Somatic mutations and T-cell clonality in patients with immunodeficiency

Common variable immunodeficiency (CVID) and other late-onset immunodeficiencies often co-manifest with autoimmunity and lymphoproliferation. The pathogenesis of most cases is elusive, as only a minor subset harbors known monogenic germline causes. The involvement of both B and T cells is, however, implicated. To study whether somatic mutations in CD4(+) and CD8(+) T cells associate with immunodeficiency, we recruited 17 patients and 21 healthy controls. Eight patients had late-onset CVID and nine patients other immunodeficiency and/or severe autoimmunity. In total, autoimmunity occurred in 94% and lymphoproliferation in 65%. We performed deep sequencing of 2,533 immune-associated genes from CD4(+) and CD8(+) cells. Deep T-cell receptor b-sequencing was used to characterize CD4(+) and CD8(+) T-cell receptor repertoires. The prevalence of somatic mutations was 65% in all immunodeficiency patients, 75% in CVID, and 48% in controls. Clonal hematopoiesis-associated variants in both CD4(+)and CD8(+) cells occurred in 24% of immunodeficiency patients. Results demonstrated mutations in known tumor suppressors, oncogenes, and genes that are critical for immuneand proliferative functions, such as STAT5B (2 patients), C5AR1 (2 patients), KRAS (one patient), and NOD2 (one patient). Additionally, as a marker of T-cell receptor repertoire perturbation, CVID patients harbored increased frequencies of clones with identical complementarity determining region 3 sequences despite unique nucleotide sequences when compared to controls. In conclusion, somatic mutations in genes implicated for autoimmunity and lymphoproliferation are common in CD4(+) and CD8(+) cells of patients with immunodeficiency. They may contribute to immune dysregulation in a subset of immunodeficiency patients.Peer reviewe

Prenatal maternal depressive symptoms are associated with smaller amygdalar volumes of four-year-old children

Prenatal maternal depressive symptoms are related to an increased offspring susceptibility to psychiatric disorders over the life course. Alterations in fetal brain development might partly mediate this association. The relation of prenatal depressive symptoms with child's amygdalar volumes is still underexplored, and this study aimed to address this gap. We explored the association of prenatal maternal depressive symptoms with amygdalar volumes in 28 4-year-old children (14 female). Amygdalar volumes were assessed using the volBrain pipeline and manual segmentation. Prenatal depressive symptoms were self-reported by mothers at gestational weeks 14, 24 and 34 (Edinburgh Postnatal Depression Scale). Sex differences were probed, and possible pre- and postnatal confounders, such as maternal general anxiety, were controlled for. We observed that elevated depressive symptoms of the early second trimester, after controlling for prenatal maternal general anxiety, were significantly related to smaller right amygdalar volumes in the whole sample. Higher depressive symptoms of the third trimester were associated with significantly smaller right amygdalar volumes in boys compared to girls. Altogether, our data suggest that offspring limbic brain development might be affected by maternal depressive symptoms in early pregnancy, and might also be more vulnerable to depressive symptoms in late pregnancy in boys compared to girls

Umbilical cord blood DNA methylation in children who later develop type 1 diabetes

Aims/hypothesis Distinct DNA methylation patterns have recently been observed to precede type 1 diabetes in whole blood collected from young children. Our aim was to determine whether perinatal DNA methylation is associated with later progression to type 1 diabetes. Methods Reduced representation bisulphite sequencing (RRBS) analysis was performed on umbilical cord blood samples collected within the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Study. Children later diagnosed with type 1 diabetes and/or who tested positive for multiple islet autoantibodies (n = 43) were compared with control individuals (n = 79) who remained autoantibody-negative throughout the DIPP follow-up until 15 years of age. Potential confounding factors related to the pregnancy and the mother were included in the analysis. Results No differences in the umbilical cord blood methylation patterns were observed between the cases and controls at a false discovery rate Conclusions/interpretation Based on our results, differences between children who progress to type 1 diabetes and those who remain healthy throughout childhood are not yet present in the perinatal DNA methylome. However, we cannot exclude the possibility that such differences would be found in a larger dataset.Peer reviewe

Genomic variation and strain-specific functional adaptation in the human gut microbiome during early life

The human gut microbiome matures towards the adult composition during the first years of life and is implicated in early immune development. Here, we investigate the effects of microbial genomic diversity on gut microbiome development using integrated early childhood data sets collected in the DIABIMMUNE study in Finland, Estonia and Russian Karelia. We show that gut microbial diversity is associated with household location and linear growth of children. Single nucleotide polymorphism- and metagenomic assembly-based strain tracking revealed large and highly dynamic microbial pangenomes, especially in the genus Bacteroides, in which we identified evidence of variability deriving from Bacteroides-targeting bacteriophages. Our analyses revealed functional consequences of strain diversity; only 10% of Finnish infants harboured Bifidobacterium longum subsp. infantis, a subspecies specialized in human milk metabolism, whereas Russian infants commonly maintained a probiotic Bifidobacterium bifidum strain in infancy. Groups of bacteria contributing to diverse, characterized metabolic pathways converged to highly subject-specific configurations over the first two years of life. This longitudinal study extends the current view of early gut microbial community assembly based on strain-level genomic variation.Peer reviewe

Mre11 modulates the fidelity of fusion between short telomeres in human cells

The loss of telomere function can result in the fusion of telomeres with other telomeric loci, or non-telomeric double-stranded DNA breaks. Sequence analysis of fusion events between short dysfunctional telomeres in human cells has revealed that fusion is characterized by a distinct molecular signature consisting of extensive deletions and micro-homology at the fusion points. This signature is consistent with alternative error-prone end-joining processes. We have examined the role that Mre11 may play in the fusion of short telomeres in human cells; to do this, we have analysed telomere fusion events in cells derived from ataxia-telangiectasia-like disorder (ATLD) patients that exhibit hypomorphic mutations in MRE11. The telomere dynamics of ATLD fibroblasts were indistinguishable from wild-type fibroblasts and they were proficient in the fusion of short telomeres. However, we observed a high frequency of insertion of DNA sequences at the fusion points that created localized sequence duplications. These data indicate that Mre11 plays a role in the fusion of short dysfunctional telomeres in human cells and are consistent with the hypothesis that as part of the MRN complex it serves to stabilize the joining complex, thereby controlling the fidelity of the fusion reaction

RNA-Seq Differentiates Tumour and Host mRNA Expression Changes Induced by Treatment of Human Tumour Xenografts with the VEGFR Tyrosine Kinase Inhibitor Cediranib.

Pre-clinical models of tumour biology often rely on propagating human tumour cells in a mouse. In order to gain insight into the alignment of these models to human disease segments or investigate the effects of different therapeutics, approaches such as PCR or array based expression profiling are often employed despite suffering from biased transcript coverage, and a requirement for specialist experimental protocols to separate tumour and host signals. Here, we describe a computational strategy to profile transcript expression in both the tumour and host compartments of pre-clinical xenograft models from the same RNA sample using RNA-Seq. Key to this strategy is a species-specific mapping approach that removes the need for manipulation of the RNA population, customised sequencing protocols, or prior knowledge of the species component ratio. The method demonstrates comparable performance to species-specific RT-qPCR and a standard microarray platform, and allowed us to quantify gene expression changes in both the tumour and host tissue following treatment with cediranib, a potent vascular endothelial growth factor receptor tyrosine kinase inhibitor, including the reduction of multiple murine transcripts associated with endothelium or vessels, and an increase in genes associated with the inflammatory response in response to cediranib. In the human compartment, we observed a robust induction of hypoxia genes and a reduction in cell cycle associated transcripts. In conclusion, the study establishes that RNA-Seq can be applied to pre-clinical models to gain deeper understanding of model characteristics and compound mechanism of action, and to identify both tumour and host biomarkers

Sex-specific association between infant caudate volumes and a polygenic risk score for major depressive disorder

Polygenic risk scores for major depressive disorder (PRS-MDD) have been identified in large genome-wide association studies, and recent findings suggest that PRS-MDD might interact with environmental risk factors to shape human limbic brain development as early as in the prenatal period. Striatal structures are crucially involved in depression; however, the association of PRS-MDD with infant striatal volumes is yet unknown. In this study, 105 Finnish mother-infant dyads (44 female, 11-54 days old) were investigated to reveal how infant PRS-MDD is associated with infant dorsal striatal volumes (caudate, putamen) and whether PRS-MDD interacts with prenatal maternal depressive symptoms (Edinburgh Postnatal Depression Scale, gestational weeks 14, 24, 34) on infant striatal volumes. A robust sex-specific main effect of PRS-MDD on bilateral infant caudate volumes was observed. PRS-MDD were more positively associated with caudate volumes in boys compared to girls. No significant interaction effects of genotype PRS-MDD with the environmental risk factor "prenatal maternal depressive symptoms" (genotype-by-environment interaction) nor significant interaction effects of genotype with prenatal maternal depressive symptoms and sex (genotype-by-environment-by-sex interaction) were found for infant dorsal striatal volumes. Our study showed that a higher PRS-MDD irrespective of prenatal exposure to maternal depressive symptoms is associated with smaller bilateral caudate volumes, an indicator of greater susceptibility to major depressive disorder, in female compared to male infants. This sex-specific polygenic effect might lay the ground for the higher prevalence of depression in women compared to men

Biomarker discovery in heterogeneous tissue samples -taking the in-silico deconfounding approach

<p>Abstract</p> <p>Background</p> <p>For heterogeneous tissues, such as blood, measurements of gene expression are confounded by relative proportions of cell types involved. Conclusions have to rely on estimation of gene expression signals for homogeneous cell populations, e.g. by applying micro-dissection, fluorescence activated cell sorting, or <it>in-silico </it>deconfounding. We studied feasibility and validity of a non-negative matrix decomposition algorithm using experimental gene expression data for blood and sorted cells from the same donor samples. Our objective was to optimize the algorithm regarding detection of differentially expressed genes and to enable its use for classification in the difficult scenario of reversely regulated genes. This would be of importance for the identification of candidate biomarkers in heterogeneous tissues.</p> <p>Results</p> <p>Experimental data and simulation studies involving noise parameters estimated from these data revealed that for valid detection of differential gene expression, quantile normalization and use of non-log data are optimal. We demonstrate the feasibility of predicting proportions of constituting cell types from gene expression data of single samples, as a prerequisite for a deconfounding-based classification approach.</p> <p>Classification cross-validation errors with and without using deconfounding results are reported as well as sample-size dependencies. Implementation of the algorithm, simulation and analysis scripts are available.</p> <p>Conclusions</p> <p>The deconfounding algorithm without decorrelation using quantile normalization on non-log data is proposed for biomarkers that are difficult to detect, and for cases where confounding by varying proportions of cell types is the suspected reason. In this case, a deconfounding ranking approach can be used as a powerful alternative to, or complement of, other statistical learning approaches to define candidate biomarkers for molecular diagnosis and prediction in biomedicine, in realistically noisy conditions and with moderate sample sizes.</p

Systematic Bias in Genomic Classification Due to Contaminating Non-neoplastic Tissue in Breast Tumor Samples

Abstract Background Genomic tests are available to predict breast cancer recurrence and to guide clinical decision making. These predictors provide recurrence risk scores along with a measure of uncertainty, usually a confidence interval. The confidence interval conveys random error and not systematic bias. Standard tumor sampling methods make this problematic, as it is common to have a substantial proportion (typically 30-50%) of a tumor sample comprised of histologically benign tissue. This "normal" tissue could represent a source of non-random error or systematic bias in genomic classification. Methods To assess the performance characteristics of genomic classification to systematic error from normal contamination, we collected 55 tumor samples and paired tumor-adjacent normal tissue. Using genomic signatures from the tumor and paired normal, we evaluated how increasing normal contamination altered recurrence risk scores for various genomic predictors. Results Simulations of normal tissue contamination caused misclassification of tumors in all predictors evaluated, but different breast cancer predictors showed different types of vulnerability to normal tissue bias. While two predictors had unpredictable direction of bias (either higher or lower risk of relapse resulted from normal contamination), one signature showed predictable direction of normal tissue effects. Due to this predictable direction of effect, this signature (the PAM50) was adjusted for normal tissue contamination and these corrections improved sensitivity and negative predictive value. For all three assays quality control standards and/or appropriate bias adjustment strategies can be used to improve assay reliability. Conclusions Normal tissue sampled concurrently with tumor is an important source of bias in breast genomic predictors. All genomic predictors show some sensitivity to normal tissue contamination and ideal strategies for mitigating this bias vary depending upon the particular genes and computational methods used in the predictor

Catalytic Mechanism of Bacteriophage T4 Rad50 ATP Hydrolysis

Spontaneous double-strand breaks (DSBs) are one of the most deleterious forms of DNA damage, and their improper repair can lead to cellular dysfunction. The Mre11 and Rad50 proteins, a nuclease and an ATPase, respectively, form a well-conserved complex that is involved in the initial processing of DSBs. Here we examine the kinetic and catalytic mechanism of ATP hydrolysis by T4 Rad50 (gp46) in the presence and absence of Mre11 (gp47) and DNA. Single-turnover and pre-steady state kinetics on the wild-type protein indicate that the rate-limiting step for Rad50, the MR complex, and the MR-DNA complex is either chemistry or a conformational change prior to catalysis. Pre-steady state product release kinetics, coupled with viscosity steady state kinetics, also supports that the binding of DNA to the MR complex does not alter the rate-limiting step. The lack of a positive deuterium solvent isotope effect for the wild type and several active site mutants, combined with pH–rate profiles, implies that chemistry is rate-limiting and the ATPase mechanism proceeds via an asymmetric, dissociative-like transition state. Mutation of the Walker A/B and H-loop residues also affects the allosteric communication between Rad50 active sites, suggesting possible routes for cooperativity between the ATP active sites