Mitotic DNA synthesis is caused by transcription-replication conflicts in BRCA2-deficient cells.

Aberrant replication causes cells lacking BRCA2 to enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS). Here, we identify genome-wide the sites where MiDAS reactions occur when BRCA2 is abrogated. High-resolution profiling revealed that these sites are different from MiDAS at aphidicolin-induced common fragile sites in that they map to genomic regions replicating in the early S-phase, which are close to early-firing replication origins, are highly transcribed, and display R-loop-forming potential. Both transcription inhibition in early S-phase and RNaseH1 overexpression reduced MiDAS in BRCA2-deficient cells, indicating that transcription-replication conflicts (TRCs) and R-loops are the source of MiDAS. Importantly, the MiDAS sites identified in BRCA2-deficient cells also represent hotspots for genomic rearrangements in BRCA2-mutated breast tumors. Thus, our work provides a mechanism for how tumor-predisposing BRCA2 inactivation links transcription-induced DNA damage with mitotic DNA repair to fuel the genomic instability characteristic of cancer cells.

Metabolic profiling of aortic stenosis and hypertrophic cardiomyopathy identifies mechanistic contrasts in substrate utilization.

Aortic stenosis (AS) and hypertrophic cardiomyopathy (HCM) are distinct disorders leading to left ventricular hypertrophy (LVH), but whether cardiac metabolism substantially differs between these in humans remains to be elucidated. We undertook an invasive (aortic root, coronary sinus) metabolic profiling in patients with severe AS and HCM in comparison with non-LVH controls to investigate cardiac fuel selection and metabolic remodeling. These patients were assessed under different physiological states (at rest, during stress induced by pacing). The identified changes in the metabolome were further validated by metabolomic and orthogonal transcriptomic analysis, in separately recruited patient cohorts. We identified a highly discriminant metabolomic signature in severe AS in all samples, regardless of sampling site, characterized by striking accumulation of long-chain acylcarnitines, intermediates of fatty acid transport across the inner mitochondrial membrane, and validated this in a separate cohort. Mechanistically, we identify a downregulation in the PPAR-α transcriptional network, including expression of genes regulating fatty acid oxidation (FAO). In silico modeling of ß-oxidation demonstrated that flux could be inhibited by both the accumulation of fatty acids as a substrate for mitochondria and the accumulation of medium-chain carnitines which induce competitive inhibition of the acyl-CoA dehydrogenases. We present a comprehensive analysis of changes in the metabolic pathways (transcriptome to metabolome) in severe AS, and its comparison to HCM. Our results demonstrate a progressive impairment of ß-oxidation from HCM to AS, particularly for FAO of long-chain fatty acids, and that the PPAR-α signaling network may be a specific metabolic therapeutic target in AS.

53BP1 Integrates DNA Repair and p53-Dependent Cell Fate Decisions via Distinct Mechanisms.

The tumor suppressor protein 53BP1, a pivotal regulator of DNA double-strand break (DSB) repair, was first identified as a p53-interacting protein over two decades ago. However, its direct contributions to p53-dependent cellular activities remain undefined. Here, we reveal that 53BP1 stimulates genome-wide p53-dependent gene transactivation and repression events in response to ionizing radiation (IR) and synthetic p53 activation. 53BP1-dependent p53 modulation requires both auto-oligomerization and tandem-BRCT domain-mediated bivalent interactions with p53 and the ubiquitin-specific protease USP28. Loss of these activities results in inefficient p53-dependent cell-cycle checkpoint and exit responses. Furthermore, we demonstrate 53BP1-USP28 cooperation to be essential for normal p53-promoter element interactions and gene transactivation-associated events, yet dispensable for 53BP1-dependent DSB repair regulation. Collectively, our data provide a mechanistic explanation for 53BP1-p53 cooperation in controlling anti-tumorigenic cell-fate decisions and reveal these activities to be distinct and separable from 53BP1's regulation of DNA double-strand break repair pathway choice.

IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses.

Polymorphisms in the gene encoding the transcription factor IRF5 that lead to higher mRNA expression are associated with many autoimmune diseases. Here we show that IRF5 expression in macrophages was reversibly induced by inflammatory stimuli and contributed to the plasticity of macrophage polarization. High expression of IRF5 was characteristic of M1 macrophages, in which it directly activated transcription of the genes encoding interleukin 12 subunit p40 (IL-12p40), IL-12p35 and IL-23p19 and repressed the gene encoding IL-10. Consequently, those macrophages set up the environment for a potent T helper type 1 (T(H)1)-T(H)17 response. Global gene expression analysis demonstrated that exogenous IRF5 upregulated or downregulated expression of established phenotypic markers of M1 or M2 macrophages, respectively. Our data suggest a critical role for IRF5 in M1 macrophage polarization and define a previously unknown function for IRF5 as a transcriptional repressor.

Using de novo assembly to identify structural variation of eight complex immune system gene regions.

Driven by the necessity to survive environmental pathogens, the human immune system has evolved exceptional diversity and plasticity, to which several factors contribute including inheritable structural polymorphism of the underlying genes. Characterizing this variation is challenging due to the complexity of these loci, which contain extensive regions of paralogy, segmental duplication and high copy-number repeats, but recent progress in long-read sequencing and optical mapping techniques suggests this problem may now be tractable. Here we assess this by using long-read sequencing platforms from PacBio and Oxford Nanopore, supplemented with short-read sequencing and Bionano optical mapping, to sequence DNA extracted from CD14+ monocytes and peripheral blood mononuclear cells from a single European individual identified as HV31. We use this data to build a de novo assembly of eight genomic regions encoding four key components of the immune system, namely the human leukocyte antigen, immunoglobulins, T cell receptors, and killer-cell immunoglobulin-like receptors. Validation of our assembly using k-mer based and alignment approaches suggests that it has high accuracy, with estimated base-level error rates below 1 in 10 kb, although we identify a small number of remaining structural errors. We use the assembly to identify heterozygous and homozygous structural variation in comparison to GRCh38. Despite analyzing only a single individual, we find multiple large structural variants affecting core genes at all three immunoglobulin regions and at two of the three T cell receptor regions. Several of these variants are not accurately callable using current algorithms, implying that further methodological improvements are needed. Our results demonstrate that assessing haplotype variation in these regions is possible given sufficiently accurate long-read and associated data. Continued reductions in the cost of these technologies will enable application of these methods to larger samples and provide a broader catalogue of germline structural variation at these loci, an important step toward making these regions accessible to large-scale genetic association studies.

Impact of spliceosome mutations on RNA splicing in myelodysplasia: dysregulated genes/pathways and clinical associations.

SF3B1, SRSF2, and U2AF1 are the most frequently mutated splicing factor genes in the myelodysplastic syndromes (MDS). We have performed a comprehensive and systematic analysis to determine the effect of these commonly mutated splicing factors on pre-mRNA splicing in the bone marrow stem/progenitor cells and in the erythroid and myeloid precursors in splicing factor mutant MDS. Using RNA-seq, we determined the aberrantly spliced genes and dysregulated pathways in CD34+ cells of 84 patients with MDS. Splicing factor mutations result in different alterations in splicing and largely affect different genes, but these converge in common dysregulated pathways and cellular processes, focused on RNA splicing, protein synthesis, and mitochondrial dysfunction, suggesting common mechanisms of action in MDS. Many of these dysregulated pathways and cellular processes can be linked to the known disease pathophysiology associated with splicing factor mutations in MDS, whereas several others have not been previously associated with MDS, such as sirtuin signaling. We identified aberrantly spliced events associated with clinical variables, and isoforms that independently predict survival in MDS and implicate dysregulation of focal adhesion and extracellular exosomes as drivers of poor survival. Aberrantly spliced genes and dysregulated pathways were identified in the MDS-affected lineages in splicing factor mutant MDS. Functional studies demonstrated that knockdown of the mitosis regulators SEPT2 and AKAP8, aberrantly spliced target genes of SF3B1 and SRSF2 mutations, respectively, led to impaired erythroid cell growth and differentiation. This study illuminates the effect of the common spliceosome mutations on the MDS phenotype and provides novel insights into disease pathophysiology.

Use of gene expression studies to investigate the human immunological response to malaria infection.

BACKGROUND: Transcriptional profiling of the human immune response to malaria has been used to identify diagnostic markers, understand the pathogenicity of severe disease and dissect the mechanisms of naturally acquired immunity (NAI). However, interpreting this body of work is difficult given considerable variation in study design, definition of disease, patient selection and methodology employed. This work details a comprehensive review of gene expression profiling (GEP) of the human immune response to malaria to determine how this technology has been applied to date, instances where this has advanced understanding of NAI and the extent of variability in methodology between studies to allow informed comparison of data and interpretation of results. METHODS: Datasets from the gene expression omnibus (GEO) including the search terms; 'plasmodium' or 'malaria' or 'sporozoite' or 'merozoite' or 'gametocyte' and 'Homo sapiens' were identified and publications analysed. Datasets of gene expression changes in relation to malaria vaccines were excluded. RESULTS: Twenty-three GEO datasets and 25 related publications were included in the final review. All datasets related to Plasmodium falciparum infection, except two that related to Plasmodium vivax infection. The majority of datasets included samples from individuals infected with malaria 'naturally' in the field (n = 13, 57%), however some related to controlled human malaria infection (CHMI) studies (n = 6, 26%), or cells stimulated with Plasmodium in vitro (n = 6, 26%). The majority of studies examined gene expression changes relating to the blood stage of the parasite. Significant heterogeneity between datasets was identified in terms of study design, sample type, platform used and method of analysis. Seven datasets specifically investigated transcriptional changes associated with NAI to malaria, with evidence supporting suppression of the innate pro-inflammatory response as an important mechanism for this in the majority of these studies. However, further interpretation of this body of work was limited by heterogeneity between studies and small sample sizes. CONCLUSIONS: GEP in malaria is a potentially powerful tool, but to date studies have been hypothesis generating with small sample sizes and widely varying methodology. As CHMI studies are increasingly performed in endemic settings, there will be growing opportunity to use GEP to understand detailed time-course changes in host response and understand in greater detail the mechanisms of NAI.

The nature and nurture of cell heterogeneity: accounting for macrophage gene-environment interactions with single-cell RNA-Seq.

BACKGROUND: Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity, despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive, only cataloging heterogeneity at one point in time, and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity, and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages. RESULTS: We introduce the programmable Polaris™ microfluidic lab-on-chip for single-cell sequencing, which performs live-cell imaging while controlling for the culture microenvironment of each cell. Using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of SAMHD1, such as an altered oxidative stress response, have a large paracrine signaling component. Furthermore, we demonstrate single-cell pathway enrichments for cell cycle arrest and APOBEC3G degradation, both associated with the oxidative stress response and altered proteostasis. Interestingly, SAMHD1 and APOBEC3G are both HIV-1 inhibitors ('restriction factors'), with no known co-regulation. CONCLUSION: As single-cell methods continue to mature, so will the ability to move beyond simple 'snapshots' of cell populations towards studying the determinants of population dynamics. By combining single-cell culture, live-cell imaging, and single-cell sequencing, we have demonstrated the ability to study cell phenotypes and microenvironmental influences. It's these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages, for example, react to inflammation and form treatment resistant HIV reservoirs.

Application of whole genome and RNA sequencing to investigate the genomic landscape of common variable immunodeficiency disorders.

Common Variable Immunodeficiency Disorders (CVIDs) are the most prevalent cause of primary antibody failure. CVIDs are highly variable and a genetic causes have been identified in <5% of patients. Here, we performed whole genome sequencing (WGS) of 34 CVID patients (94% sporadic) and combined them with transcriptomic profiling (RNA-sequencing of B cells) from three patients and three healthy controls. We identified variants in CVID disease genes TNFRSF13B, TNFRSF13C, LRBA and NLRP12 and enrichment of variants in known and novel disease pathways. The pathways identified include B-cell receptor signalling, non-homologous end-joining, regulation of apoptosis, T cell regulation and ICOS signalling. Our data confirm the polygenic nature of CVID and suggest individual-specific aetiologies in many cases. Together our data show that WGS in combination with RNA-sequencing allows for a better understanding of CVIDs and the identification of novel disease associated pathways.

DOCK8 is critical for the survival and function of NKT cells.

Patients with the dedicator of cytokinesis 8 (DOCK8) immunodeficiency syndrome suffer from recurrent viral and bacterial infections, hyper-immunoglobulin E levels, eczema, and greater susceptibility to cancer. Because natural killer T (NKT) cells have been implicated in these diseases, we asked if these cells were affected by DOCK8 deficiency. Using a mouse model, we found that DOCK8 deficiency resulted in impaired NKT cell development, principally affecting the formation and survival of long-lived, differentiated NKT cells. In the thymus, DOCK8-deficient mice lack a terminally differentiated subset of NK1.1(+) NKT cells expressing the integrin CD103, whereas in the liver, DOCK8-deficient NKT cells express reduced levels of the prosurvival factor B-cell lymphoma 2 and the integrin lymphocyte function-associated antigen 1. Although the initial NKT cell response to antigen is intact in the absence of DOCK8, their ongoing proliferative and cytokine responses are impaired. Importantly, a similar defect in NKT cell numbers was detected in DOCK8-deficient humans, highlighting the relevance of the mouse model. In conclusion, our data demonstrate that DOCK8 is required for the development and survival of mature NKT cells, consistent with the idea that DOCK8 mediates survival signals within a specialized niche. Accordingly, impaired NKT cell numbers and function are likely to contribute to the susceptibility of DOCK8-deficient patients to recurrent infections and malignant disease.

Pervasive haplotypic variation in the spliceo-transcriptome of the human major histocompatibility complex.

The human major histocompatibility complex (MHC) on chromosome 6p21 is a paradigm for genomics, showing remarkable polymorphism and striking association with immune and non-immune diseases. The complex genomic landscape of the MHC, notably strong linkage disequilibrium, has made resolving causal variants very challenging. A promising approach is to investigate gene expression levels considered as tractable intermediate phenotypes in mapping complex diseases. However, how transcription varies across the MHC, notably relative to specific haplotypes, remains unknown. Here, using an original hybrid tiling and splice junction microarray that includes alternate allele probes, we draw the first high-resolution strand-specific transcription map for three common MHC haplotypes (HLA-A1-B8-Cw7-DR3, HLA-A3-B7-Cw7-DR15, and HLA-A26-B18-Cw5-DR3-DQ2) strongly associated with autoimmune diseases including type 1 diabetes, systemic lupus erythematosus, and multiple sclerosis. We find that haplotype-specific differences in gene expression are common across the MHC, affecting 96 genes (46.4%), most significantly the zing finger protein gene ZFP57. Differentially expressed probes are correlated with polymorphisms between haplotypes, consistent with cis effects that we directly demonstrate for ZFP57 in a cohort of healthy volunteers (P = 1.2 × 10(-14)). We establish that alternative splicing is significantly more frequent in the MHC than genome-wide (72.5% vs. 62.1% of genes, P ≤ 1 × 10(-4)) and shows marked haplotypic differences. We also unmask novel and abundant intergenic transcription involving 31% of transcribed blocks identified. Our study reveals that the renowned MHC polymorphism also manifests as transcript diversity, and our novel haplotype-based approach marks a new step toward identification of regulatory variants involved in the control of MHC-associated phenotypes and diseases.

Use of multivariate analysis to suggest a new molecular classification of colorectal cancer.

Molecular classification of colorectal cancer (CRC) is currently based on microsatellite instability (MSI), KRAS or BRAF mutation and, occasionally, chromosomal instability (CIN). Whilst useful, these categories may not fully represent the underlying molecular subgroups. We screened 906 stage II/III CRCs from the VICTOR clinical trial for somatic mutations. Multivariate analyses (logistic regression, clustering, Bayesian networks) identified the primary molecular associations. Positive associations occurred between: CIN and TP53 mutation; MSI and BRAF mutation; and KRAS and PIK3CA mutations. Negative associations occurred between: MSI and CIN; MSI and NRAS mutation; and KRAS mutation, and each of NRAS, TP53 and BRAF mutations. Some complex relationships were elucidated: KRAS and TP53 mutations had both a direct negative association and a weaker, confounding, positive association via TP53-CIN-MSI-BRAF-KRAS. Our results suggested a new molecular classification of CRCs: (1) MSI(+) and/or BRAF-mutant; (2) CIN(+) and/or TP53(-) mutant, with wild-type KRAS and PIK3CA; (3) KRAS- and/or PIK3CA-mutant, CIN(+) , TP53-wild-type; (4) KRAS(-) and/or PIK3CA-mutant, CIN(-) , TP53-wild-type; (5) NRAS-mutant; (6) no mutations; (7) others. As expected, group 1 cancers were mostly proximal and poorly differentiated, usually occurring in women. Unexpectedly, two different types of CIN(+) CRC were found: group 2 cancers were usually distal and occurred in men, whereas group 3 showed neither of these associations but were of higher stage. CIN(+) cancers have conventionally been associated with all three of these variables, because they have been tested en masse. Our classification also showed potentially improved prognostic capabilities, with group 3, and possibly group 1, independently predicting disease-free survival.

Foxp2 regulates gene networks implicated in neurite outgrowth in the developing brain.

Forkhead-box protein P2 is a transcription factor that has been associated with intriguing aspects of cognitive function in humans, non-human mammals, and song-learning birds. Heterozygous mutations of the human FOXP2 gene cause a monogenic speech and language disorder. Reduced functional dosage of the mouse version (Foxp2) causes deficient cortico-striatal synaptic plasticity and impairs motor-skill learning. Moreover, the songbird orthologue appears critically important for vocal learning. Across diverse vertebrate species, this well-conserved transcription factor is highly expressed in the developing and adult central nervous system. Very little is known about the mechanisms regulated by Foxp2 during brain development. We used an integrated functional genomics strategy to robustly define Foxp2-dependent pathways, both direct and indirect targets, in the embryonic brain. Specifically, we performed genome-wide in vivo ChIP-chip screens for Foxp2-binding and thereby identified a set of 264 high-confidence neural targets under strict, empirically derived significance thresholds. The findings, coupled to expression profiling and in situ hybridization of brain tissue from wild-type and mutant mouse embryos, strongly highlighted gene networks linked to neurite development. We followed up our genomics data with functional experiments, showing that Foxp2 impacts on neurite outgrowth in primary neurons and in neuronal cell models. Our data indicate that Foxp2 modulates neuronal network formation, by directly and indirectly regulating mRNAs involved in the development and plasticity of neuronal connections.

Identification of cellular retinoic acid binding protein 2 (CRABP2) as downstream target of nuclear factor I/X (NFIX): implications for skeletal dysplasia syndromes.

Nuclear factor I/X (NFIX) mutations are associated with 2 skeletal dysplasias, Marshall-Smith (MSS) and Malan (MAL) syndromes. NFIX encodes a transcription factor that regulates expression of genes, including Bobby sox (BBX) and glial fibrillary acidic protein (GFAP) in neural progenitor cells and astrocytes, respectively. To elucidate the role of NFIX mutations in MSS, we studied their effects in fibroblast cell lines obtained from 5 MSS unrelated patients and 3 unaffected individuals. The 5 MSS NFIX frameshift mutations in exons 6-8 comprised 3 deletions (c.819-732_1079-948del, c.819-471_1079-687del, c.819-592_1079-808del), an insertion (c.1037_1038insT), and a duplication (c.1090dupG). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses using MSS and unrelated control fibroblasts and in vitro expression studies in monkey kidney fibroblast (COS-7) cells showed that frameshift mutations in NFIX exons 6-8 generated mutant transcripts that were not cleared by nonsense-mediated-decay mechanisms and encoded truncated NFIX proteins. Moreover, BBX or GFAP expression was unaffected in the majority of MSS fibroblasts. To identify novel NFIX downstream target genes, RNA sequencing and proteomics analyses were performed on mouse embryonic fibroblast (MEF) cells derived from control Nfix+/+, Nfix+/Del2, Nfix+/Del24, NfixDel24/Del24, Nfix+/Del140, and NfixDel140/Del140 mice, compared with NfixDel2/Del2 mice which had developmental, skeletal, and neural abnormalities. This identified 191 transcripts and 815 proteins misregulated in NfixDel2/Del2 MEFs with ≥2-fold-change (P <0 .05). Validation studies using qRT-PCR and western blot analyses confirmed that 2 genes, cellular retinoic acid binding protein 2 (Crabp2) and vascular cell adhesion molecule 1 (Vcam1), were misregulated at the RNA and protein levels in NfixDel2/Del2 MEFs, and that CRABP2 and VCAM1 expressions were altered in 60%-100% of MSS fibroblast cells. Furthermore, in vitro luciferase reporter assays confirmed that NFIX directly regulates CRABP2 promoter activity. Thus, these altered genes and pathways may represent possible targets for drugs as potential treatments and therapies for MSS.

Exon array data analysis using Affymetrix power tools and R statistical software.

The use of microarray technology to measure gene expression on a genome-wide scale has been well established for more than a decade. Methods to process and analyse the vast quantity of expression data generated by a typical microarray experiment are similarly well-established. The Affymetrix Exon 1.0 ST array is a relatively new type of array, which has the capability to assess expression at the individual exon level. This allows a more comprehensive analysis of the transcriptome, and in particular enables the study of alternative splicing, a gene regulation mechanism important in both normal conditions and in diseases. Some aspects of exon array data analysis are shared with those for standard gene expression data but others present new challenges that have required development of novel tools. Here, I will introduce the exon array and present a detailed example tutorial for analysis of data generated using this platform.

Analysis of CD161 expression on human CD8+ T cells defines a distinct functional subset with tissue-homing properties.

CD8(+) T lymphocytes play a key role in host defense, in particular against important persistent viruses, although the critical functional properties of such cells in tissue are not fully defined. We have previously observed that CD8(+) T cells specific for tissue-localized viruses such as hepatitis C virus express high levels of the C-type lectin CD161. To explore the significance of this, we examined CD8(+)CD161(+) T cells in healthy donors and those with hepatitis C virus and defined a population of CD8(+) T cells with distinct homing and functional properties. These cells express high levels of CD161 and a pattern of molecules consistent with type 17 differentiation, including cytokines (e.g., IL-17, IL-22), transcription factors (e.g., retinoic acid-related orphan receptor gamma-t, P = 6 x 10(-9); RUNX2, P = 0.004), cytokine receptors (e.g., IL-23R, P = 2 x 10(-7); IL-18 receptor, P = 4 x 10(-6)), and chemokine receptors (e.g., CCR6, P = 3 x 10(-8); CXCR6, P = 3 x 10(-7); CCR2, P = 4 x 10(-7)). CD161(+)CD8(+) T cells were markedly enriched in tissue samples and coexpressed IL-17 with high levels of IFN-gamma and/or IL-22. The levels of polyfunctional cells in tissue was most marked in those with mild disease (P = 0.0006). These data define a T cell lineage that is present already in cord blood and represents as many as one in six circulating CD8(+) T cells in normal humans and a substantial fraction of tissue-infiltrating CD8(+) T cells in chronic inflammation. Such cells play a role in the pathogenesis of chronic hepatitis and arthritis and potentially in other infectious and inflammatory diseases of man.

A Mouse Model with a Frameshift Mutation in the Nuclear Factor I/X (NFIX) Gene Has Phenotypic Features of Marshall-Smith Syndrome.

The nuclear factor I/X (NFIX) gene encodes a ubiquitously expressed transcription factor whose mutations lead to two allelic disorders characterized by developmental, skeletal, and neural abnormalities, namely, Malan syndrome (MAL) and Marshall-Smith syndrome (MSS). NFIX mutations associated with MAL mainly cluster in exon 2 and are cleared by nonsense-mediated decay (NMD) leading to NFIX haploinsufficiency, whereas NFIX mutations associated with MSS are clustered in exons 6-10 and escape NMD and result in the production of dominant-negative mutant NFIX proteins. Thus, different NFIX mutations have distinct consequences on NFIX expression. To elucidate the in vivo effects of MSS-associated NFIX exon 7 mutations, we used CRISPR-Cas9 to generate mouse models with exon 7 deletions that comprised: a frameshift deletion of two nucleotides (Nfix Del2); in-frame deletion of 24 nucleotides (Nfix Del24); and deletion of 140 nucleotides (Nfix Del140). Nfix +/Del2, Nfix +/Del24, Nfix +/Del140, Nfix Del24/Del24, and Nfix Del140/Del140 mice were viable, normal, and fertile, with no skeletal abnormalities, but Nfix Del2/Del2 mice had significantly reduced viability (p < 0.002) and died at 2-3 weeks of age. Nfix Del2 was not cleared by NMD, and NfixDel2/Del2 mice, when compared to Nfix +/+ and Nfix +/Del2 mice, had: growth retardation; short stature with kyphosis; reduced skull length; marked porosity of the vertebrae with decreased vertebral and femoral bone mineral content; and reduced caudal vertebrae height and femur length. Plasma biochemistry analysis revealed Nfix Del2/Del2 mice to have increased total alkaline phosphatase activity but decreased C-terminal telopeptide and procollagen-type-1-N-terminal propeptide concentrations compared to Nfix +/+ and Nfix +/Del2 mice. Nfix Del2/Del2 mice were also found to have enlarged cerebral cortices and ventricular areas but smaller dentate gyrus compared to Nfix +/+ mice. Thus, Nfix Del2/Del2 mice provide a model for studying the in vivo effects of NFIX mutants that escape NMD and result in developmental abnormalities of the skeletal and neural tissues that are associated with MSS. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Maternal high-fat diet interacts with embryonic Cited2 genotype to reduce Pitx2c expression and enhance penetrance of left-right patterning defects.

Deficiency of the transcription factor Cited2 in mice results in cardiac malformation, adrenal agenesis, neural tube, placental defects and partially penetrant cardiopulmonary laterality defects resulting from an abnormal Nodal->Pitx2c pathway. Here we show that a maternal high-fat diet more than doubles the penetrance of laterality defects and, surprisingly, induces palatal clefting in Cited2-deficient embryos. Both maternal diet and Cited2 deletion reduce embryo weight and kidney and thymus volume. Expression profiling identified 40 embryonic transcripts including Pitx2 that were significantly affected by embryonic genotype-maternal diet interaction. We show that a high-fat diet reduces Pitx2c levels >2-fold in Cited2-deficient embryos. Taken together, these results define a novel interaction between maternal high-fat diet and embryonic Cited2 deficiency that affects Pitx2c expression and results in abnormal laterality. They suggest that appropriate modifications of maternal diet may prevent such defects in humans.

RNA-seq analysis of the human surfactant air-liquid interface culture reveals alveolar type II cell-like transcriptome.

Understanding pulmonary diseases requires robust culture models that are reproducible, sustainable in long-term culture, physiologically relevant, and suitable for assessment of therapeutic interventions. Primary human lung cells are physiologically relevant but cannot be cultured in vitro long term and, although engineered organoids are an attractive choice, they do not phenotypically recapitulate the lung parenchyma; overall, these models do not allow for the generation of reliable disease models. Recently, we described a new cell culture platform based on H441 cells that are grown at the air-liquid interface to produce the SALI culture model, for studying and correcting the rare interstitial lung disease surfactant protein B (SPB) deficiency. Here, we report the characterization of the effects of SALI culture conditions on the transcriptional profile of the constituent H441 cells. We further analyze the transcriptomics of the model in the context of surfactant metabolism and the disease phenotype through SFTPB knockout SALI cultures. By comparing the gene expression profile of SALI cultures with that of human lung parenchyma obtained via single-cell RNA sequencing, we found that SALI cultures are remarkably similar to human alveolar type II cells, implying clinical relevance of the SALI culture platform as a non-diseased human lung alveolar cell model.

miR-3156-5p is downregulated in serum of MEN1 patients and regulates expression of MORF4L2.

Multiple endocrine neoplasia type 1 (MEN1), caused by mutations in the MEN1 gene encoding menin, is an autosomal dominant disorder characterised by the combined occurrence of parathyroid, pituitary and pancreatic neuroendocrine tumours (NETs). Development of these tumours is associated with wide variations in their severity, order and ages (from <5 to >80 years), requiring life-long screening. To improve tumour surveillance and quality of life, better circulating biomarkers, particularly for pancreatic NETs that are associated with higher mortality, are required. We, therefore, examined the expression of circulating miRNA in the serum of MEN1 patients. Initial profiling analysis followed by qRT-PCR validation studies identified miR-3156-5p to be significantly downregulated (-1.3 to 5.8-fold, P < 0.05-0.0005) in nine MEN1 patients, compared to matched unaffected relatives. MEN1 knock-down experiments in BON-1 human pancreatic NET cells resulted in reduced MEN1 (49%, P < 0.05), menin (54%, P < 0.05) and miR-3156-5p expression (20%, P < 0.005), compared to control-treated cells, suggesting that miR-3156-5p downregulation is a consequence of loss of MEN1 expression. In silico analysis identified mortality factor 4-like 2 (MOR4FL2) as a potential target of miR-3156-5p, and in vitro functional studies in BON-1 cells transfected with either miR-3156-5p mimic or inhibitors showed that the miR-3156-5p mimic significantly reduced MORF4L2 protein expression (46%, P < 0.005), while miR-3156-5p inhibitor significantly increased MORF4L2 expression (1.5-fold, P < 0.05), compared to control-treated cells, thereby confirming that miR-3156-5p regulates MORF4L2 expression. Thus, the inverse relationship between miR-3156-5p and MORF4L2 expression represents a potential serum biomarker that could facilitate the detection of NET occurrence in MEN1 patients.