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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6869—Methods for sequencing
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • 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

Definitions

• the present invention relates to a biomarker associated with amyotrophic lateral sclerosis (ALS) or its use or method of use.
• ALS amyotrophic lateral sclerosis
• ALS Amyotrophic lateral sclerosis
• Non-Patent Document 1 https://www.nhk.or.jp/kenko/atc_762.html.
• ALS is a neurodegenerative disease in which motor neurons (nerve cells) are damaged, and muscle atrophy and muscle weakness progress.
• ALS patients require artificial respiration or die about 2-5 years after onset.
• the prevalence of ALS in Japan is 5-7 people/100,000, with approximately 10,000 patients.
• ALS is sporadic in approximately 90% of cases and familial in approximately 10% (SOD1 mutations, etc.).
• the revised ALS Functional Rating Scale (ALSFRS-R) is known to be used to evaluate ALS symptoms.
• the ALSFRS-R is an assessment scale created to understand the daily life of ALS patients.
• the ALSFRS-R is composed of 12 items (5-point scale from 0 to 4) such as language, swallowing, daily activities, and walking, and is evaluated based on the total score (0 to 48).
• the ALSFRS-R is used as an inclusion criterion and primary endpoint for clinical trials.
• ALS many aspects of the immunological profile of peripheral blood are unknown. In particular, the profile associated with the rate of progression of symptoms has not been fully elucidated. If the immunological profile of peripheral blood could be clarified, it would be useful for diagnosis, stratification of patients in clinical trials, and as a surrogate marker for therapeutic intervention. Therefore, there is a need for biomarkers that can determine the rate of progression of ALS or the possibility of contracting the disease.
• the present inventors have conducted extensive research to solve the above problems, and have discovered a biomarker associated with amyotrophic lateral sclerosis (ALS) or a use thereof (e.g., use for determining or diagnosing the rate of progression or likelihood of developing amyotrophic lateral sclerosis (ALS) in a subject) or a method of using the same (e.g., a method for determining or diagnosing the rate of progression or likelihood of developing amyotrophic lateral sclerosis (ALS) in a subject).
• a biomarker associated with amyotrophic lateral sclerosis (ALS) or a use thereof e.g., use for determining or diagnosing the rate of progression or likelihood of developing amyotrophic lateral sclerosis (ALS) in a subject
• ALS amyotrophic lateral sclerosis
• biomarkers for use in determining or diagnosing the rate of progression or likelihood of developing amyotrophic lateral sclerosis (ALS) and/or in selecting or predicting therapeutic agents.
• ALS amyotrophic lateral sclerosis
• the inventors have also discovered a method for determining or diagnosing the rate of progression or likelihood of developing amyotrophic lateral sclerosis (ALS) in a subject, comprising determining the level of a biomarker in a sample from the subject.
• ALS amyotrophic lateral sclerosis
• a biomarker for use in determining or diagnosing the rate of progression or likelihood of developing amyotrophic lateral sclerosis (ALS), and/or in selecting or predicting therapeutic agents comprising:
• the biomarker is selected from the group consisting of IL-17A, KLRD1, KRT19, NCF2, TFF2, YTHDF3, Th17, regulatory T cells (Treg), mature CD8T, naive CD8T, exhausted CD8T, classical monocyte, memory CD4T, Th17/Treg, mature CD8T/naive CD8T, and mature CD8T/exhausted CD8T.
• the biomarker described in (1) for use in determining or diagnosing amyotrophic lateral sclerosis (ALS) with a rapid progression rate.
• (4) A method for determining or diagnosing the rate of progression or likelihood of amyotrophic lateral sclerosis (ALS) in a subject comprising: determining the level of a biomarker described in any one of (1) to (3) in a sample derived from the subject.
• the method according to (4) further comprising determining or diagnosing the rate of progression or likelihood of ALS based on the reference level of the biomarker.
• the reference level is the level of the biomarker in a sample from a subject not having ALS, a sample from a subject having ALS, a sample from a subject having ALS with a non-rapid progression rate, or a sample from a subject having ALS with a rapid progression rate.
• a biomarker for use in determining or diagnosing the rate of progression or likelihood of developing amyotrophic lateral sclerosis (ALS) and/or in selecting or predicting a treatment there is provided a method for determining or diagnosing the rate of progression or likelihood of developing amyotrophic lateral sclerosis (ALS) in a subject, comprising determining a level of a biomarker in a sample from the subject.
• RNA sequencing Single-cell RNA sequencing
• scRNA-seq single-cell RNA sequencing
• PBMCs peripheral blood mononuclear cells
• ALS non-rapid amyotrophic lateral sclerosis
• ALS rapid amyotrophic lateral sclerosis
• UMAP Uniform manifold approximation and projection
• Red dots are differentially expressed proteins (DEPs) and protein names are shown.
• ELISA enzyme-linked immunosorbent assay
• IL-17A interleukin-17A
• KLD1 killer cell lectin-like receptor D1
• KRT19 keratin 19
• NCF2 neutrophil cytoplasmic factor 2
• NPX normalized protein expression
• TFF2 trefoil factor 2
• YTHDF3 YTH N6-methyladenosine RNA-binding protein F3.
• Correlation plot of serum immune proteins and frequency/ratio of each cell type Scatter plot of serum protein expression levels and frequency/ratio of each cell type in single-cell RNA-seq analysis using Pearson's correlation coefficient. Values indicate correlation coefficients using 40 amyotrophic lateral sclerosis patient and healthy control samples.
• Annotation of each cluster with cell type markers Annotation of each cluster by cell type markers (continuation of Figure 5-1). Between-group comparison of the frequency of each cell type among all cells. Between-group comparison of the frequency of each cell type in each immune cell. Between-group comparison of cell frequency ratios.
• non-human animals include, but are not limited to, non-human primates such as monkeys, vertebrates such as sheep and dogs, and rodents such as mice, rats, and guinea pigs.
• ALS myotrophic lateral sclerosis
• ALS refers to a neurodegenerative disease that damages motor neurons (nerve cells), and that progresses with muscle atrophy and muscle weakness.
• ALS includes ALS with a "rapid rate of progression” and ALS with a “non-rapid rate of progression.”
• the "rate of progression” of ALS includes both the “rapid rate of progression” of ALS and the “non-rapid rate of progression” of ALS.
• ALS ALS Functional Rating Scale
• non-rapid progression rate ALS refers to ALS that exhibits a decline of less than one point per month based on the score on the revised ALS Functional Rating Scale (ALSFRS-R) for the assessment of ALS symptoms.
• ALSFRS-R ALS Functional Rating Scale
• non-rapid progression rate ALS, “non-rapid” ALS, and “non-rapid” ALS are used interchangeably.
• “likelihood of having” refers to the possibility of having ALS or of developing ALS.
• “Likelihood of having” includes high “likelihood of having” and low “likelihood of having.”
• a high “likelihood of having” refers to a high possibility of having ALS or of developing ALS compared to a subject who does not have ALS, a subject who does not develop ALS, or a healthy control.
• a low “likelihood of having” refers to a low or similar possibility of having ALS or of developing ALS compared to a subject who does not have ALS, a subject who does not develop ALS, or a healthy control.
• a "therapeutic agent” includes any drug used to treat, prevent, or diagnose ALS.
• a therapeutic agent may be a known drug for treating, preventing, or diagnosing ALS, or an unknown drug for treating, preventing, or diagnosing ALS.
• biomarker refers to an indicator associated with ALS, e.g., an indicator for use in determining or diagnosing the rate of progression or likelihood of ALS, and/or in selecting or predicting a treatment for ALS.
• biomarker includes any substance present in a sample that can be measured, e.g., cells (e.g., immune cells, e.g., T cells, B cells), polypeptides (e.g., immunoglobulins, peptides), polynucleotides (e.g., mRNA, DNA), and/or their metabolites.
• Biomarkers are described, for example, in Table 2, Table 4, Table 5, Table 6, Table 7, Table 8 (Table 8-1, Table 8-2), Table 9 (Table 9-1, Table 9-2, Table 9-3, Table 9-4, Table 9-5), Figure 2, Figure 3, Figure 4, Figure 5 ( Figure 5-1, Figure 5-2), Figure 6, Figure 7, and Figure 8.
• Biomarkers include biomarkers associated with ALS, ALS biomarkers, for example, rapid progression rate ALS biomarkers and/or non-rapid progression rate ALS biomarkers.
• a biomarker may be a single molecule or multiple molecules.
• the biomarker may be an indication of a relationship (e.g., a ratio) between the molecules.
• a description of "single molecule A/single molecule B" of multiple molecules indicates the ratio of single molecule A to single molecule B.
• Th17/Treg represents the ratio of Th17 and Treg levels by dividing Th17 levels by Treg levels.
• Biomarkers may be used as a single biomarker or as a combination of multiple (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) biomarkers.
• multiple biomarkers can provide a more accurate or more precise indication associated with ALS than the use of a single biomarker.
• the "level" of a biomarker refers to the concentration, expression level, and/or activity level of the biomarker. Those skilled in the art can determine the level of a biomarker as appropriate.
• IL-17A refers to interleukin-17A.
• KLRD1 refers to killer cell lectin like receptor D1.
• KRT19 refers to keratin 19.
• NCF2 neutrophil cytosolic factor 2.
• TFF2 refers to trefoil factor 2.
• YTHDF3 refers to YTH N6-methyladenosine RNA binding protein F3.
• Th17 refers to helper T17 cells.
• Treg refers to regulatory T cells.
• mature CD8 T refers to mature CD8 positive T cells.
• naive CD8 T refers to naive CD8 positive T cells.
• exhausted CD8 T refers to exhausted CD8 positive T cells.
• classical monocyte refers to a classical monocyte.
• memory CD4T refers to memory CD4 positive T cells.
• sample can be obtained from a subject or patient.
• a sample can be obtained from any source known in the art, including, but not limited to, blood, whole blood, serum, plasma, urine, interstitial fluid, tears, saliva, or skin.
• a "reference level" of a biomarker refers to the level of the biomarker in a sample from a subject without ALS, a sample from a subject with ALS, a sample from a subject with ALS that has a non-rapid rate of progression, or a sample from a subject with ALS that has a rapid rate of progression.
• a person skilled in the art can select and determine the reference level of the biomarker as appropriate.
• the subject or patient from whom the sample is obtained may be the same as or different from the subject from whom the sample providing the reference level is obtained.
• the biomarkers of the present invention can be used to determine or diagnose the rate of progression or likelihood of developing ALS.
• the invention is based on the discovery that (i) the level of a biomarker in a sample obtained from a subject with ALS that has a rapid rate of progression, (ii) the level of the same biomarker in a sample obtained from a subject with ALS that has a non-rapid rate of progression, and (iii) the level of the same biomarker in a sample obtained from a subject without ALS.
• the invention is based on the discovery that (i) the level of a biomarker in a sample obtained from a subject with ALS that has a rapid rate of progression is altered from (ii) the level of the same biomarker in a sample obtained from a subject with ALS that has a non-rapid rate of progression and/or (iii) the level of the same biomarker in a sample obtained from a subject without ALS.
• the invention is based on the discovery that (ii) the level of a biomarker in a sample obtained from a subject with ALS that has a non-rapid progression rate is altered from (i) the level of the same biomarker in a sample obtained from a subject with ALS that has a rapid progression rate and/or (iii) the level of the same biomarker in a sample obtained from a subject without ALS.
• the invention is based on the discovery that (i) the level of a biomarker in a sample obtained from a subject with ALS that has a rapid rate of progression and/or (ii) the level of the same biomarker in a sample obtained from a subject with ALS that has a non-rapid rate of progression and (iii) the level of the same biomarker in a sample obtained from a subject without ALS is altered.
• the invention provides a biomarker in which (i) the level of the biomarker in a sample obtained from a subject with ALS having a rapid rate of progression is higher or lower than (ii) the level of the same biomarker in a sample obtained from a subject with ALS having a non-rapid rate of progression and/or (iii) the level of the same biomarker in a sample obtained from a subject without ALS.
• the invention provides a biomarker in which (ii) the level of the biomarker in a sample obtained from a subject with ALS having a non-rapid progression rate is higher or lower than (i) the level of the same biomarker in a sample obtained from a subject with ALS having a rapid progression rate and/or (iii) the level of the same biomarker in a sample obtained from a subject without ALS.
• the present invention provides a biomarker in which (i) the level of the biomarker in a sample obtained from a subject with ALS having a rapid rate of progression and/or (ii) the level of the same biomarker in a sample obtained from a subject with ALS having a non-rapid rate of progression is higher or lower than (iii) the level of the same biomarker in a sample obtained from a subject without ALS.
• a therapeutic agent that (i) reduces or increases the level of a biomarker in a sample obtained from a subject with ALS with a rapid progression rate, (ii) is higher or lower than the level of the same biomarker in a sample obtained from a subject with ALS with a non-rapid progression rate and/or (iii) is higher or lower than the level of the same biomarker in a sample obtained from a subject without ALS, respectively, can be selected or predicted as a potential or effective therapeutic agent for ALS with a rapid progression rate.
• a therapeutic agent that reduces or increases, respectively, (ii) the level of a biomarker in a sample obtained from a subject with ALS having a non-rapid progression rate, higher or lower than (i) the level of the same biomarker in a sample obtained from a subject with ALS having a rapid progression rate and/or (iii) the level of the same biomarker in a sample obtained from a subject without ALS, can be selected or predicted as a potential or effective therapeutic agent for ALS having a non-rapid progression rate.
• a therapeutic agent that reduces or increases, respectively, (i) the level of a biomarker in a sample obtained from a subject with ALS with a rapid progression rate and/or (ii) the level of the same biomarker in a sample obtained from a subject with ALS with a non-rapid progression rate, (iii) higher or lower than the level of the same biomarker in a sample obtained from a subject without ALS, can be selected or predicted as a potential or effective therapeutic agent for rapid and/or non-rapid progression rate ALS.
• a candidate therapeutic agent for ALS when used to select or predict a therapeutic agent for ALS, a candidate therapeutic agent for ALS may be mixed or contacted with the biomarkers of the present invention.
• the candidate therapeutic agent for ALS may be a drug that reduces or increases the level of the biomarkers of the present invention.
• the candidate therapeutic agent for ALS may be tested or confirmed to reduce or increase the level of the biomarkers of the present invention.
• the biomarkers of the present invention can be used to determine or predict the efficacy or performance of a therapeutic agent for ALS.
• biomarkers of the present invention are described, for example, in Table 2, Table 4, Table 5, Table 6, Table 7, Table 8 (Table 8-1, Table 8-2), Table 9 (Table 9-1, Table 9-2, Table 9-3, Table 9-4, Table 9-5), Figure 2, Figure 3, Figure 4, Figure 5 ( Figure 5-1, Figure 5-2), Figure 6, Figure 7, and Figure 8.
• the biomarkers of the invention are selected from the group consisting of IL-17A, KLRD1, KRT19, NCF2, TFF2, YTHDF3, Th17, regulatory T cells (Tregs), mature CD8T, naive CD8T, exhausted CD8T, classical monocytes, memory CD4T, Th17/Treg, mature CD8T/naive CD8T, and mature CD8T/exhausted CD8T.
• the biomarkers of the invention are selected from the group consisting of NCF2-classical monocytes, TFF2-mature CD8 T cells, KLRD1-mature CD8 T cells, IL-17A-memory CD4 T cells, and IL-17A-Th17.
• the biomarkers of the invention are selected from the group consisting of TFF2-mature CD8 T cells, KLRD1-mature CD8 T cells, and IL-17A-Th17. In one embodiment, the biomarkers of the invention are selected based on partial correlation coefficients and/or P-values, for example as described in the Examples. In one embodiment, the biomarkers of the invention may be a combination of immune cells and proteins. In one embodiment, the biomarkers of the invention can be used to determine or diagnose the rate of progression (rapid or non-rapid) or likelihood of ALS (high likelihood of ALS or low likelihood of ALS). In one aspect, the biomarkers of the present invention can be used to determine or diagnose the rate of progression (rapid or non-rapid) or likelihood of ALS (high likelihood or low likelihood of ALS) based on an increase or decrease from a reference level.
• biomarkers of the present invention may be used in combination with known biomarkers (e.g., neurofilament light chain (NfL)).
• known biomarkers e.g., neurofilament light chain (NfL)
• the biomarkers of the present invention allow for the determination or diagnosis of early ALS progression rate or likelihood of onset.
• ALS patients are generally treated after a 12 week observation period.
• the biomarkers of the present invention allow for the determination or diagnosis of early (prior to the 12 week observation period) ALS progression rate or likelihood of onset.
• the determination or diagnosis of early ALS progression rate or likelihood of onset may allow for early treatment of ALS and may help to slow ALS progression.
• the use or method of use of the biomarkers of the invention is a method for determining or diagnosing the rate of progression or likelihood of developing amyotrophic lateral sclerosis (ALS) in a subject, comprising determining the level of the biomarkers of the invention in a sample from the subject.
• ALS amyotrophic lateral sclerosis
• the use or method of using the biomarkers of the invention includes determining a reference level, e.g., by determining the level of the biomarker in a sample from a subject without ALS, a sample from a subject with ALS, a sample from a subject with ALS that has a non-rapid rate of progression, or a sample from a subject with ALS that has a rapid rate of progression.
• the use or method of using the biomarkers of the present invention may include comparing the level of the biomarker in a sample from a subject to the level of the biomarker in a sample from the subject at a different time (e.g., one day, one week, one month, one year ago, etc.).
• the reference level may be pre-determined (previously).
• the present invention relates to a composition for use in determining or diagnosing the rate of progression or likelihood of developing amyotrophic lateral sclerosis (ALS) and/or in selecting or predicting a treatment for ALS, the composition comprising a biomarker of the present invention.
• ALS amyotrophic lateral sclerosis
• the present invention relates to a method for treating amyotrophic lateral sclerosis (ALS), comprising administering to a patient a therapeutic agent for ALS selected from the above aspects.
• the therapeutic agent for ALS includes, but is not limited to, riluzole and edaravone.
• the therapeutic agent can be an antibody against a biomarker of the present invention, for example, an antibody against IL-17A or IL-17 receptor.
• ALS patients and healthy controls Thirty-six sporadic ALS patients and 10 healthy volunteers were screened at Tokushima University Hospital from March 29, 2021 to October 30, 2022.
• the inclusion criteria for ALS patients were patients who were diagnosed with definite, probable, laboratory-supported probable, or possible according to the updated Awaji criteria, or ALS according to the Gold Coast criteria, and who had been diagnosed with ALS within 2 years of onset.
• Patients and healthy controls were matched for age and sex.
• the exclusion criteria for ALS patients and healthy controls are shown in Table 1. Patients found to have known ALS pathogenic mutations, such as SOD1, were preliminarily excluded from the analysis because their immunological profile differs from that of sporadic ALS.
• Rapid ALS was defined as a decline in the revised ALS Functional Rating Scale (ALSFRS-R) of 1.0 points/month or more ( ⁇ ALSFRS-R/month ⁇ 1)
• non-rapid ALS was defined as a decline in the ALSFRS-R of less than 1.0 points/month ( ⁇ ALSFRS-R/month ⁇ 1). All clinical information was collected after written informed consent was obtained from the patients. All study plans were approved by the Tokushima University Hospital Life Science and Medical Research Ethics Committee No. 3682. The investigation was conducted in accordance with the principles of the Declaration of Helsinki. Table 1. Exclusion criteria
• PBMCs Peripheral blood samples were collected from healthy donors and ALS patients at Tokushima University Hospital.
• PBMCs were prepared by density centrifugation on Ficoll-Hypaque (Lymphoprep TM , Serumwerk Bernburg AG, Germany), washed with phosphate-buffered saline (PBS), and resuspended in X-VIVO TM medium (LONZA, Basel, Switzerland) containing 5% FBS.
• PBMCs were stored at ⁇ 80°C until library preparation in CELLBANKER 1 (Nihon Zenyaku Kogyo Co., Ltd., Fukushima, Japan).
• Serum samples were collected in 8.0 ml Insepack tubes (Sekisui, Tokyo, Japan), centrifuged at 3500 rpm for 10 min, aliquoted, and frozen at ⁇ 80°C within 20 min of collection.
• scRNA-seq Single-cell RNA sequencing
• Human PBMCs were thawed, washed, and resuspended in D-PBS/BSA. After filtration, cell count and viability were assessed.
• scRNA-seq was performed using 10x Genomics. Primary analysis was performed using Cell Ranger and secondary analysis was performed using Seurat. Data normalization, variable feature selection, and integration were performed. Principal component analysis and uniform manifold approximation and projection (UMAP) were used for dimensionality reduction and cell clustering. Cell types were identified based on marker expression profiles. Differentially expressed genes (DEGs) were identified using Wilcoxon rank sum test. Visualization was performed using ggplot2. Detailed methods are described below.
• PBMC preparation Cryopreserved human peripheral blood mononuclear cells (PBMCs) were quickly lysed in 37°C water and washed with 1x D-PBS (Mg/Ca-free) containing 1% BSA. Centrifugation conditions for cell recovery were 20°C, 250 xg, 10 min in a swing-out rotor. After three washes, cells were resuspended in an appropriate volume of D-PBS/1% BSA and run through a Flowmi Cell Strainer 40 ⁇ m to remove remaining large particles. Cell count and viability were examined using a hemocytometer with trypan blue staining.
• PBMCs peripheral blood mononuclear cells
• scRNA-seq was performed using the 10x Genomics platform.
• Single-cell suspensions (10,000 cells) were loaded onto GEM generation chips using a 10x Genomics Chromium controller, and DNA libraries were prepared using Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10x Genomics, Pleasanton, CA) according to the manufacturer's instructions.
• Quality control of the prepared libraries was performed using a 4200 TapeStation D1000 ScreenTape (Agilent, Santa Clara, CA) and Qubit dsDNA Assay (Thermo Fisher Scientific, Waltham, MA) prior to sequencing.
• Gene expression libraries were sequenced using a DNBSEQ-G400 platform (MGI Tech, Shenzhen, China) at a depth of 50,000 reads per cell.
• Variable features were selected by direct modeling of the intrinsic mean-variance relationship using the Seurat function "FindVariableFeatures”, and these features were used to select integration features using the Seurat function “SelectIntegrationFeatures”. Integration features were subjected to a linear transformation and used for principal component analysis (PCA) using the Seurat functions “ScaleData” and “RunPCA”, respectively. Using reciprocal PCA, the top 50 principal components were used for anchor identification. The anchors were then used to integrate the datasets using the function "IntegrateData" in Seurat.
• PCA principal component analysis
• the cells were divided into clusters by clustering the similarities between the cells using the Louvain algorithm and Seurat's function "FindClusters". Finally, the cell types in each cluster were identified by examining the cell type-specific marker expression profiles in each cluster. Differentially expressed genes (DEGs) in each cell type between groups were identified using the nonparametric Wilcoxon rank sum test with the Seurat function "FindMarkers”. Gene expression levels in each cluster were visualized in a violin plot using the Seurat function "VlnPlot”. Differences in the frequency of each cell type between groups were identified with Tukey's range test using the "TukeyHSD" function in the stats (R default) package. Correlation tests were performed using the "cor.test” function in the stats (R default) package. Scatter plots, box plots, and bar graph visualizations were performed in R using the ggplot2 package.
• scRNA-seq revealed cell type shifts toward Th17 vs. Tregs, mature CD8 T cells vs. naive CD8 T cells, and mature natural killer cells vs. naive natural killer cells in rapidly progressing ALS.
• Serum immunoproteomics revealed that Th17-associated proteins and CD8 T cell-associated proteins were significantly elevated in rapidly progressing ALS and correlated with the rate of disease progression.
• integrated analysis of scRNA-seq and immunoproteomics showed dynamic associations between specific immune cells and proteins, such as Th17 and IL-17A, and mature CD8 T cells and KLRD1.
• Th17 can promote immunity against many pathogens, but can also promote inflammatory pathology during infection and autoimmunity.
• a few studies using flow cytometry analysis of PBMCs have shown that ALS patients have higher frequencies of Th1 and Th17, and lower frequencies of Th2 and Treg, compared with healthy controls.
• previous studies have not revealed a relationship between Th17 and the progression rate of ALS.
• Th17 we show that an increase in Th17 in CD4 T cells is associated with rapid progression of ALS.
• the frequency of Treg alone did not show a significant correlation with the progression rate. Therefore, a shift toward Th17 compared to Treg, rather than a simple decrease in Treg, may be important for the rapid progression of ALS.
• Tregs The frequency of Tregs was significantly decreased in rapid versus non-rapid ALS, but did not differ between rapid ALS and controls. Also, as mentioned above, the frequency of Tregs did not correlate significantly with the rate of progression. These results are not consistent with the idea that the decrease in Tregs is primarily related to rapid progression. The results of clinical trials of ALS using Tregs have been reported. In rapid progression, IL-17C and IL-17F were elevated, and progression and cytokine levels were not suppressed by Treg administration. This suggests that Tregs have little effect on the rapid progression associated with the increase in the IL-17 cytokine family.
• KLRD1 levels showed the highest partial correlation coefficient with progression rate among 400 immune proteins and correlated with the frequency of mature CD8 T cells. This result is consistent with the increased frequency of mature CD8 T cells in rapid ALS.
• KLRD1 has been reported as one of the genes differentially expressed in activated CD8 T cells in the CSF of ALS patients versus controls.
• TFF2 levels also correlated with the frequency of mature CD8 T cells. Previous studies have shown that CD8 T cells are increased in ALS patients, and that a higher percentage of CD8 T cells is associated with a higher risk of ALS death, but the relationship between CD8 T cells and ALS progression was unclear.
• TEMRA autoreactive, clonally expanded, terminally differentiated effector memory
• NCF2 correlated with the frequency of classical monocytes.
• NCF2 is involved in the synthesis of superoxide in neutrophils and has been reported as one of the ferroptosis- and iron metabolism-related genes that are differentially expressed in ALS and controls.
• monocytes no association was shown between monocytes and the rate of progression, but it has been reported that the ratio of classical to non-classical monocytes is elevated in ALS patients.
• the present invention provides a biomarker for use in determining or diagnosing the rate of progression or likelihood of developing amyotrophic lateral sclerosis (ALS) and/or in selecting or predicting a therapeutic agent.
• ALS amyotrophic lateral sclerosis
• the present invention can be used in the medical field, etc.

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