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L Bolund

Publications and source records attributed to L Bolund.

At least 55 records · Page 3Linked to original sources
PubMed1997

Molecular cytogenetic analysis of a nontumorigenic human breast epithelial cell line that eventually turns tumorigenic: validation of an analytical approach combining karyotyping, comparative genomic hybridization, chromosome painting, and single-locus fluorescence in situ hybridization.

The immortalized, nontumorigenic human breast epithelial cell line HMT-3522 has been used as a model for premalignant and, eventually, malignant development. During cultivation, the karyotype evolution was followed. At an early stage, a very long constant phase showed a near-diploid karyotype, with only five marker chromosomes. DNA from this phase was used for comparative genomic hybridization (CGH) analysis, confirming a previously known MYC amplification, and the integration sites were subsequently determined by single-locus fluorescence in situ hybridization (FISH). Furthermore, gains of 5q22-qter and 20q11-qter and deletion of most of chromosome 6 (6p23-qter) were detected by CGH. Because of uncertainty about some of the indicated changes, including a deletion of Ip35-pter, the CGH findings were investigated more closely by chromosome painting, leading to a revision of the karyotype: 45,XX,del(I)(p35),-6,dup(8)(pter-->qter::qter-->q24),der(12) t(6;12)(p23; p13),der(14)t(5;14)(q22;q32.3),der(17)t(8;17;20)(17pter-->17q25 ::8qter--> 8q23::8q24-->8qter::8q24-->8qter:: 8q23-->8q24.1::20q11-->20qter). Some karyotypic changes were confirmed by CGH; others had to be revised; and, in the Ip35 region, classical cytogenetics seems superior to CGH. However, CGH revealed a karyotypically unsuspected dup(20q) that might be of special relevance to breast tumor initiation or progression. Our study confirms that CGH is supplementary to current technologies, e.g., karyotyping and Southern analysis, but cannot replace them. In addition, our cell line turned out to be an excellent model for comparison among the different methods. The results imply that future cytogenetic analyses of complex karyotypes should be based on a combination of karyotyping, CGH, and FISH.

Breast↗
PubMed1996

Influence of Lewis alpha1-3/4-L-fucosyltransferase (FUT3) gene mutations on enzyme activity, erythrocyte phenotyping, and circulating tumor marker sialyl-Lewis a levels.

Fucosylated glycoproteins carrying alpha1-4 fucose residues are of importance for cell adhesion and as tumor markers. The Lewis gene, FUT3, encodes the only known alpha1-4-fucosyltransferase (FucT), and individuals who are deficient in this enzyme type as Lewis-negative on erythrocytes. We examined the mutational spectrum of the Lewis gene in Denmark and found 6 different mutations. Five, T59G, T202C, C314T, G508A, and T1067A, were frequent, and one, C445A, was only detected in one out of 40 individuals. Allele-specific polymerase chain reaction as well as cloning of FUT3 alleles showed that the 202 and 314 mutations were co-located on the same allele. COS7 cells transfected with an allele having the 202/314 mutations lacked enzyme activity. Polymerase chain reaction-cleavage assays were established for the genotyping of healthy individuals as well as 20 genuine Lewis-negative cancer patients and 10 non-genuine. The latter have Lewis-negative erythrocytes but saliva alpha1-4FucT activity. The genuine Lewis-negative individuals had mutations on both FUT3 alleles. In 66 healthy individuals, a gene dosage effect was detected as FUT3 heterozygous individuals had a lower alpha1-4FucT activity in saliva than did homozygous wild-type individuals. The lower enzyme level in heterozygous individuals resulted in a significantly (p < 0.04) lower level of circulating sialyl-Lewis a structure in serum. This has the clinical impact that cut-off levels in tumor marker assays should be defined on the basis of genotyping. In the group of non-genuine Lewis-negative cancer patients, whose erythrocytes convert from Lewis-positive to Lewis-negative during the disease, FUT3 heterozygosity was significantly (p < 0.05) more common.

ABO Blood-Group System↗
PubMed1996

Allele-specific measurement of low-density lipoprotein receptor transcript levels.

We have developed an assay for allele-specific determination of low-density lipoprotein receptor (LDLR) mRNAs. Transcript levels are measured by reverse transcription (RT), PCR, and electrophoresis on an automatic DNA sequencer using fluorescence-labeled primers and direct quantitation of the allele-specific RT-PCR products. The discrimination between the allelic products is based on the use of DNA polymorphisms located in the coding regions of the gene as markers for the individual alleles. Using this method on LDLR mRNA from heterozygous patients with familial hypercholesterolemia (FH) due to a defective LDLR protein, it is possible to relate the expression of the mutant allele directly to the expressed amounts of the normal allele, thus overcoming the problems of using artificial internal standards in the PCR. To validate the method we have measured (1) the range of normal LDLR allele transcript levels, and (2) the transcript levels in patients heterozygous for different types of mutant LDLR alleles associated with FH. The method is general in principle and can be applied in the allele-specific analysis of transcripts from all genes harbouring DNA polymorphisms in their coding regions.

Alleles↗
PubMed1996

Primed in situ labeling (PRINS). A fast method for in situ labeling of nucleic acids.

PRimed IN Situ labeling (PRINS) is a fast and sensitive alternative to fluorescence in situ hybridization (FISH) for identification of chromosome aberrations. In this article, we present the detailed protocols for detection of repeat sequences using oligonucleotides or fragments of cloned probes as primers for PRINS. We describe a multicolor PRINS procedure for simultaneous visualization of more probes in different colors on a metaphase preparation, and a PRINS-painting procedure, which combines PRINS and chromosome painting. Finally, a protocol for detection of single-copy genes is presented.

Chromosomes, Human↗
PubMed1996

Phenotypic characterization of a patient homozygous for the D558N LDL receptor gene mutation.

We describe the clinical, biochemical, and genetic features of a patient with true homozygous familial hypercholesterolemia due to the D558N low-density lipoprotein receptor gene mutation, previously designated FH Cincinnati-4. Functional flow-cytometric analysis of the LDL receptorR protein on upregulated EBV-transformed lymphocytes indicated reduction of the number of receptors on the cell surface by 87% and reduction of receptor activity by 89% compared to control cells. With drugs and a portacaval shunt operation, performed when the patient was 15 years old, serum cholesterol was reduced from about 28 to about 15 mmol/l. He died at the age of 32 of a myocardial infarction. The autopsy showed generalized atherosclerosis, especially in the coronary arteries, which were severely stenosed proximally. A rare finding was a large intracranial xanthoma that apparently had been asymptomatic.

Adult↗
PubMed1996

Primed IN situ labelling (PRINS) as a rational procedure for identification of marker chromosomes using a panel of primers differentially tagging the human chromosomes.

PRimed IN Situ labelling (PRINS) is a highly specific and sensitive technique for detecting DNA sequences on human chromosomes in situ. PRINS is currently being introduced for research and routine analysis in clinical and cancer genetics. In this paper, we report a rational PRINS procedure for the rapid identification of marker chromosomes. Using this method it is possible to test a sample from a patient with up to eight different primers simultaneously on one slide. We have synthesized oligonucleotide primers that can differentially tag the human chromosomes, and with the protocol presented in this report we are able to identify the chromosomal origin of a marker chromosome within 2 hours.

Amniocentesis↗
PubMed1996

A G-1-to-A acceptor splice site LDLR mutant allele leads to reduced relative transcript levels in patients with heterozygous familial hypercholesterolemia.

A plethora of different mutations in the gene for the low density receptor (LDLR) are responsible for the autosomal dominant inherited disorder familial hypercholesterolemia (FH). However, only a few splice site mutations have been identified in this gene. We here report a defect presumably affecting the splicing of precursor mRNA, resulting from a novel mutation, a G to A transition at the terminal nucleotide of intron 12, of the LDLR gene detected in three unrelated families with heterozygous FH. This mutation markedly reduced the steady-state transcript level of the mutant LDLR allele as compared to the corresponding normal LDLR allele in heterozygous FH patients as measured by a fluorescence based, allele-specific quantitation technique. In the FH families, the acceptor splice site mutation cosegregates with hypercholesterolemia, and it is associated with onset of ischemic heart disease in the fifth and sixth decade of life.

Adult↗
PubMed1996

Ethylmalonic aciduria is associated with an amino acid variant of short chain acyl-coenzyme A dehydrogenase.

Ethylmalonic aciduria is a common biochemical finding in patients with inborn errors of short chain fatty acid beta-oxidation. The urinary excretion of ethylmalonic acid (EMA) may stem from decreased oxidation by short chain acyl-CoA dehydrogenase (SCAD) of butyryl-CoA, which is alternatively metabolized by propionyl-CoA carboxylase to EMA. We have recently detected a guanine to adenine polymorphism in the SCAD gene at position 625 in the SCAD cDNA, which changes glycine 209 to serine (G209S). The variant allele (A625) is present in homozygous and in heterozygous form in 7 and 34.8% of the general population, respectively. One hundred and thirty-five patients from Germany, Denmark, the Czech Republic, Spain, and the United States were selected for this study on the basis of abnormal EMA excretion ranging from 18 to 1185 mmol/mol of creatinine (controls < 18 mmol/mol of creatinine). Among them, we found a significant overrepresentation of the variant allele. Eighty-one patients (60%) were homozygous for the A625 allele, 40 (30%) were heterozygous, and only 14 (10%) harbored the wild-type allele (G625) in homozygous form. By overexpressing the wild-type and variant protein (G209S) in Escherichia coli and COS cells, we showed that the folding of the variant protein was slightly compromised in comparison to the wild-type and that the temperature stability of the tetrameric variant enzyme was lower than that of the wild type. Taken together, the over-representation and the biochemical studies indicate that the A625 allele confers susceptibility to the development of ethylmalonic aciduria.

Acyl-CoA Dehydrogenase↗
PubMed1996

Rapid characterization of disease-causing mutations in the low density lipoprotein receptor (LDL-R) gene by overexpression in COS cells.

To characterize disease-causing mutations in the low density lipoprotein receptor (LDL-R) gene, COS cells are transfected with the mutant gene in an EBV-based expression vector and characterized by flow cytometry. Using antibodies against the LDL-receptor the amount of receptor protein on the cell surface is quantitated. The receptor activity is measured by incubating the cells with fluorescence labeled LDL (Dil-labelled LDL) at 37 degrees C and 4 degrees C. The transfected cells stained with anti-LDL-R antibodies can also be analysed by immunofluorescence microscopy allowing the study of the intracellular location of variants of the receptor. To evaluate these methods, we are analyzing four previously well-characterized LDL-R mutations, belonging to each of the classes 2 to 5. Preliminary data show that mutant genes belonging to class 3 and 4A give rise to receptor protein on the cell surface, but impaired LDL uptake, while mutant receptors belonging to class 2A and 5 can only be detected intracellularly. Expression of the class 2A mutation results in an ER staining pattern, whereas the class 5 mutation gives rise to an intracellular staining compatible with localization in the endosomal/lysosomal compartments. We conclude that this system is useful for a rapid functional analysis of newly discovered mutations in the LDL-R gene.

Cell Line↗
PubMed1996

A flow cytometric competition technique for measuring interaction of LDL with cellular LDL-receptors applied to patients with mutant (Arg3500-->Gln) apolipoprotein B.

We report our experience with a method to evaluate binding and uptake in cells of low density lipoprotein (LDL) from heterozygous patients with familial defective apolipoprotein B-100 (FDB-LDL) and LDL from normolipidemic subjects (nonFDB-LDL). The method is based on competition for binding/uptake in Epstein-Barr Virus (EBV)-transformed lymphocytes or COS cells overexpressing an LDL-receptor transgene between fluorescently labeled LDL and the unlabeled LDL of interest, and measurements are by flow cytometry. With EBV-lymphoblasts, the ability of FDB-LDL to displace fluorescent LDL ("Dil"-LDL) from cells at 4 degrees C (binding) was reduced to approximately 1/3 of normal. Displacement of "Dil"-LDL by FDB-LDL from cells at 20 degrees C (binding/uptake) was reduced to less than 1/2 of normal. Similar results were obtained with COS cells. Freezing of serum to -80 degrees C for 24 hours did not affect results, and we could discriminate between binding/uptake of FDB-LDL and nonFDB-LDL prepared from serum that had been stored at -80 degrees C for three months.

Adult↗
PubMed1996

Flow cytometric DNA analyses of 105 fresh hydatidiform moles, with correlations to prognosis.

Cellular DNA contents of consecutive hydatidiform moles were analysed by flow cytometry of unfixed samples, freed of maternal tissue and treated with detergent and trypsin, using two external controls. DNA-diploidy was found in 62 moles, DNA-triploidy in 42 and DNA-tetraploidy in one. Two non-molar placentas and two invasive moles were found to be DNA-diploid. In 28 of 42 DNA-triploid moles, trophoblastic hyperplasia was not noted, making the discrimination between vesicular abortions with or without trophoblastic hyperplasia unwarranted on genetic grounds. A good fit was obtained to a distribution with one G0-1 peak in 49 cases, whereas 60 cases showed two peaks. Technical artifacts, created by flow cytometry, were excluded. Differences in the nuclear accessibility for the dye and degradation of DNA are unlikely causes of this heterogeneity. The existence, in vivo, of two or more cell populations with different DNA contents is a reasonable explanation. The criteria for persistent trophoblastic disease were standardised. Eight cases of persistent trophoblastic disease were observed among the 62 DNA-diploid moles (13%); no case was observed after DNA-triploid or DNA-tetraploid moles. Combining these data, with those of other studies where ploidy was determined using optimal techniques [10,13], allows the calculation of 95%-confidence-limits for the risk of persistent trophoblastic disease after a triploid mole to 0-2.7%.

Aneuploidy↗
PubMed1995

Comparison between medium-chain acyl-CoA dehydrogenase mutant proteins overexpressed in bacterial and mammalian cells.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a potentially lethal inherited defect in the beta-oxidation of fatty acids. By comparing the behaviour of five missense MCAD mutant proteins expressed in COS cells and in Escherichia coli, we can define some of these as "pure folding mutants." Upon expression in E. coli, these mutant proteins produce activity levels in the range of the wild-type enzyme only if the chaperonins GroESL are co-overproduced. When overexpressed in COS cells, the pure folding mutants display enzyme activities comparable to the wild-type enzyme. The results suggest that the MCAD mutations can be modulated by chaperones, a phenomenon that may influence the manifestation of the MCAD disease.

Acyl-CoA Dehydrogenase↗
PubMed1995

Simultaneous detection of centromere-specific probes and chromosome painting libraries by a combination of primed in situ labelling and chromosome painting (PRINS-painting).

In situ techniques for the detection of specific chromosomes using centromeric probes and the decoration of entire chromosomes using chromosome painting are well established. However, in the deciphering of complex chromosomal aberrations it is valuable to be able to detect the centromere and the entire DNA of a specific chromosome in different colours simultaneously on the same metaphase. In this report we describe a combination of the primed in situ labelling (PRINS) technique and chromosome painting for simultaneous visualization of centromere-specific oligonucleotides and chromosome painting libraries. A key feature is that the denaturation step in the PRINS reaction is sufficient to keep the chromosomes denatured for chromosome painting. This means that PRINS and consecutive chromosome painting can be performed as a single procedure (PRINS-painting).

Centromere↗
PubMed1995

Complexity of molecular genetics of dyslipidemia in a family highly susceptible to ischemic heart disease.

In a Danish family highly susceptible to ischemic heart disease, hyperlipidemia did not simply cosegregate with a previously undescribed 10 bp deletion in the LDL receptor gene causing heterozygous familial hypercholesterolemia (FH). This mutation, designated as FH DK-4, deletes 10 nucleotides from exon 4 coding for the third cysteine-rich repeat of the ligand-binding domain. The resulting translational frameshift and stop codon corresponding to amino acid position 181 in the LDL receptor cDNA is predicted to result in a truncated LDL receptor protein. Several family members had hyperlipidemia and early onset of ischemic heart disease not due to the 10 bp deletion, and several family members had unexpectedly high serum lipoprotein(a) contributing to high concentrations of serum LDL cholesterol. The study illustrates important limitations and possibilities of molecular genetic diagnosis.

Base Sequence↗
PubMed1995

Construction of a panel of chromosome-specific oligonucleotide probes (PRINS-primers) useful for the identification of individual human chromosomes in situ.

We present the sequences of a set of oligonucleotides that, when used as primers for PRimed IN Situ (PRINS) labeling, are diagnostic for repetitive sequences in specific human chromosomes. Combined, they enable identification of all human chromosomes except 6, 19, and 20. However, as is also the case with cloned centromeric hybridization probes, chromosomes 14 and 22 are stained together. Along with the sequences of these oligonucleotides we offer a simple, universal procedures for their use. We also present an oligonucleotide that binds to both strands of alpha-satellite DNA, making it possible to specifically amplify alpha-satellite DNA by PCR with this one oligonucleotide alone as primer. The origin of the alpha-satellite DNA in the starting material (whether somatic cell hybrids, flow-sorted chromosomes, or microdissected material) can then be determined on test metaphase spreads by in situ hybridization or PRINS with the PCR product.

Base Sequence↗
PubMed1995

Molecular diagnosis and characterization of medium-chain acyl-CoA dehydrogenase deficiency.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common defect in mitochondrial beta-oxidation in humans. It is an autosomal recessive disorder which usually presents in infancy. The disease manifests itself in periods of metabolic stress to the beta-oxidation system and may be fatal. Four years ago we identified a prevalent disease-causing mutation (G985) which causes an amino acid change (K304E) in the mature MCAD protein. Using a Polymerase Chain Reaction (PCR) based assay for this mutation we have demonstrated: 1. that the G985 mutation is present in 90% of the disease alleles from patients from all over the world; 2. that the allele frequency of G985 in the general population from most European countries is very high (the carrier frequency ranges from 1/68 to 1/333); 3. that MCAD deficiency is not, as has previously been suggested, related to Sudden Infant Death Syndrome (SIDS). Moreover, investigation by Restriction Fragment Length Polymorphism (RFLP) analysis of several families with diagnosed MCAD deficiency revealed that the G985 mutation is only present in chromosomes of a particular RFLP haplotype, suggesting a common chromosomal background for this mutation. The other mutations in the MCAD gene are distributed to all known MCAD RFLP haplotypes. Because 80% of the patients are homozygous for the G985 mutation, DNA based diagnosis of most patients is now fast and easy. In order to make DNA based diagnosis possible for the remaining 20% of patients we have set up PCR/solid-phase based semi-automated sequencing of all 12 exons of the MCAD gene. We have so far identified the mutation in 33 of 45 non-G985 homozygous families with verified MCAD deficiency, thereby bringing the number of known mutations in the MCAD gene up to 26. In order to investigate in detail the molecular defects of the mutant MCAD proteins we overexpressed them in COS-7 and in an E. coli based expression system with and without co-overexpression of the molecular chaperones GroES and GroEL. The expression studies revealed that the primary effect of all the identified mutations is on formation of correct enzyme structure, and does not directly affect the catalytically active regions of the enzyme. We find that our diagnostic set up, consisting of an initial testing by the G985 assay, followed by semi-automated sequencing of DNA from those patients who were indicated to be compound heterozygous, is an important improvement to the diagnosis of MCAD deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)

Acyl-CoA Dehydrogenase↗