The association of GNB5 with Alzheimer disease revealed by genomic analysis restricted to variants impacting gene function.

Disease-associated variants identified from genome-wide association studies (GWASs) frequently map to non-coding areas of the genome such as introns and intergenic regions. An exclusive reliance on gene-agnostic methods of genomic investigation could limit the identification of relevant genes associated with polygenic diseases such as Alzheimer disease (AD). To overcome such potential restriction, we developed a gene-constrained analytical method that considers only moderate- and high-risk variants that affect gene coding sequences. We report here the application of this approach to publicly available datasets containing 181,388 individuals without and with AD and the resulting identification of 660 genes potentially linked to the higher AD prevalence among Africans/African Americans. By integration with transcriptome analysis of 23 brain regions from 2,728 AD case-control samples, we concentrated on nine genes that potentially enhance the risk of AD: AACS, GNB5, GNS, HIPK3, MED13, SHC2, SLC22A5, VPS35, and ZNF398. GNB5, the fifth member of the heterotrimeric G protein beta family encoding Gß5, is primarily expressed in neurons and is essential for normal neuronal development in mouse brain. Homozygous or compound heterozygous loss of function of GNB5 in humans has previously been associated with a syndrome of developmental delay, cognitive impairment, and cardiac arrhythmia. In validation experiments, we confirmed that Gnb5 heterozygosity enhanced the formation of both amyloid plaques and neurofibrillary tangles in the brains of AD model mice. These results suggest that gene-constrained analysis can complement the power of GWASs in the identification of AD-associated genes and may be more broadly applicable to other polygenic diseases.

Ancestry-specific high-risk gene variant profiling unmasks diabetes-associated genes.

How ancestry-associated genetic variance affects disparities in the risk for polygenic diseases and influences the identification of disease-associated genes warrant a deeper understanding. We hypothesized that the discovery of genes associated with polygenic diseases may be limited by overreliance on single-nucleotide polymorphism (SNP)-based genomic investigation, since most significant variants identified in genome-wide SNP association studies map to introns and intergenic regions of the genome. To overcome such potential limitation, we developed a gene-constrained and function-based analytical method centered on high-risk variants (hrV) that encode frameshifts, stopgains, or splice site disruption. We analyzed the total number of hrV per gene in populations of different ancestry, representing a total of 185 934 subjects. Using this analysis, we developed a quantitative index of hrV (hrVI) across 20 428 genes within each population. We then applied hrVI analysis to the discovery of genes associated with type 2 diabetes mellitus (T2DM), a polygenic disease with ancestry-related disparity. HrVI profiling and gene-to-gene comparisons of ancestry-specific hrV between the case (20 781 subjects) and control (24 440 subjects) populations in the T2DM national repository identified 57 genes associated with T2DM, 40 of which were discoverable only by ancestry-specific analysis. These results illustrate how function-based and ancestry-specific analysis of genetic variations can accelerate the identification of genes associated with polygenic diseases. Besides T2DM, such analysis may facilitate our understanding of the genetic basis for other polygenic diseases that are also greatly influenced by environmental and behavioral factors, such as obesity, hypertension, and Alzheimer's disease.

Oncogenic HPV promotes the expression of the long noncoding RNA lnc-FANCI-2 through E7 and YY1.

Long noncoding RNAs (lncRNAs) play diverse roles in biological processes, but their expression profiles and functions in cervical carcinogenesis remain unknown. By RNA-sequencing (RNA-seq) analyses of 18 clinical specimens and selective validation by RT-qPCR analyses of 72 clinical samples, we provide evidence that, relative to normal cervical tissues, 194 lncRNAs are differentially regulated in high-risk (HR)-HPV infection along with cervical lesion progression. One such lncRNA, lnc-FANCI-2, is extensively characterized because it is expressed from a genomic locus adjacent to the FANCI gene encoding an important DNA repair factor. Both genes are up-regulated in HPV lesions and in in vitro model systems of HR-HPV18 infection. We observe a moderate reciprocal regulation of lnc-FANCI-2 and FANCI in cervical cancer CaSki cells. In these cells, lnc-FANCI-2 is transcribed from two alternative promoters, alternatively spliced, and polyadenylated at one of two alternative poly(A) sites. About 10 copies of lnc-FANCI-2 per cell are detected preferentially in the cytoplasm. Mechanistically, HR-HPVs, but not low-risk (LR)-HPV oncogenes induce lnc-FANCI-2 in primary and immortalized human keratinocytes. The induction is mediated primarily by E7, and to a lesser extent by E6, mostly independent of p53/E6AP and pRb/E2F. We show that YY1 interacts with an E7 CR3 core motif and transactivates the promoter of lnc-FANCI-2 by binding to two critical YY1-binding motifs. Moreover, HPV18 increases YY1 expression by reducing miR-29a, which targets the 3' untranslated region of YY1 mRNA. These data have provided insights into the mechanisms of how HR-HPV infections contribute to cervical carcinogenesis.

Tailoring Galactose Oxidase for Self-Powered Benzyl Alcohol Sensing.

Benzyl alcohol (BnOH) is a widely-used preservative in a variety of cosmetics, but the excess addition (≥1.0 %) may cause strong symptoms such as nausea, gastrointestinal irritation, convulsion, even death, making it crucial to monitor and control the addition quantity. Herein, we have developed a test-strip-like BnOH detection method via tailoring a galactose oxidase (GOase) towards BnOH oxidation and preparing a self-powered electrochromic strip for BnOH concentration visualization. A double-substituted GOase variant (Y329S/R330F), on the basis of the reported GOase M1 , has been obtained by semi-rational design with a 24.6-fold improved activity towards BnOH compared to GOase M1 . The GOase Y329S/R330F electrode has a response to BnOH with a linear range of 0.04 to 3.25 mM (R2 =0.9985), a sensitivity of 122.78 µA mM-1 cm-2 , and a detection limit of 0.03 mM (S/N=3). Coupling an electrochromic Prussian blue (PB) cathode helps the successful sensing visualization without any further power supply. The present sensing is more convenient and user-friendly than the generally used gas chromatography (GC) and high performance liquid chromatography (HPLC), and brings a more accessible solution to the field of quality controlling.

Cell-Free DNA to Detect Heart Allograft Acute Rejection.

BACKGROUND: After heart transplantation, endomyocardial biopsy (EMBx) is used to monitor for acute rejection (AR). Unfortunately, EMBx is invasive, and its conventional histological interpretation has limitations. This is a validation study to assess the performance of a sensitive blood biomarker-percent donor-derived cell-free DNA (%ddcfDNA)-for detection of AR in cardiac transplant recipients. METHODS: This multicenter, prospective cohort study recruited heart transplant subjects and collected plasma samples contemporaneously with EMBx for %ddcfDNA measurement by shotgun sequencing. Histopathology data were collected to define AR, its 2 phenotypes (acute cellular rejection [ACR] and antibody-mediated rejection [AMR]), and controls without rejection. The primary analysis was to compare %ddcfDNA levels (median and interquartile range [IQR]) for AR, AMR, and ACR with controls and to determine %ddcfDNA test characteristics using receiver-operator characteristics analysis. RESULTS: The study included 171 subjects with median posttransplant follow-up of 17.7 months (IQR, 12.1-23.6), with 1392 EMBx, and 1834 %ddcfDNA measures available for analysis. Median %ddcfDNA levels decayed after surgery to 0.13% (IQR, 0.03%-0.21%) by 28 days. Also, %ddcfDNA increased again with AR compared with control values (0.38% [IQR, 0.31-0.83%], versus 0.03% [IQR, 0.01-0.14%]; P<0.001). The rise was detected 0.5 and 3.2 months before histopathologic diagnosis of ACR and AMR. The area under the receiver operator characteristic curve for AR was 0.92. A 0.25%ddcfDNA threshold had a negative predictive value for AR of 99% and would have safely eliminated 81% of EMBx. In addition, %ddcfDNA showed distinctive characteristics comparing AMR with ACR, including 5-fold higher levels (AMR ≥2, 1.68% [IQR, 0.49-2.79%] versus ACR grade ≥2R, 0.34% [IQR, 0.28-0.72%]), higher area under the receiver operator characteristic curve (0.95 versus 0.85), higher guanosine-cytosine content, and higher percentage of short ddcfDNA fragments. CONCLUSIONS: We found that %ddcfDNA detected AR with a high area under the receiver operator characteristic curve and negative predictive value. Monitoring with ddcfDNA demonstrated excellent performance characteristics for both ACR and AMR and led to earlier detection than the EMBx-based monitoring. This study supports the use of %ddcfDNA to monitor for AR in patients with heart transplant and paves the way for a clinical utility study. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02423070.

Interferon stimulation creates chromatin marks and establishes transcriptional memory.

Epigenetic memory for signal-dependent transcription has remained elusive. So far, the concept of epigenetic memory has been largely limited to cell-autonomous, preprogrammed processes such as development and metabolism. Here we show that IFNß stimulation creates transcriptional memory in fibroblasts, conferring faster and greater transcription upon restimulation. The memory was inherited through multiple cell divisions and led to improved antiviral protection. Of ∼2,000 IFNß-stimulated genes (ISGs), about half exhibited memory, which we define as memory ISGs. The rest, designated nonmemory ISGs, did not show memory. Surprisingly, mechanistic analysis showed that IFN memory was not due to enhanced IFN signaling or retention of transcription factors on the ISGs. We demonstrated that this memory was attributed to accelerated recruitment of RNA polymerase II and transcription/chromatin factors, which coincided with acquisition of the histone H3.3 and H3K36me3 chromatin marks on memory ISGs. Similar memory was observed in bone marrow macrophages after IFNγ stimulation, suggesting that IFN stimulation modifies the shape of the innate immune response. Together, external signals can establish epigenetic memory in mammalian cells that imparts lasting adaptive performance upon various somatic cells.

Long intergenic noncoding RNA profiles of pheochromocytoma and paraganglioma: A novel prognostic biomarker.

Many long intergenic noncoding RNAs (lincRNAs) serve as cancer biomarkers for diagnosis or prognostication. To understand the role of lincRNAs in the rare neuroendocrine tumors pheochromocytoma and paraganglioma (PCPG), we performed first time in-depth characterization of lincRNA expression profiles and correlated findings to clinical outcomes of the disease. RNA-Seq data from patients with PCPGs and 17 other tumor types from The Cancer Genome Atlas and other published sources were obtained. Differential expression analysis and a machine-learning model were used to identify transcripts specific to PCPGs, as well as established PCPG molecular subtypes. Similarly, lincRNAs specific to aggressive PCPGs were identified, and univariate and multivariate analysis was performed for metastasis-free survival. The results were validated in independent samples using RT-PCR. From a pan-cancer context, PCPGs had a specific and unique lincRNA profile. Among PCPGs, five different molecular subtypes were identified corresponding to the established molecular classification. Upregulation of 13 lincRNAs was found to be associated with aggressive/metastatic PCPGs. RT-PCR validation confirmed the overexpression of four lincRNAs in metastatic compared to non-metastatic PCPGs. Kaplan-Meier analysis identified five lincRNAs as prognostic markers for metastasis-free survival of patients in three subtypes of PCPGs. Stratification of PCPG patients with a risk-score formulated using multivariate analysis of lincRNA expression profiles, presence of key driver mutations, tumor location, and hormone secretion profiles showed significant differences in metastasis-free survival. PCPGs thus exhibit a specific lincRNA expression profile that also corresponds to the established molecular subgroups and can be potential marker for the aggressive/metastatic PCPGs.

Harnessing Filler Materials for Enhancing Biogas Separation Membranes.

Biogas is an increasingly attractive renewable resource, envisioned to secure future energy demands and help curb global climate change. To capitalize on this resource, membrane processes and state-of-the-art membranes must efficiently recover methane (CH4) from biogas by separating carbon dioxide (CO2). Composite (a.k.a. mixed-matrix) membranes, prepared from common polymers and rationally selected/engineered fillers, are highly promising for this application. This review comprehensively examines filler materials that are capable of enhancing the CO2/CH4 separation performance of polymeric membranes. Specifically, we highlight novel synthetic strategies for engineering filler materials to develop high-performance composite membranes. Besides, as the matrix components (polymers) of composite membranes largely dictate the overall gas separation performances, we introduce a new empirical metric, the "Filler Enhancement Index" ( Findex), to aid researchers in assessing the effectiveness of the fillers from a big data perspective. The Findex systematically decouples the effect of polymer matrices and critically evaluates both conventional and emerging fillers to map out a future direction for next-generation (bio)gas separation membranes. Beyond biogas separation, this review is of relevance to a broader community with interests in composite membranes for other gas separation processes, as well as water treatment applications.

Towards Understanding the Mechanism of Heavy Metals Immobilization in Biochar Derived from Co-pyrolysis of Sawdust and Sewage Sludge.

Biochar was prepared by mixing sewage sludge with sawdust via a co-pyrolysis with different mixture ratios and temperatures. The results showed that the sawdust addition resulted in a lower yield of biochar with higher C content. The total concentrations of Pb and Cd in biochar were reduced. Besides, pyrolysis can transform the potentially toxic Pb and Cd to stable fractions. However the sawdust addition had slight influence on the chemical forms of Pb and Cd in the biochar. The biochar with 50% sawdust at 600°C exhibited a remarkable reduction of the leachable metal concentrations. The possible transformation mechanisms of Pb and Cd were inferred as the formation of aluminum and silicon-containing minerals. These results provide insights into the influence of sawdust addition on the characteristics of biochar and the possible Pb and Cd immobilization mechanisms during co-pyrolysis process.

Glial cell line-derived neurotrophic factor (GDNF) mediates hepatic stellate cell activation via ALK5/Smad signalling.

OBJECTIVE: Although glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-ß superfamily, its function in liver fibrosis has rarely been studied. Here, we investigated the role of GDNF in hepatic stellate cell (HSC) activation and liver fibrosis in humans and mice. DESIGN: GDNF expression was examined in liver biopsies and sera from patients with liver fibrosis. The functional role of GDNF in liver fibrosis was examined in mice with adenoviral delivery of the GDNF gene, GDNF sgRNA CRISPR/Cas9 and the administration of GDNF-blocking antibodies. GDNF was examined on HSC activation using human and mouse primary HSCs. The binding of activin receptor-like kinase 5 (ALK5) to GDNF was determined using surface plasmon resonance (SPR), molecular docking, mutagenesis and co-immunoprecipitation. RESULTS: GDNF mRNA and protein levels are significantly upregulated in patients with stage F4 fibrosis. Serum GDNF content correlates positively with α-smooth muscle actin (α-SMA) and Col1A1 mRNA in human fibrotic livers. Mice with overexpressed GDNF display aggravated liver fibrosis, while mice with silenced GDNF expression or signalling inhibition by GDNF-blocking antibodies have reduced fibrosis and HSC activation. GDNF is confined mainly to HSCs and contributes to HSC activation through ALK5 at His39 and Asp76 and through downstream signalling via Smad2/3, but not through GDNF family receptor alpha-1 (GFRα1). GDNF, ALK5 and α-SMA colocalise in human and mouse HSCs, as demonstrated by confocal microscopy. CONCLUSIONS: GDNF promotes HSC activation and liver fibrosis through ALK5/Smad signalling. Inhibition of GDNF could be a novel therapeutic strategy to combat liver fibrosis.

Nonmuscle myosin IIB regulates epicardial integrity and epicardium-derived mesenchymal cell maturation.

Nonmuscle myosin IIB (NMIIB; heavy chain encoded by MYH10) is essential for cardiac myocyte cytokinesis. The role of NMIIB in other cardiac cells is not known. Here, we show that NMIIB is required in epicardial formation and functions to support myocardial proliferation and coronary vessel development. Ablation of NMIIB in epicardial cells results in disruption of epicardial integrity with a loss of E-cadherin at cell-cell junctions and a focal detachment of epicardial cells from the myocardium. NMIIB-knockout and blebbistatin-treated epicardial explants demonstrate impaired mesenchymal cell maturation during epicardial epithelial-mesenchymal transition. This is manifested by an impaired invasion of collagen gels by the epicardium-derived mesenchymal cells and the reorganization of the cytoskeletal structure. Although there is a marked decrease in the expression of mesenchymal genes, there is no change in Snail (also known as Snai1) or E-cadherin expression. Studies from epicardium-specific NMIIB-knockout mice confirm the importance of NMIIB for epicardial integrity and epicardial functions in promoting cardiac myocyte proliferation and coronary vessel formation during heart development. Our findings provide a novel mechanism linking epicardial formation and epicardial function to the activity of the cytoplasmic motor protein NMIIB.

Rutin, γ-Aminobutyric Acid, Gallic Acid, and Caffeine Negatively Affect the Sweet-Mellow Taste of Congou Black Tea Infusions.

The sweet-mellow taste sensation is a unique and typical feature of premium congou black tea infusions. To explore the key taste-active compounds that influence the sweet-mellow taste, a sensory and molecular characterization was performed on thirty-three congou black tea infusions presenting different taste qualities, including the sweet-mellow, mellow-pure, or less-mellow taste. An integrated application of quantitative analysis of 48 taste-active compounds, taste contribution analysis, and further validation by taste supplementation experiments, combined with human sensory evaluation revealed that caffeine, γ-aminobutyric acid, rutin, succinic acid, citric acid, and gallic acid negatively affect the sweet-mellow taste, whereas glucose, sucrose, and ornithine positively contribute to the sweet-mellow taste of congou black tea infusions. Particularly, rutin, γ-aminobutyric acid, gallic acid, and caffeine, which impart the major inhibitory effect to the manifestation of the sweet-mellow taste, were identified as the key influencing components through stepwise screening and validation experiments. A modest level of these compounds was found to be favorable for the development and manifestation of the sweet-mellow taste. These compounds might potentially serve as the regulatory targets for oriented-manufacturing of high-quality sweet-mellow congou black tea.

Enhanced performance of multi-dimensional CoS nanoflake/NiO nanosheet architecture with synergetic effect for asymmetric supercapacitor.

Multi-dimensional nanomaterials possess a porous structure and plenty of active sites, so they have promising prospects in supercapacitor applications. As the typical pseudocapacitance materials, interlaced CoS nanoflakes and two-dimensional NiO nanosheets were assembled into multi-dimensional CoS/NiO architectures. The fabricated CoS/NiO nanostructures on nickel foam can directly serve as the supercapacitor electrodes. Such multi-dimensional CoS/NiO architectures exhibit the enhanced electrochemical performances in the light of the cyclic voltammetry curves and galvanostatic charging-discharging (GCD) tests. A multi-dimensional CoS/NiO electrode releases a high specific capacitance of 1620 F g-1 at 1.0 A g-1, which is distinctly higher than those of pristine CoS and NiO electrodes. The CoS/NiO//nitrogen-doped carbon nanoarrays (NC) asymmetric supercapacitor (ASC) can operate stably at 1.6 V. The GCD curves of the ASC at diverse current densities within the voltage window of 0-1.6 V exhibit reasonable symmetry. The CoS/NiO//NC ASC shows great long-term cycling performance, it has 93.5% capacity retention after 3000 cycles. Electrochemical analyses and detailed material characterizations are performed to reveal the mechanism for the enhanced performance of capacitance.

MicroRNA-942 mediates hepatic stellate cell activation by regulating BAMBI expression in human liver fibrosis.

MicroRNA (miRNA)-mediated gene regulation contributes to liver pathophysiology, including hepatic stellate cell (HSC) activation and fibrosis progression. Here, we investigated the role of miR-942 in human liver fibrosis. The expression of miR-942, HSC activation markers, transforming growth factor-beta pseudoreceptor BMP and activin membrane-bound inhibitor (BAMBI), as well as collagen deposition, were investigated in 100 liver specimens from patients with varying degree of hepatitis B virus (HBV)-related fibrosis. Human primary HSCs and the immortalized cell line (LX2 cells) were used for functional studies. We found that miR-942 expression was upregulated in activated HSCs and correlated inversely with BAMBI expression in liver fibrosis progression. Transforming growth factor beta (TGF-ß) and lipopolyssacharide (LPS), two major drivers of liver fibrosis and inflammation, induce miR-942 expression in HSCs via Smad2/3 respective NF-κB/p50 binding to the miR-942 promoter. Mechanistically, the induced miR-942 degrades BAMBI mRNA in HSCs, thereby sensitizing the cells for fibrogenic TGF-ß signaling and also partly mediates LPS-induced proinflammatory HSC fate. In conclusion, the TGF-ß and LPS-induced miR-942 mediates HSC activation through downregulation of BAMBI in human liver fibrosis. Our study provides new insights on the molecular mechanism of HSC activation and fibrosis.

A genome landscape of SRSF3-regulated splicing events and gene expression in human osteosarcoma U2OS cells.

Alternative RNA splicing is an essential process to yield proteomic diversity in eukaryotic cells, and aberrant splicing is often associated with numerous human diseases and cancers. We recently described serine/arginine-rich splicing factor 3 (SRSF3 or SRp20) being a proto-oncogene. However, the SRSF3-regulated splicing events responsible for its oncogenic activities remain largely unknown. By global profiling of the SRSF3-regulated splicing events in human osteosarcoma U2OS cells, we found that SRSF3 regulates the expression of 60 genes including ERRFI1, ANXA1 and TGFB2, and 182 splicing events in 164 genes, including EP300, PUS3, CLINT1, PKP4, KIF23, CHK1, SMC2, CKLF, MAP4, MBNL1, MELK, DDX5, PABPC1, MAP4K4, Sp1 and SRSF1, which are primarily associated with cell proliferation or cell cycle. Two SRSF3-binding motifs, CCAGC(G)C and A(G)CAGCA, are enriched to the alternative exons. An SRSF3-binding site in the EP300 exon 14 is essential for exon 14 inclusion. We found that the expression of SRSF1 and SRSF3 are mutually dependent and coexpressed in normal and tumor tissues/cells. SRSF3 also significantly regulates the expression of at least 20 miRNAs, including a subset of oncogenic or tumor suppressive miRNAs. These data indicate that SRSF3 affects a global change of gene expression to maintain cell homeostasis.

Global intron retention mediated gene regulation during CD4+ T cell activation.

T cell activation is a well-established model for studying cellular responses to exogenous stimulation. Using strand-specific RNA-seq, we observed that intron retention is prevalent in polyadenylated transcripts in resting CD4(+) T cells and is significantly reduced upon T cell activation. Several lines of evidence suggest that intron-retained transcripts are less stable than fully spliced transcripts. Strikingly, the decrease in intron retention (IR) levels correlate with the increase in steady-state mRNA levels. Further, the majority of the genes upregulated in activated T cells are accompanied by a significant reduction in IR. Of these 1583 genes, 185 genes are predominantly regulated at the IR level, and highly enriched in the proteasome pathway, which is essential for proper T cell proliferation and cytokine release. These observations were corroborated in both human and mouse CD4(+) T cells. Our study revealed a novel post-transcriptional regulatory mechanism that may potentially contribute to coordinated and/or quick cellular responses to extracellular stimuli such as an acute infection.

Telomere attrition and candidate gene mutations preceding monosomy 7 in aplastic anemia.

The pathophysiology of severe aplastic anemia (SAA) is immune-mediated destruction of hematopoietic stem and progenitor cells (HSPCs). Most patients respond to immunosuppressive therapies, but a minority transform to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), frequently associated with monosomy 7 (-7). Thirteen SAA patients were analyzed for acquired mutations in myeloid cells at the time of evolution to -7, and all had a dominant HSPC clone bearing specific acquired mutations. However, mutations in genes associated with MDS/AML were present in only 4 cases. Patients who evolved to MDS and AML showed marked progressive telomere attrition before the emergence of -7. Single telomere length analysis confirmed accumulation of short telomere fragments of individual chromosomes. Our results indicate that accelerated telomere attrition in the setting of a decreased HSPC pool is characteristic of early myeloid oncogenesis, specifically chromosome 7 loss, in MDS/AML after SAA, and provides a possible mechanism for development of aneuploidy.

Iron, inflammation, and early death in adults with sickle cell disease.

RATIONALE: Patients with sickle cell disease (SCD) have markers of chronic inflammation, but the mechanism of inflammation and its relevance to patient survival are unknown. OBJECTIVE: To assess the relationship between iron, inflammation, and early death in SCD. METHODS AND RESULTS: Using peripheral blood mononuclear cell transcriptome profile hierarchical clustering, we classified 24 patients and 10 controls in clusters with significantly different expression of genes known to be regulated by iron. Subsequent gene set enrichment analysis showed that many genes associated with the high iron cluster were involved in the toll-like receptor system (TLR4, TLR7, and TLR8) and inflammasome complex pathway (NLRP3, NLRC4, and CASP1). Quantitative PCR confirmed this classification and showed that ferritin light chain, TLR4, and interleukin-6 expression were >100-fold higher in patients than in controls (P<0.001). Further linking intracellular iron and inflammation, 14 SCD patients with a ferroportin Q248H variant that causes intracellular iron accumulation had significantly higher levels of interleukin-6 and C-reactive protein compared with 14 matched SCD patients with the wild-type allele (P<0.05). Finally, in a cohort of 412 patients followed for a median period of 47 months (interquartile range, 24-82), C-reactive protein was strongly and independently associated with early death (hazard ratio, 3.0; 95% confidence interval, 1.7-5.2; P<0.001). CONCLUSIONS: Gene expression markers of high intracellular iron in patients with SCD are associated with markers of inflammation and mortality. The results support a model in which intracellular iron promotes inflammatory pathways, such as the TLR system and the inflammasome, identifying important new pathways for additional investigation.