Classifications

• • 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
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
  • G16B20/40—Population genetics; Linkage disequilibrium
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations

Definitions

• This invention relates to the use of a digital computer to analyze a database. More specifically, the database contains medical information. Over recent years, much progress has been made in mapping and sequencing the human genome, and it is to be expected that the full coding sequence of nearly every human gene will be known within a few years. However, determining the function of newly identified genes, or, conversely, identifying phenotypes associated with genes, has proceeded more slowly. Elucidation of function can be particularly difficult in situations in which a single gene contributes to several phenotypes and/or where multiple genes contribute to a single phenotype. Such situations may prove to be the norm rather than the exception.
• genes correlating with multiple phenotypes include ApoE (heart disease and Alzheimer's disease), the AT gene (ataxis, telangiectasias, radiation sensitivity, leukemia, breast cancer and diabetes); the brcal gene (breast and ovarian cancer); Huntington's gene (movement disorder, dementia and psychosis); and the leptin receptor (diabetes, obesity).
• ApoE heart disease and Alzheimer's disease
• the AT gene ataxis, telangiectasias, radiation sensitivity, leukemia, breast cancer and diabetes
• the brcal gene breast cancer
• Huntington's gene moving disorder, dementia and psychosis
• leptin receptor diabetes, obesity
• diseases correlated with multiple genes include heart disease, Alzheimer's disease, hypertension, diabetes, and obesity.
• the above approach has, in some instances, been extended to look for correlations with one or more additional phenotypes.
• the approach in identifying a correlation with a second phenotype has been very similar to that for the first phenotype. That is, a further population of individuals is identified that have the second phenotype. This population typically has entirely different individuals from the population having the first phenotype.
• C3 human complement C3
• the complement system contains a number of serum proteins that play a major role in immunologically-mediated inflammation (McLean and Winklestein (1984) J. Pediatr. 105:179-188).
• C3 is an acute phase reactant active in both the classic and alternative complement pathway. Its synthe ⁇ sis is increased during acute inflammation. Its fragments (C3a and C3b) have anaphylatoxic, chemotactic and histaminic actions and affect smooth muscle function.
• C3F C3F
• Activation of the complement system is usually beneficial to the host, as in resistance to infections, but may also lead to cytolysis of "normal" cells, as in autoimmune disorders.
• C3F C3F
• Sorensen and co-workers have investigated an association between the C3F allele and three disease phenotypes; multiple sclerosis, atherosclerosis and hypertension (Jans & Sorenson (1980) Acta Neurol. Scandinav. 63:237-342; Sorensen & Dissing (1975) Human Heredity 25:279-283; Schaadt et al. (1981) Clin. Sci. 61:363-365).
• populations having and lacking the respective disease symptoms were identified, and the individuals in the populations were tested for the presence of C3F and C3S alleles.
• C3F has also been reported to be increased in a variety of less common disorders such as bronchial asthma (Srivastava et al. (1985) Hum. Hered. 35:263-264), chronic renal failure (Reguerio and Arnaiz-Villena (1984) Hum. Genet. 67:437-440), renal allograft dysfunction (Andrews et al. (1995) Transplantation 60:1342-1346), Crohn's Disease (Elmgreen et al. (1984) Acta. Med. Scand. 215:375-378), hepatitis (Farhud et al.
• this invention provides a method performed in a programmable digital computer.
• the method comprises providing a database having, for each member of a subject population; a) a first value set specifying at least one polymorphic form at at least one genetic locus exhibiting polymorphism, and b) a second value set specifying a plurality of phenotypes, wherein at least one of said polymorphic forms is not known to have a statistically significant correlation with at least one of said phenotypes; and determining the statistical correlation between the at least one polymorphic form and the plurality of phenotypes.
• the step of providing the database comprises providing a nucleic acid sample for each member and determining the at least one polymorphic form from the nucleic acid samples.
• the database is, preferably, a relational database.
• the invention provides the above-described database.
• the invention provides a kit comprising a database having, for each member of a subject population, a value set specifying a plurality of phenotypes, and a DNA sample from each member of the subject population.
• the invention provides a method for determiiiing the degree of risk that a subject has or will develop a phenotype or syndrome.
• the method involves determining whether the subject has a polymorphic form shown by the method of this invention to have a statistically significant correlation with the phenotype or syndrome. Having the polymorphic form indicates the positive or negative risk of developing the phenotype or syndrome.
• the invention provides a method of determining whether a human subject is at increased risk of allergy to foods, thyroid disease, osteoporosis, arthritis and/or heart disease comprising determining whether the subject has a C3F allele, whereby the presence of the allele indicates that the subject is at increased risk.
• the invention provides a kit comprising at least one nucleic acid probe capable of detecting in a subject a polymorphic form identified by the method of this invention as having a significant positive statistical correlation with a phenotype or syndrome, and instructions indicating that the presence of the polymorphic form indicates that the subject is at positive risk of developing the phenotype or syndrome.
• this invention provides a kit comprising at least one • nucleic acid probe for distinguishing between C3F and C3S alleles; and instructions indicating that the C3F allele confers susceptibility to allergy to foods, thyroid disease, osteoporosis, arthritis and/or heart disease.
• the kit comprises two probes capable of acting as primers for amplification of genomic DNA around nucleotide 364 of exon 3 of the C3F gene allele.
• this invention provides a method of treating a subject having allergy to foods, thyroid disease, osteoporosis, arthritis and/or heart disease associated widi the C3F syndrome, comprising administering an ⁇ nmunosuppressive agent or an anti-histamine to the subject.
• this invention provides a kit comprising an immunosuppressive agent or an anti-histamine and instructions for use of the immunosuppressive agent or anti-histamine the treatment of allergy to foods, thyroid disease, osteoporosis, arthritis and/or heart disease associated with C3F syndrome.
• this invention provides a method for screening a compound for the ability to inhibit C3F protein. The method involves contacting a C3F protein with the compound; and determining whether the compound inhibits C3F- mediated complement activity. A compound that inhibits the activity of C3F is a candidate drug in the treatment of C3F syndrome.
• this invention provides a computer program product for analyzing a database, in particular a relational database, comprising: code that receives as input a database having, for each member of a subject population; a) a first value set specifying at least one polymorphic form at least one genetic locus exhibiting polymorphism, and b) a second value set specifying a plurality of phenotypes, wherein at least one of said polymorphic forms is not known to have a statistically significant correlation with at least one of said phenotypes; code that determines the statistical correlation between the at least one polymorphic form and the plurality of phenotypes; and a computer readable medium that stores the codes.
• the program product further comprises code that displays results of the statistical analyses.
• the computer program product further comprises code that displays results of the statistical analyses.
• Fig. 1 illustrates an example of a computer system used to execute Lhe software of the present invention.
• Fig. 1 shows a computer system 1 which includes a monitor 3, screen 5, cabinet 7, keyboard 9, and mouse 11.
• Mouse 11 may have one or more buttons such as mouse buttons 13.
• Cabinet 7 houses a CD-ROM drive 15 and a hard drive (not shown) that may be utilized to store and retrieve computer programs including code incorporating the present invention.
• a CD-ROM 17 is shown as the computer readable storage medium, other computer readable storage media including floppy disks, DRAM, hard drives, flash memory, tape, and the like may be utilized.
• Cabinet 7 also houses familiar computer components (not shown) such as a processor, memory, and the like.
• Fig. 2 shows a system block diagram of computer system 1 used to execute the software of the present invention.
• computer system 1 includes monitor 3 and keyboard 9.
• Computer system 1 further includes subsystems such as a central processor 102, system memory 104, I/O controller 106, display adapter 108, removable disk 112, fixed disk 116, network interface 118, and speaker 120.
• Removable disk 112 is representative of removable computer readable media like floppies, tape, CD-ROM, removable hard drive, flash memory, and the like.
• Fixed disk 116 is representative of an internal hard drive, DRAM, or the like.
• Other computer systems suitable for use with the present invention may include additional or fewer subsystems.
• another computer system could include more than one processor 102 (i.e. , a multi-processor system) or memory cache.
• Arrows such as 122 represent the system bus architecture of computer system 1. However, these arrows are illustrative of any interconnection scheme serving to link the subsystems.
• display adapter 108 may be connected to central processor 102 through a local bus or the system may include a memory cache.
• Computer system 1 shown in Fig. 2 is but an example of a computer system suitable for use with the present invention. Other configurations of subsystems suitable for use with me present invention will be readily apparent to one of ordinary skill in the art.
• the computer system is a work station from, e.g. , Sun Microsystems.
• Fig. 3 presents a flow chart for performing the analyses of this invention.
• polymorphic form refers to one of the forms of a polymorphism existing at a genetic locus in a population of organisms. At the molecular level, when two homologous segments of genetic material have different nucleotide sequences, the segments exhibit polymorphism and each different sequence is a polymorphic form.
• An example of polymorphic forms is the different sequences for a gene that encode polypeptides of different amino acids sequences, or functional RNA molecules having different sequences. The differences may reflect nucleotide substitutions, insertions or deletions between the different sequences.
• polymorphic forms are a variable number of tandem repeats (VNTR's), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats and tetranucleotide repeats.
• Polymorphic forms can be identified by different genetic markers such as restriction patterns for DNA, e.g., restriction fragment length polymorphisms ("RFLPs"). Polymorphic forms also manifest as different mendelian alleles for a gene.
• a polymorphic form can be identified within a sub-group of a population sharing a common characteristic, e.g., sex (male or female), classification (e.g. , primates).
• phenotype refers to any distinguishable trait of an organism.
• a phenotype can be a physical trait or a mental (e.g., emotional) trait.
• Physical traits include both normal (non-disease) physical variants and disease states.
• Disease states include, for example, general medical conditions (e.g., heart disease, cancer, autoimmune diseases, inflammatory disorders, diabetes), neurological conditions (e.g. , Alzheimer's disease, migraine) and psychiatric conditions (e.g., anxiety, depression).
• Non-disease phenotypes include, for example, hair color, left- or right-handedness, height and baldness. Phenotypes used in a database typically include several conditions selected from these different groupings.
• Phenotypes are usually defined by conventional criteria (e.g. , Diagnostic and Statistical 8 Manual), but can be defined by customized criteria depending on the intended use of the database.
• the value that specifies the phenotype may be binary, i.e. , an individual does or does not have the phenotype.
• the value may be sealer, such as height, weight, or blood pressure, or a selected specific trait, such as eye color.
• the data on the phenotypes are collected by conducting a thorough medical work-up and history of the subjects in the population. This can involve identifying all past and present illnesses and complaints, both mental and physical, as well as physiological tests on a variety of systems, including, for example, blood work-ups, cardiovascular tests, etc.
• the term "statistical correlation" refers to a statistical association between two values as measured by any statistical test including, for example, chi-squared analysis, ANOVA or multivariate analysis.
• the correlation between a polymorphic form and a phenotype is considered statistically significant at a P value of ⁇ 0.05, and, preferably ⁇ 0.01 or ⁇ 0.001.
• the term "syndrome” refers to a collection of phenotypes having a statistically significant correlation with a polymo ⁇ hic form.
• the polymo ⁇ hic form is said to be "associated" with the phenotypes.
• subject refers, preferably, to humans, but also to mammals and other animals, other multicellular organisms such as plants, single celled organisms or viruses.
• nucleic acid probe refers to a nucleic acid molecule which binds to a specific sequence or sub-sequence of another nucleic acid molecule.
• a probe is preferably a nucleic acid molecule which binds through complementary base pairing to me full sequence or to a sub-sequence of a target nucleic acid. It will be understood by one of skill in the art that probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions.
• the probes are preferably directly labelled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labelled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or sub-sequence.
• a “label” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
• useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, dioxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available (e.g., by inco ⁇ orating a radio-label into the peptide, and used to detect antibodies specifically reactive with the peptide).
• a label often generates a measurable signal, such as radioactivity, fluorescent light or enzyme activity, which can be used to quantitate the amount of bound label.
• a “labeled nucleic acid probe” is a nucleic acid probe that is bound, either covalently, through a linker, or through ionic, van der Waals or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe.
• code refers to human-readable source code, which is the instructions written by the programmer in a programming language, as well to executable machine code, which is the instructions of a program that are converted from source code to instructions that the computer can understand.
• relational database means a database that stores information in tables - rows and columns of data - and conducts searches by using data in specified columns of one table to find additional data in another table.
• the invention provides novel memods of using a programmable digital computer for determining the statistical correlation between a polymo ⁇ hic form of interest and each of multiple phenotypes. Correlations are achieved through the provision of a database, which stores a matrix of information.
• the database is created from a subject population. Each individual in the population is tested for the presence of a plurality of phenotypes.
• the database indicates whether each individual has or lacks each of the phenotypes.
• the database is a relational database.
• the number of individuals in the population is selected so as to yield a statistically meaningful measure of the correlation between the genotypic polymo ⁇ hism and the phenotypes to be tested. This number, in turn, depends on the frequency of the polymo ⁇ hic form in the population, the frequency of the phenotype in the population, and the power of the statistical test being used for make the correlation. For example, if die chi-squared test is used for two polymo ⁇ hic forms, each at a frequency of about 50%, and two phenotypes, each at a frequency of about 50%, then each analysis cell " needs about five data points, or twenty data points in all, for meaningful statistical analysis.
• a polymo ⁇ hic form is present at a frequency greater than about 10%, most statistical analyses will require a population of at least about 200 members. If the polymo ⁇ hic form is found in about 1 % to 10% of the population, the test population needs to have at least about 1000 members. If the polymo ⁇ hic form is found in about 0.1 % to 1 % of the population, the test population in the database requires at least about 10,000 members. Thus, the population can have at least 50, at least 100, at least 200, at least 500, at least 1000 at least 5000 or at least 10,000 members. The population of individuals to be the subject of the database is often selected without prior knowledge or testing of the phenotypes of any individual. For example, the population can be selected at random.
• the population can be selected at random with respect to the phenotypic traits. Alternatively, the population can be selected to reflect a diversity of phenotypes. Also, the population can be directed with respect to a particular phenotype, for example, 50% with the trait and 50% without.
• populations comprise unrelated individuals. For example, no two individuals can be from the same nuclear family (i.e., two parents and children of those parents.) In other databases, some individuals may be from the same family.
• the database also indicates, for each individual, the polymo ⁇ hic form at at least one genetic locus exhibiting polymo ⁇ hism and the identity of multiple phenotypes.
• the polymo ⁇ hic form typically is determined from DNA testing, although polymo ⁇ hic forms determined from mendelian analysis also are useful.
• DNA testing for each individual included in the database, a sample of DNA, usually genomic DNA, is obtained. This sample is often obtained at about the same time that phenotypes are being determined for that individual. Typically, the DNA is placed in storage until a later time when a candidate genotypic marker for investigation becomes available.
• the invention provides a database including value sets specifying a plurality of phenotypes for each subject of the population and a DNA sample for each member of the population indexed to identify the subject from whom it came. Often, one locus is analyzed at which two polymo ⁇ hic forms have been identified. However, if there are many polymo ⁇ hic forms at a locus, several can be tested. Optionally, polymo ⁇ hic forms from several loci can be tested.
• the polymo ⁇ hic form occurs within a gene or putative gene of unknown function, for example, a newly sequenced open reading frame.
• Lhe polymo ⁇ hic form may represent a variation preventing expression of a functional gene product.
• Other times the genotypic marker occurs within a gene that has been correlated with certain phenotypes, but for which it is believed that correlations with additional phenotypes can be found.
• the polymo ⁇ hic form has no known statistically significant correlation with a disease phenotype.
• the genotypic marker is not itself within a gene or non- coding sequence of any significance, but is linked to such a sequence.
• the human genome project is detecting many genes whose function is unknown.
• the polymo ⁇ hic form is for a gene of unknown function.
• the polymo ⁇ hic form for each subject in the population at the locus is determined. Naturally, heterozygotes will have two polymo ⁇ hisms. The polymo ⁇ hic forms are added to the existing database.
• the computer is programmed to perform an analysis of the database to determine the statistical correlation between a polymo ⁇ hic form at at least one locus and at least two phenotypes.
• the chosen polymo ⁇ hic form typically does not have a known statistical correlation with at least one of the phenotypes tested.
• Statistical correlation is typically determined by the chi-squared method.
• the computer can specifically identify correlations that are statistically significant. Both negative and positive statistically significant correlations are of interest. Different phenotypes can be ranked by the strength of correlation.
• a typical database usually contains information on at least about 10, 20,
• the number of subjects in me population is at least 100 and the number of phenotypes analyzed is at least 20.
• Some of the phenotypes included in the database are known or 457
• the phenotypes selected each involve a plurality of tissue types or organ systems, e.g., nervous system, circulatory system, digestive system, connective tissue, etc.
• the population is selected independently of the phenotypes, i.e., without regard for the presence or absence of any particular phenotypes.
• genotypic markers can be correlated with phenotypes by a simple extension of the above approach. For example, if mutations A and B occur at different places within the same gene, one might analyze whether the presence of A or B correlates with disease. If mutations A and B occur in different genes, which are suspected to have a similar function, one might analyze whether the presence of A and B shows stronger correlation with disease phenotype than A or B alone. Large numbers of polymo ⁇ hic forms (e.g., 5, 10, 20 or 100) can be analyzed in different combinations in Lhis manner.
• Lhis invention provides a computer program product for analyzing a database that includes code that receives as input the database code mat receives as input at least one selected polymo ⁇ hic form; code that performs statistical analyses on the selected polymo ⁇ hic form and phenotypes in the database; and a computer readable medium that stores the codes.
• the computer program product further includes code that displays results of the statistical analyses.
• the computer program product further includes code that displays results of the statistical analyses. It may, for example, select and display all statistically significant correlations.
• the database may be stored in the hard drive or a floppy disk and moved into the computer memory as needed to perform the analysis.
• the product can include code that receives as input instructions from a programmer, e.g. , for identifying polymo ⁇ hic forms or phenotypes to be correlated, or the form or contents of the display.
• the relational database has separate tables wim value sets specifying general medical history, presence or absence of migraine, psychiatric history, subject identifiers, and personal information.
• the tables are linked by a record number which also acts as an index.
• the result of this analysis is a list in which the degree of correlation between one or more polymo ⁇ hic forms and a plurality of phenotypes is determined. This information is valuable in a number of ways.
• a statistically significant positive or negative correlation between a polymo ⁇ hic form and a disease phenotype indicates that with respect to the general population, a subject has a positive or negative risk of developing the disease. In general, me greater the statistical correlation, the greater the positive or negative risk of developing the disease. Accordingly, this invention provides methods of determining whether a person is at positive or negative risk of developing a disorder, trait, or syndrome. The method involves selecting a polymo ⁇ hic form identified by the method of this invention to have a statistically significant correlation with a phenotype and determining whether the subject has the polymo ⁇ hic form.
• phenotypic forms When the method uncovers a statistically significant correlation between one or more phenotypic forms and several phenotypes, these phenotypes constitute a syndrome of traits Lhat are associated with each other. Identification of syndromes is useful designing modes of prophylaxis and treatment, and in uncovering the common source of the syndrome.
• the subject or his or her treating physician can be informed if the subject has a polymo ⁇ hic form positively correlated with a phenotype or wim a syndrome. Patients possessing such a polymo ⁇ hic form can often be counselled as to the risk of contracting the syndrome and about relatively benign prophylactic measures to take before the onset of disease. For example, Lhe risk of developing heart disease, and cancer can be reduced by changes in diet and life style.
• a further benefit of the analysis is that it identifies genes that should be given priority for further analysis. That is, genes containing genotypic markers showing strong correlations with common human diseases. The functions of these genes can be analyzed in vitro or in transgenic animal models, and compounds can be identified that antagonize or agonize the function of gene products. 7 6457
• C3F syndrome refers to disease state in which a person exhibits several or all of the disorders associated with C3F and has a C3F allele. Persons at positive risk of developing C3F syndrome can be identified by determining whether the person has the C3F allele. The person can be informed that they have the allele and that they are at increased risk of contracting one or more of the associated diseases.
• the persons also can be counseled on prophylactic measures to take to decrease the risk of contracting an associated disease.
• me subject can be counseled to change diet.
• an immunosuppressive agent can be administered to the subject.
• the C3F allele can be detected genetically by, for example, isolating genomic DNA from the subject and detecting a variation at nucleotide 364 in exon 3 of a C3 gene. A method for doing this is described in M. Botto et al. (1990), supra. Briefly, Lhe C3F and C3S variants differ in Lhe presence of an Hhal site at nucleotide 364 of exon 3.
• Primers based on the nucleotide sequence of the gene are used to amplify a stretch of genomic DNA including Lhis site. Exposure to Hhal will or will not cleave Lhe amplified fragment, depending upon whether or not the restriction site is present. The cleaved and uncleaved fragments are detectable by, for example, agarose gel electrophoresis.
• kits of this invention include nucleic acid probes for detecting in a subject a polymo ⁇ hic form identified by the memods of this invention as having a significant positive statistical correlation with a phenotype or syndrome, in particular with a disease phenotype, and instructions indicating that Lhe presence of the polymo ⁇ hic form indicates that the subject is at positive risk of developing the phenotype or syndrome.
• the probe will be labeled and will be useful for hybridizing with a restriction fragment on, for example, a Soumern blot, thereby identifying the polymo ⁇ hic form.
• the kit contains at least one nucleic acid probe for detecting the C3F allele.
• Such probes can, for example, amplify genomic DNA around nucleotide 364 of exon 3. Examples of such primers are Ex3 (SEQ ID NO:l) and Ex2 (SEQ ID NO:2).
• C3F syndrome is treatable prophylactically or therapeutically by administering immunosuppressive drugs and/or anti-histamines.
• this invention provides a kit comprising an immunosuppressive agent and/or an antihistamine and instructions to use the drug in me treatment of allergy to foods, thyroid disease, osteoporosis, arthritis and/ or heart disease, disorders of C3F syndrome associated for the first time with C3F by determining their statistical correlation using a database as described herein.
• This invention also provides methods of screening for an agent effective to treat C3F syndrome.
• the methods comprise contacting a C3F protein with a compound and determining whether the compound inhibits C3F-mediated complement activity. Inhibition indicates that the compound is a candidate as a drug to treat the syndrome.
• the C3F protein is contacted with me compound in a mixture containing cells, an antibody that specifically binds to the cells, and complement proteins Cl, C2, and C4-C9. Complement activity is determined from lysis of the cells.
• A. Clinical Genetic Relational Database Individuals were recruited for participation in a long term clinical genetic study of common medical disorders. All individuals were interviewed by a physician, nurse and/or trained clinical investigator, and all clinical interviews were reviewed by a physician. Subjects were evaluated using a semi-structured interview which included questions about demographics, personal medical history, family medical history, a detailed review of systems, medication use, and healthcare utilization. Informed consent was obtained and DNA samples collected. A random sample of 200 DNA samples from unrelated Caucasians, who were 35 years of age or older, was selected for analysis from a pre-established clinical genetic database. All clinical data were obtained independently of the genotypic data.
• Genotyping of C3 using the Hhal RFLP polymo ⁇ hism was performed in the study participants. Genomic DNA was isolated using the Puregene DNA isolation kit (Gentra Systems, Research Triangle Park, North Carolina). Genomic DNA was amplified and restriction enzyme digested as described by M. Botto et al. (1990) J. Exp. Med. 172: 1011-1017. Briefly, 1 ⁇ g of genomic DNA was amplified using 25 pM of primer Ex3 (5' ATCCCAGCCA ACAGGGAG 3' (SEQ ID NO: l)) and Ex4 (5' TAGCAGCTTG TGGTTGAC 3' (SEQ ID NO:2)), corresponding to positions 328-345 and complementary to nucleotides 514-531 30 , respectively.
• Ex3 ATCCCAGCCA ACAGGGAG 3' (SEQ ID NO: l)
• Ex4 5' TAGCAGCTTG TGGTTGAC 3' (SEQ ID NO:2)
• the amplification was performed in 25 ⁇ L of a buffer containing 10 mM Tris, pH 8.3, 1.5 mM MgCl 2 , 50 mM KCl, 200 ⁇ M each dNTP and 1 U Taq polymerase (Perkin Elmer, Foster City, CA).
• the reaction was initially denatured at 95°C for 4 minutes, followed by 30 cycles of amplification with denaturation at 95°C for 1 minute, annealing at 56°C for 1 minute, extension at 72°C for 1 minute and a final extension at 72°C for 10 minutes.
• 10 U of Hhal New England Biolabs, Beverly, MA
• the products were separated on a 1.5% agarose SFR gel (Amresco, Solon, OH). Analysis of the polymo ⁇ hic form was performed by two individuals blinded to the clinical status.
• the frequency of the C3F allele was then analyzed in a variety of common clinical disorders by screening a clinical genetic database. As shown in Table 1, a number of disorders are significantly more frequent in C3F positive individuals. Food allergies, arthritis, coronary heart disease (CHD), hypertension with CHD, osteoporosis, and thyroid disease are significantly more frequent in the C3F positive individuals man in the C3F negative individuals. Hypertension is increased in the C3F positive group but the increase does not reach statistical significance.
• Food allergies (defined as symptoms of immediate type hypersensitivity reactions to shellfish, vegetables or fruit) were reported by 42 of the 200 individuals in Lhe present smdy. Urticaria and angioedema were reported by 11 individuals following shellfish ingestion and by 31 individuals following fruit or vegetable ingestion.
• CHD defined as angiographically documented coronary artery disease
• a similar pattern is observed with hypertension. Hypertension is reported by 60 of the individuals in the present study to have been diagnosed by their personal physician.
• the difference in hypertension frequency between the C3F positive and C3F negative groups does not reach statistical significance.
• Osteoporosis was reported by 5 of Lhe 200 individuals in the present study. All 5 individuals had been diagnosed independently by their personal physician and were being treated for osteoporosis at the time of the interview. 4 of the 5 individuals wim osteoporosis are C3F positive.
• hypothyroidism was reported by 27 of the 200 individuals in the present study. All 27 individuals had been diagnosed independently by their personal physician and 25 had been treated with thyroid replacement.
• the C3F gene frequency in the thyroid disease group is 0.28, a frequency which is greater than the C3F frequency (0.19) in subjects without thyroid disease.
• the correlations discovered in this invention together with previous data support the hypothesis that activation of the complement system in C3F positive individuals, in comparison to C3F negative individuals, can lead to an increase in endomelial damage and secondary arteriosclerosis.
• the method of this invention identified a number of novel clinical associations. For example, a highly significant increase in food allergies were reported by the C3F positive vs. C3F negative individuals. Food allergy is an immunologically mediated response to an ingested food antigen. Symptoms may consist of nausea, vomiting, diarrhea, urticaria, angioedema, bronchospasm and/or rhinitis. Reactions to food antigens are more pronounced and/ or more common in C3F positive individuals compared to C3F negative individuals.
• C3F positive individuals evidence for tissue damage secondary to immune system hyperactivity in C3F positive individuals is also consistent with the present observation that an increased frequency of thyroid disease and osteoporosis is present in C3F positive individuals.
• Chronic inflammatory thyroid disease or Hashimoto's Disease
• Osteoporosis is a common disorder of elderly women in which mast cells and / 457
• C3F has been reported to be increased in a wide variety of less common disorders (Table 2).
• the diseases in which the C3F frequency has been found to be increased are similar in that the immune system and/or inflammation is believed to play a prominent role in the pathophysiology of each disorder.
• the underlying molecular mechanism that might clinically link these various disorders has yet to be elucidated.
• tissue injury secondary to a functional hyperactivity of the complement system in C3F positive individuals could be the molecular mechanism that underlies all of these otherwise unre ⁇ lated disorders.
• the increased efficacy of C3F following an immunological challenge may lead to individually subtle, but cumulatively significant, increases in vascular lesions over the course of many years.
• C3F in comparison to C3S, may lead to either increased local membrane damage or increased permeability of the endothelium following activation of the complement system. This conclusion is consistent wim the present data which indicate that the clinical morbidity resulting from the C3F allele occurs over the course of many years.
• Thyroid disease 19% 10% 0.03
• Coronary heart disease 13% 6% 0.04
• Atherosclerotic vascular disease .264 1.6 0.0005 Amerosclerotic vascular disease .272 1.6 0.0005 Bronchial asthma .059 3.3 0.001 Indian childhood cirrhosis .149 12 0.001 Essential hypertension .264 1.9 0.003 Chronic polyarthritis .252 1.3 0.01
• the present invention provides a novel method for determining the correlation between polymo ⁇ hic forms and phenotypes. While specific examples have been provided, the above description is illustrative and not restrictive. Many variations of the invention will become apparent to those of skill in the art upon review of this specification. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents.

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