Aryl Hydrocarbon Receptor Controls Monocyte Differentiation into Dendritic Cells versus Macrophages.

After entering tissues, monocytes differentiate into cells that share functional features with either macrophages or dendritic cells (DCs). How monocyte fate is directed toward monocyte-derived macrophages (mo-Macs) or monocyte-derived DCs (mo-DCs) and which transcription factors control these differentiation pathways remains unknown. Using an in vitro culture model yielding human mo-DCs and mo-Macs closely resembling those found in vivo in ascites, we show that IRF4 and MAFB were critical regulators of monocyte differentiation into mo-DCs and mo-Macs, respectively. Activation of the aryl hydrocarbon receptor (AHR) promoted mo-DC differentiation through the induction of BLIMP-1, while impairing differentiation into mo-Macs. AhR deficiency also impaired the in vivo differentiation of mouse mo-DCs. Finally, AHR activation correlated with mo-DC infiltration in leprosy lesions. These results establish that mo-DCs and mo-Macs are controlled by distinct transcription factors and show that AHR acts as a molecular switch for monocyte fate specification in response to micro-environmental factors.

Extracellular vesicles from triple negative breast cancer promote pro-inflammatory macrophages associated with better clinical outcome.

Tumor associated macrophages (TAMs), which differentiate from circulating monocytes, are pervasive across human cancers and comprise heterogeneous populations. The contribution of tumor-derived signals to TAM heterogeneity is not well understood. In particular, tumors release both soluble factors and extracellular vesicles (EVs), whose respective impact on TAM precursors may be different. Here, we show that triple negative breast cancer cells (TNBCs) release EVs and soluble molecules promoting monocyte differentiation toward distinct macrophage fates. EVs specifically promoted proinflammatory macrophages bearing an interferon response signature. The combination in TNBC EVs of surface CSF-1 promoting survival and cargoes promoting cGAS/STING or other activation pathways led to differentiation of this particular macrophage subset. Notably, macrophages expressing the EV-induced signature were found among patients' TAMs. Furthermore, higher expression of this signature was associated with T cell infiltration and extended patient survival. Together, this data indicates that TNBC-released CSF-1-bearing EVs promote a tumor immune microenvironment associated with a better prognosis in TNBC patients.

SpliceAI-visual: a free online tool to improve SpliceAI splicing variant interpretation.

SpliceAI is an open-source deep learning splicing prediction algorithm that has demonstrated in the past few years its high ability to predict splicing defects caused by DNA variations. However, its outputs present several drawbacks: (1) although the numerical values are very convenient for batch filtering, their precise interpretation can be difficult, (2) the outputs are delta scores which can sometimes mask a severe consequence, and (3) complex delins are most often not handled. We present here SpliceAI-visual, a free online tool based on the SpliceAI algorithm, and show how it complements the traditional SpliceAI analysis. First, SpliceAI-visual manipulates raw scores and not delta scores, as the latter can be misleading in certain circumstances. Second, the outcome of SpliceAI-visual is user-friendly thanks to the graphical presentation. Third, SpliceAI-visual is currently one of the only SpliceAI-derived implementations able to annotate complex variants (e.g., complex delins). We report here the benefits of using SpliceAI-visual and demonstrate its relevance in the assessment/modulation of the PVS1 classification criteria. We also show how SpliceAI-visual can elucidate several complex splicing defects taken from the literature but also from unpublished cases. SpliceAI-visual is available as a Google Colab notebook and has also been fully integrated in a free online variant interpretation tool, MobiDetails ( https://mobidetails.iurc.montp.inserm.fr/MD ).

High prevalence of Bardet-Biedl syndrome in La Réunion Island is due to a founder variant in ARL6/BBS3.

Bardet-Biedl syndrome (BBS) is a rare ciliopathy with variable retinal dystrophy, polydactyly, renal abnormalities, obesity, cognitive impairment, and hypogonadism. Biallelic pathogenic variants have been identified in 24 genes, leading to BBS in an autosomal recessive inheritance pattern. In this study, we investigated a cohort of 16 families (20 individuals) presenting with typical BBS originating from La Réunion Island using sequencing (Sanger and high-throughput methods) and SNP array. In eight families (12 individuals) we identified the same ARL6/BBS3 variation [c.535G > A, p.(Asp179Asn)]. Bioinformatics and functional analyses revealed an effect of this variant on the splicing of ARL6/BBS3. Owing to the relatively high frequency of this variant, a possible founder effect was suspected. Genotyping of six individuals revealed a common 3.8-Mb haplotype and estimated the most recent common ancestor to about eight generations confirmed by the known genealogy. Knowledge of this founder effect modifies our diagnostic strategy and enables a personalized genetic counseling for patients from La Réunion Island. Being the first description of BBS patients from La Réunion Island, we could estimate its prevalence between ~1/45000 and ~ 1/66000 individuals.

Sensitive detection methods are key to identify secondary EGFR c.2369C>T p.(Thr790Met) in non-small cell lung cancer tissue samples.

BACKGROUND: Correct identification of the EGFR c.2369C>T p.(Thr790Met) variant is key to decide on a targeted therapeutic strategy for patients with acquired EGFR TKI resistance in non-small cell lung cancer. The aim of this study was to evaluate the correct detection of this variant in 12 tumor tissue specimens tested by 324 laboratories participating in External Quality Assessment (EQA) schemes. METHODS: Data from EQA schemes were evaluated between 2013 and 2018 from cell lines (6) and resections (6) containing the EGFR c.2369C>T p.(Thr790Met) mutation. Adequate performance was defined as the percentage of tests for which an outcome was available and correct. Additional data on the used test method were collected from the participants. Chi-squared tests on contingency tables and a biserial rank correlation were applied by IBM SPSS Statistics version 25 (IBM, Armonk, NY, USA). RESULTS: In 26 of the 1190 tests (2.2%) a technical failure occurred. For the remaining 1164 results, 1008 (86.6%) were correct, 151 (12.9%) were false-negative and 5 (0.4%) included incorrect mutations. Correct p.(Thr790Met) detection improved over time and for repeated scheme participations. In-house non-next-generation sequencing (NGS) techniques performed worse (81.1%, n = 293) compared to non-NGS commercial kits (85.2%, n = 656) and NGS (97.0%, n = 239). Over time there was an increase in the users of NGS. Resection specimens performed worse (82.6%, n = 610 tests) compared to cell line material (90.9%, n = 578 tests), except for NGS (96.3%, n = 344 for resections and 98.6%, n = 312 for cell lines). Samples with multiple mutations were more difficult compared to samples with the single p.(Thr790Met) variant. A change of the test method was shown beneficial to reduce errors but introduced additional analysis failures. CONCLUSIONS: A significant number of laboratories that offer p.(Thr790Met) testing did not detect this relevant mutation compared to the other EQA participants. However, correct identification of this variant is improving over time and was higher for NGS users. Revising the methodology might be useful to resolve errors, especially for resection specimens with low frequency or multiple variants. EQA providers should include challenging resections in the scheme.

Pathogenic and likely pathogenic variants in at least five genes account for approximately 3% of mild isolated nonsyndromic thrombocytopenia.

BACKGROUND: Thrombocytopenia has a variety of different etiologies, both acquired and hereditary. Inherited thrombocytopenia may be associated with other symptoms (syndromic forms) or may be strictly isolated. To date, only about half of all the familial forms of thrombocytopenia have been accounted for in terms of well-defined genetic abnormalities. However, data are limited on the nature and frequency of the underlying causative genetic variants in individuals with mild isolated nonsyndromic thrombocytopenia. STUDY DESIGN AND METHODS: Thirteen known or candidate genes for isolated thrombocytopenia were included in a gene panel analysis in which targeted next-generation sequencing was performed on 448 French blood donors with mild isolated nonsyndromic thrombocytopenia. RESULTS: A total of 68 rare variants, including missense, splice site, frameshift, nonsense, and in-frame variants (all heterozygous) were identified in 11 of the 13 genes screened. Twenty-nine percent (N = 20) of the variants detected were absent from both the French Exome Project and gnomAD exome databases. Using stringent criteria and an unbiased approach, we classified seven predicted loss-of-function variants (three in ITGA2B and four in TUBB1) and four missense variants (one in GP1BA, two in ITGB3 and one in ACTN1) as being pathogenic or likely pathogenic. Altogether, they were found in 13 members (approx. 3%) of our studied cohort. CONCLUSION: We present the results of gene panel sequencing of known and candidate thrombocytopenia genes in mild isolated nonsyndromic thrombocytopenia. Pathogenic and likely pathogenic variants in five known thrombocytopenia genes were identified, accounting for approximately 3% of individuals with the condition.

Sequential analysis of 18 genes in polycythemia vera and essential thrombocythemia reveals an association between mutational status and clinical outcome.

Philadelphia-negative classical myeloproliferative neoplasms (MPN) are clonal diseases characterized by driver mutations of JAK2, MPL, or CALR. Additional mutations may occur in epigenetic regulators, signaling, or splicing genes that may be useful in the prognostic assessment of MPN patients. In primary myelofibrosis, molecular-based prognostic scoring systems have been recently proposed, but few data are available to date for polycythemia vera (PV) and essential thrombocythemia (ET). In this study, we used a next generation sequencing-based 18-gene panel in 50 JAK2V617F positive PV and JAK2V617F positive ET patients from an institutional cohort investigated at diagnosis and at 3-year follow-up (3y). Disease progression at 3y was defined by a composite criterion. Patients (28 PV and 22 ET) were included according to their clinical status, with or without disease progression. At diagnosis, we found 28 additional mutations in 21 of the 50 patients. Patients with disease progression were more likely to have at least one additional mutation. There was no difference between PV and ET. All patients with two or more additional mutations exhibited disease progression at 3y. No novel mutations appeared at 3y. The allele burden increase by at least one mutation at 3y was more frequent in patients with disease progression. Our data suggest that screening for additional mutations in PV and ET could identify patients at a higher risk of disease progression. © 2017 Wiley Periodicals, Inc.

A misleading false-negative result of Pneumocystis real-time PCR assay due to a rare punctual mutation: A French multicenter study.

This article describes a previously unreported mutation at position 210 (C210T) of the mitochondrial large subunit ribosomal RNA (mtLSUrRNA) gene of Pneumocystis jirovecii, which led to a false-negative result of a real-time polymerase chain reaction (PCR) assay. Since the aforementioned real-time PCR assay is widely used in France, a French multicenter study was conducted to estimate the mutation frequency and its potential impact on the routine diagnosis of Pneumocystis pneumonia (PCP). Through analysis of data obtained from eight centers, the mutation frequency was estimated at 0.28%. This low frequency should not call into question the routine use of this PCR assay. Nonetheless, the occurrence of the false-negative PCR result provides arguments for maintaining microscopic techniques combined to PCR assays to achieve PCP diagnosis.

ACTN1-related Macrothrombocytopenia: A Novel Entity in the Progressing Field of Pediatric Thrombocytopenia.

The most common cause of thrombocytopenia in children is immune thrombocytopenia. Nevertheless, some atypical cases should evoke the hypothesis of genetic thrombocytopenia. Indeed, in the past years, 30 new genes had been described in the field of inherited thrombocytopenia. We report a series of 11 cases of a newly diagnosed entity: ACTN1-related macrothrombocytopenia. Mutations in the gene ACTN1 cause mild macrothrombocytopenia characterized by elevated mean platelet volume and elevated immature platelet fraction, and low bleeding tendency. Its transmission is autosomal dominant. Molecular diagnosis is made by sequencing the ACTN1 gene. Its potential role in hematological malignancy predisposition remains unclear and should be clarified. CONCLUSION: We identified 11 patients with ACTN1-related macrothrombocytopenia diagnosed through pediatric probands. The aim was to underline the specificities of this entity, especially in children, and bring it to the knowledge of pediatricians.

Next-generation sequencing is a credible strategy for blood group genotyping.

Although several medium/high-throughput tools have been engineered for molecular analysis of blood group genes, they usually rely on the targeting of single nucleotide polymorphisms, while other variants remain unidentified. To circumvent this limitation a strategy for genotyping blood group genes by next-generation sequencing (NGS) was set up. Libraries consisting of exons, flanking introns and untranslated regions of 18 genes involved in 15 blood systems were generated by the Ion AmpliSeq(™) Library Kit 2.0 and by fragmenting polymerase chain reaction products, normalized by two different approaches, mixed and sequenced by the Ion Torrent Personal Genome Machine (PGM(™) ) Sequencer. In our conditions, defined to limit both intra- and inter-sample variability, sequences from mixed libraries were read in a single run for a total coverage of 86·03% of the coding DNA sequences, including all loci defining the most clinically relevant antigens in all genes, except ABO. Importantly, the challenging attempt to generate gene-specific data for the homologous genes was successful. This work, which combines two complementary approaches to generate libraries, defines technical conditions for genotyping blood group genes, illustrates that NGS is suitable for such an application and suggests that, after automation, this novel tool could be used for molecular typing at the laboratory level.

Semi-supervised integration of single-cell transcriptomics data.

Batch effects in single-cell RNA-seq data pose a significant challenge for comparative analyses across samples, individuals, and conditions. Although batch effect correction methods are routinely applied, data integration often leads to overcorrection and can result in the loss of biological variability. In this work we present STACAS, a batch correction method for scRNA-seq that leverages prior knowledge on cell types to preserve biological variability upon integration. Through an open-source benchmark, we show that semi-supervised STACAS outperforms state-of-the-art unsupervised methods, as well as supervised methods such as scANVI and scGen. STACAS scales well to large datasets and is robust to incomplete and imprecise input cell type labels, which are commonly encountered in real-life integration tasks. We argue that the incorporation of prior cell type information should be a common practice in single-cell data integration, and we provide a flexible framework for semi-supervised batch effect correction.

SUV39H1 Ablation Enhances Long-term CAR T Function in Solid Tumors.

Failure of adoptive T-cell therapies in patients with cancer is linked to limited T-cell expansion and persistence, even in memory-prone 41BB-(BBz)-based chimeric antigen receptor (CAR) T cells. We show here that BBz-CAR T-cell stem/memory differentiation and persistence can be enhanced through epigenetic manipulation of the histone 3 lysine 9 trimethylation (H3K9me3) pathway. Inactivation of the H3K9 trimethyltransferase SUV39H1 enhances BBz-CAR T cell long-term persistence, protecting mice against tumor relapses and rechallenges in lung and disseminated solid tumor models up to several months after CAR T-cell infusion. Single-cell transcriptomic (single-cell RNA sequencing) and chromatin opening (single-cell assay for transposase accessible chromatin) analyses of tumor-infiltrating CAR T cells show early reprogramming into self-renewing, stemlike populations with decreased expression of dysfunction genes in all T-cell subpopulations. Therefore, epigenetic manipulation of H3K9 methylation by SUV39H1 optimizes the long-term functional persistence of BBz-CAR T cells, limiting relapses, and providing protection against tumor rechallenges. SIGNIFICANCE: Limited CAR T-cell expansion and persistence hinders therapeutic responses in solid cancer patients. We show that targeting SUV39H1 histone methyltransferase enhances 41BB-based CAR T-cell long-term protection against tumor relapses and rechallenges by increasing stemness/memory differentiation. This opens a safe path to enhancing adoptive cell therapies for solid tumors. See related article by Jain et al., p. 142. This article is featured in Selected Articles from This Issue, p. 5.

Recurrent ATP1A1 variant Gly903Arg causes developmental delay, intellectual disability, and autism.

ATP1A1 encodes a sodium-potassium ATPase that has been linked to several neurological diseases. Using exome and genome sequencing, we identified the heterozygous ATP1A1 variant NM_000701.8: c.2707G>A;p.(Gly903Arg) in two unrelated children presenting with delayed motor and speech development and autism. While absent in controls, the variant occurred de novo in one proband and co-segregated in two affected half-siblings, with mosaicism in the healthy mother. Using a specific ouabain resistance assay in mutant transfected HEK cells, we found significantly reduced cell viability. Demonstrating loss of ATPase function, we conclude that this novel variant is pathogenic, expanding the phenotype spectrum of ATP1A1.

Pneumocystis jirovecii haplotypes at the internal transcribed spacers of the rRNA operon in French HIV-negative patients with diverse clinical presentations of Pneumocystis infections.

Pneumocystis jirovecii, a transmissible fungus, is the causative agent of pulmonary infections. Its genomic diversity has appeared in reports from around the world but data on P. jirovecii genotypes in France are still limited. This study describes the typing of P. jirovecii isolates from 81 HIV-negative patients monitored at Brest University Hospital, Brittany, France, 40 of whom developed Pneumocystis pneumonia (PcP), and remaining 41 patients were colonized by the fungus. The isolates were assayed at the internal transcribed spacer (ITS)1 and ITS2 under improved amplification conditions to avoid in vitro ITS recombination. P. jirovecii ITS haplotypes were identified in 56/81 patients (31 PcP patients and 25 patients who were colonized) which revealed a high diversity in that 27 different haplotypes were identified. Eg was the most frequent haplotype (31/56, 55.3%), followed by Ec and Ai (5/56, 8.9% each). In contrast, Ne, usually the second most frequent haplotype in Europe and the USA, was observed in only 2/56 patients (3.6%). Mixed infections were detected in 18/56 patients (32.1%; 12 PcP patients and six who were colonized). No significant differences were observed in haplotype diversity, frequency of peculiar haplotypes, and mixed infection occurrence, between the two patient populations. The study, conducted with the largest HIV-negative patient population investigated so far, shows that ITS typing remains an efficient method for characterizing P. jirovecii among human populations, whatever their clinical presentation of Pneumocystis infections.

T Cell Clonal Analysis Using Single-cell RNA Sequencing and Reference Maps.

T cells are endowed with T-cell antigen receptors (TCR) that give them the capacity to recognize specific antigens and mount antigen-specific adaptive immune responses. Because TCR sequences are distinct in each naïve T cell, they serve as molecular barcodes to track T cells with clonal relatedness and shared antigen specificity through proliferation, differentiation, and migration. Single-cell RNA sequencing provides coupled information of TCR sequence and transcriptional state in individual cells, enabling T-cell clonotype-specific analyses. In this protocol, we outline a computational workflow to perform T-cell states and clonal analysis from scRNA-seq data based on the R packages Seurat, ProjecTILs, and scRepertoire. Given a scRNA-seq T-cell dataset with TCR sequence information, cell states are automatically annotated by reference projection using the ProjecTILs method. TCR information is used to track individual clonotypes, assess their clonal expansion, proliferation rates, bias towards specific differentiation states, and the clonal overlap between T-cell subtypes. We provide fully reproducible R code to conduct these analyses and generate useful visualizations that can be adapted for the needs of the protocol user. Key features Computational analysis of paired scRNA-seq and scTCR-seq data Characterizing T-cell functional state by reference-based analysis using ProjecTILs Exploring T-cell clonal structure using scRepertoire Linking T-cell clonality to transcriptomic state to study relationships between clonal expansion and functional phenotype Graphical overview.

Sweet ending: When genetics prevent a dramatic CDG diagnostic mistake.

Herein, we described the case of a newborn male, from consanguineous parents, who developed, at day 11 of life, an obstructive hydrocephalus resulting from bilateral cerebellar hemorrhage without evident cause. Then, at 1 month, he developed a fulminant hepatitis with hyperammonia, hyperlactatemia and metabolic acidosis. Infectious and first line metabolic explorations were normal. Screening for congenital disorder of glycosylation (CDG) was performed using capillary electrophoresis and western blot of serum transferrin. Abnormal results were evocative of mannose-phosphate isomerase deficiency (MPI-CDG or CDG-Ib) as it can be responsible for fulminant hepatitis, digestive disease, developmental delay, and coagulopathy. However, trio whole exome sequencing revealed a pathogenic variant at the homozygous state in ALDOB, responsible for hereditary fructose intolerance (HFI), an inherited metabolic disorder with excellent prognosis under a fructose-free diet. HFI had not been previously evoked in view of the absence of diet diversification, but meticulous inquiry revealed that parents systematically added white sugar to the bottle milk of their child, unintentionally triggering potentially fatal HFI decompensations. Early genetic analysis upsetted both diagnosis and prognosis for this infant who had excellent development after fructose removal. This full-of-surprises diagnostic approach illustrates the importance of an integrative collaboration between clinicians, biochemists, and geneticists.

Description of Copy Number Variations in a Series of Children and Adolescents with FASD in Reunion Island.

BACKGROUND: Fetal Alcohol Spectrum Disorders (FASD) are the most common cause of neurocognitive impairment and social inadaptation, affecting 1 birth in 100. Despite the existence of precise diagnostic criteria, the diagnosis remains difficult, often confounded with other genetic syndromes or neurodevelopmental disorders. Since 2016, Reunion Island has been a pilot region for the identification, diagnosis, and care of FASD in France. OBJECTIVE: To evaluate the prevalence and the types of Copy Number Variations (CNV) in FASD patients. METHODS: A retrospective chart review of 101 patients diagnosed with FASD in the Reference Center for developmental anomalies and in the FASD Diagnostic Center of the University Hospital was performed. Records of all patients were reviewed to obtain their medical history, family history, clinical phenotype, and investigations, including genetic testing (CGH- or SNP-array). RESULTS: A rate of 20.8% (n = 21) of CNVs was found including 57% (12/21) of pathogenic variants and 29% (6/21) of variants of uncertain signification (VUS). CONCLUSION: A particularly high number of CNVs was found in children and adolescents with FASD. It reinforces the plea for a multidisciplinary approach for developmental disorders to explore both environmental factors, such as avoidable teratogens and intrinsic vulnerabilities, especially genetic determinants.

Biological determinants of bleeding in patients with heterozygous factor XI deficiency.

Bleeding risk is not predictable in patients with factor XI (FXI; F11) deficiency. In this prospective study, our objectives were to determine the biological determinants for bleeding risk in patients with heterozygous FXI deficiency. Patients were classified as either bleeding patients or non-bleeding patients by calculating the bleeding score (BS) described for von Willebrand disease. Primary haemostasis, thrombin generation, thromboelastometry, procoagulant proteins, inhibitors, fibrinolysis, and F11 gene mutations were compared between bleeding and non-bleeding patients. Thirty-nine patients were included. BS significantly correlated with clinical assessment (P=0·001), and a score over 3 discriminated between bleeding (n=15) and non-bleeding (n=24) patients (P=0·034). Despite normal values, von Willebrand factor (VWF) and thrombomodulin (TM) plasma levels were significantly lower in bleeding patients than non-bleeding patients [ristocetin cofactor activity (VWF:RCo)=80·6±29·7 iu/dl and 101·8±29·5iu/dl respectively, P=0·043; and VWF antigen (VWF:Ag)=84·0±28·0 iu/dl and 106·3±36·1 iu/dl respectively, P=0·035; and TM=17·7±11·7ng/ml and 23·6±9·7ng/ml respectively, P=0·043]. When considering BS as a continuous variable, only VWF:RCo remained significant (P=0·042), which accounted for 11% of the variability in BS.