Methods for diagnosing fibrotic NASH

Abstract

The present invention provides methods for diagnosing, staging, or monitoring fibrotic non-alcoholic steatohepatitis (NASH) in a subject. The invention also provides for monitoring the subject's compliance with a prescription treatment against fibrotic NASH, and for monitoring the therapeutic effect of a prescription treatment against fibrotic NASH. The invention also provides a kit suitable for use in the method of the invention.

Description

Technical Field

[0001] This invention provides a method for diagnosing, staging, or monitoring fibrotic nonalcoholic steatohepatitis (NASH) in subjects. It also provides methods for monitoring subject adherence to prescribed treatment for fibrotic NASH and for monitoring the efficacy of prescribed treatment for fibrotic NASH. Furthermore, this invention provides kits suitable for use with the methods of this invention. Background Technology

[0002] Nonalcoholic fatty liver disease (NAFLD) is a disease with an alarmingly high prevalence, associated with metabolic diseases, cardiovascular diseases, and malignant tumors. 1,2 This is a range of liver diseases, ranging from nonalcoholic solitary steatosis (whose main histological feature is lipid accumulation in hepatocytes) to nonalcoholic steatohepatitis (NASH), accompanied by other liver inflammations that lead to fibrosis, and ultimately resulting in NASH-related cirrhosis and hepatocellular carcinoma (HCC). 1 Obesity and insulin resistance are closely associated with NAFLD, both due to increased transport of free fatty acids to the liver and increased hepatic lipogenesis associated with hyperglycemia and hyperinsulinemia. 2 With the global increase in obesity and insulin resistance / type 2 diabetes, NAFLD has become the most prevalent liver disease in the world, with a prevalence rising to 25-30% of the adult population. 3,4 .

[0003] Increased liver fibrosis has been shown to be the most important feature associated with increased all-cause mortality and liver-related mortality, as well as an increased likelihood of developing liver-related complications (including HCC) and requiring liver transplantation, while disease activity itself is not related to prognosis. 5,6 .

[0004] This makes NAFLD fibrosis staging crucial for determining the severity of NAFLD during clinical examination in order to initiate the correct and timely multidisciplinary treatment plan for each patient. This can include lifestyle and dietary interventions, treatments in clinical trials, and, in some cases, consideration of bariatric surgery. 1,7 .

[0005] Liver biopsy remains the clinical reference standard for fibrosis detection and staging, but it carries the potential for complications and is subject to sampling and interpretation errors. 8,9 Over the past decade, radiographic assessment of liver stiffness, using vibration-controlled transient elastography (i.e., Fibroscan) and magnetic resonance imaging, has gained trust as a diagnostic and staging tool for liver fibrosis. 10,11These include vibration-controlled transient elastography (i.e., Fibroscan) and magnetic resonance imaging. Simplified surrogate markers for liver fibrosis include FIB-4 (age, platelet count, aminotransferases), NAFLD fibrosis score, and a serological group of combined biomarkers (such as enhancing liver fibrosis (ELF)). These methods have good accuracy in excluding advanced fibrosis and can be used to identify individuals at low risk of developing advanced disease. 11,12 However, none of these diagnostic tests can distinguish between people at risk of developing fibrosis and / or progression and those at no risk. Furthermore, recent research has shown that repeated measurements of these tests are not useful for monitoring the progression of NAFLD. 13 These are significant unmet clinical needs in the daily practice of NAFLD-NASH patients and their healthcare practitioners (including hepatologists, internists, family physicians, and diabetes nurses). 12 .

[0006] This invention aims to at least partially address these long-standing, unmet clinical needs. Summary of the Invention

[0007] This invention is based on novel biomarkers discovered by the inventors that are specific for fibrotic NASH. Surprisingly, these biomarkers can be used not only to diagnose fibrotic NASH, but also to monitor and stage it.

[0008] As shown in the Embodiments section of this disclosure, the inventors have found that patients with fibrotic NASH exhibit reduced α2,3-sialylation in most complex N-glycans. Specifically, altered α2,3-sialylation levels were also observed in N-glycans with two antennae (A2L), three antennae (A3L), and four antennae (A4L). Furthermore, the combined levels of α2,3-sialylate were reduced in both complex N-glycans and (single-antenna) mixed N-glycans, with odds ratios (ORs) of 0.36 and 0.33 in the two cohorts compared to healthy controls. Conversely, α2,6-sialylation was found to be generally increased in patients with fibrotic NASH. This altered α2,6-sialylation was most pronounced in complex N-glycans with three antennae (A3E), where the inventors noted ORs of 2.95 and 6.16 in the two cohorts compared to healthy controls.

[0009] Furthermore, as disclosed herein Figure 3 , 11As shown in Tables 2, 4, 6, and 8, the inventors discovered that changes in α2,3-sialylation and / or α2,6-sialylation levels can be proportional to the severity of fibrosis in NASH patients. In other words, patients with more advanced liver fibrosis (e.g., patients with a Brunt fibrosis score of 4) may have lower α2,3-sialylation levels and / or higher α2,6-sialylation levels compared to patients with less severe fibrosis (e.g., patients with a Brunt fibrosis score of 3, 2, or 1) or patients without fibrosis. This proportional variation in α2,3-sialylation and / or α2,6-sialylation levels makes these biomarkers useful for applications such as fibrosis staging and / or patient monitoring.

[0010] As shown in Example 2, the expansion of the replication cohort improved the statistical power of the analysis and confirmed the initial findings as shown in Example 1. Notably, the combined levels of α2,3-sialic acid in complex N-glycans and (single-antenna) mixed N-glycans were reduced, with odds ratios of 0.36 and 0.11 in the two cohorts compared to healthy controls. Conversely, α2,6-sialylation was generally increased in fibrotic NASH patients. This altered α2,6-sialylation was most pronounced in complex N-glycans with three antennas (A3E), where the inventors noted ORs of 2.95 and 11.6 in the two cohorts compared to healthy controls.

[0011] Therefore, in a first aspect, this article provides a method for diagnosing fibrotic nonalcoholic steatohepatitis (NASH) in a subject, the method comprising the following steps:

[0012] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in samples from subjects; and

[0013] • Compare the determined levels of α2,3-sialylation and / or α2,6-sialylation with reference values, where:

[0014] i) Increased α2,6-sialylation levels in the sample; and / or

[0015] ii) Decreased α2,3-sialylation level in the sample

[0016] The subjects were indicated to have fibrotic NASH.

[0017] In another aspect, this article provides a method for staging fibrotic nonalcoholic steatohepatitis (NASH) in subjects, the method comprising the following steps:

[0018] To determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in samples from subjects; and

[0019] The determined levels of α2,3-sialylation and / or α2,6-sialylation were compared with reference values ​​indicating the stage of fibrotic NASH to determine the stage of fibrotic NASH in the subject.

[0020] Suitablely, the staging can be based on the Brunt fibrosis score.

[0021] In another aspect, this article provides a method for monitoring fibrotic nonalcoholic steatohepatitis (NASH) in subjects, the method comprising the following steps:

[0022] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in the first sample from the subject; and

[0023] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in a second sample from the subject, wherein the second sample was obtained from the subject at a later time point than the first sample, wherein:

[0024] i) Compared with the first sample, an increase in α2,6-sialylation levels and / or a decrease in α2,3-sialylation levels in the second sample indicate the progression of fibrotic NASH in the subject; or

[0025] ii) The decrease in α2,6-sialylation level in the second sample and / or the increase in α2-3-sialylation level in the second sample compared to the first sample indicate the regression of fibrotic NASH in the subject.

[0026] In another aspect, this article provides a method for monitoring subject adherence to prescribed treatment for fibrotic nonalcoholic steatohepatitis (NASH), the method comprising the following steps:

[0027] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in the first sample from the subject; and

[0028] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in a second sample from the subject, wherein the second sample is obtained from the subject after prescription treatment, wherein a decrease or no change in the level of α2,6-sialylation in the second sample compared to the first sample, and / or an increase or no change in the level of α2,3-sialylation in the second sample indicates the subject's adherence to prescription treatment for fibrotic NASH.

[0029] In another aspect, this article provides a method for monitoring the therapeutic effect of prescription treatment for fibrotic nonalcoholic steatohepatitis (NASH), the method comprising the following steps:

[0030] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in the first sample from the subject; and

[0031] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in a second sample from a subject, wherein the second sample is obtained from the subject after prescribed treatment, wherein a decrease or no change in the level of α2,6-sialylation in the second sample compared to the first sample, and / or an increase or no change in the level of α2,3-sialylation in the second sample indicates the therapeutic effect of the prescribed treatment for fibrotic NASH.

[0032] Suitablely, the N-glycan may be a complex N-glycan.

[0033] Suitablely, the N-glycan may be selected from the group consisting of: biantennary N-glycans, triantennary N-glycans and tetraantennary N-glycans or combinations thereof.

[0034] Suitablely, the N-glycan may be selected from the group consisting of tri-antenna N-glycans and tetra-antenna N-glycans.

[0035] Suitablely, the sample may be a blood sample.

[0036] Suitablely, the blood sample may be a dried blood spot sample.

[0037] Suitable, the blood sample may be selected from the group consisting of whole blood, plasma and serum.

[0038] Appropriately, the subject may have been diagnosed with fibrotic NASH or identified as being at risk of developing fibrotic NASH.

[0039] Appropriately, subjects identified as being at risk for fibrotic NASH:

[0040] i) May have been diagnosed with NAFLD or nonfibrotic NASH;

[0041] ii) May have been diagnosed with type 2 diabetes or insulin resistance;

[0042] iii) Can be obese;

[0043] iv) May have been diagnosed with hypertension and / or dyslipidemia; and / or

[0044] v) May be suspected of having a genetic susceptibility factor, optionally, wherein the genetic susceptibility factor is a mutation in a gene selected from the group consisting of: PNPLA3, TM6SF2, MBOAT7, GCKR and HSD18B13.

[0045] Appropriately, the treatment may be selected from a combination of weight loss and lifestyle modifications.

[0046] Suitablely, the levels of α2,3-sialylation and / or α2,6-sialylation can be determined by methods selected from the group consisting of: mass spectrometry, high-performance liquid chromatography, capillary (gel) electrophoresis with laser-induced fluorescence detection, hydrophilic interaction liquid chromatography, lectin- or antibody-based binding assays, and ELISA-based assays.

[0047] Suitablely, the method may also include determining the subject's FIB-4 score, ELF score, and / or APRI score; and / or the subject's aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels; and / or performing liver imaging.

[0048] Suitablely, before determining the α2,3-sialylation and / or α2,6-sialylation levels of the glycan, the sample may have been subjected to release of N-glycan from a blood protein and sialidation for specific chemical derivatization, optionally wherein the protein may be selected from the group consisting of plasma, serum and whole blood proteins.

[0049] Therefore, in one aspect, the present invention provides a method for diagnosing fibrotic nonalcoholic steatohepatitis (NASH) in a subject, the method comprising the following steps:

[0050] • Samples from subjects are derivatized by releasing N-glycans from blood proteins and linking them with specific chemical sialic acid to provide derivatized samples;

[0051] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in the derivatized samples; and

[0052] • Compare the determined levels of α2,3-sialylation and / or α2,6-sialylation with reference values, where:

[0053] i) An increase in the level of α2,6-sialylation in the derivatized sample; and / or

[0054] ii) The decrease in α2,3-sialylation level in the derivatized sample

[0055] The subjects were indicated to have fibrotic NASH.

[0056] In another aspect, this article provides a method for staging fibrotic nonalcoholic steatohepatitis (NASH) in subjects, the method comprising the following steps:

[0057] • Samples from subjects are derivatized by releasing N-glycans from blood proteins and linking them with specific chemical sialic acid to provide derivatized samples;

[0058] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in the derivatized samples; and

[0059] • The α2,3-sialylation and / or α2,6-sialylation levels determined in the derivatized sample are compared with reference values ​​indicating the fibrotic NASH stage to determine the subject's fibrotic NASH stage.

[0060] Suitablely, the staging can be based on the Brunt fibrosis score.

[0061] In another aspect, this article provides a method for monitoring fibrotic nonalcoholic steatohepatitis (NASH) in subjects, the method comprising the following steps:

[0062] • First and second samples from subjects are derivatized with N-glycans from blood proteins and linked with specific chemical sialic acid to provide first and second derivatized samples;

[0063] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in the first derivatized sample; and

[0064] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in the second derivatized sample, wherein the second sample was obtained from the subject at a later time point than the first sample, wherein:

[0065] i) Compared with the first sample, the increase in α2,6-sialylation level and / or the decrease in α2,3-sialylation level in the second sample indicate the progression of fibrotic NASH in the subject;

[0066] ii) The decrease in α2,6-sialylation level and / or the increase in α2-3-sialylation level in the second sample compared to the first sample indicate the regression of fibrotic NASH in the subject.

[0067] In another aspect, this article provides a method for monitoring subject adherence to prescribed treatment for fibrotic nonalcoholic steatohepatitis (NASH), the method comprising the following steps:

[0068] • First and second samples from subjects are derivatized with N-glycans from blood proteins and linked with specific chemical sialic acid to provide first and second derivatized samples;

[0069] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in the first derivatized sample; and

[0070] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in a second derivatized sample from a subject, wherein the second sample is obtained from the subject after prescription treatment, wherein a decrease or no change in the level of α2,6-sialylation in the second derivatized sample compared to the first derivatized sample, and / or an increase or no change in the level of α2,3-sialylation in the second derivatized sample indicates subject adherence to prescription treatment for fibrotic NASH.

[0071] In another aspect, this article provides a method for monitoring the therapeutic effect of prescription treatment for fibrotic nonalcoholic steatohepatitis (NASH), the method comprising the following steps:

[0072] • First and second samples from subjects are derivatized with N-glycans from blood proteins and linked with specific chemical sialic acid to provide first and second derivatized samples;

[0073] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in the first derivatized sample; and

[0074] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in the second derivatized sample, wherein the second sample is obtained from the subject after prescription treatment, wherein a decrease or no change in the level of α2,6-sialylation in the second derivatized sample compared to the first derivatized sample, and / or an increase or no change in the level of α2,3-sialylation in the second derivatized sample indicates the therapeutic effect of the prescription treatment for fibrotic NASH.

[0075] Suitablely, any of the methods described herein may also include the step of administering fibrotic NASH treatment to a subject who has been diagnosed with NASH or identified as being at risk of developing fibrotic NASH.

[0076] In another aspect, this document provides a kit for use in the methods of the present invention, the kit comprising a detectable label of a glycan that specifically binds to α2,3-sialylated and / or α2,6-sialylated glycans.

[0077] In this article, the terms NAFLD and MASLD are used interchangeably. Due to recent changes in nomenclature, nonalcoholic fatty liver disease (NAFLD) is now referred to as metabolic dysfunction-associated fatty liver disease (MASLD).

[0078] In this article, the terms NASH and MASH are used interchangeably. Due to recent changes in nomenclature, nonalcoholic steatohepatitis (NASH) is now referred to as metabolic dysfunction-associated steatohepatitis (MASH).

[0079] In the description and claims of this specification, the words “comprising” and “containing” and variations thereof mean “including but not limited to”, and they are not intended to exclude other parts, additives, components, wholes or steps.

[0080] In the description and claims of this specification, the singular form includes the plural form unless the context otherwise requires. In particular, where the indefinite article is used, the specification should be understood to include both the plural and singular forms unless the context otherwise requires.

[0081] The features, integrals, properties, compounds, chemical parts or groups described in connection with a particular aspect, embodiment or example of the invention should be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.

[0082] The various aspects of the invention will be described in more detail below. Attached Figure Description

[0083] The embodiments of the present invention are further described below with reference to the accompanying drawings, wherein:

[0084] Figure 1 The monosaccharide components that make up human N-glycans are shown (bottom panel) and the glycosylation properties calculated therefrom, as shown in a fully sialylated tetraantennae N-glycan (top panel).

[0085] Figure 2 The repeated associations between NAFLD patients and healthy controls are shown. (a) Volcano plot based on 36 glycosylation properties calculated in the discovery cohort. The relative abundance differences between healthy controls and NAFLD were compared using glycosylation properties with negative effect sizes (be) and positive effect sizes (f, g). p-values, ORs, and 95% CIs are shown in Table 2. *, **: p-values ​​< 0.05 and 0.01, respectively.

[0086] Figure 3The duplicate associations between NAFLD patients and healthy controls, further stratified according to the NASH-related fibrosis stage (Brunt), are shown. *, **, ***: p-values ​​< 0.05, 0.01, and 0.001, respectively. The data shown correspond to the duplicate cohort.

[0087] Figure 4 Principal component analysis (PCA) based on glycosylation characteristics calculated in each cohort is shown, illustrating the distribution of samples and standards or pools. PCA score plots for the discovery cohort (a) and replicate cohort (b) illustrate the separation along PC1 and PC2 between healthy controls and NAFLD patients, as well as the clustering of standards (discovery cohort, (a)) or pools (replicated cohort, (b)). Loading plots visually show the variables leading to separation in the PCA model for each cohort. Plasma standards in discovery cohort (a) are distributed across 5 plates and have previously been shown to be randomly dispersed, indicating the absence of systematic batch effects. The tight clustering of plasma standards in the discovery cohort and pools in the replicate cohorts indicates low technical variability of the method. In the score plots, in (a), healthy controls are represented by lines, NAFLD patients by solid circles, and plasma standards by solid squares, while in (b), serum pools are represented by solid squares.

[0088] Figure 5 The age and sex distributions of different disease groups are shown: (a) is the overall age distribution of the cohort, while (b) is the age distribution stratified by sex within the cohort. (c) is the overall age distribution of the repeated cohort, while (d) is the age distribution stratified by sex within the repeated cohort. Simple statistical tests revealed significant age differences among women in the cohorts, although this effect was corrected for by logistic regression analysis, which included age, sex, and their interaction in the model as covariates.

[0089] Figure 6 The ratio of AL to AE for glycosylation characteristics is shown, illustrating the observed effects of NAFLD (ab), and more specifically, fibrosis (cd), on the blood N-glucose group as a single glycosylation characteristic. (a) shows the overall difference in relative AL / AE levels between healthy controls and NAFLD patients, while (b) shows the difference stratified by sex. (c) shows the overall difference in AL / AE between healthy controls and patients with different degrees of fibrosis (Brunt fibrosis score) in the replicate cohort, while (d) shows the difference stratified by sex. Although this ratio represents the overall effect, due to its limitations (described in the results and discussion of Example 1), all significantly relevant glycosylation characteristics are shown and described individually in Example 1. Figure 2 , 3(Tables 2 and 3). It should be noted that women (d) in the Brunt fibrosis score group did not pass the Kruskal-Wallis statistical test, although the trend was visually apparent. Since the age and sex were fairly well matched between the groups, the inventors believe that the lack of statistical significance in the women group was due to the small sample size.

[0090] Figure 7 The comparison of overall sialylation (CS) between disease groups by cohort and sex is shown. (a) shows the overall difference in relative CS levels between healthy controls and NAFLD patients, while (b) shows the difference stratified by sex. (c) shows the overall difference in CS between healthy controls and patients with different degrees of fibrosis (Brunt fibrosis score) in the replicate cohort, while (d) shows the difference stratified by sex. The non-significant results of the statistical test indicate that the observed fibrosis-specific effect is caused by changes in the relative abundance of sialyl-linked CS. Figure 6 (R) rather than being driven by changes in the relative level of CS S =-0.022; p-value=0.89). (e) is a forest plot used to compare the logistic regression models for each predictor and their corresponding odds ratios. Gradient indicator - log 10 p-value. -log of A4L and A3L. 10 The p-values ​​are 3.5 and 4, respectively. (A2L's -log) 10 The p-value is approximately 2.8. AL's -log 10 The p-value is approximately 2.5. (f) shows the ROC curves for the integrated model and the A4L model.

[0091] Figure 8 PCA based on glycosylation characteristics calculated in each cohort is shown, illustrating the distribution of samples and standards or mixtures. PCA analysis score plots for the discovery cohort (a) and the repeated anti-plasma cohort (b) illustrate the segregation along PC1 and PC2 between healthy controls and MASLD (NAFLD) patients, as well as the clustering of standards (discovery cohort, a) or mixtures (repeated cohort, b). Loading plots visually show the variables leading to segregation in the PCA model for each cohort. Plasma standards in the discovery cohort (a) were distributed across 5 plates and had previously been shown to be randomly dispersed, indicating the absence of systematic batch effects. 30 The tight clustering of plasma standards in the cohort and mixtures in the replicate cohort indicates low technical variability of the method. In the scoring plot, in (a), healthy controls are represented by lines, MASLD patients by solid circles, and plasma standards by solid squares, while in (b), serum mixtures are represented by solid squares.

[0092] Figure 9The repeated associations between MASLD (NAFLD) patients and healthy controls are shown. (a) Volcano plot based on 36 glycosylation properties calculated in the discovery cohort. (bg) Comparison of relative abundance differences between healthy controls and MASLD (NAFLD) using glycosylation properties with negative effect sizes (b, e) and positive effect sizes (f, g). p-values, ORs, and 95% CIs are shown in Table 6. *, **: p-values ​​< 0.05 and 0.01, respectively.

[0093] Figure 10 The ratio of AL to AE for glycosylation characteristics is shown, illustrating the observed effects of MASLD (NAFLD) (ab), and more specifically, fibrosis (cd), on the blood N-glycate group as a single glycosylation characteristic. (a) shows the overall difference in the AL / AE ratio between healthy controls and MASLD (NAFLD) patients in the cohort, while (b) shows the difference stratified by sex. (c) shows the overall difference in AL / AE between healthy controls and patients with different degrees of fibrosis (Brunt fibrosis score) in the replicate cohort, while (d) shows the difference stratified by sex. Although this ratio represents the overall effect, due to its limitations (described in the results and discussion of Example 2), all significantly relevant glycosylation characteristics are shown and described individually in Example 2. Figure 9 , 11 (Tables 6 and 7). It should be noted that gender stratification leads to lower power of statistical analysis, especially for women (d). Since age and gender are fairly well matched between groups, the inventors believe that the smaller difference in health & F0 vs. F1-F4 fibrosis scores between the women group is due to the small sample size. Furthermore, neither gender nor age was found to be a significant covariate in the binary classification model.

[0094] Figure 11 The duplicate associations identified between MASLD (NAFLD) patients and healthy controls stratified according to the MASH (NASH)-related fibrosis staging (Brunt) are shown. *, **, ***: p-values ​​< 0.05, 0.01, and 0.001, respectively. The data shown correspond to the duplicate cohort.

[0095] Figure 12The age and sex distributions for different disease groups are shown: (a) is the overall age distribution of the cohort, and (b) is the age distribution stratified by sex within the cohort. (c) is the overall age distribution in the replicate cohort, and (d) is the age distribution stratified by sex within the replicate cohort. Simple statistical tests revealed a significant age difference between women in the cohort and men in the replicate cohort, although this effect has been illustrated by logistic regression analysis, which included age, sex, and their interaction in the model as covariates (see "Statistics" in the Examples section). Furthermore, neither sex nor age was found to be a significant covariate in the binary classification model.

[0096] Figure 13 The effects of type 2 diabetes mellitus (T2DM) are shown. The association between glycosylation characteristics and fibrosis is shown in the T2DM stratification groups (0 (left two plots) = no T2DM; 1 (right two plots) = T2DM). Healthy patients were considered F0 and non-diabetic. For applicable groups, healthy and F0 patients were pooled for this analysis. It should be noted that further univariate statistical analysis was not possible due to the small number of F0 patients in the T2DM group and the small number of patients after further stratification by fibrosis stage. However, the direction of changes caused by fibrosis was similar in both the non-T2DM and T2DM groups, suggesting a fibrosis-specific rather than T2DM-specific effect.

[0097] Figure 14 The comparison of overall sialylation (CS) between disease groups by cohort and sex is shown. (a) shows the overall difference in relative CS levels between healthy controls and MASLD (NAFLD) patients in the cohort, while (b) shows the difference stratified by sex. (c) shows the overall difference in CS between healthy controls and patients with different degrees of fibrosis (Brunt fibrosis score) in the replicate cohort, while (d) shows the difference stratified by sex. Although statistical tests indicated significant differences between groups (cd), this effect was evident in patients with advanced fibrosis (F4; cirrhosis), and no difference was observed between healthy and / or F0 patients vs. F1–F3 fibrotic patients. These results suggest that the observed fibrosis-specific effect is caused by changes in the relative abundance of sialyl ( Figure 10 (R) rather than being driven by changes in the relative level of CS S =-0.15; p-value = 0.86).

[0098] Figure 15 The ROC analysis and associated AUC values ​​of the single predictor model including A4L are shown, illustrating its ability to predict fibrosis based on histological appearance.

[0099] The patents, scientific and technical documents mentioned herein establish knowledge available to a person skilled in the art at the time of filing this application. All disclosures of granted patents, published and pending patent applications, and other publications cited herein are incorporated herein by reference in the same manner as if each were specifically and individually identified and incorporated by reference. In any event of inconsistency, this disclosure shall prevail.

[0100] The various aspects of the invention will be described in more detail below. Detailed Implementation

[0101] In one aspect, the present invention provides a method for diagnosing fibrotic nonalcoholic steatohepatitis (NASH) in a subject.

[0102] The methods described herein are based on the inventors' discovery of novel biomarkers capable of differential diagnosis, staging, and / or monitoring of fibrotic NASH. Currently, diagnosing and monitoring fibrotic NASH without invasive liver biopsy and without further testing (such as liver imaging, e.g., Fibroscan) is difficult. Even so, differentiating certain types of liver disease, such as autoimmune hepatitis and fibrotic NASH, can sometimes be challenging. Therefore, non-invasive testing represents an unmet clinical need.

[0103] Surprisingly, the inventors have discovered biomarkers that can be used for the diagnosis, staging, and / or monitoring of fibrotic NASH. These biomarkers are unique N-glycosylation patterns. Specifically, the inventors have found that increased levels of α2,6-sialylation in samples; and / or decreased levels of α2,3-sialylation in samples indicate that a subject has fibrotic NASH.

[0104] Nonalcoholic fatty liver disease (NAFLD) refers to a condition in which fat is deposited in the liver (steatodegeneration) not due to excessive alcohol consumption. It can be caused by insulin resistance and metabolic syndrome and can respond to treatments initially developed for other insulin-resistant conditions (such as type 2 diabetes), such as weight loss, metformin, and thiazolidinediones. NAFLD ranges from simple steatosis to nonalcoholic steatohepatitis (NASH). "NASH" is the most severe form of NAFLD and occurs when the accumulation of fat in NAFLD causes inflammation and / or fibrosis leading to liver disease. The most severe form of NASH—cirrhosis—is a life-threatening condition. Depending on the severity, NASH can be fibrotic (i.e., NASH with liver fibrosis) or non-fibrotic (NASH without liver fibrosis). Non-fibrotic NASH is the mildest form of NASH. However, it can develop into fibrotic NASH.

[0105] The degree of liver inflammation and / or liver fibrosis, and the resulting severity of NASH, can be assessed using methods known in the art. One example method is the histopathological evaluation of a subject's liver biopsy using the Brunt Fibrosis Scoring System (which scores the amount of fibrosis in the liver) and / or the Brunt Activity Scoring System (which scores the amount of inflammation in the liver).

[0106] A Brunt fibrosis score of 0 indicates that the subject has no liver fibrosis. A Brunt fibrosis score of 1 indicates that the subject has portal fibrosis (minimum scarring). A Brunt fibrosis score of 2 indicates that the subject has periportal fibrosis (significant scarring extending beyond the liver area). A Brunt fibrosis score of 3 indicates that the subject has severe fibrosis (fibrosis has spread and forms bridges with other fibrotic liver areas). A Brunt fibrosis score of 4 indicates that the subject has cirrhosis (advanced scarring).

[0107] A Brunt activity level score of 0 indicates that the subject has no liver inflammation. A Brunt activity level score of 1 indicates that the subject has mild liver inflammation. A Brunt activity level score of 2 indicates that the subject has moderate liver inflammation. A Brunt activity level score of 3 indicates that the subject has severe liver inflammation.

[0108] Liver biopsy is currently the clinical reference standard for fibrosis detection and staging; however, it is an invasive procedure and subject to sampling errors because the sample is taken from a small area of ​​the liver, thus it cannot accurately represent the overall condition of the liver. The invention described herein provides a non-invasive method for diagnosing and grading the severity of fibrotic nonalcoholic steatohepatitis (NASH) in subjects. Specifically, the method for diagnosing fibrotic NASH as described herein can diagnose patients with fibrotic NASH and a Brunt fibrosis score of 1 or higher.

[0109] Tracking a subject's Brunt Fibrosis Score over a period of time can monitor NASH. A decrease in the Brunt score over time can indicate disease regression, while an increase in the Brunt score over time can indicate disease progression.

[0110] As used herein, “diagnosis” refers to assessing whether a subject, according to the method described in this invention, has fibrotic NASH.

[0111] In the context of this disclosure, "subject" can be any organism with a liver. Suitably, the subject can be a mammal or a non-mammal. More suitably, the subject can be a mammal. Most suitably, the subject can be a human. Suitably, the human subject is an adult. The human subject can be male or female. Suitably, the adult subject is 20 years of age or older. For example, the subject can be 30, 40, 50, 60, 70 years of age or older.

[0112] Suitablely, the subjects may be symptomatic or asymptomatic. Symptomatic subjects may exhibit one or more symptoms associated with NAFLD and / or NASH. By way of example only, symptoms associated with NAFLD and / or NASH may be selected from the following groups: severe itching, altered liver enzyme levels, abdominal swelling, easy abrasion and bleeding, jaundice, subcutaneous vascularization, ascites, and behavioral changes (such as confusion, fatigue, slurred speech, etc.).

[0113] Suitable, the subject may have been diagnosed with NAFLD and / or NASH (e.g., the subject may have been diagnosed with non-fibrotic or fibrotic NASH). The subject may have been diagnosed with NAFLD and / or NASH by any method known in the art.

[0114] Asymptomatic subjects may be known to be susceptible to NAFLD and / or NASH, or may be suspected of being susceptible to NAFLD and / or NASH. Subjects known to be susceptible to NAFLD and / or NASH have risk factors for NAFLD and / or NASH. Risk factors for NAFLD and / or NASH may include type 2 diabetes or insulin resistance, obesity, hypertension and / or dyslipidemia, age, and / or genetic susceptibility factors. Genetic susceptibility factors may include mutations in genes selected from the following groups: PNPLA3, TM6SF2, MBOAT7, GCKR, and HSD18B13. The PNPLA3 gene encodes adiponutrin enzyme (also known as patatin-like phospholipase domain-containing protein 3, acylglycerol O-acyltransferase, or calcium-independent phospholipase A2-ε). The TM6SF2 gene encodes transmembrane 6 superfamily 2 protein, which is involved in the secretion of triglyceride-rich lipoproteins by hepatocytes. The MBOAT7 gene encodes a protein containing a membrane-bound O-acyltransferase domain 7. The GCKR gene encodes a glucokinase regulatory factor protein. The HSD18B13 gene encodes a hydroxysteroid 17-β dehydrogenase 13 protein.

[0115] Subjects suspected of being susceptible to NAFLD and / or NASH may have a family history of NAFLD and / or NASH, and / or a family history of NAFLD and / or NASH risk factors. Methods for diagnosing fibrotic nonalcoholic steatohepatitis (NASH) in subjects include steps to determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in the subject's sample.

[0116] As will be apparent to those skilled in the art, the levels of α2,3-sialylation and / or α2,6-sialylation are typically “determined” by measuring the levels of α2,3-sialylation and / or α2,6-sialylation in a sample. Therefore, the term “determined” is used herein in place of the term “measured” or “determined by measurement.” The term “level” as used herein refers to the amount or accumulation of a biomarker. In the context of this disclosure, a biomarker is α2,3-sialylation and / or α2,6-sialylation. The level can be an absolute amount of the biomarker in a sample or a relative amount of the biomarker. A relative amount can be, for example, a percentage, a fraction, or a ratio. A ratio can be, for example, the ratio of α2,3-sialylation to α2,6-sialylation, and vice versa. As shown in the Embodiments section of this disclosure, the level of α2,3-sialylation and / or α2,6-sialylation of the glycan can be determined in samples that have undergone N-glycan release from plasma and sialic acid-linking derivatization. Samples that have undergone N-glycan release and sialic acid-linking derivatization are referred to herein as “derivative samples”.

[0117] Conventional "determining" methods may include sending clinical samples to a commercial laboratory to measure the level of a biomarker in a biological sample, or using a commercially available assay kit to measure the level of a biomarker in a biological sample. Exemplary kits and vendors will be apparent to those skilled in the art. In various instances, biomarkers may be determined, detected, and / or quantified using methods such as mass spectrometry, high-performance liquid chromatography, capillary (gel) electrophoresis with laser-induced fluorescence detection, hydrophilic interaction liquid chromatography (e.g., with fluorescence or UV detection), and binding assays based on lectins or antibodies, including but not limited to those relying on ELISA-based methods (e.g., ELISA-based assays using glycan-binding proteins such as lectins, antibodies, or aptamers). Exemplary methods for determining the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans are also provided in the Examples section of this specification. As an example only, the α2,3-sialylation of N-glycans (combinations of mono, di, tri, and tetra-antennae glycans) can be calculated as follows: α2,3-sialylation = ( ( 0 / 1 * (H3N3E1 + H3N3F1E1 + H4N3E1 + H4N3F1E1 ) + 1 / 1 * ( H4N3F2L1 ) ) / ( H3N3E1 + H3N3F1E1 + H4N3E1 + H4N3F1E1 + H4N3F2L1 ) + ( 0 / 2 * ( H3N4F1 + H4N4 + H3N5 + H4N4F1 + H5N4 + H3N5F1 + H4N5 + H5N4F1 + H4N5F1 + H5N5 + H4N4F1E1 + H5N4E1 + H5N5F1 + H5N5F1 + H5N5 + H4N4F1E1 + H5N4E1 + H5N5F1 + H5N5F1 + H5N5F1 + H5N4F1E1 + H5N4E1 + H5N5F1 + H5N5F1 + H5N5F1 + H5N4F1E1 + H5N4E1 + H5N5F1 + H5N5F1 + H5N4F1 ... H5N4F1E1 + H4N5F1E1 + H5N5E1 + H5N5E1Ac1 + H5N4E2 + H5N5F1E1 +H5N4F1E2 + H5N5E2 + H5N5F1E2 ) + 1 / 2 * ( H5N4L1E1 + H5N4F1L1E1 + H5N5F1L1E1 )+ 2 / 2 * ( H5N4L2 + H5N4F1L2 ) ) / ( H3N4F1 + H4N4 + H3N5 + H4N4F1 + H5N4 +H3N5F1 + H4N5 + H5N4F1 + H4N5F1 + H5N5 + H4N4F1E1 + H5N4E1 + H5N5F1 +H5N4F1E1 + H4N5F1E1+ H5N5E1 + H5N4L2 + H5N5E1Ac1 + H5N4L1E1 + H5N4E2 +H5N5F1E1 + H5N4F1L2 + H5N4F1L1E1 +H5N4F1E2 + H5N5E2 + H5N5F1L1E1 + H5N5F1E2) + ( 0 / 3 * ( H6N5E1 + H6N5F1E1 + H6N5E2 + H6N5F1E2 + H6N5E3 ) + 1 / 3 * ( H6N5L1E1 + H6N5F1L1E1 + H6N5L1E2 + H6N5F1L1E2 + H6N5F2L1E2 ) + 2 / 3 * (H6N5L2E1 + H6N5F1L2E1 ) + 3 / 3 * (0) ) / ( H6N5E1 + H6N5F1E1 + H6N5L1E1 + H6N5E2 + H6N5F1L1E1 + H6N5F1E2 + H6N5L2E1 + H6N5L1E2 + H6N5E3 + H6N5F1L2E1 + H6N5F1L1E2 + H6N5F2L1E2 ) + ( 0 / 4 * ( H7N6E1 + H7N6E2 + H7N6E3 ) + 1 / 4 * ( H7N6L1E2 + H7N6F1L1E2 + H7N6L1E3 + H7N6F1L1E3 ) + 2 / 4 * ( H7N6L2E1 + H7N6F1L2E1 + H7N6F2L2E1 + H7N6L2E2 + H7N6F1L2E2 + H7N6F2L2E2 ) + 3 / 4 * ( H7N6L3E1 + H7N6F1L3E1 + H7N6F2L3E1 ) + 4 / 4 * (0) ) / ( H7N6E1 + H7N6E2 + H7N6L2E1 + H7N6L1E2 + H7N6E3 + H7N6F1L2E1 + H7N6F1L1E2 + H7N6L3E1 + H7N6F2L2E1 + H7N6L2E2 + H7N6L1E3 + H7N6F1L3E1 + H7N6F1L2E2 + H7N6F1L1E3 + H7N6F2L3E1 + H7N6F2L2E2 ) ) / ( 0 / 1 * ( H3N3E1 + H3N3F1E1 + H4N3E1 + H4N3F1E1 ) + 1 / 1 * ( H4N3F2L1 ) ) / ( H3N3E1 + H3N3F1E1 + H4N3E1 + H4N3F1E1 +H4N3F2L1 ) + ( 0 / 2 * ( H3N4F1 + H4N4 + H3N5 + H4N4F1 + H5N4 + H3N5F1 + H4N5 +H5N4F1 + H4N5F1 +H5N5 + H4N4F1E1 + H5N4E1 + H5N5F1 + H5N4F1E1 + H4N5F1E1 +H5N5E1 + H5N5E1Ac1 + H5N4E2 + H5N5F1E1 + H5N4F1E2 + H5N5E2 + H5N5F1E2 ) + 1 / 2* ( H5N4L1E1 + H5N4F1L1E1 + H5N5F1L1E1 ) + 2 / 2 * ( H5N4L2 + H5N4F1L2 ) ) / (H3N4F1 + H4N4 + H3N5 + H4N4F1 + H5N4 + H3N5F1 + H4N5 + H5N4F1 + H4N5F1 + H5N5+ H4N4F1E1 + H5N4E1 + H5N5F1 + H5N4F1E1 + H4N5F1E1 + H5N5E1 + H5N4L2 +H5N5E1Ac1 + H5N4L1E1 + H5N4E2 + H5N5F1E1 + H5N4F1L2 + H5N4F1L1E1 + H5N4F1E2 +H5N5E2 + H5N5F1L1E1 + H5N5F1E2 ) + ( 0 / 3 * ( H6N5E1 + H6N5F1E1 + H6N5E2 +H6N5F1E2 + H6N5E3 ) + 1 / 3 * ( H6N5L1E1 + H6N5F1L1E1 + H6N5L1E2 + H6N5F1L1E2 +H6N5F2L1E2 ) + 2 / 3 * ( H6N5L2E1 + H6N5F1L2E1 ) + 3 / 3 * (0) ) / ( H6N5E1 +H6N5F1E1 + H6N5L1E1 + H6N5E2 + H6N5F1L1E1 + H6N5F1E2 + H6N5L2E1 + H6N5L1E2 +H6N5E3 + H6N5F1L2E1 + H6N5F1L1E2 + H6N5F2L1E2 ) + ( 0 / 4 * ( H7N6E1 + H7N6E2 +H7N6E3 ) + 1 / 4 * ( H7N6L1E2 + H7N6F1L1E2 + H7N6L1E3 + H7N6F1L1E3 ) + 2 / 4 * (H7N6L2E1 + H7N6F1L2E1 + H7N6F2L2E1 + H7N6L2E2 + H7N6F1L2E2 + H7N6F2L2E2 ) +3 / 4 * ( H7N6L3E1 + H7N6F1L3E1 +(H7N6F2L3E1) + 4 / 4 * (0)) / (H7N6E1 +H7N6E2 + H7N6L2E1 + H7N6L1E2 + H7N6E3 + H7N6F1L2E1 + H7N6F1L1E2 + H7N6L3E1 +H7N6F2L2E1 + H7N6L2E2 + H7N6L1E3 + H7N6F1L3E1 + H7N6F1L2E2 + H7N6F1L1E3 +H7N6F2L3E1 + H7N6F2L2E2) + (0 / 1 * (H4N3F2L1) + 1 / 1 * (H3N3E1 + H3N3F1E1 + H4N3E1 + H4N3F1E1)) / ( H3N3E1 + H3N3F1E1 + H4N3E1 + H4N3F1E1 + H4N3F2L1) + ( 0 / 2 * ( H3N4F1 + H4N4 + H3N5 + H4N4F1 + H5N4 + H3N5F1 + H4N5 + H5N4F1 + H4N5F1 + H5N5 + H5N5F1 + + 2 / 2 * ( H5N4E2 + H5N4F1E2 + H5N5E2 + H5N5F1E2 ) ) / ( H3N4F1 +H4N4 + H3N5 + H4N4F1 + H5N4 + H3N5F1 + H4N5 + H5N4F1 + H4N5F1 + H5N5 +H4N4F1E1 + H5N4E1 + H5N5F1 + H5N4F1E1 + H4N5F1E1 + H5N5E1 + H5N4L2 + H5N5E1Ac1 + H5N4L1E1 + H5N4E2 + H5N5F1E1 + H5N4F1L2 + H5N4F1L1E1 + H5N4F1E2 + H5N5E2 + H5N5F1L1E1 + H5N5F1E2 ) + ( 0 / 3 * (0) + 1 / 3 * ( H6N5E1 + H6N5F1E1 + H6N5L1E1 + H6N5F1L1E1 + H6N5L2E1 + H6N5F1L2E1 ) + 2 / 3 * ( H6N5E2+ H6N5F1E2 +H6N5L1E2 + H6N5F1L1E2 + H6N5F2L1E2 ) + 3 / 3 * ( H6N5E3 ) ) / ( H6N5E1 +H6N5F1E1 + H6N5L1E1 + H6N5E2 + H6N5F1L1E1 + H6N5F1E2 + H6N5L2E1 + H6N5L1E2 +H6N5E3 + H6N5F1L2E1 + H6N5F1L1E2 + H6N5F2L1E2 ) + ( 0 / 4 * (0) + 1 / 4 * (H7N6E1 + H7N6L2E1 + H7N6F1L2E1 + H7N6L3E1 + H7N6F2L2E1 + H7N6F1L3E1 +H7N6F2L3E1 ) + 2 / 4 * ( H7N6E2 + H7N6L1E2 + H7N6F1L1E2 + H7N6L2E2 + H7N6F1L2E2+ H7N6F2L2E2 ) + 3 / 4 * ( H7N6E3 + H7N6L1E3 + H7N6F1L1E3 ) + 4 / 4 * (0) ) / (H7N6E1 + H7N6E2 + H7N6L2E1 + H7N6L1E2 + H7N6E3 + H7N6F1L2E1 + H7N6F1L1E2 +H7N6L3E1 + H7N6F2L2E1 + H7N6L2E2 + H7N6L1E3 + H7N6F1L3E1 + H7N6F1L2E2 +H7N6F1L1E3 + H7N6F2L3E1 + H7N6F2L2E2 )) [H = hexose; N = N-acetaminohexose; F = deoxyhexose (fucose); L = lactone-esterified N-acetylneuraminic acid (α2,3-linked); E = ethyl-esterified N-acetylneuraminic acid (α2,6-linked). These structures may have different isomers. The positions and linkages of the monosaccharides are well known in the literature].

[0118] As an example only, the α2,6-sialylation of N-glycans (combinations of mono, di, tri, and tetraanthracans) can be calculated as follows: α2,6-sialylation = ( ( 0 / 1 * ( H4N3F2L1 ) + 1 / 1 * ( H3N3E1 + H3N3F1E1 + H4N3E1 + H4N3F1E1 ) ) / ( H3N3E1 + H3N3F1E1 + H4N3E1 + H4N3F1E1 + H4N3F2L1 ) + ( 0 / 2 * ( H3N4F1 + H4N4 + H3N5 + H4N4F1 + H5N4 + H3N5F1 + H4N5 + H5N4F1 + H4N5F1 + H5N5 + H5N5F1 + H5N4L2 + H5N4F1L2 ) + 1 / 2 * ( H4N4F1E1 + H5N4E1 + H5N4F1E1 +H4N5F1E1 + H5N5E1 + H5N5E1Ac1 + H5N4L1E1 + H5N5F1E1 + H5N4F1L1E1 + H5N5F1L1E1) + 2 / 2 * ( H5N4E2 + H5N4F1E2 + H5N5E2 + H5N5F1E2 ) ) / ( H3N4F1 + H4N4 +H3N5 + H4N4F1 + H5N4 + H3N5F1 + H4N5 + H5N4F1 + H4N5F1 + H5N5 + H4N4F1E1 +H5N4E1 + H5N5F1 + H5N4F1E1 + H4N5F1E1 + H5N5E1 + H5N4L2 + H5N5E1Ac1 +H5N4L1E1 + H5N4E2 + H5N5F1E1 + H5N4F1L2 + H5N4F1L1E1 + H5N4F1E2 + H5N5E2 +H5N5F1L1E1 + H5N5F1E2 ) + ( 0 / 3 * (0) + 1 / 3 * ( H6N5E1 + H6N5F1E1 + H6N5L1E1+ H6N5F1L1E1 + H6N5L2E1 + H6N5F1L2E1 ) + 2 / 3 * ( H6N5E2 + H6N5F1E2 + H6N5L1E2+ H6N5F1L1E2 + H6N5F2L1E2 ) + 3 / 3 * ( H6N5E3 ) ) / ( H6N5E1 + H6N5F1E1 +H6N5L1E1 + H6N5E2 + H6N5F1L1E1 + H6N5F1E2 + H6N5L2E1 + H6N5L1E2 +H6N5E3 + H6N5F1L2E1 + H6N5F1L1E2 + H6N5F2L1E2 ) + ( 0 / 4 * (0) + 1 / 4 * ( H7N6E1 + H7N6L2E1 + H7N6F1L2E1 + H7N6L3E1 + H7N6F2L2E1 + H7N6F1L3E1 + H7N6F2L3E1 ) + 2 / 4 * ( H7N6E2 + H7N6L1E2 + H7N6F1L1E2 + H7N6L2E2 + H7N6F1L2E2 + H7N6F2L2E2 ) + 3 / 4 * ( H7N6E3 + H7N6L1E3 + H7N6F1L1E3 ) + 4 / 4 * (0)) ) / ( H7N6E1 + H7N6E2 + H7N6L2E1 + H7N6L1E2 + H7N6E3 + H7N6F1L2E1 + H7N6F1L1E2 + H7N6L3E1 + H7N6F2L2E1 + H7N6L2E2 + H7N6L1E3 + H7N6F1L3E1 + H7N6F1L2E2 + H7N6F1L1E3 + H7N6F2L3E1 + H7N6F2L2E2 ) ) / ( ( 0 / 1 * ( H3N3E1 + H3N3F1E1 + H4N3E1 + H4N3F1E1 ) + 1 / 1 * ( H4N3F2L1 ) ) / ( H3N3E1 + H3N3F1E1 + H4N3E1 + H4N3F1E1 +H4N3F2L1 ) + ( 0 / 2 * ( H3N4F1 + H4N4 + H3N5 + H4N4F1 + H5N4 + H3N5F1 + H4N5 +H5N4F1 + H4N5F1 + H5N5 + H4N4F1E1 + H5N4E1 + H5N5F1 + H5N4F1E1 + H4N5F1E1 +H5N5E1 + H5N5E1Ac1 + H5N4E2 + H5N5F1E1 + H5N4F1E2 + H5N5E2 + H5N5F1E2 ) + 1 / 2* ( H5N4L1E1 + H5N4F1L1E1 + H5N5F1L1E1 ) + 2 / 2 * ( H5N4L2 + H5N4F1L2 ) ) / (H3N4F1 + H4N4 + H3N5 + H4N4F1 + H5N4 + H3N5F1 + H4N5 + H5N4F1 + H4N5F1 + H5N5+ H4N4F1E1 + H5N4E1 +H5N5F1 + H5N4F1E1 + H4N5F1E1 + H5N5E1 + H5N4L2 + H5N5E1Ac1 + H5N4L1E1 + H5N4E2 + H5N5F1E1 + H5N4F1L2 + H5N4F1L1E1 + H5N4F1E2 +H5N5E2 + H5N5F1L1E1 + H5N5F1E2 ) + ( 0 / 3 * ( H6N5E1 + H6N5F1E1 + H6N5E2 +H6N5F1E2 + H6N5E3 ) + 1 / 3 * ( H6N5L1E1 + H6N5F1L1E1 + H6N5L1E2 + H6N5F1L1E2 +H6N5F2L1E2 ) + 2 / 3 * ( (H6N5L2E1 + H6N5F1L2E1) + 3 / 3 * (0)) / (H6N5E1 + H6N5F1E1 + H6N5L1E1 + H6N5E2 + H6N5F1L1E1 + H6N5F1E2 + H6N5L2E1 + H6N5L1E2 + H6N5E3 + H6N5F1L2E1 + H6N5F1L1E2 + H6N5F2L1E2) + (0 / 4 * (H7N6E1 + H7N6E2 + H7N6E3) + 1 / 4 * (H7N6L1E2 + H7N6F1L1E2 + H7N6L1E3 + H7N6F1L1E3) + 2 / 4 * (H7N6L2E1 + H7N6F1L2E1 + H7N6F2L2E1 + H7N6L2E2 + H7N6F1L2E2 + H7N6F2L2E2 ) + 3 / 4 * ( H7N6L3E1 + H7N6F1L3E1 + H7N6F2L3E1 ) + 4 / 4 * (0) ) / ( H7N6E1 + H7N6E2 + H7N6L2E1 + H7N6L1E2 + H7N6E3 + H7N6F1L2E1 + H7N6F1L1E2 + H7N6L3E1 + H7N6F2L2E1 + H7N6L2E2 + H7N6L1E3 + H7N6F1L3E1 + H7N6F1L2E2 + H7N6F1L1E3 +H7N6F2L3E1 + H7N6F2L2E2 ) + ( 0 / 1 * ( H4N3F2L1 ) + 1 / 1 * ( H3N3E1 + H3N3F1E1 + H4N3E1 + H4N3F1E1 ) ) / ( H3N3E1 + H3N3F1E1 +H4N3E1 + H4N3F1E1 + H4N3F2L1) + ( 0 / 2 * ( H3N4F1 + H4N4 + H3N5 + H4N4F1 + H5N4 + H3N5F1 + H4N5 + H5N4F1 +H4N5F1 + H5N5 + H5N5F1 + H5N4L2 + H5N4F1L2 ) + 1 / 2 * ( H4N4F1E1 + H5N4E1 +H5N4F1E1 + H4N5F1E1 + H5N5E1 + H5N5E1Ac1 + H5N4L1E1 + H5N5F1E1 + H5N4F1L1E1 +H5N5F1L1E1 ) + 2 / 2 * ( H5N4E2 + H5N4F1E2 + H5N5E2 + H5N5F1E2 ) ) / ( H3N4F1 +H4N4 + H3N5 + H4N4F1 + H5N4 + H3N5F1 + H4N5 + H5N4F1 + H4N5F1 + H5N5 +H4N4F1E1 + H5N4E1 + H5N5F1 + H5N4F1E1 + H4N5F1E1 + H5N5E1 + H5N4L2 +H5N5E1Ac1 + H5N4L1E1 + H5N4E2 + H5N5F1E1 + H5N4F1L2 + H5N4F1L1E1 + H5N4F1E2 +H5N5E2 + H5N5F1L1E1 + H5N5F1E2 ) + ( 0 / 3 * (0) + 1 / 3 * ( H6N5E1 + H6N5F1E1 +H6N5L1E1 + H6N5F1L1E1 + H6N5L2E1 + H6N5F1L2E1 ) + 2 / 3 * ( H6N5E2 + H6N5F1E2 +H6N5L1E2 + H6N5F1L1E2 + H6N5F2L1E2 ) + 3 / 3 * ( H6N5E3 ) ) / ( H6N5E1 +H6N5F1E1 + H6N5L1E1 + H6N5E2 + H6N5F1L1E1 + H6N5F1E2 + H6N5L2E1 + H6N5L1E2 +H6N5E3 + H6N5F1L2E1 + H6N5F1L1E2 + H6N5F2L1E2 ) + ( 0 / 4 * (0) + 1 / 4 * (H7N6E1 + H7N6L2E1 + H7N6F1L2E1 + H7N6L3E1 + H7N6F2L2E1 + H7N6F1L3E1 +H7N6F2L3E1 )+ 2 / 4 * ( H7N6E2 + H7N6L1E2 + H7N6F1L1E2 + H7N6L2E2 + H7N6F1L2E2+ H7N6F2L2E2 ) + 3 / 4 * ( H7N6E3 + H7N6L1E3 + H7N6F1L1E3 ) + 4 / 4 * (0) ) / (H7N6E1 + H7N6E2 + H7N6L2E1 + H7N6L1E2 + H7N6E3 + H7N6F1L2E1 + H7N6F1L1E2 +H7N6L3E1 + H7N6F2L2E1 + H7N6L2E2 + H7N6L1E3 + H7N6F1L3E1 + H7N6F1L2E2 +H7N6F1L1E3 + H7N6F2L3E1 + H7N6F2L2E2 )) [H = hexose; N = N-acetaminohexose; F = deoxyhexose (fucose); L = lactone-esterified N-acetylneuraminic acid (α2,3-linked); E = ethyl-esterified N-acetylneuraminic acid (α2,6-linked). These structures may have different isomers. The positions and linkages of the monosaccharides are well known in the literature].

[0119] Suitablely, the α2,3-sialylation of N-glycans can be determined on two-antenna, three-antenna, and / or four-antenna N-glycans. In other words, it can be said that the levels of A2L, A3L, and / or A4L are determined separately. Suitablely, the α2,3-sialylation of N-glycans can be determined on three-antenna and / or four-antenna N-glycans.

[0120] Suitablely, α2,6-sialylation of N-glycans can be determined on tri-antenna and / or tetra-antenna N-glycans. In other words, it can be said that the levels of A3E and / or A4E are determined separately. Suitablely, α2,6-sialylation of N-glycans can be determined on tri-antenna N-glycans.

[0121] Generally, a glycan is a sugar, including monosaccharides, disaccharides, or trisaccharides; it can include oligosaccharides or polysaccharides. Oligosaccharides are oligosaccharides containing two or more sugars. The structure of an oligosaccharide is typically characterized by the specific types, sequences, bond positions (including branch points), and bond stereochemistry (α, β) of its monomers, thus having a defined molecular weight and composition. Oligosaccharides typically contain about 2 to about 20 or more sugar monomers. In polysaccharides, the types, sequences, bond positions (including branch points), and / or bond stereochemistry can vary from molecule to molecule. Proteins containing glycans can be called glycoproteins.

[0122] The glycan component of a glycoprotein can be N-linked or O-linked. N-glycans are attached to nitrogen atoms, such as the side-chain nitrogen atom of an asparagine amino acid within a peptide. O-linked glycans are attached to oxygen atoms, such as the side-chain hydroxyl oxygen atom of a hydroxyl lysine, hydroxyproline, tyrosine, serine, or threonine amino acid within a peptide. "Glycosylation" refers to the covalent attachment of at least one sugar moiety to a molecule. Glycosidic bonds include O-glycosidic bonds, N-glycosidic bonds, S-glycosidic bonds, and C-glycosidic bonds. O-glycosidic bonds form between the anodic carbon (C1) of a sugar and the oxygen atom of another molecule (such as another sugar or polypeptide), while N-glycosidic bonds form between the anodic carbon (C1) of a sugar and the nitrogen atom of another molecule. Similarly, S-glycosidic bonds and C-glycosidic bonds involve sulfur and carbon atoms from another molecule, respectively. Furthermore, glycosidic bonds are classified according to the ring position of the carbon atoms involved in the bond. For example, a 1,4 glycosidic bond forms between the first carbon (C1) of the first sugar and the fourth carbon (C4) of the second sugar, while a 1,6 glycosidic bond forms between the first carbon (C1) of the first sugar and the sixth carbon (C6) of the second sugar. As another example, a 2,3 glycosidic bond forms between the second carbon (C2) of the first sugar and the third carbon (C3) of the second sugar, while a 2,6 glycosidic bond forms between the second carbon (C2) of the first sugar and the sixth carbon (C6) of the second sugar. Depending on whether the substituents on the carbons flanking the oxygen in the sugar point in the same or opposite directions, glycosidic bonds can be further classified as α-glycosidic bonds or β-glycosidic bonds. The term “glycosylation” as used herein should be interpreted broadly to include the covalent linkage of any other carbohydrate moiety such as fucose and sialic acid, and therefore includes fucosylation or sialylation. Most N-linked glycans share a common structure called a core, typically containing three mannose residues and two N-acetylglucosamine residues. The core can contain modifications such as sulfation or phosphorylation; the core can be intact or truncated. Both end-cap and core modifications are feasible for various glycans. Core glycosylation refers to adding a glycosyl moiety to the core N-acetylglucosamine and / or mannose. Core fucosylation refers to adding a fucose residue to the core N-acetylglucosamine. The glycan can be branched or unbranched. Non-core moieties, and such as sialic acid, can also be readily modified, such as acetylated.

[0123] Suitably, the N-glycan may comprise or consist of complex N-glycans. A "complex N-glycan" is a glycan containing at least one N-acetylglucosamine on each of the two mannose branches of the core. Suitably, the complex N-glycan may be selected from the group consisting of: biantennary N-glycans, triantennary N-glycans, and tetraantennary N-glycans. More suitably, the glycan may be selected from the group consisting of: biantennary, triantennary N-glycans, and tetraantennary N-glycans. More suitably, the glycan may be selected from the group consisting of: triantennary N-glycans and tetraantennary N-glycans.

[0124] In branched polysaccharides, the monosaccharide at the branch point is covalently linked to two other sugars on carbons other than C1. For example, in addition to being linked to another monosaccharide or amino acid at C1, branched monosaccharides can also be linked to other monosaccharides at C4 and C6.

[0125] Complex polysaccharides can be, but are not limited to, biantennary (i.e., having two branches on the core structure), triantennary (i.e., having three branches on the core structure), or tetraantennary (i.e., having four branches on the core structure). One or more branches may be galactose-terminated. When the galactose moiety is attached to a sialic acid moiety, it is called sialylation.

[0126] The inventors have discovered that subjects with fibrotic NASH exhibit altered levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans compared to healthy controls or patients with NAFLD or non-fibrotic NASH. Specifically, the inventors have found decreased α2,3-sialylation levels and / or increased α2,6-sialylation levels in patients with fibrotic NASH. The term "α2,3-sialylation" refers to the linkage of sialic acid to galactose via an α2,3 bond. In humans, α2,3-sialic acid linkage is primarily determined by the activity of sialyltransferases (genes: St3Gal1, St3Gal2, St3Gal3, St3Gal4, St3Gal5, and / or St3Gal6). Similarly, the term "α2,6-sialylation" refers to the linkage of sialic acid to galactose via an α2,6 bond. In humans, the α2,6 bond is primarily determined by the activity of the sialyltransferase genes St6Gal1 and St6Gal2. Other factors may include glycosidases, which are enzymes that specifically remove sialic acid from a particular bond (sialylases; genes: NEUR1-4), or receptors that remove glycoproteins from circulation (desialylglycoprotein receptors; genes: ASGR1, ASGR2).

[0127] The method described herein includes steps for comparing the α2,3-sialylation and / or α2,6-sialylation levels of N-glycans with reference values. It should be understood that reference values ​​may be derived from suitable control samples.

[0128] Depending on the context, a reference value can be a value that allows for the determination of whether a subject has fibrotic NASH. Such a reference value can be particularly relevant in the context of the methods disclosed herein for diagnosing fibrotic NASH. In this context, the reference value can be derived from control samples having normal levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans. Suitably, said control samples can be obtained from one or more subjects who do not have NAFLD and / or NASH (e.g., fibrotic or non-fibrotic NASH) and / or are not at risk of developing NAFLD and / or NASH (referred to herein as control subjects). Suitably, the reference value can be the level of α2,3-sialylation and / or α2,6-sialylation of N-glycans in individuals or groups of individuals considered to be generally healthy (i.e., without a diagnosed condition and / or without symptoms indicative of a condition).

[0129] In another embodiment, the reference value may be a value that defines the stage of fibrotic NASH. In such an embodiment, the reference value may be obtained from a subject or subject group previously diagnosed with a specific stage of fibrosis and optionally assigned a Brunt Fibrosis Score and / or another score based on a known classification method for fibrotic NASH. Other classification methods (also referred to herein as scoring systems, and described elsewhere herein) are also possible. Exemplary methods for determining reference values ​​are provided in the Embodiments section of this disclosure.

[0130] As used in this article, “derived from” refers to levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans that substantially correspond to the levels in suitable control subjects.

[0131] Suitable, the control sample may be a sample of the same type as the test sample (i.e., a sample from a subject who was tested using the methods disclosed herein).

[0132] Suitablely, control samples can be obtained from subjects matched with the test subjects (i.e., of the same sex or age). However, surprisingly, the inventors found that the α2,3-sialylation and / or α2,6-sialylation of N-glycans are not affected by the age and sex of the subjects. Therefore, advantageously, sample matching is not required.

[0133] Control samples can be measured simultaneously with, before or after, alone or simultaneously with test samples to provide reference values.

[0134] Reference values ​​used for comparison with test samples can be values ​​calculated from the mean or median of one or more control samples (e.g., two or more, five or more, ten or more, a group, etc.). Alternatively, control samples can be samples derived from one or more control samples (i.e., mixtures thereof). Reference values ​​can be calculated as the mean or median of a group or cohort of control subjects. Suitablely, a reference value can be a single cutoff value, such as the median or mean. Alternatively, it can be a range of cutoff values ​​(or thresholds). Ranges may be more suitable as reference values ​​in methods for determining the stage of fibrosis.

[0135] The reference value can be a predetermined reference value for the α2,3-sialylation and / or α2,6-sialylation of N-glycans. Such predetermined reference values ​​can be obtained from a reference database. Therefore, it should be understood that, within the context of the methods disclosed herein, in the same determination of the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in a sample, it is not necessary to determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans to obtain reference values.

[0136] Suitablely, the reference value may be derived from the average number of α2,3- and / or α2,6-linked sialic acids for each antenna in a sample that has undergone N-glycan release and linker-specific sialic acid derivatization from blood (e.g., plasma, serum, whole blood, or dried blood) proteins. After preparing the sample as described elsewhere herein, the total number and individual quantity of α2,3- and α2,6-sialylated antennas can be determined, thereby allowing the determination of the relative abundance of α2,3- and / or α2,6-sialylation. This relative abundance can be expressed, for example, as a percentage or fraction.

[0137] α2,3-sialylation can be measured on single-antenna, two-antenna, three-antenna, and / or four-antenna N-glycans. Combinations of single-, two-, three-, and four-antenna N-glycans with α2,3-sialylation are referred to herein as “AL”. Two-antenna N-glycans with α2,3-sialylation are referred to herein as “A2L”. Three-antenna N-glycans with α2,3-sialylation are referred to herein as “A3L”. Four-antenna N-glycans with α2,3-sialylation are referred to herein as “A4L”.

[0138] Similarly, α2,6-sialylation can be measured on single-antenna, two-antenna, three-antenna, and / or four-antenna N-glycans. Combinations of single-, two-, three-, and four-antenna N-glycans with α2,6-sialylation are referred to herein as “AE”. Three-antenna N-glycans with α2,6-sialylation are referred to herein as “A3E”.

[0139] As shown in Table 4, the inventors found that the average relative abundance of AL in fibrotic NASH patients was 0.2316, compared to 0.2497 in non-fibrotic patients. This means that in samples from fibrotic patients (who underwent α2,3-sialylation of N-glycans released from blood proteins (e.g., plasma, serum, or whole blood) with sialic acid-specific chemiluminescence derivatization), 23.16% of mono, di, tri, and tetra-anthracanthanines were α2,3-sialylated, compared to 24.97% in non-fibrotic patients. In other words, it can be said that fibrotic NASH patients had 1.81% less AL glycans than non-fibrotic patients. Based on these results, the inventors have calculated an average cutoff value of 0.2406 as a suitable reference value for AL. Therefore, if a patient's relative abundance of AL is below this value, it indicates that the patient has fibrotic NASH. For clarity, in this context, a reference value of 0.2406 means that 24.06% of the sialylated N-glycan antennas belonging to the groups of mono-, di-, tri-, and tetra-glycan antennas in the sample are α2,3-sialylated.

[0140] Similarly, the inventors found that the average relative abundance of A2L in fibrotic NASH patients was 0.0292, compared to non-fibrotic patients with an A2L relative abundance of 0.0361. Based on these results, the inventors have calculated an average cutoff value of 0.0327 as a suitable reference value for A2L. Therefore, if a patient's relative abundance of A2L is lower than this value, it indicates that the patient has fibrotic NASH.

[0141] Similarly, the inventors found that the average relative abundance of A3L in fibrotic NASH patients was 0.2593, compared to a relative abundance of 0.2850 in non-fibrotic patients. Based on these results, the inventors calculated an average cutoff value of 0.2721 as a suitable reference value for A3L. Therefore, if a patient's relative abundance of A3L is lower than this value, it indicates that the patient has fibrotic NASH.

[0142] Similarly, the inventors found that the average relative abundance of A4L in fibrotic NASH patients was 0.4438, compared to a relative abundance of 0.4936 in non-fibrotic patients. Based on these results, the inventors have calculated an average cutoff value of 0.4687 as a suitable reference value for A4L. Therefore, if a patient's relative abundance of A4L is lower than this value, it indicates that the patient has fibrotic NASH.

[0143] As shown in Table 4, the inventors found that the average relative abundance of AEs in fibrotic NASH patients was 0.7684, compared to 0.7503 in non-fibrotic patients. This means that in samples from fibrotic patients (who underwent α2,6-sialylation of N-glycans and linker-specific chemicals from blood proteins (e.g., plasma, serum, whole blood, or dried blood)), 76.84% of mono, di, tri, and tetra-anthracans were α2,6-sialylated, compared to 75.03% in non-fibrotic patients. In other words, it can be said that fibrotic NASH patients had 1.81% more AE glycans than non-fibrotic patients. Based on these results, the inventors have calculated an average cutoff value of 0.7594 as a suitable reference value for AEs. Therefore, if a patient's relative abundance of AEs is higher than this value, it indicates that the patient has fibrotic NASH. For clarity, in this context, a reference value of 0.7594 means that 75.94% of the sialylated N-glycan antennas belonging to the groups of mono-, di-, tri-, and tetra-glycan antennas in the sample are α2,6-sialylated.

[0144] Similarly, the inventors found that the average relative abundance of A3E in fibrotic NASH patients was 0.6678, compared to a relative abundance of 0.6463 in non-fibrotic patients. Based on these results, the inventors have calculated an average cutoff value of 0.6571 as a suitable reference value for A3E. Therefore, if a patient's relative abundance of A3E is higher than this value, it indicates that the patient has fibrotic NASH.

[0145] As shown in Table 8, the inventors found that the average relative abundance of AL in fibrotic NASH patients was 0.215 compared to that in non-fibrotic patients (where the relative abundance was 0.227). This means that in samples from fibrotic patients (who underwent α2,3-sialylation of N-glycans released from blood proteins (e.g., plasma, serum, or whole blood)), 21.5% of mono, di, tri, and tetra-anthracans were α2,3-sialylated, compared to 22.7% in non-fibrotic patients. In other words, it can be said that fibrotic NASH patients had 1.2% less AL glycans than non-fibrotic patients. Based on these results, the inventors have calculated an average cutoff value of 0.221 as a suitable reference value for AL. Therefore, if a patient's relative abundance of AL is below this value, it indicates that the patient has fibrotic NASH. For clarity, in this context, a reference value of 0.221 means that 22.1% of the sialylated N-glycan antennas belonging to the groups of mono-, di-, tri-, and tetra-glycan antennas in the sample are α2,3-sialylated.

[0146] Similarly, the inventors found that the average relative abundance of A2L in fibrotic NASH patients was 0.031, compared to non-fibrotic patients with an A2L relative abundance of 0.039. Based on these results, the inventors have calculated an average cutoff value of 0.035 as a suitable reference value for A2L. Therefore, if a patient's relative abundance of A2L is lower than this value, it indicates that the patient has fibrotic NASH.

[0147] Similarly, the inventors found that the average relative abundance of A3L in fibrotic NASH patients was 0.272, compared to a relative abundance of 0.294 in non-fibrotic patients. Based on these results, the inventors have calculated an average cutoff value of 0.283 as a suitable reference value for A3L. Therefore, if a patient's relative abundance of A3L is lower than this value, it indicates that the patient has fibrotic NASH.

[0148] Similarly, the inventors found that the average relative abundance of A4L in fibrotic NASH patients was 0.508, compared to non-fibrotic patients with an A4L relative abundance of 0.538. Based on these results, the inventors have calculated an average cutoff value of 0.523 as a suitable reference value for A4L. Therefore, if a patient's relative abundance of A4L is lower than this value, it indicates that the patient has fibrotic NASH.

[0149] As shown in Table 8, the inventors found that the average relative abundance of AEs in fibrotic NASH patients was 0.785, compared to 0.773 in non-fibrotic patients. This means that in samples from fibrotic patients (who underwent α2,6-sialylation of N-glycans and linker-specific chemicals from blood proteins (e.g., plasma, serum, whole blood, or dried blood)), 78.5% of mono, di, tri, and tetra-anthracans were α2,6-sialylated, compared to 77.3% in non-fibrotic patients. In other words, it can be said that fibrotic NASH patients had 1.2% more AE glycans than non-fibrotic patients. Based on these results, the inventors have calculated an average cutoff value of 0.779 as a suitable reference value for AEs. Therefore, if a patient's relative abundance of AEs is higher than this value, it indicates that the patient has fibrotic NASH. For clarity, in this context, a reference value of 0.779 means that 77.9% of the sialylated N-glycan antennas belonging to the groups of mono-, di-, tri-, and tetra-glycan antennas in the sample are α2,6-sialylated.

[0150] Similarly, the inventors found that the average relative abundance of A3E in fibrotic NASH patients was 0.672, compared to non-fibrotic patients with a relative abundance of 0.652. Based on these results, the inventors have calculated an average cutoff value of 0.662 as a suitable reference value for A3E. Therefore, if a patient's relative abundance of A3E is higher than this value, it indicates that the patient has fibrotic NASH.

[0151] In the context of this disclosure, the term "change" refers to a statistically significant difference in the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in a sample obtained from a test subject compared to a reference value. This difference (or change) may suitably be an increase or decrease in the level of the biomarker compared to a control sample or a predetermined reference value. Similarly, "no change" means that there is no statistically significant difference in the levels of α2,3-sialylation and / or α2,6-sialylation in a sample obtained from a test subject compared to a reference value. No change in α2,3-sialylation and / or α2,6-sialylation levels may indicate that the subject does not have fibrotic NASH. In other embodiments, no change may indicate that fibrotic NASH has not progressed. However, given that this disclosure teaches that an increase in α2,6-sialylation levels and / or a decrease in α2,3-sialylation levels generally indicate the progression of fibrotic NASH and / or fibrotic NASH, those skilled in the art are capable of interpreting any changes (or lack thereof) observed in α2,3-sialylation and / or α2,6-sialylation levels.

[0152] The terms “increased,” “increased,” or “higher” are generally used herein to indicate an increase that is statistically significant; for the avoidance of any doubt, the terms “increased” or “increased” mean an increase of at least about 0.5% compared to a reference value, such as an increase of at least about 0.6%, or at least about 0.7%, or at least about 0.8%, or at least about 0.9%, or at least about 1%. For example, an increase compared to a reference value could be at least about 1.25%, or at least about 1.5%, or at least about 1.75%, at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4%, at least about 4.5%, at least about 5%, or more.

[0153] The terms “reduced,” “lower,” or “less” are generally used herein to indicate a statistically significant reduction; for the avoidance of any doubt, these terms mean a reduction of at least about 0.5% compared to a reference value, such as at least about 0.6%, or at least about 0.7%, or at least about 0.8%, or at least about 0.9%, or at least about 1%. For example, a reduction compared to a reference value could be at least about 1.25%, or at least about 1.5%, or at least about 1.75%, at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4%, at least about 4.5%, at least about 5%, or more.

[0154] The methods described herein can be performed on samples obtained from a subject. The term "sample" as used herein is intended to include biological material from a subject. Suitably, the sample may be blood. Suitably, the blood may be arterial blood, capillary blood, venous blood, or a mixture thereof.

[0155] In some instances, the blood sample may be selected from the group consisting of whole blood, plasma, and serum. As used herein, the term "whole blood" means blood containing all, or a large number of, the natural components, elements, or elements of blood. Therefore, whole blood will include plasma, the erythrocyte sedimentation rate (ESR) layer (white blood cells and platelets), and red blood cells. The term "plasma" (or blood plasma) refers to the fully soluble portion of blood, while the term "serum" (or blood serum) refers to plasma free of clotting factors, i.e., plasma obtained after blood has clotted.

[0156] In some instances, the blood sample may be liquid or non-liquid.

[0157] Suitably, the sample may be liquid blood. In some instances, the liquid blood sample is fresh. In this case, fresh means that the liquid blood sample was drawn from the subject less than 48 hours before being used in the methods described herein. Suitably, the sample may have been refrigerated and / or frozen for part or all of the time between sample extraction from the subject and use in the methods described herein. In some instances, the liquid blood sample has been thawed. In some instances, the liquid blood sample may be a rehydrated blood sample.

[0158] In some instances, the blood sample is non-liquid. As used herein, the term "non-liquid blood sample" is intended to include blood samples that were previously liquid but have been dried through processes such as evaporation, freeze-drying, or dehydration. In some instances, the non-liquid blood sample is a dried blood spot sample. In some instances, dried blood is rehydrated to form a liquid blood sample.

[0159] In some instances, blood samples have been subjected to N-glycan release and sialic acid-linked derivatization from blood proteins (e.g., plasma, serum, whole blood, or dried blood) prior to determining the α2,3-sialylation and / or α2,6-sialylation levels of the glycan. Therefore, the methods described herein may include a step of releasing N-glycan from blood proteins and sialic acid-linked derivatization of the sample prior to determining the α2,3-sialylation and / or α2,6-sialylation levels of the glycan. Suitably, the N-glycan release step may be performed as described in Vreeker, GCM et al. 2018 (doi:10.1021 / acs.analchem.8b02391), which is incorporated herein by reference.

[0160] As an example only, the release of N-glycans can be achieved by incubating a sample (e.g., a plasma sample) with SDS (e.g., 2% SDS). Incubation can be performed at approximately 60°C for approximately 1 minute to approximately 1 hour (e.g., 10 minutes). After incubation, the sample can be incubated with a release mixture. The release mixture may contain NP-40 (e.g., 4% NP-40), acidified PBS, and PNGase F. Incubation with the release mixture can be performed at approximately 37°C for approximately 1 hour to 24 hours, suitably, for example, approximately 8 to 10 hours.

[0161] It should be understood that when the sample is dried blood spot, the N-glycan release step can be performed before drying the blood sample. Alternatively, the N-glycan release can be performed after the dried blood sample has been rehydrated.

[0162] The methods disclosed herein can be combined with other tests used for the diagnosis, staging, and / or monitoring of fibrotic NASH and for disease surveillance. While other tests useful in this context are well known to those skilled in the art, they may, by way of example only, include determining a subject's FIB-4 score, ELF score, and / or APRI score. Additionally or optionally, the methods described herein may also include liver imaging (such as Fibroscan) to determine the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) in the subject (or samples from the subject), and / or histological analysis via liver biopsy.

[0163] In another aspect, the present invention provides a method for staging fibrotic nonalcoholic steatohepatitis (NASH) in a subject, the method comprising the following steps:

[0164] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in samples from subjects; and

[0165] • The determined α2,3-sialylation and / or α2,6-sialylation levels are compared with reference values ​​indicating the stage of fibrotic NASH to determine the subject's stage of fibrotic NASH.

[0166] As used herein, the term "staging" refers to classifying the degree or severity of a subject's disease. Suitablely, staging may include assigning a Brunt fibrosis score (e.g., score 0, 1, 2, 3, or 4) to the patient. The inventors have found that patients with different Brunt fibrosis scores exhibit different levels of α2,3-sialylation and / or α2,6-sialylation. Typically, obtaining a Brunt fibrosis score involves obtaining a liver biopsy from the subject. However, since the inventors have found that the levels of α2,3-sialylation and / or α2,6-sialylation can differ among patients with different Brunt scores, it is now possible to measure the levels of α2,3-sialylation and / or α2,6-sialylation without requiring a liver biopsy and to assign a Brunt score to the subject. While the embodiments of this application demonstrate the correlation between different Brunt scores and α2,3-sialylation and / or α2,6-sialylation levels, those skilled in the art will understand that correlations may also exist between α2,3-sialylation and / or α2,6-sialylation levels and other fibrosis staging methods, such as Brunt activity, fibrosis-4 (FIB-4), enhanced liver fibrosis (ELF), AST to platelet ratio index (APRI), and / or Fibroscan score. Therefore, in suitable embodiments, staging can be based on Brunt fibrosis score, Brunt activity score, FIB-4 score, ELF score, and / or APRI score.

[0167] Appropriate reference values ​​for different stages of fibrosis can be determined, for example, through receiver operating characteristic analysis (which assesses sensitivity and specificity at different cutoff values) and by determining the optimal cutoff value based on the area under the curve (AUC). As another example, a linear regression equation can be obtained indicating changes in any glycosylation properties as Brunt score units change. Other methods for determining reference values ​​are known to those skilled in the art.

[0168] In another aspect, the present invention provides a method for monitoring fibrotic nonalcoholic steatohepatitis (NASH) in subjects, the method comprising the following steps:

[0169] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in the first sample from the subject; and

[0170] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in a second sample from the subject, wherein the second sample was obtained from the subject at a later time point than the first sample, wherein:

[0171] i) Compared with the first sample, the increase in α2,6-sialylation level and / or the decrease in α2,3-sialylation level in the second sample indicated the progression of fibrotic NASH in the subjects;

[0172] ii) The decrease in α2,6-sialylation level and / or the increase in α2,3-sialylation level in the second sample compared to the first sample indicated the regression of fibrotic NASH in the subjects.

[0173] Appropriately, the lack of change in α2,6-sialylation and / or α2,3-sialylation levels in the second sample compared to the first sample can indicate that NASH has neither progressed nor regressed in the subject.

[0174] As used herein, the term "monitoring" refers to determining whether fibrotic NASH in a subject progresses or regresses between at least two distinct time points. Those skilled in the art will understand that, in order to monitor a subject, samples used to determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans must be obtained at two distinct time points. During the time between obtaining the first and second samples, the subject may or may not receive treatment for fibrotic NASH. In one embodiment, monitoring can also be used to determine whether the subject has been successfully treated or at least whether fibrotic NASH has improved over time with some form of treatment while the subject is receiving treatment between obtaining the first and second samples. Therefore, the foregoing methods can be applied to determine whether treatment for fibrotic NASH is effective and / or whether the subject is adhering to prescribed treatment for fibrotic NASH. These applications lead to further aspects of the invention as described below.

[0175] As used in this article, “progression” refers to the worsening of fibrotic NASH in subjects. Fibrotic NASH can be said to be progressing when an increase in α2,6-sialylation levels and / or a decrease in α2,3-sialylation levels are observed in the second sample compared to the first sample. Similarly, as used in this article, “regression” refers to the improvement of fibrotic NASH in subjects. Fibrotic NASH can be said to be regressing when a decrease in α2,6-sialylation levels and / or an increase in α2,3-sialylation levels are observed in the second sample compared to the first sample.

[0176] In another aspect, the present invention provides a method for monitoring the therapeutic effect of prescription treatment for fibrotic nonalcoholic steatohepatitis (NASH), the method comprising the following steps:

[0177] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in the first sample from the subject; and

[0178] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in a second sample obtained from the subject following prescribed treatment.

[0179] Compared with the first sample, a decrease or no change in the level of α2,6-sialylation in the second sample and / or an increase or no change in the level of α2,3-sialylation in the second sample indicated the therapeutic effect of the prescribed treatment for fibrotic NASH.

[0180] The term “prescription treatment” (also referred to herein as “treatment”) refers to any intervention designed to improve fibrotic NASH in a subject. Improvement in this article means preventing, stopping, or slowing the progression of fibrotic NASH, or completely or partially reversing fibrotic NASH.

[0181] It should be understood that this treatment can directly or indirectly improve fibrotic NASH. Indirect improvement of fibrotic NASH may be a treatment prescribed for different underlying diseases (such as type 2 diabetes) and / or conditions (such as obesity), and this treatment may also have a therapeutic effect on fibrotic NASH while treating said underlying diseases and / or conditions. Direct improvement of fibrotic NASH may be a treatment specifically prescribed to improve fibrotic NASH in the subject.

[0182] Appropriately, prescription treatment may be lifestyle changes (such as improving diet, increasing physical activity and / or losing weight) or medications (such as biologics or small molecules).

[0183] Appropriately, lifestyle changes or improvements may include a reduction in the subject's weight, an increase in the subject's physical activity, such as through increased physical exercise, and / or improvements in the subject's diet, such as an increase in the (proportion) intake of fruits and / or vegetables, and / or a decrease in the (proportion) intake of ultra-processed foods.

[0184] The method can be used as a screening tool to determine whether a drug and / or drug treatment regimen is effective against fibrotic NASH. The drug and / or drug treatment regimen tested may be a new treatment regimen or a new drug, an improved treatment regimen or an improved drug, or a known treatment regimen or a known drug that requires further testing. In this context, the term "drug treatment regimen" refers to the dosage, dosing cycle, dosing interval, and / or combination of drugs.

[0185] If, compared to the first sample, the prescribed treatment results in a decrease in the level of α2,6-sialylation and / or an increase in the level of α2,3-sialylation in the second sample, then the prescribed treatment can be identified as having a therapeutic effect.

[0186] In the context of methods for monitoring the therapeutic effects of prescribed treatments, it should be understood that the time interval between the first and second samples obtained from the subject must be sufficient to detect any potential therapeutic effects that the prescribed treatment may have. Therefore, the second sample may be obtained approximately 2 weeks, 4 weeks, 6 weeks, 3 months, 6 months, 1 year, or longer after obtaining the first sample. Additionally or optionally, the second sample may be obtained from the subject approximately 2 weeks, 4 weeks, 6 weeks, 3 months, 6 months, 1 year, or longer after the commencement of prescribed treatment. At the time the first sample is obtained from the subject, the subject may or may not have commenced prescribed treatment.

[0187] Those skilled in the art can readily determine the appropriate time interval (e.g., during the subject’s treatment) for monitoring disease status or symptom improvement, and that time interval will depend on the patient being monitored (e.g., the cause of fibrotic NASH).

[0188] In a related aspect, the present invention provides a method for monitoring subject adherence to prescribed treatment for fibrotic NASH, the method comprising the following steps:

[0189] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in the first sample from the subject; and

[0190] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in a second sample obtained from the subject following prescribed treatment.

[0191] Compared with the first sample, a decrease or no change in α2,6-sialylation levels in the second sample and / or an increase or no change in α2,3-sialylation levels in the second sample indicated subject adherence to prescription treatment for fibrotic NASH.

[0192] The term “compliance” as used in this article refers to a patient’s obedience to prescribed treatment, that is, the degree to which a patient follows the prescribed treatment.

[0193] In the context of methods used to monitor treatment efficacy or subject adherence, prescribed treatment can be a treatment designed to reverse (eliminate) fibrotic NASH. In such an implementation, it should be understood that a decrease in α2,6-sialylation levels and / or an increase in α2,3-sialylation levels in a second sample compared to a first sample can indicate therapeutic efficacy or subject adherence to treatment.

[0194] In other instances, prescription treatment may be aimed at preventing the development of fibrotic NASH or halting its progression. In such implementations, the absence of change in α2,3-sialylation and / or α2,6-sialylation levels may indicate therapeutic efficacy or subject adherence to treatment.

[0195] Furthermore, in other instances, treatment may be designed to slow the progression of NASH. In such implementations, an increase in α2,6-sialylation levels and / or a decrease in α2,3-sialylation levels can indicate therapeutic efficacy or subject adherence to treatment. In such implementations, the increase in α2,6-sialylation levels and / or the decrease in α2,3-sialylation levels may be slower than expected (e.g., slower than changes in α2,6-sialylation and / or α2,3-sialylation levels observed before the subject began prescribed treatment).

[0196] In some aspects, the present invention provides a method for diagnosing and treating patients with fibrotic NASH, the method comprising:

[0197] • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in samples from subjects;

[0198] • Compare the determined levels of α2,3-sialylation and / or α2,6-sialylation with reference values;

[0199] • If α2,6-sialylation levels are increased and / or α2,3-sialylation levels are decreased, the subject is identified as having fibrotic NASH; and

[0200] • Administer fibrotic NASH treatment to subjects who have been identified as having fibrotic NASH.

[0201] As used herein, the term “treatment” includes interventions undertaken to improve fibrotic NASH. Therefore, “treatment” refers to therapeutic procedures and preventative or preventive measures in which the aim is to prevent or alleviate (reduce) a target condition, symptom, or illness.

[0202] The methods disclosed herein can be combined with other tests used to diagnose or monitor fibrotic NASH. Such other tests are known to those skilled in the art. By way of example only, they may include determining the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) in a subject (or a sample from a subject), and / or by histological analysis of a liver biopsy, liver imaging (such as Fibroscan), liver disease scoring, etc.

[0203] In another aspect, the present invention also provides a kit for use with the methods disclosed herein. The kit may include reagents suitable for determining analyte levels in a test sample (e.g., reagents suitable for determining levels of biomarkers disclosed herein).

[0204] Suitably, the kit contains a detectable labeling agent that specifically binds to the α2,3-sialylation and / or α2,6-sialylation of the glycan.

[0205] Optionally, the kit may contain one or more control samples or references. Additionally, in some cases, the kit may include written information (indicators) providing a reference (e.g., predetermined values), wherein comparisons between biomarker levels in subjects and references (predicted values) indicate clinical status. In some cases, the kit includes software for comparing biomarker levels or events with references (e.g., predictive models). This software is typically provided in a computer-readable format (e.g., CD-ROM) but may also be available for download via the Internet. However, the kit is not limited to these, and other variations will be apparent to those skilled in the art.

[0206] The components of the kit can be packaged in containers suitable for transport.

[0207] The term "detectable labeled reagent" refers to a binding ligand that specifically interacts (i.e., binds) to a target biomarker (α2.3-sialylation and / or α2.6-sialylation) or a complex glycan, and can be detected, for example, directly (e.g., by mass labeling or fluorescence or tagging) or indirectly (e.g., by labeled secondary antibody). Therefore, a detectable labeled reagent is a selectively binding ligand of the target biomarker (and substantially does not bind to other glycans, proteins, or combinations thereof (e.g., glycopeptides or glycoproteins)). Selectively binding ligands can include antibodies that selectively bind to one of the target biomarkers.

[0208] As used herein, "specific binding to α2,3-sialylation and / or α2,6-sialylation" means that under certain conditions, a binding ligand that "specifically binds to α2,3-sialylation and / or α2,6-sialylation" will selectively bind to α2,3-sialylation and / or α2,6-sialylation without extensively binding to other protein modifications, including other types of sialylation. Therefore, the binding ligand can bind to α2,3-sialylation and / or α2,6-sialylation with an affinity at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times higher than its affinity for other glycans, peptides, proteins, and / or protein modifications.

[0209] In some instances, the kit includes a detectable labeled reagent on a continuous (e.g., solid) surface (e.g., a lateral flow surface). Alternatively, in instances containing more than one detectable labeled reagent, the detectable labeled reagent may be located in different (i.e., spatially separated) regions on a (e.g., solid) surface, such as a multi-walled microtiter plate (e.g., for ELISA assays). Other suitable surfaces and containers well known in the art may also form part of the kit described herein.

[0210] In one example, the kit also includes one or more reagents for detecting the detectable label. Suitable reagents are well known in the art, including but not limited to standard reagents and buffers required to perform any suitable detection method that can be used (and is well known in the art). In one example, the kit includes one or more of the following: a multi-well plate, ball bearings, extraction buffer, extraction bottle, and lateral flow device.

[0211] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide general dictionaries for those skilled in the art of the many terms used in this invention. Although any use of methods and materials similar to or equivalent to those described herein has been found in the practice of this invention, preferred methods and materials are described herein. Therefore, the terms defined below are described more fully by reference to the specification as a whole. Furthermore, as used herein, the singular terms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Unless otherwise stated, nucleic acids are written from left to right in a 5' to 3' orientation; amino acid sequences are written from left to right, respectively, in an amino-to-carboxyl orientation. It should be understood that the present invention is not limited to the specific methodologies, schemes, and reagents described, as these may vary depending on the context in which they are used by those skilled in the art.

[0212] The invention will be illustrated by the following non-limiting embodiments.

[0213] Example

[0214] Here, the inventors employed a semi-automated high-throughput mass spectrometry-based glycomics approach to explore N-glycosylation of blood proteins at the level of released glycans (i.e., after enzymatic removal of N-glycans from their carrier proteins). Their aim is to identify clinically translatable biomarkers that enable early, non-invasive detection of NAFLD, and particularly to address an unmet clinical need to capture the progression from non-fibrotic NASH to NASH-related fibrosis.

[0215] Example 1:

[0216] Materials and methods

[0217] Research Design

[0218] In this study, samples were obtained from the biobanks of Leiden University Medical Center (discovery cohort and replicate cohort) and Amsterdam University Medical Center (replicated cohort). The discovery cohort and replicate cohort involved plasma samples from 30 NAFLD patients and 60 presumed healthy controls, respectively, and serum samples from 45 NAFLD patients and 12 presumed healthy controls (see also Reference 30). Demographics for these cohorts can be found in Table 1. The study protocol was approved in advance by the local ethics committee (B19.071 for the discovery cohort and B21.045 for the replicate cohort). Healthy controls in the replicate cohort were obtained through the Leiden University Medical Center Voluntary Donor Service (“LUMC Vrijwillige Donoren Service”). Informed consent was obtained from all patients and healthy controls, and this study complied with the latest version of the Declaration of Helsinki.

[0219]

[0220] Table 1. Demographic and clinical characteristics of patients and healthy controls in the discovery cohort and replicate cohorts. Unless otherwise stated, median and interquartile ranges are shown.

[0221] Material

[0222] Materials and reagents used in this study were analytical grade and purchased from commercial suppliers. Type I ultrapure water (UP) manufactured by the ELGA PurelabUltra system (Elga LabWater, High Wycombe, UK) was used for solution preparation. Nonidet P-40 substitute (NP-40), super-DHB and 1-hydroxybenzotriazole monohydrate (HOBt), ammonium bicarbonate (NH4HCO3), potassium chloride (KCl), disodium hydrogen phosphate hydrate (Na2HPO4∙7H2O), and 85% phosphoric acid (H3PO4) were obtained from Sigma-Aldrich (Steinheim, Germany). Ethanol, sodium hydroxide (NaOH), sodium dodecyl sulfate (SDS), trifluoroacetic acid, disodium hydrogen phosphate dihydrate (Na2HPO4∙2H2O), potassium dihydrogen phosphate (KH2PO4), and sodium chloride (NaCl) were purchased from Merck (Darmstadt, Germany). 1-Ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) was obtained from Fluorochem (Hadfield, UK), while peptidase-N-glycosidase F (PNGase F) was purchased from Roche Diagnostics (Mannheim, Germany). HPLC-supergradient acetonitrile (ACN) and ethanol (EtOH) were obtained from Biosolve (Valkenswaard, Netherlands) and Merck (Darmstadt, Germany), respectively. Visucon-F healthy human plasma standard was obtained from Affinity Biologicals (Ancaster, Canada). Peptide calibration mixture II was obtained from Bruker Daltonics (Billerica, MA).

[0223] liver biopsy

[0224] In the repetitive queue, the fibrosis score is defined according to the Brunt scoring system. 14 The scoring system is based on histological examination of liver biopsies by two independent pathologists. F0: No fibrosis (no scarring); F1: Portal fibrosis (minimal scarring); F2: Periportal fibrosis (obvious scarring extending beyond the liver area); F3: Severe fibrosis (fibrosis spreading and forming bridges with other fibrotic liver areas); F4: Cirrhosis (advanced scarring). For patients included in the cohort, no fibrosis-specific reading was found, but the presence of cirrhosis was defined histologically, or, if histology was not applicable, by liver elastography or liver ultrasound. Decompensated cirrhosis was defined as the presence of ascites, variceal bleeding, hepatocellular carcinoma, hepato-renal syndrome, or hepatopulmonary syndrome.

[0225] Mass spectrometry glycomics and data processing

[0226] As mentioned above 15 See below for details. Total blood N-glucose analysis was performed by matrix-assisted laser desorption / ionization-Fourier transform ion cyclotron resonance-mass spectrometry (MALDI-FTICR-MS) after ligation-specific sialic acid derivatization. Following initial data preprocessing including data quality control, the results were compared with previous reports. 16 Similarly, as detailed below, the relative abundance of individual glycans in total plasma and total serum is calculated. The relative abundance of individual glycans is used to calculate glycosylation characteristics that summarize specific glycosylation features reflecting the biosynthetic pathway. Figure 1 ).

[0227] Sample preparation for high-throughput glycosylation analysis by matrix-assisted laser desorption / ionization-Fourier transform ion cyclotron resonance-mass spectrometry (MALDI-FTICR-MS).

[0228] The discovery of plasma samples from the cohort was part of a previous, larger study on autoimmune hepatitis. 30 Cases were randomly distributed across a total of five 96-well plates, with four Visucon F standards and two or three blanks on each plate. Serum samples from replicate studies were randomly distributed across one 96-well plate, and four pools were set up (i.e., pools generated by pooling equal volumes of serum from each patient in the cohort). Age and sex were considered to optimize the case and control distribution for each plate.

[0229] Glycan release, linker-specific sialic acid stabilization, and MALDI-FTICR-MS analysis in serum / plasma

[0230] The release of N-glycans from plasma proteins and the linkage of specific chemical sialic acid derivatization were performed as previously described in a similar high-throughput automated workflow, using 2 uL of plasma / serum for release. 1,2 For MALDI-FTICR-MS measurements, cover 1 μL of HILIC-purified sample onto 1 μL of sDHB matrix and allow it to air dry. 1,2 Measurements were performed in positive ion mode on a 15T Bruker Solari XR FTICR mass spectrometer equipped with a ParaCell, Smartbeam-II laser, and Combisource (Bruker Daltonics, Bremen, Germany). 2Prior to measurement, calibration was performed using a peptide calibration mixture II (Bruker Daltonics). For each point, an average spectrum was obtained from the acquisition of 10 spectra in the m / z range of 1000–5000 using 1 M data points. As will be understood by those skilled in the art, stabilization of sialic acid can be obtained as a “byproduct” required for link-specific derivatization. In general, MALDI measurements may require stabilization (otherwise sialic acid would be lost), and distinguishing between α2,3 and α2,6-bonds requires link specificity (otherwise they would be indistinguishable due to their similar mass).

[0231] Data processing

[0232] The raw MALDI-FTICR-MS spectra were converted into xy files. These raw data were then extracted using the internally developed software MassyTools3. To selectively extract glycan peaks, an analyte list was created based on manual annotations of the accumulated mass spectra. Glycan designation was based on precise mass and previous reports. 2,4-6 1 + Charge states are used for extraction. Signals are integrated by covering at least 95% of the isotopic envelope area of ​​the glycan peaks. An analyte is included in the final data analysis if its signal-to-noise ratio is higher than 27, its isotopic pattern deviates from the theoretical pattern by no more than 25%, and its quality error is within ±20 ppm. Furthermore, the same analyte must appear in at least one of the four spectra (25%) in each disease group to be included in the final data analysis. The relative intensity values ​​of the glycan compositions by quality standards are calculated by normalizing the sum of their total areas.

[0233] Calculation of glycosylation properties

[0234] Based on measured blood-derived glycan properties (discovery and repeat cohorts n = 81 and 72, respectively), glycosylation properties were calculated based on common structural features, including antenna number (A), fucosylation (F), antenna-fucosylation (Fa) bisection (B), galactose glycosylation (G), or sialylation (S). Figure 1 ).

[0235] Method repeatability and robustness

[0236] To assess the reproducibility of the MALDI-FTICR-MS method, the inter-plate coefficient of variation (COP) for the most abundant glycan peak H5N4E2 in the discovery cohort of plasma standards and the intra-plate COP for the same glycan in the replication cohort were calculated, which were 4.9% and 1.2%, respectively.

[0237] Statistical analysis

[0238] For both cohorts, a logistic regression model was used to investigate the association between healthy controls and NAFLD patients (healthy = 0; NAFLD = 1) using standardized data (subtracted from the mean and divided by SD) including age, sex, and their interaction as covariates (Table 2, methods summarized below). To compare the differences in fibrosis scores between the aforementioned significantly different glycosylation characteristics, the Kruskal-Wallis test was performed, followed by a post-hoc Dunn's test if the results were significant. Figure 3 Spearman's grading correlation was performed to assess the correlation between glycosylation characteristics and Brunt fibrosis scores and age (Table 3). Considering multiple testing, the Benjamini-Hochberg procedure with a false discovery rate (FDR) of 5% was used to evaluate the statistical significance of each statistical question (Table 2 (discovery cohort)). Statistical tests were performed in the repeated cohort using a cutoff value of p < 0.05 without multiple test correction (Table 2).

[0239] statistics

[0240] Age, sex, and their interaction were incorporated as covariates into a logistic regression model to identify a disease-specific association between HC (0) and NAFLD (1). The odds ratio (OR) was calculated using their 95% confidence intervals (CI) and represented by a single standard deviation increasing the test-derived characteristic. Multiple test correction was performed using the Benjamini-Hochberg procedure and based on a 5% false discovery rate (FDR) in the discovery cohort. Similar to genome-wide association studies, the inventors used a discovery cohort to identify potential glycomic associations between healthy individuals and NAFLD individuals. In the discovery study, multiple test correction was applied to avoid / limit the discovery of false positive associations. In contrast, a replication cohort was used to confirm associations previously found in the discovery cohort. To avoid overlooking valid associations, the inventors found that the necessity of multiple test correction in the replication cohort was reduced. Statistical analyses were performed in R version 4.2.2 (R Foundation for Statistical Computing, Vienna, Austria) and RStudio version 2022.12.0, Build 353 (RStudio, Boston, MA).

[0241] result

[0242] Mass spectrometry analysis of blood protein N-glycomics identified 81 and 72 N-glycans (80% overlap) in the discovery and repeat cohorts, respectively. The annotated glycotypes were relatively quantified and summarized into 36 glycosylation characteristics based on their structural features, which were used in the discovery and repeat cohorts. These structural features included fucosylation, antenna fucosylation, dichotomy, galactosylation, sialylation, antennalization, and N-glycan type. Figure 1 The identified glycoform is consistent with glycoforms commonly found in blood proteins, although its structure is speculative and may include a collection of isoforms. 15,17 .

[0243] Association between N-glycosylation of blood proteins and NAFLD

[0244] In the first step, the inventors aimed to explore the differences between NAFLD and healthy controls through principal component analysis, which showed that the key difference between the two groups was driven by sialic acid modifying N-glycans in a link-specific manner. Figure 4 Further statistical analysis revealed that nine glycosylation characteristics showed statistical differences between NAFLD and healthy controls in the cohort, six of which were duplicates. Figure 2 Table 2). Based on sialic acid-linked variants (i.e., α2,3- or α2,6-linked), repetitive glycosylation characteristics can be divided into two main categories ( Figure 2 (bg, Table 2).

[0245]

[0246] Table 2. Association between plasma N-glycan properties and NAFLD compared to healthy controls. Logistic regression was performed between NAFLD (1) and HC (0), including age, sex, and their interaction as covariates. Only significant associations meeting the predefined log2 advantage ratio threshold (0.5) are shown. To account for multiple tests, p-values ​​in the discovery cohort were corrected for using the Benjamini-Hochberg procedure with a 5% FDR. *P-values ​​in the discovery cohort were corrected for multiple tests using the Benjamini-Hochberg procedure (5% FDR). CI: Confidence interval.

[0247] The inventors observed that NAFLD exhibited lower α2,3-linked sialylation in most complex N-glycans. Specifically, the combined levels of α2,3-sialylation in all complex N-glycans (ALs) showed odds ratios (ORs) of 0.36 and 0.33 for the discovery cohort and the replication cohort, respectively. Figure 2 be, Table 2).

[0248] In contrast, α2,6-sialylation (AE) was generally increased in NAFLD. This was most pronounced in N-glycans with three antennas (A3E) (ORs were 2.95 and 6.46 in the discovery and replication cohorts, respectively). Figure 2 f, g). The ratio of AL to AE also reflects the significant shift from α2,6-sialylation to α2,3-sialylation (f, g). Figure 6 ).

[0249] Association of α2,3- and α2,6-sialylation with NASH-related fibrosis

[0250] Since changes in α2,3- and α2,6-sialylation appear to be characteristic of NAFLD, the inventors further investigated the relationship between these glycosylation characteristics and the degree of fibrosis in patients included in the repeat cohort. Figure 3 Patients without fibrotic scars (non-fibrotic NASH; Brunt fibrosis score 0) compared to healthy individuals with α2,3- ( Figure 3 ad) and α2,6-linked sialylation (ad) and α2,6-linked sialylation Figure 3 There was no difference in the degree of e and f). On the other hand, sialylation effects were observed in patients with histologically fibrotic presentation (NASH with varying degrees of fibrosis; Brunt fibrosis score 1-4). Importantly, these associations were not affected by individual age and sex. Figure 5 ).

[0251] Correlation between glycosylation and fibrosis stages

[0252] To further investigate the correlations found, Spearman correlation analysis was performed between repeated glycosylation properties and Brunt fibrosis scores in patients with NASH-related fibrosis. Using this method, the inventors determined that α2,3-sialylation of tri(A3L) and tetra(A4L) glycans and α2,6-sialylation of trianeglycan (A3E) were significantly associated with the progression of fibrosis, suggesting that these glycosylation properties follow a unidirectional trend and may be phenotypic indicative (Table 3). Interestingly, no correlation was found between total sialylation levels (i.e., no distinction between bond isomers) and Brunt fibrosis scores. Figure 7 ad).

[0253]

[0254] Table 3. Correlation between glycosylation characteristics and Brunt fibrosis score. The table shows Spearman's correlation coefficient (R²). S Significant correlations are highlighted in bold (p < 0.05).

[0255] Determine the cutoff value for polysaccharide biomarkers of fibrosis

[0256] Because the histological features of NASH-related fibrosis (F1-F4, Brunt fibrosis score) are consistent with lower relative levels of α2,3-sialylation and higher relative levels of α2,6-sialylation compared to healthy controls or F0 (non-fibrotic) NASH patients, the inventors aim to establish a method to define a cutoff value for fibrosis-indicative glycosylation characteristics, which can then be used to detect the occurrence and / or stage of fibrosis in a subsequent clinical setting. To establish such a threshold, the inventors first examined the median relative levels of glycosylation characteristics in the severity group. Based on the observed distribution, trends, and statistics, the inventors then merged the healthy controls and the F0 group (i.e., the non-fibrotic group). Next, the inventors decided to merge the F1-F4 groups (i.e., the fibrotic group). Based on these results, the inventors suggest using the median of the non-fibrotic group as a reference point for determining the cutoff value (Table 4). Specifically, the inventors propose that if the level of α2,3-sialylation (A2L, A3L, A4L, and / or AL) is below the cutoff value defined in the non-fibrotic group, this indicates that the patient may have fibrotic NASH. In other words, if the measured levels of A2L, A3L, A4L, and / or AL are below the average of the group cutoff values ​​for each glycosylation characteristic, this may indicate that the patient has fibrotic NASH. Similarly, the inventors propose that if the level of α2,6-sialylation (A3E and / or AE) is above the cutoff value defined in the non-fibrotic group, the patient may have fibrotic NASH. In other words, if the determined levels of A3E and / or AE are above the average of the group cutoff values ​​for each glycosylation characteristic, this may indicate that the patient has fibrotic NASH.

[0257]

[0258] Table 4. Exemplary cutoff values ​​defined by the methods described above. Cutoff values ​​must be defined for each measurement batch, targeting the corresponding connection-specific sialylation level for non-fibrotic (healthy) controls.

[0259] Binary classification model generation for fibrosis prediction

[0260] To evaluate the ability of glycosylation characteristics, both individually and in combination, to predict fibrillation, the inventors decided to conduct multiple receiver operating characteristic (ROC) analysis and compare their predictive accuracy. First, the inventors established a composite model with six predictors (A2L, A3L, A4L, AL, A3E, AE). The results of this model showed that, except for A3L and A4L, the predictor behavior exhibited a non-linear space. AE was excluded from the composite model. The obtained composite model (including A2L, A3L, A4L, AL, and A3E) had the following characteristics: AUC = 0.92, CI = 0.84–0.97; cross-validation accuracy = 0.77, accuracy SD = 0.14 (…). Figure 7 e). Despite obtaining a high AUC value, the inventors believe that the composite model is insufficient because the nonlinear behavior of many variables and multicollinearity among variables may inflate the model. Therefore, they decided to switch to a single predictor model. Using the model's "step" function, the inventors obtained the optimal model with A4L as the single predictor, with the following results: Odds ratio: 0.07, CI = 0.01-0.24, p < 0.001 ( Figure 7 f). Other model parameters are: AUC = 0.90, CI = 0.82–0.97; cross-validation accuracy = 0.79, accuracy SD = 0.13. Similar results were obtained using A3L as a single predictor. Including potential confounding factors such as age and gender did not improve the model.

[0261] Overall, these results indicate that the best-performing predictors of fibrotic NASH are A4L and A3L.

[0262] discuss

[0263] Using mass spectrometry-based methods to distinguish sialic acid bond variants 15 The inventors studied the total blood protein N-glycomics of all 72 potentially healthy controls and 75 individuals who developed different stages of NAFLD and NASH-related fibrosis. This study revealed a novel, reproducible fibrosis-specific blood N-glycomic profile in NASH patients. The inventors believe that these results have the potential to develop non-invasive diagnostic methods for detecting the transition from non-fibrotic NASH to fibrotic NASH and for fibrotic NASH.

[0264] Since reliable diagnosis and follow-up of fibrosis are based on biopsy, there is a great need for non-invasive biomarkers that can guide the selection of NAFLD patients who will benefit most from biopsy. 18 Alterations in blood protein N-glycosylation patterns can serve as biomarkers for liver disease and may contribute to its development and progression. 22In this study, the inventors discovered a feature that, to our knowledge, is a previously unreported characteristic of NASH-associated fibrosis, as the inventors demonstrated in two independent cohorts that NAFLD patients exhibited (1) lower overall α2,3-linked sialylation compared to healthy individuals, (2) higher overall α2,6-linked sialylation of their triantaneous N-glycans, and (3) a subset of the defined glycosylation characteristics that reflected the stage of NASH-associated fibrosis.

[0265] Importantly, the aforementioned glycosylation signature distinguishes fibrotic and non-fibrotic NASH in the NAFLD patient population, which is challenging for existing non-invasive diagnostic methods. Therefore, this novel glycosylation pattern potentially addresses an unmet clinical need for a non-invasive approach to identifying NAFLD patients at risk of fibrosis progression. The inventors believe that due to sample size limitations, some results, such as… Figure 3 The results shown were not statistically significant between the different fibrosis score groups. Therefore, increasing the sample size may yield statistically significant results.

[0266] Sialic acid linker isomers exhibit different functional roles, and abundance variations of one linker variant relative to another have been reported in cancer and inflammatory bowel disease. 19-21 In the field of glycomics for NAFLD, large-scale studies relying on sialic acid bond isomerism are lacking. A study of 15 patients across three fibrosis categories found a reduced ratio of fully sialylated, non-fucosylated glycans carrying a single α2,3-linked sialic acid to fucosylated glycans, concluding that the observed feature was antenna-fucosylation dependent and likely derived from the acute-phase protein α-1-antitrypsin (AAT). 23 Another research group found that the features associated with the aforementioned composition (i.e., triantaneously fully sialylated N-glycans) were related to both AAT and α-1-acid-glycoprotein (AGP), although no detailed information was available regarding the fucosylation and sialic acid linkage type. 24 In addition, composite markers such as GlycoFibroTest 25 And the recent GlycoFibroTyper 26 The analysis relied on total serum or affinity-enriched immunoglobulin G N-glycosylation assays, and both indicated that the presence of di-N-acetylglucosamine (i.e., di-) was an important glycosylation feature that distinguishes clinical phenotypes of NAFLD.

[0267] Glycomics studies reported the characterization of N-glycans associated with fibrosis and directly... 24 or indirectly 23,25 These glycans with candidate proteins 22Correlation. Notably, changes in the glycoprotein abundance of these candidate proteins (i.e., decreased or increased hepatic production) can, to some extent, explain the observed changes in relative glycoform frequencies. It is noteworthy that our data point to an overall lower α2,3-sialylation, suggesting that the observed changes may be sialyl-linked rather than protein abundance-dependent. This observation in the blood N-glycose group of fibrotic patients indicates alterations in the hepatic N-glycan biosynthetic pathway or changes in the clearance of circulating glycoproteins. Future studies on the expression of glycosyltransferases in hepatocytes or liver tissue sections could provide valuable insights into the pathophysiology of NASH-associated fibrosis. The plasma half-life of glycoproteins is regulated by glycan recognition receptors, such as the desialyl glycoprotein receptor (ASGPR), which clears non-sialylated glycoproteins from circulation. This receptor also has an affinity for α2,6-linked sialic acid. 27 Therefore, decreased ASGPR expression can also lead to increased α2,6-linked sialylation of blood proteins.

[0268] Interestingly, a study in mice using hepatocyte-specific conditional knockout of the glycosyltransferase β-galactosidase α2,6-sialotyltransferase 1 (St6gal1; encoding the enzyme responsible for adding sialic acid to the α2,6-bond) linked the absence of hepatocytes and the circulating glycoprotein α2,6-sialic acid to the spontaneous development and inflammation of fatty liver disease. 28 In contrast, in humans, the inventors found that patients with NASH-associated fibrosis had low α2,3-sialylation and high α2,6-sialylation of triantane N-glycans. Since the latter characteristic is limited to triantane N-glycans, it may be related to variations in the abundance of proteins carrying this glycoform, including but not limited to previously reported acute-phase proteins such as AAT. 24 To clarify this, it is necessary to perform sialic acid-linked specificity analysis on AAT-related glycans or glycopeptides.

[0269] Current research has potential implications for the diagnosis of fibrosis. Unfortunately, neither the clinically available GlycoFibroTest nor GlycoFibroTyper includes (connection-specific) sialylation analysis. 23,25 .

[0270] in conclusion

[0271] In summary, a reproducible fibrosis-specific blood N-glycosylation signature has been identified in NAFLD patients, enabling early detection of fibrotic NASH. The overall reduction in α2,3-sialylation (a distinctive feature of circulating proteins produced by fibrotic livers) opens the possibility of developing new non-invasive diagnostic tests, thus aiding in the early diagnosis of NASH-related fibrosis. Furthermore, this finding provides new insights into the molecular mechanisms that may play a role in the development of NASH fibrosis. The clinical translation of this glycomic signature could make diagnosis and follow-up more comfortable for NAFLD patients and physicians in the future.

[0272] Example 2

[0273] Materials and methods

[0274] The materials and methods of Example 2 are generally the same as those of Example 1 above, but with the following modifications.

[0275] Research Design

[0276] In this study, samples were obtained from the biobanks of Leiden University Medical Center (discovery cohort and replicate cohort) and Amsterdam University Medical Center (replicative cohort). The discovery cohort and replicate cohort involved plasma samples from 30 NAFLD patients and 60 presumed healthy controls, respectively, and serum samples from 102 NAFLD patients and 29 presumed healthy controls (Pongracz et al. 2024, Blood N-glycomic signature of fibrosis in metabolic-dysfunction associated steatotic liver disease shows low level of global α2,3-sialylation, unpublished manuscript). Demographics for these cohorts are available in Table 5. The study protocol was approved in advance by the local ethics committees (B19.071 for the discovery cohort and B21.045 for the replicate cohort). Healthy controls in the replicate cohort were obtained through the Leiden University Medical Center Voluntary Donor Service (“LUMC VrijwilligeDonoren Service”). Informed consent was obtained from all patients and healthy controls, and this study complied with the latest version of the Declaration of Helsinki.

[0277]

[0278]

[0279]

[0280] *Incomplete observations

[0281] Table 5. Demographic and clinical characteristics of patients and healthy controls in the discovery cohort and replicate cohorts.

[0282] Material

[0283] As described in Example 1.

[0284] liver biopsy

[0285] As described in Example 1.

[0286] Mass spectrometry glycomics and data processing

[0287] As described in Example 1.

[0288] Sample preparation for high-throughput glycosylation analysis by matrix-assisted laser desorption / ionization-Fourier transform ion cyclotron resonance-mass spectrometry (MALDI-FTICR-MS).

[0289] As described in Example 1.

[0290] Glycan release, linker-specific sialic acid stabilization, and MALDI-FTICR-MS analysis in serum / plasma

[0291] As described in Example 1.

[0292] Data processing

[0293] As described in Example 1.

[0294] Calculation of glycosylation properties

[0295] As described in Example 1.

[0296] Method repeatability and robustness

[0297] As described in Example 1.

[0298] Statistical analysis

[0299] For both cohorts, a logistic regression model was used to investigate the association between healthy controls and NAFLD patients (healthy = 0; NAFLD = 1) using standardized data (subtracted from the mean and divided by SD) including age, sex, and their interaction as covariates (Table 6, methods summarized below). To compare the differences in fibrosis scores between the aforementioned significantly different glycosylation characteristics, the Kruskal-Wallis test was performed, followed by a post-hoc Dunn's test if the results were significant. Figure 11 Spearman's grading correlation was performed to assess the correlation between glycosylation characteristics and Brunt fibrosis score, as well as age and type 2 diabetes status (Table 7). Figure 13Considering multiple tests, the Benjamini-Hochberg procedure with a false discovery rate (FDR) of 5% was used to evaluate the statistical significance of each statistical question. Figure 9 And Table 6 (Discovery Queue), Figure 15 ) Statistical tests were performed in the repeated cohort without multiple test correction using a cutoff value of p < 0.05 (Table 6).

[0300] statistics

[0301] As described in Example 1.

[0302] result

[0303] Mass spectrometry analysis of blood protein N-glycomics identified 81 and 72 N-glycans (80% overlap) in the discovery and repeat cohorts, respectively. The annotated glycotypes were relatively quantified and summarized into 36 glycosylation characteristics based on their structural features, which were used in the discovery and repeat cohorts. These structural features included fucosylation, antenna fucosylation, dichotomy, galactosylation, sialylation, antennalization, and N-glycan type. Figure 1 The identified glycoform is consistent with glycoforms commonly found in blood proteins, although its structure is speculative and may include a collection of isoforms. 15,17 .

[0304] Association between N-glycosylation of blood proteins and NAFLD

[0305] In the first step, the inventors aimed to explore the differences between NAFLD and healthy controls through principal component analysis, which showed that the key difference between the two groups was driven by sialic acid modifying N-glycans in a link-specific manner. Figure 8 Further statistical analysis revealed that nine glycosylation characteristics showed statistical differences between NAFLD and healthy controls in the cohort, six of which were duplicates. Figure 9 Table 6). Based on the sialic acid linkage variants (i.e., α2,3- or α2,6-linked), the repeating glycosylation characteristics can be divided into two main categories ( Figure 9 bg, Table 6).

[0306]

[0307]

[0308] Table 6. Association between plasma N-glycan properties and NAFLD compared to healthy controls. Logistic regression was performed between NAFLD (1) and HC (0), including age, sex, and their interaction as covariates. Only significant associations meeting the predefined ln odds ratio threshold (0.345) are shown. Considering multiple tests, p-values ​​in the cohort were corrected for 5% FDR using the Benjamini-Hochberg procedure. CI: Confidence interval.

[0309] The inventors observed that NAFLD exhibited lower α2,3-linked sialylation in most complex N-glycans. Specifically, the combined levels of α2,3-sialylation in all complex N-glycans (ALs) showed odds ratios (ORs) of 0.36 and 0.11 for the discovery cohort and the repeat cohort, respectively. Figure 9 be, Table 6).

[0310] In contrast, α2,6-sialylation (AE) was generally increased in NAFLD. This was most pronounced in N-glycans with three antennas (A3E) (ORs were 2.95 and 11.6 in the discovery and replication cohorts, respectively). Figure 9 f, g, Table 6). The ratio of AL to AE also reflects the significant shift from α2,6-sialylation to α2,3-sialylation (f, g, Table 6). Figure 10 ).

[0311] Association of α2,3- and α2,6-sialylation with NASH-related fibrosis

[0312] Since changes in α2,3- and α2,6-sialylation appear to be characteristic of NAFLD, the inventors further investigated the relationship between these glycosylation characteristics and the degree of fibrosis in patients included in the repeat cohort. Figure 11 Patients without fibrotic scars (non-fibrotic NASH; Brunt fibrosis score 0) compared to healthy individuals with α2,3- ( Figure 11 ad) and α2,6-linked sialylation (ad) and α2,6-linked sialylation Figure 11 There was no difference in the degree of e and f). On the other hand, connection-specific sialylation effects were observed in patients with histologically fibrotic presentation (NASH with varying degrees of fibrosis; Brunt fibrosis score 1–4). Importantly, these associations were independent of individual age and sex. Figure 12 ) or comorbidities (type 2 diabetes status) Figure 13 The study investigated the effects of total sialylation (i.e., no distinction was found between bond isomers) and fibrosis. Figure 14 ).

[0313] Correlation between glycosylation and fibrosis stages

[0314] To further investigate the correlations found, Spearman correlation analysis was performed between repeated glycosylation properties and Brunt fibrosis scores in patients with NASH-related fibrosis. Using this method, the inventors identified α2,3-sialylation (AL) of all complex glycans and α2,6-sialylation (AE) of all complex glycans, as well as α2,3-sialylation of di-(A2L), tri-(A3L), and tetra-antenna (A4L) glycans and α2,6-sialylation (A3E) of tri-antenna glycans, as significantly associated with the progression of fibrosis, suggesting that these glycosylation properties follow a unidirectional trend and may be phenotypic indicative (Table 7).

[0315]

[0316] Table 7. Correlation between glycosylation characteristics and Brunt fibrosis score. The table shows Spearman's correlation coefficient (R²). S All correlations were significant (p < 0.05). Healthy controls were considered F0 and were pooled with patients having normal histology (F0) for this analysis.

[0317] Determine the cutoff value for polysaccharide biomarkers of fibrosis

[0318] Because the histological features of NASH-related fibrosis (F1-F4, Brunt fibrosis score) are consistent with lower relative levels of α2,3-sialylation and higher relative levels of α2,6-sialylation compared to healthy controls or F0 (non-fibrotic) NASH patients, the inventors aim to establish a method to define a cutoff value for fibrosis-indicative glycosylation characteristics, which can then be used to detect the occurrence and / or stage of fibrosis in a subsequent clinical setting. To establish such a threshold, the inventors first examined the median relative levels of glycosylation characteristics in the severity group. Based on the observed distribution, trends, and statistics, the inventors then merged the healthy controls and the F0 group (i.e., the non-fibrotic group). Next, the inventors merged the F1-F4 groups (i.e., the fibrotic group). Based on these results, the inventors suggest using the median of the non-fibrotic group as a reference point for determining the cutoff value (Table 8). Specifically, the inventors propose that if the level of α2,3-sialylation (A2L, A3L, A4L, and / or AL) is below the cutoff value defined in the non-fibrotic group, this indicates that the patient may have fibrotic NASH. In other words, if the measured levels of A2L, A3L, A4L, and / or AL are below the average of the group cutoff values ​​for each glycosylation characteristic, this may indicate that the patient has fibrotic NASH. Similarly, the inventors propose that if the level of α2,6-sialylation (A3E and / or AE) is above the cutoff value defined in the non-fibrotic group, the patient may have fibrotic NASH. In other words, if the determined levels of A3E and / or AE are above the average of the group cutoff values ​​for each glycosylation characteristic, this may indicate that the patient has fibrotic NASH.

[0319]

[0320] Table 8. Exemplary cutoff values ​​defined by the methods described above. Cutoff values ​​must be defined for each measurement batch, targeting the corresponding connection-specific sialylation level for non-fibrotic (healthy) controls.

[0321] Binary classification model generation for fibrosis prediction

[0322] Inspired by the discovered associations, the inventors conducted receiver operating characteristic (ROC) analysis to evaluate the discriminative ability of glycosylation properties to predict the histological manifestations of fibrosis. Using a "step" function, the inventors obtained the optimal model with A4L as a single predictor. Figure 15Other model parameters were: AUC = 0.87, CI = 0.80–0.94. A3L also yielded similar results as a single predictor, indicating that A4L and A3L are the best predictors of fibrosis MASH. Including potential confounding factors such as age, sex, BMI, and type 2 diabetes status did not improve these models. Due to the limited sample size in the F0 group (n=12), the same analysis was not performed on the common diagnostic biomarkers FIB-4 and NFS.

[0323] Overall, these results indicate that the best-performing predictors of fibrotic NASH are A4L and A3L.

[0324] discuss

[0325] Using a mass spectrometry-based method15 capable of distinguishing sialic acid bond variants, the inventors investigated the total blood protein N-glycomic profile of 75 individuals who developed different stages of NAFLD and NASH-related fibrosis, compared with all 72 controls. This study reveals a novel, reproducible liver fibrosis-specific blood N-glycomic profile in NAFLD-NASH patients. These results have the potential to develop non-invasive diagnostic methods for detecting the non-fibrotic to fibrotic transition in NAFLD.

[0326] Alterations in blood protein N-glycosylation patterns can serve as biomarkers for liver disease and may contribute to its development and progression. 22 In this study, the inventors identified a previously unreported characteristic feature of fibrotic NAFLD-NASH, demonstrating in two independent cohorts that patients exhibited lower overall α2,3-linked sialylation compared to healthy individuals. Importantly, this aforementioned glycosylation feature differentiates fibrotic NAFLD from non-fibrotic counterparts in the NAFLD patient population from a histological perspective of fibrosis, an area where current non-invasive diagnostic methods are inadequate.

[0327] Sialic acid linker isomers exhibit different functional roles, and abundance variations of one linker variant relative to another have been reported in cancer, inflammatory bowel disease, and type 2 diabetes.

[0328] It is noteworthy that the data above indicate an overall decrease in α2,3-sialylation, suggesting that the observed changes are sialic acid bond-dependent rather than protein abundance-dependent, since proteomics studies supporting this hypothesis did not describe a significant decrease in the concentration of major plasma glycoproteins. Therefore, the above observations in the blood N-glycan group of fibrotic patients suggest alterations in the biosynthetic pathway of N-glycans in the liver or changes in the clearance of circulating secreted glycoproteins.

[0329] Interestingly, a study in mice using hepatocyte-specific conditional knockout of the glycosyltransferase β-galactosidase α2,6-sialotyltransferase 1 (St6gal1; encoding the enzyme responsible for adding sialic acid to the α2,6-bond) linked the absence of hepatocyte and circulating glycoprotein α2,6-sialic acid to the spontaneous development of fatty liver disease and the shift to a pro-inflammatory immunophenotype. 20 In contrast, the inventors found that patients with NASH-associated fibrosis had low α2,3-sialylation and high α2,6-sialylation of triantane N-glycans. Since the latter characteristic is limited to triantane N-glycans, it may be associated with an increased abundance of proteins carrying this glycoform, including but not limited to previously reported acute-phase proteins such as AAT. 24 To further clarify this point, it is necessary to perform sialic acid-linked specificity analysis on AAT-related glycans or glycopeptides.

[0330] Current research has potential implications for the diagnosis of fibrosis. Unfortunately, neither the clinically available GlycoFibroTest nor GlycoFibroTyper includes (connection-specific) sialylation analysis. 23,25 .

[0331] in conclusion

[0332] In summary, a reproducible fibrosis-specific blood N-glycosylation signature has been identified in NAFLD patients, enabling early detection of fibrotic NASH. The overall reduction in α2,3-sialylation (a distinctive feature of circulating proteins produced by fibrotic livers) opens the possibility of developing new non-invasive diagnostic tests, thus aiding in the early diagnosis of NASH-related fibrosis. Furthermore, this signature provides new insights into the molecular mechanisms that may play a role in the development of NASH fibrosis. The clinical translation of this glycomic signature could make diagnosis and follow-up more comfortable for NAFLD patients and physicians in the future, allowing for timely intervention and improved disease management.

[0333] Readers should note all papers and documents related to this application, submitted concurrently with or prior to this specification, which are publicly available along with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0334] All features disclosed in this specification (including any appended claims, abstract, and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, unless such combinations of at least some of the features and / or steps are mutually exclusive.

[0335] Unless otherwise expressly stated, each feature disclosed in this specification (including any appended claims, abstract, and drawings) may be replaced by alternative features for the same, equivalent, or similar purpose. Therefore, unless otherwise expressly stated, each disclosed feature is merely one example of a series of equivalent or similar features.

[0336] This invention is not limited to the details of any of the foregoing embodiments. This invention covers any novel feature or any combination of novel features disclosed in this specification (including any appended claims, abstract, and drawings), or any novel step or any combination of novel steps in any method or process so disclosed.

[0337] References

[0338] 1Ruissen, MM, Mak, AL, Beuers, U., Tushuizen, ME &Holleboom, AG Non-alcoholic fatty liver disease: a multidisciplinary approach towards a cardiometabolic liver disease. Eur J Endocrinol 183, R57-R73 (2020). https: / / doi.org:10.1530 / EJE-20-0065

[0339] 2Loomba, R., Friedman, SL & Shulman, GI Mechanisms and diseaseconsequences of nonalcoholic fatty liver disease. Cell 184, 2537-2564 (2021). https: / / doi.org:10.1016 / j.cell.2021.04.015

[0340] 3Younossi, Z. et al. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. Nat Rev Gastroenterol Hepatol 15,11-20 (2018). https: / / doi.org:10.1038 / nrgastro.2017.109

[0341] 4Estes, C. et al.Modeling NAFLD disease burden in China, France,Germany, Italy, Japan, Spain, United Kingdom, and United States for theperiod 2016-2030.J Hepatol 69, 896-904 (2018). https: / / doi.org:10.1016 / j.jhep.2018.05.036

[0342] 5Taylor, R. S. et al.Association Between Fibrosis Stage and Outcomesof Patients With Nonalcoholic Fatty Liver Disease: A Systematic Review andMeta-Analysis.Gastroenterology 158, 1611-1625 e1612 (2020). https: / / doi.org:10.1053 / j.gastro.2020.01.043

[0343] 6Dulai, P. S. et al.Increased risk of mortality by fibrosis stage innonalcoholic fatty liver disease: Systematic review and meta-analysis.Hepatology 65, 1557-1565 (2017). https: / / doi.org:10.1002 / hep.29085

[0344] 7Byrne, C. D. & Targher, G. EASL-EASD-EASO Clinical PracticeGuidelines for the management of non-alcoholic fatty liver disease: isuniversal screening appropriate? Diabetologia 59, 1141-1144 (2016). https: / / doi.org:10.1007 / s00125-016-3910-y

[0345] 8Gilmore, I. T. et al.Indications, methods, and outcomes ofpercutaneous liver biopsy in England and Wales: an audit by the BritishSociety of Gastroenterology and the Royal College of Physicians of London.Gut36, 437-441 (1995). https: / / doi.org:10.1136 / gut.36.3.437

[0346] 9Seeff, L. B. et al.Complication rate of percutaneous liver biopsiesamong persons with advanced chronic liver disease in the HALT-C trial.ClinGastroenterol Hepatol 8, 877-883 (2010). https: / / doi.org:10.1016 / j.cgh.2010.03.025

[0347] 10Eddowes, P. J. et al.Accuracy of FibroScan Controlled AttenuationParameter and Liver Stiffness Measurement in Assessing Steatosis and Fibrosisin Patients With Nonalcoholic Fatty Liver Disease.Gastroenterology 156, 1717-1730 (2019). https: / / doi.org:10.1053 / j.gastro.2019.01.042

[0348] 11Troelstra, M. A. et al.Assessment of Imaging Modalities AgainstLiver Biopsy in Nonalcoholic Fatty Liver Disease: The Amsterdam NAFLD-NASHCohort.J Magn Reson Imaging 54, 1937-1949 (2021). https: / / doi.org:10.1002 / jmri.27703

[0349] 12van Dijk, A. M., Schattenberg, J. M., Holleboom, A. G. & Tushuizen,M. E. Referral care paths for non-alcoholic fatty liver disease-Gearing upfor an ever more prevalent and severe liver disease.United EuropeanGastroenterol J 9, 903-909 (2021). https: / / doi.org:10.1002 / ueg2.12150

[0350] 13Balkhed, W., Aberg, F. O., Nasr, P., Ekstedt, M. & Kechagias, S.Repeated measurements of non-invasive fibrosis tests to monitor theprogression of non-alcoholic fatty liver disease: A long-term follow-upstudy.Liver Int 42, 1545-1556 (2022). https: / / doi.org:10.1111 / liv.15255

[0351] 14Bedossa, P. Intraobserver and Interobserver Variations in LiverBiopsy Interpretation in Patients with Chronic Hepatitis C. Hepatology 20,15-20 (1994). https: / / doi.org:10.1002 / hep.1840200104

[0352] 15Vreeker, G. C. M. et al.Automated Plasma Glycomics with Linkage-Specific Sialic Acid Esterification and Ultrahigh Resolution MS.Anal Chem 90,11955-11961 (2018). https: / / doi.org:10.1021 / acs.analchem.8b02391

[0353] 16Dotz, V. et al.Plasma protein N-glycan signatures of type 2diabetes.Biochimica et Biophysica Acta (BBA) - General Subjects 1862, 2613-2622 (2018). https: / / doi.org:https: / / doi.org / 10.1016 / j.bbagen.2018.08.005

[0354] 17Clerc, F. et al.Human plasma protein N-glycosylation.Glycoconj J33, 309-343 (2016). https: / / doi.org:10.1007 / s10719-015-9626-2

[0355] 18Wong, G. L. Non-invasive assessments for liver fibrosis: Thecrystal ball we long for.J Gastroenterol Hepatol 33, 1009-1015 (2018).https: / / doi.org:10.1111 / jgh.14103

[0356] 19Park, E. I., Yilling, M., Unverzagt, C., Gabius, H.-J. & Baenziger,J. U. The asialoglycoprotein receptor clears glycoconjugates terminating withsialic acidα2,6GalNAc.PNAS 102, 17125-17129 (2005). https: / / doi.org:https: / / doi.org / 10.1073 / pnas.050853710

[0357] 20Oswald, D. M., Jones, M. B. & Cobb, B. A. Modulation of hepatocytesialylation drives spontaneous fatty liver disease andinflammation.Glycobiology (2019). https: / / doi.org:10.1093 / glycob / cwz096

[0358] 21Cummings, R. D., Darvill, A. G., Etzler, M. E. & Hahn, M. G. inEssentials of Glycobiology (eds rd et al.) 611-625 (2015).

[0359] 22Verhelst, X. et al.Protein Glycosylation as a Diagnostic andPrognostic Marker of Chronic Inflammatory Gastrointestinal and LiverDiseases.Gastroenterology 158, 95-110 (2020). https: / / doi.org:10.1053 / j.gastro.2019.08.060

[0360] 23Hanamatsu, H. et al.Comparative Glycomic Analysis of Sialyl LinkageIsomers by Sialic Acid Linkage-Specific Alkylamidation in Combination withStable Isotope Labeling of alpha2,3-Linked Sialic Acid Residues.Anal Chem 91,13343-13348 (2019). https: / / doi.org:10.1021 / acs.analchem.9b03617

[0361] 24Ramachandran, P. et al.Serum Glycoprotein Markers in NonalcoholicSteatohepatitis and Hepatocellular Carcinoma.J Proteome Res 21, 1083-1094(2022). https: / / doi.org:10.1021 / acs.jproteome.1c00965

[0362] 25Vanderschaeghe, D. et al.GlycoFibroTest is a highly performantliver fibrosis biomarker derived from DNA sequencer-based serum proteinglycomics.Mol Cell Proteomics 8, 986-994 (2009). https: / / doi.org:10.1074 / mcp.M800470-MCP200

[0363] 26Scott, D. A. et al.GlycoFibroTyper: A Novel Method for the GlycanAnalysis of IgG and the Development of a Biomarker Signature of LiverFibrosis.Front Immunol 13, 797460 (2022). https: / / doi.org:10.3389 / fimmu.2022.797460

[0364] 27Fernandez-Poza, S. et al.Tailor-made recombinant prokaryoticlectins for characterisation of glycoproteins.Anal Chim Acta 1155, 338352(2021). https: / / doi.org:10.1016 / j.aca.2021.338352

[0365] 28Ruhaak, L. R. et al.Optimized workflow for preparation of APTS-labeled N-glycans allowing high-throughput analysis of human plasma glycomesusing 48-channel multiplexed CGE-LIF.J Proteome Res 9, 6655-6664 (2010).https: / / doi.org:10.1021 / pr100802f

[0366] 29Mitra, I. et al.Structural Characterization of Serum N-Glycans byMethylamidation, Fluorescent Labeling, and Analysis by MicrochipElectrophoresis.Anal Chem 88, 8965-8971 (2016). https: / / doi.org:10.1021 / acs.analchem.6b00882

[0367] 30Pongracz, T. et al.Autoimmune hepatitis displays distinctively highmulti-antennary sialylation on plasma N-glycans compared to other liverdiseases; J Transl Med.2024 May 14;22(1):456. doi: 10.1186 / s12967-024-05173-z

Claims

1. A method for diagnosing fibrotic nonalcoholic steatohepatitis (NASH) in a subject, the method comprising the following steps: • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in samples from subjects; and • Compare the determined levels of α2,3-sialylation and / or α2,6-sialylation with reference values, where: i) Increased α2,6-sialylation levels in the sample; and / or ii) Decreased α2,3-sialylation level in the sample The subjects were indicated to have fibrotic NASH.

2. A method for staging fibrotic nonalcoholic steatohepatitis (NASH) in subjects, the method comprising the following steps: • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in samples from subjects; and • The determined α2,3-sialylation and / or α2,6-sialylation levels were compared with reference values ​​indicating the stage of fibrotic NASH to determine the stage of fibrotic NASH in the subject.

3. The method of claim 2, wherein the staging is based on the Brunt fibrosis score.

4. A method for monitoring fibrotic nonalcoholic steatohepatitis (NASH) in subjects, the method comprising the following steps: • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in the first sample from the subject; and • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in a second sample from the subject, wherein the second sample was obtained from the subject at a later time point than the first sample, wherein: i) Compared with the first sample, an increase in α2,6-sialylation levels and / or a decrease in α2,3-sialylation levels in the second sample indicate the progression of fibrotic NASH in the subject; or ii) The decrease in α2,6-sialylation level and / or the increase in α2-3-sialylation level in the second sample compared to the first sample indicate the regression of fibrotic NASH in the subject.

5. A method for monitoring subject adherence to prescribed treatment for fibrotic nonalcoholic steatohepatitis (NASH), the method comprising the steps of: • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in the first sample from the subject; and • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in a second sample obtained from the subject following prescribed treatment. Compared with the first sample, a decrease or no change in the level of α2,6-sialylation in the second sample and / or an increase or no change in the level of α2,3-sialylation in the second sample indicate subject adherence to the prescribed treatment for fibrotic NASH.

6. A method for monitoring the therapeutic effect of prescribed treatment for fibrotic nonalcoholic steatohepatitis (NASH), the method comprising the steps of: • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in the first sample from the subject; and • Determine the levels of α2,3-sialylation and / or α2,6-sialylation of N-glycans in a second sample obtained from the subject following prescribed treatment. Compared with the first sample, a decrease or no change in the α2,6-sialylation level in the second sample and / or an increase or no change in the α2,3-sialylation level in the second sample indicate the therapeutic effect of the prescribed treatment for fibrotic NASH.

7. The method according to any one of the preceding claims, wherein the N-glycan is a complex N-glycan and / or a mixed N-glycan.

8. The method according to any one of the preceding claims, wherein the N-glycan is selected from the group consisting of: single-antenna N-glycan, two-antenna N-glycan, three-antenna N-glycan, four-antenna N-glycan, and combinations thereof.

9. The method of claim 7, wherein the N-glycan is a complex N-glycan, optionally selected from the group consisting of tri-antenna N-glycans and tetra-antenna N-glycans.

10. The method according to any one of the preceding claims, wherein the sample is a blood sample.

11. The method of claim 10, wherein the blood sample is a dried blood spot sample.

12. The method of claim 10 or 11, wherein the blood sample is selected from the group consisting of whole blood, plasma, and serum.

13. The method according to any one of the preceding claims, wherein the subject has been diagnosed with fibrotic NASH or has been identified as being at risk of developing fibrotic NASH.

14. The method of claim 13, wherein the subject identified as being at risk of developing fibrotic NASH: i) Has been diagnosed with NAFLD or nonfibrotic NASH; ii) Has been diagnosed with type 2 diabetes or insulin resistance; iii) is obese; iv) Has been diagnosed with hypertension and / or dyslipidemia; and / or v) Having or suspected of having a genetic susceptibility factor, optionally, said genetic susceptibility factor is a mutation in a gene selected from the group consisting of: PNPLA3, TM6SF2, MBOAT7, GCKR and HSD18B13.

15. The method according to any one of claims 5 to 14, wherein the treatment is selected from the group consisting of: weight loss and lifestyle improvement.

16. The method according to any one of the preceding claims, wherein the level of α2,3-sialylation and / or α2,6-sialylation is determined by a method selected from the group consisting of: mass spectrometry, high performance liquid chromatography, capillary (gel) electrophoresis with laser-induced fluorescence detection, hydrophilic interaction liquid chromatography, binding assay based on lectin or antibody, and ELISA-based assay.

17. The method according to any one of the preceding claims, wherein the method further comprises determining the subject's FIB-4 score, ELF score and / or APRI score; and / or the subject's aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels; and / or performing liver imaging.

18. The method according to any one of claims 1-17, wherein the sample has been subjected to N-glycan release from blood proteins and sialic acid-specific derivatization prior to determining the α2,3-sialylation and / or α2,6-sialylation levels of the glycan, optionally wherein the proteins are selected from the group consisting of plasma, serum and whole blood proteins.

19. A method for treating fibrotic NASH in a subject, the method comprising administering fibrotic NASH treatment to a subject who has been diagnosed with NASH or determined to be at risk of developing fibrotic NASH by the method of any one of claims 1 to 18.

20. A kit for use in the method of any one of claims 1 to 19, said kit comprising a detectable labeling reagent that specifically binds to α2,3-sialylated and / or α2,6-sialylated glycans.

Images