C. A. van Bennekom

New Polymorphic DNA Marker Close to the Fragile Site FRAXA

DNA from a human-hamster hybrid cell line, 908-K1B17, containing a small terminal portion of the long arm of the human X chromosome as well as the pericentric region of 19q was used as starting material for the isolation of an X-chromosome-specific DNA segment, RN1 (DXS369), which identifies a XmnI RFLP. Linkage analysis in fragile X families resulted in a maximum lod score of 15.3 at a recombination fraction of 0.05 between RN1 and fra(X). Analysis of recombinations around the fra(X) and distal to DXS105. Analysis of the marker content of hybrid cell line 908K1B17 suggests the localization of RN1 between DXS98 and fra(X). Heterozygosity of DXS369 is approximately 50%, which extends the diagnostic potential of RFLP analysis in fragile X families significantly.

In Vivo Induced Malignant Hyperthermia in Pigs. I. Physiological and Biochemical Changes and the Influence of Dantrolene Sodium

The effects of an induced malignant hyperthermia (MH) crisis have been studied in the intact pig. Both physiological and biochemical changes in skeletal muscle were studied. MH was induced with 3% halothane plus a bolus injection of succinylcholine. In the prechallenge period a significant difference was observed in the concentration of certain muscle metabolites, comparing the MH‐susceptible (MH+) with the non‐susceptible (MH‐) pigs. A lower level was measured for phosphocreatine (PCr), inosine monophosphate (IMP) and an increased level of lactate and creatine (Cr) in the susceptible pigs (MH+). The challenge caused a significant reduction of the level of PCr and adenosine in MH+ pigs, compared to the prechallenge period. After administration of dantrolene sodium, a significant decrease was measured in the level of lactate, compared to the prechallenge period as well as during the challenge. In contrast, in the control pigs no significant changes were observed in muscle metabolites, either after induction of MH or after the administration of dantrolene sodium. Enzyme activity determinations of muscle adenylate kinase and adenosine monophosphate (AMP)‐ deaminase did not show any difference in activity either before or during the MH crisis or after treatment with dantrolene sodium. The earliest physiological change during an induced MH crisis in our study was thrapid increase of the end‐tidal CO2. Within 5 min after MH induction, end‐tidal CO2 was doubled. It is concluded that the monitoring of the end‐tidal CO2 is essential to diagnose MH at a very early stage.

Heterozygote detection in Fabry's disease using mailed hair roots

Hair roots can successfully be employed as an easily obtainable biopsy material for heterozygote detection in inborn errors of metabolism (Gartler et al., 1971; de Bruyn, Oei & ter Haar, 1974; De Bruyne; ah, 1979). We have recently described an easy and rapid method for measuring a-galactosidase and acid phosphatase activities in individual human hair roots (Vermorken et al., 1978). It was shown that acid phosphatase is a suitable reference enzyme with respect to a-galactosidase since it has a comparable distribution in the hair follicle and it is equally stable on storage. In this study we report that mailing hair roots does not interfere with the possibility of measuring the enzyme activities and distinguishing between patients, heterozygotes and normal controls. Hair roots are plucked, preferably from the scalp, using a pincct such as nr. 805 or nr. 970 Medicon Instruments (Tuttlingen, F.G.R.). Twenty-five follicles, from each individual, with visible bulb and sheath (Fig. iA) are attached to

Metabolite profiling of human muscle extracts by isotachophoresis

Isotachophoresis provides an effective method of examining purines and pyrimidines in muscle extracts. Its application to dystrophic muscle of the Duchenne type is illustrated.

Purine Metabolism in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is the most widely known of the muscular dystrophies. It is inherited as an X-linked recessive trait (1). It becomes manifest only in males and it is transmitted by asymptomatic females, although there are exceptions (2). The primary manifestation is progressive muscle weakness, but there are more organs that are progressively affected, e.g. heart and brain. The primary gene defect is unknown but there is increasing evidence to suggest an abnormal sarcolemmal membrane (1). Leakage of ATP from muscles as found in the case of DMD patients (3) might lead to some defect in muscle purine metabolism. It was speculated that irreversible loss of purines, caused by the breakdown of ATP by enzymes in the blood, might be reduced by blocking the last enzymatic step in purine catabolism in man and by concommitant enhancement of purine reutilisation. This last step involves the xanthine oxidase reaction and allopurinol is widely used as an inhibitor of this reaction in the treatment of hyperuricemia. DMD patients receiving allopurinol were reported to improve clinically (4,5,6).

Glucose-6-phosphate Dehydrogenase Deficiency: Biochemical and Histochemical Studies on Hair Roots for Carrier Detection

Kinetic properties of human hair root glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were studied in order to optimize the assay of these enzymes in lysates from single hair roots. In contrast to previously reported methods, an excess of purified 6-phosphogluconate dehydrogenase was added to the glucose-6-phosphate dehydrogenase reaction mixtures, thus allowing a more exact quantification of glucose-6-phosphate dehydrogenase activity. Although enzyme histochemical techniques suggest a similar distribution of hair root glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, enzyme assays on hair root segments after microdissection nevertheless indicate differences in the distribution of these enzymes. Upon storage a gradual drop in the activity of both hair root enzymes was found, but the rate of decrease in enzyme activity was about equal: the enzyme activity ratio was, therefore, not affected. This opens interesting possibilities for mailing hair roots for screening purposes without any special precautions.

A Simple and Sensitive Radiochemical Assay for Plasma Guanase

A simple and sensitive radiochemical micro-assay has been developed for the determination of plasma guanase activity. The method is based upon the measurement of the conversion of 14C-labeled guanine to xanthine, catalysed by the enzyme guanase (guanine aminohydrolase;EC 3.5.4.3). Using this method, the catalytic activity in the plasma of adult healthy controls was 0.040 +/- 0.09 nmol/h.mg protein (x +/- s). In children under 5 years of age higher levels of enzyme activity were demonstrated. In adult patients with liver disease plasma guanase activities were found to be 3 to 7-fold increased as compared to the normal adult mean value.

Heterozygote detection in glucose‐6‐phosphate dehydrogenase deficiency: limitation of hair follicle analysis

Glucose‐6‐phosphate dehydrogenase deficiency was demonstrated in a case of favism. The X‐linked enzyme defect was expressed in erythrocytes but not in hair root cells. Predictably, the mother shown to be a heterozygous carrier on the basis of intermediate erythrocyte glucose‐6‐phosphate dehydrogenase activity could not be identified as a carrier by means of hair root study. It seems to be necessary to test the hair roots of at least one enzyme‐deficient member of the family to exclude false negative results, if hair root analysis is used for carrier detection. Because of the more or less clonal origin of hair roots, they remain a convenient biopsy material with which to study heterozygosity in X‐linked inborn errors of metabolism.

Malignant Hyperthermia: Adenine Incorporation and Adenine Metabolism in Human Platelets, Influenced by Halothane

Malignant hyperthermia (MH) is a pharmacogenetic disorder, elicited during general anaesthesia and carries a high mortality (1). MH is elicited in susceptible subjects by volatile anaesthetics like halothane, ether, enflurane and depolarizing muscle relaxants like succinylcholine(1). The inheritance of MH is autosomal dominant with incomplete penetrance and variable expression(1).

Enzymes of Purine Metabolism in Muscle Specimens from Patients with Duchenne-Type Muscular Dystrophy

Duchenne-type Muscular Dystrophy (DMD), the most well-known of the muscular dystrophies, is inherited as an X-linked recessive trait (1). This implicates that it becomes manifest mainly in males and it is transmitted by asymptomatic female heterozygotes (2). The primary manifestation is progressive muscle weakness, but there are more organs that are progressively affected; e.g. heart and brain. The primary gene defect is unknown, but the evidence available at present suggests a defect of the sarcolemnal membrane (1).