Core Indicators of Children’s Health and Well-Being at the Municipal Level in Greenland

The aim of this study was to expose the background for proposing core indicators for children’s health and well-being at the municipal level in Greenland; to define criteria for relevant and scientifically sound indicators; to propose a set of such indicators; to give data on their distribution, and compare the outcomes between different types of municipalities. The indicators were drawn from actual knowledge of children’s health and experiences reported in international studies. They were based on goals from the National Public Health strategy and the UN Convention on the Rights of the Child. A set of 24 indicators were identified in 4 domains (demographic and socio-economic conditions; health status and well-being; determinants of health, risk, and protective factors; and health systems and health policy). Additional indicators were proposed for later implementation, most of these lack available data. Data revealed large differences between municipalities. In general, remote communities have the most unfavourable socio-demographic and health conditions. At the same time, larger communities’ access to health care services is more favourable. A consequence is that the health care system unintentionally contributes to the increasing health gap between privileged and less privileged children. A comprehensive strategy is need to guarantee equitable health standards for all Greenlandic children and that reaches far beyond the aims stated in the present national public health programme.     

Pneumoproteins as markers of paraquat lung injury: a clinical case

OBJECTIVE: To describe the changes in lung-specific secretory proteins in biological fluids in a fatal case of paraquat ingestion and to present immunostaining data obtained on postmortem lung tissue specimens. METHODS: A 20-year-old man committed suicide by ingesting 100ml of a 20% paraquat solution. Surfactant associated proteins A (SP-A), B (SP-B) and Clara cell 16kDa protein (CC16) were determined in the serum and on broncho-alveloar lavage performed 18h after admission. Renal failure progressed rapidly and the patient died from refractory hypoxia. Immunostaining studies using antibodies directed against CC16, SP-A and SP-B were performed on postmortem lung tissue specimens. RESULTS: Serum CC16 seemed to increase gradually with the progression of renal impairment. Serum SP-A and SP-B levels increased before any significant changes in pulmonary gas exchanges. The immunostaining study showed that the labeling for SP-A and SP-B was reduced or absent following paraquat toxicity, while Clara cells were relatively preserved. CONCLUSIONS: The elevation of serum CC16 with paraquat toxicity is probably mainly related to a reduced renal clearance. The increase of serum SP-A and SP-B could reflect an increased lung to blood leakage, independently of the alteration of the renal function.     

Reference values for N-terminal pro-B-type natriuretic peptide in fetal circulation between 20 and 34 weeks of gestation

ObjectiveWe aimed to investigate the range of fetal NT-proBNP values in normal pregnancy between 20 and 34 weeks of gestation.MethodNT-proBNP was measured in 56 fetal blood samples.ResultsMean (± 2 SD) NT-proBNP concentration was 1998 (242–3754) ng/L; a significant decline occurred with advancing gestational age (p = 0.012).ConclusionsGestational age has to be taken in to consideration in the assessment of NT-proBNP. Our data may be used as reference values in fetal and neonatal medicine.     

Evaluation of Second-Generation Sequencing of 19 Dilated Cardiomyopathy Genes for Clinical Applications

Medical sequencing for diseases with locus and allelic heterogeneities has been limited by the high cost and low throughput of traditional sequencing technologies. “Second-generation” sequencing (SGS) technologies allow the parallel processing of a large number of genes and, therefore, offer great promise for medical sequencing; however, their use in clinical laboratories is still in its infancy. Our laboratory offers clinical resequencing for dilated cardiomyopathy (DCM) using an array-based platform that interrogates 19 of more than 30 genes known to cause DCM. We explored both the feasibility and cost effectiveness of using PCR amplification followed by SGS technology for sequencing these 19 genes in a set of five samples enriched for known sequence alterations (109 unique substitutions and 27 insertions and deletions). While the analytical sensitivity for substitutions was comparable to that of the DCM array (98%), SGS technology performed better than the DCM array for insertions and deletions (90.6% versus 58%). Overall, SGS performed substantially better than did the current array-based testing platform; however, the operational cost and projected turnaround time do not meet our current standards. Therefore, efficient capture methods and/or sample pooling strategies that shorten the turnaround time and decrease reagent and labor costs are needed before implementing this platform into routine clinical applications.Genetic testing for disorders with locus and allelic heterogeneity has been a challenge due to the high cost of sequencing entire coding regions of numerous genes. Classically, medical sequencing has used capillary-based “Sanger” sequencing technology and this has remained the gold standard for three decades. However, this method is expensive and has low throughput. It was not until novel technology platforms emerged that comprehensive testing became within reach. One such technology is array-based sequencing, which drastically increased the number of genes that could be analyzed simultaneously.^{1,2,3,4,5,6,7} We previously developed an array-based resequencing test for dilated cardiomyopathy (DCM), that doubled the number of genes analyzed in parallel while reducing test cost and turnaround time.⁶ However, this technology has two major drawbacks, particularly in a clinical setting. First, resequencing arrays have a poor detection rate for insertions and deletions (in/dels).^8,9 Second, the somewhat static nature of the chip design makes it time-consuming and impractical to add new content, especially in a disease area like DCM where genes are being discovered at a rapid pace. As such, novel technologies are needed to provide comprehensive sequencing of all DCM genes with high analytical sensitivity and with the goal of further reducing the cost of diagnostic testing.Second-generation sequencing (SGS) technologies, commonly referred to as “next-generation sequencing,” are based on massive parallel sequencing of millions of DNA templates through cycles of enzymatic treatment and image-based data acquisition. Several platforms have been developed in the last few years based on different biochemistries and cluster generation.^10,11,12 The most commonly used platforms include the Illumina Genome Analyzer (GAII; Solexa Technology),¹³ Roche Applied Sciences (454 sequencing),¹⁴ and ABI-Applied Biosystems (SOLiD platform).¹⁵ These technologies have been adopted for a wide variety of research applications^10,11,16 and have now matured sufficiently to be considered as robust enough for clinical applications. For example, SGS has recently been applied to resequencing the NF1 locus as well as the mitochondrial and small-cell lung cancer genomes.^17,18,19 In addition, whole exome resequencing has been conducted to discover genes underlying rare monogenic diseases.^20,21,22,23The specific advantages offered by SGS are twofold: the low cost per base and the ability to sequence millions of reads in parallel, allowing for simultaneous analysis of a large number of genes. However, these are offset by two major disadvantages: shorter reads and reduced accuracy as compared to Sanger sequencing.¹¹ Despite their disadvantages, improvements in these technologies promise to meet the technical requirements and strict quality standards of clinical diagnostics including analytical sensitivity, reproducibility and cost effectiveness.Several methodological approaches to capturing target gene regions have evolved to complement the higher capacity and throughput of novel sequencing technologies. These methods involve constructing and enriching a DNA “library” and use both PCR and/or hybridization as the mode of target selection. The classic PCR approach to library generation requires amplification of target regions, pooling, concatenation, shearing and ligation of adaptors and sequencing primers. Secondary droplet-based microfluidic technologies have evolved to facilitate high-throughput PCR in picoliter droplets.²⁴ With hybridization-based methods, libraries are constructed by shearing total gDNA followed by adaptor ligation and hybridization to oligonucleotides that are complementary to the desired target. Hybridization can be performed either on a solid surface array, on a filter, or by hybridization in solution^21,25,26,27 A third general approach to target selection uses molecular inversion probes. Molecular inversion probes consist of two primers linked together by a backbone and, similar to PCR, bind to specific target DNA. This is followed by gap filling, ligation, and enrichment steps.^28,29Important considerations in choosing a method include total amount of starting DNA, the size of the target region and the types of sequence alteration under investigation. Technical parameters such as target specificity, uniformity, and completeness of coverage also vary with each methodology.¹² Although each method has advantages or disadvantages depending on the application, we selected PCR, a well-established and robust targeted amplification method for this study.Leveraging on our array-based resequencing test for DCM,⁶ we evaluated the suitability of targeted PCR followed by Illumina GAII resequencing for a clinical testing environment. We used a number of samples enriched for a large number of substitutions as well as insertions and deletions in DCM genes to assess the analytical performance parameters. We also evaluated the cost and turnaround time of such a test and compared it to our current, array-based resequencing test.     

Patient Position Reproducibility in Fractionated Stereotactically Guided Conformal Radiotherapy Using the BrainLab? Mask System

Purpose: Dedicated mask systems nowadays allow the use of stereotactic radiotherapy in fractionated regimes, therefore combining the advantages of high precision radiotherapy with the biological benefit of fractionation. Therefore the knowledge of institution specific isocenter accuracy is essential for decision-making about margins to be allowed to form the planning target volume. Patients and Methods: Measurements of isocenter deviations during fractionated treatments were performed in 33 patients using the simulator Simulix-xy (Oldelft) in connection with the BrainLab^® angiographic localizer-box as well as port-films. In both cases repeated images were overlaid by use of anatomical landmarks with a methodical accuracy in the order of 0.5 mm. Results: Both methods yield random isocenter deviations of less then 2 mm (standard deviation) in all three directions and no significant systematic deviations. These values are in the order of the accuracy of the method, obtained by comparison of two independent investigators, as well as they are comparable with the literature. Conclusions: The accuracy of less than 2 mm indicates safety margins of 3-4 mm as sufficient for clinical routine to cover the target in 95.5% of all set-ups (2 SD). Ziel: Spezielle Maskensysteme erlauben heutzutage die Anwendung der stereotaktischen Strahlentherapie in fraktionierten Regimes und damit die Kombination der Vorteile der Hochpräzisionsbestrahlung mit dem biologischen Nutzen der Fraktionierung. Deshalb ist die Kenntnis der institutsspezifischen Genauigkeit der Isozentrumseinstellung eine notwendige Voraussetzung für die Entscheidung über die erforderlichen Sicherheitsabstände. Patienten und Methode: Die Messung der Isozentrumsgenauigkeit erfolgte bei 33 Patienten sowohl durch Simulatorkontrollen in Verbindung mit der BrainLab^®-Localizer-Box oder durch Verifikation mit Portfilmen. In beiden Fällen wurden die Filme anhand anatomischer Lankdmarken mit einer methodischen Sicherheit von unter 0,5 mm überlagert. Ergebnisse: Beide Methoden zeigten zufällige Isozentrumsabweichungen von weniger als 2 mm (Standardabweichung) in allen drei Raumebenen (Abbildung 1, Tabelle 2) und keine signifikanten systematischen Abweichungen. Damit liegen die Ergebnisse im Bereich der methodischen Genauigkeit, wie durch den Vergleich der Befunde zweier unabhängiger Untersucher gezeigt wird (Tabelle 1), und sind mit Literaturdaten gut vergleichbar. Schlussfolgerung: Die Genauigkeit von unter 2 mm zeigt, dass ein Sicherheitsabstand von 3-4 mm für die klinische Routine ausreichend ist, um bei 95,5% der Einstellung die sichere Erfassung des Targets zu garantieren.     

Patient Position Reproducibility in Fractionated Stereotactically Guided Conformal Radiotherapy Using the BrainLab® Mask System

PURPOSE: Dedicated mask systems nowadays allow the use of stereotactic radiotherapy in fractionated regimes, therefore combining the advantages of high precision radiotherapy with the biological benefit of fractionation. Therefore the knowledge of institution specific isocenter accuracy is essential for decision-making about margins to be allowed to form the planning target volume. PATIENTS AND METHOD: Measurements of isocenter deviations during fractionated treatments were performed in 33 patients using the simulator Simulix-xy (Oldelft) in connection with the BrainLab angiographic localizer-box as well as port-films. In both cases repeated images were overlaid by use of anatomical landmarks with a methodical accuracy in the order of 0.5 mm. RESULTS: Both methods yield random isocenter deviations of less then 2 mm (standard deviation) in all three directions and no significant systematic deviations. These values are in the order of the accuracy of the method, obtained by comparison of two independent investigators, as well as they are comparable with the literature. CONCLUSIONS: The accuracy of less than 2 mm indicates safety margins of 3-4 mm as sufficient for clinical routine to cover the target in 95.5% of all set-ups (2 SD).     

3-Ureidopropionate contributes to the neuropathology of 3-ureidopropionase deficiency and severe propionic aciduria: a hypothesis.

3-Ureidopropionate (3-UPA) is a physiologic metabolite in pyrimidine degradation. Pathological accumulation of 3-UPA in body fluids is found in 3-ureidopropionase deficiency and severe forms of propionic aciduria. Both diseases clinically present with a severe neuropathology involving gray and white matter as well as with a dystonic dyskinetic movement disorder. To date nothing is known about the toxic nature of this metabolite. The aim of the present study was to elucidate whether 3-UPA may act as endogenous neurotoxin. Exposure of cultured chick neurons to 3-UPA induced a concentration- and time-dependent neurodegeneration. Neuronal damage was reduced by the antioxidant alpha-tocopherol and the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801. In contrast, the non-NMDA receptor antagonist CNQX, the metabotropic glutamate receptor antagonist L-AP3, and succinate showed no protective effect. Furthermore, 3-UPA elicited an increased production of reactive oxygen species followed by a delayed increase in intracellular calcium concentrations. Activity measurement of single respiratory chain complexes I-V revealed an inhibition of complex V activity, but not of the electron-transferring complexes I-IV by 3-UPA. In contrast, 3-UPA did not affect the mitochondrial beta-oxidation of fatty acids. In conclusion, our results provide strong evidence that 3-UPA acts as endogenous neurotoxin via inhibition of mitochondrial energy metabolism, resulting in the initiation of secondary, energy-dependent excitotoxic mechanisms.