Biomedical subjects
Publications and source records attributed to N Gregersen.
A small marker chromosome was identified as an X-derived ring chromosome by in situ hybridization with a biotinylated X-chromosome specific a-satellite DNA probe. This procedure clearly determined the chromosomal origin of the marker chromosome, which had been impossible to define by conventional cytogenetic techniques including high resolution banding.
We present here an alternative approach to the study of mosaic cell lines containing dicentric chromosomes. The approach is based on chromosome-specific non-radioactive in situ hybridization with centromere (alpha satellite DNA) probes. The hybridization analysis may be used as an alternative to the C-band analysis, while at the same time to some extent replacing the Q-band analysis as well. The advantage of using in situ hybridization is mainly that it allows the very fast screening of a large number of metaphases. We illustrate this new application of the technique by using it for the analysis of two cases of isodicentric X-chromosomes. The approach is expected to be generally applicable, so that it may be applied to the scoring of other types of chromosomal mosaicism as well.
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The three first cases of isovaleric acidemia diagnosed in Scandinavia are described. The disorder is characterized by periodic vomiting, lethargy and coma accompanied by ketoacidosis and a "sweaty feet" odour. These attacks are often triggered of by upper respiratory tract infections or by ingestion of large amounts of protein. Often there are feeding difficulties because these children have aversion to protein-containing foods. Isovaleric acidemia can be subdivided into two types: an acute neonatal form and a chronic intermittent form. The basic defect is deficient activity of isovaleryl-CoA dehydrogenase, resulting in increased urinary excretion of mainly isovaleryl-glycine and 3-hydroxy-isovaleric acid. The defective gene is assigned to the long arm of chromosome 15, and at least five different mutations among 15 patients have been demonstrated. Therapy is symptomatic with correction of the metabolic acidosis and protein restriction and long term treatment with oral glycine and possibly carnitine.
Specific analysis for point mutations in genomic DNA has until recently been a difficult and time-consuming task, using large amounts of unstable, hazardous and expensive 32P. By enzymatically amplifying the mutation-bearing sequence of the DNA the sensitivity of the analysis is increased several 100-fold, making the detection possible with stable, non-radioactive and inexpensive biotinylated oligonucleotides. We have applied this method (polymerase chain reaction (PCR] to the detection of the Z-mutation in the alpha-1-antitrypsin gene. After amplification, dot-blots of amplified DNA were subjected to hybridization with allele specific biotinylated oligonucleotide probes and washed at temperatures giving allele specificity. The bound biotin was visualized with avidin conjugated alkaline phosphatase using 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium as colour reagents. The detection can be performed on less than 1 microgram genomic DNA, and is therefore applicable on small amounts of blood, fibroblasts and chorionic villus biopsies.
It is demonstrated that either general staining of the centromeric regions of all primate chromosomes, or selective staining of the centromeric region of specific chromosomes, may be obtained in preparations of metaphase chromosomes by probing specifically for different regions within the alpha satellite DNA monomer. In order to exploit observed patterns of sequence variation within the monomer for this purpose, we have developed two new DNA analysis methods. In PRimed IN Situ labelling (PRINS), synthetic oligonucleotides derived from subsections of the monomer are hybridized to the chromosomes. The oligonucleotides then serve as primers for the in situ incorporation of biotin-labelled nucleotides catalysed by Klenow polymerase. Incorporated biotin is visualized with fluorescein isothiocyanate-labelled avidin (FITC-avidin). In Primed Amplification Labelling (PAL), biotin-labelled hybridization probes are produced in a polymerase chain reaction (PCR, Saiki et al. 1985), in which two synthetic oligonucleotide primers anneal within the same monomer. With the right choice of primers libraries of labelled probes derived from most monomers present as templates are produced. If DNA from a specific chromosome is used as template, then the resulting probe mixture gives stronger and more chromosome-specific signals in in situ hybridization experiments than does a cloned alpha satellite DNA probe derived from the same chromosome. The results obtained indicate that the alpha-repeat monomer is composed of regions with different degrees of chromosome specificity.
Prenatal diagnosis of alpha 1-antitrypsin (AAT) deficiency can be performed in the 1st trimester of pregnancy. These diagnoses have been based on DNA technology using either RFLP analysis or hybridization with allele specific oligonucleotides. Several RFLPs within and flanking the AAT gene have been found to render most families informative. The polymerase chain reaction allows specific DNA sequences to be amplified up to ten million fold. Both sequences containing a specific mutation or an RFLP can be amplified by this method. We have compared conventional RFLP methods with PCR used in combination with allele specific oligonucleotides or RFLP analysis, in a case of prenatal diagnosis of AAT deficiency of the ZZ type.
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Biotinylated DNA hybridization probes offers a stable, cheap and non-radioactive alternative to probes labelled with 32P. Insufficient sensitivity has, however, up till now, been prohibitive for the use of such probes in detecting unique sequences in Southern blots of human DNA. By optimizing the steps in the procedure we have improved the sensitivity enough for such use. We have showed (1) that long probes (greater than 500 nucleotides) perform unproportionally better than short probes; (2) that a simple affinity labelling with avidin alkaline phosphatase conjugate performs better than laborious immunochemical systems; (3) that use of 3% BSA as blocking agent at 37 degrees C and the presence of 0.5 mol/l NaCl together with 1% BSA during the affinity labelling nearly eliminate background staining; (4) that a dramatic gain in sensitivity is gained by affinity labelling at pH 9.0 instead of 7.5; (5) that biotin-labelling can be highly reproducibly performed on a preparative scale with cheap and easily synthesized bio-11-dUTP in a two step nick-translation and (6) that biotinylated probes and hybridization mixtures can be stored for months and reused. The study has resulted in the presentation of a fast procedure, which is generally applicable to routine DNA diagnostic work, also in parts of the world where it is difficult to get a regular supply of 32P.
To date, seven inborn errors of mitochondrial fatty acid oxidation have been identified. A total of about 100 patients in the world have been reported. Clinically the beta-oxidation defects are more often characterized by episodic hypoglycaemia leading to a coma mimicking Reye's syndrome. The hypoglycaemia is non-ketotic since the synthesis of ketone bodies is deficient. Periods of decompensation occur when carbohydrate supply is poor, e.g. prolonged fasting, vomiting, or increased caloric requirements, as and when lipid stores are used. Defects in beta-oxidation have also been reported to be one cause of sudden infant death syndrome. The diagnosis of these inborn errors is by biochemical investigation since where symptoms suggest such a defect, the precise aetiology cannot be assessed. The biochemical diagnosis is based firstly on identification of abnormal plasma and of urinary metabolites during acute attacks. Derivatives of the omega-oxidation and omega-1-oxidation of medium chain fatty acids have been identified, as well as acylglycine and acylcarnitine conjugates. These metabolites are nearly always absent when patients are in good clinical condition. Secondly, the diagnosis must be based on the identification of the enzymatic defects: this involves global assays which allow a localization of the 'level' of the defect (i.e. the oxidation of long, medium or short chain fatty acids) and specific measurement of enzyme activities (acyl-CoA dehydrogenases and electron carriers: ETF and ETF-DH). The diagnosis of these disorders is of prime importance because of the severity of the clinical symptoms. These can be prevented, in some cases, by an appropriate diet (a high carbohydrate, low fat diet, sometimes supplemented with L-carnitine). In other cases, genetic counselling can be offered.
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The degradation of medium-chained dicarboxylic (DC) acids was investigated on purified mitochondria and peroxisomes. Intact organelles were incubated with dodecanedioic acid (DC12), suberic acid (DC8) and adipic acid (DC6), and the production of lower-chained DC-acids and of acetyl-CoA + acetyl-carnitine was monitored. It was shown, that intact peroxisomes could beta-oxidize DC12, DC10, and DC8 at least as far as DC6, while intact mitochondria readily beta-oxidized DC12, and DC10 as far as succinic acid. DC8 and DC6 were not oxidized by intact mitochondria when these two acids were presented externally to the intact organelle. When they were formed intramitochondrially from DC12 and DC10, both DC8 and DC6 were, however, to a great extent beta-oxidized as far as succinic acid. The major reason for this difference between mitochondrial oxidation of externally and internally located DC8 and DC6 seems to be an inability to transport these two acids through the mitochondrial membrane. For DC12 and DC10, the mitochondrial transport systems, which were indicated to be identical to the systems used by the corresponding monocarboxylic acids, were found to be rate-limiting in the beta-oxidation of these acids. A contributing factor to the undetectable beta-oxidation of externally located DC8 and DC6 may also be, that the Km values of DC8-CoA (460 +/- 70 mumol/l) and DC6-CoA (980 +/- 90 mumol/l) towards the acyl-CoA dehydrogenases are very high. These results imply that very high concentrations of intermediates are created intramitochondrially during beta-oxidation, concentrations which are probably only formed through formation of DC8-CoA and DC6-CoA from longer DC-acids and not by transport from outside the mitochondria. The data presented thus for the first time give evidence to a pathway for medium-chained monocarboxylic acids (especially lauric acid and decanoic acid) through cytosolic omega-oxidation followed by activation, transport over the mitochondrial membrane and beta-oxidation to succinic acid.
Apparent kinetic constants (Km and Vmax values) were determined for human liver acyl-CoA: glycine acyltransferase (glycine-N-acylase) towards isobutyryl-CoA, 2-methyl butyryl-CoA, isovaleryl-CoA, butyryl-CoA, hexanoyl-CoA, octanoyl-CoA, and decanoyl-CoA. These acyl-CoA esters were selected because of their relevance to the human diseases with cellular accumulation of these esters, i.e., especially to metabolic defects in the acyl-CoA dehydrogenation steps of the branched-chain amino acids, lysine, 5-hydroxy lysine, tryptophan, and fatty acid oxidation pathways. With the acyl-CoA ester as the fixed substrate, the Km value for glycine ranged from 0.5 to 2.9 mole/liter, and with glycine as fixed substrate, the Km values for the acyl-CoA esters varied from 0.3 to 5.6 mmole/liter. It is concluded that the substrate concentration is decisive for the glycine conjugate formation and that the occurrence in urine of acylglycines reflects an intramitochondrial accumulation of the corresponding acyl-CoA ester.
Prompted by the fact that the urinary excretion of organic acids in the riboflavin-deficient rat closely mimics that found in patients with inborn errors in the acyl-CoA dehydrogenation systems, the organelle localization and the apparent kinetic constants (Km and Vmax values) for the rat liver acyl-CoA:glycine-N-acyltransferase (glycine-N-acylase) toward isobutyryl-CoA, 2-methylbutyryl-CoA, isovaleryl-CoA, butyryl-CoA, hexanoyl-CoA, octanoyl-CoA, decanoyl-CoA, and benzoyl-CoA were determined. The studies on organelle localization demonstrated that the glycine-N-acylase is exclusively an intramitochondrial enzyme, and that no activity is present in peroxisomes, which also possess ability to produce Acyl-CoAs. The kinetic studies were done in order to elucidate whether the quantitative differences in excretion profile of acylglycines between riboflavin-deficient rats and patients with beta-oxidation defects are caused by differences in ability to conjugate the various acyl-CoAs. It was found that the Km values for the rat liver enzyme were generally somewhat lower than the values found in man, but with the same chain length profile. Consequently, the above-mentioned differences in excretion profile of acylglycines between riboflavin-deficient rats and patients with beta-oxidation defects cannot be explained by differences in affinity toward the glycine-N-acylase.
A boy with riboflavin-responsive beta-oxidation deficiency (multiple acyl-CoA dehydrogenation deficiency) was assessed clinically and biochemically after 3 years of continuous riboflavin medication. He was diagnosed at the age of three years after an attack of a Reye's syndrome-like disease. During the 3 years of assessment he has experienced no serious disease; although short episodes of fatigue and loss of appetite have been noted. His mental and physical development has been normal. Biochemically the abnormal excretion of organic acid metabolites, characteristic of the acyl-CoA dehydrogenation deficiency, has been continuously present. Quantitatively there has been a trend to a more simple picture with ethylmalonic acid as the predominant metabolite. However, because of the large within-day variation in the excretion of all the metabolites, changes following diet and riboflavin trials must be interpreted with caution in these patients.
The key reaction in the beta-oxidation of fatty acids is the acyl-CoA dehydrogenation, catalyzed by short chain, medium chain, and long chain acyl-CoA dehydrogenases. Acyl-CoA dehydrogenation reactions are also involved in the metabolism of the branched chain amino acids, where isovaleryl-CoA and 2-methylbutyryl-CoA dehydrogenases are involved and in the metabolism of lysine, 5-hydroxylysine and tryptophan, where glutaryl-CoA dehydrogenase functions. In all of these dehydrogenation systems reducing equivalents are transported to the main respiratory chain by electron transfer flavoprotein (ETF) and electron transfer flavoprotein dehydrogenase (ETFDH), which are common to all the dehydrogenation systems. The acyl-CoA dehydrogenation enzymes are dependent on flavin adenine dinucleotide (FAD) as coenzyme, for which riboflavin is the precursor. Patients with multiple acyl-CoA dehydrogenation deficiencies have been found in whom the defect has been located to ETF and/or ETFDH. A few patients with multiple acyl-CoA dehydrogenation deficiencies have been described, in whom no defects in acyl-CoA dehydrogenases, ETF or ETFDH have been found but who respond clinically and biochemically to pharmacological doses of riboflavin. This indicates a defect related to the metabolism of FAD. An uptake defect of riboflavin or a synthesis defect of FAD from riboflavin have been excluded by in vivo and in vitro studies. A mitochondrial transport defect of FAD or a defect in the binding FAD to ETF and/or ETFDH remains possible.