Search PubMed⌕ Search

Biomedical subjects

L Bolund

Publications and source records attributed to L Bolund.

At least 73 records · Page 4Linked to original sources
PubMed1994

Characterization of a disease-causing Glu119-Lys mutation in the low-density lipoprotein receptor gene in two Danish families with heterozygous familial hypercholesterolemia.

Mutations in the gene for the low-density lipoprotein receptor (LDL receptor) cause the autosomal dominant inherited disease familial hypercholesterolemia (FH). In 15 Danish patients with heterozygous FH we have screened exon 4 of the LDL receptor gene for point mutations and small rearrangements employing genomic DNA amplification and bidirectional solid-phase sequencing. Two subjects were found to be heterozygous for a guanine to adenine base substitution at nucleotide position 418 of the LDL receptor cDNA. This point mutation results in an amino acid change from glutamic acid to lysine at amino acid residue 119 in the third repeat of the cysteine-rich ligand binding domain of the mature LDL receptor. Disruption of LDL receptor function by the Glu119-Lys mutation was confirmed by site-directed mutagenesis and expression in COS-7 cells. By Western blotting the mutation was found to affect the processing of the LDL receptor protein. Using flow cytometric analysis of the transfected cells a decreased binding and internalization of LDL by the mutant receptor was documented. By means of a mutation-specific PCR-based assay the Glu119-Lys mutation was not detected in another 85 apparently unrelated Danish heterozygous FH patients. We identified six persons in the index families with the Glu119-Lys mutation cosegregating with the clinical syndrome of FH in these families. Furthermore, haplotype analysis revealed that the haplotype [SfaNI+, StuI+, AvaII-, (dTA)7] of the mutation carrying allele was the same in the two apparently unrelated patients. This indicates that the mutation has been inherited from a common ancestor.

Adult↗
PubMed1994

Characterization of wild-type human medium-chain acyl-CoA dehydrogenase (MCAD) and mutant enzymes present in MCAD-deficient patients by two-dimensional gel electrophoresis: evidence for post-translational modification of the enzyme.

Two-dimensional gel electrophoresis was used to study and compare wild-type medium-chain acyl-CoA dehydrogenase (MCAD; EC 1.3.99.3) and mis-sense mutant enzyme found in patients with MCAD deficiency. By comparing the patterns for wild-type and mutant MCAD expressed in Escherichia coli or in eukaryotic COS-7 cells we demonstrate that variants with point mutations changing the net charge of the protein can be readily resolved from the wild-type protein. After expression of the cDNA in eukaryotic cells two spots representing mature MCAD can be distinguished, one with an isoelectric point (pI) corresponding to that obtained for the mature protein expressed in E. coli and another one shifted to lower pI. This demonstrates that MCAD protein is partially modified after transport into the mitochondria and removal of the transit peptide. The observed pI shift would be compatible with phosphorylation of one aspartic acid residue per monomer. Comparison of pulse labeling and steady-state amounts of MCAD protein in overexpressing COS-7 cells confirms that K304E MCAD is synthesized and transported into mitochondria in amounts similar to the wild-type protein, but is degraded much more readily. For wild-type MCAD, the spot representing the nonmodified form predominates after pulse labeling while that representing the modified form is relatively stronger in steady state, demonstrating that the modification occurs in mitochondria after the transit peptide has been removed. For K304E mutant MCAD, the nonmodified spot is relatively stronger both in pulse labeling and in steady state, indicating that either the efficiency of modification or the stability of the modified form is affected by the K304E mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyl-CoA Dehydrogenase↗
PubMed1994

Molecular genetic characterization and urinary excretion pattern of metabolites in two families with MCAD deficiency due to compound heterozygosity with a 13 base pair insertion in one allele.

Two families with medium-chain acyl-CoA dehydrogenase (MCAD) deficiency due to compound heterozygosity are described. All patients have a 13 bp insertion in exon 11 of one allele at the MCAD gene locus. In the other allele patients in one of the families harbour the prevalent G985 mutation, and the other family possess an unidentified mutation causing reduced levels of MCAD mRNA. We demonstrate that the disease in these families is inherited as an autosomal recessive trait. Individuals heterozygous for the mutations show heterozygous/control levels of beta-oxidation activities in cultured fibroblasts (9.1-16.3 pmol/min per mg protein; control 10-17 pmol/min per mg protein), and in the excretion of the 'beta-oxidation metabolites', hexanoylglycine (< 2 mumol/mmol creatinine), suberylglycine (< 2 mumol/mmol creatinine) and phenylpropionylglycine (< 2 mumol/mmol creatinine). This shows that there is no 'negative dominance' from the mutant monomeric protein onto the normal ones, in accordance with the finding of low levels of MCAD mRNA from the allele harbouring the 13 bp insertion as well as the allele with the unidentified mutation, and the low steady-state level of enzyme protein expressed from the G985-bearing allele. In the family possessing the G985 and the 13 bp insertion mutations, two asymptomatic compound heterozygous individuals were detected. They exhibited elevated excretion of hexanoylglycine (5-15 mumol/mmol creatinine) and suberylglycine (4-13 mumol/mmol creatinine), together with beta-oxidation activity in fibroblasts in the homozygous range (2.9 pmol/min per mg protein), showing a lack of correlation between the genotype, some biochemical parameters and the clinical phenotype.

Acyl-CoA Dehydrogenase↗
PubMed1994

Gene transfer into cultured human epidermis and its transplantation onto immunodeficient mice: an experimental model for somatic gene therapy.

To try epidermis as a target for somatic gene therapy we studied transfected primary human keratinocytes grown in culture and grafted onto athymic mice. We have developed a novel technique for grafting cultured epidermal sheets onto mice. First, the graft is placed on the dorsal muscle fascia underneath the mouse skin using the latter as a bandage. Secondly, the mouse skin above the graft is removed, which exposes the grafted skin to open air and thus stimulates terminal differentiation. A novel method for the discrimination between murine and human epidermal cells is also presented, employing in situ hybridization with human Alu repeated DNA sequences. During monolayer culture the keratinocytes were lipofected with the gene for human growth hormone in an Epstein-Barr virus-based expression vector. The cells were allowed to develop a multilayered tissue for 5 d, secreting human growth hormone into the medium at a daily rate of at least 50 ng/cm2 of tissue. The transfected tissues were then grafted onto mice. We detected human growth hormone at levels of up to 2.6 ng/ml in mouse serum for 4 d, but later no human growth hormone could be found, although the transplants survived for months. To investigate the fate of the transfected cells in the transplanted tissue, we labeled them with the beta-galactosidase reporter gene. The cells staining positive for X-gal were found exclusively in the most superficial differentiated layers at 7 d after transplantation. This may be the main reason why no human growth hormone is found in the mouse circulation at this time.

Animals↗
PubMed1994

Value of chromosome painting in determining the chromosomal outcome in offspring of a 12;16 translocation carrier.

We currently use direct and reverse chromosome painting in prenatal diagnosis. In a family with a subtle 12;16 translocation, adjacent 1 segregation was diagnosed in the first child, a boy, in whom symptoms compatible with partial trisomy 16p and partial monosomy 12q were seen. In the next pregnancy, a chorionic villus biopsy was tested using chromosome painting. Only by supplementing conventional cytogenetic methods with molecular cytogenetic techniques could the true karyotype be unequivocally determined. Reverse painting, using DOP-PCR amplified, flow sorted paternal derivative chromosomes as a DNA library to paint the chorionic villus cells, was especially informative.

Abnormalities, Multiple↗
PubMed1994

Fast, sensitive multicolor detection of nucleic acids in situ by PRimed IN Situ labeling (PRINS).

PRimed IN Situ labeling (PRINS) has become an alternative to traditional fluorescence in situ hybridization (FISH) methods for detection of nucleic acids in situ. PRINS is based on sequence-specific annealing in situ of an unlabeled DNA probe. The probe serves as a primer for chain elongation in situ, catalyzed by a suitable DNA polymerase that uses labeled nucleotides as substrate. The fact that the probe is unlabeled means that high probe concentrations can be utilized, making the hybridization very fast. We describe here a fast method for detection of three different target sequences visualized in different colors with PRINS. An advantage, relative to FISH, is that even probes with different melting temperatures can be detected in the same metaphase with optimal stringency for each probe.

DNA Probes↗
PubMed1994

Disease-causing mutations in exon 11 of the medium-chain acyl-CoA dehydrogenase gene.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most commonly recognized defect of the mitochondrial beta-oxidation in humans. It is a potentially fatal, autosomal recessive inherited defect. Most patients with MCAD deficiency are homozygous for a single disease-causing mutation (G985), causing a change from lysine to glutamate at position 304 (K304E) in the mature MCAD. Only seven non-G985 mutations, all of which are rare, have been reported. Because the G985 mutation and three of the non-G985 mutations are located in exon 11, it has been suggested that this exon may be a mutational hot spot. Here we describe the results from sequence analysis of exon 11 and part of the flanking introns from 36 compound heterozygous patients with MCAD deficiency. We have identified four previously unknown disease-causing mutations (M301T, S311R, R324X, and E359X) and two silent mutations in exon 11. Our results show that exon 11 is not especially mutation prone. We demonstrate that two of the identified disease-causing mutations can be detected by restriction enzyme digestion of the PCR product from the assay for the G985 mutation, a discovery that makes this assay even more useful than before. On the basis of expression of wild-type and mutant MCAD protein in COS-7 cells, we show that the identified mutations abolish MCAD enzyme activity and that they therefore must be disease causing. The M301T, S311R, and K304E mutations are located in helix H, which makes up part of the dimer-dimer interface of the MCAD tetramer. On the basis of the three-dimensional structure of MCAD and the results from the COS-7 expression experiments, we speculate that the primary effect of the M301T and S311R mutations is on correct folding/tetramer assembly, as it has previously been observed for the K304E mutation.

Acyl-CoA Dehydrogenase↗
PubMed1993

Co-overexpression of bacterial GroESL chaperonins partly overcomes non-productive folding and tetramer assembly of E. coli-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) carrying the prevalent disease-causing K304E mutation.

The influence of co-overexpression of the bacterial chaperonins GroEL and GroES on solubility, tetramer formation and enzyme activity of three variants of heterologously-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) was analysed in order to investigate the molecular mechanism underlying MCAD deficiency caused by the prevalent K304E mutation. Depending on which of the three amino acids--lysine (wild-type), glutamic acid (K304E) or glutamine (K304Q) are present at position 304 of the mature polypeptide, three different patterns were observed in our assay system: (i) solubility, tetramer formation and yield of enzyme activity of wild-type MCAD is largely independent of GroESL co-overexpression; (ii) the larger part of the K304Q mutant is insoluble without and solubility is enhanced with GroESL co-overexpression; solubility correlates with the amount of tetramer detected and the enzyme activity measured as observed for the wild-type protein. (iii) Solubility of the K304E mutant is in a similar fashion GroESL responsive as the K304Q mutant, but the amount of tetramer observed and the enzyme activity measured do not correlate with the amount of soluble K304E MCAD protein detected in Western blotting. In a first attempt to estimate the specific activity, we show that tetrameric K304E and K304Q mutant MCAD display a specific activity in the range of the wild-type enzyme. Taken together, our results strongly suggest, that the K304E mutation primarily impairs the rate of folding and subunit assembly. Based on the data presented, we propose that lysine-304 is important for the folding pathway and that an exchange of this amino acid both to glutamine or glutamic acid leads to an increased tendency to misfold/aggregate. Furthermore, exchange of lysine-304 with an amino acid with negative charge at position 304 (glutamic acid) but not with a neutral charge (glutamine) negatively affects conversion to active tetramers. A possible explanation for this latter effect--charge repulsion upon subunit docking--is discussed.

Acyl-CoA Dehydrogenase↗
PubMed1993

Correction of steroid sulfatase deficiency by gene transfer into basal cells of tissue-cultured epidermis from patients with recessive X-linked ichthyosis.

To develop an experimental model for somatic gene therapy we have tried to correct the steroid sulfatase (STS) deficiency in tissue-cultured primary epidermal keratinocytes from patients suffering from recessive X-linked ichthyosis. An efficient Epstein-Barr virus-based vector was constructed, in which full-length steroid sulfatase cDNA is located between an SV40 early promotor and processing signals. After STS gene transfer into cultured basal cells from ichthyotic skin, the cells produce large amounts of enzymatically active steroid sulfatase protein. The subpopulation of transfected cells can be made to produce approximately 100 times more STS activity than normal keratinocytes. Keratinocytes from patients suffering from recessive X-linked ichthyosis display an abnormal phenotype when developing a multilayered tissue in culture: Initially an extensive burst of keratinization is observed, followed by rapid, premature shedding and degradation of most suprabasal cell layers, leaving a culture with hyperproliferative relatively immature keratinocytes. Transfection of these immature ichthyotic cells with the functional STS construct led to an increase in the amount of retained cell material in the culture medium, indicating an increased cell maturation. It is possible to genetically label individual transfected epidermal cells with a reporter gene. Cotransfection experiments with STS and reporter gene vectors show that the cohort of transfected cells had a tendency to develop less rapidly since they became overrepresented in the smaller size classes at the same time the total population was somewhat shifted toward higher cell sizes. We interpret these results as an indication that restoration of the enzymatic activity induces a more normal maturation of the transfected keratinocytes.

Arylsulfatases↗
PubMed1993

Genetic analysis of repeated, biparental, diploid, hydatidiform moles.

A woman presented with five consecutive pregnancies displaying molar morphology. In the fifth pregnancy, a non-malformed, liveborn infant was delivered. Genetic analyses (RFLP analysis, cytogenetics, flow cytometry) were performed in pregnancies II-V. It was demonstrated that these pregnancies originated in separate conceptions, all conceptuses were diploid, and all had maternally as well as paternally derived genetic markers. By cytogenetic analysis, aberrant heteromorphisms were noted; no other abnormalities were observed in chromosome structure or in DNA sequence. Many different causes for the abnormal development can be envisaged, environmental as well as genetic. To conform to current ideas of molar pathogenesis, it is suggested that the present conceptuses might have arisen from imbalances in imprinted genomic regions. This could be a consequence of uniparental disomy in critical regions generated by somatic crossing over. Alternatively, it could be caused by a malfunction in the generation or maintenance of imprinting.

Chromosome Aberrations↗
PubMed1993

Assignment of an autosomal sex reversal locus (SRA1) and campomelic dysplasia (CMPD1) to 17q24.3-q25.1.

We have mapped the autosomal sex reversal locus, SRA1, associated with campomelic dysplasia (CMPD1) to 17q24.3-q25.1 by three independent apparently balanced de novo reciprocal translocations. Chromosome painting indicates that the translocated segment of 17q involves about 15% of chromosome 17 in all three translocations, corresponding to a breakpoint at the interphase between 17q24-q25. All three 17q breakpoints were localized distal to the growth hormone locus (GH), and proximal to thymidine kinase (TK1). Due to the distal location of the breakpoints, previously mentioned candidate genes, HOX2 and COL1A1, can be excluded as being involved in CMPD1/SRA1. The mouse mutant tail-short (Ts) which maps to the homologous syntenic region on mouse chromosome 11, displays some of the features of CMPD1.

Abnormalities, Multiple↗
PubMed1993

Identification of a supernumerary der(18) chromosome by a rational strategy for the cytogenetic typing of small marker chromosomes with chromosome-specific DNA probes.

A case of a supernumerary der(18) marker chromosome is presented. The chromosomal origin of the marker chromosome was not evident by traditional chromosome analysis, but was determined by PRimed IN Situ labelling (PRINS) with chromosome specific centromere probes as primers for chain elongation in situ. For this purpose a strategy was developed which, in a few simple reactions, makes it possible unequivocally to determine the origin of any small marker chromosome. The approach does not require any hints about the origin of the chromosome prior to the analysis, since the chromosomal origin of the marker is established through PRINS reactions with pooled and single chromosome-specific centromere probes. Identification, mosaic screening and structural analysis require a total of 8-9 such reactions and may, due to the extreme speed of the PRINS reaction, be obtained within a single working day.

Chromosome Aberrations↗