Biomedical subjects

Niels Gregersen

Publications and source records attributed to Niels Gregersen.

At least 19 recordsLinked to original sources

PubMed2006

Vectorial modeling of near-field imaging with uncoated fiber probes: transfer function and resolving power.

Using exact 3D vectorial simulations of radiation coupling into uncoated dielectric fiber probes, we calculate amplitude transfer functions for conical single-mode fiber tips at the light wavelength of 633 nm. The coupling efficiency of glass fiber tips is determined in a wide range of spatial frequencies of the incident radiation for opening angles varying from 30 degrees to 120 degrees . The resolution in near-field imaging with these tips is considered for field distributions limited in both direct and spatial-frequency space. The characteristics of the transfer functions describing the relation between probed optical fields and near-field images are analyzed in detail. The importance of utilizing a perfectly sharp tip is also examined.

Computer Simulation↗

PubMed2006

Single-nucleotide variations in the genes encoding the mitochondrial Hsp60/Hsp10 chaperone system and their disease-causing potential.

Molecular chaperones assist protein folding, and variations in their encoding genes may be disease-causing in themselves or influence the phenotypic expression of disease-associated or susceptibility-conferring variations in many different genes. We have screened three candidate patient groups for variations in the HSPD1 and HSPE1 genes encoding the mitochondrial Hsp60/Hsp10 chaperone complex: two patients with multiple mitochondrial enzyme deficiency, 61 sudden infant death syndrome cases (MIM: #272120), and 60 patients presenting with ethylmalonic aciduria carrying non-synonymous susceptibility variations in the ACADS gene (MIM: *606885 and #201470). Besides previously reported variations we detected six novel variations: two in the bidirectional promoter region, and one synonymous and three non-synonymous variations in the HSPD1 coding region. One of the non-synonymous variations was polymorphic in patient and control samples, and the rare variations were each only found in single patients and absent in 100 control chromosomes. Functional investigation of the effects of the variations in the promoter region and the non-synonymous variations in the coding region indicated that none of them had a significant impact. Taken together, our data argue against the notion that the chaperonin genes play a major role in the investigated diseases. However, the described variations may represent genetic modifiers with subtle effects.

Butyryl-CoA Dehydrogenase↗

PubMed2006

Variations in IBD (ACAD8) in children with elevated C4-carnitine detected by tandem mass spectrometry newborn screening.

The isobutyryl-CoA dehydrogenase (IBD) enzyme is involved in the degradation of valine. IBD deficiency was first reported in 1998 and subsequent genetic investigations identified acyl-CoA dehydrogenase (ACAD) 8, now IBD, as the gene responsible for IBD deficiency. Only three individuals homozygous or compound heterozygous for variations in the IBD gene have been reported. We present IBD deficiency in an additional four newborns with elevated C(4)-carnitine identified by tandem mass spectrometry (MS/MS) screening in Denmark and the United States. Three showed urinary excretions of isobutyryl-glycine, and in vitro probe analysis of fibroblasts from two newborns indicated enzymatic IBD defect. Molecular genetic analysis revealed seven new rare variations in the IBD gene (c.348C>A, c.400G>T, c.409G>A, c.455T>C, c.958G>A, c.1000C>T and c.1154G>A). Furthermore, sequence analysis of the short-chain acyl-CoA dehydrogenase (SCAD) gene revealed heterozygosity for the prevalent c.625G>A susceptibility variation in all newborns and in the first reported IBD patient. Functional studies in isolated mitochondria demonstrated that the IBD variations present in the Danish newborn (c.409G>A and c.958G>A) together with a previously published IBD variation (c.905G>A) disturbed protein folding and reduced the levels of correctly folded IBD tetramers. Accordingly, low/no IBD residual enzyme activity was detectable when the variant IBD proteins were overexpressed in Chang cells.

Acyl-CoA Dehydrogenases↗

PubMed2006

Transfer function and near-field detection of evanescent waves.

We consider characterization of a near-field optical probe in terms of detection efficiency of different spatial frequencies associated with propagating and evanescent field components. The former are both detected with and radiated from an etched single-mode fiber tip, showing reciprocity of collection and illumination modes. Making use of a collection near-field microscope with a similar fiber tip illuminated by an evanescent field, we measure the collected power as a function of the field spatial frequency in different polarization configurations. Considering a two-dimensional probe configuration, numerical simulations of detection efficiency based on the eigenmode expansion technique are carried out for different tip apex angles. The detection roll-off for high spatial frequencies observed in the experiment and obtained during the simulations is fitted using a simple expression for the transfer function, which is derived by introducing an effective point of (dipolelike) detection inside the probe tip. It is found to be possible to fit reasonably well both the experimental and the simulation data for evanescent field components, implying that the developed approximation of the near-field transfer function can serve as a simple, rational, and sufficiently reliable means of fiber probe characterization.

Journal Article↗

PubMed2006

Topography characterization of a deep grating using near-field imaging.

Using near-field optical microscopy at the wavelength of 633 nm, we image light intensity distributions at several distances above an approximately 2-microm-deep and a 1-microm-period glass grating illuminated from below under the condition of total internal reflection. The intensity distributions are numerically modeled, and an inversion procedure based on a least-squares-fit optimization is employed to extract the grating geometry from the optical images.

Journal Article↗

PubMed2006

Protein misfolding and human disease.

Protein misfolding is a common event in living cells. In young and healthy cells, the misfolded protein load is disposed of by protein quality control (PQC) systems. In aging cells and in cells from certain individuals with genetic diseases, the load may overwhelm the PQC capacity, resulting in accumulation of misfolded proteins. Dependent on the properties of the protein and the efficiency of the PQC systems, the accumulated protein may be degraded or assembled into toxic oligomers and aggregates. To illustrate this concept, we discuss a number of very different protein misfolding diseases including phenylketonuria, Parkinson's disease, alpha-1-antitrypsin deficiency, familial neurohypophyseal diabetes insipidus, and short-chain acyl-CoA dehydrogenase deficiency. Despite the differences, an emerging paradigm suggests that the cellular effects of protein misfolding provide a common framework that may contribute to the elucidation of the cell pathology and guide intervention and treatment strategies of many genetic and age-dependent diseases.

Aging↗

PubMed2006

Heat-shock protein 70 genes and human longevity: a view from Denmark.

We have studied the association of three single nucleotide polymorphisms (SNPs) present in the three HSP70 (heat-shock protein) genes on 6p21 with human longevity. The availability of biological samples from various population cohorts in Denmark has given us the opportunity to try novel methods of gene association with human longevity. A significant association of one haplotype with male longevity was observed. Furthermore, a significant difference in the survival of the carriers of the different genotypes in females was observed. We also found an age-dependant decline in the ability of peripheral blood mononuclear cells to respond to heat stress in terms of Hsp70 induction.

Adult↗

PubMed2006

Reduced heat shock response in human mononuclear cells during aging and its association with polymorphisms in HSP70 genes.

Age-dependent changes in heat shock response (HSR) were studied in mononuclear cells (monocytes and lymphocytes) collected from young (mean age = 22.6 +/- 1.7 years) and middle-aged (mean age = 56.3 +/- 4.7 years) subjects after 1 hour of heat shock at 42 degrees C. Genotype-specific HSR was measured by genotyping the subjects for 3 single nucleotide polymorphisms, HSPA1A(A-110C), HSPA1B(A1267G), and HSPA1L(T2437C), 1 each in the 3 HSP70 genes. A significant age-related decrease in the induction of Hsp70 occurred after heat shock in both monocytes and lymphocytes. The noninducible and inducible forms of Hsp70 decreased 1.3-fold (P < 0.001) and 1.4-fold (P < 0.001), respectively, in the monocytes with age. In the young subjects, a positive association was found between HSPA1L(T2437C) polymorphism and HSR. CC carriers had a significantly lower induction than TT carriers in both monocytes (P = 0.015) and lymphocytes (P = 0.044). This polymorphism, which is present in the coding region of HSPA1L gene, can affect the chaperoning function of Hsp70. These data consolidate our other observations that the CC genotype is unfavorable for human longevity and provide a functional explanation in terms of variations in HSR.

Adult↗

PubMed2005

Short/branched-chain acyl-CoA dehydrogenase deficiency due to an IVS3+3A>G mutation that causes exon skipping.

Short/branched-chain acyl-CoA dehydrogenase deficiency (SBCADD) is an autosomal recessive disorder of L: -isoleucine catabolism. Little is known about the clinical presentation associated with this enzyme defect, as it has been reported in only a limited number of patients. Because the presence of C5-carnitine in blood may indicate SBCADD, the disorder may be detected by MS/MS-based routine newborn screening. It is, therefore, important to gain more knowledge about the clinical presentation and the mutational spectrum of SBCADD. In the present study, we have studied two unrelated families with SBCADD, both with seizures and psychomotor delay as the main clinical features. One family illustrates the fact that affected individuals may also remain asymptomatic. In addition, the normal level of newborn blood spot C5-acylcarnitine in one patient underscores the fact that newborn screening by MS/MS currently lacks sensitivity in detecting SBCADD. Until now, seven mutations in the SBCAD gene have been reported, but only three have been tested experimentally. Here, we identify and characterize an IVS3+3A>G mutation (c.303+3A>G) in the SBCAD gene, and provide evidence that this mutation is disease-causing in both families. Using a minigene approach, we show that the IVS3+3A>G mutation causes exon 3 skipping, despite the fact that it does not appear to disrupt the consensus sequence of the 5' splice site. Based on these results and numerous literature examples, we suggest that this type of mutation (IVS+3A>G) induces missplicing only when in the context of non-consensus (weak) 5' splice sites. Statistical analysis of the sequences shows that the wild-type versions of 5' splice sites in which +3A>G mutations cause exon skipping and disease are weaker on average than a random set of 5' splice sites. This finding is relevant to the interpretation of the functional consequences of this type of mutation in other disease genes.

Alternative Splicing↗

PubMed2005

Differential degradation of variant medium-chain acyl-CoA dehydrogenase by the protein quality control proteases Lon and ClpXP.

The coordinated activities of chaperones and proteases that supervise protein folding and degradation are important factors for deciding the fate of proteins whose folding is impaired by missense variations. We have studied the role of Lon and ClpXP proteases in handling of wild-type and a folding-impaired disease-associated variant (R28C) of the mitochondrial enzyme medium-chain acyl-CoA dehydrogenase (MCAD). Using an Escherichia coli model system, we co-overexpressed the MCAD variants and the respective proteases at two conditions: at 31 degrees C where R28C MCAD protein folds partially and at 37 degrees C where it misfolds and aggregates. Co-overexpression of Lon protease considerably accelerated the degradation rate of a pool of R28C variant MCAD synthesised during a 30min pulse and counteracted accumulation of aggregates at 37 degrees C, whereas increasing the amounts of ClpXP protease had no clear effect. Co-overexpression of either Lon or ClpXP protease markedly decreased the steady state levels of both wild-type and R28C mutant MCAD at 37 degrees C but not at 31 degrees C. Our results suggest that Lon is more efficient than ClpXP in elimination of non-native MCAD protein conformations, and accordingly, that Lon can recognise a broader spectrum of MCAD protein conformations.

Acyl-CoA Dehydrogenase↗

PubMed2005

DNA-based prenatal diagnosis for severe and variant forms of multiple acyl-CoA dehydrogenation deficiency.

OBJECTIVES: Multiple acyl-CoA dehydrogenation deficiency (MADD) is a clinically heterogeneous disorder of mitochondrial fatty acid, amino acid, and choline oxidation due to mutations in the genes encoding electron transfer flavoprotein (ETF) or ETF ubiquinone oxidoreductase (ETFQO). So far, prenatal diagnosis of MADD has relied mostly on second-trimester biochemical analyses of amniotic fluid or cultured amniocytes. We report here on an alternative DNA-based approach for prenatal diagnosis in pregnancies at risk of MADD. METHODS: We used our knowledge of the mutational status in three unrelated families with a history of MADD to perform direct sequencing for the familial mutations using genomic DNA isolated from chorionic villus samples (CVS) at gestational week 10 to 11. RESULTS: Within two days, we were able to carry out accurate DNA-based prenatal testing in one pregnancy at risk of severe MADD, and in two pregnancies at risk of variant forms of MADD. CONCLUSION: This is the first report of DNA-based prenatal diagnosis of MADD. Our molecular approach is suitable for fast and reliable first-trimester prenatal diagnosis in pregnancies at risk of severe and variant forms of MADD.

Acyl-CoA Dehydrogenase↗

PubMed2005

Expression of three different mutations in the arginine vasopressin gene suggests genotype-phenotype correlation in familial neurohypophyseal diabetes insipidus kindreds.

OBJECTIVE AND STUDY DESIGN: The autosomal dominant form of familial neurohypophyseal diabetes insipidus (adFNDI) is a rare disease characterized by a severe and progressive deficiency of AVP secondary to mutations in the gene encoding the AVP precursor. Whereas a number of studies have investigated the pathogenetic mechanisms behind the disease only few studies have included detailed clinical characterization of the affected patients, thereby making genotype-phenotype correlations difficult. The aims of the present study were to investigate the cellular effects of three different adFNDI mutations (A19T, L81P and C110X) by heterologous expression in a neurogenic cell line and to correlate these findings to the corresponding clinical phenotype as determined by extensive clinical tests. RESULTS: The clinical studies showed a later age of onset in the family carrying the A19T mutation (3.4 years, range 2-9 years) compared with families with the L81P and C110X mutations [0.75 year, range 0.5-1 year and 1.0 year (n = 1), respectively]. No other differences could be demonstrated in the clinical phenotype between families. Expression studies showed that each of the three mutant genes caused significant reduction of the amount of immunoreactive AVP in the cell culture medium and severe impairment of the intracellular trafficking and processing of the AVP prohormone, supporting the disease causing nature of all three mutations. However, the A19T mutation was associated with some capacity for processing and trafficking consistent with the clinical observations. Immunoflourescence studies provided evidence of reticular accumulation of protein within the ER in the A19T and C110X mutants but a unique accumulation of much larger aggregates in the L81P, which were localized both within and immediately outside the ER. CONCLUSION: The study suggests a genotype-phenotype correlation with regard to age of onset of diabetes insipidus symptoms and provides support by expression studies.

Adolescent↗

PubMed2005

Actin mutations in hypertrophic and dilated cardiomyopathy cause inefficient protein folding and perturbed filament formation.

Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are the most common hereditary cardiac conditions. Both are frequent causes of sudden death and are often associated with an adverse disease course. Alpha-cardiac actin is one of the disease genes where different missense mutations have been found to cause either HCM or DCM. We have tested the hypothesis that the protein-folding pathway plays a role in disease development for two actin variants associated with DCM and six associated with HCM. Based on a cell-free coupled translation assay the actin variants could be graded by their tendency to associate with the chaperonin TCP-1 ring complex/chaperonin containing TCP-1 (TRiC/CCT) as well as their propensity to acquire their native conformation. Some variant proteins are completely stalled in a complex with TRiC and fail to fold into mature globular actin and some appear to fold as efficiently as the wild-type protein. A fraction of the translated polypeptide became ubiquitinated and detergent insoluble. Variant actin proteins overexpressed in mammalian cell lines fail to incorporate into actin filaments in a manner correlating with the degree of misfolding observed in the cell-free assay; ranging from incorporation comparable to wild-type actin to little or no incorporation. We propose that effects of mutations on folding and fiber assembly may play a role in the molecular disease mechanism.

Actin Cytoskeleton↗

PubMed2005

Protein misfolding, aggregation, and degradation in disease.

Pathologies associated with protein misfolding have been observed in neurodegenerative diseases such as Alzheimer's disease, metabolic diseases like phenylketonuria, and diseases affecting structural proteins like collagen or keratin. Misfolding of mutant proteins in these and many other diseases may result in premature degradation, formation of toxic aggregates, or incorporation of toxic conformations into structures. We review common traits of these diverse diseases under the unifying view of protein misfolding. The molecular pathogenesis is discussed in the context of protein quality control systems consisting of molecular chaperones and intracellular proteases that assist the folding and supervise the maintenance of the folded structure. Furthermore, genetic and environmental factors that may modify the severity of these diseases are underscored.

Amino Acids↗

PubMed2004

No genetic linkage or molecular evidence for involvement of the PCSK9, ARH or CYP7A1 genes in the Familial Hypercholesterolemia phenotype in a sample of Danish families without pathogenic mutations in the LDL receptor and apoB genes.

A locus on chromosome 1p34.1-p32 has been linked to autosomal dominant Familial Hypercholesterolemia (FH) and is termed the third FH locus. We tested whether this third FH locus is linked to the FH phenotype in 20 Danish families, with 158 members, without pathogenic mutations in the genes, encoding the low-density lipoprotein (LDL) receptor or apolipoprotein B (apoB). We could exclude the third FH locus as a cause of FH by genetic linkage analysis in the families taken together. Since haplotype analysis of each family nevertheless suggested that the FH phenotype co-segregated in a manner consistent with linkage to the third FH locus in three small pedigrees, we performed sequencing analysis without being able to demonstrate mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene, the main candidate gene in the third FH locus. By the same combination of genetic linkage and molecular analysis we could also exclude mutations in the gene for the LDL receptor adaptor protein and in the gene for cholesterol-7-alpha-hydroxylase as causes of FH in our sample. Although not indicating linkage to any known loci, our data still indicate that another dominant gene may be involved in causing a FH phenotype.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Homozygosity for a severe novel medium-chain acyl-CoA dehydrogenase (MCAD) mutation IVS3-1G > C that leads to introduction of a premature termination codon by complete missplicing of the MCAD mRNA and is associated with phenotypic diversity ranging from sudden neonatal death to asymptomatic status.

Virtually all patients with medium-chain acyl-CoA dehydrogenase deficiency (MCADD) are homozygous or compound heterozygous for the 985A > G mutation, which limits the study of a possible genotype/phenotype correlation. A newborn Palestinian infant died suddenly on the second day of life. A previous sibling had also died in similar circumstances aged 3 weeks. Urine organic acid and bloodspot acylcarnitine analysis were consistent with MCADD. He was homozygous for a novel MCAD splice mutation, IVS3-1G > C. This mutation leads to deletion of 7 bp and introduction of a premature termination codon as a result of complete missplicing of MCAD mRNA. This misspliced MCAD mRNA encodes a non-functional protein and is furthermore reduced in amounts due to nonsense-mediated decay, resulting in total lack of functional MCAD enzyme. This is the first molecular identification of MCADD in an Arab patient and the first reported splice mutation in the MCAD gene that has been functionally characterized. The association of homozygosity for a null mutation with lethal neonatal presentation in the index patient and presumably the previous infant suggested a genotype/phenotype correlation. However, a 6-year-old completely asymptomatic sibling also had the characteristic MCADD biochemical phenotype and was homozygous for the same IVS3-1G > C mutation. As a first candidate to modify the disease presentation, by modulating the overlapping enzyme activity, we tested the entire family for the prevalent SCAD gene 625G > A susceptibility variant. Interestingly, all family members were 625G > A homozygous. Additional genetic and/or environmental factors must play a major role in determining the phenotypic diversity of MCADD.

Acyl-CoA Dehydrogenase↗

PubMed2004

Association between low self-rated health and heterozygosity for -110A > C polymorphism in the promoter region of HSP70-1 in aged Danish twins.

We have studied the possible association between the -110A > C polymorphism in the promoter region of one of the heat shock protein genes HSP70-1 with human longevity in a cohort of aged Danish twins. This cohort includes individuals aged between 70 and 91 years (mean = 75.6 years), who are categorized according to the presence or absence of various diseases and according to the various, age-related parameters for which a genetic component has already been defined. Four hundred DNA samples from the cohort were genotyped using real-time PCR. Aging phenotypes (diseases, physical and cognitive functioning) were compared with regard to genotype. Of all the aging phenotypes studied, self-rated health and relative self-rated health, which represent an individual's overall sense of physical well-being and which have been shown to be both predictors of survival at older ages and better indicators of future survival than objectively measured health status, were associated with the polymorphism. An association was found between low self-rated health and heterozygosity for -110A > C polymorphism in the promoter region of HSP70-1 in aged Danish twins.

Aged↗

PubMed2004

Six novel mutations in the arginine vasopressin gene in 15 kindreds with autosomal dominant familial neurohypophyseal diabetes insipidus give further insight into the pathogenesis.

Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) is caused by postnatal arginine vasopressin (AVP) deficiency resulting from mutations in the AVP gene encoding the AVP pre-prohormone. To advance the understanding of adFNDI further, we have searched for mutations in the AVP gene in 15 unrelated kindreds in which diabetes insipidus appeared to be segregating. In nine kindreds, seven different previously described mutations were identified. In each of the other six kindreds, unique novel mutations were identified. Two of these (225A>G and 227G>A) change a nucleotide in the translation initiation codon of the signal peptide, whereas the other four (1797T>C, 1884G>A, 1907T>G, and 2112C>G) predict amino-acid substitutions in the neurophysin II moiety of the AVP prohormone, namely V67A (NP36), G96D (NP65), C104G (NP73), and C116W (NP85). Among these, the mutation predicting the V67A (NP36) substitution is remarkable. It affects a region of the neurophysin II not affected by any other mutations, produces only a minor change, and its inheritance suggests an incomplete penetrance. Our findings both confirm and further extend the mutation pattern that has emerged in adFNDI, suggesting that the mutations affect amino-acid residues known or reasonably presumed to be important for the proper folding and/or dimerization of the neurophysin II moiety of the AVP prohormone.

Arginine Vasopressin↗