Maternal cell contamination in postnatal umbilical cord blood samples implies a low risk for genetic misdiagnoses.

OBJECTIVE: Maternal cell contamination (MCC) poses a risk for misdiagnosis in prenatal genetic testing, and is examined in accredited diagnostic laboratories However, the awareness of possible MCC in perinatal/postnatal genetic testing, mainly of umbilical cord blood (CB), is lower. METHOD: We investigated the rate of MCC in DNA from both umbilical CB samples and umbilical cord samples that were sent to our diagnostic laboratory for diagnostic testing between 1995 and 2021 (n = 236). RESULTS: MCC was detected in 4% of umbilical CB samples, and in one umbilical cord sample. Particularly tests enriching for a specific variant are very sensitive for low amounts of MCC, as we emphasize here with a false positive diagnosis of myotonic dystrophy type 1 in a newborn. CONCLUSIONS: Overall, with appropriate collection and use, umbilical CB and umbilical cord samples are suitable for genetic testing based on the low rates of MCC and misdiagnosis. These findings do however underline the importance of routine MCC testing in umbilical CB samples and umbilical cord samples for both requesting clinicians and diagnostic genetic laboratories.

All-in-one whole exome sequencing strategy with simultaneous copy number variant, single nucleotide variant and absence-of-heterozygosity analysis in fetuses with structural ultrasound anomalies: A 1-year experience.

OBJECTIVE: We performed a 1-year evaluation of a novel strategy of simultaneously analyzing single nucleotide variants (SNVs), copy number variants (CNVs) and copy-number-neutral Absence-of-Heterozygosity from Whole Exome Sequencing (WES) data for prenatal diagnosis of fetuses with ultrasound (US) anomalies and a non-causative QF-PCR result. METHODS: After invasive diagnostics, whole exome parent-offspring trio-sequencing with exome-wide CNV analysis was performed in pregnancies with fetal US anomalies and a non-causative QF-PCR result (WES-CNV). On request, additional SNV-analysis, restricted to (the) requested gene panel(s) only (with the option of whole exome SNV-analysis afterward) was performed simultaneously (WES-CNV/SNV) or as rapid SNV-re-analysis, following a normal CNV analysis. RESULTS: In total, 415 prenatal samples were included. Following a non-causative QF-PCR result, WES-CNV analysis was initially requested for 74.3% of the chorionic villus (CV) samples and 45% of the amniotic fluid (AF) samples. In case WES-CNV analysis did not reveal a causative aberration, SNV-re-analysis was requested in 41.7% of the CV samples and 17.5% of the AF samples. All initial analyses could be finished within 2 weeks after sampling. For SNV-re-analysis during pregnancy, turn-around-times (TATs) varied between one and 8 days. CONCLUSION: We show a highly efficient all-in-one WES-based strategy, with short TATs, and the option of rapid SNV-re-analysis after a normal CNV result.

Clinical exome sequencing-Mistakes and caveats.

Massive parallel sequencing technology has become the predominant technique for genetic diagnostics and research. Many genetic laboratories have wrestled with the challenges of setting up genetic testing workflows based on a completely new technology. The learning curve we went through as a laboratory was accompanied by growing pains while we gained new knowledge and expertise. Here we discuss some important mistakes that have been made in our laboratory through 10 years of clinical exome sequencing but that have given us important new insights on how to adapt our working methods. We provide these examples and the lessons that we learned to help other laboratories avoid to make the same mistakes.

An integrative genomics approach identifies KDM4 as a modulator of trained immunity.

Innate immune cells are able to build memory characteristics via a process termed "trained immunity." Host factors that influence the magnitude of the individual trained immunity response remain largely unknown. Using an integrative genomics approach, our study aimed to prioritize and understand the role of specific genes in trained immunity responses. In vitro-induced trained immunity responses were assessed in two independent population-based cohorts of healthy individuals, the 300 Bacillus Calmette-Guérin (300BCG; n = 267) and 200 Functional Genomics (200FG; n = 110) cohorts from the Human Functional Genomics Project. Genetic loci that influence cytokine responses upon trained immunity were identified by conducting a meta-analysis of QTLs identified in the 300BCG and 200FG cohorts. From the identified QTL loci, we functionally validated the role of PI3K-Akt signaling pathway and two genes that belong to the family of Siglec receptors (Siglec-5 and Siglec-14). Furthermore, we identified the H3K9 histone demethylases of the KDM4 family as major regulators of trained immunity responses. These data pinpoint an important role of metabolic and epigenetic processes in the regulation of trained immunity responses, and these findings may open new avenues for vaccine design and therapeutic interventions.

Seasonal and Nonseasonal Longitudinal Variation of Immune Function.

Different components of the immune response show large variability between individuals, but they also vary within the same individual because of host and environmental factors. In this study, we report an extensive analysis of the immune characteristics of 56 individuals over four timepoints in 1 single year as part of the Human Functional Genomics Project. We characterized 102 cell subsets using flow cytometry; quantified production of eight cytokines and two chemokines in response to 20 metabolic, bacterial, fungal, and viral stimuli; and measured circulating markers of inflammation. Taking advantage of the longitudinal sampling, both seasonal and nonseasonal sources of variability were studied. The circulating markers of inflammation IL-18, IL-18 binding protein, and resistin displayed clear seasonal variability, whereas the strongest effect was observed for α-1 antitrypsin. Cytokine production capacity also showed strong seasonal changes, especially after stimulation with the influenza virus, Borrelia burgdorferi, and Escherichia coli Furthermore, we observed moderate seasonality effects on immune cell counts, especially in several CD4+/CD8+ T cell subpopulations. Age of the volunteers was an important factor influencing IFN-γ and IL-22 production, which matched the strong impact of age on several T cell subsets. Finally, on average, genetics accounted for almost 50% of the interindividual variance not already explained by age, sex, and body mass index, although this varies strongly for different parameters. In conclusion, seasonality is an important environmental factor that influences immune responses, in addition to specific genetic and nongenetic host factors, and this may well explain the seasonal variation in the incidence and severity of immune-mediated diseases.

Integration of metabolomics, genomics, and immune phenotypes reveals the causal roles of metabolites in disease.

BACKGROUND: Recent studies highlight the role of metabolites in immune diseases, but it remains unknown how much of this effect is driven by genetic and non-genetic host factors. RESULT: We systematically investigate circulating metabolites in a cohort of 500 healthy subjects (500FG) in whom immune function and activity are deeply measured and whose genetics are profiled. Our data reveal that several major metabolic pathways, including the alanine/glutamate pathway and the arachidonic acid pathway, have a strong impact on cytokine production in response to ex vivo stimulation. We also examine the genetic regulation of metabolites associated with immune phenotypes through genome-wide association analysis and identify 29 significant loci, including eight novel independent loci. Of these, one locus (rs174584-FADS2) associated with arachidonic acid metabolism is causally associated with Crohn's disease, suggesting it is a potential therapeutic target. CONCLUSION: This study provides a comprehensive map of the integration between the blood metabolome and immune phenotypes, reveals novel genetic factors that regulate blood metabolite concentrations, and proposes an integrative approach for identifying new disease treatment targets.

Human recombinant interleukin-38 suppresses inflammation in mouse models of local and systemic disease.

Interleukin (IL)-38 belongs to the IL-1 family and is part of the IL-36 subfamily due to its binding to the IL-36 Receptor (IL-1R6). In the current study, we assessed the anti-inflammatory properties of IL-38 in murine models of arthritis and systemic inflammation. First, the anti-inflammatory properties of mouse and human IL-38 precursors were compared to forms with a truncated N-terminus. In mouse bone marrow derived dendritic cells (BMDC), human and mouse IL-38 precursors with a truncation of the two N-terminal amino acids (3-152) suppressed LPS-induced IL-6. Recombinant human IL-38 (3-152) was further investigated for its immunomodulatory potential using four murine models of inflammatory disease: streptococcal cell wall (SCW)-induced arthritis, monosodium urate (MSU) crystal-induced arthritis, MSU crystal-induced peritonitis, and systemic endotoxemia. In each of these models IL-38 significantly reduced inflammation. In SCW and MSU crystal-induced arthritis, joint swelling, inflammatory cell influx, and synovial levels of IL-1ß, IL-6, and KC were reduced by 50% or greater. These suppressive properties of IL-38 in SCW-induced arthritis were independent of the anti-inflammatory co-receptor IL-1R8, as IL-38 reduced arthritis equally in IL-1R8 deficient and WT mice. In MSU crystal-induced peritonitis, IL-38 reduced hypothermia, while plasma IL-6 and KC and peritoneal KC levels were reduced by 65-70%. In the LPS endotoxemia model, IL-38 pretreatment reduced systemic IL-6, TNFα and KC. Furthermore, in ex vivo cultured bone marrow, LPS-induced IL-6, TNFα and KC were reduced by 75-90%. Overall, IL-38 exhibits broad anti-inflammatory properties in models of systemic and local inflammation and therefore may be an effective cytokine therapy.

Sex-Specific Regulation of Inflammation and Metabolic Syndrome in Obesity.

OBJECTIVE: Metabolic dysregulation and inflammation are important consequences of obesity and impact susceptibility to cardiovascular disease. Anti-inflammatory therapy in cardiovascular disease is being developed under the assumption that inflammatory pathways are identical in women and men, but it is not known if this is indeed the case. In this study, we assessed the sex-specific relation between inflammation and metabolic dysregulation in obesity. Approach and Results: Three hundred two individuals were included, half with a BMI 27 to 30 kg/m2 and half with a BMI>30 kg/m2, 45% were women. The presence of metabolic syndrome was assessed according to the National Cholesterol Education Program-ATPIII criteria, and inflammation was studied using circulating markers of inflammation, cell counts, and ex vivo cytokine production capacity of isolated immune cells. Additionally, lipidomic and metabolomic data were gathered, and subcutaneous fat biopsies were histologically assessed. Metabolic syndrome is associated with an increased inflammatory profile that profoundly differs between women and men: women with metabolic syndrome show a lower concentration of the anti-inflammatory adiponectin, whereas men show increased levels of several pro-inflammatory markers such as IL (interleukin)-6 and leptin. Adipose tissue inflammation showed similar sex-specific associations with these markers. Peripheral blood mononuclear cells isolated from men, but not women, with metabolic syndrome display enhanced cytokine production capacity. CONCLUSIONS: We identified sex-specific pathways that influence inflammation in obesity. Excessive production of proinflammatory cytokines was observed in men with metabolic syndrome. In contrast, women typically showed reduced levels of the anti-inflammatory adipokine adiponectin. These different mechanisms of inflammatory dysregulation between women and men with obesity argue for sex-specific therapeutic strategies.

Platelet Integrin αIIbß3 Activation is Associated with 25-Hydroxyvitamin D Concentrations in Healthy Adults.

BACKGROUND: Cardiovascular events are associated with low circulating vitamin D concentrations, although the underlying mechanisms are poorly understood. This study investigated associations between 25-hydroxyvitamin D concentrations, platelet function, and single-nucleotide polymorphisms (SNPs) in genes influencing vitamin D biology in the 500 Functional Genomics (500FG) cohort. METHODS: In this observational study, platelet activation and function were measured by flow cytometry by binding of fibrinogen to the activated fibrinogen receptor integrin αIIbß3 and expression of P-selectin, markers of platelet aggregation and degranulation, respectively. These parameters were correlated to serum 25-hydroxyvitamin D and genotyping was performed to investigate SNPs in genes important for vitamin D biology. RESULTS: Circulating 25-hydroxyvitamin D concentrations correlated inversely with baseline platelet binding of fibrinogen to integrin αIIbß3 (Pearson's r= -0.172, p = 0.002) and platelet responses to platelet agonist cross-linked collagen-related peptide (CRP-XL) (Pearson's r= -0.196,p = 0.002). This effect was due to circulating vitamin D levels ≤50nmol/L, since no differences in platelet fibrinogen binding were observed between subjects with normal 25-hydroxyvitamin D concentrations (>75nmol/L) and a 25-hydroxyvitamin D insufficiency (50-75 nmol/L). No correlations between 25-hydroxyvitamin D concentrations and platelet P-selectin expression were found. Several SNPs in the GC region of the vitamin D binding proteingene were associated with platelet responses to CRP-XL. CONCLUSION: Low circulating vitamin D concentrations are associated with increased platelet fibrinogen binding to integrin αIIbß3 in unstimulated samples and after stimulation with CRP-XL. These findings may contribute to the increased incidence of cardiovascular events in vitamin D deficient adults and its seasonal variation. Further studies are needed to investigate causality.

A Genome-Wide Functional Genomics Approach Identifies Susceptibility Pathways to Fungal Bloodstream Infection in Humans.

BACKGROUND: Candidemia, one of the most common causes of fungal bloodstream infection, leads to mortality rates up to 40% in affected patients. Understanding genetic mechanisms for differential susceptibility to candidemia may aid in designing host-directed therapies. METHODS: We performed the first genome-wide association study on candidemia, and we integrated these data with variants that affect cytokines in different cellular systems stimulated with Candida albicans. RESULTS: We observed strong association between candidemia and a variant, rs8028958, that significantly affects the expression levels of PLA2G4B in blood. We found that up to 35% of the susceptibility loci affect in vitro cytokine production in response to Candida. Furthermore, potential causal genes located within these loci are enriched for lipid and arachidonic acid metabolism. Using an independent cohort, we also showed that the numbers of risk alleles at these loci are negatively correlated with reactive oxygen species and interleukin-6 levels in response to Candida. Finally, there was a significant correlation between susceptibility and allelic scores based on 16 independent candidemia-associated single-nucleotide polymorphisms that affect monocyte-derived cytokines, but not with T cell-derived cytokines. CONCLUSIONS: Our results prioritize the disturbed lipid homeostasis and oxidative stress as potential mechanisms that affect monocyte-derived cytokines to influence susceptibility to candidemia.

The fecal and mucosal microbiome in acute appendicitis patients: an observational study.

AIM: We aimed to study the mucosal microbiota of the appendix in a prospective appendicitis cohort and to compare the fecal microbiota of patients and controls. We hypothesized that the microbiota may be associated with susceptibility to appendicitis. PATIENTS & METHODS: The fecal microbiota of 99 patients and 106 controls were characterized using 16S-23S intergenic spacer profiling. Richness, diversity and community structure were compared. The appendiceal microbiota from 90 patients was analyzed according to the severity of appendicitis. RESULTS: Overall fecal microbial richness and diversity were similar in patients and controls, yet richness and diversity within the group of Firmicutes, Actinobacteria, Fusobacteria and Verrucomicrobia phyla were lower in patients. Discriminant analyses could correctly classify patients and controls with fair accuracy. No differences were found according to severity in appendiceal or fecal microbiota. CONCLUSION: This study demonstrates differences in the composition of intestinal microbiota of appendicitis patients and healthy individuals.

The Inter-Relationship of Platelets with Interleukin-1ß-Mediated Inflammation in Humans.

BACKGROUND: Inflammation and coagulation are key processes in cardiovascular diseases (CVDs). The Canakinumab Anti-inflammatory Thrombosis Outcome Study trial affirmed the importance of inflammation in CVD by showing that inhibition of the interleukin (IL)-1ß pathway prevents recurrent CVD. A bi-directional relationship exists between inflammation and coagulation, but the precise interaction of platelets and IL-1ß-mediated inflammation is incompletely understood. We aimed to determine the inter-relationship between platelets and inflammation-and especially IL-1ß-in a cohort of healthy volunteers. METHODS: We used data from the 500-Human Functional Genomics cohort, which consists of approximately 500 Caucasian, healthy individuals. We determined associations of plasma levels of IL-1ß and other inflammatory proteins with platelet number and reactivity, the association of platelet reactivity with ex vivo cytokine production as well as the impact of genetic variations through a genome-wide association study (GWAS). RESULTS: Platelets were associated with IL-1ß on different levels. First, platelet number was positively associated with plasma IL-1ß concentrations (p = 8.9 × 10-9) and inversely with concentrations of α-1-anti-trypsin (p = 1.04 × 10-18), which is a known antagonist of IL-1ß. Second, platelet degranulation capacity, as determined by agonist-induced P-selectin expression, was associated with ex vivo IL-1ß and IL-6 production. Third, several platelet single-nucleotide polymorphisms (SNPs) were associated with cytokine production and there was a significant platelet SNP enrichment in specific biological important pathways. Finally, platelet SNPs were enriched among SNPs earlier identified in GWAS studies in blood-related diseases and immune-mediated diseases. CONCLUSION: This comprehensive assessment of factors associated with platelet number and reactivity reinforces the important inter-relationship of platelets and IL-1ß-mediated inflammation.

Integration of multi-omics data and deep phenotyping enables prediction of cytokine responses.

The immune response to pathogens varies substantially among people. Whereas both genetic and nongenetic factors contribute to interperson variation, their relative contributions and potential predictive power have remained largely unknown. By systematically correlating host factors in 534 healthy volunteers, including baseline immunological parameters and molecular profiles (genome, metabolome and gut microbiome), with cytokine production after stimulation with 20 pathogens, we identified distinct patterns of co-regulation. Among the 91 different cytokine-stimulus pairs, 11 categories of host factors together explained up to 67% of interindividual variation in cytokine production induced by stimulation. A computational model based on genetic data predicted the genetic component of stimulus-induced cytokine production (correlation 0.28-0.89), and nongenetic factors influenced cytokine production as well.

Rewiring monocyte glucose metabolism via C-type lectin signaling protects against disseminated candidiasis.

Monocytes are innate immune cells that play a pivotal role in antifungal immunity, but little is known regarding the cellular metabolic events that regulate their function during infection. Using complementary transcriptomic and immunological studies in human primary monocytes, we show that activation of monocytes by Candida albicans yeast and hyphae was accompanied by metabolic rewiring induced through C-type lectin-signaling pathways. We describe that the innate immune responses against Candida yeast are energy-demanding processes that lead to the mobilization of intracellular metabolite pools and require induction of glucose metabolism, oxidative phosphorylation and glutaminolysis, while responses to hyphae primarily rely on glycolysis. Experimental models of systemic candidiasis models validated a central role for glucose metabolism in anti-Candida immunity, as the impairment of glycolysis led to increased susceptibility in mice. Collectively, these data highlight the importance of understanding the complex network of metabolic responses triggered during infections, and unveil new potential targets for therapeutic approaches against fungal diseases.

Gut microbiome in ADHD and its relation to neural reward anticipation.

BACKGROUND: Microorganisms in the human intestine (i.e. the gut microbiome) have an increasingly recognized impact on human health, including brain functioning. Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder associated with abnormalities in dopamine neurotransmission and deficits in reward processing and its underlying neuro-circuitry including the ventral striatum. The microbiome might contribute to ADHD etiology via the gut-brain axis. In this pilot study, we investigated potential differences in the microbiome between ADHD cases and undiagnosed controls, as well as its relation to neural reward processing. METHODS: We used 16S rRNA marker gene sequencing (16S) to identify bacterial taxa and their predicted gene functions in 19 ADHD and 77 control participants. Using functional magnetic resonance imaging (fMRI), we interrogated the effect of observed microbiome differences in neural reward responses in a subset of 28 participants, independent of diagnosis. RESULTS: For the first time, we describe gut microbial makeup of adolescents and adults diagnosed with ADHD. We found that the relative abundance of several bacterial taxa differed between cases and controls, albeit marginally significant. A nominal increase in the Bifidobacterium genus was observed in ADHD cases. In a hypothesis-driven approach, we found that the observed increase was linked to significantly enhanced 16S-based predicted bacterial gene functionality encoding cyclohexadienyl dehydratase in cases relative to controls. This enzyme is involved in the synthesis of phenylalanine, a precursor of dopamine. Increased relative abundance of this functionality was significantly associated with decreased ventral striatal fMRI responses during reward anticipation, independent of ADHD diagnosis and age. CONCLUSIONS: Our results show increases in gut microbiome predicted function of dopamine precursor synthesis between ADHD cases and controls. This increase in microbiome function relates to decreased neural responses to reward anticipation. Decreased neural reward anticipation constitutes one of the hallmarks of ADHD.

A Functional Genomics Approach to Understand Variation in Cytokine Production in Humans.

As part of the Human Functional Genomics Project, which aims to understand the factors that determine the variability of immune responses, we investigated genetic variants affecting cytokine production in response to ex vivo stimulation in two independent cohorts of 500 and 200 healthy individuals. We demonstrate a strong impact of genetic heritability on cytokine production capacity after challenge with bacterial, fungal, viral, and non-microbial stimuli. In addition to 17 novel genome-wide significant cytokine QTLs (cQTLs), our study provides a comprehensive picture of the genetic variants that influence six different cytokines in whole blood, blood mononuclear cells, and macrophages. Important biological pathways that contain cytokine QTLs map to pattern recognition receptors (TLR1-6-10 cluster), cytokine and complement inhibitors, and the kallikrein system. The cytokine QTLs show enrichment for monocyte-specific enhancers, are more often located in regions under positive selection, and are significantly enriched among SNPs associated with infections and immune-mediated diseases. PAPERCLIP.

Host and Environmental Factors Influencing Individual Human Cytokine Responses.

Differences in susceptibility to immune-mediated diseases are determined by variability in immune responses. In three studies within the Human Functional Genomics Project, we assessed the effect of environmental and non-genetic host factors of the genetic make-up of the host and of the intestinal microbiome on the cytokine responses in humans. We analyzed the association of these factors with circulating mediators and with six cytokines after stimulation with 19 bacterial, fungal, viral, and non-microbial metabolic stimuli in 534 healthy subjects. In this first study, we show a strong impact of non-genetic host factors (e.g., age and gender) on cytokine production and circulating mediators. Additionally, annual seasonality is found to be an important environmental factor influencing cytokine production. Alpha-1-antitrypsin concentrations partially mediate the seasonality of cytokine responses, whereas the effect of vitamin D levels is limited. The complete dataset has been made publicly available as a comprehensive resource for future studies. PAPERCLIP.

Linking the Human Gut Microbiome to Inflammatory Cytokine Production Capacity.

Gut microbial dysbioses are linked to aberrant immune responses, which are often accompanied by abnormal production of inflammatory cytokines. As part of the Human Functional Genomics Project (HFGP), we investigate how differences in composition and function of gut microbial communities may contribute to inter-individual variation in cytokine responses to microbial stimulations in healthy humans. We observe microbiome-cytokine interaction patterns that are stimulus specific, cytokine specific, and cytokine and stimulus specific. Validation of two predicted host-microbial interactions reveal that TNFα and IFNγ production are associated with specific microbial metabolic pathways: palmitoleic acid metabolism and tryptophan degradation to tryptophol. Besides providing a resource of predicted microbially derived mediators that influence immune phenotypes in response to common microorganisms, these data can help to define principles for understanding disease susceptibility. The three HFGP studies presented in this issue lay the groundwork for further studies aimed at understanding the interplay between microbial, genetic, and environmental factors in the regulation of the immune response in humans. PAPERCLIP.

The effect of host genetics on the gut microbiome.

The gut microbiome is affected by multiple factors, including genetics. In this study, we assessed the influence of host genetics on microbial species, pathways and gene ontology categories, on the basis of metagenomic sequencing in 1,514 subjects. In a genome-wide analysis, we identified associations of 9 loci with microbial taxonomies and 33 loci with microbial pathways and gene ontology terms at P < 5 × 10-8. Additionally, in a targeted analysis of regions involved in complex diseases, innate and adaptive immunity, or food preferences, 32 loci were identified at the suggestive level of P < 5 × 10-6. Most of our reported associations are new, including genome-wide significance for the C-type lectin molecules CLEC4F-CD207 at 2p13.3 and CLEC4A-FAM90A1 at 12p13. We also identified association of a functional LCT SNP with the Bifidobacterium genus (P = 3.45 × 10-8) and provide evidence of a gene-diet interaction in the regulation of Bifidobacterium abundance. Our results demonstrate the importance of understanding host-microbe interactions to gain better insight into human health.