WO2001007663A1 - Methods and compositions for diagnosing palmoplantar keratodermas and dysplasias and other periodontal diseases - Google Patents

Methods and compositions for diagnosing palmoplantar keratodermas and dysplasias and other periodontal diseases Download PDF

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Publication number
WO2001007663A1
WO2001007663A1 PCT/US2000/020400 US0020400W WO0107663A1 WO 2001007663 A1 WO2001007663 A1 WO 2001007663A1 US 0020400 W US0020400 W US 0020400W WO 0107663 A1 WO0107663 A1 WO 0107663A1
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ctsc
nucleic acid
encoding
alteration
codon
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PCT/US2000/020400
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French (fr)
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Thomas C. Hart
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Wake Forest University
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Priority to AU64957/00A priority Critical patent/AU6495700A/en
Publication of WO2001007663A1 publication Critical patent/WO2001007663A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the fields of genetic screening and molecular biology. More specifically, the invention provides compositions and methods that may be used to advantage to isolate and detect a palmoplantar keratoderma predisposing gene, cathepsin C (CTSC) , some mutant alleles of which cause susceptibility to certain pathological disorders, in particular Papillon-LeFevre Syndrome, Haim-Munk Syndrome and certain forms of early onset periodontal diseases. More specifically, the invention relates to germline mutations and functional polymorphisms in the CTSC gene and their use in the diagnosis of predisposition to palmoplantar ectodermal disorders/ dysplasias and periodontal diseases.
  • CTSC cathepsin C
  • the invention also relates to the therapy of palmoplantar ectodermal disorders/dysplasias and periodontal diseases which have a mutation or functional polymorphisms in the CTSC gene, including gene therapy, protein replacement therapy and protein mimetics.
  • the invention further relates to the screening of drugs for treating and alleviating disease symptoms.
  • the invention relates to the screening of the CTSC gene for disease-related mutations, which are useful for diagnosing the predisposition to additional disorders and dysplasias, including but not limited to prepubertal periodontitis, early onset periodontal disease or other forms of gum disease.
  • Periodontal disease Most forms of inflammatory periodontal disease can be successfully treated and managed. As a result, the ultimate goal of periodontal therapy has changed from that of simply arresting disease progression to one aimed at regenerating the supporting tissues. Unfortunately, not all forms of periodontal disease respond to treatment. Severe periodontitis that is resistant to conventional periodontal treatment has been recognized in a number of monogenic conditions. Certainly some of the most interesting and dentally challenging of these conditions include Papillon-Lefevre syndrome (PLS) , Haim-Munk syndrome (HMS) and periodontal diseases .
  • PLS Papillon-Lefevre syndrome
  • HMS Haim-Munk syndrome
  • Papillon and Lefevre described two siblings, the products of a first cousin mating, with a condition characterized by diffuse transgradient palmoplantar keratosis (PPK) and the premature loss of both the decidous and permanent dentitions.
  • PPK diffuse transgradient palmoplantar keratosis
  • This condition came to be known as Papillon-Lefevre syndrome and subsequently over 200 cases have been described.
  • the hallmarks of PLS are palmoplantar keratosis and rapid periodontal destruction of both dentitions.
  • An increased susceptibility to infection has been reported in approximately 20% of PLS patients.
  • Additional findings include intracranial calcifications, retardation of the somatic development, follicular hyperkeratosis and onychogryphosis .
  • Clinical findings reported in PLS patients suggest that the clinical expression of the condition is a. highly variable. Unfortunately, to date, no pathognomonic disease marker exists allowing definitive diagnosis of PLS.
  • Haim and Munk described an unusual syndrome in four siblings of a Jewish religious isolate from Cochin, India [21] .
  • other clinical findings shared by these individuals included recurrent pyogenic skin infections, acroosteolysis, atrophic changes of the nails, arachnodactyly, and a peculiar radiographic deformity of the fingers consisting of tapered pointed phalangeal ends and a clawlike volar curve. Subsequently pes planus was reported to be associated with the syndrome [24] .
  • PLS Papillon-Lefevre syndrome
  • MIM245000 Papillon-Lefevre syndrome
  • HMS was a distinct disorder.
  • PLS and HMS are classified as type IV palmoplantar ectodermal keratodermas [2] .
  • the unique presence of severe, early onset periodontitis distinguishes PLS and HMS from other PPKs and raises the question of whether they result from the variable clinical expression of a common gene mutation, are allelic mutations at the same genetic locus, or result from expression of gene mutations at separate loci.
  • Haenke [5] summarizes an extensive list of clinical findings reported in PLS affected individuals, including increased susceptibility to infections, ectopic cranial calcifications and nail anomalies [5,26] . It is unclear if these additional clinical features are coincidental findings that may be segregating in a particular family or if they are etiologically related to a syndrome with a very variable clinical expression.
  • PLS is an uncommon condition, and generally occurs only in a single generation it is difficult to determine if these occasional reports of associated clinical findings are etiologically related to PLS. Additionally, consanguinity is common among parents of PLS cases and therefore, it may be expected that an increased number of rare recessive conditions may be seen. Such is likely the case for the reports of mental retardation associated with PLS [5] .
  • PPP Pre-pubertal periodontitis
  • the condition may be localized (usually to deciduous molars) or generalized.
  • the localized form begins at approximately 4 years of age and is associated with only mild gingival inflammation in the presence of relatively little plaque.
  • the generalized form begins earlier, immediately after eruption of the deciduous teeth. It is associated with severe gingival inflammation and hyperplasia, although significant gingival recession has also been described as an associated clinical feature.
  • the attachment loss appears to be continuous rather than intermittent as with most other forms of periodontitis.
  • the present invention provides such a disease marker and methods of use thereof having diagnostic and prognostic utilities for several PPKs and many periodontal diseases .
  • the present invention provides compositions and methods which allow for genetic screening and diagnosis of certain palmoplantar keratodermas and periodontal disease states in affected individuals.
  • CSC cathepsin C gene
  • Mutations or functional polymorphisms associated with the disease state are those which give rise to a altered, truncated, misfolded or otherwise non-functional CTSC polypeptides.
  • Polymorphisms in the CTSC sequence which do not affect the nature of the encoded protein are not associated with PLS, HMS or periodontal disease.
  • a method for determining the presence of alterations in CTSC encoding nucleic acids which give rise to altered CTSC proteins.
  • the wild-type CTSC nucleic acid sequence and its encoded amino acid sequence are known.
  • CTSC mutations specifically associated with PLS, HMS and PPP are described herein and are set forth in Table 1. Accordingly in one embodiment of the invention, nucleic acid molecules encoding altered CTSC proteins are considered to be within the scope of the present invention. In a preferred embodiment of the invention, the altered CTSC nucleic acid has at least one of the alterations set forth in Table 1.
  • nucleic acid probes which specifically hybridize to the human altered CTSC-encoding nucleic acids described herein and not to wild-type CTSC encoding nucleic acids are provided.
  • the probes specifically hybridize with altered CTSC encoding nucleic acids having at least one of the alterations set forth in Table 1.
  • a mutated CTSC protein encoded by the altered CTSC encoding nucleic acids of the invention is provided.
  • CTSC proteins are encoded by a nucleic acid containing a mutation as set forth in Table 1.
  • assays for biochemically assessing altered cathepsin C activity are also contemplated to be within the scope of the present invention.
  • a method for detecting a germline alteration in a CTSC gene is provided. In a preferred embodiment the alteration is selected from the group consisting of the alterations set forth in Table 1.
  • the method comprises analyzing a sequence of a CTSC gene or CTSC RNA from a human sample or analyzing a sequence of CTSC cDNA made from mRNA from a human sample and comparing sequences so isolated to the wild type sequence encoding CTSC.
  • CTSC computed tomography
  • methods are provided for assessing the enzymatic activity of proteins encoded by nucleic acid molecules which do not possess the wild type CTSC sequence.
  • kits for detecting the presence of an altered CTSC encoding nucleic acids in a biological sample.
  • An exemplary kit comprises the following: i) oligonucleotides which specifically hybridize with CTSC encoding nucleic acids having the alterations set forth in Table 1; ii) reaction buffer; and iii) an instruction sheet.
  • Kits for detecting the presence an altered CTSC proteins in a biological sample are also provided.
  • Exemplary kits for this purpose comprise: i) antibodies immunologically specific for the altered CTSC proteins of the invention; ii) a solid support with immobilized CTSC antigens as a positive control; and iii) an instruction sheet.
  • anti-CTSC antibodies used for this purpose may contain a detectable label or tag for used in isolating or detecting immune complexes .
  • isolated nucleic acid refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it was derived.
  • the "isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a procaryote or eucaryote.
  • An "isolated nucleic acid molecule” may also comprise a cDNA molecule.
  • An isolated nucleic acid molecule inserted into a vector is also sometimes referred to herein as a Arecombinant@ nucleic acid molecule.
  • '1 nucleic acid primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above.
  • the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a "substantially pure” form (the term “substantially pure” is defined below) .
  • isolated protein or “isolated and purified protein” is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in "substantially pure” form.
  • substantially pure refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like) .
  • immunologically specific refers to antibodies that bind to one or more epitopes of a protein of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.
  • the term “specifically hybridizing” refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary”).
  • the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single- stranded nucleic acids of non-complementary sequence.
  • Appropriate conditions enabling specific hybridization of single stranded nucleic acid molecules of varying complementarity are well known in the art.
  • T m 81.5°C + 16.6 og [Na+] + 0.41(% G+C) - 0.63 (% formamide) - 600/#bp in duplex
  • promoter region refers to the transcriptional regulatory regions of a gene, which may be found at the 5' or 3 ' side of the coding region, or within the coding region, or within introns .
  • promoters for the yeast and mammalian expression systems of the invention are available and known to those of ordinary skill in the art.
  • operably linked means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to enable expression of the coding sequence. This same definition is sometimes applied to the arrangement of transcription units and other transcription control elements (e.g. enhancers) in an expression vector.
  • oligonucleotide refers to primers and probes of the present invention, and is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide.
  • probe refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe.
  • a probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • the probes herein are selected to be complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to "specifically hybridize” or anneal with their respective target strands under a set of pre-deter ined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5' or 3 ' end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically.
  • primer refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis.
  • suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and pH
  • the primer may be extended at its 3 ' terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield an primer extension product.
  • the primer may vary in length depending on the particular conditions and requirement of the application.
  • the oligonucleotide primer is typically 15-25 or more nucleotides in length.
  • the primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able anneal with the desired template strand in a manner sufficient to provide the 3 ' hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer
  • I I sequence represent an exact complement of the desired template.
  • a non-complementary nucleotide sequence may be attached to the 5 ' end of an otherwise complementary primer.
  • non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.
  • Figures 1A-1E are a series of clinical photographs showing palmoplantar keratosis and periodontal disease in PLS study patient.
  • Fig. IA palmar hyperkeratotic lesions
  • Fig. IB plantar hyperkeratotic lesions
  • Fig. IC hyperkeratotic lesions affecting the knees
  • Fig. ID periodontitis involving erupting permanent dentition
  • Fig. IE periapical radiographs showing severe alveolar bone loss affecting erupting permanent teeth.
  • Figure 2 shows haplotype data for chromosome llq short tandem repeat polymorphisms (STRP) markers spanning the PLS gene locus . Segments which are likely to be homozygous by descent are boxed. Arrows indicate recombinant events. Individuals 7 and 22 share a common haplotype for D11S1979, D11S1887, D11S1780, D11S1367, D11S931, and D11S4175.
  • STP short tandem repeat polymorphisms
  • FIG. 3 depicts pedigree and sequence analysis of
  • CTSC exon 6 The numbering of the wildtype sequence shown above the figure is based upon the genomic sequence of CTSC. See SEQ ID NO : 1. Circles represent females and squares represent males. Filled symbols indicate affected individuals. Half-shading indicates carriers based upon DNA sequencing results. All affected individuals are homozygous for the specific CTSC mutations. Arrows indicate the position of the mutation. This family has a nonsense mutation (856 C->T) at codon 286 resulting in a truncated protein of 286 amino acids.
  • Figures 4A - 4D show pedigrees and sequence analysis of CTSC exon 7 for 4 Families with PLS. Symbols are as described for Figure 3.
  • Fig. 4A Family with a single base pair deletion (1047delA) of CTSC resulting in a frameshift and premature termination.
  • Fig. 4B Family with a 2bp deletion (1028-1029delCT) of CTSC resulting in a frameshift and premature termination.
  • Fig. 4C . and Fig. 4D pedigrees of families with a nonsense mutation (1286G->A) at codon 429 resulting in a truncated protein of 428 amino acids. The father in family C is deceased and no sample was available for analysis.
  • Figures 5A-5C is a schematic diagram of the CTSC gene showing the locations of the mutations described herein.
  • Panel 5A Genomic structure of CTSC gene with introns shown as solid lines and exons depicted as boxes. The 5' and 3' untranslated regions are shown as filled boxes.
  • Panel 5B Coding region of CTSC gene. The amino acid numbers are shown at the end of each exon. Mutations listed in Table 1 are shown according to their genomic locations with Missense, ⁇ ; Nonsense, ⁇ ; Insertion, ⁇ ; and Deletion, ⁇ . The splicing site mutation is indicated by an arrow.
  • Panel 5C Panel 5C.
  • the 10 kDa pro-region is cleaved out upon activation.
  • the disulfide bond within the 13.5 pro-region is shown.
  • the glycosylation sites are indicated by filled circle and arrows indicate the active sites.
  • Figures 6A-6F show a series of micrographs
  • Fig. 6A dermal involvement of fingers in individual #34.
  • Fig. 6B Individual #34 radiograph of terminal phalanges of the fingers showing marked thinning increasing towards the distal, tapering pointed ends showing a claw-like volar bend.
  • Fig. 6C Individual #17 palmar keratosis;
  • Fig. 6D Individual #17 plantar keratosis:
  • Fig. 6E Individual #17 gingival inflammation;
  • Fig. 6F Individual #17 radiograph showing alveolar bone destruction associated with gingival inflammation shown in 6D.
  • Figure 7A shows pedigree of Cochin descendents segregating Haim Munk syndrome (HMS) . Numbered individuals have been analyzed for the current study.
  • HMS Haim Munk syndrome
  • Unshaded numbered individuals represent non-carriers based upon DNA analysis.
  • Figure 7B shows a pedigree of a Turkish family segregating PLS. Numbered individuals were available for study. Half-shading indicates carrier based upon DNA analysis. Individual 77 is a non-carrier based upon DNA sequencing.
  • Figures 8A and 8B show the results of sequence analysis of exon 6 of CTSC.
  • the numbering of the wildtype sequence is based upon the cDNA sequence of CTSC. See SEQ ID NO : 1.
  • Fig. 8A Family A (Cochin isolate diagnosed with Haim Munk syndrome) from Figure 1.
  • Affected individuals are homozygous for a 857A->G missense mutation which results in a conserved glutamine being changed to an arginine (Q286R) .
  • Representative sequences are shown for individuals #36 (affected) and #31 (carrier) .
  • Fig. 8B Family B from Figure 1.
  • Affected individuals are homozygous for a 856C->T nonsense mutation which results in a premature stop codon at position 286 (Q286X) .
  • the Q286X mutation has been previously reported in an inbred Turkish family [12] .
  • Figure 9 depicts a gel showing the results of restriction enzyme analysis of Q286R mutation in Family A of Fig. 7.
  • a 465 bp fragment of exon 6 was amplified and subjected to restriction digestion with Aval as described under methods.
  • the Q286R mutation introduces a new Aval site.
  • wildtype individuals exhibit bands of 465 bp
  • affected individuals have bands of 404 and 61 bp
  • carriers have bands of 465, 404, and 61 bp.
  • M. 1 kb ladder (Gibco) Lane 1. Individual #5 uncut, demonstrating 465bp amplicon. Lane 2. Individual #5 cut with Aval. Only the 465 bp fragment is observed.
  • individual #5 has the wildtype sequence on both alleles.
  • Lane 3. Individual #31 uncut.
  • Lane 4. Individual # 31 cut with Aval. The 465 and 404bp fragments are visible, confirming that individual #31 is a carrier of the Q286R, consistent with the sequencing results shown in Figure 3A.
  • Lane 5. Individual #34 uncut.
  • Lane 6. Individual #34 cut with Aval. The 404 and 61bp fragments are indicated by arrows.
  • the present invention relates generally to the field of human genetics. Specifically, the present invention relates to methods and materials used to isolate and detect mutated forms of the lysosomal protease cathepsin C (CTSC) gene, associated with autosomal recessive disorders characterized by palmar hyperkeratosis and/or periodontitis. More specifically, the present invention relates to germline mutations in the CTSC gene and their use in the diagnosis of predisposition to such pathological conditions. Additionally, the invention relates to germline mutations in the CTSC gene in other palmoplantar ectodermal disorders and dysplasias and their use in the diagnosis and prognosis of such pathological conditions.
  • CTSC lysosomal protease cathepsin C
  • the invention also relates to the therapy of palmoplantar ectodermal disorders and dysplasias which have a mutation or functional polymorphism in the CTSC gene, including gene therapy, protein replacement therapy and protein mimetics.
  • the invention further relates to the screening of drugs which may have therapeutic value. Biochemical assays are provided for the assessment of altered activity of aberrant CTSC enzymes encoded by the mutated CTSC encoding nucleic acids of the invention.
  • the invention relates to the screening of the CTSC gene for mutations, which are useful for diagnosing the predisposition to ectodermal disorders and dysplasias.
  • the present invention provides an isolated polynucleotide comprising all, or a portion of the CTSC locus or of a mutated CTSC locus, preferably at least eight bases and not more than about 100 kb in length. Such polynucleotides may be antisense polynucleotides.
  • the present invention also provides a recombinant construct comprising such an isolated polynucleotide, for example, a recombinant construct suitable for expression in a transformed host cell.
  • Also provided by the present invention are methods of detecting a polynucleotide comprising a portion of the CTSC locus or its expression product in an analyte. Such methods may further comprise the step of amplifying the portion of the CTSC locus, and may further include a step of providing a set of polynucleotides which are primers for amplification of said portion of the CTSC locus.
  • the method is useful for either diagnosis of the predisposition to PPKs or the diagnosis or prognosis of keratodermal disorders/dysplasias and periodontal diseases .
  • the present invention also provides isolated antibodies, preferably monoclonal antibodies, which specifically bind to an isolated polypeptide comprised of at least five amino acid residues encoded by the altered CTSC locus.
  • the present invention also provides kits for detecting in an analyte a polynucleotide comprising a portion of the CTSC locus, the kits comprising a polynucleotide complementary to the portion of the CTSC locus packaged in a suitable container, and instructions for its use.
  • the present invention further provides methods of preparing a polynucleotide comprising polymerizing nucleotides to yield a sequence comprised of at least eight consecutive nucleotides of the CTSC locus; and methods of preparing a polypeptide comprising polymerizing amino acids to yield a sequence comprising at least five amino acids encoded within the CTSC locus.
  • the present invention further provides methods of screening the CTSC gene to identify mutations. Such methods may further comprise the step of amplifying a portion of the CTSC locus, and may further include a step of providing a set of polynucleotides which are primers for amplification of said portion of the CTSC locus. Exemplary primers are set forth in Table A.
  • the method is useful for identifying mutations for use in either diagnosis of the predisposition to keratodermal disorders/dysplasias and periodontal diseases or the diagnosis of such disorders.
  • the present invention further provides methods of screening suspected CTSC mutant alleles and functional
  • the present invention provides methods of screening drugs for therapy and to identify suitable drugs for restoring CTSC gene product function.
  • the present invention provides the means necessary for production of gene-based therapies directed at aberrant cells associated with keratodermal disorders and dysplasias.
  • These therapeutic agents may take the form of polynucleotides comprising all or a portion of the CTSC locus placed in appropriate vectors or delivered to target cells in more direct ways such that the function of the CTSC protein is reconstituted.
  • Therapeutic agents may also take the form of polypeptides based on either a portion of, or the entire protein sequence of CTSC. These may functionally replace the activity of CTSC in vivo.
  • mutations in the CTSC locus in the germline are indicative of a predisposition to keratodermal disorders/dysplasias and periodontal diseases.
  • the mutational events of the CTSC locus can involve deletions, insertions and point mutations within the coding sequence and the non-coding sequence.
  • a major gene locus associated with the keratodermal disorders and dysplasias of the invention has been localized to a 2.8 cM interval on chromosome llql4 of the human genome.
  • This region contains a genetic locus, CTSC.
  • the CTSC message is expressed at high levels in a variety of immune cells including polymorphonuclear leukocytes, macrophages and their precursors. This gene is expressed in the palms, soles, knees, and oral keratinized gingiva .
  • the CTSC gene was originally reported to consist of 2 exons .
  • the sequence encoding the wild type human CTSC gene is provided below (SEQ ID NO : 1) :
  • ATTTTCTTGT CAACAGAACA AAGATTAAGT CATTTGTTAC TAAAGAAATA CCTTTTTAAC
  • CTCCGCCTCC CGGGTTTAAG CGATTCTCCT GCCTCAGTCT CCCGAGTAGC TAGGATTACG
  • CTCCCATGTC AGAGCATTAC TCTCAAACAT GGAAAAACTT TAAAATACAC AACTCTCCAG
  • TTTTTTCTTT AGGACCACAA GAAAAAAAAG TAGTGGTGTA CCTTCAGAAG CTGGATACAG CATATGATGA CCTTGGCAAT TCTGGCCATT TCACCATCAT TTACAACCAA GGCTTTGAGA
  • CTGAACACTC CCTTGGAAAA CAGTAAACAT CATTTTGGAA TGTGAACAAC CAGAGACTAC
  • CTTCTTTCAG CTTTGGTAAG CCTGAAATTA TGGGGTTATG TTTAATTCAT ATTGTCTGGG
  • CTAGAAAGAC AAATCAGGAA TGGTGCCATA TACATCTTTT TTGATTCCCT GCTCTAAAGA
  • GCCAGTTCTC CCAGCACCAT TTATTAAATA GGGAATCCTT TCCCTATTGT TTGTTTTTGT
  • AAAAATCCTA TTGTCATAGT TATGTTTTTG TGGAAAGAAC AACCTGATAG ACTATCGTGA AAAATAAATG AGTTAGTATA CATAAAACAC AGCCCATGAG ATATAGTCTG TAATTATCAT CCCTGCTTCA TTTATTTATT TATTTATTTA TTTATTTAGA GAATGGATCT GATTCTGTCA TCCAGGCCGG AGTTCAGTGG CTGGATCATT GCTCACTGTA ACCTCAAAAC ACCTGGCCTC AAGTGATTCC CCCAACCTCG GCGTTCCAAA GTGCTGGGAT TAAAGGCATG AGCCACTGTA
  • CTCTGCGTGA ATTAAACTCA TTTTCTATTG CAATTTCCCT GTCTTGATAA TCAGTTCTGT GTAGGCCGTG AGGAAGGAGA ACCCGTTGGG TGATTACGAG ACTGTGTTAC TGCCCACTAC
  • TCCCCTGGAG TCCACTTGTT CCTGTTCTCC TCTGGGTTCA AATACCTCTT CCCTCCTTTT
  • AACGGAGCAT ATCATATCCC CTTCTCAAAT TCACCAAAGT GAAGTCCTAA TGTGTCTTAA TGTATCTGCA TGAGACAGGA AGCTGAGATC TATTCAACAA CAAAAATCCA AACAAGCATC AAGAGGAGGA GTGTTAGCAC TTGAGCCTAG GGAGACTGTG GCTCCTGCCT GAAAGATGGG AGCCTCAGTC ACAGCTGCTT TACCAAGTGT CATATGCTAT GTTTCTGAGG ACTCCTGCTA AAGCTCCCTT CTCCCTCCAG CCAACCACTT TTGTTTTAGA CAAGGGCTGG GTTTATGAAG
  • TGCTGTCTGG AAACATTCTG ATTAGTCT CCTGGGAGGA TTATAAATTT ATAGTACCCA AAGANTAAAC NTGTTGTTTC CCTTTCCTAA ACTTTTAGTG NATAATNCAG TCTCTGCCGT GTCTCATTTT CATCACTTGC CCTCCANAGC TCCCATCTCA CTGAATTCTT GCAGTGTTCT GAATGTTGAG AGCCCCAANG TGGGTCTTAT AACAGCCAGT CAGCAACATT TCTGTTTTTC ATCTGACACC AAGGGTCTCG TCTCTTTGCT TTTCTACCAG TTATTCTGGG CTCTTCAGCT CTAAAGAAAG TATAGGTCCT GAAATCTTTC CCTACCTTCT CAATTTCCTG GGGAGGGCTT CTTTGGAAAG TGGGATTGGA AATAAGATAA ATTTGAAGAT AATTGAGAAA TGAATGGAAA GTGAAATTGA AGGGTCCATG TTAAGAGATT GCAAGTTATG CTATCACCAA ATAGATTT
  • AAATATTCTC AAAGAAAGTC CCATGTACTA ATGTTTGCCT TTTGATGAAA AAGGATGAAA TCTTAATGAT TGCCTTAATA
  • GTTGCATTTC AGACACCATT TATGAGCTAT TTACCTGTGT GCAGCTGGCT GTTGTTGGCA
  • GAAGATGGTC AGCTATGAAG TAATAGAGTT TGCTTAATCA TTTGTAATTC AAACATGCTA
  • the wild type CTSC protein sequence is set forth below as SEQ ID NO : 3 : MGAGPSLLLAALLLLLSGDGAVRCDTPANCTYLDLLGTWVFQVG
  • Papillon Lefevre syndrome is an autosomal recessive disorder characterized by palmoplantar hyperkeratosis and severe early onset periodontitis that results in the premature loss of the primary and secondary dentitions.
  • the 46 kb CTSC gene consists of 7 exons and is mutated in PLS patients. Sequence analysis of CTSC from PLS affected individuals from thirty-two Turkish families identified four different mutations. An exon 6 nonsense mutation (856C->T) introduces a premature stop codon at amino acid 286.
  • Three exon 2 mutations were identified including a single nucleotide deletion (1047delA) of codon 349 introducing a frameshift and premature termination codon, a two base pair deletion (1028-1029delCT) that results in introduction of a stop codon at amino acid 343, and a G->A substitution in codon 429 (1286G->A) introducing a premature termination codon.
  • All PLS affected individuals examined were homozygous for cathepsin C mutations inherited from a common ancestor. Parents and siblings heterozygous for cathepsin C mutations do not show either the palmoplantar hyperkeratosis or severe early onset periodontitis characteristic of PLS.
  • Table I summarizes CTSC mutations identified in 27 other families presenting with symptoms of PLS.
  • Haim-Munk syndrome is a rare condition associated with congenital palmoplantar keratosis, pes planus, onychogyrphosis, periodontosis, arachnodactyly and acroosteolysis .
  • a mutation in cathepsin C causing Haim-Munk Syndrome has been identified. It appears that substitution of an A for a G at CTSC nucleotide position 857 in Exon 6 is responsible for this syndrome in patients .
  • PPP prepubertal periodontitis
  • early onset periodontal disease or other forms of gum disease.
  • PPP prepubertal periodontitis
  • the invention also provides methods for screening the CTSC gene for alterations associated with these disease states.
  • Nucleic acid molecules encoding the human CTSC proteins of the invention may be prepared by two general methods: (1) synthesis from appropriate nucleotide triphosphates, or (2) isolation from biological sources. Both methods utilize protocols well known in the art.
  • the availability of nucleotide sequence information, such as a DNA having the sequence of SEQ ID NOS: 1-2 enables preparation of an isolated nucleic acid molecule of the invention by oligonucleotide synthesis.
  • Synthetic oligonucleotides may be prepared by the
  • phosphoramidite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC) .
  • HPLC high performance liquid chromatography
  • Long, double-stranded polynucleotides, such as a DNA molecule of the present invention must be synthesized in stages, due to the size limitations inherent in current oligonucleotide synthetic methods.
  • a 4.7 kb double- stranded molecule may be synthesized as several smaller segments of appropriate complementarity. Complementary segments thus produced may be annealed such that each segment possesses appropriate cohesive termini for attachment of an adjacent segment.
  • Adjacent segments may be ligated by annealing cohesive termini in the presence of DNA ligase to construct an entire 4.7 kb double-stranded molecule.
  • a synthetic DNA molecule so constructed may then be cloned and amplified in an appropriate vector. Nucleic acid sequences encoding the altered human
  • CTSC proteins of the invention may be isolated from appropriate biological sources using methods known in the art.
  • a cDNA clone is isolated from a cDNA expression library of human origin.
  • human genomic clones encoding altered CTSC proteins may be isolated.
  • Table 1 sets forth several different mutations associated with particular PPKs and PPP.
  • Altered CTSC- specific probes for identifying such sequences may be between 15 and 40 nucleotides in length. For probes longer than those shown above, the additional contiguous nucleotides are provided within SEQ ID NOS : 1 and 2. Additionally, cDNA or genomic clones having homology with human CTSC may be isolated from other species using oligonucleotide probes corresponding to predetermined sequences within the human CTSC encoding nucleic acids.
  • nucleic acids having the appropriate level of sequence homology with the protein coding region of SEQ ID NO : 1 may be identified by using hybridization and washing conditions of appropriate stringency.
  • hybridizations may be performed, according to the method of Sambrook et al . , Molecular Cloning, Cold Spring Harbor Laboratory (1989), using a hybridization solution comprising: 5X SSC, 5X Denhardt's reagent, 1.0% SDS, 100 ⁇ g/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide.
  • Hybridization is carried out at 37-42°c for at least six hours.
  • Nucleic acids of the present invention may be maintained as DNA in any convenient cloning vector.
  • clones are maintained in a plasmid cloning/expression vector, such as pBluescript (Stratagene, La Jolla, CA) , which is propagated in a suitable E. coli host cell.
  • Altered CTSC-encoding nucleic acid molecules of the invention include cDNA, genomic DNA, RNA, and fragments thereof which may be single- or double-stranded.
  • this invention provides oligonucleotides having sequences capable of hybridizing with at least one sequence of a nucleic acid molecule of the present invention, such as selected segments of the DNA having SEQ ID NO : 1.
  • oligonucleotide probes which specifically hybridize with the mutated CTSC genes of the invention while not hybridizing with the wild type sequence under high stringency conditions.
  • Primers capable of specifically amplifying the altered CTSC encoding nucleic acids described herein are also contemplated herein. As mentioned previously, such oligonucleotides are useful as probes and primers for detecting, isolating or amplifying altered CTSC genes.
  • Antisense nucleic acid molecules may be targeted to translation initiation sites and/or splice sites to inhibit the expression of the CTSC gene or production of the CTSC protein of the invention.
  • Such antisense molecules are typically between 15 and 30 nucleotides in length and often span the translational start site of CTSC encoding mRNA molecules.
  • antisense constructs may be generated which contain the entire CTSC cDNA in reverse orientation. Such antisense constructs are easily prepared by one of ordinary skill in the art.
  • variants e.g., allelic variants
  • CTSC sequences e.g., allelic variants
  • the term "natural allelic variants" is used herein to refer to various specific nucleotide sequences of the invention and variants thereof that would occur in a human population.
  • the term "substantially complementary" refers to oligonucleotide sequences that may not be perfectly matched to a target sequence, but such mismatches do not n materially affect the ability of the oligonucleotide to hybridize with its target sequence under the conditions described.
  • Full-length, altered, human CTSC proteins of the present invention may be prepared in a variety of ways, according to known methods .
  • the proteins may be purified from appropriate sources, e.g., transformed bacterial or animal cultured cells or tissues, by immunoaffinity purification. However, this is not a preferred method due to the low amount of protein likely to be present in a given cell type at any time.
  • the availability of nucleic acid molecules encoding CTSC protein enables production of the protein using in vi tro expression methods known in the art.
  • a cDNA or gene may be cloned into an appropriate in vi tro transcription vector, such as pSP64 or pSP65 for in vi tro transcription, followed by cell-free translation in a suitable cell-free translation system, such as wheat germ or rabbit reticulocyte lysates .
  • vi tro transcription and translation systems are commercially available, e.g., from Promega Biotech, Madison, Wisconsin or Gibco-BRL, Gaithersburg, Maryland.
  • larger quantities of CTSC protein may be produced by expression in a suitable prokaryotic or eukaryotic system.
  • a DNA molecule such as a DNA having SEQ ID NOS : 1 or 2 containing an alteration set forth in Table 1 may be inserted into a plasmid vector adapted for expression in a bacterial cell, such as E. coli .
  • Such vectors comprise the regulatory elements necessary for expression of the DNA in the host cell positioned in such a manner as to permit expression of the DNA in the host cell.
  • regulatory elements required for expression include promoter sequences, transcription initiation sequences and, optionally, enhancer sequences.
  • the human CTSC protein produced by gene expression in a recombinant procaryotic or eukaryotic system may be purified according to methods known in the art.
  • a commercially available expression/secretion system can be used, whereby the recombinant protein is expressed and thereafter secreted from the host cell, and readily purified from the surrounding medium.
  • an alternative approach involves purifying the recombinant protein by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant protein or nickel columns for isolation of recombinant proteins tagged with 6-8 histidine residues at their N-terminus or C- terminus .
  • Alternative tags may comprise the FLAG epitope or the hemagglutinin epitope. Such methods are commonly used by skilled practitioners.
  • the human CTSC protein of the invention prepared by the aforementioned methods, may be analyzed according to standard procedures. For example, such protein may be subjected to amino acid sequence analysis, according to known methods .
  • the present invention also provides antibodies capable of immunospecifically binding to proteins of the invention. Polyclonal antibodies directed toward altered human CTSC proteins may be prepared according to standard methods. In a preferred embodiment, monoclonal antibodies are prepared, which react immunospecifically with the various epitopes of the CTSC protein described herein. Monoclonal antibodies may be prepared according to general methods of Kohler and Milstein, following standard protocols.
  • Polyclonal or monoclonal antibodies that immunospecifically interact with altered CTSC proteins can be utilized for identifying and purifying such proteins.
  • antibodies may be utilized -5 for affinity separation of proteins with which they immunospecifically interact.
  • Antibodies may also be used to immunoprecipitate proteins from a sample containing a mixture of proteins and other biological molecules. Other uses of anti-CTSC antibodies are described below.
  • SSCP Single stranded conformational polymorphism
  • Sample preparation is relatively easy in these assays, and conditions for electrophoresis required to generate the often subtle mobility differences that form the basis for identifying the targets that contain mutations are known to those of skill in the art.
  • Another parameter to be considered is the size of the target region being screened.
  • SSCP is used to screen target regions no longer than about 200-300 bases.
  • Another type of screening technique currently in use is based on cleavage of unpaired bases in heteroduplexes formed between wild type probes hybridized to experimental targets containing point mutations.
  • the cleavage products are also analyzed by gel electrophoresis, as subfragments generated by cleavage of the probe at a mismatch generally differ significantly in size from full length, uncleaved probe and are easily detected with a standard gel system.
  • Mismatch cleavage has been effected either chemically (osmium tetroxide, hydroxylamine) or with a less toxic, enzymatic alternative, using RNase A.
  • the RNase A cleavage assay has also been used, although much less frequently, to screen for mutations in endogenous mRNA targets for detecting mutations in DNA targets amplified by PCR. A mutation detection rate of over 50% was reported for the original RNase screening method.
  • a newer method to detect mutations in DNA relies on DNA ligase which covalently joins two adjacent oligonucleotides which are hybridized on a complementary target nucleic acid. The mismatch must occur at the site of ligation. As with other methods that rely on oligonucleotides, salt concentration and temperature at hybridization are crucial. Another consideration is the amount of enzyme added relative to the DNA concentration.
  • exemplary approaches for detecting alterations in CTSC encoding nucleic acids or polypeptides /proteins include: a) comparing the sequence of nucleic acid in the sample with the wild-type CTSC nucleic acid sequence to determine whether the sample from the patient contains mutations; or b) determining the presence, in a sample from a patient, of the polypeptide encoded by the CTSC gene and, if present, determining whether the polypeptide is full length, and/or is mutated, and/or is expressed at the normal level; or c) using DNA restriction mapping to compare the restriction pattern produced when a restriction enzyme cuts a sample of nucleic acid from the patient with the restriction pattern obtained from normal CTSC gene or from known mutations thereof; or, d) using a specific binding member capable of binding to a CTSC nucleic acid sequence (either normal sequence or known mutated sequence) , the specific binding member comprising nucleic acid hybridizable with the CTSC sequence, or substances comprising an antibody domain with specificity for a
  • a “specific binding pair” comprises a specific binding member (sbm) and a binding partner (bp) which have a particular specificity for each other and which in normal conditions bind to each other in preference to other molecules.
  • specific binding pairs are antigens and antibodies, ligands and receptors and complementary nucleotide sequences. The skilled person is aware of many other examples and they do not need to be listed here. Further, the term “specific binding pair” is also applicable where either or both of the specific binding member and the binding partner comprise a part of a large molecule.
  • the specific binding pair are nucleic acid sequences, they will be of a length to hybridize to each other under conditions of the assay, preferably greater than 10 nucleotides long, more preferably greater than 15 or 20 nucleotides long.
  • the CTSC nucleic acid in the sample will initially be amplified, e.g. using PCR, to increase the amount of the analyte as compared to other sequences present in the sample. This allows the target CTSC sequences to be detected with a high degree of sensitivity if they are present in the sample. This initial step may be avoided by using highly sensitive array techniques that are becoming increasingly important in the art .
  • the identification of the CTSC gene and its association with keratodermal disorders/dysplasias and peridontal diseases paves the way for aspects of the present invention to provide the use of materials and methods, such as are disclosed and discussed above, for establishing the presence or absence in a test sample of a variant form of the gene, in particular an allele or variant specifically associated with PLS, HMS or periodontal diseases.
  • This may be for diagnosing a predisposition of an individual to PLS, HMS or periodontal disease. It may be for diagnosing PLS, HMS or periodontal disease in a patient with the disease as being associated with the altered CTSC gene.
  • This allows for planning of appropriate therapeutic and/or prophylactic measures, permitting stream-lining of diagnosis, treatment and outcome assessments. The approach further stream-lines treatment by targeting those patients most likely to benefit.
  • methods of screening drugs for therapy to identify suitable drugs for restoring CTSC product functions are provided.
  • the CTSC polypeptide or fragment employed in drug screening assays may either be free in solution, such as gingival crevicular fluid, affixed to a solid support or within a cell.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays.
  • H I determine, for example, formation of complexes between a CTSC polypeptide or fragment and the agent being tested, or examine the degree to which the formation of a complex between a CTSC polypeptide or fragment and a known ligand is interfered with by the agent being tested.
  • CTSC polypeptide is then detected by methods well known in the art .
  • a further technique for drug screening involves the use of host eukaryotic cell lines or cells (such as described above) which have a nonfunctional CTSC gene.
  • These host cell lines or cells are defective at the CTSC polypeptide level.
  • the host cell lines or cells are grown in the presence of drug compound.
  • the rate of growth of the host cells is measured to determine if the compound is capable of regulating the growth of CTSC defective cells.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g., enhance or interfere with the function of a polypeptide in vivo . See, e.g., Hodgson, (1991) Bio/Technology 9:19-21.
  • one first determines the three-dimensional structure of a protein of interest (e.g., CTSC polypeptide) or, for example, of the CTSC-substrate complex, by x-ray crystallography, by nuclear magnetic resonance, by computer modeling or most typically, by a combination of approaches. Less often, useful information regarding the structure of a polypeptide may be gained by modeling based on the structure of homologous proteins.
  • An example of rational drug design is the development of HIV protease inhibitors (Erickson et al . , (1990) Science 249:527- 533).
  • peptides e.g., CTSC polypeptide
  • peptides may be analyzed by an alanine scan (Wells, 1991) Meth. Enzym.
  • CTSC polypeptide activity e.g., improved CTSC polypeptide activity or stability or which act as inhibitors, agonists, antagonists, etc. of CTSC polypeptide activity.
  • drugs which have, e.g., improved CTSC polypeptide activity or stability or which act as inhibitors, agonists, antagonists, etc. of CTSC polypeptide activity.
  • compositions useful for treatment and diagnosis of these syndromes and conditions may comprise, in addition to one of the above substances, a pharmaceutcally acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutcally acceptable excipient e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
  • administration is preferably in a "prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • the nucleic acid encoding the authentic biologically active CTSC polypeptide could be used in a method of gene therapy, to treat a patient who is unable to synthesize the active "normal” polypeptide or unable to synthesize it at the normal o level, thereby providing the effect elicited by wild- type CTSC and suppressing the occurrence of "abnormal" CTSC associated with keratodermal disorders and dysplasias .
  • Vectors, such as viral vectors have been used in the prior art to introduce genes into a wide variety of different target cells. Typically the vectors are exposed to the target cells so that transformation can take place in a sufficient proportion of the cells to provide a useful therapeutic or prophylactic effect from the expression of the desired polypeptide.
  • the transfected nucleic acid may be permanently incorporated into the genome of each of the targeted cells, providing long lasting effect, or alternatively the treatment may have to be repeated periodically.
  • viral vectors both viral vectors and plasmid vectors are known in the art, see US Patent No. 5,252,479 and WO 93/07282.
  • viruses have been used as gene transfer vectors, including papovaviruses, such as SV40, vaccinia virus, herpes viruses including HSV and EBV, and retroviruses .
  • papovaviruses such as SV40
  • vaccinia virus vaccinia virus
  • herpes viruses including HSV and EBV
  • retroviruses retroviruses
  • Many gene therapy protocols in the prior art have employed disabled murine retroviruses.
  • Gene transfer techniques which selectively target the CTSC nucleic acid to oral tissues are preferred.
  • Examples of this include receptor-mediated gene transfer, in which the nucleic acid is linked to a protein ligand via polylysine, with the ligand being specific for a receptor present on the surface of the target cells.
  • RNA Isolation RNA Isolation, Amplification, and Tissue Expression Analysis .
  • GenBank accession numbers Full-length cDNA of CTSC (NM-001814) and full- length genomic DNA of CTSC contained within a BAC vector, Genbank accession number (AC011088) . See SEQ ID NO: 1.
  • cathepsin C activity ranges from 600-1200 ⁇ mol/min/ mg .
  • measurement of cathepsin C enzymatic activity can be used to determine whether at-risk family members are carriers. Enzymatic activity can also be used to determine if individuals marrying into a family are carriers. Carriers typically have approximately 50% of normal enzyme activity. Determination of enzymatic activity can also be used to establish a diagnosis of PLS when mutational studies of cathepsin C have been negative. This is important in assuring that a diagnosis of PLS has been properly given to an individual with clinical symptoms suggestive of PLS.
  • Viable leukocyte pellets are obtained from lithium heparinized whole blood by mixing blood with 3 volumes of 3% dextran in normal saline, and allowing the red cells to settle for 45 min at room temperature. Cells are pelleted by centrifugation at 1500 rpm for 5 min at 4*C. After washing and removal of contaminating red cells, leukocyte pellets are resuspended in dH20 and sonicated on ice for 5 sec each for total of 6 blasts using a Sonic 300 Dismembrator . Protein concentration is determined by the Lowry method.
  • Enzymatic activity is determined by measuring hydrolysis of the synthetic substrate glycyl-L-arginine-7-amido-4-methylcoumarin at a final concentration of 5 mM using a modified method. All reactions are performed in duplicate. Twenty ⁇ l of leukocyte lysate are added to 200 ⁇ l of Na 3 P0 4 buffer (0.1M, pH 6.5) in a 96 well plate and then substrate added. Reactions are allowed to proceed for 1 hr at room temperature at which time 10 ⁇ l of glycine-NaOH buffer (0.5M, pH 9.8 ) is added to stop the reaction.
  • Fluorescence is determined using a Perkin-Elmer LS50B luminescence spectrometer at 370-nm excitation and 460-nm emission. The amount of NHMec released is determined by generating a standard curve using NHMec. Cathepsin C activity is reported as ⁇ mol
  • PCR primers were designed to cover the entire cathepsin C gene in overlapping fragments, from 955 nucleotides 5' to the start codon to 240 nucleotides 3' to the termination codon using cathepsin C (DPP-I) sequence data (Accession # U79415; SEQ ID NO: 1).
  • the PCR products were prepared for sequencing by excising the bands from the agarose gel and extracting the fragments using a Qiagen Gel Clean-up Kit.
  • the sense and antisense strand of each PCR product were directly sequenced on an ABI Prism 310 Genetic Analyzer (Perkin- Elmer) using four dye terminator chemistry.
  • cathepsin C is normally expressed in epithelium from palms, soles, knees and keratinized oral gingiva from unaffected individuals
  • the cathepsin C gene spans approximately 46 kb and consists of 7 exons. Sequence analysis of exonic, intronic and the 5 ' regulatory regions of the cathepsin C gene revealed PLS affected individuals from these families were homozygous for CTSC mutations that significantly altered the cathepsin C open reading frame.
  • TGG TRP codon
  • TAG terminator codon
  • the expected truncated protein is 428 amino acids. Although these families were not known to be related, the fact that affected individuals from these two
  • Papillon Lefevre syndrome is a palmoplantar keratoderma (PPK) with the characteristic clinical features of palmoplantar hyperkeratosis ⁇ and severe periodontal destruction.
  • the PPKs are a heterogeneous group of diseases all having gross thickening of the palmoplantar skin.
  • cathepsin C both localized to the refined PLS candidate interval on chromosome llql4 and was normally expressed in epithelium from sites affected by
  • cathepsin C is a large (200 kD) oligomeric protein that consists of four identical subunits, each composed of three different polypeptide chains [12,13].
  • CSC cathepsin C
  • CTSC is expressed in pituitary gland, spinal cord, aorta, left atrium, right atrium, left ventricle, right ventricle, inter ventricular septum, apex of heart, esophagus, stomach, duodenum, jejunum, ileum, ileocecum, appendix, ascending colon, transverse colon, descending colon, rectum, kidney, skeletal muscle, spleen, thymus, peripheral blood lymphocytes, lymph node, bone marrow, trachea, lung, placenta, bladder, uterus, testis, liver, pancreas, adrenal gland, thyroid gland, salivary gland, mammary gland, fetal heart, fetal kidney, fetal liver, fetal spleen, fetal thymus, and fetal lung.
  • the CTSC message is also expressed at high levels in immune cells including polymorphonuclear leukocytes and alveolar macrophages and is also expressed at high levels in osteoclasts [11,16].
  • the pathologic clinical findings of the PLS affected individuals studied here involve severe inflammation and destruction of the gingiva as well as hyperkeratosis of the skin from palmar, plantar and knee sites.
  • cathepsin C is normally expressed in epithelial tissues from sites clinically affected by PLS.
  • cathepsin C As a lysosomal cysteine proteinase, cathepsin C is important in intracellular degradation of proteins and appears to be a central coordinator for activation of many serine proteinases in immune/inflammatory cells [11]. It is unknown if the profound periodontal disease susceptibility is a consequence of altered integrity of junctional epithelium surrounding the teeth. It is interesting that once teeth are exfoliated, and consequently the junctional epithelium is eliminated, the severe gingival inflammation resolves . A more complete understanding of the functional physiology of cathepsin C carries significant implications for understanding periodontal disease susceptibility. Identification of cathepsin C gene mutations in PLS raises the possibility of creating an animal model to study the development, treatment and prevention of hyperkeratosis and periodontitis.
  • Haim Munk syndrome are associated with premature periodontal destruction.
  • PLS and HMS share the cardinal features of PPK and severe periodontitis, a number of additional findings are reported in HMS including arachnodactyly, acroosteolysis, atrophic changes of the nails, and a radiographic deformity of the fingers. While PLS cases have been identified throughout the world, HMS has only been described among descendents of a religious isolate originally from Cochin, India. Parental consanguinity is a characteristic of many cases of both conditions.
  • HMSl keratosis palmoplantaris with periodontopathia and onychogryposis
  • PLS Papillon-Lefevre syndrome
  • HMS family genotyping results All HMS affected individuals from the Cochin kindred were found to be homozygous for all three polymorphic DNA loci (D11S1887, D11S1780 and D11S1367) flanking the cathepsin C locus. Additionally, these individuals shared a common haplotype for these polymorphic markers. These findings are consistent with inheritance of both maternal and paternal copies of this genetic interval from a common familial ancestor ("identical by descent").
  • the cathepsin C gene spans approximately 46 kb and consists of 7 exons. Sequence analysis of exonic, intronic and the 5 ' regulatory regions of the cathepsin C gene revealed that HMS affected individuals from the Cochin kindred were homozygous for a mutation in codon
  • the Q286R mutation creates an Aval restriction cleavage site. We utilized this newly created restriction site to develop a rapid test to screen for the Q286R mutation.
  • Amplification of the wildtype sequence results in a 465 bp product that is not cleaved by Aval.
  • Amplification of the mutated (857A(G) sequence results in a 465 bp product that is cleaved by Aval to yield products of 404 and 61 bp .
  • individuals who are homozygous for the wildtype sequence exhibit a 465 bp band.
  • Heterozygous individuals exhibit 3 bands: 465, 404, and 61 bp bands.
  • Individuals who are homozygous for the Q286R mutation exhibit bands of 404 and 61 bp. Restriction analysis confirmed the sequencing results of all examined individuals ( Figure 9) .
  • a suitable assay for diagnosing this disorder includes the step of differentiating harmless polymorphisms from those mutations which give rise to PPKs and periodontal disorders. These include changes in the coding sequence which give rise to decreased mRNA stability as compared to wild type CTSC mRNA. Alternatively cathepsin C enzymatic activity can be compared between altered CTSC coding sequences and nucleic acids encoding the wild type enzyme.
  • assays are well known in the art and need not be set forth here. See for example, McGuire et al . , Archives of Biochemistry and Biophysics 295:280-8, 1992; McDonald et al . , J. of Biological Chemistry
  • Puliyel JM Sridharan Iyer KS .
  • Goldschmidt E (ed) The Genetics of Migrant and Isolate Populations. Baltimore: Williams & Wilkins 1963,-352.

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Abstract

Compositions related to mutations in the CTSC gene associated with palmoplantar keratodermas are disclosed. Also provided are methods for using the compositions of the invention in diagnostic and prognostic screening assays.

Description

METHODS AND COMPOSITIONS FOR DIAGNOSING PALMOPLANTAR KΞRATODERMAS AND DYSPLASIAS AND OTHER PERIODONTAL
DISEASES
Pursuant to 35 U.S.C. §202 (c), it is acknowledged that the U.S. Government has certain rights in the invention described herein, which was made in part with funds from NIDCR, Grant Numbers: DE11601 and DE12920.
FIELD OF THE INVENTION
The present invention relates to the fields of genetic screening and molecular biology. More specifically, the invention provides compositions and methods that may be used to advantage to isolate and detect a palmoplantar keratoderma predisposing gene, cathepsin C (CTSC) , some mutant alleles of which cause susceptibility to certain pathological disorders, in particular Papillon-LeFevre Syndrome, Haim-Munk Syndrome and certain forms of early onset periodontal diseases. More specifically, the invention relates to germline mutations and functional polymorphisms in the CTSC gene and their use in the diagnosis of predisposition to palmoplantar ectodermal disorders/ dysplasias and periodontal diseases. The invention also relates to the therapy of palmoplantar ectodermal disorders/dysplasias and periodontal diseases which have a mutation or functional polymorphisms in the CTSC gene, including gene therapy, protein replacement therapy and protein mimetics. The invention further relates to the screening of drugs for treating and alleviating disease symptoms. Finally, the invention relates to the screening of the CTSC gene for disease-related mutations, which are useful for diagnosing the predisposition to additional disorders and dysplasias, including but not limited to prepubertal periodontitis, early onset periodontal disease or other forms of gum disease. BACKGROUND OF THE INVENTION
Various publications or patents may be referenced in this application by numerals in parentheses to describe the state of the art to which the invention pertains. Full citations for these references are provided at the end of the specification. Each of these publications or patents is incorporated by reference herein.
Most forms of inflammatory periodontal disease can be successfully treated and managed. As a result, the ultimate goal of periodontal therapy has changed from that of simply arresting disease progression to one aimed at regenerating the supporting tissues. Unfortunately, not all forms of periodontal disease respond to treatment. Severe periodontitis that is resistant to conventional periodontal treatment has been recognized in a number of monogenic conditions. Certainly some of the most intriguing and dentally challenging of these conditions include Papillon-Lefevre syndrome (PLS) , Haim-Munk syndrome (HMS) and periodontal diseases .
In 1924, Papillon and Lefevre described two siblings, the products of a first cousin mating, with a condition characterized by diffuse transgradient palmoplantar keratosis (PPK) and the premature loss of both the decidous and permanent dentitions. This condition came to be known as Papillon-Lefevre syndrome and subsequently over 200 cases have been described. The hallmarks of PLS are palmoplantar keratosis and rapid periodontal destruction of both dentitions. An increased susceptibility to infection has been reported in approximately 20% of PLS patients. Additional findings include intracranial calcifications, retardation of the somatic development, follicular hyperkeratosis and onychogryphosis . Clinical findings reported in PLS patients suggest that the clinical expression of the condition is a. highly variable. Unfortunately, to date, no pathognomonic disease marker exists allowing definitive diagnosis of PLS.
In 1965, Haim and Munk described an unusual syndrome in four siblings of a Jewish religious isolate from Cochin, India [21] . In addition to congenital palmoplantar keratosis and progressive early onset periodontal destruction, other clinical findings shared by these individuals included recurrent pyogenic skin infections, acroosteolysis, atrophic changes of the nails, arachnodactyly, and a peculiar radiographic deformity of the fingers consisting of tapered pointed phalangeal ends and a clawlike volar curve. Subsequently pes planus was reported to be associated with the syndrome [24] . This was the first reported association of these clinical findings, and the condition became known as Haim Munk syndrome, or keratosis palmoplantaris with periodontopathia and onychogryposis (HMS; MIM245010) [22] . Although the palmoplantar findings and severe periodontitis were suggestive of the
Papillon-Lefevre syndrome (PLS; MIM245000) [3 ] , the association of other clinical features, particularly nail deformities and arachnodactyly, argued that HMS was a distinct disorder. PLS and HMS are classified as type IV palmoplantar ectodermal keratodermas [2] . The unique presence of severe, early onset periodontitis distinguishes PLS and HMS from other PPKs and raises the question of whether they result from the variable clinical expression of a common gene mutation, are allelic mutations at the same genetic locus, or result from expression of gene mutations at separate loci. Although Haim and Munk ' s initial report proposed HMS was a distinct entity, Hacham-Zadeh and co-workers referred to the disorder as Papillon-Lefevre syndrome and cited Gorlin's suggestion that HMS was a clinical variant of PLS [23,25] . In his review of PLS cases reported in the literature, Haenke [5] summarizes an extensive list of clinical findings reported in PLS affected individuals, including increased susceptibility to infections, ectopic cranial calcifications and nail anomalies [5,26] . It is unclear if these additional clinical features are coincidental findings that may be segregating in a particular family or if they are etiologically related to a syndrome with a very variable clinical expression. Because PLS is an uncommon condition, and generally occurs only in a single generation it is difficult to determine if these occasional reports of associated clinical findings are etiologically related to PLS. Additionally, consanguinity is common among parents of PLS cases and therefore, it may be expected that an increased number of rare recessive conditions may be seen. Such is likely the case for the reports of mental retardation associated with PLS [5] .
Pre-pubertal periodontitis (PPP) is a rare and rapidly progressive disease that results in destruction of the periodontal support of the primary dentition. The condition may be localized (usually to deciduous molars) or generalized. The localized form begins at approximately 4 years of age and is associated with only mild gingival inflammation in the presence of relatively little plaque. The generalized form begins earlier, immediately after eruption of the deciduous teeth. It is associated with severe gingival inflammation and hyperplasia, although significant gingival recession has also been described as an associated clinical feature. The attachment loss appears to be continuous rather than intermittent as with most other forms of periodontitis. A varied clinical phenotype has been reported for PPP, probably reflecting the fact that the term PP describes an etiologically heterogeneous group of conditions that share an overlapping clinical presentation. Although PPP can occur as an isolated finding, many reports of PPP describe an increased systemic susceptibility to infections. Children with PPP have frequent inner ear infections and infections of the respiratory tract [39,40]. Prepubertal periodontitis is known to be associated with Papillon Lefevre syndrome and with a number of systemic disease states that share an increased susceptibility to microbial infections.
To date, no pathognomonic disease marker exists for most PPKs allowing for definitive diagnosis. The present invention provides such a disease marker and methods of use thereof having diagnostic and prognostic utilities for several PPKs and many periodontal diseases .
SUMMARY OF THE INVENTION The present invention provides compositions and methods which allow for genetic screening and diagnosis of certain palmoplantar keratodermas and periodontal disease states in affected individuals. In accordance with the present invention, it has been discovered that mutations or functional polymorphisms in the cathepsin C gene (CTSC) give rise to certain pathological conditions including PLS, HMS and periodontal diseases. Mutations or functional polymorphisms associated with the disease state are those which give rise to a altered, truncated, misfolded or otherwise non-functional CTSC polypeptides. Polymorphisms in the CTSC sequence which do not affect the nature of the encoded protein are not associated with PLS, HMS or periodontal disease.
Thus, in one embodiment of the invention, a method is provided for determining the presence of alterations in CTSC encoding nucleic acids which give rise to altered CTSC proteins. The wild-type CTSC nucleic acid sequence and its encoded amino acid sequence are known.
See SEQ ID NOS: 1-3 provided herein. This sequence information facilitates the identification of genetic changes that give rise to aberrant CTSC proteins.
CTSC mutations specifically associated with PLS, HMS and PPP are described herein and are set forth in Table 1. Accordingly in one embodiment of the invention, nucleic acid molecules encoding altered CTSC proteins are considered to be within the scope of the present invention. In a preferred embodiment of the invention, the altered CTSC nucleic acid has at least one of the alterations set forth in Table 1.
In a further embodiment of the invention, nucleic acid probes which specifically hybridize to the human altered CTSC-encoding nucleic acids described herein and not to wild-type CTSC encoding nucleic acids are provided. In a preferred embodiment, the probes specifically hybridize with altered CTSC encoding nucleic acids having at least one of the alterations set forth in Table 1.
In yet another embodiment of the invention, a mutated CTSC protein encoded by the altered CTSC encoding nucleic acids of the invention is provided. Preferably such CTSC proteins are encoded by a nucleic acid containing a mutation as set forth in Table 1. Also provided are assays for biochemically assessing altered cathepsin C activity. Antibodies immunologically specific for altered CTSC proteins are also contemplated to be within the scope of the present invention. In another aspect of the invention, a method for detecting a germline alteration in a CTSC gene is provided. In a preferred embodiment the alteration is selected from the group consisting of the alterations set forth in Table 1. The method comprises analyzing a sequence of a CTSC gene or CTSC RNA from a human sample or analyzing a sequence of CTSC cDNA made from mRNA from a human sample and comparing sequences so isolated to the wild type sequence encoding CTSC. Inasmuch as certain alterations of the CTSC coding sequence may not alter the function of CTSC, methods are provided for assessing the enzymatic activity of proteins encoded by nucleic acid molecules which do not possess the wild type CTSC sequence.
In yet another embodiment of the invention, kits are provided for detecting the presence of an altered CTSC encoding nucleic acids in a biological sample. An exemplary kit comprises the following: i) oligonucleotides which specifically hybridize with CTSC encoding nucleic acids having the alterations set forth in Table 1; ii) reaction buffer; and iii) an instruction sheet. Kits for detecting the presence an altered CTSC proteins in a biological sample are also provided. Exemplary kits for this purpose comprise: i) antibodies immunologically specific for the altered CTSC proteins of the invention; ii) a solid support with immobilized CTSC antigens as a positive control; and iii) an instruction sheet. Optionally, anti-CTSC antibodies used for this purpose may contain a detectable label or tag for used in isolating or detecting immune complexes .
Various terms relating to the biological molecules and cells of the present invention are used throughout the specifications and claims.
With reference to nucleic acids used in the invention, the term "isolated nucleic acid" is sometimes employed. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it was derived. For example, the "isolated nucleic acid" may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a procaryote or eucaryote. An "isolated nucleic acid molecule" may also comprise a cDNA molecule. An isolated nucleic acid molecule inserted into a vector is also sometimes referred to herein as a Arecombinant@ nucleic acid molecule.
With respect to RNA molecules, the term "isolated
'1 nucleic acid" primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a "substantially pure" form (the term "substantially pure" is defined below) .
With respect to protein, the term "isolated protein" or "isolated and purified protein" is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in "substantially pure" form.
The term "substantially pure" refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like) .
With respect to antibodies, the term "immunologically specific" refers to antibodies that bind to one or more epitopes of a protein of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.
With respect to single stranded nucleic acids, particularly oligonucleotides, the term "specifically hybridizing" refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed "substantially complementary"). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single- stranded nucleic acids of non-complementary sequence. Appropriate conditions enabling specific hybridization of single stranded nucleic acid molecules of varying complementarity are well known in the art.
For instance, one common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is set forth below (Sambrook et al. , 1989) :
Tm = 81.5°C + 16.6 og [Na+] + 0.41(% G+C) - 0.63 (% formamide) - 600/#bp in duplex
As an illustration of the above formula, using [Na+] = [0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the Tm is 57 °C. The Tra of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42 °C.
The term "promoter region" refers to the transcriptional regulatory regions of a gene, which may be found at the 5' or 3 ' side of the coding region, or within the coding region, or within introns . In the present invention, the use of SV40, TK, Albumin, SP6, T7 gene promoters, among others, is contemplated. Specific promoters for the yeast and mammalian expression systems of the invention are available and known to those of ordinary skill in the art.
The term "operably linked" means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to enable expression of the coding sequence. This same definition is sometimes applied to the arrangement of transcription units and other transcription control elements (e.g. enhancers) in an expression vector.
The phrase "functional polymorphism" refers to a change in wild type CTSC coding sequence giving rise to altered cathepsin C activity as assayed using conventional methods.
The term "oligonucleotide," as used herein refers to primers and probes of the present invention, and is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide.
The term "probe" as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. The probes herein are selected to be complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to "specifically hybridize" or anneal with their respective target strands under a set of pre-deter ined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5' or 3 ' end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically.
The term "primer" as used herein refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis. When presented with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and pH, the primer may be extended at its 3 ' terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield an primer extension product. The primer may vary in length depending on the particular conditions and requirement of the application. For example, in diagnostic applications, the oligonucleotide primer is typically 15-25 or more nucleotides in length. The primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able anneal with the desired template strand in a manner sufficient to provide the 3 ' hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer
I I sequence represent an exact complement of the desired template. For example, a non-complementary nucleotide sequence may be attached to the 5 ' end of an otherwise complementary primer. Alternatively, non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A-1E are a series of clinical photographs showing palmoplantar keratosis and periodontal disease in PLS study patient. Fig. IA: palmar hyperkeratotic lesions; Fig. IB: plantar hyperkeratotic lesions; Fig. IC : hyperkeratotic lesions affecting the knees; Fig. ID: periodontitis involving erupting permanent dentition and Fig. IE: periapical radiographs showing severe alveolar bone loss affecting erupting permanent teeth.
Figure 2 shows haplotype data for chromosome llq short tandem repeat polymorphisms (STRP) markers spanning the PLS gene locus . Segments which are likely to be homozygous by descent are boxed. Arrows indicate recombinant events. Individuals 7 and 22 share a common haplotype for D11S1979, D11S1887, D11S1780, D11S1367, D11S931, and D11S4175.
Figure 3 depicts pedigree and sequence analysis of
CTSC exon 6. The numbering of the wildtype sequence shown above the figure is based upon the genomic sequence of CTSC. See SEQ ID NO : 1. Circles represent females and squares represent males. Filled symbols indicate affected individuals. Half-shading indicates carriers based upon DNA sequencing results. All affected individuals are homozygous for the specific CTSC mutations. Arrows indicate the position of the mutation. This family has a nonsense mutation (856 C->T) at codon 286 resulting in a truncated protein of 286 amino acids.
Figures 4A - 4D show pedigrees and sequence analysis of CTSC exon 7 for 4 Families with PLS. Symbols are as described for Figure 3. Fig. 4A: Family with a single base pair deletion (1047delA) of CTSC resulting in a frameshift and premature termination. Fig. 4B: Family with a 2bp deletion (1028-1029delCT) of CTSC resulting in a frameshift and premature termination. Fig. 4C . and Fig. 4D: pedigrees of families with a nonsense mutation (1286G->A) at codon 429 resulting in a truncated protein of 428 amino acids. The father in family C is deceased and no sample was available for analysis.
Figures 5A-5C is a schematic diagram of the CTSC gene showing the locations of the mutations described herein. Panel 5A. Genomic structure of CTSC gene with introns shown as solid lines and exons depicted as boxes. The 5' and 3' untranslated regions are shown as filled boxes. Panel 5B. Coding region of CTSC gene. The amino acid numbers are shown at the end of each exon. Mutations listed in Table 1 are shown according to their genomic locations with Missense, ^; Nonsense, ♦; Insertion, ■ ; and Deletion, □. The splicing site mutation is indicated by an arrow. Panel 5C. Subunit structure of CTSC polypeptide with SP, signal peptide; Pi, 13.5 kDa pro-region; P2 , 10 kDa pro-region; H, heavy chain; and L, light chain. The 10 kDa pro-region is cleaved out upon activation. The disulfide bond within the 13.5 pro-region is shown. The glycosylation sites are indicated by filled circle and arrows indicate the active sites.
Figures 6A-6F show a series of micrographs
>3 depicting the clinical and radiographic findings in Haim Munk syndrome. Fig. 6A: dermal involvement of fingers in individual #34. Fig. 6B: Individual #34 radiograph of terminal phalanges of the fingers showing marked thinning increasing towards the distal, tapering pointed ends showing a claw-like volar bend. Fig. 6C : Individual #17 palmar keratosis; Fig. 6D: Individual #17 plantar keratosis: Fig. 6E: Individual #17 gingival inflammation; Fig. 6F: Individual #17 radiograph showing alveolar bone destruction associated with gingival inflammation shown in 6D.
Figure 7A shows pedigree of Cochin descendents segregating Haim Munk syndrome (HMS) . Numbered individuals have been analyzed for the current study.
Circles = females, squares = males, shaded symbols = HMS affected individuals. Double lines = consanguinity. Individuals #10,11 * = second cousins. Numbers inside circles, squares and diamonds indicate the number of additional offspring not examined in this study.
Sibships described in previous reports are indicated and referenced below the pedigrees. The subjects of Haim and Munks original report (1965) are individuals 33, 34, 35, and 36. Half-shading indicates carriers based upon DNA sequencing and/or restriction enzyme analysis.
Unshaded numbered individuals represent non-carriers based upon DNA analysis. Figure 7B shows a pedigree of a Turkish family segregating PLS. Numbered individuals were available for study. Half-shading indicates carrier based upon DNA analysis. Individual 77 is a non-carrier based upon DNA sequencing.
Figures 8A and 8B show the results of sequence analysis of exon 6 of CTSC. The numbering of the wildtype sequence is based upon the cDNA sequence of CTSC. See SEQ ID NO : 1. Fig. 8A: Family A (Cochin isolate diagnosed with Haim Munk syndrome) from Figure 1. Affected individuals are homozygous for a 857A->G missense mutation which results in a conserved glutamine being changed to an arginine (Q286R) . Representative sequences are shown for individuals #36 (affected) and #31 (carrier) . Fig. 8B. Family B from Figure 1.
Affected individuals are homozygous for a 856C->T nonsense mutation which results in a premature stop codon at position 286 (Q286X) . The Q286X mutation has been previously reported in an inbred Turkish family [12] .
Figure 9 depicts a gel showing the results of restriction enzyme analysis of Q286R mutation in Family A of Fig. 7. A 465 bp fragment of exon 6 was amplified and subjected to restriction digestion with Aval as described under methods. The Q286R mutation introduces a new Aval site. After digestion and electrophoresis through 1.8% agarose gels, wildtype individuals exhibit bands of 465 bp, affected individuals have bands of 404 and 61 bp, and carriers have bands of 465, 404, and 61 bp. M. 1 kb ladder (Gibco) . Lane 1. Individual #5 uncut, demonstrating 465bp amplicon. Lane 2. Individual #5 cut with Aval. Only the 465 bp fragment is observed. Thus individual #5 has the wildtype sequence on both alleles. Lane 3. Individual #31 uncut. Lane 4. Individual # 31 cut with Aval. The 465 and 404bp fragments are visible, confirming that individual #31 is a carrier of the Q286R, consistent with the sequencing results shown in Figure 3A. Lane 5. Individual #34 uncut. Lane 6. Individual #34 cut with Aval. The 404 and 61bp fragments are indicated by arrows.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates generally to the field of human genetics. Specifically, the present invention relates to methods and materials used to isolate and detect mutated forms of the lysosomal protease cathepsin C (CTSC) gene, associated with autosomal recessive disorders characterized by palmar hyperkeratosis and/or periodontitis. More specifically, the present invention relates to germline mutations in the CTSC gene and their use in the diagnosis of predisposition to such pathological conditions. Additionally, the invention relates to germline mutations in the CTSC gene in other palmoplantar ectodermal disorders and dysplasias and their use in the diagnosis and prognosis of such pathological conditions. The invention also relates to the therapy of palmoplantar ectodermal disorders and dysplasias which have a mutation or functional polymorphism in the CTSC gene, including gene therapy, protein replacement therapy and protein mimetics. The invention further relates to the screening of drugs which may have therapeutic value. Biochemical assays are provided for the assessment of altered activity of aberrant CTSC enzymes encoded by the mutated CTSC encoding nucleic acids of the invention. Finally, the invention relates to the screening of the CTSC gene for mutations, which are useful for diagnosing the predisposition to ectodermal disorders and dysplasias.
The present invention provides an isolated polynucleotide comprising all, or a portion of the CTSC locus or of a mutated CTSC locus, preferably at least eight bases and not more than about 100 kb in length. Such polynucleotides may be antisense polynucleotides. The present invention also provides a recombinant construct comprising such an isolated polynucleotide, for example, a recombinant construct suitable for expression in a transformed host cell.
Also provided by the present invention are methods of detecting a polynucleotide comprising a portion of the CTSC locus or its expression product in an analyte. Such methods may further comprise the step of amplifying the portion of the CTSC locus, and may further include a step of providing a set of polynucleotides which are primers for amplification of said portion of the CTSC locus. The method is useful for either diagnosis of the predisposition to PPKs or the diagnosis or prognosis of keratodermal disorders/dysplasias and periodontal diseases .
The present invention also provides isolated antibodies, preferably monoclonal antibodies, which specifically bind to an isolated polypeptide comprised of at least five amino acid residues encoded by the altered CTSC locus.
The present invention also provides kits for detecting in an analyte a polynucleotide comprising a portion of the CTSC locus, the kits comprising a polynucleotide complementary to the portion of the CTSC locus packaged in a suitable container, and instructions for its use.
The present invention further provides methods of preparing a polynucleotide comprising polymerizing nucleotides to yield a sequence comprised of at least eight consecutive nucleotides of the CTSC locus; and methods of preparing a polypeptide comprising polymerizing amino acids to yield a sequence comprising at least five amino acids encoded within the CTSC locus. The present invention further provides methods of screening the CTSC gene to identify mutations. Such methods may further comprise the step of amplifying a portion of the CTSC locus, and may further include a step of providing a set of polynucleotides which are primers for amplification of said portion of the CTSC locus. Exemplary primers are set forth in Table A.
The method is useful for identifying mutations for use in either diagnosis of the predisposition to keratodermal disorders/dysplasias and periodontal diseases or the diagnosis of such disorders.
The present invention further provides methods of screening suspected CTSC mutant alleles and functional
) '7 polymorphisms to identify mutations in the CTSC gene. In addition, the present invention provides methods of screening drugs for therapy and to identify suitable drugs for restoring CTSC gene product function. Finally, the present invention provides the means necessary for production of gene-based therapies directed at aberrant cells associated with keratodermal disorders and dysplasias. These therapeutic agents may take the form of polynucleotides comprising all or a portion of the CTSC locus placed in appropriate vectors or delivered to target cells in more direct ways such that the function of the CTSC protein is reconstituted. Therapeutic agents may also take the form of polypeptides based on either a portion of, or the entire protein sequence of CTSC. These may functionally replace the activity of CTSC in vivo.
It is a discovery of the present invention that mutations in the CTSC locus in the germline are indicative of a predisposition to keratodermal disorders/dysplasias and periodontal diseases. The mutational events of the CTSC locus can involve deletions, insertions and point mutations within the coding sequence and the non-coding sequence.
A major gene locus associated with the keratodermal disorders and dysplasias of the invention has been localized to a 2.8 cM interval on chromosome llql4 of the human genome. This region contains a genetic locus, CTSC. The CTSC message is expressed at high levels in a variety of immune cells including polymorphonuclear leukocytes, macrophages and their precursors. This gene is expressed in the palms, soles, knees, and oral keratinized gingiva .
The CTSC gene was originally reported to consist of 2 exons . US Provisional Application 60/165,016 from which the present application claims priority, describes mutations in Exons 1 and 2. The mutations described actually fall within Exons 6 and 7. Reference numerals to the altered amino acids are the same as those in US Provisional 60/165,016, only the nucleotide numbering has changed to reflect the actual genomic structure of the CTSC gene which is now known to contain 7 exons. The sequence encoding the wild type human CTSC gene is provided below (SEQ ID NO : 1) :
AGGGAGATAT AAGTGAATAA TTTGGACCTG CTCTCTTTGA ATGTTTATAA TCTGGTGGAA AAAAAATGGA CATATGAATA TTGATTTGTG ACCAGTGCAA AGGGGGCAAA AATTCATATC CCAAAGAAAA CGGGGACACA TCAGGTCTGT CTTGTTCATC ACTGTGTCCA CAGGGCCTGA CACCTAGTAG GCTCAGTGGG AGAAAGGAGC CCCAATTACC AACAAAAGCC AGGAAAGAAC GGGAGGCTCT TACGGAAAAG GGTGATACTT AAACTGAGCA AGGAGGCACC TGGAAATAGT GCCACCTAAT AATTTTTGGC GATCAGACTG ACACACTAGA ACGGTTCATA AGACCAGCCT TCTCCCATTG GCTAGCTTCC TTCCTCACCC TTCTCACCCT GGGCAAGCCG CTTCCTCTCT CTGGGCCTCT TGCTTTTCCT CTGTAACATA AAAGGGGTTG AGCAATATCA TCTCTGAGAG CGCCATGTGT GTGCGTGCCA GAGGGAAAAC CCCCACAACG CTAATACATC AAAACTGCAG GTTTGCACAA AAACTGAATT CTGCTGAATG CAAACAGGCA AACAGCATTT ACCAGGAAAC AAAACAAAAT CAAGCACATA AAAAAGTAGG AAGAGTTGGA AAACGGAAGG AAGATAAGTT CTCAAACAGC TGGAATAGTT GATGTTAGCT AGCGAAGTTT TTCAGAGGAA AAAACAAGAA GTTGGTTATG AGGCAAGTGG ACCTGAGAAA AAAGACTAAA GGGGAAGAAT AGCAAGTAAA ACAGAACTCC ACTTGCTAGA TCTCTCCCTC TGTCGCGCTC TTTCACCTGA CCCACTCCCT TATTCCCCCC ACACCCTTTC CTTCTCTCCC TACGTTACCG CACAGGAACG AAGTCTGGGT CATGTGCGGA CCGCTTGTGG CTCTTAAATC CTCTTTTTGT CACCCTGGCC GTGCAAAATT TTGAAACGTC CCTCGGCAAA AAAAATAAAA ATAAAAAAAA AAAATCTGTC CCTGGCCTCT TCCCTAGTTC TGGGTCCAGT TGCAGCCAAG TGAGGGGCAG CGCGCGCTCC CAAGTCCCCG
TTTCAGAGAC GCGCACGCGC CTGGCGCCCA ACCCCCAATC CCCTGCTGCT CAGTGACCCC GCCCACGGGT TTCCGGGCCG GCGTAGCTAT TTCAAGGCGC GCGCCTCGTG GTGGACTCAC CGCTAGCCCG CAGCGCTCGG CTTCCTGGTA ATTCTTCACC TCTTTTCTCA GCTCCCTGCA GCATGGGTGC TGGGCCCTCC TTGCTGCTCG CCGCCCTCCT GCTGCTTCTC TCCGGCGACG GCGCCGTGCG CTGCGACACA CCTGCCAACT GCACCTATCT TGACCTGCTG GGCACCTGGG
TCTTCCAGGT GGGCTCCAGC GGTTCCCAGC GCGATGTCAA CTGCTCGGTT ATGGGTAAGC CGCCGGCTCG GCAGTCCTCC GGGTCGTCCT TTCTGCCCTT GAGCCCCTAA CGCAGCGCCA CGCCAACTAC CGCTTCCCCC CAGGCAGACG CTTGTGGGTG GCCAGAGCAT CTTGACTGGA TTCGGGGACC CTTGGGGACC TTCTTCCCCG CCAGGCTCGC GAAGTTAAAG TTCATCTGCT GAGAACTTCT AACTCCACAC TTTCTTGGTT ATCTTGGGGA CTCAACACTT TGATCAAGAA CTTTTTTATT CCTCCCGCTT AATTTTGTTT GCTTTGAGAG AGACTTGGGA ACTGCAATCG TTTGGTTCTC CAGTCCGATC TGGTAGCGTT ATTTTTAAAA TTTATTTTTA TTTTTTATTA CTATTTTACT AGTGAAGATA GATGAGCTCA GAGACTCTCG AGGATATAGC ATGAAGTTTT CTCTTTTTGT TAGATGGTGG GAAAGGGACT TTCTGCCCAG CGATTTTGGT TTGAGCGGGT GTTGATGAGT ACTAGAAAAC GGCTAGTACC ACTCTGCATT GTTTCATGCA TTGCAAGGAG GTAAAAATTT TTTAAAAAAT TAATAACAAA GAAAACTTAA CTCTGAACCT AGTAATTAGA AATGCCCAGA GTCTGCACAA TGTTTGGCTC ATGGAAGGCT CTCAATAAAT ACCTAGTGTT TGAACATACT GGAGATATTC CATATGCCTT CAGATAACAT GGTTACCCCT AGAACAAAGA ACCTAGTGAA GGGGGTGGGG TGGAAAAAAG ACAATCACTA GTTAGAAGAG TCACACTGTG
GTCTACCCAA ACCCTCTTAC AGCCTGTGAC TTCTGCAGAG TCAGTAAAAA ATCAGCTATA
ATTTTCTTGT CAACAGAACA AAGATTAAGT CATTTGTTAC TAAAGAAATA CCTTTTTAAC
TGACTGTATA GCATTTCATA ATCCTGAACA TTGTAACTTT TTTTTTTTTT TTTTTGAGAA TGGAGTCTTG CTCTGTCACC CAGGCTGTAG TTCAGTGGCG GGATCTCGGC TCACTGCAAC
CTCCGCCTCC CGGGTTTAAG CGATTCTCCT GCCTCAGTCT CCCGAGTAGC TAGGATTACG
GGCACATGCC ACCACGCCCG GCTAATATTT TGTATTTTTA GTAGAGACGG GTTTCACTGT
GTTAGCCAGG ATGGTCTCCA TCTCCTGACC TCGTGATCCG CCCGCCTCGG CCTCCCAAAG
TACTGGGATT ACAGGCGGGA GTCACCACGC CTGGCCTTTT TTTTTTTTTT TTTTTTAAAT TTGAGTTTGA AGGTTGGGGC AGAAAGAGAT CAGGATTTGC ACTGCCCTGT CACATGCAAT
CTCCCATGTC AGAGCATTAC TCTCAAACAT GGAAAAACTT TAAAATACAC AACTCTCCAG
ATGGACACAG CTGAATCATT TTCAGGAAGG CTCTGCCTAT AAAATTACCT TTGGCCTTAA TTCAGTAATT AAAGGCACCC ACAGGTCTGA GCCCTCATCT ATGAAGGTTA AACGTATCAT
TCCTATCAGC ACTAATTGGT TTTATAGGAT ACAGACCTCT AGTTTGCTGA ATAAACTTTG GAAGGATTCA GATTATGAGG TTCTAAACTG TAGAGCCTTT GAGGAGAAAG GTACCCCATT
TCTTCTCCGA ATAGATCATT TTGTGTCTTC TCCTGGGCTT AGCACATTGT CTTCCTTAGG
ATCTGATAGT CTGTTTATTT ATCTTTTTGT TGTCATACCT TTTATTCTTG CATTTCCCAC
TTTTGACTAG CATTTTGCCT TTTCTCCGTT TCTGGAAGCC TGTAATTTTC AACATTCCCC
ATTTCTCCTT TTGTTCAGTG GAAAAATTTC TTCAGTGTTG GGATGCTCTG GAAAGGTACT GTAGTTTTGG GGGTCTCCCT GCCTGCTGGG CTGCATAGCA TATCTCTCTT TTTTGAGACA
AGGTCTCACT GTGTTGCCCA GGTTGGAATG CAGTTGTGCA ATCATAGCTC ACTGCAGCCT
CCAACTCCTG GGCTCAAGCA ATCCTTCCAC CTCAGCTTCA GCCGCTCAAA TAGCTGGGAC
TACAGGCAAT GCACCACCAT AACCAGCGAA TTTTTAAAAA TTATTTTTGT AGAGACAGGG TCTCACTATG TTGCCCAAAC TAGTCTTGAA GTTCTGACCT CAGCCCCGCA AAGTGCTAGG ATTACAGGCG TGAGCCACTG CTTCTGCTTG CATTATCTCA AATTTTTAGA GCCTAACTTC
ACAGTCTTGC CTCGGAGCAG GAACTGTTTG AGGCAACAAG ATGGAGCCTC TGGTTTCTTC
ACTAGGCAGA CTGTGCTCAA ACTGGGTAGC ATGAAAGGAA AGCAGCACAA TTAAAATTGA
AATTGGGGGA TTCTTTCCCC TATTGAAATC CAAATAATTT TCCTTTATTG TGCTTTTTTT
TTTTTTCTTT AGGACCACAA GAAAAAAAAG TAGTGGTGTA CCTTCAGAAG CTGGATACAG CATATGATGA CCTTGGCAAT TCTGGCCATT TCACCATCAT TTACAACCAA GGCTTTGAGA
TTGTGTTGAA TGACTACAAG TGGTTTGCCT TTTTTAAGGT TAGTTTTGTT GGAAGTTGGA
TTTACATTTT CAATGATTTG ATATCTGAAA CCTCTTCTGA TTAGTAGACC CTCAGAATTT
TAATTTTAGA TTAAGAAGAT GACCGGAATT GACACCACTC TTCCCAAGAG TAGTAGTAGT
TGGTATAATT TTGCCACTTT ATTTAAAATT ATGATTTTAG TAGGTTTACA AAATACCAGT GCTACATTTG AATATGTATA AATTATGTTT AAAATTTACA TTTTGGTAAG TATGACTAAA
TTCTTAATTT ATTTTCCTTA TTACTACCAC TTTATTTCTA AATGTTGCCA TAGTCATTTG
GCTTTGTTCT AAATCTGTAG GAAAGATAGA GAGATTACAC ATTTTGTTTT CTTGCAGTTA
CTATGCTGTC CTTCCTATCA CTACCTGTTG GCTGAGGTAG TGATAGGCCT AAATGATTCA
TTATCTTAAA TGTACTAAAT ATGTTGAGTA ATTTTTTCTT CTAAACTAAC AGAAAGAGAG AACCTAGGAG TTACTCCCTT AGGCTGGTTA AAGTGAAAGG TAGCCAAGTC AACCCAGCTT
GTTTCCTTCT CTCATTAGGA AAGAACTATT GTTCATTCTC ATAACACACT TTTTCCAATT
GCAAACATAC TCAGGGTTAA AATAGTTTAG CACAAATTGC AGCCCATTTC ATTTGTTCTT
CACAAGCTGG AACTTTTCTT GTAAGCTAAA TATTAAATGG TTCAAGTAAA TTGGATACAT
AAGCCTGAAA CTAGGCGTTT CTCATTATAC ATAGAGTATA AATTAAGACA GACTTTTTCA
_ U TGGTGAAAGG TTTACAGCCT TTAAAACATC TGGGAAGAAG TGGGAAAGTA GGGAATAACT
CTGTTAAATA TGATAAAAGA CAAAGCACCA ACAAAGGCCT AGTTCTAAAC TTGTTATAAT
TTCTCATGGG AGTTGTGGTT TGTCACAAGG TTATGGCGGT CCAAGCAAGT TTACAATATT
TTTTAGAATA ATAACTCCCA GAAATATTTT TAAAATAAGG ACCTTTCTTT AATATGGAAA AAAAAAAGAT GAAATAGAGA GGAAGAGGTT GCCTTTCCTC AACTGGATTT GCTGGTGTGA
TATGCAGCTG GTGGAAACCA GTTTGTCCAT GCTAGCTGTT GATGGCCACC AGTCCACTGA
ATTAGTGGAA ACCCATGCTT AACCAGTTGT TAACTATTTT TAGTATCATT TCCAGATGCT
ACGCATTTTT TTTGTAAAAA ACATACTTAC AATAGTATCT ATATAAATTT TTAAAATTGT
AATGTATATG TGGTATATTT AACCTGAAAA TAATCTTTGG TGTATACGTT GATGAGCCTA GGCCTTTGCA GACCTCTTAC AGTTACTCTG TCACAGCCCT TCAAAGGTTC TTTGACTTCA
GATTAAGAAT CAATTGCATG TGGAATGCAT GTGCAAAGGA AGAGATATTC AAGGCAATTG
TTACCATGTA CCATAAACCT TGTACATAAT TTTTTGTCAT TTTCTTTTCC TCTGTCTCTA
TCCCTTCTTC TTTGACACAC AATAGACACC TAGTCAATTT ATTACAAAAA AAATGAATGA
ATGAAGTGGA ATTCAGTTGG GAAATAGGTT AAATAAATTA TTTAGGAGAT GAGGAATAGG TAAAAAGAGA CAAGTACATA GTTTATTCTT TTGACTTAGA AAACTTTTGA TTCTTAAATT
CTGCAGAATT GGAGAAACTG GTGGGGAAAC TTCTAAAATC ATTATTTAAT TACCAGAGAT
GTAATAGATA TAGACAAAAG CAGTTTTCTT CTTTTATTAT TTTTTCATCA GTTAGTTCTT
AGCTTAAATA GTAGTCCAAA GCTGGTAGGG ACAGAGGGAA TTAGCTGGTG GCTGAATGAG
GAATTGTATC ACTTTTTGTG AATCACGGTG TAAGCACATT TGGTGTTTTG CCATTGCCTA AGAACATTAG TCACATTAGG TCAATAGAAA ATCACTTTTT AAAGCCAAAT AAAGTTATAT
GTGTTCCCAA CCATGAGTTG GAAAGAATTA ATATATATGC TGTTGGAGGG TAGAACCCTG CCTAATCATA TGGTTCTGGA TGGCATTGAT CGAATCCTTA TTCTTTCATT AGGAATAACA ATAGAAAAAA TACTCCTGCC CTACTGATTT CAGGATATGT CTATTTTAAA GTGCCCATTT GACAAAACCA TTATCAGGGC CATGTTTTCT TTTTCTGCAG AAAAATCAAC CACTCTGGTC AGTAGTTAGG TCTTATGACA AGCACCATAA TTTCCTTAGG CAGAGTAGAA TATAATAGGA TACTTCTTTT TGAAACTTAA TATAATCAGG TAGTTCCAGA TAAACATAGC TTGCAAAGTG ATAAAATACC ATGTTATTTT AGTAAATCCA ATTGCAAGAG TGATGGGAAA CAGAGTTTAA AAACTTAAGA AAGATATTAA GATGGAGTTG ACTTTGAATA ATAAAGTCAT CCACTGTTGA TGGGTGACAT TTATTAATAC AGAAAGTTTA CAGATTTTAC CATAAGCATC AGGGTATTTC CTGCAGCTGG GGAAACCGTG CTTGAAAGGA TGCGTAACTC AAGGAAAACA CAAGCCCATG
TAATAAGTAT TGCATGTGAG AATTGTTCCT ATAGAATTAG AAAGCATCTT TTACATTAAA ATTTATTTTT GTAAAAAGAG AAAACATCAA AACTTGAGTA GTATTTGCTA TTCAAAGAGT CTTACATAAA CGAAAACATA CTCAACCTAC TGCCATTACA GAAATATTTG ACAAATTCTT GCCATGCTTA CCTGCCATCG TTGTTGTTAT CCTACAAATC AATTGGATTT TCACGCCTCT CCACTGACTG GAACCCTACA ACTTGCTTCC TTTTATCCTC TTTATATATG CTTCAGATAT GCTTGAAGTA GATTGTTTCT TATTGTTCTT GCTGCTCGAG TTTGTTGAGT AGTTGGTACA TGCAGAGTAT TTGTATGTTA TGACATATAT AGGTTTTGTC CATGGTTCCT GGCTCATAAC TCACTCCCAA GACCCTTGTT ACAGAAACCA GAATCTCTCT CTCTGATCTT CTCCTACCCT CCTTTCATCT GCCCACTGCA GAACTCTAAT CTGATTATGG TTTCTAAGAC CCTCATACCA GAGAGTATTC TGCCCCATAC CATAGCAGAA GGAACACTGC ACAGAGACAC CAAGAAGAAT
CTGAACAGAC AGGCCTTGTT AGGTTTAGAT CATGTCCTTA TAACCTAATT ATATTTTAAC
ATGGTTATCC ATGCTTTAAT CATGTGTATT CAATGAACGC TCCATAAAAG CCCAAGAAGA
ACAGATTTGA GGGAATTCTG AAGCGCTAAA CATGTAGGGT CTGACAGGAA GGTGAAGAAG
AACTCATCAT GCTGGAAGGG TGGTCCACCC TAACTCCGCA GGGACAGAAG CTCCTGTGTC
Λ TGGGACCCTT CCAGAACTTG CTCTATGTAT CTCTTCAAGT GCCTGTGTAT TTATATCCTT TCAAATATCC TTTGTAATAA ATCATAAACA TGTTTCCCTG AGTTTTTATG CCACTCTGTC AAATTAATTG AACCCAAAGA GGGGGTTATG GGAACCCCAA TTTGAAGCCA GTCCATCAGA AGTTCTGGAG GCCTGGACTT GAGACTGGTG TCTAAAGTGG GCAGGCAGTC TTGGGGACTA AGCCCTCAAC CTACGGGATC TGAAACTGTC TCCTGGTAGG TAGCATTGGA GTTGTACTGG AGGGCACTGA GCTGGTGTCT GCTGCAGAAT TGATTGCTTG CTTGCTGGTG GGGAGAACTT CCTACATATT TTGGGGTCAC CGAAGTCTTT TGTATTGATT GTTGTTGCTG AGCTCCTATT GCTAGAAACT TGCTTTATTA AGTTCTGTTT ATTCAGGGCT TATCTGAAAG AGAAACATTT TTTATGATTT GAGATTTCTA AGCCATTTTA AAACTTGGTT TTAACAGCTT GAGAAATTGG GGGTATAGTA GGGATGGAGA TACATATATT TGGATGTGAC TTCAAGCTAG ATAAAAGTTT GGAAGGAATA AAAGTTTGAC ATCTAAAGTT TTTGCATAGT TTGAGTTGAG CAGGAGGTAC TAGGTATGTT TTTAAAATAT TTTTTTCAGC CAGGCACGGT GACTCATACC TGTAATCCCA GCACTTTGGG AGGCCGAGAC AGGCGATCAC CTGAGGTCAG GCGTTCGAGA CCAGTCTGGC CAACATGAAG AAACCCCGTC TCTACTAAAA ATACAAAAAT TAGCTGGGTG CGGTGACACA TGCCTGTAAT CCCAGCTATT CAGGAGGCTG AGGCAGGAGA ATTGCTTGAA CCCAGGAGGC AGAGGTTGCA GTGAGCCGAG ATCACACCAT TGCACTCCAG CCTGGGTGTC TCAAAATAAT AATAATAATA AAAAAATGAA AGATTTTTTC TTACTCAGCA TCCTCCAGGC ATTTTATTAT CTGAGCACTT TATGGGAGTT GCATATTACA TTTAGGGCCC ACTCAGGTGG GTGGGTATCT AAGCATTTGA AATAACCTTA TGTAAACTAA TAAGGAGTAA TCAGGCCTGT GGCAAGATGG AAACAGTCTT AGAGGCATTC AAATTCAAAT TTCCTTTAAA ACACTGGGCT GGCCAAAACA AAAGACAATA CTATCTACAG GCCAGTTTCT AAGATTATCA GATTTTAGTA GCATTTACCA TTTCATTGTA CTTGGCACAC TTTAGCAAAT TTGCACTTCT TAAAAGTACC TGCAGGCAAT CTCCTATATA AAAACACAAT GCAGGCTAGC TTGGCTCCTG CCTTTAATTC CAGCACTTTG AGAAGTTGGA GACTAGCGTG GCCAACGTGG TGAAACCTCA TCTCCACTAA AAATACAAAA ATTAGCCAGG CATAGCAGCG CACGCCTGTA GTCCCAGTTA CTTGGGAGGC CGAGGCAGTA
GAATCACTTG AACGCTGGAG ACAGAGCATG GAGTGAGCTG AGATTGCACC ACTGCACTCC ATCCTGGGTG ACAGCGTGAG ACTCTGTCTC CAAACAAAAC AAAACACACA CACACACAAT GTAACAACAC GAAACAGAAT ACTGTGAAAA TGCTTAATTA TGTCTGACTT TACATGATGG CTGGATATGT GATTATTTTT TCTTCTTTAT GCTCTTATGT ACTTTGTTCA TTTTTAATGA TGATCATGTA TAAAGCTCCT CTGTGTAGCA TTCTCTCCAC CAAATTGCCC AGAGACAGGA
AGTCCTGTAA AACAAACTAA GCTCCAAAAA ATGACCTCCT GTTGAATAGG CTTTTTTTTT TTTTTTTTTT TTTTTTTTTG AAATGGAGTC TAGCTCTGTC TCCCAGGCCC TCGCTCCTTC CACCTCCTGG GTTTAAGAGA TTGTCCTTCC TCAGCCTCCA GAGTAGATTG GATTACAGGT GCCCGCCATC ACGCCCAGCT AATTTTTGTA TTTTTAGTAG AGATGGGGTT TCACCATGTT GACCAGACTG GTCTTGAACT CCTGACCCCA AGTGATCCGC CCGCCTGGAC CTCCCCAAGT GCTGGGATTA CAGGTGTGAG CCACCACGCC CAGCCTGAAT AGGCTTTCTA ACCTACTGTT TCTCATTTTA CTTTCTCTGA GGCAGTAAAA GAAACTGACT CTAAAAGGGA GCAGTAGAGA AGGAACTCAG ATTTTATTTT GAAGATTAAG CTACTCAAGG GCTGAGGAAA TATGTAGAGG GGAAGTAGAC TCATTATGGC TGGAAATTTT ATTTGGTGAT AGTGAAGCAA ATCTTAGGGC TTTCTAATTG AGCTCTTGTT TGAAAGGCTC CAATCTTAAT AGAACTATAA GCTAAAAAAA
ATGACCATCA GTGTTTCTAA AGCAAGTTGC TACTCAAAAC AAGAACACTT TGGGAGGCTG
GGGCTGGTGG ATCACCTGAG GTCAGAAGAT GGAGTCCAGC CTCAACATGG TGAAACCCCT
CTATACTAAA AATACAAAAA GTAGCCGGGT GTGGTGGTGC ACGCCTATAG TCTCAGCCAC
CTGGGAGGCT GGGGCAGAAG AATCGCTTGA ACCCGGGAGG TGGAGGTTGC AGTGAGCTGA
2.2. CATTGTGCCA CTGCATTTCA GCCTGGGTGA CAGAGTGAGA CTCTGTCTCA AAAACAAAAC AAACAGAAAC AAGAACTCTA TGTTGAGAAA TCCATACTAG AGGGTTTAAT TTCTCACTTT GTGTGCTAGT GATTAATAAA TTTCAAGCTT ATCACACAGC CAGAAATGTC GCCTGTGTTT CTACAATAAA AGATTTGGGA ATATTGTGAC ATTTTTTCAC TGAGCCTTCT GGGTTCATTA TTAAGATATA ACAATTTTAG AAGACTTATT GAGATAGGTA TACTTTTTAA AATTTGGATT CAATATATCA AGCTCAGCAT TTTGTCTTTT TTTTGTTTTG TTTTAAACAA CTTTGGGTTT ACTTGTAAGA GTATTTCAGT GGAGGAAAAG TATCAGAAGT TTAGCACTGA GTGCCTGCCT AACTATTTTA TGTCTTCTTT CATGATGATG CTTAATCTCA ACATAATGAC TCAGTTTACC ATTGTAAACT CTTTGTAGGA TGTCACTGAT TTTATCAGTC ATTTGTTCAT GCAGCTGGGA ACTGTGGGGA TATATGATTT GCCACATCTG AGGAACAAAC TGGGTGAGCT CCATTGAAAA ATTTCTAACT TTCTTATTAA AAGCAAGCTT CCTCACTTCC TGATGGCTTA TGCAAATTAA TGCCTATTTA TTCATCAGAG GGTGACTGGA GAGATTATGG TTTTCTTATG AATTTCTTGC TTGAGCCTGC TTGTCTGCTG TTTCTAAGTT GTCTGCACTA TTTTATGTGA GTAATTTTCT ACTTTATTAT GTTCTGTTTT CACATGTCAA ATCAGCTCTC CCAAGAATGC TACTTGTAAC CTAAGTAGAC GTGAACCGAA AAGGGTAAAG ACCCAGCTAA AAAAAAGGTT GACTCAAGTT CAGTTCACAT TCGTAAAGTA GTTAGGCTGC CTTAGGTTGC TTATTCTTTT CTTGAAAAGA ATCCCTCAAG AGAGAAACAT GTGAGGCCAC AGCAGCTTAG ATCTGTCTTC CACAGAGAAG GTGGCTTTTA CAGAGAAATT GACATACTCA TCACTTATCT GACATGACCC AGCTTTGTAA AACTGGCTTC TATTAAAATA GTCTTAATAG ACTATTCACT GAGGAGGAGA GAATCTTATT CACTCTTTAC ATTCTCTTCA CATTTTCAGA ATAGATGTTT AAATCATTGC TCACACTGGA TCCATAAATG TCTAAAATGT TGATGAAGAA ATAGGTGATT AGAGAGTAAA ATTAATAGAG ACTTACCCTT CCTTGCATTT TAACATAATA TTCTTTCCCC TTTTCCTTCC TCTGTTACTT GGCTCTTAAA TACCAGAAGT GAGATATGAA AAAGGGAACT GGGAACAAGT ATTGAAAGCA CCATAGGTTT ATCTTATATT AGCATTTTCC AAACTTTATA ATGAACCAGC AGTGACCCAC ACATCTGCCA GGTAGGAATT GCTCATCAGT TGTGTCGCGT TATCTTGTTA AGCTTCAGCA
ATGTTCCATA CAGCCACTAA TAACAGATCA AAGTGAGCAT TAGGGTTGAA ATTAGTAAGC TGTTTCTTCT CAGTTCTTTC TGGTAGTTGA ATAAATATAG AATGTATTAA ATAGTTTTCT TTATTTCAGA ACTTCTCAGA GTCTGTAATA TATTGTATGG TGGTAGCTTA GGAGGAAGAT GCAATAGGAA ACTTTTCCCA GATAGGTTCA CTATTTTTTT TTTCCACGAA AAATAAGCTG TTCTCAAAAT ACAGTTTACA AAATTTTATC CTTAACTCTT CACTCTTTCT CCTAGTTAGG
GAGACCGCTC CACCAGTAGA AAAGATAAAC CCTGGTAATT TGTTGTGTAA ATGGGATAAA TAGCCTAGTA CCTAGTCATG TGGATTCAGG CAGCACTGAG CCTAAATTAA AGTTTGCAAG GTATACATGT TAATGTATCT AAGTTACTAT ATTTAGCCTG TTTCTTAAGT ATGTTTCAGA AACATATTCG TTTTTTTCAG TGGCAGTTAC CTTCAGATGC ATGTGCTTCT AAAGCATGTT GGTTGCATGT GCAGTACATG TTGCTTAAGC ATTTAGCTTC AGAATGGCAT CTTTCCTGTG AATGTCTTAA CATTTACAAA AATATACCAG GATCTCAAAT ATCAGTGCTG GTATTTTTTT TTTTTTTTAC TTAAAGAAAC TGATATGATT AAATATTAAG AGACAATATG ATCCTTGTTG GCTTGTAACC CTAGTTTTTA TTGTCTTGTA GTTATTAAAT AGAGCATCTG TTGAGGGACT CTTTTAAAAC CACAGCCATG AACAGACGTT GGGGCTAAGA GACAGAGCAG CCTGCGACAG TGTGGACCTA CCTGTAGCAG CTAGCAAAGG CCTCTAGCAG CTACAGTCCC TTCTGGAGTC
TTTATTTGCA TGCAAAATGC AAAGGAGTCC TGGTGACCTA CCTCCAAGGC AGCTGCCCTC
CTGAACACTC CCTTGGAAAA CAGTAAACAT CATTTTGGAA TGTGAACAAC CAGAGACTAC
ACAGGAGAAA GGAAAAAAAA ATTCTGAAGA TGCAAAATCT TGGGTGGCTT CACCGTTCAG
TTTTTTAATA AAAGGAACAA TATACAACAC GTTGTTCTTT TTCTCTTTTG AAATCCCTTC TATTACAGTG ATTTTTTTCT AAGATTGTCA GGATTTGAAG TGTATGTTTT GTTTTATTCA
CAGCGTAAAT TTTATTCACA GTTTAACTGT CTGCCTGAGT GTCTTTCCTT TCTCTAATTA
CCTTGAGGAA CCCAAGAGCC TGTTGTAAGG AGAAAATAAG GCCCTTGGAT CTCTTGAGAT
TCACAGATAT AAGTTATTGA AGGGAAGATG GTCCTATGGA GGACATATTT AAAGAAGGGA AAAAAGAGGC TTTCTCAGAT ATGTCAGACT GCTATAGTAT ACTTCTACAG ATTATAGACC
TCCAGTACCT CTGGCCAGAA AGATGGTATC GTAAACACCC TATTTTTTTT CTTTTCTTTT
TTCATTAGGT ACAAGCTTTG TGCTAAGAAG TTGACATACT ATAAGCTACA AAAGTTCTGT
AAAGTAGATA TAACTAGTTT CATTTTATAG ATAGAGAAAA TTAATCTCTT ACAGTGCTAA
GCTCACAGAG TTTCTAACTG TAAAATGCTA GAACTTGTCT TTCAAGCCTA AAGACTTCCT TGGGGCTAAA TAGTGAAAAA AGCCATTTCA CAAATAAGTA AATGGTATTT AGAGGCATAT
TTGGATTTCC TGGTAAATTC CAGTCTGTGA GCATCATGAA TATTAGTTTA ATGTTGCATG
GGCTCATGTT GAAGTTTTAA GAGAAGAACT GCCTTGAAGC TTAGGTTTCC TTAGCTATTA
GGCTACTGAC TTTCTTGCCT AAACCAGGGT TTTTTCATTG AAGACCAAAA CTTACCTTCT
CCTTCAGTTT GTAGTTTGGA AATTGGTAGA AGAGCTTTGT AAACTTCAAA TTAAGTACAA ACTAAGTGTC ATAGTCAAAT TTACTAATCT TAATTACAGT ATTGTTCAAC TGATTGCTAT
CTTCTAGCTC TTTCCTGCCG AATAATGGTC TTGTTTCCTG CTCTGTTGGT TTAGAGCTGA
CTTCTTTCAG CTTTGGTAAG CCTGAAATTA TGGGGTTATG TTTAATTCAT ATTGTCTGGG
TGGACTTTCC TCTCTTGCAT TTCTGCTTGA ATAGAAGAAT TTTTCTCTAG AGAGTAGTTT
GTCATCCTTA CTCTGTTGAT TCAGATGACT CTTTGTATGA TCTGAGAGGT ATACTGTTCT GCTATTCTGA GAAGAAGTAT TTCAGAAAGA TGAATTAAGA GTACAGTGGA CTGCTCCCAC
CTGGAAACTT TTATCTATCT CACCTCTGGA CCTGATAAAT TCTTTATCAC TCAGGACCTT
GATGACGCTG CTCTCTGAAA CCCTCCCCAG CTCTCTCTAT TACCGTGAGA AACATCAGAA
CTTTGGTTCC CATTGCATAT CGCAGGTACC TCTGCTTTCA TGCCATGCTG TAATGGAGTG
ATTGGGTAGC ATGTTTTCAT CTCTTTCCAG ATTGAAAATC TGTATTTCTC CCTGTATATC TTCAACACCT AATGCACATA GAACTTTGTA GGTACCTGGA AAATGCACCA CAGTTTTCTT
TTCTTTTTGC AGACTTTTCA CAAGTATTAC CAACTTACAA AGAATTAATT TTGTAGGATT
CTAGAAAGAC AAATCAGGAA TGGTGCCATA TACATCTTTT TTGATTCCCT GCTCTAAAGA
ATATTATCAG GTTACCTTCC TGCAGAGTTT TAAAAGAATT GCATATTTCA AGCTGACTTT
CAGGATGTAA ATATAACCAA AGCAACTGAT ATGTAAAAAA TATATTCAAT GGCATTCCTA GATTTTCTTC TAGGGTGTTT TATTGTTTTG GGTTTTACAT TTAAGTCTTT AATCCATCTT
GAGTTAATTT TTGTATAGGT ATAAGAAAGG GGTCCAGTTT TAATTTTCTG CGTATGGCTA
GCCAGTTCTC CCAGCACCAT TTATTAAATA GGGAATCCTT TCCCTATTGT TTGTTTTTGT
ACGGTTTGTC AAAGATTAGA TGGTTGTAGA TGTGTGGTCT TATTTCTGAG ATCTTCATTC
TCTTCCACTG GTCTATGTGT CTGTTTTTGT ACCATGCTTT TTTGGTTACT GTAGCCTTGT AGTATAGTAT GAAAGATAGC ATGATGCCTC CAGGTTTGTT CTTTTTGCTT AGGATTGTCT
TGGCTATACG AGCTTTTTTT TGGTTCTATA TGAATTTTAA AATAGTTTCT TCTAATTGTG
TGAAGAATGT TAATGGTAGT TTAATGGGAA TAGCATTGAA TCTGTGAATT GCTTTGGGCA
GTATGGCCAT TTTCATGATA TTGATTCTTC CTATCCATGA GCATGTAACG TTTTTCCCTT
CGTTTGTGTC CTCTCTCATT TCCTTGAGTA GTGGTTTGTA GTTCTCCTTG AAGAGATCCT TCACTTCTTC TGTATTCCTA GATATTTTAT TCTCTCTGTA GCTATTGGGA ATGGGAGTTC
ATTCATGATT TTGCTCTCTG CTTGCCTTTT GTTGGTGTAT AGGGATCCTG GTGACTTCTG
CACATTGATT TTGTATCCTG AGACTTTACC GAAGTTGCTT ATCAGCTTAA GAAGCTTTTG
GGCTGAGATG ATGGGGTTTT CTAGATATAG GATCATGTTA TCTTCAAACA AAGACAATTT
GACTTCCTCT CTTCCTATTT GAGTACGCTT TATTTCTTTC TCTTGCCTGA TTGCCCTGGC CAGAACTCCC AATACTATAT TGAATAAGAA TGGTGAGAGA GGGCATCCTT GTCTTGTGCC AGTTTTCACG GGGAATGCTT CCAGCTTTTG CCCATTCAGT ATGATATTAT CTGTGGGTTT CTCATAAAAA GCTCTTATTA TTTGAGATAC GTTCCTTCAA TACCTAGTTT ATTGAGAGTT TTTAACATGA AGCGATGTTG AATTGTATCG AAGGCCTTTT CTGTGTCTAT TGAGATAATC ATGTGGTTTT TGTCTTTAGT TCTGTTTATG TGATGAATGA CGTTTATTGA TTTGCATATG TTGAACCGGC CTTGCATCCT GGGGATGAAG CCAACTTGAC TGTGGTAGAT AAGCTTTTGG ATGTGCTGCT GGATTTGGTT TATCAGTATT TCATTGAGAT TTTTTGCGTC GAAGTTCATC AGGGATATTG GACTGAAGTT TTCTTTTTGT TGTCGTATCT CTGCCAGGTT TTGGTATCAG GATGATGCTG GCCTCATAAA ATGAGTTAGG GAGGAGTCCC TCCTTTTCAA TTGTTTGGAA TAGTTTCAGA AGAAAGGGTA TCAGCTCCTC TTTGTACCTC TGGTAGAATT CAACTGTAAA TCCATCTGGT CCTGGACTTT TTTTCATTAG TAGGCTATTT ATTACTGCCT CACTTTCATA ACTTGTTATT GATCTATTCA GGGATCCAAC TTCTTCCTGA TTCAGTCTTG GGAGTGTGTA TGCATCCAGG AATTTATCCA TTTCTTCTAG ATTTTCTAGT TTCTTTGCAT AGAGGTGTTT GTAGTATTTG CTGTTGGTTG TTTGTACTTC TGTGAGATCA GTGGTGGTAT CCTGTTTATC ATTTTTTATT GTGTCTGTTT GATTCTTCTC TTATTTTTGA CAAAGCTGAC AAAAAGAAGC AATAGGGAAA GGACTCTCTA TTCAATTAAT CCTACTGTAT ATCTGGCTAG CCATATGCAG AAAATTGAAA CTGTTCCTGT TTCTTAATCC ATATACGAAA ATCAACTTAC GATGGATTAA AGACTTAAAT GTAAAACCCA AAATTATAAA ACCCTGGAAT AGAATATAGG CAATATCATT CTGGACATAG GAATGGGCAA AGATTTTATG AGAAAGACAC CAAAAGCAAT TACAACAAAA GCAAAAATTG GCAAATGAGA TCTAATTAAA CTAAAGAGCT CTGCACAGCA AAAGAAACTA
CTGTCAGAGT GAACAGGCAA CCAACAGAAT GGGAGAAAAT TTTTTCAATC TATCCATATG ACAAAGGTCT AACATCCAGA ATCTACAAGG AACTTAACAA ATTTACAAGA AAAAAGGAGC CCCATTAAAA AGTTGGCAAA GAACATGAAC AGACACTTCC CAGAAGATAT TCATGTGGCC AATAAACATG AAGAAAAGCT CAACATCACT GACCATTAGA GACGTGCATA TCAAAATCAC AATGAGATAC CATCTCATGT CACAATGGTG ATTATTAAAA AGTCAAACAA CATGCTAGTG AGGTTGTAGA GAAATAAGAA CGCTTTTACA CTGTTGGTGG GAATGTCAAC TAATTCAACC ACTGTGGAAG ACAGTGTGGT GATTCCTCAA GGATTTAGAA CCAGAAATAT CATTACTGCA TATAGACCCA AAGGAATAGA AATCATTCTA TTACAAAGAT ACATGCACAT GTATGTTTAT TACAGCACTA TTCACAATAG CAAAGACATG GAATCAACCC AAATGCTCAT CAGTGATAGA CTGGAAAAAG AGAATGTGGA ACATAAACAC CATGGAATAC TATGCAGCAA TAAAAAGGAA
TGAGATCCTG TCCTTTTCAG GGACATGGAT GGAGTTGGAA GCTGTTATCC TCAGCAAACT AATGCAGGAA CAGAAAACCA ACCACCACAT GTTCTCACTT ATAAGTGGGA GCTGAACAAT AGAACACATG GGCACAGGGA GGGGAATAAC ACACACTGGG GCCAGTCAGG GGGTGGGGGG TCAAGCTGAG GGAGAGCATT AGAAAAAATA GCTAATGCAT TCTGGGCTTA ACCCATTTAT GCCTAGTGTT CCATTTCTGG AATGCTAAGC ATGTGGAAGT TCTTTATATC CTGCTCAAGG TCATTGCCAA GGTCTGATTT TTCACATTCA ACAAATTGCA ACCTCTGGCA TAAATGGGTT AATACCTAGG TGATGAGTTG ATAGGTGCAG GAAACCACCA TGGCACATGT TTATCTATGT AAGAAACCTG CACATCCTAC ACATGTACCC TGGAACTTAA AAAATTTAAA ATATATATGT ATATATATTT AATATGGAAT TTTAAAAATT ACTAATGAGT TCTTTTATCT GAGTAATTTT GCATCAACAT GCTTTTATTA TGGAAGAGAA GATTCAGTGA GTACAAAATT GCAGATACAT
GTGTCAGAAG ATCCCTGAAT ATAATAAGGC TTAGTATTCT GTGTCATAAT TGCCTGTTTG
TATTCCTCTC TGGTCTTTAA ACTTCATTAG GGCAAGGATC AACTCCATCT TACTAACCAT
TTGATTCCCT ATGTATTACA CGATATATGA CCAATAATAA GCCTTCAATA AATACTTGTA
AAATAAAGAA TGTTATGTAA TATATCATGT GGTATTGTTT TATTGATGTG TTCTTTGAAG TGTTACCTTT GTGTCCTTAG AGGCTATGGT GGGCCCTCAG GCCATATATG TTGGATGATT TAGTGGAAAA GAGATTAGAC TTTTATATTT TATTCTATTT ATTTATTTAT GTTTTAATCT TGTGTAAGCC TATAGAGGCC AAGATAGGGC TCAGAGTTAA AAACAGTGAT GTTCTAACTT TCAAAACTAC TAAAAACAAA ATTAGCTATC AAGGGAAATC CTGAGTGTTC TGACTGCAGG GGTTCCCCAA AGCCTGGAGG ACCGAAGCTT TGAAATGCTG CATGGTGTGC CTCACCTCTG TTGAGTATAG TGAGGAACAT GAGCTGAGGA GTGGATTAGA TGAACTCTTA AGATTTTTTT CCAACTTTAC GGTTTGAGTT TATGAGTAAG TAAGTGAGGA ATGAGCCCAG GGACTTTGAC TACTTTGCTG TGCAGTAAAT TGCTGTTTTC TTAGCTAATT CTGGAGTGTG TATTTTGGTT TTAATGTAAT GTCCTTTCAG CACTGGAAAT AAACAGATTT ATGACCCTCC ATTTTCACAT GTAACTTCCA GTTCCTTCAG TAAAATGCTG GAAGTTGCTT GGCTGTAACC ACTGTAACTA TTTCCTGGCA GTTTTGCATT TTATTACAAT TCGACTTTTT AAATGCATGG TTCAGTCCAG TTAATAGGCA GAAAACTATA GCTATTCCTT GGAACTGACA TAGGGAAGTT GGTTTCTTTC TGAGGAAAGA TGTGACACTC TGATGGATTT GGCAAAATAC CAATTCAGTA AAATTTGCCT GGTTCCTTTC TGACAGCAAA TGGAGGTAAT TGTACGTATA TAGATTTCTA CAGTAAACAA AGGGCAAAAA TGGAAACATT TTTTGTTTTT TAATGTTGCT GGAATATAAT ATTTCCATAT TTGGTTAATC ATCAAATCAT TCAACTCCAT GCAAAGTACT CTTTTCAAAA TTACAAATCA TCTTGATGGA TTAGACTTGG ATGTCTAAAT TTTTTATTAT GTATTCTAAA TTATCTATAG GTACATTTTG AAAATTATAT ATACATGTAT CACTGTACTA CATTTGTTAT AAAATGTGAA ATATTAAGCG ATTAAATACT TTATTAATGT CATAAAATTG TGCCCTACCC AAAATATATT TTAAGAAGAT ATCATTTGAA AATAATGTAA AATATTCTTC ATTATTATGA TTTTGTTGCC
AAAAGTAATG TTAGAGTCCC TGTTACTGAT AATTTTCATG ACTGTAAATT TTCAACATTT GGAAACAAAA TTTTCTGATG TGTTTTAAAA TCTTAAAGAA TTTCAGTAGA TTTTGACACA TTTTTGTTAA AAGTATGAAA GTGTCACAGC TTTAGAAGGA TTTAGAATAA TTATCAATTG GTATTAGGGA TGACAATTTT AATAGACAAA TTTTAACTGA AACATTTGCT TTATTGATGT CTAGGACTAA AAAAAATATT ATAGTGATGG AACCATTTCA AAACATCTGT TTTTAAAAAC TTCTGTCAAC ATGTTTGACA GAAGTCAGAA TAGGGTAATT ATAAAAAAAC TGCCATTTTC TTATGTTTGC ATCATTAGTA TTTGCAAATC CTTGTTTCTT TGCAAGTATA TTTTAAACTA CTTGTTCATT TTATGAGTGT TAGCCTGATT AATTCAGATA AGATGCTCAA GTCAGTTTAA GCAGGCAGAT GTGAAAAAGG GAATATGCTG TGCCTTAACT ATCTTATAGG GACTTACTAG CACACTGTAC ATAACAGATG AACATCTGAT ATTAGATCTG ATGGGCACAC CAATGTTGTT TCATATATTA AATAATAGTG GAAGTTTTCA ATTTAATTAG TTTATTTATT TATTTATTTA TTTTTGAGCC GGTGTTTCCC TCTTTTTGCC AAGGCTGGGG TGCAATGGCA CATGGTTGCA GCTCACTGCA ATCTCCACCT CCCAGGTTCA AAGGATTCTT CTGCCTCATC CTCCCAAGTA GCTGGGATTA CAGGCGTGTG CCACTATGCC AGGCTAATTT TTGTATTATT AGTAGAGACA AGGTTTCACC ATGTTGGTGA GGCTGCTCTC AAAATCCCGA CTTCAGGTGA TCCACCCGCC
TCAGCCTCCT AAAGTGTTGG GATTACAGGC ATGAGCCACT GCGCCTGGCC AATTGTATTT
TTATGTTAGA AATATATATG GGAGTTACAA TACTTTGTTT CCAATGTTCC GCTGGAAACC
ACTGGTTGAA AGGATAGGTC CCAGTGTCAG CTGCCCAGTT GGCAGTTTTG GATAGAATAT
AAGTTCTATG AAGGCATGGA TGAAGTATCC TTCATGGTAA TGATGGTAAT GTTAGTATAG TCACTTGTTT AATGTTGTAT AACTTTTTAA CTCTTCCAAC TTCAATTTCA TCTATATCTT
TCATAGCAGT CCAGTAAAGT AGTATCAATT ATAGATATCA TTCCTACTTT CAGATAAAGT
AACTGAGGCT CACTAGTTTT GACTTGCTCA AGGTTACGTA AATGATAAGT GACAGACTGG
ATTTAAAGGG CGGAGTCCAC TTTACTAAAC TGCCTCTGTC TTCTAGCCAA GTTTGTTATG
TAAAGTAAGT ATTCAGACAT TGGCTAAGTA TATTATTAAT CAAGAAAACA GTAGAACAGG
36 TTTAAGGAAT TTTTTTTTCC CTCTCCTCAG TGTCAAAAAG CAGTCAAGTA AAAGATCCAA GATAAATAAC TTCAGTTCCC AAAAATAACT TCAATTCCAA AATTTCCCTA TGACTAGGTA TATCTAATTG GATAGCTGAT AAAATGAGTG GGAAATTGAA GAAGGGATAT TTTGAGAATG TGTGGCAACT TAATGGTAAT ATTGAAGAAG CAAATGGATA GTTATTGAAT CATAATAGTT TTTTAAACTT AAATACTAGG GCTTGATCTT CATATTTTAG GAAATTATCT ATTTGTTGCA GTAGAAATTA GCATAAATGA ATGGAAACTT AAATTATCTC ATCTGTGCAA GGCAGTCTTC AGAGACTGAT GTTTATATAA GTGAAAACTC AGAAAAATTT TTTGGCTGCA TTTCAAGCTG GATAGCGGGG TCAAAACTTC AACTTCTTAA TTGGTGATAC AACGTCAGAA ATCACAAAAG AAGTATTTCA CAGGAAAAAA TGACATACCC CGAAAACATT CAAATCAAGA AAATCACAGG GGTACAGTGG ATGGTCCCCC TTAAGGTACT GCGTACTTGG CTTCCCCGCT GGTAAATGCC TCTCTTTTAC TAAGTTGTTG TCGTGCAGAG TGGCATTGCA GCTCTGTACT TTTACAGAAT AATTTCAGGT TCTAAAATTG ATACATTATT TCAGATTGTA AGAATAGTAT AAAATAAAAT TTTAAAAGGG AAAATAATTC CATCAATTTA ATGGAATGTG TAGGGTTTAA GTTATAACAA CAAAAACAAA GTTGTAGTTT TTTAGGATTA CTCATATAAA TAGTGTGTCT ATTAAGAATT ACTGGCTTTA TGAATATTAA ATAAGAAGGC TGGCCGTGGT GGATCATGCC TGTAATCCCA GCACTTTGGG AGGCCGAGGC AGACGGATCA CCTGAGGTCA GGAGTTCAAG ACCAGCCTGG CCAACATGGC GAAACCCTGT CTCTACTAAA AATACAAAAA TTAGCCGGGC GTGGTGACGC ATGCCTGTAA TCCCAGCTAC TCAGGCGGCT GAGGCAGGAG AATTGCTTGA ACCCGGAAGA CGGAGGTTGC AGTGAGCTGA GATCGCGCCA CTGCACTCCA GCCTGGGTGA CAGAACAAGA CTCCATCTTG AAAAAAAAAA AAAATTAAGA CAACCACAGA TATAAGGATT TGCAATTAGA AAGCTTGAAG GGAAATTTTT GGAACTTCTA GGTCAACCTA AAATTACAAA TAGGGAAGCA GCTTTGGAGA GGAACATTTG CACTTCTGAA GTAATACTGT GATTTGGACT GTCAGACATT GTGATATATT GGCATCACCA CACCTCTGTT AATTATAAAC TTACCTATTC TCAGTGCCTT GTTAACTGTT GAAATAGGAC TTGACATTTG TTGTTCTTCC TCTTCTGTTA TTCATTTATT CACTCATTTA GTAGTTAATT ACTTCCTAGT GCCAGATAGT TTTCTAGGTA CTGAATAAAC
AAAAATCCTA TTGTCATAGT TATGTTTTTG TGGAAAGAAC AACCTGATAG ACTATCGTGA AAAATAAATG AGTTAGTATA CATAAAACAC AGCCCATGAG ATATAGTCTG TAATTATCAT CCCTGCTTCA TTTATTTATT TATTTATTTA TTTATTTAGA GAATGGATCT GATTCTGTCA TCCAGGCCGG AGTTCAGTGG CTGGATCATT GCTCACTGTA ACCTCAAAAC ACCTGGCCTC AAGTGATTCC CCCAACCTCG GCGTTCCAAA GTGCTGGGAT TAAAGGCATG AGCCACTGTA
CCTGGCCATT GCTCCTGTTT TAAAGATGAG GAAACTGAAA GTACAGAGAG GAACGTGACT
TGCTCAGGAT CACACAGCCA ATCAGTGGCA GAGCAGTCTA GGCAGTCTAG GCCTGAAGGC
ATTATTCTTT CTTTCTGTTC TGCGTCAAAA ACCCTAGGCA GGCATGGTAA AAAGACTGAA
AGAGCCAAGG CAACATAGGT ACCTGAAGAA TGGGAGAACA CCTATGAGGA CTGCTAGAAA TTTTAGGCAG CCCTTTAAAG GCCTTGGGCA ACAATTTGAA CGTGACTCAT GAGTCAGTTA
ATTCTCTGCA CCTTTTTTTT TTTAGAGAAT ACAATGGATA AAGCAATGAC CTTTGTAACA
TGTAAACCTG TATTTGAATC CTTTCCTCTC CATTTAATTT TGCAGAGTCA CCTTTTCTTC
ATTAGAAAGA AATTTTATCC CAAAGAGATT ATCGTGAGGC TCAAATAATA TAGGTGGAAG
CCCTTTGGGT TTAAAAAAAA GCCTTTTCTG TCATTTCTGG GTATCTCCCG TCAGTGATTT TAGTAATTGT TTACCTCTCT GGCTCTTCGT GAGAAGACAA ATTGTAGGAA GAAGTGGAGG
ATGGCATTGG GTAGGATGCT TAGTTCTAGT TCCACCACTT CCTTAGGTCT TTTTTTTGTC
CCCCAAGACT ATATCATTTC TGTGTTCTCT TGAACCTGTA AGATTTATGA GTAATCTGTA
CAATATTTAT AAAGTATGAA ATCTCTGCCA TTGTGGCACA AAAGTAGCCA TAGACAATGT
ATAAATGAAT GGGCATGCTG TTCTCCAGTA AAACTATATT TATAGAAATA GGCAGTCTTC
■-η TCCAATTTTA GTTTGCTCAC CCCTTTCCTA TAGAACTCAG GTTTATCATA TACAATGTTA CACATTATTC CTTTGAATAA ATTGTTTACC TAATACTTTT TAAATTTGGG CATTTTCTAG GACATTCTAT TGTTTTTATT TTTAACTTGT GTTATACTGC AGTGAATGTC TTCCTGTGTG TAGCATTTTG TTTGTGTATG TGTGTCAATA TCTATAAAGT AGCTCTTAAG AACTCTGGAA ACATTGTCAG ACTGCTTCCT CAAACAGCAG AGGTGGGAGG GAGCATCGGC CTAACCCTAC CTGGCCTGCC TTACCAGCAT GCAACCAAGG AGAAGAGAAT AGGATCAAGA GCCAGTGCCA GAGAGAAGTA CATAATACAA CTTTATGGAG TTTAGGTTGC ATCCTATAAA GTTACCATGT TACACTGTTT CGATCTACCA AAGTAGAAGT TTTACATAGT TTAGTATGAA GTTAATTTTA TTAACCTTTT CACACAACAC ACCCTCTCAT ATTGCTGCCC AACAATGTCC TGGGTCTTTG TTTCCATGGT CATCATATTG AGTAGGGTTT TAGCAGGGAC TTGAGTAGTT GGGAACTGAA CTCATGCTAG TGAAGCCATT CTTCACTAGA AGCAGACATA TGGGATCCAG AATATCATGA ACAACCAGCT GGGTACAGTG GCTCACACCT GTAATCCCAG CACTTTGGGA GGCTGAAGGA AGCAGATCAC CTCAGGTCAG GAGTTTGAGA CCAGCCTGGC CAACATGGTG AAACCCTATC TCTACTAAAA ATACAAAAAT TAGCCAGGCC TGGTAGCACA TGCCTGTAGT CGCAGCTACT TGGGAGGCTG AGGCAGGAGA ATCGCTTGAA CCTGGGAGGC GGAGGTTGCA GTGAGCCGAC ATCTCGCCAG TGTACTCCAG CCTGGGCAAC AGAGTGAGAC TCTGTCTGAA AAAAAGAGAA TATCATGAAT ATCACCAAAG ATAAAAGCAA GATGCGGATA TGAGTAGCCC AGAGTAACTT TTCCTGTGCT ATATAACACA GTGAGTGGTG ATTCCCACAA TTCAAGTCCA GTTTTTCTGG ATTCTAAAGC CATACATTTT TAACTCTCCC CCTAAATATG ATTTGAATTC AGAGAGTCAG ATGCAATAAG GTAATTTTAA CTTTAAGTGT AGTTTAGCCC ACACATTTCT CTAAACTGGG GGTTGGCAAA CTACAAACCT CTTTTTGTAA GTAAAGTTTT ATTGGAACAC AGCCTAGCTC ATTCATTTAT GTATTCCCTT TGCACCTTTT AGGTTGAGTA GTTGTGACTG AGGTCTTCCA GCCTTCAAGC CTAAAATATT TACTATCTAG TCCTTCAAAA AGAAGTTTGC CAACCTCTGC AAACAAGTGA AATAATGTGT ATTAGAGTAG AGCAGGAGTC CACAAACTAC AACCTCTGGA GCCAAAACCA CCCTACTGTC TGTTTTTGTA AACAGAAATT TTATAGAAAC ATAGTTGACC CCTGTGCCAA TTTGTTTGAG TCTTGCTTTT GATGGCTTTT GTGCTACAAG GGCAGAGTTG AACAGTTGCA AGAGAACTAA GTGGCCTGCA AAGCCTAGTG GTCTTTGCTG TCTGGCCATT ATAGGAAGTG TTTACCAAAC CCTGCATTTG ATCATGGAAT CCCAGAATGT TTATACCTTT GAGGATTTTT TTTTCTACTT TCCATATTTT ACAGGCAACA GAACTAAAGC CTCGTAAAAT CTTGCCCAAG GTTCTACAAA TAGTCTTTTC TATTAAATTA AGTCAGAGCC TGAAATTGTC TGTGCCCAAG TCTCTCCTCA GAAATTTAGC ACAGCATTTT CCCTTTCTGT CTCCCACATG CTACACAGTC ATCCTGGATC ATGTGCCCCG CCTCCATGAG GAGATGTTCT AACATGAGGG TGACTAGGCT CTGGAGCCAG AGCATCTTAG TTCAAATCCT GGATCTGTTA AAAATAATAG TTATTTTTCA GTTCTAACAC TGAAATAAGA CAAAAGGCCA TATTACTGAA ATAAAGTGTA AAGAATATAT TTAATACTCA GGTTACTTTA GCCCCCTCAT GATCAGAATA TGCTAATAAA TTGAGAGATT GACAGAAAAA AAGGATTCTG TTTCACTGTG GCTCACACCT GTAATCCCAT CACTTTGGGA GGCCGAGGTG GGTGGATCAC TTGTCTGGAG TTCAAGACCA GCCTGGCCAA CATGGCAAAA CCCCGTCTCT ACTAAACAAT ACAAAAATTA GCCAGGCGTG GTGGTAAGCA CCTTTAATCC CAGCTACTCG GGAGGTTGAG GCAAGAGAAT CGCTTGAACC TGGGAGGTGC AAGTTGCAGT GAGCCAAGAT CACACCACTG CACTCCAGCC TGGGCAACAC AGCAAGACTC CATCCCTGGA AAAAAAAAGT TCATCATTTG TTCTTGTAGT TTCCTAGTGT GGCCCTTGTC TCAGTGGAAA ATTGTAGTGT GATAGACTGA GGAGAATTCT GTTTCTGCCA CTTCGCCTGT TGCCTTGGTA GGAGCTCCTT ATCTCTGAGC TGAGCTCCTT TTGTCCTTGG TAAAATGTGG TTATTATCAT CTGCATCCTC ATAGTCTTCT TGTAAGGACT GTGAAGTCAC TTTTGAAAAA TGCCTTGAAT ATGCCAGATG GTCTGTTTTC TTTCTTTTTT GTTTTGTTTT GTTTTGTTTT CCCTTGGCCT TCATTCCTGG AAGTGTTTTG TTATATACAT TTTGTTTGGT GGCCAACAGT GCTAACCACA GAAAAGTTTG TATGTCTTCT TTTTCTAGAA TAATTCTTAC AGTGTAACCT CTCAGGGTGA AGTTTTTGTT TTGAAAGGGG CACATTTACT GTGAATGAGA GCCATGGAAA TGGACCTGAA ATTTGAAGAC AGATTTTACT TGGTTTTCTT TTCTCTTATT TGTTTGCAGT ATAAAGAAGA GGGCAGCAAG GTGACCACTT ACTGCAACGA GACAATGACT GGGTGGGTGC ATGATGTGTT GGGCCGGAAC TGGGCTTGTT TCACCGGAAA GAAGGTGGGA ACTGCCTCTG AGAATGTGTA TGTCAACACA GCACACCTTA AGAATTCTCA GGAAAAGTGA GTTGCTACAA ATGAGACATA CGTTTAGTTT TGTTTTATAG TATATATGAA TATGTGTGTA CATTTTTGGA ATTTTAGTTT GATTATACAA AATATCTTTG GCTTAGAAAT ATTAGGCATG CTATGTAAAA CCTTACTGGA AAAATAAATT GACCAACATT ATTGAGAGTA TTTTTTCAAA GTGTTCCAAA AGTAATGGAC CAATGATTAC TTTAAATGAA ATCATGTAAT GGACCACAGA ATTGCAAATT ACTAATAAAG AAAAGCCATT TTGCTTATTG CCATGTAATA ACATGTTGCA TGATACAAGT AGATACGTAT GTTTATGCTG CAACAAGTAT AGGTGATACT AATTGGGCAA CTTTTAAACA AGACCATAAA TAACTGAAAT CAAAGTTCTT AGTATTTATG CAGCCTGTTG GTTTGCGAGG GCTGCCATAA CAAAGTACCA CAGACTGGGT GGCTTAAGGA AGGTTGTTTT TTCACAGTTT TGGAGGCTAG AAGTCCAAGA TTGAGGTGTT GTCAGGTTTG GTTTCTTCCA AGGCCACACT CCTTGGCTTG CAGATGTCTG CTTGCTTACT GGGCTCCTAA ACAGTCTTTT CTGATTGTCC TAATGTCCTC TTCTTATAAG GGCATCAGTC ATATTGGATT GGGGCCCACC CATATGACCT AATTTTACCT TAATGACCTC TTTAAAGCTC TGTCTCCAAA TACAGTCATA TGGCTGTGAT ACAGGGGGTT AGAATTTCAA CGTATGAATT TTGAGGGGGA CACAATTCAG ACTATAAACT GCAGTTAATG TTTACTGTTA AATTAAATCT ATAACTGACT AAACCACACA AACAGGAGAT TTCAAATTAG ACTTTATTAG TTTTGGAAGG AAGAGATGAA ATGTTGTTAC TTTTTCTGTT TTATGTAGTT GTAAAAGCCA CTAAAAATGT ACATGTACAA ATCATCCCAA GCCAGGCAAC ATTAGTAATG AATGACTGCA GCACAGAGGT AAGAGAGATG ATTTAAAGGA GGATGAATTG TCTGCAAAGG TGCTGGGCAA GATCTAGACT AATTCATCCC CTTCATTTTA AGTTTAGGTT TTAAATAGCT TTGTTTGGCT TGATCCTAGA GCTACACATT TACTTTTAAC TTGTTTACTT TGTACCCTAT CATTTAGGAT ATGCTATGTA CTATTGTACT CTATTTGATA TTTCAAACAT TCTCTCATTT AGTGAAGAAG CTCCCAACCG GAGTGCTCAG AACCTACATT GCCTCACCTG TGAAGTGAGC ATGTGGGACT GAATTGCCTT TGAGGTCTCT TTCAGCTCTT AATGATCTGT TATCCCATAG TAAGATACAT TATTTTTAAT CTCGTTGGAT CTTAAACACC AAAAATAATA GTATTTAAGA CGTAGGATGC TATCTTGTCA TTATTTTAAT GCACATGTCA ACACACGAGG TTTTGCTAGA TGTTATGATA AAGCAAGCGA AACAGCAGGC TACTGCTCCT CAAATATCTA CAGTCAATGA AATATTGACT GGCGTTGTGG GAAAATATCT CAAAAGTATT TTATTTACAA TTTAAAATAT ATTCTTTTGC CTAAAGAAAC AATGAAACAA CTATAACTTG TTTTTGTTAT TGTGTTAGTG TAGTCATACA TATGATACAG TTTTATAATC TGCTTGATAA GGGTACGTCT CTGGGTTTTT CACACTTTGC AACAAGTGGG AAAGAACTTC GACAAAAACA TTAAAACATT ATCAACCTGG TTGCACGTTT TTCTGATTAT TTTCAGTGCT CTTAGGTTTT TAGACCATAT CAAAATTCTC TCTCTACACA CATGATTTAT AAGGGAAAGA AAAGTTTGCA GTATGCCTAA AATGTTCCTC AGGTTAATTA TATCTTGTTT TGCATAGTTG AATGTTTATG GAAGTCTTAG ATAGTGAGTC ACTCTGAAAC CACAGACTCT TTTTTAATAG TAATAATATG GTGAGTTCTA TAATTCCTAG CATCTAGCAC ACAGGTATGA TAAATGTTTT AATTAAGTGA ATAATCAGCT TCCTGAATTT TTCTACTTTG TAAATATGCT CTTTGTGTGT TGAATCTATT TACATATATA TCCAAGCATT TGGCACAAGG TAAACCAATG TGGCTCAGAT TTTAATGTTA TTGTGAATCA CTTGGGGATC TTGTTTAAAA AAAAAAAAAA AAAGGAGATT CTGATTCAGG TGATCTGGGC
TGAGTACTTA CAGTATGCAT TTCTAATAAA CTTCTGGGTG TTGCTACTGA TATTGGTTCA
AGAACTAAAT TCTGAGTAAC AAGTATAAAC CGAATGTCCA TAAAGTATAA TTTCTTTTAG
TATCATATGG TTATAATGCT TGCATTTTGT ATATATGTTG CATACAGGTT TTGCTTTTCT CTTTTTTCTT ATTAAAAAGG TTATAAAGGT ACACTGTAGA AACTGTAGGG AGTAGGGGAA
AGCTTGGCAT CTCATGCCAC CATTCAGAGG TAACCATGTT TGGCATTTTA AAAATGCATT
TATAGTGTGT GTGTGTGTGT GTGTGTGTGT GTGTGTGTGC GCGCATACAT ATATATGTAT
GTATTATACA TATACGTTAA TATACATATA ATATACATAT ATTACTGTGC AAAAGTTGAT
AAATTGATTA TGATTATGTA TAAACTTTGA TATCTTCTCA TATTTAATAT AATATAGTTG TGAGCACTCT TCCATATCAT TAAATAACTT TCTAGACATT TTAAATGAAT ATATAGAAAA
TGAAACATCA TTAATCAGTC CCTTATCTTA GAATATTTAT TTCGGTTATT TACAATCTAT
TAGTAGTTTA AAAGTACTTC ATGGAAAATC CTTATACATG CATCTTAGTT AACTTGAACT
TTTATTTTTA GAGAAGACAT GCCAGGCTGT TCTAGGCTAT TGATACATAT TGCCAATTAC
CTTCCTGAAA GATTGTACCA CTTTCCACTT AGGCACAGAG TGTGAATGCC TGTTTTAATT ACTCTATTTA CATCAGTAAA GACTATATCT GAAATTATCT CATTTTTGGG GACAGGTATT
CTAATAGGCT CTACAAGTAT GATCACAACT TTGTGAAAGC TATCAATGCC ATTCAGAAGT
CTTGGACTGC AACTACATAC ATGGAATATG AGACTCTTAC CCTGGGAGAT ATGATTAGGA GAAGTGGTGG CCACAGTCGA AAAATCCCAA GGTAATCAAG CACACATTTT ATCATTAATA
AAAATATGAA TGCTGAATAC CATCCTCCCT CCTAAGCAGT CCTACCAGTG TTGCTCGCCA TTTTATTGGT CATCCCAGTT GTAACTTTTT ATGTGATCTG TTAACGATCT TTATCTCCTA
TTGATACATA TTCTGTCACT CTCCAAGTCC TATTTATGAG TTTTTCTTTC ACATGTCTGT
TGTTTCCATT GCCCCCTTTC CATTTTCTAC ACTCTGGCAT GCAGCTTTGT CCTGTTTCCC
CCATGCTATG GATTAGCCTC CTGCCGGTAA CCACCTGAGG GGTTCTTCCT GCCTGCTGCA
TAAAGAAAAA CCATGGCAAT ATAGTAGAGA AAGAGCTTAA GAGACACTAG GCTGGCCATG CCACGTGGGG GATGGAGTTC ATATTCAAAT TATCTTGTCC AAAGCTCATA GGTAGGGGTT
TTTCAAAGGC AGCTTGGGGG AAGGGGTGGG GGTGGCCAGG TAACAGATGC TTGCTGCTGA
TTGGTTGGGG TGGGTGAAAT CACAGGGAGT TGAAGCTGTC CTCCTGTGGG CTGAATTGCT
TCTAGGTGGG GCCATAGGAG TGGGGTTGCT GGGTCCAGGT AGAACCACGG GTGTCAGACA TGCAAAAATA AAATAAAATA AAATAAGATA ANGGTAAGAT AAAATAAAAT AAAATAAAAT AATAAATAAA ATAAATATAA AATTTCCCTG AAAAGATATT TCAAAAAGCC AGTCTTAGAT TCTACAATAA TGATGTTATT TGCTGGAGTA ATTGATGGAG TTGCATGTCT TATAACCTCT
GGAATAACGG CTGACAATCT CTCAAGTCTG CGCCTTAGCT GGACTCAGGT TCCTCTTCTC CCCACAGCCT GACTGCCTCC ATTAGCTTCA CAAAAGTGGT TGGGTTTCAG GGCAAGGCCC
ATTGTCATTT AAACTGTAGC CGAAATGACT TCCAAAGTTA GCTTGGCCCA ATAGCCCAGG AATATTTAAG TGGAAGGCAA GATGGGGGAT GGGTTAGCTT AGCTCTCTTT CACTCTCATA
GTTTTCTCAC TGGTATAATT TTTGCAAAGG CGGTTTCATG CCTGCCATCT CTTTCGTCGC
TACCTCTCCC AGTTCCCATT CTTAGCTGTT TTATGAAATG CTTCTAGTTT CATCCTCTTA
TACCAAGTTC TGGGAGACTG ATTTGAGTAA TAATAAAACT CCAGTTTCCC ATACAGCCGG
CTCTGCGTGA ATTAAACTCA TTTTCTATTG CAATTTCCCT GTCTTGATAA TCAGTTCTGT GTAGGCCGTG AGGAAGGAGA ACCCGTTGGG TGATTACGAG ACTGTGTTAC TGCCCACTAC
CTAATGGATA CATTTAGCCT GGTATCCAAA CCCATCTAAT TATGACCATA ACTATATTTA
TCACCTTGCT CTGCTTTCAA ACGATATGAC ACACAATGAA TGAAAACTTT CATTTTTCAT
CTTCATTTGT GCTGTTCCCT TTGCCTCAAA TAGCCCTCAA CTTGCCTACA GTAACTGTAA
AATTTGCCAC CTAAAAAAAA ATCTCAAAAT CCTCTCTATG CTTTGATGTC CAGCAAAAAA AAAAAAAAAA AAAAAAAAGT TATCAGTATA GCTCTATTAC TCTCAACCAG AGGTGGCATT
TTTCATCTTT ACCCATAAGC CCCATGGTAT ATTTCTTAGT TTGGGAAATT ATATCATAAT
ACTTTGAATC TGTCTAGTCA GAAATTTAGA TAATTTAAAA AAAAATTATT TTTCTGAAAG
TATAGCTGGA TCTAGCTTAG GATCTACATC TATCTAATAT AGTTCCTTAA CATNTTGTTA AATGGCCACN GGNATAAGTC CTGTAATGCC ATACTTTGCT TTAGGATCAT GTGACTAAGG
GGTAAGGAAT TGGAAAGCAA TGGGGAGCTA GCAGAATTTG AAAAATTATA TAGGTAGGTT
ATTTTTCCTT AATACATTGA AATAGCCTCA AATTCTCAGA GAATACAATG TTTAATCCTC
TAATATCTGT AGAGTTCATG GCTCAAATTT GTATTATTTG AATAAACTAC TAATAGATTA
ATAATTACTC AACTAAAACA CTTTGAAATG TGAGAGTTCC CTTATCTCCC TCGCAGGCAT ATGACAGAGG TGTGGCTTCT CACACCTTTG GTTGCCCTTG CCCTACCACC CAAACCCCTA
GGGGGAGCAT GCAGAGGGGC AGGTGCAGAG GCCATGGGGG GTGCTTTTGG GCTCTGGCCC
CACAGCAGTG TCTAGGAGTG GATGTTGGAG ACTCCTGAAG CCCAAGTGGG CATGTGTTAC
AGTGTGCTCT TTCAGCTTAG CCGTCTGCAG ATGGCTTGTG TTAATCAGGT CATTAGACCC
CATGCCTTAT TGCAAGGGCA GGGGTCCAAT GTGACAGCCT AAGTTCTTGC TCAGTGTACC AGAAGAATTG GATCACACGT GGGCTGGAAG GATGAGCACA AGGTTTTATT GAGTGGTGGA
GGTGGCTCTC CGCGAGACGA CTCTCAGCCA GAGAAGGAGG ATGGAGGGGA AAGGTGTTCT
TCCCCTGGAG TCCACTTGTT CCTGTTCTCC TCTGGGTTCA AATACCTCTT CCCTCCTTTT
CTGCTGTGCT GTCCCACCAC TCTCCACAGC TCTGTGCCGC TCTGTTCCTC TGCTCCTCTG
GATGTTCAGC TCCTTGTATC TGTGCCTGCT AAGATTTTGG GTTTATATGG GGGAAGGATG GGGGGCATGG CGGGCCAAAA GGCACCTTTT TTGGTGTGAA AACAGAAATG CCTGTCTTCT
CTTAGGGCCT TAGGTCTTCA GGCTTGAGGG TGGGGCCTTT GCTGAGGGAC CACCCTCTTC
TACCCAGTAT TTCCCTGTCT CCTGTCCATA TCAACATTAC TAACTTTTTC ATCTGCAGAC
TAATAATGCT AAGGTGTGGC ATTTTTCAAC TGTGAGACTA TGTGAAGGTT TTTCCTGTCC
AACTGATGGC ATCCTCCCAT AATTCTACCC CTTTCTTAAA AGAATCTTTT GCAGTATTTC TCCAAGTTTA TTCTAGAGAA TTTCTTGTCT GTGAAATGCT CTAGTTAATC AAAATTGGAA
AACGGAGCAT ATCATATCCC CTTCTCAAAT TCACCAAAGT GAAGTCCTAA TGTGTCTTAA TGTATCTGCA TGAGACAGGA AGCTGAGATC TATTCAACAA CAAAAATCCA AACAAGCATC AAGAGGAGGA GTGTTAGCAC TTGAGCCTAG GGAGACTGTG GCTCCTGCCT GAAAGATGGG AGCCTCAGTC ACAGCTGCTT TACCAAGTGT CATATGCTAT GTTTCTGAGG ACTCCTGCTA AAGCTCCCTT CTCCCTCCAG CCAACCACTT TTGTTTTAGA CAAGGGCTGG GTTTATGAAG
GACTGTTTTC ATGACTAAAG CTTTATAGAA GGTTTAAGAT AAGGAGATGG AATTGAGTGA AGTAGGAAAT ATGAAAGCAG ATATTATAAT CTGGCTTCCT GATTTTTCAC TAGCATTTTT GTTTATAAAT TAGTTCTGTT CTAAGAATCC AATGACGTAA TAGAAACTCT CAAAGATTCT TAACTTGAGA TATAGGGAGT CTTTGAAACT GCTGAAATTA CAGACAGCAT TTATTGTTTA TGAGCATTTC TGAATCTAGA GCTTTCACTA GATTTGTAAA GAATGTGGGC CAAAAGATTA AGAGCCAATC CTGTATCTTG TACTCAAAAT GTTTGTAATT CTCACCTTTT TATCCCCCAG ACTCCTCTTG CTCTCTCTTC ATTTTCACAG TGTTACTGGA AAGGGGTCCT GATCCAGACC CCAAGAGAGG GCTCTTGGAT CTTGTGCAAG AAAGAATTTA GGGCGAGTCC TCAATGCAAA GTGAAAGCAA GTTTATTAAG AAAGTAAAGG AATAAAAGAA TGGCTACTCC ATAGACAGAG CAGCCCTGAG GGCAGCTGGT TGTCCATTTT TATGGTTATT TCTTGATTGT ATGCTAAACA AGGGATGGAT TATTCATGCC TCCCCTTTTT AGACCATAGA GGGTAACTTC CTGACATTGT CATGGCATTC TTTTTTTTTT TTGACAGAGT CTCACTGTGT TGTCCAGGCT GGAGTGCGGT AAAGCAATCT TGGCTCACTG CAACCTCTGC CTCCTGGGTT TAAATGATTC TCCTGCCTCA GCCTCCCAAG TAGCTGGGAT TACAGGCATG ACCCACCATG CCCAGCCAAT TTTTGTATTT
3/ TTAGTTGAAA CAGGGTTTTG CCATGTTGGC CAGGCTGGTC TTGAACTCCT GACTTCAGGT GATCTGCCTA CCTTGGCCTC CCAAAGTGCT GCAATTACAG ATGTGAGCCA CCAAACCTGG CCTGTCATGG CATTCTTAAA CTGTCATGGT GCTGGTGGGA GTGTAGCAGT GAGGAAGACC AGAGGTCACT CTCATCGCCA TCTTGGTTTT GGTGGGTTTT AGCTGGCTTC TTTACTGCAG CCTGTCTGTT CTATCAGCAA GGTCTTTATG ACCTGAATCT TGTGCTGACC TCCTATCTCA TTCTATGACT TAGAATGCCT TAACTGTCTG GGAATGCAGC CCAGTAGGTC TCAGCCTCAT CTTACCCAGC TCCTATTCAA GATGGAGTTG CTCTGGGTCA AACACCTTTG ACAACATCAT TAAGCCTCAG TTCTCACACT GTTTTTGTTT TGTTTGTGTG TGCAATGATG GGCAAATCTC TGCCTTATAG GATGGTAGAA AAAAGGAACT TAATATTGTA ATGACTGTTT TGTGCCAGAT AATTGCTTTA AACTGTATCA GCATCTTATT TAGTCCTGTT AATGATATGA ATGTTATCTT CATAACAGCT GCCATTTTAT TAAGGACTTA TCAGAGAAAA ACACTGTTCT AAGCACTTGT TACCCATTAT TACATTGAAT TTTCATAACA ACCCTTTGAG GTAAGCATGA TTATACCCAC TTTAATAGAA GTGAACTGTA GTTTTGTAAT GTTAGGTTCC TTGCCAAATG TTACACAGAT AGTAAGTGAT AAAATCATAT GCCCTGAAAT TACATTATGC TGCCAAACTT AAATTTCTTT TTTATCCTTT ATATTAGTAT ATTCTTAGGT TTAAACAAGA CAACTAGTTA ACACATACTA GATTTTGTCC ACAGTTCCTG GCTCATAACT CCCATAGCCC TTGTCACTAT CTTTTAANCG TTGGGGCACA TTAGGCCTCA GAAGTAGGCC TCANGAAAAC AGAGTCTCTC TCTCTCTCTC TCTCTGATCT TCTTCCGCCC TCCTCTCACC TGCCCAGGGC AGCACTTTAA TCTTCTCCTG CCTTTCTGAT CTTGGGTCAT AAGACCTTCA TTTCCAAAGA TGTCCTGTGT CATACCCTAA AGGAAGGAAC ACTGAACAGA GAGAGGCTCA GAAGAATCTG GACAGGCCTT GCTGTGTTTA CATCATTCCC TTTATGTCCA GTCACATCTC TACATGGTTG TCAGTTGTGC CTATTTGATG AAGTCCCCAT ATAAGGCTCA CAAGGACAGG GTGCAGAGAG CTTCCAGATA GCTGAACAAG TGGAAGTTCC TGGAGGGTGG CGTGTTCAGG GAGGGCATGG AAGCTGTGTG CCCCTTCCCC CATACCTTGC CCTACTCATT TCTTCATCTG TTTCATTTGT AGTATCTTTT ATAATAAACC ACTAAACATT AGTTAGTATT TCTCTGAGTT CTGTGAGTCA CTCTAGCAAA TTAATTGAAC CCAAGGAGGG TGTCATAGGA TCCCCNACAT TATAGCTGGT TGGCCAGAAG CACAGGTAAA CAACCTAGGG CTTTCAATTG GCATGAGAAG TAGGGGGCAG TTTTGTGGGA CGGAGCCCTC AGCCTGTGAG ATCTGATGCC ATCTCTAAGT ACACAGTGTC AAAACTGGAT TGGAGGACAC CCAGCTAGTA TTCACTGTGA AATTGGTTGC TTGCTTGATT TGTGGGGAAA AACCCACATG CATTTGATCA CAGAAGTCTT TTGTGTTGAC AGTTGATAGT GTTCAGTGAG AGAATTAAAA AAAAATTGAG TTTCTTCTTC AACATACTCT CTCAATGTGA AACCACAGAA ACTATTTCCA TTCAAAGATG GAAATGGTTT GTTTGCATCT TAGTTTTTAT TTATACATCT TAGAAGAAAT GTCCAAGCTT TGTTTTTTCT CTCACCCTAT ATATAAAATT ACCTATGAGG CACAGATTTT TATGATCCTT GATTATATAG ACTTTGTCCA AATTGTGTGT TTTATAGCAT TACTGTAACT TGTTATAGTA ATCTTTGTGT ATATTATGTC TCTTAACATT GTCTTCCATA TTGTTAATGA CCATCTCATA TTTATCTCTG TATCATGTAT ATCTTCAACC AATGTGACTG GCTTAGGAGA AAAAATTAGT GAACAATTAA CTAGTGTTTG TGTAATCTAT ACAATTGTCA AGGTTACAAT TGCTATTTTT GAAGAAATCG TTGTTGTTTT TCTCTTTGTT TCATCTCAGT TCCATTTTGT CAAGGATTCC TTTTTTTTTT TTTTTTTTTT TTTTTGAGGC GGAGTCTTGC TCTGTCACCC GGGCTGGAGT GCAGTGGTGC AATCTCGGCT CGCTGCAAGC TCCACCTCCT GGGTTCATGC CATTCTCCTG CCTCAGCCTC CCGAGTAGCT GGGACTACAG GCACCCGCCA CCACGCCCAG CTAATTTTTT TTTGTATTTT TAGTAGGGAC GGGGTTTCAC TGTGTTAGCC AGGATGGTCT CAATCTCCTG ACCTCGTGAT CCGCCCGCCT CGGCCTCCCA ATGCTGGGAT TACAGGCGTG AGCCACCGCG CGTGGCCCCT TGTCATGTAT TCTTAACCTG TGTTATATCT AAGAGAAGGT GTGAGAGGCG GGACCATTTG TGGATAGGTG CAGAGGGCAT GAAGTAGCCC AGAAAGAATC TTTGCCATTG ACTAAATTTT AGCCTAGTAA AAAACATGTG GTAACCACTG AAAAACTAGA ACAGTGTGAT ATGACAATGC CCAGTTGAAT AAGAAATTCA AATAGTTTAT AGTAACAAAA AATAATTTTT ACCACATTGT GACTAGTGCC CTAAAAACAA ATTTGAAGGA GCAGAGAGAG AGAGGAAGTG AGTTTTCGCT GGGGGCTGGT GAGTGGAGGC TACTTTATGA GCAGTTTTGA AAATTACAAG TTAGGGAAAA ACTCTTAGGG GAGTTTTAAG TGGTGAGCAA CAGGCACTTG AGGTTACAGG AGCAGCAGCA TAAAGATATA GAACAGAGAG GCCATTTGGC AGTCTTGGGG AAACTCCAAG AAGTCGAATG TGTCTAAAGC TGTGGTTCTC TCTCTGTGTG TGTGTGTGTG TGTGTGTGTG CGCGCGTGTG TTTGTGTGTG TGTGCATGTG TATGTATGTG TGAAATCGCT AATACAGAGA AGCTGTTAGA ACAATTTTCT CACTTCCCAA AAATGTCCGT TCATTCATGG GCCTAGGCAA CTCTCCTTTG TGTGTCTTAC AGCCCAACAC TGTCATATAA GGTGTGGTTC TATAATGAAA CAGTTTTATC TGTTGTTTAC GGAGCTAGTC AGCCTGTGTT ATGCTTGTTA CTGGTTGAGG GTGTCCAGGT TCTTGGCTTC TTGAACAAAG AATTGGACAA AACTCACAAA TGAGGCAAGG AAAGAATGAA GCAACAAAAG CGGAGATTTA TTGAAAATGA AAGCACACTC CACAGGATGG GAGTGGGCCT AAGTAAATGA TTCAAGGCCC TGGTTACAGA ATTTTCTGGG GTTTCAATAC CGTCTAGAGG TTTCCCATTG GTTACCTGGT GTATGCCTTA TGTAAATGAA GAGGGTGGAG TTAAGTTACA AAGTCATTTA CTCAGTATAG GCCTTGTGTT AATGGAGAGG GTGTTACTCC TGGGGGTTGT GGCCCATGTA AACGGAGAGG ATGAAGTGAA GTGACAAAGC CCTTCGCATT CCTGCCATTG CTGAAGTGTT TCCACTTTAT TTAGTTCTAG GAAGTCAGTG TGAATTGGCC TTATGTTCCC TGCCTCCAGA ACCTGTTCTC CTGCCTCACA TAGACCCTTT TTTCCCTTTG CCAACTTCAC TTCTTTTACA GCCACCTCAC TACCAATGTG TCTGTCTCCT AAGTCAAACA TCAGTGATTC CTCTGTTTTC TCTAAACCCT TCTTATGTCT TCTACTTCTC ATCTCTTTCT TGGTCAAAAA TCTTTCAAAA ATGAGTAAGA ATGCAGCTAT TCAGGCAAAC TAAAAATAAC ATCACAGTGA TATACAAAAC CAGTGTCATT TCACAAAGGA AAATTATCAA TACTAGATCC TGAAAAAGAA ACAGCGAATG AAAGCCATTT ACACAACTCC ATTGTGTAAT TGACACATTG AATCACTCAT AAAACAGGTG CTCTGGGTCT GAATCTAGAT CCTAGCTAGT CTGGTAGCTG AAATCATAGA ATTATAGTAG AGTTTAGGAA ATCATCCTCA AAGGAAAGAT TATATGTTGA TATCAAATGT ATATTTCCTT TCTAGGCCCA AACCTGCACC ACTGACTGCT GAAATACAGC AAAAGATTTT GCATTTGCCA ACATCTTGGG ACTGGAGAAA TGTTCATGGT ATCAATTTTG TCAGTCCTGT TCGAAACCAA GGTAAAAAAA TAAGCCTAAG TTTTTTGTTA ATTTGTTTGG AACTATTTAT TGAACAGTTG CTCTGTGTGA TGGATTTCGG GGATACCTAG ATGGAATGGG CATGATCCCC TTTTTACAGA AATAGAAAAT AGGTGGCCTA TGAATTATTC TTTCCTTTTA TATCCATGAC AAACTTTAGT AAAAAATTTT CTTTTCTACT GAGCTTAGCA TTTATTCAGT ACTCTTCTCA ATATATTTTC CAGGTAGCTA GTGACAATCA GAGTGATATG TAAGACAAAC TCATTTGTCC TCCTAGTAGG AAAGATTCTA GTAGAAGCAA AGAATTGTGT ACCATTCTGC AAGTGGTTTG TTGGAATCTT TCTTTGATAC CTGTTCCTGT ATTCCCCCAC CCCCATTAAT TTAGTCATTA ATTACTACAT GAACCCGTAA AATAAATCCT TAAATTATTT TCCTGGTAGA TTTTTTGAGC TGTGTAAGGA CCTTTCAATT CACTTTACAT TAGAATACGA TATGTGATGG TAAGTATTAA CCCAGCTTCC TGAGTGATGG CGTGAGGGCG GAACCCAGGT TCATGAAAAG TATTCCTATT ATTGAATTTT ACCAGTTATT TTCAGAAGTT TGATACAATG
TGAGTTGATT TCATAACATG TCCTCTAACT GCACTTGATA GCAAATTATC TTGTTATCCT
GAGTTGTAGC CAACAATGAC TTGGAGAATC TATGCAATAC TCAGTTTTAT TACTTCTAAG
CTCATTTTGA AGATAATACT ACCCATGGCT GATTTGTTAC TATAAAATAG GTTTAGTATT
TGCTGTCTGG AAACATTCTG ATTAGTGTCT CCTGGGAGGA TTATAAATTT ATAGTACCCA AAGANTAAAC NTGTTGTTTC CCTTTCCTAA ACTTTTAGTG NATAATNCAG TCTCTGCCGT GTCTCATTTT CATCACTTGC CCTCCANAGC TCCCATCTCA CTGAATTCTT GCAGTGTTCT GAATGTTGAG AGCCCCAANG TGGGTCTTAT AACAGCCAGT CAGCAACATT TCTGTTTTTC ATCTGACACC AAGGGTCTCG TCTCTTTGCT TTTCTACCAG TTATTCTGGG CTCTTCAGCT CTAAAGAAAG TATAGGTCCT GAAATCTTTC CCTACCTTCT CAATTTCCTG GGGAGGGCTT CTTTGGAAAG TGGGATTGGA AATAAGATAA ATTTGAAGAT AATTGAGAAA TGAATGGAAA GTGAAATTGA AGGGTCCATG TTAAGAGATT GCAAGTTATG CTATCACCAA ATAGATTTTT TGTGCCTGAG AGGATTAATT CATAGTGCAT ATTATGTGTT GACTTTATCA TTGAGGTCCT GGCACATGAT AGCATTGGCA TGATATAATT TGAGCTACTG ATACTATAGT GTTGCTTCTG GTGTTGTTAC CAGAACATCT AAAATATATT AGGATTTTTT TAATGGCAGA GGAAATGAAG ACTAATATGA CATAGTCCTT GTCCTAGTGA TTTACAGTTT AGCAAGACAT ACAAGCAAAA CATTAAAGTA AAGCATGATA ACTACTTTAA TAAAGCATTT TTTAAATTCA TTGGTAACAT AAGAGAAGGT AGAAGAGTTT AGCAAACCCT TCCCAAAAGA AATGATTGAC AAGTTATATG AGATAATAAT TCAGGGAAAG GAAATTCGAC TTTCTAAAGC CAAATTATTT GACATTGGTT TTCATATAGC TTGGTAAAAG CTGTTATTTT CTCCCATGTT CTTTATTCTT TGACTGTTAT AAGTATGATT TGTACAGAGA AATGGCAATT TCAAAACAGA GGGCTTTGAT GGATTAATTG CTTTGAATTG ATCCCTCATC TACAGTATCT TGTCAGGTAC TTGAGAAAAT AATGTACTTA AAGTTTCCTC TTTTGACTTT CTTTTGGTAT TCTATACTGT AAGTTGGGGA AAAAAGTATT TTCTCTTCCT GCTAATTGGG CTACTTGAAA ATTCCCACCA ACTTTGCCAA TACCAGTGTT CTGTATAACC CAGAATTCAG AATTAGCTGC AATTAAGGGA ATTCACAGCT TTTCTGTAGT
CAGAGAGCAA ATTGAAATTA AAGAAAAAAG AAATAGTGGG AGGACAAATG AGGTTTTACC TTTACACTTG AAAACAGATT TAAGAACAAG CCTTATACCT AGATTTATTA ATACTTTGAG GTATGAGAGG GAAGAGAAGC TTAGAAATAC GGCAGAATGG GCTTTCTTTG TTCTTCTCCC AGCTATGCTG TTTTTATTTA TTATTGTATT TTTAAGAGAG AAGGGAAGTG TCTCTCCTGG GTCACATTAA TTAGGAAATA CAGAGTGTTT TCATAATGCG TAAAGTCTAG TCCATTTAAG TCTTGTTTCA AAATGCTATT TATATTATTT GAGCAGGAAG GCAGAGACCT TAAACTGCTC CACCCAATTC ATTTTACACA AAAGATTAAA AAGAAAAAAA CAGTGTCAAA AGGTCAAGTG CCCAGTGTTG CACAACTAAT GATAGGCAAA ACAAGAGAGG AGGGGAAAAA AAAGAATCCT GACTCCCGAC TCCTAGTTCA GTGTGTTCTT CACTGTTAGA AGGTGCTGCT GAACATAGTT ATACCATATG AAGATCACAC TATCTATTTG AGATTGTAGA AATTTGATTA CTGCAGAGCT
CTGGCTGGGC TAGATTCACT TCTTATTCTT TATTGCATTG CAGTATTCTT AAGAGATAAA TGGCTCTTTT AGAATCAGAT TGGCCTTGGC TGTTGAAATG AGGCATTAAT TACCTTGGTA GCTGACACAT TCTTACAGGT CAGGGGCTTG ATGAAGTTTA TCTTCTTCCC TTGTTCCTGT GATTGCTCTG TAAATAGAAC ACATTCAGAG CCCTTGAATG CACTTGCAGC CTGTGCCTCC CACAGTGATC GATGGTCAGA TAATGGGAGT TTAATGACCA GTACTGAGAG AGATTATTTC CATGGCTGCT ATGGGCCAAG AAAGCTGGGT GGTCAGAAAG GGACCTTTTC CAGACTCTCC TGGGTTGTGT TATTTCTTTG ACATTGGTTT CCTTTCATGA GGCGCCAAGG TGTATTTGTA AGTTGTCCAG TGTTGCACAG CTAATGAGGG GCAAAACAAG AAAGGAGGAA AAAAAAAGTA TCCTCACTCC TAGTTCAGTG TGTTCTTCAC TGTTGGAAGG TACTGCTGAA CACAGTTATA TGTTCAGTGT ACAATGTACA ATGTTCTTTG ACATTCCGTC ATTTGAAGGA TGGGTCCTAT GGATAGTATC ATCTGCATGG TTTTGAAAAC AAAAGATATA AGCTAATTTT GCCCTGTCTA GTGACTACGA GACAGGGAGA GAAAATCTGA ATATTTGTTA AAGTAGACAC AGACCCATAA ATTGAAAAGG ACACTAATCC TGCCTTAGGA GACAGTAAGG CACTTGTCCC TGCTATCTAT TAGCTGTGTG TCCTTGGACA GCTCATTGCT TCTTTCTGAG CCACCGTTAC ACGTTATCTA
3 Jf TCAAGTGAGC AGGTTGTACA CTAGATGAAT TCACAGGTCC TTCTTCCAAG TGCTTTTCTA
ATCTTCATAA TTTAGATAAT CTCTCAGTAG CAAAACAGTG TACAATATGG TCAATCTGAG
ATTTTTAGGG GGAGAATTTT AGGGAACATC AGAAATGGCA GTAGTTAAAA GGAAATAGGA
CTCACAGGCT GACTTCTCTC TATAACTTCA CATGGTAGAA GGGATGAGAG TTCTCTCTGG AGCCTTTTGT ATTAGGTCAC TAATCCCAAA GGCCCCACCT CCTAAGACCA TCACTTTTGG
GATTGGGGTT TTAACATGAT TTTAGAGGGA CATAAACCTT CATCTCATTG CATTAGAGAT
ATTTGAAAGC TCAGCTCACG TGTATTCCTC CACAGCTCAC ATGTATTCCT CCATCACCCA
ACCTGATGGC TTTGAACGTT GTAGACATAA ATCCTTTCAT CATTATCAAG AATATTGCCA
AAAGCTTCTC AGAAATTATG AGGGGTTTTT TTAGTTTCTA AAATATTCTC AAAGAAAGTC CCATGTACTA ATGTTTGCCT TTTGATGAAA AAGGATGAAA TCTTAATGAT TGCCTTAATA
AGCTCAACAA TGCTTGTTAG TTGAGTCTTC TTATTGTGCT GATTCTTATA AACAACAACA
TTCAGTATAA ACATTAATGT ATGTGATTCA CTAAGGTTTT TGCATGATTC TCTGTGAGGC
TTCAGATGTC TCTTGGATTA TGTGTCTTTT TTTCATCGCC AGCATCCTGT GGCAGCTGCT
ACTCATTTGC TTCTATGGGT ATGCTAGAAG CGAGAATCCG TATACTAACC AACAATTCTC AGACCCCAAT CCTAAGCCCT CAGGAGGTTG TGTCTTGTAG CCAGTATGCT CAAGGTAAGT
GTTGCATTTC AGACACCATT TATGAGCTAT TTACCTGTGT GCAGCTGGCT GTTGTTGGCA
AAGGCAAAAG GATGATGCAG TAGAGAGAGC GCAGTGTCTA TAGTCAGAAA ATCTGAGTGC
AAGTCTGGCC CTATCACTTA TTAATGGATG ATTGCTCATG GAATTTACTG TACCATCCAG
CAAAATGTCA ATAGTTACTA TATATTGAGT GAGCTCTGCT TGTTACATAT ATTGCCTAAC AATGCTCAAA ACTCTGAGAA GTAGTAAGTA TAATCCCTAT TTATGGGCGG GGAACAGGAA
CTAAGAAATT TTTCTAATAA TTTGAAGGTC TCACAGCTTT TAGCATTGGA GTTTCACTTC
TAATCATCGT CTCCAAAACC CAACTTTTAT TAAAACTATA CTAACACTGG TTTCTCTCTG
GGAGAATTTT AAAATTCTGT ACTTAGGGCT GGGCACAGTG GCTTATGCCT ATAACCCTAT
CACTTTGGGA GGCTGAGATG GGTGAATCTC TTGAGTCCTA GAGTTTGAGA CCAGCCTGGG CAACACGGCG AAACCCCTTC TCTATTAAAA ATACAAAAAA TTAGCTGGGC GTGGTGGTGT
GTGCTTGTAG TCCCAGCTAT TCAGGAGGCG GAGGTGTAAG AATCACCTGA GCCCAGGAGG
TCAAGGCTGC AGTGAGCCGA TATCATGCCA CTGCACTCCA GCCTGGGCAA ACGGAGTGAG
GCCCTGTTAT GAAAAAAAAA AAATCTGTAC TTAGGCTTTC AGATCAGGCT GTATGTGATG
TATGTCGAAA ACACAGCTAT AATTGATTGA GGGAGAAACG TTACCATTTT AAAGTTTATG CTTTCAAGCC CAGATTTGGC CACTAGGAAT TTCCCAGCTC ACTAGTGAAA CTGCTGATGA
GTGATTATTT GCCAGTGAGC CTTTCATTCT TTCTAAAATA TGTACTACTA GTTGTGACTT GTAGGCTATA GGGGCTATAA TATATCAAGA CAATCTTTAT CCTCATGAAG CTTACAGTTA AGTAAGAGAT AGAGATTAAA TAATTATAAC AACAGAGTGA AGAACAGTGA AGAAAAAGTA CAGAGTTATA ATATATATAA TAGGGCCAGG ACTGCATGAG GAAGGTAGGA AAGACATTTC GGCAAGAGGT TGTCAGGGAA AAGACTTGCT TGAGAAAGAG CCAAGTTGTG GGGTCTGGCT GCTTAGCAAT GACCATAATA CCTAACTTTT GCTATTTTTA CATGAAGTAA CTAATTTAAC CCTATGAGGA AAGTACTACT ACCATCTAGA TTTTACAGGT AAGTAAGCAG AGATACAGAG AAGTTAAACT CTTCACACGG CTTTGGCTTT AAACCTATAT AGGCTTCAGA GCCTCCCCAC TTAACCACTT TGCCATAGCT ACATCCATAT TAGGTGCTAA GTAGATATCT GTTAAGTAGA AGGAGGATGA AAGGATAGTT AGCTAGTTGG AAAATGGATG GATGAATGAA GTGATGCTTA AGCTAAGAAC AACTTTCAGG GGTAACATGC AAAGAATAAT GGAGCAAAGA AGAAAAAATA GAAAATGGGA TAATCCTTTT TCTACTAAGG GGTAACCATG TGTGTTATTC ATCTTCAGGC TGTGAAGGCG GCTTCCCATA CCTTATTGCA GGAAAGTACG CCCAAGATTT TGGGCTGGTG GAAGAAGCTT GCTTCCCCTA CACAGGCACT GATTCTCCAT GCAAAATGAA GGAAGACTGC TTTCGTTATT ACTCCTCTGA GTACCACTAT GTAGGAGGTT TCTATGGAGG CTGCAATGAA
GCCCTGATGA AGCTTGAGTT GGTCCATCAT GGGCCCATGG CAGTTGCTTT TGAAGTATAT
GATGACTTCC TCCACTACAA AAAGGGGATC TACCACCACA CTGGTCTAAG AGACCCTTTC
AACCCCTTTG AGCTGACTAA TCATGCTGTT CTGCTTGTGG GCTATGGCAC TGACTCAGCC TCTGGGATGG ATTACTGGAT TGTTAAAAAC AGCTGGGGCA CCGGCTGGGG TGAGAATGGC
TACTTCCGGA TCCGCAGAGG AACTGATGAG TGTGCAATTG AGAGCATAGC AGTGGCAGCC
ACACCAATTC CTAAATTGTA GGGTATGCCT TCCAGTATTT CATAATGATC TGCATCAGTT
GTAAAGGGGA ATTGGTATAT TCACAGACTG TAGACTTTCA GCAGCAATCT CAGAAGCTTA
CAAATAGATT TCCATGAAGA TATTTGTCTT CAGAATTAAA ACTGCCCTTA ATTTTAATAT ACCTTTCAAT CGGCCACTGG CCATTTTTTT CTAAGTATTC AATTAAGTGG GAATTTTCTG
GAAGATGGTC AGCTATGAAG TAATAGAGTT TGCTTAATCA TTTGTAATTC AAACATGCTA
TATTTTTTAA AATCAATGTG AAAACATAGA CTTATTTTTA AATTGTACCA ATCACAAGAA
AATAATGGCA ATAATTATCA AAACTTTTAA AATAGATGCT CATATTTTTA AAATAAAGTT TTAAAA
The corresponding cDNA sequence for CTSC is provided below (SEQ ID NO : 2) :
1 aattcttcac ctcttttctc agctccctgc agcatgggtg ctgggccctc cttgctgctc 61 gccgccctcc tgctgcttct ctccggcgac ggcgccgtgc gctgcgacac acctgccaac
121 tgcacctatc ttgacctgct gggcacctgg gtcttccagg tgggctccag cggttcccag
181 cgcgatgtca actgctcggt tatgggacca caagaaaaaa aagtagtggt gtaccttcag
241 aagctggata cagcatatga tgaccttggc aattctggcc atttcaccat catttacaac
301 caaggctttg agattgtgtt gaatgactac aagtggtttg ccttttttaa gtataaagaa 361 gagggcagca aggtgaccac ttactgcaac gagacaatga ctgggtgggt gcatgatgtg
421 ttgggccgga actgggcttg tttcaccgga aagaaggtgg gaactgcctc tgagaatgtg
481 tatgtcaaca cagcacacct taagaattct caggaaaagt attctaatag gctctacaag
541 tatgatcaca actttgtgaa agctatcaat gccattcaga agtcttggac tgcaactaca
601 tacatggaat atgagactct taccctggga gatatgatta ggagaagtgg tggccacagt 661 cgaaaaatcc caaggcccaa acctgcacca ctgactgctg aaatacagca aaagattttg
721 catttgccaa catcttggga ctggagaaat gttcatggta tcaattttgt cagtcctgtt
781 cgaaaccaag catcctgtgg cagctgctac tcatttgctt ctatgggtat gctagaagcg
841 agaatccgta tactaaccaa caattctcag accccaatcc taagccctca ggaggttgtg
901 tcttgtagcc agtatgctca aggctgtgaa ggcggcttcc cataccttat tgcaggaaag 961 tacgcccaag attttgggct ggtggaagaa gcttgcttcc cctacacagg cactgattct
1021 ccatgcaaaa tgaaggaaga ctgctttcgt tattactcct ctgagtacca ctatgtagga
1081 ggtttctatg gaggctgcaa tgaagccctg atgaagcttg agttggtcca tcatgggccc
1141 atggcagttg cttttgaagt atatgatgac ttcctccact acaaaaaggg gatctaccac
1201 cacactggtc taagagaccc tttcaacccc tttgagctga ctaatcatgc tgttctgctt 1261 gtgggctatg gcactgactc agcctctggg atggattact ggattgttaa aaacagctgg
1321 ggcaccggct ggggtgagaa tggctacttc cggatccgca gaggaactga tgagtgtgca
1381 attgagagca tagcagtggc agccacacca attcctaaat tgtagggtat gccttccagt
1441 atttcataat gatctgcatc agttgtaaag gggaattggt atattcacag actgtagact
3 1501 ttcagcagca atctcagaag cttacaaata gatttccatg aagatatttg tcttcagaat
1561 taaaactgcc cttaatttta atataccttt caatcggcca ctggccattt ttttctaagt
1621 attcaattaa gtgggaattt tctggaagat ggtcagctat gaagtaatag agtttgctta
1681 atcatttgta attcaaacat gctatatttt ttaaaatcaa tgtgaaaaca tagacttatt 1741 tttaaattgt accaatcaca agaaaataat ggcaataatt atcaaaactt ttaaaataga
1801 tgctcatatt tttaaaataa agttttaaaa ataactgc
The wild type CTSC protein sequence is set forth below as SEQ ID NO : 3 : MGAGPSLLLAALLLLLSGDGAVRCDTPANCTYLDLLGTWVFQVG
SSGSQRDVNCSVMGPQEKKVWYLQKLDTAYDDLGNSGHFTIIYNQGFEIVLNDYKWF AFFKYKEEGSKVTTYCNETMTGWVHDVLGRNWACFTGKKVGTASENVYVNTAHLKNSQ EKYSNRLYKYDHNFVKAINAIQKSWTATTYMEYETLTLGDMIRRSGGHSRKIPRPKPA PLTAEIQQKILHLPTSWDWRNVHGINFVSPVRNQASCGSCYSFASMGMLEARIRILTN NSQTPILSPQEWSCSQYAQGCEGGFPYLIAGKYAQDFGLVEEACFPYTGTDSPCKMK EDCFRYYSSEYHYVGGFYGGCNEALMKLELVHHGPMAVAFEVYDDFLHYKKGIYHHTG LRDPFNPFELTNHAVLLVGYGTDSASGMDYWIVKNSWGTGWGENGYFRIRRGTDECAI ESIAVAATPIPKL. Papillon Lefevre syndrome (PLS) is an autosomal recessive disorder characterized by palmoplantar hyperkeratosis and severe early onset periodontitis that results in the premature loss of the primary and secondary dentitions. The 46 kb CTSC gene consists of 7 exons and is mutated in PLS patients. Sequence analysis of CTSC from PLS affected individuals from thirty-two Turkish families identified four different mutations. An exon 6 nonsense mutation (856C->T) introduces a premature stop codon at amino acid 286. Three exon 2 mutations were identified including a single nucleotide deletion (1047delA) of codon 349 introducing a frameshift and premature termination codon, a two base pair deletion (1028-1029delCT) that results in introduction of a stop codon at amino acid 343, and a G->A substitution in codon 429 (1286G->A) introducing a premature termination codon. All PLS affected individuals examined were homozygous for cathepsin C mutations inherited from a common ancestor. Parents and siblings heterozygous for cathepsin C mutations do not show either the palmoplantar hyperkeratosis or severe early onset periodontitis characteristic of PLS. In addition to the 5 families described above, Table I summarizes CTSC mutations identified in 27 other families presenting with symptoms of PLS.
Haim-Munk syndrome is a rare condition associated with congenital palmoplantar keratosis, pes planus, onychogyrphosis, periodontosis, arachnodactyly and acroosteolysis . In an additional embodiment of the invention, a mutation in cathepsin C causing Haim-Munk Syndrome has been identified. It appears that substitution of an A for a G at CTSC nucleotide position 857 in Exon 6 is responsible for this syndrome in patients .
Based on the data presented herein, it appears that additional mutations or functional polymorphisms are associated with other pathological conditions, including, but not limited to prepubertal periodontitis (PPP) , early onset periodontal disease or other forms of gum disease. For example, as shown herein, PPP is caused a substitution of a G for an A at position 1040 in the CTSC coding sequence. Thus, the invention also provides methods for screening the CTSC gene for alterations associated with these disease states.
I. Preparation of Altered Human CTSC-Encoding Nucleic Acid Molecules, CTSC Proteins, and Antibodies
Thereto A. Nucleic Acid Molecules
Nucleic acid molecules encoding the human CTSC proteins of the invention may be prepared by two general methods: (1) synthesis from appropriate nucleotide triphosphates, or (2) isolation from biological sources. Both methods utilize protocols well known in the art. The availability of nucleotide sequence information, such as a DNA having the sequence of SEQ ID NOS: 1-2 enables preparation of an isolated nucleic acid molecule of the invention by oligonucleotide synthesis.
Synthetic oligonucleotides may be prepared by the
3 ? phosphoramidite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices. The resultant construct may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC) . Long, double-stranded polynucleotides, such as a DNA molecule of the present invention, must be synthesized in stages, due to the size limitations inherent in current oligonucleotide synthetic methods. Thus, for example, a 4.7 kb double- stranded molecule may be synthesized as several smaller segments of appropriate complementarity. Complementary segments thus produced may be annealed such that each segment possesses appropriate cohesive termini for attachment of an adjacent segment. Adjacent segments may be ligated by annealing cohesive termini in the presence of DNA ligase to construct an entire 4.7 kb double-stranded molecule. A synthetic DNA molecule so constructed may then be cloned and amplified in an appropriate vector. Nucleic acid sequences encoding the altered human
CTSC proteins of the invention may be isolated from appropriate biological sources using methods known in the art. In a preferred embodiment, a cDNA clone is isolated from a cDNA expression library of human origin. In an alternative embodiment, utilizing the sequence information provided by the cDNA sequence, human genomic clones encoding altered CTSC proteins may be isolated.
Table 1 sets forth several different mutations associated with particular PPKs and PPP. Altered CTSC- specific probes for identifying such sequences may be between 15 and 40 nucleotides in length. For probes longer than those shown above, the additional contiguous nucleotides are provided within SEQ ID NOS : 1 and 2. Additionally, cDNA or genomic clones having homology with human CTSC may be isolated from other species using oligonucleotide probes corresponding to predetermined sequences within the human CTSC encoding nucleic acids.
In accordance with the present invention, nucleic acids having the appropriate level of sequence homology with the protein coding region of SEQ ID NO : 1 may be identified by using hybridization and washing conditions of appropriate stringency. For example, hybridizations may be performed, according to the method of Sambrook et al . , Molecular Cloning, Cold Spring Harbor Laboratory (1989), using a hybridization solution comprising: 5X SSC, 5X Denhardt's reagent, 1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37-42°c for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37oc in IX SSC and 1% SDS; (4) 2 hours at 42-65oc in IX SSC and 1% SDS, changing the solution every 30 minutes. Nucleic acids of the present invention may be maintained as DNA in any convenient cloning vector. In a preferred embodiment, clones are maintained in a plasmid cloning/expression vector, such as pBluescript (Stratagene, La Jolla, CA) , which is propagated in a suitable E. coli host cell.
Altered CTSC-encoding nucleic acid molecules of the invention include cDNA, genomic DNA, RNA, and fragments thereof which may be single- or double-stranded. Thus, this invention provides oligonucleotides having sequences capable of hybridizing with at least one sequence of a nucleic acid molecule of the present invention, such as selected segments of the DNA having SEQ ID NO : 1. Also contemplated in the scope of the present invention are oligonucleotide probes which specifically hybridize with the mutated CTSC genes of the invention while not hybridizing with the wild type sequence under high stringency conditions. Primers capable of specifically amplifying the altered CTSC encoding nucleic acids described herein are also contemplated herein. As mentioned previously, such oligonucleotides are useful as probes and primers for detecting, isolating or amplifying altered CTSC genes.
Antisense nucleic acid molecules may be targeted to translation initiation sites and/or splice sites to inhibit the expression of the CTSC gene or production of the CTSC protein of the invention. Such antisense molecules are typically between 15 and 30 nucleotides in length and often span the translational start site of CTSC encoding mRNA molecules.
Alternatively, antisense constructs may be generated which contain the entire CTSC cDNA in reverse orientation. Such antisense constructs are easily prepared by one of ordinary skill in the art.
It will be appreciated by persons skilled in the art that variants (e.g., allelic variants) of CTSC sequences exist in the human population, and must be taken into account when designing and/or utilizing oligonucleotides of the invention. Accordingly, it is within the scope of the present invention to encompass such variants, with respect to the CTSC sequences disclosed herein or the oligonucleotides targeted to specific locations on the respective genes or RNA transcripts. Accordingly, the term "natural allelic variants" is used herein to refer to various specific nucleotide sequences of the invention and variants thereof that would occur in a human population. The usage of different wobble codons and genetic polymorphisms which give rise to conservative or neutral amino acid substitutions in the encoded protein are examples of such variants . Such variants would not demonstrate altered CTSC activity. Additionally, the term "substantially complementary" refers to oligonucleotide sequences that may not be perfectly matched to a target sequence, but such mismatches do not n materially affect the ability of the oligonucleotide to hybridize with its target sequence under the conditions described.
B. Proteins
Full-length, altered, human CTSC proteins of the present invention may be prepared in a variety of ways, according to known methods . The proteins may be purified from appropriate sources, e.g., transformed bacterial or animal cultured cells or tissues, by immunoaffinity purification. However, this is not a preferred method due to the low amount of protein likely to be present in a given cell type at any time. The availability of nucleic acid molecules encoding CTSC protein enables production of the protein using in vi tro expression methods known in the art. For example, a cDNA or gene may be cloned into an appropriate in vi tro transcription vector, such as pSP64 or pSP65 for in vi tro transcription, followed by cell-free translation in a suitable cell-free translation system, such as wheat germ or rabbit reticulocyte lysates . In vi tro transcription and translation systems are commercially available, e.g., from Promega Biotech, Madison, Wisconsin or Gibco-BRL, Gaithersburg, Maryland.
Alternatively, according to a preferred embodiment, larger quantities of CTSC protein may be produced by expression in a suitable prokaryotic or eukaryotic system. For example, part or all of a DNA molecule, such as a DNA having SEQ ID NOS : 1 or 2 containing an alteration set forth in Table 1 may be inserted into a plasmid vector adapted for expression in a bacterial cell, such as E. coli . Such vectors comprise the regulatory elements necessary for expression of the DNA in the host cell positioned in such a manner as to permit expression of the DNA in the host cell. Such regulatory elements required for expression include promoter sequences, transcription initiation sequences and, optionally, enhancer sequences.
The human CTSC protein produced by gene expression in a recombinant procaryotic or eukaryotic system may be purified according to methods known in the art. In a preferred embodiment, a commercially available expression/secretion system can be used, whereby the recombinant protein is expressed and thereafter secreted from the host cell, and readily purified from the surrounding medium. If expression/secretion vectors are not used, an alternative approach involves purifying the recombinant protein by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant protein or nickel columns for isolation of recombinant proteins tagged with 6-8 histidine residues at their N-terminus or C- terminus . Alternative tags may comprise the FLAG epitope or the hemagglutinin epitope. Such methods are commonly used by skilled practitioners. The human CTSC protein of the invention, prepared by the aforementioned methods, may be analyzed according to standard procedures. For example, such protein may be subjected to amino acid sequence analysis, according to known methods . The present invention also provides antibodies capable of immunospecifically binding to proteins of the invention. Polyclonal antibodies directed toward altered human CTSC proteins may be prepared according to standard methods. In a preferred embodiment, monoclonal antibodies are prepared, which react immunospecifically with the various epitopes of the CTSC protein described herein. Monoclonal antibodies may be prepared according to general methods of Kohler and Milstein, following standard protocols. Polyclonal or monoclonal antibodies that immunospecifically interact with altered CTSC proteins can be utilized for identifying and purifying such proteins. For example, antibodies may be utilized -5 for affinity separation of proteins with which they immunospecifically interact. Antibodies may also be used to immunoprecipitate proteins from a sample containing a mixture of proteins and other biological molecules. Other uses of anti-CTSC antibodies are described below.
II. DETECTION OF KERATODERMAL DISORDERS/DYSPLASIAS and PERIODONTAL DISEASE-ASSOCIATED MUTATIONS AND DIAGNOSTIC SCREENING ASSAYS THEREFORE
Currently, the most direct method for mutational analysis is DNA sequencing, however it is also the most labor intensive and expensive. It is usually not practical to sequence all potentially relevant regions of every experimental sample. Instead some type of preliminary screening method is commonly used to identify and target for sequencing only those samples that contain mutations. Single stranded conformational polymorphism (SSCP) is a widely used screening method based on mobility differences between single-stranded wild type and mutant sequences on native polyacrylamide gels. Other methods are based on mobility differences in wild type/mutant heteroduplexes (compared to control homoduplexes) on native gels (heteroduplex analysis) or denaturing gels (denaturing gradient gel electrophoresis) . Sample preparation is relatively easy in these assays, and conditions for electrophoresis required to generate the often subtle mobility differences that form the basis for identifying the targets that contain mutations are known to those of skill in the art. Another parameter to be considered is the size of the target region being screened. In general, SSCP is used to screen target regions no longer than about 200-300 bases.
Another type of screening technique currently in use is based on cleavage of unpaired bases in heteroduplexes formed between wild type probes hybridized to experimental targets containing point mutations. The cleavage products are also analyzed by gel electrophoresis, as subfragments generated by cleavage of the probe at a mismatch generally differ significantly in size from full length, uncleaved probe and are easily detected with a standard gel system. Mismatch cleavage has been effected either chemically (osmium tetroxide, hydroxylamine) or with a less toxic, enzymatic alternative, using RNase A. The RNase A cleavage assay has also been used, although much less frequently, to screen for mutations in endogenous mRNA targets for detecting mutations in DNA targets amplified by PCR. A mutation detection rate of over 50% was reported for the original RNase screening method.
A newer method to detect mutations in DNA relies on DNA ligase which covalently joins two adjacent oligonucleotides which are hybridized on a complementary target nucleic acid. The mismatch must occur at the site of ligation. As with other methods that rely on oligonucleotides, salt concentration and temperature at hybridization are crucial. Another consideration is the amount of enzyme added relative to the DNA concentration. In summary, exemplary approaches for detecting alterations in CTSC encoding nucleic acids or polypeptides /proteins include: a) comparing the sequence of nucleic acid in the sample with the wild-type CTSC nucleic acid sequence to determine whether the sample from the patient contains mutations; or b) determining the presence, in a sample from a patient, of the polypeptide encoded by the CTSC gene and, if present, determining whether the polypeptide is full length, and/or is mutated, and/or is expressed at the normal level; or c) using DNA restriction mapping to compare the restriction pattern produced when a restriction enzyme cuts a sample of nucleic acid from the patient with the restriction pattern obtained from normal CTSC gene or from known mutations thereof; or, d) using a specific binding member capable of binding to a CTSC nucleic acid sequence (either normal sequence or known mutated sequence) , the specific binding member comprising nucleic acid hybridizable with the CTSC sequence, or substances comprising an antibody domain with specificity for a native or mutated CTSC nucleic acid sequence or the polypeptide encoded by it, the specific binding member being labeled so that binding of the specific binding member to its binding partner is detectable; or, e) using PCR involving one or more primers based on normal or mutated CTSC gene sequence to screen for normal or mutant CTSC gene in a sample from a patient.
A "specific binding pair" comprises a specific binding member (sbm) and a binding partner (bp) which have a particular specificity for each other and which in normal conditions bind to each other in preference to other molecules. Examples of specific binding pairs are antigens and antibodies, ligands and receptors and complementary nucleotide sequences. The skilled person is aware of many other examples and they do not need to be listed here. Further, the term "specific binding pair" is also applicable where either or both of the specific binding member and the binding partner comprise a part of a large molecule. In embodiments in which the specific binding pair are nucleic acid sequences, they will be of a length to hybridize to each other under conditions of the assay, preferably greater than 10 nucleotides long, more preferably greater than 15 or 20 nucleotides long.
In most embodiments for screening for susceptibility alleles, the CTSC nucleic acid in the sample will initially be amplified, e.g. using PCR, to increase the amount of the analyte as compared to other sequences present in the sample. This allows the target CTSC sequences to be detected with a high degree of sensitivity if they are present in the sample. This initial step may be avoided by using highly sensitive array techniques that are becoming increasingly important in the art .
The identification of the CTSC gene and its association with keratodermal disorders/dysplasias and peridontal diseases paves the way for aspects of the present invention to provide the use of materials and methods, such as are disclosed and discussed above, for establishing the presence or absence in a test sample of a variant form of the gene, in particular an allele or variant specifically associated with PLS, HMS or periodontal diseases. This may be for diagnosing a predisposition of an individual to PLS, HMS or periodontal disease. It may be for diagnosing PLS, HMS or periodontal disease in a patient with the disease as being associated with the altered CTSC gene. This allows for planning of appropriate therapeutic and/or prophylactic measures, permitting stream-lining of diagnosis, treatment and outcome assessments. The approach further stream-lines treatment by targeting those patients most likely to benefit. According to another aspect of the invention, methods of screening drugs for therapy to identify suitable drugs for restoring CTSC product functions are provided.
The CTSC polypeptide or fragment employed in drug screening assays may either be free in solution, such as gingival crevicular fluid, affixed to a solid support or within a cell. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may
H I determine, for example, formation of complexes between a CTSC polypeptide or fragment and the agent being tested, or examine the degree to which the formation of a complex between a CTSC polypeptide or fragment and a known ligand is interfered with by the agent being tested.
Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to CTSC polypeptides and is described in detail in Geysen, PCT published application WO
84/03564, published on Sep. 13, 1984. Briefly stated, large numbers of different, small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with CTSC polypeptide and washed. Bound
CTSC polypeptide is then detected by methods well known in the art .
A further technique for drug screening involves the use of host eukaryotic cell lines or cells (such as described above) which have a nonfunctional CTSC gene.
These host cell lines or cells are defective at the CTSC polypeptide level. The host cell lines or cells are grown in the presence of drug compound. The rate of growth of the host cells is measured to determine if the compound is capable of regulating the growth of CTSC defective cells.
The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g., enhance or interfere with the function of a polypeptide in vivo . See, e.g., Hodgson, (1991) Bio/Technology 9:19-21. In one approach, one first determines the three-dimensional structure of a protein of interest (e.g., CTSC polypeptide) or, for example, of the CTSC-substrate complex, by x-ray crystallography, by nuclear magnetic resonance, by computer modeling or most typically, by a combination of approaches. Less often, useful information regarding the structure of a polypeptide may be gained by modeling based on the structure of homologous proteins. An example of rational drug design is the development of HIV protease inhibitors (Erickson et al . , (1990) Science 249:527- 533). In addition, peptides (e.g., CTSC polypeptide) may be analyzed by an alanine scan (Wells, 1991) Meth. Enzym. 202:390-411. In this technique, an amino acid residue is replaced by Ala, and its effect on the peptide 's activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide.
It is also possible to isolate a target-specific antibody, selected by a functional assay, and then to solve its crystal structure. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original molecule. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced banks of peptides . Selected peptides would then act as the pharmacore . Thus, one may design drugs which have, e.g., improved CTSC polypeptide activity or stability or which act as inhibitors, agonists, antagonists, etc. of CTSC polypeptide activity. By virtue of the availability of cloned CTSC sequences, sufficient amounts of the CTSC polypeptide may be made available to perform such analytical studies as x-ray crystallography. In addition, the knowledge of the CTSC protein sequence
4"? provided herein will guide those employing computer modeling techniques in place of, or in addition to x-ray crystallography.
Ill Therapeutics
A. Pharmaceuticals and Peptide Therapies
The discovery that mutations in the CTSC gene give rise to PLS, HMS, and periodontal disease facilitates the development of pharmaceutical compositions useful for treatment and diagnosis of these syndromes and conditions. These compositions may comprise, in addition to one of the above substances, a pharmaceutcally acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
Whether it is a polypeptide, antibody, peptide, nucleic acid molecule, small molecule or other pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
B. Methods of Gene Therapy
As a further alternative, the nucleic acid encoding the authentic biologically active CTSC polypeptide could be used in a method of gene therapy, to treat a patient who is unable to synthesize the active "normal" polypeptide or unable to synthesize it at the normal o level, thereby providing the effect elicited by wild- type CTSC and suppressing the occurrence of "abnormal" CTSC associated with keratodermal disorders and dysplasias . Vectors, such as viral vectors have been used in the prior art to introduce genes into a wide variety of different target cells. Typically the vectors are exposed to the target cells so that transformation can take place in a sufficient proportion of the cells to provide a useful therapeutic or prophylactic effect from the expression of the desired polypeptide. The transfected nucleic acid may be permanently incorporated into the genome of each of the targeted cells, providing long lasting effect, or alternatively the treatment may have to be repeated periodically.
A variety of vectors, both viral vectors and plasmid vectors are known in the art, see US Patent No. 5,252,479 and WO 93/07282. In particular, a number of viruses have been used as gene transfer vectors, including papovaviruses, such as SV40, vaccinia virus, herpes viruses including HSV and EBV, and retroviruses . Many gene therapy protocols in the prior art have employed disabled murine retroviruses.
Gene transfer techniques which selectively target the CTSC nucleic acid to oral tissues are preferred. Examples of this include receptor-mediated gene transfer, in which the nucleic acid is linked to a protein ligand via polylysine, with the ligand being specific for a receptor present on the surface of the target cells.
The following methods are provided to facilitate the practice of the present invention.
Family material and clinical diagnosis.
Five Turkish families were described previously
[8] . All available family members provided consent for i the study and were clinically examined. A diagnosis of PLS was made in individuals with severe early onset periodontitis and the clinical appearance of hyperkeratosis on the palmar and plantar surfaces. All affected individuals also had hyperkeratosis on the knees. DNA was isolated from peripheral blood samples from all available members from these nuclear families using standard techniques (Qiamp Blood Kit, Qiagen) .
RNA Isolation, Amplification, and Tissue Expression Analysis .
Total RNA was generated from fresh tissue samples (gingiva, palm, sole, knee) using TRIZOL reagent (Molecular Research Center, Inc.; Cincinnati, OH) according to the manufacturer's protocol. To determine if cathepsin C was expressed in a given tissue, single- tube RT-PCR was carried out using the Access RT-PCR System (Promega; Madison, WI ) , following the manufacturer's protocol. A portion of each reaction was visualized following agarose gel electrophoresis in the presence of ethidium bromide. Amplification primers located within exon 6 F 5 ' -AGGAGGTTGTGTCTTGTAGCC-3 ' (nt . 857-877,-SEQ ID NO : 4) and exon 7 R 5'- AGTGCCTGTGTAGGGGAAGC-3 ' (NT 981-962; SEQ ID NO: 5) produce an amplicon of 123 base pairs from cDNA. A standard PCR protocol was followed with an annealing temperature of 65 °C.
GenBank accession numbers . Full-length cDNA of CTSC (NM-001814) and full- length genomic DNA of CTSC contained within a BAC vector, Genbank accession number (AC011088) . See SEQ ID NO: 1.
Cathepsin C Activity Assay
In unafffected non-carriers, cathepsin C activity ranges from 600-1200 μmol/min/ mg . As carriers of a
SX cathepsin C mutation do not have clinical manifestations, measurement of cathepsin C enzymatic activity can be used to determine whether at-risk family members are carriers. Enzymatic activity can also be used to determine if individuals marrying into a family are carriers. Carriers typically have approximately 50% of normal enzyme activity. Determination of enzymatic activity can also be used to establish a diagnosis of PLS when mutational studies of cathepsin C have been negative. This is important in assuring that a diagnosis of PLS has been properly given to an individual with clinical symptoms suggestive of PLS.
Viable leukocyte pellets are obtained from lithium heparinized whole blood by mixing blood with 3 volumes of 3% dextran in normal saline, and allowing the red cells to settle for 45 min at room temperature. Cells are pelleted by centrifugation at 1500 rpm for 5 min at 4*C. After washing and removal of contaminating red cells, leukocyte pellets are resuspended in dH20 and sonicated on ice for 5 sec each for total of 6 blasts using a Sonic 300 Dismembrator . Protein concentration is determined by the Lowry method.
Enzymatic activity is determined by measuring hydrolysis of the synthetic substrate glycyl-L-arginine-7-amido-4-methylcoumarin at a final concentration of 5 mM using a modified method. All reactions are performed in duplicate. Twenty μl of leukocyte lysate are added to 200 μl of Na3P04 buffer (0.1M, pH 6.5) in a 96 well plate and then substrate added. Reactions are allowed to proceed for 1 hr at room temperature at which time 10 μl of glycine-NaOH buffer (0.5M, pH 9.8 ) is added to stop the reaction. Fluorescence is determined using a Perkin-Elmer LS50B luminescence spectrometer at 370-nm excitation and 460-nm emission. The amount of NHMec released is determined by generating a standard curve using NHMec. Cathepsin C activity is reported as μmol
£ NHMec released per min per mg protein.
Sequencing and mutation analysis .
PCR primers were designed to cover the entire cathepsin C gene in overlapping fragments, from 955 nucleotides 5' to the start codon to 240 nucleotides 3' to the termination codon using cathepsin C (DPP-I) sequence data (Accession # U79415; SEQ ID NO: 1). The PCR products were prepared for sequencing by excising the bands from the agarose gel and extracting the fragments using a Qiagen Gel Clean-up Kit. The sense and antisense strand of each PCR product were directly sequenced on an ABI Prism 310 Genetic Analyzer (Perkin- Elmer) using four dye terminator chemistry. Approximately 1-3 ng of purified product and 3.2 pmol primer were added to premixed reagents from the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit, FS (Perkin-Elmer) and underwent a cycle sequencing reaction in a GeneAmp PCR System 9700 (Perkin Elmer) . The linear amplification started with a 10 s denaturation at 96°C, 5 s annealing at 50°C and 4 min extension at 60°C. The fluorescently labeled sequencing products were separated from residual reaction reagents using a Centri-Sep spin column (Princeton Separations, Aldelphia NJ) and electrophoresed on POP6 capillary at 1500 V for 30 min. Sequencing data were automatically collected and analyzed by the ABI Prism 310 software.
Table A: Primers Used to Determine Genomic Organization of
Cathepsin C
Figure imgf000055_0001
Figure imgf000056_0001
Primers for cDNA templates:
F: 5 ' -GCCGCCCTCCTGCTGCTTCT-3 ' (#30)
R: 5'-GGCTTAGGATTGGGGTCTGA-3'(#31)
We analyzed raw sequence data, generated consensus sequences, and produced nucleotide/amino acid alignments (DNASIS V2.6 for Windows, Hitachi Software Engineering Co., Ltd.). Mutations were detected by creating nucleotide/amino acid alignments of reported cathepsin C sequence data versus affected PLS patients sequence data using the Higgins-Sharpe UPGMA Numbers in parentheses are SEQ ID NOS:.
Example I PLS families
Parents of most families were consanguineous. Linkage studies localized a PLS gene in these five families to chromosome llql4 [8] . Most affected individuals were homozygous for SSTR markers within the PLS candidate interval on chromosome llql4, consistent with inheritance of both maternal and paternal copies of this genetic interval from a common familial ancestor ("identical by descent") . Four different haplotypes for short sequence tandem repeat (SSTR) markers spanning the critical region were identified (Fig. 2), consistent with four independent mutations in the gene responsible for PLS.
Analysis of cathepsin C
Using RT-PCR, we found cathepsin C is normally expressed in epithelium from palms, soles, knees and keratinized oral gingiva from unaffected individuals
(data not shown) . The cathepsin C gene spans approximately 46 kb and consists of 7 exons. Sequence analysis of exonic, intronic and the 5 ' regulatory regions of the cathepsin C gene revealed PLS affected individuals from these families were homozygous for CTSC mutations that significantly altered the cathepsin C open reading frame.
Exon 6 : Two affected individuals from one family were found to have an exon 6 nonsense mutation (856C->T) which introduces a stop codon at amino acid 286 (Fig.
3) . Exon 7 : Three different exon 7 mutations were detected (Fig. 4) . A deletion of a single nucleotide
(1047delA) of codon 349 was found that introduced a frame shift and an early termination codon (TGA) 27 bases downstream. This mutation would result in a mutated protein of 358 amino acids, compared to the normal (wild type) 463 amino acids. A deletion of 2 bases of codon 343 (1028-1029delCT) resulting in the introduction of an early termination codon (TGA) , and a truncated protein of 342 amino acids was identified in another family. A G->A substitution in codon 429 (1286G-
>A) that altered the original TRP codon (TGG) to a terminator codon (TAG) was identified in two affected individuals (#7 and #22) from two additional families.
The expected truncated protein is 428 amino acids. Although these families were not known to be related, the fact that affected individuals from these two
Turkish families are homozygous for a common cathepsin C
61 gene mutation and also share a common haplotype for SSTR markers in the PLS candidate interval flanking the cathepsin C gene (D11S931 - D11S1311) suggests that these individuals have inherited the same cathepsin C gene mutation from a common ancestor. Additional mutations were also identified in Exons 2, 3, 4, 5, 6 and 7. Summaries of the mutations identified to date are set forth in Table I and locations are shown in Figure 5. Papillon Lefevre syndrome is a palmoplantar keratoderma (PPK) with the characteristic clinical features of palmoplantar hyperkeratosis^ and severe periodontal destruction. The PPKs are a heterogeneous group of diseases all having gross thickening of the palmoplantar skin. Clinically, the finding that distinguishes PLS from other PPKs is severe, early onset periodontal destruction. In affected individuals, the development and eruption of the primary teeth proceed normally, but the eruption of these teeth into the oral cavity is associated with gingival inflammation and subsequent rapid destruction of the periodontium. This form of destructive periodontitis is characteristically unresponsive to traditional periodontal treatment modalities, and consequently, the primary dentition is usually exfoliated prematurely. After exfoliation, the inflammation subsides, and the gingiva resumes a healthy appearance. However, with the eruption of the permanent dentition the process is usually repeated, resulting in the premature exfoliation of the permanent dentition, although the third molars are sometimes spared [5] . Destruction of the alveolar bone in PLS is usually severe, resulting in generalized atrophy of the alveolar ridges, further complicating dental therapy.
Because cathepsin C both localized to the refined PLS candidate interval on chromosome llql4 and was normally expressed in epithelium from sites affected by
PLS it was evaluated as a candidate gene for PLS.
„2T7 Cathepsin C, or dipeptidyl aminopeptidase I (EC 3.4.14.1), is a lysosomal cysteine protease capable of removing dipeptides from the terminus of protein substrates, but at higher pH it also exhibits dipeptidyl transferase activity [10] . The cathepsin C gene spans approximately 46 kb and consists of 7 exons that encode a 463-amino acid polypeptide with predicted features of the papain family of cysteine proteases [11] . Unlike cathepsin B, H, L, and S, which are small monomeric enzymes, cathepsin C is a large (200 kD) oligomeric protein that consists of four identical subunits, each composed of three different polypeptide chains [12,13]. Expression of cathepsin C (CTSC) is tissue dependent [14] . CTSC is expressed in pituitary gland, spinal cord, aorta, left atrium, right atrium, left ventricle, right ventricle, inter ventricular septum, apex of heart, esophagus, stomach, duodenum, jejunum, ileum, ileocecum, appendix, ascending colon, transverse colon, descending colon, rectum, kidney, skeletal muscle, spleen, thymus, peripheral blood lymphocytes, lymph node, bone marrow, trachea, lung, placenta, bladder, uterus, testis, liver, pancreas, adrenal gland, thyroid gland, salivary gland, mammary gland, fetal heart, fetal kidney, fetal liver, fetal spleen, fetal thymus, and fetal lung. The CTSC message is also expressed at high levels in immune cells including polymorphonuclear leukocytes and alveolar macrophages and is also expressed at high levels in osteoclasts [11,16]. The pathologic clinical findings of the PLS affected individuals studied here involve severe inflammation and destruction of the gingiva as well as hyperkeratosis of the skin from palmar, plantar and knee sites. In unaffected individuals, cathepsin C is normally expressed in epithelial tissues from sites clinically affected by PLS.
Most parents of the PLS affected individuals in this study are consanguineous. As a result, most PLS affected individuals in each family are homozygous for cathepsin C mutations inherited from a common ancestor. Yet parents and several siblings who are heterozygous carriers for cathepsin C mutations do not appear to show either the palmoplantar hyperkeratosis or severe early onset periodontitis characteristic of PLS. It appears that the presence of one wild type cathepsin C gene is sufficient to prevent PPK and periodontal destruction in most patients. However, 1 mutation identified to date, (1047 A->G) appears to be associated with the presence of dermatological lesions. A consistent finding in the three linkage reports to date is the lack of a common haplotype among affected individuals from different families. The present report describes 20 different cathepsin C gene mutations associated with PLS. These findings suggest that the CTSC mutations responsible for PLS have arisen independently. Several mutations reported here result in the introduction of premature stop codons. While the W429X mutation encodes a protein shortened by only 35 amino acids, the introduced stop codon is 1 amino acid from the asparagine residue in the active site (Fig. 5) . It is likely that such a mutation would cause a conformational alteration that may decrease or abolish activity. Additionally, we have also identified single nucleotide changes that result in missense amino acid changes in several additional PLS affected individuals from other populations, suggesting that structural alterations of cathepsin C may cause PLS. In addition to the cardinal features of PLS, reports suggest some PLS patients have an increased susceptibility to infections [5]. This generalized increased susceptibility to infection may reflect the more deleterious effects of specific cathepsin C mutations, or may reflect the epigenetic effects of other gene loci . A variety of immunological findings have been reported in PLS affected individuals including
5<? decreased onocyte chemotaxis, decreased neutrophil chemotaxis, impaired neutrophil phagocytosis, altered superoxide production, and decreased blastogenic response, but it has been difficult to extrapolate results of these studies. Consequently, the underlying pathogenesis of PLS has been poorly understood [17]. Immunological findings previously reported for affected individuals from the current families includes decreased PMN chemotaxis and elevated CDllb expression [18,19]. The pathologic clinical findings associated with PLS suggest that cathepsin C is functionally important in the structural growth and development of skin and in susceptibility to periodontal disease. As a lysosomal cysteine proteinase, cathepsin C is important in intracellular degradation of proteins and appears to be a central coordinator for activation of many serine proteinases in immune/inflammatory cells [11]. It is unknown if the profound periodontal disease susceptibility is a consequence of altered integrity of junctional epithelium surrounding the teeth. It is interesting that once teeth are exfoliated, and consequently the junctional epithelium is eliminated, the severe gingival inflammation resolves . A more complete understanding of the functional physiology of cathepsin C carries significant implications for understanding periodontal disease susceptibility. Identification of cathepsin C gene mutations in PLS raises the possibility of creating an animal model to study the development, treatment and prevention of hyperkeratosis and periodontitis.
Classification of the PPKs based upon histological findings, epidermolysis and localization of lesions within the skin (diffuse, linear or focal) has not been helpful in understanding the pathomechanism of disease [20] . Identification of mutations in specific genes has led to development of a revised nosology of these diseases in which PLS is grouped with the palmoplantar ectodermal dysplasias [2] . In addition to providing insight into both normal as well abnormal epithelial growth and development, identification of mutations in cathepsin C associated with PLS will contribute to the overall nosology of the PPKs.
EXAMPLE II CTSC Mutation in Haim-Munk Syndrome
Of the many palmoplantar keratoderma (PPK) conditions, only Papillon Lefevre syndrome (PLS) and
Haim Munk syndrome (HMS) are associated with premature periodontal destruction. Although both PLS and HMS share the cardinal features of PPK and severe periodontitis, a number of additional findings are reported in HMS including arachnodactyly, acroosteolysis, atrophic changes of the nails, and a radiographic deformity of the fingers. While PLS cases have been identified throughout the world, HMS has only been described among descendents of a religious isolate originally from Cochin, India. Parental consanguinity is a characteristic of many cases of both conditions. Although autosomal recessive transmission of PLS is evident, a more "complex" autosomal recessive pattern of inheritance with phenotypic influences from a closely linked modifying locus has been hypothesized for HMS. As set forth in Example I, mutations of the cathepsin C gene have been identified as the underlying genetic defect in PLS. To determine if a cathepsin C mutation is also responsible for HMS, we sequenced the gene in affected and unaffected individuals from families with HMS. Here we report identification a mutation of cathepsin C (exon 6, 857A->G) that changes a highly conserved amino acid in the cathpesin C peptide. This mutation segregates with HMS in four nuclear families. Additionally, the existence of a shared common haplotype for genetic loci flanking the cathepsin C gene suggests that affected individuals descended from the Cochin isolate are homozygous for a mutation inherited "identical by descent" from a common ancestor. This finding supports simple autosomal recessive inheritance for HMS in these families. As described above, we also report a mutation of the same exon 6 CTSC codon
(856C->T) in a Turkish family with classic PLS. These findings provide evidence that PLS and HMS are allelic variants of cathepsin C gene mutations.
In addition to congenital palmoplantar keratosis and progressive early onset periodontal destruction, other clinical findings shared by these individuals included recurrent pyogenic skin infections, acroosteolysis , atrophic changes of the nails, arachnodactyly, and a peculiar radiographic deformity of the fingers consisting of tapered pointed phalangeal ends and a clawlike volar curve (Figures 6A and 6B) . Subsequently pes planus was reported to be associated with the syndrome [24] . This was the first reported association of these clinical findings, and the condition became known as Haim Munk syndrome, or keratosis palmoplantaris with periodontopathia and onychogryposis (HMSl; MIM245010) [22] . Although the palmoplantar findings and severe periodontitis were suggestive of the Papillon-Lefevre syndrome (PLS; MIM245000) [3] , the association of other clinical features, particularly nail deformities and arachnodactyly, argued that HMS was a distinct disorder. In contrast to PLS, the skin manifestations in HMS were reported to be more severe and extensive. In addition to a marked palmoplantar keratosis (Figure 6C, 6D) , affected individuals had scaly erythematous and circumscribed patches on the elbows, knees, forearms, shins and dorsum of the hands. While the periodontium in HMS was reported to be less severely affected than in PLS, gingival inflammation and alveolar bone destruction are present and severe (Figure 6E, 6F) . In a subsequent genetic study of this extended family, Hacham-Zadeh and coworkers [25] concluded that the syndrome might not behave as a simple autosomal recessive trait. Based upon their estimate of the disease allele frequency in this population (0.1), the absence of the condition in other kindreds of the Cochin isolate, and an inability to document consanguinity for many of the parents of affected individuals, they hypothesized that a "complex" autosomal recessive inheritance pattern with a closely linked dominant modifier locus may be responsible for the HMS phenotype.
HMS families
Pedigrees of the reported familial relationships for the Cochin descendents are shown in Figure 7A. Descendents of the Cochin isolate studied include sibships 2, 3, 4 and 5 in the kindred pedigree originally described by Hacham-Zadeh and coworkers [25] .
HMS family genotyping results All HMS affected individuals from the Cochin kindred were found to be homozygous for all three polymorphic DNA loci (D11S1887, D11S1780 and D11S1367) flanking the cathepsin C locus. Additionally, these individuals shared a common haplotype for these polymorphic markers. These findings are consistent with inheritance of both maternal and paternal copies of this genetic interval from a common familial ancestor ("identical by descent").
Analysis of cathepsin C in HMS
The cathepsin C gene spans approximately 46 kb and consists of 7 exons. Sequence analysis of exonic, intronic and the 5 ' regulatory regions of the cathepsin C gene revealed that HMS affected individuals from the Cochin kindred were homozygous for a mutation in codon
286 of exon 6 (857A->G) which results in substitution of a conserved glutamine residue at position 286 by an
( 3 arginine: Q286R (Figure 8). This glutamine residue is normally completely conserved in wild type cathepsin C from at least five species (data not shown) . This was the only sequence change different from the reported, highly conserved, wild type CTSC sequence (GenBank
Accession No.: AC011088; SEQ ID NO: 1). All available parents of HMS affected individuals were found to be heterozygous for the mutated (857A->G) allele and the wild type allele. None of the parents or siblings heterozygous for the mutated (857A->G) allele and the wild type allele manifested clinically identifiable characteristics of PPK or had a history of severe, early onset periodontitis.
Restriction Analysis
The Q286R mutation creates an Aval restriction cleavage site. We utilized this newly created restriction site to develop a rapid test to screen for the Q286R mutation. After amplification of a 465bp fragment encompassing the 3 ' end of exon 6 using primers: Forward 5 ' -GTATGCTAGAAGCGAGAATCCGTAT-3 ' (SEQ ID NO: 32) and Reverse 5 ' -CCAATGCTAAAACTTGTTGAGACC-3 ' (SEQ ID NO: 33), the PCR products were purified using the Promega PCR kit according to the manufacturer's instructions. Purified products were eluted in 20 μl water. Approximately 5-10 μl of purified product was digested with 5U Aval (New England Biolabs) in a total volume of 15 μl for 1.5 hr at 37 °C. Following digestion, the products were separated by electrophoresis through an 1.8 % agarose gel.
Amplification of the wildtype sequence results in a 465 bp product that is not cleaved by Aval. Amplification of the mutated (857A(G) sequence results in a 465 bp product that is cleaved by Aval to yield products of 404 and 61 bp . Accordingly, individuals who are homozygous for the wildtype sequence exhibit a 465 bp band. Heterozygous individuals exhibit 3 bands: 465, 404, and 61 bp bands. Individuals who are homozygous for the Q286R mutation exhibit bands of 404 and 61 bp. Restriction analysis confirmed the sequencing results of all examined individuals (Figure 9) .
EXAMPLE III Genetic Screening for PPK-Associated Mutations The foregoing findings provide the basis for screening and diagnostic assays for assessing patients for the presence of mutations in the CTSC gene related to the pathological conditions described herein. A summary of the mutations in CTSC identified as associated with PPKs are set forth in Table 1.
(p Table 1. Phenotype correlations with CTSC mutations
Figure imgf000067_0001
1 . cDNA numbenng considering the initiator Met codon as nucleotide +1. 2 . Phenotype symbols: PLS, Papillon-Lefevre syndrome; PPP, Prepubertal penodontitis;
HMS, Haim Munk syndrome; RP, Retinitis pigmentosa
While the mutations described in the previous examples are associated with certain pathological conditions, it is important to note that the CTSC gene contains many polymorphisms. Many of these genetic changes are not associated with the disease state. The genetic changes assessed by the methods of the present invention must be associated with the production of an aberrant CTSC protein. Accordingly, a suitable assay for diagnosing this disorder includes the step of differentiating harmless polymorphisms from those mutations which give rise to PPKs and periodontal disorders. These include changes in the coding sequence which give rise to decreased mRNA stability as compared to wild type CTSC mRNA. Alternatively cathepsin C enzymatic activity can be compared between altered CTSC coding sequences and nucleic acids encoding the wild type enzyme. Such assays are well known in the art and need not be set forth here. See for example, McGuire et al . , Archives of Biochemistry and Biophysics 295:280-8, 1992; McDonald et al . , J. of Biological Chemistry
244:2693-26709, 1969; Metroione et al, Biochemistry 5:1597-1604, 1966; and Vanha-Perttula et al . , Histochemie 5:170-181, 1965.
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9. Hart TC, Bowden DW, Hart PS, Walker SJ, Callison SA, Bobby PL, Firatli E. An integrated physical and genetic map of the PLS locus interval on chromosome llql4. Mammalian Genome In Press lO.Kirschke, H., Barrett, A.J., Rawlings, N.D. Proteinases 1: Lysosomal cysteine proteinases . In: Protein Profile Vol.2, P. Sheterline, ed. (London, UK: Academic Press Ltd. ) 1995 ; 1587-1643.
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19. Firatli, E., Gurel, N. , Efeoglu, A., Badur, S. Clinical and immunological findings in 2 siblings with Papillon-Lefevre syndrome. J" Periodontol 1996 ; 67 : 1210- 5.
20. Itin, P.H. Classification of autosomal dominant palmoplantar keratoderme: past-present-future. Dermatol 1992;185:163-165. 21. Haim, S., Munk J: Keratosis palmo-plantaris congenita, with periodontosis , arachnodactyly and peculiar deformity of the terminal phalanges. Br J Dermatol 1965;77:42-45. 22. Online Mendelian Inheritance in Man, OMIM (TM) .
Johns Hopkins University, Baltimore, MD . MIM Number: {MIM 245010}: {5/20/97}: World Wide Web URL : http: //www.ncbi .nlm.nih. gov/omim/ 23.Gorlin, R. J., Pindborg, J. J., Cohen, M. M. , Jr.
Syndromes of the Head and Neck. New York: McGraw-Hill (pub.) (2nd ed.) 1976. Pp. 373-376.
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25. Hacham-Zadeh S, Schaap T, Cohen MM. A genetic analysis of the Papillon-Lefevre syndrome in a Jewish family from Cochin. Am J Med Genet 1978;2:153-157.
26 Gorlin RJ, Cohen MM jr., Levin LS . Syndromes of the head and neck . 3rd edn. New York. Oxford University Press, 1990:853-854 (Oxford Monographs on Medical Genetics, No.19).
27. Hart T, Hart PS, Bowden D, Walker S, Callison S, Michalec M, Firatli E. Mutations of the cathepsin C gene are responsible for Papillon Lefevre syndrome. In Press J Med Genet
28. Hart TC, Stabholz A, Meyle J, Shapira L, Van Dyke TE, Cutler CW, Soskolne WA. Genetic studies of syndromes with severe periodontitis and palmoplantar hyperkeratosis. J Periodont Res 1997;32:81-89.
29. Weissenbach J, Gyapay G, Dib C, Vignal A, Morissette J, Millasseau P, Vaysseix G, Lathrop M. A second-generation linkage map of the human genome. Nature 1992 ; 359 : 794- 801 .
30. Lucker GP, Van de Kerkhof PC, Stei len PM. The hereditary palmoplantar keratoses : an updated review and classification. Brit J Derm 1994 ; 131 (1) : 1-14.
31. Smith P, Rosenzweig KA. Seven cases of Papillon Lefevre Syndrome. Periodontics 1967;5:42-6. 32. Cohen T, Bloch N. Immigrant Jews from Cochin. In
Goldschmidt E (ed) The Genetics of Migrant and Isolate Populations. Baltimore: Williams & Wilkins 1963,-352.
33. Wilkie AO . Craniosynostosis : genes and mechanisms. Hum Molee Genet 1997;6:1647-56.
34. Hola-Jamriska L, Tort JF, Dalton JP, Day SR, Fan J, Asskov J and Brindley PJ. Cathepsin C from Schistosoma japonicum; cDNA encoding the preproenzyme and its phylogenetic relationships. Eur J Biochem 1998;255:527-534.
35. Wolters PJ, Raymond WW, Blount JL, Caughey GH. Regulated expression, processing, and secretion of dog mast cell dipeptidyl peptidase I. J Biol Chem 1998;25:15514-15520.
36. Mackenzie IC, Rittman G, Gao Z, Leigh I, Lane EB . Patterns of cytokeratin expression in human gingival epithelia. J Periodont Res 1991 ; 26468-478.
37. Hormia M, Sahlberg C, Thesleff I, Airenne T. The epithelium-tooth interface--a basal lamina rich in laminin-5 and lacking other known laminin isoforms. J Den Res (1998) ; 77 : 1479-85.
38. Stabholz A, Taichman NS, Soskolne WA. Occurrence of Actinobacillus actinomycetemcomitans and anti leukotoxin antibodies in some members of an extended family affected by Papillon-Lefevre syndrome. J Periodontol 1995;66:653-57.
39. Page RC . Altman LC . Ebersole JL . Vandesteen GE . Dahlberg WH. Williams BL . Osterberg SK. Rapidly progressive periodontitis. A distinct clinical condition. Journal of Periodontology. 54 (4) : 197-209 , 1983
40. Page RC . Bowen T. Altman L. Vandesteen E. Ochs H. Mackenzie P. Osterberg S. Engel LD. Williams BL.
Prepubertal periodontitis. I. Definition of clinical disease entity. Journal of Periodontology. 54 (5 ): 257-71, 1983 7 While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.
7/

Claims

What is claimed is:
1. An nucleic acid molecule encoding an altered CTSC protein, said nucleic acid having at least one of the alterations set forth in Table 1.
2. A nucleic acid probe specifically hybridizable to a human altered CTSC-encoding nucleic acid and not to wild-type CTSC encoding nucleic acids, said altered CTSC encoding nucleic acid having one of the alterations set forth in Table 1.
3. The nucleic acid probe of claim 2 wherein said altered CTSC DNA has the alteration comprising a substitution of a C for a T at nucleotide position 856 in Exon 6, thereby replacing a codon encoding glutamine for a stop codon.
4. The nucleic acid probe of claim 2 wherein said altered CTSC DNA has the alteration comprising a substitution of an A for a G at nucleotide position 857 in Exon 6, thereby replacing a codon encoding glutamine for an arginine encoding codon.
5. The nucleic acid probe of claim 2 wherein said altered CTSC DNA has the alteration comprising a deletion of an A at nucleotide position 1047 in Exon 7, thereby causing a frameshift and a premature stop codon.
6. The nucleic acid probe of claim 2 wherein said altered CTSC DNA has the alteration comprising a deletion of a dinucleotide CT at nucleotide positions 1028 and 1029 in Exon 7, thereby causing a premature stop codon.
7. The nucleic acid probe of claim 2 wherein said altered CTSC DNA has the alteration comprising a substitution of a G for a A at nucleotide position 1286 in Exon 7, thereby replacing a tryptophan codon with a premature stop codon.
8. The nucleic acid probe of claim 2 wherein said altered CTSC DNA has the alteration comprising a substitution of a C for a T at nucleotide position 1015 in Exon 7, thereby replacing a codon encoding arginine for a cysteine encoding codon.
9. The nucleic acid probe of claim 2 wherein said altered CTSC DNA has the alteration comprising a substitution of an A for a G at nucleotide position 1019 in Exon 7, thereby replacing a codon encoding tyrosine for a cysteine encoding codon.
10. The nucleic acid probe of claim 2 wherein said altered CTSC DNA has the alteration comprising a substitution of an A for a G at nucleotide position 1040 in Exon 7, thereby replacing a codon encoding tyrosine for a cysteine encoding codon.
11. A mutated CTSC protein encoded by a CTSC encoding nucleic acid, said nucleic acid containing a mutation as set forth in Table 1.
12. An antibody immunologically specific for the protein of claim 11.
13. A method for detecting a germline alteration in a CTSC gene, said alteration selected from the group consisting of the alterations set forth in Table 1 in a human, said method comprising analyzing a sequence of a CTSC gene or CTSC RNA from a human sample or analyzing a sequence of CTSC cDNA made from mRNA from said human sample.
14. The method of claim 13 which comprises analyzing CTSC RNA from the subject.
15. The method of claim 14 wherein a germline alteration is detected by hybridizing a CTSC gene probe which specifically hybridizes to nucleic acids containing at least one of said alterations and not to wild-type CTSC sequences to RNA isolated from said human sample and detecting the presence of a hybridization product, wherein the presence of said product indicates the presence of said alteration in said RNA and thereby the presence of said germline alteration in said sample.
16. The method of claim 13 wherein a germline alteration is detected by obtaining a first CTSC gene fragment from a CTSC gene isolated from said human sample and a second CTSC gene fragment from a wild-type CTSC gene, said second fragment corresponding to said first fragment, forming single-stranded DNA from said first CTSC gene fragment and from said second CTSC gene fragment, electrophoresing said single-stranded DNAs on a non-denaturing polyacrylamide gel, comparing the mobility of said single-stranded DNAs on said gel to determine if said single-stranded DNA from said first CTSC gene fragment is shifted relative to said second CTSC gene fragment and sequencing said single-stranded DNA from said first CTSC gene fragment having a shift in mobility.
17. The method of claim 13 wherein a germline alteration is detected by hybridizing a CTSC probe which specifically hybridizes to nucleic acids containing at least one of said alterations and not to wild-type CTSC sequences to genomic DNA isolated from said sample and detecting the presence of a hybridization product, wherein a presence of said product indicates the presence of said germline alteration in the sample.
The method of claim 13 wherein a germline alteration is detected by amplifying all or part of a CTSC gene in said sample using a set of primers specific for a wild-type CTSC gene to produce amplified CTSC nucleic acids and sequencing the amplified CTSC nucleic acids
19. The method of claim 13 wherein a germline alteration is detected by amplifying all or part of a CTSC gene in said sample using a primer specific for an allele having for one of said alterations and detecting the presence of an amplified product, wherein the presence of said product indicates the presence of said allele in the sample.
20. The method of claim 13 wherein a germline alteration is detected by molecularly cloning all or part of a CTSC gene in said sample to produce a cloned nucleic acid and sequencing the cloned nucleic acid.
21. The method of claim 13 wherein a germline alteration is detected by forming a heteroduplex consisting of a first strand of nucleic acid selected from the group consisting of CTSC gene genomic DNA fragment isolated from said sample, CTSC RNA fragment isolated from said sample and CTSC cDNA fragment made from mRNA from said sample and a second strand of a nucleic acid consisting of a corresponding human wild-type CTSC gene fragment, analyzing for the presence of a mismatch in said heteroduplex, and sequencing said first strand of nucleic acid having a mismatch.
22. The method of claim 13 wherein a germline alteration is detected by amplifying CTSC gene nucleic acids in said sample, hybridizing the amplified nucleic acids to a CTSC DNA probe which specifically hybridizes to nucleic acids containing at least one of said alterations and not to wild-type CTSC sequences and detecting the presence of a hybridization product, n - wherein a presence of said product indicates the presence of said germline alteration.
23. The method of claim 13 wherein a germline alteration is detected by analyzing the sequence of a CTSC gene in said sample for one of the mutations set forth in Table 1.
24. The method of claim 13 wherein a germline alteration is detected by obtaining a first CTSC gene fragment from a CTSC gene isolated from said human sample and a second CTSC gene fragment from a CTSC allele specific for one of said alterations, said second fragment corresponding to said first fragment, forming single-stranded DNA from said first CTSC gene fragment and from said second CTSC gene fragment, electrophoresing said single-stranded DNAs on a non-denaturing polyacrylamide gel and comparing the mobility of said single-stranded DNAs on said gel to determine if said single-stranded DNA from said first CTSC gene fragment is shifted relative to said second CTSC gene fragment, wherein no shift in electrophoretic mobility indicates the presence of said alteration in said sample.
26. The method of claim 13 wherein a germline alteration is detected by obtaining a first CTSC gene fragment from (a) CTSC gene genomic DNA isolated from said sample, (b) CTSC RNA isolated from said sample or (c) CTSC cDNA made from mRNA isolated from said sample and a second CTSC gene fragment from a CTSC allele specific for one of said alterations, said second fragment corresponding to said first fragment, forming single-stranded DNA from said first CTSC gene fragment and from said second CTSC gene fragment, forming a heteroduplex consisting of single-stranded DNA from said first CTSC gene fragment and single-stranded DNA from
74 said second CTSC gene fragment and analyzing for the presence of a mismatch in said heteroduplex, wherein no mismatch indicates the presence of said alteration.
27. A method as claimed in claim 13, wherein said germline alteration comprises a substitution of a C for a T at nucleotide position 856 in Exon 6, thereby replacing a codon encoding glutamine for a stop codon.
28. A method as claimed in claim 13, wherein said germline alteration comprises a substitution of an A for a G at nucleotide position 857 in Exon 6, thereby replacing a codon encoding glutamine for an arginine encoding codon.
29. A method as claimed in claim 13, wherein said germline alteration comprises a deletion of an A at nucleotide position 1047 in Exon 7, thereby causing a frameshift and a premature stop codon.
30. A method as claimed in claim 13, wherein said germline alteration comprises a deletion of a dinucleotide CT at nucleotide positions 1028 and 1029 in Exon 7, thereby causing a premature stop codon.
31. A method as claimed in claim 13, wherein said germline alteration comprises a substitution of a G for a A at nucleotide position 1286 in Exon 7, thereby replacing a tryptophan codon with a premature stop codon.
32. A method as claimed in claim 13, wherein said germline alteration comprises a substitution of a C for a T at nucleotide position 1015 in Exon 7, thereby replacing a codon encoding arginine for a cysteine encoding codon.
33. A method as claimed in claim 13, wherein said germline alteration comprises a substitution of an A for a G at nucleotide position 1019 in Exon 7, thereby replacing a codon encoding tyrosine for a cysteine encoding codon.
34. A method as claimed in claim 13, wherein said germline alteration comprises a substitution of an A for a G at nucleotide position 1040 in Exon 7, thereby replacing a codon encoding tyrosine for a cysteine encoding codon.
35. A method for detecting a germline alteration in a CTSC human encoding nucleic acid, said method comprising comparing a sequence of a CTSC DNA or CTSC
RNA from a human sample with an isolated wild type CTSC sequence as provided in SEQ ID NO : 1.
36. A method as claimed in claim 35, wherein stability of said altered CTSC mRNA is compared with stability of wild type CTSC mRNA.
37. A method as claimed in claim 35, further comprising expressing an altered CTSC protein from said altered CTSC encoding nucleic acid and comparing cathepsin C enzymatic activity of said altered CTSC protein to enzymatic activity of wild-type cathepsin C.
38. A kit for detecting the presence of an altered CTSC encoding nucleic acid in a biological sample, comprising: i) oligonucleotides which specifically hybridize with CTSC encoding nucleic acids having the alterations set forth in Table 1; ii) reaction buffer; and iii) an instruction sheet.
•?r
39. A kit as claimed in claim 38, wherein said oligonucleotide contains a tag.
40. A kit for detecting the presence an altered CTSC encoding nucleic acid in a biological sample, comprising: i) antibodies immunologically specific for the altered CTSC proteins of the invention; ii) a solid support with immobilized CTSC antigens as a positive control; and iii) an instruction sheet.
41. A kit as claimed in claim 40, wherein said antibody contains a tag.
7<
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US7736875B2 (en) 2000-09-08 2010-06-15 Prozymex A/S Dipeptidyl peptidase I crystal structure and its uses

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