Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1996-11-22
2003-02-11
Caputa, Anthony C. (Department: 1642)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023500, C536S024310, C536S024330, C536S024500
Reexamination Certificate
active
06518411
ABSTRACT:
BACKGROUND OF THE INVENTION
Rieger syndrome (RS) is an autosomal dominant disorder of morphogenesis characterized by abnormalities of the anterior segment of the eye and dental hypoplasia. Other systemic anomalies have been reported in association with the syndrome, the most common of which are mild craniofacial dysmorphism, including maxillary hypoplasia and failure of involution of the periumbilical skin and omphalocele. Gastrointestinal defects reported include Meckels diverticulum (Krespi, Y. P. and D. Pertsemlidis (1979)
Am. J. Gastroenter
. 71:608-610) colon atresia (Rogers, R. C. (1988)
Proc. Greenwood. Genet. Center
7:9-13) and anal stenosis (Crawford, R. D. (1967) Brit. J. Ophthalmol. 51:438-440) The ocular features can include a prominent, anteriorly displaced Schwalbe line, iris processes which insert into the Schwalbe line, hypoplasia of the anterior iris stroma or corectopiea and pseudopolycoria. Roughly one-half of the patients will develop glaucoma, usually by young adulthood. The dental features associated with the condition include oligodontia and microdontia.
There is general agreement that patients with both the ocular features and systemic abnormalities (dental, umbilical, craniofacial) should be designated Rieger syndrome. The nomenclature of patients with only the ocular features, however, is confusing and disputed. For example, the condition of only ocular abnormalities is sometimes referred to as Rieger syndrome and other times referred to as Axenfeld-Rieger anomaly.
The underlying genetic defect and the chromosomal localization of RS is uncertain. Cases with Axenfeld-Rieger anomaly have been reported with various aberrations of chromosomes 4, 6, 10, 13, 16, and 22 (Wilcox, L. M. et al., (1978)
Am. J. Ophthalmol
. 86:834-839; Tabbara, K. F. et al. (1973)
Canad. J. Ophthal
. 8:488-491; Herve, J. et al. 1984)
Ann. Pediatr
. 31:77-80; Stathacopoulas, R. A. et al. (1987)
J. Ped. Ophthalmol. Strabismus
24:198-203; Akazawa, K. et al. (1981)
J. Ophthalmol
. 105:323; Furguson, J. G. and E. L. Hicks (1987)
Arch. Ophthal
. 105:323; Heinemann, M. et al. (1979)
Br. J. Ophthalmol
. 63:40-44). Four cases of fully developed RS, including the dental and umbilical anomalies, have been associated with deletions in the region of chromosome 4q23-26. One case of a deletion in the region of 4q26 which did not have RS has also been reported. These cases indicate that a gene for RS lies in the region of 4q25.
In RS, a high risk of developing increased intraocular pressures and subsequent glaucomatous damage necessitates vigorous screening procedures beginning in infancy. Current screening for the ocular abnormalities of RS requires slit lamp examination, tonometry, gonioscopy and a dilated examination of the optic nerve head.
SUMMARY OF THE INVENTION
The present invention is based on the discovery of novel molecules, referred to herein as RIEG nucleic acids and RIEG or “Solurshin” polypeptide molecules. The human RIEG gene, which is approximately 18 kb, consists of four exons, 222, 49, 206 and 1306 nucleotides in length. The ATG initiation codon is located in the second exon and the homeobox region in the third and fourth exons. Translation of the open reading frame yields a protein of about 271 amino acids.
The gene is expressed in oral epithelium, the umbilicus, and periocular mesoderm, consistent with the phenotypic abnormalities seen in Rieger syndrome patients. In addition, the gene is expressed in Rathke's pouch and the vitelline artery.
In one aspect, the invention features isolated vertebrate RIEG nucleic acid molecules. The disclosed molecules can be non-coding, (e.g. probe, antisense or ribozyme molecules) or can encode a functional RIEG polypeptide (e.g. a polypeptide which specifically modulates, e.g., by acting as either an agonist or antagonist, at least one bioactivity of the human RIEG polypeptide). In one embodiment, the nucleic acids of the present invention can hybridize to a vertebrate RIEG gene or to the complement of a vertebrate RIEG gene. In a further embodiment, the claimed nucleic acid hybridizes with the coding sequence designated in at least one of SEQ ID Nos: 1 or 3 or to the complement to the coding sequence designated in at least one of SEQ ID Nos: 1 or 3. In a preferred embodiment, the hybridization is conducted under mildly stringent or stringent conditions.
In further embodiments, the nucleic acid molecule is an RIEG nucleic acid that is at least 70%, preferably 80%, more preferably 85%, and even more preferably at least 95% homologous in sequence to the nucleic acids shown as SEQ ID Nos: 1 or 3 or to the complement of the nucleic acids shown as SEQ ID Nos: 1 or 3. In another embodiment, the RIEG nucleic acid molecule encodes a polypeptide that is at least 90% and more preferably at least 94% similar in sequence to the polypeptide shown in SEQ ID No: 2.
The invention also provides probes and primers comprising substantially purified oligonucleotides, which correspond to a region of nucleotide sequence which hybridizes to at least 6 consecutive nucleotides of the sequences set forth as SEQ ID Nos: 1 or 3 or complements of the sequences set forth as SEQ ID Nos: 1 or 3, or naturally occurring mutants thereof. In preferred embodiments, the probe/primer further includes a label group attached thereto, which is capable of being detected.
For expression, the subject RIEG nucleic acids can include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter (e.g., for constitutive expression or inducible expression) or transcriptional enhancer sequence, which regulatory sequence is operably linked to the RIEG gene sequence. Such regulatory sequences in conjunction with a RIEG nucleic acid molecule can be useful vectors for gene expression. This invention also describes host cells transfected with said expression vector whether prokaryotic or eukaryotic and in vitro (e.g. cell culture) and in vivo (e.g. transgenic) methods for producing RIEG proteins by employing said expression vectors.
In another aspect, the invention features isolated RIEG or Solurshin polypeptides, preferably substantially pure preparations e.g. of plasma purified or recombinantly produced RIEG polypeptides. In preferred embodiments, the polypeptide is a functional transcription factor.
In one embodiment, the polypeptide is identical to or similar to a RIEG protein represented in SEQ ID No: 2. Related members of the vertebrate and particularly the mammalian RIEG family are also within the scope of the invention. Preferably, a RIEG polypeptide has an amino acid sequence at least 60% homologous and preferably at least 80% homologous to the polypeptide represented in SEQ ID No: 2. The subject RIEG proteins also include modified protein, which are resistant to post-translation modification, as for example, due to mutations which alter modification sites (such as tyrosine, threonine, serine or aspargine residues), or which prevent glycosylation of the protein, or which prevent interaction of the protein with intracellular proteins involved in signal transduction.
The RIEG polypeptide can comprise a full length protein, such as represented in SEQ ID No: 2, or it can comprise a fragment corresponding to one or more particular motifs/domains, or to arbitrary sizes, e.g., at least 5, 10, 25, 50, 100, 150, 175, 200, 225, 250 or 260 amino acids in length.
Another aspect of the invention features chimeric molecules (e.g. fusion proteins) comprised of a RIEG protein. For instance, the RIEG protein can be provided as a recombinant fusion protein which includes a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated (heterologous) to the RIEG polypeptide (e.g. the second polypeptide portion is glutathione-S-transferase, an enzymatic activity such as alkaline phosphatase or an epitope tag).
Yet another aspect of the present invention concerns an immunogen comprising a RIEG polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for a RIEG polypeptide; e.g. a h
Murray Jeffrey C.
Semina Elena
Canella Karen A.
Caputa Anthony C.
Clauss Isabelle
Foley & Hoag LLP
University of Iowa
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