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Timothy Vyse and colleagues report the results of a large-scale association study of systemic lupus erythematosus (SLE). They identify ten new susceptibility loci and implicate aberrant regulation of innate and adaptive immunity genes in disease pathogenesis. Systemic lupus erythematosus (SLE) is a genetically complex autoimmune disease characterized by loss of immune tolerance to nuclear and cell surface antigens. Previous genome-wide association studies (GWAS) had modest sample sizes, reducing their scope and reliability. Our study comprised 7,219 cases and 15,991 controls of European ancestry, constituting a new GWAS, a meta-analysis with a published GWAS and a replication study. We have mapped 43 susceptibility loci, including ten new associations. Assisted by dense genome coverage, imputation provided evidence for missense variants underpinning associations in eight genes. Other likely causal genes were established by examining associated alleles for cis -acting eQTL effects in a range of ex vivo immune cells. We found an over-representation ( n = 16) of transcription factors among SLE susceptibility genes. This finding supports the view that aberrantly regulated gene expression networks in multiple cell types in both the innate and adaptive immune response contribute to the risk of developing SLE.

Key Points There are intense continuing efforts to increase the throughput and accuracy, and reduce the costs, of methods for genotyping single nucleotide polymorphisms (SNPs). This is driven by the hope that SNPs can act as markers for identifying the genes that underlie multifactorial disorders. PCR, invented in the 1980s, allows the sensitivity and specificity required for genotyping SNPs in large diploid genomes. The PCR step is the principal limiting factor in the throughput of current SNP-genotyping assays. The large number of different SNP assays are based on a small number of reaction principles that have been combined with solid-phase or solution-based assay formats. Fluorescence is the most frequently used detection method. Allele-specific oligonucleotides as probes or PCR primers are used to achieve high throughput in homogeneous solution phase assays that are monitored in real time during PCR. Alternatively, large numbers of oligonucleotide probes are immobilized at high density on microarrays to allow parallel analysis of many SNPs. The most promising methods for accurate genotyping of SNPs involve nucleic-acid-modifying enzymes as genotyping tools. Frequently used enzymes are DNA polymerases, ligases and endonucleases. Assays based on primer extension catalysed by a DNA polymerase are robust and have been adapted to various assay formats and detection strategies. These include colorimetric detection in microtitre plates, fluorescence detection using DNA sequencers, mass spectrometric detection and microarray-based assays with fluorescence detection. Assays based on DNA ligation, or cleavage by FLAP endonucleases, have led to the development of SNP-genotyping methods in which a PCR amplification step is avoided. Instead, an enzymatic signal amplification scheme is used to obtain sufficient sensitivity. Future SNP assays could be based on PCR carried out in microcapillaries streamlined with one of the enzymatic detection principles, and the assays could be multiplexed by combinatorial fluorescent labels. Understanding the relationship between genetic variation and biological function on a genomic scale is expected to provide fundamental new insights into the biology, evolution and pathophysiology of humans and other species. The hope that single nucleotide polymorphisms (SNPs) will allow genes that underlie complex disease to be identified, together with progress in identifying large sets of SNPs, are the driving forces behind intense efforts to establish the technology for large-scale analysis of SNPs. New genotyping methods that are high throughput, accurate and cheap are urgently needed for gaining full access to the abundant genetic variation of organisms.

BackgroundThe aim of this work was to identify new genetic causes of Rett-like phenotypes using array comparative genomic hybridisation and a whole exome sequencing approach.Methods and resultsWe studied a cohort of 19 Portuguese patients (16 girls, 3 boys) with a clinical presentation significantly overlapping Rett syndrome (RTT). Genetic analysis included filtering of the single nucleotide variants and indels with preference for de novo, homozygous/compound heterozygous, or maternally inherited X linked variants. Examination by MRI and muscle biopsies was also performed. Pathogenic genomic imbalances were found in two patients (10.5%): an 18q21.2 deletion encompassing four exons of the TCF4 gene and a mosaic UPD of chromosome 3. Variants in genes previously implicated in neurodevelopmental disorders (NDD) were identified in six patients (32%): de novo variants in EEF1A2, STXBP1 and ZNF238 were found in three patients, maternally inherited X linked variants in SLC35A2, ZFX and SHROOM4 were detected in two male patients and one homozygous variant in EIF2B2 was detected in one patient. Variants were also detected in five novel NDD candidate genes (26%): we identified de novo variants in the RHOBTB2, SMARCA1 and GABBR2 genes; a homozygous variant in EIF4G1; compound heterozygous variant in HTT.ConclusionsNetwork analysis reveals that these genes interact by means of protein interactions with each other and with the known RTT genes. These findings expand the phenotypical spectrum of previously known NDD genes to encompass RTT-like clinical presentations and identify new candidate genes for RTT-like phenotypes.

In the 1980s, my research career begun with microbial DNA diagnostics at Orion Pharmaceutica in Helsinki, Finland, where I was part of an innovative team that developed novel methods based on the polymerase chain reaction (PCR) and the biotin–avidin interaction. One of our key achievements during this time was the invention of the solid-phase minisequencing method for genotyping single nucleotide polymorphisms (SNPs). In the 1990s, I shifted focus to human genetics, investigating mutations of the ‘Finnish disease heritage’. During this period, I also developed quantitative methods using PCR and minisequencing of mitochondrial mutations and for forensic analyses. In the late 1990s and early 2000s, microarray-based SNP genotyping became a major topic for my research, first in Helsinki and later with my research group at Uppsala University in Sweden. By the mid-2000s, I began collaborating with leading clinicians on genetics of autoimmune disease, specifically systemic lupus erythematosus and later worked on the classification and clinical outcome of pediatric acute lymphoblastic leukemia, when large-scale genomics and epigenomics emerged. These collaborations, which focused on integrating genomics into clinical practice, lasted almost two decades until I retired from research in 2022. In parallel with my research activities, I led the SNP/DNA Technology Platform in the Wallenberg Consortium North program from 2001 to 2006. I continued as Director of the SNP&SEQ Technology Platform, which expanded rapidly during the 2010s, and became part of Science for Life Laboratory in 2013. Today (in 2024), the SNP&SEQ Technology Platform is one of the largest units of the Swedish National Genomics Infrastructure hosted by SciLifeLab. The present article provides a personal perspective on nearly four decades of research, highlighting projects and methods I found particularly exciting or important.

To perform a cross-comparative analysis of DNA methylation in patients with systemic lupus erythematosus (SLE), patients with primary Sjögren's syndrome (pSS), and healthy controls addressing the question of epigenetic sharing and aiming to detect disease-specific alterations. DNA extracted from peripheral blood from 347 cases with SLE, 100 cases with pSS, and 400 healthy controls were analyzed on the Human Methylation 450k array, targeting 485,000 CpG sites across the genome. A linear regression model including age, sex, and blood cell type distribution as covariates was fitted, and association -values were Bonferroni corrected. A random forest machine learning classifier was designed for prediction of disease status based on DNA methylation data. We established a combined set of 4,945 shared differentially methylated CpG sites (DMCs) in SLE and pSS compared to controls. In pSS, hypomethylation at type I interferon induced genes was mainly driven by patients who were positive for Ro/SSA and/or La/SSB autoantibodies. Analysis of differential methylation between SLE and pSS identified 2,244 DMCs with a majority of sites showing decreased methylation in SLE compared to pSS. The random forest classifier demonstrated good performance in discerning between disease status with an area under the curve (AUC) between 0.83 and 0.96. The majority of differential DNA methylation is shared between SLE and pSS, however, important quantitative differences exist. Our data highlight neutrophil dysregulation as a shared mechanism, emphasizing the role of neutrophils in the pathogenesis of systemic autoimmune diseases. The current study provides evidence for genes and molecular pathways driving common and disease-specific pathogenic mechanisms.

Here we describe the SweGen data set, a comprehensive map of genetic variation in the Swedish population. These data represent a basic resource for clinical genetics laboratories as well as for sequencing-based association studies by providing information on genetic variant frequencies in a cohort that is well matched to national patient cohorts. To select samples for this study, we first examined the genetic structure of the Swedish population using high-density SNP-array data from a nation-wide cohort of over 10 000 Swedish-born individuals included in the Swedish Twin Registry. A total of 1000 individuals, reflecting a cross-section of the population and capturing the main genetic structure, were selected for whole-genome sequencing. Analysis pipelines were developed for automated alignment, variant calling and quality control of the sequencing data. This resulted in a genome-wide collection of aggregated variant frequencies in the Swedish population that we have made available to the scientific community through the website https://swefreq.nbis.se. A total of 29.2 million single-nucleotide variants and 3.8 million indels were detected in the 1000 samples, with 9.9 million of these variants not present in current databases. Each sample contributed with an average of 7199 individual-specific variants. In addition, an average of 8645 larger structural variants (SVs) were detected per individual, and we demonstrate that the population frequencies of these SVs can be used for efficient filtering analyses. Finally, our results show that the genetic diversity within Sweden is substantial compared with the diversity among continental European populations, underscoring the relevance of establishing a local reference data set.

ObjectivesSystemic lupus erythematosus (SLE) is an autoimmune disease with extensive heterogeneity in disease presentation between patients, which is likely due to an underlying molecular diversity. Here, we aimed at elucidating the genetic aetiology of SLE from the immunity pathway level to the single variant level, and stratify patients with SLE into distinguishable molecular subgroups, which could inform treatment choices in SLE.MethodsWe undertook a pathway-centred approach, using sequencing of immunological pathway genes. Altogether 1832 candidate genes were analysed in 958 Swedish patients with SLE and 1026 healthy individuals. Aggregate and single variant association testing was performed, and we generated pathway polygenic risk scores (PRS).ResultsWe identified two main independent pathways involved in SLE susceptibility: T lymphocyte differentiation and innate immunity, characterised by HLA and interferon, respectively. Pathway PRS defined pathways in individual patients, who on average were positive for seven pathways. We found that SLE organ damage was more pronounced in patients positive for the T or B cell receptor signalling pathways. Further, pathway PRS-based clustering allowed stratification of patients into four groups with different risk score profiles. Studying sets of genes with priors for involvement in SLE, we observed an aggregate common variant contribution to SLE at genes previously reported for monogenic SLE as well as at interferonopathy genes.ConclusionsOur results show that pathway risk scores have the potential to stratify patients with SLE beyond clinical manifestations into molecular subsets, which may have implications for clinical follow-up and therapy selection.

ObjectivesGenetic variations in TNFAIP3 (A20) de-ubiquitinase (DUB) domain increase the risk of systemic lupus erythematosus (SLE) and rheumatoid arthritis. A20 is a negative regulator of NF-κB but the role of its DUB domain and related genetic variants remain unclear. We aimed to study the functional effects of A20 DUB-domain alterations in immune cells and understand its link to SLE pathogenesis.MethodsCRISPR/Cas9 was used to generate human U937 monocytes with A20 DUB-inactivating C103A knock-in (KI) mutation. Whole genome RNA-sequencing was used to identify differentially expressed genes between WT and C103A KI cells. Functional studies were performed in A20 C103A U937 cells and in immune cells from A20 C103A mice and genotyped healthy individuals with A20 DUB polymorphism rs2230926. Neutrophil extracellular trap (NET) formation was addressed ex vivo in neutrophils from A20 C103A mice and SLE-patients with rs2230926.ResultsGenetic disruption of A20 DUB domain in human and murine myeloid cells did not give rise to enhanced NF-κB signalling. Instead, cells with C103A mutation or rs2230926 polymorphism presented an upregulated expression of PADI4, an enzyme regulating protein citrullination and NET formation, two key mechanisms in autoimmune pathology. A20 C103A cells exhibited enhanced protein citrullination and extracellular trap formation, which could be suppressed by selective PAD4 inhibition. Moreover, SLE-patients with rs2230926 showed increased NETs and increased frequency of autoantibodies to citrullinated epitopes.ConclusionsWe propose that genetic alterations disrupting the A20 DUB domain mediate increased susceptibility to SLE through the upregulation of PADI4 with resultant protein citrullination and extracellular trap formation.

The mechanisms driving clonal heterogeneity and evolution in relapsed pediatric acute lymphoblastic leukemia (ALL) are not fully understood. We performed whole genome sequencing of samples collected at diagnosis, relapse(s) and remission from 29 Nordic patients. Somatic point mutations and large-scale structural variants were called using individually matched remission samples as controls, and allelic expression of the mutations was assessed in ALL cells using RNA-sequencing. We observed an increased burden of somatic mutations at relapse, compared to diagnosis, and at second relapse compared to first relapse. In addition to 29 known ALL driver genes, of which nine genes carried recurrent protein-coding mutations in our sample set, we identified putative non-protein coding mutations in regulatory regions of seven additional genes that have not previously been described in ALL. Cluster analysis of hundreds of somatic mutations per sample revealed three distinct evolutionary trajectories during ALL progression from diagnosis to relapse. The evolutionary trajectories provide insight into the mutational mechanisms leading relapse in ALL and could offer biomarkers for improved risk prediction in individual patients.

In a genomewide association study of North Americans of European descent, followed by a Swedish replication analysis, investigators showed that in addition to the previously described loci at HLA-DRB1, IRF5, and STAT4, two new loci confer susceptibility to systemic lupus erythematosus. These loci are close to genes that encode B lymphoid tyrosine kinase and integrin alpha M. A genomewide association study showed that two new loci confer susceptibility to systemic lupus erythematosus. These loci are close to genes that encode B lymphoid tyrosine kinase and integrin alpha M. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with strong genetic and environmental components. 1 – 3 Autoantibodies play an important role in the pathogenesis of SLE, and the diverse clinical manifestations of the disease are caused by the deposition of antibody-containing immune complexes in blood vessels, leading to inflammation in the kidney, brain, and skin. Direct pathogenic effects of the autoantibodies contribute to hemolytic anemia and thrombocytopenia. During the past 20 years, many linkage and candidate-gene studies have been performed to identify genetic factors contributing to a susceptibility to SLE. For example, haplotypes carrying the HLA class II alleles DRB1*0301 . . .