Genetic tests for primary immunodeficiency disorders (PIDs) are expensive, time-consuming, and not easily accessible in developing countries. Therefore, we studied the feasibility of a customized ...single nucleotide variant (SNV) microarray that we developed to detect disease-causing variants and copy number variation (CNV) in patients with PIDs for only 40 Euros.
Probes were custom-designed to genotype 9,415 variants of 277 PID-related genes, and were added to the genome-wide Illumina Global Screening Array (GSA). Data analysis of GSA was performed using Illumina GenomeStudio 2.0, Biodiscovery Nexus 10.0, and R-3.4.4 software. Validation of genotype calling was performed by comparing the GSA with whole-genome sequencing (WGS) data of 56 non-PID controls. DNA samples of 95 clinically diagnosed PID patients, of which 60 patients (63%) had a genetically established diagnosis (by Next-Generation Sequencing (NGS) PID panels or Sanger sequencing), were analyzed to test the performance of the GSA. The additional SNVs detected by GSA were validated by Sanger sequencing.
Genotype calling of the customized array had an accuracy rate of 99.7%. The sensitivity for detecting rare PID variants was high (87%). The single sample replication in two runs was high (94.9%). The customized GSA was able to generate a genetic diagnosis in 37 out of 95 patients (39%). These 37 patients included 29 patients in whom the genetic variants were confirmed by conventional methods (26 patients by SNV and 3 by CNV analysis), while in 8 patients a new genetic diagnosis was established (6 patients by SNV and 2 patients suspected for leukemia by CNV analysis). Twenty-eight patients could not be detected due to the limited coverage of the custom probes. However, the diagnostic yield can potentially be increased when newly updated variants are added.
Our robust customized GSA seems to be a promising first-line rapid screening tool for PIDs at an affordable price, which opens opportunities for low-cost genetic testing in developing countries. The technique is scalable, allows numerous new genetic variants to be added, and offers the potential for genetic testing not only in PIDs, but also in many other genetic diseases.
Pathogenic gain-of-function mutations in the gene encoding phosphoinositide 3-kinase delta (PI3Kδ) cause activated PI3Kδ syndrome (APDS), a disease characterized by humoral immunodeficiency, ...lymphadenopathy, and an inability to control persistent viral infections including Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infections. Understanding the mechanisms leading to impaired immune response is important to optimally treat APDS patients. Immunosenescence of CD8
T cells was suggested to contribute to APDS pathogenesis. However, the constitutive activation of T cells in APDS may also result in T cell exhaustion. Therefore, we studied exhaustion of the CD8
T cell compartment in APDS patients and compared them with healthy controls and HIV patients, as a control for exhaustion. The subset distribution of the T cell compartment of APDS patients was comparable with HIV patients with decreased naive CD4
and CD8
T cells and increased effector CD8
T cells. Like in HIV
patients, expression of activation markers and inhibitory receptors CD160, CD244, and programmed death receptor (PD)-1 on CD8
T cells was increased in APDS patients, indicating exhaustion. EBV-specific CD8
T cells from APDS patients exhibited an exhausted phenotype that resembled HIV-specific CD8
T cells in terms of inhibitory receptor expression. Inhibition of PD-1 on EBV-specific CD8
T cells from APDS patients enhanced
proliferation and effector cytokine production. Based on these results, we conclude that total and EBV-specific CD8
T cells from APDS patients are characterized by T cell exhaustion. Furthermore, PD-1 checkpoint inhibition may provide a possible therapeutic approach to support the immune system of APDS patients to control EBV and CMV.
Breast cancer has for long been recognized as a highly diverse tumor group, but the underlying genetic basis has been elusive. Here, we report an extensive molecular characterization of a collection ...of 41 human breast cancer cell lines. Protein and gene expression analyses indicated that the collection of breast cancer cell lines has retained most, if not all, molecular characteristics that are typical for clinical breast cancers. Gene mutation analyses identified 146 oncogenic mutations among 27 well-known cancer genes, amounting to an average of 3.6 mutations per cell line. Mutations in genes from the p53, RB and PI3K tumor suppressor pathways were widespread among all breast cancer cell lines. Most important, we have identified two gene mutation profiles that are specifically associated with luminal-type and basal-type breast cancer cell lines. The luminal mutation profile involved E-cadherin and MAP2K4 gene mutations and amplifications of Cyclin D1, ERBB2 and HDM2, whereas the basal mutation profile involved BRCA1, RB1, RAS and BRAF gene mutations and deletions of p16 and p14ARF. These subtype-specific gene mutation profiles constitute a genetic basis for the heterogeneity observed among human breast cancers, providing clues for their underlying biology and providing guidance for targeted pharmacogenetic intervention in breast cancer patients.
Analysis of polymorphisms of canine Cytochrome P 450‐CYP2D15 Hagen, Marjan A. E.; Schipper, Louska; Oosterveer‐van der Doelen, Marjolein A. M. ...
Journal of veterinary pharmacology and therapeutics,
November 2020, Letnik:
43, Številka:
6
Journal Article
Recenzirano
Odprti dostop
Cytochrome P450 (CYP) proteins constitute a large ancient family of oxidative enzymes essential for the efficient elimination of a wide variety of clinically used drugs. Polymorphic variants of human ...CYP2D6 are associated with the conversion rate and efficacy of several drugs such as antidepressants. Polymorphisms of the canine orthologue CYP2D15 are of interest because these antidepressants are also used in dogs with behavioral problems and the outcome of the treatment is variable. However, the annotated CYP2D15 gene is incomplete and inaccurately assembled in CanFam3.1, hampering DNA sequence analysis of the gene in individual dogs. We elucidated the complete exon–intron structure of CYP2D15 to enable comprehensive genotyping of the gene using genomic DNA. We surveyed variations of the gene in four diverse dog breeds and identified novel polymorphisms in exon 2 in border collies. Further investigation to establish the impact of these canine CYP2D15 polymorphisms on interindividual variability in expression and function of this metabolizing enzyme is now feasible. Further knowledge of CYP pharmacogenetics will help individualize therapy and thereby increase therapeutic efficacy, especially in the use of antidepressants in veterinary behavioral medicine.
The highly homologous 4Fe-4S containing fumarases FumA and FumB, sharing 90% amino acid sequence identity, from Escherichia coli are differentially regulated, which suggests a difference in their ...physiological function. The ratio of FumB over FumA expression levels increases by one to two orders of magnitude upon change from aerobic to anaerobic growth conditions.
To understand this difference in terms of structure-function relations, catalytic and thermodynamic properties were determined for the two enzymes obtained from homologous overexpression systems. FumA and FumB are essentially identical in their Michaelis-Menten kinetics of the reversible fumarate to L-malate conversion; however, FumB has a significantly greater catalytic efficiency for the conversion of D-tartrate to oxaloacetate consistent with the requirement of the fumB gene for growth on D-tartrate. Reduction potentials of the 4Fe-4S(2+) Lewis acid active centre were determined in mediated bulk titrations in the presence of added substrate and were found to be approximately -290 mV for both FumA and FumB.
This study contradicts previously published claims that FumA and FumB exhibit different catalytic preferences for the natural substrates L-malate and fumarate. FumA and FumB differ significantly only in the catalytic efficiency for the conversion of D-tartrate, a supposedly non-natural substrate. The reduction potential of the substrate-bound 4Fe-4S active centre is, contrary to previously reported values, close to the cellular redox potential.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The introduction of rapid exome sequencing (rES) for critically ill neonates admitted to the neonatal intensive care unit has made it possible to impact clinical decision-making. Unbiased prospective ...studies to quantify the impact of rES over routine genetic testing are, however, scarce. We performed a clinical utility study to compare rES to conventional genetic diagnostic workup for critically ill neonates with suspected genetic disorders. In a multicenter prospective parallel cohort study involving five Dutch NICUs, we performed rES in parallel to routine genetic testing for 60 neonates with a suspected genetic disorder and monitored diagnostic yield and the time to diagnosis. To assess the economic impact of rES, healthcare resource use was collected for all neonates. rES detected more conclusive genetic diagnoses than routine genetic testing (20% vs. 10%, respectively), in a significantly shorter time to diagnosis (15 days (95% CI 10–20) vs. 59 days (95% CI 23–98,
p
< 0.001)). Moreover, rES reduced genetic diagnostic costs by 1.5% (€85 per neonate).
Conclusion
: Our findings demonstrate the clinical utility of rES for critically ill neonates based on increased diagnostic yield, shorter time to diagnosis, and net healthcare savings. Our observations warrant the widespread implementation of rES as first-tier genetic test in critically ill neonates with disorders of suspected genetic origin.
What is Known:
• Rapid exome sequencing (rES) enables diagnosing rare genetic disorders in a fast and reliable manner, but retrospective studies with neonates admitted to the neonatal intensive care unit (NICU) indicated that genetic disorders are likely underdiagnosed as rES is not routinely used.
• Scenario modeling for implementation of rES for neonates with presumed genetic disorders indicated an expected increase in costs associated with genetic testing.
What is New:
• This unique prospective national clinical utility study of rES in a NICU setting shows that rES obtained more and faster diagnoses than conventional genetic tests.
• Implementation of rES as replacement for all other genetic tests does not increase healthcare costs but in fact leads to a reduction in healthcare costs.
Introduction Sarcoidosis is a multi-system inflammatory disease of unknown origin with heterogeneous clinical manifestations varying from a single organ non-caseating granuloma site to chronic ...systemic inflammation and fibrosis. Gene expression studies have suggested several genes and pathways implicated in the pathogenesis of sarcoidosis, however, due to differences in study design and variable statistical approaches, results were frequently not reproducible or concordant. Therefore, meta-analysis of sarcoidosis gene-expression datasets is of great importance to robustly establish differentially expressed genes and signalling pathways. Methods We performed meta-analysis on 22 published gene-expression studies on sarcoidosis. Datasets were analysed systematically using same statistical cut-offs. Differentially expressed genes were identified by pooling of p -values using Edgington’s method and analysed for pathways using Ingenuity Pathway Analysis software. Results A consistent and significant signature of novel and well-known genes was identified, those collectively implicated both type I and type II interferon mediated signalling pathways in sarcoidosis. In silico functional analysis showed consistent downregulation of eukaryotic initiation factor 2 signalling, whereas cytokines like interferons and transcription factor STAT1 were upregulated. Furthermore, we analysed affected tissues to detect differentially expressed genes likely to be involved in granuloma biology. This revealed that matrix metallopeptidase 12 was exclusively upregulated in affected tissues, suggesting a crucial role in disease pathogenesis. Discussion Our analysis provides a concise gene signature in sarcoidosis and expands our knowledge about the pathogenesis. Our results are of importance to improve current diagnostic approaches and monitoring strategies as well as in the development of targeted therapeutics.
Cell signaling is dependent on the balance between phosphorylation of proteins by kinases and dephosphorylation by phosphatases. This balance if often disrupted in colorectal cancer (CRC), leading to ...increased cell proliferation and invasion. For many years research has focused on the role of kinases as potential oncogenes in cancer, while phosphatases were commonly assumed to be tumor suppressive. However, this dogma is currently changing as phosphatases have also been shown to induce cancer growth. One of these phosphatases is protein tyrosine phosphatase 1B (PTP1B). Here we report that the expression of PTP1B is increased in colorectal cancer as compared to normal tissue, and that the intrinsic enzymatic activity of the protein is also enhanced. This suggests a role for PTP1B phosphatase activity in CRC formation and progression. Furthermore, we found that increased PTP1B expression is correlated to a worse patient survival and is an independent prognostic marker for overall survival and disease free survival. Knocking down PTP1B in CRC cell lines results in a less invasive phenotype with lower adhesion, migration and proliferation capabilities. Together, these results suggest that inhibition of PTP1B activity is a promising new target in the treatment of colorectal cancer and the prevention of metastasis.
PDF
