Publications by authors named "Marleen C D G Huigen"

13 Publications

  • Page 1 of 1

Amadori rearrangement products as potential biomarkers for inborn errors of amino-acid metabolism.

Commun Biol 2021 Mar 19;4(1):367. Epub 2021 Mar 19.

Radboud University, Institute for Molecules and Materials, FELIX Laboratory, Toernooiveld 7, Nijmegen, the Netherlands.

The identification of disease biomarkers plays a crucial role in developing diagnostic strategies for inborn errors of metabolism and understanding their pathophysiology. A primary metabolite that accumulates in the inborn error phenylketonuria is phenylalanine, however its levels do not always directly correlate with clinical outcomes. Here we combine infrared ion spectroscopy and NMR spectroscopy to identify the Phe-glucose Amadori rearrangement product as a biomarker for phenylketonuria. Additionally, we find analogous amino acid-glucose metabolites formed in the body fluids of patients accumulating methionine, lysine, proline and citrulline. Amadori rearrangement products are well-known intermediates in the formation of advanced glycation end-products and have been associated with the pathophysiology of diabetes mellitus and ageing, but are now shown to also form under conditions of aminoacidemia. They represent a general class of metabolites for inborn errors of amino acid metabolism that show potential as biomarkers and may provide further insight in disease pathophysiology.
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http://dx.doi.org/10.1038/s42003-021-01909-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979741PMC
March 2021

Monitoring phenylalanine concentrations in the follow-up of phenylketonuria patients: An inventory of pre-analytical and analytical variation.

JIMD Rep 2021 Mar 22;58(1):70-79. Epub 2020 Nov 22.

Translational Metabolic Laboratory, Department of Laboratory Medicine Radboud University Medical Centre Nijmegen The Netherlands.

Background: Reliable measurement of phenylalanine (Phe) is a prerequisite for adequate follow-up of phenylketonuria (PKU) patients. However, previous studies have raised concerns on the intercomparability of plasma and dried blood spot (DBS) Phe results. In this study, we made an inventory of differences in (pre-)analytical methodology used for Phe determination across Dutch laboratories, and compared DBS and plasma results.

Methods: Through an online questionnaire, we assessed (pre-)analytical Phe measurement procedures of seven Dutch metabolic laboratories. To investigate the difference between plasma and DBS Phe, participating laboratories received simultaneously collected plasma-DBS sets from 23 PKU patients. In parallel, 40 sample sets of DBS spotted from either venous blood or capillary fingerprick were analyzed.

Results: Our data show that there is no consistency on standard operating procedures for Phe measurement. The association of DBS to plasma Phe concentration exhibits substantial inter-laboratory variation, ranging from a mean difference of -15.5% to +30.6% between plasma and DBS Phe concentrations. In addition, we found a mean difference of +5.8% in Phe concentration between capillary DBS and DBS prepared from venous blood.

Conclusions: The results of our study point to substantial (pre-)analytical variation in Phe measurements, implicating that bloodspot Phe results should be interpreted with caution, especially when no correction factor is applied. To minimize variation, we advocate pre-analytical standardization and analytical harmonization of Phe measurements, including consensus on application of a correction factor to adjust DBS Phe to plasma concentrations.
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http://dx.doi.org/10.1002/jmd2.12186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7932865PMC
March 2021

The role of clinical response to treatment in determining pathogenicity of genomic variants.

Genet Med 2021 Mar 22;23(3):581-585. Epub 2020 Oct 22.

PerkinElmer Genomics, Duluth, GA, USA.

Purpose: The 2015 American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines for the interpretation of sequence variants provide a framework to standardize terminology in the classification of variants uncovered through genetic testing. We aimed to assess the validity of utilizing clinical response to therapies specifically targeted to a suspected disease in clarifying variant pathogenicity.

Methods: Five families with disparate clinical presentations and different genetic diseases evaluated and treated in multiple diagnostic settings are summarized.

Results: Extended evaluations indicated possible genetic diagnoses and assigned candidate causal variants, but the cumulative clinical, biochemical, and molecular information in each instance was not completely consistent with the identified disease. Initiation of treatment specific to the suspected diagnoses in the affected individuals led to clinical improvement in all five families.

Conclusion: We propose that the effect of therapies that are specific and targeted to treatable genetic diseases embodies an in vivo physiological response and could be considered as additional criteria within the 2015 ACMG/AMP guidelines in determining genomic variant pathogenicity.
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http://dx.doi.org/10.1038/s41436-020-00996-9DOI Listing
March 2021

Bi-allelic GOT2 Mutations Cause a Treatable Malate-Aspartate Shuttle-Related Encephalopathy.

Am J Hum Genet 2019 09 15;105(3):534-548. Epub 2019 Aug 15.

On behalf of "United for Metabolic Diseases," 1105AZ Amsterdam, the Netherlands; Department of Laboratory Medicine, Translational Metabolic Laboratory, Radboud University Medical Centre, 6525 GA Nijmegen, the Netherlands. Electronic address:

Early-infantile encephalopathies with epilepsy are devastating conditions mandating an accurate diagnosis to guide proper management. Whole-exome sequencing was used to investigate the disease etiology in four children from independent families with intellectual disability and epilepsy, revealing bi-allelic GOT2 mutations. In-depth metabolic studies in individual 1 showed low plasma serine, hypercitrullinemia, hyperlactatemia, and hyperammonemia. The epilepsy was serine and pyridoxine responsive. Functional consequences of observed mutations were tested by measuring enzyme activity and by cell and animal models. Zebrafish and mouse models were used to validate brain developmental and functional defects and to test therapeutic strategies. GOT2 encodes the mitochondrial glutamate oxaloacetate transaminase. GOT2 enzyme activity was deficient in fibroblasts with bi-allelic mutations. GOT2, a member of the malate-aspartate shuttle, plays an essential role in the intracellular NAD(H) redox balance. De novo serine biosynthesis was impaired in fibroblasts with GOT2 mutations and GOT2-knockout HEK293 cells. Correcting the highly oxidized cytosolic NAD-redox state by pyruvate supplementation restored serine biosynthesis in GOT2-deficient cells. Knockdown of got2a in zebrafish resulted in a brain developmental defect associated with seizure-like electroencephalography spikes, which could be rescued by supplying pyridoxine in embryo water. Both pyridoxine and serine synergistically rescued embryonic developmental defects in zebrafish got2a morphants. The two treated individuals reacted favorably to their treatment. Our data provide a mechanistic basis for the biochemical abnormalities in GOT2 deficiency that may also hold for other MAS defects.
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http://dx.doi.org/10.1016/j.ajhg.2019.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6732527PMC
September 2019

Organic anion transporters 1 and 3 influence cellular energy metabolism in renal proximal tubule cells.

Biol Chem 2019 09;400(10):1347-1358

Department of Pharmacology and Toxicology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, P.O. Box 9101, NL-6500HB, Nijmegen, The Netherlands.

Organic anion transporters (OATs) 1 and 3 are, besides being uptake transporters, key in several cellular metabolic pathways. The underlying mechanisms are largely unknown. Hence, we used human conditionally immortalized proximal tubule epithelial cells (ciPTEC) overexpressing OAT1 or OAT3 to gain insight into these mechanisms. In ciPTEC-OAT1 and -OAT3, extracellular lactate levels were decreased (by 77% and 71%, respectively), while intracellular ATP levels remained unchanged, suggesting a shift towards an oxidative phenotype upon OAT1 or OAT3 overexpression. This was confirmed by increased respiration of ciPTEC-OAT1 and -OAT3 (1.4-fold), a decreased sensitivity to respiratory inhibition, and characterized by a higher demand on mitochondrial oxidative capacity. In-depth profiling of tricarboxylic acid (TCA) cycle metabolites revealed reduced levels of intermediates converging into α-ketoglutarate in ciPTEC-OAT1 and -OAT3, which via 2-hydroxyglutarate metabolism explains the increased respiration. These interactions with TCA cycle metabolites were in agreement with metabolomic network modeling studies published earlier. Further studies using OAT or oxidative phosphorylation (OXPHOS) inhibitors confirmed our idea that OATs are responsible for increased use and synthesis of α-ketoglutarate. In conclusion, our results indicate an increased α-ketoglutarate efflux by OAT1 and OAT3, resulting in a metabolic shift towards an oxidative phenotype.
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http://dx.doi.org/10.1515/hsz-2018-0446DOI Listing
September 2019

Next-generation metabolic screening: targeted and untargeted metabolomics for the diagnosis of inborn errors of metabolism in individual patients.

J Inherit Metab Dis 2018 05 16;41(3):337-353. Epub 2018 Feb 16.

Department of Laboratory Medicine, Translational Metabolic Laboratory (TML), Radboud University Medical Center, Geert Groote Plein Zuid 10, 6525, GA, Nijmegen, The Netherlands.

The implementation of whole-exome sequencing in clinical diagnostics has generated a need for functional evaluation of genetic variants. In the field of inborn errors of metabolism (IEM), a diverse spectrum of targeted biochemical assays is employed to analyze a limited amount of metabolites. We now present a single-platform, high-resolution liquid chromatography quadrupole time of flight (LC-QTOF) method that can be applied for holistic metabolic profiling in plasma of individual IEM-suspected patients. This method, which we termed "next-generation metabolic screening" (NGMS), can detect >10,000 features in each sample. In the NGMS workflow, features identified in patient and control samples are aligned using the "various forms of chromatography mass spectrometry (XCMS)" software package. Subsequently, all features are annotated using the Human Metabolome Database, and statistical testing is performed to identify significantly perturbed metabolite concentrations in a patient sample compared with controls. We propose three main modalities to analyze complex, untargeted metabolomics data. First, a targeted evaluation can be done based on identified genetic variants of uncertain significance in metabolic pathways. Second, we developed a panel of IEM-related metabolites to filter untargeted metabolomics data. Based on this IEM-panel approach, we provided the correct diagnosis for 42 of 46 IEMs. As a last modality, metabolomics data can be analyzed in an untargeted setting, which we term "open the metabolome" analysis. This approach identifies potential novel biomarkers in known IEMs and leads to identification of biomarkers for as yet unknown IEMs. We are convinced that NGMS is the way forward in laboratory diagnostics of IEMs.
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http://dx.doi.org/10.1007/s10545-017-0131-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5959972PMC
May 2018

Hyperammonemia due to Adult-Onset N-Acetylglutamate Synthase Deficiency.

JIMD Rep 2017 5;31:95-99. Epub 2016 May 5.

Department of Internal Medicine, Radboud University Medical Center, 9101, 6500 HB, Nijmegen, The Netherlands.

A 59-year-old woman, with a medical history of intellectual disability after perinatal asphyxia, was admitted because of coma due to hyperammonemia after she was treated for a fracture of the pelvis. The ammonia level was 280 μM. Acquired disorders as explanation for the hyperammonemia were excluded. Metabolic investigations showed an elevated glutamine and alanine and low citrulline, suspect for a urea cycle defect (UCD). Orotic acid could not be demonstrated in urine. DNA investigations were negative for mutations or deletions in the OTC and CPS1 gene, but revealed a homozygous c.603G>C mutation in exon 2 of the N-acetylglutamate synthase (NAGS) gene (NM_153006.2:c.603G>C), which mandates p.Lys201Asn. This is a novel mutation in the NAGS gene.After the diagnosis of NAGS deficiency was made carbamylglutamate was started in a low dose. In combination with mild protein restriction the ammonia level decreased to 26 μM.This is one of the first patients in literature in whom the diagnosis of a UCD is made at such an advanced age. It is important for the adult physician to consider a metabolic disorder at every age.
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http://dx.doi.org/10.1007/8904_2016_565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5272844PMC
May 2016

Cerebral lipid accumulation in Chanarin-Dorfman Syndrome.

Mol Genet Metab 2015 Jan 4;114(1):51-4. Epub 2014 Nov 4.

Department of Paediatric Neurology, Radboud University Medical Center, Donders Institute for Brain, Cognition, and Behaviour, Geert Grooteplein zuid 10, route 801, 6525 GA Nijmegen, The Netherlands. Electronic address:

Chanarin-Dorfman Syndrome (CDS) is caused by a defect in the CGI-58/ABHD5 gene resulting in a deficiency of CGI-58 and in intracellular accumulation of triacylglycerol in skin and liver. Patients are mainly characterized by congenital ichthyosis, but the clinical phenotype is very heterogeneous. Distinct brain involvement has never been described. We present a clinical description of two patients with congenital ichthyosis. On suspicion of Sjögren-Larsson syndrome (SLS) single-voxel 1H-MR spectroscopy of the brain was performed and biochemical testing of fatty aldehyde dehydrogenase (FALDH) to establish this diagnosis gave normal results. Vacuolisation in a peripheral blood smear has led to the CDS suspicion. In both patients the diagnosis CDS was confirmed by ABHD5 mutation analysis. Interestingly, a clear lipid accumulation in the cerebral white matter, cortex and basal ganglia was demonstrated in both CDS-patients. These results demonstrate, for the first time, cerebral involvement in CDS and give new insights in the complex phenotype. Since the clinical implications of this abnormal cerebral lipid accumulation are still unknown, further studies are warranted.
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http://dx.doi.org/10.1016/j.ymgme.2014.10.016DOI Listing
January 2015

Identification of a novel resistance (E40F) and compensatory (K43E) substitution in HIV-1 reverse transcriptase.

Retrovirology 2008 Feb 13;5:20. Epub 2008 Feb 13.

Department of Medical Microbiology, University Medical Center Utrecht, The Netherlands.

Background: HIV-1 nucleoside reverse transcriptase inhibitors (NRTIs) have been used in the clinic for over twenty years. Interestingly, the complete resistance pattern to this class has not been fully elucidated. Novel mutations in RT appearing during treatment failure are still being identified. To unravel the role of two of these newly identified changes, E40F and K43E, we investigated their effect on viral drug susceptibility and replicative capacity.

Results: A large database (Quest Diagnostics database) was analysed to determine the associations of the E40F and K43E changes with known resistance mutations. Both amino acid changes are strongly associated with the well known NRTI-resistance mutations M41L, L210W and T215Y. In addition, a strong positive association between these changes themselves was observed. A panel of recombinant viruses was generated by site-directed mutagenesis and phenotypically analysed. To determine the effect on replication capacity, competition and in vitro evolution experiments were performed. Introduction of E40F results in an increase in Zidovudine resistance ranging from nine to fourteen fold depending on the RT background and at the same time confers a decrease in viral replication capacity. The K43E change does not decrease the susceptibility to Zidovudine but increases viral replication capacity, when combined with E40F, demonstrating a compensatory role for this codon change.

Conclusion: In conclusion, we have identified a novel resistance (E40F) and compensatory (K43E) change in HIV-1 RT. Further research is indicated to analyse the clinical importance of these changes.
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http://dx.doi.org/10.1186/1742-4690-5-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2276231PMC
February 2008

Evolution of a novel 5-amino-acid insertion in the beta3-beta4 loop of HIV-1 reverse transcriptase.

Virology 2007 Aug 23;364(2):395-406. Epub 2007 Apr 23.

Department of Medical Microbiology, University Medical Center Utrecht, Heidelberglaan 100, Utrecht, The Netherlands.

HIV-1 isolates harbouring an insertion in the beta3-beta4 loop of reverse transcriptase (RT) confer high-level resistance to nucleoside analogues. We have identified a novel 5-amino-acid insertion (KGSNR amino acids 66-70) in a patient on prolonged nucleoside combination therapy (didanosine and stavudine) and investigated which factors were responsible for its outgrowth. Remarkably, only small fold increases in drug resistance to nucleoside analogues were observed compared to wild type. The insertion variant displayed a reduced replicative capacity in the absence of inhibitor, but had a slight replicative advantage in the presence of zidovudine, didanosine or stavudine, resulting in the selection and persistence of this insertion in vivo. Mathematical analyses of longitudinal samples indicated a 2% in vivo fitness advantage for the insertion variant compared to the initial viral population. The novel RT insertion variant conferring low levels of resistance was able to evolve towards a high-level resistant replication-competent variant.
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http://dx.doi.org/10.1016/j.virol.2007.03.028DOI Listing
August 2007

Insertions in the beta3-beta4 loop of reverse transcriptase of human immunodeficiency virus type 1 and their mechanism of action, influence on drug susceptibility and viral replication capacity.

Antiviral Res 2007 Aug 26;75(2):93-103. Epub 2007 Mar 26.

Department of Medical Microbiology, University Medical Center Utrecht, The Netherlands.

Introduction of antiretroviral therapy combining protease and reverse transcriptase (RT) inhibitors has dramatically improved the quality of life and survival of patients infected with the human immunodeficiency virus (HIV). However, effective long-term therapy of HIV-infection has been severely hampered by the development of drug resistance. Resistance to antiretroviral drugs is generally conferred by specific amino acid substitutions in the target gene of the drug. Yet, occasionally gene insertions are being observed. The most commonly observed insertion is seen during substrate analogue RT inhibitor therapy and is selected in the beta3-beta4 loop of the RT enzyme. This flexible loop is located in the fingers subdomain of the enzyme and plays an important role in substrate binding. The acquisition of drug resistance related mutations or insertions might come at a price, which is reduced performance of the enzyme resulting in a diminished replication capacity of the virus. Various types of insertions have been described, and, in this review, we have summarized these data and discussed the mechanism of action of the RT inserts and their impact on both drug susceptibility and replication capacity.
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http://dx.doi.org/10.1016/j.antiviral.2007.03.001DOI Listing
August 2007

Spectrum of antiviral activity of o-(acetoxyphenyl)hept-2-ynyl sulphide (APHS).

Int J Antimicrob Agents 2005 May;25(5):419-26

Eijkman-Winkler Center, Hp G04.614, University Medical Center Utrecht, Heidelberglaan 100, NL-3584 CX Utrecht, The Netherlands.

Since some antiviral drugs have a broad spectrum of action, the aim of this study was to assess whether o-(acetoxyphenyl)hept-2-ynyl sulphide (APHS), a recently described inhibitor of human immunodeficiency virus type 1 (HIV-1) replication, has an effect on the replication of other retroviruses, (-) and (+) RNA viruses and DNA viruses. APHS did not affect the replication of feline immunodeficiency virus, HIV-2 and a HIV-1 strain resistant to non-nucleoside reverse transcriptase inhibitors (NNRTI). APHS could also not inhibit the replication of the RNA viruses, respiratory syncytium virus or mouse hepatitis virus. In contrast, APHS did inhibit the replication of wild-type herpes simplex virus type 1 (HSV-1) as well as acyclovir-resistant HSV-1 and HSV-2 mutant. These results suggest that APHS is a NNRTI of HIV-1 replication, but not HIV-2 replication, and that APHS is an inhibitor of both HSV-1 and HSV-2 replication.
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http://dx.doi.org/10.1016/j.ijantimicag.2004.11.011DOI Listing
May 2005

Aspirin-like molecules that inhibit human immunodeficiency virus 1 replication.

Antiviral Res 2003 May;58(3):253-63

Eijkman-Winkler Center, HpG04.614, University Medical Center Utrecht, Heidelberglaan 100, NL-3584, CX Utrecht, The Netherlands.

Some anti-inflammatory molecules are also known to possess anti-human immunodeficiency virus (HIV) activity. We found that o-(acetoxyphenyl)hept-2-ynyl sulfide (APHS), a recently synthesized non-steroidal anti-inflammatory molecule can inhibit HIV-1 replication. The aim of this study was to clarify the mechanism of action of APHS. When administered during the first steps of the infection, APHS was capable of inhibiting the replication of several HIV-1 strains (macrophage-tropic and/or lymphocytotropic) in a dose-dependent manner in both peripheral blood mononuclear cells (PBMC), monocyte-derived macrophages and peripheral blood lymphocytes with 50% inhibitory concentration values of approximately 10 microM. The 50% toxic concentration of APHS varied between 100 and 200 microM in the different primary cells tested. APHS did not affect HIV-1 replication once the provirus was already inserted into the cellular genome. APHS also did not inhibit HIV-1 entry into the host cells as determined by quantification of gag RNA inside PBMC 2h after infection. However, APHS did inhibit gag DNA synthesis during reverse transcription in primary cells, which indicates that APHS may target the reverse transcription process.
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http://dx.doi.org/10.1016/s0166-3542(03)00006-8DOI Listing
May 2003