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AssigneeNone recorded
InventorsAlain Hovnanian, Stephane Chavanas, William Cookson, Miriam Moffatt, Andrew Walley
Estimating the term…
The invention relates to the identification of the SPINK5 gene as the gene which is mutated in the autosomal recessive genetic skin condition Netherton's Syndrome and as a susceptibility gene for atopic disease in general. Genetic screens, therapeutic products and Nucleic acids and proteins corresponding to mutant versions of the SPINK5 cDNA and expression product are all described.
A method of determining whether an individual is susceptible or predisposed to atopic disease which comprises screening the genome of the individual for the presence or absence of one or more polymorphic variants of the SPINK5 gene and/or screening for the expression of a variant LEKTI protein.
A method of determining whether an individual is susceptible or predisposed to atopic disease which comprises screening a tissue sample from the individual for the expression of a variant LEKTI protein.
A method of determining the or any genetic basis of atopic disease in a patient previously diagnosed with such disease, which method comprises screening the genome of the individual for the presence or absence of one or more polymorphic variants of the SPINK5 gene.
A method according to any one of the preceding claims wherein the atopic disease is asthma, eczema or hay fever.
An isolated variant LEKTI polypeptide comprising the complete amino acid sequence illustrated in SEQ ID NO:
An isolated nucleic acid molecule comprising the complete nucleotide sequence illustrated in SEQ ID NO:
A kit for use in screening for human subjects for susceptibility or predisposition to atopic disease comprising at least one oligonucleotide probe or primer specific for a SPINK5 polymorphic variant associated with susceptibility or predisposition to atopic disease.
A genetic screening method for use in determining the carrier status of an individual for Netherton's syndrome or in diagnosing Netherton's syndrome in a patient, which method comprises screening the genome of the individual or patient for the presence of loss-of-function mutations in the SPINK5 gene.
An isolated mutant SPINK5 allele containing a mutation selected from the group consisting of:
An isolated variant SPINK5 allele containing at least one polymorphic variant selected from the group consisting of:
A screening method for use in determining the carrier status of an individual for Netherton's syndrome or in diagnosing Netherton's syndrome in a patient which method comprises screening for expression of a protein product of the SPINK5 gene in a tissue sample from the individual or patient and thereby determining whether the protein is mutated and/or whether it is expressed at non-wild type levels.
A screening method for use in determining the carrier status of an individual for Netherton's syndrome or in diagnosing Netherton's syndrome in a patient, which method comprises determining the activity of any serine protease inhibitor encoded by the SPINK5 gene in a tissue sample from the individual or patient.
A method of determining the nature of the disease-causing mutation in a patient suffering or suspected of suffering from Netherton's syndrome, which method comprises comparing the nucleotide sequence of all or a part of the SPINK5 alleles carried by the patient with the wild type SPINK5 nucleotide sequence, wherein differences between the patient alleles and the wild-type alleles identify a disease-causing mutation.
A substance comprising a serine protease inhibitor having the amino acid sequence illustrated in SEQ ID NO:
A method of treating atopic disease or Netherton's syndrome in a human patient which comprises administering to a patient in need thereof an effective amount of a medicament comprising a pharmaceutically active substance comprising a serine protease inhibitor having the amino acid sequence illustrated in SEQ ID NO:
A method of treating atopic disease or Netherton's syndrome in a human patient which comprises administering to a patient in need thereof an effective amount of a medicament comprising an expression vector suitable for directing expression of a serine protease inhibitor having the amino acid sequence illustrate in SEQ ID NO:
An expression vector comprising nucleic acid encoding a serine protease inhibitor comprising the amino acid sequence illustrated in SEQ ID NO:
A method of screening for compounds with potential pharmacological activity in the treatment of atopic disease or Netherton's syndrome, which method comprises:
A nucleic acid molecule having the complete nucleotide sequence illustrated in SEQ ID NO:
A method of identifying a compound with potential pharmacological activity in the treatment of atopic disease or Netherton's syndrome, which method comprises:
Dependencies are inferred from the claim text.
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Serine protease inhibitors: kazal type inhibitors, e.g. pancreatic secretory inhibitor, ovomucoid
Medicinal preparations containing peptides
Peptides
Medicines and medical preparations
Term end not estimated
The present invention relates to the identification of the SPINK5 gene as both the gene which is mutated in Netherton's syndrome, a rare, autosomal recessive genetic skin condition, and a susceptibility gene for atopic disease in general, and to genetic screens and therapeutic agents arising from this finding.
Netherton syndrome (which may be abbreviated herein to NS) is characterised by the association of abnormal keratinization, trichorrexis invaginata and atopic manifestations. Life-threatening complications such as microbial infections and hypernatraemic dehydration result in high postnatal mortality in infancy.
Using linkage analysis and homozygosity mapping, the present inventors have mapped the NS gene locus to the interval D5S463-D5S2013 on chromosome 5q31-33, 8 Mb distal to the cytokine gene cluster (CGC) (Frazer et al. 1997). This region has been associated with high IgE levels, atopy, asthma and autosomal dominant familial eosinophilia (Meyers et al. 1994; Cookson, 1999; Rioux et al. 1998).
The present invention will be further understood with reference to the following non-limiting experimental examples and the accompanying drawings in which:
FIGS. 1 to 5 schematic diagrams of the cloning strategy for cloning of DNA sequences encoding the LEKTI protein and various fragments thereof into expression vectors.
Search for Candidate Genes or Transcripts in the NS Gene Locus.
Database searching of the consensus map Genemap '99 identified 8 genes and 38 ESTs in the D5S463-D5S2013 interval. Of these genes, PDGFRB, SPARC and CDX1 which encode the Platelet-Derived Growth Factor Receptor β, osteonectin and the caudal type homeo box transcription factor 1, respectively, had been mapped distal to BT1 on the published physical maps of the Diastrophic Dysplasia and the Treacher Collins syndrome regions (Hastbacka et al. 1994; Superti-Furga et al. 1996), and thus could be excluded from the NS region. Of the remaining 5 genes, none appeared to be obvious candidates for the NS gene. Two were involved in human diseases sharing no common features with NS: the PDE6A gene which encodes the α subunit of retinal rod cGMP phosphodiesterase is mutated in autosomal recessive retinitis pigmentosa (Huang et al. 1995); and the DTDST gene coding for a transmembrane sulfate transporter is defective in Diastrophic Dysplasia and achondrogenesis type 1B (Hastbacka et al. 1994; Superti-Furga et al. 1996). The three other genes encode the casein kinase Iα (CSNK1A1), the adrenergic receptor β2 (ADRB2) and the regulator of mitotic spindle assembly (RMSA1). The casein kinase Iα is an ubiquitously expressed serine/threonine protein kinase which is involved in the regulation of G-protein-coupled receptors (Tobin et al. 1997), cell cycle (Gross et al. 1997), and DNA and RNA synthesis (Ceglieska and Virshup 1993). Polymorphisms in ADRB2 have been associated with susceptibility to nocturnal asthma and obesity (Turki et al. 1995; Large et al. 1997), and targeted disruption of the ADRB2 gene in the mouse leads to abnormal vascular tone and impaired energy metabolism during exercise (Chruscinski et al. 1999). Lastly, the RMSA1 gene product is required for spindle assembly and chromosomal segregation (Yeo et al. 1994).
Computer-assisted EST walking was performed for all of the 38 non-redundant ESTs mapping within the NS interval, using the Medical Research Council Human Genome Mapping Project (HGMP) EST-blast program. ESTs were found with homologies to the serine-protease inhibitor precursor VAKTI, the P1 chain of alcohol dehydrogenase class II, the chemokine RANTES, the leulkosialin precursor, and the rat arylsulfatase B (stSG28807, WI-22716, stSG53883, H14645, stSG15286, respectively). These ESTs were detectable by RT-PCR analysis from cultured keratinocytes and were therefore further evaluated.