Optimization of performance of Dutch newborn screening for cystic fibrosis.

BACKGROUND: Dutch newborn screening (NBS) for Cystic Fibrosis (CF) introduced in 2011 showed a sensitivity of 90% and a positive predictive value (PPV) of 63%. We describe a study including an optimization phase and evaluation of the modified protocol. METHODS: Dutch protocol consists of four steps: determination of immunoreactive trypsinogen (IRT) and pancreatitis-associated protein (PAP), DNA analysis by INNO-LiPA and extended gene analysis (EGA). For the optimization phase we used results of 556,952 newborns screened between April 2011 and June 2014 to calculate effects of 13 alternative protocols on sensitivity, specificity, PPV, ratios of CF to other diagnoses, and costs. One alternative protocol was selected based on calculated sensitivity, PPV and costs and was implemented on 1st July 2016. In this modified protocol DNA analysis is performed in samples with a combination of IRT ≥60 µg/l and PAP ≥3.0 µg/l, IRT ≥100 µg/l and PAP ≥1.2 µg/l or IRT ≥124 µg/l and PAP not relevant. Results of 599,137 newborns screened between 1st July 2016 and 31st December 2019 were similarly evaluated as in the optimization phase. RESULTS: The modified protocol showed a sensitivity of 95%, PPV of 76%, CF to CF transmembrane conductance regulator-related metabolic syndrome/CF screen positive, inconclusive diagnoses (CRMS/CFSPID) ratio 12/1, CF/CF carrier ratio 4/1. Costs per screened newborn were slightly higher. Eleven children, of whom five with classic CF, would not have been referred with the previous protocol. CONCLUSIONS: The modified protocol results in acceptable sensitivity (95%) and good PPV of 76% with minimal increase in costs.

[Successful treatment of clinical manifestations of Muckle-Wells syndrome with anakinra]. / Erfolgreiche Behandlung der klinischen Manifestationen des Muckle-Wells-Syndroms mit Anakinra.

HISTORY: A 58-year-old woman and her 18-year-old daughter were first seen in our rheumatology centre after having experienced many years of periodic fever, arthralgia and urticaria. Some months ago a diagnosis of Muckle-Wells syndrome (MWS) had been made and confirmed by genetic testing. The mother had developed partial deafness and substantial loss of vision. Her daughter had been suffering mainly from urticaria and fever at least once daily. THERAPY: Because of the established role of interleukin (IL)-1 in this hereditary disease and some positive case reports, we decided to treat these patients with the IL-1 receptor antagonist anakinra. The patients reported a great success of this treatment with virtually complete absence of any acute MWS-associated symptoms. CONCLUSION: Therapy of MWS with anakinra seems to be highly efficacious for several clinical manifestations of this disease, including laboratory markers for inflammation. It is possible that organ destruction may be prevented by this medication.

Peripheral blood lymphocyte appearance in a case of I cell disease.

In general, peripheral blood smears are performed to obtain information with regard to various morphological features as an aid in the diagnosis of infection or malignancy. This report presents a patient with I cell disease (inclusion cell disease), a fatal lysosomal storage disorder caused by a defect in an enzyme responsible for the transfer of mannose-6-phosphate ligands to precursor lysosomal enzymes. As a consequence, most lysosomal enzymes are transported outside the cell instead of being correctly targeted into the lysosomes, resulting in the storage of macromolecules in lysosomes. I cell disease, with its heterogeneous clinical presentation, can be diagnosed by the presence of intracellular vacuole-like inclusions in lymphocytes and fibroblasts, high serum lysosomal enzyme activities, and a defect of N-acetylglucosamine-1-phosphotransferase. This report describes the morphological aspects of peripheral lymphocytes in a blood smear of a patient, the first clue to the final diagnosis of I cell disease. The observed vacuole-like inclusions in lymphocytes of this patient were negative for periodic acid Schiff (PAS) and Sudan black B staining, in contrast to earlier reports.

Membrane decoration by amphiphilic block copolymers in bicontinuous microemulsions.

The effect of various amphiphilic block copolymers of different molar masses on the structure and phase behavior of ternary amphiphilic systems (water, oil, and nonionic surfactant) is investigated. Small amounts of PEP-PEO block copolymer lead to a dramatic increase in the volumes of oil and water, which can be solubilized in a bicontinuous microemulsion. High-precision neutron scattering experiments with a sophisticated contrast variation technique demonstrate that the polymers form uniformly distributed mushroom conformations on the surfactant membrane. Based on these observations, we propose a universal mechanism for the swelling behavior, which is due to the variation of the membrane curvature elasticity.

Chromosomal integration of tcb chlorocatechol degradation pathway genes as a means of expanding the growth substrate range of bacteria to include haloaromatics.

The tcbR-tcbCDEF gene cluster, coding for the chlorocatechol ortho-cleavage pathway in Pseudomonas sp. strain P51, has been cloned into a Tn5-based minitransposon. The minitransposon carrying the tcb gene cluster and a kanamycin resistance gene was transferred to Pseudomonas putida KT2442, and chromosomal integration was monitored by selection either for growth on 3-chlorobenzoate or for kanamycin resistance. Transconjugants able to utilize 3-chlorobenzoate as a sole carbon source were obtained, although at a >100-fold lower frequency than kanamycin-resistant transconjugants. The vast majority of kanamycin-resistant transconjugants were not capable of growth on 3-chlorobenzoate. Southern blot analysis revealed that many transconjugants selected directly on 3-chlorobenzoate contained multiple chromosomal copies of the tcb gene cluster, whereas those selected for kanamycin resistance possessed a single copy. Subsequent selection of kanamycin resistance-selected single-copy transconjugants for growth on 3-chlorobenzoate yielded colonies capable of utilizing this carbon source, but no amplification of the tcb gene cluster was apparent. Introduction of two copies of the tcb gene cluster without prior 3-chlorobenzoate selection resulted in transconjugants able to grow on this carbon source. Expression of the tcb chlorocatechol catabolic operon in P. putida thus represents a useful model system for analysis of the relationship among gene dosage, enzyme expression level, and growth on chloroaromatic substrates.

New method for faecal fat determination by mid-infrared spectroscopy, using a transmission cell: an improvement in standardization.

Current techniques used in clinical laboratories for faecal fat determination, such as the Van de Kamer method, are not very accurate or precise. This became apparent when results obtained by different laboratories were compared, and could explain the disappointing performance of near-infrared and mid-infrared spectroscopy since the accuracy of these techniques depends upon the accuracy of the calibration used (i.e. inaccurate wet chemical analysis). In order to improve standardization, we developed and tested a new quantitative method in three laboratories, based on Fourier transform infrared (FT-IR) spectroscopy. Fatty acids were extracted from faecal samples with acidified petroleum ether-ethanol and the extracts were dried and dissolved in chloroform. An infrared spectrum of the extracts was recorded in the range 4000-650 cm(-1), using an infrared transmission cell. Standard mixtures of stearic and palmitic acids (65:35) were used for calibration. Quantification was based on the absorbance band of the CH2 group (2855 cm(-1)) of free fatty acids and fatty acid glycerol esters. The calibration curve showed excellent linearity. The correlation coefficient between the titrimetric Van de Kamer and FT-IR methods was 0.96 (y = 1.12x-0.02, standard error of prediction = 0.89 g% fat). No significant difference was found when the FT-IR results of 28 faecal samples from patients were compared between two different university hospital laboratories. The new FT-IR method, using primary standards, is simple and rapid, and provides satisfactory intra- and inter-laboratory precision for the diagnosis and monitoring of steatorrhoea.

Histology of magnesium-deficient Norway spruce needles influenced by nitrogen source.

Effects of magnesium deficiency and variation in nitrate to ammonium ratio on needle histology and chlorophyll concentration were investigated in current-year and one-year-old needles of clonal Norway spruce trees (Picea abies (L.) Karst.). Six-year-old trees were grown for one year in sand culture with circulating nutrient solutions containing a sufficient (0.2 mM) or a limiting (0.04 mM) concentration of Mg. The nitrogen concentration was not varied (5 mM), but the NO(3) (-)/NH(4) (+)-ratio was adjusted to 0.76 in Mg-sufficient and to 1.86, 0.76 or 0.035 in Mg-limited plants. Visible symptoms of Mg deficiency occurred only in current-year needles, indicating adequate Mg nutrition before the experiment. Under conditions of Mg limitation, chlorophyll and Mg concentrations were lowest in needles of trees supplied with NH(4) (+) as the major nitrogen source and highest in needles of trees supplied with NO(3) (-) as the major nitrogen source. In current-year and one-year-old needles, starch accumulation induced by Mg deficiency was increased when NH(4) (+) was the major nitrogen source. The accumulation of tannin spherules in current-year needles, which occurred in response to Mg deficiency, also increased with decreasing NO(3) (-)/NH(4) (+)-ratios. Deficient Mg supply caused premature aging in tissues of the vascular bundle, as indicated by modifications of the cambium and increased amounts of collapsed sieve cells. The number of collapsed sieve cells was slightly lower in needles grown in a NH(4) (+)-dominated nutrient regime than in needles grown in a NO(3) (-)-dominated nutrient regime. We conclude that was not directly toxic to Norway spruce trees at the applied concentrations. However, effects of Mg deficiency were considerably greater in an NH(4) (+)-dominated nutrient regime than in a NO(3) (-)-dominated nutrient regime.

Rapid and reliable measurement of highly elevated blood ammonia concentrations in children.

Newborns and children may suffer from extremely high ammonia levels in their blood. We evaluated a fast, reliable micromethod, based on the Blood Ammonia Checker II (BAC II) in combination with the dilution with fresh whole blood. Comparison of the proposed method with an enzymatic method revealed a statistically significant correlation. We conclude that the dilution of patient's blood with fresh whole blood extends the measuring range of ammonia on the BAC II analyzer from 286 mumol/l to about 700 mumol/l.

Cotranscription of a GTPase gene from the cyanobacterium Synechocystis PCC 6803 and a P-type Ca(2+)-ATPase gene.

A GTPase gene adjacent to the Ca(2+)-ATPase gene from Synechocystis PCC 6803 has been sequenced. It encodes for a protein of 456 amino acids revealing high homology to so-called 50K proteins of Bacillus subtilis and Pseudomonas putida. Cotranscription of GTPase and Ca(2+)-ATPase genes has been shown by reverse transcription PCR.

Metabolic aspects of peroxisomal disorders.

In recent years an increasing number of inherited diseases in man have been identified in which there is an impairment in one or more peroxisomal functions. This paper discusses the current state of knowledge on these disorders with particular emphasis on the metabolic abnormalities in these diseases.

The influence of magnesium deficiency on carbohydrate concentrations in Norway spruce (Picea abies) needles.

Both short- and long-term effects of Mg deficiency on carbohydrate metabolism were investigated in 6-year-old clonal Norway spruce (Picea abies (L.) Karst.) trees cultivated in sand culture with an optimal supply of nutrients, except for Mg which was supplied at 0.203, 0.041 and 0.005 mM to provide optimal, moderately deficient and severely deficient Mg supply, respectively. Annual changes in carbohydrate concentrations (starch, sucrose, glucose and fructose) were analyzed and diurnal changes were investigated on a single day during the summer. Older needles of trees in the moderate Mg-deficiency treatment developed tip-yellowing symptoms, whereas current-year needles remained green. The severe Mg-deficiency treatment led to pronounced yellowing symptoms in needles of all ages. Increased carbohydrate concentrations were observed before needle yellowing occurred. Diurnal and annual changes in carbohydrates were similar in all treatments; however, carbohydrate concentrations were influenced by Mg supply. In both Mg-deficiency treatments, starch concentrations increased in needles, especially during summer and autumn. Starch accumulation was more pronounced at the beginning of the Mg-deficiency treatments than at the end of the treatments. Sucrose, and to a minor extent, glucose and fructose concentrations tended to increase in response to Mg deficiency. The consequences of Mg deficiency on carbohydrate metabolism are discussed with respect to reduced plant growth and decreased transport rates of carbohydrates to sink organs.

Fatty acid beta-oxidation in peroxisomes and mitochondria: the first, unequivocal evidence for the involvement of carnitine in shuttling propionyl-CoA from peroxisomes to mitochondria.

We have investigated how [1-14C]propionyl-CoA, which is the first product of the peroxisomal beta-oxidation of [1-14C] pristanic acid, is transported to mitochondria for further oxidation in human skin fibroblasts from patients with a defect in the mitochondrial carnitine/acylcarnitine translocase and carnitine-palmitoyltransferase II (CPT II) (EC 2.3.1.21), respectively. Oxidation of pristanic acid was found to be partially deficient in both types of mutant cells. More important, 14CO2 production was completely deficient in the carnitine/acylcarnitine translocase deficient cells but not in the carnitine-palmitoyltransferase II deficient cells. These results strongly suggest that formation of 14CO2 in the Krebs cycle from [1-14C]propionyl-CoA as generated in peroxisomes requires the active participation of the mitochondrial carnitine/acylcarnitine translocase. The results described in this paper provide the first evidence suggesting that propionyl-CoA leaves the peroxisome as a carnitine ester and strongly suggest that the commonly accepted concept that peroxisomal beta-oxidation is not dependent on carnitine is incorrect.

Beta-oxidation of fatty acids in cultured human skin fibroblasts devoid of the capacity for oxidative phosphorylation.

Prolonged treatment of cultured cells with ethidium bromide results in loss of the capacity for oxidative phosphorylation. Because of the tight coupling between mitochondrial beta-oxidation of fatty acids and the activity of the respiratory chain, such cells may be used to study the contribution of mitochondria and peroxisomes to fatty acid beta-oxidation. To investigate this, human skin fibroblasts were cultured in the presence of ethidium bromide for at least 10 cell generations, resulting in a virtually complete absence of oxidative phosphorylation as demonstrated directly in digitonin-permeabilized fibroblasts. The cells showed a lowered ATP/ADP ratio, most likely as the consequence of the inability to generate ATP via oxidative phosphorylation. The loss of the capacity for oxidative phosphorylation was also reflected in an increased cytosolic NADH/NAD+ ratio: the cells showed a highly elevated lactate/pyruvate ratio in the suspending medium when incubated with glucose. The beta-oxidation of octanoic and palmitic acid was dramatically decreased, suggesting that the beta-oxidation of these fatty acids takes place predominantly (> 90%) in mitochondria, at least in the cells studied. In contrast, the rates of pristanic and cerotic acid beta-oxidation were only slightly decreased, suggesting that this is mainly a peroxisomal process. The reduction of beta-oxidation of cerotic and pristanic acid, 27% and 15%, respectively, is most likely due to a lowered ATP level and an increased NADH/NAD(+)-redoxstate in these cells. We conclude that fibroblasts subjected to prolonged treatment with ethidium bromide can be used as a model system to study the substrate specificity and functional characteristics of the peroxisomal beta-oxidation system.

X-linked adrenoleukodystrophy: biochemical diagnosis and enzyme defect.

The adrenoleukodystrophies refer to three genetically distinct disorders all characterized by the accumulation of very long-chain fatty acids. In this paper we will review the biochemical aspects of these leukodystrophies with particular emphasis on the methods used to measure very long-chain fatty acid levels in plasma and their reliability. Furthermore, we will concentrate on the primary defect in the X-linked form of adrenoleukodystrophy.

Conclusive evidence that very-long-chain fatty acids are oxidized exclusively in peroxisomes in human skin fibroblasts.

We have investigated the contribution of peroxisomes and mitochondria to the beta-oxidation of palmitate (C16:0) and cerotate (C26:0) in intact human skin fibroblasts. The oxidation of both fatty acids was found to be inhibited by rotenone plus antimycin and cyanide, respectively, although to a different extent. When 2-[5-(4-chlorophenyl)pentyl]-oxirane-2-carboxylate (POCA) was used to specifically block carnitine palmitoyltransferase I, it was found that palmitate beta-oxidation was inhibited almost completely whereas cerotate beta-oxidation was not affected. Since carnitine palmitoyltransferase is essential for the oxidation of fatty acids in mitochondria this result provides conclusive evidence that oxidation of very-long-chain fatty acids is initiated in peroxisomes and not in mitochondria.