Single-cell resolution of longitudinal blood transcriptome profiles in rheumatoid arthritis, systemic lupus erythematosus and healthy control pregnancies.

OBJECTIVES: Comparative longitudinal analyses of cellular composition and peripheral blood gene expression in Rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and healthy pregnancies. METHODS: In total, 335 whole blood samples from 84 RA, SLE and healthy controls before pregnancy, at each trimester, 6 weeks, 6 months and 12 months post partum were analysed. We combined bulk and single cell RNA analyses for cell-type estimation, validated by flow cytometry, before combining this in a cell-type adjusted analysis for an improved resolution of unrecognised gene expression changes associated with RA and SLE pregnancies. RESULTS: Patients were well regulated throughout pregnancy, and few had pregnancy complications. In SLE, the interferon signature was augmented during pregnancy, and the pregnancy signature was continued post partum. An altered cell type composition strongly influences the profile. In the pregnancy signature, transcripts involved in galactosylation potentially altering the effector functions of autoantibodies became more evident. Several genes in the adjusted RA signature are expressed in mucosal associated invariant T cells. CONCLUSION: We found distinct RA, SLE and pregnancy signatures, and no expression patterns could be attributed to medication or disease activity. Our results support the need for close postpartum follow-up of patients with SLE. Gene expression patterns in RA were closer to healthy controls than to SLE, and primarily became evident after cell-type adjustment. Adjusting for cell abundance unravelled gene expression signatures less associated with variation in cell-composition and highlighted genes with expression profiles associated with changes in specialised cell populations.

Low serum lipocalin-2 in pregnant women with systemic lupus erythematosus.

OBJECTIVES: Systemic lupus erythematosus (SLE) pregnancies are considered high-risk due to risk of disease flare and pregnancy complications. A more in-depth understanding of the immunological alterations in SLE patients during pregnancy and identification of predictive biomarkers may help to achieve stable disease and to avoid pregnancy complications. Lipocalin-2 (LCN2) has been implicated as a potential biomarker for rheumatic diseases and preeclampsia, but remains unexplored in SLE pregnancies. METHODS: We measured LCN2 levels in serum samples from SLE pregnancies (n=25) at seven different time points. Samples were taken preconception, in each trimester, at 6 weeks, 6 months and 12 months postpartum. Serum LCN2 levels were compared to samples from rheumatoid arthritis (RA) (n=27) and healthy (n=18) pregnancies at each time point using t-test, and for all time points using a linear mixed effects model. In addition, we investigated the association between LCN2 levels and disease activity, CRP, kidney function, BMI, treatment regimen and adverse pregnancy outcome for SLE and RA patients. RESULTS: We found significantly lower serum LCN2 levels throughout pregnancy in SLE patients with quiescent disease compared to RA and healthy pregnancies. We did not find an association between serum LCN2 and disease activity or adverse pregnancy outcome in SLE pregnancies. CONCLUSIONS: In a population of SLE women with low disease activity we have not found evidence that serum LCN2 levels predict disease activity or adverse pregnancy outcomes. Further studies are needed to elucidate a possible biological role of low LCN2 levels in SLE pregnancies.

Epitope-resolved profiling of the SARS-CoV-2 antibody response identifies cross-reactivity with endemic human coronaviruses.

The SARS-CoV-2 proteome shares regions of conservation with endemic human coronaviruses (CoVs), but it remains unknown to what extent these may be cross-recognized by the antibody response. Here, we study cross-reactivity using a highly multiplexed peptide assay (PepSeq) to generate an epitope-resolved view of IgG reactivity across all human CoVs in both COVID-19 convalescent and negative donors. PepSeq resolves epitopes across the SARS-CoV-2 Spike and Nucleocapsid proteins that are commonly targeted in convalescent donors, including several sites also recognized in some uninfected controls. By comparing patterns of homologous reactivity between CoVs and using targeted antibody-depletion experiments, we demonstrate that SARS-CoV-2 elicits antibodies that cross-recognize pandemic and endemic CoV antigens at two Spike S2 subunit epitopes. We further show that these cross-reactive antibodies preferentially bind endemic homologs. Our findings highlight sites at which the SARS-CoV-2 response appears to be shaped by previous CoV exposures and which have the potential to raise broadly neutralizing responses.

Epitope-resolved profiling of the SARS-CoV-2 antibody response identifies cross-reactivity with an endemic human CoV.

A high-resolution understanding of the antibody response to SARS-CoV-2 is important for the design of effective diagnostics, vaccines and therapeutics. However, SARS-CoV-2 antibody epitopes remain largely uncharacterized, and it is unknown whether and how the response may cross-react with related viruses. Here, we use a multiplexed peptide assay ('PepSeq') to generate an epitope-resolved view of reactivity across all human coronaviruses. PepSeq accurately detects SARS-CoV-2 exposure and resolves epitopes across the Spike and Nucleocapsid proteins. Two of these represent recurrent reactivities to conserved, functionally-important sites in the Spike S2 subunit, regions that we show are also targeted for the endemic coronaviruses in pre-pandemic controls. At one of these sites, we demonstrate that the SARS-CoV-2 response strongly and recurrently cross-reacts with the endemic virus hCoV-OC43. Our analyses reveal new diagnostic and therapeutic targets, including a site at which SARS-CoV-2 may recruit common pre-existing antibodies and with the potential for broadly-neutralizing responses.

Phosphatase of regenerating liver-3 is expressed in acute lymphoblastic leukemia and mediates leukemic cell adhesion, migration and drug resistance.

Phosphatase of regenerating liver-3 (PRL-3/PTP4A3) is upregulated in multiple cancers, including BCR-ABL1- and ETV6-RUNX-positive acute lymphoblastic leukemia (ALL). With this study, we aim to characterize the biological role of PRL-3 in B cell ALL (B-ALL). Here, we demonstrate that PRL-3 expression at mRNA and protein level was higher in B-ALL cells than in normal cells, as measured by qRT-PCR or flow cytometry. Further, we demonstrate that inhibition of PRL-3 using shRNA or a small molecular inhibitor reduced cell migration towards an SDF-1α gradient in the preB-ALL cell lines Reh and MHH-CALL-4. Knockdown of PRL-3 also reduced cell adhesion towards fibronectin in Reh cells. Mechanistically, PRL-3 mediated SDF-1α stimulated calcium release, and activated focal adhesion kinase (FAK) and Src, important effectors of migration and adhesion. Finally, PRL-3 expression made Reh cells more resistance to cytarabine treatment. In conclusion, the expression level of PRL-3 was higher in B-ALL cells than in normal cells. PRL-3 promoted adhesion, migration and resistance to cytarabine. PRL-3 may represent a novel target in the treatment of B-ALL.

Validity of the diagnosis of pre-eclampsia in the Medical Birth Registry of Norway.

OBJECTIVE: Evaluating the validity of pre-eclampsia registration in the Medical Birth Registry of Norway (MBRN) according to both broader and restricted disease definitions. DESIGN: Retrospective nested cohort study. SETTING: Multicenter study. POPULATION: In this study, two cohorts of women with pre-eclamptic pregnancies registered in the MBRN were selected. Study group 1 contained 966 pregnancies from 1967 to 2002. Concomitant participation in the Nord-Trøndelag Health Study 2 was required. Study group 2 comprised 1138 pregnancies recorded in 1967-2005, examined as a pre-eclampsia biobank was established. METHODS: Diagnostic criteria vary. The broader criteria for pre-eclampsia, used by the MBRN, are one measurement of hypertension and proteinuria (Criterion A). Criteria used internationally today require two measurements of hypertension and proteinuria (Criterion B). The diagnostic validities in Study groups 1 and 2 were judged against medical records according to Criterion A and B, respectively. MAIN OUTCOME MEASURES: Positive predictive value (PPV) and trend analyses. RESULTS: The diagnosis was confirmed in 88.3% of pregnancies in Study group 1, and in 63.6% in Study group 2. PPV was high for Study group 1 throughout the period. For Study group 2, results improved significantly after 1986. CONCLUSIONS: This study ascertains high PPV of pre-eclampsia in the MBRN using broader traditional criteria, although the PPV decreases through assessment using restricted modern criteria. This illustrates how inclusion of direct measurements may improve registration of complex disorders defined by changing diagnostic criteria.

Genetic dissection of the pre-eclampsia susceptibility locus on chromosome 2q22 reveals shared novel risk factors for cardiovascular disease.

Pre-eclampsia is an idiopathic pregnancy disorder promoting morbidity and mortality to both mother and child. Delivery of the fetus is the only means to resolve severe symptoms. Women with pre-eclamptic pregnancies demonstrate increased risk for later life cardiovascular disease (CVD) and good evidence suggests these two syndromes share several risk factors and pathophysiological mechanisms. To elucidate the genetic architecture of pre-eclampsia we have dissected our chromosome 2q22 susceptibility locus in an extended Australian and New Zealand familial cohort. Positional candidate genes were prioritized for exon-centric sequencing using bioinformatics, SNPing, transcriptional profiling and QTL-walking. In total, we interrogated 1598 variants from 52 genes. Four independent SNP associations satisfied our gene-centric multiple testing correction criteria: a missense LCT SNP (rs2322659, P = 0.0027), a synonymous LRP1B SNP (rs35821928, P = 0.0001), an UTR-3 RND3 SNP (rs115015150, P = 0.0024) and a missense GCA SNP (rs17783344, P = 0.0020). We replicated the LCT SNP association (P = 0.02) and observed a borderline association for the GCA SNP (P = 0.07) in an independent Australian case-control population. The LRP1B and RND3 SNP associations were not replicated in this same Australian singleton cohort. Moreover, these four SNP associations could not be replicated in two additional case-control populations from Norway and Finland. These four SNPs, however, exhibit pleiotropic effects with several quantitative CVD-related traits. Our results underscore the genetic complexity of pre-eclampsia and present novel empirical evidence of possible shared genetic mechanisms underlying both pre-eclampsia and other CVD-related risk factors.

OP006. A preeclampsia genome-wide linkage scan in norwegian families.

INTRODUCTION: Several maternal susceptibility loci for preeclampsia (PE) have been discovered amongst Icelandic, Australian/New Zealand, Dutch and Finnish family cohorts, implicating locus heterogeneity. Through candidate gene studies, allele-specific heterogeneity in different populations is also evident. It is therefore likely that numerous population specific PE susceptibility variants exist, differing in their effect size. Despite on-going efforts to identify susceptibility genes for PE, the causal genetic variants still remain obscure. OBJECTIVES: The aim of this study is to interrogate the genetic architecture of PE susceptibility by performing a genome-wide linkage scan in a novel familial cohort from Norway. METHODS: A total of 480 DNA samples from The Norwegian PE Family Biobank were genotyped at Genomic Core Facility at NTNU. Genome-wide genotyping was performed with the Infinium HumanExome BeadChip (>240,000 markers) (Illumina, USA) that delivers focused coverage of exonic regions of the human genome. RESULTS: A total of 137 families are represented with 222 women with a valid PE diagnosis (SBP⩾140mmHg DBP⩾90mmHg, ⩾2 measurements at least 4h apart with documented proteinuria at ⩾2 occasions occurring after 20weeks of pregnancy), 44 with self-reported PE and 72 women with a healthy pregnancy. The genotyping has just recently been completed with an average call rate of 99.96%. Data and statistical analysis is now underway using MERLIN, R and SOLAR. A description of the Norwegian PE familial cohort plus preliminary results will be presented at the Congress. CONCLUSION: To our knowledge this is the first SNP-based genome-wide linkage study on PE, and the first performed in a novel Norwegian PE family cohort. By using an approach focusing on functionally relevant markers we anticipate the identification of susceptibility loci that are of substantial importance for disease development.

Identification of ACOX2 as a shared genetic risk factor for preeclampsia and cardiovascular disease.

Preeclampsia (PE) is a serious complication of pregnancy, which is highly correlated with later life cardiovascular disease (CVD). Many risk factors are common for both diseases, but the contribution of shared genes remains to be determined. In this study, we used an integrative strategy to assess lipid traits as risk factors for PE and CVD by whole genome transcriptional profiling performed on Norwegian decidua basalis tissues (N = 95) from preeclamptic and normal pregnancies and on blood lymphocytes (N = 1240) from the San Antonio Family Heart Study (SAFHS). Among 222 genes that were differentially expressed (false discovery rate (FDR) P-value <0.05) between the PE, cases and controls, we found one gene, ACOX2 (acyl-coenzyme A oxidase 2, branched chain), that was downregulated in PE whose transcription was also inversely correlated with triglyceride levels (P = 5.6 × 10(-7); FDR P-value = 0.0002) in SAFHS. We further report associations between SNPs in the ACOX2 gene and the transcription level (P-value = 0.0045) of the gene, as well as with triglyceride levels (P-value = 0.0051). ACOX2 is involved in bile acid production, a process that has been associated with both oxidative stress and regulation of triglyceride levels. Oxidative stress and increased triglyceride levels are known risk factors for CVD and both have also been associated with PE. Our results suggest that downregulation of ACOX2 is a shared risk factor for PE and CVD.

Partial correlation network analyses to detect altered gene interactions in human disease: using preeclampsia as a model.

Differences in gene expression between cases and controls have been identified for a number of human diseases. However, the underlying mechanisms of transcriptional regulation remain largely unknown. Beyond comparisons of absolute or relative expression levels, disease states may be associated with alterations in the observed correlational patterns among sets of genes. Here we use partial correlation networks aiming to compare the transcriptional co-regulation for 222 genes that are differentially expressed in decidual tissues between preeclampsia (PE) cases and non-PE controls. Partial correlation coefficients (PCCs) have been calculated in cases (N = 37) and controls (N = 58) separately. For all PCCs, we tested if they were significant non-zero in the cases and controls separately. In addition, to examine if a given PCC is different between the cases and controls, we tested if the difference between two PCCs were significant non-zero. In the group with PE cases, only five PCCs were significant (FDR p value ≤ 0.05), of which none were significantly different from the PCCs in the controls. However, in the controls we identified a total of 56 statistically significant PCCs (FDR p value ≤ 0.05), of which 31 were also significantly different (FDR p value ≤ 0.05) from the PCCs in the PE cases. The identified partial correlation networks included genes that are potentially relevant for developing PE, including both known susceptibility genes (EGFL7, HES1) and novel candidate genes (CFH, NADSYN1, DBP, FIGLA). Our results might suggest that disturbed interactions, or higher order relationships between these genes play an important role in developing the disease.

A transcriptional profile of the decidua in preeclampsia.

OBJECTIVE: We sought to obtain insight into possible mechanisms underlying preeclampsia using genomewide transcriptional profiling in decidua basalis. STUDY DESIGN: Genomewide transcriptional profiling was performed on decidua basalis tissue from preeclamptic (n = 37) and normal (n = 58) pregnancies. Differentially expressed genes were identified and merged into canonical pathways and networks. RESULTS: Of the 26,504 expressed transcripts detected, 455 were differentially expressed (P < .05; false discovery rate, P < .1). Both novel (ARL5B, SLITRK4) and previously reported preeclampsia-associated (PLA2G7, HMOX1) genes were identified. Pathway analysis revealed that tryptophan metabolism, endoplasmic reticulum stress, linoleic acid metabolism, notch signaling, fatty acid metabolism, arachidonic acid metabolism, and NRF2-mediated oxidative stress response were overrepresented canonical pathways. CONCLUSION: In the present study single genes, canonical pathways, and gene-gene networks that are likely to play an important role in the pathogenesis of preeclampsia have been identified. Future functional studies are needed to accomplish a greater understanding of the mechanisms involved.

Genetic and molecular functional characterization of variants within TNFSF13B, a positional candidate preeclampsia susceptibility gene on 13q.

BACKGROUND: Preeclampsia is a serious pregnancy complication, demonstrating a complex pattern of inheritance. The elucidation of genetic liability to preeclampsia remains a major challenge in obstetric medicine. We have adopted a positional cloning approach to identify maternal genetic components, with linkages previously demonstrated to chromosomes 2q, 5q and 13q in an Australian/New Zealand familial cohort. The current study aimed to identify potential functional and structural variants in the positional candidate gene TNFSF13B under the 13q linkage peak and assess their association status with maternal preeclampsia genetic susceptibility. METHODOLOGY/PRINCIPAL FINDINGS: The proximal promoter and coding regions of the positional candidate gene TNFSF13B residing within the 13q linkage region was sequenced using 48 proband or founder individuals from Australian/New Zealand families. Ten sequence variants (nine SNPs and one single base insertion) were identified and seven SNPs were successfully genotyped in the total Australian/New Zealand family cohort (74 families/480 individuals). Borderline association to preeclampsia (p = 0.0153) was observed for three rare SNPs (rs16972194, rs16972197 and rs56124946) in strong linkage disequilibrium with each other. Functional evaluation by electrophoretic mobility shift assays showed differential nuclear factor binding to the minor allele of the rs16972194 SNP, residing upstream of the translation start site, making this a putative functional variant. The observed genetic associations were not replicated in a Norwegian case/control cohort (The Nord-Trøndelag Health Study (HUNT2), 851 preeclamptic and 1,440 non-preeclamptic women). CONCLUSION/SIGNIFICANCE: TNFSF13B has previously been suggested to contribute to the normal immunological adaption crucial for a successful pregnancy. Our observations support TNFSF13B as a potential novel preeclampsia susceptibility gene. We discuss a possible role for TNFSF13B in preeclampsia pathogenesis, and propose the rs16972194 variant as a candidate for further functional evaluation.