Droplet single-cell RNA-sequencing (dscRNA-seq) has enabled rapid, massively parallel profiling of transcriptomes. However, assessing differential expression across multiple individuals has been hampered by inefficient sample processing and technical batch effects. Here we describe a computational tool, demuxlet, that harnesses natural genetic variation to determine the sample identity of each cell and detect droplets containing two cells. These capabilities enable multiplexed dscRNA-seq experiments in which cells from unrelated individuals are pooled and captured at higher throughput than in standard workflows. Using simulated data, we show that 50 SNPs per cell are sufficient to assign 97% of singlets and identify 92% of doublets in pools of up to 64 individuals. Given genotyping data for each of 8 pooled samples, demuxlet correctly recovers the sample identity of >99% of singlets and identifies doublets at rates consistent with previous estimates. We apply demuxlet to assess cell type-specific changes in gene expression in 8 pooled lupus patient samples treated with IFN-β and perform eQTL analysis on 23 pooled samples.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease. Knowledge of circulating immune cell types and states associated with SLE remains incomplete. We profiled more than 1.2 million peripheral blood mononuclear cells (162 cases, 99 controls) with multiplexed single-cell RNA sequencing (mux-seq). Cases exhibited elevated expression of type 1 interferon–stimulated genes (ISGs) in monocytes, reduction of naïve CD4 + T cells that correlated with monocyte ISG expression, and expansion of repertoire-restricted cytotoxic GZMH + CD8 + T cells. Cell type–specific expression features predicted case-control status and stratified patients into two molecular subtypes. We integrated dense genotyping data to map cell type–specific cis–expression quantitative trait loci and to link SLE-associated variants to cell type–specific expression. These results demonstrate mux-seq as a systematic approach to characterize cellular composition, identify transcriptional signatures, and annotate genetic variants associated with SLE.

SLE is a complex autoimmune disease that results from the interplay of genetics, epigenetics and environmental exposures. DNA methylation is an epigenetic mechanism that regulates gene expression and tissue differentiation. Among all the epigenetic modifications, DNA methylation perturbations have been the most widely studied in SLE. It mediates processes relevant to SLE, including lymphocyte development, X-chromosome inactivation and the suppression of endogenous retroviruses. The establishment of most DNA methylation marks occurs in utero; however, a small percentage of epigenetic marks are dynamic and can change throughout a person’s lifetime and in relation to exposures. In this review, we discuss the current understanding of the biology of DNA methylation and its regulators, the measurement and interpretation of methylation marks, the effects of genetics on DNA methylation and the role of environmental exposures with relevance to SLE. We also summarise research findings associated with SLE disease risk and heterogeneity. The robust finding of hypomethylation of interferon-responsive genes in patients with SLE and new associations beyond interferon-responsive genes such as cell-specific methylation abnormalities are described. We also discuss methylation changes associated with lupus nephritis, autoantibody status and disease activity. Lastly, we explore future research directions, emphasising the need for longitudinal studies, cell tissue and context-specific profiling, as well as integrative approaches. With new technologies, DNA methylation perturbations could be targeted and edited, offering novel therapeutic approaches.

Systemic lupus erythematous (SLE) is a heterogeneous autoimmune disease in which outcomes vary among different racial groups. Here, we aim to identify SLE subgroups within a multiethnic cohort using an unsupervised clustering approach based on the American College of Rheumatology (ACR) classification criteria. We identify three patient clusters that vary according to disease severity. Methylation association analysis identifies a set of 256 differentially methylated CpGs across clusters, including 101 CpGs in genes in the Type I Interferon pathway, and we validate these associations in an external cohort. A cis-methylation quantitative trait loci analysis identifies 744 significant CpG-SNP pairs. The methylation signature is enriched for ethnic-associated CpGs suggesting that genetic and non-genetic factors may drive outcomes and ethnic-associated methylation differences. Our computational approach highlights molecular differences associated with clusters rather than single outcome measures. This work demonstrates the utility of applying integrative methods to address clinical heterogeneity in multifactorial multi-ethnic disease settings.

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ObjectiveAfrican Americans, East Asians, and Hispanics with systemic lupus erythematous (SLE) are more likely to develop lupus nephritis (LN) than are SLE patients of European descent. The etiology of this difference is not clear, and this study was undertaken to investigate how genetic variants might explain this effect.MethodsIn this cross-sectional study, 1244 SLE patients from multiethnic case collections were genotyped for 817,810 single-nucleotide polymorphisms (SNPs) across the genome. Continental genetic ancestry was estimated utilizing the program ADMIXTURE. Gene-based testing and pathway analysis was performed within each ethnic group and meta-analyzed across ethnicities. We also performed candidate SNP association tests with SNPs previously established as risk alleles for SLE, LN, and chronic kidney disease (CKD). Association testing and logistic regression models were performed with LN as the outcome, adjusted for continental ancestries, sex, disease duration, and age.ResultsWe studied 255 North European, 263 South European, 238 Hispanic, 224 African American and 264 East Asian SLE patients, of whom 606 had LN (48.7%). In genome-wide gene-based and candidate SNP analyses, we found distinct genes, pathways and established risk SNPs associated with LN for each ethnic group. Gene-based analyses showed significant associations between variation in ZNF546 (p = 1.0E-06), TRIM15 (p = 1.0E-06), and TRIMI0 (p = 1.0E-06) and LN among South Europeans, and TTC34 (p = 8.0E-06) was significantly associated with LN among Hispanics. The SNP rs8091180 in NFATC1 was associated with LN (OR 1.43, p = 3.3E-04) in the candidate SNP meta-analysis with the highest OR among African-Americans (OR 2.17, p = 0.0035).ConclusionDistinct genetic factors are associated with the risk of LN in SLE patients of different ethnicities. CKD risk alleles may play a role in the development of LN in addition to SLE-associated risk variants. These findings may further explain the clinical heterogeneity of LN risk and response to therapy observed between different ethnic groups.

Objective. Health-related quality of life (HRQoL) is reduced in systemic lupus erythematosus (SLE), partly driven by comorbid depression. Among patients with SLE, the association between major depression and HRQoL, measured using the NIH's Patient-Reported Outcomes Measurement Information System (PROMIS), is not well characterized. The objective was to determine an association between major depression and HRQoL as measured by PROMIS. Methods. Cross-sectional data were obtained from the California Lupus Epidemiology Study, a cohort of adults in the San Francisco Bay Area with SLE. We studied the association between major depression (score ≥10 on the Patient Health Questionnaire 8 depression scale) and T scores (scaled to population mean ± SD of 50 ± 10) on 12 PROMIS domains representing physical, mental, and social health. Mean T scores in depressed and nondepressed individuals were compared using multiple linear regression models adjusting for age, sex, race/ethnicity, disease activity, damage, body mass index, and household income. Results. Mean age of the 326 participants was 45 years; ~89% were women, 29% White, 23% Hispanic, 10% African American, and 36% Asian. One-fourth met the criteria for major depression. In multivariable analyses, major depression was independently associated with worse T scores on all 12 PROMIS domains (P < 0.001); compared with those without major depression, depressed individuals scored >10 points (1 SD) worse on fatigue, sleep impairment, negative psychosocial impact of illness, satisfaction in discretionary social activities, and satisfaction in social roles. Conclusion. In individuals with SLE, major depression is associated with markedly worse PROMIS scores in physical, mental, and social domains. Diagnosing and treating depression may help improve HRQoL in individuals with SLE.

Objective Adverse childhood experiences (ACEs) are associated with poor adult health and immune dysregulation. The impact of ACEs on patients with autoimmune disease is unknown. The present study was undertaken to compare the prevalence of ACEs in patients with systemic lupus erythematosus (SLE) to a population‐based survey estimate and to investigate relationships between ACEs and SLE outcomes. Methods Data derived from the California Lupus Epidemiology Study (CLUES), a sample of adult patients with SLE. Participants completed a 10‐item ACE questionnaire covering 3 domains (abuse, neglect, household challenges). We estimated ACE prevalence in 269 CLUES participants compared to geographically matched respondents from the 2015 California Behavioral Risk Factor Surveillance System (BRFSS), which was standardized to CLUES participant characteristics (age, sex, race/ethnicity). We examined associations of patient‐reported and physician‐assessed health status measures with overall ACE levels and domains using multivariable linear regression, controlling for sociodemographics, nephritis, and juvenile‐onset SLE. Results Although specific domains varied, overall ACE levels were similar for CLUES and BRFSS respondents. Among SLE patients, 63.2% had ≥1 ACE, and 19.3% had ≥4. ACEs were more prevalent in those who were older, women, Latino or African American, and without college degrees, but not in those with lupus nephritis. In adjusted models, higher ACE levels and ACE domains were associated with worse patient‐reported SLE activity, depression, and health status but were not significantly associated with physician‐assessed SLE activity, damage, or severity. Conclusion Given the association between ACE levels and important patient‐reported outcomes in SLE, our study reinforces the need for prevention of ACEs in childhood and for clinical interventions to promote resilience among adults who have experienced ACEs.