Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Hormones – e.g. – prolactin – thymosin – growth factors – etc.
Reexamination Certificate
1999-06-29
2002-03-12
Crouch, Deborah (Department: 1632)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Hormones, e.g., prolactin, thymosin, growth factors, etc.
C530S350000
Reexamination Certificate
active
06355782
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acid sequences and proteins involved in hypohidrotic ectodermal dysplasia (HED) and hair follicle induction.
BACKGROUND OF THE INVENTION
Hereditary ectodermal dysplasia is an inherited disorder that affects the development of ectodermally derived structures, such as the hair, teeth and sweat glands. The hidrotic form of the disease is characterized by poorly developed teeth and hair. The anhidrotic or hypohidrotic form of the disease further affects the development of sweat glands, which interferes with the ability to sweat, and the maintenance of thermoregulatory homeostasis. Both X-linked and autosomal dominant and recessive forms of the disease have been described.
X-linked hypohidrotic ectodermal dysplasia (XLHED; McKusick's number 305100), the most common form of the ectodermal dysplasias, results in the abnormal development of teeth, hair and eccrine sweat glands. Identification of the gene that is defective in this disease would help explain the molecular basis of XLHED, as well as the molecular mechanisms involved in normal tooth, hair and eccrine sweat gland development. Identification of the gene would also permit mutation testing for XLHED in potentially affected males and carrier females.
Heterozygote carriers of XLHED may have minor or moderate degrees of hypodontia, hypotrichosis and hypohidrosis, although many show no obvious clinical manifestations. This clinical variation, presumably caused by random X-inactivation (Lyonization), makes accurate diagnosis of carrier females difficult. Although indirect testing for carrier status is possible by linkage analysis in informative families (Zonana 1993), carrier detection by this method is impossible in families with single affected individuals, male or female, whose disorder may be the result of a de novo mutation. Detection of mutations within the EDA1 gene would also be advantageous in families with only a single affected sibship, because a rarer autosomal recessive form of the disorder (ARHED) is clinically indistinguishable from XLHED in affected males (Munoz et al. 1997).
A gene identified as EDA1 has been isolated by positional cloning (Kere et al. 1996). A single 858 bp cDNA, representing a full length transcript composed of 2 exons, was identified from an adult sweat gland cDNA library. In situ analysis showed that the EDA1 gene was expressed in hair follicles and the epidermis of adult skin. The putative gene product is a 135 amino acid protein, which has no clear homology to other proteins (see U.S. Pat. No. 5,700,926). The protein is predicted to contain a single transmembrane domain, and fractionation studies of transfected cell lines showed that the protein product is localized to the plasma membrane (Ezer et al. 1997). Yeast artificial chromosomes (YACs) containing at least a portion of the human EDA1 gene were disclosed in U.S. Pat. No. 5,556,786.
A syndrome similar to HED, with anhidrosis and absence of sweat glands, is known in the mouse, in which the mutant gene is called Tabby (Ta). Consistent with the map position in humans, the Ta gene has been mapped in the syntenically corresponding region in the X chromosome of the mouse (Brockdorff et al. 1991). Other murine forms of the disease include those found in the downless (dl) mutants, in which the disorder is not X-linked.
SUMMARY OF THE INVENTION
The present invention has been made possible by the discovery that there are previously unidentified alternative transcripts of the EDA1 gene. The inventors recognized that while the full length of the EDA1-I cDNA was 858 bp, a predominant 5-6 kb transcript was detected in several human tissues by Northern analysis. Mutation screening of 173 unrelated families with XLHED showed that only 7% of the families had likely mutations within exon 1, and none had variants within exon 2. Moreover, cDNAs from the homologous murine gene, Tabby (Ta), were found to include alternative exons.
The present invention includes an EDA1 cDNA splice-form (Seq. I.D. No. 1) that is homologous to the Ta cDNA (Seq. I.D. No. 3), and codes for a second isoform of the EDA1 protein (isoform II or EDA1-II). Nearly all of the mutations associated with XLHED are located within the exons identified in this new splice-form. These results show that EDA1 isoform II is essential for hair, tooth and eccrine sweat gland morphogenesis. In addition, the identification of the additional exons permits direct molecular diagnostic testing for XLHED by mutation analysis.
Also disclosed is the nucleic acid sequence encoding the human gene EDA1-II sequence (Seq. ID. NOS. 5-11), which is predicted to encode a 391 amino acid protein (Seq. I.D. No. 2). The EDA1-II gene is predicted to encode a protein that is related to the TNF family of proteins, and acts as either a membrane-associated or soluble ligand. The biologically active domains of the EDA1-II protein are within approximately the C-terminal 240 amino acids (and particularly residues 133-391, and especially residues 239-391 of EDA1-II) and includes a Furin recognition sequence, a Gly-X-Y repeat, and a region of structural homology to TNF proteins. This predicted active region is unique to this isoform, with none of the amino acids included within the previously described EDA1 sequence. The DNA sequence that codes for this isoform (Seq. I.D. No. 1) is also unique.
The EDA1-II protein is an essential component of a signaling pathway that is required for the normal development of hair follicles, teeth, sweat glands and mammary glands. A form of the protein is present in humans, mice, cow and dog, and is likely to serve the same or nearly identical roles in each of these organisms. The invention includes the use of the human EDA1-II gene sequence, as it applies to the use of commercial and clinical diagnostic testing for ectodermal dysplasia (ED). The invention also includes use of an EDA1-II gene sequence for the production of EDA1-II protein, as a therapeutic substance stimulating the growth of hair, teeth, skin, and sweat glands. Potential medical or cosmetic benefits of EDA1-II include the stimulation of hair growth, including cases of alopecia (balding) or skin grafts. As such, EDA1-II may be applied as a purified protein or nucleic acid (DNA or RNA), delivered as topical substance, or injected into the skin, alone or in combination with a pharmaceutical carrier.
The EDA1-II isoform (or active subsequences thereof, particularly the C terminal 240 amino acid residues, or residues 133-391, particularly 239-391), may also be used as a stimulant for tooth growth, either in cases of tooth loss or of natural absence of teeth. The protein may be used to stimulate tooth growth in humans directly, or alternatively in tissue culture (artificial) conditions, with subsequent introduction of teeth into humans or other organisms. The EDA1-II protein or EDA1-II gene may also be useful for the stimulation of eccrine sweat gland development, for example in individuals for whom the normal sweating mechanism is compromised by disease or surgery.
The protein may also be used to stimulate the growth of mammary epithelial tissue, either for reconstructive or cosmetic purposes. Alternatively, methods that block the production of the protein, for example antisense or antibody approaches, may be useful for inhibiting breast epithelial cell proliferation. Blocking EDA1-II activity may provide an effective therapeutic approach to slow or to inhibit the spread of breast cancer malignancies. The EDA1-II gene and gene product itself may also be useful in promoting or maintaining differentiation of breast epithelium.
The present invention also provides an isolated human nucleic acid molecule which may be able to correct the cellular defect characteristic of HED, including XLHED. It is shown that XLHED patients carry mutations in the genomic copies of this nucleic acid molecule. Orthologs of the disclosed nucleic acid molecule from other species are also provided, which may be able to correct the effects of the mutation. Such homologous proteins may have 95% or 98% i
Ferguson Betsy M.
Headon Denis
Overbeek Paul
Zonana Jonathan
Baker Anne-Marie
Baylor College of Medicine
Crouch Deborah
Klarquist & Sparkman, LLP
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