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Biomedical subjects

Frank Baas

Publications and source records attributed to Frank Baas.

At least 37 records · Page 2Linked to original sources
PubMed2004

Rab6 membrane association is dependent of Presenilin 1 and cellular phosphorylation events.

Processing of the amyloid precursor protein (APP) by alpha-secretase precludes the formation of beta-amyloid (Abeta). Therefore, the increase of cleavage by alpha-secretase upon stimulation by protein kinase C (PKC) is of potential therapeutic interest for Alzheimer's disease (AD). Unknown is whether phosphorylation by PKC increases alpha-secretase-mediated cleavage directly or indirectly, for example, by modulation of APP trafficking. Because modulation of Rab6-mediated transport has been shown to affect APP processing, we investigated the regulation of Rab6 membrane association by PKC and its relation to PS1. We show that in fibroblasts, Rab6 membrane association is PKC dependent, an effect strongly potentiated by inhibition of calcineurin. Moreover, we demonstrate that this regulation of Rab6 membrane association is dependent on PS1. The possible implications for APP processing and AD are discussed.

Animals↗
PubMed2004

Neutrophils in Barth syndrome (BTHS) avidly bind annexin-V in the absence of apoptosis.

Barth syndrome (BTHS) is a rare X-linked disease characterized by a triad of dilated cardiomyopathy, skeletal myopathy, and neutropenia. The disease is associated with mutations of the TAZ gene, resulting in defective cardiolipin (CL), an important inner mitochondrial membrane component. Untreated boys die in infancy or early childhood from septicemia or cardiac failure. To date, neutrophil function has never been studied. Directed motility and killing activity of neutrophils was investigated in 7 BTHS patients and found normal in those tested. The circulating neutrophils and eosinophils (but not monocytes or lymphocytes) showed annexin-V binding, suggesting phosphatidylserine (PS) exposure due to apoptosis. However, caspase activity was absent in fresh BTHS cells. Unexpectedly, the near absence of CL impacted neither the mitochondrial mass and shape in fresh BTHS neutrophils nor mitochondrial clustering and Bax translocation upon apoptosis. Annexin-V binding to BTHS neutrophils was not caused by phospholipid scrambling. Moreover, freshly purified BTHS neutrophils were not phagocytosed by macrophages. In sum, a massive number of circulating annexin-V-binding neutrophils in the absence of apoptosis can be demonstrated in BTHS. These neutrophils expose an alternative substrate for annexin-V different from PS and not recognized by macrophages, excluding early clearance as an explanation for the neutropenia.

Adolescent↗
PubMed2003

Expression of complement components in the peripheral nervous system.

We have generated a SAGE (serial analysis of gene expression) library of normal sciatic nerve and found tags encoding for mRNAs of the complement system highly represented. RNA (RT-PCR and northern blot hybridization) and protein (western blot analysis and immunohistochemistry) studies confirmed these findings. High expression of classical pathway components, alternative pathway components and inhibitory components was observed in specific regions of the sciatic nerve. The first components of complement were found in axons, whereas the inhibitory components were detected in the perineurium, thereby protecting the nerve from a complement attack. Immunoreactivity towards activated complement factors was noted in post traumatic neuromas and after acute crush injury, which exemplify nerve regeneration and degeneration. We propose that local production of complement in the peripheral nervous system participates in the protection of healthy nerve and is needed for efficient clearance of myelin after injury: a prerequisite for normal regeneration and remyelination of the peripheral nerve.

Animals↗
PubMed2003

Dihydropyrimidinase deficiency and severe 5-fluorouracil toxicity.

Dihydropyrimidinase (DHP) is the second enzyme in the catabolism of 5-fluorouracil (5FU), and it has been suggested that patients with a deficiency of this enzyme are at risk from developing severe 5FU-associated toxicity. In this study, we demonstrated for the first time that in one patient the severe toxicity, after a treatment with 5FU, was attributable to a partial deficiency of DHP. Analysis of the DHP gene showed that the patient was heterozygous for the missense mutation 833G>A (G278D) in exon 5. Heterologous expression of the mutant enzyme in Escherichia coli showed that the G278D mutation leads to a mutant DHP enzyme without residual activity. An analysis for the presence of this mutation in 96 unrelated Dutch Caucasians indicates that the allele frequency in the normal population is <0.5%. Our results show that a partial DHP deficiency is a novel pharmacogenetic disorder associated with severe 5FU toxicity.

Amidohydrolases↗
PubMed2003

Partial deficiency of the C-terminal-domain phosphatase of RNA polymerase II is associated with congenital cataracts facial dysmorphism neuropathy syndrome.

Congenital cataracts facial dysmorphism neuropathy (CCFDN) syndrome (OMIM 604168) is an autosomal recessive developmental disorder that occurs in an endogamous group of Vlax Roma (Gypsies; refs. 1-3). We previously localized the gene associated with CCFDN to 18qter, where a conserved haplotype suggested a single founder mutation. In this study, we used recombination mapping to refine the gene position to a 155-kb critical interval. During haplotype analysis, we found that the non-transmitted chromosomes of some unaffected parents carried the conserved haplotype associated with the disease. Assuming such parents to be completely homozygous across the critical interval except with respect to the disease-causing mutation, we developed a new 'not quite identical by descent' (NQIBD) approach, which allowed us to identify the mutation causing the disease by sequencing DNA from a single unaffected homozygous parent. We show that CCFDN is caused by a single-nucleotide substitution in an antisense Alu element in intron 6 of CTDP1 (encoding the protein phosphatase FCP1, an essential component of the eukaryotic transcription machinery), resulting in a rare mechanism of aberrant splicing and an Alu insertion in the processed mRNA. CCFDN thus joins the group of 'transcription syndromes' and is the first 'purely' transcriptional defect identified that affects polymerase II-mediated gene expression.

Amino Acid Sequence↗
PubMed2003

Two families with familial amyotrophic lateral sclerosis are linked to a novel locus on chromosome 16q.

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset disease in which motor neurons in the brain and spinal cord degenerate by largely unknown mechanisms. ALS is familial (FALS) in 10% of cases, and the inheritance is usually dominant, with variable penetrance. Mutations in copper/zinc super oxide dismutase (SOD1) are found in 20% of familial and 3% of sporadic ALS cases. Five families with ALS and frontotemporal dementia (ALS-FTD) are linked to 9q21, whereas one family with pure ALS is linked to 18q21. We identified two large European families with ALS without SOD1 mutations or linkage to known FALS loci and conducted a genomewide linkage screen using 400 microsatellite markers. In both families, two-point LOD scores >1 and a haplotype segregating with disease were demonstrated only across regions of chromosome 16. Subsequent fine mapping in family 1 gave a maximum two-point LOD score of 3.62 at D16S3137 and a three-point LOD score of 3.85 for markers D16S415 and D16S3137. Haplotype analysis revealed no recombination > approximately 30 cM, (flanking markers at D16S3075 and D16S3112). The maximum two-point LOD score for family 2 was 1.84 at D16S415, and the three-point LOD score was 2.10 for markers D16S419 and D16S415. Definite recombination occurred in several individuals, which narrowed the shared haplotype in affected individuals to a 10.1-cM region (flanking markers: D16S3396 and D16S3112). The region shared by both families on chromosome 16q12 corresponds to approximately 4.5 Mb on the Marshfield map. Bioinformatic analysis of the region has identified 18 known genes and 70 predicted genes in this region, and sequencing of candidate genes has now begun.

Adult↗
PubMed2003

Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients.

The dystrophin deficiency leading to the severely progressing muscle degeneration in Duchenne muscular dystrophy (DMD) patients is caused by frame-shifting mutations in the DMD gene. We are developing a reading frame correction therapy aimed at the antisense-induced skipping of targeted exons from the pre-mRNA. Despite introducing a (larger) deletion, an in-frame transcript is generated that allows the synthesis of a slightly shorter, but largely functional dystrophin as found in the mostly milder Becker muscular dystrophy (BMD). We have recently demonstrated both the efficacy and high efficiency of the antisense-induced skipping of numerous exons from the DMD transcript in control muscle cells. In principle, this would restore the reading frame in over 75% of the patients reported in the Leiden DMD mutation database. In this study, we in fact demonstrate the broad therapeutic applicability of this strategy in cultured muscle cells from six DMD patients carrying different deletions and a nonsense mutation. In each case, the specific skipping of the targeted exon was induced, restoring dystrophin synthesis in over 75% of cells. The protein was detectable as soon as 16 h post-transfection, then increased to significant levels at the membrane within 2 days, and was maintained for at least a week. Finally, its proper function was further suggested by the restored membranal expression of four associated proteins from the dystrophin-glycoprotein complex. These results document important progress towards a clinically applicable, small-molecule based therapy.

Blotting, Western↗
PubMed2003

In vivo tumor growth inhibition and biodistribution studies of locked nucleic acid (LNA) antisense oligonucleotides.

Locked nucleic acids (LNA) are novel high-affinity DNA analogs that can be used as genotype-specific drugs. The LNA oligonucleotides (LNA PO ODNs) are very stable in vitro and in vivo without the need for a phosphorothiolated backbone. In this study we tested the biological fate and the efficacy in tumor growth inhibition of antisense oligonucleotides directed against the gene of the large subunit of RNA polymerase II (POLR2A) that are completely synthesized as LNA containing diester backbones. These full LNA oligonucleotides strongly reduce POLR2A protein levels. Full LNA PO ODNs appeared to be very stable compounds when injected into the circulation of mice. Full LNA PO ODNs were continuously administered for 14 days to tumor-bearing nude mice. Tumor growth was inhibited sequence specifically at dosages from 1 mg/kg/day. LNA PO ODNs appeared to be non-toxic at dosages <5 mg/kg/day. Biodistribution studies showed the kidneys to have the highest uptake of LNA PO ODNs and urinary secretion as the major route of clearance. This report shows that LNA PO ODNs are potent genotype-specific drugs that can inhibit tumor growth in vivo.

Animals↗
PubMed2003

Disease course of Charcot-Marie-Tooth disease type 2: a 5-year follow-up study.

BACKGROUND: Charcot-Marie-Tooth disease (CMT) type 2 is the axonal variant of an inherited, sensorimotor polyneuropathy. To our knowledge, the clinical course of CMT type 2 has never been prospectively studied in a large group of patients. OBJECTIVE: To prospectively evaluate the disease course of patients with CMT type 2. METHODS: We prospectively evaluated the disease course in patients with CMT type 2. Forty-three patients (24 men) of 27 families with CMT type 2 were included. All patients were analyzed by the same investigator at entry and after 5 years. The standardized protocol included manual muscle testing, which could lead to a motor sum score of 140 points, and quantification of sensory deficit. Disability was assessed using the modified Rankin scale, and quality of life was assessed using the RAND 36-item health survey questionnaire. Eighteen families were tested for known mutations in the MPZ, PMP22, and GJB1 genes. RESULTS: At entry, the mean +/- SD age of the patients was 52 +/- 14 years, and the mean +/- SD duration of disease was 12 +/- 8 years. The median motor sum score deteriorated from 135 to 128 points (P =.02). Progression was never rapid. There was no sensory deterioration. The Rankin score decreased by 1 point in 16 patients. At follow-up, more patients needed walking aids, but most patients remained ambulant. The number of patients with claw toes increased, whereas the number of patients with foot deformities such as pes cavus and short calf muscles remained stable. There was no correlation between deterioration and age. Analysis of quality of life did not show any changes. In one family, a mutation in the GJB1 gene was found. CONCLUSION: This prospective study shows a slow deterioration of muscle strength and increase in disability in CMT type 2 during a 5-year follow-up period.

Adult↗
PubMed2003

Transcriptional profile of the human peripheral nervous system by serial analysis of gene expression.

The peripheral nerve contains both nonmyelinating and myelinating Schwann cells. The interactions between axons, surrounding myelin, and Schwann cells are thought to be important for the correct functioning of the nervous system. To get insight into the genes involved in human myelination and maintenance of the myelin sheath and nerve, we performed a serial analysis of gene expression of human sciatic nerve and cultured Schwann cells. In the sciatic nerve library, we found high expression of genes encoding proteins related to lipid metabolism, the complement system, and the cell cycle, while cultured Schwann cells showed mainly high expression of genes encoding extracellular matrix proteins. The results of our study will assist in the identification of genes involved in maintenance of myelin and peripheral nerve and of genes involved in inherited peripheral neuropathies.

Blotting, Northern↗
PubMed2003

Autoimmunoreactivity to Schwann cells in patients with inflammatory neuropathies.

Inflammatory demyelinating neuropathies are characterized by a loss of peripheral nerve myelin. Myelin breakdown is thought to result from an autoimmune reaction towards nerve components. Schwann cells play a crucial role in the synthesis and maintenance of peripheral nerve myelin. An immune attack targeting Schwann cells could therefore affect myelin integrity, leading to disease. We studied the reactivity of sera from patients with Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) towards Schwann cells using immunofluorescence microscopy. We found 24% of the GBS (56 out of 233) and 26% of the CIDP (12 out of 46) patients to have circulating immunoglobulin G autoantibodies against proliferating, non-myelinating human Schwann cells. In contrast, healthy donors showed positive staining in only two out of 34 sera. No reaction was found with sera from patients with non-inflammatory neurological disorders. Immunofluorescence was localized at the distal tips (leading lamella) of the Schwann cell processes. Distal tips of neurites (nerve-growth-cones) of in vitro differentiated non-myelinated hNT2 neurons also stained strongly. GBS and CIDP serum immunoreactivity was also observed in teased nerve fibre preparations. These data suggest that, at least part of the immunoreactivity is not directed against myelin, but towards non-myelin proteins and epitopes possibly involved in Schwann cell-axon interaction.

Adolescent↗
PubMed2002

Characterisation of mutations in 77 patients with X-linked myotubular myopathy, including a family with a very mild phenotype.

X-linked myotubular myopathy is characterised by neonatal hypotonia, muscle weakness and respiratory distress in affected males, leading often to early death, although prolonged survival is observed in milder forms, or as a result of prolongation of ventilation support. It is caused by mutations in the MTM1 gene, which encodes a phosphatase called myotubularin, which has been highly conserved during evolution, down to yeasts ( S. cerevisiae and S. pombe). To date, 251 mutations have been identified in unrelated families, corresponding to 158 different disease-associated mutations, which are widespread throughout the gene. We have found additional mutations in 77 patients, including 35 novel ones. We identified a missense mutation N180K in a 67-year-old grandfather (the oldest known patient with an MTM1 mutation), previously suspected to have autosomal centronuclear myopathy, and in his two grandsons also mildly affected. Mild and moderate phenotypes associated with novel missense mutations and with a translation initiation defect mutation are discussed, as well as severe phenotypes associated with particular novel mutations. With the present report, 192 different mutations in the MTM1 gene have been described in 328 families. The spectrum of mutations is now enlarged from the very severe classic neonatal phenotype to very mild phenotype allowing survival to the age of 67 years.

Adolescent↗
PubMed2002

Statistical evaluation of SAGE libraries: consequences for experimental design.

Since the introduction of serial analysis of gene expression (SAGE) as a method to quantitatively analyze the differential expression of genes, several statistical tests have been published for the pairwise comparison of SAGE libraries. Testing the difference between the number of specific tags found in two SAGE libraries is hampered by the fact that each SAGE library is only one measurement: the necessary information on biological variation or experimental precision is not available. In the currently available tests, a measure of this variance is obtained from simulation or based on the properties of the tag distribution. To help the user of SAGE to decide between these tests, five different pairwise tests have been compared by determining the critical values, that is, the lowest number of tags that, given an observed number of tags in one library, needs to be found in the other library to result in a significant P value. The five tests included in this comparison are SAGE300, the tests described by Madden et al. (Oncogene 15: 1079-1085, 1997) and by Audic and Claverie (Genome Res 7: 986-995, 1997), Fisher's Exact test, and the Z test, which is equivalent to the chi-squared test. The comparison showed that, for SAGE libraries of equal as well as different size, SAGE300, Fisher's Exact test, Z test, and the Audic and Claverie test have critical values within 1.5% of each other. This indicates that these four tests will give essentially the same results when applied to SAGE libraries. The Madden test, which can only be used for libraries of similar size, is, with 25% higher critical values, more conservative, probably because the variance measure in its test statistic is not appropriate for hypothesis testing. The consequences for the choice of SAGE library sizes are discussed.

Databases, Genetic↗
PubMed2002

Inactivating mutations in the gene for thyroid oxidase 2 (THOX2) and congenital hypothyroidism.

BACKGROUND: Several genetic defects are associated with permanent congenital hypothyroidism. Immunologic, environmental, and iatrogenic (but not genetic) factors are known to induce transient congenital hypothyroidism, which spontaneously resolves within the first months of life. We hypothesized that molecular defects in the thyroid oxidase system, which is composed of at least two proteins, might be involved in the pathogenesis of permanent or transient congenital hypothyroidism in babies with defects in iodide organification, for which the oxidase system is required. METHODS: Nine patients were recruited who had idiopathic congenital hypothyroidism (one with permanent and eight with transient hypothyroidism) and an iodide-organification defect and who had been identified by the screening program for congenital hypothyroidism. The DNA of the patients and their relatives was analyzed for mutations in the genes for thyroid oxidase 1 (THOX1 ) and 2 (THOX2 ). RESULTS: The one patient with permanent and severe thyroid hormone deficiency and a complete iodide-organification defect had a homozygous nonsense mutation in the THOX2 gene that eliminates all functional domains of the protein. Three of the eight patients with mild transient congenital hypothyroidism and a partial iodide-organification defect had heterozygous mutations in the THOX2 gene that prematurely truncate the protein, thus abolishing its functional domains. CONCLUSIONS: Biallelic inactivating mutations in the THOX2 gene result in complete disruption of thyroid-hormone synthesis and are associated with severe and permanent congenital hypothyroidism. Monoallelic mutations are associated with milder, transient hypothyroidism caused by insufficient thyroidal production of hydrogen peroxide, which prevents the synthesis of sufficient quantities of thyroid hormones to meet the large requirement for thyroid hormones at the beginning of life.

Congenital Hypothyroidism↗
PubMed2002

Tumor genotype-specific growth inhibition in vivo by antisense oligonucleotides against a polymorphic site of the large subunit of human RNA polymerase II.

Loss of heterozygosity (LOH) reduces genes to hemizygosity in cancer cells and presents an absolute difference between normal and cancer cells. The regions of LOH are usually much larger than the tumor suppressor gene, which is lost, and are expected to contain genes that are essential for cell survival. Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation in man, often giving rise to two or more allelic forms of most genes. SNPs of essential genes that are frequently affected by LOH can be used as a target for a novel therapy against cancer cells with LOH. The SNPs can be targeted by antisense oligonucleotides (ODNs) that will discriminate between two alleles. We have designed allele-specific phosphorothioate ODNs against the gene of the large subunit of RNA polymerase II (POLR2A), a gene located in close proximity to the tumor suppressor gene p53, which frequently shows LOH in cancer cells. This report shows that phosphorothioate antisense ODNs directed against POLR2A can inhibit tumor growth in vivo as efficiently as a well-described antitumor antisense ODN directed against Ha-ras. In addition, we show that a single bp mismatch can be sufficient to obtain allele-specific inhibition of tumor growth, demonstrating that the effects observed are true antisense effects.

Alleles↗
PubMed2002

Expression and distribution of id helix-loop-helix proteins in human astrocytic tumors.

The Id family of helix-loop-helix proteins is involved in a variety of processes, such as development, proliferation, and angiogenesis. In this study, we investigated the expression pattern of Id1, Id2, and Id3 in surgical specimens of human glial tumors. Western blot analysis demonstrated that all three Id proteins were expressed in astrocytic tumors. Expression levels in high-grade tumors were higher than in low-grade tumors. Immunohistochemical analysis confirmed that many of the tumor astrocytes exhibited strong Id1-3 IR. In contrast, in adult human normal brain, Id expression was low both in resting astrocytes and in endothelial cells. In tumor cells, Id proteins displayed cytoplasmic as well as nuclear localization. Id1-3 IR scores in tumor cells were positively correlated with proliferation indices. Moreover, Id1-3 IR was detected in endothelial cells of the astrocytic tumor blood vessels. The vascular Id1-3 expression correlated positively with tumor vascularity and grade. These results support the role of the Id gene family in the enhanced proliferative potential of tumor astrocytes. The evidence also supports the involvement of the Id gene family in tumor angiogenesis, a process that critically influences the malignant behavior of glial tumors.

Astrocytes↗
PubMed2002

Ribonuclease H1 maps to chromosome 2 and has at least three pseudogene loci in the human genome.

We have analyzed the genomic structure of ribonuclease H1 (RNase H1) loci in the human genome. Human PAC library screening combined with database searches indicated that several loci are present. The transcribed gene is localized on chromosome 2p25. This was confirmed by RNA analysis of a monochromosomal hybrid cell line that expressed human chromosome 2. These data contradict a previous report, as well as the current Human Genome Project (HGP) annotation, which had placed the gene on chromosome 17p11.2. This location represents a pseudogene. Another highly similar pseudogene is present at a separate locus located more distal on chromosome 17p, while a third pseudogene is localized on chromosome 1q.

Base Sequence↗