Genome editing in human cells with targeted nucleases now enables diverse experimental and therapeutic genome engineering applications, but extension to primary human B cells remains limited. Here we report a method for targeted genetic engineering in primary human B cells, utilizing electroporation of CRISPR-Cas9 ribonucleoproteins (RNPs) to introduce gene knockout mutations at protein-coding loci with high efficiencies that in some cases exceeded 80%. Further, we demonstrate knock-in editing of targeted nucleotides with efficiency exceeding 10% through co-delivery of oligonucleotide templates for homology directed repair. We delivered Cas9 RNPs in two distinct in vitro culture systems to achieve editing in both undifferentiated B cells and activated B cells undergoing differentiation, reflecting utility in diverse experimental conditions. In summary, we demonstrate a powerful and scalable research tool for functional genetic studies of human B cell biology that may have further applications in engineered B cell therapeutics.

Both a lack of biomarkers and relatively ineffective treatments constitute impediments to management of lupus nephritis (LN). Here we used gene expression microarrays to contrast the transcriptomic profiles of active SLE patients with and without LN to identify potential biomarkers for this condition. RNA isolated from whole peripheral blood of active SLE patients was used for transcriptomic profiling and the data analyzed by linear modeling, with corrections for multiple testing. Results were validated in a second cohort of SLE patients, using NanoString technology. The majority of genes demonstrating altered transcript abundance between patients with and without LN were neutrophil-related. Findings in the validation cohort confirmed this observation and showed that levels of RNA abundance in renal remission were similar to active patients without LN. In secondary analyses, RNA abundance correlated with disease activity, hematuria and proteinuria, but not renal biopsy changes. As abundance levels of the individual transcripts correlated strongly with each other, a composite neutrophil score was generated by summing all levels before examining additional correlations. There was a modest correlation between the neutrophil score and the blood neutrophil count, which was largely driven by the dose of glucocorticosteroids and not the proportion of low density and/or activated neutrophils. Analysis of longitudinal data revealed no correlation between baseline neutrophil score or changes over the first year of follow-up with subsequent renal flare or treatment outcomes, respectively. The findings argue that although the neutrophil score is associated with LN, its clinical utility as a biomarker may be limited.

Objective

We previously demonstrated the utility of the Automated Neuropsychological Assessment Metrics (ANAM) for screening cognitive impairment (CI) in patients with systemic lupus erythematosus (SLE) and developed composite indices for interpreting ANAM results. Our objectives here were to provide further support for the ANAM's concurrent criterion validity against the American College of Rheumatology neuropsychological battery (ACR-NB), identify the most discriminatory subtests and scores of the ANAM for predicting CI, and provide a new approach to interpret ANAM results using Classification and Regression Tree (CART) analysis.

Methods

300 adult SLE patients completed an adapted ACR-NB and ANAM on the same day. As per objectives, six models were built using combinations of ANAM subtests and scores and submitted to CART analysis. Area under the curve (AUC) was calculated to evaluate the ANAM's criterion validity compared to the adapted ACR-NB; the most discriminatory ANAM subtests and scores in each model were selected, and performance of models with the highest AUCs were compared to our previous composite indices; decision trees were generated for models with the highest AUCs.

Results

Two models had excellent AUCs of 86 and 89%. Eight most discriminatory ANAM subtests and scores were identified. Both models demonstrated higher AUCs against our previous composite indices. An adapted decision tree was created to simplify the interpretation of ANAM results.

Conclusion

We provide further validity evidence for the ANAM as a valid CI screening tool in SLE. The decision tree improves interpretation of ANAM results, enhancing clinical utility.

Systemic lupus erythematosus (SLE) is an autoimmune disease with marked gender and ethnic disparities. We report a large transancestral association study of SLE using Immunochip genotype data from 27,574 individuals of European (EA), African (AA) and Hispanic Amerindian (HA) ancestry. We identify 58 distinct non-HLA regions in EA, 9 in AA and 16 in HA (∼50% of these regions have multiple independent associations); these include 24 novel SLE regions (P<5 × 10^-8), refined association signals in established regions, extended associations to additional ancestries, and a disentangled complex HLA multigenic effect. The risk allele count (genetic load) exhibits an accelerating pattern of SLE risk, leading us to posit a cumulative hit hypothesis for autoimmune disease. Comparing results across the three ancestries identifies both ancestry-dependent and ancestry-independent contributions to SLE risk. Our results are consistent with the unique and complex histories of the populations sampled, and collectively help clarify the genetic architecture and ethnic disparities in SLE.