WO2001079552A1 - Haplotypes du gene hsd3b1 - Google Patents

Haplotypes du gene hsd3b1 Download PDF

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Publication number
WO2001079552A1
WO2001079552A1 PCT/US2001/011945 US0111945W WO0179552A1 WO 2001079552 A1 WO2001079552 A1 WO 2001079552A1 US 0111945 W US0111945 W US 0111945W WO 0179552 A1 WO0179552 A1 WO 0179552A1
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hsd3b1
ofthe
gene
haplotype
individual
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PCT/US2001/011945
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English (en)
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Anne Chew
Julie Y. Choi
Beena Koshy
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Genaissance Pharmaceuticals, Inc.
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Priority to AU2001257020A priority Critical patent/AU2001257020A1/en
Publication of WO2001079552A1 publication Critical patent/WO2001079552A1/fr

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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
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • This invention relates to variation in genes that encode pharmaceutically-important proteins.
  • this invention provides genetic variants ofthe human hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1) gene and methods for identifying which variant(s) of this gene is/are possessed by an individual.
  • HSD3B1 human hydroxy-delta-5-steroid dehydrogenase
  • haplotype is the ordered combination of polymorphisms in the sequence of each form of a gene that exists in the population. Because haplotypes represent the variation across each form of a gene, they provide a more accurate and reliable measurement of genetic variation than individual polymorphisms. For example, while specific variations in gene sequences have been associated with a particular phenotype such as disease susceptibility (Roses AD supra; Ulbrecht M et al. 2000 Am JRespir Crit Care Med , 161: 469-74) and drug response (Wolfe CR et al.
  • HSD3B1 hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1) gene or its encoded product.
  • HSD3B1 is the type 1 isozyme of 3 beta-HSD, which catalyzes the formation of delta 4-3-ketosteroids from delta 5-3 beta- hydroxysteroids, an obligate step in the biosynthesis not only of androgens and estrogens but also of mineralocorticoids and glucocorticoids (Mason et al., Steroids 1997; 62:164-168).
  • HSD3B1 is expressed at high levels in syncytial trophoblast and in sebaceous glands.
  • the complexity of 3 beta-HSD expression through multiple signaling pathways acting on a multigene family of enzymes may contribute importantly to the diverse patterns and locations of steroid hormone biosynthesis. Studies have demonstrated linkage between a Bglll RFLP in the HSD3B1 gene and mutations in the HSD3B2 gene (Rheaume et al., Nat. Genet. 1992; 1:239-245).
  • HSD3B2 Mutations in the HSD3B2 gene cause classical 3 beta-HSD deficiency, which is responsible for CAHII, a severe form of congenital adrenal hyperplasia (CAH) that impairs steroidogenesis in both the adrenals and gonads (Shnard et al., J. Steroid Biochem. Mol. Biol. 1995; 53:127-138). Newborns affected by 3 beta-HSD deficiency exhibit signs and symptoms of adrenal insufficiency of varying degrees associated with pseudohermaphroditism in males.
  • CAH congenital adrenal hyperplasia
  • the hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 gene is located on chromosome lpl3-pl 1 and contains 4 exons that encode a 373 amino acid protein.
  • a reference sequence for the HSD3B1 gene is shown in Figure 1. This figure is based up GenBank Accession No. M38180.1, but has been edited to conform to the reference coding sequence (Geru3ankAccession No. NM_000862.1) shown in Figure 2.
  • a reference sequence for the protein is shown in Figure 3 (SEQ ID NO:3).
  • a polymorphism of adenine or guanine at a position corresponding to nucleotide 5771 in Figure 1 results in an amino acid variation of isoleucine or valine at a position corresponding to amino acid 79 in Figure 3 (Cargill et al., Nat. Genet. 1999; 22:231-238).
  • a polymorphism of thymine or cytosine at a position corresponding to nucleotide 8559 in Figure 1 results in an amino acid variation of phenylalanine or leucine at a position corresponding to amino acid 286 in Figure 3 (Cargill et al., Nat. Genet. 1999; 22:231-238).
  • HSD3B 1 Because of the potential for variation in the HSD3B 1 gene to affect the expression and function ofthe encoded protein, it would be useful to know whether additional polymorphisms exist in the HSD3B1 gene, as well as how such polymorphisms are combined in different copies ofthe gene. Such information could be applied for studying the biological function of HSD3B1 as well as in identifying drugs targeting this protein for the treatment of disorders related to its abnormal expression or function.
  • PS polymorphic sites
  • PSI polymorphic sites
  • PS2 corresponds to the following nucleotide positions in Figure 1 : 1854 (PSI), 1900 (PS2), 2017 (PS3), 5552 (PS4), 5564 (PS5), 5686 (PS6), 5782 (PS9) 5986 (PS11), 7958 (PS12), 8642 (PS14) and 8803 (PS16).
  • the polymorphisms at these sites are guanine or thymine at PSI, adenine or cytosine at PS2, adenine or guanine at PS3, adenine or guanine at PS4, cytosine or thymine at PS5, cytosine or guanine at PS6, cytosine or tiiymine at PS9, adenine or guanine at PSI 1, guanine or adenine at PS12, guanine or adenine at PS14 and cytosine or adenine at PS16.
  • the inventors have determined the identity ofthe alleles at these sites, as well as at the previously identified sites at nucleotide positions 5764 (PS7), 5771 (PS8), 5806 (PS10), 8559 (PS13), 8715 (PS 15) and 8864 (PS 17) in Figure 1, in a human reference population of 79 unrelated individuals self- identified as belonging to one of four major population groups: African descent, Asian, Caucasian and Hispanic/Latino. From this information, the inventors deduced a set of haplotypes and haplotype pahs for PS 1-PS 17 in the HSD3B 1 gene, which are shown below in Tables 5 and 4, respectively. Each of these HSD3B1 haplotypes defines a naturally-occurring isoform (also referred to herein as an "isogene") ofthe HSD3B1 gene that exists in the human population.
  • isogene also referred to herein as an "isogene
  • the invention provides a method, composition and kit for genotyping the HSD3B1 gene in an individual.
  • the genotyping method comprises identifying the nucleotide pah that is present at one or more polymorphic sites selected from the group consisting of PSI, PS2, PS3, PS4, PS5, PS6, PS9, PSI 1, PS12, PS14 and PS16 in both copies ofthe HSD3B1 gene from the individual.
  • a genotyping composition ofthe invention comprises an oligonucleotide probe or primer which is designed to specifically hybridize to a target region containing, or adjacent to, one of these novel HSD3B1 polymorphic sites.
  • a genotyping kit ofthe invention comprises a set of oligonucleotides designed to genotype each of these novel HSD3B1 polymorphic sites.
  • the genotyping kit comprises a set of oligonucleotides designed to genotype each of PS 1-PS 17.
  • the genotyping method, composition, and kit are useful in determining whether an individual has one ofthe haplotypes in Table 5 below or has one ofthe haplotype pahs in Table 4 below.
  • the invention also provides a method for haplotyping the HSD3B 1 gene in an individual.
  • the haplotyping method comprises determining, for one copy ofthe HSD3B1 gene, the identity ofthe nucleotide at one or more polymorphic sites selected from the group consisting of PSI, PS2, PS3, PS4, PS5, PS6, PS9, PS11, PS12, PS14 and PS16.
  • the haplotyping method comprises determining whether one copy ofthe individual's HSD3B1 gene is defined by one ofthe HSD3B1 haplotypes shown in Table 5, below, or a sub-haplotype thereof.
  • the haplotyping method comprises determining whether both copies ofthe individual's HSD3B1 gene are defined by one ofthe HSD3B1 haplotype pahs shown in Table 4 below, or a sub-haplotype pah thereof.
  • the method for establishing the HSD3B1 haplotype or haplotype pair of an individual is useful for improving the efficiency and reliability of several steps in the discovery and development of drugs for treating diseases associated with HSD3B1 activity, e.g., disorders related to steroid biosynthesis such as congenital adrenal hyperplasia.
  • the haplotyping method can be used by the pharmaceutical research scientist to validate HSD3B 1 as a candidate target for treating a specific condition or disease predicted to be associated with HSD3B 1 activity. Determining for a particular population the frequency of one or more ofthe individual HSD3B 1 haplotypes or haplotype pahs described herein will facilitate a decision on whether to pursue HSD3B1 as a target for treating the specific disease of interest. In particular, if variable HSD3B1 activity is associated with the disease, then one or more HSD3B1 haplotypes or haplotype pahs will be found at a higher frequency in disease cohorts than in appropriately genetically matched controls.
  • variable HSD3B1 activity has little, if any, involvement with that disease.
  • the pharmaceutical research scientist can, without a priori knowledge as to the phenotypic effect of any HSD3B1 haplotype or haplotype pah, apply the information derived from detecting HSD3B1 haplotypes in an individual to decide whether modulating HSD3B1 activity would be useful in treating the disease.
  • the claimed invention is also useful in screening for compounds targeting HSD3B1 to treat a specific condition or disease predicted to be associated with HSD3B1 activity. For example, detecting which ofthe HSD3B1 haplotypes or haplotype pahs disclosed herein are present in individual members of a population with the specific disease of interest enables the pharmaceutical scientist to screen for a compound(s) that displays the highest deshed agonist or antagonist activity for each ofthe most frequent HSD3B1 isoforms present in the disease population.
  • the claimed haplotyping method provides the scientist with a tool to identify lead compounds that are more likely to show efficacy in clinical trials.
  • the method for haplotyping the HSD3B1 gene in an individual is also useful in the design of clinical trials of candidate drugs for treating a specific condition or disease predicted to be associated with HSD3B1 activity. For example, instead of randomly assigning patients with the disease of interest to the treatment or control group as is typically done now, determining which ofthe HSD3B1 haplotype(s) disclosed herein are present in individual patients enables the pharmaceutical scientist to distribute HSD3B 1 haplotypes and/or haplotype pahs evenly to treatment and control groups, thereby reducing the potential for bias in the results that could be introduced by a larger frequency of a HSD3B1 haplotype or haplotype pair that had a previously unknown association with response to the drug being studied in the trial. Thus, by practicing the claimed invention, the scientist can more confidently rely on the information learned from the trial, without first determining the phenotypic effect of any HSD3B1 haplotype or haplotype pah.
  • the invention provides a method for identifying an association between a trait and a HSD3B 1 genotype, haplotype, or haplotype pah for one or more ofthe novel polymorphic sites described herein.
  • the method comprises comparing the frequency ofthe HSD3B1 genotype, haplotype, or haplotype pah in a population exhibiting the trait with the frequency ofthe HSD3B1 genotype, haplotype, or haplotype pah in a reference population.
  • a higher frequency ofthe HSD3B1 genotype, haplotype, or haplotype pah in the trait population than in the reference population indicates the trait is associated with the HSD3B1 genotype, haplotype, or haplotype pah.
  • the trait is susceptibility to a disease, severity of a disease, the staging of a disease or response to a drug.
  • the HSD3B1 haplotype is selected from the haplotypes shown in Table 5, or a sub-haplotype thereof.
  • the invention provides an isolated polynucleotide comprising a nucleotide sequence which is a polymorphic variant of a reference sequence for the HSD3B 1 gene or a fragment thereof.
  • the reference sequence comprises SEQ ID NO: 1 and the polymorphic variant comprises at least one polymorphism selected from the group consisting of thymine at PSI, cytosine at PS2, guanine at PS3, guanine at PS4, thymine at PS5, guanine at PS6, thymine at PS9, guanine at PSI 1, adenine at PS12, adenine at PS14 and adenine at PS16.
  • the polymorphic variant comprises one or more additional polymorphisms selected from the group consisting of thymine at PS7, guanine at PS8, adenine at PS10, cytosine at PS13, thymine at PS15 and thymine at PS17.
  • a particularly preferred polymorphic variant is an isogene ofthe HSD3 1 gene.
  • a HSD3B 1 isogene ofthe invention comprises guanine or thymine at PSI, adenine or cytosine at PS2, adenine or guanine at PS3, adenine or guanine at PS4, cytosine or thymine at PS5, cytosine or guanine at PS6, cytosine or thymine at PS7, adenine or guanine at PS8, cytosine or thymine at PS9, thymine or adenine at PS10, adenine or guanine at PSI 1, guanine or adenine at PS12, thymine or cytosine at PS13, guanine or adenine at PS14, cytosine or thymine at PS15, cytosine or adenine at PS16 and guanine or thymine at PS 17.
  • the invention also
  • the invention provides a polynucleotide comprising a polymorphic variant of a reference sequence for a HSD3B1 cDNA or a fragment thereof.
  • the reference sequence comprises SEQ ID NO:2 (Fig.2) and the polymorphic cDNA comprises at least one polymorphism selected from the group consisting of guanine at a position corresponding to nucleotide 150, thymine at a position corresponding to nucleotide 246, adenine at a position corresponding to nucleotide 939 and adenine at a position corresponding to nucleotide 1100.
  • the polymorphic variant comprises one or more additional polymorphisms selected from the group consisting of thymine at a position corresponding to nucleotide 228, guanine at a position corresponding to nucleotide 235, adenine at a position corresponding to nucleotide 270, cytosine at a position corresponding to nucleotide 856 and thymine at a position corresponding to nucleotide 1012.
  • a particularly preferred polymorphic cDNA variant comprises the coding sequence of a HSD3B 1 isogene defined by haplotypes 1- 2 and 4-17.
  • HSD3B1 genomic and cDNA variants are also provided by the invention. It is believed that polymorphic variants ofthe HSD3Bl-gene will be useful in studying the expression and function of HSD3B1, and in expressing HSD3B1 protein for use in screening for candidate drugs to treat diseases related to HSD3B1 activity.
  • the invention provides a recombinant expression vector comprising one ofthe polymorphic genomic variants operably linked to expression regulatory elements as well as a recombinant host cell transformed or transfected with the expression vector.
  • the recombinant vector and host cell may be used to express HSD3B1 for protein structure analysis and drug binding studies.
  • the invention provides a polypeptide comprising a polymorphic variant of a reference amino acid sequence for the HSD3B 1 protein.
  • the reference amino acid sequence comprises SEQ ID NO:3 (Fig.3) and the polymorphic variant comprises asparagine at a position corresponding to amino acid position 367.
  • the polymorphic variant also comprises at least one variant amino acid selected from the group consisting of valine at a position corresponding to amino acid position 79 and leucine at a position corresponding to amino acid position 286.
  • a polymorphic variant of HSD3B1 is useful in studying the effect ofthe variation on the biological activity of HSD3B1 as well as on the binding affinity of candidate drugs targeting HSD3B1 for the treatment of disorders related to steroid biosynthesis such as congenital adrenal hyperplasia.
  • the present invention also provides antibodies that recognize and bind to the above polymorphic HSD3B 1 protein variant. Such antibodies can be utilized in a variety of diagnostic and prognostic formats and therapeutic methods.
  • the present invention also provides nonhuman transgenic animals comprising one ofthe HSD3B 1 polymorphic genomic variants described herein and methods for producing such animals.
  • the transgenic animals are useful for studying expression ofthe HSD3B1 isogenes in vivo, for in vivo screening and testing of drugs targeted against HSD3B1 protein, and for testing the efficacy of therapeutic agents and compounds for disorders related to steroid biosynthesis such as congenital adrenal hyperplasia in a biological system.
  • the present invention also provides a computer system for storing and displaying polymorphism data determined for the HSD3B1 gene.
  • the computer system comprises a computer processing unit; a display; and a database containing the polymorphism data.
  • the polymorphism data includes the polymorphisms, the genotypes and the haplotypes identified for the HSD3B 1 gene in a reference population.
  • the computer system is capable of producing a display showing HSD3B1 haplotypes organized according to theh evolutionary relationships.
  • Figure 1 illustrates a reference sequence for the HSD3B1 gene (Genbank Accession Number M38180.98; contiguous lines; SEQ ED NO:l), with the start and stop positions of each region of coding sequence indicated with a bracket ([ or ]) and the numerical position below the sequence and the polymorphic site(s) and polymorphism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymorphic site in the sequence.
  • Figure 2 illustrates a reference sequence for the HSD3B1 coding sequence (contiguous lines; . SEQ ED NO:2) with the polymorphic site(s) and polymorphism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymorphic site in the sequence.
  • Figure 3 illustrates a reference sequence for the HSD3B1 protein (contiguous lines; SEQ ED NO:3), with the variant amino acid(s) caused by the polymorphism(s) of Figure 2 positioned below the polymorphic site in the sequence.
  • the present invention is based on the discovery of novel variants ofthe HSD3B1 gene.
  • the inventors herein discovered 19 isogenes ofthe HSD3B1 gene by characterizing the HSD3B1 gene found in genomic DNAs isolated from an Index Repository that contains immortalized cell lines from one chimpanzee and 93 human individuals.
  • the human individuals included a reference population of 79 unrelated individuals self-identified as belonging to one of four major population groups: Caucasian (22 individuals) (CA), African descent (20 individuals) (AF), Asian (20 individuals) (AS), or Hispanic/Latino (17 individuals) (HL).
  • CA Caucasian
  • AF African descent (20 individuals)
  • AS Asian (20 individuals)
  • HL Hispanic/Latino (17 individuals)
  • the Index Repository contains three unrelated indigenous American Indians (AM) (one from each of North, Central and South America), one three-generation Caucasian family (from the CEPH Utah cohort) and one two-generation African- American family.
  • AM indigenous American Indians
  • the HSD3B1 isogenes present in the human reference population are defined by haplotypes for 17 polymorphic sites in the HSD3B1 gene, 11 of which are believed to be novel.
  • the HSD3B1 polymorphic sites identified by the inventors are referred to as PS1-PS17 to designate the order in which they are located in the gene (see Table 3 below), with the novel polymorphic sites referred to as PSI, PS2, PS3, PS4, PS5, PS6, PS9, PSI 1, PS12, PS14 and PS16.
  • PSI novel polymorphic sites
  • the human genotypes and haplotypes found in the repository for the HSD3B1 gene include those shown in Tables 4 and 5, respectively.
  • the polymorphism and haplotype data disclosed herein are useful for vahdating whether HSD3B1 is a suitable target for drugs to treat disorders related to steroid biosynthesis such as congenital adrenal hyperplasia, screening for such drugs and reducing bias in clinical trials of such drugs.
  • Allele - A particular form of a genetic locus, distinguished from other forms by its particular nucleotide sequence.
  • Candidate Gene - A gene which is hypothesized to be responsible for a disease, condition, or the response to a treatment, or to be correlated with one of these.
  • Genotype An unphased 5' to 3' sequence of nucleotide pah(s) found at one or more polymorphic sites in a locus on a pah of homologous chromosomes in an individual.
  • genotype includes a full-genotype and/or a sub-genotype as described below.
  • Sub-genotype The unphased 5 ' to 3 ' sequence of nucleotides seen at a subset ofthe known polymorphic sites in a locus on a pah of homologous chromosomes in a single individual.
  • Genotyping A process for determining a genotype of an individual.
  • Haplotype A 5' to 3' sequence of nucleotides found at one or more polymorphic sites in a locus on a single chromosome from a single individual.
  • haplotype includes a full- hapldtype and/or a sub-haplotype as described below.
  • Full-haplotype The 5' to 3' sequence of nucleotides found at all known polymorphic sites in a locus on a single chromosome from a single individual.
  • Sub-haplotype The 5 ' to 3 ' sequence of nucleotides seen at a subset ofthe known polymorphic sites in a locus on a single chromosome from a single individual.
  • Haplotype pair The two haplotypes found for a locus in a single individual.
  • Haplotyping A process for determining one or more haplotypes in an individual and includes use of family pedigrees, molecular techniques and/or statistical inference.
  • Haplotype data Information concerning one or more ofthe following for a specific gene: a listing ofthe haplotype pahs in each individual in a population; a listing ofthe different haplotypes in a population; frequency of each haplotype in that or other populations, and any known associations between one or more haplotypes and a trait.
  • Isoform - A particular form of a gene, mRNA, cDNA or the protein encoded thereby, distinguished from other forms by its particular sequence and/or structure.
  • Isogene - One ofthe isoforms of a gene found in a population.
  • An isogene contains all ofthe polymorphisms present in the particular isoform ofthe gene.
  • Isolated - As applied to a biological molecule such as RNA, DNA, oligonucleotide, or protein, isolated means the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth media. Generally, the term “isolated” is not intended to refer to a complete absence of such material or to absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with the methods ofthe present invention.
  • Locus - A location on a chromosome or DNA molecule corresponding to a gene or a physical or phenotypic feature.
  • Naturally-occurring A term used to designate that the object it is applied to, e.g., naturally- occurring polynucleotide or polypeptide, can be isolated from a source in nature and which has not been intentionally modified by man.
  • Nucleotide pair The nucleotides found at a polymo ⁇ hic site on the two copies of a chromosome from an individual.
  • phased As applied to a sequence of nucleotide pahs for two or more polymo ⁇ hic sites in a locus, phased means the combination of nucleotides present at those polymo ⁇ hic sites on a single copy ofthe locus is known.
  • PS Polymorphic site
  • Polymorphism The sequence variation observed in an individual at a polymo ⁇ hic site.
  • Polymo ⁇ hisms include nucleotide substitutions, insertions, deletions and microsatellites and may, but need not, result in detectable differences in gene expression or protein function.
  • Polymorphism data Information concerning one or more ofthe following for a specific gene: location of polymo ⁇ hic sites; sequence variation at those sites; frequency of polymo ⁇ hisms in one or more populations; the different genotypes and/or haplotypes determined for the gene; frequency of one or more of these genotypes and/or haplotypes in one or more populations; . any known association(s) between a trait and a genotype or a haplotype for the gene.
  • Polymorphism Database A collection of polymo ⁇ hism data arranged in a systematic or methodical way and capable of being individually accessed by electronic or other means.
  • Polynucleotide - A nucleic acid molecule comprised of single-stranded RNA or DNA br comprised of complementary, double-stranded DNA.
  • Reference Population A group of subjects or individuals who are predicted to be representative ofthe genetic variation found in the general population.
  • the reference population represents the genetic variation in the population at a certainty level of at least 85%, preferably at least 90%, more preferably at least 95% and-even more preferably at least 99%.
  • SNP Single Nucleotide Polymorphism
  • Subject - A human individual whose genotypes or haplotypes or response to treatment or disease state are to be determined.
  • Treatment - N stimulus administered internally or externally to a subject.
  • Unphased - As applied to a sequence of nucleotide pahs for two or more polymo ⁇ hic sites in a locus, unphased means the combination of nucleotides present at those polymo ⁇ hic sites on a single copy ofthe locus is not known.
  • the invention also provides compositions and methods for detecting the novel HSD3B1 polymo ⁇ hisms and haplotypes identified herein.
  • compositions comprise at least one HSD3B1 genotyping oligonucleotide.
  • a HSD3B1 genotyping oligonucleotide is a probe or primer capable of hybridizing to a target region that is located close to, or that contains, one ofthe novel polymo ⁇ hic sites described herein.
  • the term "oligonucleotide” refers to a polynucleotide molecule having less than about 100 nucleotides.
  • a preferred oligonucleotide ofthe invention is 10 to 35 nucleotides long. More preferably, the oligonucleotide is between 15 and 30, and most preferably, between 20 and 25 nucleotides in length.
  • oligonucleotide may be comprised of any phosphorylation state of ribonucleotides, deoxyribonucleotides, and acyclic nucleotide derivatives, and other functionally equivalent derivatives.
  • oligonucleotides may have a phosphate- free backbone, which may be comprised of linkages such as carboxymethyl, acetamidate, carbamate, polyamide (peptide nucleic acid (P ⁇ A)) and the like (Varma, R. in Molecular Biology and Biotechnology, A Comprehensive Desk Reference, Ed. R. Meyers, VCH Publishers, Inc.
  • Oligonucleotides ofthe invention may be prepared by chemical synthesis using any suitable methodology known in the art, or may be derived from a biological sample, for example, by restriction digestion.
  • the oligonucleotides may be labeled, according to any technique known in the art, including use of radiolabels, fluorescent labels, enzymatic labels, proteins, haptens, antibodies, sequence tags and the like.
  • Genotyping oligonucleotides ofthe invention must be capable of specifically hybridizing to a target region of a HSD3B1 polynucleotide, i.e., a HSD3B1 isogene.
  • specific hybridization means the oligonucleotide forms an anti-parallel double-stranded structure with the target region under certain hybridizing conditions, while failing to form such a structure when incubated with a non-target region or a non-HSD3Bl polynucleotide under the same hybridizing conditions.
  • the oligonucleotide specifically hybridizes to the target region under conventional high stringency conditions.
  • a nucleic acid molecule such as an oligonucleotide or polynucleotide is said to be a "perfect” or “complete” complement of another nucleic acid molecule if every nucleotide of one ofthe molecules is complementary to the nucleotide at the corresponding position ofthe other molecule.
  • a nucleic acid molecule is "substantially complementary" to another molecule if it hybridizes to that molecule with sufficient stability to remain in a duplex form under conventional low-stringency conditions.
  • Conventional hybridization conditions are described, for example, by Sambrook J. et al., in Molecular Cloning, A Laboratory Manual, 2 nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) and by Haymes, B.D. et al. in Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985). While perfectly complementary oligonucleotides are preferred for detecting polymorphisms, departures from complete complementarity are contemplated where such departures do not prevent the molecule from specifically hybridizing to the target region.
  • an oligonucleotide primer may have a non-complementary fragment at its 5 ' end, with the remainder ofthe primer being complementary to the target region.
  • non-complementary nucleotides may be interspersed into the oligonucleotide probe or primer as long as the resulting probe or primer is still capable of specifically hybridizing to the target region.
  • Preferred genotyping oligonucleotides ofthe invention are allele-specific oligonucleotides.
  • ASO allele-specific oligonucleotide
  • allele-specificity will depend upon a variety of readily optimized stringency conditions, including salt and formamide concentrations, as well as temperatures for both the hybridization and washing steps.
  • Allele-specific oligonucleotides ofthe invention include ASO probes and ASO primers.
  • ASO probes which usually provide good discrimination between different alleles are those in which a central position ofthe oligonucleotide probe aligns with the polymo ⁇ hic site in the target region (e.g., approximately the 7 th or 8 th position in a 15mer, the 8 th or 9 th position in a 16mer, and the 10 th or 11 th position in a 20mer).
  • An ASO primer ofthe invention has a 3' terminal nucleotide, or preferably a 3' penultimate nucleotide, that is complementary to only one nucleotide of a particular SNP, thereby acting as a primer for polymerase-mediated extension only if the allele containing that nucleotide is present.
  • ASO probes and primers hybridizing to either the coding or noncoding strand are contemplated by the invention.
  • a preferred ASO probe for detecting HSD3B1 gene polymo ⁇ hisms comprises a nucleotide sequence, listed 5' to 3', selected from the group consisting of:
  • CAGAACTSCAGAACA (SEQ ID NO: 9) and its complement
  • AAGGAGAMCCTGAAG (SEQ ID NO 14) and its complement.
  • a preferred ASO primer for detecting HSD3B1 gene polymo ⁇ hisms comprises a nucleotide sequence, listed 5 ' to 3', selected from the group consisting of:
  • genotyping oligonucleotides ofthe invention hybridize to a target region located one to several nucleotides downstream of one ofthe novel polymo ⁇ hic sites identified herein. Such oligonucleotides are useful in polymerase-mediated primer extension methods for detecting one ofthe novel polymo ⁇ hisms described herein and therefore such genotyping oligonucleotides are referred to herein as "primer-extension oligonucleotides”.
  • the 3 '-terminus of a primer-extension oligonucleotide is a deoxynucleotide complementary to the nucleotide located immediately adjacent to the polymo ⁇ hic site.
  • a particularly preferred oligonucleotide primer for detecting HSD3B 1 gene polymo ⁇ hisms by primer extension terminates in a nucleotide sequence, listed 5' to 3', selected from the group consisting of:
  • GCATGTATGT (SEQ ID NO 37) CATCTCCCCC • (SEQ ID NO:38) TTTGTAGCCA (SEQ 'ID NO- 39) TGGCTTAGAT (SEQ ID NO:4 ' 0) GCCCTGACCC (SEQ ID NO 41) TCTCAAGTTC (SEQ.
  • a composition contains two or more differently labeled genotyping oligonucleotides for simultaneously probing the identity of nucleotides at two or more polymo ⁇ hic sites. It is also contemplated that primer compositions may contain two or more sets of allele-specific primer pahs to allow simultaneous targeting and amplification of two or more regions containing a polymo ⁇ hic site.
  • HSD3B1 genotyping oligonucleotides ofthe invention may also be immobilized on or synthesized on a solid surface such as a microchip, bead, or glass slide (see, e.g., WO 98/20020 and WO 98/20019). Such immobilized genotyping oligonucleotides may be used in a variety of polymo ⁇ hism detection assays, including but not limited to probe hybridization and polymerase extension assays.
  • Immobilized HSD3B1 genotyping oligonucleotides ofthe invention may comprise an ordered array of oligonucleotides designed to rapidly screen a DNA sample for polymo ⁇ hisms in multiple genes at the same time.
  • the invention provides a kit comprising at least two genotyping oligonucleotides packaged in separate containers.
  • the kit may also contain other components such as hybridization buffer (where the oligonucleotides are to be used as a probe) packaged in a separate container.
  • the kit may contain, packaged in separate containers, a polymerase and a reaction buffer optimized for primer extension mediated by the polymerase, such as PCR.
  • the above described oligonucleotide compositions and kits are useful in methods for genotyping and/or haplotyping the HSD3B1 gene in an individual.
  • the terms "HSD3B1 genotype” and “HSD3B1 haplotype” mean the genotype or haplotype contains the nucleotide pah or nucleotide, respectively, that is present at one or more ofthe novel polymo ⁇ hic sites described herein and may optionally also include the nucleotide pah or nucleotide present at one or more additional polymo ⁇ hic sites in the HSD3B1 gene.
  • the additional polymo ⁇ hic sites may be currently known polymo ⁇ hic sites or sites that are subsequently discovered.
  • One embodiment of the genotyping method involves isolating from the individual a nucleic acid sample comprising the two copies ofthe HSD3B1 gene, or a fragment thereof, that are present in the individual, and determining the identity ofthe nucleotide pah at one or more polymo ⁇ hic sites selected from the group consisting of PSI, PS2, PS3, PS4, PS5, PS6, PS9, PSI 1, PS12, PS14 and PS 16 in the two copies to assign a HSD3B1 genotype to the individual.
  • the two "copies" of a gene in an individual may be the same allele or may be different alleles.
  • the identity ofthe nucleotide pah at one or more ofthe polymo ⁇ hic sites selected from the group consisting of PS7, PS8, PS10, PS 13, PS 15 and PS 17 is also determined.
  • the genotyping method comprises determining the identity ofthe nucleotide pah at each of PS1-PS17.
  • the nucleic acid sample is isolated from a biological sample taken from the individual, such as a blood sample or tissue sample.
  • tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hah.
  • the nucleic acid sample may be comprised of genomic DNA, mRNA, or cDNA and, in the latter two cases, the biological sample must be obtained from a tissue in which the HSD3B1 gene is expressed.
  • mRNA or cDNA preparations would not be used to detect polymo ⁇ hisms located in introns or in 5' and 3' untranslated regions. If a HSD3B1 gene fragment is isolated, it must contain the polymo ⁇ hic site(s) to be genotyped.
  • One embodiment ofthe haplotyping method comprises isolating from the individual a nucleic acid sample containing only one ofthe two copies ofthe HSD3B1 gene, or a fragment thereof, that is present in the individual and determining in that copy the identity ofthe nucleotide at one or more polymo ⁇ hic sites selected from the group consisting of PSI, PS2, PS3, PS4, PS5, PS6, PS9, PS11, PS 12, PS 14 and PS 16 in that copy to assign a HSD3B 1 haplotype to the individual.
  • the nucleic acid may be isolated using any method capable of separating the two copies ofthe HSD3B1 gene or fragment such as one ofthe methods described above for preparing HSD3B1 isogenes, with targeted in vivo cloning being the preferred approach.
  • any individual clone will only provide haplotype information on one ofthe two HSD3B1 gene copies present in an individual. If haplotype information is deshed for the individual's other copy, additional HSD3B1 clones will need to be examined. Typically, at least five clones should be examined to have more than a 90% probability of haplotyping both copies ofthe HSD3B1 gene in an individual.
  • the haplotyping method also comprises identifying the nucleotide at one or more polymo ⁇ hic sites selected from the group consisting of PS7, PS8, PS10, PS13, PS15 and PS17. In a particularly preferred embodiment, the nucleotide at each of PS . 1-PS17 is identified.
  • the haplotyping method comprises determining whether an individual has one or.more ofthe HSD3B1 haplotypes shown in Table 5. This can be accomplished by identifying, for one or both copies ofthe individual's HSD3B1 gene, the phased sequence of nucleotides present at each of PS1-PS17.
  • the present invention also contemplates that typically only a subset of PS 1-PS 17 will need to be directly examined to assign to an individual one or more ofthe haplotypes shown in Table 5. This is because at least one polymo ⁇ hic site in a gene is frequently in strong linkage disequilibrium with one or more other polymo ⁇ hic sites in that gene (Drysdale, CM et al.
  • a HSD3B 1 haplotype pair is determined for an individual by identifying the phased sequence of nucleotides at one or more polymo ⁇ hic sites selected from the group consisting of PSI, PS2, PS3, PS4, PS5, PS6, PS9, PSll, PS12, PS14 and PS16 in each copy of the HSD3B1 gene that is present in the individual.
  • the haplotyping method comprises identifying the phased sequence of nucleotides at each of PS 1-PS 17 in each copy ofthe HSD3B1 gene. When haplotyping both copies ofthe gene, the identifying step is preferably performed with each copy ofthe gene being placed in separate containers.
  • first and second copies ofthe gene are labeled with different first and second fluorescent dyes, respectively, and an allele-specific oligonucleotide labeled with yet a third different fluorescent dye is used to assay the polymo ⁇ hic site(s), then detecting a combination ofthe first and third dyes would identify the polymo ⁇ hism in the first gene copy while detecting a combination ofthe second and third dyes would identify the polymo ⁇ hism in the second gene copy.
  • the identity of a nucleotide (or nucleotide pair) at a polymo ⁇ hic site(s) may be determined by amplifying a target region(s) containing the polymo ⁇ hic site(s) directly from one or both copies ofthe HSD3B1 gene, or a fragment thereof, and the sequence ofthe amplified region(s) determined by conventional methods. It will be readily appreciated by the skilled artisan that only one nucleotide will be detected at a polymo ⁇ hic site in individuals who are homozygous at that site, while two different nucleotides will be detected if the individual is heterozygous for that site.
  • the polymo ⁇ hism may be identified directly, known as positive-type identification, or by inference, referred to as negative-type identification.
  • a site may be positively determined to be either guanine or cytosine for an individual homozygous at that site, or both guanine and cytosine, if the individual is heterozygous at that site.
  • the site may be negatively determined to be not guanine (and thus cytosine/cytosine) or not cytosine (and thus guanine/guanine).
  • the target region(s) may be amplified using any oligonucleotide-directed amplification method, including but not limited to polymerase chain reaction (PCR) (U.S. Patent No. 4,965,188), ligase chain reaction (LCR) (Barany et al., Proc. Natl. Acad. Sci. USA 88:189-193, 1991; WO90/01069), and oligonucleotide ligation assay (OLA) (Landegren et al., Science 241:1077-1080, 1988).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • OLA oligonucleotide ligation assay
  • nucleic acid amplification procedures may be used to amplify the target region including transcription-based amplification systems (U.S. Patent No. 5,130,238; EP 329,822; U.S. Patent No. 5,169,766, WO89/06700) and- isothermal methods (Walker et al., Proc. Natl. Acad. Sci. USA 89:392-396, 1992).
  • a polymo ⁇ hism in the target region may also be assayed before or after amplification using one of several hybridization-based methods known in the art.
  • allele-specific oligonucleotides are utilized in performing such methods.
  • the allele-specific oligonucleotides may be used as differently labeled probe pahs, with one member ofthe pair showing a perfect match to one variant of a target sequence and the other member showing a perfect match to a different variant.
  • more than one polymo ⁇ hic site may be detected at once using a set of allele- specific oligonucleotides or oligonucleotide pairs.
  • the members ofthe set have melting temperatures within 5°C, and more preferably within 2°C, of each other when hybridizing to each of the polymo ⁇ hic sites being detected.
  • Hybridization of an allele-specific oligonucleotide to a target polynucleotide may be performed with both entities in solution, or such hybridization may be performed when either the oligonucleotide or the target polynucleotide is covalently or noncovalently affixed to a solid support. Attachment may be mediated, for example, by antibody-antigen interactions, poly-L-Lys, streptavidin or avidin-biotiri, salt bridges, hydrophobic interactions, chemical linkages, UV cross-linking baking, etc. Allele-specific oligonucleotides may be synthesized directly on the solid support or attached to the solid support subsequent to synthesis.
  • Solid-supports suitable for use in detection methods ofthe invention include substrates made of silicon, glass, plastic, paper and the like, which may be formed, for example, into wells (as in 96-well plates), slides, sheets, membranes, fibers, chips, dishes, and beads.
  • the solid support may be treated, coated or derivatized to facilitate the immobilization ofthe allele-specific oligonucleotide or target nucleic acid.
  • the genotype or haplotype for the HSD3B 1 gene of an individual may also be determined by hybridization of a nucleic acid sample containing one or both copies ofthe gene, or fragment(s) thereof, to nucleic acid arrays and subarrays such as described in WO 95/11995.
  • the arrays would contain a battery of allele-specific oligonucleotides representing each ofthe polymo ⁇ hic sites to be included in the genotype or haplotype. .
  • polymo ⁇ hisms may also be determined using a mismatch detection technique, including but not limited to the RNase protection method using riboprobes (Winter et al., Proc. Natl. Acad. Sci. USA 82:7575, 1985; Meyers et al., Science 230:1242, 1985) and proteins which recognize nucleotide mismatches, such as the E. coli mutS protein (Modrich, P. Ann. Rev. Genet. 25:229-253, 1991).
  • riboprobes Winter et al., Proc. Natl. Acad. Sci. USA 82:7575, 1985; Meyers et al., Science 230:1242, 1985
  • proteins which recognize nucleotide mismatches such as the E. coli mutS protein (Modrich, P. Ann. Rev. Genet. 25:229-253, 1991).
  • variant alleles can be identified by single strand conformation polymo ⁇ hism (SSCP) analysis (Orita et al., Genomics 5:874-879, 1989; Humphries et al., in Molecular Diagnosis of Genetic Diseases, R. Elles, ed., pp. 321-340, 1996) or denaturing gradient gel electrophoresis (DGGE) (Wartell et al., Nucl. Acids Res. 18:2699-2706, 1990; Sheffield et al., Proc. Natl. Acad. Sci. USA ⁇ 86:232-236, 1989).
  • SSCP single strand conformation polymo ⁇ hism
  • DGGE denaturing gradient gel electrophoresis
  • a polymerase-mediated primer extension method may also be used to identify the polymo ⁇ hism(s).
  • Several such methods have been described in the patent and scientific literature and include the "Genetic Bit Analysis” method (W092/15712) and the ligase/poly ⁇ erase mediated genetic bit analysis (U.S. Patent 5,679,524.
  • Related methods are disclosed in WO91/02087, WO90/09455, W095/17676, U.S. Patent Nos. 5,302,509, and 5,945,283.
  • Extended primers containing a polymo ⁇ hism may be detected by mass spectrometry as described in U.S. Patent No. 5,605,798.
  • Another primer extension method is allele-specific PCR (Ruano et al., Nucl. Acids Res. 17:8392, 1989; Ruano et al, Nucl. Acids Res. 19, 6877-6882, 1991; WO 93/22456; Turki et al., J. Clin. Invest. 95:1635-1641, 1995).
  • multiple polymo ⁇ hic sites may be investigated by simultaneously amplifying multiple regions ofthe nucleic acid using sets of allele-specific primers as described in Wallace etui. (WO89/10414).
  • the identity ofthe allele(s) present at any ofthe novel polymo ⁇ hic sites described herein may be indirectly determined by genotyping another polymo ⁇ hic site that is in linkage disequilibrium with the polymo ⁇ hic site that is of interest.
  • an individual's HSD3B1 haplotype pah is predicted from its HSD3B1 genotype using information on haplotype pahs known to exist in a reference population.
  • the haplotyping prediction method comprises identifying a HSD3B1 genotype for the individual at two or more HSD3B1 polymo ⁇ hic sites described herein, enumerating all possible haplotype pahs which are consistent with the genotype, accessing data containing HSD3B1 haplotype pahs identified in a reference population, and assigning a haplotype pair to the individual that is consistent with the data.
  • the reference haplotype pahs include the HSD3B 1 haplotype pairs shown in Table 4.
  • the reference population should be composed of randomly-selected individuals representing the major ethnogeographic groups ofthe world.
  • a preferred reference population allows the detection of any haplotype whose frequency is at least 10% with about 99% certainty and comprises about 20 unrelated individuals from each ofthe four population groups named above.
  • a particularly preferred reference population includes a 3-generation family representing one or more ofthe four population groups to serve as controls for checking quality of haplotyping procedures.
  • the haplotype frequency data for each ethnogeographic group is examined to determine whether it is consistent with Hardy-Weinberg equilibrium.
  • a statistically significant difference between the observed and expected haplotype frequencies could be due to one or more factors including significant inbreeding in the population group, strong selective pressure on the gene, sampling bias, and/or errors in the genotyping process. If large deviations from Hardy- Weinberg equilibrium are observed in an ethnogeographic group, the number of individuals in that group can be increased to see if the deviation is due to a sampling bias. If a larger sample size • ' does not reduce the difference between observed and expected haplotype pah frequencies, then one may wish to consider haplotyping the individual using a direct haplotyping method such as, for example, CLASPER System TM technology (U.S. Patent No. 5,866,404), single molecule dilution, or allele-specific long-range PCR (Michalotos-Beloin et al., Nucleic Acids Res. 24:4841-4843, 1996).
  • CLASPER System TM technology U.S. Patent No. 5,866,404
  • single molecule dilution single
  • the assigning step involves performing the following analysis. First, each ofthe possible haplotype pahs is compared to the haplotype pahs in the reference population. Generally, only one ofthe haplotype pahs in the reference population matches a possible haplotype pah and that pah is assigned to the individual. Occasionally, only one haplotype represented in the reference haplotype pairs is consistent with a possible haplotype pah for an individual, and in such cases the individual is assigned a haplotype pah containing this known haplotype and a new haplotype derived by subtracting the known haplotype from the possible haplotype pah.
  • the haplotype pah in an individual may be predicted from the individual's genotype for that gene using reported methods (e.g., Clark et al. 1990 Mol Bio Evol 7: 111-22) or through a commercial haplotyping service such as offered by Genaissance Pharmaceuticals, Inc. (New Haven, CT).
  • the individual is preferably haplotyped using a dhect molecular haplotyping method such as, for example, CLASPER System TM technology (U.S. Patent No. 5,866,404), SMD, or allele-specific long-range PCR (Michalotos-Beloin et al., supra).
  • the invention also provides a method for determining the frequency of a HSD3B1 genotype, haplotype, or haplotype pah in a population.
  • the method comprises, for each member ofthe population, determining the genotype or the haplotype pair for the novel HSD3B 1 polymo ⁇ hic sites described herein, and calculating the frequency any particular genotype, haplotype, or haplotype pah is found in the population.
  • the population may be a reference population, a family population, a same sex population, a population group, or a trait population (e.g., a group of individuals exhibiting a trait of interest such as a medical condition or response to a therapeutic treatment).
  • frequency data for HSD3B1 genotypes, haplotypes, and/or haplotype pahs are determined in a reference population and used in a method for identifying an association between a trait and a HSD3B1 genotype, haplotype, or haplotype pah.
  • the trait may be any detectable phenotype, including but not limited to susceptibility to a disease or response to a treatment.
  • the method involves obtaining data on the frequency ofthe genotype(s), haplotype(s), or haplotype pah(s) of interest in a reference population as well as in a population exhibiting the trait.
  • Frequency data for one or both ofthe reference and trait populations may be obtained by genotyping or haplotyping each individual in the populations using one ofthe methods described above.
  • the haplotypes for the trait population may be determined directly or, alternatively, by the predictive genotype to haplotype approach described above.
  • the frequency data for the reference and/or trait populations is obtained by accessing previously determined frequency data., which may be in written or electronic form.
  • the frequency data may be present in a database that is accessible by a computer. Once the frequency data is obtained, the frequencies ofthe genotype(s), haplotype(s), ' or haplotype pair(s) of interest in the reference and trait populations are compared.
  • the frequencies of all genotypes, haplotypes, and or haplotype pahs observed in the populations are compared. If a particular HSD3B1 genotype, haplotype, or haplotype pair is more frequent in the trait population than in the reference population at a statistically significant amount, then the trait is predicted to be associated with that HSD3B1 genotype, haplotype, or haplotype pah.
  • the HSD3B1 genotype, haplotype, or haplotype pah being compared in the trait and reference populations is selected from the full-genotypes and full-haplotypes shown in Tables 4 and 5, or from sub-genotypes and sub-haplotypes derived from these genotypes and haplotypes.
  • the trait of interest is a clinical response exhibited by a patient to some therapeutic treatment, for example, response to a drug targeting HSD3B1 or response to a therapeutic treatment for a medical condition.
  • medical condition includes but is not limited to any condition or disease manifested as one or more physical and/or psychological symptoms for which treatment is deshable, and includes previously and newly identified diseases and other disorders.
  • clinical response means any or all ofthe following: a quantitative measure ofthe response, no response, and adverse response (i.e., side effects).
  • clinical population data on the clinical responses exhibited by a population of individuals who received the treatment.
  • This clinical data may be obtained by analyzing the results of a clinical trial that has already been run and/or the clinical data may be obtained by designing and carrying out one or more new clinical trials.
  • clinical trial means any research study designed to collect clinical data on responses to a particular treatment, and includes but is not limited to phase I, phase II and phase m clinical trials. Standard methods are used to define the patient population and to enroll subjects.
  • the individuals included in the clinical population have been graded for the existence ofthe medical condition of interest. This is important in cases where the symptom(s) being presented by the patients can be caused by more than one underlying condition, and where treatment ofthe underlying conditions are not the same. An example of this would be where patients experience breathing difficulties that are due to either asthma or respiratory infections. If both sets were treated with an asthma medication, there would be a spurious group of apparent non-responders that did not actually have asthma. These people would affect the ability to detect any correlation between haplotype and treatment outcome.
  • This grading of potential patients could employ a standard physical exam or one or more lab tests. Alternatively, grading of patients could use haplotyping for situations where there is a strong correlation between haplotype pah and disease susceptibility or severity.
  • the therapeutic treatment of interest is administered to each individual in the trial population and each individual's response to the treatment is measured using one or more predetermined criteria. It is contemplated that in many cases, the trial population will exhibit a range of responses and that the investigator will choose the number of responder groups (e.g., low, medium, high) made up by the various responses.
  • the HSD3B 1 gene for each individual in the trial population is genotyped and/or haplotyped, which may be done before or after administering the treatment.
  • correlations between individual response and HSD3B1 genotype or haplotype content are created. Correlations may be produced in several ways. In one method, individuals are grouped by theh HSD3B1 genotype or haplotype (or haplotype pah) (also referred to as a polymo ⁇ hism group), and then the averages and standard deviations of clinical responses exhibited by the members of each polymo ⁇ hism group are calculated.
  • a second method for finding correlations between HSD3B1 haplotype content and clinical responses uses predictive models based on error-minimizing optimization algorithms.
  • One of many possible optimization algorithms is a genetic algorithm (R. Judson, "Genetic Algorithms and Theh Uses in Chemistry” in Reviews in Computational Chemistry, Vol. 10, pp. 1-73, K. B. Lipkowitz and D. B. Boyd, eds. (NCH Publishers, New York, 1997). Simulated annealing (Press et al., "Numerical Recipes in C: The Art of Scientific Computing", Cambridge University Press (Cambridge) 1992, Ch. 10), neural networks (E. Rich and K.
  • Correlations may also be analyzed using analysis of variation (ANOVA) techniques to determine how much ofthe variation in the clinical data is explained by different subsets ofthe " polymo ⁇ hic sites in the HSD3B1 gene.
  • ANOVA analysis of variation
  • PCT/USOO/17540 ANOVA is used to test hypotheses about whether a response variable is caused by or correlated with one or more traits or variables that can be measured (Fisher and vanBelle, supra, Ch. 10).
  • a mathematical model may be readily constructed by the skilled artisan that predicts clinical response as a function of HSD3B1 genotype or haplotype content.
  • the model is validated in one or more follow-up clinical trials designed to test the model.
  • the identification of an association between a clinical response and a genotype or haplotype (or haplotype pah) for the HSD3B 1 gene may be the basis for designing a diagnostic method to determine those individuals who will or will not respond to the treatment, or alternatively, will respond at a lower level and thus may require more treatment, i.e., a greater dose of a drug.
  • the diagnostic method may take one of several forms: for example, a dhect DNA test (i.e., genotyping or haplotyping one or more ofthe polymo ⁇ hic sites in the HSD3B1 gene), a serological test, or a physical exam measurement.
  • a dhect DNA test i.e., genotyping or haplotyping one or more ofthe polymo ⁇ hic sites in the HSD3B1 gene
  • serological test i.e., a serological test
  • a physical exam measurement i.e., a physical exam measurement.
  • this diagnostic method uses the predictive haplotyping
  • the invention provides an isolated polynucleotide comprising a polymo ⁇ hic variant ofthe HSD3B1 gene or a fragment ofthe gene which contains at least one ofthe novel polymorphic sites described herein.
  • the nucleotide sequence of a variant HSD3B1 gene is identical to the reference genomic sequence for those portions ofthe gene examined, as described in the Examples below, except that it comprises a different nucleotide at one or more ofthe novel polymo ⁇ hic sites PSI, PS2, PS3, PS4, PS5, PS6, PS9, PSll, PS12, PS14 and PS16, and may also comprise one or more additional polymo ⁇ hisms selected from the group consisting of thymine at PS7, guanine at PS8, adenine at PS10, cytosine at PS13, ymine at PS15 and thymine at PS17.
  • the nucleotide sequence of a variant fragment ofthe HSD3B1 gene is identical to the corresponding portion ofthe reference sequence except for having a different nucleotide at one or more ofthe novel polymo ⁇ hic sites described herein.
  • the invention specifically does not include polynucleotides comprising a nucleotide sequence identical to the reference sequence ofthe HSD3B1 gene, which is defined by haplotype 3, (or other reported HSD3B1 sequences) or to portions ofthe reference sequence (or other reported HSD3B1 sequences), except for genotyping oligonucleotides as described below.
  • polymo ⁇ hism in a variant gene or fragment is identified by aligning its sequence against SEQ ID NO: 1.
  • the polymo ⁇ hism is selected from the group consisting of thymine at PSI, cytosine at PS2, guanine at PS3, guanine at PS4, thymine at PS5, guanine at PS6, thymine at PS9, guanine at PSll, adenine at PS12, adenine at PS14 and adenine at PS16.
  • the polymo ⁇ hic variant comprises a naturally-occurring isogene ofthe HSD3B1 gene which is defined by any one of haplotypes 1- 2 and 4-17 shown in Table 5 below.
  • Polymo ⁇ hic variants ofthe invention may be prepared by isolating a clone containing the HSD3B 1 gene from a human genomic library.
  • the clone may be sequenced to determine the identity ofthe nucleotides at the novel polymo ⁇ hic sites described herein. Any particular variant claimed herein could be prepared from this clone by performing in vitro mutagenesis using procedures well- known in the art.
  • HSD3B1 isogenes may be isolated using any method that allows separation ofthe two "copies" ofthe HSD3B1 gene present in an individual, which, as readily understood by the skilled artisan, may be the same allele or different alleles. Separation methods include targeted in vivo cloning (TINC) in yeast as described in WO 98/01573, U.S. Patent No. 5,866,404, and U.S. Patent No. 5,972,614. Another method, which is described in U.S. Patent No. 5,972,614, uses an allele specific oligonucleotide in combination with primer extension and exonuclease degradation to generate hemizygous DNA targets.
  • TTC targeted in vivo cloning
  • Another method which is described in U.S. Patent No. 5,972,614, uses an allele specific oligonucleotide in combination with primer extension and exonuclease degradation to generate hemizygous DNA targets.
  • the invention also provides HSD3B1 genome anthologies, which are collections of HSD3B1 isogenes found in a given population.
  • the population may be any group of at least two individuals, including but not limited to a reference population, a population group, a family population, a clinical population, and a same sex population.
  • a HSD3B1 genome anthology may comprise individual .
  • HSD3B1 isogenes stored in separate containers such as microtest tubes, separate wells of a microtitre plate and the like.
  • two or more groups ofthe HSD3B1 isogenes in the anthology may be stored in separate containers.
  • a preferred HSD3B 1 genome anthology of the invention comprises a set of isogenes defined by the haplotypes shown in Table 5 below.
  • An isolated polynucleotide containing a polymo ⁇ hic variant nucleotide sequence ofthe invention may be operably linked to one or more expression regulatory elements in a recombinant expression vector capable of being propagated and expressing the encoded HSD3B 1 protein in a prokaryotic or a eukaryotic host cell.
  • expression regulatory elements which may be used include, but are not limited to, the lac system, operator and promoter regions of phage lambda, yeast promoters, and promoters derived from vaccinia virus, adenovirus, retroviruses, or SN40.
  • regulatory elements include, but are not limited to, appropriate leader sequences, termination codons, polyadenylation signals, and other sequences requhed for the appropriate transcription and subsequent translation ofthe nucleic acid sequence in a given host cell.
  • the expression vector contains any additional elements necessary for its transfer to and subsequent replication in the host cell. Examples of such elements include, but are not limited to, origins of replication and selectable markers.
  • Such expression vectors are commercially available or are readily constructed using methods known to those in the art (e.g., F. Ausubel et al., 1987, in "Current Protocols in Molecular Biology", John Wiley and Sons, New York, New York).
  • Host cells which may be used to express the variant HSD3B1 sequences ofthe invention include, but are not limited to, eukaryotic and mammalian cells, such as animal, plant, insect and yeast cells, and prokaryotic cells, such as E. coli, or algal cells as known in the art.
  • the recombinant expression vector may be introduced into the host cell using any method known to those in the art including, but not limited to, microinjection, electroporation, particle bombardment, transduction, and transfection Using DEAE- dextran, lipofection, or calcium phosphate (see e.g., Sambrook et al. (1989) in "Molecular Cloning. A , Laboratory Manual", Cold Spring Harbor Press, Plainview, New York).
  • eukaryotic expression vectors that function in eukaryotic cells, and preferably mammalian cells, are used.
  • Non-limiting examples of such vectors include vaccinia virus vectors, adenovirus vectors, he ⁇ es virus vectors, and baculovirus transfer vectors.
  • Preferred eukaryotic cell lines include COS cells, CHO cells, HeLa cells, NIH/3T3 cells, and embryonic stem cells (Thomson, J. A. et al., 1998 Science 282: 1145-1147).
  • Particularly preferred host cells are mammalian cells.
  • HSD3B1 mRNAs varying from each other at any polymo ⁇ hic site retained in the spliced and processed mRNA molecules.
  • These mRNAs can be used for the preparation of a HSD3B1 cDNA comprising a nucleotide sequence which is a polymo ⁇ hic variant of the HSD3B1 reference coding sequence shown in Figure 2.
  • the invention also provides HSD3B1 mRNAs and corresponding cDNAs which comprise a nucleotide sequence that is identical to SEQ ED NO: 2 (Fig.
  • RNA sequence except for having one or more polymo ⁇ hisms selected from the group consisting of guanine at a position corresponding to nucleotide 150, thymine at a position corresponding to nucleotide 246, adenine at a position corresponding to nucleotide 939 and adenine at a position corresponding to nucleotide 1100, and may also comprise one or more additional polymo ⁇ hisms selected from the group consisting of thymine at a position corresponding to nucleotide 228, guanine at a position corresponding to nucleotide 235, adenine at a position corresponding to nucleotide 270, cytosine at a position corresponding to nucleotide 856 and thymine at a position corresponding to nucleotide 1012.
  • a particularly preferred polymo ⁇ hic cDNA variant comprises the coding sequence of a HSD3B1 isogene defined by haplotypes 1- 2 and 4-17. Fragments of these variant mRNAs and cDNAs are included in the scope of the invention, provided they contain the novel polymo ⁇ hisms described herein.
  • the invention specifically excludes polynucleotides identical to previously identified and characterized HSD3B1 cDNAs and fragments thereof.
  • Polynucleotides comprising a variant RNA or DNA sequence may be isolated from a biological sample using well-known molecular biological procedures or may be chemically synthesized.
  • a polymo ⁇ hic variant of a HSD3B1 gene fragment comprises at least one novel polymo ⁇ hism identified herein and has a length of at least 10 nucleotides and may range up to the full length ofthe gene.
  • such fragments are between 100 and 3000 nucleotides in length, and more preferably between 200 and 2000 nucleotides in length, and most preferably between 500 and 1000 nucleotides in length.
  • nucleic acid molecules containing the HSD3B1 gene may be complementary double stranded molecules and thus reference to a particular site on the sense strand refers as well to the corresponding site on the complementary antisense strand.
  • reference may be made to the same polymo ⁇ hic site on either strand and an oligonucleotide may be designed to hybridize specifically to either strand at a target region containing the polymo ⁇ hic site.
  • the invention also includes single-stranded polynucleotides which are complementary to the sense strand ofthe HSD3B1 genomic variants - described herein.
  • Polynucleotides comprising a polymo ⁇ hic gene variant or fragment may be useful for therapeutic pu ⁇ oses.
  • an expression vector encoding the isoform may be administered to the patient.
  • the patient may be one who lacks the HSD3B 1 isogene encoding that isoform or may already have at least one copy of that isogene.
  • HSD3B1 isogene expression of a particular HSD3B1 isogene may be turned off by transforming a targeted organ, tissue or cell population with an expression vector that expresses high levels of untranslatable mRNA for the isogene.
  • oligonucleotides dhected against the regulatory regions (e.g., promoter, introns, enhancers, 3 ' untranslated region) ofthe isogene may block transcription. Oligonucleotides targeting the transcription initiation site, e.g., between positions -10 and +10 from the start site are preferred.
  • inhibition of transcription can be achieved using oligonucleotides that base-pah with region(s) ofthe isogene DNA to form triplex DNA (see e.g., Gee et al. in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., 1994).
  • Antisense ohgonucleotides may also be designed to block translation of HSD3B1 mRNA transcribed from a particular isogene. It is also contemplated that ribozymes may be designed that can catalyze the specific cleavage of HSD3B 1 mRNA transcribed from a particular isogene.
  • the oligonucleotides may be delivered to a target cell or tissue by expression from a vector introduced into the cell or tissue in vivo or ex vivo.
  • the oligonucleotides may be formulated as a pharmaceutical composition for administration to the patient.
  • Oligoribonucleotides and/or oligodeoxynucleotides intended for use as antisense oligonucleotides may be modified to increase stability and half-life.
  • Possible modifications include, but are not limited to phosphorothioate or 2' O-methyl linkages, and the inclusion of nontraditional bases such as inosine and queosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytosine, guanine, thymine, and uracil which are not as easily recognized by endogenous nucleases.
  • the invention also provides an isolated polypeptide comprising a polymo ⁇ hic variant of the reference HSD3B1 amino acid sequence shown in Figure 3.
  • the location of a variant amino acid in a HSD3B1 polypeptide or fragment ofthe invention is identified by aligning its sequence against SEQ ID NO:3 (Fig.3).
  • a HSD3B1 protein variant ofthe invention comprises an amino acid sequence identical to SEQ ID NO:3 except for having, asparagine at a position corresponding to amino acid position 367, and may also comprise one or more additional variant amino acids selected from the group consisting of valine at a position corresponding to amino acid position 79 and leucine at a position corresponding to amino acid position 286.
  • HSD3B 1 protein variants included within the invention comprise all amino acid sequences based on SEQ ID NO: 3 and having the combination of amino acid variations described in Table 2 below.
  • a HSD3B1 protein variant ofthe invention is encoded by an isogene defined by one ofthe observed haplotypes shown in Table 5.
  • Table 2 Novel Polymo ⁇ hic Variants of HSD3B1
  • the invention also includes HSD3B1 peptide variants, which are any fragments of a HSD3B1 protein variant that contain asparagine at a position corresponding to amino acid position 367.
  • a HSD3B1 peptide variant is at least 6 amino acids in length and is preferably any number between 6 and 30 amino acids long, more preferably between 10 and 25, and most preferably between 15 and 20 amino acids long.
  • Such HSD3B1 peptide variants may be useful as antigens to generate antibodies specific for one ofthe above HSD3B 1 isoforms.
  • the HSD3B 1 peptide variants may be useful in drug screening assays.
  • a HSD3B 1 variant protein or peptide ofthe invention may be prepared by chemical synthesis or by expressing one ofthe variant HSD3B1 genomic and cDNA sequences as described above.
  • the HSD3B 1 protehi variant may be isolated from a biological sample of an individual having a HSD3B1 isogene which encodes the variant protein.
  • a particular HSD3B 1 isoform ofthe invention can be isolated by immunoaffinity chromatography using an antibody which specifically binds to that particular HSD3B1 isoform but does not bind to the other HSD3B1 isoform.
  • HSD3B1 protein may be detected by methods known in the art, including Coomassie blue staining, silver staining, and Western blot analysis, using antibodies specific for the isoform ofthe HSD3B1 protein as discussed further below.
  • HSD3B1 variant proteins can be purified by standard protein purification procedures known in the art, including differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrc-phoresis, affinity and immunoaffinity chromatography and the like. (Ausubel et. al., 1987, In Current Protocols in Molecular Biology John Wiley and Sons, New York, New York). In the case of immunoaffinity chromatography, antibodies specific for a particular polymo ⁇ hic variant may be used.
  • a polymo ⁇ hic variant HSD3B 1 gene ofthe invention may also be fused in frame with a heterologous sequence to encode a chimeric HSD3B1 protein.
  • the non-HSD3Bl portion ofthe chimeric protein may be recognized by a commercially available antibody.
  • the chimeric protein may also be engineered to contain a cleavage site located between the HSD3B 1 and non- HSD3B1 portions so that the HSD3B1 protein may be cleaved and purified away from the non- HSD3B1 portion.
  • An additional embodiment ofthe invention relates to using a novel HSD3B1 protehi isoform in any of a variety of drug screening assays.
  • Such screening assays may be performed to identify agents that bind specifically to all known HSD3B1 protein isoforms or to only a subset of one or more of these isoforms.
  • the agents may be from chemical compound libraries, peptide libraries and the like.
  • the HSD3B1 protein or peptide variant may be free in solution or affixed to a solid support. In .
  • high throughput screening of compounds for binding to a HSD3B1 variant may be accomplished using the method described in PCT application WO84/03565, in which large numbers of test compounds are synthesized on a solid substrate, such as plastic pins or some other surface, contacted with the HSD3B 1 protein(s) of interest and then washed. Bound HSD3B 1 protein(s) are then detected using methods well-known in the art.
  • a novel HSD3B1 protein isoform may be used in assays to measure the binding affinities of one or more candidate drugs targeting the HSD3B1 protein.
  • a particular HSD3B1 haplotype or group of HSD3B1 haplotypes encodes a HSD3B1 protein variant with an amino acid sequence distinct from that of HSD3B1 protein isoforms encoded by other HSD3B1 haplotypes
  • detection of that particular HSD3B1 haplotype or group of HSD3B1 haplotypes may be accomplished by detecting expression of the encoded HSD3B1 protein variant using any ofthe methods described herein or otherwise commonly known to the skilled artisan.
  • the invention provides antibodies specific for and immunoreactive with one or more ofthe novel HSD3B1 variant.proteins described herein.
  • the antibodies may be either monoclonal or polyclonal in origin.
  • the HSD3B1 protein or peptide variant used to generate the antibodies may be from natural or recombinant sources or produced by chemical synthesis using synthesis techniques known in the art. If the HSD3B1 protein variant is of insufficient size to be antigenic, it may be conjugated, complexed, or otherwise covalently linked to a carrier molecule to enhance the antigenicity ofthe peptide.
  • carrier molecules include, but are not limited to, albumins (e.g., human, bovine, fish, ovine), and keyhole limpet hemocyanin (Basic and Clinical Immunology, 1991, Eds. D.P. Stites, and A.I. Terr, Appleton and Lange, Norwalk Connecticut, San Mateo, California).
  • albumins e.g., human, bovine, fish, ovine
  • keyhole limpet hemocyanin Basic and Clinical Immunology, 1991, Eds. D.P. Stites, and A.I. Terr, Appleton and Lange, Norwalk Connecticut, San Mateo, California.
  • an antibody specifically immunoreactive with one ofthe novel protein isoforms described herein is administered to an individual to neutralize activity ofthe HSD3B1 isoform expressed by that individual.
  • the antibody may be formulated as a pharmaceutical composition which includes a pharmaceutically acceptable carrier.
  • Antibodies specific for and immunoreactive with one ofthe noyel protein isoforms described herein may be used to immunoprecipitate the HSD3B1 protein variant from solution as well as react with HSD3B1 protein isoforms on Western or immunoblots of polyacrylamide gels on membrane supports or substrates.
  • the antibodies will detect HSD3B1 protein isoforms in paraffin or frozen tissue sections, or in cells which have been fixed or unfixed and • prepared on slides, coverslips, or the like, for use in immunocytochemical, immunohistochemical, and immunofluorescence techniques.
  • an antibody specifically immunoreactive with one ofthe novel HSD3B1 protein variants described herein is used in immunoassays to detect this variant in biological samples.
  • an antibody ofthe present invention is contacted with a biological sample and the formation of a complex between the HSD3B 1 protein variant and the antibody is detected.
  • suitable immunoassays include radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme linked immunoassay (ELISA), chemiluminescent assay, immunohistochemical assay, immunocytochemical assay, and the like (see, e.g., Principles and Practice of Immunoassay, 1991, Eds. Christopher P. Price and David J.
  • Neoman Stockton Press, New York, New York; Current Protocols in Molecular Biology, 1987, Eds. Ausubel et al., John Wiley and Sons, New York, New York).
  • Standard techniques known in the art for ELISA are described in Methods in Immunodiagnosis, 2nd Ed., Eds. Rose and Bigazzi, John Wiley and Sons, New York 1980; and Campbell et al., 1984, Methods in Immunology, W.A. Benjamin, Inc.).
  • Such assays may be dhect, indirect, competitive, or noncompetitive as described in the art (see, e.g., Principles and Practice of Immunoassay, 1991, Eds. Christopher P. Price and David J.
  • Proteins may be isolated from test specimens and biological samples by conventional methods, as described in Current Protocols in Molecular Biology, supra.
  • antibody molecules for use in the detection and therapy methods ofthe present invention are intact immunoglobulin molecules, substantially intact hnmunoglobulhi molecules, or those portions of immunoglobulin molecules that contain the antigen binding site.
  • Polyclonal or monoclonal antibodies may be produced by methods conventionally known in the art (e.g., Kohler and Milstein, 1975, Nature, 256:495-497; Campbell Monoclonal Antibody Technology, the Production and Characterization of Rodent and Human Hybridomas, 1985, In: Laboratory Techniques in Biochemistry and Molecular Biology, Eds. Burdon et al., Volume 13, Elsevier Science Publishers, Amsterdam).
  • the antibodies or antigen binding fragments thereof may also be produced by genetic engineering.
  • the technology for expression of both heavy and light chain genes in E. coli is the subject of PCT patent applications, publication number WO 901443, WO 901443 and WO 9014424 and in Huse et al., 1989, Science, 246:1275-1281.
  • the antibodies may also be humanized (e.g., Queen, C. et al. 1989 Proc. Natl. Acad. Sci.USA 86;10029).
  • HSD3B1 Effect(s) ofthe polymo ⁇ hisms identified herein on expression of HSD3B1 may be investigated by preparing recombinant cells and/or nonhuman recombinant organisms, preferably recombinant animals, containing a polymo ⁇ hic variant ofthe HSD3B1 gene.
  • expression includes but is not limited to one or more ofthe following: transcription ofthe gene into precursor mRNA; splicing and other processing ofthe precursor mRNA to produce mature mRNA; mRNA stability; translation ofthe mature mRNA into HSD3B1 protein (including codon usage and tRNA availability); and glycosylation and or other modifications ofthe translation product, if requhed for proper expression and function.
  • the deshed HSD3B 1 isogene may be introduced into the cell in a vector such that the isogene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location.
  • the HSD3B1 isogene is introduced into a pell in such a way that it recombines with the endogenous HSD3B1 gene present in the cell. Such recombination requires the occurrence of a double recombination event, thereby resulting in the deshed HSD3B1 gene polymo ⁇ hism.
  • Vectors for the introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector or vector construct may be used in the invention. Methods such as electroporation, particle bombardment, calcium phosphate co-precipitation and viral transduction for introducing DNA into cells are known in the art; therefore, the choice of method may lie with the competence and preference- ofthe skilled practitioner.
  • Examples of cells into which the HSD3B1 isogene may be introduced include, but are not limited to, continuous culture cells, such as COS, NIH/3T3, and primary or culture cells ofthe relevant tissue type, i.e., they express the HSD3B1 isogene. Such recombinant cells can be used to compare the biological activities ofthe different protein variants.
  • Recombinant nonhuman organisms i.e., transgenic animals, expressing a variant HSD3B1 gene are prepared using standard procedures known in the art.
  • a construct comprising the variant gene is introduced into a nonhuman animal or an ancestor ofthe animal at an embryonic stage, i.e., the one-cell stage, or generally not later than about the eight-cell stage.
  • Transgenic animals carrying the constructs ofthe invention can be made by several methods known to those having skill in the art.
  • One method involves transfecting into the embryo a retrovirus constructed to contain one or more insulator elements, a gene or genes of interest, and other components known to those skilled in the art to provide a complete shuttle vector harboring the insulated gene(s) as a transgene, see e.g., U.S. Patent No. 5,610,053.
  • Another method involves directly injecting a transgene into the embryo.
  • a third method involves the use of embryonic stem cells. Examples of animals into which the HSD3B1 isogenes may be introduced include, but are not limited to, mice, rats, other rodents, and nonhuman primates (see "The Introduction of Foreign Genes into Mice" and the cited references therein, In: Recombinant DNA, Eds. J.D. Watson, M.
  • Transgenic animals stably expressing a human HSD3B1 isogene and producing human HSD3B1 protein can be used as biological models for studying diseases related to abnormal HSD3B1 expression and or activity, and for screening and assaying various candidate drugs, compounds, and treatment regimens to reduce the symptoms or effects of these diseases.
  • An additional embodiment ofthe invention relates to pharmaceutical compositions for treating disorders affected by expression or function of a novel HSD3B1 isogene described herein.
  • the pharmaceutical composition may comprise any ofthe following active ingredients: a polynucleotide comprising one of these novel HSD3B1 isogenes; an antisense oligonucleotide directed against one of the novel HSD3B1 isogenes, a polynucleotide encoding such an antisense oligonucleotide, or another compound which inhibits expression of a novel HSD3B1 isogene described herein.
  • the composition contains the active ingredient in a therapeutically effective amount.
  • composition also comprises a pharmaceutically acceptable carrier, examples of which include, but are not limited to, saline, buffered saline, dextrose, and water.
  • a pharmaceutically acceptable carrier examples of which include, but are not limited to, saline, buffered saline, dextrose, and water.
  • Those skilled in the art may employ a formulation most suitable for the active ingredient, whether it is a polynucleotide, oligonucleotide, protein, peptide or small molecule antagonist.
  • the pharmaceutical composition may be administered alone or in combination with at least one other agent, such as a stabilizing compound.
  • Administration ofthe pharmaceutical composition may be by any number of routes including, but not limited to orai, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, intradermal, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).
  • determination ofthe therapeutically effective dose of active ingredient ⁇ and/or the appropriate route of administration is well witliin the capability of those skilled in the art.
  • the dose can be estimated initially either in cell culture assays or in animal models.
  • the animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. The exact dosage will be determined by the practitioner, in light of factors relating to the patient requiring treatment, including but not limited to severity ofthe disease state, general health, age, weight and gender ofthe patient, diet, time and frequency of administration, other drugs being taken by the patient, and tolerance/response to the treatment.
  • any or all analytical and mathematical operations involved in practicing the methods ofthe present invention may be implemented by a computer.
  • the computer may execute a program that generates views (or screens) displayed on a display device and with which the user can interact to view and analyze large amounts of information relating to the HSD3B1 gene and its genomic variation, including chromosome location, gene structure, and gene family, gene expression data, polymo ⁇ hism data, genetic sequence data, and clinical data population data (e.g., data on ethnogeographic origin, clinical responses, genotypes, and haplotypes for one or more populations).
  • the HSD3B1 polymo ⁇ hism data described herein may be stored as part of a relational database (e.g., an instance of an Oracle database or a set of ASCII flat files). These polymo ⁇ hism data may be stored on the computer's hard drive or may, for example, be stored on a CD-ROM or on one or more other storage devices accessible by the computer. For example, the data may be stored on one or more databases in communication with the computer via a network.
  • a relational database e.g., an instance of an Oracle database or a set of ASCII flat files.
  • These polymo ⁇ hism data may be stored on the computer's hard drive or may, for example, be stored on a CD-ROM or on one or more other storage devices accessible by the computer.
  • the data may be stored on one or more databases in communication with the computer via a network.
  • EXAMPLES The Examples herein are meant to exemplify the various aspects of carrying out the invention and are not intended to limit the scope ofthe invention in any way.
  • the Examples do not include detailed descriptions for conventional methods employed, such as in the performance of genomic DNA isolation, PCR and sequencing procedures. Such methods are well-known to those skilled in the art and are described in numerous publications, for example, Sambrook, Fritsch, and Maniatis, "Molecular Cloning: A Laboratory Manual", 2 nd Edition, Cold Spring Harbor Laboratory Press, USA, (1989).
  • EXAMPLE 1 This example illustrates examination of various regions ofthe HSD3B1 gene for polymo ⁇ hic sites.
  • the following target regions ofthe HSD3B1 gene were amplified.using PCR primer pahs.
  • the primers used for each region are represented below by providing the nucleotide positions of theh initial and final nucleotides, which correspond to positions in Figure 1.
  • Fragment 6 8597-8622 . 9118-9095 522 nt -
  • Amplification profile 97°C - 2 min. 1 cycle
  • the PCR products were purified using a Whatman Polyfiltronics 100 ⁇ l 384 well unifilter plate essentially according to the manufacturers protocol.
  • the purified DNA was eluted in 50 ⁇ l of distilled water.
  • Sequencing reactions were set up using Applied Biosystems Big Dye Terminator chemistry essentially according to the manufacturers protocol.
  • the purified PCR products were sequenced in both dhections using the primer sets, described previously or those represented below by the nucleotide positions of theh initial and final nucleotides, which correspond to positions in Figure 1.
  • Reaction products were purified by isopropanol precipitation, and run on an Applied Biosystems 3700
  • Fragment 6 8635-8653 9064-9045
  • the different genotypes containing these polymo ⁇ hisms that were observed in the reference population are shown in Table 4 below, with the haplotype pah indicating the combination of haplotypes determined for the individual using the haplotype derivation protocol described below.
  • Table 4 homozygous positions are indicated by one nucleotide and heterozygous positions are indicated by two nucleotides. Missing nucleotides in any given genotype in Table 4 were inferred based on linkage disequilibrium and/or Mendelian inheritance.
  • haplotype pahs shown in Table 4 were estimated from the unphased genotypes using a computer-hnplemented extension of Clark's algorithm (Clark, A.G. 1990 Mol Bio Evol 7, 111-122) for assigning haplotypes to unrelated individuals in a population sample.
  • haplotypes are assigned directly from individuals who are homozygous at all sites or heterozygous at no more than one ofthe variable sites.
  • Thislist of haplotypes is augmented with haplotypes obtained from two families (one three-generation Caucasian family and one two-generation African- American family) and then used to deconvolute the unphased genotypes in the remaining (multiply heterozygous) individuals.
  • Table 6 shows the number of chromosomes seen for each haplotype arranged by the ethnic background ofthe subjects in the Index Repository.
  • Table 7 shows the number of subjects assigned.to each haplotype pah arranged by the ethnic background ofthe subjects in the Index Repository.

Abstract

L'invention concerne des nouveaux polymorphismes mononucléotidiques dans le gène humain hydroxy-delta-5-stéroïde déhydrogénase, 3 beta- et stéroïde delta-isomérase 1 (HSD3B1). L'invention concerne également divers génotypes, haplotypes et paires d'haplotypes pour le gène HSD3B1 existant dans la population. L'invention concerne également des compositions et des procédés permettant d'haplotyper et/ou de génotyper le gène HSD3B1 dans un individu. L'invention concerne également des polynucléotides renfermant un ou plusieurs des polymorphismes du HSD3B1 susmentionnés.
PCT/US2001/011945 2000-04-12 2001-04-12 Haplotypes du gene hsd3b1 WO2001079552A1 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
US7858306B2 (en) * 2005-07-07 2010-12-28 Mayo Foundation For Medical Education And Research HSD3B1 sequence variants
CN107338319A (zh) * 2017-08-25 2017-11-10 天津艾至恩医疗科技有限公司 一种核苷酸引物及预测前列腺癌去势术预后的基因多态性检测试剂盒

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Title
LACHANCE ET AL.: "Characterization of human 3B hydroxysteroid dehydrogenase/delta 5-delta4 isomerase gene and its expression in mammalian cells", J. BIOL. CHEM., vol. 265, no. 33, 25 November 1990 (1990-11-25), pages 20469 - 20475, XP002944255 *
MCBRIDE ET AL.: "Cloning expression and physical mapping of the 3B hydroxysteroid dehydrogenase gene cluster (HSD3BP1-HSD3BP5) in human", GENOMICS, vol. 61, 1999, pages 277 - 284, XP002944254 *
MOISAN ET AL.: "New insight into the molecular basis of 3B hydroxysteroid dehydrogenase deficiency: identification of eight mutations in the HSD3B2 gene in eleven patients from seven new families and comparison of the functional properties of twenty five mutant enzymes", J. CLIN. ENDOCRINOL. METABOL., vol. 84, no. 12, December 1999 (1999-12-01), pages 4410 - 4425, XP002944253 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7858306B2 (en) * 2005-07-07 2010-12-28 Mayo Foundation For Medical Education And Research HSD3B1 sequence variants
CN107338319A (zh) * 2017-08-25 2017-11-10 天津艾至恩医疗科技有限公司 一种核苷酸引物及预测前列腺癌去势术预后的基因多态性检测试剂盒

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