73 results for ""hydrogenase+cluster"+"metal+ion+uptake"+flanking+aquatic+gamma-proteobacteria"

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• Genome Studies in Hereditary Spastic Paraplegia

  Next-generation sequencing technology is opening up new opportunities to rethink the way we identify disease causing genetic variation. An early application, whole exome sequencing, has now been established by a small number of research labs, including ours. Exome sequencing allows obtaining a near complete set of protein coding genomic variation in single individuals for less than $5,000. Promising targets for exome sequencing studies are Mendelian diseases, such as hereditary spastic paraplegias (HSP). HSP comprise a genetically very heterogeneous set of neurological disorders with currently 39 different HSP chromosomal loci being reported; yet, the identified genes explain only 60% of the genetic effect at best. Traditional methods of gene identification require linkage analysis of large families, but face increasing difficulties to identify such extended pedigrees for rare HSP forms. However, the innovative approach described in this application will overcome some of these limitations and utilize relatively small pedigrees for highly effective gene identification. We will apply exome sequencing, which will characterize all coding changes and flanking exonic variation in two individuals of a family. We have developed a multi-tiered strategy to reduce the number of identified novel variants to the very causative change in an individual family. We propose to study at least 60 HSP families, which are too small to yield conclusive results with linkage analysis. If the developing technology permits we will consider a larger sample or perform whole genome sequencing. Beyond the important benefit to genetics of HSP, this study will allow us to further establish this new method, which will benefit a large range of additional disease studies.

  Study phs001080

• Genesis of Two Most Prevalent Variants Causing Combined Pituitary Hormone Deficiency in 21 Populations

  Two variants (c.[301_302delAG];[301_302delAG] and c.[150delA];[150delA]) in the PROP1 gene are the most common genetic causes of recessively inherited combined pituitary hormones deficiency (CPHD). Our objective was to analyze in detail the origin of the two most prevalent variants. In the multicentric study were included 237 patients with CPHD and their 15 relatives carrying c.[301_302delAG];[301_302delAG] or c.[150delA];[150delA] or c.[301_302delAG];[ 150delA]. They originated from 21 different countries worldwide. We genotyped 21 single nucleotide variant markers flanking the 9.6Mb region around the PROP1 gene that are not in mutual linkage disequilibrium in the general populations - a finding of a common haplotype would be indicative of ancestral origin of the variant. Haplotypes were reconstructed by Phase and Haploview software, and the variant age was estimated using an allelic association method. We demonstrated ancestral origin of both variants: c.[301_302delAG] was carried on 0.2Mb-long haplotype in a majority of European patients arising approximately 101 generations ago (confidence interval 90.1-116.4). Patients from the Iberian Peninsula displayed a different haplotype which was estimated to have emerged 23.3 (20.1-29.1) generations ago. Subsequently, the data indicated that both haplotypes were transmitted to Latin American patients approximately 13.8 (12.2-17.0) and 16.4 (14.4-20.1) generations ago, respectively. The c.[150delA] variant that was carried on a haplotype spanning about 0.3Mb was estimated to appear 43.7 (38.4-52.7) generations ago. We present strong evidence that the most frequent variants in the PROP1 gene are not a consequence of variant hot-spots as previously assumed, but are founder variants.

  Study EGAS00001001165

• Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) Lipidomics Study

  The GOLDN study was initiated to assess how genetic factors interact with environmental (diet and drug) interventions to influence blood levels of triglycerides and other atherogenic lipid species and inflammation markers (registered at clinicaltrails.gov, number NCT00083369). The study recruited Caucasian participants primarily from three-generational pedigrees from two NHLBI Family Heart Study (FHS) field centers (Minneapolis, MN and Salt Lake City, UT). Only families with at least two siblings were recruited and only participants who did not take lipid-lowering agents (pharmaceuticals or nutraceuticals) for at least 4 weeks prior to the initial visit were included. A total of 1048 GOLDN participants were included in the diet intervention. The diet intervention followed the protocol of Patsch et al. (1992). The whipping cream (83% fat) meal had 700 Calories/m2 body surface area (2.93 MJ/m2 body surface area): 3% of calories were derived from protein (instant nonfat dry milk) and 14% from carbohydrate (sugar). The ratio of polyunsaturated to saturated fat was 0.06 and the cholesterol content of the average meal was 240 mg. The mixture was blended with ice and flavorings. Blood samples were drawn immediately before (fasting) and at 3.5 and 6 hours after consuming the high-fat meal. For the GOLDN lipidomics study, sterols and fatty acids were measured from stored plasma (-80 degrees Celsius) collected at fasting and 3.5 hours after the diet intervention using TrueMass Panels from Lipomics (West Sacramento, CA). A total of 11 sterols were quantified in nmols/gram of sample including total cholesterol, 7-dehydrocholesterol, desmosterol, lanosterol, lathasterol, cholestanol, coprostanol, beta-sitosterol, campesterol, stigmasterol, and 7alpha-hydroxycholesterol. A total of 35 fatty acids were quantified in nmols/gram of sample inlcuding myristic acid (14:0); pentadecanoic acid (15:0); palmitic acid (16:0); stearic acid (18:0); arachidic acid (20:0); behenic acid (22:0); lignoceric acid (24:0); myristoleic acid (14:1n5); palmitoleic acid (16:1n7); palmitelaidic acid (t16:1n7); oleic acid (18:1n9); elaidic acid (t18:1n9); vaccenic acid (18:1n7); linoleic acid (18:2n6); gamma-linolenic acid (18:3n6); alpha-linolenic acid (18:3n3); stearidonic acid (18:4n3); eicosenoic acid (20:1n9); eicosadienoic acid (20:2n6); mead acid (20:3n9); di-homo-gamma-linolenic acid (20:3n6); arachidonic acid (20:4n6); eicsoatetraenoic acid (20:4n3); eicosapentaenoic acid (20:5n3); erucic acid (22:1n9); docosadienoic acid (22:2n6); adrenic acid (22:4n6); docosapentaenoic acid (22:5n6); docosapentaenoic acid (22:5n3); docosahexaenoic acid (22:6n3); nervonic acid (24:1n9); and plasmalogen derivatives of 16:0, 18:0, 18:1n9, and 18:1n7.

  Study phs000741

• Exploring the role of mtDNA variation in Multiple Sclerosis in a large cohort of discordant monozygotic twins

  Several lines of evidence indicate that mitochondrial DNA (mtDNA) variants might predispose to multiple sclerosis (MS). We examined this in 49 monozygotic (MZ) twin pairs clinically discordant for MS at study entry. Since the nuclear DNA of MZ twins is identical, our study provides a unique setting to functionally associate unique mtDNA variants and skewed heteroplasmy with MS development. In order to identify mtDNA variants in the twin cohort, we deeply sequenced the mitochondrial genome in blood of all 49 MZ twin pairs using next generation sequencing (Illumina, HiSeq or MiSeq) with an average coverage depth of ~25,000 sequences per base. Heteroplasmic variants were validated using targeted deep sequencing (TDS) (Illumina, MiSeq) in DNA isolated from blood and buccal swabs. In addition, whole blood was also available of 5 additional MS-affected or healthy siblings and 1 dizygotic twin pair discordant for MS, comprising in total 6 non-identical sib pairs. Moreover, all cases were screened for pathogenic mtDNA polymerase gamma (POLG) mutations. The data provides valuable insights in the possible involvement of mtDNA variants in the pathogenesis of MS, and gives information on the segregation of heteroplasmic variants within MZ twins and non-identical siblings and across different tissues.

  Study EGAS00001001240

• Chromatin remodeling enhances MAP3K8 expression in HAM: a key pathogenesis for therapeutic intervention

  Human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy (HAM) is a debilitating neuroinflammatory disease with no available effective treatments. A hallmark of HAM is the transformation of HTLV-1-infected cells into T helper type 1 (Th1)-like cells, characterized by excessive interferon (IFN)-gamma production that drives chronic inflammation. However, the molecular mechanisms fuel this aberrant Th1-like transformation and sustained inflammation remain poorly understood. We hypothesized that HAM-characteristic chromatin remodeling plays a pivotal role in the overexpression of key genes driving inflammatory pathogenesis. Using transcriptomic analysis, chromatin accessibility profiling, and biomarker evaluations across HTLV-1-related diseases, we identify MAP3K8 as a key gene that defines the unique inflammatory profile of HAM. MAP3K8 overexpression promotes Th1-like differentiation and constitutively activates the MEK-ERK signaling pathway. Furthermore, we elucidate the mechanism by which HTLV-1 Tax, Fosl2, and c-Jun collaboratively induce HAM-characteristic chromatin remodeling at the enhancer region of the MAP3K8 locus. Crucially, we demonstrate that mitogen-activated protein kinase kinase (MEK) inhibitors effectively suppress the MAP3K8-MEK signaling cascade and significantly mitigated inflammatory pathogenesis in an ex vivo culture assay. Our findings provide critical insights into the virus-host interactions underpinning HAM and propose the MAP3K8-MEK-ERK axis as a promising therapeutic target for this challenging condition.

  Study JGAS000835

• Comprehensive Genomic Characterization of Gene Therapy-Induced T-cell Acute Lymphoblastic Leukemia (H007)

  Six out of ten patients in a hematopoietic stem cell gene therapy (GT) trial for Wiskott-Aldrich syndrome (WAS) developed T-cell acute lymphoblastic leukemia (T-ALL) associated with gamma-retroviral vector integration at the LMO2 locus. We hypothesized that detailed molecular analysis of T-ALL driven by temporally defined first deregulation of LMO2 might offer valuable insights into the pathogenesis of GT-associated and sporadic T-ALL (HIPO project 007). In all six patients whole-genome and transcriptome sequencing demonstrated that in addition to oncogenic chromosomal translocations involving the T-cell receptor loci, retrovirally induced leukemias faithfully recapitulated secondary genomic events observed in sporadic T-ALL. Multiple genetic events culminated in the development of a rapidly proliferating leukemic clone and onset of clinical manifestation at 1.3 to 5 years after infusion of the gene corrected transplant. These genetic alterations affected NOTCH1 and PI3K signaling, cell cycle regulation as wells as T-cell transcription factors LEF1 and TCF1. Two of the six cases displayed a phenotype reminiscent of early immature T-ALLs, and lacked additional T-ALL typical alterations. Our analysis shows that insertional mutagenesis involving the LMO2 locus triggers T-ALL development due to diverse genetic alterations in large parts, but not exclusively, in known T-ALL driver genes, demonstrating the molecular diversity of GT-associated T-ALL.

  Study EGAS00001003870

• Identification of intronic sequences promoting natural skipping of ABCA4 exon 15 using long-read transcriptomics and midigene assays

  Stargardt disease is an inherited retinal disease that leads to progressive loss of vision, and is caused by biallelic variants in the ABCA4 gene. A significant proportion of ABCA4 variants of variable penetrance affect pre-mRNA splicing. Previously, we observed the exclusion of certain ABCA4 exons into the final transcript in the absence of any disease-causing variants. In this study, we explored this phenomenon that we coined natural exon skipping (NES) for the entire ABCA4 transcript, using targeted RT-PCR analysis as well as long-read transcriptome sequencing of control retina samples. For the most abundant NES event, which is skipping of ABCA4 exon 15, overlapping ABCA4 deletion constructs were employed to identify responsible cis-regulatory sequence motifs. Subsequently, antisense oligonucleotides (AONs) were designed and tested to reverse NES and restore fully-spliced ABCA4 mRNA. We identified intronic regions on each side of exon 15 that, when deleted, increased the inclusion of exon 15 in the final transcripts. Interestingly, these motifs appear to work in a synergistic manner. Further fine-mapping of these regions revealed several cis-regulatory motifs that may be responsible for inducing NES, on each side of the exon. AONs targeting these motifs unfortunately did not decrease the skipping of exon 15. In conclusion, we discovered an intriguing phenomenon that, in the absence of disease-causing variants, results in the skipping of one or more exons in the ABCA4 transcript. These NES events appear to be regulated by cis-regulatory motifs within the flanking introns. This work captured the extend of NES throughout ABCA4 transcript and the role of cis-regulatory motifs in pre-mRNA splicing.. The use of AONs targeting cis-regulatory motifs is an innovative strategy for splicing modulation, suitable for complementing future variant-specific splicing modulation therapies in ABCA4 retinopathies.

  Study EGAS50000000071

• Combination pembrolizumab and radiotherapy induces systemic anti-tumor immune responses in immunologically-cold non-small cell lung cancer

  The abscopal effects of radiation may sensitize immunologically “cold” tumors to immune checkpoint inhibition (ICI). We investigated the immunostimulatory effects of radiotherapy leveraging multi-omic analyses of serial tissue and blood biospecimens (n=293) from a phase 2 clinical trial of stereotactic body radiation therapy (SBRT) followed by pembrolizumab in metastatic non-small cell lung cancer (NSCLC; NCT02492568). Patients with immunologically-cold tumors (low tumor mutation burden, null PD-L1 expression, WNT-pathway mutated) in the SBRT arm had significantly longer progression-free survival compared to ICI alone (P<0.05). Induction of interferon-gamma, interferon-alpha, and antigen processing and presentation gene sets was significantly enriched post SBRT in non-irradiated tumor sites (FDR adjusted P<0.01). Significant on-therapy expansions of new and pre-existing TCR clones in both the tumor and blood compartments were noted in the SBRT arm (P<0.05). These findings support the systemic anti-tumor effects of immuno-radiotherapy and may open a therapeutic window of opportunity to overcome resistance to ICI.

  Dataset EGAD50000000404

• Addiction of Primary Cutaneous Gamma-Delta T-cell Lymphomas to JAK-STAT Signaling

  Primary cutaneous &#947;&#948; T-cell lymphoma (PCGDTL) is a mature T-cell lymphoma (TCL) of the skin with a median survival of 31 months and no effective therapies. PCGDTLs frequently possess mutations in the JAK/STAT pathway, including in JAK1, JAK3, STAT3, STAT5B, and SOCS1. We hypothesized that single-agent JAK inhibition may be effective in PCGDTLs, particularly those with targetable JAK/STAT pathway mutations. Herein, we demonstrate the near-complete response of two PCGDTL cases to single-agent JAK inhibitors, ruxolitinib or cerdulatinib. However, both patients eventually relapsed with acquired mutations in JAK1, JAK3, or STAT5B.

  Study phs003837

• Massively parallel nanowell-based single-cell gene expression profiling

  Technological advances have enabled transcriptome characterization of cell types at the single-cell level providing new biological insights. New methods that enable simple yet high-throughput single-cell expression profiling are highly desirable. Here we report a novel nanowell-based single-cell RNA sequencing system, ICELL8, which enables processing of thousands of cells per sample. The system employs a 5184-nanowell-containing microchip to capture ~1300 single cells and process them. Each nanowell contains preprinted oligonucleotides encoding poly-d(T), a unique well barcode, and a unique molecular identifier. The ICELL8 system uses imaging software to identify nanowells containing viable single cells and only wells with single cells are processed into sequencing libraries. Here, we report the performance and utility of ICELL8 using samples of increasing complexity from cultured cells to mouse solid tissue samples. Our assessment of the system to discriminate between mixed human and mouse cells showed that ICELL8 has a low cell multiplet rate (&lt; 3%) and low cross-cell contamination. We characterized single-cell transcriptomes of more than a thousand cultured human and mouse cells as well as 468 mouse pancreatic islets cells. We were able to identify distinct cell types in pancreatic islets, including alpha, beta, delta and gamma cells. Overall, ICELL8 provides efficient and cost-effective single-cell expression profiling of thousands of cells, allowing researchers to decipher single-cell transcriptomes within complex biological samples.

  Study EGAS00001002320