WO2017009214A1

WO2017009214A1 - Early and non invasive method for assessing a subject's risk of having a papillon-lefevre syndrome

Info

Publication number: WO2017009214A1
Application number: PCT/EP2016/066239
Authority: WO
Inventors: Francis Gauthier; Brice KORKMAZ; Sandrine DALLET-CHOISY; Yveline HAMON
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Université De Tours François Rabelais
Priority date: 2015-07-10
Filing date: 2016-07-08
Publication date: 2017-01-19

Abstract

The present invention relates to a non-invasive method for assessing a subject's risk of having a Papillon-Lefèvre syndrome, by the identification of the presence or absence of the urinary biomarker associated with Papillon-Lefèvre syndrome.

Description

EARLY AND NON INVASIVE METHOD FOR ASSESSING A SUBJECT'S RISK OF HAVING A PAPILLON-LEFEVRE SYNDROME

FIELD OF THE INVENTION:

The present invention relates to a non-invasive method for assessing a subject's risk of having a Papillon-Lefevre syndrome, by the identification of the presence or absence of the urinary biomarker associated with Papillon-Lefevre syndrome. BACKGROUND OF THE INVENTION:

Papillon-Lefevre syndrome (PLS) is a rare inherited autosomal recessive disorder characterized by a palmoplantar hyperkeratosis and an early and severe periodontitis, causing loss of both the primary and permanent teeth. The prevalence of PLS is 1-4 cases per million persons and the carrier rate is 2 to 4 per 1000. There is no gender predilection but parental consanguinity has been reported in more than 50% of the cases (1,2). The onset of keratoderma may appear at birth or 1 to 2 months of age, but more generally begins simultaneously with periodontitis between the 6th month and the 4th year of life, coinciding with the eruption of the first tooth (3). Other symptoms can also occur such as mental retardation, intracranial calcifications, recurrent skin infections, hyperhidrosis and liver abscesses (2,4). The etiology of the disease has remained misunderstood until its association with mutations in the gene of cathepsin C (CTSC) was demonstrated (5,6). To date, 75 mutations of the CTSC gene, impairing the enzymatic function of cathepsin C (CatC), have been identified (7). Among these 68% were reported in a homozygous form in PLS patients. Fifty percent of homozygous mutations were missense, 25% nonsense, 23% frameshift, and 2% were other types of mutations (7). In addition to the classic form of PLS, 6 cases with late-onset periodontal disease and/or late-onset palmoplantar-lesions were reported (8). But CatC mutations have been also identified in the closely PLS-related Haim-Munk syndrome characterized by the presence of arachnodactyly and pes planus which are absent in PLS (9). The diagnosis of PLS is based on clinical signs and is generally confirmed by a genetic analysis. This latter however may be lacking because of its cost relative to the low socioeconomic status of the patient and/or the unavailability of an appropriate platform of analysis (10,11). Therefore, biomarker in PLS have essentially been relegated to invasive procedures, and the use of biomarker could be substantially facilitated if a non-invasive test were available. The inventors have recently shown that a proteolytically active CatC, a lysosomal cysteine exopeptidase, is secreted by activated neutrophils in lung fluids from patients with chronic inflammatory lung diseases, which makes it a marker of neutrophilic lung inflammation. Inventors also observed that pro-CatC, but not the mature protease, was secreted by bronchial epithelial cells and that MCF-7 epithelial cells secrete both mature and proCatC.

In the present invention it has been demonstrated that urinary and/or renal epithelial cells could well secrete CatC constitutively, so that its absence in urine of PLS patient is an early, simple, reliable, low cost and easy-to perform method of diagnosis of the disease.

SUMMARY OF THE INVENTION:

The present invention relates to a method for assessing a subject's risk of having or developing a PLS comprising the step of measuring in a urinary sample obtained from said subject the presence or absence of CatC, wherein the presence or absence of CatC protein is correlated with the risk of said subject of having or developing a PLS.

The absence of CatC protein in the urine sample is predictive of a high risk of having a

PLS.

Accordingly a subject with CatC protein in their urine sample does not have a PLS. DETAILED DESCRIPTION OF THE INVENTION:

Diagnostic method:

The present inventors have assayed for a statistical association between CatC biomarker detected in urine and PLS using a cohort of subjects. More precisely, the present inventors have assayed for a statistical association between CatC biomarker which is not contained and detected in urine samples from a cohort of LPS patient subjects when 100% of urine samples of control subjects of any age and of both sexes contain measurable amounts of active CatC.

As disclosed in the examples herein, the inventors have screened urine samples of a characterized cohort of LPS subject and the inventors have found that there is a highly statistical linkage between (1) the absence of pro- and/or mature CatC in subject urine, and (2) the risk to develop PLS.

More precisely, the inventors found that the absence of urinary CatC was correlated with presence of mutation in the CTSC gene associated with LPS (either nonsense, frameshift or missense mutations) as shown in Example and Table 1. In contrast, Pro and mature CatC are constitutively present in 100% of the urine of healthy subjects. In conclusion, the inventors found here that the lack of CatC or its proform in the urine is a strong indicator for a reliable non-invasive and early diagnosis of PLS.

The invention provides a method for assessing a subject's risk of having or developing a PLS comprising the step of measuring in a urinary sample obtained from said subject the presence or absence of CatC, wherein the presence or absence of CatC protein is correlated with the risk of said subject of having or developing a PLS.

PLS.

The presence of CatC protein in the urine sample is predictive of a low or zero risk of having a PLS.

Accordingly a subject with CatC protein in their urine sample does not have a PLS.

The invention provides a method for assessing a subject's risk of having or developing a PLS comprising the step of measuring in a urinary sample obtained from said subject the presence or absence of CatC, wherein the absence of CatC protein in the urine sample is predictive of a high risk of having a PLS.

PLS is a rare inherited autosomal recessive disorder characterized by a palmoplantar hyperkeratosis and an early and severe periodontitis, causing loss of both the primary and permanent teeth. The prevalence of PLS is 1-4 cases per million persons and the carrier rate is 2 to 4 per 1000. There is no gender predilection but parental consanguinity has been reported in more than 50% of the cases (1,2). The onset of keratoderma may appear at birth or 1 to 2 months of age, but more generally begins simultaneously with periodontitis between the 6th month and the 4th year of life, coinciding with the eruption of the first tooth (3). Other symptoms can also occur such as mental retardation, intracranial calcifications, recurrent skin infections, hyperhidrosis and liver abscesses (2,4). The etiology of the disease has remained misunderstood until its association with mutations in the gene of CatC (CTSC) was demonstrated (5,6). To date, 75 mutationsof the CTSC gene, impairing the enzymatic function of CatC, have been identified (7). Among these 68% were reported in a homozygous form in PLS patients. Fifty percent of homozygous mutations were missense, 25% nonsense, 23% frameshift, and 2% were other types of mutations (7).

The term "Cathepsin C" also called "CatC" also known as "dipeptidyl peptidase I (DPPI)" should be understood broadly, it encompasses the mature and propeptide CatC, isoforms thereof. CatC, is a lysosomal cysteine exopeptidase belonging to the papain superfamily of cysteine peptidase (12). The gene encoding CatC contains seven exons and six introns (6,13). Functional CatC is a tetrameric enzyme consisting of four identical subunits linked together by non-covalent bonds with a total molecular mass of approximately 200 kDa. Each subunit is composed of three polypeptides chains: a N-terminal fragment or exclusion domain (-13 kDa), a heavy chain (-23 kDa) and a light chain (-7 kDa) (14,15). CatC subunits are synthetized as single chain pro-enzymes of about -60 kDa that are rapidly processed to generate the mature form and remove an about 15 kDa endopropeptide (14,15). The regulation of this processing however is still unknown. CatC is expressed mostly in myeloid cells like neutrophils, macrophages and their precursors but also in the lung, spleen, kidney and liver (13). It has an important role in the activation of various granular serine proteases from neutrophils, mast cells, cytotoxic T- lymphocytes and natural killer cells (16).

The sequence of said protein (human) may be found under the NCBI Reference: ENZYME entry: EC 3.4.14.1.

"Risk" in the context of the present invention, relates to the probability that an event will occur over a specific time period, as in the conversion to PLS, and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l- p) where p is the probability of event and (1- p) is the probability of no event) to no conversion. Alternative continuous measures which may be assessed in the context of the present invention include time to PLS conversion and therapeutic PLS conversion risk reduction ratios.

In the context of the present invention the "risk" is associated with biomarker CatC and PLS phenotypes which in turn may be a risk for developing a PLS. Such methods comprise contacting a urinary sample obtained from the subject to be tested under conditions allowing detection of CatC protein. Once the urinary sample from the subject is prepared, the level of inflammatory biomarkers may be measured by any known method in the art.

For example, the presence of CatC may be measured by using standard immunodiagnostic techniques using anti-CatC antibody, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots, agglutination tests, enzyme-labeled and mediated immunoassays such as ELISA, biotin/avidin type assays, radioimmunoassays, Immunoelectrophoresis, immunoprecipitation .

Specifically, the Anti-CatC antibodies are commercially available:

http://www.labome.com/gene/human/cathepsin-C-antibody.html ;

http://www.scbt.com/datasheet-74590-cathepsin-c-d-6-antibody.html ;

http://www.rndsystems.com/Products/AF1071 ;

http://everestbiotech.com/product/goat-anti-cathepsin-c-dppl-aa238-252-antibody/

In another embodiment, the presence of CatC may also be measured by using standard immunochemical methods (Western-blotting) (17) and enzyme assays (18) in order to detect the proteolytic activity of CatC as described in the Example.

In some embodiments, the present invention provides devices that are useful to detect and/or visualize the presence of CatC in a liquid sample (like urine). These devices may comprise a surface and the anti-CatC antibody and/or a reactant of the enzyme assay. Solid- phase assay devices include microtiter plates, flow-through assay devices (e.g., lateral flow immunoassay devices), dipsticks, and immunocapillary or immunochromatographic immunoassay devices. A particularly useful assay format is a lateral flow immunoassay format. In some embodiments, the device includes a solid support that contains a sample application zone and a capture zone. The lateral flow immunoassay (LFA) is a particular embodiment that allows the user to perform a complete immunoassay within 10 minutes or less. Those skilled in the art know many embodiments and variations of the lateral flow format, including: a variety of porous materials including nitrocellulose, polyvinylidene difluoride, paper, and fiber glass; a variety of test strip housings; colored and fluorescent particles for signal detection including colloidal metals, sols, and polymer latexes; a variety of labels, binding chemistries, and other variations. Various known formats exist for immunochromatographic test strips for detecting analytes in liquid samples (like urine). One format of LFA uses a direct binding "sandwich" assay, wherein the analyte is bound by two specific binding molecules which can thus include anti-CatC antibody. Examples of LFA format are described in U.S. Pat. No. 4,861,711; H. Friesen et al. (1989), which discloses a solid-phase diagnostic device for the determination of biological substances; U.S. Pat. No. 4,740,468; L. Weng et al. (1988) which discloses a solid phase specific binding method and device for detecting an analyte; U.S. Pat. No. 4,168,146; A. Grubb et al. (1979) which discloses a solid phase method and strip with bound antibodies and U.S. Pat. No. 4,435,504; R. Zuk (1984) which discloses a chromatographic immunoassay employing a ligand-binding molecule and a label conjugate. In one type of this format, described in U.S. Pat. No. 4,959,307; J. Olson (1990), the result is revealed as two lines (positive result) or one line (negative result). Another particularly useful assay format is a flow-through immunoassay format. Flow-through immunoassay devices involve a capture reagent (i.e. anti-CatC antibody) bound to a porous membrane or filter to which a liquid sample is added. As the liquid flows through the membrane, target analyte (i.e. CatC) binds to the capture reagent. The addition of sample is followed by (or made concurrent with) addition of detector reagent, such as labeled (e.g., gold-conjugated or colored latex particle-conjugated protein). Alternatively, the detector reagent may be placed on the membrane in a manner that permits the detector to mix with the sample and thereby label the analyte. The visual detection of detector reagent provides an indication of the presence of target analyte in the sample. Representative flow-through assay devices are described in U.S. Pat. Nos. 4,246,339; 4,277,560; 4,632,901; 4,812,293; 4,920,046; and 5,279,935; U.S. Patent Application Publication Nos. 20030049857 and 20040241876; and WO 08/030,546. Migration assay devices usually incorporate within them reagents that have been attached to colored labels, thereby permitting visible detection of the assay results without addition of further substances. See, for example, U.S. Pat. No. 4,770,853; PCT Publication No. WO 88/08534 and European Patent No. EP-A 0 299 428. There are a number of commercially available lateral flow type tests and patents disclosing methods for the detection of large analytes (MW greater than 1,000 Daltons). U.S. Pat. No. 5,229,073 describes a semi-quantitative competitive immunoassay lateral flow method for measuring liquid protein levels. This method utilizes a plurality of capture zones or lines containing immobilized antibodies to bind both the labeled and free protein to give a semi-quantitative result. In addition, U.S. Pat. No. 5,591,645 provides a chromatographic test strip with at least two portions. The first portion includes a movable tracer and the second portion includes an immobilized binder capable of binding to the analyte. Additional examples of lateral flow tests for large analytes are disclosed in the following patent documents: U.S. Pat. Nos. 4,168,146; 4,366,241; 4,855,240; 4,861,711; and 5,120,643; European Patent No. 0296724; WO 97/06439; WO 98/36278; and WO 08/030,546. Devices described herein generally include a strip of absorbent material (such as a microporous membrane), which, in some instances, can be made of different substances each joined to the other in zones, which may be abutted and/or overlapped. In some examples, the absorbent strip can be fixed on a supporting non-interactive material (such as nonwoven polyester), for example, to provide increased rigidity to the strip. Zones within each strip may differentially contain the specific binding partner(s) and/or other reagents required for the detection and/or quantification of the particular analyte being tested for, for example, one or more CatC disclosed herein (Pro or mature form). Thus these zones can be viewed as functional sectors or functional regions within the test device. In general, a fluid sample is introduced to the strip at the proximal end of the strip, for instance by dipping or spotting. The fluid migrates distally through all the functional regions of the strip. The final distribution of the fluid in the individual functional regions depends on the adsorptive capacity and the dimensions of the materials used.

Lateral flow devices are commonly known in the art. Briefly, a lateral flow device is an analytical device having as its essence a test strip, through which flows a test sample fluid that is suspected of containing an analyte of interest. The test fluid and any suspended analyte can flow along the strip to a detection zone in which the analyte (CatC if present) interacts with a capture agent (i.e. the anti-CatC antibody) and a detection agent to indicate a presence, absence and/or quantity of the analyte. Numerous lateral flow analytical devices have been disclosed, and include those shown in U.S. Pat. Nos. 4,313,734; 4,435,504; 4,775,636; 4,703,017; 4,740,468; 4,806,311; 4,806,312; 4,861,711; 4,855,240; 4,857,453; 4,943,522; 4,945,042; 4,496,654; 5,001,049; 5,075,078; 5,126,241; 5,451,504; 5,424,193; 5,712,172; 6,555,390; 6,258,548; 6,699,722; 6,368,876 and 7,517,699; EP 0810436; and WO 92/12428; WO 94/01775; WO 95/16207; and WO 97/06439, each of which is incorporated by reference. Many lateral flow devices are one-step lateral flow assays in which a biological fluid is placed in a sample area on a bibulous strip (though non-bibulous materials can be used, and rendered bibulous, e.g., by applying a surfactant to the material), and allowed to migrate along the strip until the liquid comes into contact with a specific binding partner (such as an antibody) that interacts with an analyte (such as one or more molecules) in the liquid. Once the analyte interacts with the binding partner, a signal (such as a fluorescent or otherwise visible dye) indicates that the interaction has occurred. Multiple discrete binding partners (such as the polypeptide of the present invention) can be placed on the strip (for example in parallel lines) to detect multiple analytes (such as two or more molecules) in the liquid. The test strips can also incorporate control indicators, which provide a signal that the test has adequately been performed, even if a positive signal indicating the presence (or absence) of an analyte is not seen on the strip. The construction and design of lateral flow devices is very well known in the art, as described, for example, in Millipore Corporation, A Short Guide Developing Immunochromatographic Test Strips, 2nd Edition, pp. 1-40, 1999, available by request at (800) 645-5476; and Schleicher & Schuell, Easy to Work with Bioscience, Products and Protocols 2003, pp. 73-98, 2003, 2003, available by request at Schleicher & Schuell Bioscience, Inc., 10 Optical Avenue, Keene, N.H. 03431, (603) 352-3810; both of which are incorporated herein by reference.

In a preferred embodiment the CatC protein is detected using a rapid Lateral flow devices such as a urine test strip, a basic diagnostic tool used to determine pathological changes in a patient' s urine in standard urinalysis.

Indeed, besides having discovered that CatC is present and detectable in urine of healthy subject, the inventors have also demonstrated that the urinary absence of CatC is strictly correlated with the presence of mutation in CTSC gene (coding for CatC). When the inventors detected in 2 patients the presence of pro-CatC in their urine (but the concentration of mature CatC was abnormally low) the subsequent genetic analysis confirmed the absence of mutation of the CatC gene.

Accordingly the invention also provides a method for assessing in a subject the presence of mutation in CatC gene (CTSC) which affected the expression of the CatC gene comprising the step of measuring in a urinary sample obtained from said subject the level of CatC, wherein :

The absence of CatC protein is predictive of a high risk of the presence of mutation in CatCgene (CTSC) which affected the expression of the CatC gene.

The presence of CatC protein is predictive of a low risk of the presence of mutation in CatC gene (CTSC) which affected the expression of the CatC gene.

The method according to the invention may also be combined with other methods for assessing the risk to have a PLS. Examples of such methods are well-known in the art. Classical methods for evaluating risk factors include, but are not limited to, assessing mutation(s) in the CatC gene (CTSC) that affect its expression. Mutations of the CTSC gene, impairing the enzymatic function of CatC, have been described in (6,15). The invention also relates to the use of urinary form of CatC as a marker of PLS risk.

The invention also provides an in vitro method for diagnosing PLS in a subject, said method comprising the step of detecting in a urinary sample obtained from the subject the presence or absence of CatC, wherein the presence or absence of CatC protein is correlated with the diagnosis of a PLS.

The absence of CatC protein in the urine sample is indicative of a PLS in the subject.

The presence of CatC protein in the urine sample is indicative of the absence of a PLS in the subject.

In this method, the absence of detection of CatC in the urine sample may be indicative of early PLS in the subject.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: CatC in urine from healthy subjects. Immunoblots of 20 fold- concentrated samples of urine collected from healthy subjects, as revealed using anti-CatC Abl which recognizes an epitope on the heavy chain (A), Ab2 directed against the propeptide (B) and AB3 which recognize another epitope on the heavy chain of CatC (C). Recombinant CatC was used as a control.

Figure 2: Constitutive and constant over time presence of CatC in urine from healthy children and adults. (A) Immunoblots of 20 fold-concentrated samples of urine collected from healthy children (from 3 months (MO) to 3 years (YO)) compared to a healthy adult, as revealed using anti-CatC Abl. (B) Immunoblots of 20 fold-concentrated samples of urine collected at different time of the day from one healthy woman and two healthy men, as revealed using anti-CatC Abl Recombinant CatC was used as a control. (C) CatC activity in 20 fold-concentrated urines from 50 healthy subjects using the fluorogenic substrate Gly-Phe- AMC (50 μΜ) and inhibition in presence of E64c (100 μΜ) and L-Thi-L-Phe (5 μΜ).

Figure 3: Absence of CatC in urine from PLS patients. Summarized protocol of urine samples treatment (A). Immunoblots of 20 fold-concentrated samples of urine collected from two healthy subjects (controls Ctl/Ct2) and of 120 fold-concentrated samples of urine collected from one representative PLS patient (P#16), as revealed using anti-CatC Abl (B), Ab2 (C) and AB3 (D). CatC activity in a 20 fold-concentrated urines from one healthy subject (control Ctl) and of 120 fold-concentrated samples of urine collected from one representative PLS patient (P#16) using the fluorogenic substrate Gly-Phe-AMC (50 μΜ) (E). Recombinant CatC was used as a control.

Figure 4: CatC in urine from a patient with a PLS-like phenotype (P#21) without CTSC mutation. (A) Pedigree of the french family of P#21. (B) Immunoblotting analysis with anti-CatC Abl antibody of 120 fold-concentrated urines from P#21 and his mother and of 20 fold-concentrated urines from a healthy control (Ct). (C) Western blotting of white blood cell lysates from P#21, his mother and a healthy control (Ct).

Figure 5: CatC in urine and white cell lysate from a patient with a PLS-like phenotype (P#22) without CTSC mutation. (A) Immunoblotting analysis with anti-CatC Abl antibody of a 120 fold-concentrated urine sample from patient P#22 and of a 20 fold- concentrated urine sample collected from a healthy subject (Ct). (B) Immunoblots of white blood cell (0,5.10⁶ cells) and purified PMN lysate from patient P#16 and from a healthy subject (Ct), as revealed using anti-CatC and anti-haptoglobin Abs.

EXAMPLE:

Material & Methods

Reagents

Recombinant CatC and aminopeptidase N (APN) were from Unizyme Laboratories

(H0rsholm, Denmark) and R&D systems (Lille, France), respectively. antiCatC antibodies were used in this study: the mouse monoclonal anti-CatC Ab (Abl) (Santa Cruz Biotechnology, Heidelberg, Germany), the goat polyclonal anti-CatC Ab (Ab2) (R&D Systems) and the goat polyclonal anti-CatC (Ab 3) (Everest Biotech, Oxforshire, UK). The mouse monoclonal anti-Aminopeptidase N Ab was from Santa Cruz Biotechnology. Gly-Phe- AMC was from Enzyme Systems Products (Illkirch, France) whereas H-Ala-AMC was supplied by Bachem (Weil am Rhein, Germany). The cysteine protease inhibitor E-64c ((2S,3S)-iraw5^,-Epoxysuccinyl-L-leucylamido-3-methylbutane) was from Sigma-Aldrich (St Louis, MO) and the specific inhibitor of CatC, L-Thi-L-Phe was kindly provided by Dr Lesner (University of Gdansk, Poland). EDTA or Ethylenediaminetetraacetic acid was from Merk (Darmstadt, Germany) and the specific inhibitor of Aminopeptidase N, Bestatin was from Santa Cruz. Urine collection and analysis

Urine samples were collected from 22 PLS patients from various countries and from 75 healthy volunteers. Samples were obtained after written informed consent and after ethics committee approval (Comite de Protection des Personnes, CPP OUEST-1, Tours, France). Demographics and clinical data from PLS patients are summarized in Table I.

Urine supernatants were collected after centrifugation (3000 g, 15 min), then concentrated 20 or 120 times (Vivaspin 15R concentrators cutoff, 10 kDa (Sartorius, Goettingen, Germany) and stored at 4°C. Cell counting was made after cytospin centrifugation (CytoSpin™ 4 Cytocentrifuge (ThermoScientific)) and microscopic analysis of May-Grunwald-Giemsa- stained slides. For other experiments, the cell pellets obtained after centrifugation (10000 g, 15 min at 4°C) were lysed in PBS containing 0.5 % nonidet P- 40(IGEPAL-630), and stored at -20°C until use.

Western blotting analysis

Briefly, urine samples were electrophoretically separated on a 10% SDS-PAGE in reducing conditions for CatC analysis and in non-reducing conditions for APN analysis, and then transferred to a nitrocellulose membrane (Amersham Biosciences, Uppsala, Suede). After saturation, membranes were incubated with anti-CatC Abs (Ab 1 diluted 1:750; Ab 2 diluted 1: 1000; Ab 3 diluted 1:500) or with anti-APN mAb (diluted 1:500). After washing, membranes were incubated with peroxidase-conjugated anti-mouse IgG (diluted 1: 10000) or anti-goat IgG Abs (diluted 1:20000) (Sigma-Aldrich), when appropriate. Bound Abs were detected by chemiluminescence (ECL Plus Western Blotting Kit Detection Reagents, GE Healthcare, UK) according to the manufacturer's instruction.

Enzyme assays Assays were carried out at 37°C in 50 mM sodium acetate, 30 mM NaCl, 1 mM EDTA, 2 mM DTT, pH 5,5 for CatC and in 50 mM Tris, pH 7 for APN. Proteolytic activity was measured using 5 μΐ_^ of urine supernatants with 30 μΜ Gly-Phe-AMC in a total volume of 60 μΐ. for CatC or with 50 μΜ H-Ala-AMC in a total volume of 100 μΐ. for APN (excitation wavelength = 340 nm, emission wavelength = 460 nm; Spectramax Gemini (Molecular Devices, Sunnyvale, CA)). Recombinant DPPI and APN were used as controls, respectively. For some experiments, urine samples were preincubated with 100 μΜ E64c, 5 μΜ L-Thi-L-Phe, 5 mM EDTA or 100 μΜ bestatine for 30 min at 37°C before to measure the proteolytic activity.

Genetic analysis

Patient's genomic DNA and when available parents' DNA was extracted from EDTA blood samples with the automated method EVO100 ReliaPrep (Tecan, Promega).

PCR and sequencing reaction. The in vitro amplification and sequencing of all CTSC exons and intron-exon boundaries were performed as described in (19). The in silico analysis of missense mutations and the database queries were conducted through the Alamut Interface (Alamut v2.3). Mutations were described in accordance with the CTSC cDNA sequence GenBank NM_001814.2 and HGVS recommendations.

Custom array CGH Custom microarrays (8 x 60K) were designed with e-array web software (Agilent Technologies, CA) using the Similarity Score Filter in order to select highly specific probes. A total of 3141 probes were distributed: about one probe every 100 bp in the CTSC gene and 50 Kb around the gene then one probe every 350 bp in the 300 Kb regions apart. DNA was labelled (cyanin 3 or cyanin 5) using the Sure Tag DNA labeling kit from Agilent Technologies and hybridized onto the microarrays according to the manufacturer's instructions. DNA was analyzed in comparative genomic hybridization experiments with fluorochrome swapping, in a trio along with DNA from two subjects not affected by the PLS. Scanning of the microarrays was performed using a G2565CA scanner (Agilent Technologies). Data analysis was carried out with softwares from Agilent Technologies, namely Feature Extraction 10.7.3.1 and Agilent Genomic Workbench 6.0.130.24.

Results

Clinical signs of PLS become apparent by the age of one to five years when dry, scaly patches appear on the skin of palms and soles, and severe inflammation starts to affect the surrounding of primary teeth, leading to their rapid lost. The lack of expression of CatC is responsible of these clinical signs but the relationship between protease activity and dermal and periodontal pathogenesis still remains to be clarified. CatC is expressed as a proprotease by epithelial and myeloid cells and their precursors and is activated by a multistep mechanism possibly involving several proteases (13,18,20). Thus pro- and/or mature CatC may be constitutively present in a variety of cells and tissues including renal and/ or bladder epithelial cells. We made here the hypothesis that urine from healthy individuals might well contain pro- or active CatC in sufficient amount to be systematically and easily detected by enzymatic and/or immunochemical techniques. The absence of urinary CatC would thus be indicative of a strong suspicion of PLS.

Pro and mature CatC are constitutively present in urine of healthy subjects

We first analysed by immunoblotting the putative presence of CatC in 20-fold concentrated urine supernatants from 50 healthy individuals aged 3 months to 80 years, using three different anti-CatC commercial antibodies (Abl, Ab2, Ab3). Abl and Ab3 recognized two different epitopes on the heavy chain whereas Ab2 recognized one epitope on the propeptide region. We observed two immunoreactive bands with an apparent molecular mass of -23 kDa and -60 kDa in 100% of the urine samples. These bands corresponded to the heavy chain of mature CatC and to pro-CatC respectively (Figure 1). The presence of pro and mature CatC did not depend on the time of urine sampling, on the age and sex of the donor (Figure 2). All of the 50 urine samples hydrolysed the cat C substrate Gly-Phe-MCA and all peptidic activities were fully inhibited by the selective CatC nitrile inhibitor L-Thi-L-Phe-CN (21) indicating that the measured activity was specifically due to CatC. Based on the rate of hydrolysis of Gly-Phe-MCA by recCat C, we estimated the concentration of active CatC in normal urine in the 1-10 nanomolar range. Next question was to document the origin of urinary CatC. Since tetrameric CatC would hardly cross the kidney filtration barrier because of its Mr of -240,000, there is little chance that urinary catC is filtrated from the circulation. Moreover we did not detect any pro or mature CatC in plasma by immunoblotting {data not shown). We found however that epithelial bladder cells T24/T24M produced and secreted proCatC but not the mature protease. We then compared the ratio proCatC /active CatC in a urine samples before and after a 24h incubation time at 37°C to know whether proCatC could be matured into active CatC in urine. We observed no change which means that proCat C is activated in bladder or renal cells before its secretion in urine.

CatC is not present in urine of PLS patients 20 urine samples were collected from notified or suspected PLS patients issuing from different European, American and Asian countries (Table I). Urine samples were centrifuged upon receipt, concentrated (xl20) and analysed for the presence and activity of CatC as described for controls. A genetic-based PLS diagnosis was previously established for 8 patients allowing the identification of either nonsense, frameshift or missense mutations in the CTSC gene (Table I). In spite of a greater concentration of PLS urine samples (xl20 vs x20 in controls), no pro or active CatC was detected in any of these patients whatever the type of mutation. We used the measurement of aminopeptidase N activity as a positive control to check the quality of the urine sample.

We then analysed CatC in the urine of 7 patients whose PLS diagnosis was established only on clinical features (Table 1). Again, no CatC was detected in their urine and we confirmed the PLS diagnosis by a genetic analysis allowing detection of the mutation (Figure 3, Table 1). Urinary Cat C profile of patients with a PLS phenotype

We first analysed the urine from a 16-year-old French teenager (P#21) who presented cutaneous manifestations suggesting a case of PLS but the genetic analysis revealed no mutation of the CatC gene. Indeed, we showed the presence of pro- and mature CatC in his urine and that of her mother. Urinary CatC activity was fully inhibited by the specific CatC inhibitor L-Thi-L-Phe-CN (21). Further, proteolytically active elastase-like proteases were present in the white blood cells lysates of the patient and her mother indicating that functional CatC was present in cells and tissues.

We also analyzed the urine from a 15-year-old Turkish boy (P#22) with classical dental and dermatologic characteristics of PLS but no genetic analysis had been performed. He also had bilateral, corneal leukomas (22). We observed the presence of pro and mature CatC in his urine. We were unable to fully inhibit the activity measured using the substrate GF-AMC in presence of the specific inhibitor of CatC, L-Thi-L-Phe, perhaps due to another unidentified protease cleaving the substrate. We also observed the presence and the activity of mature CatC in his white blood cell lysate. White blood cells from P#22 also contained proteolytically active proteinase 3 (PR3) and huge amounts of myloperoxydase. As expected, we found no mutation in the CatC gene.

The genetic etiology of PLS has now been clearly established but the reason why mutations in the CatC gene result in dermatological lesions and destructive periodontitis remains to be clarified. Evidence has been recently provided that the lack of neutrophil protease 3 activation by CatC in PLS patients lead to the deficit of antimicrobial and immunomodulatory functions of the antimicrobial peptide LL-37 in the gingiva, allowing for infection with A. actinomycetemcomitans and the development of severe periodontal disease (23). Several diseases however can mimic PLS so that dermatological lesions and destructive periodontitis may not be related to PLS and may occur as independent diseases (24). Because of the variable extent of periodontal breakdown, PLS may be misdiagnosed or not acknowledged and could be far more prevalent than documented (25). The diagnosis and the care of this syndrome would thus need a multidisciplinary approach as suggested by the use of radiological examinations. But we found here that the lack of CatC or its proform in the urine was a strong indicator for a reliable and early diagnosis of PLS. In this regard the extremely rare Haim-Munk syndrome, also characterized by a lack of catC but a slightly different phenotype, should be classified as a PLS-like pathology because of its common origin.

Unlike saliva or gingival crevicular fluid that retain no significant CatC activity in control subjects, 100% of urine samples of control subjects of any age and of both sexes contain measurable amounts of active CatC whereas urines of PLS patients do not contain any. The importance of an early treatment to avoid the progression of periodontotitis and skin lesions and to improve the quality of life of PLS patients has been highlighted (26). Demonstrating the absence of urinary CatC activity soon after birth and before the clinical signs of PLS have appeared certainly goes in this direction. For this purpose CatC detection strips that could measure both the immunoreactivity and the enzymatic activity of the protease could well be developed.

Patients Ethnicity Gender Age Clinical manifestations Mutation Urinary Cat C

proCat Mature

C Cat C

P#l French F 55 c.96T>G

(p.Y32X)

Nonsense

P#2 Indian M 15 Transgradient c.912C>A

palmoplantar (p.Y304X)

keratoderma, Nonsense

periodontitis

P#3 Hungarian F 4 Palmoplantar c.681delCAT

hyperkeratosis, ACAT

periodontitis (p.T188fsX19

9)

Frameshift

P#4 Hungarian F 13 Palmoplantar c.681delCAT

hyperkeratosis, severe ACAT

periodontitis (p.T188fsX19

9)

Frameshift

P#5 Italian F 42 Palmoplantar c. l l41delC

hyperkeratosis, severe (p.L381fsX39

periodontitis 3)

Frameshift

P#6 Hungarian F c.901G>A

(p.G301S)

Missense

P#7 Hungarian F c.901G>A

(p.G301S)

Missense

P#8 Erythrea M 12 Palmoplantar c.755A>T

hyperkeratosis, severe (p.Q252L)

periodontitis, Tinea Missense

capitis

P#9 Erythrea M 15 Palmoplantar c.755A>T

hyperkeratosis, severe (p.Q252L)

periodontitis Missense

P#10 Morocco M 19 Mild palmoplantar c.854C>T

hyperkeratosis, severe (p.P285L)

periodontitis Missense P#l l* German M 48 Severe palmoplantar c.322A>T7

hyperkeratosis, late c.436delT

onset of severe (p.K108X /

periodontitis (22 years p.S146fsl53X

of age), liver abscess )

Nonsense /

Frameshift

P#12 German M 27 Palmoplantar comp.

hyperkeratosis, severe heterozyg

periodontitis c.947T>G

(pL316R)

c. l268G>C

(_PW423S)

P#13 Morocco M 35 Palmoplantar c.854C>T

hyperkeratosis, severe (p.P285L)

periodontitis, Missense

edentulous by now

P#14 German F 24 Palmoplantar c.566-572Del

hyperkeratosis, severe (T189FS199X

periodontitis, )

edentulous by now

P#15 Puerto F 31 Palmoplantar c. l l6G>C

Rican hyperkeratosis, severe (p.W39S)

periodontitis Missense

P#16 Persian F 33 Palmoplantar c.815G>C

hyperkeratosis, severe (p.R272P)

periodontitis Missense

P#17 Persian M 9 Palmoplantar c.815G>C

hyperkeratosis, (p.R272P)

periodontitis Missense

P#18 Persian M 4 c.815G>C

(p.R272P)

Missense

P#19 Saudi M 17 c.815G>C

arabian (p.R272P)

Missense

P#20 Saudi M 24 c.815G>C

arabian (p.R272P)

Missense

P#21 French M 20 Palmoplantar No mutation ++++ ++++ psoriasiform

appearance, anomalies

of deciduous teeth with

no net periodontitis

P#22 Turkish M 15 Palmoplantar No mutation ++++ ++++

hyperkeratosis, severe

periodontitis

TABLE I

Patients' demographic and clinical data

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

1. Gorlin, R. J., Sedano, H., and Anderson, V. E. (1964) The Syndrome of Palmar- Plantar Hyperkeratosis and Premature Periodontal Destruction of the Teeth. A Clinical and Genetic Analysis of the PapiUon-Lefevre Syndrome. The Journal of pediatrics 65, 895-908

2. Haneke, E. (1979) The PapiUon-Lefevre syndrome: keratosis palmoplantaris with periodontopathy. Report of a case and review of the cases in the literature. Human genetics 51, 1-35

3. Dhanrajani, P. J. (2009) PapiUon-Lefevre syndrome: clinical presentation and a brief review. Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics

108, el -7

4. Almuneef, M., Al Khenaizan, S., Al Ajaji, S., and Al-Anazi, A. (2003) Pyogenic liver abscess and PapiUon-Lefevre syndrome: not a rare association. Pediatrics 111, e85-88

5. Hart, T. C, Hart, P. S., Bowden, D. W., Michalec, M. D., Callison, S. A., Walker, S.

J., Zhang, Y., and Firatli, E. (1999) Mutations of the cathepsin C gene are responsible for PapiUon-Lefevre syndrome. Journal of medical genetics 36, 881-887

6. Toomes, C, James, J., Wood, A. J., Wu, C. L., McCormick, D., Lench, N., Hewitt, C, Moynihan, L., Roberts, E., Woods, C. G., Markham, A., Wong, M., Widmer, R., Ghaffar, K. A., Pemberton, M., Hussein, I. R., Temtamy, S. A., Davies, R., Read, A. P., Sloan, P., Dixon, M. J., and Thakker, N. S. (1999) Loss-of-function mutations in the cathepsin C gene result in periodontal disease and palmoplantar keratosis. Nature genetics 23, 421-424

7. Nagy, N., Valyi, P., Csoma, Z., Sulak, A., Tripolszki, K., Farkas, K., Paschali, E., Papp, F., Toth, L., Fabos, B., Kemeny, L., Nagy, K., and Szell, M. (2014) CTSC and PapiUon-Lefevre syndrome: detection of recurrent mutations in Hungarian patients, a review of published variants and database update. Molecular genetics & genomic medicine 2, 217-228

8. Pilger, U., Hennies, H. C, Truschnegg, A., and Aberer, E. (2003) Late-onset PapiUon- Lefevre syndrome without alteration of the cathepsin C gene. Journal of the American Academy of Dermatology 49, S240-243

9. Hart, T. C, Hart, P. S., Michalec, M. D., Zhang, Y., Firatli, E., Van Dyke, T. E., Stabholz, A., Zlotogorski, A., Shapira, L., and Soskolne, W. A. (2000) Haim-Munk syndrome and PapiUon-Lefevre syndrome are allelic mutations in cathepsin C. Journal of medical genetics 37, 88-94

10. Keskin-Yildirim, Z., Simsek-Derelioglu, S., Kantarci, M., Yilmaz, Y., and Buyukavci, M. (2012) PapiUon-Lefevre syndrome: report of three cases in the same family. The Turkish journal of pediatrics 54, 171-176

11. Kord Valeshabad, A., Mazidi, A., Kord Valeshabad, R., Imani, E., Kord, H., Koohkan, M., Sayinar, Z., and Al-Talib, K. (2012) Papillon-lefevre syndrome: a series of six cases in the same family. ISRN dermatology 2012, 139104

12. Rawlings, N. D., and Barrett, A. J. (1993) Evolutionary families of peptidases. The Biochemical journal 290 ( Pt 1), 205-218 13. Rao, N. V., Rao, G. V., and Hoidal, J. R. (1997) Human dipeptidyl-peptidase I. Gene characterization, localization, and expression. The Journal of biological chemistry 272, 10260-10265

14. Dolenc, I., Turk, B., Pungercic, G., Ritonja, A., and Turk, V. (1995) Oligomeric structure and substrate induced inhibition of human cathepsin C. The Journal of biological chemistry 270, 21626-21631

15. Turk, D., Janjic, V., Stern, I., Podobnik, M., Lamba, D., Dahl, S. W., Lauritzen, C, Pedersen, J., Turk, V., and Turk, B. (2001) Structure of human dipeptidyl peptidase I (cathepsin C): exclusion domain added to an endopeptidase framework creates the machine for activation of granular serine proteases. The EMBO journal 20, 6570-6582

16. Pham, C. T., and Ley, T. J. (1999) Dipeptidyl peptidase I is required for the processing and activation of granzymes A and B in vivo. Proceedings of the National Academy of Sciences of the United States of America 96, 8627-8632

17. Korkmaz, B., Lesner, A., Letast, S., Mahdi, Y. K., Jourdan, M. L., Dallet-Choisy, S., Marchand-Adam, S., Kellenberger, C, Viaud-Massuard, M. C, Jenne, D. E., and

Gauthier, F. (2013) Neutrophil proteinase 3 and dipeptidyl peptidase I (cathepsin C) as pharmacological targets in granulomatosis with polyangiitis (Wegener granulomatosis). Seminars in immunopathology 35, 411-421

18. Dahl, S. W., Halkier, T., Lauritzen, C, Dolenc, I., Pedersen, J., Turk, V., and Turk, B.

(2001) Human recombinant pro-dipeptidyl peptidase I (cathepsin C) can be activated by cathepsins L and S but not by autocatalytic processing. Biochemistry 40, 1671- 1678

19. Jouary, T., Goizet, C, Coupry, I., Redonnet-Vernhet, I., Levade, T., Burgelin, I., Toutain, A., Delaporte, E., Douillard, C, Lacombe, D., Taieb, A., and Arveiler, B. (2008) Detection of an intragenic deletion expands the spectrum of CTSC mutations in

PapiUon-Lefevre syndrome. The Journal of investigative dermatology 128, 322-325

20. Mallen-St Clair, J., Shi, G. P., Sutherland, R. E., Chapman, H. A., Caughey, G. H., and Wolters, P. J. (2006) Cathepsins L and S are not required for activation of dipeptidyl peptidase I (cathepsin C) in mice. Biological chemistry 387, 1143-1146 21. Methot, N., Rubin, J., Guay, D., Beaulieu, C, Ethier, D., Reddy, T. J., Riendeau, D., and Percival, M. D. (2007) Inhibition of the activation of multiple serine proteases with a cathepsin C inhibitor requires sustained exposure to prevent pro-enzyme processing. The Journal of biological chemistry 282, 20836-20846

22. Saatci, P., Arli, A. O., Demir, K., Saatci, A. O., and Kavakcu, S. (2006) Corneal involvement in PapiUon-Lefevre syndrome. Journal of pediatric ophthalmology and strabismus 43, 167-169

23. Eick, S., Puklo, M., Adamowicz, K., Kantyka, T., Hiemstra, P., Stennicke, H., Guentsch, A., Schacher, B., Eickholz, P., and Potempa, J. (2014) Lack of cathelicidin processing in PapiUon-Lefevre syndrome patients reveals essential role of LL-37 in periodontal homeostasis. Orphanet journal of rare diseases 9, 148

24. Shanavas, M., Chatra, L., Shenai, P., Kumar, S., Bilahari, N., and Balakrishnan, S.

(2014) Partial expression of the PapiUon-Lefevre syndrome. Journal of the College of Physicians and Surgeons—Pakistan : JCPSP 24 Suppl 3, S230-232

25. Jose, J., Bartlett, K., Salgado, C, and Gutierrez, N. M. (2014) PapiUon-Lefevre Syndrome: Review of Imaging Findings and Current Literature. Foot & ankle specialist

26. Ragunatha, S., Ramesh, M., Anupama, P., Kapoor, M., and Bhat, M. (2015) PapiUon- Lefevre syndrome with homozygous nonsense mutation of cathepsin C gene presenting with late-onset periodontitis. Pediatric dermatology 32, 292-294

Claims

CLAIMS:

1. A method for assessing a subject's risk of having or developing a Papillon-Lefevre syndrome comprising the step of measuring in a urinary sample obtained from said subject the presence or absence of Cathepsin C protein, wherein the absence of Cathepsin C protein in the urine sample is predictive of a high risk of having a Papillon-Lefevre syndrome.

2. A method according to claim 1 wherein the presence of Cathepsin C protein in the urine sample is predictive of a low or zero risk of having a Papillon-Lefevre syndrome.

3. A method for assessing in a subject the presence of mutation in Cathepsin C gene (CTSC) which affected the expression of the Cathepsin C gene comprising the step of measuring in a urinary sample obtained from said subject the level of Cathepsin C, wherein :

The absence of Cathepsin C protein is predictive of a high risk of the presence of mutation in Cathepsin C gene (CTSC) which affected the expression of the Cathepsin C gene;

The presence of Cathepsin C protein is predictive of a low risk of the presence of mutation in Cathepsin C gene (CTSC) which affected the expression of the Cathepsin C gene.

4. Use of urinary form of Cathepsin C as a marker of Papillon-Lefevre syndrome risk.

5. Devices useful to detect and/or visualize the presence of Cathepsin C in a urine sample.

6. Devices according to claim 5, wherein it is rapid Lateral flow devices.

7. An in vitro method for diagnosing Papillon-Lefevre syndrome in a subject, said method comprising the step of detecting in a urinary sample obtained from the subject the presence or absence of Cathepsin C, wherein the absence of Cathepsin C protein in the urine sample is indicative of a PapiUon-Lefevre syndrome in the subject.

8. An in vitro method according to claim 7, wherein PapiUon-Lefevre syndrome is detected at an early stage.