Collagen COL4A6: Gene, Protein and Method of Detecting Collagen Deficiency

The present invention relates, in general, to a genetic method for detecting collagen deficiencies associated with Alport's syndrome, and to a type IV collagen protein designated .alpha.6(IV), and a gene COL4A6.

Alport's syndrome ("AS") is disorder characterized by progressive kidney disease (glomerulonephritis), progressive hearing loss (sensorineural deafness) and eye lesions (lenticonus). Alport's syndrome is usually dominantly inherited and occurs in the population at a frequency of about 1/5000 persons, with greater frequency in males. Progressive kidney failure causes death in many AS patients.

Alport's syndrome is a progressive disease, usually inherited as an X-chromosome-linked dominant trait, but autosomal forms have been described. In AS, ultrastructural defects are present in the glomerular basement membrane ("BM"), a complex network of glycoproteins and collagens, which serves an important function in filtration in the kidney.

Recently, mutations in the collagen gene COL4A5, a specific component of the BM within the kidney, were correlated with Alport's syndrome in three kindred patients. Both point mutations and intragenic deletions of COL4A5, which maps to the X chromosome, have been implicated in Alport's syndrome. (See, Barker et al., Science 248:1224 (1990); Zhou et al., Genomics 8:10 (1991); Antignac et al., Kidney Int. 42:1178 (1992)).

In some families, AS cosegregates with the occurrence of diffuse leiomyomatosis ("DL"), a benign proliferation of smooth muscle in the esophagus, female genitalia, and trachea. These benign tumors found in DL are commonly known as fibroids.

Recently, three out of three AS-DL patients were shown to have deletions that include the 5' end of COL4A5. The association of AS and DL in patients raised the possibility that COL4A5 or another gene was exerting pleiotropic effects in causing DL, or that a tumor or suppressor gene might exist in the vicinity upstream of COL4A5.Alternatively, the fact that familial leiomyomatosis, a non-AS linked disorder, occurs with autosomal dominant inheritance (as opposed to X-linked) urged that an unrelated gene was at work. Despite these hypotheses, there exists no coherent understanding in the field of the causative factor(s) behind AS-DL.

It is therefore an object of the present invention to provide a polynucleotide molecule, COL4A6, encoding .alpha.6(IV). Assays according to the invention, employ such a gene and a protein encoded thereby to detect a collagen type IV-associated pathology, preferably Alport's syndrome, and more preferably, AS-DL. These assays are based on (a) structural and sequence evaluation of an .alpha.6(IV) gene and (b) evaluation of its expression patterns, either at the DNA, RNA, and/or protein levels.

A further objective is to provide a gene for use in gene therapy of a patient having a collagen type IV-associated pathology, by administering to a patient a vector containing an .alpha.6(IV)-encoding polynucleotide molecule such that transfection with the vector induces production of an effective amount of .alpha.6(IV) in the patient. Preferably, the collagen type IV-associated pathology is Alport's syndrome, or is a subset of Alport's syndrome, AL-DS.

In accomplishing these and other objects of the invention, in accordance with one aspect of the present invention, an isolated COL4A6 polynucleotide molecule comprising a DNA sequence encoding a .alpha.6(IV) polypeptide is provided.

Another object of the invention is to provide a method for detecting a collagen type IV-associated pathology comprising the step of determining the presence or absence of a structural alteration in COL4A6 polynucleotide molecule in a biological sample by comparing said sample COL4A6 with a standard that is indicative of an unaltered COL4A6 polynucleotide molecule.

Another object of the present invention is to provide an isolated .alpha.6(IV) collagen polypeptide. An .alpha.6(IV) polypeptide provided according to the invention is useful for generating monoclonal or polyclonal antibodies having specificity for .alpha.6(IV) polypeptide, preferably an antibody that is not cross-reactive with other collagen proteins, including at least, .alpha.1(IV), .alpha.2(IV), .alpha.3(IV), .alpha.4(IV) or .alpha.5(IV) collagens.

.alpha.6(IV)--specific antibodies, and in particular, labeled monoclonal .alpha.6(IV)-specific antibodies, are useful for detection of collagen type IV-associated pathologies.

FIGS. 1A-1Q show a COL4A6 polynucleotide molecule (SEQ. ID NO: 1, and below, .alpha.6(IV) polypeptide molecule (SEQ ID NO: 2). Within the full-length polynucleotide spanning 5102 nucleotides and the corresponding polypeptide molecule spanning 1700 amino acids, a fragment molecule also is identified spanning amino acids 1-1460 inclusive and nucleotides 1-4380 inclusive, respectively.

FIG. 2 shows a restriction map of lambdaLA226 which contains the 5' ends of both COL4A6 and COL4A5. The striped bars represent phage arms; H. Hind III. The DNA sequence of the region containing the first exon of each gene (SEQ ID NOS: 3 and 5) is shown as well as the deduced amino acid sequences of the first exons (SEQ ID NOS: 4 and 6).

FIGS. 3A-3B show deduced amino acid sequences of the cDNA clone JZ-3 of the human .alpha.6(IV) chain (SEQ ID NO: 7) and comparison of JZ-3 with the amino acid sequences of the human .alpha.1(IV) (SEQ ID NO: 9) , .alpha.2(IV) (SEQ ID NO: 8) and .alpha.5(IV) (SEQ ID NO: 10) chains. Dashes indicate amino acid identity, and conserved cysteine residues are indicated by arrows. The locations of cysteines in the mature chains are identical in .alpha.6(IV) and .alpha.2(IV). Interruptions in the collagenous repeat are underlined and numbered (I through IX).

FIG. 4 shows an analysis of collagen gene deletions in five patients with AS-DL of Example 2. The three exons of .alpha.6(IV) and two exons of .alpha.5(IV) shown as black and striped boxes, respectively, are placed on a genomic map of Eco RI sites (RI). The open bars below show the minimum extent of the deletion in each AS-DL patient. The filed bars show the minimum extent of the nondeleted regions. The shaded bars represent ambiguities in mapping the deletion boundaries.

According to the present invention, a human polynucleotide molecule, COL4A6, and a corresponding human polypeptide, .alpha.6(IV), encoded by COL4A6, are disclosed. Human COL4A6 polynucleotide molecule is expressed primarily by cells that synthesize specialized basement membranes of the body, particularly in the kidney glomerulus, inner ear and esophagus. Also, COL4A6 polynucleotide molecule is expressed in heart, skeletal muscle, lung and placenta (term). Type IV collagens, of which is .alpha.6(IV) is a member, are ultrastructural proteins found in the BM and are implicated in adhesion and motility of cells during tumor invasion. According to the present invention, importantly, a variety of alterations to the COL4A6 gene are correlated with "collagen type IV-associated pathologies," a term defined to include, but not be limited to, diseases of the BM, such as Alport's syndrome, AS-DL, and formation and proliferation of benign tumors.

The distribution of the .alpha.5(IV) and .alpha.6(IV) transcripts were studied in the fetus. An .alpha.6(IV) mRNA of .apprxeq.7.0 kb was found in meninges and esophagus and was just detectable in fetal choroid plexus and stomach. An .apprxeq.7.0 kb transcript of .alpha.5(IV) was present in many tissues but was most abundant in choroid plexus, meninges, and esophagus. This distribution is in agreement with the suggestion that interactions between cells and the underlying basement membrane substrate play a vital role in embryonic morphogenesis.

It is believed that, as a result of this role in development, deletions of both COL4A5 and COL4A6, which were found in all four independent AS-DL kindreds, cause AS-DL based on the following mechanism. Signals from BM proteins, transduced by members of the integrin family of cell surface receptors, regulate cell growth and differentiation and influence cell shape by affecting the cytoskeleton. Several BM components interact with integrins and can affect differentiation of epithelial, endothelial, and mesenchymal cells. Type IV collagens contain binding sites within the triple-helical and NCI domains for several cell types including myocytes. In the presence of antibodies to a .beta. integrin, chicken embryo myoblasts continue to replicate, fail to fuse, and have abnormal morphology. Vandenberg et al., J. Cell Bio. 13:1475 (1991). Although the cited studies are based on collagen sources rich in the ubiquitous .alpha.1(IV) and .alpha.2(IV) chains, the data presented herein suggest that the .alpha.5(IV) and .alpha.6(IV) chains may play similar roles in cell-matrix interactions in tissues involved in the AS-DL syndrome. Likewise, their absence may disrupt normal morphogenesis and lead to uncontrolled proliferation of distorted smooth muscle cells.

Mutations of neither COL4A5 alone nor COL4A6 alone have been observed in AS-DL. It is possible that only the .alpha.6(IV) chain is critical for normal smooth muscle differentiation. If so, our failure to observe mutations of COL4A6 alone in AS-DL might merely reflect the small sample size. Linkage studies have shown that X-linked AS mutations are all tightly linked to markers in the Xq22 region where both COL4A5 and COL4A6 are located. COL4A6 is the probably site for the .apprxeq.50% of X-linked AS mutations that have not been found in COL4A5. Therefore, the absence of DL in these patients suggest that simultaneous mutation of both COL4A5 and COL4A6 is required for the development of DL.

According to the present invention, structural alterations in a type IV collagen gene, in particular COL4A6 gene, were detected in a subset of patients exhibiting collagen type IV associated pathologies. The experiments below also show that structural alterations in a type IV collagen can result in cell proliferation resulting in benign tumors, for example, such as leiomyomas.

"Structural alterations" include constitutional rearrangements, including deletion(s), duplicate(s), inversion(s) or point mutation(s) and somatic mutations (in tumors) of the coding and/or regulatory regions, that affect a cell's ability to make functional .alpha.6(IV). Thus, the term does not include natural polymorphisms which do not affect the cell's ability to make functional .alpha.6(IV). One such functional .alpha.6(IV), for example, was identified from cells not exhibiting collagen type IV associated pathologies, shown as the full-length polypeptide in FIG. 1 (SEQ ID NO: 2).

"Altered expression" of .alpha.6(IV) or .alpha.6(IV) RNA (mRNA) includes a decreased expression of .alpha.6(IV) or .alpha.6(IV) RNA relative to that found in "normal" cells derived from an individual not exhibiting collagen type IV-associated pathologies. For example, altered expression level of .alpha.6(IV) or .alpha.6(IV) RNA may include a range from one-half to less than one-half of an amount identified as a median standard expression level.

According to the invention, assays are provided to detect a collagen type IV-associated pathology, based on (a) structural and sequence evaluation of .alpha.6(IV) gene and (b) evaluation of related expression patterns, at any of the DNA, RNA, and/or protein levels.

For analyzing genomic structural alterations, known methods are applied, such as utilized by Antignac et al., Kidney Int. 42:1178 (1992), incorporated herein by reference in its entirety. A .alpha.6(IV) polynucleotide molecule is used to screen for alterations by hybridization in the sample. Thereafter, the .alpha.6(IV) gene can be analyzed, for example, by Southern blot, polymerase chain reaction sequence identification of cloned material, single strand conformational polymorphism (SSCP), or by DNA sequencing of the locus.

A probe useful in this context is a labeled fragment of .alpha.6(IV) gene or preferably an approximately full-length COL4A6 DNA molecule (useful for detection of deletions) which will hybridize to RNA or DNA from normal cells. In addition to a whole gene or fragment thereof, a probe can include a genomic or cDNA clone, an adjacent region, or a regulatory region of the .alpha.6(IV) gene. Such a marker may be labeled by a standard method, such as radiolabelling.

For example, detection is performed first by extracting DNA from peripheral blood leukocytes using standard procedures. Next, DNA digestion is performed using a variety of restriction endonucleases and the resulting fragments are run on gel electrophoresis, followed by transfer to a suitable nylon filter or other support matrix for hybridization. Next, hybridization with a radiolabelled cDNA of COL4A6 polynucleotide molecule, preferably spanning the entire polynucleotide molecule of FIG. 1 (SEQ ID NO: 1) or preferably, nucleotides 1-4380 of FIG. 1, is performed.

Hybridization is performed under stringent conditions (for example, 10% dextran sulfate, 1M NaCl, 10% SDS and 100 .mu.g/ml of sonicated denatured salmon sperm DNA, at a temperature of 42.degree. C. Fragments hybridizing to the genomic marker are isolated and subcloned for sequencing using conventional methods. Nucleotide sequencing is performed, e.g. by dideoxy termination method using universal and sequence-specific primers. The reaction products are analyzed by commercially available automated DNA sequencer.

The resulting sequence information then is analyzed for structural alterations, such as deletions and point mutations in coding and/or regulatory sequences.

RNA ("Northern") blotting is employed, for example, using a COL4A6 polynucleotide molecule of the invention. According to this method, RNA is isolated from tissue by any of a number of standard procedures (Lehrach, H., Biochemistry, 16: 4743 (1975)). RNA is subjected to denaturing gel electrophoresis, followed by transfer to nitrocellulose or other support matrix. The COL4A6 mRNA can be detected byhybridization of radioactively or non-radioactively labelled COL4A6, or COL4A6 fragments, preferably under high stringency conditions, such as recognized by a scientist in this field. The amount of hybridization can be quantified by densitometric methods.

In yet another embodiment of the present invention, the polymerase chain reaction ("PCR") is used to detect .alpha.6(IV) DNA or RNA in a sample. For example, a pair of COL4A6 sequence specific primers is employed, which hybridize to opposite strands of the .alpha.6(IV) gene at offset positions on the double helix. Such primers, taken from the COL4A6 polynucleotide sequences provided in accordance with the invention, represent fragments which preferably are unique to .alpha.6(IV), e.g., sequences having low homology with other proteins than .alpha.6(IV).

Primers provide initiation points for DNA synthesis. In the presence of DNA polymerase, the four nucleotide triphosphates ("NTPs") and other necessary co-factors, all of which are well known to the art, new DNA strands are synthesized complementary to the templates which hybridized with the primers. Several rounds of synthesis are carried out, with allowance for denaturation of the double-stranded products between rounds. Preferably, a thermal stable DNA polymerase is used so that it is not necessary to add enzyme anew for each round of synthesis.

The PCR produces a double stranded DNA amplification product which has the same sequence as the original stretch of the DNA defined by the ends of the primer pair sequences. PCR can be modified such that it quanitates the amount of .alpha.6(IV) DNA or RNA in the sample. See, for example, U.S. Pat. No. 5,219,727. The product can be detected by a variety of methods well-known in the art. Where such products are produced in a test tube, or the like, they can be resolved by agarose or polyacrylamide electrophoresis and detected by fluorescence staining, such as ethidium bromide. Alternatively, one of the NTPs may be labelled and the PCR products may be determined by measuring incorporation of the labeled NTP. A variety of other methods for resolving, detecting and measuring the amount of PCR product are well-known to the art that are suitable for use in the present invention.

PCR may be rendered specific for DNA or RNA in situ and in liquid PCRs. For instance, RNAse or DNAse may be used to remove one template or the other from the sample, and the use of primers that distinguish between the gene and the message, for example, a primer that hybridizes to a sequence in the untranscribed region of the .alpha.6(IV) promoter will be gene specific.

Other techniques suitable to the claimed methods are readily apparent to the skilled artisan and can include Nuclease Protection Assays, ELISA and Western blotting. Several assay techniques which are based upon immunological reactions between antigens and antibodies are contemplated by the invention, as well. In particular, assays which use antibodies having specificity for .alpha.6(IV) protein are useful to detect cells which produce .alpha.6(IV) protein.

The full sequence cDNA shown in FIG. 1 (SEQ ID NO: 1) also allows production of the .alpha.6(IV) polypeptide via known recombinant DNA techniques. Recombinant production methods will allow the polypeptide to be obtained in a purified, isolated form, which will permit further study of the .alpha.6(IV) polypeptide structure and function. It is further envisioned that such protein production could be scaled up for making large quantities of .alpha.6(IV) polypeptide for commercial purposes. .alpha.6(IV) polypeptide can be incorporated in drug delivery devices or wound dressings, such as used in surgical applications and burn treatment.

Antibodies having specificity for .alpha.6(IV)--expressing cells are obtained by stimulating the immune system of an animal with .alpha.6(IV) protein. In this context, the term "antibody" encompasses monoclonal and polyclonal antibodies. Such an antibody can belong to any antibody class (IgG, IgM, IgA, etc.). According to the present invention, an entire .alpha.6(IV) polypeptide is injected into an animal for the purpose of obtaining polyclonal antibodies, or for obtaining lymphocytes or spleen cells for production of monoclonal antibodies.

The general techniques of monoclonal antibody (Mab) production, such as described by Kohler and Milstein, Nature 256:495 (1975), are applied to produce a monoclonal antibody having specificity for .alpha.6(IV) protein. This procedure includes the steps of isolating lymphocytes of an animal which has been sensitized or injected with .alpha.6(IV) polypeptide, fusing them with myeloma cells to produce hybridomas, then screening the hybridomas for production of "anti-.alpha.6(IV) antibodies" which bind preferentially to or exhibit binding specificity for .alpha.6(IV) polypeptide. Preferably such an antibody is screened to eliminate those antibodies that are cross reactive with other collagen types, such as .alpha.1(IV) or .alpha.5(IV). Either the full length polypeptide molecule shown in FIG. 1 (SEQ. ID NO: 2), or a fragment such as a fragment spanning amino acids 1-1460, inclusive are used to sensitize the animal.

"Antibody" also encompasses fragments, like Fab and F(ab').sub.2, of anti-.alpha.6(IV) antibodies, and conjugates of such fragments, and so-called "antigen binding proteins" (single-chain antibodies) which are based on anti-.alpha.6(IV) antibodies, in accordance, for example, with U.S. Pat. No. 4,704,692, the contents of which are hereby incorporated by reference. Alternatively, Mabs or a fragment thereof within the present invention can be produced using conventional procedures via the expression of isolated DNA which codes for variable regions of such an Mab in host cells like E. coli, see, e.g., Ward et al., Nature 341:544-546 (1989), or transfected murine myeloma cells. See Verhoyen et al., BioAssays 8:74 (1988); Gillies et al., Biotechnol. 7:799-804 (1989); Nakatani et al., Biotechnol. 7:805-10 (1989).

Assays in which the above antibodies are employed can include enzyme-linked immunosorbent assay (ELISA), radioimmunoassays, immunoelectrophoresis, and the like. Also useful diagnostically are immunohistochemical techniques which employ monoclonal antibodies of known, specific reactivities.

Diagnostic applications of these antibodies are exemplified, according to the present invention, by the use of a kit containing an anti-.alpha.6(IV) antibody, which undergoes a reaction with a biological sample to detect .alpha.6(IV) protein expression. Such a reaction involves the binding of anti-.alpha.6(IV) antibody to .alpha.6(IV) antigen, under conditions permissive of binding. The observation of an antibody-antigen complex in a biological sample indicates a positive result. A kit of this sort could be used to detect the extent of expression of .alpha.6(IV) in a particular biological sample from an individual, animal, or cell line.

Such an immunodiagnostic kit can include anti-.alpha.6(IV) antibody and a receptacle for containing the antibody in a sterilized form. The kit can further include anti-isotype serum antibody which recognizes the anti-.alpha.6(IV) antibody (Fc portion) and which is conjugated to a label, such as an enzyme or fluorescent moiety.

Another embodiment of the present invention concerns gene therapy applications of the human COL4A6 polynucleotide molecule to treat a patient having a collagen type IV-associated pathology. An adenoviral vector or other suitable vector containing a copy of COL4A6 polynucleotide molecule, for example, provides one means for ex vivo gene transfer in the clinical setting. A preferred vector is characterized by its ability to stably integrate into at least the targeted collagen producing cells and to induce production of an effective amount of .alpha.6(IV) in such cells of the patient.

A method for gene therapy includes the step of administering to a patient a vector containing human COL4A6 polynucleotide molecule such that transfection with the vector induces production of an effective amount of .alpha.6(IV) in the patient.

The present invention is further described with reference to the following, illustrative examples. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the invention, the preferred methods and materials have been described. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies known to the art. The materials, methods and examples are illustrative only and not limiting.

To isolate COL4A6, an X-chromosome library was screened with JZ-4, an .alpha.5(IV) cDNA clone obtained according to the method of Zhou et al., J. Biol. Chem. 267:12475 (1992). ›This reference is incorporated by reference in its entirety! A 14.1-kb clone, 2LA226, was isolated, as shown in FIG. 2 (SEQ ID NOS: 3-6). This clone contained exon 1 of COL4A5 and an upstream 2.8-kb Hind III fragment, lambda-LA226-H6, that displayed cross-species hybridization with mouse or cow DNA.

Next, lambda-LA226-H6 was used to probe an adult kidney cDNA library. Three identical clones, JZK-1, JZK-2, and JZ-3, each contained an open reading frame (1643 bp) encoding a 21-amino acid signal peptide, a 25-amino acid noncollagenous segment, and a 502-amino acid collagenous domain with nine interruptions (see FIG. 3) that are believed to confer flexibility in type IV collagens.

The deduced translation product, termed .alpha.6(IV), is a type IV collagen that has not previously been detected genetically or biochemically. Sequence analysis clearly places .alpha.6(IV) in the .alpha.2(IV)-like class (FIG. 3, SEQ ID NOS 7-10).

In normal adult kidney, COL4A6 is expressed, making this collagen gene a good candidate for a second X-linked AS gene. The first known of such genes, COL4A5, harbors mutations in relatively low frequency in AS patients, supporting the likelihood that some other factor, now identified as COL4A6, participates in the X-linked disease. This possibility was confirmed by Southern (DNA) blot analysis 140 AS patients, including four unrelated AS-DL patients (13), with JZ-3. Genomic DNA samples (.apprxeq.5 .mu.g) from patients and controls were digested with Eco RI and hybridized with JZ-3-FR5, a cDNA fragment containing the first four exons of .alpha.6(IV).

Analysis of collagen gene deletions in five patients with AS-DL revealed an abnormal pattern in the AS-DL patients, shown in FIG. 4. There was a loss of bands in males and a 50% reduction in the intensity of some bands in females, but the pattern was complex. A fragment containing exons 1 to 4 of COL4A6, JZ-3-FR5, was used to map the deletions more precisely. The 1.4-kb and 5.6-kb Eco RI fragments, which contain exons 1 and 2 of COL4A6, were absent in males and reduced in intensity by .apprxeq.50% in females; exons 3 and 4 were intact. Hybridization with JZ-4 demonstrated the loss of exon 1 of COL4A5. Exons 2 to 10 of COL4A5 were present in four of the five patients. Therefore, the smallest AS-DL deletions involve part of intron 1 and all of exon 1 of COL4A5, the intergenetic region, and exons 1 and 2 and part of intron 2 of COL4A6.