Patexia. Research
Patent No. US 08114592
Issue Date Feb 14, 2012
Claim this patent

Patent 08114592 - Genetic markers associated with age-related macular degeneration, methods of detection and uses thereof > Description

Description

CROSS-REFERENCE PARAGRAPH

This application claims the benefit of U.S. Provisional Application No. 61/037,411 filed on Mar. 18, 2008, the entire content of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the area of genetic testing, drug discovery, and Age-Related Macular Degeneration. In particular, it relates to genetic variants found within the complement cascade C3 gene which increase the risk of Age-Related Macular Degeneration.

BACKGROUND OF THE INVENTION

Age-related macular degeneration (AMD) causes progressive impairment of central vision and is the leading cause of irreversible vision loss in older Americans(1). The most severe form of AMD involves neovascular/exudative (wet) and/or atrophic (dry) changes to the macula. Although the etiology of AMD remains largely unknown, implicated risk factors include age, ethnicity, smoking, hypertension, obesity and diet (2). Familial aggregation (3), twin studies (4), and segregation analysis(5) suggest that there is also a significant genetic contribution to the disease. The candidate gene approach and genome-wide association studies have consistently implicated the CFH, ARMS2 and C2/BF genes, all members of the complement-mediated inflammatory cascade.

Age-related macular degeneration (AMD) is a common complex disorder that affects the central region of the retina (macula) and is the leading cause of legal blindness in older American adults. The prevalence of AMD and its significant morbidity will rise sharply as the population ages. AMD is a clinically heterogeneous disorder with a poorly understood etiology. Population-based longitudinal studies(6-8) have established that the presence of extracellular protein/lipid deposits (drusen) between the basal lamina of the retinal pigment epithelium (RPE) and the inner layer of Bruchs' membrane is associated with an increased risk of progressing to an advanced form of AMD, either geographic atrophy or exudative disease. The presence of large and indistinct (soft) drusen coupled with RPE abnormalities is considered an early form of the disorder and is often referred to as age-related maculopathy (ARM).

Epidemiology: AMD is a complex disorder with contributions of environmental factors as well as genetic susceptibility(2). Many environmental and lifestyle factors have been postulated, but by far the most consistently implicated non-genetic risk factor for AMD is cigarette smoking (6). Much progress has been made in identifying and characterizing the genetic basis of AMD. In a remarkable example of the convergence of methods for disease gene discovery, multiple independent research efforts identified the Y402H variant in the complement factor H(CFH [(MIM 134370]) gene on chromosome 1q32 as the first major AMD susceptibility allele (9-14). While one of the studies was able to pinpoint CFH on the basis of a whole-genome association study (11), most studies focused on the 1q32 region because it had consistently been implicated by several whole-genome linkage scans. Disease associated haplotypes within the CFH gene are also associated with AMD (15). A second genomic region with similarly consistent linkage evidence is chromosome 10q26, which was identified as the single most promising region by a recent meta-analysis of published linkage screens (16).

Two studies have suggested specific AMD susceptibility genes located on chromosome 10q26. One used a combination of family-based and case-control analyses to implicate the PLEKHA1 gene (pleckstrin homology domain containing, family A (phosphoinositide binding specific) member 1 [MIM 607772]) and the predicted ARMS2 gene (14;17;18). ARMS2 appears to be a mitochondrial membrane protein involved in inflammation (19) A second study using two independent case-control datasets concluded that the T allele of SNP rs10490924 in ARMS2, a coding change (Ala69Ser) in exon 1 of this gene, was the most likely AMD susceptibility allele (16). Both studies reported that the chromosome 10q26 variant confers an AMD risk similar in magnitude to that of the Y402H variant in CFH. A locus with less strong association, but reproducible association with AMD is the complement component 2 (C2) and Factor B (C2/BF) locus within the major histocompatability complex III locus found on chromosome 6 The L9H variant of BF and the E318D variant of C2, as well as a variant in intron 10 of C2 and the R32Q variant of BF, confer a significantly reduced risk of AMD (20).

SUMMARY OF THE INVENTION

Here, we describe highly significant association of SNPs within the C3 gene (NCBI GeneID: 718), specifically rs2230199 (Arg102Gly) found on chromosome 19 with age related macular degeneration and its use, alone or in combination, in predicting predisposition to this disease (21). We have thus established that identification of the nucleotide residue at rs2230199 can predict the predisposition of an individual to AMD. Related findings have since been published by Maller et al. (22).

According to some embodiments of the invention, a method is provided for assessing increased risk of Age Related Macular Degeneration. The identity is determined of at least one nucleotide residue of the genomic germ-line C3 coding sequence of an individual The nucleotide residue is identified as normal or variant by comparing it to a normal genomic germ-line sequence of C3 coding sequence as shown in SEQ ID NO:1 (coding sequence) or SEQ ID NO: 3 (genomic sequence). A normal nucleotide residue is identical to the corresponding nucleotide residue in the normal genomic germ-line sequence of C3. A variant nucleotide residue is not identical to the corresponding nucleotide residue in the normal genomic germ-line sequence of C3. A variant C3 coding sequence may contain at least one variant nucleotide residue relative to the normal C3 coding sequence. An individual with a variant sequence has a higher risk of Age Related Macular Degeneration than an individual with a normal sequence.

According to some embodiments, a method is provided for assessing increased risk of Age Related Macular Degeneration. The identity is determined of at least one amino acid residue of the C3 protein of an individual. The at least one amino acid residue is identified as normal or variant by comparing it to a normal sequence of the C3 protein as shown in SEQ ID NO: 2. A person with a variant sequence has a higher risk of Age Related Macular Degeneration than a person with a normal sequence.

Further embodiments of the invention provide a method to assess risk of AMD in an individual. The presence of a G or C allele at the single nucleotide polymorphism (SNP)rs 2230199 within the genomic sequence is determined in an individual. The person is identified as being at high risk of AMD if the patient has one or two copies of the G allele on the negative genomic strand at this SNP (or conversely one or two copies of the C allele on the positive genomic strand) in relation to the March 2006 human reference sequence (NCBI Build 36.1). The SNP rs2230199 is found in the first position of codon 102 (corresponding to position 366 in the C3 coding sequence of SEQ ID NO: 1 or 304 nucleotides downstream of the start of the initiation codon). SNP rs2230199 is located at position 6669387 on human chromosome 19 ((NCBI Build 36.1). The G allele changes the amino acid specified from arginine to glycine. The patient is identified as being at lower risk of AMD if the patient does not have one or two copies of the G allele at rs2230199.

Further embodiments provide a method for assessing increased risk of Age Related Macular Degeneration. The identity of the residue at position 102 of the pro-C3 protein sequence or position 80 of the mature C3 protein sequence is determined in an individual. The residue is identified as normal or variant by comparing it to a normal sequence of the pro-C3 protein or C3 protein as shown in SEQ ID NO: 2. An individual with a variant sequence has a higher risk of Age Related Macular Degeneration than an individual with a normal sequence. For example, an individual with Gly at position 102 has a higher risk of Age Related Macular Degeneration than an individual with Arg at position 102.

While not being bound by any theory, this marker, or one in linkage disequilibrium, may change the composition, function or abundance of the elements of cellular constituents resulting in a predisposition to age related macular degeneration. Measuring this marker in individuals who do not ostensibly have age related macular degeneration may identify those at heightened risk for the subsequent development of age related macular degeneration, providing benefit for, but not limited to, individuals, insurers, care givers and employers. Information obtained from the detection of SNPs associated with age related macular degeneration is of great value in the treatment and prevention of this condition.

In the context of this invention, a marker is said to be in “linkage disequilibrium” with the residue at rs2230199 when the correlation coefficient (r2) between the marker and rs2230199 is >0.5 (23).

Further scope of the applicability of the present invention will become apparent from the detailed description provided below. It should be understood however, that the following detailed description and examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modification within the spirit and scope of the invention will become apparent to those skilled in the art from the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have discovered that polymorphic variants in the C3 gene, which is shown in sequences, SEQ ID NOs: 1-3 are associated with an altered risk of developing age related macular degeneration in subjects. The present invention thus provides a SNP associated with age related macular degeneration, nucleic acid molecules containing the SNP, methods and reagents for the detection of the SNP disclosed herein, uses of this SNP for the development of detection reagents, and assays or kits that utilize such reagents. The age related macular degeneration-associated SNP disclosed herein may be useful for diagnosing, screening for, and evaluating predisposition to age related macular degeneration and related pathologies in humans.

The age related macular degeneration-associated SNP has been identified by genotyping DNA from 1548 individuals, 847 of these individuals having been previously diagnosed with age related macular degeneration and 701 being “control” or individuals thought to be free of age related macular degeneration.

Aspects of the present invention thus provides an individual SNP associated with age related macular degeneration, genomic sequences (SEQ ID NO: 3) containing SNPs, transcript sequences (SEQ ID NO: 1) and amino acid sequences (SEQ ID NO: 2). Aspects of the invention include methods of detecting these polymorphisms in a test sample, methods of determining the risk of an individual of having or developing age related macular degeneration, methods of using the disclosed SNPs to select a treatment strategy, and methods of using the SNPs of the present invention for human identification.

When the presence in the genome of an individual of a particular base, e.g., guanine, at a particular location in the genome (e.g. the SNP rs2230199) correlates with an increased probability of that individual contracting age related macular degeneration vis-à-vis a population not having that base at that location in the genome, that individual is said to be at “increased risk” of contracting age related macular degeneration, i.e., to have an increased susceptibility. In the present case, such increased probability exists when the base is present in one or the other or both alleles of the individual. Furthermore, the probability is increased when the base is present in both alleles of the individual rather than one allele of the individual.

When the presence in the genome of an individual of a particular base, e.g., cytosine, at a particular location in the genome (e.g. the SNP rs2230199) decreases the probability of that individual contracting age related macular degeneration vis-à-vis a population not having that base at that location in the genome, that individual is said to be at “decreased risk” of contracting age related macular degeneration, i.e., to have a decreased susceptibility. Such an allele is sometimes referred to in the art as being “protective”. As with increased risk, it is also possible for a decreased risk to be characterized as dominant or recessive.

An “altered risk” means either an increased or a decreased risk.

The genetic analysis detailed below linked age related macular degeneration with a SNP in the human genome. A SNP is a particular type of polymorphic site, a polymorphic site being a region in a nucleic acid sequence at which two or more alternative nucleotides are observed in a significant number of individuals from a population. A polymorphic site may be a nucleotide sequence of two or more nucleotides, an inserted nucleotide or nucleotide sequence, a deleted nucleotide or nucleotide sequence, or a microsatellite, for example. A polymorphic site that is two or more nucleotides in length may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, 20 or more, 30 or more, 50 or more, 75 or more, 100 or more, 500 or more, or about 1000 nucleotides in length, where all or some of the nucleotide sequences differ within the region. The specific polymorphic site found in the genomic sequences identified as SEQ ID NOs: 1 and 3 is a “single nucleotide polymorphism” or a “SNP” i.e. a polymorphic site which is one nucleotide in length.

Where there are two, three, or four alternative nucleotide sequences at a polymorphic site, each nucleotide sequence is referred to as a “polymorphic variant” or “nucleic acid variant.” Where two polymorphic variants exist, for example, the polymorphic variant represented in a majority of samples from a population is sometimes referred to as a “prevalent allele” and the polymorphic variant that is less prevalently represented is sometimes referred to as an “uncommon allele.” An individual who possesses two prevalent alleles or two uncommon alleles is “homozygous” with respect to the polymorphism, and an individual who possesses one prevalent allele and one uncommon allele is “heterozygous” with respect to the polymorphism. Individuals who are homozygous with respect to one allele are sometimes predisposed to a different phenotype as compared to individuals who are heterozygous or homozygous with respect to another allele.

A genotype or polymorphic variant may also be expressed in terms of a “haplotype,” which refers to the identity of two or more polymorphic variants occurring within genomic DNA on the same strand of DNA. For example, two SNPs may exist within a gene where each SNP position may include a cytosine variation or an adenine variation. Certain individuals in a population may carry an allele (heterozygous) or two alleles (homozygous) having the gene with a cytosine at each SNP position. As the two cytosines corresponding to each SNP in the gene travel together on one or both alleles in these individuals, the individuals can be characterized as having a cytosine/cytosine haplotype with respect to the two SNPs in the gene.

A “phenotype” is a trait which can be compared between individuals, such as presence or absence of a condition, for example, occurrence of age related macular degeneration.

Polymorphic variants are often reported without any determination of whether the variant is represented in a significant fraction of a population. Some reported variants are sequencing errors and/or not biologically relevant. Thus, it is often not known whether a reported polymorphic variant is statistically significant or biologically relevant until the presence of the variant is detected in a population of individuals and the frequency of the variant is determined.

A polymorphic variant may be detected on either or both strands of a double-stranded nucleic acid. Also, a polymorphic variant may be located within an intron or exon of a gene or within a portion of a regulatory region such as a promoter, a 5′ untranslated region (UTR), a 3′ UTR, and in DNA (e.g., genomic DNA (gDNA) and complementary DNA (cDNA)), RNA (e.g., mRNA, tRNA, and rRNA), or a polypeptide. Polymorphic variations may or may not result in detectable differences in gene expression, polypeptide structure, or polypeptide function.

In our genetic analysis associating age related macular degeneration with the polymorphic variants set forth in Table 1, samples from individuals diagnosed with age related macular degeneration and individuals not having age related macular degeneration were allelotyped and genotyped. The allele frequency for each polymorphic variant among cases and controls was determined. These allele frequencies were compared in cases and controls, or combinations. Particular SNPs were thus found to be associated with age related macular degeneration when genotype and haplotype frequency differences calculated between case and control pools were established to be statistically significant.

As mentioned above, polymorphic variants can travel together. Such variants are said to be in “linkage disequilibrium” so that heritable elements e.g., alleles that have a tendency to be inherited together instead of being inherited independently by random assortment are in linkage disequilibrium. Alleles are randomly assorted or inherited independently of each other if the frequency of the two alleles together is the product of the frequencies of the two alleles individually. For example, if two alleles at different polymorphic sites are present in 50% of the chromosomes in a population, then they would be said to assort randomly if the two alleles are present together on 25% of the chromosomes in the population. A higher percentage would mean that the two alleles are linked. For example, a first polymorphic site P1 having two alleles, e.g. A and C—each appearing in 50% of the individuals in a given population, is said to be in linkage disequilibrium with a second polymorphic site P2 having two alleles e.g. G and T—each appearing in 50% of the individuals in a given population, if particular combinations of alleles are observed in individuals at a frequency greater than 25% (if the polymorphic sites are not linked, then one would expect a 50% chance of an individual having A at P1 and a 50% chance of having G at P2 thus leading to a 25% chance of having the combination of A at P1 and G at P2 together). Heritable elements that are in linkage disequilibrium are said to be “linked” or “genetically linked” to each other.

One can see that in the case of a group of SNPs that are in linkage disequilibrium with each other, knowledge of the existence of all such SNPs in a particular individual generally provides redundant information. Thus, when identifying an individual who has an altered risk for developing age related macular degeneration according to this invention, it is necessary to detect only one SNP of such a group of SNPs associated with an altered risk of developing age related macular degeneration.

The data set out below shows that one or more SNPs in the C3 genomic sequences identified herein as SEQ ID NOs: 1 and 3 are associated with the occurrence of age related macular degeneration. Thus, featured herein are methods for identifying a risk of age related macular degeneration in a subject, which includes detecting the presence or absence of a polymorphic variant at one or more of the SNPs described herein in a human nucleic acid sample. For example, the presence or absence of a polymorphic variant at rs2230199 (e.g. the G allele) may be detected in a human nucleic acid sample.

Three different analyses were performed for each marker and significant results reported below as follows: (a) a test of trend across the 3 genotypes(24), (b) a dominant model where the homozygous genotype for allele “B” is combined with the prevalent heterozygote genotype; and (c) a recessive model where the homozygous genotype for allele “A” is combined with the heterozygous genotype. An empirical p-value for the largest of these three test statistics was calculated by permutations. In addition, a Mantel-Haenszel odds ratio measuring the change in risk associated with each additional copy of allele B is also calculated and reported.

Pertinent results for the SNP are summarized in Table 1: Chromosomal number and position- using the International Human Genome Sequencing Consortium build 35 (http://www.ncbi.nlm.nih.gov/genome/seq/) as made available by the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Bethesda, Md. 20894 U.S.A., gene marker name-using the nomenclature of the NCBI dbSNP (URLf[colon][slash][slash]www[dot]ncbi[dot]nlm[dot]nih[dot]gov[slash]SNP[slash]) and gene name-using the unigene naming convention. Under the “Case Flag” the number 1 designates Cases and the number 0 designates Controls. The identity of the base designated “A” in the analysis is indicated where 1=A (adenine), 2=C (cytosine), 3=G (guanine) and 4=T (thymidine). “B” indicates the polymorphic allele. AA, AB, BB are the counts of the number of individuals with the given genotype, by cases/controls. The odds ratio is the Mantel-Haenszel odds ratio across the three genotypes.

It has been discovered that polymorphic variation at SNPs in the C3 genomic sequences which are identified herein as SEQ ID NOs: 1 or 3 is associated with the occurrence of age related macular degeneration. Thus, featured herein are methods for identifying a risk of age related macular degeneration in a subject, which comprises detecting the presence or absence of one or more of the polymorphic variations described herein in a human nucleic acid sample. The polymorphic variations and SNPs are detailed in the table. In some embodiments, the presence of a polymorphic variant at rs2230199 is indicative of an altered risk of age related macular degeneration. For example, the presence of the uncommon G allele at rs2230199 may be indicative of an increased risk of age related macular degeneration, relative to individuals with the prevalent C allele at rs2230199.

Methods for determining whether a subject is at risk of age related macular degeneration are provided herein. These methods include detecting the presence or absence of one or more polymorphic variations at SNPs which are associated with age related macular degeneration, in a sample from a subject.

SNPs may be associated with a disease state such as AMD, in humans or in animals. The association can be direct, as in conditions where the substitution of a base results in alteration of the protein coding sequence of a gene which contributes directly to the pathophysiology of the condition. Common examples of this include diseases such as sickle cell anemia and cystic fibrosis. The association can be indirect when the SNP plays no role in the disease, but is located close to the defective gene such that there is a strong association between the presence of the SNP and the disease state. Because of the high frequency of SNPs within the genome, there is a greater probability that a SNP will be linked to a genetic locus of interest than other types of genetic markers.

Disease-associated SNPs may occur in coding and non-coding regions of the genome. When located in the coding region altered function of the ensuing protein sequence may occur. For example, polymorphic variation at SNP rs2230199 may alter the amino acid residue at position 102 of the C3 pro-protein. If it occurs in the regulatory region of a gene it may affect expression of the protein. If the protein is involved in protecting the body against pathological conditions this can result in disease susceptibility.

Numerous methods exist for the measurement of specific SNP genotypes. Individuals carrying mutations in one or more SNPs of the present invention may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and autopsy material.

The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR prior to analysis (25). RNA or cDNA may also be used in the same ways. As an example, PCR primers complementary to the nucleic acid of one or more SNPs of the present invention can be used to identify and analyze the presence or absence of the SNP. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled SNP RNA of the present invention or alternatively, radiolabeled SNP antisense DNA sequences of the present invention. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.

Sequence differences between a reference gene and genes having mutations also may be revealed by direct DNA sequencing. In addition, cloned DNA segments may be employed as probes to detect specific DNA segments. The sensitivity of such methods can be greatly enhanced by appropriate use of PCR or another amplification method. For example, a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures(26).

Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method(27).

Thus, the detection of a specific DNA sequence may be achieved by methods which include, but are not limited to, hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., restriction fragment length polymorphisms (“RFLP”) and Southern blotting of genomic DNA).

Hybridisation may be carried out under stringent hybridization conditions, for example for detection of sequences that are about 80-90% identical suitable conditions include hybridization overnight at 42° C. in 0.25M Na2HPO4, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55° C. in 0.1×SSC, 0.1% SDS. For detection of sequences that are greater than about 90% identical, suitable conditions include hybridization overnight at 65° C. in 0.25M Na2HPO4, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60° C. in 0.1×SSC, 0.1% SDS.

In addition to more conventional gel-electrophoresis and DNA sequencing, mutations also can be detected by in situ analysis.

Genetic mutations can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes(28;29). For example, genetic mutations can be identified in two-dimensional arrays containing light-generated DNA probes as described in Cronin et al., supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene. Specific mutations can also be determined through direct sequencing of one or both strands of DNA using dideoxy nucleotide chain termination chemistry, electrophoresis through a semi-solid matrix and fluorescent or radioactive chain length detection techniques. Further mutation detection techniques may involve differential susceptibility of the polymorphic double strand to restriction endonuclease digestion, or altered electrophoretic gel mobility of single or double stranded gene fragments containing one polymorphic form. Other techniques to detect specific DNA polymorphisms or mutation may involve evaluation of the structural characteristics at the site of polymorphism using nuclear magnetic resonance or x-ray diffraction techniques.

These genetic tests are useful for prognosing and/or diagnosing age related macular degeneration and often are useful for determining whether an individual is at an increased or decreased risk of developing or having age related macular degeneration.

Thus, the invention includes a method for identifying a subject at risk of age related macular degeneration, which includes detecting in a nucleic acid sample from the subject the presence or absence of a polymorphic variant at a SNP associated with age related macular degeneration in a nucleotide sequence identified as SEQ ID NOs:1 and 3.

For example, the presence of one or two copies of the G allele at SNP rs2230199 may be indicative of the subject being at risk of age related macular degeneration i.e. an individual at risk of AMD may be heterozygous (genotype GC) or homozygous (genotype GG) at SNP rs2230199 in the C3 gene,

Results from prognostic tests may be combined with other test results to diagnose age related macular degeneration. For example, prognostic results may be gathered, a patient sample may be ordered based on a determined predisposition to age related macular degeneration, the patient sample analyzed, and the results of the analysis may be utilized to diagnose age related macular degeneration. Also age related macular degeneration diagnostic methods can be developed from studies used to generate prognostic/diagnostic methods in which populations are stratified into subpopulations having different progressions of age related macular degeneration. In some embodiments, prognostic results may be gathered; a patient's risk factors for developing age related macular degeneration analyzed (e.g., age, family history, smoking); and a patient sample may be ordered based on a determined predisposition to age related macular degeneration. In some embodiments, the results from predisposition analyses may be combined with other test results, epidemiologic or genetic in nature, indicative of age related macular degeneration, which were previously, concurrently, or subsequently gathered with respect to the predisposition testing. In these embodiments, the combination of the prognostic test results with other test results can be probative of age related macular degeneration, and the combination can be utilized as a age related macular degeneration diagnostic.

Risk of age related macular degeneration sometimes is expressed as a probability, such as an odds ratio, percentage, or risk factor. The risk is based upon the presence or absence of the SNP variant described herein, and also may be based in part upon phenotypic traits of the individual being tested. Methods for calculating risk based upon patient data are well known (30). Allelotyping and genotyping analyses may be carried out in populations other than those exemplified herein to enhance the predictive power of the prognostic method. These further analyses are executed in view of the exemplified procedures described herein, and may be based upon the same polymorphic variations or additional polymorphic variations. Risk determinations for age related macular degeneration are useful in a variety of applications. In some embodiments, age related macular degeneration risk determinations may be used by clinicians to direct appropriate detection, preventative and treatment procedures to subjects who most require these. In other embodiments, age related macular degeneration risk determinations may be used by health insurers for preparing actuarial tables and for calculating insurance premiums.

The nucleic acid sample typically is isolated from a biological sample obtained from a subject. For example, nucleic acid can be isolated from blood, saliva, sputum, urine, cell scrapings, and biopsy tissue. The nucleic acid sample can be isolated from a biological sample using standard techniques. The nucleic acid sample may be isolated from the subject and then directly utilized in a method for determining the presence of a polymorphic variant, or alternatively, the sample may be isolated and then stored (e.g., frozen) for a period of time before being subjected to analysis.

The presence or absence of a polymorphic variant may be determined using one or both chromosomal complements represented in the nucleic acid sample. Determining the presence or absence of a polymorphic variant in both chromosomal complements represented in a nucleic acid sample is useful for determining the zygosity of an individual for the polymorphic variant (i.e., whether the individual is homozygous or heterozygous for the polymorphic variant). For example, a homozygous individual having the GG genotype at SNP rs2230199 (i.e. the G allele in both copies of the C3 gene) may have an increased risk of AMD relative to a heterozygous individual having the GC genotype at SNP rs2230199 (i.e. the G allele in one copies of the C3 gene and the C allele in the other)

Any oligonucleotide-based diagnostic may be utilized to determine whether a sample includes the presence or absence of a polymorphic variant in a sample. For example, primer extension methods, ligase sequence determination methods (e.g., U.S. Pat. Nos. 5,679,524 and 5,952,174, and WO 01/27326), mismatch sequence determination methods (e.g., U.S. Pat. Nos. 5,851,770; 5,958,692; 6,110,684; and 6,183,958), microarray sequence determination methods, restriction fragment length polymorphism (RFLP), single strand conformation polymorphism detection (SSCP) (e.g., U.S. Pat. Nos. 5,891,625 and 6,013,499), PCR-based assays (e.g., TAQMAN™ PCR System (Applied Biosystems)), and nucleotide sequencing methods may be used.

Oligonucleotide extension methods typically involve providing a pair of oligonucleotide primers in a polymerase chain reaction (PCR) or in other nucleic acid amplification methods for the purpose of amplifying a region from the nucleic acid sample that comprises the polymorphic variation. One oligonucleotide primer is complementary to a region 3′ of the polymorphism and the other is complementary to a region 5′ of the polymorphism. A PCR primer pair may be used in methods disclosed in U.S. Pat. Nos. 4,683,195; 4,683,202, 4,965,188; 5,656,493; 5,998,143; 6,140,054; WO 01/27327; and WO 01/27329 for example. PCR primer pairs may also be used in any commercially available machines that perform PCR, such as any of the GENEAMP™, systems available from Applied Biosystems. Also, those of ordinary skill in the art will be able to design oligonucleotide primers based upon the nucleotide sequences set forth in SEQ ID NOs: 1 and 3.

Also provided is an extension oligonucleotide that hybridizes to the amplified fragment adjacent to the polymorphic variation. An adjacent fragment refers to the 3′ end of the extension oligonucleotide being often 1 nucleotide from the 5′ end of the polymorphic site, and sometimes 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 5′ end of the polymorphic site, in the nucleic acid when the extension oligonucleotide is hybridized to the nucleic acid. The extension oligonucleotide then is extended by one or more nucleotides, and the number and/or type of nucleotides that are added to the extension oligonucleotide determine whether the polymorphic variant is present. Oligonucleotide extension methods are disclosed, for example, in U.S. Pat. Nos. 4,656,127; 4,851,331; 5,679,524; 5,834,189; 5,876,934; 5,908,755; 5,912,118; 5,976,802; 5,981,186; 6,004,744; 6,013,431; 6,017,702; 6,046,005; 6,087,095; 6,210,891; and WO 01/20039. Oligonucleotide extension methods using mass spectrometry are described, for example, in U.S. Pat. Nos. 5,547,835; 5,605,798; 5,691,141; 5,849,542; 5,869,242; 5,928,906; 6,043,031; and 6,194,144. Multiple extension oligonucleotides may be utilized in one reaction, which is referred to as multiplexing.

A microarray can be utilized for determining whether a SNP is present or absent in a nucleic acid sample. A microarray may include any oligonucleotides described herein, and methods for making and using oligonucleotide microarrays suitable for diagnostic use are disclosed in U.S. Pat. Nos. 5,492,806; 5,525,464; 5,589,330; 5,695,940; 5,849,483; 6,018,041; 6,045,996; 6,136,541; 6,142,681; 6,156;501; 6,197,506; 6,223,127; 6,225,625; 6,229,911; 6,239,273; WO 00/52625; WO 01/25485; and WO 01/29259. The microarray typically comprises a solid support and the oligonucleotides may be linked to this solid support by covalent bonds or by non-covalent interactions. The oligonucleotides may also be linked to the solid support directly or by a spacer molecule. A microarray may comprise one or more oligonucleotides complementary to a nucleotide sequence which includes a SNP set forth in Table 1. The one or more oligonucleotides may for example, hybridise specifically to a nucleotide sequence which comprises a particular polymorphic variant at the SNP, but not to nucleotide sequences which comprise other polymorphic variants at the SNP. A kit also may be utilized for determining whether a polymorphic variant is present or absent in a nucleic acid sample. A kit may include one or more pairs of oligonucleotide primers useful for amplifying a fragment of a nucleotide sequence of interest, where the fragment includes a polymorphic site. The kit may comprise a polymerizing agent, for example, a thermostable nucleic acid polymerase such as one disclosed in U.S. Pat. No. 4,889,818 or 6,077,664. Also, the kit may comprise an elongation oligonucleotide that hybridizes to the nucleotide sequence in a nucleic acid sample adjacent to the polymorphic site. Where the kit includes an elongation oligonucleotide, it may also comprise chain elongating nucleotides, such as dATP, dTTP, dGTP, dCTP, and dITP, including analogs of dATP, dTTP, dGTP, dCTP and dITP, provided that such analogs are substrates for a thermostable nucleic acid polymerase and can be incorporated into a nucleic acid chain elongated from the extension oligonucleotide. Along with chain elongating nucleotides may be one or more chain terminating nucleotides such as ddATP, ddTTP, ddGTP, ddCTP. The kit may comprise one or more oligonucleotide primer pairs, a polymerizing agent, chain elongating nucleotides, at least one elongation oligonucleotide, and one or more chain terminating nucleotides. Kits optionally include buffers, vials, microtiter plates, and instructions for use.

An individual identified as being susceptible to age related macular degeneration may be heterozygous or homozygous with respect to the allele associated with an increased risk of age related macular degeneration, as indicated in the table. For example, the individual may be heterozygous or homozygous with respect to the G allele of rs2230199 which is shown herein to be associated with an increased risk of age related macular degeneration. A subject homozygous for an allele associated with an increased risk of age related macular degeneration is at a comparatively high risk of age related macular degeneration as far as that SNP is concerned whether or not the allelic effect has been determined to be dominant or recessive. A subject who is heterozygous for an allele associated with an increased risk of age related macular degeneration, in which the allelic effect is recessive would likely be at a comparatively reduced risk of age related macular degeneration predicted by that SNP. The allelic effect of the G allele of rs2230199 is shown herein to be dominant and an individual who is heterozygous for the G allele may be at an increased risk of age related macular degeneration relative to individuals who lack the G allele.

Individuals carrying mutations in one or more SNP of the present invention may be detected at the protein level by a variety of techniques. Cells suitable for diagnosis may be obtained from a patient's blood, urine, saliva, tissue biopsy and autopsy material.

Also featured are methods for determining risk of age related macular degeneration and/or identifying a subject at risk of age related macular degeneration by contacting a polypeptide or protein encoded by a nucleotide sequence from a subject with an antibody that specifically binds to an epitope associated with an altered, usually increased risk of age related macular degeneration in the polypeptide.

Another aspect of the invention provides an isolated nucleic acid molecule comprising at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15, or at least 16, or at least 17, or at least 18, or at least 19, or at least 20, or at least 21, or at least 22, or at least 23, or at least 24, or at least 25, or at least 26, or at least 27, or at least 28, or at least 29, or at least 30, or at least 31, or at least 32, or at least 33, or at least 34, or at least 35, or at least 36, or at least 37, or at least 38, or at least 39, or at least 40, or at least 41, or at least 42, or at least 43, or at least 44, or at least 45, or at least 46, or at least 47, or at least 48, or at least 49, or at least 50, or at least 51, or at least 52, or at least 53, or at least 54, or at least 55, or at least 56, or at least 57, or at least 58, or at least 59, or at least 60, or at least 61, or at least 62, or at least 63, or at least 64, or at least 65, or at least 66, or at least 67, or at least 68, or at least 69, or at least 70, or at least 71, or at least 72, or at least 73, or at least 74, or at least 75, or at least 76, or at least 77, or at least 78, or at least 79, or at least 80, or at least 81, or at least 82, or at least 83, or at least 84, or at least 85, or at least 86, or at least 87, or at least 88, or at least 89, or at least 90, or at least 91, or at least 92, or at least 93, or at least 94, or at least 95, or at least 96, or at least 97, or at least 98, or at least 99, or at least 100 contiguous nucleotides from any one of SEQ NOS: 1 or 3 wherein one of the nucleotides is located at the site of single nucleotide polymorphism (SNP) corresponding to single nucleotide polymorphism (SNP) at rs2230199 on human chromosome 19 as set out herein or the complement thereof, and optionally, wherein the isolated nucleic acid molecule has a maximum length of 100 said contiguous nucleotides, or a maximum length of 90 said contiguous nucleotides, or a maximum length of 80 said contiguous nucleotides, or a maximum length of 70 said contiguous nucleotides, or a maximum length of 60 said contiguous nucleotides, or a maximum length of 50 said contiguous nucleotides, or a maximum length of 40 said contiguous nucleotides, or a maximum length of 30 said contiguous nucleotides, or a maximum length of 20 said contiguous nucleotides.

Oligonucleotides can be linked to a second moiety, which can be another nucleic acid molecule to provide, for example, a tail sequence (e.g., a polyadenosine tail), an adapter sequence (e.g., phage M13 universal tail sequence), etc. Alternatively, the moiety might be one that facilitates linkage to a solid support or a detectable label, e.g., a radioactive label, a fluorescent label, a chemiluminescent label, a paramagnetic label, etc.

Nucleic acid sequences shown in SEQ ID NO: 1, 3 or 4, or fragments thereof, may be used for diagnostic purposes for detection of polypeptide expression.

DNA encoding a polypeptide can also be used in the diagnosis of age related macular degeneration. For example, the nucleic acid sequence can be used in hybridization assays of biopsies or autopsies to polymorphic variants associated with increased risk of AMD (e.g., Southern or Northern blot analysis, in situ hybridization assays).

Expression of a polypeptide during embryonic development can also be determined using nucleic acid encoding the polypeptide, particularly production of a functionally impaired polypeptide that is the cause of age related macular degeneration. In situ hybridizations using a polypeptide as a probe can be employed to predict problems related to age related macular degeneration.

Included as part of this invention are nucleic acid vectors, often expression vectors, which contain a nucleotide sequence set forth in the SEQ ID NO:1 or 3, or a fragment thereof. A vector is a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid, or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors may include replication defective retroviruses, adenoviruses and adeno-associated viruses for example.

A vector can include a nucleotide sequence from SEQ ID NO: 1 or 3 or a fragment thereof, in a form suitable for expression of an encoded protein or nucleic acid in a host cell. The recombinant expression vector generally includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. A regulatory sequence includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. Expression vectors can be introduced into host cells to produce the desired polypeptides, including fusion polypeptides.

Recombinant expression vectors can be designed for expression of polypeptides in prokaryotic or eukaryotic cells. For example, the polypeptides can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further by Goeddel (31). A recombinant expression vector can also be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of polypeptides in prokaryotes can be carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide. Such fusion vectors typically serve to increase expression of recombinant polypeptide, to increase the solubility of the recombinant polypeptide and/or to aid in the purification of the recombinant polypeptide by acting as a ligand during purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety after purification of the fusion polypeptide. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.

Purified fusion polypeptides can be used in screening assays and to generate antibodies specific for polypeptides.

Expressing a polypeptide in host bacteria with an impaired capacity to proteolytically cleave the recombinant polypeptide can be used to maximize recombinant polypeptide expression (32). The nucleotide sequence of the nucleic acid to be inserted into an expression vector can be changed so that the individual codons for each amino acid are those preferentially utilized in E. coli (33).

When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. Recombinant mammalian expression vectors can be capable of directing expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Examples of suitable tissue-specific promoters include an albumin promoter(34), lymphoid-specific promoters (35) (36), promoters of immunoglobulins(37;38), neuron-specific promoters (39), pancreas-specific promoters (40), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are sometimes utilized, for example, the murine hox promoters(41) and the .alpha.-fetopolypeptide promoter(42).

Vectors can be introduced into host cells via conventional transformation or transfection techniques. The terms transformation and transfection refer to a variety of techniques known for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, transduction/infection, DEAE-dextran-mediated transfection, lipofection, or electroporation.

A host cell can be used to produce a polypeptide. Accordingly, methods for producing a polypeptide using the host cells are included as part of this invention. Such a method can include culturing host cells into which a recombinant expression vector encoding a polypeptide has been introduced in a suitable medium such that the polypeptide is produced. The method can further include isolating the polypeptide from the medium or the host cell.

Polypeptides can be expressed in transgenic animals or plants by introducing a nucleic acid encoding the polypeptide into the genome of an animal. In certain embodiments the nucleic acid is placed under the control of a tissue specific promoter, e.g., a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal. Also included is a population of cells from a transgenic animal.

Isolated polypeptides encoded by a nucleotide sequence from SEQ ID NO: 1 or 3, or a fragment thereof, can be synthesized. Isolated polypeptides include both the full-length polypeptide and the mature polypeptide (i.e., the polypeptide minus the signal sequence or propeptide domain). An isolated, or purified, polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or is substantially free from chemical precursors or other chemicals when chemically synthesized. Substantially free means a preparation of a polypeptide having less than about 5% (by dry weight) of contaminating protein, or of chemical precursors or non-target chemicals. When the desired polypeptide is recombinantly produced, it is typically substantially free of culture medium, specifically, where culture medium represents less than about 10% of the polypeptide preparation.

Also, polypeptides may exist as chimeric or fusion polypeptides. As used herein, a “target chimeric polypeptide” or “target fusion polypeptide” includes a target polypeptide linked to a different polypeptide. The target polypeptide in the fusion polypeptide can correspond to an entire or nearly entire polypeptide as it exists in nature or a fragment thereof. The other polypeptide can be fused to the N-terminus or C-terminus of the target polypeptide.

Fusion polypeptides can include a moiety having high affinity for a ligand. For example, the fusion polypeptide can be a GST-target fusion polypeptide in which the target sequences are fused to the C-terminus of the GST sequences, or a polyhistidine-target fusion polypeptide in which the target polypeptide is fused at the N- or C-terminus to a string of histidine residues. Such fusion polypeptides can facilitate purification of recombinant target polypeptide. Expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide), and a nucleotide sequence from SEQ ID NO: 1, 3 or 4, or a fragment thereof, or a substantially identical nucleotide sequence thereof, can be cloned into an expression vector such that the fusion moiety is linked in-frame to the target polypeptide. Further, the fusion polypeptide can be a target polypeptide containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression, secretion, cellular internalization, and cellular localization of a target polypeptide can be increased through use of a heterologous signal sequence. Fusion polypeptides can also include all or a part of a serum polypeptide (e.g., an IgG constant region or human serum albumin).

Target polypeptides can be used as immunogens to produce anti-target antibodies in a subject, to purify the polypeptide ligands or binding partners.

Polypeptides can be differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any known modification including specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc. may be used. Additional post-translational modifications include, for example, N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. The polypeptide fragments may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the polypeptide.

Pharmacogenomics is a discipline that involves tailoring a treatment for a subject according to the subject's genotype. For example, based upon the outcome of a prognostic test, a clinician or physician may target pertinent information and preventative or therapeutic treatments to a subject who would be benefited by the information or treatment and avoid directing such information and treatments to a subject who would not be benefited (e.g., the treatment has no therapeutic effect and/or the subject experiences adverse side effects). As therapeutic approaches for age related macular degeneration continue to evolve and improve, the goal of treatments for age related macular degeneration related disorders is to intervene even before clinical signs manifest themselves. Thus, genetic markers associated with susceptibility to age related macular degeneration prove useful for early diagnosis, prevention and treatment of age related macular degeneration.

The following is an example of a pharmacogenomic embodiment. A particular treatment regimen can exert a differential effect depending upon the subject's genotype. Where a candidate therapeutic exhibits a significant beneficial interaction with a prevalent allele and a comparatively weak interaction with an uncommon allele (e.g., an order of magnitude or greater difference in the interaction), such a therapeutic typically would not be administered to a subject genotyped as being homozygous for the uncommon allele, and sometimes not administered to a subject genotyped as being heterozygous for the uncommon allele. In another example, where a candidate therapeutic is not significantly toxic when administered to subjects who are homozygous for a prevalent allele but is comparatively toxic when administered to subjects heterozygous or homozygous for an uncommon allele, the candidate therapeutic is not typically administered to subjects who are genotyped as being heterozygous or homozygous with respect to the uncommon allele.

Methods of the invention are applicable to pharmacogenomic methods for detecting, preventing, alleviating and/or treating age related macular degeneration. For example, a nucleic acid sample from an individual may be subjected to a genetic test. Where one or more polymorphic variants associated with increased risk of age related macular degeneration are identified at SNPs in the individual, information for detecting, preventing or treating age related macular degeneration and/or one or more age related macular degeneration detection, prevention and/or treatment regimens then may be directed to and/or prescribed to that individual.

In certain embodiments, a detection, preventative and/or treatment regimen is specifically prescribed and/or administered to individuals who will most benefit from it based upon their risk of developing age related macular degeneration assessed by the methods described herein. Methods are thus provided for identifying a subject at risk of age related macular degeneration and then prescribing a detection, therapeutic or preventative regimen to individuals identified as being at increased risk of age related macular degeneration. Thus, certain embodiments are directed to methods for treating age related macular degeneration in a subject, reducing risk of age related macular degeneration in a subject, or early detection of age related macular degeneration in a subject, which comprise: detecting the presence or absence of a polymorphic variant associated with age related macular degeneration at a SNP in a nucleotide sequence set forth in SEQ ID NOs:1 and 3, and prescribing or administering an age related macular degeneration treatment regimen, preventative regimen and/or detection regimen to a subject from whom the sample originated where the presence of polymorphic variants associated with age related macular degeneration are detected at one or more SNPs in the nucleotide sequence. In these methods, genetic results may be utilized in combination with other test results to diagnose age related macular degeneration as described above.

The use of certain age related macular degeneration treatments are known in the art, and include surgery, chemotherapy and/or radiation therapy. Any of the treatments may be used in combination to treat or prevent age related macular degeneration (e.g., surgery followed by radiation therapy or chemotherapy).

Pharmacogenomics methods also may be used to analyze and predict a response to a age related macular degeneration treatment or a drug. For example, if pharmacogenomics analysis indicates a likelihood that an individual will respond positively to a age related macular degeneration treatment with a particular drug, the drug may be administered to the individual. Conversely, if the analysis indicates that an individual is likely to respond negatively to treatment with a particular drug, an alternative course of treatment may be prescribed. A negative response may be defined as either the absence of an efficacious response or the presence of toxic side effects. The response to a therapeutic treatment can be predicted in a background study in which subjects in any of the following populations are genotyped: a population that responds favorably to a treatment regimen, a population that does not respond significantly to a treatment regimen, and a population that responds adversely to a treatment regiment (e.g., exhibits one or more side effects). These populations are provided as examples and other populations and subpopulations may be analyzed. Based upon the results of these analyses, a subject is genotyped to predict whether he or she will respond favorably to a treatment regimen, not respond significantly to a treatment regimen, or respond adversely to a treatment regimen.

The methods described herein also are applicable to clinical drug trials. Polymorphic variants indicative of response to an agent for treating age related macular degeneration or to side effects to an agent for treating age related macular degeneration may be identified at one or more SNPs. Thereafter, potential participants in clinical trials of such an agent may be screened to identify those individuals most likely to respond favorably to the drug and exclude those likely to experience side effects. In that way, the effectiveness of drug treatment may be measured in individuals who respond positively to the drug, without lowering the measurement as a result of the inclusion of individuals who are unlikely to respond positively in the study and without risking undesirable safety problems.

Thus, another embodiment is a method of selecting an individual for inclusion in a clinical trial of a treatment or drug comprising the steps of: (a) obtaining a nucleic acid sample from an individual; (b) determining the identity of a polymorphic variant which is associated with a positive response to the treatment or the drug, or a polymorphic variant which is associated with a negative response to the treatment or the drug at least one SNP in the nucleic acid sample, and (c) including the individual in the clinical trial if the nucleic acid sample contains the polymorphic variant associated with a positive response to the treatment or the drug or if the nucleic acid sample lacks said polymorphic variant associated with a negative response to the treatment or the drug. The SNP may be in a sequence selected individually or in any combination from the C3 genomic sequence disclosed in the table. Step (c) can also include administering the drug or the treatment to the individual if the nucleic acid sample contains the polymorphic variant associated with a positive response to the treatment or the drug and the nucleic acid sample lacks the polymorphic variant associated with a negative response to the treatment or the drug.

A peptide nucleic acid, or PNA, refers to a nucleic acid mimic such as a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. Synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described, for example, in Hyrup et al. (71), and Perry-O'Keefe et al.(70).

PNA nucleic acids can be used in prognostic and diagnostic applications. For example, PNAs can be used in the analysis of SNPs in a gene, (e.g., by PNA-directed PCR clamping); as artificial restriction enzymes when used in combination with other enzymes, (e.g., S1 nucleases(71) or as probes or primers for DNA sequencing or hybridization (71;72).

A further aspect of the invention provides an antibody molecule that binds specifically to a variant C3 polypeptide i.e. a polypeptide encoded by a nucleotide sequence comprising polymorphic variants at one or more SNPs described herein. For example, an antibody molecule may bind specifically to the C3F polypeptide which comprises an R102G substitution which is encoded by the coding sequence comprising the G allele of SNP rs2230199. Such an antibody binds preferentially to the C3F polypeptide relative to C3S polypeptide which lacks the R102G substitution.

A method of identifying and/or obtaining an antibody specific for C3F polypeptide may comprise;

    • providing a population of antibody molecules specific for C3F polypeptide,
    • contacting said population with a C3S polypeptide,
    • identifying one or more members of said population which bind preferentially to C3F relative to C3S polypeptide.

Antibody molecules may be useful both in the diagnosis of AMD, in accordance with the invention.

Antibodies that are specific for a C3 polypeptide may be obtained using techniques that are standard in the art. An immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal). An appropriate immunogenic preparation can contain, for example, recombinantly expressed chemically synthesized polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulatory agent.

Amino acid polymorphisms can be detected using antibodies specific for the altered epitope by western analysis after the electrophoresis of denatured proteins. Protein polymorphism can also be detected using fluorescently identified antibodies which bind to specific polymorphic epitopes and detected in whole cells using fluorescence activated cell sorting techniques (FACS). Polymorphic protein sequence may also be determined by NMR spectroscopy or by x-ray diffraction studies. Further, determination of polymorphic sites in proteins may be accomplished by observing differential cleavage by specific or non specific proteases.

An antibody is an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. Examples of 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. An antibody can be polyclonal, monoclonal, or recombinant (e.g., a chimeric or humanized), fully human, non-human (e.g., murine), or a single chain antibody.

A full-length polypeptide or antigenic peptide fragment encoded by a target nucleotide sequence can be used as an immunogen or can be used to identify antibodies made with other immunogens, e.g., cells, membrane preparations, and the like. An antigenic peptide often includes at least 8 amino acid residues of the amino acid sequences encoded by a nucleotide sequence of one of SEQ ID NOs:1 and 3, and encompasses an epitope. Antigenic peptides sometimes include 10 or more amino acids, 15 or more amino acids, 20 or more amino acids, or 30 or more amino acids. Hydrophilic and hydrophobic fragments of polypeptides sometimes are used as immunogens.

Epitopes encompassed by the antigenic peptide are regions located on the surface of the polypeptide (e.g., hydrophilic regions) as well as regions with high antigenicity. For example, an Emini surface probability analysis of the human polypeptide sequence can be used to indicate the regions that have a particularly high probability of being localized to the surface of the polypeptide and are thus likely to constitute surface residues useful for targeting antibody production. The antibody may bind an epitope on any domain or region on polypeptides for use in the invention.

An antibody (e.g., monoclonal antibody) can be used to isolate target polypeptides by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, an antibody can be used to detect a target polypeptide (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the polypeptide. Antibodies can be used diagnostically to monitor polypeptide levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H. Also, an antibody can be utilized as a test molecule for determining whether it can treat age related macular degeneration, and as a therapeutic for administration to a subject for treating age related macular degeneration .

An antibody can be made by immunizing with a purified antigen, or a fragment thereof, a membrane associated antigen, tissues, e.g., crude tissue preparations, whole cells, preferably living cells, lysed cells, or cell fractions.

Included as part of this invention are antibodies which bind only a native polypeptide, only denatured or otherwise non-native polypeptide, or which bind both, as well as those having linear or conformational epitopes. Conformational epitopes sometimes can be identified by selecting antibodies that bind to native but not denatured polypeptide. Also featured are antibodies that specifically bind to a polypeptide variant associated with age related macular degeneration.

Preferably, an antibody displays increased binding to the C3F polypeptide relative to the C3S polypeptide.

The examples set forth below are intended to illustrate but not limit the invention.

Age-related macular degeneration is the most common cause of blindness in Western populations. Susceptibility is influenced by age and by genetic and environmental factors. Complement activation is implicated in the pathogenesis. We tested for an association between age-related macular degeneration and 13 single nucleotide polymorphisms (SNPs) spanning the complement genes C3 and C5 in case subjects and control subjects from the southeastern region of England. All subjects were examined by an ophthalmologist and had independent grading of fundus photographs to confirm their disease status. To test for replication of the most significant findings, we genotyped a set of Scottish cases and controls. The common functional polymorphism rs2230199 (Arg102Gly) in the C3 gene, corresponding to the electrophoretic variants C3S (slow) and C3F (fast), was strongly associated with age-related macular degeneration in both the English group (603 cases and 350 controls, P=5.9×1-5) and the Scottish group (244 cases and 351 controls, P=5.0×10-5). The odds ratio for age-related macular degeneration in C3 S/F heterozygotes as compared with S/S homozygotes was 1.7 (95% confidence interval [CI], 1.3 to 2.1); for F/F homozygotes, the odds ratio was 2.6 (95% CI, 1.6 to 4.1). The estimated population attributable risk for C3F was 22%. Complement C3 is important in the pathogenesis of age-related macular degeneration. This finding further underscores the influence of the complement pathway in the pathogenesis of this disease.

The inventors of the present invention have discovered a single base pair polymorphism that is present in a highly significant percentage of the genetic DNA of individuals affected with age related macular degeneration while only present in a smaller percentage of individuals who are not known to be affected by the disease.

For individuals with age-related macular degeneration, the distribution of polymorphic alleles at position 6669387 of chromosome 19, found within the C3 gene, was different from those without age-related macular degeneration (Table 1). The trend test for risk associated with carrying the C allele (on the positive reference strand of the human genome) had an empirical p-value of 0.000059225, and the corresponding Mantel-Haenszel odds ratio for trend is 1.600 (Table 1). These data further suggest that this marker, located within the C3 gene, is associated with age-related macular degeneration risk and that the C allele at position 6669387 of chromosome 19 is associated with an increased risk of developing age-related macular degeneration. The C allele at position 6669387 of the positive strand corresponds to the G allele within the negative strand, in which is found the coding sequence for C3.

TABLE 1rs no.2230199Chromosome; Position19; 6669387Gene NameC3SEQ ID NO; Position3; 2274Genotype; Phenotypen = C; increased risk (positive strand relative tothe human reference sequence version 36.1)Hardy-Weinberg0.86594CaseAllelep-OddsFlagBAAABBBModelValueRatio0C22310914Trend0.0000591.6001C30324245

The present invention has been described in detail by way of illustration and example in order to acquaint others skilled in the art with the invention, its principles and its practical application. Particular formulations and processes of the present invention are not limited to the descriptions of the specific embodiments presented, but rather the descriptions and examples should be viewed in terms of the claims that follow and their equivalents. While some of the examples and descriptions above include some conclusions about the way the invention may function, the inventors do not intend to be bound by those conclusions and functions, but put them forth only as possible explanations.

It is to be further understood that the specific embodiments of the present invention as set forth are not intended as being exhaustive or limiting of the invention, and that many alternatives, modifications and variations will be apparent to those of ordinary skill in the art in light of the foregoing examples and detailed description. Accordingly, this invention is intended to embrace all such alternatives, modifications and variations that fall within the spirit and scope of the following claims.

REFERENCE LIST

  • (1) Swaroop A, Branham K E, Chen W, Abecasis G. Genetic susceptibility to age-related macular degeneration: a paradigm for dissecting complex disease traits. Hum Mol Genet. 2007 Oct. 15; 16 Spec No 2:R174-82:R174-R182.
  • (2) Ambati J, Ambati B K, Yoo S H, Ianchulev S, Adamis A P. Age-related macular degeneration: etiology, pathogenesis, and therapeutic strategies. Surv Opthalmol 2003 May; 48(3):257-93.
  • (3) Klaver C C, Wolfs R C, Vingerling J R, Hofman A, de Jong P T. Age-specific prevalence and causes of blindness and visual impairment in an older population: the Rotterdam Study. Arch Opthalmol 1998 May; 116(5):653-8.
  • (4) Hammond C J, Webster A R, Snieder H, Bird A C, Gilbert C E, Spector T D. Genetic influence on early age-related maculopathy: a twin study. Opthalmology 2002 April; 109(4):730-6.
  • (5) Heiba I M, Elston R C, Klein B E, Klein R. Sibling correlations and segregation analysis of age-related maculopathy: the Beaver Dam Eye Study. Genet Epidemiol 1994; 11(1):51-67.
  • (6) Smith W, Assink J, Klein R, Mitchell P, Klaver C C, Klein B E, et al. Risk factors for age-related macular degeneration: Pooled findings from three continents. Opthalmology 2001 April; 108(4):697-704.
  • (7) van L R, Klaver C C, Vingerling J R, Hofman A, de Jong P T. Epidemiology of age-related maculopathy: a review. Eur J Epidemiol 2003; 18(9):845-54.
  • (8) Huang G H, Klein R, Klein B E, Tomany S C. Birth cohort effect on prevalence of age-related maculopathy in the Beaver Dam Eye Study. Am J Epidemiol 2003 April 15; 157(8):721-9.
  • (9) Haines J L, Hauser M A, Schmidt S, Scott W K, Olson L M, Gallins P, et al. Complement factor H variant increases the risk of age-related macular degeneration. Science 2005 Apr. 15; 308(5720):419-21.
  • (10) Hageman G S, Anderson D H, Johnson L V, Hancox L S, Taiber A J, Hardisty L I, et al. A common haplotype in the complement regulatory gene factor H(HF1/CFH) predisposes individuals to age-related macular degeneration. Proc Natl Acad Sci USA 2005 May 17; 102(20):7227-32.
  • (11) Klein R J, Zeiss C, Chew E Y, Tsai J Y, Sackler R S, Haynes C, et al. Complement factor H polymorphism in age-related macular degeneration. Science 2005 Apr. 15; 308(5720):385-9.
  • (12) Edwards A O, Ritter R, III, Abel K J, Manning A, Panhuysen C, Farrer L A. Complement factor H polymorphism and age-related macular degeneration. Science 2005 Apr. 15; 308(5720):421-4.
  • (13) Zareparsi S, Branham K E, Li M, Shah S, Klein R J, Ott J, et al. Strong association of the Y402H variant in complement factor H at 1q32 with susceptibility to age-related macular degeneration. Am J Hum Genet. 2005 Jul.; 77(1):149-53.
  • (14) Jakobsdottir J, Conley Y P, Weeks D E, Mah T S, Ferrell R E, Gorin M B. Susceptibility genes for age-related maculopathy on chromosome 10q26. Am J Hum Genet. 2005 September.; 77(3):389-407.
  • (15) Li M, tmaca-Sonmez P, Othman M, Branham K E, Khanna R, Wade M S, et al. CFH haplotypes without the Y402H coding variant show strong association with susceptibility to age-related macular degeneration. Nat Genet. 2006 September.; 38(9):1049-54.
  • (16) Rivera A, Fisher S A, Fritsche L G, Keilhauer C N, Lichtner P, Meitinger T, et al. Hypothetical LOC387715 is a second major susceptibility gene for age-related macular degeneration, contributing independently of complement factor H to disease risk. Hum Mol Genet. 2005 Nov. 1; 14(21):3227-36.
  • (17) DeWan A, Liu M, Hartman S, Zhang S S, Liu D T, Zhao C, et al. HTRA1 promoter polymorphism in wet age-related macular degeneration. Science 2006 Nov. 10; 314(5801):989-92.
  • (18) Yang Z, Camp N J, Sun H, Tong Z, Gibbs D, Cameron D J, et al. A variant of the HTRA1 gene increases susceptibility to age-related macular degeneration. Science 2006 Nov. 10; 314(5801):992-3.
  • (19) Kanda A, Chen W, Othman M, Branham K E, Brooks M, Khanna R, et al. A variant of mitochondrial protein LOC387715/ARMS2, not HTRA1, is strongly associated with age-related macular degeneration. Proc Natl Acad Sci USA 2007 Oct. 9; 104(41):16227-32.
  • (20) Gold B, Merriam J E, Zemant J, Hancox L S, Taiber A J, Gehrs K, et al. Variation in factor B (BF) and complement component 2 (C2) genes is associated with age-related macular degeneration. Nat Genet. 2006 April; 38(4):458-62.
  • (21) Yates J R, Sepp T, Matharu B K, Khan J C, Thurlby D A, Shahid H, et al. Complement C3 variant and the risk of age-related macular degeneration. N Engl J Med 2007 Aug. 9; 357(6):553-61.
  • (22) Maller J B, Fagerness J A, Reynolds R C, Neale B M, Daly M J, Seddon J M. Variation in complement factor 3 is associated with risk of age-related macular degeneration. Nat Genet. 2007 October; 39(10):1200-1.
  • (23) Johnson G C, Esposito L, Barratt B J, Smith A N, Heward J, Di G G, et al. Haplotype tagging for the identification of common disease genes. Nat Genet. 2001 October; 29(2):233-7.
  • (24) Sasieni P D. From genotypes to genes: doubling the sample size. Biometrics 1997 December; 53(4):1253-61.
  • (25) Saiki R K, Bugawan T L, Horn G T, Mullis K B, Erlich H A. Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature 1986 Nov. 13; 324(6093):163-6.
  • (26) Myers R M, Larin Z, Maniatis T. Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:DNA duplexes. Science 1985 Dec. 13; 230(4731):1242-6.
  • (27) Cotton R G, Rodrigues N R, Campbell R D. Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proc Natl Acad Sci USA 1988 June; 85(12):4397-401.
  • (28) Cronin M T, Fucini R V, Kim S M, Masino R S, Wespi R M, Miyada C G. Cystic fibrosis mutation detection by hybridization to light-generated DNA probe arrays. Hum Mutat 1996; 7(3):244-55.
  • (29) Kozal M J, Shah N, Shen N, Yang R, Fucini R, Merigan T C, et al. Extensive polymorphisms observed in HIV-1 clade B protease gene using high-density oligonucleotide arrays. Nat Med 1996 July; 2(7):753-9.
  • (30) Agresti A. Exact inference for categorical data: recent advances and continuing controversies. Stat Med 2001 Sep. 15; 20(17-18):2709-22.
  • (31) Goeddel D V. Systems for heterologous gene expression. Methods Enzymol 1990; 185:3-7:3-7.
  • (32) Gottesman S. Minimizing proteolysis in Escherichia coli: genetic solutions. Methods Enzymol 1990; 185:119-29:119-29.
  • (33) Wada K, Wada Y, Ishibashi F, Gojobori T, Ikemura T. Codon usage tabulated from the GenBank genetic sequence data. Nucleic Acids Res 1992 May 11; 20 Suppl:2111-8:2111-8.
  • (34) Pinkert C A, Ornitz D M, Brinster R L, Palmiter R D. An albumin enhancer located 10 kb upstream functions along with its promoter to direct efficient, liver-specific expression in transgenic mice. Genes Dev 1987 May; 1 (3):268-76.
  • (35) Calame K, Eaton S. Transcriptional controlling elements in the immunoglobulin and T cell receptor loci. Adv Immunol 1988; 43:235-75:235-75.
  • (36) Winoto A, Baltimore D. A novel, inducible and T cell-specific enhancer located at the 3′ end of the T cell receptor alpha locus. EMBO J. 1989 March; 8(3):729-33.
  • (37) Banerji J, Olson L, Schaffner W. A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes. Cell 1983 July; 33(3):729-40.
  • (38) Queen C, Baltimore D. Immunoglobulin gene transcription is activated by downstream (sequence elements. Cell 1983 July; 33(3):741-8.
  • (39) Byrne G W, Ruddle F H. Multiplex gene regulation: a two-tiered approach to transgene regulation in transgenic mice. Proc Natl Acad Sci USA 1989 July; 86(14):5473-7.
  • (40) Edlund T, Walker M D, Barr P J, Rutter W J. Cell-specific expression of the rat insulin gene: evidence for role of two distinct 5′ flanking elements. Science 1985 Nov. 22; 230(4728):912-6.
  • (41) Kessel M, Gruss P. Murine developmental control genes. Science 1990 Jul. 27; 249(4967):374-9.
  • (42) Camper S A, Tilghman S M. Postnatal repression of the alpha-fetoprotein gene is enhancer independent. Genes Dev 1989 April; 3(4):537-46.
  • (43) Malik F, Delgado C, Knusli C, Irvine A E, Fisher D, Francis G E. Polyethylene glycol (PEG)-modified granulocyte-macrophage colony-stimulating factor (GM-CSF) with conserved biological activity. Exp Hematol 1992 September; 20(8):1028-35.
  • (44) Zuckermann R N, Martin E J, Spellmeyer D C, Stauber G B, Shoemaker K R, Kerr J M, et al. Discovery of nanomolar ligands for 7-transmembrane G-protein-coupled receptors from a diverse N-(substituted)glycine peptoid library. J Med Chem 1994 August; %19; 37(17):2678-85.
  • (45) Lam K S. Application of combinatorial library methods in cancer research and drug discovery. Anticancer Drug Des 1997 April; 12(3):145-67.
  • (46) DeWitt S H, Kiely J S, Stankovic C J, Schroeder M C, Cody D M, Pavia M R. “Diversomers”: an approach to nonpeptide, nonoligomeric chemical diversity. Proc Natl Acad Sci USA 1993 Aug. 1; 90(15):6909-13.
  • (47) Erb E, Janda K D, Brenner S. Recursive deconvolution of combinatorial chemical libraries. Proc Natl Acad Sci USA 1994 Nov. 22; 91(24):11422-6.
  • (48) Cho C Y, Moran E J, Chemy S R, Stephans J C, Fodor S P, Adams C L, et al. An unnatural biopolymer. Science 1993 Sep. 3; 261(5126):1303-5.
  • (49) Gallop M A, Barrett R W, Dower W J, Fodor S P, Gordon E M. Applications of combinatorial technologies to drug discovery. 1. Background and peptide combinatorial libraries. J Med Chem 1994 Apr. 29; 37(9):1233-51.
  • (50) Houghten R A, Appel J R, Blondelle S E, Cuervo J H, Dooley C T, Pinilla C. The use of synthetic peptide combinatorial libraries for the identification of bioactive peptides. Biotechniques 1992 September; 13(3):412-21.
  • (51) Lam K S, Salmon S E, Hersh E M, Hruby V J, Kazmierski W M, Knapp R J. A new type of synthetic peptide library for identifying ligand-binding activity. Nature 1991 Nov. 7; 354(6348):82-4.
  • (52) Fodor S P, Rava R P, Huang X C, Pease A C, Holmes C P, Adams C L. Multiplexed biochemical assays with biological chips. Nature 1993 Aug. 5; 364(6437):555-6.
  • (53) Cull M G, Miller J F, Schatz P J. Screening for receptor ligands using large libraries of peptides linked to the C terminus of the lac repressor. Proc Natl Acad Sci USA 1992 Mar. 1; 89(5):1865-9.
  • (54) Scott J K, Smith G P. Searching for peptide ligands with an epitope library. Science 1990 Jul. 27; 249(4967):386-90.
  • (55) Devlin J J, Panganiban L C, Devlin P E. Random peptide libraries: a source of specific protein binding molecules. Science 1990 Jul. 27; 249(4967):404-6.
  • (56) Cwirla S E, Peters E A, Barrett R W, Dower W J. Peptides on phage: a vast library of peptides for identifying ligands. Proc Natl Acad Sci USA 1990 August; 87(16):6378-82.
  • (57) Felici F, Castagnoli L, Musacchio A, Jappelli R, Cesareni G. Selection of antibody ligands from a large library of oligopeptides expressed on a multivalent exposition vector. J Mol Biol 1991 November; %20; 222(2):301-10.
  • (58) Gautier C, Morvan F, Rayner B, Huynh-Dinh T, Igolen J, Imbach J L, et al. Alpha-DNA. IV: Alpha-anomeric and beta-anomeric tetrathymidylates covalently linked to intercalating oxazolopyridocarbazole. Synthesis, physicochemical properties and poly (rA) binding. Nucleic Acids Res 1987 Aug. 25; 15(16):6625-41.
  • (59) Inoue H, Hayase Y, Imura A, Iwai S, Miura K, Ohtsuka E. Synthesis and hybridization studies on two complementary nona(2′-O-methyl)ribonucleotides. Nucleic Acids Res 1987 Aug. 11; 15(15):6131-48.
  • (60) Inoue H, Hayase Y, Iwai S, Ohtsuka E. Sequence-dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H. FEBS Lett 1987 May 11; 215(2):327-30.
  • (61) Haseloff J, Gerlach W L. Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature 1988 Aug. 18; 334(6183):585-91.
  • (62) Bartel D P, Szostak J W. Isolation of new ribozymes from a large pool of random sequences [see comment]. Science 1993 Sep. 10; 261(5127):1411-8.
  • (63) Helene C. The anti-gene strategy: control of gene expression by triplex-forming-oligonucleotides. Anticancer Drug Des 1991 December; 6(6):569-84.
  • (64) Helene C, Thuong N T, Harel-Bellan A. Control of gene expression by triple helix-forming oligonucleotides. The antigene strategy. Ann NY Acad Sci 1992 Oct. 28; 660:27-36:27-36.
  • (65) Maher L J, III. DNA triple-helix formation: an approach to artificial gene repressors? Bioessays 1992 December; 14(12):807-15.
  • (66) Bosher J M, Labouesse M. RNA interference: genetic wand and genetic watchdog. Nat Cell Biol 2000 February; 2(2):E31-E36.
  • (67) Caplen N J, Parrish S, Imani F, Fire A, Morgan R A. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc Natl Acad Sci USA 2001 Aug. 14; 98(17):9742-7.
  • (68) Elbashir S M, Harborth J, Weber K, Tuschl T. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 2002 February; 26(2):199-213.
  • (69) Caplen N J, Parrish S, Imani F, Fire A, Morgan R A. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc Natl Acad Sci USA 2001 Aug. 14; 98(17):9742-7.
  • (70) Abderrahmani A, Steinmann M, Plaisance V, Niederhauser G, Haefliger J A, Mooser V, et al. The transcriptional repressor REST determines the cell-specific expression of the human MAPK8IP1 gene encoding IBI (JIP-1). Mol Cell Biol 2001 November; 21(21):7256-67.
  • (71) Hyrup B, Nielsen P E. Peptide nucleic acids (PNA): synthesis, properties and potential applications. Bioorg Med Chem 1996 January; 4(1):5-23.
  • (72) Perry-O'Keefe H, Yao X W, Coull J M, Fuchs M, Egholm M. Peptide nucleic acid pre-gel hybridization: an alternative to southern hybridization. Proc Natl Acad Sci USA 1996 Dec. 10; 93(25):14670-5.
  • (73) Letsinger R L, Zhang G R, Sun D K, Ikeuchi T, Sarin P S. Cholesteryl-conjugated oligonucleotides: synthesis, properties, and activity as inhibitors of replication of human immunodeficiency virus in cell culture. Proc Natl Acad Sci USA 1989 September; 86(17):6553-6.
  • (74) Lemaitre M, Bayard B, Lebleu B. Specific antiviral activity of a poly(L-lysine)-conjugated oligodeoxyribonucleotide sequence complementary to vesicular stomatitis virus N protein mRNA initiation site. Proc Natl Acad Sci USA 1987 February; 84(3):648-52.
  • (75) van der Krol A R, Mol J N, Stuitje A R. Modulation of eukaryotic gene expression by complementary RNA or DNA sequences. Biotechniques 1988 November; 6(10):958-76.
  • (76) Zon G. Oligonucleotide analogues as potential chemotherapeutic agents. Pharm Res 1988 September; 5(9):539-49.
  • (77) Better M, Chang C P, Robinson R R, Horwitz A H. Escherichia coli secretion of an active chimeric antibody fragment. Science 1988 May; %20; 240(4855):1041-3.
  • (78) Liu A Y, Robinson R R, Hellstrom K E, Murray E D, Jr., Chang C P, Hellstrom I. Chimeric mouse-human IgG1 antibody that can mediate lysis of cancer cells. Proc Natl Acad Sci USA 1987 May; 84(10):3439-43.
  • (79) Liu A Y, Robinson R R, Murray E D, Jr., Ledbetter J A, Hellstrom I, Hellstrom K E. Production of a mouse-human chimeric monoclonal antibody to CD20 with potent Fc-dependent biologic activity. J Immunol 1987 Nov. 15; 139(10):3521-6.
  • (80) Sun L K, Curtis P, Rakowicz-Szulczynska E, Ghrayeb J, Chang N, Morrison S L, et al. Chimeric antibody with human constant regions and mouse variable regions directed against carcinoma-associated antigen 17-1A. Proc Natl Acad Sci USA 1987 January; 84(1):214-8.
  • (81) Nishimura Y, Yokoyama M, Araki K, Ueda R, Kudo A, Watanabe T. Recombinant human-mouse chimeric monoclonal antibody specific for common acute lymphocytic leukemia antigen. Cancer Res 1987 Feb. 15; 47(4):999-1005.
  • (82) Wood C R, Boss M A, Kenten J H, Calvert J E, Roberts N A, Emtage J S. The synthesis and in vivo assembly of functional antibodies in yeast. Nature 1985 Apr. 4; 314(6010):446-9.
  • (83) Shaw D R, Khazaeli M B, LoBuglio A F. Mouse/human chimeric antibodies to a tumor-associated antigen: biologic activity of the four human IgG subclasses. J Natl Cancer Inst 1988 Dec. 7; 80(19):1553-9.
  • (84) Morrison S L. Transfectomas provide novel chimeric antibodies. Science 1985 September; %20; 229(4719): 1202-7.
  • (85) Verhoeyen M, Milstein C, Winter G. Reshaping human antibodies: grafting an antilysozyme activity. Science 1988 Mar. 25; 239(4847):1534-6.
  • (86) Beidler C B, Ludwig J R, Cardenas J, Phelps J, Papworth C G, Melcher E, et al. Cloning and high level expression of a chimeric antibody with specificity for human carcinoembryonic antigen. J Immunol 1988 Dec. 1; 141(11):4053-60.
  • (87) Lonberg N, Huszar D. Human antibodies from transgenic mice. Int Rev Immunol 1995; 13(1):65-93.
  • (88) Jespers L S, Roberts A, Mahler S M, Winter G, Hoogenboom H R. Guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen. Biotechnology (NY) 1994 September; 12(9):899-903.
  • (89) Colcher D, Pavlinkova G, Beresford G, Booth B J, Batra S K. Single-chain antibodies in pancreatic cancer. Ann NY Acad Sci 1999 Jun. 30; 880:263-80:263-80.
  • (90) Reiter Y, Pastan I. Antibody engineering of recombinant Fv immunotoxins for improved targeting of cancer: disulfide-stabilized Fv immunotoxins. Clin Cancer Res 1996 February; 2(2):245-52.
  • (91) McConnell H M, Owicki J C, Parce J W, Miller D L, Baxter G T, Wada H G, et al. The cytosensor microphysiometer: biological applications of silicon technology. Science 1992 Sep. 25; 257(5078):1906-12.
  • (92) Sjolander S, Urbaniczky C. Integrated fluid handling system for biomolecular interaction analysis. Anal Chem 1991 Oct. 15; 63(20):2338-45.
  • (93) Szabo A, Stolz L, Granzow R. Surface plasmon resonance and its use in biomolecular interaction analysis (BIA). Curr Opin Struct Biol 1995 October; 5(5):699-705.
  • (94) Rivas G, Minton A P. New developments in the study of biomolecular associations via sedimentation equilibrium. Trends Biochem Sci 1993 August; 18(8):284-7.
  • (95) Current Protocols in Molecular Biology. New York: Wiley; 1999.
  • (96) Heegaard N H. Capillary electrophoresis for the study of affinity interactions. J Mol Recognit 1998; 11(1-6):141-8.
  • (97) Zervos A S, Gyuris J, Brent R. Mxi1, a protein that specifically interacts with Max to bind Myc-Max recognition sites. Cell 1993 Jan. 29; 72(2):223-32.
  • (98) Madura K, Dohmen R J, Varshavsky A. N-recognin/Ubc2 interactions in the N-end rule pathway. J Biol Chem 1993 Jun. 5; 268(16):12046-54.
  • (99) Bartel P, Chien C T, Stemglanz R, Fields S. Elimination of false positives that arise in using the two-hybrid system. Biotechniques 1993 June; 14(6):920-4.
  • (100) Iwabuchi K, Li B, Bartel P, Fields S. Use of the two-hybrid system to identify the domain of p53 involved in oligomerization. Oncogene 1993 June; 8(6):1693-6.
  • (101) Remington's Pharmaceutical Sciences. Mack; 2005.
  • (102) Cruikshank W W, Doctrow S R, Falvo M S, Huffman K, Maciaszek J, Viglianti G, et al. A lipidated anti-Tat antibody enters living cells and blocks HIV-1 viral replication. J Acquir Immune Defic Syndr Hum Retrovirol 1997 Mar. 1; 14(3):193-203.
  • (103) Chen S H, Shine H D, Goodman J C, Grossman R G, Woo S L. Gene therapy for brain tumors: regression of experimental gliomas by adenovirus-mediated gene transfer in vivo. Proc Natl Acad Sci USA 1994 Apr. 12; 91(8):3054-7.
  • (104) Osborne S E, Matsumura I, Ellington A D. Aptamers as therapeutic and diagnostic reagents: problems and prospects. Curr Opin Chem Biol 1997 June; 1(1):5-9.
  • (105) Patel D J. Structural analysis of nucleic acid aptamers. Curr Opin Chem Biol 1997 June; 1(1):32-46.
  • (106) Herlyn D, Birebent B. Advances in cancer vaccine development. Ann Med 1999 February; 31(1):66-78.
  • (107) Bhattacharya-Chatterjee M, Foon K A. Anti-idiotype antibody vaccine therapies of cancer. Cancer Treat Res 1998; 94:51-68:51-68.