WO2002051859A2 - Haplotypes of the ptafr gene - Google Patents
Haplotypes of the ptafr gene Download PDFInfo
- Publication number
- WO2002051859A2 WO2002051859A2 PCT/US2001/047441 US0147441W WO02051859A2 WO 2002051859 A2 WO2002051859 A2 WO 2002051859A2 US 0147441 W US0147441 W US 0147441W WO 02051859 A2 WO02051859 A2 WO 02051859A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- ptafr
- haplotype
- seq
- gene
- polymoφhic
- Prior art date
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Classifications
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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
-
- 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)
Definitions
- This invention relates to variation in genes that encode pharmaceutically-important proteins.
- this invention provides genetic variants of the human platelet activating factor receptor (PTAFR) gene and methods for identifying which variant(s) of this gene is/are possessed by an individual.
- PTAFR platelet activating factor receptor
- 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.
- PTAFR platelet activating factor receptor
- PAF platelet activating factor receptor
- PAF stimulates endothelial cell migration and angiogenesis and can modulate blood pressure by affecting the renal vascular circulation.
- PAF is thereby implicated in a variety of coronary pathologies, including hypotension, cardiac dysfunctions and myocardial ischemia-reperfusion injury (Montrucchio et al., Physiol Rev 2000 Oct;80(4): 1669-99).
- the platelet activating factor receptor gene is located on chromosome 1 and contains 1 exon that encodes a 342 amino acid protein.
- a reference sequence for the PTAFR gene is shown in the contiguous lines of Figure 1 (Genaissance Reference No. 23196521; SEQ ID NO: 1); Reference sequences for the coding sequence (GenBank Accession No. NM_000952.1) and protein are shown in Figures 2 (SEQ ID NO: 2) and 3 (SEQ ID NO: 3), respectively.
- PS3 and PS5 Two polymorphisms of the PTAFR gene have been previously identified. These single nucleotide polymorphisms correspond to the sites named PS3 and PS5 herein. Specifically, the variation which corresponds to PS3 consists of a cytosine or adenine at nucleotide position 4765 in Figure 1 (NCBI SNP ID: rs5938). This polymorphism is expressed in the coding sequence at nucleotide position 671 in Figure 2, and results in an amino acid variation of alanine or aspartate at position 224 in Figure 3.
- the variation which corresponds to PS5 consists of an adenine or guanine at nucleotide position 5107 in Figure 1 (NCBI SNP ID: rs5939). This polymorphism is expressed in the coding sequence at nucleotide position 1013 in Figure 2, and results in an amino acid variation of asparagine or serine at position 338 in Figure 3.
- PS polymorphic sites
- PSI polymorphisms at these sites are guanine or adenine at PSI, cytosine or thymine at PS2 and cytosine or thymine at PS4.
- the inventors have determined the identity of the alleles at these sites, as well as at the previously identified sites at nucleotide positions 4765 (PS3) and 5107 (PS5), 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.
- each of these PTAFR haplotypes constitutes a code that defines the variant nucleotides that exist in the human population at this set of polymorphic sites in the PTAFR gene.
- each PTAFR haplotype also represents a naturally-occurring isoform (also referred to herein as an "isogene") of the PTAFR gene.
- the frequency of each haplotype and haplotype pair within the total reference population and within each of the four major population groups included in the reference population was also determined.
- the invention provides a method, composition and kit for genotyping the PTAFR 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 PSI, PS2 and PS4 in both copies of the PTAFR 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 PTAFR polymorphic sites.
- a genotyping kit of the invention comprises a set of oligonucleotides designed to genotype each of these novel PTAFR polymorphic sites.
- the genotyping kit comprises a set of oligonucleotides designed to genotype each of PS1-PS5.
- 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 PTAFR gene in an -individual.
- the haplotyping method comprises determining, for one copy of the PTAFR gene, the identity of the nucleotide at one or more polymorphic sites selected from the group consisting of PS 1 , PS2 and PS4.
- the haplotyping method comprises determining whether one copy of the individual's PTAFR gene is defined by one of the PTAFR haplotypes shown in Table 5, below, or a sub-haplotype thereof.
- the haplotyping method comprises determining whether both copies of the individual's PTAFR gene are defined by one of the PTAFR haplotype pairs shown in Table 4 below, or a sub-haplotype pair thereof. Establishing the PTAFR 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 PTAFR activity, e.g., inflammatory disease, coronary disease and cancer.
- the haplotyping method can be used by the pharmaceutical research scientist to validate PTAFR as a candidate target for treating a specific condition or disease predicted to be associated with PTAFR activity. Determining for a particular population the frequency of one or more of the individual PTAFR haplotypes or haplotype pairs described herein will facilitate a decision on whether to pursue PTAFR as a target for treating the specific disease of interest. In particular, if variable PTAFR activity is associated with the disease, then one or more PTAFR haplotypes or haplotype pairs will be found at a higher frequency in disease cohorts than in appropriately genetically matched controls.
- variable PTAFR 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 PTAFR haplotype or haplotype pair, apply the information derived from detecting PTAFR haplotypes in an individual to decide whether modulating PTAFR activity would be useful in treating the disease.
- the claimed invention is also useful in screening for compounds targeting PTAFR to treat a specific condition or disease predicted to be associated with PTAFR activity.
- detecting which of the PTAFR 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 PTAFR isoforms present in the disease population, or for only the most frequent PTAFR 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.
- Haplotyping the PTAFR 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 PTAFR 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 of the PTAFR haplotype(s) disclosed herein are present in individual patients enables the pharmaceutical scientist to distribute PTAFR 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 PTAFR haplotype or haplotype pair that is associated with response to the drug being studied in the trial, even if this association was previously unknown.
- the scientist can more confidently rely on the information learned from the trial, without first determining the phenotypic effect of any PTAFR haplotype or haplotype pair.
- the invention provides a method for identifying an association between a trait and a PTAFR genotype, haplotype, or haplotype pair for one or more of the novel polymorphic sites described herein. The method comprises comparing the frequency of the PTAFR genotype, haplotype, or haplotype pair in a population exhibiting the trait with the frequency of the PTAFR genotype or haplotype in a reference population.
- a higher frequency of the PTAFR genotype, haplotype, or haplotype pair in the trait population than in the reference population indicates the trait is associated with the PTAFR 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 PTAFR 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 PTAFR gene or a fragment thereof.
- the reference sequence comprises the contiguous sequences shown in Figure 1 and the polymorphic variant comprises at least one polymorphism selected from the group consisting of adenine at PSI, thymine at PS2 and thymine at PS4.
- the polymorphic variant comprises one or more additional polymorphisms selected from the group consisting of adenine at PS3 and guanine at PS5.
- a particularly preferred polymorphic variant is an isogene of the PTAFR gene.
- a PTAFR isogene of the invention comprises guanine or adenine at PSI, cytosine or thymine at PS2, cytosine or adenine at PS3, cytosine or thymine at PS4 and adenine or guanine at PS5.
- the invention also provides a collection of PTAFR isogenes, referred to herein as a PTAFR genome anthology.
- the invention provides a polynucleotide comprising a polymorphic variant of a reference sequence for a PTAFR 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 thymine at a position corresponding to nucleotide 82 and thymine at a position corresponding to nucleotide 735.
- the polymorphic variant comprises one or more additional polymorphisms selected from the group consisting of adenine at a position corresponding to nucleotide 671 and guanine at a position corresponding to nucleotide 1013.
- a particularly preferred polymorphic cDNA variant comprises the coding sequence of a PTAFR isogene defined by haplotypes 2 and 4-6.
- Polynucleotides complementary to these PTAFR genomic and cDNA variants are also provided by the invention. It is believed that polymorphic variants of the PTAFR gene will be useful in studying the expression and function of PTAFR, and in expressing PTAFR protein for use in screening for candidate drugs to treat diseases related to PTAFR activity.
- the invention provides a recombinant expression vector comprising one of the polymorphic genomic and cDNA 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 PTAFR 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 PTAFR protein.
- the reference amino acid sequence comprises SEQ ID NO:3 (Fig.3) and the polymorphic variant comprises phenylalanine at a position corresponding to amino acid position 28.
- the polymo ⁇ hic variant also comprises at least one variant amino acid selected from the group consisting of aspartic acid at a position corresponding to amino acid position 224 and serine at a position corresponding to amino acid position 338.
- a polymo ⁇ hic variant of PTAFR is useful in studying the effect of the variation on the biological activity of PTAFR as well as on the binding affinity of candidate drugs targeting
- PTAFR for the treatment of inflammatory disease, coronary disease and cancer.
- the present invention also provides antibodies that recognize and bind to the above polymo ⁇ hic PTAFR 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 or more of the PTAFR polymo ⁇ hic genomic variants described herein and methods for producing such animals.
- the transgenic animals are useful for studying expression of the PTAFR isogenes in vivo, for in vivo screening and testing of drugs targeted against PTAFR protein, and for testing the efficacy of therapeutic agents and compounds for inflammatory disease, coronary disease and cancer in a biological system.
- the present invention also provides a computer system for storing and displaying polymo ⁇ hism data determined for the PTAFR gene.
- the computer system comprises a computer processing unit; a display; and a database containing the polymo ⁇ hism data.
- the polymo ⁇ hism data includes one or more of the following: the polymo ⁇ hisms, the genotypes, the haplotypes, and the haplotype pairs identified for the PTAFR gene in a reference population.
- the computer system is capable of producing a display showing PTAFR haplotypes organized according to their evolutionary relationships .
- Figure 1 illustrates a reference sequence for the PTAFR gene (Genaissance Reference No. 23196521; contiguous lines), 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 polymo ⁇ hic site(s) and polymo ⁇ hism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the p ⁇ lymo ⁇ hic site in the sequence.
- SEQ ID NO:21 is a modified version of SEQ ID NO: 1 thatshows the context sequence of each polymo ⁇ hic site, PS1-PS5, in a uniform format to facilitate electronic searching.
- SEQ ID NO:21 contains a block of 60 bases of the nucleotide sequence encompassing the centrally-located polymo ⁇ hic site at the 30 th position, followed by 60 bases of unspecified sequence to represent that each PS is separated by genomic sequence whose composition is defined elsewhere herein.
- Figure 2 illustrates a reference sequence for the PTAFR coding sequence (contiguous lines; SEQ ID NO:2), with the polymo ⁇ hic site(s) and polymo ⁇ hism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymo ⁇ hic site in the sequence.
- Figure 3 illustrates a reference sequence for the PTAFR protein (contiguous lines; SEQ ID NO:2)
- the present invention is based on the discovery of novel variants of the PTAFR gene.
- 6 isogenes of the PTAFR gene by characterizing the PTAFR 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 (21 individuals), African descent (20 individuals), Asian (20 individuals), or Hispanic/Latino (18 individuals). To the extent possible, the members of this reference population were organized into population subgroups by their self-identified ethnogeographic origin as shown in Table 1 below.
- the Index Repository contains three unrelated indigenous American Indians (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 PTAFR isogenes present in the human reference population are defined by haplotypes for 5 polymo ⁇ hic sites in the PTAFR gene, 3 of which are believed to be novel.
- the PTAFR polymo ⁇ hic sites identified by the inventors are referred to as PS1-PS5 to designate the order in which they are located in the gene (see Table 3 below), with the novel polymo ⁇ hic sites referred to as
- the inventors herein Using the genotypes identified in the Index Repository for PS1-PS5 and the methodology described in the Examples below, the inventors herein also determined the pair of haplotypes for the PTAFR gene present in individual human members of this repository.
- the human genotypes and haplotypes found in the repository for the PTAFR gene include those shown in Tables 4 and 5, respectively.
- the polymo ⁇ hism and haplotype data disclosed herein are useful for validating whether PTAFR is a suitable target for drugs to treat inflammatory disease, coronary disease and cancer, 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.
- Gene - A segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.
- Genotype An unphased 5' to 3 ' sequence of nucleotide pair(s) found at one or more polymo ⁇ hic 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.
- Sub-genotype The unphased 5 ' to 3 ' sequence of nucleotides seen at a subset of the polymo ⁇ hic sites examined herein 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 polymo ⁇ hic 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 polymo ⁇ hic sites examined herein 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 polymo ⁇ hic sites examined herein 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, coding sequence or the protein encoded thereby, distinguished from other forms by its particular sequence and/or structure.
- Isogene - One of the isoforms (e.g., alleles) of a gene found in a population.
- An isogene (or allele) contains all of the polymo ⁇ hisms present in the particular isoform of the 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 of the present invention.
- Locus - A location on a chromosome or DNA molecule corresponding to a gene or a physical or phenotypic feature, where physical features include polymo ⁇ hic sites.
- 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 pairs 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 of the 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 of the 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 or comprised of complementary, double-stranded DNA.
- Reference Population A group of individuals sharing a common ethnogeographic origin.
- 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%.
- SNP Single Nucleotide Polymorphism
- 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 polymo ⁇ hic sites in a locus, unphased means the combination of nucleotides present at those polymo ⁇ hic sites on a single copy of the locus is not known.
- the invention also provides compositions and methods for detecting the novel
- PTAFR polymo ⁇ hisms, haplotypes and haplotype pairs identified herein.
- compositions comprise at least one oligonucleotide for detecting the variant nucleotide or nucleotide pair located at a novel PTAFR polymo ⁇ hic site in one copy or two copies of the PTAFR gene.
- oligonucleotides are referred to herein as PTAFR haplotyping oligonucleotides or genotyping oligonucleotides, respectively, and collectively as PTAFR oligonucleotides.
- a PTAFR haplotyping or genotyping oligonucleotide is a probe or primer capable of hybridizing to a target region that contains, or that is located close to, one of the novel polymo ⁇ hic sites described herein.
- oligonucleotide refers to a polynucleotide molecule having less than about 100 nucleotides.
- a preferred oligonucleotide of the invention is 10 to 35 nucleotides long.
- the oligonucleotide is between 15 and 30, and most preferably, between 20 and 25 nucleotides in length.
- the exact length of the oligonucleotide will depend on many factors that are routinely considered and practiced by the skilled artisan.
- the 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. (1995), pages 617-620).
- 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.
- Haplotyping or genotyping oligonucleotides of the invention must be capable of specifically hybridizing to a target region of a PTAFR polynucleotide.
- the target region is located in a PTAFR 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 another region in the PTAFR polynucleotide or with a non-PTAFR 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 Hay es, 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 probe or primer as long as the resulting probe or primer is still capable of specifically hybridizing to the target region.
- Preferred haplotyping or 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 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 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.
- a preferred ASO probe for detecting PTAFR gene polymo ⁇ hisms comprises a nucleotide sequence, listed 5' to 3', selected from the group consisting of:
- a preferred ASO primer for detecting PTAFR gene polymo ⁇ hisms comprises a nucleotide sequence, listed 5 ' to 3 ', selected from the group consisting of:
- CCAGAATTTTTGTRT (SEQ ID NO: 7); GCCTGGAAGACCAYA (SEQ ID NO: 8); CATCATCTTTGTGYT (SEQ ID NO: 9); GCAATGACCCCGARC (SEQ ID NO: 10); TCATCATCTGCTTYG (SEQ ID NO:ll); and CGTGGTGGGGCACRA (SEQ ID NO:12).
- oligonucleotides of the invention hybridize to a target region located one to several nucleotides downstream of one of the novel polymo ⁇ hic sites identified herein. Such oligonucleotides are useful in polymerase-mediated primer extension methods for detecting one of the novel polymo ⁇ hisms described herein and therefore such 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 PTAFR gene polymo ⁇ hisms by primer extension terminates in a nucleotide sequence, listed 5 ' to 3 ', selected from the group consisting of:
- GAATTTTTGT SEQ ID NO:13
- TGGAAGACCA SEQ ID NO:14
- CATCTTTGTG SEQ ID NO:15
- ATGACCCCGA SEQ ID NO:16
- TCATCTGCTT SEQ ID NO:17
- GGTGGGGCAC SEQ ID NO:18
- a composition contains two or more differently labeled PTAFR oligonucleotides for simultaneously probing the identity of nucleotides or nucleotide pairs at two or more polymo ⁇ hic 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 polymo ⁇ hic site.
- PTAFR 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 oligonucleotides may be used in a variety of polymo ⁇ hism detection assays, including but not limited to probe hybridization and polymerase extension assays. Immobilized PTAFR oligonucleotides of the 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 PTAFR 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 PTAFR gene in an individual.
- the terms "PTAFR genotype” and “PTAFR haplotype” mean the genotype or haplotype contains the nucleotide pair or nucleotide, respectively, that is present at one or more of the novel polymo ⁇ hic sites described herein and may optionally also include the nucleotide pair, or nucleotide present at one or more additional polymo ⁇ hic sites in the PTAFR gene.
- the additional polymo ⁇ hic sites may be currently known polymo ⁇ hic sites or sites that are subsequently discovered.
- One embodiment of a genotyping method of the invention involves isolating from the individual a nucleic acid sample comprising the two copies of the PTAFR gene, mRNA transcripts thereof or cDNA copies thereof, or a fragment of any of the foregoing, that are present in the individual, and determining the identity of the nucleotide pair at one or more polymo ⁇ hic sites selected from the group consisting of PSI, PS2 and PS4 in the two copies to assign a PTAFR genotype to the individual.
- the two "copies" of a gene, mRNA or cDNA (or fragment of such PTAFR molecules) in an individual may be the same allele or may be different alleles.
- a genotyping method of the invention comprises determining the identity of the nucleotide pair at each of PS1-PS5.
- 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 PTAFR 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 not present in the mRNA or cDNA. If a PTAFR gene fragment is isolated, it must contain the polymo ⁇ hic site(s) to be genotyped.
- One embodiment of a haplotyping method of the invention comprises isolating from the individual a nucleic acid sample containing only one of the two copies of the PTAFR gene, mRNA or cDNA, or a fragment of such PTAFR molecules, that is present in the individual and determining in that copy the identity of the nucleotide at one or more polymo ⁇ hic sites selected from the group consisting of PSI, PS2 and PS4 in that copy to assign a PTAFR haplotype to the individual.
- the nucleic acid used in the above haplotyping methods of the invention may be isolated using any method capable of separating the two copies of the PTAFR gene or fragment such as one of the methods described above for preparing PTAFR isogenes, with targeted in vivo cloning being the preferred approach.
- any individual clone will typically only provide haplotype information on one of the two PTAFR gene copies present in an individual. If haplotype information is desired for the individual's other copy, additional PTAFR clones will usually need to be examined. Typically, at least five clones should be examined to have more than a 90% probability of haplotyping both copies of the PTAFR gene in an individual.
- the haplotype for the other allele may be inferred if the individual has a known genotype for the polymo ⁇ hic sites of interest or if the haplotype frequency or haplotype pair frequency for the individual's population group is known.
- the PTAFR haplotype is assigned to the individual by also identifying the nucleotide at one or more polymo ⁇ hic sites selected from the group consisting of PS3 and PS5. In a particularly preferred embodiment, the nucleotide at each of PS1-PS5 is identified.
- the haplotyping method comprises determining whether an individual has one or more of the PTAFR haplotypes shown in Table 5. This can be accomplished by identifying, for one or both copies of the individual's PTAFR gene, the phased sequence of nucleotides present at each of PS1-PS5. This identifying step does not necessarily require that each of PS1-PS5 be directly examined. Typically only a subset of PS1-PS5 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 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. 2000 PNAS 97: 10483-10488; Rieder MJ et al.
- a PTAFR 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 and PS4 in each copy of the PTAFR gene that is present in the individual.
- the haplotyping method comprises identifying the phased sequence of nucleotides at each of PS1-PS5 in each copy of the PTAFR gene.
- the identifying step is preferably performed with each copy of the gene being placed in separate containers.
- the two copies are labeled with different tags, or are otherwise separately distinguishable or identifiable, it could be possible in some cases to perform the method in the same container.
- 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 polymo ⁇ hic site(s), then detecting a combination of the first and third dyes would identify the polymo ⁇ hism in the first gene copy while detecting a combination of the 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 of the PTAFR 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 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;
- PCR polymerase chain reaction
- LCR ligase chain reaction
- WO90/01069 oligonucleotide ligation assay
- OVA oligonucleotide ligation assay
- Other known 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 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 polymo ⁇ hic 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 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-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 PTAFR gene of an individual may also be determined by hybridization of a nucleic acid sample containing one or both copies of the gene, mRNA, cDNA 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 polymo ⁇ hic sites to be included in the genotype or haplotype.
- the identity of 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).
- 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 (WO92/15712) and the ligase/polymerase mediated genetic ' bit analysis (U.S. Patent 5,679,524.
- Related methods are disclosed in WO91/02087, WO90/09455, WO95/17676, U.S. Patent Nos. 5,302,5.09, 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. Gin. Invest. 95:1635-1641, 1995).
- .multiple polymo ⁇ hic sites may be investigated by simultaneously amplifying multiple regions of the nucleic acid using sets of allele-specific primers as described in Wallace et al. (WO89/10414).
- the identity of the allele(s) present at any of the novel polymo ⁇ hic sites described herein may be indirectly determined by haplotyping or genotyping another polymo ⁇ hic site that is in linkage disequilibrium with the polymo ⁇ hic site that is of interest.
- Polymo ⁇ hic sites in linkage disequilibrium with the presently disclosed polymo ⁇ hic sites may be located in regions of the gene or in other genomic regions not examined herein.
- Detection of the allele(s) present at a polymo ⁇ hic site in linkage disequilibrium with the novel polymo ⁇ hic 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 polymo ⁇ hic site.
- an individual's PTAFR haplotype pair is predicted from its PTAFR genotype using information on haplotype pairs known to exist in a reference population.
- the haplotyping prediction method comprises identifying a PTAFR genotype for the individual at two or more PTAFR polymo ⁇ hic sites described herein, accessing data , containing PTAFR haplotype pairs identified in a reference population, and assigning a haplotype pair to the individual that is consistent with the genotype data.
- the reference haplotype pairs include the PTAFR haplotype pairs shown in Table 4.
- the PTAFR haplotype pair can be assigned by comparing the individual's genotype with the genotypes corresponding to the haplotype pairs known to exist in the general population or in a specific population group, and determining which haplotype pair is consistent with the genotype of the individual.
- the comparing step may be performed by visual inspection (for example, by consulting Table 4).
- frequency data such as that presented in Table, 7 may be used to determine which of these haplotype pairs is most likely to be present in the individual. This determination may also be performed in some embodiments by visual inspection, for example by consulting Table 7.
- the comparison may be made by a computer-implemented algorithm with the genotype of the individual and the reference haplotype data stored in computer-readable formats.
- one computer-implemented algorithm to perform this comparison entails enumerating all possible haplotype pairs which are consistent with the genotype, > accessing data containing PTAFR haplotype pairs frequency data determined in a reference population to determine a probability that the individual has a possible haplotype pair, and analyzing the determined probabilities to assign a haplotype pair to the individual.
- 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.
- 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.
- 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; copending PCT USOl/12831 filed April 18, 2001 ) 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 SystemTM technology (U.S. Patent No. 5,866,404), SMD, or allele-specific long-range PCR (Michalotos-Beloin et al., supra).
- a direct molecular haplotyping method such as, for example, CLASPER SystemTM 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 PTAFR 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 PTAFR polymo ⁇ hic sites described herein, and calculating the frequency any particular genotype, haplotype, or haplotype pair is found in the population.
- the population may be e.g., a reference population, a family population, a same gender 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 PTAFR 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 PTAFR 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 or more of the methods described above.
- the haplotypes for the trait population may be determined directly or, alternatively, by a predictive genotype to haplotype approach as 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.
- the frequencies of all genotypes, haplotypes, and/or haplotype pairs observed in the populations are compared. If a particular PTAFR 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 PTAFR genotype, haplotype or haplotype pair.
- the PTAFR 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.
- Sub-genotypes useful in the invention preferably do not include sub-genotypes solely for any one of PS3 and PS5 or for any combination thereof.
- the trait of interest is a clinical response exhibited by a patient to some therapeutic treatment, for example, response to a drug targeting PTAFR 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/or adverse response (i.e., side effects).
- clinical population In order to deduce a correlation between clinical response to a treatment and a PTAFR 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 but 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 III 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.
- the PTAFR 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 PTAFR genotype or haplotype content are created. Correlations may be produced in several ways. In one method, individuals are grouped by their PTAFR genotype or haplotype (or haplotype pair) (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 PTAFR 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 Then- 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 polymo ⁇ hic sites in the PTAFR gene.
- ANOVA analysis of variation
- a mathematical model may be readily constructed by the skilled artisan that predicts clinical response as a function of PTAFR 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 PTAFR 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 polymo ⁇ hic sites in the PTAFR 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 PTAFR genotype or haplotype that is in turn correlated with the clinical response.
- this diagnostic method uses the predictive haplotyping method described above.
- the invention provides an isolated polynucleotide. comprising a polymo ⁇ hic variant of the PTAFR gene or a fragment of the gene which contains at least one of the novel polymo ⁇ hic sites described herein.
- the nucleotide sequence of a variant PTAFR 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 polymo ⁇ hic sites PSI, PS2 and PS4, and may also comprise one or more additional polymo ⁇ hisms selected from the group consisting of adenine at PS3 and guanine at PS5.
- nucleotide sequence of a variant fragment of the PTAFR gene is identical to the corresponding portion of the reference sequence except for haying a different nucleotide at one or more of the novel polymo ⁇ hic sites described herein.
- the invention specifically does not include polynucleotides comprising a nucleotide sequence identical to the reference sequence of the PTAFR gene, which is defined by haplotype 3, (or other reported PTAFR sequences) or to portions of the reference sequence (or other reported PTAFR sequences), except for the haplotyping and genotyping oligonucleotides described above.
- the location of a polymo ⁇ hism in a variant PTAFR gene or fragment is preferably identified by aligning its sequence against SEQ ID NO: 1.
- the polymo ⁇ hism is selected from the group consisting of adenine at PSI, thymine at PS2 and thymine at PS4.
- the polymo ⁇ hic variant comprises a naturally-occurring isogene of the PTAFR gene which is defined by any one of haplotypes 1- 2 and 4 - 6 shown in Table 5 below.
- Polymo ⁇ hic variants of the invention may be prepared by isolating a clone containing the PTAFR gene from a human genomic library.
- the clone may be sequenced to determine the identity of the nucleotides at the novel polymo ⁇ hic sites described herein.
- Any particular variant or fragment thereof, that is claimed herein could be prepared from this clone by performing in vitro mutagenesis using procedures well-known in the art.
- Any particular PTAFR variant or fragment thereof may also be prepared using synthetic or semi-synthetic methods known in the art.
- PTAFR isogenes, or fragments thereof may be isolated using any method that allows separation of the two "copies" of the PTAFR 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 (TIVC) in yeast as described in WO 98/01573, U.S. Patent No.
- TIVC targeted in vivo cloning
- 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. Yet other methods are single molecule dilution (SMD) as described in Ruano et al., Proc. Natl. Acad. Sci. 87:6296-6300, 1990; and allele specific PCR (Ruano et al., 1989, supra; Ruano et al., 1991, supra; Michalatos-Beloin et al., supra).
- SMD single molecule dilution
- the invention also provides PTAFR genome anthologies, which are collections of at least two PTAFR 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 gender population.
- a PTAFR genome anthology may comprise individual PTAFR 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 PTAFR isogenes in the anthology may be stored in separate containers.
- a preferred PTAFR 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 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 PTAFR 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 PTAFR 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, 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.
- polymo ⁇ hic variants of the PTAFR gene will produce PTAFR 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 PTAFR cDNA comprising a nucleotide sequence which is a polymo ⁇ hic variant of the PTAFR reference coding sequence shown in Figure 2.
- the invention also provides PTAFR mRNAs and corresponding cDNAs which comprise a nucleotide sequence that is identical to SEQ ID NO:2 (Fig.
- polymo ⁇ hisms selected from the group consisting of thymine at a position corresponding to nucleotide 82 and thymine at a position corresponding to nucleotide 735, and may also comprise one or more additional polymo ⁇ hisms selected from tlie group consisting of adenine at a position corresponding to nucleotide 671 and guanine at a position corresponding to nucleotide 1013.
- a particularly preferred polymo ⁇ hic cDNA variant comprises the coding sequence of a PTAFR isogene defined by any one of haplotypes 2 and 4-6. Fragments of these variant mRNAs and cDNAs are included in the scope of the invention, provided they contain one or more of the novel polymo ⁇ hisms described herein.
- the invention specifically excludes polynucleotides identical to previously identified PTAFR mRNAs or cDNAs, and previously described fragments thereof.
- Polynucleotides comprising a variant PTAFR 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 PTAFR gene, mRNA or cDNA 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 PTAFR gene or cDNA 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 of the PTAFR genomic, mRNA and cDNA variants described herein.
- Polynucleotides comprising a polymo ⁇ hic gene variant or fragment of the invention 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 PTAFR isogene encoding that isoform or may already have at least one copy of that isogene.
- PTAFR isogene In other situations, it may be desirable to decrease or block expression of a particular PTAFR isogene. Expression of a PTAFR 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 or antisense RNA for the isogene or fragment thereof. Alternatively, 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.
- regulatory regions e.g., promoter, introns, enhancers, 3' untranslated region
- oligonucleotides mat 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 PTAFR mRNA transcribed from a particular isogene. It is also contemplated that ribozymes may be designed that can catalyze the specific cleavage of PTAFR mRNA transcribed from a particular isogene.
- the untranslated.mRNA, antisense RNA or antisense 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. Alternatively, such molecules 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 (a) the reference PTAFR amino acid sequence shown in Figure 3 or (b) a fragment of this reference sequence.
- the location of a variant amino acid in a PTAFR polypeptide or fragment of the invention is preferably identified by aligning its sequence against SEQ ID NO:3 (Fig. 3).
- a PTAFR protein variant of the invention comprises an amino acid sequence identical to SEQ JJD NO:3 for those regions of SEQ ID NO: 3 that are encoded by examined portions of the PTAFR gene (as described in the
- a PTAFR fragment of the invention also referred to herein as a PTAFR peptide variant, is any fragment of a PTAFR protein variant that contains phenylalanine at a position corresponding to amino acid position 28.
- the invention specifically excludes amino acid sequences identical to those previously identified for PTAFR, including SEQ ID NO:3, and previously described fragments thereof.
- PTAFR 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 PTAFR protein variant of the invention is encoded by an isogene defined by one of the observed haplotypes, 2 and 4-6, shown in Table 5.
- a PTAFR peptide variant of the invention 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 PTAFR peptide variants may be useful as antigens to generate antibodies specific for one of the above PTAFR isoforms.
- the PTAFR peptide variants may be useful in drug screening assays.
- a PTAFR variant protein or peptide of the invention may be prepared by chemical synthesis or by expressing an appropriate variant PTAFR genomic or cDNA sequence described above.
- the PTAFR protein variant may be isolated from a biological sample of an individual having a PTAFR isogene which encodes the variant protein. Where the sample contains two different PTAFR isoforms (i.e., the individual has different PTAFR isogenes), a particular PTAFR isoform of the invention can be isolated by immunoaffinity chromatography using an antibody which specifically binds to that particular PTAFR isoform but does not bind to the other PTAFR isoform.
- the expressed or isolated PTAFR protein or peptide may be detected by methods known in the art, including C ⁇ omassie blue staining, silver staining, and Western blot analysis using antibodies specific for the isoform of the PTAFR protein or peptide as discussed further below.
- PTAFR variant proteins and peptides 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 immunoaffinity chromatography and the like. (Ausubel et. al., 1987, In Current Protocols in Molecular Biology John Wiley and Sons, New York,
- antibodies specific for a particular polymo ⁇ hic variant may be used.
- a polymo ⁇ hic variant PTAFR gene of the invention may also be fused in frame with a heterologous sequence to encode a chimeric PTAFR protein.
- the non-PTAFR 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 PTAFR and non-PTAFR portions so that the PTAFR protein may be cleaved and purified away from the non-PTAFR portion.
- An additional embodiment of the invention relates to using a novel PTAFR protein isoform, or a fragment thereof, in any of a variety of drug screening assays.
- screening assays may be performed to identify agents that bind specifically to all known PTAFR 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 PTAFR protein or peptide variant may be free in solution or affixed to a solid support.
- high throughput screening of compounds for binding to a PTAFR 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 PTAFR protein(s) of interest and then washed. Bound PTAFR protein(s) are then detected using methods well-known in the art.
- a novel PTAFR protein isoform may be used in assays to measure the binding affinities of one or more candidate drugs targeting the PTAFR protein.
- a particular PTAFR haplotype or group of PTAFR haplotypes encodes a PTAFR protein variant with an amino acid sequence distinct from that of PTAFR protein isoforms encoded by other PTAFR haplotypes
- detection of that particular PTAFR haplotype or group of PTAFR haplotypes may be accomplished by detecting expression of the encoded PTAFR 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 PTAFR protein or peptide variants described herein.
- the antibodies may be either monoclonal or polyclonal in origin.
- the PTAFR protein or peptide variant used to generate the antibodies may be from natural or recombinant sources (in vitro or in vivo) or produced by chemical synthesis or semi-synthetic synthesis using synthesis techniques known in the art. If the PTAFR protein or peptide variant is of insufficient size to be antigenic, it may be concatenated or 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,
- an antibody specifically immunoreactive with one of the novel protein or peptide variants described herein is administered to an individual to neutralize activity of the PTAFR 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 PTAFR protein variant from solution as well as react with PTAFR protein isoforms on Western or immunoblots of polyacrylamide gels on membrane supports or substrates.
- the antibodies will detect PTAFR 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 PTAFR 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 PTAFR protein variant and the antibody is detected.
- suitable immunoassays include radioimmunoassay, Western blot assay, immunofluoresce ⁇ t 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. Burdoii et al., Volume 13, Elsevier Science Publishers,
- 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, W ⁇ 901443 and W ⁇ 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 PTAFR may be investigated by various means known in the art, such as by in vitro translation of mRNA transcripts of the PTAFR gene, cDNA orfragment thereof, or by preparing recombinant cells and/or nonhuman recombinant organisms, preferably recombinant animals, containing a polymo ⁇ hic variant of the PTAFR 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(s) into PTAFR protein(s) (including effects of polymo ⁇ hihsms on codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
- the desired PTAFR isogene, cDNA or coding sequence may be introduced into the cell in a vector such that the isogene, cDNA or coding sequence remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location.
- the PTAFR isogene, cDNA or coding sequence is introduced into a cell in such a way that it recombines with the endogenous PTAFR gene present in the cell. Such recombination requires the occurrence of a double recombination event, thereby resulting in the desired PTAFR 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 PTAFR isogene, cDNA or coding sequence may be introduced include, but are not limited to, continuous culture cells, such as COS, . CHO, NEH/3T3, and primary or culture cells of the relevant tissue type, i.e., they express the PTAFR isogene, cDNA or coding sequence. 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 PTAFR gene, cDNA or coding sequence are prepared using standard procedures known in the art.
- a construct comprising the variant gene, cDNA or coding sequence 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 lafer than about the eight-cell stage.
- Transgenic animals carrying the constructs of the 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 (or cDNA or coding sequence) 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.
- mice Examples of animals into which the PTAFR isogene, cDNA or coding sequences 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. Gilriian, J. Witkowski, and M. Zoller; W.H. Freeman and Company, New York, pages 254-272).
- Transgenic animals stably expressing a human PTAFR isogene, cDNA or coding sequence and producing the encoded human PTAFR protein can be used as biological models for studying diseases related to abnormal PTAFR 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 PTAFR isogene described herein.
- the pharmaceutical composition may comprise any of the following active ingredients: a polynucleotide comprising one of these novel PTAFR isogenes (or cDNAs or coding sequences); an antisense oligonucleotide directed against one of the novel PTAFR isogenes, a polynucleotide encoding such an antisense oligonucleotide, or another compound which inhibits expression of a novel PTAFR 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 administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).
- determination of the therapeutically effective dose of active ingredient and/or the appropriate route of administration is well within 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 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.
- any or all analytical and mathematical operations involved in practicing the methods of the 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 PTAFR 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 PTAFR 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).
- 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.
- EXAMPLE 1 This example illustrates. examination of various regions of the PTAFR gene for polymorphic sites.
- the following target regions were amplified using either the PCR primers represented below or 'tailed' PCR primers, each of which includes a universal sequence forming a noncomplementary 'tail' attached to the 5' end of each unique sequence in the PCR primer pairs.
- the universal 'tail' sequence for the forward PCR primers comprises the sequence 5'-TGTAAAACGACGGCCAGT-3' (SEQ ID NO: 19) and the universal 'tail' sequence for the reverse PCR primers comprises the sequence
- Fragment 2 4000-4023 complement of 4417-4396 418 nt
- Fragment 3 4000-4023 complement of 4466-4446 467 nt
- Fragment 4 4187-4208 complement of 4778-4758 592 nt
- Fragment 6 1 4719-4740 complement of 5244-5223 526 nt These primer pairs were used in PCR reactions containing genomic DNA isolated from immortalized cell lines for each member of the Index Repository. The PCR reactions were carried out under the following conditions:
- Amplification profile 97°C - 2 min. 1 cycle
- 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 either the primer sets represented below with the positions of their first and last nucleotide corresponding to positions in Figure 1, or the appropriate universal 'tail' sequence as a primer. Reaction products were purified by isopropanol precipitation, and run on an Applied Biosystems 3700 DNA Analyzer.
- Fragment No. Forward Primer Reverse Primer Fragment 1 3973-3992 complement of 4465-4446 Fragment 2 Tailed Primer Fragment 3 4079-4091 complement of 4418-4399 Fragment 4 4218-4238 complement of 4727-4708 Fragment 5 4515-4533 complement of 4998-4979 Fragment 6 Tailed Primer
- Polyld is a unique identifier assigned to each PS by Genaissance Pharmaceuticals, Inc. (R) Reported previously.
- This example illustrates analysis of the PTAFR polymo ⁇ hisms identified in the Index Repository for human genotypes and haplotypes.
- the different genotypes containing these polymo ⁇ hisms that were observed in unrelated members of 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 a d 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. Table 4. Genotypes and Haplotype Pairs Observed for PTAFR Gene
- 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 7, 111-122) for assigning haplotypes to unrelated individuals in a population sample, as described in PCT/USOl/12831, filed April 18, 2001.
- haplotypes are assigned directly from individuals who are homozygous at all sites or heterozygous at no more than one of the variable sites.
- This list of haplotypes is then used to deconvolute the unphased genotypes in the remaining (multiply heterozygous) individuals.
- the list of haplotypes was augmented with haplotypes obtained from two families (one three-generation Caucasian family and one two-generation African-American family).
- a PTAFR isogene defined by a full-haplotype shown in Table 5 below comprises the regions of the SEQ ID NOS indicated in Table 5, with their corresponding set of polymo ⁇ hic locations and identities, which are also set forth in Table 5.
- Region examined represents the nucleotide positions defining the start and stop positions within SEQ ID NO: 1 of the regions sequenced;
- SEQ ID NO:l refers to Figure 1, with the two alternative allelic variants of each polymo ⁇ hic site indicated by the appropriate nucleotide symbol.
- SEQ ID NO:21 is a modified version of SEQ ID NO:
- SEQ ID NO:21 that shows the context sequence of each of PS1-PS5 in a uniform format to facilitate electronic searching of the PTAFR haplotypes.
- SEQ ID NO:21 contains a block of 60 bases of the nucleotide sequence encompassing the centrally-located polymo ⁇ hic site at the 30 th position, followed by 60 bases of unspecified sequence to represent that each p ⁇ lymo ⁇ hic site is separated by genomic sequence whose composition is defined elsewhere herein.
- the size and composition of the Index Repository were chosen to represent the genetic diversity across and within four major population groups comprising the general United States population.
- this repository contains approximately equal sample sizes of African-descent, Asian-American, European-American, and Hispanic-Latino population groups. Almost all individuals representing each group had all four grandparents with the same ethnogeographic background.
- the number of unrelated individuals in the Index Repository provides a sample size that is sufficient to detect SNPs and haplotypes that occur in the general population with high statistical certainty. For instance, a haplotype that occurs with a frequency of 5% in the general population has a probability higher than 99.9% of being observed in a sample of 80 individuals from the general population.
- a haplotype that occurs with a frequency of 10% in a specific population group has a 99% probability of being observed in a sample of 20 individuals from that population group.
- the size and composition of the Index Repository means that the relative frequencies determined therein for the haplotypes and haplotype pairs of the PTAFR gene are likely to be similar to the relative frequencies of these PTAFR haplotypes and haplotype pairs in the general U.S. population and in the four population groups represented in the Index Repository. The genetic diversity observed for the three Native Americans is presented because it is of scientific interest, but due to the small sample size it lacks statistical significance.
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Non-Patent Citations (2)
Title |
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CHASE ET AL.: 'Cloning of a human platelet-activating factor receptor gene: evidence for an intron in the 5'-untranslated region' AMERICAN JOURNAL OF RESPIRATORY AND MOLECULAR BIOLOGY vol. 8, no. 3, March 1993, pages 240 - 244, XP002951953 * |
DATABASE GENBANK [Online] May 2000 BIRREN ET AL.: 'Homo sapiens chromosome 1 clone RP11-1C6 map 1 working draft sequence', XP002951952 Retrieved from EMBL Database accession no. (AC027421.3) * |
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