AU2019101124A4 - Method of diagnosing systemic lupus erythematosus - Google Patents
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
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- C—CHEMISTRY; METALLURGY
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
- C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
- C07H5/06—Aminosugars
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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- G01N2800/10—Musculoskeletal or connective tissue disorders
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Abstract
A method of diagnosing systemic lupus erythematosus comprising a step of qualitative or quantitative detecting an oligosaccharide fragment or its variant originated by an 5 enzyme dependent cleavage in a sample, wherein the oligosaccharide fragment comprises a first part of Man3-GlcNAc2-Fuc, and a second part comprising one to three GlcNAc units, and one to five additional monosaccharide units linked to the GlcNAc units.
Description
A method of diagnosing systemic lupus erythematosus comprising a step of qualitative or quantitative detecting an oligosaccharide fragment or its variant originated by an 5 enzyme dependent cleavage in a sample, wherein the oligosaccharide fragment comprises a first part of Man3-GlcNAc2-Fuc, and a second part comprising one to three GIcNAc units, and one to five additional monosaccharide units linked to the GIcNAc units.
METHOD OF DIAGNOSING SYSTEMIC LUPUS ERYTHEMATOSUS
TECHNICAL FIELD
The invention relates to a method of diagnosing an autoimmune disease, particularly systemic lupus erythematosus, by detecting an oligosaccharide fragment or its variant.
BACKGROUND OF THE INVENTION
Systemic lupus erythematosus (SLE) is a common autoimmune disease characterized by diverse autoantibodies and wide heterogeneity of clinical manifestations. In 2017, despite many progresses in managing these patients, patients with SLE still have a higher mortality risk and 1 year clinical remission reaches in less than 30%. To facilitate higher remissions of disease-modifying therapies for SLE, which are expected to be more efficacious at the earliest and mildest stages of the disease, supportive biomarker information is necessary. Antinuclear antibody (ANA), anti-double stranded DNA (dsDNA) antibody and anti-Smith (Sm) antibody are used in the classification criteria of SLE; however, they are lack any definitive role in the diagnosis with low sensitivity or specificity (<60%) and not useful in evaluating disease activity. Also, it is of a great challenge to detect IgG for diagnosis, especially for the low-abundance species with important biological significance.
Accordingly, there remains a need for an improved method for determining the pathological or health condition of a subject who is at risk of or suffering from an autoimmune disease particularly SLE for early treatment.
SUMMARY OF THE INVENTION
The invention provides a method of diagnosing systemic lupus erythematosus comprising a step of qualitative or quantitative detecting an oligosaccharide fragment or its variant originated by an enzyme dependent cleavage in a sample, wherein the oligosaccharide fragment comprises a first part of Man3-GlcNAc2-Fuc, and a second part comprising one to three GIcNAc units, and one to five additional monosaccharide units linked to the GIcNAc units.
2019101124 27 Sep 2019
In an embodiment, the second part comprises a galactose (Gal) unit, a 3-deoxy-Dmanno-oct-2-ulosonic acid (Kdo) unit and/or a sialic acid (Sia) unit in addition to the GIcNAc unit.
In an embodiment, the oligosaccharide fragment consists of 8 to 12 monosaccharide units. Particularly, the oligosaccharide fragment consists of 9 or 10 monosaccharide units.
In an embodiment, the oligosaccharide fragment is a mono-antennary glycan or a diantennary glycan.
In an embodiment, the oligosaccharide fragment is a di-antennary glycan, and the second part of the oligosaccharide fragment has bisecting GIcNAc units each being linked to the first part, in which one of the bisecting GIcNAc units is further linked to a Gal unit.
In an embodiment, the oligosaccharide fragment is a sialylated mono-antennary glycan, and the second part comprises Sia-Kdo-GIcNAc.
Preferably, the method comprises the steps of:
(I) isolating immunoglobulins from the sample;
(ii) conducting enzyme dependent cleavage to cleave an oligosaccharide fragment or its variant from the immunoglobulins under conditions suitable for enzymatic reaction; and (ill) determining the presence and/or amount of the oligosaccharide fragment or its variant.
In an embodiment, the immunoglobulin is IgG.
In an embodiment, the step (ill) comprises conducting a high-performance liquid chromatography chip-triple quadrupole-mass spectrometry (HPLC chip-QQQ-MS) for the determination. Preferably, a titanium dioxide-porous graphitized carbon (T1O2-PGC) chip is applied in the step (ill).
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention 2 includes all such variations and modifications. The invention also includes all steps and features referred to or indicated in the specification, individually or collectively, and any and all combinations of the steps or features.
Other features and aspects of the invention will become apparent by consideration of the following detailed description and accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1a shows the schematic representation of an oligosaccharide fragment, TruG1, of an embodiment of the present invention.
Fig. 1 b shows the corresponding sugar residues of the oligosaccharide fragment of Fig. 1a.
Fig. 2a shows the schematic representation of another oligosaccharide fragment, TruG2, of an embodiment of the present invention.
Fig. 2b shows the corresponding sugar residues of the oligosaccharide fragment of Fig. 2a.
Fig. 3 is a graph showing the receiver operating characteristic (ROC) curves of the oligosaccharide fragment TruG1.
Fig. 4 is a graph showing the receiver operating characteristic (ROC) curves of the oligosaccharide fragment TruG2.
Fig. 5 is a boxplot showing the relative abundance of the oligosaccharide fragment TruG1 in samples obtained from patients suffering from SLE or from healthy controls (HCs). The dotted lines represent the cut-off values determined based on the maximum values generated using the formula (sensitivity + specificity - 1) in the analysis.
Fig. 6 is a boxplot showing the relative abundance of the oligosaccharide fragment TruG2 in samples obtained from patients suffering from SLE or from healthy controls (HCs). The dotted lines represent the cut-off values determined based on the maximum values generated using the formula (sensitivity + specificity - 1) in the analysis.
Fig. 7a is a boxplot showing the relative abundance of the oligosaccharide fragment TruG1 in samples obtained from patients suffering from rheumatic arthritis (RA) or from healthy controls (HCs).
Fig. 7b is a boxplot showing the relative abundance of the oligosaccharide fragment TruG2 in samples obtained from patients suffering from rheumatic arthritis (RA) or from healthy controls (HCs).
Fig. 8a is a boxplot showing the relative abundance of the oligosaccharide fragment TruG1 in samples obtained from patients suffering from primary Sjogren's syndrome (pSS) or from healthy controls (HCs).
Fig. 8b is a boxplot showing the relative abundance of the oligosaccharide fragment TruG2 in samples obtained from patients suffering from primary Sjogren's syndrome (pSS) or from healthy controls (HCs).
Fig. 9a is a boxplot showing the relative abundance of the oligosaccharide fragment TruG1 in samples obtained from patients suffering from systemic sclerosis (SSc) or from healthy controls (HCs).
Fig. 9b is a boxplot showing the relative abundance of the oligosaccharide fragment TruG2 in samples obtained from patients suffering from systemic sclerosis (SSc) or from healthy controls (HCs).
Fig. 10 shows the relative abundance of 12 altered oligosaccharide fragments including altered TruG1 and TruG2 fragments (X-TruG1 , X- TruG2) in SLE patients before and after treatment with a low-dose of interleukin-2 (IL-2) (n=15) and in HCs (n=15).
Fig. 11a shows the relative abundance of TruG1 and TruG2 oligosaccharide fragments in SLE patients with low or normal level of complement 3.
Fig. 11b shows the relative abundance of TruG1 and TruG2 oligosaccharide fragments in SLE patients with low or normal level of complement 4.
2019101124 27 Sep 2019
Fig. 11c shows the relative abundance of TruG1 and TruG2 oligosaccharide fragments in SLE patients with low or high erythrocyte sedimentation rate (ESR).
Fig. 12a shows the relative abundance of TruG1 and TruG2 oligosaccharide fragments in SLE patients with negative, low concentration (<1:320) or high concentration ( > 1:320) of ANA.
Fig. 12b shows the relative abundance of TruG1 and TruG2 oligosaccharide fragments in SLE patients with negative, low concentration (25-100 IU/ML) or high concentration (>100 IU/ML) of Anti-dsDNA.
Fig. 12c shows the relative abundance of TruG1 and TruG2 oligosaccharide fragments in SLE patients with normal, negative and positive Anti- ribonucleoprotein (RNP).
Fig. 12d shows the relative abundance of TruG1 and TruG2 oligosaccharide fragments in SLE patients with normal, negative and positive Anti-Sm.
DETAILED DESCRIPTION OF THE EMBODIMENTS
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one skilled in the art to which the invention belongs.
As used herein, “comprising” means including the following elements but not excluding others. “Essentially consisting of” means that the material consists of the respective element along with usually and unavoidable impurities such as side products and components usually resulting from the respective preparation or method for obtaining the material such as traces of further components or solvents. “Consisting of” means that the material solely consists of, i.e. is formed by the respective element. As used herein, the forms “a,” “an,” and “the,” are intended to include the singular and plural forms unless the context clearly indicates otherwise.
The present invention in the first aspect provides a method of diagnosing an autoimmune disease by identifying or detecting the presence or absence of an oligosaccharide fragment or its variant in a sample. The oligosaccharide fragment or its variant is preferably a product derived after an enzyme dependent cleavage, and is 5
2019101124 27 Sep 2019 produced under artificial conditions using reagents. Whilst the oligosaccharide fragment or its variant may be present in a conjugated form with a protein or lipid in some embodiments, the present invention is able to identify and detect the isolated pure form of the oligosaccharide fragment or its variant for subsequent determination of health 5 conditions of a subject who provides the sample.
The sample is preferably a biological sample from a subject such as a mammal, preferably a human and can comprise, for example, blood or serum. Preferably, the sample is a serum sample.
The method herein is particularly useful in diagnosing systemic lupus erythematosus (abbreviated as SLE). In some other embodiments, the method may be applied to diagnose other autoimmune diseases such as, but is not limited to, rheumatic arthritis (abbreviated as RA), primary Sjogren's syndrome (abbreviated as pSS), systemic 15 sclerosis (abbreviated as SSc) and the like, to determine the risk of suffering from an autoimmune disease, and/or to determine the drug efficacy on the disease after administration.
The “oligosaccharide fragment” as used herein refers to a short saccharide polymer 20 having from about 2 to about 14 monosaccharide units. In an embodiment herein, the oligosaccharide fragment or its variant of interest comprises or consists of about 8 to 12 monosaccharide units, or preferably comprises or consists of about 9 or 10 monosaccharide units. In a particular embodiment, the oligosaccharide fragment or its variant of interest consists of 9 monosaccharide units. It would be appreciated that the 25 oligosaccharide fragment may be subjected to immaterial alternation to enhance the specificity for its detection. The immaterial alternation may include, for example but not exclusively, substitution, deletion or addition of one or more monosaccharide units to the oligosaccharide fragment derived from the sample.
In the method herein, the oligosaccharide fragment or its variant is originated by an enzyme dependent cleavage preferably under artificial conditions. During the cleavage, the oligosaccharide fragment or its variant is converted to an artificial product existed as a pure and isolated form which does not normally present in the sample. In a particular embodiment, the oligosaccharide fragment or its variant is preferably a N35 glycan derived from an immunoglobulin, particularly IgG, and it may be presented as a mono-antennary glycan or a di-antennary glycan.
2019101124 27 Sep 2019
The oligosaccharide fragment preferably comprises or consists of two parts, i.e. a first part of Man3-GlcNAc2-Fuc, and a second part comprising one to three GIcNAc units, and one to five additional monosaccharide units linked to the GIcNAc units, in which 5 “Man” denotes a mannose unit, “GIcNAc” denotes a N-acetyl-D-glucosamine unit, and “Fuc” denotes a D-fucose unit. It would be appreciated that the first part and second part are linked via glycosidic bond. Also, the oligosaccharide fragment may comprise 3 to 5 GIcNAc units, 3 GIcNAc units, or 4 GIcNAc units in total.
In an embodiment, the oligosaccharide fragment comprises the aforesaid first part of Man3-GlcNAc2-Fuc being linked to a second part containing 1-5 GIcNAc units together with a galactose (Gal) unit, a 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) unit and/or a sialic acid (Sia) unit.
In one embodiment, the oligosaccharide fragment is a di-antennary glycan, and the second part of the oligosaccharide fragment has bisecting GIcNAc units each being linked to the first part, in which one of the bisecting GIcNAc units is further linked to a Gal unit. A particular embodiment of such an oligosaccharide fragment is further illustrated in Fig. 1a and 1b, and denoted as “TruG1” throughout the disclosure herein. 20 The inventors found that this oligosaccharide fragment is useful in determination of health and pathological conditions of a subject who is at risk of or suffering from an autoimmune disease particularly SLE. For example, the subject suffering from or at risk of SLE may have an abnormally lower level of TruG1 in the serum, compared to a healthy subject.
In another embodiment, the oligosaccharide fragment is a sialylated mono-antennary glycan, and the second part comprises or consists of Sia-Kdo-GIcNAc. The second part is linked via glycosidic bond to the aforesaid first part. A particular embodiment of such an oligosaccharide fragment is illustrated in Fig. 2a and 2b, and denoted as “TruG2” 30 throughout the disclosure herein. The inventors found that this oligosaccharide fragment is exceptionally effective in determination of health and pathological conditions of a subject who is at risk of or suffering from an autoimmune disease particularly SLE. For example, the subject suffering from or at risk of SLE may have an abnormally higher level of TruG2 in the serum, compared to a healthy subject. Further, 35 the oligosaccharide fragment TruG2 is also effective in monitoring the development or treatment of the disease. It was found that the level of TruG2 in the serum sample of a
2019101124 27 Sep 2019
SLE patient is responsive to treatment, and therefore the level of TruG2 can act as an indicator for a physician to monitor the progress of disease and to adapt suitable therapies according to the patient’s conditions in a more objective manner.
It was also found that the oligosaccharide fragments TruG1 and TruG2 are particularly specific to SLE, with promising sensitivities and specificities towards SLE, among other autoimmune diseases. It is thus believed that these oligosaccharide fragments or their variants are potential markers targeting SLE for clinical and research purposes.
The term “variant” herein means that a saccharide polymer containing one or more alternation on the monosaccharide units compared to the oligosaccharide fragment of concern. The alternation may be substitution, deletion of addition of one or more monosaccharide units. The variant also encompasses any complexes or elements that include the oligosaccharide fragment of concern, e.g. an artificially complex conjugated 15 with said fragment, and it may include, but not limited to, a probe conjugated with said fragment.
Turning to the method, since the oligosaccharide fragment or its variant may be present in relatively low abundance in the subject, the method requires a comprehensive 20 approach to quantitative or qualitative detect the oligosaccharide fragment or its variant of concern. Preferably, the method comprises steps of (i) isolating immunoglobulins such as IgG from the sample;
(ii) conducting enzyme dependent cleavage to cleave an oligosaccharide fragment or its variant from the immunoglobulins under conditions suitable for enzymatic reaction; and (iii) determining the presence and/or amount of the oligosaccharide fragment or its variant.
Step (i) may be conducted by using a protein or a conjugated complex or compound 30 which is capable of binding immunoglobulins. For example, Protein A or a bead conjugated with Protein A can be used to bind the immunoglobulins for isolation. In an embodiment, the sample is incubated in a mixture containing beads conjugated with Protein A for a period of time, e.g. for 5 to 1 h, at room temperature. The beads are then isolated from the mixture and optionally washed with a washing buffer. The resultant 35 mixture may then be subjected to purification by using, for example, a column or filter.
Step (ii) includes an enzyme dependent cleavage process using an enzyme which is capable of cleaving the isolated immunoglobulins to release one or more oligosaccharides or their variants. The enzyme may be PNGase. Particularly, the isolated immunoglobulins are incubated with a mixture containing the enzyme for a period of time. The mixture may further comprise a buffer such as ammonium bicarbonate buffer to facilitate the enzymatic reaction. After that, it may be followed by a step of purification using column such as a cartridge or a filter to remove unreacted substances and de-glycosylated proteins.
After step (ii), it is optional to further modify the derived oligosaccharide fragment for subsequent detection. For example, the derived oligosaccharide fragment may be subjected to monosaccharide unit modification including substitution, deletion or addition, or binding with a probe such as a fluorescent probe, a DNA/RNA probe. These modifications may be useful to improve the detection in the subsequent step. The modified oligosaccharide fragment is also considered as a variant of the oligosaccharide fragment.
Step (iii) is then performed to quantitatively or qualitatively detect the derived oligosaccharide fragment or its variant. Preferably, it comprises conducting a liquid chromatography-mass spectrometry particularly a high-performance liquid chromatography triple quadrupole-mass spectrometry such as a high-performance liquid chromatography chip-triple quadrupole-mass spectrometry (HPLC chip-QQQMS).
A titanium dioxide-porous graphitized carbon (TiCk-PGC) chip composed of a sandwich-like enrichment column, e.g. a T1O2 column positioned between two PGC trapping columns may be used to differentiate the oligosaccharide fragment or its variant of concern from the rest of the oligosaccharide fragments derived from the previous steps. The chip is advantageous to concentrate the amount of fragments of concerns so as to facilitate the liquid chromatography-mass spectrometry (LC-MS) detection.
The presence and/or amount of the oligosaccharide fragment or its variant of concern is then determined based on the mass spectrometry (MS) data. As described above, the oligosaccharide fragment or its variant of the present invention is particularly useful
2019101124 27 Sep 2019 to diagnose an autoimmune disease particularly SLE. It is therefore believed that the method herein can be applied in clinical and research applications.
It would thus be appreciated that the present invention also pertains to use of the oligosaccharide fragment or its variant in the diagnosis and other clinical applications.
The present invention may further pertain to a kit comprising a probe capable of binding to the oligosaccharide or its variant as described above. The kit may further include an enzyme facilitating the cleavage of the fragment from the immunoglobulins extracted from a sample.
The inventors conducted experiments to prove the specificity and sensitivity of the oligosaccharide fragments as discussed in this invention. The followings are some discussion on the examples for illustration purposes and are not provided to limit the scope of protection.
EXAMPLES
Identification of oligosaccharide fragments
Target subject groups
Subjects with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), primary Sjogren’s syndrome (pSS), systemic sclerosis (SSc) and healthy controls (HCs) were enrolled from four hospitals in China including: Peking University People’s Hospital (Beijing, China), First Teaching Hospital of Tianjin University of Traditional Chinese Medicine (Tianjin, China), Guangdong General Hospital of Guangdong Academy of Medical Sciences (Guangzhou, China) and Zhuhai Hospital of Integrated Traditional Chinese and Western Medicine (Zhuhai, China). All subjects provided informed consent, and ethical approval was obtained from the local institutional committee approval of the relevant hospitals (2015PHB219-01).
Patients with SLE: A total of 227 patients with SLE and 164 HCs from three independent cohorts (including a training dataset and two validation datasets), were analyzed in this study. The average disease duration for SLE patients were 5 years. The training dataset consisted of 133 patients with SLE and 89 HCs. The average age for individuals with SLE was 37.3 ± 13.0, comparable to HCs (37.9 ± 13.4). The female rate for SLE and HCs are 91.7% and 92.1% respectively. The first validation dataset consisted of 58 10
2019101124 27 Sep 2019 patients with SLE and 39 age and gender matched HCs and the second validation dataset consisted of 36 patients with SLE and 36 age and gender matched HCs. All patients with SLE fulfilled the 1997 revised classification criteria of the American College of Rheumatology. Besides, 15 patients with SLE were followed up for 10 weeks and their paired serum samples were collected from the same patients before and after treatment with corticosteroids, antimalarials, immunosuppressants and low dose of interleukin-2 (IL-2) from the Department of Rheumatology and Immunology of Peking University People’s Hospital (Beijing, China).
Patients with RA: Samples of patients with RA (n = 33) and HCs (n = 32) were also enrolled. The RA patients met the 2010 ACR/EULAR classification criteria for RA
Patients with pSS. In total, 49 patients with pSS and 49 age- and sex-matched HCs were enrolled in this study. The pSS patients were classified according to the 2016 American College of Rheumatology (ACR)ZEuropean League Against Rheumatism (EULAR) classification criteria for pSS.
Patients with SSc: Twenty-six patients with SSc and 25 age- and sex-matched HCs were also enrolled. The SSc patients were classified according to the American College of Rheumatology (ACR)ZEuropean League Against Rheumatism (EULAR) criteria for the classification of SSc.
Human serum samples were obtained from the hospital following the same protocol. All samples were stored at -80 °C prior to use.
Analysis of oligosaccharide fragments, i.e. N-glycans, isolated from IgG
IgGs were isolated from serum samples, as discussed above and N-glycans were released and analyzed by high-performance liquid chromatography chip-triple quadrupole-mass spectrometry (HPLC chip-QQQ-MS). Wang, J.-R. et al Nature communications 8, 631 (2017) also discussed a possible approach to isolate IgG and oligosaccharides.
Statistical analysis
Two-sided p values <0.05 in the wilcoxon rank-sum test were considered to indicate statistical significance. Continuous variables are presented as the mean ± s.d. The sample size was not determined in advance using statistical methods. All data points 11
2019101124 27 Sep 2019 were included in the analysis. The method of ComBat (Johnson, W. E., et al. Biostatistics 8, (2007)) (implemented in the R package “SVA” (Leek, J. T. et al. Bioinformatics 28, (2012)) was applied to adjust for batch effects using an empirical Bayes framework. The classification ability of the N-glycan biomarkers identified was 5 assessed using ROC curve analysis (implemented in the “pROC” (Robin, X. et al. BMC
Bioinformatics 12 (2011)) package in R) for individual markers and the combination of predictors, and the diagnostic value of the N-glycan biomarkers was evaluated on the basis of AUCs. Cut-off values were determined according to the maximum values generated using the formula (sensitivity + specificity - 1) in our analyses. Confidence 10 intervals (95%CI) for AUC, sensitivity and specificity of ROC curves were obtained using the method proposed by DeLong and his colleagues25. In addition, p values corrected for multiple testing by setting the false discovery rate at 0.05 are shown throughout. All statistical analyses were performed using RStudio version 1.0.153 (RStudio incorporated corporation, Boston, USA).
RESULTS
Two high potential N-glycan biomarkers for the classification of SLE
A total of 114 N-glycans from serum IgG were identified for SLE patients, including 53 20 neutral glycans and 61 acidic N-glycans. The inventors then used correlation-based feature subset selection method (CfsSubsetEval) in WEKA to select informative Nglycans. Four neutral and eight acidic N-glycans were selected as potential biomarkers for the classification of SLE in the training set (SLE patients, n=133; healthy controls (HCs), n=89). These 12 biomarkers were then used individually to generate a diagnostic 25 model using logistic regression (LR) for evaluating their sensitivities and specificities.
According to receiver operating characteristic (ROC)-analysis, the sensitivity values of the 12 N-glycans varied from 46.6% to 83.5%, the specificity values ranged from 67.4% to 100%, and area under the curve (AUC) values ranged from 0.702 to 0.895 (Table 1).
The inventors further examined the capability of the 12 N-glycan biomarkers to classify of SLE patients in two validation cohorts (Validation set 1: SLE patients, n=58; HCs, n=39. Validation set 2: SLE patients, n=36; HCs, n=36) (Table 1) and the results were well consistent with data for the training set. Diagnostic performances and levels of two potential N-glycan biomarkers for systemic lupus erythematosus (SLE) are shown in 35 Fig. 3 to Fig. 6. The sample sizes for analysis are SLE (n=133) and healthy controls (HCs) (n=89) in training set, SLE (n=58) and HCs (n=39) in validation set1 and SLE (n=36) and HCs (n=36) in validation set2.
Among the 12 biomarkers, TruG1 and TruG2, demonstrated relatively high prediction capacity for SLE. TruG1, with one galactose (G1), was significantly decreased in SLE patients, while TruG2 was significantly increased (P<0.0001), both of which had high AUC (0.765-0.902), sensitivities (75%~87.9%) and specificities (78%~88.3%) for the training and validation sets (Fig. 1b, c, e, f, Table 1). Additionally, the prediction accuracy for TruG1 was 79%-84% and for TruG2 was 81%-83% (Table 2).
Specificity of two identified N-glycan biomarkers for the diagnosis of SLE
To confirm the specificity of TruG1 and TruG2, the inventors detected changes in the level of OLI-1 and OLI-2 in patients with other common autoimmune diseases including RA, pSS and SSc, which share high sensitivity of ANA. As shown in Fig, 7a and 7b, the abundance of TruG1 and TruG2 does not show significant changes between RA patients. Similar results are obtained for pSS (Fig. 8a and 8b), and SSc (Fig. 9a and 9b) patients. Accordingly, the results show that TruG1 and TruG2 are less specific towards RA, pSS and SSc. Table 3 demonstrates the numerical results.
Treatment response of SLE patients before and after treatment
The inventors performed a glycomics analysis on 15 patients with SLE before and after treatment with a low-dose of IL-2, in which their paired serum samples that were collected from the same patients. Results are shown in Fig. 10 and Table 4 regarding alterations in the 12 selected glycans. The level of glycan TruG2 was significantly decreased after IL-2 treatment, indicating that glycan TruG2 might be served as both a diagnostic marker and a response marker for treatment.
Correlation between the two identified glycans and the disease phenotypes
The inventors next correlated the TruG1 and TruG2 with multiple disease phenotypes. The complement system has a dual role in SLE, both mediating pathogenesis and preventing the disease, and N-glycosylation alterations were previously shown to promote the production of pro-inflammatory mannose-dependent glycan ligands in part, which activate and cleave complement (C) 3 and C4. In line with these previous reports, TruG2 was significantly up-regulated in patients with low levels of C3, C4, whereas TruG1 was not (Fig. 11a and 11b). Of note, TruG2, a sialylated IgG glycan, was also positively related to the ESR (Fig. 11c) with ability to predict disease activity.
According to Fig. 12a to 12c, it is shown that there is no correlation between TruG1 and TruG2 and titers of serum autoantibodies including ANA, dsDNA, anti-RNP and antiSm. Accordingly, TruG1 and TruG2 are independent diagnostic biomarkers, that may act as “on and off” switches or as “analog regulators” in the pathogenesis of SLE.
CONCLUSION
The oligosaccharide fragments TruG1 and TruG2 are potential biomarkers for indicating the health conditions and pathological conditions of a subject particularly a human at risk of or suffering from SLE. They have high sensitivity and high specificity for diagnosis of SLE. TruG1 and TruG2 can be developed for clinical and diagnosis applications.
CM
P
CD
QC
Table 2. The prediction accuracy of the two potential N-glycan biomarkers for SLE N-glycans Training set Validation set 1 Validation set 2
2019101124 27 Sep 2019
Claims (12)
1. A method of diagnosing systemic lupus erythematosus comprising a step of qualitative or quantitative detecting an oligosaccharide fragment or its variant originated by an enzyme dependent cleavage in a sample, wherein the oligosaccharide fragment comprises a first part of Man3-GlcNAc2-Fuc, and a second part comprising one to three GIcNAc units, and one to five additional monosaccharide units linked to the GIcNAc units.
2. The method of claim 1, wherein the second part comprises a galactose (Gal) unit, a 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) unit and/or a sialic acid (Sia) unit in addition to the GIcNAc unit.
3. The method of claim 1, wherein the oligosaccharide fragment consists of 8 to 12 monosaccharide units.
4. The method of claim 1, wherein the oligosaccharide fragment consists of 9 or 10 monosaccharide units.
5. The method of claim 1, wherein the oligosaccharide fragment is a monoantennary glycan or a di-antennary glycan.
6. The method of claim 1, wherein the oligosaccharide fragment is a di-antennary glycan, and the second part of the oligosaccharide fragment has bisecting GIcNAc units each being linked to the first part, in which one of the bisecting GIcNAc units is further linked to a Gal unit.
7. The method of claim 1, wherein the oligosaccharide fragment is a sialylated mono-antennary glycan, and the second part comprises Sia-Kdo-GIcNAc.
8. The method of claim 1 comprises the steps of (I) isolating immunoglobulins from the sample;
(ii) conducting enzyme dependent cleavage to cleave an oligosaccharide fragment or its variant from the immunoglobulins under conditions suitable for enzymatic reaction; and
2019101124 27 Sep 2019 (iii) determining the presence and/or amount of the oligosaccharide fragment or its variant.
9. The method of claim 8, wherein the immunoglobulin is IgG.
10. The method of claim 8, wherein the step (iii) comprises conducting a highperformance liquid chromatography chip-triple quadrupole-mass spectrometry (HPLC chip-QQQ-MS) for the determination.
10
11. The method of claim 10, wherein a titanium dioxide-porous graphitized carbon chip is applied in the step (iii).
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