WO2001077125A2 - Haplotypes du gene ccr5 - Google Patents

Haplotypes du gene ccr5 Download PDF

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Publication number
WO2001077125A2
WO2001077125A2 PCT/US2001/010708 US0110708W WO0177125A2 WO 2001077125 A2 WO2001077125 A2 WO 2001077125A2 US 0110708 W US0110708 W US 0110708W WO 0177125 A2 WO0177125 A2 WO 0177125A2
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ccr5
gene
haplotype
individual
nucleotide
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PCT/US2001/010708
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WO2001077125A3 (fr
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Julie Y. Choi
Stefanie E. Kliem
Beena Koshy
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Genaissance Pharmaceuticals, Inc.
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Priority to AU2001249798A priority Critical patent/AU2001249798A1/en
Publication of WO2001077125A2 publication Critical patent/WO2001077125A2/fr
Publication of WO2001077125A3 publication Critical patent/WO2001077125A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates to variation in genes that encode pharmaceutically-important proteins.
  • this invention provides genetic variants of the human chemokine (C-C motif) receptor 5 (CCR5) gene and methods for identifying which variant(s) of this gene is/are possessed by an individual.
  • C-C motif human chemokine receptor 5
  • 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.
  • CCR5 chemokine receptor 5
  • CCR5 is part of a family of trans-membrane proteins that transduce signals via heterotrimeric G-proteins (Horuk, Trends Pharmacol. Sci. 1994; 15:159-165).
  • Chemokines are a family of structurally similar proteins that help elicit a proinflammatory response by binding to receptors on leuckocytes.
  • CXC chemokines act on neutrophils, while the CC chemokines act on monocytes, lymphocytes, basophils and eosonophils (Choe et al., Cell 1996; 85:1135-1148).
  • CCR5 was identified as a coreceptor for the human immunodeficiency virus type 1 (HTV-1), and the presence of this protein renders rionpermissive CD4+ cells susceptible to HTV-1 strains (Deng et al., Nature 1996; 381:661-666; Dragic et al., Nature 1996; 381:667-673).
  • CCR5 has also been shown to enhance HTV-1 infection in HeLa cells (Choe et al., Cell 1996; 85: 1135-1148). Although HTV binding and internalization can be mediated by CD4 acting together with several members of the chemokine receptor family, CCR5 appears to be the critical receptor used by HTV glycoproteins during the early stages of infection (OMIM 601373, ). Mutations that alter the CCR5 gene product have been associated with resistance to HTV- 1. In vitro fusion assays have shown that a deletion resulting in truncated CCR5 do not allow fusion of CD4+ cells with viral propagating cells expressing env protein.
  • the truncated protein was not detected at the surface of cells that normally have the receptor (Samson et al., Nature 1996; 382:722-725; Liu et al., Cell 1996; 86:367-377).
  • Zimmerman et al. found the CCR5 deletion mutation, which they designated CCR5-2, in random blood donors from North America, Asia, and Africa, and observed that homozygosity for the inactive allele was an HTV-1 resistance factor, while heterozygotes demonstrate a slower rate of disease progression.
  • the chemokine (C-C motif) receptor 5 gene is located on chromosome 3 ⁇ 21 and contains 2 exons that encode a 352 amino acid protein.
  • a reference sequence for the CCR5 gene is shown in Figure 1 (GenBank Accession No. U95626; 1 ; SEQ ID NO: 1).
  • Reference sequences for the coding sequence (GenBankAccession No. NM_000579.1) and protein are shown in Figures 2 (SEQ ID NO:2) and 3 (SEQ ID NO:3), respectively.
  • the SNP corresponding to nucleotide 59029 in Figure 1 results in a slowed progression to AIDS following seroconversion (McDermott et al., Lancet 1998; 352:866-870).
  • the SNPs corresponding to nucleotide 59356 and 59402 in Figure 1 are responsible for increasing the rate of parental HIV-1 transmission (Kostrikis et al., J. Virol. 1999; 73:10264-10271) (Kostrikis et al., Nat. Med. 1998; 4:350-353)
  • polymorphic sites correspond to the following nucleotide positions in the indicated GenBank Accession Number: 59802 (PS6), 61370 (PS7), 61803 (PS10) and 62028 (PSl 1) in U95626.1.
  • PS6 polymorphic sites
  • PS7 polymorphic sites
  • PS10 polymorphic sites
  • PSl 1 polymorphic sites
  • the polymorphisms at these sites are adenine or cytosine at PS6, guanine or thymine at PS7, cytosine or thymine at PS 10 and thymine or guanine at PS 11.
  • the inventors have determined the identity of the alleles at these sites, as well as at the previously identified sites at nucleotide positions 59029 (PSl), 59353 (PS2), 59356 (PS3), 59402 (PS4), 59653 (PS5), 61646 (PS8), 61707 (PS9) and 62150 (PS12) in U95626.1, 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 pairs for PSl -PS 12 in the CCR5 gene, which are shown below in Tables 5 and 4, respectively. Each of these CCR5 haplotypes defines a naturally-occurring isoform (also referred to herein as an "isogene") of the CCR5 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 CCR5 gene in an individual.
  • the genotyping method comprises identifying the nucleotide pair that is present at one or more polymorphic sites selected from the group consisting of PS6, PS7, PS10 and PSl 1 in both copies of the CCR5 gene from the individual.
  • a genotyping composition of the 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 CCR5 polymorphic sites.
  • a genotyping kit of the invention comprises a set of oligonucleotides designed to genotype each of these novel CCR5 polymorphic sites.
  • the genotyping kit comprises a set of oligonucleotides designed to genotype each of PS1-PS12.
  • the genotyping method, composition, and kit are useful in determining whether an individual has one of the haplotypes in Table 5 below or has one of the haplotype pairs in Table 4 below.
  • the invention also provides a method for haplotyping the CCR5 gene in an individual.
  • the haplotyping method comprises determining, for one copy of the CCR5 gene, the identity of the nucleotide at one or more polymorphic sites selected from the group consisting of PS6, PS7, PS 10 and PSl 1.
  • the haplotyping method comprises detennining whether one copy of the individual's CCR5 gene is defined by one of the CCR5 haplotypes shown in Table 5, below, or a sub-haplotype thereof.
  • the haplotyping method comprises determining whether both copies of the individual's CCR5 gene are defined by one of the CCR5 haplotype pairs shown in Table 4 below, or a sub-haplotype pair thereof.
  • the method for establishing the CCR5 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 CCR5 activity, e.g., HTV infection and ADDS.
  • the haplotyping method can be used by the pharmaceutical research scientist to validate CCR5 as a candidate target for treating a specific condition or disease predicted to be associated with CCR5 activity. Determining for a particular population the frequency of one or more of the individual CCR5 haplotypes or haplotype pairs described herein will facilitate a decision on whether to pursue CCR5 as a target for treating the specific disease of interest. In particular, if variable CCR5 activity is associated with the disease, then one or more CCR5 haplotypes or haplotype pairs will be found at a higher frequency in disease cohorts than " in appropriately genetically matched controls.
  • variable CCR5 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 CCR5 haplotype or haplotype pair, apply the information derived from detecting CCR5 haplotypes in an individual to decide whether modulating CCR5 activity would be useful in treating the disease.
  • the claimed invention is also useful in screening for compounds targeting CCR5 to treat a specific condition or disease predicted to be associated with CCR5 activity. For example, detecting which of the CCR5 haplotypes or haplotype pairs 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 desired agonist or antagonist activity for each of the most frequent CCR5 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 CCR5 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 CCR5 activity. For example, instead of randomly assigning patients with the disease of mterest to the treatment or control group as is typically done now, determining which of the CCR5 haplotype(s) disclosed herein are present in individual patients enables the pharmaceutical scientist to distribute CCR5 haplotypes and/or haplotype pairs 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 CCR5 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 CCR5 haplotype or haplotype pair.
  • the invention provides a method for identifying an association between a trait and a CCR5 genotype, haplotype, or haplotype pair for one or more of the novel polymorphic sites described herein.
  • the method comprises comparing the frequency of the CCR5 genotype, haplotype, or haplotype pair in a population exhibiting the trait with the frequency of the CCR5 genotype, haplotype, or haplotype pair in a reference population. A higher frequency of the CCR5 genotype, haplotype, or haplotype pair in the trait population than in the reference population indicates the trait is associated with the CCR5 genotype, haplotype, or haplotype pair.
  • the trait is susceptibility to a disease, severity of a disease, the staging of a disease or response to a drug.
  • the CCR5 haplotype is selected from the haplotypes shown in Table 5, or a sub-haplotype thereof. Such methods have applicability in developing diagnostic tests and therapeutic treatments for HTV infection and AIDS.
  • the invention provides an isolated polynucleotide comprising a nucleotide sequence which is a polymorphic variant of a reference sequence for the CCR5 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 cytosine at PS6, thymine at PS7, thymine at PS 10 and guanine at PS 11.
  • the polymorphic variant comprises one or more additional polymorphisms selected from the group consisting of adenine at PSl, cytosine at PS2, thymine at PS3, adenine at PS4, thymine at PS5, adenine at PS8, cytosine at PS9 and adenine at PS 12.
  • a particularly preferred polymorphic variant is an isogene of the CCR5 gene.
  • a CCR5 isogene of the invention comprises guanine or adenine at PSl, thymine or cytosine at PS2, cytosine or thymine at PS3, guanine or adenine at PS4, cytosine or thymine at PS5, adenine or cytosine at PS6, guanine or thymme at PS7, thymine or adenine at PS8, thymine or cytosine at PS9, cytosine or thymine at PS10, thymine or guanine at PSl 1 and guanine or adenine at PS12.
  • the invention also provides a collection of CCR5 isogenes, referred to herein as a CCR5 genome anthology.
  • the invention provides a polynucleotide comprising a polymorphic variant of a reference sequence for a CCR5 cDNA or a fragment thereof.
  • the reference sequence comprises SEQ ED NO:2 (Fig.2) and the polymorphic cDNA comprises at least one polymorphism selected from the group consisting of thymine at a position corresponding to nucleotide 321 and guanine at a position corresponding to nucleotide 546.
  • the polymorphic variant comprises one or more additional polymorphisms selected from the group consisting of adenine at a position corresponding to nucleotide 164, cytosine at a position corresponding to nucleotide 225 and adenine at a position corresponding to nucleotide 668.
  • a particularly preferred polymorphic cDNA variant comprises the coding sequence of a CCR5 isogene defined by haplotypes 2-11.
  • Polynucleotides complementary to these CCR5 genomic and cDNA variants are also provided by the invention. It is believed that polymorphic variants of the CCR5-gene will be useful in studying the expression and function of CCR5, and in expressing CCR5 protein for usd in screening for candidate drugs to treat diseases related to CCR5 activity.
  • the invention provides a recombinant expression vector comprising one of the 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 CCR5 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 CCR5 protein.
  • the reference aminp acid sequence comprises SEQ ID NO:3 (Fig.3) and the polymorphic variant comprises leucine at a position corresponding to amino acid position 182.
  • the polymorphic variant also comprises at least one variant amino acid selected from the group consisting of glutamine at a position corresponding to amino acid position 55 and glutamine at a position corresponding to amino acid position 223.
  • a polymorphic variant of CCR5 is useful in studying the effect of the variation on the biological activity of CCR5 as well as on the binding affinity of candidate drugs targeting CCR5 for the treatment of HTV infection and AIDS .
  • the present invention also provides antibodies that recognize and bind to the above polymorphic CCR5 protein variant. Such antibodies can be utilized in a variety of diagnostic and prognostic formats and therapeutic methods.
  • the present invention also provides nohhuman transgenic animals comprising one of the CCR5 polymorphic genomic variants described herein and methods for producing such animals.
  • the transgenic animals are useful for studying expression of the CCR5 isogenes in vivo, for in vivo screening and testing of drugs targeted against CCR5 protein, and for testing the efficacy of therapeutic agents and compounds for HTV infection and ADDS in a biological system.
  • the present invention also provides a computer system for storing and displaying polymorphism data determined for the CCR5 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 CCR5 gene in a reference population.
  • the computer system is capable of producing a display showing CCR5 haplotypes organized according to their evolutionary relationships.
  • Figure 1 illustrates a reference sequence for the CCR5 gene (Genbank Accession Number U95626.1; contiguous lines; SEQ ID 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 CCR5 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 CCR5 protein (contiguous lines; SEQ ID 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 of the CCR5 gene.
  • the inventors herein discovered 11 isogenes of the CCR5 gene by characterizing the CCR5 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 (CA)(22 individuals), African descent (AF)(20 individuals), Asian (AS)(20 individuals), or Hispanic/Latino (17 individuals)(HL).
  • CA Caucasian
  • AF African descent
  • AS Asian
  • the members of this reference population were organized into population subgroups by the self-identified ethnogeographic origin of their four grandparents as shown in Table 1 below.
  • 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.
  • the CCR5 isogenes present in the human reference population are defined by haplotypes for 12 polymorphic sites in the CCR5 gene, 4 of which are believed to be novel.
  • the CCR5 polymorphic sites identified by the inventors are referred to as PS1-PS12 to designate the order in which they are located in the gene (see Table 3 below), with the novel polymorphic sites referred to as PS6, PS7, PS 10 and PSl 1.
  • the inventors herein Using the genotypes identified in the Index Repository for PS1-PS12 and the methodology described in the Examples below, the inventors herein also determined the pair of haplotypes for the CCR5 gene present in individual human members of this repository.
  • the human genotypes and haplotypes found in the repository for the CCR5 gene include those shown in Tables 4 and 5, respectively.
  • the polymorphism and haplotype data disclosed herein are useful for validating whether CCR5 is a suitable target for drugs to treat HTV infection and AIDS, 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 pair(s) found at one or more polymorphic sites in a locus on a pair of homologous chromosomes in an individual.
  • genotype includes a full-genotype and/or a sub-genotype as described below.
  • Full-genotype The unphased 5' to 3' sequence of nucleotide pairs found at all known polymorphic sites in a locus on a pair of homologous chromosomes in a single individual.
  • Sub-genotype The unphased 5' to 3' sequence of nucleotides seen at a subset of the known polymorphic sites in a locus on a pair 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- haplotype 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 of the 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 of the following for a specific gene: a listing of the haplotype pairs in each individual in a population; a listing of the 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 of the isoforms of a gene found in a population.
  • An isogene contains all of the polymorphisms present in the particular isoform of the gene.
  • 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 of the 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 polymorphic site on the two copies of a chromosome from an individual.
  • Polymorphic site (PS) - A position within a locus at which at least two alternative sequences are found in a population, the most frequent of which has a frequency of no more than 99%.
  • Polymorphism The sequence variation observed in an individual at a polymorphic site. Polymorphisms 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 of the following for a specific gene: location of polymorphic sites; sequence variation at those sites; frequency of polymorphisms 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 polymorphism data arranged in a systematic or methodical way and capable of being individually accessed by electronic or other means.
  • Reference Population A group of subjects or individuals who are predicted to be representative of the 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%.
  • Subject A human individual whose genotypes or haplotypes or response to treatment or disease state are to be determined.
  • Treatment A stimulus administered internally or externally to a subject.
  • Unphased - As applied to a sequence of nucleotide pairs for two or more polymorphic sites in a locus, unphased means the combination of nucleotides present at those polymorphic sites on a single copy of the locus is not known.
  • the invention also provides compositions and methods for detecting the novel CCR5 polymorphisms and haplotypes identified herein.
  • compositions comprise at least one CCR5 genotyping oligonucleotide.
  • a CCR5 genotyping oligonucleotide is a probe or primer capable of hybridizing to a target region that is located close to, or that contains, one of the novel polymorphic sites described herein.
  • the term "oligonucleotide” refers to a polynucleotide molecule having less than about 100 nucleotides.
  • a preferred oligonucleotide of the 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 (PNA)) and the like (Varma, R. in Molecular Biology and Biotechnology, A Comprehensive Desk Reference, Ed. R. Meyers, VCH Publishers, Inc.
  • Oligonucleotides of the 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 of the invention must be capable of specifically hybridizing to a target region of a CCR5 polynucleotide, i.e., a.CCR5 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-CCR5 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 of the molecules is complementary to the nucleotide at the corresponding position of the 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.
  • an oligonucleotide primer may have a non-complementary fragment at its 5' end, with the remainder of the 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 of the 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 of the invention include ASO probes and ASO primers.
  • ASO probes which usually provide good discrimination between different alleles are those in which a central position of the oligonucleotide probe aligns with the polymorphic 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 of the 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.
  • GAGCAATMGTATTTT (SEQ ID NO: 4) and its complement,.
  • CTCATTTKCCATACA (SEQ ID NO: 7) and its complement.
  • a preferred ASO primer for detecting CCR5 gene polymorphisms comprises a nucleotide sequence, listed 5' to 3', selected from the group consisting of:
  • TAAAGGGAGCAATMG SEQ ID NO: 8
  • GTTATTAA ⁇ TACKA SEQ ID NO: 9
  • genotyping oligonucleotides of the invention hybridize to a target region located one to several nucleotides downstream of one of the novel polymorphic sites identified herein. Such oligonucleotides are useful in polymerase-mediated primer extension methods for detecting one of the novel polymorphisms 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 polymorphic site.
  • a particularly preferred oligonucleotide primer for detecting CCR5 gene polymorphisms by primer extension terminates in a nucleotide sequence, listed 5' to 3 ', selected from the group consisting of:
  • a composition contains two or more differently labeled genotyping oligonucleotides for simultaneously probing the identity of nucleotides at two or more polymorphic sites. It is also contemplated that primer compositions may contain two or more sets of allele-specific primer pairs to allow simultaneous targeting and amplification of two or more regions containing a polymorphic site.
  • CCR5 genotyping oligonucleotides of the 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 polymorphism detection assays, including but not limited to probe hybridization and polymerase extension assays.
  • Immobilized CCR5 genotyping oligonucleotides of the invention may comprise an ordered array of oligonucleotides designed to rapidly screen a DNA sample for polymorphisms 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 additional polymorphic sites may be currently known polymorphic 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 of the CCR5 gene, or a fragment thereof, that are present in the individual, and determining the identity of the nucleotide pair at one or more polymorphic sites selected from the group consisting of PS6, PS7, PS 10 and PSl 1 in the two copies to assign a CCR5 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 of the nucleotide pair at one or more of the polymorphic sites selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS8, PS9 and PS12 is also determined.
  • the genotyping method comprises determining the identity of the nucleotide pair at each of PS1-PS12.
  • 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 hair.
  • 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 CCR5 gene is expressed.
  • mRNA or cDNA preparations would not be used to detect polymorphisms located in introns or in 5' and 3' untranslated regions. If a CCR5 gene fragment is isolated, it must contain the polymorphic site(s) to be genotyped.
  • One embodiment of the haplotyping method comprises isolating from the individual a nucleic acid sample containing only one of the two copies of the CCR5 gene, or a fragment thereof, that is present in the individual and determining in that copy the identity of the nucleotide at one or more polymorphic sites selected from the group consisting of PS6, PS7, PS 10 and PS 11 in that copy to assign a CCR5 haplotype to the individual.
  • the nucleic acid may be isolated using any method capable of separating the two copies of the CCR5 gene or fragment such as one of the methods described above for preparing CCR5 isogenes, with targeted in vivo cloning being the preferred approach.
  • the haplotyping method also comprises identifying the nucleotide at one or more polymorphic sites selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS8, PS9 and PS12. In a particularly preferred embodiment, the nucleotide at each of PS1-PS12 is identified.
  • the haplotyping method comprises determining whether an individual has one or more of the CCR5 haplotypes shown in Table 5. This can be accomplished by identifying, for one or both copies of the individual's CCR5 gene, the phased sequence of nucleotides present at each of PS1-PS12.
  • the present invention also contemplates that typically only a subset of PS1-PS12 ' will need to be directly examined to assign to an individual one or more of the haplotypes shown in Table 5. This is because at least one polymorphic site in a gene is frequently in strong linkage disequilibrium with one or more other polymorphic sites in that gene (Drysdale, CM et al.
  • a CCR5 haplotype pair is determined for an individual by identifying the phased sequence of nucleotides at one or more polymorphic sites selected from the group consisting of PS6, PS7, PS 10 and PSl 1 in each copy of the CCR5 gene that is present in the individual.
  • the haplotyping method comprises identifying the phased sequence of nucleotides at each of PS1-PS12 in each copy of the CCR5 gene.
  • the identifying step is preferably performed with each copy of the gene being placed in separate containers. However, it is also envisioned that if the two copies are labeled with different tags, or are otherwise separately distinguishable or identifiable, it could be .
  • first and second copies of the 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 polymorphic site(s), then detecting a combination of the first and third dyes would identify the polymorphism in the first gene copy while detecting a combination of the second and third dyes would identify the polymorphism in the second gene copy.
  • the identity of a nucleotide (or nucleotide pair) at a polymorphic site(s) may be determined by amplifying a target region(s) containing the polymorphic site(s) directly from one or both copies of the CCR5 gene, or a fragment thereof, and the sequence of the 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 polymorphic 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 polymorphism 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 polymorphism 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 pairs, with one member of the 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 polymorphic site may be detected at once using a set of allele- specific oligonucleotides or oligonucleotide pairs.
  • the members of the set have melting temperatures within 5°C, and more preferably within 2°C, of each other when hybridizing to each of the polymorphic 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-biotin, 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 of the 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 of the allele- specific oligonucleotide or target nucleic acid.
  • the genotype or haplotype for the CCR5 gene of an individual may also be determined by hybridization of a nucleic acid sample containing one or both copies of the 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 of the polymorphic sites to be included in the genotype or haplotype.
  • polymorphisms 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 polymorphism (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 polymorphism
  • DGGE denaturing gradient gel electrophoresis
  • a polymerase-mediated primer extension method may also be used to identify the polymorphism ⁇ ).
  • Several such methods have been described in the patent and scientific literature and include the "Genetic Bit Analysis” method (W092/15712) and the ligase/polymerase 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 polymorphism may be detected by mass spectrometry as described in U.S. Patent No. 5,605,798.
  • Another primer extension method is allele-specific PCR (Ruaiio et al., Nucl.
  • the identity of the allele(s) present at any of the novel polymorphic sites described herein may be indirectly determined by genotyping another polymorphic site that is in linkage disequilibrium with the polymorphic site that is of interest.
  • Polymorphic sites in linkage disequilibrium with the presently disclosed polymorphic sites may be located in regions of the gene or in other genomic regions not examined herein. Genotyping of a polymorphic site in linkage disequilibrium with the novel, polymorphic sites described herein may be performed by, but is not limited to, any of the above-mentioned methods for detecting the identity of the allele at a polymorphic site.
  • an individual's CCR5 haplotype pair is predicted from its CCR5 genotype using information on haplotype pairs known to exist in a reference population.
  • the haplotyping prediction method comprises identifying a CCR5 genotype for the individual at two or more CCR5 polymorphic sites described herein, enumerating all possible haplotype pairs which are consistent with the genotype, accessing data containing CCR5 haplotype pairs identified in a reference population, and assigning a haplotype pair to the individual that is consistent with the data.
  • the reference haplotype pairs include the CCR5 haplotype pairs shown in Table 4:
  • the reference population should be composed of randomly-selected individuals representing the major ethnogeographic groups of the 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 of the four population groups named above.
  • a particularly preferred reference population includes a 3-generation family representing one or more of the 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 pair 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 molecule dil
  • the assigning step involves performing the following analysis. First, each of the possible haplotype pairs is compared to the haplotype pairs in the reference population. Generally, only one of the haplotype pairs in the reference population matches a possible haplotype pair and that pair is assigned to the individual. Occasionally, only one haplotype represented in the reference haplotype pairs is consistent with a possible haplotype pair for an individual, and in such cases the individual is assigned a haplotype pair containing this known haplotype and a new haplotype derived by subtracting the known haplotype from the possible haplotype pair.
  • the haplotype pair 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 direct 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 determming the frequency of a CCR5 genotype, haplotype, or haplotype pair in a population.
  • the method comprises, for each member of the population, determining the genotype or the haplotype pair for the novel CCR5 polymorphic sites described herein, and calculating the frequency any particular genotype, haplotype, or haplotype pair 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 CCR5 genotypes, haplotypes, and/or haplotype pairs are determined in a reference population and used in a method for identifying an association between a trait and a CCR5 genotype, haplotype, or haplotype pair.
  • 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 of the genotype(s), haplotype(s), or haplotype pair(s) of interest in a reference population as well as in a population exhibiting the trait.
  • Frequency data for one or both of the reference and trait populations may be obtained by genotyping or haplotyping each individual in the populations using one of the 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 of the genotype(s), haplotype(s), or haplotype pair(s) of interest in the reference and trait populations are compared. In a preferred embodiment, the frequencies of all genotypes, haplotypes, and/or haplotype pairs observed in the populations are compared.
  • the trait is predicted to be associated with that CCR5 genotype, haplotype, or haplotype pair.
  • the CCR5 genotype, haplotype, or haplotype pair 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 CCR5 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 desirable, and includes previously and newly identified diseases and other disorders.
  • clinical response means any or all of the following: a quantitative measure of the response, no response, and adverse response (i.e., side effects).
  • clinical population In order to deduce a correlation between clinical response to a treatment and a CCR5 genotype, haplotype, or haplotype pair, it is necessary to obtain data on the clinical responses exhibited by a population of individuals who received the treatment, hereinafter the "clinical population".
  • 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.
  • the term "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 IH 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 of the 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 of the 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 pair 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. In addition, the CCR5 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 CCR5 genotype or haplotype content are created. Correlations may be produced in several ways. In one method, individuals are grouped by their CCR5 genotype or haplotype (or haplotype pair) (also referred to as a polymorphism group), and then the averages and standard deviations of clinical responses exhibited by the members of each polymorphism group are calculated.
  • a second method for finding correlations between CCR5 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 Their Uses in Chemistry” in Reviews in Computational Chemistry, Vol. 10, pp. 1-73, K. B. Lipkowitz and D. B. Boyd, eds. (VCH 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 of the variation in the clinical data is explained by different subsets of the polymorphic sites in the CCR5 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 CCR5 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 pair) for the CCR5 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 direct DNA test (i.e., genotyping or haplotyping one or more of the polymorphic sites in the CCR5 gene), a serological test, or a physical exam measurement. The only requirement is that there be a good correlation between the diagnostic test results and the underlying CCR5 genotype or haplotype that is in turn correlated with the clinical response. In a preferred embodiment, this diagnostic method uses the predictive haplotyping method described above.
  • the invention provides an isolated polynucleotide comprising a polymorphic variant of the CCR5 gene or a fragment of the gene which contains at least one of the novel polymorphic sites described herein.
  • the nucleotide sequence of a variant CCR5 gene is identical to the reference genomic sequence for those portions of the gene examined, as described in the Examples below, except that it comprises a different nucleotide at one or more of the novel polymorphic sites PS6, PS7, PS 10 and PSl 1, and may also comprise one or more additional polymorphisms selected from the group consisting of adenine at PSl, cytosine at PS2, thymine at PS3, adenine at PS4, thymine at PS5, adenine at PS8, cytosine at PS9 and adenine at PS 12.
  • nucleotide sequence of a variant fragment of the CCR5 gene is identical to the corresponding portion of the reference sequence except for having a different nucleotide at one or more of the novel polymorphic sites described herein.
  • the invention specifically does not include polynucleotides comprising a nucleotide sequence identical to the reference sequence of the CCR5 gene, which is defined by haplotype 1, (or other reported CCR5 sequences) or to portions of the reference sequence (or other reported CCR5 sequences), except for genotyping oligonucleotides as described below.
  • polymorphism in a variant gene or fragment is identified by aligning its sequence against SEQ ID NO:l.
  • the polymorphism is selected from the group consisting of cytosine at PS6, thymine at PS7, thymine at PS 10 and guanine at PS 11.
  • the polymorphic variant comprises a naturally-occurring isogene of the CCR5 gene which is defined by any one of haplotypes 2-11 shown in Table 5 below.
  • Polymorphic variants of the invention may be prepared by isolating a clone containing the CCR5 gene from a human genomic library.
  • the clone may be sequenced to determine the identity of the nucleotides at the novel polymorphic 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.
  • CCR5 isogenes may be isolated using any method that allows separation of the two "copies" of the CCR5 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 (TTVC) 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.
  • TTVC 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 CCR5 genome anthologies, which are collections of CCR5 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 CCR5 genome anthology may comprise individual CCR5 isogenes stored in separate containers such as microtest tubes, separate wells of a microtitre plate and the like. Alternatively, two or more groups of the CCR5 isogenes in the anthology may be stored in separate containers.
  • a preferred CCR5 genome anthology of the invention comprises a set of isogenes defined by the haplotypes shown in Table 5 below.
  • An isolated polynucleotide containing a polymorphic variant nucleotide sequence of the 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 CCR5 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 SV40.
  • regulatory elements include, but are not limited to, appropriate leader sequences, termination codons, polyadenylation signals, and other sequences required for the appropriate transcription and subsequent translation of the 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 CCR5 sequences of the 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, herpes 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.
  • CCR5 mRNAs can be used for the preparation of a CCR5 cDNA comprising a nucleotide sequence which is a polymo ⁇ hic variant of the CCR5 reference coding sequence shown in Figure 2.
  • the invention also provides CCR5 mRNAs and corresponding cDNAs which comprise a nucleotide sequence that is identical to SEQ ED NO: 2 (Fig.
  • a particularly preferred polymo ⁇ hic cDNA variant comprises the coding sequence of a CCR5 isogene defined by haplotypes 2-11.
  • 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 CCR5 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 CCR5 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 of the 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 CCR5 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 miay 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 of the CCR5 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 CCR5 isogene encoding that isoform or may already have at least one copy of that isogene.
  • CCR5 isogene In other situations, it may be desirable to decrease or block expression of a particular CCR5 isogene.
  • Expression of a CCR5 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 directed against the regulatory regions (e.g., promoter, introns, enhancers, 3' untranslated region) of the 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-pair with region(s) of the 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 oligonucleotides may also be designed to block translation of CCR5 mRNA transcribed from a particular isogene. It is also contemplated that ribozymes may be designed that can catalyze the specific cleavage of CCR5 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 CCR5 amino acid sequence shown in Figure 3.
  • the location of a variant amino acid in a CCR5 polypeptide or fragment of the invention is identified by aligning its sequence against SEQ ID NO:3 (Fig.3).
  • a CCR5 protein variant of the invention comprises an amino acid sequence identical to SEQ ED NO:3 except for having leucine at a position corresponding to amino acid position 182, and may also comprise one or more additional variant amino acids selected from the group consisting of glutamine at a position corresponding to amino acid position 55 and glutamine at a position corresponding to amino acid position 223.
  • CCR5 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 CCR5 protein variant of the invention is encoded by an isogene defined by one of the observed haplotypes shown in Table 5.
  • Table 2 Novel Polymorphic Variants of CCR5
  • the invention also includes CCR5 peptide variants, which are any fragments of a CCR5 protein variant that contain leucine at a position corresponding to amino acid position 182.
  • a CCR5 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 CCR5 peptide variants may be useful as antigens to generate antibodies specific for one of the above CCR5 isoforms.
  • the CCR5 peptide variants may be useful in drug screening assays.
  • a CCR5 variant protein or peptide of the invention may be prepared by chemical synthesis or by expressing one of the variant CCR5 genomic and cDNA sequences as described above.
  • the CCR5 protein variant may be isolated from a biological sample of an individual having a CCR5 isogene which encodes the variant protein. Where the sample contains two different CCR5 isoforms (i.e., the individual has different CCR5 isogenes), a particular CCR5 isoform of the invention can be isolated by immunoaffinity chromatography using an antibody which specifically binds to that particular CCR5 isoform but does not bind to the other CCR5 isoform.
  • CCR5 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 of the CCR5 protein as discussed further below.
  • CCR5 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 electrophoresis, affinity and immunoaffimty 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 CCR5 gene of the invention may also be fused in frame with a heterologous sequence to encode a chimeric CCR5 protein.
  • the non-CCR5 portion of the 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 CCR5 and non-CCR5 portions so that the CCR5 protein may be cleaved and purified away from the non-CCR5 portion.
  • An additional embodiment of the invention relates to using a novel CCR5 protein 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 CCR5 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 CCR5 protein or peptide variant may be free in solution or affixed to a solid support.
  • high throughput screening of compounds for binding to a CCR5 variant may be accompUshed 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 CCR5 protein(s) of interest and then washed. Bound CCR5 protein(s) are then detected using methods well-known in the art.
  • a novel CCR5 protein isoform may be used in assays to measure the binding affinities of one or more candidate drugs targeting the CCR5 protein.
  • a particular CCR5 haplotype or group of CCR5 haplotypes encodes a CCR5 protein variant with an amino acid sequence distinct from that of CCR5 protein isoforms encoded by other CCR5 haplotypes
  • detection of that particular CCR5 haplotype or group of CCR5 haplotypes maybe accomplished by detecting expression of the encoded CCR5 protein variant using any of the methods described herein or otherwise commonly known to the skilled artisan.
  • the invention provides antibodies specific for and immunoreactive with one or more of the novel CCR5 variant proteins described herein.
  • the antibodies may be either monoclonal or polyclonal in origin.
  • the CCR5 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 CCR5 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 of the 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 of the novel protein isoforms described herein is administered to an individual to neutralize activity of the CCR5 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 of the novel protein isoforms described herein may be used to immunoprecipitate the CCR5 protein variant from solution as well as react with GCR5 protein isoforms on Western or immunoblots of polyacrylamide gels on membrane supports or substrates.
  • the antibodies will detect CCR5 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 of the novel CCR5 protein variants described herein is used in immunoassays to detect this variant in biological samples.
  • an antibody of the present invention is contacted with a biological sample and the formation of a complex between the CCR5 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 direct, 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.
  • Exemplary antibody molecules for use in the detection and therapy methods of the present invention are intact immunoglobulin molecules, substantially intact immunoglobulin 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).
  • Effect(s) of the polymo ⁇ hisms identified herein on expression of CCR5 may be investigated by preparing recombinant cells and or no ⁇ human recombinant organisms, preferably recombinant animals, containing a polymo ⁇ hic variant of the CCR5 gene.
  • expression includes but is not limited to one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into CCR5 protein (including codon usage and tRNA availability); and glycosylation and or other modifications of the translation product, if required for proper expression and function.
  • the desired CCR5 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 CCR5 isogene is introduced into a cell in such a way that it recombines with the endogenous CCR5 gene present in the cell. Such recombination requires the occunence of a double recombination event, thereby resulting in the desired CCR5 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 of the skilled practitioner.
  • Examples of cells into which the CCR5 isogene may be introduced include, but are not limited to, continuous culture cells, such as COS, NIH/3T3, and primary or culture cells of the relevant tissue type, i.e., they express the CCR5 isogene. Such recombinant cells can be used to compare the biological activities of the different protein variants.
  • Recombinant nonhuman organisms i.e., transgenic animals, expressing a variant CCR5 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 of the 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 of the invention can be made by several methods known to those ha ⁇ dng 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 CCR5 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 CCR5 isogene and producing human CCR5 protein can be used as biological models for studying diseases related to abnormal CCR5 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.
  • compositions for treating disorders affected by expression or function of a novel CCR5 isogene described herein.
  • the pharmaceutical composition may comprise any of the following active ingredients: a polynucleotide comprising one of these novel CCR5 isogenes; an antisense oligonucleotide directed against one of the novel CCR5 isogenes, a polynucleotide encoding such an antisense oligonucleotide, or another " compound which inhibits expression of a novel CCR5 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 of the pharmaceutical composition may be by any number of routes including, but not limited to oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, intradermal, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Further details on techniques for formulation and admimstration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).
  • 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 of the disease state, general health, age, weight and gender of the patient, diet, time and frequency of administration, other drugs being taken by the patient, and tolerance/response to the treatment.
  • EXAMPLE 1 This example illustrates examination of various regions of the CCR5 gene for polymo ⁇ hic sites.
  • the following target regions of the CCR5 gene were amplified using PCR primer pairs.
  • the primers used for each region are represented below by providing the nucleotide positions of their initial and final nucleotides, which correspond to positions in the indicated GenBank Accession Number.
  • 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 directions using the primer sets described previously or those represented below by the nucleotide positions of their initial and final nucleotides, which correspond to positions in the indicated GenBank Accession Number. Reaction products were purified by isopropanol precipitation, and run on an Applied Biosystems 3700 DNA Analyzer.
  • This example illustrates analysis of the CCR5 polymo ⁇ hisms identified in the Index Repository for human genotypes and haplotypes.
  • the different genotypes containing these polymo ⁇ hisms that were observed in the reference population are shown in Table 4 below, with the haplotype pair 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.
  • the haplotype pairs shown in Table 4 were estimated from the unphased genotypes using a computer-implemented extension of Clark's algorithm (Clark, A.G. 1990 Mol Bio Evol 1, 111-122) for

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Abstract

L'invention concerne des nouveaux polymorphismes de nucléotide simple dans le gène récepteur 5 (présentant le motif C-C) (CCR5) de la chimiokine de l'homme. De plus, l'invention concerne divers génotypes, divers haplotypes et diverses paires d'haplotypes destinés au gène CCR5 existant dans la population. L'invention concerne également des compositions et des procédés permettant d'haplotyper et/ou de génotyper le gène CCR5 dans un individu. On décrit également des polynucléotides renfermant au moins un des polymorphismes du gène CCR5.
PCT/US2001/010708 2000-04-05 2001-04-04 Haplotypes du gene ccr5 WO2001077125A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7393634B1 (en) * 1999-10-12 2008-07-01 United States Of America As Represented By The Secretary Of The Air Force Screening for disease susceptibility by genotyping the CCR5 and CCR2 genes

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CARRINGTON ET AL.: 'Novel alleles of the chemokine-receptor gene CCR5' AMERICAN J. OF HUMAN GENETICS vol. 61, 1997, pages 1261 - 1267, XP002948878 *
KOSTRIKIS ET AL.: 'A chemokine receptor CCR2 allele delays HIV-1 disease progression and is associated with a CCR5 promoter mutation' NATURE MEDICINE vol. 4, no. 3, March 1998, pages 350 - 353, XP002948876 *
KOSTRIKIS ET AL.: 'A polymorphism in the regulatory region of the CC-chemokine receptor 5 gene influences perinatal transmission of HIV-1 to African-American infants' J. OF VIROLOGY vol. 73, no. 12, December 1999, pages 10264 - 10271, XP002948875 *
MARTIN ET AL.: 'Genetic acceleration of AIDS progression by a promoter variant of CCR5' SCIENCE vol. 282, pages 1907 - 1911, XP002948877 *
MCDERMOTT ET AL.: 'CCR5 promoter polymorphism and HIV-1 disease progression' THE LANCET vol. 352, 12 September 1998, pages 866 - 870, XP002948874 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7393634B1 (en) * 1999-10-12 2008-07-01 United States Of America As Represented By The Secretary Of The Air Force Screening for disease susceptibility by genotyping the CCR5 and CCR2 genes

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