FIELD OF THE INVENTIONThis invention relates to screening methods for complement-mediated diseases such as age-related macular degeneration and vascular diseases. The invention finds application in the fields of biology and medicine.
BACKGROUND OF THE INVENTIONComplement Factor H (CFH) is a multifunctional protein that acts as a key regulator of the complement system. See
Zipfel, 2001, "Factor H and disease: a complement regulator affects vital body functions" Semin Thromb Hemost. 27:191-9. The Factor H protein activities include: (1) binding to C-reactive protein (CRP), (2) binding to C3b, (3) binding to heparin, (4) binding to sialic acid; (5) binding to endothelial cell surfaces, (6) binding to cellular integrin receptors (7) binding to pathogens, including microbes (see
Figure 3 of
U.S. patent publication No. 20070020647), and (8) C3b co-factor activity.Factor H has been associated with atypical haemolytic uremic syndrome; see
Perez-Caballero et al, 2001, "Clustering of missense mutations in the C-terminal region of Factor H in atypical haemolytic uremic syndrome" Am J. Hum. Genet. 68:478-484; and
Heinen et al, 2006, "De novo gene conversion in the RCA gene cluster (1q32) causes mutations in complement Factor H associated with atypical haemolytic uremic syndrome" Human Mutation 27(3): 292-293. Factor H has also been associated with membranoproliferative glomerulonephritis, see
Abrem-Abeleda et al, 2006, "Variations in the complement regulatory genes Factor H (CFH) and factor H related 5 (CFHR5) are associated with membranoproliferative glomerulonephritis type II (dense deposit disease)" J. Med Genet 43: 582-589. Factor H has been shown to be associated with AMD: see
Hageman et al, 2005, "A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration" Proc. Natl. Acad. Sci. USA, 102(20):7227-7232;
Haines et al, 2005, "Complement Factor H variant increases the risk of age-related macular degeneration" Science, 308: 419-421;
Klein et al, 2005, "Complement Factor H polymorphism in age-related macular degeneration" Science, 308: 385-389;
Edwards et al, 2005, "Complement Factor H polymorphism in age-related macular degeneration" Science, 308: 421-424; and
WO 2007/144621 (which falls under Article 54(3) EPC). The Factor H gene, known as
HFI,
CFH and
HF, is located on human chromosome 1, at position 1q32. The 1q32 locus contains a number of complement pathway-associated genes. One group of these genes, referred to as the regulators of complement activation (RCA) gene cluster, contains the genes that encode Factor H, five Factor H-related proteins (FHR-1, FHR-2, FHR-3, FHR-4 and FHR-5 or CFHR1, CFHR2,CFHR3, CFHR4 and CFHR5, respectively), and the gene encoding the beta subunit of coagulation factor XIII. The Factor H and Factor H related proteins are composed almost entirely of short consensus repeats (SCRs). Factor H and FHL1 are composed of SCRs 1-20 and 1-7, respectively. FHR-1, FHR-2, FHR-3, FHR-4 and FHR-5 are composed of 5, 4, 5, 5 and 8 SCRs, respectively. The order of genes, from centromere to telomere is
FH/
FHL1,
FHR-3,
FHR-1,
FHR-4,
FHR-2 and
FHR-5.
The Factor H cDNA encodes a polypeptide 1231 amino acids in length having an apparent molecular weight of 155 kDa (seeRipoche et al., 1988, Biochem J 249:593-602). There is an alternatively spliced form of Factor H known as FHL-1 (and also has been referred to as HFL1 or CFHT). FHL-1 corresponds essentially to exons 1 through 9 of Factor H (seeRipoche et al., 1988, Biochem J 249:593-602). The FHL1 cDNA encodes a polypeptide 449 amino acids in length having an apparent molecular weight of 45-50 kDa. The first 445 amino acids of FH1 and FHL1 are identical, with FHL1 having four unique C-terminal amino acids (encoded by alternative exon 10A, which is located in the intron between exon 9 and exon 10. cDNA and amino acid sequence data for human Factor H and FHL1 are found in the EMBL/GenBank Data Libraries under accession numbers Y00716 and X07523, respectively. The 3926 base nucleotide sequence of the reference form of human Factor H cDNA has GenBank accession number Y00716 and the polypeptide has GenBank accession number Y00716. The 1658 base nucleotide sequence of the reference form of HFL1, the truncated form of the human Factor H, has GenBank accession number X07523, and the polypeptide sequence has GenBank accession number X07523. The Factor H gene sequence (150626 bases in length) has GenBank accession number AL049744. The Factor H promoter is located 5' to the coding region of the Factor H gene.
FHR-1 GeneThe FHR-1 gene is also known asCFHR1,CFHL1,CFHL,FHR1 andHFL1. The FHR-1 cDNA encodes a polypeptide 330 amino acids in length having an predicted molecular weight of 39 kDa (seeEstaller et al., 1991, J. Immunol. 146:3190-3196). cDNA and amino acid sequence data for human FHR-1 are found in the EMBL/GenBanlc Data Libraries under accession number M65292. The FHR-1 gene sequence is found under GenBank accession number AL049741.
FHR-2 GeneThe FHR-2 gene is also known asCFHR2,CFHL2,FHR2 andHFL3. The FHR-2 cDNA encodes a polypeptide 270 amino acids in length having a predicted molecular weight of 31 kDa (seeStrausberg et al., Proc. Natl. Acad. Sci USA 99:16899-16903). cDNA and amino acid sequence data for human FHR-2 are found in the EMBL/GenBank Data Libraries under accession number BC022283. The FHR-2 gene sequence is found under GenBank accession number AL139418.
FHR-3 GeneThe FHR-3 gene is also known asCFHR3,CFHL3,FHR3 andHLF4. The FHR-3 cDNA encodes a polypeptide 330 amino acids in length having a predicted molecular weight of 38 kDa (seeStrausberg et al., Proc. Natl. Acad. Sci USA 99:16899-16903). cDNA and amino acid sequence data for human FHR-3 are found in the EMBL/GenBank Data Libraries under accession number BC058009. The FHR-3 gene sequence is found under GenBank accession number AL049741.
FHR-4 GeneThe FHR-4 gene is also known asCFHR4,CFHL4 andFHR4. The FHR-4 cDNA encodes a polypeptide 331 amino acids in length having a predicted molecular weight of 38 kDa (seeSkerka et al., 1991, J. BioL Chem. 272:5627-5634). cDNA and amino acid sequence data for human FHR-4 are found in the EMBL/GenBank Data Libraries under accession number X98337. The FHR-4 gene sequence is found under GenBank accession numbers AF190816 (5' end), AL139418 (3' end) and BX248415.
FHR-5 GeneThe FHR-5 gene is also known asCFHR5,CFHL5 andFHR5. The CFHR5 cDNA encodes a polypeptide 569 amino acids in length having an apparent molecular weight of 65 kDa (seeMcRae et al., 2001, J. Biol. Chem. 276:6747-6754). cDNA and amino acid sequence data for human CFER5 are found in the EMBL/GenBank Data Libraries under accession number AF295327. The 2821 base nucleotide sequence of the reference form of humanCFHR5 has GenBank accession number AF295327, and the polypeptide sequence has GenBank accession number AAK15619. The CFHR5 genomic sequence is found under GenBank accession numbers AL139418 (5' end) and AL353809 (3' end). The FHR-5 promoter is located 5' to the coding region of the CFHR5 gene.
BRIEF SUMMARY OF THE INVENTIONThe present invention provides the following Embodiments ("E"):
- E 1. A screening method for determining a human subject's propensity to develop a vascular disorder and/or age-related macular degeneration (AMD) comprising:detecting the presence or absence in chromosome 1 of the subject of a deletion that encompasses at least a portion of the complement Factor H-related 3 (CFHR3) gene and at least a portion of the Factor H-related 1 (CFHR1) genewherein the presence of the deletion indicates the subject is at increased risk of developing a vascular disorder and is at decreased risk of developing AMD.
- E 2. The method of E 1 wherein entire protein coding region of the CFHR3 gene is deleted and wherein entire protein coding region of the CFHR1 gene is deleted.
- E 3. The method of E 1 or E 2 wherein the method comprises assaying for a CFHR1 gene product, where the absence of a gene product, or a reduced level of expression of the gene product, is indicative of the deletion.
- E 4. The method of any preceding E wherein the method comprises assaying for a CFHR3 gene product, where the absence of a gene product, or a reduced level of expression of the gene product, is indicative of the deletion.
- E 5. The method of E 3 or E 4 wherein the gene product is a protein.
- E 6. The method of E 5 wherein the protein is detected using an immunoassay or mass spectroscopy.
- E 7. The method of E 1 wherein the method comprises assaying for a truncated CFHR1 gene product, where detection of a truncated gene product is indicative of the deletion.
- E 8. The method of E 1 or E 7 wherein the method comprises assaying for a truncated CFHR3 gene product, where detection of a truncated gene product is indicative of the deletion.
- E 9. The method of E 1 or E 2 wherein the presence or absence of the deletion is detected by analyzing a chromosome or nucleic acid from the subject.
- E 10. The method of E 9 wherein the nucleic acid is DNA or RNA.
- E 11. The method of E 1 wherein the method comprises detecting a deletion of an intragenic sequence selected from a sequence between the CFHR3 gene and the CFHR1 gene and a sequence between the CFHR1 gene and the CFHR4 gene.
- E 12. The method of E 1 wherein the subject is homozygous for the deletion.
- E 13. The method or E 1 wherein the subject has been diagnosed with AMD.
- E 14. The method of E 1 wherein the method comprises analyzing a blood, serum, urine or tissue sample obtained from the patient.
- E 15. The method of E 1 wherein the subject has a genotype of T at position 1277 of the coding region of the CFH gene of the chromosome comprising the deletion.
- E 16. The method of E 1 further comprising detecting genetic variants of complement factor H (CFH) gene comprising detecting one or a plurality of polymorphic sites selected from the group consisting of:a) any one or more of rs529825; rs800292; rs3766404; rs1061147; rs1061170; and rs203674;b) any one or more of intron 2 (IVS2 or insTT); rs2274700; exon 10A; and rs375046;c) one or both of rs529825 and rs800292;d) one or more of rs1061147, rs1061170 and rs203674;e) at least one of rs529825 and rs800292; and rs3766404; and at least one of rs1061147; rs1061170; and rs203674;f) at least rs529825, rs800292, rs3766404, rs1061170 and rs203674;g) exon 22 (R1210C); andh) exon 22 (R1210C) and any of (a)-(g).
- E 17. The method of E 1 further comprising detecting a macular degeneration-associated molecule selected from the group consisting of fibulin-3, vitronectin, β-crystallin A2, β-crystallin A3, β-rystallin A4, β-crystallin S, glucose-regulated protein 78kD (GRP-78), calreticulin, 14-3-3 protein epsilon, serotransferrin, albumin, keratin, pyruvate carboxylase, villin 2, complement 1 q binding protein/hyaluronic acid binding protein ("complement 1 q complement"), amyloid A (al amyloid A), amyloid P component, C5 and C5b-9 terminal complexes, HLA-DR, fibrinogen, Factor X, prothrombin, complements 3, 5 and 9, complement reactive protein (CRP), HLA-DR, apolipoprotein A, apolipoprotein E, antichymotrypsin, p2 microgobulin, thrombospondin, elastin, collagen, ICAM-1, LFA1, LFA3, B7, IL-1, IL-6, IL-12, TNF-alpha, GM-CSF, heat shock proteins, colony stimulating factors (GM-CSF, M-CSFs), and IL-10.
- E 18. The method of E 1 further comprising detecting genetic variants of the HTRA1 gene comprising detecting a polymorphic site selected from the group consisting of:at least one of rs10490924; rs11200638, rs760336, and rs763720.
- E 19. The method of E 1 further comprising detecting genetic variants of the complement factor B (BF) gene and/or the complement component 2 (C2) gene comprising detecting a polymorphic site selected from the group consisting of:a) A or G at rs641153 of the BF gene, or R or Q at position 32 of the BF protein;b) A or T at rs4151667 of the BF gene, or L or H at position 9 of the BF protein;c) G or T at rs547154 of the C2 gene; andd) C or G at rs9332379 of the C2 gene, or E of D at position 318 of the C2 protein.
BRIEF DESCRIPTION OF THE FIGURESFigure 1 is a diagram showing the organization of the regulators-of-complement-activation (RCA) gene cluster on chromosome 1q32 and the arrangement of approximately 60-amino acid domains known as short consensus repeats (SCRs) in complement Factor H (CFH), Factor H-Like 1 (CFHL1) and Factor H-Related 1, 2, 3, 4 and 5 (CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5). CFH has 20 SCRs. The interacting partners with some of these SCRs has been determined and is shown on the top right (CRP, C reactive protein; Hep, heparin). Complement factor H-like 1 (CFHL1) is a splice isoform of CFH, while complement factor H-related proteins 1-5 (CFHR1-5) are each encoded by a unique gene (CFHR1-5). The SCRs of CFHR1 -5 are similar to some of the SCRs in CFH, as denoted by the numbers in the ovals. For example, CFHR5 has 9 SCRs, with the first two being similar to SCRs 6 and 7 of Factor H and therefore having CRP and heparin binding properties. SCRs 5-7 of CFHR5 have the numbers 12-14 within the corresponding ovals because these SCRs are similar to SCRs 12-14 of Factor H and have C3b and heparin binding properties.
Figure 2 shows regions of homology (genomic duplications) in the genes encoding CFH and the Factor H-related proteins. Exons are indicated as vertical lines. Regions labeled with the same letter (e.g., A, A', and A") have substantially identical sequences.
Figure 3 shows a Western blot of serum proteins from seven patients using an anti-human CFH antibody. FHL-1, CFHR1 and CFHR2 indicate the positions of the truncated form of CFH, CFHR1 and CFHR2, respectively. The anti-human CFH antibody employed also cross-reacts with CFHR1 and CFHR2. No CFHR1 is detected in the serum of two patients (197-02 and 325-02) that have a homozygous deletion of theCFHR3 andCFHR1 genes, as determined by SSCP analysis and direct DNA sequencing.
Figure 4 shows a SSCP analysis of theCFH,CFHR3 andCFHR1 genes. 1, 2, 3, and 4 indicate four different SSCP patterns observed using primers from exon 22 of theCFH gene to PCR amplify DNA. SSCP patterns 1, 2 and 3 correspond to homozygous non-deletion or heterozygous deletion ofCFHR3 andCFHR1, and pattern 4 corresponds to homozygous deletion ofCFHR3 andCFHR1.
Figure 5 shows a PCR analysis of theCFH andCFH-related genes 1 to 5 in leukocytes from 20 patients that are separated into four groups according to the SSCP patterns using theCFH exon 22 primers (patterns 1-4 are as described inFigure 4). From left to right, in each panel (gel), 5 leukocyte-derived DNA samples each from patients displaying SSCP patterns 1, 2, 3 and 4 were subjected to PCR using primers specific forCFH, CFHR1, CFHR2, CFHR3, CFHR4 andCFHR5, as indicated. When SSCP analysis and direct DNA sequencing show a homozygous deletion of theCFHR3 andCFHR1 genes, no PCR amplifiableCFHR3 andCFHR1 DNA are detected.
Figure 6 shows an amino acid alignment of the CFH (SEQ ID NO: 2), CFHR1 (SEQ ID NO: 4), and CFHR3 (SEQ ID NO: 6) proteins.
Figure 7 shows a nucleotide alignment of the CFH (SEQ ID NO: 1), CFHR1 (SEQ ID NO: 3), and CFHR3 genes (SEQ ID NO: 5).
DETAILED DESCRIPTION1. DefinitionsThe following definitions are provided to aid in understanding the invention. Unless otherwise defined, all terms of art, notations and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the arts of medicine and molecular biology. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not be assumed to represent a substantial difference over what is generally understood in the art.
A "vascular disorder" is a disease or condition of the vascular system. One type of vascular disorder is an aneurysm such as abdominal aortic aneurysm or brain intracranial aneurysm. Other types of vascular disorder include hypertension, cerebral vascular accidents, trans-ischemic accidents (e.g., stroke). Still other types of vascular disorders include coronary artery disease, peripheral artery disease, varicose veins, and peripheral vascular disease.
A "nucleic acid", "polynucleotide" or "oligonucleotide" is a polymeric form of nucleotides of any length, may be DNA or RNA, and may be single- or double-stranded. Nucleic acids may include promoters or other regulatory sequences. Oligonucleotides are usually prepared by synthetic means. A reference to the sequence of one strand of a double-stranded nucleic acid defines the complementary sequence and except where otherwise clear from context, a reference to one strand of a nucleic acid also refers to its complement. For certain applications, nucleic acid (e.g., RNA) molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. Modified nucleic acids include peptide nucleic acids (PNAs) and nucleic acids with nontraditional bases such as inosine, queosine and wybutosine and acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
"Hybridization probes" are nucleic acids capable of binding in a base-specific manner to a complementary strand of nucleic acid. Such probes include nucleic acids and peptide nucleic acids (Nielsenet al., 1991). Hybridization may be performed under stringent conditions which are known in the art. For example, see,e.g.,Berger and Kimmel (1987) METHODS IN ENZYMOLOGY, VOL. 152: GUIDE To MOLECULAR CLONING TECHNIQUES, San Diego: Academic Press, Inc.;Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Vols. 1-3, Cold Spring Harbor Laboratory; Sambook (2001) 3rd Edition;Rychlik, W. and Rhoads, R.E., 1989, Nucl. Acids Res. 17, 8543;Mueller, P.R. et al. (1993) In: CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 155, Greene Publishing Associates, Inc. and John Wiley and Sons, New York; andAnderson and Young, QUANTITATIVE FILTER HYBRIDIZATION IN NUCLEIC ACID HYBRIDIZATION (1985)). As used herein, the term "probe" includes primers. Probes and primers are sometimes referred to as "oligonucleotides."
The term "primer" refers to a single-stranded oligonucleotide capable of acting as a point of initiation of template-directed DNA synthesis under appropriate conditions, in an appropriate buffer and at a suitable temperature. The appropriate length of a primer depends on the intended use of the primer but typically ranges from 15 to 30 nucleotides. A primer sequence need not be exactly complementary to a template but must be sufficiently complementary to hybridize with a template. The term "primer site" refers to the area of the target DNA to which a primer hybridizes. The term "primer pair" means a set of primers including a 5' upstream primer, which hybridizes to the 5' end of the DNA sequence to be amplified and a 3' downstream primer, which hybridizes to the complement of the 3' end of the sequence to be amplified.
Exemplary hybridization conditions for short probes and primers is about 5 to 12 degrees C below the calculated Tm. Formulas for calculating Tm are known and include: Tm = 4°C x (number of G's and C's in the primer) + 2°C x (number of A's and T's in the primer) for oligos <14 bases and assumes a reaction is carried out in the presence of 50mM monovalent cations. For longer oligos, the following formula can be used: Tm = 64.9°C + 41°C x (number of G's and C's in the primer - 16.4)/N, where N is the length of the primer. Another commonly used formula takes into account the salt concentration of the reaction (Rychlik, supra, Sambrook, supra, Mueller, supra.): Tm = 81.5°C + 16.6°C x (log10[Na+] + (K+]) + 0.41°C x (%GC) - 675/N, where N is the number of nucleotides in the oligo. The aforementioned formulae provide a starting point for certain applications; however, the design of particular probes and primers may take into account additional or different factors. Methods for design of probes and primers for use in the methods of the invention are well known in the art.
The term "polymorphism" refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A "polymorphic site" is the locus at which sequence divergence occurs. Polymorphic sites have at least two alleles. A diallelic polymorphism has two alleles. A triallelic polymorphism has three alleles. Diploid organisms may be homozygous or heterozygous for allelic forms. A polymorphic site may be as small as one base pair. Examples of polymorphic sites include: restriction fragment length polymorphisms (RFLPs); variable number of tandem repeats (VNTRs); hypervariable regions; minisatellites; dinucleotide repeats; trinucleotide repeats; tetranucleotide repeats; and simple sequence repeats. As used herein, reference to a "polymorphism" can encompass a set of polymorphisms (i.e., a haplotype).
A "single nucleotide polymorphism (SNP)" occurs at a polymorphic site occupied by a single nucleotide, which is the site of variation between allelic sequences. The site is usually preceded by and followed by highly conserved sequences of the allele. A SNP usually arises due to substitution of one nucleotide for another at the polymorphic site. Replacement of one purine by another purine or one pyrimidine by another pyrimidine is called a transition. Replacement of a purine by a pyrimidine or vice versa is called a transversion. A synonymous SNP refers to a substitution of one nucleotide for another in the coding region that does not change the amino acid sequence of the encoded polypeptide. A non-synonymous SNP refers to a substitution of one nucleotide for another in the coding region that changes the amino acid sequence of the encoded polypeptide. A SNP may also arise from a deletion or an insertion of a nucleotide or nucleotides relative to a reference allele.
The term "deletion," when referring to a nucleic acid sequence, has the usual meaning in genetics of an allele in which one or more bases are missing compared to a reference or wild-type sequence. Deletions may be as short as one base-pair. Deletions detected in the present invention may be longer, such as a deletion of at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, at least 1000 bp, at least 1100 bp, at least 1200 bp, at least 1300 bp, at least 1400 bp, at least 1500 bp, at least 1600 bp, at least 1700 bp, at least 1800 bp, at least 1900 bp, at least 2000 bp, at least 2500 bp, at least 3000 bp, at least 3500 bp, at least 4000 bp, at least 4500 bp, at least 5000 bp, at least 6000 bp, at least 7000 bp, at least 8000 bp, at least 9000 bp, at least 10,000 bp, at least 15,000 bp, at least 20,000 bp, at least 30,000 bp, at least 40,000 bp, at least 50,000 bp, at least 75,000 bp, at least 100,000 bp, at least 125,000 bp, at least 150,000 bp, at least 200,000 bp or at least 250,000 bp.
The term "haplotype" refers to the designation of a set of polymorphisms or alleles of polymorphic sites within a gene of an individual. For example, a "112" Factor H haplotype refers to the Factor H gene comprising allele 1 at each of the first two polymorphic sites and allele 2 at the third polymorphic site. A "diplotype" is a haplotype pair.
An "isolated" nucleic acid means a nucleic acid species that is the predominant species present in a composition. Isolated means the nucleic acid is separated from at least one compound with which it is associated in nature. A purified nucleic acid comprises (on a molar basis) at least about 50, 80 or 90 percent of all macromolecular species present.
Two amino acid sequences are considered to have "substantial identity" when they are at least about 80% identical, preferably at least about 90% identical, more preferably at least about 95%, at least about 98% identical or at least about 99% identical. Percentage sequence identity is typically calculated by determining the optimal alignment between two sequences and comparing the two sequences. Optimal alignment of sequences may be conducted by inspection, or using the local homology algorithm ofSmith and Waterman, 1981, Adv. Appl. Math. 2: 482, using the homology alignment algorithm ofNeedleman and Wunsch, 1970, J. Mol. Biol. 48: 443, using the search for similarity method ofPearson and Lipman, 1988, Proc. Natl. Acad. Sci. U.S.A. 85: 2444, by computerized implementations of these algorithms (e.g., in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.) using default parameters for amino acid comparisons (e.g., for gap-scoring,etc.). It is sometimes desirable to describe sequence identity between two sequences in reference to a particular length or region (e.g., two sequences may be described as having at least 95% identity over a length of at least 500 basepairs). Usually the length will be at least about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 amino acids, or the full length of the reference protein. Two amino acid sequences can also be considered to have substantial identity if they differ by 1, 2, or 3 residues, or by from 2-20 residues, 2-10 residues, 3-20 residues, or 3-10 residues.
"Linkage" describes the tendency of genes, alleles, loci or genetic markers to be inherited together as a result of their location on the same chromosome. Linkage can be measured by percent recombination between the two genes, alleles, loci or genetic markers. Typically, loci occurring within a 50 centimorgan (cM) distance of each other are linked. Linked markers may occur within the same gene or gene cluster. "Linkage disequilibrium" or "allelic association" means the preferential association of a particular allele or genetic marker with a specific allele or genetic marker at a nearby chromosomal location more frequently than expected by chance for any particular allele frequency in the population. A marker in linkage disequilibrium can be particularly useful in detecting susceptibility to disease, even if the marker itself does not cause the disease.
The terms "susceptibility," "propensity," and "risk" refer to either an increased or decreased likelihood of an individual developing a disorder (e.g., a condition, illness, disorder or disease) relative to a control population. In one example, the control population may be individuals in the population (e.g., matched by age, gender, race and/or ethnicity) without the disorder, or without the genotype or phenotype assayed for. In some contexts, the terms diagnosing and screening are used interchangeably (e.g., a person skilled in the art can diagnose a propensity to develop the disease).
The term "diagnose" and "diagnosis" refer to the ability to determine or identify whether an individual has a particular disorder (e.g., a condition, illness, disorder or disease).
The term "screen" or "screening" as used herein has a broad meaning. It includes processes intended for the diagnosis or for determining the susceptibility, propensity, risk, or risk assessment of an asymptomatic subject for developing a disorder later in life. Screening also includes the prognosis of a subject, i.e., when a subject has been diagnosed with a disorder, determinating in advance the progress of the disorder as well as the assessment of efficacy of therapy options to treat a disorder.
The terms "portion," "fragment" and/or "truncated form" when used in reference to a Factor H-related gene product (e.g., CFHR3 or CFHR1 gene product), refers to a nucleic acid or polypeptide sequence that is less than the full-length sequence (i.e., a portion of the full-length gene or polypeptide). A portion or fragment or truncated form of CFHR3 or CFHR1 gene or polypeptide can be at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, or at least 300 nucleotides or amino acids in length. Typically the portion includes at least 1, often at least two, and sometimes at least 3 or 4 complete SCRs.
As used herein, the term "gene product" means an RNA (e.g., mRNA) or protein that is encoded by the gene. A "protein coding region" is a region of DNA/RNA sequence within a gene that encodes a polypeptide or protein.
An "assay" is a procedure wherein the presence or amount or a property of a test substance, e.g., a nucleic acid or gene product, is detected or measured.
The terms "inhibit" and "reduce" refer to any inhibition, reduction, or decrease in expression or activity including partial or complete inhibition of gene expression or gene product activity.
2. Association of Polymorphisms in the CFHR1 And CFHR3 Genes and Risk Of Developing AMD and Vascular DisordersA correlation between polymorphic sites and haplotypes in the
CFH gene and the likelihood of developing AMD has been discovered. See
Hageman et al., 2005, Proc. Natl. Acad Sci. U.S.A. 102:7227-32;
Haines et al., 2005, Science 308:419-21;
Klein et al., 2005, Science 308:385-9;
Edwards et al., 2005, Science 308:421-4 and
U.S. patent publication No. 20070020647.Both
CFH risk haplotypes and
CFH protective haplotypes are known. Polymorphisms particularly associated with increased risk include a variant allele at: rs 1061170 (402H; exon 9); rs203674 (intron 10) and the polymorphism at residue 1210 (1210C; exon 22). Polymorphisms particularly associated with decreased risk include the protective H2 haplotype, which includes a variant allele in IVS6 (intron 6, rs3766404) and the H4 haplotype, which includes a variant allele in IVS I (intron 1, rs529825) and a variant allele (162) (exon 2, rs800292).
It has now been discovered that an AMD protective haplotype is genetically linked to deletions in the DNA sequence between the 3' end of exon 22 of the complement factor H (CFH) gene and the 5' end of exon I of complement Factor H-related 4(CFHR4) gene on human chromosome I (i.e., the DNA sequence encoding the CFHR1 and CFHR3 proteins). See Example 1,infra. The discovery that deletions at theCFHR1 andCFHR3 loci are associated with decreased risk of developing AMD has a number of specific applications, including screening individuals to ascertain risk of developing AMD and identification of new and optimal therapeutic approaches for individuals afflicted with, or at increased risk of developing, AMD. As discussued in Example 1, below, the deletion genotype is predominantly associated with theCFH H4 haplotype. SeeHageman et al., 2005, Proc. Natl. Acad Sci. U.S.A. 102:7227-32. Thus, this deletion acts as a marker for decreased risk of conditions for which the H4 haplotype is protective.
Moreover, it has now been discovered that deletions in the DNA sequence between the 3' end of exon 22 of the complement factor H (CFH) gene and the 5' end of exon 1 of complement Factor H-related 4 (CFHR4) gene on human chromosome I (i.e., the DNA sequence encoding the CFHR1 and CFHR3 proteins) are associated with increased risk of developing a vascular disease such as aortic aneurysm. See Example 1,infra. The discovery that deletions at theCFHR1 andCFHR3 loci are associated with increased risk of developing a vascular disorder has a number of specific applications, including screening individuals to ascertain risk of developing a vascular disorder and identification of new and optimal therapeutic approaches for individuals afflicted with, or at increased risk of developing, vascular disorders.
3. Screening MethodsBased on the discoveries described herein, a subject's risk for AMD or vascular disease can be assessed by determining whether or not a the subject has a deletion within the region of chromosome 1 lying between the 3' end of exon 22 of the complement factor H(CFH) gene and the 5' end of exon 1 of complement Factor H-related 4 (CFHR4). The extent of the deletion may vary in different individuals or populations. For example, in one embodiment the all of most of the region between CFH exon 22 and CFHR4 exon 1 is deleted. Alternatively, a portion of the region may be deleted, such as, for example, a deletion of less than the entire region between CFH exon 22 and CFHR4 exon 1 but including a portion of the CFHR1 encoding sequence, and including a portion of the CFHR3 encoding sequence.An individual may be homozygous for deletion (both chromosomes 1 have a deletion in the region) or may be heterozygous for deletion.
For example and not limitation, the homozygous deletion ofCFHR1 and/orCFHR3 can be detected from the absence of CFHR1 and/or CFHL3 protein in a body fluid or tissue sample (seeFigure 3), by the absence of RNA encoded in the region between the 3' end of CFH exon 22 and the 5' end of CFHR4 exon 1 (e.g., absense of absence of CFHR1 and/or CFHL3 mRNAs), or by absense of genomic DNA in the region in the region between the 3' end of CFH exon 22 and the 5' end of CFHR4 exon 1. The present or absense of DNA or RNA sequences can be determined using art known methods, such as PCR. The absense of a nucleic acid sequence is deduced from the absense of an amplified PCR product in an assay of a tissue sample (seeFigure 5). It will be understood that, although PCR is frequently cited herein as a method for genetic analysis, many other analytical methods are known and are suitable for detection of a deletion. For example and not limitation several are described below in the section captioned "Analysis of Nucleic Acid Samples."
The heterozygous deletion ofCFHR1 and/orCFHR3 can be determined, for illustration and not limitation, (1) from a reduction in the amount of protein in a body fluid or tissue sample as compared to the amount from a control having both alleles ofCFHR1 and/orCFHR3 genes, (2) from a reduction in the amount of RNA, DNA, or amplified PCR product in a tissue sample as compared to the amount from similar sample of a homozygote without the deletion, or (3) by an assay using direct DNA sequencing, quantitative PCR or other methods known in the art. For example, the amount of a gene product may be reduced in a heterozygote by at least 10%, at least 20%, at least 30%, about 50% or or more compared to a homozygote without the deletion. Quantitative PCR and methods are available that would be able to detect a two-fold difference in mRNA or DNA in a sample.
As noted, a deletion lies in the region between CFH exon 22 and CFHR4 exon 1 but need not span the entire region. Deletions of a portion of the CFHR1 and/or CFHR3 genes ("partial deletions") may result in truncated forms of CFHR1 and/or CFHR3 RNAs and polypeptides. Such partial deletions can be identified by a difference in size of a protein in a body fluid or tissue sample compared to the full-length protein, by detecting a size difference in the RNA, and by various methods well known in the art, including PCR amplification of DNA or RNA in a biological sample using primers selected to distinguish between a nucleic acid comprising a deletion and a nucleic acid not containg a deletion. Methods known in the art can be used to distinguish homozygotes from heterozygotes (see, e.g., Example 1).
The selection, design and manufacture of suitable primers or probes for analysis of nucleic acid is well known in the art. A person of ordinary skill in the art can use suitable combinations of primers to detect deletions. In an embodiment, the primers or probes are designed to hybridize at any position in the DNA sequence between the 3' end of exon 22 of the complement factor H(CFH) gene and the 5' end of exon 1 of complement Factor H-related 4(CFHR4) gene. For instance, both primers may be located in the CHFR3 gene to detect its presence or absence. In another example. In other examples, one or more primers are located within intergenic (non-coding) sequence,e.g., intergenic sequence between betweenCFHR3 andCFHR1 or betweenCFHR1 andCFHR4.
In another embodiment, the invention includes a method of detecting a nonreciprocal transfer of genetic information, such as gene conversion. In one instance, the gene conversion results in replacement of a 3' portion of the CFH gene with a portion of the 3' CFHR1 gene, such that a chimeric protein with sequence derived from both the CFH gene and the CFHR1 gene is produced.
3.1 Analysis of Nucleic Acid SamplesMethods for detection of polymorphisms and deletions in genetic sequences are well known in the art and can be adapted for use in the present invention.
In one embodiment, genomic DNA is analyzed. For assay of genomic DNA, virtually any biological sample containing genomic DNA or RNA,e.g., nucleated cells, is suitable. For example, genomic DNA can be obtained from peripheral blood leukocytes collected from case and control subjects (QIAamp DNA Blood Maxi kit, Qiagen, Valencia, CA). Other suitable samples include saliva, cheek scrapings, biopsies of retina, kidney, skin, or liver or other organs or tissues; amniotic fluid, cerebral spinal fluid (CSF) samples; and the like. Alternatively RNA or cDNA can be assayed. Methods for purification or partial purification of nucleic acids from patient samples for use in diagnostic or other assays are well known
Methods for detecting polymorphisms and deletions in nucleic acids include, without limitation, Southern blot analysis (see
Kees et al., "Homozygous Deletion of the p16/MTS1 Gene in Pediatric Acute Lymphoblastic Leukemia Is Associated With Unfavorable Clinical Outcome," Blood 89:4161-4166,
Fizzotti et al., "Detection of homozygous deletions of the cyclin-dependent kinase 4 inhibitor (p16) gene in acute lymphoblastic leukemia and association with adverse prognostic features," Blood 85(10):2685-2690,
Kitada et al., "Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism," Nature 392 (9):605-608); Northern Blot Analysis (see
Fieschi et al., "A novel form of complete IL-12/IL-23 receptor b1 deficiency with cell surface-expressed nonfunctional receptors," Immunobiology 104(7):2095-2101) and amplification based method such as PCR-based methods are used to detect deletions in samples. PCR primers may be designed to target DNA sequences flanking a known mutation, in which a change in PCR product size in comparison to amplification reactions using WT DNA identifies a mutant template. Primers may also be targeted to deleted sequences, wherein an absence of a PCR product identifies a mutant template (
Kitada et al., "Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism," Nature 392:605-608) including multiplex PCR (
Chong et al., "Single-tube multiplex-PCR screen for common deletional determinants of α-thalassemia," Blood 95 (1):360-362).
Polymorphisms (e.g., deletions) can also be detected using allele-specific probes; use of allele-specific primers; direct sequence analysis; denaturing gradient gel electropohoresis (DGGE) analysis; single-strand conformation polymorphism (SSCP) analysis; and denaturing high performance liquid chromatography (DHPLC) analysis. Other well known methods to detect polymorphisms in DNA include use of: Molecular Beacons technology (see,
e.g.,Piatek et al., 1998; Nat. Biotechnol. 16:359-63;
Tyagi, and Kramer, 1996, Nat. Biotechnology 14:303-308; and
Tyagi, et al., 1998, Nat. Biotechnol. 16:49-53), Invader technology (see,
e.g.,Neri et al., 2000, Advances in Nucleic Acid and Protein Analysis 3826:117-125 and
U.S. Patent No. 6,706,471), nucleic acid sequence based amplification (Nasba) (Compton, 1991), Scorpion technology (
Thelwell et al., 2000, Nuc. Acids Res, 28:3752-3761 and
Solinas et al., 2001, "Duplex Scorpion primers in SNP analysis and FRET applications" Nuc. Acids Res, 29:20), restriction fragment length polymorphism (RFLP) analysis, and the like.
The design and use of allele-specific probes for analyzing polymorphisms are described by
e.g., Saiki
et al., 1986;
Dattagupta, EP 235,726; and
Saiki, WO 89/11548. Briefly, allele-specific probes are designed to hybridize to a segment of target DNA from one individual but not to the corresponding segment from another individual, if the two segments represent different polymorphic forms. Hybridization conditions are chosen that are sufficiently stringent so that a given probe essentially hybridizes to only one of two alleles. Typically, allele-specific probes are designed to hybridize to a segment of target DNA such that the polymorphic site aligns with a central position of the probe.
Exemplary probes for analyzing deletions and polymorphisms are shown in Table 1 of Example 1, but many others may be designed by one of skill.
Allele-specific probes are often used in pairs, one member of a pair designed to hybridize to the reference allele of a target sequence and the other member designed to hybridize to the variant allele. Several pairs of probes can be immobilized on the same support for simultaneous analysis of multiple polymorphisms within the same target gene sequence.
The design and use of allele-specific primers for analyzing polymorphisms are described by, e.g.,
WO 93/22456 and Gibbs, 1989. Briefly, allele-specific primers are designed to hybridize to a site on target DNA overlapping a polymorphism and to prime DNA amplification according to standard PCR protocols only when the primer exhibits perfect complementarity to the particular allelic form. A single-base mismatch prevents DNA amplification and no detectable PCR product is formed. The method works best when the polymorphic site is at the extreme 3'-end of the primer, because this position is most destabilizing to elongation from the primer.
Amplification products generated using PCR can be analyzed by the use of denaturing gradient gel electrophoresis (DGGE). Different alleles can be identified based on sequence-dependent melting properties and electrophoretic migration in solution. SeeErlich, ed., PCR Technology, Principles and Applications for DNA Amplification, Chapter 7 (W.H. Freeman and Co, New York, 1992).
Alleles of target sequences can be differentiated using single-strand conformation polymorphism (SSCP) analysis. Different alleles can be identified based on sequence- and structure-dependent electrophoretic migration of single stranded PCR products (Oritaet al., 1989). Amplified PCR products can be generated according to standard protocols, and heated or otherwise denatured to form single stranded products, which may refold or form secondary structures that are partially dependent on base sequence.
Alleles of target sequences can be differentiated using denaturing high performance liquid chromatography (DHPLC) analysis. Different alleles can be identified based on base differences by alteration in chromatographic migration of single stranded PCR products (Frueh and Noyer-Weidner, 2003, Clin Chem Lab Med. 41 (4):452-61). Amplified PCR products can be generated according to standard protocols, and heated or otherwise denatured to form single stranded products, which may refold or form secondary structures that are partially dependent on the base sequence.
Direct sequence analysis of polymorphisms can be accomplished using DNA sequencing procedures that are well-known in the art. SeeSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL (2nd Ed., CSHP, New York 1989) andZyskind et al., RECOMBINANT DNA LABORATORY MANUAL (Acad. Press, 1988).
Homozygote deletions can be identified by a variety of methods known in the art. For example, in one approach DNA samples are amplified for further analysis. In an embodiment, two CFHR1-specific primer pairs are used, for instance, ("CFHL1 ex6.F" [5'- AGTCGGTTTGGACAGTG -3' (SEQ ID NO: 7)] & "CFHL1ex6R" [5'- GCACAAGTTGGATACTCC -3' (SEQ ID NO: 8)]; and/or "CHFL1ex6.F2" [5'- CATAGTCGGTTTGGACAGTG -3' (SEQ ID NO: 9)] & "CFHL1ex6.R" [5'-GCACAAGTTGGATACTCC -3' (SEQ ID NO: 8)]). In another embodiment, CFHR3-specific primer pairs are used. for instance, ("CFHL3ex3.F" [5'- TCATTGCTATGTCCTTAGG -4' (SEQ ID NO: 10)] & "CFHL3ex3.R" [5'- TCTGAGACTGTCGTCCG -3' (SEQ ID NO: 11)]; and/or "CFHL3ex3seq.F" [5'-TTTTGGATGTTTATGCG -3' (SEQ ID NO: 12)] & "CFHL3ex3seq.R" [5'- AAATAGGTCCGTTGGC -3' (SEQ ID NO: 13)]). Absence of the correct-sized PCR product indicates that the CFHL1 and/or CFHL3 gene(s) are deleted.
Similarly, heterozygote deletions can be identified by a variety of methods known in the art. For example, in one approach DNA samples are amplified for further analysis, for example with the same primers listed above, followed by direct sequencing. Heterozygotes are characterized, for instance, by chromatograms in which one peak is approximately half the height of the second peak (in contrast to equal sized peaks) at the SNP positions (rs460897, rs16840561, rs4230, rs414628 for CFHR1; rs1061170 for CFHR3). In another embodiment, a protocol employing ParAllele genotyping data, a copy number analysis is performed, in which samples that fail to genotype key markers (MRD_3855, MRD_3856, MRD_3857, rs385390, rs389897) in the region of these two genes are identified. All samples assigned a copy number of 0 (designated CN0) allow the haplotypes that contain the deletion to be defined. Having defined a deletion haplotype, linkage disequilibrium is used to infer whether samples could not carry a deletion. Specifically, if a sample is homozygous for a different allele than one that defines the haplotype, then it does not carry a deletion.
3.2 Analysis of Protein SamplesMethods for protein analysis that can be adapted for detection of proteins such as the CFHR1 and CFHR3 gene products and variants or fragments thereof are well known. These methods include analytical biochemical methods such as electrophoresis (including capillary electrophoresis and one- and two-dimensional electrophoresis), chromatographic methods such as high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, mass spectrometry, and various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmnunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Western blotting and others.
For example, a number of well established immunological binding assay formats suitable for the practice of the invention are known (see,e.g.,Harlow, E.; Lane, D. ANTIBODIES: A LABORATORY MANUAL. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory; 1988; andAusubel et al., (2004) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York NY. The assay may be, for example, competitive or non-competitive. Typically, immunological binding assays (or immunoassays) utilize a "capture agent" to specifically bind to and, often, immobilize the analyte. In one embodiment, the capture agent is a moiety that specifically binds to a variant or wild-type CFHR1 or CFHR3 polypeptide or subsequence (e.g., a fragment or truncated form of CFHR1 or CFHR3). Thebound protein may be detected using, for example, a detectably labeled anti.CFHR1 or enti-GFHR3 antibody. Such antibodies are described in more detail below.
3.3 Screening Using Multiple Polymorphisms and MarkersIn diagnostic methods, analysis of
CFHR1 and/or
CFHR3 polymorphisms can be combined with analysis of polymorphisms in other genes associated with AMD or vascular disease (e.g., AAA), detection of protein markers of AMD (see,
e.g.,
Hageman et al., patent publications US 20030017501;
US 20020102581;
WO0184149; and
WO0106262; and
US patent applications 11/706,154 (entitled "Protective Complement Proteins and Age-Related Macular Degeneration") and
11/706,074 (entitled "Variants in Complement Regulatory Genes Predict Age-Related Macular Regeneration");
Gorin el al., US20060281120; and
Hoh, WO2007/044897, assessment of other risk factors of AMD or vascular disease (such as family history).
For example, analysis of
CFHR1 and/or
CFHR3 polymorphisms (e.g., deletions) can be combined with the analysis of polymorphisms in the Complement Factor H gene (CFH). Genetic variants of the
CFH gene that may be detected include, but are not limited to, a genotype of a T at position 1277 of the coding region of human
CFH, any one or more of rs529825; rs800292; rs3766404; rs1061147; rs1061170; and rs203674; any one of more of intron 2 (IVS2 or insTT); rs2274700; exon 10A; and rs375046; one or both of rs529825 and rs800292; one or more of rs1061147, rs1061170 and rs203674; at least one of rs529825 and rs800292; and rs3766404; and at least one of rs1061147, rs1061170 and rs203674; at least rs529825, rs800292, rs3766404, rs1061170, and rs203674; and/or exon 22 (R1210C). See. e.g.,
Hartman et al., 2006, "HTRA1 promoter polymorphism in wet age-related macular degeneration" Science 314:989-92.
In certain embodiments, the analysis of CFHR1 and/or CFHR3 polymorphisms can be combined with analysis of polymorphisms in the HTRA1 gene (also known as the PRESS11 gene), the complement factor B (BF) gene, and/or the complement component 2 (C2) gene. Genetic variants of the HTRA1 gene that may be detected include, but are not limited to, at least one of rs10490924, rs 1200638, rs760336, and rs763720. Each of the single nucleotide polymorphisms (SNPs) within the HTRA1 gene are associated with increased risk of developing AMD. The genetic variants of the BF gene that may be detected include the presence of an A or G at rs641153 of the BF gene, or an R or Q at position 32 of the BF protein; and/or an A or T at rs4151667 of the BF gene, or L or H at position 9 of the BF protein. The genetic variants of the C2 protein that may be detected include a G or T at rs547154 of the C2 gene; and/or a C or G at rs9332379 of the C2 gene, or E of D at position 318 of the C2 protein. See, e.g.,
Gold et al., 2006 "Variation in factor B (BF) and complement component 2 (C2) genes is associated with age-related macular degeneration"Nat Genet. 38:458-62.
In addition, the analysis of CFHR1 and/or CFHR3 polymorphisms can be combined with an analysis of protein markers associated with AMD. The protein markers may include, but are not limited to, fibulin-3, vitronectin, β-crystallin A2, β-crystallin A3, β-crystallin A4, β-crystallin S, glucose-regulated protein 78 kD (GRP-78), calreticulin, 14-3-3 protein epsilon, serotransferrin, albumin, keratin, pyruvate carboxylase, villin 2, complement I q binding protein/hyaluronic acid binding protein ("Complement 1q component"), amyloid A (al amyloid A), amyloid P component, C5 and CSb-9 terminal complexes, HLA-DR, fibrinogen, Factor X, prothrombin, complements 3,5 and 9, complement reactive protein (CRP), HLA-DR, apolipoprotein A, apolipoprotein E, antichymotrypsin, p2 microglobulin, thrombospondin, elastin, collagen, ICAM-1, LFA1, LFA3, 87, IL-1, IL-6, IL-12, TNF-alpha, GM-CSF, heat shock proteins, colony stimulating factors (GM-CSF, M-CSFs), and IL-10.
4. Antibodies.Anti-CFHR1 or anti-CFHR3 binding agents (e.g., antibodies) are discussed in Section 3.2 above.
In one embodiment, an anti-CFHR1 antibody specifically binds an epitope of CFHR1, in particular human CFHR1. In certain embodiments, an anti-CFHR1 antibody specifically binds an epitope located within the amino-terminus of a CFHR1 polypeptide. In particular, an anti-CFHR1 antibody specifically binds an epitope located between amino acids 1-143 of SEQ ID NO: 4 as shown inFigure 6. In other embodiments, an anti-CFHR1 antibody specifically binds an epitope within the CFHR1 short consensus repeats (SCRs) 6 and/or 7 as shown inFigure 1. The amino acid sequence of CFHR1 SCR6 is 35% homologous to the corresponding CFH SCR, and the amino acid sequence of CFHR SCR7 is 45% homologous to the corresponding CFH SCR. Anti-CFHR1 antibodies of the invention specifically bind CFHR1 and do not cross-react with CFH or other factor H related proteins including CFHT, CFHR2, CFHR3, CFHR4, or CFHR5. A variety of immunoassay formats may be used to select antibodies that are specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with an antigen. SeeHarlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York. Epitope mapping of the CFHR1 protein is within the skill of the art to determine epitopes that are most immunogenic for the generation of anti-CFHR1 antibodies.
In another embodiment, an anti-CFHR3 antibody specifically binds an epitope of CFHR3, in particular human CFHR3. In certain embodiments, an anti-CFHR3 antibody specifically binds an epitope located within the carboxyl-terminus of a CFHR3 polypeptide. For example, an anti-CFHR3 antibody may specifically bind to an epitope between amino acids 144-330 of SEQ ID NO: 6 as shown inFigure 6. In other embodiments, an anti-CFHR3 antibody specifically binds an epitope within the CHFR3 SCRs 8, 19 and/or 20 as shown inFigure 1. The amino acid sequence of CFHR3 SCR8 is 63% homologous to the corresponding CRH SCR, the amino acid sequence of CFHR3 SCR19 is 62% homologous to the corresponding CFH SCR, and the amino acid sequence of CFHR3 SCR20 is 36% homologous to the corresponding CFH SCR. Anti-CFHR3 antibodies specifically bind CFHR3 and do not cross-react with CFH or other factor H related proteins including CFHT, CFHR1, CFHR2, CFHR4, or CFHR5. Epitope mapping of the CFHR3 protein is within the skill of the art to determine epitopes that may be immunogenic for the generation of anti-CFHR3 antibodies.
It is understood that each of the antibodies discussed above can be an intact antibody, for example, a monoclonal antibody. Alternatively, the binding protein can be an antigen binding fragment of an antibody, or can be a biosynthetic antibody binding site. Antibody fragments include Fab, Fab', (Fab')
2 or Fv fragments. Techniques for making such antibody fragments are known to those skilled in the art. A number of biosynthetic antibody binding sites are known in the art and include, for example, single Fv or sFv molecules, described, for example, in
U.S. Patent Nos. 5,476,786. Other biosynthetic antibody binding sites include bispecific or bifunctional binding proteins, for example, bispecific or bifunctional antibodies, which are antibodies or antibody fragments that bind at least two different antigens. For example, bispecific binding proteins can bind CFHR1, CFHR3, and/or another antigen. Methods for making bispecific antibodies are known in art and, include, for example, by fusing hybridomas or by linking Fab' fragments. See, e.g.,
Songsivilai et al. (1990) CLIN. EXP. IMMUNOL. 79: 315-325;
Kostelny et al. (1992) J. IMMINOL. 148: 1547-1553.
Anti-CFHR1 and anti-CFHR3 antibodies can be produced using techniques well known in the art. Monoclonal antibodies can be produced using standard fusion techniques for forming hybridoma cells. SeeG. Kohler, et al., Nature, 256:456 (1975). Alternatively, monoclonal antibodies can be produced from cells by the method ofHuse, et al., Science, 256:1275 (1989).
It is understood that the CDRs of the antibodies described herein confer the binding specificity to CFHR1 or CFHR3. The antibodies described herein can be used as diagnostic agents. It is understood that the antibodies of the invention can be modified to optimize performance depending upon the intended use of the antibodies.
Various techniques for reducing the antigenicity of antibodies and antibody fragments are known in the art. These techniques can be used to reduce or eliminate the antigenicity of the antibodies of the invention. For example, the antibodies may be engineered to reduce their antigenicity in humans. This process often is referred to as humanization. Preferably, the humanized binding proteins have the same or substantially the same affinity for the antigen as the original non-humanized binding protein it was derived from.
In one well known humanization approach, chimeric proteins are created in which immunoglobulin constant regions of antibodies from one species, e.g., mouse, are replaced with immunoglobulin constant regions from a second, different species, e.g., a human. In this example, the resulting antibody is a mouse-human chimera, where the human constant region sequences, in principle, are less immunogenic than the counterpart murine sequences. This type of antibody engineering is described, for example,
Morrison, et al. (1984) Proc. Nat. Acad. Sci. 81: 6851-6855,
Neuberger et al., 1984, Nature 312: 604-608;
U.S. Patent Nos. 6,893,625 (Robinson);
5,500,362 (Robinson); and
4,816,567 (Cabilly).
In another approach, known as CDR grafting, the CDRs of the light and heavy chain variable regions of an antibody of interest are grafted into frameworks (FRs) from another species. For example, murine CDRs can be grafted into human FR sequences. In some embodiments, the CDRs of the light and heavy chain variable regions of an anti-CFHR1 antibody or an anti-CFHR3 antibody are grafted into human FRs or consensus human FRs. In order to create consensus human FRs, FRs from several human heavy chain or light chain amino acid sequences are aligned to identify a consensus amino acid sequence. CDR grafting is described, for example, in
U.S. Patent Nos. 7,022,500 (Queen);
6,982,321 (Winter);
6,180,370 (Queen);
6,054,297 (Carter);
5,693,762 (Queen);
5,859,205 (Adair);
5,693,761 (Queen);
5,565,332 (Hoogenboom);
5,585,089 (Queen);
5,530,101 (Queen);
Jones et al. (1986) NATURE 321: 522-525;
Riechmann et al. (1988) NATURE 332: 323-327;
Verhoeyen et al. (1988) SCIENCE 239: 1534-1536; and
Winter (1998) FEBS LETT 430: 92-94.
In addition, it is possible to create fully human antibodies in mice. In this approach, human antibodies are prepared using a transgenic mouse in which the mouse's antibody-producing genes have been replaced by a substantial portion of the human antibody producing genes. Such mice produce human immunoglobulin instead of murine immunoglobulin molecules. See, e.g.,
WO 98/24893 (Jacobovitz et al.) and
Mendez et al., 1997, Nature Genetics 15: 146-156. Fully human anti-CFHR1 and/or anti-CFHR3 monoclonal antibodies can be produced using the following approach. Transgenic mice containing human immunoglobulin genes are immunized with the antigen of interest, e.g., CFHR1 or CFHR3. Lymphatic cells from the mice then are obtained from the mice, which are then fused with a myeloid-type cell line to prepare immortal hybridoma cell lines. The hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to CFHR1 or CFHR3.
The disclosure describes reagents, devices and kits for detectingCFHR1 orCFHR3 deletions. A number of assay systems are known in the art, and it is within the skill of the art to arrive at means to determine the presence of variations associated with vascular disorders or AMD. The kit reagents, such as multiple primers, multiple probes, combinations of primers, or combinations of probes, may be contained in separate containers prior to their use for diagnosis or screening. The kit can contain a first container containing a probe, primer, or primer pair for a firstCFHR1 orCFHR3 allele described herein, and a second container containing a probe, primer, or primer pair for a secondCFHR1 orCFHR3 allele described herein.
The kits may contain one or more pairs ofCFHR1 and/orCFHR3 allele-specific oligonucleotides hybridizing to different forms of a polymorphism. The allele-specific oligonucleotides may be provided immobilized on a substrate.
The disclosure also provides devices and reagents useful for diagnostic, prognostic, drug screening, and other methods are provided. Also described is a device comprising immobilized primer(s) or probe(s) specific for detecting deletions in the
CFHR1 and/or
CFHR3 genes and optionally also including immobilized primer(s) or probe(s) specific for detecting polymorphic sites in
CFH that are associated with AMD. Exemplary probes and polymorphic sites are described in
U.S. patent publication No. 20070020647.
Also described is a device comprising immobilized primer(s) or probe(s) specific for one or more Factor H and/or CFBR5 and/or CFHR1 and/or CFHR3 gene products (polynucleotides or proteins) is provided. The primers or probes can bind polynucleotides (e.g., based on hybridization to specific polymorphic sites) or polypeptides (e.g., based on specific binding to a variant polypepdde).
In one instance, an array format is used in which a plurality (at least 2, usually at least 3 or more) of different primers or probes are immobilized. The term "array" is used in its usual sense and means that each of a plurality of primers or probes, usually immobilized on a substrate, has a defined location (address)
e.g., on the substrate. The number of primers or probes on the array can vary depending on the nature and use of the device. For example, a dipstick format array can have as few as 2 distinct primers or probes, although usually more than 2 primers or probes, and often many more, will be present. One method for attaching the nucleic acids to a surface is by making high-density oligonucleotide arrays (see,
Fodor et al., 1991, Science 251:767-73;
Lockhart et al., 1996, Nature Biotech 14:1675; and
U.S. Pat. Nos. S,S78,832;
5,556,752; and
5,510,270). It is also contemplated that, in some embodiments, a device comprising a single immobilized probe can be used.
In one instance, an array format is used in which a plurality (at least 2, usually at least 3 or more) of different primers or probes are immobilized. The term "array" is used in its usual sense and means that each of a plurality of primers or probes, usually immobilized on a substrate, has a defined location (address)e.g., on the substrate. The number of primers or probes on the array can vary depending on the nature and use of the device.
In one instance the immobilized probe is an antibody or other CFHR1 or CFHR3 binding moiety.
It will be apparent to the skilled practitioner guided by this disclosure than various polymorphisms and haplotypes can be detected, and used in combination with a deletion in the DNA sequence between the 3' end of exon 22 of the complement factor H (CFH) gene and the 5' end of exon 1 of complement Factor H-related 4 (CFHR4) gene on human chromosome 1, to assess the propensity of an individual to develop a Factor H related condition. Examples ofCFH polymorphisms that may be assayed for include the following SNPs and combinations of SNPs: rs529825; rs800292; rs3766404; rs1061147; rs1061170; rs203674; and optionally including exon 22 (R1210C). In one embodiment the array includes primers or probes to determine the allele at at least one of the following polymorphic sites: rs529825; rs800292; intron 2 (IVS2 or insTT); rs3766404; rs1061147; rs1061170; exon 10A; rs203674; rs375046; and optionally including exon 22 (R1210C). In an embodiment the array includes primers or probes to determine the allele at at least one of the following polymorphic sites: (a) rs3753394; (b) rs529825; (c) rs800292; (d) intron 2 (IVS2 or insTT); (e) rs3766404; (f) rs1061147; (g) rs1061170; (h) rs2274700; (i) rs203674; (j) rs3753396; (j) rs1065489; and optionally including exon 22 (R1210C). In one embodiment, the array includes primers or probes to determine the allele at at least one of the following polymorphic sites: rs800292 (162V); IVS 2 (-18insTT); rs 1061170 (Y402H); and rs2274700 (A473A). In one instance, the array includes primers or probes to determine the allele at at least one of the following polymorphic sites: rs9427661 (-249T>C); rs9427662 (-20T>C); and rs 12097550 (P46S).
The array can include primers or probes to determine the allele at two of the above sites, at least three, at least four, at least five or at least six. In one instance the primers or probes distinguish alleles at rs529825. In one instance the primers or probes distinguish alleles at rs800292. In one embodiment the primers or probes distinguish alleles at rs3766404. In one instance the primers or probes distinguish alleles at rs1061147. In one instance the primers or probes distinguish alleles at rs1061170. In one instance the primers or probes distinguish alleles at rs203674. In one instance the primers or probes distinguish alleles at exon 22 (R1210C). In one instance the primers or probes distinguish alleles at rs529825 and rs800292. In one instance the primers or probes distinguish alleles at two or three of rs1061147, rs1061170 and rs203674. In one instance the primers or probes distinguish alleles at rs529825 and rs800292, at rs3766404, two or three of rs1061147, rs1061170 and rs203674. In one instance the primers or probes distinguish alleles at rs529825, rs800292, rs3766404, rs1061170 and rs203674. In one instance, the primers or probes distinguish alleles at exon 22 (R1210C) and at rs529825; at rs800292; at rs3766404; at rs1061147; at rs1061170; at rs203674; at rs529825 and rs800292; at two or three of rs1061147, rs1061170 and rs203674; at rs529825 and rs800292, rs3766404, and two or three of rs1061147, rs1061170 and rs203674; or at rs529825, rs800292, rs3766404, rs1061170 and rs203674, In one instance, the primers or probes distinguish alleles at (a) any one or more of rs529825; rs800292; rs3766404; rs1061147; rs1061170; and rs203674; (b)any one of more of intron 2 (IVS2 or insTT); rs2274700; exon 10A; and rs375046; (c) one or both of rs529825 and rs800292; (d) one or more of rs1061147, rs1061170 and rs203674; (e) at least one of rs529825 and rs800292; and rs3766404; and at least one of rs1061147, rs1061170 and rs203674; (f) at least rs529825, rs800292, rs3766404, rs1061170, and rs203674; (g) exon 22 (R1210C); (h) exon 22 (R1210C) and any of (a)-(g); or (i) any one or more of rs529825; rs800292; rs3766404; rs1061147; rs1061170; rs203674; intron 2 (IVS2 or insTT); rs2274700; exon 10A; rs375046; and exon 22 (R1210C) and any one or more of rs9427661, rs9427662 and rs12097550.
The array can include primers or probes to determine the allele at two of the above sites, at least three, at least four, at least five or at least six. In one instance the primers or probes distinguish alleles at rs529825. In one instance the primers or probes distinguish alleles at rs800292. In one instance the primers or probes distinguish alleles at intron 2 (IVS2 or insTT). In one the primers or probes distinguish alleles at rs3766404. In one instance the primers or probes distinguish alleles at rs1061147. In one instance the primers or probes distinguish alleles at rs1061170. In one instance the primers or probes distinguish alleles at exon 10A. In one instance the primers or probes distinguish alleles at rs2274700. In one instance the primers or probes distinguish alleles at rs203674. In one- instance the primers or probes distinguish alleles at rs375046. In one instance the primers or probes distinguish alleles at exon 22 (R1210C). In one instance the primers or probes distinguish alleles at rs529825 and rs800292. In one instance the primers or probes distinguish alleles at two or three of rs1061147, rs1061170 and rs203674. In one instance the primers or probes distinguish alleles at of rs529825 and rs800292, at intron 2, at rs3766404, at two or three of rs1061147, rs1061170 and rs203674, at exon 10A, at rs2274700, and at rs375046. In one instance the primers or probes distinguish alleles at rs529825, rs800292, intron 2 (IVS2 or insTT), rs3766404, rs1061170, exon 10A, rs2274700, rs203674, and rs375046. In one Instance, the primers or probes distinguish alleles at exon 22 (R1210C) and at either at rs529825; at rs800292; at intron 2 (IVS2 or insTT); at rs3766404; at rs1061147; at rs1061170; at rs2274700, at exon 10A; at rs203674; at rs375046; at rs529825 and rs800292; at two or three of rs1061147, rs1061170 and rs203674; at rs529825 and rs800292, intron 2 (IVS2 or insTT), rs3766404, two or three of rs10C1147, rs1061170 and rs203674, rs2274700, exon 10A, and rs375046; or at rs52982S, rs800292, intron 2 (IVS2 or insTT), rs3766404, rs1061170, rs2274700, exon 10A, rs203674, and rs375046. In one instance the device distinguishes any combination of allelles at the sites listed above in the context of kits.
In one instance, the substrate comprises fewer than about 1000 distinct primers or probes, often fewer than about 100 distinct primers or probes, fewer than about 50 distinct primers or probes, or fewer than about 10 distinct primers or probes. As used in this context, a primer is "distinct" from a second primer if the two primers do not specifically bind the same polynucleotide (i.e., such as cDNA primers for different genes). As used in this context, a probe is "distinct" from a second probe if the two probes do not specifically bind the same polypeptide or polynucleotide (i.e., such as cDNA probes for different genes). Primers or probes may also be described as distinct if they recognize different alleles of the same gene (i.e., CFHor CFHR5). Thus, the diagnostic devices can detect alleles ofCFH only,CFHR5 only,CFH andCFHR5 only, orCFH, CFHR5 and up to 20, preferably up to 10, or preferably up to 5 genes other thanCFH and/orCFHR5. That is, the device is particularly suited to screening for AMD and related complement-associated diseases. In one instance, the device comprises primers or probes that recognizeCFH and/or one or more ofCFHR1-5 only. In a related instance, the device contains primers and probes for up to 20, preferably up to 10, or preferably up to 5 other genes thanCFH or CFHR1-5.
In one instance, the immobilized primer(s) is/are an allele-specific primer(s) that can distinguish between alleles at a polymorphic site in the Factor H or
CHRF5 gene. Exemplary allele-specific primers to identify alleles at polymorphic sites in the Factor H gene are shown in TABLE 16A of
U.S. patent publication No. 20070020647. The immobilized allele-specific primers hybridize preferentially to nucleic acids, either RNA or DNA, that have sequences complementary to the primers. The hybridization may be detected by various methods, including single-base extension with fluorescence detection, the oligonucleotide ligation assay, and the like (see
Shi, M.M., 2001, Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies" Clin. Chem. 47(2):164-172). Microarray-based devices to detect polymorphic sites are commercially available, including Affymetrix (Santa Clara, CA), Protogene (Menlo Park, CA), Genometrix (The Woodland, TX), Motorola BioChip Systems (Northbrook, IL), and Perlegen Sciences (Mountain View. CA).
The disclosure describes reagents and kits for detecting CFHR1 and/or CFHR3 proteins. A number of assay systems are known in the art, and it is within the skill of the art to arrive at means to determine the presence or absence of CFHR1 and/or CFHR3, or variant or truncated forms thereof, associated with vascular disorders or AMD. The kit reagents, such as anti-CFHR3 or CFHR1 antibodies or other CFH3 or CFHR1 binding moieties, may be contained in separate containers prior to their use for diagnosis or screening. In an instance, the kit contains a first container containing an antibody or binding moiety that specifically binds to CFHR1 protein, or a variant or truncated form thereof, and a second container containing an antibody or binding moiety that specifically binds to CFHR3 protein, or a variant or truncated form thereof. In some instances the binding moieties is an aptamer, such as a nucleic acid aptamer. Aptamers are RNA or DNA molecules selected
in vitro from vast populations of random sequence that recognize specific ligands by forming binding pockets. Aptamers are nucleic acids that are capable of three dimensional recognition that bind specific proteins or other molecules. See, e.g.,
US20050176940 "Aptamers and Antiaptamers"
Thus, the disclosure describes reagents for conducting the screening methods of the invention, comprising a binding moiety capable of specifically binding CFHR1 and/or CFHR3 protein or a portion thereof (e.g., a labeled binder that reacts preferentially with CFHR1 and/or CFHR3 protein or a portion thereof or a labeled binder that reacts preferentially with CFHR1 mRNA and/or CFHR3 mRNA or a portion thereof, or a labeled binder that reacts preferentially with CFHR1 DNA and/or CFHR3 DNA). The binding moiety may comprise, for example, a member of a ligand-receptor pair, i.e., a pair of molecules capable of having a specific binding interaction (such as antibody-antigen, protein-protein, nucleic acid-nucleic acid, protein-nucleic acid, or other specific binding pair known in the art). Optionally the binding moiety is labled (e.g., directly labeled) or is accompanied by a labeled molecule that reacts with the binding moiety (indirectly labeled). Detectable labels can be directly attached to or incorporated into the detection reagent by chemical or recombinant methods. Examples of detectable labels include, but are not limited to, radioisotopes, fluorophores, chromophores (e.g., colored particles), mass labels, electron dense particles, magnetic particles, spin labels, and molecules that emit chemiluminescence. Methods for labeling are well known in the art.
The kits may contain an instruction manual with instructions how to use the anti-CFHR3 or CFHR1 antibodies or other CFHR3 or CFHR1 binding moieties to detect CFHR3 or CFHR1 proteins in body fluids or in tissue samples.
The kits may contain a control antibody or binding moiety. An example of a control antibody or binding moiety is an antibody that specifically binds to CFH protein.
The kits may contain one or more pairs of antibodies or binding moieties that specifically bind to different (i.e., not wild-type or full-length) forms (e.g., variant or truncated) of CFHR1 or CFHR3 proteins.
In one instance, the antibodies or binding moieties are immobilized to a solid support such as an ordered array.
In one instance, the antibodies or binding moieties are used in Western blots.
EXAMPLESExample 1:Polymerase chain reaction (PCR) amplification, single-strand conformation polymorphism (SSCP) analysis and direct DNA sequencing were used to characterize a deletion in the
CFHR3 and
CFHR1 genes located between the
CFH and
CFHR4 genes on chromosome 1. Examples of primers that can be used for PCR amplification of the
CFH gene and
CFH-related genes 1 to 5 are shown in Table 1A. Examples of primers that can be used for SSCP analysis of the
CFH and
CFHR3 genes are shown in Table 1B. Examples of primers that can be used for direct DNA sequencing of the
CFH, CFHR1 and
CFHR3 genes are shown in Table 1 C and I D.
Table 1. Primers Used for Detecting the and Genes| A. PCR Primers |
| Forward 5'-3' | Reverse 5'-3' | Product Size (bp) |
| CFH ex22 | GGTTTGGATAGTGTTTTGAG (SEQ ID NO:14) | ACCGTTAGTTTTCCAGG (SEQ ID NO:15) | 521 |
| CFHR1 ex6 | AGTCGGTTTGGACAGTG (SEQ ID NO:7) | GCACAAGTTGGATACTCC (SEQ ID NO:8) | 321 |
| CFHR2 ex4 | TGTGTTCATTCAGTGAG (SEQ ID N0:16) | ATAGACATTTGGTAGGC (SEQ ID NO:17) | 510 |
| CFHR3 ex3 | TCATTGCTATGTCCTTAGG (SEQ ID NO: 10) | TCTGAGACTGTCGTCCG (SEQ ID NO:11) | 263 |
| CFHR4 ex3 | CTACAATGGGACTTTCTTAG (SEQ ID NO:18) | TTCACACTCATAGGAGGAC (SEQ ID NO:19) | 378 |
| CFHR5 ex2 | AACCCTTTTTCCCAAG (SEQ ID NO:20) | CACATCCTTCTCTATTCAC (SEQ ID NO:21) | 193 |
|
Table 1. Primers Used for Detecting the and Genes| B. SSCP Primers |
| Forward 5'-3' | Reverse 5'-3' | Product Size (bp) |
| CFH ex22 | GGTTTGGATAGTGTTTTGAG (SEQ ID NO:14) | ATGTTGTTCGCAATGTG (SEQ ID NO:22) | 283 |
| CFHR3 ex3 | TCATTGCTATGTCCTTAGG (SEQ ID NO:10) | TCTGAGACTGTCGTCCG (SEQ ID NO: 11) | 263 |
|
Table 1. Primers Used for Detecting the and Genes| C. Sequencing Primers |
| Forward 5'-3' | Reverse 5'-3' | Product Size (bp) |
| CFH ex22 | GGTTTGGATAGTGTTTTGAG (SEQ ID NO:14) | ACCGTTAGTTTTCCAGG (SEQ ID NO:15) | 521 |
| CFHR3 ex3 seq | TTTTGGATGTTTATGCG (SEQ ID NO:12) | AAATAGGTCCGTTGGC (SEQ ID NO: 13) | 420 |
| CFHR1 ex6 | AGTCGGTITGGACAGTG (SEQ ID NO:7) | GCACAAGTTGGATACTCC (SEQ ID NO:8) | 321 |
| | | |
Table 1. Primers Used for Detecting the and Genes |
| Forward 5'- 3' | Reverse 5'- 3' | Product |
| CFH (ex22) | GGTTTGGATAGTGTTTTGAG (SEQ ID NO: 14) | ATGTTGTTCGCAATGTG (SEQ ID NO:22) | Yes |
| CFH (ex22) | GGTTTGGATAGTGTTTGAG (SEQ ID NO:14) | ACCGTTAGTTTTCCAGG (SEQ ID NO: 15) | Yes |
| IVS 5' to CFHR3 | CACGCTATTTGAAAGACAAACTT (SEQ ID NO:23) | AAGCAACCCTGCTCTACAATGT (SEQ ID NO:24) | Yes |
| IVS 5' to CFHR3 | GGAACCACATGGGTCAAATG (SEQ ID NO:25) | GCACAACAAATAAAACTAGCAAATCAT (SEQ ID NO:26) | Yes |
| IVS 5' to CFHR3 | ATTGCTGCAATCTCAGAAGAAAA (SEQ ID NO:27) | TCAAAACGAACAAACAAACAGG (SEQ ID NO:28) | No |
| CFHR3 (ex2) | TGCGTAGACCATACTTTCCAG (SEQ ID NO:29) | CTCTCTTTAATCTTTTAAAGTTTTATACATGTG (SEQ ID NO:30) | No |
| CFHR3 (ex3) | TTTTGGATGTTTATGCG (SEQ ID NO: 12) | AAATAGGTCCGTTGGC (SEQ ID NO:13) | No |
| CFHR3 (ex3) | TCATTGCTATGTCCTTAGG (SEQ ID NO: 10) | TCTGAGACTGTCGTCCG (SEQ ID NO: 11) | No |
| CFHR1 (ex2) | TAAAGTGCTGTGTTTGTATTTGC (SEQ ID NO:31) | GTGATTATTTTGTTACCAACAGC (SEQ ID NO:32) | No |
| CFHR1 (ex6) | AGTCGGTTTGGACAGTG (SEQ ID NO:7) | GCACAAGTTGGATACTCC (SEQ ID NO:8) | No |
| CFHR1 (ex6) | CATAGTCGGTTTGGACAGTG (SEQ ID NO:9) | GCACAAGTTGGATACTCC (SEQ ID NO:8) | No |
| CFHR2 | TCCTTTTCTAGTTCATTAACATA (SEQ m N0:33) | AGTGATATGACACATGCTGAC (SEQ ID NO:34) | Yes |
| CFHR2 | CTACAGACTAACTTTCAATAATTT (SEQ ID N0:35) | GATACTTTTACATTTTCTTATGAT (SEQ ID NO:36) | Yes |
| CFHR2 | ACATAGTTATATGATCGTTTTGAGT (SEQ ID NO:37) | ACAGAGAAAGAACTTACTAATTG (SEQ ID NO:38) | Yes |
| CFHR2 | TGTGTTCATTCAGTGAG (SEQ ID NO:16) | ATAGACATTTGGTAGGC (SEQ ID NO: 17) | Yes |
| CFHR4 | AGTATTAAATTGTTCAGTCCAG (SEQ m N0:39) | AAACTAGTGTAAGAATGTATGAT (SEO ID NO:40) | Yes |
| CFHR4 | TAAGTTGAAAGAGATCTAAACAC (SEQ ID NO:41) | ACTGTATGTAAGATTATGAAAGTAT (SEQ ID NO:42) | Yes |
| CFHR4 | CTACAATGGGACTTTCTTAG (SEQ ID NO:18) | TTCACACTCATAGGAGGAC (SEQ ID NO: 19) | Yes |
| CFHR5 | AACCCTTTTTCCCAAG (SEQ ID NO:20) | CACATCCTTCTCTATTCAC (SEQ ID NO:21) | Yes |
In a study directed toward further characterization ofCFH and its associated haplotypes on chromosome 1q, a complete deletion of the entireCFHL1 andCFHL3 genes was identified. In examining SSCP gels generated usingCFH exon 22 primers (Table 1), several additional patterns of variation were observed due to the amplification ofCFHR1 in addition toCFH. By designing another set of CFH-specific primers, it was determined that there were no variations in exon 22 ofCFH. CFHR1-specific primers were generated and used to identify a deletion ofCFHR1. Further analysis of theCFHR1, CFHR2, CFHR3, CFHR4 and CFHR5 genes and intervening sequence 5' to CFHR3 (Table 1D) using specific primers revealed a deletion that extends across the entire length of theCFHR1 andCFHR3 genes. The precise boundaries of the complete deletion have not be determined, but the mapping of the boundaries is within the skill of the art.
SSCP analysis and direct DNA sequencing was used to determine the frequency of the homozygous deletion of the
CFHR3 and
CFHR1 genes in a set of 1074 patients with and without a clinical history of AMD. The cohort included patients who had other systemic diseases, including vascular diseases, irrespective of their AMD status. As shown in Table 2, homozygous deletion of the
CFHR1 and
CFHR3 genes was found in ∼2.7% of the persons tested.
Table 2. Frequency of homozygous deletion of and genes| Genotype* | Count | Percent |
| +/+, +/A | 1046 | 97.3% |
| Δ/Δ | 28 | 2.7% |
| +/+, +/Δ, Δ/Δ | 1074 | 100% |
Table 2. Frequency of homozygous deletion of and genesinitial analysis suggested that the deletion homozygotes were more common in control individuals than in AMD cases. To determine whether there was an association of the homozygous deletion of the
CFHR3 and
CFHR1 genes with AMD, a subset of the above patient population was analyzed by SSCP analysis and direct DNA sequencing. As shown in Table 3, in a study of 576 AMD patients and 352 age-matched non-AMD control patients, deletion homozygotes make up 5.1 % of controls and 1.2% of cases. The homozygous deletion of
CFHR1 and
CFHR3 is strongly associated with controls, with χ2 = 10.2 and P value = 0.0014, demonstrating a highly significant protective effect of the homozygous
CFHR1/
CFHR3 deletion for AMD.
Table 3. Association of homozygous deletion of and genes with non-AMD | Genotype | Non-AMD patients | AMD patients |
| Count | +/+, +/Δ | 352 | 576 |
| Count | Δ/Δ | 18 | 7 |
| Frequency | +/+, +/Δ | 0.951 | 0.988 |
| Frequency | Δ/Δ | 0.049 | 0.012 |
Table 3. Association of homozygous deletion of and genes with non-AMDTo determine whether there was an association of the homozygous deletion of the
CFHR3 and
CFHR1 genes with vascular disorders, two subsets of the above patient population were analyzed by SSCP analysis and direct DNA sequencing. As shown in Table 4A, a study of 26 patients with abdominal aortic aneurysm (AAA) and 133 non-AAA patients revealed that the homozygous deletion of
CFHR1 and
CFHR3 was strongly associated with AAA, with χ2 = 6.982329 and P = 0.0082. As shown in Table 4B, a second study of 86 patients with abdominal aortic aneurysm (AAA) and 221 non-AAA patients revealed that the homozygous deletion of
CFHR1 and
CFHR3 was associated with AAA, with χ2 = 4.05 and P = 0.0442.
|
| Genotype | Controls | AAA |
| Count | +/+, +/Δ | 126 | 19 |
| Count | Δ/Δ | 7 | 7 |
| +/+, +/A, Δ/Δ | 133 | 26 |
|
|
| Genotype | Controls | AAA |
| Count | +/+, +/Δ | 221 | 86 |
| Count | Δ/Δ | 12 | 11 |
| Total | +/+, +/Δ Δ/Δ | 233 | 97 |
To determine whether previously identified protective haplotypes in the
CFH gene were associated with the del (Δ)
CFHR1 allele, haplotype analysis was performed. As shown in Tables 5A-5E, the relationship between the del (Δ)
CFHR1 allele and SNPs in the
CFH gene revealed strong linkage disequilibrium. The SNPs used in this haplotype analysis are described in
U.S. patent publication No. 20070020647. In the table, letters refer to genotypes and numbers refer to SSCP shift patterns.
Table 5. haplotype analysis in subjects with the del/del (Δ/Δ) allele| A. Promoter 1 to Exon 3 |
| Promoter | Promoter 4 | Exon 2 | Exon 3a | Exon 3 |
| | rs3753394 | rs800292 | | same SNP as 3a |
| | | I62V | | |
| | C-257T | G184A | IVS2-18insTT | |
| 1 | AA | TT | GG | SS | SS |
| 2 | AA | CC | GG | SS | SS |
| 3 | AA | CT | GG | SS | SS |
| 4 | AA | CC | GG | SS + G100R het | SS + G100R het |
| 5 | AA | CT | GG | SS | SS |
| 6 | AA | CT | GG | SS | SS |
| 7 | AA | CC | GG | SS | SS |
| 8 | AA | TT | GG | SS | SS |
| 9 | AA | CT | GG | SS | SS |
| 10 | AA | CC | GG | SS | SS |
| 11 | AA | CC | GG | SS | SS |
| 12 | AA | CC | GG | SS | SS |
| 13 | AA | CT | GG | SS | SS |
| 14 | | | GG | | SS |
| 15 | | | GG | | SS |
| 16 | | | GG | | SS |
| 17 | | | GG | | SS |
| 18 | | | GG | | SS |
| 19 | | | GG | | SS |
| 20 | | | GG | | SS |
| 21 | | | GA | | SS |
| 22 | | | GG | | SS |
| 23 | | | | | SS |
| 24 | | | | | SS |
| 25 | | | | | SS |
| 26 | | | | | SS |
Table 5. haplotype analysis in subjects with the del/del (Δ/Δ) allele| B. IVS 6 to Exon 7b |
| IVS 6 | IVS 6 | IVS6 | IVS6 | Exon 7b |
| shift | N or Del | rs16840419 | rs3766404 | rs1061147 |
| | | | | A307A |
| | | | | A921C |
| 1 | 3 | NN | GA | CT | CC |
| 2 | 5 | NDel | 5(GG) | 5 (CC?) | CC |
| 3 | 2 | NN | GG | CC | CC |
Table 5. haplotype analysis in subjects with the del/del (Δ/Δ) allele | IVS 6 | IVS 6 | IVS6 | IVS6 | Exon 7b |
| shift | N or Del | rs16840419 | rs3766404 | ras1061147 |
| 4 | 2 | NN | GG | CC | CC |
| 5 | 3 | NN | GA | CT | CC |
| 6 | 1 | NN | AA | TT | AC |
| 7 | 5 | NDel | 5 (GG) | 5 (CC?) | CC |
| 8 | 1 | NN | AA | TT | CC |
| 9 | 3 | NN | GA | CT | CC |
| 10 | 2 | NN | GG | CC | CC |
| 11 | No DNA (3) | NN | No DNA | No DNA (CT) | AC |
| 12 | 2 | NN | GG | CC | CC |
| 13 | 1 | NN | AA | TT | CC |
| 14 | | | | | |
| 15 | | | | | |
| 16 | | | | | |
| 17 | | | | | |
| 18 | | | | | AA |
| 19 | | | | | |
| 20 | | | | | |
| 21 | | | | | |
| 22 | | | | | |
| 23 | | | | | |
| 24 | | | | | |
| 25 | | | | | |
| 26 | | | | | |
Table 5. haplotype analysis in subjects with the del/del (Δ/Δ) allele| C. Exon 9 to Exon 16b |
| Exon 9 | Exon 10A | Exon 10a | Exon 13b | Exon 16b |
| rs1061170 | | rs2274700 | rs3753396 | rs375046 |
| Y402H | | A473A | Q672Q | IVS 15 |
| C1204T | CFHtrunc | G2016A | A2089G | |
| 1 | TT | 1 | AA | AA | |
| 2 | TT | | | AA | AA |
| 3 | TT | 1 | AA | AA | AA |
| 4 | TT | 1 | AA | AA | AA |
| 5 | TT | 1 | AA | AA | 4 |
| 6 | CT | 1 | GA | AA | |
| 7 | TT | | | | AA |
| 8 | TT | 1 | AA | AA | AA |
| 9 | TT | 1 | AA | AA | AA |
| 10 | TT | 1 | AA | AA | AC? |
| 11 | CT | 1 | GA | AA | |
| 12 | TT | | | AA | |
| 13 | TT | | | | |
| 14 | TT | 1 | AA | | |
| 15 | TT | I | AA | | |
| 16 | CT | 1 | GA | | |
Table 5. haplotype analysis in subjects with the del/del (Δ/Δ) allele | Exon 9 | Exon 10A | Exon 10a | Exon 13b | Exon 16b |
| rs1061170 | | rs2274700 | rs3753396 | rs375046 |
| Y402H | | A473A | Q672Q | IVS15 |
| C1204T | CFHtrunc | G2016A | A2089G | |
| 17 | TT | 1 | AA | AA | CC |
| 18 | CC | 1 | GG | | |
| 19 | TT | 1µ | AA | | |
| 20 | TT | 1 | AA | | |
| 21 | TT | 1 | AA | | |
| 22 | TT | 1 | AA | | |
| 23 | TT | | | | |
| 24 | TT | | | | |
| 25 | TT | | | | |
| 26 | TT | | | | |
Table 5. haplotype analysis in subjects with the del/del (Δ/Δ) allele| D. Exon 17a to Exon 19a |
| Exon 17a | Exon 17b | Exon 18a | Exon 18b | Exon 19a |
| | | rs1065489 | rs1065489 | rs534399 |
| | A892V | E936D | E936D | V1007L |
| | C2748T | G2881T | G2881T | G3092T |
| 1 | 1 | CC | GG | GG | GG |
| 2 | 1 | CC | | | |
| 3 | 1 | CC | GG | GG | GG |
| 4 | 1 | CC | GG | GG | GG |
| 5 | 1 | CC | GG | GG | GG |
| 6 | 3 | CC | GG | GG | TT |
| 7 | 1 | | | | |
| 8 | 1 | CC | GG | GG | GG |
| 9 | 1 | CC | GG | GG | GG |
| 10 | 1 | CC | GG | GG | GG |
| 11 | 1 | CC | GG | GG | GG |
| 12 | 1 | CC | | | |
| 13 | 1 | CC | | | |
| 14 | | | | GG | |
| 15 | | | | GG | |
| 16 | | | | GG | |
| 17 | | | | GG | |
| 18 | 1 | CC | GG | GG | GG |
| 19 | 0 | | | GG | |
| 20 | | | | GG | |
| 21 | | | | GG | |
| 22 | | | | GG | |
| 23 | | | | GG | |
| 24 | | | | GG | |
| 25 | | | | GG | |
Table 5. haplotype analysis in subjects with the del/del (Δ/Δ) allele | Exon 17a | Exon 17b | Exon 18a | Exon 18b | Exon 19a |
| | rs1065489 | rs1065489 | rs534399 |
| A892V | E936D | E936D | V1007L |
| C2748T | G2881T | G2881T | G3092T |
| 26 | | | | GG | |
Table 5. haplotype analysis in subjects with the del/del (Δ/Δ) allele |
| | Exon 20b | Exon 22b | Exon 22split | |
| |
| | 1191/1197/1210 | 1197 | |
| | | | |
| 1 | 4 | 4 | 4 | |
| 2 | | | 4 | |
| 3 | 4 | 4 | 4 | |
| 4 | 2 | 4 | 4 | |
| 5 | 4 | 4 | 4 | |
| 6 | 4 | 4 | 4 | |
| 7 | | | 4 | |
| 8 | 4 | 4 | 4 | |
| 9 | 4 | 4 | 4 | |
| 10 | 6 | 4 | 4 | |
| 11 | 4 | 4 | 4 | |
| 12 | | | 4 | |
| 13 | | | 4 | |
| 14 | | 4 | | |
| 15 | 4 | 4 | | |
| 16 | 4 | 4 | | |
| 17 | | 4 | | |
| 18 | 4 | 4 | | |
| 19 | 4 | 4 | | |
| 20 | 6 | 4 | | |
| 21 | 4 | 4 | | |
| 22 | 4 | 4 | | |
| 23 | 4 | 4 | | |
| 24 | 4 | 4 | | |
| 25 | 4 | 4 | | |
| 26 | 4 | | |
As shown in Table 6, in two studies it was found that the deletion of the
CFHR1 and
CFHR3 genes was associated with 402T-containing haplotypes. This deletion is almost never found on the same 402C-containing haplotype as the major
CFH risk allele, Y402H. The del (A)
CFHR1 mutation is predominantly associated with the
CFH H4 haplotype, a haplotype with T at position 1277 of the coding region of CFH (codon 402) shown previously shown to be protective for AMD. However, not every del (A)
CFHR1 chromosome is on H4, and the protection of del/del (A/A)
CFHR1 homozygotes for AMD is even stronger than H4 homozygotes. Heterozygous deletion of the
CFHR3 and
CFHR1 genes was detected by direct DNA sequencing of the
CFH, CFHR1 and
CFHR3 genes using a
CFH exon 22 primer.
Table 6. Association of homozygous deletion of and genes with the TT genotype at position 1277 of the coding region of CFH (codon 402)| A. Study 1 |
| | | CFH402 Genotype | |
| Genotype | TT | TC | CC |
| Count | +/+, +/Δ | 102 | 209 | 50 |
| Count | Δ/Δ | 11 | 2 | 0 |
| Count | +/+, +/Δ, Δ/Δ | 113 | 211 | 150 |
|
| **CFH402 Genotype refers to the nucleotide on both alleles at position 1277 of the coding region of human CFH. A T results in a tyrosine at codon 402, whereas a C results in a histidine at codon 402. |
|
Table 6. Association of homozygous deletion of and genes with the TT genotype at position 1277 of the coding region of CFH (codon 402)| B. Study 2 |
| | | CFH402 Genotype | |
| Genotype | TT | TC | CC |
| Count | +/+, +/A | 192 | 393 | 283 |
| Count | Δ/Δ | 23 | 3 | 0 |
| Count | +/+, +/Δ, Δ/Δ | 215 | 396 | 283 |
| *Genotype refers to the deletion (A) or non-deletion (+) of the CFHR1 and CFHR3 genes by SSCP analysis and direct sequencing. |
| **CFH402 Genotype refers to the nucleotide on both alleles at position 1277 of the coding region of human CFH. A T results in a tyrosine at codon 402, whereas a C results in a histidine at codon 402. |
By Western blotting, it was determined that CFHR1 protein, normally an abundant serum protein, is absent in sera derived from individuals homozygous for theCFHR1/CFHR3 deletion.Figure 3 shows a representative Western blot of serum proteins from seven (out of a sample set of 52) patients using an anti-human CFH antibody. Serum proteins were separated by one-dimensional SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. After transfer, the membrane was blocked with 5% non-fat dry milk, washed, and then incubated with a goat anti-human CFH (Calbiochem, 1:1000 dilution). After incubation, the membrane was washed, and then incubated with horse radish peroxidase-conjugated rabbit anti-goat Ig antibody (Abcam, 1:4000 dilution). After incubation, the membrane was washed, and then incubated with extravidin (1:1500 dilution). Samples 197-02 and 325-02 were from patients with a TT 402 genotype (protective CFH H4 haplotype) and have homozygous deletion ofCFHR1 andCFHR3 genes, as determined by SSCP analysis and direct sequencing.Figure 3 shows that no CFHR1 is detected in the serum from patients having a homozygous deletion of theCFHR1 andCFHR3 genes.
Western blotting using the same anti-human CFH antibody was used to detect CFH and CFHR1 in serum from an additional 40 patients, separated according to SSCP patterns using theCFH exon 22 primers. Patterns 1-3 correspond to homozygous, or heterozygous for, non-deletion ofCFHR1 andCFHR3 (+/+, +/Δ), and pattern 4 corresponds to homozygous deletion ofCFHR1 andCFHR3 (Δ/Δ) (seeFigure 4). All 10 of the serum samples from patients displaying SSCP pattern 4 show no CFHR1, whereas all 30 of the serum samples from patients displaying SSCP patterns 1-3 show at least some CFHR1 (data not shown). Thus, analysis of serum from individuals with aCFHR1 del/del (Δ/Δ) genotype shows that they lack any detectable CFHR1 protein. This protein analysis confirms that these individuals lack both theCFHR1 gene and encoded protein. Individuals who are heterozygous for deletion ofCFHR1 andCFHR3 can be recognized by protein analysis of serum samples by virtue of the intensity of the band corresponding to CFHR1 being roughly half the intensity in heterozygous (+/Δ) patients as compared to homozygous non-deletion (+/+) patients.
PCR experiments using leukocyte-derived DNA were performed to confirm that patients having a homozygous deletion ofCFHR1 andCFHR3 do not haveCFHR1 andCFHR3 DNA.Figure 5 shows a PCR analysis ofCFH andCFHR1-5 from DNA samples from 20 patients, separated into four groups according to SSCP patterns using theCFH exon 22 primers mentioned above. Patterns 1-3 correspond to homozygous non-deletion or heterozygous deletion ofCFHR1 andCFHR3 (+/+, +/Δ), and pattern 4 corresponds to homozygous deletion ofCFHR1 andCFHR3 (Δ/Δ). From left to right, 5 samples each from patients displaying SSCP patterns 1, 2, 3 and 4 were subjected to PCR using primers specific forCFH, CFHR1, CFHR2, CFHR3, CFHR4 andCFHR5, as indicated. This figure shows thatCFH, CFHR4 andCFHR5 DNA are amplified in all of the samples, whereasCFHR1 andCFHR3 DNA are amplified in samples from patients displaying SSCP patterns 1-3, but not from patients displaying SSCP pattern 4. TheCFHR2 DNA was amplified in some, but not all, of the samples. Thus, when SSCP and direct sequencing show a homozygous deletion of theCFHR1 andCFHR3 genes, no PCR amplifiableCFHR1 andCFHR3 DNA are detected in samples.
Example 2: Production of Anti-CFHR1 and Anti-CFHR3 Monoclonal AntibodiesMice will be immunized with recombinant human CFHR1 or CFHR3. Two mice with sera displaying the highest anti-CFHR1 and anti-CFHR3 activity by Enzyme Linked Immunosorbent Assay (ELISA) will be chosen for subsequent fusion and spleens and lymph nodes from the appropriate mice will be harvested. B-cells will be harvested and fused with an myeloma line. Fusion products will be serially diluted on one or more plates to near clonality. Supernatants from the resulting fusions will be screened for their binding to hCFHR1 or hCFHR3 by ELISA. Supernatants identified as containing antibodies to CFHR1 or CFHR3 will be further characterized byin vitro functional testing as discussed below. A panel of hybridomas will be selected and the hybridomas will be subcloned and expanded. The monoclonal antibodies will then be purified by affinity chromatography on Protein A/G resin under standard conditions.
Anti-CFHR1 and anti-CFHR3 antibodies may be further characterized byin vitro functional testing using complement activation assays well known in the art. For example, complement activation assays may be conducted in solution (e.g., fluid phase in blood) or on immobilized surfaces. Exemplary assays may measure the ability of the anti-CFHR1 and/or anti-CFHR3 antibodies to block or reduce CFH, C3b, heparin and/or C-reactive protein (CRP) binding to a substrate.
SEQUENCE LISTING- <110> University of Iowa Research Foundation
- <120> METHODS AND REAGENTS FOR TREATMENT AND DIAGNOSIS OF VASCULAR DISORDERS AND AGE-RELATED MACULAR DEGENERATION
- <130> P33367EP-PCT
- <140> EP07812932.7<141> 2007-07-13
- <150> US 60/831,018<151> 2006-07-13
- <150> US 60/840,073<151> 2006-08-23
- <160> 42
- <170> PatentIn version 3.4
- <210> 1<211> 3853<212> DNA<213> Homo sapiens
- <400> 1
- <210> 2<211> 1231<212> PRT<213> Homo sapiens
- <400> 2
- <210> 3<211> 1189<212> DNA<213> Homo sapiens
- <400> 3
- <210> 4<211> 330<212> PRT<213> Homo sapiens
- <400> 4
- <210> 5<211> 1246<212> DNA<213> Homo sapiens
- <400> 5
- <210> 6<211> 331<212> PRT<213> Homo sapiens
- <400> 6
- <210> 7<211> 17<212> DNA<213> Artificial
- <220><223> Synthetic CFHL1ex6.F, CFHR1 ex6 forward primer
- <400> 7agtcggtttg gacagtg 17
- <210> 8<211> 18<212> DNA<213> Artificial
- <220><223> Synthetic CFHL1ex6 CFHR1 ex6 reverse primer
- <400> 8gcacaagttg gatactcc 18
- <210> 9<211> 20<212> DNA<213> Artificial
- <220><223> Synthetic CFHL1ex6. F2, CFHR1 (ex6) forward primer
- <400> 9catagtcggt ttggacagtg 20
- <210> 10<211> 19<212> DNA<213> Artificial
- <220><223> Synthetic CFHL3ex3.F, CFHR3 ex3 forward primer
- <400> 10tcattgctat gtccttagg 19
- <210> 11<211> 17<212> DNA<213> Artificial
- <220><223> Synthetic CFHL3ex3.R, CFHR3 ex3 reverse primer
- <400> 11tctgagactg tcgtccg 17
- <210> 12<211> 17<212> DNA<213> Artificial
- <220><223> Synthetic CFHL3ex3seq.F, CFHR3 ex3 seq forward primer
- <400> 12ttttggatgt ttatgcg 17
- <210> 13<211> 16<212> DNA<213> Artificial
- <220><223> Synthetic CFHL3ex3seq.R, CFHR3 ex3 seq reverse primer
- <400> 13aaataggtcc gttggc 16
- <210> 14<211> 20<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFH ex22 forward
- <400> 14ggtttggata gtgttttgag 20
- <210> 15<211> 17<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFH ex22 reverse
- <400> 15accgttagtt ttccagg 17
- <210> 16<211> 17<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFHR2 ex4 forward
- <400> 16tgtgttcatt cagtgag 17
- <210> 17<211> 17<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFHR2 ex4 reverse
- <400> 17atagacattt ggtaggc 17
- <210> 18<211> 20<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFHR4 ex3 forward
- <400> 18ctacaatggg actttcttag 20
- <210> 19<211> 19<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFHR4 ex3 reverse
- <400> 19ttcacactca taggaggac 19
- <210> 20<211> 16<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFHR5 ex2 forward
- <400> 20aacccttttt cccaag 16
- <210> 21<211> 19<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFHR5 ex2 reverse
- <400> 21cacatccttc tctattcac 19
- <210> 22<211> 17<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFH ex22 reverse
- <400> 22atgttgttcg caatgtg 17
- <210> 23<211> 23<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer IVS 5' to CFHR3 forward
- <400> 23cacgctattt gaaagacaaa ctt 23
- <210> 24<211> 22<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer IVS 5' to CFHR3 reverse
- <400> 24aagcaaccct gctctacaat gt 22
- <210> 25<211> 20<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer IVS 5' to CFHR3 forward
- <400> 25ggaaccacat gggtcaaatg 20
- <210> 26<211> 27<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer IVS 5' to CFHR3 reverse
- <400> 26gcacaacaaa taaaactagc aaatcat 27
- <210> 27<211> 23<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer IVS 5' to CFHR3 forward
- <400> 27attgctgcaa tctcagaaga aaa 23
- <210> 28<211> 22<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer IVS 5' to CFHR3 reverse
- <400> 28tcaaaacgaa caaacaaaca gg 22
- <210> 29<211> 21<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFHR3 (ex2) forward
- <400> 29tgcgtagacc atactttcca g 21
- <210> 30<211> 33<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFHR3 (ex2) reverse
- <400> 30ctctctttaa tcttttaaag ttttatacat gtg 33
- <210> 31<211> 23<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFHR1 (ex2) forward
- <400> 31taaagtgctg tgtttgtatt tgc 23
- <210> 32<211> 23<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFHR1 (ex2) reverse
- <400> 32gtgattattt tgttaccaac agc 23
- <210> 33<211> 23<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFHR2 forward
- <400> 33tccttttcta gttcattaac ata 23
- <210> 34<211> 21<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFHR2 reverse
- <400> 34agtgatatga cacatgctga c 21
- <210> 35<211> 24<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFHR2 forward <400> 35ctacagacta actttcaata attt 24
- <210> 36<211> 24<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFHR2 reverse
- <400> 36gatactttta cattttctta tgat 24
- <210> 37<211> 25<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFHR2 forward
- <400> 37acatagttat atgatcgttt tgagt 25
- <210> 38<211> 23<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFHR2 reverse
- <400> 38acagagaaag aacttactaa ttg 23
- <210> 39<211> 22<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFHR4 forward
- <400> 39agtattaaat tgttcagtcc ag 22
- <210> 40<211> 23<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFHR4 reverse
- <400> 40aaactagtgt aagaatgtat gat 23
- <210> 41<211> 23<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFHR4 forward
- <400> 41taagttgaaa gagatctaaa cac 23
- <210> 42<211> 25<212> DNA<213> Artificial
- <220><223> Synthetic PCR primer CFHR4 reverse
- <400> 42actgtatgta agattatgaa agtat 25