Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-09-28
2003-05-13
Benzion, Gary (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S022100, C536S023100, C536S023500, C536S024330, C435S252300, C530S350000, C530S388200
Reexamination Certificate
active
06562957
ABSTRACT:
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 OWR
1
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 &bgr; (TGF-&bgr;) 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-&bgr;binding protein on endothelial cells (Cheifetz et al., J. Biol. Chem. 267:19027-19030, 1992). In the presence of TGF-&bgr; ligand, endoglin can associate with the two TGF-&bgr; signaling receptors RI and RII (Yamashita et al., J. Biol. Chem. 269:1995-2001, 1994). TGF-&bgr; 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 more preferably has the sequence of
FIG. 5
(SEQ ID NOs:9-19). 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-&bgr; type I receptor (RI), TGF-&bgr; type II receptor (RII), or TGF-&bgr;/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, 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 (e.g., the vascular endothelial cells of a newborn's placenta or umbilical cord) which normally expresses a gene of the TGF-&bgr; receptor complex; 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
Letarte Michelle
Marchuk Douglas A.
McAllister Kimberly
Benzion Gary
Fish & Richardson P.C.
HSC Research & Development Limited Partnership
Tung Joyce
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