Endometriosis-associated gene

Details Endometriosis-associated gene Endometriosis-associated gene

2000-11-29

2003-07-01

Ketter, James (Department: 1636)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C530S350000

Reexamination Certificate

active

06586569

ABSTRACT:

DESCRIPTION
The present invention relates to a gene associated with invasive processes, for example endometriosis, to a polypeptide encoded by it, to an antibody directed against the polypeptide, and to the pharmaceutical application of the nucleic acid, the polypeptide and the antibody.
Endometriosis is the second most common disease in women and is defined as the occurrence of endometrial cells outside the womb. Endometriosis affects about one in five women of reproductive age, and as many as one in two women with fertility problems.
In normal circumstances the endometrium is only found in the womb. In endometriosis, tissue with a histological appearance resembling the endometrium is found outside the womb, for example externally on the womb, on the intestine or even in the pancreas or the lung. Although these endometriotic foci are located outside the womb, they also bleed during menstruation, thus they are influenced by hormones of the female cycle. Since endometriotic foci like the endometrium go through volume changes during the cycle, these changes may cause pain depending on location. Moreover, the body reacts to endometriotic cells with an inflammatory response which again causes pain. Furthermore, inflammation leads to adhesions in the area of the ovaries and fallopian tubes and, as a result of these, is responsible for a so-called mechanical sterility of affected women. Apparently however, in endometriosis messengers are released as well (e.g. cytokines, prostaglandins) which can reduce the fertility of affected women even in the absence of adhesions.
In view of their pathobiological properties, endometriotic cells could be classified as being between normal cells and tumor cells: on the one hand they show no neoplastic behavior, on the other hand, however, they are, like metastasizing tumor cells, capable of moving across organ boundaries in the organism and of growing into other organs, i.e. they show invasive behavior. For this reason endometriotic cells are defined as “benign tumor cells” in the literature, although up until now no tumor-specific-mutations in proto-oncogenes have been found in cells of this type.
Since the pathogenesis of endometriosis is still not clarified completely, there are as yet no effective options for the therapy or prevention of endometriosis-associated diseases.
It was the object of the invention to identify novel genes which play a role in invasive processes and which may be associated with the pathophysiological phenotype of endometriosis.
This object is achieved according to the invention by identifying, cloning and characterizing a gene which is called an endometriosis-associated gene and which codes for a polypeptide. This gene sequence was discovered with the aid of differential display RT-PCR (Liang and Pardee, Science 257 (1992), 967-971). For this, invasive and noninvasive variants of an endometriotic cell line were compared with each other. In the process a cDNA sequence was found which is specific for the invasive variant of endometriotic cells. An associated RNA of 4 kb in length was found. A corresponding cDNA isolated from a cDNA phage bank has an open reading frame (ORF) of 302 amino acids.
The present invention relates to a nucleic acid which comprises
(a) the nucleotide sequences depicted in SEQ ID NO. 1, 3 or/and 5, a combination or a protein-encoding segment thereof,
(b) a nucleotide sequence corresponding to the sequence in (a) within the scope of the degeneracy of the genetic code or
(c) a nucleotide sequence hybridizing with the sequences in (a) and/or (b) under stringent conditions.
The nucleic acids preferably code for a polypeptide associated with invasive processes or a segment thereof.
The following nucleotide sequences have been deposited in the EMBL EST database with the following accession numbers: Z98886, Ac003017, AL023586, Aa52993, Aa452856. These sequences do not represent nucleic acids according to the invention. The first two of these sequences are DNAs which were isolated from human brain and show over 90% identical bases to SEQ. ID NO. 1 in the segments from nucleotide 970 to about 2000 and from 760 to about 1450, respectively, or in the segments from nucleotide 1054 to 2084 and from 844 to about 1534 in relation to SEQ ID NO. 3 which has 84 additional bases at the 5′ end. AL023586 is also a human sequence which is very similar to Z98885 and also has homology with SEQ ID NO. 1 in the region from 970 to about 2000.
Sequences Aa452993 and Aa452856 originate from mouse embryos and show base identity with the nucleotides (nt) from about 1060 to about 1450 and from about 24 to 440, respectively, of SEQ. ID NO. 1, or from about 1144 to about 1534 and from about 108 to about 524, respectively, according to the nucleotide positions in SEQ. ID NO. 3. Up until now no reading frame or function has been assigned to any of these 4 sequences.
The nucleotide sequence depicted in SEQ. ID NO. 1 contains an open reading frame which corresponds to a polypeptide having a length of 302 amino acids. This polypeptide is indicated in the amino acid sequence depicted SEQ. ID NO. 2. SEQ. ID NO. 3 shows a nucleotide sequence as in SEQ. ID NO. 1, but it has 84 additional nucleotides at the 5′ end. As a result, the positions of the nucleotides corresponding to each other shift by 84 nucleotides in each case. The polypeptide encoded by SEQ. ID NO. 3 therefore has 28 additional amino acids at the N terminus and is depicted in SEQ. ID NO. 4 with its total of 330 amino acids. SEQ. ID NO. 2 and 4 depict a C-terminal segment of the native polypeptide.
For illustration purposes reference is made to
FIG. 1
which shows a diagrammatic representation of the cDNA of the endometriosis-associated gene according to the invention. Five exons, E1 to E5, and the position of fragment 1 (394 nt) used as a probe in DDRT-PCR are shown. The positions of the PCR primers (see example 4, table 1) used for RT-PCR are also shown.
Not shown in
FIG. 1
is a further exon 4a whose nucleotide sequence is shown in SEQ. ID NO. 5. This exon 4a may be present. If it is present, it is found between exon 4 and exon 5. This corresponds to the position between nt1054 and nt1055 in SEQ. ID NO. 3. A combination of the sequences SEQ. ID NO. 1/3 with SEQ. ID NO. 5 is accordingly, for example, a sequence which contains the sequence of the exon 4a at said position.
Besides the nucleotide sequences shown in SEQ. ID NO. 1, 3 and 5 and combinations thereof such as the sequence of SEQ. ID NO. 3, which has the sequence of SEQ. ID NO. 5 between nt1054 and 1055 and to a nucleotide sequences which corresponds to the sequences within the scope of the degeneracy of the genetic code, the present invention also includes nucleotide sequences which hybridize with one of the sequences mentioned before. The term “hybridization” according to the present invention is used by Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), 1.101-1.104). Preferably a hybridization is called stringent if a positive hybridization signal is still observed after washing for one hour with 1×SSC and 0.1% SDS at 50° C., preferably at 55° C., particularly preferably at 62° C. and most preferably at 68° C., in particular for 1 h in 0.2×SSC and 0.1% SDS at 55° C., preferably at 55° C., particularly preferably at 62° C. and most preferably at 68° C. A nucleotide sequence hybridizing under these washing conditions with one or more of the nucleotide sequences depicted in SEQ ID NO. 1, 3 and 5, or with a nucleic sequence corresponding to these sequences within the scope of the degeneracy of the genetic code, is a nucleotide sequence according to the invention.
The nucleotide sequence according to the invention is preferably a DNA. However, it can also include an RNA or a nucleic acid analog such as a peptidic nucleic acid, for example. Particularly preferably the nucleic acid according to the invention includes a protein-encoding segment of the nucleotide sequences depicted in SEQ ID NO. 1, 3 and/or 5 or a sequence having a homology of more

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