EP1552012A2 - Susceptibility gene for human stroke; methods of treatment - Google Patents
Susceptibility gene for human stroke; methods of treatmentInfo
- Publication number
- EP1552012A2 EP1552012A2 EP03770392A EP03770392A EP1552012A2 EP 1552012 A2 EP1552012 A2 EP 1552012A2 EP 03770392 A EP03770392 A EP 03770392A EP 03770392 A EP03770392 A EP 03770392A EP 1552012 A2 EP1552012 A2 EP 1552012A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- stroke
- pde4d
- nucleic acid
- gene
- polypeptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/172—Haplotypes
Definitions
- Stroke is a common and serious disease. Each year in the United States more than 600,000 individuals suffer a stroke and more than 160,000 die from stroke- related causes (Sacco, R.L. et al, Stroke 28, 1507-17 (1997)). In western countries stroke is the leading cause of severe disability and the third leading cause of death (Bonita, R., Lancet 339, 342-4 (1992)). The lifetime risk of those who reach the age of 40 exceeds 10%.
- the clinical phenotype of stroke is complex but is broadly divided into ischemic (accounting for 80-90%) and hemorrhagic stroke (10-20%) (Caplan, L.R. Caplan 's Stroke: A Clinical Approach, 1-556 (Butterworth-Heinemann, 2000)).
- Ischemic stroke is further subdivided into large vessel occlusive disease (referred to here as carotid stroke), usually due to atherosclerotic involvement of the common and internal carotid arteries, small vessel occlusive disease, thought to be a non- atherosclerotic narrowing of small end-arteries within the brain, and cardiogenic stroke due to blood clots arising from the heart usually on the background of atrial fibrillation or ischemic (atherosclerotic) heart disease (Adams, H.P., Jr. et al., Stroke 24, 35-41 (1993)). Therefore, it appears that stroke is not one disease but a heterogeneous group of disorders reflecting differences in the pathogenic mechanisms (Alberts, M .
- a locus conferring susceptibility to ischemic stroke to chromosome 5ql2 in the Icelandic population has been mapped and the identification of phosphodiesterase 4D (PDE4D) as the gene at 5ql2 contributing to the risk of ischemic stroke has been reported.
- PDE4D phosphodiesterase 4D
- This locus was extensively fine mapped and tested for association to stroke. Most striking is that haplotypes can be classified into three distinct groups: wild type, at- risk and protective. Additionally, a significant disregulation of multiple PDE4D isoforms in stroke patients was observed. The strongest association was within the PDE4D, especially to the two major subtypes of ischemic stroke, carotid and cardiogenic stroke.
- the invention relates to methods of diagnosing a predisposition to stroke.
- the methods of diagnosing a predisposition to stroke in an individual include detecting the presence of a polymorphism in PDE4D, as well as detecting alterations in expression of a PDE4D polypeptide or isoform, such as the presence of, or relative expression of different splicing variants of PDE4D polypeptides. For example, it may be that the ratio of certain splice variants could be used as a diagnostic marker for stroke predisposition.
- an abnormal splice form can be detected (that is one that is not normally expressed but is created from a DNA sequence mutation that leads to an abnormal splice form to be created from the primary transcript) may be created from mutations in the PDE4D gene.
- new splice sites might be created from a single base substitution within an intron that is inappropriately used as a splice acceptor or donor site, resulting in an abnormal message which is likely to have a premature stop codon leading to a truncated form of PDE4D protein.
- the alterations in expression can be quantitative, qualitative, or both quantitative and qualitative.
- the methods of the invention allow the accurate diagnosis of stroke at or before disease onset, thus reducing or minimizing the debilitating effects of stroke.
- predisposition to stroke or susceptibility to stroke can be assessed by determining PDE4D isoform levels in the individual compared to control levels, wherein a difference in isofoim expression is indicative of predisposition or susceptibility to stroke.
- the level of expression of PDE4D7 and/or PDE4D9 is assessed.
- the invention additionally relates to an assay for identifying agents that alter (e.g., enhance or inhibit) the activity or expression or transcription of one or more PDE4D polypeptides or isoforms.
- agents that alter e.g., enhance or inhibit
- Such an assay may also identify agents that alter the relative expression of one or more PDE4D isoforms with respect to other isoforms at either the mRNA level or polypeptide level.
- a cell, cellular fraction, or solution containing a PDE4D polypeptide or a fragment or derivative thereof can be contacted with an agent to be tested, and the level of PDE4D polypeptide expression or activity can be assessed.
- a cell, or cell with artificial DNA construct with part or all of the PDE4D gene with or without a reporter gene can be used to identify agents that may directly affect transcription at one or more of the many alternative PDE4D promoters upstream of the alternative 5 prime exons or splicing efficiency of the primary transcript to one or more mRNA isoforms.
- the activity or expression of more than one PDE4D polypeptides can be assessed concurrently (or the corresponding reporter gene activity) (e.g., the cell, cellular fraction, or solution can contain more than one type of PDE4D polypeptide, such as different splicing variants, and the levels of the different polypeptides or splicing mRNA variants can be assessed).
- Agents that enhance or inhibit PDE4D mRNA or polypeptide expression or activity are also included in the current invention, as are methods of altering (enhancing or inhibiting) PDE4D mRNA or polypeptide expression or activity by contacting a cell containing PDE4D gene, mRNA, and/or polypeptide, or by contacting the PDE4D gene, mRNA, and/or polypeptide, with an agent that enhances or inhibits expression or activity of PDE4D mRNA or polypeptide.
- isoform mRNA and/or protein levels can be altered, compared to control levels, using the agents of the invention.
- the invention pertains to pharmaceutical compositions comprising the nucleic acids of the invention, the polypeptides of the invention, and/or the agents that alter activity of PDE4D polypeptide.
- the invention further pertains to methods of treating stroke, by administering PDE4D therapeutic agents, such as nucleic acids of the invention, polypeptides of the invention, the agents that alter activity of PDE4D polypeptide, or compositions comprising the nucleic acids, polypeptides, and/or the agents that alter activity of PDE4D polypeptide.
- the invention further relates to methods for preventing the occurrence of stroke in an individual in need thereof by regulating a PDE4D mRNA and/or polypeptide isoform level compared to control levels, whereby the regulated isoform level mimics the level of a healthy individual.
- Isoform expression at the mRNA and/or polypeptide level can be regulated using the agents and pharmaceutical compositions of the invention, by genetic alteration, by altering the ratio of isoforms and/or their absolute expression.
- isoforms PDE4D7 and/or PDE4D9 can be regulated.
- the invention further provides a method of diagnosing susceptibility to stroke in an individual.
- This method comprises screening for one of the at-risk haplotypes in the phosphodiesterase 4D gene that is more frequently present in an individual susceptible to stroke, compared to the frequency of its presence in the general population, wherein the presence of an at-risk haplotype is indicative of a susceptibility to stroke.
- An "at-risk haplotype” is intended to embrace one or a combination of haplotypes described herein over the PDE4D gene that show high correlation to stroke.
- the at-risk haplotype is characterized by the presence of at least one single nucleotide polymorphism at nucleic acid positions at risk haplotype 1 is G at nucleic acid position 142780 respectively, relative to SEQ ID NO: 1 and allele 0 of microsatellite marker AC0088181-1.
- the at-risk haplotype 2 is characterized by the presence of at least one single nucleotide polymo ⁇ hism and microsatellite marker at nucleic acid positions 142780, 135112, 132562, 131865, 129361, 129360, 125304, 123426, 123312, 120628, 118914, 111781, 111252, 109301, 107849, 105225, 104552, 102977, 100795, 99035, 88614, 88456, 83119, 82244, 80127, 78552, relative to SEQ ID NO: 1 and allele 0 microsatellite marker AC0088181-1.
- the at-risk haplotype 3 is characterized by the presence of at least one polymo ⁇ hism at nucleic acid positions 138806, 131865, 129361, 120628, 91470 relative to SEQ ID NO: 1.
- Also described are methods for diagnosing susceptibility to stroke in an individual comprising screening for an at-risk haplotype in the phosphodiesterase 4D gene that is more frequently present in an individual susceptible to stroke (affected), compared to the frequency of its presence in a healthy individual (control) wherein the screening for the presence of an at-risk haplotype within or near PDE4D that significantly correlates with at least one of the haplotypes described herein or stroke susceptibility.
- a simple test for correlation would be a Fisher-exact test on a two by two table. Given a cohort of chromosomes the two by two table is constructed out of the number of chromosomes that include both of the haplotypes, one of the haplotype but not the other and neither of the haplotypes.
- a protective haplotype is intended to embrace one or a combination of haplotypes described herein over the PDE4D gene that show a protective characteristic or property of a reduced risk of stroke.
- the particular combination of genetic markers (haplotypes) are present at a higher than expected frequency in controls than patients.
- Individuals with a protective allele or haplotype are about 30% less likely to have a stroke compared to the general population.
- a protective haplotype is characterized by the presence of at least one single nucleotide polymo ⁇ hism, such as the allele A at nucleotide position 142780 relative to SEQ ID NO: 1.
- the presence of the polymo ⁇ hisms that comprise the at- risk haplotype or protective haplotype can be determined by electrophoretic analysis, restriction length polymo ⁇ hism analysis, fluorescence energy transfer detection, kinetic PCR, allele specific PCR, sequence analysis, hybridization analysis or other known techniques.
- Kits for diagnosing susceptibility to stroke in an individual comprise primers for nucleic acid amplification of a region of PDE4D comprising the at-risk haplotype and/or protective haplotype.
- the first major application of the current invention involves prediction of those at higher risk of developing a stroke. Diagnostic tests that define genetic factors contributing to stroke might be used together with or independent of the known clinical risk factors to define an individual's risk relative to the general population. Better means for identifying those individuals at risk for stroke should lead to better prophylactic and treatment regimens, including more aggressive management of the current clinical risk factors such as hypertension, diabetes, hypercholesterolemia, hypertriglyceridemia, obesity, and inflammatory components as reflected by increased C-reactive protein levels or other inflammatory markers. Information on genetic risk may be used by physicians to help convince particular patients to adjust life style and quit smoking. This invention provides the means to define a genetic component that doubles an individual's risk for stroke.
- the second major application of the current invention is the specific identification of a rate-limiting pathway involved in stroke. While many have attempted to find genes that are over-expressed or under-expressed in atherosclerosis plaques in the carotid arteries, the vast majority of the changes seen in diseased blood vessels compared to normal blood vessels are simply a reaction to the underlying process of atherosclerosis and stroke predisposition and are not the underlying cause.
- a disease gene with genetic variation that is significantly more common in stroke patients as compared to controls represents a specifically validated causative step in the pathogenesis of stroke. That is, the uncertainty about whether a gene is causative or simply reactive to the disease process is eliminated.
- the protein encoded by the disease gene defines a rate-limiting molecular pathway involved in the biological process of stroke predisposition.
- the proteins encoded by such stroke genes or its interacting proteins in its molecular pathway may represent drug targets that may be selectively modulated by small molecule, protein, antibody, or nucleic acid therapies. Such specific information is greatly needed since stroke prevention and treatment is a major unmet medical need that affects over a half-million Americans each year. Also useful is determining the gene that is protective against stroke.
- the proteins encoded by the protective gene and the biological pathway that it is a member may represent another target selectively modulated by small molecule, protein antibody or nucleic acid therapies.
- a third application of the current invention is its use to predict an individual's response to a particular drug, even drugs that do not act on PDE4D or its pathway. It is a well-known phenomenon that in general, patients do not respond equally to the same drug. Much of the differences in drug response to a given drug is thought to be based on genetic and protein differences among individuals in certain genes and their corresponding pathways. Our invention defines the PDE4D pathway and its effect on cAMP levels in cells where it is expressed as one key molecular pathway involved in stroke risk. Some current or future therapeutic agents may be able to affect this pathway directly or indirectly and therefore, be effective in those patients whose stroke risk is in part determined by PDE4D pathway genetic variation.
- PDE4D variation or haplotypes may be used as a pharmacogenomic diagnostic to predict drug response and guide choice of therapeutic agent in a given individual.
- the invention helps meet the unmet medical needs in at least two major ways: 1) it provides a means to define patients at higher risk for stroke than the general population who can be more aggressively managed by their physicians in an effort to prevent stroke; and 2) it defines a drug target that can be used to screen and develop therapeutic agents that can be used to prevent stroke before it happens or prevent a second stroke in those who have already suffered a stroke or transient ischemic attack.
- FIGS. 1.1 and 1.2 show two family pedigrees each affected by several of the stroke subtypes, including hemorrhagic stroke.
- FIGS. 2.1, 2.2 and 2.3 show the genetic, combined and physical maps for locating the PDE4D gene using 30 polymo ⁇ hic markers.
- all markers have been assigned in the genetic and physical map unless otherwise indicated (* indicates marker only assigned in the physical map; ** indicates markers only assigned in genetic map).
- FIG. 3 shows the schematic representations of PDE4D splice variants.
- Splice variants PDE4D9 are novel, as well as exons D7A-1, D7A-2, D7A-3, D8 and D9.
- Splice variants 4DN1, 4DN2 and 4DN3 (Miro, et al, Biochem. Biophys. Res. Comm., 274: 415-421 (2002), and 4D1, 4D2, 4D3, 4D4 and 4D5 are known (Bolger et al, Biochem. J. pt. 2: 539-548 (1997).
- FIG. 4 is a graphic representation showing PDE4D isoform expression in EBV transformed cells (expression of PDE4D3 and PDE4D9 below detection limits).
- FIG. 5 is a graphic representation showing expression of PDE4D isoforms in EBV transfo ⁇ ned cells from patients with or without the stroke-associated haplotype.
- FIG. 6 is a graphic representation showing expression of PDE4D isoforms in EBV cells from controls with or without the stroke-associated haplotype.
- FIGS. 7.1 to 7.10 show the amino acid sequences for the isoforais of the PDE4D gene.
- SEQ ID NO: 2 is D4;
- SEQ ID NO: 3 is N2;
- SEQ ID NO: 4 is D5;
- SEQ ID NO: 5 is N3;
- SEQ ID NO: 6 is D3;
- SEQ ID NO: 7 is NI;
- SEQ ID NO: 8 is D8;
- SEQ ID NO: 9 is DI;
- SEQ ID NO: 10 is D2.
- FIGS. 8.1 and 8.2 list all publicly available PDE4D mRNAs and novel cDNA segments identified by deCODE genetics.
- FIGS. 9.1 to 9.351 show the genomic sequence of the human PDE4D gene.
- FIGS. 10.1 to 10.3 show a graphic representation showing the single marker allelic association within the PDE4D gene.
- FIG. 10.1 is a schematic showing the gene structures.
- FIG. 10.2 shows graphic representation of the microsatellite and SNP distribution within thePDE4D gene.
- FIG. 10.3 shows graphic representation of the single marker allelic association across the PDE4D gene for both microsatellites (filled circles) and SNPs (open circles); negative log p-valve versus the physical location in kilobases.
- FIGS. 11.1 to 11.3 graphically depict the haplotype association for carotid and cardiogenic stroke combined.
- FIG. 11.1 is a comparison of groups of haplotypes constructed from SNP45 and AC008818-1, two markers separated by 6kb.
- X is a composite allele that denotes jointly all alleles of AC008818-1 except allele 0.
- haplotype A0 that is not found in our samples, other haplotypes can be grouped into three groups with distinct risks.
- Each arrow corresponds to a comparison between two groups and RR is the estimated risk of the group the arrow is pointing at relative to the other group.
- the difference between 1 and the information (Info) is a measure of the fraction of information that is lost due to uncertainty in phase and missing genotypes.
- FIG. 11.1 is a comparison of groups of haplotypes constructed from SNP45 and AC008818-1, two markers separated by 6kb.
- X is a composite allele that denotes jointly all alleles of AC008818-1 except allele 0.
- haplotype A0 that is not found in our samples, other haplotypes can
- H is the at- risk haplotype, identified in FIG. 13 and Lc is a composite haplotype that denotes jointly all haplotypes of the 25 SNPs except H c . Together with AC008818-1 and SNP45, the haplotypes here span 64kb.
- Haplotype GO in A is split into extended haplotypes GOHc and GOLc- GOHc has significantly higher risk than G0Z,c, and the risk of GOJc is not distinguishable from the wild type GX.
- FIG. 11.3 shows a refinement of the groupings in A — GOJc is moved from the at-risk group to the wild type group. Also noted is that the extended haplotype AXHc does not exist indicating that blocks B and C are in LD.
- FIG. 12 is a schematic representation of the physical map o ⁇ STRKl interval showing all genes and mRNAs in region. Markers identified with an asterisk (*) indicate those with significant single marker association.
- FIGS 13.1 to 13.3 show a graphical depiction of the linkage disequilibrium (LD) and haplotypes in the 5'end of PDE4D gene.
- FIG. 13.1 shows pairwise linkage disequilibrium between SNPs in a 600 kb region in the 5' end of PDE4D. The markers are plotted equidistant. Two measures of LD are shown: D' in the upper left triangle and p-values in the lower right triangle. This region can be divided into three blocks of strong LD, each with limited haplotype diversity, block A, block B and block C. The lines indicate the position of the three exons D7-1, D7-2 and D7-3 and the microsatellite marker AC008818-1.
- FIG. 13.2 show all common haplotypes identified within each of the three blocks. Association results for all the haplotypes are presented in Table 2C.
- FIG. 13.3 depicts the percentage of chromosomes within each block that match one of the common haplotypes
- the first major stroke locus was mapped to 5ql2 using a genome- wide search for susceptibility genes in the common forms of stroke.
- a broad but rigorous definition of the phenotype was used including patients with ischemic stroke, transient ischemic attack (TIA), and hemorrhagic stroke.
- the lod score increased to 4.9 after the hemorrhagic stroke patients were removed, suggesting that the gene at the locus is primarily important for ischemic stroke.
- the most promising region harboring a stroke susceptibility gene was narrowed down to a segment less than 6 cM (approximately 3.8 Mb), from D5S1474 to D5S398, as defined by a decrease of one in LOD score (will be referred to as the "one-LOD interval" hereafter).
- Haplotype analyses show that the most significant haplotype extends over an area of 260 kb covering the first exon of the PDE4D gene.
- the haplotype is significantly associated to carotid and cardiogenic stroke with a relative risk of 2.3 and approximately 47 % of carotid/cardiogenic stroke patients carry at least one copy of this haplotype.
- This same haplotype has a relative risk of 1.8 for stroke in general.
- This haplotype extends over the 5 'exon unique to the PDE4D7 isoform and the presumed promoter region of this isoform suggesting that the functional variation may be involved in transcriptional regulation. This hypothesis is also supported by our PDE4D expression analysis that shows that there is significant correlation between the disease associated haplotype and the level of PDE4D7 message.
- Atherosclerosis is a chronic progressive disease characterized by accumulation of lipids, fibrous, and cellular elements within the large arteries. These lesions can grow sufficiently large to impede blood flow and, more importantly, their surfaces can rupture leading to local thrombus formation occluding the blood vessel and causing a stroke or myocardial infarction.
- the major pathological process for the two ischemic subtypes, carotid and cardiogenic stroke is atherosclerosis.
- Atrial fibrillation may occur on the background of other diseases such as valvular disease, hyperthyroidism, and hypertension
- ischemic heart disease remains one of the most important causes.
- Ischemic stroke resulting from occlusion of small penetrating arteries within the brain is generally thought to result from local endothelial proliferation since atherosclerosis only occurs in larger arteries.
- PDE4D does not show association to small vessel stroke, consistent with it role in atherosclerosis.
- atherosclerosis accounts for the majority of all strokes, particularly carotid and cardiogenic stroke, two subphenotypes that show the strongest association to the PDE4D gene.
- An individual at risk for stroke is an individual who has at least one risk factor, such as previous stroke or TIA, an at-risk haplotype in one or more stroke risk genes, an at-risk haplotype for the PDE4D gene; a polymo ⁇ hism in a PDE4D gene; disregulation of PDE4D isoform expression; diabetes; hypertension; hypercholesterolemia; elevated lp(a); obesity; a past or current smoker; an elevated inflammatory marker (e.g., a marker such as C-reactive protein (CRP), serum amyloid A, fibrinogen, tissue necrosis factor-alpha, a soluble vascular cell adhesion molecule (sVCAM), a soluble mtervascular adhesion molecule (sICAM), E-selectin, matrix metalloprotease type-1, matrix metalloprotease type-2, matrix metalloprotease type-3, and matrix metalloprotease type-9); increased LDL cholesterol and/or decreased HDL cholesterol; and/or at least one previous myocardial
- an individual who has a protective haplotype is one who is less likely to have a stroke.
- an individual who is at risk for stroke is an individual who has a polymo ⁇ hism in a PDE4D gene, in which the presence of the polymo ⁇ hism is indicative of a susceptibility to stroke.
- An individual who has a protective haplotype and less likely to have a stroke is an individual who has a polymo ⁇ hism in a PDE4D gene such as the A allele at nucleotide position 142780 relative to SEQ ID NO: 1, in which the presence of the polymo ⁇ hism is indicative of a protection from stroke.
- gene refers to not only the sequence of nucleic acids encoding a polypeptide, but also the promoter regions, transcription enhancement elements, splice donor/acceptor sites, splice enhancer and silencer sequences and other regulators of splicing, and other non-transcribed nucleic acid elements.
- Representative polymo ⁇ hisms include those presented in Table 11, below.
- an individual who is at risk for stroke is an individual who has an at-risk haplotype in PDE4D, as described herein, particularly but not limited to ischemic stroke.
- Increased risk for the two major subtypes of ischemic stroke, carotid and cardiogenic stroke can be assessed by screening for at-risk haplotype that comprises SNP5PDM361194, SNP5PDM368135, SNP5PDM370640, SNP5PDM379372 and SNP5PDM408531 at the 5' UTR of PDE4D7. Results reported herein indicate that PDE4D is involved in pathogenesis of stroke through atherosclerosis. The major pathological process for carotid stroke and cardiogenic stroke is atherosclerosis. Thus, an individual who is at-risk for atherosclerosis, peripheral arterial occlusive disease, or myocardial infarction can also benefit from the teachings of the invention.
- haplotype refers to a combination of genetic markers ("alleles"), such as those set forth in Tables 1, 2C, 4A and 4B.
- the haplotype can comprise one or more alleles, two or more alleles, three or more alleles, four or more alleles, or five or more alleles.
- the genetic markers are particular "alleles” at "polymo ⁇ hic sites” associated with PDE4D.
- a nucleotide position at which more than one sequence is possible in a population is referred to herein as a "polymo ⁇ hic site”.
- a polymo ⁇ hic site is a single nucleotide in length
- the site is referred to as a single nucleotide polymo ⁇ hism ("SNP").
- SNP single nucleotide polymo ⁇ hism
- polymo ⁇ hic site can allow for differences in sequences based on substitutions, insertions or deletions.
- Each version of the sequence with respect to the polymo ⁇ hic site is referred to herein as an "allele" of the polymo ⁇ hic site.
- the SNP allows for both an adenine allele and a thymine allele.
- a reference sequence is referred to for a particular sequence. Alleles that differ from the reference are referred to as “variant” alleles.
- the reference PDE4D sequence is described herein by SEQ ID NO: 1.
- the term, "variant PDE4D”, as used herein, refers to a sequence that differs from SEQ ID NO: 1, but is otherwise substantially similar.
- the genetic markers that make up the haplotypes described herein are PDE4D variants.
- Additional variants can include changes that affect a polypeptide, e.g., the PDE4D polypeptide.
- sequence differences when compared to a reference nucleotide sequence, can include the insertion or deletion of a single nucleotide, or of more than one nucleotide, resulting in a frame shift; the change of at least one nucleotide, resulting in a change in the encoded amino acid; the change of at least one nucleotide, resulting in the generation of a premature stop codon; the deletion of several nucleotides, resulting in a deletion of one or more amino acids encoded by the nucleotides; the insertion of one or several nucleotides, such as by unequal recombination or gene conversion, resulting in an interruption of the coding sequence of a reading frame; duplication of all or apart of a sequence; transposition; or a rearrangement of a nucleotide sequence, as described in detail above.
- Such sequence changes alter the polypeptide encoded by a PDE4D nucleic acid.
- the change in the nucleic acid sequence causes a frame shift
- the frame shift can result in a change in the encoded amino acids, and/or can result in the generation of a premature stop codon, causing generation of a truncated polypeptide.
- a polymo ⁇ hism associated with stroke or a susceptibility to stroke can be a synonymous change in one or more nucleotides (i.e., a change that does not result in a change in the amino acid sequence).
- polymo ⁇ hism can, for example, alter splice sites, affect the stability or transport of mRNA, or otherwise affect the transcription or translation of the polypeptide.
- the polypeptide encoded by the reference nucleotide sequence is the "reference" polypeptide with a particular reference amino acid sequence, and polypeptides encoded by variant alleles are referred to as "variant" polypeptides with variant amino acid sequences.
- Haplotypes are a combination of genetic markers, e.g., particular alleles at polymo ⁇ hic sites.
- the haplotypes described herein e.g., having markers such as those shown in Table 3, Table 4A and 4B, are found more frequently in individuals with stroke than in individuals without stroke. Therefore, these haplotypes have predictive value for detecting stroke or a susceptibility to stroke in an individual.
- the haplotypes described herein are a combination of various genetic markers, e.g., SNPs and microsatellites. Therefore, detecting haplotypes can be accomplished by methods known in the art for detecting sequences at polymo ⁇ hic sites, such as the methods described above.
- an individual who is at risk for stroke is an individual in whom an at-risk haplotype is identified.
- the at- risk haplotype is one that confers a significant risk of stroke.
- significance associated with a haplotype is measured by an odds ratio.
- the significance is measured by a percentage, hi one embodiment, a significant risk is measured as an odds ratio of at least about 1.2, including but not limited to: 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 and 1.9.
- an odds ratio of at least 1.2 is significant.
- an odds ratio of at least about 1.5 is significant.
- a significant increase in risk is at least about 1.7 is significant.
- a significant increase in risk is at least about 20%, including but not limited to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 98%. In a further embodiment, a significant increase in risk is at least about 50%. It is understood however, that identifying whether a risk is medically significant may also depend on a variety of factors, including the specific disease, the haplotype, and often, environmental factors.
- An at-risk haplotype in, or comprising portions of, the PDE4D gene is one where the haplotype is more frequently present in an individual at risk for stroke (affected), compared to the frequency of its presence in a healthy individual (control), and wherein the presence of the haplotype is indicative of stroke or susceptibility to stroke.
- a protective haplotype in or comprising portions of the PDE4D gene is one where the haplotype is more frequently present in an individual where the haplotype is protective against being affected by stroke compared to the frequency of its presence in an individual with stroke. The presence of the haplotype is indicative of a protection from stroke or protection from susceptibility to stroke as described above.
- the method comprises assessing in an individual the presence or frequency of SNPs and/or microsatellites in, comprising portions of, the PDE4Dgene, wherein an excess or higher frequency of the SNPs and/or microsatellites compared to a healthy control individual is indicative that the individual has stroke, or is susceptible to stroke.
- an at-risk haplotype can include microsatellite markers and/or SNPs such as those set forth in Table 2C, Table 4B and 4B.
- the presence of the haplotype is indicative of stroke, or a susceptibility to stroke, and therefore is indicative of an individual who falls within a target population for the treatment methods described herein.
- Haplotype analysis first involves defining a candidate susceptibility locus using LOD scores. The defined regions are then ultra-fine mapped with microsatellite markers with an average spacing between markers of less than lOOkb. All usable microsatellite markers that found in public databases and mapped within that region can be used. In addition, microsatellite markers identified within the deCODE genetics sequence assembly of the human genome can be used. The frequencies of haplotypes in the patient and the control groups using an expectation- maximization algorithm can be estimated (Dempster A. et al, 1977. J. R. Stat. Soc. B, 39:1-389). An implementation of this algorithm that can handle missing genotypes and uncertainty with the phase can be used.
- At-risk-haplotypes in the 1-lod drop or protective haplotypes for example, association of all possible combinations of genotyped markers is studied, provided those markers span a practical region.
- the combined patient and control groups can be randomly divided into two sets, equal in size to the original group of patients and controls.
- the haplotype analysis is then repeated and the most significant p-value registered is determined. This randomization scheme can be repeated, for example, over 100 times to construct an empirical distribution of p- values.
- the at-risk haplotype is characterized by the presence of the polymo ⁇ hism(s) represented by one or a combination of single nucleotide polymo ⁇ hisms at nucleic acid positions 1425923, 1415979, 1414804, 1371388, 1307403 and 1257206, relative to SEQ ID NO: 1.
- a diagnostic method for susceptibility to stroke can comprise determining the presence of at-risk haplotype represented by one or a combination of single nucleotide polymo ⁇ hisms and microsatellite markers at nucleic acid positions 263539, 252772, 189780, 175259, 171240, 136550 and 120628, relative to SEQ ID NO: 1.
- the at-risk haplotype is characterized by the following SNPs: SNP5PDM361194, SNP5PDM368135, SNP5PDM370640, SNP5PDM379372, and SNP5PDM408531.
- the protective haplotype comprises the A allele of SNP45 at position 142780 relative to SEQ ID NO: 1. This haplotype is particularly useful for assessing susceptibility to the two major subtypes of ischemic stroke, carotid and cardiogenic stroke.
- an at-risk haplotype, particularly for carotid and cardiogenic stroke is characterized by use of microsatellite marker AC008818-1 to define the presence of an at-risk allele.
- a nucleic acid of the invention in another embodiment, can be used in "antisense" therapy, in which a nucleic acid (e.g., an oligonucleotide) which specifically hybridizes to the mRNA and/or genomic DNA of a nucleic acid is administered or generated in situ.
- a nucleic acid e.g., an oligonucleotide
- the antisense nucleic acid that specifically hybridizes to the mRNA and/or DNA inhibits expression of the polypeptide encoded by that mRNA and or DNA, e.g., by inhibiting translation and/or transcription. Binding of the antisense nucleic acid can be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interaction in the major groove of the double helix.
- An antisense construct can be delivered, for example, as an expression plasmid as described above. When the plasmid is transcribed in the cell, it produces RNA that is complementary to a portion of the mRNA and/or DNA that encodes a PDE4D polypeptide.
- the antisense construct can be an oligonucleotide probe that is generated ex vivo and introduced into cells; it then inhibits expression by hybridizing with the mRNA and/or genomic DNA of the polypeptide.
- the oligonucleotide probes are modified oligonucleotides that are resistant to endogenous nucleases, e.g., exonucleases and/or endonucleases, thereby rendering them stable in vivo.
- Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Patent Nos. 5,176,996, 5,264,564 and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy are also described, for example, by Van der Krol et al.
- oligodeoxyribonucleotides derived from the translation mitiation site are preferred.
- oligonucleotides are designed that are complementary to mRNA encoding the polypeptide.
- the antisense oligonucleotides bind to mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required.
- a sequence "complementary" to a portion of an RNA, as referred to herein, indicates that a sequence has sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
- the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid, as described in detail above. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures.
- the oligonucleotides used in antisense therapy can be DNA, RNA, or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
- the oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
- the oligonucleotides can include other appended groups such as peptides (e.g. for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, Proc. Natl. Acad. Sci. USA 86:6553-6556 (1989); emai re et al, Proc. Natl. Acad. Sci. USA 84:648-652 (1987); PCT International Publication No.
- oligonucleotide may be conjugated to another molecule (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent).
- another molecule e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent.
- the antisense molecules are delivered to cells that express a PDE4D polypeptide in vivo.
- a number of methods can be used for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systematically.
- antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systematically.
- antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be
- a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong promoter (e.g., pol III or pol II).
- a strong promoter e.g., pol III or pol II.
- the use of such a construct to transfect target cells in the patient results in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous transcripts and thereby prevent translation of the mRNA.
- a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
- Such vectors can be constructed by recombinant DNA technology methods standard in the art and described above.
- a plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct that can be introduced directly into the tissue site.
- viral vectors can be used which selectively infect the desired tissue, in which case administration may be accomplished by another route (e.g., systemically).
- RNA interference small double-stranded interfering RNA
- RNAi is a post-transcription process, in which double-stranded RNA is introduced, and sequence-specific gene silencing results, though catalytic degradation of the targeted mRNA. See, e.g., Elbashir, S.M. et al, Nature 411:494-49% (2001); Lee, N.S., Nature Biotech. iP:500-505 (2002); Lee, S-K. et al, Nature Medicine 8(7):681-6S6 (2002); the entire teachings of these references are inco ⁇ orated herein by reference.
- Endogenous expression of a gene product can also be reduced by inactivating or "knocking out” the gene or its promoter using targeted homologous recombination (e.g., see Smithies et al, Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al, Cell 5:313-321 (1989)).
- targeted homologous recombination e.g., see Smithies et al, Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al, Cell 5:313-321 (1989)
- an altered, non-functional gene flanked by DNA homologous to the endogenous gene (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the gene in vivo.
- the recombinant DNA constructs can be directly administered or targeted to the required site in vivo using appropriate vectors, as described above.
- expression of non-altered genes can be increased using a similar method: targeted homologous recombination can be used to insert a DNA construct comprising a non-altered functional gene, or the complement thereof, or a portion thereof, in place of an gene in the cell, as described above.
- targeted homologous recombination can be used to insert a DNA construct comprising a nucleic acid that encodes a polypeptide variant that differs from that present in the cell.
- endogenous expression of a gene product can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region (i.e., the promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells in the body.
- deoxyribonucleotide sequences complementary to the regulatory region i.e., the promoter and/or enhancers
- triple helical structures that prevent transcription of the gene in target cells in the body.
- the antisense constructs described herein by antagonizing the normal biological activity of the gene product, can be used in the manipulation of tissue, e.g., tissue differentiation, both in vivo and for ex vivo tissue cultures.
- tissue e.g., tissue differentiation
- the anti- sense techniques e.g., microinjection of antisense molecules, or transfection with plasmids whose transcripts are anti-sense with regard to a nucleic acid RNA or nucleic acid sequence
- Such techniques can be utilized in cell culture, but can also be used in the creation of transgenic animals.
- the therapeutic agents as described herein can be delivered in a composition, as described above, or alone. They can be administered systemically, or can be targeted to a particular tissue.
- the therapeutic agents can be produced by a variety of means, including chemical synthesis; recombinant production; in vivo production (e.g., a transgenic animal, such as U.S. Patent No.
- the current invention also pertains to methods of monitoring the effectiveness of treatment on the regulation of expression (e.g., relative or absolute expression) of one or more PDE4D isoforms at the RNA or protein level or its enzymatic activity.
- PDE4D message or protein or enzymatic activity can be measured in a sample of peripheral blood or cells derived therefrom.
- An assessment of the levels of expression or activity can be made before and during treatment with PDE4D therapeutic agents.
- an individual who is a member of the target population can be assessed for response to treatment with a PDE4D inhibitor, by examining cAMP levels or PDE4D enzymatic activity or absolute and/or relative levels of PDE4D protein or mRNA isoforais in peripheral blood in general or specific cell sub fractions or combination of cell subtractions.
- variation such as haplotypes or mutations within or near (within 100 to 200kb) of the PDE4D gene may be used to identify individuals who are at higher risk for stroke or TIA to increase the power and efficiency of clinical trials for pharmaceutical agents to prevent or treat first or subsequent stroke.
- haplotypes and other variations may be used to exclude or fractionate patients in a clinical trial who are likely to have non-cAMP or non-PDE4D pathway involvement in their stroke risk in order to enrich patients who have other pathways involved and boost the power and sensitivity of the clinical trial.
- Such variation may be used as a pharmacogenomic test to guide selection of pharmaceutical agents for individuals.
- NUCLEIC ACIDS OF THE INVENTION Nucleic Acids, Portions and Variants All nucleotide positions are relative to SEQ ID NO: 1.
- the nucleic acids, polypeptides and antibodies described herein can be used in methods of diagnosis of susceptibility to stroke, as well as in kits useful for diagnosis of a susceptibility to stroke.
- the invention pertains to isolated nucleic acid molecules comprising a human PDE4D nucleic acid.
- PDE4D nucleic acid refers to an isolated nucleic acid molecule encoding PDE4D polypeptide.
- the PDE4D nucleic acid molecules of the present invention can be RNA, for example, mRNA, or DNA, such as cDNA and genomic DNA.
- DNA molecules can be double-stranded or single-stranded; single stranded RNA or DNA can be either the coding, or sense strand or the non-coding, or antisense strand.
- the nucleic acid molecule can include all or a portion of the coding sequence of the gene or nucleic acid and can further comprise additional non-coding sequences such as introns and non-coding 3' and 5' sequences (including regulatory sequences, for example, as well as promoters, transcription enhancement elements, splice donor/acceptor sites, etc.).
- a PDE4D nucleic acid can comprise the nucleic acid of SEQ ID NO: 1 which may optionally comprise at least one polymo ⁇ hism as shown in Tables 11 and 12, the complement thereof, or to a portion or fragment of such an isolated nucleic acid molecule (e.g. , cDNA or the nucleic acid) that encodes PDE4D polypeptide.
- SEQ ID NO: 1 which may optionally comprise at least one polymo ⁇ hism as shown in Tables 11 and 12, the complement thereof, or to a portion or fragment of such an isolated nucleic acid molecule (e.g. , cDNA or the nucleic acid) that encodes PDE4D polypeptide.
- nucleic acid molecules of the invention can be fused to a marker sequence, for example, a sequence that encodes a polypeptide to assist in isolation or purification of the polypeptide.
- a marker sequence for example, a sequence that encodes a polypeptide to assist in isolation or purification of the polypeptide.
- sequences include, but are not limited to, those that encode a glutathione-S-transferase (GST) fusion protein and those that encode a hemagglutinin A (HA) polypeptide marker from influenza.
- GST glutathione-S-transferase
- HA hemagglutinin A
- an "isolated" nucleic acid molecule is one that is separated from nucleic acids that normally flank the gene or nucleotide sequence (as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in an RNA library).
- an isolated nucleic acid of the invention may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
- the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix.
- an isolated nucleic acid molecule comprises at least about 50, 80 or 90% (on a molar basis) of all macromolecular species present.
- genomic DNA the term “isolated” also can refer to nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated.
- the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotides which flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid molecule is derived.
- nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.
- recombinant DNA contained in a vector is included in the definition of "isolated” as used herein.
- isolated nucleic acid molecules include recombinant DNA molecules in heterologous host cells, as well as partially or substantially purified DNA molecules in solution.
- isolated nucleic acid molecules also encompass in vivo and in vitro RNA transcripts of the DNA molecules of the present invention.
- An isolated nucleic acid molecule or nucleotide sequence can include a nucleic acid molecule or nucleotide sequence that is synthesized chemically or by recombinant means.
- isolated DNA contained in a vector is included in the definition of "isolated” as used herein.
- isolated nucleotide sequences include recombinant DNA molecules in heterologous organisms, as well as partially or substantially purified DNA molecules in solution.
- RNA transcripts of the DNA molecules of the present invention are also encompassed by “isolated" nucleotide sequences.
- Such isolated nucleotide sequences are useful in the manufacture of the encoded polypeptide, as probes for isolating homologous sequences (e.g., from other mammalian species), for gene mapping (e.g., by in situ hybridization with chromosomes), or for detecting expression of the gene in tissue (e.g., human tissue), such as by Northern blot analysis.
- homologous sequences e.g., from other mammalian species
- gene mapping e.g., by in situ hybridization with chromosomes
- tissue e.g., human tissue
- the present invention also pertains to variant nucleic acid molecules which are not necessarily found in nature but which encode a PDE4D polypeptide (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14), or another splicing variant of PDE4D polypeptide or polymo ⁇ hic variant thereof.
- a PDE4D polypeptide e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14
- DNA molecules which comprise a sequence that is different from the naturally-occurring nucleotide sequence but which, due to the degeneracy of the genetic code, encode a PDE4D polypeptide of the present invention are also the subject of this invention.
- the invention also encompasses nucleotide sequences encoding portions (fragments), or encoding variant polypeptides such as analogues or derivatives of the PDE4D polypeptide.
- variants can be naturally-occurring, such as in the case of allelic variation or single nucleotide polymo ⁇ hisms, or non-naturally-occurring, such as those induced by various mutagens and mutagenic processes.
- Intended variations include, but are not limited to, addition, deletion and substitution of one or more nucleotides that can result in conservative or non-conservative amino acid changes, including additions and deletions.
- nucleotide (and/or resultant amino acid) changes are silent or conserved; that is, they do not alter the characteristics or activity of the PDE4D polypeptide.
- nucleotide sequences are fragments that comprise one or more polymo ⁇ hic microsatellite markers.
- nucleotide sequences are fragments that comprise one or more single nucleotide polymo ⁇ hisms in the PDE4D gene.
- nucleic acid molecules of the invention can include, for example, labeling, methylation, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates), charged linkages (e.g., phosphorothioates, phosphorodithioates), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids).
- synthetic molecules that mimic nucleic acid molecules in the ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
- the invention also pertains to nucleic acid molecules that hybridize under high stringency hybridization conditions, such as for selective hybridization, to a nucleotide sequence described herein (e.g., nucleic acid molecules which specifically hybridize to a nucleotide sequence encoding polypeptides described herein, and, optionally, have an activity of the polypeptide).
- the invention includes variants described herein which hybridize under high stringency hybridization conditions (e.g., for selective hybridization) to a nucleotide sequence comprising a nucleotide sequence selected from SEQ ID NO: 1 which may optionally comprise at least one polymo ⁇ hism as shown in Tables 11 and 12 or the complement thereof.
- the invention includes variants described herein which hybridize under high stringency hybridization conditions (e.g., for selective hybridization) to a nucleotide sequence encoding an amino acid sequence selected from SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14 or polymo ⁇ hic variant thereof.
- the protein product of the variant that hybridizes under high stringency conditions has an activity of PDE4D.
- Such nucleic acid molecules can be detected and/or isolated by specific hybridization (e.g., under high stringency conditions).
- Specific hybridization refers to the ability of a first nucleic acid to hybridize to a second nucleic acid in a manner such that the first nucleic acid does not hybridize to any nucleic acid other than to the second nucleic acid (e.g., when the first nucleic acid has a higher similarity to the second nucleic acid than to any other nucleic acid in a sample wherein the hybridization is to be performed).
- “Stringency conditions" for hybridization is a te ⁇ n of art which refers to the incubation and wash conditions, e.g., conditions of temperature and buffer concentration, which permit hybridization of a particular nucleic acid to a second nucleic acid; the first nucleic acid may be perfectly (i.e., 100%) complementary to the second, or the first and second may share some degree of complementarity which is less than perfect (e.g., 70%, 75%, 85%, 95%). For example, certain high stringency conditions can be used which distinguish perfectly complementary nucleic acids from those of less complementarity.
- the exact conditions which determine the stringency of hybridization depend not only on ionic strength (e.g., 0.2XSSC, 0.1XSSC), temperature (e.g., room temperature, 42°C, 68°C) and the concentration of destabilizing agents such as formamide or denaturing agents such as SDS, but also on factors such as the length of the nucleic acid sequence, base composition, percent mismatch between hybridizing sequences and the frequency of occmrence of subsets of that sequence within other non-identical sequences. Thus, equivalent conditions can be determined by varying one or more of these parameters while maintaining a similar degree of identity or similarity between the two nucleic acid molecules.
- conditions are used such that sequences at least about 60%, at least about 70%, at least about 80%), at least about 90% or at least about 95% or more identical to each other remain hybridized to one another.
- hybridization conditions from a level of stringency at which no hybridization occurs to a level at which hybridization is first observed, conditions which will allow a given sequence to hybridize (e.g., selectively) with the most similar sequences in the sample can be determined.
- washing conditions are described in Krause, M.H. and S.A. Aaronson, Methods in Enzymology, 200:546-556 (1991). Also, in, Ausubel, et al, "Current Protocols in Molecular Biology” , John Wiley & Sons, (1998), which describes the determination of washing conditions for moderate or low stringency conditions. Washing is the step in which conditions are usually set so as to determine a minimum level of complementarity of the hybrids. Generally, starting from the lowest temperature at which only homologous hybridization occurs, each °C by which the final wash temperature is reduced (holding SSC concenfration constant) allows an increase by 1% in the maximum extent of mismatching among the sequences that hybridize. Generally, doubling the concentration of SSC results in an increase in T m of ⁇ 17°C. Using these guidelines, the washing temperature can be determined empirically for high, moderate or low stringency, depending on the level of mismatch sought.
- a low stringency wash can comprise washing in a solution containing 0.2XSSC/0.1% SDS for 10 min at room temperature;
- a moderate stringency wash can comprise washing in a prewarmed solution (42°C) solution containing 0.2XSSC/0.1% SDS for 15 min at 42°C;
- a high stringency wash can comprise washing in prewarmed (68°C) solution containing O.lXSSC/O.P/oSDS for 15 min at 68°C.
- washes can be performed repeatedly or sequentially to obtain a desired result as known in the art.
- Equivalent conditions can be determined by varying one or more of the parameters given as an example, as known in the art, while maintaining a similar degree of identity or similarity between the target nucleic acid molecule and the primer or probe used.
- the percent homology or identity of two nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison pvuposes (e.g., gaps can be introduced in the sequence of a first sequence for optimal alignment).
- nucleic acid or amino acid “homology” is equivalent to nucleic acid or amino acid "identity"
- the length of a sequence aligned for comparison pu ⁇ oses is at least 30%, for example, at least 40%, in certain embodiments at least 60%, and in other embodiments at least 70%, 80%, 90% or 95% of the length of the reference sequence.
- the actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm.
- One, non-limiting example of such a mathematical algorithm is described in Karlin et al, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993).
- NBLAST and XBLAST programs version 2.0 as described in Altschul et al, Nucleic Acids Res. 25:389-3402 (1997).
- NBLAST the default parameters of the respective programs
- Such an algorithm is inco ⁇ orated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
- a PAM120 weight residue table When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5; and FASTA described in Pearson and Lipman (1988) PNAS, ⁇ 5:2444-8.
- the percent identity between two amino acid sequences can be accomplished using the GAP program in the GCG software package (Accelrys, Cambridge, UK) using either a Blossom 63 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4.
- the percent identity between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package, using a gap weight of 50 and a length weight of 3.
- the present invention also provides isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleotide sequence comprising a nucleotide sequence selected from SEQ ID NO: 1 which may optionally comprise at least one polymo ⁇ hism as shown in Tables 11 and 12 and the complement thereof, and also provides isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleotide sequence encoding an amino acid sequence selected from SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14, or polymo ⁇ hic variant thereof.
- the nucleic acid fragments of the invention are at least about 15, preferably at least about 18, 20, 23 or 25 nucleotides, and can be 30, 40, 50, 100, 200 or more nucleotides in length. Longer fragments, for example, 30 or more nucleotides in length, which encode antigenic polypeptides described herein are particularly useful, such as for the generation of antibodies as described below.
- probes and Primers are oligonucleotides that hybridize in a base-specific manner to a complementary strand of nucleic acid molecules.
- base specific manner is meant that the two sequences must have a degree of nucleotide complementarity sufficient for the primer or probe to hybridize. Accordingly, the primer or probe sequence is not required to be perfectly complementary to the sequence of the template. Non- complementary bases or modified bases can be interspersed into the primer or probe, provided that base substitutions do not inhibit hybridization.
- the nucleic acid template may also include "non-specific priming sequences" or “nonspecific sequences” to which the primer or probe has varying degrees of complementarities.
- probes and primers include polypeptide nucleic acids, as described in Nielsen et al, Science, 254, 1497-1500 (1991).
- a probe or primer comprises a region of nucleic acid that hybridizes to at least about 15, for example about 20-25, and in certain embodiments about 40, 50 or 75, consecutive nucleotides of a nucleic acid of the invention, such as a nucleic acid comprising a contiguous nucleic acid sequence of SEQ ID NO: 1 or the complement of SEQ ID NO: 1, or a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14, or polymo ⁇ hic variant thereof.
- a probe or primer comprises 100 or fewer nucleotides, in certain embodiments, from 6 to 50 nucleotides, for example, from 12 to 30 nucleotides.
- the probe or primer is at least 70% identical to the contiguous nucleic acid sequence or to the complement of the contiguous nucleotide sequence, for example, at least 80% identical, in certain embodiments at least 90% identical, and in other embodiments at least 95% identical, or even capable of selectively hybridizing to the contiguous nucleic acid sequence or to the complement of the contiguous nucleotide sequence.
- the probe or primer further comprises a label, e.g., radioisotope, fluorescent compound, enzyme, or enzyme co-factor.
- nucleic acid molecules of the invention such as those described above can be identified and isolated using standard molecular biology techniques and the sequence information provided herein.
- nucleic acid molecules can be amplified and isolated by the polymerase chain reaction using synthetic oligonucleotide primers designed based on one or more of the sequences provided in SEQ ID NO: 1 which may optionally comprise at least one polymo ⁇ hism shown in Tables 11 and 12, and/or the complement thereof, or designed based on nucleotides based on sequences encoding one or more of the amino acid sequences provided herein. See generally PCR Technology: Principles and Applications for DNA
- nucleic acid molecules can be amplified using cDNA, mRNA or genomic DNA as a template, cloned into an appropriate vector and characterized by DNA sequence analysis.
- LCR ligase chain reaction
- NASBA nucleic acid based sequence amplification
- the latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.
- the amplified DNA can be labeled (e.g. , with radiolabel or other reporter molecule) and used as a probe for screening a cDNA library derived from human cells, mRNA in zap express, ZIPLOX or other suitable vector.
- Conesponding clones can be isolated, DNA can obtained following in vivo excision, and the cloned insert can be sequenced in either or both orientations by art recognized methods to identify the conect reading frame encoding a polypeptide of the appropriate molecular weight.
- the direct analysis of the nucleotide sequence of nucleic acid molecules of the present invention can be accomplished using well-known methods that are commercially available. See, for example, Sambrook et al, Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind et al, Recombinant DNA Laboratory Manual, (Acad. Press, 1988)). Using these or similar methods, the polypeptide and the DNA encoding the polypeptide can be isolated, sequenced and further characterized.
- Antisense nucleic acid molecules of the invention can be designed using the nucleotide sequences of SEQ ID NO: 1 and/or the complement of SEQ ID NO: 1, and or a portion of SEQ ID NO: 1 or the complement of SEQ ID NO: 1 and/or a sequence encoding the amino acid sequences or SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 and/or 14, or encoding a portion of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 and/or 14, (wherein any one of these may optionally comprise at least one polymo ⁇ hism as shown in Tables 11 and 12) and constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
- an antisense nucleic acid molecule e.g., an antisense oligonucleotide
- an antisense nucleic acid molecule can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
- the antisense nucleic acid molecule can be produced biologically using an expression vector into which a nucleic acid molecule has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid molecule will be of an antisense orientation to a target nucleic acid of interest).
- the isolated nucleic acid sequences of the invention can be used as molecular weight markers on Southern gels, and as chromosome markers that are labeled to map related gene positions.
- the nucleic acid sequences can also be used to compare with endogenous DNA sequences in patients to identify genetic disorders (e.g., a predisposition for or susceptibility to stroke), and as probes, such as to hybridize and discover related DNA sequences or to subtract out known sequences from a sample.
- the nucleic acid sequences can further be used to derive primers for genetic finge ⁇ rinting, to raise anti-polypeptide antibodies using DNA immunization techniques, and as an antigen to raise anti-DNA antibodies or elicit immune responses.
- nucleotide sequences identified herein can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. Additionally, the nucleotide sequences of the invention can be used to identify and express recombinant polypeptides for analysis, characterization or therapeutic use, or as markers for tissues in which the conesponding polypeptide is expressed, either constitutively, during tissue differentiation, or in diseased states.
- nucleic acid sequences can additionally be used as reagents in the screening and/or diagnostic assays described herein, and can also be included as components of kits (e.g., reagent kits) for use in the screening and/or diagnostic assays described herein.
- kits e.g., reagent kits
- nucleic acid constructs containing a nucleic acid molecule selected from the group consisting of SEQ ID NO: 1 which may optionally comprise at least one polymo ⁇ hism shown in Tables 11 and 12 and the complement thereof (or a portion thereof).
- the constructs comprise a vector (e.g., an expression vector) into which a sequence of the invention has been inserted in a sense or antisense orientation.
- vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
- plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
- viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
- Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
- vectors e.g., non-episomal mammalian vectors
- Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
- certain vectors, expression vectors are capable of directing the expression of genes to which they are operably linked.
- expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
- the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) that serve equivalent functions.
- Prefened recombinant expression vectors of the invention comprise a nucleic acid molecule of the invention in a form suitable for expression of the nucleic acid molecule in a host cell.
- the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed.
- "operably or operatively linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
- regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transforaied and the level of expression of polypeptide desired.
- the expression vectors of the invention can be introduced into host cells to thereby produce polypeptides, including fusion polypeptides, encoded by nucleic acid molecules as described herein.
- the recombinant expression vectors of the invention can be designed for expression of a polypeptide of the invention in prokaryotic or eukaryotic cells, e.g., bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, supra.
- the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
- host cell and "recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
- a host cell can be any prokaryotic or eukaryotic cell.
- a nucleic acid molecule of the invention can be expressed in bacterial cells (e.g., E. coli), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
- bacterial cells e.g., E. coli
- insect cells e.g., insect cells
- yeast or mammalian cells such as Chinese hamster ovary cells (CHO) or COS cells.
- Other suitable host cells are known to those skilled in the art.
- Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
- transformation and “transfection” are intended to refer to a variety of art- recognized techniques for introducing a foreign nucleic acid molecule (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, D ⁇ A ⁇ -dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al, (supra), and other laboratory manuals.
- a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
- selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.
- Nucleic acid molecules encoding a selectable marker can be introduced into a host cell on the same vector as the nucleic acid molecule of the invention or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid molecule can be identified by drug selection (e.g., cells that have inco ⁇ orated the selectable marker gene will survive, while the other cells die).
- a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a polypeptide of the invention.
- the invention further provides methods for producing a polypeptide using the host cells of the invention, hi one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide is produced. In another embodiment, the method further comprises isolating the polypeptide from the medium or the host cell.
- a host cell of the invention can also be used to produce nonhuman transgenic ammals.
- a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a nucleic acid molecule of the invention has been introduced (e.g., an exogenous PDE4D gene, or an exogenous nucleic acid encoding PDE4D polypeptide).
- a nucleic acid molecule of the invention e.g., an exogenous PDE4D gene, or an exogenous nucleic acid encoding PDE4D polypeptide.
- Such host cells can then be used to create non-human transgenic animals in which exogenous nucleotide sequences have been introduced into the genome or homologous recombinant animals in which endogenous nucleotide sequences have been altered.
- transgenic animal is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal include a transgene.
- rodent such as a rat or mouse
- transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens and amphibians.
- a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
- an "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
- Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al. (1997) Nature, 555:810-813 and PCT Publication Nos. WO 97/07668 and WO 97/07669.
- the present invention also pertains to isolated polypeptides encoded by PDE4D ("PDE4D polypeptides”) and fragments and variants thereof, as well as polypeptides encoded by nucleotide sequences described herein (e.g., other splicing variants).
- PDE4D polypeptides encoded by PDE4D
- polypeptides encoded by nucleotide sequences described herein e.g., other splicing variants.
- polypeptide refers to a polymer of amino acids, and not to a specific length; thus, peptides, ohgopeptides and proteins are included within the definition of a polypeptide.
- a polypeptide is said to be “isolated” or “purified” when it is substantially free of cellular material when it is isolated from recombinant and non-recombinant cells, or free of chemical precursors or other chemicals when it is chemically synthesized.
- a polypeptide can be joined to another polypeptide with which it is not normally associated in a cell (e.g., in a "fusion protein") and still be “isolated” or “purified.”
- polypeptides of the invention can be purified to homogeneity. It is understood, however, that preparations in which the polypeptide is not purified to homogeneity are useful. The critical feature is that the preparation allows for the desired function of the polypeptide, even in the presence of considerable amounts of other components. Thus, the invention encompasses various degrees of purity.
- the language "substantially free of cellular material” includes preparations of the polypeptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10%) other proteins, or less than about 5%> other proteins.
- a polypeptide When a polypeptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20%, less than about 10%, or less than about 5% of the volume of the polypeptide preparation.
- the language "substantially free of chemical precursors or other chemicals” includes preparations of the polypeptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the polypeptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5%> chemical precursors or other chemicals.
- a polypeptide of the invention comprises an amino acid sequence encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 which may optionally comprise at least one polymo ⁇ hism shown in Tables 11 and 12 and complements and portions thereof, e.g., SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14, or a portion or polymo ⁇ hic variant thereof.
- the polypeptides of the invention also encompass fragment and sequence variants. Variants include a substantially homologous polypeptide encoded by the same genetic locus in an organism, i.e., an allelic variant, as well as other splicing variants.
- Variants also encompass polypeptides derived from other genetic loci in an organism, but having substantial homology to a polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 which may optionally comprise at least one polymo ⁇ hism shown in Tables 11 and 12 and complements and portions thereof, or having substantial homology to a polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of nucleotide sequences encoding SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14, or polymo ⁇ hic variants thereof.
- Variants also include polypeptides substantially homologous or identical to these polypeptides but derived from another organism, i.e., an ortholog. Variants also include polypeptides that are substantially homologous or identical to these polypeptides that are produced by chemical synthesis. Variants also include polypeptides that are substantially homologous or identical to these polypeptides that are produced by recombinant methods.
- two polypeptides are substantially homologous or identical when the amino acid sequences are at least about 45-55%), in certain embodiments at least about 70-75%, and in other embodiments at least about 80-85%, and in others greater than about 90%> or more homologous or identical.
- a substantially homologous amino acid sequence will be encoded by a nucleic acid molecule hybridizing to SEQ ID NO: 1 which may optionally comprise at least one polymo ⁇ hism shown in Tables 11 and 12, or portion thereof, under stringent conditions as more particularly described above, or will be encoded by a nucleic acid molecule hybridizing to a nucleic acid sequence encoding SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14, portion thereof or polymo ⁇ hic variant thereof, under stringent conditions as more particularly described thereof.
- the invention also encompasses polypeptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by a polypeptide encoded by a nucleic acid molecule of the invention. Similarity is determined by conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Conservative substitutions are likely to be phenotypically silent.
- conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe and Tyr.
- Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al, Science 247:1306-1310 (1990).
- variant polypeptide can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these. Further, variant polypeptides can be fully functional or can lack function in one or more activities. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree. Nonfunctional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.
- Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al. , Science, 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity in vitro, or in vitro proliferative activity. Sites that are critical for polypeptide activity can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffmity labeling (Smith et al, J. Mol. Biol, 224:899-904 (1992); de Vos et al, Science, 255:306-312 (1992)).
- the invention also includes polypeptide fragments of the polypeptides of the invention. Fragments can be derived from a polypeptide encoded by a nucleic acid molecule comprising SEQ ID NO: 1 which may optionally comprise at least one polymo ⁇ hism shown in Tables 11 and 12 or a portion thereof and the complements • thereof (e.g., SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14, or other splicing variants). However, the invention also encompasses fragments of the variants of the polypeptides described herein. As used herein, a fragment comprises at least 6 contiguous amino acids. Useful fragments include those that retain one or more of the biological activities of the polypeptide as well as fragments that can be used as an immunogen to generate polypeptide-specific antibodies. Biologically active fragments (peptides which are, for example, 6, 9, 12, 15,
- 16, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length can comprise a domain, segment, or motif that has been identified by analysis of the polypeptide sequence using well-known methods, e.g., signal peptides, extracellular domains, one or more transmembrane segments or loops, ligand binding regions, zinc finger domains, DNA binding domains, acylation sites, glycosylation sites, or phosphorylation sites.
- Fragments can be discrete (not fused to other amino acids or polypeptides) or can be within a larger polypeptide. Further, several fragments can be comprised within a single larger polypeptide.
- a fragment designed for expression in a host can have heterologous pre- and pro-polypeptide regions fused to the amino terminus of the polypeptide fragment and an additional region fused to the carboxyl terminus of the fragment.
- the invention thus provides chimeric or fusion polypeptides. These comprise a polypeptide of the invention operatively linked to a heterologous protein or polypeptide having an amino acid sequence not substantially homologous to the polypeptide. "Operatively linked" indicates that the polypeptide and the heterologous protein are fused in-frame.
- the heterologous protem can be fused to the N-terminus or C-terminus of the polypeptide.
- the fusion polypeptide does not affect function of the polypeptide per se.
- the fusion polypeptide can be a GST-fusion polypeptide in which the polypeptide sequences are fused to the C-terminus of the GST sequences.
- Other types of fusion polypeptides include, but are not limited to, enzymatic fusion polypeptides, for example ⁇ -galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions and Ig fusions.
- fusion polypeptides can facilitate the purification of recombinant polypeptide.
- expression and/or secretion of a polypeptide can be increased by using a heterologous signal sequence. Therefore, in another embodiment, the fusion polypeptide contains a heterologous signal sequence at its N-terminus.
- EP-A-O 464 533 discloses fusion proteins comprising various portions of immunoglobulin constant regions.
- the Fc is useful in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262).
- human proteins have been fused with Fc portions for the pu ⁇ ose of high-throughput screening assays to identify antagonists. Bennett et al, Journal of Molecular Recognition, 5:52-58 (1995) and Johanson et al, The Journal of Biological Chemistry, 270,16:9459-9471 (1995).
- this invention also encompasses soluble fusion polypeptides containing a polypeptide of the invention and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE).
- a chimeric or fusion polypeptide can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques.
- the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
- PCR amplification of nucleic acid fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive nucleic acid fragments which can subsequently be annealed and re-amplified to generate a chimeric nucleic acid sequence (see Ausubel et al, Current Protocols in Molecular Biology, 1992).
- fusion moiety e.g., a GST protem
- a nucleic acid molecule encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the polypeptide.
- the isolated polypeptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods, hi one embodiment, the polypeptide is produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the polypeptide is cloned into an expression vector, the expression vector introduced into a host cell and the polypeptide expressed in the host cell. The polypeptide can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques.
- polypeptides of the present invention can be used as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using art-recognized methods.
- the polypeptides of the present invention can be used to raise antibodies or to elicit an immune response.
- the polypeptides can also be used as a reagent, e.g., a labeled reagent, in assays to quantitatively determine levels of the polypeptide or a molecule to which it binds (e.g., a receptor or a ligand) in biological fluids.
- the polypeptides can also be used as markers for cells or tissues in which the conesponding polypeptide is preferentially expressed, either constitutively, during tissue differentiation, or in a diseased state.
- the polypeptides can be used to isolate a conesponding binding agent, e.g., receptor or ligand, such as, for example, in an interaction trap assay, and to screen for peptide or small molecule antagonists or agonists of the binding interaction.
- a conesponding binding agent e.g., receptor or ligand
- Polyclonal and/or monoclonal antibodies that specifically bind one form of the gene product but not to the other form of the gene product are also provided.
- Antibodies are also provided that bind a portion of either the variant or the reference gene product that contains the polymo ⁇ hic site or sites.
- the invention provides antibodies to the polypeptides and polypeptide fragments of the invention, e.g., having an amino acid sequence encoded by SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14, or a portion thereof, or having an amino acid sequence encoded by a nucleic acid molecule comprising all or a portion of SEQ ID NO: 1 which may optionally comprise at least one polymo ⁇ hism shown in Tables 11 and 12 (e.g., SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14, or another splicing variant or portion thereof).
- antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen.
- a molecule that specifically binds to a polypeptide of the invention is a molecule that binds to that polypeptide or a fragment thereof, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide.
- immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
- the invention provides polyclonal and monoclonal antibodies that bind to a polypeptide of the invention.
- a monoclonal antibody composition thus typically displays a single binding affinity for a particular polypeptide of the invention with which it immunoreacts.
- Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a desired immunogen, e.g., polypeptide of the invention or fragment thereof.
- a desired immunogen e.g., polypeptide of the invention or fragment thereof.
- the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide.
- ELISA enzyme linked immunosorbent assay
- the antibody molecules directed against the polypeptide can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protem A chromatography to obtain the IgG fraction.
- antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature, 256:495-497, the human B cell hybridoma technique (Kozbor et al (1983) Immunol Today, 4:72), the EBN-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or frioma techniques.
- standard techniques such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature, 256:495-497, the human B cell hybridoma technique (Kozbor et al (1983) Immunol Today, 4:72), the EBN-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or frioma techniques
- hybridomas The technology for producing hybridomas is well known (see generally Current Protocols in Immunology (1994) Coligan et ⁇ l (eds.) John Wiley & Sons, Inc., New York, NY). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds a polypeptide of the invention.
- lymphocytes typically splenocytes
- a monoclonal antibody to a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide to thereby isolate immunoglobulin library members that bind the polypeptide.
- Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SwfZAPTM Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S.
- recombinant antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
- Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.
- antibodies of the invention e.g., a monoclonal antibody
- a polypeptide-specific antibody can facilitate the purification of natural polypeptide from cells and of recombinantly produced polypeptide expressed in host cells.
- an antibody specific for a polypeptide of the invention can be used to detect the polypeptide (e.g., in a cellular lysate, cell supernatant, or tissue sample) in order to evaluate the abundance and pattern of expression of the polypeptide.
- Antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Coupling the antibody to a detectable substance can facilitate detection.
- detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
- suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
- suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
- suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
- an example of a luminescent material includes luminol;
- bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 1251, 1311, 35S or 3H.
- kits useful for diagnosis of stroke or a susceptibility to stroke or to a disease or condition associated with PDE4D comprises primers as described herein, wherein the primers contain one or more of the SNPs identified herein.
- definition of stroke risk associated with PDE4D/cAMP pathway is useful and novel to define subgroups of individuals who would be best treated by pharmaceutical agents acting on PDE4D and/ cAMP pathways (and vice versa).
- diagnosis of stroke or susceptibility to stroke is made by detecting a polymo ⁇ hism in a PDE4D nucleic acid as described herein.
- the polymo ⁇ hism can be an alteration in a PDE4D nucleic acid, such as the ⁇ insertion or deletion of a single nucleotide, or of more than one nucleotide, resulting in a frame shift alteration; the change of at least one nucleotide, resulting in a change in the encoded amino acid; the change of at least one nucleotide, resulting in the generation of a premature stop codon; the deletion of several nucleotides, resulting in a deletion of one or more amino acids encoded by the nucleotides; the insertion of one or several nucleotides, such as by unequal recombination or gene conversion, resulting in an interruption of the coding sequence of the gene or nucleic acid; duplication of all or a part of the gene or nucleic acid; transposition of all or a part of t the gene or nucleic acid; or reanangement of all or a part of the gene or nucleic acid.
- More than one such alteration may be present in a single gene or nucleic acid.
- Such sequence changes cause an alteration in the polypeptide encoded by a PDE4D nucleic acid.
- the alteration is a frame shift alteration
- the frame shift can result in a change in the encoded amino acids, and/or can result in the generation of a premature stop codon, causing generation of a truncated polypeptide.
- a polymo ⁇ hism associated with a disease or condition associated with a PDE4D nucleic acid or a susceptibility to a disease or condition associated with a PDE4D nucleic acid can be a synonymous alteration in one or more nucleotides (i.e., an alteration that does not result in a change in the polypeptide encoded by a PDE4D nucleic acid).
- Such a polymo ⁇ hism may alter splicing sites, affect the stability or transport of mRNA, or otherwise affect the transcription or translation of the nucleic acid.
- a PDE4D nucleic acid that has any of the alteration described above is refened to herein as an "altered nucleic acid.”
- hybridization methods such as Southern analysis, Northern analysis, or in situ hybridizations, can be used (see Current Protocols in Molecular Biology, Ausubel, F. et al, eds., John Wiley & Sons, including all supplements through 1999).
- a biological sample from a test subject (a "test sample") of genomic DNA, RNA, or cDNA, is obtained from an individual suspected of having, being susceptible to or predisposed for, or ca ⁇ ying a defect for, a susceptibility to a disease or condition associated with a PDE4D nucleic acid (the "test individual").
- the individual can be an adult, child, or fetus.
- the test sample can be from any source which contains genomic DNA, such as a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs.
- genomic DNA such as a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs.
- a test sample of DNA from fetal cells or tissue can be obtained by appropriate methods, such as by amniocentesis or chorionic villus sampling.
- the DNA, RNA, or cDNA sample is then examined to detennine whether a polymo ⁇ hism in a stroke nucleic acid is present, and/or to detennine which splicing variant(s) encoded by the PDE4D is present.
- nucleic acid probe can be a DNA probe or an RNA probe; the nucleic acid probe can contain at least one polymo ⁇ hism in a PDE4D nucleic acid or contains a nucleic acid encoding a particular splicing variant of a PDE4D nucleic acid.
- the probe can be any of the nucleic acid molecules described above (e.g., the nucleic acid, a fragment, a vector comprising the nucleic acid, a probe or primer, etc.).
- a hybridization sample is formed by contacting the test sample containing PDE4D, with at least one nucleic acid probe.
- a prefened probe for detecting mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA sequences described herein.
- the nucleic acid probe can be, for example, a full-length nucleic acid molecule, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to appropriate mRNA or genomic DNA.
- the nucleic acid probe can be all or a portion of SEQ ID NO: 1 which may optionally comprise at least one polymo ⁇ hism shown in Tables 11 and 12, or the complement thereof, or a portion thereof; or can be a nucleic acid encoding a portion of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14.
- Other suitable probes for use in the diagnostic assays of the invention are described above (see e.g., probes and primers discussed under the heading, "Nucleic Acids of the Invention").
- the hybridization sample is maintained under conditions that are sufficient to allow specific hybridization of the nucleic acid probe to PDE4D.
- Specific hybridization indicates exact hybridization (e.g., with no mismatches). Specific hybridization can be performed under high stringency conditions or moderate stringency conditions, for example, as described above. In a particularly prefened embodiment, the hybridization conditions for specific hybridization are high stringency.
- Specific hybridization if present, is then detected using standard methods. If specific hybridization occurs between the nucleic acid probe and PDE4D in the test sample, then PDE4D has the polymo ⁇ hism, or is the splicing variant, that is present in the nucleic acid probe. More than one nucleic acid probe can also be used concunently in this method. In one embodiment, specific hybridization of at least one of the nucleic acid probes is indicative of a polymo ⁇ hism in PDE4D, or of the presence of a particular splicing variant encoding PDE4D and is therefore diagnostic for a susceptibility to stroke. In Northern analysis (see Cunent Protocols in Molecular Biology, Ausubel,
- RNA from the individual is obtained from the individual by appropriate means. Specific hybridization of a nucleic acid probe, as described above, to RNA from the individual is indicative of a polymo ⁇ hism in PDE4D, or of the presence of a particular splicing variant encoded by PDE4D, and is therefore diagnostic for a susceptibility to stroke.
- nucleic acid probes For representative examples of use of nucleic acid probes, see, for example, U.S. Patents No. 5,288,611 and 4,851,330.
- a peptide nucleic acid (PNA) probe can be used instead of a nucleic acid probe in the hybridization methods described above.
- PNA is a DNA mimic having a peptide-like, inorganic backbone, such as N-(2-aminoethyl)glycine units, with an organic base (A, G, C, T or U) attached to the glycine nitrogen via a methylene carbonyl linker (see, for example, Nielsen, P.E. et al, Bioconjugate Chemistry, 1994, 5, American Chemical Society, p. 1 (1994).
- the PNA probe can be designed to specifically hybridize to a gene having a polymo ⁇ hism associated with a susceptibility to stroke. Hybridization of the PNA probe to PDE4D is diagnostic for a susceptibility to stroke.
- mutation analysis by restriction digestion can be used to detect a mutant gene, or genes containing a polymo ⁇ hism(s), if the mutation or polymo ⁇ hism in the gene results in the creation or elimination of a restriction site. If a restriction site is not naturally created, one can be created by PCR that depends on the polymo ⁇ hism and allows genotyping. A test sample containing genomic DNA is obtained from the individual. Nucleic acid amplification methods, including but not limited to Polymerase chain reaction (PCR), Transcription Mediated Amplifications (TMA), and Ligase Mediate Amplification (LMA), can be used to amplify PDE4D.
- PCR Polymerase chain reaction
- TMA Transcription Mediated Amplifications
- LMA Ligase Mediate Amplification
- the digestion pattern of the relevant DNA fragment indicates the presence or absence of the mutation or polymo ⁇ hism in PDE4D, and therefore indicates the presence or absence of this susceptibility to stroke.
- RFLP analysis is conducted as described (see Cunent Protocols in Molecular Biology, supra). Amplification techniques based upon detection of sequence of interest using reverse dot blot technology (linear anay or strips) can be used and are described, for example, in U.S. Patent No. 5,468,613. Sequence analysis can also be used to detect specific polymo ⁇ hisms in PDE4D. A test sample of DNA or RNA is obtained from the test individual. PCR or other appropriate methods can be used to amplify the gene, and/or its flanking sequences, if desired.
- the sequence of PDE4D, or a fragment of the gene, or cDNA, or fragment of the cDNA, or mRNA, or fragment of the mRNA is determined, using standard methods.
- the sequence of the gene, gene fragment, cDNA, cDNA fragment, mRNA, or mRNA fragment is compared with the known nucleic acid sequence of the gene, cDNA (e.g., SEQ ID NO: 1 which may optionally comprise at least one polymo ⁇ hism shown in Tables 11 and 12, or a nucleic acid sequence encoding SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14, or a fragment thereof) or mRNA, as appropriate.
- the presence of at least one of the polymo ⁇ hisms in PDE4D indicates that the individual has a susceptibility to stroke.
- Allele-specific oligonucleotides can also be used to detect the presence of a polymo ⁇ hism in PDE4D, through the use of dot-blot hybridization of amplified oligonucleotides with allele-specific oligonucleotide (ASO) probes (see, for example, Saiki, R. et al, (1986), Nature (London) 324:163-166).
- ASO allele-specific oligonucleotide
- an “allele-specific oligonucleotide” (also refened to herein as an “allele-specific oligonucleotide probe”) is an oligonucleotide of approximately 10-50 base pairs, preferably approximately 15-30 base pairs, that specifically hybridizes to PDE4D, and that contains a polymo ⁇ hism associated with a susceptibility to stroke.
- An allele-specific oligonucleotide probe that is specific for particular polymo ⁇ hisms in PDE4D can be prepared, using standard methods (see Cunent Protocols in Molecular Biology, supra). To identify polymo ⁇ hisms in the gene that are associated with a susceptibility to stroke, a test sample of DNA is obtained from the individual.
- PCR can be used to amplify all or a fragment of PDE4D, and its flanking sequences.
- the DNA containing the amplified PDE4D (or fragment of the gene) is dot-blotted, using standard methods (see Cunent Protocols in Molecular Biology, supra), and the blot is contacted with the oligonucleotide probe. The presence of specific hybridization of the probe to the amplified PDE4D is then detected. Specific hybridization of an allele-specific oligonucleotide probe to DNA from the individual is indicative of a polymo ⁇ hism in PDE4D, and is therefore indicative of a susceptibility to stroke.
- the invention further provides allele-specific oligonucleotides that hybridize to the reference or variant allele of a nucleic acid comprismg a single nucleotide polymo ⁇ hism or to the complement thereof. These oligonucleotides can be probes or primers.
- An allele-specific primer hybridizes to a site on target DNA overlapping a polymo ⁇ hism and only primes amplification of an allelic form to which the primer exhibits perfect complementarity. See Gibbs, Nucleic Acid Res. 17, 2427-2448 (1989). This primer is used in conjunction with a second primer that hybridizes at a distal site. Amplification proceeds from the two primers, resulting in a detectable product that indicates the particular allelic form is present.
- a control is usually performed with a second pair of primers, one of which shows a single base mismatch at the polymo ⁇ hic site and the other of which exhibits perfect complementarity to a distal site.
- the single-base mismatch prevents amplification and no detectable product is formed.
- the method works best when the mismatch is included in the 3'- most position of the oligonucleotide aligned with the polymo ⁇ hism because this position is most destabilizing to elongation from the primer (see, e.g., WO 93/22456).
- LNAs locked nucleic acids
- oxy-LNA O-methylene
- thio-LNA S-methylene
- amino-LNA amino methylene
- oxy-LNA nonamers have been shown to have melting temperatures of 64 °C and 74 ° C when in complex with complementary DNA or RNA, respectively, as opposed to 28 °C for both DNA and RNA for the conesponding DNA nonamer.
- Substantial increases in T m are also obtained when LNA monomers are used in combination with standard DNA or RNA monomers.
- the T m could be increased considerably.
- anays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual can be used to identify polymo ⁇ hisms in PDE4D.
- an oligonucleotide linear anay can be used.
- Oligonucleotide anays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. These oligonucleotide arrays, also described as "Genechips.TM.,” have been generally described in the art, for example, U.S. Patent No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and 92/10092.
- anays can generally be produced using mechanical synthesis methods or light directed synthesis methods that inco ⁇ orate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods. See Fodor et al, Science, 251:767-777 (1991), Pirrung et al, U.S. Patent No. 5,143,854 (see also PCT Application No. WO 90/15070) and Fodor et al, PCT Publication No. WO 92/10092 and U.S. Patent No. 5,424,186, the entire teachings of each of which are inco ⁇ orated by reference herein. Techniques for the synthesis of these anays using mechanical synthesis methods are described in, e.g., U.S. Patent No. 5,384,261, the entire teachings of which are inco ⁇ orated by reference herein. In another embodiment, linear anays or microanays can be utilized.
- a nucleic acid of interest is hybridized with the anay and scanned for polymo ⁇ hisms.
- Hybridization and scanning are generally carried out by methods described herein and also in, e.g., Published PCT Application Nos. WO 92/10092 and WO 95/11995, and U.S. Patent No. 5,424,186, the entire teachings of which are inco ⁇ orated by reference herein.
- a target nucleic acid sequence that includes one or more previously identified polymo ⁇ hic markers is amplified by well-known amplification techniques, e.g., PCR.
- Asymmetric PCR techniques may also be used.
- Amplified target generally inco ⁇ orating a label, is then hybridized with the anay under appropriate conditions.
- the array is scanned to determine the position on the anay to which the target sequence hybridizes.
- the hybridization data obtained from the scan is typically in the form of fluorescence intensities as a function of location on the anay.
- anays can include multiple detection blocks, and thus be capable of analyzing multiple, specific polymo ⁇ hisms.
- detection blocks may be grouped within a single anay or in multiple, separate anays so that varying, optimal conditions may be used during the hybridization of the target to the anay. For example, it may often be desirable to provide for the detection of those polymo ⁇ hisms that fall within G-C rich stretches of a genomic sequence, separately from those falling in A-T rich segments. This allows for the separate optimization of hybridization conditions for each situation.
- nucleic acid analysis can be used to detect polymo ⁇ hisms in PDE4D or splicing variants encoding by PDE4D.
- Representative methods include direct manual sequencing (Church and Gilbert, (1988), Proc. Natl. Acad. Sci. USA 81:1991-1995; Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. 74:5463-5467; Beavis et al, U.S. Patent No.
- diagnosis of a disease or condition associated with PDE4D e.g., stroke
- a susceptibility to a disease or condition associated with PDE4D e.g., sfroke
- This technique utilizing TaqMan ® or Lightcycler ® can be used to allow the identification of polymo ⁇ hisms and whether a patient is homozygous or heterozygous.
- the technique can assess the presence of an alteration in the expression or composition of the polypeptide encoded by a PDE4D nucleic acid or splicing variants encoded by a PDE4D nucleic acid. Further, the expression of the variants can be quantified as physically or functionally different.
- diagnosis of a susceptibility to stroke can also be made by examining expression and or composition of an PDE4D polypeptide, by a variety of methods, including enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
- ELISAs enzyme linked immunosorbent assays
- a test sample from an individual is assessed for the presence of an alteration in the expression and/or an alteration in composition of the polypeptide encoded by PDE4D, or for the presence of a particular variant (e.g. , an isoform) encoded by PDE4D.
- An alteration in expression of a polypeptide encoded by PDE4D can be, for example, an alteration in the quantitative polypeptide expression (i.e., the amount of polypeptide produced); an alteration in the composition of a polypeptide encoded by PDE4D is an alteration in the qualitative polypeptide expression (e.g., expression of a mutant PDE4D polypeptide or of a different splicing variant or isoform).
- detecting a particular splicing variant encoded by that PDE4D, or a particular pattern of splicing variants makes diagnosis of the disease or condition associated with PDE4D or a susceptibility to a disease or condition associated with PDE4D.
- An "alteration" in the polypeptide expression or composition refers to an alteration in expression or composition in a test sample, as compared with the expression or composition of polypeptide by PDE4D in a control sample.
- a control sample is a sample that conesponds to the test sample (e.g., is from the same type of cells), and is from an individual who is not affected by sfroke.
- An alteration in the expression or composition of the polypeptide in the test sample, as compared with the control sample is indicative of a susceptibility to stroke.
- the presence of one or more different splicing variants or isoforms in the test sample, or the presence of significantly different amounts of different splicing variants in the test sample, as compared with the confrol sample, is indicative of a susceptibility to stroke.
- Various means of examining expression or composition of the polypeptide encoded by PDE4D can be used, including spectroscopy, colorimetry, electrophoresis, isoelectric focusing, and immunoassays (e.g., David et al, U.S. Patent No. 4,376,110) such as immunoblotting (see also Cunent Protocols in Molecular Biology, particularly chapter 10).
- an antibody capable of binding to the polypeptide e.g., as described above
- Antibodies can be polyclonal, or more preferably, monoclonal.
- An intact antibody, or a fragment thereof e.g., Fab or F(ab') 2
- the term "labeled", with regard to the probe or antibody is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
- Western blotting analysis using an antibody as described above that specifically binds to a polypeptide encoded by a mutant PDE4D, or an antibody that specifically binds to a polypeptide encoded by a non-mutant gene, or an antibody that specifically binds to a particular splicing variant encoded by PDE4D, can be used to identify the presence in a test sample of a particular splicing variant or isoform, or of a polypeptide encoded by a polymo ⁇ hic or mutant PDE4D, or the absence in a test sample of a particular splicing variant or isoform, or of a polypeptide encoded by a non-polymo ⁇ hic or non-mutant gene.
- the presence of a polypeptide encoded by a polymo ⁇ hic or mutant gene, or the absence of a polypeptide encoded by a non-polymo ⁇ hic or non-mutant gene, is diagnostic for a susceptibility to stroke, as is the presence (or absence) of particular splicing variants encoded by the PDE4D gene.
- the level or amount of polypeptide encoded by PDE4D in a test sample is compared with the level or amount of the polypeptide encoded by PDE4D in a control sample.
- a level or amount of the polypeptide in the test sample that is higher or lower than the level or amount of the polypeptide in the control sample, such that the difference is statistically significant is indicative of an alteration in the expression of the polypeptide encoded by PDE4D, and is diagnostic for a susceptibility to stroke.
- the composition of the polypeptide encoded by PDE4D in a test sample is compared with the composition of the polypeptide encoded by PDE4D in a control sample (e.g., the presence of different splicing variants).
- a difference in the composition of the polypeptide in the test sample, as compared with the composition of the polypeptide in the control sample, is diagnostic for a susceptibility to stroke.
- both the level or amount and the composition of the polypeptide can be assessed in the test sample and in the confrol sample.
- a difference in the amount or level of the polypeptide in the test sample, compared to the control sample; a difference in composition in the test sample, compared to the control sample; or both a difference in the amount or level, and a difference in the composition is indicative of a susceptibility to stroke.
- assessment of the splicing variant or isoform(s) of a polypeptide encoded by a polymo ⁇ hic or mutant PDE4D can be performed.
- the assessment can be performed directly (e.g., by examining the polypeptide itself), or indirectly (e.g., by examining the mRNA encoding the polypeptide, such as through mRNA profiling).
- probes or primers as described herein can be used to determine which splicing variants or isoforms are encoded by PDE4D mRNA, using standard methods.
- the presence in a test sample of a particular splicing variant(s) or isoform(s) associated with stroke or risk of stroke, or the absence in a test sample of a particular splicing variant(s) or isoform(s) not associated with stroke or risk of stroke, is diagnostic for a disease or condition associated with a PDE4D gene or a susceptibility to a disease or condition associated with a PDE4D gene.
- the absence in a test sample of a particular splicing variant(s) or isoform(s) associated with stroke or risk of stroke, or the presence in a test sample of a particular splicing variant(s) or isoform(s) not associated with sfroke or risk of stroke is diagnostic for the absence of disease or condition associated with a PDE4D gene or a susceptibility to a disease or condition associated with a PDE4D gene.
- differential expression of isoforms PDE4D7, PDE4D9 and combinations thereof can be assessed and compared to confrol individuals. Decreased expression of these isoforms is indicative of susceptibility to stroke, particularly carotid sfroke and/or cardiogenic stroke.
- the invention further pertains to a method for the diagnosis and identification of susceptibility to stroke in an individual, by identifying an at-risk haplotype in PDE4D.
- the at-risk haplotype is a haplotype for which the presence of the haplotype increases the risk of stroke significantly.
- identifying whether a risk is significant may depend on a variety of factors, including the specific disease, the haplotype, and often, environmental factors, the significance may be measured by an odds ratio or a percentage, h a further embodiment, the significance is measured by a percentage.
- a significant risk is measured as an odds ratio of at least about 1.2, including but not limited to: 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 and 1.9. In a further embodiment, an odds ratio of at least 1.2 is significant. In a further embodiment, an odds ratio of at least about 1.5 is significant. In a further embodiment, a significant increase in risk is at least about 1.7 is significant. In a further embodiment, a significant increase in risk is at least about 20%, including but not limited to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%o and 98%>. In a further embodiment, a significant increase in risk is at least about 50%. It is understood however, that identifying whether a risk is medically significant may also depend on a variety of factors, including the specific disease, the haplotype, and often, environmental factors.
- the invention also pertains to methods of diagnosing stroke or a susceptibility to stroke in an individual, comprising screening for an at-risk haplotype in the PDE4D nucleic acid that is more frequently present in an individual susceptible to stroke (affected), compared to the frequency of its presence in a healthy individual (control), wherein the presence of the haplotype is indicative of stroke or susceptibility to stroke.
- Standard techniques for genotyping for the presence of SNPs and/or microsatellite markers that are associated with stroke can be used, such as fluorescent-based techniques (Chen, et al, Genome Res. 9, 492 (1999), PCR, LCR, Nested PCR and other techniques for nucleic acid amplification.
- the method comprises assessing in an individual the presence or frequency of SNPs and/or microsatellites in the PDE4D nucleic acid that are associated with stroke, wherein an excess or higher frequency of the SNPs and/or microsatellites compared to a healthy control individual is indicative that the individual has stroke or is susceptible to stroke.
- SNPs and markers that comprise haplotypes that can be used as screening tools. See also, Table 5, Table 6, Table 11 and Table 12 that set forth previously known SNP and novel microsatellite markers and their counte ⁇ art sequence ID reference numbers. SNPs and markers from these lists represent at-risk haplotypes and can be used to design diagnostic tests for determining a susceptibility to stroke.
- Kits useful in the methods of diagnosis comprise components useful in any of the methods described herein, including for example, hybridization probes or primers as described herein (e.g., labeled probes or primers), reagents for detection of labeled molecules, restriction enzymes (e.g., for RFLP analysis), allele-specific oligonucleotides, antibodies which bind to altered or to non- altered (native) PDE4D polypeptide, means for amplification of nucleic acids comprising PDE4D, or means for analyzing the nucleic acid sequence of PDE4D or for analyzing the amino acid sequence of an PDE4D polypeptide, etc.
- hybridization probes or primers as described herein e.g., labeled probes or primers
- restriction enzymes e.g., for RFLP analysis
- allele-specific oligonucleotides e.g., antibodies which bind to altered or to non- altered (native) PDE4D polypeptide
- a kit for diagnosing susceptibility to stroke can comprise primers for nucleic acid amplification of a region in the PDE4D gene comprismg an at-risk haplotype that is more frequently present in an individual susceptible to stroke.
- the primers can be designed using portions of the nucleic acids flanking SNPs that are indicative of stroke.
- the primers are designed to amplify regions of the PDE4D gene associated with an at-risk haplotype for stroke, shown in Tables 8 A and 8B.
- a kit for diagnosing susceptibility to stroke can further comprise probes designed to hybridize to regions of the PDE4D gene associated with an at-risk haplotype for sfroke, shown in Table 5 and table 6 and/or generated from SEQ TD Nos: 85-102.
- the invention provides methods (also refened to herein as "screening assays") for identifying the presence of a nucleotide that hybridizes to a nucleic acid of the invention, as well as for identifying the presence of a polypeptide encoded by a nucleic acid of the invention.
- the presence (or absence) of a nucleic acid molecule of interest (e.g., a nucleic acid that has significant homology with a nucleic acid of the invention) in a sample can be assessed by contacting the sample with a nucleic acid comprising a nucleic acid of the invention (e.g., a nucleic acid having the sequence of SEQ ID NO: 1 which may optionally comprise at least one polymo ⁇ hism shown in Tables 11 and 12, or the complement thereof, or a nucleic acid encoding an amino acid having the sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14, or a fragment or variant of such nucleic acids), under stringent conditions as described above, and then assessing the sample for the presence (or absence) of hybridization.
- a nucleic acid comprising a nucleic acid of the invention e.g., a nucleic acid having the sequence of SEQ ID NO: 1 which may optionally comprise at least one polymo ⁇ hism shown in Tables
- high stringency conditions are conditions appropriate for selective hybridization.
- a sample containing the nucleic acid molecule of interest is contacted with a nucleic acid containing a contiguous nucleotide sequence (e.g., a primer or a probe as described above) that is at least partially complementary to a part of the nucleic acid molecule of interest (e.g., a PDE4D nucleic acid), and the contacted sample is assessed for the presence or absence of hybridization, h another embodiment, the nucleic acid containing a contiguous nucleotide sequence is completely complementary to a part of the nucleic acid molecule of interest.
- a nucleic acid containing a contiguous nucleotide sequence e.g., a primer or a probe as described above
- the nucleic acid containing a contiguous nucleotide sequence is completely complementary to a part of the nucleic acid molecule of interest.
- all or a portion of the nucleic acid of interest can be subjected to amplification prior to performing the hybridization.
- the presence (or absence) of a polypeptide of interest, such as a polypeptide of the invention or a fragment or variant thereof, in a sample can be assessed by contacting the sample with an antibody that specifically hybridizes to the polypeptide of interest (e.g., an antibody such as those described above), and then assessing the sample for the presence (or absence) of binding of the antibody to the polypeptide of interest.
- the invention provides methods for identifying agents (e.g., fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes) that alter (e.g., increase or decrease) the activity of the polypeptides described herein, or which otherwise interact with the polypeptides herein.
- agents e.g., fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes
- such agents can be agents which bind to polypeptides described herein (e.g., PDE4D binding agents); which have a stimulatory or inhibitory effect on, for example, activity of polypeptides of the invention; or which change (e.g., enhance or inhibit) the ability of the polypeptides of the invention to interact with PDE4D binding agents (e.g., receptors or other binding agents); or which alter posttranslational processing of the PDE4D polypeptide (e.g., agents that alter proteolytic processing to direct the polypeptide from where it is normally synthesized to another location in the cell, such as the cell surface); agents that alter proteolytic processing such that more polypeptide is released from the cell, etc.
- PDE4D binding agents e.g., PDE4D binding agents
- PDE4D binding agents e.g., receptors or other binding agents
- alter posttranslational processing of the PDE4D polypeptide e.g., agents that alter proteolytic processing to direct the polypeptide
- the invention provides assays for screening candidate or test agents that bind to or modulate the activity of polypeptides described herein (or biologically active portion(s) thereof), as well as agents identifiable by the assays.
- Test agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
- the biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des., 12:145).
- a cell, cell lysate, or solution containing or expressing a PDE4D polypeptide e.g., SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14, or another splicing variant encoded by PDE4D), or a fragment or derivative thereof (as described above)
- a PDE4D polypeptide e.g., SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14, or another splicing variant encoded by PDE4D
- a fragment or derivative thereof as described above
- the level (amount) of PDE4D activity is assessed (e.g., the level (amount) of PDE4D activity is measured, either directly or indirectly), and is compared with the level of activity in a control (i.e., the level of activity of the PDE4D polypeptide or active fragment or derivative thereof in the absence of the agent to be tested). If the level of the activity in the presence of the agent differs, by an amount that is statistically significant, from the level of the activity in the absence of the agent, then the agent is an agent that alters the activity of PDE4D polypeptide. An increase in the level of PDE4D activity relative to level of the control, indicates that the agent is an agent that enhances (is an agonist of) PDE4D activity.
- a decrease in the level of PDE4D activity relative to level of the control indicates that the agent is an agent that inhibits (is an antagonist of) PDE4D activity.
- the level of activity of a PDE4D polypeptide or derivative or fragment thereof in the presence of the agent to be tested is compared with a confrol level that has previously been established. A level of the activity in the presence of the agent that differs from the confrol level by an amount that is statistically significant indicates that the agent alters PDE4D activity.
- the present invention also relates to an assay for identifying agents which alter the expression of the PDE4D gene (e.g., antisense nucleic acids, fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes) which alter (e.g., increase or decrease) expression (e.g., transcription or translation) of the gene or which otherwise interact with the nucleic acids described herein, as well as agents identifiable by the assays.
- agents which alter the expression of the PDE4D gene e.g., antisense nucleic acids, fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes
- alter e.g., increase or decrease expression (e.g., transcription or translation) of the gene or which otherwise interact with the nucleic acids described herein, as well as agents identifiable by the assays.
- the solution can comprise, for example, cells containing the nucleic acid or cell lysate containing the nucleic acid; alternatively, the solution can be another solution that comprises elements necessary for transcription/translation of the nucleic acid. Cells not suspended in solution can also be employed, if desired.
- the level and/or pattern of PDE4D expression e.g., the level and/or pattern of mRNA or of protein expressed, such as the level and/or pattern of different splicing variants
- a confrol i.e., the level and or pattern of the PDE4D expression in the absence of the agent to be tested.
- the agent is an agent that alters the expression of PDE4D.
- Enhancement of PDE4D expression indicates that the agent is an agonist of PDE4D activity.
- inhibition of PDE4D expression indicates that the agent is an antagonist of PDE4D activity.
- the level and/or pattern of PDE4D polypeptide(s) e.g., different splicing variants
- the level and/or pattern of PDE4D polypeptide(s) is compared with a control level and/or pattern that have previously been established.
- a level and/or pattern in the presence of the agent that differs from the control level and/or pattern by an amount or in a manner that is statistically significant indicates that the agent alters PDE4D expression.
- agents that can alter expression levels of isoforms PDE4D7 and/or PDE4D9 can be assessed, preferably to complement the expression levels to approximate the ratios of a healthy individual.
- agents which alter the expression of the PDE4D gene or which otherwise interact with the nucleic acids described herein can be identified using a cell, cell lysate, or solution containing a nucleic acid encoding the promoter region of the PDE4D gene operably linked to a reporter gene.
- the level of expression of the reporter gene e.g., the level of mRNA or of protein expressed
- a control i.e., the level of the expression of the reporter gene in the absence of the agent to be tested.
- the agent is an agent that alters the expression of PDE4D, as indicated by its ability to alter expression of a gene that is operably linked to the PDE4D gene promoter. Enhancement of the expression of the reporter indicates that the agent is an agonist of PDE4D activity. Similarly, inhibition of the expression of the reporter indicates that the agent is an antagonist of PDE4D activity.
- the level of expression of the reporter in the presence of the agent to be tested is compared with a confrol level that has previously been established. A level in the presence of the agent that differs from the confrol level by an amount or in a manner that is statistically significant indicates that the agent alters PDE4D expression.
- Agents which alter the amounts of different splicing variants encoded by PDE4D e.g., an agent which enhances activity of a first splicing variant, and which inhibits activity of a second splicing variant
- agents which are agonists of activity of a first splicing variant and antagonists of activity of a second splicing variant can easily be identified using these methods described above.
- assays can be used to assess the impact of a test agent on the activity of a polypeptide in relation to a PDE4D binding agent.
- a cell that expresses a compound that interacts with PDE4D (herein refened to as a "PDE4D binding agent", which can be a polypeptide or other molecule that interacts with PDE4D, such as a receptor) is contacted with PDE4D in the presence of a test agent, and the ability of the test agent to alter the interaction between PDE4D and the PDE4D binding agent is determined.
- a cell lysate or a solution containing the PDE4D binding agent can be used.
- test agents can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. It is also within the scope of this invention to determine the ability of a test agent to interact with the polypeptide without the labeling of any of the interactants.
- a microphysiometer can be used to detect the interaction of a test agent with PDE4D or a PDE4D binding agent without the labeling of either the test agent, PDE4D, or the PDE4D binding agent. McConnell, H.M. et al. (1992) Science, 257:1906-1912.
- a "microphysiometer” e.g., CytosensorTM
- LAPS light- addressable potentiometric sensor
- Changes in this acidification rate can be used as an indicator of the interaction between ligand and polypeptide.
- these receptors can be used to screen for compounds that are PDE4D receptor agonists for use in treating stroke or PDE4D receptor antagonists for studying sfroke.
- the linkage data provided herein, for the first time, provides such connection to stroke.
- Drugs could be designed to regulate PDE4D receptor activation that in turn can be used to regulate signaling pathways and transcription events of genes downstream, such as Cbfal .
- assays can be used to identify polypeptides that interact with one or more PDE4D polypeptides, as described herein.
- a yeast two-hybrid system such as that described by Fields and Song (Fields, S. and Song, O., Nature 340:245-246 (1989)) can be used to identify polypeptides that interact with one or more PDE4D polypeptides.
- vectors are constructed based on the flexibility of a transcription factor that has two functional domains (a DNA binding domain and a transcription activation domain).
- transcriptional activation can be achieved, and transcription of specific markers (e.g., nutritional markers such as His and Ade, or color markers such as lacZ) can be used to identify the presence of interaction and transcriptional activation.
- specific markers e.g., nutritional markers such as His and Ade, or color markers such as lacZ
- a first vector which includes a nucleic acid encoding a DNA binding domain and also an PDE4D polypeptide, splicing variant, fragment or derivative thereof
- a second vector is used which includes a nucleic acid encoding a transcription activation domain and also a nucleic acid encoding a polypeptide which potentially may interact with the PDE4D polypeptide, splicing variant, or fragment or derivative thereof (e.g., a PDE4D polypeptide binding agent or receptor).
- incubation of yeast containing the first vector and the second vector under appropriate conditions e.g., mating conditions such as used in the MatchmakerTM System from Clontech
- yeast containing the first vector and the second vector under appropriate conditions (e.g., mating conditions such as used in the MatchmakerTM System from Clontech) allows identification of colonies which express the markers of interest. These colonies can be examined to identify the polypeptide(s) that interact with the PDE4D polypeptide or fragment or derivative thereof.
- binding agent to the polypeptide, or interaction of the polypeptide with a binding agent in the presence and absence of a test agent, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
- a fusion protein e.g., a glutathione-S-transferase fusion protein
- a fusion protein e.g., a glutathione-S-transferase fusion protein
- modulators of expression of nucleic acid molecules of the invention are identified in a method wherein a cell, cell lysate, or solution containing a nucleic acid encoding PDE4D is contacted with a test agent and the expression of appropriate mRNA or polypeptide (e.g., splicing variant(s)) in the cell, cell lysate, or solution, is determined.
- appropriate mRNA or polypeptide e.g., splicing variant(s)
- the level of expression of appropriate mRNA or polypeptide(s) in the presence of the test agent is compared to the level of expression of mRNA or polypeptide(s) in the absence of the test agent.
- the test agent can then be identified as a modulator of expression based on this comparison.
- the test agent when expression of mRNA or polypeptide is greater (statistically significantly greater) in the presence of the test agent than in its absence, the test agent is identified as a stimulator or enhancer of the mRNA or polypeptide expression.
- the test agent when expression of the mRNA or polypeptide is less (statistically significantly less) in the presence of the test agent than in its absence, the test agent is identified as an inhibitor of the mRNA or polypeptide expression.
- the level of mRNA or polypeptide expression in the cells can be detennined by methods described herein for detecting mRNA or polypeptide.
- This invention further pertains to novel agents identified by the above- described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
- an agent identified as described herein e.g., a test agent that is a modulating agent, an antisense nucleic acid molecule, a specific antibody, or a polypeptide-binding agent
- an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
- an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
- this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
- an agent identified as described herein can be used to alter activity of a polypeptide encoded by PDE4D, or to alter expression of PDE4D, by contacting the polypeptide or the gene (or contacting a cell comprising the polypeptide or the gene) with the agent identified as described herein.
- the present invention also pertains to pharmaceutical compositions comprising agents described herein, particularly nucleotides encoding the polypeptides described herein; comprising polypeptides described herein (e.g., one or more of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14); and/or comprising other splicing variants encoded by PDE4D; and/or an agent that alters (e.g., enhances or inhibits) PDE4D gene expression or PDE4D polypeptide activity as described herein.
- agents described herein particularly nucleotides encoding the polypeptides described herein; comprising polypeptides described herein (e.g., one or more of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14); and/or comprising other splicing variants encoded by PDE4D; and/or an agent that alters (e.g., enhances or inhibits) PDE4D gene expression or PDE4D polypeptide activity as described herein.
- a polypeptide, protein e.g., an PDE4D receptor
- an agent that alters PDE4D gene expression or a PDE4D binding agent or binding partner, fragment, fusion protein or prodrug thereof, or a nucleotide or nucleic acid construct (vector) comprismg a nucleotide of the present invention, or an agent that alters PDE4D polypeptide activity
- a physiologically acceptable carrier or excipient can be formulated with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition.
- the carrier and composition can be sterile. The formulation should suit the mode of administration.
- Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof.
- the pharmaceutical preparations can, if desired, be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active agents.
- auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active agents.
- the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
- the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
- the composition can be formulated as a suppository, with traditional binders and carriers such as trigly
- compositions of introduction of these compositions include, but are not limited to, intradermal, intramuscular, intraperitoneal, intraocular, intravenous, subcutaneous, topical, oral and intranasal.
- Other suitable methods of introduction can also include gene therapy (as described below), rechargeable or biodegradable devices, particle acceleration devises ("gene guns") and slow release polymeric devices.
- the pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other agents.
- compositions for intravenous administration typically are solutions in sterile isotonic aqueous buffer.
- the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection.
- the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent.
- the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water.
- an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
- nonsprayable forms viscous to semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water
- Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, enemas, lotions, sols, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc.
- the agent may be inco ⁇ orated into a cosmetic formulation.
- sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., pressurized air.
- a pressurized volatile, normally gaseous propellant e.g., pressurized air.
- Agents described herein can be formulated as neutral or salt forms.
- Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.
- the agents are administered in a therapeutically effective amount.
- the amount of agents which will be therapeutically effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques, h addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges.
- the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the symptoms of stroke, and should be decided according to the judgment of a practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
- the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
- Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use of sale for human administration.
- the pack or kit can be labeled with information regarding mode of administration, sequence of drug administration (e.g., separately, sequentially or concurrently), or the like.
- the pack or kit may also include means for reminding the patient to take the therapy.
- the pack or kit can be a single unit dosage of the combination therapy or it can be a plurality of unit dosages, hi particular, the agents can be separated, mixed together in any combination, present in a single vial or tablet. Agents assembled in a blister pack or other dispensing means is prefened.
- unit dosage is intended to mean a dosage that is dependent on the individual pharmacodynamics of each agent and administered in FDA approved dosages in standard time courses.
- the present invention encompasses methods of treatment (prophylactic and/or therapeutic) for stroke or a susceptibility to stroke, such as individuals in the target populations described herein particularly ischemic (e.g., carotid and cardiogenic strokes) and TIA, using a PDE4D therapeutic agent.
- a "PDE4D therapeutic agent” is an agent that alters (e.g., enhances or inhibits) PDE4D polypeptide (enzymatic activity) and/or PDE4D gene expression, as described herein (e.g., a PDE4D agonist or antagonist).
- PDE4D therapeutic agents can alter PDE4D polypeptide activity or nucleic acid expression by a variety of means, such as, for example, by providing additional PDE4D polypeptide or by upregulating the transcription or translation of the PDE4D gene; by altering posttranslational processing of the PDE4D polypeptide; by altering transcription of PDE4D splicing variants; or by interfering with PDE4D polypeptide activity (e.g., by binding to a PDE4D polypeptide), or by downregulating the transcription or translation of the PDE4D gene.
- means such as, for example, by providing additional PDE4D polypeptide or by upregulating the transcription or translation of the PDE4D gene; by altering posttranslational processing of the PDE4D polypeptide; by altering transcription of PDE4D splicing variants; or by interfering with PDE4D polypeptide activity (e.g., by binding to a PDE4D polypeptide), or by downregulating the transcription or translation
- the invention relates to methods of treatment for stroke or susceptibility to stroke (for example, for individuals in an at-risk population such as those described herein); as well as to methods of treatment for myocardial infarction, atherosclerosis, acute coronary syndrome (e.g., unstable angina, non-ST-elevation myocardial infarction (NSTEMI) or ST-elevation myocardial infarction (STEMI)); for decreasing risk of a second myocardial infarction; for atherosclerosis, such as for patients requiring treatment (e.g., angioplasty, stents, coronary artery bypass graft) to restore blood flow in arteries (e.g. , coronary arteries) and peripheral arterial occlusive disease.
- NSTEMI non-ST-elevation myocardial infarction
- ST-elevation myocardial infarction ST-elevation myocardial infarction
- atherosclerosis such as for patients requiring treatment (e.g.,
- Representative PDE4D therapeutic agents include the following: nucleic acids or fragments or derivatives thereof described herein, particularly nucleotides encoding the polypeptides described herein and vectors comprising such nucleic acids (e.g., a gene, cDNA, and/or mRNA, double-stranded interfering RNA, a nucleic acid encoding a PDE4D polypeptide or active fragment or derivative thereof, or an oligonucleotide; for example, SEQ ID NO: 1 which may optionally comprise at least one polymo ⁇ hism shown in Tables 11 and 12 or a nucleic acid encoding SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14, or fragments or derivatives thereof), antisense nucleic acids or small double-stranded interfering RNA; polypeptides described herein (e.g., one or more of SEQ ID NO: 2, 3, 4, 5, 6,
- PDE4D polypeptides
- PDE4D receptors polypeptides
- PDE4D binding agents peptidomimetics
- fusion proteins or prodrugs thereof antibodies (e.g., an antibody to a mutant PDE4D polypeptide, or an antibody to a non-mutant PDE4D polypeptide, or an antibody to a particular splicing variant encoded by PDE4D, as described above); ribozymes; other small molecules; and other agents that alter (e.g., inhibit or antagonize) PDE4D gene expression or polypeptide activity, or that regulate transcription of PDE4D splicing variants (e.g., agents that affect which splicing variants are expressed, or that affect the amount of each splicing variant that is expressed).
- PDE4D receptors polypeptides
- PDE4D binding agents e.g., an antibody to a mutant PDE4D polypeptide, or an antibody to a non-mutant PDE4D polypeptide
- More than one PDE4D therapeutic agent can be used concurrently, if desired.
- the PDE4D therapeutic agent that is a nucleic acid is used in the freatment of sfroke.
- the term, "freatment” as used herein, refers not only to ameliorating symptoms associated with the disease, but also preventing or delaying the onset of the disease, and also lessening the severity or frequency of symptoms of the disease, preventing or delaying the occunence of a second episode of the disease or condition; and/or also lessening the severity or frequency of symptoms of the disease or condition.
- treatment also refers to a minimization or reversal of the development of plaques.
- the therapy is designed to alter (e.g., inhibit or enhance), replace or supplement activity of a PDE4D polypeptide in an individual.
- a PDE4D therapeutic agent can be administered in order to upregulate or increase the expression or availability of the PDE4D gene or of specific splicing variants of PDE4D, or, conversely, to downregulate or decrease the expression or availability of the PDE4D gene or specific splicing variants of PDE4D.
- Upregulation or increasing expression or availability of a native PDE4D gene or of a particular splicing variant could interfere with or compensate for the expression or activity of a defective gene or another splicing variant; downregulation or decreasing expression or availability of a native PDE4D gene or of a particular splicing variant could minimize the expression or activity of a defective gene or the particular splicing variant and thereby minimize the impact of the defective gene or the particular splicing variant.
- the PDE4D therapeutic agent(s) are administered in a therapeutically effective amount (i.e., an amount that is sufficient to treat the disease, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or also lessening the severity or frequency of symptoms of the disease).
- a therapeutically effective amount i.e., an amount that is sufficient to treat the disease, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or also lessening the severity or frequency of symptoms of the disease.
- the amount wliich will be therapeutically effective in the treatment of a particular individual's disorder or condition will depend on the symptoms and severity of the disease, and can be determined by standard clinical techniques.
- in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges.
- Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
- a nucleic acid of the invention e.g., a nucleic acid encoding a PDE4D polypeptide, such as SEQ ID NO: 1 which may optionally comprise at least one polymo ⁇ hism shown in Tables 11 and 12; or another nucleic acid that encodes a PDE4D polypeptide or a splicing variant, derivative or fragment thereof, such as a nucleic acid encoding SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14
- SEQ ID NO: 2 e.g., a nucleic acid encoding a PDE4D polypeptide, such as SEQ ID NO: 1 which may optionally comprise at least one polymo ⁇ hism shown in Tables 11 and 12; or another nucleic acid that encodes a PDE4D polypeptide or a splicing variant, derivative or fragment thereof, such as a nucleic acid encoding SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14
- PDE4D or a cDNA encoding the PDE4D polypeptide can be introduced into cells (either in vitro or in vivo) such that the cells produce native PDE4D polypeptide. If necessary, cells that have been transformed with the gene or cDNA or a vector comprising the gene or cDNA can be introduced (or re-introduced) into an individual affected with the disease.
- cells which, in nature, lack native PDE4D expression and activity, or have mutant PDE4D expression and activity, or have expression of a disease-associated PDE4D splicing variant can be engineered to express PDE4D polypeptide or an active fragment of the PDE4D polypeptide (or a different variant of PDE4D polypeptide).
- nucleic acid encoding the PDE4D polypeptide, or an active fragment or derivative thereof can be infroduced into an expression vector, such as a viral vector, and the vector can be introduced into appropriate cells in an animal.
- an expression vector such as a viral vector
- Other gene transfer systems including viral and nonviral transfer systems, can be used.
- nonviral gene transfer methods such as calcium phosphate coprecipitation, mechanical techniques (e.g., microinjection); membrane fusion-mediated transfer via liposomes; or direct DNA uptake, can also be used.
- a nucleic acid of the invention a nucleic acid complementary to a nucleic acid of the invention; or a portion of such a nucleic acid (e.g., an oligonucleotide as described below), can be used in "antisense" therapy, in which a nucleic acid (e.g., an oligonucleotide) which specifically hybridizes to the mRNA and/or genomic DNA of PDE4D is administered or generated in situ.
- the antisense nucleic acid that specifically hybridizes to the mRNA and/or DNA inhibits expression of the PDE4D polypeptide, e.g., by inhibiting translation and/or transcription. Binding of the antisense nucleic acid can be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interaction in the major groove of the double helix.
- An antisense construct of the present invention can be delivered, for example, as an expression plasmid as described above. When the plasmid is transcribed in the cell, it produces RNA that is complementary to a portion of the mRNA and/or DNA that encodes PDE4D polypeptide.
- the antisense construct can be an oligonucleotide probe that is generated ex vivo and introduced into cells; it then inhibits expression by hybridizing with the mRNA and/or genomic DNA of PDE4D.
- the oligonucleotide probes are modified oligonucleotides that are resistant to endogenous nucleases, e.g., exonucleases and/or endonucleases, thereby rendering them stable in vivo.
- Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Patent Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy are also described, for example, by Van der Krol et al.
- oligodeoxyribonucleotides derived from the translation initiation site e.g., between the -10 and +10 regions of PDE4D sequence, are prefened.
- oligonucleotides are designed that are complementary to mRNA encoding PDE4D.
- the antisense oligonucleotides bind to PDE4D mRNA transcripts and prevent translation.
- Absolute complementarity although prefened, is not required, a sequence "complementary" to a portion of an RNA, as refened to herein, indicates that a sequence has sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
- the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid, as described in detail above. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures.
- the oligonucleotides used in antisense therapy can be DNA, RNA, or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
- the oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
- the oligonucleotides can include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci.
- the oligonucleotide may be conjugated to another molecule (e.g., a peptide, hybridization triggered cross- linking agent, transport agent, hybridization-triggered cleavage agent).
- another molecule e.g., a peptide, hybridization triggered cross- linking agent, transport agent, hybridization-triggered cleavage agent.
- the antisense molecules are delivered to cells that express PDE4D in vivo.
- a number of methods can be used for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systematically.
- a recombinant DNA construct is utilized in which the antisense oligonucleotide is placed under the control of a strong promoter (e.g., pol III or pol II).
- a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA.
- Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
- Such vectors can be constructed by recombinant DNA technology methods standard in the art and described above.
- a plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct that can be introduced directly into the tissue site.
- viral vectors can be used which selectively infect the desired tissue, in which case administration may be accomplished by another route (e.g., systemically).
- RNA inhibitor refers to an inhibitory RNA that silences expression of the target protein by RNA interference (McManus, M.T. and Sha ⁇ , P.A., 2002. Nat. Rev. Genet.
- RNA interference is conserved throughout evolution, from C. elegans to humans, and is believed to function in protecting cells from invasion by RNA viruses.
- Dicer an RNaselll-type enzyme termed Dicer.
- the Dicer enzyme "dices" the RNA into short duplexes of 21 nucleotides, termed short-interfering RNAs or siRNAs, composed of 19 nucleotides of perfectly paired ribonucleotides with two unpaired nucleotides on the 3' end of each strand.
- siRNAs short-interfering RNAs
- These short duplexes associate with a multiprotein complex termed RISC, and direct this complex to mRNA transcripts with sequence similarity to the siRNA.
- RISC multiprotein complex
- nucleases present in the RISC complex cleave the mRNA transcript, thereby abolishing expression of the gene product, h the case of viral infection, this mechanism would result in destruction of viral transcripts, thus preventing viral synthesis.
- siRNAs are double-stranded, either strand has the potential to associate with RISC and direct silencing of transcripts with sequence similarity. Recently, it was determined that gene silencing could be induced by presenting the cell with the siRNA, mimicking the product of Dicer cleavage
- Synthetic siRNA duplexes maintain the ability to associate with RISC and direct silencing of mRNA transcripts, thus providing researchers with a powerful tool for gene silencing in mammalian cells.
- Yet another method to introduce the dsRNA for gene silencing is shRNA, for short hai ⁇ in RNA (Paddison, P.J., et al., 2002. Genes Dev.
- a desired siRNA sequence is expressed from a plasmid (or virus) containing an "shRNA" gene having an inverted repeat with an intervening loop sequence to form a hai ⁇ in structure.
- the resulting shRNA transcript containing the hai ⁇ in is subsequently processed by Dicer to produce siRNAs for silencing.
- Plasmid-based shRNAs can be expressed stably in cells, allowing long-term gene silencing in cells, or even in animals (McCaffrey,A.P., et al, 2002. Nature 418:38-9; Xia, H., et al, 2002. Nat. Biotech. 20:1006-10; Lewis, D.L., et al, 2002. Nat. Genetics 32:107-8; Rubinson, D.A., et al., 2003. Nat. Genetics 33:401-6; Tiscornia, G., et al, (2003) Proc. Natl. Acad. Sci. U.S.A. 100:1844-8).
- R ⁇ A interference has been successfully used therapeutically to protect mice from fulminant hepatitis (Song, E., et al., 2003. Nat. Medicine 9:347-51).
- Endogenous PDE4D expression can be also reduced by inactivating or "knocking out” PDE4D or its promoter using targeted homologous recombination (e.g., see Smithies et al. (1985) Nature 317:230-234; Thomas & Capecchi (1987) Cell 51:503-512; Thompson et al. (1989) Cell 5:313-321).
- targeted homologous recombination e.g., see Smithies et al. (1985) Nature 317:230-234; Thomas & Capecchi (1987) Cell 51:503-512; Thompson et al. (1989) Cell 5:313-321).
- a mutant, non-functional PDE4D flanked by DNA homologous to the endogenous PDE4D (either the coding regions or regulatory regions of PDE4D) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express PDE4D in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of PDE4D.
- the recombinant DNA constructs can be directly administered or targeted to the required site in vivo using appropriate vectors, as described above.
- targeted homologous recombination can be used to insert a DNA construct comprising a non-mutant, functional PDE4D (e.g., a gene having SEQ ID NO: 1 which may optionally comprise at least one polymo ⁇ hism shown in Tables 11 and 12), or a portion thereof, in place of a mutant PDE4D in the cell, as described above.
- targeted homologous recombination can be used to insert a DNA construct comprismg a nucleic acid that encodes a PDE4D polypeptide variant that differs from that present in the cell.
- endogenous PDE4D expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of PDE4D (i.e., the PDE4D promoter and/or enhancers) to form triple helical structures that prevent transcription of PDE4D in target cells in the body.
- deoxyribonucleotide sequences complementary to the regulatory region of PDE4D i.e., the PDE4D promoter and/or enhancers
- the antisense constructs described herein by antagonizing the normal biological activity of one of the PDE4D proteins, can be used in the manipulation of tissue, e.g., tissue differentiation, both in vivo and for ex vivo tissue cultures.
- tissue e.g., tissue differentiation
- the anti- sense techniques e.g., microinjection of antisense molecules, or transfection with plasmids whose transcripts are anti-sense with regard to a PDE4D mRNA or gene sequence
- Such techniques can be utilized in cell culture, but can also be used in the creation of transgenic animals.
- PDE4D therapeutic agents as described herein can also be used in the treatment or prevention of stroke.
- the therapeutic agents can be delivered in a composition, as described above, or by themselves. They can be administered systemically, or can be targeted to a particular tissue.
- the therapeutic agents can be produced by a variety of means, including chemical synthesis; recombinant production; in vivo production (e.g., a transgenic animal, such as U.S. Patent No. 4,873,316 to Meade et ⁇ l.), for example, and can be isolated using standard means such as those described herein.
- a combination of any of the above methods of treatment e.g., administration of non-mutant PDE4D polypeptide in conjunction with antisense therapy targeting mutant PDE4D mRNA; administration of a first splicing variant encoded by PDE4D in conjunction with antisense therapy targeting a second splicing encoded by PDE4D
- administration of non-mutant PDE4D polypeptide in conjunction with antisense therapy targeting mutant PDE4D mRNA administration of a first splicing variant encoded by PDE4D in conjunction with antisense therapy targeting a second splicing encoded by PDE4D
- EXAMPLE 1 PDE4D VARIATIONS AND HAPLOTYPES INCREASE RISK FOR STROKE
- the database uses a patient list, with encrypted personal identifiers, as input, and recursive algorithms to find all ancestors in the database who are related to any member on the input list within a given number of generations back (Gulcher, J.R., and Stefansson, K., Clin. Chem. Lab. Med. 36:523 (1998)) covering the whole Icelandic nation.
- the cluster function then searches for ancestors who are common to any two or more members of the input list.
- hemonhagic stroke patients clustered with ischemic stroke and TIA patients, and there were no families with a striking preponderance of hemonhagic stroke or of the subtypes of ischemic stroke.
- Patients with ischemic sfroke were reclassified according to the TOAST (Trial of Org 10172 in Acute Stroke Treatment) sub-classification system for stroke (Adams, H.P., Jr., et al, Stroke, 24:34-41 (1993)).
- This system includes five categories: (1) large-artery atherosclerosis, (2) cardioembolism, (3) small-artery occlusion (lacune), (4) stroke of other determined etiology and (5) stroke of undetermined etiology.
- the diagnoses were based on clinical features and on data from ancillary diagnostic studies.
- Patients defined with large-artery atherosclerosis had clinical and brain imaging findings of cerebral cortical dysfunction and either significant (>70%>) stenosis (this is a stricter criteria than used in TOAST where 50% stenosis is the cut-off) or occlusion of a major brain artery or branch cortical artery.
- Potential sources of cardiogenic embolism were excluded.
- the category cardioembolism included patients with at least one cardiac source for an embolus and potential large-artery sources of thromobosis and embolism was eliminated.
- Patients with small-artery occlusion had one of the traditional clinical lacunar syndromes and no evidence of cerebral cortical dysfunction.
- FIG. 1 displays two pedigrees each affected by several of the sfroke subtypes, including hemonhagic sfroke. Apparently what is inherited in stroke is the broadly defined phenotype.
- a genome-wide scan was performed using a framework map of about 1000 microsatellite markers.
- the DNA samples were genotyped using approximately 1000 fluorescently labelled primers.
- a microsatellite screening set based in part on the ABI Linkage Marker (v2) screening set and the ABI Linkage Marker (v2) intercalating set in combination with 500 custom-made markers were developed. All markers were extensively tested for robustness, ease of scoring, and efficiency in 4X multiplex PCR reactions.
- the average spacing between markers was approximately 4 cM with no gaps larger than 10 cM. Marker positions were obtained from the Marshfield map, except for a three-marker putative inversion on chromosome 8 (Jonsdottir, G.M., et al, Am. J.
- the PCR amplifications were set up, run and pooled on Perkin Elmer/ Applied Biosystems 877 Integrated Catalyst Thermocyclers with a similar protocol for each marker.
- the reaction volume used was 5 ⁇ l and for each PCR reaction 20 ng of genomic DNA was amplified in the presence of 2 pmol of each primer, 0.25 U AMPLITAQ GOLD (DNA polymerase; trademark of Roche Molecular Systems), 0.2 mM dNTPs and 2.5 mM MgC12 (buffer was supplied by manufacturer).
- the PCR conditions used were 95°C for 10 minutes, then 37 cycles of 15 s at 94°C, 30s at 55°C and 1 min at 72°C.
- the PCR products were supplemented with the internal size standard and the pools were separated and detected on Applied Biosystems model 377 Sequencer using v3.0 GENESCAN (peak calling software; trademark of Applied Biosystems).
- Alleles were called automatically with the TRUEALLELE (computer program for alleles identification; trademark of Cybergenetics, hie.) program, and the program, DECODE-GT (computer editing program that works downstream of the TRUEALLELE program; trademark of deCODE genetics), was used to fractionate according to quality and edit the called genotypes (Palsson, B., et al, Genome Res. 9:1002 (1999)). At least 180 Icelandic controls were genotyped to derive allelic frequencies.
- the allele sharing lod scores for the genome scan using the framework map showed three regions that achieved a lod score above 1.0. Two of these regions are on chromosome 5q. The first peak is at approximately 69 cM with a lod score of 2.00. The second peak is at 99 cM with a lod score of 1.14. The third region is on chromosome 14q at 55 cM with a lod score of 1.24.
- the information for linkage at the 5q locus was increased by genotyping an additional 45 markers over a 45 cM segment which spanned both peaks. The information used here is defined by Hoffman (D. L.
- ALLEGRO deCODE genetics
- the marker order and inter-marker distances were improved by constructing high density physical and genetic maps over a 20 cM region between markers D5S474 and D5S2046.
- a combination of data from coincident hybridizations of BAC membranes using a high density of STSs and the Finge ⁇ rinting Contig database was used to build large contigs of BACs from the RPCI -11 library.
- the order of the linkage markers was also confirmed by high-resolution genetic mapping using the stroke families supplemented with over 112 other large nuclear families. High resolution genetic mapping was used both to anchor and place in order contigs found by physical mapping as well as to obtain accurate inter-marker distances for the conectly ordered markers.
- BAC contigs were generated by a method that combines coincident primer hybridization with data mining.
- the RPCI-11 human male BAC library segments 1 & 2 (Pieter de Jong, Children's Hospital Oakland Research Institute) containing about 200,000 clones with a 12X coverage, were gridded using a 6x6 double offset pattern in 23 cm x 23 cm membranes with a BioGrid robot (Biorobotics Ltd., Cambridge, UK).
- hybridizations were performed with markers in the region of interest according to their location in the Weizmann Institute Unified Database. Primer sequences were analyzed and discarded according to their content of known repeats, E. coli and vector sequences (the analysis was performed using software developed at deCODE genetics). One hundred and fifty markers in the region (30 polymo ⁇ hic markers used in linkage and 120 generated from STSs) separated by an average of 130 kb were used. The selected markers were used to generate two 32 P labelled probes, F that contained the pooled forward primers and R that contained the pooled reverse primers. Reading of positive signals was performed automatically from digitized images of resulting autoradiograms by informatics tools developed at deCODE genetics.
- the coincident signals in both hybridizations were selected as positive clones.
- a set of overlapping clones was assembled through a combination of hybridization and BAC finge ⁇ rint walking. Finge ⁇ rints of positive clones were analyzed using the FPC database developed at the Sanger Center. Data from FPC contigs prebuilt with a cutoff of 3e-12 and from sequence datamining was integrated with the hybridization results. BACs in the region detected by data mining and hybridization were re-anayed using a Multiprobe Ilex robot (Packard, Meriden, CT). Small membranes (8 cm x 12 cm) were gridded in 6x6 double offset pattern and individually hybridized with the markers of interest.
- Positive patterns were transfened using transparencies to an Excel file containing macros to provide BAC to marker associations.
- a visual map was generated by combining the hybridization, finge ⁇ rinting and sequence data. New markers were generated from BAC end sequences to close the gap. After several rounds of hybridization positive BACs were assembled into 7 contigs covering approximately 20 Mb. Thirty of the polymo ⁇ hic markers used in linkage were assigned to four of the contigs. Estimation of contig lengths and distance between markers assigned to them was based on the FPC program. Twenty-seven of our 30 linkage markers mapped to three contigs in the
- the second P-value was calculated by comparing the observed LOD-score with its complete data sampling distribution under the null hypothesis (Gudbjartsson et al, Nat. Genet. 25:12-3, 2000). When the data consist of more than a few families, as is the case here, these two P- values tend to be very similar.
- DG5S397 Three of the tests, one for cardiogenic stroke (AC008818-1), one for carotid stroke (DG5S397), and one for the combination of carotid and cardiogenic stroke (AC008818-1) were significant even after conecting for multiple testing (Table 1).
- the marker DG5S397 is located within the PDE4D gene and AC008818-1 is in the 5' end of PDE4D and in the overlapping gene Prostate androgen-regulated transcript (PARTI) whose transcript is on the other strand going in the opposite direction.
- PDE4D is an important regulator of intracellular levels of cAMP and is expressed widely. PARTI encodes a putative protein with unknown function predominantly expressed in the prostate gland and in several cancer cell lines.
- Microsatellite allelic association analysis of the two-lod drop of the STRKl locus All microsatellites that show association with a p-value less than 0.01 for all stroke, all stroke excluding hemorrhagic stroke, cardiogenic stroke, carotid stroke, small vessel disease and
- allele 1 is 1 bp longer than the lower allele in the CEPH sample 1347-02
- allele 2 is 2 bp longer than the lower allele in the CEPH sample 1347-02
- allele 3 is 3 bp longer than the lower allele in the CEPH sample 1347-02
- allele 4 is 4 bp longer than the lower allele in the CEPH sample 1347-02
- allele -1 is 1 bp shorter than the lower allele in the CEPH sample 1347-02
- allele -2 is 2 bp shorter than the lower allele in the CEPH sample 1347-02
- so on Note that this same CEPH sample is a standard that is widely used throughout the world for calibration and comparison of alleles.
- CEPH 1347-02, family 137, individual 02. Swedish patients have also been genotyped and microsatellite single and multimarker association has been analyzed using the E-M algorithm.
- PDE4D gene a functional variant in the PDE4D gene might be the cause of our observed microsatellite association.
- the PDE4D gene encodes eight protein isoforms and has at least seven promoters. All isoforms identified have an identical C-terminal catalytic domain but differ at the N-terminal regulatory domain (FIG. 2).
- Markers AA change exon Allele p-value Aff. % Ctrl. % # Aff # Ctrl
- Table 2C SNP identification, single marker association andLD mapping of the DE4D region
- SNP 26 SNP5PDM379372 rs40512 59775992 59775992 120628 120628
- SNP 1 SNP5PDM421449 rs248911 59818063 59818063 78552 78552 D5S1990 60945599 60945816 D5S1359 63542603 63542894 D5S2089 65914315 65914496 D5S647 66217674 66218065 D5S2121 66584091 66584385
- the SNPs were identified in the public NCBI SNP database or by sequencing selected intronic and flanking regions in the gene in at least 94 patients and 94 controls. We initially identified 637 SNPS. Many of these SNPs were completely correlated so we removed many redundant SNPs from further genotyping.
- SNP5PDM361545 SNP41
- the two most significant SNPs, SNP45 and SNP41, are within 6 kb of the microsatellite marker AC008818-1, and the at-risk alleles of all three genetic markers are in strong linkage disequilibrium withD' > 0.9 and p-value nearly zero.
- the square of the correlation (R 2 ) is very high between the two SNPs ( ⁇ 0.93), but is substantially lower (- 0.08) between each SNP and the at-risk allele of the microsatellite. This is due to the fact that the frequency of the at-risk alleles of the two SNPs are similar, and much more frequent than that for the at-risk allele of the microsatellite.
- the LD block structure around the 5' end of PDE4D is displayed in FIG. 13.1. We delineate three blocks A, B and C encompassing the first three exons ⁇ PDE4D and its immediate downstream region.
- Exons D7-3 and D7-2 are both in block A, while D7-1 (the first exon) is in block B, but close to its border with block C. Given this block structure we were prepared to investigate the haplotype associated susceptibility to stroke in this region. Table 2D. All SNPs that show association with a p-value less than 0.01 for all stroke patients, all patients excluding hemorrhagic stroke and the combined cardiogenic and carotid stroke.
- haplotype analysis involves using likelihood-based inference applied to NEsted MOdels.
- the method is implemented in our program NEMO, which allows for many polymorphic markers, SNPs and microsatellites.
- the method and software are specifically designed for case-control studies where the purpose is to identify haplotype groups that confer different risks. It is also a tool for studying LD structures.
- haplotypes When investigating haplotypes constructed from many markers, apart from looking at each haplotype individually, meaningful summaries often require putting haplotypes into groups.
- a particular partition of the haplotype space is a model that assumes haplotypes within a group have the same risk, while haplotypes in different groups can have different risks.
- Two models/partitions are nested when one, the alternative model, is a finer partition compared to the other, the null model, i.e, the alternative model allows some haplotypes assumed to have the same risk in the null model to have different risks.
- the models are nested in the classical sense that the null model is a special case of the alternative model. Hence traditional generalized likelihood ratio tests can be used to test the null model against the alternative model.
- haplotypes hi and h j are assumed to have the same risk, it corresponds to assuming that fi/pi ⁇ f/pj where/and y? denote haplotype frequencies in the affected population and the control population respectively.
- One common way to handle uncertainty in phase and missing genotypes is a two-step method of first estimating haplotype counts and then treating the estimated counts as the exact comits, a method that can sometimes be problematic (e.g., see the information measure section below) and may require randomization to properly evaluate statistical significance, hi NEMO, maximum likelihood estimates, likelihood ratios and p-values are calculated directly, with the aid of the EM algorithm, for the observed data treating it as a missing-data problem.
- NEMO allows complete flexibility for partitions. For example, the first haplotype problem described in the Methods section on Statistical analysis considers testing whether h ⁇ has the same risk as the other haplotypes fa, ..., fa.
- the alternative grouping is [h ⁇ ], [h 2 , ..., h k ] and the null grouping is [fa, ..., fa].
- the alternative grouping is [fa], [fa], [fa] and the null grouping is [fa, fa], [fa]-
- the actual problem we faced in FIG. 11.1 is actually slightly more complicated because allele X is a composite allele that includes five alleles other than allele 0, and hence GX and AX each correspond to five haplotypes.
- GX should be understood as the composite of all haplotypes including the G nucleotide of SNP45 except for the GO haplotype.
- A0 haplotype does not exist. This suggests that allele 0 originated in a haplotype background with allele G of SNP45, and since then no recombination has occurred between those two markers for chromosomes that carried allele 0.
- AX, GO and GX have significantly distinct risks for the combined carotid and cardiogenic stroke phenotype.
- GX as the wild type because it is the most common (53.4% in controls) and also because it has the intermediate level risk that is not too different from the population risk.
- the haplotype GO has increased risk and AX is protective, with risks of 1.46 and 0.70 relative to the wild type, respectively.
- the GO risk is 2.07 times that of the protective haplotype AX.
- Each of the three pairwise comparisons is highly significant, with p- values ranging from 0.006 to 7.2x10 "8 . It is interesting to observe that even though both AX and GX are composite haplotypes, the AX haplotype can be simply summarized by the allele A of SNP45, since the A0 haplotype does not exist. For a similar reason, the GO haplotype is completely determined by the 0 allele of
- AC008818-1 Also displayed in FIG. 11.1 is the information content (Info) of each test.
- Info is a measure of the information that is lost due to the uncertainty with phase and missing genotypes.
- Info is very close to 1 for each of the three tests in FIG. 11.1. That is a result of SNP45 and AC008818-1 being in very strong LD.
- tests presented later in FIG. 11.2 and 11.3, involving longer haplotypes have lower information content.
- Block A 300 kb
- Block B 200 kb
- block C 60 kb
- All haplotypes within each block with an estimated frequency in the population of 2% or greater have been identified.
- a brief schematic of the identified haplotypes are displayed in FIG. 13.2 and the risks and frequencies of these haplotypes are available in Table 3.
- haplotypes within block A no common haplotype has greater risk than SNP87 alone. The strongest signals were for haplotypes in block B and C. Each block contained a haplotype significantly associated with the combination of carotid and cardiogenic stroke and having relative risk around 1.5.
- the common at-risk haplotype in block B is the SNP background of the GO haplotype previously identified.
- the extended at-risk haplotype GOHc (8.8% in controls) and protective composite haplotype AXXc (21.1% in controls), have, respectively, relative risks of 1.98 and 0.68 compared to the wild type (70.1% in controls).
- the at-risk haplotype GOHc spans a region of about 64kb. While it is possible that the increased risk is due to multiple polymorphisms over that region, the results are also consistent with a relatively recent mutation, as yet to be identified, which occurred in that haplotype background, and since then no recombination has occurred in that extended region for chromosomes carrying the mutation.
- the protective composite haplotype AXXc can be simply represented by allele A of SNP45.
- allele A of SNP45 is the functional protective variant, although it is possible that the functional variant is simply in strong LD with allele A of SNP45 and has yet to be identified. Indeed, statistically, the effects of SNP45 and SNP41 are indistinguishable from each other. Statistical analysis.
- relative risk and the population attributable risk (PAR) were calculated assuming a multiplicative model (haplotype relative risk model), (Terwilliger, J.D. & Ott, J., Hum Hered, 42, 337-46 (1992) and Falk, CT. & Rubinstein, P, Ann Hum Genet 51 ( Pt 3), 227-33 (1987)), i.e., that the risks of the two alleles/haplotypes a person carries multiply.
- haplotypes are independent, i.e., in Hardy- Weinberg equilibrium, within the affected population as well as within the control population.
- haplotype counts of the affecteds and controls each have multinomial distributions, but with different haplotype frequencies under the alternative hypothesis.
- haplotype frequencies are estimated by maximum likelihood and tests of differences between cases and controls are performed using a generalized likelihood ratio test (Rice, J.A. Mathematical Statistics and Data Analysis, 602 (International Thomson Publishing, (1995)).
- NEMO haplotype analysis program
- A 2 [£(r,p 1 , ⁇ 2 , ... k- ⁇ ) - e(l.P ⁇ , h. -'- k- ⁇ ) ]
- 2 denotes log e likelihood
- ⁇ and ⁇ denote maximum likelihood estimates under the null hypothesis and alternative hypothesis respectively.
- ⁇ has asymptotically a chi-square distribution with 1-df, under the null hypothesis and it was used to compute p-values presented in Table 3.
- the tests presented in FIG. 11 have slightly more complicated null and alternative hypotheses. For the results in FIG. 11, let fa be GO, fa be GX and fa be AX.
- X>' and R 2 were extended to include microsatellites by averaging over the values for all possible allele combination of the two markers weighted by the marginal allele probabilities.
- all marker combination When plotting all marker combination to elucidate the LD structure in a particular region, we plot X>' in the upper left corner and the p-value in the lower right corner. In the LD plots we present the markers are plotted equidistant rather than according to their physical location.
- Table 3 Haplotype diversity at the 5'end of the PDE4D gene.
- the SNP haplotype is more specific in the sense that it has a higher relative risk, i.e., 2.3. This haplotype is carried by 47% of the patients and has the same population attributable risk (PAR) of 0.25.
- PAR population attributable risk
- microsatellite markers are as follows:
- this single SNP haplotype (which is only one haplotype of the several found above but is probably the most tightly associated to stroke) more than doubles an individual's risk for cardiogenic and carotid stroke and accounts for 25% of such strokes in Iceland.
- the other haplotypes described above provide additional risk for stroke.
- the magnitude of this risk haplotype is comparable or higher than the well-known clinical risk factors for stroke such as hypertension, diabetes, hyperlipidemia, and smoking.
- Table 5 and Table 6 show previously known microsatellite markers and novel microsatellites in sequence. Forward and reverse primers are shown.
- the magnitude of the risk and the frequency of the disease haplotypes in the general population confirm that we have mapped a gene for the common forms of stroke and not some rare form of stroke. This gene almost doubles one's risk for stroke in . ' general, and more than doubles one's risk for the two most common subtypes of stroke, carotid and cardiogenic stroke.
- the most common disease haplotype has. a population attributed risk of 25% (which means it accounts for 25% of the patients) and there are other haplotypes that we describe herein that are less common that accounts for other patients.
- PDE4D is a major cause of stroke and its relative risk rivals those of hypertension, smoking, diabetes, and hyperlipidemia.
- PDE4D shows tighter correlation to the forms of stroke dependent on atherosclerosis (carotid and cardiogenic stroke) and it is expressed in cell types known to be important for atherosclerosis such as vascular smooth muscle cells, macrophages, and endothelial cells. This suggests that the strong effect that PDE4D variation has on stroke risk is through its role in the vascular biology of atherosclerosis (see discussion at the end of the examples).
- Example 2 details our sequencing of the entire PDE4D gene and the definition of its exon-intron structure based on new and old cDNAs, and Example 3 shows that the expression pattern of PDE4D isoforms correlates with a stroke associated haplotype.
- EXAMPLE 2 SEQUENCING AND CHARACTERIZATION OF THE HUMAN GENE AND ITS RNA PROTEIN ISOFORMS
- BACs (bacterial artificial clones ⁇ (RP11-164A5, RP11-188I15, RP11-313P15, RP11-631M6, RP11-103A15, RP11- 489L13, RP11-621C19, RP11-113C1, RP11-567M18, RP11-412M9, RP11-151G2, RP11-151F7, RP11-281M3, RP11-421L6, RP11-1A7, RP11-68E13, RP11-379P8, and RP11-422K3) covering the minimum tiling path of the one LOD interval were analysed using shotgun cloning and sequencing.
- Dye terminator (ABI PRISM BigDye) chemistry was used for fluorescent automated DNA sequencing.
- ABI prism 377 sequences were used to collect data and the Phred/Phrap/Consed software package in combination with the Polyphred software were used to assemble sequences (See Table 9A and 9B)
- Publicly available sequences AC008836, AC073546, AC021603, AC008498, AC016435, AC021601, AC016591, AC008818, AC008879, AC008934, AC011929, AC027322, AC008111, AC020924, AC026693, AC012315, AC08804, AC008791, AC020975, AC008833, AC008829, AC022125, AC008790, AC026095, AC066693, AC008852, AC016642, AC034250, AC025179, AC08814, AC008926, AC010391, AC016635 and AC016604) from this region were
- the BAC clones we sequenced are from the RCPI-11 Human BAC library (Pieter deJong, Roswell Park).
- the vector used was pBACe3.6.
- the clones were picked into a 94 well microtiter plate containing LB/chloramphenicol (25 ⁇ g/ml)/glycerol (7.5%o) and stored at -80°C after a single colony has been positively identified through sequencing.
- the clones can then be streaked out on a LB agar plate with the appropriate antibiotic, chloramphenicol (25 ⁇ g/ml)/sucrose (5%).
- the gene, human cAMP specific phosphodiesterase 4D was identified in the sequenced region by BLAST of our novel genomic sequence with the cDNAs/EST databases from GenBank.
- RT-PCR reactions and 5 prime and 3 prime RACE reactions using cDNA libraries generated from a variety of tissues including human aorta.
- the primer sites used corresponded to known or exons predicted from our genomic sequence using Genscan, and Fgene.
- the genomic sequence covering all known and novel exons in PDE4D so far is approximately 1,550,000 bases in length.
- the PDE4D gene contains 22 exons over at least 1.5 Mb and overlaps with the PARTI gene whose transcript is on the other strand at the 5' end.
- the PDE4D gene has at least 7 promoters and encodes 8 protein isoforms. All isoforms have an identical C-terminal catalytic domain but differ at the N-terminal regulatory domain. Six of the 8 forms are so called long isoforms. Each of them have unique N- terminal regulatory domains but they are all characterized by two highly conserved regions found in all PDE4 subfamilies, i.e. upstream conserved regions 1 and 2 (UCR 1 and 2).
- the six long forms differ from each other by unique alternative 5 prime exons which predicts six alternative promoters that are each upstream of the corresponding 5 prime exon.
- the remaining two are the so-called short forms, variants that lack the UCR 1 (Houslay, M.D. & Adams, D.R., Biochem J, 370, 1-18 (2003)).
- the five previously known isoforms are encoded by 17 exons distributed over a segment of 0.9 Mb.
- the three new exons D7A-1 , D7A-2 and D7A-3 are spliced to one another and together splice onto exon LF1 forming the splice variant we named PDE4D7 (FIG. 3).
- Exon D7-1 is non-coding.
- Exons D8 and D9 are spliced by themselves onto exon LF1 forming two splice variants we named PDE4D8 and PDE4D9, respectively (FIG. 3).
- the D7A exon extends the 5' end of PDE4D by 590,000 bp, and the D8 and D9 exons lie between exons D3 and LFl(physical position of exons presented in Table 2C).
- the new PDE4D7 isoform has an open reading frame extending into LF1, resulting in additional 91 amino acids at the N-terminus of the predicted protein.
- the D8 and D9 5 ' exons contain a long 5 ' UTR, followed by an ATG near the end of the exons that extends an ORF into LF1 resulting in a novel N-terminal segments of 22 and 30 amino acids in the PDE4D8 and PDE4D9 predicted proteins, respectively.
- the new splice variants were verified by RT-PCR on different cDNA tissue panels and subsequent cloning and sequencing of the products.
- the PDE4D gene encodes at least eight different isoforms. Six of the eight forms are the so-called long isoforms.
- accession numbers AF536975 isoform named PDE4D6
- AF536976 isoform named PDE4D7
- AF536977 isoform named PDE4D8
- the sequence AF536977 corresponds to our earlier reported PDE4D6 isoform
- AF536976 corresponds partly to our earlier reported PDE4D7 isoform, however the first untranslated exon we named D7-1 is missing from this sequence.
- the sequence AF536975 is a new short PDE4D isoform.
- PDE4D6 is now called PDE4D8
- PDE4D7 is now called PDE4D7
- PDE4D8 is now called PDE4D9.
- GenBank accesion numbers AY245866 and AY245867, respectively.
- Primers designed from these exons were used in conjunction with primers from LF1 and exon3 for RT PCR in the hope of identifying novel exons.
- Novel exons were in turn used to design primers for various RT-PCR reactions.
- 5 'RACE primers designed from the known exons upstream of LF1.
- We have to date identified 14 new exons, including exons belonging to UniGene Cluster Hs.343602 that have now been connected to LF1.
- For the 5 ' RACE reactions we used cDNA made from heart, SkNAS (neuroblastoma cell line) and HVAEnd 5050 (endothelial cell line).
- SkNAS neuroroblastoma cell line
- HVAEnd 5050 endothelial cell line
- RT-PCR reactions a number of cDNAs made of total RNA were used (see below)
- TCGCTGTTGCTAGTTCCAG (SEQ ID NO: 94) >4D7-7
- CAATCCCCCATGGATACCAAG (SEQ ID NO: 96)
- CAAAAGA SEQ ID NO: 98
- RT4D7 4D7-1 + 4D7-2 + 4D7-3 + LF1
- RT4 4D7-1 + 4D7-2 + 4D7-7 + 4D7-3 + 4D9-1 + 4D9-3.1
- RT5 4D7- 1 + 4D7-2 + 4D7-3 + 4D9-2 + 4D9-3.1
- RT9 4D7-1 + 4D7-6 + 4D7-2
- RT10 4D9-1 + 4D9-3.1 + LF1
- Exon 4D9-3 contains stop codons in all 3 reading frames and Variants RT10, 11,12,13, and 14 having their ATG initiation codon inside LFl . It is not clear whether variants RT4 and RT5, which contain exon 4D9-3 extend to LFl or have their 3' at 4D9-3 (the latter possibility is supported by the EST data).
- exons contain Alu repetitive element sequences: 4D7-5 and 4D7- C.
- the gene specific reverse (3 ') primer was designed for PDE4D exon LFl (5' GGCAATGGAGGAGTTCCGGGACA TA-3 '; SEQ ID NO: 87 origin from Homo sapiens).
- a contig for the incomplete genomic sequence of the PDE4D gene was submitted by others in November 2000 (GenBank entry NT_023193 by International Human Genome Project collaborators).
- the size of the contig is 614 481 bp (including gaps) whereas our novel genomic sequence for the whole PDE4D region (i.e., from the first exon for PDE4D variant) is close to 1,690,000 bp and contains no gaps.
- the contig NT_023193 comprises only 11 exons of the PDE4D gene (in FIG. 3, exons 4D1/D2 - 11) and the 5' differently spliced exons are missing in the contig (in FIG. 3, exons D4, D5, D3, D8, D9, D7A-1, D7A-2, D7A-3, LFl, LF2, LF3 and LF4).
- D7A-1 ATAGTTGGCGTACCCTGAGGCCTGCCAGTTCCTGCCTTAATGCATATGTAGT CGTAATTGAGTTCTGACACGGCCTTGGATGTTTCTGTCCTAAATAGCTGACA TTGCATCTTCAAGACTGT
- D7A-2 CATTCCAGTTGGCTTTTGAGTGGATACGTGCAGTGAGATCATTGACACTGGA AACACTAGTTCCCATTTTAATTACTTAAAACACCACGATGAAAAGAAATACC TGTGATTTGCTTTCTCGGAGCAAAAGT
- D7A-3 GCCTCTGAGGAAACACTACATTCCAGTAATGAAGAGGAAGACCCTTTCCGC GGAATGGAACCCTATCTTGTCCGGAGACTTTCATGTCGCAATATTCAGCTTC CCCCTCTCGCCTTCAGACAGTTGGAACAAGCTGACTTGAAAAGTGAATCAGA GAACATTCAACGACCAACCAGCCTCCCCCTGAAGATTCTGCCGCTGATTGCT ATCACTTCTGCAGAATCCAGTGG (SEQ ID NO: 11; includes D7A-1, D7A-2 and D7A-3)
- Example 2 Here we present the first complete genomic sequence of human PDE4D, two novel mRNA protein isoforms of PDE4D and their corresponding exons, and the intron- exon structure of known and novel isoforms.
- the basis for phosphodiesterases is the mammalian homolog of the "dunce" gene in Drosophila melanogaster, implicated in learning and memory (Davis, R.L. and B. Dauwalder, Trends Genet, 7(7):224-229 (1991)).
- PDEs are members of a large superfamily of isoenzymes subdivided into 9 and possibly 10 distinct families (Conti, M. and S.L. Jin, Prog. Nucleic Acid Res. Mol.
- PDE4A a gene that encodes the type 5 PDEs
- PDE4B a gene that encodes the type 5 PDEs
- PDE4C a group of enzymes characterized by high affinity for cAMP.
- the gene for PDE4D was assigned to human chromosome 5ql2 (Milatovich, A., et al,
- the pattern of splicing and different promoter usage is highly conserved during evolution indicating an important physiological role (Nemoz, G., et al, FEBS Lett, 384(1):97 -102 (1996)).
- the PDE4D variants are generated at two major boundaries present in the gene. The first boundary corresponds to the junction of exon 2. Differential splicing in this region generates the 2 short variants PDE4D1 (586 a.a.) and PDE4D2 (508 a.a.) (FIG. 3). This splicing boundary is conserved in mouse, rat and between different human PDE4 genes.
- the splicing variant PDE4D2 is generated by the removal of 256 bp from the PDE4D1 sequence.
- the initiation codon in the PDE4D2 variant lies within exon D1/D2.
- Data demonstrates that the expression of the short PDE4D variants is under the control of an internal promoter regulated by cAMP (Nicini, E. and M. Conti, Mol. Endocrinol, 11 (7):839-850 (1997)).
- the second major splicing boundary is also conserved during evolution and is identical to that described in the Drosophila dunce gene. Splicing occurs at the intron/exon boundary at the LFl exon (FIG. 3).
- the PDEs serve at least four major functions in the cell. They can (1) act as effector of signal transduction by interacting with receptors and G-proteins; (2) integrate ⁇ the cyclic nucleotide-dependent pathway with other signal transduction pathways; (3) function as homeostatic regulators, playing a role in feedback mechanisms controlling cyclic nucleotide levels during hormone and neurotransmitter stimulation; (4) play an important role in controlling the diffusion of cyclic nucleotides and in creating subcellular domains or channeling cyclic nucleotide signaling (Conti, M. and S.L. Jin, Prog. Nucleic Acid Res. Mol Biol, 63: 1-38.(1999)). Inhibition of PDE has long been recognized as an effective pharmacological strategy to alter intracellular cyclic nucleotide levels (Flamm, E.S., et al, Arch. Neurol, 32(8):569-71 (1975)).
- PDE4 is the predominant isozyme regulating vascular tone mediated by cAMP hydrolysis in cerebral vessels (Willette, R. ⁇ ., et al, J. Cereb. Blood Flow Metab., 17(2):210-9 (1997)).
- a recent study on mice with targeted disruption of PDE4D gene has demonstrated a crucial role of PDE4D in the control of smooth muscle contraction and muscarinic cholinergic receptor signaling but not in the control of airway inflammation.
- the lung phenotype of the PDE4D-/- mice demonstrates that this gene plays a nonredundant role in cAMP homeostasis.
- PDE4D serves a unique, nonoverlapping functions in cell signalling.
- No clear link between an established inherited disorder and known PDE loci has emerged, with the exception of PDE6.
- Inhibitors of PDEs have been shown to affect airway responsiveness and pulmonary allergic inflammation (Schudt, C, et al, Pulm.
- vasorelaxation modulated by PDE4 (not mentioned whether it is A, B, C or D gene family) is compromised in chronic cerebral vasospasm associated with subarachnoid hemorrhage (Willette, R.N., et al, J. Cereb. Blood Flow Metab., 17(2):210-9 (1997)). PDE4D itself has not been linked to stroke before.
- PDE4Ds are expressed in human peripheral mononuclear cells (Nemoz, G., et al, FEBS Lett, 384(1):97 -102 (1996)), brain (Bolger, G., et al, Mol. Cell Biol, 13(10):6558-71 (1993)), heart (Kostic, M.M., et al, J. Mol. Cell Cardiol, 29(11):3135- 46 (1997)) and vascular smooth muscle cells (Liu, H. and D.H. Maurice, J Biol. Chem., 274(15):10557-65 (1999)).
- the haplotype was constructed out of the at-risk allele for the microsatellite marker AC008818-1 and SNP45 (SNP5PDM357221) and SNP41 (SNP5PDM361545).
- This haplotype acts as a surrogate for the disease-associated haplotype we have identified in LD block B (Table 3).
- Patients with the haplotype had a significantly decreased expression of the PDE4D7 and PDE4D9 isoforms (FIG. 5).
- Several other isoforms of PDE4D were expressed but did not show correlation to the disease haplotype.
- the PDE4D7 correlation was also present in controls but only marginally significant (FIG. 6). Of interest, this at-risk haplotype covers the 5' exon specific to PDE4D7 and presumably its promoter.
- GAPDH "Assay- On-Demand" was obtained from Applied Biosystems and used as a housekeeping gene. PDE assays were tested and optimized for 384 well high throughput expression analysis using ABI 7900 Instrument. A final concentration of 200 nM probes, 900 nM primers and 2 ng/mcl cDNA was used in a lOmcl reaction volume. Each plate was run twice and an average for each sample calculated. ABI7900 instrument was used to calculate CT (Threshold Cycle) values. Samples displaying a greater than 1 deltaCT between duplicates were not used in our analysis. Quantity was obtained using the formula 2 " ⁇ CT where ⁇ CT represents the difference of CT values between target and housekeeping assay.
- This haplotype is present in 47% of the carotid/cardiogenic stroke patients, compared to 21% in the control group with more than two-fold stroke risk for the carriers of this haplotype. It has a population attributed risk of 25%.
- haplotypes covering the first exon of PDE4D can be classified into three groups with clearly distinct risks. Relative to the protective group, the population attributed risk of the at-risk and wild type groups combined is estimated to be 55%. Approximately 16% of the population carries one copy of the at-risk haplotype in FIG. 12.3.
- the disease-associated haplotype extends over the 5'exon unique to the new PDE4D7 variant and the presumed promoter region of this isoform suggesting that the functional variation may be involved in transcriptional regulation. This hypothesis is also supported by our PDE4D expression analysis that shows significant correlation between the disease associated haplotype and the level of PDE4D7 message.
- cardiac thrombi which shed emboli to the brain most commonly occur on the background of coronary artery disease, such as following acute myocardial infarction or ischemic cardiomyopathy, and/or due to atrial fibrillation on the basis of poor compliance of ischemic ventricles (diastolic dysfunction/stiffening).
- atrial fibrillation may occur on the background of other diseases such as valvular disease, hyperthyroidism, and hypertension, in the age group that tends to suffer from stroke, ischemic heart disease remains one of the most important causes.
- Ischemic stroke resulting from occlusion of small penetrating arteries within the brain is generally thought to result from endothelial proliferation since atherosclerosis only occurs in larger arteries.
- PDE4D does not show association to small vessel stroke, consistent with its role in atherosclerosis.
- Carotid and cardiogenic stroke together account for the majority of .
- ischemic stroke note that our number for carotid is lower since we used a more stringent cutoff of stenosis).
- PDE4D selectively degrades second messenger cAMP (Kong, A. et al, Nat
- vascular smooth muscle cells vascular smooth muscle cells
- monocytes monocytes
- macrophages T-lymphocytes
- VSCM vascular smooth muscle cells
- monocytes monocytes
- macrophages monocytes
- T-lymphocytes T-lymphocytes
- Cyclic AMP is a key signalling-molecule in these cells (Landells, L.J. et al, Br J Pharmacol 133, 722-9 (2001); Fukumoto, S. et al, CircRes 85, 985-91. (1999); Ogawa, S. et /., Am J Physiol 262, C546-54 (1992)).
- PDE4 PDE4
- cAMP In monocytes and T-lymphocytes, accumulation of cAMP is generally associated with inhibition of immune functions such as proliferation and cytokine secretion (Indolfi, C. et al, J Am Coll Cardiol 36, 288-93. (2000)). It is attractive to postulate that the regulation of cAMP through absolute or relative expression of one or more PDE4D isoforms may differ in individuals susceptible to stroke; some stioke patients may have increased PDE4D activity and, consequently lower cAMP levels in any of the above cell types, leading to development of the atherosclerotic plaque and/or its instability. However, contiary to what one might expect we see decreased expression in some of the PDE4D isoforms in EBV cell lines from stroke patients.
- association analyses single marker and haplotype analyses
- PDE4D single marker and haplotype analyses
- this gene is involved in the pathogenesis of stroke through atherosclerosis.
- PDE4D is expressed in cell types important in atherosclerosis and regulates a second messenger with a central role to processes important in the pathogenesis of atherosclerosis.
- Inhibition of PDE4D in general or specifically one or more isoforms, by a small molecule drug or other pharmacological agent might decrease the risk of stroke in general, and especially those who are predisposed to stroke through variation in the PDE4D gene.
Abstract
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US10/419,723 US20040014099A1 (en) | 2001-03-19 | 2003-04-18 | Susceptibility gene for human stroke; methods of treatment |
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CN109298133B (en) * | 2018-07-18 | 2021-07-13 | 重庆邮电大学 | Detector module production yield improvement method based on edge channel correction |
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WO2000023091A2 (en) * | 1998-10-15 | 2000-04-27 | Bioimage A/S | Specific therapeutic interventions obtained by interference with redistribution and/or targeting of cyclic nucleotide phosphodiesterases of i-kappa-b kinases |
WO2000040714A2 (en) * | 1998-12-30 | 2000-07-13 | Oligos Etc. Inc. | Therapeutic phosphodiesterase inhibitors |
WO2001000851A1 (en) * | 1999-06-25 | 2001-01-04 | Memory Pharmaceutical Corporation | Cyclic amp phosphodiesterase isoforms and methods of use |
WO2002074992A2 (en) * | 2001-03-19 | 2002-09-26 | Decode Genetics Ehf. | Phosphodiesterase 4d genes related to human stroke |
-
2003
- 2003-09-25 AU AU2003278877A patent/AU2003278877A1/en not_active Abandoned
- 2003-09-25 CA CA002499320A patent/CA2499320A1/en not_active Abandoned
- 2003-09-25 EP EP03770392A patent/EP1552012A4/en not_active Withdrawn
- 2003-09-25 JP JP2004540173A patent/JP2006500068A/en active Pending
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WO2000023091A2 (en) * | 1998-10-15 | 2000-04-27 | Bioimage A/S | Specific therapeutic interventions obtained by interference with redistribution and/or targeting of cyclic nucleotide phosphodiesterases of i-kappa-b kinases |
WO2000040714A2 (en) * | 1998-12-30 | 2000-07-13 | Oligos Etc. Inc. | Therapeutic phosphodiesterase inhibitors |
WO2001000851A1 (en) * | 1999-06-25 | 2001-01-04 | Memory Pharmaceutical Corporation | Cyclic amp phosphodiesterase isoforms and methods of use |
WO2002074992A2 (en) * | 2001-03-19 | 2002-09-26 | Decode Genetics Ehf. | Phosphodiesterase 4d genes related to human stroke |
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GRAEME B BOLGER ET AL: "Characterization of five different proteins produced by alternatively spliced mRNAs from the human cAMP-specific phosphodiesterase PDE4D gene" BIOCHEMICAL JOURNAL, PORTLAND PRESS, LONDON, GB, vol. 328, 1997, pages 539-548, XP002150449 ISSN: 0264-6021 * |
GRETARSDOTTIR SOLVEIG ET AL: "The gene encoding phosphodiesterase 4D confers risk of ischemic stroke." NATURE GENETICS, vol. 35, no. 2, October 2003 (2003-10), pages 131-138, XP002376110 ISSN: 1061-4036 * |
GRETARSDOTTIR SOLVEIG ET AL: "Localization of a susceptibility gene for common forms of stroke to 5q12" AMERICAN JOURNAL OF HUMAN GENETICS, vol. 70, no. 3, March 2002 (2002-03), pages 593-603, XP002376109 ISSN: 0002-9297 * |
KOSTIC M M ET AL: "Altered expression of PDE1 and PDE4 cyclic nucleotide phosphodiesterase isoforms in 7-oxo-prostacyclin-preconditioned rat heart" JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY, vol. 29, no. 11, November 1997 (1997-11), pages 3135-3146, XP002278927 ISSN: 0022-2828 * |
PEREZ-TORRES S ET AL: "Phosphodiesterase type 4 isozymes expression in human brain examined by in situ hybridization histochemistry and (3H)rolipram binding autoradiography comparison with monkey and rat brain" JOURNAL OF CHEMICAL NEUROANATOMY, CHICHESTER, GB, vol. 20, no. 3-4, December 2000 (2000-12), pages 349-374, XP002275597 ISSN: 0891-0618 * |
See also references of WO2004028341A2 * |
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AU2003278877A8 (en) | 2004-04-19 |
CA2499320A1 (en) | 2004-04-08 |
JP2006500068A (en) | 2006-01-05 |
EP1552012A4 (en) | 2006-06-07 |
AU2003278877A1 (en) | 2004-04-19 |
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