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1.
Brain ; 142(11): 3382-3397, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31637422

RESUMO

CTP:phosphoethanolamine cytidylyltransferase (ET), encoded by PCYT2, is the rate-limiting enzyme for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. Phosphatidylethanolamine is one of the most abundant membrane lipids and is particularly enriched in the brain. We identified five individuals with biallelic PCYT2 variants clinically characterized by global developmental delay with regression, spastic para- or tetraparesis, epilepsy and progressive cerebral and cerebellar atrophy. Using patient fibroblasts we demonstrated that these variants are hypomorphic, result in altered but residual ET protein levels and concomitant reduced enzyme activity without affecting mRNA levels. The significantly better survival of hypomorphic CRISPR-Cas9 generated pcyt2 zebrafish knockout compared to a complete knockout, in conjunction with previously described data on the Pcyt2 mouse model, indicates that complete loss of ET function may be incompatible with life in vertebrates. Lipidomic analysis revealed profound lipid abnormalities in patient fibroblasts impacting both neutral etherlipid and etherphospholipid metabolism. Plasma lipidomics studies also identified changes in etherlipids that have the potential to be used as biomarkers for ET deficiency. In conclusion, our data establish PCYT2 as a disease gene for a new complex hereditary spastic paraplegia and confirm that etherlipid homeostasis is important for the development and function of the brain.

2.
J Inherit Metab Dis ; 41(3): 479-487, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-28849344

RESUMO

Peroxisomes play an important role in a variety of metabolic pathways, including the α- and ß-oxidation of fatty acids, and the biosynthesis of ether phospholipids. Single peroxisomal enzyme deficiencies (PEDs) are a group of peroxisomal disorders in which either a peroxisomal matrix enzyme or a peroxisomal membrane transporter protein is deficient. To investigate the functional consequences of specific enzyme deficiencies on the lipidome, we performed lipidomics using cultured skin fibroblasts with different defects in the ß-oxidation of very long-chain fatty acids, including ABCD1- (ALD), acyl-CoA oxidase 1 (ACOX1)-, D-bifunctional protein (DBP)-, and acyl-CoA binding domain containing protein 5 (ACBD5)-deficient cell lines. Ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry revealed characteristic changes in the phospholipid composition in fibroblasts with different fatty acid ß-oxidation defects. Remarkably, we found that ether phospholipids, including plasmalogens, were decreased. We defined specific phospholipid ratios reflecting the different enzyme defects, which can be used to discriminate the PED fibroblasts from healthy control cells.


Assuntos
Fibroblastos/química , Fibroblastos/metabolismo , Lipídeos/análise , Metabolômica/métodos , Transtornos Peroxissômicos/diagnóstico , Estudos de Casos e Controles , Células Cultivadas , Cromatografia Líquida de Alta Pressão/métodos , Ácidos Graxos/metabolismo , Humanos , Metabolismo dos Lipídeos , Espectrometria de Massas/métodos , Oxirredução , Transtornos Peroxissômicos/metabolismo , Peroxissomos/metabolismo , Pele/citologia , Pele/metabolismo
3.
J Inherit Metab Dis ; 41(3): 489-498, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29209936

RESUMO

Peroxisomes are ubiquitous cell organelles that play an important role in lipid metabolism. Accordingly, peroxisomal disorders, including the peroxisome biogenesis disorders and peroxisomal single-enzyme deficiencies, are associated with aberrant lipid metabolism. Lipidomics is an emerging tool for diagnosis, disease-monitoring, identifying lipid biomarkers, and studying the underlying pathophysiology in disorders of lipid metabolism. In this study, we demonstrate the potential of lipidomics for the diagnosis of peroxisomal disorders using plasma samples from patients with different types of peroxisomal disorders. We show that the changes in the plasma profiles of phospholipids, di- and triglycerides, and cholesterol esters correspond with the characteristic metabolite abnormalities that are currently used in the metabolic screening for peroxisomal disorders. The lipidomics approach, however, gives a much more detailed overview of the metabolic changes that occur in the lipidome. Furthermore, we identified novel unique lipid species for specific peroxisomal diseases that are candidate biomarkers. The results presented in this paper show the power of lipidomics approaches to enable the specific diagnosis of different peroxisomal disorders.


Assuntos
Lipídeos/sangue , Metabolômica/métodos , Transtornos Peroxissômicos/diagnóstico , Biomarcadores/análise , Biomarcadores/sangue , Análise Química do Sangue/métodos , Ácidos Graxos/metabolismo , Humanos , Metabolismo dos Lipídeos , Transtornos Peroxissômicos/sangue , Peroxissomos/metabolismo
4.
J Lipid Res ; 57(8): 1447-54, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27284103

RESUMO

Peroxisomes are subcellular organelles involved in various metabolic processes, including fatty acid and phospholipid homeostasis. The Zellweger spectrum disorders (ZSDs) represent a group of diseases caused by a defect in the biogenesis of peroxisomes. Accordingly, cells from ZSD patients are expected to have an altered composition of fatty acids and phospholipids. Using an LC/MS-based lipidomics approach, we show that the phospholipid composition is characteristically altered in cultured primary skin fibroblasts from ZSD patients when compared with healthy controls. We observed a marked overall increase of phospholipid species containing very long-chain fatty acids, and a decrease of phospholipid species with shorter fatty acid species in ZSD patient fibroblasts. In addition, we detected a distinct phosphatidylcholine profile in ZSD patients with a severe and mild phenotype when compared with control cells. Based on our data, we present a set of specific phospholipid ratios for fibroblasts that clearly discriminate between mild and severe ZSD patients, and those from healthy controls. Our findings will aid in the diagnosis and prognosis of ZSD patients, including an increasing number of mild patients in whom hardly any abnormalities are observed in biochemical parameters commonly used for diagnosis.


Assuntos
Fibroblastos/metabolismo , Fosfolipídeos/metabolismo , Síndrome de Zellweger/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Humanos , Metabolismo dos Lipídeos , Metabolômica , Síndrome de Zellweger/patologia
5.
PLoS One ; 6(1): e16118, 2011 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-21283679

RESUMO

In 5-40% of respiratory infections in children, the diagnostics remain negative, suggesting that the patients might be infected with a yet unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA) is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR. However, direct discovery of unknown pathogens in nasopharyngeal swabs is difficult due to the high concentration of ribosomal RNA (rRNA) that acts as competitor. In the current study we optimized VIDISCA by adjusting the reverse transcription enzymes and decreasing rRNA amplification in the reverse transcription, using hexamer oligonucleotides that do not anneal to rRNA. Residual cDNA synthesis on rRNA templates was further reduced with oligonucleotides that anneal to rRNA but can not be extended due to 3'-dideoxy-C6-modification. With these modifications >90% reduction of rRNA amplification was established. Further improvement of the VIDISCA sensitivity was obtained by high throughput sequencing (VIDISCA-454). Eighteen nasopharyngeal swabs were analysed, all containing known respiratory viruses. We could identify the proper virus in the majority of samples tested (11/18). The median load in the VIDISCA-454 positive samples was 7.2 E5 viral genome copies/ml (ranging from 1.4 E3-7.7 E6). Our results show that optimization of VIDISCA and subsequent high-throughput-sequencing enhances sensitivity drastically and provides the opportunity to perform virus discovery directly in patient material.


Assuntos
Técnicas de Diagnóstico do Sistema Respiratório/normas , Reação em Cadeia da Polimerase/métodos , Infecções Respiratórias/diagnóstico , Vírus/isolamento & purificação , DNA Complementar/genética , DNA Viral/análise , Sequenciamento de Nucleotídeos em Larga Escala , RNA Ribossômico/genética , Infecções Respiratórias/virologia , Sensibilidade e Especificidade , Carga Viral/métodos , Vírus/genética
6.
BMC Bioinformatics ; 11: 598, 2010 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-21156038

RESUMO

BACKGROUND: Bioinformatics is confronted with a new data explosion due to the availability of high throughput DNA sequencers. Data storage and analysis becomes a problem on local servers, and therefore it is needed to switch to other IT infrastructures. Grid and workflow technology can help to handle the data more efficiently, as well as facilitate collaborations. However, interfaces to grids are often unfriendly to novice users. RESULTS: In this study we reused a platform that was developed in the VL-e project for the analysis of medical images. Data transfer, workflow execution and job monitoring are operated from one graphical interface. We developed workflows for two sequence alignment tools (BLAST and BLAT) as a proof of concept. The analysis time was significantly reduced. All workflows and executables are available for the members of the Dutch Life Science Grid and the VL-e Medical virtual organizations All components are open source and can be transported to other grid infrastructures. CONCLUSIONS: The availability of in-house expertise and tools facilitates the usage of grid resources by new users. Our first results indicate that this is a practical, powerful and scalable solution to address the capacity and collaboration issues raised by the deployment of next generation sequencers. We currently adopt this methodology on a daily basis for DNA sequencing and other applications. More information and source code is available via http://www.bioinformaticslaboratory.nl/


Assuntos
Biologia Computacional/métodos , Armazenamento e Recuperação da Informação/métodos , Análise de Sequência de DNA/métodos , Sistemas de Computação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Alinhamento de Sequência , Software , Fluxo de Trabalho
7.
BMC Evol Biol ; 6: 84, 2006 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-17040564

RESUMO

BACKGROUND: With the increased availability of sequenced genomes there have been several initiatives to infer evolutionary relationships by whole genome characteristics. One of these studies suggested good congruence between genome synteny, shared gene content, 16S ribosomal DNA identity, codon usage and the genome signature in prokaryotes. Here we rigorously test the phylogenetic signal of the genome signature, which consists of the genome-specific relative frequencies of dinucleotides, on 334 sequenced prokaryotic genome sequences. RESULTS: Intrageneric comparisons show that in general the genomic dissimilarity scores are higher than in intraspecific comparisons, in accordance with the suggested phylogenetic signal of the genome signature. Exceptions to this trend, (Bartonella spp., Bordetella spp., Salmonella spp. and Yersinia spp.), which have low average intrageneric genomic dissimilarity scores, suggest that members of these genera might be considered the same species. On the other hand, high genomic dissimilarity values for intraspecific analyses suggest that in some cases (e.g. Prochlorococcus marinus, Pseudomonas fluorescens, Buchnera aphidicola and Rhodopseudomonas palustris) different strains from the same species may actually represent different species. Comparing 16S rDNA identity with genomic dissimilarity values corroborates the previously suggested trend in phylogenetic signal, albeit that the dissimilarity values only provide low resolution. CONCLUSION: The genome signature has a distinct phylogenetic signal, independent of individual genetic marker genes. A reliable phylogenetic clustering cannot be based on dissimilarity values alone, as bootstrapping is not possible for this parameter. It can however be used to support or refute a given phylogeny and resulting taxonomy.


Assuntos
Genoma Bacteriano , Oligodesoxirribonucleotídeos/genética , Filogenia , Bactérias/classificação , Bactérias/genética , Cromossomos Bacterianos/genética , DNA Bacteriano/genética , DNA Ribossômico/genética , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Especificidade da Espécie
8.
FEMS Microbiol Lett ; 262(1): 77-84, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16907742

RESUMO

Anomalous DNA (aDNA) in prokaryotic genomes, identified by its aberrant nucleotide composition, generally represents horizontally acquired DNA. Previous studies showed that frequent DNA transfer occurs between commensal Neisseriae and Neisseria meningitidis. Currently, it is unknown whether aDNA regions are also transferred between these species. The genome of Neisseria lactamica strain 892586 was assessed by a strategy that enables the selective isolation of aDNA, using endonucleases with recognition sites that are overrepresented in aDNA. Of eight regions with aDNA, five displayed similarity to virulence-associated meningococcal sequences. Of three aDNA fragments with limited or no similarity to neisserial sequences, one encodes a novel putative autotransporter/adhesin. The remaining two fragments are adjacent in the N. lactamica genome, and encode a novel putative ATPase/subtilisin-like protease operon. A similar operon is present in the genomes of different respiratory tract pathogens. The identification of aDNA from N. lactamica with similarity to meningococcal aDNA shows that genetic exchange between the Neisseriae is not limited to the neisserial core genome. The discovery of aDNA in N. lactamica similar to a locus in other pathogens substantially expands the neisserial gene pool.


Assuntos
DNA Bacteriano/genética , Transferência Genética Horizontal , Neisseria lactamica/genética , Adenosina Trifosfatases/genética , Adesinas Bacterianas/genética , Sequência de Aminoácidos , Proteínas de Transporte/genética , Biologia Computacional , DNA Bacteriano/química , Genoma Bacteriano , Dados de Sequência Molecular , Neisseria lactamica/patogenicidade , Neisseria meningitidis/genética , Óperon/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/genética , Virulência/genética
9.
BMC Genomics ; 7: 26, 2006 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-16480495

RESUMO

BACKGROUND: Most plasmids depend on the host replication machinery and possess partitioning genes. These properties confine plasmids to a limited range of hosts, yielding a close and presumably stable relationship between plasmid and host. Hence, it is anticipated that due to amelioration the dinucleotide composition of plasmids is similar to that of the genome of their hosts. However, plasmids are also thought to play a major role in horizontal gene transfer and thus are frequently exchanged between hosts, suggesting dinucleotide composition dissimilarity between plasmid and host genome. We compared the dinucleotide composition of a large collection of plasmids with that of their host genomes to shed more light on this enigma. RESULTS: The dinucleotide frequency, coined the genome signature, facilitates the identification of putative horizontally transferred DNA in complete genome sequences, since it was found to be typical for a certain genome, and similar between related species. By comparison of the genome signature of 230 plasmid sequences with that of the genome of each respective host, we found that in general the genome signature of plasmids is dissimilar from that of their host genome. CONCLUSION: Our results show that the genome signature of plasmids does not resemble that of their host genome. This indicates either absence of amelioration or a less stable relationship between plasmids and their host. We propose an indiscriminate lifestyle for plasmids preserving the genome signature discordance between these episomes and host chromosomes.


Assuntos
Composição de Bases , Cromossomos/química , Cromossomos/genética , Interações Hospedeiro-Patógeno/genética , Plasmídeos/química , Plasmídeos/genética , Borrelia burgdorferi/genética , Cromossomos Bacterianos/química , Cromossomos Bacterianos/genética , DNA/química , DNA/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Transferência Genética Horizontal , Genômica , Células Procarióticas
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