Differential transcriptomics in sarcoidosis lung and lymph node granulomas with comparisons to pathogen-specific granulomas.

RATIONALE: Despite the availability of multi-"omics" strategies, insights into the etiology and pathogenesis of sarcoidosis have been elusive. This is partly due to the lack of reliable preclinical models and a paucity of validated biomarkers. As granulomas are a key feature of sarcoidosis, we speculate that direct genomic interrogation of sarcoid tissues, may lead to identification of dysregulated gene pathways or biomarker signatures. OBJECTIVE: To facilitate the development sarcoidosis genomic biomarkers by gene expression profiling of sarcoidosis granulomas in lung and lymph node tissues (most commonly affected organs) and comparison to infectious granulomas (coccidiodomycosis and tuberculosis). METHODS: Transcriptomic profiles of immune-related gene from micro-dissected sarcoidosis granulomas within lung and mediastinal lymph node tissues and compared to infectious granulomas from paraffin-embedded blocks. Differentially-expressed genes (DEGs) were profiled, compared among the three granulomatous diseases and analyzed for functional enrichment pathways. RESULTS: Despite histologic similarities, DEGs and pathway enrichment markedly differed in sarcoidosis granulomas from lymph nodes and lung. Lymph nodes showed a clear immunological response, whereas a structural regenerative response was observed in lung. Sarcoidosis granuloma gene expression data corroborated previously reported genomic biomarkers (STAB1, HBEGF, and NOTCH4), excluded others and identified new genomic markers present in lung and lymph nodes, ADAMTS1, NPR1 and CXCL2. Comparisons between sarcoidosis and pathogen granulomas identified pathway divergences and commonalities at gene expression level. CONCLUSION: These findings suggest the importance of tissue and disease-specificity evaluation when exploring sarcoidosis genomic markers. This relevant translational information in sarcoidosis and other two histopathological similar infections provides meaningful specific genomic-derived biomarkers for sarcoidosis diagnosis and prognosis.

Genome-Wide Association Study in African Americans with Acute Respiratory Distress Syndrome Identifies the Selectin P Ligand Gene as a Risk Factor.

RATIONALE: Genetic factors are involved in acute respiratory distress syndrome (ARDS) susceptibility. Identification of novel candidate genes associated with increased risk and severity will improve our understanding of ARDS pathophysiology and enhance efforts to develop novel preventive and therapeutic approaches. OBJECTIVES: To identify genetic susceptibility targets for ARDS. METHODS: A genome-wide association study was performed on 232 African American patients with ARDS and 162 at-risk control subjects. The Identify Candidate Causal SNPs and Pathways platform was used to infer the association of known gene sets with the top prioritized intragenic SNPs. Preclinical validation of SELPLG (selectin P ligand gene) was performed using mouse models of LPS- and ventilator-induced lung injury. Exonic variation within SELPLG distinguishing patients with ARDS from sepsis control subjects was confirmed in an independent cohort. MEASUREMENTS AND MAIN RESULTS: Pathway prioritization analysis identified a nonsynonymous coding SNP (rs2228315) within SELPLG, encoding P-selectin glycoprotein ligand 1, to be associated with increased susceptibility. In an independent cohort, two exonic SELPLG SNPs were significantly associated with ARDS susceptibility. Additional support for SELPLG as an ARDS candidate gene was derived from preclinical ARDS models where SELPLG gene expression in lung tissues was significantly increased in both ventilator-induced (twofold increase) and LPS-induced (5.7-fold increase) murine lung injury models compared with controls. Furthermore, Selplg-/- mice exhibited significantly reduced LPS-induced inflammatory lung injury compared with wild-type C57/B6 mice. Finally, an antibody that neutralizes P-selectin glycoprotein ligand 1 significantly attenuated LPS-induced lung inflammation. CONCLUSIONS: These findings identify SELPLG as a novel ARDS susceptibility gene among individuals of European and African descent.

LPS-induced Acute Lung Injury Involves NF-κB-mediated Downregulation of SOX18.

One of the early events in the progression of LPS-mediated acute lung injury in mice is the disruption of the pulmonary endothelial barrier resulting in lung edema. However, the molecular mechanisms by which the endothelial barrier becomes compromised remain unresolved. The SRY (sex-determining region on the Y chromosome)-related high-mobility group box (Sox) group F family member, SOX18, is a barrier-protective protein through its ability to increase the expression of the tight junction protein CLDN5. Thus, the purpose of this study was to determine if downregulation of the SOX18-CLDN5 axis plays a role in the pulmonary endothelial barrier disruption associated with LPS exposure. Our data indicate that both SOX18 and CLDN5 expression is decreased in two models of in vivo LPS exposure (intraperitoneal, intratracheal). A similar downregulation was observed in cultured human lung microvascular endothelial cells (HLMVECs) exposed to LPS. SOX18 overexpression in HLMVECs or in the mouse lung attenuated the LPS-mediated vascular barrier disruption. Conversely, reduced CLDN5 expression (siRNA) reduced the HLMVEC barrier-protective effects of SOX18 overexpression. The mechanism by which LPS decreases SOX18 expression was identified as transcriptional repression through binding of NF-κB (p65) to a SOX18 promoter sequence located between -1,082 and -1,073 bp with peroxynitrite contributing to LPS-mediated NF-κB activation. We conclude that NF-κB-dependent decreases in the SOX18-CLDN5 axis are essentially involved in the disruption of human endothelial cell barrier integrity associated with LPS-mediated acute lung injury.

The effect of non-coding DNA variations on P53 and cMYC competitive inhibition at cis-overlapping motifs.

Non-coding DNA variations play a critical role in increasing the risk for development of common complex diseases, and account for the majority of SNPs highly associated with cancer. However, it remains a challenge to identify etiologic variants and to predict their pathological effects on target gene expression for clinical purposes. Cis-overlapping motifs (COMs) are elements of enhancer regions that impact gene expression by enabling competitive binding and switching between transcription factors. Mutations within COMs are especially important when the involved transcription factors have opposing effects on gene regulation, like P53 tumor suppressor and cMYC proto-oncogene. In this study, genome-wide analysis of ChIP-seq data from human cancer and mouse embryonic cells identified a significant number of putative regulatory elements with signals for both P53 and cMYC. Each co-occupied element contains, on average, two COMs, and one common SNP every two COMs. Gene ontology of predicted target genes for COMs showed that the majority are involved in DNA damage, apoptosis, cell cycle regulation, and RNA processing. EMSA results showed that both cMYC and P53 bind to cis-overlapping motifs within a ChIP-seq co-occupied region in Chr12. In vitro functional analysis of selected co-occupied elements verified enhancer activity, and also showed that the occurrence of SNPs within three COMs significantly altered enhancer activity. We identified a list of COM-associated functional SNPs that are in close proximity to SNPs associated with common diseases in large population studies. These results suggest a potential molecular mechanism to identify etiologic regulatory mutations associated with common diseases.

Plasma metabolomic profiles enhance precision medicine for volunteers of normal health.

Precision medicine, taking account of human individuality in genes, environment, and lifestyle for early disease diagnosis and individualized therapy, has shown great promise to transform medical care. Nontargeted metabolomics, with the ability to detect broad classes of biochemicals, can provide a comprehensive functional phenotype integrating clinical phenotypes with genetic and nongenetic factors. To test the application of metabolomics in individual diagnosis, we conducted a metabolomics analysis on plasma samples collected from 80 volunteers of normal health with complete medical records and three-generation pedigrees. Using a broad-spectrum metabolomics platform consisting of liquid chromatography and GC coupled with MS, we profiled nearly 600 metabolites covering 72 biochemical pathways in all major branches of biosynthesis, catabolism, gut microbiome activities, and xenobiotics. Statistical analysis revealed a considerable range of variation and potential metabolic abnormalities across the individuals in this cohort. Examination of the convergence of metabolomics profiles with whole-exon sequences (WESs) provided an effective approach to assess and interpret clinical significance of genetic mutations, as shown in a number of cases, including fructose intolerance, xanthinuria, and carnitine deficiency. Metabolic abnormalities consistent with early indications of diabetes, liver dysfunction, and disruption of gut microbiome homeostasis were identified in several volunteers. Additionally, diverse metabolic responses to medications among the volunteers may assist to identify therapeutic effects and sensitivity to toxicity. The results of this study demonstrate that metabolomics could be an effective approach to complement next generation sequencing (NGS) for disease risk analysis, disease monitoring, and drug management in our goal toward precision care.

Defective Store-Operated Calcium Entry Causes Partial Nephrogenic Diabetes Insipidus.

Store-operated calcium entry (SOCE) is the mechanism by which extracellular signals elicit prolonged intracellular calcium elevation to drive changes in fundamental cellular processes. Here, we investigated the role of SOCE in the regulation of renal water reabsorption, using the inbred rat strain SHR-A3 as an animal model with disrupted SOCE. We found that SHR-A3, but not SHR-B2, have a novel truncating mutation in the gene encoding stromal interaction molecule 1 (STIM1), the endoplasmic reticulum calcium (Ca(2+)) sensor that triggers SOCE. Balance studies revealed increased urine volume, hypertonic plasma, polydipsia, and impaired urinary concentrating ability accompanied by elevated circulating arginine vasopressin (AVP) levels in SHR-A3 compared with SHR-B2. Isolated, split-open collecting ducts (CD) from SHR-A3 displayed decreased basal intracellular Ca(2+) levels and a major defect in SOCE. Consequently, AVP failed to induce the sustained intracellular Ca(2+) mobilization that requires SOCE in CD cells from SHR-A3. This effect decreased the abundance of aquaporin 2 and enhanced its intracellular retention, suggesting impaired sensitivity of the CD to AVP in SHR-A3. Stim1 knockdown in cultured mpkCCDc14 cells reduced SOCE and basal intracellular Ca(2+) levels and prevented AVP-induced translocation of aquaporin 2, further suggesting the effects in SHR-A3 result from the expression of truncated STIM1. Overall, these results identify a novel mechanism of nephrogenic diabetes insipidus and uncover a role of SOCE in renal water handling.

Adult genetic risk screening.

Recent advances in genetic analysis especially DNA sequencing technology open a new strategy for adult disease prevention by genetic screening. Physicians presently treat disease pathology with less emphasis on disease risk prevention/reduction. Genetic screening has reduced the incidence of untreatable childhood genetic diseases and improved the care of newborns. The opportunity exists to expand screening programs and reduce the incidence of adult onset diseases via genetic risk identification and disease intervention. This article outlines the approach, challenges, and benefits of such screening for adult genetic disease risks.

Personalized genomic disease risk of volunteers.

Next-generation sequencing (NGS) is commonly used for researching the causes of genetic disorders. However, its usefulness in clinical practice for medical diagnosis is in early development. In this report, we demonstrate the value of NGS for genetic risk assessment and evaluate the limitations and barriers for the adoption of this technology into medical practice. We performed whole exome sequencing (WES) on 81 volunteers, and for each volunteer, we requested personal medical histories, constructed a three-generation pedigree, and required their participation in a comprehensive educational program. We limited our clinical reporting to disease risks based on only rare damaging mutations and known pathogenic variations in genes previously reported to be associated with human disorders. We identified 271 recessive risk alleles (214 genes), 126 dominant risk alleles (101 genes), and 3 X-recessive risk alleles (3 genes). We linked personal disease histories with causative disease genes in 18 volunteers. Furthermore, by incorporating family histories into our genetic analyses, we identified an additional five heritable diseases. Traditional genetic counseling and disease education were provided in verbal and written reports to all volunteers. Our report demonstrates that when genome results are carefully interpreted and integrated with an individual's medical records and pedigree data, NGS is a valuable diagnostic tool for genetic disease risk.

Lymphatic abnormalities are associated with RASA1 gene mutations in mouse and man.

Mutations in gene RASA1 have been historically associated with capillary malformation-arteriovenous malformation, but sporadic reports of lymphatic involvement have yet to be investigated in detail. To investigate the impact of RASA1 mutations in the lymphatic system, we performed investigational near-infrared fluorescence lymphatic imaging and confirmatory radiographic lymphangiography in a Parkes-Weber syndrome (PKWS) patient with suspected RASA1 mutations and correlated the lymphatic abnormalities against that imaged in an inducible Rasa1 knockout mouse. Whole-exome sequencing (WES) analysis and validation by Sanger sequencing of DNA from the patient and unaffected biological parents enabled us to identify an early-frameshift deletion in RASA1 that was shared with the father, who possessed a capillary stain but otherwise no overt disease phenotype. Abnormal lymphatic vasculature was imaged in both affected and unaffected legs of the PKWS subject that transported injected indocyanine green dye to the inguinal lymph node and drained atypically into the abdomen and into dermal lymphocele-like vesicles on the groin. Dermal lymphatic hyperplasia and dilated vessels were observed in Rasa1-deficient mice, with subsequent development of chylous ascites. WES analyses did not identify potential gene modifiers that could explain the variability of penetrance between father and son. Nonetheless, we conclude that the RASA1 mutation is responsible for the aberrant lymphatic architecture and functional abnormalities, as visualized in the PKWS subject and in the animal model. Our unique method to combine investigatory near-infrared fluorescence lymphatic imaging and WES for accurate phenoptyping and unbiased genotyping allows the study of molecular mechanisms of lymphatic involvement of hemovascular disorders.

Somatic mutations affect key pathways in lung adenocarcinoma.

Determining the genetic basis of cancer requires comprehensive analyses of large collections of histopathologically well-classified primary tumours. Here we report the results of a collaborative study to discover somatic mutations in 188 human lung adenocarcinomas. DNA sequencing of 623 genes with known or potential relationships to cancer revealed more than 1,000 somatic mutations across the samples. Our analysis identified 26 genes that are mutated at significantly high frequencies and thus are probably involved in carcinogenesis. The frequently mutated genes include tyrosine kinases, among them the EGFR homologue ERBB4; multiple ephrin receptor genes, notably EPHA3; vascular endothelial growth factor receptor KDR; and NTRK genes. These data provide evidence of somatic mutations in primary lung adenocarcinoma for several tumour suppressor genes involved in other cancers--including NF1, APC, RB1 and ATM--and for sequence changes in PTPRD as well as the frequently deleted gene LRP1B. The observed mutational profiles correlate with clinical features, smoking status and DNA repair defects. These results are reinforced by data integration including single nucleotide polymorphism array and gene expression array. Our findings shed further light on several important signalling pathways involved in lung adenocarcinoma, and suggest new molecular targets for treatment.

Genome-wide association study of a lipedema phenotype among women in the UK Biobank identifies multiple genetic risk factors.

Lipedema is a common disorder characterized by excessive deposition of subcutaneous adipose tissue (SAT) in the legs, hips, and buttocks, mainly occurring in adult women. Although it appears to be heritable, no specific genes have yet been identified. To identify potential genetic risk factors for lipedema, we used bioelectrical impedance analysis and anthropometric data from the UK Biobank to identify women with and without a lipedema phenotype. Specifically, we identified women with both a high percentage of fat in the lower limbs and a relatively small waist, adjusting for hip circumference. We performed a genome-wide association study (GWAS) for this phenotype, and performed multiple sensitivity GWAS. In an independent case/control study of lipedema based on strict clinical criteria, we attempted to replicate our top hits. We identified 18 significant loci (p < 5 × 10-9), several of which have previously been identified in GWAS of waist-to-hip ratio with larger effects in women. Two loci (VEGFA and GRB14-COBLL1) were significantly associated with lipedema in the independent replication study. Follow-up analyses suggest an enrichment of genes expressed in blood vessels and adipose tissue, among other tissues. Our findings provide a starting point towards better understanding the genetic and physiological basis of lipedema.

Examination of eQTL Polymorphisms Associated with Increased Risk of Progressive Complicated Sarcoidosis in European and African Descent Subjects.

Background: A limited pool of SNPs are linked to the development and severity of sarcoidosis, a systemic granulomatous inflammatory disease. By integrating genome-wide association studies (GWAS) data and expression quantitative trait loci (eQTL) single nuclear polymorphisms (SNPs), we aimed to identify novel sarcoidosis SNPs potentially influencing the development of complicated sarcoidosis. Methods: A GWAS (Affymetrix 6.0) involving 209 African-American (AA) and 193 European-American (EA, 75 and 51 complicated cases respectively) and publicly-available GWAS controls (GAIN) was utilized. Annotation of multi-tissue eQTL SNPs present on the GWAS created a pool of ~46,000 eQTL SNPs examined for association with sarcoidosis risk and severity (Logistic Model, Plink). The most significant EA/AA eQTL SNPs were genotyped in a sarcoidosis validation cohort (n=1034) and cross-validated in two independent GWAS cohorts. Results: No single GWAS SNP achieved significance (p<1x10-8), however, analysis of the eQTL/GWAS SNP pool yielded 621 eQTL SNPs (p<10-4) associated with 730 genes that highlighted innate immunity, MHC Class II, and allograft rejection pathways with multiple SNPs validated in an independent sarcoidosis cohort (105 SNPs analyzed) (NOTCH4, IL27RA, BTNL2, ANXA11, HLA-DRB1). These studies confirm significant association of eQTL/GWAS SNPs in EAs and AAs with sarcoidosis risk and severity (complicated sarcoidosis) involving HLA region and innate immunity. Conclusion: Despite the challenge of deciphering the genetic basis for sarcoidosis risk/severity, these results suggest that integrated eQTL/GWAS approaches may identify novel variants/genes and support the contribution of dysregulated innate immune responses to sarcoidosis severity.

The finished DNA sequence of human chromosome 12.

Human chromosome 12 contains more than 1,400 coding genes and 487 loci that have been directly implicated in human disease. The q arm of chromosome 12 contains one of the largest blocks of linkage disequilibrium found in the human genome. Here we present the finished sequence of human chromosome 12, which has been finished to high quality and spans approximately 132 megabases, representing approximately 4.5% of the human genome. Alignment of the human chromosome 12 sequence across vertebrates reveals the origin of individual segments in chicken, and a unique history of rearrangement through rodent and primate lineages. The rate of base substitutions in recent evolutionary history shows an overall slowing in hominids compared with primates and rodents.

MicroRNA and protein-coding gene expression analysis in idiopathic pulmonary fibrosis yields novel biomarker signatures associated to survival.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease of unknown etiology that poses significant challenges in early diagnosis and prediction of progression. Analyses of microRNA and gene expression in IPF have yielded potentially predictive information. However, the relationship between microRNA/gene expression and quantitative phenotypic value in IPF remains controversial, as is the added value of this approach to current molecular signatures in IPF. To identify biomarkers predictive of survival in IPF via a microRNA-driven strategy. We profiled microRNA and protein-coding gene expression in peripheral blood mononuclear cells from 70 IPF subjects in a discovery cohort. We linked the microRNA/gene expression level with the quantitative phenotypic variation in IPF, including diffusing capacity of the lung for carbon monoxide and the forced vital capacity percent predicted. In silico analyses of expression profiles and quantitative phenotypic data allowed the generation of 2 sets of IPF molecular signatures (unique for microRNAs and protein-coding genes) that predict IPF survival. Each signature performed well in a validation cohort comprised of IPF patients aggregated from distinct patient populations recruited from different sites. Resampling test suggests that the protein-coding gene based signature is comparable and potentially superior to published IPF prognostic gene signatures. In conclusion, these results highlight the utility of microRNA-driven peripheral blood molecular signatures as valuable and novel biomarkers associated to individuals at high survival risk and for potentially facilitating individualized therapies in this enigmatic disorder.

Whole-Blood Mitochondrial DNA Copies Are Associated With the Prognosis of Acute Respiratory Distress Syndrome After Sepsis.

Acute respiratory distress syndrome (ARDS) is an inflammatory process of the lungs that develops primarily in response to pulmonary or systemic sepsis, resulting in a disproportionate death toll in intensive care units (ICUs). Given its role as a critical activator of the inflammatory and innate immune responses, previous studies have reported that an increase of circulating cell-free mitochondrial DNA (mtDNA) is a biomarker for fatal outcome in the ICU. Here we analyzed the association of whole-blood mtDNA (wb-mtDNA) copies with 28-day survival from sepsis and sepsis-associated ARDS. We analyzed mtDNA data from 687 peripheral whole-blood samples within 24 h of sepsis diagnosis from unrelated Spanish patients with sepsis (264 with ARDS) included in the GEN-SEP study. The wb-mtDNA copies were obtained from the array intensities of selected probes, with 100% identity with mtDNA and with the largest number of mismatches with the nuclear sequences, and normalized across the individual-probe intensities. We used Cox regression models for testing the association with 28-day survival. We observed that wb-mtDNA copies were significantly associated with 28-day survival in ARDS patients (hazard ratio = 3.65, 95% confidence interval = 1.39-9.59, p = 0.009) but not in non-ARDS patients. Our findings support that wb-mtDNA copies at sepsis diagnosis could be considered an early prognostic biomarker in sepsis-associated ARDS patients. Future studies will be needed to evaluate the mechanistic links of this observation with the pathogenesis of ARDS.

Transcriptomic profiles in pulmonary arterial hypertension associate with disease severity and identify novel candidate genes.

Using RNAseq, we identified a 61 gene-based circulating transcriptomic profile most correlated with four indices of pulmonary arterial hypertension severity. In an independent dataset, 13/61 (21%) genes were differentially expressed in lung tissues of pulmonary arterial hypertension cases versus controls, highlighting potentially novel candidate genes involved in pulmonary arterial hypertension development.

Low Dose Carbon Monoxide Exposure in Idiopathic Pulmonary Fibrosis Produces a CO Signature Comprised of Oxidative Phosphorylation Genes.

Compelling preclinical studies indicate that low-dose carbon monoxide (CO) abrogates experimental lung fibrosis. We recently reported the results of a multicenter, double-blinded, clinical trial of inhaled CO in patients with idiopathic pulmonary fibrosis (IPF). Identifying no significantly changes in metalloproteinase-7 (MMP7) serum concentration, or secondary endpoints of physiologic measurements, hospitalization, death, or patient-reported outcomes. In the present study, we evaluated the effect of low dose CO exposure (100-200 ppm) for 12 weeks on genome-wide gene expression in peripheral blood mononuclear cells (PBMC) derived from these IPF study subjects. We conducted transcriptome profiling on 38 IPF subjects with time points available at 0, 12, and 24 weeks. Total RNA isolated from PBMCs was hybridized onto the Affymetrix Human Gene 2.0 ST Array. We identified 621 genes significantly upregulated in the 24-week CO exposed group compared with the 12-week. Pathway analysis demonstrated association with Oxidative Phosphorylation (adjusted P < 0.05). We identified a clear CO signature dominated with 23 oxidative phosphorylation-related genes (FDR <10%). We confirmed the expression of nine selected gene products using Nanostring's nCounter analysis system. These findings suggest this signature may serve as a potential genomic biomarker for CO exposure and for potential titration of dosage to allow precision testing of therapies in future low dose CO therapeutic studies in IPF.

Fecal virome composition of migratory wild duck species.

The fecal virome comprises a complex diversity of eukaryotic viruses, phages and viruses that infect the host. However, little is known about the intestinal community of viruses that is present in wild waterfowl, and the structure of this community in wild ducks has not yet been studied. The fecal virome compositions of six species of wild dabbling ducks and one species of wild diving duck were thus analyzed. Fecal samples were collected directly from the rectums of 60 ducks donated by hunters. DNA and RNA virus particles were purified and sequenced using the MiSeq Illumina platform. The reads obtained from the sequencing were analyzed and compared with sequences in the GenBank database. Viral-related sequences from the Herpesviridae, Alloherpesviridae, Adenoviridae, Retroviridae and Myoviridae viral families showed the highest overall abundances in the samples. The virome analysis identified viruses that had not been found in wild duck feces and revealed distinct virome profiles between different species and between samples of the same species. This study increases our understanding of viruses in wild ducks as possible viral reservoirs and provides a basis for further studying and monitoring the transmission of viruses from wild animals to humans and disease outbreaks in domestic animals.

New cases of Glucose-6-Phosphate Dehydrogenase deficiency in Pulmonary Arterial Hypertension.

Pulmonary Arterial Hypertension (PAH) is a fatal disorder with limited treatment options and reduced life expectancy after diagnosis. Complex genetic backgrounds in PAH complicates identification of causative mutations that is essential for an understanding of the disease diagnostics and etiology especially for idiopathic PAH (iPAH). Hemolysis has been implicated as contributing to the pathobiology of PAH. Glucose-6-Phosphate Dehydrogenase (G6PD) expression and activity define erythrocyte's antioxidant capacity, and its decrease contributes to erythrocyte fragility. As G6PD deficiency was previously reported in a limited number of PAH cases, we tested whether iPAH patients exhibit underlying G6PD alterations in erythrocytes. A cohort of 22 PAH patients and 8 non-PAH patients were recruited for this study. DNA isolated from Peripheral Blood Mononuclear Cells (PBMC) was used for detection of mutations in the coding region of the G6PD gene. RNA isolated from PBMC was used for determination of G6PD mRNA expression level. G6PD activity was measured in Red Blood Cell (RBC) pellets. Three patients had missense mutations in G6PD (Val291Met, Asn126Asp, Asp194Glu), however, only one mutation (Val291Met) results in a severe G6PD deficiency. A single patient with mutation (Asn126Asp) showed a 21% decrease in G6PD activity, two subjects showed G6PD deficiency without mutations, and one patient had a decreased level of G6PD mRNA and reduced enzyme levels. This study demonstrates that a moderate decrease in G6PD activity is associated with PAH. Screening for G6PD activity and mutations in the G6PD gene may provide early detection of individuals predisposed to PAH.

Robust Extracellular pH Modulation by Candida albicans during Growth in Carboxylic Acids.

The opportunistic fungal pathogen Candida albicans thrives within diverse niches in the mammalian host. Among the adaptations that underlie this fitness is an ability to utilize a wide array of nutrients, especially sources of carbon that are disfavored by many other fungi; this contributes to its ability to survive interactions with the phagocytes that serve as key barriers against disseminated infections. We have reported that C. albicans generates ammonia as a byproduct of amino acid catabolism to neutralize the acidic phagolysosome and promote hyphal morphogenesis in a manner dependent on the Stp2 transcription factor. Here, we report that this species rapidly neutralizes acidic environments when utilizing carboxylic acids like pyruvate, α-ketoglutarate (αKG), or lactate as the primary carbon source. Unlike in cells growing in amino acid-rich medium, this does not result in ammonia release, does not induce hyphal differentiation, and is genetically distinct. While transcript profiling revealed significant similarities in gene expression in cells grown on either carboxylic or amino acids, genetic screens for mutants that fail to neutralize αKG medium identified a nonoverlapping set of genes, including CWT1, encoding a transcription factor responsive to cell wall and nitrosative stresses. Strains lacking CWT1 exhibit retarded αKG-mediated neutralization in vitro, exist in a more acidic phagolysosome, and are more susceptible to macrophage killing, while double cwt1Δ stp2Δ mutants are more impaired than either single mutant. Together, our observations indicate that C. albicans has evolved multiple ways to modulate the pH of host-relevant environments to promote its fitness as a pathogen. IMPORTANCE: The fungal pathogen Candida albicans is a ubiquitous and usually benign constituent of the human microbial ecosystem. In individuals with weakened immune systems, this organism can cause potentially life-threatening infections and is one of the most common causes of hospital-acquired infections. Understanding the interactions between C. albicans and immune phagocytic cells, such as macrophages and neutrophils, will define the mechanisms of pathogenesis in this species. One such adaptation is an ability to make use of nonstandard nutrients that we predict are plentiful in certain niches within the host, including within these phagocytic cells. We show here that the metabolism of certain organic acids enables C. albicans to neutralize acidic environments, such as those within macrophages. This phenomenon is distinct in several significant ways from previous reports of similar processes, indicating that C. albicans has evolved multiple mechanisms to combat the harmful acidity of phagocytic cells.