra-assay reproducibility was determined by measuring T 4 G in ten separate incubations with the same HLM pool. The coefficient of variation was 3.2%. Inter-assay reproducibility was determined by incubating four pooled HLM samples each day for three days. The coefficient of variation was 10.2%. Screening of Recombinant Microsomes Expressing Human UGT Isoforms for T 4 G Formation
  • UGTlAlO UGTlAlO
  • the membrane was washed with tris buffered saline containing 0.05% tween 20 and incubated with horseradish peroxidase conjugated goat anti-rabbit secondary antibody (1:500 dilution in blotto) for 1 hour. Immunocomplexes were visualized with chemiluminescence using a ChemiDoc (BioRad, Hercules, CA) and quantified by Biolmage Visage 110s (Genomic Solution Inc., Ann Arbor, Michigan).
  • liver samples had been previously genotyped for UGTlAl -53(TA) 6>7 (UGTlAl *28) and UGT1A9 -118T 9>10 (UGT1A9*22) polymorphisms (Innocenti et al, 2002; Innocenti et al, 2005).
  • T 4 Glucuronidation in HLM T 4 G formation rates are linear with microsomal protein concentration up to 1.5 mg/mL and time up to 2 hours.
  • T 4 glucuronidation follows Michaelis-Menten kinetics through 300 ⁇ M T 4 .
  • T 4 G formation slowly decreased at T 4 concentrations higher than 300 ⁇ M.
  • kinetic parameters were determined using the Michaelis- Menten equation for 10-300 ⁇ M.
  • the apparent K m , V max and intrinsic clearance were determined to be 85.7 ⁇ 14.6 ⁇ M (mean ⁇ SE), 6.7 ⁇ 0.4 pmol/min/mg (mean ⁇ SE), and 0.078 ml/min/mg (FIG. 6).
  • Interindividual variation in T 4 G formation was analyzed in 54 Caucasian HLMs.
  • the median T 4 G formation was 2.6 pmol/min/mg, range 0.8 - 10.5 pmol/min/mg.
  • Minor T 4 G production was observed with UGTl A7 (4.7 pmol/min/mg protein) and UGTl A9 (4.1 pmol/min/mg protein).
  • Negligible T 4 glucuronidation was observed with UGT1A4, UGT1A6, UGT2B4, UGT2B7, UGT2B15 and UGT2B17 (FIG. 7A- B).
  • V max was normalized for relative protein expression by western blot.
  • the UGTlA standard was designated as the basal UGT expression level.
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. AU such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • Patent 5,910,407 U.S. Patent 5,912,124 U.S. Patent 5,912,145 U.S. Patent 5,912,148 U.S. Patent 5,916,776 U.S. Patent 5,916,779 U.S. Patent 5,919,626 U.S. Patent 5,919,630 U.S. Patent 5,922,574 U.S. Patent 5,925,517 U.S. Patent 5,925,525 U.S. Patent 5,928,862 U.S. Patent 5,928,869 U.S. Patent 5,928,870 U.S. Patent 5,928,905 U.S. Patent 5,928,906 U.S. Patent 5,929,227 U.S. Patent 5,932,413 U.S. Patent 5,932,451 U.S.

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Abstract

La présente invention concerne des méthodes et des compositions permettant de déterminer le taux de glucuronidation de la L-thyroxine chez un patient. Dans certains exemples de l'invention, le nombre de répétitions TA dans le gène UGT1A1 est déterminé ou évalué de façon à prédire le taux d'activité. Les méthodes et les compositions de l'invention sont utilisées pour optimiser le dosage de la L-thyroxine, un médicament connu pour le traitement de maladies thyroïdiennes, y compris l'hyperthyroïdie.
PCT/US2006/003846 2005-02-01 2006-02-01 Methodes et compositions destinees au dosage de la l-thyroxine Ceased WO2006084133A2 (fr)

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Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
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BOSMA P J ET AL: "THE GENETIC BASIS OF THE REDUCED EXPRESSION OF BILIRUBIN UDP-GLUCURONOSYLTRANSFERASE 1 IN GILBERT'S SYNDROME" NEW ENGLAND JOURNAL OF MEDICINE, THE, MASSACHUSETTS MEDICAL SOCIETY, WALTHAM, MA, US, vol. 333, no. 18, 2 November 1995 (1995-11-02), pages 1171-1175, XP002040437 ISSN: 0028-4793 cited in the application *
FANG J-L ET AL: "corellation between the UDP-Glucuronosyltransferase (UGT1a1) TATAA box polymorphism and carcinogen detoxification phenotype: significantly decreased glucuronidating activity against Benzo(a) pyrene-7,8-dihydrodiol(-) in liver microsomes from subjects with UGT1A1*28 variant" CANCER EPIDEMIOLOGY, BIOMARKERS AND PREVENTION, AMERICAN ASSOCIATION FOR CANCER RESEARCH,, US, vol. 13, January 2004 (2004-01), pages 102-109, XP002331168 ISSN: 1055-9965 *
FINDLAY K A ET AL: "Characterization of the uridine diphosphate-glucuronosyltransferase-c atalyzing thyroid hormone glucuronidation in man." THE JOURNAL OF CLINICAL ENDOCRINOLOGY AND METABOLISM. AUG 2000, vol. 85, no. 8, August 2000 (2000-08), pages 2879-2883, XP002392384 ISSN: 0021-972X cited in the application *
IYER L ET AL: "PHENOTYPE-GENPTYPE CORRELATION OF IN VITRO SN-38 (ACTIVE METABOLITE OF IRINOTECAN) AND BILIRUBIN GLUCURONIDATION IN HUMAN LIVER TISSUE WITH UGT1A1 PROMOTER POLYMORPHISM" CLINICAL PHARMACOLOGY & THERAPEUTICS, MOSBY-YEAR BOOK, ST LOUIS, MO, US, vol. 65, no. 5, 1999, pages 576-582, XP002909099 ISSN: 0009-9236 cited in the application *
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