Hereditary haemolytic anaemias are genetically and phenotypically heterogeneous disorders characterized by increased red cell destruction, with consequences ranging from innocuous to severe life-threatening anaemia. Diagnostic laboratories endeavour to assist clinicians reach the exact patient diagnosis by using tests principally based on morphological and biochemical techniques. However, these routine studies may be inconclusive, particularly in newborn infants and when transfusions have recently been administered. Large numbers and size of the potentially involved genes also impose a practical challenge for molecular diagnosis using routine sequencing approaches. To overcome these diagnostic shortcomings, we have utilized next-generation sequencing to provide a high-throughput, highly sensitive assay. We developed a panel interrogating 28 genes encoding cytoskeletal proteins and enzymes with sequencing coverage of the coding regions, splice site junctions, deep intronic and regulatory regions. We then evaluated 19 samples, including infants with unexplained extreme hyperbilirubinaemia and patients with transfusion-dependent haemolytic anaemia. Where possible, inheritance patterns of pathogenic mutations were determined by sequencing of immediate relatives. We conclude that this next-generation sequencing panel could be a cost-effective approach to molecular diagnosis of hereditary haemolytic anaemia, especially when the family history is uninformative or when routine laboratory testing fails to identify the causative haemolytic process.

The online version of this article contains a supplementary appendix. BackgroundMany different techniques have been designed for the quantification of JAK2V617F allelic burden, sometimes producing discrepant results. Design and MethodsJAK2V617F quantification techniques were compared among 16 centers using 11 assays based on quantitative polymerase chain reaction (with mutation-specific primers or probes, or fluorescent resonance energy transfer/melting curve analysis), allele-specific polymerase chain reaction, conventional sequencing or pyrosequencing. ResultsA first series of blinded samples (granulocyte DNA, n=29) was analyzed. Seven assays (12 centers) reported values inside the mean±2SD; the mean coefficient of variation was 31%. Sequencing techniques lacked sensitivity, and strong discrepancies were observed with four techniques, which could be attributed to inadequate standards or to different modes of expression of results. Indeed, quantification of JAK2V617F in relation to another control gene produced higher than expected values, suggesting the possibility of more than two JAK2 copies/cell. After calibration of assays with common 1% to 100% JAK2V617F standards (dilutions of UKE-1 cells in normal leukocytes), 14 centers tested ten new samples. JAK2V617F allelic burdens greater or equal than 1% were then reliably quantified by five techniques -one allele specific-polymerase chain reaction and four TaqMan allele-specific quantitative polymerase chain reaction assays, including one previously giving results outside the mean±2SD -with a lower mean coefficient of variation (21%). Of these, only the two TaqMan allele-specific quantitative polymerase chain reaction assays with primerbased specificity could detect 0.2% JAK2V617F. ConclusionsTechniques expressing the allelic burden as JAK2V617F/total JAK2 and using a common set of standards produced similar quantification results but with variable sensitivity. Calibration to a reference standard improved reproducibility.Key words: JAK2V617F, quantification, standardization, allele-specific PCR, myeloproliferative diseases, multicenter study. Citation

Most Tibetans are protected from polycythemia while living in high altitude. An EGLN1 co-adapted haplotype, EGLN1 c.12C>G, c.380G>C is uniquely Tibetan. The Tibetan EPAS1 haplotype has introgressed from the Denisovan genome. While EGLN1 and EPAS1 genotypes lower Hb, this study indicates additional Hb modifiers.

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BackgroundPulmonary hypertension and left ventricular diastolic dysfunction are complications of sickle cell disease. Pulmonary hypertension is associated with hemolysis and hypoxia, but other unidentified factors are likely involved in pathogenesis as well.Design and MethodsPlasma concentrations of three angiogenic markers (fibroblast growth factor, platelet derived growth factor–BB [PDGF-BB], vascular endothelial growth factor [VEGF]) and seven inflammatory markers implicated in pulmonary hypertension in other settings were determined by Bio-Plex suspension array in 237 children and adolescents with sickle cell disease at steady state and 43 controls. Tricuspid regurgitation velocity (which reflects systolic pulmonary artery pressure), mitral valve E/Edti ratio (which reflects left ventricular diastolic dysfunction), and a hemolytic component derived from four markers of hemolysis and hemoglobin oxygen saturation were also determined.ResultsPlasma concentrations of interleukin-8, interleukin-10 and VEGF were elevated in the patients with sickle cell disease compared to controls (P≤0.003). By logistic regression, greater values for PDGF-BB (P = 0.009), interleukin-6 (P = 0.019) and the hemolytic component (P = 0.026) were independently associated with increased odds of elevated tricuspid regurgitation velocity while higher VEGF concentrations were associated with decreased odds (P = 0.005) among the patients with sickle cell disease. These findings, which are consistent with reports that PDGF-BB stimulates and VEGF inhibits vascular smooth muscle cell proliferation, did not apply to E/Etdi.ConclusionsCirculating concentrations of angiogenic and pro-Inflammatory markers are altered in sickle cell disease children and adolescents with elevated tricuspid regurgitation velocity, a subgroup that may be at risk for developing worsening pulmonary hypertension. Further studies to understand the molecular changes in these children are indicated.

Manganese peroxidase (MnP) produced by Phanerochaete chrysosporium, which catalyzes the oxidation of Mn(2+) to Mn(3+) by hydrogen peroxide, was shown to be susceptible to thermal inactivation due to the loss of calcium [Sutherland, G. R. J.; Aust, S. D. Arch. Biochem. Biophys. 1996, 332, 128-134]. The recombinant enzyme, lacking glycosylation, was found to be more susceptible [Nie, G.; Reading, N. S.; Aust, S. D. Arch. Biochem. Biophys. 1999, 365, 328-334]. On the basis of the properties and structure of peanut peroxidase, we have engineered a disulfide bond near the distal calcium binding site of MnP by means of the double mutation A48C and A63C. The mutant enzyme had activity and spectral properties similar to those of native, glycosylated MnP. The thermostabilities of native, recombinant, and mutant MnP were studied as a function of temperature and pH. MnPA48C/A63C exhibited kinetics of inactivation similar to that of native MnP. The addition of calcium decreased the rate of thermal inactivation of the enzymes, while EGTA increased the rate of inactivation. Thermally treated MnPA48C/A63C mutant was shown to contain one calcium, and it retained a percentage of its original manganese oxidase activity; native and recombinant MnP were inactivated by the removal of calcium from the protein.

haemoglobin concentration in sickle cell anaemia by a mechanism other than increased haemolysis.