Identification of kallikrein 1 and transthyretin as faecal biomarkers for diagnosis and prognosis of non-alcoholic steatohepatitis

Abstract

The present invention refers to the field of medical diagnostics. In particular, it concerns the evaluation of faecal levels of two identified biological markers, whose modulation allows reliable differentiation between clinical liver disease states distinguishing between NAFLD and NASH. It is identified as a biomarker for non-invasive diagnosis and prognosis of non-alcoholic liver steatosis (NAFLD) and its most severe form, Steatohepatitis (NASH), the protein pair, Transthyretin and Kallikrein 1 that can be detected through a specific antibody-linked immunosorbent assay (ELISA) of faecal samples obtained from animals with NAFLD / NASH and whose statistically significant variation from healthy controls allows both diagnosis and prognosis to be provided in a simple and low-cost manner. The identification of these novel diagnostic / prognostic faecal biomarkers paves the way for the development of non-invasive methodologies for early and low-cost diagnosis that can make a major contribution to the control of high-impact-social diseases such as NAFLD / NASH and other liver diseases.

Description

[0001] IDENTIFICATION OF KALLIKREIN 1 AND TRANSTHYRETIN AS FAECAL BIOMARKERS FOR DIAGNOSIS AND PROGNOSIS OF NON-ALCOHOLIC STEATOHEPATITIS DESCRIPTION SUMMARY OF THE INVENTION

[0002] The present finding relates to the identification of two biomarkers for non-invasive diagnosis and prognosis of non-alcoholic hepatic steatosis (NAFLD) and its most severe form, Steatohepatitis (NASH), Kallikrein 1 and Transthyretin that were detected through a proteomic analysis of faecal samples obtained from animals with NAFLD / NASH and whose statistically significant variation compared to healthy controls and in NASH compared to NAFLD, allows both simple and low-cost diagnosis and prognosis to be provided.

[0003] Based on the evidence that liver disease can also be reflected in the composition of the faecal content, a mouse pre-clinical model of NAFLD and NASH was used, i.e. mice on a diet high in fat and sugar for 13 weeks as a model of NAFLD and mice on a high fat and sugar diet and on 5 cycles of dextran sulphate treatment to induce intestinal inflammation aggravating liver damage, as a model of NASH. Proteomic analysis was performed on faecal samples of the two groups of animals with NAFLD and NASH, respectively, and a control group consisting of standard diet-fed mice. This proteomic analysis identified a total of 215 host-derived proteins. Statistical analyses of the identified proteins revealed, imposing a three-fold increase or decrease threshold, a statistically significant variation of 85 proteins in total, of which 23 were altered in comparison between NAFLD mice and control, 38 between NASH mice and control, and 24 between NAFLD and NASH. In order to identify non-invasive biomarkers for these pathologies, the most elevated proteins in the transition between form at onset (NAFLD) and the mostsevere disease (NASH), the two proteins: Kallikrein 1 and Transthyretin increased by 35 and 41 -fold, respectively, and both related to liver function, were considered. Validation of the results of proteomics analysis by ELISA of both proteins in a larger number of faecal samples from mice with NAFLD and NASH showed a significant increase of Kallikrein 1 alone in NAFLD (as compared to healthy controls) and a significant increase of both proteins in the most severe form NASH. This results in proposing simultaneous analysis of protein pair as a valuable both diagnostic and prognostic marker.

[0004] STATE OF ART

[0005] Non-alcoholic fatty liver steatosis (NAFLD), characterised by a build-up of fats in the liver, and its most severe form Steatohepatitis (NASH), characterised by inflammation and fibrosis, are steadily increasing diseases in western countries, especially in the youth age. The new nomenclature for NAFLD and NASH is MASLD (steatotic liver disease associated with metabolic dysfunction) and MASH (Steatohepatitis associated with metabolic dysfunction) respectively, to better reflect the aetiology and clinical implications of these conditions. Consequently, in the present case, reference will be made to NASH and NAFLD, indicating respectively MASLD and MASH. NASH is closely associated with obesity and type 2 diabetes and has serious complications ranging from cirrhosis to liver cancer. It is therefore evident how NAFLD / NASH represent an epidemiological, clinical problem with a strong impact on the patient's quality of life and high costs for SSN.

[0006] Despite the high prevalence of NAFLD / NASH and the importance of early diagnosis, biochemical biomarkers are lacking and there are no solidly validated clinical signs of disease for diagnosis, prognosis and monitoring of disease progression in response to therapeutic treatments. Diagnosis is conducted by highly invasive and expensive liverimaging and biopsy. Furthermore, since there are no valid approved therapies, early diagnosis is crucial, in particular to identify that subgroup of patients with NAFLD progressing in NASH; therefore, new non-invasive techniques for clinical diagnosis are needed.

[0007] There is therefore an urgent need to develop reliable biomarkers and non-invasive diagnostic tests. This requirement is particularly relevant since the usefulness of liver biopsy is limited by its invasive nature, poor patient acceptability and variability of sampling, as well as being very costly. Plasma levels of the Cytocheratin 18 (CK18) fragment, a hepatocyte apoptosis marker, represent the most widely evaluated Steatohepatitis biomarker, although specificity / sensitivity is modest. Several genetic polymorphisms (such as those in PNPLA3 (Crisps like phosholipase containing 3) and TM6SF2 (Transmembrane 6 superfamily member 2) have been shown, but also those for the genes KLF6 (Kruppel-like factor 6), LPIN1 (Mg (2+ )-dependent phosphatidic acid (PA) phosphohydrolase, SOD2 (Superoxide dismutase (MnSOD) manganese-dependent) are related to NAFLD genetics and its severity, but their role in patient evaluation remains to be established with greater precision. Due to the risks associated with liver biopsy, high costs and inability to apply it on a large scale, it is necessary to identify non-invasive biomarkers that can reliably identify patients at increased risk of progression. Several research groups are seeking to address this problem using omics approaches, including lipidomics, metabolomics and profiling of RNA molecules such as microRNAs and noncoding RNAs, in patients' blood.

[0008] A series of diagnostic and prognostic markers including metabolites and lipids in faeces are identified in NANJING UNIVERSITY patent document CN116660543 A, while protein biomarkers are identified and protected only in serum and urine but do not include the two markers that fall within the scope of protection of this invention.In Oxford University patent document WO 2018 / 037229: Apolipoprotein F as a biomarker for non-alcoholic hepatic steatosis, a method is described for the diagnosis, prognosis, monitoring or staging of non-alcoholic hepatic steatosis (NAFLD) progression using biomarkers in unspecified biological samples. No proteins of this patent are mentioned.

[0009] A non-invasive method of diagnosing non-alcoholic hepatic steatosis and distinguishing between non-alcoholic hepatic steatosis (NAFL) and non-alcoholic Steatohepatitis (NASH) is described in the EP 32822256 Al patent document of the SOCIAL WELFARE ORGANIZATION SAISEIKAI IMP GIFT FOUNDATION, which includes: (1) a measurement step of the amounts of markers contained in the blood taken by a subject; (2) a determination of an index value from a standard score calculated on the basis of the amounts of markers belonging to the same group; and (3) a phase of determination of the possible presence of NASH in the case where the benchmark is higher than a reference value. Transthyretin is present in the blood, but the faecal sample is not mentioned.

[0010] In the patent document WO2023238881 Al of the NITTO-DENKO Corporation (JP) the problem of providing a marker for the diagnosis of a non-alcoholic liver steatosis (NAFLD) is solved by a marker or the like for the diagnosis of a non-alcoholic fatty liver disease (NAFLD), where the marker is at least a type of protein selected by the group consisting of an endopeptidase, hydrolase, a protein that binds calcium ions, a cytoskeletric protein, a transferase, a protein that binds the receptor, a protein that binds the antigen, a protein that binds the RNA, a reductase and a protein that binds the cadherin or a protein that encodes the cadherin. The patent extends to all biological fluids including faeces, but listed proteins do not include proteins that fall within the scope of protection of this invention.US 10495648 B2 of GILEAD SCIENCES INC: Describes a method to diagnose treat and monitor liver disease by detecting levels of certain microRNAs in human body fluids including faeces: There is no overlap with biomarkers of this application.

[0011] WO 202438901 Al of TOKYO METROPOLITAN INSTITUTE OF MEDICAL SCIENCE + NATIONAL HOSPITAL ORGANIZATION + KAGOSHIMA UNIVERSITY describes a biomarker, a method and a program for detecting a biomarker belonging to the class of microRNAs in biological fluids including stools of patients with liver diseases. In particular, the biomarker is selected in the SEQ ID NOS: 1-125 group.

[0012] In recent years, studies have identified and characterised the relationship between a condition of steatosis and Steatohepatitis and an altered permeability of the intestinal barrier and microbiota composition, known as the gut-liver axis. However, using a faecal sample to identify protein-based biomarkers in liver disease is an innovative approach In this context, the current invention aims to identify and develop new diagnostic / prognostic biomarkers to help early diagnosis, monitor disease progression and the response to therapies, to be detected in faecal samples, which from the trials carried out, make it possible to overcome the limitations of non- specificity and invasiveness of biological samples currently in use such as serum and biopsy.

[0013] According to one aspect of the current invention, using omics screening (to analyse the proteome) on faecal samples from NAFLD and NASH mice and comparing them with healthy mice, potential biomarkers have been identified that need to be validated on a large number of patients. Two proteins were selected from the identified markers based on their level of increase, significance and role in liver function and validated on a larger number of animals. Large-scale markers identified and validated can be used for the development of large-scale diagnostic kits for use by analytical laboratories or even by the patient himself, at low cost. This innovative approach allows early detection of the disease and especiallymonitoring of disease progression as well as patient response to therapy, also in order to reduce the number and frequency of invasive and expensive investigations such as liver biopsy.

[0014] To achieve this goal, a mouse pre-clinical model of NAFLD and NASH, mice on a high fat and high sugar diet for 13 weeks, was used to model NAFLD and mice on a high fat and high sugar diet and undergoing five courses of dextran sulphate treatment to induce intestinal inflammation that aggravates liver damage as a model of NASH. Through proteomic analysis on faecal samples from the two groups of animals and a control group (CTRL) consisting of standard diet-fed mice, a total of 215 host-derived proteins were identified, of which 135 were statistically significantly modulated. Considering the three test groups, the following comparisons were made, and proteins were selected showing an increase or decrease of at least three times

[0015] • comparing subjects with NAFLD compared to controls, 23 proteins (of which 11 increased and 12 decreased in NAFLD) (tab.l) are statistically significantly modulated.

[0016] • Comparing subjects with NASH versus controls results in statistically significant modulation of 38 proteins (26 increased and 12 decreased in NASH) (Tab. 2). • Comparing NASH versus NAFLD subjects results in statistically significant modulation of 24 proteins (16 increased and 8 decreased in NASH) (tab.3).

[0017] Since some proteins are present in more than one comparison, the total protein modulated amounts to 49. In order to identify possible markers of the pathology and its progression, proteins that showed a significant increase in the most severe condition (NASH) compared to the mild form (NAFLD) were examined and 16 proteins were identified (Tab.3). Amongst these, Kallikrein 1 (Klkl) and Transthyretin (Ttr) were selected for validation by ELISA based on their role in liver function and degree ofincrease. The results obtained (Fig.3) revealed a significant increase in Klkl in both NAFLD and NASH mice while Ttr was increased only in the strictest form of disease, so it is intended to propose as diagnostic biomarkers and prognosis the analysis of protein pair at the same time.

[0018] A list of publications that are relevant, but do not overlap with the current invention, is given in the Bibliography. In particular, the documents found relate to studies carried out on the faecal sample in NASH, but for the analysis of the microbiome and metabolome and the effects of nutrients or drugs on the faecal metabolome and microbiome and do not present the study of the proteome. Specifically, regarding the proteins studied in faeces, refs 8, 11 and 12 refer to calprotectin (ref. No. 8,11) and zonulin (ref. n.12) proteins respectively that do not overlap with those identified in the present invention. Also, in ref.

[0019] 2 the name refers to the Trefoil factor 2, Colipase and Chymotrypsin-like elastase as associated with the dog's hepatobiliary disorders, which do not include NAFLD / NASH. In a work (ref. 4) on faecal proteomics in neonates with biliary atresia, Kallikrein 11 is identified as more present in the stools of patients, whereas in our case it is the Kallikrein 1 isoform of tissue origin and in a different pathology. A recent publication (ref.l) reports an analysis of faecal proteome in patients with alcoholic hepatitis, but the identified proteins do not overlap the proteins identified by us. In another work (ref. 22) plasma Transthyretin is proposed as a new potential biomarker of liver function, but faecal samples are not mentioned. In another work (ref.23) faecal metaproteomic analysis is carried out in patients with NASH with or without hepatocellular carcinoma in which the Trefoil factor 3, included in our lists, is mentioned but the selected proteins Kallikrein 1 and Transthyretin are not included. Finally, the study carried out by Mazzini et al. (ref. 24) reports a metabolic analysis on stools of individuals with NASH, but proteins are not mentioned.

[0020] Based on the research carried out, the uniqueness and originality of this patentproposal is evident.

[0021] DETAILED DESCRIPTION OF THE INVENTION

[0022] The rational of the research that led to the present invention is based on evidence that liver pathologies can also be reflected in the composition of faecal content. This directed the inventors to identify biomarkers for NAFLD / NASH diagnosis and prognosis through proteomic analysis of faecal samples obtained from animals with NAFLD / NASH against healthy controls to enable the development of a sensitive, specific and low-cost diagnostic test. The identification of novel diagnostic / prognostic faecal biomarkers paves the way for the development of non-invasive methodologies for early and low-cost diagnosis that can make a strong contribution to the control of high-impact-social diseases such as NAFLD / NASH and other liver diseases.

[0023] The description of the invention will be better followed by reference to the attached figures in which:

[0024] Figure.1 is a graph of the variation of proteins that show an increase or decrease of at least three times comparing the three test groups (NAFLD vs CTRL; NASH vs CTRL; NASH vs NAFLD)

[0025] fig. 2 A and B are respectively the analysis of the main components (A) and "heatmap" (B) of proteins modulated in the three groups of mice;

[0026] Figure 3 shows the validation of Kallikreina 1 (Klkl) and Transthyretin (Ttr) by the Elisa assays using the three groups of mice.

[0027] In the experiment conducted by the inventor, omics analysis was performed from faecal samples of 3 groups of mice (n= 3 / group): a diet fed group with high fat and sugar content for 13 weeks to induce a NAFLD, a high fat and sugar diet group supplemented with 5 cycles of dextran sulphate treatment, to induce a NASH and a group of standard diet control mice. Diagnosis of NAFLD and NASH was performed by histological analysis ofliver biopsies.

[0028] In faecal samples from animals with NAFLD / NASH, proteomic analysis detected a total of 215 proteins. Among these, considering a three-fold variation threshold, the statistically modulated ones are given in the following Tables 1-3, where:

[0029] Table 1 contains 11 proteins increased in NAFLD compared to controls and 12 proteins decreased in NAFLD compared to controls

[0030] Table 2 shows 26 proteins increased in NASH compared to controls and 12 proteins decreased in NASH compared to controls. 18 proteins are already present in the tables above. Therefore, a further 20 proteins are identified;

[0031] Table 3 shows No 16 proteins increased in NAFLD compared to NASH and No 8 proteins decreased in NASH compared to NAFLD, of which 18 are already present in the tables above. Then a further 6 proteins are identified.

[0032] As specified, some proteins are modulated in more than one comparison between the different test groups, so even if the total of the proteins reported in the different tables is 85, due to multiple occurrences, 49 proteins are identified, highlighted in bold.

[0033] Table 1: List of proteins identified by proteomic analysis and modulated in NAFLD compared to controls.

[0034] Description Symbol of t Anova(p) Fold change (NAFLD / CTRL) Ig heavy chain V region 93G7 HVM02 3,01E-04 + 136,62 Ig kappa chain V-V region HP R16.7 KV5AB l,93E-03 + 88,83 Chymotrypsin-like elastase family member 3 B Cela3b 2,00E-09 + 21,51 Trefoil factor 3 Tff3 3,17E-11 + 16,96 Sphingosine kinase 1 Sphkl 3,73E-06 + 13,01

[0035] O-mannosyl-transferase protein transmembrane Tmtc3 6,47 E-03 + 7,91 Solute carrier family 2_facilitated glucose transporter mer Slc2al2 6,23E-10 + 4,55 Pancreatic secretory granule membrane major < unk> Gp2 2,93 E-07 + 4,37 Hydrolyspondin type-1 domain-containing protein 7 A Thsd7a l,02E-05 + 3,27

[0036]

[0037] CUB and zona pellucida-like domain-containing protein 1 Cuzdl 7,74E-09 + 3,19 Adenylate kinase lactate 5 Ak5 l,77E-03 + 3,12 Septin-7 Septin7 6,73E-12 - Infinity H-2 class II histotrophic antigen_A-D beta chain H2 -Abl 6,19E-05 - 431,53 Lymphocyte c subunit 5B_ < unk>l Cox5b 7.44E-08 - 398,97 Serine / threonine-protein cytokines 1 regulatory subunit PpplrlO 2,17E-06 - 106,27 Angiogenin-4 Ang4 2,54E-07 - 48,60 Protein TCL1B2 Tcllb2 1,18E-11 - 33,33 Keratin type 1 cytoskeletal Krtl4 5,2E-12 - 33,03 Protein b5 domain-containing protein 1 Cyb5dl 6,33E-06 - 15,89 ATP-dependent RNA helicase DDX17 Ddxl7 2,37E-10 - 15,32 Ras-related protein Rab-13 Rabl3 9,87E-11 - 12,60 Glutamyl aminopeptidase Enpep 6.04E-10 - 3,64 Glutamate-tRNA ligase_ < unk>l Ears2 5,llE-06 - 3,61

[0038]

[0039] Table 2: List of proteins identified by proteomic analysis and modulated in NASH compared to controls

[0040] Description Symbol of the g Anova(p) Fold change (NASH / CTRL) Ig kappa chain V-V region HP R16.7 KV5AB 4,52E-12 + 3759,64 Trefoil factor 2 Tff2 2,51E-07 + 2973,23 Ig heavy chain V region 93G7 HVM02 7,62E-05 + 2103,85 Kallikrein 1 Klkl 2,22E-16 + 44,67 Hemoglobin Alpha subunits Hba 1,08 E-02 + 41,36 Transthyretin TTR l,87E-03 + 40,37 Superoxide dismutase [Cu-Zn] Sodl 5,26E-04 + 28,71 Chymotrypsin-like elastase family member 3 B Cela3b 4,27E-05 + 14,47 Trefoil factor 3 Tff3 l,50E-04 + 13,72 Alpha-l-antitrypsin 1-1 Serpinla 1,76E-12 + 13,48 Immunoglobulin kappa constant Igkc 3,80E-06 + 8,34

[0041] Ig heavy chain V region AC38205.12 HVM51 2,78E-10 + 6,89 Peroxi redoxin-2 Prdx2 5,72E-03 + 6,80 Pancreatic secretory granule membrane major < unk: Gp2 l,19E-07 + 6,21 Solute carrier family 2_facilitated glucose transportei Slc2al2 l,44E-10 + 5,90 12

[0042] Colipase Clps l,17E-08 + 5,75 Pancreatic triacylglycerol lipase Pnlip 6,38E-09 + 5,38

[0043]

[0044] Hydrolyspondin type-1 domain-containing protein 7 Thsd7a 2,63E-09 + 5,30 CUB and zona pellucida-like domain-containing prote Cuzdl 1,37 E-08 + 5,15 Pregnancy zone protein Pzp 4,18E-05 + 4,72 Hemopexin Hpx 4,19E-02 + 4,56

[0045] Ig heavy chain V region MOPC 104E HVM12 3,15E-05 + 3,92

[0046] ATP-dependent RNA helicase DDX41 Ddx41 l,07E-05 + 3,58 Centromere protein J Cenpj 2,llE-06 + 3,47 Alpha-l-antitrypsin 1-4 Serpinld 4,22E-07 + 3,33 Adenylate kinase lactate 5 Ak5 6,10E-08 + 3,09 Lymphocyte c subunit 5B_ < unk>l Cox5b 7,09 E-08 - Infinity H-2 class II histotrophic antigen_A-D beta chain H2 -Abl 5,98E-05 - Infinity Protein TCL1B2 Tcllb2 4,24E-14 - 105,79 Angiogenin-4 Ang4 3,84E-07 - 25,24

[0047] Ras-related protein Rab-13 Rabl3 2,86E-12 - 25,03 Keratin_type 1 cytoskeletal Krtl4 4,24E-10 - 14,02 Protein b5 domain-containing protein 1 Cyb5dl l,04E-05 - 12,79 Keratin type II cuticular Hbl Krt81 7,17E-03 - 12,61 Major urinary protein 3 Mup3 4,12E-06 - 10,84 ATP-dependent RNA helicase DDX17 Ddxl7 l,21E-07 - 7,36 Keratin type i cuticular Ha5 Krt35 2,85 E-04 - 4,44 Septin-7 Septin7 l,22E-03 - 3,18

[0048]

[0049] Table 3: Protein list identified by proteomic analysis and modulated in NASH versus NAFLD

[0050] Description Symbol of the g Anova(p) Fold change (NASH / NAFLD) Serine / threonine-protein cytokines 1 regulatory sub PpplrlO 2,51E-06 + 80,34

[0051] Ig kappa chain V-V region MOPC 173 KV5AA 4,70E-02 + 60,26 Ig kappa chain V-V region HP R16.7 KV5AB 1,68E-11 + 42,32 Transthyretin TTR l,84E-03 + 41,51 Kallikrein 1 Klkl 1,11E-15 + 35,47 Ig heavy chain V region 93G7 HVM02 2,64E-04 + 15,40 Trefoil factor 2 Tff2 l,12E-06 + 13,99 Superoxide dismutase [Cu-Zn] Sodl 1,49 E-03 + 13,43 Pregnancy zone protein Pzp 2,15E-07 + 6,34 Peroxiredoxin-2 Prdx2 7,90E-03 + 5,74

[0052]

[0053] Alpha-l-antitrypsin 1 Serpinla 6,46E-12 + 5,61 Lysosomal alpha-mannosidase Man2bl 5,94E-10 + 5,01 ATP-dependent RNA helicase DDX41 Ddx41 2,33 E-07 + 4,66 Anionic trypsin 2 Prss2 3,90E-02 + 4,10 ALK tyrosine kinase receptor Aik 3,01E-06 + 3,41 Ig heavy chain V region AC38205.12 HVM51 l,70E-07 + 3,03 Keratin_type II cuticular Hbl Krt81 l,73E-02 - 37,28 Sphingosine kinase 1 Sphkl 5,77E-05 - 7,51 O-mannosyl-transferase protein transmembrane Tmtc3 8.37E-03 - 6,80 Major urinary protein 3 Mup3 2,60E-03 - 5,78 Glycerophosphodiester p < unk> domain-containing Gdpd4 2,56 E-04 - 5,03 Keratin_type i cuticular Hal Krt31 l,54E-02 - 4,83 Keratin_type i cuticular Ha5 Krt35 7,97E-03 - 3,28 Protein TCL1B2 Tcllb2 5,29E-04 - 3,17

[0054]

[0055] As can be seen in Figure 2 A and B, the analysis of the main components (PC As) and the "heatmap" of proteins revealed a good separation between the three experimental groups.

[0056] The following proteins were selected for validation using the ELISA assay: Kallikrein 1 (Klkl), and Transthyretin (Ttr).

[0057] The proteins chosen for validation and selected as possible markers represent those most increased in the transition between the onset form (NAFLD) and the most severe form (NASH) of disease by eliminating immunoglobulins and serine / threonine phosphatase less specific to the pathology under investigation. Tissue Klkl is present in various tissues and metabolised by the liver. Kallikreins degrade kininogen, releasing bradykinin, a powerful inflammatory mediator. Ttr is a liver-derived protein that is the retinol transporter and has recently been reported to be involved in cholesterol metabolism and appears to be increased in the plasma of subjects with NAFLD. As shown in Fig.3, validation using the ELISA assay on all mice belonging to the 3 groups (n.7 NAFLD, n.7 NASH and n.4 CTRL) showed an increase in Kallikrein 1 in both NAFLD and NASH,while Transthyretin has been significantly increased only in NASH.

[0058] So far, there is no diagnostic and / or prognostic test for NAFLD / NASH that is carried out from the faecal sample, so the use of faecal sample for this type of investigation is particularly innovative. The proposed analyses on the faecal sample and subject of this invention involve several advantages as they are repeatable over time, therefore, useful for monitoring the course of the disease or the response to therapy, relatively inexpensive and well tolerated by the patient who should not undergo invasive investigations.

[0059] In conclusion, with the present invention we have shown that the use of biomarkers identified in faecal samples is a viable route for liver pathologies such as NAFLD / NASH and we have identified two proteins, which we examine at the same time, represent potential biomarkers usable in diagnostics and prognostics.

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Claims

CLAIMS1) Method for diagnosis of liver diseases NAFLD and NASH, including:(a) determination of faecal levels of biological markers Kallikrein 1 and Transthyretin;and(b)the assignment of the subject to a selected clinical status between NAFLD and NASH, based on the coordinated modulation of the above markers in a statistically significant manner.2) Method according to Claim 1, in which NAFLD is identified by an increase in the marker Kallikrein 1 as compared to healthy subjects.3) Method according to Claim 1, where NASH is identified by an increase in both markers, Kallikrein 1 and Transthyretin compared to healthy subjects.4) Concomitant use of Kallikrein 1 (Klkl) and Transthyretin (Ttr) as biomarkers for diagnostic, prognostic detection and monitoring of hepatic Steatosis and nonalcoholic Steatohepatitis in a faecal sample of a human patient, based on their statistically significant coordinated modulation.5) Use of techniques such as immunoblotting, proteomic analysis and specific antibody-linked immunosorbent assay (ELISA) to qualitatively and quantitatively determine the presence of the two proteins in a faecal sample in a statistically significant manner.6) The use of the two coupled proteins for use according to claims 4 and 5 as statistically significant! modulated biomarkers in combination with other proteins identified by the performed proteomic analysis and in combinations with parameters already used (body mass index (BMI), plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), cholesterol, triglycerides, cytocheratin 18 (K18), imaging techniques and genetic polymorphisms).7) The use of Kallikrein 1 and Transthyretin proteins for use according to claim 4 extended in terms of protection to (i) other liver disorders such as alcoholic Steatohepatitis, viral hepatitis, cirrhosis etc. and (ii) other biological samples or fluids such as saliva, urine, interstitial fluid, lymph, tears, and sweat.8) An in vitro diagnostic kit for diagnosis and prognosis of liver disease distinguishing between NAFLD and NASH, including:a) an initial detection for the determination of the Kallikrein 1 biomarker;b) a second detection for the determination of the Transthyretin biomarker; and c) instructions describing:- the identification of the presence of NAFLD through an increase in the Kallikrein 1 biomarker, and- determination of the NASH stage by an increase of both Kallikrein 1 and Transthyretin markers.9) The Kit according to the previous claim, where the detection media include antibodies or ligands specific to the Kallikrein 1 and Transthyretin biomarkers.

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