Diagnosis of and therapy for hereditary haemorrhagic telangiectasia

BACKGROUND OF THE INVENTION
The field of the invention is diagnosis and genetic therapy for inherited diseases.
Hereditary haemorrhagic telangiectasia (HHT) or Osler-Weber-Rendu disease (OMIM #18730) is an autosomal dominant disorder characterized by multisystemic vascular dysplasia and recurrent haemorrhage. The disorder is named after the recurrent hemorrhage from vascular lesions, especially in the nasal mucosa and gastrointestinal tract, and for the presence of mucosal, dermal and visceral telangiectases. Pulmonary arteriovenous malformations (PAVMs) occur in approximately 20% of patients and are associated with serious complications including stroke and brain abscess. Other neurological manifestations include cerebral arteriovenous malformation, aneurysm and migraine headache.
Ultrastructural analyses of the vascular dysplasia seen in affected individuals have failed to demonstrate a unique pathological abnormality that might suggest the nature of the primary biochemical defect. Studies indicate that the dilated channels of telangiectases are lined by a single layer of endothelium attached to a continuous basement membrane (Jahnke, Arch. Ototaryngol. 91:262-265, 1970; Hashimoto and Pritzker, Oral Surg., Oral Med., Oral Pathol. 34:751-768, 1972). The earliest event in the formation of telangiectases appears to be dilation of post-capillary venules (Braverman et al., J. Invest Dermatol. 95:422-427, 1990). Eventually the dilated venules connect to enlarging arterioles through capillary segments which later disappear, creating direct arteriolar-venular connections. This sequence of events is associated with a perivascular mononuclear infiltrate (Braverman et al., J. Invest Dermatol. 95:422-427, 1990). Various explanations have been put forward to explain the angiodysplasia seen in HHT, including endothelial cell degeneration (Manafee et al., Arch. Ototaryngol. 101:246-251, 1975), defects in endothelial junctions (Hashimoto and Pritzker, Oral Surg., Oral Med., Oral Pathol. 34:751-768, 1972), lack of elastic fibers and incomplete smooth muscle cell coating of the vessels (Jahnke, Arch. Otolaryngol. 91:262-265, 1970), and weak connective tissue surrounding the vessel (Manafee et al., Arch. Otolaryngol. 101:246-251, 1975).
Genetic linkage for some HHT families was recently established to markers on chromosome 9q33-q34 (McDonald et al., Nature Genet. 6:197-204, 1994; Shovlin et al., Nature Genet. 6:205-209, 1994) and the locus was named OWR1. Genetic heterogeneity was established with the identification of some families clearly not linked to this region (Shovlin et al., Nature Genet. 6:205-209, 1994). The identification of key obligate recombinants in affected individuals allowed refinement of the OWR1 locus and placed the most likely candidate interval between D9S60 and D9S61 in a 2 centiMorgan (cM) interval (Shovlin et al., Nature Genet. 6:205-209, 1994).
SUMMARY OF THE INVENTION
The present invention is based upon the discovery that various defects in the gene encoding endoglin, a transforming growth factor .beta. (TGF-.beta.) binding protein, are responsible for some cases of HHT. Endoglin is a homodimeric integral membrane glycoprotein expressed at high levels on human vascular endothelial cells of capillaries, arterioles and venules in all tissues examined (Gougos and Letarte, J. Biol. Chem. 265:8361-8364, 1990; and Bellon et al., Eur. J. Immunol. 23:2340-2345, 1993). The cDNA sequence of human endoglin (SEQ ID NO:1) is shown in FIG. 1. In the work discussed herein, the genomic DNA of human endoglin has been cloned, its exon/intron structure determined, and the characteristics of certain HHT-associated mutations ascertained.
Endoglin is the most abundant TGF-.beta. binding protein on endothelial cells (Cheifetz et al., J. Biol. Chem. 267:19027-19030, 1992). In the presence of TGF-.beta. ligand, endoglin can associate with the two TGF-.beta. signaling receptors RI and RII (Yamashita et al., J. Biol. Chem. 269:1995-2001, 1994). TGF-.beta. is the prototype of a family of at least 25 growth factors which regulate growth, differentiation, motility, tissue remodeling, wound repair and programmed cell death in many cell types (Massague et al., Trends Cell Biol. 4:172-178, 1994, herein incorporated by reference).
The invention features an isolated DNA comprising a human genomic DNA sequence encoding endoglin. The genomic DNA preferably includes a nucleotide sequence corresponding to any one or more of SEQ ID NOs:9-19 and 42, and more preferably has the sequence of FIG. 5 (SEQ ID NOs:9-19 and 42). By "isolated DNA" is meant a DNA that is not immediately contiguous with at least one of the two genes with which it is immediately contiguous (i.e., one at the 5' end and one at the 3' end) in the naturally-occurring human genome. The term thus encompasses, for example, a genomic DNA fragment produced by PCR or restriction endonuclease treatment, whether such fragment is incorporated into a vector, integrated into the genome of a cell (at a site other than the naturally-occurring site), or independent of any other DNA sequences. The DNA may be double-stranded or single-stranded, sense or antisense.
Also within the invention is a single-stranded oligonucleotide 14-50 nucleotides in length having a nucleotide sequence identical to that of a portion of a strand of a human endoglin gene, which portion is within an intron of the gene and preferably borders an adjacent exon. Such an oligonucleotide can be paired with a second single-stranded oligonucleotide also 14-50 nucleotides length which is identical to a fragment of the strand complementary to the first strand (i.e., where the first strand is the sense strand, the second strand would be the antisense strand, and vice versa), which fragment is within (a) a second intron, (b) the 5' untranslated region immediately adjacent to the translation start signal, or (c) the 3' untranslated region immediately adjacent to the termination signal of the gene. The pair of oligonucleotides could serve as PCR primers selected to prime the PCR amplification of a single exon of the gene, or up to three exons (with intervening introns).
Described herein is a diagnostic method useful for determining whether a patient or a fetus bears a gene that would make the patient or fetus susceptible to HHT. This method includes the steps of obtaining a sample of genomic DNA from the patient (e.g., in the form of a blood sample) or fetus (e.g., by amniocentesis or chorionic villi sampling), and determining whether the DNA contains a mutation in a gene encoding endoglin, betaglycan, TGF-.beta. type I receptor (RI), TGF-.beta. type II receptor (RII), or TGF-.beta./activin type I receptor (TSR-I), such a mutation being an indication that the patient or fetus bears a gene making the patient or fetus susceptible to HHT. The method may include the step of treating the sample of genomic DNA with a restriction enzyme (e.g., MaeIII or BbvI where the gene of interest is endoglin). Alternatively or in addition, the method may include the step of subjecting the sample to PCR, using (1) a forward PCR primer complementary to a portion of the antisense strand of the gene, such portion being within (a) a first intron of said gene, or (b) the 5' untranslated region adjacent to the start codon of the gene; and (2) a reverse PCR primer complementary to a fragment of the sense strand of the gene, such fragment being within (a) a second intron of the gene, or (b) the 3' untranslated region adjacent to the termination codon of the gene.
Another diagnostic method of the invention includes the steps of identifying an individual suspected of being genetically predisposed to developing the HHT phenotype; obtaining from that individual a sample of mRNA from a tissue that normally expresses a gene of the TGF-.beta. receptor complex, such as endoglin (e.g., cells of a newborn's placenta or umbilical cord or cells from the peripheral blood, vascular endothelium or mesenchyme of older individuals); subjecting the mRNA to RT-PCR to produce amplified cDNA having a sequence corresponding to that of a portion of the mRNA of the gene of interest; and determining whether the amplified cDNA includes a mutation responsible for the HHT phenotype, and thus indicates that the individual is genetically predisposed to developing the HHT phenotype.
The leukocyte antigen "CD 105" was assigned to endoglin at a recent Leukocyte typing workshop (Leukocyte Typing V, 1994). CD105/endoglin is expressed by cells within the "buffy coat" of peripheral blood. The "buffy coat" consists of leukocytes that are isolated by density centrifugation of whole blood. When mRNA is isolated from these cells, reverse transcribed, and amplified by a single round (35 cycles) of PCR, endoglin cDNA is reliably produced in quantities sufficient for mutational analysis by, for example, restriction digest. Unlike the umbilical cord or placenta, cells within peripheral blood provide a readily available source of endoglin mRNA.
The invention also includes compositions and methods for treating or preventing the symptoms of HHT by means of genetic therapy. Such methods would include identifying a patient who has inherited a defective gene that encodes a component of the TGF-.beta. receptor complex (e.g., endoglin), and introducing an isolated DNA that encodes that component into the patient's cells. The targeted cells could include any of those that normally express genes in the TGF-.beta. receptor complex, such as vascular endothelial cells, macrophages, or mesenchymal cells, and the DNA could be expressed by operably linking the cDNA or genomic DNA to a regulatory sequence that would direct expression in the targeted cells. The isolated DNA is preferably within (a) a vector suitable for introducing DNA into cells that would normally express endoglin, or (b) liposomes suitable for introducing DNA into these cells. The vector can include the endoglin cDNA sequence of SEQ ID NO:1 or a degenerate variant thereof, and may be introduced into the patient by intravenous or intra-arterial injection or by intravenous or intra-arterial catheter.
One could alternatively treat HHT by increasing the amount of TGF-.beta. available at the cell surface. The object of this treatment would be to overcome the resistance of TGF-.beta.-responsive cells caused by a malfunctioning TGF-.beta. receptor complex. This could be accomplished by applying a pharmaceutical preparation containing TGF-.beta. (preferably .beta.1 or .beta.3) to the skin or mucous membrane at the site of a telangiectasis (e.g., to the membranes lining the patient's nose), or by injection.
The invention further encompasses an animal model for HHT, such as a non-human transgenic animal (e.g., a rodent such as a mouse or rat) bearing a transgene encoding a truncated or otherwise mutant form of endoglin; a mouse, each of the cells of which has only one allele encoding wildtype endoglin; or a mouse which has no allele encoding wildtype endoglin.
Other features and advantages of the invention will be apparent from the detailed description that follows, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a representation of the genomic structure of endoglin. The cDNA sequence of endoglin (SEQ ID NO:1) is shown with the deduced amino acid sequence (SEQ ID NO:2) below. The nucleotide and amino acid positions are based on numbering the A in the ATG start codon of the full-length L-form of endoglin (Bellon et al., Eur. J. Immunol. 23:2340-2345, 1993) as nucleotide number 1. Exons 1 through 14 are labelled above the cDNA sequence in bold and the intron/exon borders are marked with arrows. Exon 7 codes for amino acids 273-331; exon 11 spans residues 477-562. The four potential N-linked glycosylation sites are in boldface, italicized type and are underlined. The membrane spanning domain is double underlined. The positions of the mutations described in this report are shown in relation to the gene structure; the C to G change at nucleotide 831 is indicated by a star and the positions of the two deletions are underlined (nucleotides 882-920 and 1553-1554). The 2 bp deletion creates a premature termination codon, which is indicated by bold type.
FIG. 2a is an heteroduplex analysis showing a shift in an affected proband (sample 1159; lane 1) next to two samples (lanes 2 and 3) not displaying this anomaly.
FIG. 2b is a photograph of a sequencing gel comparing the sequences of representative clones revealing the normal (C) and mutant sequence (G) of amplified exon 7 in sample 1159.
FIG. 2c is a comparison of the normal (SEQ ID NO:3) and mutant (SEQ ID NO:4) DNA and amino acid sequences in the region of the mutation in sample 1159. This C to G substitution converts a tyrosine to a stop codon at amino acid 277.
FIG. 3a is a comparison of the normal (SEQ ID NO:5) and mutant (SEQ ID NO:6) DNA and amino acid sequences in the region of the deletion mutation in sample 8019 from the 9q3-linked Family 3186. This 39 bp deletion, which is located at nucleotide positions 882 through 920 in exon 7, removes 13 amino acids from the protein and alters the first amino acid (position 307) in a potential N-linked glycosylation site.
FIG. 3b is an illustration of the inheritance of HHT by the members of Family 3186, showing segregation of the 39 bp deletion. Agarose gel analysis of amplified exon 7 from each member of Family 3186 reveals the presence of a lower band (the 39 bp deletion product) in affected family members only. Preferential amplification of the smaller fragment is sometimes observed (see individual 13).
FIG. 4a is an heteroduplex analysis of sample 2061 showing a shift in an affected proband (sample 2061; lane 1) next to two samples (lanes 2 and 3) not displaying this anomaly.
FIG. 4b is a photograph of gels indicating the sequence of the 2 bp deletion in sample 2061, compared to the normal sequence. The sequences of the two independently cloned PCR products of affected individual sample 2061 reveal the normal sequence and the 2 bp deletion in exon 11 beginning at nucleotide position 1553.
FIG. 4c is a representation of the normal (SEQ ID NO:7) and mutant (SEQ ID NO:8) DNA and amino acid sequences in the region of the 2 bp deletion in sample 2061. The two deleted nucleotides are underlined in the normal sequence. This mutation causes a frameshift and a premature termination after an additional seven amino acids.
FIG. 4d is an illustration of the inheritance of HHT by the members of Family 63, and an agarose gel electrophoresis analysis of amplified exon 11 from each family member, showing segregation of the 2 bp deletion. The 2 bp deletion creates an additional MaeIII restriction site. Affected family members exhibit an additional novel fragment visible as the middle band in each lane, with half the intensity of the other two bands. A second novel band produced by digestion at this site is not visible on this gel.
FIG. 5 is a representation of a partial genomic DNA sequence of the wildtype human endoglin gene (SEQ ID NOs:9-19 and 42). Capital letters indicate coding sequence (exons), while lower-case letters indicate introns. Dashes indicate portions of the introns for which the nucleotide sequence has not yet been determined.
FIG. 6a is a photograph of a gel illustrating the sequence of an exon 10 mutation in sample 1247 of family 32. Direct sequencing reveals both the normal (C) and the mutant (T) at nucleotide 1414, which creates a premature termination at codon 472, CAG to TAG.
FIG. 6b is a representation of the pedigree of family 32 denoting affected (closed symbols) and unaffected (open symbols) individuals. The numbers beneath the symbols refer to lane position in FIG. 6c.
FIG. 6c is a photograph of a gel, each lane of which represents BbvI-digested DNA of amplified exon 10 from a member of family 32. This mutation removes a BbvI site in the amplification product. All unaffected members reveal the digested doublet bands, whereas all affected individuals show the presence of three bands, corresponding to the uncut (mutant) and cut (normal) fragments of exon 10.
FIG. 7 is a schematic illustration of the endoglin protein, shown to scale with contributions from each exon shown by the boxes numbered below. Amino acid residues from the first of a portion of the second exon are cleaved from the mature protein and are shown separated from the main body of the protein. Exons 2-12 encode the extracellular domain, exon 13 encodes the transmembrane (TM) domain, and exon 14 encodes the cytoplasmic domain. The positions of the cysteine residues are marked with C. The approximate positions of truncation mutations, whether nonsense or frameshifts, are shown with the asterisks. The bracket delineates the betaglycan-related amino-terminal domain of endoglin, which may correspond to the TGF-.beta. binding domain.

DETAILED DESCRIPTION
The endoglin gene previously was mapped to human chromosome 9q34 using fluorescence in situ hybridization (FISH; Fernandez-Ruiz et al., Cytogenet. Cell Genet. 64:204-207, 1993).
Genomic structure of endoglin
As an initial screen for gross abnormalities in the endoglin gene in affected HHT individuals, Southern blots of DNA from the probands of 33 unrelated families having family members with HHT were probed with a nearly complete cDNA of endoglin, the clone 18A of Gougos and Letarte, supra. This analysis using three restriction endonucleases revealed no gross abnormalities of the endoglin gene in these samples. Reverse transcription-PCR (RT-PCR) was attempted using RNA prepared from several Epstein-Barr virus-transformed lymphoblast lines established from our patient cohort, but expression levels of endoglin appeared to be too low to allow routine amplification in a single round (35 cycles) of PCR. As the expression of endoglin is restricted to endothelial cells, activated monocytes (Lastres et al., Eur. J. Immunol. 22:393-397, 1992), syncytiotrophoblast (Gougos et al., Int'l. Immunol. 4:83-92, 1992), and certain stromal cells (St. Jacques et al., Endocrinology 134:2645-2657, 1994), screening for mutations within endoglin cDNA was not feasible. The genomic structure of endoglin was therefore studied.
A gridded cosmid chromosome 9 library was screened with the 18A cDNA probe, and 17 cosmids were obtained. Southern analysis of these clones in comparison with total genomic DNA revealed that one cosmid, 21c10, appeared to contain most of the gene. This cosmid was subcloned into a phagemid library which was screened for positive plaques with the 18A cDNA probe. Hybridizing clones were converted to plasmids and sequenced using vector primers flanking the cloning site to identify intron-exon borders.
Preliminary sequence analysis suggests that the coding region of endoglin contains 14 exons (FIGS. 1 and 7). one or more splice junctions may remain unidentified within the 5' end of the gene, as the sequence denoted exon 1, which contains the putative signal peptide, was found to be missing in the 21c10 cosmid. There is also evidence for alternative splicing variants of the endoglin transcript. Since only one variant was used to identify subclones for genomic sequencing, it is possible that additional exons exist within the depicted coding region (FIG. 1). (The exon number assignments must be regarded as preliminary until the entire gene structure is resolved.)
The 14 exons are sufficiently small to allow for PCR amplification of each as a single unit (Table 1). The smallest is exon 12 which contains the complete membrane spanning domain and is 55 basepairs (bp) in length. The longest exon completely contained within the coding region is exon 11 (258 bp). Exon 14 contains at least 429 bp but contains only 125 bases of coding information, the remainder being the 3' untranslated region.
TABLE 1__________________________________________________________________________PCR assays for endoglin exons Size Forward Primer Reverse Primer PCR Annealing # ofExon (pb) Sequence (5'->3') Sequence (5'->3') Prod. Size Buffer [MgCl2] Temp cycles__________________________________________________________________________1 >672 152 cctcataaggtggctgtgatgatg catctgccttggagcttcctct 413 BHB 1.5 mM 60.degree. 35 SEQ ID NO: 20 SEQ ID NO: 213 1414 163 ttcctgacctcctacatgg ctcttggtgcccaagttt 330 TNK 50 1.0 mM 51.degree. 30 SEQ ID NO: 22 SEQ ID NO: 235 166 cgggctctgttaggtgcag gggtggggctttataaggga 294 TNK 25 1.0 mM 57.degree. 35 SEQ ID NO: 24 SEQ ID NO: 256 127 ctgtccgcttcagtgttccatc ggaaacttccctgatccagaggtt 230 TNK 100 1.5 mM 59.degree. 40 SEQ ID NO: 26 SEQ ID NO: 277 176 gaggcctggcataaccct gtggccactgatccaagg 315 BMB 1.5 mM 60.degree. 35 SEQ ID NO: 28 SEQ ID NO: 298 141 acacatatcacacagtgaccagc ctaggggaggaaccagatgtc TNK 50 1.0 mM 55.degree. 30 SEQ ID NO: 30 SEQ ID NO: 319 17810 117 agattgaccaagtctccctccc aggctgtctccctcctgactct 227 TNK 50 1.0 mM 61.degree. 35 SEQ ID NO: 32 SEQ ID NO: 3311 258 actcaggggtgggaactctt ccttccatgcaaaccacag 430 TNK 50 1.0 mM 57.degree. 32 SEQ ID NO: 34 SEQ ID NO: 3512 55 gagtaaacctggaagccgc gccactagaacaaacccgag 164 TNK 100 1.0 mM 55.degree. 35 SEQ ID NO: 36 SEQ ID NO: 3713 111 ccagcacaacagggtaggggat ctcagaggcttcactgggctcc 255 TNK 50 1.0 mM 61.degree. 35 SEQ ID NO: 38 SEQ ID NO: 3914 >429 tgaagcctctgagggattgagg gagttcacaccagtgctcccag 267 TNK 50 1.0 mM 57.degree. 40 SEQ ID NO: 40 SEQ ID NO: 41__________________________________________________________________________
Identification of HHT mutations
In an initial screen for mutations, primers located within the introns flanking exons 7 and 11 (the first exons to be identified) were designed to establish PCR assays for each exon (see Methodology). A panel of 68 DNA samples was used for the mutation screen. These were collected from probands of unrelated families, most of which were members of kindreds with PAVM involvement, increasing the likelihood that the individuals would harbour mutations at the OWR1 locus. The panel of DNA samples used in the initial mutation screen included affected individuals from nine families previously linked to 9q34 (McDonald et al., Nature Genet. 6:197-204, 1994; Porteous et al., J. Med. Genet. 31:925-926, 1994; McAllister et al., J. Med. Genet. 31:927-932, 1994; Heutink et al., J. Med. Genet. 31:933-936, 1994). Heteroduplex analysis was performed on amplified products from this cohort as a screen for potential mutations. Abnormal PCR products seen on these gels were directly sequenced for further analysis.
With the initial screen, three mutations in affected individuals were identified. The first mutation was identified by a heteroduplex shift in the exon 7 PCR product from sample 1159 (FIG. 2a). The products of two independent PCR reactions were directly sequenced, whereupon a C (normal) and a G (mutant) at nucleotide position 831 were clearly visible. PCR products amplified from this individual were then cloned and individual clones sequenced to validate the results of the direct sequencing (FIG. 2b). This change converts a tyrosine at codon 277 to a termination codon (FIG. 2c). This mutation is present in the proband of a pedigree with multiple affected members having documented PAVMs. However, additional members of this family were not available for analysis. The truncated protein resulting from this mutation would comprise only half of the extracellular domain and lack the membrane spanning and cytoplasmic domains.
Amplification of exon 7 in sample 8019 revealed a second mutation, in a family (Family 3186) previously linked to 9q3. A second PCR fragment smaller than the wild-type fragment was visible in both agarose gels and heteroduplex analysis, suggesting the existence of a deletion. The smaller fragment was not seen in 278 normal chromosomes and is unlikely to be a polymorphism. Sequence analysis of the PCR products revealed a 39 bp deletion in the exon beginning at nucleotide position 882 of endoglin (FIG. 3a). This in-frame deletion removes 13 amino acids (amino acids 295 to 307) and alters the first amino acid of a potential N-linked glycosylation site (see FIG. 1). Amplification of this exon revealed the presence of the deletion in all affected family members, but no unaffected members (FIG. 3b).
Heteroduplex analysis of amplified exon 11 revealed a very pronounced band in sample 2061 that was not visible with agarose gel electrophoresis (FIG. 4a). Independent clones of the PCR product were sequenced, revealing the wild-type sequence and a 2 bp deletion beginning with nucleotide 1553 of endoglin (FIG. 4b). This deletion creates a MaeIII restriction site. This sample was from the proband of a family with multiple affected members displaying PAVMs. Exon 11 was amplified from all available family members and digested with MaeIII. All affected family members share the additional MaeIII site, whereas the unaffected members do not, establishing linkage of this mutation to the disease phenotype in this family (FIG. 4d). The mutation creates a frame shift that results in a premature termination codon 8 amino acids beyond the deletion (FIG. 4c). The predicted truncated protein would lack the membrane spanning and cytoplasmic domains of endoglin.
A subsequent screen detected six additional mutations. Primers were designed to amplify 11 of the 14 endoglin exons, heteroduplex analysis was performed, and PCR products were directly sequenced. Mutations were found in exons 5, 7, 8, 10 and 11, and all but one of these mutations would produce a truncated protein if translated. Each of the mutations was seen only in a single chromosome in over 160 total chromosomes analyzed. Where additional family members were available, the mutations were shown to co-segregate with the disease phenotype.
One of the mutations detected in the second screen includes a C to T base pair substitution, creating a stop codon in exon 10 in a previously described ORW1 family (McAllister et al., J. Med. Genet. 31:927-932, 1994). The normal and mutant bases were seen in direct sequencing of the PCR product (FIG. 6a). This point mutation removes a BbvI restriction site. Exon 10 was amplified from all available family members and digested with BbvI. All unaffected family members revealed the resulting digested fragments (normal allele), whereas all affected family members revealed the uncut fragment (mutant allele) in addition to the digested fragments (FIGS. 6b and 6c). A summary of all nine currently defined endoglin mutations appears in Table 2.
TABLE 2__________________________________________________________________________Endoglin Mutation SummaryExon Sample No. Family No. Type of Mutation__________________________________________________________________________5 1152 Substitution at position 587 creates stop condon TGG to TAG7 58019 3186 (5) Deletion (39 bp) at position 882 (6)7 1159 Substitution at position 831, creates stop condon TAC to TAG (6)8 1275 56 (4) Substitution at position 1050 creates stop condon TGT to TGA8 1160 Insertion (1 bp) at position 1111, frameshift creates premature stop condon10 1247 32 (4) Substitution at position 1414, creates stop condon CAG to TAG11 102061 Deletion (2 bp) at position 1553, frameshift creates premature stop condon (6)11 1162 Deletion (1 bp) at position 1655, frameshift creates premature stop condon11 2241 Deletion (2 bp) at position 1550, frameshift creates premature stop condon__________________________________________________________________________
DISCUSSION
These results, in which nine independent mutations in affected HHT individuals are identified, establish the endoglin gene as the HHT1 disease locus mapping to 9q3. The gene maps to the tightest HHT1 candidate interval on 9q33-q34, based on evidence from the mouse and human genetic and physical maps. The restricted tissue distribution of endoglin and its expression at high levels on the surface of endothelial cells is consistent with the pathology of the disorder.
One of the nine mutations described in this report would remove 13 amino acids from the extracellular domain of endoglin and could have a deleterious effect on receptor function. A defect in a cell surface TGF-.beta. binding protein would account for the limited and localized nature of the vascular lesions present in this disease.
The eight remaining mutations create premature termination codons and would be expected to lead to reduced message levels that, if translated, would encode severely truncated proteins, suggestive of loss-of-function alleles. The apparent clustering of premature termination mutations in exons 5-11 is also consistent with a dominant-negative model. This model would suggest that the normal endoglin function is disrupted as a consequence of an effect caused by the mutant protein. Endoglin is isolated from cells as a homodimer that can be dissociated by reducing agents (Gougos and Letarte, J. Biol. Chem. 265:8361-8364, 1990; Gougos and Letarte, J. Immunol. 141:1934-1940, 1988). In affected individuals it is possible that normal/mutant hetero-dimers, which do not function properly or which lead to rapid turnover of the receptor complex, are assembled. Three cysteines are present in the processed endoglin protein prior to the first known truncation mutation in exon 5. One or more of these may be required for inter-molecular disulfide linkage, which might explain the apparent lack of truncation mutations prior to exon 5. Binding of TGF-.beta. may also be required for the dominant-negative effect. Deletion of the amino-terminal endoglin-related domain of betaglycan abolishes the TGF-.beta. binding activity of betaglycan (Lopez-Casillas et al., J. Cell Biol. 124:557-568, 1994). It is proposed that the corresponding region of endoglin constitutes the TGF-.beta. binding domain of endoglin.
Alternatively, the mutant endoglin allele might produce a soluble polypeptide which can bind and sequester the TGF-.beta. ligand. Deletion mutants of betaglycan which remove the transmembrane and cytoplasmic domains produce soluble polypeptides which can bind TGF-.beta. and inhibit TGF-.beta. binding to membrane receptors (Lopez-Casillas et al., J. Cell Biol. 124:557-568). In the same way, soluble endoglin may act as an antagonist of TGF-.beta. signaling.
TGF-.beta. in vivo is a potent angiogenic factor and a mediator of vascular remodelling as it controls extracellular matrix production by endothelial cells, smooth muscle cells and pericytes (Madri et al., Endothelial cell dysfunctions (eds. Simionescu & Simionescu) (Plenum Press, New York, 1992). Following soft tissue injury or in response to angiogenic factors, microvascular endothelial cells detach from their basement membrane, migrate and proliferate in the interstitial stroma, and form new microvessels. When grown in vitro in three-dimensional gels and in the presence of TGF-.beta., these endothelial cells form tube-like cellular aggregates with a lumen and tight junctions, and deposit an organized basement membrane, mimicking vessel formation (Madri et al., supra). However, TGF-.beta., almost exclusively in the .beta.1 isoform, will inhibit the proliferation of endothelial cells grown on plastic (Madri et al., supra; Jennings et al., J. Cell Physiol. 137:167-172, 1988). The response of endothelial cells to TGF-.beta. depends on the interaction with the surrounding extracellular matrix via integrins expressed on their surface (Luscinkas et al., FASEB J. 8:929-938, 1994). The production of matrix protein by stromal interstitial cells, smooth muscle cells, pericytes and endothelial cells and the expression of integrins on endothelial cells are also regulated by TGF-.beta. (Madri et al., supra; Luscinkas et al., FASEB J. 8:929-938, 1994).
Endothelial cells lacking endoglin may respond poorly to TGF-.beta.1 and thus form abnormal blood vessels, particularly in response to injury. TGF-.beta. signalling is mediated by TGF-.beta. receptors RI and RII, which form a heteromeric complex upon binding TGF-.beta. (Wrana et al., Cell 71:1003-1014, 1992; Wrana et al., Nature 370:341-347, 1994). Endoglin, which binds TGF-.beta.1 and -.beta.3 with high affinity but does not bind TGF-.beta.2 (Cheifetz et al., J. Biol. Chem. 267:19027-19030, 1992), is structurally related to betaglycan, which binds all three isoforms of TGF-.beta. (Lopez-Casillas et al., Cell 67:785-795, 1991; Wang et al., Cell 67:797-805, 1991). Betaglycan in the presence of ligand interacts with the signaling kinase complex of RI and RII and potentiates the response to all three isoforms of the growth factor (Lopez-Casillas et al., Cell 73:1435-1444, 1993). Endoglin also interacts with the kinase complex, suggesting a potentiating role similar to that of betaglycan. Endothelial cells express very low levels of betaglycan, which may explain their poor response to TGF-.beta.2 (Cheifetz et al., J. Biol. Chem. 265:20533-20538, 1990). Thus endoglin-deficient endothelial cells, as observed in HHT1-linked patients, would express only the signaling RI and RII complex and would lack the regulatory co-receptor capable of controlling the response. This might alter cell adhesion properties, leading to the vascular anomalies seen in this disorder. Stromal cells in several tissues (St. Jacques et al., Endocrinology 134:2645-2657, 1994) and activated monocytes (Lastres et al., Eur. J. Immunol. 22:393-397, 1992) also express endoglin and could be impaired in their response to TGF-.beta.1 in the vascular lesions of HHT1 patients.
METHODOLOGY
Clinical evaluation. The diagnostic criteria used for collection of family members was as described (McDonald et al., Nature Genet. 6:197-204, 1994).
Genomic sequence determination. A nearly complete cDNA sequence of endoglin (18A) was used to screen a gridded chromosome 9 cosmid library (Los Alamos National Laboratory). One subclone that contained nearly all hybridizing bands that are seen with genomic DNA was subcloned using Lambda ZAP Express system (Stratagene). Plaque screens were performed by hybridization with the 18A cDNA probe to identify positive clones. Intron-exon borders were identified by sequencing these clones using the Sequenase Ver. 2.0 DNA sequencing kit (United States Biochemical) using both vector and exon primers.
PCR amplification on exons. Primers were designed from intron genomic sequences flanking exons 7 and 11 of endoglin. For exon 7 (nts 817-992), the forward primer is 5'-GAGGCCTGGCATAACCCT (SEQ ID NO:28), and the reverse primer is 5'-GTGGCCACTGATCCAAGG (SEQ ID NO:29). The 315 bp product was amplified using a buffer consisting of 10 mM Tris-HCl, pH 8.3; 1.5 mM MgCl.sub.2 ; and 50 mM KCl. After initial denaturation, 35 cycles of the following program were run: 94.degree. C. for 30 s; 60.degree. C. for 60 s; 72.degree. C. for 30 s. For exon 11 (nts 1429-1686), the forward primer is 5'ACTCAGGGGTGGGAACTCTT (SEQ ID NO:34) and the reverse is 5'-CCTTCCATGCAAACCACAG (SEQ ID NO:35). The 430 bp product was amplified in 10 mM Tris-HCl, pH 8.3; 1 mM MgCl.sub.2 ; 50 mM KCl; and 5 mM NH.sub.4 Cl. After initial denaturation, 32 cycles of the following program were run: 94.degree. C. for 50 sec., 57.degree. C. for 60 sec., 72.degree. C. for 30 sec.
Each exon can be amplified using conditions described in Table 1. The amplification reaction contains 100 ng of genomic DNA, 100 ng of each oligonucleotide primer, 0.20 mM of each dNTP, and 1.25 U of Taq DNA polymerase in final volume of 25 .mu.l. Reaction conditions were optimized individually for each primer pair by adjusting annealing temperatures and buffer conditions as described (Blanchard et al., PCR Meth. Applic. 2:234-240, 1993), using the Taq polymerase buffer supplied by Boehringer Mannheim Biochemicals, Indianapolis, Ind. (BMB).
Mutation analysis. Heteroduplex analysis was carried out as described using MDE gel mix (AT Biochem) with the addition of 15% urea. Samples were denatured for 5 min and allowed to slow cool before fragments were separated by electrophoresis on non-denaturing gels. Products were visualized by ethidium bromide staining. Altered PCR products detected by heteroduplex analysis were directly sequenced using AmpliTaq Cycle sequencing kit (Perkin Elmer). Primers were end-labelled and samples run on 6% polyacrylamide gels. PCR products of the individuals containing the identified stop codon and the 2 bp deletion were cloned into pCR-Script Direct SK(+) cloning vector using pCR-Script Direct SK(+) Directional Cloning Kit (Stratagene) and sequenced using Sequenase Ver. 2.0 (United State Biochemical).
EXAMPLE 1
Diagnosis
The discovery that a defect in a component of the TGF-.beta. receptor complex underlies the HHT phenotype means that individuals (in particular, those with a family history of the disease) can be tested for inheritance of the disease gene even before symptoms appear. This will permit appropriate genetic counseling of those individuals who have inherited the disease. In addition, individuals diagnosed with the genetic defect can be closely monitored for the appearance of symptoms, permitting early intervention, including genetic therapy, as appropriate. Analysis can be carried out on any suitable genomic DNA sample from the individual to be tested. Typically, a blood sample from an adult or child, or a sample of placental or umbilical cord cells of a newborn would be used; alternatively, one could utilize a fetal sample obtained by amniocentesis or chorionic villi sampling.
It is expected that standard genetic diagnostic methods can be used. For example, PCR (polymerase chain reaction) can be utilized in the manner described above, to identify the presence of a deletion, addition, or substitution of one or more nucleotides within any one of the exons of endoglin, RI, RII, or betaglycan. Following the PCR reaction, the PCR product can be analyzed by methods as described above, such as the heteroduplex detection technique based upon that of White et al. (Genomics 12:301-306, 1992), or by techniques such as cleavage of RNA-DNA hybrids using RNase A (Myers et al., Science 230:1242-1246, 1985); single-stranded conformation polymorphism (SSCP) analysis (Orita et al., Genomics 10:298-299, 1989); and denaturing gradient gel electrophoresis (DGGE; Myers et al., Methods Enzymol. 155:501-527, 1987). The PCR may be carried out using a primer which adds a G+C-rich sequence (termed a "GC-clamp") to one end of the PCR product, thus improving the sensitivity of the subsequent DGGE procedure (Sheffield et al., Proc. Natl. Acad. Sci. USA 86:232-236, 1989). If the particular mutation present in the patient's family is known to have removed or added a restriction site, or to have significantly increased or decreased the length of a particular restriction fragment, a protocol based upon restriction fragment length polymorphism (RFLP) analysis (perhaps combined with PCR) may be appropriate. The apparent genetic heterogeneity of the HHT phenotype means that the nature of the particular mutation carried by affected individuals in the patient's family may have to be ascertained (e.g., by methods as described above) prior to attempting genetic diagnosis of the patient. Alternatively, a battery of tests designed to identify any of several mutations known to result in HHT may be utilized to screen individuals without a defined familial genotype. The analysis can be carried out on any genomic DNA derived from the patient, typically from a blood sample.
As discussed above, the genetic defect underlying the HHT phenotype may be in an endoglin gene, or in any of the other components of the TGF-.beta. receptor complex. A given family may harbor a defective gene encoding the type I receptor (Frazen et al., Cell 75:681-692, 1993), type II receptor (Lin et al., Cell 68:775-785, 1992), betaglycan (Wang et al., Cell 67:797-805, 1991; Lopez-Casillas et al., Cell 67:785-795, 1991; Moren et al., Biochem. Biophys. Res. Commun. 189:356-362, 1992), or the type I TGF-.beta./activin receptor (TSR-I; Attisano et al., Cell 75:671-680, 1993). Mutations in each of these genes may be assayed by methods similar to those described herein. If a given family's particular defect has not yet been characterized, selection of the gene most likely to be the source of the defect may be guided by genetic linkage analysis, using appropriate markers.
Instead of basing the diagnosis of HHT on analysis of the genomic DNA of a patient, one could seek evidence of the mutation in the level or nature of the relevant expression products. Unlike genomic DNA-based diagnostic methods, this approach permits detection of defects resulting in a decrease in the level of expression of the affected gene (e.g., endoglin) which do not involve mutations in the coding sequence itself. An analysis of expression requires use of cells that normally express the gene of interest in detectable amounts. For endoglin, that has meant vascular endothelial cells of capillaries, arterioles and venules, or possibly fibroblasts. A useful source of such cells would be the umbilical cord and/or placenta of a newborn, which could be harvested at birth and stored frozen until needed for the diagnostic tests. Recently, endoglin was also found to be expressed in cells within the "buffy coat" of peripheral blood, i.e., white blood cells isolated by density centrifugation of whole blood. This readily available source of endoglin mRNA has obvious advantages over tissues that must be harvested perinatally and stored.
Well-known techniques for analyzing expression include mRNA-based methods, such as Northern blots and in situ hybridization (using a nucleic acid probe derived from the relevant cDNA), and quantitative PCR (as described in St-Jacques et al., Endocrinology 134:2645-2657, 1994). One also could employ polypeptide-based methods, including use of antibodies specific for the polypeptide of interest. These techniques permit quantitation of the amount of expression of a given gene in the tissue of interest, at least relative to positive and negative controls. One would expect an individual who is heterozygous for a genetic defect affecting level of expression of endoglin to show up to a 50% loss of expression of this gene in such a hybridization or antibody-based assay. An antibody specific for the carboxy terminal end would be likely to pick up (by failure to bind to) most or all frame-shift and premature termination signal mutations, as well as deletions of the carboxy terminal sequence. Use of a battery of monoclonal antibodies specific for different epitopes of endoglin would be useful for rapidly screening cells to detect those expressing mutant forms of endoglin (i.e., cells which bind to some endoglin-specific MAbs, but not to others), or for quantifying the level of endoglin on the surface of the cells.
Another type of polypeptide-based assay would measure loss of function. For example, one might determine whether the patient's endoglin-expressing cells bind and/or respond to TGF-.beta. in a manner similar to cells from a normal individual. Binding of TGF-.beta. could be measured using a radiolabelled ligand or ELISA (for example, see Cheifetz et al., J. Biol. Chem. 269:1995-2001, 1994), while response to the ligand could be measured by a standard TGF-.beta. biological assay, e.g. as discussed in Madri et al., Annals of the New York Academy of Science 593:243-258, 1990 (herein incorporated by reference). A significant degree of loss of function (e.g., at least 50%) indicates that the patient bears at least one genetic defect in a gene involved in the TGF-.beta. response, and is indicative of HHT. Since endoglin forms homodimers, it may be that one defective endoglin gene acts as a dominant negative mutant, suppressing the activity of the normal allele. If so, the level of functional dimeric endoglin would be something less than 50% of the normal level (e.g., 10-30%), even though only one allele is defective.
Finally, one could use a protein truncation assay (Heim et al., Nature Genetics 8:218-219, 1994) to screen for any genetic defect which results in the production of a truncated polypeptide instead of the wildtype protein.
EXAMPLE 2
Genetic Therapy
A patient with HHT can be treated by supplying a functional gene encoding the defective component of the TGF-.beta. receptor complex. Following diagnosis to determine which gene of the complex is defective in a given patient, a DNA (e.g., a cDNA) is prepared which encodes the wildtype form of the gene operably linked to expression control elements (e.g., promoter and enhancer) that induce expression in endothelial cells, peripheral blood cells or any other affected cells. Examples of expression control elements that would be useful in targeting endothelial cells include those associated with the genes encoding endoglin; tie-2 (Schnurch and Risau, Development 119:957-968, 1993); the vascular endothelial growth factor (VEGF) receptor Flk-1 (Millauer et al., Cell 72:835-846, 1993); and the Tek endothelial cell receptor (Dumont et al., Genes & Development 8:1897-1909, 1994). The DNA may be incorporated into a vector appropriate for transforming the cells, such as a retrovirus, adenovirus or adeno-associated virus. Alternatively, one of the many other known types of techniques for introducing DNA into cells in vivo may be used: e.g., liposomes. The presence of the target endothelial cells in the lining of the patient's blood vessels means that the DNA may be administered to the patient by the simple means of intravenous injection, whereupon it should travel throughout the bloodstream and contact essentially all of the intended target cells. In such a protocol, it is expected that approximately 1 to 100 .mu.g/kg body weight would be an effective amount of cDNA.
One could instead rely on local injections into the surface lesions and catheter-delivered infusions directly into the deeper lesions (e.g., PAVMs), repeating the treatment as necessary to achieve the desired response. The catheter could be coupled with a pair of balloons to trap the infusion in a particular region of the blood vessel until the DNA can enter the endothelial cells at the site of the telangiecstasis. Approximately 1 to 10 molecules of DNA per endothelial cell, or about 10.sup.3 to 10.sup.6 per telangiectasis, would be an adequate dose.
EXAMPLE 3
TGF-.beta. Treatment
Because the defect in HHT is attributable to the inability of vascular endothelial cells to respond to TGF-.beta., it is expected that the function of these cells can be improved by increasing the amount of TGF-.beta. present at the cellular surface. Thus, patients suffering from the symptoms of HHT can be treated with TGF-.beta., preferably by local injection at the site of the telangiecstasis, or by applying a TGF-.beta.-containing ointment or dressing to the skin or mucous membrane at the site of the lesion. This may be particularly appropriate for treating the frequent nosebleeds which often accompany HHT. Where the genetic defect is in an endoglin gene, TGF-.beta.1 or TGF-.beta.3 may be used.
EXAMPLE 4
Animal Model
A line of transgenic animals (e.g., mice, rats, guinea pigs, hamsters, rabbits, or other mammals) could be produced bearing a transgene encoding a defective form of endoglin which retains the ability to dimerize with the wildtype monomer, but which is biologically inactive and forms inactive dimers. Such a mutant form of endoglin would act as a dominant-negative mutant, suppressing the activity of the wildtype alleles and permitting use of the animal as a model system for studying HHT and potential therapy therefor. Standard methods of generating such transgenic animals would be used, e.g. as described in Leder et al., U.S. Pat. No. 4,736,866.
Alternatively, standard methods of producing null mice could be used to generate a mouse which bears one defective and one wildtype allele encoding endoglin. It is expected that such a mouse would be susceptible to developing the symptoms which characterize HHT in humans heterozygous for a mutant endoglin gene, and so would serve as a useful animal model for the disease. If desired, two such heterozygous mice could be crossed to produce offspring which are homozygous for the mutant allele.
Other embodiments are within the following claims.
Under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure, deposit of cosmid 21c10 has been made with the American Type Culture Collection (ATCC) of Manassas, Va., where the deposit was given Accession Number 98685.
Applicants' assignees, Duke University and HSC Research & Development Limited Partnership, represent that the ATCC is a depository affording permanence of the deposit and ready accessibility thereto by the public if a patent is granted. All restrictions on the availability to the public of the material so deposited have been irrevocably removed. The material will be available during the pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 C.F.R. .sctn.1.14 and 35 U.S.C. .sctn.1.22. The deposited material will be maintained with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposited material, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of the patent, whichever period is longer. Applicants acknowledge their duty to replace the deposit should the depository be unable to furnish a sample when requested due to the condition of the deposit.
__________________________________________________________________________# SEQUENCE LISTING- (1) GENERAL INFORMATION:- (iii) NUMBER OF SEQUENCES: 42- (2) INFORMATION FOR SEQ ID NO:1:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 2025 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (ix) FEATURE: (A) NAME/KEY: Coding Se - #quence (B) LOCATION: 1...1974- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:- ATG GAC CGC GGC ACG CTC CCT CTG GCT GTT GC - #C CTG CTG CTG GCC AGC 48Met Asp Arg Gly Thr Leu Pro Leu Ala Val Al - #a Leu Leu Leu Ala Ser# 15- TGC AGC CTC AGC CCC ACA AGT CTT GCA GAA AC - #A GTC CAT TGT GAC CTT 96Cys Ser Leu Ser Pro Thr Ser Leu Ala Glu Th - #r Val His Cys Asp Leu# 30- CAG CCT GTG GGC CCC GAG AGG GGC GAG GTG AC - #A TAT ACC ACT AGC CAG 144Gln Pro Val Gly Pro Glu Arg Gly Glu Val Th - #r Tyr Thr Thr Ser Gln# 45- GTC TCG AAG GGC TGC GTG GCT CAG GCC CCC AA - #T GCC ATC CTT GAA GTC 192Val Ser Lys Gly Cys Val Ala Gln Ala Pro As - #n Ala Ile Leu Glu Val# 60- CAT GTC CTC TTC CTG GAG TTC CCA ACG GGC CC - #G TCA CAG CTG GAG CTG 240His Val Leu Phe Leu Glu Phe Pro Thr Gly Pr - #o Ser Gln Leu Glu Leu# 80- ACT CTC CAG GCA TCC AAG CAA AAT GGC ACC TG - #G CCC CGA GAG GTG CTT 288Thr Leu Gln Ala Ser Lys Gln Asn Gly Thr Tr - #p Pro Arg Glu Val Leu# 95- CTG GTC CTC AGT GTA AAC AGC AGT GTC TTC CT - #G CAT CTC CAG GCC CTG 336Leu Val Leu Ser Val Asn Ser Ser Val Phe Le - #u His Leu Gln Ala Leu# 110- GGA ATC CCA CTG CAC TTG GCC TAC AAT TCC AG - #C CTG GTC ACC TTC CAA 384Gly Ile Pro Leu His Leu Ala Tyr Asn Ser Se - #r Leu Val Thr Phe Gln# 125- GAG CCC CCG GGG GTC AAC ACC ACA GAG CTG CC - #A TCC TTC CCC AAG ACC 432Glu Pro Pro Gly Val Asn Thr Thr Glu Leu Pr - #o Ser Phe Pro Lys Thr# 140- CAG ATC CTT GAG TGG GCA GCT GAG AGG GGC CC - #C ATC ACC TCT GCT GCT 480Gln Ile Leu Glu Trp Ala Ala Glu Arg Gly Pr - #o Ile Thr Ser Ala Ala145 1 - #50 1 - #55 1 -#60- GAG CTG AAT GAC CCC CAG AGC ATC CTC CTC CG - #A CTG GGC CAA GCC CAG 528Glu Leu Asn Asp Pro Gln Ser Ile Leu Leu Ar - #g Leu Gly Gln Ala Gln# 175- GGG TCA CTG TCC TTC TGC ATG CTG GAA GCC AG - #C CAG GAC ATG GGC CGC 576Gly Ser Leu Ser Phe Cys Met Leu Glu Ala Se - #r Gln Asp Met Gly Arg# 190- ACG CTC GAG TGG CGG CCG CGT ACT CCA GCC TT - #G GTC CGG GGC TGC CAC 624Thr Leu Glu Trp Arg Pro Arg Thr Pro Ala Le - #u Val Arg Gly Cys His# 205- TTG GAA GGC GTG GCC GGC CAC AAG GAG GCG CA - #C ATC CTG AGG GTC CTG 672Leu Glu Gly Val Ala Gly His Lys Glu Ala Hi - #s Ile Leu Arg Val Leu# 220- CCG GGC CAC TCG GCC GGG CCC CGG ACG GTG AC - #G GTG AAG GTG GAA CTG 720Pro Gly His Ser Ala Gly Pro Arg Thr Val Th - #r Val Lys Val Glu Leu225 2 - #30 2 - #35 2 -#40- AGC TGC GCA CCC GGG GAT CTC GAT GCC GTC CT - #C ATC CTG CAG GGT CCC 768Ser Cys Ala Pro Gly Asp Leu Asp Ala Val Le - #u Ile Leu Gln Gly Pro# 255- CCC TAC GTG TCC TGG CTC ATC GAC GCC AAC CA - #C AAC ATG CAG ATC TGG 816Pro Tyr Val Ser Trp Leu Ile Asp Ala Asn Hi - #s Asn Met Gln Ile Trp# 270- ACC ACT GGA GAA TAC TCC TTC AAG ATC TTT CC - #A GAG AAA AAC ATT CGT 864Thr Thr Gly Glu Tyr Ser Phe Lys Ile Phe Pr - #o Glu Lys Asn Ile Arg# 285- GGC TTC AAG CTC CCA GAC ACA CCT CAA GGC CT - #C CTG GGG GAG GCC CGG 912Gly Phe Lys Leu Pro Asp Thr Pro Gln Gly Le - #u Leu Gly Glu Ala Arg# 300- ATG CTC AAT GCC AGC ATT GTG GCA TCC TTC GT - #G GAG CTA CCG CTG GCC 960Met Leu Asn Ala Ser Ile Val Ala Ser Phe Va - #l Glu Leu Pro Leu Ala305 3 - #10 3 - #15 3 -#20- AGC ATT GTC TCA CTT CAT GCC TCC AGC TGC GG - #T GGT AGG CTG CAG ACC1008Ser Ile Val Ser Leu His Ala Ser Ser Cys Gl - #y Gly Arg Leu Gln Thr# 335- TCA CCC GCA CCG ATC CAG ACC ACT CCT CCC AA - #G GAC ACT TGT AGC CCG1056Ser Pro Ala Pro Ile Gln Thr Thr Pro Pro Ly - #s Asp Thr Cys Ser Pro# 350- GAG CTG CTC ATG TCC TTG ATC CAG ACA AAG TG - #T GCC GAC GAC GCC ATG1104Glu Leu Leu Met Ser Leu Ile Gln Thr Lys Cy - #s Ala Asp Asp Ala Met# 365- ACC CTG GTA CTA AAG AAA GAG CTT GTT GCG CA - #T TTG AAG TGC ACC ATC1152Thr Leu Val Leu Lys Lys Glu Leu Val Ala Hi - #s Leu Lys Cys Thr Ile# 380- ACG GGC CTG ACC TTC TGG GAC CCC AGC TGT GA - #G GCA GAG GAC AGG GGT1200Thr Gly Leu Thr Phe Trp Asp Pro Ser Cys Gl - #u Ala Glu Asp Arg Gly385 3 - #90 3 - #95 4 -#00- GAC AAG TTT GTC TTG CGC AGT GCT TAC TCC AG - #C TGT GGC ATG CAG GTG1248Asp Lys Phe Val Leu Arg Ser Ala Tyr Ser Se - #r Cys Gly Met Gln Val# 415- TCA GCA AGT ATG ATC AGC AAT GAG GCG GTG GT - #C AAT ATC CTG TCG AGC1296Ser Ala Ser Met Ile Ser Asn Glu Ala Val Va - #l Asn Ile Leu Ser Ser# 430- TCA TCA CCA CAG CGG AAA AAG GTG CAC TGC CT - #C AAC ATG GAC AGC CTC1344Ser Ser Pro Gln Arg Lys Lys Val His Cys Le - #u Asn Met Asp Ser Leu# 445- TCT TTC CAG CTG GGC CTC TAC CTC AGC CCA CA - #C TTC CTC CAG GCC TCC1392Ser Phe Gln Leu Gly Leu Tyr Leu Ser Pro Hi - #s Phe Leu Gln Ala Ser# 460- AAC ACC ATC GAG CCG GGG CAG CAG AGC TTT GT - #G CAG GTC AGA GTG TCC1440Asn Thr Ile Glu Pro Gly Gln Gln Ser Phe Va - #l Gln Val Arg Val Ser465 4 - #70 4 - #75 4 -#80- CCA TCC GTC TCC GAG TTC CTG CTC CAG TTA GA - #C AGC TGC CAC CTG GAC1488Pro Ser Val Ser Glu Phe Leu Leu Gln Leu As - #p Ser Cys His Leu Asp# 495- TTG GGG CCT GAG GGA GGC ACC GTG GAA CTC AT - #C CAG GGC CGG GCG GCC1536Leu Gly Pro Glu Gly Gly Thr Val Glu Leu Il - #e Gln Gly Arg Ala Ala# 510- AAG GGC AAC TGT GTG AGC CTG CTG TCC CCA AG - #C CCC GAG GGT GAC CCG1584Lys Gly Asn Cys Val Ser Leu Leu Ser Pro Se - #r Pro Glu Gly Asp Pro# 525- CGC TTC AGC TTC CTC CTC CAC TTC TAC ACA GT - #A CCC ATA CCC AAA ACC1632Arg Phe Ser Phe Leu Leu His Phe Tyr Thr Va - #l Pro Ile Pro Lys Thr# 540- GGC ACC CTC AGC TGC ACG GTA GCC CTG CGT CC - #C AAG ACC GGG TCT CAA1680Gly Thr Leu Ser Cys Thr Val Ala Leu Arg Pr - #o Lys Thr Gly Ser Gln545 5 - #50 5 - #55 5 -#60- GAC CAG GAA GTC CAT AGG ACT GTC TTC ATG CG - #C TTG AAC ATC ATC AGC1728Asp Gln Glu Val His Arg Thr Val Phe Met Ar - #g Leu Asn Ile Ile Ser# 575- CCT GAC CTG TCT GGT TGC ACA AGC AAA GGC CT - #C GTC CTG CCC GCC GTG1776Pro Asp Leu Ser Gly Cys Thr Ser Lys Gly Le - #u Val Leu Pro Ala Val# 590- CTG GGC ATC ACC TTT GGT GCC TTC CTC ATC GG - #G GCC CTG CTC ACT GCT1824Leu Gly Ile Thr Phe Gly Ala Phe Leu Ile Gl - #y Ala Leu Leu Thr Ala# 605- GCA CTC TGG TAC ATC TAC TCG CAC ACG CGT TC - #C CCC AGC AAG CGG GAG1872Ala Leu Trp Tyr Ile Tyr Ser His Thr Arg Se - #r Pro Ser Lys Arg Glu# 620- CCC GTG GTG GCG GTG GCT GCC CCG GCC TCC TC - #G GAG AGC AGC AGC ACC1920Pro Val Val Ala Val Ala Ala Pro Ala Ser Se - #r Glu Ser Ser Ser Thr625 6 - #30 6 - #35 6 -#40- AAC CAC AGC ATC GGG AGC ACC CAG AGC ACC CC - #C TGC TCC ACC AGC AGC1968Asn His Ser Ile Gly Ser Thr Gln Ser Thr Pr - #o Cys Ser Thr Ser Ser# 655- ATG GCA TAGCCCCGGC CCCCCGCGCT CGCCCAGCAG GAGAGACTGA GC - #AGCCGCCA G2025Met Ala- (2) INFORMATION FOR SEQ ID NO:2:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 658 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (v) FRAGMENT TYPE: internal- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:- Met Asp Arg Gly Thr Leu Pro Leu Ala Val Al - #a Leu Leu Leu Ala Ser# 15- Cys Ser Leu Ser Pro Thr Ser Leu Ala Glu Th - #r Val His Cys Asp Leu# 30- Gln Pro Val Gly Pro Glu Arg Gly Glu Val Th - #r Tyr Thr Thr Ser Gln# 45- Val Ser Lys Gly Cys Val Ala Gln Ala Pro As - #n Ala Ile Leu Glu Val# 60- His Val Leu Phe Leu Glu Phe Pro Thr Gly Pr - #o Ser Gln Leu Glu Leu# 80- Thr Leu Gln Ala Ser Lys Gln Asn Gly Thr Tr - #p Pro Arg Glu Val Leu# 95- Leu Val Leu Ser Val Asn Ser Ser Val Phe Le - #u His Leu Gln Ala Leu# 110- Gly Ile Pro Leu His Leu Ala Tyr Asn Ser Se - #r Leu Val Thr Phe Gln# 125- Glu Pro Pro Gly Val Asn Thr Thr Glu Leu Pr - #o Ser Phe Pro Lys Thr# 140- Gln Ile Leu Glu Trp Ala Ala Glu Arg Gly Pr - #o Ile Thr Ser Ala Ala145 1 - #50 1 - #55 1 -#60- Glu Leu Asn Asp Pro Gln Ser Ile Leu Leu Ar - #g Leu Gly Gln Ala Gln# 175- Gly Ser Leu Ser Phe Cys Met Leu Glu Ala Se - #r Gln Asp Met Gly Arg# 190- Thr Leu Glu Trp Arg Pro Arg Thr Pro Ala Le - #u Val Arg Gly Cys His# 205- Leu Glu Gly Val Ala Gly His Lys Glu Ala Hi - #s Ile Leu Arg Val Leu# 220- Pro Gly His Ser Ala Gly Pro Arg Thr Val Th - #r Val Lys Val Glu Leu225 2 - #30 2 - #35 2 -#40- Ser Cys Ala Pro Gly Asp Leu Asp Ala Val Le - #u Ile Leu Gln Gly Pro# 255- Pro Tyr Val Ser Trp Leu Ile Asp Ala Asn Hi - #s Asn Met Gln Ile Trp# 270- Thr Thr Gly Glu Tyr Ser Phe Lys Ile Phe Pr - #o Glu Lys Asn Ile Arg# 285- Gly Phe Lys Leu Pro Asp Thr Pro Gln Gly Le - #u Leu Gly Glu Ala Arg# 300- Met Leu Asn Ala Ser Ile Val Ala Ser Phe Va - #l Glu Leu Pro Leu Ala305 3 - #10 3 - #15 3 -#20- Ser Ile Val Ser Leu His Ala Ser Ser Cys Gl - #y Gly Arg Leu Gln Thr# 335- Ser Pro Ala Pro Ile Gln Thr Thr Pro Pro Ly - #s Asp Thr Cys Ser Pro# 350- Glu Leu Leu Met Ser Leu Ile Gln Thr Lys Cy - #s Ala Asp Asp Ala Met# 365- Thr Leu Val Leu Lys Lys Glu Leu Val Ala Hi - #s Leu Lys Cys Thr Ile# 380- Thr Gly Leu Thr Phe Trp Asp Pro Ser Cys Gl - #u Ala Glu Asp Arg Gly385 3 - #90 3 - #95 4 -#00- Asp Lys Phe Val Leu Arg Ser Ala Tyr Ser Se - #r Cys Gly Met Gln Val# 415- Ser Ala Ser Met Ile Ser Asn Glu Ala Val Va - #l Asn Ile Leu Ser Ser# 430- Ser Ser Pro Gln Arg Lys Lys Val His Cys Le - #u Asn Met Asp Ser Leu# 445- Ser Phe Gln Leu Gly Leu Tyr Leu Ser Pro Hi - #s Phe Leu Gln Ala Ser# 460- Asn Thr Ile Glu Pro Gly Gln Gln Ser Phe Va - #l Gln Val Arg Val Ser465 4 - #70 4 - #75 4 -#80- Pro Ser Val Ser Glu Phe Leu Leu Gln Leu As - #p Ser Cys His Leu Asp# 495- Leu Gly Pro Glu Gly Gly Thr Val Glu Leu Il - #e Gln Gly Arg Ala Ala# 510- Lys Gly Asn Cys Val Ser Leu Leu Ser Pro Se - #r Pro Glu Gly Asp Pro# 525- Arg Phe Ser Phe Leu Leu His Phe Tyr Thr Va - #l Pro Ile Pro Lys Thr# 540- Gly Thr Leu Ser Cys Thr Val Ala Leu Arg Pr - #o Lys Thr Gly Ser Gln545 5 - #50 5 - #55 5 -#60- Asp Gln Glu Val His Arg Thr Val Phe Met Ar - #g Leu Asn Ile Ile Ser# 575- Pro Asp Leu Ser Gly Cys Thr Ser Lys Gly Le - #u Val Leu Pro Ala Val# 590- Leu Gly Ile Thr Phe Gly Ala Phe Leu Ile Gl - #y Ala Leu Leu Thr Ala# 605- Ala Leu Trp Tyr Ile Tyr Ser His Thr Arg Se - #r Pro Ser Lys Arg Glu# 620- Pro Val Val Ala Val Ala Ala Pro Ala Ser Se - #r Glu Ser Ser Ser Thr625 6 - #30 6 - #35 6 -#40- Asn His Ser Ile Gly Ser Thr Gln Ser Thr Pr - #o Cys Ser Thr Ser Ser# 655- Met Ala- (2) INFORMATION FOR SEQ ID NO:3:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 21 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:#21 TCAA G- (2) INFORMATION FOR SEQ ID NO:4:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 21 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:#21 TCAA G- (2) INFORMATION FOR SEQ ID NO:5:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 57 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:- CTCCCAGACA CACCTCAAGG CCTCCTGGGG GAGGCCCGGA TGCTCAATGC CA - #GCATT 57- (2) INFORMATION FOR SEQ ID NO:6:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 18 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:# 18 TT- (2) INFORMATION FOR SEQ ID NO:7:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 39 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:# 39 TGTC CCCAAGCCCC GAGGGTGAC- (2) INFORMATION FOR SEQ ID NO:8:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 36 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:# 36 TCCC CAAGCCCCGA GGGTGA- (2) INFORMATION FOR SEQ ID NO:9:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 67 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:- ATGGACCGCG GCACGCTCCC TCTGGCTGTT GCCCTGCTGC TGGCCAGCTG CA - #GCCTCAGC 60# 67- (2) INFORMATION FOR SEQ ID NO:10:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 484 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:- AATCCATGGA ACGAATATAA TGATATCACC TCATAAGGTG GCTGTGATGA TG - #CAGGAAAG 60- CNGTTAGCTC ATGTCAAGTC CCTAGGAGAC GTTTGGAAAG TAGGAGTCAT TG - #TCATCACC 120- TTATTCTCAC CTGGCCTCTT TCCGGATGTT TCTCCAACAG GTCTTGCAGA AA - #CAGTCCAT 180- TGTGACCTTC AGCCTGTGGG CCCCGAGAGG GGCGAGGTGA CATATACCAC TA - #GCCAGGTC 240- TCGAAGGGCT GCGTGGCTCA GGCCCCCAAT GCCATCCTTG AAGTCCATGT CC - #TCTTCCTG 300- GAGTTCCCAA CGGTGAGTGT CCCATGGCAG GGTCGGGTGG GGGCTCAGAG GA - #AGCTCCAA 360- GGCAGATGGG GTGAGGGGTG CCTTCCTTGT GGCTGTCCCT GGGGCAGTGG CT - #GAGTCCTC 420- GTTAGCCCCC TGCCAAGAGA GTGATGTGGG CATCTCACAG GGCCCATAAG AG - #GTGGCATT 480# 484- (2) INFORMATION FOR SEQ ID NO:11:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 141 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:- GGCCCGTCAC AGCTGGAGCT GACTCTCCAG GCATCCAAGC AAAATGGCAC CT - #GGCCCCGA 60- GAGGTGCTTC TGGTCCTCAG TGTAAACAGC AGTGTCTTCC TGCATCTCCA GG - #CCCTGGGA 120# 141TA C- (2) INFORMATION FOR SEQ ID NO:12:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 496 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:- GGCCTCTTTC TCTCAGCCAA TGGGCTGACT CCACAAATTA CTTCCTGACC TC - #CTACATGG 60- GATAGAGAGG GCACAGGGCA GGAACAGCGT GCTGAGCCTC CACATGTCTC CC - #CAGAATTC 120- CAGCCTGGTC ACCTTCCAAG AGCCCCCGGG GGTCAACACC ACAGAGCTGC CA - #TCCTTCCC 180- CAAGACCCAG ATCCTTGAGT GGGCAGCTGA GAGGGGCCCC ATCACCTCTG CT - #GCTGAGCT 240- GAATGACCCC CAGAGCATCC TCCTCCGACT GGGCCAAGGT CAGTTTCCCC AG - #CAACCTCT 300- CTGGGCCTCA TGATACTGCT CAGGAGGAAT CTGAGCTCCT CTGGCCCACA CC - #TCAAACTT 360- GGGCACCAAG AGTGCAGGAG GGGACACGCT GTGCCACAGT TCACATGCCA CA - #AGCCAGTG 420- CTGCCTTGGG ACAGTGATGG CTCCTCCACC AAATATCAGA TTGAAGCATG TG - #GAATATGC 480# 496- (2) INFORMATION FOR SEQ ID NO:13:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 135 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:- TTGGCAGGTA GTGGTGGAAG GGAAGTTCGA ACCTAGGTCC TCTGAGCCTC TC - #CCCTCTGC 60- AGCACCGTCC TGCCTGCCCC ACCACTATCT TTGGCTGTGG GTGAGGGCGG GC - #TCTGTTAG 120# 135- (2) INFORMATION FOR SEQ ID NO:14:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1817 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:- CCCAGGGGTC ACTGTCCTTC TGCATGCTGG AAGCCAGCCA GGACATGGGC CG - #CACGCTCG 60- AGTGGCGGCC GCGTACTCCA GCCTTGGTCC GGGGCTGCCA CTTGGAAGGC GT - #GGCCGGCC 120- ACAAGGAGGC GCACATCCTG AGGGTCCTGC CGGGCCACTC GGCCGGGTAT GG - #CTCTCGCC 180- CCGCCCCTGA CACTAGTCCC CACCCCGAGA GACCACCCCC CTGACCCCCC CC - #CGCCCCCT 240- CTCCGGTCCC TTATAAAGCC CCACCCCAGT CCCAGACCCA GCCCCGCCGC AG - #CCCTGTGA 300- GAGCACAGTC GCTTTCTCCT ACTCTAGGCT ACGCCCCCTA TGGGCCCCTT CC - #CTTTGGGC 360- ACAAGCCTGG CCCCAGTCCC ATCCCTATCC CATAAACCCA CACCTGGCCA GG - #TAAGAGTG 420- CAGCCGCCGC CCACCCGACG CCAGGCCTCG CTCCCCGCCT GGCCTGTCCG CT - #TCAGTGTT 480- CCATCCGCGT CTGTCTCCCC GCAGGCCCCG GACGGTGACG GTGAAGGTGG AA - #CTGAGCTG 540- CGCACCCGGG GATCTCGATG CCGTCCTCAT CCTGCAGGGT CCCCCCTACG TG - #TCCTGGCT 600- CATCGACGCC AACCACAACA TGCAGATCTG GGTGAGTTGT GCGCAGCTCC CG - #GGACACAA 660- AACCCAAACT CCCAACCTCT GGATCAGGGA AGTTTCCTGG AAAGGTGAAC CC - #CCGAGTTG 720- AGCTGAAGGA CAAATCACCT ATGCCCATAC GTGAGGGAAG GGGCCAGGCA GA - #AGACGCAG 780- CAGGAGTGGG GACACAGCAG GACCGAGGCC TGGCATAACC CTGGCTGGCC TG - #CTGTGGCA 840- CAGACTGTGT CCATGGCCCC CTGTTCTGCC TCTCTCCCCA CCATTAGACC AC - #TGGAGAAT 900- ACTCCTTCAA GATCTTTCCA GAGAAAAACA TTCGTGGCTT CAAGCTCCCA GA - #CACACCTC 960- AAGGCCTCCT GGGGGAGGCC CGGATGCTCA ATGCCAGCAT TGTGGCATCC TT - #CGTGGAGC1020- TACCGCTGGC CAGCATTGTC TCACTTCATG CCTCCAGCTG CGGTGAGCAC CC - #TTCCCCTG1080- CCCCTCCCTT CCCTTCCCCT CCCTTGGATC AGTGGCCACA CTGTTGGTGA AG - #CACCTCTG1140- TGTGAGCTTG GGCAAGGTAC ATCAGCCTCT CTGAGCCTCA TTTTTCTCAT CT - #GCACATGG1200- GAACAATGGG AGTAGCTAAT CATAGAAGAG CCTGAGAATC GCTTGAACCT GG - #GAGATGGA1260- GGTTGCAGTG AGCCAAGATC GTGCCACTGC ACTCCCAGCC CGGGTAACAG AG - #CAAAACTC1320- CGTCTCAAAA AAAAAAAAAA AAAAAAAAAA GCCTGGTGCG GCACACATAT CA - #CACAGTGA1380- CCAGCCGCCT GGCCTGCCTC TNCNACCCCA CAGGTGGTAG GCTGCAGACC TC - #ACCCGCAC1440- CGATCCAGAC CACTCCTCCC AAGGACACTT GTAGCCCGGA GCTGCTCATG TC - #CTTGATCC1500- AGACAAAGTG TGCCGACGAC GCCATGACCC TGGTACTAAA GAAAGAGCTT GT - #TGCGGTAA1560- GGGAACTCCT GCCCCTCTGG CTCAGGATGA CATGGACATC TGGTTCCTCC CC - #TAGCCAAG1620- ACTCTTGGGG TCCTAGCCCA GGCAGGGGGG CAAGTCACGT CCCTCTGCAA GC - #CTTAGTTT1680- TCCCACTTGT ATAATGGAAT TGATAATGGT ACCTACCACG TGGTGAGAAT TA - #AAGGCAGT1740- CTGACAGGCC AATCACGTGG CACAGTAAGA TGTGGTACAT AGTAAGTGCT TA - #GTAAATAA1800# 1817 T- (2) INFORMATION FOR SEQ ID NO:15:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 139 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:- GCATTTGAAG TGCACCATCA CGGGCCTGAC CTTCTGGGAC CCCAGCTGTG AG - #GCAGAGGA 60- CAGGGGTGAC AAGTTTGTCT TGCGCAGTGC TTACTCCAGC TGTGGCATGC AG - #GTGTCAGC 120#139 GAG- (2) INFORMATION FOR SEQ ID NO:16:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 39 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:# 39 TGTC GAGCTCATCA CCACAGCGG- (2) INFORMATION FOR SEQ ID NO:17:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 313 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:- GATCGCACCA CTGCACTCCA GCCTGGGCGA CAGAGCGAGA CTCCGTCTCA AA - #AAAAAAAA 60- AAAAGAGAGT CAGGCAACTC CACAGGGCCA TGATGCCTGT TCCTCCCCAC AC - #CCCTCGCC 120- CTCCTGGCTG GCGCCGCCAG ATTGACCAAG TCTCCCTCCC GTCCTCCCCC AG - #AAAAAGGT 180- GCACTGCCTC AACATGGACA GCCTCTCTTT CCAGCTGGGC CTCTACCTCA GC - #CCACACTT 240- CCTCCAGGCC TCCAACACCA TCGAGCCGGG GCAGCAGAGC TTTGTGCAGG TA - #CCTGGCAT 300# 313- (2) INFORMATION FOR SEQ ID NO:18:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 654 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:- TTCCTCTCCG CCTTTCTTCC CACCATGACT CCAGAGGAGA TGAGACTCCC AG - #AGTCAGGA 60- GGGAGACAGC CTGGGTGCAC AGGGAGAGGG AGAGACAGAG AAGGCATTGC TC - #AGGGACAC 120- TGACAAGGAT GTGGCCCTGT CCTCCTCCTC TGCCCCAGTA CAGGTCCATG TC - #TTTCTTTC 180- CACTGTGAGG ACTCAGGGGT GGGAACTCTT AATTCTAGCC GATATTTGAA GG - #CAGCAGGT 240- GGGGTGGGGT GAAGAGCAGC TGCCCATGCC GNGTGGCCCT ACCTACCCAT GC - #AGGTCAGA 300- GTGTCCCCAT CCGTCTCCGA GTTCCTGCTC CAGTTAGACA GCTGCCACCT GG - #ACTTGGGG 360- CCTGAGGGAG GCACCGTGGA ACTCATCCAG GGCCGGGCGG CCAAGGGCAA CT - #GTGTGAGC 420- CTGCTGTCCC CAAGCCCCGA GGGTGACCCG CGCTTCAGCT TCCTCCTCCA CT - #TCTACACA 480- GTACCCATAC CCAAAACCGG CACCCTCAGC TGCACGGTAG CCCTGCGTCC CA - #AGACCGGG 540- TCTCAAGACC AGGTGAGTGG GGCCTGGGCG GCCAGCTTCA AGTGGGAGCT TC - #CAGGTCTG 600- TGGTTTGCAT GGAAGGGACA TGGCAGCCCA CAGGATGTGG CCAGCTGGTG AG - #GG 654- (2) INFORMATION FOR SEQ ID NO:19:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 319 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:- GATCTTCCAG GACTCACCCA GAGGCATCCA GCTACGAAGC GGTGGAGATG GG - #ATTCAAAG 60- CCAAGGCTCT AGGGTGGGCT GGGGTCACGG AGCCAGGGAG TAAACCTGGA AG - #CCGCCTCC 120- CAAAGGTGCC ACATACTGCT CTCTCTTTCT CCTCCAGGAA GTCCATAGGA CT - #GTCTTCAT 180- GCGCTTGAAC ATCATCAGCC CTGACCTGTC TGGTGAGCTC CCTCCAGGTC TC - #TCGGGTTT 240- GTTCTAGTGG CTGAGGTCAC AGTAGGGCAC AGCGGGCAGC CCTGAGAACG GC - #CTGGCACA 300#319 GGA- (2) INFORMATION FOR SEQ ID NO:20:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 24 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:# 24TGAT GATG- (2) INFORMATION FOR SEQ ID NO:21:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 22 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:# 22CCT CT- (2) INFORMATION FOR SEQ ID NO:22:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 19 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:# 19 TGG- (2) INFORMATION FOR SEQ ID NO:23:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 18 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:# 18 TT- (2) INFORMATION FOR SEQ ID NO:24:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 19 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:# 19 CAG- (2) INFORMATION FOR SEQ ID NO:25:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 20 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:# 20 GGGA- (2) INFORMATION FOR SEQ ID NO:26:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 22 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:# 22CCA TC- (2) INFORMATION FOR SEQ ID NO:27:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 24 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:# 24CAGA GGTT- (2) INFORMATION FOR SEQ ID NO:28:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 18 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:# 18 CT- (2) INFORMATION FOR SEQ ID NO:29:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 18 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:# 18 GG- (2) INFORMATION FOR SEQ ID NO:30:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 23 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:# 23GACC AGC- (2) INFORMATION FOR SEQ ID NO:31:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 21 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:#21 ATGT C- (2) INFORMATION FOR SEQ ID NO:32:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 22 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:# 22CTC CC- (2) INFORMATION FOR SEQ ID NO:33:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 22 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:# 22ACT CT- (2) INFORMATION FOR SEQ ID NO:34:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 20 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:# 20 TCTT- (2) INFORMATION FOR SEQ ID NO:35:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 19 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:# 19 CAG- (2) INFORMATION FOR SEQ ID NO:36:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 19 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:# 19 CGC- (2) INFORMATION FOR SEQ ID NO:37:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 20 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:# 20 CGAG- (2) INFORMATION FOR SEQ ID NO:38:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 22 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:# 22GGG AT- (2) INFORMATION FOR SEQ ID NO:39:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 22 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:# 22GCT CC- (2) INFORMATION FOR SEQ ID NO:40:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 22 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:# 22TGA GG- (2) INFORMATION FOR SEQ ID NO:41:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 22 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:# 22CCC AG- (2) INFORMATION FOR SEQ ID NO:42:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1249 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:- CCCTCTAGGT GGACAGTCCT AGCAACCATG GCTCAATNNC AGGCCTGGCT GT - #GATGAGCC 60- CGTTTGCTGC AAGAGGAGAC TGAGGTTCAG AGAAGTCGAG GGTCCATGGC TC - #AGCAGAGC 120- TGGCACCAAA CCCACATGGG CCAGCACAAC AGGGTAGGGG ATGGGGCAGG GG - #CAGAGTGG 180- CAGTGCTGAT GGCGTCGGCC CTCTCTAGGT TGCACAAGCA AAGGCCTCGT CC - #TGCCCGCC 240- GTGCTGGGCA TCACCTTTGG TGCCTTCCTC ATCGGGGCCC TGCTCACTGC TG - #CACTCTGG 300- TACATCTACT CGCACACGCG TGAGTACCCC AGGCCCCCAC AGTGAGCATG CC - #GGGCCCCT 360- CCATCCACCC GGGGGAGCCC AGTGAAGCCT CTGAGGGATT GAGGGGCCCT GG - #CAGGACCC 420- TGACCTCCGC CCCTGCCCCC GCTCCCGCTC CCAGGTTCCC CCAGCAAGCG GG - #AGCCCGTG 480- GTGGCGGTGG CTGCCCCGGC CTCCTCGGAG AGCAGCAGCA CCAACCACAG CA - #TCGGGAGC 540- ACCCAGAGCA CCCCCTGCTC CACCAGCAGC ATGGCATAGC CCCGGCCCCC CG - #CGCTCGCC 600- CAGCAGGAGA GACTGAGCAG CCGCCAGCTG GGAGCACTGG TGTGAACTCA CC - #CTGGGAGC 660- CAGTCCTCCA CTCGACCCAG AATGGAGCCT GCTCTCCGCG CCTACCCTTC CC - #GCCTCCCT 720- CTCAGAGGCC TGCTGCCAGT GCAGCCACTG GCTTGGAACA CCTTGGGGTC CC - #TCCACCCC 780- ACAGAACCTT CAACCCAGTG GGTCTGGGAT ATGGCTGCCC AGGAGACAGA CC - #ACTTGCCA 840- CGCTGTTGTA AAAACCCAAG TCCCTGTCAT TTGAACCTGG ATCCAGCACT GG - #TGAACTGA 900- GCTGGGCAGG AAGGGAGAAC TTGAAACAGA TTCAGGCCAG CCCAGCCAGG CC - #AACAGCAC 960- CTCCCCGCTG GGAAGAGAAG AGGGCCCAGC CCAGAGCCAC CTGGATCTAT CC - #CTGCGGCC1020- TCCACACCTG AACTTGCCTA ACTAACTGGC AGGGGAGACA GGAGCCTAGC GG - #AGCCCAGC1080- CTGGGAGCCC AGAGGGTGGC AAGAACAGTG GGCGTTGGGA GCCTAGCTCC TG - #CCACATGG1140- AGCCCCCTCT GCCGGTCGGG CAGCCAGCAG AGGGGGAGTA GCCAAGCTGC TT - #GTCCTGGG1200# 1249TCACC ACCAATAAAT CAGACCATGA AACCTGAAA__________________________________________________________________________

Diagnosis of and therapy for hereditary haemorrhagic telangiectasia

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