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1.  Genome-wide linkage analysis is a powerful prenatal diagnostic tool in families with unknown genetic defects 
Genome-wide linkage analysis is an established tool to map inherited diseases. To our knowledge it has not been used in prenatal diagnostics of any genetic disorder. We present a family with a severe recessive mental retardation syndrome, where the mother wished pregnancy termination to avoid delivering another affected child. By genome-wide scanning using the Affymetrix (Santa Clara, CA, USA) 10k chip we were able to establish the disease haplotype. Without knowing the exact genetic defect, we excluded the condition in the fetus. The woman finally gave birth to a healthy baby. We suggest that genome-wide linkage analysis – based on either SNP mapping or full-genome sequencing – is a very useful tool in prenatal diagnostics of diseases.
doi:10.1038/ejhg.2012.198
PMCID: PMC3598323  PMID: 23032112
linkage analysis; prenatal diagnostics; mental retardation
2.  NSUN4 Is a Dual Function Mitochondrial Protein Required for Both Methylation of 12S rRNA and Coordination of Mitoribosomal Assembly 
PLoS Genetics  2014;10(2):e1004110.
Biogenesis of mammalian mitochondrial ribosomes requires a concerted maturation of both the small (SSU) and large subunit (LSU). We demonstrate here that the m5C methyltransferase NSUN4, which forms a complex with MTERF4, is essential in mitochondrial ribosomal biogenesis as mitochondrial translation is abolished in conditional Nsun4 mouse knockouts. Deep sequencing of bisulfite-treated RNA shows that NSUN4 methylates cytosine 911 in 12S rRNA (m5C911) of the SSU. Surprisingly, NSUN4 does not need MTERF4 to generate this modification. Instead, the NSUN4/MTERF4 complex is required to assemble the SSU and LSU to form a monosome. NSUN4 is thus a dual function protein, which on the one hand is needed for 12S rRNA methylation and, on the other hand interacts with MTERF4 to facilitate monosome assembly. The presented data suggest that NSUN4 has a key role in controlling a final step in ribosome biogenesis to ensure that only the mature SSU and LSU are assembled.
Author Summary
Mitochondria perform a number of essential functions in the cell, including synthesis of ATP via the oxidative phosphorylation (OXPHOS) system. Normal mitochondrial function requires coordinated expression of two genomes: mitochondria's own genome (mtDNA), which encodes 13 respiratory chain subunits with essential structural and functional roles for the OXPHOS system, and the nuclear genome encoding the remaining ∼80 subunits. The mtDNA-encoded polypeptides are synthesized on mitochondrial ribosomes (mitoribosomes) located in the mitochondrial matrix. Biogenesis, maintenance and regulation of the complex mitochondrial translation apparatus are poorly understood despite its fundamental importance for cellular energy homeostasis. Here, we show that inactivation of the Nsun4 gene, encoding a mitochondrial m5C-methyltransferase, causes embryonic lethality, whereas tissue-specific disruption of Nsun4 in the heart causes cardiomyopathy with mitochondrial dysfunction. By performing sequencing of bisulfite-treated RNA we report that NSUN4 methylates C911 in 12S rRNA of the small ribosomal subunit. Surprisingly, NSUN4 can on its own perform this rRNA modification, whereas interaction with its partner protein MTERF4 is required for assembly of functional ribosomes. NSUN4 thus has dual roles in ribosome maturation and performs an important final quality control step to ensure that only mature mitoribosomal subunits are assembled into functional ribosomes.
doi:10.1371/journal.pgen.1004110
PMCID: PMC3916286  PMID: 24516400
3.  Interferon α interferes with immunological tolerance 
Oncoimmunology  2014;2(12):e27528.
The ability of regulatory T cells (Tregs) to promote immunological tolerance represents an important obstacle in cancer immunotherapy. We have recently discovered that the clinically established immunotherapeutic agent interferon α (IFNα) inactivates the suppressive functions of human Tregs. Here, we outline the mechanisms whereby IFNα mediates this important function and discuss its therapeutic implications for cancer immunotherapy.
doi:10.4161/onci.27528
PMCID: PMC3926876  PMID: 24575381
regulatory T cells; IFN-alpha; cAMP; cancer; tolerance; NK cells; PDE
4.  Correction: Kinetics of IL-6 Production Defines T Effector Cell Responsiveness to Regulatory T Cells in Multiple Sclerosis 
PLoS ONE  2013;8(11):10.1371/annotation/0e76c09e-75b2-493a-90fd-cda3187a0888.
doi:10.1371/annotation/0e76c09e-75b2-493a-90fd-cda3187a0888
PMCID: PMC3838305  PMID: 24282490
5.  Comprehensive RNA-Seq Expression Analysis of Sensory Ganglia with a Focus on Ion Channels and GPCRs in Trigeminal Ganglia 
PLoS ONE  2013;8(11):e79523.
The specific functions of sensory systems depend on the tissue-specific expression of genes that code for molecular sensor proteins that are necessary for stimulus detection and membrane signaling. Using the Next Generation Sequencing technique (RNA-Seq), we analyzed the complete transcriptome of the trigeminal ganglia (TG) and dorsal root ganglia (DRG) of adult mice. Focusing on genes with an expression level higher than 1 FPKM (fragments per kilobase of transcript per million mapped reads), we detected the expression of 12984 genes in the TG and 13195 in the DRG. To analyze the specific gene expression patterns of the peripheral neuronal tissues, we compared their gene expression profiles with that of the liver, brain, olfactory epithelium, and skeletal muscle. The transcriptome data of the TG and DRG were scanned for virtually all known G-protein-coupled receptors (GPCRs) as well as for ion channels. The expression profile was ranked with regard to the level and specificity for the TG. In total, we detected 106 non-olfactory GPCRs and 33 ion channels that had not been previously described as expressed in the TG. To validate the RNA-Seq data, in situ hybridization experiments were performed for several of the newly detected transcripts. To identify differences in expression profiles between the sensory ganglia, the RNA-Seq data of the TG and DRG were compared. Among the differentially expressed genes (> 1 FPKM), 65 and 117 were expressed at least 10-fold higher in the TG and DRG, respectively. Our transcriptome analysis allows a comprehensive overview of all ion channels and G protein-coupled receptors that are expressed in trigeminal ganglia and provides additional approaches for the investigation of trigeminal sensing as well as for the physiological and pathophysiological mechanisms of pain.
doi:10.1371/journal.pone.0079523
PMCID: PMC3832644  PMID: 24260241
6.  Kinetics of IL-6 Production Defines T Effector Cell Responsiveness to Regulatory T Cells in Multiple Sclerosis 
PLoS ONE  2013;8(10):e77634.
In multiple sclerosis (MS) autoaggressive T effector cells (Teff) are not efficiently controlled by regulatory T cells (Treg) but the underlying mechanisms are incompletely understood. Proinflammatory cytokines are key factors facilitating Teff activity in chronic inflammation. Here we investigated the influence of IL-6 on Treg sensitivity of Teff from therapy-naïve MS patients with or without active disease. Compared to healthy volunteers and independent of disease course CD4+ and especially CD8+ MS-Teff were insensitive against functional active Treg from healthy controls. This unresponsiveness was caused by accelerated production of IL-6, elevated IL-6 receptor expression and phosphorylation of protein kinase B (PKB)/c-Akt in MS-Teff. In a positive feedback loop, IL-6 itself induced its accelerated synthesis and enhanced phosphorylation of PKB/c-Akt that finally mediated Treg resistance. Furthermore, accelerated IL-6 release especially by CD8+ Teff prevented control of surrounding Teff, described here as “bystander resistance”. Blockade of IL-6 receptor signaling or direct inhibition of PKB/c-Akt phosphorylation restored Treg responsiveness of Teff and prevented bystander resistance. In Teff of healthy controls (HC) exogenous IL-6 also changed the kinetics of IL-6 production and induced Treg unresponsiveness. This modulation was only transient in Teff from healthy volunteers, whereas accelerated IL-6 production in MS-Teff maintained also in absence of IL-6. Hence, we showed that the kinetics of IL-6 production instead of elevated IL-6 levels defines the Teff responsiveness in early Treg-T cell communication in MS independent of their disease course and propose IL-6 and associated PKB/c-Akt activation as effective therapeutic targets for modulation of Teff activity in MS.
doi:10.1371/journal.pone.0077634
PMCID: PMC3796502  PMID: 24155968
7.  Clinal distribution of human genomic diversity across the Netherlands despite archaeological evidence for genetic discontinuities in Dutch population history 
Background
The presence of a southeast to northwest gradient across Europe in human genetic diversity is a well-established observation and has recently been confirmed by genome-wide single nucleotide polymorphism (SNP) data. This pattern is traditionally explained by major prehistoric human migration events in Palaeolithic and Neolithic times. Here, we investigate whether (similar) spatial patterns in human genomic diversity also occur on a micro-geographic scale within Europe, such as in the Netherlands, and if so, whether these patterns could also be explained by more recent demographic events, such as those that occurred in Dutch population history.
Methods
We newly collected data on a total of 999 Dutch individuals sampled at 54 sites across the country at 443,816 autosomal SNPs using the Genome-Wide Human SNP Array 5.0 (Affymetrix). We studied the individual genetic relationships by means of classical multidimensional scaling (MDS) using different genetic distance matrices, spatial ancestry analysis (SPA), and ADMIXTURE software. We further performed dedicated analyses to search for spatial patterns in the genomic variation and conducted simulations (SPLATCHE2) to provide a historical interpretation of the observed spatial patterns.
Results
We detected a subtle but clearly noticeable genomic population substructure in the Dutch population, allowing differentiation of a north-eastern, central-western, central-northern and a southern group. Furthermore, we observed a statistically significant southeast to northwest cline in the distribution of genomic diversity across the Netherlands, similar to earlier findings from across Europe. Simulation analyses indicate that this genomic gradient could similarly be caused by ancient as well as by the more recent events in Dutch history.
Conclusions
Considering the strong archaeological evidence for genetic discontinuity in the Netherlands, we interpret the observed clinal pattern of genomic diversity as being caused by recent rather than ancient events in Dutch population history. We therefore suggest that future human population genetic studies pay more attention to recent demographic history in interpreting genetic clines. Furthermore, our study demonstrates that genetic population substructure is detectable on a small geographic scale in Europe despite recent demographic events, a finding we consider potentially relevant for future epidemiological and forensic studies.
doi:10.1186/2041-2223-4-9
PMCID: PMC3707805  PMID: 23687922
Population substructure; Genetic cline; Genome-wide diversity; SNP; Europe; Netherlands
8.  Human Trace Amine-Associated Receptor TAAR5 Can Be Activated by Trimethylamine 
PLoS ONE  2013;8(2):e54950.
In addition to the canonical olfactory receptors, TAARs were currently suggested to be a second class of chemosensory receptors in the olfactory epithelium of vertebrates. In contrast to several deorphanized murine TAARs, agonists for the intact human TAAR genes 2, 5, 6, 8 and 9 that are potentially expressed in the human olfactory epithelium have not been determined so far. Moreover, the physiological relevance of TAARs still remains elusive. We present the first successful functional expression of a human TAAR and agonists of human TAAR5. We performed a ligand screening using recombinantly expressed human TAAR5 in HANA3A cells and Xenopus laevis oocytes. In order to measure receptor activity, we used a cAMP-dependent reporter gene assay and two-electrode voltage clamp technique. As a result, human TAAR5 can be activated in a concentration-dependent manner by trimethylamine and with less efficacy by dimethylethylamine. It could neither be activated by any other of the tested single amines with a related chemical structure (42 in total), nor by any of the tested odorant mixtures. The hypothesis that Single Nucleotide Polymorphisms (SNP) within the reading frame of an olfactory receptor gene can cause a specific anosmia, formed the basis for clarifying the question, if anosmia for trimethylamine is caused by a SNP in a TAAR coding sequence. All functional human TAAR gene reading frames of subjects with specific anosmia for trimethylamine were amplified and products analyzed regarding SNP distribution. We demonstrated that the observed specific anosmia for trimethylamine is not correlated with a SNP in the coding sequence of one of the putatively functional human TAAR genes.
doi:10.1371/journal.pone.0054950
PMCID: PMC3564852  PMID: 23393561
9.  A Molecular Mechanism for Direct Sirtuin Activation by Resveratrol 
PLoS ONE  2012;7(11):e49761.
Sirtuins are protein deacetylases regulating metabolism, stress responses, and aging processes, and they were suggested to mediate the lifespan extending effect of a low calorie diet. Sirtuin activation by the polyphenol resveratrol can mimic such lifespan extending effects and alleviate metabolic diseases. The mechanism of Sirtuin stimulation is unknown, hindering the development of improved activators. Here we show that resveratrol inhibits human Sirt3 and stimulates Sirt5, in addition to Sirt1, against fluorophore-labeled peptide substrates but also against peptides and proteins lacking the non-physiological fluorophore modification. We further present crystal structures of Sirt3 and Sirt5 in complex with fluorogenic substrate peptide and modulator. The compound acts as a top cover, closing the Sirtuin’s polypeptide binding pocket and influencing details of peptide binding by directly interacting with this substrate. Our results provide a mechanism for the direct activation of Sirtuins by small molecules and suggest that activators have to be tailored to a specific Sirtuin/substrate pair.
doi:10.1371/journal.pone.0049761
PMCID: PMC3504108  PMID: 23185430
10.  Circulating Endothelial Progenitor Cells Are Up-Regulated in a Mouse Model of Endometriosis 
The American Journal of Pathology  2011;178(4):1782-1791.
Endometriosis is a debilitating disease characterized by the growth of ectopic endometrial tissue. It is widely accepted that angiogenesis plays an integral part in the establishment and growth of endometriotic lesions. Recent data from a variety of angiogenesis-dependent diseases suggest a critical role of bone marrow–derived endothelial progenitor cells (EPCs) in neovascularization. In this study we examined the blood levels of EPCs and mature circulating endothelial cells in a mouse model of surgically induced endometriosis. Fluorescence-activated cell sorting analysis revealed elevated levels of EPCs in the blood of mice with endometriosis compared with control subject that underwent a sham operation. EPC concentrations positively correlated with the amount of endometriotic tissue and peaked 1 to 4 days after induction of disease. In a green fluorescent protein bone marrow transplant experiment we found green fluorescent protein–positive endothelial cells incorporated into endometriotic lesions but not eutopic endometrium, as revealed by flow cytometry and immunohistochemistry. Finally, treatment of endometriosis-bearing mice with the angiogenesis inhibitor Lodamin, an oral nontoxic formulation of TNP-470, significantly decreased EPC levels while suppressing lesion growth. Taken together, our data indicate an important role for bone marrow–derived endothelial cells in the pathogenesis of endometriosis and support the potential clinical use of anti-angiogenic therapy as a novel treatment modality for this disease.
doi:10.1016/j.ajpath.2010.12.037
PMCID: PMC3070089  PMID: 21435458
11.  Genome-wide association study with DNA pooling identifies variants at CNTNAP2 associated with pseudoexfoliation syndrome 
Genetic and nongenetic factors contribute to development of pseudoexfoliation (PEX) syndrome, a complex, age-related, generalized matrix process frequently associated with glaucoma. To identify specific genetic variants underlying its etiology, we performed a genome-wide association study (GWAS) using a DNA-pooling approach. Therefore, equimolar amounts of DNA samples of 80 subjects with PEX syndrome, 80 with PEX glaucoma (PEXG) and 80 controls were combined into separate pools and hybridized to 500K SNP arrays (Affymetrix). Array probe intensity data were analyzed and visualized with expressly developed software tools GPFrontend and GPGraphics in combination with GenePool software. For replication, independent German cohorts of 610 unrelated patients with PEX/PEXG and 364 controls as well as Italian cohorts of 249 patients and 190 controls were used. Of 19, 17 SNPs showing significant allele frequency difference in DNA pools were confirmed by individual genotyping. Further single genotyping at CNTNAP2 locus revealed association between PEX/PEXG for two SNPs, which was confirmed in an independent German but not the Italian cohort. Both SNPs remained significant in the combined German cohorts even after Bonferroni correction (rs2107856: Pc=0.0108, rs2141388: Pc=0.0072). CNTNAP2 was found to be ubiquitously expressed in all human ocular tissues, particularly in retina, and localized to cell membranes of epithelial, endothelial, smooth muscle, glial and neuronal cells. Confirming efficiency of GWAS with DNA-pooling approach by detection of the known LOXL1 locus, our study data show evidence for association of CNTNAP2 with PEX syndrome and PEXG in German patients.
doi:10.1038/ejhg.2010.144
PMCID: PMC3025781  PMID: 20808326
pseudoexfoliation syndrome; DNA pooling; association study
12.  Boosting regulatory T cell function by CD4 stimulation enters the clinic 
Understanding tolerance mechanisms at the cellular and molecular level holds the promise to establish novel immune intervention therapies in patients with allergy or autoimmunity and to prevent transplant rejection. Administration of mAb against the CD4 molecule has been found to be exceptionally well suited for intentional tolerance induction in rodent and non-human primate models as well as in humanized mouse models. Recent evidence demonstrated that regulatory T cells (Treg) are directly activated by non-depleting CD4 ligands and suggests Treg activation as a central mechanism in anti-CD4-mediated tolerance induction. This review summarizes the current knowledge on the role of Treg in peripheral tolerance, addresses the putative mechanisms of Treg-mediated suppression and discusses the clinical potential of harnessing Treg suppressive activity through CD4 stimulation.
doi:10.3389/fimmu.2012.00164
PMCID: PMC3376463  PMID: 22719741
anti-CD4; cAMP; monoclonal antibody; regulatory T cells; tolerance
13.  COQ6 mutations in human patients produce nephrotic syndrome with sensorineural deafness  
The Journal of Clinical Investigation  2011;121(5):2013-2024.
Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of end-stage renal failure. Identification of single-gene causes of SRNS has generated some insights into its pathogenesis; however, additional genes and disease mechanisms remain obscure, and SRNS continues to be treatment refractory. Here we have identified 6 different mutations in coenzyme Q10 biosynthesis monooxygenase 6 (COQ6) in 13 individuals from 7 families by homozygosity mapping. Each mutation was linked to early-onset SRNS with sensorineural deafness. The deleterious effects of these human COQ6 mutations were validated by their lack of complementation in coq6-deficient yeast. Furthermore, knockdown of Coq6 in podocyte cell lines and coq6 in zebrafish embryos caused apoptosis that was partially reversed by coenzyme Q10 treatment. In rats, COQ6 was located within cell processes and the Golgi apparatus of renal glomerular podocytes and in stria vascularis cells of the inner ear, consistent with an oto-renal disease phenotype. These data suggest that coenzyme Q10–related forms of SRNS and hearing loss can be molecularly identified and potentially treated.
doi:10.1172/JCI45693
PMCID: PMC3083770  PMID: 21540551
14.  Circulating Endothelial Progenitor Cells Are Up-Regulated in a Mouse Model of Endometriosis 
The American Journal of Pathology  2011;178(4):1782-1791.
Endometriosis is a debilitating disease characterized by the growth of ectopic endometrial tissue. It is widely accepted that angiogenesis plays an integral part in the establishment and growth of endometriotic lesions. Recent data from a variety of angiogenesis-dependent diseases suggest a critical role of bone marrow–derived endothelial progenitor cells (EPCs) in neovascularization. In this study we examined the blood levels of EPCs and mature circulating endothelial cells in a mouse model of surgically induced endometriosis. Fluorescence-activated cell sorting analysis revealed elevated levels of EPCs in the blood of mice with endometriosis compared with control subject that underwent a sham operation. EPC concentrations positively correlated with the amount of endometriotic tissue and peaked 1 to 4 days after induction of disease. In a green fluorescent protein bone marrow transplant experiment we found green fluorescent protein–positive endothelial cells incorporated into endometriotic lesions but not eutopic endometrium, as revealed by flow cytometry and immunohistochemistry. Finally, treatment of endometriosis-bearing mice with the angiogenesis inhibitor Lodamin, an oral nontoxic formulation of TNP-470, significantly decreased EPC levels while suppressing lesion growth. Taken together, our data indicate an important role for bone marrow–derived endothelial cells in the pathogenesis of endometriosis and support the potential clinical use of anti-angiogenic therapy as a novel treatment modality for this disease.
doi:10.1016/j.ajpath.2010.12.037
PMCID: PMC3070089  PMID: 21435458
15.  Molecular karyotyping in patients with mental retardation using 100K single‐nucleotide polymorphism arrays 
Journal of Medical Genetics  2007;44(10):629-636.
Background
Using array techniques, it was recently shown that about 10% of patients with mental retardation of unknown origin harbour cryptic chromosomal aneusomies. However, data analysis is currently not standardised and little is known about its sensitivity and specificity.
Methods
We have developed an electronic data analysis tool for gene‐mapping SNP arrays, a software tool that we call Copy Number Variation Finder (CNVF). Using CNVF, we analysed 104 unselected patients with mental retardation of unknown origin with a genechip mapping 100K SNP array and established an optimised set of analysis parameters.
Results
We detected deletions as small as 20 kb when covered by at least three single‐nucleotide polymorphisms (SNPs) and duplications as small as 150 kb when covered by at least six SNPs, with only one false‐positive signal in six patients. In 9.1% of patients, we detected apparently disease‐causing or de novo aberrations ranging in size from 0.4 to 14 Mb. Morphological anomalies in patients with de novo aberrations were equal to that of unselected patients when measured with de Vries score.
Conclusion
Our standardised CNVF data analysis tool is easy to use and has high sensitivity and specificity. As some genomic regions are covered more densely than others, the genome‐wide resolution of the 100K array is about 400–500 kb for deletions and 900–1000 kb for duplications. The detection rate of about 10% of de novo aberrations is independent of selection of patients for particular features. The incidental finding in two patients of heterozygosity for the 250 kb recurrent deletion at the NPH1 locus, associated with autosomal recessive juvenile nephronophthisis, which was inherited from a healthy parent, highlights the fact that inherited aberrations might be disease‐related even though not causal for mental retardation.
doi:10.1136/jmg.2007.050914
PMCID: PMC2597959  PMID: 17601928
molecular karyotyping; 100K SNP array; mental retardation; copy number variation finder; NPH1
16.  Genes that determine immunology and inflammation modify the basic defect of impaired ion conductance in cystic fibrosis epithelia 
Journal of Medical Genetics  2010;48(1):24-31.
Background
The cystic fibrosis (CF) basic defect, caused by dysfunction of the apical chloride channel CFTR in the gastrointestinal and respiratory tract epithelia, has not been employed so far to support the role of CF modifier genes.
Methods
Patients were selected from 101 families with a total of 171 F508del-CFTR homozygous CF patients to identify CF modifying genes. A candidate gene based association study of 52 genes on 16 different chromosomes with a total of 182 genetic markers was performed. Differences in haplotype and/or diplotype distribution between case and reference CF subpopulations were analysed.
Results
Variants at immunologically relevant genes were associated with the manifestation of the CF basic defect (0.01
Conclusions
The inherited capabilities of the innate and adaptive immune system determine the manifestation of the CF basic defect. Variants on F508del-CFTR chromosomes contribute to the observed patient-to-patient variability among F508del-CFTR homozygotes. A survivor effect, manifesting as a transmission disequilibrium at many loci, is consistent with the improvement of clinical care over the last decades, resulting in a depletion of risk alleles at modifier genes. Awareness of non-genetic factors such as improvement of patient care over time is crucial for the interpretation of CF modifier studies.
doi:10.1136/jmg.2010.080937
PMCID: PMC3003880  PMID: 20837493
Cystic fibrosis; modifier genes; association study; affected sib pairs; CF basic defect; genetics; immunology (including allergy); infectious diseases; epidemiology
The autosomal recessive kidney disease nephronophthisis (NPHP) constitutes the most frequent genetic cause of terminal renal failure in the first 3 decades of life. Ten causative genes (NPHP1–NPHP9 and NPHP11), whose products localize to the primary cilia-centrosome complex, support the unifying concept that cystic kidney diseases are “ciliopathies”. Using genome-wide homozygosity mapping, we report here what we believe to be a new locus (NPHP-like 1 [NPHPL1]) for an NPHP-like nephropathy. In 2 families with an NPHP-like phenotype, we detected homozygous frameshift and splice-site mutations, respectively, in the X-prolyl aminopeptidase 3 (XPNPEP3) gene. In contrast to all known NPHP proteins, XPNPEP3 localizes to mitochondria of renal cells. However, in vivo analyses also revealed a likely cilia-related function; suppression of zebrafish xpnpep3 phenocopied the developmental phenotypes of ciliopathy morphants, and this effect was rescued by human XPNPEP3 that was devoid of a mitochondrial localization signal. Consistent with a role for XPNPEP3 in ciliary function, several ciliary cystogenic proteins were found to be XPNPEP3 substrates, for which resistance to N-terminal proline cleavage resulted in attenuated protein function in vivo in zebrafish. Our data highlight an emerging link between mitochondria and ciliary dysfunction, and suggest that further understanding the enzymatic activity and substrates of XPNPEP3 will illuminate novel cystogenic pathways.
doi:10.1172/JCI40076
PMCID: PMC2827951  PMID: 20179356
PLoS ONE  2009;4(9):e7158.
Background
In humans and mice naturally occurring CD4+CD25+ regulatory T cells (nTregs) are a thymus-derived subset of T cells, crucial for the maintenance of peripheral tolerance by controlling not only potentially autoreactive T cells but virtually all cells of the adaptive and innate immune system. Recent work using Dicer-deficient mice irrevocably demonstrated the importance of miRNAs for nTreg cell-mediated tolerance.
Principal Findings
DNA-Microarray analyses of human as well as murine conventional CD4+ Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations. Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3. In order to address the functional relevance of elevated miR-155 levels, we transfected miR-155 inhibitors or mature miR-155 RNAs into freshly-isolated human and mouse primary CD4+ Th cells and nTregs and investigated the resulting phenotype in nTreg suppression assays. Whereas miR-155 inhibition in conventional CD4+ Th cells strengthened nTreg cell-mediated suppression, overexpression of mature miR-155 rendered these cells unresponsive to nTreg cell-mediated suppression.
Conclusion
Investigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4+ Th cells to nTreg cell-mediated suppression.
doi:10.1371/journal.pone.0007158
PMCID: PMC2743997  PMID: 19777054
Genetic matching potentially provides a means to alleviate the effects of incomplete Mendelian randomization in population-based gene–disease association studies. We therefore evaluated the genetic-matched pair study design on the basis of genome-wide SNP data (309 790 markers; Affymetrix GeneChip Human Mapping 500K Array) from 2457 individuals, sampled at 23 different recruitment sites across Europe. Using pair-wise identity-by-state (IBS) as a matching criterion, we tried to derive a subset of markers that would allow identification of the best overall matching (BOM) partner for a given individual, based on the IBS status for the subset alone. However, our results suggest that, by following this approach, the prediction accuracy is only notably improved by the first 20 markers selected, and increases proportionally to the marker number thereafter. Furthermore, in a considerable proportion of cases (76.0%), the BOM of a given individual, based on the complete marker set, came from a different recruitment site than the individual itself. A second marker set, specifically selected for ancestry sensitivity using singular value decomposition, performed even more poorly and was no more capable of predicting the BOM than randomly chosen subsets. This leads us to conclude that, at least in Europe, the utility of the genetic-matched pair study design depends critically on the availability of comprehensive genotype information for both cases and controls.
doi:10.1038/ejhg.2008.266
PMCID: PMC2986489  PMID: 19156175
population structure; matching; association; ancestry; microarray
Case Reports in Medicine  2009;2009:132452.
Acute cholecystitis is a major health problem. There are multiple etiologies to be considered and early recognition of the condition is important to optimize management and outcome. We report the first case in the medical literature of symptomatic acute cholecystitis triggered by ceftriaxone-associated gallbladder sludge formation and, importantly, solid ceftriaxone gallstone formation in an adult patient with underlying mineral and pigment cholecystolithiasis, necessitating cholecystectomy. This case serves as a reminder for physicians to keep this uncommon cause of cholecystolithiasis and cholecystitis in mind in patients who receive prolonged ceftriaxone therapy. These patients should be cautioned to promptly report to their physicians any signs or symptoms of cholecystitis in order to ensure timely and appropriate evaluation.
doi:10.1155/2009/132452
PMCID: PMC2729472  PMID: 19707473
Summary
Background
Blood products derived from donors on medication can contain drugs which might pose a risk for the recipients or influence the quality of the product itself.
Material and Methods
To judge the eligibility of blood donors on medication, 4 drug classes have been formed with respect to their pharmacological properties, and blood products have been divided in accordance with their single-donor plasma contents.
Results
For drugs with dose-dependent pharmacodynamics, no deferral periods are necessary for donation of blood products containing less than 50 ml single-donor plasma for application to adults. Waiting periods of tmax + 5 t1/2 were calculated for the other blood products. Teratogenic drugs do not require special considerations (exception: retinoids, thalidomide and lenalidomide, dutasteride or finasteride with waiting periods for all blood products). A deferral period of tmax + 24 t1/2 is proposed for every blood product from blood donors on genotoxic drugs. Drugs without systemic effects can be neglected. Irreversible inhibitors of platelet function cause a 10-day waiting period if production of platelet concentrates is intended.
Conclusion
Donors on medication are allowed to donate blood for blood products containing less than 50 ml plasma of a single donor, like red blood cell concentrates, for the use in adults without deferral periods, except those taking retinoids, thalidomide, lenalidomide, dutasteride, finasteride, or genotoxic drugs.
doi:10.1159/000203355
PMCID: PMC2928823  PMID: 20823991
Blood donation; Donor deferral; Drug therapy; Pharmacokinetics; Pharmacology
PLoS Genetics  2009;5(1):e1000353.
The identification of recessive disease-causing genes by homozygosity mapping is often restricted by lack of suitable consanguineous families. To overcome these limitations, we apply homozygosity mapping to single affected individuals from outbred populations. In 72 individuals of 54 kindred ascertained worldwide with known homozygous mutations in 13 different recessive disease genes, we performed total genome homozygosity mapping using 250,000 SNP arrays. Likelihood ratio Z-scores (ZLR) were plotted across the genome to detect ZLR peaks that reflect segments of homozygosity by descent, which may harbor the mutated gene. In 93% of cases, the causative gene was positioned within a consistent ZLR peak of homozygosity. The number of peaks reflected the degree of inbreeding. We demonstrate that disease-causing homozygous mutations can be detected in single cases from outbred populations within a single ZLR peak of homozygosity as short as 2 Mb, containing an average of only 16 candidate genes. As many specialty clinics have access to cohorts of individuals from outbred populations, and as our approach will result in smaller genetic candidate regions, the new strategy of homozygosity mapping in single outbred individuals will strongly accelerate the discovery of novel recessive disease genes.
Author Summary
Many childhood diseases are caused by single-gene mutations of recessive genes, in which a child has inherited one mutated gene copy from each parent causing disease in the child, but not in the parents who are healthy heterozygous carriers. As the two mutations represent the disease cause, gene mapping helped understand disease mechanisms. “Homozygosity mapping” has been particularly useful. It assumes that the parents are related and that a disease-causing mutation together with a chromosomal segment of identical markers (i.e., homozygous markers) is transmitted to the affected child through the paternal and the maternal line from an ancestor common to both parents. Homozygosity mapping seeks out those homozygous regions to map the disease-causing gene. Homozygosity mapping requires families, in which the parents are knowingly related, and have multiple affected children. To overcome these limitations, we applied homozygosity mapping to single affected individuals from outbred populations. In 72 individuals with known homozygous mutations in 13 different recessive disease genes, we performed homozygosity mapping. In 93% we detected the causative gene in a segment of homozygosity. We demonstrate that disease-causing homozygous mutations can be detected in single cases from outbred populations. This will strongly accelerate the discovery of novel recessive disease genes.
doi:10.1371/journal.pgen.1000353
PMCID: PMC2621355  PMID: 19165332
Bead-based assay systems offer the possibility of measuring several specific antibodies in one sample simultaneously. This study evaluated a vaccine panel of a multianalyte system that measures antibodies to tetanus toxin, diphtheria toxin, and pertussis toxin (PT) from Bordetella pertussis. The antibody concentrations of human immunoglobulin G (IgG) to PT, tetanus toxin, and diphtheria toxin were measured in 123 serum pairs (total of 246 sera) from a vaccine study. The multianalyte bead assay was compared to a standardized in-house IgG- anti-PT enzyme-linked immunosorbent assay (ELISA) of the German reference laboratory for bordetellae, as well as to various commercially available ELISAs for anti-PT IgG, anti-tetanus IgG, and anti-diphtheria IgG. The results of the multiplex assay regarding the antibodies against diphtheria toxin compared favorably with a regression coefficient of 0.938 for values obtained with an ELISA from the same manufacturer used as a reference. Similarly, antibodies to tetanus toxin showed a correlation of 0.910 between the reference ELISA and the multianalyte assay. A correlation coefficient of 0.905 was found when an “in-house” IgG anti-PT and the multiplex assay were compared. Compared to single ELISA systems from two other manufacturers, the multiplex assay performed similarly well or better. The multianalyte assay system was a robust system with fast and accurate results, analyzing three parameters simultaneously in one sample. The system was well suited to quantitatively determine relevant vaccine induced antibodies compared to in-house and commercially available single-antigen ELISA systems.
doi:10.1128/CVI.00225-07
PMCID: PMC2394830  PMID: 18321880
Molecular Vision  2008;14:1081-1093.
Purpose
The goal of this study was to identify mutations in X-chromosomal genes associated with retinitis pigmentosa (RP) in patients from Germany, The Netherlands, Denmark, and Switzerland.
Methods
In addition to all coding exons of RP2, exons 1 through 15, 9a, ORF15, 15a and 15b of RPGR were screened for mutations. PCR products were amplified from genomic DNA extracted from blood samples and analyzed by direct sequencing. In one family with apparently dominant inheritance of RP, linkage analysis identified an interval on the X chromosome containing RPGR, and mutation screening revealed a pathogenic variant in this gene. Patients of this family were examined clinically and by X-inactivation studies.
Results
This study included 141 RP families with possible X-chromosomal inheritance. In total, we identified 46 families with pathogenic sequence alterations in RPGR and RP2, of which 17 mutations have not been described previously. Two of the novel mutations represent the most 3’-terminal pathogenic sequence variants in RPGR and RP2 reported to date. In exon ORF15 of RPGR, we found eight novel and 14 known mutations. All lead to a disruption of open reading frame. Of the families with suggested X-chromosomal inheritance, 35% showed mutations in ORF15. In addition, we found five novel mutations in other exons of RPGR and four in RP2. Deletions in ORF15 of RPGR were identified in three families in which female carriers showed variable manifestation of the phenotype. Furthermore, an ORF15 mutation was found in an RP patient who additionally carries a 6.4 kbp deletion downstream of the coding region of exon ORF15. We did not identify mutations in 39 sporadic male cases from Switzerland.
Conclusions
RPGR mutations were confirmed to be the most frequent cause of RP in families with an X-chromosomal inheritance pattern. We propose a screening strategy to provide molecular diagnostics in these families.
PMCID: PMC2426717  PMID: 18552978

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