Massive and parallel expression profiling using microarrayed single-cell sequencing

Single-cell transcriptome analysis overcomes problems inherently associated with averaging gene expression measurements in bulk analysis. However, single-cell analysis is currently challenging in terms of cost, throughput and robustness. Here, we present a method enabling massive microarray-based barcoding of expression patterns in single cells, termed MASC-seq. This technology enables both imaging and high-throughput single-cell analysis, characterizing thousands of single-cell transcriptomes per day at a low cost (0.13 USD/cell), which is two orders of magnitude less than commercially available systems. Our novel approach provides data in a rapid and simple way. Therefore, MASC-seq has the potential to accelerate the study of subtle clonal dynamics and help provide critical insights into disease development and other biological processes

High-throughput total RNA sequencing in single cells using VASA-seq.

Most methods for single-cell transcriptome sequencing amplify the termini of polyadenylated transcripts, capturing only a small fraction of the total cellular transcriptome. This precludes the detection of many long non-coding, short non-coding and non-polyadenylated protein-coding transcripts and hinders alternative splicing analysis. We, therefore, developed VASA-seq to detect the total transcriptome in single cells, which is enabled by fragmenting and tailing all RNA molecules subsequent to cell lysis. The method is compatible with both plate-based formats and droplet microfluidics. We applied VASA-seq to more than 30,000 single cells in the developing mouse embryo during gastrulation and early organogenesis. Analyzing the dynamics of the total single-cell transcriptome, we discovered cell type markers, many based on non-coding RNA, and performed in vivo cell cycle analysis via detection of non-polyadenylated histone genes. RNA velocity characterization was improved, accurately retracing blood maturation trajectories. Moreover, our VASA-seq data provide a comprehensive analysis of alternative splicing during mammalian development, which highlighted substantial rearrangements during blood development and heart morphogenesis

Serum neurofilament light chain for individual prognostication of disease activity in people with multiple sclerosis: a retrospective modelling and validation study.

BACKGROUND Serum neurofilament light chain (sNfL) is a biomarker of neuronal damage that is used not only to monitor disease activity and response to drugs and to prognosticate disease course in people with multiple sclerosis on the group level. The absence of representative reference values to correct for physiological age-dependent increases in sNfL has limited the diagnostic use of this biomarker at an individual level. We aimed to assess the applicability of sNfL for identification of people at risk for future disease activity by establishing a reference database to derive reference values corrected for age and body-mass index (BMI). Furthermore, we used the reference database to test the suitability of sNfL as an endpoint for group-level comparison of effectiveness across disease-modifying therapies. METHODS For derivation of a reference database of sNfL values, a control group was created, comprising participants with no evidence of CNS disease taking part in four cohort studies in Europe and North America. We modelled the distribution of sNfL concentrations in function of physiological age-related increase and BMI-dependent modulation, to derive percentile and Z score values from this reference database, via a generalised additive model for location, scale, and shape. We tested the reference database in participants with multiple sclerosis in the Swiss Multiple Sclerosis Cohort (SMSC). We compared the association of sNfL Z scores with clinical and MRI characteristics recorded longitudinally to ascertain their respective disease prognostic capacity. We validated these findings in an independent sample of individuals with multiple sclerosis who were followed up in the Swedish Multiple Sclerosis registry. FINDINGS We obtained 10 133 blood samples from 5390 people (median samples per patient 1 [IQR 1-2] in the control group). In the control group, sNfL concentrations rose exponentially with age and at a steeper increased rate after approximately 50 years of age. We obtained 7769 samples from 1313 people (median samples per person 6·0 [IQR 3·0-8·0]). In people with multiple sclerosis from the SMSC, sNfL percentiles and Z scores indicated a gradually increased risk for future acute (eg, relapse and lesion formation) and chronic (disability worsening) disease activity. A sNfL Z score above 1·5 was associated with an increased risk of future clinical or MRI disease activity in all people with multiple sclerosis (odds ratio 3·15, 95% CI 2·35-4·23; p<0·0001) and in people considered stable with no evidence of disease activity (2·66, 1·08-6·55; p=0·034). Increased Z scores outperformed absolute raw sNfL cutoff values for diagnostic accuracy. At the group level, the longitudinal course of sNfL Z score values in people with multiple sclerosis from the SMSC decreased to those seen in the control group with use of monoclonal antibodies (ie, alemtuzumab, natalizumab, ocrelizumab, and rituximab) and, to a lesser extent, oral therapies (ie, dimethyl fumarate, fingolimod, siponimod, and teriflunomide). However, longitudinal sNfL Z scores remained elevated with platform compounds (interferons and glatiramer acetate; p<0·0001 for the interaction term between treatment category and treatment duration). Results were fully supported in the validation cohort (n=4341) from the Swedish Multiple Sclerosis registry. INTERPRETATION The use of sNfL percentiles and Z scores allows for identification of individual people with multiple sclerosis at risk for a detrimental disease course and suboptimal therapy response beyond clinical and MRI measures, specifically in people with disease activity-free status. Additionally, sNfL might be used as an endpoint for comparing effectiveness across drug classes in pragmatic trials. FUNDING Swiss National Science Foundation, Progressive Multiple Sclerosis Alliance, Biogen, Celgene, Novartis, Roche

Serum Glial Fibrillary Acidic Protein Compared With Neurofilament Light Chain as a Biomarker for Disease Progression in Multiple Sclerosis.

IMPORTANCE There is a lack of validated biomarkers for disability progression independent of relapse activity (PIRA) in multiple sclerosis (MS). OBJECTIVE To determine how serum glial fibrillary acidic protein (sGFAP) and serum neurofilament light chain (sNfL) correlate with features of disease progression vs acute focal inflammation in MS and how they can prognosticate disease progression. DESIGN, SETTING, AND PARTICIPANTS Data were acquired in the longitudinal Swiss MS cohort (SMSC; a consortium of tertiary referral hospitals) from January 1, 2012, to October 20, 2022. The SMSC is a prospective, multicenter study performed in 8 centers in Switzerland. For this nested study, participants had to meet the following inclusion criteria: cohort 1, patients with MS and either stable or worsening disability and similar baseline Expanded Disability Status Scale scores with no relapses during the entire follow-up; and cohort 2, all SMSC study patients who had initiated and continued B-cell-depleting treatment (ie, ocrelizumab or rituximab). EXPOSURES Patients received standard immunotherapies or were untreated. MAIN OUTCOMES AND MEASURES In cohort 1, sGFAP and sNfL levels were measured longitudinally using Simoa assays. Healthy control samples served as the reference. In cohort 2, sGFAP and sNfL levels were determined cross-sectionally. RESULTS This study included a total of 355 patients (103 [29.0%] in cohort 1: median [IQR] age, 42.1 [33.2-47.6] years; 73 female patients [70.9%]; and 252 [71.0%] in cohort 2: median [IQR] age, 44.3 [33.3-54.7] years; 156 female patients [61.9%]) and 259 healthy controls with a median [IQR] age of 44.3 [36.3-52.3] years and 177 female individuals (68.3%). sGFAP levels in controls increased as a function of age (1.5% per year; P < .001), were inversely correlated with BMI (-1.1% per BMI unit; P = .01), and were 14.9% higher in women than in men (P = .004). In cohort 1, patients with worsening progressive MS showed 50.9% higher sGFAP levels compared with those with stable MS after additional sNfL adjustment, whereas the 25% increase of sNfL disappeared after additional sGFAP adjustment. Higher sGFAP at baseline was associated with accelerated gray matter brain volume loss (per doubling: 0.24% per year; P < .001) but not white matter loss. sGFAP levels remained unchanged during disease exacerbations vs remission phases. In cohort 2, median (IQR) sGFAP z scores were higher in patients developing future confirmed disability worsening compared with those with stable disability (1.94 [0.36-2.23] vs 0.71 [-0.13 to 1.73]; P = .002); this was not significant for sNfL. However, the combined elevation of z scores of both biomarkers resulted in a 4- to 5-fold increased risk of confirmed disability worsening (hazard ratio [HR], 4.09; 95% CI, 2.04-8.18; P < .001) and PIRA (HR, 4.71; 95% CI, 2.05-9.77; P < .001). CONCLUSIONS AND RELEVANCE Results of this cohort study suggest that sGFAP is a prognostic biomarker for future PIRA and revealed its complementary potential next to sNfL. sGFAP may serve as a useful biomarker for disease progression in MS in individual patient management and drug development

Serum Glial Fibrillary Acidic Protein Compared With Neurofilament Light Chain as a Biomarker for Disease Progression in Multiple Sclerosis

Importance: There is a lack of validated biomarkers for disability progression independent of relapse activity (PIRA) in multiple sclerosis (MS). Objective: To determine how serum glial fibrillary acidic protein (sGFAP) and serum neurofilament light chain (sNfL) correlate with features of disease progression vs acute focal inflammation in MS and how they can prognosticate disease progression. Design, Setting, and Participants: Data were acquired in the longitudinal Swiss MS cohort (SMSC; a consortium of tertiary referral hospitals) from January 1, 2012, to October 20, 2022. The SMSC is a prospective, multicenter study performed in 8 centers in Switzerland. For this nested study, participants had to meet the following inclusion criteria: cohort 1, patients with MS and either stable or worsening disability and similar baseline Expanded Disability Status Scale scores with no relapses during the entire follow-up; and cohort 2, all SMSC study patients who had initiated and continued B-cell-depleting treatment (ie, ocrelizumab or rituximab). Exposures: Patients received standard immunotherapies or were untreated. Main Outcomes and Measures: In cohort 1, sGFAP and sNfL levels were measured longitudinally using Simoa assays. Healthy control samples served as the reference. In cohort 2, sGFAP and sNfL levels were determined cross-sectionally. Results: This study included a total of 355 patients (103 [29.0%] in cohort 1: median [IQR] age, 42.1 [33.2-47.6] years; 73 female patients [70.9%]; and 252 [71.0%] in cohort 2: median [IQR] age, 44.3 [33.3-54.7] years; 156 female patients [61.9%]) and 259 healthy controls with a median [IQR] age of 44.3 [36.3-52.3] years and 177 female individuals (68.3%). sGFAP levels in controls increased as a function of age (1.5% per year; P <.001), were inversely correlated with BMI (-1.1% per BMI unit; P =.01), and were 14.9% higher in women than in men (P =.004). In cohort 1, patients with worsening progressive MS showed 50.9% higher sGFAP levels compared with those with stable MS after additional sNfL adjustment, whereas the 25% increase of sNfL disappeared after additional sGFAP adjustment. Higher sGFAP at baseline was associated with accelerated gray matter brain volume loss (per doubling: 0.24% per year; P <.001) but not white matter loss. sGFAP levels remained unchanged during disease exacerbations vs remission phases. In cohort 2, median (IQR) sGFAP z scores were higher in patients developing future confirmed disability worsening compared with those with stable disability (1.94 [0.36-2.23] vs 0.71 [-0.13 to 1.73]; P =.002); this was not significant for sNfL. However, the combined elevation of z scores of both biomarkers resulted in a 4- to 5-fold increased risk of confirmed disability worsening (hazard ratio [HR], 4.09; 95% CI, 2.04-8.18; P <.001) and PIRA (HR, 4.71; 95% CI, 2.05-9.77; P <.001). Conclusions and Relevance: Results of this cohort study suggest that sGFAP is a prognostic biomarker for future PIRA and revealed its complementary potential next to sNfL. sGFAP may serve as a useful biomarker for disease progression in MS in individual patient management and drug development.