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Systemic lupus erythematosus (SLE) is a life-threatening autoimmune disease characterized by adaptive immune system activation, formation of double-stranded DNA autoantibodies and organ inflammation. Five patients with SLE (four women and one man) with a median (range) age of 22 (6) years, median (range) disease duration of 4 (8) years and active disease (median (range) SLE disease activity index Systemic Lupus Erythematosus Disease Activity Index: 16 (8)) refractory to several immunosuppressive drug treatments were enrolled in a compassionate-use chimeric antigen receptor (CAR) T cell program. Autologous T cells from patients with SLE were transduced with a lentiviral anti-CD19 CAR vector, expanded and reinfused at a dose of 1 × 10 6 CAR T cells per kg body weight into the patients after lymphodepletion with fludarabine and cyclophosphamide. CAR T cells expanded in vivo, led to deep depletion of B cells, improvement of clinical symptoms and normalization of laboratory parameters including seroconversion of anti-double-stranded DNA antibodies. Remission of SLE according to DORIS criteria was achieved in all five patients after 3 months and the median (range) Systemic Lupus Erythematosus Disease Activity Index score after 3 months was 0 (2). Drug-free remission was maintained during longer follow-up (median (range) of 8 (12) months after CAR T cell administration) and even after the reappearance of B cells, which was observed after a mean (±s.d.) of 110 ± 32 d after CAR T cell treatment. Reappearing B cells were naïve and showed non-class-switched B cell receptors. CAR T cell treatment was well tolerated with only mild cytokine-release syndrome. These data suggest that CD19 CAR T cell transfer is feasible, tolerable and highly effective in SLE. Results from a study of five patients with refractory systemic lupus erythematosus, who were treated with anti-CD19 CAR T cell therapy under a compassionate-use program, demonstrate remission of SLE disease with follow-up of up to 17 months.

Short stature is a common pediatric disorder affecting 3% of the population. However, the clinical variability and genetic heterogeneity prevents the identification of the underlying cause in about 80% of the patients. Recently, heterozygous mutations in the ACAN gene coding for the proteoglycan aggrecan, a main component of the cartilage matrix, were associated with idiopathic short stature. To ascertain the prevalence of ACAN mutations and broaden the phenotypic spectrum in patients with idiopathic short stature we performed sequence analyses in 428 families. We identified heterozygous nonsense mutations in four and potentially disease-causing missense variants in two families (1.4%). These patients presented with a mean of −3.2 SDS and some suggestive clinical characteristics. The results suggest heterozygous mutations in ACAN as a common cause of isolated as well as inherited idiopathic short stature.

The kidneys integrate information from continuous systemic processes related to the absorption, distribution, metabolism and excretion (ADME) of metabolites. To identify underlying molecular mechanisms, we performed genome-wide association studies of the urinary concentrations of 1,172 metabolites among 1,627 patients with reduced kidney function. The 240 unique metabolite–locus associations (metabolite quantitative trait loci, mQTLs) that were identified and replicated highlight novel candidate substrates for transport proteins. The identified genes are enriched in ADME-relevant tissues and cell types, and they reveal novel candidates for biotransformation and detoxification reactions. Fine mapping of mQTLs and integration with single-cell gene expression permitted the prioritization of causal genes, functional variants and target cell types. The combination of mQTLs with genetic and health information from 450,000 UK Biobank participants illuminated metabolic mediators, and hence, novel urinary biomarkers of disease risk. This comprehensive resource of genetic targets and their substrates is informative for ADME processes in humans and is relevant to basic science, clinical medicine and pharmaceutical research. Genome-wide association analysis of 1,172 urinary metabolites identifies 240 metabolite–locus associations that when combined with UK Biobank data suggest novel metabolic mediators of disease and markers of disease risk.

High throughput sequencing has greatly advanced disease gene identification, especially in heterogeneous entities. Despite falling costs this is still an expensive and laborious technique, particularly when studying large cohorts. To address this problem we applied Exome Pool-Seq as an economic and fast screening technology in neurodevelopmental disorders (NDDs). Sequencing of 96 individuals can be performed in eight pools of 12 samples on less than one Illumina sequencer lane. In a pilot study with 96 cases we identified 27 variants, likely or possibly affecting function. Twenty five of these were identified in 923 established NDD genes (based on SysID database, status November 2016) (ACTB, AHDC1, ANKRD11, ATP6V1B2, ATRX, CASK, CHD8, GNAS, IFIH1, KCNQ2, KMT2A, KRAS, MAOA, MED12, MED13L, RIT1, SETD5, SIN3A, TCF4, TRAPPC11, TUBA1A, WAC, ZBTB18, ZMYND11), two in 543 (SysID) candidate genes (ZNF292, BPTF), and additionally a de novo loss-of-function variant in LRRC7, not previously implicated in NDDs. Most of them were confirmed to be de novo, but we also identified X-linked or autosomal-dominantly or autosomal-recessively inherited variants. With a detection rate of 28%, Exome Pool-Seq achieves comparable results to individual exome analyses but reduces costs by >85%. Compared with other large scale approaches using Molecular Inversion Probes (MIP) or gene panels, it allows flexible re-analysis of data. Exome Pool-Seq is thus well suited for large-scale, cost-efficient and flexible screening in characterized but heterogeneous entities like NDDs.

Although tissue-resident memory T cells (T RM cells) have been shown to regulate host protection in infectious disorders, their function in inflammatory bowel disease (IBD) remains to be investigated. Here we characterized T RM cells in human IBD and in experimental models of intestinal inflammation. Pro-inflammatory T RM cells accumulated in the mucosa of patients with IBD, and the presence of CD4 + CD69 + CD103 + T RM cells was predictive of the development of flares. In vivo, functional impairment of T RM cells in mice with double knockout of the T RM -cell-associated transcription factors Hobit and Blimp-1 attenuated disease in several models of colitis, due to impaired cross-talk between the adaptive and innate immune system. Finally, depletion of T RM cells led to a suppression of colitis activity. Together, our data demonstrate a central role for T RM cells in the pathogenesis of chronic intestinal inflammation and suggest that these cells could be targets for future therapeutic approaches in IBD. Tissue-resident memory T cells (T RM cells) have well-described functions in the protective response to infectious agents. Neurath and colleagues demonstrate that intestinal T RM cells can also have key pathogenic roles in inflammatory bowel disease.

Background The TUBA1A -associated tubulinopathy is clinically heterogeneous with brain malformations, microcephaly, developmental delay and epilepsy being the main clinical features. It is an autosomal dominant disorder mostly caused by de novo variants in TUBA1A. Results In three individuals with developmental delay we identified heterozygous de novo missense variants in TUBA1A using exome sequencing. While the c.1307G > A, p.(Gly436Asp) variant was novel, the two variants c.518C > T, p.(Pro173Leu) and c.641G > A, p.(Arg214His) were previously described. We compared the variable phenotype observed in these individuals with a carefully conducted review of the current literature and identified 166 individuals, 146 born and 20 fetuses with a TUBA1A variant. In 107 cases with available clinical information we standardized the reported phenotypes according to the Human Phenotype Ontology. The most commonly reported features were developmental delay (98%), anomalies of the corpus callosum (96%), microcephaly (76%) and lissencephaly (agyria-pachygyria) (70%), although reporting was incomplete in the different studies. We identified a total of 121 specific variants, including 15 recurrent ones. Missense variants cluster in the C-terminal region around the most commonly affected amino acid position Arg402 (13.3%). In a three-dimensional protein model, 38.6% of all disease-causing variants including those in the C-terminal region are predicted to affect the binding of microtubule-associated proteins or motor proteins. Genotype-phenotype analysis for recurrent variants showed an overrepresentation of certain clinical features. However, individuals with these variants are often reported in the same publication. Conclusions With 166 individuals, we present the most comprehensive phenotypic and genotypic standardized synopsis for clinical interpretation of TUBA1A variants. Despite this considerable number, a detailed genotype-phenotype characterization is limited by large inter-study variability in reporting.

In 90% of pancreatic ductal adenocarcinoma cases, genetic alteration of the proto-oncogene Kras has occurred, leading to uncontrolled proliferation of cancerous cells. Targeting Kras has proven to be difficult and the battle against pancreatic cancer is ongoing. A promising approach to combat cancer was the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, which can be used to genetically modify cells. To assess the potential of a CRISPR/CRISPR-associated protein 9 (Cas9) method to eliminate Kras mutations in cells, we aimed to knock-out the c.35G>A (p.G12D) Kras mutation. Therefore, three cell lines with a heterozygous Kras mutation (the human cell lines SUIT-2 and Panc-1 and the cell line TB32047 from a KPC mouse model) were used. After transfection, puromycin selection and single-cell cloning, proteins from two negative controls and five to seven clones were isolated to verify the knock-out and to analyze changes in key signal transduction proteins. Western blots showed a specific knock-out in the KrasG12D protein, but wildtype Kras was expressed by all of the cells. Signal transduction analysis (for Erk, Akt, Stat3, AMPKα, and c-myc) revealed expression levels similar to the wildtype. The results described herein indicate that knocking-out the KrasG12D mutation by CRISPR/Cas9 is possible. Additionally, under regular growth conditions, the knock-out clones resembled wildtype cells.

Terminal differentiation of villous cytotrophoblasts (CT) ends in formation of the multinucleated syncytiotrophoblast representing the fetal-maternal interface. Aberrations during this cell-fusion process are associated with Intrauterine Growth Restriction (IUGR), Preeclampsia (PE) and High Elevated Liver and Low Platelets (HELLP) Syndrome. Syncytin-1, the envelope gene of the human Endogenous Retrovirus ERVW-1, is one of the most important genes involved in cell-fusion and showed decreased gene expression during these pathological pregnancies. The aim of this study was to determine the methylation pattern of the entire promoter of ERVW-1 and to correlate these findings with the expression profile of Syncytin-1 in the placental syndromes. 14 isolated villous cytotrophoblasts from control (n = 3), IUGR (n = 3), PE (n = 3), PE/IUGR (n = 3) and HELLP/IUGR (n = 2) placentae were used to determine the mean methylation level (ML) for the ERVW-1 promoter region. ML rose significantly from 29% in control CTs to 49% in IUGR, 53% in PE, 47% in PE/IUGR and 64% in HELLP/IUGR indicating an epigenetic down-regulation of Syncytin-1 by promoter hypermethylation. DNA demethylation of the trophoblast like cell lines BeWo, JEG-3 and JAR with 5-AZA-2'desoxycytidine (AZA) showed an increased Syncytin-1 expression and fusion ability in all cell lines. Promoter activity of the 5'LTR could be inhibited by hypermethylation 42-fold using a luciferase based reporter-gene assay. Finally overexpression of the methyltransferases DNMT3a and LSH could be responsible for a decreased Syncytin-1 expression by promoter hypermethylation of ERVW-1. Our study linked decreased Syncytin-1 expression to an epigenetic hypermethylation of the entire promoter of ERVW-1. Based on our findings we are predicting a broad aberrant epigenetic DNA-methylation pattern in pathological placentae affecting placentogenesis, but also the development of the fetus and the mother during pregnancy.

Spermatogenesis is an efficient and complex system of continuous cell differentiation. Previous studies investigating the transcriptomes of different cell populations in the testis relied either on sorting cells, cell depletion, or juvenile animals where not all stages of spermatogenesis have been completed. We present single-cell RNA sequencing (scRNA-Seq) data of 2,500 cells from the testes of two 8-week-old C57Bl/6J mice. Our dataset includes all spermatogenic stages from preleptotene to condensing spermatids as well as individual spermatogonia, Sertoli and Leydig cells. The data capture the full continuity of the meiotic and postmeiotic stages of spermatogenesis, and is thus ideally suited for marker discovery, network inference and similar analyses for which temporal ordering of differentiation processes can be exploited. Furthermore, it can serve as a reference for future studies involving single-cell RNA-Seq in mice where spermatogenesis is perturbed.

Chromosomal rearrangements and specific aneuploidy patterns are initiating events and define subgroups in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Here we analyzed 250 BCP-ALL cases and identified a novel subgroup (‘PAX5-plus’, n = 19) by distinct DNA methylation and gene expression profiles. All patients in this subgroup harbored mutations in the B-lineage transcription factor PAX5, with p.P80R as hotspot. Mutations either affected two independent codons, consistent with compound heterozygosity, or suffered LOH predominantly through chromosome 9p aberrations. These biallelic events resulted in disruption of PAX5 transcriptional programs regulating B-cell differentiation and tumor suppressor functions. Homozygous CDKN2A/B deletions and RAS-activating hotspot mutations were highly enriched as cooperating events in the genomic profile of PAX5-plus ALL. Together, this defined a specific pattern of triple alterations, exclusive to the novel subgroup. PAX5-plus ALL was observed in pediatric and adult patients. Although restricted by the limited sample size, a tendency for more favorable clinical outcome was observed, with 10 of 12 adult PAX5-plus patients achieving long-term survival. PAX5-plus represents the first BCP-ALL subgroup defined by sequence alterations in contrast to gross chromosomal events and exemplifies how deregulated differentiation (PAX5), impaired cell cycle control (CDKN2A/B) and sustained proliferative signaling (RAS) cooperatively drive leukemogenesis.