Publications by authors named "Lambert P van den Heuvel"

103 Publications

Therapeutic concentrations of calcineurin inhibitors do not deregulate glutathione redox balance in human renal proximal tubule cells.

PLoS One 2021 30;16(4):e0250996. Epub 2021 Apr 30.

Laboratory of Peroxisome Biology and Intracellular Communication, Department of Cellular and Molecular Medicine, University of Leuven, Leuven, Belgium.

The calcineurin inhibitors (CNI) cyclosporine A and tacrolimus comprise the basis of immunosuppressive regimes in all solid organ transplantation. However, long-term or high exposure to CNI leads to histological and functional renal damage (CNI-associated nephrotoxicity). In the kidney, proximal tubule cells are the only cells that metabolize CNI and these cells are believed to play a central role in the origin of the toxicity for this class of drugs, although the underlying mechanisms are not clear. Several studies have reported oxidative stress as an important mediator of CNI-associated nephrotoxicity in response to CNI exposure in different available proximal tubule cell models. However, former models often made use of supra-therapeutic levels of tissue drug exposure. In addition, they were not shown to express the relevant enzymes (e.g., CYP3A5) and transporters (e.g., P-glycoprotein) for the metabolism of CNI in human proximal tubule cells. Moreover, the used methods for detecting ROS were potentially prone to false positive results. In this study, we used a novel proximal tubule cell model established from human allograft biopsies that demonstrated functional expression of relevant enzymes and transporters for the disposition of CNI. We exposed these cells to CNI concentrations as found in tissue of stable solid organ transplant recipients with therapeutic blood concentrations. We measured the glutathione redox balance in this cell model by using organelle-targeted variants of roGFP2, a highly sensitive green fluorescent reporter protein that dynamically equilibrates with the glutathione redox couple through the action of endogenous glutaredoxins. Our findings provide evidence that CNI, at concentrations commonly found in allograft biopsies, do not alter the glutathione redox balance in mitochondria, peroxisomes, and the cytosol. However, at supra-therapeutic concentrations, cyclosporine A but not tacrolimus increases the ratio of oxidized/reduced glutathione in the mitochondria, suggestive of imbalances in the redox environment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0250996PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8087105PMC
April 2021

Implications of genetic variation in the complement system in age-related macular degeneration.

Prog Retin Eye Res 2021 Feb 19:100952. Epub 2021 Feb 19.

Department of Ophthalmology, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, 6525, GA, Nijmegen, the Netherlands; Department of Human Genetics, Radboud University Medical Center, 6525, GA, Nijmegen, the Netherlands. Electronic address:

Age-related macular degeneration (AMD) is the main cause of vision loss among the elderly in the Western world. While AMD is a multifactorial disease, the complement system was identified as one of the main pathways contributing to disease risk. The strong link between the complement system and AMD was demonstrated by genetic associations, and by elevated complement activation in local eye tissue and in the systemic circulation of AMD patients. Several complement inhibitors have been and are being explored in clinical trials, but thus far with limited success, leaving the majority of AMD patients without treatment options to date. This indicates that there is still a gap of knowledge regarding the functional implications of the complement system in AMD pathogenesis and how to bring these towards clinical translation. Many different experimental set-ups and disease models have been used to study complement activation in vivo and in vitro, and recently emerging patient-derived induced pluripotent stem cells and genome-editing techniques open new opportunities to study AMD disease mechanisms and test new therapeutic strategies in the future. In this review we provide an extensive overview of methods employed to understand the molecular processes of complement activation in AMD pathogenesis. We discuss the findings, advantages and challenges of each approach and conclude with an outlook on how recent, exciting developments can fill in current knowledge gaps and can aid in the development of effective complement-targeting therapeutic strategies in AMD.
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http://dx.doi.org/10.1016/j.preteyeres.2021.100952DOI Listing
February 2021

Quantitative multiplex profiling of the complement system to diagnose complement-mediated diseases.

Clin Transl Immunology 2020 9;9(12):e1225. Epub 2020 Dec 9.

Laboratory of Medical Immunology Department of Laboratory Medicine Radboud Institute for Molecular Life Sciences Radboud University Medical Center Nijmegen The Netherlands.

Objectives: Complement deficiencies are difficult to diagnose because of the variability of symptoms and the complexity of the diagnostic process. Here, we applied a novel 'complementomics' approach to study the impact of various complement deficiencies on circulating complement levels.

Methods: Using a quantitative multiplex mass spectrometry assay, we analysed 44 peptides to profile 34 complement proteins simultaneously in 40 healthy controls and 83 individuals with a diagnosed deficiency or a potential pathogenic variant in 14 different complement proteins.

Results: Apart from confirming near or total absence of the respective protein in plasma of complement-deficient patients, this mass spectrometry-based profiling method led to the identification of additional deficiencies. In many cases, partial depletion of the pathway up- and/or downstream of the absent protein was measured. This was especially found in patients deficient for complement inhibitors, such as angioedema patients with a C1-inhibitor deficiency. The added value of complementomics was shown in three patients with poorly defined complement deficiencies.

Conclusion: Our study shows the potential clinical utility of profiling circulating complement proteins as a comprehensive read-out of various complement deficiencies. Particularly, our approach provides insight into the intricate interplay between complement proteins due to functional coupling, which contributes to the better understanding of the various disease phenotypes and improvement of care for patients with complement-mediated diseases.
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http://dx.doi.org/10.1002/cti2.1225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724921PMC
December 2020

Primary Human Derived Blood Outgrowth Endothelial Cells: An Appropriate In Vitro Model to Study Shiga Toxin Mediated Damage of Endothelial Cells.

Toxins (Basel) 2020 07 29;12(8). Epub 2020 Jul 29.

Cell Biology Program, Research Institute, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada.

Hemolytic uremic syndrome (HUS) is a rare disease primarily characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. Endothelial damage is the hallmark of the pathogenesis of HUS with an infection with the Shiga toxin (Stx) producing (STEC-HUS) as the main underlying cause in childhood. In this study, blood outgrowth endothelial cells (BOECs) were isolated from healthy donors serving as controls and patients recovered from STEC-HUS. We hypothesized that Stx is more cytotoxic for STEC-HUS BOECs compared to healthy donor control BOECs explained via a higher amount of Stx bound to the cell surface. Binding of Shiga toxin-2a (Stx2a) was investigated and the effect on cytotoxicity, protein synthesis, wound healing, and cell proliferation was studied in static conditions. Results show that BOECs are highly susceptible for Stx2a. Stx2a is able to bind to the cell surface of BOECs with cytotoxicity in a dose-dependent manner as a result. Pre-treatment with tumor necrosis factor alpha (TNF-α) results in enhanced Stx binding with 20-30% increased lactate dehydrogenase (LDH) release. Endothelial wound healing is delayed in a Stx2a-rich environment; however, this is not caused by an effect on the proliferation rate of BOECs. No significant differences were found between control BOECs and BOECs from recovered STEC-HUS patients in terms of Stx2a binding and inhibition of protein synthesis.
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http://dx.doi.org/10.3390/toxins12080483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472281PMC
July 2020

Effect of rare coding variants in the CFI gene on Factor I expression levels.

Hum Mol Genet 2020 08;29(14):2313-2324

Department of Ophthalmology, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.

Factor I (FI) is one of the main inhibitors of complement activity, and numerous rare coding variants have been reported in patients with age-related macular degeneration, atypical hemolytic uremic syndrome and C3 glomerulopathy. Since many of these variants are of unknown clinical significance, this study aimed to determine the effect of rare coding variants in the complement factor I (CFI) gene on FI expression. We measured FI levels in plasma samples of carriers of rare coding variants and in vitro in the supernatants of epithelial cells expressing recombinant FI. FI levels were measured in 177 plasma samples of 155 individuals, carrying 24 different rare coding variants in CFI. In carriers of the variants p.Gly119Arg, p.Leu131Arg, p.Gly188Ala and c.772G>A (r.685_773del), significantly reduced FI plasma levels were detected. Furthermore, recombinant FI expression levels were determined for 126 rare coding variants. Of these variants 68 (54%) resulted in significantly reduced FI expression in supernatant compared to wildtype (WT). The recombinant protein expression levels correlated significantly with the FI level in plasma of carriers of CFI variants. In this study, we performed the most comprehensive FI expression level analysis of rare coding variants in CFI to date. More than half of CFI variants lead to reduced FI expression, which might impair complement regulation in vivo. Our study will aid the interpretation of rare coding CFI variants identified in clinical practice, which is in particular important in light of patient inclusion in ongoing clinical trials for CFI gene supplementation in AMD.
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http://dx.doi.org/10.1093/hmg/ddaa114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7424754PMC
August 2020

Biosynthetic homeostasis and resilience of the complement system in health and infectious disease.

EBioMedicine 2019 Jul 29;45:303-313. Epub 2019 Jun 29.

Section Pediatric Infectious Diseases, Laboratory of Medical Immunology, Department of Laboratory Medicine, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands; Radboud Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, The Netherlands.

Background: The complement system is a central component of the innate immune system. Constitutive biosynthesis of complement proteins is essential for homeostasis. Dysregulation as a consequence of genetic or environmental cues can lead to inflammatory syndromes or increased susceptibility to infection. However, very little is known about steady state levels in children or its kinetics during infection.

Methods: With a newly developed multiplex mass spectrometry-based method we analyzed the levels of 32 complement proteins in healthy individuals and in a group of pediatric patients infected with bacterial or viral pathogens.

Findings: In plasma from young infants we found reduced levels of C4BP, ficolin-3, factor B, classical pathway components C1QA, C1QB, C1QC, C1R, and terminal pathway components C5, C8, C9, as compared to healthy adults; whereas the majority of complement regulating (inhibitory) proteins reach adult levels at very young age. Both viral and bacterial infections in children generally lead to a slight overall increase in complement levels, with some exceptions. The kinetics of complement levels during invasive bacterial infections only showed minor changes, except for a significant increase and decrease of CRP and clusterin, respectively.

Interpretation: The combination of lower levels of activating and higher levels of regulating complement proteins, would potentially raise the threshold of activation, which might lead to suppressed complement activation in the first phase of life. There is hardly any measurable complement consumption during bacterial or viral infection. Altogether, expression of the complement proteins appears surprisingly stable, which suggests that the system is continuously replenished. FUND: European Union's Horizon 2020, project PERFORM, grant agreement No. 668303.
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http://dx.doi.org/10.1016/j.ebiom.2019.06.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6642076PMC
July 2019

A Novel Choroidal Endothelial Cell Line Has a Decreased Affinity for the Age-Related Macular Degeneration-Associated Complement Factor H Variant 402H.

Invest Ophthalmol Vis Sci 2018 02;59(2):722-730

Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.

Purpose: Choroidal endothelial cells play a central role in the pathogenesis of age-related macular degeneration (AMD). Protocols for isolating primary choroidal endothelial cells have been described but require access to human donor eyes, which is a limiting factor. Therefore, a conditionally immortalized choroidal endothelial cell (ciChEnC) line has been established.

Methods: Choroidal endothelial cells were selected by magnetic-activated cell sorting and conditionally immortalized using temperature-sensitive simian virus 40 large T antigen and human telomerase. The cell line obtained was characterized based on expression of endothelial marker proteins and endothelial cell-specific responses to various stimuli. Binding of AMD-associated and non-AMD variants of complement factor H in the context of a recombinant CCP6-8 (complement control protein domains 6-8) construct was determined using ELISA.

Results: ciChEnCs maintained morphology and von Willebrand factor and vascular endothelial cadherin expression for up to 27 passages. The cells internalized acetylated low-density lipoprotein, formed tubes on Matrigel, and increased intercellular adhesion molecule-1 expression in response to tumor necrosis factor-α. Cells grew into dense monolayers with barrier function and showed characteristics of choriocapillary cells, such as expression of plasmalemma vesicle-associated protein, human leukocyte antigen ABC, carbonic anhydrase IV, and membrane indentations reflecting fenestrations. ciChEnCs synthesized glycosaminoglycans chondroitin sulfate and the complement factor H ligand heparan sulfate. Interestingly, binding of the AMD-associated 402H variant of factor H to ciChEnC was significantly decreased compared to the 402Y variant.

Conclusions: A novel ciChEnC cell line with choriocapillary characteristics has been established and should greatly facilitate investigation of the pathogenesis of AMD in the context of the choriocapillary microenvironment.
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http://dx.doi.org/10.1167/iovs.IOVS-17-22893DOI Listing
February 2018

The genetics of atypical hemolytic uremic syndrome.

Med Genet 2018 21;30(4):400-409. Epub 2018 Dec 21.

2Cell Biology Program, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada.

Atypical hemolytic uremic syndrome (aHUS) is a disorder characterized by thrombocytopenia and microangiopathic hemolytic anemia due to endothelial injury. aHUS is felt to be caused by defective complement regulation due to underlying genetic mutations in complement regulators or activators, most often of the alternative pathway. Mutations causing aHUS can be subdivided into two groups, loss of function mutations (affecting factor H, factor H-related proteins, membrane co-factor protein, and factor I), and gain of function mutations (affecting factor B and C3). As more information becomes available on the relationship between specific mutations and clinical outcome, complete genetic workup of aHUS patients becomes more and more important. In this review, we will discuss the genetic background of aHUS, the role of complement for aHUS pathogenesis, and the different groups of specific mutations known to be involved in the pathogenesis of aHUS.
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http://dx.doi.org/10.1007/s11825-018-0216-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404389PMC
December 2018

ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism.

Brain 2017 Jun;140(6):1595-1610

Department of Pediatrics, Saitama Medical University, Moroyama-machi, Iruma-gun, Saitama, Japan.

Although mitochondrial disorders are clinically heterogeneous, they frequently involve the central nervous system and are among the most common neurogenetic disorders. Identifying the causal genes has benefited enormously from advances in high-throughput sequencing technologies; however, once the defect is known, researchers face the challenge of deciphering the underlying disease mechanism. Here we characterize large biallelic deletions in the region encoding the ATAD3C, ATAD3B and ATAD3A genes. Although high homology complicates genomic analysis of the ATAD3 defects, they can be identified by targeted analysis of standard single nucleotide polymorphism array and whole exome sequencing data. We report deletions that generate chimeric ATAD3B/ATAD3A fusion genes in individuals from four unrelated families with fatal congenital pontocerebellar hypoplasia, whereas a case with genomic rearrangements affecting the ATAD3C/ATAD3B genes on one allele and ATAD3B/ATAD3A genes on the other displays later-onset encephalopathy with cerebellar atrophy, ataxia and dystonia. Fibroblasts from affected individuals display mitochondrial DNA abnormalities, associated with multiple indicators of altered cholesterol metabolism. Moreover, drug-induced perturbations of cholesterol homeostasis cause mitochondrial DNA disorganization in control cells, while mitochondrial DNA aggregation in the genetic cholesterol trafficking disorder Niemann-Pick type C disease further corroborates the interdependence of mitochondrial DNA organization and cholesterol. These data demonstrate the integration of mitochondria in cellular cholesterol homeostasis, in which ATAD3 plays a critical role. The dual problem of perturbed cholesterol metabolism and mitochondrial dysfunction could be widespread in neurological and neurodegenerative diseases.
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http://dx.doi.org/10.1093/brain/awx094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5445257PMC
June 2017

First Successful Conception Induced by a Male Cystinosis Patient.

JIMD Rep 2018 13;38:1-6. Epub 2017 Apr 13.

Department of Pediatric Nephrology, University Hospitals Leuven, Herestraat 49, 3000, Leuven, Belgium.

Cystinosis is a rare autosomal recessive lysosomal storage disease characterized by multi-organ cystine accumulation, leading to renal failure and extra-renal organ dysfunction. Azoospermia of unknown origin is the main cause of infertility in all male cystinosis patients. Although spermatogenesis has shown to be intact at the testicular level in some patients, no male cystinosis patient has been reported yet to have successfully induced conception.We present the first successful conception ever reported, induced by a 27-year-old male renal transplant infantile nephropathic cystinosis patient through percutaneous epididymal sperm aspiration (PESA) followed by intracytoplasmatic sperm injection (ICSI). After 36 weeks and 6 days of an uncomplicated pregnancy, a dichorial diamniotic (DCDA) twin was born with an appropriate weight for gestational age and in an apparently healthy status. Moreover, we demonstrate that the sperm of epididymal origin in selected male cystinosis patients can be viable for inducing successful conception.Our observation opens a new perspective in life for many male cystinosis patients whom nowadays have become adults, by showing that despite azoospermia fathering a child can be realized. In addition, our findings raise questions about the possibility of sperm cryopreservation at a young age in these patients.
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http://dx.doi.org/10.1007/8904_2017_19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874217PMC
April 2017

Serological and genetic complement alterations in infection-induced and complement-mediated hemolytic uremic syndrome.

Pediatr Nephrol 2017 02 7;32(2):297-309. Epub 2016 Oct 7.

Department of Pediatric Nephrology (804), Radboud University Medical Center, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands.

Background: The role of complement in the atypical form of hemolytic uremic syndrome (aHUS) has been investigated extensively in recent years. As the HUS-associated bacteria Shiga-toxin-producing Escherichia coli (STEC) can evade the complement system, we hypothesized that complement dysregulation is also important in infection-induced HUS.

Methods: Serological profiles (C3, FH, FI, AP activity, C3d, C3bBbP, C3b/c, TCC, αFH) and genetic profiles (CFH, CFI, CD46, CFB, C3) of the alternative complement pathway were prospectively determined in the acute and convalescent phase of disease in children newly diagnosed with STEC-HUS or aHUS. Serological profiles were compared with those of 90 age-matched controls.

Results: Thirty-seven patients were studied (26 STEC-HUS, 11 aHUS). In 39 % of them, including 28 % of STEC-HUS patients, we identified a genetic and/or acquired complement abnormality. In all patient groups, the levels of investigated alternative pathway (AP) activation markers were elevated in the acute phase and normalized in remission. The levels were significantly higher in aHUS than in STEC-HUS patients.

Conclusions: In both infection-induced HUS and aHUS patients, complement is activated in the acute phase of the disease but not during remission. The C3d/C3 ratio displayed the best discrepancy between acute and convalescent phase and between STEC-HUS and aHUS and might therefore be used as a biomarker in disease diagnosis and monitoring. The presence of aberrations in the alternative complement pathway in STEC-HUS patients was remarkable, as well.
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http://dx.doi.org/10.1007/s00467-016-3496-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5203860PMC
February 2017

Complement Factor H Serum Levels Determine Resistance to Pneumococcal Invasive Disease.

J Infect Dis 2016 06 21;213(11):1820-7. Epub 2016 Jan 21.

Laboratory of Pediatric Infectious Diseases Division of Pediatric Infectious Diseases and Immunology, Department of Pediatrics, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.

Streptococcus pneumoniae is a major cause of life-threatening infections. Complement activation plays a vital role in opsonophagocytic killing of pneumococci in blood. Initial complement activation via the classical and lectin pathways is amplified through the alternative pathway amplification loop. Alternative pathway activity is inhibited by complement factor H (FH). Our study demonstrates the functional consequences of the variability in human serum FH levels on host defense. Using an in vivo mouse model combined with human in vitro assays, we show that the level of serum FH correlates with the efficacy of opsonophagocytic killing of pneumococci. In summary, we found that FH levels determine a delicate balance of alternative pathway activity, thus affecting the resistance to invasive pneumococcal disease. Our results suggest that variation in FH expression levels, naturally occurring in the human population, plays a thus far unrecognized role in the resistance to invasive pneumococcal disease.
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http://dx.doi.org/10.1093/infdis/jiw029DOI Listing
June 2016

Differential Expression of Specific Dermatan Sulfate Domains in Renal Pathology.

PLoS One 2015 31;10(9):e0134946. Epub 2015 Aug 31.

Nephrology Research Laboratory, Radboud Institute for Molecular Life Sciences, Department of Nephrology, Radboud University Medical Center, Nijmegen, The Netherlands.

Dermatan sulfate (DS), also known as chondroitin sulfate (CS)-B, is a member of the linear polysaccharides called glycosaminoglycans (GAGs). The expression of CS/DS and DS proteoglycans is increased in several fibrotic renal diseases, including interstitial fibrosis, diabetic nephropathy, mesangial sclerosis and nephrosclerosis. Little, however, is known about structural alterations in DS in renal diseases. The aim of this study was to evaluate the renal expression of two different DS domains in renal transplant rejection and glomerular pathologies. DS expression was evaluated in normal renal tissue and in kidney biopsies obtained from patients with acute interstitial or vascular renal allograft rejection, patients with interstitial fibrosis and tubular atrophy (IF/TA), and from patients with focal segmental glomerulosclerosis (FSGS), membranous glomerulopathy (MGP) or systemic lupus erythematosus (SLE), using our unique specific anti-DS antibodies LKN1 and GD3A12. Expression of the 4/2,4-di-O-sulfated DS domain recognized by antibody LKN1 was decreased in the interstitium of transplant kidneys with IF/TA, which was accompanied by an increased expression of type I collagen, decorin and transforming growth factor beta (TGF-β), while its expression was increased in the interstitium in FSGS, MGP and SLE. Importantly, all patients showed glomerular LKN1 staining in contrast to the controls. Expression of the IdoA-Gal-NAc4SDS domain recognized by GD3A12 was similar in controls and patients. Our data suggest a role for the DS domain recognized by antibody LKN1 in renal diseases with early fibrosis. Further research is required to delineate the exact role of different DS domains in renal fibrosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0134946PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4556443PMC
May 2016

Uremic Toxins Induce ET-1 Release by Human Proximal Tubule Cells, which Regulates Organic Cation Uptake Time-Dependently.

Cells 2015 Jun 26;4(3):234-52. Epub 2015 Jun 26.

Department of Pediatric Nephrology, Radboudumc 6525 GA Nijmegen, The Netherlands.

In renal failure, the systemic accumulation of uremic waste products is strongly associated with the development of a chronic inflammatory state. Here, the effect of cationic uremic toxins on the release of inflammatory cytokines and endothelin-1 (ET-1) was investigated in conditionally immortalized proximal tubule epithelial cells (ciPTEC). Additionally, we examined the effects of ET-1 on the cellular uptake mediated by organic cation transporters (OCTs). Exposure of ciPTEC to cationic uremic toxins initiated production of the inflammatory cytokines IL-6 (117 ± 3%, p < 0.001), IL-8 (122 ± 3%, p < 0.001), and ET-1 (134 ± 5%, p < 0.001). This was accompanied by a down-regulation of OCT mediated 4-(4-(dimethylamino)styryl)-N-methylpyridinium-iodide (ASP+) uptake in ciPTEC at 30 min (23 ± 4%, p < 0.001), which restored within 60 min of incubation. Exposure to ET-1 for 24 h increased the ASP+ uptake significantly (20 ± 5%, p < 0.001). These effects could be blocked by BQ-788, indicating activation of an ET-B-receptor-mediated signaling pathway. Downstream the receptor, iNOS inhibition by (N(G)-monomethyl-l-arginine) l-NMMA acetate or aminoguanidine, as well as protein kinase C activation, ameliorated the short-term effects. These results indicate that uremia results in the release of cytokines and ET-1 from human proximal tubule cells, in vitro. Furthermore, ET-1 exposure was found to regulate proximal tubular OCT transport activity in a differential, time-dependent, fashion.
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http://dx.doi.org/10.3390/cells4030234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4588034PMC
June 2015

Development of a living membrane comprising a functional human renal proximal tubule cell monolayer on polyethersulfone polymeric membrane.

Acta Biomater 2015 Mar 17;14:22-32. Epub 2014 Dec 17.

Department of Biomaterials Science and Technology, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands. Electronic address:

The need for improved renal replacement therapies has stimulated innovative research for the development of a cell-based renal assist device. A key requirement for such a device is the formation of a "living membrane", consisting of a tight kidney cell monolayer with preserved functional organic ion transporters on a suitable artificial membrane surface. In this work, we applied a unique conditionally immortalized proximal tubule epithelial cell (ciPTEC) line with an optimized coating strategy on polyethersulfone (PES) membranes to develop a living membrane with a functional proximal tubule epithelial cell layer. PES membranes were coated with combinations of 3,4-dihydroxy-l-phenylalanine and human collagen IV (Coll IV). The optimal coating time and concentrations were determined to achieve retention of vital blood components while preserving high water transport and optimal ciPTEC adhesion. The ciPTEC monolayers obtained were examined through immunocytochemistry to detect zona occludens 1 tight junction proteins. Reproducible monolayers were formed when using a combination of 2 mg ml(-1) 3,4-dihydroxy-l-phenylalanine (4 min coating, 1h dissolution) and 25 μg ml(-1) Coll IV (4 min coating). The successful transport of (14)C-creatinine through the developed living membrane system was used as an indication for organic cation transporter functionality. The addition of metformin or cimetidine significantly reduced the creatinine transepithelial flux, indicating active creatinine uptake in ciPTECs, most likely mediated by the organic cation transporter, OCT2 (SLC22A2). In conclusion, this study shows the successful development of a living membrane consisting of a reproducible ciPTEC monolayer on PES membranes, an important step towards the development of a bioartificial kidney.
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http://dx.doi.org/10.1016/j.actbio.2014.12.002DOI Listing
March 2015

Mutations in COA6 cause cytochrome c oxidase deficiency and neonatal hypertrophic cardiomyopathy.

Hum Mutat 2015 Jan 18;36(1):34-8. Epub 2014 Nov 18.

Department of Pediatrics, Nijmegen Centre for Mitochondrial Disorders, Radboud University Medical Centre, GA Nijmegen, The Netherlands; Department of General Pediatrics, Neonatology and Pediatric Cardiology, University Children's Hospital, Heinrich-Heine-University Duesseldorf, Duesseldorf, Germany.

COA6/C1ORF31 is involved in cytochrome c oxidase (complex IV) biogenesis. We present a new pathogenic COA6 variant detected in a patient with neonatal hypertrophic cardiomyopathy and isolated complex IV deficiency. For the first time, clinical details about a COA6-deficient patient are given and patient fibroblasts are functionally characterized: COA6 protein is undetectable and steady-state levels of complex IV and several of its subunits are reduced. The monomeric COX1 assembly intermediate accumulates. Using pulse-chase experiments, we demonstrate an increased turnover of mitochondrial encoded complex IV subunits. Although monomeric complex IV is decreased in patient fibroblasts, the CI/CIII2 /CIVn -supercomplexes remain unaffected. Copper supplementation shows a partial rescue of complex IV deficiency in patient fibroblasts. We conclude that COA6 is required for complex IV subunit stability. Furthermore, the proposed role in the copper delivery pathway to complex IV subunits is substantiated and a therapeutic lead for COA6-deficient patients is provided.
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http://dx.doi.org/10.1002/humu.22715DOI Listing
January 2015

Genetic testing for podocyte genes in sporadic focal segmental glomerulosclerosis.

Nephrol Dial Transplant 2014 Nov 21;29(11):1985-6. Epub 2014 Jul 21.

Department of Paediatric Nephrology, Radboud University Nijmegen Medical Center, Nijmegen, Netherlands.

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http://dx.doi.org/10.1093/ndt/gfu247DOI Listing
November 2014

SDHA mutations causing a multisystem mitochondrial disease: novel mutations and genetic overlap with hereditary tumors.

Eur J Hum Genet 2015 Feb 30;23(2):202-9. Epub 2014 Apr 30.

Nijmegen Center for Mitochondrial Disorders (NCMD) at the Department of Pediatrics, Radboud University Medical Center (Radboudumc), Nijmegen, The Netherlands.

Defects in complex II of the mitochondrial respiratory chain are a rare cause of mitochondrial disorders. Underlying autosomal-recessive genetic defects are found in most of the 'SDHx' genes encoding complex II (SDHA, SDHB, SDHC, and SDHD) and its assembly factors. Interestingly, SDHx genes also function as tumor suppressor genes in hereditary paragangliomas, pheochromocytomas, and gastrointestinal stromal tumors. In these cases, the affected patients are carrier of a heterozygeous SDHx germline mutation. Until now, mutations in SDHx associated with mitochondrial disease have not been reported in association with hereditary tumors and vice versa. Here, we characterize four patients with isolated complex II deficiency caused by mutations in SDHA presenting with multisystem mitochondrial disease including Leigh syndrome (LS) and/or leukodystrophy. Molecular genetic analysis revealed three novel mutations in SDHA. Two mutations (c.64-2A>G and c.1065-3C>A) affect mRNA splicing and result in loss of protein expression. These are the first mutations described affecting SDHA splicing. For the third new mutation, c.565T>G, we show that it severely affects enzyme activity. Its pathogenicity was confirmed by lentiviral complementation experiments on the fibroblasts of patients carrying this mutation. It is of special interest that one of our LS patients harbored the c.91C>T (p.Arg31*) mutation that was previously only reported in association with paragangliomas and pheochromocytomas, tightening the gap between these two rare disorders. As tumor screening is recommended for SDHx mutation carriers, this should also be considered for patients with mitochondrial disorders and their family members.
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http://dx.doi.org/10.1038/ejhg.2014.80DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4297908PMC
February 2015

Compound heterozygous mutations in the C6 gene of a child with recurrent infections.

Mol Immunol 2014 Apr 30;58(2):201-5. Epub 2013 Dec 30.

Department of Pediatric Infectious Diseases & Immunology (HP 804), Radboud University Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands; Nijmegen Institute for Infection, Immunity and Inflammation (HP 804), Radboud University Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Electronic address:

The complement system plays an important role in both the innate and adaptive immune system. Patients with inherited complement deficiencies have an increased risk of systemic bacterial infections. Deficiencies of the terminal complement pathway are especially associated with invasive meningococcal disease. Here, we report a case of a boy that presented with arthritis and recurrent bacterial and viral infections. Extensive analyses revealed decreased complement activity of both classical and alternative pathway, indicating a deficiency of C3 or one of the factors of the terminal complement pathway. Mutational analysis of the C6 gene identified two compound heterozygous mutations. An unknown missense aberration was found that involves the loss of a cysteine, possibly affecting the 3D structure of the protein. Furthermore, a known splice site variation was identified that results in a 14% shorter protein, due to transcription of amino acids that are normally intronic until a stop codon is reached (exon-intron boundary defect). It is known that the protein with this latter aberration is still functionally active when present with other C6 mutations and therefore, the consequences of the combination of the identified variations have been studied. Quantitative ELISAs showed that at least one allele produced a circulating C6 molecule that can be incorporated in the membrane attack complex, likely the truncated protein. In the present case we observed relapsing bacterial and viral infections, but no meningococcal disease. The reduced complement activity can be explained by the identified genetic variations in C6, as recombinant C6 supplementation corrected complement function in vitro.
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http://dx.doi.org/10.1016/j.molimm.2013.11.023DOI Listing
April 2014

Analysis of 953 human proteins from a mitochondrial HEK293 fraction by complexome profiling.

PLoS One 2013 23;8(7):e68340. Epub 2013 Jul 23.

Department of Laboratory Medicine, Laboratory of Genetic Endocrine and Metabolic Disorders, Radboud University Medical Centre, Nijmegen, The Netherlands.

Complexome profiling is a novel technique which uses shotgun proteomics to establish protein migration profiles from fractionated blue native electrophoresis gels. Here we present a dataset of blue native electrophoresis migration profiles for 953 proteins by complexome profiling. By analysis of mitochondrial ribosomal complexes we demonstrate its potential to verify putative protein-protein interactions identified by affinity purification-mass spectrometry studies. Protein complexes were extracted in their native state from a HEK293 mitochondrial fraction and separated by blue native gel electrophoresis. Gel lanes were cut into gel slices of even size and analyzed by shotgun proteomics. Subsequently, the acquired protein migration profiles were analyzed for co-migration via hierarchical cluster analysis. This dataset holds great promise as a comprehensive resource for de novo identification of protein-protein interactions or to underpin and prioritize candidate protein interactions from other studies. To demonstrate the potential use of our dataset we focussed on the mitochondrial translation machinery. Our results show that mitoribosomal complexes can be analyzed by blue native gel electrophoresis, as at least four distinct complexes. Analysis of these complexes confirmed that 24 proteins that had previously been reported to co-purify with mitoribosomes indeed co-migrated with subunits of the mitochondrial ribosome. Co-migration of several proteins involved in biogenesis of inner mitochondrial membrane complexes together with mitoribosomal complexes suggested the possibility of co-translational assembly in human cells. Our data also highlighted a putative ribonucleotide complex that potentially contains MRPL10, MRPL12 and MRPL53 together with LRPPRC and SLIRP.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0068340PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3720734PMC
March 2014

Cationic uremic toxins affect human renal proximal tubule cell functioning through interaction with the organic cation transporter.

Pflugers Arch 2013 Dec 29;465(12):1701-14. Epub 2013 Jun 29.

Department of Pediatric Nephrology, Radboud University Nijmegen Medical Centre, 6525 GA, Nijmegen, The Netherlands.

Several organic cations, such as guanidino compounds and polyamines, have been found to accumulate in plasma of patients with kidney failure due to inadequate renal clearance. Here, we studied the interaction of cationic uremic toxins with renal organic cation transport in a conditionally immortalized human proximal tubule epithelial cell line (ciPTEC). Transporter activity was measured and validated in cell suspensions by studying uptake of the fluorescent substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium-iodide (ASP(+)). Subsequently, the inhibitory potencies of the cationic uremic toxins, cadaverine, putrescine, spermine and spermidine (polyamines), acrolein (polyamine breakdown product), guanidine, and methylguanidine (guanidino compounds) were determined. Concentration-dependent inhibition of ASP(+) uptake by TPA, cimetidine, quinidine, and metformin confirmed functional endogenous organic cation transporter 2 (OCT2) expression in ciPTEC. All uremic toxins tested inhibited ASP(+) uptake, of which acrolein required the lowest concentration to provoke a half-maximal inhibition (IC50 = 44 ± 2 μM). A Dixon plot was constructed for acrolein using three independent inhibition curves with 10, 20, or 30 μM ASP(+), which demonstrated competitive or mixed type of interaction (K i = 93 ± 16 μM). Exposing the cells to a mixture of cationic uremic toxins resulted in a more potent and biphasic inhibitory response curve, indicating complex interactions between the toxins and ASP(+) uptake. In conclusion, ciPTEC proves a suitable model to study cationic xenobiotic interactions. Inhibition of cellular uptake transport was demonstrated for several uremic toxins, which might indicate a possible role in kidney disease progression during uremia.
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http://dx.doi.org/10.1007/s00424-013-1307-zDOI Listing
December 2013

A complex V ATP5A1 defect causes fatal neonatal mitochondrial encephalopathy.

Brain 2013 May 18;136(Pt 5):1544-54. Epub 2013 Apr 18.

Department of Paediatrics, Nijmegen Centre for Mitochondrial Disorders, Laboratory for Genetic, Endocrine, and Metabolic Disorders, Radboud University Medical Centre, Nijmegen, The Netherlands.

Whole exome sequencing is a powerful tool to detect novel pathogenic mutations in patients with suspected mitochondrial disease. However, the interpretation of novel genetic variants is not always straightforward. Here, we present two siblings with a severe neonatal encephalopathy caused by complex V deficiency. The aim of this study was to uncover the underlying genetic defect using the combination of enzymatic testing and whole exome sequence analysis, and to provide evidence for causality by functional follow-up. Measurement of the oxygen consumption rate and enzyme analysis in fibroblasts were performed. Immunoblotting techniques were applied to study complex V assembly. The coding regions of the genome were analysed. Three-dimensional modelling was applied. Exome sequencing of the two siblings with complex V deficiency revealed a heterozygous mutation in the ATP5A1 gene, coding for complex V subunit α. The father carried the variant heterozygously. At the messenger RNA level, only the mutated allele was expressed in the patients, whereas the father expressed both the wild-type and the mutant allele. Gene expression data indicate that the maternal allele is not expressed, which is supported by the observation that the ATP5A1 expression levels in the patients and their mother are reduced to ∼50%. Complementation with wild-type ATP5A1 restored complex V in the patient fibroblasts, confirming pathogenicity of the defect. At the protein level, the mutation results in a disturbed interaction of the α-subunit with the β-subunit of complex V, which interferes with the stability of the complex. This study demonstrates the important value of functional studies in the diagnostic work-up of mitochondrial patients, in order to guide genetic variant prioritization, and to validate gene defects.
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http://dx.doi.org/10.1093/brain/awt086DOI Listing
May 2013

A novel mutation in COQ2 leading to fatal infantile multisystem disease.

J Neurol Sci 2013 Mar 21;326(1-2):24-8. Epub 2013 Jan 21.

Laboratory of Clinical Chemistry and Hematology, St. Elisabeth Hospital, Tilburg, The Netherlands.

Coenzyme Q10 (ubiquinone or CoQ10) serves as a redox carrier in the mitochondrial oxidative phosphorylation system. The reduced form of this lipid-soluble antioxidant (ubiquinol) is involved in other metabolic processes as well, such as preventing reactive oxygen species (ROS) induced damage from the mitochondrial membrane. Primary coenzyme Q10 deficiency is a rare, autosomal recessive disorder, often presenting with neurological and/or muscle involvement. Until now, five patients from four families have been described with primary coenzyme Q10 deficiency due to mutations in COQ2 encoding para-hydroxybenzoate polyprenyl transferase. Interestingly, four of these patients showed a distinctive renal involvement (focal segmental glomerular sclerosis, crescentic glomerulonephritis, nephrotic syndrome), which is only very rarely seen in correlation with mitochondrial disorders. The fifth patient deceases due to infantile multi organ failure, also with renal involvement. Here we report a novel homozygous mutation in COQ2 (c.905C>T, p.Ala302Val) in a dizygotic twin from consanguineous Turkish parents. The children were born prematurely and died at the age of five and six months, respectively, after an undulating disease course involving apneas, seizures, feeding problems and generalized edema, alternating with relative stable periods without the need of artificial ventilation. There was no evidence for renal involvement. We would like to raise awareness for this potentially treatable disorder which could be under diagnosed in patients with fatal neonatal or infantile multi-organ disease.
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http://dx.doi.org/10.1016/j.jns.2013.01.004DOI Listing
March 2013

Bartter syndrome type III and congenital anomalies of the kidney and urinary tract: an antenatal presentation.

Clin Nephrol 2012 Dec;78(6):492-6

Department of Pediatric Nephrology, VU University Medical Center, Amsterdam, The Netherlands.

Bartter syndrome encompasses a variety of inheritable renal tubular transport disorders characterized by hypokalemia and hypochloremic metabolic alkalosis. Bartter syndrome Type III is caused by genetic alterations in the chloride channel kidney B (CLCNKB) gene and often presents in the first 2 years of life, known as classic Bartter syndrome. However, in rare cases Bartter syndrome Type III has an antenatal presentation with polyhydramnios, premature delivery and severe dehydration in the first weeks of life. Associations between congenital anomalies of the kidney and urinary tract and Bartter syndrome are extremely rare. This case report presents a girl with Bartter syndrome Type III due to a homozygous CLCNKB mutation and bilateral congenital anomalies of the kidney and urinary tract. In addition, we describe the antenatal presentation as well as its perinatal management.
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http://dx.doi.org/10.5414/cn107206DOI Listing
December 2012

The challenge of managing hemophilia A and STEC-induced hemolytic uremic syndrome.

Pediatr Nephrol 2013 Feb 23;28(2):349-52. Epub 2012 Sep 23.

Department of Pediatric Nephrology, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, the Netherlands.

Background: The hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy leading to acute kidney injury in children. In most cases it is triggered by an infection caused by Shiga-like toxin-producing Escherichia coli (STEC). Endothelial damage plays a central role in the pathogenesis of disease. Hemophilia A is a genetic disorder leading to factor VIII (FVIII) deficiency, an important factor in the coagulation system.

Case: Here we describe a hemophilia A patient who developed HUS due to a STEC O26 infection. The patient developed not only acute kidney injury, but also severe gastro-intestinal and neurological complications. Increased amounts of recombinant FVIII (rFVIII) had to be administered during the acute phase of the disease to reach acceptable blood levels of FVIII, in order to control the hemorrhagic colitis and to prevent severe neurological complications.

Conclusion: The patient's treatment schedule of rFVIII during the HUS period was a serious challenge, and we cannot exclude that it contributed to the severity of the HUS by enhancing the thrombotic microangiopathic process. The role of factor VIII administration in the severe outcome of this disease is discussed.
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http://dx.doi.org/10.1007/s00467-012-2312-8DOI Listing
February 2013

A comprehensive full factorial LC-MS/MS proteomics benchmark data set.

Proteomics 2012 Aug;12(14):2276-81

Nijmegen Centre for Mitochondrial Disorders, Department of Laboratory Medicine, Laboratory of Genetic, Endocrine and Metabolic Diseases, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

An important prerequisite for the development and benchmarking of novel analysis methods is a well-designed comprehensive LC-MS/MS data set. Here, we present our data set consisting of 59 LC-MS/MS analyses of 50 protein samples extracted individually from Escherichia coli K12 and spiked with different concentrations of bovine carbonic anhydrase II and/or chicken ovalbumin, according to a 2 × 3 full factorial design. Using the well-annotated and commonly used E. coli proteome as the sample background ensures that the complexity of the data is on a par with most current proteomic analyses. Data were acquired over a 2-month period using multiple reversed-phase columns and instrument calibrations to include real-life challenges faced when analyzing large proteomics data sets. Moreover, so-called "ground truth" data, comprised by LC-MS/MS measurements of the pure spikes are included in the data set. The current manuscript elaborates this comprehensive benchmark data set for future development and evaluation of analysis methods and software.
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http://dx.doi.org/10.1002/pmic.201100284DOI Listing
August 2012

Molecular base of biochemical complex I deficiency.

Mitochondrion 2012 Sep 20;12(5):520-32. Epub 2012 Jul 20.

Department of Paediatrics, Nijmegen Centre for Mitochondrial Disorders, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

The oxidative phosphorylation (OXPHOS) system, consisting of five enzyme complexes (I-V) together with 2 electron carriers, has an important role in the energy metabolism of the cell. With 45 subunits, complex I is the first and largest complex of the respiratory chain. It is under bigenomic control and a proper interaction between the mitochondrial and the nuclear genome is important for a good biogenesis and functioning of the complex. Isolated complex I deficiency is the most frequently diagnosed form of mitochondrial disorders caused by the disturbance of the OXPHOS system. It has a wide clinical variety and, at present, in many patients the underlying genetic cause of the complex I deficiency is still not known. In this review, the role of complex I in the oxidative phosphorylation and the localization and function of the different complex I subunits will be described. Furthermore, a brief overview of the assembly process and biochemical studies, performed when a patient is suspected of a mitochondrial disorder is given. Finally, the present knowledge for molecular base of complex I deficiency is described and the findings in a research cohort of patients with complex I deficiency are reported. Identifying new genes encoding proteins involved in complex I biogenesis is challenging and in the near future new powerful techniques will make high throughput screening possible. Progress in elucidating the genetic defect causing complex I deficiencies is important for a better genetic counseling, prenatal diagnostic possibilities and further development of new treatment strategies to cure the complex I deficiencies in the future.
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http://dx.doi.org/10.1016/j.mito.2012.07.106DOI Listing
September 2012

Atypical hemolytic uremic syndrome and genetic aberrations in the complement factor H-related 5 gene.

J Hum Genet 2012 Jul 24;57(7):459-64. Epub 2012 May 24.

Department of Pediatric Nephrology, Radboud University Medical Centre, Nijmegen, The Netherlands.

Atypical hemolytic uremic syndrome (aHUS) is a severe renal disorder that is associated with mutations in genes encoding proteins of the alternative complement pathway. Previously, we identified pathogenic variations in genes encoding complement regulators (CFH, CFI and MCP) in our aHUS cohort. In this study, we screened for mutations in the alternative pathway regulator CFHR5 in 65 aHUS patients by means of PCR on genomic DNA and sequence analysis. Potential pathogenicity of genetic alterations was determined by published data on CFHR5 variants, evolutionary conservation and in silico mutation prediction programs. Detection of serum CFHR5 was performed by western blot analysis and enzyme-linked immunosorbent assay. A potentially pathogenic sequence variation was found in CFHR5 in three patients (4.6%). All variations were located in short consensus repeats that might be involved in binding to C3b, heparin or C-reactive protein. The identified CFHR5 mutations require functional studies to determine their relevance to aHUS, but they might be candidates for an altered genetic profile predisposing to the disease.
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http://dx.doi.org/10.1038/jhg.2012.57DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3407369PMC
July 2012

Impaired ubiquitin-proteasome-mediated PGC-1α protein turnover and induced mitochondrial biogenesis secondary to complex-I deficiency.

Proteomics 2012 May;12(9):1349-62

Nijmegen Center for Mitochondrial Disorders (NCMD), Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

Most eukaryotic cells depend on mitochondrial OXidative PHOSphorylation (OXPHOS) in their ATP supply. The cellular consequences of OXPHOS defects and the pathophysiological mechanisms in related disorders are incompletely understood. Using a quantitative proteomics approach we provide evidence that a genetic defect of complex-I of the OXPHOS system may associate with transcriptional derangements of mitochondrial biogenesis through stabilization of the master transcriptional regulator PPARγ co-activator 1α (PGC-1α) protein. Chronic oxidative stress suppresses the gene expression of PGC-1α but concomitant inhibition of the ubiquitin-proteasome system (UPS) can stabilize this co-activator protein, thereby inducing its downstream metabolic gene expression programs. Thus, mitochondrial biogenesis, which lays at the heart of the homeostatic control of energy metabolism, can be deregulated by secondary impairments of the protein turnover machinery.
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http://dx.doi.org/10.1002/pmic.201100326DOI Listing
May 2012

Clinical features and heteroplasmy in blood, urine and saliva in 34 Dutch families carrying the m.3243A > G mutation.

J Inherit Metab Dis 2012 Nov 9;35(6):1059-69. Epub 2012 Mar 9.

Department of Pediatrics, Nijmegen Centre for Mitochondrial Disorders, Radboud University Nijmegen Medical Centre, Huispost 804, Geert Grooteplein 10, 6500, HB, PO BOX 9101, Nijmegen, The Netherlands.

The m.3243A>G mutation has become known as the MELAS mutation. However, many other clinical phenotypes associated with this mutation have been described,most frequently being Maternally Inherited Diabetes and Deafness (MIDD). The m.3243A>G mutation, can be detected in virtually all tissues, however heteroplasmy differs between samples. Recent reports indicate, a preference to perform mutation analysis in Urinary Epithelial Cells(UEC). To test this, and to study a correlation between the mutational load in different tissues with two mitochondrial scoring systems (NMDAS and NPMDS) we investigated 34 families carrying the m.3243A>G mutation. Heteroplasmy was determined in three non-invasively collected samples,namely leucocytes, UEC and buccal mucosa. We included 127 patients, of which 82 carried the m.3243A>G mutation.None of the children (n011) had specific complaints. In adults(n071), a median NMDAS score of 15 (IQR 10-24) was found. The most prevalent symptoms were hearing loss(68 %), gastro-intestinal problems (59 %), exercise intolerance(54 %) and glucose intolerance (52 %). Ten patients had neurologic involvement. Buccal mucosa had the best correlation with the NMDAS in all adults (r00.437, p<0.001),whereas UEC had the strongest correlation with the NMDAS in severely affected patients (r00.593, p00.002). Heteroplasmy declined significantly with increasing age in all three samples (leucocytes r0-0.705 (p<0.001), UEC r0-0.374 (p00.001), buccal mucosa r0-0.460 (p<0.001). In our cohort of 82 patients, the m.3243A>Gmutation causes a wide variety of signs and symptoms, MIDD being far more prevalent than MELAS. Looking at the characteristics of the three noninvasively available tissues for testing heteroplasmy we confirm that UEC are the preferred sample to test [corrected].
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http://dx.doi.org/10.1007/s10545-012-9465-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3470685PMC
November 2012